Brain Research, 542 (1991) 77-82
77
Elsevier BRES 16343
Bombesin immunoreactive neurons in the hypothalamic paraventricular nucleus innervate the dorsal vagal complex in the rat Joseph F. Costello 1, Marvin R. Brown 2 and Thackery S. Gray 1 1Department of Cell Biology, Neurobiology and Anatomy, Loyola Stritch School of Medicine, Maywood, 1L 60153 (U.S.A.)and 2Autonomic Physiology Laboratory, Department of Medicine, University of California at San Diego, San Diego, CA 92037 (U.S.A.) (Accepted 4 September 1990)
Key words: Nucleus tractus solitarii; Dorsal vagal nucleus; Cardiovascular response; Retrograde tracing; Immunohistochemistry
Bombesin-like immunoreactivity has been localized within neuronal cell bodies of the hypothalamus and nerve terminals within the dorsal vagai complex. The possibility that the hypothalamus is a source for bombesin-like immunoreactive terminals within the dorsal vagal complex was examined using the combined retrograde tracing and immunohistochemical technique. After injections of retrograde tracer were made into the dorsal vagal complex, cells in the hypothalamus labeled with both retrograde tracer and bombesin immunoreactivity were localized in the parvocellular part of the paraventricular nucleus. In the paraventricular nucleus most of the vagal projecting bombesin immunoreactive neurons were located within the medial parvocellular subdivision. Approximately 30% of the bombesin immunoreactive neurons in this subnucleus projected to the dorsal vagal complex. The results suggest that the paraventricular hypothalamic nucleus is a major source of bombesin terminals within the dorsal vagal complex. This pathway may mediate some of the autonomic nervous system changes that are observed when bombesin is injected within the central nervous system. Additionally, this data adds to a growing amount of evidence supporting the role of bombesin as a peptide neurotransmitter. INTRODUCTION Bombesin, a t e t r a d e c a p e p t i d e , was originally isolated from the skin of the E u r o p e a n amphibian, Bombina bombina 1. M o r e recently a bombesin-like p e p t i d e has been identified within the central nervous system of m a m m a l s 2'9. This b o m b e s i n - l i k e p e p t i d e resembles and may be a gastrin-releasing peptide. The m a m m a l i a n bombesin has fulfilled a n u m b e r of the criteria to be considered as a n e u r o t r a n s m i t t e r or n e u r o m o d u l a t o r . Bombesin-like immunoreactivity has been localized within neuronal cell bodies and/or terminals in the amygdala, hypothalamus, brainstem and spinal cord 1°, 11,14 • R a d i o i m m u n o a s s a y studies also reveal bombesin immunoreactivity in the above areas 2'5. Bombesin receptors have been identified in various brain regions including the hippocampus,, hypothalamus and medulla/pons. Studies have suggested that bombesin-like immunoreactivity is located in presynaptic nerve terminals in close proximity to postsynaptic bombesin receptors s. B o m b e sin-like peptides are released from brain slices by potassium- and veratridine-induced depolarizations in the presence of calcium a. Bombesin has a n u m b e r of biologically significant actions when injected into the brain. The central effects
of bombesin are p r o b a b l y m e d i a t e d by the autonomic nervous system rather than the n e u r o e n d o c r i n e system 19. Bombesin acts within the central nervous system to cause elevated plasma levels of e p i n e p h r i n e and glucagon with resulting hyperglycemia 3. Intracisternal injections of bombesin have anti-ulcerogenic effects in the stomach 17, 18. Also, central administration of bombesin results in increased grooming, hyperactivity, analgesia, hypothermia and suppression of feeding 3'6. Previous studies have identified n u m e r o u s bombesin immunoreactive cells within the h y p o t h a l a m u s 12. The dorsal vagal complex receives a m a j o r input from several hypothalamic nuclei 15. In addition, there is a dense plexus of bombesin immunoreactive terminals within the dorsal vagal complex H. Thus, the present study investigated whether hypothalamic bombesin cells are an endogenous source of bombesin within the dorsal vagal complex.
