Frontiers in Cellular Neuroscience

Prostaglandin E2 (PGE2) is one of the major contributors to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of the nociceptive axons is incompletely understood. We used a microfluidic cell culture platform to investigate the changes in responsiveness of sensory axons following treatment with PGE2. We show that the application of PGE2 to fluidically isolated axons leads to sensitization of their responses to depolarising stimuli, and the inclusion of zatebradine, a blocker of HCN channels, blocks this enhancement. However, unexpectedly, we also found that the application of PGE2 to the axons elicited a direct and persistent spiking activity in the sensory neurons. We demonstrate that this persistent activity is due to a direct depolarization of axons by PGE2, which is inhibited by Nav1.8 sodium channel blockers but is mainly refractory to Nav1.7 channel blockade. Both the persistent activity and the membrane depolarization in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Our data indicate that PGE2/EP4/cAMP pathway culminates in a sustained depolarization in the sensory axons, leading to the generation of action potentials propagating to the soma. PGE2 therefore, not only mediates nociceptor sensitization but can directly elicit discharges in nociceptive axons, hence redefining its role as a pain mediator in inflammatory conditions. One Sentence SummaryProstaglandin E2 can depolarise nociceptive axons in the absence of any noxious stimuli leading to a sustained activation of pain sensing neurons.

Purpose: It has been known for some time that normal retinal signaling is disrupted early on in diabetes, before the onset of the vascular pathologies associated with diabetic retinopathy. There is growing evidence that levels of retinal dopamine, a neuromodulator that mediates light adaptation, may also be reduced in early diabetes. Previously, we have shown that after six weeks of diabetes in a mouse model, light adaptation is impaired at the level of ON-sustained (ON-s) ganglion cells. The purpose of this study was to determine whether changes in dopamine receptor sensitivity contribute to this dysfunction. Here we used single cell retinal patch-clamp recordings from the mouse retina to determine how activating dopamine type D4 receptors (D4Rs) changes the light-evoked and spontaneous excitatory inputs to ON-s ganglion cells, in both control and diabetic animals. We also used in-situ fluorescent hybridization to assess whether D4R expression was impacted by diabetes. We found that D4R activation had a smaller impact on light-evoked excitatory inputs to ON-s ganglion cells in diabetic retinas compared to controls. This impaired D4R signaling is not attributable to a decline in D4R expression, as we found increased D4R mRNA density in the outer plexiform layer in diabetic retinas. This suggests that the cellular machinery of dopaminergic signaling is itself disrupted in early diabetes and may be amenable to chronic dopamine supplementation therapy.

Myelinating Schwann cell (SC)- dorsal root ganglion (DRG) neuron cocultures have been an important technique over the last four decades in understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. While methods using rat SCs and rat DRG neurons are commonplace, there are no established protocols in the field describing the use of mouse SCs with mouse DRG neurons in dissociated myelinating cocultures. There is a great need for such a protocol as this would allow the use of cells from many different transgenic mouse lines. Here we describe a protocol to coculture dissociated mouse SCs and DRG neurons and induce robust myelination. Use of microfluidic chambers permits fluidic isolation for drug treatments, allows cultures to be axotomised to study injury responses, and cells can readily be transfected with lentiviruses to permit live imaging. We used this model to quantify the rate of degeneration after traumatic axotomy in the presence and absence of myelinating SCs and axon aligned SCs that were not induced to myelinate. We find that SCs, irrespective of myelination status, are axo-protective and delay axon degeneration early on. At later time points after injury, we use live imaging of cocultures to show that once axonal degeneration has commenced SCs break up, ingest, and clear axonal debris. Summary statementA novel compartmentalised dissociated mouse myelinating SC-DRG coculture system reveals distinct axo-protective and axo-destructive phases of Schwann cells on axon integrity after trauma.

