Background:
Inflammation-induced sensitization of primary afferents is associated with a decrease in K
+
current. However, the type of K
+
current and basis for the decrease varies as a function of ...target of innervation. Because glabrous skin of the rat hindpaw is used often to assess changes in nociception in models of persistent pain, the purpose of the present study was to determine the type and extent to which K
+
currents contribute to the inflammation-induced sensitization of cutaneous afferents. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion neurons from naïve and inflamed (3 days post complete Freund's adjuvant injection) rats were studied with whole cell and perforated patch techniques.
Results:
Inflammation-induced sensitization of small diameter cutaneous neurons was associated with an increase in action potential duration and rate of decay of the afterhyperpolarization. However, no changes in voltage-gated K
+
currents were detected. In contrast, Ca
2+
modulated iberiotoxin sensitive and paxilline sensitive K
+
(BK
Ca
) currents were significantly smaller in small diameter IB4+ neurons. This decrease in current was not associated with a detectable change in total protein levels of the BK
Ca
channel α or β subunits. Single cell PCR analysis revealed a significant change in the pattern of expression of α subunit splice variants and β subunits that were consistent, at least in part, with inflammation-induced changes in the biophysical properties of BK
Ca
currents in cutaneous neurons.
Conclusions:
Results of this study provide additional support for the conclusion that it may be possible, if not necessary to selectively treat pain arising from specific body regions. Because a decrease in BK
Ca
current appears to contribute to the inflammation-induced sensitization of cutaneous afferents, BK
Ca
channel openers may be effective for the treatment of inflammatory pain.
Summary
Purpose: γ‐Aminobutyric acid (GABA)ergic transmission plays an important role in the initiation of epileptic activity and the generation of ictal discharges. The functional alterations in ...the epileptiform hippocampus critically depend on GABAergic mechanisms and cation‐chloride cotransporters.
Methods: To understand the cellular basis of specific functional alterations in the epileptic hippocampus, we studied physiologic characteristics and pharmacologically isolated evoked GABAA receptor–mediated inhibitory postsynaptic currents (IPSCs) recorded from principal neurons in hippocampal slices from status epilepticus (SE) and control rats using whole‐cell and gramicidin perforated patch‐clamp recordings.
Key Findings: Whereas the resting membrane potential and input resistance were not significantly different between control and epileptic tissue, the reversal potential (EGABA) of IPSCs was significantly shifted to more positive values in SE rats with regard to the resting membrane potential. Pharmacologic experiments and quantitative reverse transcriptase polymerase chain reaction (RT‐PCR) showed that the observed changes in the epileptic tissue were due to a decreased ratio of the main Cl− extrusion transporter (K+‐Cl− cotransporter, KCC2) to the main Cl− uptake transporter (Na+‐K+‐2Cl− cotransporter, NKCC1).
Significance: Our results suggest that alterations of cation‐chloride cotransporter functions, comprising a higher NKCC1 action, contribute to hyperexcitability within the hippocampus following SE.
GABA exerts excitatory actions on embryonic and neonatal cortical neurons, but the in vivo function of this GABA excitation is essentially unknown. Using in utero electroporation, we eliminated the ...excitatory action of GABA in a subpopulation of rat ventricular progenitors and cortical neurons derived from these progenitors by premature expression of the Cl- transporter KCC2, as confirmed by the changes in the reversal potential of GABA-induced currents and the resting membrane potential after GABA(A) receptor blockade. We found that radial migration to layer II/III of the somatosensory cortex of neurons derived from the transfected progenitors was not significantly affected, but their morphological maturation was markedly impaired. Furthermore, reducing neuronal excitability of cortical neurons in vivo by overexpressing an inward-rectifying K+ channel, which lowered the resting membrane potential, mimicked the effect of premature KCC2 expression. Thus, membrane depolarization caused by early GABA excitation is critical for morphological maturation of neonatal cortical neurons in vivo.
