The aim of the present study was to identify the central structures involved in the organization of the swallowing reflex in the rat. Using concentric bipolar electrodes, the medulla and pons were systematically explored in order to determine which central areas responded to stimulation by inducing swallowing. These areas, which were located in the dorsal medulla oblongata, were the solitary tract, the nucleus of the solitary tract (NST) and the adjacent reticular formation. Stimulation of the ventral ponto-medullary regions was ineffective with regard to the initiation of the swallowing reflex. The activity of medullary swallowing neurons was recorded using extracellular microelectrodes. These swallowing neurons responded with a burst of spikes (swallowing activity) which was closely linked to the swallowing reflex elicited by stimulation of the superior laryngeal nerve (SLN). Under SLN stimulation, the activity of some of the swallowing neurons furthermore showed an initial response consisting of 1 or 2 spikes after a brief latency. According to their location and the latency of their initial response, swallowing neurons were divided into two groups. Group I neurons were located in a dorsal area of the medulla oblongata corresponding to the NST and the adjacent reticular formation. All these neurons exhibited an initial response with a very short latency (1 to 4 ms), the swallowing activity of most of these neurons started before the onset of the swallowing motor sequence. Group II neurons were located either in a ventral area corresponding to the nucleus ambiguus and the surrounding reticular formation or in a dorsal and medial area corresponding to the hypoglossal nucleus and its vicinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium influxes through ionotropic glutamate receptors (AMPA and NMDA receptors, AMPARs and NMDARs) are considered to be critical for the shaping and refinement of neural circuits during synaptogenesis. Using a combined morphological and electrophysiological approach, we evaluated this hypothesis at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. We confirmed that in the NTS, the first excitatory synapses appeared at embryonic day 18. We next characterized the biophysical properties of NTS AMPARs. Throughout perinatal development, both evoked and miniature EPSCs recorded in the presence of an NMDAR blocker were insensitive to polyamines and had linear current-voltage relationships. This demonstrated that AMPARs at NTS excitatory synapses were calcium-impermeable receptors composed of a majority of GluR 2 subunits. We then investigated the influence of calcium influxes through NMDARs on the development of NTS synaptic transmission. We found that NMDAR expression at synaptic sites did not precede AMPAR expression. Moreover, NMDAR blockade in utero did not prevent the development of AMPAR synaptic currents and the synaptic clustering of GluR 2 subunits. Thus, our data support an alternative model of synaptogenesis that does not depend on calcium influxes through either AMPARs or NMDARs. This model may be particularly relevant to the formation of neural networks devoted to basic behaviours required at birth for survival.

Astrocytes are now considered as essential partners of neurons. In particular, they play important roles in glutamatergic transmission, including transmitter inactivation by uptake. Here, we investigated the organization of astroglia in the Nucleus Tractus Solitarii (NTS), a sensory nucleus located in the caudal medulla. Special attention was given to perisynaptic astroglial processes. Investigations were performed at the light and electron microscope levels, using immunodetection of glial glutamate transporters, stereological methods, and serial reconstruction. In the NTS, the main glutamate transporter expressed by astrocytes was GLT1. The volume fraction of astrocyte processes and the density of astrocyte membranes reached 15% and 2.8 μm(2) μm(-3) , respectively. In spite of the relative abundance of astrocyte processes, we found that NTS glutamatergic synapses were not entirely surrounded by glia. Measurements were performed on 43 reconstructed asymmetric junctions which were either single synapses (n = 22) or synapses involved in multisynaptic arrangements (n = 21). Single synapses had 58% of their perimeter contacted by astrocyte processes on average. In multisynaptic arrangement, glial coverage was restricted to the outer part of synaptic diameters and amounted to 50% of this outer part on average. Incomplete glial coverage of NTS synapses may allow glutamate to diffuse out of the synaptic cleft and to activate extrasynaptic receptors as well as receptors from neighboring synapses. Especially, in multisynaptic arrangements, the lack of intervening glia may favor functional coupling between individual contacts.

