Excitatory Interneurons Research Articles

Antisense oligonucleotides restore excitability, GABA signalling and sodium current density in a Dravet syndrome model.

Dravet syndrome is an intractable developmental and epileptic encephalopathy caused by de novo variants in SCN1A resulting in haploinsufficiency of the voltage-gated sodium channel Nav1.1. We showed previously that administration of the antisense oligonucleotide STK-001, also called ASO-22, generated using targeted augmentation of nuclear gene output technology to prevent inclusion of the nonsense-mediated decay, or poison, exon 20N in human SCN1A, increased productive Scn1a transcript and Nav1.1 expression and reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy in a mouse model of Dravet syndrome. Here, we investigated the mechanism of action of ASO-84, a surrogate for ASO-22 that also targets splicing of SCN1A exon 20N, in Scn1a+/- Dravet syndrome mouse brain. Scn1a +/- Dravet syndrome and wild-type mice received a single intracerebroventricular injection of antisense oligonucleotide or vehicle at postnatal Day 2. We examined the electrophysiological properties of cortical pyramidal neurons and parvalbumin-positive fast-spiking interneurons in brain slices at postnatal Days 21-25 and measured sodium currents in parvalbumin-positive interneurons acutely dissociated from postnatal Day 21-25 brain slices. We show that, in untreated Dravet syndrome mice, intrinsic cortical pyramidal neuron excitability was unchanged while cortical parvalbumin-positive interneurons showed biphasic excitability with initial hyperexcitability followed by hypoexcitability and depolarization block. Dravet syndrome parvalbumin-positive interneuron sodium current density was decreased compared to wild-type. GABAergic signalling to cortical pyramidal neurons was reduced in Dravet syndrome mice, suggesting decreased GABA release from interneurons. ASO-84 treatment restored action potential firing, sodium current density and GABAergic signalling in Dravet syndrome parvalbumin-positive interneurons. Our work suggests that interneuron excitability is selectively affected by ASO-84. This new work provides critical insights into the mechanism of action of this antisense oligonucleotide and supports the potential of antisense oligonucleotide-mediated upregulation of Nav1.1 as a successful strategy to treat Dravet syndrome.

Social memory deficit caused by dysregulation of the cerebellar vermis

Social recognition memory (SRM) is a key determinant of social interactions. While the cerebellum emerges as an important region for social behavior, how cerebellar activity affects social functions remains unclear. We selectively increased the excitability of molecular layer interneurons (MLIs) to suppress Purkinje cell firing in the mouse cerebellar vermis. Chemogenetic perturbation of MLIs impaired SRM without affecting sociability, anxiety levels, motor coordination or object recognition. Optogenetic interference of MLIs during distinct phases of a social recognition test revealed the cerebellar engagement in the retrieval, but not encoding, of social information. c-Fos mapping after the social recognition test showed that cerebellar manipulation decreased brain-wide interregional correlations and altered network structure from medial prefrontal cortex and hippocampus-centered to amygdala-centered modules. Anatomical tracing demonstrated hierarchical projections from the central cerebellum to the social brain network integrating amygdalar connections. Our findings suggest that the cerebellum organizes the neural matrix necessary for SRM.

Network Model of Nociceptive Processing in the Superficial Spinal Dorsal Horn Reveals Mechanisms of Hyperalgesia, Allodynia, and Spinal Cord Stimulation.

The spinal dorsal horn (DH) processes sensory information and plays a key role in transmitting nociception to supraspinal centers. Loss of DH inhibition during neuropathic pain unmasks a pathway from non-nociceptive Aβ-afferent inputs to superficial dorsal horn (SDH) nociceptive-specific (NS) projection neurons, and this change may contribute to hyperalgesia and allodynia. We developed and validated a computational model of SDH neuronal circuitry that linked non-nociceptive Aβ-afferent inputs in lamina II/III to a NS projection neuron in lamina I via a network of excitatory interneurons. The excitatory pathway and the NS projection neuron were in turn gated by inhibitory interneurons with connections based on prior patch clamp recordings. Changing synaptic weights in the computational model to replicate neuropathic pain states unmasked a low-threshold excitatory pathway to NS neurons similar to experimental recordings. Spinal cord stimulation (SCS) is an effective therapy for neuropathic pain, and accumulating experimental evidence indicates that NS neurons in the SDH also respond to SCS. Accounting for these responses may inform therapeutic improvements, and we quantified responses to SCS in the SDH network model and examined the role of different modes of inhibitory control in modulating NS neuron responses to SCS. We combined the SDH network model with a previously published model of the deep dorsal horn (DDH) and identified optimal stimulation frequencies across different neuropathic pain conditions. Finally, we found that SCS-generated inhibition did not completely suppress model NS activity during simulated pinch inputs, providing an explanation of why SCS does not eliminate acute pain.

