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Title:
The role of norepinephrine in learning cerebellar motor learning in rats
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English
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Paredes, Daniel A
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University of South Florida
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Neurotransmitters
Eyeblink conditioning
Cerebellum
Classical conditioning
Microdialysis
Aging
Dissertations, Academic -- Molecular Pharmacology and Physiology -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Delay classical eyeblink conditioning is an important model of associative, cerebellar dependent learning. Norepinephrine (NE) plays a significant modulatory role in the acquisition of learning; other neurotransmitter systems are also at play. The goal of this dissertation was to determine whether NE, GABA and glutamate (Glu) release is observed in cerebellar cortex during delay eye blink conditioning, and whether such release was selectively associated with training and not due only to stimulatory sensory input. The data support the hypothesis of noradrenergic and GABAergic system involvement in motor learning with NE as a modulator of early responding and GABA as a mediator of the learned response. In addition to neurotransmitter levels, we found that the local administration into the cerebellum of Rp-cAMP and propranolol impair the consolidation of learning when administered post training on the eyeblink conditioning task indicating that the B-adrenergic receptor and the cAMP downstream signaling cascade are essential for memory consolidation. These results support the hypothesis of NE acting as a neuromodulator in the cerebellum for the acquisition of motor learning. A similar experimental design was applied to aged animals and the neurochemical pattern of release was haracterized by a delay in the response to eyeblink conditioning and smaller amounts of the neurotransmitter evoked by the paired US-CS. It is hypothesized that the impairment in aging could be due to excitotoxicity caused by chronic inflammation. The present study also approached this issue by targeting the pro-inflammatory cytokine TNF-a and we found that suppression of TNF-a in aged animals improved learning.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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by Daniel A. Paredes.
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Document formatted into pages; contains 144 pages.
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Includes vita.

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ABSTRACT: Delay classical eyeblink conditioning is an important model of associative, cerebellar dependent learning. Norepinephrine (NE) plays a significant modulatory role in the acquisition of learning; other neurotransmitter systems are also at play. The goal of this dissertation was to determine whether NE, GABA and glutamate (Glu) release is observed in cerebellar cortex during delay eye blink conditioning, and whether such release was selectively associated with training and not due only to stimulatory sensory input. The data support the hypothesis of noradrenergic and GABAergic system involvement in motor learning with NE as a modulator of early responding and GABA as a mediator of the learned response. In addition to neurotransmitter levels, we found that the local administration into the cerebellum of Rp-cAMP and propranolol impair the consolidation of learning when administered post training on the eyeblink conditioning task indicating that the B-adrenergic receptor and the cAMP downstream signaling cascade are essential for memory consolidation. These results support the hypothesis of NE acting as a neuromodulator in the cerebellum for the acquisition of motor learning. A similar experimental design was applied to aged animals and the neurochemical pattern of release was haracterized by a delay in the response to eyeblink conditioning and smaller amounts of the neurotransmitter evoked by the paired US-CS. It is hypothesized that the impairment in aging could be due to excitotoxicity caused by chronic inflammation. The present study also approached this issue by targeting the pro-inflammatory cytokine TNF-a and we found that suppression of TNF-a in aged animals improved learning.
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The Role Of Norepinephrine in Learning: Cerebellar Motor Learning in Rats by Daniel A. Paredes A dissertation submitted in partial fulfillment of the requirement s for the degree of Doctor of Philosophy Department of Molecular P harmacology and Physiology College of Medicine University of South Florida Co-Major Professor: Paula Bickford, Ph.D. Co-Major Professor: Lynn Wecker, Ph.D. Jahanshah Amin Ph.D. Javier Cuevas Ph.D. Cheryl Kirstein Ph.D. David Morgan Ph.D. Date of Approval: March 28, 2007 Keywords: Neurotransmitters, eyeblink conditi oning, cerebellum, classical conditioning, microdialysis, aging Copyright 2007, Daniel A. Paredes

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DEDICATION This work is dedicated to my mother, father, sister and every person I have encountered in my journey. To the memory of Vicente Gallardo S ilva and Laszlo Szilasi, who taught me the meaning of seeking true reas on. And to my role model as a scientist and human being, Dr. Luis Francisco Hernandez.

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ACKNOWLEDGEMENTS Very special thanks to my major professo r, Dr. Paula Bickford, for her guidance and cheering words every time I needed them and for being an exceptional human. I really appreciate the constant support from Dr. Jahanshah and Dr. Cuevas, whom always encouraged me to pursue the best. Special thanks to all the people in the animal facility who have done an excellent job making possible that each rat used in this study lived wit h dignity. To my soul and life mate, Briony. My sincere gratitude to: All the lab mates. The undergraduate students who parti cipated in the experiments. VAH research personnel, in especially to John Soto whose bright energy brings encouragement to anybody open to it. This work was supported by NIH grants AG15490 and AG18478.

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i TABLE OF CONTENTS LIST OF FIGURES ......iv ABSTRACT .vi CHAPTER 1: Introduction ...1 1.1 Eyeblink Conditioning .....3 1.2 Norepinephrine and its involvement on motor learning ..7 1.2.1 NE and memory 1.2.2 The action of NE in the cerebellum 1.2.3 Behavioral effects of NE 1.2.4 The NE signal transduction cascade and learning in the cerebellum 1.2.5 Cerebellar moto r learning: LTD and LTP 1.3 Intracerebral Microdialysis ...19 1.4 Other neurotransmitters ...20 1.4.1 Glutamate 1.4.2 GABA 1.5 Aging as a process in which the modulatory effect of NE on PC is compromised....23 CHAPTER 2: Norepinephrine, GABA and Glutamate as a Substrate of Memory Formation in Cerebellar Eye Blink Conditioning 2.1 Abstract........26 2.2 Introduction ..27 2.3 Methods....30 2.3.1 Animals and surgery 2.3.2 Microdialysis procedure 2.3.3 Training

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ii 2.3.4 Microdialysis and ey e-blink conditioning procedure 2.3.5 Neurochemical analysis 2.3.6 HPLC: Norepinephrine 2.3.7 Capillary electrophoresis: Glu and GABA 2.3.8 Derivatization 2.3.9 Histology 2.3.10 Statistical analyses 2.4 Results 2.4.1 Young Rats 2.4.1.1 Norepinephrine 2.4.1.2 GABA 2.4.1.3 Glutamate 2.4.1.4 Tetrodotoxin (TTX) effect on neurotransmitter release 2.4.2 Aging Rats 2.4.2.1 Norepinephrine 2.4.2.2 GABA 2.4.2.3 Glutamate 2.5 Conclusions 2.5.1 Norepinephrine 2.5.2 GABA 2.5.3 Glutamate 2.5.4 Effect of TTX on neurotransmitter release 2.5.5 Conclusions CHAPTER 3: Beta-Noradrene rgic Receptors in the Cerebellum Are Involved in Acquisition of Delay Classical Conditioning in Rats: Timing of Disruption ...64 3.1 Abstract ......64 3.2 Introduction 3.3 Methods....68 3.3.1 Animals and surgery 3.3.2 Training 3.3.3 Design and Analysis 3.4 Results.. 3.5 Conclusions ...

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iii CHAPTER 4: TNFinactivation improves learning in aged rats whereas the activation of TNFin young rats depletes learning ...83 4.1 Abstract ..83 4.2 Introduction.. 4.3 Methods....87 4.3.1 Animals and surgery 4.3.2 Training of behavior in a delay classical eyeblink conditioning task 4.3.3. Treatment with rrTNFand anti-rat TNF4.3.4 Design and Analysis 4.4 Results.. 4.4.1 Young 4.4.2 Aging 4.5 Conclusions.. 4.5.1 TNFin young rats 4.5.2 TNFin aging rats CHAPTER 5: Conclusions ...101 REFERENCES ABOUT THE AUTHOR .end page

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iv LIST OF FIGURES CHAPTER 1. Introduction FIGURE 1.1 Rat in eyeblink chamber 4 FIGURE 1.2 Norepinephrine sign al transduction cascade 17 FIGURE 1.3Microdialysis coupled to eyeblink conditioning 20 CHAPTER 2. Norepinephrine and its involvement on motor learning FIGURE 2.1 Effect of TTX on cerebellar dependent eyelid conditioning. 36 FIGURE 2.2. Temporal release of norepinephrine during eyelid conditioning. 38 FIGURE 2.3. Temporal rel ease of GABA during eyelid conditioning. 41 FIGURE 2.4. Temporal release of glutamate during eyelid conditioning. 43 FIGURE 2.5. Tetrodotoxin (TTX ) effect on neurotransmitter levels during eyelid conditioning. 46 FIGURE 2.6. Cerebel lar dependent eyeblink conditioning is impaired in aging. 48 FIGURE 2.7. Temporal rel ease of norepinephrine during eyelid conditioning in aged rats. 50 FIGURE 2.8. Temporal rel ease of GABA during eyelid conditioning in aged rats. 51 FIGURE 2.9. Temporal release of glutamate during eyelid conditioning in aged rats. 53

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v CHAPTER 3. Beta-Noradrenergic Receptors in the Cerebellum Are Involved in Acquisition of Delay Classical Conditioning in Rats: Timing of Disruption FIGURE 3.1 Temporal dynami cs of propanolol on eyeblink conditioning: Conditioned responses. 72 FIGURE 3.2. Temporal dynam ics of propanolol on eyeblink conditioning: Amplitude. 74 FIGURE 3.3. The effect of Rp -cAMP on eyeblink conditioning. 75 CHAPTER 4. TNFinactivation improves l earning in aged rats whereas the activation of TNFin young rats depletes learning FIGURE 4.1 Effects of TNFin young rats. 92 FIGURE 4.2. Effects of blockade of TNFin aging rats. 95

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vi The Role Of Norepinephrine in Learning: Cerebellar Motor Learning in Rats Daniel A. Paredes ABSTRACT Delay classical eyeblink conditioning is an import ant model of associative, cerebellar dependent lear ning. Norepinephrine (NE) plays a significant modulatory role in t he acquisition of learning; other neurotransmitter systems are also at pl ay. The goal of this dissertation was to determine whether NE, GABA and glutamate (Glu) release is observed in cerebellar cortex duri ng delay eye blink conditioning, and whether such release was selectively associated with training and not due only to stimulatory sensory input. The data support the hypothesis of noradrenergic and GABAerg ic system involvement in motor learning with NE as a modulator of early re sponding and GABA as a mediator of the learned response. In addition to neurotransmitter levels, we found that the local administration into the cerebellum of Rp-cAMP and propranolol impair the cons olidation of learning wh en administered post training on the eyeblink condit ioning task indicating that the adrenergic receptor and the cAMP downstream signaling cascade are

PAGE 10

vii essential for memory consolidat ion. These results support the hypothesis of NE acting as a neuromodulator in the cerebellum for the acquisition of motor learning. A similar experimental design was applied to aged animals and the neuroche mical pattern of release was haracterized by a delay in the res ponse to eyeblink conditioning and smaller amounts of the neurotransmitte r evoked by the paired US-CS. It is hypothesized that the impairm ent in aging could be due to excitotoxicity caused by chronic infl ammation. The present study also approached this issue by targeting the pro-inflammatory cytokine TNFand we found that suppression of TNFin aged animals improved learning.

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1 CHAPTER 1 Introduction In this dissertation it is hypothesized that the release of norep inephrine (NE) in the cerebellum is necessary to activate the -adrenergic receptor and its signal transduction cascade which are critic al for cerebellar motor learning consolidation. This thesis has focused on the behavioral paradigm of classical eyeblink conditioning which is a well c haracterized cerebellar-dependent motor learning task. Despite the accumulati on of work showi ng that NE in the cerebellum modulates the rate of acquisi tion of cerebellar dependent learning, little is known about the nor adrenergic downstream tempor al events, specifically the role of cyclic adenosine monophosph ate (cAMP) dependent protein kinase A (PKA). Other neurotransmitters such as ga mma-aminobutyric acid (GABA) and glutamate (Glu) are hypothesized to play an important role in cerebellar motor learning. We hypothesized that GABA acts as a mediator of the learned response in the cerebellar cortex durin g the training and th e pattern of GABA release becomes strengthened and sharpened in response to the behavioral task following several days of training. Furthe rmore, we hypothesize that increases in extracellular glutamate levels ar e observed as a consequence of the somatosensory projections to the cerebellu m during training. Interestingly, there is an age-related decline in memory func tion in which neurotransmitter release is compromised perhaps as a result of chroni c inflammation. For this reason we hypothesized that TNFinactivation improves learning in aged rats whereas the

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2 activation of TNFin young rats impairs learni ng since inflammation and more specifically TNFmay be a critical factor that is involved in the decline in classical conditioning behavio r during the aging process. Evidence of memory formation shows that distinct molecular mechanisms contribute to the multiphasic process of memory formation, however, little is known about the biochemical pathw ays underlying the induction and maintenance of these different memory phases. It has been shown that NE in the cerebellum modulates the rate of ac quisition for a variet y of motor learning tasks among them delayed classi cal eyelid conditioning. Many studies in diverse animal models lead to a convergence in the temporal dynamics of signaling cascades and how these cascades contribute to the distinct aspects of learning. Norepinephrine has been shown to have a modulatory role in cerebellar dependent learning. Noradrenergi c inputs from the locus co eruleous (LC) inhibit spontaneous discharge but app ear to augment the signal-t o noise ratio for both excitatory and inhibitory neurotransmissi on, however little is known regarding noradrenergic upstream and down stream temporal events including the signal transduction cascades with t he implication of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in both long and short-term memory formation in numerous parts of the brain and spec ially in cerebellum which is our major focus. Our lab has recently show n that discrete localized blockade of adrenergic receptors or PKA in the cerebel lum caused a significant deficit in acquisition of eyelid classical conditioni ng. As a consequence this result has led

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3 us to our current interest in studyi ng this mechanism of cerebellar memory formation. This dissertation co mbined pharmacological and neurochemical approaches to uncover different aspects of memory forma tion and molecular mechanisms using delay eyeblink conditioning as the behavioral task. The delay form of eyelid conditioning is a well-characterized system in which t he implication of the cerebellum is clear and localiz ed effects of learning can be measured before, during and after acquisition of tr aining in different discrete cerebellar areas such as lobule HVI and interpositus nucleus (see (Steinmetz, 2000)). 1.1. Eyeblink Conditioning Classical conditioning of the eye blink is a type of Pavlovian conditioning which is one of the simplest forms of associativ e learning by which animals, including humans, learn relations among events in the world so that thei r future behaviors are better adapted to their environments. In this method a discrete conditioned stimulus (CS) is paired with a discrete unconditioned stimulus (US) with particular temporal relationships between the CS a nd US. The untrained animal exhibits the eyeblink response only to the US; this unlearned behavior to the US is referred to as the unconditioned response (UR). Over the course of training sessions, the animal develops a conditi oned response (CR) to the CS that mimics the UR, precedes the US in onse t time, and peaks at about the time of US onset. As only two stimuli are involved, the learning or a ssociation of CS and US has to occur at the brain sites wher e the two forms of information converge (Rescorla, 2003; Kim & Thom pson, 1997). The majority of work in this model

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4 system has been performed in rabbits usin g the nictitating membrane, however more recently work has been performed in ra ts and mice. In the present studies we examine delay eyeblink conditioning in ra ts. Figure 1.1 is an illustration of the headgear design where the rats move freely in a training chamber that sits inside a sound-attenuating box. The discovery of a high proportion of cerebellar inhibitory synapses was the highlight of the cerebellar neuronal circ uitry described in over 14 articles published between 1961 and 1966 by John C Eccles (Eccles et al., 1961; Eccles, 1967; Eccles, 1986). Eccles established basis to dissect out the neuronal circuitry of the cerebellum by taki ng advantage of recording and stimulation techniques with a high degree of temporal re solution (for review see (Ito, 2006). Air puff EEG recording wire Figure 1.1. Rat in eye blink chamber with the head stage holding the EEG recording wire and the connection to the air puff tubing.

