A-type Currents Research Articles

Inhibition of A-Type K+ Channels by Urotensin-II Induces Sensory Neuronal Hyperexcitability Through the PKCα-ERK Pathway.

Previous studies have implicated urotensin-II in the nociception of sensory neurons. However, to date the relevant mechanisms remain unknown. In the current study we determined the role of urotensin-II in the regulation of transient outward A-type potassium currents (IA) and neuronal excitability in trigeminal ganglion (TG) neurons. We found that application of urotensin-II to small-diameter TG neurons decreased IA in a dose-dependent manner, whereas the delayed rectifier potassium current was unaffected. The IA decrease induced by urotensin-II depended on the urotensin-II receptor (UT-R) and was associated with a hyperpolarizing shift in the steady-state inactivation curve. Exposure of TG cells to urotensin-II markedly increased protein kinase C (PKC) activity, and PKC inhibition eliminated the UT-R-mediated IA decrease. Antagonism of PKCα, either pharmacologically or genetically, but not of PKCβ prevented the decrease in IA induced by urotensin-II. Analysis of phospho-extracellular signal-regulated kinase (p-ERK) revealed that urotensin-II significantly increased the expression level of p-ERK, whereas p-p38 and p-c-Jun N-terminal kinase remained unchanged. Inhibition of mitogen-activated protein kinase/ERK signaling by the kinase antagonist U0126 and PD98059 completely abolished the UT-R-mediated IA decrease. Moreover, urotensin-II significantly increased the action potential firing rate of small TG neurons; pretreatment with 4-aminopyridine prevented this effect. In summary, our findings suggest that urotensin-II selectively attenuated IA through stimulation of the PKCα-dependent ERK1/2 signaling pathway. This UT-R-dependent mechanism might contribute to neuronal hyperexcitability in TG neurons.

The Distributions of Voltage-Gated K+ current Subtypes in Different Cell Sizes from Adult Mouse Dorsal Root Ganglia.

Voltage-gated K+ (KV) currents play a crucial role in regulating pain by controlling neuronal excitability, and are divided into transient A-type currents (IA) and delayed rectifier currents (IK). The dorsal root ganglion (DRG) neurons are heterogeneous and the subtypes of KV currents display different levels in distinct cell sizes. To observe correlations of the subtypes of KV currents with DRG cell sizes, KV currents were recorded by whole-cell patch clamp in freshly isolated mouse DRG neurons. Results showed that IA occupied a high proportion in KV currents in medium- and large-diameter DRG neurons, whereas IK possessed a larger proportion of KV currents in small-diameter DRG neurons. A lower correlation was found between the proportion of IA or IK in KV currents and cell sizes. These data suggest that IA channels are mainly expressed in medium and large cells and IK channels are predominantly expressed in small cells.

Behavioral and SCN neurophysiological disruption in the Tg-SwDI mouse model of Alzheimer's disease

Disruption of circadian rhythms is commonly reported in individuals with Alzheimer's disease (AD). Neurons in the primary circadian pacemaker, the suprachiasmatic nucleus (SCN), exhibit daily rhythms in spontaneous neuronal activity which are important for maintaining circadian behavioral rhythms. Disruption of SCN neuronal activity has been reported in animal models of other neurodegenerative disorders; however, the effect of AD on SCN neurophysiology remains unknown. In this study we examined circadian behavioral and electrophysiological changes in a mouse model of AD, using male mice from the Tg-SwDI line which expresses human amyloid precursor protein with the familial Swedish (K670N/M671L), Dutch (E693Q), Iowa (D694N) mutations. The free-running period of wheel-running behavior was significantly shorter in Tg-SwDI mice compared to wild-type (WT) controls at all ages examined (3, 6, and 10 months). At the SCN level, the day/night difference in spike rate was significantly dampened in 6–8 month-old Tg-SwDI mice, with decreased AP firing during the day and an increase in neuronal activity at night. The dampening of SCN excitability rhythms in Tg-SwDI mice was not associated with changes in input resistance, resting membrane potential, or action potential afterhyperpolarization amplitude; however, SCN neurons from Tg-SwDI mice had significantly reduced A-type potassium current (IA) during the day compared to WT cells. Taken together, these results provide the first evidence of SCN neurophysiological disruption in a mouse model of AD, and highlight IA as a potential target for AD treatment strategies in the future.