MATERIALS AND METHODS Long-Evans male rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The rats were subsequently mounted in a stereotaxic frame and the floor of the fourth ventricle was visualized through an incision in the atlantooccipital membrane. Injections of 2.0% Fast blue retrograde tracer were administered via a Hamilton syringe fitted with a glass
Correspondence: T. S. Gray, Department of Cell Biology, Neurobiology and Anatomy, Loyola Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153, U.S.A.
0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
78 micropipette tip (10 /zm outside diameter). The glass tip was lowered into the dorsal brainstem at a level of the area postrema approximately 1.0 mm rostral to the obex. The pipette tip was lowered 0.7 mm below the dorsal surface of the brainstem. Seventy-five nanoliters of Fast blue were injected over 10 min. The rats were allowed to survive for 14 days. On the twelfth day of this survival period 60/zg (20 ~g//A dH20 ) of colchicine was injected into each lateral ventricle. After a 36-48 h survival period each rat was perfused through the ascending aorta with 250 ml of 0.01 M phosphate buffered saline (PBS) 40 °C, followed by 500 ml of cold (4 °C) 4% paraformaldehyde in 0.167 M sodium phosphate buffer, pH 7.6. The brains were allowed to postfix for 1 h and were cut into 25-/~m-thick coronal sections. The sections were cut in the coronal plane approximating the Paxinos and Watson13 rat brain atlas via a Lancer vibratome. Sections cut from the injection site were mounted onto slides and then coverslipped. One set of sections from the forebrain was mounted and coverslipped. Three sets of sections from the forebrain were rinsed 30-60 min in scintillation vials containing 0.01 M PBS, 0.1% Triton X-100 (Malinkrodt). The sections were then placed in one of 3 vials of primary antibodies diluted 1:1000 in 1.5 ml PBS containing 1.0% donkey serum. One vial contained a bombesin antiserum (Lys4-Bom TG-4-4) that was raised in rabbits immunized with a Lys4-bombesin-thyroglobin complex conjugated using glutaraldehyde. The tetradecapeptide [lyse 4]-bombesin was synthesized and provided to us by Jean Rivier, The Salk Institute, LaJolla, Ca. The [lyse 4]-bombesin antibody when used in a radioimmunoassay does not recognize substance P. Another vial contained a bombesin antibody that was purchased commercially (Incstar) and was generated in a rabbit against synthetic bombesin conjugated to thyroglobulin. The third vial contained substance P antiserum that was purchased commercially (Incstar) and was generated in rabbit against a keyhole limpet hemocyanin conjugate. Sections were incubated in primary antibodies for 16-24 h. Next, the sections were rinsed in 0.01 M PBS for 15 min. The sections were then incubated in biotinylated donkey anti-rabbit immunogammaglobulin (50/A/10 ml PBS, Bethesda Res. Labs) for 30 min followed by a 15 min cold PBS rinse. The sections were placed in a solution of 30/~1of streptavidin conjugated to Texas red (Bethesda Res. Labs) in 10 ml PBS for 30 min. The sections were rinsed in cold PBS, mounted on slides and allowed to dry. The slides were coverslipped using Depex mounting media (BDH Chemicals). Immunoreactive cells were visualized using the green filter system and retrograde fluorescent cells were observed with the UV filter system on an Olympus AHS Vanox microscope. Immunoadsorption controls involved processing two sets of tissue with each antibody (1.5 ml 1:1000 dilution) with l0/~g of synthetic bombesin or substance P (provided by Jean Rivier). Substance P and bombesin antigen blocked immunostaining when placed in their corresponding antisera. Bombesin antigen failed to block substance P antibody staining and substance P did not abolish bombesin antibody staining.