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5 David Marr published ‘A theor y of cerebellar cortex’ in which he proposed two forms of input-output relations, one su itable for learning movements and the other for the maintenance of reflexes su ch as postures and balance (Marr, 1969). Marr’s theory predicted that synapses from parallel fibers to Purkinje cell (PC) are facilitated by the conjunc tion of presynaptic and climbing fiber activity. On the other hand, Eccles proposed that only tw o kinds of afferent fibers convey information to the cerebellum, the clim bing fibers and mossy fibers with only one type of efferent fiber from the cerebellum, the PC which projects to the deep cerebellar nuclei (Eccles, 1967). Eccles’s es tablished that the climbing fiber is uniquely distributed to a single PC on whic h it has a powerful excitatory action whereas the mossy fiber input is characte rized by enormous divergence and it has excitatory as well as inhibitory ac tions on PC’s. Interestingly, Eccles assumed that in the cerebellar cortex th e transfer of information from each small zone (beam) to another is not significant because the only association pathways would be by the Purkinje axon collateral and the basket cells which are weakly inhibitory. Later, evidence was establishe d for noradrenergic central inhibition in the cerebellum when the pathway from LC to rat cerebellar PC are activated, this finding elucidated anot her important cerebellar input fr om the LC (Hoffer et al., 1973). Following this, several studies examined the effect of NE on the cerebellar PC. For instance, NE applied by microiontophoresis to rat cerebellar PC selectively depressed spontaneous neur onal discharges showing that NE increased the responsiveness of PC to affer ent inputs, also it was found that NE exerted its acti on on PC through -adrenergic receptors (Parfitt et al., 1990;

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6 Parfitt et al., 1988; Freedman et al., 1976; Freedman et al., 1977; Moises et al., 1979; Woodward et al., 1991b). Several decades after the anatomy and dynami cs of the cerebellar circuitry were established there are still general disagr eements in regard to whether learning occurs in the cerebellum. The plasticity for cerebellar learning is shared between the cortex and deep nuclei, but exactly how much each of these structures contribute to memory formation is still under debate. Extensive work has been done using very small and discrete intervent ions to impair either the cerebellar cortex or interpositus nucleus of the ce rebellum. The issue is not whether the memory occurs in the cerebellum, but whet her it happens in the cerebellar cortex or in the deep nuclei or may be in both. In order to address these questions several approaches have been used, such as electrolytic lesions using kainic acid and temporary inactivation (cooling, muscimol, anisomycin) to localize the learning process to the interpositus nucleus (Lavond, 2002) concluded that the interpositus nucleus is res ponsible and this is also s upported by published works (Mojtahedian et al., 2007; Thompson et al., 1997). However, other studies claim that memo ry formation in the cerebellum during eyeblink conditioning might be mediated mostly in the cerebellar cortex (Sanchez et al., 2002; Yeo, 2004; De Zeeuw & Yeo, 2005). In either case, lesions of the cerebellar cortex, interposit us nuclei or inferior olive prevent or diminish the expression of conditioned responses. Nevertheless, there is

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7 agreement that all these structures are interconnected and rely on each other to perform the overall process of conditi oned learning (Attwell et al., 2002b) 1.2. Norepinephrine and its in volvement on motor learning NE chemically known as 4-(2-Amino-1-hy droxyethyl)benzene-1,2-diol is both a hormone and neurotransmitter. In the peripheral nervous system NE is secreted by the adrenal medulla and the nerve endings of the sympathetic nervous system to cause vasoconstriction, an increase in heart rate, blood pressure, and blood glucose. The LC is the major brain ar ea containing NE in the central nervous system (CNS) (Loughlin et al., 1986; Foot e et al., 1983; Luppi et al., 1995; AstonJones et al., 1991). Projections from the LC innervate multiple brain areas which are involved with process such as l earning, memory, attention and anxiety (Vavrejnova et al., 1990). Human clinical therapies in traumatic stress relate NE to enhanced encoding of memory for arousing stimuli as well as aversive events. Therapeutic approaches using drugs such as propranolol ( -adrenergic antagonist), MAO inhibitors and tricyc lic antidepressants are currently and effectively being implemented (for re view see(Southwick et al., 1999). Multiple activities have been associated to the activation of adrenergic receptors in the CNS. There are ten different types of adrenergic receptors in the nervous system; 1A, 1B, 1C, 1D, 2A, 2B, 2C, 1, 2 and 3. Depending on the type of adrenergic receptor activated by NE, the spatial distribution, relative density of receptors subtypes, affinities and selectivity will determine when and

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8 how the adrenergic receptors will be par t of the memory formation process (Gibbs & Summers, 2002). Cerebellar NE has been extensively studi ed in relation to associative motor learning in rats (Mason & Iversen, 1977; Bickford, 1993) and has been proposed to mediate cerebellar synapses (Bloom et al., 1972; Freedman et al., 1977). Also pharmacological evidence of noradr energic central inhibition has been established by activating LC and recordi ng in rat cerebellar PC (Hoffer et al., 1973). It has been shown that the local administration of propranolol injected prior to NE decreased cerebellar cycl ic GMP (Haidamous et al., 1980). Noradrenergic receptors are known to be involved in cer ebellar learning by increasing the "signal to noise" ratio of afferent inputs to cerebellar Purkinje neurons (Yeh & Woodward, 19 83b; Woodward et al., 1991a). Most recently it has been suggested that cerebellar NE enhances GABA release by 1adrenoceptors, which are ex pressed in presynaptic terminals and somatodendritic domains, whereas NE suppresses the excitability of interneurons by 2-adrenoceptors, which are expressed in presynaptic somatodendritic domains. This finding reveals the dual modulation of GABAergic inputs from interneurons to PCs as a possible mechan ism for the fine-tuning of information flow in the cerebellar cortex (Hirono & Obata, 2006). On the other hand, studies using halothane-anesthetized rats have sh own that within t he medial septum, stimulation of the beta-adrenergic rec eptors by isoproterenol can mimic the

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9 arousing effect of LC stimulation, while antagonist infusion blocks this effect (Berridge et al., 1996). Motor skill learning requires repetitive training sessions, and there is a great amount of evidence that associative me mory is a cerebellar dependent process (Jirenhed et al., 2007). Neuroimaging studi es have reported clear cerebellar activation during the acquisition of a moto r skill, followed by low activation after prolonged practice on a proc edural learning or skill ac quisition (Petersen et al., 1998; van Mier & Petersen, 2002). Using t he vestibule-ocular reflex task (VOR), the modulatory role of NE has been shown. In these studies the administration of a -adrenergic antagonist sign ificantly reduced the adapt ation of the VOR gain showing that the blockade of -adrenergic receptors significantly decrease the ability to produce adaptative ch anges in VOR gain (Van et al., 1990; Heron et al., 1996). Brain areas other than the cerebellum are also in volved in the acquisition and retention of skilled motor tasks (Sanes, 2000). For example, in the rotarod task mice show differential corticostriatal plasticity during fast and slow motor skill learning (Costa et al., 2004). These reports are consistent with the hypothesis that the -adrenergic receptor is involved in t he acquisition of novel motor tasks. 1.2.1. NE and memory A role for norepinephrine in learning and memory has been elusive and controversial. A longstanding hypothesis states that the adrenergic nervous system mediates enhanced memory consolidat ion of emotional events and it this

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10 hypothesis has been tested using seve ral learning tasks in mutant mice conditionally lacking norepi nephrine and epinephr ine, as well as control mice and rats treated with adrenergic receptor agonists and antagonists. Murchison and colleagues found that adrenergi c signaling is critical for the retrieval of intermediate-term contextual and spatial memories, but is not necessary for the retrieval or consolidation of emotional memories in general (Murchison et al., 2004). The role of NE in retrieval requires signaling through the 1-adrenergic receptor in the hippocampus. Murchi son demonstrated that mechanisms of memory retrieval can vary over time and can be different from those required for acquisition or consolidation. These fi ndings may be relev ant to symptoms in several neuropsychiatric disorders as well as the treatment of cardiac failure with -blockers. Activation of the central noradrenergic system is associated with increased arousal, orienting to novel st imuli, selective attention, enhanced memory, and cardiovascular responses (Southwick et al., 2003). In rats, eliminating the central nor adrenergic system leads to impaired fear conditioning (Neophytou et al., 2001), and in humans pharmacological suppression of noradrenergic responses with a -adrenoceptor (AR) ant agonist impairs memory of emotional events (van Stegeren et al., 1 998). NE seems to play rather a role in the maintenance as well as for the induc tion of long term potentiation (LTP) in the dentate gyrus. Application of NE or -adrenergic receptor agonists can induce LTP phenomena in the DG (Dahl & Sarvey, 1989; Stanton et al., 1989; Dahl & Li, 1994) and NE depletion or -adrenergic receptor blockade have been shown to result in impaired DG-LTP in brain slices and anaesthetized animals

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11 (Swanson-Park et al., 1999; Bliss et al., 1983; Stanton & Sarvey, 1987; Stanton & Sarvey, 1985; Bramham et al., 1997). It is clear that NE plays an important role in memory formation as well as arousal states. 1.2.2 The action of NE in the cerebellum A number of modulatory signa ls are needed for powerful cerebellar learning and control in particular NE. The NE input to the cerebellum is the second largest modulatory input and distributes to all part of the cerebellar cortex with a patchy innervation pattern. The nor adrenergic fibers project to all parts of the cerebellar cortex and originate from t he dorsal and ventral parts of the LC. These fibers are found both around the glomer uli, making close contacts with granule cell dendrites, and around the PC dendr ites (Kimoto et al., 1978) It is known that cultured cerebellar granule cells express ad renergic receptors (Dillon-Carter & Chuang, 1989) but due to the difficulty of recording from granule cells, there are not significant data sh owing the direct effect of NE upon granule cells. However, NE has been shown to increase cAMP in granule cells as well as astrocytes an important key messenger of neuronal plasticity (Morton & Bredt, 1998). Furthermore NE inhibits spontaneous PC discharge (Bickford et al., 1985a) possibly through both presynaptic adrenergi c receptors on basket cells (Mitoma & Konishi, 1996) an d enhancement of spontaneous sp ike firing of basket cells (Saitow & Konishi, 2000). Ho wever, unlike serotonin, and relative to the change in spontaneous activity, norepinephrine in creases the responsiv eness of the PC to its afferent excitatory inputs (Freedman et al., 1976 ). J.E. Cheun and H.H.

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12 Yeh., showed that noradrenergic potentiati on of cerebellar PC responses to GABA is mediated by cyclic AMP as intracel lular intermediary, in other words NE exerts two types of long-term influences on PC, the activation of the betaadrenergic receptors resulting in a raised intracellular leve ls of cyclic AMP, which leads to an increase of cyclic AMP-depe ndent protein kinase activity (Cheun & Yeh, 1996). This action of NE suggests t hat it can enhance r ebound potentiation, which requires elevation of intracellular cAMP le vel (Kano et al., 1992b), and antagonize the suppression of rebound potentiation, which requires the suppression of cAMP level (Kano et al., 1992a). On the other hand, NE increases the expression of immediate-early genes, such as c-fos and Jun-B, in PC. Induction of immediate-early genes coul d then represent a mechanism by which sustained inputs are transformed into l ong-term biochemical changes that are required for the maintenance of cerebellar long-term plastici ty, such as long term depression (LTD) (Pompeiano, 1998). NE receptors have also been found in the cerebellar nuclei, and NE modulates t he GABAergic neurons inhibition of deep cerebellar neurons (Gould et al., 1997c). 1.2.3. Behavioral effects of NE Naka et al, (2002) have had shown that an enriched environm ent specifically increases NE concentration in the parieto-temporo-occi pital cortex, cerebellum and pons/medulla oblongata (by 17 .0–37.5%) without changing the concentrations of NE, 5-HT or DA in ot her brain regions, including the frontal cortex and hippocampus. This finding agr ees with our working hypothesis of NE

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13 playing a important role on cerebellar moto r learning, also NE has been shown to facilitate synaptic plasticity to regulate oc ular dominance plasticity (Brocher et al., 1992) and to enhance learning and memory vi a the activation of beta-adrenergic receptors (Devauges & Sara, 1991). Accord ingly, it is likely that the increased concentrations of extracellular NE wi ll be observed following delay eyeblink conditioning which must be involved in synaptic plasticity and possible changes in leaning and memory. Several author s (Watson & McElligott, 1983; Watson & McElligott, 1984; McElligott & Keller, 1984); by using motor learning paradigms have established a modulatory role of NE in cerebellar dependent motor learning behaviors. On the other hand, either depletion of NE or blockade of -adrenergic receptors impairs the ability of rats to improve performance on a runway task where the rats have to learn to walk on varying patterns of pegs that protrude from the runway walls (W atson & McElligott, 1983; Watson & McElligott, 1984; Bickford et al., 1992). Selective depletio n of cerebellar NE produces a decline in acquisition of this task (Watson & Mc Elligott, 1984) and furthermore the data from our lab shows that infusions of pr opranolol into the paramedian lobe of the cerebellum impair learning (Cartford et al., 2004b). Motor l earning impairments in aged rats are correlated with the loss of cerebellar -adrenergic receptor sensitivity (Bickford, 1993; Bickford, 1995). The behavioral impairment is noticed as a decrease in the rate of acquisition on the task, rather than a complete blockade of learning. Another motor le arning task is the classical eyelid conditioning which has been linked to ce rebellar modulation when the delayed paradigm is used. In this task the uncond itioned stimulus (US) is either an air

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14 puff or eye shock, the signal from whic h enters the cerebellum via the climbing fibers. The conditioned st imulus (CS) is usually a tone and is relayed via the granule cell-parallel fiber synaps e. Subjects learn to link or associate the air puff with the tone and a new response is forme d (a conditioned response) to the tone that anticipates the air puff. Conditioning related activity has been established for neurons within lobule HVI and the interposit us nucleus in rabbits as well as in rats (Berthier & Moore, 1986; Gould & St einmetz, 1994; Rogers et al., 2001b). Lesions of the cerebellar lobule HVI (Y eo et al., 1985; Perrett et al., 1993; Nordholm et al., 1993); and interpositus nu cleus (Clark et al., 1992; Krupa et al., 1993b); abolish the response of the cla ssical eyelid conditioning. Mice containing PC mutant (pcd), which have a complete lack of cerebellar PCs, still acquire the eyelid response (Chen et al ., 1996). Also jaundiced Gunn rats (a mutant with loss of PC) have normal to el evated levels of NE innervation and functional activity in cerebellar cortex and deep nuclei after degeneration of the PC layer (Onozuka et al., 1990; Kostrzewa & Harston, 1986; Ghetti et al., 1981; Clark et al., 1997; Clark & Lavond, 1996; Rogers et al., 2001a). By using electrophysiological techniques to examine the action of NE, NE seems to modulate the activity of other neurotransmitters in disc rete cerebellar areas such as cortex and deep nuclei (Gould et al., 19 97b). On the other hand, the fact of NE modulating this motor learning task is also supported by the capability of propranolol to delay the acquisition of t he eyelid conditioning in rabbits (Gould, 1998) as well as in rats (Cartford et al, 2002). Early studies also showed that by doing electrolytic lesions of the LC a resi stance to extinction in delay conditioning

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15 in rabbits has been observed (McCormi ck & Thompson, 1982). Winsky and Harvey demonstrated that bilateral intraventricular administration of 6hydroxydopamine causes retarded acquisition but not performance of conditioned eyeblink responses (Winsky & Harvey, 1992). Another cerebellar learning paradigm is the vestibulo-ocular reflex (VOR), has been demonstrated to be influence by cerebellar NE (McElligo tt & Freedman, 1988) and appears to be to mediated by -adrenergic receptors since the -adrenergic antagonist sotalol decreases the adaptation of the VOR gain when microinj ected into the cerebellar flocullus of the rabbit (Pompeiano et al ., 1991). As we can see there is an accumulation of evidence that clearly sh ows a significant role of NE in the acquisition phase of cerebellar dependent l earning. It has been established that PCs receive excitatory inputs from par allel and climbing fibers, inhibitory GABAergic inputs from basket and stellate interneurons, and a noradrenergic input from the pontine nucleus LC (Eccles 1967; Hoffer et al., 1973). Later on, NE was proposed as a "modulatory” input due to its capability to induce synaptic plasticity in PCs by selectively improvi ng the “signal to noise” ratio of evoked versus spontaneous activity (Freedman et al., 1977). This enhances the sensitivity of cerebellar neurons to both ex citatory and inhibitory afferent inputs by inhibiting spontaneous discharge, spec ifically with regard to action of NE on inhibitory neurotransmission withi n the cerebellum, when applied iontophoretically or via activation of the LC and potentiates GABA-induced inhibition of cerebellar Purkinje neur ons (Parfitt & Bickford-Wimer, 1990; Woodward et al., 1979; Y eh & Woodward, 1983c; Cheun & Yeh, 1992). Based

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16 upon the Marr-Albus theories of cerebella r motor learning and NE's modulatory action on neurotransmitter function Gilber t proposed that NE should have a role in the consolidation of memory withi n the cerebellum (Gilbert, 1975). The modulatory role of NE in the cerebellum is mediated through the -adrenergic receptor (Yeh & Woodward, 1983d). 1.2.4. The NE signal transduction cascade and learning in the cerebellum. As mentioned above NE is known to be a neuromodulator and can increase the "signal to noise" ratio of afferent inputs to cerebellar Purkinje neurons. This effect is mediated through the -adrenergic receptor (Y eh & Woodward, 1983e; Woodward et al., 1991a) involving the adrenergic signaling cascade which involves the familiar G protein coupled in which adenyl cyclase, cAMP, and PKA are activated and lead to such well studied phenomena as the downstream phosphorylation of cAMP responsive el ement binding protein (CREB) and subsequent expression of genetic material required for protein synthesis (see figure 1.2). The fact that CREB phosphorylation can al so be mediated via Ca++ signaling mechanisms and protein kinase C (PKC) and that multiple signal cascades have been implicated to play a role in learning and memory (see (Selcher et al., 2002; Ma & Huang, 2002; Kornhauser & Greenberg, 1997) and paragraph below on LTD) it is likely that more than on e molecular mechanism is necessary for learning and consolidation of memories in any given brain location.

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17 In other brain areas (such as hippoc ampus), cAMP, PKA and phosphorylated CREB (pCREB) have been implicated as majo r players in the establishment of synaptic changes necessary for both shor t-term and long-term memory formation (Vianna et al., 2000; Baldwin et al., 2002; Taylor et al., 1999; Muller, 2000; NE G protein A C C A M PAKAP PKA cc GABA a Receptors Ad r e n e r g i c r e c e pt o r Purkinje cell Noradrenergic cell (from LC) Figure 1.2 The hypothesis predicts that release of NE from presynaptic terminals acts upon -adrenergic receptors to in crease c-AMP and activation of PKA by releasing the ca talytic subunit (c) from the regulatory subunits. There is a potential regulator y role of AKAP's in this process that is unknown at this time. One known effect of th is cascade is a modulation of GABAa neurotransmission via PKA that is observed in Purkinje cells and also in the cells in the interpositus nucleus (Goul d et al., 1997d). There is a short term increase in GABA neurotransmission (NE is also known to modulate mossy fiber (MF) and parallel fiber (PF) inputs). A second role for PKA activation relates to a potential phosphorylation of CREB and subsequent transcription of proteins that modulate neuronal activity or lead to new synapse formation.

