Threshold For Action Potential Generation Research Articles

Targeted blockade of aberrant sodium current in a stem cell-derived neuron model of SCN3A encephalopathy.

Missense variants in SCN3A encoding the voltage-gated sodium (Na+) channel α subunit Nav1.3 are associated with SCN3A-related neurodevelopmental disorder (SCN3A-NDD), a spectrum of disease that includes epilepsy and malformation of cortical development. How genetic variation in SCN3A leads to pathology remains unclear, as prior electrophysiological work on disease-associated variants has been performed exclusively in heterologous cell systems. To further investigate the mechanisms of SCN3A-NDD pathogenesis, we used CRISPR/Cas9 gene editing to modify a control human induced pluripotent stem cell (iPSC) line to express the recurrent de novo missense variant SCN3A c.2624T>C (p.Ile875Thr). With the established Ngn2 rapid induction protocol, we generated glutamatergic forebrain-like neurons (iNeurons), which we showed to express SCN3A mRNA and Nav1.3-mediated Na+ currents. We performed detailed whole-cell patch clamp recordings to determine the effect of the SCN3A-p.Ile875Thr variant on endogenous Na+ currents in, and intrinsic excitability of, human neurons. Compared to control iNeurons, variant-expressing iNeurons exhibit markedly increased slowly-inactivating/persistent Na+ current, abnormal firing patterns with paroxysmal bursting and plateau-like potentials with action potential failure, and a hyperpolarized voltage threshold for action potential generation. We then validated these findings using a separate iPSC line generated from a patient harbouring the SCN3A-p.Ile875Thr variant compared to a corresponding CRISPR-corrected isogenic control line. Finally, we found that application of the Nav1.3-selective blocker ICA-121431 normalizes action potential threshold and aberrant firing patterns in SCN3A-p.Ile1875Thr iNeurons; in contrast, consistent with action as a Na+ channel blocker, ICA-121431 decreases excitability of control iNeurons. Our findings demonstrate that iNeurons can model the effects of genetic variation in SCN3A yet reveal a complex relationship between gain-of-function at the level of the ion channel versus impact on neuronal excitability. Given the transient expression of SCN3A in the developing human nervous system, selective blockade or suppression of Nav1.3-containing Na+ channels could represent a therapeutic approach towards SCN3A-NDD.

Sodium channel endocytosis drives axon initial segment plasticity.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

The Na<sup>+</sup>/K<sup>+</sup>-ATPase Inhibitor Ouabain Has Different Effects on the Electrophysiological Properties of Excitatory and Inhibitory Neurons in the Entorhinal Cortex E

Na+/K+-ATPase maintains the neuron’s resting potential and the transmembrane gradient of K+ and Na+ cations, thus regulating ion transport and cellular volume. Mutations in Na+/K+-ATPase genes that impair its function can cause significant impairments in the nervous system function, including the development of epilepsy, if not lethal. Different forms of Na+/K+-ATPase are expressed in various classes of neurons and exhibit different characteristics. Thus, the impaired function of Na+/K+-ATPase may differentially affect the functioning of inhibitory and excitatory neurons. This study aims to determine the effects of the Na+/K+-ATPase antagonist ouabain on the electrophysiological characteristics of pyramidal cells and fast-spiking interneurons, as well as its impact on synaptic transmission. The results indicate that exposure to 5 µM ouabain results in depolarization of the resting membrane potential by 5 mV, as well as decreased amplitude and increased duration of the action potential of pyramidal neurons. Furthermore, ouabain caused a decrease in the amplitude of afterhyperpolarization in fast-spiking i-nterneurons. Moreover, both types of neurons exhibited a decrease in the threshold of action potential generation and the current at which depolarization block occurs. The addition of ouabain did not alter other electrophysiological characteristics of neurons. Furthermore, ouabain rapidly attenuates GABAergic transmission without affecting e-xcitatory synaptic transmission. These new findings on the effects of ouabain on excitatory pyramidal neurons and inhibitory interneurons contribute to the understanding of the mechanism underlying changes in the balance of excitation and inhibition in neural networks under Na+/K+-ATPase function impairment.

