Whole-cell Current Clamp Research Articles

Measuring Feedforward Inhibition and Its Impact on Local Circuit Function.

This protocol describes a series of approaches to measure feedforward inhibition in acute brain slices from the cerebellar cortex. Using whole-cell voltage and current clamp recordings from Purkinje cells in conjunction with electrical stimulation of the parallel fibers, these methods demonstrate how to measure the relationship between excitation and inhibition in a feedforward circuit. This protocol also describes how to measure the impact of feedforward inhibition on Purkinje cell excitability, with an emphasis on spike timing.

The effect of IL-1β on synaptophysin expression and electrophysiology of hippocampal neurons through the PI3K/Akt/mTOR signaling pathway in a rat model of mesial temporal lobe epilepsy

Background: The inflammation induced by interleukin-1β (IL-1β) is a critical factor in the pathogenesis of mesial temporal lobe epilepsy (MTLE). Synaptophysin (SYN) and other changes, including neuron electrophysiology, participate in the pathophysiological processes of MTLE. Phosphatidylinositol 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling pathway may play a critical role in regulating SYN expression and electrophysiology of hippocampal neurons.Methods: We used lithium-pilocarpine-treated rats as model of human MTLE, detecting epileptic seizures with digital video-EEG, and evaluating the proteins related to the PI3K/Akt/mTOR signaling pathway by western blot (WB). Then, we cultured primary neuron and established a neuronal epilepsy model using Mg2+-free media. Immunocytochemistry and WB were used to investigate SYN expression, and whole-cell current clamp recording techniques were used to detect the electrophysiological properties of cultured neurons.Results: We have demonstrated that IL-1β can activate the PI3K/Akt/mTOR signaling pathway in primary hippocampal neurons, and we speculate that IL-1β may affect SYN expression and neuron electrophysiology through PI3K/Akt/mTOR signaling pathway.Conclusion: We confirmed that IL-1β stimulated SYN expression and epileptiform discharges, and that blocking the PI3K/Akt/mTOR pathway alleviated these phenomena. Therefore, activation of the PI3K/Akt/mTOR signaling pathway by IL-1β contributes to the pathogenesis of MTLE, and modulating this pathway is a promising strategy of study for therapies to prevent or reverse the cellular and molecular mechanisms of epileptogenesis in MTLE.

Distinct Neural Properties in the Low-Frequency Region of the Chicken Cochlear Nucleus Magnocellularis.

Topography in the avian cochlear nucleus magnocellularis (NM) is represented as gradually increasing characteristic frequency (CF) along the caudolateral-to-rostromedial axis. In this study, we characterized the organization and cell biophysics of the caudolateral NM (NMc) in chickens (Gallus gallus). Examination of cellular and dendritic architecture first revealed that NMc contains small neurons and extensive dendritic processes, in contrast to adendritic, large neurons located more rostromedially. Individual dye-filling study further demonstrated that NMc is divided into two subregions, with NMc2 neurons having larger and more complex dendritic fields than NMc1. Axonal tract tracing studies confirmed that NMc1 and NMc2 neurons receive afferent inputs from the auditory nerve and the superior olivary nucleus, similar to the adendritic NM. However, the auditory axons synapse with NMc neurons via small bouton-like terminals, unlike the large end bulb synapses on adendritic NM neurons. Immunocytochemistry demonstrated that most NMc2 neurons express cholecystokinin but not calretinin, distinct from NMc1 and adendritic NM neurons that are cholecystokinin negative and mostly calretinin positive. Finally, whole-cell current clamp recordings revealed that NMc neurons require significantly lower threshold current for action potential generation than adendritic NM neurons. Moreover, in contrast to adendritic NM neurons that generate a single-onset action potential, NMc neurons generate multiple action potentials to suprathreshold sustained depolarization. Taken together, our data indicate that NMc contains multiple neuron types that are structurally, connectively, molecularly, and physiologically different from traditionally defined NM neurons, emphasizing specialized neural properties for processing low-frequency sounds.

Muscarinic Receptors Are Responsible for the Cholinergic Modulation of Projection Neurons in the Song Production Brain Nucleus RA of Zebra Finches.