Elevated susceptibility to exogenous seizure triggers and impaired interneuron excitability in a mouse model of Leigh syndrome epilepsy

Mutations in the NADH dehydrogenase (ubiquinone reductase) iron‑sulfur protein 4 (NDUFS4) gene, which encodes for a key structural subunit of the OXFOS complex I (CI), lead to the most common form of mitochondrial disease in children known as Leigh syndrome (LS). As in other mitochondrial diseases, epileptic seizures constitute one of the most significant clinical features of LS. These seizures are often very difficult to treat and are a sign of poor disease prognosis. Mice with whole-body Ndufs4 KO are a well-validated model of LS; they exhibit epilepsy and several other clinical features of LS. We have previously shown that mice with Ndufs4 KO in only GABAergic interneurons (Gad2-Ndufs4-KO) reproduce the severe epilepsy phenotype observed in the global KO mice. This observation indicated that these mice represent an excellent model of LS epilepsy isolated from other clinical manifestations of the disease. To further characterize this epilepsy phenotype, we investigated seizure susceptibility to selected exogenous seizure triggers in Gad2-Ndufs4-KO mice. Then, using electrophysiology, imaging, and immunohistochemistry, we studied the cellular, physiological, and neuroanatomical consequences of Ndufs4 KO in GABAergic interneurons. Homozygous KO of Ndufs4 in GABAergic interneurons leads to a prominent susceptibility to exogenous seizure triggers, impaired interneuron excitability and interneuron loss. Finally, we found that the hippocampus and cortex participate in the generation of seizure activity in Gad2-Ndufs4-KO mice. These findings further define the LS epilepsy phenotype and provide important insights into the cellular mechanisms underlying epilepsy in LS and other mitochondrial diseases.

X-linked neuronal migration disorders: Gender differences and insights for genetic screening.

Cortical development depends on neuronal migration of both excitatory and inhibitory interneurons. Neuronal migration disorders (NMDs) are conditions characterised by anatomical cortical defects leading to varying degrees of neurocognitive impairment, developmental delay and seizures. Refractory epilepsy affects 15 million people worldwide, and it is thought that cortical developmental disorders are responsible for 25% of childhood cases. However, little is known about the epidemiology of these disorders, nor are their aetiologies fully understood, though many are associated with sporadic genetic mutations. In this review, we aim to highlight X-linked NMDs including lissencephaly, periventricular nodular heterotopia and polymicrogyria because of their mostly familial inheritance pattern. We focus on the most prominent genes responsible: including DCX, ARX, FLNA, FMR1, L1CAM, SRPX2, DDX3X, NSHDL, CUL4B and OFD1, outlining what is known about their prevalence among NMDs, and the underlying pathophysiology. X-linked disorders are important to recognise clinically, as females often have milder phenotypes. Consequently, there is a greater chance they survive to reproductive age and risk passing the mutations down. Effective genetic screening is important to prevent and treat these conditions, and for this, we need to know gene mutations and have a clear understanding of the function of the genes involved. This review summarises the knowledge base and provides clear direction for future work by both scientists and clinicians alike.

A Continuum of Response Properties across the Population of Unipolar Brush Cells in the Dorsal Cochlear Nucleus.

