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Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons
Brain Research, 494 (1989) 267-275 Elsevier
267
BRES 14706
Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons Linda L. Wright and Jennifer I. Luebke Department of Anatomy, Boston University School of Medicine, Boston, MA 02118 (U.S.A.) (Accepted 17 January 1989)
Key words." Sympathetic; Target-specific neuron; Neuropeptide; Adult rat; Gender difference Studies combine the use of the retrograde tracer, fluorogold, and immunocytochemical staining to determine whether superior cervical ganglion (SCG) neurons projecting to the iris or submandibular gland (SMG) in adult male and female rats show distinctive immunoreactivity to somatostatin (SS), vasoactive intestinal polypeptide (VIP), or neuropeptide Y. Overall, more SMG-projecting neurons than eye-projecting neurons contain VIP-like immunoreactivity (VIP-LI), and more eye-projecting neurons than SMG-projecting neurons contain SS-LI and VIP-LI. Thus, postganglionic neurons of the SCG that project to specific target tissues are heterogeneous in their peptide content, and there are differences in the pattern of peptide-immunoreactivity between neurons projecting to these two target tissues. In addition, the results indicate that there may be gender differences in the expression of these neuropeptides. INTRODUCTION Much of what we know about postganglionic sympathetic neurons is based on studies of whole ganglia. However, it is becoming increasingly apparent that the neurons of the sympathetic nervous system are as heterogeneous as the types of target tissues innervated. The rat superior cervical ganglion (SCG) innervates blood vessels of the head and neck, including cerebral I and pituitary vasculature 9' 44 the pineal gland, choroid plexus 49, thyroid gland, submandibular gland, iris and Muller's muscle 6. Principal neurons that innervate different tissue types are intermixed within the ganglion with only very limited regional localization. For example, neurons innervating the pineal tend to lie in the rostral part of the ganglion, but are intermixed with other neurons projecting out the internal carotid nerve 5. Postganglionic neurons give off a single axon that does not branch within the ganglion even in neonates 43. There is no evidence of a single neuron innervating two tissue types 15"36 although some
rearrangement of postganglionic axons among nearby tissues may occur during development 52. Sympathetic neurons are also heterogeneous in their neuropeptide content ~6"17. In addition to norepinephrine, some postganglionic sympathetic neurons also contain substance P (SP) 2-4,21,24,25, vasoactive intestinal polypeptide (VIP) 16"19"22,23,35,50, 7aminobutyric acid ( G A B A ) 12'53, somatostatin (SS) 3' 18.19.25.34.35, Met- and Leu-enkephalins (ENK) 2.3, 29.30.45,vasopressin (VP) 11"20'26"27,neuropeptide Y (NPY) 16'35"38~40"41, bombesin 46 or pancreatic polypeptide (pp)37.38.42.51. Principal neurons of the rat SCG have also been found to contain serotonin and GABA-synthesizing enzymes t2,14, although serotonin is apparently only present during the neonatal period. An intrinsic population of G A B A - l i k e immunoreactive cells persists into adulthood 53. There are morphological differences between neurons that project to different tissues: SCG neurons that innervate the submandibular gland are significantly larger and have a more elaborate dendritic structure than those that innervate the
Correspondence: L.L. Wright, Department of Anatomy, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
268
iris 47~4~. Thus, SCG neurons are heterogeneous in size, transmitter/peptide content, and tissues innervated, but the relationships between neuron characteristics and tissue innervated have not been fully explored. The studies described here investigate the expression of immunoreactivity to 3 peptides (NPY, SS and VIP) in SCG neurons that innervate the eye and submandibular gland to determine: (1) whether the neurons projecting to a given tissue are homogeneous or heterogeneous in peptide expression; and (2) whether the peptide expression is different in neurons projecting to these different target tissues, MATERIALS AND METHODS SCG neurons innervating the submandibular gland (SMG) were identified with retrogradely transported fluorogold, and their immunoreactivity to NPY, VIP, or SS determined immunocytochemically. Vectastain ABC kits for glucose oxidase staining, obtained from Vector Laboratories, were used for immunocytochemistry. The polyclonal antibodies used were obtained from Amersham, and displayed no significant cross-reactivity with other neuropeptides. Adult male and female SpragueDawley rats were anesthetized either with ether or chloral