Cerebellar Purkinje cells (PCs) exhibit two types of discharge activities: simple spike (SS) and complex spike (CS). Previous studies found that noradrenaline (NA) can inhibit CS and bidirectionally regulate SS, but the enhancement of NA on SS is overwhelmed by the strong inhibition of excitatory molecular layer interneurons. However, the mechanism underlying the effect of NA on SS discharge frequency is not clear. Therefore, in the present study, we examined the mechanism underlying the increasing effect of NA on SS firing of PC in mouse cerebellar cortex in vivo and in cerebellar slice by cell-attached and whole-cell recording technique and pharmacological methods. GABA_A receptor was blocked by 100 µmol/L picrotoxin in the whole process. In vivo results showed that NA significantly reduced the number of spikelets of spontaneous CS and enhanced the discharge frequency of SS, but did not affect the discharge frequency of CS. In vitro experiments showed that NA reduced the number of CS spikelets and after hyperpolarization potential (AHP) induced by electrical stimulation, and increased the discharge frequency of SS. NA also reduced the amplitude of excitatory postsynaptic current (EPSC) of parallel fiber (PF)-PC and significantly increased the paired-pulse ratio (PPR). Application of yohimbine, an antagonist of α2-adrenergic receptor (AR), completely eliminated the enhancing effect of NA on SS. The α2-AR agonist, UK14304, also increased the frequency of SS. The β-AR blocker, propranolol, did not affect the effects of NA on PC. These results suggest that in the absence of GABA_A receptors, NA could attenuate the synaptic transmission of climbing fiber (CF)-PC via activating α2-AR, inhibit CS activity and reduce AHP, thus enhancing the SS discharge frequency of PC. This result suggests that NA neurons of locus coeruleus can finely regulate PC signal output by regulating CF-PC synaptic transmission.
Action Potentials/physiology* ; Animals ; Cerebellar Cortex/metabolism* ; Cerebellum/metabolism* ; Mice ; Norepinephrine/pharmacology* ; Purkinje Cells/metabolism* ; Receptors, Adrenergic, alpha-2/metabolism* ; Receptors, GABA-A/metabolism*

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.
Animals ; Bicuculline ; pharmacology ; Disease Models, Animal ; Glycine ; metabolism ; Hyperalgesia ; drug therapy ; etiology ; metabolism ; Imidazoles ; pharmacology ; Inhibitory Postsynaptic Potentials ; drug effects ; physiology ; Male ; Mice, Inbred C57BL ; Neurons ; drug effects ; metabolism ; Neurotransmitter Agents ; pharmacology ; Peripheral Nerve Injuries ; drug therapy ; metabolism ; Phenanthrolines ; pharmacology ; Potassium Channels, Inwardly Rectifying ; antagonists & inhibitors ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, Glycine ; metabolism ; Strychnine ; pharmacology ; Synaptic Transmission ; drug effects ; physiology ; Tissue Culture Techniques ; Touch

Exploring the transition from inter-ictal to ictal epileptiform discharges (IDs) and how GABA receptor-mediated action affects the onset of IDs will enrich our understanding of epileptogenesis and epilepsy treatment. We used Mg-free artificial cerebrospinal fluid (ACSF) to induce epileptiform discharges in juvenile mouse hippocampal slices and used a micro-electrode array to record the discharges. After the slices were exposed to Mg-free ACSF for 10 min-20 min, synchronous recurrent seizure-like events were recorded across the slices, and each event evolved from inter-ictal epileptiform discharges (IIDs) to pre-ictal epileptiform discharges (PIDs), and then to IDs. During the transition from IIDs to PIDs, the duration of discharges increased and the inter-discharge interval decreased. After adding 3 μmol/L of the GABA receptor agonist muscimol, PIDs and IDs disappeared, and IIDs remained. Further, the application of 10 μmol/L muscimol abolished all the epileptiform discharges. When the GABA receptor antagonist bicuculline was applied at 10 μmol/L, IIDs and PIDs disappeared, and IDs remained at decreased intervals. These results indicated that there are dynamic changes in the hippocampal network preceding the onset of IDs, and GABA receptor activity suppresses the transition from IIDs to IDs in juvenile mouse hippocampus.
Animals ; Animals, Newborn ; Bicuculline ; pharmacology ; Disease Models, Animal ; Epilepsy ; pathology ; GABA-A Receptor Agonists ; pharmacology ; GABA-A Receptor Antagonists ; therapeutic use ; Hippocampus ; drug effects ; metabolism ; physiopathology ; In Vitro Techniques ; Magnesium ; metabolism ; pharmacology ; Male ; Membrane Potentials ; drug effects ; Mice ; Mice, Inbred C57BL ; Muscimol ; pharmacology ; Nerve Net ; drug effects ; Receptors, GABA-A ; metabolism

