Developmental Brain Research, 68 (1992) 9-15 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-3806/92/$05.00 BRESD 51471
Neurogenesis of the amygdaloid nuclear complex in the Rhesus monkey Jeffrey H. K o r d o w e r
a,
Patricia Piecinski b a n d P a s k o R a k i c c
a Department of Neurological Sciences, and Rush Alzheimer's Disease Center, Rush Presbyterian / St. Lukes Medical Center, Chicago, IL 60612 (USA), b Department of Anatomy and Cell Biology, University of Illinois School of Medicine, Chicago, IL 60612 (USA) and c Section of Neurobiology, Yale School of Medicine, New Haven, CT 06510 (USA) (Accepted 3 March 1992)
Key words: Amygdala; Neurogenesis; [3H]Thymidine; Non-human primate
The time course of neurogenesis for neurons which comprise the amygdaloid complex in Rhesus monkeys was determined using tritiated thymidine autoradiography. Fourteen pregnant monkeys received injections of tritiated thymidine between embryonic days 27 (E27) and 56 of their 165 day gestation and offspring were sacrificed during the early postnatal period. The first neurons destined for the amygdaloid complex were generated at E33 making them among the earliest postmitotic neurons in the telencephalon. Neurogenesis peaked within all nuclei of the amygdaloid complex between E38 and E48 and had ceased between E50 and E56. While amygdaloid neurogenesis in postnatally sacrificed monkeys displayed a dorsal-to-ventral gradient of radiolabeled neurons, the considerable rotation of the temporal lobe during the latter stages of primate development indicates that neurogenesis in the embryo, during the first third of gestation, actually occurs across a medial-to-lateral gradient. This medial-to-lateral gradient occurs as a smooth wave across the amygdaloid nuclei and does not respect neuroanatomical subdivisions or patterns of connectivity of the amygdaloid nuclei in the Rhesus monkey.
INTRODUCTION Neuroanatomical, electrophysiological, and behavioral studies have demonstrated that the amygdaloid complex participates in the regulation of neuroendocrine 3,4,t6, reproductive 9 and autonomic27'3° functions. Its position in iimbic circuitry has led to a role for the amygdala in the processing of emotional and social information as well (see review in ref. 31). For many years it has been postulated that the amygdala plays a role in memory function 29'32'33 although this role has recently been questioned 51. It is unfortunate that the amygdala, a brain region which integrates diverse essential information also undergoes severe age- and disease-related pathology. In this regard, neuropathological changes are observed within the amygdala of aged monkeys 45 and humans 2°. Furthermore, this region is a focus for seizures 17'is'5° and undergoes severe neuropathological consequences in diseases such as Alzheimer's disease 5'6'24'49, schizophrenia 44 and infantile autism ~°.
The amygdaloid complex in primates occupies a central position in the anterior temporal lobe, lying posterior to the temporal pole, anterior to the hippocampal formation and entorhinal cortex, and inferior to the striatum at the level of the decussation of the anterior commissure. Based upon phylogenetic considerations, as well as cytoarchitecture, connectivity and transmitter content, the amygdala can be divided into two main subregions comprised of seven cytoarchitecturally distinct nuclear groups which are separated by populations of intercalated cells and fiber bundles 21 (see refs. 12, 14 for review). The phylogeneticaUy older corticomedial subgroup is comprised of the cortical, medial and central nuclei. The more recently evolved basolateral group is comprised of the basal accessory, basal medial, basolateral, and lateral nuclei. The topographic connections between the amygdala and other structures across the neuraxis respect, for the most part, cytoarchitectonically defined subnuclei within the amygdala. For example, direct inputs from the olfactory bulb innervate the corticomedial nuclei
Correspondence: J.H. Kordower, Department of Neurological Sciences, Rush Presbyterian/St. Lukes Medical Center, 2242 West Harrison St., Chicago, IL 60612, USA. Fax: (1) (312) 633-1586.
10 whereas olfactory information integrated through the prepyriform cortex innervate the basolateral amygdaloid nuclei 1L13"14'19'25'43. Efferent projections emanate from select subregions of the amygdaloid complex with the corticomedial group innervating forebrain structures through the stria terminalis and the basolateral group providing descending influences via the ventral amygdalofugal pathway 12.14. Comparativei~ little is known regarding the development of this important region, especially in primates. It has been reported that the amygdaloid complex develops between gestational (E) days E l l and El5 in the mouse 's, El5 and El9 in the rat 2 and between El2 and El7 in the Chinese hamster 4s. In humans, the neurons within the medial amygdaloid nucleus can first be observed within the sixth week of fetal development 22 while the lateral nucleus appears to be generated between weeks 12.5 and 16 of gestation 34. The aim of the present study was to carry out a systematic examination of the development of the amygdaloid complex in the nonhuman primate (Rhesus monkeys) using tritiated thymidine as a marker for the birthdate of developing neurons. MATERIALS AND METHODS Sections through the amygdaloid complex of fourteen perinatal Rhesus monkeys (Macaca mulatta)were evaluated in this study. During gestation, each monkey was exposed to tritiated thymidine via a single injection to their respective mothers (10 p,Ci/kg: New England Nuclear). The pregnant dams were injected between days 27 and 56 of gestation (E) of the Rhesus monkey 165-day gestational period. Following normal delivery, the offspring were sacrificed between days 7 and II0 of the postnatal (P) period (Table I). This material is part of the Rakic collection (e,g, refs. 37-42) and are the same set of sections used by us for a previous report on the neurogenesis of the non-human primate basal forebrain :''~. The details of the injection protocols including the method of timed pregnancies have been reported previously (see review in ref. 40).