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18 Shobe, 2002). Behavioral studies in long term potentiation (LTP) and long term depression (LTD) support this finding (Nayak et al., 1998; Rotenberg et al., 2000; Huang & Kandel, 1996a; Hu ang et al., 1994). It has been shown in eyelid conditioning that blockade of protein kinas es impairs acquisition but not retention in rabbits (Chen & Steinmetz, 2000). 1.2.5. Cerebellar motor learning: long-term depression (LTD) and long term potentiation (LTP) In the past decade there have been advanc es in understanding the cellular mechanims of LTD at paralle l fiber-purkinje cell synapses. LTD in the cerebellum has been established as a mechanism the activation of voltage-gated Ca+2 channels, ionotropic (AMPA) and metabotropic (mGluR1) glutamate receptors as well as the stimulation of the PKC and nitr ic oxide (NO) formation. Thus, LTD is now well supported by recent experiments on transgenic mice (for review see (Daniel et al., 1998). A prominent, but still controversial hypothesis is that LTD of parallel fiber-PC synapses is at least one substrate for synaptic plasticity that underlies motor learning in the cerebellum. Most of the work done in this area has pointed to an accepted role fo r Ca++, PKC, MAP kinases, protein phosphatases, metabotropic glutamate rec eptors, nitric oxide, among other signaling pathways, yet the vast majority of studies have found no role for PKA in either short term or late-phase LTD (Har tell et al., 2001; Linden, 1994; Linden, 1996; Kawasaki et al., 1999). Late phase LTD is protein synthesis dependent and requires CaMKIV and CRE B (Ahn et al., 1999). A ve ry convincing argument

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19 for the role of LTD and PKC in motor l earning comes from a study of transgenic mice with PC specific inhibition of PKC. These mice do not demonstrate LTD and do not show modulation of gain in VOR (De Zeeuw et al., 1998), but yet, the adaptation mechanism to compensate the no natural condition implicit in the transgenic mice should still be considered. This is in contrast to an obvious role for NE and PKA in at least some fo rms of LTP in the hippocampus (Heginbotham & Dunwiddie, 1991; Huang & Kandel, 1996b; Frey et al., 1993; Roberson et al., 1996). PKA also plays a role in LTP in the cerebellum (Salin et al., 1996; Jacoby et al., 2001; Kimura et al., 1998) specifically, the action of PKA is proposed to be in cerebellar granule ce lls (Linden & Ahn, 1999) however, the regulation of PKA by NE has not been determined. 1.3. Intracerebral Microdialysis Microdialysis is a technique for the in vivo assessment of cerebral neurotransmitters and their metabolit es. The technique has been improved dramatically to reduce probe size in order to target smaller brain regions such as the anterior interpositus nuc leus of the cerebellum, re duce the amount of gliosis which improves the diffusion barrier and hence the levels of neurotransmitters recovered and even extends to the use of multiple probes (Hernandez et al., 1986b). During microdialysis artificial cer ebral spinal fluid (aCSF) is perfused through the internal tube, flows down in to the microdialysis probe and back up the outer tube where it is collected fo r assay. Neurotransmitters and their metabolites diffuse from an area of hi gh concentration to an area of low

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20 concentration (through probe cellulose memb rane) and flow with the perfusate up the outer tubing to the coll ection vial where it is im mediately refrigerated. In order to correlate memory to neurotransmi tter levels in vivo microdialysis was coupled to delay eye blink conditioning (s ee figure 1.3 for illustration of head gear and microdialysis setup). 1.4. Other neurotransmitters Other neurotransmitter systems are also implicated in delay eyeblink conditioning tasks. Administration of the glutam ate AMPA receptor ant agonist CNQX or the GABA-A receptor antagonist picrotoxin into the cerebellar cortex completely and reversibly impairs fully established c onditioned response’s (CR’s), suggesting Head stage-air puff microdialysis probe Figure 1.3. Rat in the eye-blink conditioning chamber with the head stage-air puff and the microdialysis probe tar g etin g the cerebellum.

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21 that GABAergic and glutamatergic trans mission are involved in CR performance (Attwell et al., 2002b). It has been sugges ted that the basal GABAergic output from the cortex onto the in terpositus nucleus modulat es CR expression, whereas timing of CR's is modulated by the stimulus activated inhibition (Bao et al., 2002). The expression of CR’s can be interrupt ed by both GABA mediated inactivation of the interpositus neurons with muscimol as well as up-regulation of activity with picrotoxin (Aksenov et al., 2004). 1.4.1 Glutamate Glutamate (GLU) is an excitatory neurot ransmitter which is essential in the development of synaptic plasticity and is involved in cognitive functions like learning and memory. Glutamate is ubiquitious throughout the mammalian brain and is involved in cellular metabolism (A ttwell, 2000; Petrof f, 2002). GLU is formed by the precusors glutamine and -ketoglutarate and subsequently packaged into vesicles for fu ture release into the synaptic cleft (Tapiero et al., 2002). Glu is released from vesicles in presynaptic terminals by a Ca2+dependent mechanism that involves voltage-dependent calcium channels (Anderson & Swanson, 2000; Meldrum, 2000). The synaptic release of GLU is controlled by a wide range of presynapt ic receptors (Anderson & Swanson, 2000). These include both Group II and Group III metabotropic GLU receptors and also cholinergic (nicotinic and mu scarinic) receptors, adenosine (A1), opioid, GABAB, cholecystokinin and neuropeptide Y (Y2) receptors (Anderson & Swanson, 2000). Once released into the synaptic cleft, Glu is bound to either

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22 preor post-synaptic receptors, r euptaken via the glutamate transporter and repackaged, diffuses away from the synapt ic cleft, or is internalized by GLU transporters on glial cells (Anderson & Swan son, 2000; Attwell, 2000; Daikhin & Yudkoff, 2000). GLU is converted into the inhibitory GABA with the enzyme glutamic acid decarboxylase (GAD) whic h is highly abundan t in the cerebellum (Sherif, 1994). 1.4.2 GABA In 1965, GABA was established as a major inhibitory neurotransmitter in the CNS (Curtis & Watkins, 1965). GABA is syn thesized by the enzyme GAD and taken up by glial-cells and neuronal mitochondria where it is transaminated to succinic semialdehyde and subsequently oxidized to su ccinic acid then enters the citric acid cycle in the glial cell (Sherif, 1994). Once GABA is released, it binds to GABA receptors which have been classified into three major subtypes (GABAA, GABAB and GABAC) on the basis of pharmacol ogical and physiological data (Bowery et al., 1984; Mody et al., 1994) The major output pathway of the cerebellum is via the PC which are GABAergic. It has been suggested that the basal GABAergic output from the cerebella r cortex onto the interpositus nucleus modulates CR expression, whereas timing of CR's is modulated by the stimulus activated inhibition (Bao et al., 2002). The expression of CR’s can be interrupted by both GABA mediated inactivation of the interpositus neurons with muscimol as well as up-regulation of activity wit h picrotoxin (Aksenov et al., 2004).

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23 1.5. Aging as a process in which t he modulatory effect of NE on PC is compromised Aging-associated deficits on motor learning have been linked to dysfunction of the noradrenergic system (Cartford et al., 2004a; Bickford et al., 1985b; Bickford et al., 1986) which is thought to be ca used by the loss of noradrenergic enhancement of the relative responsiveness of PCs to afferent inputs in aged animals. Age-related pathologies are characte rized by a pronounced imbalance in immune functions like glial hyperactivity with altered antigen expression of microglia in aged rodents (Perry et al., 2003a; Cunningham et al., 2007b). Chronic inflammation is known as one of the multiple age-re lated pathologies that involves the activity of several pr oducts, including cytokines (Murray et al., 1997b; Murray & Lynch, 1998a). Cytokines are proteins that mediate the response of the body’s defense syst em to injury and mediate diverse inflammatory processes. The presence of altered levels of cytokines in the central nervous system has been im plicated several aged-related and neurodegenerative diseases (Benveniste & Benos, 1995). Cytokines are secreted by activated microglia and can be either pro-inflammatory cytokines, among them tumor necrosis factor alpha (TNF), interleukin 1 beta (IL1), antiinflammatory cytokines such as inte rleukin 10 (IL-10) and transforming growth factor beta 1 (Uccelli et al., 2005b). Proinflammatory cytokines are chronically increased in the aging brain (Godbout et al., 2005b) and more specifically, TNF

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24 and TNFare significantly elevated in the cerebellum of aged rats (Gemma et al., 2002). In this study rats were fed an anti-oxidant enriched diet for six weeks which resulted in a significant reduction in both TNFand TNFlevels. These diets have also been shown to improv e classical eyeblink conditioning performance in aged rats and are direct ly correlated with a reduction in the expression of TNFand TNFlevels in the cerebellum (Cartford et al., 2002b). The pro-inflammatory cytokine TNFbinds TNFreceptors that are expressed on both neurons and glial cells (Benveniste & Benos, 1995). TNFis synthesized and released by astrocytes microglia and some neurons (Lieberman et al., 1989; Chung & Benveniste, 1990; Morganti-Kossman et al., 1997). TNFlevels are usually increased in many CNS disorders, including ischemia (Liu et al., 1994), trauma (Goodman et al., 1990) and multiple scl erosis (Rieckmann et al., 1995). In these pathological condition s, the expression and release of TNFcan occur as soon as one hour after an in sult to the brain and long before neuronal death (Liu et al., 1994; Wang et al., 1994; Allan & Rothwell, 2001). Given the evidence stated above for a role of inflammation in aging and specifically the role for an increase in TNFin the cerebellum with age, one goal of the studies outlined in this dissertation was to ex amine the impact of TNFon cerebellar dependent learning and the concomitant release of NE using microdialysis.

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25 In summary, NE is implicated as an important factor in cerebellar plasticity that underlies motor learning behaviors. Specific ally, it has been postulated that NE plays a role in consolidation of the performed behavior (Gilbert, 1975). One goal of this dissertation was to characterize the presynaptic release of NE during eyeblink conditioning in the rat and to inve stigate the timing of the influence of NE on acquisition of the learned response. Furthermore, there is evidence for declines in cerebellar dependent motor lear ning with age, as well as changes in NE signal transduction. One aspect of aging that may impact NE signaling and motor learning is an increase in inflam matory cytokines, specifically TNF. Thus, another goal of this dissertation was to examine the impact of TNFon cerebellar motor learning and NE signaling.

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26 CHAPTER 2 Norepinephrine, GABA and Glutamate as a Substrate of Memory Formation in Cerebellar Eye Blink Conditioning 2.1 Abstract Delay classical eyeblink conditioning (EBC) is an important model of associative, cerebellar dependent learning. Norepi nephrine (NE) plays a significant modulatory role in the acquisition of le arning; however, other neurotransmitters are also involved. The goal was to determine whether NE, Gamma-aminobutyric acid (GABA) and Glutamate (Glu) releas e are observed in cerebellar cortex during EBC, and whether such release was selectively associated with training. In vivo microdialysis coupled to EBC was pe rformed. NE release was observed in EBC and peaked on day 1 then diminis hed with subsequent days of training. No changes in baseline NE release we re observed in pseudo-conditioning indicating that NE release is directly re lated to the associat ive learning process. GABA release was also observed only dur ing paired training but increased in magnitude over days of training. Glu release was observed during both paired and unpaired training. These data support t he hypothesis that NE is a modulator of early responding and GABA is a mediator of the learned response. Age related deficits in eyeblink condition ing are linked to the loss of noradrenergic activity in the cerebellum. The present study used eyeblink conditioning coupled to microdialysis to demonstrate that, in c ontrast to young rats, NE, GABA and Glu

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27 levels that occur during ey eblink conditioning show diffe rent patterns of release which appear to be correlated to the age-re lated impairment in the acquisition of learning. 2.2 Introduction Delay eyelid conditioning is an excellent model of cer ebellar motor learning. NE is known to play a modulatory role in cerebellar-dependent learning. NE induces synaptic plasticity in Purkinje neurons by selectively improving the signal to noise ratio of evoked versus spontaneous activity, enhancing the sensitivity of cerebellar neurons to both excitatory and inhibitory afferent inputs (Siegel & Freedman, 1988). Based upon the Marr-Albus theories of cerebellar learning Gilbert proposed that NE should have a ro le in the consolidation of memory within the cerebellum (Gilbert, 1975). Behavioral evidence which shows the involvement of NE in memory consoli dation in the cerebellum is observed in several cerebellar dependent paradigms. For example in rod running motor learning, a cerebellar-dependent task, the abi lity of rats to learn is reduced following lesion of the locus coeruleus (LC) (Khachaturian et al., 1983). This effect is localized to cerebellar NE (Wat son & McElligott, 1984) and is specific to the -adrenergic receptor (Bickford et al ., 1992). Adaptation of the vestibulo ocular reflex (VOR) is modulated by noradrenergic inputs (McElligott & Freedman, 1988) and appears to be mediated by the -noradrenergic receptor (Pompeiano et al., 1991). Cerebellar del ay classical conditioning has been shown to be modulated by NE. Electrolytic lesions of the LC induce resistance to

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28 extinction in rabbits (McCormick & T hompson, 1982) and 6-hy roxydopamine (6OHDA) has been shown to retard acquisit ion but not performance of conditioned eyelid responses in rabbits (Winsky & Harvey, 1992). Blockade of the adrenergic receptor retards the acquisi tion of learned responses in delay conditioning tasks in rats (Cartford et al., 2002a; Cartford et al., 2004b). NE is also implicated in non-ce rebellar dependent tasks. LC neurons fire prior to a target cue in a vigilance task in monkeys and during reversal of task contingency, the LC response to the new stimuli precedes behavioral responding (AstonJones et al., 1997) Other neurotransmitter systems are also implicated in delay eyeblink conditioning tasks. Administration of the Glu AMPA receptor ant agonist CNQX or the GABAA receptor antagonist picrotoxin into the cerebellar cortex completely and reversibly impairs fully established c onditioned response’s (CR’s), suggesting that GABAergic and glutamatergic trans mission are involved in CR performance (Attwell et al., 2002b). It has been sugges ted that the basal GABAergic output from the cortex onto the in terpositus nucleus modulat es CR expression, whereas timing of CR's is modulated by the stimulus activated inhibition (Bao et al., 2002). The expression of CR’s can be interr upted by both GABA mediated inactivation of the interpositus neurons with muscimol as well as up-regulation of activity with picrotoxin (Aksenov et al., 2004). Agin g-related deficits have been reported for the acquisition of CR in a variety of motor learning task. In particular, loss of cerebellar noradrenergic functi on has been directly corre lated with the decreased

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29 ability to learn the rod walking task (Bi ckford, 1995). Currently, the correlation between aged-related impairments in mo tor plasticity and specific neurophysiological deficits in the cerebella r PC is accepted (Bickford, 1993). Eyeblink conditioning in particular has been shown to be a good paradigm to evaluate the effects of antio xidant-enriched diets on cognitive ability of aged rats (Cartford et al., 2002b; Gemma et al., 2002). In these studies the authors showed that anti-oxidant enriched di ets can reverse the learning impairment while the levels of the cytokine TNFwere reduced in aged rats. This suggests that the high levels of TNFfound in aged rats might be one of the major reasons learning deficits are observed during aging. To date work exam ining the role of neurotransmitters in delay eyeblink cond itioning has used either the application of agonists or antagonists to investigat e the postsynaptic effects of these neurotransmitters (Bao et al., 2002; Krupa & Thompson, 1997; Mamounas et al., 1987; Schreurs & Alkon, 1993). Howe ver, questions remain regarding presynaptic release of neurotransmitte rs during performance of the delay conditioning task. The pres ent study uses in vivo mi crodialysis to examine the temporal patterns of release of NE, G ABA, and Glu during eyeblink conditioning training to examine critical presynapt ic events and whether the aged-related deficits in learning are reflected in th e neurochemical orc hestration within the cerebellum during the ey eblink conditioning.

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30 2.3 Methods 2.3.1 Animals and surgery Male F344 rats four and twenty two months of age were used in this study. Room temperature was kept at 72 F and the dark/light circle was 12-h (lights were on from 7:00 AM to 7:00 PM). An imal number was the minimum required for reliable statistical test results. Ra ts were anesthetized with pentobarbital (10 mg/kg, i.p.) and ketamine (25 mg/kg, i.p.) and placed in a stereotaxic instrument. Anesthesia level was monito red every 10 min and maintained in such a way that the withdrawal reflex to paw pinch was absent dur ing surgery. If the experimenter noted any spontaneous move ment or minute vocalization an additional charge of ketamine (10 mg/kg) and pentobarbital (5 mg/kg) was given. A 10 mm long guide shaft made of 21-gauge stainless-steel tubing (Plastics One) was inserted into the cerebellum. The guide shaft was attached to the skull by jeweler screws and cemented with dental acrylic. In young rats the coordinates to implant the guide cannulae for the micr odialysis probe into the cerebellar lobule HVI (simplex, and interpositus nucleus) we re AP-11.3, ML +2.5 and DV -2.5 mm in reference to bregma for young rats, while in aged rats were AP -11.8, ML +2.5 and DV -2.5 mm. In the same surgical session rats were prepared for eyelid training by fixing a small ITT/Cannon connecto r strip to their skull to hold gold pin connectors to EMG wires that were run under the left eyelid. This method has been previously published by ou r lab (Cartford et al., 2002b). Rats were allowed to recover for one week after the surger y procedure before starting the eyeblink conditioning training and microdialysis. Each animal was used for only one

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31 experimental condition. All procedures were carried out in accordance with the institutional guidelines (IA CUC) and with USA National In stitute of Health Guide for the Care and Use of Laboratory Animals. 2.3.2 Microdialysis procedure Microdialysis probes were lab-made with cellulose hollow fiber (MW 13,000) attached to stainless steel tubing, with a 45 cm length of fused silica capillary (internal diameter [I.D.] 76 m; outside diameter [O.D.] 150 m) inserted into the cellulose tube (Hernandez et al., 1986a). T he effective length of the dialysis piece was 3 mm. 2.3.3 Training The rats were habituated to the traini ng chamber and headstage cable for three days. The training consisted of 50 trials each training tr ial consisted of a 250 ms baseline, a 400 ms CS period, and a 100 ms US period. The t one was 500 ms in duration and overlapped the airpuff for 100 ms. The training tone was 3 kHz and the airpuff 10 psi. Hardware and softwar e used to train and analyze data were manufactured by J.Tracy, J.Green and Jo e Steinmetz, (Bloom ington, Indiana). Eyelid EMG data was collected, amplif ied, rectified, and integrated. Learned responses were determined using a 10 st andard deviation criterion for eyelid amplitude elevated during t he CS period when compared to the baseline. Alpha responses to the tone were excluded fr om learned response a nalysis by using a 70 ms discrimination/exclusion wi ndow. Learning was measured as the

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32 percentage of learned (c onditioned) responses (CR’s) made in each training session. 2.3.4 Microdialysis and eyeblink conditioning procedure The night prior to the experiment the pr obe was inserted into the cerebellum of rats via the guide cannula. The inlet of the probe was connected to a syringe pump filled with artificial cerebrospina l fluid (aCSF) solution (134.9 mM NaCl, 3.7 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2, and 10 mM NaHCO3 at pH 7.4) and the perfusion flow rate was set at 0.1 l/min during 12 hours (overnight) to allow recovery from the probe insertion damage. The next morning the flow rate was set at 2 l/min for 2 hours and the head stage (ITT/Cannon connector ) was coupled to the rat head stage for the EEG recording and airpuff delivery, microdialysates were collected every 10 minutes. Microdialysis samples were collected for one hour prior to training base line (B), during training (T)(18-20 min) and for two hrs post-training (PT). 2.3.5 Neurochemical analysis Immediately after each sample collection, one microliter of sample was taken from the vial and placed into another vial for GLU and GABA analysis, the rest of the sample (nineteen microlit ers) was acidified with 2 l of 0.1 M HCL and all the samples immediately frozen for High Performance Liquid Chromography (HPLC) and Capillary Zone electrophoresis (CZE) analysis.

