Dianne E. Dewey

Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons. The spatial organization of postsynaptic receptors is likely to determine many functional parameters at these synapses and is probably related to the integrative capabilities of different neurons. In the present study, we have investigated the organization of gephyrin expression along the dendritic membranes of α‐ and γ‐motoneurons, Ia inhibitory interneurons, and Renshaw cells. Gephyrin is a protein responsible for the postsynaptic clustering of glycine receptors, and the features of gephyrin and glycine receptor α1‐subunit immunofluorescent clusters displayed similar characteristics on ventral horn spinal neurons. However, the density of clusters and their topographical organization and architecture varied widely in different neurons and in different dendritic regions. For motoneurons and Ia inhibitory interneurons, cluster size and complexity increased with distance from the soma, perhaps as a mechanism to enhance the influence of distal synapses. Renshaw cells were special in that they displayed an abundant complement of large and morphologically complex clusters concentrated in their somas and proximal dendrites. Serial electron microscopy confirmed that the various immunoreactivity patterns observed with immunofluorescence accurately parallel the variable organization of pre‐ and postsynaptic active zones of glycinergic synapses. Finally, synaptic boutons from single‐labeled axons of glycinergic neurons (Ia inhibitory interneurons) were also associated with postsynaptic receptor clusters of variable shapes and configurations. Our results indicate that mechanisms regulating receptor clustering do so primarily in the context of the postsynaptic neuron identity and localization in the dendritic arbor. J. Comp. Neurol. 379:150‐170, 1997.

Distribution of 5-Hydroxytryptamine-Immunoreactive Boutons on Alpha-Motoneurons in the Lumbar Spinal Cord of Adult Cats

Recent studies have shown that at least some of the functional effects of serotonin (5‐HT) on motoneuron excitability are direct and are mediated via postsynaptic 5‐HT receptors on motoneurons. To determine the spatial distribution of direct inputs from the serotonin system on the proximal and distal dendrites of individual motoneurons, we examined identified motoneurons in vivo with a combination of immunohistochemical localization of 5‐HT‐immunoreactive boutons and intracellular staining with horseradish peroxidase.

Distribution of Cholinergic Contacts on Renshaw Cells in the Rat Spinal Cord: A Light Microscopic Study

1 Cholinergic terminals in the rat spinal cord were revealed by immunohistochemical detection of the vesicular acetycholine transporter (VAChT). In order to determine the relationships of these terminals to Renshaw cells, we used dual immunolabelling with antibodies against gephyrin or calbindin D28k to provide immunohistochemical identification of Renshaw cells in lamina VII of the ventral horn. 2 A total of 50 Renshaw cells were analysed quantitatively using a computer‐aided reconstruction system to provide accurate localization of contact sites and determination of somatic and dendritic surface area. Dendrites could be traced for up to 413 μm from the soma in calbindin D28k‐identified Renshaw cells and up to 184 μm in gephyrin‐identified cells. 3 A total of 3330 cholinergic terminals were observed on 50 Renshaw cells, with a range of 21–138 terminal appositions per cell (mean 66.6 ± 25.56 contacts per cell). The vast majority (83.5%) of the terminals were apposed to dendrites rather than the soma. The overall density of cholinergic contacts increased from a little above 1 per 100 μm2 on the soma and initial 25 μm of proximal dendrites to 4–5 per 100 μm2 on the surface of dendritic segments located 50–250 μm from the soma. Single presynaptic fibres frequently formed multiple contacts with the soma and/or dendrites of individual Renshaw cells. 4 VAChT‐immunoreactive terminals apposed to Renshaw cells varied in size from 0.6 to 6.9 μm in diameter (mean 2.26 ± 0.94; n= 986) and were on average smaller than the cholinergic C‐terminals apposed to motoneurones, but larger than VAChT‐immunoreactive terminals contacting other ventral horn interneurones. 5 The high density and relatively large size of many cholinergic terminals on Renshaw cells presumably correlates with the strong synaptic connection between motoneurones and Renshaw cells. The fact that the majority of contacts are distributed over the dendrites makes the motoneurone axon collateral input susceptible to inhibition by the prominent glycinergic inhibitory synapses located on the soma and proximal dendrites. The relative positions and structural features of the excitatory cholinergic and inhibitory glycinergic synapses may explain why Renshaw cells, although capable of firing at very high frequency following motor axon stimulation, appear to fire at relatively low rates during locomotor activity.

Downregulation of metabotropic glutamate receptor 1a in motoneurons after axotomy.

AXOTOMIZED motoneurons display drastic modifications in synaptic structure and function related to their disconnection from the periphery and establishment of a regenerative metabolic functional mode. The molecular basis of these modifications is not fully understood. Here we describe changes in metabotropic glutamate receptor 1a (mGluR1a)-immunoreactivity 3, 7 or 14 days after unilateral sciatic transection. mGluR1a-immunoreactivity was distributed throughout the somatic cytoplasm and somatodendritic membrane of uninjured motoneurons and was significantly reduced in axotomized motoneurons. This reduction was observed at 3 days and grew progressively over 2 weeks. These findings suggest that downregulation of mGluR1a could contribute to reduced excitatory neurotransmission in axotomized motoneurons.