hydrate, and approximately 5 j~l of a 1% solution of fluorogold in distilled water was injected bilaterally into the submandibular gland or into the anterior chamber of the eye. After survival times of between 2 and 7 days following injection, animals were re-anesthetized, perfused with 4% paraformatdehyde, and their SCGs removed. Ganglia were postfixed in the same fixative for 1-2 h, placed in 30% sucrose at 4 °C for 2-24 h, frozen, and cryostat sections were taken at 8-16 ~m. Sections were collected in vials of cold phosphate buffer, pH 7.4, and then preincubated with 3% normal goat serum in 0.4% Triton X-100. Sections from each ganglion were divided into vials to be incubated with each of the 3 antisera, and control sections were incubated in the absence of primary or secondary antiserum. The sections were incubated with one of the 3 primary antisera for 24 h at 4 °C. The dilutions used were 1:500 for all 3 antisera. Sections were exposed to a biotinylated secondary antibody for 1 h at room temperature, followed by the avidin:biotinylated glucose oxidase complex for 30 min at room tern-
perature, and the glucose oxidase substrate for 30 min in the dark. Sections were then mounted on slides, air dried and coverslipped with Fluoromount. SCG sections containing fluorogold label were viewed first with fluorescence microscopy to demonstrate the fluorogold (i.e. target-specific neurons), then with brightfield to determine whether each target-specific neuron was immunoreactive for the specific peptide. Counts of the number of fluorogold-labeled neurons and neurons containing both fluorogold and immunoreactivity were tallied either directly from the microscope or from photomicrographs. Control sections incubated in the absence of primary or secondary antisera consistently showed no immunoreactivity. Fluorogold and immunoreactive neurons were counted in 648 sections from SCGs of a total of 17 adult male and 18 adult female rats injected with fluorogold in the submandibular gland or anterior chamber of the eye. The total sample size was 2503 SCG neurons projecting to the eye and 13,357 SCG neurons projecting to the submandibular gland. Counts of the number of fluorogold-labeled neurons that were immunoreactive for each peptide were tallied in each section, and the results were expressed as the percentage of fluorogold labeled neurons (i.e. eye- or SMG-projecting neurons) that were immunoreactive for each peptide. RESULTS A 3-factor A N O V A (gender x peptide x target) showed significant main effects of peptide, target and gender, as well as significant interactions. Taking males and females together, significantly (P < 0.01) more SMG-projecting neurons than eyeprojecting neurons contain vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) (eye = 36%, SMG = 57%). More eye-projecting neurons than SMG-projecting neurons contain somatostatinlike immunoreactivity (SS-LI) (eye = 75%, SMG = 54%) and neuropeptide Y-like immunoreactivity (NPY-L1 (eye = 42%, SMG = 26%). Thus, SCG neurons projecting to the eye and SMG differ significantly in their expression of VIP-LI, SS-LI and NPY-LI. Among neurons projecting to the eye, 36% contain VIP-LI, 75% contain SS-LI and 42% contain NPY-LI. Among neurons projecting to the SMG,
269 57% contain V I P - L I , 54% contain SS-LI and 26%
MALE 1oo
contain N P Y - L I . In both cases, since the total percentages add up to m o r e than 100%, co-expression of two or 3 of these peptides is certain. It is also clear that the expression of these 3 n e u r o p e p t i d e s is h e t e r o g e n e o u s even within target-specific subpopulations of S C G neurons. A priori pairwise t comparisons were made where the main effects were significant. The experiment-
Mean~ of 80 targetvo 60 scG Neurons That Are rmmunereactive $0. 20! 1°o:
wise e r r o r rate was set at 0.05 (ref. 28). W h e n the
Fig. 2. Bars represent the mean percentage of fluorogold-
p e p t i d e immunoreactivity of S C G neurons of males and females was evaluated separately, some differ-
labeled SCG neurons projecting to either the eye or the SMG of adult male rats that showed NPY-like immunoreactivity (-LI), VIP-LI or SS-LI. Error bars are standard errors of the mean. A significantly (P < 0.0001) larger percentage of SMG-projecting neurons than of eye-projecting neurons showed VIP-LI. A significantly (P < 0.0001) larger percentage of eye-projecting neurons than of submandibular glandprojecting neurons showed NPY-LI. The difference between eye- and SMG-projecting neurons showing SS-LI is not statistically significant. Asterisks indicate significant differences between eye- and SMG-projecting neurons. Exclamation points indicate significant differences between that value and the corresponding value seen in females, shown in Fig. 1.