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33 2.3.6. HPLC: Norepinephrine Catecholamine Analysis: The microdialysis samples that were frozen at -80 C were unfrozen for the analysis in the HPLC. This method is published and routinely used by our laboratory (Hall et al., 1986; Bowenkamp et al., 1997). The detection of NE, 3,4-Dihydroxyphenylaceti c acid (DOPAC), was performed using an isocratic HPLC system (Beckman, Inc., Fullerton, CA), at a flow rate of 1 ml/min. This system is coupled to a d ual-channel electrochemical array detector (model 5100A, ESA, Inc., Chelmsford, MA), E1 = +0.35 mV and E2 = -0.25 mV, using an ESA model 5011 dual analytical cell. The compounds of interest were separated with reverse-phase chromatography using a C18 column (4.6 mm x 100 mm, 3 m particles, ODS Thermo Hyper sil; Keystone Scientific, Bellefonte, PA) with a pH 4.1 citrateacetate mobile phase, containing 10% methanol and 0.45 mM 1-octane-sulfonic acid. Data were quantified using Totalchrom software V6.2 (Perkin Elmer) based on peak ar ea, in comparison with an external standard calibration curve. 2.3.7. Capillary electr ophoresis: Glu and GABA This method has been published (Rada et al ., 2003) and used by our laboratory. A capillary electrophoresis system equippe d with an argon laser tuned to 488 nm was used (Model R2D2, Meridialysis Co ., Merida, Venezuela). A carbonate buffer (20 mM carbonate/bicarbonate) was the running buffer to transport the microdialysis sample through the capilla ry when detecting glutamate. Detection

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34 of GABA from the samples required a di fferent running buffer consisting of 23 mM borate with 120 mM sodium dodecyl sulf ate and 1% methanol. The samples or standards were sucked into the anodi c end by applying a negative pressure (19 psi or 1.34 kg/cm for 0.5 s) at the cathodic end of the capillary. Electrophoretic separation was achieved by applying a high vo ltage between the anode and the cathode for 12 min, 22 kV fo r glutamate and 26 kV for GABA. 2.3.8. Derivatization Fluorescein isothiocyanate (FITC) was conjugated with glutamate and GABA as the fluorescent chromophore. Optimal concen trations of FITC and the calibration curves for both amino acids have been reported previously Dialysates and standards were derivatized mixing 1 l (sample or standard solution) with 1 l of a solution containing FITC (1 mM/ac etone) carbonate buffer (20 mM) 1:1 mixture. A syringe loaded with FI TC–carbonate mixture was placed in a precision pump, and 1 l of the mixture wa s delivered into a tube containing 1 l of microdialysis sample. The samples reacted overnight (14 hr) at room temperature in a water-saturated ch amber that minimized evaporation. Homoglutamine (10–5 M) was used as an in ternal standard and was mixed in the carbonate buffer used to derivatize samples and standards. 2.3.9. Histology After completing the experiment, the animals were overdosed with sodium pentobarbital and decapitated a nd the brains were dissected, placed in 10%

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35 formalin for 24 h and then transferred to 15% sucrose with 10% formalin for 24 hrs. The brains were then frozen, sectioned (40 m) on a freezing microtome and stained with cresyl violet fo r verification of the probe placement and extensions of the lesion. Only animals with pr obe placements verified through cerebellar cortex and lobule HVI (sim plex, and interpositus nucl eus) region were used for data analysis, also animals that presented high leve l of damage or hemorrhage due to the microdialysis probe inserti on were discarded from the study. 2.3.10. Statistical analyses Analyses were performed using conditioned responses (CR’s) and area under the curve (AUC) of neurotransmitter c oncentration as the dependent measures. Data from experiments were subjected to a two-way analysis of variance of either conditioned responses or area under the curve (AUC) for the neurotransmitter, followed by subsequent post hot tests. Supernova was the statistical software utilized in these analyses. A p value of <0.05 was considered to be statistically significant. 2.4 Results 2.4.1 Young Rats Microdialysis was performed in the cerebella r cortex on rats during training in the delay eyelid conditioning task. Rats learn this task over days and were significantly different from ps eudo-conditioned performance and days 2 – 5 (Figure 2.1). Neurotransmitter release wa s examined on all five days of training.

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36 However the microdialysis performed and plo tted for neurotransmitter release on each day was done with independent groups. Figure 2.1 Effect of TTX on cerebe llar dependant eyelid conditioning. Eyelid conditioning was performed over 5 days. Rats received either vehicle or TTX twenty minutes prior to eyelid conditioning on the first day of training. The Y-axis shows the percentage of conditioned respon se (% CR’s), the x-axis represents daily training sessions of 50 trials. Bo th TTX and vehicle treated rats learned this task over days (represented by higher % CR) whereas rats who under went psuedoconditioning did not learn the task. Black squares represent TTX treated rats, open squares represent control rats and X represents pseudoconditioning. Control and TTX treated rats learned si gnificantly more than psedoconditioning treated rats ( p <0.05). 2.4.1.1 Norepinephrine NE release was observed in the microdialys ate on all 5 days of training, however the temporal pattern and magnitude of release changed over days of training (Figure 2.2A-E). On day one of training there was an increase in NE detected in the microdialysate that peaked at the end of the behavioral training and remained

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37 significantly above baseline for 70 minute s after the training session. A 2 (Treatment: conditioning, pseudoconditioni ng) x 5 (Day: 1-5) between subjects ANOVA revealed a significant main effect of treatment, [F (1, 27) = 84.20, MSe = 91975.60], with conditioning resultin g in higher AUC of NE than pseudoconditioning (Fig 2.2F). This indica ted that the NE release was specific for the learning condition and was not the result of sensory stimulation alone. There was also a significant main effe ct of day, [F (4, 27) = 4.30], with AUC generally decreasing over days. The Fi sher’s LSD test revealed that day 1 resulted in significantly hi gher AUC than days 2, 4 and 5. As shown in Figure 2.2F, there was no significant interacti on between day and treatment [F (4, 26) = 2.20]. NE release was signif icantly greater in the cond itioning gro up on days 1-5 compared to the pseudocondit ioning group. These find ings can be observed in Figures 2.2A-E which illustrates the time window of NE release once the training started. On all days a clear increase in NE levels were observed with the onset of training in the conditioni ng group. Interestingly, ov er days NE levels returned to baseline levels faster, indicating a larger amount of re lease with a longer duration was observed on the beginning da ys of training. In contrast, the pseudoconditioning group did not show this pa ttern of transient NE release which indicates that the NE release observed (c onditioning group) is di rectly related to the learning process in the eyeblink task.

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38 Figure 2.2. Temporal release of norepinephrine during eyelid conditioning Microdialysis was performed in the cerebella r cortex on rats during training in the delay eyelid conditioning task. The ti me window of NE release once eyelid

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39 conditioning starts can be observed on day 1 (A), day 2 (B), da y 3 (C), day 4 (D) and day 5 (E). On day one of training (A ) there was an increase in NE that peaked at the end of the behavioral trai ning and remains significantly above baseline for 70 minutes after the training session. On all days (A-E) a clear increase in NE levels was obs erved with the onset of tr aining in the conditioning group. Data are expressed as NE [nM] (y-a xis) over time in 10 minute dialysate samples (x-axis). Black squares repres ent conditioning, open squares represent pseudoconditioning. F) Area under the curve (AUC units) representation of NE release for each day of training during Ey elid conditioning. Note that the pseudoconditioning group (Ps-Cd) did not s how this pattern of transient NE release (indicates that the NE release observed (conditioning group) is directly related to the learning process in the eyeblink task.) 2.4.1.2 GABA GABA release was also examined over da ys of training and it can be observed in Figure 2.3 that the amplit ude of GABA release increas ed over days of training and the time course shortened. Data are expressed as percent of baseline and mean basal levels were 0.12 M ( 0.1). A (treatment: conditioning, pseudoconditioning) x 5 (day: 1-5) bet ween subjects ANOVA revealed a significant main effect of treatment, [F (1 27) = 92.70, MSe = 33192.80], where conditioning resulted in significantly more GABA release (expressed as AUC units) than the pseudoconditioning (see figure 2.3F). There was also a significant interaction between treatm ent and day, [F (4, 27) = 4.70], which revealed that GABA release was significantly greater in the conditioning group on day 1 compared to days 2, 3 and 5 ( p <0.05), one reason for this difference is observed when looking at t he time course of the GABA release over days of training. The maximum amplitude of rel ease was lower on Day 1, yet the release is spread over time, thus leading to an overall larger amount of GABA measured over baseline. Figures 2.3A-E illustrat es the time window of GABA release once

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40 the training starts. The peak magnitude of release increased over time and the time frame of the response sharpened so that GABA release is sharply timed with the performance of CR’s by day 3 and sharpens further up to day 5. As observed with NE, the condit ioning group resulted in si gnificantly higher AUC on all days except day 5, when compared to the pseudoconditioning group ( p <0.05), suggesting that the GABA release is a ssociated with learning of the conditioned response.

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41 Figure 2.3 Temporal release of G ABA during eyelid conditioning Microdialysis was performed through the cerebellar cort ex and interpositus nuclei on rats during training in the delay eyelid cond itioning task. The time window of GABA

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42 release once eyelid conditioning starts can be observed on day 1 (A), day 2 (B), day 3 (C), day 4 (D) and day 5 (E). GABA release was significantly greater in the conditioning group on day 1 co mpared to days 2, 3 and 5 ( p <0.05). Peak magnitude of release increases over time and the time frame of the response sharpens so that GABA release is shar ply timed with the performance of CR’s by day 3 and sharpens further up to day 5. Data are expressed as percent of baseline (% baseline) (y-axis). Black squares represent conditioning, open squares represent pseudoconditioning. (F ) Area under the curve representation of GABA release for each day of traini ng during eyeblink conditioning. As was observed with NE, the conditio ning group resulted in signi ficantly higher AUC for GABA on all days except day 5, when co mpared to the pseudoconditioning group (Ps-Cd) ( p <0.05), suggesting that the GABA rel ease is associated with learning of the conditioned response. 2.4.1.3 Glutamate As can be seen in figure 2.4F, a 2 (tr eatment: conditioning, pseudoconditioning) x 5 (day: 1-5) between subjects ANOVA of the AUC of Glu did not reveal main effects of day, [F (4,31) = 0.40, MSe = 2854.80] or treatment, [F(1,31) = 0.0036], or any interaction between treatment and day, [F(4,31) = 1.50]. Interestingly, figures 2.4A-E shows that comparable levels of Glu were released during training on the eyeblink task for both the conditi oning and the pseudoconditioning groups.

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43 Figure 2.4 Temporal release of glutamat e during eyelid conditioning. Microdialysis was performed in the cerebella r cortex on rats during training in the delay eyelid conditioning task. The re lease of Glu over time during eyelid

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44 conditioning is shown on day 1 (A), day 2 (B ), day 3 (C), day 4 (D) and day 5 (E). Comparable levels of glutamate were released during training on the eyeblink task for both the conditioning and the pseudoconditioning groups. Data are expressed as Glu [M] (y-axis). Bla ck squares represent conditioning, open squares represent pseudoconditioning. (F ) Area under the curve representation of Glu release for each day of trai ning during eyelid conditioning and pseudoconditioning (Ps-Cd, average of days). There were no differences in AUC of Glu. 2.4.1.4 Tetrodotoxin (TTX) effe ct on neurotransmitter release In a subset of young rats TTX was adminis tered twenty minutes prior to training on the first day of training and administra tion continued for the duration of the microdialysis collection. TTX was admin istered to test the impulse dependence of neurotransmitter release with microdial ysis. The behavior of the rats was analyzed and there were no significant diff erences in learning between groups (figure 2.5). There was a decrease in amplitude of the uncond itioned response on the TTX treatment group in two of the six rats, suggesting that there was a suppression of performance of the blink response for those two animals (data not shown). The microdialysates were analyz ed for NE, GABA and Glu. The effect of TTX on NE release is observed in Figure 2.5A-B, where TTX reduced the AUC of NE compared with controls demonstr ating an impulse depend ent nature of NE release (p<.05). The effect of TTX on GABA release was also a reduction in the AUC for GABA (Figures 2.5D). A sim ilar effect was obser ved with glutamate (Figures 2.5E-F). TTX was administ ered twenty minutes before the eyeblink conditioning training started, and it is notable that t he extracellular levels for every neurotransmitter (NE, GABA and Glu) were depleted from their basal levels, indicating tonic release for NE, GABA and Glu. Despite the depletion in

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45 basal extracellular levels of neurotransmitters, once t he training sessions started each particular neurotransmitter was rele ased following the same temporal pattern as the non-TTX groups. Howe ver the total amount of release was significantly lower compared to the control groups. It is interesting to see that there were not significant differences on percent CR’s reached on day one of training. This behavioral outcome wa s unexpected, however it should be considered that the low concentration of TTX used might have not been enough to impair the circuitry flow of sensory inputs carrying the US and CS. Even with the reduction in tonic release (due to the presence of TTX) of NE, GABA and Glu, it is also possible that the signal to noise ratio on the PC was sufficient to relay essential information for memory fo rmation as a greater percentage of CR’s were reached on day two of training. The data show that TTX depleted baseline neurotransmitter levels and attenuated the neurochemical response to training. However, taking into consideration chan ges in evoked neurotransmitter levels relative to baseline levels it is appar ent that neurotransmitter release for both TTX and non-TTX are similar. Therefore, despite t he depletion on presynaptic activity through the cerebellar cortex and inte rpositus nuclei, if the signal to noise ratio remains the same throughout the trai ning sessions, it mi ght still be possible that memory encoding occurs. This wo uld explain why in aged animals, which have attenuated levels of NE, GABA and Glu, if training persists the rats develop CR’s. Therefore, it is not surprising that the TTX group sh ow levels of CRs comparable to the non-TTX group. Mo re experiments need to be conducted to

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46 clarify this explanation, and if it is shown to be true it will support the concept of NE signaling facilitating the signal to noise ratio of the PC.

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47 Figure 2.5 Tetrodotoxin (TTX) effect on neur otransmitter release during eyelid conditioning TTX was administered 20 minutes prior to training on the first day of training (indicated by arrow) and administration cont inued for the duration of the microdialysis collection. Training began at time point 0 (indicated by arrow) and lasted approximately 20 minutes. (A) Sh ows extracellular NE levels [nM] over time (minutes) on day 1 of condi tioning and (B) shows the area under the curve (AUC) of NE release during eyeblin k conditioning on day 1. TTX reduced the amount of NE in the microdialysate when compared with control demonstrating an impulse depen dent nature of NE releas e. (C) Extracellular GABA levels (% baseline) over time (minut es) on day 1 of conditioning. Note the reduction in basal levels after TTX adminis tration compared to vehicle. (D) AUC of GABA release during eyeblink condition ing on day 1. There was a slight reduction in the GABA levels after TTX. (E ) Extracellular Glu levels [M] over time on day 1 of conditioning. (F) AUC of Glu release during eyeblink conditioning on day 1. 2.4.2 Aged Animals Microdialysis was performed in the cerebella r cortex on rats during training in the delay eyelid conditioning task. As can be seen in figure 2.6 aging and young rats performed significantly better than pseu doconditioning, however young rats learned the task faster with more accuracy compared to aging rats ( p <0.05). The pattern of release follows a different dynam ic with might reflex why the rats takes longer (need more training sessions) to learn a CR comparable to young rats. These changes include a de lay in the release in respond to the training conditioning sessions and also the magnit ude for the release is significantly smaller compare to young rats. Long last ing release is also observed in aged rats, which could reflect impairments in the metabolism (clearance) of the neurotransmitters such as the re-uptake systems for Glu and NE.

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48 Figure 2.6 Cerebellar dependant eyelid c onditioning is impaired in aging Eyelid conditioning was performed over 5 days. The Y-axis shows the percentage of conditioned response (% CR’s ), the x-axis represents 5 daily training sessions of 50 trials. Black circ les represent young co nditioning, open circles are young pseudoconditioning, black squared represent aging conditioning, open squared ar e aging pseudoconditioning. Young rats learned this task over days (represented by hig her % CR) whereas ra ts who under went did not learn the task. Both aging and young rats performed significantly better than pseudoconditioning, however young rats learned the task faster with more accuracy compared to aging rats ( p <0.05). indicates difference between young Cd and young Ps-Cd. 2.4.2.1 Norepinephrine NE release was examined on days 1, 3 and 5 of training. NE release was observed in the microdialysate on all 3 da ys of training, however the temporal pattern and magnitude of re lease changed over days of tr aining (Figure 2.7A-C). In contrast to what is observed in yo ung rats, NE levels increased faster and

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49 return to baseline sooner after days of tr aining. A 2 (treatment: conditioning, pseudoconditioning) x 3 ( day: 1,3,5) between subjects ANOVA revealed a significant main effect of learning, [F (1,9) = 29, p<. 05], with conditioning resulting in higher AUC of NE than pseudoconditioning (Fig 2.7D). This indicates that NE release is specific for t he learning condition and is not the result of sensory stimulation alone. There was also a significant main effect of day, [F (2,9) = 7.2], with AUC generally increasing over days in the aged an imals. Figure 2.7D illustrates a significant in teraction between day and treatm ent [F (2,9) = 5.6] with NE release being significantly greater in the conditioning group on days 3 and 5 compared to the pseudocondit ioning group. These find ings can be observed in Figures 2.7A-C which illustrate the time window of NE release once the training starts. On all days an increase in NE levels are observed in the conditioning group whereas, the pseudoconditioning group did not show this pattern of transient NE release which indicates t hat the NE release observed (conditioning group) is directly related to the l earning process in the eyeblink task.

