Lack of sigma-1 receptor exacerbates ALS progression in mice.

The function of the sigma-1 receptor (S1R) has been implicated in modulating the activity of various ion channels. In the CNS S1R is enriched in cholinergic postsynaptic densities in spinal cord motoneurons (MNs). Mutations in S1R have been found in familial cases of amyotrophic lateral sclerosis (ALS). In this study we show that a knockout of S1R in the SOD1*G93A mouse model of ALS significantly reduces longevity (end stage). Electrophysiological experiments demonstrate that MN of mice lacking S1R exhibit increased excitability. Taken together the data suggest the S1R acts as a brake on excitability, an effect that might enhance longevity in an ALS mouse model.

Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN.

The sigma-1 receptor is enriched in postsynaptic sites of C-terminals in mouse motoneurons. An anatomical and behavioral study.

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.

Distinct roles of glycinergic and GABAergic inhibition in coordinating locomotor-like rhythms in the neonatal mouse spinal cord.

The primary objective of our study was to examine the role of the inhibitory neurotransmitters glycine and GABA in modulating spontaneous activity and coordinating neurochemically induced locomotor-like rhythms in the mouse spinal cord. Motor outputs were recorded in lumbar ventral roots of 1-4-day old neonatal mice, and the function of glycinergic and GABAergic synapses in regulating spontaneous and induced activities was examined by suppressing synaptic inhibition using selective glycine or GABAA receptor antagonists. Strychnine (0.5 microM), a glycine receptor antagonist, did not change the pattern of spontaneous activity that consisted of random single spikes and discharges of variable durations and intervals. In contrast, blocking GABAA receptors with either picrotoxin (10 microM) or bicuculline (5 microM) triggered bilaterally synchronous, non-rhythmic discharges. These findings suggested that GABAergic synapses suppressed excitatory synapses, and their disinhibition synchronized spontaneous discharges between the two sides of the spinal cord. Locomotor-like rhythms alternating between the two sides of the spinal cord were triggered by the neurotransmitter agonists 5-HT, N-methyl-D,L-aspartic acid and dopamine. Blocking glycine receptors increased tonic discharges, and in most preparations it reduced the phase correlation between the alternating rhythms. Inhibiting GABAA receptor-mediated synapses synchronized the onset and prolonged the duration of rhythmic discharges. Intraburst alternating peaks were evident and those were suppressed by strychnine, suggesting that they were mediated via glycinergic synapses. Our findings indicated that GABAergic and glycinergic synapses played different roles in modulating neurochemically induced locomotion rhythms. GABAergic inhibition regulated the onset and duration of neurochemically induced locomotor-like rhythms, and glycinergic inhibition stabilized the pattern of the alternating rhythms.

Transition from GABAergic to glycinergic synaptic transmission in newly formed spinal networks.

The role of glycinergic and GABAergic systems in mediating spontaneous synaptic transmission in newly formed neural networks was examined in motoneurons in the developing rat spinal cord. Properties of action potential-independent miniature inhibitory postsynaptic currents (mIPSCs) mediated by glycine and GABA(A) receptors (GlyR and GABA(A)R) were studied in spinal cord slices of 17- to 18-day-old embryos (E17-18) and 1- to 3-day-old postnatal rats (P1-3). mIPSC frequency and amplitude significantly increased after birth, while their decay time decreased. To determine the contribution of glycinergic and GABAergic synapses to those changes, GlyR- and GABA(A)R-mediated mIPSCs were isolated based on their pharmacological properties. Two populations of pharmacologically distinct mIPSCs were recorded in the presence of glycine or GABA(A) receptors antagonists: bicuculline-resistant, fast-decaying GlyR-mediated mIPSCs, and strychnine-resistant, slow-decaying GABA(A)R-mediated mIPSCs. The frequency of GABA(A)R-mediated mIPSCs was fourfold higher than that of GlyR-mediated mIPSCs at E17-18, indicating that GABAergic synaptic sites were functionally dominant at early stages of neural network formation. Properties of GABA(A)R-mediated mIPSC amplitude fluctuations changed from primarily unimodal skewed distribution at E17-18 to Gaussian mixtures with two to three discrete components at P1-3. A developmental shift from primarily long-duration GABAergic mIPSCs to short-duration glycinergic mIPSCs was evident after birth, when the frequency of GlyR-mediated mIPSCs increased 10-fold. This finding suggested that either the number of glycinergic synapses or the probability of vesicular glycine release increased during the period studied. The increased frequency of GlyR-mediated mIPSCs was associated with more than a twofold increase in their mean amplitude, and in the number of motoneurons in which mIPSC amplitude fluctuations were best fitted by multi-component Gaussian curves. A third subpopulation of mIPSCs was apparent in the absence of glycine and GABA(A) receptor antagonists: mIPSCs with both fast and slow decaying components. Based on their dual-component decay time and their suppression by either strychnine or bicuculline, we assumed that these were generated by the activation of co-localized postsynaptic glycine and GABA(A) receptors. The contribution of mixed glycine-GABA synaptic sites to the generation of mIPSCs did not change after birth. The developmental switch from predominantly long-duration GABAergic inhibitory synaptic currents to short-duration glycinergic currents might serve as a mechanism regulating neuronal excitation in the developing spinal networks.

