A robust surge of gonadotropin-releasing hormone (GnRH) release triggers the luteinizing hormone surge that induces ovulation. The GnRH surge is attributable to estradiol feedback, but the mechanisms are incompletely understood. Voltage-gated calcium channels (VGCCs) regulate hormone release and neuronal excitability, and may be part of the surge-generating mechanism. We examined VGCCs of GnRH neurons in brain slices from a model exhibiting daily luteinizing hormone surges. Mice were ovariectomized (OVX), and a subset was treated with estradiol implants (OVXϩE). OVXϩE mice exhibit negative feedback in the A.M. and positive feedback in the P.M. GnRH neurons express prominent high-voltage-activated (HVA) and small low-voltage-activated (LVA) macroscopic (whole-cell) Ca currents (I Ca ). LVA-mediated currents were not altered by estradiol or time of day. In contrast, in OVXϩE mice, HVA-mediated currents varied with time of day; HVA currents in cells from OVXϩE mice were lower than those in cells from OVX mice in the A.M. but were higher in the P.M. These changes were attributable to diurnal alternations in L-and N-type components. There were no diurnal changes in any aspect of HVA-mediated I Ca in OVX mice. Acute in vitro treatment of cells from OVX and OVXϩE mice with estradiol rapidly increased HVA currents primarily through L-and R-type VGCCs by activating estrogen receptor ␤ and GPR30, respectively. These results suggest multiple mechanisms contribute to the overall feedback regulation of HVA-mediated I Ca by estradiol. In combination with changes in synaptic inputs to GnRH neurons, these intrinsic changes in GnRH neurons may play critical roles in estradiol feedback.

Key points• M-type potassium channels play a key role in modulating neuronal excitability. However, the effects of M-channel activation on synaptic transmission are poorly understood.• This study found that an M1 receptor agonist and M-channel blockers increased action potential-independent glutamate release at Schaffer collateral-CA1 pyramidal neuron synapses in acute hippocampus slices.• This enhancement was dependent on Ca 2+ influx from extracellular space but not intracellular calcium stores.• Inhibition of M-channels results in the depolarization of CA3 pyramidal neurons and activated presynaptic voltage-gated P/Q-and N-type calcium channels, which in turn causes Ca 2+ influx and increased glutamate release.• Thus, M1 muscarinic agonists modulate action potential-independent glutamatergic synaptic transmission in the hippocampus by inhibition of presynaptic M-channels.Abstract Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q-and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents -Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates Abbreviations M1, muscarinic type 1; mEPSCs, miniature excitatory postsynaptic currents; CNQX, 6-cyano-7-nitroquinoxalene-2,3-dione; K-S, Kolmogorov-Smirnov; NcN-A-343, (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride; sEPSC, spontaneous excitatory postsynaptic current; XE-991,10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone.

The axon initial segment (AIS) is the site of action potential (AP) initiation, thus a crucial regulator of neuronal activity. In excitatory pyramidal neurons, the high density of voltage-gated sodium channels (NaV1.6) at the distal AIS regulates AP initiation. A surrogate AIS marker, ankyrin-G (ankG) is a structural protein regulating neuronal functional via clustering voltage-gated ion channels. In neuronal circuits, changes in presynaptic input can alter postsynaptic output via AIS structural-functional plasticity. Recently, we showed experimental mild traumatic brain injury (mTBI) evokes neocortical circuit disruption via diffuse axonal injury (DAI) of excitatory and inhibitory neuronal systems. A key finding was that mTBI-induced neocortical electrophysiological changes involved non-DAI/ intact excitatory pyramidal neurons consistent with AIS-specific alterations. In the current study we employed Thy1-yellow fluorescent protein (YFP)-H mice to test if mTBI induces AIS structural and/or functional plasticity within intact pyramidal neurons 2 days after mTBI. We used confocal microscopy to assess intact YFP+ pyramidal neurons in layer 5 of primary somatosensory barrel field (S1BF), whose axons were continuous from the soma of origin to the subcortical white matter (SCWM). YFP+ axonal traces were superimposed on ankG and NaV1.6 immunofluorescent profiles to determine AIS position and length. We found that while mTBI had no effect on ankG start position, the length significantly decreased from the distal end, consistent with the site of AP initiation at the AIS. However, NaV1.6 structure did not change after mTBI, suggesting uncoupling from ankG. Parallel quantitative analysis of presynaptic inhibitory terminals along the postsynaptic perisomatic domain of these same intact YFP+ excitatory pyramidal neurons revealed a significant decrease in GABAergic bouton density. Also within this non-DAI population, patch-clamp recordings of intact YFP+ pyramidal neurons showed AP acceleration decreased 2 days post-mTBI, consistent with AIS functional plasticity. Simulations of realistic pyramidal neuron computational models using experimentally determined AIS lengths showed a subtle decrease is NaV1.6 density is sufficient to attenuate AP acceleration. Collectively, these findings highlight the complexity of mTBI-induced neocortical circuit disruption, involving changes in extrinsic/presynaptic inhibitory perisomatic input interfaced with intrinsic/postsynaptic intact excitatory neuron AIS output.

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of alpha1-adrenergic and sigma1 receptors, but in the hippocampus it might be dependent on sigma1 receptors only. The activation of alpha1-adrenergic receptors synergized sigma1 receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.