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50 Figure 2.7 Temporal release of norepinephrine during eye lid conditioning in aged rats. Microdialysis was performed in t he cerebellar cortex on aged rats during training in the delay eyelid cond itioning task. The time window of NE release once eyelid conditioning starts can be observed on day 1 (A), day 3 (B) and day 5 (C). Data are expressed as NE [nM] (y-axis) over time in 10 minute dialysate samples (x-axis). Black squar es represent conditioning, open squares represent pseudoconditioning. D) Area under the curve (AUC units) representation of NE release for each day of training during eyelid conditioning. 2.4.2.2 GABA GABA release was also examined over da ys of training and it can be observed in Figure 2.8 that the amplit ude of GABA release increas ed over days of training and the GABA levels remained elevat ed above baseline. A (treatment: conditioning, pseudoconditioning) x 3 (day: 1,3,5) between subjects ANOVA revealed a significant main effect of tr eatment, [F (1,8) = 85.0, p<0.05], where -40 -20 0 20 40 60 80 100 120 0 10 20 30 40 50 60Conditioning Pseudo-Conditioning A.Time (minutes)NE [nM] -40 -20 0 20 40 60 80 100 120 0 10 20 30 40 50 60Conditioning Pseudo-Conditioning C.Time (minutes)NE [nM] -40 -20 0 20 40 60 80 100 120 0 10 20 30 40 50 60Conditioning Pseudo-Conditioning B.Time (minutes)NE [nM] 135Ps-Cd 0 400 800 1200 1600 2000 2400 2800D. *Day.AUC units

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51 conditioning resulted in significantly more GABA release (expressed as AUC units) than the pseudoconditioning (see figure 2.8D). There was also a significant interaction between treatment and day, [F (2,8) = 3.7, p<0.05], which revealed that GABA release was significantly greater in the conditioning group on day 1 compared to day 5 ( p <0.05), one reason for this difference is observed when looking at the time course of the GABA release over days of training. The pattern of release changes across days so that by day 5 GABA remains elevated 2 hours after training. This can be observe d in figures 2.8A-C which illustrates the time window of GABA release once training starts. Figure 2.8 Temporal release of GABA duri ng eyelid conditioning in aged rats Microdialysis was performed in the cerebe llar cortex on aged rats during training in the delay eyelid conditioning task. The time window of GABA release once -40 -20 0 20 40 60 80 100 120 90 100 110 120 130 140 150 160 170Conditioning Pseudo-Conditioning A.Time (minutes)GABA (% baseline) -40 -20 0 20 40 60 80 100 120 90 100 110 120 130 140 150 160 170Conditioning Pseudo-Conditioning B.Time (minutes)GABA (% baseline) -40 -20 0 20 40 60 80 100 120 90 100 110 120 130 140 150 160 170Conditioning Pseudo-Conditioning C.Time (minutes)GABA (% baseline) 135Ps-Cd 0 400 800 1200 1600 2000 2400 2800D. * *Day.AUC units

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52 eyelid conditioning starts can be observed on day 1 (A), day 3 (B) and day 5 (C). GABA release was significantly greater in the conditioning group on day 1 compared to day 5 ( p <0.05). The pattern of release in aged rats changes across days so that by day 5 GABA remains elev ated 2 hours after training. Data are expressed as percent of bas eline (% baseline) (y-axis). Black squares represent conditioning, open squares represent ps eudoconditioning. (D) Area under the curve representation of GABA release fo r each day of training during eyeblink conditioning 2.4.2.3 Glutamate As can be seen in figure 2.9D, a 2 (treat ment: conditioning, pseudoconditioning) x 3 (day: 1,3,5) between subj ects ANOVA of the AUC of Glu did not detect main effects of day, [F (2,6) = 2.2] or lear ning, [F(1,6) = 0.041], or any interaction between treatment and day, [F(2,6) = 0.66]. Interestingly, figures 2.9A-D shows that comparable levels of glutamate ar e released during training on the eyeblink task for both the conditioning and the pseudoconditioning groups.

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53 Figure 2.9 Temporal release of glutamate during eyelid conditioning in aged rats. Microdialysis was perform ed in the cerebellar cortex on aged rats during training in the delay eyelid conditioning task. The release of Glu over time during eyelid conditioning is shown on day 1 (A), day 3 (B) and day 5 (C). Comparable levels of glutamate are re leased during training on t he eyeblink task for both the conditioning and the pseudoconditioning gr oups. Data are expressed as Glu [M] (y-axis). Black squares represent conditioning, open squares represent pseudoconditioning. (D) Area under the cu rve representation of Glu release for each day of training during eyelid co nditioning and pseudocon ditioning (Ps-Cd, average of days). There were no di fferences in AUC of Glu.

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54 2.5 Conclusions The overall results show a significant role of the neurotransmitters NE, GABA and GLU occurring during the acquisiti on of the CR in the delay eyelid conditioning paradigm. The literature s hows that these neurotransmitters participate in different ways and are essential for memory formation, consolidation and extinction (Attwell et al., 2002a; Yeo, 2004; De Zeeuw & Yeo, 2005; Weis et al., 2004; Farley & Alkon, 1985). To date, there is no direct evidence showing presynaptic release in vivo while the animals perform the learning task. In this study, the extracel lular levels of neurotr ansmitters reported correspond to those captured by the acti ve zone of the microdialysis probe from the lobus simplex and inter positus nucleus. We show the temporal pattern of release which occurs during training on the delay eyeblink conditioning task and the fact that there were no significant changes in NE and GABA release for the pseudo-conditioning groups shows the s pecificity of NE and GABA release related to the associative learning task and that it is not due to sensory stimulus activation by the CS and/or US, which wa s the case for GLU release. Our data also show that aged rats have a deficit in the neurotransmitte rs NE, GABA and Glu associated to the learning of the CR of eyeblink conditioning. This deficit seems to be directly associated with t he impairments on the acquisition of CR’s observed in aged rats.

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55 2.5.1 Norepinephrine. We demonstrate that extrac ellular levels of NE are increased in the cerebellum during training sessions of eyeblink conditi oning from day 1 through day 5. On day 1, NE release remains increased over baseline for 60 minutes, whereas, as training progresses over days, the peak becomes smaller and NE remains elevated for a shorter time period so that by day 4 the NE signal returns to baseline within 30 minutes. In cont rast, when pseudo-conditioning was examined there is no change in NE release during or after training on any day of training. This clearly indicates that the increase in NE which was observed during and after CS-US paired training is spec ifically linked to the combination of both the CS and US. NE induces synaptic plasticity in purkinj e neurons by select ively improving the signal to noise ratio of evoked versus spontaneous activity, enhancing the sensitivity of cerebellar neurons to both ex citatory and inhibitory afferent inputs (Moises et al., 1979). The importance of NE during acquisition of motor learning tasks is supported by reports that 6-hydr oxydopamine induced lesions of the LC disrupt cerebellar motor learning on a runway task (Watson & McElligott, 1983; Bickford, 1995) and classical eyeblink co nditioning (Winsky & Harvey, 1992). Furthermore, blockade of postsynaptic -adrenergic receptors with systemic administration of propranol ol disrupts acquisition of the delay eyeblink conditioning in both the rabbit (Gould & St einmetz, 1996) and rat (Cartford et al., 2002a). Both propranolol and Rp-cAM PS (activate cAMP-dependent PKA)

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56 directly administered into the cerebellum impair acquisit ion of CRs (Cartford et al., 2004b). Interestingly, once the CR has been established, neither propranolol nor Rp-cAMPS block performance of acquir ed CRs. Overall, the amount of NE released decreases across days of training consistent with a role of NE early in acquisition and possibly in consolidation (Gil bert, 1974). This is consistent with work showing the firing of LC neurons demonstrates good discrimination within the first 500 trials of reversal of cont ingency in a visual discrimination task in monkeys (Kubiak et al., 1998) (monke ys are still showing a high number of errors), again suggesting that NE is im portant during the acquisition phase of learning of this task. Deficits in the NE re-uptake system of aged rats have been reported and could be associated to the delay in NE releas e observed and also with the long lasting overflow We did not observe t hat NE returned to basal levels at least during the period of ti me that we collected the microdialysis samples. Our data agree with previous reported data showing deficits in the noradrenergic system in aged animals which been sugges ted to be directly related with the defic it in learning. 2.5.2. GABA. As was observed with NE, GABA was released in a lear ning dependent manner and was specifically associated with paired conditioning and not pseudoconditioning. As GABA is the predominant neurotransmitte r in the cerebellum it was expected that if the cerebellum and inte rpositus nucleus are involved in the learned response, GABA release would be observed during the time

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57 corresponding to the training period. Interestingly, duri ng the first days of training, the increase above baseline in extracellular levels of GABA extends beyond the training period for over 60 minutes. This pattern changes across days of training where the time frame of GABA release shortens and at a time where the behavioral response is nearly maximal the GABA release is now primarily only observed during t he behavioral testing. Much of the work examining GABAe rgic transmission using either GABA agonists or antagonists has been directed at examining the functional role of the cerebellar cortex and cerebellar nuclei in regard to the memory formation and timing. Discrete infusions of GABA a gonists and antagonists into the cerebellar cortex and interpositus nucleus suppor t the hypothesis that both areas are involved in plasticity (Bao et al., 2002; Krupa & Thompson, 1997; Mamounas et al., 1987; Schreurs & Alkon, 1993). For example, t he expression of CRs is mediated by LTD at the granule to purki nje synapses and by LTP at the mossy fiber synapses in the cerebellar nuclei (Mauk et al., 1998). GABAergic circuitry in the cerebellar cortex has al so been postulated to play a role in post training memory consolidation (Cooke et al., 2004). Authors demonstrate that delayed infusions of muscimol into the cerebel lar cortex at 5 or 45 minutes produced significant impairments of consolidation, suggesting that about 1 hour after training there is a period of cortical activi ty that is necessary for consolidation of the learned response. This time window is similar to that observed for GABA release (and NE release) observed in this report on the first few days of training.

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58 GABA release likely reflects the activity of the cerebellar circuitry following training. During the later phase of training (days 4 and 5) there is a more discrete release of GABA which shows a hi gher peak while the timing is confined to the training session. At this time the behavioral re sponse is well trained and the GABA release pattern likely reflects t he concomitant activity of the cerebellar circuitry that is associated with the CR. In contrast, GABA release (in aged rats) was significantly delayed compared to the young rats and this delay might indicate that not just the noradrenergic system is impaired but also the activity of the PCs. This could be an indication of over activi ty of the incoming signaling carried out by the climbing and mossy fibers, however this does not rule out the possibility that other factors such as impairments in postsynaptic currents on the PC are contributing to produce this delay in GABA release. 2.5.3. Glutamate. The GLU release measured in this repo rt likely reflects the inputs to the cerebellar cortex and interpositus whic h is activated in both paired and unpaired conditions and explains why we observe Glu release in both conditions. A different scenario is observed for t he NE and GABA as described above. Changes in the dynamics of NE release ov er time suggest that NE plays a major role in the first days of training. Th e change in timing of the GABA response correlates with the learned response in the rat and possibly reflects the activity of purkinje neurons and interpositus nucleus n eurons that show entrained firing that develops over days of training. Glutam ate has been shown to have significant

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59 post-synaptic effects during training. For example, infusion of the NMDA antagonist AP5 disrupts eyeblink condit ioning (Chen & St einmetz, 2000). However, our results only reflect the pr e-synaptic release of glutamate from afferents to the cerebellar cortex and support the idea that learning of the conditioned response does not take place before the signals reach the cerebellum as there is no change in affer ent activity across days of training. It is interesting that Glu release happens in both, paired and unpai red trials of the conditioning sessions and the time period fo r the release is concomitant with the time period in which the sensory input to the cerebellum is happening. However in aged rats it is observed that even afte r the training finish ed extracellular Glu remains above the basal levels. This fi nding suggests a possible deficit in the Glu re-uptake system, which could be the cause of the prolonged release observed for NE and GABA once the training starts. Specifically, Glu levels in aged rats could be the result of over activity of microg lia which would lead to a deficit in the Glu re-uptake system. This possibility sti ll remains as one possible cause until more experiments are done to cl arify the causes for this pattern of release of Glu in aged rats. 2.5.4. Effect of TTX on ne urotransmitter release. In a subset of young rats TTX was admin istered (through reverse dialysis) twenty minutes prior to training on the first day of training and administration continued for the duration of the microdialysis collect ion. TTX was administered to test the impulse dependence of neurotransmitter rel ease with microdialysis. The behavior

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60 of the rats was analyzed an d there were no significan t differences in learning between groups (figure 2.1). There was a decrease in amplitude of the unconditioned response in the TTX treat ment group in two of the six rats, suggesting that there was a suppression of performance of the blink response for those two animals (data not shown). T he microdialysates were analyzed for NE, GABA and Glu. The effect of TTX on NE release is observed in figure 2.5A-B, where TTX reduced the amount of NE in the microdialysate when compared with control demonstrating an impul se dependent nature of NE re lease. The effect of TTX on GABA release was also a reduction in the AUC for GABA (Figures 2.5CD). A similar effect is observed with glutamate (Figures 2.5E-F). TTX depleted NE, GABA and Glu basal levels, indicating tonic release for the measured neurotransmitters. Despite the depl etion in basal extracellular levels of neurotransmitters, once the training sessions started each particular neurotransmitter was released with simila r temporal patterns as the non-TTX groups. However the total amount of rel ease was significantly lower compared to baseline levels of the control groups. It is interesting to see that there were not significant differences in the CR’s levels reached on day one of training. This behavioral outcome was unexpected, however we should consider that the low concentration of TTX used might not hav e been enough to impair the sensory inputs carrying information about the US and CS. Even with the reduction in tonic release (due to the presence of TT X) of NE, GABA and Glu, it is also possible that the signal to noise ratio on the PC was sufficient to relay essential

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61 information for memory formation as a gr eater percentage of CR’s were reached on day two of training. The data show that TTX depleted baseline neurotransmitter levels and attenuated the neur ochemical response to training. However, taking into consideration chan ges in evoked neurotransmitter levels relative to baseline levels it is appar ent that neurotransmitter release for both TTX and non-TTX are similar. Therefore, despite t he depletion on presynaptic activity through the cerebellar cortex and inte rpositus nuclei, if the signal to noise ratio remains the same throughout the trai ning sessions, it mi ght still be possible that memory encoding occurs. This wo uld explain why in aged animals, which have attenuated levels of NE, GABA and Glu, if training persists the rats develop CR’s. Therefore, it is not surprising that the TTX group sh ow levels of CRs comparable to the non-TTX group. Mo re experiments need to be conducted to clarify this explanation, and if it is shown to be true it will support the concept of NE signaling facilitating the signal to noise ratio of the PC. 2.5.5 Conclusions. In conclusion, this study monitored t he dynamics of noradrenergic, gabaergic and glutamatergic release as a consequence of delay classical eyeblink conditioning in the cerebellum. These data show t hat NE and GABA release vary over time and are specific to the pair ed training while Glu release is unaltered over days of training and is also obser ved with unpaired training. The timing of the NE response is consistent with previous st udies demonstrating a role for NE to facilitate acquisition. The timing of the GABA response is consistent with a

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62 period of about 1 hour post trai ning during which the cerebellar circuitry is active which may reflect a period of consolidation. The fact that NE is also elevated after training is consistent with a possi ble role for NE in the consolidation process. Furthermore, the data show an al teration in the patte rn of release for NE, GABA and Glu in aged rats, strongly s uggesting that the deficit observed in the acquisition of the CR’s in aged rats is due to a lost in the synchronization of neurochemical patterns during th e acquisition of CR on t he eyeblink conditioning. Also, we have to consider that t he changes in the neurochemical pattern observed in the aged animals could be a consequence of processes underlying aging, such as oxidative stress for which the radical theory of aging have become more accepted (Orr & Sohal, 1994; Orr et al., 1992). A decline in the capacity of normal antioxidant defense mechanism has been postulated as a causative factor in aging related decline in the no rmal activity of different physiological systems (HARMAN, 1956a; Ames et al., 1993a). Damage to proteins, DNA and membranes has also been reported in a ssociation with aging (Ames et al., 1993b; Bickford, 1993; Davies & Goldberg, 1987; HARMAN, 1956b; Gutteridge & Stocks, 1976). Chronic inflammation leads to an over activation of microglia and high levels of pro-inflammatory cyt okines and has been shown to deplete learning in the eyeblink conditioning ta sk (Cartford et al., 2002b). The present report agrees with previous data whic h has demonstrated that deficits in noradrenergic signaling seen in aging are dire ctly associated to the decline in learning capabilities (Bickford et al., 1999) More experiments are required to address the different aspects associated with cognitive deficits observed during

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63 aging. We have shown with microdialysis that ex tracellular levels of NE increase at the beginning of traini ng and remain elevated above basal levels for up to 80 minutes after the training session has finish ed. If we consider the release of NE directly into the cerebellar cortex during t he critical time for consolidation, it is reasonable to suggest that NE might also ha ve a critical role providing a source of energy when it is requir ed for encoding memory in the cerebellum. Therefore, we propose that in addition to improvi ng the signal to noise ratio, NE also activates glycogenolysis, a source of ener gy, while the consolidation of the new memory is taking place. It will be necessary to do specific experiments to elucidate in more details how NE acts on astrocytes to provide the energy necessary for the novo protein synthesis as well as calcium mobilization within the cells which must be also critical during the consolidation of the memory process. The findings from these experim ents could likely help to understand an important aspect of aged-relat ed memory deficits in which there is a deficit in NE signaling.