Songbirds are a useful model for the study of learned vocal behavior in vertebrates. The robust nucleus of the arcopallium (RA) is a premotor nucleus in the vocal motor pathway. It receives excitatory synaptic inputs from the anterior forebrain pathway. RA also receives cholinergic inputs from the ventral paleostriatum of the basal forebrain. Our previous study showed that carbachol, a non-selective cholinergic receptor agonist, modulates the electrophysiology of RA projection neurons (PNs), indicating that cholinergic modulation of RA may play an important role in song production. However, the receptor mechanisms underlying these effects are poorly understood. In the present study, we investigated the electrophysiological properties of two acetylcholine receptors on the RA PNs of adult male zebra finches using in vitro whole-cell current clamp. Our results demonstrate that activation of muscarinic acetylcholine receptors (mAChRs) simulate the effects of carbachol. Both carbachol and the mAChR agonist muscarine produced a decrease in the excitability of RA PNs and a hyperpolarization of the membrane potential. The mAChR antagonist atropine blocked the effects of carbachol. Activation of nicotinic acetylcholine receptors (nAChRs) with nAChR agonist nicotine or DMPP had no effect on the excitability of RA PNs, and the nAChR antagonist mecamylamine failed to inhibit the effects of carbachol. These results suggest that mAChRs, but not nAChRs, primarily modulate the effects of carbachol on the activity of RA PNs. Collectively, these findings contribute to our understanding of the mechanism of cholinergic modulation in the vocal nuclei of songbirds.

Engineering thiophene-based nanoparticles to induce phototransduction in live cells under illumination.

We report that nanoparticles prepared from appropriately functionalized polythiophenes once administered to live cells can acquire phototransduction properties under illumination, becoming photoactive sites able to absorb visible light and convert it to an electrical signal through cell membrane polarization. Amine-reactive fluorescent nanoparticles with pendant N-succinimidyl-ester groups (NPs-NHS) are prepared from polythiophenes alternating unsubstituted and 3-(2,5-dioxopyrrolidin-1-yl-8-octanoate)-substituted thiophenes by a nanoprecipitation method. By 1H NMR of nanoparticles prepared using THF-d8/D2O (solvent/non-solvent) we demonstrate that the hydrolysis of the N-succinimidyl-ester group to free N-hydroxysuccinimide takes place slowly over several hours. NPs-NHS reactivity towards primary amine groups is tested towards the NH2 of d- and l-enantiomers of tryptophan. We show that the formation of a tryptophan-nanoparticle amidic bond creates a chiral shell displaying opposite CD signals for the nanoparticles bound to d or l enantiomers. The interaction of NPs-NHS with live HEK-293 cells is monitored via LSCM. We show that the NPs-NHS are not internalized but remain docked on the cell membrane. We assume that this is mainly the result of the reaction of the NHS groups in the external layer with NH2 groups present in cell membrane proteins, although the contribution of alternative mechanisms cannot be excluded. To support this assumption LSCM experiments show that nanoparticles of comparable size obtained from poly(3-hexylthiophene), NPs-P3HT, are rapidly internalized by live HEK-293 cells. Finally, using the whole-cell current clamp technique under light illumination we demonstrate that NPs-NHS can polarize the cell membrane upon light irradiation while NPs-P3HT cannot.

Heantos-4, a natural plant extract used in the treatment of drug addiction, modulates T-type calcium channels and thalamocortical burst-firing.

