Ultrastructural evidence for an olfactory-autonomic pathway through the rat central amygdaloid nucleus

Neuroscience Letters, 133 (1991) 100 104 © 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50

100

NSL 08209

Ultrastructural evidence for an olfactory-autonomic pathway through the rat central amygdaloid nucleus M.D. Cassell a n d L. R o b e r t s Department of Anatomy, University of lowa, Iowa City, IA 52245 (U.S.A.) (Received 26 July 1991; Revised version received 22 August 1991; Accepted 24 August 1991)

Key words: Autonomic pathway; Olfaction; Amygdala; Retrograde horseradish peroxidase transport; Electron microscopy The innervation of medullary projection neurons in the central amygdaloid nucleus (Ce) by afferents from the ventral taenia tecta (VTT) was investigated using combined lesion-induced axonal degeneration and retrograde transport of horseradish peroxidase-conjugated wheat germ agglutinin (HRP-WGA). Injections of HRP-WGA into the nucleus tractus solitarii resulted in retrograde labeling of neurons in the medial Ce. Ultrastructurally HRP-WGA reaction product was identifiable in the perikarya and proximal dendrites of Ce neurons. Degenerating terminals, probably due to damage at the HRP-WGA injection site, were few and confined to the ventral Ce. Electrolytic coagulation of the VTT resulted in approximately 9% of terminals in medial Ce showing signs of degeneration at 5 days post-lesion. Of the terminals sampled, slightly more than 40% were in contact with the dendrites of retrogradely labeled neurons. Where evident, these terminals formed exclusively symmetrical synaptic contacts. These data provide evidence for an oligosynaptic olfactory-autonomic pathway in the rat that may mediate olfactory influences on gastric and cardiovascular aspects of autonomic function.

The capacity for olfactory stimuli to elicit profound changes in cardiovascular and gastrointestinal aspects of autonomic function has been well known since the classic studies of Pavlov [19] and Allen [1]. Several anatomical substrates have been proposed to link olfactory structures with CNS structures involved in autonomic regulation, one of the most widely offered being the connections passing from the olfactory bulb and piriform cortex, via the anterior cortical and medial amygdaloid nuclei, to the hypothalamus [e.g. 2,5,6,12,20,30]. Though the anterior cortical and medial amygdaloid nuclei project heavily to the hypothalamus [13,14,21], their influence on brainstem-mediated autonomic function is likely to be indirect since neither they nor their hypothalamic targets appear to project strongly to brainstem autonomic regions [22-24]. The central amygdaloid nucleus (Ce), on the other hand, projects directly to the parabrachial complex, nucleus tractus solitarii, dorsal vagal motor nucleus and ventral medulla [e.g. 11,17,26,29]. Moreover, physiological studies have demonstrated a prominent role for the Ce in central autonomic regulation, particularly in relation to aversive or stressful stimuli (see ref. 7 for review). Unlike other

Correspondence." M.D. Cassell, Department of Anatomy, University of Iowa, Iowa City, IA 52245, U.S.A.

amygdaloid nuclei, the Ce does not receive direct projections from either primary olfactory cortex or from the main and accessory olfactory bulbs [9,10,25]. The Ce does, however, receive projections from the ventral taenia tecta (VTT), a forebrain region with heavy, reciprocal connections with both primary olfactory cortex and the olfactory bulb [4,18]. Previous studies in this laboratory [4] had demonstrated that the termination of the VTT input to the central nucleus (and to the amygdala as a whole) is restricted to its medial subdivision where the bulk of the neurons projecting to the dorsal and ventral medulla arise [e.g. 3,28,29]. Clearly, the VTT-Cemedulla pathway offers a more direct route for olfactory information to access autonomic regulatory centers. However, since the medial Ce also contains neurons projecting to the hypothalamus, bed nucleus of the stria terminalis, midbrain and parabrachial complex [3,8,29], it cannot be assumed that VTT inputs to the Ce are directly related to medullary projection neurons. The present study was undertaken to determine whether VTT projections to medial Ce directly contact neurons projecting to the dorsal medulla. Fourteen adult, male rats (Harlan Sprague-Dawley), weighing 200-250 g, were utilized in this study. Under Nembutal anesthesia (40 mg/kg), 6 animals received small, electrolytic lesions of the ventral taenia tecta. Lesions were made by passing a 1 mA anodal current for

