GABAA receptor subunit composition and competition at synapses are tuned by GABAB receptor activity.

GABABRs have a well-established role in controlling neuronal excitability and presynaptic neurotransmitter release. We examined the role of GABABR activity in modulating the number and lateral diffusion of GABAARs at inhibitory synapses. Changes in diffusion of GABAARs at synapses were observed when subunit heterogeneity was taken into account. While α1-GABAARs were unaffected, α2- and α5-GABAARs showed inverse changes in enrichment and diffusion. The intracellular TM3-4 loop of α2 was sufficient to observe the changes in diffusion by GABABR activity, whereas the loop of α5 was not. The opposing effect on α2- and α5-GABAARs was caused by a competition between GABAARs for binding slots at synapses. Receptor immobilization by cross-linking revealed that α5-GABAAR trapping at synapses is regulated by modulation of α2-GABAAR mobility. Finally, PKC activity was determined to be part of the signaling pathway through which GABABR activity modulates α2-GABAAR diffusion at synapses. These results outline a novel mechanism for tuning inhibitory transmission in a subunit-specific manner, and for the first time describe competition between GABAARs with different subunit compositions for binding slots at synapses.

Wavelet analysis for single molecule localization microscopy.

Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses.

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.

Distribution of glycine receptors at central synapses: an immunoelectron microscopy study.

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.

Effect of serotonergic afferents on quantal release at central inhibitory synapses.

Although most examples of modulation of synaptic transmission have been obtained from excitatory rather than from inhibitory connections, serotonin (5HT) is now shown to cause a presynaptic facilitation of release of the inhibitory neurotransmitter glycine. Brief local injections of this amine, or application of a 5HT uptake blocker, produce a long-lasting enhancement of both spontaneous and evoked inhibitory currents in the teleost Mauthner cell. Quantal analysis showed that the probability of release is increased. Focal recording indicated that 5HT acts directly on the inhibitory terminals, possibly reducing potassium conductances. Double staining with specific antibodies demonstrated a morphological substrate for this effect. Nerve endings that contain 5HT contact inhibitory terminals directly apposed to postsynaptic glycine receptors.

Fluctuating responses at a central synapse: n of binomial fit predicts number of stained presynaptic boutons.

Binomial predictions provided a better description than the Poisson law of fluctuating unitary inhibitory postsynaptic potentials evoked in the goldfish Mauthner cell by impulses in presynaptic interneurons. The number of terminal boutons established on this target cell by each horseradish peroxidase-filled interneuron corresponded to the value of the binomial parameter n.

Fast and reversible trapping of surface glycine receptors by gephyrin.

Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

GABA(A) receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1.

Controlling the number of functional gamma-aminobutyric acid A (GABA(A)) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABA(A) receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABA(A) receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABA(A) receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABA(A) receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.

Alpha subunit-dependent glycine receptor clustering and regulation of synaptic receptor numbers.

Accumulation of glycine receptors at synapses requires the interaction between the beta subunit of the receptor and the scaffold protein gephyrin. Here, we questioned whether different alpha subunits could modulate the receptors' diffusion and propensity to cluster at spinal cord synapses. Using quantitative photoactivated localisation microscopy we found that alpha-1 and alpha-3 containing glycine receptors display the same α3:ß2 stoichiometry and gephyrin binding. Despite these similarities, alpha-3 containing receptors are less mobile and cluster at higher density compared to alpha-1, with 1500 versus 1100 complexes µm-2, respectively. Furthermore, we identified a subunit-specific regulation of glycine receptor copy numbers at synapses: when challenged with interleukin 1ß, the synaptic occupancy of alpha-1 but not alpha-3 receptors was reduced. This mechanism may play a role in the cell-type dependent regulation of glycinergic currents in response to interleukin 1ß and highlights the capacity of the alpha subunits to affect receptor-gephyrin binding at synapses.

IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells.