Ethanol reduces neuronal excitability and excitatory synaptic transmission in the developing rat spinal cord.

Effects of acute ethanol (EtOH) exposure on motoneuron excitability and properties of synaptic transmission were examined in spinal cords of postnatal rats. Whole-cell patch clamp recordings and intracellular recordings with high-resistance electrodes were carried out in motoneurons of 1- to 4-day-old postnatal rats. To determine the effects of extracellular EtOH on action potential waveform, properties of current-evoked soma action potentials and motoneuron ability to generate repetitive action potential firing were examined. During a brief EtOH (70 mM) exposure, larger depolarizing current was required for action potential generation, the duration of the after hyperpolarizing potential increased, and fewer action potentials were produced during a prolonged intracellular current injection. These effects were reversed within 20 min of EtOH removal from the extracellular solution. To determine whether the reduced probability of action potential generation was associated with changes in synaptic transmission, properties of evoked synaptic potentials and spontaneous synaptic currents were investigated. In the presence of EtOH, the amplitude of dorsal root-evoked synaptic potentials was reduced, the frequency of spontaneous excitatory postsynaptic currents decreased, while the frequency of inhibitory postsynaptic currents increased. Our data suggested that acute EtOH exposure suppressed motoneuron electrical activity by decreasing motoneuron excitability and shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition.

Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons.

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.

Physiological functions of GABA-induced depolarizations in the developing rat spinal cord.

Gamma-aminobutyric acid (GABA) is one of the principle inhibitory neurotransmitters in the mature spinal cord. It effectively suppresses synaptic transmission by mechanisms of postsynaptic and presynaptic inhibition. The function of GABA is less well understood early in spinal cord development, when the amino acid is transiently expressed in most neurons, and it depolarizes instead of hyperpolarizes neurons. This article reviews the possible physiological roles of GABA in modulating synaptic transmission, promoting neuronal development, and regulating neuronal pH during early stages of spinal cord differentiation. It is proposed that despite its depolarizing action, GABA acts as an inhibitory neurotransmitter that may also function as a neurotrophic agent.

Development of spontaneous synaptic transmission in the rat spinal cord.

Dorsal root afferents form synaptic connections on motoneurons a few days after motoneuron clustering in the rat lumbar spinal cord, but frequent spontaneous synaptic potentials are detected only after birth. To increase our understanding of the mechanisms underlying the differentiation of synaptic transmission, we examined the developmental changes in properties of spontaneous synaptic transmission at early stages of synapse formation. Spontaneous postsynaptic currents (PSCs) and tetrodotoxin (TTX)-resistant miniature PSCs (mPSCs) were measured in spinal motoneurons of embryonic and postnatal rats using whole cell patch-clamp recordings. Spontaneous PSC frequencies were higher than mPSC frequencies in both embryonic and postnatal motoneurons, suggesting that even at embryonic stages, when action-potential firing rate was low, presynaptic action potentials played an important role in triggering spontaneous PSCs. After birth, the twofold increase in spontaneous PSC frequency was attributed to an increase in action-potential-independent quantal release rather than to a higher rate of action-potential firing. In embryonic motoneurons, the fluctuations in peak amplitude of spontaneous PSCs were normally distributed around single peaks with modal values similar to those of mPSCs. These data indicated that early in synapse differentiation spontaneous PSCs were primarily composed of currents generated by quantal release. After birth, mean mPSC amplitude increased by 50% but mean quantal current amplitude did not change. Synchronous, multiquantal release was apparent in postnatal motoneurons only in high-K+ extracellular solution. Comparison of the properties of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) demonstrated that mean mEPSC frequency was higher than mIPSC frequency, suggesting that either excitatory synapses outnumbered inhibitory synapses or that the probability of excitatory transmitter release was higher than the release of inhibitory neurotransmitters. The finding that mIPSC duration was several-fold longer than mEPSC duration implied that despite their lower frequency, inhibitory currents could modulate motoneuron synaptic integration by shunting incoming excitatory inputs for prolonged time intervals.

