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Cortical inhibitory circuits are formed by γ-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how

Mitochondria play a prominent role in mechanosensory hair cell damage and death. Although hair cells are thought to be energetically demanding cells, how mitochondria respond to these demands and how this might relate to cell death is largely unexplored. Using genetically encoded indicators, we found that mitochondrial calcium flux and oxidation are regulated by mechanotransduction and demonstrate that hair cell activity has both acute and long-term consequences on mitochondrial function. We tested whether variation in mitochondrial activity reflected differences in the vulnerability of hair cells to the toxic drug neomycin. We observed that susceptibility did not correspond to the acute level of mitochondrial activity but rather to the cumulative history of that activity., Competing Interests: SP, ET, JS, TL, RE, DH, DR No competing interests declared, (© 2018, Pickett et al.)

We report functional and structural evidence for GluA2-lacking Ca 2+ -permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca 2+ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca 2+ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca 2+ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca 2+ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss. SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca 2+ imaging approaches in evolutionarily divergent species, we demonstrate that Ca 2+ -permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca 2+ accumulation and swelling that can be prevented by blocking CP-AMPARs. We demonstrate that CP-AMPARs mediate transmission at this first-order sensory synapse and that limiting Ca 2+ accumulation in the terminal may protect against hearing loss., (Copyright © 2017 the authors 0270-6474/17/376162-14$15.00/0.)

Activation of metabotropic GABAB receptors (GABABRs) enhances tonic GABA current and substantially increases the frequency of spontaneous seizures. Despite the and pro-epileptic consequences of GABABR activation, mice lacking functional GABAB receptors (GABAB1R KO mice) exhibit clonic and rare absence seizures. To examine these mutant mice further, we recorded excitatory and inhibitory synaptic inputs and tonic mutant GABA currents from Layer 2 neocortical pyramidal neurons of GABAB1R WT and KO mice (P30-40). Tonic current was increased while the frequency of synaptic inputs was unchanged in KO mice relative to WT littermates. The neocortical laminar distribution of interneuron subtypes derived from the medial ganglionic eminence (MGE) was also not statistically different in KO mice relative to WT while the number of calretinin-positive, caudal GE-derived cells in Layer 1 was reduced. Transplantation of MGE progenitors obtained from KO mice lacking functional GABAB1R did not increase tonic inhibition in the host brain above that of media-injected controls. Taken together, these results suggest a complex role for GABAB receptors in mediating neocortical circuit function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Most cortical interneurons originate in a region of the embryonic subpallium called the medial ganglionic eminence (MGE). When MGE cells are transplanted into cerebral cortex, these progenitors migrate extensively and differentiate into functional inhibitory neurons. Although MGE progenitors have therapeutic potential following transplantation, it is unknown precisely how these cells distribute within neocortical lamina of the recipient brain. Here we transplanted mouse embryonic day 12.5 MGE progenitors into postnatal neocortex and evaluated laminar distribution of interneuron subtypes using double- and triple-label immunohistochemistry. Studies were performed using wild type (WT) or donor mice lacking a metabotropic GABA(B) receptor subunit (GABA(B1)R KO). MGE-derived neurons from WT and GABA(B1)R KO mice preferentially and densely distributed in neocortical layers 2/3, 5 and 6. As expected, MGE-derived neurons differentiated into parvalbumin+ and somatostatin+ interneurons within these neocortical lamina. Our findings provide insights into the anatomical integration of MGE-derived interneurons following transplantation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

LIS1 gene mutations lead to a rare neurological disorder, classical lissencephaly, characterized by brain malformations, mental retardation, seizures, and premature death. Mice heterozygous for Lis1 (Lis1(+/-)) exhibit cortical malformations, defects in neuronal migration, increased glutamate-mediated synaptic transmission, and spontaneous electrographic seizures. Recent work demonstrated that in utero treatment of Lis1(+/-) mutant dams with ALLN, a calpain inhibitor, partially rescues neuronal migration defects in the offspring. Given the challenges of in utero drug administration, we examined the therapeutic potential of ALLN on postnatal lissencephalic cells. Voltage- and current-clamp studies were performed with acute hippocampal slices obtained from Lis1 mutant mice and age-matched littermate control mice. Specifically, we determined whether postnatal ALLN treatment can reverse excitatory synaptic transmission deficits, namely, an increase in spontaneous and miniature excitatory postsynaptic current (EPSC) frequency, on CA1 pyramidal neurons observed in tissue slices from Lis1(+/-) mice. We found that acute application of ALLN restored spontaneous and miniature EPSC frequencies to wild-type levels without affecting inhibitory postsynaptic synaptic current. Furthermore, Western blot analysis of protein expression, including proteins involved in excitatory synaptic transmission, demonstrated that ALLN blocks the cleavage of the calpain substrate αII-spectrin but does not rescue Lis1 protein levels in Lis1(+/-) mutants.