Kv4.2 channel activity controls intrinsic firing dynamics of arcuate kisspeptin neurons

Key points Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development.Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known.In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation.The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is.Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development. Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole‐cell patch‐clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc), in brain slices isolated from adult male Kiss‐Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A‐type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular‐spiking cortical interneurons, in which the A‐type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arc neurons, A‐type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single‐cell RT‐PCR. In addition, firing irregularity was correlated to the density of A‐type current in the membrane. Using conductance injection, we demonstrated that adding Kv4‐like potassium conductance (gKv4) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4 has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arc neurons, shaping their physiological function in gonadotropin release.

Inhibitory Effects of Honokiol on the Voltage-Gated Potassium Channels in Freshly Isolated Mouse Dorsal Root Ganglion Neurons.

Voltage-gated potassium (KV) currents, subdivided into rapidly inactivating A-type currents (I A) and slowly inactivating delayed rectifier currents (I K), play a fundamental role in modulating pain by controlling neuronal excitability. The effects of Honokiol (Hon), a natural biphenolic compound derived from Magnolia officinalis, on KV currents were investigated in freshly isolated mouse dorsal root ganglion neurons using the whole-cell patch clamp technique. Results showed that Hon inhibited I A and I K in concentration-dependent manner. The IC50 values for block of I A and I K were 30.5 and 25.7µM, respectively. Hon (30µM) shifted the steady-state activation curves of I A and I K to positive potentials by 17.6 and 16.7mV, whereas inactivation and recovery from the inactivated state of I A were unaffected. These results suggest that Hon preferentially interacts with the active states of the I A and I K channels, and has no effect on the resting state and inactivated state of the I A channel. Blockade on K+ channels by Hon may contribute to its antinociceptive effect, especially anti-inflammatory pain.

Nongenomic Glucocorticoid Suppression of a Postsynaptic Potassium Current via Emergent Autocrine Endocannabinoid Signaling in Hypothalamic Neuroendocrine Cells following Chronic Dehydration.

Glucocorticoids rapidly stimulate endocannabinoid synthesis and modulation of synaptic transmission in hypothalamic neuroendocrine cells via a nongenomic signaling mechanism. The endocannabinoid actions are synapse-constrained by astrocyte restriction of extracellular spatial domains. Exogenous cannabinoids have been shown to modulate postsynaptic potassium currents, including the A-type potassium current (IA), in different cell types. The activity of magnocellular neuroendocrine cells is shaped by a prominent IA. We tested for a rapid glucocorticoid modulation of the postsynaptic IK and IA in magnocellular neuroendocrine cells of the hypothalamic paraventricular nucleus (PVN) using whole-cell recordings in rat brain slices. Application of the synthetic glucocorticoid dexamethasone (Dex) had no rapid effect on the IK or IA amplitude, voltage dependence, or kinetics in magnocellular neurons in slices from untreated rats. In magnocellular neurons from salt-loaded rats, however, Dex application caused a rapid suppression of the IA and a depolarizing shift in IA voltage dependence. Exogenously applied endocannabinoids mimicked the rapid Dex modulation of the IA, and CB1 receptor antagonists and agonists blocked and occluded the Dex-induced changes in the IA, respectively, suggesting an endocannabinoid dependence of the rapid glucocorticoid effect. Preincubation of control slices in a gliotoxin resulted in the partial recapitulation of the glucocorticoid-induced rapid suppression of the IA. These findings demonstrate a glucocorticoid suppression of the postsynaptic IA in PVN magnocellular neurons via an autocrine endocannabinoid-dependent mechanism following chronic dehydration, and suggest a possible role for astrocytes in the control of the autocrine endocannabinoid actions.