Using combined morphological and electrophysiological approaches, we have determined the composition of inhibitory synapses of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. Immunohistochemical experiments demonstrate that, in adult rat, GABA axon terminals are present throughout the NTS while mixed GABA-glycine axon terminals are strictly located to the lateral part of the NTS within subnuclei surrounding the tractus solitarius. Purely glycine axon terminals are rare in the lateral part of the NTS and hardly detected in its medial part. Electrophysiological experiments confirm the predominance of GABA inhibition throughout the NTS and demonstrate the existence of a dual inhibition involving the co-release of GABA and glycine restricted to the lateral part of NTS. Since GABA A and glycine receptors are co-expressed postsynaptically in virtually all the inhibitory axon terminals throughout the NTS, it suggests that the inhibition phenotype relies on the characteristics of the axon terminals. Our results also demonstrate that glycine is mostly associated with GABA within axon terminals and raise the possibility of a dynamic regulation of GABA/glycine release at the presynaptic level. Our data provide new information for understanding the mechanisms involved in the processing of visceral information by the central nervous system in adult animals.

Striatin, SG2NA and zinedin, the three mammalian members of the striatin family are multimodular WD-repeat, calmodulin and calveolin-binding proteins. These scaffolding proteins, involved in both signaling and trafficking, are highly expressed in neurons. Using ultrastructural immunolabeling, we showed that, in Purkinje cells and hippocampal neurons, SG2NA is confined to the somatodendritic compartment with the highest density in dendritic spines. In cultured hippocampal neurons, SG2NA is also highly concentrated in dendritic spines. By expressing truncated forms of HA-tagged SG2NAb, we demonstrated that the coiled-coil domain plays an essential role in the targeting of SG2NA within spines. Furthermore, co-immunoprecipitation experiments indicate that this coiled-coil domain is also crucial for the homo-and hetero-oligomerization of these proteins. Thus, oligomerization of the striatin family proteins is probably an obligatory step for their routing to the dendritic spines, and hetero-oligomerization explains why all these proteins are often co-expressed in the neurons of the rat brain and spinal cord. In the central nervous system, most excitatory terminals contact dendritic actin-rich protrusions called dendritic spines. The formation and maintenance of spines, including the postsynaptic components, requires precise targeting and coordinated activation of structural and signaling molecules (1-3). The mechanisms by which scaffolding proteins are sorted into pre-or postsynaptic compartments remain largely unclear. Multi-protein complexes, vesicles and organelles found at axon terminals and dendritic spines are transported by motor proteins along microtubules (4,5). Numerous proteins located in the postsynaptic density (PSD) undergo vesicular transport to dendritic spines in association with neurotransmitter receptors (6). Although the routing motifs of several trans-membrane proteins such as ionotropic glutamate receptors have been identified (7,8), much less is known about the targeting of soluble cytoplasmic proteins to dendritic spines, especially for proteins that do not belong to the PSD. To investigate this problem, we analyzed the routing mechanisms of cytoplasmic, non-PSD-associated, dendritic spine proteins: the striatin family.In mammals, this family consists of three scaffolding proteins composed of striatin, SG2NA and zinedin. They are mainly expressed in the cytoplasm of neurons of both the central and the peripheral nervous system (9-11). Ultrastructural studies show that striatin is strictly localized to the somatodendritic compartment of neurons and highly concentrated within the spines of the striatal GABAergic neurons (12). Information concerning the physiological role of the striatin family is beginning to emerge. First, we have shown that striatin expression is crucial for both dendritic growth in cultured rat embryonic motoneurons and for the control of motor function in adult rats (13). Second, striatin and SG2NA have been proposed to be regulatory subunits of protein phosphatase 2A (14). I...

NMDA-only synapses, called silent synapses, are thought to be the initial step in synapse formation in several systems. However, the underlying mechanism and the role in circuit construction are still a matter of dispute. Using combined morphological and electrophysiological approaches, we searched for silent synapses at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibers. Silent synapses were detected at birth by using electrophysiological recordings and minimal stimulation protocols. However, anatomical experiments indicated that nearly all, if not all, NTS synapses had AMPA receptors. Based on EPSC fluctuation measurements and differential blockade by low-affinity competitive and noncompetitive glutamate antagonists, we then demonstrated that NTS silent synapses were better explained by glutamate spillover from neighboring fibers and/or slow dynamic of fusion pore opening. Glutamate spillover at immature NTS synapses may favor crosstalk between active synapses during development when glutamate transporters are weakly expressed and contribute to synaptic processing as well as autonomic circuit formation.

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