Heantos-4 is a refined combination of plant extracts currently approved to treat opiate addiction in Vietnam. In addition to its beneficial effects on withdrawal and prevention of relapse, reports of sedation during clinical treatment suggest that arousal networks in the brain may be recruited during Heantos administration. T-type calcium channels are implicated in the generation of sleep rhythms and in this study we examined whether a Heantos-4 extraction modulates T-type calcium channel currents generated by the Cav3.1, Cav3.2 and Ca3.3 subtypes. Utilizing whole-cell voltage clamp on exogenously expressed T-type calcium channels we find that Heantos inhibits Cav3.1 and Cav3.3 currents, while selectively potentiating Cav3.2 currents. We further examined the effects of Heantos-4 extract on low-threshold burst-firing in thalamic neurons which contribute to sleep oscillations. Using whole-cell current clamp in acute thalamic brain slices Heantos-4 suppressed rebound burst-firing in ventrobasal thalamocortical neurons, which express primarily Cav3.1 channels. Conversely, Heantos-4 had no significant effect on the burst-firing properties of thalamic reticular neurons, which express a mixed population of Cav3.2 and Cav3.3 channels. Examining Heantos-4 effects following oral administration in a model of absence epilepsy revealed the potential to exacerbate seizure activity. Together, the findings indicate that Heantos-4 has selective effects both on specific T-type calcium channel isoforms and distinct populations of thalamic neurons providing a putative mechanism underlying its effects on sedation and on the thalamocortical network.

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na+ currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance.

Aortic Baroreceptors Display Higher Mechanosensitivity than Carotid Baroreceptors.

Arterial baroreceptors are mechanical sensors that detect blood pressure changes. It has long been suggested that the two arterial baroreceptors, aortic and carotid baroreceptors, have different pressure sensitivities. However, there is no consensus as to which of the arterial baroreceptors are more sensitive to changes in blood pressure. In the present study, we employed independent methods to compare the pressure sensitivity of the two arterial baroreceptors. Firstly, pressure-activated action potential firing was measured by whole-cell current clamp with a high-speed pressure clamp system in primary cultured baroreceptor neurons. The results show that aortic depressor neurons possessed a higher percentage of mechano-sensitive neurons. Furthermore, aortic baroreceptor neurons show a lower pressure threshold than that of carotid baroreceptor neurons. Secondly, uniaxial stretching of baroreceptor neurons, that mimics the forces exerted on blood vessels, elicited a larger increase in intracellular Ca2+ rise in aortic baroreceptor neurons than in carotid baroreceptor neurons. Thirdly, the pressure-induced action potential firing in the aortic depressor nerve recorded in vivo was also higher. The present study therefore provides for a basic physiological understanding on the pressure sensitivity of the two baroreceptor neurons and suggests that aortic baroreceptors have a higher pressure sensitivity than carotid baroreceptors.

Low-intensity repetitive magnetic stimulation lowers action potential threshold and increases spike firing in layer 5 pyramidal neurons in vitro

Repetitive transcranial magnetic stimulation (rTMS) has become a popular method of modulating neural plasticity in humans. Clinically, rTMS is delivered at high intensities to modulate neuronal excitability. While the high-intensity magnetic field can be targeted to stimulate specific cortical regions, areas adjacent to the targeted area receive stimulation at a lower intensity and may contribute to the overall plasticity induced by rTMS. We have previously shown that low-intensity rTMS induces molecular and structural plasticity in vivo, but the effects on membrane properties and neural excitability have not been investigated. Here we investigated the acute effect of low-intensity repetitive magnetic stimulation (LI-rMS) on neuronal excitability and potential changes on the passive and active electrophysiological properties of layer 5 pyramidal neurons in vitro. Whole-cell current clamp recordings were made at baseline prior to subthreshold LI-rMS (600 pulses of iTBS, n=9 cells from 7 animals) or sham (n=10 cells from 9 animals), immediately after stimulation, as well as 10 and 20min post-stimulation. Our results show that LI-rMS does not alter passive membrane properties (resting membrane potential and input resistance) but hyperpolarises action potential threshold and increases evoked spike-firing frequency. Increases in spike firing frequency were present throughout the 20min post-stimulation whereas action potential (AP) threshold hyperpolarization was present immediately after stimulation and at 20min post-stimulation. These results provide evidence that LI-rMS alters neuronal excitability of excitatory neurons. We suggest that regions outside the targeted region of high-intensity rTMS are susceptible to neuromodulation and may contribute to rTMS-induced plasticity.

