Ontogenesis of presynaptic GABAB receptor-mediated inhibition in the CA3 region of the rat hippocampus

gamma-Aminobutyric acid-B(GABAB) receptor-dependent and -independent components of paired-pulse depression (PPD) were investigated in the rat CA3 hippocampal region. Intracellular and whole cell recordings of CA3 pyramidal neurons were performed on hippocampal slices obtained from neonatal (5-7 day old) and adult (27-34 day old) rats. Electrical stimulation in the hilus evoked monosynaptic GABAA postsynaptic currents (eIPSCs) isolated in the presence of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)2-amino-5-phosphovaleric acid (-AP5, 50 microM) with 2(triethylamino)-N-(2,6-dimethylphenyl) acetamine (QX314) filled electrodes. In adult CA3 pyramidal neurons, when a pair of identical stimuli was applied at interstimulus intervals (ISIs) ranging from 50 to 1,500 ms the amplitude of the second eIPSC was depressed when compared with the first eIPSC. This paired-pulse depression (PPD) was partially blocked by P-3-aminoprophyl -P-diethoxymethylphosphoric acid (CGP35348, 0.5 mM), a selective GABAB receptor antagonist. In neonates, PPD was restricted to ISIs shorter than 200 ms and was not affected by CGP35348. The GABAB receptor agonist baclofen reduced the amplitude of eIPSCs in a dose-dependent manner with the same efficiency in both adults and neonates. Increasing the probability of transmitter release with high Ca2+ (4 mM)/low Mg2+ (0.3 mM) external solution revealed PPD in neonatal CA3 pyramidal neurons that was 1) partially prevented by CGP35348, 2) independent of the membrane holding potential of the recorded cell, and 3) not resulting from a change in the reversal potential of GABAA eIPSCs. In adults the GABA uptake blocker tiagabine (20 microM) increased the duration of eIPSCs and the magnitude of GABAB receptor-dependent PPD. In neonates, tiagabine also increased duration of eIPSCs but to a lesser extent than in adult and did not reveal a GABAB receptor-dependent PPD. These results demonstrate that although GABAB receptor-dependent and -independent mechanisms of presynaptic inhibition are present onGABAergic terminals and functional, they do not operate at the level of monosynaptic GABAergic synaptic transmission at early stages of development. Absence of presynaptic autoinhibition of GABA release seems to be due to the small amount of transmitter that can access presynaptic regulatory sites.     

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1. The release of endogenous gamma-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours. 2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K(+)-evoked overflows of both amino acids were almost totally dependent on the presence of Ca(2+) in the superfusion medium. 3. The GABAB receptor agonist (-)-baclofen (1, 3 or 10 microM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 microM of the agonist ranged from 45-50%. 5. The effect of three selective GABAB receptor antagonists on the inhibition of the K(+)-evoked GABA and glutamate overflows elicited by 10 microM (-)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 microM; almost totally at 300 microM) the effect of (-)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (-)-baclofen on the K(+)-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 microM); however, CGP 35348 (10 or 100 microM) antagonized (-)-baclofen (complete blockade at 100 microM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 microM, blocked the GABAB autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52423 was lost at 30 microM, as the compound, at this concentration, inhibited completely the (-)-baclofen effect on both GABA and glutamate release. 5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABAB receptors.     