TABLE I
Autoradiographic material used to assess the neurogenesis of the amygdata Case number
Embryonic(E) day of injection
Postnatal(P) day of sacrifice
Date of injection
1 2 3 4 5 6 7 8 9 l0 Il 12 13 14
E27 E30 E31 E32 E33 E36 E38 E38 E40 E43 E45 E48 E50 E56
P67 P76 P56 P07 P64 P54 P72 P60 P62 P70 P58 Pi l0 P61 P66
09/27/76 10/16/73 05/26/77 04/29/72 05/22/74 07/26/73 10/17/74 12/05/74 03/13/73 12/05/74 09/23/71 08/08/72 05/16/73 10/11/76
Briefly, the offspring were perfused with a buffered solution containing a mixture of paraformaldehyde and glutaraldehyde. Tissue blocks were imbedded in polyester wax and cut in 8 #m thickness in the coronal plane. The sections were mounted on slides and processed for autoradiography according to established protocols as previously described 37-39 (see review in ref. 40). The sections were the~ counterstained for Nissl substance with Toluidine blue to delineat,i the nuclear cytoarchitecture of the central nervous system
(c~s). Sections through the amygdaloid complex were examined using both dark and bright field illumination with an Olympus Vanox mr~roscope. Neurons with greater than half the maximum grain count found over the most intensely labeled neuron on that particular specimen were designated as heavily labeled and were therefore considered to have undergone their final mitotic division close to the time of the tritiated thymidine injection. Sections were then traced on an Aus ,lena illuminator and heavily labeled neurons from each animal were mapped from four equispaced sections through the amygdaloid complex. The nomenclature of Amaral and Price I for the monkey amygdaloid complex was employed in this study.
RESULTS
In most specimens, the corticomedial and basolateral amygdaloid nuclei can be delineated in Nisslstained sections. However, in the very young perinatal specimens, the boundaries between nuclei were sometimes difficult to discern due to the significant proliferation of glia which is in progress during this period. Therefore, only nuclei for which clear boundaries could be discerned were mapped in this study. As illustrated in Fig. 1, both heavily and lightly labeled neurons could easily be discerned within the amygdala. The signal-to.noise ratio of tritiated thymidine labeling was high and criterion for the classification into lightly and heavily labeled nuclei was determined separately for each case 37-a~. In specimens which received tritiated thymidine before E30 of gestation, heavily radiolabeled neurons were not observed within any amygdaloid nuclei (Fig. 2). Neurogenesis within the amygdaloid complex was first observed in a specimen injected with tritiated thymidine on E33 of gestation (Fig. 3). In this case, heavily labeled neurons were principally seen within the central amygdaloid nucleus. A few scattered, but heavily labeled, neurons were also seen within the lateral and basolateral amygdaloid nuclei at this time point, as well as the magnocellular and parvicellular subdivisions of the basal accessory nucleus. Between E33 and E38, there was a gradual increase in the number of heavily labeled neurons. By E38, numerous heavily labeled neurons were observed within all amygdaloid nuclei. In particular, the central and medial amygdaloid nuclei contained dense populations of heavily labeled neurons. In contrast, there was a relative attenuation of labeled cells within the parvicellular division of the basolateral nucleus compared to the other nuclei. While labeled neurons could be seen
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Figs. 2-7. Schematic illustrations of the distribution of heavily labeled neurons within the amygdaloid complex at four equispaced levels between E30 and E53 of gestation. No heavily labeled neurons were observed at E30 and this panel serves to illustrate the cytoarchitecture of the amygdaloid complex.
scattered throughout each nucleus, there appeared to be a preferential distribution of cells within the more dorsal aspects within the lateral and basolateral nuclei (Fig. 4). This pattern of neurogenesis was maintained until E43. At E43 intensely labeled neurons were still seen throughout most of the amygdaloid nuclei with the parvicellular division of the basolateral nucleus again displaying fewer heavily labeled neurons. In con-
Fig. 4.