New tools in optogenetics and molecular biology have culminated in recent studies which mark immediate-early gene (IEG)-expressing neurons as memory traces or engrams. Although the activity-dependent expression of IEGs has been successfully utilised to label memory traces, their roles in engram specification is incompletely understood. Outstanding questions remain as to whether expression of IEGs can interplay with network properties such as functional connectivity and also if neurons expressing different IEGs are functionally distinct. We investigated the expression of Arc and c-Fos, two commonly utilised IEGs in memory engram specification, in cultured hippocampal neurons. After pharmacological induction of long-term potentiation (LTP) in the network, we noted an emergent network property of refinement in functional connectivity between neurons, characterized by a global down-regulation of network connectivity, together with strengthening of specific connections. Subsequently, we show that Arc expression correlates with the effects of network refinement, with Arc-positive neurons being selectively strengthened. Arc positive neurons were also found to be located in closer physical proximity to each other in the network. While the expression pattern of IEGs c-Fos and Arc strongly overlaps, Arc was more selectively expressed than c-Fos. These IEGs also act together in coding information about connection strength pruning. These results demonstrate important links between IEG expression and network connectivity, which serve to bridge the gap between cellular correlates and network effects in learning and memory.

Elevated intraocular pressure (IOP) triggers glaucoma by damaging the output neurons of the retina called retinal ganglion cells (RGCs). This leads to the loss of RGC signaling to visual centers of the brain such as the dorsolateral geniculate nucleus (dLGN), which is critical for processing and relaying information to the cortex for conscious vision. In response to altered levels of activity or synaptic input, neurons can homeostatically modulate postsynaptic neurotransmitter receptor numbers, allowing them to scale their synaptic responses to stabilize spike output. While prior work has indicated unaltered glutamate receptor properties in the glaucomatous dLGN, it is unknown whether glaucoma impacts dLGN inhibition. Here, using DBA/2J mice, which develop elevated IOP beginning at 6-7 months of age, we tested whether the strength of inhibitory synapses on dLGN thalamocortical relay neurons is altered in response to the disease state. We found an enhancement of feed-forward disynaptic inhibition arising from local interneurons along with increased amplitude of quantal inhibitory synaptic currents. A combination of immunofluorescence staining for the GABAA-1 receptor subunit, peak-scaled nonstationary fluctuation analysis, and measures of homeostatic synaptic scaling indicated this was the result of an approximately 1.4-fold increase in GABA receptor number at post-synaptic inhibitory synapses, although several pieces of evidence strongly indicate a non-uniform scaling across inhibitory synapses within individual relay neurons. Together, these results indicate an increase in inhibitory synaptic strength in the glaucomatous dLGN, potentially pointing toward homeostatic compensation for disruptions in network and neuronal function triggered by increased IOP. Significance StatementElevated eye pressure in glaucoma leads to loss of retinal outputs to the dorsolateral geniculate nucleus (dLGN), which is critical for relaying information to the cortex for conscious vision. Alterations in neuronal activity, as could arise from excitatory synapse loss, can trigger homeostatic adaptations to synaptic function that attempt to maintain activity within a meaningful dynamic range, although whether this occurs uniformly at all synapses within a given neuron or is a non-uniform process is debated. Here, using a mouse model of glaucoma, we show that dLGN inhibitory synapses undergo non-uniform upregulation due to addition of post-synaptic GABA receptors. This is likely to be a neuronal adaptation to glaucomatous pathology in an important sub-cortical visual center.

Presynaptic voltage-gated Ca2+ channels (CaV) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system, most presynaptic terminals are CaV2.1 dominant with a developmental reduction in CaV2.2 and CaV2.3 levels, and CaV2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV2 subtype levels are largely unsolved. Since the CaV2 1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV2.1 1 subunits containing swapped motifs with the CaV2.2 and CaV2.3 1 subunit on a CaV2.1/CaV2.2 null background at the calyx of Held presynaptic terminal. We found that expression of CaV2.1 1 subunit chimeras containing the CaV2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV2.1 1 subunit. However, the Ca2+ current sensitivity to the CaV2.1 blocker Agatoxin-IVA, was the same between the chimeras and the CaV2.1 1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV2.1 loop II-III and CT do not individually regulate CaV2.1 preference, but these motifs control CaV2.1 levels and the CaV2.3 CT contains motifs that negatively regulate presynaptic CaV2.3 levels. We propose that the motifs controlling presynaptic CaV2.1 preference are distinct from those regulating CaV2.1 levels and may act synergistically to impact pathways regulating CaV2.1 preference and abundance. Key points summaryO_LIPresynaptic CaV2 subtype abundance regulates neuronal circuit properties, however the mechanisms regulating presynaptic CaV2 subtype abundance and preference remains enigmatic. C_LIO_LIThe CaV 1 subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. C_LIO_LIThe CaV2.1 1 subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV2.1 abundance but do not regulate preference. C_LIO_LIThe CaV2.3 1 subunit cytoplasmic C-terminus negatively regulates presynaptic CaV2 subtype abundance but not preference while the CaV2.2 1 subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV2 subtype abundance or preference. C_LIO_LIThe CaV2 1 subunit motifs determining the presynaptic CaV2 preference are distinct from abundance. C_LI