Presynaptic GABAB autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons

GABA is the primary transmitter released by neurons of the suprachiasmatic nucleus (SCN), the circadian clock in the brain. Whereas GABAB receptor agonists exert a significant effect on circadian rhythms, the underlying mechanism by which GABAB receptors act in the SCN has remained a mystery. We found no GABAB receptor-mediated effect on slow potassium conductance, membrane potential, or input resistance in SCN neurons in vitro using whole-cell patch-clamp recording. In contrast, the GABAB receptor agonist baclofen (1-100 microM) exerted a large and dose-dependent inhibition (up to 100%) of evoked IPSCs. Baclofen reduced the frequency of spontaneous IPSCs but showed little effect on the frequency or amplitude of miniature IPSCs in the presence of tetrodotoxin. The activation of GABAB receptors did not modulate postsynaptic GABAA receptor responses. The depression of GABA release by GABAB autoreceptors appeared to be mediated primarily through a modulation of presynaptic calcium channels. The baclofen inhibition of both calcium currents and evoked IPSCs was greatly reduced (up to 100%) by the P/Q-type calcium channel blocker agatoxin IVB, suggesting that P/Q-type calcium channels are the major targets involved in the modulation of GABA release. To a lesser degree, N-type calcium channels were also involved. The inhibition of GABA release by baclofen was abolished by a pretreatment with pertussis toxin (PTX), whereas the inhibition of whole-cell calcium currents by baclofen was only partially depressed by PTX, suggesting that G-protein mechanisms involved in GABAB receptor modulation at the soma and axon terminal may not be identical. We conclude that GABAB receptor activation exerts a strong presynaptic inhibition of GABA release in SCN neurons, primarily by modulating P/Q-type calcium channels at axon terminals.     

Mechanism of early anoxia-induced suppression of the GABAA-mediated inhibitory postsynaptic current

1. We investigated the mechanism of hypoxia-induced depression of gamma-aminobutyric acid-A (GABAA)-mediated inhibitory postsynaptic currents (IPSCs) in CA1 neurons of hippocampal slices from 21- to 28-day-old rats. Cells were examined by whole-cell patch-clamp recording and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% CO2. 2. Synaptic responses evoked by stimulation of the Schaffer collateral-commissural projection at a fixed holding potential (VH = -60 mV) during anoxia revealed that the IPSC appeared more sensitive than the excitatory postsynaptic current to anoxia-induced depression. All subsequent studies examined the GABAA-mediated IPSC synaptic responses in isolation by direct stimulation of GABA interneurons in the stratum radiatum in the presence of extracellular 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (20 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 microM) to block glutamatergic currents and intracellular QX-314 (lidocaine N-ethyl bromide, 1 mM) to block GABAB-mediated currents. When studied in this manner (VH = -60 mV) the GABAA-mediated IPSC appeared to change from an outward to inward current after exposure to anoxia. 3. The current-voltage relationship of GABAA-mediated IPSCs revealed that these changes resulted from a positive shift in the IPSC reversal potential without a significant change in the conductance. Thus under patch clamp apparent IPSC inhibition may result from a decrease in the extracellular concentration of chloride ions. Similar findings were observed with micropipettes that contained high intracellular chloride concentrations. 4. Miniature spontaneous IPSCs were examined in the presence of tetrodotoxin (1 microM) with micropipettes containing high intracellular chloride concentrations. The miniature IPSCs (mIPSCs) appeared as spontaneous transient inward currents. Consistent with an anoxia-induced decrease in extracellular chloride, the mean amplitude of the mIPSCs increased after the onset of anoxia. A significant decrease in rise and decay time was also noted during anoxia. The frequency of the mIPSCs also increased by approximately 300%. 5. The resting input resistance of the cells was examined by measuring the current resulting from a 20-mV hyperpolarizing pulse. A significant reduction in resistance was observed 2 min after the onset of anoxia. This still occurred, although to a lesser degree, in the presence of glutamatergic blockers (20 microM CPP plus 50 microM CNQX). In the presence of both GABAergic (picrotoxin, 100 microM) and glutamatergic blockers no significant reduction in resting input resistance was apparent after 2 min of anoxia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell-specific alterations in synaptic properties of hippocampal CA1 interneurons after kainate treatment

