Substance P release in the dorsal horn assessed by receptor internalization: NMDA receptors counteract a tonic inhibition by GABA(B) receptors

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.     

Molecular and functional properties of neurons in the human lateral amygdala

Neuronal properties were investigated through patch-clamp recording in situ in surgical specimens of the human lateral amygdala (LA) obtained from patients with intractable temporal lobe epilepsy. Projection neurons displayed spiny dendrites, action potentials with varying degree of frequency adaptation, and an inwardly rectifying K+ (Kir) conductance coupled to GABA(B) receptors. In interneurons, dendrites were spineless or sparsely spiny, action potentials were shorter than those in projection neurons and often occurred spontaneously, and GABA(B) receptor-mediated responses were lacking. Single-cell RT-PCR demonstrated expression of Kir channel subunits Kir3.1 and Kir3.2 and of vesicular glutamate transporters VGLUT1 and VGLUT2 in projection neurons. It is concluded that projection neurons and interneurons of the human LA can be distinguished based upon morphological, electrophysiological, and molecular biological criteria. The most striking difference relates to the expression of postsynaptic GABA(B) receptors coupled to Kir3 channels in projection neurons and the lack of functional GABA(B) receptors in interneurons.     

The weaver mutation reverses the function of dopamine and GABA in mouse dopaminergic neurons.

In the present study, we characterized the intrinsic electrophysiological properties and the membrane currents activated by dopamine (DA) D(2) and GABA(B) receptors in midbrain dopaminergic neurons, maintained in vitro in a slice preparation, from wild-type and homozygous weaver (wv/wv) mice. By using patch-clamp techniques, we found that membrane potential, apparent input resistance, and spontaneous firing of wv/wv dopaminergic neurons were similar to those of dopamine-containing cells recorded from nonaffected (+/+) animals. More interestingly, the wv/wv neurons were excited rather than inhibited by dopamine and the GABA(B) agonist baclofen. This neurotransmitter-mediated excitation was attributable to the activation of a G-protein-gated inward current that reversed polarity at a membrane potential of approximately -30 mV. We suggest that the altered behavior of the receptor-operated wv G-protein-gated inwardly rectifying K(+) channel 2 (GIRK2) might be related to the selective degeneration of the dopaminergic neurons. In addition, the wv GIRK2 would not only suppress the autoreceptor-mediated feedback inhibition of DA release but could also establish a feedforward mechanism of DA release in the terminal fields.     

Gamma-aminobutyric acid type B receptors facilitate L-type and attenuate N-type Ca(2+) currents in isolated hippocampal neurons

Activation of presynaptic gamma-aminobutyric acid type B (GABA(B)) receptors inhibits neurotransmitter release at many synapses (both excitatory and inhibitory), and activation of postsynaptic GABA(B) receptors leads to a general inhibition of the postsynaptic cell in mature neurons. Although the action of GABA(B) receptors at the soma of excitatory hippocampal pyramidal cells has been resolved to be regulation of a potassium or calcium conductance, it is not clear that all neurons in the hippocampus demonstrate similar effects of GABA(B) receptor activation. In the current study, GABA(B) receptor-mediated effects on calcium currents in acute cultures composed of heterogeneous cells from the superior region of neonatal hippocampi were studied. In 54.5% of cells, the GABA(B) receptor agonist baclofen (10 microM) attenuated the whole-cell calcium current by 21.0% +/- 1.1%. In 29.9% of cells, baclofen facilitated the calcium current by 43.5% +/- 8.1%. The component of current attenuated by baclofen was blocked by the N-type calcium channel antagonist omega-conotoxin GVIA (3 microM). The component of current facilitated by baclofen was blocked by the L-type channel antagonist nimodipine (20 microM). For cells that showed calcium current facilitation, baclofen shifted the half-maximal activation by approximately -14 mV. The data indicate that activation of GABA(B) receptors in neurons of the superior hippocampus attenuates current through N-type channels and facilitates current through L-type channels. The two opposing effects of GABA(B) receptor activation may reflect the heterogeneity of the cultured cells or may be a developmentally regulated phenomenon.     

