- Email: [email protected]
The amygdalo-brainstem pathway: Selective innervation of dopaminergic, noradrenergic and adrenergic cells in the rat
.,Veuro,science Letters'. 97 (1989) 252 25.~ Elsevier Scientific Publishers Ireland Ltd
252
NSL 05893
The amygdalo-brainstem pathway: selective innervation of dopaminergic, noradrenergic and adrenergic cells in the rat Donna M. Wallace, Debra J. Magnuson and Thackery S. Gray Department o['Anatomy, Loyola Stritch School o['Medicine, Maywood, 1L 60153 ( U.S.A. ) (Received 6 September 1988; Revised version received 15 October 1988; Accepted 17 October 1988)
Key words.
Tyrosine hydroxylase; phenylethanolamine N-methyltransferase; Dopamine/¢-hydroxylase; Phaseotus vulgaris leucoagglutinin lectin; Immunocytochemistry
The present study investigated the organization and distribution of amygdaloid axons within the various brainstem dopaminergic, noradrenergic and adrenergic cell groups. This was accomplished via Phaseolus vulgaris leucoagglutinin lectin (PHA-L) anterograde tracing technique combined with glucose-oxidase immunocytochemistry to catecholamine markers (i.e. tyrosine hydroxylase, dopamine//-hydroxylase, and phenylethanolamine N-methyltransferase). Injections of PHA-L within the medial part of the central amygdaloid nucleus resulted in axonal labeling within most catecholamine containing cell groups within the brainstem. The most heavily innervated catecholaminergic groups were the A9 (lateral) cells of the substantia nigra, the A8 dopaminergic cells of the retrorubrat field and the C2 adrenergic cells of nucleus of the solitary tract. Amygdaloid terminals frequently contacted cells within these regions. A moderate amount of amygdaloid terminals were located within the rostral A6 (locus coeruleus) and A2 (nucleus of the solitary tract) groups. Amygdaloid terminal contacts were apparent on the majority of the rostral A6 and A2 neurons. Light or no amygdaloid terminal labeling was observed within the other brainstem catecholaminergic cell groups. Thus, the amygdala mainly innervates the A8 and lateral A9 dopaminergic cells of midbrain, rostral locus coeruleus (A6) noradrenergic neurons and the adrenergic (C2) and noradrenergic (A2) cells within the nucleus of the solitary tract. Selective innervation of these brainstem catecholaminergic systems may be important for integration of amygdaloid-mediated defensive and stressinduced behaviors.
The amygdala is involved in the integration of somatomotor and autonomic responses associated with defensive and 'stress-induced' behaviors. Electrical or chemical stimulation of the central nucleus of the amygdala (Ce) produces behavioral arousal, increases in heart rate, blood pressure and respiratory rate, and an elevation in plasma adrenaline and noradrenaline levels [2, 6, 8, 12, 24]. Stimulation of the Ce also enhances acoustic startle responses [22]. Activation of the Ce results in increases in heart rate and blood pressure strikingly similar to responses occurring during conCorrespondence: T.S. Gray, Department of Anatomy, Loyola Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153, U.S.A. 0304-3940/89/$ 03.50 ,~'3 1989 Elsevier Scientific Publishers Ireland Ltd.
253 ditioned cardiovascular responses [12]. Amygdaloid lesions attenuate learned cardiovascular and intestinal responses to aversive stimuli (cf. refs. 12, 27) and inhibit the development of stress-induced gastric ulcers [9]. Central and peripheral release of catecholamines occurs concomitantly in response to a variety of stressors (for review see ref. 25). For example, acute or repeated stress increases phenylethanolamine N-methyltransferase (PNMT) activity within the brainstem, but not within the hypothalamus [23]. The amygdala could affect the various components of stress or defense responses through its efferent projections to the brainstem which contains numerous catecholaminergic cell groups [10, 11, 15, 26]. Among these regions are the substantia nigra/ventral tegmental region, parabrachial area, dorsal vagal complex and ventrolateral medulla. These regions have also been implicated in autonomic and/or somatomotor responses that are also elicitable from amygdaloid stimulation [16]. The purpose of the present study was to determine which brainstem catecholaminergic cells are innervated by amygdala. Eight male Long-Evans male rats were anesthetized with sodium pentobarbital (45 mg/kg) and placed in a stereotaxic apparatus. Injections of Phaseolus vulgaris leucoagglutinin lectin (PHA-L; Vector Labs) were delivered iontophoretically via glass micropipette tips 10-15 pM in diameter. Coordinates were derived from the Paxinos and Watson rat brain atlas [19]. The primary amygdaloid target was the Ce, because it is almost the sole source of amygdaloid brainstem efferents [11, 25]. A 5.0/~A cathodal current was delivered in 7 s pulses every 14 s over a 30 min period using a Midgard Electronics constant current device. Postinjection survival periods were 10 14 days. Animals were then administered a lethal dose of sodium pentobarbital (0.5 cc). Their brains were fixed using the dual pH method [1] and sections were immunocytochemically processed according to the method of Gerfen and Sawchenko [7]. Rabbit antibodies to PHA-L (Dako) were used at a 1:1000 dilution. Biotinylated donkey antirabbit antibodies (Jackson lmmunoResearch) were diluted 1:5000. Avidin bound horseradish peroxidase (Bethesda Research Labs) was used at a 