Cell-specific alterations in synaptic properties of hippocampal CA1 interneurons after kainate treatment. J. Neurophysiol. 80: 2836-2847, 1998. Hippocampal sclerosis and hyperexcitability are neuropathological features of human temporal lobe epilepsy that are reproduced in the kainic acid (KA) model of epilepsy in rats. To assess directly the role of inhibitory interneurons in the KA model, the membrane and synaptic properties of interneurons located in 1) stratum oriens near the alveus (O/A) and 2) at the border of stratum radiatum and stratum lacunosum-moleculare (LM), as well as those of pyramidal cells, were examined with whole cell recordings in slices of control and KA-lesioned rats. In current-clamp recordings, intrinsic cell properties such as action potential amplitude and duration, amplitude of fast and medium duration afterhyperpolarizations, membrane time constant, and input resistance were generally unchanged in all cell types after KA treatment. In voltage-clamp recordings, the amplitude and conductance of pharmacologically isolated excitatory postsynaptic currents (EPSCs) were significantly reduced in LM interneurons of KA-treated animals but were not significantly changed in O/A and pyramidal cells. The rise time of EPSCs was not significantly changed in any cell type after KA treatment. In contrast, the decay time constant of EPSCs was significantly faster in O/A interneurons of KA-treated rats but was unchanged in LM and pyramidal cells. The amplitude and conductance of pharmacologically isolated gamma-aminobutyric acid-A (GABAA) inhibitory postsynaptic currents (IPSCs) were not significantly changed in any cell type of KA-treated rats. The rise time and decay time constant of GABAA IPSCs were significantly faster in pyramidal cells of KA-treated rats but were not significantly changed in O/A and LM interneurons. These results suggest that complex alterations in synaptic currents occur in specific subpopulations of inhibitory interneurons in the CA1 region after KA lesions. A reduction of evoked excitatory drive onto inhibitory cells located at the border of stratum radiatum and stratum lacunosum-moleculare may contribute to disinhibition and polysynaptic epileptiform activity in the CA1 region. Compensatory changes, involving excitatory synaptic transmission on other interneuron subtypes and inhibitory synaptic transmission on pyramidal cells, may also take place and contribute to the residual, functional monosynaptic inhibition observed in principal cells after KA treatment.     

Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells

The purpose of the present work was to test the hypothesis that no more than one vesicle of transmitter can be liberated by an action potential at a single release site. Spontaneous and evoked IPSCs were recorded from interneurons in the molecular layer of cerebellar slices. Evoked IPSCs were obtained using either extracellular stimulation or paired recordings of presynaptic and postsynaptic neurons. Connections were identified as single-site synapses when evoked current amplitudes could be grouped into one peak that was well separated from the background noise. Peak amplitudes ranged from 30 to 298 pA. Reducing the release probability by lowering the external Ca2+ concentration or adding Cd2+ failed to reveal smaller quantal components. Some spontaneous IPSCs (1.4-2.4%) and IPSCs evoked at single-site synapses (2-6%) were followed within <5 msec by a secondary IPSC that could not be accounted for by random occurrence of background IPSCs. Nonlinear summation of closely timed events indicated that they involved activation of a common set of receptors and therefore that several vesicles could be released at the same release site by one action potential. An average receptor occupancy of 0.70 was calculated after single release events. At some single-site connections, two closely spaced amplitude peaks were resolved, presumably reflecting single and double vesicular release. Consistent with multivesicular release, kinetics of onset, decay, and latency were correlated to IPSC amplitude. We conclude that the one-site, one-vesicle hypothesis does not hold at interneuron-interneuron synapses.     

Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABA(B) receptors in rat sensorimotor cortex

At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of gamma-aminobutyric acid (GABA) activates postsynaptic GABA(A) receptors but not pre- or postsynaptic GABA(B) receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABA(B) receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABA(B) receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABA(B) receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.     

Human brain cholecystokinin: release of cholecystokinin-like immunoreactivity (CCK-LI) from isolated cortical nerve endings and its modulation through GABA(B) receptors