ences were found. In both males and females, a significantly ( P > 0.001) higher percentage of eyeprojecting neurons than S M G - p r o j e c t i n g neurons were N P Y - L I (Figs. 1 and 2). In males but not females, significantly ( P < 0.0001) m o r e S M G projecting neurons than eye-projecting neurons were VIP-LI. In females but not males, significantly ( P < 0.0001) m o r e of the eye-projecting neurons than S M G - p r o j e c t i n g neurons contained SS-LI. W h e n the d a t a from ganglia of males and females were c o m p a r e d , gender differences were also evident. The percentage of S C G neurons projecting to the S M G that contained V I P - L I was significantly greater in males than in females ( P < 0.0001). The p e r c e n t a g e of S M G - p r o j e c t i n g neurons containing
FEMALE ,00
9O
That Are Immunoreactive
1
, "
E%IpSMG
q
EVE S~G N PY
EYE SIdG SS
SS was greater in females than in males ( P < 0.0001). There was no significant gender difference in percentage of SMG-projecting neurons containing NPY-LI. In neurons projecting to the eye, females had a higher percentage of neurons that contained SS than did males, while the percentage of neurons showing NPYLI and VIP-LI was not different in males and females. Figs. 3 - 6 show photomicrographs of fluorogold labeled and immunolabeled SCG neurons.
!
80 70
Mean X of TargetSpecific 60 SCG Neurons ~o
90
*I ~ T
I r__z~t
DISCUSSION
iiiIiii I
40 ~ 30 2o
10 o
EYE SMG EYE SMG EYE SMG VIP NPY SS Fig. 1. Bars represent the mean percentage of fluorogoldlabeled SCG neurons projecting to either the eye or the SMG of adult female rats that showed NPY-like immunoreactivity (-LI), VIP-LI or SS-LI. Error bars are standard errors of the mean. A significantly (P < 0.0001) larger percentage of eye-projecting neurons than of submandibular gland-projecting neurons showed NPY-LI and SS-LI. There was no difference between eye- and SMG-projecting neurons in VIP-LI. Asterisks indicate significant differences between eyeand SMG-projecting neurons. Exclamation points indicate significant differences between that value and the corresponding value seen in males, shown in Fig. 2.
The studies described here show differences in the n e u r o p e p t i d e s expressed by sympathetic neurons projecting to the eye and s u b m a n d i b u l a r gland. Overall, more S M G - p r o j e c t i n g neurons than eyeprojecting neurons contain VIP, and m o r e eyeprojecting neurons than S M G - p r o j e c t i n g neurons contain SS and NPY. The postganglionic neurons that project to specific target tissues are heterogeneous in their peptide content. In addition, these results indicate that there m a y be gender differences in the expression of n e u r o p e p t i d e s . F u r t h e r studies need to be done to d o c u m e n t the pattern of co-localization of these p e p t i d e s in targetspecific subpopulations of S C G neurons, as well as
270
the role of these neuropeptides in each target tissue,
expression of these neuropeptides. Additional stud-
The presence of immunoreactivity to these (or
ies are underway to determine the role of the target
similar) peptides is not sufficient evidence to establish them as neurotransmitters or neuromodulators,
in the development of peptide expression in SCG neurons.
