Constantino Sotelo

Lack of Barrels in the Somatosensory Cortex of Monoamine Oxidase A–Deficient Mice: Role of a Serotonin Excess during the Critical Period

In a transgenic mouse line (Tg8) deficient for the gene encoding monoamine oxidase A (MAOA), we show that the primary somatosensory cortex (S1) lacks the characteristic barrel-like clustering of layer IV neurons, whereas normal pattern formation exists in the thalamus and the trigeminal nuclei. No barrel-like patterns were visible with tenascin or serotonin immunostaining or with labeling of thalamocortical axons. An excess of brain serotonin during the critical period of barrel formation appears to have a causal role in these cortical abnormalities, since early administration of parachlorophenylalanine, an inhibitor of serotonin synthesis, in Tg8 pups restored the formation of barrels in S1, whereas inhibition of catecholamine synthesis did not. Transient inactivation of MAOA in normal newborns reproduced a barrelless phenotype in parts of S1.

Direct Immunohistochemical Evidence of the Existence of 5-HT1A Autoreceptors on Serotoninergic Neurons in the Midbrain Raphe Nuclei

Physiological, pharmacological and radioautographic binding studies have suggested the presence of the 5‐HT1A autoreceptors on midbrain serotoninergic neurons. The recent production of specific anti‐rat 5‐HT1A receptor antibodies in rabbits injected with a synthetic peptide has provided a tool to examine this problem directly. Using the immunoperoxidase method to localize the receptor protein, neurons of all the sizes and forms characterizing the neuronal populations in the dorsal and median raphe nuclei were stained. Reaction product was distributed along the neuronal surface, outlining the contours of perikarya and dendrites in a continuous but uneven manner. Intracellular staining was scarce and confined to the perinuclear region. Double immunohistochemical staining using the anti‐5‐HT1A receptor antibodies and an anti‐serotonin (5‐HT) antiserum showed that all the 5‐HT1A receptor immunoreactive neurons in the dorsal raphe, and the vast majority of them in the median raphe, are serotoninergic neurons. These data provide the first direct demonstration of the existence of 5‐HT1A autoreceptors on the perikarya and dendrites of serotoninergic neurons in the anterior raphe nuclei.

SLIT2-MEDIATED CHEMOREPULSION AND COLLAPSE OF DEVELOPING FOREBRAIN AXONS

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig‐1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit‐responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up‐regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain. J. Comp. Neurol. 442:130–155, 2002.

Transient biochemical compartmentalization of Purkinje cells during early cerebellar development

It has recently been observed that during early cerebellar development--from embryonic Day 17 to postnatal Day 3 in the rat--only certain discrete clusters of Purkinje cells (PCs) are immunoreactive to cyclic GMP-dependent protein kinase (cGK). In contrast, at later stages and in the adult, all the PCs are immunoreactive. These results obtained with cGK suggest a transitory intrinsic heterogeneity in the immature cerebellar cortex. It seemed therefore interesting to investigate the distribution of other PC markers during early development in the rat and in other species. The results presented here were obtained with two other antibodies--against vitamin D-dependent calcium binding protein and against Purkinje cell specific glycoprotein--which, like cGK, label all adult PCs. Each antibody gave a different and reproducible mosaic of positive and negative clusters of PCs in the perinatal cerebellum, thus indicating a transient biochemical compartmentalization resulting from the differential expression of parts of the same genotype by clusters of PCs. This compartmentalization in concomitant with the ingrowing of the cerebellar afferents. Once synaptogenesis starts, the biochemical heterogeneity of PCs disappears.

Multiple Roles for Slits in the Control of Cell Migration in the Rostral Migratory Stream

The subventricular zone (SVZ) contains undifferentiated cells, which proliferate and generate olfactory bulb (OB) interneurons. Throughout life, these cells leave the SVZ and migrate along the rostral migratory stream (RMS) to the OB where they differentiate. In vitro, the septum and the choroid plexus (CP) secrete repulsive factors that could orient the migration of OB precursors. Slit1 and Slit2, two known chemorepellents for developing axons, can mimic this effect. We show here that the Slit receptors Robo2 and Robo3/Rig-1 are expressed in the SVZ and the RMS and that Slit1 and Slit2 are still present in the adult septum. Using Slit1/2-deficient mice, we found that Slit1 and Slit2 are responsible for both the septum and the CP repulsive activity in vitro. In adult mice lacking Slit1, small chains of SVZ-derived cells migrate caudally into the corpus callosum, supporting a role for Slits in orienting the migration of SVZ cells. Surprisingly, in adult mice, Slit1 was also expressed by type A and type C cells in the SVZ and RMS, suggesting that Slit1 could act cell autonomously. This hypothesis was tested using cultures of SVZ explants or isolated neurospheres from Slit1-/- or Slit1+/- mice. In both types of cultures, the migration of SVZ cells was altered in the absence of Slit1. This suggests that the regulation of the migration of OB precursors by Slit proteins is complex and not limited to repulsion.