injected into the fourth ventricle. Two animals had Fast blue injections that were located within the hypoglossal nucleus with limited spread into the dorsal vagal nucleus, nucleus of the solitary tract and surrounding reticular formation. Consistent retrograde labeling within the hypothalamus occurred only with Fast blue injections centered in the dorsal vagal complex. The distribution of retrogradely labeled cells in the hypothalamic paraventricular nucleus (PVN) was the same as previously reported and will only be described briefly ~5. Retrograde labeling was most notable in the PVN, and in the lateral hypothalamic, arcuate and retrochiasmatic nuclei. The vast majority of retrogradely labeled cells were located within the medial and lateral parvocellular parts of the PVN (Figs. 2 and 3). Fewer retrogradely labeled cells were observed within the periventricular and magnocellular subdivisions. Only a few retrogradely labeled cells were observed in the dorsal parvocellular part of the PVN indicating that tracer injections did not involve fibers of passage en route to the spinal cord. Injections of tracer in the dorsal vagal complex also produced consistent retrograde labeling in other forebrain areas that are known to project to the dorsal vagal complex including the central nucleus of the amygdala, bed nucleus of the stria terminalis and insular cortex 2°.
Distribution of immunoreactive cells. The distribution of b o m b e s i n and substance P (SP) within the hypothalamus was t h e same as previously described t2. Bombesin immunoreactive cells were located in the medial and anterior parvocellular and periventricular subdivisions of the PVN. Most bombesin immunoreactive cells were located within the ventral
RESULTS
Distribution of retrogradely labeled cells A total of 12 rats had injections of Fast blue located within the dorsal brainstem. Six animals had bilateral injections that centered within the nucleus of the solitary tract with spread to the dorsal vagal nucleus and dorsal part of the reticular formation and nucleus gracilis. Fig. 1 is a photomicrograph of a typical bilateral injection of Fast blue injection in the dorsal vagal complex. Two animals had unilateral injections that were centered within the gracile nuclei with some spread to the nucleus of the solitary tract. In two animals the Fast blue was
Fig. 1. Fluorescent photomicrographs of a representative bilateral injection of Fast blue tracer into the dorsal vagal complex. Sol, nucleus of the solitary tract; 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; Gr, nucleus gracilis. Bar = 250/~m.
79
Fig. 2. Fluorescent photomicrographs of examples of retrogradely labeled bombesin-immunoreactivecells in the medial parvocellular part of the hypothalamicparaventricular nucleus (MP) of a rat. Fast blue retrogradely labeling is presented in A and C and Texas red bombesin-like immunofluorescent cells are shown in B and D. Arrows indicate the location of cells that are doubly-labeled. 3V, third ventricle; bar = 100 ~tm.
parts of the medial parvocellular subdivision (Figs. 2 and 3). Bombesin immunoreactive cells were also present in adjacent hypothalamic areas including the lateral, ventromedial, and retrochiasmatic nuclei. Bombesin immunoreactive cells were also seen in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala. Consistent with previous findings 12, the distribution of SP immunoreactive neurons did not overlap the distribution of bombesin immunoreactive cells. Overall very few SP immunoreactive neurons were observed within the PVN compared to bombesin immunoreactive neurons. Substance P immunoreactive neurons were located along the dorsal border of the posterior magnocellular subdivision and within the periventricular subdivision of the PVN.
Distribution of retrogradely labeled-bombesin immunoreactive cells In two animals (NFC16 and NFC19) the total number of bombesin immunoreactive, retrogradely labeled and
retrogradely labeled immunoreactive cells were counted (see Table I). In both of these cases the injections of retrograde tracer were bilateral. The data in Table I represent raw counts from 1 out of every 4 sections sampled through the PVN. Cells labeled with both retrograde tracer and bombesin-like immunoreactivity (i.e. 'double-labeled') were seen primarily in the medial parvocellular PVN (Figs. 2 and 3). Fifteen to 37% of the total bombesin-immunoreactive cells in the PVN were labeled with retrograde tracer. Approximately 1-6% of the retrogradely labeled cells were bombesin immunoreactive. Double labeled cells observed in the lateral and anterior parvocellular subdivisions contributed only a very small percentage to the total number of double labeled cells in the PVN. In one case a pair of double labeled cells was observed in the retrochiasmatic area. Retrograde labeling was not observed within the bombesin immunoreactive cells of the amygdala or bed nucleus of the stria terminalis. No differences in the distribution of bombesin immunoreactive cells were observed between the two different bombesin antisera.