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Altered GABA-mediated inhibition is proposed to play a role in the pathogenesis of epilepsy. Previous studies have demonstrated a loss of somatostatin-containing GABAergic interneurons innervating granule cells in epileptic animals. However, the reorganization of synapses between interneurons and granule cells has not been investigated. We studied synapse organization in an animal model of temporal lobe epilepsy (TLE) using continuous hippocampal stimulation. The distribution of axon terminals and inhibitory synapses on granule cell dendrites was studied using a combination of immunohistochemistry and pre-embedding electron microscopy techniques. A whole-cell patch-clamp technique was applied to study the functional changes in GABAergic input from different interneurons. In epileptic animals, the density of cholecystokinin (CCK)-immunoreactive (IR) fibers and α2 subunit containing GABAA receptors in the inner molecular layer of the dentate gyrus was reduced. Quantitative immuno-electron microscopy study revealed that the ratio of CCK-containing symmetric synapses to the total symmetric synapses was reduced. The frequency of GABAergic synaptic currents (sIPSC) was decreased and their amplitude was increased. The inhibitory effect of the activation of cannabinoid 1(CB1) receptors was also reduced in epileptic animals. Isolation of CCK- and parvalbumin (PV)-containing GABAergic inputs by N- and P/Q-type calcium channel blockers respectively suggested that GABA release from CCK-containing interneurons was selectively reduced in epileptic rats. This study found that there was a loss of CCK-containing GABAergic synapses to granule cells both morphologically and functionally. These studies add to our understanding of the mechanisms that contribute to altering GABAergic inhibition of granule cells in TLE.

Activity-dependent changes in the properties of the motor system underlie the necessary adjustments in its responsiveness on the basis of the environmental and developmental demands of the organism. Although plastic changes in the properties of the spinal cord have historically been neglected because of the archaic belief that the spinal cord is constituted by a hardwired network that simply relays information to muscles, plenty of evidence has been accumulated showing that synapses impinging on spinal motoneurons undergo short- and long-term plasticity. In the brain, brief changes in the activity level of the network have been shown to be paralleled by changes in the intrinsic excitability of the neurons and are suggested to either reinforce or stabilize the changes at the synaptic level. However, rapid activity-dependent changes in the intrinsic properties of spinal motoneurons have never been reported. In this study, we show that in neonatal mice the intrinsic excitability of spinal motoneurons is depressed after relatively brief but sustained changes in the spinal cord network activity. Using electrophysiological techniques together with specific pharmacological blockers of KCNQ/Kv7 channels, we demonstrate their involvement in the reduction of the intrinsic excitability of spinal motoneurons. This action results from an increased M-current, the product of the activation of KCNQ/Kv7 channels, which leads to a hyperpolarization of the resting membrane potential and a decrease in the input resistance of spinal motoneurons. Computer simulations showed that specific up-regulations in KCNQ/Kv7 channels functions lead to a modulation of the intrinsic excitability of spinal motoneurons as observed experimentally. These results indicate that KCNQ/Kv7 channels play a fundamental role in the activity-dependent modulation of the excitability of spinal motoneurons.

Mitochondria are central to cell survival and Ca2+ homeostasis due to their intracellular buffering capabilities. Mitochondrial dysfunction resulting in mitochondrial permeability transition pore (mPTP) opening has been reported after mild traumatic brain injury (mTBI). Cyclosporine A provides protection against the mPTP opening through its interaction with cyclophilin-D (CypD). A recent study has found that the extent of axonal injury after mTBI was diminished in neocortex in cyclophilin-D knockout (CypDKO) mice. Here we tested whether this CypDKO could also provide protection from the increased intrinsic and synaptic neuronal excitability previously described after mTBI in a mild central fluid percussion injury mice model. CypDKO mice were crossed with mice expressing yellow fluorescent protein (YFP) in layer V pyramidal neurons in neocortex to create CypDKO/YFP-H mice. Whole cell patch clamp recordings from axotomized (AX) and intact (IN) YFP+ layer V pyramidal neurons were made 1 and 2 days after sham or mTBI in slices from CypDKO/YFP-H mice. Both excitatory post synaptic currents (EPSCs) recorded in voltage clamp and intrinsic cellular properties, including action potential (AP), afterhyperpolarization (AHP), and depolarizing after potential (DAP) characteristics recorded in current clamp were evaluated. There was no significant difference between sham and mTBI for either spontaneous or miniature EPSC frequency, suggesting that CypDKO ameliorates excitatory synaptic abnormalities. There was a partial amelioration of intrinsic properties altered by mTBI. Alleviated were the increased slope of the AP frequency vs. injected current plot, the increased AP, AHP and DAP amplitudes. Other properties that saw a reversal that became significant in the opposite direction include the current rheobase and AP overshoot. The AP threshold remained depolarized and the input resistance remained increased in mTBI compared to sham. Additional altered properties suggest that the CypDKO likely has a direct effect on membrane properties, rather than producing a selective reduction of the effects of mTBI. These results suggest that inhibiting CypD after TBI is an effective strategy to reduce synaptic hyperexcitation, making it a continued target for potential treatment of network abnormalities.