Expression of 5-HT(2A) receptor mRNA in some nuclei of brain stem enhanced in monoarthritic rats

The present study was designed to investigate the expression of 5-hydroxytryptamine (5-HT) (2A) receptor mRNA in the nucleus of raphe magnus (NRM), ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) in a monoarthritic rat model using an in situ hybridization technique. 5-HT(2A) receptor mRNA was expressed with low to moderate levels in NRM, vlPAG and DRN. After complete Freund's adjuvant (CFA)-induced monoarthritis, a significant increase in 5-HT(2A) receptor mRNA expression was observed in bilateral NRM, vlPAG and DRN, and the change lasted, at least, for 2 weeks. This result indicates that the synthesis of 5-HT(2A) receptor is enhanced in bilateral NRM, vlPAG and DRN of monoarthritic rats, suggesting that 5-HT(2A) receptor is involved in the central regulation of pain transmission following CFA-induced chronic inflammation.     

Alterations of GABAA receptor subunit mRNA levels associated with increases in punished responding induced by acute alprazolam administration: an in situ hybridization study

Changes in the mRNA encoding α1, α2, β2 and γ2 subunits of the GABA_A receptor associated with the anxiolytic effects of alprazolam were measured in 20 brain regions using in situ hybridization techniques. Compared to non-punished controls, punishment decreased α1 mRNA levels in two nuclei of the amygdala, the cerebral cortex, and the mediodorsal thalamic nucleus and decreased α2 mRNA levels in the hippocampus. Punishment increased β2 mRNA levels in ventroposterior thalamic nucleus and γ2 mRNA levels in the CA2 area of the hippocampus. All of these effects were reversed when alprazolam increased punished responding, while alprazolam alone had no effect on either non-punished responding or GABA_A receptor subunit regulation in these brain regions. Some brain regions that were unaffected by punishment were altered by alprazolam plus punishment. These results demonstrate that punishment and alprazolam can produce reciprocal changes in the mRNA levels for some subunits of the GABA_A receptor. These changes may alter GABAergic synaptic inhibition by altering the density of GABA_A receptors or their efficacy to bind drugs. They suggest that the underlying mechanisms by which drugs affect behavior can depend upon the conditions under which behavior is assessed.     

Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area

We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.     

Origins of GABA(B) receptor-like immunoreactive terminals in the rat spinal dorsal horn

By means of immunohistochemistry for gamma-aminobutyric acid receptor B subtype (GABA(B)R), the origins of GABA(B)R-like immunoreactive (GABA(B)R-LI) terminals in the rat spinal dorsal horn were investigated. After dorsal root rhizotomy and/or spinal cord hemisection, the densities of GABA(B)R-LI terminals were remarkably depleted in the ipsilateral superficial dorsal horn of relevant segments, whereas GABA(B)R-LI neurons and sparsely distributed GABA(B)R-LI terminals remained. After injection of Fluoro-Gold (FG) into the left side of superficial lumbar dorsal horn, FG retrograde-labeled neurons were mainly observed in the ipsilateral rostral ventromedial medulla (RVM) and brainstem raphe nuclei. Some of the FG-labeled neurons, especially in the RVM, exhibited GABA(B)R-like immunoreactivity. Additionally, immunofluorescence histochemical double-staining revealed that the majority of GABA(B)R-LI neurons in the periaqueductal gray (PAG), RVM and brainstem raphe nuclei showed 5-hydroxytryptamine (5-HT)-like immunoreactivity. The present study morphologically proves that GABA(B)R-LI terminals in the spinal dorsal horn originate from peripheral afferents, intrinsic neurons and supraspinal structures; GABA(B)R and 5-HT co-exist in many neurons in the PAG, RVM and brainstem raphe nuclei. Considering that PAG, RVM, brainstem raphe nuclei and spinal dorsal horn are important structures involved in the pain modulation, we suggest that the descending pain modulation system might be mediated, at least in part, by GABA(B)R.     

