Whole-cell Patch-clamp Approach Research Articles

Acute effect of delta-9-tetrahydrocannabinol (THC) on neuromuscular transmission and locomotive behaviours in larval zebrafish.

Given the increasing trend of cannabis use for recreational and therapeutic purposes, a comprehensive examination of cannabis effects is warranted. The principal psychoactive constituent of cannabis, delta-9-tetrahydrocannabinol (THC), is a potent disrupter of neurodevelopment. Nevertheless, the impact of acute exposure to THC on developing motor systems is not well-investigated. In this study, using a neurophysiological whole-cell patch clamp approach we demonstrated that a 30 min exposure to THC can alter spontaneous synaptic activities at neuromuscular junctions of 5-day post-fertilized zebrafish. An increased frequency of synaptic activity and altered decay kinetic properties were documented in the THC-treated larvae. Locomotive behaviours, including swimming activity rate and C-start escape response to sound were also affected by THC. While the THC-treated larvae displayed hyperactivity of their basal swimming levels, their escape response rate to sound stimuli was reduced. These findings suggest that the acute exposure to THC can disrupt neuromuscular transmission and locomotor-driven responses in developing zebrafish.

Signaling pathways involved in NMDA-induced suppression of M-channels in corticotropin-releasing hormone neurons in central amygdala.

Glutamate N-methyl-d-aspartate (NMDA) receptors (NMDARs) and Kv7/M channels are importantly involved in regulating neuronal activity involved in various physiological and pathological functions. Corticotropin-releasing hormone (CRH)-expressing neurons in the central nucleus of the amygdala (CeA) critically mediate autonomic response during stress. However, the interaction between NMDA receptors and Kv7/M channels in the CRHCeA neurons remains unclear. In this study, we identified rat CRHCeA neurons through the expression of an AAV viral vector-mediated enhanced green fluorescent protein (eGFP) driven by the rat CRH promoter. M-currents carried by Kv7/M channels were recorded using the whole-cell patch-clamp approach in eGFP-tagged CRHCeA neurons in brain slices. Acute exposure to NMDA significantly reduced M-currents recorded from the CRHCeA neurons. NMDA-induced suppression of M-currents was eliminated by chelating intracellular Ca2+ , supplying phosphatidylinositol 4,5-bisphosphate (PIP2) intracellularly, or blocking phosphoinositide3-kinase (PI3K). In contrast, inhibiting protein kinase C (PKC) or calmodulin did not alter NMDA-induced suppression of M-currents. Sustained exposure of NMDA decreased Kv7.3 membrane protein levels and suppressed M-currents, while the Kv7.2 expression levels remained unaltered. Pre-treatment of brain slices with PKC inhibitors alleviated the decreases in Kv7.3 and reduction of M-currents in CRHCeA neurons induced by NMDA. PKC inhibitors did not alter Kv7.2 and Kv7.3 membrane protein levels and M-currents in CRHCeA neurons. These data suggest that transient activation of NMDARs suppresses M-currents through the Ca2+ -dependent PI3K-PIP2 signaling pathway. In contrast, sustained activation of NMDARs reduces Kv7.3 protein expression and suppresses M-currents through a PKC-dependent pathway.

Activation of NMDA receptors suppresses Kv7 channel activity in corticotropin‐releasing hormone neurons in Amygdala

