GABA-mediated Synaptic Transmission Research Articles

Quantitative GABA magnetic resonance spectroscopy as a measure of motor learning function in the motor cortex after subarachnoid hemorrhage.

The neural mechanisms underlying gross and fine motor dysfunction after subarachnoid hemorrhage (SAH) remain unknown. The γ-aminobutyric acid (GABA) deficit hypothesis proposes that reduced neuronal GABA concentrations and the subsequent lack of GABA-mediated inhibition cause motor impairment after SAH. This study aimed to explore the correlation between GABA levels and a behavioral measure of motor performance in patients with SAH. Motor cortical GABA levels were assessed in 40 patients with SAH and 10 age-matched healthy controls using proton magnetic resonance spectroscopy. The GABA and N-acetylasparate (NAA) ratio was measured in the normal gray matter within the primary motor cortex. The relationship between GABA concentration and hand-motor performance was also evaluated. Results showed significantly lower GABA levels in patients with SAH's left motor cortex than in controls (GABA/NAA ratio: 0.282 ± 0.085 vs. 0.341 ± 0.031, respectively; p = 0.041). Reaction times (RTs), a behavioral measure of motor performance potentially dependent on GABAergic synaptic transmission, were significantly longer in patients than in controls (936.8 ± 303.8 vs. 440.2 ± 67.3 ms, respectively; p < 0.001). Moreover, motor cortical GABA levels and RTs exhibited a significant positive linear correlation among patients (r = 0.572, rs = 0.327, p = 0.0001). Therefore, a decrease in GABA levels in the primary motor cortex after SAH may lead to impaired cortical inhibition of neuronal function and indicates that GABA-mediated synaptic transmission in the motor cortex is critical for RT.

Defective AMPA-mediated synaptic transmission and morphology in human neurons with hemizygous SHANK3 deletion engrafted in mouse prefrontal cortex

Genetic abnormalities in synaptic proteins are common in individuals with autism; however, our understanding of the cellular and molecular mechanisms disrupted by these abnormalities is limited. SHANK3 is a postsynaptic scaffolding protein of excitatory synapses that has been found mutated or deleted in most patients with 22q13 deletion syndrome and about 2% of individuals with idiopathic autism and intellectual disability. Here, we generated CRISPR/Cas9-engineered human pluripotent stem cells (PSCs) with complete hemizygous SHANK3 deletion (SHANK3+/–), which is the most common genetic abnormality in patients, and investigated the synaptic and morphological properties of SHANK3-deficient PSC-derived cortical neurons engrafted in the mouse prefrontal cortex. We show that human PSC-derived neurons integrate into the mouse cortex by acquiring appropriate cortical layer identities and by receiving and sending anatomical projections from/to multiple different brain regions. We also demonstrate that SHANK3-deficient human neurons have reduced AMPA-, but not NMDA- or GABA-mediated synaptic transmission and exhibit impaired dendritic arbors and spines, as compared to isogenic control neurons co-engrafted in the same brain region. Together, this study reveals specific synaptic and morphological deficits caused by SHANK3 hemizygosity in human cortical neurons at different developmental stages under physiological conditions and validates the use of co-engrafted control and mutant human neurons as a new platform for studying connectivity deficits in genetic neurodevelopmental disorders associated with autism.

Long-Term Stress Disrupts the Structural and Functional Integrity of GABAergic Neuronal Networks in the Medial Prefrontal Cortex of Rats.

