Action Potential Threshold Shifts Research Articles

Cation leak underlies neuronal excitability in an HCN1 developmental and epileptic encephalopathy.

Pathogenic variants in HCN1 are associated with developmental and epileptic encephalopathies. The recurrent de novo HCN1 M305L pathogenic variant is associated with severe developmental impairment and drug-resistant epilepsy. We engineered the homologue Hcn1 M294L heterozygous knock-in (Hcn1M294L) mouse to explore the disease mechanism underlying an HCN1 developmental and epileptic encephalopathy. The Hcn1M294L mouse recapitulated the phenotypic features of patients with the HCN1 M305L variant, including spontaneous seizures and a learning deficit. Active epileptiform spiking on the electrocorticogram and morphological markers typical of rodent seizure models were observed in the Hcn1M294L mouse. Lamotrigine exacerbated seizures and increased spiking, whereas sodium valproate reduced spiking, mirroring drug responses reported in a patient with this variant. Functional analysis in Xenopus laevis oocytes and layer V somatosensory cortical pyramidal neurons in ex vivo tissue revealed a loss of voltage dependence for the disease variant resulting in a constitutively open channel that allowed for cation 'leak' at depolarized membrane potentials. Consequently, Hcn1M294L layer V somatosensory cortical pyramidal neurons were significantly depolarized at rest. These neurons adapted through a depolarizing shift in action potential threshold. Despite this compensation, layer V somatosensory cortical pyramidal neurons fired action potentials more readily from rest. A similar depolarized resting potential and left-shift in rheobase was observed for CA1 hippocampal pyramidal neurons. The Hcn1M294L mouse provides insight into the pathological mechanisms underlying hyperexcitability in HCN1 developmental and epileptic encephalopathy, as well as being a preclinical model with strong construct and face validity, on which potential treatments can be tested.

Interneuron Dysfunction in a New Mouse Model of SCN1A GEFS.

Advances in genome sequencing have identified over 1300 mutations in the SCN1A sodium channel gene that result in genetic epilepsies. However, it still remains unclear how most individual mutations within SCN1A result in seizures. A previous study has shown that the K1270T (KT) mutation, linked to genetic epilepsy with febrile seizure plus (GEFS+) in humans, causes heat-induced seizure activity associated with a temperature-dependent decrease in GABAergic neuron excitability in a Drosophila knock-in model. To examine the behavioral and cellular effects of this mutation in mammals, we introduced the equivalent KT mutation into the mouse (Mus musculus) Scn1a (Scn1aKT) gene using CRISPR/Cas9 and generated mutant lines in two widely used genetic backgrounds: C57BL/6NJ and 129X1/SvJ. In both backgrounds, mice homozygous for the KT mutation had spontaneous seizures and died by postnatal day (P)23. There was no difference in mortality of heterozygous KT mice compared with wild-type littermates up to six months old. Heterozygous mutants exhibited heat-induced seizures at ∼42°C, a temperature that did not induce seizures in wild-type littermates. In acute hippocampal slices at permissive temperatures, current-clamp recordings revealed a significantly depolarized shift in action potential threshold and reduced action potential amplitude in parvalbumin (PV)-expressing inhibitory CA1 interneurons in Scn1aKT/+ mice. There was no change in the firing properties of excitatory CA1 pyramidal neurons. These results suggest that a constitutive decrease in inhibitory interneuron excitability contributes to the seizure phenotype in the mouse model.

In vivo patch-clamp recordings reveal distinct subthreshold signatures and threshold dynamics of midbrain dopamine neurons

The in vivo firing patterns of ventral midbrain dopamine neurons are controlled by afferent and intrinsic activity to generate sensory cue and prediction error signals that are essential for reward-based learning. Given the absence of in vivo intracellular recordings during the last three decades, the subthreshold membrane potential events that cause changes in dopamine neuron firing patterns remain unknown. To address this, we established in vivo whole-cell recordings and obtained over 100 spontaneously active, immunocytochemically-defined midbrain dopamine neurons in isoflurane-anaesthetized adult mice. We identified a repertoire of subthreshold membrane potential signatures associated with distinct in vivo firing patterns. Dopamine neuron activity in vivo deviated from single-spike pacemaking by phasic increases in firing rate via two qualitatively distinct biophysical mechanisms: 1) a prolonged hyperpolarization preceding rebound bursts, accompanied by a hyperpolarizing shift in action potential threshold; and 2) a transient depolarization leading to high-frequency plateau bursts, associated with a depolarizing shift in action potential threshold. Our findings define a mechanistic framework for the biophysical implementation of dopamine neuron firing patterns in the intact brain.