1:300 dilution. The tissue was then washed in phosphate-buffered saline for 4 days. Alternate adjacent sections were processed for tyrosine hydroxylase (TH), dopamine fl-hydroxylase (DBH) and P N M T immunoreactivity according to the methodology of Piekut [20]. Sheep anti-TH (donated by John Haycock), rabbit anti-DBH (Incstar) and rabbit anti-PNMT (Incstar) were used at a 1:1000 dilution. Biotinylated donkey anti-sheep and anti-rabbit antibodies were diluted as indicated above and avidin bound glucose oxidase reagents (Jackson ImmunoResearch) were used at 1:330. Sections were washed in phosphate-buffered saline, mounted on glass slides, dried and cover-slipped with Permount (Fischer Scientific) mounting media. Of the 8 animals injected with PHA-L into the amygdala, 4 had injections restricted to the medial central nucleus, two had injections centered within the lateral central nucleus and two had injections localized within the dorsal tip of the medial amygdaloid nucleus. Maximal brainstem labeling was observed in animals that had injections of PHA-L centered within the medial central nucleus (Fig. 1). The maximum diameter of this injection site was approximately 0.5 mm. Considerably less la-
254
Fig. 1. Light field low (a) and high (b) power photomicrographs ofa PHA-L injection site within the medial part of the central amygdaloid nucleus (CeM). CeL, lateral part of central amygdaloid nucleus: ot, optic tract; asterisk, stria terminalis. Bar in a = 250/~M, in b = 50/tin. Fig. 2. Light field low (a) power photomicrograph of the A8 dopaminergic cell group (indicated by open arrow), within the brainstem of a rat. The solid black arrow in (a) indicates the position of two cells illustrated in the high power light field photomicrograph of (b). Arrows in (b) indicate examples of amygdaloid terminal boutons that appear to be contacting the two tyrosine hydroxylase-immunoreactive cells. Aq, cerebral aqueduct; xscp, decussation of the superior cerebellar peduncle. Bar = 250/~M in (al, bar = 10/tM in (b).
beling was o b s e r v e d in a n i m a l s with injections centered within the lateral central nucleus. N o a n t e r o g r a d e labeling within the b r a i n s t e m c a t e c h o l a m i n e regions was o b s e r v e d in a n i m a l s with injections o f P H A - L centered within the d o r s a l tip o f the m e d i a l a m y g d a l o i d nucleus. A p r e v i o u s study in o u r l a b o r a t o r y has d e m o n s t r a t e d that injections o f P H A - L d o r s a l , ventral, lateral, m e d i a l o r c a u d a l to the central nucleus resulted in m i n i m a l o r no P H A - L i m m u n o r e a c t i v e a x o n a l labeling within the b r a i n s t e m [3]. P H A - L i m m u n o r e a c t i v e cell b o d i e s at the P H A - L injection sites c o u l d be recognized by the b r o w n r e a c t i o n p r o d u c t d i s t r i b u t e d within the cell bodies, d e n d r i t e s a n d a x o n s within the a m y g d a l a (Fig. 1). The P H A - L i m m u n o r e a c t i v e a x o n s c o u l d be recognized clearly as b r o w n labeled p u n c t u a t e a n d varicose processes. I m m u n o r e a c t i vity to P N M T , T H a n d D B H c o u l d be readily identified by blue r e a c t i o n p r o d u c t that a p p e a r e d to fill the c y t o p l a s m o f cell b o d i e s a n d d e n d r i t e s within restricted re-
255
gions of the brainstem. The distribution of PNMT, TH and DBH immunoreactive cells within the brainstem was identical to that described by H6kfelt et al. [10~ 15] and for purposes of description we have adopted their terminology. Labeled amygdaloid fiber density and the relative frequency of contacts between amygdaloid axon terminals and catecholamine cell bodies varied among the brainstem catecholamine cell groups. Dense projections of labeled amygdaloid fibers and presumed terminals were present within the A8 dopaminergic cells of the retrorubral field (Fig. 2) and the lateral portion of the A9 dopaminergic cell groups. Relatively dense amygdaloid axonal labeling was also observed within the C2 adrenergic cell group (Fig. 3) of the nucleus of the solitary tract NTS). Numerous close appositions
b
" m
4¥
I.C
Fig. 3. Light field low power photomicrograph (a) of the C2 adrenergic cell group within the nucleus of the solitary tract of a rat. The solid black arrows in (a) indicate the position of two cells with the medial part of the nucleus of the solitary tract (SolM) that are illustrated in the high power light field photomicrograph of(b). Arrows in (b) indicate examples of amygdaloid terminal boutons that appear to be contacting the two PNMT-immunoreactive cells. SOIL, lateral part of the nucleus of the solitary tract; 4v, fourth ventricle: asterisk, solitary tract. B a r - 100 l~M in (a), b a r - 25 uM in (b). Fig. 4. Light field low (a) power photomicrograph of the A6 noradrenergic cell group (indicated by open arrow) within the rostral locus coeruleus of a rat. The solid black arrow in (a) indicates the position of two cells that are illustrated in the high-power light-field photomicrograph of (b). Arrows in (b) indicate examples of amygdaloid terminal boutons that appear to be contacting the two dopamine ~-hydroxylase immunoreactive cells. LC, locus coeruleus; 4v, fourth ventricle: LPB, lateral parabrachial nucleus: scp, superior cerebellar peduncle; me5, mesencephalic tract of the trigeminal nucleus. Bar= 100 itM in (a), b a r = 2 5 / t M in (b).
