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Brain Research, 598 (1992) 107-113 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18329

Colocalization of peptide- and tyrosine hydroxylase-like immunoreactivities with Fos-immunoreactive neurons in rat central amygdaloid nucleus after immobilization stress Jari Honkaniemi Department of Biomedical Sciences, University of Tampere, Tampere (Finland) (Accepted 14 July 1992)

Key words: Brain; Hypothalamus; Brainstem; Immediate early gene; Proto-oncogene; Transcription factor

The central amygdaloid nucleus (ACe) is part of the amygdaloid body, and it has been shown to participate in several stress related reactions. The ACe is densely innervated by tyrosine hydroxylase- (TH), corticotropin releasing factor- (CRF), calcitonin gene-related peptide- (CGRP), neurotensin- (NT), somatostatin- (SOM), enkephalin- (ENK), substance P- (SP), vasoactive intestinal polypeptide- (VIP) and cholecystokinin(CCK) immunoreactive (IR) nerve terminals. In addition, the ACe contains numerous CRF-, NT-, SOM-, ENK- and SP-IR perikarya. In previous studies it has been shown that stress stimulates the expression of the immediate early gene c-los in the ACe. The aim of this study was to demonstrate the colocalization of the Fos-IR neurons with the peptide- and TH-IR structures using an immunocytochemical double staining technique. In intact animals the ACe contained only a few Fos-IR neurons. After immobilization stress about 100 Fos-IR neurons were seen per section. They were mainly located in the area, which was enriched by peptide- and TH-IR nerve terminals. The close contacts observed between the Fos-IR neurons and the peptide- and TH-IR nerve endings suggest that the Fos-IR neurons were innervated by these nerve terminals. Furthermore, several NT-, ENK-, SOM- and CRF-IR neurons were observed and the vast majority of these cells exhibited Fos-like immunoreactivity. These results suggest that stress enhances the synaptic activity of the ACe, which stimulates the expression of c-los. Subsequently, Fos may regulate the expression of the NT, ENK, SOM and CRF genes and thus affect the peptidergic efferents from the ACe.

INTRODUCTION

Stress induces several metabolic changes in the brain areas involved in behavioral, neuroendocrine and visceral responses, especially in the hypothalamus and limbic system. The amygdaloid complex is part of the limbic system, which regulates stress-related endocrine and autonomic responses 2 (see refs. 9 and 24 for review). The amygdaloid complex can be divided into subnuclei, which differ from each other morphogenetically, histochemically and functionally 6'9'29. The central amygdaloid nucleus (ACe) is an amygdaloid subnucleus, which participates in several stress-related functions 2 (see refs. 9 and 24 for review). The ACe receives synaptic input by nerve terminals containing corticotropin releasing factor (CRF), calcitonin gene-related peptide (CGRP), neurotensin (NT), somatostatin (SOM), enkephalin (ENK), substance P (SP), vasoac-

tive intestinal polypeptide (VIP), cholecystokinin (CCK) and catecholamines 7"23'3t'39 and these neurotransmitters have been proposed to regulate the function of the ACe 2'9'28. In addition, the ACe contains numerous CRF-, NT-, SOM-, ENK- and SP-immunoreactive (IR) neurons. The ACe can be further divided into subnuclei (lateral capsular (CLC), lateral (LC) and medial (CM) subdivision), which differ from each other in their contents of neurotransmitter found in nerve terminals and somata 42. We have previously demonstrated, that after stress the neurons of the ACe express high amounts of the immediate early gene cofos 16. The protein product of the c-los (Fos) is a transcription factor, which has proved to be a useful marker in detecting changes in intracellular metabolic activity 32. In the brain the basal expression of c-fos is relatively low, but it is induced rapidly and transiently after several stressful stimuli 3.

Correspondence: J. Honkaniemi, Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101, Tampere, Finland. Fax: (358) (31) 156 170.

108 The Fos protein forms dimers with other transcription factors and acts as an enhancer by binding to a specific sequence called the AP-1 binding site in the target cell genome. It is thereby capable of repressing or stimulating gene transcription (see ref. 37 for review). Since synaptic activity has been shown to stimulate the expression of c-fos in neurons 27 it was of interest to study the possible colocalization of the peptide- and T H - I R structures with the Fos-IR neurons in the ACe. MATERIALS