101

8 s through a 000 gauge insect pin inserted stereotaxically into the brain. A further 6 animals received electrolytic lesions either in the infralimbic cortex (n = 3), anterior olfactory nucleus (n = 1) or the anterior hippocampal rudiment (dorsal taenia tecta) (n = 2). Three days following lesioning, all these animals received pressure injections of 50 nl of 0.1% HRP-WGA (Sigma) into the nucleus tractus solitarii (NTS). Injections were made under direct vision through an incision in the atlanto-occipital membrane. The HRP-WGA solution was delivered through a glass micropipette (tip diameter 50-100 mm) fitted to a Hamilton syringe. To control for the presence of degeneration due to injection site damage, two unlesioned animals received similar injections of HRP-WGA into the NTS. Following an additional two days, animals were deeply anesthetized and perfused with 100 ml of saline followed by 500 ml of an ice-cold fixative composed of 2% glutaraldehyde , 1% paraformaldehyde in 0.167 M phosphate buffer (pH 7.2). Brains were removed and post-fixed for 24 h in the fixative. Sections 50 mm thick were cut through the brain on a Vibratome. HRP activity was detected in sections through the amygdaloid complex and brainstem using the 3,3'-tetramethylbenzidine (TMB) procedure of Mesulam [16] followed by a 30 min incubation at 37°C in a solution containing 0.1% 3,3'-diaminobenzidine (DAB), 0.025% nickel ammonium sulphate and cobaltous chloride, and 0.003% hydrogen peroxide in phosphate buffer (pH 5.5). Sections were subsequently post-fixed in 1% OSO4, dehydrated and flat embedded in Spurr's medium. Pieces of tissue containing the Ce were removed from 3 thick sections (representing roughly the rostral, mid- and caudal levels of the Ce) from each animal and mounted for sectioning. Ultrathin sections were cut from the region of the Ce, mounted on 400 mesh grids, and examined using a Hitachi 7000 electron microscope at 10,000 and 25,000 x magnification. All degenerating terminals in one ultrathin section from each thick section from VTT animals were photographed at 10,000× (total micrographs = 100): degenerating terminals in contact with HRP labeled profiles were additionally photographed at 25,000x (n=68). An additional 100 micrographs (10,000 × ) were made of degenerating terminals from ultrathin sections of the Ce from the control-lesioned animals. The numbers of normal and degenerating terminals present in each micrograph were counted. Sections through the brain anterior to the anterior commissure were mounted on slides and stained with 0.5% Cresyl violet and mounted in Eukitt. Sections through the amygdala from the brains of two additional animals with VTT lesions, but without HRP-WGA injections, were also incubated in TMB and the DAB solution to control for endogenous peroxidase activity. These sec-

tions were also osmicated and flat embedded in Spurr's medium. In all 6 animals with combined VTT lesions and HRPWGA injections, the foci of the lesions were located between 2 and 3 mm in front of the anterior commissure (Fig. 1A). Necrosis and gliosis were present over a radius of about 1 mm from the lesion focus. Most of the VTT, and usually the adjacent dorsal peduncular cortex, were destroyed. In two cases, extensive damage to the medial part of the anterior olfactory nucleus was also present. Within the Ce, numbers of degenerating terminals were observed, usually characterized by the aggregation of vesicles, increases in electron density and the presence of electron-lucent glial processes surrounding the terminal. In the sample of 100 micrographs, approximately 1 in 11 (9%) terminals was degenerating. In sections from animals with lesions in the infralimbic cortex, anterior olfactory nucleus and in the anterior hippocampal rudiment, approximately 1 in 25 (4%) terminals was degenerating in a similar sample of 100 micrographs from the Ce. This percentage is similar to the number of degenerating terminals found in unlesioned animals (unpublished observations). Under light microscopic examination, HRP-WGA labeled neurons were identified throughout the medial Ce (Fig. 1B). These neurons were characterized by a dense, black reaction product filling the perikarya and proximal dendrites. Up to 300/~m of the dendritic tree was often filled with reaction product (Fig. 1B), this extent representing approximately 50% of the average total dendritic length of medial Ce neurons [15]. Ultrastructurally, HRP-WGA labeled perikarya and dendritic profiles were clearly identifiable by the presence of either TMB crystals (Fig. 1C), an electron dense reaction product associated with intracellular membranous and tubular structures (Fig. 1D), or large numbers of round, electron-dense inclusions. In sections from non-HRPinjected animals reacted with TMB and DAB, no crystals or reaction product were observed, though occasional, electron-dense inclusions were seen in perikarya. Numerous degenerating terminals (77 out of 186 degenerating terminals sampled) were observed in close contact with HRP-WGA labeled dendrites. In general, the typical, labeled postsynaptic profile was a dendrite between 1 and 2/tm in diameter, though approximately 25% of these labeled profiles were less than 1/tm in diameter. Though approximately 5% (9 out of 186 degenerating profiles sampled) were seen to contact perikarya, none were seen in contact with HRP-WGA-labeled perikarya. In several instances, 2 or 3 degenerating terminals were observed in the same section contacting the same dendrite (Fig. 1C). Synaptic specializations were clearly evident in 11 of the 77 terminals seen in contact