256
or contacts between amygdaloid axon terminals and T H immunoreactive (AS, A9) and P N M T immunoreactive (C2) cell bodies and dendrites were observed within these regions. Moderate to light densities of amygdaloid fibers were present within AI0, the medial and ventral areas of A9, A6, A5 and A2. The majority of the DBHimmunoreactive cells of A2 (nucleus of the solitary tract) and the rostral portion of A6 (central gray-rostral locus coeruleus) were innervated by amygdaloid terminals (Fig. 4). Fewer contacts were observed between amygdaloid axon terminals and A10 TH-immunoreactive, the A6 (main part of locus coeruleus) and A5 DBH-immunoreactive cells. Labeled amygdaloid axons were not observed contacting the TH immunoreactive cells of the ventral area of A9. Sparse amygdaloid fiber labeling was present within the lateral dorsal area of A9, A7, the ventral region of A6 (subcoeruleus), C3 and CI. Amygdaloid axon contacts were observed on CI adrenergic cell bodies, but contacts upon PNMT-immunoreactive dendrites within the ventrolateral reticular formation were more commonly observed. Occasional contacts between an amygdaloid axon varicosity and the cells of the A7 and ventral A6 and C1 regions were also observed. The TH-immunoreactive cells of the lateral dorsal region of A9 and the PNMT-immunoreactive neurons of C3 were not innervated by amygdaloid terminals. Amygdaloid axonal labeling was rarely observed in the A4, the dorsal region of A2 (area postrema), and A 1 noradrenergic cell groups. The results demonstrate that the Ce projects heavily to the lateral portion of A9 (pars lateralis) and A8 dopaminergic cell groups. The lateral portion of A9 and the A8 cells contribute dopaminergic fibers to the mesostriatal pathway and the mesolimbocortical pathway [15, 17]. Both the mesostriatal and mesolimbocortical systems have postulated roles during stress. The mesolimbocortical pathway, which projects back to the amygdala, is involved in the maintenance of gastric mucosal integrity in the presence of stressors [17, 21]. This protective effect may be related to amygdaloid dopamine neuronal processing of reward-related behaviors [18]. Dopamine levels are increased in the prefrontal cortex in response to various types of stressors presumably through activation of the mesocorticolimbic dopaminergic system [4]. The mesostriatal pathway is involved in the central control of thermoregulation by its action upon heat dissipation mechanisms [14] which could be related to temperature changes observed during stress. Amygdaloid terminals frequently contacted cells of the adrenergic C2 and noradrenergic A2 cell groups of the nucleus of the solitary tract. Stimulation of the Ce attenuates the baroreceptor reflex [24] and injections of adrenaline or noradrenaline within the NTS decrease the activity of neurons that participate in the baroreceptor reflex [5]. Thus, it is possible that Ce neurons could excite adrenergic and/or noradrenergic neurons within the NTS causing the release of catecholamines which have an inhibitory action on the baroreceptive modulating neurons. This postulated amygdala-mediated baroreceptor reflex inhibition could explain the cardiovascular effects observed when the amygdala is activated. In summary, the results of this study demonstrate that the Ce sends efferents to selected subgroups of dopaminergic, noradrenergic and adrenergic neurons in the brainstem. Selective innervation of these catecholaminergic cell groups may be a sub-
257
strate for integration of individual or multiple components of amygdaloid-mediated defensive behaviors. This study was supported by N | H Grant NS 20041, ONR Grant N00014-88-k0010 and a grant from the Potts Fotmdation. The authors thank Diane Piekut for technical advice.