Cues intrinsic to the spinal cord determine the pattern and timing of primary afferent growth.

We have used organotypic cultures of embryonic rat spinal cord and dorsal root ganglia (DRG) to study the development of central projections of primary sensory afferent axons that express calcitonin gene-related peptide (CGRP). In vivo, small- and medium-diameter CGRP-positive primary afferents terminate in laminae I, II, and V of the spinal cord and do not enter the ventral horn. A similar pattern of CGRP-positive axonal projections was observed in spinal cord slices of Day 16 embryos (E16) maintained in culture for 6 days. Both intact and dissociated DRG neurons showed the same pattern of central arborization, indicating that complex intercellular interactions between DRG neurons are not required for laminar specific targeting. Furthermore, targeting to the dorsal horn and avoidance of the ventral horn was observed in isolated dorsal and ventral hemicords, suggesting that separate mechanisms mediate the avoidance of CGRP-positive axons from the ventral horn and the elaboration of the afferent arbors within the dorsal horn. CGRP-positive afferents can grow into the dorsal horn only during a brief time window. Cultures of age-matched (isochronic) DRG and spinal cord from E14, E16, and E18 animals showed the characteristic pattern of CGRP-positive axon arborization, while cultures from E20 and neonatal animals did not. Heterochronic cultures indicate that it is the age of the spinal cord, and not the age of the DRG, that determines the ability of the CGRP-positive afferents to arborize within the dorsal horn. Together these results demonstrate that cues intrinsic to the spinal cord can direct sensory projections to appropriate locations in the spinal cord.

Blocking Ca(2+)-dependent synaptic release delays motoneuron differentiation in the rat spinal cord.

Development of motoneuron electrical properties and excitability was studied in spinal cord explants of rat embryos cultured for 1-3 weeks. The morphological organization of the spinal cord and synaptic inputs onto motoneurons were maintained in organ culture. The rate of differentiation of motoneuron resting potential and increase in membrane excitability was similar in vitro and in vivo, suggesting that these properties were regulated by cellular signals or extracellular differentiation-promoting factors that were preserved in culture. However, maturation of input resistance, action potential threshold and action potential maximum rate of rise was slower than in vivo. Culturing spinal cord explants with their dorsal root ganglia attached did not facilitate motoneuron differentiation. The role of newly formed synaptic pathways in regulating the changes in motoneuron electrical properties was studied in the presence of blockers of synaptic transmission. Motoneuron differentiation was delayed in spinal cords cultured in the presence of TTX, indicating that electrical activity influenced the time course of their development. However, blocking synaptic transmission with antagonists of glutamate, glycine, and GABAA receptors did not affect the rate of motoneuron differentiation, suggesting that maturation of motoneuron phenotype was independent of activation of these transmitter-gated channels. Incubating spinal cords in medium containing high-K+, which increased the frequency of spontaneous potentials, reversed the inhibitory effect of TTX. Similar to TTX action, motoneuron development was retarded when synaptic release was chronically blocked with either tetanus toxin or omega-conotoxin, a Ca2+ channel blocker. These findings suggested that electrical activity in spinal cord explants modulated motoneuron differentiation via Ca(2+)-dependent synaptic release of neurotransmitters or neurotrophic factors.

Development of glycine- and GABA-gated currents in rat spinal motoneurons.