Factors regulating AMPA‐type glutamate receptor subunit changes induced by sciatic nerve injury in rats

Excitatory glutamatergic neurotransmission at Ia afferent‐motoneuron synapses is enhanced shortly after physically severing or blocking impulse propagation of the afferent and/or motoneuron axons. We considered the possibility that these synaptic changes occur because of alterations in the number or properties of motoneuron α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptors. Therefore, we quantitatively analyzed glutamate receptor (GluR)1, GluR2/3, and GluR4 AMPA subunit immunoreactivity (ir) in motoneurons 3, 7, or 14 days after axotomy or continuous tetrodotoxin (TTX) block of the sciatic nerve. GluR1‐ir remained low in experimental and control motoneurons with either treatment and at any date. However, there was a large reduction of GluR2/3‐ir (peak at 7 days >60% reduced) and a smaller, but statistically significant, reduction of GluR4‐ir (around 10% reduction at days 3, 7, and 14) in axotomized motoneurons. TTX sciatic blockade did not affect AMPA subunit immunostainings. Axonal injury or interruption of the trophic interaction between muscle and spinal cord, but not activity disruption, appears therefore more likely responsible for altering AMPA subunit immunoreactivity in motoneurons. These findings also suggest that synaptic plasticity induced by axotomy or TTX block, although similar in the first week, could be related to different mechanisms. The effects of axotomy or TTX block on motoneuron expression of the metabotropic glutamate receptor mGluR1a were also studied. mGluR1a‐ir was also strongly decreased after axotomy but not after TTX treatment. The time course of the known stripping of synapses from the cell somas of axotomized motoneurons was studied by using synaptophysin antibodies and compared with AMPA and mGluR1a receptor changes. Coverage by synaptophysin‐ir boutons was only clearly decreased 14 days post axotomy and not at shorter intervals or after TTX block. J. Comp. Neurol. 426:229–242, 2000.

EXPRESSION OF GLYCINE RECEPTORS BY IDENTIFIED ALPHA AND GAMMA MOTONEURONES

The neurotransmitter role of glycine is well established and has recently been reviewed by Aprison (1990). Glycine is a major inhibitory neurotransmitter in the mammalian spinal cord, and it exerts its postsynaptic effects via increases in the chloride permeability of the postsynaptic membrane. Glycine receptors, like GABAA receptors, are multimeric ligand-gated chloride channels whose structural and functional properties have been elucidated in great detail in recent years (Betz, 1991). Glycine receptor subunit mRNAs are expressed in widespread areas of the central nervous system (CNS; e.g. Malosio, Marqueze, Kuhse & Betz, 1991; Kirsch, Malosio, Wolters & Betz, 1993a). Advances in understanding of the molecular composition of glycine receptors have also led to the use of monoclonal antibodies against subunits of the glycine receptor complex (Pfeiffer, Simler, Grenningloh & Betz, 1984) to study the general localisation of glycine receptors in the CNS (e.g. Triller, Cluzead, Pfeiffer, Betz & Korn, 1985; Triller, Cluzead & Korn 1987; van den Pol & Gorcs, 1988). One of the antibodies used for such immuno-localisation studies is directed against gephyrin, a 93 kDa glycine receptor-associated, tubulin binding protein which is required for glycine receptor clustering and localisation at synaptic specialisations (Kirsch, Wolters, Triller & Betz, 1993b). In the mammalian ventral horn, gephyrin immunoreactivity has been shown to be localised to the sub-membrane cytoplasm, coincident with immunoreactivity against ligand binding subunits of the glycine receptor, and, importantly, is localised at sites corresponding to synaptic specialisations (see Triller et al., 1985 and unpublished observations from our laboratory). These properties make gephyrin a particularly useful marker for use in probing the organisation and distribution of glycine receptors in identified central neurones, as we will describe in this presentation.

Control of HCO 3 -Dependent Exchangers by Cyclic Nucleotides in Vascular Smooth Muscle Cells

A variety of membrane transport systems responsible for the regulation of intracellular pH (pHi) have been identified in smooth muscle (Aickin, 1986; Aalkjaer and Cragoe, 1988; Wray, 1988; Kikeri et al., 1990) and smooth musclelike cells (Boyarsky et al.,1988a,b; Putnam,1990). These include the ubiquitous Na/H exchanger and at least two HC03-dependent transport systems (Fig. 1): i) a putative alkalinizing (Na + HCO3)/C1 exchanger (although the role of Cl in this exchanger is still at issue) (Aickin and Brading, 1984; Aalkjaer and Mulvany, 1988); and ii) an acidifying Cl/HCO3 exchanger. While these exchangers are important for determining steady state pHi (Aalkjaer and Cragoe, 1988; Boyarsky et al., 1988a; Wray, 1988; Kikeri et al., 1990; Putnam and Grubbs, 1990), defending pHi against acid/base disturbances (Aalkjaer and Cragoe, 1988; Boyarsky et al., 1988b; Putnam, 1990) and mediating cellular responses to external signals (Berk et al., 1987; Ganz et al., 1989), only the Na/H exchanger has been extensively studied in regard to the factors which regulate its activity. In fact, the regulation of the HCO3-dependent transport systems is poorly studied in any cell.