Corticotropin releasing factor neurons are innervated by calcitonin gene-related peptide terminals in the rat central amygdaloid nucleus

Brain Research Bulletin, Vol. 33, No. 5, pp. 529-534, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + Ml

Pergamon 0361-9230(93)EOOlO-J

Corticotropin Releasing Factor Neurons are Innervated by Calcitonin Gene-related Peptide Terminals in the Rat Central Amygdaloid Nucleus ELIZABETH

A. HARRIGAN, DEBRA J. MAGNUSON, GAYLE AND THACKERY S. GRAY’

M. THUNSTEDT

Department of Cell Biology, Neurobiology and Anatomy, Loyola Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153 Received

29 July 1993; Accepted

4 October

1993

HARRIGAN, E. A., D. J. MAGNUSON, G. M. THUNSTEDT AND T. S. GRAY. Corticotropin releasing factor neurons are innervated by calcitonin gene-relatedpeptide terminals in the rat central amygdaloid nucleus. BRAIN RES BULL 33(5) 529534, 1994.-The central nucleus of the rat amygdala (CeA) contains many corticotropin releasing factor (CRF) immunoreactive neurons. Previous studies have demonstrated that these CRF neurons project to brain stem regions responsible for modulation of autonomic outflow. Calcitonin gene-related peptide (CGRP) terminals overlap the distribution of CRF cell bodies in the CeA. These CGRP terminals mainly originate from cell bodies that are located in the pontine parabrachial nucleus. The present study examined the possibility that CRF cell bodies are innervated by CGRP terminals. The results suggest that over 35% of the CRF neurons in the CeA are contacted by CGRP terminals as judged by the indiscernible distances between the terminals and cell bodies and or dendrites. In addition, a dual-labeled electron microscopic technique demonstrates that CGRP terminals form synaptic contacts with CRF cell bodies and dendrites. This suggests that CGRP neurons in the parabrachial nucleus can modulate the activity of CRF amygdaloid brain stem efferents. Previous studies have shown that CRF, when administered into the central nervous system, produces increases in heart rate, blood pressure, and plasma catecholamines. CGRP administration into the amygdala has been shown to have a similar effect on the autonomic nervous system. It is, therefore, possible that CGRP could exert these effects via an amygdaloid CRF pathway. Stress

Parabrachial

nucleus

Autonomic

Immunocytochemistry

THE amygdala is a limbic system structure that regulates autonomic and endocrine functions that occur in response to stressful stimuli. Studies have shown that electrical or chemical stimulation of the central nucleus of the amygdala (CeA) in awake, nonanesthetized cats and rats produces autonomic changes that resemble a stress response. Stimulation of the CeA produces increases in heart rate, mean arterial pressure, and plasma catecholamines, and alters respiratory, gastric, and behavioral activity (2,12,13,17,27,36). In addition, lesions of the CeA block or attenuate autonomic, neuroendocrine, and behavioral response to various conditioned stressors (8,20,22,40,45). The central nucleus of the amygdala (CeA) is populated by a large number of neurons that express corticotropin releasing factor (CRF) (9,37). These CRF-containing neurons have projections to autonomic brain stem nuclei including the dorsal vagal complex, central grey, and parabrachial region (14,26,41). Corticotropin releasing factor has been shown to have physiological actions in the central nervous system similar to amygdaloid stimulation. Administration of CRF into the central nervous system produces elevations in heart rate, mean arterial pressure, and