trast to earlier gestational time points, however, there was a clear reduction of heavily labeled neurons within the central and medial amygdaloid nuclei at E43 (Fig. 5). Between E43 and E48 neurogenesis within the amygdaloid complex appeared to slow and by E48, there was a general reduction in labeling throughout most of the amygdaloid nuclei (Fig. 6). At this time in
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daloid nuclei and only a sparse pattern of heavily labeled neurons were seen within the parvicellular division of the basolateral amygdaloid nucleus. Within the other amygdaloid nuclei, there again was a clear preferential distribution of heavily labeled neurons within the ventral portion of the nuclei. This was especially true for the lateral, basolateral, and magnocellular division of the basal accessory nuclei where few heavily labeled neurons were seen within the dorsal half of each nucleus. Far fewer labeled amygdaloid neurons were seen after E50 and by E56 no heavily labeled neurons were observed within any amygdaloid nucleus. DISCUSSION
Fig. 6.
gestation, few heavily labeled neurons were seen within the central and amygdaloid nuclei at all. Within the other nuclear groups, most heavily labeled neurons were seen predominantly within the ventral aspect of the nuclei. This pattern was most obvious within the basolateral and basal accessory amygdaloid nuclei. At E50 of gestation, there was a significant diminution in the number of heavily labeled neurons (Fig. 7). No labeling was seen within the central and medial amyg-
A
Fig. 7.
B
The present study demonstrates that neurons of the amygdaloid complex are generated between E30 and E50 of the of the Rhesus monkey 165-day gestational period. This makes the amygdaloid complex, like the magnocellular basal forebrain 23, among the earliest developing structures of the primate telencephalon. For example, the nucleus accumbens, cerebral neocorte.~ and neostriatum do not begin neurogenesis until E36 s, E40 3s and E437, respectively. The differences in the age of cell birth between brain regions are quite reliable since these studies haw been conducted on the same set of animals. It is interesting that the hippocampus, an archicortical structure also located within the temporal lobe, begins neurogenesis at a similar early time point (E33) 42. However, while the amygdaL completes neurogenesis during a 3-week period, the hippocampus develops over a much more protracted time course 42. The early development of the Rhesus monkey amygdaloid complex is similar to that assumed from the histological studies conducted in humans. The timing of neurogenesis seei: in non-human primates, however, is in contrast to the timing reported for non-primates. The amygdaloid complex is generated during E l l - E 1 5 of the mouse 18-day gestational period 2s. In the rat, a tritiated thymidine analysis demonstrated that neurogenesis occurs late in mid-to-late gestation, between El3 and El9 2. Similarly, generation of the amygdaloid complex occurs during the last half of the 21-day gestation period of the Chinese hamster 4s. In contrast, the human amygdaloid complex is generated early in gestation. The development of the human lateral amygdaloid nucleus, the largest and most differentiated amygdaloid nucleus in man, begins in the first trimester at the twelfth emb~onic week 34 while the medial amygdaloid nucleus can be seen as early as the sixth embryonic week 22. These data, taken in conjunction
14 with the present findings, provide further evidence that the developmental pattern of many telencephalic brain regions in primates occurs earlier in the gestational period than their homologues in non-primate species. Indeed, this pattern of early development in primates is not restricted to forebrain structures. For example, brainstem moneamine neurons are generated early in gestation in monkeys 26 and humans a5'36 but develop within the second half of gestation in rodents (e.g., ref. 46). In rodents, intranuclear and internuclear gradients of neurogenesis occur within the amygdaloid complex. in mice, rats, and Chinese hamsters a clear rostrocaudal gradient across amygdaloid nuclei has been reported 2'28'48. A complex series of five internuclear gradients has been described in the rat as well 2. In the monkey, a rostral-to-caudal gradient across the amygdaloid nuclei was not observed. However, a dorsal-toventral gradient was seen in the perinatal specimens. This gradient could be discerned within most amygdaloid nuclei but was most pronounced in the basolateral complex. In a,,~sessing the significance of these gradients, it is importa~ to note that the primate temporal lobe rotates co,,!siderably during development s~muitaneously along two axes. Early in the developing primate brain, the amygdaloid nuclei are initially located in a ventrolateral position within the expanding temporal lobe. Then, as the temporal lobe grows in size, it begins a coanterclockwise rotation (as viewed from the front) resulting in a shift in the amygdaloid nuclei. Ultimately the amygdala becomes positioned in the dorsomedial aspect of the temporal lobe. In humans this complex rotation occurs principally between the third and sixth lunar month 47. Using backwards extrapolation from the older to younger specimens it is evident that this rotation occurs between E66 and E132 of the Rhesus monkey 165-day gestational period (Fig. 8). Thus the dorsal-to-ventral gradient of neurogenesis observed in the perinatal specimens actually reflects a medial-tolateral gradient of neuron generation that occurred in the early embryonic brain. In either case, the gradient of neurogenesis does not respect the nuclear subdivisions of the amygdaloid complex nor the afferent or efferent patterns of connectivity. Rather, the gradients occur as a smooth wave across the amygdaloid complex. This finding represents another example that gradients of neurogenesis are not necessarily sufficient determinants of brain compartmentalization or hodology 41. In summary, the present study demonstrates the early neurogenesis of the amygdaloid complex in Rhesus monkeys between E33 and E50 of the 165 day
DA¥33 DAY 165
DAYe6
Y132
ModifiM From Sidman andRakic, '82 Fig. 8. Schematic figure illustrating the rotation of the amygdala and temporal lobe during development of the Rhesus monkey (Modified from Sidman and Rakic47).
gestational period. The generation of neurons appears to occur at similar time points throughout each of the amygdaloid nuclei across a medial-to-lateral gradient. Ackr,owledgements. This research was supported by Grants NS14841 and NS22807 (P.R.).
