GABAB binding sites in early adult and aging rat brain

The effect of aging on GABA_B binding was investigated in rat brain. Receptor autoradiography was used to investigate both GABA_B and GABA_A binding at 2 months, 3 months, 13 months, and 23 months. GABA_B binding decreases significantly between 2 months and 23 months of age, as does GABA_A binding, with was investigated in rat brain. Receptor autoradiography was used to investigate both GABA_B and GABA_A binding at 2 months, 3 months, 13 months, and 23 months. GABA_B binding decreases significantly between 2 months and 23 months of age, as does GABA_A binding, with greatest decrease between 2 and 3 months. The decrease in GABA_B binding appears to be due to a decrease in binding site affinity rather than a decrease in receptor density. The noncompetitive GABA_B antagonist zinc, the competitive GABA_B antagonist CGP 35348, and the guanyl nucleotide analogue GTP-γ-S all inhibit GABA_B binding identically in 2 month and 23 month brain. These data indicate subtle age-related changes in the GABA_B binding in early adult life but little change with senescence.     

GABA(A) receptor facilitation of neurokinin release from primary afferent terminals in the rat spinal cord

Our goal was to test the following hypotheses: 1) GABA_A receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABA_B receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABA_A receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC₅₀] 3 μM) and isoguvacine (EC₅₀ 4.5 μM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABA_A receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC₅₀] 2 μM) and picrotoxin (IC₅₀ 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABA_A receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl⁻, low Ca²⁺, Ca²⁺ channel blockers and N-methyl-d-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABA_A receptors. The GABA_B agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC₅₀ 1.5 μM), whereas the GABA_B receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC₅₀ 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABA_B receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABA_A receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABA_B receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABA_A receptors and inhibiting GABA_B receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.     

Age-related decrease in GABAB receptor binding in the Fischer 344 rat i inferior colliculus

Quantitative receptor autoradiography was used to asses GABA_B receptor binding in three primary subdivisions of the inferior colliculus (IC): dorsal cortex (DCIC), external cortex (ECIC), and the central nucleus (CIC) of 3-, 18–20- and 26-month-old Fischer 344 rats. GABA_B binding sites were localized using [³H]GABA in the presence of a saturating concentration of isoguvacine, a selective GABA_A receptor agonist, to displace [³H]GABA bound to GABA_A receptor sites. In the three IC subdivisions examined, GABA_B receptor binding was significantly reduced in 26-month-old rats when compared to 3-month-old rats (DCIC, −44%; ECIC, −36%; CIC, −32%; p .05 For comparison, GABA_B binding was determined in the portion of cerebellum located in the recess of the IC. In the molecular layer of this region, there were no statistically significant differences in receptor binding between 3, 18–20- and 26-month-old rats. In addition, there was not a significant age-related change in the cross-sectional area of the IC. These findings provide additional evidence to support the existence of selective age-related changes in GABA neurotransmitter function in the rat IC.     

Inhibitory responses of rat basolateral amygdaloid neurons recorded in vitro

The purpose of the present study was to characterize the ionic and pharmacological basis of the actions of synaptically released and exogenously applied GABA in basolateral amygdaloid pyramidal cells in vitro. Stimulation of forebrain afferents to pyramidal neurons in the basolateral amygdala evoked an excitatory postsynaptic potential followed by early and late inhibitory postsynaptic potentials. The early inhibitory postsynaptic potential had a reversal potential near −70 mV, was sensitive to changes in the chloride gradient across the membrane and was blocked by the GABA_A antagonists picrotoxin and bicuculline methiodide but not by the GABA_B antagonists phaclofen or 2-hydroxysaclofen. In contrast, the late inhibitory postsynaptic potential had a reversal potential of approximately −95 mV and was markedly reduced or abolished by GABA_B antagonists.

Pressure application of GABA to the surface of the slice typically elicited a triphasic response in basolateral amygdaloid pyramidal neurons consisting of a short-latency hyperpolarization that preceded or was superimposed on a membrane depolarization followed by a longer latency hyperpolarization. Each of the responses was associated with an increase in membrane conductance. Determinations of the reversal potential, ionic dependency and sensitivity to pharmacological blockade of each component of the GABA-induced response revealed that the initial hyperpolarizing (E_rev approximately −70 mV) and depolarizing (E_rev approximately −55 mV) responses were mediated by a GABA_A-mediated increase in chloride conductance, whereas the late hyperpolarizing response (E_rev approximately −82 mV) to GABA arose from a GABA_B-mediated increase in potassium conductance. Experiments in which GABA was applied at various locations on the cell suggested that the short-latency hyperpolarization resulted from activation of somatic GABA receptors, whereas the depolarizing and late hyperpolarizing responses were generated primarily in the dendrites. In contrast to the complex membrane response profile elicited by GABA, pressure ejection of the GABA_B agonist baclofen produced only membrane hyperpolarizations.