Calcium stores regulate excitability in cultured rat hippocampal neurons.

Extracellular calcium ions support synaptic activity but also reduce excitability of central neurons. In the present study, the effect of calcium on excitability was explored in cultured hippocampal neurons. CaCl2 injected by pressure in the vicinity of a neuron that is bathed only in MgCl2 as the main divalent cation caused a depolarizing shift in action potential threshold and a reduction in excitability. This effect was not seen if the intracellular milieu consisted of Cs+ instead of K-gluconate as the main cation or when it contained ruthenium red, which blocks release of calcium from stores. The suppression of excitability by calcium was mimicked by caffeine, and calcium store antagonists cyclopiazonic acid or thapsigargin blocked this action. Neurons taken from synaptopodin-knockout mice show significantly reduced efficacy of calcium modulation of action potential threshold. Likewise, in Orai1 knockdown cells, calcium is less effective in modulating excitability of neurons. Activation of small-conductance K (SK) channels increased action potential threshold akin to that produced by calcium ions, whereas blockade of SK channels but not big K channels reduced the threshold for action potential discharge. These results indicate that calcium released from stores may suppress excitability of central neurons. NEW & NOTEWORTHY Extracellular calcium reduces excitability of cultured hippocampal neurons. This effect is mediated by calcium-gated potassium currents, possibly small-conductance K channels. Release of calcium from internal stores mimics the effect of extracellular calcium. It is proposed that calcium stores modulate excitability of central neurons.

Nitric oxide in glutamate-induced compound action potential threshold shifts

Objective: Investigate the role of NO as a neurotransmitter in the gerbil cochlea and the effects of (7-NI) on compound action potential (CAP) threshold elevations induced by l-glutamate, an agonist at the NMDA glutamate receptor subtype, to further elucidate the role of NO in cochlear excitotoxicity. Method: In anesthetized gerbils, CAP thresholds were recorded before and after cochlear perfusions with a control solution of artificial perilymph (APS) and a test solution of l-glutamate (GA) in three experimental groups. Results: The control group showed no CAP threshold elevations ( p < 0.05) when APS was perfused after systemic pre-treatment with 7-NI. GA perfusion alone caused significant elevation ( p < 0.05) of the mean cochlear CAP threshold (25 dB SPL ± 5.8 dB to 78 dB SPL ± 19.5 dB). The CAP threshold elevation was prevented ( p < 0.05) when the animals were pretreated with 7-NI before GA perfusion (24 dB SPL ± 4.2 dB to 27 dB SPL ± 6.7 dB). Conclusion: NO mediates excitotoxicity when the cochlea is perfused with l-glutamate.

Therapeutic efficacy of intra-cochlear administration of methylprednisolone after acoustic trauma caused by gunshot noise in guinea pigs

The therapeutic efficacy of cochlear infusion of methylprednisolone (MP) after an impulse noise trauma (170 dB SPL peak) was evaluated in guinea pigs. The compound action potential threshold shifts were measured over a 14 days recovery period after the gunshot exposure. For each animal, one of the cochlea was perfused directly into the scala tympani with MP during 7 days via a mini-osmotic pump, whereas the other cochlea was not pump-implanted. The functional study of hearing was supplemented by histological analysis. Forty eight hours after the trauma, significant differences between auditory threshold shifts in the implanted and non-implanted ears were observed for frequencies above 8 kHz. At day 7, the difference was significant for only one frequency and no difference was observed after 14 days recovery. Cochleograms showed that the hair cell losses were significantly lower in the MP treated ears. This work indicates that direct infusion of MP into perilymphatic space accelerates hearing recovery, reduces hair cell losses after impulse noise trauma but does not limit permanent threshold shifts.

Dehydroepiandrosterone Sulfate Reduces Acoustic Injury of the Guinea-Pig Cochlea

The present study was performed to determine effects of dehydroepiandrosterone sulfate (DHEAS), a neurosteroid, on acoustic injury. Albino guinea pigs were exposed to a 2 kHz pure tone of 120 or 125 dB sound pressure level for 10 min immediately after intravenous administration of DHEAS. Statistically significant improvement in the compound action potential threshold shifts and in amplitude reduction of distortion-product otoacoustic emissions was observed 1 week after the acoustic overexposure in the animals treated with DHEAS. The present results suggest that DHEAS has a protective effect against acoustic injury of the cochlea.