MicroRNA cluster miR-17-92 regulates multiple functionally related voltage-gated potassium channels in chronic neuropathic pain

miR-17-92 is a microRNA cluster with six distinct members. Here, we show that the miR-17-92 cluster and its individual members modulate chronic neuropathic pain. All cluster members are persistently upregulated in primary sensory neurons after nerve injury. Overexpression of miR-18a, miR-19a, miR-19b and miR-92a cluster members elicits mechanical allodynia in rats, while their blockade alleviates mechanical allodynia in a rat model of neuropathic pain. Plausible targets for the miR-17-92 cluster include genes encoding numerous voltage-gated potassium channels and their modulatory subunits. Single-cell analysis reveals extensive co-expression of miR-17-92 cluster and its predicted targets in primary sensory neurons. miR-17-92 downregulates the expression of potassium channels, and reduced outward potassium currents, in particular A-type currents. Combined application of potassium channel modulators synergistically alleviates mechanical allodynia induced by nerve injury or miR-17-92 overexpression. miR-17-92 cluster appears to cooperatively regulate the function of multiple voltage-gated potassium channel subunits, perpetuating mechanical allodynia.

Synaptic transmission: Changing the (potassium) channel?

The endocannabinoid 2-arachidonoylglycerol increases the tonic and evoked firing rate of mouse dopamine midbrain neurons by inhibiting A-type potassium currents through a direct lipid interaction with Kv4.3 channels.

Cell-Autonomous Excitation of Midbrain Dopamine Neurons by Endocannabinoid-Dependent Lipid Signaling

The major endocannabinoid in the mammalian brain is the bioactive lipid 2-arachidonoylglycerol (2-AG). The best-known effects of 2-AG are mediated by G-protein-coupled cannabinoid receptors. In principle, 2-AG could modify neuronal excitability by acting directly on ion channels, but such mechanisms are poorly understood. Using a preparation of dissociated mouse midbrain dopamine neurons to isolate effects on intrinsic excitability, we found that 100nM 2-AG accelerated pacemaking and steepened the frequency-current relationship for burst-like firing. In voltage-clamp experiments, 2-AG reduced A-type potassium current (IA) through a cannabinoid receptor-independent mechanism mimicked by arachidonic acid, which has no activityon cannabinoid receptors. Activation of orexin, neurotensin, and metabotropic glutamate Gq/11-linked receptors mimicked the effects of exogenous2-AG and their actions were prevented by inhibiting the 2-AG-synthesizing enzyme diacylglycerol lipaseα. The results show that 2-AG and related lipid signaling molecules can directly tune neuronal excitability in a cell-autonomous manner by modulating IA.

Enhanced Sensitivity to Hyperpolarizing Inhibition in Mesoaccumbal Relative to Nigrostriatal Dopamine Neuron Subpopulations.