Shunting and hyperpolarizing GABAergic inhibition in the high-potassium model of ictogenesis in the developing rat hippocampus

Ontogenesis of GABAergic signaling may play an important role in developmental changes in seizure susceptibility in the high-potassium model of ictogenesis in vitro. The age-dependent effects of [K(+)](o) on the reversal potential of the GABA(A)-mediated responses and membrane potential in hippocampal slices in vitro were compared with the effect of GABA(A)-receptors antagonists and GABA(A) modulators on high-potassium induced seizures in the CA3 pyramidal layer of rat hippocampus in vivo. GABA(A) responses were depolarizing at P8-12 and hyperpolarizing at P17-21. In P8-12 rats, GABA(A) responses switch their polarity from depolarizing to hyperpolarizing upon elevation of extracellular potassium. At approximately 10 mM [K(+)](o), activation of GABA(A) receptors produced an isoelectric, purely shunting response characterized by no changes in the membrane potential but an increase in the membrane conductance. In P17-21 rats, the hyperpolarizing GABA(A) driving force progressively increased with elevation of [K(+)](o). In P8-12 rats in vivo, GABA(A)-receptor antagonists did not affect the occurrence of ictal discharges induced by intrahippocampal injection of 10 mM [K(+)](o), but significantly increased seizure duration. Diazepam and isoguvacine completely prevented seizures induced by 10 mM [K(+)](o). In P17-21 rats, GABA(A)-receptor antagonists strongly increased the occurrence of ictal activity induced both by 10 mM [K(+)](o). Taken together, these results suggest that anticonvulsive effects of GABA are because of the combination of shunting and hyperpolarizing actions of GABA. Although shunting GABA is already efficient in the young age group, a developmental increase in the hyperpolarizing GABA(A) driving force likely contributes to the increase in the GABAergic control of seizures upon maturation.     

Inwardly rectifying K(+) (Kir) channels antagonize ictal-like epileptiform activity in area CA1 of the rat hippocampus

Reactive glial cells, for example, from patients with temporal lope epilepsy have a reduced density of inward rectifying K(+) (Kir) channels and thus a reduced K(+) buffering capacity. Evidence is accumulating that this downregulation of Kir channels could be implicated in epileptogenesis. In rat hippocampal brain slices, prolonged exposure to the nonselective Kir channel antagonist, Cs(+) (5 mM), gives rise to an epileptiform field potential (Cs-FP) in area CA1 composed of an initial positive (interictal-like) phase followed by a prolonged negative (ictal-like) phase. We have previously shown that the interictal-like phase depends on synaptic activation. The present study extends these findings by showing that the ictal-like phase of the Cs-FP is (i) sensitive to osmotic expansion of the extracellular space, (ii) reversed very quickly during wash out of Cs(+), and (iii) re-established in the presence of Ba(2+) (30-200 microM) or isosmotic low extracellular concentration of Na(+) ([Na(+)](o), 51.25 mM). The interictal-like phase showed less or no sensitivity to these treatments. In the complete absence of Cs(+), the Cs-FP could be fully reconstructed by the combined application of 4-aminopyridine (0.5 mM), an isosmotic high extracellular concentration of K(+) ([K(+)](o), 7 mM), and low [Na(+)](o) (51.25 mM). These results suggest that the interictal-like phase is initiated through synaptic activation and results from an unspecific increase in neuronal excitability, whereas the ictal-like phase is entirely dependent on blockade of Kir channels in CA1. We propose that glial dysfunction-related loss of Kir channels may not alone be sufficient for starting the induction process, but will likely increase the tendency of an epileptogenic process to proceed into seizure activity.     

Topology of ionotropic glutamate receptors in brains of heterozygous and homozygous weaver mutant mice.

In weaver mice, mutation of a G-protein inwardly rectifying K(+) channel leads to a cerebellar developmental anomaly characterized by granule and Purkinje cell loss and, in addition, degeneration of dopaminergic neurons. To evaluate other deficits, ionotropic glutamate receptors sensitive to N-methyl-D-aspartate (NMDA), amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainic acid (KA) were examined by autoradiography with [(3)H]MK-801, [(3)H]AMPA, and [(3)H]KA. These surveys were carried out in selected areas of cerebral cortex, hippocampus and related limbic regions, basal ganglia, thalamus, hypothalamus, brainstem, and cerebellum from heterozygous (wv/+) and homozygous (wv/wv) weaver mutants, and compared to wild-type (+/+) mice. In wv/+ and wv/wv mutants, NMDA receptor levels were lower in cortical areas, septum, hippocampus, subiculum, neostriatum, nucleus accumbens, superior colliculus, and in the cerebellar granular layer. Densities of KA receptors were lower in cortical areas, hippocampus, limbic system structures, neostriatum, nucleus accumbens, thalamus and hypothalamus, superior and inferior colliculi, and cerebellar cortex of wv/wv mutants. Levels of AMPA receptors in the weaver were higher than in +/+ mice, particularly in somatosensory and piriform cortices and periaqueductal gray of wv/+, and in somatosensory cortex, CA1 field of Ammon's horn and cerebellar granular layer of wv/wv. Abnormal developmental signals, aberrant cellular responses, or a distorted balance between neurotransmitter interactions may underlie such widespread and reciprocal glutamate receptor alterations, while in the case of cerebellar cortex, NMDA receptors are lacking due to a massive disappearance of cerebellar granule cells and some loss of Purkinje neurons.     

