Patch Recording Configuration Research Articles

Sniffer patch laser uncaging response (SPLURgE): an assay of regional differences in allosteric receptor modulation and neurotransmitter clearance

Allosteric modulators exert actions on neurotransmitter receptors by positively or negatively altering the effective response of these receptors to their respective neurotransmitter. γ-Aminobutyric acid (GABA) type A ionotropic receptors (GABAARs) are major targets for allosteric modulators such as benzodiazepines, neurosteroids, and barbiturates. Analysis of substances that produce similar effects has been hampered by the lack of techniques to assess the localization and function of such agents in brain slices. Here we describe measurement of the sniffer patch laser uncaging response (SPLURgE), which combines the sniffer patch recording configuration with laser photolysis of caged GABA. This methodology enables the detection of allosteric GABAAR modulators endogenously present in discrete areas of the brain slice and allows for the application of exogenous GABA with spatiotemporal control without altering the release and localization of endogenous modulators within the slice. Here we demonstrate the development and use of this technique for the measurement of allosteric modulation in different areas of the thalamus. Application of this technique will be useful in determining whether a lack of modulatory effect on a particular category of neurons or receptors is due to insensitivity to allosteric modulation or a lack of local release of endogenous ligand. We also demonstrate that this technique can be used to investigate GABA diffusion and uptake. This method thus provides a biosensor assay for rapid detection of endogenous GABAAR modulators and has the potential to aid studies of allosteric modulators that exert effects on other classes of neurotransmitter receptors, such as glutamate, acetylcholine, or glycine receptors.

Effects of noradrenaline on the GABA response in rat isolated spiral ganglion neurons in culture

In the present study, the modulatory effects of noradrenaline (NA) on the GABA response were investigated in the isolated cultured spiral ganglion neurons of rat by using nystatin perforated patch recording configuration under voltage-clamp conditions. NA reversibly depressed GABA response in a concentration-dependent manner and neither changed the reversal potential of the GABA response nor affected the apparent affinity of GABA to its receptor. alpha2-adrenoceptor agonist and antagonist, clonidine and yohimbine mimicked and blocked the NA action on the GABA response, respectively. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89), a protein kinase A inhibitor, mimicked the effect of NA on the GABA response. NA failed to affect the GABA response in the presence of both cAMP and protein kinase A modulator. However, NA still depressed the GABA response even in the presence of both phorbol-12-myristate-13-acetate, a protein kinase C activator and chelerythrine, a protein kinase C inhibitor. These results suggest that the NA suppression of the GABA response is mediated by alpha2-adrenoceptor which reduces intracellular cAMP formation through the inhibition of adenylyl cyclase. Therefore, NA input to the spiral ganglion neurons may modulate the auditory transmission by affecting the GABA response.

Potentiation of inhibitory amino acid receptors-mediated responses by lanthanum in rat sacral dorsal commissural neurons

Lanthanum is one of rare earth cations with extremely active chemical property and has been reported to influence neuronal transmitter systems. To date, little attention has been directed towards the sacral dorsal commissural nucleus (SDCN), which serves as a relay of sensory information from the pelvic viscera in the spinal cord. Therefore, the effect of lanthanum on the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and glycine (Gly) responses in neurons acutely dissociated from the rat SDCN was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. At a holding potential of − 40 mV, La 3+ reversibly potentiated GABA (3 μM)-activated currents ( I GABA) in a concentration-dependent manner over the concentration range of 10 μM to 30 mM, with the EC 50 value of 67.3 ± 16.4 μM. Similarly, La 3+ reversibly potentiated glycine (10 μM)-activated currents ( I Gly) in a concentration-dependent manner over the concentration range of 1 μM to 1 mM, with the EC 50 value of 52.3 ± 10.9 μM. The effects of La 3+ on I GABA and I Gly were voltage-independent. Moreover, both of the potentiations were not use-dependent and were overcome by increasing the concentration of agonist. Our results indicate that La 3+ potentiates the inhibitory amino acid receptors-mediated responses in SDCN, which may reduce the transmission of the pelvic visceral information. The information provided by this work may help to elucidate the mechanisms and effects of lanthanum on brain functions.

Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

Common Determinants of Single Channel Conductance within the Large Cytoplasmic Loop of 5-Hydroxytryptamine Type 3 and α4β2 Nicotinic Acetylcholine Receptors

GABA-induced response in spiral ganglion cells acutely isolated from guinea pig cochlea

The physiological and pharmacological properties of γ-aminobutyric acid (GABA)-induced responses were investigated in acutely isolated spiral ganglion cells (SGCs) of guinea pig by using either a nystatin-perforated patch recording configuration or a conventional whole-cell patch recording mode combined with rapid drug application. GABA and GABA A subtype receptor agonist, muscimol, induced inward currents in a concentration-dependent manner in 74% of all cells. The current-voltage relationship for the GABA response indicated the GABA-induced current in SGCs is carried by Cl −. Bicuculline (BIC), strychnine (STR), and picrotoxin (PTX) suppressed the GABA response in a concentration-dependent manner. BIC and STR, and PTX blocked the GABA response in a competitive manner and in a non-competitive manner, respectively. For inorganic antagonists, Cd 2+ and Ni 2+ also inhibited the GABA response. On the other hand, Zn 2+ failed to suppress the GABA response in SGCs. An antibiotic, benzylpenicillin, suppressed the GABA response. The GABA response was augmented by both a barbiturate derivative, pentobarbital (PB), and a benzodiazepine derivative, diazepam. The results suggest clearly that the physiological and pharmacological characteristics of GABA A receptor on acutely isolated guinea pig SGCs are quite similar to the common GABA A receptor found in other sensory ganglion cells.

Selective Modulation of Neuronal Nicotinic Acetylcholine Receptor Channel Subunits by Go-Protein Subunits

G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTPgammaS increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTPgammaS. In inside-out membrane patches, the open probability (NP(o)) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mm GTPgammaS relative to control but was unchanged in the presence of GDPbetaS. The modulation of nAChR-mediated whole-cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTPgammaS. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or Gbetagamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nm Gbetagamma increased the open probability of ACh-activated single-channel currents fivefold, whereas G(o)alpha (50 nm) produced no significant increase in NP(o). Neuronal nAChR subunits alpha3-alpha5 and beta2 exhibited a positive interaction with G(o)alpha and Gbetagamma, whereas beta4 and alpha7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

Potentiation, activation and blockade of GABA A receptors by etomidate in the rat sacral dorsal commissural neurons

Etomidate (ET), an imidazole general anesthetic, has been medically widely used. Recent evidence suggests that the inhibitory neurotransmitter GABA receptor may be one of the important molecular target(s) of general anesthetics. Up to date, little attention has been directed toward the sacral dorsal commissural nucleus (SDCN), which serves as a relay of sensory information from the pelvic viscera in the spinal cord. Therefore, the effect of ET on GABA A receptor function in neurons acutely dissociated from the SDCN was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. At a holding potential of −40 mV, ET (above 10 #x00A0;μM) induced an inward ET-activated current ( I ET) with the EC 50 value of 33±3 #x00A0;μM, which was reversibly blocked by bicuculline and picrotoxin. The reversal potential of I ET was close to the Cl − equilibrium potential. ET also displayed a biphasic modulatory effect on GABA responses. At lower concentrations (0.1–100 #x00A0;μM), ET reversibly potentiated GABA (1 #x00A0;μM)-activated Cl − currents in a bell-shaped manner, with the maximal facilitative effect at 10 #x00A0;μM, whereas at concentrations >100 #x00A0;μM, the peak of the ET-induced current was suppressed in the absence or presence of GABA (1 #x00A0;μM). These results suggest that in SDCN, in addition to the potentiation of GABA A receptor-mediated responses at low concentrations and the direct activation of GABA A receptors at moderate concentrations as expected, ET produced a fast blocking action at high concentrations. The general anesthetic-induced effects in SDCN, at least the potentiation of GABA responses, may significantly contribute to anesthesia of pelvic viscera during the general anesthesia.

Noradrenaline-induced cation currents in isolated rat paratracheal ganglion neurons

