Changes In Neuronal Excitability Research Articles

Impaired synaptic plasticity and decreased glutamatergic neuron excitability induced by SIRT1/BDNF downregulation in the hippocampal CA1 region are involved in postoperative cognitive dysfunction

BackgroundPostoperative cognitive dysfunction (POCD) is a common complication after anesthesia/surgery, especially among elderly patients, and poses a significant threat to their postoperative quality of life and overall well-being. While it is widely accepted that elderly patients may experience POCD following anesthesia/surgery, the exact mechanism behind this phenomenon remains unclear. Several studies have indicated that the interaction between silent mating type information regulation 2 homologue 1 (SIRT1) and brain-derived neurotrophic factor (BDNF) is crucial in controlling cognitive function and is strongly linked to neurodegenerative disorders. Hence, this research aims to explore how SIRT1/BDNF impacts cognitive decline caused by anesthesia/surgery in aged mice.MethodsOpen field test (OFT) was used to determine whether anesthesia/surgery affected the motor ability of mice, while the postoperative cognitive function of 18 months old mice was evaluated with Novel object recognition test (NORT), Object location test (OLT) and Fear condition test (FC). The expressions of SIRT1 and other molecules were analyzed by western blot and immunofluorescence staining. The hippocampal synaptic plasticity was detected by Golgi staining and Long-term potentiation (LTP). The effects of SIRT1 and BDNF overexpression as well as chemogenetic activation of glutamatergic neurons in hippocampal CA1 region of 18 months old vesicular glutamate transporter 1 (VGLUT1) mice on POCD were further investigated.ResultsThe research results revealed that older mice exhibited cognitive impairment following intramedullary fixation of tibial fracture. Additionally, a notable decrease in the expression of SIRT1/BDNF and neuronal excitability in hippocampal CA1 glutamatergic neurons was observed. By increasing levels of SIRT1/BDNF or enhancing glutamatergic neuron excitability in the CA1 region, it was possible to effectively mitigate synaptic plasticity impairment and ameliorate postoperative cognitive dysfunction.ConclusionsThe decline in SIRT1/BDNF levels leading to changes in synaptic plasticity and neuronal excitability in older mice could be a significant factor contributing to cognitive impairment after anesthesia/surgery.Graphical

GluN2A or GluN2B subunits of the NMDA receptor contribute to changes in neuronal excitability and impairments in LTP in the hippocampus of aging mice but do not mediate detrimental effects of oligomeric Aβ (1–42)

Studies in rodent models have revealed that oligomeric beta-amyloid protein [Aβ (1–42)] plays an important role in the pathogenesis of Alzheimer’s disease. Early elevations in hippocampal neuronal excitability caused by Aβ (1–42) have been proposed to be mediated via enhanced activation of GluN2B-containing N-methyl-D-aspartate receptors (NMDAR). To what extent GluN2A or GluN2B-containing NMDAR contribute to Aβ (1–42)-mediated impairments of hippocampal function in advanced rodent age is unclear. Here, we assessed hippocampal long-term potentiation (LTP) and neuronal responses 4–5 weeks after bilateral intracerebral inoculation of 8–15 month old GluN2A+/− or GluN2B+/− transgenic mice with oligomeric Aβ (1–42), or control peptide. Whole-cell patch-clamp recordings in CA1 pyramidal neurons revealed a more positive resting membrane potential and increased total spike time in GluN2A+/−, but not GluN2B+/−-hippocampi following treatment with Aβ (1–42) compared to controls. Action potential 20%-width was increased, and the descending slope was reduced, in Aβ–treated GluN2A+/−, but not GluN2B+/− hippocampi. Sag ratio was increased in Aβ–treated GluN2B+/−-mice. Firing frequency was unchanged in wt, GluN2A+/−, and GluN2B+/−hippocampi after Aβ–treatment. Effects were not significantly different from responses detected under the same conditions in wt littermates, however. LTP that lasted for over 2 h in wt hippocampal slices was significantly reduced in GluN2A+/− and was impaired for 15 min in GluN2B+/−-hippocampi compared to wt littermates. Furthermore, LTP (>2 h) was significantly impaired in Aβ–treated hippocampi of wt littermates compared to wt treated with control peptide. LTP induced in Aβ–treated GluN2A+/− and GluN2B+/−-hippocampi was equivalent to LTP in control peptide-treated transgenic and Aβ–treated wt animals. Taken together, our data indicate that knockdown of GluN2A subunits subtly alters membrane properties of hippocampal neurons and reduces the magnitude of LTP. GluN2B knockdown reduces the early phase of LTP but leaves later phases intact. Aβ (1–42)-treatment slightly exacerbates changes in action potential properties in GluN2A+/−-mice. However, the vulnerability of the aging hippocampus to Aβ–mediated impairments of LTP is not mediated by GluN2A or GluN2B-containing NMDAR.

Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice

BackgroundChronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM.MethodsA mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated.ResultsRepeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE.ConclusionEE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM.

Aberrant cortical activity, functional connectivity, and neural assembly architecture after photothrombotic stroke in mice.

Despite substantial progress in mapping the trajectory of network plasticity resulting from focal ischemic stroke, the extent and nature of changes in neuronal excitability and activity within the peri-infarct cortex of mice remains poorly defined. Most of the available data have been acquired from anesthetized animals, acute tissue slices, or infer changes in excitability from immunoassays on extracted tissue, and thus may not reflect cortical activity dynamics in the intact cortex of an awake animal. Here, in vivo two-photon calcium imaging in awake, behaving mice was used to longitudinally track cortical activity, network functional connectivity, and neural assembly architecture for 2 months following photothrombotic stroke targeting the forelimb somatosensory cortex. Sensorimotor recovery was tracked over the weeks following stroke, allowing us to relate network changes to behavior. Our data revealed spatially restricted but long-lasting alterations in somatosensory neural network function and connectivity. Specifically, we demonstrate significant and long-lasting disruptions in neural assembly architecture concurrent with a deficit in functional connectivity between individual neurons. Reductions in neuronal spiking in peri-infarct cortex were transient but predictive of impairment in skilled locomotion measured in the tapered beam task. Notably, altered neural networks were highly localized, with assembly architecture and neural connectivity relatively unaltered a short distance from the peri-infarct cortex, even in regions within 'remapped' forelimb functional representations identified using mesoscale imaging with anaesthetized preparations 8 weeks after stroke. Thus, using longitudinal two-photon microscopy in awake animals, these data show a complex spatiotemporal relationship between peri-infarct neuronal network function and behavioral recovery. Moreover, the data highlight an apparent disconnect between dramatic functional remapping identified using strong sensory stimulation in anaesthetized mice compared to more subtle and spatially restricted changes in individual neuron and local network function in awake mice during stroke recovery.

Aberrant cortical activity, functional connectivity, and neural assembly architecture after photothrombotic stroke in mice

Despite substantial progress in mapping the trajectory of network plasticity resulting from focal ischemic stroke, the extent and nature of changes in neuronal excitability and activity within the peri-infarct cortex of mice remains poorly defined. Most of the available data have been acquired from anesthetized animals, acute tissue slices, or infer changes in excitability from immunoassays on extracted tissue, and thus may not reflect cortical activity dynamics in the intact cortex of an awake animal. Here, in vivo two-photon calcium imaging in awake, behaving mice was used to longitudinally track cortical activity, network functional connectivity, and neural assembly architecture for 2 months following photothrombotic stroke targeting the forelimb somatosensory cortex. Sensorimotor recovery was tracked over the weeks following stroke, allowing us to relate network changes to behavior. Our data revealed spatially restricted but long-lasting alterations in somatosensory neural network function and connectivity. Specifically, we demonstrate significant and long-lasting disruptions in neural assembly architecture concurrent with a deficit in functional connectivity between individual neurons. Reductions in neuronal spiking in peri-infarct cortex were transient but predictive of impairment in skilled locomotion measured in the tapered beam task. Notably, altered neural networks were highly localized, with assembly architecture and neural connectivity relatively unaltered a short distance from the peri-infarct cortex, even in regions within ‘remapped’ forelimb functional representations identified using mesoscale imaging with anaesthetized preparations 8 weeks after stroke. Thus, using longitudinal two-photon microscopy in awake animals, these data show a complex spatiotemporal relationship between peri-infarct neuronal network function and behavioral recovery. Moreover, the data highlight an apparent disconnect between dramatic functional remapping identified using strong sensory stimulation in anaesthetized mice compared to more subtle and spatially restricted changes in individual neuron and local network function in awake mice during stroke recovery.