In the rat cerebellum, Golgi cells receive serotonin-evoked inputs from Lugaro cells (L-IPSCs), in addition to spontaneous inhibitory inputs (S-IPSCs). In the present study, we analyze the pharmacology of these IPSCs and show that S-IPSCs are purely GABAergic events occurring at basket and stellate cell synapses, whereas L-IPSCs are mediated by GABA and glycine. Corelease of the two transmitters at Lugaro cell synapses is suggested by the fact that both GABA(A) and glycine receptors open during individual L-IPSCs. Double immunocytochemical stainings demonstrate that GABAergic and glycinergic markers are coexpressed in Lugaro cell axonal varicosities, together with the mixed vesicular inhibitory amino acid transporter. Lugaro cell varicosities are found apposed to glycine receptor (GlyR) clusters that are localized on Golgi cell dendrites and participate in postsynaptic complexes containing GABA(A) receptors (GABA(A)Rs) and the anchoring protein gephyrin. GABA(A)R and GlyR/gephyrin appear to form segregated clusters within individual postsynaptic loci. Basket and stellate cell varicosities do not face GlyR clusters. For the first time the characteristics of GABA and glycine cotransmission are compared with those of GABAergic transmission at identified inhibitory synapses converging onto the same postsynaptic neuron. The ratio of the decay times of L-IPSCs and of S-IPSCs is a constant value among Golgi cells. This indicates that, despite a high cell-to-cell variability of the overall IPSC decay kinetics, postsynaptic Golgi cells coregulate the kinetics of their two main inhibitory inputs. The glycinergic component of L-IPSCs is responsible for their slower decay, suggesting that glycinergic transmission plays a role in tuning the IPSC kinetics in neuronal networks.

Dynamics of glycine receptor insertion in the neuronal plasma membrane.

The exocytosis site of newly synthesized glycine receptor was defined by means of a morphological assay to characterize its export from the trans-Golgi Network to the plasma membrane. This was achieved by expressing in transfected neurons an alpha1 subunit bearing an N-terminal tag selectively cleavable from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly occurred at nonsynaptic sites in the cell body and the initial portion of dendrites. At the time of cell surface insertion, the receptors existed as discrete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regions. This localization resulted from diffusion in the plasma membrane and not from exocytosis of transport vesicles directed to dendrites. Kinetic analysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent intermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses.

Synaptic control of glycine and GABA(A) receptors and gephyrin expression in cultured motoneurons.

We have evaluated the influence of the secretory phenotype of presynaptic boutons on the accumulation of postsynaptic glycine receptors (GlyRs), type A GABA receptors (GABA(A)Rs), and gephyrin clusters. The cellular distribution of these components was analyzed on motoneurons cultured either alone or with glycinergic and/or GABAergic neurons. In motoneurons cultured alone, we observed gephyrin clusters at nonsynaptic sites and in front of cholinergic boutons, whereas glycine and GABA(A) receptors formed nonsynaptic clusters. These receptors are functionally and pharmacologically similar to those found in cultures of all spinal neurons. Motoneurons receiving GABAergic innervation from dorsal root ganglia neurons displayed postsynaptic clusters of gephyrin and GABA(A)Rbeta but not of GlyRalpha/beta subunits. In motoneurons receiving glycinergic and GABAergic innervation from spinal interneurons, gephyrin, GlyRalpha/beta, and GABA(A)Rbeta formed mosaics at synaptic loci. These results indicate that (1) the transmitter phenotype of the presynaptic element determines the postsynaptic accumulation of specific receptors but not of gephyrin and (2) the postsynaptic accumulation of gephyrin alone cannot account for the formation of GlyR-rich microdomains.

Dopamine D3 receptors expressed by all mesencephalic dopamine neurons.

A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D(3) receptor (D(3)R). Using transfected cells expressing recombinant D(3)R, but not D(2) receptor, this antibody labeled 45-80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [(125)I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D(3)R binding, revealed using [(125)I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin ([(125)I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D(3)R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D(3)R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D(3)R binding in both AccSh and SN/VTA. D(3)R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D(3) autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D(3)R-deficient mice.