Corticotropin-releasing hormone (CRH)-expressing neurons in the central nucleus of the amygdala (CeA) are critically involved in autonomic response during stress. Glutamate N-methyl-D-aspartate (NMDA) receptors and Kv7 channels regulate the neuronal activity. However, the interaction between NMDA receptors and Kv7 channels remains unclear. CeA-CRH neurons were identified by an AAV virus-mediated expressing enhanced green fluorescent protein (eGFP) driven by the rat CRH promoter. M-currents carried by Kv7 channels were recorded by whole-cell patch-clamp approach in eGFP-tagged CeA-CRH neurons and Kv7 proteins were measured by western-blot. Acute application of NMDA significantly reduced M-currents recorded from CeA-CRH neurons. NMDA-induced suppression of M-currents was eliminated by chelating intracellular Ca2+ or supplying phosphatidylinositol 4,5-bisphosphate (PIP2) intracellularly but not by inhibiting protein kinase C (PKC), calmodulin, or phosphoinositide3-kinase (PI3K). Incubation of brain slices with NMDA decreased Kv7.3 membrane protein levels and M-currents, while the Kv7.2 expression levels was not altered. Treatment brain slice with PKC inhibitor alleviated the decreases in surface Kv7.3 levels and M-currents in CeA-CRH neurons induced by NMDA incubation. PKC inhibitor alone did not alter surface Kv7 levels and M-currents in CeA-CRH neurons. These data suggest that activation of NMDA receptors suppresses Kv7 channel activity through Ca2+-dependent PIP2 signaling in acute phase and PKC-dependent pathway in chronic phase.

Mechanisms of noradrenergic modulation of synaptic transmission and neuronal excitability in ventral horn neurons of the rat spinal cord

Noradrenaline (NA) modulates the spinal motor networks for locomotion and facilitates neuroplasticity, possibly assisting neuronal network activation and neuroplasticity in the recovery phase of spinal cord injuries. However, neither the effects nor the mechanisms of NA on synaptic transmission and neuronal excitability in spinal ventral horn (VH) neurons are well characterized, especially in rats aged 7 postnatal days or older. To gain insight into NA regulation of VH neuronal activity, we used a whole-cell patch-clamp approach in late neonatal rats (postnatal day 7–15). In voltage-clamp recordings at −70 mV, NA increased the frequency and amplitude of excitatory postsynaptic currents via the activation of somatic α1- and β-adrenoceptors of presynaptic neurons. Moreover, NA induced an inward current through the activation of postsynapticα1- and β-adrenoceptors. At a holding potential of 0 mV, NA also increased frequency and amplitude of both GABAergic and glycinergic inhibitory postsynaptic currents via the activation of somatic adrenoceptors in presynaptic neurons. In current-clamp recordings, NA depolarized resting membrane potentials and increased the firing frequency of action potentials in VH neurons, indicating that it enhances the excitability of these neurons. Our findings provide new insights that establish NA-based pharmacological therapy as an effective method to activate neuronal networks of the spinal VH in the recovery phase of spinal cord injuries.

Voltage-Dependent Interaction of Capsaicine and Protons on TRPV1-Receptors

The interaction of TRPV1-receptors agonists (capsaicin and protons) has been studied on cultured CHO cells transfected by TRPV1-receptors. Using the whole-cell patch-clamp approach, it was shown that summation of the currents induced by agonist application was dependent on the membrane potential. The TRPV1-mediated currents induced by the pH and Capsaicin demonstrated arithmetical summation at potentials between 40--40 mV, while they were potentiated at potentials below -40 mV. Currents induced by the pH and Capsaicin combined were higher in comparison with the arithmetic sum of the currents induced by the pH and Capsaicin applied separately at such potentials. Such a potential dependence seems to be a base of the sensitization that is induced by inflammation or pain, when concentrations of proinflammatory mediators acting as TRPV1 agonists are increasing. Further depolarization induced by TRPV1 activation doesn't generate potentiation, which might serve as a protective mechanism to restrict their activity.

Hydrogen peroxide modulates synaptic transmission in ventral horn neurons of the rat spinal cord.

