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University MicrxSilms International 300 N. ZEEB RD., ANN ARBOR. Ml 48106 8128990

D iT ir r o, F r a n k Je r a u l d

IMMUNOHISTOCHEMICAL STUDIES OF SPINAL PEPTIDE AND SEROTONIN ELEMENTS IN THE NORTH AMERICAN OPOSSUM, DIDELPHIS VIRGINIAN A. I. THE DISTRIBUTION OF SOMATOSTATIN, METHIONINE-ENKEPHALIN AND SEROTONIN IMMUNOREACTIVITIES IN THE SPINAL CORD OF THE ADULT OPOSSUM. II. THE ONTOGENY OF SPINAL PEPTIDERGIC AND SEROTONINERGIC IMMUNOREACTIVE ELEMENTS IN THE OPOSSUM.

The Ohio State University Ph.D. 1981

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University Microfilms International IMMUNOHISTOCHEMICAL STUDIES OF SPINAL PEPTIDE AND SEROTONIN

ELEMENTS IN THE NORTH AMERICAN OPOSSUM, Didelphis virginiana.

I. THE DISTRIBUTION OF SOMATOSTATIN, METHIONINE-ENKEPHALIN

AND SEROTONIN IMMUNOREACTIVITIES IN THE SPINAL CORD

OF THE ADULT OPOSSUM.

II. THE ONTOGENY OF SPINAL PEPTIDERGIC AND SEROTONINERGIC

IMMUNOREACTIVE ELEMENTS IN THE OPOSSUM.

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Frank Jerauld DiTirro, B.S., M.Sc.

******

The Ohio State University

1981

Reading Committee: Approved By

Raymond H. Ho, Ph.D., Adviser

George F. Martin, Ph.D., Adviser

Albert 0. Humbertson, Jr., Ph.D. Advisers Department of Anatomy Dedicated to my best friends,

Ellen, Fred and Fran.

ii ACKNOWLEDGEMENTS

The author wishes to sincerely thank Dr. Raymond H. Ho for his continual concern and support, but most of all, for his compassion expressed in his professional and personal guidance.

He also wishes to thank Dr. George F. Martin for an inexhaustable source of motivation, knowledge and for his literary expertise.

To Drs. Raymond H. Ho and Robert P. Elde who provided a seemingly endless supply of antisera, he is indebted.

The author expresses his deep appreciation for aid, advice and the camraderie provided him by Mr. Karl B. Rubin. Thanks also go out to Ms. Malinda E. Amspaugh for the typing of the manuscript.

Finally, to the faculty, staff and students of the Department of Anatomy, especially Dr. James S. King, many thanks for making his graduate experience, an experience.

iil VITA

NAME: Frank Jerauld DiTirro

BORN: December 12, 1953

PLACE OF BIRTH: Cleveland, Ohio

EDUCATION: Grade School and High School

Council Rock Elementary School - 1958-1963 Brighton Junior High School - 1963-1967 Brighton High School - 1967-1971 Rochester, New York 14618

Colleges and Degrees

Denison University (1971-1975) Granville, Ohio B.S., Biology Ohio State University (1975-1977) Columbus, Ohio Post-Baccalaureate Studies, Continuing Education Ohio State University (1977-1979) Columbus, Ohio Department of Anatomy, M.S. Ohio State University (1979-present) Columbus, Ohio, Department of Anatomy, Ph.D. Candidate

TEACHING EXPERIENCE: Graduate Teaching Associate - Gross Anatomy to Allied Medical School Students - Autumn 1977, Winter and Spring 1978, Spring 1979, Winter, Spring and Autumn 1980, Winter and Spring 1981. Graduate Teaching Associate - Dental Anatomy, Gross Anatomy, Histology and Neuroanatomy - Summer and Autumn 1978, Winter 1979. Graduate Teaching Associate - Medical School, Gross Anatomy, Histology and Neuroanatomy - Summer and Autumn 1979. Graduate Teaching Associate - Graduate Gross Anatomy, Autumn 1980.

SCIENTIFIC SOCIETIES: Society for Neuroscience

iv OFFICES AND COMMITTEES: Association of Anatomy Graduate Students President (1978-1979) Curriculum Committee (1979-1980) Departmental Self-Study Committee (1979-1980)

PUBLICATIONS

DiTirro, F.J., R. Ho and G.F. Martin 1979 Substance-P (SP) immuno- reactivity in the spinal cord of the North American opossum. Anat. Rec., 193: 525 (abstract).

DiTirro, F.J., R.H. Ho and G.F. Martin 1979 Substance-P, somato­ statin and methionine-enkephalin immunoreactivity in the spinal cord of the North American opossum. Neurosci. Abstr., 5: 2431.

DiTirro, F.J. and R.H. Ho 1980 Distribution of substance-P (SP), somatostatin (SOM) and methionine-enkephalin (ENK) in the spinal cord of the domestic rabbit. Anat. Rec., 196: 47A (abstract).

DiTirro, F.J., R.H. Ho and G.F. Martin 1980 Serotonin (5-HT) immuno­ reactivity in the spinal cord of the North American opossum, Didelphis virginiana. Neurosci. Abstr., 10: 383.

Ho, R.H., A.L. LaValley and F.J. DiTirro 1980 Preliminary studies of 5-hydroxytryptamine-like immunoreactivity in the spinal cord of the domestic fowl. Neurosci. Abstr., 10: 382.

Martin, G.F., T. Cabana, F.J. DiTirro, R.H. Ho and A.0. Humbertson, Jr. 1981 Evidence for heterogeneity in the raphe-spinal projections of the North American opossum. Anat. Rec., 199: 161 (abstract).

Cabana, T., F.J. DiTirro, R.H. Ho and G.F. Martin 1981 The develop­ ment of serotoninergic pathways within the spinal cord. Studies using the North American opossum as an experimental model. Neurosci. Abstr., 11.

Full papers

DiTirro, F.J., R.H. Ho and G.F. Martin 1981 Immunohistochemical localization of substance-P, somatostatin and methionine-enkephalin in the spinal cord and dorsal root ganglia of the North American opossum, Didelphis virginiana. J. Comp. Neur., 198: 351-363.

v Martin, G.F., T. Cabana, F.J. DiTirro, R.H. Ho and A.O. Humbertson, Jr. 1981 The organization and development of spinal projections from reticular, raphe and rubral neurons and their control by the motor cortex. In: J.E. Desmedt (ed.) Motor Control in Man: Mechanisms and Clinical Applications. Raven Press (in press).

Martin, G.F., T. Cabana, F.J. DiTirro, R.H. Ho and A.O. Humbertson, Jr. 1981 The development of descending spinal connections. Studies using the North American opossum. In: H.G.J.M. Kuypers and G.F. Martin (eds.) Progress in Brain Research series on Descending Spinal Control, Elsevier Press, Amsterdam.

Martin, G.F., T. Cabana, F.J. DiTirro, R.H. Ho and A.O. Humbertson, Jr. 1981 Reticular and raphe projections to the spinal cord of the North American opossum. Evidence for connectional heterogeneity. In: H.G.J.M. Kuypers and G.F. Martin (eds.) Progress in Brain Research series on Descending Spinal Control, Elsevier Press, Amsterdam.

PRESENTATIONS

Substance-P (SP) immunoreactivity in the spinal cord of the North American opossum. Ninety-second Annual Session of American Association of Anatomists, Miami, Florida, 1979.

Substance-P, somatostatin and methionine-enkephalin immunoreactivity in the spinal cord of the North American opossum. Ninth Annual Meeting, Society for Neuroscience, Atlanta, Georgia, 1979.

Distribution of substance-P (SP), somatostatin (SOM) and methionine- enkephalin (ENK) in the spinal cord of the domestic rabbit. Ninety-Third Annual Session of American Association of Anatomists, Omaha, Nebraska, 1980.

Serotonin (5-HT) immunoreactivity in the spinal cord of the North American opossum, Didelphis virginiana. Tenth Annual Meeting, Society for Neuroscience, Cincinnati, Ohio, 1980.

vi TABLE OF CONTENTS

Page

DEDICATION...... ii

ACKNOWLEDGMENTS...... iii

VITA ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES...... ix

CHAPTER 1 ...... 1

INTRODUCTION...... 1 MATERIALS AND METHODS ...... 4 RESULTS ...... 10 DISCUSSION...... 15 ILLUSTRATIONS ...... 21

CHAPTER I I ...... 40

INTRODUCTION...... 40 MATERIALS AND METHODS ...... 42 RESULTS ...... 47 DISCUSSION...... 55 ILLUSTRATIONS ...... 63

APPENDIX A ...... 99

LITERATURE CITED ...... 106

vii LIST OF TABLES

Table Page

1 The cross-reactivity of the substance-P antibody with SP, its various fragments, as well as with other substances as evidenced by radioimmunoassay. . . 102

viii LIST OF FIGURES

Figure Page

1 Plots of substance-P (SP), somatostatin (SOM), methionine-enkephalin (ENK) and serotonin (5-HT) imraunohistofluorescence at four representative levels of the adult opossum spinal cord. Note the sparse amounts of SP and SOM elements within the ventral horn and the relative abundance of ENK and 5-HT ele­ ments within comparable regions. The curved arrow indicates the intermediolateral cell column and the stars denote the location of SOM perikarya...... 23

2 Photomicrograph of SOM immunoreactive elements in laminae I (I) and II (II). The dorsal funiculus is indicated (DF). The bar represents lOOpm for figures 2 - 5 ...... 25

3 Photomicrograph of a transverse section of the opossum cord at the cervical enlargement. The arrows indicate punctate and/or varicose structures containing SOM immunoreactivity. The central canal is indicated (CC)...... 25

4 Photomicrograph of a transverse section from the thoracic cord of the opossum showing SOM elements within the intermediolateral cell column (arrow). The lateral funiculus is labelled (LF)...... 25

5 Photomicrograph of a transverse section of the opossum's sacral cord. The open arrows indicate SOM immunorective elements within the sacral para­ sympathetic area. The lateral funiculus is indicated (LF)...... 25

6 Micrograph of a transverse section from the thoracic cord of the opossum. The dorsal horn exhibits enkepha­ lin (ENK) immunoreactivity within laminae I and II. The dorsal funiculus is indicated (DF) and the bar represents 80ym for figures 6 - 9 ...... 27

ix Figure Page

7 Micrograph of a transverse section of opossum spinal cord taken at the cervical level. ENK immunofluores­ cence can be seen around the central canal (CC). The dorsal funiculus is labelled (DF) ...... 27

8 Micrograph of a transverse section from the thoracic cord of the opossum showing a high magnification of the intermediolateral cell column. ENK elements can be seen within the same area. The lateral funiculus is labelled (LF) ...... 27

9 Micrograph of a transverse section from the lumbar cord of the opossum showing sparse ENK immunostaining within the ventral horn. Lateral funiculus is labelled (LF)...... 27

10 Photomicrograph of ENK within the dorsal horn of a midthoracic section incubated with antisera specific for M-ENK. ENK immunoreactive elements can be ob­ served within lamina I (I) and lamina II (II). The dorsal funiculus (DF) is labelled and the bar repre­ sents 80pm...... 29

11 Photomicrograph of a semiadjacent section (opossum) incubated with preabsorbed antisera for ENK. The control antisera failed to localize ENK to comparable areas when processed in parallel with routine staining procedures. Laminae I (I) and II (II) as well as the dorsal funiculus (DF) are indicated. The bar repre­ sents 80pm...... 29

12,13 Semiadjacent transverse sections of the opossum's sacral cord which were processed in parallel by the indirect immunofluorescent method of Coons. Section shown in figure 10 was incubated with serotonin (5-HT) antiserum (194D). Note the punctate fluorescent vari­ cosities that stained for 5-HT immunoreactivity in the superficial laminae of the dorsal horn. The section shown in figure 11 was incubated with antiserum (194D) pretreated with an excess of synthetic serotonin. Note the absence of immunostaining in comparable areas of the dorsal horn. Laminae I (I) and II (II) and the dorsal funiculus (DF) are indicated. The bar repre­ sents 80pm...... 31

14 Micrograph of a transverse section from the thoracic cord of the opossum showing the intermediolateral cell column. Serotonin immunostaining can be seen. Lateral funiculus (LF). The bar represents 80pra...... 31

x Figure Page

15 Micrograph of a transverse section of opossum spinal cord taken from a cervical level. 5-HT immunofluores­ cence can be seen around the central canal (CC). The dorsal funiculus is indicated (DF) and the bar repre­ sents 80pm...... 32

16 Micrograph of transverse section from the lumbar cord of the opossum showing 5-HT immunostaining within the ventral horn. The white arrow indicates serotoninergic elements surrounding a presumed motor neuron. The lateral funiculus (LF) is indicated. The bar repre­ sents 80pm...... 32

17 Photomicrograph of a transverse section of the opossum's cervical cord showing 5-HT varicosities adjacent to the dorsal spinocerebellar tract (dsc). The bar represents 80pm...... 32

18 Plots of SP, ENK and 5-HT immunoreactivities at five representative levels of the spinal cord following a spinal transection at the eighth thoracic level (T8). Note that the distributions of SP and ENK are unchanged when compared with their normal distributions (fig. 1), while 5-HT elements are virtually absent caudal to the lesion. C8=eighth cervical segment; T6=sixth thoracic segment; Tll=eleventh thoracic segment; and L3=third lumbar segment...... 34

19 Photomicrograph of a transverse section from the fourth thoracic segment of an opossum that received a spinal transection at the eighth thoracic level (T8). The dorsal horn exhibits SP immunoreactivity within laminae I (I) and II (II). The arrow indicates a bundle of SP fibers within the dorsolateral fasciculus. The dorsal funiculus (DF) is indicated and the bar repre­ sents 100pm for figures 1 9 - 2 4 ...... 36