102

with HRP-WGA labeled dendrites. These appeared to be symmetrical in nature, having a cleft 25-30 nm wide

separating the terminal and dendrite and with narrow pre- and postsynaptic densities (Fig. 1D). The presynap-

B

q •

I

~

tl

ql

•

'41,

I tl

I

Fig. 1. A: location of typical electrolytic lesion (arrowheads) in the ventral taenia tecta. IL, infralimbic cortex; cc, corpus callosum. Bar = 1 mm. B: appearance of H R P - W G A labeled central nucleus (Ce) neurons in an osmicated 50 g m section, fiat embedded in Spurr's medium, st, stria terminalis. Bar = 100 gm. C: 3 degenerating terminals (d) in contact with an H R P - W G A labeled dendrite of a central nucleus neuron 5 days after VTT lesioning. The typical appearance of DAB-intensified T M B crystals can be clearly seen (arrowheads). Bar = 1/tm. D: degenerating terminal (d) forming a symmetrical synaptic contact (arrow) with an H R P - W G A labeled dendrite of a central nucleus neuron. Bar = 0.5/~m.

103 tic terminals appeared to contain exclusively spherical vesicles, 40-60 n m in diameter (Fig. 1C,D). In sections f r o m unlesioned animals with H R P - W G A injections into the N T S , the pattern and appearance o f reaction p r o d u c t resembled closely that seen in lesioned animals. Occasional labeled or degenerating terminals were observed in the Ce but n o t in contact with retrogradely labeled neurons. In fact, m o s t o f the terminals in these two cases were observed in regions where no labeled dendrites or perikarya were present, a finding consistent with the reported termination pattern o f N T S projections in the ventral Ce [27]. The present data provide strong evidence that projections f r o m the V T T directly innervate central a m y g d a loid neurons projecting to the dorsal medulla. The presence o f degenerating terminals in association with retrogradely labeled neurons does not seem attributable to d a m a g e due to the injection o f H R P - W G A into the N T S since the few degenerating terminals seen in the unlesioned cases were never seen in contact with labeled structures. Since the dorsal peduncular cortex was invariably involved in the V T T lesions, it is possible that some or all o f the degeneration observed in Ce is due to d a m a g e to this region, and not the VTT. However, in the cases with control lesions in the infralimbic cortex, where the dorsal peduncular cortex, but not the VTT, was also damaged, very little degeneration was observed in the Ce. Moreover, H R P injections into the infralimbic and dorsal peduncular cortices [4] p r o d u c e anterograde labeling surrounding, but not within, Ce. The significance o f the present findings lies in the evidence that olfactory information can be relayed via the a m y g d a l a directly to medullary a u t o n o m i c centers without an additional relay in the h y p o t h a l a m u s , as previously p r o p o s e d [e.g. 2,20,30]. The VTT, t h r o u g h their reciprocal connections with p r i m a r y olfactory cortex and the olfactory bulbs, are capable o f providing integrated olfactory input to central a u t o n o m i c centers via the Ce and its projections to the dorsal medulla. The presence o f this direct o l f a c t o r y - a u t o n o m i c route could thus provide the rat a means o f rapidly altering a u t o n o mic activity in response to olfactory stimuli. The finding that the majority o f terminals o f V T T origin contact large dendritic profiles is suggestive, t h o u g h n o t conclusive, that contact is preferentially m a d e with proximal dendrites. I f this is the case, the V T T input to Ce neurons is powerfully placed to influence their activity. This study was supported by N I H G r a n t NS 25139. The assistance o f the University o f I o w a Central E M Facility is acknowledged, as is the excellent p h o t o graphic assistance o f Paul R e i m a n n and C h a d K o p p e n haver.

