Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning.

Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity-changes in myelin lipid synthesis and composition-may also be associated with motor learning. We combined a motor learning task in mice with in vivo two-photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across individual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte-specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning.

TRPC Channels Activated by G Protein-Coupled Receptors Drive Ca2+ Dysregulation Leading to Secondary Brain Injury in the Mouse Model.

Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to Gαq-type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca2+ reporter demonstrated that the majority of the Ca2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels (Trpc3KO compared with wildtype (WT)). This Ca2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP-Trpc3KO reporter brain slice model). Contribution of the Gαq-coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3KO, and Trpc1/3/6/7 quadruple knockout (TrpcQKO), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3KO and TrpcQKO models, with the TrpcQKO showing greatest neuroprotection. These findings demonstrate the contribution of the Gαq-coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.

Unpacking and validating the "cell membrane" core concept of physiology by an Australian team.

A task force of physiology educators from 25 Australian universities generated an Australia-wide consensus on seven core concepts for physiology curricula. One adopted core concept was "cell membrane," defined as "Cell membranes determine what substances enter or leave the cell and its organelles. They are essential for cell signaling, transport, and other cellular functions." This concept was unpacked by a team of 3 Australian physiology educators into 4 themes and 33 subthemes arranged in a hierarchical structure up to 5 levels deep. The four themes related to defining the cell membrane, cell membrane structure, transport across cell membranes, and cell membrane potentials. Subsequently, 22 physiology educators with a broad range of teaching experience reviewed and assessed the 37 themes and subthemes for importance for students to understand and the level of difficulty for students on a 5-point Likert scale. The majority (28) of items evaluated were rated as either Essential or Important. Theme 2: cell membrane structure was rated as less important than the other three themes. Theme 4: membrane potential was rated most difficult, while theme 1: defining cell membranes was rated as the easiest. The importance of cell membranes as a key aspect of biomedical education received strong support from Australian educators. The unpacking of the themes and subthemes within the cell membrane core concept provides guidance in the development of curricula and should facilitate better identification of the more challenging aspects within this core concept and help inform the time and resources required to support student learning.NEW & NOTEWORTHY The "cell membrane" core concept was unpacked by a team of Australian physiology educators into a conceptual framework to provide guidance for students and educators. Key themes in the cell membrane core concept were cell membrane definition and structure, transport across cell membranes, and membrane potentials. Australian educators reviewing the framework identified cell membrane as an essential yet relatively simple core concept, suggesting that this is well-placed in foundational physiology courses across a diverse range of degrees.

Microglia enable cross-modal plasticity by removing inhibitory synapses.

Cross-modal plasticity is the repurposing of brain regions associated with deprived sensory inputs to improve the capacity of other sensory modalities. The functional mechanisms of cross-modal plasticity can indicate how the brain recovers from various forms of injury and how different sensory modalities are integrated. Here, we demonstrate that rewiring of the microglia-mediated local circuit synapse is crucial for cross-modal plasticity induced by visual deprivation (monocular deprivation [MD]). MD relieves the usual inhibition of functional connectivity between the somatosensory cortex and secondary lateral visual cortex (V2L). This results in enhanced excitatory responses in V2L neurons during whisker stimulation and a greater capacity for vibrissae sensory discrimination. The enhanced cross-modal response is mediated by selective removal of inhibitory synapse terminals on pyramidal neurons by the microglia in the V2L via matrix metalloproteinase 9 signaling. Our results provide insights into how cortical circuits integrate different inputs to functionally compensate for neuronal damage.

Controlled activation of cortical astrocytes modulates neuropathic pain-like behaviour.

Chronic pain is a major public health problem that currently lacks effective treatment options. Here, a method that can modulate chronic pain-like behaviour induced by nerve injury in mice is described. By combining a transient nerve block to inhibit noxious afferent input from injured peripheral nerves, with concurrent activation of astrocytes in the somatosensory cortex (S1) by either low intensity transcranial direct current stimulation (tDCS) or via the chemogenetic DREADD system, we could reverse allodynia-like behaviour previously established by partial sciatic nerve ligation (PSL). Such activation of astrocytes initiated spine plasticity to reduce those synapses formed shortly after PSL. This reversal from allodynia-like behaviour persisted well beyond the active treatment period. Thus, our study demonstrates a robust and potentially translational approach for modulating pain, that capitalizes on the interplay between noxious afferents, sensitized central neuronal circuits, and astrocyte-activation induced synaptic plasticity.