ID: 216598 Effects of Sodium Channel Clustering on Activation Thresholds of Nonmyelinated Axons

It is hypothesized that some neurostimulation therapies, such as dorsal root ganglion stimulation (DRGS), may provide therapeutic benefit by activating nonmyelinated neurons (e.g., C-neurons). However, conventional neurostimulation theory suggests that extracellular electrical stimulation preferentially activates large-diameter myelinated neurons over small-diameter nonmyelinated neurons. Interestingly, a recent study of DRGS in rats demonstrated that the activation thresholds of nonmyelinated C-neurons are only ∼1.5 times greater than the activation thresholds of myelinated Abeta-neurons (Chao et al., 2020). It is possible that certain electrophysiological features of C-type neurons unaccounted for in previous computational models could reduce activation thresholds to ranges comparable to the thresholds of myelinated neurons. For example, Nav1.8 sodium channels, which underlie most of the sodium current during the upstroke of action potentials in some C-neurons, have been shown to be distributed in clusters along the axons of C-type neurons forming so-called “micro-nodes” (Pristerà et al., 2012). In this study, we developed a computational model to investigate the impact of Nav1.8-channel clustering on the threshold for action potential generation in nonmyelinated axons.

Heparan Sulfates Regulate Axonal Excitability and Context Generalization through Ca2+/Calmodulin-Dependent Protein Kinase II.

Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 h after injection in mice. Patch clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 h after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting the key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.

S-ketamine enhances thalamocortical and corticocortical synaptic transmission in acute murine brain slices via increased AMPA-receptor-mediated pathways.

Despite ongoing research efforts and routine clinical use, the neuronal mechanisms underlying the anesthesia-induced loss of consciousness are still under debate. Unlike most anesthetics, ketamine increases thalamic and cortical activity. Ketamine is considered to act via a NMDA-receptor antagonism-mediated reduction of inhibition, i.e., disinhibition. Intact interactions between the thalamus and cortex constitute a prerequisite for the maintenance of consciousness and are thus a promising target for anesthetics to induce loss of consciousness. In this study, we aim to characterize the influence of s-ketamine on the thalamocortical network using acute brain-slice preparation. We performed whole-cell patch-clamp recordings from pyramidal neurons in cortical lamina IV and thalamocortical relay neurons in acute brain slices from CB57BL/6N mice. Excitatory postsynaptic potentials (EPSPs) were obtained via electrical stimulation of the cortex with a bipolar electrode that was positioned to lamina II/III (electrically induced EPSPs, eEPSPs) or via optogenetic activation of thalamocortical relay neurons (optogenetically induced EPSPs, oEPSPs). Intrinsic neuronal properties (like resting membrane potential, membrane threshold for action potential generation, input resistance, and tonic action potential frequency), as well as NMDA-receptor-dependent and independent spontaneous GABAA-receptor-mediated inhibitory postsynaptic currents (sIPSCs) were evaluated. Wilcoxon signed-rank test (level of significance < 0.05) served as a statistical test and Cohen's U3_1 was used to determine the actual effect size. Within 20 min, s-ketamine (5 μM) significantly increased both intracortical eEPSPs as well as thalamocortical oEPSPs. NMDA-receptor-mediated intracortical eEPSPs were significantly reduced. Intrinsic neuronal properties of cortical pyramidal neurons from lamina IV and thalamocortical relay neurons in the ventrobasal thalamic complex were not substantially affected. Neither a significant effect on NMDA-receptor-dependent GABAA sIPSCs (thought to underly a disinhibitory effect) nor a reduction of NMDA-receptor independent GABAA sIPSCs was observed. Both thalamocortical and intracortical AMPA-receptor-mediated EPSPs were significantly increased.In conclusion, our findings show no evidence for a NMDA-receptor antagonism-based disinhibition, but rather suggest an enhanced thalamocortical and intracortical synaptic transmission, which appears to be driven via increased AMPA-receptor-mediated transmission.