The release of cholecystokinin-like immunoreactivity (CCK-LI) in human brain was investigated using synaptosomes prepared from neocortical specimens removed during neurosurgery. CCK-LI basal release from superfused synaptosomes was increased 3 to 4-fold during depolarization with 15 mM KCI. The K(+)-evoked overflow of CCK-LI was strictly Ca(++)-dependent. The gamma-aminobutyric acidB (GABA(B)) receptor agonist (-)baclofen (0.3-100 microM) inhibited CCK-LI overflow in a concentration-dependent manner (EC50 = 2.20 microM; maximal effect: 45%). The novel GABA(B) receptor ligand CGP 47656 mimicked (-)baclofen (EC50 = 2.45 microM; maximal effect: 50%), whereas the GABA(A) agonist muscimol was ineffective up to 100 microM. The inhibitory effect of 10 microM (-)baclofen on the CCK-LI overflow was concentration-dependently prevented by two selective GABA(B) receptor antagonists, CGP 35348 (IC50 = 13.91 microM) and CGP 52432 (IC50 = 0.08 microM). The effect of 10 microM CGP 47656 was abolished by 1 microM CGP 52432. In experiments on [3H]GABA release, CGP 47656 behaved as an antagonist at the GABA(B) autoreceptors: added at 10 microM, it prevented the inhibitory effect of 10 microM (-)baclofen on the K+ (15 mM)-evoked release of [3H]GABA from human synaptosomes. We conclude that 1) the release of CCK-LI evoked from human brain tissue appears of neuronal origin; 2) the CCK-releasing terminal possess inhibitory presynaptic GABA(B) receptors; 3) these receptors differ pharmacologically from human neocortex GABA(B) autoreceptors, which are CGP 35348-insensitive (Fassio et al., 1994) but can be blocked by CGP 47656; 4) because cholecystokinin has been implicated in anxiety, the GABA(B) receptors here characterized may represent targets for novel anxiolytic agents.     

Monosynaptic GABA-mediated inhibitory postsynaptic potentials in CA1 pyramidal cells of hyperexcitable hippocampal slices from kainic acid-treated rats

To examine the mechanisms underlying chronic epileptiform activity, field potentials were first recorded to identify hyperexcitable hippocampal slices from kainic acid-treated rats. Intracellular recordings were then obtained from CA1 pyramidal cells in the hyperexcitable areas. Twenty-two of the 47 cells responded to electrical stimulation of the stratum radiatum with a burst of two or more action potentials and reduced early inhibitory postsynaptic potentials, and were considered hyperexcitable. The remaining 25 cells were not hyperexcitable, displaying a single action potential and biphasic inhibitory postsynaptic potentials after stimulation, like control cells (n = 20). A long duration, voltage-sensitive component was associated with subthreshold excitatory postsynaptic potentials in the majority of hyperexcitable (12/15) and non-hyperexcitable (3/5) cells examined from kainic acid-treated animals, but not from cells (1/10) of control animals. Stimulation of stratum radiatum during pharmacological blockade of ionotropic excitatory amino acid synaptic transmission elicited biphasic monosynaptic inhibitory postsynaptic potentials in all hyperexcitable (n = 9) and non-hyperexcitable (n = 9) cells tested from kainate-treated animals, as well as in control cells (n = 8). The mean amplitude, latency to peak, equilibrium potential, and conductance changes of early and late monosynaptic inhibitory postsynaptic potentials were not different between cells of kainic acid-treated and control animals. In seven hyperexcitable cells tested, the early component of monosynaptic inhibitory postsynaptic potentials was significantly reduced by the GABAA receptor antagonist bicuculline (100-200 microM). The late component was significantly decreased by the GABAB receptor antagonist 2-hydroxysaclofen (1-2 mM; n = 3). Comparable effects were observed on early and late monosynaptic inhibitory postsynaptic potentials in non-hyperexcitable cells (n = 4) from kainic acid-treated animals and control cells (n = 5). These results suggest that GABAergic synapses on hyperexcitable hippocampal pyramidal cells of kainate-treated rats are intact and functional. Therefore, epileptiform activity in the kainate-lesioned hippocampus may not arise from a disconnection of GABAergic synapses made by inhibitory interneurons on pyramidal cells. The hyperexcitability may be due to underactivation of inhibitory interneurons and/or reorganization of excitatory inputs to pyramidal cells since, in kainate-treated animals, pyramidal cells appear to express additional excitatory mechanisms.     