20 Photomicrograph of a transverse section from the tenth thoracic segment of an opossum that received a spinal transection at T8. The dorsal horn exhibits SP immuno­ staining within laminae I (I) and II (II). The bundle of SP fibers referred to in figure 19 appears markedly enlarged (arrow) in this and other thoracic sections, caudal to the lesion. The dorsal funiculus (DF) is indicated...... 36

xi Figure Page

21 Photomicrograph of 5-HT immunoreactive elements in the intermediolateral cell column. This section was taken from the thoracic cord, rostral to the spinal transec­ tion. The lateral funiculus (LF) is indicated. . . . 36

22 Photomicrograph of a transverse section from the thora­ cic cord caudal to a spinal transection. Note the lack of 5-HT elements within the intermediolateral cell column (arrow). The lateral funiculus (LF) is indic a t e d...... 37

23 Photomicrograph of a transverse section of the cervical cord taken at a level rostral to a spinal transection. Note the aggregation of 5-HT fibers (arrow) in the peri­ phery of the dorsolateral funiculus (DLF) ...... 37

24 Photomicrograph of a transverse section of the thoracic cord taken at a level caudal to a spinal transection. Note the lack of 5-HT immunostaining within the dorso­ lateral funiculus (DLF) ...... 37

25,26 Micrographs of SP immunoreactivity within the dorsal horn of the third lumbar segment. Figure 26 illus­ trates the pattern of SP staining 5 days after transec­ tion of adjacent dorsal roots on that side, while figure 25 illustrates the pattern of SP immunostaining within the dorsal horn of the unoperated side. Note the close similarity in the distribution of SP immunoreac­ tivity within laminae I (I) and II (II). The dorsal funiculus (DF) is labelled and the bar represents 80ym for figures 25 and 26...... 39

Schematic representation of substance-P (SP), somato­ statin (SOM), methionine-enkephalin (ENK) and serotonin (5-HT) immunoreactivities at three representative levels from a newborn opossum (day 1) spinal cord. Note the lack of SOM. The solid arrow (left side) indicates the SP in the dorsolateral marginal zone. The open arrow (right side) indicates 5-HT elements within the same area...... 65

xii Figure Page

28 Plots illustrating the spinal distribution of sub- stance-P (SP), somatostatin (SOM), methionine-enkepha­ lin (ENK) and serotonin (5-HT) at three representative levels from a 5 day old pouch-young opossum. Note the rostral-caudal gradient of SOM elements and the paucity of ENK elements within the intermediate zone. The stars indicate immunoreactive perikarya. SOM cell bodies are present within the dorsal horn at this stage, but they have not been plotted for the sake of clarity (see Results) ...... 67

29 Drawings of the distributions of substance-P (SP), somatostatin (SOM), methionine-enkephalin (ENK) and serotonin (5-HT) at three representative spinal levels from a 15 day old pouch-young opossum. Note the abundance of 5-HT elements within the intermediate zone at all spinal levels. Serotonin (5-HT) elements abutting presumed motoneurons can be observed (curved arrow). The stars indicate the position of 5-HT cell bodies. SOM and ENK cell bodies are also present within superficial areas of the dorsal horn, but they have not been included for the sake of clarity (see Results)...... 69

30 Schematic representation of substance-P (SP), somato­ statin (SOM), methionine-enkephalin (ENK) and serotonin (5-HT) distributions at three representative levels of the spinal cord from a 30 day old opossum. Note the abundance of SP and SOM within the superficial laminae of the dorsal horn and the lack of ENK and 5-HT elements within comparable regions. The solid arrow (left side) indicates a bundle of SP-containing fibers. The curved arrow indicates SP elements within the inter­ mediolateral cell column. 5-HT elements are aggregated within comparable areas (right side). Cell bodies are indicated by the stars. SOM and ENK cell bodies are present in superficial areas of the dorsal horn, but they have not been included for the sake of clarity (see R e s u l t s ) ...... 71

xiii Plots illustrating the spinal distribution of sub­ stance-P (SP), somatostatin (SOM), methionine-enkeph­ alin (ENK) and serotonin (5-HT) elements at three representative levels from a 50 day old opossum. Note that ENK and 5-HT elements are present for the first time within the superficial laminae of the dorsal horn. Also, note that 5-HT elements are ag­ gregated within lamina IX. The open arrow indicates a bundle of SP fibers. SOM cell bodies are denoted by stars. SOM and ENK cell bodies are also present within superficial laminae of the dorsal horn but they have not been included for the sake of clarity (see Results) ......

Chartings of the distributions of substance-P (SP), somatostatin (SOM), methionine-enkephalin (ENK) and serotonin (5-HT) at three spinal levels from a 75 day old opossum. Note the dense aggregation of SP and 5-HT (curved arrows) elements within the intermedio­ lateral cell column. SOM cell bodies are denoted by stars. SOM and ENK cell bodies are also present within superficial laminae of the dorsal horn but they have not been included for the sake of clarity (see Results) ......

Plots of substance-P (SP), somatostatin (SOM), methi­ onine-enkephalin (ENK) and serotonin (5-HT) distribu­ tions at four representative levels of the spinal cord from an adult opossum. Note the sparse amounts of SP and SOM elements within the ventral horn and the rela­ tive abundance of ENK and 5-HT elements within com­ parable regions. The curved arrow indicates the intermediolateral cell column and the stars denote the location of SOM perikarya ......

Photomicrograph of the dorsal horn from a 24mm opossum. The arrow indicates a cluster of SP elements within the developing superficial laminae. The dorsal funi­ culus is indicated (DF) and the bar represents lOOym for figures 34-37 ......

Photomicrograph of a transverse section from the cer­ vical cord of a 57mra pouch-young opossum. SP imrauno- reactivity can be seen within presumptive laminae I (I) and II (II), as well as in deeper laminae of the dorsal horn. The curved arrow indicates a bundle of SP fibers. The dorsal funiculus is indicated (DF). . Figure Page

36 Photomicrograph of SP elements within presumptive lamina X of a 57ram opossum at the cervical level. The central canal is labelled (CC). 79

37 Photomicrograph of SP immunoreactivity within the intermediolateral cell column (curved arrow) of a 57ram opossum. The lateral funiculus (LF) is labelled. . . 79

38 Micrograph of SOM immunoreactivity (arrow) within the dorsolateral and lateral marginal (MAR) zone of the cervical cord from a 24mra pouch-young opossum. Note the virtual lack of immunostaining within the inter­ mediate (INT) zone. The bar represents 100pm for figures 38-41 ...... 82

39 Micrograph of a transverse section of the cervical cord taken from a 57mm opossum. Note the presence of SOM perikarya in the upper right. The arrows indicate two of the sparse SOM elements within pre­ sumptive lamina X. The central canal (CC) is indicated...... 82

AO Micrograph of a transverse section of the cervical cord from a 57mm opossum. Punctate SOM elements are present within laminae I (I) and II (II) as well as SOM perikarya which are more obvious. The arrow indicates an SOM cell body that is also shown at a higher magnification (inset). The inset bar repre­ sents 10pm, and the dorsal funiculus is indicated (DF)...... 82

41 Micrograph of a transverse section of the cervical cord from a 127mm opossum. Note the predominance of punctate and varicose immunoreactivity and the relative lack of SOM cell bodies. Laminae I (I) and II (II) as well as the dorsal funiculus (DF) are indicated ...... 82

Photomicrograph of SOM iramunoreactive elements in laminae I (I) and II (II) of the adult opossum. The dorsal funiculus is indicated (DF). The bar repre­ sents 100pm for figures 42-45 ...... 84

xv Figure Page

43 Photomicrograph of a transverse section from the cer­ vical enlargement of an adult opossum. The arrows indicate punctate and/or varicose iramunoreactive structures which contain SOM. The central canal is indicated (CC)...... 84

44 Photomicrograph of a transverse section from the thoracic cord showing SOM elements within the inter­ mediolateral cell column of the adult opossum (arrow). The lateral funiculus is labelled (LF)...... 84

45 Photomicrograph of a transverse section of the sacral cord from an adult opossum. The open arrows indicate SOM immunoreactive elements within the sacral parasym­ pathetic area. The lateral funiculus is indicated (LF)...... 84

46 Micrograph of ENK immunoreactivity within the dorso­ lateral and lateral marginal (MAR) zone of an 18mm opossum at the cervical level. Note the lack of immunostaining within the intermediate (INT) zone. The arrows indicate ENK elements interpreted as fibers in cross-section. The bar represents lOOym for figures 46, 47 and 4 9 ...... 87

47 Micrograph of a transverse section from the cervical spinal cord of a 57ram opossum. Punctate and varicose ENK immunoreactive elements are apparent throughout presumptive lamina X. The central canal (CC) is indicated...... 87

48 Micrograph of ENK immunoreactivity within the super­ ficial laminae of the dorsal horn of a 57mm opossum (cervical cord). Note that the sparse punctate ENK elements are restricted primarily to lamina II (II) while lamina I (I) exhibits little, if any, staining. The arrow indicates an ENK cell body within lamina II that is shown at a higher magnification (inset). The bar in figure 48 represents 50ym and the inset bar equals lOym ...... 87

Micrograph of ENK immunoreactivity within laminae I (I) and II (II) of the thoracic cord from a 127mm opossum. Note the predominance of varicose ENK elements and the lack of ENK cell bodies. The dorsal funiculus (DF) is indicated...... 87

xvi Figure Page

50 Photomicrograph of 5-HT immunoreactivity within the dorsolateral and lateral marginal zone (MAR) of the cervical level from an 18mm pouch young opossum. Note the lack of 5-HT elements in the intermediate zone (INT). The arrow points to 5-HT immunoreactive elements aggregated within the peripheralmost part of the marginal zone. The bar represents 100pm for figures 50-54 ...... 90

51 Photomicrograph of a transverse section of the cer­ vical cord from a 35ram pouch-young opossum. Note the presence of 5-HT elements within the ventral funicu­ lus (VF) which can be followed with the ventral horn. The arrow indicates 5-HT elements surrounding a pre­ sumed motoneuron...... 90

52 Photomicrograph of 5-HT immunostaining within pre­ sumptive lamina X of the cervical cord from a 57mm opossum. Fine caliber 5-HT varicosities are apparent throughout the regions adjacent to the central canal (CC)...... 90

53 Photomicrograph of a transverse section from the thoracic cord of a 57mm pouch-young opossum. 5-HT elements are present within the developing gray matter (open arrow) and are particularly numerous within the intermediolateral cell column (curved arrow). The lateral funiculus (LF) is indicated ...... 90

54 Photomicrograph of the sparse 5-HT immunostaining present within laminae I (I) and II (II) of the cer­ vical cord from an 84mra opossum. The open arrows indicate 5-HT elements within the superficial laminae of the dorsal horn. Some immunostaining is present within the deeper laminae. The dorsal funiculus is labelled (DF) ...... 90

Photomicrograph of a trnasverse section of the cer­ vical cord from a 57mm pouch-young opossum. Note the presence of 5-HT varicosities as well as 5-HT peri­ karya (arrows). The sulco-marginal funiculus (SM) and a blood vessel (BV) are labelled. The bar rep­ resents 50ym...... 90

xvil Photomicrograph of SP immunoreactivity within the dorsal horn of a midthoracic section from a 50 day o1 d opossum that was incubated with the antiserum specific for SP. SP immunoreactivity can be observed in laminae I (I) and II (II) as well as within more ventral regions. The dorsal funiculus (DF) is indi­ cated and the bar represents lOOym for figures 56-59 ......

Photomicrograph of a semiadjacent midthoracic sec­ tion that was incubated with the preabsorbed anti­ sera for SP. The control antisera failed to localize SP to comparable areas when processed in parallel with routine staining procedures. Laminae I (I) and II (II) as well as the dorsal funiculus (DF) are indicated ......

Photomicrograph of SOM immunoreactivity within the dorsal horn of a cervical section from a 30 day old opossum that was incubated with the antiserum specific for SOM. SOM immunoreactivity can be observed mainly in laminae I (I) and II (II). The dorsal funiculus (DF) is labelled......

Photomicrograph of a semiadjacent cervical section that was incubated with the preabsorbed antiserum for SOM. The control antisera failed to localize SOM to comparable areas when processed in parallel with routine staining procedures. Laminae I (I) and II (II) as well as the dorsal funiculus (DF) are labelled......

Photomicrograph of ENK immunoreactivity within lamina X of a cervical section from a 30 day old opossum that was incubated with the antiserum specific for ENK. ENK immunoreactive neuronal elements are represented by varicose or punctate structures (arrows). The central canal (CC) is labelled and the bar represents 100pm for figures 60-63 ......

Photomicrograph of a semiadjacent cervical section that was incubated with the preabsorbed antisera for ENK. The control antisera failed to localize ENK to comparable areas when processed in parallel with routine staining procedures. The central canal (CC) is labelled ......

xviii Figure Page

62 Photomicrograph of 5-HT immunoreactivity within lamina X of a cervical section from a 30 day old opossum that was incubated with the antiserum specific for 5-HT. Serotonin immunostaining can be observed as punctate and varicose structures throughout the regions adjacent to the central canal (CC)...... 96

63 Photomicrograph of a semiadjacent cervical section that was incubated with the preabsorbed antisera for 5-HT. The control antisera failed to localize 5-HT to comparable areas when processed in parallel with routine staining procedures. The central canal (CC) is l a b e l l e d ...... 96

Graphic representation of the sequence of peptidergic and serotoninergic innervation of specific spinal re­ gions. VH = ventral horn; IMLC = intermediolateral cell column; LX = lamina X; DH = dorsal horn; MZ = marginal zone; A = adult. The snout-rump (S-R) lengths are indicated in millimeters...... 98

xix IMMUNOHISTOCHEMICAL STUDIES OF SPINAL PEPTIDE AND SEROTONIN

ELEMENTS IN THE NORTH AMERICAN OPOSSUM, Didelphis virginiana.

I. THE DISTRIBUTION OF SOMATOSTATIN, METHIONINE-ENKEPHALIN

AND SEROTONIN IMMUNOREACTIVITIES IN THE SPINAL CORD

OF THE ADULT OPOSSUM.