Kindling alters entorhinal cortex-hippocampal interaction by increased efficacy of presynaptic GABA(B) autoreceptors in layer III of the entorhinal cortex

We studied the effect of kindling, a model of temporal lobe epilepsy, on the frequency-dependent information transfer from the entorhinal cortex to the hippocampus in vitro. In control rats repetitive synaptic activation of layer III projection cells resulted in a frequency dependent depression of the synaptic transfer of action potentials to the hippocampus. One-to-two-days after kindling this effect was strongly reduced. Although no substantial change in synaptic inhibition upon single electrical stimulation was detected in kindled rats, there was a significant depression in the prolonged inhibition following high frequency stimulation. In kindled animals, paired-pulse depression (PPD) of stimulus-evoked IPSCs in layer III neurons was significantly stronger than in control rats. The increase of PPD is most likely caused by an increased presynaptic GABA(B) receptor-mediated autoinhibition. In kindled animals activation of presynaptic GABA(B) receptors by baclofen (10 microM) suppressed monosynaptic IPSCs significantly more than in control rats. In contrast, activation of postsynaptic GABA(B) receptors by baclofen was accompanied by comparable changes of the membrane conductance in both animal groups. Thus, in kindled animals activation of the layer III-CA1 pathway is facilitated by an increased GABA(B) receptor-mediated autoinhibition leading to an enhanced activation of the monosynaptic EC-CA1 pathway.     

GABA(A) receptor subunit and gephyrin protein changes differ in the globus pallidus in Huntington's diseased brain

Immunoreactivity of GABA(A) receptor subunits and the receptor anchoring protein gephyrin was investigated in the human globus pallidus using antibodies raised against the alpha(1) and gamma(2) subunits of the GABA(A) receptor complex and gephyrin. The results revealed increased GABA(A) receptor subunit immunoreactivity and unchanged levels of gephyrin immunoreactivity in Huntington's diseased (HD) globus pallidus (GP). The results demonstrate that gephyrin immunoreactivity did not change in unison with GABA(A) receptor changes in HD, suggesting that the receptor anchoring protein gephyrin is unaltered and maintains a stable lattice structure in the face of GABA(A) receptor changes in HD.     

Subcellular regulation of metabotropic GABA receptors in the developing cerebellum

Our understanding of GABAergic and glutamatergic neurotransmission in the CNS has been greatly influenced with the discovery and subsequent investigations of the metabotropic gamma-aminobutyric acid (B) (GABA(B)) receptors. These G-protein coupled receptors mediate slow inhibitory neurotransmission and are widely expressed and distributed in the cerebellum, where they play critical roles in neuronal excitability and modulation of synaptic neurotransmission. Their function is modulated by interaction with effector ion channels, notably inwardly rectifying K(+) channels and voltage-gated Ca(2+) channels. The receptors are encoded by two distinct subunits, GABA(B1) and GABA(B2), both of which are required in order to function normally in vivo, as shown in recombinant expression systems and in GABA(B1) -/- mice. The GABA(B1) and GABA(B2) subunits exhibit overlapping distributions in the cerebellar cortex, both at pre- and postsynaptic sites, during development and adulthood. They are in particular abundant in Purkinje cells prior to synaptogenesis and throughout postnatal development. Using high-resolution immunohistochemical techniques at the electron microscopic level in combination with quantitative analysis and three-dimensional reconstructions, it has recently been demonstrated that GABA(B) receptors undergo changes in localization on the surface of Purkinje cell dendrites and spines during postnatal development in association with the establishment and maturation of excitatory synapses. Due to this dynamic regulation, the highest densities of GABA(B1) and GABA(B2) subunits occur around the glutamatergic synapses between Purkinje cell spines and parallel fibre varicosities. This review highlights recent studies that have shed further light on the subcellular localization during postnatal development and the cell surface dynamics of GABA(B) receptors.     

Opposite effects of a GABA(B) antagonist in two models of epileptic seizures in developing rats

The action of a GABA(B) antagonist CGP 35348 and a GABA(B) agonist baclofen was studied in two models of epileptic seizures characterized by EEG spike-and-wave rhythm in freely moving immature rats. Rhythmic metrazol activity (RMA, model of human absences) was induced by low systemic dose of pentylenetetrazol (PTZ) in 18- and 25-day-old rats, epileptic after discharges (ADs, model of human myoclonic seizures) were elicited by electrical stimulation of sensorimotor cortex in rat pups 12, 18, and 25 days old. CGP 35348 (50, 100 and 200 mg/kg i.p.) suppressed RMA in both age groups in a dose-dependent manner. Simultaneously it increased the incidence of clonic seizures, potentiating thus an effect of PTZ. Baclofen (1, 3 and 6 mg/kg i.p.) augmented markedly RMA in 25-day-old rats. On the contrary, baclofen suppressed RMA in a part of 18-day-old animals. Incidence of seizures was not changed by baclofen in either age group. As ADs are concerned CGP 35348 (100 and 200 mg/kg i.p.) exhibited a proconvulsant action, baclofen (3, 6 or 12 mg/kg i.p.) was anticonvulsant, but again an irregularity of action was found in 18-day-old rats. The role of GABA(B)-mediated inhibition in epileptogenesis depends on the type of seizures and also on the stage of maturation.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of NOS inhibitor