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Whole-cell recordings were made in the nucleus tractus solitarii (NTS) in transverse brainstem slices from rats. Monosynaptic GABAA-receptor-mediated inhibitory postsynaptic currents (IPSCs) or potentials (IPSPs) were evoked (0.1-0.2 Hz) by electrical stimulation within and medial to the tractus solitarius in the presence of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) or 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microM) and D-amino-5-phosphonopentanoic acid (APV; 50 microM). A brief period of tetanic stimulation (20 Hz, 2 s) resulted in posttetanic (< 5 min, 69 of 73 recordings) and sustained potentiation (> 15 min, 31 of 73 recordings) of the IPSP/Cs. Sustained potentiation was not due to alterations in the reversal potential of IPSP/Cs. Both pre- and post-tetanus IPSP/Cs were completely blocked by the GABAA antagonist bicuculline (10 microM). Postsynaptic responses to pressure ejection of the GABAA-receptor agonist muscimol were unaltered in cells displaying sustained potentiation. Sustained potentiation of IPSP/Cs could be induced by tetanus in the presence of either metabotropic glutamate receptor antagonists or bicuculline. However, sustained potentiation could not be induced in the presence of the GABAB-receptor antagonists 2-OH-saclofen (400 microM) or CGP35348 (3-amino-propyl-(diethoxymethyl)phosphinic acid, 100 microM), although a subsequent tetanus following washout induced sustained potentiation. Posttetanic potentiation was unaffected by GABAB-receptor antagonists. These data suggest that neuronal or terminal excitability of GABAergic interneurons in the NTS is enhanced following brief periods of increased frequency of activation in vitro. This novel phenomenon within the rat medulla may be involved in the temporal modulation of autonomic reflex sensitivity observed during certain behavioral states, such as the defense reaction.     

Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAA receptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.     

GABAB receptor-mediated inhibition of GABAA receptor calcium elevations in developing hypothalamic neurons

In the CNS, gamma-aminobutyric acid (GABA) affects neuronal activity through both the ligand-gated GABAA receptor channel and the G protein-coupled GABAB receptor. In the mature nervous system, both receptor subtypes decrease neural excitability, whereas in most neurons during development, the GABAA receptor increases neural excitability and raises cytosolic Ca2+ levels. We used Ca2+ digital imaging to test the hypothesis that GABAA receptor-mediated Ca2+ rises were regulated by GABAB receptor activation. In young, embryonic day 18, hypothalamic neurons cultured for 5 +/- 2 days in vitro, we found that cytosolic Ca2+ rises triggered by synaptically activated GABAA receptors were dramatically depressed (>80%) in a dose-dependent manner by application of the GABAB receptor agonist baclofen (100 nM-100 microM). Coadministration of the GABAB receptor antagonist 2-hydroxy-saclofen or CGP 35348 reduced the inhibitory action of baclofen. Administration of the GABAB antagonist alone elicited a reproducible Ca2+ rise in >25% of all synaptically active neurons, suggesting that synaptic GABA release exerts a tonic inhibitory tone on GABAA receptor-mediated Ca2+ rises via GABAB receptor activation. In the presence of tetrodotoxin the GABAA receptor agonist muscimol elicited robust postsynaptic Ca2+ rises that were depressed by baclofen coadministration. Baclofen-mediated depression of muscimol-evoked Ca2+ rises were observed in both the cell bodies and neurites of hypothalamic neurons taken at embryonic day 15 and cultured for three days, suggesting that GABAB receptors are functionally active at an early stage of neuronal development. Ca2+ rises elicited by electrically induced synaptic release of GABA were largely inhibited (>86%) by baclofen. These results indicate that GABAB receptor activation depresses GABAA receptor-mediated Ca2+ rises by both reducing the synaptic release of GABA and decreasing the postsynaptic Ca2+ responsiveness. Collectively, these data suggest that GABAB receptors play an important inhibitory role regulating Ca2+ rises elicited by GABAA receptor activation. Changes in cytosolic Ca2+ during early neural development would, in turn, profoundly affect a wide array of physiological processes, such as gene expression, neurite outgrowth, transmitter release, and synaptogenesis.     