Electrophysiological complexity in the rat dorsomedial hypothalamus and its susceptibility to daily rhythms and high-fat diet.

The dorsomedial hypothalamus (DMH) in amongst the most important brain structures involved in the regulation of feeding behaviour and metabolism. In contrast to other hypothalamic centres, its main role is related to the circadian rhythmicity of food intake and energy homeostasis; both reported to be disrupted in obesity. In modern world, overweight and obesity reached global epidemic proportions. Thus, not only is it important to study their negative implications but also the mechanism responsible for their development. Here, we exposed rats to short-term (2-4weeks) high-fat diet (HFD)-not long enough to induce obesity. Next, we performed electrophysiological patch-clamp recordings ex vivo from neurons in the DMH either during the day or at night. Our results showed a day-to-night change in the firing frequency of DMH cells, with higher activity during the dark phase. This was abolished by HFD consumption, resulting in a decreased threshold for action potential generation during the day and therefore increased electrical activity at this phase. We propose this electrophysiological disturbance as a mechanism for the induction of abnormal daytime feeding, previously observed for HFD-fed animals, which might in turn contribute to the development of obesity. In addition, we provide an electrophysiological characteristic of DMH neurons with a separation into three anatomically and functionally distinct subpopulations, namely, the compact part, separating the structure into the ventral and dorsal divisions. Our study is the first to show electrophysiological complexity of the DMH with its sensitivity to diet and daily rhythms.

Sevoflurane excites nociceptive sensory neurons by inhibiting K+ conductances in rats

Sevoflurane, which is preferentially used as a day-case anesthetic based on its low blood solubility, acts on the central nervous system and exerts analgesic effects. However, it still remains unknown whether sevoflurane affects the excitability of nociceptive sensory neurons. Therefore, we conducted this study to examine the effects of sevoflurane on the excitability of small-sized dorsal root ganglion (DRG) neurons of rats using the whole-cell patch-clamp technique. In a voltage-clamp condition, sevoflurane elicited the membrane current in a concentration-dependent manner, in which the reversal potential was similar to the equilibrium potential of K+. In a current-clamp condition, sevoflurane directly depolarized the membrane potentials in a concentration-dependent manner. Moreover, at a clinically relevant concentration, sevoflurane decreased the threshold for action potential generation. These findings suggest that sevoflurane acts on the leak K+ channels to increase the excitability of DRG neurons. Sevoflurane increased the half-width of single action potentials, which resulted from the inhibition of voltage-gated K+ currents, including the fast inactivating A-type and non-inactivating delayed rectifier K+ currents. Our study indicates that sevoflurane could exhibit pronociceptive effects on nociceptive sensory neurons by inhibiting K+ conductances.

Activation of GABAB receptors results in excitatory modulation of calyx terminals in rat semicircular canal cristae.

Previous studies have found GABA in vestibular end organs. However, existence of GABA receptors or possible GABAergic effects on vestibular nerve afferents has not been investigated. The current study was conducted to determine whether activation of GABAB receptors affects calyx afferent terminals in the central region of the cristae of semicircular canals. We used patch-clamp recording in postnatal day 13-18 (P13-P18) Sprague-Dawley rats of either sex. Application of GABAB receptor agonist baclofen inhibited voltage-sensitive potassium currents. This effect was blocked by selective GABAB receptor antagonist CGP 35348. Application of antagonists of small (SK)- and large-conductance potassium (BK) channels almost completely blocked the effects of baclofen. The remaining baclofen effect was blocked by cadmium chloride, suggesting that it could be due to inhibition of voltage-gated calcium channels. Furthermore, baclofen had no effect in the absence of calcium in the extracellular fluid. Inhibition of potassium currents by GABAB activation resulted in an excitatory effect on calyx terminal action potential firing. While in the control condition calyces could only fire a single action potential during step depolarizations, in the presence of baclofen they fired continuously during steps and a few even showed repetitive discharge. We also found a decrease in threshold for action potential generation and a decrease in first-spike latency during step depolarization. These results provide the first evidence for the presence of GABAB receptors on calyx terminals, showing that their activation results in an excitatory effect and that GABA inputs could be used to modulate calyx response properties.NEW & NOTEWORTHY Using in vitro whole cell patch-clamp recordings from calyx terminals in the vestibular end organs, we show that activation of GABAB receptors result in an excitatory effect, with decreased spike-frequency adaptation and shortened first-spike latencies. Our results suggest that these effects are mediated through inhibition of calcium-sensitive potassium channels.

