USF Libraries

An anatomical investigation of higher visual structures in the pigeon (columba livia)

MISSING IMAGE

Material Information

Title:
An anatomical investigation of higher visual structures in the pigeon (columba livia)
Physical Description:
Book
Language:
English
Creator:
Patton, Tadd
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
Publication Date:

Subjects

Subjects / Keywords:
Telencephalon
Lateralization
Birds
Rotundus
Zenk
Dissertations, Academic -- Psychology -- Masters -- USF   ( lcsh )
Genre:
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: Early visual processing in the avian brain has been studied extensively, the retina and midbrain in particular. However, a clear understanding of the higher visual centers in the forebrain (the telencephalon and thalamus) remains poor. Two structures located within the avian visual telencephalon, the entopallium (E) and the lateral portion of the intermediate nidopallium (NIL), merit extensive investigation based on their critical role in visual processing. The goal of the current study was to further clarify the anatomical characteristics of E and NIL. Visual information that reaches these telencephalic structures is mostly from the contralateral retina. Thus, blocking visual input on one side affects the opposite hemisphere, but leaves the hemisphere on the same side largely unaffected. This unique property of the avian visual system was used in order to emphasize neurochemical expression in the higher visual structures. After blocking visual input to one hemisphere either by monocular occlusion or unilateral lesion of the nucleus rotundus we examined the expression of specific neuroanatomical markers; namely cytochrome oxidase (CO) and the calcium binding protein, parvalbumin (PV) in E and ZENK protein expression in NIL. . Our results showed there were significantly more PV-ir cell bodies in the inner region of E compared to the outer region. In terms of PV-ir neuropil, regional differences within E were significant. In particular, the ventrolateral E tended to have a higher density of PV-ir neuropil than other regions except the most ventromedial area which was also high in PV-ir. Differential CO staining patterns were observed as well. However, unlike PV-ir neuropil the ventro-intermedial area showed a low level of CO staining compared to the other areas. Finally, there was a significant reduction of ZENK-ir cell bodies on NIL in the experimental hemisphere compared to control side. Based on this differential expression of ZENK, it was possible for the first time to visualize the location of NIL. The findings presented here are discussed in terms of their relevance to the identification of the size and extent of NIL and the heterogeneous nature of E.
Thesis:
Dissertation (PHD)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
System Details:
Mode of access: World Wide Web.
System Details:
System requirements: World Wide Web browser and PDF reader.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains X pages.
Statement of Responsibility:
by Tadd Patton.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
usfldc doi - E14-SFE0004594
usfldc handle - e14.4594
System ID:
SFS0027909:00001


This item has the following downloads:


Full Text

PAGE 1

An Anatomical Investigation Of Higher Visual Structures In The Pigeon ( Columba l ivia ) by Tadd B. Patton A d issertation submitted in partial fulfillment of the requirements for the degree of Doctor of P hilosophy Department of Psychology Colleg e of Arts and Sciences University of South Florida Major Professor: Toru Shimizu, Ph.D. Michael Brannick, Ph.D. Michael Coovert, Ph.D. Cheryl Kirstein, Ph.D. Thomas Sanocki, Ph.D. Date of Approval: June 2 2010 Keywords: telencephalon, lateralization, birds, rotundus, zenk Copyright 2010, Tadd B. Patton

PAGE 2

Dedication This work is dedicated to the people in my life who understood how important it was for me to finish what I started. Completing this dissertation represents more than achieving a c ertain credential; it shows that with perseverance, the sky is the limit. I dedicate this dissertation to those who, like me, believe in doing something to the best of their ability and not settling for anything less. I owe a tremendous debt of gratitude t o a very special person in my life, Sophi e She never stopped believing in me even when my self -confidence wavered, as I often did. Thank you, Sophie. This ones for you.

PAGE 3

Acknowledgments I would like to thank the following students for helping me with various aspects of this project: Jeremy Brenner, Amanda Bunton, Frank Fishburn, Asef Mahm u d David Nicholson, and Justine Vandenbosche. Their relentless desire to assist me with this seemingly never ending project will always be greatly appreciated. I would also like to thank Dr. Byeong Cha from the Lisa Muma Weitz Advanced Microscopy & Cell Imaging Core Laboratory in USFs College of Medicine for allowing me to use his microscope. Dr. Cha was generous with his time and resources, both of which greatly facil itated the completion of this project. I would be remiss if I did not acknowledge the very patient and accommodating Dissertation committee members: Dr. Michael Brannick, Dr. Michael Coovert, Dr. Cheryl Kirstein, and Dr. Thomas Sanocki. I have no doubt tha t each of them has eagerly awaited the completion of my graduate work. Last, but not least, I would like to thank my Major advisor and mentor, Dr. Toru Shimizu. Without his patient and thoughtful guidance, none of this would have been possible and I would not be the person I am today.

PAGE 4

i Table of Contents List of Figures iii List of Tables vii i Abstract i x Introduction 1 General Background 2 Visuo Cognitive Abilities of Birds 2 Two Major Ascending Visual Pathways 5 Functions of Early Visual Processing 7 Background for Specific Aim One 9 Functional Significance of E 9 Heterogeneity of E 10 Anterior -Posterior Organization of E 10 Internal External Organization of E 12 Background for Specific Aim Two 14 Definition of NIL 14 NIL Connections are Reminiscent of NCM 16 Background for Specific Aim Three 17 Neuromarkers Used to Identify Visual Structures 17 Parvalbumin (PV) 17 Cytochrome Oxidase (CO) 18 ZENK 19 Rationale 22 Method 24 Subjects 24 Unilateral Lesions 2 4 Monocular Occlusions 25 Exposure to Live Stimulus 25 Perfusions 26 Parvalbumin and ZENK Immunohistochemistry 27 Cytochrome Oxidase Histology 27 Additional Histology 28 Parvalbumin and Cytochrome Oxidase Image Capturing and Analysis 28 ZENK Image Capturing and Analysis 30

PAGE 5

ii Lesion Reconstructions 33 Statistical Analyses 33 Results 35 Lesion Reconstructions 35 Individual Lesion Reconstruction Cases 36 PV Immunohistochemistry 40 Individual PV Cases 41 Number of PV -ir Neurons in E 49 De nsity of PV ir Neuropil in E 53 CO Histochemistry 58 Individual CO Cases 59 Density of CO Staining in E 65 ZENK Immunohistochemistry 70 Individual ZENK Cases 71 Number of ZENK -ir Neurons in NIL 82 Discussion 83 List of References 93 Appendi ces 102 Appendix A: A protocol for Conducting Optical Density Analysis 103 Appendix B: A New Digital Imaging Protocol for Signal Detection and Distribution 104 About The Author END PAGE

PAGE 6

iii List of Figures Figure 1. Schematic view of the tectofugal pa thway of the pigeon 7 Figure 2. Schematic view of pigeon brain ( sagittal view ) 11 Figure 3. Microphotographs showing PV and CO a ctivity in the entopallium (E) 14 Figure 4. Schematic showing the spatial relationship between E and NIL 15 Figure 5. Schema tic s showing the inner and outer target regions where the number of PV ir cells was counted 29 Figure 6. Schematic s showing the six target regions where the density of PV -ir neuropil and CO staining was measured 30 Figure 7. Schematic s showing two trans verse sections of the avian brain where NIL is thought to be located 31 Figure 8. Schematics showing the location of the nucleus rotundus (Rt) 36 Figure 9. Rt damage reconstruction for subject PG 236 36 Figure 10. Rt damage reconstruction for subject PG2 39 37 Figure 11. Rt damage reconstruction for subject PG234 38 Figure 12. Rt damage reconstruction for subject PG259 38 Figure 13. Rt damage reconstruction for subject PG254 39 Figure 14. Rt damage reconstruction for subject PG 47 40 Figure 15. Microphotographs of PG234 showing the distribution pattern of PV in E after unilateral lesion 41 Figure 16. Microphotographs of PG259 showing the distribution pattern of PV in E after unilateral lesion 42 Figure 17. Microphotographs of PG254 showing t he distribution pattern of PV in E after unilateral lesion 43

PAGE 7

iv Figure 18. Microphotographs of PG47 showing the distribution pattern of PV in E after unilateral lesion 44 Figure 19. Microphotographs of PG253 showing the distribution pattern of PV in E afte r monocular occlusion 45 Figure 20. Microphotographs of PG263 showing the distribution pattern of PV in E after monocular occlusion 46 Figure 21. Microphotographs of PG256 showing the distribution pattern of PV in E after monocular occlusion 47 Figure 2 2. Microphotographs of PG260 showing the distribution pattern of PV in E after monocular occlusion 48 Figure 23. Graph showing the mean number of PV -ir cells counted in the inner and outer regions of E 49 Figure 24. Graph showing the mean number of PV -ir cells in regions of E at each of the four locations 50 Figure 25. Graph showing the mean number of PV -ir cells in E for each treatment condition, hemisphere, and region 51 Figure 26. Graph showing the mean number of PV -ir cells by hemisphere, region, an d location 52 Figure 27. Graph showing the mean number of PV -ir cells by regions, treatment, and location 52 Figure 28. Graph showing the mean density value of PV -ir neuropil in the six target regions of E across treatment condition, hemisphere, and loca tions 54 Figure 29. Graph showing the mean density value of PV -ir neuropil for both Treatment conditions (lesion versus occlusion) across hemispheres, regions in E, and locations 55 Figure 30. Graph showing the mean density value of PV -ir neuropil after unilateral lesion and monocular occlusion across the different regions in E and all locations (anterior -posterior coordinates) 56 Figure 31. Graph showing the mean density value of PV -ir neuropil after unilateral lesion and monocular occlusion for each he misphere at each of the different regions within E 56

PAGE 8

v Figure 32 Graph showing the mean density value of PV -ir neuropil at each of the regions in E and all four locations (anterior -posterior coordinates) across treatment condition and hemisphere 57 Figu re 33. Microphotographs of PG234 showing the distribution pattern of CO in E after unilateral lesion 59 Figure 34. Microphotographs of PG259 showing the distribution pattern of CO in E after unilateral lesion 60 Figure 35. Microphotographs of PG254 showing the distribution pattern of CO in E after unilateral lesion 61 Figure 36. Microphotographs of PG264 showing the distribution pattern of CO in E after monocular occlusion 62 Figure 37. Microphotographs of PG263 showing the distribution pattern of CO in E after monocular occlusion 63 Figure 38. Microphotographs of PG256 showing the distribution pattern of CO in E after monocular occlusion 64 Figure 39. Microphotographs of PG260 showing the distribution pattern of CO in E after monocular occlusion 65 F igu re 40. Graph showing the mean density value of CO activity in the six target regions of E across treatment condition, hemisphere, and locations (anterior posterior coordinates) 66 Figure 41. Graph showing mean density value of CO activity after unilateral lesion and monocular occlusion across all six regions within E and across all four locations (anterior posterior coordinates) 67 Figure 42. Graph showing the mean density value of CO activity for each hemisphere, treatment condition, and the six target r egions of E across locations (anterior -posterior coordinates) 68 Figure 43. Graph showing the mean density value of CO activity at each of the six regions within E and at all locations (anterior posterior coordinates) across treatment condition and hemisp here 69 Figure 44. Graph showing the mean density value of CO and PV activity at each of the six regions within E and at all locations (anterior -posterior coordinates) across treatment condition and hemisphere 69

PAGE 9

vi Figure 45. Graph showing the mean density value of CO activity at each of the six Regions within E for both hemispheres. Mean values and standard error are shown 70 Figure 46. Microphotographs and corresponding transformed images of PG234 showing the distribution pattern of ZENK in NIL after uni lateral lesion. 71 Figure 47. Microphotographs and corresponding transformed images of PG236 showing the distribution pattern of ZENK in NIL after unilateral lesion 72 Figure 48. Microphotographs and corresponding transformed images of PG239 showing the distribution pattern of ZENK in NIL after unilateral lesion 73 Figure 49. Microphotographs and corresponding transformed images of PG254 showing the distribution pattern of ZENK in NIL after unilateral lesion 74 Figure 50. Microphotographs and correspond ing transformed images of PG259 showing the distribution pattern of ZENK in NIL after unilateral lesion 75 Figure 51. Microphotographs and corresponding transformed images of PG253 showing the distribution pattern of ZENK in NIL after monocular occlusion 76 Figure 52. Microphotographs and corresponding transformed images of PG264 showing the distribution pattern of ZENK in NIL after monocular occlusion 77 Figure 53. Microphotographs and corresponding transformed images of PG170 showing the distribution pattern of ZENK in NIL after monocular occlusion 78 Figure 54. Microphotographs and corresponding transformed images of PG263 showing the distribution pattern of ZENK in NIL after monocular occlusion 79 Figure 55. Microphotographs and corresponding transf ormed images of PG256 showing the distribution pattern of ZENK in NIL after monocular occlusion 80

PAGE 10

vii Figure 56. Microphotographs and corresponding transformed images of PG260 showing the distribution pattern of ZENK in NIL after monocular occlusion 81 F igu re 57. Graph showing the mean number of ZENK ir cells in NIL after unilateral lesions and monocular occlusions 82

PAGE 11

viii List of Tables Table 1. Table showing results of pairwise comparisons of each of the regions within E for PV ir neuropil 54 Table 2. Tab le showing results of pairwise comparisons of each of the regions within E for CO staining 67

PAGE 12

ix An Anatomical Investigation of Higher Visual Structures in the Pigeon ( Columba livia ) Tadd B. Patton Abstract Early visual processing in the avian brain ha s been studied extensively, the retina and midbrain in particular. However, a clear understanding of the higher visual centers in the forebrain (the telencephalon and thalamus) remains poor. Two structures located within the avian visual telencephalon, the entopallium (E) and the lateral portion of the intermediate nidopallium (NIL), merit extensive investigation based on their critical role in visual processing. The goal of the current study was to further clarify the anatomical characteristics of E and NI L. Visual information that reaches these telencephalic structures is mostly from the contralateral retina. Thus, blocking visual input on one side affects the opposite hemisphere, but leaves the hemisphere on the same side largely unaffected. This unique property of the avian visual system was used in order to emphasize neurochemical expression in the higher visual structures. After blocking visual input to one hemisphere either by monocular occlusion or unilateral lesion of the nucleus rotundus we examined the expression of specific neuroanatomical markers; namely cytochrome oxidase (CO) and the calcium binding protein, parvalbumin (PV) in E and Z ENK protein expression in NIL. Our results showed there were significantly more PV ir cell bodies in the inner region of E compared to the outer region. In terms of PV ir neuropil, r egional difference s within E w ere significant In particular, the ventrolateral E tended to have a higher density of PV ir neuropil than other regions except the most

PAGE 13

x ventromedial area which was also high in PV -ir Differential CO staining patterns were observed as well. However, unlike PV ir neuropil the ventro -intermedial area showed a low level of CO staining compared to the other areas. Finally, t here was a significant reduction of ZENK ir cell bodies on NIL in the experimental hemisphere compared to control side. Based on th is differential expression of ZENK, it was possible for the first time to visualize the location of NIL. The findings presented here are discussed in terms of th e ir relevance to the identification of the size and extent of NIL and the heterogeneous nature of E.