Dopaminergic dendrites in the pars reticulata of the rat substantia nigra and their striatal input. combined immunocytochemical localization of tyrosine hydroxylase and anterograde degeneration

An antiserum prepared in the rabbit against bovine adrenal gland tyrosine hydroxylase has been used to identify by the immunoperoxidase method dopaminergic neurons in the rat substantia nigra. The purpose of this identification was (i) to assess the storing compartments and the release sites in the dopamine-containing processes of the pars reticulata; (ii) to determine if these processes receive a direct input from the neostriatum. Immunoreactive neurons were present in the three divisions of the substantia nigra (pars compacta, pars lateralis and pars reticulata), but they were much more numerous in pars compacta. The caudal half and the most rostral end of pars reticulata contained single and small clusters of reactive neurons, which were absent from the remaining regions. Processes emerging from the positive neurons, exhibiting also immunoperoxidase reactivity, spread throughout the whole pars reticulata. The ultrastructural study was limited to the region of the pars reticulata free of reactive perikarya, in order to analyze the processes that originate from neurons located in the pars compacta. Five hundred and eighty well-preserved immunoreactive processes were analyzed. Almost all of them (578) displayed cytological features allowing their identification as dendrites. Two of them corresponded to thin unmyelinated, non-synaptic segments of axons, probably in their way to their terminal fields outside the substantia nigra. The large majority of the reactive dendrites (82%) were postsynaptic to one or several axon terminals and did not establish direct appositions with other dendritic elements. Only 4.35% of the labeled dendrites were directly apposed to other reactive or unreactive dendrites. Two of the labeled dendrites (0.35%) contained synaptic-like vesicles. In one of them, the vesicles were clustered against a restricted area of the plasma-membrane, forming an active zone. In two animals, kainic acid was used to destroy neurons located within the central region of the main body of the neostriatum. Their projections were traced to the ipsilateral substantia nigra, in which dopaminergic neurons were visualized by the immunoperoxidase method. The axons originating from the injured neurons in the striatum established direct synaptic contacts with the immunoreactive dendrites in pars reticulata. These findings indicate that (i) there is no dopaminergic recurrent collateral axonal plexus in pars reticulata; (ii) the dopamine-storing compartment in the dendritic processes is not vesicular; the cisterns of the smooth endoplasmic reticulum might be such a compartment; (iii) The differentiation of presynaptic dendrites which establish typical junctional synaptic complexes does not occur in the dopaminergic dendrites present in pars reticulata; (iv) The proportion of presynaptic release sites observed in dopaminergic dendrites (1 active zone out of the 578 analyzed dendrites) is too low to account for the dendritic release revealed by biochemical analysis (Nieoullou, Cheramy & Glowinski, 1977a). Therefore, the modality of transmitter release from dopaminergic dendrites must be different from that supposed in the vesicular theory; (v) combined anterograde degeneration and immunocytochemistry has allowed us to demonstrate a direct striatal input to the dopamine-containing dendrites present within the pars reticulata.

Injection of 6-hydroxydopamine into the substantia nigra of the rat. II. Diffusion and specificity

Characteristics of the microinjection method described in the present work were analyzed by examining the pattern of the radioactivity distribution after local administration of [3H]DA in the substantia nigra (SN) of the rat. Small volumes of [3H]DA solutions not exceeding 2 μl gave reproducible results and are appropriate for pharmacological studies since most of the diffusion region was limited to the SN. The local distribution of [3H]DA in the SN, used as an index of 6-OH-DA diffusion, was larger than the extent of both specific and unspecific lesions induced by the drug as shown in a previous morphological study14. Small injected volumes of 6-OH-DA (< 4 μg in 2 μl) very likely allow a specific degeneration of catecholaminergic neurones in the SN as shown by the predominant elective neuronal damage observed in these conditions. Concentrations of 6-OH-DA above 4 μg (2 μl) induced important unspecific destruction of brain tissue, and some of the nervous parenchyma surrounding the SN was also destroyed. The estimation of DA in the neostriatum 10 days after the local administration of various doses of 6-OH-DA in the ipsilateral SN showed the existence of a good correlation between the DA decline and the extent of the lesion. It therefore appears possible to predict the quantity of 6-OH-DA required to induce a quantified destruction of the nigro-neostriatal pathway.

Purkinje Cell Ontogeny: Formation and Maintenance of Spines

Publisher Summary This chapter discusses the morphological analysis of microenvironmental factors influencing the molding of the Purkinje cell dendritic tree. Emphasis is given on the problem of the formation and maintenance of dendritic spines, because they are, by far, the most numerous receptor sites in these neurons. During normal development, Purkinje cell differentiation occurs in a concomitant manner with the development of its synaptic inputs. Climbing fibers reach Purkinje cells, establishing early axo-somatic synapses that are already functional postnatally by day three. This pericellular climbing plexus originates from 3–4 different climbing fibers. The further development of the Purkinje cell dendritic tree parallels the growth of climbing fibers and the maturation of their synapses. By day 12–15, the climbing fiber varicosities make synapses on thorns emerging from the thick Purkinje cell dendritic branches. At this stage, the immature multiple innervation is transformed into the definitive one-to-one relationship, which characterizes the climbing fiber-Purkinje cell synapses in adult animals. During the 2nd and 3rd postnatal weeks, migration of the bulk of granule cells takes place, giving rise to the millions of parallel fibers found in the molecular layer. The development of parallel fibers is accompanied by the formation of thin terminal branches of the Purkinje cell dendritic arborization—the spiny brundzlets—and by the achievement of the espalier arrangement of these dendritic trees that give the Purkinje cells their unique tridimensional shape.

Chemoattraction and Chemorepulsion of Olfactory Bulb Axons by Different Secreted Semaphorins

During development, growth cones can be guided at a distance by diffusible factors, which are attractants and/or repellents. The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic, motor, and forebrain axons. We found that in rat embryos, the olfactory epithelium releases a diffusible factor that repels olfactory bulb axons. In addition, Sema A and Sema IV, but not Sema III, Sema E, or Sema H, are able to orient in vitrothe growth of olfactory bulb axons; Sema IV has a strong repulsive action, whereas Sema A appears to attract those axons. The expression patterns of sema A and sema IV in the developing olfactory system confirm that they may play a cooperative role in the formation of the lateral olfactory tract. This also represents a further evidence for a chemoattractive function of secreted semaphorins.