Unipolar brush cells (UBCs) in the cerebellum and dorsal cochlear nucleus (DCN) perform temporal transformations by converting brief mossy fiber bursts into long-lasting responses. In the cerebellar UBC population, mixing inhibition with graded mGluR1-dependent excitation leads to a continuum of temporal responses. In the DCN, it has been thought that mGluR1 contributes little to mossy fiber responses and that there are distinct excitatory and inhibitory UBC subtypes. Here, we investigate UBC response properties using noninvasive cell-attached recordings in the DCN of mice of either sex. We find a continuum of responses to mossy fiber bursts ranging from 100 ms excitation to initial inhibition followed by several seconds of excitation to inhibition lasting for hundreds of milliseconds. Pharmacological interrogation reveals excitatory responses are primarily mediated by mGluR1 Thus, UBCs in both the DCN and cerebellum rely on mGluR1 and have a continuum of response durations. The continuum of responses in the DCN may allow more flexible and efficient temporal processing than can be achieved with distinct excitatory and inhibitory populations.SIGNIFICANCE STATEMENT UBCs are specialized excitatory interneurons in cerebellar-like structures that greatly prolong the temporal responses of mossy fiber inputs. They are thought to help cancel out self-generated signals. In the DCN, the prevailing view was that there are two distinct ON and OFF subtypes of UBCs. Here, we show that instead the UBC population has a continuum of response properties. Many cells show suppression and excitation consecutively, and the response durations vary considerably. mGluR1s are crucial in generating a continuum of responses. To understand how UBCs contribute to temporal processing, it is essential to consider the continuous variations of UBC responses, which have advantages over just having opposing ON/OFF subtypes of UBCs.

Effect of Non-invasive Spinal Stimulation on Self-sustained Firing Motoneuron Model: In-Silico Study Using Human Body Model.

Transcutaneous spinal electrical stimulation (tSCS) is a non-invasive neuromodulation approach using a low intensity direct current. Recent developments in the technique have opened the possibility that tSCS can help restore motor function after spinal cord injury (SCI). However, the exact mechanism of action tSCS has on the spinal circuits is still unknown. Due to the complexity of experimental synthesis in a human model to delineate the mechanisms, models that link the stimulation paradigm and circuit behaviors are advantageous. Thus, this study aims to simulate the underlying changes in motor circuit firing rates in response to external stimuli induced by tSCS. Serial stimulations combining a high-fidelity finite element model with the human torso and spinal cord with a lumped motor neuron model is constructed. The parameters for both components of the model were derived from previous studies. We focused our analysis on a lumped motor neuron model that describes sustained firing behavior of the motor neuron driven primarily by persistent inward current (PIC), a signature behavior of the motor neuron after SCI. Modulation of the PIC behaviors was achieved by stimulating voltage-dependent calcium and sodium channels in the dendrite using a tSCS-induced electric field (E-field) expressed at different a spatial locations of the motor neuron in the gray matter. The PIC behaviors of spinal motor neurons in the left ventral horn were suppressed, while for the most part invariant in the right ventral horn. These initial simulations will provide a steppingstone for future examinations that incorporate additional neuronal models of inhibitory and excitatory interneurons to access the circuit-level effect of spinal stimulation.

Acute alcohol and chronic drinking bidirectionally regulate the excitability of prefrontal cortex vasoactive intestinal peptide interneurons

The prefrontal cortex (PFC) regulates drinking behaviors and affective changes following chronic alcohol use. PFC activity is dynamically modulated by local inhibitory interneurons (INs), which can be divided into non-overlapping groups with distinct functional roles. Within deeper layers of neocortex, INs that express either parvalbumin or somatostatin directly inhibit pyramidal cells. By contrast, the plurality of all remaining INs express vasoactive intestinal peptide (VIP), reside within superficial layers, and preferentially target other types of INs. While recent studies have described adaptations to PFC parvalbumin-INs and somatostatin-INs in alcohol use models, whether ethanol or drinking affect the physiology of PFC VIP-INs has not been reported. To address this gap, we used genetically engineered female and male mice to target VIP-INs in layers 1–3 of prelimbic PFC for whole-cell patch-clamp electrophysiology. We found that ethanol (20 mM, ∼0.09 BEC/90 mg/dL) application to PFC brain slices enhances VIP-IN excitability. We next examined effects following chronic drinking by providing mice with 4 weeks of intermittent access (IA) ethanol two-bottle choice in the home cage. In these studies, VIP-INs from female and male IA ethanol mice displayed reduced excitability relative to cells from water-only controls. Finally, we assessed whether these effects continue into abstinence. After 7–13 days without ethanol, the hypo-excitability of VIP-INs from male IA ethanol mice persisted, whereas cells from female IA ethanol mice were not different from their controls. Together, these findings illustrate that acute ethanol enhances VIP-IN excitability and suggest these cells undergo pronounced homeostatic changes following long-term drinking.