Kv4.2 channels, which mediate A-type K+ current, exert significant influence on synaptic input signals and synaptic plasticity in the principal cells of the hippocampus. While their influence on activity-dependent regulation of synaptic response is well-established, the impact of Kv4.2 channels on baseline synaptic strength remains elusive. To investigate this, we selectively inhibited postsynaptic Kv4.2 by introducing Kv4.2 antibodies into the hippocampal granule cells and evaluated its impact on the baseline synaptic transmission. Our results demonstrated that Kv4.2 inhibitions led to notable increase in the amplitude of AMPA receptor (AMPAR)-mediated synaptic currents, and this effect was in parallel with the Kv4.2 expression level at dendritic regions. This Kv4.2-dependent synaptic potentiation was effectively abolished by intracellular 10 mM BAPTA or block of R-type calcium channels (RTCC) and downstream signaling molecules including protein kinase A (PKA) and protein kinase C (PKC). Importantly, Kv4.2 inhibitions did not occlude further synaptic strengthening high frequency stimulation, suggesting that synaptic strength regulation by Kv4.2 s distinct from the mechanism of long-term potentiation. Our study highlights the role of Kv4.2 in regulating the baseline synaptic strength, where Kv4.2-mediated inhibition of RTCC is crucial.

Vertebrate axons can be wrapped by myelin produced by oligodendrocytes. This cellular interaction ensures fast and accurate propagation of action potentials, but the physiology of the myelin sheath is almost completely unknown. To investigate the physiology of the myelin sheath, we implemented an imaging strategy that allowed optical measurements of myelin membrane voltage, with the aim to identify physiological changes of the myelin membrane during neuronal firing. We expressed the genetically encoded voltage indicator ASAP3 in mouse oligodendrocytes in vivo and subsequently investigated myelin physiology by optically measuring myelin membrane voltage. We found that myelin depolarizes during neuronal activity, which is blocked by inhibiting neuronal action potentials. Pharmacological and knock-out experiments of Kir4.1 showed that potassium uptake channels mediate action potential induced depolarization. Blocking myelin dependent potassium uptake and direct application of high potassium to identified axons induced axonal initiated and antidromic propagating action potentials. Our study shows that myelin is not an electrically passive insulator, but exhibits ion dynamics and its physiological response is fine tuned to neuronal activity. By facilitating potassium removal during action potentials, myelin supports high precision axonal firing. Genetically encoded sensors are thus a useful tool to study physiological properties of myelin, inaccessible by classical techniques. HighlightsO_LIOptical imaging of myelin membrane potential C_LIO_LIMyelin sheaths exhibit depolarization in response to neuronal firing C_LIO_LIDepolarizations are partially mediated through Kir channels C_LIO_LIPotassium originates from axonal Kv channels C_LI

Loss of function mutations of Cx32, which is expressed in Schwann cells, cause X-linked Charcot Marie Tooth disease, a slowly progressive peripheral neuropathy. Cx32 is thus essential for the maintenance of myelin. During action potential propagation, Cx32 hemichannels in the Schwann cell paranode are thought to open and release ATP. As Cx32 hemichannels are directly sensitive to CO2, we have tested whether CO2 produced in the axonal node, as a consequence of the energetic demands of action potential propagation, might gate Cx32 hemichannels. Using isolated sciatic nerve from the mouse, we have shown that the critical components required for intercellular CO2 signalling are present (nodal mitochondria, the source of CO2; a CO2-permeable aquaporin, AQP1; paranodal Cx32; and carbonic anhydrase). We have used a membrane impermeant fluorescent dye FITC, which can permeate Cx32 hemichannels, to demonstrate the opening of Cx32 in Schwann cells in response to an external CO2 stimulus or during action potential propagation in the isolated nerve. Pharmacological blockade of APQ1 or allosteric enhancement of carbonic anhydrase activity greatly reduced Cx32 gating during action potential firing. By contrast, inhibition of carbonic anhydrase with acetazolamide greatly increased Cx32 gating. Cx32 gating was unaffected by the G-protein blocker GDP{beta}S, indicating that it was not mediated by G protein coupled receptors. This CO2-dependent opening of Cx32 also mediates an activity dependent Ca2+ influx into the paranode and, by increasing the leak current across the myelin sheath, slows the conduction velocity. Our data demonstrate that CO2 can act via connexins to mediate neuron-to-glia signalling and that CO2 permeable aquaporins and carbonic anhydrase are key components of this signalling mechanism.

