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Amygdalotegmental projections in the rat, cat and rhesus monkey
Neu:,o~cience Letters, 1 (1975) 263--270
263
© Els,_~vier/North-HoHand, Amsterdam -- Printed in The Netherlands
AMYGDALOTEGMENTAL RHESUS MONKEY
PROJECTIONS
IN T H E RAT, C A T A N D
DAVID A~ HOPKINS
Department o f Anatomy, Erasmus University Rotterdam, Rotterdam (The Netherlands) (Received November 10th, 197 5) (Accepted November 17 th, 197 5)
SUMMARY
Horseradish peroxid~e was injected in different parts of the mesencephalic, pontine and medullary " ~ m e n t u m in the rat, cat and rhesus monkey. In each species, large numbers of retrogradely labeled neurons were observed in the ipsilateral amygdala mainly in the central nucleus. These amygdaloid neurons, therefore, give rise So a hitherto unreported descending pathway which proceeds as far caudally as the medulla oblongata.
The amygdala sends fiber~ to the hypothalamus by way of the stria terminaiis and the ventral amygdalofugal pathway [1,3,6,12,14 ]. Anatomical studies generally agree that in mammals efferent fibers of the amygdala cannot be traced further caudally than the posterior hypothalamus. However, Gloor's [4] electrophysiological evidence suggested the existence of a direct projection to the rostral mesencephalon in the cat in addition to multisynaptic connections. In the present study the retrograde intraaxonal transport of horseradish peroxidase (HRP) has been used to determine to what extent the amygdala projects to the tegmentum in the rat, cat and rhesus monkey. In 27 rats 0 . 0 5 - 0.3 #l of 10 -- 30% HRP (Sigma or Boehringer) were injected in the brain stem or spinal cord under Nembutal anesthesia. The enzyme was injected by means of a Hamilton syringe and delivered over 5 - 30 rain periods. In 21 rats unilateral injections were made in different parts of the mesencephalic, pontine and medullary tegmentum using different stereotaxic approaches. In three rats unilateral injections were made in the cervical spinal cord, levelsC3 to C6. In three other rats injections were made bilaterallyin the pontine tegmentum. In these ca~s unilateral electrolytic lesions were placed in the striaterminalis prior to the ir.jections.Control injections of H R P in the cortex, thalamus and hypothalamus were available from another study [9]. In one cat 0.4/~I of 3 0 % H R P were iniected in the pontine tegmentum In
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Fig.1. D~stribution of retrogradely labeled neurons in the amygdala in the rat (A) after unilateral injection of HRP in the medullary tegmentum, and in the cat (B) after injection in the pontine and medullary Legmentum. Heavy injection area indicated by solid black, lighter injection or diffusion indicated by horizontal lines. C: dark-field photomicrograph of labeled neurons in the central nucleus in the cat. Abbreviations of amygdaloid nuclei: AA, anterior amygdaloid area; AL, lateral; B, basal; BM, basom~dlai; BL, b~olateral; CE, central; CO, cortical; M, medial. Other abbreviations: CI, i#-~rnal capsule; E, entopeduncular nucleus; F, fornix; GP, globns pallidus; ICP, inferior cerebellar peduncle; LT, lateral tegmental field; MFB, medial forebrain bundle; MT, medial ~gmental field; OT, optic traot;P, caudate-putamen, putamen; PT, pyramidal tract; PV, paraventricular nucleus of hypothalamus; PYR, pyriform cortex; RT, reticular nucleus of thalamus; STC, spinal trigeminal complex; VC, vestibular complex.