80 However, bombesin antiserum raised in the laboratory i m m u n o s t a i n e d many more cells as compared to com-
DISCUSSION
mercially purchased antiserum. No retrogradely labeled SP-immunoreactive cells were
The results suggest that the hypothatamic paraventricular nucleus is a major source of bombesin immunoreactive terminals that are located in the dorsal vagai complex. This projection is topographically organized in
observed within the PVN.
DP
Pe
ox -1.40mm
-2.12mm
Y -1.80mm
-2.30ram
Fig. 3. Line drawings of 4 coronal sections through the hypothalamic paraventricular nucleus illustrating the distribution of bombesin immunoreactive neurons (open circles) and regradely labeled bombesin immunoreactive neurons (solid circles) in an animal that had Fast blue tracer injected into the dorsal vagal complex. Abbreviations are from the Paxinos and Watson rat brain atlas13. Coronal plane section levels in mm relative to bregma are indicated below each drawing.
81 TABLE I Number and per cent of bombesin immunoreactive and retrogradely labeled cells in hypothalamic paraventricular nucleus
a, Bombesin raised in laboratory; b, Bombesin from Incstar.
NFC16 a b NFC19 a b
Number of bombesin immunoreactive cells
Number of retrogradely labeled cells
Number of doublelabeled cells
% ofretrogradely labeled bombesin immunoreactive cells
% ofbombesin immunoreactive retrogradely labeled cells
Left
Right
Left
Right
Left
Right
Left
Left
Right
46 16
42 10
444 508
405 535
17 6
11 2
4 1
3
77
Elsevier BRES 16343
Bombesin immunoreactive neurons in the hypothalamic paraventricular nucleus innervate the dorsal vagal complex in the rat Joseph F. Costello 1, Marvin R. Brown 2 and Thackery S. Gray 1 1Department of Cell Biology, Neurobiology and Anatomy, Loyola Stritch School of Medicine, Maywood, 1L 60153 (U.S.A.)and 2Autonomic Physiology Laboratory, Department of Medicine, University of California at San Diego, San Diego, CA 92037 (U.S.A.) (Accepted 4 September 1990)
Key words: Nucleus tractus solitarii; Dorsal vagal nucleus; Cardiovascular response; Retrograde tracing; Immunohistochemistry
Bombesin-like immunoreactivity has been localized within neuronal cell bodies of the hypothalamus and nerve terminals within the dorsal vagai complex. The possibility that the hypothalamus is a source for bombesin-like immunoreactive terminals within the dorsal vagal complex was examined using the combined retrograde tracing and immunohistochemical technique. After injections of retrograde tracer were made into the dorsal vagal complex, cells in the hypothalamus labeled with both retrograde tracer and bombesin immunoreactivity were localized in the parvocellular part of the paraventricular nucleus. In the paraventricular nucleus most of the vagal projecting bombesin immunoreactive neurons were located within the medial parvocellular subdivision. Approximately 30% of the bombesin immunoreactive neurons in this subnucleus projected to the dorsal vagal complex. The results suggest that the paraventricular hypothalamic nucleus is a major source of bombesin terminals within the dorsal vagal complex. This pathway may mediate some of the autonomic nervous system changes that are observed when bombesin is injected within the central nervous system. Additionally, this data adds to a growing amount of evidence supporting the role of bombesin as a peptide neurotransmitter. INTRODUCTION Bombesin, a t e t r a d e c a p e p t i d e , was originally isolated from the skin of the E u r o p e a n amphibian, Bombina bombina 1. M o r e recently a bombesin-like p e p t i d e has been identified within the central nervous system of m a m m a l s 2'9. This b o m b e s i n - l i k e p e p t i d e resembles and may be a gastrin-releasing peptide. The m a m m a l i a n bombesin has fulfilled a n u m b e r of the criteria to be considered as a n e u r o t r a n s m i t t e r or n e u r o m o d u l a t o r . Bombesin-like immunoreactivity has been localized within neuronal cell bodies and/or terminals in the amygdala, hypothalamus, brainstem