Motoneurons are under strong pressure to maintain stable motor output throughout an individual life, through homeostatic regulation of their electrical properties. Dysregulated spinal motoneuron excitability has long been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent work in SOD1G93A mice suggests that the homeostatic response of motoneurons becomes dysregulated as cellular processes are disrupted by the disease, causing fluctuations in motoneuron electrical properties. Yet, few studies directly test whether ALS motoneurons respond differently than wild type motoneurons to a common chronic perturbation. Here, we used in vivo electrophysiology to test whether motoneurons from pre-symptomatic SOD1G93A mice modulate excitability differently than wild type motoneurons in response to the same homeostatic perturbation: chronic inhibition exerted by the benzodiazepine diazepam. Using linear mixed-effects statistical models, we assessed whether diazepam treatment differentially modulated passive properties, firing behavior, spike properties, and/or synaptic inputs in SOD1G93A versus wild type motoneurons. We identified a significant genotype x treatment interaction effect selectively for properties related to passive membrane integration and spike initiation, including membrane time constant, peak input resistance, and recruitment current. In contrast, firing gain, spike waveform characteristics, and synaptic inputs were largely unaffected. These findings indicate that sustained inhibitory perturbation selectively triggered overactive intrinsic compensatory mechanisms in SOD1G93A motoneurons rather than inducing widespread changes in firing or synaptic transmission. Together, our results provide direct evidence for over-active homeostatic control of motoneuron excitability and support a view of motoneuron dysfunction in ALS as a problem of altered feedback regulation rather than simply hyper- or hypo-excitability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/725609v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@25f125org.highwire.dtl.DTLVardef@faf2c9org.highwire.dtl.DTLVardef@15993a8org.highwire.dtl.DTLVardef@1ed006a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Clinically relevant mutations to voltage-gated ion channels, called channelopathies, alter ion channel function, properties of ionic current and neuronal firing. The effects of ion channel mutations are routinely assessed and characterized as loss of function (LOF) or gain of function (GOF) at the level of ionic currents. Emerging personalized medicine approaches based on LOF/GOF characterization have limited therapeutic success. Potential reasons are that the translation from this binary characterization to neuronal firing especially when considering different neuronal cell types is currently not well understood. Here we investigate the impact of neuronal cell type on the firing outcome of ion channel mutations with simulations of a diverse collection of neuron models. We systematically analyzed the effects of changes in ion current properties on firing in different neuronal types. Additionally, we simulated the effects of mutations in the KCNA1 gene encoding the KV1.1 potassium channel subtype associated with episodic ataxia type 1 (EA1). These simulations revealed that the outcome of a given change in ion channel properties on neuronal excitability is cell-type dependent. As a result, cell-type specific effects are vital to a full understanding of the effects of channelopathies on neuronal excitability and present an opportunity to further the efficacy and precision of personalized medicine approaches. Significance StatementAlthough the genetic nature of ion channel mutations as well as their effects on the biophysical properties of an ion channel are routinely assessed experimentally, determination of their role in altering neuronal firing is more difficult. In particular, cell-type dependency of ion channel mutations on firing has been observed experimentally, and should be accounted for. In this context, computational modelling bridges this gap and demonstrates that the cell type in which a mutation occurs is an important determinant in the effects of neuronal firing. As a result, classification of ion channel mutations as loss or gain of function is useful to describe the ionic current but should not be blindly extend to classification at the level of neuronal firing.