1. Developmental changes in glycine- and gamma-aminobutyric acid (GABA)-activated currents were studied in spinal motoneurons of embryonic and neonatal rats with the use of whole cell recording techniques. 2. Pressure ejection of glycine or GABA onto motoneuron somata produced Cl(-)-mediated inward currents and membrane depolarizations. During embryonic development, the average amplitude of GABA-gated currents was threefold larger than that of glycine-gated currents, but as a result of a large eightfold postnatal increase in glycine-activated currents, similar currents were produced by both amino acids after birth. 3. At all ages the decay of glycine- and GABA-gated currents best fit one-exponential curve, and their time constants were similar. The average decay time constant decreased by twofold after birth. 4. The ionic specificity of glycine- and GABA-gated channels was studied to determine whether the large amplitude of GABA-activated currents in embryonic motoneurons resulted from the contribution of an outward HCO-3 movement. Manipulations of Cl- and HCO-3 concentrations produced changes in the reversal potentials of glycine and GABA that were similar to the calculated changes in the equilibrium potentials of Cl-. This suggested that glycine- and GABA-gated currents were Cl- specific, and HCO-3 movement did not contribute more to the current generated by GABA than that produced by glycine. 5. Glycine- and GABA-gated currents were associated with severalfold increases in membrane conductance. The conductance increase generated by GABA in embryonic motoneurons was sevenfold larger than that generated by glycine, but similar conductance changes were produced by both amino acids after birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of transient inappropriate sensorimotor synapses in developing rat spinal cords.

The specificity of the convergence of primary afferent projections from ankle muscles onto motoneurons that innervate these muscles was studied in lumbar spinal cords of embryonic and neonatal rats. The connectivity pattern was determined for each motoneuron by stimulating nerves from ankle flexor and extensor muscles and recording the synaptic potentials in identified motoneurons. In mature mammals, muscle spindle afferents make direct excitatory connections with motoneurons that innervate homonymous and synergistic muscles, and with interneurons that inhibit motoneurons innervating antagonistic muscles. Therefore, appropriate primary afferent-motoneuron connections were identified when stimulation of homonymous and synergistic muscle nerves evoked monosynaptic EPSPs. Two criteria were used for identification of EPSPs as monosynaptic potentials: (1) the monosynaptic potentials were evoked at the shortest latency, and (2) they were more resistant to fatigue by repetitive nerve stimulation than the longer-latency, polysynaptic potentials. Functionally inappropriate primary afferent-motoneuron contacts were identified when stimulation of an antagonistic muscle nerve produced monosynaptic EPSPs instead of polysynaptic IPSPs in homonymous motoneurons. At days 18-21 of gestation, about 30% of motoneurons were innervated by primary afferents of antagonist muscles. Such functionally inappropriate synapses persisted at birth, but their percentage was significantly reduced within 3-5 d after birth. The findings suggested that in the developing spinal cord of the rat, a significant percentage of motoneurons were initially innervated by inappropriate primary afferents of antagonistic muscles. The decrease in percentage of such inappropriate connections was correlated temporally with the increase in the frequency of spontaneous activity and the onset of myelination.

Development of chemosensitivity in serotonin-deficient spinal cords of rat embryos.

The characteristics of serotonin-induced potentials change during normal development of spinal cords of rat embryos. These changes are temporally correlated with the growth of serotonin projections into the spinal cord. To determine whether serotonin (5-hydroxytryptamine, 5-HT) in the growing projections is responsible for modulating the changes in neuronal responses to 5-HT, 5-HT synthesis was blocked, and motoneuron responses to exogenous 5-HT were studied 1-2 weeks later. Starting at Day 9 of gestation, prior to the generation of 5-HT neurons in the medulla, pregnant rats were injected daily with p-chlorophenylalanine (p-CPA) which suppressed 5-HT synthesis. p-CPA was effective in reducing 5-HT concentrations because immunoreactive 5-HT projections were absent in embryonic spinal cords of p-CPA-treated rats. However, suppression of 5-HT synthesis did not affect the onset and time course of development of 5-HT-induced potentials. Furthermore, in the absence of 5-HT, the potentials generated by 5-HT were significantly larger than those produced in motoneurons of control rats. These findings indicated that the number of receptors or their binding affinity increased in the absence of 5-HT. The increased responses in p-CPA-treated rats were mediated via 5-HT2/5-HT1C receptors, while activation of other 5-HT1 receptor subtypes and 5-HT3 receptors induced similar depolarizations in p-CPA-treated and untreated rats. Our study suggested that 5-HT was not required for the onset of receptor expression in developing spinal neurons, but it may be responsible for receptor down-regulation.