Clinical and experimental data suggest that fronto-cortical GABAergic deficits contribute to the pathophysiology of major depressive disorder (MDD). To further test this hypothesis, we used a well characterized rat model for depression and examined the effect of stress on GABAergic neuron numbers and GABA-mediated synaptic transmission in the medial prefrontal cortex (mPFC) of rats. Adult male Wistar rats were subjected to 9-weeks of chronic mild stress (CMS) and based on their hedonic-anhedonic behavior they were behaviorally phenotyped as being stress-susceptible (anhedonic) or stress-resilient. Post mortem quantitative histopathology was used to examine the effect of stress on parvalbumin (PV)-, calretinin- (CR), calbindin- (CB), cholecystokinin- (CCK), somatostatin-(SST) and neuropeptide Y-positive (NPY+) GABAergic neuron numbers in all cortical subareas of the mPFC (anterior cingulate (Cg1), prelimbic (PrL) and infralimbic (IL) cortexes). In vitro, whole-cell patch-clamp recordings from layer II–III pyramidal neurons of the ventral mPFC was used to examine GABAergic neurotransmission. The cognitive performance of the animals was assessed in a hippocampal-prefrontal-cortical circuit dependent learning task. Stress exposure reduced the number of CCK-, CR- and PV-positive GABAergic neurons in the mPFC, most prominently in the IL cortex. Interestingly, in the stress-resilient animals, we found higher number of neuropeptide Y-positive neurons in the entire mPFC. The electrophysiological analysis revealed reduced frequencies of spontaneous and miniature IPSCs in the anhedonic rats and decreased release probability of perisomatic-targeting GABAergic synapses and alterations in GABAB receptor mediated signaling. In turn, pyramidal neurons showed higher excitability. Anhedonic rats were also significantly impaired in the object-place paired-associate learning task. These data demonstrate that long-term stress results in functional and structural deficits of prefrontal GABAergic networks. Our findings support the concept that fronto-limbic GABAergic dysfunctions may contribute to emotional and cognitive symptoms of MDD.

IgSF21 promotes differentiation of inhibitory synapses via binding to neurexin2\u03b1

Coordinated development of excitatory and inhibitory synapses is essential for higher brain function, and impairment in this development is associated with neuropsychiatric disorders. In contrast to the large body of accumulated evidence regarding excitatory synapse development, little is known about synaptic adhesion and organization mechanisms underlying inhibitory synapse development. Through unbiased expression screens and proteomics, we identified immunoglobulin superfamily member 21 (IgSF21) as a neurexin2α-interacting membrane protein that selectively induces inhibitory presynaptic differentiation. IgSF21 localizes postsynaptically and recruits axonal neurexin2α in a trans-interaction manner. Deleting IgSF21 in mice impairs inhibitory presynaptic organization, especially in the hippocampal CA1 stratum radiatum, and also diminishes GABA-mediated synaptic transmission in hippocampal CA1 neurons without affecting their excitatory synapses. Finally, mice lacking IgSF21 show a sensorimotor gating deficit. These findings suggest that IgSF21 selectively regulates inhibitory presynaptic differentiation through interacting with presynaptic neurexin2α and plays a crucial role in synaptic inhibition in the brain.

Persistent Interneuronopathy in the Prefrontal Cortex of Young Adult Offspring Exposed to Ethanol In Utero.

The significance of this study is twofold. First, we demonstrate that a time-delimited binge-type ethanol exposure in utero during early gestation alters corticopetal tangential migration of GABAergic interneurons in the fetal brain. Second, our study is the first to integrate neuroanatomical, electrophysiological, and behavioral evidence that this "interneuronopathy" persists in the young adult offspring and contributes to enduring changes in (1) the distribution of parvalbumin-expressing GABAergic cortical interneurons in the medial prefrontal cortex, (2) GABA-mediated synaptic transmission that resulted in an inhibitory/excitatory synaptic imbalance, and (3) behavioral flexibility. These findings alert women of child-bearing age that fetal alcohol spectrum disorders can be rooted very early in fetal brain development, and reinforce evidence-based counseling against binge drinking even at the earliest stages of pregnancy.

Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission

Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signaling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain’s energy supply affects inhibitory signaling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABAA receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABAA receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly-recruited α3-containing GABAA receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identifies mROS as a putative homeostatic signaling molecule coupling cellular metabolism to the strength of inhibitory transmission.

Properties of a Distinct Subpopulation of GABAergic Commissural Interneurons That Are Part of the Locomotor Circuitry in the Neonatal Spinal Cord