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria

BackgroundExcess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes.MethodsThe split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing.ResultsExploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1–JAK2–FGF14–Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection.ConclusionsFGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Mitochondrial malfunction and atrophy of astrocytes in the aged human cerebral cortex

How aging affects cells of the human brain active milieu remains largely unknown. Here, we analyze astrocytes and neurons in the neocortical tissue of younger (22–50 years) and older (51–72 years) adults. Aging decreases the amount of reduced mitochondrial cytochromes in astrocytes but not neurons. The protein-to-lipid ratio decreases in astrocytes and increases in neurons. Aged astrocytes show morphological atrophy quantified by the decreased length of branches, decreased volume fraction of leaflets, and shrinkage of the anatomical domain. Atrophy correlates with the loss of gap junction coupling between astrocytes and increased input resistance. Aging is accompanied by the upregulation of glial fibrillary acidic protein (GFAP) and downregulation of membrane-cytoskeleton linker ezrin associated with leaflets. No significant changes in neuronal excitability or spontaneous inhibitory postsynaptic signaling is observed. Thus, brain aging is associated with the impaired morphological presence and mitochondrial malfunction of cortical astrocytes, but not neurons.

Changes in neuronal excitability in the rat hippocampus in a prolonged febrile seizures model

Febrile seizures (FSs) are a prevalent neurological disorder among children aged 3 months to 5 years, with the highest incidence observed in the second year of life [1]. Considering that neuronal and glial cell development and synaptic contact formation are ongoing during this period [2, 3], FSs can potentially influence these processes. However, the present data on the effect of FSs on brain development remain inconsistent. This study aims to examine the impact of extended seizures on the attributes of hippocampal pyramidal neurons in rats of varying ages. Wistar rats were used in this study. FSs were induced on postnatal day (P)10 through placing pups onto the bottom of a glass chamber for 30 minutes and exposing them to a controlled stream of heated air, causing their body temperature to rise to 39 °C and trigger the occurrence of FSs. Only animals that underwent FSs that persisted for at least 15 minutes were included in the study. Littermates were employed as controls and were placed away from the nest for the same duration but kept at room temperature. At postnatal days 12, 21–23, and 51–55, the rats were decapitated, and their brains were extracted. Horizontal brain slices (400 micrometers) were sliced. The study examined the biophysical properties of CA1 pyramidal neurons using the whole-cell patch-clamp method. 1.5-second current pulses were injected, and subthreshold membrane properties such as resting membrane potential, input resistance, membrane time constant, and intrinsic firing properties, were assessed. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum of the hippocampus to assess the efficacy of synaptic neurotransmission at CA3-CA1 pyramidal neuron synapses. The amplitude and fiber volley (FV) amplitude of each fEPSP were measured by applying high amplitude currents to each slice. A sigmoidal Gompertz function was used to evaluate the efficacy of neurotransmission. Paired-pulse stimulation was used to examine potential alterations in short-term synaptic plasticity. Paired pulses were administered at intervals spanning from 10 to 500 ms, and the paired-pulse ratio (PPR) was assessed as the proportion of the amplitude of the second to the first fEPSP for each interval. Maximum electroshock seizure threshold (MEST) was determined two months after FS to assess the animals’ susceptibility to seizures. The lowest current at which tonic hind limb extension was exhibited was determined for every animal. No significant changes in subthreshold firing properties were observed in P12 or P21 rats following FSs. However, we did observe several changes in intrinsic firing properties. For example, the maximum firing frequency decreased by 23% in P12 rats exposed to FSs, relative to age-matched control rats. Additionally, we noticed that firing frequency adaptation was significantly less pronounced in P12 rats that underwent FSs, compared to control rats of the same age. Seizure-induced alterations in firing characteristics were absent in P21 rats. No significant variations in fEPSP or FV amplitudes were found among the different groups of P12 and P55 rats at different current intensities. However, a significant increase in FV amplitudes and decrease in neuronal input-output (I/O) relationships between fEPSP and FV amplitudes were observed in P21 rats following FSs. At P12, short-term synaptic plasticity was disrupted, evidenced by a significant increase in PPR in rats two days post-FS. However, at P21 and P55, experimental and control groups were not significantly different. MEST test displayed a significant rise in the hind limb extension threshold in rats two months following FS as compared to control animals. Overall, these findings suggest alterations in neuronal excitability after prolonged FS. Two days after FS, neuronal excitability transiently decreased while PPR increased, indicating a decrease in the probability of presynaptic glutamate release in hippocampal neurons. The efficiency of synaptic neurotransmission in CA3-CA1 was reduced in three-week-old animals. Additionally, the MEST test demonstrated that rats, two months following FS, exhibited a higher hind limb extension threshold in comparison to control animals.