Midbrain dopamine neurons recorded in vivo pause their firing in response to reward omission and aversive stimuli. While the initiation of pauses typically involves synaptic or modulatory input, intrinsic membrane properties may also enhance or limit hyperpolarization, raising the question of how intrinsic conductances shape pauses in dopamine neurons. Using retrograde labeling and electrophysiological techniques combined with computational modeling, we examined the intrinsic conductances that shape pauses evoked by current injections and synaptic stimulation in subpopulations of dopamine neurons grouped according to their axonal projections to the nucleus accumbens or dorsal striatum in mice. Testing across a range of conditions and pulse durations, we found that mesoaccumbal and nigrostriatal neurons differ substantially in rebound properties with mesoaccumbal neurons displaying significantly longer delays to spiking following hyperpolarization. The underlying mechanism involves an inactivating potassium (IA) current with decay time constants of up to 225 ms, and small-amplitude hyperpolarization-activated currents (IH), characteristics that were most often observed in mesoaccumbal neurons. Pharmacological block of IA completely abolished rebound delays and, importantly, shortened synaptically evoked inhibitory pauses, thereby demonstrating the involvement of A-type potassium channels in prolonging pauses evoked by GABAergic inhibition. Therefore, these results show that mesoaccumbal and nigrostriatal neurons display differential responses to hyperpolarizing inhibitory stimuli that favors a higher sensitivity to inhibition in mesoaccumbal neurons. These findings may explain, in part, observations from in vivo experiments that ventral tegmental area neurons tend to exhibit longer aversive pauses relative to SNc neurons.SIGNIFICANCE STATEMENT Our study examines rebound, postburst, and synaptically evoked inhibitory pauses in subpopulations of midbrain dopamine neurons. We show that pauses in dopamine neuron firing, evoked by either stimulation of GABAergic inputs or hyperpolarizing current injections, are enhanced by a subclass of potassium conductances that are recruited at voltages below spike threshold. Importantly, A-type potassium currents recorded in mesoaccumbal neurons displayed substantially slower inactivation kinetics, which, combined with weaker expression of hyperpolarization-activated currents, lengthened hyperpolarization-induced delays in spiking relative to nigrostriatal neurons. These results suggest that input integration differs among dopamine neurons favoring higher sensitivity to inhibition in mesoaccumbal neurons and may partially explain in vivo observations that ventral tegmental area neurons exhibit longer aversive pauses relative to SNc neurons.

Physiological roles of Kv2 channels in entorhinal cortex layer II stellate cells revealed by Guangxitoxin-1E.

Kv2 channels underlie delayed-rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia. Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space-representing grid cells. We used the new Kv2 blocker Guangxitoxin-1E (GTx) to study Kv2 functions in these neurons. Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed-rectifier K+ current but not transient A-type current. In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after-depolarizations; (iii) reducing the fast and medium after-hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering. GTx is a useful tool for studying Kv2 channels and their functions in neurons. The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin-1E (GTx; 10-100nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond -30mV but not transient A-type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after-depolarization (ADP); (iii) reduced fast and medium after-hyperpolarizations (AHPs); (iv) strongly enhanced burst firing and increased excitability at moderate spike rates but reduced spiking at high rates; and (v) reduced spike clustering and rebound potentials. The changes in bursting and excitability were related to the altered ADPs and AHPs. Kv2 channels strongly shape the activity of mEC SCs by affecting spike repolarization, after-potentials, excitability and spike patterns. GTx is a useful tool and may serve to further clarify Kv2 channel functions in neurons. We conclude that Kv2 channels in mEC SCs are important determinants of intrinsic properties that allow these neurons to produce spatial representation. The results of the present study may also be important for the accurate modelling of grid cells.

Abstract P273: Increased Inhibition of Potassium Channel Currents by Angiotensin II in Sympathetic Neurons may Contribute to a Sustained Blood Pressure Elevation in (mRen2)27 Rats.