Biology of the postsynaptic glycine receptor.

Glycine is one of the major inhibitory neurotransmitters, and upon binding to its receptor it activates chloride conductances. Receptors are accumulated immediately opposite release sites, at the postsynaptic differentiations, where they form functional microdomains. This review describes recent advances in our understanding of the structure-function relationships of the glycine receptor, a member of the ligand-gated ion channel superfamily. Following purification of the receptor complex and identification of its integral and peripheral membrane protein components, molecular cloning has revealed the existence of several subtypes of the ligand-binding subunit. This heterogeneity is responsible for the distinct pharmacological and functional properties displayed by the various receptor configurations that are differentially expressed and assembled during development. This review also focuses on the molecular aspects of glycinergic synaptogenesis, highlighting gephyrin, the peripheral component of the receptor. The role of this cytoplasmic protein in anchoring and maintaining the channel complex in postsynaptic clusters is discussed. The glycine receptor recently moved into the spotlight as a paradigm in the approach to cell biology of the formation of the postsynaptic membrane.

Variability of axonal arborizations hides simple rules of construction: a topological study from HRP intracellular injections.

Intracellular staining with horseradish peroxidase (HRP) allows the analysis of the extent and diversity of axonal field, as the Golgi techniques did for dendritic fields. In this study, we have used such HRP injections to investigate possible rules of construction that underlie the variability in the observed morphological patterns of axons. The Mauthner cell of the teleost provides a suitable material for such a work since it receives inhibitory inputs from two distinct classes of cells that can be identified physiologically prior to their intracellular staining: those that contribute to a recurrent collateral network and those that are part of the commissural vestibulovestibular pathway. The distribution of their terminal boutons over the M-cell surface was quantified and their axonal arborizations were analyzed topologically with the help of a centripetal method of terminal ordering. The results indicated that both types of interneurons have qualitatively a similar distribution of boutons over this target, encompassing the soma, initial portions of the main dendrites, and the cap-dendrites, and that furthermore, the terminals have a tendency to form clusters, the proportion of which is relatively constant regardless of the total number of boutons established by a given afferent cell. In respect to their projection on the M-cell, the two populations differ mainly in the number of established contacts, which averaged 44.1 +/- 28.2 (n = 10) and 14.4 +/- 11.3 (n = 43) for collateral and commissural interneurons, respectively. These differences reflect the variations in axonal arborizations. A topological analysis has revealed that branching occurs mainly as bifurcations, whereas in contrast three or four segments issuing from a branch point are only occasionally observed at the level of the last segment; for each cell, the distribution of boutons within orders is not random and rather follows an unimodal distribution, centered on the mean order (theta); the terminals of each subset, or class, of neurons have their own pattern of distribution within orders; and finally the mean order (theta) is linked to the number of terminal segments (nT) by the relation theta = 1.28 log2 nT. This parameter is of importance: it characterizes the axonal arborization and it allows one to predict the number of terminal segments of individual neurons. Similar analysis of camera lucida reconstruction of axonal arborizations of various cell types studied by other investigators suggests that this relation can be generalized.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneous distribution of glycinergic and GABAergic afferents on an identified central neuron.

