Martin Lévesque

Newly generated neurons in the amygdala and adjoining cortex of adult primates

The subventricular zone remains mitotically active throughout life in rodents. Studies with tritiated thymidine, which is incorporated into the DNA of mitotic cells, have revealed that the rodent subventricular zone produces neuroblasts that migrate toward the olfactory bulb along the rostral migratory stream. A similar migratory stream has been documented in monkeys by using the thymidine analogue BrdUrd. The same approach showed that neurogenesis occurred in the dentate gyrus of adult primates, including humans. In the present study, experiments combining injections of BrdUrd and the dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine, with the immunostaining for molecular markers of neurogenesis (polysialylated neural cell adhesion molecule, β-tubulin-III, collapsin response mediator protein-4, neuronal nuclear protein) in New World (Saimiri sciureus) and Old World (Macaca fascicularis) monkeys have revealed that new neurons are produced in the amygdala, piriform cortex, and adjoining inferior temporal cortex in adult primates. These newborn neurons expressed the antiapoptotic protein Bcl-2 and formed a more-or-less continuous pathway that extended from the tip of the temporal ventricular horn to the deep portion of the temporal lobe. The production of newborn neurons in the amygdala, piriform cortex, and inferior temporal cortex seems to parallel the continuing addition of neurons in the olfactory bulb. These two concomitant phenomena may ensure structural stability and functional plasticity to the primate olfactory system and temporal lobe.

Organization of the basal ganglia: the importance of axonal collateralization

Recent neuroanatomical data obtained with single-axon or single-cell labeling procedures in both rodents and primates have revealed the presence of various types of projection neurons with profusely collateralized axons within each of the major components of the basal ganglia. Such findings call for a reappraisal of current concepts of the anatomical and functional organization of the basal ganglia,which play such a crucial role in the control of motor behavior. The basal ganglia now stand as a widely distributed neuronal network, whose elements are endowed with a highly patterned set of axon collaterals. The elucidation of this finely tuned network is needed to understand the complex spatiotemporal sequence of neural events that ensures the flow of cortical information through the basal ganglia.

Single‐axon tracing study of neurons of the external segment of the globus pallidus in primate

Axonal projections arising from the external segment of the globus pallidus (GPe) in cynomolgus monkeys (Macaca fascicularis) were mapped after labeling small pools (5–15 cells) of neurons with biotinylated dextran amine. Seventy‐six single axons were reconstructed from serial sagittal sections with a camera lucida. The majority of labeled GPe cells displayed long, aspiny, and poorly branched dendrites that arborized mostly along the sagittal plane, whereas others showed dendrites radiating in all directions. Numerous GPe axons emitted short, intranuclear collaterals that arborized close to their parent cell body. Based on their axonal targets, four distinct types of GPe projection neurons have been identified: 1) neurons that project to the internal segment of the globus pallidus (GPi), the subthalamic nucleus (STN), and the substantia nigra, pars reticulata (SNr; 13.2%); 2) neurons that target the GPi and the STN (18.4%); 3) neurons that project to the STN and the SNr (52.6%); and 4) neurons that target the striatum (15.8%). Labeled GPe axons displayed large varicosities that often were closely apposed to the somata and proximal dendrites of STN, GPi, and SNr neurons. At striatal levels, however, GPe axons displayed small axonal varicosities that did not form perineuronal nets. These results suggest that the GPe is an important integrative locus in primate basal ganglia. This nucleus harbors several subtypes of projection neurons that are endowed with a highly patterned set of collaterals. This organization allows single GPe neurons to exert a multifarious effect not only on the STN, which is the claimed GPe target, but also on the two major output structures of the basal ganglia, the SNr and the GPi. J. Comp. Neurol. 417:17–31, 2000. ©2000 Wiley‐Liss, Inc.

Two types of projection neurons in the internal pallidum of primates: single-axon tracing and three-dimensional reconstruction.