and spinal cord 1°, 11,14 • R a d i o i m m u n o a s s a y studies also reveal bombesin immunoreactivity in the above areas 2'5. Bombesin receptors have been identified in various brain regions including the hippocampus,, hypothalamus and medulla/pons. Studies have suggested that bombesin-like immunoreactivity is located in presynaptic nerve terminals in close proximity to postsynaptic bombesin receptors s. B o m b e sin-like peptides are released from brain slices by potassium- and veratridine-induced depolarizations in the presence of calcium a. Bombesin has a n u m b e r of biologically significant actions when injected into the brain. The central effects
of bombesin are p r o b a b l y m e d i a t e d by the autonomic nervous system rather than the n e u r o e n d o c r i n e system 19. Bombesin acts within the central nervous system to cause elevated plasma levels of e p i n e p h r i n e and glucagon with resulting hyperglycemia 3. Intracisternal injections of bombesin have anti-ulcerogenic effects in the stomach 17, 18. Also, central administration of bombesin results in increased grooming, hyperactivity, analgesia, hypothermia and suppression of feeding 3'6. Previous studies have identified n u m e r o u s bombesin immunoreactive cells within the h y p o t h a l a m u s 12. The dorsal vagal complex receives a m a j o r input from several hypothalamic nuclei 15. In addition, there is a dense plexus of bombesin immunoreactive terminals within the dorsal vagal complex H. Thus, the present study investigated whether hypothalamic bombesin cells are an endogenous source of bombesin within the dorsal vagal complex.
MATERIALS AND METHODS Long-Evans male rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The rats were subsequently mounted in a stereotaxic frame and the floor of the fourth ventricle was visualized through an incision in the atlantooccipital membrane. Injections of 2.0% Fast blue retrograde tracer were administered via a Hamilton syringe fitted with a glass
Correspondence: T. S. Gray, Department of Cell Biology, Neurobiology and Anatomy, Loyola Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153, U.S.A.
0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
78 micropipette tip (10 /zm outside diameter). The glass tip was lowered into the dorsal brainstem at a level of the area postrema approximately 1.0 mm rostral to the obex. The pipette tip was lowered 0.7 mm below the dorsal surface of the brainstem. Seventy-five nanoliters of Fast blue were injected over 10 min. The rats were allowed to survive for 14 days. On the twelfth day of this survival period 60/zg (20 ~g//A dH20 ) of colchicine was injected into each lateral ventricle. After a 36-48 h survival period each rat was perfused through the ascending aorta with 250 ml of 0.01 M phosphate buffered saline (PBS) 40 °C, followed by 500 ml of cold (4 °C) 4% paraformaldehyde in 0.167 M sodium phosphate buffer, pH 7.6. The brains were allowed to postfix for 1 h and were cut into 25-/~m-thick coronal sections. The sections were cut in the coronal plane approximating the Paxinos and Watson13 rat brain atlas via a Lancer vibratome. Sections cut from the injection site were mounted onto slides and then coverslipped. One set of sections from the forebrain was mounted and coverslipped. Three sets of sections from the forebrain were rinsed 30-60 min in scintillation vials containing 0.01 M PBS, 0.1% Triton X-100 (Malinkrodt). The sections were then placed in one of 3 vials of primary antibodies diluted 1:1000 in 1.5 ml PBS containing 1.0% donkey serum. One vial contained a bombesin antiserum (Lys4-Bom TG-4-4) that was raised in rabbits immunized with a Lys4-bombesin-thyroglobin complex conjugated using glutaraldehyde. The tetradecapeptide [lyse 4]-bombesin was synthesized and provided to us by Jean Rivier, The Salk Institute, LaJolla, Ca. The [lyse 4]-bombesin antibody when used in a radioimmunoassay does not recognize substance P. Another vial contained a bombesin antibody that was purchased