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Catlow, Briony J.
Involvement of the 5-HT2A receptor in the regulation of hippocampal-dependent learning and neurogenesis
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by Briony J. Catlow.
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Dissertation (Ph.D.)--University of South Florida, 2008.
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ABSTRACT: Aberrations in brain serotonin (5-HT) neurotransmission have been implicated in psychiatric disorders including anxiety, depression and deficits in learning and memory. Many of these disorders are treated with drugs which promote the availability of 5-HT in the synapse. Selective serotonin uptake inhibitors (SSRIs) are known to stimulate the production of new neurons in the hippocampus (HPC) by increasing synaptic concentration of serotonin (5-HT). However, it is not clear which of the 5-HT receptors are involved in behavioral improvements and enhanced neurogenesis. The current study aimed to investigate the effects of 5HT[subscript 2A] agonists psilocybin and 251-NBMeO and the 5HT[subscript 2A/C] antagonist ketanserin on neurogenesis and hippocampal-dependent learning. Agonists and an antagonist to the 5-HT2A receptor produced alterations in hippocampal neurogenesis and trace fear conditioning. Future studies should examine the temporal effects of acute and chronic psilocybin administration on hippocampal-dependent learning and neurogenesis.
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Involvement of 5-HT2A Recept or in the Regulation of Hippocampal-Dependent Learning and Neurogenesis by Briony J Catlow A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Psychology College of Arts and Sciences University of South Florida Major Professor: Cheryl Kirstein, Ph.D. Michael Brannick, Ph.D. Cindy Cimino, Ph.D. Juan Sanchez-Ramos, M.D. Toru Shimizu, Ph.D. Date of Approval: November 7, 2008 Keywords: neurogenesis, serotonin, hippoc ampus, fear conditioning, psilocybin Copyright 2008, Briony J Catlow
To anyone who overcomes obstacles to live out their dreamsÂ…
Acknowledgements First and foremost thank you to Dr Kirstein and Dr Sanchez-Ramos for their complete support during my graduate studies. Dr Kirstein, you fought for me from the beginning and I am so gratef ul because I know none of this would have been possible without your advice and support. Dr Sanchez, you have taught me to think about the brain in a holistic way and with your passion for knowledge I learned that neur oscience is more than just a career, it is a way of life. Dr Paula Bickford, you are one of the most generous people I know, thank you for support and guidance and blessing me in so many ways. To Dr Brannick, Dr Cimino and Dr Shimizu, thank you fo r taking the time to serve on my committee and for your thoughtful consideration of my projects. I would also like to thank Dr Naomi Yavneh for chairing my defense. On a personal level I am so grateful to have had the support of my family. To my Grandma, Nana, Mum, Dad, Pa m, Steve, Jodi and Joani e you have all supported me in ways only family can and hope that I can do the same for you. To Anne, you have always been a role model to me with beauty, smarts and positivity and I am so lucky to have you in my life. To Danielito, you o pened my eyes and held my hand, ahora vamos!
i Table of Contents List of T ables ........................................................................................................ iii List of Fi gures ....................................................................................................... iv Abstract ................................................................................................................ vi Chapter One: In troducti on .................................................................................... 1 Anatomy of Hippocampal Neurogenes is ................................................... 2 Regulation of Neurogenesis in the Dentat e Gyru s ..................................... 5 Hippocampal Neurogenes is and Learni ng ............................................... 13 Assessing Neur ogenesis ......................................................................... 18 Serotonergic Innervation in the Dentate Gyrus ........................................ 22 Serotonin and Neurogenesis in the Dentat e Gyrus .................................. 23 Psilocybin ................................................................................................. 25 Specific Aims ........................................................................................... 28 Specific Aim 1 ............................................................................... 28 Specific Aim 2 ............................................................................... 28 Chapter Two: Invo lvement of the 5HT2A Receptor in the Regulation of Adult Neurogenesis in the Hippocam pus ...................................................... 29 Abstract .................................................................................................... 29 Introducti on .............................................................................................. 30 Materials and Methods ............................................................................. 32 Subjects ................................................................................................... 32 Drugs ....................................................................................................... 32 General pr ocedure ................................................................................... 32 Immunofluore scence ............................................................................... 33
ii Quantitat ion ............................................................................................. 34 Design and Analyses ............................................................................... 34 Result s ..................................................................................................... 35 Effects of Acute Administration of 5-HT2A receptor agonists and an antagonist in vivo on Cell Survival and Neurogenesis in the Hi ppocampus. ....................................... 35 Effects of Repeated Intermi ttent PSOP Administration on Progenitor Cell Survival and Neurogenesis in the Hippocampus ........................................................................... 45 Discussio n ............................................................................................... 50 Chapter Three: The Effects of Psilo cybin on Hippocampal Neurogenesis. ....... 55 Abstract .................................................................................................... 55 Introducti on .............................................................................................. 56 Materials and Methods ............................................................................. 58 Subjects ................................................................................................... 58 General Pr ocedure ................................................................................... 59 Design and Analyses ............................................................................... 61 Result s ..................................................................................................... 61 Acquisiti on ................................................................................................ 61 Contextual Fear Conditioning ................................................................... 64 Cue Fear Cond itioning ............................................................................. 66 Discussion ................................................................................................ 69 References ......................................................................................................... 73 About the Author ...................................................................................... End Page
iii List of Tables Table 1.1 Antibodies used to assess phenotypic fate of progenitors .................. 20
iv List of Figures FIGURE 1.1 Anatomy of the Hippoc ampus ......................................................... 3 FIGURE 1.2 Neurogenesis in the D entate Gyrus of t he Hippocam pus ............... 4 FIGURE 1.3 Chemical structure of Ps ilocybin, Psilocin an d Serotonin .............. 26 FIGURE 2.1 Effect of Acute PSO P Administration on Hippocampal Neurogenesis ............................................................................... 36 FIGURE 2.2. Represent ative photomicrographs s howing the effects of acute PSOP on hippocam pal neurogene sis .................................. 38 FIGURE 2.3. Effect of the selective 5-HT2A receptor agonist, 251-NBMeO on Hippocampal N eurogenesis ..................................................... 40 FIGURE 2.4. Representat ive photomicrographs showi ng the effects of the selective 5-HT2A receptor agonist 251-NBMeO on hippocampal neuroge nesis ........................................................... 42 FIGURE 2.5. Acute Admi nistration of the 5-HT2A/c receptor antagonist ketanserin negatively regulates cell survival and neurogenesis in the Hippocampu s ................................................ 44 FIGURE 2.6. Representat ive photomicrographs showi ng the effects of the 5-HT2A/C receptor antagonist ketans erin on neurogenesis in the dentate gy rus .......................................................................... 45 FIGURE 2.7. Effect of Chroni c PSOP Administration on Hippocampal Neurogenesis ................................................................................ 47 FIGURE 2.8. Represent ative photomicrographs s howing the effects of chronic PSOP or Ketanserin administration on neurogenesis in the dentate gyrus ....................................................................... 49 FIGURE 3.1 Schematic representation of the Trace Fear Conditioning Paradigm ....................................................................................... 60
v FIGURE 3.2 Effects of Psilocybin on the Acquisition of Trace Fear Conditioni ng .................................................................................. 63 FIGURE 3.3 Contextual Fear Conditi oning ........................................................ 65 FIGURE 3.4 Effect of Acute PSO P on Cue Fear C onditionin g .......................... 68
vi Involvement of the 5-HT2A Receptor In The Regulation of Hippocampal-Dependent Learning and Neurogenesis Briony J Catlow ABSTRACT Aberrations in brain serotonin (5 -HT) neurotransmission have been implicated in psychiatric disorders in cluding anxiety, depression and deficits in learning and memory. Many of these disorders are treated with drugs which promote the availability of 5-HT in the synapse. Selective serotonin uptake inhibitors (SSRIs) are known to stimul ate the production of new neurons in the hippocampus (HPC) by increasing synaptic concentration of serotonin (5-HT). However, it is not clear which of the 5-HT receptors are involved in behavioral improvements and enhanced neur ogenesis. The current study aimed to investigate the effects of 5HT2A agonists psilocybin and 251-NBMeO and the 5HT2A/C antagonist ketanserin on neuro genesis and hippocampal-dependent learning. Agonists and an antagonist to the 5-HT2A rec eptor produced alterations in hippocampal neurogenesis and trace fear conditioning. Future studies should examine the temporal effects of acut e and chronic psilocybin administration on hippocampal-d ependent learning and neurogenesis.
1 Chapter One Introduction The idea of new neurons forming in the adult central nervous system (CNS) is a relatively new one. In t he 1960Â’s Joseph Altman published the first evidence of neurogenesis, or the birth of new neurons in the adult mammalian brain (Altman, 1962; Altman, 1963; Altman & Das, 1965) Utilizing the tritiated thymidine method (Sidman et al., 1959; Me ssier et al., 1958; Messier & Leblond, 1960) Joseph Altman was able to demonstr ate that the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus (HPC) produce new neurons throughout t he lifespan (Altman, 1962; Altman & Das, 1965; Altman, 1969). For years fo llowing AltmanÂ’s discovery scientists acknowledged the possibi lity of the generation of new glial cells in the adult brain but rejected the concept of new born neurons. With the advent of new technologies such as the bromodeoxyuridine (BrdU) method of birth dating cells and double labeling using immunofluorescence, adu lt neurogenesis has been identified in many mammali an species including mice (Kempermann et al., 1998), rats (Kaplan & Hinds, 1977), hamsters (H uang et al., 1998), tree shrews (Gould et al., 1997), nonhuman primates (Gould et al., 1999b; Bernier et al., 2002) and humans (Eriksson et al., 1998). Peter Erik ssonÂ’s discovery of new neurons in the human HPC changed the perce ption of neurogenesis in the scientific community
2 so now the fact that new neurons are produced in the adult brain is firmly established. The Anatomy of Hippocampal Neurogenesis The HPC is divided into four areas: DG (also called area dentate, or fascia dentata), cornu ammonis (CA, furt her divided into CA1, CA2, CA3 and CA4), the presubiculum and t he subiculum. This anatom ical description of the HPC has been confirmed by both gene ex pression and fiber connections. The DG and areas CA form a trisynaptic circui try within the HPC (see Figure 1.1). Neurons in the entorhinal co rtex (EC) project to dendrit es of the granule cells in the DG forming the perforant pathway. The granule cells extend their axons (Ramon, 1952) to pyramidal neurons in area CA3, forming the mossy fiber tract (Ribak et al., 1985). CA3 pyramidal neur ons project to the contralateral (via associational commissural pathway) and t he ipsilateral CA1 region forming the Shaffer collateral pathway. Pyramidal neurons in CA1 extend axons to the subiculum and from the subiculum back to EC (for detailed descriptions of hippocampal circuitry see (Witter, 1993)).