The aim of the present study was to observe whether dopamine receptor (DR) was involved in the effects of sodium salicylate (SS) on the expressions of N-methyl-D-aspartic acid (NMDA) and γ-aminobutyric acid (GABA) receptors in rat cochlear spiral ganglion neurons (SGNs). Forty-eight hours after primary culture of rat SGNs, immunofluorescence technique was applied to detect expressions of DR1 and DR2, the two subtypes of dopamine receptors. Western blot was performed to assess NMDA receptor NR1 subunit and GABAreceptor subunit α2 (GABRα2) protein expressions in the SGNs after the treatments of SS alone or in combination with DR antagonists. The results demonstrated that: (1) The DR1 and DR2 were expressed in the bodies and axons of the SGN; (2) After the treatment with SS, the surface protein expressions of GABRα2 and NR1 were decreased by 44.69% and 21.57%, respectively, while the total protein expressions showed no significant changes; (3) Neither SS + SCH23390 (DR1 antagonist) group nor SS + Eticlopride (DR2 antagonist) group showed significant differences in GABRα2 and NR1 surface protein expressions compared with the control group. These results suggest that SS regulates the surface GABAand NMDA receptors trafficking on SGN, and the mechanism may involve DR mediation.
Animals ; Benzazepines ; pharmacology ; Cells, Cultured ; Cochlea ; cytology ; Neurons ; drug effects ; Rats ; Receptors, Dopamine ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Sodium Salicylate ; toxicity ; Spiral Ganglion ; drug effects

Analgesics, Opioid ; chemistry ; pharmacology ; Animals ; Cattle ; Diazepam ; chemistry ; pharmacology ; Diazepam Binding Inhibitor ; chemical synthesis ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Mice ; Morphine ; chemistry ; pharmacology ; Receptors, GABA-A ; metabolism ; Swine

BACKGROUNDPropofol and etomidate are the most important intravenous general anesthetics in the current clinical use and that mediate gamma-aminobutyric acid's (GABAergic) synaptic transmission. However, their long-term effects on GABAergic synaptic transmission induced by neonatal propofol or etomidate exposure remain unclear. We investigated the long-term GABAergic neurotransmission alterations, following neonatal propofol and etomidate administration.

METHODSSprague-Dawley rat pups at postnatal days 4-6 were underwent 6-h-long propofol-induced or 5-h-long etomidate-induced anesthesia. We performed whole-cell patch-clamp recording from pyramidal cells in the cornus ammonis 1 area of acute hippocampal slices of postnatal 80-90 days. Spontaneous and miniature inhibitory GABAergic currents (spontaneous inhibitory postsynaptic currents [sIPSCs] and miniature inhibitory postsynaptic currents [mIPSCs]) and their kinetic characters were measured. The glutamatergic tonic effect on inhibitory transmission and the effect of bumetanide on neonatal propofol exposure were also examined.

RESULTSNeonatal propofol exposure significantly increased the frequency of mIPSCs (from 1.87 ± 0.35 Hz to 3.43 ± 0.51 Hz, P< 0.05) and did not affect the amplitude of mIPSCs and sIPSCs. Both propofol and etomidate slowed the decay time of mIPSCs kinetics (168.39 ± 27.91 ms and 267.02 ± 100.08 ms vs. 68.18 ± 12.43 ms; P< 0.05). Bumetanide significantly blocked the frequency increase and reversed the kinetic alteration of mIPSCs induced by neonatal propofol exposure (3.01 ± 0.45 Hz and 94.30 ± 32.56 ms).