Commissural inhibitory interneurons (INs) are integral components of the locomotor circuitry that coordinate left-right motor activity during movements. We have shown that GABA-mediated synaptic transmission plays a key role in generating alternating locomotor-like activity in the mouse spinal cord (Hinckley et al., 2005a). The primary objective of our study was to determine whether properties of lamina VIII (LVIII) GABAergic INs in the spinal cord of GAD67::GFP transgenic mice fit the classification of rhythm-coordinating neurons in the locomotor circuitry. The relatively large green fluorescent protein-expressing (GFP(+)) INs had comparable morphological and electrophysiological properties, suggesting that they comprised a homogenous neuronal population. They displayed multipolar and complex dendritic arbors in ipsilateral LVII-LVIII, and their axonal projections crossed the ventral commissure and branched into contralateral ventral, medial, and dorsal laminae. Putative synaptic contacts evident as bouton-like varicosities were detected in close apposition to lateral motoneurons, Renshaw cells, other GFP(+) INs, and unidentified neurons. Exposure to a rhythmogenic mixture triggered locomotor-like rhythmic firing in the majority of LVIII GFP(+) INs. Their induced oscillatory activity was out-of-phase with bursts of contralateral motoneurons and in-phase with bouts of ipsilateral motor activity. Membrane voltage oscillations were elicited by rhythmic increases in excitatory synaptic drive and might have been augmented by three types of voltage-activated cationic currents known to increase neuronal excitability. Based on their axonal projections and activity pattern, we propose that this population of GABAergic INs forms a class of local commissural inhibitory interneurons that are integral component of the locomotor circuitry.

GABA Neuron Alterations, Cortical Circuit Dysfunction and Cognitive Deficits in Schizophrenia

Schizophrenia is a brain disorder associated with cognitive deficits that severely affect the patients' capacity for daily functioning. Whereas our understanding of its pathophysiology is limited, postmortem studies suggest that schizophrenia is associated with deficits of GABA-mediated synaptic transmission. A major role of GABA-mediated transmission may be producing synchronized network oscillations which are currently hypothesized to be essential for normal cognitive function. Therefore, cognitive deficits in schizophrenia may result from a GABA synapse dysfunction that disturbs neural synchrony. Here, we highlight recent studies further suggesting alterations of GABA transmission and network oscillations in schizophrenia. We also review current models for the mechanisms of GABA-mediated synchronization of neural activity, focusing on parvalbumin-positive GABA neurons, which are altered in schizophrenia and whose function has been strongly linked to the production of neural synchrony. Alterations of GABA signaling that impair gamma oscillations and, as a result, cognitive function suggest paths for novel therapeutic interventions.

NRG1 and Synaptic Function in the CNS

Recent genetic evidence indicates that neuregulin 1 (NRG1) and its receptor erbB4 may be susceptibility genes in schizophrenia, but their function in CNS synaptic transmission and circuitry is not well understood. In this issue of Neuron, studies from Li et al. and Woo et al. show that NRG1 and erbB4 regulate transmission at brain glutamate and GABA synapses. These findings raise the possibility of synaptic defects in schizophrenia.

Role of GABA in generating the 10‐Hz rhythm in sympathetic nerve discharge

The 10-Hz rhythm in SND of the cat is dependent on neurons located in the caudal and rostral ventral lateral medulla (VLM) and medullary raphe (Barman and Gebber, J Biol Rhythms 15: –379, 2000). We sought to determine whether GABA-mediated synaptic transmission in these regions is involved in 10-Hz rhythm generation. For this purpose, we recorded the discharges of the left and right inferior cardiac or the left vertebral and right inferior cardiac postganglionic sympathetic nerves in baroreceptor-denervated cats anesthetized with urethane. Unilateral microinjection of GABA-A receptor antagonists (50 nl of 1- mM solution of either SR-95531 or bicuculline) into the VLM eliminated the 10-Hz rhythm bilaterally. This was the case for injections made in single or multiple tracks in the VLM between the obex and 6 mm rostral. Recovery occurred within 3 h, and 10-Hz SND was unchanged after microinjection of vehicle (phosphate buffered saline). In contrast, the 10-Hz rhythm in SND was enhanced by multiple microinjections of the GABA antagonists into the raphe from 0 to 6 mm rostral to the obex. These results indicate that GABA-mediated synaptic inhibition in the VLM is critical for 10-Hz rhythm generation. Moreover, the data support the view that networks of neurons located bilaterally throughout the VLM act as a unit to generate the 10-Hz rhythm in SND. (Supported by NIH grant HL33266.)