3 Figure 1.1. Anatomy of the Hippocampus. The HPC forms a trisynaptic pathway with inputs from the Entorhinal Cortex (E C) that projects to the Dentate Gyrus (DG) and CA3 pyramidal neuron s via the perforant pathwa y. Granule cells in the DG project to CA3 via the mossy fiber pathway. Pyramidal neurons in CA3 project to both the contralateral (asso ciational commissural pathway) and the ipsilateral CA1 region via the Schaffe r Collateral Pathway. CA1 pyramidal neurons send their axons to t he Subiculum (Sb) which in turn projects out of the HPC back to the EC. In normal physiological conditions, neur ogenesis that occurs in the HPC is found only in the DG and results in the generation of new granule cells. Within the DG, progenitor cells reside in a na rrow band between the DG and the hilus (also called CA4 or plexiform layer) ca lled the subgranular zone (SGZ) which is AC EC CA3 CA1 DG Sb EC
4 approximately 2-3 cells thick (20-25 M) (s ee Figure 1.2). Neural progenitor cells (1) divide and form clusters of proliferating cells (2). Proliferat ing cells exit from the cell cycle and begin to differentiate into immature neurons (3). The immature granule cell forms sodium currents, ex tends dendrites and an axon to make connections with other cells and form synapses to become a mature neuron (4). The SGZ contains many cell types includin g astrocytes (Seri et al., 2001; Filippov et al., 2003; Fukuda et al., 2003), several types of glial and neuronal progenitor cells (Filippov et al., 2003; Fukuda et al., 2003; Kronenberg et al., 2003; Seri et al., 2004) and neurons in all st ages of differentiation an d maturation (Brandt et al., 2003; Ambrogini et al., 2004). Figure 1.2. Neurogenesis in the Dentat e Gyrus of the Hippocampus. Neural stem cells exist the SGZ of the DG, these cells then divide, differentiate and mature into their phenotypic fate. Neural progenitor cells (1) divide and form clusters of proliferating ce lls (2). Proliferating cells exit from the cell cycle and begin to differentiate into i mmature neurons (3). The im mature granule cell forms sodium currents, dendrites and extends an axon out to make connections with other cells and form synapses to become a mature granule cell (4).
5 Regulation of Neurogenesis in the Dentate Gyrus The proliferation and surv ival of neural progenitors in the adult HPC can be influenced in a positive and negative manner by a variet y of stimuli. Factors as diverse as stress, odors, neurotrophins, psychoactive drugs such as antidepressants, opioids and alcohol, electroconvulsive therapy, seizures, ischemia, cranial irradiat ion, physical activity, learning, hormones and age amongst many others have been linked to the regulation of neurogenesis (Kempermann et al., 1998; V an et al., 1999b; Malberg et al., 2000; Malberg & Duman, 2003; Tanapat et al., 2001). So me of these factors have been studied extensively and their role in the regulati on of neurogenesis is well defined. For example, environmental enr ichment and physical activity are strong positive regulators of neurogenesis (Van et al., 1999b; Kempermann et al., 1997), while stress and age (Cameron et al ., 1993; Kempermann et al., 1998) appear to be negative regulators of neurogenesis. The first report of any factor in creasing neurogenesis in the mammalian brain was an enriched environment. In an experimental setting a rodent enriched environment typically consists of a larg e cage, a large number of animals, toys and a tunnel system. In order to maintain the enrichment aspect novel toys are introduced and tunnel system is rea rranged on a regular basis. Mice (Kempermann et al., 1997) and rats (Nilss on et al., 1999) living in an enriched environment exhibited a strong up-regul ation of cell proliferation and
6 neurogenesis in the DG of the HPC. The proneurogenic effects of environmental enrichment can be increased further depen ding on the age of the animal when exposure occurs. When late adolescent/y oung adult animals li ve in an enriched environment it enhances the ability of env ironmental enrichment to up-regulate cellular proliferati on and neurogenesis in the DG. In fact, when the morphology of the HPC was examined later in life, a greater number of absolute granule cells was observed (Kempermann et al., 1997). In aged animals (typically 18 months or older in rodents) lower levels of neurogenesis have been observed, however living in an enriched envir onment counteracts the e ffects of aging (Kempermann et al., 1998). Kempermann and coll eagues demonstrated environment enrichment during aging increas es cell proliferation and neurogenesis in the DG (Kempermann et al., 1998). Furthermore if animals live in an enriched environment during mid age, basal levels of neurogenesis increase as much as five fold in old age. When experimentation with environment al enrichment began, novel foods were included as apart of the environmental enrichm ent experience. When similar food was given to mice living ei ther an enriched or control environment, the effect of environmental enrichment was still present. One type of diet however, has been found to have positive effects on neurogenesis specifically, caloric restriction (Lee et al., 2000). As an experimental m anipulation caloric restriction usually consists of limiting the amount of food an animal can eat by a third. Caloric restriction is the only fa ctor that has been shown experimentally to
7 increase the life span of anima ls and it is thought that caloric restriction actually acts as a mild stressor. It is of intere st to note while environmental enrichment is a strong positive regulator of adult hi ppocampal neurogenesis, it does not affect adult neurogenesis in the olfactory system (Brown et al., 2003). Exposure to an enriched environment increases neurogenesis in the DG of adult rodents, however, environmental en richment typically includes a running wheel and increased ph ysical activity. Physical activity is known to up-regulate cell proliferation and neurogenesis in the DG. Rodents will take full advantage of the opportunity to exercise on a running wheel during their active phase of their day. Mice have been reported to run betw een 3 and 8 km per night on a running wheel (Van et al., 1999a; Van et al., 1999b). Voluntary physical activity has been shown to increase the number of progenitor cells and new neurons in the DG of the HPC (Van et al., 1999a; Van et al., 1999b). The effect of running on neurogenesis is acute so that running must continue to effect neurogenesis and once the animal no longer uses the runni ng wheel the effect on neurogenesis will decline. The up-regulatio n of adult neurogenesis by physical activity also increases long term potentiation (LTP) in the DG and enhanc es performance on the Morris water maze (MWM) (Van et al ., 1999a). The MWM is a behavioral task that assesses memory and learning, but as part of the task the animals are placed into a pool of water and forced to swim. Some have argued that swimming, being physical activity, could al so influence the outcome of the task. This point was addressed by Ehninger et al who found involuntary physical
8 activity (swimming in radial arm wa ter maze (RAWM)) had no effect on hippocampal neurogenesis (Ehni nger & Kempermann, 2003). The mechanisms underlying the incr ease in neurogenesis by physical activity are unknown however, growth factor s such as insulin growth factor -1 (IGF-1), vascular endothelial growth factor (VEGF), and brain derived neurotrophic factor (BDNF) have been str ongly implicated. IGF-1 levels are increased in the HPC of running anima ls and running induced increases in cellular proliferation and neurogenesis (Carro et al., 2001; Carro et al., 2000; Trejo et al., 2001). This increase in neurogenesis is blocked by scavenging circulating IGF-1 absent in IGF-1 mut ants (Carro et al., 2000). VEGF is necessary for the effects of running on adult hippocampal neurogenesis. Blocking peripheral VEGF abolished the runninginduced induction of neurogenesis, however there were no detectable effects on baseline neurogenesis in non-running animal (Fabel et al., 2003). Quantitative polymerase chain reaction analysis revealed BDNF mRNA levels are significantly increased in the DG of running rats (Farme r et al., 2004). BDNF is a key factor involved in modulating neuroplasticity incl uding LTP and neurogenesis. Infusions of BDNF into the lateral ventricles induced neurogenesis orig inating in the SVZ (Pencea et al., 2001) and BDNF knockout (K O) mice have diminished levels of neurogenesis in the DG (Lee et al., 2002) Like environmental enrichment, physical activity up-regulates adult hipp ocampal neurogenesis, however, it does not affect adult neurogenesis in the ol factory system (Brown et al., 2003).
9 Stress severely impairs hippocampal neurogenesis. One of the first studies to link stress to hippocampal neurogenesis was conducted by Gould and colleagues (1992). They found stress increased the number of dying cells in the HPC but that the total number of granul e cells in the dentat e was not different from non-stressed controls and conclud ed neurogenesis must be occurring to maintain cellular balance (Gould et al ., 1992). They postulated the stress hormone, cortisol in humans and corticosterone in ro dents mediates the stress effect on neurogenesis and went on to discover adrenalectomy (removing the adrenal gland hence the source of endogenous corticosterone) led to an upregulation of neurogenesis and exogenous corticosterone down-regulated cellular proliferati on and neurogenesis in the DG (C ameron et al., 1993). Since these early experiments, severe stre ss has been shown to downregulate cell proliferation and consec utive stages of neuronal development using many different paradigms. Prenatal stress caused learning deficits and had detrimental effects on neurogenesis that la sted well into adulthood (Lemaire et al., 2000). The effects of psychosocial stress on neurogenesis were demonstrated using the resident-intruder model of territorial tr ee shrews (Gould et al., 1997). Tree shrews are extremely territo rial and guard their envi ronment so the introduction of an intruder to the resident Â’s cage is extremely stressf ul. The territorial tree shrews compete for dominance and soon after the introduction of an intruder a dominant-subordinate relationship is establis hed resulting in elevated cortisol and decreased neurogenesis in the subordinate tree shrew (Gould et al., 1997).
