Regular firing of a single output neuron reduces its own inhibition through endocannabinoids in substantia nigra pars reticulata of juvenile mice.

Depolarization-induced suppression of inhibition in substantia nigra pars reticulata suggests that burst-like activity but not regular firing suffices to activate presynaptic endocannabinoid CB1 receptors. To more closely determine the type of activity required, we applied gramicidin perforated patch recording under visual control to substantia nigra slices of juvenile mice. We found that evoked inhibitory postsynaptic currents (eIPSCs) were reduced in amplitude by the spontaneous firing of a neuron under study, whereas silencing this neuron enhanced inhibitory responses. Autonomous firing reduced eIPSCs to 78%+/-2% in a time- but not frequency-dependent manner. The phenomenon which we termed firing-induced suppression of inhibition was cannabinoid receptor subtype 1-dependent, whereas adenosine A1 receptors played only a minor role. Depletion of intracellular Ca(2+) stores abolished the firing-induced suppression of inhibition suggesting that Ca(2+) release from internal stores is necessary for the production of endocannabinoids during autonomous firing. We suggest that the Ca(2+) influx during autonomous activity of pars reticulata neurons suffices to selectively dampen incoming inhibition from striatal neurons because it is amplified by ryanodine receptor-mediated Ca(2+) release from intracellular stores.

Subcellular localization of type 1 cannabinoid receptors in the rat basal ganglia.

Endocannabinoids, acting via type 1 cannabinoid receptors (CB1), are known to be involved in short-term synaptic plasticity via retrograde signaling. Strong depolarization of the postsynaptic neurons is followed by the endocannabinoid-mediated activation of presynaptic CB1 receptors, which suppresses GABA and/or glutamate release. This phenomenon is termed depolarization-induced suppression of inhibition (DSI) or excitation (DSE), respectively. Although both phenomena have been reported to be present in the basal ganglia, the anatomical substrate for these actions has not been clearly identified. Here we investigate the high-resolution subcellular localization of CB1 receptors in the nucleus accumbens, striatum, globus pallidus and substantia nigra, as well as in the internal capsule, where the striato-nigral and pallido-nigral pathways are located. In all examined nuclei of the basal ganglia, we found that CB1 receptors were located on the membrane of axon terminals and preterminal axons. Electron microscopic examination revealed that the majority of these axon terminals were GABAergic, giving rise to mostly symmetrical synapses. Interestingly, preterminal axons showed far more intense staining for CB1, especially in the globus pallidus and substantia nigra, whereas their terminals were only faintly stained. Non-varicose, thin unmyelinated fibers in the internal capsule also showed strong CB1-labeling, and were embedded in bundles of myelinated CB1-negative axons. The majority of CB1 receptors labeled by immunogold particles were located in the axonal plasma membrane (92.3%), apparently capable of signaling cannabinoid actions. CB1 receptors in this location cannot directly modulate transmitter release, because the release sites are several hundred micrometers away. Interestingly, both the CB1 agonist, WIN55,212-2, as well as its antagonist, AM251, were able to block action potential generation, but via a CB1 independent mechanism, since the effects remained intact in CB1 knockout animals. Thus, our electrophysiological data suggest that these receptors are unable to influence action potential propagation, thus they may not be functional at these sites, but are likely being transported to the terminal fields. The present data are consistent with a role of endocannabinoids in the control of GABA, but not glutamate, release in the basal ganglia via presynaptic CB1 receptors, but also call the attention to possible non-CB1-mediated effects of widely used cannabinoid ligands on action potential generation.

Two pathways for the activation of small-conductance potassium channels in neurons of substantia nigra pars reticulata.