another cat 2.0 gl of HRP were injected with four separate penetrations i n ~ the pontine and medullary tegmentum using a caudaI infratentofial approach. Similar injections were made in one rhesus monkey. Following 2- or 3-day m~¢ival periods, the animals were deeply anesthetized and peffused with Macrodex followed by a buffered fixative containing 0.5% paraformaldehyde and 2.5% glutaraldehyde. The brains were removed and placed in a sodium cacodyhte buffer and 3 0 % sucrose for 12 -- 18 h. Subsequently, 40 ~ m frozen sections were cut and every third to fifth section was incubated according to the Graham-Kaznovsky method [5]. S o m e sections
266
were
~ m m ~ m n e u w]m c~vJy~ v~oJe~. ~ne Joca~on o~ mvema neurons the aid of an X-Y ploRer linked t o the stage of a miczoscope
concentrated m t h e nucleus of the a m y g d a ~ Scatt~ed HRP-positive neurons ~ e ~ at rostral levels in the anterior, lateral and basal nucleL 1 ~ . 1A shows the distribution of labeled neurons in the amygdala of a rat afl~er a 0.2/d ~jection of HRP in the medulla oblongata at the level of the ~ nucleus. Fig. 1B shows the distribution in the cat after injection of 2.0/~1 of H R P i n the pontine and medullary reticular formation and Fig. 2 shows the distribution in the monkey after a 2.5 #1 injection in the tegmentum. Lof labeled ne-arons in the central mdal level of the amygdala. The majority of H sitive neurons in the central nucle,ds were 15 -- 20 #m in diameter in the rat and from 20 to 30 ~m in diameter r a t h e cat and monkey. Fig. 1C s h o w s a dark-field photomicrograph of labeled neurons in the central nucleus in the cat. Injections both laterally a n d medially in the mesencephalon resulted in large numbers of labeled neurons in the amygdala. However, after injections in the m e d a l parts of the bulbar reticular formation of the rat, relatively few labeled neurons were present in the amygdala as compared to injections situated more laterally. These findings suggest that the central nucleus of the amygdala sends fibers to widespread areas of the mesencephalon and primarily to central and lateral parts of the bulbar reticular formation. Further, in spite of a complete lesion of the stria terminalis prior to tegmental injections in one rat many labeled neurons were still present in the amygdala which suggests that the efferent fibers from the central nucleus to the brain stem do not pass in the stria terminalis but probably follow the ventral amygdalofugal pathway (cf. ref. 2). In two rats, a single labeled neuron was observed in the mygdala after spinal cord injections. No labeled neurons were observed in e periamygdaloid cortex after tegmental injections. However, after tegmental injections labeled neurons did occur in the bed nucleus of the stria terminalis, the hypothalamus and the zona incerta. The distribution of these ~ells will be described in detail in another study which deals with cells of origin of the medial forebrain bundle (in preparation.). The resultsobtained afterH R P injectionsin thebrain stem demonstrate the existence of a hitherto unreported direct oroiection from.the opnt~l IS
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Fig.2. Distribution o f retrogradely iabel~.t neurons in, the m o n k e y after injection o f HRP in the tegmentum.
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Fig.3. Low-power dark-field photomicrograph of retrogradely labeled neurons in the central nucleus in the monkey at a caudal level of the amygdala.
stem. This may have been due to the fact that the corticomedial areas of the amygdala were not as systematically investigated, and the central nucleus was therefore probably not explored. Similarly~ in many of the previous degeneration studies which failed to reveal this descending pathway only minimal damage was usually inflicted on the central! nucleus except in those cases in which amygdaloid lesions'were accompani~ by substantial cortical damage~ In one instance where heavy degeneration ,Has observed in the ventral amygda]ofugal pathway and lateral hypothalamus after a lesion of the central nucleus the brain stem was apparently not examined [2]. The differences in the distributions of labeled neurons following injections in different mediolateral parts of the tegmentum indicate that the majority of the d e s c e n d i n g fibers from the amygdala pass through or terminate laterally and medially in the rostral tegmentum and in central and lateral parts of the bulbar reticular formation. This suggests that the distribution of amygdalotegmental fibers overlaps considerably with that of lateralcomponents of descending projections of the limbic system [13,16]. The amygdala has frequently been assigned a role in modulating motivation and emotion (see ref. 11 for a review). For example, in a number of species the area of the central nucleus has been specificallyimplicated in the defense
