Robert E.W. Fyffe

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.

The Continuing Case for the Renshaw Cell

Renshaw cell properties have been studied extensively for over 50 years, making them a uniquely well‐defined class of spinal interneuron. Recent work has revealed novel ways to identify Renshaw cells in situ and this in turn has promoted a range of studies that have determined their ontogeny and organization of synaptic inputs in unprecedented detail. In this review we illustrate how mature Renshaw cell properties and connectivity arise through a combination of activity‐dependent and genetically specified mechanisms. These new insights should aid the development of experimental strategies to manipulate Renshaw cells in spinal circuits and clarify their role in modulating motor output.

Diversity of structure and function at mammalian central synapses

Our appreciation of the relationship between synaptic structure and function, and in particular our understanding of quantal synaptic transmission, is derived from classical studies on the neuromuscular junction. However, physiological studies of quantal transmission at mammalian CNS synapses have produced a variety of results, and thus no consensus of opinion has emerged. This variability could be due, in part, to experimental and analytical limitations or to differences in the structural and functional features of central synapses, or both. Some of the experimental limitations have recently been overcome by the use of novel preparations that permit direct measurement of quantal synaptic events in the CNS. Although these studies reveal similarities between the synaptic mechanisms of the neuromuscular junction and CNS synapses, important differences and specializations are also evident. The purpose of this review is to highlight the structural and functional diversity of synapses in the mammalian CNS, and to discuss the potential relevance of structural features to synaptic function.

Focal aggregation of voltage-gated, Kv2.1 subunit-containing, potassium channels at synaptic sites in rat spinal motoneurones

Delayed rectifier K+ currents are involved in the control of α‐motoneurone excitability, but the precise spatial distribution and organization of the membrane ion channels that contribute to these currents have not been defined. Voltage‐activated Kv2.1 channels have properties commensurate with a contribution to delayed rectifier currents and are expressed in neurones throughout the mammalian central nervous system. A specific antibody against Kv2.1 channel subunits was used to determine the surface distribution and clustering of Kv2.1 subunit‐containing channels in the cell membrane of α‐motoneurones and other spinal cord neurones. In α‐motoneurones, Kv2.1 immunoreactivity (‐IR) was abundant in the surface membrane of the soma and large proximal dendrites, and was present also in smaller diameter distal dendrites. Plasma membrane‐associated Kv2.1‐IR in α‐motoneurones was distributed in a mosaic of small irregularly shaped, and large disc‐like, clusters. However, only small to medium clusters of Kv2.1‐IR were observed in spinal interneurones and projection neurones, and some interneurones, including Renshaw cells, lacked demonstrable Kv2.1‐IR. In α‐motoneurones, dual immunostaining procedures revealed that the prominent disc‐like domains of Kv2.1‐IR are invariably apposed to presynaptic cholinergic C‐terminals. Further, Kv2.1‐IR colocalizes with immunoreactivity against postsynaptic muscarinic (m2) receptors at these locations. Ultrastructural examination confirmed the postsynaptic localization of Kv2.1‐IR at C‐terminal synapses, and revealed clusters of Kv2.1‐IR at a majority of S‐type, presumed excitatory, synapses. Kv2.1‐IR in α‐motoneurones is not directly associated with presumed inhibitory (F‐type) synapses, nor is it present in presynaptic structures apposed to the motoneurone. Occasionally, small patches of extrasynaptic Kv2.1‐IR labelling were observed in surface membrane apposed by glial processes. Voltage‐gated potassium channels responsible for the delayed rectifier current, including Kv2.1, are usually assigned roles in the repolarization of the action potential. However, the strategic localization of Kv2.1 subunit‐containing channels at specific postsynaptic sites suggests that this family of voltage‐activated K+ channels may have additional roles and/or regulatory components.

Topographic Organization in the Auditory Brainstem of Juvenile Mice is Disrupted in Congenital Deafness

There is an orderly topographic arrangement of neurones within auditory brainstem nuclei based on sound frequency. Previous immunolabelling studies in the medial nucleus of the trapezoid body (MNTB) have suggested that there may be gradients of voltage‐gated currents underlying this tonotopic arrangement. Here, our electrophysiological and immunolabelling results demonstrate that underlying the tonotopic organization of the MNTB is a combination of medio‐lateral gradients of low‐and high‐threshold potassium currents and hyperpolarization‐activated cation currents. Our results also show that the intrinsic membrane properties of MNTB neurones produce a topographic gradient of time delays, which may be relevant to sound localization, following previous demonstrations of the importance of the timing of inhibitory input from the MNTB to the medial superior olive (MSO). Most importantly, we demonstrate that, in the MNTB of congenitally deaf mice, which exhibit no spontaneous auditory nerve activity, the normal tonotopic gradients of neuronal properties are absent. Our results suggest an underlying mechanism for the observed topographic gradient of neuronal firing properties in the MNTB, show that an intrinsic neuronal mechanism is responsible for generating a topographic gradient of time‐delays, and provide direct evidence that these gradients rely on spontaneous auditory nerve activity during development.