commercially (Incstar) and was generated in a rabbit against synthetic bombesin conjugated to thyroglobulin. The third vial contained substance P antiserum that was purchased commercially (Incstar) and was generated in rabbit against a keyhole limpet hemocyanin conjugate. Sections were incubated in primary antibodies for 16-24 h. Next, the sections were rinsed in 0.01 M PBS for 15 min. The sections were then incubated in biotinylated donkey anti-rabbit immunogammaglobulin (50/A/10 ml PBS, Bethesda Res. Labs) for 30 min followed by a 15 min cold PBS rinse. The sections were placed in a solution of 30/~1of streptavidin conjugated to Texas red (Bethesda Res. Labs) in 10 ml PBS for 30 min. The sections were rinsed in cold PBS, mounted on slides and allowed to dry. The slides were coverslipped using Depex mounting media (BDH Chemicals). Immunoreactive cells were visualized using the green filter system and retrograde fluorescent cells were observed with the UV filter system on an Olympus AHS Vanox microscope. Immunoadsorption controls involved processing two sets of tissue with each antibody (1.5 ml 1:1000 dilution) with l0/~g of synthetic bombesin or substance P (provided by Jean Rivier). Substance P and bombesin antigen blocked immunostaining when placed in their corresponding antisera. Bombesin antigen failed to block substance P antibody staining and substance P did not abolish bombesin antibody staining.
injected into the fourth ventricle. Two animals had Fast blue injections that were located within the hypoglossal nucleus with limited spread into the dorsal vagal nucleus, nucleus of the solitary tract and surrounding reticular formation. Consistent retrograde labeling within the hypothalamus occurred only with Fast blue injections centered in the dorsal vagal complex. The distribution of retrogradely labeled cells in the hypothalamic paraventricular nucleus (PVN) was the same as previously reported and will only be described briefly ~5. Retrograde labeling was most notable in the PVN, and in the lateral hypothalamic, arcuate and retrochiasmatic nuclei. The vast majority of retrogradely labeled cells were located within the medial and lateral parvocellular parts of the PVN (Figs. 2 and 3). Fewer retrogradely labeled cells were observed within the periventricular and magnocellular subdivisions. Only a few retrogradely labeled cells were observed in the dorsal parvocellular part of the PVN indicating that tracer injections did not involve fibers of passage en route to the spinal cord. Injections of tracer in the dorsal vagal complex also produced consistent retrograde labeling in other forebrain areas that are known to project to the dorsal vagal complex including the central nucleus of the amygdala, bed nucleus of the stria terminalis and insular cortex 2°.
Distribution of immunoreactive cells. The distribution of b o m b e s i n and substance P (SP) within the hypothalamus was t h e same as previously described t2. Bombesin immunoreactive cells were located in the medial and anterior parvocellular and periventricular subdivisions of the PVN. Most bombesin immunoreactive cells were located within the ventral
RESULTS
Distribution of retrogradely labeled cells A total of 12 rats had injections of Fast blue located within the dorsal brainstem. Six animals had bilateral injections that centered within the nucleus of the solitary tract with spread to the dorsal vagal nucleus and dorsal part of the reticular formation and nucleus gracilis. Fig. 1 is a photomicrograph of a typical bilateral injection of Fast blue injection in the dorsal vagal complex. Two animals had unilateral injections that were centered within the gracile nuclei with some spread to the nucleus of the solitary tract. In two animals the Fast blue was
Fig. 1. Fluorescent photomicrographs of a representative bilateral injection of Fast blue tracer into the dorsal vagal complex. Sol, nucleus of the solitary tract; 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus; Gr, nucleus gracilis. Bar = 250/~m.