Age-dependent decrease in inhibitory drive on the excitatory superficial spinal dorsal horn neurons

The excitatory and inhibitory interneurons of superficial laminae I-II of the spinal dorsal horn (SDH) receive and process pain-related information from the primary afferents and transmit it to the brain via the projection neurons. Thus, the interaction between excitatory and inhibitory SDH interneurons is crucial in determining the output from the spinal cord network. Disruption of this interaction in pathological conditions leads to increased SDH output to the higher brain centers, which could underlie pathological pain. Here, we examined whether the changes in the intrinsic SDH connectivity also occur with age, possibly underlying age-related increase in pain sensitivity. Using Vgat;tdTomato transgenic mouse line, we compared the spontaneous inhibitory postsynaptic currents (sIPSCs) in inhibitory tdTomato+ and excitatory tdTomato− interneurons between adult (3–5 m.o.) and aged (12–13 m.o.) mice. We demonstrate that in adult mice, the amplitude and frequency of the sIPSCs on the excitatory interneurons were significantly higher than on inhibitory interneurons. These differences were annulled in aged mice. Further, we show that in aged mice, excitatory neurons receive less inhibition than in adult mice. This could lead to overall disinhibition of the SDH network, which might underlie increased pain perception among the aged population.

Modulation of SK Channels via Calcium Buffering Tunes Intrinsic Excitability of Parvalbumin Interneurons in Neuropathic Pain: A Computational and Experimental Investigation.

Parvalbumin-expressing interneurons (PVINs) play a crucial role within the dorsal horn of the spinal cord by preventing touch inputs from activating pain circuits. In both male and female mice, nerve injury decreases PVINs' output via mechanisms that are not fully understood. In this study, we show that PVINs from nerve-injured male mice change their firing pattern from tonic to adaptive. To examine the ionic mechanisms responsible for this decreased output, we used a reparametrized Hodgkin-Huxley type model of PVINs, which predicted (1) the firing pattern transition is because of an increased contribution of small conductance calcium-activated potassium (SK) channels, enabled by (2) impairment in intracellular calcium buffering systems. Analyzing the dynamics of the Hodgkin-Huxley type model further demonstrated that a generalized Hopf bifurcation differentiates the two types of state transitions observed in the transient firing of PVINs. Importantly, this predicted mechanism holds true when we embed the PVIN model within the neuronal circuit model of the spinal dorsal horn. To experimentally validate this hypothesized mechanism, we used pharmacological modulators of SK channels and demonstrated that (1) tonic firing PVINs from naive male mice become adaptive when exposed to an SK channel activator, and (2) adapting PVINs from nerve-injured male mice return to tonic firing on SK channel blockade. Our work provides important insights into the cellular mechanism underlying the decreased output of PVINs in the spinal dorsal horn after nerve injury and highlights potential pharmacological targets for new and effective treatment approaches to neuropathic pain.SIGNIFICANCE STATEMENT Parvalbumin-expressing interneurons (PVINs) exert crucial inhibitory control over Aβ fiber-mediated nociceptive pathways at the spinal dorsal horn. The loss of their inhibitory tone leads to neuropathic symptoms, such as mechanical allodynia, via mechanisms that are not fully understood. This study identifies the reduced intrinsic excitability of PVINs as a potential cause for their decreased inhibitory output in nerve-injured condition. Combining computational and experimental approaches, we predict a calcium-dependent mechanism that modulates PVINs' electrical activity following nerve injury: a depletion of cytosolic calcium buffer allows for the rapid accumulation of intracellular calcium through the active membranes, which in turn potentiates SK channels and impedes spike generation. Our results therefore pinpoint SK channels as potential therapeutic targets for treating neuropathic symptoms.

Belly roll, a GPI-anchored Ly6 protein, regulates Drosophila melanogaster escape behaviors by modulating the excitability of nociceptive peptidergic interneurons.

Appropriate modulation of escape behaviors in response to potentially damaging stimuli is essential for survival. Although nociceptive circuitry has been studied, it is poorly understood how genetic contexts affect relevant escape responses. Using an unbiased genome-wide association analysis, we identified an Ly6/α-neurotoxin family protein, Belly roll (Bero), which negatively regulates Drosophila nociceptive escape behavior. We show that Bero is expressed in abdominal leucokinin-producing neurons (ABLK neurons) and bero knockdown in ABLK neurons resulted in enhanced escape behavior. Furthermore, we demonstrated that ABLK neurons responded to activation of nociceptors and initiated the behavior. Notably, bero knockdown reduced persistent neuronal activity and increased evoked nociceptive responses in ABLK neurons. Our findings reveal that Bero modulates an escape response by regulating distinct neuronal activities in ABLK neurons.