Relationship between acupoint sensitization and changes in dorsal root ganglion neuron excitability in myocardial ischemia mice

To investigate the relationship between the sensitization state of acupoints on the surface of the myocardial ischemia (MI) model mice and the changes in the electrophysiological properties of the dorsal root ganglion (DRG) neurons in the corresponding spinal cord segment, and its underlying mechanism. Sixty-eight male C57BL/6J mice were randomly divided into control and model groups (34 mice in each group). The model group received an intraperitoneal injection of 160 mg/kg isoproterenol (ISO) to establish the MI model, and the control group received an injection of the same dose of normal saline as the model group. After modeling for about 6 days, MI proportion was measured by HE staining to verify the pathological changes in the heart tissue. Evans blue (EB) dye was injected into the tail vein of mice to reflect the size, location, distribution, and number of exudates on the body surface. Then, whole-cell membrane currents, intrinsic excitability and membrane properties of different types of DRG neurons were evaluated by electrophysiological experiment in vitro. Compared with the control group, the heart size was larger, with pathological outcomes showing enlarged myocardial hypertrophy, destroyed structure of cardiomyocytes, with mononuclear cell infiltration among the cardiomyocytes in the model group. Compared with the control group, the number of EB exudation points was significantly increased (P<0.01), which were mainly concentrated in the epidermis near the T1-T5 segment of the spinal cord, "Feishu" (BL13), "Jueyinshu" (BL14) and "Xinshu" (BL15) in the model group. Compared with the control group, the rheobase and action potential amplitude (APA) of DRG medium-sized neurons were obviously decreased (P<0.01, P<0.05), while the whole-cell membrane currents, the spike numbers, the average instantaneous frequency, and the average discharge frequency were markedly increased (P<0.01). There were no significant alterations in the membrane properties and intrinsic excitability induced by depolarized currents of small-sized neurons between groups. Compared with the control group, the whole-cell membrane currents, spike numbers, and the average instantaneous frequency were significantly increased in the model group(P<0.05, P<0.01) while rheobase was significantly decreased (P<0.05) in DRG medium-sized neurons labeled with biotin and CGRP. After the mice were modeled by ISO, the DRG medium-size neurons in the T1-T5 segment of the spinal cord may mediate the sensitization of acupoints on the body surface through their different neuronal membrane properties and intrinsic excitabilities.

Molecular machines stimulate intercellular calcium waves and cause muscle contraction.

Intercellular calcium waves (ICW) are complex signalling phenomena that control many essential biological activities, including smooth muscle contraction, vesicle secretion, gene expression and changes in neuronal excitability. Accordingly, the remote stimulation of ICW could result in versatile biomodulation and therapeutic strategies. Here we demonstrate that light-activated molecular machines (MM)-molecules that perform mechanical work on the molecular scale-can remotely stimulate ICW. MM consist of a polycyclic rotor and stator that rotate around a central alkene when activated with visible light. Live-cell calcium-tracking and pharmacological experiments reveal that MM-induced ICW are driven by the activation of inositol-triphosphate-mediated signalling pathways by unidirectional, fast-rotating MM. Our data suggest that MM-induced ICW can control muscle contraction in vitro in cardiomyocytes and animal behaviour in vivo in Hydra vulgaris. This work demonstrates a strategy for directly controlling cell signalling and downstream biological function using molecular-scale devices.