10 Predator odor triggered a stress response in prey and had detrimental effects on cell proliferation. In rodent models, fox odor has been shown to decrease cell proliferation and ne urogenesis in the DG (Tanapat et al., 2001). Both acute and chronic restraint stress have been shown to affect the rate of adult hippocampal neurogenesis. Pham and colleagues dem onstrated that 6 weeks of daily restraint stress suppressed cell proliferat ion and attenuated survival of the newly born cells, resulting in a 47% reduction of granule cell neurogenesis (Pham et al., 2003). Neurogenesis is not only affected by environmental stimuli, the absence of stimuli, such as social isolati on, negatively regulated neurogenesis. Young rats reared in social isolation for 4-8 weeks showed decreased performance on the MWM and decreased hippocampal neur ogenesis (Lu et al., 2003). In the learned helplessness model of depression animals are exposed to an inescapable foot shock using avoidance test ing. Exposure to inescapable shock decreased cell proliferation in the HPC, extending previous studies demonstrating downregulation of neurogene sis by exposure to acute stressors (Malberg & Duman, 2003). The key mechanism underlying the negat ive impact that stress has on neuroplasticity appears to be stress hormone (glucocorticoid) secretion (Cameron et al., 1993). Acute, severe and sometimes traumatic stress leads to chronically high levels of glucocorti coids and alters the functioning of the hypothalamic-adrenal-pituitary (HPA) ax is resulting in disregulation of glucocorticoid secretion and receptor expr ession. Depression is an example of a
11 clinical condition associ ated with disturbed regulatio n of the HPA axis which upsets the circadian rhythm of hormone secretion resulting in chronically elevated glucocorticoid levels and decreas ed neurogenesis (Jacobs et al., 2000). Aging is another factor known to have a strong negative influence on neurogenesis. This has bee n known since the discovery of adult neurogenesis by Altman and Das in 1965. In the original study a progr essive decrease in the levels of neurogenesis was observed afte r puberty and continued into old age (Altman & Das, 1965) and this finding has been since replicated in both rats (Seki & Arai, 1995; Kuhn et al., 1996; Cameron & McKay, 1999; Bizon & Gallagher, 2003), mice (Kempermann et al., 1998) and humans (Eriksson et al., 1998). The highest levels of adult neurogenesis occu rred in young adultho od and steadily decreased over the lifespan. In old age (typically 18 mont hs or older in rodents) baseline levels of neurogenesis are extrem ely low, however, there are ways to enhance neurogenesis in the aging hippo campus. Environment enrichment during aging increases cell proliferation an d neurogenesis in the DG, however, the effect of an enriched environment is more robust in young animals (Kempermann et al., 1998). Animals th at lived in an enriched environment starting at mid age had five fold increases in basal levels of neurogenesis in old age (Kempermann et al., 1998). Cortisol (or corticosterone in rodents) levels are elevated in aging which like ly reduces baseline prolifer ation and neurogenesis. Adrenalectomy in aged animal s restored adult neurogenesis in the DG to a level comparable to that of a much younger age, demonstrat ing corticosterone is at
12 least in part responsible for the decline in neurogenesis observed in aging (Cameron & McKay, 1999). IGF-1 levels are increased in the HPC running animals and running induced increases in cellular proliferati on and neurogenesis (Carro et al., 2001; Carro et al., 2000; Trejo et al., 2001) Similarly, aged animals administered exogenous IGF-1 to restor e endogenous IGF-1 levels to that of a younger age and induced neurogenesis above controls thus counteracted the negative effect of aging on neurogenesis (Lichtenwalner et al., 2001). BDNF is considered a crit ical secreted factor modu lating brain plasticity. Physical activity, which is known to pos itively regulate neuroge nesis and induce LTP, induces hippocampal BDNF mRNA expr ession. It is thought that BDNF may modulate the effect that physica l activity has on LTP and neurogenesis (Farmer et al., 2004). Infusions of BDNF into the lateral ventricles induced neurogenesis originating in the SVZ (Pen cea et al., 2001) and BDNF KO mice have diminished levels of hippocampal neurogenesis (Lee et al., 2002). In pathological conditions such as depressi on, BDNF blocks neurogenesis (which is opposite to healthy animals) and it is now under stood one of the critical functions of BDNF is to keep neur ogenesis within a physiological range. BDNF function has been implicated in the neurogenesis hypothesis of depression, the idea being that the antidepressants enhanc e neurogenesis, and BDNF is a key regulator of this mechanism (Jacobs et al., 2000; D'Sa & Duman, 2002). Antidepressants (including selective sero tonin reuptake inhibitors (SSRIs)) induce the phosphorylation of CREB, af ter which CREB binds to the BDNF
13 promoter and induces BDNF transcription. 5-HT2A receptor agonists, such as 2,5-dimethoxy-4-io doamphetamine (DOI), increase B DNF mRNA expression in the HPC (Vaidya et al., 1997 ). BDNF is involved in inducing neuronal differentiation possibly through the induc tion of neuronal nitric oxide synthase (nNOS) which has been shown to stop proliferation and promote differentiation. In vitro BDNF is a differentiation fact or that can down-regulate precursor cell proliferation (Cheng et al., 2003). Hippocampal Neuroge nesis and Learning Memory involves the encoding, storing and recalling of information. The HPC plays a critical role in learning and memory by converting short-term memories into long-term memories and is pivotal in the encod ing, consolidation and retrieval of episodic memory (Squire et al., 1992; Squire, 1992). Several studies have investigated the connec tion between learning and hippocampal neurogenesis. Hippocampal mediated learning and me mory has been shown to be related to the generation of new neuron s in the adult DG (Van et al., 2002; Nilsson et al., 1999). It has been postulated only learning tasks which are hippocampal dependant affect progenitor cell proliferation and neurogenesis in the DG (Gould et al., 1999a). This idea has since been demonstrated using a learning task that is easily manipulated to be either hippocampal dependent or independent. Hippocampal-dependent learning can be assessed using trace eye blink conditioning. In trace conditioning the conditioned stimulus (CS), a tone, sounds
14 for 5 seconds, then after a 100-1000 ms in terval, the unconditioned stimulus (US) an airpuff or eyelid shock is activated. In this way the CS and US do not overlap. Hippocampal-independent learning can be assessed using delay eyeblink conditioning. In delay eyeblink conditi oning the tone (CS) sounds for 5 seconds and in the last 20 ms of the tone sounding the airpuff (US) is activated. In this way the CS and US overlap. Shor s and colleagues used both trace and delay eyeblink conditioning to demonstrate th at trace eyeblink conditioning, a hippocampal dependent task, is affect ed by neurogenesis whereas delay eyeblink conditioning is not. Mice we re treated with methylazoxymethanol acetate (MAM), an anti-mitotic agent wh ich wipes out the progenitor cell population in the DG and ad ministered BrdU to bi rth date the cells then performed either trace or del ay eyeblink conditioning. In both trace and delay eyeblink conditioning, saline treated mi ce performed well on the task and had similar numbers of BrdU posit ive cells in the DG. This is in contrast to mice treated with MAM which produced different results for trace and delay eyeblink conditioning. In trace eyeblink condition ing MAM severely impaired learning and obliterated BrdU incorporati on in the DG, whereas, no im pairment in learning was observed after delay eyeblink conditioni ng despite mice being treated with MAM, thus obliterating the progenitor pool and re sulted in a dramatic reduction of BrdU positive cells in the DG (Shors et al., 2001). These results clearly indicate that newly generated neurons in the adult DG are affected by the formation of hippocampal-dependent memory.
15 Only certain types of hippocampal dependent tasks have been shown to be involved in hippocampal neurogenesis (Shors et al., 2002). This was demonstrated using two different learning paradigms known to require the HPC, the spatial navigation task and trace fear conditioning. Similar to the study mentioned earlier, mice we re treated with MAM and Br dU then performance on either behavioral task was assessed. T he spatial navigation task is performed in the MWM and required the mouse to use spat ial cues in the environment (like a black square on a wall) to navigate to and find the platform. Over trials mice learned where the platform was located a nd spent less time trying to find it. MAM failed to result in impairment in escape latency but did significantly decreased BrdU+ cells in t he SGZ, demonstrating that hippocampal progenitor cell proliferation is not essential for this hippocampal-dependent task (Shors et al., 2002). In a separate gr oup of mice trace fear condi tioning, which like trace eyeblink conditioning involves a time gap between CS and US presentation was performed. In trace fear conditioning MAM severely impaired learning and significantly diminished BrdU incorporat ion in the DG, thus providing more evidence for the involvement of trace conditioning in hi ppocampal neurogenesis (Shors et al., 2002). The above experim ents clearly demonstr ate that some forms of learning are depend ent on the HPC but not all hippocampal-dependent learning tasks require neurogenesis. The HPC is involved in the formati on and expression of memory in the passive avoidance task in rats (Cahill & McGaugh, 1998). The logic underlying
16 the passive avoidance (PA) task is that animals associate a particular environment with an unpleasant foot shock and learn by avoiding the environment they can avoid the aversive fo ot shock. Consequently, an increase in response latency is thought to reflec t the strength of the memory for the aversive event (Sahgal & Mason, 1985). Specifically the multi-herbal formula BR003 increased response latency, and henc e the memory of the foot shock while also increasing the num ber of BrdU positive cells in the DG (Oh et al., 2006). The PA task is relatively quick and si mple but is limited in the information it provides regarding memory, that la tencies increase following shock. A modified version of PA, the active av oidance paradigm meas ures acquisition (learning), retention (memory) and the ex tinction of the conditioned response. Active avoidance is a fear-motivated associative avoidance task. In this task the mouse has to learn to predict the occurrence of an aversive event (shock) based on the presentation of a specif ic stimulus (tone), in order to avoid the aversive event by moving to a different compartment. The measures recorded include number of avoidances (the mouse crossing to the other compartment during the warning signal), number of non-responses (the mouse failing to cross to the other compartment during the trial), response latency (latency to avoid or escape), number of in tertrial responses (i.e., crossing the barrier within the intertrial interval), and serve as an index of learning which allows memory to be assessed. Many studies supported the role of the HPC in active avoidance learning.
17 LTP via electrical stimulation to the perfo rant pathway is negatively correlated to learning in the shuttle box avoidance task, suggesting active avoidance training lowered the threshold frequency to induce LTP in the DG (Ramirez & Carrer, 1989). Active avoidance learning incr eased the length of the postsynaptic density in the molecular cell layer of the DG (Van et al., 1992) and increased immunoreactivity for muscarinic receptors in the granular cell layer (Van der Zee & Luiten, 1999). Two-way Active avoi dance also increased synthesis of BDNF (Ulloor & Datta, 2005) and cAMP response el ement binding (CREB) in the dorsal HPC (Saha & Datta, 2005). Rats that l earned the active shock avoidance task (responders) had similar levels of Brdu pos itive and Ki67 positive cells in the DG as non-responders, suggesting ASA has no effect on hippocampal progenitor cell proliferation (Van der et al., 2005). Active avoidance testing is commonly used following ex posure to severe inescapable foot shock in the learned helplessness model of depression. Exposure to inescapable foot shock decre ased progenitor cell pr oliferation in the DG and this effect is reversed by chr onic treatment with fluoxetine (Malberg & Duman, 2003). One target of antid epressant treatment is BDNF since antidepressants not only increase t he expression of CREB in the rat HPC (Nibuya et al., 1996) but also increase the expression of BDNF (Nibuya et al., 1995). BDNF produced antidepressant like effects in the learned helplessness model of depression (Shirayama et al., 2002).
18 Assessing Neurogenesis The systemic injection of thymidine, radioactively labeled with tritium was the first method developed to la bel dividing cells (Messier et al., 1958). Once in the bloodstream, tritiated thymidine com petes with endogenous thymidine in all cells in the S phase of cell division and is permanently in corporated into the DNA. Labeled thymidine has a short half-life in vivo and labels all cells in the process of cell division when the label is injected. At a later time point, tissue sections are prepared and coated with a phot o emulsion. The radiation from the labeled thymidine molecules blackens the photo em ulsion, thus making visible the typical grains of thymidine autoradiography. Ut ilizing the tritiated thymidine method (Sidman et al., 1959; Messier et al., 1958; Messier & Leblond, 1960) Joseph Altman was able to demonstrate that t he SVZ and the DG of the HPC produce new neurons throughout the lifespan (Altman, 1962; Altm an & Das, 1965; Altman, 1969). BrdU is a false base that com petes with endogenous thymidine and becomes permanently incorporated in t he DNA during the S phase of the cell cycle. BrdU is typically administered via in jections of usually 50 250 mg/kg in a single bout or over several days depend ing on the experimental paradigm (Corotto et al., 1993). BrdU is advant ageous because it is a permanent marker so any cells that express BrdU can be di rectly related to the time BrdU was administered thus providing th e birth date of the cell. It is important to note BrdU can be incorporated into cells that ar e on the verge of dy ing when cell death
19 related mechanisms trigger DNA repair, therefore proper controls need to be included such as immunohistochemical stains for apoptosis (caspase-3 or TUNEL) and proliferative marker s (Ki67) to determine if a ce ll is truly proliferative. The rate of proliferation can be differ entiated from the rate of survival by manipulating time between BrdU injection and sacrifice so proliferating cells can be determined by sacrificing anima ls 24 hours after a BrdU injection. In this way BrdU has time to incorporate into t he cell but the cell does not have time to differentiate into a neuron, a process which takes a minimum of 72 hours. Survival can be determined by taking the br ains of animals days, weeks or even months after BrdU injection. The phenotypic fate of th e cell is determined in the survival condition by double labeling BrdU with another marker. Table 1 presents a summary of the common markers used to determine the phenotype of neural progenitors.