The actions of noradrenaline (NA) on the neurons acutely isolated from paratracheal ganglia of rats and the ionic mechanisms involved were studied with nystatin-perforated patch recording configuration. Under current-clamp conditions, application of 10 μM NA produced membrane depolarization followed by repetitive action potentials. NA evoked an inward cationic current under voltage-clamp conditions at a holding potential of −60 mV. Transient tail inward (‘hump’) current was also induced by washout of NA. The NA-induced current was reduced by extracellular Ca 2+ and Mg 2+, with half-maximal concentrations of 0.7 and 2.6 mM for Ca 2+ and Mg 2+, respectively. Phenylephrine, an α 1-adrenoceptor agonist, mimicked the NA-induced current, but the ‘hump’ current did not occur upon washout of phenylephrine. The NA-induced current was inhibited by prazosin and WB-4101, α 1-adrenoceptor antagonists. In contrast, in the presence of yohimbine, an α 2-adrenoceptor antagonist, the NA-induced current was potentiated and the washout of NA failed to evoke the ‘hump’ current. The pretreatment of paratracheal neurons with pertussis toxin also potentiated the NA-induced current. The NA-induced inward current was inhibited by pretreatment with U73122, a phospholipase C inhibitor, and xestospongin-C, a membrane-permeable IP 3 receptor antagonist. On the other hand, thapsigargin, BAPTA-AM and calmidazolium had no effect on the NA-induced current, suggesting that release of Ca 2+ from intracellular Ca 2+ stores via IP 3 receptors is not involved in the NA action. The cationic channels activated by NA play an important physiological role in neuronal membrane depolarization in rat paratracheal ganglia.

Gramicidin-perforated Patch Recording Revealed the Oscillatory Nature of Secretory Cl− Movements in Salivary Acinar Cells

Elevations of cytoplasmic free calcium concentrations ([Ca2+]i) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl− through Ca2+-activated Cl− channels, while Cl− enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na+-K+-2Cl− cotransporter (NKCC) and/or parallel operations of Cl−-HCO3 − and Na+-H+ exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl− secretion, we analyzed carbachol (CCh)-activated Cl− currents in submandibular acinar cells using the “gramicidin-perforated patch recording configuration.” Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl− currents in the gramicidin-perforated patch recording were carried by Cl− efflux via Cl− channels, dependent upon Cl− entry through Cl− transporters expressed in the acinar cells. CCh-evoked oscillatory Cl− currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl− currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO3 − had significant effects, suggesting that the cotransporter rather than parallel operations of Cl−-HCO3 − and Na+-H+ exchangers is the primary Cl− uptake pathway. Pharmacological manipulation of the activities of the Ca2+-activated Cl− channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl− currents, while adjusting to the rate imposed by the Ca2+-activated Cl− channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl− movements may effectively provide a driving force for fluid secretion in intact acinar cells.

Adenosine suppresses GABA A receptor-mediated responses in rat sacral dorsal commissural neurons

The modulatory effect of adenosine on γ-aminobutyric acid (GABA)-activated whole-cell currents were investigated in the neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions. The results showed that: (1) GABA acted on GABA A receptor and elicited inward Cl − currents ( I GABA) at a holding potential ( V H) of −40 mV; (2) adenosine suppressed GABA-induced Cl − current without affecting the reversal potential of I GABA and the apparent affinity of GABA to its receptor; (3) N6-cyclohexyladenosine mimicked the suppression effect of adenosine on I GABA, whereas 8-cyclopentyl-1,3-dipropylxanthine blocked the suppression effect of adenosine; (4) adenosine fails to suppress I GABA on the neurons that were pretreated with bisindolylmaleimide I (BIM), while after pretreatment with H-89, the inhibitory effect of adenosine on I GABA were not affected; (5) the suppression effect of adenosine on I GABA remained in the presence of BAPTA-AM. The present results indicate that the suppression of adenosine on I GABA is mediated by adenosine A 1 receptor and through a Ca 2+-independent protein kinase C transduction pathway, and that the interactions between adenosine and GABA might participate in the modulation of nociceptive information transmission at the SDCN.

The modulation of action potential generation by calcium-induced calcium release is enhanced by mitochondrial inhibitors in mudpuppy parasympathetic neurons

Previously, we demonstrated that outward currents activated by calcium-induced calcium release (CICR) opposed depolarization-induced action potential (AP) generation in dissociated mudpuppy parasympathetic neurons [J Neurophysiol 88 (2002) 1119]. In the present study, we tested whether AP generation by depolarizing current ramps could be altered by dissipating the mitochondrial membrane potential and thus interrupting mitochondrial Ca 2+ buffering. Exposure to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 μM) alone or in combination with the mitochondrial ATP synthase inhibitor oligomycin (8 μg/ml), increased the latency to AP generation. Exposure to the electron transport chain inhibitor rotenone (10 μM) alone or in combination with oligomycin (8 μg/ml) similarly increased the latency to AP generation. CCCP and oligomycin or rotenone and oligomycin treatment caused rhodamine 123 loss from mitochondria within a few minutes, confirming that the mitochondrial membrane potential was dissipated during drug exposure. Oligomycin alone had no effect on the latency to AP generation and did not cause loss of rhodamine 123 from mitochondria. The increase in latency induced by CCCP and oligomycin was similar when recordings were made with either the perforated patch or standard whole cell patch recording configuration. Exposure to the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (1 μM), decreased the latency to AP generation. In cells pretreated with thapsigargin to eliminate CICR, CCCP and oligomycin had no effect on AP latency. Pretreatment with iberiotoxin (IBX; 100 nM), an inhibitor of large conductance, calcium- and voltage-activated potassium channels, reduced the extent of the CCCP- and oligomycin-induced increase in latency to AP generation. These results indicate that treatment with CCCP or rotenone to dissipate the mitochondrial membrane potential, a condition which should minimize sequestration of Ca 2+ by mitochondria, facilitated the Ca 2+-induced Ca 2+ release activation of IBX-sensitive and IBX-insensitive conductances that regulate AP generation.