Neurons in substantia nigra pars reticulata express the messenger RNA for SK2 but not for SK3 subunits that form small-conductance, Ca2+-dependent K+ channels in dopamine neurons. To determine pathways for the activation of small-conductance, Ca2+-dependent K+ channels in substantia nigra pars reticulata neurons of rats and mice, we studied effects of the selective blocker of small-conductance, Ca2+-dependent K+ channels, apamin (0.01 or 0.3 microM). Apamin diminished the afterhyperpolarization following each action potential and induced burst discharges in substantia nigra pars reticulata neurons. Apamin had a robust effect already at a low (10 nM) concentration consistent with the expression of the SK2 subunit. Afterhyperpolarizations were also reduced by the Ca2+ channel blockers Ni2+ (100 microM) and omega-conotoxin GVIA (1 microM). Depletion of intracellular Ca2+ stores did not change the afterhyperpolarization. However, we observed outward current pulses that occurred independently from action potentials and were abrogated by apamin. Apart from a faster time course, they shared all properties with spontaneous hyperpolarizations or outward currents that ryanodine receptor-mediated Ca2+ release from intracellular stores induces in juvenile dopamine neurons. Sensitization of ryanodine receptors by caffeine silenced substantia nigra pars reticulata neurons. This effect was abolished by the depletion of intracellular Ca2+ stores. We conclude that SK2 channels in substantia nigra pars reticulata neurons are activated by Ca2+ influx through at least two types of Ca2+ channels in the membrane and by ryanodine receptor-mediated Ca2+ release from intracellular stores. Ryanodine receptors do not amplify small-conductance, Ca2+-dependent K+ channel activation by the Ca2+ influx during a single spike. Yet, ryanodine receptor-mediated Ca2+ release and, thereby, an activation of small-conductance, Ca2+-dependent K+ channels by intracellular Ca2+ are available for excitability modulation in these output neurons of the basal ganglia system.

Retrograde signaling changes the venue of postsynaptic inhibition in rat substantia nigra.

Both endocannabinoids through cannabinoid receptor type I (CB1) receptors and dopamine through dopamine receptor type D1 receptors modulate postsynaptic inhibition in substantia nigra by changing GABA release from striatonigral terminals. By recording from visually identified pars compacta and pars reticulata neurons we searched for a possible co-release and interaction of endocannabinoids and dopamine. Depolarization of a neuron in pars reticulata or in pars compacta transiently suppressed evoked synaptic currents which were blocked by GABA(A) receptor antagonists (inhibitory postsynaptic currents [IPSCs]). This depolarization-induced suppression of inhibition (DSI) was abrogated by the cannabinoid CB1 receptor antagonist AM251 (1 microM). A correlation existed between the degree of DSI and the degree of reduction of evoked IPSCs by the CB1 receptor agonist WIN55,212-2 (1 microM). The cholinergic receptor agonist carbachol (0.5-5 microM) enhanced DSI, but suppression of spontaneous IPSCs was barely detectable pointing to the existence of GABA release sites without CB1 receptors. In dopamine, but not in GABAergic neurons DSI was enhanced by the dopamine D1 receptor antagonist SCH23390 (3-10 microM). Both the antagonist for CB1 receptors and the antagonist for dopamine D1 receptors enhanced or reduced, respectively, the amplitudes of evoked IPSCs. This tonic influence persisted if the receptor for the other ligand was blocked. We conclude that endocannabinoids and dopamine can be co-released. Retrograde signaling through endocannabinoids and dopamine changes inhibition independently from each other. Activation of dopamine D1 receptors emphasizes extrinsic inhibition and activation of CB1 receptors promotes intrinsic inhibition.

Insulin-like growth factor 1 and a cytosolic tyrosine kinase activate chloride outward transport during maturation of hippocampal neurons.

The development of hyperpolarizing inhibition is an important step in the maturation of neuronal networks. Hyperpolarizing inhibition requires Cl(-) outward transport that is accomplished by KCC2, a K(+)/Cl(-) cotransporter. We show that cultured hippocampal neurons initially contain an inactive form of the KCC2 protein, which becomes activated during subsequent maturation of the neurons. We also show that this process is accelerated by transient stimulation of IGF-1 receptors. Because the transporter can be rapidly activated by coapplication of IGF-1 and an Src kinase and can be deactivated by membrane-permeable protein tyrosine kinase inhibitors, we suggest that activation of K(+)/Cl(-) cotransporter function by endogenous protein tyrosine kinases mediates the developmental switch of GABAergic responses to hyperpolarizing inhibition.