CONCLUSIONSNeonatal propofol and etomidate exposure has long-term effects on inhibitory GABAergic transmission. Propofol might act at pre- and post-synaptic GABA receptor A (GABAA) receptors within GABAergic synapses and impairs the glutamatergic tonic input to GABAergic synapses; etomidate might act at the postsynaptic site.

Animals ; CA1 Region, Hippocampal ; drug effects ; metabolism ; Electrophysiology ; Etomidate ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Neurons ; drug effects ; metabolism ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; metabolism ; Synaptic Transmission ; drug effects ; gamma-Aminobutyric Acid ; metabolism

The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.
Animals ; Female ; Ganglia, Spinal ; physiology ; Male ; Patch-Clamp Techniques ; Protein Kinase C-epsilon ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; physiology ; Signal Transduction ; Substance P ; physiology

The present study was designed to investigate the inhibitory effects of intravenous general anesthetic propofol (0.1-3.0 mmol/L) on excitatory synaptic transmission in supraoptic nucleus (SON) neurons of rats, and to explore the underlying mechanisms by using intracellular recording technique and hypothalamic slice preparation. It was observed that stimulation of the dorsolateral region of SON could elicit the postsynaptic potentials (PSPs) in SON neurons. Of the 8 tested SON neurons, the PSPs of 7 (88%, 7/8) neurons were decreased by propofol in a concentration-dependent manner, in terms of the PSPs' amplitude (P < 0.01), area under curve, duration, half-width and 10%-90% decay time (P < 0.05). The PSPs were completely and reversibly abolished by 1.0 mmol/L propofol at 2 out of 7 tested cells. The depolarization responses induced by pressure ejection of exogenous glutamate were reversibly and concentration-dependently decreased by bath application of propofol. The PSPs and glutamate-induced responses recorded simultaneously were reversibly and concentration-dependently decreased by propofol, but 0.3 mmol/L propofol only abolished PSPs. The excitatory postsynaptic potentials (EPSPs) of 7 cells increased in the condition of picrotoxin (30 µmol/L, a GABA(A) receptor antagonist) pretreatment. On this basis, the inhibitory effects of propofol on EPSPs were decreased. These data indicate that the presynaptic and postsynaptic mechanisms may be both involved in the inhibitory effects of propofol on excitatory synaptic transmission in SON neurons. The inhibitory effects of propofol on excitatory synaptic transmission of SON neurons may be related to the activation of GABA(A) receptors, but at a high concentration, propofol may also act directly on glutamate receptors.
Anesthetics, Intravenous ; pharmacology ; Animals ; Excitatory Postsynaptic Potentials ; drug effects ; GABA-A Receptor Antagonists ; pharmacology ; Glutamic Acid ; pharmacology ; In Vitro Techniques ; Neurons ; drug effects ; Propofol ; pharmacology ; Rats ; Receptors, Glutamate ; metabolism ; Supraoptic Nucleus ; cytology

BACKGROUNDOur previous study has confirmed that one bout of exhaustion (Ex) can cause hippocampus neurocyte damage, excessive apoptosis, and dysfunction. Its initial reason is intracellular calcium overload in hippocampus triggered by N-methyl-D-aspartic acid receptor (NMDAR) over-activation. NMDAR activation can be suppressed by γ-aminobutyric acid (A) receptor (GABAAR). Whether GABAAR can prevent intense exercise-induced hippocampus apoptosis, damage, or dysfunction will be studied in this study.

METHODSAccording to dose test, rats were randomly divided into control (Con), Ex, muscimol (MUS, 0.1 mg/kg) and bicuculline (BIC, 0.5 mg/kg) groups, then all rats underwent once swimming Ex except ones in Con group only underwent training. Intracellular free calcium concentration ([Ca2+]i) was measured by Fura-2-acetoxymethyl ester; glial librillary acidic protein (GFAP) and synaptophysin (SYP) immunofluorescence were also performed; apoptosis were displayed by dUTP nick end labeling (TUNEL) stain; endoplasmic reticulum stress-induced apoptosis pathway was detected by Western blotting analysis; Morris water maze was used to detect learning ability and spatial memory.