Suppression of Kainate-Evoked AMPA Receptor Mediated Responses by Lanthanum in Rat Sacral Dorsal Commissural Neurons

The effect of lanthanum (La) on kainate (KA) responses in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The responses to KA were mediated by activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in SDCN neurons. La³⁺ reversibly inhibited KA (100 µM) activated currents (I_KA) in a concentration-dependent manner over the range from 30 µM to 30 mM, with IC₅₀ values of 0.64 ± 0.06 mM at a holding potential (V_H) of –40 mV. Our further study indicated that the effects of La³⁺ on I_KA were voltage independent. Moreover, the inhibition was not use dependent and was not overcome by increasing the concentration of agonist. These findings indicate that La³⁺ is an efficacious inhibitor of AMPA receptor mediated responses which may contribute to its cytotoxic effect.

Biphasic action of midazolam on GABA A receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of the benzodiazepine agonist midazolam on γ-aminobutyric acid A (GABA A) receptor-mediated currents was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. Midazolam displayed a biphasic effect on GABA responses. Low concentrations of midazolam (1 nM–10 μM) reversibly potentiated GABA (3 μM)-activated Cl − currents ( I GABA) in a bell-shaped manner, with the maximal facilitary effect at 0.1 μM; whereas at higher concentrations (above 10 μM), midazolam had an antagonistic effect on I GABA. Our further study indicated that midazolam changed GABA A receptor affinity to GABA and the effects of midazolam on I GABA were voltage-independent. The benzodiazepine receptor antagonist, flumazenil, abolished the facilitary effect of low concentrations of midazolam rather than the antagonism of I GABA induced by high doses of midazolam. In addition, activation of protein kinase C prevented the inhibitory effect of midazolam at higher concentrations, but did not influence the effect of midazolam at low concentrations. These results indicate that midazolam interacts with another distinct site other than the central benzodiazepine receptors on GABA A receptors as an antagonist at higher concentrations in SDCN neurons.

Opioid peptides modulate the response of neurons of the superficial laminae of the rat spinal dorsal horn to GABA

The modulatory effects of methionine-enkephalin (M-ENK) and selective opioid-receptor agonists on GABA-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae of the rat spinal dorsal horn using nystatin-perforated patch recording configuration under voltage-clamp conditions. The results show that: (1) GABA acted on GABA A receptors and elicited inward Cl − currents ( I GABA) at −60 mV; (2) M-ENK depressed I GABA in ∼65% of the tested neurons and potentiated I GABA in ∼15% of the neurons tested; (3) the agonists of μ-, κ-, and δ-opioid receptors—[ d-AIa 2, N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO), dynorphin-A (Dyn-A), and [ d-Pen 2, d-Pen 5]-enkephalin (DPDPE) also depressed the I GABA, and the order of agonist potency was DAMGO > Dyn-A > DPDPE; and (4) naloxone blocked the inhibitory effects of M-ENK on I GABA. The antagonists of μ-, κ-, and δ-opioid receptors—β-funaltrexamine (β-FNA), nor-binaltorphimine (nor-BNI), and naltrindole (NTI) prevented the DAMGO-, Dyn-A-, and DPDPE-induced depression of I GABA. The results suggest that M-ENK downregulates I GABA principally through μ- and κ-opioid receptors, and thus exerts its modulating effects indirectly on the transmission of noxious information at the spinal level.