PAGE 14

1 Introduction Birds have superb visual abilities that are on par and sometimes better than those of highly visual primates (Hodos, 1993; Frost & Sun, 1997; Shimizu, Patton, & Szafranski, 2008) Birds use these abilities to locate food sources, find a potential mate, remain vigilant against predators, and avoid obstacles during flight. Such well developed vision makes the avian brain an excellent model for i nvestigating the underlying mechanisms which drive these abilities and for the comparative analysis of complex visual systems in general. The goal of this investigation was to clarify the anatomical characteristics of two important visual structures in the avian cerebrum (telencephalon). There are numerous anatomical and physiological studies that have investigated the early stages of visual processing in birds, the retina and structures within the midbrain in particular. In contrast, a clear understanding of the higher visual centers in the forebrain (the telencephalon and thalamus) remains unclear. Two structures located within the avian visual telencephalon, the entopallium (E) and the lateral portion of the intermediate nidopallium (NIL), merit extensive investigation based on their critical role in visual processing. The current study focused on anatomical properties of these two structures. This investigation had three main objectives: 1) to clarify the anatomical and physiological subgroups of E, 2) t o anatomically define the higher visual structure, NIL, and 3) to determine the degree to which neural activity in E and NIL are dependent on visual information. Each of these specific aims was investigated through the examination

PAGE 15

2 of neural activity detect ed by the expression of various neurochemicals (i.e., a mitochondrial enzyme, a calcium -binding protein, and an immediate -early gene). This set of experiments was one of the first systematic anatomical studies to employ lesion and occlusion techniques to directly analyze the higher visual structures, E and NIL. Furthermore, the results reported here are important because they provide information that can be used to examine the similarities and differences of the visual system s between phylogenetically remo te animal classes, birds and mammals Furthermore, based on such comparisons with the mammalian system, the findings in the current study can be used to understand the general and specific neural principles for visual processing. General Background Visuo -cognitive abilities of birds. The remarkable visual and cognitive abilities of pigeons have been demonstrated both in the laboratory and field. Using operant conditioning techniques researchers have shown that birds can be trained to categorize a variety of visual stimuli. Pigeons (Columba livia ) have been trained to respond differently to the presence or absence of photographic images of humans (Herrnstein & Loveland, 1964), trees, bodies of water, a particular person (Herrnstein, Loveland, & Cable, 1976), and different styles of painting (Watanabe, Sakamoto, & Masumi, 1995; see Huber, 2001 for review). Based on these studies, some authors argue that pigeons can form concepts of these objects through discrimination training (Lubow, 1974; Cook, Cavoto, & Cavoto, 1995; Watanabe, Lea, & Dittrich, 1993).

PAGE 16

3 Several bird species also have remarkable visual memor y ability For example, it is well known that food -caching birds, such as the Western Scrub jay ( Aphelocoma californica ), will harvest food and store it i n different locations when food is abundant and then return to these caches at a later time as food becomes scarce (Sherry, Krebs, & Cowie, 1981; Shettleworth, 1983; Sherry, 1984; Shettleworth & Krebs, 1986). One particularly impressive example comes from the behavior of the Clarks nutcracker (Nucifraga columbiana). In the late summer and early fall, these birds will store over 30,000 seeds in various locations and recover the majority of them later in the winter (Vander Wall & Balda, 1977). Discrimination studies in laboratories have shown that even non -food storing birds, such as pigeons, also have extraordinary visual memories (Vaughan & Greene, 1984; Cook, Levision, Gillet, & Blaisdell, 2005). In the study by Cook et al. (2005), pigeons were trained to remember large numbers of photographs, each of which were randomly selected to be associated with the choice stimulus on the left or right side. The results showed that pigeons can memorize between 800 and 1,200 picture response associations. It is clear w hy food -storing birds have such remarkable memory capacity as this ability is crucial to their survival. But, why do pigeons and other birds have such remarkable visuo -cognitive abilities? In addition to the foraging purpose for food-storing birds, one pos sible important reason for these abilities is that birds use them to survive in a highly developed social world. Birds must be able to quickly tell the difference between predator and prey, as well as locate a potential mate. Success at each of these behav iors requires the ability to recognize which animals are members of their own species and which are not. As described above, many discrimination studies use operant conditioning

PAGE 17

4 techniques to investigate how pigeons process complex natural objects, includi ng social stimuli. However, because these operant procedures essentially require birds to discriminate such stimuli for access to a food reward, it is possible that the birds discriminate the stimuli using strategies that are very different from those they may use in their natural setting. Under the operant conditioning paradigm, birds may be able to discriminate photographs of conspecifics based on small details (Brown & Dooling, 1992, 1993). But again, one cannot conclude that birds use such details to di scriminate individuals in the real world. An instructive alternative to the operant conditioning paradigm is to measure a subjects natural responses to ecologically relevant stimuli. A previous investigation conducted in our laboratory (Shimizu, 1998) sh owed that male pigeons exhibit courtship behavior in response to video playbacks of a female pigeon. In particular, male pigeons reacted with strong courtship display even when the body of the female was covered, as long as the head remained visible. In contrast, only weak courtship responses were observed when the head of the video pigeon was covered, so that only the female body was visible. This study (Shimizu, 1998) suggested the overwhelming significance of the head in conspecific detection and mate selection in birds just as in humans. Based on this finding, Patton, Szafranski, and Shimizu (2010) examined the significance of different facial features, such as the beak and eyes, by analyzing the preference of male pigeon subjects between images of femal es whose facial features were digitally manipulated The results showed that the beak is important since males preferred females with an abnormally large beak. In contrast, the presence or lack of the eyes had less of an e ffect on their preference. This fi nding is interesting since humans and other primates tend to innately pay specific attention to eyes in the face

PAGE 18

5 (Farroni, Massaccesi, Menon, & Johnson, 2006). Moreover, t he spatial rearrangement of these facial features did not affect preference, suggesti ng that the configural relationship of the features is not essential. This is also different from the human data that indicate we are sensitive to the spatial arrangement of the facial features (Gauthier & Logothetis, 2000) These results suggest that alth ough the face and facial features are important to birds and humans, the salience of the facial features and thus the meaning of these features are fundamentally different between them. These perceptual differences between birds and mammals are likely a result of the differences in the underlying neural mechanisms associated with such complex visual behavior. The current study was an extension of this previous research on avian visual cognition to study the underlying neural mechanism for visual processing of complex stimuli. Unlike the case of human and nonhuman primates, it is unclear exactly which higher avian brain structures are involved in these sophisticated visuo-cognitive functions. Two specific visual structures, E and NIL, were the focus of this study. Two major ascending visual pathways. In all amniotes (reptiles, mammals, and birds), visual information is sent to the telencephalon along two ascending pathways: the tectofugal pathway and the thalamofugal pathway (Butler & Hodos, 1996; Shimizu & Bowers, 1999). The E and NIL are two primary telencephalic structures associated with the tectofugal pathway, which is the major route for visual processing in birds. Based on connection pattern s the avian tectofugal pathway has been compared to the retino colliculo -pulvinar cortical system in mammals, whereas the thalamofugal pathway is comparable to the mammalian retino -

PAGE 19

6 geniculo -cortical pathway (Shimizu & Karten, 1993). In birds, the majority (over 90%) of the retinal fibers are sent to the optic tectum (TeO) (the tectofugal pathway) rather than directly to the thalamic center of the thalamofugal pathway. This pattern is in contrast to that of highly visual mammals, such as primates, in which the retino -geniculo -cortical pathway is often a prominent pathway (Shimizu & Bowers, 1999). In the avian tectofugal pathway the TeO, the avian equivalent of the mammalian superior colliculus (Gntrkn, 2000; Shimizu, 2001), is a large structure which protrudes laterally between the cerebellum and the cerebrum. The TeO is highly laminated, with the superficial layers receiving retinotopically organized input and the deeper layers giving rise to ascending efferents. The TeO projects bilaterally and nontopographically to the largest thalamic structure, the nucleus rot undus (Rt), the avian equivalent of the mammalian pulvinar (Shimizu & Karten, 1991). Multiple subdivisions have been identified within Rt (i.e., anterior, central, and posterior Rt) based on analysis of cytoarchitecture, neurochemicals, and connections (Be nowitz & Karten, 1976; Nixdorf & Bischof, 1982; Karten, Cox, & Mpdozis, 1997; Hellmann & Gntrkn, 1999; Laverghetta & Shimizu, 2003). Visual information from Rt is further sent to a large nuclear mass in the telencephalon, E, which in turn projects to mu ltiple telencephalic structures, including NIL (see Figure 1).

PAGE 20

7 Figure 1 Schematic view of the tectofugal pathway of the pigeon. Information from each eye is sent to the midbrain of the opposite hemisphere. In the midbrain is the optic tectum (TeO ). TeO from each hemisphere is connected by tectal commissure (CT). Information from TeO is sent to the thalamus bilaterally via the supraoptic decussation (DSO). From Rt, visual information is sent ipsilaterally to the primary telencephalic target, E. Fro m E, information is sent to several higher visual structures such as the lateral portion of the intermediate nidopallium (NIL). Functions of early visual processing. Investigations of the response properties of individual neurons (electrophysiology) and lesion studies have shown that neurons in the tectofugal pathway are sensitive and/or selective to different aspects of visual stimuli, such as motion, direction, luminance, and color (Engelage & Bischof, 1993). Many TeO neurons respond preferentially to certain aspects of moving stimuli. For example, some TeO neurons show responses to objects moving out of phase with the background and tend to be inhibited when the stimulus object is moving with the background, suggesting that these cells play

PAGE 21

8 a role in fi gure -ground segregation or the discrimination of motion of an object as opposed to self induced optical motion (Frost & Nakayama, 1983). In general, neurons in Rt have similar response characteristics to TeO cells. However, Rt is not a homogenous structure but is composed of multiple subdivisions along the rostral -caudal axis. The anterior Rt has neurons that are sensitive to changes in luminance and color (Granda & Yazulla, 1971; Maxwell & Granda, 1979; Wang, Jiang, & Frost, 1993). Neurons in the central and posterior Rt are sensitive to features of motion such as, changes in stimulus size, direction, and velocity (Revzin, 1979). Some neurons in the posterior Rt are sensitive to approaching stimuli suggesting that they act as collision detectors (Wang & F rost, 1992). Destruction, by way of lesions, to structures within the tectofugal pathway results in critical deficits in intensity, color, pattern, acuity, and movement discrimination (Hodos, 1993) Severe and often irreversible deficits in a variety of v isual discrimination tasks, such as stimulus intensity, pattern, and location discriminations result when TeO is damaged (Jarvis, 1974). Similar deficits can be seen when lesions are made in Rt. Rotundal lesions cause significant deficits in discriminatio n of intensity difference thresholds (Hodos & Bonbright, 1974), visual acuity thresholds (Macko & Hodos, 1984), and size threshold differences (Kertzman & Hodos, 1988). In contrast to the tectofugal pathway, the functional significance of the thalamofugal pathway remains elusive. However, there is some evidence that this pathway is important for processing the binocular portion of the visual field, especially for frontal -eyed birds such as most raptors when they are in the process of capturing prey (Davies & Green, 1990) In studies of lateral -eyed birds, such as pigeons, lesions to the thalamofugal pathway cause little to no

PAGE 22

9 discrimination deficits (Watanabe, 1992; Gntrkn Miceli, & Watanabe, 1993). Specifically, virtually no deficits have been found in visual acuity or brightness intensity thresholds after lesions of the thalamic structure, nucleus opticus principalis thalami (OPT complex) ( Macko & Hodos, 1984) or the telencephalic target, Wulst ( Pasternak & Hodos, 1977; Hodos, Macko, & Bessette, 1984). These findings suggest that the tecotofugal pathway plays a crucial role in processing visual information that is essential to higher cognition compared to the thalamofugal pathway. Despite extensive studies in the lower visual structures of the tectofuga l pathway (TeO and Rt), the structure and function of higher visual structures are not as well understood as the early visual stations. The first specific aim will be to examine the subgroups of the primary visual telencephalic structure, E. Background fo r Specific Aim One Functional significance of E. The primary telencephalic visual structure in the tectofugal pathway is E. Studies have shown that lesions to E cause similar deficits in visual discrimination tasks as seen after lesions to Rt. Bilateral le sions of E cause significant deficits in discrimination of intensity difference thresholds (Hodos, Weiss, & Bessette, 1988), visual acuity thresholds (Hodos, Macko, & Bessette, 1984), size threshold differences (Hodos, Weiss, & Bessette, 1986), as well as intensity, color, and pattern discrimination ( Bessette & Hodos, 1989). Hodos et al. (1988) demonstrated that lesions to E caused a severe decline (50% to 83% of the preoperative performance) in pigeons ability to discriminate differences in

PAGE 23

10 l uminous inten sity. These results indicate that E, similar to lower stations of the tectofugal pathway, processes multiple aspects of the visual stimulus in parallel. However, it may also be involved with processing higher, more complex aspects of visual stimuli, such as object class or membership (Watanabe, 1992, 1996). Watanabe (1992) trained pigeons to discriminate between two bird species (pigeon vs. quail) and individual birds within a species (pigeon vs. pigeon). After successful training, bilateral lesions were a pplied to E and the birds were tested again on both tasks. The results showed that when E has been destroyed, birds can still perform the species discrimination task, but not the individual discrimination task. This finding suggests that object classificat ions such as species membership is at least in part controlled by E. Heterogeneity of E. For many years, E was considered to be an anatomically homogenous structure based on cytoarchitecture and neurochemistry Specifically, early anatomical studies showe d that E was composed of a core region (E) and an outer belt or shell region (Ep) based on connections (Ritchie & Cohen, 1977; Watanabe, Ito & Ikushima, 1985). Furthermore, projections from Rt extend directly to E, not Ep (Karten & Hodos, 1970). Ther e is now evidence from electrophysiological, hodological, and lesion studies indicating that E is composed of multiple subgroups in a similar fashion to Rt. Anterior-posterior organization of E. As described above, Rt is organized in a parallel fashion i n terms of cytoarchitecture, response characteristics, and connections. That is, processing of distinct

PAGE 24

11 visual features takes place in the anterior, central, and posterior portions of Rt. This parallel organization appears to be extended to E because the projection from Rt to E is topographically organized along the anterior posterior axis (i.e., the anterior central, and posterior E receives inputs from anterior central, and posterior Rt, respectively) (Benowitz & Karten, 1976; Nixdorf & Bischof, 1982; Laverghetta & Shimizu, 2003). This projection pattern has not been easily recognized in the past, partly due to the fact that brain sections used in many previous studies were made based on standard bird brain atlases that are significantly rotated in the anterior direction ( Laverghetta & Shimizu, 2003) (see Figure 2). Figure 2 Schematic view of pigeon brain (sagittal view). Schematic shows rotundal (Rt) projections to the core region of E. The projections from the anterior portion of Rt (Da) project to the anterior portion of E, whereas neurons in the central portion of Rt (Ce) and the posterior region of Rt (Post) project to the central and posterior regions of E, respectively. The top horizontal line represents the horizontal plane. The vertical lin e in the middle of the figure (*) represents the transverse plane typically used for cutting the pigeon brain. Note that the presumptive axis of E subdivisions is not parallel to the horizontal plane. Instead, E subdivisions appear to be arranged along an axis rotated in the anterior direction. This figure was taken from Laverghetta and Shimizu (2003).

PAGE 25

12 Early electrophysiological studies have shown that many neurons in E respond to motion stimuli. In particular, many of the cells within E fire preferentially when an object in the receptive field moves in an upward/downward or fore/aft fashion (Revzin, 1970; Kimberly, Holden, & Bamborough, 1971). A more recent study showed that a group of cells in the caudal portion of E respond to looming stimuli similar to t he cells that have been identified in Rt (Xiao, Li, & Wang, 2006). This finding suggests that looming sensitive cells in Rt send their information directly to the looming sensitive cells in E, which lends further support to the notion that information from Rt is transferred in an orderly and parallel manner to E. The majority of previous lesion studies destroyed the entire E and there were no clear data showing functional segregation among possible subdivisions. However, a study conducted by Nguyen et al., (2004) showed that cells in E can be functionally grouped along the rostral caudal axis. In their study, pigeons that received lesions to the anterior portion of E performed poorly on spatial pattern discrimination tasks, but showed no deficits on motion discrimination tasks. In contrast, pigeons which received lesions restricted to the caudal portion of E showed deficits in visual motion discrimination, but showed little or no deficits on the spatial pattern discrimination. The double dissociation found in this study suggests that visual information in the avian brain may be processed in at least two major parallel streams, similar to that which has been demonstrated in primates (DeYoe & Van Essen, 1988; Goodale & Milner, 1992).

PAGE 26

13 Internal -external organi zation of E. Various anatomical staining techniques have been used to visualize the physical characteristics of E, including its size, shape, and subgroups (Hellmann, Waldmann, & Gntrkn, 1995; Krtzfeldt & Wild, 2004; Krtzfeldt & Wild, 2005). Hellmann et al. (1995) used cytochrome oxidase (CO), a metabolic activity marker which can be used to identify highly active groups of neurons, to examine the neural activity within E. The results showed that in terms of cellular activity, E is not homogenous. High levels of CO activity were found, the medial, central and ventrolateral areas, while CO activity in the centroventral and dorsolateral areas were weak. These CO parcellations in the intact E strongly suggest that multiple cellular subdivisions exist here and perhaps these cells perform different visual functions. Krtzfeldt and Wild (2005) used tract tracing methods to visualize Rt efferents and, histochemical methods to examine the calcium -binding protein (CaBP) parvalbumin (PV) and CO activity. There wer e three main findings in this study which have important implications regarding the accurate definition of the core and shell region of E. Specifically, the results showed that 1) E can be accurately defined by the overlap of the pattern of CO activity and the rotundal efferents, 2) Ep receives few rotundal efferents, but sends most of its efferents to other areas of the ipsilateral hemisphere, including NIL, and 3) based on the pattern of PV immunoreactivity, the core region of E can be divided into two se parate regions, an internal division (Ei) and an external division (Ex) (see Figure 3A).