INTRODUCTION

Somatostatin (SOM) was first identified as an inhibitory factor for the release of somatotrophin and thyrotropin (Brazeau et al., *73;

Labrie et al., ’78) and was isolated, purified from extracts of ovine hypothalami and sequenced by Brazeau and co-workers. Somatostatin has also been demonstrated within ganglion cells of dorsal roots (the rat,

Hokfelt et al., ’76, ’76) and shown experimentally to be transported centrally through their axons (Hokfelt et al., ’76). Somatostatinergic elements have since been identified within the central nervous system of several other species including the monkey (HSkfelt et al., ’75), the cat (Hokfelt et a l ., ’ 77), the guinea pig (Elde, ’78), Japanese dancing mouse (Forssmann, ’78) and domestic fowl (LaValley and Ho, ’80).

The distribution of enkephalinergic elements (ENK) within the rat spinal cord has been studied extensively (Elde et al., ’76;

1 Simantov et al., ’77; Hokfelt et al., '77; Simantov and Snyder, '76;

Johansson et al., '78; Sar et al., '78 and Uhl et al., '79). Unlike

SP and SOM, spinal enkephalins have been considered to originate mainly from intrinsic neurons (HUkfelt et al., '77; Sar et al., '78; Uhl et al., '79). Using various double-labelling techniques, however, ENK neurons within certain reticular and raphe nuclei have also been shown to innervate the spinal cord (HUkfelt et al., '79; Bowker et al., '81).

Spinal enkephalins are of particular interest because of their opiate­ like characteristics. Both ligands of the enkephalins (leucine- enkephalin and methionine-enkephalin) have been shown to be agonists to opiate receptor sites (Hughes et al., '75), suggesting a role in analgesia. Jessel and Iversen (’77) have demonstrated that low con­ centrations of enkephalins suppress the release of SP from substantia nigra neurons following potassium ion application. Results from experiments by Yaksh and Rudy (’76) corroborate these data, wherein intrathecal administration of opiates produced analgesia in the lumbar cord of the rat. Using immunohistochemical methods, Johansson et al.

(’78) have demonstrated ENK-containing neuronal elements throughout the rat spinal gray matter.

Serotonin (5-HT) has been implicated in various functions within the central nervous system of mammals (see Chase and Murphy, '73, for review), among which is a role in the elicitation of endogenous analgesia (Basbaum and Fields, *78). To date, spinal 5-HT has been studied by biochemical techniques (Amin, Crawford and Gaddum, '54;

Bogdanski, Weinbach and Underfriend, '57) and identified in neuronal processes by the aldehyde-induced fluorescent methods of Falck-Hillarp (Falck et al., *62; Dahlstrom and Fuxe, '64; Fuxe, ’65). In addition,

investigators have examined 5-HT elements within other regions of the

central nervous system using the autoradiographic technique at the

light (Azmitia and Segal, '78; Conrad et al., ’74) and electron micro­

scopic levels (Bloom et al., '72; Chan-Palay, ’75). Very recently,

Steinbusch and co-workers ('81) have described the distribution of

serotonin-containing neuronal elements within the central nervous

system of the rat using the indirect immunofluorescent method of Coons

('58).

To date, SOM, ENK and 5-HT have been studied only in selected birds and mammals and it is premature to conclude that the distribution

of these elements is identical in all species. The purpose of this

report is to describe the spinal distribution of these elements in the

North American opossum, a marsupial, and to compare it with that seen

in other species. The data reported herein also establish the adult

organization of these elements as a baseline for studies of their

development. MATERIALS AND METHODS

Mapping of Somatostatin, Enkephalin and Serotonin Immunoreactlvltles

Five adult opossums, Dldelphis vlrginiana, were anesthetized with sodium pentobarbital (i.p. 30mg/Kg) and sacrificed by intracardiac perfusion with phosphate buffered saline (PBS) followed by either freshly prepared, ice-cold 4% paraformaldehyde in phosphate buffer

(Pease, *62) or Zamboni's fixative (Stephanini et al., '67) at room temperature (RT) for thirty minutes. Spinal cords were promptly removed and placed in the same fixative for 4-6 hours (4°C), followed by immersion in 5% sucrose/Sorensen's phosphate buffer solution at 4°C for 1-2 days. Ten micrometer sections were cut transversely using a cryostat from representative levels of the spinal cord and mounted on chrome-alum-gelatin coated slides. The tissues were then processed for the localization of SOM, ENK and 5-HT by both the indirect immuno- fluorescent technique (FITC) of Coons (’58) and the indirect antibody peroxidase-anti-peroxidase method (indirect antibody PAP technique) of Sternberger (’70).

Tissues processed by the FITC technique were rehydrated using

PBS and incubated overnight with either anti-SOM (diluted 1:100), anti-

ENK (diluted 1:100) or anti-5-HT (diluted 1:100) sera or with the

4 appropriate control sera in a humid environment at 4°C. The diluent for all antisera used in the FITC method was PBS containing 0.3% Triton

X-100 (Octyl Phenoxy Polyethoxyethanol). Sections were rinsed and immersion-washed in PBS and subsequently incubated with fluorescein isothiocyanate conjugated goat-anti-rabbit IgG serum (GAR) (diluted

1:30) for one hour in a humid environment at room temperature. Follow­ ing a PBS rinse and immersion wash, the sections were coverslipped with a glycerin-PBS solution (3:1) and observed and photographed under a

Zeiss photomicroscope with reflected illumination (excitation wave­ length 440-490nm).

Tissue sections that were processed according to the indirect antibody PAP technique (Sternberger, '70) were rehydrated using borate buffered saline (BBS) and incubated consecutively with the following antisera: (1) either anti-SOM serum (diluted 1:500), anti-ENK serum

(diluted 1:500), anti-5-HT serum (diluted 1:1000) or the appropriate control serum for 48 hrs. at 4°C in a humid environment; (2) sheep- anti-rabbit immunoglobulin G (SAR) antiserum (diluted 1:300) for 1 hour at room temperature; (3) rabbit peroxidase-anti-peroxidase (PAP) complex (diluted 1:500) for 1 hour at room temperature. The diluent for all antisera used in the PAP technique was 0.5% bovine serum albumin (BSA) in borate buffered saline containing 0.3% Triton X-100

(Octyl Phenoxy Polyethoxyethanol), except the anti-5-HT serum whose diluent was 1.0% bovine serum albumin (BSA) in borate buffered saline containing 0.3% Triton X-100 (Octyl Phenoxy Polyethoxyethanol). The diluent for the PAP complex was BBS-BSA. Sections were rinsed and immersion-washed after each incubation in BBS. After incubation with the PAP complex, sections were immersion- washed in BBS and immersed in a PBS solution (RT) containing 3,3'- diamino-benzidine tetrachloride (DAB) (0.3g/600ml) and 0.006% hydrogen peroxide for 5-10 minutes. The sections were immersed in BBS, dehy­ drated in a series of graded alcohols, cleared with xylene and coverslipped with Permount. Processed materials were observed and photographed using a Zeiss photomicroscope.

Plots illustrating the distribution of peptide and indolamine immunoreactivities were constructed on drawings of adjacent or semi- adjacent Nissl stained sections. Sections from representative levels of the cord were drawn using a Tri-Simplex projector and the subsequent plotting was accomplished by direct observation of the processed sections.

Experimental Procedures

In an attempt to determine the origin of the axons containing the substances described in this and other (DiTirro et al., ’81) accounts, several surgical procedures were performed. Under sodium pentobarbital anesthesia (40mg/kg animal), a spinal hemisection (n^l) and a spinal transection (n=l) were performed at the fourth and eighth thoracic level, respectively. The animals were allowed to survive for ten days to allow for axonal degeneration caudal to the lesions. They were perfused and their spinal cords processed in a manner identical to that described in the previous section. Sections from segments proximal and distal to the lesions were examined and compared with 7 those processed from unoperated animals in order to determine if the peptides (SP, SOM and ENK) or 5-HT were depleted.

Dorsal rhizotomies (n*“3), involving three or four dorsal roots, were performed unilaterally at caudal thoracic/rostral lumbar levels.

The operated animals were maintained for either five or seven days to allow for degeneration of the central processes of dorsal root ganglion cells. In addition, dorsal rhizotomies were performed bilaterally at caudal thoracic levels involving two roots on each side (n=l). This animal was allowed to survive for four days. All animals were perfused and processed for the localization of peptide (SP, SOM and ENK) and indolamine elements according to the FITC or the indirect antibody PAP technique. Sections from the levels of the rhizotomies were compared with sections from unoperated (normal) spinal levels of the same animal and with sections from non-operated controls.

The use of colchicine to increase cellular concentrations of neurotransmitters by inhibiting axonal transport is well-documented

(Dahlstrom, ’68; Hokfelt et al., ’77). Six adult opossums received colchicine injections in an attempt to localize peptidergic and indola­ mine rgic perikarya within the spinal cord and brainstem. In four of them 600yg/3.0mg of colchicine, dissolved in 0.9% saline (2.5yg/yl- lOyg/yl) were delivered into the cistema magna via an intrathecal catheter (PE-10 tubing). The other two opossums received colchicine injections into the subarachnoid space over the spinal cord. In the latter cases 800yg of colchicine was dissolved in 2.0yg/yl and delivered by an intrathecal catheter. The opossums were sacrificed

18-48 hours after the injection. The brainstems from animals receiving 8 cisternal injections were sectioned using the cryostat (10ym) from caudal medulla to the trapezoid body and stained for the localization of peptide and indolamine perikarya. The spinal cords of animals receiving spinal injections were processed and examined in a similar manner for the presence of immunoreactive cells.

Antisera

Rabbit anti-SOM serum (11C) was obtained from the third bleeding of rabbit #11 (Ho and DePalatis, ’80) which received a somatostatin- keyhole limpet hemocyanin conjugate. Rabbit anti-ENK serum (18G)

(DiTirro et al., ’81) was obtained from the seventh bleeding of rabbit

#18 which was immunized with a methionine-enkephalin thyroglobulin conjugate. The rabbit anti-5-HT serum (194D) was obtained from the fourth bleeding of rabbit $194 (Holets et al., ’81; Holets and Elde, submitted for publication) which was immunized with a 5-HT bovine serum albumin conjugate. The anti-5-HT serum was provided by Dr. Robert Elde

(University of Minnesota). The secondary antisera for both, the FITC and indirect antibody PAP method, fluorescein isothiocyanate conjugated goat-anti-rabbit immunoglobulin G (GAR) antiserum and the sheep-anti- rabbit immunoglobulin G (SAR) antiserum, respectively, were purchased from Antibodies Incorporated, The rabbit peroxidase-anti-peroxidase complex (PAP) was purchased from Cappel Laboratories,

In order to establish the specificity of immunostaining, adja­ cent or semiadjacent sections from all representative levels of the spinal cord were incubated with control serum and processed in parallel with the experimental sections. Control sera consisted of the diluted anti-ENK, anti-SOM and anti-5-HT sera pretreated overnight

(4°C) with an excess of synthetic ENK, SOM and 5-HT (20yg/ml of diluted antiserum) respectively. Photomicrographs illustrating the results of the control stainings are shown in figures 11 and 13.

For further antibody information, please see Appendix A. RESULTS

Distribution of Presumed Neuronal Elements Containing

Peptide and Serotonin (5-HT) Immunoreactivities

Substance-P

The distribution of SP immunoreactive elements within the adult opossum spinal cord has been reported elsewhere (DiTirro et al., ?81).

A plot illustrating the pattern of SP elements is included in figure 1.

Somatostatin

SOM immunoreactive elements were most abundant within the outer portion of lamina II (Rexed, '52), although they were also present within lamina I, deeper regions of lamina II, as well as within subja­ cent regions of the dorsal horn at all spinal levels (figs. 1 and 2).

Moderate aggregations of SOM varicosities were also observed in areas adjacent to the central canal (lamina X) (figs. 1 and 3) especially at sacral levels (fig. 1). At thoracic and rostral lumbar levels, SOM immunostaining was present within the intermediolateral cell column

(nucleus intermediolateralis thoracolumbalis, of Petras and Cummings,

’72) (curved arrows in figs. 1 and 4) and at sacral levels, it was aggregated along the lateral aspect of the dorsal horn (figs. 1 and 5)

10 11

In a region comparable in position to the visceral afferent area described for the cat (DeGroat et al., *78). SOM immunoreactivity was sparse within the ventral horn (laminae VIII and IX) at all spinal levels (fig. 1). Immunostaining for SOM was also present within the dorsolateral funiculi adjacent to the lateral aspect of the dorsal horn (lateral regions of laminae IV-VI) throughout the spinal cord

(fig. 1), as well as within the sulcomarginal funiculus at sacral levels (fig. 1).

Me thionine-Enkephali n

ENK immunoreactivity was present within most areas of the spinal gray (fig. 1). At all spinal levels, ENK-containing fibers and/or their terminals were most numerous within lamina I and outer regions of lamina II (figs. 1 and 6). ENK elements were sparsely distributed within deeper laminae of the dorsal horn (laminae IV-VI) (fig. 1) and in selected cervical, lumbar and sacral sections, they were aggregated laterally. Fluorescent structures representing ENK immunoreactivity were present within lamina X at all spinal levels (figs. 1 and 7) where they were clustered dorsolateral to the central canal. ENK- containing neuronal elements were apparent within the intermediolateral cell column (figs. 1 and 8) and within the sacral parasympathetic area of the sacral dorsal horn, as described by DeGroat et al. (’78)

(fig. 1). Fine-caliber ENK varicosities were evenly distributed throughout the ventral horn at all spinal levels (figs. 1 and 9).

Figure 10 shows ENK immunofluorescence in laminae I and II and 12 figure 11 Illustrates the lack of fluorescence In the same area from a semiadjacent absorption control.

ENK Immunostaining was present within the dorsolateral funiculi throughout the cord (fig. 1) and found occasionally within the lateral and ventral funiculi at cervical, thoracic and sacral levels.