Chloride ions play a critical role in neuronal inhibition through the activity of chloride-permeable GABAA receptor channels. Ion transporters, chloride channels, and immobile ion species tightly regulate intracellular chloride concentrations. Several studies related to epilepsy suggest that chloride extrusion function may decrease in an activity-dependent manner. Consequently, it is crucial to investigate whether intense neuronal activity, as observed during status epilepticus, could lead to sustained increases in intracellular chloride levels in neurons, which in turn could contribute to epilepsy-associated hyperexcitability. This study utilized the chloride sensitive indicator (6-Methoxyquinolinio) acetic acid ethyl ester bromide (MQAE) combined with fluorescence lifetime imaging (FLIM) to examine whether application of the convulsant, pilocarpine, a muscarinic acetylcholine receptor agonist, could induce synchronous epileptiform activity and elevate intracellular chloride concentrations in hippocampal slice cultures. Using a Gaussian mixture model, we identified a multimodal distribution of intracellular chloride levels among neurons, with a significant subset of these cells exhibiting massive and prolonged (days) chloride accumulation. The combination of multicellular imaging and statistical analysis served as a powerful tool for studying the emergence of multiple, distinct populations of neurons in pathological conditions, in contrast to homogeneous populations evident under control conditions. HighlightsO_LIMaintaining low [Cl-]in is important for inhibitory function, however, hyperactivity, such as that seen in epilepsy, may lead to elevated [Cl-]in. C_LIO_LIPilocarpine induces hyperactivity in dentate granule cells (DGCs) in hippocampal organotypic slice cultures. C_LIO_LIMulticellular imaging using a chloride sensing dye with a fluorescence lifetime imaging approach revealed that [Cl-]in is elevated in a subpopulation of DGCs. C_LIO_LIGaussian mixture model analysis is a powerful tool for studying the emergence of cellular heterogeneity in a pathological condition. C_LI

Dopamine plays a pivotal role in adjusting the flow of information across the retina as luminance changes from night to day. Here we show, under dim photopic conditions, that both dopamine and a D1-like receptor (D1R) agonist hyperpolarized the resting membrane potential (Vm) of AII amacrine cells (AII-ACs). Surprisingly, in the presence of glutamatergic and GABAergic synaptic blockers that isolate glycinergic synapses, D1R agonists are without effect. However, a D1R antagonist depolarized Vm and reduced the input resistance of AII-ACs in wild type mice, but not in Cx36-/- mice. Accordingly, D1R antagonists enhanced tonic glycinergic transmission to type-2 OFF-cone bipolar cells (OFF-CBCs). D1Rs thus adjust the Vm and excitability of AII-ACs and, thereby, the level of glycine release to OFF-CBCs by regulating gap junction coupling with ON cone bipolar cells. Our findings provide insights into how the retina may use dopamine to adapt crossover inhibitory microcircuits during changes in luminance.

Dopamine neurons, which are critical in movement, cognition, and reward learning, are vulnerable to oxidative stress during aging, drugs of abuse, and viral infection and can lead to neurodegeneration. Previous work used computational modeling to study dopamine neuron function based on experimental findings from rodent brain slices containing dopamine neurons. Here we show for the first time the feasibility and utility of applying such computational models of DA neurons to the analysis of experimental findings from in-vitro cultured cells. We used DCFH-DA (and DHE) and time-lapse, Fura-2 ratiometric imaging to measure changes in ROS levels and changes in intracellular calcium (Ca2+) levels, respectively, in two dopaminergic cell models: differentiated SH-SY5Y and differentiated human neural progenitor cells. We investigated how peroxide-dependent changes in the behavior of the L-type channel might alter the excitability of the dopaminergic cell. We found that L-type channels mediated clusters of calcium spikes (or oscillations) and that our model suggested that such increased excitability could be explained by changes in the voltage-dependence of activation of the L-type channels in response to exogenous peroxide. Our findings suggested that L-type channels link oxidative stress responses to modulation of excitability. We conclude that the Ca2+ channel blocker nicardipine may help disrupt this link by reducing oxidative stress and preventing channel activation at more hyperpolarized potentials, thus reducing plasma membrane excitability.