Primary cilia TRP channel regulates hippocampal excitability

Polycystins (PKD2, PKD2L1, and PKD2L2) are members of the transient receptor potential family, which form ciliary ion channels. Most notably, PKD2 dysregulation in the kidney nephron cilia is associated with polycystic kidney disease, but the function of PKD2L1 in neurons is undefined. In this report, we develop animal models to track the expression and subcellular localization of PKD2L1 in the brain. We discover that PKD2L1 localizes and functions as a Ca2+ channel in the primary cilia of hippocampal neurons that apically radiate from the soma. Loss of PKD2L1 expression ablates primary ciliary maturation and attenuates neuronal high-frequency excitability, which precipitates seizure susceptibility and autism spectrum disorder-like behavior in mice. The disproportionate impairment of interneuron excitability suggests that circuit disinhibition underlies the neurophenotypic features of these mice. Our results identify PKD2L1 channels as regulators of hippocampal excitability and the neuronal primary cilia as organelle mediators of brain electrical signaling.

Excitatory somatostatin interneurons in the dentate gyrus drive a widespread seizure network in cortical dysplasia

Seizures due to cortical dysplasia are notorious for their poor prognosis even with medications and surgery, likely due to the widespread seizure network. Previous studies have primarily focused on the disruption of dysplastic lesions, rather than remote regions such as the hippocampus. Here, we first quantified the epileptogenicity of the hippocampus in patients with late-stage cortical dysplasia. We further investigated the cellular substrates leading to the epileptic hippocampus, using multiscale tools including calcium imaging, optogenetics, immunohistochemistry and electrophysiology. For the first time, we revealed the role of hippocampal somatostatin-positive interneurons in cortical dysplasia-related seizures. Somatostatin-positive were recruited during cortical dysplasia-related seizures. Interestingly, optogenetic studies suggested that somatostatin-positive interneurons paradoxically facilitated seizure generalization. By contrast, parvalbumin-positive interneurons retained an inhibitory role as in controls. Electrophysiological recordings and immunohistochemical studies revealed glutamate-mediated excitatory transmission from somatostatin-positive interneurons in the dentate gyrus. Taken together, our study reveals a novel role of excitatory somatostatin-positive neurons in the seizure network and brings new insights into the cellular basis of cortical dysplasia.

Optogenetic Generation of Neural Firing Patterns with Temporal Shaping of Light Pulses

The fundamental process of information processing and memory formation in the brain is associated with complex neuron firing patterns, which can occur spontaneously or be triggered by sensory inputs. Optogenetics has revolutionized neuroscience by enabling precise manipulation of neuronal activity patterns in specified neural populations using light. However, the light pulses used in optogenetics have been primarily restricted to square waveforms. Here, we present a detailed theoretical analysis of the temporal shaping of light pulses in optogenetic excitation of hippocampal neurons and neocortical fast-spiking interneurons expressed with ultrafast (Chronos), fast (ChR2), and slow (ChRmine) channelrhodopsins. Optogenetic excitation has been studied with light pulses of different temporal shapes that include square, forward-/backward ramps, triangular, left-/right-triangular, Gaussian, left-/right-Gaussian, positive-sinusoidal, and left-/right-positive sinusoidal. Different light shapes result in significantly different photocurrent amplitudes and kinetics, spike-timing, and spontaneous firing rate. For short duration stimulations, left-Gaussian pulse results in larger photocurrent in ChR2 and Chronos than square pulse of the same energy density. Time to peak photocurrent in each opsin is minimum at right-Gaussian pulse. The optimal pulse width to achieve peak photocurrent for non-square pulses is 10 ms for Chronos, and 50 ms for ChR2 and ChRmine. The pulse energy to evoke spike in hippocampal neurons can be minimized on choosing square pulse with Chronos, Gaussian pulse with ChR2, and positive-sinusoidal pulse with ChRmine. The results demonstrate that non-square waveforms generate more naturalistic spiking patterns compared to traditional square pulses. These findings provide valuable insights for the development of new optogenetic strategies to better simulate and manipulate neural activity patterns in the brain, with the potential to improve our understanding of cognitive processes and the treatment of neurological disorders.