Calbindin D28k Expression in Immunohistochemically Identified Renshaw Cells

Double immunofluorescence was utilized to determine whether Renshaw cells contain calbindin D28k immunoreactivity. Renshaw cells were identified by their characteristic expression patterns of gephyrin immunoreactivity in sections of rat and cat lumbar spinal cord. In the rat, all neurons classified as Renshaw cells (n = 487) also contained calbindin D28k-immunoreactivity, and all calbindin D28k-immunoreactive cells located in the ventral-most region of lamina VII expressed the characteristic gephyrin labeling and morphology of Renshaw cells. In the cat, fewer than half of the Renshaw cells (47%; n = 128) were double-labeled. In both species, occasional calbindin D28k-immunoreactive Renshaw cells were identified within motor nuclei in lamina IX. The distinctive immunolabeling of Renshaw cells allowed us to estimate that there are about 250 Renshaw cells in each ventral horn of the fourth lumbar segment of rat spinal cord, and about 750 cells per ventral horn in the L6 segment of the cat. We conclude that the functional properties of Renshaw cells, including their ability to fire action potentials at high rates, likely require specific homeostatic mechanisms including strong intracellular calcium buffering, the precise mechanisms of which may vary between species.

Stathmin-deficient mice develop an age-dependent axonopathy of the central and peripheral nervous systems.

Stathmin is a cytosolic protein that binds tubulin and destabilizes cellular microtubules, an activity regulated by phosphorylation. Despite its abundant expression in the developing mammalian nervous system and despite its high degree of evolutionary conservation, stathmin-deficient mice do not exhibit a developmental phenotype.(1) Here we report that aging stathmin(-/-) mice develop an axonopathy of the central and peripheral nervous systems. The pathological hallmark of the early axonal lesions was a highly irregular axoplasm predominantly affecting large, heavily myelinated axons in motor tracts. As the lesions progressed, degeneration of axons, dysmyelination, and an unusual glial reaction were observed. At the functional level, electrophysiology recordings demonstrated a significant reduction of motor nerve conduction velocity in stathmin(-/-) mice. At the molecular level, increased gene expression of SCG 10-like protein, a stathmin-related gene with microtubule destabilizing activity, was detected in the central nervous system of aging stathmin(-/-) mice. Together, these findings suggest that stathmin plays an essential role in the maintenance of axonal integrity.

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.

The Cav2.1/alpha1A (P/Q-type) voltage-dependent calcium channel mediates inhibitory neurotransmission onto mouse cerebellar Purkinje cells.

The effects of voltage‐dependent calcium channel (VDCC) antagonists on spontaneous inhibitory postsynaptic currents (sIPSCs) in mouse Purkinje cells were examined using in vitro cerebellar slices. The inorganic ion Cd2+ reduced sIPSC amplitude and frequency. No additional block was seen with the Na+ channel antagonist tetrodotoxin (TTX) suggesting that all action potential‐evoked inhibitory GABA release was mediated by high‐voltage‐activated VDCCs. No evidence was found for involvement of Cav1/α1C and α1D (L‐type), Cav2.2/α1B (N‐type) or Cav2.3/α1E (R‐type) high‐voltage‐activated VDCCs or low‐voltage‐activated Cav3/α1G, α1H and α1I (T‐type) VDCCs in mediating presynaptic GABA release. Blockade of sIPSCs by 200 nmω‐agatoxin IVA implicated the Cav2.1/α1A (P/Q‐type) subtype of high‐voltage‐activated VDCCs in mediating inhibitory transmission. Inhibition by ω‐agatoxin IVA was similar to that seen with Cd2+ and TTX. Selective antibodies directed against the Cav2.1 subunit revealed staining in regions closely opposed to Purkinje cell somata. Cav2.1 staining was colocalized with staining for antibodies against glutamic acid decarboxylase and corresponded well with the pericellular network formed by GABAergic basket cell interneurons. Antibody labelling of Cav2.3 revealed a region‐specific expression. In the cerebellar cortex anterior lobe, Cav2.3 staining was predominantly somatodendritic; whilst in the posterior lobe, perisomatic staining was seen primarily. However, electrophysiological data was not consistent with a role for the Cav2.3 subunit in mediating presynaptic GABA release. No consistent staining was seen for other Cav (α1) subunits. Electrophysiological and immunostaining data support a predominant role for Cav2.1 subunits in mediating action potential‐evoked inhibitory GABA release onto mouse Purkinje cells.