Effects of steroids on NMDA receptors and excitatory synaptic transmission in neonatal motoneurons in rat spinal cord slices

The effect of steroids on NMDA receptors and excitatory postsynaptic transmission was studied in fluorescence-labelled motoneurons in thin spinal cord slices. In outside-out patches, NMDA-induced responses were potentiated by 79% in the presence of 20-oxopregn-5-en-3beta-yl sulfate (PS), while in the presence of 20-oxo-5alpha-pregnan-3alpha-yl sulfate (3alpha5alphaS) and 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) they were diminished by 57% and 66%, respectively. PS and 3alpha5betaS had no effect on the amplitude of single NMDA receptor channel openings, however, both compounds altered relative distribution of the openings to individual conductance levels. In control cases, the most frequent openings of the NMDA receptor channels were at the 70 pS conductance level, while in the presence of PS or 3alpha5betaS, the most frequent openings were at the 55 pS conductance level. Analysis of the mean current transferred by NMDA receptor channel openings at individual conductance levels indicated that in the presence of PS, the mean current induced by 55 pS conductance openings was significantly increased. In the presence of 3alpha5betaS, the mean currents induced by 55 pS and 70 pS conductance openings were significantly decreased. The amplitude of NMDA receptor-mediated EPSCs was potentiated by 54% in the presence of PS and the deactivation kinetics slowed. Neither the amplitude nor the kinetics of NMDA receptor-mediated EPSCs was significantly changed in the presence of 3alpha5betaS. The results of our experiments indicate that neurosteroids affect NMDA receptors in motoneurons. The effect appears to be influenced by the receptor subunit composition.     

Characterization of the single-channel properties of NMDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord

The single-channel properties of native NMDA receptors in laminae I and II of the dorsal horn of the neonatal rat spinal cord were studied using outside-out patch-clamp techniques. These receptors were found to have several features that distinguish them from native NMDA receptors elsewhere in the CNS. Single-channel currents activated by NMDA (100 nm) and glycine (10 microm) exhibited five distinct amplitude components with slope-conductance values of 19.9 +/- 0.8, 32.9 +/- 0.6, 42.2 +/- 1.1, 53.0 +/- 1.0 and 68.7 +/- 1.5 pS. Direct transitions were observed between all conductance levels but transitions between 69-pS openings and 20-, 33- and 42-pS openings were rare. There was no significant difference in the frequency of direct transitions from 42- to 20-pS compared to 20- to 42-pS transitions. The Kb (0 mV) for Mg2+ was 89 microm. The Mg2+ unblocking rate constant was similar to other reported values. However, the Mg2+ blocking rate constant was larger than other reported values, suggesting an unusually high sensitivity to Mg2+. The NR2B subunit-selective antagonist, ifenprodil, had no significant effect on overall channel activity but significantly decreased the mean open time of 53-pS openings. These results suggest neonatal laminae I and II NMDA receptors are not simply composed of NR1 and NR2B subunits or NR1 and NR2D subunits. It is possible that these properties are due to an as yet uninvestigated combination of two NR2 subunits with the NR1 subunit or a combination of NR3A, NR2 and NR1 subunits.     

Excitatory synaptic transmission between interneurons and motoneurons in chick spinal cord cell cultures

We have examined the development of synaptic transmission between interneurons and motoneurons in spinal cord cell cultures. Unitary excitatory synaptic currents and complex bursts of excitatory currents develop rapidly: EPSCs (excitatory postsynaptic currents) were detected in 100% of the motoneurons by the 4th day after plating. Inhibitory synaptic currents develop more slowly: IPSCs (inhibitory postsynaptic currents) were detected in only 10% of the motoneurons on day 5 and 40% on day 8. During the 1st and 2nd days in vitro, 24% of the motoneurons tested were dye (Lucifer Yellow) coupled to nearby interneurons. The incidence of dye coupling declined during the first week in culture. No coupling was observed between motoneurons. Our data imply that both G1 and G2 receptors are activated at each synapse. The amplitude of spontaneous excitatory synaptic currents did not change when the motoneuron was hyperpolarized from -50 to -80 mV. This behavior is similar to that of currents induced by glutamate, an agonist that activates 2 types of receptors (G1 and G2) on motoneurons. In addition, a concentration of 2-amino-5-phosphonovaleric acid sufficient to inhibit all G1 receptors only partially inhibited the excitatory synaptic currents. Given the conductance of G1 and G2 channels and the ratio of channels activated during unitary EPSCs, we estimate that as few as 25 G1 channels and 5 G2 channels may mediate excitatory interaction between interneurons and motoneurons during the first week in culture.     