RETRACTED: Increased Activation of the TRESK K+ Mediates Vago-Vagal Reflex Malfunction in Diabetic Rats

Patients with diabetes have defects in the vagal afferent pathway that result in abnormal gastrointestinal function. We investigated whether selective increased activation of the 2-pore domain potassium channel TRESK (2-pore-domain weak inward-rectifying potassium channel-related spinal cord potassium channel) contributes to nodose ganglia (NG) malfunction, disrupting gastrointestinal function in diabetic rats. We conducted whole-cell current-clamp and single-unit recordings in NG neurons from diabetes-prone BioBreeding/Worcester rats and streptozotocin-induced diabetic (STZ-D) rats and compared them with control rats. NG neurons in rats or cultured NG neurons were exposed to pharmacologic antagonists and/or transfected with short hairpin or small interfering RNAs that reduced expression of TRESK. We then made electrophysiologic recordings and studied gastrointestinal functions. We observed reduced input resistance, hyperpolarized membrane potential, and increased current threshold to elicit action potentiation in NG neurons of STZ-D rats compared with controls. NG neuron excitability was similarly altered in diabetes-prone rats. Invivo single-unit NG neuronal discharges in response to 30 and 60 pmol cholecystokinin octapeptide were significantly lower in STZ-D rats compared with controls. Reducing expression of the TRESK K+ channel restored NG excitability invitro and invivo, as well as cholecystokinin 8-stimulated secretion of pancreatic enzymes and secretin-induced gastrointestinal motility, which are mediated by vago-vagal reflexes. These abnormalities resulted from increased intracellular Ca2+ in the NG, activating calcineurin, which, in turn, bound to an nuclear factor of activated T cell-like docking site on the TRESK protein, resulting in neuronal membrane hyperpolarization. In 2 rate models of diabetes, we found that activation of the TRESK K+ channel reduced NG excitability and disrupted gastrointestinal functions.

Atorvastatin enhances kainate-induced gamma oscillations in rat hippocampal slices.

Atorvastatin has been shown to affect cognitive functions in rodents and humans. However, the underlying mechanism is not fully understood. Because hippocampal gamma oscillations (γ, 20-80Hz) are associated with cognitive functions, we studied the effect of atorvastatin on persistent kainate-induced γ oscillation in the CA3 area of rat hippocampal slices. The involvement of NMDA receptors and multiple kinases was tested before and after administration of atorvastatin. Whole-cell current-clamp and voltage-clamp recordings were made from CA3 pyramidal neurons and interneurons before and after atorvastatin application. Atorvastatin increased γ power by ~50% in a concentration-dependent manner, without affecting dominant frequency. Whereas atorvastatin did not affect intrinsic properties of both pyramidal neurons and interneurons, it increased the firing frequency of interneurons but not that of pyramidal neurons. Furthermore, whereas atorvastatin did not affect synaptic current amplitude, it increased the frequency of spontaneous inhibitory post-synaptic currents, but did not affect the frequency of spontaneous excitatory post-synaptic currents. The atorvastatin-induced enhancement of γ oscillations was prevented by pretreatment with the PKA inhibitor H89, the ERK inhibitor U0126, or the PI3K inhibitor wortmanin, but not by the NMDA receptor antagonist D-AP5. Taken together, these results demonstrate that atorvastatin enhanced the kainate-induced γ oscillation by increasing interneuron excitability, with an involvement of multiple intracellular kinase pathways. Our study suggests that the classical cholesterol-lowering agent atorvastatin may improve cognitive functions compromised in disease, via the enhancement of hippocampal γ oscillations.

Characterization of Rett Syndrome-like phenotypes in Mecp2-knockout rats.