’ To whom requests

concentrations of catecholamines (4,lO). Thus, the central actions of CRF resemble the constellation of responses observed after stimulation of the CeA. This suggests a role for amygdaloid CRF neurons in autonomic nervous system function. The CeA is densely innervated by nerve terminals containing calcitonin gene-related peptide (CGRP) (35). Retrograde labeling and anterograde degeneration studies demonstrate that CGRP terminals projecting to central amygdaloid subnuclei mainly originate from cell bodies in the parabrachial nucleus of the pons (28,33). A few CGRP axon terminals arise from cells located in the thalamus (44). Direct injection of CGRP into the central nucleus produces increases in blood pressure, heart rate, and plasma catecholamines (525). Furthermore, studies have shown that the CeA contains a high density of radiolabeled %CGRP binding sites (29,34). The CeA is one of the few brain regions that contains a comparable number of CGRP binding sites in relation to the density of nerve fibers found there (34). Thus, the above data suggest important site specific biological activity for CGRP release in the amygdala. The distribution of CRF neurons overlaps that of CGRP terminals in the CeA. This suggests the possibility that CGRP terplasma

for reprints should be addressed.

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ET AL.

FIG. 1. Bright-field photomicrographs illustrating the distribution of CRF immunoreactive cell bodies and CGRP terminals in the CeA sampled from a 40 micron (A and B) and a 1.0 micron thick coronal section each sampled from a midrostrocaudal level of the CeA. Large arrows on left (A and C) indicate CRP cell bodies that are contacted by CGRP terminals indicated by small arrows on the right (B and D). Bars for A = 100 pm, Bars in B, C, and D = 25 pm. minals modulate CRF neurons in the CeA. The present study investigated whether or not CGRP terminals synapse on CRF neurons, and, if so, what percentage of CRF neurons are innervated. METHOD

The subjects of this study were 200-250 g Long-Evans, biack-holed male rats. Animals were overdosed with sodium pentobarbital (0.5 cc 45 mg/kg). Their brains were fixed using the dual pH fixative method (1). Animals were perfused through the ascending aorta with 100 ml of PBS (pH 7.6 at 37°C). This was foIlowed by 250 ml of 4.0% parafo~~dehyde and 0.05% glutaraldehyde in 0.167 M phosphate buffer (PH 6.5 at 4.O”C) and then 250 ml of 4.0% paraformaldehyde and 0.05% glutaraldehyde in 0.167 M phosphate buffer @H 8.5 at 4.O”C). The brains were removed and placed in 4.0% parafo~aldehyde in phosphate buffer @H 8.5) for 20-60 min. Brains were cut into 20-25 pm thick sections using a vibratome (Lancer). Every section was saved and placed sequentially into one of four vials. Sections were first washed in PBS with 0.25% Triton-X (PBStx) with 2.5% normal donkey serum (NDS) for 30 min, and then placed in 0.3% hydrogen peroxide in PBS @H 7.6) for 30 min. Then the sections were rinsed in PBS-tx with 2.5% NDS for 30 min. Tissue was processed for dual staining of CCZP and CRF immunoreactivity according to the methods of Hancock (15) for

Iight microscopic and semithin sections or Lakes and Basbaum (21) for electron microscopic analysis. The tissue was immersed for 18-24 h in rabbit antibodies to CGRP (courtesy of W. Vale, Salk Institute) diluted 1:lOOO in a solution of 1.0% normal donkey serum and PBS-tx. Sections were washed in PBS-tx with 2.5% NDS for 30 min and placed in donkey antirabbit immunogammaglobulin conjugated to biotin (Jackson Immunoresearch Labs) diluted 1:5000 in PBS-tx for 30 min. Sections were then washed in PBS-tx for 15 min and placed in a solution of avidin-bound horseradish peroxidase (Bethesda Res. Labs) diluted in 1:300 PBS-tx with 2.5% NDS for 30 min. The tissue was washed 1 min 0.1 M phosphate buffer (pH 7.4) washed for 10 min in 0.1 M acetate buffer, pH 6.0, and reacted using 8.75 mg of diaminobenzidine (DAB, Hach) in 25 ml 0.1 M acetate buffer @H 6.0) plus 625 mg nickel ammonium sulfate to which 20 ~10.3% Hz02 were added. The reaction was stopped by placing the sections in acetate buffer. For electron micros~py, Triton-X 100 detergent was not used in any of the solutions with one exception, the CGRP antibody solution. Sections are rinsed with 0.1 M Tris buffer (pH 7.6) for 60 min and then placed in a 50 ml solution containing 25 mg of DAB in Tris buffer. Then 50 ,~l of 0.3% H202 is added to initiate the reaction. The tissue is washed in cold phosphate buffer to stop the reaction, and is washed in PBS for at least 1 h. For light microscopy, tissue was processed for visualization of CRF immunoreactivity according to the methodology of Han-