These data do show that there is a relationship between the type of tissue innervated and the
There is evidence in other autonomic systems that peptide-specific subpopulations of n e u r o n s project
Fig. 3. Photomicrographs of sections of the superior cervical ganglion of an adult male rat injected with fluorogold in the submandibular gland and processed for immunoreactivity to NPY. A: combined fluorescence microscopy and brightfield to show NPY-like immunoreactivity. B: the same field as in A under brightfield only. This fluorogold-lab'eled neuron projects to the submandibulargland, and does not show NPY-like immunoreactivity, as seen in several neighboring neurons. Magnification: x 2400. Arrowheads point to the same cell in A and B.
271
Fig. 4. Photomicrographs of sections of the superior cervical ganglion of an adult female rat injected with fluorogold in the anterior chamber of the eye and processed for immunoreactivity to NPY. A: combined fluorescence microscopy and brightfield to show NPY-like immunoreactivity. B: the same field as in A under brightfield only. The fluorogold-labeled (eye-projecting) neuron shows NPY-like immunoreactivity. Magnification: x2210. Arrowheads point to the same cell in A and B.
272
Fig. 5. Photomicrographs of sections of the superior cervical ganglion of an adult male rat injected with fluorogold in the submandibular gland and processed for immunoreactivity to VIP. A: combined fluorescence microscopy and brightfield to show VIP-like immunoreactivity. B: the same field as in A under brightfield only. These fluorogold-labeled neurons project to the submandibular gland and also show VIP-like immunoreactivity. Magnification: x2210. Arrowheads point to the same cells in A and B.
273 tO specific targets. A well-known instance of targetspecificity of neurotransmitters is the postganglionic sympathetic neurons that innervate sweat glands,
which develop noradrenergic, then cholinergic traits 3~'33, without entirely losing their n o r a d r e n e r g i c traits 32. In the guinea-pig celiac-superior mesenteric
Fig. 6. Photomicrographs of a section of the superior cervical ganglion of an adult male rat injected with fluorogold in the submandibular gland and processed for immunoreactivity to SS. A: combined fluorescence microscopy and brightfield to show SS-like immunoreactivity. B: the same field as in A under brightfield only. This fluorogold-labeled neuron projects to the submandibular gland, but does not show SS-like immunoreactivity, as seen in several neighboring neurons. Magnification: x2400. Arrowheads point to the same cell in A and B.
274 ganglion complex there are two main populations of
are heterogeneous in their levels of NPY-LI, VIP-LI
norepinephrine (NE)-containing postganglionic neurons: some contain NPY-L1, and others contain
and SS-LI. There are also differences in the peptide 'profile' between n e u r o n s projecting to the eye and
SS-LI. The NE/NPY-LI neurons 7~ project mainly to
submandibular gland. Thus, there is heterogeneity
intestinal blood vessels, in agreement with studies on other peripheral tissues showing a close relation
within the population of n e u r o n s projecting to a single target tissue, but there are also differences
between NPY-LI neurons and the vascular system 9'
between the populations of SCG neurong innervat-
3s. In contrast, the NE/SS-LI neurons project to the
ing different target tissues.
submucous ganglia and the mucosa. A third population of noradrenergic neurons lacking both pep-
ACKNOWLEDGEMENTS
tides projects to the myenteric ganglia35. A m o n g parasympathetic vagal neurons, those that are SS-LI project to the lung, whereas most of VIP-LI vagal
This work was supported in part by a B R S G grant from Boston University School of Medicine to
neurons project to the esophagus 1°. The present studies show that in the adult rat,
L.L.W. The careful work done by Mr. Patrick Calecas and Ms. Catherine Skelton is gratefully
SCG neurons projecting to the eye or to the SMG
acknowledged.