I Berod, A., Hartman, B.K. and Pujol, J.F., hnportance of fixation in imnmnohistochemistry: use of 11)rmaldehyde solutions at variable pH for the localization of tyrosine hydroxylase, J, Histochem. Cytochem., 29 (1981) 844 850. 2 Brown, M.R. and Gray, T.S., Peptide injections into the amygdaIa of conscious rats: effects on blood pressure, heart rate and plasma catecholamines, Regul. Pept., 21 (1988) 95 106. 3 Danielsen, E.H., Magnuson, D.J. and Gray, T.S., Amygdala projections to the dorsal vagal complex in rat: a re-examination using the PHA-L anterograde tracing method, Anat. Rec., 220 (1988) 28A. 4 Dunn, A.J. and Kramarcy, M.R., Neurochemical responses during stress: relationships between the hypothalamic-pituitary-adrenal and catecholamine systems. In L.l. lversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 18, 1984, pp. 455 515. 5 Felman, P.D. and Moises, H.C., Adrenergic responses of baroreceptor cells in the nucleus tractus solitarii of the rat: a microiontophoretic study, Brain Res., 420 (1987) 351 361. 6 Galeno, T.M. and Brody, M., Hemodynamic responses to amygdaloid stimulation in spontaneously hypertensive rats, Am. J. Physiol., 245 (1983) 281 286. 7 Gerfen, C.R. and Sawchenko, P., An anterograde neuroanatomical tracing mcthod that shows Ihc detailed morphology of neurons, their axons and terminals: lmmunohistochemical localization of an axonally transported plant lectin, Phaseolus Vulgaris leucoagglutinin (PtIA-L), Brain Res.. 290 (1984) 219 238. 8 Harper, R.M., Frysinger, R.C., Trelease, R.B. and Marks, J.D., State dependent alteration of respiratory cycle timing by stimulation of the central nucleus of amygdala, Brain Res., 306 (1984) I 8. 9 Henke, P.G.. The telencephalic limbic system and experimental gastric pathology: a review, Neurosci. Biobefiav. Rev., 6 (1982) 381 390. 10 H6kfelt, T., Martensson, R., Bj6rklund, A., Kleinau, S, and Goldstein, M., Distributional maps oF tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. Bj6rklund and T. t I6kfclt (Eds,), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part 1. Elsevier. New York, 1984, pp. 277 379. 11 Hopkins, D.A., Amygdalotegmental projections in the rat, cat and rhesus monkey, Neurosci. Lett.. 1 (1975) 263 270. 12 lwata, J., Chida, K. and LeDoux, J.E., Cardiovascular responses elicited by stimulation of neurons in the central nucleus of amygdala in awake but not anesthetized rats resemble conditioned emotional responses, Brain Res., 418 (1987) 183 188. 13 Kapp, B.S., Pascoe, J.P. and Bixler, M.A., The amygdala: a neuroanatomical systems approach to its contribution to aversive conditioning. In N. Butters and L. Squire (Eds.). The Neurophysiology of Memory, Guilford, New York, 1984, pp. 473 488. 14 Lee, T.F., Mora, F. and Myers, R.D., Dopamine and thermoregulation: an evaluation with special reference to dopaminergic pathways, Neurosci. Biobehav. Rev., 9 (1985) 589 598. 15 Lindvall, O. and Bj6rklund, A., Dopamine and norepinephrine-containing neuron systems: their anatomy in the rat brain. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983. 16 Locwy, A.D., Anatomic aspects of central nervous cardiovascular regulation. In T. Stober, K. Schimirtk. D. Ganten and D.G. Sherman (Eds. I, Central Nervous System Control of the Heart, Nijhoff, The Netherlands, 1986, 1 18. 17 Loughlm. S.E. and Fallon, J.H., Dopaminergic projections to amygdala from substantia nigra and ventral tegmental area, Brain Res., 262 (1983) 334 338.
258 18 Nakano, Y., Lenard, L., Oomura, Y., Nishino, H., Aou, S. and Yamamoto, T., Functional involvemerit of catecholamines in reward-related neuronal activity of the monkey amygdala, J. Neurophysiol.. 57 (1987) 72 91. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1986. 20 Piekut, D,T., Interactions of immunostained ACTH ~ 39fibers and CRF neurons in the paraventricular nucleus of rat hypothalamus: application of avidin-glucose oxidase to dual immunostaining procedures, J. Histochem. Cytochem., 35 (1987) 261 265. 21 Ray, A., Henke, P.G. and Sullivan, R.M., Central dopamine systems and gastric stress pathology m rats, Physiol. Behav., 42 (1988) 359 364. 22 Rosen, J.B. and Davis, M., Enhancement of acoustic startle by electrical stimulation of the amygdala, Beh. Neurosci., 102 (1988) 195 202. 23 Saavedra, J.M., Fernandez-Pardal, J., Torda, T., Reis, D. and Ross, C., Dissociation between rat hypothalamic and brainstem PNMT after stress and between hypothalamic catecholamines and PNMT after midbrain hemitransections. In E. Usdin, E. Kvetnansky and J. Axelrod (Eds.), Stress, the Role of Catecholamines and Other Neurotransmitters, Vol. 1, 1984, pp. 137 146. 24 Stock, G., Rupprecht, U., Stumpf, H. and Schl6r, K.H., Cardiovascular changes during arousal elicited by stimulation of amygdala, hypothalamus and locus coeruleus, J. Auton. Nerv. Syst., 3 (1981) 503 510. 25 Tilders, F.J.H. and Berkenbosch, F., CRF and catecholamines; their place in central and peripheral regulation of the stress response, Acta Endocrinol., Suppl. 276 (1986) 63 75. 26 Van der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R. and Bloom, F.E., The organization of projections from the cortex, amygdala and hypothalamus to the nucleus of the solitary tract in rat, J. Comp. Neurol., 224(1984) 1 24. 27 Vanderwolf, C.H., Kelly, M.E., Kraemer, P. and Streather, A., Are emotion and motivation localized in the limbic system and nucleus accumbens'?, Beh. Brain Res., 27 (1988) 45 58.