CGRP AMYDGALOID

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in PBS containing 1.0% normal donkey serum for 16-24 h. Sections were washed for 30 min in PBS, incubated in biotinylated donkey antirabbit IgG (Jackson Res., diluted 1:2500 in PBS) for 60 min and washed in PBS for 15 min. Next, sections were incubated in streptavidin-HRP (Bethesda Res. Labs) diluted 1:300 in PBS for 60 min. Sections are next rinsed in 0.01 M phosphate buffer, pH 6.8, for 30 min. The tissue was reacted in a 0.01 M phosphate buffer solution @H 6.8) containing 0.1% BDHC, 0.25% sodium nitroprusside, and 0.005% H202. The reaction was stopped by immersing the sections in 0.01 M phosphate buffer, pH 6.8. Tissue prepared for electron microscopic and semithin analysis was osmium treated, dehydrated in a series of alcohols, and embedded in Epon. Semithin and thin sections were produced according to standard sectioning procedures. Immunoadsorption of each antibody with homologous antigen and primary antibody omission served as a control for immunocytochemical specificity. RESULTS The individual distribution of CGRP and CRF immunoreactivity in the CeA has been published previously (7,28,35). The present findings are essentially the same as previous reports. Thus, only a brief summary of the location and organization of CGRP and CRF immunoreactive cell bodies, fibers, and terminals is presented. For purposes of anatomical description the terminology of McDonald (23) and Cassell et al. (7) is used. Light Microscopy

Bregma-3.14mm FIG.2.Line drawing illustrating the distribution of corticotropin releasing factor (CRF) cells (0) and calcitonin gene-related (CGRP) fibers and terminals immunoreactive terminals (dashes) at three rostrocaudal levels of the central nucleus of the amygdala (CeA). Coronal levels relative to bregma correspond to the Paxinos and Watson rat brain atlas. CLC = lateral capsular division of CeA, CM = medial division of CeA, CL = lateral division of CeA, st = stria terminalis.

cock (15). Briefly, sections were placed in a rabbit anti-CRF (donated by the late Tom O’Donohue) for 18-24 h. PBS-tx at a 1:2000 dilution. Sections were washed and placed in biotinylated donkey antirabbit and diluted as described above for 45 min. Sections were again washed and placed in avidin bound horseradish peroxidase for 45 min. Again the tissue was washed 1 h in 0.1 M phosphate buffer (pH 7.6) and reacted using 8.75 mg of DAB in 25 ml 0.1 M phosphate buffer @H 7.4) to which 50 ~1 0.3% H202 were added. The reaction was stopped by immersion in PBS. Sections were mounted on slides and cover slipped using DePex mounting media. For electron microscopy, second stage immunohistochemistry was performed using benzidine dihydrochloride (BDHC) as a chromogen. Tissue was placed in rabbit anti-CRF diluted 1:2000