Cortical inhibitory circuits are formed by γ-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis, is that cortical interneurons are overproduced, and then after their migration into cortex the excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we characterize the developmental cell death of mouse cortical interneurons in vivo, in vitro and after transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax (Bcl-2-associated X)-dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by neurons of the central nervous system. Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Taken together, our findings indicate that interneuron cell death is determined intrinsically, either cell-autonomously or through a population-autonomous competition for survival signals derived from other interneurons.

Of the nearly 3 million Americans diagnosed with epilepsy, approximately 30% are unresponsive to current medications. Recent data has shown that early postnatal transplantation of interneuronal precursor cells increases GABAergic inhibition in the host brain and dramatically suppresses seizure activity in epileptic mice. In this review, we will highlight findings from seizure-prone mice and humans that demonstrate the link between dysfunctional GABAergic inhibition and hyperexcitability. In particular, we will focus on rodent models of temporal lobe epilepsy, the most common and difficult to treat form of the disease, and interneuronopathies, an emerging classification. A wealth of literature showing a causal link between reduced GABA-mediated inhibition and seizures has directed our efforts to recover the loss of inhibition via transplantation of interneuronal precursors. Numerous related studies have explored the anticonvulsant potential of cell grafts derived from a variety of brain regions, yet the mechanism underlying the effect of such heterogeneous cell transplants is unknown. In discussing our recent findings and placing them in context with what is known about epilepsy, and how related transplant approaches have progressed, we hope to initiate a frank discussion of the best path toward the translation of this approach to patients with intractable forms of epilepsy., (Copyright © 2010 Wiley Periodicals, Inc.)

Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl(-) reversal potential (E(Cl)). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA(A) receptors (GABA(A) Rs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2-4] then decreases dramatically by the second postnatal week (P7-10 and P30-40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA(A) R α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 μM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E(Cl) recorded from P2-5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Epilepsy, a disease characterized by abnormal brain activity, is a disabling and potentially life-threatening condition for nearly 1% of the world population. Unfortunately, modulation of brain excitability using available antiepileptic drugs can have serious side effects, especially in the developing brain, and some patients can only be improved by surgical removal of brain regions containing the seizure focus. Here, we show that bilateral transplantation of precursor cells from the embryonic medial ganglionic eminence (MGE) into early postnatal neocortex generates mature GABAergic interneurons in the host brain. In mice receiving MGE cell grafts, GABA-mediated synaptic and extrasynaptic inhibition onto host brain pyramidal neurons is significantly increased. Bilateral MGE cell grafts in epileptic mice lacking a Shaker-like potassium channel (a gene mutated in one form of human epilepsy) resulted in significant reductions in the duration and frequency of spontaneous electrographic seizures. Our findings suggest that MGE-derived interneurons could be used to ameliorate abnormal excitability and possibly act as an effective strategy in the treatment of epilepsy.

Respiratory neurons are synchronized on a long time scale to generate inspiratory and expiratory-phase activities that are critical for respiration. Long time scale synchrony within the respiratory network occurs on a time scale of more than hundreds of milliseconds to seconds. During inspiration, neurons are synchronized on a short time scale to produce synchronous oscillations, which shape the pattern of inspiratory motor output. This latter form of synchrony within the respiratory network spans a shorter time range of tens of milliseconds. In the neonatal mouse rhythmically active medullary slice preparation, we recorded bilateral inspiratory activity from hypoglossal (XII) rootlets to study where in the slice synchronous oscillations are generated. Based on previous work that proposed the origin of these oscillations, we tested the pre-Bötzinger complex (PreBötC) and the XII motor nucleus. Unilateral excitation of the PreBötC, via local application of a perfusate containing high K(+), increased mean inspiratory burst frequency bilaterally (296+/-66%; n=10, P<0.01), but had no effect on the relative power of oscillations. In contrast, unilateral excitation of the XII nucleus increased both mean peak integrated activity bilaterally (ipsilateral: 41+/-10%, P<0.01; contralateral: 17+/-7%; P<0.05, n=10) and oscillation power in the ipsilateral (50+/-17%, n=7, P<0.05), but not in the contralateral rootlet. Cross-correlation analysis of control inspiratory activity recorded from the left and right XII rootlets produced cross-correlation histograms with significant peaks centered around a time lag of zero and showed no subsidiary harmonic peaks. Coherence analysis of left and right XII rootlet recordings demonstrated that oscillations are only weakly coherent. Together, the findings from local application experiments and cross-correlation and coherence analyses indicate that short time scale synchronous oscillations recorded in the slice are likely generated in or immediately upstream of the XII motor nucleus.