BackgroundPrevious work showed that electrically-evoked inhibition to Rod Bipolar Cells (RBC) is reduced in a mouse model of early diabetes. It is hypothesized that this is due to impaired Ca2+ handling in the presynaptic amacrine cell, either through increased Ca2+ buffering or decreased influx. To test this hypothesis and develop a mechanism for this effect, a model where direct optogenetic activation of inhibitory amacrine cells that expressed the light-activated channel ChR2 was used to isolate amacrine cell inputs to RBCs. Application of selective Ca2+ channel blockers could then assess potential locations of amacrine Ca2+ disruption. Using whole cell patch clamp electrophysiology, recordings were made from a 6 week diabetic population (DM) and vehicle injected non-DM animals. ResultsRobust GABAC receptor inhibitory currents were recorded from RBCs after ChR2 stimulus that were significantly diminished by the application of nifedipine to block L-type Ca2+ channels in both DM and non-DM conditions. There were significant differences in the peak amplitude of these responses between DM and non-DM groups (p = 0.0146). However, in the non-DM group the decay tau of the response to the 50ms stimulus was significantly diminished by nifedipine ({tau} p =0.0498, n = 5), but this was not seen in the DM group ({tau} p = 0.9498, n=7). A 1s nifedipine-reduced response saw its decay tau increase in the DM group but not the non-DM. Ca2+ - induced Ca2+ release (CICR) blockade with ryanodine decreased responsivity equally between groups in the 1s stimulus but showed no significant kinetic changes. CICR blockade for a 50ms stimulus response showed significant kinetic changes in diabetes but otherwise reduced the response equally between DM and non-DM. Blockade of the mitochondrial Ca2+ uniporter (MCU) had little effect on the optogenetic response. ConclusionThis study presents evidence that diabetes alters amacrine cell output to the RBC unmasked through blockade of the L-type calcium channel, and the Endoplasmic Reticulum (ER). An apparent explanation for our results is that DM calcium buffering is dysregulated, leading to prolonged responses. The underlying mechanism for this alteration is complex and not yet clearly elucidated.

BackgroundSynaptic dysfunction is an early feature of Alzheimers disease (AD) and a significant contributor to cognitive decline and neurodegeneration. Proper localization of proteins involved in pre-and post-synaptic composition is dependent on endosomal recycling and trafficking. Alterations in trafficking complexes, such as retromer, have been shown to impair neuronal synaptic function. The SORL1 gene has been strongly implicated in AD pathogenesis and its protein product, SORLA, is an endosomal receptor that works in conjunction with retromer to regulate endosomal recycling. MethodsWe utilized our established human induced pluripotent stem cell (hiPSC) derived excitatory cortical neuron model to examine SORL1s role in synaptic protein composition and neuronal function. We used Quantitative Multiplex co-Immunoprecipitation (QMI), a mesoscale proteomics assay to measure synaptic protein interactions, immunocytochemistry to assay synapses and AMPA receptor subunits, and multi-electrode arrays (MEAs) to measure neuronal function of SORL1 KO and isogenic control hiPSC derived neurons. ResultsWe show that loss of SORL1 expression significantly changes many synaptic protein-protein interactions and patterns of expression. We demonstrate that SORL1 deficient neurons are hyperactive and that the increased activity is driven by glutamatergic neurotransmission. Hyperexcitability has been seen in other models of AD with familial AD variants in amyloid precursor protein and presenilin genes, due to the increases in amyloid beta (A{beta}) peptides. In the case of SORL1 deficiency, the hyperexcitability we observe is primarily due to mis-trafficking of synaptic proteins, rather than an overall increase in A{beta}. Finally, we find that SORL1 deficient neurons have impaired synaptic plasticity. ConclusionsThese findings further support a growing body of literature implicating early endosomal recycling defects as drivers of AD pathogenesis. Furthermore, our work supports further emphasis on exploring the SORL1-retromer pathway for therapeutic development in AD.

Synaptic plasticity is a fundamental mechanism of memory storage in the brain. Among the various rules governing changes in synaptic strength, Spike Timing-Dependent Plasticity (STDP) stands out for its strong physiological relevance in vivo. Ubiquitous across brain regions and neuronal types, STDP is a complex and multifactorial process influenced by factors such as neuromodulation, extracellular calcium levels, and activity patterns. However, one relatively understudied factor is the role of astrocytes, despite their well-established involvement in regulating synaptic transmission and neuronal excitability through gliotransmitter release. While some factors have garnered significant attention, others, like S100{beta}, have remained relatively underexplored despite their potential importance in regulating synaptic plasticity. S100{beta} is a calcium-binding protein, allowing it to influence extracellular Ca{superscript 2} concentration and potentially all Ca2+-dependent plasticity processes. Building on our previous research in the visual cortex, where we examined the regulation of neuronal excitability by S100{beta}, we chose to further investigate the role of astrocytes and S100{beta} in synaptic plasticity at layer 2/3-layer 5 synapses in the visual cortex. We demonstrated that S100{beta} is an important gliotransmitter to consider, capable of regulating long-term potentiation.