Taken together, these results suggest that inhibitory responses that are recorded in basolateral amygdaloid pyramidal cells are mediated by activation of both GABA_A and GABA_B receptors. Consistent with findings elsewhere in the CNS, the early inhibitory postsynaptic potential and initial hyperpolarization and depolarizing response to local GABA application appear to involve a GABA_A-mediated increase in chloride conductance, whereas the late inhibitory postsynaptic potential and the late hyperpolarizing response to GABA arise from a GABA_B-mediated increase in potassium conductance.     

GABAA and GABAB antagonists prevent the opioid inhibition of endogenous acetylcholine release evoked by glutamate from rat neostriatal slices

We have examined the role of the GABAergic system in the opioid inhibition of endogenous acetylcholine (ACh) release from rat neostriatal slices by blocking either γ-aminobutyric acid-A (GABA_A) or GABA_B receptors. GABAergic antagonists (bicuculline or phaclofen) completely blocked μ- (morphine or DAGO) and δ-opioid (DPDPE) inhibition of glutamate-evoked endogenous ACh release in a concentration-dependent manner. However, GABA antagonists were ineffective in blocking the opioid inhibition of potassium-evoked endogenous ACh release. These findings point to the important role of the GABAergic system in the regulation of μ- and δ-opioid inhibition of cholinergic neurons stimulated by glutamate.     

精氨酸加压素对大鼠视前区GABA_A受体亚单位磷酸化的影响

目的:研究精氨酸加压素(AVP)对大鼠视前区γ-氨基丁酸(GABA)A型受体(GABA_A受体)亚单位(α、β和γ2)表达和磷酸化的影响。方法:实验分为对照组、AVP组、V1a受体抑制剂+AVP组和V1a受体抑制剂组(均n=10);腹腔注射AVP或V1a受体抑制剂0.5 h后,采用RT-qPCR和Western blot法检测视前区GABA_A受体亚单位(α、β和γ2)表达及磷酸化的变化。结果:与对照组相比,AVP或V1a受体抑制剂组大鼠视前区GABA_A受体亚单位表达均无显著变化;AVP能显著上调视前区GABA_A受体γ2亚单位的磷酸化水平(P0.05);AVP显著增加蛋白激酶C(PKC)和钙/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)表达和磷酸化(P0.01)。结论:外源性AVP不影响GABA_A受体亚单位(α、β和γ2)表达,但主要通过V1a受体激活PKC和CaMKⅡ,影响γ2亚单位磷酸化水平,从而调制视前区GABA_A受体介导的抑制性突触传递。     

Shortened-duration GABAA receptor-mediated synaptic potentials underlie enhanced CA1 excitability in a chronic model of temporal lobe epilepsy

Intracellular recording techniques were used to examine GABA_A receptor-mediated synaptic inhibition in pyramidal cells of the CA1 region of the rat hippocampus in the post-self sustaining limbic status epilepticus model of temporal lobe epilepsy. Orthodromically evoked, monosynaptic inhibitory postsynaptic potentials were recorded in vitro following pharmacological blockade of ionotropic glutamate and GABA_B receptors. Inhibitory postsynaptic potentials from epileptic tissue were kinetically altered relative to controls; both the 10–90% rise-time and width (measured at half-maximum amplitude) were reduced by approximately 50% resulting in significant shortening of duration. The degree of pyramidal cell hyperexcitability, assessed before pharmacological treatment as the number of action potentials evoked by maximum intensity afferent stimulation, correlated significantly with the magnitude of synaptic potential duration reduction determined following blockade of glutamatergic neurotransmission. Bath application of the benzodiazepine type 1 receptor agonist zolpidem reduced post-self sustaining limbic status epilepticus CA1 pyramidal cell hyperexcitability substantially (but not completely) via a marked increase in inhibitory postsynaptic potential area. Post-self sustaining limbic status epilepticus inhibitory postsynaptic potentials which exhibited the most pronounced shortening were augmented by zolpidem to a greater degree than longer duration synaptic potentials. In contrast, zolpidem-induced augmentation of control inhibitort postsynaptic potential area was much less robust.