REFERENCES
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267
BRES 14706
Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons Linda L. Wright and Jennifer I. Luebke Department of Anatomy, Boston University School of Medicine, Boston, MA 02118 (U.S.A.) (Accepted 17 January 1989)
Key words." Sympathetic; Target-specific neuron; Neuropeptide; Adult rat; Gender difference Studies combine the use of the retrograde tracer, fluorogold, and immunocytochemical staining to determine whether superior cervical ganglion (SCG) neurons projecting to the iris or submandibular gland (SMG) in adult male and female rats show distinctive immunoreactivity to somatostatin (SS), vasoactive intestinal polypeptide (VIP), or neuropeptide Y. Overall, more SMG-projecting neurons than eye-projecting neurons contain VIP-like immunoreactivity (VIP-LI), and more eye-projecting neurons than SMG-projecting neurons contain SS-LI and VIP-LI. Thus, postganglionic neurons of the SCG that project to specific target tissues are heterogeneous in their peptide content, and there are differences in the pattern of peptide-immunoreactivity between neurons projecting to these two target tissues. In addition, the results indicate that there may be gender differences in the expression of these neuropeptides. INTRODUCTION Much of what we know about postganglionic sympathetic neurons is based on studies of whole ganglia. However, it is becoming increasingly apparent that the neurons of the sympathetic nervous system are as heterogeneous as the types of target tissues innervated. The rat superior cervical ganglion (SCG) innervates blood vessels of the head and neck, including cerebral I and pituitary vasculature 9' 44 the pineal gland, choroid plexus 49, thyroid gland, submandibular gland, iris and Muller's muscle 6. Principal neurons that innervate different tissue types are intermixed within the ganglion with only very limited regional localization. For example, neurons innervating the pineal tend to lie in the rostral part of the ganglion, but are intermixed with other neurons projecting out the internal carotid nerve 5. Postganglionic neurons give off a single axon that does not branch within the ganglion even in neonates 43. There is no evidence of a single neuron innervating two tissue types 15"36 although some
rearrangement of postganglionic axons among nearby tissues may occur during development 52. Sympathetic neurons are also heterogeneous in their neuropeptide content ~6"17. In addition to norepinephrine, some postganglionic sympathetic neurons also contain substance P (SP) 2-4,21,24,25, vasoactive intestinal polypeptide (VIP) 16"19"22,23,35,50, 7aminobutyric acid ( G A B A ) 12'53, somatostatin (SS) 3' 18.19.25.34.35, Met- and Leu-enkephalins (ENK) 2.3, 29.30.45,vasopressin (VP) 11"20'26"27,neuropeptide Y (NPY) 16'35"38~40"41, bombesin 46 or pancreatic polypeptide (pp)37.38.42.51. Principal neurons of the rat SCG have also been found to contain serotonin and GABA-synthesizing enzymes t2,14, although serotonin is apparently only present during the neonatal period. An intrinsic population of G A B A - l i k e immunoreactive cells persists into adulthood 53. There are morphological differences between neurons that project to different tissues: SCG neurons that innervate the submandibular gland are significantly larger and have a more elaborate dendritic structure than those that innervate the
Correspondence: L.L. Wright, Department of Anatomy, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
268
iris 47~4~. Thus, SCG neurons are heterogeneous in size, transmitter/peptide content, and tissues innervated, but the relationships between neuron characteristics and tissue innervated have not been fully explored. The studies described here investigate the expression of immunoreactivity to 3 peptides (NPY, SS and VIP) in SCG neurons that innervate the eye and submandibular gland to determine: (1) whether the neurons projecting to a given tissue are homogeneous or heterogeneous in peptide expression; and (2) whether the peptide expression is different in neurons projecting to these different target tissues, MATERIALS AND METHODS SCG neurons innervating the submandibular gland (SMG) were identified with retrogradely transported fluorogold, and their immunoreactivity to NPY, VIP, or SS determined immunocytochemically. Vectastain ABC kits for glucose oxidase staining, obtained from Vector Laboratories, were used for immunocytochemistry. The polyclonal antibodies used were obtained from Amersham, and displayed no significant cross-reactivity with other neuropeptides. Adult male and female SpragueDawley rats were anesthetized either with ether or chloral hydrate, and approximately 5 j~l of a 1% solution