ABBREVIATIONS Brag basolateral nucleus of the amygdala magnocellular subdivision Bpc basolateral nucleus of the amygdala parvocellular subdivision BAmg basal accessory nucleus of the amygdala magnocellular subdivision BApc basal accessory nucleus of the amygdala parvocellular subdivision C central nucleus of the amygdala L lateral nucleus of the amygdala M medial nucleus of the amygdala
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15 6 Brashear, H.R., Godec, M.S. and Carlsen, J., The distribution of neuritic plaques and acetylcholinesterase staining in the amygdala in Alzheimer's disease, Neurology, 38 (1988) 1694-1699. 7 Brand, S. and Rakic, P., Genesis of the primate neostriatum: [3H]thymidine autoradiographic analysis of the time of origin in the rhesus monkey, Neuroscience, 4 (1979) 767-778. 8 Brand, S. and Rakic, P., Neurogenesis of the nucleus accumbens septi and neighboring septal nuclei in the rhesus monkey: a combined [3H]thymidine and electron microscopic study, Neuroscience, 5 (1980) 2125-2138. 9 Carrer, H.F., Masco, D.H. and Aoiki, A., Neural structures involved in the control of female sexual behavior. In A. Cummings, J.W. Funder and F.A.O. Mendehon (Eds.), Endocrinology, Australian Academy of Science, Canberra, Australia, 1980, pp. 611-614. 10 Cook, E.H., Autism: a review of neurochemicai investigations, Synapse, 6 (1990) 292-308. 11 Cowan, W.M., Raisman, G. and Powell, T.P.S., The connections of the amygdala, J. Neurol. Neurosurg. Psychiatry, 28 (1965) 137-151. 12 de Gimps, J., Amygdaloid nuclear gray complex. In G. Paxinos (Ed.), The Human Nervous System, Academic Press, Australia, 1990, pp. 583-710. 13 de Gimps, J., The amygdaloid projection field in the rat as studies with the cupric-silver method. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum, New York, 1972, pp. 145-204. 14 de Olmos, J., Alheid, G.F. and Beltramino, C., Amygdala. In G. Paxinos (Ed.), The Rat Nervous System, Vol. 1, Academic Press,
Australia, 1985, pp. 223-334. 15 Dunn, J.D., Plasma corticosterone responses to electrical stimulation of the bed nucleus of the stria terminalis, Brain Res., 407 (1987) 327--331. 16 Dunn, J.D. and Whitener, J., Plasma corticosterone in response to electrical stimulation of the amygdaloid complex: cytoarchitectural specificity, Neuroendocrinology, 42 (1986) 211-217. 17 Gigli, G.L., Effects of seizures and carbamazeprine on interictal spiking in amygdala kindled cats, Epilepsy Res., 8 (1991) 204-212. 18 Gioanni, Y., Gioanni, H. and Mitrovic, N., Seizures can be triggered by stimulating noncortieal structures in the quaking mouse, Epilepsy Res., 9 (1991) 19-31. 19 Hall, E.A., Efferent connections of the basal and lateral nuclei of the amygdala in the cat, Am. I. Anat., 113 (1963) 139-151.
20 Herzog, A.C. and Kemper, T.C., Amygdaloid changes in aging and dementia, Arch. Neurol., 37 (1980) 626-629. 21 Humphrey, T., The telencephalon in the bat. 1. The noncortical nuclear muses and certain pertinent fiber connections, J. Comp. Neurol., 65 (1936) 603-711. 22 Humphrey, T., The development of the human amygdala during early embryonic life, I. Comp. Neurol., 132 (1968) 135-165. 23 Kordower, J.H. and Rakic, P., Neurogenesis of the magnocellular basal forebrain nuclei in the rhesus monkey. I. Comp. Neurol., 292 (1990) 637-653. 24 Kromer-Vogt, L.J., Hyman, B.T., Van Hoesen, G.W. and Damasio, A.R., Pathological alterations in the amygdala in Alzheimer's disease, Neuroscience, 37 (1990) 377-385. 25 Lammers, H.J., The neural connections of the amygdaloid complex it, mammals. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum, New York, 1972, pp. 123-144. 26 Levitt, P. and Rakic, P., The time of genesis, embryonic origin, and differentiation of the brain stem monoaminergic neurons in the rhesus monkey, Dev. Brain Res., 4 (1982) 35-57. 27 Loewy, A.D. and McKellar, S., The neuroanatomical basis of cardiovascular control, Fed. Proc., 39 (1980) 2495-2503. 28 McConnell, J. and Angevine Jr. J.B., Time of origin in the amygdaloid complex of the mouse, Brain Res., 272 (1983) 150-156. 29 Mishkin, M., Memory in monkeys is severely impaired by com-