Global transgenic upregulation of KCC2 confers enhanced diazepam efficacy in treating sustained seizures.

Reduced anticonvulsant efficacy of benzodiazepines is a problem in the treatment of status epilepticus, with up to 50% of patients failing to respond to their first dose. KCC2 is a neuronal K+ -Cl- co-transporter that helps set and maintain intracellular Cl- concentrations. KCC2 functional downregulation is a potential contributor to benzodiazepine resistance. We tested this idea using male and female doxycycline-inducible, conditional transgenic mice to increase the functional expression of KCC2 in pyramidal neurons. We administered mice with two doses of the chemoconvulsant kainic acid (5 mg/kg, i.p.) 60 min apart and quantified the resultant seizures with electroencephalography (EEG) recordings. Overexpression of KCC2 prior to the chemoconvulsant challenge did not affect seizure latency or other measures of seizure severity, but it did increase diazepam's efficacy in stopping EEG seizures. Spike rate, time in seizure, and EEG spectral power following diazepam (5 mg/kg, i.p) were all significantly lower in KCC2 overexpression mice as compared to control mice. Our results indicate that, in the context of benzodiazepine resistance during sustained seizures, addressing impaired Cl- homeostasis alone appreciably improves the efficacy of γ-aminobutyric acid (GABA)ergic inhibition. We therefore suggest the simultaneous targeting of KCC2 and GABAA receptors as a pathway for improving current anticonvulsant therapeutic strategies.

Pain induces stable, active microcircuits in the somatosensory cortex that provide a therapeutic target.

Sustained neuropathic pain from injury or inflammation remains a major burden for society. Rodent pain models have informed some cellular mechanisms increasing neuronal excitability within the spinal cord and primary somatosensory cortex (S1), but how activity patterns within these circuits change during pain remains unclear. We have applied multiphoton in vivo imaging and holographic stimulation to examine single S1 neuron activity patterns and connectivity during sustained pain. Following pain induction, there is an increase in synchronized neuronal activity and connectivity within S1, indicating the formation of pain circuits. Artificially increasing neuronal activity and synchrony using DREADDs reduced pain thresholds. The expression of N-type voltage-dependent Ca2+ channel subunits in S1 was increased after pain induction, and locally blocking these channels reduced both the synchrony and allodynia associated with inflammatory pain. Targeting these S1 pain circuits, via inhibiting N-type Ca2+ channels or other approaches, may provide ways to reduce inflammatory pain.

Maternal immune activation induces sustained changes in fetal microglia motility.

Maternal infection or inflammation causes abnormalities in brain development associated with subsequent cognitive impairment and in an increased susceptibility to schizophrenia and autism spectrum disorders. Maternal immune activation (MIA) and increases in serum cytokine levels mediates this association via effects on the fetal brain, and microglia can respond to maternal immune status, but consensus on how microglia may respond is lacking and no-one has yet examined if microglial process motility is impaired. In this study we investigated how MIA induced at two different gestational ages affected microglial properties at different developmental stages. Immune activation in mid-pregnancy increased IL-6 expression in embryonic microglia, but failed to cause any marked changes in morphology either at E18 or postnatally. In contrast MIA, particularly when induced earlier (at E12), caused sustained alterations in the patterns of microglial process motility and behavioral deficits. Our research has identified an important microglial property that is altered by MIA and which may contribute to the underlying pathophysiological mechanisms linking maternal immune status to subsequent risks for cognitive disease.

Activity-Dependent Global Downscaling of Evoked Neurotransmitter Release across Glutamatergic Inputs in Drosophila.

Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This "presynaptic downscaling" maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long-range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

Dual microglia effects on blood brain barrier permeability induced by systemic inflammation.

Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood-brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.

Physiological Implications of Microglia-Synapse Interactions.

Microglia are the sole immune responding cells in the central nervous system. Their role as neuroimmune cells in the pathogenesis of various neurodegenerative and infectious diseases of the brain have been extensively studied. Upon brain disease and infection, they adopt an activated phenotype associated with the release of cytokines and neurotrophic factors and resulting in neuroprotective or neurotoxic outcomes. However, microglia are resident also in the healthy or physiological brain, but much less is known about their role(s) in the healthy brain, partly due to technical limitations regarding investigation of these highly reactive cells in the intact brain. Recent developments in molecular probes and in vivo optical imaging techniques has now helped to characterize microglia in the physiological or healthy brain. In vivo two-photon imaging of fluorescently labeled microglia have revealed that they are highly motile cells in the healthy brain, extending and retracting their processes that extend from a largely stationary cell soma. In this chapter, we briefly summarize some of the physiological functions of microglia in the uninjured brain, with a focus on interactions they have with synapses.

In Vivo Two-Photon Imaging of Microglial Synapse Contacts.

Microglia are traditionally known as immune sentinels of the brain and as key player in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson disease, or amyotrophic lateral sclerosis. Recently, they were also identified as synaptic organizer, promoting formation and maturation of synapses as well as modifying synaptic activity. Interestingly, microglia-mediated synaptic pruning and microglia-mediated changes in synaptic plasticity were observed both during brain development and in neurodegenerative diseases, stressing the key role of microglia-synapse interaction in these processes. Here we descried a technique for noninvasive in vivo monitoring of microglia-synapse interactions by means of two-photon microscopy.

Making light work of fine-tuning channelrhodopsins.

The development of genetically engineered proteins that can control cell excitability with light have revolutionized our understanding of the nervous system. The most widely used of these optogenetic tools is the light-gated ion channel, channelrhodopsin 2 (ChR2). A new study by Cho et al. describes the development of ChR2 variants with improved photocurrents and more selective ion permeability using an automated multifaceted fluorescence-based screening. This methodological framework holds promise not only in refining features of ChR2, but also for other proteins in which fluorescence phenotyping is possible.

Microglia Enhance Synapse Activity to Promote Local Network Synchronization.

Microglia are highly motile immunoreactive cells that play integral roles in the response to brain infection and damage, and in the progression of various neurological diseases. During development, microglia also help sculpt neural circuits, via both promoting synapse formation and by targeting specific synapses for elimination and phagocytosis. Microglia are also active surveyors of neural circuits in the mature, healthy brain, although the functional consequences of such microglia-neuron contacts under these conditions is unclear. Using in vivo imaging of neurons and microglia in awake mice, we report here the functional consequences of microglia-synapse contacts. Direct contact between a microglial process and a single synapse results in a specific increase in the activity of that contacted synapse, and a corresponding increase in back-propagating action potentials along the parent dendrite. This increase in activity is not seen for microglia-synapse contacts when microglia are activated by chronic lipopolysaccharide (LPS) treatment. To probe how this microglia-synapse contact affects neural circuits, we imaged across larger populations of motor cortical neurons. When microglia were again activated by LPS (or partially ablated), there was a decrease in the extent to which neuronal activity was synchronized. Together, our results demonstrate that interactions between physiological or resting microglia and synapses in the mature, healthy brain leads to an increase in neuronal activity and thereby helps to synchronize local populations of neurons. Our novel findings provide a plausible physical basis for understanding how alterations in immune status may impact on neural circuit plasticity and on cognitive behaviors such as learning.

Cortical astrocytes prime the induction of spine plasticity and mirror image pain.