252
NSL 05893
The amygdalo-brainstem pathway: selective innervation of dopaminergic, noradrenergic and adrenergic cells in the rat Donna M. Wallace, Debra J. Magnuson and Thackery S. Gray Department o['Anatomy, Loyola Stritch School o['Medicine, Maywood, 1L 60153 ( U.S.A. ) (Received 6 September 1988; Revised version received 15 October 1988; Accepted 17 October 1988)
Key words.
Tyrosine hydroxylase; phenylethanolamine N-methyltransferase; Dopamine/¢-hydroxylase; Phaseotus vulgaris leucoagglutinin lectin; Immunocytochemistry
The present study investigated the organization and distribution of amygdaloid axons within the various brainstem dopaminergic, noradrenergic and adrenergic cell groups. This was accomplished via Phaseolus vulgaris leucoagglutinin lectin (PHA-L) anterograde tracing technique combined with glucose-oxidase immunocytochemistry to catecholamine markers (i.e. tyrosine hydroxylase, dopamine//-hydroxylase, and phenylethanolamine N-methyltransferase). Injections of PHA-L within the medial part of the central amygdaloid nucleus resulted in axonal labeling within most catecholamine containing cell groups within the brainstem. The most heavily innervated catecholaminergic groups were the A9 (lateral) cells of the substantia nigra, the A8 dopaminergic cells of the retrorubrat field and the C2 adrenergic cells of nucleus of the solitary tract. Amygdaloid terminals frequently contacted cells within these regions. A moderate amount of amygdaloid terminals were located within the rostral A6 (locus coeruleus) and A2 (nucleus of the solitary tract) groups. Amygdaloid terminal contacts were apparent on the majority of the rostral A6 and A2 neurons. Light or no amygdaloid terminal labeling was observed within the other brainstem catecholaminergic cell groups. Thus, the amygdala mainly innervates the A8 and lateral A9 dopaminergic cells of midbrain, rostral locus coeruleus (A6) noradrenergic neurons and the adrenergic (C2) and noradrenergic (A2) cells within the nucleus of the solitary tract. Selective innervation of these brainstem catecholaminergic systems may be important for integration of amygdaloid-mediated defensive and stressinduced behaviors.
The amygdala is involved in the integration of somatomotor and autonomic responses associated with defensive and 'stress-induced' behaviors. Electrical or chemical stimulation of the central nucleus of the amygdala (Ce) produces behavioral arousal, increases in heart rate, blood pressure and respiratory rate, and an elevation in plasma adrenaline and noradrenaline levels [2, 6, 8, 12, 24]. Stimulation of the Ce also enhances acoustic startle responses [22]. Activation of the Ce results in increases in heart rate and blood pressure strikingly similar to responses occurring during conCorrespondence: T.S. Gray, Department of Anatomy, Loyola Stritch School of Medicine, 2160 S. First Ave., Maywood, IL 60153, U.S.A. 0304-3940/89/$ 03.50 ,~'3 1989 Elsevier Scientific Publishers Ireland Ltd.
253 ditioned cardiovascular responses [12]. Amygdaloid lesions attenuate learned cardiovascular and intestinal responses to aversive stimuli (cf. refs. 12, 27) and inhibit the development of stress-induced gastric ulcers [9]. Central and peripheral release of catecholamines occurs concomitantly in response to a variety of stressors (for review see ref. 25). For example, acute or repeated stress increases phenylethanolamine N-methyltransferase (PNMT) activity within the brainstem, but not within the hypothalamus [23]. The amygdala could affect the various components of stress or defense responses through its efferent projections to the brainstem which contains numerous catecholaminergic cell groups [10, 11, 15, 26]. Among these regions are the substantia nigra/ventral tegmental region, parabrachial area, dorsal vagal complex and ventrolateral medulla. These regions have also been implicated in autonomic and/or somatomotor responses that are also elicitable from amygdaloid stimulation [16]. The purpose of the present study was to determine which brainstem catecholaminergic cells are innervated by amygdala. Eight male Long-Evans male rats were anesthetized with sodium pentobarbital (45 mg/kg) and placed in a stereotaxic apparatus. Injections of Phaseolus vulgaris leucoagglutinin lectin (PHA-L; Vector Labs) were delivered iontophoretically via glass micropipette tips 10-15 pM in diameter. Coordinates were derived from the Paxinos and Watson rat brain atlas [19]. The primary amygdaloid target was the Ce, because it is almost the sole source of amygdaloid brainstem efferents [11, 25]. A 5.0/~A cathodal current was delivered in 7 s pulses every 14 s over a 30 min period using a Midgard Electronics constant current device. Postinjection survival periods were 10 14 days. Animals were then administered a lethal dose of sodium pentobarbital (0.5 cc). Their brains were fixed using the dual pH method [1] and sections were immunocytochemically processed according to the method of Gerfen and Sawchenko [7]. Rabbit antibodies to PHA-L (Dako) were used at a 1:1000 dilution. Biotinylated donkey antirabbit antibodies (Jackson lmmunoResearch) were diluted 1:5000. Avidin bound horseradish peroxidase (Bethesda Research Labs) was used at a 1:300 dilution. The tissue was then washed in phosphate-buffered saline for 4 days. Alternate adjacent sections