Resveratrol noncompetitively inhibits glycine receptor-mediated currents in neurons of rat central auditory neurons.

Resveratrol, a naturally occurring stilbene found in red wine, is known to modulate the activity of several types of ion channels and membrane receptors, including Ca2+, K+, and Na+ ion channels. However, little is known about the effects of resveratrol on some important receptors, such as glycine receptors and GABAA receptors, in the central nervous system (CNS). In the present study, the effects of resveratrol on glycine receptor or GABAA receptor-mediated currents in cultured rat inferior colliculus (IC) and auditory cortex (AC) neurons were studied using whole-cell voltage-clamp recordings. Resveratrol itself did not evoke any currents in IC neurons but it reversibly decreased the amplitude of glycine-induced current (IGly) in a concentration-dependent manner. Resveratrol did not change the reversal potential of IGly but it shifted the concentration-response relationship to the right without changing the Hill coefficient and with decreasing the maximum response of IGly. Interestingly, resveratrol inhibited the amplitude of IGly but not that of GABA-induced current (IGABA) in AC neurons. More importantly, resveratrol inhibited GlyR-mediated but not GABAAR-mediated inhibitory postsynaptic currents in IC neurons using brain slice recordings. Together, these results demonstrate that resveratrol noncompetitively inhibits IGly in auditory neurons by decreasing the affinity of glycine to its receptor. These findings suggest that the native glycine receptors but not GABAA receptors in central neurons are targets of resveratrol during clinical administrations.

Cross-talk pattern between GABAA- and glycine-receptors in CNS neurons is shaped by their relative expression levels.

GABAA receptors (GABAARs) and glycine receptors (GlyRs) are two principal inhibitory chloride ion channels in the central nervous system. The two receptors do not function independently but cross-talk to each other, i.e., the activation of one receptor would inhibit the other. This cross-talk is present in different patterns across various regions in the central nervous system; however, the factor that determines these patterns is not understood. Here, we show that the pattern of cross-talk between the two receptors is shaped by their relative expression level in a neuron: a higher expression level correlates with louder talk. In line with a tendency of decrease in expression level of GlyRs and increase in expression level of GABAARs from the spinal cord, the brainstem to the neocortex, GlyRs talked much louder (i.e. produced greater inhibition) than GABAARs (one-way pattern) in spinal cord neurons, about equally loud as GABAARs (symmetric pattern) in inferior colliculus neurons and less loud (i.e. less inhibition) than GABAARs (asymmetric pattern) in auditory cortex neurons. Overexpression of GlyRs in inferior colliculus neurons produced an asymmetric pattern that should otherwise have been observed in spinal cord neurons. These expression level-dependent patterns of cross-talk between the two receptors may suggest how the central nervous system uses an alternative mechanism to maintain a delicate level of inhibition through adjusting the proportion of the two receptors in a neuron along its pathway.

Persistent pain facilitates response to morphine reward by downregulation of central amygdala GABAergic function.

Opioid-based analgesics are widely used for treating chronic pain, but opioids are highly addictive when repeatedly used because of their strong rewarding effects. In recent years, abuse of prescription opioids has dramatically increased, including incidences of misuse of opioid drugs prescribed for pain control. Despite this issue in current clinical pain management, it remains unknown how pain influences the abuse liability of prescription opioids. Pain as aversive experience may affect opioid reward of positive emotion through common brain sites involved in emotion processing. In this study, on a rat model of chronic pain, we determined how persistent pain altered behavioral responses to morphine reward measured by the paradigm of unbiased conditioned place preference (CPP), focusing on GABAergic synaptic activity in neurons of the central nucleus of the amygdala (CeA), an important brain region for emotional processing of both pain and reward. We found that pain reduced the minimum number of morphine-conditioning sessions required for inducing CPP behavior. Both pain and morphine conditioning that elicited CPP inhibited GABA synaptic transmission in CeA neurons. Pharmacological activation of CeA GABAA receptors reduced the pain and inhibited CPP induced both by an effective dose of morphine and by a sub-threshold dose of morphine under pain condition. Furthermore, inhibition of CeA GABAA receptors mimicked the pain effect, rendering the sub-threshold dose of morphine effective in CPP induction. These findings suggest that pain facilitates behavioral responses to morphine reward by predisposing the inhibitory GABA function in the CeA circuitry involved in the behavior of opioid reward.

Brain-derived neurotrophic factor-mediated downregulation of brainstem K+-Cl- cotransporter and cell-type-specific GABA impairment for activation of descending pain facilitation.