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64 CHAPTER 3 Beta-Noradrenergic Receptors in the Cerebellum Are Involved in Acquisition of Delay Classi cal Conditioning in Rats: Timing of Disruption 3.1 Abstract The delay classical conditioning task in rats is a paradigm that depends primarily on the cerebellum. Several neuronal pathways are involved in the memory formation process for this task, including the noradrenergic pr ojections from the locus coeruleus to the cerebellum. Pr evious evidence from our lab has shown that blocking beta-noradrener gic receptors with propranolol prior to training sessions significantly impairs acquisition in the eye blink task in rats. This was observed with both intraperitoneal injections and infusion directly into the cerebellar interpositus and lobule HVI. In a recent microdialysis study we have shown that there is a significant elev ation in the extracellular levels of norepinephrine (NE) which occurs during t he training sessions. Interestingly, the increased overflow of NE remains elevat ed for about 60 minutes after the training session has finished on day one, whereas this long lasting pattern of NE release decreases over days of trai ning. Based upon these resu lts we hypothesized that NE overflow that outlasts the tr aining session might be involved in the consolidation of the learned behavior. In order to test this hypothesis, 1 uL of propranolol (100 uM) or vehicle (aCSF) was ipsilaterally infused into the cerebellar lobule HVI 5, 60 or 120 minutes after each training session. The rats which received a local infusion of propranolol showed impaired learning

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65 compared to the control group (aCSF). In addition, activation of cAMPdependent PKA was blocked with the lo cal administrati on of Rp-cAMPS assessed at the same time points (5, 60 and 120 minutes post training). The results show a significant decrease in the acquisition of CR’s. Indicating that both, -adrenergic receptors and PKA signali ng are essential during memory consolidation of delay eyeblink condition ing. These data also show that NE overflow, which remains elevated after the training session may be playing an important role in the process of memory consolidation by prolonging -adrenergic activation beyond the training session period. 3.2 Introduction Delay eyelid conditioning is a cerebellar learning ta sk in which norepinephrine (NE) is known to play a modulatory role NE induces synaptic plasticity in Purkinje neurons by selectively improvin g the signal to noise ratio of evoked versus spontaneous activity, enhancing the sensitivity of cerebellar neurons to both excitatory and inhibitory afferent inputs (Freedman et al., 1977; Moises et al., 1980). Based upon the Marr-Albus theor ies of cerebellar learning Gilbert proposed that NE should have a role in the consolidat ion of memory within the cerebellum (Gilbert, 1975). The involvemen t of NE in memory consolidation in the cerebellum is observ ed in several cerebellar dependent paradigms. For example, in a rod running motor learni ng task the ability of rats to improve performance is reduced following lesion of the locus coeruleus (LC) (Watson & McElligott, 1983). This effect is localized to cerebellar NE (W atson & McElligott,

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66 1984) and is specific to the -adrenergic receptor (Heron et al., 1996). Adaptation of the vestibulo ocular re flex (VOR) is also modulated by noradrenergic inputs (McElligott & Freedm an, 1988) and appears to be mediated by the -noradrenergic receptor (Pompeiano et al., 1991). Cerebellar delay classical conditioning has also been shown to be modulated by NE. Electrolytic lesions of the LC induce resistance to ex tinction in delay conditioning in rabbits (McCormick & Thompson, 1982) and 6-hyroxydopamine (6-OHDA) has been shown to retard acquisition but not perfo rmance of conditioned eyelid responses in rabbits (Winsky and Harv ey 1992). This effect is also mediated by the adrenergic receptor as propranol ol (administered either i.p. or locally into the cerebellum) retards the acquisition of lear ned responses in a delay conditioning task in rats (Cartford et al., 2002a; Cartford et al., 2004b). NE is also implicated in non-cerebellar dependent tasks. LC neurons fire prior to a target cue in a vigilance task in monkeys (Aston-Jones et al., 1994) and during reversal of task contingency, the LC response to the new stimuli precedes behav ioral responding (Aston-Jones et al., 1997). The acti vation of adenylyl cyclase-cAMP-protein kinase A (PKA) intracellular signaling cascade has been shown to be essential for long-term memory consolidation in diverse brain areas including the hippocampal formation and prefrontal co rtex (Squire & Zola-Morgan, 1991; Goldman-Rakic, 1987; Arnsten & GoldmanRakic, 1987). However, the role of PKA cascade in the consolidat ion process is still under debate. In this study we manipulated the activity of the -adrenergic receptors and PKA activity by administering microinjections of propranol ol and Rp-cAMP directly into the

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67 cerebellar cortex at specific post traini ng periods within the ti me period that NE overflow remains elevated. The time period post training (in which we see elevated levels of NE) is though to be critical for memory consolidation (De Zeeuw & Yeo, 2005; Y eo, 2004; Krupa et al., 1993a; Bao et al., 2002; Nolan et al., 2002; Rogers et al., 2001c; Gould & Steinmetz, 1996) Other neurotransmitter systems are also implicated in delay eyeblink conditioning tasks. Administration of the glutam ate AMPA receptor ant agonist CNQX or the GABA-A receptor antagonist picrotoxin into the cerebellar cortex completely and reversibly impairs fully established CR’s, suggesting that GABAergic and glutamatergic transmission are equal, and essential for the CR performance (Attwell et al., 2002b). It has been sugges ted that the basal GABAergic output from the cortex onto the interpositus nucleus modulates conditioned response (CR) expression, whereas timing of CR's is modulated by the stimulus activated inhibition (Bao et al., 2002). The expre ssion of CR’s can be interrupted by both GABA mediated inactivation of the inte rpositus neurons with muscimol (GABA-A agonist) as well as up-regulat ion of activity with picr otoxin (GABA-A antagonist) (Aksenov et al., 2004). To date much of the work examining th e role of neurotransmitters in delay eyeblink conditioning have us ed either the application of agonists or antagonists to investigate the postsynaptic effects of these neurotransmitters. We have also recently demonstrated that NE is released in the cerebellum during training on

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68 the delay eyeblink task and that the leve l of NE remains above background for up to 80 minutes post training. An impor tant question that remains unanswered concerns the timing in wh ich presynaptic release of neurotransmitters in the delay conditioning task are critical fo r consolidation of the new learned experience. This study uses delay eyeblin k conditioning to examine critical postsynaptic events relating to the NE signal transduction cascade at different time points post training. It is hypothesized t hat the release of NE in the cerebellum during and post training activates -adrenergic receptors and the subsequent PKA signal transduction cascade, which is critical for the consolidation of cerebellar motor learning. 3.3 Methods 3.3.1 Animals and surgery Male F344 rats weighing 270–320 g were used in this study. Room temperature was kept at 72 F and the dar k/light circle was 12-h (li ght was on from 7:00 AM to 7:00 PM). Animal number wa s the minimum required for reliable statistical test results. Rats were anesthetized with Isoflurane and placed in a stereotaxic instrument. A 10 mm long guide shaft made of 21-gauge stai nless-steel tubing was inserted into the cerebellum. The guide shaft was attached to the skull by jeweler screws and cemented with dental acrylic. The coordinates AP-11.4, ML +2.4 and DV -1.7 in reference to bregma was used to implant the guide cannulae for the microinjection into the cerebellar lobule HVI (s implex, and interpositus nucleus). In the same surgical session ra ts were prepared for eyelid training by

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69 fixing a small ITT/Cannon connector strip to thei r skull to hold gold pin connectors to EMG wires that are run under the left eyelid. This method has been previously published by our lab (Cartf ord et al., 2002a). Rats are allowed to recover for one week after the sur gery procedure before st art the eyeblink conditioning training and microdialysis beg an. Each animal was used for only one experimental condition. All procedures were carri ed out in accordance with the institutional guidelines (IACUC) and with USA National Inst itute of Health Guide for the Care and Use of Laboratory Animals. 3.3.2 Training The rats were habituated to the traini ng chamber and headstage cable for three days. The training consisted of 50 trials each training tr ial consisted of a 250 ms baseline, a 400 ms CS period, and a 100 ms US period. The tone was 500 ms in duration and overlapped the airpuff for 100 ms. The tr aining tone was 3kHz and the airpuff 10 psi. Hardware and softwar e used to train and analyze data were manufactured by J.Tracy, J.Green and Joe Steinmetz, (Bl oomington, Indiana). Eyelid EMG data was collected, amplif ied, rectified, and integrated. Learned responses were determined using a 10 st andard deviation criterion for eyelid amplitude elevated during t he CS period when compared to the baseline. Alpha responses to the tone ar e excluded from learned res ponse analysis by using a 70 ms discrimination/exclusion wi ndow. Learning was measured as the percentage of learned (c onditioned) responses (CR’s) made in each training session.

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70 On every experimental session 1 L of propranolol (100 M) or Rp-cAMP (80 nM) was administered over five minutes through the guide cannula with an infusion pump at intervals of 5, 60 or 120 minutes after the training. The control group received artificial cerebral spinal fluid at pH: 7.4. 3.3.3 Design and Analysis To analyze behavior for the eyeblink conditioning task, separate mixed model analyses of variance were used to analyz e Drug and Session e ffects at each of the drug administration time points. Data from dr ug administration 5 minutes after training was analyzed using a three-way mixed m odel analyses of variance. For the analyses of the other time poi nts 60 and 120 minutes data were analyzed using a two-way mixed model analyses of variance {[ Drug (2): (Propanolol, aCSF)] x [Session (6): Day 1-3 AM and PM]}. Data were analyzed using a threeway mixed model analyses of variance {[Dr ug (2): (aCSF, Rp-cAMP)] x [Time of drug delivery (3): (5, 60, and 120 minutes)] x [Session (6): 1, 2, 3, 4, 5, 6]}. Post Hoc Analyses (Dunnett’s) were used to test for Drug and Time effects. Comparisons were determined significant at the 0. 05 alpha level. Percent Conditioned Response ( CR %) and Amplitude of re sponse (Conditioned and unconditioned) were the dependent measures.

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71 3.4 Results Propranolol when administered 5, 60 or 120 minutes following training significantly disrupts learning of CR’s. Figure 3.1A i llustrates the attenuation of learning (shown as % CR) when propranol ol is administered 5 minutes post training {significant drug x sessi on interaction [F(5,80) = 2.72, p <0.05]}. The effect of propranolol is most pronounced at 5 minutes post training with rats learning significantly less than aCSF on se ssions 3, 4, 5 and 6. A similar pattern is observed in rats treated with propranol ol 60 minutes post training, however the magnitude of the differenc e is reduced. As can been seen in Figure 3.1B administration of propranol ol 60 minutes after eyeblink conditioning reduced learning in sessions 3 and 4 {significant drug x session interaction [F(5,40) = 2.58, p <0.05]}. However, w hen propranolol was adminis tered 120 minutes post training there was no difference with aCSF in learning acquisition interaction (see figure 1C) [F(7,35) = 0.81, p = 0.812]. These data show robust learning in aCSF treated rats and partial learni ng in propranolol treated rats when administered 5 or 60 minutes post training. The effect of pseudoconditioning on CR’s did not differ over sessions or as a result of dr ug administration.

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72

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73 Figure 3.1 Propranolol administered 5, 60 or 120 minutes following training significantly disrupts learni ng of CR’s. The X-axis sh ows training sessions over time, with session 1 being Day 1 AM, 2 Da y 1 PM, 3 Day 2 AM, 4 Day 2 PM, 5 Day 3 AM, and 6 Day 3 PM. A) illustrate s the attenuation of learning (shown as % CR) when propanolol is administered 5 minutes post training, with rats learning significantly less than aCSF on se ssions 3, 4, 5 and 6 (indicated by *). Pseudoconditioning did not differ over sessions or as a result of drug administration. B) Propanolol administration 60 minutes after eyeblink conditioning reduced CRs relative to aCSF in sessions 3 and 4 (indicated by *). C) When propanolol was administered 120 mi nutes post training there was no difference with aCSF in learning acquisition. These data show robust learning in aCSF treated rats and partial learni ng in propranolol treated rats when administered 5 or 60 minutes post training. Figure 3.2 shows the amplit ude of the conditioned re sponse (A, C, E) and the unconditioned response (B, D, F). Analyse s of amplitude of both CR and UR for drug treatments 5, 60 and 120 minutes post training failed to detect any significant difference between aCSF and propr anolol. The timing of the CR was also not affected by drug treatment. Neither CR onset or CR peak amplitude times were significantly different in any of the drug treatm ent groups (data not shown).

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74 Figure 3.2. The amplitude of the conditioned response (A,C,E) and the unconditioned response (B, D, F) during eyeblink condi tioning. The X-axis shows training sessions over time, wit h session 1 being Day 1 AM, 2 Day 1 PM, 3 Day 2 AM, 4 Day 2 PM, 5 Day 3 AM, and 6 Day 3 PM. Analyses of amplitude of both CR and UR for drug tr eatments 5, 60 and 120 minut es post training failed to detect any significant differenc e between aCSF and propanolol. A) CR 5 minutes post training. B) UR 5 minutes post training. C) CR 60 minutes post training. D) UR 60 minutes pos t training. E) CR 120 mi nutes post training. F) UR 120 minutes post training. Rp-cAMP when administered 5, 60 or 120 minutes followin g training significantly disrupts learning of CR’s. Animals which received infusions of ACSF improved from session 1 (mean = 29%) to session 6 (mean = 71%), demonstrating

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75 learning (shown as % CR) over time, independent of time of drug delivery. Figure 3.3 illustrates the attenuati on of learning when Rp-cAMP was administered 5, 60 or 120 minutes post trai ning {significant dr ug x session x time of drug interaction [F(10,120) = 2.20, p <0.05]}. The effect of Rp-cAMP was most pronounced when administered 5 or 120 minutes post training with rats learning significantly less than aCSF on sessions 3, 4, 5 and 6. A similar pattern was observed in rats treated with Rp-cAMP 60 minutes post training, however the magnitude of the difference was reduced. These data s how robust learning in aCSF treated rats and partial learni ng in Rp-cAMP treated rats when administered after training. Figure 3.3 The Effect of Rp-cAMP on Eye Blink Conditioning. Rp-cAMP or aCSF was administered via local infusion into the interpositus nucleus of the cerebellum 5, 60 or 120 minutes post traini ng on the eye blink co nditioning task. Rats which received aCSF (open square) significantly improved learning over

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76 sessions ( p <0.05). Partial learning in Rp-c AMP treated rats was observed when administered 5, 60 or 120 minutes after training ( p <0.05). Y axis shows percent conditioned responses (% CR). n=5 per condition. 3.5 Conclusions For several decades there have been investigations into the role of the cerebellum in the process of memory c onsolidation in the classical eyeblink conditioning task (Thompson et al., 1997). Despite the findings which have shown that the cerebellar cortex, cerebellar nuclei and inferior olive are all required in the processing of conditioned learning, le ss work has examined how noradrenergic inputs from the LC contribute to such processes. What is clear to date is that the noradrenergic inputs to t he cerebellum from the LC excerpt both stimulatory and inhibitory effects when NE binds to receptors in the cerebellar cortex and the literature also supports a role of NE in arousal, emotion and learning (Cooke et al., 2004). The alph a and beta adrenoceptor have been found to be present in PC of the cerebellum (Rusakov et al., 2005), in which NE selectively inhibits the spontaneous activi ty of PC at the same time climbing fibers evoked activity remains either unaffected or augmented with NE action, thus increasing the signal to noise ratio of the evoked activity onto the PC (Luft et al., 2004). A vast body of data suppor ts that aged-related decreases in catecholamines may contribute to impai rments in learning acquisition on motor learning tasks (Luft & Buitrago, 2005; O'Dowd et al., 1994). It is well known that NE has profound effects in the cerebellum while a significant sensory input is

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77 activated such as CS and US (thr ough climbing and mossy fibers) during eyeblink conditioning which has led to a great interest in dissecting out the precise or discrete areas of the cerebe llum in which memory formation takes place. In this study we showed how the disrupt ion of beta-adrenergic signaling in the cerebellar cortex diminished learning of conditioned responses (CR’s) when propranolol was locally administered into the cerebellar cortex at 5 and 60 minutes post-training. The data show that noradrenergic signaling inputs to the cerebellar cortex are necessary for the consolidation proce ss by which CR’s are formed. Previous studies from our lab have shown that local administration of propranolol into the cerebellum prior to t he training sessions disrupts the normal rate of learning of CR’s (Cartford et al., 2002a). However in this study we showed that there is still significant activi ty of NE receptors even after the training session has finished indicating the possibili ty that noradrenergic activity is part of the overall process of memory consolidation. Our findings concur with previous data indicating that the dep letion of NE signaling in the cerebellum of aged animals might be the primary cause of the learning deficit but not of the performance of CR’s. The local administration of propranolol at 5 a nd 60 minutes post training had the largest impact depleting the CR’s acquisition while pr opranolol administered 120 min post training session did not cause a si gnificant impairment in the acquisition

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78 of CR’s. It is important to note that when propranolol was administered 5 and 60 minutes post-training despite the depletio n of CR’s the rats still learned (see figures 3.1 and 3.2), indicating that ev en when the beta-noradrenergic signaling is blocked there must be other neurot ransmitters or factors driving the consolidation of memory. It also has been shown that the blockade of nonNMDA receptors by CNQX infusion into the cerebellar cortex is also capable of disrupting learning (Attwell et al., 2001). In addition, the activation of GABA-A receptors in the cerebellar nuclei using muscimol can disrupt the acquisition of CR’s (Hardiman et al., 1996; Krupa et al., 1993c; Ramnani & Yeo, 1996; Yeo et al., 1997). Most recently, Cooke and coll eagues published an experiment where they demonstrated that cortical infu sions of the GABA-A receptor agonist muscimol delayed by 5 or 45 minutes after a conditioning session disrupted learning, suggesting a role of the cerebellar cortex in consolidation (Cooke et al., 2004). Our data concur with this report prov iding evidence that there is critical post-training memory consolidation peri od which is mediated by the cerebellar cortex. Together these finding sug gest that in addition to various neurotransmitters playing a role in the acquisition of CR’s th ere are critical temporal effects which have been elucidat ed for GABA and NE which are critical for the acquisition of CR’s. The data presented in this study show t hat when beta-adrenerg ic receptors are blocked in the cerebellar cortex 5 and 60 mi nutes post-training, the acquisition of CR’s are impaired, however, the rats still learn the task. Propranolol may

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79 interfere with the consolidation process by the following mechanisms: First, it is known that NE is a neuromodul ator in that it selectivel y improves the signal to noise ratio of evoked versus spontaneous activity which enhances the sensitivity of the cerebellar neurons to excitatory and inhibitory afferent inputs (Eccles, 1967; Hoffer et al., 1973). It is possibl e that the inhibition of the betanoradrenergic signaling makes it difficult to enhance the required sensitivity of the cerebellar neurons to the sensory input carrying the US and CS signal through the climbing fibers and mossy fibers. In other words, the lack of the noradrenergic signal (elicit ed by propranolol administration) impairs the improvement of the signal to noise rati o of evoked versus spontaneous activity which may explain why the rats would requ ire more training sessions in order to reach a significant condition ing response. Even when t he rats received only six conditioning training sessions it is plausible to pred ict that after a few more training sessions the rats could show better CR’s. In figure 3.1 there was a significant difference in the groups t hat received aCSF and propranolol which started in session 3, however a trend toward s learning remained for the rest of the sessions. When propranolol was adm inistered 60 minutes post-training, a significant impairment was observed (com pared to aCSF – see figure 3.1) on sessions 3 and 4 but sessions 5 and 6 did not show significant differences. These results demonstrate that when pr opranolol is administered into the cerebellar cortex, the rats might require more training sessions in order to develop similar levels of CR’s as observed in the control group.