1 Allen, W.F., Effect of various inhaled vapors on respiration and blood pressure in anesthetized, unanesthetized, sleeping and anosmic subjects, Am. J. Physiol., 88 (1929) 620-632. 2 Cain, D.P. and Bindra, D., Responses of amygdala single units to odors in the rat, Exp. Neurol., 35 (1972) 98-110. 3 Cassell, M.D., Gray, T.S. and Kiss, J.Z., Neuronal architecture in the rat central nucleus of the amygdala: a cytological, hodological and immunocytochemical study, J. Comp. Neurol., 246 (1986) 478499. 4 Cassell, M.D. and Wright, D.J., Topography of projections from the medial prefrontal cortex to the amygdala in the rat, Brain Res. Bull., 17 (1986) 321-333. 5 Ellendorf, F. and Parvizi, H., Neuroendocrinology of amygdaloid control of gonadotropin secretion. In Y. Ben Ari (Ed.), The Amygdaloid Complex, Elsevier, Amsterdam, 1981, pp. 239-250. 6 Feldman, S. and Conforti, N., Amygdalectomy inhibits adrenocorticotrophin responses to somatosensory and olfactory stimuli, Neuroendocrinology, 5 (1981) 1323-1329. 7 Gray, T.S., Autonomic neuropeptide connections ofthe amygdala. In Y. TachS, J.E. Morley and M. Brown (Eds), Neuropeptides and Stress. Symposia Hans Selge, Springer, New York, 1989, pp. 92106. 8 Gray, T.S., Carney, M.E. and Magnuson, D.J., Direct projections from the central amygdaloid nucleus to the hypothalamic paraventricular nucleus: possible role in stress-induced adrenocorticotropin release, Neuroendocrinology, 50 (1989) 433-446. 9 Haberly, L.B. and Price, J.L., Association and commissural fiber systems of the olfactory cortex of the rat. I. Systems originating in the piriform cortex and adjacent areas, J. Comp. Neurol., 178 (1978) 711-741. 10 Haberly, L.B. and Price, J.L., Association and commissural fiber systems of the olfactory peduncle. II. Systems originating in the olfactory peduncle, J. Comp. Neurol., 181 (1978) 781-808. 11 Hopkins, D.A., Amygdalotegmental projections in the rat, cat and monkey, Neurosci. Lett., 1 (1975) 263-270. 12 Kaada, B.R., Somatomotor, autonomic and electrocorticographic responses to electrical stimulation of 'rhinencephalic' and other structures in primates, cat and dog, Acta Physiol. Scand. (Suppl. 83), 24 (1951) 1-285. 13 Kita, M. and Oomura, Y., An HRP study of the afferent connections to the rat medial hypothalamic region, Brain Res. Bull., 8 (1982) 53-62. 14 Krettek, J.E. and Price, J.L., Amygdaloid projections to subeortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol., 178 (1978) 225-254. 15 McDonald, A.J., Cytoarchitecture of the central amygdaloid nucleus of the rat, J. Comp. Neurol., 208 (1982) 401-418. 16 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase histochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) 106-I 17. 17 Moga, M. and Gray, T.S., Evidence for corticotrophin-releasing factor, neurotensin and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus, J. Comp. Neurol., 241 (1985) 275-284. 18 Ottersen, O.P., Connections of the amygdala of the rat. IV: Corticoamygdaloid and intraamygdaloid connections as studied with axonal transport of horseradish peroxidase, J. Comp. Neurol., 205 (1982) 30-48. 19 Pavlov, I.P., Conditioned Reflexes. An Investigation of the Physiological Activity of the Cerebral Cortex, Oxford University Press, London, 1927, 427 pp.

104 20 Powell, T.P.S., Cowan, W.M. and Raisman, G., The central olfactory connections, J. Anat., 99 (1965) 791-813. 21 Price, J.L., Slotnick, B.M. and Revial, M.-F., Olfactory projections to the hypothalamus, J. Comp. Neurol., 306 (I 991 ) 447~,61. 22 Saper, C.B., Swanson, L.W. and Cowan, W.M., The efferent connections of the ventromedial nucleus of the hypothalamus of the rat, J. Comp. Neurol., 169 (1976) 409~42. 23 Saper, C.B., Loewy,, A.D., Swanson, L.W. and Cowan, W.M., Direct hypothalamo-autonomic connections, Brain Res., 117 (1976) 305-12. 24 Saper, C.B., Swanson, L.W. and Cowan, W.M., An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat, J. Comp. Neurol., 183 (1979) 689-706. 25 Scalia, F. and Winans, S.S., The differential projections of the olfactory and accessory olfactory bulb in mammals, J. Comp. Neurol., 161 (1975)31-56. 26 Schwaber, J.S., Kapp, B.S. and Higgins, G.A., The origin and extent of direct amygdala projections to the region of the dorsal

27

28

29

30

motor nucleus of the vagus and nucleus of the solitary tract, Neurosci. Lett., 20 (1980) 15-20. Ter Horst, G.J., DeBoer, P., Luiten, P.G.M. and Van Willigen, J.D., Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus vulgaris lectin tracing study in the rat, Neuroscience, 31 (1989) 785 797. Thompson, R.L. and Cassell, M.D., Differential distribution and non-collateralization of central amygdaloid neurons projecting to different medullary regions, Neurosci. Lett., 97 (1989) 235-251. Veening, J.G., Swanson, L.W. and Sawchenko, P.E., The organization of projections from the central nucleus of amygdala to brainstem sites involved in central autonomic regulation: a combined retrograde transport-immunohistochemical study, Brain Res., 303 (1984) 337 357. Werka, T., Skar, J. and Ursin, H., Exploration and avoidance in rats with lesions in amygdala and piriform cortex, J. Comp. Physiol. Psychol., 92 (1978) 672~i81.