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons.

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate receptor subunit expression after spinal cord injury in young rats

To investigate the possibility that glutamate receptor levels in the spinal cord are altered following injury to young rats, we used a previously characterized model of spinal cord contusion that produces a reliable injury in rats at postnatal day 14-15. Quantitative Western blot analysis was used to measure relative amounts of protein for several glutamate receptor subunits acutely (24 h) and chronically (28 days) after spinal cord injury (SCI). Acutely after injury significant decreases were observed in the GluR1, GluR2, and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor, and the NR2A and NR2B subunits, but not the NR1 subunit, of the N-methyl-d-aspartate (NMDA) receptor. However, 28 days after injury only one subunit (GluR4) was shown to be altered. These widespread changes that occur acutely in receptor subunit expression may be an attempt to protect cells from glutamate-induced death. The injured spinal cord in these young animals, however, appears to have the capacity to regulate receptor subunit levels to normal within a month of injury.     

NR2 to NR3B subunit switchover of NMDA receptors in early postnatal motoneurons

The NR3B NMDA receptor subunit is selective to somatic motoneurons in the adult nervous system. Here we report its developmental expression in the mouse brain and spinal cord by in situ hybridization. NR3B mRNA was detected in few neural regions during embryonic and neonatal periods. It first appeared in motoneurons at postnatal day (P)10-P14, and attained the maximal level at P21 and adult stage. This developmental profile was reciprocal with that of NR2 subunits, of which NR2A mRNA was most predominant in embryonic and neonatal motoneurons and downregulated by P14. Interestingly, mRNA of the NR1 subunit, which is required for functional NMDA receptors, displayed a 'V'-shaped change, decreasing with the early postnatal decline of NR2 mRNAs and increasing with the subsequent appearance of NR3B mRNA. Therefore, the major regulatory subunit of NMDA receptors is likely to switch from NR2 to NR3B in somatic motoneurons during the early postnatal period.     

Cloning and characterization of a novel NMDA receptor subunit NR3B: a dominant subunit that reduces calcium permeability

We report the cloning and characterization of a novel NMDA receptor subunit cDNA, which encodes a predicted polypeptide of 1003 amino acids. Phylogenic analysis indicates that this new subunit is most closely related to NR3A. Therefore, we term it NR3B. Important functional domains of glutamate receptors, such as the ligand-binding domain, the channel pore, and the channel gate, are conserved in NR3B. NR3B mRNA was expressed highly in pons, midbrain, medulla, and the spinal cord, but at low levels in the forebrain and the cerebellum. Although NR3A mRNA expression decreases sharply after the second postnatal weeks, NR3B mRNA expression levels in whole brain were constant during postnatal development and into adult. Coimmunoprecipitation analysis showed that NR3B could form NMDA receptor complex with NR1a and NR2A subunits in heterologous cells. Although expression of NR3B alone did not reconstitute functional NMDA receptors, coexpression of NR3B reduced the Ca(2+) permeability of glutamate-induced currents in cells expressing NR1a and NR2A. These results indicate that NR3B is a dominant modulatory subunit that can modify the function of NMDA receptors. Since high Ca(2+) permeability of NMDA receptors is thought to be a key feature for NMDA receptors to play critical roles in neurodevelopment, synaptic plasticity, and neuronal death, NR3B may contribute to the regulation of these physiological and pathological processes.     