Excessive production of reactive oxygen species (ROS) is implicated in many central nervous system disorders; however, the physiological role of ROS in spinal ventral horn (VH) neurons remains poorly understood. We investigated how pathological levels of H2O2, an abundant ROS, regulate synaptic transmission in VH neurons of rats using a whole-cell patch clamp approach. H2O2 increased the release of glutamate and GABA from presynaptic terminals. The increase in glutamate release involved N-type voltage-gated calcium channels (VGCCs), ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3 Rs); the increase in GABA release, which inhibited glutamatergic transmission, involved IP3 R. Inhibiting N-type VGCCs and RyRs attenuates excitotoxicity resulting from increased glutamatergic activity while preserving the neuroprotective effects of GABA, and may represent a novel strategy for treating H2O2-induced motor neuron disorders resulting from trauma or ischaemia-reperfusion injury. Excessive production of reactive oxygen species (ROS) is a critical component of the cellular and molecular pathophysiology of many central nervous system (CNS) disorders, including trauma, ischaemia-reperfusion injury, and neurodegenerative diseases. Hydrogen peroxide (H2O2), an abundant ROS, modulates synaptic transmission and contributes to neuronal damage in the CNS; however, the pathophysiological role of H2O2 in spinal cord ventral horn (VH) neurons remains poorly understood, despite reports that these neurons are highly vulnerable to oxidative stress and ischaemia. This was investigated in the present study using a whole-cell patch clamp approach in rats. We found that exogenous application of H2O2 increased the release of glutamate from excitatory presynaptic terminals and γ-aminobutyric acid (GABA) from inhibitory presynaptic terminals. The increase of glutamate release was induced in part by an increase in Ca(2+) influx through N-type voltage-gated calcium channels (VGCCs) as well as by ryanodine receptor (RyR)- and inositol trisphosphate receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER). In inhibitory presynaptic neurons, increased IP3 R-mediated Ca(2+) release from the ER increased GABAergic transmission, which served to rescue VH neurons from excessive release of glutamate from presynaptic terminals. These findings indicate that inhibiting N-type VGCCs or RyRs may attenuate excitotoxicity resulting from increased glutamatergic activity while preserving the neuroprotective effects of GABA, and may therefore represent a novel and targeted strategy for preventing and treating H2O2-induced motor neuron disorders.

Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats.

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with μ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24–30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 μM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 μM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at −95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.

Polypeptide Modulators of TRPV1 Produce Analgesia without Hyperthermia

Transient receptor potential vanilloid 1 receptors (TRPV1) play a significant physiological role. The study of novel TRPV1 agonists and antagonists is essential. Here, we report on the characterization of polypeptide antagonists of TRPV1 based on in vitro and in vivo experiments. We evaluated the ability of APHC1 and APHC3 to inhibit TRPV1 using the whole-cell patch clamp approach and single cell Ca2+ imaging. In vivo tests were performed to assess the biological effects of APHC1 and APHC3 on temperature sensation, inflammation and core body temperature. In the electrophysiological study, both polypeptides partially blocked the capsaicin-induced response of TRPV1, but only APHC3 inhibited acid-induced (pH 5.5) activation of the receptor. APHC1 and APHC3 showed significant antinociceptive and analgesic activity in vivo at reasonable doses (0.01–0.1 mg/kg) and did not cause hyperthermia. Intravenous administration of these polypeptides prolonged hot-plate latency, blocked capsaicin- and formalin-induced behavior, reversed CFA-induced hyperalgesia and produced hypothermia. Notably, APHC3’s ability to inhibit the low pH-induced activation of TRPV1 resulted in a reduced behavioural response in the acetic acid-induced writhing test, whereas APHC1 was much less effective. The polypeptides APHC1 and APHC3 could be referred to as a new class of TRPV1 modulators that produce a significant analgesic effect without hyperthermia.

Presynaptic and postsynaptic ion channel expression in vestibular nuclei neurons after unilateral vestibular deafferentation