It is suggested that a deficiency in post-self sustaining limbic status epilepticus GABA_A receptor-mediated synaptic inhibition contributes to a state of partial disinhibition which is a major factor in enhanced CA1 excitability in chronic limbic epilepsy. Possible mechanisms underlying post-self sustaining limbic status epilepticus kinetic abnormalities are discussed.     

Activation of μ-opioid receptor modulates GABAA receptor-mediated currents in isolated spinal dorsal horn neurons

Whole-cell voltage-clamp technique was used to examine the effects of a μ-opioid receptor agonist DAGO (Tyr-D-Ala-Gly-Me-Phe-Gly-ol-enkephalin) on GABA-induced currents in acutely isolated spinal dorsal horn (DH) neurons from laminae I-IV of young rats. We found that a bicuculline-sensitive GABA-induced current was potentiated by DAGO (0.5–500 nM), in a dose-dependent manner, in 62% of the tested cells. The elevated GABA responses outlasted the period of DAGO application, and either recovered within 10 min after the removal of the peptide or persisted for up to 50 min. The potentiating effect of DAGO was reduced or prevented by naloxone and the μ-opioid receptor-selective antagonist β-funaltrexamine. A similar enhancing effect on the membrane currents activated by administration of muscimol, a GABA_A receptor-specific agonist, was produced by DAGO. In addition, a transient depression of GABA responses was observed in 25% of the cells tested. These results indicate that the μ-opioid agonist DAGO modulates the sensitivity of postsynaptic GABA_A receptors in a large proportion of spinal neurons from laminae I–IV, with the major effect being facilitation. The DAGO action could contribute to the regulation of the strength of primary afferent neurotransmission, including nociception.     

The GABAC receptor is present in cone-horizontal cell axon terminals isolated from catfish retina

Whole cell voltage-clamp recordings were performed on isolated terminals and somata from catfish retina to compare the distribution of excitatory and inhibitory receptors in both structures. Saturating concentrations of glutamate or kainate produced small currents in axon terminals, averaging less than 8% of the current evoked in the soma. In contrast, application of high concentrations of gamma-aminobutyric acid (GABA) produced approximately similar current amplitudes in both structures. Based on estimates of membrane surface area, GABA-induced current densities were around 0.05 pA/μm² for both structures. The GABA-activated current in the axon terminal was not blocked by bicuculline or SR95531, but was completely inhibited by picrotoxin. Baclofen did not mimic the GABA effect, but trans-4-aminocrotonic acid (TACA, 300 μM) and muscimol (1 mM) elicited currents of 100 and 40 pA, respectively. These results suggest that the axon terminals of cone-horizontal cells possess GABA_C receptors at a high density, do not possess GABA_A or GABA_B receptors, and have few glutamate receptors. The GABA_C receptors could function as postsynaptic receptors in the inner plexiform layer or as autoreceptors.     

GABAA receptor subunits in the rat hippocampus I: Immunocytochemical distribution of 13 subunits

The GABA_A receptor is a ligand-operated chloride channel. It has a pentameric structure. In mammalian brain different subunits are recruited from four gene subfamilies. Using immunocytochemistry, we investigated the distribution of the 13 GABA_A receptor subunits in the hippocampus of the rat. GABA_A receptor subunits were heterogeneously distributed within different hippocampal subfields. High concentrations of ₁-, ₂-, ₄-, β₃-, γ₂- and δ-immunoreactivities were observed within the molecular layer of the dentate gyrus, representing the dendritic area of the granule cells. In the hippocampus proper, the predominant GABA_A receptor subunits were ₁, ₂, ₅, β₃ and γ₂ that were located throughout the strata radiatum and oriens of CA1 to CA3. Immunocytochemical staining was there less prominent for ₄-, β₁-, β₂-, γ₃- and δ- subunits. In the hippocampus proper, the β₁ subunit was preferentially located in CA2. The ₄- and δ-subunits were somewhat more abundant in CA1 than in CA3. Numerous local circuit neurons in the hippocampus proper and the hilus of the dentate gyrus contained ₁-, β₂-, γ₂- and/or δ-subunits. ₃ and γ₁ were present only in minute amounts and no ₆-IR was detected in the hippocampal formation.