Serotonin (5-HT)

Serotonin immunoreactivity occurred within laminae I and II, as well as within laminae IV-VI of the dorsal horn (figs. 1 and 12). It was extremely sparse within lamina III, however (figs. 1 and 12). 5-HT elements were particularly abundant within the intermediolateral cell column at thoracolumbar levels and found within all of the subdivisions described by Petras and Cummings ('72) (figs. 1, 14 and 15). Only sparse amounts of 5-HT immunoreactivity were present in the parasympa­ thetic regions of the sacral cord (fig. 1). Serotoninergic elements were scattered throughout laminae VII-VIII of the ventral horn (fig. 1), but they were numerous within lamina IX (figs. 1 and 16) where they surrounded motoneurons (fig. 16).

5-HT fibers could also be identified within the white matter where they were aggregated within an area ventrolateral to the dorso- spinocerebellar tract (figs. 1 and 17) and numerous within peripheral areas of the dorsolateral, lateral and ventrolateral funiculi. 5-HT elements were also found within ventral and sulcomarginal regions of the white matter (fig. 1). 13

Distribution of Peptide and Serotonin

Cell Bodies Within the Spinal Cord

Perikarya exhibiting SOM immunoreactivity were localized within laminae V-VIII and occasionally within lamina X at all of the spinal levels examined, although they occurred most often at lumbar and sacral levels (fig. 1). Although we observed both ENK and 5-HT perikarya within the spinal cord of the developing opossum (see Chapter II), we were unable to demonstrate them in the adult animal.

Experiments Designed to Determine the Origin of

Peptides and Serotonin in the Spinal Cord

There was no obvious depletion of SP or ENK either rostral or caudal to spinal transection (fig. 18). Sections were not processed for SOM because at that time we were not able to demonstrate it in the spinal cord of the normal animal (see DiTirro et al., '81). The only lesion-dependent change was enlargement of a bundle of SP fibers within the dorsolateral fasciculus at thoracic levels. At levels caudal to the lesion in the hemisected case, the bundle in question appeared larger in comparison to its counterpart on the unoperated side

(figs. 19 and 20).

Sections taken from the spinal cord of the transected animal were also processed for the localization of 5-HT. Caudal to the lesion, there was almost total depletion of 5-HT immunoreactivity (figs. 18,

21-24). In contrast, there was no apparent change rostral to the lesion (fig. 18). 14

There was no change in SP or ENK elements at the levels of the spinal cord innervated by the dorsal roots sectioned in our experiments

(figs. 25 and 26). The density and distribution of peptide elements appeared comparable to that in sections taken from unoperated levels of the same animal and from unoperated controls.

Brainstem and spinal sections from opossums receiving either cisternal or spinal injections of colchicine failed to demonstrate cell bodies exhibiting peptide immunoreactivity. Serotoninergic peri­ karya were observed in both colchicine treated and untreated animals within the brainstem. In either case, 5-HT cell bodies were not demonstrable within the spinal cord.

Control Sera

Our control sera failed to localize immunoreactive elements on sections adjacent or semi-adjacent to those processed with the non­ pretreated antisera (figs. 10-13). The possibility that our methionine- enkephalin antisera cross-reacts with leucine-enkephalin has not been ruled out. Thus, some of the ENK immunostaining may be demonstrating leucine-enkephalin immunoreactivity. DISCUSSION

It was our intent to study the spinal distribution of SOM, ENK and 5-HT in the North American opossum employing both the immunofluo- rescent method of Coons (’58) and the indirect antibody technique of

Stemberger (’70). While comparable results were obtained for ENK and

5-HT using either the immunofluroescent method with paraformaldehyde as the fixative or the PAP technique utilizing Zamboni's fixative

(Stephanini et al., '67), it was necessary to employ the latter protocol to demonstrate SOM immunoreactivity (DiTirro et al., ’81).

One advantage of the PAP technique is that it provides permanently stained and mounted material that can be conveniently handled and stored. Both techniques allowed us to examine adjacent or semi-adjacent sections from each animal for the localization of the three peptides and serotonin minimizing the possibility of inter-animal differences.

It appears that the spinal distribution of SOM, ENK and 5-HT is similar in the opossum and rat. In the opossum, SOM neuronal elements are abundant within the outer portion of lamina II (Rexed, ’52) although they are also present in laminae I and III. Such elements have been demonstrated within comparable areas of the rat's dorsal horn (Hokfelt et al., '75, '76) and shown to be primarily of dorsal

15 16 root origin (Hokfelt et al., *76). While this is probably the case in the opossum, preliminary results from ontogenetic studies (see Chapter

II) demonstrated SOM cell bodies in laminae II and III and they probably account for some of the immunoreactivity within the superficial laminae of the dorsal horn. Somatostatin has been shown to slow the firing rate of dorsal horn neurons that were activated by nociceptive input

(Randic and Miletic", ’78).

SOM elements were also observed within lamina X, the intermedio­ lateral cell column and the sacral parasympathetic area of the opossum's cord. Since such areas are not innervated by dorsal root axons

(Hokfelt et al., ’76), it is likely that SOM elements within them are associated with neurons of spinal or supraspinal origin. Sawchenko and Swanson (’80) have demonstrated that SOM neurons within the para­ ventricular nucleus of the hypothalamus project to all levels of the spinal cord. It is possible that such neurons provide some of the SOM innervation of autonomic nuclei described herein.

Sparse SOM immunostaining was observed in the ventral horn of the opossum's spinal cord. Since most, if not all, of the dorsal root axons transporting SOM distribute to the dorsal horn (Hokfelt et al.,

'76), the SOM within the ventral horn is probably associated with spinal or supraspinal neurons. SOM neurons are present within laminae

VII-IX of the spinal cord and it is likely that they contribute to such innervation.

SOM immunostaining was present within the dorsolateral funiculus at all spinal levels, an area known to contain bulbospinal axons in the opossum (Martin et al., '79) and rat (Watkins et al., '80). Although 17 we have Identified SOM neurons within reticular nuclei of the brainstem, most of them are located within areas that only Innervate cervical and thoracic levels of the spinal cord (Martin et al., '79, ’81). It is possible, of course, that SOM neurons are present in other areas but were not demonstrated because of inadequate colchicine treatment.

In contrast to SOM elements, ENK immunoreactivity was only sparse in laminae I and II. Comparable results have been reported for the rat

(Elde et al., '76; Simantov et al., '77; Hokfelt et al., '77; Simantov and Snyder, '76; Johansson et al., '78; Sar et al., '78; Uhl et al.,

'79). In the rat, ENK elements have been shown to originate from neurons within both the spinal cord (Hokfelt et al., '77; Sar et al.,

'78; Uhl et al., '79) and brainstem (Hokfelt et al., '79; Bowker et al.,

'81). We have been able to demonstrate ENK cell bodies within presump­ tive laminae I-III of the developing opossum spinal cord, but they were not demonstrable in the adult. Whether these ENK perikarya are lost during development or they become more difficult to demonstrate remains unclear. If present in the adult opossum, they probably con­ tribute to the ENK immunoreactivity within laminae I and II. In the rat, Hokfelt et al. ('79) and Bowker et al. ('81) have shown that ENK neurons are located within certain regions of the raphe and adjacent reticular formation. The apparent homologues of these brainstem nuclei innervate laminae I and II of the opossum (Martin et al., '79).

Enkephalins have been implicated as interneuronal or descending com­ ponents in circuitry related to centrally induced analgesia (Basbaum and Fields, ’78). 18

ENK elements were sparse, but evenly distributed, within lamina

X, the intermediolateral cell column and laminae VII-IX of the ventral horn. Hokfelt et al. (’79) have demonstrated that ENK neurons within

the nucleus reticularis gigantocellularis pars alpha of the rat project

to the spinal cord. Since Martin et al. (’79) have described bulbo­ spinal axons projecting to the intermediolateral cell column, as well as to laminae IX and X which arise within homologous regions of the opossum's brainstem, some of them may be enkephalinergic.

Although serotonin cell bodies are present in the spinal cord of the developing opossum (see Chapter II), no evidence for them was detected in the adult. These data and the apparent depletion of 5-HT elements caudal to a spinal transection (figs. 18, 21-24) suggest that the serotonin elements observed in the cord arise mainly, if not exclusively, within the brainstem.

Unlike, SOM, but similar to ENK, 5-HT neuronal elements were sparse within laminae I and II. These data parallel those reported for the rat (Steinbusch et al., '81). In the opossum, projections to laminae I and II originate within the nucleus reticularis pontis pars ventralis, the nucleus reticularis gigantocellularis pars ventralis and the adjacent nucleus magnus raphae (Martin et al., '78, '81;

Goode et al., '80). All of the above nuclei have been shown to contain serotonin neurons (Crutcher and Humbertson, '78), some of which project to the spinal cord (Martin et al., '81). Descending

5-HT pathways have been reported to play a role in nociception modulation (Basbaum and Fields, '78). 19

The densest aggregation of 5-HT elements occurred within laminae

IX, X and the intermediolateral cell column. Within lamina IX, particu­ larly at mid-cervical and mid-lumbar levels, 5-HT varicosities surround motoneurons. Strong projections to laminae IX, X as well as to spinal autonomic areas originate within the nucleus reticularis gigantocellu­ laris pars ventralis (Martin et a l ., '79a), caudal parts of the nucleus magnus raphae (Martin et al., ’79b), the nucleus reticularis giganto­ cellularis (Martin et al., ’81) and the nuclei obscurus and pallidus raphae (Martin et al., ’81). Several investigators have demonstrated that serotonin has a facilatory effect on motoneurons (Chase and

Murphy, ’73; Amaral and Sinnamon, ’77).

While most of the results from our experimental procedures failed to provide evidence for the origin of spinal peptide elements, the spinal transection resulted in a virtual depletion of 5-HT caudal to the lesion. This strongly suggests its supraspinal origin. With regard to the peptides, inappropriate survival times most logically explains the lack of change in their normal distribution following spinal or dorsal root lesions. The one lesion-dependent change in the dsitribution of peptidergic elements was the enlargement of a bundle of SF fibers caudal to the lesion. This bundle appeared to be within the ventrolateral extreme of the tract of Lissauer. Coggeshall et al.

(’81) have recently shown that a species-dependent percentage of primary afferent axons is contained in the dorsolateral fasciculus

(of Lissauer). In a previous study (DiTirro et al., ’81), we demon­ strated SP-containing cell bodies within the dorsal root ganglia of the adult opossum. It is possible that the enlargement of the SP 20

bundle In question resulted from an accumulation of axoplasm and

organelles within either axons of dorsal root ganglion cells ascending within the tract of Lissauer or axons from propriospinal neurons.

While our material establishes close similarities In the spinal

distribution of SOM, ENK and 5-HT elements of the opossum and the rat,

our primary motivation for undertaking these studies was to establish

the adult pattern of these elements for use as baseline data in studies of their development. Our results emphasize the presence of both peptides and serotonin within spinal areas related to sensory processing, autonomic function and motor activity. ILLUSTRATIONS 22

Figure 1 Plots of substance-P (SP), somatostatin (SOM),

methionine-enkephalin (ENK) and serotonin (5-HT)

immunohistofluorescence at four representative

levels of the adult opossum spinal cord. Note the

sparse amounts of SP and SOM elements within the

ventral horn and the relative abundance of ENK and

5-HT elements within comparable regions. The curved

arrow indicates the intermediolateral cell column

and the stars denote the location of SOM perikarya. ADULT

SACRAL 24

PLATE I

Figure 2 Photomicrograph of SOM immunoreactive elements in

laminae I (I) and II (II). The dorsal funiculus is

indicated (DF). The bar represents 100pm for

figures 2-5.

Figure 3 Photomicrograph of a transverse section of the opossum

cord at the cervical enlargement. The arrows indicate

punctate and/or varicose structures containing SOM

immunoreactivity. The central canal is indicated

(CC).

Figure 4 Photomicrograph of a transverse section from the

thoracic cord of the opossum showing SOM elements

within the intermediolateral cell column (arrow).

The lateral funiculus is labelled (LF).

Figure 5 Photomicrograph of a transverse section of the

opossum’s sacral cord. The open arrows indicate

SOM immunoreactive elements within the sacral para­

sympathetic area. The lateral funiculus is

indicated (LF). © 26

. PLATE II

Figure 6 Micrograph of a transverse section from the thoracic

cord of the opossum. The dorsal horn exhibits

enkephalin (ENK) immunoreactivity within laminae I

and II. The dorsal funiculus is Indicated (DF) and

the bar represents 80ym for figures 6-9.

Figure 7 Micrograph of a transverse section of opossum spinal

cord taken at the cervical level. ENK immunofluores­

cence can be seen around the central canal (CC). The

dorsal funiculus is labelled (DF).

Figure 8 Micrograph of a transverse section from the thoracic

cord of the opossum showing a high magnification of

the intermediolateral cell column. ENK elements can

be seen within the same area. The lateral funiculus

is labelled (LF).

Figure 9 Micrograph of a transverse section from the lumbar

cord of the opossum showing sparse ENK immunostain­

ing within the ventral horn. Lateral funiculus is

labelled (LF). DF

c c

NJ 28

PLATE III

Figure 10 Photomicrograph of ENK within the dorsal horn of a

midthoracic section incubated with antisera specific

for M-ENK. ENK immunoreactive elements can be

observed within lamina I (I) and lamina II (II).

The dorsal funiculus (DF) is labelled and the bar

represents 80ym.

Figure 11 Photomicrograph of a semiadjacent section (opossum)

incubated with preabsorbed antisera for ENK. The

control antisera failed to localize ENK to comparable

areas when processed in parallel with routine staining

procedures. Laminae I (I) and II (II) as well as the

dorsal funiculus (DF) are indicated. The bar repre­

sents 80ym. 29

o PLATE IV

Figures 12, Semiadjacent transverse sections of the opossum's

sacral cord which were processed in parallel by the

indirect immunofluorescent method of Coons. Section

shown in figure 10 was incubated with serotonin (5-HT)

antiserum (194D). Note the punctate fluorescent

varicosities that stained for 5-HT immunoreactivity

in the superficial laminae of the dorsal horn. The

section shown in figure 11 was incubated with anti­

serum (194D) pretreated with an excess of synthetic

serotonin. Note the absence of immunostaining in

comparable areas of the dorsal horn. Laminae I (I)

and II (II) and the dorsal funiculus (DF) are

indicated. The bar represents 80ym.

