Changes In Neuronal Excitability Research Articles

Second hand smoke exposure depresses neuronal excitability and enhances 4‐AP sensitive channel function of cardiac vagal neurons in the nucleus ambiguus

BACKGROUNDSecond hand smoke (SHS) is a significant risk factor for cardiac dysfunction including arrhythmias and sudden cardiac death. Previously we showed that environmentally relevant concentrations of SHS reduce heart rate variability (HRV), an index of autonomic function. We also showed that SHS exposure significantly reduces cardiac vagal neuronal excitability in the nucleus ambiguus (NA), the final vagal cardiac output regulating HRV. We further showed that SHS‐induced attenuation of SK channel activity that may serve as a partial compensatory mechanism to the reduction in overall neuronal excitability. Here, we sought to test the hypothesis that SHS exposure, at an environmentally relevant concentration that reduces HRV, depresses neuronal excitability of cardiac vagal neurons in the NA by enhancing 4‐AP sensitive potassium channel function.METHODSAdult male C57BL6/J mice underwent open chest surgeries to retrograde label cardiac vagal neurons by applying a fluorescent dye (DiI) to the sinoatrial node. After two weeks of recovery from surgery, mice were randomly assigned to either filtered air (FA) or SHS (3 mg/m3, 6 hr/d, 5 d/wk) for 4‐, 8‐ or 12‐weeks. Whole‐cell current clamp recordings in brainstem slices were performed on anatomically identified cardiac vagal neurons in the NA with fluorescent labeling.RESULTSSHS exposure significantly (p < 0.05) reduced spiking response to depolarizing current injections after 4‐ and 12‐weeks – but not 8‐weeks – of exposure. Perfusion with 1 mM of 4‐AP, a voltage‐activated potassium channel blocker, similarly increased spiking response by 5–10 spikes/s across all time points. The 4‐AP induced changes in spiking responses were not different between SHS and FA (p > 0.05). SHS exposure also significantly (p < 0.05) increased voltage and current threshold for action potential generation for all time points tested (4‐, 8‐, and 12‐weeks). 4‐AP significantly lowered the voltage threshold; these effects were significantly (p < 0.05) bigger in the SHS group (−1.2 ± 0.5 mV vs. −3.7 ± 0.4 mV, FA vs. SHS respectively). In contrast, 4‐AP similarly lowered current threshold for action potential generation in both FA and SHS groups (−80 ± 13 pA vs. −103 ± 12 pA, FA vs. SHS, respectively).CONCLUSIONSWe conclude that environmentally relevant SHS exposure induces time‐dependent changes in neuronal excitability of cardiac vagal neurons. Some of these changes may be the result of increased voltage‐activated potassium channel function.Support or Funding InformationR01 ES025229; T32 HL086350This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lysozyme over‐expression during nerve injury excites Aδ & C fibres in a fibre specific manner to incite neuropathic pain

Human lysozyme is one of the most extensively studied proteins yet its exact physiological functions still remain a mystery. Here, we highlight a hitherto unrecognized role of lysozyme in neuropathic pain and neuronal excitability. Injection of lysozyme alone in healthy rats leads to heightened sensitivity in them towards both mechanical and thermal pain stimuli. Also, in rat models of neuropathic pain, lysozyme is over – expressed and its down – regulation by siRNA or inhibition of its activity by its disaccharide inhibitor ‐ chitobiose relieves pain confirming a causal role of lysozyme in neuropathic pain. Moreover, as the catalytically incompetent lysozyme or its chemically inhibited counterparts fail to provoke pain indicate that it deploys its catalytic site for eliciting pain. Bath application of lysozyme remarkably increased both the spontaneous excitatory potentials and currents (sEPSPs & sEPSCs) and the amplitudes of afferent mediated evoked excitatory synaptic inputs (eFPSPs & eEPSCs) in Aδ fibres in a fibre specific manner in the dorsal horn neurons. It also increased paired pulsed ratio only in the C‐fibres. When complexed with chitobiose it does not lead to any change in neuronal excitability. Thus, lysozyme excites neurons by deploying its catalytic region in a fibre specific manner. Further, we show that it deploys its catalytic site to recruit Annexin A2 to transduce TLR4→NFkβ signaling pathway which may culminate in the fusion of neurotransmitter vesicles at the synapse to manifest neuronal excitability and eventually pain. Thus, glycan inhibitors of lysozyme and antagonists of the pathway recruited by it to inflict pain pave not only the way for novel approaches for dissecting the molecular mechanism of neuropathic pain but also for treating this unmet medical need efficaciously.Support or Funding InformationCouncil of Scientific and Industrial Research, India to ASThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A305 THE PRONOCICEPTIVE EFFECT OF HIGH DOSE OPIOIDS ON MOUSE DRG NEURONS IS MEDIATED BY DELTA OPIOID RECEPTORS