Astrocytes are well known for homeostatic roles in energy maintenance, neurotransmitter recovery and immunoreactivity, however their contributing role in active modulation of neuronal activity remains controversial. Recent evidence highlights astrocytes as a potential signalling partner in ongoing high-order neuronal activity, and a modulator of baseline activity. Here we utilise an iPSC-derived in vitro cortical network model to describe a slow-wave oscillation and examine the contributions of astrocytes to development of this oscillatory activity, dependant upon generation of UP/DOWN state phenomena. To examine the role of astrocytes we invoke an acetylcholinergic oscillation in the neuronal population by addition of carbachol, which proved to be a robust mechanism for eliciting prolonged and synchronised network bursting. Pharmalogical interrogation determined oscillatory maintenance was dependent on both glutamatergic and purinergic signalling pathways. Upon further interrogation, we determined that astrocytic calcium signalling was essential to timing of the oscillatory signal. By utilising chemogenetic actuators we showed that whilst neurons are essential and sufficient for instigating the oscillatory signal, astrocytes played a key role in timing. This previously unreported mechanism may contribute to initial development of brain activity, and may underlie a basal activity present later during adulthood. In collaboration with recent results highlighting the role of active gliotransmission, this highlights astrocytes as an important research target for understanding brain activity alterations during development and disease.

3.Reproducibility of research data is a significant problem with more than 60% of biological and medical researchers reporting they have failed to reproduce published data. General acceptance of incorrect results can mean that future data is incorrectly interpreted and progress significantly interrupted. Thus, replication studies play an essential role in corroborating research findings and validating future research objectives. Here, we attempted to replicate data demonstrating the neurotransmitter glutamate, as well as NMDA and AP5, acts as positive allosteric modulators of the inhibitory glycine receptor. Notably, it was shown that the amplitude of miniature glycinergic currents recorded in spinal cord slices were reversibly enhanced when extracellular glutamate concentrations were increased by the glutamate transporter antagonist TBOA. This finding indicates that endogenous fluctuations in extracellular [glutamate] permits cross-talk between excitatory and inhibitory synapses and likely plays a role in setting the spinal inhibitory glycinergic tone and modulating baseline neurotransmission. We re-evaluated the data in primary cultured spinal cord neurons, spinal cord slice and Xenopus laevis oocytes expressing recombinant glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5 or NMDA positively modulate glycine receptor currents. We paid careful attention to key aspects of the original study design, ensured rapid drug exposure by using fast-flow application and took into account receptor saturation and protocol deviations such as animal species. This study refutes the finding that glycine receptors are directly modulated by glutamate spill-over and suggests that glycinergic tone is independent of changes in excitatory activity. 4. Significance StatementGlutamate spill-over onto inhibitory synapses has been reported to positively modulate glycine receptors and alter the inhibitory tone of the spinal cord. This finding has important implications for baseline spinal transmission and could play a role when chronic pain develops. However, we failed to replicate these results and did not observe any modulation of native or recombinant glycine receptor-mediated currents by AP5, NMDA or glutamate. This indicates that inhibitory glycine receptors operate independently of fluctuations in extracellular [glutamate]. 5. Visual AbstractN/A

Caldendrin is a calmodulin-like Ca2+ binding protein that is expressed primarily in neurons and regulates multiple effectors including Cav1 L-type Ca2+ channels. Here, we tested the hypothesis that caldendrin regulates Cav1-dependent pathways that repress neurite growth in dorsal root ganglion neurons (DRGNs). By immunofluorescence, caldendrin was localized in medium- and large-diameter DRGNs. Consistent with an inhibitory effect of caldendrin on neurite growth, neurite initiation and growth was enhanced in dissociated DRGNs from caldendrin knockout (KO) mice compared to those from wild type (WT) mice. In an in vitro axotomy assay, caldendrin KO DRGNs grew longer neurites via a mechanism that was more sensitive to inhibitors of transcription as compared to WT DRGNs. Strong depolarization, which normally represses neurite growth through activation of Cav1 channels, had no effect on neurite growth in DRGN cultures from female caldendrin KO mice. Remarkably, DRGNs from caldendrin KO males were no different from those of WT males in terms of depolarization-dependent neurite growth repression. We conclude that caldendrin opposes neurite regeneration and growth, and this involves coupling of Cav1 channels to growth-inhibitory pathways in DRGNs of females but not males. Our findings suggest that caldendrin KO mice represent an ideal model in which to interrogate the transcriptional pathways controlling neurite regeneration and how these pathways may differ in males and females.