Optimized monophasic pulses with equivalent electric field for rapid-rate transcranial magnetic stimulation

Objective. Transcranial magnetic stimulation (TMS) with monophasic pulses achieves greater changes in neuronal excitability but requires higher energy and generates more coil heating than TMS with biphasic pulses, and this limits the use of monophasic pulses in rapid-rate protocols. We sought to design a stimulation waveform that retains the characteristics of monophasic TMS but significantly reduces coil heating, thereby enabling higher pulse rates and increased neuromodulation effectiveness. Approach. A two-step optimization method was developed that uses the temporal relationship between the electric field (E-field) and coil current waveforms. The model-free optimization step reduced the ohmic losses of the coil current and constrained the error of the E-field waveform compared to a template monophasic pulse, with pulse duration as a second constraint. The second, amplitude adjustment step scaled the candidate waveforms based on simulated neural activation to account for differences in stimulation thresholds. The optimized waveforms were implemented to validate the changes in coil heating. Main results. Depending on the pulse duration and E-field matching constraints, the optimized waveforms produced 12%–75% less heating than the original monophasic pulse. The reduction in coil heating was robust across a range of neural models. The changes in the measured ohmic losses of the optimized pulses compared to the original pulse agreed with numeric predictions. Significance. The first step of the optimization approach was independent of any potentially inaccurate or incorrect model and exhibited robust performance by avoiding the highly nonlinear behavior of neural responses, whereas neural simulations were only run once for amplitude scaling in the second step. This significantly reduced computational cost compared to iterative methods using large populations of candidate solutions and more importantly reduced the sensitivity to the choice of neural model. The reduced coil heating and power losses of the optimized pulses can enable rapid-rate monophasic TMS protocols.

Systems-level analysis of local field potentials reveals differential effects of lysergic acid diethylamide and ketamine on neuronal activity and functional connectivity.

Psychedelic substances have in recent years attracted considerable interest as potential treatments for several psychiatric conditions, including depression, anxiety, and addiction. Imaging studies in humans point to a number of possible mechanisms underlying the acute effects of psychedelics, including changes in neuronal firing rates and excitability as well as alterations in functional connectivity between various brain nodes. In addition, animal studies using invasive recordings, have suggested synchronous high-frequency oscillations involving several brain regions as another key feature of the psychedelic brain state. To better understand how the imaging data might be related to high-resolution electrophysiological measurements, we have here analyzed the aperiodic part of the local field potential (LFP) in rodents treated with a classic psychedelic (LSD) or a dissociative anesthetic (ketamine). In addition, functional connectivity, as quantified by mutual information measures in the LFP time series, has been assessed with in and between different structures. Our data suggest that the altered brain states of LSD and ketamine are caused by different underlying mechanisms, where LFP power shifts indicate increased neuronal activity but reduced connectivity following ketamine, while LSD also leads to reduced connectivity but without an accompanying change in LFP broadband power.

Neuromodulatory control of complex adaptive dynamics in the brain.

How is the massive dimensionality and complexity of the microscopic constituents of the nervous system brought under sufficiently tight control so as to coordinate adaptive behaviour? A powerful means for striking this balance is to poise neurons close to the critical point of a phase transition, at which a small change in neuronal excitability can manifest a nonlinear augmentation in neuronal activity. How the brain could mediate this critical transition is a key open question in neuroscience. Here, I propose that the different arms of the ascending arousal system provide the brain with a diverse set of heterogeneous control parameters that can be used to modulate the excitability and receptivity of target neurons-in other words, to act as control parameters for mediating critical neuronal order. Through a series of worked examples, I demonstrate how the neuromodulatory arousal system can interact with the inherent topological complexity of neuronal subsystems in the brain to mediate complex adaptive behaviour.

Human tau-overexpressing mice recapitulate brainstem involvement and neuropsychiatric features of early Alzheimer’s disease