Immunocytochemical methods were used on serial sections to study the glycine- and gamma-amino butyric acid (GABA)ergic innervations of the teleost Mauthner (M) cell. We found different distributions for the boutons containing the two amino acids. Endings filled with GABA predominate on the distal portion of the lateral dendrite (LD) while glycine-positive profiles are more abundant on the soma and within the axon cap (AC), a specialized neuropil surrounding the M-cell initial segment. A few endings containing both transmitters are present on the soma and on the small dendrites issuing ventrally from it. At this level some glutamic acid decarboxylase (GAD)-containing boutons face glycine receptor-93 kD-associated protein, an observation suggesting that the associated glycine functions as a neurotransmitter. Elsewhere on the M-cell, where glycine and GABA are not colocalized, GAD-positive profiles were never observed in front of postsynaptic differentiations with 93 kD labelling. GABA was detected in the small vesicle boutons (SVBs), most of them, following the classification of Tuttle et al., J. Comp. Neurol. 265:254-274, 1987, belonging to the A-type, while glycine was found in the unmyelinated club endings in the AC, and in C- and B-type SVBs, outside this region. All terminals established symmetrical synapses and were filled with a population of pleiomorphic vesicles. Boutons with GABA also contained numerous dense-core vesicles suggesting the presence of an associated peptide(s). A quantitative study of the transmitter content based on the number of the gold particles revealed a variable intensity of the labelling over certain profiles. For GABA, it was maximum at the tip of the LD and it decreased proximally. In contrast, the staining density was constant for glycine along all parts of the cell, except for the ventral dendrite (VD) where it decreased progressively. Taken together, these data suggest that the amino acid content varies, depending upon the location of the synapses on their target neuron.

Morphology of the release site of inhibitory synapses on the soma and dendrite of an identified neuron.

Synapses are complex arrangements of pre- and postsynaptic differentiations involved in neural communication. A key element in this synaptic transmission is the presynaptic active zone where the release of neurotransmitter occurs. Active zones can be visualized and analyzed after staining with ethanolic phosphotungstic acid (EPTA) on semithin (0.5 micron) sections. This staining has been used in association with postembedding immunogold labeling for the neurotransmitters glycine or GABA, to investigate the organization of chemically defined inhibitory active zones, viewed in their full extent, on different regions of the goldfish Mauthner (M-) cell. With this approach, a marked variability in size and shape was observed for the release sites contacting the different parts of the postsynaptic neuron. In the axon cap and on the soma, glycinergic afferent terminals have small presynaptic grids (0.066 +/- 0.029 micron2, n = 30 and 0.076 +/- 0.037 micron2, n = 46, respectively). These grids are quite circular and they include 12 to 13 presynaptic dense projections (PDPs). The situation is different on the lateral dendrite, where glycinergic and GABAergic active zones display a greater variability in their surface areas (mean = 0.147 +/- 0.100 micron2, n = 115 and 0.139 +/- 0.080 micron2, n = 125, respectively), and their number of PDPs (mean = 19 +/- 9) per individual grid. Similarly, the shape of the release sites over the dendrite is more complex (annular, horseshoe-shaped) when compared to those on the soma. These differences of dendritic versus somatic release sites could represent a structural basis to maximize the shunting effect of glycinergic and GABAergic inhibitory junctions, i.e., close to excitatory inputs. We also observed that the proportion of endings containing 1 or more active zones also varies. More precisely, 96% and 82% of glycinergic terminals in the axon cap and on the soma, respectively, display only one active zone. On the dendrite, their proportion falls to 65.5% for both glycine- and GABA-containing boutons. The remaining inhibitory terminals contain 2 (30%) and 3 to 4 (4.5%) presynaptic grids. These results reveal a greater variability of morphology and organization of the inhibitory release sites at dendritic versus somatic locations. The functional significance of this observation for the synaptic transmission is discussed.

Gephyrin accumulates at specific plasmalemma loci during neuronal maturation in vitro.

The distribution of glycine receptor (GlyR)-associated gephyrin has been investigated in rat spinal cord neurons maintained in vitro by means of immunocytochemical techniques. Gephyrin, which is crucial for the stabilization of postsynaptic GlyR microdomains, is present in mature neurons at postsynaptic differentiations. With immunofluorescence, discontinuous patches of gephyrin were detected within the neuronal soma of spinal cord neurons on the 1st day after plating. Subsequently, gephyrin was present at membrane areas that correspond to points of contact between cells or with the culture dish. By the 5th day, gephrin was mostly associated with the MAP2-positive somatodendritic compartment. With immunoelectron microscopy, gephyrin blobs detected at the earliest stages (1-3 days after plating) were found within the cytoplasm or associated with the plasma membrane. Asymmetrically immunostained intercellular contacts were only detected after 5 days, and gephyrin was found in association with clearly differentiated postsynaptic membranes at 7 days. At later stages, we observed gephyrin immunoreactivity only at some synapses. Our results suggest that gephyrin accumulates initially at the locus of cell-to-cell contacts involved in adhesion processes. These localizations may define hot spots for later accumulation of the GlyR and possibly other receptors.