The distribution of the GABA_A receptor subunits indicates the existence of heterogenously constituted GABA_A receptor complexes within various hippocampal subfields, which may exert different physiological or pharmacological properties upon stimulation by GABA or its agonists.     

Decline in striatal dopamine D1 and D2 receptor activation in aged F344 rats

Aging differentially affects receptor function. In the present electrophysiological study we compared neuronal responsiveness to locally applied dopamine D₁ and D₂ receptor agonist in the striatum of female Fischer 344 rats aged 3 and 26–27 months. In a subgroup of the old rats, the nigrostriatal dopamine bundle was destroyed unilaterally with 6-hydroxydopamine (6-OHDA) to assess receptor plasticity in response to denervation. Spontaneous firing rate of striatal neurons was higher in aged compared to young rats. Higher doses of the D₁ agonist SKF 38393 or the D₂ agonist quinpirole were required to elicit a 50% change in firing rate in aged compared to young rats. No difference with SKF 38393 or quinpirole was detected between 6-OHDA denervated and control (nonlesioned) striatum in aged rats. Supersensitivity to D₂ agonists has been reported following 6-OHDA lesions in young rats. These observations suggest that D₂ receptors in aged rat striatum might not be as plastic as in younger rats.     

Modulation by nitric oxide of rat brain GABAA receptors

The effect of nitric oxide (NO) on the function of GABA_A receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABA_A receptors were studied by preparing microsacs and evaluating ³⁶Cl⁻ accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor arginine there was a 15–25% reduction in the V_max for GABA-stimulated ³⁶Cl⁻ accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor . GABA_A receptor mediated Cl⁻ currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and -arginine reduced the Cl⁻ current elicited by 10 μM GABA. The -arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABA_A receptor function in cerebellar granule cells.     

Multiple effects of drugs acting on benzodiazepine receptors

The actions on [³H]noradrenaline release of chlordiazepoxide and FG 7142 were investigated in rat hippocampal synaptosomes. The release evoked by GABA, an action mediated by GABA_A receptors, was enhanced by chlordiazepoxide and depressed by FG 7142. K⁺-evoked release, however, was depressed by both chlordiazepoxide and FG 7142 and occurred in the absence of GABA. The actions on both GABA-evoked and K⁺-evoked release were blocked by Ro 15-1788. The results suggest that the distinction between agonist and inverse agonist applies to the GABA-dependent but not to the GABA-independent action of benzodiazepines.     

Motor activity induced by disinhibition of the primary motor cortex of the rat is blocked by a non-NMDA glutamate receptor antagonist

Application of a GABA_A (γ-aminobutyric acid-A) receptor antagonist through a microdialysis probe into the forelimb primary motor cortex of ketamine-anesthetized rats induced electromyographic activity in the contralateral forelimb. This activity consisted of spontaneous forelimb movements with a frequency of 0.8 ± 0.2 Hz. The motor activity induced by GABA_A receptor blockade was suppressed by application through the dialysis probe of a non-NMDA ( ) receptor antagonist, but not by an NMDA receptor antagonist. Glutamate eliminated the blocking effect of the non-NMDA receptor antagonist upon GABA_A receptor blockade mediated activity. In conclusion, the results show that an excitatory input to the motor cortical output is mediated through a non-NMDA receptor, therefore the effects of cortical disinhibition may be controlled by non-NMDA receptors.     

Ionotropic glutamate receptors regulating labeled acetylcholine release from rat striatal tissue in vitro: possible involvement of receptor modulation in magnesium sensitivity

This study evaluated the role of glutamate ionotropic receptors on the control of [3H]acetylcholine ([3H]ACh) release by the intrinsic striatal cholinergic cells. [3H]-choline previously taken up by chopped striatal tissue and converted to [3H]ACh, was released under stimulation by glutamate, N-methyl-d-aspartate (NMDA), kainate and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Experiments were conducted in the absence of choline uptake inhibitors or acetylcholinesterase inhibitors. A paradigm of two stimulations was employed, the first in control conditions and the second after 9 min of perfusion with the test agents MK-801, 2-amino-5-phosphonopentanoic acid (AP-5), tetrodotoxin (TTX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]quinoxaline-7-sulfonamide (NBQX), glycine and magnesium. Our results support that (1) in the absence of Mg2+, NMDA is the most effective agonist to stimulate [3H]ACh release from striatal slices (2) magnesium effectively antagonized kainate and AMPA stimulation suggesting that at least part of the kainate and AMPA effects might be attributed to glutamate release (3) besides NMDA, kainate receptors showed a more direct involvement in [3H]ACh release control based on the smaller dependence on Mg2+ and less inhibition by TTX and (4) stimulation of ionotropic glutamate receptors may induce long lasting biochemical changes in receptor/ion channel function since the effects of TTX and/or Mg2+ ions on [3H]ACh release were modified by previous exposure of the tissue to agonists.     