Development of Effective Therapeutics Targeting the GABAA Receptor: Naturally Occurring Alternatives

The enhancement of GABA-mediated synaptic transmission underlies the pharmacotherapy of various neurological diseases. GABAA receptors are thus targets for neuroactive drugs, including classical benzodiazepines, mediating their anxiolytic, hypnotic and anticonvulsant effects via the benzodiazepine site (BZS). Based on findings that low intrinsic efficacy and subtype selectivity can greatly improve the specificity of drugs targeting the BZS, recent research has identified possible drug leads with apparently little side effects. In particular, drug leads of natural sources have been identified as promising candidates. This review describes the advances in the design of effective therapeutics targeting the GABAA receptor, focusing on the more recent research on naturally occurring drug leads. This includes discussion on the isolation of neuroactive alkaloids and flavonoids from herbal medicines and their rational development based on structure-activity relationships studies. Interest in the development of effective therapeutics from natural sources is clear and awaits to be seen whether their medicinal potential can be fulfilled.

GABA uptake into astrocytes is not associated with significant metabolic cost: implications for brain imaging of inhibitory transmission.

Synaptically released glutamate has been identified as a signal coupling excitatory neuronal activity to increased glucose utilization. The proposed mechanism of this coupling involves glutamate uptake into astrocytes resulting in increased intracellular Na+ (Nai+) and activation of the Na+/K+-ATPase. Increased metabolic demand linked to disruption of Nai+ homeostasis activates glucose uptake and glycolysis in astrocytes. Here, we have examined whether a similar neurometabolic coupling could operate for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), also taken up by Na+-dependent transporters into astrocytes. Thus, we have compared the Nai+ response to GABA and glutamate in mouse astrocytes by microspectrofluorimetry. The Nai+ response to GABA consisted of a rapid rise of 4-6 mM followed by a plateau that did not, however, significantly activate the pump. Indeed, the GABA transporter-evoked Na+ influxes are transient in nature, almost totally shutting off within approximately 30 sec of GABA application. The metabolic consequences of the GABA-induced Nai+ response were evaluated by monitoring cellular ATP changes indirectly in single cells and measuring 2-deoxyglucose uptake in astrocyte populations. Both approaches showed that, whereas glutamate induced a robust metabolic response in astrocytes (decreased ATP levels and glucose uptake stimulation), GABA did not cause any measurable metabolic response, consistent with the Nai+ measurements. Results indicate that GABA does not couple inhibitory neuronal activity with glucose utilization, as does glutamate for excitatory neurotransmission, and suggest that GABA-mediated synaptic transmission does not contribute directly to brain imaging signals based on deoxyglucose.

Temporal shaping of phasic neuronal responses by GABA- and non-GABA-mediated mechanisms in the somatosensory thalamus of the rat

Trapezoidal mechanical movement of whiskers was used to study the responses of 44 single thalamic ventral posteromedial (VPM) neurons to dynamic and static stimulus components in urethane-anesthetized rats. The effects of local administration of the GABAA receptor antagonist, bicuculline, and the GABAB receptor antagonist, 2-hydroxysaclofen, were tested to determine whether and to what extent the responses altered when GABA-mediated inhibitory synaptic transmission was blocked. Two classes of phasically responding neurons were identified, ON/OFF and movement-sensitive types. Bicuculline enhanced the magnitudes of the responses from both types by 2.5-fold and ON/OFF responses were converted to movement-sensitive ones in 17 (43%) of the 40 ON/OFF neurons. 2-hydroxysaclofen either had no effect or appeared to act like a GABA agonist. In 21 (48%) neurons, a significantly reduced responsiveness was observed during a 100-ms period following the ON and OFF responses. This discharge suppression was especially prominent during the plateau phase of the stimulus, and in some cases extended for several 100 ms following its onset. This suppression was overcome neither by the GABA receptor antagonists, nor by ejection of AMPA or glutamate at currents that otherwise produced vigorous excitation. These results suggest that one functional role for GABAA-receptor-mediated synaptic inhibition in the somatosensory thalamus is the intramodal regulation of the form of expression of phasically responding neurons. Other thalamic inhibitory processes not mediated by GABAA or GABAB receptors that help to shape the expression of the responses of certain phasic neurons to maintained stimulation may exist. Overall, these mechanisms appear to mediate the precision of timing of thalamic neuronal firing in response to the rat's tactile environment.