In this study, CRF immunoreactive neurons were stained amber brown using the DAB staining technique. They typically exhibited a pyramiform shape and numerous DAB-stained dendrites were clearly apparent (Fig. 1). The majority of neurons immunoreactive for CRF were located in the lateral subdivision (CL) of the CeA. However, many CRF immunoreactive cells were also found in the medial subdivision (CM) and ventral subdivision. This distribution pattern was maintained throughout rostra1 and caudal levels of the CeA (Fig. 2). Previously, it was estimated that 1750 CRF immunoreactive neurons are located in the CeA (14). Terminals containing CRF were observed in the same region as the CRF immunoreactive cell bodies (i.e., primarily in the CL). The CGRP fibers and terminals were stained dark black by the nickel-intensified DAB reaction. At higher magnification, numerous varicose fibers with distinct boutons and terminals were apparent (Fig. 1). CGRP-immunoreactive fibers and presumed terminals are most heavily concentrated in the lateral capsular (CLC) subdivision, but were also found in the CL and CM (Fig. 2). Many fibers formed clusters around both unstained and CRF immunoreactive neurons. No CGRP immunoreactive cell bodies were observed in the central nucleus, Many CGRP immunoreactive terminals appeared to contact CRF immunoreactive neurons in the CeA. A terminal contact was defined as an indiscernible distance between a CGRP terminal and CRF cell body or dendrite (Fig. 1). The contacts between CGRP terminals and CRF neurons were observed most frequently in the CL, the region of greatest overlap of CRF cell bodies and CGRP terminals. Very few contacts were observed in the CM. In three animals we counted the total number of CRF neurons that received contacts by CGRP terminals. One out of four sections were sampled throughout the rostrocaudal extent of the CeA. The percentage of CRF immunonreactive cell bodies and dendrites that had CGRP contacts was calculated. In these three animals, 35-42% of the CRF cell bodies or dendrites were associated with at least one or more CGRP terminal. Commonly

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FIG. 3. Low (A) and high (B) power electronmicrographs illustrating a CGRP terminal synapsing on a CRF immunoreactive cell body in the CeA. Large arrows indicate the synaptic contacts. Small arrows indicate examples of BDHC crystals in the cytoplasm of the CRF immunoreactive cell body. Bars for A and B = 1.0 pm.

CRF immunoreactive cells received as many as three to four contacts. The innervation of CRF neurons by CGRP terminals was most apparent on the perikarya or proximal dendrites. However, smaller dendrites were contacted as well. Neurons imm~oreactive for CRF are also contacted by CRF immunoreactive terminals. Terminals immunopositive for CRF can be distinguished from CGRP terminals by their translucent appearance and brown color. The CRF terminals are most densely distributed in the CL where most of the CRF cell bodies were also located. CRF cell bodies and dendrites were also in-

nervated by CRF terminals in the CM and ventral parts of the CeA. Electron microscopy

In thin sections prepared for electron microscopy, CGRP terminals were observed forming symmetric axo-somatic synapses with CRF immunoreactive (Fig. 3) and nonreactive neurons. The terminals were usually large and were comprised of several separate synapses (Fig. 3). The DAB reaction product found in the

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terminals was usually diffuse, agranular, and more electron dense than the surrounding tissue. The DAB stain was distributed throughout the CGRP axons and terminals, but was more densely clumped around synaptic vesicles and membranes. The CRF neurons were identified by the presence of cytoplasmic BDHC reactive crystals (Fig. 3). These crystals had a columnar shape and vary considerably in their size. The number of crystals per CRF immunoreactive cells also varied greatly. Crystals were observed singly or in large electron-dense clumps or aggregates. As previously reported, in densely immunoreactive neurons the crystals frequently formed parallel cross bridges (21). Very few BDHCstained CRF terminals were apparent in our sections. Those found were characterized by heavy crystalline deposits associated with a few vesicles and shredded membranes. Presumably the formation of the crystals severely damaged the morphology of the CGRP terminals that were reactive. The structural integrity of the DAB-stained terminals was preserved much better than that of the CRF terminals. As a result, the majority of recognizable immunostained terminals observed under the electron microscope proved to be CGRP immunoreactive.