AND METHODS

Adult male Sprague-Dawley rats (n = 16) were used. Since stress induced expression of c-los has a diurnal variation in some brain areas 4 the experimental procedures were performed at the same time of the day. The rats were immobilized in plastic tubes for 2-5 h beginning at 08.00 h. Control experiments included (i) rats killed at 08.00 h, (ii) rats killed at 10.00-13.00 h and (iii) rats that had no drinking water or food for 2-5 h prior to death. The rats were perfused transcardially under chloralhydrate anesthesia (250 mg/kg) first with 100 ml of saline and then for 3 min with an ice-cold fixative containing 2% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Subsequently, the brains were excised and further fixed by immersion at 4°C in the same fixative for 60 min. The samples were cryoprotected (20% sucrose in PBS), frozen in liquid nitrogen, and coronal sections (10/zm thick) were cut with a Microm HM 500 cryostat. Several sections representing different levels in the antero-posterior orientation of the ACe were further processed to demonstrate Fos-, TH- and peptide-like immunoreactivities (LI). For the demonstration of Fos-LI with ABC-method, a rabbit polyclonal antibody was used (dilution 1 : 6,000). This antiserum has been raised against amino acids 128-152 of the rat Fos 45. This is a conserved region between different members of the Fos family and other AP-1 binding transcription factors such as LRF-117 and A T F / C R E B protein families 14. Therefore the labelling observed using this antibody may result from a variety of AP-1 binding proteins. After incubating the samples with the Fos antiserum for 12-24 h at 4°C, the sections were rinsed and incubated with biotinylated goat anti-rabbit antibody (1:200; Vector Labs., Burlingame, USA) followed by ABC-complex (Vector Labs.) for 30 min each. Diaminobenzidine was used as a chromogen to visualize Fos-LI. For double staining of Fos- and peptide-LIs the sections were further incubated for 12-24 h at 4°C with one of the following rabbit antisera: anti-NT (1:800; Peninsula Labs., Belmont, CA, USA), anti-SOM (1:400) 21, anti-SP (1:400; Merseyside Labs., Merseyside, UK), anti-VIP (1:2,000; Milab, Maim6, Sweden), anti-CGRP (1:800; Peninsula Labs.), anti-CCK (1:600) 8, anti-CRF (1"4.00) 34 and anti-met-ENK (1:600; Merseyside Labs.), A mouse TH antiserum was used to demonstrate TH-LI (1:1,000, Incstar Corp., Stillwater, USA). After

two washes the sections were incubated with a rhodamine-conjugated goat anti-rabbit antibody (1:200; Boehringer Mannheim Chemicals, Mannheim, FRG) or with a biotinylated sheep anti-mouse antibody (1:200; Amersham Int. Buckinghamshire, UK) followed by a rhodamine-conjugated avidin (1:200; Vector Labs.) at 37°C for 30 min each. All the antibodies were diluted in PBS containing 1% BSA and 0.3% Triton X-100. After staining the sections were embedded in a mixture of glycerol and PBS (3:1) containing 0.1% paraphenylenediamine. The sections were photographed with Nikon Microphot FXA microscope. Controls included the omission of the primary and secondary antibodies, staining with non-immunized rabbit serum (1:6,000 for the ABC-method and 1 : 400 for fluorescence), non-immunized mouse serum (1:1,000 for fluorescence) and peptide antisera preabsorbed with appropriate, commercially available peptides. None of the immunoreactivities described below were observed in the controls. The specificity of the Fos antiserum was controlled by incubating the antibody with corresponding M-peptide, carboxyterminal peptide (amino acids 246-266) and N-terminal peptide (amino acids 9-28) at 1/zM concentration before incubating sections with antibody. Preabsorbtion with the corresponding peptide abolished all labelling, whereas preabsoption with either C-terminal or N-terminal peptide did not influence the staining. Fos-antiserum did not stain any of the structures labelled by peptide or TH antisera and vice versa showing that there was no cross-reactivity between the primary or the secondary antibodies used in the first and the second staining sequences. RESULTS

In intact animals the ACe contained only some weakly labelled Fos-IR neurons located mainly in the CL (Fig. 1). After immobilization stress about 100 labelled neurons per section were observed (Fig. 2a). They were mainly concentrated in the CL (n = 50-80) and CLC (n = 10-20). In these subnuclei about 70% of the neurons were Fos-IR. About one third (n -- 10-20) of the neurons in the CM exhibited Fos-IR. The Fos-LI was confined to the nuclei of the labelled neurons. The number of Fos-IR neurons was about the same after 2 or 5 h immobilization. The CGRP-, NT- and SP-IR nerve terminals formed dense networks in the CL, while the CLC was even denser innervated by these peptides. A moderately dense network of CCK- and E N K - I R nerve terminals was observed in the CL and CLC. Nerve terminals containing C G R P - (Fig. 2a-b), NT- (Fig. 3), SP- (Fig.