BackgroundRett Syndrome (RTT) is a neurodevelopmental disease caused by the disruption of the MECP2 gene. Several mouse models of RTT have been developed with Mecp2 disruptions. Although the mouse models are widely used in RTT research, results obtained need to be validated in other species. Therefore, we performed these studies to characterize phenotypes of a novel Mecp2−/Y rat model and compared them with the Mecp2tm1.1Bird mouse model of RTT.MethodsRTT-like phenotypes were systematically studied and compared between Mecp2−/Y rats and Mecp2−/Y mice. In-cage conditions of the rats were monitored. Grip strength and spontaneous locomotion were used to evaluate the motor function. Three-chamber test was performed to show autism-type behaviors. Breathing activity was recorded with the plethysmograph. Individual neurons in the locus coeruleus (LC) were studied in the whole-cell current clamp. The lifespan of the rats was determined with their survival time.ResultsMecp2−/Y rats displayed growth retardation, malocclusion, and lack of movements, while hindlimb clasping was not seen. They had weaker forelimb grip strength and a lower rate of locomotion than the WT littermates. Defects in social interaction with other rats were obvious. Breathing frequency variation and apnea in the null rats were significantly higher than in the WT. LC neurons in the null rats showed excessive firing activity. A half of the null rats died in 2 months. Most of the RTT-like symptoms were comparable to those seen in Mecp2−/Y mice, while some appeared more or less severe. The findings that most RTT-like symptoms exist in the rat model with moderate variations and differences from the mouse models support the usefulness of both Mecp2−/Y rodent models.ConclusionsThe novel Mecp2−/Y rat model recapitulated numerous RTT-like symptoms as Mecp2−/Y mouse models did, which makes it a valuable alternative model in the RTT studies when the body size matters.

L-Phosphinothricin modulation of inwardly rectifying K(+) channels increased excitability in striatal medium-sized spiny neurons.

Phosphinotricin (L-PPT) is the active compound of a broad-spectrum herbicide. Acute poisoning with L-PPT has various clinical manifestations, including seizures and convulsions. However, the exact mechanism of L-PPT toxicity remains unclear. The present study addressed the role of L-PPT, in the excitability of striatal medium-sized spiny neurons (MSNs). In whole-cell current-clamp experiments, L-PPT increased the input resistance (Ri), decreased the rheobase and increased the firing frequency of action potentials. In voltage-clamp experiments, L-PPT inhibited the inward-rectifying potassium (Kir) currents. Finally, the effects of L-PPT mimicked the inhibition of Kir channels with Ba(2+) on neuronal excitability. Altogether, these results suggest that the herbicide L-PPT is a modulator of Kir channels in MSNs. Thereby, Kir channels are potent regulators of the excitability of MSNs and reduced open probability of these channels would generate a powerful upregulation of neuronal output. This effect may represent a possible mechanism for L-PPT dependent neuronal toxicity.

Olanzapine Treatment of Adolescent Rats Alters Adult D2 Modulation of Cortical Inputs to the Ventral Striatum.

Background:The striatal dopamine system undergoes vast ontogenetic changes during adolescence, making the brain vulnerable to drug treatments that target this class of neurotransmitters. Atypical antipsychotic drugs are often prescribed to children and adolescents for off-label treatment of neuropsychiatric disorders, yet the long-term impact this treatment has on brain development remains largely unknown.Methods:Adolescent male rats were treated with olanzapine or vehicle for 3 weeks (during postnatal day 28–49) using a dosing condition designed to approximate closely D2 receptor occupancies in the human therapeutic range. We assessed D2 receptor modulation of corticostriatal inputs onto medium spiny neurons in the adult ventral striatum using in vitro whole-cell current clamp recordings.Results:The D2/D3 agonist quinpirole (5 µM) enhanced cortically driven medium spiny neuron synaptic responses in slices taken from adult rats treated with vehicle during adolescence, as in untreated adult rats. However, in slices from mature rats treated with olanzapine during adolescence, quinpirole reduced medium spiny neuron activation. The magnitude of decrease was similar to previous observations in untreated, prepubertal rats. These changes may reflect alterations in local inhibitory circuitry, as the GABA-A antagonist picrotoxin (100 µM) reversed the effects of quinpirole in vehicle-treated slices but had no impact on cortically evoked responses in olanzapine-treated slices.Conclusions:These data suggest that adolescent atypical antipsychotic drug treatment leads to enduring changes in dopamine modulation of corticostriatal synaptic function.