bined but not separate removal of the amygdala and hippocampus, Nature, 273 (1978) 297-298. 30 Moga, M.M. and Gray, T.S., Peptidergic efferent from the intercalated nuclei of the amygdala to the parabrachial nucleus, Neurosci. Left., 61 (1985) 13-18. 31 Murray, E.A., Contributions of the amygdalar complex to behavior in macaque monkeys, Prog. Brain Res., 87 t1991) 167-180. 32 Murray, E.A. and Mishkin, M., Severe tactual as well as visual memory deficits following combined removal of the amygdala and hippocampus. J. Neurosci., 4 (1984) 2565-2580. 33 Murray, E.A. and Mishkin, M., Amygdalectomy impairs crossmodal association in monkeys, Science, 228 (1985) 604-606. 34 Nikoli~, I. and Kostovi~, I., Development of the lateral amygdaloid nucleus of the human fetus: transient presence of discrete cytoarchitectonic units, Anat. Embryol., 174 (1986) 355-360. 35 Pearson, J., Brandeis, L. and Goldstein, M., Appearance of tyrosine hydroxylase in the human embryo, Det'. Neurosci., 3 (1980) 140-150. 36 Pickel, V.M., Specht, L.A., Sumal, K.K., Joh, T., Reis, D.J. and Hervonen, A., Immunocytochemical localization of tyrosine hydroxylase in the human fetal nervous system, J. Comp. Neurol., 194 (1980) 465-474. 37 Rakic, P., Kinetics of proliferation and latency between final division and onset of differentation of the cerebeilar stellate and basket neurons. J. Comp. NeuroL, 147 (1973) 523-546. 38 Rakic, P., Neurons in the rhesus monkey visual cortex: systematic relation between the time of origin and eventual disposition, Science, 183 (1974) 425-427. 39 Rakic, P. Prenatal development of the visual system in the rhesus monkey, Phil. Trans. R. Soc. Lond. B, 278 (1977) 245-260. 40 Rakic, P., DNA synthesis and cell division in the adult primate brain, Ann. N.Y. Acad. Sci., 457 (1984) 193-211. 41 Rakic, P., Specifications of cerebral cortical areas, Science, 241 (1988) 170-176. 42 Rakic, P. and Nowakowski, R.S., The time of origin of neurons in the hippocampal region of the rhesvs monkey, J. Comp. NeuroL, 196 (1981) 99-128. 43 Ralsman, G., Neural connections of the hypothalamus, Br. Mud. Bull., 22 (1966) 197--201. 44 Roberts, G.W., Schizophrenia: a cellular biology of a functional psychosis, Trends Neurosci., 13 (1990) 207-21 I. 45 Schwam, E., Walker, L.C. Buckwald, B., Garcia, G. and Sepinwall, J., Senile plaques and behavior in aged squirrel monkeys, Soc. Neurosci. Abstr., 13 (1987) 441. 46 Seiger, A. and Olson, L. Late prenatal ontogeny of centl'ai mo1~oamine neurons in the rat, Z. Anat. EntwickL-Gesch., 140 (1973) 281-318. 47 Sidman, R.L. and Rakic, P., Development of the human central nervous system. In W. Haymaker and R.D. Adams (Eds.), ltistology and Histopathoiogy of the Nervous Sy~tctn, C.C. Thomas, Springfield, MA, 1982, pp. 3-145. 48 Ten Donkelaar, H., Lammers, G. and Gribnau, A.A.M., Neurogenesis of the amygdaloid complex in the rodent (Chinese hamster), Brain Res., 165 (1979) 348-353. 49 Unger, J.W., McNeil, T., Lapham, L.L. and Hamill, R.W., Neuropeptides and neuropathology in the amygdala in AIzheimer's disease: relationship between somatostatin, neuropeptide Y, and subregional distribution of neuritic plaques, Brain Res., 452 (1988) 293-302. 50 Wilson, C.L., Isokawa, M., Babb, T.L. and Crandall, P.H. Functional connection in human temporal lobe II: evidence for a loss of functional linkage between contralateral limbic structures, Exp. Brain Res., 85 (1991) 174-187. 51 Zola-Morgan, S., Squire, L.R. and Amaral, D.G., Lesions of the amygdala that spare adjacent cortical regions do not impair memory or exacerbate the impairment following lesions of the hippocampal tbrmation, J. Neurosci., 9 (1989) 1922-1936.