AbstractBackgroundOpioid drugs can be efficacious in the treatment of abdominal pain, but escalating doses of these drugs can also induce pronociceptive signalling in patients and paradoxically worsen pain. The analgesic actions of opioid drugs on dorsal root ganglia (DRG) neurons innervating the intestine are largely mediated by mu opioid receptors (MOR) but there is growing evidence that delta opioid receptors (DOR) could be important targets for pain signaling and to prevent opioid tolerance. However, it is unknown whether MOR and DOR are co-expressed on colonic DRG neurons and whether DOR play a role in pain signaling in the gut.AimsThe present study examined whether exposure to low and high concentrations of the opioid receptor agonist DAMGO had differing effects on the excitability of nociceptive DRG neurons, and if so, whether these actions involved MOR and/or DOR signaling.MethodsSingle unit afferent recordings were obtained in situ from mouse colons to determine the effects of the MOR agonist DAMGO (100nM) and the DOR agonist DADLE (100nM) on mechanosensitive responses. To examine the effects of high and low dose opioids, nociceptive mouse DRG neurons were dissociated from control mice and incubated overnight with 10 nM or 10 µM DAMGO or media alone. Changes in neuronal excitability were recorded by measuring the rheobase using patch clamp recordings. To determine whether changes in cell excitability were mediated by MOR or DOR, neurons were incubated with the MOR antagonist CTOP (100 nM) or DOR antagonist SDM25N (1 µM) 30 min prior to the incubation with DAMGO.ResultsSingle unit recordings from mouse colon were inhibited by both MOR and DOR agonists in 38% of afferents (3/8). Overnight incubation with 10 nM DAMGO inhibited neurons (rheobase increased by 43%, p <0.001) whereas 10 µM DAMGO had an excitatory effect (rheobase decreased by 31%, p <0.01). The inhibitory effect of low dose DAMGO was blocked by the MOR antagonist but the DOR antagonist had no effect. In contrast, the excitatory effect of the high dose DAMGO was completely blocked by the DOR antagonist.ConclusionsMany colonic DRG neurons co-express MOR and DOR. High concentrations of opioids paradoxically increase excitability of nociceptive DRG neurons, potentially by signaling to MOR-DOR heterodimers. This could lead to increased pain signaling and targeting DOR may be a potential target to mitigate this action.Funding AgenciesCCC