Tetanic stimulation of schaffer collaterals induces rhythmic bursts via NMDA receptor activation in rat CA1 pyramidal neurons

Exploring the principles that regulate rhythmic membrane potential (Vm) oscillations and bursts in hippocampal CA1 pyramidal neurons is essential to understanding the theta rhythm (theta). Recordings were performed in vitro in hippocampal slices from young rats, and a group of the recorded CA1 pyramidal cells were dye-filled with carboxifluorescein and immunolabeled for the R1 subunit of the NMDA receptor. Tetanic stimulation of Schaffer collaterals (SCs) and iontophoresis of glutamate evoked rhythmic Vm oscillations and bursts (approximately 10 mV, approximately 7 Hz, 2-5 spikes per burst) in cells (31%) placed close to the midline ("medial cells"). Rhythmic bursts remained under picrotoxin (10 microM) and Vm oscillations persisted with tetrodotoxin (1.5 microM), but bursts were blocked by AP5 (25 microM) and Mg2+-free solutions. Depolarization and AMPA never induced rhythmic bursts. The rest of the neurons (69%), recorded closer to the CA3 region ("lateral cells"), discharged rhythmically single repetitive spikes under SC stimulation and glutamate in control conditions, but fired rhythmic bursts under similar stimulation, both when NMDA was applied and when non-NMDA receptors were blocked with CNQX (20 microM). Medial cells exhibited a larger NMDA current component and a higher NMDAR1 density at the apical dendritic shafts than lateral cells, suggesting that these differences underlie the dissimilar responses of both cell groups. We conclude that the "theta-like" rhythmic oscillations and bursts induced by glutamate and SC stimulation relied on the activation of NMDA receptors at the apical dendrites of medial cells. These results suggest a role of CA3 pyramidal neurons in the generation of CA1 theta via the activation of NMDA receptors of CA1 pyramidal neurons.     

Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels

Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl(-) channels.     

Allopregnanolone modulates spontaneous GABA release via presynaptic Cl- permeability in rat preoptic nerve terminals

The endogenous neurosteroid 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone) affects presynaptic nerve terminals and thereby increases the frequency of spontaneous GABA release. The present study aimed at clarifying the mechanisms underlying this presynaptic neurosteroid action, by recording the frequency of spontaneous GABA-mediated inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic nucleus (MPN) of rat. Acutely dissociated neurons with functional adhering nerve terminals were studied by perforated-patch recording under voltage-clamp conditions. It was shown that the sIPSC frequency increased with the external K(+) concentration ([K(+)](o)). Further, the effect of allopregnanolone on the sIPSC frequency was strongly dependent on [K(+)](o). In a [K(+)](o) of 5 mM, 2.0 microM allopregnanolone caused a clear increase in sIPSC frequency. However, the effect declined rapidly with increased [K(+)](o) and at high [K(+)](o) allopregnanolone reduced the sIPSC frequency. The effect of allopregnanolone was also strongly dependent on the external Cl(-) concentration ([Cl(-)](o)). In a reduced [Cl(-)](o) (40 mM, but with a standard [K(+)](o) of 5 mM), the effect on sIPSC frequency was larger than that in the standard [Cl(-)](o) of 146 mM. The dependence of the effect of allopregnanolone on [K(+)](o) and on estimated presynaptic membrane potential was also altered by the reduction in [Cl(-)](o). As in standard [Cl(-)](o), the effect in low [Cl(-)](o) declined when [K(+)](o) was raised, but reversed at a higher [K(+)](o). The GABA(A) receptor agonist muscimol also potentiated the sIPSC frequency. Altogether, the results suggest that allopregnanolone exerts its presynaptic effect by increasing the presynaptic Cl(-) permeability, most likely via GABA(A) receptors.     