Alpha1-Adrenoceptor-activated cation currents in neurones acutely isolated from rat cardiac parasympathetic ganglia

The noradrenaline (NA)-induced cation current was investigated in neurones freshly isolated from rat cardiac parasympathetic ganglia using the nystatin-perforated patch recording configuration. Under current-clamp conditions, NA depolarized the membrane, eliciting repetitive action potentials. NA evoked an inward cation current under voltage-clamp conditions at a holding potential of −60 mV. The NA-induced current was inhibited by extracellular Ca2+ or Mg2+, with a half-maximal concentration of 13 μm for Ca2+ and 1.2 mm for Mg2+. Cirazoline mimicked the NA response, and prazosin and WB-4101 inhibited the NA-induced current, suggesting the contribution of an α1-adrenoceptor. The NA-induced current was inhibited by U73122, a phospholipase C (PLC) inhibitor. The membrane-permeable IP3 receptor blocker xestospongin-C also blocked the NA-induced current. Furthermore, pretreatment with thapsigargin and BAPTA-AM could inhibit the NA response while KN-62, phorbol 12-myristate 13-acetate (PMA) and staurosporine had no effect. These results suggest that NA activates the extracellular Ca2+- and Mg2+-sensitive cation channels via α1-adrenoceptors in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation mechanism also involves phosphoinositide breakdown, release of Ca2+ from intracellular Ca2+ stores and calmodulin. The cation channels activated by NA may play an important role in neuronal membrane depolarization in rat cardiac ganglia.

Alpha 1-adrenoceptor-activated cation currents in neurones acutely isolated from rat cardiac parasympathetic ganglia.

The noradrenaline (NA)-induced cation current was investigated in neurones freshly isolated from rat cardiac parasympathetic ganglia using the nystatin-perforated patch recording configuration. Under current-clamp conditions, NA depolarized the membrane, eliciting repetitive action potentials. NA evoked an inward cation current under voltage-clamp conditions at a holding potential of -60 mV. The NA-induced current was inhibited by extracellular Ca2+ or Mg2+, with a half-maximal concentration of 13 microM for Ca2+ and 1.2 mM for Mg2+. Cirazoline mimicked the NA response, and prazosin and WB-4101 inhibited the NA-induced current, suggesting the contribution of an alpha1-adrenoceptor. The NA-induced current was inhibited by U73122, a phospholipase C (PLC) inhibitor. The membrane-permeable IP3 receptor blocker xestospongin-C also blocked the NA-induced current. Furthermore, pretreatment with thapsigargin and BAPTA-AM could inhibit the NA response while KN-62, phorbol 12-myristate 13-acetate (PMA) and staurosporine had no effect. These results suggest that NA activates the extracellular Ca2+- and Mg2+-sensitive cation channels via alpha 1-adrenoceptors in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation mechanism also involves phosphoinositide breakdown, release of Ca2+ from intracellular Ca2+ stores and calmodulin. The cation channels activated by NA may play an important role in neuronal membrane depolarization in rat cardiac ganglia.

β-ALANINE ACTS ON GLYCINE RECEPTORS IN THE RAT SACRAL DORSAL COMMISSURAL NEURONS

Whole-cell current responses to bath application of g -alanine ( g -ALA) were investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under voltage-clamp conditions. g -ALA activated inward currents in a concentration-dependent manner over the range of 10 3000 µM with an EC50 of 80.8 µM. The reversal potential of g -ALA-activated currents (I g -ALA) was close to the Cl equilibrium potential. Strychnine and the chlo ride channel blocker picrotoxin suppressed the I g -ALA in a concentration-dependent manner with the IC50 values of 0.19 µM and 343.6 µM, respectively. At the concentration of 3 µM, strychnine was sufficient to completely block 100 µM g -ALA response, whereas it did not show a suppression of GABA response. In contrast, bicuculline, a potent GABAA receptor antag onist, at concentrations up to 10 µM, a dose that could block the GABA response completely, had little or no effect on I g -ALA. Further more, the I g -ALA was not affected by a preceding GABA response, but rather cross-desensitized with that evoked by glycine. The results indicate that g -ALA activates the strychnine-sensitive glycine receptors in the SDCN neurons, and suggest that g -ALA may act as a functional neurotransmitter in the mammalian SDCN.