Lamotrigine reduces spontaneous and evoked GABA A receptor-mediated synaptic transmission in the basolateral amygdala: implications for its effects in seizure and affective disorders : Lamotrigine and inhibition in the amygdala

Lamotrigine (LTG) is an antiepileptic drug that is also effective in the treatment of certain psychiatric disorders. Its anticonvulsant action has been attributed to its ability to block voltage-gated Na + channels and reduce glutamate release. LTG also affects GABA-mediated synaptic transmission, but there are conflicting reports as to whether inhibitory transmission is enhanced or suppressed by LTG. We examined the effects of LTG on GABA A receptor-mediated synaptic transmission in slices from rat amygdala, a brain area that is particularly important in epileptogenesis and affective disorders. In intracellular recordings, LTG (100 μM) reduced GABA A receptor-mediated IPSPs evoked by electrical stimulation in neurons of the basolateral nucleus. In whole-cell recordings, LTG (10, 50 and 100 μM) decreased the frequency and amplitude of spontaneous IPSCs, as well as the amplitude of evoked IPSCs, but had no effect on the kinetics of these currents. LTG also had no effects on the frequency, amplitude or kinetics of miniature IPSCs recorded in the presence of TTX. These results suggest that in the basolateral amygdala, LTG suppresses GABA A receptor-mediated synaptic transmission by a direct and/or indirect effect on presynaptic Ca ++ influx. The modulation of inhibitory synaptic transmission may be an important mechanism underlying the psychotropic effects of LTG.

CNQX increases GABA-mediated synaptic transmission in the cerebellum by an AMPA/kainate receptor-independent mechanism

GABA A receptor-mediated inhibitory synaptic transmission within the CNS is often studied in the presence of glutamate receptor antagonists. However, for nearly a decade it has been known that, in the hippocampus, one of the most commonly used α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), can increase the frequency of spontaneous GABA A receptor-mediated postsynaptic currents (sIPSCs). In the present study we examined the effect of CNQX and related compounds on GABA-mediated synaptic transmission in the cerebellum. At various stages of development, low concentrations of CNQX increased the frequency of sIPSCs recorded from granule cells. This effect was independent of the blocking action of CNQX on ionotropic glutamate receptors, as it was not observed with the broad-spectrum glutamate receptor antagonist kynurenate. No increase in sIPSC frequency was observed with the NMDA receptor antagonists d-AP5 or 7-ClK, the selective AMPA receptor antagonists GYKI 52466 or GYKI 53655, or the kainate receptor antagonist NS-102. In contrast, two other quinoxaline derivatives, NBQX and DNQX, were capable of increasing sIPSC frequency. These results demonstrate that the novel excitatory action of CNQX, unrelated to blockade of ionotropic glutamate receptors, is not restricted to the hippocampus and can be observed with structurally related compounds.

Adenosine-mediated inhibition of striatal GABAergic synaptic transmission during in vitro ischaemia.

Several reports have shown that energy deprivation, as a result of hypoxia, hypoglycaemia or ischaemia, depresses excitatory synaptic transmission in virtually all brain areas. How this pathological condition affects inhibitory synaptic transmission is still unclear. In the present in vitro study, we coupled whole-cell patch clamp recordings from striatal neurones with focal stimulation of GABAergic nerve terminals in order to characterize the electrophysiological effects of combined oxygen and glucose deprivation (in vitro ischaemia) on inhibitory postsynaptic currents (IPSCs) in this brain area. We found that brief periods (2-5 min) of in vitro ischaemia invariably caused a marked depression of IPSC amplitude. This inhibitory effect was fully reversible on removal of the ischaemic challenge. It was coupled with an increased paired-pulse facilitation, suggesting the involvement of presynaptic mechanisms. Accordingly, the ischaemic inhibition of striatal GABAergic IPSCs was not caused by a shift in the reversal potential of GABA(A)-receptor mediated synaptic currents, and was independ- ent of postsynaptic ATP concentrations. Endogenous adenosine, acting on A1 receptors, appeared responsible for this presynaptic action as the ischaemic depression of IPSCs was prevented by CPT [8-(4-chlorophenylthio) adenosine] and DPCPX, two adenosine A1 receptor antagonists, and mimicked by the application of adenosine in the bathing solution. Conversely, ATP-sensitive potassium channels were not involved in the inhibition of IPSCs by ischaemia, as demonstrated by the fact that tolbutamide and glipizide, two blockers of these channels, were ineffective in preventing this electrophysiological effect. The early depression of GABA-mediated synaptic transmission might play a role in the development of irreversible neuronal injury in the course of brain ischaemia.