It is well established that the increased sympathetic tone may contribute to initiation and progression of various forms of hypertension. Several lines of evidence suggest a link between the renin-angiotensin system and sympathetic nerve activity in hypertension, and the previous studies in animal models demonstrated increased sympathetic output in the presence of Angiotensin II (AngII). To elucidate potential underlying molecular mechanisms of such phenomenon, we compared the effect of AngII on the whole-cell potassium channel currents in superior cervical ganglia (SCG) neurons isolated from hypertensive (mRen2)27 rats with overexpression of renin gene, and control Sprague Dawley® (SD) rats. In both groups, the whole-cell potassium channel currents were identified as rapidly-activating, 4-Aminopyridine-sensitive transient A-type currents, as well as slowly-activating tetraethylammonium-sensitive delayed rectifier currents. When the cell membrane was depolarized to -40, -30 and -20 mV from a holding potential of -80 mV, AngII (100 nM) profoundly inhibited A-type current, but the magnitude of such inhibition was not significantly different between neurons isolated from (mRen2)27 (38.1±6.2%, 47.8±5.7% and 52.1±5.7%; n=11) and SD rats (37.2±4.6%, 44.±4.5% and 46.1±4.8%; n = 13). Delayed rectifier potassium channel currents were isolated by holding cells at -40 mV, which resulted in complete elimination of the transient A-type current. In contrast to transient current, inhibition of the delayed rectifier current by AngII in the range of membrane potentials between +20 and +80 mV was significantly greater (p<0.05) in neurons obtained from (mRen2)27 rats (11.0±3.2% to 25.0±2.9%, n=12) when compared to SD rats (4.7±1.5% to 16.3±2.7%, n = 12). In both groups, inhibition of both channel types was completely abolished by 10 uM Losartan, indicating involvement of AT1 receptors. Our results suggest that in (mRen2)27 hypertensive rats, the increased inhibitory effect of AngII on delayed rectifier potassium channel currents could possibly lead to lowering spike threshold, which, in turn, could elevate sympathetic outflow and lead to sustained blood pressure elevation.

MicroRNA-Mediated Downregulation of the Potassium Channel Kv4.2 Contributes to Seizure Onset

Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 proteininvitro and invivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

Operation of a homeostatic sleep switch.

Sleep disconnects animals from the external world, at considerable risks and costs that must be offset by a vital benefit. Insight into this mysterious benefit will come from understanding sleep homeostasis: to monitor sleep need, an internal bookkeeper must track physiological changes that are linked to the core function of sleep1. In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex2,3. Artificial activation of these cells induces sleep2, whereas reductions in excitability cause insomnia3,4. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent3. Correlative evidence thus supports the simple view that homeostatic sleep control works by switching sleep-promoting neurons between active and quiescent states3. Here we demonstrate state switching by dFB neurons, identify dopamine as a neuromodulator that operates the switch, and delineate the switching mechanism. Arousing dopamine4–8 caused transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects were transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involved the downregulation of voltage-gated A-type currents carried by Shaker and Shab and the upregulation of voltage-independent leak currents through a two-pore domain potassium channel we term Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreased or increased sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state.

Using a Semi-Automated Strategy to Develop Multi-Compartment Models That Predict Biophysical Properties of Interneuron-Specific 3 (IS3) Cells in Hippocampus.

Determining how intrinsic cellular properties govern and modulate neuronal input–output processing is a critical endeavor for understanding microcircuit functions in the brain. However, lack of cellular specifics and nonlinear interactions prevent experiments alone from achieving this. Building and using cellular models is essential in these efforts. We focus on uncovering the intrinsic properties of mus musculus hippocampal type 3 interneuron-specific (IS3) cells, a cell type that makes GABAergic synapses onto specific interneuron types, but not pyramidal cells. While IS3 cell morphology and synaptic output have been examined, their voltage-gated ion channel profile and distribution remain unknown. We combined whole-cell patch-clamp recordings and two-photon dendritic calcium imaging to examine IS3 cell membrane and dendritic properties. Using these data as a target reference, we developed a semi-automated strategy to obtain multi-compartment models for a cell type with unknown intrinsic properties. Our approach is based on generating populations of models to capture determined features of the experimental data, each of which possesses unique combinations of channel types and conductance values. From these populations, we chose models that most closely resembled the experimental data. We used these models to examine the impact of specific ion channel combinations on spike generation. Our models predict that fast delayed rectifier currents should be present in soma and proximal dendrites, and this is confirmed using immunohistochemistry. Further, without A-type potassium currents in the dendrites, spike generation is facilitated at more distal synaptic input locations. Our models will help to determine the functional role of IS3 cells in hippocampal microcircuits.