269 reaction [ 8 , 1 1 ] . However, in rats stimulation has also been found to be rewarding in the region of the central nucleus [ 1 7 ] . The cells of orig~m of the direct amygdalotegmental projection or those o f t h e closely adjacent stria terminalis m a y play a role in one or b o t h o f these behaviors since b o t h can also be obtained by h y p o t h a l a m i c and brain stem stimulation [ 7 , 1 0 , 1 5 ] . Knowledge of the existence of thi~ p a t h w a y should facilitate functional analyses of the amygdala and aid in differentiating effects governed by the central nucleus and the cells of origin of the stria terminalis. Autoradiographic tracing studies are currently u n d e r w a y in order to determine the precise area of t e r m i n a t i o n of this amygdalotegmental pathway. ACKNOWLEDGEMENTS This investigation was supported by Grants 13-46-08A ~,nd B of the Dutch Organization for Fundamental Research in Medicine (FUNGO). The a u t h o r thanks Dr. H.G.J.M. Kuypers for his helpful c o m m e n t s on the text and Mr. E. Dalm, Miss P. Deifos, Miss F. Kwee and Mr. W. v.d. Oudenalder for technical assistance. REFERENCES
1 Cowan, W.M., Raisman, G., and Powell, T.P.S., The connexions of the amygdala, J. Neurol. Neurosurg. Psychiat., 28 (1965) 137--151. 2 De Oimos, J.S., The amygdaloid projection field in the rat as studied with the cupricsilver method. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum Press, New York, 1972, pp. )45--204. 3 De Olmos, J.S., and Ingrain, W.R., The pro~ection field of the stria terrainalis in the rat brain. An experimental study, J comp. Neurol., 46 (1972) 303--234. 4 Gloor, P., Electrophysiological studies of the connections of the amygdaloid nucleus in the cat. Part I: the neuronal organization of the amygdaloid projection system, Electroenceph. din. Neurophysiol., 7 (1955) 223--242. 5 Graham, R.C., and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the oroximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302. 6 Hall, E.A., Efferent connections of the basal and lateral nuclei of the amygdala in the cat, Amer. J. Anat., 113 (19~8) 139--151. 7 Hess, W.R., The Functional Organization of the Diencepha{on, Grune and Stratton, New York, 1957. 8 Hilton, S.M., and Zbrozyna, A.W., Amygdaloid region for defence reactions and its efferent pathway to the brain ~tem, J. Physiol. (Lond.), 165 (1963) 160---173. 9 Hopkins, D.A., Hypothalam{~"and brain stem connections of self-stimulation pathways studied using the retrograde intraaxonal transport of horseradish perox;dase in the rat, Anat. Rec., 181 (1975) 379. 10 Hunsperger, R.W., Affektreaktionen auf e{ektri~che Reizung ira Hir,nstamm der Katze, Helv. physiol. Acta, 14 (1956) 70--92. 11 Kaada, B.R., Stimulation and regional ablation of the amygdalold complex with l'eference to functional representations. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum Press, New York, 1972, pp. 205--281.
270 12 L e o n , C.M., and Scott, J.W., Origin and distribution of the amygdalofugaI pathways in the rat: an e~l~n'~ent~! r~uroanatomical study, J. eomp. NeuroL, 141 (1971) 313--330. 13 Nauta, W.J.H., Hippoeampal projections and related neural pathways to the mid-brain in the eat, Brain, 81 (1958) 319--340. 14 Nauta, W.J.H, Fiber degeneration following lesions of the amygdaloid complex in the monkey, J. Anat. (Lond.), 95 (1961) 515--531. 15 Routtenberg, A., Intraeranial s~lf-sfimulation pathways as substrate for stimulus response integration. In J.D. ~ (F.~i.), Efferent Organization and the Integration of Behavior, Academic Press, New York, 1973, pp. 264--318. 16 Vaienstein, E.S., and Nauta, W.J.H., A comparison of the distribution of the fornix system in the rat, guinea pig, cat, and monkey, J. comp. Nenrol., 113 (1959) 337--363. 17 Wurtz, R.H., and Olds, J., Amygdaloid stimulation and operant reinforcement in the rat, J. comp. physiol. Psychol., 56 (!963) 941--949.