Reduced long-term potentiation in hippocampal slices prepared using sucrose-based artificial cerebrospinal fluid

Sucrose-based artificial cerebrospinal fluid (aCSF) is sometimes used to prepare brain slices for in vitro electrophysiological experiments. This study compared the effect of preparing brain slices using chilled sucrose-based aCSF versus the conventional method using chilled aCSF on hippocampal synaptic plasticity. Brain slices from each treatment group were transferred to normal aCSF before electrophysiological recordings were made. The stimulus–response relationship of field excitatory postsynaptic potentials (fEPSPs) in the CA1 region was indistinguishable between the two treatment groups. However, the amount of LTP induced by either a θ-burst (four stimuli at 100 Hz repeated ten times at 200 ms intervals) or tetanic stimulation (100 Hz for 1 s) was significantly reduced in slices that had been prepared using sucrose-based aCSF. This was associated with reduced facilitation of the fEPSPs during the high frequency stimulus, reduced post-tetanic potentiation and short-term potentiation. In sucrose-cut slices the fEPSPs were slightly shorter in duration (29%, P<0.01), and during paired-pulse stimulation the broadening of the second fEPSP was enhanced. The LTP deficit in sucrose-cut slices was reversed by blocking GABA A receptor function with picrotoxin. These data suggest that the use of sucrose based aCSF better preserves GABA-mediated synaptic transmission, which limits the induction of LTP in hippocampal brain slices.

Effects of differential modulation of μ-, δ- and κ-opioid systems on bicuculline-induced convulsions in the mouse

The present study investigated the effects of μ-, δ- and κ-opioid receptor agonists on seizures produced by blockade of γ-aminobutyric acid (GABA)-mediated synaptic transmission in the mouse. The selective GABA A receptor antagonist bicuculline (1.25–3 mg/kg) given subcutaneously caused dose-dependent clonic–tonic convulsions. These convulsions were potentiated by the prototypic μ-opioid receptor agonist morphine given subcutaneously 20 min prior to a subconvulsive dose of bicuculline. The potentiation by morphine was completely reversed by pretreatment intraventricularly with the selective μ-opioid receptor antagonist β-funaltrexamine (0.5 μg/mouse). Pretreatment intraventricularly with the selective δ-opioid receptor agonists 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aβ-octahydro-quinolino[2,3,3-g]isoquinoline ((−)TAN-67) or [D-Pen 2,5]-enkephalin (DPDPE) showed a dose-dependent increase in the incidence of convulsions. Pretreatment with naltrindole (2 mg/kg, s.c.), a selective δ-opioid receptor antagonist, abolished the enhancement of the bicuculline-induced convulsions by DPDPE. In contrast, pretreatment with the selective κ-opioid receptor agonist U-50,488H (0.6–80 mg/kg, subcutaneously or 25–100 μg/mouse, intraventricularly) produced a dose-dependent suppression of the bicuculline-induced convulsions. The inhibitory effect of U-50,488H was completely blocked by pretreatment subcutaneously with nor-binaltorphimine (5 mg/kg), a selective κ-opioid receptor antagonist. This study demonstrates that activation of both μ- and δ-opioid receptors increases the incidence of convulsions produced by blockade of GABA-mediated synaptic transmission, while stimulation of κ-opioid receptors has an anticonvulsive effect.