Brain-derived neurotrophic factor (BDNF) modulates inhibitory, but not excitatory, transmission in the CA1 region of the hippocampus

Brain-derived neurotrophic factor (BDNF) modulates inhibitory, but not excitatory, transmission in the CA1 region of the hippocampus. J. Neurophysiol. 80: 3383-3386, 1998. Brain-derived neurotrophic factor (BDNF) has been reported to have rapid effects on synaptic transmission in the hippocampus. We report here that bath application of BDNF causes a small but significant decrease in stimulus-evoked inhibitory postsynaptic currents (IPSCs) on CA1 pyramidal cells, which is prevented by the tyrosine kinase inhibitor lavendustin A. BDNF causes a decrease in the 1/CV2 of the IPSC, and also reduces paired-pulse depression of the IPSC, suggesting a presynaptic site of action. In contrast, BDNF did not have a detectable effect on field excitatory postsynaptic potentials measured in stratum radiatum. We conclude that BDNF has a selective depressant action on inhibitory transmission in the hippocampus, due at least in part to a presynaptic mechanism.     

Developmental regulation of basket/stellate cell-->Purkinje cell synapses in the cerebellum

We used paired recordings to study the development of synaptic transmission between inhibitory interneurons of the molecular layer and Purkinje cells in the cerebellar cortex of the rat. The electrophysiological data were combined with a morphological study of the recorded cells using biocytin or Lucifer yellow staining. Thirty-one interneuron-Purkinje cell pairs were obtained, and 11 of them were recovered morphologically. The age of the rats ranged from 11 to 31 d after birth. During this period synaptic maturation resulted in an 11-fold decrease in the average current evoked in a Purkinje cell by a spike in a presynaptic interneuron. Unitary IPSCs in younger animals exhibited paired-pulse depression, whereas paired-pulse facilitation was found in more mature animals. These data suggest that reduction in transmitter release probability contributed to the developmental decrease of unitary IPSCs. However, additional mechanisms at both presynaptic and postsynaptic loci should also be considered. The decrease of the average synaptic current evoked in a Purkinje cell by an action potential in a single interneuron suggests that as development proceeds interneuron activities must be coordinated to inhibit efficiently Purkinje cells.     

Electrophysiological and pharmacological characterization of the direct perforant path input to hippocampal area CA3

Monosynaptic perforant path responses evoked by subicular stimulation were recorded from CA3 pyramidal cells of rat hippocampal slices. These monosynaptic responses were isolated by using low intensities of stimulation and by placing a cut through the mossy fibers. Perforant path-evoked responses consisted of both excitatory and inhibitory components. Excitatory postsynaptic currents (EPSCs) were mediated by both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidreceptors (AMPAR) and N-methyl--aspartate receptors (NMDAR). Inhibitory postsynaptic currents consisted of gamma-aminobutyric acid-A (GABAA-) and -B (GABAB)-receptor-mediated components. At membrane potentials more positive than -60 mV and at physiological [Ca2+]/[Mg2+] ratios, >30% of perforant path evoked EPSC was mediated by NMDARs. This value varied as a function of the membrane voltage and external [Mg2+]. Two types of responses were observed after low-intensity stimulation of the perforant path. The first type of response showed paired-pulse facilitation and was reduced by 2-amino-4-phosphonobutyric acid (AP4). The second type of response showed paired-pulse depression and was reduced by baclofen. Electrophysiological and pharmacological characteristics of these two types of responses are similar to the properties of lateral and medial perforant path-evoked EPSPs in the dentate gyrus.     