of fluorogold in distilled water was injected bilaterally into the submandibular gland or into the anterior chamber of the eye. After survival times of between 2 and 7 days following injection, animals were re-anesthetized, perfused with 4% paraformatdehyde, and their SCGs removed. Ganglia were postfixed in the same fixative for 1-2 h, placed in 30% sucrose at 4 °C for 2-24 h, frozen, and cryostat sections were taken at 8-16 ~m. Sections were collected in vials of cold phosphate buffer, pH 7.4, and then preincubated with 3% normal goat serum in 0.4% Triton X-100. Sections from each ganglion were divided into vials to be incubated with each of the 3 antisera, and control sections were incubated in the absence of primary or secondary antiserum. The sections were incubated with one of the 3 primary antisera for 24 h at 4 °C. The dilutions used were 1:500 for all 3 antisera. Sections were exposed to a biotinylated secondary antibody for 1 h at room temperature, followed by the avidin:biotinylated glucose oxidase complex for 30 min at room tern-
perature, and the glucose oxidase substrate for 30 min in the dark. Sections were then mounted on slides, air dried and coverslipped with Fluoromount. SCG sections containing fluorogold label were viewed first with fluorescence microscopy to demonstrate the fluorogold (i.e. target-specific neurons), then with brightfield to determine whether each target-specific neuron was immunoreactive for the specific peptide. Counts of the number of fluorogold-labeled neurons and neurons containing both fluorogold and immunoreactivity were tallied either directly from the microscope or from photomicrographs. Control sections incubated in the absence of primary or secondary antisera consistently showed no immunoreactivity. Fluorogold and immunoreactive neurons were counted in 648 sections from SCGs of a total of 17 adult male and 18 adult female rats injected with fluorogold in the submandibular gland or anterior chamber of the eye. The total sample size was 2503 SCG neurons projecting to the eye and 13,357 SCG neurons projecting to the submandibular gland. Counts of the number of fluorogold-labeled neurons that were immunoreactive for each peptide were tallied in each section, and the results were expressed as the percentage of fluorogold labeled neurons (i.e. eye- or SMG-projecting neurons) that were immunoreactive for each peptide. RESULTS A 3-factor A N O V A (gender x peptide x target) showed significant main effects of peptide, target and gender, as well as significant interactions. Taking males and females together, significantly (P < 0.01) more SMG-projecting neurons than eyeprojecting neurons contain vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) (eye = 36%, SMG = 57%). More eye-projecting neurons than SMG-projecting neurons contain somatostatinlike immunoreactivity (SS-LI) (eye = 75%, SMG = 54%) and neuropeptide Y-like immunoreactivity (NPY-L1 (eye = 42%, SMG = 26%). Thus, SCG neurons projecting to the eye and SMG differ significantly in their expression of VIP-LI, SS-LI and NPY-LI. Among neurons projecting to the eye, 36% contain VIP-LI, 75% contain SS-LI and 42% contain NPY-LI. Among neurons projecting to the SMG,
269 57% contain V I P - L I , 54% contain SS-LI and 26%
MALE 1oo
contain N P Y - L I . In both cases, since the total percentages add up to m o r e than 100%, co-expression of two or 3 of these peptides is certain. It is also clear that the expression of these 3 n e u r o p e p t i d e s is h e t e r o g e n e o u s even within target-specific subpopulations of S C G neurons. A priori pairwise t comparisons were made where the main effects were significant. The experiment-
Mean~ of 80 targetvo 60 scG Neurons That Are rmmunereactive $0. 20! 1°o:
wise e r r o r rate was set at 0.05 (ref. 28). W h e n the
Fig. 2. Bars represent the mean percentage of fluorogold-
p e p t i d e immunoreactivity of S C G neurons of males and females was evaluated separately, some differ-
labeled SCG neurons projecting to either the eye or the SMG of adult male rats that showed NPY-like immunoreactivity (-LI), VIP-LI or SS-LI. Error bars are standard errors of the mean. A significantly (P < 0.0001) larger percentage of SMG-projecting neurons than of eye-projecting neurons showed VIP-LI. A significantly (P < 0.0001) larger percentage of eye-projecting neurons than of submandibular glandprojecting neurons showed NPY-LI. The difference between eye- and SMG-projecting neurons showing SS-LI is not statistically significant. Asterisks indicate significant differences between eye- and SMG-projecting neurons. Exclamation points indicate significant differences between that value and the corresponding value seen in females, shown in Fig. 1.