Vestibular compensation refers to the recovery of function occurring after unilateral vestibular deafferentation, but some patients remain uncompensated. Similarly, more than half of the operated chickens compensate three days after unilateral vestibular ganglionectomy (UVG), but the rest remain uncompensated. This review focuses on the studies performed on the principal cells of the chick tangential nucleus after UVG. The tangential nucleus is a major avian vestibular nucleus whose principal cells are all second-order, vestibular reflex projection neurons participating in the vestibuloocular and vestibulocollic reflexes controlling posture, balance, and eye movements. Using whole-cell patch-clamp approach in brain slice preparations, spontaneous spike firing, ionic conductances, and spontaneous excitatory postsynaptic currents (sEPSCs) are recorded in principal cells from controls and operated chickens three days after UVG. In compensated chickens, the proportion of spontaneous spike firing principal cells and their spike discharge rate are symmetric on the lesion and intact sides, with the rates increased over controls. However, in the uncompensated chickens, the spike discharge rate increases on the lesion side, but not on the intact side, where only silent principal cells are recorded. In all the experimental groups, including controls, silent principal cells are distinguished from spontaneous spiking cells by smaller persistent sodium conductances and higher activation thresholds for the fast sodium channel. In addition, silent principal cells on the intact side of uncompensated chickens have larger dendrotoxin-sensitive potassium conductances, with a higher ratio of immunolabeling for surface/cytoplasmic expression of a dendrotoxin-sensitive, potassium channel subunit, Kv1.1. Finally, in compensated chickens, sEPSC frequency is symmetric bilaterally, but in uncompensated chickens sEPSC frequency increased only on the lesion side, where the expression of Kv1.2 decreased in synaptotagmin-labeled terminal profiles on the principal cell bodies. Altogether, the specific sodium and potassium channels important for the development of spike firing pattern and/or presynaptic glutamate release on vestibular reflex projection neurons may be critically involved in changing postsynaptic neuron excitability after vestibular deafferentation.

The combined disruption of microfilaments and microtubules affects the distribution and function of GABA A receptors in rat cerebellum granule cells in culture

The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular distribution and function of GABA A receptors has been studied in rat cerebellar granule cells in culture. The disruption of either the microfilaments or the microtubules structures did not result in detectable changes in the receptors distribution, as assessed by immunocytochemistry, or in their function, as assessed by the whole-cell patch-clamp approach. A distinct disruption of both the subcellular distribution and the function of the GABA A receptors was found only if both microfilaments and microtubules were destroyed. The results suggest that, in the short term, the plasma membrane localization/stabilization and function of these receptors in granule cells are largely independent from microfilaments and microtubules individually, although they obviously depend on the presence of an organized cellular framework.

Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.

GABA B receptor activation protects GABA A receptor from cyclic AMP-dependent down-regulation in rat cerebellar granule cells

Interaction between GABA A and GABA B receptors was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. Our data show that the GABA B agonist (−)baclofen is not able, per se, to significantly change the muscimol-activated chloride current. However, (−)baclofen dose-dependently prevents the reduction of GABA A receptor function by forskolin, an activator of adenylate cyclase. The effect of baclofen is mediated by a pertussis toxin-sensitive G protein. In fact, in cells treated with pertussis toxin, baclofen and forskolin, the toxin is able to block baclofen action, allowing forskolin to act fully. The protective effect by GABA B receptor activation under these circumstances is most probably related to the prevention of cyclic AMP increases after forskolin treatment. In fact, in these neurons cyclic AMP and protein kinase A activation result in a down-regulation of GABA A receptor function. On the whole, the data indicate the presence of complex modulation of GABA A receptors by GABA B receptor types in cerebellum granule cells.

Regulation of gaba a receptor in cerebellar granule cells in culture: Differential involvement of kinase activities

GABA A receptor function was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. The data show that GABA activates Cl − currents in these neurons which reverse at the appropriate membrane potential and are blocked by picrotoxin. The GABA-activated currents desensitize with time of application of the neurotransmitter at concentrations ≥10 −6M. The dose-response curve for the peak Cl − current gives a K a value of 2.3 μM with a Hill coefficient of 1.2. The peak Cl − current elicited by GABA decreases with time of cell registration, with a time-constant of 7.3 min. Residual responsiveness though is maintained thereafter. This “run-down” phenomenon can be completely prevented by adding adenosine-5′-triphosphate + Mg 2+ in the pipette solution. Treatments which directly (8-bromoadenosine-3,5'-cyclicmonophosphate; adenosine-3', 5'-cyclicmonophosphate) or indirectly (forskolin, isobutylmethylxanthine) increase the adenosine-3',5'-cyclicmonophosphate intracellular content reduce the GABA-induced Cl − current. Conversely, treatment with the protein kinase A and C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine potentiates the effect of GABA. On the whole, the data indicate that different protein kinase activities modulate the functional state of the GABA A receptors on granule cells from the rat cerebellum.