Deterministic and stochastic neuronal contributions to distinct synchronous CA3 network bursts

Computational studies have suggested that stochastic, deterministic, and mixed processes all could be possible determinants of spontaneous, synchronous network bursts. In the present study, using multicellular calcium imaging coupled with fast confocal microscopy, we describe neuronal behavior underlying spontaneous network bursts in developing rat and mouse hippocampal area CA3 networks. Two primary burst types were studied: giant depolarizing potentials (GDPs) and spontaneous interictal bursts recorded in bicuculline, a GABA(A) receptor antagonist. Analysis of the simultaneous behavior of multiple CA3 neurons during synchronous GDPs revealed a repeatable activation order from burst to burst. This was validated using several statistical methods, including high Kendall's coefficient of concordance values for firing order during GDPs, high Pearson's correlations of cellular activation times between burst pairs, and latent class analysis, which revealed a population of 5-6% of CA3 neurons reliably firing very early during GDPs. In contrast, neuronal firing order during interictal bursts appeared homogeneous, with no particular cells repeatedly leading or lagging during these synchronous events. We conclude that GDPs activate via a deterministic mechanism, with distinct, repeatable roles for subsets of neurons during burst generation, while interictal bursts appear to be stochastic events with cells assuming interchangeable roles in the generation of these events.     

Gabapentin actions on Kir3 currents and N-type Ca2+ channels via GABAB receptors in hippocampal pyramidal cells

Gabapentin is a clinically effective anticonvulsant with an unclear mechanism of action. It was described as a GABA(B(1a,2)) receptor subtype-selective agonist, activating postsynaptic K(+) currents and inhibiting postsynaptic Ca(2+) channels in CA1 pyramidal cells, but without presynaptic actions. These activities appeared controversial and we therefore sought to further clarify gabapentin actions in rat hippocampal slices by characterizing K(+) currents and Ca(2+) channels targeted by gabapentin using whole-cell recording and multiphoton Ca(2+) imaging. 1) We found that gabapentin and baclofen induced inwardly rectifying K(+) currents (K(Gbp) and K(Bac), respectively), sensitive to Ba(2+) and Cs(+). 2) A constitutively active K(IR) current, independent of GABA(B) receptor activation and sensitive to Ba(2+) and Cs(+) was also present. 3) K(Gbp), K(Bac), and K(IR) currents showed some differences in sensitivity to Ba(2+) and Cs(+), indicating the possible activation of distinct Kir3 currents, independent of K(IR), by gabapentin and baclofen. 4) Gabapentin inhibition of Ca(2+) channels was abolished by omega-conotoxin GVIA, but not by omega-agatoxin IVA and nimodipine, indicating a predominant action of gabapentin on N-type Ca(2+) channels. 5) Gabapentin actions were linked to activation of pertussis toxin-sensitive G-proteins since N-ethylmaleimide (NEM) blocked K(Gbp) activation and Ca(2+) channel inhibition by gabapentin. 6) Finally, gabapentin reduced epileptiform discharges in slices via GABA(B) receptor activation. The anticonvulsant actions of gabapentin in hippocampal cells may thus involve GABA(B) receptor coupling to G-proteins and modulation of Kir3 and N-type Ca(2+) channels. Moreover, gabapentin and baclofen activation of GABA(B) receptors may couple to distinct cellular targets.     

Spontaneous, low frequency (approximately 2-3 Hz) field activity generated in rat ventral hippocampal slices perfused with normal medium

This study demonstrates that transverse slices taken from the ventral hippocampus of adult rats perfused with a medium of normal ionic composition sustain spontaneous periodic field potentials due to the synchronous activity of a population of neurons. This ventral hippocampus spontaneous synchronous activity (VHSSA) in CA1 stratum pyramidale consisted of positive potentials (approximately 0.12 mV, 55 ms) occurring at a frequency of 2.8 +/- 0.2 Hz for hours without interruption. VHSSA was most frequently observed in slices taken 1-3 mm from the ventral end of hippocampus, and was absent in slices taken from tissue more than 4.5 mm away from it. Stimulation of Schaffer collaterals primed the appearance of potentials, which were similar to VHSSA and clearly distinguishable from excitatory postsynaptic potentials. In view of the known relative proneness of ventral hippocampus to epilepsy, we perfused ventral slices with high-[K(+)](o) medium (8 mM). Albeit reduced in amplitude, VHSSA persisted during the high-[K(+)](o) induced interictal-like epileptiform activity. We could not document any temporal relationship between the two phenomena. Low concentrations of the antagonist of gamma-amino-butyric acid receptors, type A, bicuculline (2-3 microM), which enhanced the high-[K(+)](o) induced epileptiform activity, reversibly blocked the VHSSA. We conclude that under standard in vitro conditions small circuits in the ventral hippocampus are most often and for long periods of time engaged in synchronous quasi-rhythmic low-frequency activity, generated locally by mechanisms substantially differing from those supporting epileptiform discharges.     