In the present study, we have investigated the effects of prolonged inhibition of nitric oxide synthase (NOS) by infusion of NOS inhibitor, L-nitroarginine, to examine the pentobarbital-induced sleep, modulation of GABA(A) receptor binding, and GABA(A) receptor subunit mRNA level in rat brain. Pre-treatment with L-nitroarginine 30 min before pentobarbital treatment (60 mg/kg, i.p.) significantly increased the duration of sleep in rats. However, the duration of pentobarbital-induced sleep was shortened by the prolonged infusion of L-nitroarginine into ventricle. We have investigated the effect of NOS inhibitor on GABA(A) receptor binding characteristics in discrete areas of brain regions by using autoradiographic and in situ hybridization techniques. Rats were infused with L-nitroarginine (10, 100 pmol/10 microl/h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps. The levels of [(3)H]muscimol and [(3)H]flunitrazepam binding were markedly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, there was no change in the level of [(35)S]TBPS binding. The levels of beta2-subunit were elevated in the cortex, brainstem, and cerebellar granule layers. By contrast, the levels of beta3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in L-nitroarginine-infused rats. Following L-nitroarginine treatment, the levels of alpha6- and delta-subunits which were strictly localized to the cerebellum, were not changed in the cerebellar granule layer. These results show that the prolonged inhibition of NOS by L-nitroarginine-infusion markedly elevates [(3)H]muscimol and [(3)H]flunitrazepam binding throughout the brain, and alters GABA(A) receptor subunit mRNA levels in different directions. Chronic inhibition of NO generation has differential effects on the various expressions of GABA(A) receptor subunits. These suggest the involvement of different regulatory mechanisms for the NO-induced expression of GABA(A) receptor.     

Circadian changes in PACAP type 1 (PAC1) receptor mRNA in the rat suprachiasmatic and supraoptic nuclei

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP), denoted as PAC₁, is expressed in the suprachiasmatic nuclei (SCN). Since the circadian clock demonstrates phase-dependent sensitivity to PACAP, we have used in situ hybridization histochemistry to examine whether PAC₁ mRNA is differentially expressed in the rat SCN across the 24-h cycle. There was a significant variation in PAC₁ mRNA within the SCN and supraoptic nuclei during the light–dark cycle and in constant darkness, with peaks at the middle of both the real and subjective day and night; no significant variation was observed in the cingulate cortex. The results suggest that the phase-dependent actions of PACAP on the clock may involve phase-specific changes in the availability of PAC₁ receptors within the SCN.     

Changes of GABA(A) receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of 6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

GABA(A) and GABA(B) receptors mediated inhibition affect the pattern adaptation of relay cells in the dorsal lateral geniculate nucleus (LGNd) of cats

Pattern adaptation is very important for visual function, while the mechanisms that mediate pattern adaptation, especially in the dorsal lateral geniculate nucleus (LGNd), are still unclear. Iontophoresis of the antagonists and agonists of GABA receptors were employed to separately investigate the contribution of GABA(A) and GABA(B) receptors to pattern adaptation of LGNd cells. When GABA(A) receptors were blocked by bicuculline both the response amplitude of LGNd cells and the degree of adaptation increased significantly. Many neurons showing no pattern adaptation under the normal condition became adapted to a prolonged stimulus. Moreover, the proportion of cells showing adaptation doubled (from 40 to 88%). The mean adaptation index (AI, adapted response amplitude/original response amplitude) was 0.82 during bicuculline application, compared with 0.92 under the control condition. In additional, iontophoresis of baclofen, a selective GABA(B) receptor agonist, decreased the mean response amplitude to grating stimuli to 53% of normal. Nearly half of the neurons increased their adaptation index following baclofen administration and the mean AI increased from 0.89 to 1.01. Iontophoresis of GABA(B) receptor antagonist (CGP35348) could abolish this effect, though it had no significant effect on visual response amplitude and pattern adaptation itself. Iontophoresis of another GABA(B) receptor antagonist, 2-OH-saclofen, also had no significant effect on visual response amplitude and pattern adaptation. These results suggest that both GABA(A) receptors and GABA(B) receptors modulate the pattern adaptation of LGNd cells and are involved in synaptic plasticity.     