MicroRNA-204 suppresses epileptiform discharges through regulating TrkB-ERK1/2-CREB signaling in cultured hippocampal neurons

MicroRNAs (miRs) have been increasingly recognized as post-transcriptional regulators involved in the pathogenesis of epilepsy. Extensive evidence suggests that inhibition of tropomyosin related kinase type B (TrkB) signaling limits epileptogenesis. It has been reported that miR-204 directly targeted and downregulated TrkB protein in a variety of cancers to suppress tumorigenesis, but its effect on epileptogenesis is unknown. Here we showed that miR-204 was downregulated in cultured hippocampal neurons following status epilepticus (SE) induced by Mg2+-free treatment. Overexpression of miR-204 using a transfection technique negatively regulated the expression of TrkB mRNA. Insight into the downstream signaling pathway activated by TrkB may provide valuable clues to the underlying mechanisms of epileptogenesis. Biochemical measures revealed activation of phospholipase Cγ1 (PLCγ1), extracellular signal-regulated kinase1/2 (ERK1/2) and cAMP response-element binding protein (CREB) signaling molecules in the Mg2+-free model in hippocampal neurons, of which ERK1/2-CREB but not PLCγ1 signaling pathway was able to be inhibited by miR-204. Epileptiform discharges assessed using whole-cell current–clamp techniques were suppressed by miR-204 in cultured hippocampus neurons of SE. These results suggest that miR-204 exerts an antiepileptogenic effect in the Mg2+-free model in hippocampal neurons through regulating TrkB and downstream ERK1/2-CREB signaling pathway.

Expanding neurochemical investigations with multi-modal recording: simultaneous fast-scan cyclic voltammetry, iontophoresis, and patch clamp measurements.

Multi-modal recording describes the simultaneous collection of information across distinct domains. Compared to isolated measurements, such studies can more easily determine relationships between varieties of phenomena. This is useful for neurochemical investigations which examine cellular activity in response to changes in the local chemical environment. In this study, we demonstrate a method to perform simultaneous patch clamp measurements with fast-scan cyclic voltammetry (FSCV) using optically isolated instrumentation. A model circuit simulating concurrent measurements was used to predict the electrical interference between instruments. No significant impact was anticipated between methods, and predictions were largely confirmed experimentally. One exception was due to capacitive coupling of the FSCV potential waveform into the patch clamp amplifier. However, capacitive transients measured in whole-cell current clamp recordings were well below the level of biological signals, which allowed the activity of cells to be easily determined. Next, the activity of medium spiny neurons (MSNs) was examined in the presence of an FSCV electrode to determine how the exogenous potential impacted nearby cells. The activities of both resting and active MSNs were unaffected by the FSCV waveform. Additionally, application of an iontophoretic current, used to locally deliver drugs and other neurochemicals, did not affect neighboring cells. Finally, MSN activity was monitored during iontophoretic delivery of glutamate, an excitatory neurotransmitter. Membrane depolarization and cell firing were observed concurrently with chemical changes around the cell resulting from delivery. In all, we show how combined electrophysiological and electrochemical measurements can relate information between domains and increase the power of neurochemical investigations.

Altered intrinsic excitability of hippocampal CA1 pyramidal neurons in aged PDAPP mice.

Amyloidopathy involves the accumulation of insoluble amyloid β (Aβ) species in the brain’s parenchyma and is a key histopathological hallmark of Alzheimer’s disease (AD). Work on transgenic mice that overexpress Aβ suggests that elevated Aβ levels in the brain are associated with aberrant epileptiform activity and increased intrinsic excitability (IE) of CA1 hippocampal neurons. In this study we examined if similar changes could be observed in hippocampal CA1 pyramidal neurons from aged PDAPP mice (20–23 month old, Indiana mutation: V717F on APP gene) compared to their age-matched wild-type littermate controls. Whole-cell current clamp recordings revealed that sub-threshold intrinsic properties, such as input resistance, resting membrane potential and hyperpolarization activated “sag” were unaffected, but capacitance was significantly decreased in the transgenic animals. No differences between genotypes were observed in the overall number of action potentials (AP) elicited by 500 ms supra-threshold current stimuli. PDAPP neurons, however, exhibited higher instantaneous firing frequencies after accommodation in response to high intensity current injections. The AP waveform was narrower and shorter in amplitude in PDAPP mice: these changes, according to our in silico model of a CA1/3 pyramidal neuron, depended on the respective increase and reduction of K+ and Na+ voltage-gated channels maximal conductances. Finally, the after-hyperpolarization, seen after the first AP evoked by a +300 pA current injection and after 50 Hz AP bursts, was more pronounced in PDAPP mice. These data show that Aβ-overexpression in aged mice altered the capacitance, the neuronal firing and the AP waveform of CA1 pyramidal neurons. Some of these findings are consistent with previous work on younger PDAPP; they also show important differences that can be potentially ascribed to the interaction between amyloidopathy and ageing. Such a change of IE properties over time underlies that the increased incidence of seizure observed in AD patients might rely on different mechanistic pathways during progression of the disease.