Heterogeneity in use-dependent depression of inhibitory postsynaptic potentials in the rat neostriatum in vitro

"Minimal stimulation" was applied to evoke responses in an "all-or-none" fashion in presumed medium spiny neurons of rat neostriatal slices in the presence of antagonists for glutamatergic excitation. For comparison, responses were evoked in the same cells by compound stimulation. Bicuculline (30 microM) blocked responses evoked by minimal stimulation, indicating that they were gamma-aminobutyric acid-A (GABAA)-receptor-mediated inhibitory postsynaptic potentials (IPSPS), whereas responses evoked by compound stimulation were only reduced in amplitude. Likewise, R(-)baclofen (1-20 microM) blocked IPSPS evoked by minimal stimulation in all but one cell. On the contrary, responses evoked by compound stimulation were always reduced in amplitude but never blocked. Paired-pulse depression (PPD) of averaged responses to minimal and compound stimulation was observed at a stimulus interval of 300 ms. The GABAB receptor antagonist CGP55845A (0.5 microM) had no effect on PPD evoked by compound stimulation but abolished PPD evoked by minimal stimulation. In a second set of experiments, the two stimulation paradigms were used to evoke responses in neostriatal slices continuously bathed in R(-)baclofen (10-20 microM). In R(-)baclofen a strong PPD was evoked by minimal and by compound stimulation. The amplitude of the response to compound stimulation increased on application of CGP55845A (0.5 microM). At the same time, PPD evoked by compound stimulation decreased. On the contrary, IPSP amplitude and PPD evoked by minimal stimulation remained unchanged. We conclude that two types of GABAergic terminals exist in the rat neostriatum, only one of which is regulated by GABAB receptors. However, the other class of terminals, not regulated by GABAB receptors, displays a much more pronounced PPD.     

L-arginine potentiates GABA-mediated synaptic transmission by a nitric oxide-independent mechanism in rat dopamine neurons

Effects of L-arginine in the nervous system are often attributed to nitric oxide. Using whole-cell patch pipettes to record membrane currents in voltage-clamp from dopamine neurons in the rat midbrain slice, the present studies found that L-arginine potentiates GABA-dependent membrane currents via a nitric oxide-independent mechanism. L-Arginine (0.3-10 mM) increased the peak amplitude, half-width duration and time constant of decay of GABA(B) receptor-mediated inhibitory postsynaptic currents in a concentration-dependent manner. In the presence of CGP 35348 (300 microM), a GABA(B) receptor antagonist, L-arginine also prolonged the duration of inhibitory postsynaptic currents mediated by GABA(A) receptors, but their amplitudes were reduced. L-Arginine (10 mM) also evoked 17+/-3 pA of outward current (at -60 mV) which was significantly increased in the presence of exogenous GABA (100 microM). Pressure-ejection of GABA from micropipettes produced outward currents mediated by GABA(B) receptors (recorded in bicuculline) or GABA(A) receptors (recorded in CGP 35348); both types of receptor-mediated currents were increased by L-arginine (10 mM). In contrast, outward currents evoked by baclofen, a GABA(B) receptor agonist, were not potentiated by L-arginine. The GABA transport inhibitors NO 711 (1 microM) and nipecotic acid (1 mM) significantly increased the half-width duration and time-constant of decay of GABA(B)-mediated inhibitory postsynaptic currents, thus mimicking effects of L-arginine. However, nitric oxide donors failed to mimic effects of L-arginine on GABA(B) inhibitory postsynaptic currents, and inhibitors of nitric oxide synthesis failed to selectively block the action of L-arginine. These findings suggest that L-arginine potentiates GABA synaptic transmission by a nitric oxide-independent mechanism. Similarities between effects of L-arginine, NO 711 and nipecotic acid suggest that L-arginine inhibits a GABA transporter.     

GABAB receptor-mediated inhibition of tetrodotoxin-resistant GABA release in rodent hippocampal CA1 pyramidal cells

