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 subunitcontaining 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.

Key points• Spinal cord α-motoneurons display strong membrane immunoreactivity (IR) against small-conductance calcium-activated potassium channel (SK) isoform SK2, and a specific subpopulation of motoneurons also express SK3-IR.• Rat α-motoneurons expressing SK3-IR are significantly smaller, have significantly longer after-hyperpolarization half-decay time, significantly larger after-hyperpolarization amplitude and significantly slower axon conduction velocity than α-motoneurons that lack SK3-IR.• Motoneuron pools innervating slow-twitch muscles have a higher percentage of SK3-IR α-motoneurons than those innervating fast-twitch muscles.• Expression of SK3 may contribute to variability in after-hyperpolarization duration and amplitude across different types of rat α-motoneurons and may be a molecular factor differentiating between slow-and fast-type motoneurons.• In the soma and proximal dendrites of α-motoneurons, large clusters of SK2 and SK3 channel subunits appose cholinergic C-boutons and colocalize with muscarinic type 2 receptors and Kv2.1 channels, which suggests a novel cellular mechanism for state-dependent regulation of neuronal excitability.Abstract Small-conductance calcium-activated potassium (SK) channels mediate medium after-hyperpolarization (AHP) conductances in neurons throughout the central nervous system. However, the expression profile and subcellular localization of different SK channel isoforms in lumbar spinal α-motoneurons (α-MNs) is unknown. Using immunohistochemical labelling of rat, mouse and cat spinal cord, we reveal a differential and overlapping expression of SK2 and SK3 isoforms across specific types of α-MNs. In rodents, SK2 is expressed in all α-MNs, whereas SK3 is expressed preferentially in small-diameter α-MNs; in cats, SK3 is expressed in all α-MNs. Function-specific expression of SK3 was explored using post hoc immunostaining of electrophysiologically characterized rat α-MNs in vivo. These studies revealed strong relationships between SK3 expression and medium AHP properties. Motoneurons with SK3-immunoreactivity exhibit significantly longer AHP half-decay times (24.67 vs. 11.02 ms) and greater AHP amplitudes (3.27 vs. 1.56 mV) than MNs lacking SK3-immunoreactivity. We conclude that the differential expression of SK isoforms in rat and mouse spinal cord may contribute to the range of medium AHP durations across specific MN functional types and may be a molecular factor distinguishing between slow-and fast-type α-MNs in rodents. Furthermore, our results show that SK2-and SK3-immunoreactivity is enriched in distinct postsynaptic domains that contain Kv2.1 channel clusters associated with cholinergic C-boutons on the soma and proximal dendrites of α-MNs. We suggest that this remarkably specific subcellular membrane localization of SK channels is likely to represent the basis for a cholinergic mechanism for effective regulation of channel function and cell excitability.

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.

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.

Cerebellar granule neurons (CGNs) are one of the most populous cells in the mammalian brain. They express an outwardly rectifying potassium current, termed a ''standing-outward'' K ؉ current, or IK SO, which does not inactivate. It is active at the resting potential of CGNs, and blocking IK SO leads to cell depolarization. IKSO is blocked by Ba 2؉ ions and is regulated by activation of muscarinic M 3 receptors, but it is insensitive to the classical broad-spectrum potassium channel blocking drugs 4-aminopyridine and tetraethylammonium ions. The molecular nature of this important current has yet to be established, but in this study, we provide strong evidence to suggest that IK SO is the functional correlate of the recently identified two-pore domain potassium channel TASK-1. We show that IKSO has no threshold for activation by voltage and that it is blocked by small extracellular acidifications. Both of these are properties that are diagnostic of TASK-1 channels. In addition, we show that TASK-1 currents expressed in Xenopus oocytes are inhibited after activation of endogenous M 3 muscarinic receptors. Finally, we demonstrate that mRNA for TASK-1 is found in CGNs and that TASK-1 protein is expressed in CGN membranes. This description of a functional two-pore domain potassium channel in the mammalian central nervous system indicates its physiological importance in controlling cell excitability and how agents that modify its activity, such as agonists at G protein-coupled receptors and hydrogen ions, can profoundly alter both the neuron's resting potential and its excitability.

C-boutons are important cholinergic modulatory loci for state-dependent alterations in motoneuron firing rate. m2 receptors are concentrated postsynaptic to C-boutons, and m2 receptor activation increases motoneuron excitability by reducing the action potential afterhyperpolarization. Here, using an intensive review of the current literature as well as data from our laboratory, we illustrate that C-bouton postsynaptic sites comprise a unique structural/functional domain containing appropriate cellular machinery (a “signaling ensemble”) for cholinergic regulation of outward K+ currents. Moreover, synaptic reorganization at these critical sites has been observed in a variety of pathologic states. Yet despite recent advances, there are still great challenges for understanding the role of C-bouton regulation and dysregulation in human health and disease. The development of new therapeutic interventions for devastating neurological conditions will rely on a complete understanding of the molecular mechanisms that underlie these complex synapses. Therefore, to close this review, we propose a comprehensive hypothetical mechanism for the cholinergic modification of α-MN excitability at C-bouton synapses, based on findings in several well-characterized neuronal systems.

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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 alpha- and gamma-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 alpha(1)-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.