ences were found. In both males and females, a significantly ( P > 0.001) higher percentage of eyeprojecting neurons than S M G - p r o j e c t i n g neurons were N P Y - L I (Figs. 1 and 2). In males but not females, significantly ( P < 0.0001) m o r e S M G projecting neurons than eye-projecting neurons were VIP-LI. In females but not males, significantly ( P < 0.0001) m o r e of the eye-projecting neurons than S M G - p r o j e c t i n g neurons contained SS-LI. W h e n the d a t a from ganglia of males and females were c o m p a r e d , gender differences were also evident. The percentage of S C G neurons projecting to the S M G that contained V I P - L I was significantly greater in males than in females ( P < 0.0001). The p e r c e n t a g e of S M G - p r o j e c t i n g neurons containing
FEMALE ,00
9O
That Are Immunoreactive
1
, "
E%IpSMG
q
EVE S~G N PY
EYE SIdG SS
SS was greater in females than in males ( P < 0.0001). There was no significant gender difference in percentage of SMG-projecting neurons containing NPY-LI. In neurons projecting to the eye, females had a higher percentage of neurons that contained SS than did males, while the percentage of neurons showing NPYLI and VIP-LI was not different in males and females. Figs. 3 - 6 show photomicrographs of fluorogold labeled and immunolabeled SCG neurons.
!
80 70
Mean X of TargetSpecific 60 SCG Neurons ~o
90
*I ~ T
I r__z~t
DISCUSSION
iiiIiii I
40 ~ 30 2o
10 o
EYE SMG EYE SMG EYE SMG VIP NPY SS Fig. 1. Bars represent the mean percentage of fluorogoldlabeled SCG neurons projecting to either the eye or the SMG of adult female rats that showed NPY-like immunoreactivity (-LI), VIP-LI or SS-LI. Error bars are standard errors of the mean. A significantly (P < 0.0001) larger percentage of eye-projecting neurons than of submandibular gland-projecting neurons showed NPY-LI and SS-LI. There was no difference between eye- and SMG-projecting neurons in VIP-LI. Asterisks indicate significant differences between eyeand SMG-projecting neurons. Exclamation points indicate significant differences between that value and the corresponding value seen in males, shown in Fig. 2.
The studies described here show differences in the n e u r o p e p t i d e s expressed by sympathetic neurons projecting to the eye and s u b m a n d i b u l a r gland. Overall, more S M G - p r o j e c t i n g neurons than eyeprojecting neurons contain VIP, and m o r e eyeprojecting neurons than S M G - p r o j e c t i n g neurons contain SS and NPY. The postganglionic neurons that project to specific target tissues are heterogeneous in their peptide content. In addition, these results indicate that there m a y be gender differences in the expression of n e u r o p e p t i d e s . F u r t h e r studies need to be done to d o c u m e n t the pattern of co-localization of these p e p t i d e s in targetspecific subpopulations of S C G neurons, as well as
270
the role of these neuropeptides in each target tissue,
expression of these neuropeptides. Additional stud-
The presence of immunoreactivity to these (or
ies are underway to determine the role of the target
similar) peptides is not sufficient evidence to establish them as neurotransmitters or neuromodulators,
in the development of peptide expression in SCG neurons.