Four weeks of environmentally relevant second hand smoke exposure decreased neuronal excitability of cardiac vagal neurons in the dorsal motor nucleus of vagus

BACKGROUNDSecond hand smoke (SHS), a main indoor air pollutant, is a significant risk factor for cardiovascular morbidity and mortality. We previously showed that exposure to SHS, for as short as four weeks, significantly reduced heart rate variability (HRV), suggesting a decreased vagal regulation of the heart. We further showed that, in cardiac vagal neurons in the nucleus ambiguus (NA), SHS exposure significantly reduced spiking response to depolarizing current injections and increased current‐ and voltage‐threshold for action potential generation. However, we have no information on the effects of SHS exposure on cardiac vagal neurons in the dorsal motor nucleus of vagus (DMNV). Here, we sought to test the hypothesis that SHS exposure decreases neuronal excitability of cardiac vagal neurons in the DMNV.METHODSSeven‐week old male C57BL/6J mice underwent surgeries to retrograde label cardiac vagal neurons by applying a fluorescent dye (DiI) to the sinoatrial node. Two weeks after surgery, mice were randomly assigned to either filtered air‐ (FA, n = 14) or SHS‐exposed (n = 16) group. The SHS exposure (6 hr/d, 5 d/wk) was set at a level that is similar to that of a smoky bar (3 mg/m3). After four weeks of FA or SHS exposure, whole‐cell current clamp recordings in brainstem slices were performed on anatomically identified cardiac vagal neurons in the DMNV with fluorescent labeling.RESULTSDMNV cardiac vagal neurons from the SHS‐exposed group were significantly more hyperpolarized than the FA control group (−53.3 ± 0.9 mV vs. −55.7 ± 0.7 mV, FA vs. SHS respectively, p &lt; 0.05). Four weeks of SHS exposure also significantly decreased neuronal spontaneous spiking activity at a holding potential of −45 mV (1.6 ± 0.4 Hz vs. 0.8 ± 0.2 Hz, FA vs. SHS respectively, p &lt; 0.05). The decreased spontaneous activity was not associated with the spiking response to depolarizing current injections from a holding potential of −60 mV, current and voltage threshold for action potential generation, action potential peak and afterhyperpolarization. Furthermore, there was no significant difference in the neuronal input‐output relationship of DMNV cardiac vagal neurons between FA control and SHS‐exposed groups, suggesting an unchanged dynamic responsiveness to stimuli.CONCLUSIONSFour weeks exposure to environmentally relevant level of SHS decreased neuronal excitability of cardiac vagal neurons in a region‐dependent manner – reduced resting membrane potential and spontaneous spiking activity in the DMNV and reduced spiking response to depolarizing current injections and increased threshold for action potential generation in the NA.Support or Funding InformationFunding: R01 ES025229

Platelet-derived growth factor activates nociceptive neurons by inhibiting M-current and contributes to inflammatory pain.