Figure 14 Micrograph of a transverse section from the thoracic

cord of the opossum showing the intermediolateral

cell column. Serotonin immunostaining can be seen.

Lateral funiculus (LF). The bar represents 80pm.

Figure 15 Micrograph of a transverse section of opossum spinal

cord taken from a cervical level. 5-HT immunofluores­

cence can be seen around the central canal (CC). The

dorsal funiculus is Indicated (DF) and the bar

represents 80pm. 31

PLATE IV (CONTINUED)

Figure 16 Micrograph of transverse section from the lumbar cord

of the opossum showing 5-HT immunostaining within the

ventral horn. The white arrow indicates serotoniner-

gic elements surrounding a presumed motor neuron.

The lateral funiculus (LF) is indicated. The bar

represents 80ym.

Figure 17 Photomicrograph of a transverse section of the

opossum's cervical cord showing 5-HT varicosities

adjacent to the dorsal spinocerebellar tract (dsc).

The bar represents 80pm.

33

Figure 18 Plots of SP, ENK and 5-HT immunoreactivities at

five representative levels of the spinal cord

following a spinal transection at the eighth thoracic

level (T8). Note that the distributions of SP and

ENK are unchanged when compared with their normal

distributions (fig. 1), while 5-HT elements are

virtually absent caudal to the lesion. C8=eighth

cervical segment; T6=sixth thoracic segment;

Tll=eleventh thoracic segment; and L3=third lumbar

segment. 5-HT

C8

T6

LESION LESION

T tl

L3

FIG. 18 SACRAL SACRAL 35

PLATE V

Figure 19 Photomicrograph of a transverse section from the

fourth thoracic segment of an opossum that received

a spinal transection at the eighth thoracic level

(T8). The dorsal horn exhibits SP immunoreactivity

within laminae I (I) and II (II). The arrow indi­

cates a bundle of SP fibers within the dorsolateral

fasciculus. The dorsal funiculus (DF) is indicated

and the bar represents 100pm for figures 19-24.

Figure 20 Photomicrograph of a transverse section from the tenth

thoracic segment of an opossum that received a spinal

transection at T8. The dorsal horn exhibits SP

immunostaining within laminae I (I) and II (II). The

bundle of SP fibers referred to in figure 19 appears

markedly enlarged (arrow) in this and other thoracic

sections, caudal to the lesion. The dorsal funiculus

(DF) is indicated.

Figure 21 Photomicrograph of 5-HT immunoreactive elements in

the intermediolateral cell column. This section was

taken from the thoracic cord, rostral to the spinal

transection. The lateral funiculus (LF) is

indicated. 36

Figure 22 Photomicrograph of a transverse section from the

thoracic cord caudal to a spinal transection. Note

the lack of 5-HT elements within the intermediolateral

cell column (arrow). The lateral funiculus (LF) is

indicated.

Figure 23 Photomicrograph of a transverse section of the

cervical cord taken at a level rostral to a spinal

transection. Note the aggregation of 5-HT fibers

(arrow) in the periphery of the dorsolateral

funiculus (DLF).

Figure 24 Photomicrograph of a transverse section of the thora­

cic cord taken at a level caudal to a spinal transec­

tion. Note the lack of 5-HT immunostaining within

the dorsolateral funiculus (DLF). 37

DLF 38

PLATE VI

Figures 25, 26 Micrographs of SP immunoreactivity within the dorsal

horn of the third lumbar segment. Figure 26 illus­

trates the pattern of SP staining 5 days after tran­

section of adjacent dorsal roots on that side, while

figure 25 illustrates the pattern of SP immunostain-

ing within the dorsal horn of the unoperated side.

Note the close similarity in the distribution of SP

immunoreactivity within laminae I (I) and II (II).

The dorsal funiculus (DF) is labelled and the bar

represents 80ym for figures 25 and 26. 39

o IMMUNOHISTOCHEMICAL STUDIES OF SPINAL PEPTIDE AND SEROTONIN

ELEMENTS IN THE NORTH AMERICAN OPOSSUM, Didelphls virginiana.

II. THE ONTOGENY OF SPINAL PEPTIDERGIC AND SEROTONINERGIC

IMMUNOREACTIVE ELEMENTS IN THE OPOSSUM.

INTRODUCTION

Although spinal peptidergic elements have been studied in a variety of adult animals (see Hokfelt et al., '77, '78; Ljungdahl et al., '78; Sar et al., '78; and DiTirro et al., '81 for a historical perspective) little is known about their development. Substance-P (SP) has been found in the rat spinal cord by embryonic day sixteen

(Gilbert and Emson, '79) and within dorsal root ganglion of the same species 1 day after birth (Kessler and Black, '80). Since radioimmuno­ assay (RIA) techniques were used for these studies they did not provide information regarding the growth of SP-containing neurites within specific areas at different stages of development. In similar studies,

Ho et al. ('78) have revealed RIA demonstrable levels of somatostatin

(SOM) in the spinal cord of the neonatal rat. These results were further substantiated by morpological studies utilizing the immuno- fluorescent technique of Coons (’58) (Ho et al., '78). To date, there

40 Al have been no reports on the ontogeny of enkephalinergic (ENK) elements and developmental studies of serotonin (5-HT) spinal elements are also lacking.

In light of the presumed Interaction between peptide and indola- mine systems in centrally induced analgesia (see review by Basbaum and

Fields, ’78) as well as other functions, we have sought to determine their developmental history in the spinal cord. The opossum has been used for our studies because of its early birth (12+1 days after con­ ception) and its protracted development (see Martin et al., *78 and

Humbertson and Martin, '79). The latter factor may have allowed us to observe sequences of development that may be undetectable in species possessing a more accelerated maturation. The adult organization of spinal peptides (DiTirro et al., *81) as well as serotonin (DiTirro et al., '80) has been reported elsewhere. materials and methods

The pouch-young opossums (Dldelphis virginlana) used for this

study were either bred in captivity at The Ohio State University or

taken from females captured in the wild. In the former case, the exact

age of the specimens was known, whereas in the latter it was estimated

from snout-rump (S-R) lengths using growth curves published by Cutts

et al. (’78) and corroborated by our own data. The snout-rump lengths were measured after straightening the animal as much as possible without

injuring it. Although only seven stages are indicated by our plots and photomicrographs pouch-young opossums were sacrificed, processed and

examined at each 10mm (S-R length) interval between 18mm and 127mm.

Thirty-three pouch-young opossums were used in this study. Most of them were anesthetized by Metaphane (2,2-Dichloro-l,1-difluoro ethyl methyl ether) inhalation and sacrificed by intracardiac perfusion with phosphate buffered saline (PBS) (4°C) followed by Zamboni's fixative

(Stephanlni et al., *67) for 30 minutes at room temperature (RT).

Their spinal cords and brains were promptly removed and placed in

Zamboni's fixative for 4-6 hours (4°C) followed by immersion in a 5% sucrose Sorensen’s phosphate buffer solution at 4°C for 1-2 days.

Opossums that were too small to be perfused effectively (i.e. less

42 than 35mm S-R length) were decapitated, eviscerated and Immersed In

Zamboni's fixative overnight at 4°C. Their spinal cords were left within the vertebral column and processed in a manner similar to that described above. Cryostat sections were cut transversely at lOpm from cervical, thoracic and lumbar levels and mounted on chrome-alum gelatin-coated slides. These sections were processed for peptides and serotonin (5-HT) using the indirect antibody peroxidase-anti- peroxidase (PAP) technique of Stemberger ('70) for all animals and the indirect immunofluorescent (FITC) method of Coons ('58) for selected sections from some animals. In order to minimize the possi­ bility of inter-animal differences, adjacent or semi-adjacent sections from each animal were processed for both the peptides and serotonin.

Tissues processed using the PAP technique were rehydrated with borate buffered saline (BBS) and incubated with either anti-SP serum

(diluted 1:1000), anti-SOM serum (diluted 1:500), anti-ENK serum

(diluted 1:500), anti-5-HT serum (diluted 1:1000) or the appropriate control serum for 48 hrs. at 4°C in a humid environment. The sections were then incubated consecutively with sheep-anti-rabbit immunoglobulin

G (SAR) antisera (diluted 1:300) for 1 hour at room temperature and a rabbit peroxidase-anti-peroxidase (PAP) complex (diluted 1:500) for

1 hour at room temperature. The sections were rinsed and immersion- washed in BBS after each incubation. The diluent for all antisera used in the PAP technique was 0.5% bovine serum albumin (BSA) in borate buffered saline containing 0.3% Triton X-100 (Octyl Phenoxy Polyethoxy- ethanol), with the exception of the anti-5-HT serum whose diluent was

1.0% bovine serum albumin in borate buffered saline containing 0.3% 44

Triton X-100 (Octyl Phenoxy Polyethoxyethanol). The diluent for the

PAP complex was BBS-BSA.

Following incubation with the PAP complex, sections were immer­

sion-washed in BBS and placed in a phosphate buffered saline solution

(room temperature) containing 3,3'-diamino-benzidine tetrachloride (DAB)

(0.3g/600ml) and 0.096% hydrogen peroxide for 5-10 minutes. The sections

were then immersion-washed in BBS, dehydrated in a series of graded

alcohols, cleared with xylene and coverslipped with Permount. Pro­

cessed sections were examined and photographed using a Zeiss photo­

microscope. Plots illustrating the distribution of peptide and

serotonin (5-HT) immunoreactivities were constructed on drawings of

adjacent or semi-adjacent Nissl stained sections from each of the processed spinal cords.

Tissues processed by the FITC method were rehydrated with phos­

phate buffered saline (PBS) and incubated for 24 hrs. (4°C) with either

anti-SP serum (diluted 1:100), anti-SOM (diluted 1:100), anti-ENK serum

(diluted 1:100), anti-5-HT serum (diluted 1:100) or the appropriate

control serum. The diluent for all anti-sera used in the FITC tech­

nique was PBS containing 0.3% Triton X-100. Sections were rinsed and

immersion-washed in PBS following each incubation. Subsequent to incu­

bation with the primary antisera, the tissues were exposed to

fluorescein isothiocyanate conjugated goat-anti-rabbit (GAR) Immuno­

globulin G anti-sera (diluted 1:30) for 1 hour at room temperature.

After incubation with GAR, the sections were rinsed and immersion-

washing in PBS and coverslipped with a glycerin-PBS solution (3:1).

The processed materials were observed and photographed using a Zeiss 45 photomicroscope with reflected illumination (excitation wavelength

440-490nm).

Antisera

Rabbit anti-SP serum (6B and 6C) was obtained from the second and third bleeding of rabbit //6 (Ho and DePalatis, ’80) which had been immunized with a synthetic SP-thyroglobulin conjugate. Rabbit anti-SOM serum (11C) was acquired from the third bleeding of rabbit #11 (Ho and

DePalatis, '80) which had received a synthetic SOM-keyhole limpet hemocyanin conjugate. Rabbit anti-ENK serum (18G) (DiTirro et al.,

’81) was obtained from the seventh bleeding of rabbit #18 which had been immunized with a synthetic methionine-enkephalin-thyroglobulin conjugate. The serum raised against 5-HT (194D) was obtained from the fourth bleeding of rabbit #194 (Holets et al., '81; Holets and

Elde, submitted for publication) which had received a synthetic serotonin-bovine serum albumin conjugate. The anti-5-HT serum was kindly provided by Dr. Robert Elde (University of Minnesota). The

SAR and GAR sera were purchased from Antibodies Incorporated and the

PAP complex was obtained from Cappel Laboratories.

In order to establish the specificity of immunostaining, sections from all representative levels of the spinal cord were taken from every pouch-young opossum examined and incubated with each of the control sera and processed in parallel with the experimental sections. Control sera consisted of the diluted anti-SP, anti-SOM, anti-ENK and anti-5-HT sera pretreated with an excess of synthetic SP, SOM, ENK and 5-HT (20yg/ml of diluted anti­ serum) (overnight at A°C) respectively. For further antibody informa­ tion, please see Appendix A. RESULTS

Development of Presumed Neuronal Processes Containing

Peptides and Serotonin (5-HT) Immunoreactlvitles

Substance-P (SP)

A small amount of substance-P immunoreactivity is present in the

spinal cord of newborn opossums (18mm S-R length, 12-13 days after con­

ception) . A few SP elements are aggregated at the edge of the dorso­ lateral marginal zone (arrow, left side of fig. 27), especially at

cervical and thoracic levels, and an occasional one is found within more dorsal and ventral areas (fig. 27). There is no evidence for SP in the intermediate (mantle) zone. By postnatal day 5' (24mm S-R length)

SP varicosities are present within the intermediate zone (fig. 28) where they are most numerous within the superficial areas of the dorsal horn

(presumptive laminae I and II of Rexed, ’52) (open arrow, fig. 34), within lateral regions of the dorsal horn (presumptive lamina X)

(fig. 28). A few varicosities are scattered more ventrally where they occupy areas corresponding in position to laminae VII-VIII in the adult animal (fig. 28). By day 30 (57mm S-R length) SP elements are more numerous within superficial areas of the dorsal horn (figs. 30 and 35) and within lamina X (figs. 30 and 36) and are found for the first time

47 in an area that can be Identified as interxnedlolateral cell column

(curved arrows, left side of figs. 30 and 37). Presumed SP fibers

are also aggregated in a bundle located within the ventral-most part

of the dorsolateral fasciculus at thoracic levels (solid arrow, left

side of fig. 30, curved arrow, fig. 35). In addition, SP varicosities

are obvious within areas adjacent to the central canal (presumptive

lamina X) (figs. 30 and 36). This general pattern of immunostalning

within the grey matter persists with minor variations in the density

of varicosities until adulthood (fig. 33). SP is relatively abundant

in the marginal zone of the developing opossum but its density appears

to diminish by 127mm.