Alzheimer’s disease (AD) poses an ever-increasing public health concern as the population ages, affecting more than 6 million Americans. AD patients present with mood and sleep changes in the prodromal stages that may be partly driven by loss of monoaminergic neurons in the brainstem, but a causal relationship has not been firmly established. This is due in part to a dearth of animal models that recapitulate early AD neuropathology and symptoms. The goal of the present study was to evaluate depressive and anxiety-like behaviors in a mouse model of AD that overexpresses human wild-type tau (htau) prior to the onset of cognitive impairments and assess these behavior changes in relationship to tau pathology, neuroinflammation, and monoaminergic dysregulation in the dorsal raphe nucleus (DRN) and locus coeruleus (LC). We observed depressive-like behaviors at 4 months in both sexes and hyperlocomotion in male htau mice. Deficits in social interaction persisted at 6 months and were accompanied by an increase in anxiety-like behavior in males. The behavioral changes at 4 months coincided with a lower density of serotonergic (5-HT) neurons, downregulation of 5-HT markers, reduced excitability of 5-HT neurons, and hyperphosphorylated tau in the DRN. Inflammatory markers were also upregulated in the DRN along with protein kinases and transglutaminase 2, which may promote tau phosphorylation and aggregation. Loss of 5-HT innervation to the entorhinal cortex and dentate gyrus of the hippocampus was also observed and may have contributed to depressive-like behaviors. There was also reduced expression of noradrenergic markers in the LC along with elevated phospho-tau expression, but this did not translate to a functional change in neuronal excitability. In total, these results suggest that tau pathology in brainstem monoaminergic nuclei and the resulting loss of serotonergic and/or noradrenergic drive may underpin depressive- and anxiety-like behaviors in the early stages of AD.

A17 EVIDENCE OF PROTEASE-MEDIATED PRO-NOCICEPTIVE EFFECTS OF FECAL SUPERNATANTS FROM CROHN'S DISEASE PATIENTS

Abstract Background Abdominal pain is a debilitating symptom of Crohn’s disease (CD). Despite the current treatment options for this disease, abdominal pain is an unresolved problem that commonly persists in the absence of active inflammation. This suggests that something other than inflammation is driving the pain during the quiescent phase. We have previously reported that microbial proteases can directly modulate the excitability of dorsal root ganglia (DRG) neurons, many of which are pain-sensing. We hypothesize that luminal proteases of CD patients are contributing to their abdominal pain. Purpose Determine whether luminal mediators in CD fecal samples induce changes in pain signalling. Method The effects of patient (active CD [n = 3] and healthy volunteer (HV) [n = 3]) fecal supernatant (FS) samples on pain-sensing neurons were assessed using ex-vivo single unit afferent nerve recordings from mouse colons. Each sample was tested in colonic preparations from a least 5 mice. To further examine cellular mechanisms, DRG neurons were isolated and incubated overnight in media containing CD FS or HV FS media. Changes in neuronal excitability were recorded by determining the rheobase (lower rheobase=increased excitability) using patch clamp recordings (n ≥ 9 DRG neurons/group). Protease inhibitors were applied in both bioassays to determine whether these inhibited the excitatory effect of FS. Lastly, total proteolytic activity in the CD and HV fecal samples was calculated using a casein colorimetric protease detection assay. Result(s) FS from HV had no effect on afferent nerve excitability (p = 0.8920). FS from active CD patients increased action potential discharge from colonic afferent nerves by 85% (p&amp;lt;0.0001) and selectively increased the activation of high-threshold units, which are putative nociceptors, by 44% (p=0.0074). A protease inhibitor cocktail (1:1000) and protease-activated receptor (PAR)-2 antagonist GB83 (10µM) both blocked the excitatory effects of CD FS (p&amp;lt;0.05). Overnight incubation with CD FS also had an excitatory effect on DRG neurons compared to HV FS (rheobase decreased by 46%, p&amp;lt;0.05). The effect of CD FS was blocked by GB83 (10µM) (p&amp;lt;0.001) and a serine protease inhibitor (FUT175; 100µM) (p&amp;lt;0.05) independently, but the activity was not blocked by E64 (30nM) a cysteine protease inhibitor. A 200-fold increase (p&amp;lt;0.0001) in total proteolytic activity was found in CD FS compared to HV FS. Conclusion(s) Luminal serine proteases, but not cysteine proteases, appear to be driving nociceptive signalling in CD patients. This provides insight into the generation of pain in CD patients and may be a potential target to mitigate this action. Further research is required to elucidate whether these pro-nociceptive proteases are of bacterial or host origin and their effects in the quiescent phase. Disclosure of Interest None Declared

Investigating genotype-phenotype relationship of extreme neuropathic pain disorders in a UK national cohort.

