Hiroshi Nishimaru

Spontaneous motoneuronal activity mediated by glycine and GABA in the spinal cord of rat fetuses in vitro.

1. Spontaneous motoneuronal activity was monitored from the lumbar ventral roots in an isolated spinal cord preparation from rat fetuses at embryonic days (E) 13.5‐18.5. 2. Spontaneous bursts that were synchronized in both left and right ventral roots were observed periodically (mean interval, 1.5‐2.6 min) from E14.5 to 17.5. This activity was abolished in Ca(2+)‐free saline or by application of tetrodotoxin (1 microM), indicating that it was synaptically mediated. 3. The glutamate receptor blocker kynurenate (4 mM) failed to block spontaneous bursts at E14.5‐15.5, though it completely abolished them at E17.5. The glycine receptor antagonist strychnine (10 microM) completely blocked spontaneous bursts at E14.5‐15.5. Bicuculline, a GABAA receptor antagonist, reduced the amplitude of the spontaneous bursts. 4. At E15.5, a brief application of glycine (250 microM to 2 mM) evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. Such glycine‐induced responses were not observed under Ca(2+)‐free conditions, suggesting that they were synaptically evoked. These synaptic responses were not blocked by kynurenate (4 mM), but they were abolished by strychnine (10 microM). 5. It is concluded that glycine and GABA generate the earliest spontaneous motor activity of the fetus and function transiently as excitatory transmitters in the embryonic spinal cord.

Formation of the central pattern generator for locomotion in the rat and mouse

It is well known that in the neonatal rat spinal cord preparation, alternating rhythmic bursts in the left and right ventral roots in a given lumbar segment can be induced by bath-application of N-methyl-D-aspartate or 5-hydroxytryptamine. Alternation between L2 and L5 ventral roots on the same side, representing the activity of flexor and extensor muscles, respectively, can be observed as well. In the prenatal period in the rat, alternation between the left and right ventral roots is established between embryonic day (E) 16.5 and E18.5. The alternation between the L2 and L5 ventral roots emerges at E20.5. Recent findings show that locomotor-like rhythmic activity with similar characteristics can be induced in the neonatal mouse preparation. In the lumbar spinal cord in the neonatal mouse, it is likely that the rhythm-generating network is distributed throughout the lumbar region with a rostro-caudal gradient, a situation similar to that in the neonatal and fetal rat spinal cord. With this review we hope to highlight the dramatic changes that neuronal networks generating locomotor-like activity undergo during the prenatal development of the rat. Moreover, the distribution of the neuronal network generating the locomotor rhythm in the neonatal rat and mouse spinal cord is compared.

5-Hydroxytryptamine-induced locomotor rhythm in the neonatal mouse spinal cord in vitro

We examined the 5-hydroxytryptamine (5-HT)-induced locomotor rhythm in isolated spinal cord preparations taken from neonatal mice on postnatal day (P) 0-3. Motor activity was recorded from L2 and L5 ventral roots. Bath application of 5-HT (15-100 microM) evoked rhythmic bursts that alternated between the two sides, and the bursts in the L2 ventral root alternated with those in the ipsilateral L5 ventral root. After transection of the mid-lumbar cord, the locomotor rhythm in L2 persisted, while that in the L5 ventral root was abolished. This suggests that the upper lumbar region has a greater ability to generate a locomotor rhythm than the lower lumbar spinal cord. Kynurenate, a broad-spectrum glutamate receptor antagonist, blocked the 5-HT-induced locomotor rhythm indicating that ionotropic glutamate receptors are required for the rhythm to be generated.

Activity of Renshaw Cells during Locomotor-Like Rhythmic Activity in the Isolated Spinal Cord of Neonatal Mice

In the present study, we examine the activity patterns of and synaptic inputs to Renshaw cells (RCs) during fictive locomotion in the newborn mouse using visually guided recordings from GABAergic cells expressing glutamic acid decarboxylase 67–green fluorescent protein (GFP). Among the GFP-positive neurons in the lumbar ventral horn, RCs were uniquely identified as receiving ventral root-evoked short-latency EPSPs that were markedly reduced in amplitude by nicotinic receptor blockers mecamylamine or tubocurarine. During locomotor-like rhythmic activity evoked by bath application of 5-HT and NMDA, 50% of the recorded RCs fired in-phase with the ipsilateral L2 flexor-related rhythm, whereas the rest fired in the extensor phase. Each population of RCs fired throughout the corresponding locomotor phase. All RCs received both excitatory and inhibitory synaptic inputs during the locomotor-like rhythmic activity. Blocking nicotinic receptors with mecamylamine markedly reduced the rhythmic excitatory drive, indicating that these rhythmic inputs originate mainly from motor neurons (MNs). Inhibitory synaptic inputs persisted in the presence of the nicotinic blocker. Part of this inhibitory drive and remaining excitatory drive could be from commissural interneurons because the present study also shows that RCs receive direct crossed inhibitory and excitatory synaptic inputs. However, rhythmic synaptic inputs in RCs were also observed in hemicord preparations in the presence of mecamylamine. These results show that, during locomotor activity, RC firing properties are modulated not only by MNs but also by the ipsilateral and contralateral locomotor networks.

Development of locomotor activity induced by NMDA receptor activation in the lumbar spinal cord of the rat fetus studied in vitro.