Somatostatin (SOM)

The spinal cords of newborn opossums fail to show SOM immunoreac­

tivity (fig. 1). However, by postnatal day 5 (24mm S-R length) SOM is

present within all of the presumptive funiculi of the marginal zone

and within the intermediate zone (figs. 28 and 38). Within the inter-

V mediate zone SOM elements are most numerous dorsally (presumptive

laminae I and II) and there is a suggestion of a rostral to caudal

gradient (compare cervical and lumbar levels in fig. 28). At cervical

levels SOM is also found within lateral areas of the dorsal horn

(lateral parts of presumptive laminae IV-VI) and lateral to the

ependyma (fig. 28). Only a few scattered varicosities are seen in more ventral areas of the intermediate zone and most of them are

present at cervical levels (fig. 28). SOM cell bodies are also

demonstrable at this stage (see section on cell bodies). By day 15 49

(35mm S-R length), SOM iumnmoreactivity within the developing grey matter is generally comparable at all levels (fig. 29). By approxi­ mately 30 days in the pouch (57mm S-R length) SOM elements are present within presumptive lamina X (fig. 30; open arrows, fig. 39) and a few are found in the lntermediolateral cell column (fig. 30). Subsequent to day 30 the density of immunostalning increases within the developing grey matter, but appears to decrease within the white matter. For example, by day 50 (84mm S-R length), there is almost no SOM within the white matter although it is fairly abundant in the spinal grey

(fig. 31). The adult distribution of SOM (figs. 33, 42-45) is present by at least 75 days in the pouch (127mm S-R length) (figs. 32 and 41).

Although SOM is abundant in the sacral parasympathetic area (DeGroat et al., ’78) of the adult opossum (figs. 33 and 45) our series does not include enough sacral sections to speak definitely concerning its development.

Enkephalin

A small amount of ENK immunoreactivity is present within the spinal cords of 1-day-old opossums (18mm S-R length) where it is restricted to lateral and ventral parts of the marginal zone at cervical and thoracic levels (fig. 27). ENK elements are not ob­ served within the intermediate zone until about 20 days after con­ ception (fig. 28). At the same age, ENK varicosities are fairly abundant within lateral and ventral areas of the marginal zone

(fig. 28, open arrows, fig. 46). By postnatal day 15 (35mm S-R length) a small amount of ENK immunoreactivity is present within all 50 areas of the spinal gray described as containing it in the adult animal (DiTirro et al., ’81) with the exception of the superficial laminae of the dorsal horn. Such areas include the deeper areas of the dorsal horn, lateral portions of the dorsal and ventral horns, the intermediolateral cell column and the ventral horn (fig. 29). At this age, ENK elements are also present within presumptive lamina X where they become fairly abundant by 30 days in the pouch (57mm S-R length)

(figs. 30 and 47). Unlike the case in the adult, however (fig. 33),

ENK elements are also present within the dorsal funiculus (fig. 30).

Such varicosities persist until approximately 58 days after birth

(95mm S-R length). At about 50 days of age (84mm S-R length) ENK varicosities can be observed for the first time within superficial areas of the dorsal horn (laminae I and II). With the onset of such innervation (fig. 31), the adult pattern of ENK innervation is essen­ tially established. Only the density of immunostaining is greater at

127mm (figs. 32 and 49) and within the adult animal (fig. 33).

Serotonin (5-HT)

Serotonin immunoreactivity is remarkably abundant within the marginal zone of the cervical cord in newborn opossums (open arrow, right side of fig. 27, open arrow, fig. 50). A few 5-HT varicosities are even present in the intermediate zone. At thoracic and lumbar levels, however, immunostaining is sparse to absent (fig. 27). At cervical levels, serotonin (5-HT) elements are found within dorsal

(although sparse), dorsolateral, lateral and ventral areas of the marginal zone, whereas more caudally, the few which are present are 51

limited to lateral and ventral areas (fig. 27). By postnatal day 5

(24mm S-R length) 5-HT elements are scattered throughout most of the

developing gray matter, particularly at cervical and thoracic levels,

except for the superficial areas of the dorsal horn (presumptive

laminae I and II) (fig. 28). There is evidence for a rostral to caudal

gradient for the growth of 5-HT elements into the grey matter although

5-HT immunoreactive elements are fairly numerous within most areas of

the marginal zone at all levels. By day 15 (35mm S-R length) 5-HT elements are abundant within deep areas of the dorsal horn (presumptive laminae IV-VI), adjacent to the ependymal layer (presumptive lamina X) and within the ventral horn (presumptive laminae VII-IX) (fig. 29).

In the latter region, some of the varicosities approximate the perikarya of motoneurons (curved arrow, fig. 29, open arrow, fig. 51). By approximately 30 days in the pouch (57mm S-R length) aggregations of

5-HT elements are even more conspicuous within presumptive lamina IX

(fig. 30) and are found in an area extending from the central canal laterally to the intermediolateral cell column (solid arrow, fig. 30, curved arrow, fig. 53). The perineuronal nature of the 5-HT varicosi­ ties within lamina IX is even more apparent than at 35mm. The distri­ bution of 5-HT immunoreactivity remains seemingly unchanged until approximately 50 days in the pouch (84mm S-R length) at which time

5-HT elements are first detectable within superficial regions of the dorsal horn (laminae I and II) (fig. 31, open arrows, fig. 54). With the appearance of such innervation, the general distribution of 5-HT immunoreactivity resembles that in the adult (fig. 33). For comparison, 52

plots of serotonin iunnunoreactivity are shown for both the 127mm S-R

length and adult animals (figs. 32 and 33).

Distribution of Peptide and Serotonin Immunoreactive Cell Bodies

Within the Spinal Cord at Different Stages of Development

Somatostatin

As early as 5’ days after birth (24mm S-R length) SOM-containing perikarya are present within superficial areas of the dorsal horn (pre­ sumptive laminae I and II). They are characterized by brown reaction product within their cytoplasm which abuts a negatively-stained nucleus.

By approximately 30 days of age (57mm S-R length) SOM immunoreactivity is found within an area interpreted to be the axon hillock (inset,

fig. 40). Although SOM cell bodies are present within the dorsal horn at 24, 35 and 57mm, there are relatively few varicosities (fig. 40).

By postnatal day 50 (84mm S-R length) the number of stainable SOM perikarya has diminished markedly. This phenomenon is accompanied by an increase in the density of SOM varicosities. In the 127mm adult animals, the superficial laminae of the dorsal horn are dominated by

the presence of SOM varicosities and SOM cell bodies are only present occasionally (fig. 41).

SOM cell bodies are not apparent in laminae IV-X until approxi­ mately 15 days after birth (35mm S-R length) where they are found at cervical levels (fig. 29). By postnatal day 30 (57mm S-R length)

SOM-containing perikarya are present at all of the spinal levels examined (figs. 30 and 39). This distribution of cell bodies remains 53 virtually unchanged during development and continues throughout adulthood (figs. 29-33, 39).

Enkephalin

ENK perikarya are first detectable within the superficial laminae of the dorsal horn (presumptive laminae I and II) at approxi­ mately 30 days after birth (57mm S-R length) (inset, fig. 48). At that stage, ENK varicosities are sparse within the same areas. By approximately 37 days in the pouch (65mm S-R length), ENK perikarya are particularly numerous within presumptive laminae I and II. When the opossums are about 42 days old (74mm S-R length), the number of

ENK cell bodies appears to decrease markedly in laminae I and II while the density of ENK varicosities within the same areas increases.

By postnatal day 75 (127mm S-R length) ENK perikarya are almost unde­ tectable, whereas ENK varicosities are present throughout the super­ ficial laminae of the dorsal horn (figs. 32 and 49). A comparable distribution of ENK elements can be observed within laminae I and II of the adult animal (fig. 33).

Serotonin

Although 5-HT-containing perikarya are not seen in the spinal cord at birth (18mm S-R length), they are present within ventral areas of the cervical and thoracic marginal zone by day 5 (24mm S-R length)

(fig. 28). By postnatal day 15 (35mm S-R length) 5-HT cell bodies are present within an area extending from the sulco-marginal region to the central canal at cervical and thoracic and lumbar levels (fig. 29). 54

By day 50 (84mm S-R length) they are limited to cervical levels only

(fig. 31) and by postnatal day 75 (127mm S-R length) they are restricted to rostral cervical levels (figs. 32 and 55). The spinal cords of adult opossums fail to exhibit 5-HT perikarya at any level.

Control Sera

Our control sera failed to localize immunoreactive elements on sections adjacent or semi-adjacent to those processed with the non­ pretreated antisera (figs. 56-63). The possibility that our methionine- enkephalin antiserum cross-reacts with leucine-enkephalin can not be ruled out. Thus, some of the ENK immunostaining may be demonstrating leucine-enkephalin. DISCUSSION

We have employed primarily the indirect antibody PAP technique

rather than the immunohistofluorescent method because it provides

permanently stained and mounted material that is conveniently handled

and stored. Selected spinal sections were also processed by the immuno-

fluorescent method of Coons (’58), however, yielding generally comparable

results. In our hands the PAP technique was superior for revealing

small amounts of the peptides studied. Although 5-HT innervation can

also be studied by the Falck-Hillarp method, indolamine fluorescence

decays rapidly when exposed to ultraviolet light making it difficult

to produce accurate reproductions and photomicrographs in young animals.

We employed Zamboni's fixative (Stephanini et al., '67) because it

allowed us to demonstrate peptide containing elements especially at younger ages than the more conventionally used paraformaldehyde. In

fact, SOM immunoreactivity is not demonstrable in the spinal cords of

the adult opossum using paraformaldehyde as the fixative (DiTirro

et al., ’81).

Our results demonstrate the early appearance (day 1, 18mm S-R

length) of both SP and ENK elements within the marginal zone of the spinal cord. Although a substantial portion of SP containing axons

55 56 within the spinal cord are of dorsal root origin (the cat, Hokfelt et al., ’77; the rat, Barber et al., ’79), descending SP pathways have been demonstrated (Sjolund et al., '77; Kanazawa et al., ’79).

HSkfelt et al. (’77) showed that spinal ENK in the rat is mostly con­ tained within axons of interneurons although descending ENK pathways exist (Hokfelt et al., ’79). While we have not been able to demonstrate either SP or ENK perikarya within the brainstem of newborn opossums, the presence of these peptides in the periphery of the dorsolateral marginal zone (a region of the white matter shown by Martin et al.,

'78, to contain descending axons in the developing opossum) suggests that they are contained, at least partly, in neurites of brainstem origin.

The coexistence of SP, SOM and ENK within the superficial laminae of the dorsal horn together with their purported physiological effect upon dorsal horn neurons (see Basbaum and Fields, '78, for review) indicates that they participate in sensory processing. It is inter­ esting, therefore, to note the innervation sequence of the dorsal horn by these peptides during development. By 5 days after birth (24mm S-R length) SP and SOM are present within the superficial laminae of the dorsal horn (presumptive laminae I and II). Although sparse, SP immunostaining resembles that in the adult animal (DiTirro et al., '81) and is located within elements interpreted to be axons and their terminals. In contrast, SOM immunostaining appears to be mainly within the cytoplasm of small cells restricted to presumptive laminae

I and II. Until approximately one month after birth (57mm S-R length)

SOM staining in the dorsal horn remains primarily within perikarya. 57

Subsequent to this time, the number of SOM-containing perikarya

decreases, while the density of varicose SOM elements increases.

Whether many of the SOM cell bodies die during development or whether

they simply become more difficult to demonstrate is unclear. Nonethe­

less, they probably contribute, at least in part, to the SOM immuno-

staining during development.

In contrast to the early appearance of SP and SOM, ENK elements

are not detectable within the dorsal horn until at least one month

after birth. At that time (approximately 57mm S-R length), an occa­

sional cell body exhibiting ENK immunostaining can be found. Shortly

following the appearance of these cell bodies, ENK elements interpreted

to be axons are also present and the dorsal horn staining resembles

that of the adult animal (DiTirro et al., ’81). Although most ENK-

elements within the spinal cord originate from interneurons, HSkfelt

et al. ('79) have demonstrated that ENK neurons within the nucleus

reticularis gigantocellularis pars alpha project spinalward. Martin

et al. (’81) have shown that neurons within a seemingly comparable

region of the opossum brainstem project to lamina I and the outer

region of lamina II and it is possible that some of them provide ENK

innervation. The possible significance of the relatively late inner­ vation of the dorsal horn by ENK elements and its relationship to the

appearance of serotonin elements within the same areas will be dis­

cussed later.

SP, SOM and ENK elements are obvious within lamina X and the

intermediolateral cell column by 1 month after birth (57mm S-R length), but they are probably present earlier. These areas have been shown to 58

receive axons from raphe and reticular neurons in the adult opossum

(Martin et al., '79, '81) and some of them are indolaminergic. Since

other studies (HSkfelt et al., '79; Bowker et al., ’81) have shown that

SP and ENK neurons of the raphe and reticular formation innervate the

spinal cord and that at least SP and 5-HT can exist within the same

neuron, it is tempting to speculate that some of the peptides within

the intermediolateral cell column are transports within bulbar axons.

Of course they may also be associated with spinal interneurons.

To date, descending SOM pathways arising from nuclei within the

hindbrain have not been demonstrated. However, Sawchenko and Swanson

('80) have revealed spinal projections from SOM-containing neurons

within the paraventricular nucleus of the rat. Axons from the para­

ventricular nucleus are known to innervate the intermediolateral cell

column (Saper et al., ’76) and some of them may transport SOM. In

addition, some of the SOM cell bodies within laminae IV-X (Hancock and

Pevets, ’79) may contribute to the innervation of the intermediolateral

cell column.

We have reported previously that only sparse amounts of SP and

SOM elements are present within the ventral horn of the adult opossum

(DiTirro et al., '81). Our results indicate that the adult patterns

of ventral horn immunostaining for both SP and SOM are well-developed by at least 30 days after birth (57mm S-R length). Hifkfelt et al.

('78) and Bowker et al. (’80) have revealed SP-containing perikarya within raphe and reticular areas of the brainstem which project to the

spinal cord. Since Martin et al. (*81) have shown that the ventral horn (especially lamina IX) is innervated by neurons within comparable 59

areas of the opossum, it is possible that some of the ventral horn SP

is associated with bulbar axons. Although descending SOM projections

to the ventral horn have not been demonstrated it is possible that they

exist. SOM-perikarya are present within the spinal cord of the devel­

oping and adult opossum, however, and they probably contribute to the

innervation of the ventral horn.