The aims of our study were to use whole genome sequencing in a cross-sectional cohort of patients to identify new variants in genes implicated in neuropathic pain, to determine the prevalence of known pathogenic variants and to understand the relationship between pathogenic variants and clinical presentation. Patients with extreme neuropathic pain phenotypes (both sensory loss and gain) were recruited from secondary care clinics in the UK and underwent whole genome sequencing as part of the National Institute for Health and Care Research Bioresource Rare Diseases project. A multidisciplinary team assessed the pathogenicity of rare variants in genes previously known to cause neuropathic pain disorders and exploratory analysis of research candidate genes was completed. Association testing for genes carrying rare variants was completed using the gene-wise approach of the combined burden and variance-component test SKAT-O. Patch clamp analysis was performed on transfected HEK293T cells for research candidate variants of genes encoding ion channels. The results include the following: (i) Medically actionable variants were found in 12% of study participants (205 recruited), including known pathogenic variants: SCN9A(ENST00000409672.1): c.2544T>C, p.Ile848Thr that causes inherited erythromelalgia, and SPTLC1(ENST00000262554.2):c.340T>G, p.Cys133Tr variant that causes hereditary sensory neuropathy type-1. (ii) Clinically relevant variants were most common in voltage-gated sodium channels (Nav). (iii) SCN9A(ENST00000409672.1):c.554G>A, pArg185His variant was more common in non-freezing cold injury participants than controls and causes a gain of function of NaV1.7 after cooling (the environmental trigger for non-freezing cold injury). (iv) Rare variant association testing showed a significant difference in distribution for genes NGF, KIF1A, SCN8A, TRPM8, KIF1A, TRPA1 and the regulatory regions of genes SCN11A, FLVCR1, KIF1A and SCN9A between European participants with neuropathic pain and controls. (v) The TRPA1(ENST00000262209.4):c.515C>T, p.Ala172Val variant identified in participants with episodic somatic pain disorder demonstrated gain-of-channel function to agonist stimulation. Whole genome sequencing identified clinically relevant variants in over 10% of participants with extreme neuropathic pain phenotypes. The majority of these variants were found in ion channels. Combining genetic analysis with functional validation can lead to a better understanding as to how rare variants in ion channels lead to sensory neuron hyper-excitability, and how cold, as an environmental trigger, interacts with the gain-of-function NaV1.7 p.Arg185His variant. Our findings highlight the role of ion channel variants in the pathogenesis of extreme neuropathic pain disorders, likely mediated through changes in sensory neuron excitability and interaction with environmental triggers.

KCNQ4 potassium channel subunit deletion leads to exaggerated acoustic startle reflex in mice.

The potassium voltage-gated channel subfamily Q member 4 (KCNQ4) subunit forms channels responsible for M-current, a muscarine-sensitive potassium current regulating neuronal excitability. In contrast to other KCNQ subunits, its expression is restricted to the cochlear outer hair cells, the auditory brainstem and other brainstem nuclei in a great overlap with structures involved in startle reflex. We aimed to show whether startle reflexis affected by the loss of KCNQ4 subunit and whether these alterations are similar to the ones caused by brainstem hyperexcitability. Young adult KCNQ4 knockout mice and wild-type littermates, as well as mice expressing hM3D chemogenetic actuator in the pontine caudal nucleus and neurons innervating it were used for testing acoustic startle. The acoustic startle reflex was significantly increased in knockout mice compared with wild-type littermates. When mice expressing human M3 muscarinic (hM3D) in nuclei related to startle reflex were tested, a similar increase of the first acoustic startle amplitude and a strong habituation of the further responses was demonstrated. We found that the acoustic startle reflex is exaggerated and minimal habituation occurs in KCNQ4 knockout animals. These changes are distinct from the effects of the hyperexcitability of nuclei involved in startle. One can conclude that the exaggerated startle reflex found with the KCNQ4 subunit deletion is the consequence of both the cochlear damage and the changes in neuronal excitability of startle networks.