Parvalbumin-Positive Interneurons in the Medial Prefrontal Cortex Regulate Stress-Induced Fear Extinction Impairments in Male and Female Rats.

Stress has profound effects on fear extinction, a form of learning that is essential to behavioral therapies for trauma-related and stressor-related disorders. Recent work reveals that acute footshock stress reduces medial prefrontal cortex (mPFC) activity that is critical for extinction learning. Reductions in mPFC activity may be mediated by parvalbumin (PV)-containing interneurons via feedforward inhibition imposed by amygdala afferents. To test this hypothesis, footshock stress-induced Fos expression was characterized in PV+ and PV- neurons in the prelimbic (PL) and infralimbic (IL) cortices. Footshock stress increased the proportion of PV+ cells expressing Fos in both male and female rats; this effect was more pronounced in IL compared with PL. To determine whether PV+ interneurons in the mPFC mediate stress-induced extinction impairments, we chemogenetically silenced these neurons before an immediate extinction procedure in PV-Cre rats. Clozapine-N-oxide (CNO) did not affect conditioned freezing during the extinction procedure. However, CNO exacerbated extinction retrieval in both male and female rats with relatively high PL expression of designer receptors exclusively activated by designer drugs (DREADD). In contrast, in rats with relatively high IL DREADD expression, CNO produced a modest facilitation of extinction in the earliest retrieval trials, but in male rats only. Conversely, excitation of IL PV interneurons was sufficient to impair delayed extinction in both male and female rats. Finally, chemogenetic inhibition of IL-projecting amygdala neurons reduced the immediate extinction deficit in male, but not female rats. These results reveal that PV interneurons regulate extinction learning under stress in a sex-dependent manner, and this effect is mediated by amygdaloprefrontal projections.SIGNIFICANCE STATEMENT Stress significantly impairs the memory of fear extinction, a type of learning that is central to behavioral therapies for trauma-based and anxiety-based disorders (e.g., post-traumatic stress disorder). Here we show that acute footshock stress recruits parvalbumin (PV) interneurons in the medial prefrontal cortex (mPFC) of male and female rats. Silencing mPFC PV interneurons or mPFC-projecting amygdala neurons during immediate extinction influenced extinction retrieval in a sex-dependent manner. This work highlights the role for PV-containing mPFC interneurons in stress-induced impairments in extinction learning.

Spinal Basis of Direction Control during Locomotion in Larval Zebrafish.

Navigation requires steering and propulsion, but how spinal circuits contribute to direction control during ongoing locomotion is not well understood. Here, we use drifting vertical gratings to evoke directed "fictive" swimming in intact but immobilized larval zebrafish while performing electrophysiological recordings from spinal neurons. We find that directed swimming involves unilateral changes in the duration of motor output and increased recruitment of motor neurons, without impacting the timing of spiking across or along the body. Voltage-clamp recordings from motor neurons reveal increases in phasic excitation and inhibition on the side of the turn. Current-clamp recordings from premotor interneurons that provide phasic excitation or inhibition reveal two types of recruitment patterns. A direction-agnostic pattern with balanced recruitment on the turning and nonturning sides is primarily observed in excitatory V2a neurons with ipsilateral descending axons, while a direction-sensitive pattern with preferential recruitment on the turning side is dominated by V2a neurons with ipsilateral bifurcating axons. Inhibitory V1 neurons are also divided into direction-sensitive and direction-agnostic subsets, although there is no detectable morphologic distinction. Our findings support the modular control of steering and propulsion by spinal premotor circuits, where recruitment of distinct subsets of excitatory and inhibitory interneurons provide adjustments in direction while on the move.SIGNIFICANCE STATEMENT Spinal circuits play an essential role in coordinating movements during locomotion. However, it is unclear how they participate in adjustments in direction that do not interfere with coordination. Here we have developed a system using larval zebrafish that allows us to directly record electrical signals from spinal neurons during "fictive" swimming guided by visual cues. We find there are subsets of spinal interneurons for coordination and others that drive unilateral asymmetries in motor neuron recruitment for direction control. Our findings suggest a modular organization of spinal premotor circuits that enables uninterrupted adjustments in direction during ongoing locomotion.