Cellular mechanisms underlying the rhythmic bursts induced by NMDA microiontophoresis at the apical dendrites of CA1 pyramidal neurons

This article reports the cellular mechanisms underlying a form of intracellular "theta-like" (theta-like) rhythm evoked in vitro by microiontophoresis of N-methyl-D-aspartate (NMDA) at the apical dendrites of CA1 pyramidal neurons. Rhythmic membrane potential (Vm) oscillations and action potential (AP) bursts (approximately 6 Hz; approximately 20 mV; approximately 2-5 APs) were evoked in all cells. The response lasted approximately 2 s, and the initial oscillations were usually small (< 20 mV) and below AP threshold. Rhythmic bursts were never evoked by imposed depolarization in the absence of NMDA. Block of Na+ conductance with tetrodotoxin (TTX) (1.5 microM), of non-NMDA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and of synaptic inhibition by bicuculline (50 microM) and picrotoxin (50 microM) did not prevent NMDA oscillation. Inhibition of the voltage dependence of the NMDA conductance in Mg2+-free Ringer's solution blocked oscillations. Preventing Ca2+ influx with Ca2+-free and Co2+ (2-mM) solutions and block of the slow Ca2+-dependent afterhyperpolarization (sAHP) by carbamilcholine (5 microM), isoproterenol (10 microM), and intracellular BAPTA blocked NMDA oscillations. Inhibition of L-type Ca2+ conductance with nifedipine (30 microM) reduced oscillation amplitude. Block of tetraethylammonium (TEA) (10 mM) and 4AP (10 mM)-sensitive K+ conductance increased the duration and amplitude, but not the frequency, of oscillations. In conclusion, theta-like bursts relied on the voltage dependence of the NMDA conductance and on high-threshold Ca2+ spikes to initiate and boost the depolarizing phase of oscillations. The repolarization is initiated by TEA-sensitive K+ conductance and is controlled by the sAHP. These results suggest a role of interactions between NMDA conductance and intrinsic membrane properties in generating the CA1 theta-rhythm.     

Postsynaptic dopamine D3 receptor modulation of evoked IPSCs via GABA(A) receptor endocytosis in rat hippocampus

Dopamine is known to be an important modulator of learning and memory processes, but its mechanisms of action at the cellular level are diverse and are not fully characterized. In the hippocampus, pharmacologically isolated monosynaptic IPSCs were measured using the whole-cell voltage-clamp recording technique. Both electrically evoked and spontaneous miniature GABA(A) receptor currents were recorded from CA1 pyramidal neurons in slices obtained from mature rats in the presence of the D3-selective agonist PD128907. The activation of D3 receptors inhibited synaptic GABAergic input without affecting presynaptic function or passive membrane properties. Inhibition of IPSCs evoked from stratum radiatum occurred via regulation of dynamin-dependent trafficking of the GABA(A) receptor, as inclusion of dynamin inhibitory peptide (50 microM) in the recording solution prevented the inhibitory effects of PD128907 (1 microM). This effect of D3 receptor activation could be prevented by intracellular application of either an inhibitor of protein kinase A (PKI, 20 microM) or an activator of protein kinase A (8-OH-cAMP, 50 microM). Neither synchronous IPSCs evoked from the stratum oriens nor asynchronous miniature IPSCs recorded from the stratum radiatum were affected by D3 agonist. The induction of long-term potentiation (LTP) of the extracellular field response in both the stratum radiatum and stratum oriens demonstrated that only potentiation in the stratum radiatum was significantly enhanced by PD128907 (1 microM). Our results suggest that the activation of D3 receptors can modulate GABA(A) receptor endocytosis in the hippocampus in a lamina specific manner, and thereby alter the efficacy of GABAergic transmission in the stratum radiatum of the CA1 region through a postsynaptic mechanism of action.     