Changes in serotonin-induced potentials during spinal cord development.

1. Motoneuron responses to serotonin (5-hydroxytryptamine, 5-HT), and the growth pattern of 5-HT projections into the ventral horn were studied in the isolated spinal cord of embryonic and neonatal rats. 2. 5-HT projections first appeared in lumbar spinal cord at days 16-17 of gestation (E16-E17) and were localized in the lateral and ventral funiculi. By E18, the projections had grown into the ventral horn, and at 1-2 days after birth they were in close apposition to motoneuron somata. 3. At E16-E17, slow-rising depolarizing potentials of 1-4 mV were recorded intracellularly in lumbar motoneurons in response to bath application of 5-HT. These potentials were not apparent after E18; at that time 5-HT generated long-lasting depolarizations with an average amplitude of 6 mV, and an increase of 11% in membrane resistance. Starting at E18, 5-HT also induced high-frequency fast-rising potentials that were blocked by antagonists of glutamate, gamma-aminobutyric acid, and glycine. 4. Motoneuron responses to 5-HT increased significantly after birth, when 5-HT produced an average depolarization of 19 mV and repetitive firing of action potentials. 5. Tetrodotoxin and high Mg2+ did not reduce the amplitude of the long-lasting depolarizations, which suggested that they were produced by direct action of 5-HT on motoneuron membrane. 6. At all developmental ages, 5-HT reduced the amplitude of dorsal root-evoked potentials. The suppressed responses were neither due to 5-HT-induced depolarization nor the result of a decrease in motoneuron excitability. 7. The pharmacological profile of 5-HT-induced potentials was studied with the use of various agonists and antagonists of 5-HT. The findings indicated that the actions of 5-HT on spinal neurons were mediated via multiple 5-HT receptor subtypes. 8. Our results suggested that 5-HT excited spinal neurons before 5-HT projections grew into the ventral horn. The characteristics of 5-HT-induced potentials changed, however, at the time when the density of 5-HT projections increased in the motor nuclei.

Early development of glycine- and GABA-mediated synapses in rat spinal cord.

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.

Reinnervation of developing rat muscle by non-axotomized motoneurons.

To study the ability of developing motoneurons to reinnervate their denervated muscle, axotomized motoneurons in rat neonates and pups were retrogradely labeled with two fluorescent tracers. Fluorogold (FG), a long-lasting fluorescent dye, was injected into intercostal muscle T8 to retrogradely label the motoneurons that innervated it. Two days later intercostal nerves T7-T9 were cut. The intercostal muscle denervated at birth was reinnervated within 10-20 days, as evidenced by nerve-evoked muscle contraction. Three weeks following axotomy, tetramethylrhodamine isothiocyanate (TRITC) was injected into the same muscle to label the motoneurons that reinnervated it. The motoneurons double-labeled with FG and TRITC were, therefore, axotomized motoneurons that regenerated to reinnervate T8. In neonates, axotomy resulted in a significant reduction in the number of FG-labeled motoneurons, which suggests that axon transection at early postnatal days causes a massive motoneuron death. The percentage of double-labeled motoneurons was significantly smaller than that in non-axotomized rats. TRITC-labeled motoneurons constituted the majority of stained motoneurons; these were located in different nuclei than the intercostal motoneurons. These findings suggest that muscle reinnervation is, at least in part, by motoneurons which originally did not innervate intercostal muscle T8. Unlike axotomy at birth, axotomy performed 2-3 weeks after birth did not result in a significant motoneuron loss. The number of stained motoneurons labeled with both FG and TRITC was significantly smaller, however, than in non-axotomized spinal cords. Our data indicate that in pups only a small percentage of axotomized motoneurons reinnervated the denervated muscle.

NMDA receptors mediate poly- and monosynaptic potentials in motoneurons of rat embryos.