-c> Fig. 1. Demonstration of Fos-LI in the ACe of a nontreated rat. Arrowheads indicate a few labelled nerve cell nuclei distributed mainly in the CL. Bar = 100 ~zm. Fig. 2. a,b: colocalization of CGRP-IR nerve terminals and Fos-IR neurons in the ACe after immobilization stress. The majority of the Fos-IR neurons are located in the CL, whereas several labelled neurons are located in the CLC. Some positive neurons can be observed in the CM (a). Bar represents 100/~m. Arrowheads indicate three Fos-IR neurons in the CL, which are densely surrounded by CGRP-IR nerve terminals (b). Bar = 50 p,m. Fig. 3. Three NT-positive neurons, which exhibit also Fos-LI are indicated by arrows. A dense network of Fos-IR nerve terminals surrounds Fos-IR neurons. Bar = 50 tzm. Fig. 4. A dense network of SP-IR nerve terminals surrounds the Fos-IR neurons (arrowheads) in the CL. Bar = 50 p.m.

110 4), CCK- (Fig. 5a,b) and ENK-LI (Fig. 6a,b) formed close contacts with the Fos-IR neurons in the CL and CLC. Especially the CGRP-positive nerve terminals encapsulated the Fos-IR neurons (Fig. 2b). The SOM(Fig. 7a,b), CRF- (Fig. 8), VIP- (Fig. 9) and T H - I R (Fig. 10) nerve terminals were the densest in the CL, while the CLC was only sparsely innervated by these peptides. Nerve terminals containing these peptides were in close contact with the Fos-IR neurons in the CL. All the peptide- and T H - I R nerve terminals innervated only sparsely the neurons in the CM. Several NT- (n = 32) and SOM-IR (n = 69) neurons and also some neurons exhibiting ENK-LI (n = 22) were observed in the CL. A total of 82 CRF-IR neurons were observed in the CLC and CL. No labelled neurons for the other peptides studied were seen. The vast majority (90-95%) of the NT- (Fig. 3), ENK- (Fig. 6a,b), SOM- (Fig. 7a,b) and CRF-IR (Fig. 8) neurons in the ACe exhibited Fos-LI after stress. Immobilization stress did not result in any clear changes in the number of immunoreactive perikarya or nerve terminals. DISCUSSION In previous studies the ACe has been connected with several stress related changes, e.g., in the formation of gastric stress ulcers (see ref. 9 for review), regulation of blood pressure 2 and secretion of glucocorticoids 6 and catecholamines 2 from the adrenal gland. The present data provide further evidence that the ACe is activated in stress since a large number of the neurons of the ACe exhibited Fos-LI after immobilization stress. I observed no clear difference in the number of Fos-IR neurons after immobilization lasting for 2 or 5 h. Sharp et al. 36 have demonstrated using a Fos specific antibody that Fos-LI begins to disappear after 4 h of a stressful stimulus. As the Fos antiserum used in this study recognizes several members of the Fos family, the Fos-LI observed after 5 h of immobilization may represent other Fos-related proteins.

The precise signal inducing the expression of c-los is uncertain. Stress stimulates the secretion of glucocorticoid hormones and catecholamines from the adrenal gland, which both are capable to affect the expression of c-fos 13'19. Thus, part of the induction of c-fos may result from the influence of humoral factors. Similarly, synaptic excitation caused by electrical nerve stimulation TM or by treating neuronal cultures with agents that stimulate synaptic activity 27 enhances the expression of c-los. In this study, the Fos-IR neurons were mainly located in the CL and CLC, which received a strong peptide- and T H - I R input. Although synapses can not be demonstrated at light microscopic level, the close contacts observed between the Fos-IR neurons and the peptide- and catecholaminergic nerve terminals suggest that the Fos-IR neurons are innervated by these nerve terminals. Therefore, the stress induced expression of c-los may be due to the peptide- and T H - I R innervation of these neurons. Stress increases the release of noradrenaline in the amygdala 4°, and systemic I and local 38 adiministration of noradrenaline has been shown to stimulate the expression of c-los. Thus, stress induced increase in the synaptically released noradrenaline may stimulate the expression of c-fos also in the ACe. In addition, it has been shown that CRF adiministered intracerebroventricularly stimulates the synthesis of c-los in the amygdala 5. It is not known, however, whether the release of CRF or other neuropeptides present in nerve terminals in the ACe is stimulated by stress. The ACe receives synaptic input from several sources, especially from the parabrachial nucleus (PB), bed nucleus of stria terminalis (BNST) and hypothalamus (see ref. 30 for review). The pontine PB receives substantial autonomic input 2°'46 and both the PB and ACe participate in stress-related reactions. Previously it has been demonstrated that the SP-, CGRP- and NT-IR neurons in the PB project to the A C e 35'43'44. When C G R P is injected into the ACe, it influences the blood pressure and pulse rate 2'28. Injections of NT into