Endogenous inflammatory mediators contribute to the pathogenesis of pain by acting on nociceptors, specialized sensory neurons that detect noxious stimuli. Here, we describe a new factor mediating inflammatory pain. We show that platelet-derived growth factor (PDGF)-BB applied in vitro causes repetitive firing of dissociated nociceptor-like rat dorsal root ganglion neurons and decreased their threshold for action potential generation. Injection of PDGF-BB into the paw produced nocifensive behavior in rats and led to thermal and mechanical pain hypersensitivity. We further detailed the biophysical mechanisms of these PDGF-BB effects and show that PDGF receptor-induced inhibition of nociceptive M-current underlies PDGF-BB-mediated nociceptive hyperexcitability. Moreover, in vivo sequestration of PDGF or inhibition of the PDGF receptor attenuates acute formalin-induced inflammatory pain. Our discovery of a new pain-facilitating proinflammatory mediator, which by inhibiting M-current activates nociceptive neurons and thus contributes to inflammatory pain, improves our understanding of inflammatory pain pathophysiology and may have important clinical implications for pain treatment.

Acute inflammation sensitizes knee-innervating sensory neurons and decreases mouse digging behavior in a TRPV1-dependent manner

Ongoing, spontaneous pain is characteristic of inflammatory joint pain and reduces an individual's quality of life. To understand the neural basis of inflammatory joint pain, we made a unilateral knee injection of complete Freund's adjuvant (CFA) in mice, which reduced their natural digging behavior. We hypothesized that sensitization of knee-innervating dorsal root ganglion (DRG) neurons underlies this altered behavior. To test this hypothesis, we performed electrophysiological recordings on retrograde labeled knee-innervating primary DRG neuron cultures and measured their responses to a number of electrical and chemical stimuli. We found that 24-h after CFA-induced knee inflammation, knee neurons show a decreased action potential generation threshold, as well as increased GABA and capsaicin sensitivity, but have unaltered acid sensitivity. The inflammation-induced sensitization of knee neurons persisted for 24-h in culture, but was not observed after 48-h in culture. Through immunohistochemistry, we showed that the increased knee neuron capsaicin sensitivity correlated with enhanced expression of the capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) in knee-innervating neurons of the CFA-injected side. We also observed an increase in the co-expression of TRPV1 with tropomyosin receptor kinase A (TrkA), which is the receptor for nerve growth factor (NGF), suggesting that NGF partially induces the increased TRPV1 expression. Lastly, we found that systemic administration of the TRPV1 antagonist, A-425619, reversed the decrease in digging behavior induced by CFA injection, further confirming the role of TRPV1, expressed by knee neurons, in acute inflammatory joint pain.

Isolated nociceptors reveal multiple specializations for generating irregular ongoing activity associated with ongoing pain.

Ongoing pain has been linked to ongoing activity (OA) in human C-fiber nociceptors, but rodent models of pain-related OA have concentrated on allodynia rather than ongoing pain, and on OA generated in non-nociceptive Aβ fibers rather than C-fiber nociceptors. Little is known about how ongoing pain or nociceptor OA is generated. To define neurophysiological alterations underlying nociceptor OA, we have used isolated dorsal root ganglion neurons that continue to generate OA after removal from animals displaying ongoing pain. We subclassify OA as either spontaneous activity generated solely by alterations intrinsic to the active neuron or as extrinsically driven OA. Both types of OA were implicated previously in nociceptors in vivo and after isolation following spinal cord injury, which produces chronic ongoing pain. Using novel automated algorithms to analyze irregular changes in membrane potential, we have found, in a distinctive, nonaccommodating type of probable nociceptor, induction by spinal cord injury of 3 alterations that promote OA: (1) prolonged depolarization of resting membrane potential, (2) a hyperpolarizing shift in the voltage threshold for action potential generation, and (3) an increase in the incidence of large depolarizing spontaneous fluctuations (DSFs). Can DSFs also be enhanced acutely to promote OA in neurons from uninjured animals? A low dose of serotonin failed to change resting membrane potential but lowered action potential threshold. When combined with artificial depolarization to model inflammation, serotonin also strongly potentiated DSFs and OA. These findings reveal nociceptor specializations for generating OA that may promote ongoing pain in chronic and acute conditions.