PAGE 27

14 Figure 3 Microphotographs showing PV and CO activity in E. A. Microphotograph showing two divisions of E, an internal division (Ei) where a great number of PV ir cells are located and an external division (Ex) which consists of fewer PV -ir cells. This photograph was taken from Krtzfeldt and Wild (2005). B. Microphotograph showing CO expression in similar location of E. Regions of weak staining can be seen in the c entroventral and dorsolateral areas (arrows). Both microphotographs are of transverse sections through the right hemisphere E. However, the definition of E based on PV immunoreactivity is not congruent with the shape and size of this structure as defined by CO immunoreactivity (see Figure 3B). Due to the importance of E for visual processing, it is necessary to clarify the anatomical boundaries of this structure. To date, there has been no comprehensive investigation to map the organization of E, combini ng these data described above. Background for Specific Aim Two Definition of NIL. In addition to E, several studies have analyzed the relationship between the development of other forebrain regions and various cognitive abilities (Timmermans, Lefebvre, Boire, & Basu, 2000; Jarvis et al., 2005). There are specific areas within the forebrain called the mesopallium and nidopallium, the size of which varies among different types of birds. Sophisticated behaviors which require cognitive flexibility or

PAGE 28

15 innova tiveness such as, using novel food foraging strategies or tool use seem to be dependent on these brain regions. Birds that possess a large mesopallium and nidopallium tend to exhibit innovative behaviors compared to birds with relatively small forebrain s (Timmermans et al., 2000). The NIL is one specific region within the nidopallium. However, there is currently very little information regarding the anatomical and functional characteristics of NIL. In the next section, I discuss what is currently known a bout the higher visual system of birds The NIL is the main target of projections from E. When Husband and Shimizu (1999) injected anterograde tract tracers into E, the majority of the efferents projecting from E terminated in Ep and Ep2 (an additional cel l layer located immediately dorsal to Ep). Most projections from Ep terminated in Ep2, which in turn, sent projections to diverse regions of the telencephalon, including the frontolateral portion of the nidopallium (NFL), area temporo -parieto occipitalis (TPO), and NIL. Figure 4 Schematic showing the spatial relationship between E and NIL. This figure was taken from Husband & Shimizu (1999). NIL is thought to be located intermediately in the anterior -posterior axis and adja cent laterally to TPO and dorsally to the mesopallium. NIL is only a part of the

PAGE 29

16 nidopallium, which includes several hodologically and functionally distinct subdivisions. However, the cytoarchitecture of the nidoplallium is rather homogeneous in general and it is difficult to differentiate among these subdivisions based solely on cell types. In particular, the size and extent of NIL with the anterior, posterior, medial, and ventral neighboring nidopallium are ambiguous and have not been examined systematica lly. NIL connections are reminiscent of NCM. Currently, there has been no report of a physiological investigation of NIL. One possible hint to the function of this structure comes from its connectional similarities to auditory telencephalic structures, s pecifically, the caudomedial neostriatum (NCM). Note that NIL receives efferents from the primary visual area E (Husband & Shimizu, 1999). In a similar fashion, the NCM receives input from a primary (L2) and secondary (L3) auditory areas (Vates, Broome, Mello, & Nottebohm, 1996). In the songbird, NCM is involved in the processing of new songs and the formation of songrelated memories. Studies have also shown that, in this brain region, certain immediate early genes (IEGs) are induced in response to complex auditory stimuli (Mello, Vicario, & Clayton, 1992; Mello & Clayton, 1995; Nastiuk, Mello, George, & Clayton, 1994). It has been suggested by some researchers (Husband & Shimizu, 1999) that NIL serves a similar function in the visual system. That is, perha ps NIL is involved with the formation of visual memories. In the above referenced songbird studies, the expression of an IEG known as zenk was examined. Z enk induction and the expression of its protein product, ZENK, have been associated with the formatio n of long -term memories and it has been used as a neuroanatomical marker for cellular activation in direct response to specific stimuli.

PAGE 30

17 Furthermore, zenk expression has been used for mapping brain regions associated with the perception of species -specific song stimuli in the songbird (Mello et al. 1992). In th at study, adult canaries and zebra finches were presented with audio playbacks of their conspecific or heterospecific song and then the distribution pattern of zenk was analyzed. The results showed t hat zenk expression in the song learning regions of the brain was much higher when the subject bird listened to a playback of a conspecific song than for a heterospecific song, suggesting that neurons in these regions w ere selective ly responding to biologi cally and socially relevant stimuli. In the current study ZENK expression was examined in NIL after presentation of species -specific visual stimuli. Background for Specific Aim Three Neuromarkers used to identify visual structures. Several neuromarkers ( some of which have been discussed already) have been used to delineate visual structures that are cytoarchitectonically difficult to define. Among them, three different classes of neuronal markers have proven to be particularly useful: 1) the CaBP, PV, 2) CO, and 3) the IEG, zenk These neuromarkers reflect directly and indirectly the cellular changes associated with neural activity, and they have been used extensively for studies of the avian neural system. Parvalbumin (PV). One class of neuromarkers are calcium -binding proteins (CaBPs), which are a large class of proteins that have been studied extensively for their roles in neuronal regulatory processes (Baimbridge, Miller, & Parkes, 1982) as well as their potential role

PAGE 31

18 in neuronal plasticity and devel opment (Celio & Heizmann, 1981; Celio, 1990). In avian literature, a specific family of CaBPs, known as the EF Hand family, includes parvalbumin (PV), calbindin D 28k (CB), and calretinin (CR). These CaBPs have been a major focus of studies because they ar e abundant in the avian fore brain (Braun, Scheich, Schachner, & Heizmann, 1985a; Braun, Scheich, Schachner, & Heizmann, 1985b; Braun, Scheich, Braun, Rogers & Heizmann, 1991). A lthough the precise function of many of these CaBPs remains elusive PV is of p articular interest in that it may play a role in neuronal plasticity because of its association with developing neurons. A transient presence of PV ir has been observed in some of the structures associated with auditory filial imprinting in birds, suggesti ng that this protein is in some way involved in neural plasticity necessary for long term imprinting behavior (Braun et al., 1991). Similarly, PV containing neurons are found in the higher visual structures in birds (Krtzfeldt & Wild, 2004, 2005; Husband & Shimizu, 1999). Often, a reduction of PV expression is observed in a particular brain region after destruction of its afferents, suggesting that the expression of PV is dependant on this afferent information (Tigges & Tigges, 1991, 1993). Such effects ha ve been observed in the avian telencephalon after lesions in Rt (unpublished observation). Cytochrome oxidase (CO). Neural activity can also be determined by examining the expression of the enzyme, CO. Like all eukaryote cells, neurons rely on cellular respiration to convert oxygen into energy. Cells that make up vital organs, such as the brain depend heavily on the process of oxidative energy to survive. Specifically, a biochemical reaction called the

PAGE 32

19 electron transport chain takes place within the mito chondria of the cell. This biochemical process of respiration produces the energy source adenosine triphosphate (ATP), which is used for several cellular processes including the sodium -potassium pump, fast axonal transport, and the synthesis of some neurot ransmitters ( Wong Riley 1989). The enzyme CO is a byproduct of the respiration process. Therefore, when activity is required of the neuron, more ATP is produced, which in turn, causes more CO to accumulate inside of the cell. Neurons containing high conce ntrations of CO relative to other neurons are considered to be more active. Histochemical procedures have been used to visualize the expression of CO, particularly as a reliable marker for long -term cellular activity ( Wong Riley 1989). Several studies ha ve shown that the expression pattern of CO can be used to identify subregions of avian brain structures, which may be difficult to differentiate cytoarchitectonically (Hellmann, Waldmann, & Gntrkn 1995; Krtzfeldt & Wild, 2004; Krtzfeldt & Wild, 2005) As mentioned earlier, CO expression revealed anatomical subdivisions of E (Hellmann et al ., 1995; Krtzfeldt & Wild, 2005). These studies show that CO is a neuromarker that can be very useful in physiological studies aimed at clarifying poorly understood brain structures Z ENK. The IEGs, such as zenk c jun and c -fos are among the first class of genes to be regulated following neuronal activation (Goelet, Castelucci, Schacher, & Kandel, 1986; Morgan & Curran, 1989; Sheng & Greenberg, 1990; Tischmeyer & Grimm, 1999; Clayton, 2000 ). Many proteins encoded by IEGs are important because they act as

PAGE 33

20 transcriptional regulators that activate late response genes thought to be involved in longterm cellular changes associated with learning and memory (Chaudhuri, 1997; Tischmeyer & Grimm, 1999). ZENK (an acronym for zif 268, egr 1, NGFI -A, and Krox 24) is the protein product of the IEG, zenk. Activation patterns of this particular IEG have been studied extensively in the avian brain, including the analysis of the so ng control system in songbirds (Mello et al., 1992; Jarvis, Mello, & Nottebohm 1995; Mello & Ribeiro, 1998) sexual behavior in quail and starlings ( Ball & Balthazart, 2001; Can, Domjan, & Delville, 2007), sexual imprinting in finches ( Lieshoff, Grosse -Op hoff, & Bischof, 2004; Huchzermeyer, Husemann, Lieshoff, & Bischof 2006), and homing behavior in pigeons (Shimizu et al., 2004) Moreover, IEGs, such as zenk can be induced by a variety of different stimuli. In addition, they can be used as endogenous mar kers of neuronal activity and, consequently, to map functional activity within the vertebrate brain (Chaudhuri, 1997; Herdegen and Leah, 1998; Tischmeyer and Grimm, 1999). Immunohistochemistry for ZENK was used in this study to identify differential brain activity in response to a biologically and socially relevant visual stimulus Although these neurochemicals are useful markers to define higher visual structures, E and NIL, it is also important to understand whether or not the normal expression of the neurochemicals is dependent on a truly visual signal per se It was hypothesized that i f the expression of these markers is indeed associated with the neuronal activity of the regions, they will be extremely valuable to understand the functional significance of these structures. However, it is also possible that these chemical markers are not correlated to neural activity in the regions. No systematic studies on this issue had previously been carried out. A major obstacle to conduct such

PAGE 34

21 studies is that they w ould require tissue samples of different brains, each of which would be placed in a different condition. While processing tissues from different brains, there are subtle, but inevitable, differences in parameters of histological procedures (e.g., tissue th ickness, incubation duration, and temperature), which make it difficult to determine whether the differences are truly due to the experimental condition. The avian brain provides the researcher with an excellent opportunity to avoid this problem and to compare the control and the experimental condition in a single brain. In birds, retinal fibers from one eye project almost completely to the contralateral hemisphere ( Shimizu & Karten 1991, 1993). In addition, unlike placental mammals, b irds do not have a m assive interhemispheric commissure, the corpus callosum. With exception of a few small commissures and decussations, information from one visual field is processed in the opposite hemisphere. Because of this neural design, it is rather easy to block the vi sual input from one eye reaching E and NIL. Despite previous research, the exact function of the interhemispheric connections is not clear. Nevertheless, they are discussed briefly here. The left and right Te O are connected by two commissures the tectal commissure (CT) and the posterior commissures (CP), which are located adjacently (Ehrlich & Saleh, 1982). Although the exact function of CT is not clear, results from electrophysiology studies indicate that this commissure plays an inhibitory role. The oth er commissure is the supraoptic decussation (DSO). The deeper layers of TeO send bilateral projections to the thalamic structure, Rt. The contralateral projection ascends to Rt through the DSO

PAGE 35

22 Rationale As described above, the anatomy of higher visual structures had not been investigated sufficiently until now although they must play essential roles in visual and cognitive abilities. There is only scarce information about the organization and subdivisions of E (Specific Aim 1), the anatomical border and organization of NIL (Specific Aim 2), and the significance of visual signal to E and NIL (Specific Aim 3). The present study capitalized on the unique organization of visual projections of birds, in which there is no corpus callosum and virtually complete decussation of retinal fibers at the optic chiasm. This preparation created a unique opportunity to examine each hemisphere separately by administering a different treatment in each visual route to the left and right hemispheres. These treatments included lesions in Rt and occlusion of eyes, which interrupted the visual signal to higher visual areas (E and NIL) at the level of the thalamus and retina, respectively. By carrying out these treatments unilaterally, E and NIL in only one hemisphere were affected by the procedures, whereas the other hemisphere was used as control. This enabled me to evaluate the effect of the procedures in one hemisphere compared to the other hemisphere within the same brain. For Specific Aim 1, detailed analyses of differential patterns of CO and PV were used to clarify the physical characteristics (shape and extent) of the subregions in E. The information provided here served as the first step towards mapping this very important region for visual processing. A detailed atlas of anatomical subdivisions within E can be used for highly specific lesions which, in turn, will allow investigators to more clearly define the functional divisions within this structure.

PAGE 36

23 For Specific Aim 2, the expression pattern of ZENK immunoreactivity was examined in the nidopallium in order to provide a clear anatomical definition of NIL based on neural activity. The information will be important for future studies of this elusive, but functionally important structure, such as physiological recording o f NIL neurons and the understanding of the role of NIL in visual and cognitive functions. For Specific Aim 3, the expression of CO, PV, and ZENK was compared between the two hemispheres after occlusion or lesion was made unilaterally. The results clarifi ed the significance of neural signal for chemical expression in higher visual areas. The information was essential to confirm whether or not these neural markers are dependent on visual signal per se. It was hypothesized that if the visual signal had signi ficant effects, E and NIL could be distinguished from the surrounding regions.

PAGE 37

24 Method Subjects In total, twelve adult white Carneaux pigeons ( Columba livia ) obtained from Palmetto Pigeon Plant, South Carolina were used in this study. The sex of each bir d was determined behaviorally before e xperimentation by observing their behavioral responses to a live conspecific, and was verified post -mortem by visual inspection of the reproductive organs A ll birds were housed individually in cages, separated by opaque partitions to restrict visual and physical interaction with other birds throughout the entire duration of the study. The vivarium was maintained on a constant 12h light: 12 h dark cycle. The birds had free access to water and a balanced diet of mixed gr ains and were weighed weekly to ensure their health and maintenance of a stable weight. All procedures outlined in this study were conducted in accordance with the NIH guidelines and approved by the University of South Florida Institutional Animal Care and Use Committee Unilateral Lesions The subjects were divided into two groups: Lesion and Occlusion groups (n = 6 each). Pigeons in the Lesion group received a unilateral electrolytic lesion to destroy the thalamic structure, Rt. For the lesion surgery, p igeons were deeply anesthetized with ketamine (40mg/kg of body weight, i.m. Aveco Co., Inc., Fort Dodge, IA ) and xylazine (10 mg/kg of body weight, i.m. Lloyd Laboratories., Shenandoah, IA ), and were then

PAGE 38

25 placed in a stereotaxic apparatus (David Kopf Instr uments, Tujunga, CA). All lesions were administered through an insulated electrode attached to a D.C. Constant Current Lesion Maker (Grass model # D.C. LM5A, Quincy, MA) at a current of 1mA for 20 seconds. The location of lesion target was specified accord ing to coordinates in the stereotaxic atlas of the pigeon brain (A: 6.10, L: 3.00, and V: 5.50; Karten & Hodos, 1967). Lesion electrodes were made by coating insect pins with epoxy (EPOXYLITE Corp., Irvine, CA) and then exposing the tip (0.5 mm). The side of lesion (left or right) was counterbalanced among subjects. After surgery, the pigeon was returned to its cage for recovery and monitored daily for seven days. Monocular Occlusions One eye of each subject in the Occlusion group was blocked by applying a small opaque cap made of polyvinyl chloride (1.5 cm internal diameter, 2 mm thickness) around the outside of one pigeon eye using a non-toxic surgical tissue adhesive (3M Vetbond). The open end was covered with opaque tape (black electrical tape) to keep light from entering. After the eye -cap was applied, the subject was returned to its cage and monitored daily for seven days to make sure this occlusion procedure did not cause any undue stress and that the eye -cap remained in place. Exposure to Live Stim ulus Following a seven-day period of post lesion recovery for the Lesion group or eye cap application for the Occlusion group, all subjects were sacrificed immediately after they were exposed to a live conspecific stimulus. The purpose of the exposure to

PAGE 39

26 c onspecifics was to maximize the ZENK expression in the telencephalon (Mello & Ribeiro, 1998; Patton, Husband, & Shimizu, 2009). The subject was first placed in a testing apparatus (90 X 90 X 90 cm) in complete darkness for approximately 12 hours (overnight ). After the period of darkness, an overhead light was turned on and a live pigeon (male) was be placed in front of the subject for 1.5 hours. Although the subject and the live conspecific stimulus did not have physical contact, the separation by a clear P lexiglas window permitted visual, auditory, and olfactory interaction. A wire -mesh cover on the top of the chamber was used to prevent flight and escape, but in no other way inhibited the birds behavior. The subject was sacrificed following the end of the stimulus exposure session. The timeframe of 1.5 hours of live stimulus exposure was to allow for peak expression of ZENK (Mello & Ribeiro, 1998). Perfusions Pigeons were deeply anesthetized via ketamine (at least 40mg/kg of body weight, i.m.) and xylazin e (at least 10 mg/kg of body weight, i.m.) and then perfused transcardially with a 0.9% saline solution followed by 4% ice -cold paraformaldehyde in phosphate buffer (0.1 M PB, pH 7.4). Brains were post fixed for 12 hours at 4 C, followed by immersion in a 30% sucrose solution for 24 hours at 4 C. Brains were then frozen in dry ice and cut in 40 m transverse sections on a sliding microtome. Sections were washed three times at 10 min. each in PB in preparation for further histological processing.