263
© Els,_~vier/North-HoHand, Amsterdam -- Printed in The Netherlands
AMYGDALOTEGMENTAL RHESUS MONKEY
PROJECTIONS
IN T H E RAT, C A T A N D
DAVID A~ HOPKINS
Department o f Anatomy, Erasmus University Rotterdam, Rotterdam (The Netherlands) (Received November 10th, 197 5) (Accepted November 17 th, 197 5)
SUMMARY
Horseradish peroxid~e was injected in different parts of the mesencephalic, pontine and medullary " ~ m e n t u m in the rat, cat and rhesus monkey. In each species, large numbers of retrogradely labeled neurons were observed in the ipsilateral amygdala mainly in the central nucleus. These amygdaloid neurons, therefore, give rise So a hitherto unreported descending pathway which proceeds as far caudally as the medulla oblongata.
The amygdala sends fiber~ to the hypothalamus by way of the stria terminaiis and the ventral amygdalofugal pathway [1,3,6,12,14 ]. Anatomical studies generally agree that in mammals efferent fibers of the amygdala cannot be traced further caudally than the posterior hypothalamus. However, Gloor's [4] electrophysiological evidence suggested the existence of a direct projection to the rostral mesencephalon in the cat in addition to multisynaptic connections. In the present study the retrograde intraaxonal transport of horseradish peroxidase (HRP) has been used to determine to what extent the amygdala projects to the tegmentum in the rat, cat and rhesus monkey. In 27 rats 0 . 0 5 - 0.3 #l of 10 -- 30% HRP (Sigma or Boehringer) were injected in the brain stem or spinal cord under Nembutal anesthesia. The enzyme was injected by means of a Hamilton syringe and delivered over 5 - 30 rain periods. In 21 rats unilateral injections were made in different parts of the mesencephalic, pontine and medullary tegmentum using different stereotaxic approaches. In three rats unilateral injections were made in the cervical spinal cord, levelsC3 to C6. In three other rats injections were made bilaterallyin the pontine tegmentum. In these ca~s unilateral electrolytic lesions were placed in the striaterminalis prior to the ir.jections.Control injections of H R P in the cortex, thalamus and hypothalamus were available from another study [9]. In one cat 0.4/~I of 3 0 % H R P were iniected in the pontine tegmentum In
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Fig.1. D~stribution of retrogradely labeled neurons in the amygdala in the rat (A) after unilateral injection of HRP in the medullary tegmentum, and in the cat (B) after injection in the pontine and medullary Legmentum. Heavy injection area indicated by solid black, lighter injection or diffusion indicated by horizontal lines. C: dark-field photomicrograph of labeled neurons in the central nucleus in the cat. Abbreviations of amygdaloid nuclei: AA, anterior amygdaloid area; AL, lateral; B, basal; BM, basom~dlai; BL, b~olateral; CE, central; CO, cortical; M, medial. Other abbreviations: CI, i#-~rnal capsule; E, entopeduncular nucleus; F, fornix; GP, globns pallidus; ICP, inferior cerebellar peduncle; LT, lateral tegmental field; MFB, medial forebrain bundle; MT, medial ~gmental field; OT, optic traot;P, caudate-putamen, putamen; PT, pyramidal tract; PV, paraventricular nucleus of hypothalamus; PYR, pyriform cortex; RT, reticular nucleus of thalamus; STC, spinal trigeminal complex; VC, vestibular complex.