Neurotropin inhibits neuronal activity through potentiation of sustained Kv currents in primary cultured DRG neurons

Neurotropin (NTP) is a Japanese analgesic agent for treating neuropathic pain; however, its method of action remains unclear. This study examined the effects of NTP on the activity of small dorsal root ganglion (DRG) neurons using whole-cell patch clamp recordings. After 3 days of treatment, NTP decreased current injection-induced firing activity of cultured DRG neurons by raising the current threshold for action potential generation. Additionally, NTP increased the sustained component of voltage-gated potassium (Kv) channel currents without affecting other K+ currents. These results suggest that NTP inhibits the firing activity of DRG neurons through augmentation of sustained Kv current.

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

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

Spontaneous EPSP of Command Neurons in the Common Snail during Heterosynaptic Potentiation

The presynaptic mechanism of short-term plasticity of synaptic transmission was studied by analyzing the effects of rhythmic orthodromic stimulation of the intestinal nerve, inducing short-term potentiation of evoked EPSP, on spontaneous EPSP in defensive behavior command neurons in the common snail. Rhythmic stimulation had no effect on the amplitude of spontaneous EPSP but led to significant increases in the number of spontaneous EPSP. The increase in the frequency of spontaneous EPSP suggested a role for a presynaptic mechanism in the short-term potentiation of synaptic transmission. This presynaptic mechanism may include a decrease in the action potential generation threshold (AP) in presynaptic neurons, which leads to an increase in the proportion of spontaneously active neurons and, thus, supports an increase in the number of spontaneous presynaptic AP.

Spike threshold dynamics in spinal motoneurons during scratching and swimming.

Action potential threshold can vary depending on firing history and synaptic inputs. We used an ex vivo carapace-spinal cord preparation from adult turtles to study spike threshold dynamics in motoneurons during two distinct types of functional motor behaviour - fictive scratching and fictive swimming. The threshold potential depolarizes by about 10mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Slow synaptic integration resulting in a wave of membrane potential depolarization is the factor influencing the threshold potential within firing bursts during motor behaviours. Depolarization of the threshold potential decreases the excitability of motoneurons and may provide a mechanism for stabilization of the response of a motoneuron to intense synaptic inputs to maintain the motor commands within an optimal range for muscle activation. During functional spinal neural network activity motoneurons receive intense synaptic input, and this could modulate the threshold for action potential generation, providing the ability to dynamically adjust the excitability and recruitment order for functional needs. In the present study we investigated the dynamics of action potential threshold during motor network activity. Intracellular recordings from spinal motoneurons in an ex vivo carapace-spinal cord preparation from adult turtles were performed during two distinct types of motor behaviour - fictive scratching and fictive swimming. We found that the threshold of the first spike in episodes of scratching and swimming was the lowest. The threshold potential depolarizes by about 10mV within each burst of spikes generated during scratch and swim network activity and recovers between bursts to a slightly depolarized level. Depolarization of the threshold potential results in decreased excitability of motoneurons. Synaptic inputs do not modulate the threshold of the first action potential during episodes of scratching or of swimming. There is no correlation between changes in spike threshold and interspike intervals within bursts. Slow synaptic integration that results in a wave of membrane potential depolarization rather than fast synaptic events preceding each spike is the factor influencing the threshold potential within firing bursts during motor behaviours.

Neural hyperactivity in the amygdala induced by chronic treatment of rats with analgesics may elucidate the mechanisms underlying psychiatric comorbidities associated with medication-overuse headache

