Action Potential Firing Activity Research Articles

Vigilance and Behavioral State-Dependent Modulation of Cortical Neuronal Activity throughout the Sleep/Wake Cycle.

GABAergic inhibitory neurons, through their molecular, anatomic, and physiological diversity, provide a substrate for the modulation of ongoing cortical circuit activity throughout the sleep/wake cycle. Here, we investigated neuronal activity dynamics of parvalbumin (PV), vasoactive intestinal polypeptide (VIP), and somatostatin (SST) neurons in naturally sleeping head-restrained mice at the level of layer 2/3 of the primary somatosensory barrel cortex of mice. Through calcium imaging and targeted single-unit loose-patch or whole-cell recordings, we found that PV action potential firing activity was largest during both rapid eye movement (REM) and nonrapid eye movement (NREM) sleep stages, that VIP neurons were most active during REM sleep, and that the overall activity of SST neurons remained stable throughout the sleep/wake cycle. Analysis of neuronal activity dynamics uncovered rapid decreases in PV cell firing at wake onset followed by a progressive recovery during wake. Simultaneous local field potential (LFP) recordings further revealed that except for SST neurons, a large proportion of neurons were modulated by ongoing delta and theta oscillations. During NREM sleep spindles, PV and SST activity increased and decreased, respectively. Finally, we uncovered the presence of whisking behavior in mice during REM sleep and show that the activity of VIP and SST is differentially modulated during awake and sleeping whisking bouts, which may provide a neuronal substrate for internal brain representations occurring during sleep.SIGNIFICANCE STATEMENT In the sensory cortex, the balance between excitation and inhibition is believed to be highly dynamic throughout the sleep/wake cycle, shaping the response of cortical circuits to external stimuli while allowing the formation of newly encoded memory. Using in vivo two-photon calcium imaging or targeted single-unit recordings combined with LFP recordings, we describe the vigilance state and whisking-behavior-dependent activity of excitatory pyramidal and inhibitory GABAergic neurons in the supragranular layers of mouse somatosensory cortex. Interneuronal activity was found to be differentially modulated by ongoing delta and theta waves, sleep spindles, and a novel type of whisking observed during REM sleep, potentially providing a neuronal substrate for internal brain representations occurring during sleep.

Effects of subanesthetic ketamine and (2R,6R) hydroxynorketamine on working memory and synaptic transmission in the nucleus reuniens in mice

RationaleAcute cognitive impairment and abuse potential of ketamine incentivizes the search for alternatives to ketamine for clinical management of treatment-resistant depression. Recently, (2R,6R) hydroxynorketamine ((2R,6R)-HNK), a metabolite of ketamine, has shown promise due to its reported lack of ketamine-like reinforcing properties. Nonetheless, the effect of (2R,6R)-HNK on cognition has not been reported. MethodAdult male mice were placed in a Y-maze to measure spatial working memory (SWM) 24 h after treatment with either a single or repeated subanesthetic dose of (2R,6R)-HNK or ketamine. To determine the effect of the drug regimens on synaptic mechanisms in neural circuits deemed critical for SWM, we conducted patch-clamp electrophysiological recordings from neurons in the midline thalamic nucleus reuniens (RE) in response to optogenetic stimulation of medial prefrontal cortex (mPFC) inputs in acutely prepared brain slices. ResultsSingle or repeated treatment with a 10 mg/kg dose of either drug did not impact performance in a Y-maze. However, single administration of a ½-log higher dose (32 mg/kg) of ketamine significantly reduced SWM. The same dose of (2R,6R)-HNK did not produce SWM deficits. Interestingly, repeated administration of either drugs at the 32 mg/kg had no effect on SWM performances. Concomitant to these effects on SWM, only single injection of 32 mg/kg of ketamine was found to increase the mPFC-driven action potential firing activity in the RE neurons. Conversely, both single and repeated administration of the 32 mg/kg dose of (2R,6R)-HNK but not ketamine, increased the input resistance of the RE neurons. ConclusionOur results indicate that acute treatment of ketamine at 32 mg/kg increases mPFC-driven firing activity of RE neurons, and this contributes to the ketamine-mediated cognitive deficit. Secondly, sub-chronic treatment with the same dose of ketamine likely induces tolerance. Although single or repeated administration of the 32 mg/kg dose of (2R,6R)-HNK can alter intrinsic properties of RE neurons, this dose does not produce cognitive deficit or changes in synaptic mechanism in the RE.This article is part of the special Issue on ‘Stress, Addiction and Plasticity'.