Postnatal development of the inferior olivary complex in the rat. II. Topographic organization of the immature olivocerebellar projection.

The state of organization of the olivocerebellar projection in newborn and 5-day-old rats has been analyzed by autoradiography of anterogradely transported 3H-leucine, as well as by retrograde transport of horseradish peroxidase. The efferent axons of the inferior olivary neurons are already present and already highly organized in the cerebellum of newborn rats. Most of the autoradiographic labelling subsequent to the injection of 3H-leucine into the inferior olive is seen in the subcortical medullary zone. Labelled axons only partially invade the gray matter, where they reach the zone occupied by randomly distributed Purkinje cells. At this immature stage, olivocerebellar projections are already entirely crossed and distributed according to a pattern which is similar to the adult. At the fifth postnatal day olivocerebellar projections have moved from the medullary zone toward the interface between the molecular and the granular layers where Purkinje cells have arranged in a monolayer. Evidence for translocation of climbing fibers from their perisomatic to their peridendritic position is already distinct in these young cerebella. Combination of anterograde and retrograde fiber system tracing experiments discloses the following crossed topography of olivocerebellar projections: The caudal half of the medial accessory olive projects mainly to the vermis of the posterior lobe, whereas its rostral half projects to the flocculus, paraflocculus, and the intermediate cortex. The principal olive, ventral and dorsal lamellae, supplies climbing fiber inputs to the hemispheric cortex. The caudal half of the dorsal accessory olive projects to the lateral portion of the vermis of the anterior lobe, whereas neurons in its rostral half send their axons toward the intermediate cortex. This topographic arrangement is, therefore, similar to that reported for adult mammals. The present results, alone or when compared with those obtained during other studies on the synaptogenesis between climbing fibers and Purkinje cells, allow the following conclusions: The climbing fibers enter the cerebellar cortex before Purkinje cells have reached the developmental phase compatible with synaptogenesis. They wait in the medullary white matter until appropriate maturation of their cellular targets. Olivocerebellar topography is roughly similar in newborn, 5-day -old, and adult rats. Synaptogenesis between climbing fibers and Purkinje cells, which is known not to start before the second postnatal day, is not necessary for the establishment of the topographic organization of the olivocerebellar projection.

Differential distribution of serotoninergic inputs on the goldfish Mauthner cell.

The morphology and distribution of the serotoninergic (5-HT) input to the Mauthner cell (M cell) of a teleost, Carassius auratus, were analyzed at the light microscopic level. Immunohistochemical methods revealed that 1) most fibers innervating the M cell originate from the ventral and lateroventral regions of the rhombencephalon; 2) two groups of fibers contribute to this innervation, thick ones (type I, 0.4-0.7 microns in diameter) with terminal endings and thin ones (type II, less than 0.2 microns) that issue numerous beaded varicosities 4-10 microns from the target cell and only occasional side endings contacting it; 3) the density of immunoreactive profiles is uneven over the whole cell and predominates on the ventral dendrite; and 4) the two sets of axons, although overlapping, do not have the same distribution. Specifically, both classes are present on the ventral dendrite, whereas type II fibers are the only ones observed on the soma, in the region of the initial segment of the axon, and in the vicinity of the lateral dendrite. Functionally identified inputs on the M cell also have a regionalized distribution, depending, for example, on whether they belong to excitatory or inhibitory networks. Thus we propose that 5-HT inputs have specific influences that are a function of their respective localization.