20 Marker Significance Reference Ki67 Proliferation; late G1, S, G2 and M phases nuclear (Scholzen & Gerdes, 2000) Doublecortin (DCX) Immature neuron; microtubuleassociated protein enriched in migratory neuronal cells. Early neuronal marker with lineage determined and limited self-renewal dendritic (Meyer et al., 2002) III -tubulin (Tuj1) Immature neuron; Tubulin protein soma and processes (Uittenbogaard & Chiaramello, 2002) Calretinin (CRT) Immature neurons; calcium binding protein transiently (Brandt et al., 2003) Neuronal nuclei (NeuN) Mature neurons; mostly in nuclei but can be detected in cytoplasm nucleus (Mullen et al., 1992) Glial Fibrillary Acidic Protein (GFAP) Intermediate filament protein expressed in astrocytes. (Fuchs & Weber, 1994) Table 1. Antibodies used to assess phenotypic fate of progenitors If the cell expresses Ki67 it is an ear ly progenitor since Ki67 is a protein expressed the G1, S, G2 and M phases of the cell cycle (Scholzen & Gerdes, 2000). Cells that express doublecortin (DCX), -tubulin III (III -tubulin, Tuj1), or calretinin (CRT) are immatu re neurons. DCX is a micr otubule associated protein transiently expressed in immature neur ons (Meyer et al., 2002), Tuj1 marks tubulin in microtubules (Uittenbogaard & Chiaramello 2002) and CRT is a
21 calcium binding protein transiently ex pressed in immature neurons and is expressed in the developi ng neuron at a sta ge where DCX expr ession dissipates (Brandt et al., 2003). The best and most wid ely used marker to identify mature neurons is neuronal nuclei (N euN) (Mullen et al., 1992). The expression of NeuN is restricted to post-mitotic neurons and is predominately located in the nucleus of neurons although it can oc casionally be observed in the neurites. In order to convincingly demonstrate neurogenesis, cells are double labeled with BrdU plus NeuN, which clearly demonstrates that t he cell was born around the time of BrdU injection and survived to differentiate into a neuron. Cell survival depends on many factors including the ability of the cell to form dendrites, an axon, synthesize neurotransmitter, receptors and establish f unctional connections with other cells. Cells that donÂ’t establish f unctional connections will most likely die. It is possible to assess neurogenesi s using methods ot her than the BrdU and tritiated thymidine methods. Using immunohistochemical and immunofluorescent techniques, cells ca n be stained for markers of immature neurons that are transient and only present in newly formed neurons. Brandt and colleagues (2003) elegantly dem onstrated this method by defining time periods of development in which cells express particular markers double-labeled with BrdU (Brandt et al., 2003). Br dU is still the only way to birth date cells so for establishing the method it was essential to know the exact age of cells. The expression of CRT plus BrdU positive cells was greatest 1 to 2.5 weeks after BrdU injection and the number of double-labe led cells was negligible at 4 weeks,
22 demonstrating CRT is transient. If a cell expressed DCX or CRT, that cell can be positively identified as an immature neur on, thus estimates of DCX or CRT positive cells in the DG repres ent estimates of neurogenesis. Serotonergic Innervation in the Dentate Gyrus Serotonin (5-HT) is a modulatory neur otransmitter in the central nervous system which is important in the regulat ion of vital brain functions such as feeding (Lucki, 1992), thermoregulation (F eldberg & Myers, 1964), sleep (Jouvet, 1967) and aggression (Sheard, 1969). In psychopathological states such as depression (Pinder & Wieringa, 1993), eat ing disorders (Leibowitz & ShorPosner, 1986) and anxiety serotoner gic signaling is disturbed. In the mammalian brain 5-HT is produced by neurons in the raphe nucleus (RN) which project to many areas of t he brain via the medial forebrain bundle (MFB) (Azmitia & Segal, 1978; Parent et al., 1981). N eurons from RN innervate virtually all brain areas with dense inner vation occurring in the HPC, cerebral cortex, striatum, hypothalamus, thalamus septum and olfactory bulb (Jacobs & Azmitia, 1992; Leger et al., 2001). The inner vation of serotonergic fibers to areas within the HPC is variable (Moore & Halari s, 1975; Vertes et al., 1999; Bjarkam et al., 2003). The DG is innervated with sero tonergic fibers in both the molecular layer and the hilus with particularly dense innervation projecting to the SGZ where they synapse with interneu rons (Halasy & Somogyi, 1993). 5-HT activates fifteen known receptors, many of which are expressed in the DG (el et al., 1989; Tecott et al., 1993; Vilaro et al., 1996; Djavadian et al.,
23 1999; Clemett et al., 2000; Kinsey et al ., 2001). Most of the 5-HT receptors interact with G proteins except for the 5-HT3A receptors, which are ligand-gated ion channel recept ors. The 5-HT3 receptors (subtypes 5-HT3A and 5-HT3B) are ligand-gated Na+ ion channels and their activation leads to the depolarization of neurons (Barnes & Shar p, 1999). The 5-HT1 family of receptors (including subtypes 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F) are coupled to the Gi protein which, when activated decreases the activity of adenylyl cyclase thus decreasing the rate of formation of cyclic adenosine monophosphate (cAMP). Activation of 5-HT1 receptors can lead indirect ly to the opening of K+ channels therefore increasing t he conductance of the cell membrane for K+ ions. Activation of 5-HT4, 5-HT6, 5-HT7, receptors are coupled to Gs proteins which have the opposite effect. They increase t he activity of adenylyl cyclase, increase the rate of cAMP formation and decrease K+ conductance (Thomas et al., 2000; Raymond et al., 2001). The 5-HT2 receptors (including subtypes 5-HT2A, 5-HT2B, 5-HT2C) are coupled to Gq proteins and activate phospholipase C (PLC), increasing the rate of formati on of inositol triphosphate (IP3) and diacylglyerol leading to the increased formation of pr otein kinase C (PKC) (Kurrasch-Orbaugh et al., 2003; Ananth et al., 1987). Serotonin and Neurogenesis in the Dentate Gyrus While several factors regulate the rate of generation of new cells in the adult DG, one of the most important k nown factors is 5-HT. Malberg and colleagues found increased levels of 5-HT resulted in the increased rate of
24 proliferation of neural progenitors in the DG (Malber g et al., 2000). Administering 5,7-dihydrosytryptamine (5 ,7-DHT), a serotonergic ne urotoxin into the RN and caused the destruction of axons and se rotonergic cells a nd resulted in a decreased in the number of BrdU-labeled cells in the DG (Brezun & Daszuta, 1999). The 5,7-DHT lesion resulted in around a 60% d epletion of the serotonergic innervation to the DG which lasted for one month. After two months, reinnervation to the DG was obser ved with the sprouting of serotonergic axons so that by the third month ther e was no observable difference between the 5,7-DHT and vehicle in newly generated ce lls or serotonergic innervation (Brezun & Daszuta, 2000). Many serotonergic receptors have been implicated in the regulation of neurogenesis in the DG. In vitro, when the 5-HT1A receptor agonist, 8-OH-DPAT was added to a medium in which cultur ed fibroblasts transfected with the 5-HT1A receptor were present, the ra te of cell divisions increas ed (Varrault et al., 1992). In vivo, 5-HT1A receptor antagonists (NAN-190, p -MPPI and WAY-100635) decreased the number of progenitors in t he DG by approximately 30% (Radley & Jacobs, 2002) and injections of 5-HT1A receptor agonists increased the number of BrdU positive cells in the DG (Santarelli et al., 200 3). Similarly, Banasr and colleagues showed various 5-HT1 receptor agonists incr ease the number of BrdU labeled cells in the subgranular layer. Acute administration of the 5-HT2A/C receptor agonist 2,5-dimethox y-4-iodoamphetamine (DOI), 5-HT2C receptor agonist RO 600175, and 5-HT2C receptor antagonist SB 206553 had no effect of
25 cell proliferation in th e HPC, whereas the 5-HT2A/C receptor antagonist ketanserin produced a 63% decrease in BrdU incorporation (Banasr et al., 2004). A recent study found acute ketanserin decreased proliferation wh ereas chronic ketanserin increased proliferation in the DG (Jha et al., 2008). No effect on proliferation in the DG was observed after DOI or lyse rgic acid diethylamide (LSD) were administered either acutely or once daily for seven consecutive days (chronic) (Jha et al., 2008). The 5-HT2A receptor is involved in the regulation of BDNF in the HPC (Vaidya et al., 1997). D OI alone and in combinatio n with selective 5-HT2A and 5HT2C receptor antagonists decreased the expre ssion of BDNF mRNA in the HPC. Interestingly, the decrease in BDNF mR NA expression was blocked by the 5HT2A receptor antagonist but not the 5-HT2C receptor antagonist, implicating the 5-HT2A receptor in the regulatio n of BDNF expression. In addition, the stressinduced reduction in BDNF expression in the HPC was blocked by a 5-HT2A/C receptor antagonist (Vaidya et al., 1997). Psilocybin (PSOP) PSOP (4-phosphoryloxy-N,N-dimethyltrypta mine) is the main active agent in Â“magic mushroomsÂ” and is categorized as a indole hallucinogen. First isolated from psilocybe mexicana a mushroom from Central America by Albert Hofmann in 1957, PSOP was then produced synthetic ally in 1958 (Hofm ann et al., 1958a; Hofmann et al., 1958b). PSOP is converted in to the active metabolite psilocin (4hydroxy-N,N-dimethyltryptamine) which may produce some of the psychoactive
26 effects of PSOP. The chem ical structure of PSOP (C12H17N2O4P) and the metabolite, psilocin (C12H16N2O) are similar to 5-HT (C10H12N2O), the main neurotransmitter which they affect (see Figure 1.3). Figure 1.3. Chemical structure of Psilocybin, Psilocin and Serotonin. In vivo studies in mice have shown the LD50 of PSOP via intravenous administration to be 280 mg/kg (Cerletti & Konzett, 1956; Cerletti, 1959). Autonomic effects of 10 mg/kg/sc in mi ce, rats, rabbits, ca ts and dogs include mydriasis, piloerection, irregulari ties in heart and breathing rate and hyperglycemic and hypertonic effects (Cerle tti & Konzett, 1956; Cerletti, 1959). Psilocybin OH P OH O O N H N Serotonin HO N H N H H OH N H N Psilocin
27 These effects were interpreted as an ex citatory syndrome caused by stimulation of the sympathetic nervous system with one large exception being the absence of hyperlocomotion (Monnier, 1959). PSOP exerts psychoactive effects by altering serotonergic neurotransmission by binding to 5-HT1A, 5-HT1D, 5-HT2A and 5-HT2C receptor subtypes (Passie et al., 2002). PSOP binds to the 5-HT2A receptor (Ki = 6 nM) with high affinity and to a mu ch lesser extent to the 5-HT1A receptor subtype (Ki = 190 nM) (McKenna et al., 1990). However, PSOP has a lower affinity for 5-HT2A and 5-HT2C receptors compared to lysergic acid diethylamide (LSD), a similar indole hallucinogen ( Nichols, 2004). In contrast to LSD, PSOP has a very low affinity to DA receptors and only extremel y high doses affect NE receptors. PSOP has been shown to induce schizop hrenia-like psychosis in humans, a phenomenon attributed to the ac tion of PSOP through 5-HT2A receptor action. Specifically, human volunteers were pretre ated with ketanserin, an antagonist to the 5-HT2A/C receptor, then administered 0.25 mg/kg p.o. PSOP and the psychotomimetic effects of PSOP were co mpletely blocked (Vollenweider et al., 1998). Since blocking the 5-HT2A receptor prevented the psychotropic effect of PSOP it appears as though the actions of PSOP are mediated via the activation of 5-HT2A receptors. A recent study test ed PSOP-induced stimulus control and found 5-HT2A receptor antagonists prevented rats from recognizing PSOP in a drug discrimination task, an effect which was not observed with 5-HT1A receptor antagonists (Winter et al., 2007). R epeated daily administration of PSOP
28 selectively downregulated 5-HT2A receptors in the rat brain (Buckholtz et al., 1990; Buckholtz et al., 1988; Buckholtz et al., 1985). PSOP binds to the 5-HT2A receptor and stimulates arachidonic acid and consequently, the PI pathway resulting in the activation of PKC (Kurrasch-Orbaugh et al., 2003). This dissertation sought to evaluate the involvement of the 5-HT2A receptor in the regulation of hippocampal neurogenesis and hippocam pal-dependent learning. Specific Aims The present study invest igated the role of 5-HT2A receptor on hippocampal neurogenesis and hippocampal-dependent learning The effects of acute and chronic 5-HT2A receptor agonists and an antagon ist on the survival and phenotypic fate of progenitor cells in the DG were assessed using immunofluroescent techniques. In additi on the effects of acute PSOP on trace fear conditioning were used to assess learning and memory. Specific Aim 1. To evaluate the effe ct of acute 5-HT2A receptor agonists and an antagonist on the surv ival and phenotypic fate of hippocampal progenitor cells. It was hypothesized PSOP and 251-NBMeO, both 5-HT2A receptor agonists positively regulate neurogenesis in the DG of the HPC, and ketanserin, a 5-HT2A/C receptor antagonist downregul ates hippocampal neurogenesis. Specific Aim 2. To elucidate whether PSOP affects learning and memory using the trace fear conditioning paradigm It was hypothesized acute exposure to PSOP would enhance hippocampal-depe ndent learning and ketanserin would impair learning on the trace fear conditioning task.