Protective effects of α-tocopherol and tiopronin against cisplatin-induced ototoxicity

ObjectiveTo investigate the possible protective effects of α-tocopherol and tiopronin against cisplatin-induced cochlear damage. Cisplatin ototoxicity and nephrotoxicity seem to result from the inhibition of cochlear antioxidant defences, causing an increase in the amount of reactive oxygen species. Antioxidants, such as α-tocopherol and tiopronin, are able to suppress lipid peroxidation, thus attenuating tissue damage. Material and MethodsHartley albino guinea pigs were used. The animals were treated for 7 consecutive days with either (I) cisplatin alone, (II) cisplatin+α-tocopherol acetate, (III) cisplatin+tiopronin, (IV) cisplatin+α-tocopherol acetate+tiopronin, (V) α-tocopherol acetate alone or (VI) tiopronin alone. Changes in cochlear function were characterized by means of compound action potential threshold shifts. After the functional testing, tympanic bullae were removed and processed for morphological examination of the sensorineural epithelium. Renal function was evaluated by measuring serum blood urea nitrogen and creatinine levels. ResultsCisplatin induced progressive high-frequency hearing loss of 40–50 dB SPL. α-Tocopherol and tiopronin co-therapy significantly slowed the progression of hearing loss. Treatment with α-tocopherol acetate or tiopronin alone was less effective. Morphological observations showed an important loss of outer hair cells and degeneration of the organ of Corti in the basal and middle turns. Injection of both α-tocopherol and tiopronin reduced cochlear outer hair cell loss more than treatment with a single drug. Beneficial effects of α-tocopherol and tiopronin on cisplatin-induced nephrotoxicity were observed. Conclusion This study supports the hypothesis that α-tocopherol and tiopronin interfere with cisplatin-induced damage, and suggests that concurrent treatment with the two drugs can be useful in protecting against hearing loss.

Cisplatin ototoxicity in the guinea pig: vestibular and cochlear damage

The aim of the present study was to investigate both vestibular and cochlear cisplatin toxicity. Twelve albino guinea pigs were divided into an experimental ( n=8) and a control saline group ( n=4) and were treated with cisplatin at a daily dose of 2.5 mg/kg for 6 consecutive days. Vestibular dysfunction was evaluated by computing the gain of the vestibular ocular reflex (VOR) evoked by stimulation in the horizontal (HVOR) and vertical (VVOR) planes. Changes in cochlear function were characterised as compound action potential threshold shifts. After the functional testing, tympanic bullae were removed and processed for morphological examination of the sensorineural epithelium. The onset of vestibular functional impairment was observed on the third day, although the VOR gain decrease was not significant. The impairment of the vestibular function progressed until the sixth day becoming statistically significant particularly at VVOR mid frequencies of stimulation. At these frequencies both macula and crista ampullaris functions are involved. Concomitantly a progressive auditory threshold shift was observed at all stimulus frequencies. The decline of the auditory function was statistically significant from the third day of treatment and it was more evident at high frequencies. Morphological observations showed a massive loss of outer hair cells and a degeneration of the organ of Corti in the basal/middle turns and only a slight loss of hair cells of the cristae ampullares and maculae. In conclusion, functional and morphological data provide evidence that the toxic effect of cisplatin is more pronounced in the organ of Corti than in the vestibular epithelium.

Effect of ketamine, dextromethorphan, and MK-801 on cochlear dysfunction induced by transient ischemia.

Overstimulation of the N-methyl-D-aspartate (NMDA) glutamate receptor has been implicated as a factor in the pathogenesis of hypoxic-ischemic injury in the central nervous system. To evaluate the role played by NMDA antagonists in ischemia-reperfusion injury of the cochlea, 3 noncompetitive NMDA antagonists--ketamine, dextromethorphan, and MK-801--were administered to 53 albino guinea pigs subjected to transient ischemia of 30 minutes' duration, and the threshold shifts of the compound action potential were compared with those of nontreated animals 4 hours after the onset of recirculation. Ketamine and dextromethorphan moderately ameliorated the compound action potential threshold shifts, whereas MK-801, the most potent NMDA receptor antagonist among these 3 agents, did not show any protective effect. These results indicate that the action antagonizing the NMDA receptor has no protective effect against ischemia-reperfusion injury of the cochlea, and that ketamine and dextromethorphan act as protective agents for the cochlea via other pathways.