The development of neuronal circuits generating locomotor activity was characterized in an isolated lumbar spinal cord preparation from of fetal and neonatal rats. Locomotor activity induced by bath application of the NMDA receptor agonists, NMA and NMDA, was monitored from both sides of the corresponding lumbar ventral roots. Activation of NMDA receptors first evoked rhythmic motor activity at E15.5. NMA-induced rhythmic motor activity was not observed under synaptic blockade by TTX or cadmium ions, suggesting that this activity was evoked by synaptic drive from the interneuronal circuits in the spinal cord. At E15.5-E16.5, the rhythmic motor activity on both sides was synchronized. Phase relationship of the rhythmic motor activity between both sides was variable at E17.5-E19.5. The rhythmic motor activity was alternating on both sides at E20.5. Mid-sagittal splitting of the spinal cord did not affect the rhythm generation at all stages examined, suggesting the existence of independent rhythm-generating circuits on each side. The rhythmic motor activity in the presence of strychnine was synchronized on both sides at all stages examined. These results indicate that the changes in rhythm pattern are mediated by development of glycinergic inhibitory pathways, while the basic rhythm can be generated without the glycinergic inhibitory pathways.

Development of the spatial pattern of 5-HT-induced locomotor rhythm in the lumbar spinal cord of rat fetuses in vitro

Developmental changes in the 5-hydroxytryptamine (5-HT)-induced locomotor rhythm were examined in isolated spinal cord preparations taken from fetal rats at embryonic day (E) 16.5, E18.5 and E20.5. Motor activity was recorded from L2/L3 and L5 ventral roots. Bath application of 5-HT evoked rhythmic bursts that were synchronized in all ventral roots studied at E16.5. At E18.5, 5-HT evoked rhythmic bursts that alternated between the two sides, while the bursts in the L2/L3 ventral root were synchronous with those in the ipsilateral L5 ventral root. At E20.5, 5-HT evoked alternate rhythmic bursts in L2/L3 and L5 ventral roots, representing activity in flexors and extensors, respectively. In the presence of strychnine, 5-HT induced rhythmic bursts that were synchronized in all ventral roots studied at E18.5 and E20.5, suggesting that the change in the pattern of rhythmic motor activity that occurs with age is due to the development of glycine-mediated inhibition.

Reorganization of Locomotor Activity during Development in the Prenatal Rata

Abstract: Development of neuronal circuits generating locomotor activity was studied using an isolated lumbar spinal cord preparation from fetal and neonatal rats. Bath application of N‐methyl‐d‐aspartate (NMDA) or 5‐HT evoked patterned motor activity resembling that seen during normal fictive locomotion on embryonic day (E) 20.5. Glycine‐mediated inhibition was essential to the formation of this coordinated motor activity. In preparations from fetuses at the earlier stages (E14.5‐E16.5), we observed spontaneous motoneuronal activity and chemically induced rhythmic bursts, which were synchronized on the two sides in the corresponding ventral roots. The spontaneous activity was not blocked by kynurenate, the glutamate receptor blocker, although it was completely abolished by strychnine, the glycine receptor antagonist. A brief application of glycine evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. It is concluded that glycine functions transiently as excitatory transmitters at these stages. These results suggest that functional change in glycine‐induced responses during development plays an important role in differentiation of the neuronal circuits generating locomotion.

The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice

BackgroundThe vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them.ResultsVGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected.ConclusionVGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes.

Spinal Glutamatergic Neurons Defined by EphA4 Signaling Are Essential Components of Normal Locomotor Circuits

EphA4 signaling is essential for the spatiotemporal organization of neuronal circuit formation. In mice, deletion of this signaling pathway causes aberrant midline crossing of axons from both brain and spinal neurons and the complete knock-outs (KOs) exhibit a pronounced change in motor behavior, where alternating gaits are replaced by a rabbit-like hopping gait. The neuronal mechanism that is responsible for the gait switch in these KO mice is not known. Here, using intersectional genetics, we demonstrate that a spinal cord-specific deletion of EphA4 signaling is sufficient to generate the overground hopping gait. In contrast, selective deletion of EphA4 signaling in forebrain neurons, including the corticospinal tract neurons, did not result in a change in locomotor pattern. The gait switch was attributed to the loss of EphA4 signaling in excitatory Vglut2+ neurons, which is accompanied by an increased midline crossing of Vglut2+ neurons in the ventral spinal cord. Our findings functionally define spinal EphA4 signaling in excitatory Vglut2+ neurons as required for proper organization of the spinal locomotor circuitry, and place these cells as essential components of the mammalian locomotor network.

Developmental changes in rhythmic spinal neuronal activity in the rat fetus

In the developing rat spinal cord, formation and differentiation of the central pattern generator for locomotion occur during the prenatal period. Early on, excitatory synaptic transmission mediated by glycine receptors plays a leading role for rhythmogenesis, at a later stage, followed by glutamate-receptor-mediated synaptic transmission becoming dominant. The maturation of inhibitory circuitry in the spinal cord, mediated largely by glycinergic synapses, is crucial for the generation of alternating activity between left/right limbs and flexor/extensor muscles. Formation of left/right alternation is presumably due to developmental changes in the properties of the postsynaptic neurons, themselves, whereas flexor/extensor alternation requires the additional emergence of inhibitory synaptic functions in the spinal cord.