Changes in GABA(B) receptor mRNA expression in the rodent basal ganglia and thalamus following lesion of the nigrostriatal pathway

Loss of striatal dopaminergic innervation in Parkinson's disease (PD) is accompanied by widespread alterations in GABAergic activity within the basal ganglia and thalamus. Accompanying changes in GABA_B receptor binding have been noted in some basal ganglia regions in parkinsonian primates, suggesting that plasticity of this receptor may also occur in PD. However, the molecular mechanisms underlying the changes in receptor binding and the manner and extent to which different GABA_B receptor mRNA subunits and splice-variants are affected remain unknown. This study used in situ hybridisation to examine the full profile of changes in expression of the known rat GABA_B receptor genes and gene variants in the basal ganglia and thalamus of rats, brought about by degeneration of the nigrostriatal tract. All of the GABA_B mRNA species examined showed unique expression patterns throughout the basal ganglia and thalamus. In addition, all exhibited a marked loss of expression (between 46 and 80%) in the substantia nigra pars compacta of animals bearing a complete 6-hydroxydopamine-induced lesion of the nigrostriatal tract, confirming the presence of these variants in dopaminergic neurones in this region. Further analysis of autoradioagrams revealed additional changes only in GABA_B(1a) mRNA in discrete anatomical regions. Expression of the GABA_B(1a) variant was significantly increased in the substantia nigra pars reticulata (33±2%), entopeduncular nucleus (26±1%) and the subthalamic nucleus (16±1%). Since these regions all receive reduced GABAergic innervation following nigrostriatal tract lesioning, it is possible that the increased expression occurs as a compensatory measure. In conclusion, these data demonstrate that GABA_B receptor genes exhibit regional- and subunit/variant-specific plasticity at the molecular level under parkinsonian conditions.     

Central CRF, urocortins and stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors delays gastric transit in mice

Recently characterized selective agonists and developed antagonists for the corticotropin releasing factor (CRF) receptors are new tools to investigate stress-related functional changes. The influence of mammalian CRF and related peptides injected intracerebroventricularly ( i.c.v. ) on gastric and colonic motility, and the CRF receptor subtypes involved and their role in colonic response to stress were studied in conscious mice. The CRF₁/CRF₂ agonists rat urocortin 1 (rUcn 1) and rat/human CRF (r/h CRF), the preferential CRF₁ agonist ovine CRF (oCRF), and the CRF₂ agonist mouse (m) Ucn 2, injected i.c.v. inhibited gastric emptying and stimulated distal colonic motor function (bead transit and defecation) while oCRF_9–33OH (devoid of CRF receptor affinity) showed neither effects. mUcn 2 injected peripherally had no colonic effect. The selective CRF₂ antagonist astressin₂-B ( i.c.v. ), at a 20 : 1 antagonist: agonist ratio, blocked i.c.v. r/hCRF and rUcn 1 induced inhibition of gastric transit and reduced that of mUcn 2, while the CRF₁ antagonist NBI-35965 had no effect. By contrast, the colonic motor stimulation induced by i.c.v. r/hCRF and rUcn 1 and 1h restraint stress were antagonized only by NBI-35965 while stimulation induced by mUcn 2 was equally blocked by both antagonists. None of the CRF antagonists injected i.c.v. alone influenced gut transit. These data establish in mice that brain CRF₁ receptors mediate the stimulation of colonic transit induced by central CRF, urocortins (1 and 2) and restraint stress, while CRF₂ receptors mediate the inhibitory actions of these peptides on gastric transit.     

Irreversible blockade of D2 dopamine receptors by fluphenazine-N-mustard increases glutamic acid decarboxylase mRNA in rat striatum

The influence of dopaminergic activity on the function of GABAergic neurons in striatum was examined by administrating rats the irreversible D₂ dopamine receptor antagonist, fluphenazine-N-mustard (FNM), and determining the level of glutamic acid decarboxylase (GAD) mRNA in striatum. Rats were given either an acute single injection or chronic daily injections of FNM (20 gmmol/kg, i.p.) for 6 days. The level of GAD mRNA in striatum was determined by in situ hybridization histochemistry. The results showed that acute treatment with FNM failed to significantly change striatal GAD mRNA. However, chronic FNM treatment significantly increased in the level of striatal GAD mRNA. These results demonstrate that irreversible blockade of D₂ dopamine receptors increases the expression of GAD mRNA in rat striatum.     