We determined the contribution of glutamate receptor subtypes to developing excitatory synaptic transmission in isolated spinal cord of rat embryos. Using electrophysiological and morphological techniques, we studied the pattern of development of synapses between dorsal root afferents and motoneurons in lumbar spinal cords of 15- to 21-d-old rat embryos. Motoneuron dendritic fields and afferent projections onto motoneurons were identified by labeling with HRP. Afferents first entered the gray matter at Day 15 of gestation, and by Day 16 they terminated close to motoneuron dendritic trees. Afferent axons projected onto motoneuron dendritic fields at Day 17, when boutons were detected on motoneuron dendrites that were crossed by afferent axons. To determine the time course of formation of functional sensorimotor synapses and their pharmacological properties, a dorsal root was stimulated while recording intracellularly from segmental motoneurons. At Day 16, excitatory postsynaptic potentials (EPSPs) with long latencies, slow rates of rise, and long durations were recorded. The amplitudes of these EPSPs increased with membrane depolarization and in the absence of extracellular Mg2+. These EPSPs were blocked by D-2-amino-5-phosphonovalerate (APV) and ketamine, which are selective antagonists of N-methyl-D-aspartate (NMDA) receptors. These findings suggest that initial synaptic transmission in embryonic motoneurons is mediated solely by NMDA receptors. Short-latency EPSPs with fast rates of rise were first recorded in most motoneurons by Day 17. These EPSPs were composed of fast- and slow-rising potentials. The slow component was blocked by APV, while the fast component was eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione and kynurenate. This indicates that the short-latency EPSPs are mediated by both NMDA and non-NMDA receptors. Dose-response curves of motoneurons to L-glutamate, NMDA, and kainate demonstrated that motoneurons are sensitive to these agonists prior to the formation of synapses between afferents and motoneurons. Motoneuron responses to NMDA and kainate increased immediately after the onset of short-latency EPSPs. This increased sensitivity could be due to extracellular factors influenced by growing sensory axons or intrinsic properties of differentiating motoneurons.

Physiological and morphological changes in developing peripheral nerves of rat embryos.

Physiological and morphological properties of intercostal nerves were studied in rat embryos of 13-21 days of gestation (birth is at 21-22 days). Rat intercostal nerves emerged from the spinal cord at day 13 of gestation when they were surrounded by a few Schwann cells. The nerve bundles contained growth cones, and large and small diameter unmyelinated profiles. At 13-17 days of gestation, large diameter profiles and irregularly shaped growth cones made up a significant portion of the axon population in intercostal nerves. At day 13 electrical stimulation of the nerve evoked action potentials. Thus, intercostal nerves are excitable at day 13 in utero, prior to the formation of functional nerve-muscle contacts (day 14-15). A functional nerve-muscle junction is, therefore, not a prerequisite for conduction of action potentials. From the onset of excitability Na+ was the major carrier of the action potential inward current, but there was also a small Ca2+ current that lasted until 18 days of gestation. At 17-18 days of gestation, Schwann cell proliferation and extension of Schwann cell cytoplasm into the nerve subdivided it into numerous smaller bundles. Axons located in the periphery of many bundles were wrapped by Schwann cell cytoplasm and were probably the first ones to become myelinated. During the same period extracellularly recorded action potentials consisted of multiple negative peaks, and conduction velocity increased about 2-fold. Myelination began at day 22 and was completed within 3 weeks after birth when the ratio of myelinated-to-unmyelinated axons reached its adult value.

Electrical properties of motoneurons in the spinal cord of rat embryos.

Electrical properties of immature motoneurons were studied in vitro using isolated segments of spinal cords of rat embryos aged 14-21 days of gestation. Stable resting potentials and evoked synaptic potentials were recorded for more than 9 hr, indicating that motoneurons remain viable for many hours. Motoneurons are electrically excitable at 14 days of gestation and from the onset of excitability the action potentials are Na+-dependent but slow rising long-duration Ca2+-dependent action potentials can be evoked if K+ conductance is reduced. Thus, during embryonic development the regenerative potential inward current is Na+-and Ca2+-dependent. During motoneurons' differentiation there are some changes in their electrical properties: resting membrane potential increases, input resistance decreases, input capacitance increases, threshold for action potential decreases, and maximum rate of rise of action potential increases. Afferent motoneuron contacts are formed at 16-18 days of gestation when excitatory synaptic potentials can first be evoked in response to dorsal root stimulation. The changes in input capacitance and threshold for action potential occur at the onset of functional afferent motoneuron contacts, but it is not known whether these changes are autonomous or are influenced by the newly formed sensory inputs.