Dopamine increases excitability of pyramidal neurons in primate prefrontal cortex.

Dopaminergic modulation of neuronal networks in the dorsolateral prefrontal cortex (PFC) is believed to play an important role in information processing during working memory tasks in both humans and nonhuman primates. To understand the basic cellular mechanisms that underlie these actions of dopamine (DA), we have investigated the influence of DA on the cellular properties of layer 3 pyramidal cells in area 46 of the macaque monkey PFC. Intracellular voltage recordings were obtained with sharp and whole cell patch-clamp electrodes in a PFC brain-slice preparation. All of the recorded neurons in layer 3 (n = 86) exhibited regular spiking firing properties consistent with those of pyramidal neurons. We found that DA had no significant effects on resting membrane potential or input resistance of these cells. However DA, at concentrations as low as 0.5 microM, increased the excitability of PFC cells in response to depolarizing current steps injected at the soma. Enhanced excitability was associated with a hyperpolarizing shift in action potential threshold and a decreased first interspike interval. These effects required activation of D1-like but not D2-like receptors since they were inhibited by the D1 receptor antagonist SCH23390 (3 microM) but not significantly altered by the D2 antagonist sulpiride (2.5 microM). These results show, for the first time, that DA modulates the activity of layer 3 pyramidal neurons in area 46 of monkey dorsolateral PFC in vitro. Furthermore the results suggest that, by means of these effects alone, DA modulation would generally enhance the response of PFC pyramidal neurons to excitatory currents that reach the action potential initiation site.

ROMK1 (Kir1.1) causes apoptosis and chronic silencing of hippocampal neurons.

Lentiviral vectors were constructed to express the weakly rectifying kidney K(+) channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2-4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K(+) loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at -144 mV in 5.4 mM external K(+) blockable by 500 microM Ba(2+), ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba(2+)-induced depolarization of 7 +/- 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 +/- 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K(+) has implications for the use of K(+) channels in gene therapy.

Tetramethylammonium for in vivo marking of the cross-sectional area of the scala media in the guinea pig cochlea.

A physiologic technique was developed to measure endolymphatic cross-sectional area in vivo using tetramethylammonium (TMA) as a volume marker. The technique was evaluated in guinea pigs as an animal model. In the method, the cochlea was exposed surgically and TMA was injected into endolymph of the second turn at a constant rate by iontophoresis. The concentration of TMA was monitored during and after the injection using ion-selective electrodes. Cross-section estimates derived from the TMA concentration measurements were compared in normal animals and animals in which endolymphatic hydrops had been induced by ablation of the endolymphatic duct and sac 8 weeks earlier. The method demonstrated a mean increase in cross-sectional area of 258% in the hydropic group. Individually measured area values were compared with action potential threshold shifts and the magnitude of the endocochlear potential (EP). Hydropic animals typically showed an increase in threshold to 2 kHz stimuli and a decrease in EP. However, the degree of threshold shift or EP decrease did not correlate well with the degree of hydrops present.

Hearing Loss in Experimental Cytomegalovirus Infection of the Guinea Pig Inner Ear: Prevention by Systemic Immunity

Guinea pig cytomegalovirus (GPCMV) has been used to establish a reproducible model of viral labyrinthitis and hearing loss. Cochlear function was assessed by electrophysiological recordings of cochlear microphonic (CM) and eighth nerve N1 compound action potential (AP) thresholds prior to and up to eight days following inoculation of the scala tympani. Inner ear inoculation of seronegative subjects with live GPCMV produced profound elevations in CM and AP thresholds: 70% of these subjects had their thresholds raised to the limits of the sound system throughout the tested frequency range of 0.10 to 32 kHz. Histopathologic effects associated with CM and AP threshold shifts were primarily limited to the perilabyrinthine compartment, and were greatest in the most basal cochlear turns. Systemic infection with GPCMV produced an immune response, but did not affect CM or AP thresholds. Subsequent inoculation of the inner ear of these seropositive animals with live GPCMV did not result in either CM or AP threshold shifts, or cochlear histopathology. Inoculations of inactivated virus into the inner ears of seronegative and seropositive animals produced only moderate CM and AP threshold effects. Primary GPCMV labyrinthitis thus results in significant cochlear dysfunction and histopathologic changes which are prevented by prior systemic infection with GPCMV.