Chronic pain is thought to be partly caused by a loss of GABAergic inhibition and resultant neuronal hyperactivation in the central pain-modulating system, but the underlying mechanisms for pain-modulating neurons in the brain are unclear. In this study, we investigated the cellular mechanisms for activation of brainstem descending pain facilitation in rats under persistent pain conditions. In the nucleus raphe magnus (NRM), a critical relay in the brain's descending pain-modulating system, persistent inflammatory pain induced by complete Freund's adjuvant decreased the protein level of K(+)-Cl(-) cotransporter (KCC2) in both total and synaptosomal preparations. Persistent pain also shifted the equilibrium potential of GABAergic inhibitory postsynaptic current (EIPSC) to a more positive level and increased the firing of evoked action potentials selectively in µ-opioid receptor (MOR)-expressing NRM neurons, but not in MOR-lacking NRM neurons. Microinjection of brain-derived neurotrophic factor (BDNF) into the NRM inhibited the KCC2 protein level in the NRM, and both BDNF administration and KCC2 inhibition by furosemide mimicked the pain-induced effects on EIPSC and excitability in MOR-expressing neurons. Furthermore, inhibiting BDNF signaling by NRM infusion of tyrosine receptor kinase B-IgG or blocking KCC2 with furosemide prevented these pain effects in MOR-expressing neurons. These findings demonstrate a cellular mechanism by which the hyperactivity of NRM MOR-expressing neurons, presumably responsible for descending pain facilitation, contributes to pain sensitization through the signaling cascade of BDNF-KCC2-GABA impairment in the development of chronic pain.

Inhibitory effects of amiloride on the current mediated by native GABA(A) receptors in cultured neurons of rat inferior colliculus.

1. The diuretic amiloride is known to modulate the activity of several types of ion channels and membrane receptors in addition to its inhibitory effects on many ion transport systems. However, the effects of amiloride on some important ion channels and receptors, such as GABA(A) receptors, in the central nervous system have not been characterized. 2. In the present study, we investigated the functional action of amiloride on native GABA(A) receptors in cultured neurons of rat inferior colliculus using whole-cell patch-clamp recordings. 3. Amiloride reversibly inhibited the amplitude of the GABA-induced current (I(GABA)) in a concentration-dependent manner (IC(50) 454 +/- 24 micromol/L) under conditions of voltage-clamp with a holding potential at -60 mV. The inhibition depended on drug application mode and was independent of membrane potential. Amiloride did not change the reversal potential of I(GABA). Moreover, amiloride induced a parallel right-ward shift in the concentration-response curve for I(GABA) without altering the maximal value and Hill coefficient. 4. The present study shows that amiloride competitively inhibits the current mediated by native GABA(A) receptors in the brain region, probably via a direct action on GABA-binding sites on the receptor. The findings suggest that the functional actions of amiloride on GABA(A) receptors may result in possible side-effects on the central nervous system in the case of direct application of this drug into the cerebrospinal fluid for treatment of diseases such as brain tumours.

Fluoxetine inhibition of glycine receptor activity in rat hippocampal neurons.

Fluoxetine is a selective serotonin reuptake inhibitor widely used for treating depression. However, fluoxetine treatment may lead to seizures at higher doses, which underlying mechanism remains largely unknown. In this study, we examined the effects of fluoxetine on glycine receptor (GlyR) activity. Using the whole-cell patch-clamp recording method, we found that fluoxetine and its metabolite norfluoxetine inhibited glycine-induced currents in cultured rat hippocampal neurons. This inhibition was dose-dependent, and voltage-independent. Fluoxetine shifted the glycine concentration-response curve to the right without altering the maximal current. Both Lineweaver-Burk and Schild plots suggest competitive inhibition. The amount of fluoxetine inhibition significantly increased when homomeric GlyRs were selectively inhibited with picrotoxin. Moreover, fluoxetine inhibited the current mediated by heteromeric alpha2beta- but not homomeric alpha2-GlyRs transiently expressed in HEK293T cells. These results suggest that fluoxetine is a competitive and subtype-selective GlyR inhibitor, which may explain its capacity to induce seizures.

Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex.

Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.

Amiloride attenuates glycine-induced currents in cultured neurons of rat inferior colliculus.

Amiloride, a potassium sparing diuretic, is well known to interact with many ion transport systems and modulate the activity of several membrane receptors. However, relatively little information is available as to how amiloride affects membrane receptors of neurons in the brain areas. In the present study, we investigated the effects of amiloride on glycine-induced currents (I(Gly)) in cultured neurons of rat inferior colliculus with whole-cell patch-clamp recordings. Amiloride itself did not activate any current across the neuronal membrane but it reversibly inhibited the amplitude of the I(Gly) in a reversible and concentration-dependent manner, with an IC(50) of 487.4+/-25.3microM (n=5). Amiloride shifted the concentration-response relationship to the right without changing Hill coefficient and without changing the maximum response of the I(Gly). The pre-perfusion of amiloride produced an inhibitory effect on the I(Gly). In addition, amiloride was shown with a voltage ramp protocol to significantly reduce the conductance induced by glycine but not to change the reversal potential of the I(Gly). These results demonstrate that amiloride competitively inhibits the I(Gly) in rat inferior colliculus neurons by decreasing the affinity of glycine to its receptor. Our finding suggests that attention should be paid to the possible side effects of amiloride used as a drug on brain functions in the case of a defective blood-brain barrier and in the case of direct application of this drug into the cerebrospinal fluid for treatment of brain tumors.