ENK innervation of the ventral horn occurs relatively early in

development (no later than 15 days after birth, approximately 35mm S-R

length) and is well developed 30 days after birth (57mm S-R length).

Although ENK axons within the spinal cord are thought to originate mainly from interneurons in the rat (Hokfelt et al., ’77), a descending bulbar component has been revealed which originates within the nucleus reticularis gigantocellularis and adjacent raphe (Hbkfelt et al., ’79;

Bowker et al., *80). Martin et al. ('81) have shown that neurons within

the nucleus reticularis gigantocellularis project directly to the ventral horn of the opossum and it is possible that some of them transport enkephalins.

Serotonin is present in the spinal cord of the newborn opossum.

Since 5-HT cell bodies were found in the brainstem at the same age

(unpublished results), and not the spinal cord, it appears that most, if not all, of the spinal 5-HT immunoreactivity is associated with bulbar axons.

By the 2nd week in the pouch, 5-HT neurites have reached speci­ fic targets within the spinal gray matter. Humbertson and Martin ('79) have reported that monoamine (probably catecholamine) fluorescence can be detected in the ventral intermediate zone (developing ventral horn) as early as 16 days after birth (38mm S-R length) and that It Is well- developed In lamina IX by at least day 22 (approximately 46mm S-R length). In that report the authors pointed out that they were not able to demonstrate indolamine fluorescence with certainty because of its transient nature when exposed to ultraviolet light. Using the PAP technique we demonstrated 5-HT varicosities within the ventral horn as early as postnatal day 5 (24mm S-R length) although their characteris­ tic perineuronal relationship to presumed motoneurons was not obvious until somewhat later (postnatal day 15, 35mm S-R length). Experiments in the adult opossum indicate that indolamine projections to lamina IX originate within the nuclei obscurus and caudal magnus raphae as well as within the adjacent reticular formation. As reported by Chase and

Murphy (’73) and Amaral and Sinnamon ('77), 5-HT may exert a facila- tory effect upon motoneurons.

Martin et al. (’78) have described three stages in the develop­ ment of the pouch-young opossum based on the onset of and development of hindlimb motility. Direct motoneuronal innervation by 5-HT neurites is apparently not related to the first appearance of spontaneous limb motility but it may have a role in its later refinement.

Monoamine innervation of lamina X and the intermediolateral cell column also occurs early in development (Humbertson and Martin, *79).

Our material provides evidence for 5-HT innervation of such areas by at least postnatal day 30 (57mm S-R length) and probably earlier.

Catecholamine projections to the same area have been reported for 38mm specimens (Humbertson and Martin, '79). 61

One of the major findings of this study is the relatively late

innervation of laminae I and II by 5-HT projections. Comparable results

have been reported for catecholamine projections to the same laminae

(Humbertson and Martin, ’79). In the adult opossum (Martin et al., '79,

’81), the former connections arise within the nucleus magnus raphae,

the nucleus reticularis gigantocellularis pars ventralis and the

nucleus pontis pars ventralis (terminology of Oswaldo-Cruz and Rocha-

Mlranda, ’68). Indolamine innervation of laminae I and II has been

implicated in centrally induced analgesia (see review by Basbaum and

Fields, ’78). Based on the developmental sequences of 5-HT reported herein, it might be predicted that indolamine control of somatic motor

and autonomic activity precedes modulation of somatosensory processing.

Interestingly, ENK varicosities were not demonstrable within

laminae I and II until just prior to the appearance of 5-HT elements.

Basbaum and Fields (f78) have suggested that ENK interneurons play a

role in the circuitry which elicits opiate analgesia.

Our ability to demonstrate peptides in cell bodies was age de­

pendent. For example, SOM perikarya were present within superficial

laminae of the dorsal horn (laminae I and II) throughout much of the

developmental sequence, but they became less obvious in the older animals. Although enkephalin cell bodies were detectable at certain stages of development they were not demonstrable in the adult animal.

Serotonin was also found in neuronal somata of pouch-young animals, but not in the adult. It is not known whether the neurons in question

are lost during development or whether they simply become more diffi­

cult to demonstrate. In summary, our study shows that peptides known to be trans­ ported in dorsal root axons (SP and SOM) reach their definitive targets in the dorsal horn early in development. In contrast, the development of ENK systems, thought to originate either within the spinal cord or brainstem, is somewhat more protracted. Although 5-HT projections appear early in development, they do not innervate laminae I and II until long after they have grown into more ventral spinal areas. These data suggest that the circuitry involved in centrally induced analgesia

(Basbaum and Fields, ’78) appears relatively late in development. ILLUSTRATIONS

63 64

Figure 27 Schematic representation of substance-P (SP), somato­

statin (SOM), methionine-enkephalin (ENK) and serotonin

(5-HT) immunoreactivities at three representative

levels from a newborn opossum (day 1) spinal cord.

Note the lack of SOM. The solid arrow (left side)

indicates the SP in the dorsolateral marginal zone.

The open arrow (right side) indicates 5-HT elements

within the same area. 18 MM SOM ENK

THORACIC

LUMBAR

0.3MM FIG. 27

Figure 28 Plots illustrating the spinal distribution of

substance-P (SP), somatostatin (SOM), methionine-

enkephalin (ENK) and serotonin (5-HT) at three rep­

resentative levels from a 5 day old pouch-young

opossum. Note the rostral-caudal gradient of SOM

elements and the paucity of ENK elements within the

intermediate zone. The stars indicate immunoreactive

perikarya. SOM cell bodies are present within the

dorsal horn at this stage, but they have not been

plotted for the sake of clarity (see Results). 24 MM SOM ENK __ 5-HT

X

CERVICAL

THORACIC

LUMBAR

0.3MM FIG. 28 68

Figure 29 Drawings of the distributions of substance-P (SP),

somatostatin (SOM), methionine-enkephalin (ENK) and

serotonin (5-HT) at three representative spinal levels

from a 15 day old pouch-young opossum. Note the

abundance of 5-HT elements within the intermediate

zone at all spinal levels. Serotonin (5-HT) elements

abutting presumed motoneurons can be observed (curved

arrow). The stars indicate the position of 5-HT cell

bodies. SOM and ENK cell bodies are also present

within superficial areas of the dorsal horn, but

they have not been included for the sake of clarity

(see Results). 35 MM SOM ENK 5-HT

LUMBAR 0.3MM FIG. 29

ON V£> 70

Figure 30 Schematic representation of substance-P (SP), somato­

statin (SOM), methionine-enkephalin (ENK) and seroto­

nin (5-HT) distributions at three representative

levels of the spinal cord from a 30 day old opossum.

Note the abundance of SP and SOM within the superfi­

cial laminae of the dorsal horn and the lack of ENK

and 5-HT elements within comparable regions. The

solid arrow (left side) indicates a bundle of SP-

containing fibers. The curved arrow indicates SP

elements within the intermediolateral cell column.

5-HT elements are aggregated within comparable areas

(right side). Cell bodies are indicated by the stars.

SOM and ENK cell bodies are present in superficial

areas of the dorsal horn, but they have not been

included for the sake of clarity (see Results). 57MM SOM ENK 5-HT

CERVICAL

THORACIC

LUMBAR

0.5mm FIG. 30 72

Figure 31 Plots illustrating the spinal distribution of sub-

stance-P (SP), somatostatin (SOM), methionine-

enkephalin (ENK) and serotonin (5-HT) elements at

three representative levels from a 50 day old opossum.

Note that ENK and 5-HT elements are present for the

first time within the superficial laminae of the

dorsal horn. Also, note that 5-HT elements are

aggregated within lamina IX. The open arrow indi­

cates a bundle of SP fibers. SOM cell bodies are

denoted by stars. SOM and ENK cell bodies are also

present within superficial laminae of the dorsal

horn but they have not been included for the sake

of clarity (see Results). 84 MM SOM ENK

CERVICAL

THORACIC

LUMBAR 74

Figure 32 Chartings of the distributions of substance-P (SP),

somatostatin (SOM), methionine-enkephalin (ENK) and

serotonin (5-HT) at three spinal levels from a 75 day

old opossum. Note the dense aggregation of SP and

5-HT (curved arrows) elements within the intermedio­

lateral cell column. SOM cell bodies are denoted by

stars. SOM and ENK cell bodies are also present

within superficial laminae of the dorsal horn but

they have not been included for the sake of clarity

(see Results). 127MM SOM ENK 5-HT

CERVICAL 76

Figure 33 Plots of substance-P (SP), somatostatin (SOM),

methionine-enkephalin (ENK) and serotonin (5-HT)

distributions at four representative levels of the

spinal cord from an adult opossum. Note the sparse

amounts of SP and SOM elements within the ventral

horn and the relative abundance of ENK and 5-HT

elements within comparable regions. The curved

arrow indicates the intermediolateral cell column

and the stars denote the location of SOM perikarya. C4

T7 SP SOM ENK

MM

L3

FIG. 33 PLATE VII

Figure 34 Photomicrograph of the dorsal horn from a 24mm opossum.

The arrow indicates a cluster of SP elements within

the developing superficial laminae. The dorsal funi­

culus is indicated (DF) and the bar represents 100pm

for figures 34-37.

Figure 35 Photomicrograph of a transverse section from the cer­

vical cord of a 57mm pouch-young opossum. SP immuno-

reactivity can be seen within presumptive laminae I

(I) and II (II), as well as in deeper laminae of the

dorsal horn. The curved arrow indicates a bundle of

SP fibers. The dorsal funiculus is indicated (DF).

Figure 36 Photomicrograph of SP elements within presumptive

lamina X of a 57mm opossum at the cervical level.

The central canal is labelled (CC).

Figure 37 Photomicrograph of SP immunoreactivity within the

intermediolateral cell column (curved arrow) of a

57mm opossum. The lateral funiculus (LF) is labelled. 79 80

PLATE VIII

Figure 38 Micrograph of SOM innnunoreactivity (arrow) within the

dorsolateral and lateral marginal (MAR) zone of the

cervical cord from a 24mm pouch-young opossum. Note

the virtual lack of immunostaining within the inter­

mediate (INT) zone. The bar represents lOOym for

figures 38-41.

Figure 39 Micrograph of a transverse section of the cervical

cord taken from a 57mm opossum. Note the presence

of SOM perikarya in the upper right. The arrows

indicate two of the sparse SOM elements within pre­

sumptive lamina X. The central canal (CC) is

indicated.

Figure 40 Micrograph of a transverse section of the cervical

cord from a 57mm opossum. Punctate SOM elements are

present within laminae I (I) and II (II) as well as

SOM perikarya which are more obvious. The arrow

indicates an SOM cell body that is also shown at a

higher magnification (inset). The inset bar repre­

sents lOym, and the dorsal funiculus is indicated

(DF). 81

PLATE VIII (CONTINUED)

Figure 41 Micrograph of a transverse section of the cervical

cord from a 127mm opossum. Note the predominance

of punctate and varicose immunoreactivity and the

relative lack of SOM cell bodies. Laminae I (I) and

II (II) as well as the dorsal funiculus (DF) are

indicated. 82

mm

* • fc-^V'V'TV h W m # ? ' ; ' - * 83

PLATE IX

Figure 42 Photomicrograph of SOM immunoreactive elements in

laminae I (I) and II (II) of the adult opossum. The

dorsal funiculus is indicated (DF). The bar repre­

sents 100pm for figures 42-45.

Figure 43 Photomicrograph of a transverse section from the cer­

vical enlargement of an adult opossum. The arrows

indicate punctate and/or varicose immunoreactive

structures which contain SOM. The central canal

is indicated (CC).

Figure 44 Photomicrograph of a transverse section from the

thoracic cord showing SOM elements within the inter-

mediolateral cell column of the adult opossum

(arrow). The lateral funiculus is labelled (LF).

Figure 45 Photomicrograph of a transverse section of the sacral

cord from an adult opossum. The open arrows indicate

SOM immunoreactive elements within the sacral para­

sympathetic area. The lateral funiculus is

indicated (LF). 84

^ - Ns • * T v % , \ j t .. ‘ ’ n» ‘- *• v, v ••: *:■*

'W-*' r, * . * ■■ •*

^ v ' ;■ r r i V :.W-*:;v ’• '' • V . ' V ' V i ... ’r .** -»*. *■• , > - % t v •* ■■■ •■ . v v .*• ■ • ‘ . ►.vt' . J "• \»ri * •; '• ; • v * ■” • * s * ^ * ■4 -lT;#r • . , • x.v ■*■:■■' »■ ■• ' v^c.-v- r ,f •'- - v J:C'- . !)■>it.* •■ •h / i. ,i • * » . . r. .* - Vi

\

\ •!rV';‘^S j .. ».»;** -w - ~ a- a x V-i«n,s i*

sfe ? * -' ■; ■

O 85

PLATE X

Figure 46 Micrograph of ENK inmunoreactivity within the dorso­

lateral and lateral marginal (MAR) zone of an 18mm

opossum at the cervical level. Note the lack of

immunostaining within the intermediate (INT) zone.

The arrows indicate ENK elements interpreted as

fibers in cross-section. The bar represents lOOym

for figures 46, 47 and 49.

Figure 47 Micrograph of a transverse section from the cervical

spinal cord of a 57mm opossum. Punctate and

varicose ENK immunoreactive elements are apparent

throughout presumptive lamina X. The central canal

(CC) is indicated.

Figure 48 Micrograph of ENK immunoreactivity within the super­

ficial laminae of the dorsal horn of a 57mm opossum

(cervical cord). Note that the sparse punctate ENK

elements are restricted primarily to lamina II (II)

while lamina I (I) exhibits little, if any, staining.