were processed for tyrosine hydroxylase (TH), dopamine fl-hydroxylase (DBH) and P N M T immunoreactivity according to the methodology of Piekut [20]. Sheep anti-TH (donated by John Haycock), rabbit anti-DBH (Incstar) and rabbit anti-PNMT (Incstar) were used at a 1:1000 dilution. Biotinylated donkey anti-sheep and anti-rabbit antibodies were diluted as indicated above and avidin bound glucose oxidase reagents (Jackson ImmunoResearch) were used at 1:330. Sections were washed in phosphate-buffered saline, mounted on glass slides, dried and cover-slipped with Permount (Fischer Scientific) mounting media. Of the 8 animals injected with PHA-L into the amygdala, 4 had injections restricted to the medial central nucleus, two had injections centered within the lateral central nucleus and two had injections localized within the dorsal tip of the medial amygdaloid nucleus. Maximal brainstem labeling was observed in animals that had injections of PHA-L centered within the medial central nucleus (Fig. 1). The maximum diameter of this injection site was approximately 0.5 mm. Considerably less la-
254
Fig. 1. Light field low (a) and high (b) power photomicrographs ofa PHA-L injection site within the medial part of the central amygdaloid nucleus (CeM). CeL, lateral part of central amygdaloid nucleus: ot, optic tract; asterisk, stria terminalis. Bar in a = 250/~M, in b = 50/tin. Fig. 2. Light field low (a) power photomicrograph of the A8 dopaminergic cell group (indicated by open arrow), within the brainstem of a rat. The solid black arrow in (a) indicates the position of two cells illustrated in the high power light field photomicrograph of (b). Arrows in (b) indicate examples of amygdaloid terminal boutons that appear to be contacting the two tyrosine hydroxylase-immunoreactive cells. Aq, cerebral aqueduct; xscp, decussation of the superior cerebellar peduncle. Bar = 250/~M in (al, bar = 10/tM in (b).
beling was o b s e r v e d in a n i m a l s with injections centered within the lateral central nucleus. N o a n t e r o g r a d e labeling within the b r a i n s t e m c a t e c h o l a m i n e regions was o b s e r v e d in a n i m a l s with injections o f P H A - L centered within the d o r s a l tip o f the m e d i a l a m y g d a l o i d nucleus. A p r e v i o u s study in o u r l a b o r a t o r y has d e m o n s t r a t e d that injections o f P H A - L d o r s a l , ventral, lateral, m e d i a l o r c a u d a l to the central nucleus resulted in m i n i m a l o r no P H A - L i m m u n o r e a c t i v e a x o n a l labeling within the b r a i n s t e m [3]. P H A - L i m m u n o r e a c t i v e cell b o d i e s at the P H A - L injection sites c o u l d be recognized by the b r o w n r e a c t i o n p r o d u c t d i s t r i b u t e d within the cell bodies, d e n d r i t e s a n d a x o n s within the a m y g d a l a (Fig. 1). The P H A - L i m m u n o r e a c t i v e a x o n s c o u l d be recognized clearly as b r o w n labeled p u n c t u a t e a n d varicose processes. I m m u n o r e a c t i vity to P N M T , T H a n d D B H c o u l d be readily identified by blue r e a c t i o n p r o d u c t that a p p e a r e d to fill the c y t o p l a s m o f cell b o d i e s a n d d e n d r i t e s within restricted re-
255
gions of the brainstem. The distribution of PNMT, TH and DBH immunoreactive cells within the brainstem was identical to that described by H6kfelt et al. [10~ 15] and for purposes of description we have adopted their terminology. Labeled amygdaloid fiber density and the relative frequency of contacts between amygdaloid axon terminals and catecholamine cell bodies varied among the brainstem catecholamine cell groups. Dense projections of labeled amygdaloid fibers and presumed terminals were present within the A8 dopaminergic cells of the retrorubral field (Fig. 2) and the lateral portion of the A9 dopaminergic cell groups. Relatively dense amygdaloid axonal labeling was also observed within the C2 adrenergic cell group (Fig. 3) of the nucleus of the solitary tract NTS). Numerous close appositions
b
" m
4¥
I.C
Fig. 3. Light field low power photomicrograph (a) of the C2 adrenergic cell group within the nucleus of the solitary tract of a rat. The solid black arrows in (a) indicate the position of two cells with the medial part of the nucleus of the solitary tract (SolM) that are illustrated in the high power light field photomicrograph of(b). Arrows in (b) indicate examples of amygdaloid terminal boutons that appear to be contacting the two PNMT-immunoreactive cells. SOIL, lateral part of the nucleus of the solitary tract; 4v, fourth ventricle: asterisk, solitary tract. B a r - 100 l~M in (a), b a r - 25 uM in (b). Fig. 4. Light field low (a) power photomicrograph of the A6 noradrenergic cell group (indicated by open arrow) within the rostral locus coeruleus of a rat. The solid black arrow in (a) indicates the position of two cells that are illustrated in the high-power light-field photomicrograph of (b). Arrows in (b) indicate examples of amygdaloid terminal boutons that appear to be contacting the two dopamine ~-hydroxylase immunoreactive cells. LC, locus coeruleus; 4v, fourth ventricle: LPB, lateral parabrachial nucleus: scp, superior cerebellar peduncle; me5, mesencephalic tract of the trigeminal nucleus. Bar= 100 itM in (a), b a r = 2 5 / t M in (b).