R1617Q epilepsy mutation slows NaV 1.6sodium channel inactivation and increases the persistent current and neuronal firing.

A human NaV 1.6 construct was established to study the biophysical consequences of the R1617Q mutation on NaV 1.6 identified in patients with unclassified epileptic encephalopathy and severe intellectual disability. The R1617Q mutation disrupts the inactivation process of the channel, and more specifically, slows the current decay, increases the persistent sodium current that was blocked by tetrodotoxin and riluzole, and disrupts the inactivation voltage-dependence and increases the kinetics of recovery. In native hippocampal neurons, the R1617Q mutation exhibited a significant increase in action potentials triggered in response to stimulation and a significant increase in the number of neurons that exhibited spontaneous activity compared to neurons expressing WT channels that were inhibited by riluzole. The abnormally persistent current activity caused by the disruption of the channel inactivation process in NaV 1.6/R1617Q may result in epileptic encephalopathy in patients. The voltage-gated sodium channel NaV 1.6 is the most abundantly expressed sodium channel isoform in the central nervous system. It plays a critical role in saltatory and continuous conduction. Although over 40 NaV 1.6mutations have been linked to epileptic encephalopathy, only a few have been functionally analysed. In the present study, we characterized a NaV 1.6mutation (R1617Q) identified in patients with epileptic encephalopathy and intellectual disability. R1617Q substitutes an arginine for a glutamine in the S4segment of domain IV, which plays a major role in coupling the activation and inactivation of sodium channels. We used patch-clamp to show that R1617Q is a gain-of-function mutation. It is typified by slower inactivation kinetics and a loss of inactivation of voltage-dependence, which result in a 2.5-fold increase in the window current. In addition, sodium currents exhibited an enhanced rate of recovery from inactivation, most likely due to the destabilization of the inactivation state. The alterations in the fast inactivation caused a significant increase in the persistent sodium current. Overexpression of R1617Q in rat hippocampal neurons resulted in an increase in action potential firing activity that was inhibited by riluzole, consistent with the gain-of-function observed. We conclude that the R1617Q mutation causes neuronal hyperexcitability and may result in epileptic encephalopathy.

Regulation of the firing activity by PKA-PKC-Src family kinases in cultured neurons of hypothalamic arcuate nucleus.

The cAMP‐dependent protein kinase A family (PKAs), protein kinase C family (PKCs), and Src family kinases (SFKs) are found to play important roles in pain hypersensitivity. However, more detailed investigations are still needed in order to understand the mechanisms underlying the actions of PKAs, PKCs, and SFKs. Neurons in the hypothalamic arcuate nucleus (ARC) are found to be involved in the regulation of pain hypersensitivity. Here we report that the action potential (AP) firing activity of ARC neurons in culture was up‐regulated by application of the adenylate cyclase activator forskolin or the PKC activator PMA, and that the forskolin or PMA application‐induced up‐regulation of AP firing activity could be blocked by pre‐application of the SFK inhibitor PP2. SFK activation also up‐regulated the AP firing activity and this effect could be prevented by pre‐application of the inhibitors of PKCs, but not of PKAs. Furthermore, we identified that forskolin or PMA application caused increases in the phosphorylation not only in PKAs at T197 or PKCs at S660 and PKCα/βII at T638/641, but also in SFKs at Y416. The forskolin or PMA application‐induced increase in the phosphorylation of PKAs or PKCs was not affected by pre‐treatment with PP2. The regulations of the SFK and AP firing activities by PKCs were independent upon the translocation of either PKCα or PKCβII. Thus, it is demonstrated that PKAs may act as an upstream factor(s) to enhance SFKs while PKCs and SFKs interact reciprocally, and thereby up‐regulate the AP firing activity in hypothalamic ARC neurons.