CaV3.1 T-type calcium channels are important for spatial memory processing in the dorsal subiculum

The dorsal subiculum (dSub) is one of the key structures responsible for the formation of hippocampal memory traces but the contribution of individual ionic currents to its cognitive function is not well studied. Although we recently reported that low-voltage-activated T-type calcium channels (T-channels) are crucial for the burst firing pattern regulation in the dSub pyramidal neurons, their potential role in learning and memory remains unclear. Here we used in vivo local field potential recordings and miniscope calcium imaging in freely behaving mice coupled with pharmacological and genetic tools to address this gap in knowledge. We show that the CaV3.1 isoform of T-channels is critically involved in controlling neuronal activity in the dSub in vivo. Altering neuronal excitability by inhibiting T-channel activity markedly affects calcium dynamics, synaptic plasticity, neuronal oscillations and phase-amplitude coupling in the dSub, thereby disrupting spatial learning. These results provide an important causative link between the CaV3.1 channels, burst firing of dSub neurons and memory formation, thus further supporting the notion that changes in neuronal excitability regulate memory processing. We posit that subicular CaV3.1 T-channels could be a promising novel drug target for cognitive disorders.

Transcranial direct current stimulation as a preventive treatment in multiple sclerosis? Preclinical evidence

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system, presenting with optic neuritis in about 20–30% of cases. Optic nerve demyelination, associated with delay of visual evoked potentials (VEPs), is also observed prior to motor signs in the preclinical MS model Experimental Autoimmune Encephalomyelitis (EAE). Transcranial direct current stimulation (tDCS), inducing polarity-dependent changes in neuronal excitability, is widely used to promote neuroplasticity in several neurological disorders. However, its potential effects on inflammation and demyelination are largely unknown. We tested the effectiveness of a preventive, 5-day tDCS treatment started 3 days post-immunization, in reducing the severity of VEP delays observed in early EAE. In mice undergoing cathodal tDCS (n = 6/26 eyes) VEPs were significantly less delayed compared with eyes from EAE-Sham (n = 24/32 eyes) and EAE-Anodal (n = 22/32 eyes). Optic nerve immunohistochemistry revealed a significantly lower cell density of microglia/macrophages, and less axonal loss in EAE-Cathodal vs EAE-Sham and EAE-Anodal, while the percent demyelination with Luxol-fast blue staining was comparable among EAE groups. Considering the latter result, immunofluorescence paranodal staining was performed, revealing a significantly higher number of complete paranode domains in EAE-Cathodal, closer to healthy mice, compared with EAE-Sham and EAE-Anodal groups. These results were reflected by the negative correlation between the number of complete paranode domains and VEP latency increase with respect to pre-immunization. Finally, cathodal tDCS was associated with a lower number, closer to healthy, of single paranodes in contrast to EAE-Sham. The effects of cathodal stimulation in preventing VEPs delays and optic nerve myelin damage were already observed in the pre-motor onset EAE stage, and were associated with a lower density of inflammatory cells. These findings suggest that tDCS may exert an anti-inflammatory effect with potential therapeutic application to be further explored in autoimmune demyelinating diseases.

Development of Neuronal Excitability of the Bushy Cells in Anteroventral Cochlear Nucleus of Rats

The ultrafast and precise single-onset action potential (AP) of the bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN) plays an important role in precise processing of temporal auditory information for localizing sound sources and communication cues. The specialized properties of high conductance of the low-voltage-activated potassium (K⁺_LVA) channel contribute to generate ultrafast and precise single-onset APs in BCs. However, the developmental changes of K⁺_LVA distribution and their contributions to shape neuronal excitability of BCs remain unclear. Therefore, we investigated the developmental changes in neuronal excitability of BCs and K⁺_LVA distribution at different developmental periods. Using electrophysiological recording, we first characterized the firing pattern of BCs in response to a sequence of current injections at different developmental periods. The expression of the K⁺_LVA subunit Kv1.1 in AVCN was examined with Western blot. The results indicated that BCs showed single-onset AP firing patterns and paused multiple APs firing patterns at the postnatal time of day 7 (P7) and were then refined into single-onset firing patterns at P14 and P21. With development, the active membrane properties, including latency and half-width of AP, and passive membrane properties, including capacitance, input resistance, and time constant, were significantly decreased. Furthermore, the refinement of firing patterns in BCs was correlated with the upregulation of the Kv1.1 channel in AVCN. In summary, the present study indicated that BCs optimize precise and single-onset firing with development, possibly driven by the changes in membrane properties and upregulation of Kv1.1 in AVCN.