29 Chapter Two Involvement of the 5HT2A receptor in t he Regulation of Adult Neurogenesis in the Mouse Hippocampus Abstract Selective serotonin uptake inhibitors (SSRIs) are known to stimulate the production of new neurons in the hippoc ampus (HPC) by increasing synaptic concentration of serotonin (5-HT). The delay in the appearance of antidepressant effects corresponds to the ti me required to generate new neurons. However, it is not clear which of the m any serotonergic recept ors in the HPC are responsible for the enhanced neurogenesis. The current study evaluated the effects of the acute and chr onic administration of 5HT2A agonists psilocybin and 251-NBMeO and the 5HT2A/C antagonist ketanserin on hippocampal neurogenesis. To investigate the effect s of acute drug administration mice received a single injection of vary ing doses of psilocybin, 251-NBMeO, ketanserin or saline followed by i.p. in jections of 75 mg/kg bromodeoxyuridine (BrdU) for 4 consecutive days followed by euthanasia two weeks later. For chronic administration 4 injections of psilocybin, ketanserin or saline were administered weekly over the course of one month. On da ys following drug injections mice received an injection of 75 mg/kg BrdU and were euthanized two weeks after the last drug injection. Unbiased estimates of BrdU+ and
30 BrdU/NeuN+ cells in the dentate gyru s (DG) revealed a significant dose dependent reduction in the level of neurogenesis after acute 5HT2A receptor agonist or antagonist administrat ion. Interestingly, ch ronic administration of psilocybin increased the number of newborn neurons in the DG while the antagonist suppressed hippocampal neurogenesis, suggesting the 5HT2A receptor appears to be involved in the regulation of hippocampal neurogenesis. Introduction Evidence suggests neurogenesis occurs throughout the lifespan in two specific regions of the adult brain, the subventricular zone (SVZ) and the subgranular zone (SGZ) of the DG (Altman J, 1962; Altman & Das, 1965; Altman J, 1969). The proliferation and survival of neural progenitors in the adult HPC can be influenced by a variety of stimuli including stress, age, physical activity and depression (Gould et al., 1992; Kempe rmann et al., 1998; Van et al., 1999b; Malberg et al., 2000). Antidepr essant medications such as selective 5-HT uptake inhibitors (SSRIs) enhance the production of new born neurons in the DG of the HPC (Malberg et al., 2000). However, this effect is time specific with chronic administration (14 days or more) enhancing neurogenesis but not acute treatment (Malberg et al., 2000) Interestingly, there is a delay in the appearance of antidepressant effects which corresponds to the time required to generate new neurons (Santarelli et al., 2003) sugges ting an enhancement of neurogenesis may mediate the behavioral e ffects of antidepressants. The requirement of chronic administr ation of antidepressant medications
31 to enhance neurogenesis is likely due to number of factors. Antidepressant treatments upregulated the ex pression of brain-derived neurotrophic factor (BDNF) in the HPC (Nibuya et al., 1995). BDNF knockout mice have diminished levels of neurogenesis in the DG (Lee et al., 2002) and infusions of BDNF into the lateral ventricles induc ed neurogenesis originating in the SVZ (Pencea et al., 2001). The involvement of 5HT in the regulation of neurogenesis may be mediated through different 5-HT receptor subtypes expressed on cells in the neurogenic microniche (Barnes & Sharp, 1999). The 5-HT2A receptor is involved in the regulation of BDNF in the HPC (Vaidya et al., 1997). 2,5-dimethoxy-4iodoamphetamine (DOI), a 5-HT2A/C receptor agonist decreas ed the expression of BDNF mRNA in the HPC (Vaidya et al., 1997). Interestingly, the decrease in BDNF mRNA expression wa s blocked by the 5-HT2A receptor antagonist but not the 5-HT2C receptor antagonist, im plicating the 5-HT2A receptor in the regulation of BDNF expression in the HPC (Vaidya et al., 1997). Acute administration of DOI, 5-HT2C receptor agonist RO 600175, or the 5-HT2C receptor antagonist SB206553 had no effect on cell prolif eration, whereas the 5-HT2A/C receptor antagonist ketanserin produc ed a 63% decrease in BrdU incorporation (Banasr et al., 2004). A recent study found acut e ketanserin decreased proliferation whereas chronic ketanserin in creased proliferation in the DG (Jha et al., 2008). No effect on proliferation in the DG was observed afte r DOI or lysergic acid diethylamide (LSD) were administered ei ther acutely or once daily for seven
32 consecutive days (chronic) (Jha et al., 200 8). However, daily doses of LSD or psilocybin (PSOP) produce rapid tolerance to the drug and resulted in a selective downregulation of the 5HT2A receptor (Buckholtz et al., 1990; Buckholtz et al., 1985). Therefore, in order to in vestigate the role of the 5HT2A receptor in the regulation of hippocampal neurogenesis the current study evaluated the effects of acute and repeated intermi ttent administration of 5HT2A agonists and the 5HT2A/C antagonist ketanserin on hippocampal neurogenesis. Materials and Methods Subjects. C57BL/6J male mice (30-40 g) were housed in standard laboratory cages and left undisturbed for 1 week after arrival at the animal facility. All mice had unlimited access to water and laboratory chow and were maintained in a temperature and humidity controll ed room on a 12:12 li ght/dark cycle with light onset at 7:00 AM. All National Institutes for Health (NIH) gui delines for the Care and Use of Laboratory Animals were followed (National Inst itutes of Health, 2002). Drugs 251-NBMeO was synthesized in th e laboratory of Dr David Nichols (Braden et al., 2006). PSOP was provid ed by Dr Francisco Moreno from University of Arizona. Ketanserin (+)tartrate salt (#S006, St. Louis, MO) and 5Bromo-2 -deoxyuridine (#B5002, St. Louis, MO ) were supplied by Sigma-Aldrich Inc. General Procedure. Acute Administration: A total of 48 C57BL/6 mice received a single injection of 0.1 mg/kg PSOP (n=6), 0.5 mg/kg PSOP (n=6), 1.0
33 mg/kg PSOP (n=6), 0.1 mg/kg 251-NBMeO (n=6), 0.3 mg/kg 251-NBMeO (n=6), 1.0 mg/kg 251-NBMeO (n=6), 1.0 mg/kg ket anserin (n=6) or saline (n=6). Mice received an intraperitoneal (i.p.) injection of 75 mg/kg BrdU once daily for 4 days following drug administration and were euthanized two weeks after the last drug injection. Mice were euthanized with nembutal then transcardially perfused with 0.9% saline followed by 4% paraformal dehyde. Brains were stored in 4% paraformaldehyde, transferred to 20% sucrose solution and sectioned coronally using a cryostat (Leica, Germany) at 30M in a 1:6 series and stored in 24-well plates in cryoprotectant at -20C. Repeated Intermittent Administration: A total of 31 C57BL/6 mice received 4 i.p. injections of either 0.5 mg/kg PSOP (n=6), 1.0 mg/kg PSOP (n=7), 1.5 mg/kg PSOP (n=6), 1.0 mg/kg ketanserin (n=6) or 0.9% saline solution (n=6) over the course of one month on days 1, 8, 15, and 22. Each day following drug adminis tration 75 mg/kg BrdU was injected i.p. All mice were euthanized two weeks a fter the last drug injection according to the above procedures. Immunofluorescence For the double labeling of progenitor cells in the DG free-floating sections were denatured us ing 2N HCl and neutralized in 0.15M borate buffer then washed in PBS. Ti ssue was blocked in PBS+ (PBS, 10% normal goat serum, 1% 100x Triton X, 10% BSA) for 1 hour at 4C and incubated for 48 hours at 4C in an antibody cockt ail of rat monoclonal anti-BrdU (AbD Serotec, Raleigh NC, #OBT0030G, 1:100) plus mouse anti-NeuN (Chemicon, 1:100) in PBS. Sections were washed in PBS and incubated in a secondary
34 antibody cocktail of goat anti-rat IgG Al exa Fluor 594 (1:1000, Invitrogen, Eugene OR) plus goat anti-mouse (1:400, Invi trogen) and coated with vectorshield mounting medium (Invitrogen). Quantitation. For the quantification of doubled labeled cells using immunofluroescence, the number of BrdU+ and BrdU/N euN+ labeled cells were estimated using every 6th section taken throughout the DG (every 180 microns). To avoid counting partial cells a modifica tion to the optical dissector method was used so that cells on the upper and lower planes were not counted. The number of BrdU+ cells counted in every 6th section was multiplied by 6 to get the total number of BrdU+ or BrdU/N euN+ cells in the DG (Shors et al 2002). Positive labeling was verified by confocal microscopy (Zeiss). Design and analyses. Separate one-way analyse s of variance (ANOVA) were used to evaluate the acut e and chronic effects of 5-HT2A receptor agonists and an antagonist on hippocampal neurogenesis. For acute drug administration separate one-way ANOVA was used to det ermine the effects of Drug [PSOP (saline, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg /kg), 251-NBMeO (saline, 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg)] and a two-tailed t-te st (saline, 1.0 mg/kg ketanserin) was used to establish differences in cell survival and neurogenesis. For chronic drug administration a separate oneway ANOVA was used to determine the effects of Dose (saline, 0.5 mg/kg PSOP, 1.0 mg /kg PSOP, 1.5 mg/kg PSOP, 1.0 mg/kg ketanserin) on cell survival and neurogenes is. When appropriate, post hoc
35 analyses such as Bonferroni were used to isolate Drug effects. All statistical analyses were determined signific ant at the 0.05 alpha level. Results Effects of Acute Administration of 5-HT2A receptor agonists and an antagonist in vivo on Cell Survival and Neurogenesis in the Hippocampus In order to investigate the effects of acut e PSOP administration on cell survival and neurogenesis mice (n = 6-7 per condition) we re injected with PSOP (0.1, 0.5, or 1.0 mg/kg), 251-NBMeO (0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg), ketanserin (1.0 mg/kg) or 0.9% saline so lution then received 75 mg/kg BrdU once daily for 4 days following drug administration follow ed by euthanasia two weeks after the last drug injection. A one-way ANOVA de tected significant differences in the total number of surviving BrdU+ cells in the DG as a resu lt of acute PSOP treatment [ F (3,20)=6.64, p =0.003]. As can be seen in Figure 2.1A a significant decrease in the number of surviving Br dU+ cells was observed after 1.0 mg/kg PSOP compared to saline (indicated by *). The phenotypic fate of surv iving cells was determined by immunofluorescent labeli ng of BrdU and NeuN. A one way ANOVA revealed a significant effect of dose on the number of double labeled neurons in the DG [ F (3,20)=10.26, p =0.0003]. As can be seen in Fi gure 2.1B, acute administration of 1.0 mg/kg PSOP significantly dimini shed the number of BrdU/NeuN+ cells compared to saline ( p <0.05). These data suggest acute administration of 1.0 mg/kg PSOP, a 5HT2A agonist downregulated neur ogenesis in the DG.