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80 Another explanation for the depletion of C R’s after blocking the beta-adrenergic receptor at different time points post-trai ning could be associat ed with the activity that NE exerts by activating the betaadrenergic receptors which leads to the activation of cAMP/PKA signaling cascade. Acti vation of the cAMP/PKA signaling pathway has been shown to enhanc e intracellular calcium and promote the release of vesicles containing G ABA (Saitow et al., 2005). Furthermore, increases in intracellular calcium in G ABAergic synapses are also modulated by AMPA receptors (Rusakov et al., 2005; Sata ke et al., 2004). Interestingly, Luft and colleagues demonstrated that moto r skill learning dep ends on the novo synthesis of proteins in motor cortex after training. Based on the authors work they hypothesize that consolidation (of mo tor learning) require s modifications in neuronal circuitry or plastic changes in neuronal structure and established that the disruption of protei n synthesis in inter trials can diminish consolidation (Luft et al., 2004; Luft & Buitrago, 2005). In this case, protein synthesis may theref ore require a pool of energy in the form of ATP in order to assemble and reloca te the new synthesized proteins. Thus, it is essential that a reliable source of ener gy during the critical period of protein synthesis is available for intracellula r signaling to occur and induce synaptic plastic changes. In this regard, NE has been shown to enhance glycogenolysis through astrocytes therefore providing the energy necessary to undergo memory consolidation (O'Dowd et al., 1994). It also has been shown that betaadrenoceptors are highly localized in as trocytes (Stone & Ar iano, 1989b; Aoki &

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81 Pickel, 1992; McCarthy, 1983). It has been suggested that the noradrenergic action on glial cells might be the source of production an d release of glucose for neural energy production (Stone & Ariano, 1989a). We have shown that extracellular levels of NE increased at the beginning of training and remained elevated above basal levels for up to 80 minutes after the training session (see chapter 2). If we consider the release of NE directly into the cerebellar cortex during the critical time for consolidation, it is reasonable to suggest that NE might also have a critical role providing a source of energy when it is required for encoding memory in the cerebellum. Ther efore, we propose t hat in addition to improving the signal to noise ratio, NE al so activates glycogenolysis, a source of energy, while the consolidati on of the new memory is ta king place. NE also activates glycogenolysis, a source of ener gy, while the consolidation of the new memory is taking place. NE exer ts its facilitatory effect through -adrenergic receptors and the activation of cAMP/PKA signaling cascade. Our results clearly demonstrate that when cAMP/PKA is bl ocked at different time points post training, the acquisition of CR is depleted indicating that the cAMP/PKA signaling cascade is an essential pathway in memory consolidation of eyeblink conditioning. It is important to consider that both PKC and PKA mediate histamine receptors which are dependen t on their respective downstream pathways, Ca2+-PKC and cAMP-PKA (Sakhalkar et al., 2005a). Rp-cAMP has been used as a tyrosine kinase inhibito r to study the downstream signaling cascade of histamine receptors (Sakhalkar et al., 2005b). In addition, histamine receptors have been shown to play a ro le in performance on both the rota-rod

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82 and balance beam tasks through Histamine2 receptors in the cerebellar interpositus nucleus. Therefore, it is possible that they are also involved in memory consolidation of eyeblink condi tioning (Song et al., 2006). Futhermore, functional regulation of Ca2+ channels relies on phosphorylation processes. Protein kinase A and C and the Rp-cAMP c ould have affected Ca2+ channels that depend of the activity of PKA (Christ et al., 2004). It is necessary in the future to conduct experiments that utilize more specific antagonists to target the noradrenergic signaling cascade. It will be necessary to do specific experiments to elucidate in more details how NE acts on astrocytes to provide t he energy necessary for the novo protein synthesis as well as calcium mobilizat ion within the cells which must be also critical during the consolidation of me mory process. The findings from the proposed experiments could likely help to understand an important aspect of aged-related memory deficits in which ther e is a deficit in NE signaling.

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83 CHAPTER 4 TNFinactivation improves l earning in aged rats whereas the activation of TNFin young rats depletes learning 4.1 Abstract Tumor necrosis factor alpha (TNF) is a multifunctional proinflammatory cytokine, which is a critical inflamma tory mediator involved in aging and neurodegenerative diseases of aging. Previous work has shown that diets enriched with antioxidants r educe levels of the proinflammatory cytokine TNFin the central nervous system of aged rats These diets have also been shown to improve classical eyeblink conditioning performance in aged rats. Therefore we tested the hypothesis that infla mmation and more specifically TNFmay be a critical factor that modulates cla ssical conditioning behavior. If TNFincreased cerebellar levels negatively affect perfo rmance on the eyeblink conditioning task, then exogenous admini stration of TNFin young rats should result in an impairment in the acquisition and/or ret ention of eyeblink conditioning memory. On the other hand, the reduction of t he age-related increase in cerebellar TNFlevels in aged rats should result in an improvement in memory. To address this question in experiment one young (3 month old) F344 rats were pretreated (via infusions into the cerebellar cortex lobus simplex) with 2 L of recombinant rat (rr)TNF(50 ng) one day prior to training and then daily 3 h prior to eyeblink conditioning coupled to microdialysis for 5 consecutive training sessions with one session per day. The control group receiv ed the same dose of denatured rrTNF

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84 (heated at 90 C for 15 minutes). The re sults showed that young rats treated with rrTNFhave a decreased rate of learning compared to the control group. The neurochemical prof ile of neurotransmitter release measured with microdialysis on day 1 of training from young rats resembled that observed in aged rats. A significant sustained rel ease of norepinephrine was observed after the training session of eyeblink condition ing. In a second experiment aged (22 month old) F344 rats were pretreated with intracerebellar microinjection a 2 uL of anti-rat TNF0.3 pg/ml three times a week for 4 weeks prior to eyeblink conditioning training with microdialysis. Aged rats showed a better performance in the conditioned responses when compared to controls. The release of NE in this group reached basal leve ls sooner than the control group but not as early as the young rats. The results of thes e experiments demonstrate a critical correlation between TNFand the rate of conditioned learning acquisition. 4.2 Introduction NE is released from the LC to the cer ebellar cortex (among other brain areas) (Foote et al., 1983; Aston-Jones et al., 198 6) and exerts a modulatory effect on the action of other neurotransmitters in the cortex and deep nuclei of the cerebellum (Gould et al., 1997a). This l eads to amplification of the afferent inputs to the cerebellar purkinje cells (P C) and is thought to occur through the action on -noradrenergic receptors (Yeh & W oodward, 1983a; Woodward et al., 1991b). Motor learning has been widely associated with noradrenergic innervation to the cerebellar cortex in rats (Bickford et al ., 1992; Heron et al.,

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85 1996; Cartford et al., 2004b; Bickford et al., 1986; Watson & McElligott, 1983; Bickford, 1995; Pompeiano et al., 1991; McCormick & T hompson, 1982). Delay classical conditioning is a well esta blished cerebellar-dependent learning paradigm and is modulated in part by NE (McCormick & Thompson, 1982; Harvey et al., 1993). When the -adrenergic receptor antagonist, propranolol is administered systemic or locally (cer ebellum), the acquisition of learned response on the delay classical conditioning in rats is impaired (Cartford et al., 2002a; Cartford et al., 2004b). Furt hermore, the administration of 6hydroxydopamine depletes NE storage and prevents animals from regaining proficiency on a motor learning task (Watson & McElligott, 1983; Watson & McElligott, 1984). Aging-associated deficit s on motor learning have been linked to dysfunction of the noradr energic system (Cartford et al., 2004a; Bickford et al., 1985b; Bickford et al., 1986) which is thought to be caused by the loss of noradrenergic enhancement of the relative responsivene ss of purkinje neurons to afferent inputs in aged animals. Age-related pathologies are characte rized by a pronounced imbalance in immune functions like glial hyperactivity with altered antigen expression of microglia in aged rodents (Perry et al., 2003b; Cunningham et al., 2007a). Chronic inflammation is known as one of the multiple age-re lated pathologies that involves the activity of several pr oducts, including cytokines (Murray et al., 1997a; Murray & Lynch, 1998b). Cytokines are proteins that mediate the response of the body’s defense syst em to injury and mediate diverse

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86 inflammatory processes. The presence of altered levels of cytokines in the central nervous system has been implic ated in several aged-related and neurodegenerative diseases (Benveniste & Benos, 1995). Cytokines are secreted by activated microglia and can be either pro-inflammatory cytokines, among them tumor necrosis factor alpha (TNF), interleukin 1 beta (IL1), and anti-inflammatory cytokines such as interleukin 10 (IL-10) and transforming growth factor beta 1 (Uccelli et al., 2005a) Pro-inflammatory cytokines are chronically increased in the aging brain (Godbout et al., 2005a). TNFand TNFare significantly elevated in the ce rebellum of aged rats (Gemma et al., 2002) and diet rich in ant i-oxidant reduced both TNFand TNFlevels. In addition, feeding aged F344 rats a diet enr iched in spinach im proves cerebellar -adrenergic receptor functi on and improves motor learning that was associated with a decrease in oxidized gluthathione and the pro-inflammatory cytokine TNF(Cartford et al., 2002b). The present study extended on previo us findings to determine whether inflammation and more specifically TNFmay be a critical factor that modulates classical conditioning behavior during the agi ng process. In this experiment TNF was administered to young rats and bl ocked in aged rats young rats prior to eyeblink conditioning coupled microdialys is. The results of these experiments demonstrate a critical correlation between TNF, aging and learning.

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87 4.3 Materials and Methods 4.3.1 Animals and surgery Male F344 rats weighing 3 and 20 months old were used in this study. Room temperature was kept at 72 F and the da rk/light circle was 12-h (light was on from 7:00 AM to 7:00 PM). Animal number was the minimum requ ired for reliable statistical test results. Rats were anesthetized with isoflur ane and placed in a stereotaxic instrument. A double 10 mm long guide shaft made of 21 and 26 gauge stainless-steel tubi ng (separated by 1mm) were inserted into the cerebellum. The guide shaft was attached to the skull by jeweler screws and cemented with dental acrylic. The coordinates AP-11.4 ML +2.4 and DV -1.7 in reference to bregma was used to implant the guide cannulae for the microinjection into the cerebellar lobule HV I (simplex, and interpositus nucleus). In the same surgical session rats were prepared for eyelid training by fixing a small ITT/Cannon connector strip to thei r skull to hold gold pin connectors to EMG wires that are run under the left eyelid. This method has been previously published by our lab (Cartford et al. 2002). Rats are allowed to recover for one week after the surgery procedure before st art the eyeblink conditioning training and microdialysis began. Each anima l was used for only one experimental condition. All procedures were carried out in accordance wit h the institutional guidelines (IACUC) and with USA National In stitute of Health Guide for the Care and Use of Laboratory Animals.

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88 4.3.2. Treatment with rrTNFand anti-rat TNFOn completion of surgery young and ag ed rats were randomly assigned to different treatment groups. Young (3 mont h old) F344 rats were pretreated (via infusions into the cerebellar cortex lo bus simplex) with 2 uL (50ng) of rrTNFone day prior to training and then daily 3 h prior to eyeblink conditioning coupled to microdialysis for 5 consecutive traini ng sessions with one session per day. The control group received the same dose of denatured rrTNF(heated at 90 C for 15 minutes). The results show that young rats treated with rrTNFhave a decreased rate of learning compared to the control group. The neurochemical profile of neurotransmitter release meas ured with microdialysis on day 1 of training from young rats resembles that observed in aged rats. A significant sustained release of norepinephrine is obs erved after the training session of eyeblink conditioning. In a second expe riment aged (22 month old) F344 rats were pretreated with intracerebellar microinjection a 2 uL of anti-rat TNF0.3 pg/ml three times a week for 4 weeks pr ior to eyeblink conditioning training with microdialysis. 4.3.3 Training of behavior in a dela y classical eyeblink conditioning task The rats were habituated to the traini ng chamber and headstage cable for three days. The training consisted of 50 trials each training tr ial consisted of a 250 ms baseline, a 400 ms CS period, and a 100 ms US period. The tone was 500 ms in duration and overlapped the airpuff for 100 ms the training tone was 3 kHz and the airpuff 10 psi. Hardware and softwar e used to train and analyze data were

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89 manufactured by J. Tracy, J. Green and J. Steinmetz, (Bloom ington, Indiana). Eyelid EMG data was collected, amplif ied, rectified, and integrated. Learned responses were determined using a 10 st andard deviation criterion for eyelid amplitude elevated during t he CS period when compared to the baseline. Alpha responses to the tone ar e excluded from learned res ponse analysis by using a 70 ms discrimination/exclusion wi ndow. Learning was measured as the percentage of learned (condi tioned) responses (CR’s) made in each training session. 4.3.4 Design and Analysis To analyze behavior for the eyeblink conditioning task, separate two-way mixed model analyses of variance were used to analyze Drug and Day effects {[Drug (2): (YOUNG: Control, rrTNF) or (AGED: IgG, Anti-TNF)] [Day (5): 1-5]}. For the analyses of NE release separat e two-way mixed model analyses of variance were used to analyze Drug and Ti me effects {[Drug (2): (YOUNG: Control, rrTNF) or (AGED: IgG, Anti-TNF)] [Time (18): -30 to 140 minutes]}. Post Hoc Analyses (Dunnett’s) were used to test for Drug and Time effects. Comparisons were determined significant at the 0. 05 alpha level. Percent Conditioned Response (CR %) was used as the dependent measure.

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90 4.4 Results 4.4.1 Young In figure 4.1A it is shown that infusions of rrTNF(50ng) into the interpositus nucleus 24 and 3 hours before each day of training blocked learning on the classical eyeblink conditioning task (shown as percentage of conditioned response (% CR)) in young rats {significant drug x day interacti on [F(4,40) = 5.8, p <0.05]}. Rats which received control infusions (denatured rrTNF) showed progressive learning (incr eases in % CR) over 5 da ys. On day 1 % CR was significantly less than days 3, 4 and 5. Also on day 3 % CR was significantly less than days 4 and 5. Rats given rrTNFinfusions did not show a significant improvement in % CR over 5 days. These data suggest that rrTNFinjected into the cerebellum of young rats significant ly impairs the rats ability to learn the eyeblink conditioning task. The time course of NE release in young rats during classical eyeblink conditioning is shown in figur e 4.1B {significant drug x time interaction [F(17,153) = 3.0, p <0.05]}. Microdialysis was performed on day 1 of eyeblink conditioning and started 30 minutes before training and continued for 140 minutes once training started. Samples were co llected every 10 minutes. NE release significantly increased above baseline leve ls for 60 minutes after training began in both control and rrTNF, suggesting that NE plays a critical role during the acquisition of learning. NE release reac hed baseline levels 70 minutes after the beginning of training. Infusion of rrTNFinto the interpostius nucleus

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91 significantly decrease the release of NE du ring the 20 minutes of training and 10 minutes following training (p<.05) These data show that TNFattenuates NE release in the cerebellum of young rats.