RESULTSThe appropriate dose was 0.1 mg/kg for MUS and 0.5 mg/kg for BIC. Ex group showed significantly increased [Ca2+]i and astrogliosis; TUNEL positive cells and levels of GFAP, B cell lymphoma-2 (Bcl-2) associated X protein (Bax), caspase-3, caspase-12 cleavage, CCAAT/enhancer binding protein homologous protein (CHOP), and p-Jun amino-terminal kinase (p-JNK) in Ex group also raised significantly compared to Con group, while SYP, synapse plasticity, and Bcl-2 levels in Ex group were significantly lower than those in Con group. These indexes were back to normal in MUS group. BIC group had the highest levels of [Ca2+]i, astrogliosis, TUNEL positive cell, GFAP, Bax, caspase-3, caspase-12 cleavage, CHOP, and p-JNK, it also gained the lowest SYP, synapse plasticity, and Bcl-2 levels among all groups. Water maze test showed that Ex group had longer escape latency (EL) and less quadrant dwell time than Con group; all indexes between MUS and Con groups had no significant differences; BIC had the longest EL and least quadrant dwell time among all groups.

CONCLUSIONSActivation of GABAA R could prevent intense exercise-induced synapses damage, excessive apoptosis, and dysfunction of hippocampus.

Animals ; Apoptosis ; physiology ; Body Weight ; physiology ; Endoplasmic Reticulum Stress ; physiology ; Hippocampus ; metabolism ; Male ; Physical Exertion ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA ; genetics ; metabolism ; Synapses ; pathology

OBJECTIVE: To investigate mRNA expression of GABAa receptor(GABAaR) subunits in the rat cochlear spiral ganglion neurons (SGN) and explore the effect of sodium salicylate (SS) on the expression of GABAaR subunits. METHOD: The realtime fluorescent quantitative PCR (FQ-PCR) was used to detect mRNA expression of twelve GABAaR subunits in the newborn rat SGN and then investigate mRNA expression of GABAaR subunits after treatment with 5 mmol/L SS for 15 min, 30 min, 1 h, 3 h and 6 h in the primary culture SGN. RESULT: (1) GABAaR subunits of α1-6, β1-3, and γ1-3 were detected in the SGN, and the expression of GABAaR subunits was lower than those in the cerebral cortex. In the subunit α family of GABAaR, the expression rank was α2>α3/α5>α4>a1>α6, and the expression of α3 and α5 had no difference (P>0. 05). In the subunit β family, the expression rank was β3>β2>β1. In the subunit γ family, the expression rank was γ1>γ2>γ3. (2) The expression of all subunits of GABAa receptor was obviously fluctuated excepting subunit α5 after treatment with SS. At 15 min post-SS, α1, α2 , β1 and γ1-3 were upregulated, and α3 was downregulated; At 30 min post-SS, α3, β1 and β3 were upregulated, and γ1 was downregulated; At 1 h post-SS, β2 was upregulated and γ3 was downregulated; At 3 h post-SS, β1 and β2 were upregulated, and α3 and γ2 were downregulated; At 6 h post-SS, αl, α3 ,β2, β3 and γ1 were upregulated, and α2, α4 and β1 were downregulated. CONCLUSION The mRNA of GABAaR was expressed in the rat SGN, and the expression of GABAaR subunits was lower in SGN than the cerebral cortex. SS could alter the GABAaR expression quantity in rat SGN; Most of the subunits expression were elevated obviously in the early post SS (15 min), followed by a slight fluctuation.
Animals ; Cells, Cultured ; Cochlea ; cytology ; In Situ Hybridization ; Neurons ; drug effects ; RNA, Messenger ; Rats ; Receptors, GABA-A ; metabolism ; Sodium Salicylate ; pharmacology ; Spiral Ganglion ; drug effects