Periodic oscillatory activity in parahippocampal slices maintained in vitro

Brain slices maintained in vitro have been extensively used for studying neuronal synchronization. However, the validity of this approach may be questioned since pharmacological procedures are usually required to elicit spontaneous events similar to the EEG activity recorded in vivo. Here, we report that when superfused with control medium, rat brain slices comprising the entorhinal and perirhinal cortices along with a portion of the basolateral/lateral nuclei of the amygdala can synchronously generate periodic oscillatory activity at 5–11 Hz every 5–30 s. The periodic events: (i) correspond intracellularly to synaptic depolarizations in regularly firing neurons analyzed in the three areas; (ii) have no fixed site of onset; (iii) spread with time lags of 8–20 ms; and (iv) continue to occur asynchronously after their surgical isolation. NMDA receptor antagonism reduced the duration of the oscillatory events, while glutamatergic non-NMDA receptor antagonism abolished them. Activation of μ-opioid receptors, a procedure that hyperpolarizes interneurons thus decreasing GABA release, reversibly decreased the rate of occurrence of periodic oscillatory activity (POA). However, periodic events continued to occur during application of GABA_A or GABA_B receptor antagonists as well as in the presence of the cholinergic agent carbachol. We also found that POA was abolished by baclofen and irreversibly reduced by the gap junction decoupler carbenoxolone.

These findings demonstrate that parahippocampal networks in a brain slice preparation can generate periodic, synchronous activity under quasi-physiological conditions. These network oscillations (i) reflect the activation of ionotropic glutamatergic and GABAergic receptors, (ii) are contributed by gap-junction interactions, and (iii) are controlled by GABA_B receptors that are presumably located presynaptically.     

Modulation of electrically evoked acetylcholine release through cannabinoid CB1 receptors: evidence for an endocannabinoid tone in the human neocortex

Cannabinoids are known to inhibit neurotransmitter release in the CNS through CB1 receptors. The present study compares the effects of synthetic cannabinoids on acetylcholine (ACh) release in human and mice neocortex. We further investigated a possible endocannabinoid tone on CB1 receptors in human neocortex caused by endogenous agonists like anandamide or 2-arachidonylglycerol. Brain slices, incubated with [3H]-choline, were superfused and stimulated electrically under autoinhibition-free conditions to evoke tritium overflow assumed to represent ACh release. The first series of experiments was performed with 26 pulses, 60 mA, at 0.1 Hz. In mice neocortical slices, the cannabinoid receptor agonist WIN55212-2 decreased ACh release (pIC50=6.68, I(max)=67%). In the human neocortex the concentration-response curve of WIN55212-2 was bell-shaped and flat (I(max observed) approximately 30%). The estimated maximum possible inhibition, however, was much larger: I(max derived)=79%. Lec, the negative logarithm (lg) of the biophase concentration of endocannabinoids in 'WIN55212-2 units,' was -6.52, the pKd of WIN55212-2 was 7.47. The CB1 receptor antagonist/inverse agonist SR141716 enhanced ACh release in the human neocortex (by 38%) and prevented the inhibitory effect of WIN55212-2. The concentration-response curve of WIN55212-2 was changed in its shape including a shift to the right due to the presence of SR141716. A pA2 of this antagonist between 11.60 and 11.18 was obtained. SR141716 alone had no effect in mice neocortical slices. A partial agonist without inverse agonistic activity, O-1184, enhanced ACh release in the human neocortex. The endocannabinoid uptake-inhibitor AM404 decreased ACh release in human, but not in mice, neocortical slices. Change of the stimulation parameters (eight trains of pseudo-one-pulse bursts (4 pulses, 76 mA, 100 Hz), spaced by 45 s intervals) led to a stronger inhibitory effect of WIN55212-2, and abolished the disinhibitory effect of SR141716 and O-1184. The results show that activation of CB1 cannabinoid receptors leads to inhibition of ACh release in the human and mouse neocortex. The endocannabinoid tone is high in the human, but not in the mouse neocortex and is dependent on neuronal activity. SR141716 acts as a competitive CB1 receptor antagonist.