DISCUSSION

This study examines the significant overlap in the distribution of CRF neurons and CGRP terminals in the CeA. Earlier reports suggested that CGRP-CRF synaptic contacts in the CeA were likely. Electron microscopic studies have shown that CGRP-like immunoreactive neurons form axo-dendritic and axo-somatic contacts in the lateral and lateral capsular regions of the central nucleus (30). Honkaniemi et al. (18) have shown via electron microscopy that CGRP terminals form synaptic contacts with neurotensin cells in the CeA. Neurotensin has been localized within some of the CRF expressing neurons in the CeA (31). This prompted us to attempt electron microscopic studies, which have shown that CGRP terminal labeling does occur on CRF neurons in the CeA. Light microscopic analysis revealed that nerve cell terminals immunopositive for CGRP are in close association with as many as 42% of the CRF cell bodies in the CeA. Electron microscopic analysis demonstrated that these CGRP terminals form synaptic contacts with CRF immunoreactive neurons. The distinct ultrastructural features of BDHC reaction product as compared to DAB reaction product made it possible to identify CGRP terminals in synaptic contact with CRF neurons. In light microscope sections we also observed many CRF neurons in the CeA being contacted by CRF immunoreactive terminals. Corticotropin releasing factor immunoreactive terminals contact many unstained cells as well. The CRF-CRF innervation was variable: at caudal levels, CRF neurons in the CM were contacted almost exclusively by CRF terminals as compared to lateral parts of the CeA where CGRP and CRF contacts both occurred. This data confirms recent reports demonstrating that amygdaloid CRF neurons are innervated by CRF terminals (39).

A dense plexus of CRF terminals was reported in the region of CRF neurons of the CeA. Some of the CRF terminals formed synaptic contacts with CRF dendrites and perikarya (39). The source of these terminals are from cells bodies located in the lateral hypothalamus, dorsal raphe and intrinsic CRF cells in the CeA. An additional source of CRF terminals may be from neurons in the medial geniculate complex (19). It has been suggested that the CRF found within the medial geniculate nucleus and its projections play a role in the acoustic aspects of the stress response (19). Many CRF immunoreactive cells are contacted by both CRF and CGRP terminals. Because CGRP and substance P coexist in neurons in the parabrachial nucleus that project to the CeA, substance P may be located in CGRP immunoreactive terminals that synapse on CRF neurons in the CeA (43). In addition, many CGRP and CRF immunoreactive terminals appear to innervate unstained neurons in these experiments. Some of these neurons may contain enkephalin because CGRP-containing terminals also form synaptic contacts with enkephalin immunoreactive cell bodies in the CeA (32). Multiple innervation of CeA neurons by other peptidergic terminals has been previously observed (42). It is likely that CRF neurons are innervated by many other peptidergic terminal types. The strong correspondence between CGRP terminals and receptors in the CeA suggests an important biological function for CGRP as a neurotransmitter. Potassium-evoked release of CGRP has been demonstrated in vitro from slices of the amygdala (16). This release was blocked in calcium-free buffer. Thus, the data strongly supports a neurotransmitter role for CGRP terminals in the CeA. Electrophysiological studies have shown that CGRP, when administered into the central nervous system, has a predominantly inhibitory effect on the membrane excitability of neurons in the rat hypothalamus and basal forebrain (38). The effects of CGRP upon the actions of individual neurons in the CeA has not been investigated. Both CGRP and CRF have central nervous system effects that resemble changes in the autonomic nervous system that occur during stress. Administration of CRF into the lateral ventricle causes an increase in heart rate, mean arterial pressure, and catecholamines (6,ll). CRF injection into the CeA produces an elevation in plasma norepinephrine levels, but no change in heart rate or blood pressure (3,24). Elevations in heart rate, blood pressure, and norepinephrine occur in response to direct microinjection of CGRP into the CeA ($25). Overall, the data collected to date suggest study indicated that CGRP terminals can act directly upon CRF neurons in the CeA. Thus, CGRP terminals are strategically located to affect the function of CRF neurons that project to widespread regions of the brain stem. The function of these connections may be important in expression of centrally mediated autonomic responses to stress-evoking stimuli. ACKNOWLEDGEMENTS

This study was supported by NIH grant NS 20041 and a grant from the Potts Foundation.

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