The arrow indicates an ENK cell body within lamina II

that is shown at a higher magnification (inset). The

bar in figure 48 represents 50ym and the inset bar

equals lOym. 86

PLATE X (CONTINUED)

Figure 49 Micrograph of ENK immunoreactivity within laminae I

(I) and II (II) of the thoracic cord from a 127mm

opossum. Note the predominance of varicose ENK

elements and the lack of ENK cell bodies. The dorsal

funiculus (DF) is indicated. v • • \ */• * .f • . . , f * . * • ‘ *\ -■ >; . ; . J.'\ ; ;*;

‘\ ^ '*•: .y^:''

'vr ;Yf-,. \’;^.-vr.:>'’.v> v* 1 •; iy>■/. - f e ‘ • • •: * ••-..> .:•> .*> *.V' • '^VO

• A*

00 88

PLATE XI

Figure 50 Photomicrograph of 5-HT immunoreactivity within the

dorsolateral and lateral marginal zone (MAR) of the

cervical level from an 18mm pouch young opossum.

Note the lack of 5-HT elements in the intermediate

zone (INT). The arrow points to 5-HT immunoreactive

elements aggregated within the peripheralmost part

of the marginal zone. The bar represents 100pm for

figures 50-54.

Figure 51 Photomicrograph of a transverse section of the cer­

vical cord from a 35mm pouch-young opossum. Note the

presence of 5-HT elements within the ventral funi­

culus (VF) which can be followed with the ventral

horn. The arrow indicates 5—HT elements surrounding

a presumed motoneuron.

Figure 52 Photomicrograph of 5-HT immunostaining within pre­

sumptive lamina X of the cervical cord from a 57mm

opossum. Fine caliber 5-HT varicosities are

apparent throughout the regions adjacent to the

central canal CCC). PLATE XI (CONTINUED)

Figure 53 Photomicrograph of a transverse section from the

thoracic cord of a 57mm pouch-young opossum. 5-HT

elements are present within the developing gray

matter (open arrow) and are particularly numerous

within the intermediolateral cell column (curved

arrow). The lateral funiculus (LF) is indicated.

Figure 54 Photomicrograph of the sparse 5-HT immunostaining

present within laminae I (I) and II (II) of the cer­

vical cord from an 84mm opossum. The open arrows

indicate 5-HT elements within the superficial

laminae of the dorsal horn. Some immunostaining

is present within the deeper laminae. The dorsal

funiculus is labelled (DF).

Figure 55 Photomicrograph of a transverse section of the cer­

vical cord from a 57mm pouch-young opossum. Note

the presence of 5-HT varicosities as well as 5-HT

perikarya (arrows). The sulco-marginal funiculus

(SM) and a blood vessel (BV) are labelled. The

bar represents 50ym. 90 PLATE XII

Figure 56 Photomicrograph of SP immunoreactivity within the

dorsal horn of a midthoracic section from a 50 day

old opossum that was incubated with the antiserum

specific for SP. SP immunoreactivity can be ob­

served in laminae I (.1) and II (II) as well as

within more ventral regions. The dorsal funiculus

(DF) is indicated and the bar represents 100pm for

figures 56-59.

Figure 57 Photomicrograph of a semiadjacent midthoracic sec­

tion that was incubated with the preabsorbed anti­

sera for SP. The control antisera failed to localize

SP to comparable areas when processed in parallel

with routine staining procedures. Laminae I (I) and

II (II) as well as the dorsal funiculus (DF) are

indicated.

Figure 58 Photomicrograph of SOM immunoreactivity within the

dorsal horn of a cervical section from a 30 day old

opossum that was incubated with the antiserum

specific for SOM, SOM immunoreactivity can be ob­

served mainly In laminae I (I) and II (XI)« The

dorsal funiculus (DF) is labelled. 92

PLATE XII (CONTINUED)

Figure 59 Photomicrograph of a semiadjacent cervical section

that was incubated with the preabsorbed antiserum

for SOM. The control antisera failed to localize

SOM to comparable areas when processed in parallel

with routine staining procedures. Laminae I (I)

and II (II) as well as the dorsal funiculus (DF)

are labelled. 93

\ t •

■ ' --V.'j'‘*'-",4»>- PLATE XIII

Figure 60 Photomicrograph of ENK immunoreactivity within

lamina X of a cervical section from a 30 day old

opossum that was incubated with the antiserum

specific for ENK. ENK immunoreactive neuronal

elements are represented by varicose or punctate

structures (arrows). The central canal (CC) is

labelled and the bar represents 100pm for figures

60-63.

Figure 61 Photomicrograph of a semiadjacent cervical section

that was incubated with the preabsorbed antisera for

ENK. The control antisera failed to localize ENK to

comparable areas when processed in parallel with

routine staining procedures. The central canal (CC)

is labelled.

Figure 62 Photomicrograph of 5-HT immunoreactivity within

lamina X of a cervical section from a 30 day old

opossum that was incubated with the antiserum

specific for 5-HT. Serotonin immunostaining can he

observed as punctate and varicose structures

throughout the regions adjacent to the central

canal (CC). 95

PLATE XIII (CONTINUED)

Figure 63 Photomicrograph of a semiadjacent cervical section

that was incubated with the preabsorbed antisera for

5-HT. The control antisera failed to localize 5-HT

to comparable areas when processed in parallel with

routine staining procedures. The central canal (CC)

is labelled. 96 97

Figure 64 Graphic representation of the sequence of peptidergic

and serotoninergic innervation of specific spinal

regions. VH = ventral horn; IMLC = intermediolateral

cell column; LX “ lamina X; DH ■ dorsal horn; MZ =

marginal zone; A = adult. The snout-rump (S-R)

lengths are indicated in millimeters. SP-SP E-ENK S-SOM 5 - 5-HT SP,S E,5

SP,S E,5

S -R LENGTH FIG. 64

vO CO APPENDIX A

Antibody Generation:

SOM was coupled to keyhole limpet hemocyanin by the carbodiimide condensation reaction (Skowsky and Fisher, ’72). Methionine enkephalin and SP were coupled to thyroglobulin using glutaraldehyde according to

Odell et al. (’72) and 5-HT creatinine sulphate was conjugated to bovine serum albumin using formaldehyde according to Ranadive and Sehon

(’67) and Grota and Brown (’74). Each resulting polymer was emulsified with Freund's complete adjuvant and injected subcutaneously at several sites along the mammillary lines of six New Zealand white rabbits. The animals were stimulated initially with lmg. equivalent of the respec­ tive antigen, boosted every 14 days with 0.5mg. equivalent of the anti­ gen and bled via the ear artery every 14 days after the third booster injection. The serum obtained from each bleeding was tested for the presence of antibody and its adequacy for immunohistochemical studies.

Our experience with SP, SOM, and ENK antibody generation was that serum from rabbits after the third booster injection was usable and that serum from many subsequent bleedings is still very good. Additional boostering was normally unnecessary.

99 100

Characterization of Antibodies Used In Iimminohlstochemlcal Localizations;

Antisera directed against the three peptides and 5-HT were char­ acterized and used on rabbit, cat, chicken, and rat spinal cords. No non-specific staining was observed in any of the above mentioned cases.

With respect to immunohistochemical evaluations, we will only include a description of characterization of our antibodies on opossum spinal cords.

Anti-SP Antibody:

This antibody was generated in rabbit #6. Bleeding C from rabbit //6 was used in the described studies. When rat spinal cord sections are processed for the localization of SP elements utilizing anti-SP serum 6C, the distribution of SP elements, their density and intensity of staining compares very well with the best published localizations (Ljungdahl et al., *78). Pretreatraent of the primary antibody with an excess of SP completely abolished the staining thus establishing the specificity of immunostaining of the antibody for SP immunoreactivity. Opossum spinal sections processed for SP localiza­ tion by either the immunofluorescent method of Coons ('58) or the indirect antibody peroxidase-anti-peroxidase technique of Stemberger

(’70) demonstrated comparable results.

The distribution of SP in the opossum spinal cord, the density and the intenstiy of staining as well as the morphology of the SP con­ taining neuronal elements are unique when compared to the distribution of SOM and ENK. We have also shown that our SP antibody does not cross- react with SOM, ENK, partially purified neurophysins and LH-RH (Ho and 101

DePalatls, ’80). In addition, our laboratory and that of Dr. Robert

Elde's have used the SP antibody to examine numerous central nervous

system areas. In all cases, the distribution and staining character­

istics agreed with results from Hokfelt's laboratory (Ljungdahl et al.,

'78).

Dr. Robert Elde has tested our anti-SP sera using both the in­

direct immunofluorescent and the PAP techniques using beads that were

coated with a variety of peptides. Briefly, cyanogen bromide activated

sepharose beads were coated with methionine-enkephalin, leucine-enkepha-

lin, substance-P, neurotensin, somatostatin, “-MSH, ocytocin, arginine

vasopressin, lysine vasopressin, neurophysin, 0-endorphin, and “-endor­

phin. Our antibodies only stained those beads that were coated with

substance-P.

The SP antibody has been used in radioimmunoassays* (RIA) at a

final dilution of 1:80,000. The binding to labelled SP was approximately

30%. The range of the linear portion of the standard curve was 3pg/tube-

200pg/tube. Table 1 expresses the cross-reactivity of the antibody with SP, its various fragments, as well as with other substances.

From the table on the following page, it is concluded that under

the RIA experimental conditions the SP antibody does not cross-react with a wide range of substances tested. The antibody binds specifically with SP and its larger fragments on the N-terminal.

*These studies were carried out in Dr. Samuel M. McCann's laboratory (University of Texas, Southwestern Medical School) by Dr. Louis R. • DePalatis. TABLE 1

Substance Tested % Crossreactivity

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (Substance P) 100 Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 100 ------Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 99 ------Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 100 ------Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Substance P 70 ------Phe-Phe-Gly-Leu-Met-NH2 Fragments 13 , ------Phe-Gly-Leu-Met-NH2 0.01 ------Gly-Leu-Met-NH2 X Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly------X

Eledolsln 0.14 Bombesin 0.1 Luteinizing Hormone - Releasing Hormone (LH-RH) X Somatostatin X Thyrotropin-Releasing Hormone X B-endorphin X Basoactive Intestinal Peptide X Bradykinin X Neurotensin X Arginine-vasopressin X Arginlne-vasotocin X Cholecystokinin Octapeptide X

X = less than 0.01%

o N 3 103

Antl-SOM Antibody:

This antibody was generated In rabbit #11. Bleeding C was used

in the described studies. The distribution of SOM elements, its den­ sity and intensity of immunostaining compares well with the few available published localizations. Pretreatment of the primary antibody with an excess of SOM completely abolished the staining ability of the primary antibody thus establishing the specificity of immunostaining for SOM immunoreactivity.

The distribution of SOM in the opossum spinal cord, the density and intensity of staining as well as the morphology of the SOM containing neuronal elements are unique when compared to the distribution of SP,

ENK, and 5-HT.

The SOM antibody has been used in RIAs* at a final dilution of

1:80,000. The binding to the labelled SOM was approximately 35%. The range of the linear portion of the standard curve was 12.5pg/tube-

200pg/tube. The following expresses the crossreactivity of the anti­ body with a variety of substances tested:

Substance Tested % Crossreactivity

SOM 100 LH-RH X Substance P X Thyrotropin-Releasing Hormone X Neurotensin X Bombesin X Vasoactive Intestinal Peptide X B-endorphin X Cholecystokinin Octapeptide X (X ■ less than 0.01%)

*These studies were carried out in Dr. Samuel M. McCann's laboratory (University of Texas, Southwestern Medical School) by Dr. Louis R. DePalatis. 104

It can be concluded that the SOM antibody does not cross-react with the substances tested.

ENK Antibody;

This antibody was generated in rabbit //18. We have employed bleeding G from rabbit //18. When rat spinal sections are processed for immunohistochemical localization of ENK neuronal elements, the distribution of ENK elements, its density and intensity of staining compares well with the available published localizations. Pretreat­ ment of the primary antibody with an excess of ENK abolished all of the expected immunostaining.

The distribution of ENK in the opossum spinal cord, the density and intensity of staining as well as the morphology of the ENK- containing neuronal elements are unique when compared to the distribu­ tion of SOM (figs. 3, 7), SP (figs. 1, 6), and 5-HT (figs. 5, 9).

5-HT Antibody:

This antibody (R194D) was supplied to us by Dr. Robert Elde.

The distribution of spinal 5-HT elements in the rat, its density and intensity of staining compares very well with the best published results.

Pretreatment of the primary antibody with an excess of 5-HT completely eliminated the staining ability of the antiserum.

The distribution of 5-HT in the opossum spinal cord, the density and intensity of staining as well as the morphology of the 5-HT elements are unique when compared to the distribution of SOM and ENK. 105

Immunohlstochemlcal Localizations of 5-HT on Pharmacologically

Manipulated Neural Tissues:

(These studies were performed in Dr. Robert Elde's laboratory.)

The distribution of 5-HT in the rat brain as revealed by the

indirect innnunofluorescent technique is in close agreement with that

revealed by the Falck-Hillarp technique. However, the use of the anti­

body technique did reveal more details in its distribution in a number

of brain regions. To test the 5-HT antibody's staining ability to

localize 5-HT, rats were treated with drugs (reserpine, parachloro-

phenylalanine (PCPA) and 5,7-dihydroxytryptamine) that are known to

deplete 5-HT. The staining of hypothalamic nerve fibers and terminals

from drug-treated animals was markedly reduced. Conversely, hypothalami

from animals treated with L-tryptophan exhibited increased staining

intensity of hypothalamic 5-HT neuronal elements.

Further studies included treatment of rats with 6-hydroxydopa-

mine, a catecholamine depleter. The staining characteristics of hypo­

thalamic 5-HT elements in this group of animals was unaffected.

A natural concern for using an antibody raised against 5-HT is

its possible crossreactivity with tryptamine and melatonin. Since

tryptamine content is increased by PCPA treatment, the fact that the hypothalamic tissues from PCPA-treated rats exhibited markedly reduced

staining for 5-HT suggests that the antibody does not cross-react with

tryptamine.

L-DOPA is known to increase melatonin but decrease 5-HT contents

in the pineal. Treatment of rats with L-DOPA resulted in a decreased

5-HT staining in the pineal. Thus, it is very likely that the 5-HT antibody does not cross-react with melatonin. LIST OF REFERENCES

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