The axonal projections of the internal pallidum (GPi) in cynomolgus monkeys (Macaca fascicularis) were studied by labeling small pools of neurons with biotinylated dextran amine. Fifty‐two axons were reconstructed entirely from serial sections with a camera lucida. Two types of projection neurons were identified in the GPi on the basis of their target sites. The abundant and centrally located type I neurons gave rise to a long axonal branch that descended directly to the pedunculopontine tegmental nucleus, where it arborized discretely. Other branches ascended to the thalamus and broke into 10–15 thinner collaterals that ran through most of the ventral anterior nucleus, where they terminated as typical plexuses. About half of these axons gave rise to collaterals that arborized in both components of the centre médian/parafascicular thalamic complex. The less numerous and peripherally located type II neurons had an axon that climbed the rostral thalamic pole, coursed along the stria medullaris, and arborized profusely within the lateral habenular nucleus, which stood out as the most densely innervated pallidal target. Some type II axons provided collaterals to the anterior thalamic nuclei. A small proportion of axons of both types had branches that crossed the midline and terminated in contralateral GPi target structures. Three‐dimensional reconstruction showed that type I axons arborized principally along the sagittal plane. These data reveal that GPi neurons of type I act through a widely distributed axonal network upon thalamic and brainstem premotor neurons, whereas type II neurons act in a much more focused manner upon lateral habenular neurons. J. Comp. Neurol. 439:162–175, 2001.

Axonal branching pattern of neurons of the subthalamic nucleus in primates

Axonal projections arising from the subthalamic nucleus (STN) in cynomolgus monkeys (Macaca fascicularis) were traced after labeling small pools (5–15 cells) of neurons with biotinylated dextran amine. Seventy‐five single axons were reconstructed from serial sagittal sections with a camera lucida. Most of the STN labeled cells displayed five to eight long, sparsely spined dendrites that arborized mostly along the main axis of the nucleus. Based on their axonal targets, five distinct types of STN projection neurons have been identified: 1) neurons projecting to the substantia nigra pars reticulata (SNr), the internal (GPi) and external (GPe) segments of the globus pallidus (21.3%); 2) neurons targeting SNr and GPe (2.7%); 3) neurons projecting to GPi and GPe (48%); 4) neurons targeting GPe only (10.7 %); and 5) neurons with axons that coursed toward the sriatum, but whose terminal arborization could not be visualized in detail (17.3%). Axons of the first two types bifurcated into rostral subthalamopallidal and caudal pallidonigral branches. However, the majority of STN axons had only a single branch that coursed rostrally toward the pallidum and striatum. These results reveal that, in contrast to current beliefs, the primate STN is not a monolithic entity. This nucleus harbors several subtypes of projection neurons, each endowed with a highly patterned set of collaterals. This organization allows STN neurons to exert a multifarious effect not only on the GPe, with which the STN is reciprocally connected, but also on the two major output structures of the basal ganglia, the SNr and the GPi. J. Comp. Neurol. 424:142–152, 2000.

Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation.

Hedgehog (Hh) proteins regulate important developmental processes, including cell proliferation and differentiation. Although Patched acts as the main Hh receptor in Drosophila, Hh signaling absolutely requires the additional Hh-binding proteins Ihog and Boi. Here we show that, unexpectedly, cerebellar granule neuron progenitors (CGNPs) lacking Boc and Cdon, the vertebrate orthologs of Ihog and Boi, still proliferate in response to Hh. This is because in their absence, Gas1, an Hh-binding protein not present in Drosophila, mediates Hh signaling. Consistently, only CGNPs lacking all three molecules-Boc, Cdon, and Gas1-have a complete loss of Hh-dependent proliferation. In a complementary manner, we find that a mutated Hh ligand that binds Patched1 but not Boc, Cdon, or Gas1 cannot activate Hh signaling. Together, this demonstrates an absolute requirement for Boc, Cdon, and Gas1 in Hh signaling and reveals a distinct requirement for ligand-binding components that distinguishes the vertebrate and invertebrate Hh receptor systems.