37 Figure 2.1. Effect of Acute PSOP Admi nistration on Hippocampal Neurogenesis. Mice (n = 6 per condition) were injected with PSOP (0.1, 0.5, or 1.0 mg/kg) or saline then received 75 mg/kg BrdU onc e daily for 4 days following drug administration followed by euthanasia two weeks after drug injection. A) The total number of BrdU+ cells in the DG were significant diminished after a single injection of 1.0 mg/kg PSOP ( p <0.05). B) Acute administration of 1.0 mg/kg PSOP significantly diminished the number of BrdU/NeuN+ cells compared to saline ( p <0.05). These data suggest acute adm inistration of 1.0 mg/kg PSOP, a 5HT2A agonist downregulated neurogenes is in the DG of the HPC. indicates a significant difference from saline.
38 Figure 2.2. Representative photomicr ographs showing the effects of acute PSOP on hippocampal neurogenesis. NeuN+ cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (A -C), 0.1 mg/kg PSOP (D-F), 0.5 mg/kg PSOP (G-I) and 1.0 mg/kg PSO P (J-L). Scale = 50 M
39 A one-way ANOVA detected significant di fferences in the total number of surviving BrdU+ cells in the DG as a result of acute 251-NBMeO treatment [ F (3,20)=9.00, p =0.0004]. There was a significant decrease in the number of surviving BrdU+ cells after acute adminis tration of 0.1, 0.3 and 1.0 mg/kg 251NBMeO compared to saline (see fi gure 2.3A, indicated by *). In addition, there was a significant e ffect of drug on th e number of double labeled neurons in the DG [ F (3,20)=3.00, p =0.03]. As can be seen in Figure 2.3B, 1.0 mg/kg 251-NBMeO significantly diminished the number of new born neurons in the DG compared to saline ( p <0.05, indicated by *). These data suggest acute administrati on of the selective 5-HT2A receptor agonist, 251NBMeO, attenuated hippoc ampal neurogenesis.
41 Figure 2.3. Effect of the selective 5-HT2A receptor agonist, 251-NBMeO on hippocampal neurogenesis. Mice (n = 6 per condition) were injected with 251NBMeO (0.1, 0.3, or 1.0 mg/kg) or saline then received 75 mg/kg BrdU once daily for 4 days following drug administ ration followed by euthanasia two weeks after drug injection. A) There was a sign ificant decrease in the total number of BrdU+ cells in the DG after t he administration of 251-NBMeO ( p <0.05). B) The number of new born neurons was significantly decreased after 1.0 mg/kg of 251NBMeO compared to saline ( p <0.05), suggesting acute administration of the 5HT2A receptor agonist downregulated neuro genesis in the DG of the HPC. indicates a significant difference from saline.
42 Figure 2.4. Representative photomicr ographs showing the effects of the selective 5-HT2A receptor agonist 251-NBMeO on hippocampal neurogenesis. NeuN+ cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (AC), 0.1 mg/kg 251-NBMeO (D-F), 0.3 mg/kg 251-NBMeO (G-I ) and 1.0 mg/kg 251-NBMeO (J-L). Scale = 100 M
43 Interestingly, acute admi nistration of the 5-HT2A/C receptor antagonist ketanserin produced similar effects on neurogenesis as high doses of the 5-HT2A receptor agonists. As can be seen in fi gure 2.5, the total num ber of BrdU+ cells in the DG was significantly decr eased after 1.0 mg/kg ketanserin [ t (10)=3.0, p =0.008], suggesting that the 5-HT2A/C receptor is involved in the regulation of cell survival in the HPC. Furthermore, acut e ketanserin decreased the total number of BrdU/NeuN positive ce lls compared to saline [ t (10)=3.0, p =0.02] demonstrating that an tagonism of the 5-HT2A/C receptor negatively regulated the number of new born n eurons generated in t he DG of the HPC.
44 Figure 2.5. Acute Admi nistration of the 5-HT2A/C receptor antagonist ketanserin negatively regulated cell survival and neurogenes is in the HPC. Mice (n = 6 per condition) were injected with 1.0 mg/kg ke tanserin or saline then received 75 mg/kg BrdU once daily for 4 days follo wing drug followed by euthanasia two weeks after drug injection. Ketanserin decreased the total number of BrdU+ (A) and BrdU/NeuN positive cells (B) s uggesting that antagonism of the 5-HT2A/C receptor negatively regulated cell su rvival and neurogenesis in the HPC.
45 Figure 2.6. Representativ e photomicrographs showing the effects of the 5-HT2A/C receptor antagonist ketans erin on neurogenesis in the dentate gyrus. NeuN+ cells (left), BrdU+ cells (center) and NeuN /BrdU+ cells (right). Saline (A-C), 1.0 mg/kg ketanserin (D-F). Scale = 100 M Effects of Repeated Intermittent PSO P Administration on Progenitor Cell Survival and Neurogenesis in the Hippocampus. In order to investigate the effects of repeated intermittent PSOP adminis tration on cell survival mice (n = 67 per condition) were in jected with PSOP (0.5, 1.0, or 1.5 mg/kg), 1.0 mg/kg ketanserin or 0.9% saline solution once weekly for 4 w eeks. Each day following drug administration, mice were administe red 75 mg/kg BrdU and sacrificed two weeks after last drug injection. ANOVA fa iled to reveal differences in the total number of BrdU+ cells in the DG as a result of repeated intermittent drug
46 treatment [ F (4,26)=2.20, p =0.09]. As can be seen in Figure 2.7A a trend toward an increase in the number of surviving cells was observed after high doses of PSOP. The phenotypic fate of surv iving cells was determined by immunofluorescent labeling of BrdU and NeuN. ANOVA revealed a significant effect of Dose on the number of double labeled neurons in the DG [ F (4,26)=3.15, p =0.02]. As can be seen in Figure 2.7B chronic administration of 1.5 mg/kg PSOP significantly increased the number of BrdU/NeuN+ cells compared to saline and ketanserin ( p <0.05) (indicated by *). These data suggest repeated intermittent administrat ion of PSOP, a 5HT2A agonist upregulated neurogenesis in the DG of the HPC.
48 Figure 2.7. Effect of Repeated In termittent PSOP Administration on Hippocampal Neurogenesis. Mice (n = 6-7 per condition) were injected with PSOP (0.5, 1.0, or 1.5 mg/kg), 1.0 mg/kg ketanserin or saline once weekly for 4 weeks. Each day following drug adminis tration, mice were administered 75 mg/kg BrdU and sacrificed two weeks after last drug injection. A) The total number of BrdU+ cells in the DG did not di ffer as a result of repeated intermittent drug treatment, however, a trend toward an increase in the number of surviving cells was observed after high doses of PSOP. B) Repeated intermittent administration of 1.5 mg/kg PSOP sign ificantly increased the number of BrdU/NeuN+ cells compared to saline and ketanserin ( p <0.05). These data suggest repeated intermittent administrati on of high doses of PSOP, a 5HT2A agonist upregulated neurogenesis in the DG. indicates a significant difference from saline and ketanserin.
49 Figure 2.8. Representati ve photomicrographs showing the effects of repeated intermittent PSOP or ketanserin admin istration on neurogenesis in the DG.