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92 Figure 4.1 Effects of TNFin young rats. Performance on eyeblink conditioning task (A) and the time course of NE releas e (B) in young F344 rats. A) Rats which received control infusions (rrTNFheated) showed progressive learning

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93 (increases in %CR) over 5 days. Whereas, rats given rrTNFinfusions did not show significant improvement in % CR over days, suggesting that rrTNFsignificantly impairs the young rats ability to learn the eyeblink conditioning task. B) Microdialysis was performed in young ra ts and the time course of NE was recorded during eyeblink conditioning. To obtain basal level of NE, microdialysates were collected for 30 mi nutes before training (baseline) and continued for 140 minutes from the beginning of training (time points 0-20). NE release significantly increased above bas eline levels during training in both control and rrTNF, reaching baseline levels 70 minutes after training began. However, infusions of rrTNFinto the interpositus nucleus significantly lowered NE levels at time points 10-30 minutes (indicated by *) compared to controls, demonstrating that TNFattenuates NE release in the cerebellum of young rats. 4.4.2 Aging Figure 4.2A shows learning (s hown as increases in % CR over days) in aged rats which received infusions of IgG (control) or Anti-TNFinto the interpositus nucleus {significant drug x day interaction [F(4,40) = 3.6, p <0.05]}. Anti-rat TNFwas infused (0.3pg/ml in 2l, during 5 mi nutes) three times a week for 4 weeks prior to eyeblink conditioning. Both groups demonstrate progr essive learning (increases in %CR) over days. The percentage of conditio ned responses was significantly lower on day 1 than days 2 5. Rats injected with anti-TNFperformed significantly better (higher %CR) on days 4 and 5 compared to controls. These data suggest that the blockage of TNFin aged rats can improve learning on the ey eblink conditioning task. The time course of NE release in aged rats which received infusions of IgG (control) or anti-TNFinto the interpositus nucleus is shown in Figure 4.2B {significant drug x time in teraction [F(17,136) = 1.8, p <0.05]}. NE release was significantly elevated in both conditions compared to baseline NE levels. Chronic

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94 anti-rat TNFinfusion resulted in significant increases above baseline NE levels (see figure 4.2B). The insert to figure 4.2B shows the AUC for NE release from time point 0 to 50 minutes from the beginni ng of training. Note that in the antiTNFgroup NE release appeared to reach a higher peak of NE release sooner than the IgG group.

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95 Figure 4.2 Effects of the blockade of TNFin aged rats. Performance on eyeblink conditioning task (A) and the time course of NE release (B) in 22 month

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96 old F344 rats. A) Progressive learning over days (shown as increases in % CR) was shown in both IgG and anti-TNFinjected aged rats. Interestingly, aged rats injected with anti-TNFperformed significantly better (higher % CR) on days 4 and 5 compared to IgG controls (i ndicated by *), suggesting that the blockade of TNFin aged rats can improve lear ning on the eyeblink conditioning task. B) Shows the time course of NE release in aged rats which received infusions of IgG (control) or Anti-TNFinto the interpositus nucleus. NE release was significantly elevated in both conditions compared to baseline. Chronic antirat TNFinfusion resulted in a significant increase above baseline NE levels. Insert shows the AUC for NE release from time points 0-50 minutes. Note that in the anti-rat TNFgroup NE release appeared to reach a higher peak sooner than the IgG group. 4.5 Discussion The main goal of this study was evaluate whether increases in TNFlevels which are part of the aging process are to some degree responsible for the decline on memory formation capabilities. In order to evaluate this hypothesis we did unilateral infusions of rrTNFthrough the deep nuclei and cortex in rat cerebellum and have found that the administration of rrTNFin young rats, prior to the training sessions of delay eyeblink conditioning significantly interf eres with acquisition of CR’s. On the other hand, when aged rats were treated with anti-TNFimprovements in the acquisition of CR’s where observed demons trating that they were capable of learning faster than controls. The resu lts show that pharmacological intervention targeting high levels of TNFpresent in the cerebellum of aged animals leads to an improvement in learning capabilities Suggesting that the activity of TNFin some ways affects the processing of information to the cerebellum and hence interferes with the ac quisition of CR’s.

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97 These results support the theory that cere bellar physiology is to some degree vulnerable to the presenc e of high levels of TNFwhich is evident since the local administration of recombinant TNFin young rats disrupts normal acquisition of CR’s. The experimental des ign used with the young rats parallels an acute insult occurring just 5 minutes before the animals undergo eyeblink conditioning training. Chap ter 2 discussed how there is a post-training timeline which is a process by which memory consolidation happens and pharmacological or molecular interventions during the cons olidation process ca n interfere with the dynamics of memory consolidation (Mint z et al., 1994; Lavond et al., 1985; De Zeeuw & Yeo, 2005; Cooke et al ., 2004; Attwell et al., 2002a). TNFin young rats In our experimental design where young animals were pre-treated 5 minutes before training sessions it is po ssible that the presence of TNFimpacts the cerebellar region through the ac tion on its receptors. TNFhas two subtypes of receptors which have a broad spectrum of effects and have been reported to exist in areas such as cortex, brains tem, cerebellum and basal ganglia among other brain areas (Kinouchi et al., 1991) Cytokines, specifically recombinant human TNFhas been reported to induce concentration-dependent and reversible alterations in the electr ophysiological properties of axons in mammalian spinal cord (Davies et al., 2006a) This study provides evidence that elevated concentrations of TNFinduce reversible depolarization of the

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98 compound membrane potential (CAP) and reduction in CAP amplitude, sometimes to the point of extinction of the CAP, suggestive of impaired axonal conduction. Based on this report, it is pl ausible that local administration of TNFinto the cerebellum might have impaired ax onal conduction in a critical time in which the rats were receiving the ey eblink conditioning training and even for a critical period of time (pos t training session) for memory consolidation, in which case the depletion on the CR’s acquisiti on occurred. Proinflammatory cytokines, specifically TNFhave been reported to induce, through the classical I kappa B degradation pathway, a repres sion in excitatory amino acid transporter two (EAAT2) on astrocytes and increases the expression of AMPA receptors on synapses, which leads to elevated extracel lular glutamate concentrations and in consequence facilitates the risk of glutam atergic neuronal toxicity (Sitcheran et al., 2005). In this case, glutamatergic neur otoxicity due to ex cessive glutamate activation might be involved in the depl etion of CR’s acquisition observed when the rats received a direct injection of TNFdirectly into the cerebellum. Given the previous facts it is very likely that the effect observed with TNFin young rats is due to an impairment on memory consolidation, in this case more experiments would have to be conducted to test this hypothesis, which can be assessed by administering the TNFat critical times after the training sessions of eyeblink conditioning. Another possi ble cause could be over saturation of signal input through the climbing and mossy fibers due to the glutamatergic overdrive and perhaps affect ing the appropriate signal to noise ratio required to trigger a significant signal on the PC nec essary to lead memory formation.

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99 Anti-TNFin aged rats All the possible mechanisms stated to explain the effect of TNFin young rats also apply to aged rats. For aged rats we mu st consider that chronic exposure to high levels of TNFare reported in aged rats (Gemma et al., 2002), which changes the scenario compared to young ra ts. Interestingly we observed a behavioral improvement regardi ng the CR’s acquisition in aged rats by training day four and five showing that pr etreatment with th e antibody anti-TNFhas reversed the cognitive impairment normall y seemed in aged rats. This to a certain degree rules out neurotoxicity ( due to high levels of Glu and apoptosis normally triggered by TNF) as a major cause of t he CR impairment observed in aged animals (Sitcheran et al., 2005). This idea is supported by the fact that microdialysis performed in aged rats duri ng eyeblink conditionin g training showed (see chapter 1 of this dissertation) l ong lasting increases in extracellular glutamate compared to young rats. This c ould be partially due to the effect of TNFin the glutamate transporter syste m leading to a prolonged time for clearance of glutamate from the extracellular space. Another possible mechanism by which treatment with anti-TNFis acting could due to an improvement on t he impaired axonal conducti on, since it has been reported that TNFalter the electrophysiologic al properties of axons in mammalian spinal cord (Davies et al., 2006b). Based on our results we can appreciate that NE release during traini ng on eyeblink conditioning shifts (to an

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100 earlier release pattern) as a result of anti-TNFtreatment. This pattern of NE release peaks earlier and returns to bas eline sooner, showing an improvement compared to the control group (see fig 4.2B ). In addition, CR’s improve on days 4 and 5 after treatment with anti-TNFsupporting the idea of reversing the age related impairment, which could be due to an improvement in the axonal conduction as well as better re-uptak e for extracellular glutamate by the glutamate transporter system. In addition to the finding presents in the current report there is still more to be done to understand the mechanisms by which treatment with antiTNFis improving the capability of learning which we have demonstrated in this research.

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101 CHAPTER 5 Conclusions Using eyeblink conditioning coupled to microdialysis we have been able to monitor the activity of NE, GABA and Glu while the animals undergo classical delay eyeblink conditioni ng. Experiments were co nducted in both young and aged rats, which gave us the opportunity to understand basic concepts about memory formation that have remained unclear until now. Delay eyeblink conditioning is a paradigm claimed to be m ediated by the interplay of NE, GABA and Glu within the cerebellar network. The experimental design used in this research allowed the study of the comp lex neuronal circuitry in the cerebellum. Given the knowledge of t he anatomy and physiology of the cerebellum, it is an excellent structure to expl ore in details the different mechanisms involved in neuronal plasticity underlying memory form ation. The hypotheses tested in this dissertation utilized several decades of research and used behavioral pharmacology combined with neurochemical analytical techniques enabling us to answer exciting basic questions regarding the dynamics of me mory formation. We showed that the temporal pattern of NE and GABA release th at occurs during training on the delay eyeblink condition ing task is linked to the associative learning task and that it is not due to sensory stimulus activation by the CS and/or US, which is the case for Glu releas e. Our results agree with the literature which shows that these neurotransmitters participate in diffe rent ways and are essential for memory formation, consoli dation and extinction (Attwell et al.,

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102 2002a; Yeo, 2004; De Zeeuw & Yeo, 2005; Weis et al ., 2004; Farley & Alkon, 1985). It has been postulated that NE which is releas ed in the cerebellum from the LC induces synaptic plasticity in purk inje neurons by selectively improving the signal to noise ratio of evoked versus spontaneous activity, enhancing the sensitivity of cerebellar neurons to both ex citatory and inhibitory afferent inputs (Moises et al., 1979). Our data show that in fact NE is actively released in an impulse dependent manner into the lobus si mplex and interpositus nucleus. This is the first direct evidence showing presynaptic release in vivo while the animals perform the learning task. An interesting aspect of our findings are that the amount of NE released decreased across days of traini ng which is consistent wit h a role of NE early in acquisition and possibly in consolidation (Gilbert, 1974). In addition, our findings agree with published work which showed th at firing of LC ne urons demonstrates good discrimination within the first 500 trials of a visual discrimination task in monkeys suggesting that NE is important during the acquisition or early phase of learning of this task (Kubiak et al., 1998). According to the pattern of release for NE during training sessions across days that we found, it can be inferred that the cerebellar circuit requires higher extracellu lar levels of NE in the early phase of training. On the other hand, NE as well as GABA were released in a learning dependent manner and were specifically a ssociated with paired conditioning and not pseudo-conditioning which strengthens the idea that both neurotransmitters in play a role in the process of CR acquisition.

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103 Much of the work examining GABAergic transmission has been done using a pharmacological approach by either admin istering GABA agonists or antagonists in order uncover the functional role of th e cerebellar cortex and cerebellar nuclei and how it is involved in memory fo rmation and timing (Bao et al., 2002; Krupa & Thompson, 1997; Mamounas et al., 1987; Schreurs & Alkon, 1993). In this dissertation we directly measured extracel lular GABA levels by microdialysis. Interestingly, during the first days of training, the increas e above baseline in extracellular levels of GABA extended beyond the training period for over 60 minutes. This pattern changed across days of training where the time frame of GABA release shortens and th e amplitude of release increase after few days of training when the rats reached the maximal performance in CR’s, at that time the release of GABA showed a higher peak while the timing was confined to the training session, at this time the G ABA release pattern might reflect the concomitant activity of the cerebellar circ uitry that is associated with the CR. Our data agrees with other’s works in which has suggested that GABA plays a role in post training memory consolidation (Cooke et al., 2004). According to our results Glu release reflec ts the inputs to the cerebellar cortex and interpositus which are activated in both paired and unpai red conditions on the eyeblink training the pr e-synaptic release of glutam ate from afferents to the cerebellar cortex and supports the idea t hat learning of t he conditioned response does not take place before the signals r each the cerebellum as there are no changes in the pattern of release across da ys of training. Our results agree with

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104 those indicating that Glu has significant post-synaptic effe cts during training (Chen & Steinmetz, 2000). The administr ation of TTX through reverse dialysis decreased the amount of neurot ransmitter observed in the microdialysate for all three neurotransmitters indicating impu lse dependent release for all three neurotransmitters. In another set of simila r experiments but on aged rats, it was found that there was decreas ed capability to acquire CR’s n eyeblink conditioning which is paralleled by a distorted pattern of NE, GABA and Glu release. When this pattern is compared to young animals it occurs with a significant delay in relation to the moment in which training sessions start. Also, the magnitude of release is depleted about 40% compared to young animals. The time-dependent change that occurs for NE and GABA in young rats doesn’t happen in aged rats. These findings suggest an impairment of the orchestration carried by the neurotransmitters NE, GABA and Gl u during memory formation and consolidation. The data show that t he neurochemical networ k in the cerebellum becomes compromised with aging. More experiments need to be done in order to elucidate the causes of the neuroc hemical imbalance found in aged animals. Furthermore, blockade of postsynaptic -adrenergic receptors with discrete local administration of propranolol into the cerebellum disrupt s acquisition of the CR when it is administered after the animal ha s been on training. This result clearly indicates the participation of -adrenergic receptors during the consolidation process of the new memories. More ex periments need to be performed to clarify the exact mechanism by which the -adrenergic receptor s are participating

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105 during the memory consolidation proces s. Similar work has been done by administering propranolol before the training, this way it is clear that the activity of -adrenergic receptors are critical during eyeblink conditioning (Gould & Steinmetz, 1996; Cartford et al., 2002a) Our findings show that noradrenergic inputs to the cerebellar cortex are nece ssary for the consolidation process by which CR’s are formed. The local administration of propranolol at 5 and 60 minutes post training had a large impact in depleting the CR’s acquisition while propranolol administered 120 min post training session did not cause a si gnificant impairment in the acquisition of CR’s. It is known that NE is a neurom odulator in that it selectively improves the signal to noise ratio of evoked versus spontaneous activity which enhances the sensitivity of the cerebellar neurons to excitatory and inhibi tory afferent inputs (Eccles et al 1967; Hoffer et al 1973). Th e effect of propranolol could be due to the lack of the noradrenergi c signal (elicited by pr opranolol administration) impairs the improvement of the signal to noise ra tio of evoked versus spontaneous activity which ma y explain why the rats woul d require more training sessions in order to reach a significant conditioning re sponse. Another possible explanation for the depletion of CR’s afte r blocking the beta-adrenergic receptor post-training could be associat ed with the activity that NE exerts by activating the beta-adrenergic receptors which leads to the activation of cAMP/PKA signaling cascade which has been shown to enhance in tracellular calcium and promote the release of vesicles containing GABA (Saito w et al., 2005). Given the fact that NE

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106 activates glycogenolysis, we are proposing that the blockade of -adrenergic receptors might be interferi ng with the pool of energy in the form of ATP required to assemble and relocate the new syn thesized proteins during the memory consolidation. This idea needs to be addressed with specific experiments to elucidate whether NE acts on astrocytes to provide the energy necessary for the novo protein synthesis as well as calc ium mobilization within the cells as a essential part of the memory consolidat ion and how it correla te with aged-related memory deficits in which there is a deficit in NE signaling. It has been postulated that in aged-related memory deficits an increase in byproducts of oxidative stress is responsib le for some of the damages found in the central nervous system in mammals (Ha rman, 1956; Leibovitz and Siegel, 1980), and perhaps triggering the re lease of cytokines. TNFhas been found to be elevated in aged rats, and previous work has demonstrated that the elevated expression of TNFin aged rats can be reversed by diets rich in anti-oxidants and result in a reduction of TNFlevels. Also, anti-oxidant diets improve the acquisition of CR’s using eyeblink conditioning. We addressed some experiments to determine whether the acti vity of this cytokine can directly interfere with the acquisition of CR’s. The findings showed that elevated levels of TNFin young rats depletes the acquis ition of CR and the neurochemical pattern of release for the neurotransmi tter NE become compromised. These results indicate that chronic neuroinfl amation can be a leading factor by which aged animals present impaired capabilities of learning. Furthermore, we also

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107 showed that intervention wi th antibodies anti-TNFcan lead to an improvement in the ability of aged rats to acquire CR’s. We also show ed that with this pharmacological intervention the neurochemica l pattern of release for NE can be slightly modified and look mo re like the patter release of young rats. This finding has tremendous impact in un derstanding which factors dur ing aging can lead to depletion on cognitive abilities.

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108 Reference List Ahn, S., Ginty, D. D., & Linden, D. J. (1999). A late phase of cerebellar long-term depression requires activation of CaMKIV and CREB. Neuron, 23, 559568. Aksenov, D., Serdyukova, N., Irwin, K., & Bracha, V. (2004). GABA neurotransmission in the cerebellar in terposed nuclei: involvement in classically conditioned ey eblinks and neuronal activity. J.Neurophysiol., 91, 719-727. Allan, S. M. & Rothwell, N. J. (2001). Cytokines and acute neurodegeneration. Nat.Rev.Neurosci., 2, 734-744. Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993a). Oxidant s, antioxidants, and the degenerative diseases of aging. Proc.Natl.Acad.Sci.U.S.A, 90, 7915-7922. Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993b). Oxidant s, antioxidants, and the degenerative diseases of aging. Proc.Natl.Acad.Sci.U.S.A, 90, 7915-7922. Anderson, C. M. & Swanson, R. A. (2000) Astrocyte glutamat e transport: review of properties, regulation, and physiological functions. Glia, 32, 1-14. Aoki, C. & Pickel, V. M. (1992). C-terminal tail of beta-adrenergic receptors: immunocytochemical localization within astrocytes and their relation to

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144 ABOUT THE AUTHOR Daniel Paredes studied chemistry and ethnobotanics and was working on the pharmacological properties of natural products wh ich originated in Indian communities in the rainforest of South Am erica. He was actively involved in neuroscience research and participated in the development of capillary electrophoresis with laser induced fluorescence technology under the tuterlage of Dr. Luis Hernandez at the Universidad de los Andes (ULA) in Merida Venezuela. Later he joined the PhD program Univer sity of South Florida and has been involved in the study of neurochemistry und erling cerebellar motor learning under the mentorship of Dr. Paula Bickford.