Long-Term Recordings of Arcuate Nucleus Kisspeptin Neurons Reveal Patterned Activity That Is Modulated by Gonadal Steroids in Male Mice.

Pulsatile release of gonadotropin-releasing hormone (GnRH) is key to fertility. Pulse frequency is modulated by gonadal steroids and likely arises subsequent to coordination of GnRH neuron firing activity. The source of rhythm generation and the site of steroid feedback remain critical unanswered questions. Arcuate neurons that synthesize kisspeptin, neurokinin B, and dynorphin (KNDy) may be involved in both of these processes. We tested the hypotheses that action potential firing in KNDy neurons is episodic and that gonadal steroids regulate this pattern. Targeted extracellular recordings were made of green fluorescent protein-identified KNDy neurons in brain slices from adult male mice that were intact, castrated, or castrated and treated with estradiol or dihydrotestosterone (DHT). KNDy neurons exhibited marked peaks and nadirs in action potential firing activity during recordings lasting 1 to 3.5 hours. Peaks, identified by Cluster analysis, occurred more frequently in castrated than intact mice, and either estradiol or DHT in vivo or blocking neurokinin type 3 receptor in vitro restored peak frequency to intact levels. The frequency of peaks in firing rate and estradiol regulation of this frequency is similar to that observed for GnRH neurons, whereas DHT suppressed firing in KNDy but not GnRH neurons. We further examined the patterning of action potentials to identify bursts that may be associated with increased neuromodulator release. Burst frequency and duration are increased in castrated compared with intact and steroid-treated mice. The observation that KNDy neurons fire in an episodic manner that is regulated by steroid feedback is consistent with a role for these neurons in GnRH pulse generation and regulation.

ALX 1393 inhibits spontaneous network activity by inducing glycinergic tonic currents in the spinal ventral horn

BackgroundStrychnine-sensitive glycine receptors are activated by glycine and facilitate chloride influx into neurons. Glycinergic transmission might be either mediated by synaptic or extrasynaptic glycine receptors. While phasic neurotransmission is provided by a synaptic pathway, activation of extrasynaptic glycine receptors induces tonic inhibition. The glycine transporter 2 (GlyT2) regulates the uptake of glycine into presynaptic boutons. It is not determined yet, whether inhibition of GlyT2 by ALX 1393 can produce inhibition of spinal motoric networks and, whether phasic or tonic glycinergic inhibition is mostly enhanced. MethodsWe investigated the effect of ALX 1393 on spontaneous action potential firing activity by extracellular recordings in the ventral horn area of organotypic spinal cultures. Additionally, using the whole-cell patch-clamp technique, we defined the influence of GlyT2 inhibition on tonic and phasic glycinergic transmission in commissural interneurons of the ventral horn. ResultsGlyT2 inhibition by ALX 1393 potently reduced neuronal action potential activity in a concentration-dependent manner (n=211). The half maximal effect of ALX 1393 was observed at 100±31nM. Moreover, 88.3±2.6% of the action potential activity was suppressed at 1μM. Whole-cell patch-clamp recordings unveiled that ALX 1393 (200nM) induced a tonic current (−45.7±11.6pA, n=5) that was significantly reversed by application of the competitive glycine receptor antagonist strychnine. Contrastingly, phasic glycinergic transmission was not augmented by GlyT2 inhibition (charge transferred per time period for control conditions: 1.1±0.1pC, n=7, for ALX 1393: 0.9±0.2pC, n=7, p>0.05). ConclusionGlyT2 inhibition induced glycinergic tonic currents, which might be the underlying mechanism for the observed suppression of spontaneous action potential activity by ALX 1393 in the spinal ventral horn. Silencing neuronal action potential activity by blocking GlyT2 might be a novel principle to inhibit locomotor circuits in the ventral horn area and to induce muscle relaxation.