Two distinct subpopulations of neurons in the thalamic intergeniculate leaflet identified by subthreshold currents

The intergeniculate leaflet (IGL) is a flat retinorecipient thalamic structure implicated in orchestrating circadian rhythm, historically considered to be a subdivision of the neighboring ventrolateral geniculate nucleus (VLG). IGL consists of two main neuronal subpopulations: enkephalinergic and neuropeptide Y (NPY)-synthesizing cells. These cell types have different functions, connectivity and firing pattern in vivo, which suggest that they have different membrane currents to support their functional differences. We therefore performed patch-clamp experiments combined with immunohistochemical staining to clarify possible differences in the subthreshold currents of IGL neurons. Our results suggest that IGL neurons can be divided into two subpopulations based on two ionic currents. A T-type calcium current (IT) was identified in neurons that do not synthesise NPY, whereas all NPY-positive neurons were found to express a marked A-type potassium current (IA). Due to the fact that the clear electrophysiological discriminants between IGL and VLG are lacking, we decided to compare the amplitudes of the identified currents between those two structures. Our data suggest that VLG neurons can be characterized by a high amplitude IT and a low IA. Finally, we compared both currents with WAG/Rij rats, a well-established model of absence epilepsy, with co-occurring retinal pathologies, sleep-onset disturbances, and seizures exhibiting circadian rhythmicity. Data presented in this study uncovered pathologies in the IT exhibiting neurons of the IGL and VLG. In conclusion, the data presented here suggest that different subthreshold current expression supports the functional differences of thalamic nuclei. Those differences are promising for possible pharmacological manipulations of specified cell types in pathophysiologies including absence epilepsy.

Endocannabinoid 2-Arachidonoylglycerol Suppresses LPS-Induced Inhibition of A-Type Potassium Channel Currents in Caudate Nucleus Neurons Through CB1 Receptor

Inflammation plays a pivotal role in the pathogenesis of many diseases in the central nervous system. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from some proinflammatory stimuli. However, the neuroprotective mechanism of 2-AG is complex and has not been fully understood. A-type K(+) channels critically regulate neuronal excitability and have been demonstrated to be associated with some nervous system diseases. The aim of this study was to explore whether A-type K(+) channels were involved in neurotoxicity of lipopolysaccharides (LPS) and the neuroprotective mechanism of 2-AG in CN neurons. Whole cell patch clamp recording was used to investigate the influence of LPS on the function of A-type K(+) channels and its modulation by 2-AG in primary cultured rat CN neurons. Our findings showed that in cultured CN neurons, LPS significantly decreased the A-type potassium currents (I A) in a voltage-insensitive way. The further data demonstrated that an elevation of 2-AG levels by directly applying exogenous 2-AG or inhibiting monoacylglycerol lipase (MAGL) to prevent 2-AG hydrolysis was capable of suppressing the LPS-induced inhibition of IA and the action of 2-AG is mediated through CB1 receptor-dependant way. The study provides a better understanding of inflammation-related neurological disorders and suggests the therapeutic potential for 2-AG for the treatment of these diseases.

Hydroxysafflor Yellow A Protects Neurons From Excitotoxic Death through Inhibition of NMDARs.