These data do show that there is a relationship between the type of tissue innervated and the
There is evidence in other autonomic systems that peptide-specific subpopulations of n e u r o n s project
Fig. 3. Photomicrographs of sections of the superior cervical ganglion of an adult male rat injected with fluorogold in the submandibular gland and processed for immunoreactivity to NPY. A: combined fluorescence microscopy and brightfield to show NPY-like immunoreactivity. B: the same field as in A under brightfield only. This fluorogold-lab'eled neuron projects to the submandibulargland, and does not show NPY-like immunoreactivity, as seen in several neighboring neurons. Magnification: x 2400. Arrowheads point to the same cell in A and B.
271
Fig. 4. Photomicrographs of sections of the superior cervical ganglion of an adult female rat injected with fluorogold in the anterior chamber of the eye and processed for immunoreactivity to NPY. A: combined fluorescence microscopy and brightfield to show NPY-like immunoreactivity. B: the same field as in A under brightfield only. The fluorogold-labeled (eye-projecting) neuron shows NPY-like immunoreactivity. Magnification: x2210. Arrowheads point to the same cell in A and B.
272
Fig. 5. Photomicrographs of sections of the superior cervical ganglion of an adult male rat injected with fluorogold in the submandibular gland and processed for immunoreactivity to VIP. A: combined fluorescence microscopy and brightfield to show VIP-like immunoreactivity. B: the same field as in A under brightfield only. These fluorogold-labeled neurons project to the submandibular gland and also show VIP-like immunoreactivity. Magnification: x2210. Arrowheads point to the same cells in A and B.
273 tO specific targets. A well-known instance of targetspecificity of neurotransmitters is the postganglionic sympathetic neurons that innervate sweat glands,
which develop noradrenergic, then cholinergic traits 3~'33, without entirely losing their n o r a d r e n e r g i c traits 32. In the guinea-pig celiac-superior mesenteric
Fig. 6. Photomicrographs of a section of the superior cervical ganglion of an adult male rat injected with fluorogold in the submandibular gland and processed for immunoreactivity to SS. A: combined fluorescence microscopy and brightfield to show SS-like immunoreactivity. B: the same field as in A under brightfield only. This fluorogold-labeled neuron projects to the submandibular gland, but does not show SS-like immunoreactivity, as seen in several neighboring neurons. Magnification: x2400. Arrowheads point to the same cell in A and B.
274 ganglion complex there are two main populations of
are heterogeneous in their levels of NPY-LI, VIP-LI
norepinephrine (NE)-containing postganglionic neurons: some contain NPY-L1, and others contain
and SS-LI. There are also differences in the peptide 'profile' between n e u r o n s projecting to the eye and
SS-LI. The NE/NPY-LI neurons 7~ project mainly to
submandibular gland. Thus, there is heterogeneity
intestinal blood vessels, in agreement with studies on other peripheral tissues showing a close relation
within the population of n e u r o n s projecting to a single target tissue, but there are also differences
between NPY-LI neurons and the vascular system 9'
between the populations of SCG neurong innervat-
3s. In contrast, the NE/SS-LI neurons project to the
ing different target tissues.
submucous ganglia and the mucosa. A third population of noradrenergic neurons lacking both pep-
ACKNOWLEDGEMENTS
tides projects to the myenteric ganglia35. A m o n g parasympathetic vagal neurons, those that are SS-LI project to the lung, whereas most of VIP-LI vagal
This work was supported in part by a B R S G grant from Boston University School of Medicine to
neurons project to the esophagus 1°. The present studies show that in the adult rat,
L.L.W. The careful work done by Mr. Patrick Calecas and Ms. Catherine Skelton is gratefully
SCG neurons projecting to the eye or to the SMG
acknowledged.
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