50 NeuN+ cells (left), BrdU+ cells (center) and NeuN/BrdU+ cells (right). Saline (AC), 0.5 mg/kg PSOP (D-F), 1.0 mg/kg PSO P (G-I), 1.5 mg/kg PSOP (J-L), and 1.0 mg/kg Ketanserin (M-O). Scale = 100 M Discussion The present investigation illustrat es the involvement of the 5-HT2A receptor in the regulation of neurogenesis in the DG of the HPC. Acute administration of low doses of PSOP (0.1 and 0.5 mg/kg) did not alter neurogenesis, however, higher doses of PSOP (1.0 mg/kg) decreased neurogenesis two weeks after drug exposure (Figure 2.1). In addition, acute administration of the potent 5-HT2A receptor agonist 251-NBMeO (Figure 2.3) and the 5-HT2A/C receptor antagonist ketanserin (Figure 2.5) decreased hippoc ampal neurogenesis. Acute ketanserin (1-5 mg/kg) administered within 4 hours of sacrifice decreased the number of BrdU+ cells in the DG, indicating a reducti on in cell proliferat ion (Banasr et al., 2004; Jha et al., 2008). The present study extends these findings by demonstrating that acute ketanserin decreases the number of BrdU+ and BrdU/NeuN+ cells 2 weeks a fter drug administration, indi cating a reduction in cell survival and neurogenesis after exposure to acute ketanserin. The current study reports that repeated intermittent administ ration of high doses of PSOP (1.5 mg/kg) increased n eurogenesis in the DG (see Figure 2.7). A recent study investigated the effects of chronic DOI, LSD and ketanserin administration on the number of Brdu+ cells in the DG (Jha et al., 2008). They
51 report no effect of chronic DOI or LS D on progenitor cell proliferation but observed an increase in the number of BrdU+ cells in the DG after chronic ketanserin. There are several methodolog ical differences between the studies which may account for the different resu lts, namely drug admin istration protocol, doses of compounds administer ed and administration prot ocol of BrdU. Jha and colleagues administered DOI (8 mg/kg), LSD (0 .5 mg/kg) or ketanserin (5 mg/kg) once daily for seven consecutive days fo r the chronic drug administration protocol (Jha et al., 2008). The current investigation administered PSOP (0.5, 1.0 or 1.5 mg/kg) or ketanserin (1.0 mg /kg) 4 times over the course of one month so that injections were giv en one week apart. This was a critical consideration in our experimental design given that daily doses of LSD, PSOP or other 5-HT2A receptor agonists produce rapid tolerance to the drug and results in a selective downregulation of the 5HT2A receptor (Buckholtz et al., 1990; Buckholtz et al., 1985; Buckholtz et al., 1988). Jha and colleagues administered BrdU (200 mg/kg) 2 hours after the last injection of DOI, LSD or ketanserin and sacrificed the animals 24 hours later (Jha et al., 2008) In the current study BrdU (75 mg/kg) was administered 24 hours afte r each drug injection and mice were sacrificed two weeks after the last drug in jection so that time between the first BrdU injection and sacrifice was 6 weeks. This allowed for the assessment of neurogenesis giving time for the birth-dated cells (BrdU labeled) to mature into neurons. Brain derived neurotropic factor (BDNF) has been implicated in synaptic
52 plasticity (Kang et al., 1997; Pang et al., 2004; Tyler et al., 2002) through the modulation of synapse formation and dendritic spine growth in the HPC (Bamji et al., 2006; Tyler & Pozzo-Miller, 2001; Tyler & Pozzo-Miller, 2003). Chronic administration of 5-HT agonists (inc luding SSRIs) upregul ate BDNF mRNA expression in the HPC (Nibuya et al., 1995; Nibuya et al., 1996). Evidence suggests that the 5-HT2A receptor is involved in th e regulation of BDNF in the HPC (Vaidya et al., 1997). Specifically DOI, a 5-HT2A/C receptor agonist decreased BDNF mRNA expression in the gr anule cell layer of the DG but not in the CA subfields of the HPC. Interestingly, the decrease in BDNF mRNA expression was blocked by the 5-HT2A receptor antagonist but not the 5-HT2C receptor antagonist, im plicating the 5-HT2A receptor in the regulation of BDNF expression (Vaidya et al., 1997). PSOP and 251-NBMeO exert their e ffects through binding to 5-HT receptors. PSOP binds to the 5-HT2A receptor (Ki = 6 nM) with high affinity and to a much lesser extent to the 5-HT1A receptor subtype (K i = 190 nM) (McKenna et al., 1990). The synthetic phenethy lamine 251-NBMeO binds to 5-HT2A receptors (Ki = 0.044 nM) with an extremel y high affinity (Braden et al., 2006). 5-HT2A receptors are highly express ed throughout the HPC in the DG, hilus, CA1, and CA3 and are colocalized on GABAergic neurons, pyramidal and granular cells (Cornea-Hebert et al., 1999; Morilak et al., 1993; Pompeiano et al., 1994; Shen & Andrade, 1998; Luttgen et al ., 2004; Morilak et al., 1994). 5-HT2A receptor agonists stimulate arachi donic acid and consequently, the
53 phosphoinositide (PI) pathway resulting in the activation of protein kinase C (PKC) (Kurrasch-Orbaugh et al., 2003; Ananth et al., 1987). Electrophysiological evidence suggests that 5-HT2A receptors stimulate GABAergic interneurons in the HPC (Shen & Andrade, 199 8) and GABAergic interneur ons in the hilus form connections with progenitor cells in t he SGZ (Wang et al., 2005). When progenitor cells are less than 2 weeks ol d the GABAergic input exerts an excitatory influence on the progenitor cells and as the cells establish glutamatergic synapses the GABAergic interneurons becom e inhibitory (Wang et al., 2005; Zhao et al., 2006; Aimone et al., 2006). Given that 5-HT2A receptor agonists administered chronically downr egulates receptor expression, and evidence suggests that the 5-HT2A receptor excites the GABAergic interneurons which stimulate progenitor cells in the SGZ, one might anticipate a reduction in neurogenesis after chronic PSOP. On the contrary, the present study reports high doses of PSOP upregulates neurogenesis. Based on this finding it is plausible to suggest the increase in neur ogenesis observed may be attributed to the administration paradigm in which PSOP was given 4 times over the course of one month so that injections were given once a week. In this administration paradigm alterations in receptor levels may not have occurred to the extent that occurs with daily exposure and give this highly plastic microni che time to adapt between drug exposures. The results shown provide evidence t hat the 5-HT2A receptor is involved in the regulation of hippoc ampal neurogenesis. The dat a suggest that acute
54 administration of 5-HT2A receptor agonists and an antagonist downregulated neurogenesis in the DG. Whereas, chroni c administration of high doses of 5HT2A receptor agonists enhance hippocampal n eurogenesis in the DG. Future studies should investigate the effects of chronic administ ration of PSOP and ketanserin on 5-HT2A receptor levels and neuroplasticity in the HPC. Acknowledgments: This work was su pported by the Hele n Ellis Research Endowment (JSR). Thanks to David Ni chols Ph.D. for donating the selective 5HT2A agonist 251-NBMeO and Dr Francisco Mo reno from University of Arizona and Rick Doblin Ph.D. of the Multidiscipli nary Association for Psychedelic Studies (MAPS) for donating the PSOP.
55 Chapter Three The Effects of Psilocybin on Trace Fear Conditioning Abstract Aberrations in brain serotonin (5 -HT) neurotransmission have been implicated in psychiatric disorders incl uding anxiety, depression and deficits in learning and memory. Many of these disorders are treated with drugs which promote the availability of 5HT in the synapse. However, it is not clear which of the 5-HT receptors are invo lved in behavioral improvem ents. The current study aimed to investigate the effects of psilocybin, a 5HT2A receptor agonist on hippocampal-dependent learning. Mice rece ived a single inject ion of psilocybin (0.1, 0.5, 1.0 or 1.5 mg/kg), ketanserin (a 5HT2A/C antagonist) or saline 24 hours before habituation to the env ironment and subsequent training and testing on the fear conditioning task. Trace fear c onditioning is a hip pocampal-dependent task in which the presentation of the conditio ned stimulus (CS, tone) is separated in time by a trace interval to the uncondit ioned stimulus (US, shock). All mice developed contextual and c ued fear conditioning; ho wever, mice treated with psilocybin extinguished the cued fear conditioning more rapidly than saline treated mice. Interestingly, mice given the 5HT2A/C receptor antagonist ketanserin showed less of cued fear res ponse than saline and psilocybin treated
56 mice. Future studies should examine t he temporal effects of acute and chronic psilocybin administration on hipp ocampal-dependent learning tasks. Introduction The hippocampus (HPC) plays a critical role in learning tasks that involve temporal encoding of stimuli (Squire et al., 1992; Squire, 1992). The trace classical conditioning paradigm require s temporal processing because the conditioned stimulus (CS) and the unconditioned stimulus (US) are separated in time by a trace interval. Lesions to the HPC prevent trace conditioning, indicating that it is a hippocampal-dependent task (McEchron et al., 1998; Weiss et al., 1999). The serotonergic system has been imp licated in hippoc ampal-dependent learning. Administration of selective se rotonin (5-HT) uptake inhibitors (SSRIs) produce alterations in performance on lear ning tasks that require the HPC (Flood & Cherkin, 1987; Hua ng et al., 2004). In a knockout (KO) mouse model, central 5-HT deficient mice deve loped heightened contextual fear conditioning which was reversed by intracerebroventricular micr oinjection of 5-HT (D ai et al., 2008). An impairment in learni ng on the morris water maze was observed in 5-HT1A KO mice along with functional abnormalities in the HPC (Sarnyai et al., 2000). Activation of 5-HT1A receptors in the medial septum alters encoding and consolidation in a hippoc ampal-dependent memory task (K oenig et al., 2008). In addition, Lysergic acid di ethylamide (LSD), a 5-HT2A receptor agonist facilitated
57 learning of a brightness di scrimination reversal problem (King et al., 1972; King et al., 1974). Evidence suggests that performanc e on hippocampal-dependent learning tasks is influenced by neurogenesi s in the dentate gyrus (D G) of the HPC (Van et al., 2002; Nilsson et al., 1999; Shors et al., 2001; Shors et al., 2002; Gould et al., 1999a; Gould et al., 1999c). This was elegantly demonstrat ed by Shors and colleagues by treating animals with me thylazoxymethanol acetate (MAM), an anti-mitotic agent which er adicates the progenitor ce ll population in the DG before testing mice on hippocampaldependent and hippocampal-independent learning tasks (Shors et al., 2001; Shors et al., 2002). MAM treated animals had significantly fewer BrdU+ cells in the subgranular zone (SGZ) of the DG but showed no impairment in the spatial nav igation task (HPC-depe ndent) or delay eyeblink conditioning task (HPC-i ndependent) demonstrating that the hippocampal progenitor cell pop ulation is not essential for these particular tasks (Shors et al., 2002; Shors et al., 2001). In contrast MAM severely impaired performance on trace fear c onditioning and trace eyeblin k conditioning, providing evidence for the involvement of progenitor cells in the DG in trace classical conditioning (Shors et al., 2002; Shors et al., 2001). In addition, hippocampal neurogenesis is influenced by serotonergi c agonists. Specifically, SSRIs enhance the production of new born neurons in the DG of the HPC (Malberg et al., 2000; Santarelli et al., 2003). Psilocybin (PSOP), a tryptamine alkalo id, exerts psychoactive effects by
58 altering serotonergic neurotransmission (P assie et al., 2002). PSOP binds to the 5-HT2A receptor (Ki = 6 nM) with high affinity and to a much lesser extent to the 5-HT1A receptor subtype (Ki = 190 nM) (McKenna et al., 1990). 5-HT2A receptors are highly expressed throughout the HP C in the DG, hilus, CA1, and CA3 (Cornea-Hebert et al., 1999; Morilak et al., 1993; Pompeiano et al., 1994; Shen & Andrade, 1998; Luttgen et al., 2004; Morilak et al., 1994). 5-HT2A receptor agonists, including PSOP, stim ulate arachidonic acid (AA) and consequently, the phosphoinositide (PI) pathway resulting in the activation of protein kinase C (PKC) (Kurrasch-Orbaugh et al., 2003; Ananth et al., 1987). Electrophysiological evidence suggests that 5-HT2A receptors stimulate GABAergic interneurons in the HPC (Shen & Andrade, 199 8) and GABAergic interneur ons in the hilus form connections with progenitor cells in the SGZ (Wang et al., 2005). The present study aimed to inve stigate the effects of the 5HT2A receptor agonist PSOP on hippocampal-dependent lear ning. Mice received a single injection of psilocybin (0.1, 0.5, 1.0 or 1.5 mg/kg), 1.0 mg /kg ketanserin (a 5HT2A/C antagonist) or saline 24 h ours before habituation to the environment and subsequent training and testing on t he trace fear conditioning task. Materials and Methods Subjects. C57BL/6J male mice (30-40 g) were housed in standard laboratory cages and left undisturbed for 1 week after arrival at the animal facility. All mice had unlimited access to water and laboratory chow and were maintained in a temperature and humidity controll ed room on a 12:12 li ght/dark cycle with
59 light onset at 7:00 AM. All National Institutes for Health (NIH) gui delines for the Care and Use of Laboratory Animals were followed (National Inst itutes of Health, 2002). General Procedure. Mice (n=9-10/condition) received an intraperitoneal (i.p.) injection of PSOP (0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1. 5 mg/kg), ketanserin (1.0 mg/kg) or 0.9% saline and 24 h late r were habituated to the testing chamber for 30 min. The fear conditioning envir onment consisted of two chambers each placed inside a larger soundproof cham ber. The 35.6 (W) x 38.1 (D) x 31.8 (H) cm freeze monitor box (San Diego Instru ments, San Diego, CA) is a clear Plexiglas chamber with a removable lid whic h contains a metal grid floor (0.3 cm grids spaced 0.8 cm apart) through which a foot shock can be delivered. Photobeam activity within the chamber recor ded the vertical and horizontal movements of mice. Two minutes into the habituat ion period a baseline (BL) measure of movement was recorded for 3 minutes and served as the habituation BL measure. Freeze monitor boxes were cleaned with quatricide between each mouse to prevent olfactory cues. Mice were returned to their home cage after habituation. The next day mice were returned to the same freeze monitor chamber and underwent training to form CS Â– US associ ations. After a 2 minute acclimation period, mice were exposed to 10 trials of trace fear conditioning which is illustrated in Figure 3.1. Each trial c onsisted of the CS (tone, 82 dB, 15 s) followed by a trace interval (30 s) and ended with the present ation of the US
60 (shock, 0.5 s, 1 mA) delivered through the grid flooring. After each trial ended there was a 210 s intertrial interval (ITI ). Freeze monitor boxes were cleaned with quatricide between each mouse to prev ent olfactory cues and mice were returned to their home cage after training. Figure 3.1. Schematic repr esentation of the Trace Fear Conditioning Paradigm. Trace fear conditioning is a hippoc ampal-dependent task in which the presentation of the conditio ned stimulus (CS, tone) is separated in time by a trace interval to the unconditi oned stimulus (US, shock) On day 3 of the task, testing of the fear conditioning response was assessed in 2 phases. First, mice were placed in the freeze monitor box for 5 minutes and movement was recorded for t he last 3 minutes and used to assess the measure of fear associated to the tr aining context. Mice were then returned to the home cage for 1 hour. Second, the context was altered by replacing the grid floors with black Plexiglas floori ng and adding a cotton ball with 1ml vanilla essence inside the sound attenuated chamber Mice were placed inside the novel chamber and after 2 minutes, move ments were recorded for the next 3 minutes. Next 10 trials wit h the presentation of the CS only were delivered with an ITI of 240 s. Cue fear conditioni ng was measure by the percent freezing
61 during the CS (tone; 15s), dur ing the trace interval (30s ) and after the trace when the US would have o ccurred. Conditioned fear wa s defined as an increase in percent immobility during the cue test. Percent immobility was calculated by dividing time spent immobile during stimuli (CS, trace or after trace) by the length of time the stimuli la sted multiplied by 100. Design and analyses. Se parate two-way repeated measure analyses of variance (ANOVA) were used to evaluate the effect of Dose and Trial on each dependent variable in the tr ace fear conditioning task. Dependent measures recorded included percent fr eezing during CS, during trac e, after trace, during habituation BL, during the context text and in response to the novel environment. When appropriate, post hoc analyses such as Bonforr eoni were used to isolate effects. All statistical analyses will be determined significant at the 0.05 alpha level. Results Acquisition. The acquisition of the freez ing response is displayed in Figure 3.2 showing both the response to the CS (Figure 3.2A) and during the trace (Figure 3.2B). Using percent immobilit y in response to the CS for the first 3 trials of training, ANOVA showed that regardless of drug tr eatment there was a significant improvement across trial [ F (2, 108) = 40.0, p <0.0001]. Specifically, immobility in response to the CS increased from trial 1 to trial 3 indicating the learned association between the stimuli during training ( p <0.05). Additionally, ANOVA of the percent immobility during th e trace interval revealed a significant
62 effect of trial [ F (2, 108) = 20.0, p <0.0001]. There was a st riking increase in the amount of time spent immobile during th e trace period from trial 1 to trial 3 demonstrating that the association betwe en the CS and US promoted immobility in anticipation of the shock.