Excessive glutamate release causes overactivation of N-methyl d-aspartate receptors (NMDARs), leading to excitatory neuronal damage in cerebral ischemia. Hydroxysafflor yellow A (HSYA), a compound extracted from Carthamus tinctorius L., has been reported to exert a neuroprotective effect in many pathological conditions, including brain ischemia. However, the underlying mechanism of HSYA's effect on neurons remains elusive. In the present study, we conducted experiments using patch-clamp recording of mouse hippocampal slices. In addition, we performed Ca2+ imaging, Western blots, as well as mitochondrial-targeted circularly permuted yellow fluorescent protein transfection into cultured hippocampal neurons in order to decipher the physiological mechanism underlying HSYA's neuroprotective effect.Through the electrophysiology experiments, we found that HSYA inhibited NMDAR-mediated excitatory postsynaptic currents without affecting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and γ-aminobutyric acid A-type receptor-mediated currents. This inhibitory effect of HSYA on NMDARs was concentration dependent. HSYA did not show any preferential inhibition of either N-methyl d-aspartate receptor subtype 2A- or N-methyl d-aspartate receptor subtype 2B- subunit-containing NMDARs. Additionally, HSYA exhibits a facilitatory effect on paired NMDAR-mediated excitatory postsynaptic currents. Furthermore, HSYA reduced the magnitude of NMDAR-mediated membrane depolarization currents evoked by oxygen-glucose deprivation, and suppressed oxygen-glucose deprivation–induced and NMDAR-dependent ischemic long-term potentiation, which is believed to cause severe reperfusion damage after ischemia. Through the molecular biology experiments, we found that HSYA inhibited the NMDA-induced and NMDAR-mediated intracellular Ca2+ concentration increase in hippocampal cultures, reduced apoptotic and necrotic cell deaths, and prevented mitochondrial damage. Together, our data demonstrate for the first time that HSYA protects hippocampal neurons from excitotoxic damage through the inhibition of NMDARs. This novel finding indicates that HSYA may be a promising pharmacological candidate for the treatment of brain ischemia.

Amitriptyline and carbamazepine utilize voltage-gated ion channel suppression to impair excitability of sensory dorsal horn neurons in thin tissue slice: An in vitro study

Amitriptyline, carbamazepine and gabapentin are often used for the treatment of neuropathic pain. However, their analgesic action on central sensory neurons is still not fully understood. Moreover, the expression pattern of their target ion channels is poorly elucidated in the dorsal horn of the spinal cord. Thus, we performed patch-clamp investigations in visualized neurons of lamina I–III of the spinal cord. The expression of the different voltage-gated ion channels, as the targets of these drugs, was detected by RT-PCR and immunohistochemistry. Neurons of the lamina I–III express the TTX-sensitive voltage-gated Na+ as well as voltage-gated K+ subunits assembling the fast inactivating (A-type) currents and the delayed rectifier K+ currents. Our pharmacological studies show that tonically-firing, adapting-firing and single spike neurons responded dose-dependently to amitriptyline and carbamazepine. The ion channel inhibition consecutively reduced the firing rate of tonically-firing and adapting-firing neurons. This study provides evidence for the distribution of voltage-gated Na+ and K+ subunits in lamina I–III of the spinal cord and for the action of drugs used for the treatment of neuropathic pain. Our work confirms that modulation of voltage-gated ion channels in the central nervous system contributes to the antinociceptive effects of these drugs.

Decreased voltage-gated potassium currents in rat dorsal root ganglion neurons after chronic constriction injury.

Voltage-gated potassium channels (KV) regulate pain transmission by controlling neuronal excitability. Changes in KV expression patterns may thus contribute toward hyperalgesia following nerve injury. The aim of this study was to characterize KV current density in dorsal root ganglion (DRG) neurons following chronic constriction injury (CCI) of the right sciatic nerve, a robust model of post-traumatic neuropathic pain. The study examined changes in small-diameter potassium ion currents (<30 µm) in neurons in the L4-L6 DRG following CCI by whole-cell patch-clamping and the association with post-CCI mechanical and thermal nociceptive thresholds. Compared with the control group, 7 days after CCI, the mechanical force and temperature required to elicit ipsilateral foot withdrawal decreased significantly, indicating tactile allodynia and thermal hyperalgesia. Post-CCI neurons had a significantly lower rheobase current and depolarized resting membrane potential than controls, suggesting KV current downregulation. Some ipsilateral DRG neurons also had spontaneous action potentials and repetitive firing. There was a 55% reduction in the total KV current density caused by a 55% decrease in the sustained delayed rectifier potassium ion current (IK) density and a 17% decrease in the transient A-type potassium ion current (IA) density. These results indicated that changes in DRG neuron IK and IA current density and concomitant afferent hyperexcitability may contribute toward neuropathic pain following injury. The rat CCI model may prove valuable for examining pathogenic mechanisms and potential therapies, such as KV channel modulators.