One of the signs of Alzheimer's disease (AD) is the formation of β-amyloid plaques, which ultimately lead to the dysfunction of neurons with subsequent neurodegeneration. Although extensive ...researches have been conducted on the effects of different amyloid conformations such as oligomers and fibrils on neuronal function in isolated cells and circuits, the exact contribution of extracellular beta-amyloid on neurons remains incompletely comprehended. In our experiments, we studied the effect of β-amyloid peptide (Aβ1-42) on the action potential (APs) generation in isolated CA1 hippocampal neurons in perforated patch clamp conditions. Our findings demonstrate that Aβ1-42 affects the generation of APs differently in various hippocampal neurons, albeit with a shared effect of enhancing the firing response of the neurons within a minute of the start of Aβ1-42 application. In the first response type, there was a shift of 20-65% toward smaller values in the firing threshold of action potentials in response to inward current. Conversely, the firing threshold of action potentials was not affected in the second type of response to the application of Aβ1-42. In these neurons, Aβ1-42 caused a moderate increase in the frequency of spiking, up to 15%, with a relatively uniform increase in the frequency of action potentials generation regardless of the level of input current. Obtained data prove the absence of direct short-term negative effect of the Aβ1-42 on APs generation in neurons. Even with increasing the APs generation frequency and lowering the neurons' activation threshold, neurons were functional. Obtained data can suggest that only the long-acting presence of the Aβ1-42 in the cell environment can cause neuronal dysfunction due to a prolonged increase of APs firing and predisposition to this process.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. ...Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (
) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in "ventricular-like" and "atrial-like" hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial
(computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic
through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon
injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic
. In conclusion, the dynamic clamp-based approach of
injection has broad applicability for detailed AP measurements in hiPSC-CMs.
Intracellular signaling pathways directly and indirectly regulate neuronal activity. In cellular electrophysiological measurements with sharp electrodes or whole-cell patch clamp recordings, there is ...a great risk that these signaling pathways are disturbed, significantly altering the electrophysiological properties of the measured neurons. Perforated-patch clamp recordings circumvent this issue, allowing long-term electrophysiological recordings with minimized impairment of the intracellular milieu. Based on previous studies, we describe a superstition-free protocol that can be used to routinely perform perforated patch clamp recordings for current and voltage measurements.
The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying ...mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 μM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.
•Histamine depolarized rat intracardiac ganglion neurons via histamine H1 receptors.•Histamine inhibited M-type K+ channels and activated TRPC cation channels.•The histamine-induced current was mediated by intracellular Ca2+ release.•The histamine-induced depolarization depended largely on TRPC channel activation.•Intracardiac neurons may participate in the cardiovascular action of histamine.
Abstract
Background
Intracellular Ca
2+
modulates several microglial activities, such as proliferation, migration, phagocytosis, and inflammatory mediator secretion. Extracellular ATP, the levels of ...which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca
2+
concentration (Ca
2+
i
). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca
2+
-mediated response triggered by purinergic signaling.
Methods
Fura-2 based fluorescence microscopy was used to measure Ca
2+
i
in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca
2+
-dependent whole-cell currents.
Results
In human microglia extracellular ATP evoked Ca
2+
i
increases depend on Ca
2+
entry from the extracellular space and on Ca
2+
mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when Ca
2+
i
increases were prevented by removing external Ca
2+
and using an intracellular Ca
2+
chelator. TRAM-34, a selective K
Ca
3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl
−
in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca
2+
transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca
2+
mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca
2+
-evoked K
+
current led to a less sustained ATP-evoked Ca
2+
entry, as shown by the faster Ca
2+
transient kinetics observed in LPS-treated microglia.
Conclusions
Our study confirms a functional role for K
Ca
3.1 channels in human microglia, linking ATP-evoked Ca
2+
transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the K
Ca
3.1-mediated microglial response to purinergic signaling may be reduced.
The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical ...access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca
), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.
Changes in chloride reversal potential in rat spinal cord neurons have previously been associated with persistent pain in nerve injury and inflammation models. These changes correlate with a decrease ...in the expression of the potassium chloride transporter, KCC2, and with increases in neuronal excitability. Here, we test the hypothesis that similar changes occur in mice with neuropathic pain induced by chronic constriction injury of the trigeminal infraorbital nerve (CCI-ION). This model allows us to distinguish an acute pain phase (3-5 days after injury) from a persistent pain phase (12-14 days after CCI-ION). Chronic constriction injury of the trigeminal infraorbital nerve induced significant decreases in mechanical pain thresholds in both the acute and persistent phases. To estimate GABAA reversal potentials in neurons from trigeminal nucleus caudalis, we obtained perforated patch recordings in vitro. GABAA reversal potential decreased by 8% during the acute phase in unidentified neurons, but not in GABAergic interneurons. However, at 12 to 14 days after CCI-ION, GABAA reversal potential recovered to normal values. Quantitative real-time polymerase chain reaction analysis revealed no significant changes, at either 3 to 5 days or 12 to 14 days after CCI-ION, in either KCC2 or NKCC1. These findings suggest that CCI-ION in mice results in transient and modest changes in chloride reversal potentials, and that these changes may not persist during the late phase. This suggests that, in the mouse model of CCI-ION, chloride dysregulation may not have a prominent role in the central mechanisms leading to the maintenance of chronic pain.
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