PAGE 40

27 Parvalbu min and ZENK Immunohistochem i stry For visualization of parvalbumin (PV) and ZENK, the appropriate antibodies against them were used in the primary incubation. For PV, the primary antibody was a monoclonal mouse anti -PV (dilution 1:10000; Sigma, St. Louis, MO). For ZENK, the antibody was a polyclonal rabbit anti -Egr 1 (dilution 1:10000; Santa Cruz Biotechnology, Santa Cruz, CA). The tissue sections were incubated in the respective antibody solution along with PB with 0.3% Triton X 100 at 4 e tissues were then washed in PB (3 x 10 min.) followed by 1 hour of incubation in biotinylated secondary antibodies (anti -mouse for PV, and anti -rabbit for ZENK, both 1:200, Vector Laboratories, Burlingame, CA ) and 0.3% Triton X 100 in PB for 1 hour at room temperature. The tissues were washed again in PB (3 x 10 m). Antibody binding was visualized with an avidin -biotin reagent ( 1:200 dilution of the Vectastain ABC Elite kit, Vector Laboratories) followed by incubation in a 0.025% solution of 3,3 diaminob enzidine (DAB, Polysciences, Warrington, PA) and hydrogen peroxide. Once stained, tissue sections were mounted onto glass slides and coverslipped. Cytochrome Oxidase Histology Tissues were incubated in a centrifuge tube containing 4.5 mL 0.1M PB, 0.0025g DAB, 0.0025 g cytochrome C (equine heart, Sigma C7752), and 0.2 g sucrose at 37 the dark, for 2 hours. Following staining, tissues were washed in PB (3 x 10 min.) at room temperature, mounted onto glass slides, and coverslipped.

PAGE 41

28 Additional Histology Adjacent tissue sections from each brain were also stained with cresyl violet to show major cell groups and fiber tracts for precisely locating anatomical boundaries of the relevant structures. Parvalbumin and Cytochrome Oxidase Image Capturing and An alysis All tissues stained for PV and CO were examined and photographed using a macroscope (Wild Makroskop) or a microscope (Nikon Microphot FX). The d igital images were acquired using a NIKON CCD camera (DXM 1200) or SPOT Insight QE camera (Diagnostic In struments, Sterling Heights, MI) which can be mounted on each of these scopes. The images were then loaded to Adobe Photoshop software as black and white positive images (Adobe Systems Inc., Mountain View, CA) using a Dell Optiplex PC. Brightness and contr ast were adjusted for the final images. No additional filtering or manipulation of the images was performed. The distribution of PV ir cell bodies, PV -ir neuropil, and CO staining in E was mapped by using quantitative analysis at specific tissue sections through the anterior posterior extent of E were selected. Specifically, E was examined at atlas sections A10.50, A10.00, A9.50, and A9.00 according to the pigeon stereotaxic brain atlas by Karten and Hodos (1967) Within each tissue section of E, two squa re target regions each 250 X 250 m (0.0625 mm2) were designated to measure the number of PV ir cell bodies in the inner region and the outer region of E. Figure 5 shows the location of the inner and outer target regions.

PAGE 42

29 Figure 5 Schematics sh owing the inner (triangles) and outer (circles) target regions of E where the number of PV ir cells were counted. The anterior coordinates of the sections shown in the figure from the left are: A10.50, 10.00, 9.50, and 9.00. The density analysis of PV -ir neuropil and CO staining was conducted as follows. Microphotographic images taken at constant illumination were converted to grayscale using Photoshop (CS3), and then the o ptical density values were acquired for each target region via NIH ImageJ (v. 1.41) All analyses were conducted blind to the treatment condition and hemisphere. The total density values from each subject were then used to calculate summary statistics and perform subsequent statistical tests. Similar to the procedure used to count PV -ir c ell bodies, target regions were designated within each tissue section of E. However, instead of two regions, six square target regions each 250 X 250 m (0.0625 mm2) were designated to measure the density of PV -ir neuropil and CO staining in E. Figure 6 sh ows the location of the six regions. Processed sections from all brains were under constant illumination and digitally recorded using a CCD camera (Nikon DXM1200) mounted on a macroscope (Wild M420). Using ImageJ (v. 1.41, Abramoff, Magelhaes, & Ram, 2004) the six target regions of E were delineated and calibrated according to a Kodak step tablet (Rasband, 1997 2009). The relative density measurements were compared to examine the regional differences within E. The protocol for this density analysis can be found in Appendix A.

PAGE 43

30 b c f d e b c f d e a b c f d e a b c f d e a a Figure 6 Schematic s showing the six target regions within E where the density of PV -ir neuropil and CO staining was measured in lower magnification (top) and higher magnification (bottom). The anterior coordinates of the sections shown in the figure from the left are: A10.50, 10.00, 9.50, and 9.00. The PV ir cell bodies were counted as follows. A signal was counted as a cell if staining within the neurons revealed a complete soma perimeter, and it was c learly differentiated from background. Any potential bias was eliminated by the blind counting procedure. Each neuron was marked with a paint spot using CANVAS (v.11.0, Deneba Systems) graphic software and the spots were counted with ImageJ software. The t otal cell counts from each subject were then used to calculate summary statistics and perform subsequent statistical tests. ZENK Image Capturing and Analysis For all tissues stained for ZENK, low -power microphotographs were taken of the entire forebrain i n the left and right hemisphere simultaneously using a motorized Nikon TE 2000U inverted microscope to capture ZENK ir patterns across both hemispheres simultaneously. Based on the images, the presumptive borders of NIL were identified

PAGE 44

31 visually by comparin g the quantity of ZENK ir cells in the left and right hemispheres. This procedure was based on the assumptions that 1) NIL is relatively homogeneous in terms of ZENK expression and that 2) the level of ZENK expression in NIL was significantly reduced by le sions in the ipsilateral Rt and by occlusions of the contralateral eyes. Because the exact size and shape of NIL was not known in advance, it was necessary to be conservative in assuming which group of ZENK ir cells belonged to NIL. To do this, NIL was ten tatively defined to be located dorsolateral to the caudal end of E, and ventromedial to the tractus arcopallium dorsalis (DA). Figure 7 is an illustration, which shows the location of the putative NIL. For quantitative analysis, the central portion of NIL was suggested to be at A7.00 and A6.00 (Husband & Shimizu, 1999) in each of which a square target region of 750 X 750 m (0.5625 mm2) was used to count the number of ZENK ir cells. Figure 7 shows schematic drawings illustrating the areas for quantitative analysis of the ZENK expression. Figure 7 Schematic showing two transverse sections of the avian brain where NIL is thought to be located. The dotted line shows the region thought to be NIL and the square target region shows where ZENK ir cells were cou nted. The elongated hatched region dorsolateral to the putative NIL is the tractus arcopallium dorsalis (DA). The locations (anterior posterior coordinates) shown in the figure from the left are: A7.00 and 6.00.

PAGE 45

32 The number of ZENK -ir neurons was counted u sing the following method. When ZENK proteins are stained, the chromatin remains in the nucleus of the neuron and not in the dendrites or axons. Therefore, ZENK protein expression is morphologically highly similar from cell to cell; ZENK -ir cells can be di fferentiated from artifact staining and counted quickly. Using existing optical analysis functions in ImageJ, Mahmud (2008 ) developed a new image analysis technique. This new method of analysis called, A New Digital Imaging Protocol for Signal Detection and Distribution Analysis in Histological Samples uses a Find Maxima function to identify and label ZENK -ir cells as localized spikes of chromatic value on an image. Thus, the function works by recognizing any pixel with a high chromatic value relative to its neighboring pixels. Specifically, the Find Maxima function was used to detect signals based on spikes of chromatic values present in the tissue sample. The concept is logical, since signals are traditionally localized dark spots against a lighter backgr ound, which is the exact definition of Maxima according to ImageJ. In addition, since the function only looks at local color values, it is not subject to overall optical inconsistencies such as uneven staining, lighting or weak signal strength. For instanc e, staining patterns often clump up near the edges of samples masking signals in a generally dark color that visual and even threshold -based analysis can not analyze. The threshold function actually views the entire region as one dark signal due to its rel ative darkness in comparison with the rest of the slide instead of a region of multiple signals. Once the Find Maxima function was executed, the image was reproduced with only the maxima highlighted. Then, a function known as Dilation was used on this repr oduced image, which simulated a form of signal extraction. The concept is based on

PAGE 46

33 the fact that once the maxima are dilated, the signals have a finite black area. Therefore, the more signals that are present in any given space, the more area is colored bl ack in those squares. Once the squares are averaged, darker squares can be correlated with higher signal count. The protocol is included in Appendix B. Lesion Reconstructions Rotundal lesion reconstruction was carried out on a series of standard plates de rived from the atlas of the pigeon brain of Karten and Hodos (1967). Gliosis and necrosis in the diencephalon was recorded. The extent of each lesion was determined from measurements of reconstructions made with camera and graphics software (CANVAS. v.11.0). Statistical Analyses For Specific Aim One, cell count data were statistically analyzed using 4 -way (t reatment x h emisphere x r egion of E x l ocation) mixed analysis of variance (ANOVA), where the h emisphere ( c ontrol versus e xperimental), r egion of E ( i n ner versus o uter), and l ocation (anterior -posterior coordinates) were repeated (within subject) measures and t reatment ( l esion versus o cclusion) was an independent variable (see Figure 5). Similar tests were conducted for analysis of PV -ir neuropil and CO staining intensity where the h emisphere ( c ontrol versus e xperimental), r egion within E ( six different target regions of E), and l ocation (anterior -posterior coordinates) were repeated (within subject) measures and t reatment (l esion versus o cclusion) was an independent variable (see Figure 6)

PAGE 47

34 For Specific Aim Two, the differences in the mean number of ZENK ir cells in NIL were analyzed by conducting a 2 x 2 factorial mixed model ANOVA where h emisphere ( c ontrol versus e xperimental) was a repeated (within sub ject) measure and t reatment ( l esion versus o cclusion) was an independent variable. The statistical designs conducted for the first two specific aims also addressed the questions of Specific Aim Three. For each of these statistical analyses, Significant 3 -w ay ANOVAs (alpha = 0.05) were followed up with Fishers LSD (protected t) tests

PAGE 48

35 Results In the present study, the brain tissues from the same animals were processed and analyzed for, not only one specific Aim, but sometimes for other Aims as well. In or der to avoid presenting redundant data, the results section is organized by presenting the lesion reconstruction data (Specific Aims 1, 2, and 3), followed by data from PV immunohistocehmistry (Specific Aims 1 and 3), CO -histochemistry (Specific Aims 1 and 3), and ZENK immunohistochemistry (Specific Aims 2 and 3). Lesion Reconstructions Overall, six pigeons received a unilateral lesion to the thalamic nucleus, Rt (PG236, PG239, PG234, PG259, PG254, and PG47). In each of the lesion cases, Rt was either most ly or completely destroyed in one hemisphere. In some cases the lesion site was large enough to include neighboring structures, such as n. triangularis (T). Below is a brief description of each lesion case starting from the smallest lesions to the most ext ensive lesions.

PAGE 49

36 Figure 8. Schematics showing the location of the nucleus rotundus (Rt). Only the left hemisphere of each anterior -posterior coordinate is shown here. Individual l esion r econstruction c ases. Figure 9 illustrates the degree of lesion dama ge for subject PG236. Overall, the lesion was relatively small, extending just from anterior coordinate 7.00 through 6.50. The lesion was mainly localized in the anterior portion of Rt leaving intact a large portion of the posterior sections (Figure 9 D H) Figure 9 Rt damage reconstruction for subject PG236. Ablated or damaged tissue in the Rt is indicated by solid black areas.

PAGE 50

37 Figure 10 illustrates the degree of lesion damage for subject PG239. The damage overall was moderate, extending from A7.00 th rough A6.25. The lesions were seen in the rostral portion of Rt, but left all of the caudal sections intact (Figure 10 E H). Damage to other structures was restricted to some moderate damage in the adjacent T (Figure 10 C D ). Figure 10. Rt damage reconst ruction for subject PG239. Figure 11 illustrates the degree of lesion damage for subject PG234. The damage overall was more substantial compared the previous two cases. The lesions included the entire rostral portion of Rt, leaving only a small portion of the caudal sections (Figure 11 G H). Damage to other structures was restricted to some moderate damage in the adjacent T (Figure 11 C -F).

PAGE 51

38 Figure 11. Rt damage reconstruction for subject PG234. Figure 12 illustrates the degree of lesion damage for sub ject PG259. The damage overall was also substantial from A7.00 through A5.50. The lesions included the entire rostral portion of Rt, sparing the most caudal sections (Figure 12 G H). Damage to other structures was restricted to some moderate damage in the adjacent T (Figure 12 C -F). Figure 12. Rt damage reconstruction for subject PG259.

PAGE 52

39 Figure 13 illustrates the size of the lesion for subject PG254. The damage overall was substantial extending the entire anter ior posterior axis of Rt. Damage to other str uctures was restricted to some moderate damage in the adjacent T (Figure 1 3 C-F). Figure 13. Rt damage reconstruction for subject PG254. Figure 14 illustrates the degree of lesion damage for subject PG47. The damage overall was also substantial. It extended from anterior coordinate A7.00 through A5.25 and was centered in Rt.

PAGE 53

40 Figure 14. Rt damage reconstruction for subject PG47. PV I mmunohistochemistry In total, the brain tissues of eight birds ( four in the unilateral lesion condition and four in th e monocular occlusion condition) were processed to detect PV ir cell bodies and neuropil in E. Below are microphotographs showing E on the control and experimental hemispheres at each of the four different anterior -posterior locations for all of the birds.

PAGE 54

41 Individual PV c ases. PG234 (Unilateral Lesion) PV expression in E (Figure 15). Figure 15. Microphotographs of PG234 showing the distribution pattern of PV in E after unilateral lesion. For this case, the left Rt was destroyed. Scale bar = 500 m.

PAGE 55

42 PG 259 (Unilateral Lesion) PV expression in E (Figure 16). Figure 16. Microphotographs of PG259 showing the distribution pattern of PV in E after unilateral lesion. For this case, the right Rt was destroyed. Scale bar = 500 m.

PAGE 56

43 PG254 (Unilateral Lesion) P V expression in E (Figure 17). Figure 17. Microphotographs of PG254 showing the distribution pattern of PV in E after unilateral lesion. For this case, the right Rt was destroyed. Scale bar = 500 m.

PAGE 57

44 PG47 (Unilateral Lesion) PV expression in E (Figure 18). Figure 18. Microphotographs of PG47 showing the distribution pattern of PV in E after unilateral lesion. For this case, the right Rt was destroyed. Scale bar = 500 m.

PAGE 58

45 PG253 (Monocular Occlusion) PV expression in E (Figure 19). Figure 19. Mi crophotographs of PG253 showing the distribution pattern of PV in E after monocular occlusion. For this case, the left eye was occluded. Scale bar = 500 m.

PAGE 59

46 PG263 (Monocular Occlusion) PV expression in E (Figure 20). Figure 20. Microphotographs of PG263 showing the distribution pattern of PV in E after monocular occlusion. For this case, the left eye was occluded. Scale bar = 500 m.

PAGE 60

47 PG256 (Monocular Occlusion) PV expression in E (Figure 21). Figure 21. Microphotographs of PG256 showing the distri bution pattern of PV in E after monocular occlusion. For this case, the left eye was occluded. Scale bar = 500 m.

PAGE 61

48 PG260 (Monocular Occlusion) PV expression in E (Figure 22). Figure 22. Microphotographs of PG260 showing the distribution pattern of PV in E after monocular occlusion. For this case, the left eye was occluded. Scale bar = 500 m.

PAGE 62

49 Number of PV ir n eurons in E. Eight pigeons were used for Specific Aim One. Four birds received unilateral lesions to the thalamic nucleus, Rt (PG234, PG259, PG254, and PG47) and four birds received monocular occlusions (PG253, PG263, PG256, and PG260). A factorial mixed design ANOVA was conducted to determine whether or not there was a significant difference in the mean number of PV ir cells in E based on: treatm ent (lesion versus occlusion), hemisphere (control versus experimental), region of E (inner versus outer), and location (anterior -posterior coordinates). In terms of simple main effects, the results showed that there was a significant difference in the mea n number of PV ir cells depending on the region of E ( i nner versus o uter) F (1, 6) = 119.43 p < 0. 001. Specifically, the mean number of PV -ir cells was significantly higher for the inner region of E ( M = 38.00) compared to the outer region of E ( M = 8.48; s ee Figure 23). 0 10 20 30 40 50 I nne r O ut e r P V i r C e l l s i n E R e gi on of E Figure 23. Graph showing the mean number of PV -ir cells counted in the i nner and o uter regions of E, across t reatment ( l esion versus o cclusion), h emisphere ( c ontrol versus e xperimental), and l ocation (anterior posterior coordinates). Mean values and standard error are shown.

PAGE 63

50 No other simple main effects were significant. There was no significant main effect for treatment (l esion versus o cclusion) F (1, 6) = 0.01, p = 0. 920, hemisphere F (1, 6) = 39.38, p = 0. 160, or location (anterior -posterior coordinates) F (3 8 ) = 2.3 1 p = 0. 111. In terms of interaction effects, the R egion of E x L ocation interaction was significant F (3 18) = 3.59, p = 0. 034; see Figure 24). 0 10 20 30 25 35 A 9. 00 A 9. 50P V i r C e l l s i n E L o c a t i on ( a n t e r i or pos t e r i o r c oor di na t e s ) 15 5 A 10. 00 A 10. 50 L e s i on O c c l us i on Figure 24. Graph showi ng the mean number of PV ir cells in both regions of E (inner versus outer) at each of location (anterior posterior coordinates). Mean values and standard error are shown. No other two -way interaction effects were significant. Specifically, R egion of E x T reatment F (1, 6) = 3.98, p = 0. 093, H emisphere x T reatment F (1, 6) = 0.62, p = 0. 460, Region of E x H emisphere F (1, 6) = 0.8 7 p = 0. 388, and L ocation x T reatment F (3 18) = 3.07, p = 0.054). Note, that this last interaction effect approached significance

PAGE 64

51 The following three -way interaction effects were significant, R egion of E x H emisphere x T reatment F (1, 6) = 6.77, p = 0. 041, Region of E x H emisphere x L ocation F (3 18) = 4.21, p = 0. 020, and R egion of E x T reatment x L ocation F (3 18) = 3.38, p = 0. 0 41. Figure 25 graphically depicts the R egion of E x H emisphere x T reatment interaction. Specifically, note that the number of PV ir cells in E differs as a function of treatment condition and region. 0 10 20 40 30 50 L e s i on O c c l us i onP V i r C e l l s i n E T r e a t m e nt C on di t i on I n n e r R e g i o n C o n t r o l I n n e r R e g i o n E x p e r i m e n t a l O u t e r R e g i o n C o n t r o l O u t e r R e g i o n E x p e r i m e n t a l Figure 25. Graph showing the mean number of PV -ir cells in E for each t reatment condition ( l esion versus o cclusion), h emisphere ( c ontrol versus e xperimental), and region of E (i nner versus o uter) across l ocation (anterior -posterior coordinates). Mean values and standard error are shown. The Region of E x Hemisphere x Location interaction effect can be seen in Figure 26. Specifically, note that the number of PV ir cells in E differs as a function of hemisphere and region.

PAGE 65

52 0 10 20 40 30 50 A 9. 00 A 10. 50P V i r C e l l s i n E L o c a t i on ( a n t e r i or pos t e r i o r c oor di na t e s ) I n n e r R e g i o n C o n t r o l I n n e r R e g i o n E x p e r i m e n t a l O u t e r R e g i o n C o n t r o l O u t e r R e g i o n E x p e r i m e n t a l 60 A 9. 50 A 10. 00 Figure 26. Graph showing the m ean number of PV -ir cells by h emisphere (control versus experimental), r egion of E (inner versus outer), and l ocation (anterior -posterior coordinates) across both t reatment conditions (lesion versus occlusion) Mean values and standard error are shown. T he Region of E x Treatment x Location interaction effect can be seen in Figure 2 7. 0 10 20 40 30 50 A 9. 00 A 10. 50P V i r C e l l s i n E L oc a t i o n ( a nt e r i or pos t e r i or c oor d i na t e s ) L e s i o n I n n e r R e g i o n L e s i o n O u t e r R e g i o n O c c l u s i o n I n n e r R e g i o n O c c l u s i o n O u t e r R e g i o n 60 A 9. 50 A 10. 00 Figure 27. Graph showing the mean number of PV -ir cells by r egion of E (inner versus outer), t reatment (lesion versus occlusion), and l ocation ( anterior -posterior coordinates) across both hemispheres Mean values and standard error are shown.

PAGE 66

53 The following interaction effects were not significant: H emisphere x L ocation x T reatment F (3 18) = 0.13, p = 0.936, R egion of E x H emisphere x L ocation x T reatment F (3 18) = 0.25, p = 0.856. Regarding cell morphology, a cursory glance showed that no obvious physical differences in cell size and shape were observed between the inner region of E and those the outer region. However, it was apparent that there were differences in the density of PV fiber connections (neuropil). Therefore, an optical density analysis procedure was used to measure the density level of several different regions within E. Density of PV ir n europil in E. In order to determine if the density of PV ir neuropil in E varied as a function of treatment condition, hemisphere, regions within E, and anterior -posterior location, a factorial mixed design ANOVA was conducted. The between subjects variable was treatment condition (lesion versus occlusion) and the repeated measures were hemisphere (control versus experimental), region within E (sample areas a f ), and location (anterior posterior coordinates: A10.50, 10.00, 9.50, 9.00), There were two significant main effects First, the main eff ect of region within E (a f ) F (5 30) = 12.32, p < 0. 001 was significant (Figure 28) Post hoc comparisons using the Fisher LSD test revealed that the mean PV ir neuropil density values of specific regions within E were different from one another. Region a (M = 0.63, SD = 0.11) had significantly more PV -ir neuropil than that found in region e (M = 0.53, SD = 0.11; t (30) = 2.614, p = .014, twotailed). Also, region d (M = 0.65, SD = 0.14) had significantly more PV -ir neuropil than that found in region b (M = 0.56, SD = 0.11; t (30) = 2.567, p

PAGE 67

54 = .015, twotailed), region e (M = 0.53, SD = 0.11; t (30) = 3.217, p = .003, twotailed), and region f (M = 0.56, SD = 0.11; t (30) = 2.565, p = .015, twotailed). A summary of these comparisons can be found in Table 1. In addition, the main effect of treatment (lesion versus occlusion) was significant F (1, 6) = 11.08, p = 0.0 16; see Figure 29). b0 0. 2 0. 4 0. 6 0. 5 0. 7P V i r N e ur o pi l i n E R e gi on W i t hi n E 0. 8 c d e f 0. 1 0. 3 a Figure 28. Graph showing the mean density value of PV -ir neuropil in the six target region s within E across t reatment condition, h emisphere, and l ocation. Mean values and standard error are shown. Table 1 Pairwise Comparisons of Regions Within E Region Within E M SD a 0.63 A 0.11 b 0.56 A 0.11 c 0.59 A 0.12 d 0.65A 0.14 e 0.53 B 0.11 f 0.56B 0.11 Table 1. Means with the same letter in their superscripts do not differ significantly from one another according to a Fishers LSD test.

PAGE 68

55 0 0. 2 0. 4 0. 6 0. 5 0. 7P V i r N e ur op i l i n E T r e a t m e nt 0. 8 0. 1 0. 3 L e s i o n O c c l u s i o n Figure 29. Graph showing the mean density value of PV -ir neuropil for both t reatment conditions ( l esion versus o cclusion) across h emisphere, r egion within E, and l ocation. There was not a significant effect for hemisphere (c ontrol versus e xperimental) F (1, 6) = 4.1 9 p = 0. 087 or location (anterior -posterior coordinates) F (3 18) = 0.08, p = 0. 967. However, there were significant interaction effects for T reatment x H emisphere F (1, 6) = 16.25, p = 0. 007. Specifically, the mean density value of PV ir neuropil was greater for the control hemisphere than the experimental hemisphere fo r the lesion treatment condition. There was not a difference between the control and experimental hemispheres for the monocular occlusions (see Figure 30).

PAGE 69

56 0 0. 5 0. 7D e ns i t y of P V i r N e ur op i l T rea t ment 0. 9 0. 1 0. 3 C o n t r o l E x p e r i m e n t a l Occlusio n L esio n 0. 2 0. 4 0. 6 0. 8 Figure 30. Graph showing the mean density value of PV -ir neuropil after unilateral lesion and monocular occlusion across the different r egion s within E and all l ocation s (anterior posterior coordinates). Mean values and standard error are shown. Fig ure 31. further shows the density differences of six regions within E. 0 10 20 40 30 50 bP V i r N e ur opi l i n E T r e a t m e nt C ondi t i o n L e s i o n C o n t r o l L e s i o n E x p e r i m e n t a l O c c l u s i o n C o n t r o l O c c l u s i o n E x p e r i m e n t a l a c d e f Figure 31. Graph showing the mean density value of PV -ir neuropil after unilateral lesion and monocular occlusion for each h emisphere at each of the different r egions within E. Mean values and standard error are shown.

PAGE 70

57 In a ddition, the L ocation x Region within E interaction effect was significant F (15, 90) = 2.2 5 p = 0. 010; see Figure 32). b a c d e f0P V i r N e u r opi l i n E L oc a t i on ( a nt e r i or pos t e r i or c oor di na t e s ) 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 b a c d e f b a c d e f b a c d e f0 A 9 0 0 A 9 5 0 A 1 0 0 0 A 1 0 5 0 Figure 32. Graph showing the mean density value of PV -ir neuropil at each of the r egions with in E and all four l ocations (anterior posterior coordinates) across t reatment condition and h emisphere. Mean values and standard error are shown. The following interactions were not significant : Location x T reatment F (3 18 ) = 0.63, p = 0.605, Region within E x T reat ment F (5 30) = 0.46, p = 0.801, H emisphere x location F (3 18) = 0.19, p = 0.900, H emisphere x L ocation x T reatment F (3 18) = 0.49, p = 0.693, H emisphere x R egion within E F (5 30) = 0.17, p = 0.971, H emisphere x Region within E x T reatment F (5 30) = 0. 62, p = 0.682, L ocation x Region within E x T reatment F (15, 90) = 0.99, p = 0.471, H emisphere x L ocation x R egion within E F (15, 90) = 0.52, p = 0.922, and H emisphere x L ocation x R egion within E x T reatment F (15, 90) = 1.38, p = 0.174.

PAGE 71

58 CO H istochemistr y In total, ten cases were processed for CO. However, the tissue in some of these cases was not suitable for analysis. Therefore, the brain tissues of seven birds (three in the unilateral lesion condition and four in the monocular occlusion condition) were processed to detect CO staining in E. Below are microphotographs showing E on the control and experimental hemispheres at each of the four different locations (anterior -posterior coordinates) for all of the birds.

PAGE 72

59 Individual CO c ases. PG234 (Unilateral L esion) CO density (Figure 33). Figure 33. Microphotographs of PG234 showing the distribution pattern of CO in E after unilateral lesion. For this case, the left Rt was destroyed. Scale bar = 500 m.

PAGE 73

60 PG259 (Unilateral Lesion) CO density (Figure 34). Figure 34. Microphotographs of PG259 showing the distribution pattern of CO in E after unilateral lesion. For this case, the left Rt was destroyed. Scale bar = 500 m.

PAGE 74

61 PG254 (Unilateral Lesion) CO density (Figure 35). Figure 35. Microphotographs of PG 254 showing the distribution pattern of CO in E after unilateral lesion. For this case, the right Rt was destroyed. Scale bar = 500 m.

PAGE 75

62 PG264 (Monocular Occlusion) CO (Figure 36). Figure 36. Microphotographs of PG264 showing the distribution pattern of CO in E after monocular occlusion. For this case, the right eye was occluded. Scale bar = 500 m.

PAGE 76

63 PG263 (Monocular Occlusion) CO (Figure 37). Figure 37. Microphotographs of PG263 showing the distribution pattern of CO in E after monocular occlusion. For this case, the left eye was occluded. Scale bar = 500 m.

PAGE 77

64 PG256 (Monocular Occlusion) CO (Figure 38). Figure 38. Microphotographs of PG256 showing the distribution pattern of CO in E after monocular occlusion. For this case, the left eye was occl uded. Scale bar = 500 m.

PAGE 78

65 PG260 (Monocular Occlusion) CO (Figure 39). Figure 39. Microphotographs of PG260 showing the distribution pattern of CO in E after monocular occlusion. For this case, the right eye was occluded. Scale bar = 500 m. Density of CO staining in E In order to determine if the density of CO activity normally present in E varied as a function of t reatment condition, h emisphere, r egion with in E, and l ocation (anterior posterior coordinates), a factorial mixed design ANOVA was conduct ed. The between subjects variable was treatment c ondition ( l esion versus o cclusion) and the repeated

PAGE 79

66 measures were hemisphere ( c ontrol versus e xperimental), region within E (a f ), and location (anterior -posterior coordinates: A10.50, 10.00, 9.50, 9.00). There was a significant main effect for region within E (a f ) F (5, 25) = 6.51 p = 0. 001 indicating that there are differences in the mean density value of CO staining in the six different target regions within E across treatment condition, hemisphere, a nd l ocation (anterior posterior coordinates ; see Figure 40). Post hoc comparisons using the Fisher LSD test revealed that the mean CO staining density value of specific regions within E were different from one another. Region a ( M = 0.88, SD = 0.20) had si gnificantly more CO staining than that found in region e (M = 0.62, SD = 0.22; t (25) = 2. 432, p = .0 22, two tailed ) Also, region e ( M = 0.62, SD = 0.22) had significantly less CO staining than that found in region c ( M = 0.85, SD = 0.23; t (25) = .188, p = .0 38, two tailed ) and region f (M = 0.84, SD = 0.27; t (25) = 2.094, p = .0 46, two tailed ). A summary of these comparisons can be found in Table 2 b0 0. 2 0. 4 0. 8 0. 7 0. 9C O S t a i ne d C e l l s i n E R e gi on W i t hi n E 1. 0 c d e f 0. 1 0. 3 a 0. 5 0. 6 Figure 40. Graph showing the mean density value of CO activity in the six targ et r egions with in E across t reatment condition, h emisphere, and l ocation (anterior posterior coordinates). Mean values and standard error are shown.

PAGE 80

67 Table 2 Pairwise Comparisons of Regions Within E Region Within E M SD a 0.88 A 0.20 b 0.74A 0.18 c 0.85A 0.23 d 0.82 B 0.29 e 0.62 B 0.22 f 0.84 A 0.27 Table 2. Means with the same letter in their superscripts do not differ significantly from one another according to a Fishers LSD test. The following main effects were not significant: treatme nt (l esion versus o cclusion) F (1 5 ) = 0.14, p = 0. 726, hemisphere ( c ontrol versus e xperimental) F (1 5 ) = 1.60, p = 0. 262, and location F (3 15) = 0.9 6 p = 0. 437. However, t here was a significant H emisphere x T reatment interaction effect F (1 5 ) = 11.14, p = 0. 021), meaning that the difference in CO activity between hemispheres was dependent on the treatment condition (see Figure 41). 0 0. 5 0. 7C O S t a i ne d C e l l s i n E T r e a t m e nt 0. 9 0. 1 0. 3 C o n t r o l E x p e r i m e n t a l Occlusio n L esio n 0. 2 0. 4 0. 6 0. 8 1. 0 Figure 41. Graph showing mean density value of CO activity after unilateral lesion and mono cular occlusion across all six regions within E and across all four l ocations (anterior posterior coordinates). Mean values and standard error are shown.

PAGE 81

68 Furthermore, the H emisphere x T reatment x R egion within E interaction effect was significant F (5 25) = 7.84, p < 0. 001). Figure 42 shows the density differences of CO in six different regions. 0C O S t a i ne d C e l l s i n E Re g i on W i t h i n E 0 2 0 4 0 6 0 8 1 1 2a b c d e f L e s i o n C o n t r o l L e s i o n E x p e r i m e n t a l O c c l u s i o n C o n t r o l O c c l u s i o n E x p e r i m e n t a l Figure 42. Graph showing the mean density value of CO activity for each h emisphere, t reatment condition, and the six target r egion s within E across l ocation (anterior -posterior coordinates). Mean values and standard error are shown. The following interaction effects were also significant: location had an interaction effect with region within E F (15, 75) = 2.34, p = 0. 008) (see Figure 43), and hemisphere interacted with region within E F (15, 25) = 3.165, p = 0. 024) (see Figure 44). No other interactions were significant.

PAGE 82

69 0C O S t a i ne d C e l l s i n E L oc a t i on ( a nt e r i or pos t e r i or c oor di na t e s 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 b a c d e fA 9 0 0 A 9 5 0 A 1 0 0 0 A 1 0 5 0b a c d e f b a c d e f b a c d e f Figure 43. Graph showing the mean density value of CO activity at each of the six reg ions within E and at all l ocations (anterior posterior coordinates) across t reatment condition and h emisphere. Mean values and standard error are shown. Below, is a graph showing the mean density values of CO and PV together. Notice that the staining patt ern is more differential for CO than for PV. A 9. 00 A 10. 50 A 10. 00 A 9. 50 0. 4 1. 2 1. 0 0. 8 0. 6 0. 2 0 a f e d c b a f e d c b a f e d c b a f e d c b C O S t a i ni ng P V i r N e ur opi l C O a nd P V D e ns i t y i n E Figure 44. Graph showing the mean density value of CO and PV activity at each of the six r egions within E and at all l ocations (anterior posterior coordinates) across t reatment c ondition and h emisphere. Mean values and standard error are shown.

PAGE 83

70 0 0. 4 0. 8 1. 2 1. 0 a bP V i r C e l l s i n E R e gi on W i t hi n E 0. 6 0. 2 c d C ont r ol E x pe r i m e nt a l e f Figure 45. Graph showing the mean density value of CO activity at each of the six regions within E for both hemispheres across treatment condition (lesion ver sus occlusion) and location (anterior -posterior coordinates) Mean values and standard error are shown. ZENK I mmunohistochemistry Eleven pigeons (five in the unilateral lesion condition and six in the monocular occlusion condition) were used to visualize ZENK ir cells of NIL. Below, composite microphotographs (consisting 42 original photos) of ZENK -ir for each hemisphere are presented for individual cases. They are accompanied by corresponding images showing ZENK ir distribution patterns visualized by the Maxima transformation method. Based on these digitized images, the putative NIL is specified as the region surrounded by DA and the caudal end of E which extended from A 7.00 to A6.00.

PAGE 84

71 Individual ZENK c ases. PG234 (Unilateral Lesion) ZENK ir cells in NI L (Figure 46). Figure 46. Microphotographs and corresponding transformed images of PG234 showing the distribution pattern of ZENK in the telencephalon after unilateral lesion. The putative NIL is specified by dotted lines in the digitized images. For th is case, the left Rt was destroyed. Scale bar = 1 mm.

PAGE 85

72 PG236 (Unilateral Lesion) ZENK ir cells in NIL (Figure 47). Figure 47. Microphotographs of PG236 showing the distribution pattern of ZENK in the telencephalon after unilateral lesion. The putative N IL is specified by dotted lines in the digitized images. For this case, the right Rt was destroyed. Scale bar = 1 mm.

PAGE 86

73 PG239 (Unilateral Lesion) ZENK ir cells in NIL (Figure 48). Figure 48. Microphotographs of PG239 showing the distribution pattern of Z ENK in the telencephalon after unilateral lesion. The putative NIL is specified by dotted lines in the digitized images. For this case, the left Rt was destroyed. Scale bar = 1 mm.

PAGE 87

74 PG254 (Unilateral Lesion) ZENK ir cells in NIL (Figure 49). Figure 49. Microphotographs of PG254 showing the distribution pattern of ZENK in the telencephalon after unilateral lesion. The putative NIL is specified by dotted lines in the digitized images. For this case, the right Rt was destroyed. Scale bar = 1 mm.

PAGE 88

75 PG259 (Uni lateral Lesion) ZENK ir cells in NIL (Figure 50). Figure 50. Microphotographs of PG259 showing the distribution pattern of ZENK in the telencephalon after unilateral lesion. The putative NIL is specified by dotted lines in the digitized images. For this case, the left Rt was destroyed. Scale bar = 1 mm.

PAGE 89

76 PG253 (Monocular Occlusion) ZENK ir cells in NIL (Figure 51). Figure 51. Microphotographs of PG253 showing the distribution pattern of ZENK in the telencephalon after monocular occlusion. The putative NIL is specified by dotted lines in the digitized images. For this case, the left eye was occluded. Scale bar = 1 mm.

PAGE 90

77 PG264 (Monocular Occlusion) ZENK ir cells in NIL (Figure 52). Figure 52. Microphotographs of PG264 showing the distribution pattern of ZENK in the telencephalon after monocular occlusion. The putative NIL is specified by dotted lines in the digitized images. For this case, the right eye was occluded. Scale bar = 1 mm.

PAGE 91

78 PG170 (Monocular Occlusion) ZENK ir cells in NIL (Figure 53). Fig ure 53. Microphotographs of PG170 showing the distribution pattern of ZENK in the telencephalon after monocular occlusion. The putative NIL is specified by dotted lines in the digitized images. For this case, the left eye was occluded. Scale bar = 1 mm.

PAGE 92

79 P G263 (Monocular Occlusion) ZENK ir cells in NIL (Figure 54). Figure 54. Microphotographs of PG263 showing the distribution pattern of ZENK in the telencephalon after monocular occlusion. The putative NIL is specified by dotted lines in the digitized ima ges. For this case, the left eye was occluded. Scale bar = 1 mm.

PAGE 93

80 PG256 (Monocular Occlusion) ZENK ir cells in NIL (Figure 55). Figure 55. Microphotographs of PG256 showing the distribution pattern of ZENK in the telencephalon after monocular occlusion. The putative NIL is specified by dotted lines in the digitized images. For this case, the left eye was occluded. Scale bar = 1 mm.

PAGE 94

81 PG260 (Monocular Occlusion) ZENK ir cells in NIL (Figure 56). Figure 56. Microphotographs of PG260 showing the distributi on pattern of ZENK in the telencephalon after monocular occlusion. The putative NIL is specified by dotted lines in the digitized images. For this case, the right eye was occluded. Scale bar = 1 mm.

PAGE 95

82 Number of ZENK ir n eurons in NIL In order to determine if the number of ZENK -ir neurons in NIL varied as a function of treatment condition and hemisphere a 2 x 2 mixed groups factorial ANOVA was conducted. The between subjects variable was the treatment with two levels ( l esion and o cclusion) and the within subject measure was hemisphere ( c ontrol and e xperimental). Data from two anterior -posterior locations (A7.00 and A6.00) were combined for the analysis since ZENK distribution patterns were similar between them. Figure 57 shows the mean number of ZENK -ir neur ons found in the NIL target regions after unilateral lesion and monocular occlusion. The results showed that there was a main effect for hemisphere F (1 9 ) = 94.14, p < 0. 001) meaning that regardless of the treatment (lesion or occlusion), there was a diff erence between the mean number of ZENK -ir cells in the control NIL versus the experimental NIL. There was not a significant effect for treatment F (1 9 ) = 0. 50, p = 0. 498). The H emisphere x T reatment interaction effect was not significant either F (1 9 ) = 1.30, p = 0. 283) 0 400 600 800 700 900 L e s i onZ E N K i r C e l l s i n N I L T r e a t m e nt 500 100 O c c l us i on C ont r ol E xpe r i m e nt a l 200 300 Figure 57. Graph showing the mean number of ZENK ir cells counted in the control and experimental hemispheres for both treatment groups (lesion and occlusion). Mean value s and standard error are shown.

PAGE 96

83 Disc ussion The specific aims of this study were to: 1) identify distinct anatomical subregions within the primary visual structure (E) in the telencephalon, 2) define the size and extent of another telencephalic structure NIL, and 3) determine the extent to wh ich activity in these two telencephalic structures is dependent on neural input and visual signal. Each of these specific aims was addressed by analyzing the distribution patterns of various cellular activity markers. Overall, these activity patterns showe d that visual cell groups in the telencephalon were differentially affected not only between the various treatment conditions (lesions versus occlusions), but also within these conditions. In particular, PV and CO facilitated the identification of specific subregions in E (Aim One). Furthermore, mapping the cellular activity of ZENK in the telencephalon made it possible to determine the size and shape of NIL (Aim Two). Lesion and occlusion procedures had differential effects on the expression of PV ir and C O staining in E, but not the ZENK expression in NIL (Aim Three). These results provided new information about the anatomical definitions and suggest possible functional segregations of the telencephalic visual areas. F or Specific Aim One, there were sign ificantly more PV -ir cell bodies in the inner region of E compared to the outer region. These results are congruent with the findings by Krtzfeldt and Wild (2005), who showed that E can be separated based on the levels of PV ir expression While their con clusion was based on non quantitative visual observations t he present study confirmed their observation based on quantitative analysis.

PAGE 97

84 The statistical results showed that the interaction effect between Region within E and Location was significant, sugges ting that the inner vs. outer regional differentiation varied by the location along the anterior -posterior axis. Furthermore, the three -way interaction analyses showed that Region variables interacted with 1) Hemisphere and Treatment, 2) Hemisphere and Location, and 3) Treatment and Location variables. These results suggest that the regional differences of PV ir cells were more or less affected by different variables, including the anterior vs. poster ior locations, lesion vs. occlusion conditions, and targe ted vs. control hemispheres. However, in all experimental conditions analyzed in the study, the inner region had consistently more PV ir cells than the outer region. Therefore, the PV -ir expression difference between the inner and outer regions is consiste nt regardless of these variables. The present results found that cells in the inner and outer regions were not obviously different in cell size and type. Thus, the difference of PV ir cells between inner and outer regions is primarily in density, not in morphology. In terms of the PV -ir neuropil, the results were slightly different from the cell body pattern. When the PV ir neuropil density was compared between the outer regions (b and c; mean = 0.576) and inner regions (e and f ; M = 0.5 5 ), no clear differe nces were observed, unlike the PV -ir cell bodies. The results showed that the expression patterns of PV ir cell bodies and neuropil in E are not identical, suggesting that they were controlled by different mechanisms. The regional difference s among the si x areas within E w ere significant as well as the Region within E x Location (anterior -posterior coordinates) interaction. In particular, the ventrolateral E (region d ) tended to have a higher density of PV ir neuropil than other regions except the most ven tromedial area (region a ) which was also high in PV ir

PAGE 98

85 (Figure 28). This difference between regions was mo re obvious in the anterior locations than in the posterior locations (Figure 32). It is possible that the intensity difference among areas is related to a topographical organization of the Rt E projection pattern. For instance, the region d may receive distinct projections from a specific part of Rt. However, further studies are necessary to understand the significance of the specific subregion since li ttle is known about the internal organization of E, including this region. Region a appeared to be distinct in terms of PV ir neuropil in that it did not appear to be as affected by either treatment condition compared to the other five regions (Figure 28) It is likely that this region receives input from some location other than Rt. A study by Gamlin and Cohen (1986) used anterograde autoradiographic and retrograde pathway tracing techniques to show an additional projection from the optic tectum to the nu cleus dorsolateralis posterior of the thalamus (DLP). One of the main findings of this study showed that anterograde tracers injected into the caudal portion of DLP (DLPc) project to a discrete region of the ipsilateral telencephalon. This cytoarchitectura lly distinct cell group matches with region a in the current study. Thus, it is likely that the lesion to Rt had little effect on region a due to the likelihood that this region is receiving DLP terminations. As for CO staining, the overall pattern of the current study agrees with the results of Hellmann et al. (1995) The six different regions of E examined in Hellmann et al. (1995) roughly corresponded to the six regions that were analyzed in the present study. The current findings are similar to theirs in the respect that many of these areas showed similarly intense staining patterns. Furthermore, both studies found that the ventro intermedial area (region e) showed a low level of CO staining compared to the other

PAGE 99

86 areas. It is possible that the low CO in tensity in this region is associated with the fact that massive thalamo telencephalic projection fibers are passing through this region. Thus, CO activity may not be expressed much in these passing fibers in the region e. Hellmann et al. (1995) found that the dorsolateral area (region c ) also had low CO staining intensity whereas the present study did not show such a pattern. A closer look at this region suggests that the dorsolateral border of E in the present study was defined more conservatively than th e previous study, which encroached into a further dorsal area including dense efferent fiber passages of E. In the Hellmann et al. (1995) study, the CO activity in this region was low because this dorsolateral region included passing fibers whereas the pre sent study did not. As in the PV ir neuropil, no clear difference was found for CO staining patterns to separate between the inner and outer regions. The results of CO staining patterns, together with those of PV ir cell and neuropil patterns, suggest tha t E consists of multiple distinct subregions. Specifically, the present study concludes that the density of PV ir cell bodies can be used to clearly distinguish the inner and outer subregions within E. The PV ir neuropil and CO activity patterns show that further subregions exists within the inner and outer regions, indicating a complex heterogeneity within E. The current findings will be used to further refine the identity of E subdivisions, providing a map of these subregions that may, in turn, reflect ph ysiologically and functionally distinct cell groups within E. For Specific Aim Two, the expression of protein product of zenk was examined in the nidopallium by comparing the hemispheres in both treatment conditions (lesions and occlusions). There was a si gnificant reduction of ZENK ir cell bodies on NIL in the

PAGE 100

87 experimental hemisphere compared to control side. Based on the differential expression of ZENK, it was possible for the first time to visualize the location of NIL. To accomplish the visualization, the newly developed image analysis was used (Mahmud, 2008), and, the distribution patterns of ZENK positive cells were more accurately identified in low -power ima ges than any previous analysis Based on these images, the present study showed that the nidop allial area dorsocaudal to E, ventromedial to DA, in the control hemisphere had a significantly high density of ZENK positive cells (Figures 46 56) compared to the corresponding area in the control side (Figure 57). The putative NIL is 3 4 mm in the an terior posterior axis, 2 2.5 mm in the dorsal ventral axis, and 1 1.5 mm along the lateral -medial axis. This definition is rather conservative because the borders with the surrounding regions are difficult to differentiate cytoarchitectonically. The ZENK expression might also be more heterogeneous within NIL than assumed in the present study. Thus, the real NIL may even larger than the putative NIL discussed in the study. Although the exact functional significance of NIL remains unclear, the fact that Z ENK expression in NIL was significantly influenced by disrupting visual input suggests that this structure is important for higher order visual processing. The expression of zenk gene and its protein product have been used for identifying higher -order audi tory and vocal brain areas in songbirds. For example, the level of the mRNA transcribed from zenk is expressed strongly in certain structures within the specific auditory areas in the telencephalon during song acquisition by juvenile birds ( Jin & Clayton, 1997) and the perception of song by adults (Mello et al., 1992; Mello & Clayton, 1995 ; Jarvis et al., 1995). Z enk genes are also known to play a major role in the consolidation of longterm

PAGE 101

88 auditory memories in these higher auditory regions (Tischmeyer & G rimm, 1999; Davis, Bozon, & Laroche, 2003). Based on the similarities with the higher auditory areas in songbirds in terms of the connection and ZENK expression patterns, it is possible that NIL is similarly involved in the higher order visual learning and memory of biologically relevant events such as encounters with potential mates. If so, the results suggest that biologically relevant auditory and visual information is processed in a similar manner in the avian telencephalon. The finding will be of impor tance for understanding the general neural principles of sensory information processing. Using the n ew digital imaging protocol for signal detection and distribution analysis (Mahmud 2008) the results showed that ZENK immunoreactivity was only found in th e nucleus region of each positive cell and thus characterized by a morphologically highly uniform appearance from cell to cell. The new protocol took advantage of this uniformity and succeeded in accurately generating a low -power graphical representation o f signal density patterns. This method proved to be extremely useful for the present study, and should be valuable for analyses in similar quantitative studies, especially when the target signals are relatively uniform in terms of size and morphology. In contrast to NIL, much fewer ZENK positive cells were observed in E, as well as other primary sensory areas such as Field L and n. basolateralis. The lack of ZENK expression in the primary sensory areas is consistent with previous studies (Ruscio & Adkins -Regan, 2004; Thode, Bock, Braun, & Darlison, 2005).

PAGE 102

89 Regarding specific Aim Three, the results showed that Treatment (lesions vs. occlusions) had significant effects for PV -ir neuropil and CO staining in E b ut no differential effects were found for ZENK ir in NIL. As for PV -ir, the numbers of cell bodies were not affected by either lesion or occlusion treatment (Figure 25). However, lesions caused a significant reduction of PV ir neuropil density in the experimental hemisphere, but not in the control hemisphere. This effect was not observed after occlusions. As for the CO patterns, the results also showed that lesions caused a significant reduction of CO staining in the experimental hemisphere, but not in the control hemisphere. This effect was not observed after occlusions. In addition, there were differential staining patterns for CO at each of the six different regions of E. These regional differences were significant and were dependent on the location along the anterior posterior axis and hemisphere. Why did occlusions not have a significant effect compared to lesions in the PV ir neuropil and CO staining? In the lesion condition, visual processing of E in the experimental hemisphere was disrupted by destroying the unilateral Rt and thus destroying the entire thalamic input to E. In the occlusion condition, visual processing of E in the experimental side was interrupted by occluding the contralateral eye, but the thalamic connection from Rt to E remained physically intact. Thus, E could still receive visua l input from the ipsilateral eye via the inter -hemispheric connections. It is most likely that the ipsilateral input played a role to maintain the PV expression in E for the occlusion condition, whereas no such input existed in the lesion condition.

PAGE 103

90 Howev er, there are at least two other possible explanations for the results. It is possible that PV in E was transported from cell bodies of Rt to terminals in E, and thus Rt lesions, but not monocular occlusions, caused the reduction effect. However this possi bility is unlikely because no PV was observed in cell bodies of Rt although there were abundant PV -ir neuropil. In terms of CO activity in E, they might be present mainly in terminals of Rt fibers, but not intrinsic fibers in E. Thus, lesions of the thalam ic fibers caused more significant effects in the CO activity than occlusions. Another possibility is that lesions had more acute and quick impacts than occlusions in the PV ir and CO in E. It is possible that the difference in the effect of treatment condi tion might be similar if the occlusion period was longer than one week. That is, perhaps the visual information must be blocked for a longer period of time in order to significantly alter the levels of PV and CO. Could the size of the lesion affect the pa ttern of PV immunoreactivity and CO staining in E ? It is possible, but not likely, due to the fact that the four cases analyzed for this specific aim (PG234, PG254, PG259, PG47) received extensive lesions covering most, if not all, of Rt (see Figures 11 14). As for ZENK -ir in NIL, both the lesion and occlusion treatments resulted in significant reductions of ZENK expression in NIL. The results are similar to a previous study ( Hara, Kubikova, Hessler, & Jarvis, 2009) in which monocular occlusions ha d been p erformed to investigate visual telencephalic brain structures. The present study is the first to use thalamic lesions as well as monocular occlusions to show similar results in ZENK ir in the visual telencephalon. Because there were no clear treatment effe cts, the results provide three important insights about the high visual telencephalic structures in

PAGE 104

91 birds. First, the similar findings in both treatment conditions suggest that visual input to NIL from the ipsilateral thalamus through E is essential for th e expression of ZENK -ir cells in NIL. Under the assumption that ZENK is involved in the long-term memory formation, this indicates that the possible learning and memory function of NIL is dependent upon the ipsilateral thalamic input. Second, the presence of interhemispheric connections in the occlusion treatment did not contribute to the maintenance of a high level of ZENK ir in NIL. The fact that occlusions caused the same pattern of ZENK -ir as lesions suggests that information via any interhemispheric connections did not trigger sufficiently the ZENK expression in NIL. Finally, the results suggest that ZENK expression is more sensitively affected by the event that occurred just prior to the treatment compared to PV ir or CO -staining, which were not signif icantly influenced by the occlusion procedure. It is also important to note that the occlusion procedure in the most peripheral organ affected the protein expression in one of the highest visual areas in the avian visual system. In this sense, the analysis of ZENK distribution is one of the most powerful techniques to study molecular consequences of real time events on different brain regions, even in the higher telencephalic regions as reported here. In conclusion, the present study revealed the complex he terogeneity of the avian visual telencephalon, which had been considered to be a rather homogenous entity. This anatomical finding provides new information about how visual processing is carried out in the non -mammalian cerebrum, and will be the foundation for future anatomical, physiological, and functional studies. In particular, the present study suggests that subdivisions of E are organized in a topographic manner, and a more detailed topographical map of E, as well as NIL, will be useful in order to de lineate the whole

PAGE 105

92 picture of the avian visual telencephalon. The significance of visual information exchange between the left and right hemispheres needs to be further explored as well. In the avian brain, there are multiple inter -hemispheric connections i n the tectofugal visual route, including the tectal commissure, supraoptic decussation, and extra telencephalic descending routes (see Figure 1). The role of these visual connections must be studied individually and compared with the mammalian counterparts These studies will eventually reveal the general and specific neural rules associated with visual processing in vertebrates.

PAGE 106

93 References Abramoff, M.D., Magelhaes, P.J., & Ram, S.J. (2004). Image processing with ImageJ. Biophotonics International 11, 36 42. Baimbridge, K.G., Miller, J.J., & Parkes, C.O. (1982). Calcium -binding protein distribution in the rat brain. Brain Research 239, 519525. Ball, G.F. & Balthazart, J. (2001). Ethological concepts revisited: Immediate early gene induction in resp onse to sexual stimuli in birds. Brain, Behavior, and Evolution, 57, 252270. Benowitz, L.I. & Karten, H.J. (1976). Organization of the tectofugal pathway in the pigeon: A retrograde transport study. Journal of Comparative Neurology 167 503520. Besset te, B.B. & Hodos, W. (1989). Intensity, color, and pattern discrimination deficits after lesions of the core and belt regions of the ectostriatum Visual Neuroscience 2 27 34. Braun, K., Scheich, H., Braun, S., Rogers, J.H., & Heizmann, C.W. (1991). Par valbumin -, calretinin and calbindin D28kimmunoreactivity and GABA in a forebrain region involved in auditory filial imprinting. Brain Research, 539, 31 44. Braun, K., Scheich, H., Schachner, M., & Heizmann, C.W. (1985). Distribution of parvalbumin, cyto chrome oxidase activity and [14] C 2 deoxyglucose uptake in the brain of the zebra finch. I. Auditory and vocal motor systems. Cell Tissue Research 240, 101 115. Braun, K., Scheich, H., Schachner, M., & Heizmann, C.W. (1985). Distribution of parvalbumin, cytochrome oxidase activity and [14] C 2 deoxyglucose uptake in the brain of the zebra finch. II. Visual system. Cell Tissue Research 240, 117127. Brown, S.D. & Dooling, R.J. (1992). Perception of conspecific faces by budgerigars (Melopsittacus undulat us ): I. natural faces. Journal of Comparative Psychology 106, 203216. Brown, S.D. & Dooling, R.J. (1993). Perception of conspecific faces by budgerigars (Melopsittacus undulatus ): II. Synthetic models. Journal of Comparative Psychology, 107, 48 60.

PAGE 107

94 Bu tler, A.B. & Hodos, W. (1996). Comparative vertebrate neuroanatomy: Evolution and Adaptation. New York: Wiley Liss. Can, A., Domjan, M., & Delville, Y. (2007). Sexual experience modulates neuronal activity in male Japanese quail. Hormones and Behavior 52, 590599. Celio, M.R. (1990). Calbindin D 28k and parvalbumin in the rat nervous system. Neuroscience 35, 375475. Celio, M.R. & Heizmann, C.W. (1981). Calcium binding protein parvalbumin as a neuronal marker. Nature 293, 300302. Chaudhuri, A. (1997). Neural activity mapping with inducible transcription factors. Neuroreport, 8 iii -vii. Clayton, D.F. (2000). The genomic action potential. Neurobiology of Learning and Memory 74, 185216. Cook, R.G., Cavoto, K.K., & Cavoto, B.R. (1995). Same/differen t texture discrimination and concept learning in pigeons. Journal of Experimental Psychology: Animal Behavior Processes, 21 253260. Cook, R., Levision, D.G., Gillet, S.R., & Blaisdell, A.P. (2005). Capacity and limits of associative memory in pigeons. P sychonomic Bulletin & Review 12, 350358. Davies, M.N.O. & Green, P.R. (1990). Optic flow -field variables trigger landing in hawk but not in pigeons. Naturwissenschaften, 77, 142144. Davis, S., Bozon, B., & Laroche, S. (2003). How necessary is the acti vation of the immediate early gene zif268 in synaptic plasticity and learning? Behavioural Brain Research 142, 17 30. DeYoe, E.A. & Van Essen, D.C. (1988). Concurrent processing streams in monkey visual cortex. Trends in Neurosciences 11, 219226. Enge lage, J, Bischof H J (1993). The Organization of the tectofugal pathway in birds: A comparative review. In H.P. Zeigler & H J. Bischof (Eds.), Vision, brain, and behavior in birds (pp 137158). Cambridge, MA: MIT. Ehrlich, D. & Saleh, C.N. (1982). Composit ion of the tectal and posterior commissures of the chick. Neuroscience Letters 33, 115121. Farroni, T., Massaccesi, S., Menon, E., & Johnson, M.H. (2006). Direct gaze modulates face recognition in young infants. Cognition, 102, 396404. Frost, B.J. & Na kayama, K. (1983). Single visual neurons code opposing motion independent of direction. Science 220, 744745.

PAGE 108

95 Frost, B.J. & Sun, H. (1997). Visual motion processing for figure/ground segregation, collision avoidance, and optic flow analysis in the pigeon. In M.V. Srinivasan, & S. Venkatesh (Eds.), From living eyes to seeing machines (pp. 80 103). New York: Oxford University Press. Gamlin, P.D.R. & Cohen, D.H. (1986). A second ascending visual pathway from the optic tectum to the telencephalon in the pigeo n (C olumba livia ). The Journal of Comparative Neurology, 250, 296 310. Gauthier, I. & Logothetis, N.K. (2000). Is face recognition not so unique after all? Cognitive Neuropsychology 17, 125142. Goelet, P., Castellucci, V.F., Schacher, S., & Kandel, E.R (1986). The long and the short of long-term memory a molecular framework. Nature 322, 419422. Goodale, M.A. & Milner, A.D. (1992). Separate visual pathways for perception and action. Trends in Neurosciences 15, 20 25. Granda, A.M. & Yazulla, S. (197 1). The spectral sensitivity of single units in the nucleus rotundus of pigeon, Colubmia livia. Journal of General Physiology, 57, 363384. Gntrkn, O. (2000). Sensory physiology: vision. In: G.C. Whittow (Ed.), Sturkies avian physiology 5th edn (pp. 1 19). New York: Academic. Gntrkn, O, Miceli D, Watanabe M (1993) Anatomy of the avian thalamofugal pathway. In H.P. Zeigler & H J. Bischoff (Eds.), Vision, brain, and behavior in birds (pp. 115136). Cambridge, MA: MIT. Hara, E., Kubikova, L., Hessler N.A., & Jarvis, E.D. (2009). Assessing visual requirements for social context dependent activation of the songbird song system. Proceedings of the Royal Society B 276, 279289. Hellmann, B. & Gntrkn, O. (1999). Visual -field specific heterogeneity wi thin the tecto rotundal projection of the pigeon. European Journal of Neuroscience, 11, 26352650. Hellmann, B., Waldmann, C., & Gntrkn, O. (1995). Cytochrome oxidase activity reveals parcellations of the pigeons ectostriatum. Neuroreport 6 881885. Herdegen, T. & Leah, J.D. (1998). Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Research. Brain Research Reviews 28, 370490. Herrnstein, R.J., & Loveland, D.H. (1964). Complex visual concept in the pigeon. Science 146 549551.

PAGE 109

96 Herrnstein, R.J., Loveland, D.H., & Cable, C. (1976). Natural concepts in pigeons. Journal of Experimental Psychology: Animal Behavior Processes 2 285302. Hodos W. (1993). The visual capabilities of birds. In H.P. Zeigler & H. J. Bischof (Eds.), Vision, brain, and behavior in birds (pp. 77 98). Cambridge, MA: MIT Press. Hodos, W. & Bonbright, J.C., Jr. (1974). Intensity difference thresholds in pigeons after l esions of the tectofugal and thalamofugal visual pathways to the telencephalon. Journal of Comparative Physiology and Psychology 87, 10131031. Hodos, W. Macko, K.A., & Bissette, B.B. (1984). Near -field acuity changes after visual system lesions in pigeo ns, II. Telencephalon. Behavioral Brain Research, 13, 15 30. Hodos, W., Weiss, S.R.B., & Bessette, B.B. (1986). Size -threshold changes after lesions of the visual telencephalon in pigeons. Behavioral Brain Research, 28, 203214. Hodos, W., Weiss, S.R.B., & Bessette, B.B. (1988). Intensity difference thresholds after lesions of ectostriatum in pigeons. Behavioral Brain Research, 30, 43 53. Huber, L. (2001). Visual categorization in pigeons. In: R.G. Cook (Ed.), Avian Visual Cognition. Retrieved from Tufts University, Psychology Department Web Site: www.pigeon.psy.tufts.edu/avc/huber/ Huchzermeyer, C., Husemann, P., Lieshoff, C., & Bischof, H.J. (2006). ZENK expression in a restricted forebrain ar ea correlates negatively with preference for an imprinted stimulus. Behavioural Brain Research, 171 154161. Husband, S.A. & Shimizu, T. (1999). Efferent projections of the ectostriatum in the pigeon ( Columba livia ). The Journal of Comparative Neurology 406, 329345. Jarvis, C.D. (1974). Visual discrimination and spatial localization deficits after lesions of the tectofugal pathway in pigeons. Brain, Behavior, and Evolution, 9 195212. Jarvis, E.D., Mello, C.V., & Nottebohm, F. (1995). Associative lea rning and stimulus novelty influence the songinduced expression of an immediate early gene in the canary forebrain. Learning & Memory 2 6280.

PAGE 110

97 Jarvis, E.D., Gunturkun, O., Bruce, L., Csillag, A. Karten, H., Kuenzel, W., Medina, L., Paxinos, G., Pe rkel, D.J., Shimizu, T. Streidter, G., Wild, J.M., Ball, G.F., Dugas Ford, J., Durand, S.E., Hough, G.E., Husband, S. Kubikova, L. Lee, D.W., Mello, C.V., Powers, A., Siang, C., Smulders, T.V., Wada, K., White, S.A., Yamamoto, K., Yu, J., Reiner, A., & But ler, A. (2005). Avian brains and a new understanding of vertebrate brain evolution. Nature Reviews Neuroscience, 6 151159. Jin, H. & Clayton, D.F. (1997). Localized changes in immediate early gene regulation during sensory and motor learning in zebra fi nches. Neuron, 19, 10491059. Karten, H.J., Cox, K., & Mpdozis, J. (1997). Two distinct populations of tectal neurons have unique connections within the retinotectorotundal pathway of the pigeon (Columba livia ). Journal of Comparative Neurology 26, 4494 65. Karten, H.J. & Hodos, W. (1967). A Stereotaxic Atlas of the Brain of the Pigeon (Columba livia). Baltimore: Johns Hopkins Press. Karten, H.J. & Hodos, W. (1970). Telencephalic projections of the nucleus rotundus in the pigeon (Columba livia). Journal of Comparative Neurology 140, 35 51. Kertzman, C. & Hodos, W. (1988). Size difference thresholds after lesions of thalamic visual nuclei in pigeons. Visual Neuroscience 1 83 92. Krtzfeldt, N.O.E. & Wild, J.M. (2004). Definition of novel connections of the entopallium in the zebra finch ( Taeniopygia guttata). The Journal of Comparative Neurology 468 452465. Krtzfeldt, N.O.E. & Wild, J.M. (2005). Definition of novel connections of the entopallium in the pigeon ( Columba livia ). The Journal of Com parative Neurology 490 40 56. Kimberly, R.P., Holden, A.L., & Bamborough, P. (1971). Response characteristics of pigeon forebrain cells to visual stimulation. Vision Research 11, 475478. Laverghetta, A.V. & Shimizu, T. (2003). Organization of the ect ostriatum based on afferent connections in the zebra finch ( Taeniopygia guttata). Brain Research, 963, 101112. Lieshoff, C., Grosse -Ophoff, J. & Bischof, H.J. (2004). Sexual imprinting leads to lateralized and non lateralized expression of the immediate early gene zenk in the zebra finch brain. Behavioural Brain Research, 148 145155. Lubow, R.E. (1974). High-order concept formation in the pigeon. Journal of the Experimental Analysis of Behavior, 21, 475483.

PAGE 111

98 Macko, K.A., & Hodos, W. (1984). Near -field acuity after visual system lesions in pigeons: I. Thalamus. Behavioural Brain Research, 13, 1 14. Mahmud, A. (2008). A new digital imaging protocol for signal detection and distribution analysis in histological samples (Unpublished honors thesis) Univer sity of South Florida, Tampa, FL. Maxwell, J.H. & Granda, A.M. (1979). Receptive fields of movement -sensitive cells in the pigeon thalamus. In A.M. Granda and J.H. Maxwell (Eds.), Neural Mechanisms of Behavior in the Pigeon (pp. 177 197). New York :Plenum Press. Mello, C.V. & Clayton, D.F. (1995). Diffeprential induction of the ZENK gene in the avian forebrain and song control circuit after metrazole induced depolarization. Journal of Neurobiology 26, 145 161. Mello, C.V. & Ribeiro, S. (1998). ZENK prot ein regulation by song in the brain of songbirds. Journal of Comparative Neurology 393 426438. Mello, C.V., Vicario, D.S., & Clayton, D.F. (1992). Song presentation induces gene expression in the songbird forebrain. Neurobiology 89, 68186822. Morgan, J. I. & Curran, T. (1989). Stimulus transcription coupling in neurons: Role of cellular immediate -early genes. Trends in Neurosciences 12, 459462. Nastiuk, K.L., Mello, C.V., George, J.M., & Clayton, D.F. (1994). Immediate -early gene responses in the avian song control system: cloning and expression analysis of the canary c jun cDNA. Brain Research. Molecular Brain Research, 27, 299309. Nguyen, A.P., Spetch, M.L., Crowder, N.A., Winship, I.R., Hurd, P.L., & Wylie, D.R. (2004). A dissociation of motion and spatial -pattern vision in the avian telencephalon: Implications for the evolution of visual streams. Journal of Neuroscience 24, 49624970. Nixdorf, B.E. & Bischof, H.J. (1982). Afferent connections of the ectostriatum and visual wulst in the zeb ra finch ( Taeniopygia guttata castanotis Gould) an HRP study. Brain Research 248, 9 17. Pasternak, T. & Hodos, W. (1977). Intensity difference thresholds after lesions of the visual wulst in pigeons. Journal of Comparative Physiology and Psychology 91, 485497. Patton, T.B., Husband, S.A., & Shimizu, T. (2009). Female stimuli trigger gene expression in male pigeons. Social Neuroscience 4 2839.

PAGE 112

99 Patton, T.B., Szafranski, G., & Shimizu, T. (2010). Male pigeons react differentially to altered facial fea tures of female pigeons. Behaviour 147, 757773. Rasband, W.S. (19972009). ImageJ [Internet] US National Institutes of Health, Bethesda. Optical Density Calibration Retrieved from: ht tp://rsb.info.nih.gov/ij/docs/examples/calibration/ Revzin, A.M. (1970). Some characteristics of wide-field units in the brain of the pigeon. Brain Behavior and Evolution, 3 195204. Revzin, A.M. (1979).Functional localization in the nucleus rotundus. In A.M. Granda and J.H. Maxwell (Eds.), Neural Mechanisms of Behavior in the Pigeon (pp. 165175). New York:Plenum Press. Ritchie, T.C. & Cohen, D.H. (1977). The avian tectofugal visual pathway: Projections of its telencephalic target the ectostriatal co mplex. Social Neuroscience Abstracts 3 94. Ruscio, M.G. & Adkins -Regan, E. (2004). Immediate early gene expression associated with induction of brooding behavior in Japanese quail. Hormones and Behavior 46, 19 29. Sheng, M. & Greenberg, M. E. (1990). The regulation and function of c -fos and other immediate early genes in the nervous system. Neuron, 4 477485. Sherry, D.F. (1984). Food storage by black -capped chickadees: Memory for the location and contents of caches. Animal Behaviour 32, 451464. S herry, D.F., Krebs, J.R., & Cowie, R.J. (1981). Memory for the location of stored food in marsh tits. Animal Behaviour 29, 12601266. Shettleworth, S.J. (1983). Memory in food hoarding birds. Scientific American 248, 102110. Shettleworth, S.J. & Kreb s, J.R. (1986). Stored and encountered seeds: A comparison of two spatial memory tasks in marsh tits and chickadees. Journal of Experimental Psychology: Animal Behavior Processes 12, 248257. Shimizu, T. (1998). Conspecific recognition in pigeons ( Columba livia ) using dynamic video images. Behaviour 133, 43 53. Shimizu T (2001) Evolution of the forebrain in tetrapods. In: G. Roth & M.F. Wulliman (Eds.), Brain E volution and Cognition (pp. 135184). New York :Wiley/Spektrum.

PAGE 113

100 Shimizu, T. & Bowers, A.N. (1 999). Visual circuits of the avian telencephalon: Evolutionary implications. Behavioural Brain Research, 98, 183191. Shimizu, T., Bowers, A.N., Budzynski, C.A., Kahn, M.C., & Bingman, V.P. (2004). What does a pigeon ( Columba livia ) brain look like durin g homing? Selective examination of ZENK expression. Behavioral Neuroscience 118, 845851. Shimizu, T. & Karten, H.J. (1991). Central visual pathways in reptiles and birds: Evolution of the visual system. In J.R. CronlyDillon & R.L. Gregory (Eds.), Evolu tion of the Eye and Visual System, Volume 2 (pp. 421441). Boca Raton: CRC Press. Shimizu, T, Karten, H.J. (1993) The avian visual system and the evolution of the neocortex. In H.P. Zeigler & H J. Bischof (Eds.), Vision, brain, and behavior in birds (pp 1 37158). Cambridge, MA: MIT. Shimizu, T., Patton, T.B., & Szafranski, G. (2008) Evolution of the visual system in birds. In M. D. Binder, N. Hirokawa, U. Windhorst, & M. C. Hirsch (Eds.), Encyclopedic Reference of Neuroscience. Heidelberg Germany: Springer Thode, C., Bock, J., Braun, K. & Darlson, M.G. (2005). The chicken immediate early gene zenk is expressed in the medo -rostral neostriatum/hyperstriatum ventrale a brain region involved in acoustic imprinting, and is upregulated after exposure to an auditory stimulus. Neuroscience, 130, 611617. Tigges, M. & Tigges, J. (1991). Parvalbumin immunoreactivity of the lateral geniculate nucleus in adult rhesus monkey s after monocular eye enucleation. Visual Neuroscience 6 375382. Tigges, M. & Tigges, J. (1993). Parvalbumin immunoreactivity in the lateral geniculate nucleus of rhesus monkeys raised under monocular and binocular deprivation conditions. Visual Neuros cience 10, 10431053. Timmermans, S., Lefebvre, L., Boire, D., & Basu, P. (2000). Relative size of the hyperstriatum ventrale is the best predictor of feeding innovation rate in birds. Brain, Behavior and Evolution, 56, 196203. Tischmeyer, W. & Grimm, R. (1999). Activation of immediate early genes and memory formation. Cellular and Molecular Life Sciences 4 564574. Vander Wall, S.B. & Balda, R.P. (1977). Coadaptation of the Clarks nutcracker and the pinyon pine for efficient seed harvest and disper sal. Ecological Monographs 47, 89111.

PAGE 114

101 Vates, G.E., Broome, B.M., Mello, C.V., & Nottebohm, F. (1996). Auditory pathways of caudal telencephalon and their relation to the song system of adult male zebra finches ( Taenopygia guttata). Journal of Comparati ve Neurology 366, 613 642. Vaughan, W. & Greene, S.L. (1984). Pigeon visual memory capacity. Journal of Experimental Psychology: Animal Behavior Processes 10, 256271. Wang, Y. & Frost, B.J. (1992). Time to collision is signaled by neurons in the nucl eus rotundus of the pigeon. Nature (London), 356, 236238. Wang, Y.C., Jiang, S. & Frost, B.J. (1993). Visual processing in pigeon nucleus rotundus: luminance, color, motion, and looming subdivisions. Visual Neuroscience 10 21 30. Watanabe, S. (1992). E ffect of lesions in the ectostriatum and Wulst on species and individual discrimination in pigeons. Behavioural Brain Research, 49, 197203. Watanabe, S. (1996). Effects of ectostriatal lesions on discriminations of conspecific, species and familiar objec ts in pigeons. Behavioural Brain Research, 81, 183 188. Watanabe, M., Ito, H., Ikushima, M. (1985). Cytoarchitecture and ultrastructure of the avian ectostriatum: afferent terminals from the dorsal telencephalon and some nuclei in the thalamus. The Journal of Comparative Neurology 236 241257. Watanabe, S., Lea, S.E.G., & Dittrich W.H. (1993). What can we learn from experiments on pigeon concept discrimination? In H.P. Zeigler & H J. Bischof (Eds.), Vision, brain, and behavior in birds (pp 351 376). Cam bridge, MA: MIT. Watanabe, S., Sakamoto, J., Masumi, W. (1995). Pigeons discrimination of painting by Monet and Picasso. Journal of Experimental Analysis of Behavior 63, 165-174. Wong -Riley, M.T.T. (1989). Cytochrome oxidase: An endogenous metabolic ma rker for neuronal activity. Trends In Neurosciences 12, 94 101. Xiao, Q., Li, D.P., & Wang, S.R. (2006). Looming -sensitive responses and receptive field organization of the telencephalic neurons in the pigeon. Brain Research Bulletin 68, 322328.

PAGE 115

102 Ap pendices

PAGE 116

103 Appendix A A protocol for conducting optical density analysis. Photographs All photographs were taken using Wild Makroscope. Apo lens Magnification: 25 Aperture: half Light source: 9 V Protocol 1 No Normalization Method Open macrophoto of section in ImageJ In Image menu, change Type to -bit In Analyze menu, select Calibrate In Calibrate window, select Open In file menu, select OD calibration file (I/Willottlab/NIAD/CO/OD analysis/OD calibration); change Function to Rodbard, and for Unit type O.D. -Use Rectangular selections tool to make a 250 X 250 m (0.0625 mm2) for measuring the different regions within E In Analyze menu, select Measure The measurement Results window data can be saved in Excel for mat (.xls)

PAGE 117

104 Appendix B A New Digital Imaging Protocol for Signal Detection and Distribution Analysis Protocol Instructions: 1. Open ImageJ 2. Open Digital Photograph of Stained Slide 3. If Noise Tolerance Calibration is necessary follow Step 4 if not go to step 12 4. Zoom in on picture until signals are visible 5. Using the Rectangle Select Tool select a segment within the image 6. Manually count and make note of every signal 7. Select Find Maxima under Binary sub -menu within Process menu 8. Sele ct Preview Point Selection 9. Enter various numbers within the Noise Tolerance field until every signal manually counted is correctly labeled (with no extras or none missing) make note of the value 10. Close Maxima Window 11. Deselect Selection (sing le -click any part of picture) 12. Select Find Maxima under Binary sub -menu within Process menu 13. Enter value in Noise Tolerance based on the previous calibration step 14. Select Light Background if slide has a lighter background compared to the signa ls 15. Select Single Points under the Output Type drop-down menu 16. Click OK 17. Select Dilate under Binary sub -menu within Process menu 18. Repeat step 17 until dots are the same size as a typical signal 19. Save the Maxima image as either TIFF or JPEG A New Digital Imaging Protocol 22 20. Open GIMP 21. Open Maxima image from step 19 22. Select Mosaic under Distorts sub -menu within Filters menu 23. Set and Select following parameters: Square tiling, 0 tile height, 1.0 tile spacing, 1.0 tile neatness 0 light direction, 0 color variation, checked antialiasing, checked color averaging, unchecked tile splitting, unchecked pitted surfaces and checked FG/BG lighting (make sure foreground and background colors are black and white respectively). 24. Set Ti le Size 25. Click OK 26. Select RGB under Mode sub -menu within Image menu 27. Select gradient in gradient menu of toolbox 28. Select Gradient Map under Map sub -menu within Color menu

PAGE 118

About the Author Tadd Patton earned his bachelors degree of Psychology from the University of South Florida in 1994. After taking some graduate level coursework at Georgia State University and working in private industry, he made the decision to further his education of the brain. This decision led Tadd Patton back to his Alma mater to complete his doctoral degree in Psychology with a special emphasis in neuroscience. Throughout his graduat e training, Tadd has taught several courses, including Psychology of Learning and Physiology of Behavior. During this period he also conducted scholarly research and presented his findings at international conferences, including the Society for Neuroscienc e and the Conference on Comparative Cognition. In addition he traveled to Keio University in Tokyo, Japan to conduct research under the direction of Dr. Shigeru Watanabe. Several of Tadds research efforts during his graduate training have been published, including one ba sed on his Masters Thesis work in which he investigated the face features important for conspecific recognition and mate selection in male pigeons.


xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam 22 Ka 4500
controlfield tag 007 cr-bnu---uuuuu
008 s2010 flu s 000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0004594
035
(OCoLC)
040
FHM
c FHM
049
FHMM
090
XX9999 (Online)
1 100
Patton, Tadd.
0 245
An anatomical investigation of higher visual structures in the pigeon (_columba livia_)
h [electronic resource] /
by Tadd Patton.
260
[Tampa, Fla] :
b University of South Florida,
2010.
500
Title from PDF of title page.
Document formatted into pages; contains X pages.
502
Dissertation (PHD)--University of South Florida, 2010.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
Mode of access: World Wide Web.
System requirements: World Wide Web browser and PDF reader.
3 520
ABSTRACT: Early visual processing in the avian brain has been studied extensively, the retina and midbrain in particular. However, a clear understanding of the higher visual centers in the forebrain (the telencephalon and thalamus) remains poor. Two structures located within the avian visual telencephalon, the entopallium (E) and the lateral portion of the intermediate nidopallium (NIL), merit extensive investigation based on their critical role in visual processing. The goal of the current study was to further clarify the anatomical characteristics of E and NIL. Visual information that reaches these telencephalic structures is mostly from the contralateral retina. Thus, blocking visual input on one side affects the opposite hemisphere, but leaves the hemisphere on the same side largely unaffected. This unique property of the avian visual system was used in order to emphasize neurochemical expression in the higher visual structures. After blocking visual input to one hemisphere either by monocular occlusion or unilateral lesion of the nucleus rotundus we examined the expression of specific neuroanatomical markers; namely cytochrome oxidase (CO) and the calcium binding protein, parvalbumin (PV) in E and ZENK protein expression in NIL. Our results showed there were significantly more PV-ir cell bodies in the inner region of E compared to the outer region. In terms of PV-ir neuropil, regional differences within E were significant. In particular, the ventrolateral E tended to have a higher density of PV-ir neuropil than other regions except the most ventromedial area which was also high in PV-ir. Differential CO staining patterns were observed as well. However, unlike PV-ir neuropil the ventro-intermedial area showed a low level of CO staining compared to the other areas. Finally, there was a significant reduction of ZENK-ir cell bodies on NIL in the experimental hemisphere compared to control side. Based on this differential expression of ZENK, it was possible for the first time to visualize the location of NIL. The findings presented here are discussed in terms of their relevance to the identification of the size and extent of NIL and the heterogeneous nature of E.
590
Advisor: Toru Shimizu, Ph.D.
653
Telencephalon
Lateralization
Birds
Rotundus
Zenk
690
Dissertations, Academic
z USF
x Psychology
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.4594