another cat 2.0 gl of HRP were injected with four separate penetrations i n ~ the pontine and medullary tegmentum using a caudaI infratentofial approach. Similar injections were made in one rhesus monkey. Following 2- or 3-day m~¢ival periods, the animals were deeply anesthetized and peffused with Macrodex followed by a buffered fixative containing 0.5% paraformaldehyde and 2.5% glutaraldehyde. The brains were removed and placed in a sodium cacodyhte buffer and 3 0 % sucrose for 12 -- 18 h. Subsequently, 40 ~ m frozen sections were cut and every third to fifth section was incubated according to the Graham-Kaznovsky method [5]. S o m e sections
266
were
~ m m ~ m n e u w]m c~vJy~ v~oJe~. ~ne Joca~on o~ mvema neurons the aid of an X-Y ploRer linked t o the stage of a miczoscope
concentrated m t h e nucleus of the a m y g d a ~ Scatt~ed HRP-positive neurons ~ e ~ at rostral levels in the anterior, lateral and basal nucleL 1 ~ . 1A shows the distribution of labeled neurons in the amygdala of a rat afl~er a 0.2/d ~jection of HRP in the medulla oblongata at the level of the ~ nucleus. Fig. 1B shows the distribution in the cat after injection of 2.0/~1 of H R P i n the pontine and medullary reticular formation and Fig. 2 shows the distribution in the monkey after a 2.5 #1 injection in the tegmentum. Lof labeled ne-arons in the central mdal level of the amygdala. The majority of H sitive neurons in the central nucle,ds were 15 -- 20 #m in diameter in the rat and from 20 to 30 ~m in diameter r a t h e cat and monkey. Fig. 1C s h o w s a dark-field photomicrograph of labeled neurons in the central nucleus in the cat. Injections both laterally a n d medially in the mesencephalon resulted in large numbers of labeled neurons in the amygdala. However, after injections in the m e d a l parts of the bulbar reticular formation of the rat, relatively few labeled neurons were present in the amygdala as compared to injections situated more laterally. These findings suggest that the central nucleus of the amygdala sends fibers to widespread areas of the mesencephalon and primarily to central and lateral parts of the bulbar reticular formation. Further, in spite of a complete lesion of the stria terminalis prior to tegmental injections in one rat many labeled neurons were still present in the amygdala which suggests that the efferent fibers from the central nucleus to the brain stem do not pass in the stria terminalis but probably follow the ventral amygdalofugal pathway (cf. ref. 2). In two rats, a single labeled neuron was observed in the mygdala after spinal cord injections. No labeled neurons were observed in e periamygdaloid cortex after tegmental injections. However, after tegmental injections labeled neurons did occur in the bed nucleus of the stria terminalis, the hypothalamus and the zona incerta. The distribution of these ~ells will be described in detail in another study which deals with cells of origin of the medial forebrain bundle (in preparation.). The resultsobtained afterH R P injectionsin thebrain stem demonstrate the existence of a hitherto unreported direct oroiection from.the opnt~l IS
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Fig.2. Distribution o f retrogradely iabel~.t neurons in, the m o n k e y after injection o f HRP in the tegmentum.
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Fig.3. Low-power dark-field photomicrograph of retrogradely labeled neurons in the central nucleus in the monkey at a caudal level of the amygdala.
stem. This may have been due to the fact that the corticomedial areas of the amygdala were not as systematically investigated, and the central nucleus was therefore probably not explored. Similarly~ in many of the previous degeneration studies which failed to reveal this descending pathway only minimal damage was usually inflicted on the central! nucleus except in those cases in which amygdaloid lesions'were accompani~ by substantial cortical damage~ In one instance where heavy degeneration ,Has observed in the ventral amygda]ofugal pathway and lateral hypothalamus after a lesion of the central nucleus the brain stem was apparently not examined [2]. The differences in the distributions of labeled neurons following injections in different mediolateral parts of the tegmentum indicate that the majority of the d e s c e n d i n g fibers from the amygdala pass through or terminate laterally and medially in the rostral tegmentum and in central and lateral parts of the bulbar reticular formation. This suggests that the distribution of amygdalotegmental fibers overlaps considerably with that of lateralcomponents of descending projections of the limbic system [13,16]. The amygdala has frequently been assigned a role in modulating motivation and emotion (see ref. 11 for a review). For example, in a number of species the area of the central nucleus has been specificallyimplicated in the defense