64 Figure 3.2. Effects of Ps ilocybin on the Acquisition of Trace Fear Conditioning. Mice underwent training to form CS Â– US a ssociations by exposure to 10 trials of trace fear conditioning. Ea ch trial consisted of the pr esentation of the CS (tone, 15-s) followed by a trace interval (30-s) and ended with the US (s hock, 0.5-s). A) Percent immobility during presentation of t he 15-s CS during the first three trials of CS Â– US pairing. B) Percent immob ility during the 30-s trace interval during the first three trials of CS Â– US pairing. Contextual Fear Conditioning. Contextual fear co nditioning was assessed by comparing percent immobility in the freeze monitor box on habituation day to percent immobility during the context te st. There was no interaction between Dose and Trial [ F (5,90) = 0.95, p =0.45] and no effect of Dose during the habituation BL or context test [ F (5,90) = 1.15, p =0.34]. However, there was a significant effect of Trial [ F (1,90) = 105.85, p <0.0001] indicating that mice spent significantly more time freez ing after the CS Â– US pairi ngs during the context test compared to habituation BL. Figure 3.3 illustrates percent immobility during exposure to the freeze monitor box duri ng the habituation test (A) and during the contextual fear conditioning test (B).
65 Figure 3.3. Contextual Fear Conditioni ng. Percent immobility expressed during
66 exposure to the freeze monitor box durin g habituation (A) and during contextual fear conditioning (B). All mice expres sed contextual fear conditioning as indicated by a significant increase in per cent immobility during the context test ( p <0.05). Cue Fear Conditioning. Freezing responses (% immobility) during the CS only (tone) test are illustrated in Figure 3.4. There was a significant effect of Trial [ F (2,102) = 7.83, p <0.0007] with trials 2 and 3 elicit ing significantly more freezing in response to the CS compared to trial 1 (Figure 3.4A). There was also a significant effect of Dose [ F (5,51) = 4.96, p <0.0009] revealing that control mice showed a greater fear response to the cue compared to 0.1 mg/kg PSOP, 1.0 mg/kg PSOP, 1.5 mg/kg PSOP and 1.0 mg/k g ketanserin. ANOVA revealed a significant effect of Dose during the trace interval [ F (5,51) = 2.41, p <0.05]. The fear associated with the trace interval durin g the first three trials on test day was reduced in mice treated with 1.0 mg/kg PSOP compared to saline and 1.5 mg/kg PSOP (Figure 3.4B). Figure 3.4C illustrates the f ear response after the trace interval which coincides with the timing the US (shock) was delivered during the acquisition of the CS US pairing phase. A two-wa y repeated measures ANOVA revealed a significant interaction between Dose and Trial [ F (10,100) = 3.53, p <0.0005]. Interestingly, mice adminis tered low doses of PSOP (0.1 and 0.5 mg/kg) froze more on trial 1 compared to trial 2 and 3 suggesti ng they are more apt to adapt to the absence of the US so t hat the fear response is diminished as
67 the US is extinguished. This patte rn was reversed in mice treated with ketanserin who increased fear responses fr om trials 1 to 3, indicating the robust memory for the US even in its absence. Taken together, these data suggest differential effects of PSOP on trace fear conditioning.
69 Figure 3.4. Effect of Acute PSOP on Cue Fear C onditioning. Cue fear conditioning was examined 24 hours after the training period in which mice were exposed to 10 trials of CS Â– US pair ings. A) Percent immobility during presentation of the 15-s CS during the first three CS-only trials. B) Percent immobility during the 30s trace interval during the first three trials. C) Percent immobility after the trace interval which coincides with the timing the US (shock) was delivered during the acquisition of the CS US pairing phase. Discussion The present investigation illustrat es the involvement of the 5-HT2A receptor in trace fear conditioning, a hippocam pal-dependent learning task. During the acquisition of the freezing response ther e was a striking increase in the amount of time spent freezing during the present ation of the CS and during the trace period from trial 1 to trial 3 (see Figur e 3.2). These data demonstrate that the association between the CS and US prom oted freezing in anticipation of the shock, however, the ac quisition of learning wa s not altered by acute administration of PSOP or ketanserin. All conditions displayed similar loco motor activity levels in the freeze monitor box during the habit uation baseline exposure and during re-exposure to the same environment during the context ual fear conditioning test. As expected mice froze substantially more during re-e xposure to the same context after CS Â– US associations were formed compared to the habituation baseline, indicating
70 that all groups formed contex tual fear conditioning (Figure 3.3). It is well known that contextual fear conditioning is a hippocampal-dependent learning task (Kim & Fanselow, 1992; McNish et al., 1997; Hir sh, 1974; Esclassan et al., 2008; Frohardt et al., 1999). The serotonergi c system has been implicated in performance on the contextual fear conditi oning task (Dai et al., 2008). The present investigati on found that the 5-HT2A receptor does not alter contextual fear conditioning since no differences were observed between controls and mice treated with PSOP or ketanserin. The current study reports alterations in cue associated fear conditioning mediated by the 5-HT2A receptor. Independent of drug administered all mice developed cue-induced freezing during the presentation of th e tone on the CSonly trial (see Figure 3.4). At the time coinciding wi th the expected US (shock) presentation low doses of PSOP (0.1 and 0.5 mg/kg) elicited a heightened freezing response on trial 1 compared to other trials suggesting they are more apt to adapt to the absence of the US so t hat the fear response is diminished as the US is extinguished. This patte rn was reversed in mice treated with ketanserin who increased fear responses fr om trials 1 to 3, indicating the robust memory for the US even in its absence. Synaptic plasticity in the HPC is critical for the ac quisition of learning and memory. Brain derived n eurotropic factor (BDNF) has been implicated in synaptic plasticity and memory processi ng (Kang et al., 1997; Pang et al., 2004; Tyler et al., 2002) through the modulatio n of synapse formation and dendritic
71 spine growth in the HPC (Bamji et al ., 2006; Tyler & Pozzo-Miller, 2001; Tyler & Pozzo-Miller, 2003). Chronic administration of 5-HT agonists (including SSRIs) upregulate BDNF mRNA expression in the HPC (Nibuya et al., 1995; Nibuya et al., 1996). Evidence suggests that the 5-HT2A receptor is involved in the regulation of BDNF in the HPC (Vaidya et al ., 1997). Specifically DOI, a 5-HT2A/C receptor agonist decreased BDNF mRNA expression in the granule cell layer of the DG but not in the CA subfields of the HPC. Interestingly, the decrease in BDNF mRNA expression was blocked by the 5-HT2A receptor antagonist but not the 5HT2C receptor antagonist, implicating the 5-HT2A receptor in the regulation of BDNF expression (Vaidya et al., 1997). 5-HT2A receptors are highly express ed throughout the HPC in the DG, hilus, CA1, and CA3 and are colocalized on GABAergic neurons, pyramidal and granular cells (Cornea-Hebert et al., 1999; Morilak et al., 1993; Pompeiano et al., 1994; Shen & Andrade, 1998; Luttgen et al., 2004; Morilak et al., 1994). Agonists to the 5-HT2A receptor stimulate AA and consequently, the PI pathway resulting in the activation of PKC (K urrasch-Orbaugh et al., 2003; Ananth et al., 1987). Electrophysiological ev idence suggests that 5-HT2A receptors stimulate GABAergic interneurons in the HPC (S hen & Andrade, 1998). Aberrations in GABAergic function in the HPC has been im plicated in learning and memory due to the role of hippocampal GABA in tempor ospatial integration (W allenstein et al., 1998)
72 Taken together, the data r eported in the present inve stigation implicate the 5-HT2A receptor in hippocampal-dependent lear ning. The present study reports that prior exposure to PSOP altered responsivity in a novel environment indicating an absence of a fear response, an effect not elicited by control mice. Furthermore, low doses of PSOP heightened cue elicited fear conditioning and antagonists to the 5-HT2A/C receptor diminished fear conditioning to the cue. Results of this study rais e the possibility that 5-HT2A receptor activity could lead alterations hippocampal-depen dent learning and memory. Acknowledgments: This work was su pported by the Hele n Ellis Research Endowment (JSR). Thanks to Dr Franci sco Moreno from University of Arizona and Rick Doblin Ph.D. of the Multidiscipli nary Association for Psychedelic Studies (MAPS) for donating the PSOP.
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About the Author Briony Catlow was born on November 7, 1978 in Auck land, New Zealand. She moved to the United States in 1995 to comp lete a year of study in high school. During that year she began volunteering for Coastal Expeditions a kayak tour company in Charleston, SC and fell in love with the Carolina lowcountry. She graduated from Wando High School in Mt Pleasant, SC then entered the College of Charleston in SC where she majored in Biology. After graduating, she moved to Tampa, FL to pursue her Doctorate at the Universi ty of South Florida.