Modulation of embryonic chick motoneuron glutamate sensitivity by interneurons and agonists

Embryonic chick motoneurons grown in culture together with other spinal cord cells are more sensitive to L-glutamate than are sorted motoneurons grown in isolation. After 6 d in vitro, the difference in peak sensitivity reached 6-fold. Comparable increases in aspartate and kainate currents were observed, indicating that both G1 and G2 amino acid receptors were affected. Elimination of proliferating non-neuronal cells from mixed spinal cord cell cultures by addition of cytosine arabinoside (ara C) did not prevent the increase in motoneuron chemosensitivity, so the induction is probably due to the presence of interneurons. In contrast to their effect on glutamate response, interneurons did not affect the sensitivity of motoneurons to the inhibitory neurotransmitters GABA and glycine. Glutamate receptors expressed by sorted and unsorted motoneurons are identical in terms of their ED50, reversal potential, mean channel open time, and conductance, implying that the increased sensitivity of motoneurons in mixed cultures is due to an increase in the number of open channels. In addition to an increase in the number of channels, the distribution of glutamate sensitivity over the surface of individual motoneurons was altered in interneuron-containing cultures. The sensitivity of isolated motoneurons was greatest at the soma and decreased with distance along major processes, but the sites of highest sensitivity on motoneurons in mixed cultures occurred along their processes. Sharp peaks identified by focal iontophoresis of glutamate were separated by areas of lower sensitivity. The inductive effect of interneurons cannot be due to glutamate, the most likely excitatory interneuron-motoneuron transmitter in 6 d chick cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of excitatory amino acid receptors expressed by embryonic chick motoneurons in vitro

We have examined the effect of L-glutamate and other excitatory amino acids on embryonic chick motoneurons maintained in cell culture along with other types of spinal cord cells. When the motoneuron membrane is clamped at -50 mV, glutamate induces a dose-dependent inward current. Although the dose-response curve is hyperbolic with an ED50 of 78 microM, glutamate apparently activates 2 types of receptors on motoneurons. The first, G1, is activated by N-methyl-D-aspartate (NMDA) and aspartate and inhibited by 2-amino-5-phosphonovaleric acid (2-APV). The second, G2, is activated by kainate and quisqualate and is not inhibited by 2-APV. At -50 mV, 38% of the glutamate current is due to activation of G1 receptors and the remaining 62% to G2 activation. In contrast to motoneurons grown with other spinal cord cells, sorted motoneurons grown in isolation apparently exhibit only G2 receptor-mediated currents. Both G1 and G2 currents reverse polarity between -10 and -5 mV. However, they could be distinguished when the membrane was hyperpolarized. G2 currents increased but G1 currents decreased when the membrane potential was increased beyond -50 mV. Consistent with the mixed agonist action of glutamate, glutamate currents remained nearly constant on hyperpolarization. No evidence was obtained that the G2 class of receptors on motoneurons could be subdivided: Quisqualate and kainate apparently compete for the same sites; gamma-glutamylglycine blocked quisqualate as effectively as it blocked kainate currents when the different potencies of the 2 agonists were taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of the N-methyl-d-aspartate glutamate receptor subunit NR2A in control and amyotrophic lateral sclerosis spinal cord

The distribution of the different glutamate receptor subunits in human spinal cord has yet to be fully elucidated. The aim of this study was to examine the distribution of the N-methyl-d-aspartate (NMDA) glutamate receptor modulatory subunit NR2A, in control human spinal cord and to examine in parallel the expression of the mRNA in amyotrophic lateral sclerosis (ALS). The aetiology of ALS is poorly understood, although abnormalities in glutamate and glycine transport have been reported as well as alterations in NMDA receptors including the NRl subunit; suggesting a role for glutamate in the disease process. We have used the technique of in situ hybridisation to localise this receptor subunit to the laminae of human spinal cord and have found that it shows a widespread distribution similar to that previously reported for the universal NMDA receptor subunit NR1. Quantitation of mRNA expression in control and ALS cases showed a significant widespread loss of NR2A from both dorsal and ventral horns with losses of 55% and 78%, respectively. in ALS as compared to control, These results were substantiated by analysis of spinal cord homogenates, which showed a significant total decrease of 50% in ALS spinal cord as compared to control.     