Serotonin potentiation of glycine-activated whole-cell currents in the superficial laminae neurons of the rat spinal dorsal horn is mediated by protein kinase C

The modulatory effects of serotonin (5-HT) on glycine (Gly)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (I and II) of the rat spinal dorsal horn using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Our results demonstrate that (1) Gly acted on strychnine (STR)-sensitive Gly receptors and elicited inward Cl − currents ( I Gly) at a holding potential of −40 mV; (2) 5-HT potentiated I Gly without affecting the reversal potential of I Gly; (3) the agonist ( α-methyl-5-HT) and antagonist (ketanserine) of 5-HT 2 receptor mimicked and blocked the potentiating effect of 5-HT on I Gly, respectively; (4) bisindolylmaleimide I (BIM), a selective inhibitor of protein kinase C (PKC), reduced the potentiating effect of 5-HT on I Gly; and (5) 5-HT-induced enhancement of I Gly was not affected by pretreatment with 1,2-bis-(2-aminophenoxy) ethane- N, N, N′, N′-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA AM), a Ca 2+ chelator. These results indicate that (1) the potentiation of 5-HT on I Gly is mediated by 5-HT 2 receptor and through Ca 2+-independent PKC intracellular signal transduction pathway; and (2) the interactions between 5-HT and Gly might modulate the transmission of nociceptive information through the spinal cord.

An electrophysiological study of muscarinic and nicotinic receptors of rat paratracheal ganglion neurons and their inhibition by Z-338.

1. To study the mechanisms involved in the action of Z-338, a newly synthesized gastroprokinetic agent, experiments were performed with the paratracheal ganglion cells acutely dissociated from 2-week-old Wistar rats. The effects of Z-338 on both nicotinic and muscarinic responses of the ganglion cells were studied by nystatin perforated patch recording configuration under the current- and voltage-clamp conditions. 2. Acetylcholine (ACh) or nicotine, and muscarine or oxotremorine-M (OX-M) induced membrane depolarization with rapid and slow time courses respectively, followed by repetitive generation of action potentials in the ganglion cell. Corresponding to the membrane depolarization induced by cholinergic agents, ACh induced biphasic inward currents with rapid and slow time courses under the voltage-clamp condition. Nicotine and muscarine or OX-M evoked inward currents with rapid and slow time courses, respectively. The rapid and slow inward currents were accompanied by increase and decrease in the membrane conductance, respectively. In addition, OX-M dose-dependently suppressed the M-type K(+) current evoked in response to hyperpolarizing voltage-steps from V(H) of -25 mV to -50 mV, indicating that the activation of muscarinic acetylcholine receptors inhibits M-type K(+) current, thus inducing inward current in the ganglion cell. 3. Z-338 competitively suppressed the inward currents induced by OX-M through M(1) ACh receptor, and uncompetitively suppressed the currents induced by nicotine. 4. The inhibitory actions of Z-338 on the membrane depolarization and corresponding inward currents mediated by M(1)-muscarinic and neuronal nicotinic ACh receptors in the isolated ganglion cells were discussed in relation to the inhibitory actions on autoreceptors in the parasympathetic nerve terminals, which would explain the gastroprokinetic actions of Z-338.

Presynaptic inhibition of GABAergic miniature currents by metabotropic glutamate receptor in the rat CNS

The modulation of spontaneous miniature GABAergic inhibitory postsynaptic currents (mIPSC) by the metabotropic glutamate receptors was investigated in the mechanically dissociated rat nucleus basalis of Meynert neurons using the conventional whole-cell patch recording configuration. An application of (±)-1-aminocyclopentane- trans-1,3-dicarboxylic acid (tACPD) reversibly reduced the frequency of mIPSC without affecting the current amplitude distribution. The application of K + channel blockers such as 4-aminopyridine, Cs +, Ba 2+ or tetraethylammonium increased the mIPSC frequency, but failed to inhibit the tACPD action on mIPSC. Although the removal of Ca 2+ from the extracellular solution reduced the mIPSC frequency, the inhibitory effect of tACPD on mIPSC was unaltered. These results suggested that neither voltage-dependent K + or Ca 2+ channels are involved in the inhibitory effect of tACPD on mIPSC frequency. Forskolin, an activator of adenylate cyclase, facilitated the mIPSC frequency in a concentration-dependent manner and inhibited the tACPD-induced suppression of mIPSC frequency. 8-Br-cAMP, a membrane permeable analog of cAMP, also prevented the inhibitory action of tACPD. However, Sp-cAMP, an activator of protein kinase A, could not prevent the inhibitory action of tACPD. L-CCG-I and (2 R,4 R)-APDC, group II mGluR agonists, mimicked the tACPD action on mIPSC frequency, but L-AP4, a group III mGluR agonist, had no such effect. MCCG, a group II mGluR antagonist, fully blocked the tACPD action. It was concluded that the activation of group II mGluR on the GABAergic presynaptic nerve terminals projecting to the rat nucleus basalis of Meynert neurons therefore inhibits the GABA release by reducing the activity of the cAMP-dependent pathway.