The rostral migratory stream in adult squirrel monkeys: contribution of new neurons to the olfactory tubercle and involvement of the antiapoptotic protein Bcl‐2

The subventricular zone (SVZ) lying along the ependymal layer of lateral ventricle is known to generate neural progenitor cells throughout adulthood in specific areas of the mammalian brain. In rodents, the anterior region of the SVZ produces neuroblasts that migrate in chain toward the olfactory bulb along the so‐called rostral migratory stream (RMS). In the present study, the organization of the RMS in a representative of New World primates – the squirrel monkey (Saimiri sciureus) – was studied by using bromodeoxyuridine (BrdU), a thymidine analogue that incorporates itself into the DNA of cells undergoing mitotic division. Double and triple immunofluorescence labelling with a confocal microscope served to visualize cells that expressed BrdU as well as molecular markers of neurogenesis. Numerous newborn (BrdU+) cells, many ensheated in glial (GFAP+) tubes, were scattered along the entire RMS in squirrel monkeys. Some of these BrdU+ cells expressed molecular markers for early committed neurons (TuJ1), postmitotic granular neuroblasts (TUC‐4) or mature neurons (MAP‐2, NeuN), and virtually all of them expressed the antiapoptotic protein Bcl‐2. A significant number of BrdU+ cells were found to deviate from the main stream of the RMS. Instead of reaching the olfactory bulb, these cells migrated ventrally into the olfactory tubercle, where they expressed a mature neuronal phenotype (MAP‐2). These findings reveal that the RMS in New World monkeys is mitotically robust and markedly extended and suggest that Bcl‐2 might play a role in the survival and/or differentiation of newborn neurons destined to olfactory bulb and olfactory tubercle in primates.

EXTRASTRIATAL DOPAMINERGIC INNERVATION OF HUMAN BASAL GANGLIA

A tyrosine-hydroxylase immunohistochemical analysis of the brains of normal human individuals has revealed nigrostriatal axons providing collaterals that arborize in the pallidum and subthalamic nucleus. These thin and varicose collaterals emerge from thick and smooth axons that course backward along the main output pathways of the basal ganglia, including the ansa lenticularis, the lenticular fasciculus and Wilsons pencils. Many of these fibers run within pallidal medullary laminae before reaching the putamen, whereas others climb along the reticular thalamic nucleus to reach the caudate nucleus. This extrastriatal innervation, which allows nigral dopaminergic neurons to directly affect the pallidum and subthalamic nucleus, may play a crucial role in the functional organization of human basal ganglia, in both health and disease.

Proliferating cells can differentiate into neurons in the striatum of normal adult monkey

In this study we used bromodeoxyuridine (BrdU), a thymidine analogue that is incorporated into the DNA of mitotic cells, to study the cytogenesis status of the striatum in normal, adult, squirrel monkeys (Saimiri sciureus). Three weeks following BrdU injection, numerous BrdU-labeled (+) cells were encountered within both the dorsal and the ventral striatum, including the nucleus accumbens. Their number ranged from 5 to 50 per 40 microm-thick section. These BrdU+ cells were more abundant medially than laterally and displayed a rostrocaudal-decreasing gradient in the caudate nucleus and putamen. Double-immunofluorescence confocal studies have revealed that about 5-10% of the BrdU+ striatal cells expressed the neuronal nuclear antigen (NeuN), a marker for mature neurons. These findings suggest that new neurons are produced throughout adult life in the striatum of normal, adult primates. This result raises the possibility of experimentally enhancing the recruitment of these newborn neurons as a means to alleviate the symptoms of neurodegenerative diseases that affect the striatum.

The axonal arborization of single nigrostriatal neurons in rats.

Neurons of the substantia nigra pars compacta (SNc) were iontophoretically injected with biotin dextran and their anterogradely labeled axons individually reconstructed from serial sagittal sections. Most nigrostriatal axons travelled directly to the striatum, where they branched abundantly. Other axons arborized profusely in various extrastriatal structures, including the globus pallidus, the entopeduncular and subthalamic nuclei, and branched only sparsely in the striatum. This heterogeneous organization of the nigrostriatal projection allows single SNc neurons to influence differently striatal neurons and to act directly upon extrastriatal components of the basal ganglia via a highly patterned set of collaterals.