A304 FOOD ANTIGEN-STRESS INTERACTION INCREASES PERIPHERAL PAIN SIGNALING IN A MOUSE MODEL OF IBS

Abstract Background Food and stress are common triggers of symptoms in irritable bowel syndrome (IBS). However, it is unknown whether a specific food antigen alone can induce symptoms such as abdominal pain or if a second trigger, such as stress, is required for a food antigen to increase pain signaling. Aims To determine if a food antigen (ovalbumin) can induce hyperexcitabiity in nociceptive neurons alone and/or in combination with stress. Methods Mice were exposed to water avoidance stress (WAS; 1 hr for 6 days). On day 2, mice were gavaged ovalbumin (20mg) daily after completing WAS (WAS/OVA). These were compared to 3 groups: no WAS and no ovalbumin (control); WAS but no ovalbumin (WAS); and ovalbumin (OVA) alone. On day 8, WAS, OVA and WAS/OVA mice were given ovalbumin (50 μg) subcutaneously; 3 hours later euthanized. Colons were removed and incubated with ovalbumin (100 μg/ml) for 4 hrs. Supernatants were collected and DRG neurons from control mice were incubated overnight with supernatants from each of the four groups. Changes in neuronal excitability were examined by measuring the rheobase (minimal current to evoke an action potential) using perforated patch clamp recording techniques. Stress effects on intestinal permeability in the ileum and colon were assessed in Ussing chambers. One way ANOVA and Bonferroni post hoc test or unpaired t test were used to analyze the data. Results Incubation with supernatants obtained from WAS/OVA mice evoked hyperexcitability in DRG neurons compared to incubation with control supernatant (rheobase: control = 82 ± 10 pA vs WAS/OVA = 56 ± 4 pA, p &amp;lt; 0.05). Similarly, WAS/OVA supernatant decreased the rheobase compared to both OVA mice (OVA = 80 ± 8 pA, p&amp;lt;0.05) and WAS mice (WAS = 81 ± 9 pA, p&amp;lt;0.05). The effect of supernatants from OVA mice and WAS mice did not differ from controls. In a separate series of experiments, the PAR2 antagonist GB83 inhibited the effect of WAS/OVA supernatant (p &amp;lt; 0.01). There was no effect of GB83 on WAS or OVA supernatants. Stress decreased tissue resistance in ileum (p&amp;lt;0.05) but not in colon. Conclusions The food antigen ovalbumin induced hyperexcitability in DRG nociceptive neurons only when combined with stress. This action was PAR2 dependent suggesting a role of tissue proteases. Stress may cause a loss of oral tolerance to ovalbumin by increasing mucosal permeability in the small intestine. The interaction of food antigens and stress may be a mechanism of meal induced increase in abdominal pain in IBS. Funding Agencies CIHR

Axon Initial Segment Structural Plasticity is Involved in Seizure Susceptibility in a Rat Model of Cortical Dysplasia.

Cortical dysplasia is the most common etiology of intractable epilepsy. Both excitability changes in cortical neurons and neural network reconstitution play a role in cortical dysplasia epileptogenesis. Recent research shows that the axon initial segment, a subcompartment of the neuron important to the shaping of action potentials, adjusts its position in response to changes in input, which contributes to neuronal excitability and local circuit balance. It is unknown whether axon initial segment plasticity occurs in neurons involved in seizure susceptibility in cortical dysplasia. Here, we developed a "Carmustine"- "pilocarpine" rat model of cortical dysplasia and show that it exhibits a lower seizure threshold, as indicated by behavior studies and electroencephalogram monitoring. Using immunofluorescence, we measured the axon initial segment positions of deep L5 somatosensory neurons and show that it is positioned closer to the soma after acute seizure, and that this displacement is sustained in the chronic phase. We then show that Nifedipine has a dose-dependent protective effect against axon initial segment displacement and increased seizure susceptibility. These findings further our understanding of the pathophysiology of seizures in cortical dysplasia and suggests Nifedipine as a potential therapeutic agent.

(133) - Homeostatic regulation of intrinsic plasticity in mouse and human peripheral nociceptors

Stabilization of neuronal activity is crucial for normal nervous system regulation. When changes in the environment chronically alter neuronal activity, homeostatic plasticity creates compensatory shifts (i.e., regulation of intrinsic excitability) to return the system to a physiological level. This plasticity helps prevent circuits from becoming either hyper- or hypoexcitable. Despite this protective mechanism, evidence suggests that the development and maintenance of many chronic pain conditions depend on chronically altered sensory afferent activity. This led us to consider the possibility that malfunction of homeostatic mechanisms may contribute to chronic pain. Since homeostatic plasticity has been primarily studied in the central nervous system, we first tested whether this plasticity also occurs in peripheral nociceptors [i.e., small diameter dorsal root ganglia (DRG) sensory afferent neurons]. We hypothesized that mouse and human primary sensory afferents undergo homeostatic regulation of intrinsic plasticity, evidenced by compensatory changes in neuronal excitability in response to chronic stimuli. Using a combination of pharmacologic and optogenetic approaches with whole-cell electrophysiology, we found that sustained, 24 hour depolarization of dissociated mouse DRG neurons leads to a decrease in excitability of small diameter, putative nociceptors as shown by decreased input resistance, increased rheobase and decreased number of action potentials in response to suprathreshold current injections. These effects rebounded as an increase in excitability with a 24 hour recovery. Human DRG neurons, extracted and dissociated from donors, show a similar pattern of responses. Our results suggest that both mouse and human putative nociceptors undergo homeostatic regulation of intrinsic excitability in response to sustained, pharmacological depolarization or sustained stimulation of activity. Ongoing experiments are being performed to determine whether mechanisms of this plasticity are altered during chronic pain. This work was supported by grants R01 NS042595 and T32 DA007261.