256
or contacts between amygdaloid axon terminals and T H immunoreactive (AS, A9) and P N M T immunoreactive (C2) cell bodies and dendrites were observed within these regions. Moderate to light densities of amygdaloid fibers were present within AI0, the medial and ventral areas of A9, A6, A5 and A2. The majority of the DBHimmunoreactive cells of A2 (nucleus of the solitary tract) and the rostral portion of A6 (central gray-rostral locus coeruleus) were innervated by amygdaloid terminals (Fig. 4). Fewer contacts were observed between amygdaloid axon terminals and A10 TH-immunoreactive, the A6 (main part of locus coeruleus) and A5 DBH-immunoreactive cells. Labeled amygdaloid axons were not observed contacting the TH immunoreactive cells of the ventral area of A9. Sparse amygdaloid fiber labeling was present within the lateral dorsal area of A9, A7, the ventral region of A6 (subcoeruleus), C3 and CI. Amygdaloid axon contacts were observed on CI adrenergic cell bodies, but contacts upon PNMT-immunoreactive dendrites within the ventrolateral reticular formation were more commonly observed. Occasional contacts between an amygdaloid axon varicosity and the cells of the A7 and ventral A6 and C1 regions were also observed. The TH-immunoreactive cells of the lateral dorsal region of A9 and the PNMT-immunoreactive neurons of C3 were not innervated by amygdaloid terminals. Amygdaloid axonal labeling was rarely observed in the A4, the dorsal region of A2 (area postrema), and A 1 noradrenergic cell groups. The results demonstrate that the Ce projects heavily to the lateral portion of A9 (pars lateralis) and A8 dopaminergic cell groups. The lateral portion of A9 and the A8 cells contribute dopaminergic fibers to the mesostriatal pathway and the mesolimbocortical pathway [15, 17]. Both the mesostriatal and mesolimbocortical systems have postulated roles during stress. The mesolimbocortical pathway, which projects back to the amygdala, is involved in the maintenance of gastric mucosal integrity in the presence of stressors [17, 21]. This protective effect may be related to amygdaloid dopamine neuronal processing of reward-related behaviors [18]. Dopamine levels are increased in the prefrontal cortex in response to various types of stressors presumably through activation of the mesocorticolimbic dopaminergic system [4]. The mesostriatal pathway is involved in the central control of thermoregulation by its action upon heat dissipation mechanisms [14] which could be related to temperature changes observed during stress. Amygdaloid terminals frequently contacted cells of the adrenergic C2 and noradrenergic A2 cell groups of the nucleus of the solitary tract. Stimulation of the Ce attenuates the baroreceptor reflex [24] and injections of adrenaline or noradrenaline within the NTS decrease the activity of neurons that participate in the baroreceptor reflex [5]. Thus, it is possible that Ce neurons could excite adrenergic and/or noradrenergic neurons within the NTS causing the release of catecholamines which have an inhibitory action on the baroreceptive modulating neurons. This postulated amygdala-mediated baroreceptor reflex inhibition could explain the cardiovascular effects observed when the amygdala is activated. In summary, the results of this study demonstrate that the Ce sends efferents to selected subgroups of dopaminergic, noradrenergic and adrenergic neurons in the brainstem. Selective innervation of these catecholaminergic cell groups may be a sub-
257
strate for integration of individual or multiple components of amygdaloid-mediated defensive behaviors. This study was supported by N | H Grant NS 20041, ONR Grant N00014-88-k0010 and a grant from the Potts Fotmdation. The authors thank Diane Piekut for technical advice.