Neurogenesis of the amygdaloid nuclear complex in the Rhesus monkey Jeffrey H. K o r d o w e r
a,
Patricia Piecinski b a n d P a s k o R a k i c c
a Department of Neurological Sciences, and Rush Alzheimer's Disease Center, Rush Presbyterian / St. Lukes Medical Center, Chicago, IL 60612 (USA), b Department of Anatomy and Cell Biology, University of Illinois School of Medicine, Chicago, IL 60612 (USA) and c Section of Neurobiology, Yale School of Medicine, New Haven, CT 06510 (USA) (Accepted 3 March 1992)
Key words: Amygdala; Neurogenesis; [3H]Thymidine; Non-human primate
The time course of neurogenesis for neurons which comprise the amygdaloid complex in Rhesus monkeys was determined using tritiated thymidine autoradiography. Fourteen pregnant monkeys received injections of tritiated thymidine between embryonic days 27 (E27) and 56 of their 165 day gestation and offspring were sacrificed during the early postnatal period. The first neurons destined for the amygdaloid complex were generated at E33 making them among the earliest postmitotic neurons in the telencephalon. Neurogenesis peaked within all nuclei of the amygdaloid complex between E38 and E48 and had ceased between E50 and E56. While amygdaloid neurogenesis in postnatally sacrificed monkeys displayed a dorsal-to-ventral gradient of radiolabeled neurons, the considerable rotation of the temporal lobe during the latter stages of primate development indicates that neurogenesis in the embryo, during the first third of gestation, actually occurs across a medial-to-lateral gradient. This medial-to-lateral gradient occurs as a smooth wave across the amygdaloid nuclei and does not respect neuroanatomical subdivisions or patterns of connectivity of the amygdaloid nuclei in the Rhesus monkey.
INTRODUCTION Neuroanatomical, electrophysiological, and behavioral studies have demonstrated that the amygdaloid complex participates in the regulation of neuroendocrine 3,4,t6, reproductive 9 and autonomic27'3° functions. Its position in iimbic circuitry has led to a role for the amygdala in the processing of emotional and social information as well (see review in ref. 31). For many years it has been postulated that the amygdala plays a role in memory function 29'32'33 although this role has recently been questioned 51. It is unfortunate that the amygdala, a brain region which integrates diverse essential information also undergoes severe age- and disease-related pathology. In this regard, neuropathological changes are observed within the amygdala of aged monkeys 45 and humans 2°. Furthermore, this region is a focus for seizures 17'is'5° and undergoes severe neuropathological consequences in diseases such as Alzheimer's disease 5'6'24'49, schizophrenia 44 and infantile autism ~°.
The amygdaloid complex in primates occupies a central position in the anterior temporal lobe, lying posterior to the temporal pole, anterior to the hippocampal formation and entorhinal cortex, and inferior to the striatum at the level of the decussation of the anterior commissure. Based upon phylogenetic considerations, as well as cytoarchitecture, connectivity and transmitter content, the amygdala can be divided into two main subregions comprised of seven cytoarchitecturally distinct nuclear groups which are separated by populations of intercalated cells and fiber bundles 21 (see refs. 12, 14 for review). The phylogeneticaUy older corticomedial subgroup is comprised of the cortical, medial and central nuclei. The more recently evolved basolateral group is comprised of the basal accessory, basal medial, basolateral, and lateral nuclei. The topographic connections between the amygdala and other structures across the neuraxis respect, for the most part, cytoarchitectonically defined subnuclei within the amygdala. For example, direct inputs from the olfactory bulb innervate the corticomedial nuclei
Correspondence: J.H. Kordower, Department of Neurological Sciences, Rush Presbyterian/St. Lukes Medical Center, 2242 West Harrison St., Chicago, IL 60612, USA. Fax: (1) (312) 633-1586.
10 whereas olfactory information integrated through the prepyriform cortex innervate the basolateral amygdaloid nuclei 1L13"14'19'25'43. Efferent projections emanate from select subregions of the amygdaloid complex with the corticomedial group innervating forebrain structures through the stria terminalis and the basolateral group providing descending influences via the ventral amygdalofugal pathway 12.14. Comparativei~ little is known regarding the development of this important region, especially in primates. It has been reported that the amygdaloid complex develops between gestational (E) days E l l and El5 in the mouse 's, El5 and El9 in the rat 2 and between El2 and El7 in the Chinese hamster 4s. In humans, the neurons within the medial amygdaloid nucleus can first be observed within the sixth week of fetal development 22 while the lateral nucleus appears to be generated between weeks 12.5 and 16 of gestation 34. The aim of the present study was to carry out a systematic examination of the development of the amygdaloid complex in the nonhuman primate (Rhesus monkeys) using tritiated thymidine as a marker for the birthdate of developing neurons. MATERIALS AND METHODS Sections through the amygdaloid complex of fourteen perinatal Rhesus monkeys (Macaca mulatta)were evaluated in this study. During gestation, each monkey was exposed to tritiated thymidine via a single injection to their respective mothers (10 p,Ci/kg: New England Nuclear). The pregnant dams were injected between days 27 and 56 of gestation (E) of the Rhesus monkey 165-day gestational period. Following normal delivery, the offspring were sacrificed between days 7 and II0 of the postnatal (P) period (Table I). This material is part of the Rakic collection (e,g, refs. 37-42) and are the same set of sections used by us for a previous report on the neurogenesis of the non-human primate basal forebrain :''~. The details of the injection protocols including the method of timed pregnancies have been reported previously (see review in ref. 40).