Memory formation depends on both synapse-specific modifications of synaptic strength and cell-specific increases in excitability.

The modification of synaptic strength produced by long-term potentiation (LTP) is widely thought to underlie memory storage. Indeed, given that hippocampal pyramidal neurons have >10,000 independently modifiable synapses, the potential for information storage by synaptic modification is enormous. However, recent work suggests that CREB-mediated global changes in neuronal excitability also play a critical role in memory formation. Because these global changes have a modest capacity for information storage compared with that of synaptic plasticity, their importance for memory function has been unclear. Here we review the newly emerging evidence for CREB-dependent control of excitability and discuss two possible mechanisms. First, the CREB-dependent transient change in neuronal excitability performs a memory-allocation function ensuring that memory is stored in ways that facilitate effective linking of events with temporal proximity (hours). Second, these changes may promote cell-assembly formation during the memory-consolidation phase. It has been unclear whether such global excitability changes and local synaptic mechanisms are complementary. Here we argue that the two mechanisms can work together to promote useful memory function.

Layer- and Cell Type-Specific Modulation of Excitatory Neuronal Activity in the Neocortex.

From an anatomical point of view the neocortex is subdivided into up to six layers depending on the cortical area. This subdivision has been described already by Meynert and Brodmann in the late 19/early 20. century and is mainly based on cytoarchitectonic features such as the size and location of the pyramidal cell bodies. Hence, cortical lamination is originally an anatomical concept based on the distribution of excitatory neuron. However, it has become apparent in recent years that apart from the layer-specific differences in morphological features, many functional properties of neurons are also dependent on cortical layer or cell type. Such functional differences include changes in neuronal excitability and synaptic activity by neuromodulatory transmitters. Many of these neuromodulators are released from axonal afferents from subcortical brain regions while others are released intrinsically. In this review we aim to describe layer- and cell-type specific differences in the effects of neuromodulator receptors in excitatory neurons in layers 2–6 of different cortical areas. We will focus on the neuromodulator systems using adenosine, acetylcholine, dopamine, and orexin/hypocretin as examples because these neuromodulator systems show important differences in receptor type and distribution, mode of release and functional mechanisms and effects. We try to summarize how layer- and cell type-specific neuromodulation may affect synaptic signaling in cortical microcircuits.

Atypical changes in DRG neuron excitability and complex pain phenotype associated with a Nav1.7 mutation that massively hyperpolarizes activation

Sodium channel Nav1.7 plays a central role in pain-signaling: gain-of-function Nav1.7 mutations usually cause severe pain and loss-of-function mutations produce insensitivity to pain. The Nav1.7 I234T gain-of-function mutation, however, is linked to a dual clinical presentation of episodic pain, together with absence of pain following fractures, and corneal anesthesia. How a Nav1.7 mutation that produces gain-of-function at the channel level causes clinical loss-of-function has remained enigmatic. We show by current-clamp that expression of I234T in dorsal root ganglion (DRG) neurons produces a range of membrane depolarizations including a massive shift to >−40 mV that reduces excitability in a small number of neurons. Dynamic-clamp permitted us to mimic the heterozygous condition via replacement of 50% endogenous wild-type Nav1.7 channels by I234T, and confirmed that the I234T conductance could drastically depolarize DRG neurons, resulting in loss of excitability. We conclude that attenuation of pain sensation by I234T is caused by massively depolarized membrane potential of some DRG neurons which is partly due to enhanced overlap between activation and fast-inactivation, impairing their ability to fire. Our results demonstrate how a Nav1.7 mutation that produces channel gain-of-function can contribute to a dual clinical presentation that includes loss of pain sensation at the clinical level.

CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory.

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBN mutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7 ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN–associated ID.

Altered Homeostatic Functions in Reactive Astrocytes and Their Potential as a Therapeutic Target After Brain Ischemic Injury.

Brain ischemic injury represents one of the greatest medical challenges for the aging population in developed countries, yet despite strong efforts, possibilities to treat ischemic injury still remain poor. Stroke, the most common type of brain ischemic injury in humans, is caused by brain artery occlusion, and represents a focal form of ischemia, which leads to neuronal loss in the ischemic core, and glial scar formation in the penumbral region around the core. Such glial scar mainly comprises reactive astrocytes, reactive NG2 glia and activated microglia. Reactive astrocytes display distinct features when compared to healthy astroglia, including changes in their morphology, metabolism, gene expression profiles, production of extracellular matrix proteins or proliferation rate. Similarly to astrocytes in the healthy nervous tissue, reactive astrocytes surrounding the glial scar strongly influence the activity of surviving neurons around the ischemic lesion. Bringing insight into pathophysiological functions of reactive astrocytes within the glial scar might thus open new possibilities for stroke treatment. Here, we summarize the properties of reactive astrocytes, with emphasis on the expression and function of ion channels, transporters and neurotransmitter receptors; all of which possess the ability to change the functional state of astrocytes, such as the membrane equilibrium potentials for different ions. This may have major effects on the functioning of surviving neurons, consequently leading to changes in neuronal excitability and progression of secondary pathologies, such as epilepsy. Moreover, we provide possible clues for therapy, based on functional modulation of astrocytic ion transporting mechanisms.

SNC80 and naltrindole modulate voltage-dependent sodium, potassium and calcium channels via a putatively delta opioid receptor-independent mechanism.

SNC80 was designed as a highly selective nonpeptide delta opioid receptor (DOR) agonist. Antidepressant-like and antinociceptive effects of this compound were demonstrated in animal models. Naltrindole was synthetized as a highly selective DOR antagonist. Its antitussive and antinociceptive effects were reported. Observed effects of SNC80 and naltrindole may be accompanied by changes in neuronal excitability including modulation of voltage-dependent ion channels. We investigated possible DOR-independent modulation of neuronal sodium, calcium and potassium currents by both agents. NG108-15 cells lacking expression of DOR protein were used as model of neuronal cells. Cells were differentiated into neuronal phenotype by exposure to dibutyryl cyclic-AMP (dbcAMP). Lack of DORs expression in NG108-15 cells and the presence of DOR expression in brain and neuronal cultures were demonstrated by Western blot analysis. Both SNC80 and naltrindole exerted low to moderate modulatory effects on voltage-dependent ion currents. SNC80 weakly inhibited sodium current, potentiated calcium current, and did not act on potassium channels. Naltrindole inhibited sodium current, did not act on calcium current and inhibited potassium current at a high concentration. Such effects should be taken into account when these compounds are used for investigation of DOR-mediated signaling pathways.

Behavioral tagging: Synaptic event or cellular alteration?

Recently a number of studies were published addressing the translation of the synaptic tagging and capture hypothesis (STCH), developed and tested in hippocampal slices, into learning and long-term memory mechanisms in intact animals. Based on behavioral observations, these studies show similar results as in the original STCH experiments. A weak memory, induced by weak training in one hippocampus dependent behavioral task is converted into a long-term memory, by timely related induction of a long-term memory, induced by strong training in a second hippocampus dependent task. The induction of long-term memory should result in the synthesis of plasticity related proteins (PRP), which can be captured by the synapses involved in the weak memory trace and thus be converted into a long-term memory. Here it is outlined, that these results can alternatively be explained by non-synaptic mechanisms like changes in neuronal intrinsic excitability (IE). The plasticity of short and long-term IE show some common features with synaptic plasticity. Thus, behavioral tagging experiments, purely based on behavioral observations, do not necessarily support that synaptic tagging and capture mechanisms are involved in memory formations, unless it has been shown that the weak and strong training protocols result in synaptic early and late potentiation, or the STCH could be extended to short and long-term forms of intrinsic excitability.