Tight-seal whole-cell recordings from CA1 pyramidal cells of rodent hippocampus were performed to study GABAB receptor-mediated inhibition of tetrodotoxin (TTX)-resistant IP-SCs. IPSCs were recorded in the presence of TTX and glutamate receptor antagonists. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs by a presynaptic action. The inhibition by (R)-(-)-baclofen was concentration-dependent, was not mimicked by the less effective enantiomer (S)-(+)-baclofen, and was blocked by the GABAB receptor antagonist CGP 55845A, suggesting a specific effect on GABAB receptors. The inhibition persisted in the presence of the Ca2+ channel blocker Cd2+. There was no requirement for an activation of K+ conductances by (R)-(-)-baclofen, because the inhibition of TTX-resistant IPSCs persisted in Ba2+ and Cd2+. Because the time courses of TTX-resistant IPSCs were not changed by (R)-(-)-baclofen, there was no evidence for a selective inhibition of quantal release from a subgroup of GABAergic terminals. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs in guinea pigs and Wistar rats, whereas the inhibition was much smaller in Sprague Dawley rats. In Cd2+ and Ba2+, beta-phorbol-12,13-dibutyrate and forskolin enhanced the frequency of TTX-resistant IPSCs. Only beta-phorbol-12, 13-dibutyrate reduced the inhibition by (R)-(-)-baclofen. We conclude that GABAB receptors inhibit TTX-resistant GABA release through a mechanism independent from the well known effects on Ca2+ or K+ channels. The inhibition of quantal GABA release can be reduced by an activator of protein kinase C.     

Increased opioid inhibition of GABA release in nucleus accumbens during morphine withdrawal

The nucleus accumbens is a key component of the reward pathway that plays a role in addiction to many drugs of abuse, including psychostimulants and opioids. The effects of withdrawal from chronic morphine were examined in the nucleus accumbens using brain slices from morphine-treated animals. Recordings were made from interneurons in the shell of nucleus accumbens, and the presynaptic inhibition of GABA-A IPSCs by opioids was examined. In slices from control animals, opioids caused a maximal inhibition of 50%, forskolin increased the IPSC amplitude by less than twofold, and the maximal inhibition by opioids in the presence of forskolin was not changed. During withdrawal, however, forskolin caused approximately a fourfold increase in the amplitude of the IPSC, and the maximal inhibition by opioids was increased to 80%. The results indicate that transmitter release is increased during opioid withdrawal, particularly after the activation of adenylyl cyclase. The cAMP-dependent increase in transmitter release is potently inhibited by opioids, such that the overall effect of opioids is augmented during withdrawal. The induction of an opioid-sensitive cAMP-dependent mechanism that regulates transmitter release may be a critical component of acute opioid withdrawal.     

Acetylcholine activates an alpha-bungarotoxin-sensitive nicotinic current in rat hippocampal interneurons, but not pyramidal cells

The effects of acetylcholine on both pyramidal neurons and interneurons in the area CA1 of the rat hippocampus were examined, using intracellular recording techniques in an in vitro slice preparation. In current-clamp mode, fast local application of acetylcholine (ACh) to the soma of inhibitory interneurons in stratum radiatum resulted in depolarization and rapid firing of action potentials. Under voltage-clamp, ACh produced fast, rapidly desensitizing inward currents that were insensitive to atropine but that were blocked by nanomolar concentrations of the nicotinic alpha7 receptor-selective antagonists alpha-bungarotoxin (alphaBgTx) and methyllycaconitine. Nicotinic receptor antagonists that are not selective for alpha7-containing receptors had little (mecamylamine) or no effect (dihydro-beta-erythroidine) on the ACh-induced currents. Glutamate receptor antagonists had no effect on the ACh-evoked response, indicating that the current was not mediated by presynaptic facilitation of glutamate release. However, the current could be desensitized almost completely by bath superfusion with 100 nM nicotine. In contrast to those actions on interneurons, application of ACh to the soma of CA1 pyramidal cells did not produce a detectable current. Radioligand-binding experiments with [125I]-alphaBgTx demonstrated that stratum radiatum interneurons express alpha7-containing nAChRs, and in situ hybridization revealed significant amounts of alpha7 mRNA. CA1 pyramidal cells did not show specific binding of [125I]-alphaBgTx and only low levels of alpha7 mRNA. These results suggest that, in addition to their proposed presynaptic role in modulating transmitter release, alpha7-containing nAChRs also may play a postsynaptic role in the excitation of hippocampal interneurons. By desensitizing these receptors, nicotine may disrupt this action and indirectly excite pyramidal neurons by reducing GABAergic inhibition.