High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture

We examined the membrane action of the endogenous dipeptide and putative neurotransmitter N-acetylaspartylglutamate (NAAG) on the excitatory amino acid receptors of cultured mouse spinal cord neurons using electrophysiological methods. Responses to NAAG (1 microM-5 mM) were compared to those elicited by N-methyl-D-aspartate (1 microM-1 mM) and L-glutamate (0.5-500 microM). Under voltage clamp, concentration-response curves of agonist-evoked currents demonstrated that NAAG was much less potent than either L-glutamate or N-methyl-D-aspartate (NMDA), so that inward currents could be evoked only at NAAG concentrations above 300 microM. Analysis of the dipeptide by high-pressure liquid chromatography showed no evidence of contamination by excitatory amino acids, suggesting that NAAG has an intrinsic, although weak, neuroexcitatory action on spinal neurons. Previous studies have shown that activation of NMDA receptors produces a voltage-dependent response. The current-voltage relationship of responses evoked by NAAG was also voltage-dependent. The peptide-activated conductance decreased with hyperpolarization in the presence of extracellular Mg2+, such that little inward current could be evoked at a membrane potential of -80 mV. In addition, responses to NAAG were completely antagonized by 250 microM DL-2-amino-5-phosphonovaleric acid, a specific NMDA-receptor antagonist. Application of NAAG in Mg2+-free medium resulted in an inward current with a large increase in membrane current noise. The spectral density function of this current noise could be fitted with a single Lorentzian with a decay time constant near 5 msec and a calculated single-channel conductance of 50-60 pS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative measurement of glutamate receptor subunit protein expression in the postnatal rat spinal cord

Glutamate is the major excitatory neurotransmitter in the CNS and its effects on neurons are dependent on the type and composition of glutamate receptors with which it interacts. In this study, the protein expression levels of several ionotropic glutamate receptor subunits (N-methyl-D-aspartate (NMDA) subunits NR1, NR2A, NR2B, and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits GluR1, GluR2, GluR4) were quantified in particulate preparations from rat spinal cord at various ages after birth. We found that all six subunits showed high expression in the early postnatal period, followed by a subsequent decline as the rats matured to adults. The levels of two subunits (NR2A and GluR4) were found to initially increase during the first postnatal week prior to the decline to adult levels. The high levels of expression observed of these subunits in the early postnatal period may have implications for mechanisms of neural injury and cell death in the immature nervous system that involve cation influx through ionotropic glutamate receptors.     

Interaction between metabotropic and ionotropic glutamate receptors regulates neuronal network activity.

Experimental and computational techniques have been used to investigate the group I metabotropic glutamate receptor (mGluR)-mediated increase in the frequency of spinal cord network activity underlying locomotion in the lamprey. Group I mGluR activation potentiated the amplitude of NMDA-induced currents in identified motoneurons and crossed caudally projecting network interneurons. Group I mGluRs also potentiated NMDA-induced calcium responses. This effect was blocked by a group I mGluR-specific antagonist, but not by blockers of protein kinase A, C, or G. The effect of group I mGluRs activation was also tested on NMDA-induced oscillations known to occur during fictive locomotion. Activation of these receptors increased the duration of the plateau phase and decreased the duration of the hyperpolarizing phase. These effects were blocked by a group I mGluR antagonist. To determine its role in the modulation of NMDA-induced oscillations and the locomotor burst frequency, the potentiation of NMDA receptors by mGluRs was simulated using computational techniques. Simulating the interaction between these receptors reproduced the modulation of the plateau and hyperpolarized phases of NMDA-induced oscillations, and the increase in the frequency of the locomotor rhythm. Our results thus show a postsynaptic interaction between group I mGluRs and NMDA receptors in lamprey spinal cord neurons, which can account for the regulation of the locomotor network output by mGluRs.     

Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Transient neonatal expression of NR2B/2D subunit mRNAs of the N-methyl-D-aspartate receptor in the parasympathetic preganglionic neurons in the rat spinal cord

Physiological studies have shown that lower urinary tract function is regulated through glutamate receptors at the levels of spinal and supraspinal cord. Of the receptor family, N-methyl-D-aspartate (NMDA) receptors mediate activity-dependent changes of synaptic efficacy, underlying synaptic plasticity and synapse development. To know the ontogenic changes of NMDA receptor expression in the visceromotor system innervating pelvic organs, including the bladder, we employed double labeling technique of retrograde neuronal tracing and in situ hybridization for detecting NMDA subunit mRNAs in preganglionic neurons (PGNs) of the lumbosacral cord. Rats at postnatal day 7 (P7), 14 (P14), 21 (P21), and adult were used. In situ hybridization was conducted using 35S-labeled antisense oligonucleotides specific to mRNAs for NMDA receptor subunits. Hybridizing signals in PGNs were detected by a dark-field microscope equipped fluorescence detector. PGNs showed strong signals for NR1 subunit mRNA at each developmental stage examined. Moderate signals for the NR2B and NR2D subunit mRNAs were found in PGNs at P7. However, their expression levels decreased thereafter, reaching the minimal level in adults. No significant signals for NR2A and NR2C subunit mRNAs were detected at any stages. This temporal pattern of expression suggests a possible involvement of NMDA receptors in the development of micturitional neural circuit through activity-dependent mechanisms.     

The potential role of phrenic nucleus glutamate receptor subunits in mediating spontaneous crossed phrenic activity in neonatal rat

Cervical spinal cord hemisection rostral to the phrenic nucleus leads to paralysis of the ipsilateral hemidiaphragm in adult rats. Respiratory function can be restored to the paralyzed hemidiaphragm by activating a latent respiratory motor pathway. The latent pathway is called the crossed phrenic pathway. In adult rats, the pathway can be activated by drug-induced upregulation of NMDA receptor NR2A subunit and AMPA receptor GluR1 subunit in the phrenic nucleus following hemisection. In neonatal rats, this pathway is not latent as shown by the spontaneous expression of activity in the ipsilateral hemidiaphragm following hemisection. We hypothesized that the NR2A and GluR1 subunits may be highly expressed naturally on phrenic motoneurons of neonatal rats and may play a potential role in mediating the spontaneous expression of activity in the ipsilateral hemidiaphragm after hemisection. To test this hypothesis, the protein levels of NR2A and GluR1 in different age rats were assessed via Western blot analysis immediately following C2 hemisection and EMG recording of crossed phrenic activity. The protein levels of NR2A and GluR1 were transiently high in postnatal day 2 (P2) rats and then was significantly reduced in P7 and P35 animals. An immunofluorescence study qualitatively supported these findings. The present results indicate that the developmental downregulation of the phrenic nucleus glutamate receptor subunits correlates with the conversion of the crossed phrenic pathway in older postnatal animals from an active state to a latent state.     

Chloride channels gated by extrajunctional glutamate receptors (H-receptors) on locust leg muscle

Outside-out patches of extrasynaptic membrane were isolated from leg muscles of locusts. L-Glutamate and its agonists were applied to such patches either continuously or in rapidly switched pulses. When the pipette contained a high chloride concentration, 2.5 x 10(-5) M glutamate triggered single-channel currents (gated by H-receptors) with a conductance of 25 pS which were carried by chloride, in addition to cationic channels (gated by D-receptors). For the chloride channels, the distribution of channel open times had components of about 2 and 12 ms. Pulses of higher glutamate concentrations elicited many superimposed channel openings, and the approximately saturating concentration of 10(-3) M glutamate opened 100-200 channels simultaneously. When the pipette contained low chloride, channel conductance was reduced, and the current voltage relation was shifted towards the now negative chloride equilibrium potential. H-Receptor-gated chloride channels were activated by glutamate, ibotenate and aspartate, but not by GABA, quisqualate, kainate, N-methyl-D-aspartate and carbachol. The currents declined in the continued presence of agonist showing a time constant of desensitization greater than 1 s. Recovery from desensitization after removal of the agonist was tested with double pulses and was found to have a time constant of about 300 ms.     

Changes in the expression of parvalbumin immunoreactivity in the lumbar spinal cord of the rat following neonatal nerve injury

Nerve injury in newborn animals results in the loss of motoneurons and dorsal root ganglion neurons and long-term changes in reflex activation of surviving motoneurons. Parvalbumin has been previously shown to be found in large-diameter primary afferent axons and interneurons in the spinal cord, and was used here to study the changes in parvalbumin-immunoreactive appositions onto identified tibialis anterior/extensor digitorum longus (TA/EDL) motoneurons, during both normal development and following neonatal nerve injury in the rat spinal cord. During normal development, there was a decrease in the number of parvalbumin-immunoreactive appositions onto TA/EDL motoneurons. Thus, at postnatal day 7 (P7), there were 72.8 +/- 17.5 (mean +/- SD) appositions per motoneuron and by P14, it had decreased to 38.8 +/- 13.2 (mean +/- SD; p > 0.05). Following neonatal nerve injury at P2, there were fewer parvalbumin-positive afferent appositions close to the TA/EDL motoneurons than normal, so that at P7, there were 53.5 +/- 17.1 (mean +/- SD), and at P14, it further decreased to 25.8 +/- 8.6 (mean +/- SD; p > 0.05). This injury-induced reduction in the number of parvalbumin-immunoreactive boutons apposing TA/EDL motoneurons may result, at least in part, from the death of dorsal root ganglion cells with the consequent loss of their central projections. The alterations in the number of parvalbumin-positive appositions close to motoneurons observed in this study may contribute to the changes in the pattern of reflex activity observed in the developing spinal cord both during normal development and following neonatal injury.     

Chronically saturating levels of endogenous glycine disrupt glutamatergic neurotransmission and enhance synaptogenesis in the CA1 region of mouse hippocampus

Glycine serves a dual role in neurotransmission. It is the primary inhibitory neurotransmitter in the spinal cord and brain stem and is also an obligatory coagonist at the excitatory glutamate, N-methyl-D-aspartate receptor (NMDAR). Therefore, the postsynaptic action of glycine should be strongly regulated to maintain a balance between its inhibitory and excitatory inputs. The glycine concentration at the synapse is tightly regulated by two types of glycine transporters, GlyT1 and GlyT2, located on nerve terminals or astrocytes. Genetic studies demonstrated that homozygous (GlyT1-/-) newborn mice display severe sensorimotor deficits characterized by lethargy, hypotonia, and hyporesponsivity to tactile stimuli and ultimately die in their first postnatal day. These symptoms are similar to those associated with the human disease glycine encephalopathy in which there is a high level of glycine in cerebrospinal fluid of affected individuals. The purpose of this investigation is to determine the impact of chronically high concentrations of endogenous glycine on glutamatergic neurotransmission during postnatal development using an in vivo mouse model (GlyT1+/-). The results of our study indicate the following; that compared with wild-type mice, CA1 pyramidal neurons from mutants display significant disruptions in hippocampal glutamatergic neurotransmission, as suggested by a faster kinetic of NMDAR excitatory postsynaptic currents, a lower reduction of the amplitude of NMDAR excitatory postsynaptic currents by ifenprodil, no difference in protein expression for NR2A and NR2B but a higher protein expression for PSD-95, an increase in their number of synapses and finally, enhanced neuronal excitability.