Prefrontal cortical-specific differences in behavior and synaptic plasticity between adolescent and adult mice.

Adolescence is a highly vulnerable period for the emergence of major neuropsychological disorders and is characterized by decreased cognitive control and increased risk-taking behavior and novelty-seeking. The prefrontal cortex (PFC) is involved in the cognitive control of impulsive and risky behavior. Although the PFC is known to reach maturation later than other cortical areas, little information is available regarding the functional changes from adolescence to adulthood in PFC, particularly compared with other primary cortical areas. This study aims to understand the development of PFC-mediated, compared with non-PFC-mediated, cognitive functions. Toward this aim, we performed cognitive behavioral tasks in adolescent and adult mice and subsequently investigated synaptic plasticity in two different cortical areas. Our results showed that adolescent mice exhibit impaired performance in PFC-dependent cognitive tasks compared with adult mice, whereas their performance in non-PFC-dependent tasks is similar to that of adults. Furthermore, adolescent mice exhibited decreased long-term potentiation (LTP) within upper-layer synapses of the PFC but not the barrel cortex. Blocking GABAA receptor function significantly augments LTP in both the adolescent and adult PFC. No change in intrinsic excitability of PFC pyramidal neurons was observed between adolescent and adult mice. Finally, increased expression of the NR2A subunit of the N-methyl-d-aspartate receptors is found only in the adult PFC, a change that could underlie the emergence of LTP. In conclusion, our results demonstrate physiological and behavioral changes during adolescence that are specific to the PFC and could underlie the reduced cognitive control in adolescents. NEW & NOTEWORTHY This study reports that adolescent mice exhibit impaired performance in cognitive functions dependent on the prefrontal cortex but not in cognitive functions dependent on other cortical regions. The current results propose reduced synaptic plasticity in the upper layers of the prefrontal cortex as a cellular correlate of this weakened cognitive function. This decreased synaptic plasticity is due to reduced N-methyl-d-aspartate receptor expression but not due to dampened intrinsic excitability or enhanced GABAergic signaling during adolescence.

Evaluation of Postspike Changes in Neuronal Excitability by Comparing Ordinary and Shuffled Autocorrelation Functions

Responses of single neurons to tonal signals amplitude-modulated by repeating segments of lowfrequency noise were studied in the dorsal (cochlear) medullary nucleus and midbrain auditory center (torus semicircularis) of the grass frog Rana temporaria. An autocorrelation function of the response to a total presentation and a shuffled autocorrelation function were derived. The latter was obtained by correlating the impulse response to each segment of the modulated signal with responses to all other segments with the exception of the initial one. After the necessary normalization, the function differed from the initial autocorrelation only in lacking postspike changes in excitability. A delay dependence of the ratio of the two functions directly demonstrated the time course of the postspike change in excitability of the studied cell. The majority of second-order neurons, which are in the dorsal nucleus of the medulla oblongata, were characterized only by brief intervals of absolute and relative refractoriness. However, cells with excitability that was markedly facilitated immediately after the refractory period were observed even in this nucleus. Neurons with a complex pattern of postspike changes in excitability were detected in the torus semicircularis. In these cells, a comparatively long postspike decrease in excitability was usually interrupted by intervals in which the neuron sensitivity was significantly higher than normal. The results demonstrate that spike generation has a marked effect on subsequent activity in brainstem auditory units. The effects may play an important role in the formation of the temporal pattern of neuronal responses to auditory signals.

Changes in the Excitability of Neocortical Neurons in a Mouse Model of Amyotrophic Lateral Sclerosis Are Not Specific to Corticospinal Neurons and Are Modulated by Advancing Disease.