I Berod, A., Hartman, B.K. and Pujol, J.F., hnportance of fixation in imnmnohistochemistry: use of 11)rmaldehyde solutions at variable pH for the localization of tyrosine hydroxylase, J, Histochem. Cytochem., 29 (1981) 844 850. 2 Brown, M.R. and Gray, T.S., Peptide injections into the amygdaIa of conscious rats: effects on blood pressure, heart rate and plasma catecholamines, Regul. Pept., 21 (1988) 95 106. 3 Danielsen, E.H., Magnuson, D.J. and Gray, T.S., Amygdala projections to the dorsal vagal complex in rat: a re-examination using the PHA-L anterograde tracing method, Anat. Rec., 220 (1988) 28A. 4 Dunn, A.J. and Kramarcy, M.R., Neurochemical responses during stress: relationships between the hypothalamic-pituitary-adrenal and catecholamine systems. In L.l. lversen, S.D. Iversen and S.H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 18, 1984, pp. 455 515. 5 Felman, P.D. and Moises, H.C., Adrenergic responses of baroreceptor cells in the nucleus tractus solitarii of the rat: a microiontophoretic study, Brain Res., 420 (1987) 351 361. 6 Galeno, T.M. and Brody, M., Hemodynamic responses to amygdaloid stimulation in spontaneously hypertensive rats, Am. J. Physiol., 245 (1983) 281 286. 7 Gerfen, C.R. and Sawchenko, P., An anterograde neuroanatomical tracing mcthod that shows Ihc detailed morphology of neurons, their axons and terminals: lmmunohistochemical localization of an axonally transported plant lectin, Phaseolus Vulgaris leucoagglutinin (PtIA-L), Brain Res.. 290 (1984) 219 238. 8 Harper, R.M., Frysinger, R.C., Trelease, R.B. and Marks, J.D., State dependent alteration of respiratory cycle timing by stimulation of the central nucleus of amygdala, Brain Res., 306 (1984) I 8. 9 Henke, P.G.. The telencephalic limbic system and experimental gastric pathology: a review, Neurosci. Biobefiav. Rev., 6 (1982) 381 390. 10 H6kfelt, T., Martensson, R., Bj6rklund, A., Kleinau, S, and Goldstein, M., Distributional maps oF tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. Bj6rklund and T. t I6kfclt (Eds,), Handbook of Chemical Neuroanatomy, Vol. 2, Classical Transmitters in the CNS, Part 1. Elsevier. New York, 1984, pp. 277 379. 11 Hopkins, D.A., Amygdalotegmental projections in the rat, cat and rhesus monkey, Neurosci. Lett.. 1 (1975) 263 270. 12 lwata, J., Chida, K. and LeDoux, J.E., Cardiovascular responses elicited by stimulation of neurons in the central nucleus of amygdala in awake but not anesthetized rats resemble conditioned emotional responses, Brain Res., 418 (1987) 183 188. 13 Kapp, B.S., Pascoe, J.P. and Bixler, M.A., The amygdala: a neuroanatomical systems approach to its contribution to aversive conditioning. In N. Butters and L. Squire (Eds.). The Neurophysiology of Memory, Guilford, New York, 1984, pp. 473 488. 14 Lee, T.F., Mora, F. and Myers, R.D., Dopamine and thermoregulation: an evaluation with special reference to dopaminergic pathways, Neurosci. Biobehav. Rev., 9 (1985) 589 598. 15 Lindvall, O. and Bj6rklund, A., Dopamine and norepinephrine-containing neuron systems: their anatomy in the rat brain. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983. 16 Locwy, A.D., Anatomic aspects of central nervous cardiovascular regulation. In T. Stober, K. Schimirtk. D. Ganten and D.G. Sherman (Eds. I, Central Nervous System Control of the Heart, Nijhoff, The Netherlands, 1986, 1 18. 17 Loughlm. S.E. and Fallon, J.H., Dopaminergic projections to amygdala from substantia nigra and ventral tegmental area, Brain Res., 262 (1983) 334 338.
258 18 Nakano, Y., Lenard, L., Oomura, Y., Nishino, H., Aou, S. and Yamamoto, T., Functional involvemerit of catecholamines in reward-related neuronal activity of the monkey amygdala, J. Neurophysiol.. 57 (1987) 72 91. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, New York, 1986. 20 Piekut, D,T., Interactions of immunostained ACTH ~ 39fibers and CRF neurons in the paraventricular nucleus of rat hypothalamus: application of avidin-glucose oxidase to dual immunostaining procedures, J. Histochem. Cytochem., 35 (1987) 261 265. 21 Ray, A., Henke, P.G. and Sullivan, R.M., Central dopamine systems and gastric stress pathology m rats, Physiol. Behav., 42 (1988) 359 364. 22 Rosen, J.B. and Davis, M., Enhancement of acoustic startle by electrical stimulation of the amygdala, Beh. Neurosci., 102 (1988) 195 202. 23 Saavedra, J.M., Fernandez-Pardal, J., Torda, T., Reis, D. and Ross, C., Dissociation between rat hypothalamic and brainstem PNMT after stress and between hypothalamic catecholamines and PNMT after midbrain hemitransections. In E. Usdin, E. Kvetnansky and J. Axelrod (Eds.), Stress, the Role of Catecholamines and Other Neurotransmitters, Vol. 1, 1984, pp. 137 146. 24 Stock, G., Rupprecht, U., Stumpf, H. and Schl6r, K.H., Cardiovascular changes during arousal elicited by stimulation of amygdala, hypothalamus and locus coeruleus, J. Auton. Nerv. Syst., 3 (1981) 503 510. 25 Tilders, F.J.H. and Berkenbosch, F., CRF and catecholamines; their place in central and peripheral regulation of the stress response, Acta Endocrinol., Suppl. 276 (1986) 63 75. 26 Van der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R. and Bloom, F.E., The organization of projections from the cortex, amygdala and hypothalamus to the nucleus of the solitary tract in rat, J. Comp. Neurol., 224(1984) 1 24. 27 Vanderwolf, C.H., Kelly, M.E., Kraemer, P. and Streather, A., Are emotion and motivation localized in the limbic system and nucleus accumbens'?, Beh. Brain Res., 27 (1988) 45 58.