D Fig. 5. a,b: arrowheads indicate three Fos-IR neurons, which are densely surrounded by CCK-IR nerve terminals. Bar = 50 ~tm. Fig. 6. Arrowheads indicate three E N K - I R neurons, which exhibit also Fos-Ll. Several Fos-IR neurons can be seen, and the ENK - I R nerve terminals are in close contact with these neurons. Bar = 50 tzm. Fig. 7. a,b: six SOM-positive neurons are indicated by arrowheads. All these neurons exhibit also Fos-LI. SOM-IR nerve terminals can be seen in close contact with Fos-IR neurons. Bar = 50/zm. Fig. 8. Arrowheads indicate four C R F - I R neurons, which exhibit also Fos-LI. C R F - I R nerve terminals form close contacts with Fos-IR neurons. Bar = 50/xm. Fig. 9. VIP-IR nerve terminals are in close contact with Fos-positive neurons (arrowheads). Bar = 50 ~zm. Fig. 10. A dense network of TH-IR nerve terminals surrounds the Fos-IR neurons (arrowheads) in the CL. Bar = 50 ~tm.

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112 the ACe also decrease the formation of gastric stress ulcers (see ref. 9 for review). Since the SP-, CGRPand NT-IR nerve terminals were seen in close contact with the Fos-IR neurons, these peptides may mediate autonomic information during stress from the PB, which further activates the neurons in the ACe resulting in stress-related visceral responses. The dopaminergic and noradrenergic nerve terminals of the ACe originate from the brainstem 7 and the enkephalinergic nerve terminals from the BNST 41. Furthermore, there is evidence that part of the SOM-, SP- and ENK-IR nerve terminals in the ACe are of intrinsic origin 1°'11. When injected into the ACe, SOM has cardiovascular effects 2, enkephalins and dopamine decrease the formation of stress induced gastric ulcers (see ref. 9 for review), C G R P and CRF alter plasma norepinephrine levels and SOM influences plasma epinephrine levels 2. Since these neurotransmitters innervated the Fos-IR neurons in the ACe, it is likely that also these peptides mediate stress induced information from several nuclei in the brainstem and hypothalamus as well as from intra-amygdaloid sources to the ACe. When injected into the ACe, VIP and CCK have no effects on blood pressure, plasma catecholamines or formation of gastric stress ulcers 2'9, but the dense innervation of Fos-IR neurons by nerve terminals immunoreactive for these peptides suggests that VIP and CCK may have some currently unknown effects on the ACe. For instance, electrical stimulation of the ACe has clear effects on plasma glucocorticoid levels (see ref. 24 for review), but there are no reports describing whether transmitters found in nerve terminals in the ACe are involved in the regulation of glucocorticoid secretion. Several CRF-, SOM-, ENK- and NT-IR neurons were observed in the ACe. Since nearly all the peptide positive neurons exhibited also Fos-LI after stress the transcription of these peptides may be directly affected by the AP-1 binding proteins. Stress caused no significant change in the number of the labelled neurons but nevertheless it is possible that the expression of these peptides is altered in stress since immunocytochemistry is a poor method for quantitive analysis. It is also likely that the activity of the CRF-, SOM-, ENK- and NT-IR efferent pathways from the ACe are altered in stress. These peptidergic neurons of the ACe have shown to project to various nuclei in the hypothalamus and brainstem 12'15'22'25'26'33'41. Thus, these peptidergic efferents may mediate stress-related information from the ACe to the hypothalamus and brainstem. In conclusion, the finding that the peptide- and T H - I R nerve terminals formed close contacts with the

Fos-IR neurons suggest that the expression of c-los is at least partly induced by peptide- and catecholaminergic input. Since the vast majority of the CRF-, SOM-, ENK- and NT-IR neurons in the ACe also exhibited Fos-LI it seems that the peptidergic efferent connections from the ACe to the hypothalamic and brainstem nuclei are activated in stress. Acknowledgements. I thank Dr. R.P. Elde, University of Minnesota, Minneapolis, USA, Dr. P. Frey, Sandoz Research Institute, Bern, Switzerland, Prof. T. H6kfelt, Department of Neurobiology and Histology, Karotinska Institute, Stockholm, Sweden, Dr. M. ladarola, NIDR, NIH, USA and Dr. W. Vale, Salk Institute, La Jolla, CA, USA for generous supply of antisera. The skilful technical assistance of Mrs. Hannele Ylitie is greatly appreciated. This study was supported by a grant from Emil Aaltonen Foundation.

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Colocalization of peptide- and tyrosine hydroxylase-like immunoreactivities with Fos-immunoreactive neurons in rat central amygdaloid nucleus after immobilization stress.

The central amygdaloid nucleus (ACe) is part of the amygdaloid body, and it has been shown to participate in several stress related reactions. The ACe...
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