79
Fig. 2. Fluorescent photomicrographs of examples of retrogradely labeled bombesin-immunoreactivecells in the medial parvocellular part of the hypothalamicparaventricular nucleus (MP) of a rat. Fast blue retrogradely labeling is presented in A and C and Texas red bombesin-like immunofluorescent cells are shown in B and D. Arrows indicate the location of cells that are doubly-labeled. 3V, third ventricle; bar = 100 ~tm.
parts of the medial parvocellular subdivision (Figs. 2 and 3). Bombesin immunoreactive cells were also present in adjacent hypothalamic areas including the lateral, ventromedial, and retrochiasmatic nuclei. Bombesin immunoreactive cells were also seen in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala. Consistent with previous findings 12, the distribution of SP immunoreactive neurons did not overlap the distribution of bombesin immunoreactive cells. Overall very few SP immunoreactive neurons were observed within the PVN compared to bombesin immunoreactive neurons. Substance P immunoreactive neurons were located along the dorsal border of the posterior magnocellular subdivision and within the periventricular subdivision of the PVN.
Distribution of retrogradely labeled-bombesin immunoreactive cells In two animals (NFC16 and NFC19) the total number of bombesin immunoreactive, retrogradely labeled and
retrogradely labeled immunoreactive cells were counted (see Table I). In both of these cases the injections of retrograde tracer were bilateral. The data in Table I represent raw counts from 1 out of every 4 sections sampled through the PVN. Cells labeled with both retrograde tracer and bombesin-like immunoreactivity (i.e. 'double-labeled') were seen primarily in the medial parvocellular PVN (Figs. 2 and 3). Fifteen to 37% of the total bombesin-immunoreactive cells in the PVN were labeled with retrograde tracer. Approximately 1-6% of the retrogradely labeled cells were bombesin immunoreactive. Double labeled cells observed in the lateral and anterior parvocellular subdivisions contributed only a very small percentage to the total number of double labeled cells in the PVN. In one case a pair of double labeled cells was observed in the retrochiasmatic area. Retrograde labeling was not observed within the bombesin immunoreactive cells of the amygdala or bed nucleus of the stria terminalis. No differences in the distribution of bombesin immunoreactive cells were observed between the two different bombesin antisera.
80 However, bombesin antiserum raised in the laboratory i m m u n o s t a i n e d many more cells as compared to com-
DISCUSSION
mercially purchased antiserum. No retrogradely labeled SP-immunoreactive cells were
The results suggest that the hypothatamic paraventricular nucleus is a major source of bombesin immunoreactive terminals that are located in the dorsal vagai complex. This projection is topographically organized in
observed within the PVN.
DP
Pe
ox -1.40mm
-2.12mm
Y -1.80mm
-2.30ram
Fig. 3. Line drawings of 4 coronal sections through the hypothalamic paraventricular nucleus illustrating the distribution of bombesin immunoreactive neurons (open circles) and regradely labeled bombesin immunoreactive neurons (solid circles) in an animal that had Fast blue tracer injected into the dorsal vagal complex. Abbreviations are from the Paxinos and Watson rat brain atlas13. Coronal plane section levels in mm relative to bregma are indicated below each drawing.
81 TABLE I Number and per cent of bombesin immunoreactive and retrogradely labeled cells in hypothalamic paraventricular nucleus
a, Bombesin raised in laboratory; b, Bombesin from Incstar.
NFC16 a b NFC19 a b
Number of bombesin immunoreactive cells
Number of retrogradely labeled cells
Number of doublelabeled cells
% ofretrogradely labeled bombesin immunoreactive cells
% ofbombesin immunoreactive retrogradely labeled cells
Left
Right
Left
Right
Left
Right
Left
Left
Right
46 16
42 10
444 508
405 535
17 6
11 2
4 1
3