Neurons within the intact respiratory network produce bursts of action potentials that cause inspiration or expiration. Within inspiratory bursts, activity is synchronized on a shorter timescale to generate clusters of action potentials that occur in a set frequency range and are called synchronous oscillations. We investigated how GABA and glycine modulate synchronous oscillations and respiratory rhythm during postnatal development. We recorded inspiratory activity from hypoglossal nerves using the in vitro rhythmically active mouse medullary slice preparation from P0-P11 mice. Average oscillation frequency increased with postnatal development, from 17 +/- 12 Hz in P0-P6 mice (n = 15) to 38 +/- 7 Hz in P7-P11 mice (n = 37) (P < 0.0001). Bath application of GABAA and GlyR antagonists significantly reduced oscillation power in neonates (P0-P6) and juveniles (P7-P10) and increased peak integrated activity in both age groups. To test whether elevating slice excitability is sufficient to reduce oscillation power, Substance P was bath applied alone. Substance P, although increasing peak integrated activity, had no significant effect on oscillation power. Prolonging the time course of GABAergic synaptic currents with zolpidem decreased the median oscillation frequency in P9-P10 mouse slices. These data demonstrate that oscillation frequency increases with postnatal development and that both GABAergic and glycinergic transmission contribute to synchronization of activity. Further, the time course of synaptic GABAergic currents is a determinant of oscillation frequency.

We studied the effects of GABA(B) receptor activation on either glycine or GABA(A) receptor-mediated synaptic transmission to hypoglossal motoneurons (HMs, P8-13) using a rat brainstem slice preparation. Activation of GABA(B) receptors with baclofen, a GABA(B) receptor agonist, inhibited the amplitude of evoked glycine and GABA(A) receptor-mediated inhibitory postsynaptic currents. Additionally, with blockade of postsynaptic GABA(B) receptors baclofen decreased the frequency of both glycine and GABA(A) receptor-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs), indicating a presynaptic site of action. Conversely, the GABA(B) receptor antagonist CGP 35348 increased the frequency of glycine receptor-mediated mIPSCs. Application of the GABA transport blocker SKF 89976A decreased the frequency of glycinergic mIPSCs. Lastly, we compared the effects of baclofen on the frequency of glycine and GABA(A) receptor-mediated mIPSC during HM development. At increased postnatal ages (P8-13 versus P1-3) mIPSC frequency was more strongly reduced by baclofen. These results show that presynaptic GABA(B) receptors inhibits glycinergic and GABAergic synaptic transmission to HMs, and the presynaptic sensitivity to baclofen is increased in P8-13 versus P1-3 HMs. Further, endogenous GABA is capable of modulating inhibitory synaptic transmission to HMs.

Ethanol potentiates glycinergic synaptic transmission to hypoglossal motoneurons (HMs). This effect on glycinergic transmission changes with postnatal development in that juvenile HMs (P9-13) are more sensitive to ethanol than neonate HMs (P1-3). We have now extended our previous study to investigate ethanol modulation of synaptic GABA(A) receptors (GABA(A)Rs), because both GABA and glycine mediate inhibitory synaptic transmission to brain stem motoneurons. We tested the effects of ethanol on GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded from neonate and juvenile rat HMs in an in vitro slice preparation. Bath application of 30 mM ethanol had no significant effect on the GABAergic mIPSC amplitude or frequency recorded at either age. At 100 mM, ethanol significantly decreased the GABAergic mIPSC amplitude recorded from neonate (6 +/- 3%, P < 0.05) and juvenile (16 +/- 3%, P < 0.01) HMs. The same concentration of ethanol increased the GABAergic mIPSC frequency recorded from neonate (64 +/- 17%, P < 0.05) and juvenile (40 +/- 15%, n.s.) HMs. In contrast, 100 mM ethanol robustly potentiated glycinergic mIPSC amplitude in neonate (31 +/- 3%, P < 0.0001) and juvenile (41 +/- 7%, P < 0.001) HMs. These results suggest that glycine receptors are more sensitive to modulation by ethanol than GABA(A) receptors and that 100 mM ethanol has the opposite effect on GABA(A)R-mediated currents in juvenile HMs, that is, inhibition rather than enhancement. Further, comparing ethanol's effects on GABAergic mIPSC amplitude and frequency, ethanol modulates GABAergic synaptic transmission to HMs differentially. Presynaptically, ethanol enhances mIPSC frequency while postsynaptically it decreases mIPSC amplitude.