Cell type-specific changes in neuronal excitability have been proposed to contribute to the selective degeneration of corticospinal neurons in amyotrophic lateral sclerosis (ALS) and to neocortical hyperexcitability, a prominent feature of both inherited and sporadic variants of the disease, but the mechanisms underlying selective loss of specific cell types in ALS are not known. We analyzed the physiological properties of distinct classes of cortical neurons in the motor cortex of hSOD1G93A mice of both sexes and found that they all exhibit increases in intrinsic excitability that depend on disease stage. Targeted recordings and in vivo calcium imaging further revealed that neurons adapt their functional properties to normalize cortical excitability as the disease progresses. Although different neuron classes all exhibited increases in intrinsic excitability, transcriptional profiling indicated that the molecular mechanisms underlying these changes are cell type specific. The increases in excitability in both excitatory and inhibitory cortical neurons show that selective dysfunction of neuronal cell types cannot account for the specific vulnerability of corticospinal motor neurons in ALS. Furthermore, the stage-dependent alterations in neuronal function highlight the ability of cortical circuits to adapt as disease progresses. These findings show that both disease stage and cell type must be considered when developing therapeutic strategies for treating ALS.SIGNIFICANCE STATEMENT It is not known why certain classes of neurons preferentially die in different neurodegenerative diseases. It has been proposed that the enhanced excitability of affected neurons is a major contributor to their selective loss. We show using a mouse model of amyotrophic lateral sclerosis (ALS), a disease in which corticospinal neurons exhibit selective vulnerability, that changes in excitability are not restricted to this neuronal class and that excitability does not increase monotonically with disease progression. Moreover, although all neuronal cell types tested exhibited abnormal functional properties, analysis of their gene expression demonstrated cell type-specific responses to the ALS-causing mutation. These findings suggest that therapies for ALS may need to be tailored for different cell types and stages of disease.

Metallo-β-lactamase Domain-Containing Protein 1 (MBLAC1) Is a Specific, High-Affinity Target for the Glutamate Transporter Inducer Ceftriaxone.

Ceftriaxone, a β-lactam antibiotic, has been reported to act independently of its antimicrobial actions to normalize perturbed central nervous system glutamate levels, principally by elevating expression of glial glutamate transporters. Identification of a specific, high-affinity target for ceftriaxone could significantly impact therapeutic development for multiple brain disorders, ranging from neurodegenerative disorders to addiction. Recently, we identified a glial-expressed Caenorhabditis elegans gene, swip-10, that encodes a metallo-β-lactamase domain-containing protein, and limits glutamate-dependent changes in dopamine neuron excitability. Bioinformatic analyses identified MBLAC1 as the likely mammalian orthologue of swip-10. Using cyanogen bromide immobilized ceftriaxone for affinity capture experiments and backscattering interferometry to monitor MBLAC1 binding of unmodified ceftriaxone, we obtained evidence for specific, high affinity (KD = 2.2 μM) binding of ceftriaxone to MBLAC1. We discuss our findings with respect to MBLAC1 as a potentially exclusive, high-affinity binding partner of ceftriaxone in the CNS, and the path forward in the development of novel, MBLAC1-based therapeutics.

Interictal oscillations and focal epileptic disorders.

Neuronal network oscillations represent a main feature of the brain activity recorded in the EEG under normal and pathological conditions such as epilepsy. Specific oscillations occur between seizures in patients and in animal models of focal epilepsy, and thus, they are termed interictal. According to their shape and intrinsic signal frequency, interictal oscillations are classified as spikes and high-frequency oscillations (HFOs). Interictal spikes are recorded in the 'wideband' EEG signal and consist of large-amplitude events that usually last less than 1s; HFOs, in contrast, are extracted by amplifying the appropriately filtered EEG signal and are usually categorized as ripples (80-200Hz) and fast ripples (250-500Hz). Interictal spikes and HFOs are used in clinical practice to localize the seizure onset zone in focal epileptic disorders, which is fundamental for performing successful surgical interventions in epileptic patients not responding to anti-epileptic drug therapy. Both types of interictal oscillations have been widely studied in animal models of focal epilepsy to identify the mechanisms underlying their generation as well as to establish their role in ictogenesis and epileptogenesis. In this review, we will address the cellular mechanisms underlying the generation of interictal spikes and HFOs in animal models of epileptiform synchronization and of focal epilepsy. Moreover, we will highlight invitro and invivo evidence indicating that these interictal oscillations mirror specific, dynamic changes in neuronal network excitability causing seizure generation (i.e. ictogenesis) and leading to a chronic epileptic condition (i.e. epileptogenesis).

