Motoneuron responses to the inhibitory amino acids glycine and GABA, and the contribution of inhibitory synapses to developing sensorimotor synapses were studied in rat spinal cords during the last week in utero. In differentiating motoneurons, glycine and GABA induced Cl(-)-dependent membrane depolarizations and large decreases in membrane resistance. These responses gradually decreased during embryonic development, and at birth they were significantly smaller than in embryos. In motoneurons of embryos and neonates, dorsal root stimulation produced only depolarizing potentials, some of which reversed at -50 mV membrane potential. Reduction of extracellular Cl- concentrations increased the amplitude of these potentials, suggesting that they are generated by Cl- current. Contribution of Cl(-)-dependent potentials to compound dorsal root-evoked potentials was studied by determining the effects of glycine and GABA antagonists on them. In motoneurons of embryos at days 16-17 of gestation (D16-D17), strychnine or bicuculline blocked dorsal root-evoked potentials. This suppression was neither the result of a decrease in neuronal excitability nor the inhibition of glutamate receptors. Strychnine-evoked depression was not blocked by atropine, indicating that it was not due to disinhibition of muscarinic synapses. By D19, strychnine and bicuculline significantly increased dorsal root-evoked potentials rather than blocking them. This reversed function did not result from an increase in neuronal excitability or changes in the specificity of strychnine and bicuculline antagonism. The number of glycine- and GABA-immunoreactive cells increased 20% between D17 and D19. The number of immunoreactive cells and fibers significantly increased in the motor nuclei and dorsal horn laminae. These morphological changes may contribute to establishment of new synaptic contacts on motoneurons, thus changing the actions of strychnine and bicuculline on dorsal root-evoked potentials.

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.

The sigma-1 receptor regulates various ion channel activity and possesses protein chaperone function. Using an antibody against the full sequence of the sigma-1 receptor we detected immunostaining in wild type but not in knockout mice. The receptor was found primarily in motoneurons localized to the brainstem and spinal cord. At the subcellular level the receptor is restricted to large cholinergic postsynaptic densities on the soma of motoneurons and is colocalized with the Kv2.1 potassium channel and the muscarinic type 2 cholinergic receptor. Ultrastructural analysis of the neurons indicates that the immunostained receptor is located close but separate from the plasma membrane, possibly in subsurface cisternae formed from the endoplasmic reticulum (ER), which are a prominent feature of cholinergic postsynaptic densities. Behavioral testing on a rotorod revealed that Sigma-1 receptor knockout mice remained on the rotorod for significantly less time (a shorter latency period) compared to the wild type mice. Together these data indicate that the sigma-1 receptor may play a role in the regulation of motor behavior.

Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons. J. Neurophysiol. 80: 3047-3061, 1998. Differentiation of the ionic mechanism underlying changes in action potential properties was investigated in spinal motoneurons of embryonic and postnatal rats using whole cell voltage- and current-clamp recordings. Relatively slow-rising, prolonged, largely Na+-dependent action potentials were recorded in embryonic motoneurons, and afterdepolarizing potentials were elicited in response to prolonged intracellular injections of depolarizing currents. Action potential amplitude, as well as its rates of rise and repolarization significantly increased, and an afterhyperpolarizing potential (AHP) became apparent immediately after birth. Concurrently, repetitive action potential firing was elicited in response to a prolonged current injection. To determine the ionic mechanism underlying these changes, the properties of voltage-gated macroscopic Na+, Ca2+, and K+ currents were examined. Fast-rising Na+ currents (INa) and slow-rising Ca2+ currents (ICa) were expressed early in embryonic development, but only INa was necessary and sufficient to trigger an action potential. INa and ICa densities significantly increased while the time to peak INa and ICa decreased after birth. The postnatal increase in INa resulted in overshooting action potential with significantly faster rate of rise than that recorded before birth. Properties of three types of outward K+ currents were examined: transient type-A current (IA), noninactivating delayed rectifier-type current (IK), and Ca2+-dependent K+ current (IK(Ca)). The twofold postnatal increase in IK and IK(Ca) densities resulted in shorter duration action potential and the generation of AHP. Relatively large IA was expressed early in neuronal development, but unlike IK and IK(Ca) its density did not increase after birth. The three types of K+ channels had opposite modulatory actions on action potential firing behavior: IK and IA increased the firing rate, whereas IK(Ca) decreased it. Our findings demonstrated that the developmental changes in action potential waveforms and the onset of repetitive firing were correlated with large increases in the densities of existing voltage-gated ion channels rather than the expression of new channel types.

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