TABLE I
Autoradiographic material used to assess the neurogenesis of the amygdata Case number
Embryonic(E) day of injection
Postnatal(P) day of sacrifice
Date of injection
1 2 3 4 5 6 7 8 9 l0 Il 12 13 14
E27 E30 E31 E32 E33 E36 E38 E38 E40 E43 E45 E48 E50 E56
P67 P76 P56 P07 P64 P54 P72 P60 P62 P70 P58 Pi l0 P61 P66
09/27/76 10/16/73 05/26/77 04/29/72 05/22/74 07/26/73 10/17/74 12/05/74 03/13/73 12/05/74 09/23/71 08/08/72 05/16/73 10/11/76
Briefly, the offspring were perfused with a buffered solution containing a mixture of paraformaldehyde and glutaraldehyde. Tissue blocks were imbedded in polyester wax and cut in 8 #m thickness in the coronal plane. The sections were mounted on slides and processed for autoradiography according to established protocols as previously described 37-39 (see review in ref. 40). The sections were the~ counterstained for Nissl substance with Toluidine blue to delineat,i the nuclear cytoarchitecture of the central nervous system
(c~s). Sections through the amygdaloid complex were examined using both dark and bright field illumination with an Olympus Vanox mr~roscope. Neurons with greater than half the maximum grain count found over the most intensely labeled neuron on that particular specimen were designated as heavily labeled and were therefore considered to have undergone their final mitotic division close to the time of the tritiated thymidine injection. Sections were then traced on an Aus ,lena illuminator and heavily labeled neurons from each animal were mapped from four equispaced sections through the amygdaloid complex. The nomenclature of Amaral and Price I for the monkey amygdaloid complex was employed in this study.
RESULTS
In most specimens, the corticomedial and basolateral amygdaloid nuclei can be delineated in Nisslstained sections. However, in the very young perinatal specimens, the boundaries between nuclei were sometimes difficult to discern due to the significant proliferation of glia which is in progress during this period. Therefore, only nuclei for which clear boundaries could be discerned were mapped in this study. As illustrated in Fig. 1, both heavily and lightly labeled neurons could easily be discerned within the amygdala. The signal-to.noise ratio of tritiated thymidine labeling was high and criterion for the classification into lightly and heavily labeled nuclei was determined separately for each case 37-a~. In specimens which received tritiated thymidine before E30 of gestation, heavily radiolabeled neurons were not observed within any amygdaloid nuclei (Fig. 2). Neurogenesis within the amygdaloid complex was first observed in a specimen injected with tritiated thymidine on E33 of gestation (Fig. 3). In this case, heavily labeled neurons were principally seen within the central amygdaloid nucleus. A few scattered, but heavily labeled, neurons were also seen within the lateral and basolateral amygdaloid nuclei at this time point, as well as the magnocellular and parvicellular subdivisions of the basal accessory nucleus. Between E33 and E38, there was a gradual increase in the number of heavily labeled neurons. By E38, numerous heavily labeled neurons were observed within all amygdaloid nuclei. In particular, the central and medial amygdaloid nuclei contained dense populations of heavily labeled neurons. In contrast, there was a relative attenuation of labeled cells within the parvicellular division of the basolateral nucleus compared to the other nuclei. While labeled neurons could be seen
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Figs. 2-7. Schematic illustrations of the distribution of heavily labeled neurons within the amygdaloid complex at four equispaced levels between E30 and E53 of gestation. No heavily labeled neurons were observed at E30 and this panel serves to illustrate the cytoarchitecture of the amygdaloid complex.
scattered throughout each nucleus, there appeared to be a preferential distribution of cells within the more dorsal aspects within the lateral and basolateral nuclei (Fig. 4). This pattern of neurogenesis was maintained until E43. At E43 intensely labeled neurons were still seen throughout most of the amygdaloid nuclei with the parvicellular division of the basolateral nucleus again displaying fewer heavily labeled neurons. In con-
Fig. 4.
trast to earlier gestational time points, however, there was a clear reduction of heavily labeled neurons within the central and medial amygdaloid nuclei at E43 (Fig. 5). Between E43 and E48 neurogenesis within the amygdaloid complex appeared to slow and by E48, there was a general reduction in labeling throughout most of the amygdaloid nuclei (Fig. 6). At this time in
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daloid nuclei and only a sparse pattern of heavily labeled neurons were seen within the parvicellular division of the basolateral amygdaloid nucleus. Within the other amygdaloid nuclei, there again was a clear preferential distribution of heavily labeled neurons within the ventral portion of the nuclei. This was especially true for the lateral, basolateral, and magnocellular division of the basal accessory nuclei where few heavily labeled neurons were seen within the dorsal half of each nucleus. Far fewer labeled amygdaloid neurons were seen after E50 and by E56 no heavily labeled neurons were observed within any amygdaloid nucleus. DISCUSSION
Fig. 6.
gestation, few heavily labeled neurons were seen within the central and amygdaloid nuclei at all. Within the other nuclear groups, most heavily labeled neurons were seen predominantly within the ventral aspect of the nuclei. This pattern was most obvious within the basolateral and basal accessory amygdaloid nuclei. At E50 of gestation, there was a significant diminution in the number of heavily labeled neurons (Fig. 7). No labeling was seen within the central and medial amyg-
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The present study demonstrates that neurons of the amygdaloid complex are generated between E30 and E50 of the of the Rhesus monkey 165-day gestational period. This makes the amygdaloid complex, like the magnocellular basal forebrain 23, among the earliest developing structures of the primate telencephalon. For example, the nucleus accumbens, cerebral neocorte.~ and neostriatum do not begin neurogenesis until E36 s, E40 3s and E437, respectively. The differences in the age of cell birth between brain regions are quite reliable since these studies haw been conducted on the same set of animals. It is interesting that the hippocampus, an archicortical structure also located within the temporal lobe, begins neurogenesis at a similar early time point (E33) 42. However, while the amygdaL completes neurogenesis during a 3-week period, the hippocampus develops over a much more protracted time course 42. The early development of the Rhesus monkey amygdaloid complex is similar to that assumed from the histological studies conducted in humans. The timing of neurogenesis seei: in non-human primates, however, is in contrast to the timing reported for non-primates. The amygdaloid complex is generated during E l l - E 1 5 of the mouse 18-day gestational period 2s. In the rat, a tritiated thymidine analysis demonstrated that neurogenesis occurs late in mid-to-late gestation, between El3 and El9 2. Similarly, generation of the amygdaloid complex occurs during the last half of the 21-day gestation period of the Chinese hamster 4s. In contrast, the human amygdaloid complex is generated early in gestation. The development of the human lateral amygdaloid nucleus, the largest and most differentiated amygdaloid nucleus in man, begins in the first trimester at the twelfth emb~onic week 34 while the medial amygdaloid nucleus can be seen as early as the sixth embryonic week 22. These data, taken in conjunction
14 with the present findings, provide further evidence that the developmental pattern of many telencephalic brain regions in primates occurs earlier in the gestational period than their homologues in non-primate species. Indeed, this pattern of early development in primates is not restricted to forebrain structures. For example, brainstem moneamine neurons are generated early in gestation in monkeys 26 and humans a5'36 but develop within the second half of gestation in rodents (e.g., ref. 46). In rodents, intranuclear and internuclear gradients of neurogenesis occur within the amygdaloid complex. in mice, rats, and Chinese hamsters a clear rostrocaudal gradient across amygdaloid nuclei has been reported 2'28'48. A complex series of five internuclear gradients has been described in the rat as well 2. In the monkey, a rostral-to-caudal gradient across the amygdaloid nuclei was not observed. However, a dorsal-toventral gradient was seen in the perinatal specimens. This gradient could be discerned within most amygdaloid nuclei but was most pronounced in the basolateral complex. In a,,~sessing the significance of these gradients, it is importa~ to note that the primate temporal lobe rotates co,,!siderably during development s~muitaneously along two axes. Early in the developing primate brain, the amygdaloid nuclei are initially located in a ventrolateral position within the expanding temporal lobe. Then, as the temporal lobe grows in size, it begins a coanterclockwise rotation (as viewed from the front) resulting in a shift in the amygdaloid nuclei. Ultimately the amygdala becomes positioned in the dorsomedial aspect of the temporal lobe. In humans this complex rotation occurs principally between the third and sixth lunar month 47. Using backwards extrapolation from the older to younger specimens it is evident that this rotation occurs between E66 and E132 of the Rhesus monkey 165-day gestational period (Fig. 8). Thus the dorsal-to-ventral gradient of neurogenesis observed in the perinatal specimens actually reflects a medial-tolateral gradient of neuron generation that occurred in the early embryonic brain. In either case, the gradient of neurogenesis does not respect the nuclear subdivisions of the amygdaloid complex nor the afferent or efferent patterns of connectivity. Rather, the gradients occur as a smooth wave across the amygdaloid complex. This finding represents another example that gradients of neurogenesis are not necessarily sufficient determinants of brain compartmentalization or hodology 41. In summary, the present study demonstrates the early neurogenesis of the amygdaloid complex in Rhesus monkeys between E33 and E50 of the 165 day
DA¥33 DAY 165
DAYe6
Y132
ModifiM From Sidman andRakic, '82 Fig. 8. Schematic figure illustrating the rotation of the amygdala and temporal lobe during development of the Rhesus monkey (Modified from Sidman and Rakic47).
gestational period. The generation of neurons appears to occur at similar time points throughout each of the amygdaloid nuclei across a medial-to-lateral gradient. Ackr,owledgements. This research was supported by Grants NS14841 and NS22807 (P.R.).
ABBREVIATIONS Brag basolateral nucleus of the amygdala magnocellular subdivision Bpc basolateral nucleus of the amygdala parvocellular subdivision BAmg basal accessory nucleus of the amygdala magnocellular subdivision BApc basal accessory nucleus of the amygdala parvocellular subdivision C central nucleus of the amygdala L lateral nucleus of the amygdala M medial nucleus of the amygdala
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