Peripheral nerve injury causes maladaptive plasticity in the central nervous system and induces chronic pain. In addition to the injured limb, abnormal pain sensation can appear in the limb contralateral to the injury, called mirror image pain. Because synaptic remodeling in the primary somatosensory cortex (S1) has critical roles in the induction of chronic pain, cortical reorganization in the S1 ipsilateral to the injured limb may also accompany mirror image pain. To elucidate this, we conducted in vivo 2-photon calcium imaging of neuron and astrocyte activity in the ipsilateral S1 after a peripheral nerve injury. We found that cross-callosal inputs enhanced the activity of both S1 astrocytes and inhibitory neurons, whereas activity of excitatory neurons decreased. When local inhibitory circuits were blocked, astrocyte-dependent spine plasticity and allodynia were revealed. Thus, we propose that cortical astrocytes prime the induction of spine plasticity and mirror image pain after peripheral nerve injury. Moreover, this result suggests that cortical synaptic rewiring could be sufficient to cause allodynia on the uninjured periphery.

Focal Ischaemic Infarcts Expand Faster in Cerebellar Cortex than Cerebral Cortex in a Mouse Photothrombotic Stroke Model.

It is generally accepted that the cerebellum is particularly vulnerable to ischaemic injury, and this may contribute to the high mortality arising from posterior circulation strokes. However, this has not been systematically examined in an animal model. This study compared the development and resolution of matched photothrombotic microvascular infarcts in the cerebellar and cerebral cortices in adult 129/SvEv mice of both sexes. The photothrombotic lesions were made using tail vein injection of Rose Bengal with a 532 nm laser projected onto a 2 mm diameter aperture over the target region of the brain (with skull thinning). Infarct size was then imaged histologically following 2 h to 30-day survival using serial reconstruction of haematoxylin and eosin stained cryosections. This was complemented with immunohistochemistry for neuron and glial markers. At 2 h post-injury, the cerebellar infarct volume averaged ~ 2.7 times that of the cerebral cortex infarcts. Infarct volume reached maximum in the cerebellum in a quarter of the time (24 h) taken in the cerebral cortex (4 days). Remodelling resolved the infarcts within a month, leaving significantly larger residual injury volume in the cerebellum. The death of neurons in the core lesion at 2 h was confirmed by NeuN and Calbindin immunofluorescence, alongside activation of astrocytes and microglia. The latter persisted in the region within and surrounding the residual infarct at 30 days. This comparison of acute focal ischaemic injuries in cerebellar and cerebral cortices provides direct confirmation of exacerbation of neuropathology and faster kinetics in the cerebellum.

Activation-Dependent Rapid Postsynaptic Clustering of Glycine Receptors in Mature Spinal Cord Neurons.

Inhibitory synapses are established during development but continue to be generated and modulated in strength in the mature nervous system. In the spinal cord and brainstem, presynaptically released inhibitory neurotransmitter dominantly switches from GABA to glycine during normal development in vivo. While presynaptic mechanisms of the shift of inhibitory neurotransmission are well investigated, the contribution of postsynaptic neurotransmitter receptors to this shift is not fully elucidated. Synaptic clustering of glycine receptors (GlyRs) is regulated by activation-dependent depolarization in early development. However, GlyR activation induces hyperpolarization after the first postnatal week, and little is known whether and how presynaptically released glycine regulates postsynaptic receptors in a depolarization-independent manner in mature developmental stage. Here we developed spinal cord neuronal culture of rodents using chronic strychnine application to investigate whether initial activation of GlyRs in mature stage could change postsynaptic localization of GlyRs. Immunocytochemical analyses demonstrate that chronic blockade of GlyR activation until mature developmental stage resulted in smaller clusters of postsynaptic GlyRs that could be enlarged upon receptor activation for 1 h in the mature stage. Furthermore, live cell-imaging techniques show that GlyR activation decreases its lateral diffusion at synapses, and this phenomenon is dependent on PKC, but neither Ca2+ nor CaMKII activity. These results suggest that the GlyR activation can regulate receptor diffusion and cluster size at inhibitory synapses in mature stage, providing not only new insights into the postsynaptic mechanism of shifting inhibitory neurotransmission but also the inhibitory synaptic plasticity in mature nervous system.

Microglia contact induces synapse formation in developing somatosensory cortex.

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8-10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca(2+) transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia-axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.