BackgroundPatients with medication-overuse headache suffer not only from chronic headache, but often from psychiatric comorbidities, such as anxiety and depression. The mechanisms underlying these comorbidities are unclear, but the amygdala is likely to be involved in their pathogenesis. To investigate the mechanisms underlying the comorbidities we used elevated plus maze and open field tests to assess anxiety-like behavior in rats chronically treated with analgesics. We measured the electrical properties of neurons in the amygdala, and examined the cortical spreading depression (CSD)-evoked expression of Fos in the trigeminal nucleus caudalis (TNC) and amygdala of rats chronically treated with analgesics. CSD, an analog of aura, evokes Fos expression in the TNC of rodents suggesting trigeminal nociception, considered to be a model of migraine.ResultsIncreased anxiety-like behavior was seen both in elevated plus maze and open field tests in a model of medication overuse produced in male rats by chronic treatment with aspirin or acetaminophen. The time spent in the open arms of the maze by aspirin- or acetaminophen-treated rats (53 ± 36.1 and 37 ± 29.5 s, respectively) was significantly shorter than that spent by saline-treated vehicle control rats (138 ± 22.6 s, P < 0.001). Chronic treatment with the analgesics increased the excitability of neurons in the central nucleus of the amygdala as indicated by their more negative threshold for action potential generation (–54.6 ± 5.01 mV for aspirin-treated, –55.2 ± 0.97 mV for acetaminophen-treated, and –31.50 ± 5.34 mV for saline-treated rats, P < 0.001). Chronic treatment with analgesics increased the CSD-evoked expression of Fos in the TNC and amygdala [18 ± 10.2 Fos-immunoreactive (IR) neurons per slide in the amygdala of rats treated with aspirin, 11 ± 5.4 IR neurons per slide in rats treated with acetaminophen, and 4 ± 3.7 IR neurons per slide in saline-treated control rats, P < 0.001].ConclusionsChronic treatment with analgesics can increase the excitability of neurons in the amygdala, which could underlie the anxiety seen in patients with medication-overuse headache.

Dynamic DNA methylation regulates neuronal intrinsic membrane excitability.

Epigenetic modifications, such as DNA cytosine methylation, contribute to the mechanisms underlying learning and memory by coordinating adaptive gene expression and neuronal plasticity. Transcription-dependent plasticity regulated by DNA methylation includes synaptic plasticity and homeostatic synaptic scaling. Memory-related plasticity also includes alterations in intrinsic membrane excitability mediated by changes in the abundance or activity of ion channels in the plasma membrane, which sets the threshold for action potential generation. We found that prolonged inhibition of DNA methyltransferase (DNMT) activity increased intrinsic membrane excitability of cultured cortical pyramidal neurons. Knockdown of the cytosine demethylase TET1 or inhibition of RNA polymerase blocked the increased membrane excitability caused by DNMT inhibition, suggesting that this effect was mediated by subsequent cytosine demethylation and de novo transcription. Prolonged DNMT inhibition blunted the medium component of the after-hyperpolarization potential, an effect that would increase neuronal excitability, and was associated with reduced expression of the genes encoding small-conductance Ca(2+)-activated K(+) (SK) channels. Furthermore, the specific SK channel blocker apamin increased neuronal excitability but was ineffective after DNMT inhibition. Our results suggested that DNMT inhibition enables transcriptional changes that culminate in decreased expression of SK channel-encoding genes and decreased activity of SK channels, thus providing a mechanism for the regulation of neuronal intrinsic membrane excitability by dynamic DNA cytosine methylation. This study has implications for human neurological and psychiatric diseases associated with dysregulated intrinsic excitability.

The influence of increased membrane conductance on response properties of spinal motoneurons

During functional spinal neural network activity motoneurons receive massive synaptic excitation and inhibition, and their membrane conductance increases considerably – they are switched to a high-conductance state. High-conductance states can substantially alter response properties of motoneurons. In the present study we investigated how an increase in membrane conductance affects spike frequency adaptation, the gain (i.e., the slope of the frequency-current relationship) and the threshold for action potential generation. We used intracellular recordings from adult turtle motoneurons in spinal cord slices. Membrane conductance was increased pharmacologically by extracellular application of the GABAA receptor agonist muscimol. Our findings suggest that an increase in membrane conductance of about 40–50% increases the magnitude of spike frequency adaptation, but does not change the threshold for action potential generation. Increased conductance causes a subtractive rather than a divisive effect on the initial and the early frequency-current relationships and may have not only a subtractive but also a divisive effect on the steady-state frequency-current relationship.