Regulation of Arcuate Neurons Coexpressing Kisspeptin, Neurokinin B, and Dynorphin by Modulators of Neurokinin 3 and κ-Opioid Receptors in Adult Male Mice

Pulsatile GnRH release is essential to fertility and is modulated by gonadal steroids, most likely via steroid-sensitive afferents. Arcuate neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) are steroid-sensitive and have been postulated to both generate GnRH pulses and mediate steroid feedback on pulse frequency. KNDy neurons are proposed to interact with one another via NKB and dynorphin to activate and inhibit the KNDy network, respectively, and thus alter kisspeptin output to GnRH neurons. To test the roles of NKB and dynorphin on KNDy neurons and the steroid sensitivity of these actions, targeted extracellular recordings were made of Tac2(NKB)-GFP-identified neurons from castrate and intact male mice. Single-cell PCR confirmed most of these cells had a KNDy phenotype. The neurokinin 3 receptor (NK3R) agonist senktide increased action potential firing activity of KNDy neurons. Dynorphin reduced spontaneous KNDy neuron activity, but antagonism of κ-opioid receptors (KOR) failed to induce firing activity in quiescent KNDy neurons. Senktide-induced activation was greater in KNDy neurons from castrate mice, whereas dynorphin-induced suppression was greater in KNDy neurons from intact mice. Interactions of dynorphin with senktide-induced activity were more complex; dynorphin treatment after senktide had no consistent inhibitory effect, whereas pretreatment with dynorphin decreased senktide-induced activity only in KNDy neurons from intact but not castrate mice. These data suggest dynorphin-mediated inhibition of senktide-induced activity requires gonadal steroid feedback. Together, these observations support the hypotheses that activation of NK3R and KOR, respectively, excites and inhibits KNDy neurons and that gonadal steroids modulate these effects.

Neuropeptide‐mediated calcium signaling in the suprachiasmatic nucleus network

Neuroactive peptides and the intracellular calcium concentration ([Ca(2+) ](i) ) play important roles in light-induced modulation of gene expression in the suprachiasmatic nucleus (SCN) neurons that ultimately control behavioral rhythms. Vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) are expressed rhythmically within populations of SCN neurons. Pituitary adenylate cyclase-activating peptide (PACAP) is released from retinohypothalamic tract (RHT) terminals synapsing on SCN neurons. Nociceptin/orphanin FQ (OFQ) receptors are functionally expressed in the SCN. We examined the role of several neuropeptides on Ca(2+) signaling, simultaneously imaging multiple neurons within the SCN neural network. VIP reduced the [Ca(2+) ](i) in populations of SCN neurons during the day, but had little effect at night. Stimulation of the RHT at frequencies that simulate light input signaling evoked transient [Ca(2+) ](i) elevations that were not altered by VIP. AVP elevated the [Ca(2+) ](i) during both the day and night, PACAP produced variable responses, and OFQ induced a reduction in the [Ca(2+) ](i) similar to VIP. During the day, VIP lowered the [Ca(2+) ](i) to near nighttime levels, while AVP elevated [Ca(2+) ](i) during both the day and night, suggesting that the VIP effects on [Ca(2+) ](i) were dependent, and the AVP effects independent of the action potential firing activity state of the neuron. We hypothesize that VIP and AVP regulate, at least in part, Ca(2+) homeostasis in SCN neurons and may be a major point of regulation for SCN neuronal synchronization.

Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus

Zolpidem is a widely prescribed sleep aid with relative selectivity for GABA(A) receptors containing alpha1-3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABA(A) receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABA(A) receptor-mediated tonic current (I(tonic)) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that alpha1-3 subunits are expressed in synaptic GABA(A) receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABA(A) receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABA(A) receptors containing alpha1-3 subunits contribute to the I(tonic).

