To effect movement, motoneurons must respond appropriately to motor commands. Their responsiveness to these inputs, or excitability, is regulated by neuromodulators. Possible sources of modulation include the abundant cholinergic ''C boutons'' that surround motoneuron somata. In the present study, recordings from motoneurons in spinal cord slices demonstrated that cholinergic activation of m 2-type muscarinic receptors increases excitability by reducing the action potential afterhyperpolarization. Analyses of isolated spinal cord preparations in which fictive locomotion was elicited demonstrated that endogenous cholinergic inputs increase motoneuron excitability during locomotion. Anatomical data indicate that C boutons originate from a discrete group of interneurons lateral to the central canal, the medial partition neurons. These results highlight a unique component of spinal motor networks that is critical in ensuring that sufficient output is generated by motoneurons to drive motor behavior.T o generate movement, it is necessary for motoneurons (MNs) to integrate the inputs (motor commands) they receive and produce an output sufficient to effect muscular contraction. The relationship of input to output is determined by neuronal excitability, which in the case of MNs is known to be regulated by identified descending modulatory systems (1). Given that these descending systems are disrupted after spinal cord injury, strategies aimed at restoring movement need to address not only premotor circuits that provide motor commands but also any modulatory systems that ensure MNs are sufficiently excitable to respond to these commands. Spinal premotor circuits for locomotion can be activated after spinal transection (2) and provide one clear target for treatments designed to produce functional recovery. Should an intrinsic spinal modulatory system exist, this would be an important additional target for such strategies.The somata and proximal dendrites of MNs are contacted by large cholinergic varicosities named ''C boutons'' (3-11). It has been known since 1972 (12) that C boutons originate from spinal cord neurons, but the location of these cells remains unknown (10). Although the C bouton synapse has been anatomically characterized and shown to be associated with postsynaptic type 2 muscarinic (m 2 ) receptors (8-10), neither the physiological effects of m 2 receptor activation on MNs nor the roles of C boutons in motor activity are known. In the absence of motor behavior, exogenous application of cholinergic agonists affects MN excitability via undefined mechanisms (13-17). We therefore studied the possibility that the intrinsic spinal neurons that give rise to the C boutons regulate MN excitability via activation of m 2 receptors, and that this system is used during motor behavior. ResultsThe Effects of Muscarinic Receptor Activation on Spinal MNs. Because C boutons are closely associated with postsynaptic m 2 receptors by the second postnatal week (8-10), we investigated the effects of muscarinic receptor activatio...

Developmental studies identified four classes (V0, V1, V2, V3) of embryonic interneurons in the ventral spinal cord. Very little however is known about their adult phenotypes. In order to further characterize interneuron cell types in the adult, the location, neurotransmitter phenotype, calciumbuffering protein expression and axon distributions of V1-derived neurons in the mouse spinal cord was determined. In the mature (P20 and older) spinal cord, most V1-derived neurons are located in lateral LVII and in LIX, few in medial LVII and none in LVIII. Approximately 40% express calbindin and/or parvalbumin, while few express calretinin. Of seven groups of ventral interneurons identified according to calcium-buffering protein expression, two groups (1 and 4) correspond with V1-derived neurons. Group 1 are Renshaw cells and intensely express calbindin and coexpress parvalbumin and calretinin. They represent 9% of the V1 population. Group 4 express only parvalbumin and represent 27% of V1-derived neurons. V1-derived group 4 neurons receive contacts from primary sensory afferents and are therefore proprioceptive interneurons and the most ventral neurons in this group receive convergent calbindin-IR Renshaw cell inputs. This subgroup resembles Ia inhibitory interneurons (IaINs) and represents 13% of V1-derived neurons. Adult V1-interneuron axons target LIX and LVII and some enter the deep dorsal horn. V1-axons do not cross the midline. V1 derived axonal varicosities were mostly (>80%) glycinergic and a third were GABAergic. None were glutamatergic or cholinergic. In summary, V1 interneurons develop into ipsilaterally projecting, inhibitory interneurons that include Renshaw cells, Ia inhibitory interneurons and other unidentified proprioceptive interneurons.Keywords inhibitory interneurons; engrailed-1; motor control; GABA; glycine; calbindin; parvalbumin; calretinin; motoneurons; ventral horn; spinal cord; development; V1 During the past 50 years, classical studies in the spinal cord have uncovered a wealth of details about the organization of interneuronal networks that control motoneuron firing, segmental reflexes and generate locomotor patterns in mammals (Jankowska et al., 1992). These studies, which relied heavily on the physiological identification of synaptic inputs and (Renshaw 1946;Eccles et al., 1954). IaINs are characterized by inputs from sensory Ia muscle afferents, provide reciprocal inhibition to antagonistic motor pools (Eccles et al., 1956) and are modulated by RCs (Hultborn et al., 1971). Despite enormous progress in further identification of interneurons using these methods, much is unknown and this approach is not without limitations (Edgley, 2001;Jankowska, 2001). Thus a comprehensive classification of ventral interneurons into major subclasses has not yet emerged.Recent studies indicate that distinct cell types in the adult spinal cord are derived from genetically-discrete populations of embryonic neurons (Jessell, 2000;Briscoe and Ericson, 2001;Lee and Pfaff, 2001;Goulding et al., 2002, Sa...

The properties of mammalian spinal interneurons that underlie rhythmic locomotor networks remain poorly described. Using postnatal transgenic mice in which expression of green fluorescent protein is driven by the promoter for the homeodomain transcription factor Hb9, as well as Hb9 -lacZ knock-in mice, we describe a novel population of glutamatergic interneurons located adjacent to the ventral commissure from cervical to midlumbar spinal cord levels. Hb9 ϩ interneurons exhibit strong postinhibitory rebound and demonstrate pronounced membrane potential oscillations in response to chemical stimuli that induce locomotor activity. These data provide a molecular and physiological delineation of a small population of ventral spinal interneurons that exhibit homogeneous electrophysiological features, the properties of which suggest that they are candidate locomotor rhythm-generating interneurons.

Electrophysiological and morphological properties of genetically identified spinal interneurons were examined to elucidate their possible contribution to locomotor-like rhythmic activity in 1- to 4-day-old mice. In the transgenic mice used in our study, the HB9 promotor controlled the expression of the reporter gene enhanced green fluorescent protein (eGFP), giving rise to GFP+ motoneurons and ventral interneurons. However, only motoneurons and a small group of bipolar, GFP+ interneurons expressed the HB9 protein. The HB9(+)/GFP+ interneurons were clustered close to the medial surface in lamina VIII along segments L1-L3. The correlation between activity pattern in these visually identified interneurons and motoneuron output was examined using simultaneous whole cell and ventral root recordings. Neurochemically induced rhythmic membrane depolarizations in HB9/GFP interneurons were synchronous with ventral root rhythms, indicating that the interneurons received synaptic inputs from rhythm-generating networks. The frequency of excitatory postsynaptic currents significantly increased during ventral root bursts, but there was no change in the frequency of inhibitory postsynaptic currents during the cycle period. These data implied that HB9/GFP interneurons received primarily excitatory inputs from rhythmogenic interneurons. Neurobiotin-filled axon terminals were in close apposition to other neurons in the cluster and to motoneuron dendrites, raising the possibility that HB9/GFP interneurons formed synaptic connections with each other and with motoneurons. The expression of the vesicular glutamate transporter 2 in axon terminals of HB9/GFP interneurons indicated that these were glutamatergic interneurons. Our findings suggest that the visually identified HB9/GFP interneurons are premotor excitatory interneurons and putative constituents of networks generating locomotor rhythms in the mammalian spinal cord.

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