Delayed Cryptochrome Degradation Asymmetrically Alters the Daily Rhythm in Suprachiasmatic Clock Neuron Excitability.

Suprachiasmatic nuclei (SCN) neurons contain an intracellular molecular circadian clock and the Cryptochromes (CRY1/2), key transcriptional repressors of this molecular apparatus, are subject to post-translational modification through ubiquitination and targeting for proteosomal degradation by the ubiquitin E3 ligase complex. Loss-of-function point mutations in a component of this ligase complex, Fbxl3, delay CRY1/2 degradation, reduce circadian rhythm strength, and lengthen the circadian period by ∼2.5 h. The molecular clock drives circadian changes in the membrane properties of SCN neurons, but it is unclear how alterations in CRY1/2 stability affect SCN neurophysiology. Here we use male and female Afterhours mice which carry the circadian period lengthening loss-of-function Fbxl3Afh mutation and perform patch-clamp recordings from SCN brain slices across the projected day/night cycle. We find that the daily rhythm in membrane excitability in the ventral SCN (vSCN) was enhanced in amplitude and delayed in timing in Fbxl3Afh/Afh mice. At night, vSCN cells from Fbxl3Afh/Afh mice were more hyperpolarized, receiving more GABAergic input than their Fbxl3+/+ counterparts. Unexpectedly, the progression to daytime hyperexcited states was slowed by Afh mutation, whereas the decline to hypoexcited states was accelerated. In long-term bioluminescence recordings, GABAA receptor blockade desynchronized the Fbxl3+/+ but not the Fbxl3Afh/Afh vSCN neuronal network. Further, a neurochemical mimic of the light input pathway evoked larger shifts in molecular clock rhythms in Fbxl3Afh/Afh compared with Fbxl3+/+ SCN slices. These results reveal unanticipated consequences of delaying CRY degradation, indicating that the Afh mutation prolongs nighttime hyperpolarized states of vSCN cells through increased GABAergic synaptic transmission.SIGNIFICANCE STATEMENT The intracellular molecular clock drives changes in SCN neuronal excitability, but it is unclear how mutations affecting post-translational modification of molecular clock proteins influence the temporal expression of SCN neuronal state or intercellular communication within the SCN network. Here we show for the first time, that a mutation that prolongs the stability of key components of the intracellular clock, the cryptochrome proteins, unexpectedly increases in the expression of hypoexcited neuronal state in the ventral SCN at night and enhances hyperpolarization of ventral SCN neurons at this time. This is accompanied by increased GABAergic signaling and by enhanced responsiveness to a neurochemical mimic of the light input pathway to the SCN. Therefore, post-translational modification shapes SCN neuronal state and network properties.

Effect of hypoxia on BOLD fMRI response and total cerebral blood flow in migraine with aura patients

Experimentally induced hypoxia triggers migraine and aura attacks in patients suffering from migraine with aura (MA). We investigated the blood oxygenation level-dependent (BOLD) signal response to visual stimulation during hypoxia in MA patients and healthy volunteers. In a randomized double-blind crossover study design, 15 MA patients were allocated to 180 min of normobaric poikilocapnic hypoxia (capillary oxygen saturation 70–75%) or sham (normoxia) on two separate days and 14 healthy volunteers were exposed to hypoxia. The BOLD functional MRI (fMRI) signal response to visual stimulation was measured in the visual cortex ROIs V1–V5. Total cerebral blood flow (CBF) was calculated by measuring the blood velocity in the internal carotid arteries and the basilar artery using phase-contrast mapping (PCM) MRI. Hypoxia induced a greater decrease in BOLD response to visual stimulation in V1–V4 in MA patients compared to controls. There was no group difference in hypoxia-induced total CBF increase. In conclusion, the study demonstrated a greater hypoxia-induced decrease in BOLD response to visual stimulation in MA patients. We suggest this may represent a hypoxia-induced change in neuronal excitability or abnormal vascular response to visual stimulation, which may explain the increased sensibility to hypoxia in these patients leading to migraine attacks.