Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Developmental Formation of the GABAergic and Glycinergic Networks in the Mouse Spinal Cord

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine co-releasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play key roles in the regulation of pain, locomotive movement, and respiratory rhythms. Herein, we first describe GABAergic and glycinergic transmission and inhibitory networks, which consist of three types of terminals, in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many of GABAergic neurons convert to co-releasing state. The co-releasing neurons and terminals remain in the dorsal horn, whereas many of co-releasing ones ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Identification of a stereotypic molecular arrangement of endogenous glycine receptors at spinal cord synapses

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs µm-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

Single cell and ensemble activity of lumbar intermediate and ventral horn interneurons in the spinal air-stepping cat

We explored the relationship between population interneuronal network activation and motor output in the adult, in-vivo, air stepping, spinal cat. By simultaneously measuring the activity of large numbers of spinal interneurons, we explored ensembles of coherently firing interneurons and their relation to motor output. Additionally, the networks were analyzed in relation to their spatial distribution along the lumbar enlargement for evidence of localized groups driving particular phases of the locomotor step cycle. We simultaneously recorded hindlimb EMG activity during stepping and extracellular signals from 128 channels across two polytrodes inserted within lamina V-VII of two separate lumbar segments. Results indicated that spinal interneurons participate in one of two ensembles that are highly correlated with the flexor or the extensor muscle bursts during stepping. Interestingly, less than half of the isolated single units were significantly unimodally tuned during the step cycle while >97% of the single units of the ensembles were significantly correlated with muscle activity. These results show the importance of population scale analysis in neural studies of behavior as there is a much greater correlation between muscle activity and ensemble firing than between muscle activity and individual neurons. Finally, we show that there is no correlation between interneurons' rostrocaudal locations within the lumbar enlargement and their preferred phase of firing or ensemble participation. These findings indicate that spinal interneurons of lamina V-VII encoding for different phases of the locomotor cycle are spread throughout the lumbar enlargement in the adult spinal cord.

Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR

IntroductionLY294002 has been validated as a PI3K pan-inhibitor in the pathogenesis of airway inflammation, which attenuated IL-25-induced asthma-like AHR. Liriodendrin was proved to play essential roles in attenuating endometriosis-associated pain. This work aimed to gain a mechanical insight into the therapeutic efficiency of Liriodendrin administration on attenuating endometriosis-associated pain.Material and methodsVon Frey filament test and thermal hyperalgesia test were carried out to evaluate the acute and daily efficiency of drug administration. Western blot was used to analyze the expression of substance P, PI3K/AKT/mTOR, p-PI3K/p-AKT/p-mTOR, and CGRP. ELISA was performed to examine interleukin-1, interleukin-2, interleukin-6, TNF-α and PGE2 levels.ResultsAs a result, Liriodendrin significantly attenuated pain in endometriosis rats and restored the up-regulation of p-PI3K/p-AKT/p-mTOR in the endometriosis rats. The increased interleukin-1, interleukin-2, interleukin-6, TNF-α, and PGE2 levels were remarkably restored by Liriodendrin in endometriosis rats. Moreover, the activated expression of substance P in the ventral horn of the spinal cord of endometriosis rats was notably restored by Liriodendrin in addition to CGRP. Furthermore, Liriodendrin effectively restored the LPS induced up-regulation of p-PI3K/p-AKT/p-mTOR protein as well as the LPS activated IL-6, TNF-α, and IL-1β mRNA and protein expression in 12Z cells.ConclusionsWe found that compared with LY294200, Liriodendrin exerted a more evident therapeutic effect in the treatment of endometriosis-associated pain by suppressing the secretion of pro-inflammatory cytokines.

Downstream projection of Barrington's nucleus to the spinal cord in mice

Barrington's nucleus (Bar) which controls micturition behavior through downstream projections to the spinal cord contains two types of projection neurons BarCRH and BarESR1 that have different functions and target different spinal circuitry. Both types of neurons project to the L6-S1 spinal intermediolateral (IML) nucleus while BarESR1 neurons also project to the dorsal commissural nucleus (DCN). To obtain more information about the spinal circuits targeted by Bar, we used patch-clamp recording in spinal slices from adult mice in combination with optogenetic stimulation of Bar terminals. Recording of opto-evoked excitatory post synaptic currents (oEPSCs) in DiI-labeled lumbosacral preganglionic neurons (LS-PGN) revealed that both Bar neuronal populations make strong glutamatergic monosynaptic connections with LS-PGN, while BarESR1 neurons also elicited smaller amplitude glutamatergic polysynaptic oEPSCs or polysynaptic inhibitory post synaptic currents (oIPSCs) in some LS-PGN. Optical stimulation of BarCRH and BarESR1 terminals also elicited monosynaptic oEPSCs and polysynaptic oIPSCs in sacral DCN neurons, some of which must include interneurons projecting either to the IML or ventral horn. Application of capsaicin increased opto-evoked firing during repetitive stimulation of Bar terminals through the modulation of spontaneous post synaptic currents in LS-PGN. In conclusion, our experiments have provided insights into the synaptic mechanisms underlying the integration of inputs from Bar to autonomic circuitry in the lumbosacral spinal cord that may control micturition.

hnRNP A1B, a Splice Variant of HNRNPA1, Is Spatially and Temporally Regulated

RNA binding proteins (RBPs) play a key role in cellular growth, homoeostasis and survival and are tightly regulated. A deep understanding of their spatiotemporal regulation is needed to understand their contribution to physiology and pathology. Here, we have characterized the spatiotemporal expression pattern of hnRNP A1 and its splice variant hnRNP A1B in mice. We have found that hnRNP A1B expression is more restricted to the CNS compared to hnRNP A1, and that it can form an SDS-resistant dimer in the CNS. Also, hnRNP A1B expression becomes progressively restricted to motor neurons in the ventral horn of the spinal cord, compared to hnRNP A1 which is more broadly expressed. We also demonstrate that hnRNP A1B is present in neuronal processes, while hnRNP A1 is absent. This finding supports a hypothesis that hnRNP A1B may have a cytosolic function in neurons that is not shared with hnRNP A1. Our results demonstrate that both isoforms are differentially expressed across tissues and have distinct localization profiles, suggesting that the two isoforms may have specific subcellular functions that can uniquely contribute to disease progression.

Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brain stem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in native spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that GlyRs exhibit equal receptor-scaffold occupancy and constant absolute packing densities of about 2000 GlyRs µm−2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory PSDs rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.