The Gonadotropin-Releasing Hormone (GnRH) Neuronal Population Is Normal in Size and Distribution in GnRH-Deficient and GnRH Receptor-Mutant Hypogonadal Mice

Hypothalamic GnRH neurons are essential for initiation and regulation of reproductive function. In addition to pituitary gonadotrope stimulation, activity of GnRH through its receptor (GnRHR) has been suggested to include autocrine regulation of the GnRH neuron. Two hypogonadal mouse strains, the Gnrh1 mutant (hpg) mice and Gnrhr mutant mice were used to investigate the potential role of GnRH signaling in the proper development and maintenance of GnRH neurons. Immunocytochemical analysis of heterozygous hpg mice revealed a GnRH neuron population that was normal in size and distribution, indicating no effect from reduced Gnrh1 gene dosage on the neurons themselves. To visualize GnRH neurons in homozygous GnRH-deficient hpg mice, heterozygous hpg mice were crossed with GnRH-green fluorescent protein (GFP) transgenic mice with targeted expression of the GFP reporter gene in GnRH neurons. Analysis of forebrains of homozygous hpg/GFP-positive mice immunostained for GFP revealed a normal population size and appropriate distribution of GnRH neurons in hpg mice, with immunoreactive neuronal processes present at the median eminence. Similarly, adult mice deficient in functional GnRHR possessed a full complement of GnRH neurons in the basal forebrain that was indistinguishable from the distribution of GnRH neurons in their wild-type counterparts. Moreover, hpg/GFP neurons retained the ability to generate spontaneous bursts of action potential firing activity, suggesting that GnRH peptide is not required for this function. These data establish that autocrine-paracrine GnRH-signaling is not a prerequisite for the developmental migration of GnRH neurons into the brain or for the projection of GnRH neurosecretory axons.

Peptide YY: obesity's cause and cure?

RECENTLY, the United States Food and Drug Administration (FDA) decided for the present against approving the cannabinoid receptor antagonist Rimonabant® for the treatment of obesity in the United States. This decision mainly resulted from concerns based on study results showing that the drug’s benefits would not outweigh its risks for side effects. This decision further diminished already limited hopes, that a potent and safe anti-obesity drug would soon emerge and remove one of the most serious health threats to industrialized and developing societies of today and tomorrow. What would a new drug candidate have to become a perfect anti-obesity agent? Such a perfect anti-obesity molecule should not only lead to sustained loss of body fat, but also induce the necessary negative energy balance without causing undesired side effects. What’s more, a perfect anti-obesity agent would preferably be a naturally occurring, endogenous, molecule and it should target a known specific mechanism. Finally, it should remove the cause for obesity rather than treating symptoms of the disease. Looking at these requirements, it seems that such a silver bullet may already be available: the gastrointestinal hormone, peptide YY (PYY). Based on published data, peptide YY may be the only currently known molecule which matches these criteria without exception: PYY has been reported to induce a negative energy balance by potently decreasing food intake in rodents (1) and humans (2) without causing any undesired side effects (1,2). This gut peptide is a naturally occurring hormone which is secreted by endocrine L-cells of the colon (3). The reported mechanism of action of its predominantly circulating form, PYY3-36, is based on the

PYY 3–36 inhibits the action potential firing activity of POMC neurons of arcuate nucleus through postsynaptic Y 2 receptors

Intracerebroventricular administration of gut peptide PYY3-36 stimulates food intake. In contrast, peripheral administration inhibits food intake, suggesting that the peptide has the opposite effect by virtue of accessing a unique subset of brain sites. A previous study suggested that peripheral PYY3-36 activates anorexigenic POMC neurons in the arcuate nucleus, and this was proposed to be the mechanism underlying the peptide's anorexigenic activity. Here, we demonstrate in an electrophysiological slice preparation that, in contrast to the original model, PYY3-36 potently and reversibly inhibits POMC neurons via postsynaptic Y2 receptors. These data show a complex role for Y2 receptors in regulation of the NPY/POMC circuitry, as they are present as inhibitory receptors on both the orexigenic NPY neurons as well as the anorexigenic POMC neurons. Secondly, these data argue against a direct role of POMC neurons in mediating the anorexigenic response to administration of peripheral PYY3-36.