Persistent activation of TNFR1 on spinal cord interneurons contributes to immune dysfunction after spinal cord injury in rodents

Abstract Spinal cord injury (SCI) is a devastating condition with 250,000 to 500,000 new cases globally each year. Respiratory infections, e.g., pneumonia and influenza, are the leading cause of death after SCI. For instance, an individual with SCI is 37 times more likely to become infected and hospitalized from the flu. Yet, the field has a poor understanding of how immune dysfunction arises after trauma to the central nervous system (CNS) and how altered neuro-immune communication impacts an individual’s outcome to infection. In humans and rodents, SCI leads to maladaptive changes in the spinal-sympathetic reflex (SSR) circuit which is crucial to sympathetic function. Soluble Tumor Necrosis Factor (sTNF) is a pro-inflammatory cytokine that is elevated in the CNS after SCI and remains elevated for several months after injury. We recently demonstrated that blocking TNFR1 activation in the CNS, using XPro1595, decreased the maladaptive excitability of SpINs in the SSR circuit leading to improved immune response to infection after SCI. We hypothesize that hyper-excitability of glutamatergic SpINs after SCI due to persistent TNFR1 activation contributes to immune dysfunction. To test this, we utilized viral mediated targeted knock-down of TNFR1 on excitatory SpINs, and demonstrated that inhibiting TNFR1 on excitatory interneurons contributes to improved immune function, including an increase in virus-specific T cells and decrease in viral load remaining in the lung after infection. Supported by grants from NIH (R01 NS111761) Supported by grants from NIH (R01 NS111761)

Differential Modulation of Cerebellar Flocculus Unipolar Brush Cells during Vestibular Compensation.

Vestibular compensation is a natural behavioral recovery process following unilateral vestibular injury. Understanding the mechanism can considerably enhance vestibular disorder therapy and advance the adult central nervous system functional plasticity study after injury. The cerebellum, particularly the flocculonodular lobe, tightly modulates the vestibular nucleus, the center for vestibular compensation; however, it is still unclear if the flocculus on both sides is involved in vestibular compensation. Here we report that the unipolar brush cells (UBCs) in the flocculus are modulated by unilateral labyrinthectomy (UL). UBCs are excitatory interneurons targeting granule cells to provide feedforward innervation to the Purkinje cells, the primary output neurons in the cerebellum. According to the upregulated or downregulated response to the mossy fiber glutamatergic input, UBC can be classified into ON and OFF forms of UBCs. Furthermore, we discovered that the expression of marker genes of ON and OFF UBCs, mGluR1α and calretinin, was increased and decreased, respectively, only in ipsilateral flocculus 4-8 h after UL. According to further immunostaining studies, the number of ON and OFF UBCs was not altered during UL, demonstrating that the shift in marker gene expression level in the flocculus was not caused by the transformation of cell types between UBCs and non-UBCs. These findings imply the importance of ipsilateral flocculus UBCs in the acute response of UL, and ON and OFF UBCs may be involved in vestibular compensation in opposite directions.

Characterisation of NPFF-expressing neurons in the superficial dorsal horn of the mouse spinal cord

Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75–80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.

Synaptic Targets of Glycinergic Neurons in Laminae I-III of the Spinal Dorsal Horn.

A great deal of evidence supports the inevitable importance of spinal glycinergic inhibition in the development of chronic pain conditions. However, it remains unclear how glycinergic neurons contribute to the formation of spinal neural circuits underlying pain-related information processing. Thus, we intended to explore the synaptic targets of spinal glycinergic neurons in the pain processing region (laminae I-III) of the spinal dorsal horn by combining transgenic technology with immunocytochemistry and in situ hybridization accompanied by light and electron microscopy. First, our results suggest that, in addition to neurons in laminae I-III, glycinergic neurons with cell bodies in lamina IV may contribute substantially to spinal pain processing. On the one hand, we show that glycine transporter 2 immunostained glycinergic axon terminals target almost all types of excitatory and inhibitory interneurons identified by their neuronal markers in laminae I-III. Thus, glycinergic postsynaptic inhibition, including glycinergic inhibition of inhibitory interneurons, must be a common functional mechanism of spinal pain processing. On the other hand, our results demonstrate that glycine transporter 2 containing axon terminals target only specific subsets of axon terminals in laminae I-III, including nonpeptidergic nociceptive C fibers binding IB4 and nonnociceptive myelinated A fibers immunoreactive for type 1 vesicular glutamate transporter, indicating that glycinergic presynaptic inhibition may be important for targeting functionally specific subpopulations of primary afferent inputs.