269 reaction [ 8 , 1 1 ] . However, in rats stimulation has also been found to be rewarding in the region of the central nucleus [ 1 7 ] . The cells of orig~m of the direct amygdalotegmental projection or those o f t h e closely adjacent stria terminalis m a y play a role in one or b o t h o f these behaviors since b o t h can also be obtained by h y p o t h a l a m i c and brain stem stimulation [ 7 , 1 0 , 1 5 ] . Knowledge of the existence of thi~ p a t h w a y should facilitate functional analyses of the amygdala and aid in differentiating effects governed by the central nucleus and the cells of origin of the stria terminalis. Autoradiographic tracing studies are currently u n d e r w a y in order to determine the precise area of t e r m i n a t i o n of this amygdalotegmental pathway. ACKNOWLEDGEMENTS This investigation was supported by Grants 13-46-08A ~,nd B of the Dutch Organization for Fundamental Research in Medicine (FUNGO). The a u t h o r thanks Dr. H.G.J.M. Kuypers for his helpful c o m m e n t s on the text and Mr. E. Dalm, Miss P. Deifos, Miss F. Kwee and Mr. W. v.d. Oudenalder for technical assistance. REFERENCES
1 Cowan, W.M., Raisman, G., and Powell, T.P.S., The connexions of the amygdala, J. Neurol. Neurosurg. Psychiat., 28 (1965) 137--151. 2 De Oimos, J.S., The amygdaloid projection field in the rat as studied with the cupricsilver method. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum Press, New York, 1972, pp. )45--204. 3 De Olmos, J.S., and Ingrain, W.R., The pro~ection field of the stria terrainalis in the rat brain. An experimental study, J comp. Neurol., 46 (1972) 303--234. 4 Gloor, P., Electrophysiological studies of the connections of the amygdaloid nucleus in the cat. Part I: the neuronal organization of the amygdaloid projection system, Electroenceph. din. Neurophysiol., 7 (1955) 223--242. 5 Graham, R.C., and Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the oroximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302. 6 Hall, E.A., Efferent connections of the basal and lateral nuclei of the amygdala in the cat, Amer. J. Anat., 113 (19~8) 139--151. 7 Hess, W.R., The Functional Organization of the Diencepha{on, Grune and Stratton, New York, 1957. 8 Hilton, S.M., and Zbrozyna, A.W., Amygdaloid region for defence reactions and its efferent pathway to the brain ~tem, J. Physiol. (Lond.), 165 (1963) 160---173. 9 Hopkins, D.A., Hypothalam{~"and brain stem connections of self-stimulation pathways studied using the retrograde intraaxonal transport of horseradish perox;dase in the rat, Anat. Rec., 181 (1975) 379. 10 Hunsperger, R.W., Affektreaktionen auf e{ektri~che Reizung ira Hir,nstamm der Katze, Helv. physiol. Acta, 14 (1956) 70--92. 11 Kaada, B.R., Stimulation and regional ablation of the amygdalold complex with l'eference to functional representations. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum Press, New York, 1972, pp. 205--281.
270 12 L e o n , C.M., and Scott, J.W., Origin and distribution of the amygdalofugaI pathways in the rat: an e~l~n'~ent~! r~uroanatomical study, J. eomp. NeuroL, 141 (1971) 313--330. 13 Nauta, W.J.H., Hippoeampal projections and related neural pathways to the mid-brain in the eat, Brain, 81 (1958) 319--340. 14 Nauta, W.J.H, Fiber degeneration following lesions of the amygdaloid complex in the monkey, J. Anat. (Lond.), 95 (1961) 515--531. 15 Routtenberg, A., Intraeranial s~lf-sfimulation pathways as substrate for stimulus response integration. In J.D. ~ (F.~i.), Efferent Organization and the Integration of Behavior, Academic Press, New York, 1973, pp. 264--318. 16 Vaienstein, E.S., and Nauta, W.J.H., A comparison of the distribution of the fornix system in the rat, guinea pig, cat, and monkey, J. comp. Nenrol., 113 (1959) 337--363. 17 Wurtz, R.H., and Olds, J., Amygdaloid stimulation and operant reinforcement in the rat, J. comp. physiol. Psychol., 56 (!963) 941--949.