Involvement of GABA(A) receptor in modulation of jaw muscle activity evoked by mustard oil application to the rat temporomandibular joint

The effect of intrathecal administration of the GABA(A) receptor antagonist bicuculline methylbromide on jaw muscle electromyographic (EMG) activity evoked by mustard oil injection into the rat temporomandibular joint was studied. Bicuculline given prior to mustard oil augmented the EMG activity evoked by mustard oil, and "rekindling" of EMG activity was induced by bicuculline given 30 min after mustard oil. These results suggest that central GABA(A) receptors modulate reflex responses to noxious craniofacial stimuli.     

Transition to absence seizures and the role of GABA(A) receptors

Absence seizures appear to be initiated in a putative cortical 'initiation site' by the expression of medium-amplitude 5-9Hz oscillations, which may in part be due to a decreased phasic GABA(A) receptor function. These oscillations rapidly spread to other cortical areas and to the thalamus, leading to fully developed generalized spike and wave discharges. In thalamocortical neurons of genetic models, phasic GABA(A) inhibition is either unchanged or increased, whereas tonic GABA(A) inhibition is increased both in genetic and pharmacological models. This enhanced tonic inhibition is required for absence seizure generation, and in genetic models it results from a malfunction in the astrocytic GABA transporter GAT-1. Contradictory results from inbred and transgenic animals still do not allow us to draw firm conclusions on changes in phasic GABA(A) inhibition in the GABAergic neurons of the nucleus reticularis thalami. Mathematical modelling may enhance our understanding of these competing hypotheses, by permitting investigations of their mechanistic aspects, hence enabling a greater understanding of the processes underlying seizure generation and evolution.     

GABA(B) receptor activation inhibits N- and P/Q-type calcium channels in cultured lamprey sensory neurons

In lamprey, sensory transmission from mechanosensory receptors (dorsal cells) to central neurons is presynaptically inhibited by GABA(B) receptor activation. The mechanisms underlying this effect were investigated using isolated dorsal cells, where voltage-dependent calcium currents were recorded in the whole-cell configuration. Activation of GABA(B) receptors by baclofen decreased the peak amplitude of high voltage-activated (HVA) calcium currents and slowed the activation phase. The role of G-proteins in mediating the effects of baclofen was examined. Intracellular dialysis of GTPgammaS occluded the effects of baclofen. Intracellular dialysis of GDPbetaS and preincubation in pertussis toxin both attenuated the effect of baclofen. Specific calcium channel blockers were used to study the types of HVA calcium channels involved in the GABA(B)-mediated modulation. The baclofen-induced inhibition was not affected by the L-type calcium channel antagonist nimodipine, but was partially blocked by the N-type blocker omega-conotoxin GVIA, and completely occluded by omega-conotoxin MVIIC, a blocker of both N- and P/Q-type channels. The pharmacology of dorsal cell GABA(B) receptors was studied using two agonists, baclofen and CGP 27492, and four antagonists, CGP 35348, CGP 55845, phaclofen and saclofen. The inhibition induced by either of the two agonists was blocked by CGP 55845, phaclofen and saclofen. The antagonist CGP 35348 completely blocked the inhibition of HVA calcium current induced by the agonist CGP 27492, but had no effect on baclofen-induced GABA(B) receptor activation. This study thus demonstrates that GABA(B) receptor activation in lamprey mechanosensory neurons inhibits N- and P/Q-type calcium channels in a voltage- and G-protein-dependent manner.     

Glutamic acid decarboxylase mRNA in the suprachiasmatic nucleus of rats housed in constant darkness

This study demonstrates that the levels of the mRNAs encoding the two isoforms of glutamic acid decarboxylase (GAD) (i.e., GAD65 and GAD67) do not differ over the circadian activity cycle in the suprachiasmatic nucleus (SCN) of rats housed in constant darkness. These data indicate that the rhythmic expression of GAD56 mRNA previously observed in animals housed in a light:dark cycle [K.L. Huhman, A.C. Hennessey, H.E. Albers, Rhythms of glutamic acid decarboxylase mRNA in the suprachiasmatic nucleus, J. Biol. Rhythms 11 (1996) 311-316.] is the result of the activity of retinal afferents.