Aging-Related Hyperexcitability in CA3 Pyramidal Neurons Is Mediated by Enhanced A-Type K+ Channel Function and Expression.

Aging-related impairments in hippocampus-dependent cognition have been attributed to maladaptive changes in the functional properties of pyramidal neurons within the hippocampal subregions. Much evidence has come from work on CA1 pyramidal neurons, with CA3 pyramidal neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing in the hippocampal circuit. Here, we use whole-cell current-clamp to demonstrate that aged rat (29-32 months) CA3 pyramidal neurons fire significantly more action potentials (APs) during theta-burst frequency stimulation and that this is associated with faster AP repolarization (i.e., narrower AP half-widths and enlarged fast afterhyperpolarization). Using a combination of patch-clamp physiology, pharmacology, Western blot analyses, immunohistochemistry, and array tomography, we demonstrate that these faster AP kinetics are mediated by enhanced function and expression of Kv4.2/Kv4.3 A-type K(+) channels, particularly within the perisomatic compartment, of CA3 pyramidal neurons. Thus, our study indicates that inhibition of these A-type K(+) channels can restore the intrinsic excitability properties of aged CA3 pyramidal neurons to a young-like state. Significance statement: Age-related learning deficits have been attributed, in part, to altered hippocampal pyramidal neuronal function with normal aging. Much evidence has come from work on CA1 neurons, with CA3 neurons receiving comparatively less attention despite its age-related hyperactivation being postulated to interfere with spatial processing. Hence, we conducted a series of experiments to identify the cellular mechanisms that underlie the hyperexcitability reported in the CA3 region. Contrary to CA1 neurons, we demonstrate that postburst afterhyperpolarization is not altered with aging and that aged CA3 pyramidal neurons are able to fire significantly more action potentials and that this is associated with faster action potential repolarization through enhanced expression of Kv4.2/Kv4.3 A-type K(+) channels, particularly within the cell bodies of CA3 pyramidal neurons.

Tumor necrosis factor-α enhances voltage-gated Na⁺ currents in primary culture of mouse cortical neurons.

BackgroundPrevious studies showed that TNF-α could activate voltage-gated Na+ channels (VGSCs) in the peripheral nervous system (PNS). Since TNF-α is implicated in many central nervous system (CNS) diseases, we examined potential effects of TNF-α on VGSCs in the CNS.MethodsEffects of TNF-α (1–1000 pg/mL, for 4–48 h) on VGSC currents were examined using whole-cell voltage clamp and current clamp techniques in primary culture of mouse cortical neurons. Expression of Nav1.1, Nav1.2, Nav1.3, and Nav1.6 were examined at both the mRNA and protein levels, prior to and after TNF-α exposure.ResultsTNF-α increased Na+ currents by accelerating the activation of VGSCs. The threshold for action potential (AP) was decreased and firing rate were increased. VGSCs were up-regulated at both the mRNA and protein levels. The observed effects of TNF-α on Na+ currents were inhibited by pre-incubation with the NF-κB inhibitor BAY 11–7082 (1 μM) or the p38 mitogen-activated protein kinases (MAPK) inhibitor SB203580 (1 μM).ConclusionsTNF-α increases Na+ currents by accelerating the channel activation as well as increasing the expression of VGSCs in a mechanism dependent upon NF-κB and p38 MAPK signal pathways in CNS neurons.

Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes.

Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology. The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel. The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry. A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern. Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic.