Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells by Motor Axon Excitatory Inputs

Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/ GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for 2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9 –P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABAA receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABAA receptors

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made

Postnatal Maturation of Gephyrin/Glycine Receptor Clusters on Developing Renshaw Cells

Adult mammalian Renshaw cells express large and complex postsynaptic gephyrin/glycine receptor clusters on their surface. Larger gephyrin clusters correlate with more “efficacious” inhibitory synapses, in terms of larger postsynaptic quantal size amplitudes, in part because they likely contain more postsynaptic receptors (Lim et al. [1999] J. Physiol. (Lond.) 516:505–512; Oleskevich et al. [1999] J. Neurophysiology 82:312–319). Here, we studied the postnatal development of the gephyrin/glycine receptor cluster size on Renshaw cells. Renshaw cells were identified by their calbindin immunoreactivity, location and morphology, and presence of cholinergic input. The populations of clusters over developing Renshaw cells immunoreactive to gephyrin or glycine receptor α1 subunits were comparable in number, size, and complexity and displayed a high degree of colocalization (\u3e90%) at all ages. Quantitative morphologic analysis was performed on gephyrin-immunoreactive clusters. In neonatal animals, Renshaw cells expressed small punctate gephyrin-immunoreactive clusters (mean cluster size ± SD = 0.19 ± 0.19 μm2at 2 days; 0.22 ± 0.19 μm2at 5 days). By 10 and 15 days of age, Renshaw cells exhibited gephyrin-immunoreactive clusters that were larger and more complex (0.32 ± 0.19 μm2 at 10 days; 0.41 ± 0.32 μm2 at 15 days). Cluster growth reached a plateau in 25- and 60-day-old Renshaw cells (0.45 ± 0.43 μm2; 0.56 ± 0.55 μm2, respectively). By using electron microscopy, we confirmed that gephyrin-immunoreactive clusters were located at postsynaptic sites at both early and late postnatal ages on Renshaw cells. The potential significance of this gephyrin/glycine receptor cluster size maturation that sets Renshaw cells apart from other interneurons is discussed. J. Comp. Neurol. 426:130–142, 2000. © 2000 Wiley-Liss, Inc

Postnatal Maturation of Gephyrin/Glycine Receptor Clusters on Developing Renshaw Cells

Adult mammalian Renshaw cells express large and complex postsynaptic gephyrin/glycine receptor clusters on their surface. Larger gephyrin clusters correlate with more “efficacious” inhibitory synapses, in terms of larger postsynaptic quantal size amplitudes, in part because they likely contain more postsynaptic receptors (Lim et al. [1999] J. Physiol. (Lond.) 516:505–512; Oleskevich et al. [1999] J. Neurophysiology 82:312–319). Here, we studied the postnatal development of the gephyrin/glycine receptor cluster size on Renshaw cells. Renshaw cells were identified by their calbindin immunoreactivity, location and morphology, and presence of cholinergic input. The populations of clusters over developing Renshaw cells immunoreactive to gephyrin or glycine receptor α1 subunits were comparable in number, size, and complexity and displayed a high degree of colocalization (\u3e90%) at all ages. Quantitative morphologic analysis was performed on gephyrin-immunoreactive clusters. In neonatal animals, Renshaw cells expressed small punctate gephyrin-immunoreactive clusters (mean cluster size ± SD = 0.19 ± 0.19 μm2at 2 days; 0.22 ± 0.19 μm2at 5 days). By 10 and 15 days of age, Renshaw cells exhibited gephyrin-immunoreactive clusters that were larger and more complex (0.32 ± 0.19 μm2 at 10 days; 0.41 ± 0.32 μm2 at 15 days). Cluster growth reached a plateau in 25- and 60-day-old Renshaw cells (0.45 ± 0.43 μm2; 0.56 ± 0.55 μm2, respectively). By using electron microscopy, we confirmed that gephyrin-immunoreactive clusters were located at postsynaptic sites at both early and late postnatal ages on Renshaw cells. The potential significance of this gephyrin/glycine receptor cluster size maturation that sets Renshaw cells apart from other interneurons is discussed. J. Comp. Neurol. 426:130–142, 2000. © 2000 Wiley-Liss, Inc

Glycine and GABA\u3csub\u3eA\u3c/sub\u3e Receptor Subunits on Renshaw Cells: Relationship with Presynaptic Neurotransmitters and Postsynaptic Gephyrin Clusters

Inhibitory synapses with large and gephyrin-rich postsynaptic receptor areas are likely indicative of higher synaptic strength. We investigated the presynaptic inhibitory neurotransmitter content (GABA, glycine, or both) and the presence and subunit composition of GABAA and glycine postsynaptic receptors in one example of gephyrin-rich synapses to determine neurochemical characteristics that could also contribute to enhance synaptic strength. Hence, we analyzed subunit receptor expression in gephyrin patches located on Renshaw cells, a type of spinal interneuron that receives powerful excitatory and inhibitory inputs and displays many large gephyrin patches on its surface. GABAA and glycine receptors were almost always colocalized inside Renshaw cell gephyrin clusters. According to the subunit-immunoreactivities detected, the composition of GABAA receptors was inferred to be either α3β(2or3)γ2, α5β(2or3)γ2, α3α5β(2or3)γ2 or a combination of these. The types of neurotransmitters contained inside boutons presynaptic to Renshaw cell gephyrin patches were also investigated. The majority (60–75%) of terminals presynaptic to Renshaw cell gephyrin patches contained immunocytochemical markers for GABA as well as glycine, but a proportion contained markers only for glycine. Significantly, 40% of GABAA receptor clusters were opposed to presynaptic boutons that contained only glycinergic markers. We postulate that GABA and glycine corelease, and the presence of α3-containing GABAA receptors can enhance the postsynaptic current and contribute to strengthen inhibitory input on Renshaw cells. In addition, a certain degree of imprecision in the localization of postsynaptic GABAA receptors in regard to GABA release sites onto adult Renshaw cells was also found. J. Comp. Neurol. 444:275–289, 2002. © 2002 Wiley-Liss, Inc

Glycine and GABA\u3csub\u3eA\u3c/sub\u3e Receptor Subunits on Renshaw Cells: Relationship with Presynaptic Neurotransmitters and Postsynaptic Gephyrin Clusters

Inhibitory synapses with large and gephyrin-rich postsynaptic receptor areas are likely indicative of higher synaptic strength. We investigated the presynaptic inhibitory neurotransmitter content (GABA, glycine, or both) and the presence and subunit composition of GABAA and glycine postsynaptic receptors in one example of gephyrin-rich synapses to determine neurochemical characteristics that could also contribute to enhance synaptic strength. Hence, we analyzed subunit receptor expression in gephyrin patches located on Renshaw cells, a type of spinal interneuron that receives powerful excitatory and inhibitory inputs and displays many large gephyrin patches on its surface. GABAA and glycine receptors were almost always colocalized inside Renshaw cell gephyrin clusters. According to the subunit-immunoreactivities detected, the composition of GABAA receptors was inferred to be either α3β(2or3)γ2, α5β(2or3)γ2, α3α5β(2or3)γ2 or a combination of these. The types of neurotransmitters contained inside boutons presynaptic to Renshaw cell gephyrin patches were also investigated. The majority (60–75%) of terminals presynaptic to Renshaw cell gephyrin patches contained immunocytochemical markers for GABA as well as glycine, but a proportion contained markers only for glycine. Significantly, 40% of GABAA receptor clusters were opposed to presynaptic boutons that contained only glycinergic markers. We postulate that GABA and glycine corelease, and the presence of α3-containing GABAA receptors can enhance the postsynaptic current and contribute to strengthen inhibitory input on Renshaw cells. In addition, a certain degree of imprecision in the localization of postsynaptic GABAA receptors in regard to GABA release sites onto adult Renshaw cells was also found. J. Comp. Neurol. 444:275–289, 2002. © 2002 Wiley-Liss, Inc

Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells By Motor Axon Excitatory Inputs

Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for ∼2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9-P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABAA receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABAA receptors

Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells By Motor Axon Excitatory Inputs

Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for ∼2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9-P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABAA receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABAA receptors

\u3cem\u3ePax6\u3c/em\u3e and \u3cem\u3eEngrailed 1\u3c/em\u3e Regulate Two Distinct Aspects of Renshaw Cell Development

Many of the interneuron cell types present in the adult spinal cord contribute to the circuits that control locomotion and posture. Little is known, however, about the embryonic origin of these cell types or the molecular mechanisms that control their differentiation. Here we provide evidence that V1 interneurons (INs), an embryonic class of interneurons that transiently express the En1 transcription factor, differentiate as local circuit inhibitory interneurons and form synapses with motor neurons. Furthermore, we show that a subset of V1 INs differentiates as Renshaw cells, the interneuronal cell type that mediates recurrent inhibition of motor neurons. We analyze the role that two V1 IN-related transcription factor genes play in Renshaw cell development. Pax6 ( paired box gene 6) is necessary for an early step in Renshaw cell development, whereas Engrailed 1 (En1), which is genetically downstream of Pax6, regulates the formation of inhibitory synapses between Renshaw cells and motor neurons. Together, these results show that Pax6 and En1 have essential roles in establishing the recurrent inhibitory circuit between motor neurons and Renshaw cells

\u3cem\u3ePax6\u3c/em\u3e and \u3cem\u3eEngrailed 1\u3c/em\u3e Regulate Two Distinct Aspects of Renshaw Cell Development

Many of the interneuron cell types present in the adult spinal cord contribute to the circuits that control locomotion and posture. Little is known, however, about the embryonic origin of these cell types or the molecular mechanisms that control their differentiation. Here we provide evidence that V1 interneurons (INs), an embryonic class of interneurons that transiently express the En1 transcription factor, differentiate as local circuit inhibitory interneurons and form synapses with motor neurons. Furthermore, we show that a subset of V1 INs differentiates as Renshaw cells, the interneuronal cell type that mediates recurrent inhibition of motor neurons. We analyze the role that two V1 IN-related transcription factor genes play in Renshaw cell development. Pax6 ( paired box gene 6) is necessary for an early step in Renshaw cell development, whereas Engrailed 1 (En1), which is genetically downstream of Pax6, regulates the formation of inhibitory synapses between Renshaw cells and motor neurons. Together, these results show that Pax6 and En1 have essential roles in establishing the recurrent inhibitory circuit between motor neurons and Renshaw cells