The pro-convulsant actions of corticotropin-releasing hormone in the hippocampus of infant rats

Whole-cell patch-clamp and extracellular field recordings were obtained from 450- μm-thick brain slices of infant rats (10–13 days postnatal) to determine the actions of corticotropin-releasing hormone on glutamate- and GABA-mediated synaptic transmission in the hippocampus. Synthetic corticotropin-releasing hormone (0.15 μM) reversibly increased the excitability of hippocampal pyramidal cells, as determined by the increase in the amplitude of the CA1 population spikes evoked by stimulation of the Schaffer collateral pathway. This increase in population spike amplitude could be prevented by the corticotropin-releasing hormone receptor antagonist α-helical (9–41)-corticotropin-releasing hormone (10 μM). Whole-cell patch-clamp recordings revealed that, in the presence of blockers of fast excitatory and inhibitory synaptic transmission, corticotropin-releasing hormone caused only a small (1–2 mV) depolarization of the resting membrane potential in CA3 pyramidal cells, and it did not significantly alter the input resistance. However, corticotropin-releasing hormone, in addition to decreasing the slow afterhyperpolarization, caused an increase in the number of action potentials per burst evoked by depolarizing current pulses. Corticotropin-releasing hormone did not significantly change the frequency, amplitude or kinetics of miniature excitatory postsynaptic currents. However, it increased the frequency of the spontaneous excitatory postsynaptic currents in CA3 pyramidal cells, without altering their amplitude and single exponential rise and decay time constants. Corticotropin-releasing hormone did not change the amplitude of the pharmacologically isolated (i.e. recorded in the presence of GABA A receptor antagonist bicuculline) excitatory postsynaptic currents in CA3 and CA1 pyramidal cells evoked by stimulation of the mossy fibers and the Schaffer collaterals, respectively. Current-clamp recordings in bicuculline-containing medium showed that, in the presence of corticotropin-releasing hormone, mossy fiber stimulation leads to large, synchronized, polysynaptically-evoked bursts of action potentials in CA3 pyramidal cells. In addition, the peptide caused a small, reversible decrease in the amplitude of the pharmacologically isolated (i.e. recorded in the presence of glutamate receptor antagonists) evoked inhibitory postsynaptic currents in CA3 pyramidal cells, but it did not significantly alter the frequency, amplitude, rise and decay time constants of spontaneous or miniature inhibitory postsynaptic currents. These data demonstrate that corticotropin-releasing hormone, an endogenous neuropeptide whose intracerebroventricular infusion results in seizure activity in immature rats, has diverse effects in the hippocampus which may contribute to epileptogenesis. It is proposed that the net effect of corticotropin-releasing hormone is a preferential amplification of those incoming excitatory signals which are strong enough to reach firing threshold in at least a subpopulation of CA3 cells. These findings suggest that the actions of corticotropin-releasing hormone on neuronal excitability in the immature hippocampus may play a role in human developmental epilepsies.

Comparison of responses of spontaneously active cells in the cerebellar purkinje layer to parallel fibre stimulation in slice preparations and urethane-anaesthetised rats: Effects of benzodiazepine receptor ligands

1. 1. GABA-mediated inhibitory responses were induced in spontaneously active Purkinje cells by parallel fibre stimulation in cerebellar slices or in urethane-anaesthetised rats. Effects of agonist and inverse agonist benzodiazepine (BDZ) receptor ligands were compared in the preparations. 2. 2. Purkinje cells fired simple spikes at higher rates in slice preparations while complex spikes were seldom ( in vivo) or never observed (slice). Cells fired more regularly in vivo resulting in the occurrence of rhythmic postinhibitory responses in the PSTH analysis in some preparations. 3. 3. Single pulse stimulation of parallel fibres at just suprathreshold intensity induced inhibition of Purkinje cell activity in both preparations. At lower firing rates there was a marked increase in the duration of this response, which was more evident in vivo where more slowly firing cells were encountered. 4. 4. BDZ receptor ligands modified inhibitory responses in slice preparations with only weak effects on the firing rates of the cells. These compounds predominately induced changes in firing rate in the anaesthetised rat with little evidence of direct modification of GABA-mediated synaptic transmission. 5. 5. In a few experiments, following injection of the partial inverse agonists β-CCE and β-CCM, block of the inhibitory response was observed independent of changes in firing rate. Bidirectional efficacy of BDZ receptor ligand (agonists decrease firing and increase inhibitory response, inverse agonists increase firing and decrease inhibitory response) was demonstrated for modulation of inhibitory responses in slices and for changes in firing rate in vivo. The increased firing rate response in vivo was biphasic the magnitude of the later phase being correlated with efficacy of inverse agonists. 6. 6. It is concluded that cerebellar slice preparations are more appropriate for studying direct effects of BDZ receptor ligands on GABA-mediated synaptic inhibition than in vivo preparations.