Comparison of astrocytic morphology, proliferation, marker profile and response to neurons in wild-type and weaver mutant mouse cerebella in culture

In serum-free monolayer cultures of early postnatal weaver (wv/wv) cerebellum granule neurons show decreased attachment, survival and neurite outgrowth when compared to wild-type (+/+) littermate cultures. wv/wv Astrocytes display a more epithelioid morphology and altered proliferation. However, both morphology and proliferation of wv/wv astrocytes were reversed to a normal phenotype by addition of purified small neurons from early postnatal cerebella from +/+ animals. Attachment of +/+ neurons to wv/wv astrocytes was not significantly different from that of +/+ astrocytes and antigenic marker profiles of wv/wv and +/+ astrocytes differed only slightly. Attempts failed to revert the abnormal wv/wv phenotype in neurons by addition of gangliosides, triiodothyronine T3, prostaglandin A2, medium containing 1% horse serum, conditioned medium from +/+ cerebellar cultures, or by cocultivation with +/+ astrocytes. We would like to suggest that the primary defect of the wv/wv mutation is predominantly an abnormality in granule cell neurons, but not of the vast majority of astrocytes.     

Presynaptic GABA(B) receptors on glutamatergic terminals of CA1 pyramidal cells decrease in efficacy after partial hippocampal kindling

We tested the hypothesis that presynaptic GABA(B) receptors on glutamatergic terminals (GABA(B) heterosynaptic receptors) decreased in efficacy after partial hippocampal kindling. Rats were implanted with chronically indwelling electrodes and 15 hippocampal afterdischarges were evoked by high-frequency electrical stimulation of hippocampal CA1. Control rats were implanted with electrodes but not given high-frequency stimulations. One to 21 days after the last afterdischarge, excitatory postsynaptic potentials (EPSPs) were recorded in CA1 of hippocampal slices in vitro, following stimulation of the stratum radiatum. Field EPSPs (fEPSPs) were recorded in CA1 stratum radiatum and intracellular EPSPs (iEPSPs) were recorded from CA1 pyramidal cells. GABA(B) receptor agonist +/- baclofen (10 microM) in the bath suppressed the fEPSPs significantly more in control than kindled rats, at 1 or 21 days after kindling. Similarly, baclofen (10 microM) suppressed iEPSPs more in the control than the kindled group of neurons recorded at 1 day after kindling. Suppression of the fEPSPs by 1 microM N(6)-cyclopentyladenosine, which acted on presynaptic A1 receptors, was not different between kindled and control rats. Activation of the GABA(B) heteroreceptors by a conditioning burst stimulation of CA3 afferents suppressed the iEPSPs evoked by a test pulse. The suppression of the iEPSPs at 250-500 ms condition-test interval was larger in control than kindled groups of neurons. It was concluded that the efficacy of presynaptic GABA(B) receptors on the glutamatergic terminals was reduced after partial hippocampal kindling. The reduction in heterosynaptic presynaptic GABA(B) receptor efficacy will increase glutamate release and seizure susceptibility, particularly during repeated neural activity.     

Effect of catecholamines on the hyperpolarization-activated cationic Ih and the inwardly rectifying potassium I(Kir) currents in the rat substantia nigra pars compacta

Whole-cell ruptured-patch and perforated-patch recordings were used in principal neurons of the rat substantia nigra pars compacta (SNc) to study the effect of catecholamines both on the hyperpolarization-activated cationic (Ih) and the inwardly rectifying potassium (I(Kir)) currents. In internal potassium, a 2 min bath application of noradrenaline (NA; 50 microM) or dopamine (DA; 50 microM) both inhibited Ih and induced an outward current associated with an increase in I(Kir) conductance. These two effects recovered poorly after wash-out. Protein kinase A (PKA), protein kinase C (PKC) and phosphatases 1 and 2A inhibitors did not modify the NA and DA effects on the amplitude of Ih and I(Kir) currents. They also had no effect on the recovery of the catecholamine responses. In perforated-patch experiments, NA and DA also induced an inhibition of Ih and revealed an outward current associated with an increase in conductance. However, both effects recovered in less than 5 min following the wash-out. These results indicate that neither PKA, PKC, nor phosphatases 1 or 2A were required in the NA and DA modulation of these two currents and that an intracellular factor, that could be either washed-out or inversely up-regulated in the ruptured-patch configuration, was implicated in the recovery of both effects. In the presence of external barium (300 microM) or internal caesium which both blocked the outward current and the increase in conductance, neither NA nor DA affected Ih, suggesting that the effect on Ih observed is secondary to the activation of the I(Kir) channels. Increasing chloride conductance of the cell by activation of GABA(A) receptors also induced an inhibition of Ih. All together these results suggest that the NA or DA induced inhibition of Ih could result from an occlusion of Ih by a space-clamp effect.