Gamma-aminobutyric acid(B) autoreceptors in substantia nigra and neostriatum of the weaver mutant mouse.

The weaver mutation causes cell loss in the center of the substantia nigra, pars compacta. We compared the depression of gamma-aminobutyric acid (GABA)(A) synaptic currents by the GABA(B) agonist R-baclofen in pars compacta neurons of weaver mice which were largely spared from cell degeneration and of wild-type mice. In weaver neurons the suppression of GABA(A) synaptic currents by R-baclofen was reduced compared to wild-type neurons. The EC(50) of R-baclofen was 6.3 times higher in weaver than in wild-type mice. In the neostriatum, which is not a target of the mutation, such a difference did not exist. We conclude that in the pars compacta the weaver mutation leads to a reduced presynaptic autoinhibition through GABA(B) receptors which may promote survival of a subset of weaver neurons in the pars compacta.

Impaired inhibition of epileptiform activity by baclofen, but not by adenosine in the weaver hippocampus.

The weaver defect results in a loss of baclofen- and adenosine-gated K+ conductance in the hippocampus of adult homozygous (wv/wv) mice. In addition, suppression of hippocampal epileptiform activity by baclofen is impaired (Jarolimek, W., Bäurle, J., Misgeld, U., 1998. Pore mutation in a G protein-gated inwardly rectifying K+ channel subunit causes loss of K+ dependent inhibition in weaver hippocampus. Journal of Neuroscience 18, 4001-4007). We used wv/wv and wild-type (+/+) mice to determine whether K+ conductance increases are essential for the suppression of epileptiform activity by R-baclofen and adenosine in disinhibited hippocampal slices. In wv/wv mice R-baclofen was less potent by two orders of magnitude in reducing the frequency of spontaneous synchronous burst discharges than in +/+ mice. Endogenous adenosine and adenosine A1 receptor agonists differed only slightly in their efficacy to inhibit spontaneous synchronous burst discharges in wv/wv and +/+ mice. The findings on adenosine A1 receptors suggest that the varied efficacy of R-baclofen in wv/wv and +/+ mice may not be explained solely on the basis of a loss of ligand-gated K+ conductance. Therefore, we investigated the affinity of GABA(B) receptors for the antagonist CGP55845A in wv/wv and +/+ hippocampi. Schild plot analysis revealed a K(D) for the GABA(B) antagonist CGP55845A 10 fold higher in wv/wv than in +/+ mice. The data suggest that an alteration of GABA(B) receptors could contribute to the reduced efficacy of R-baclofen to suppress hippocampal epileptiform activity in weaver mice, while the suppression by adenosine remains largely unaffected.

A furosemide-sensitive K+-Cl- cotransporter counteracts intracellular Cl- accumulation and depletion in cultured rat midbrain neurons.

Efficacy of postsynaptic inhibition through GABAA receptors in the mammalian brain depends on the maintenance of a Cl- gradient for hyperpolarizing Cl- currents. We have taken advantage of the reduced complexity under which Cl- regulation can be investigated in cultured neurons as opposed to neurons in other in vitro preparations of the mammalian brain. Tightseal whole-cell recording of spontaneous GABAA receptor-mediated postsynaptic currents suggested that an outward Cl- transport reduced dendritic [Cl-]i if the somata of cells were loaded with Cl- via the patch pipette. We determined dendritic and somatic reversal potentials of Cl- currents induced by focally applied GABA to calculate [Cl-]i during variation of [K+]o and [Cl-] in the patch pipette. [Cl-]i and [K+]o were tightly coupled by a furosemide-sensitive K+-Cl- cotransport. Thermodynamic considerations excluded the significant contribution of a Na+-K+-Cl- cotransporter to the net Cl- transport. We conclude that under conditions of normal [K+]o the K+-Cl- cotransporter helps to maintain [Cl-]i at low levels, whereas under pathological conditions, under which [K+]o remains elevated because of neuronal hyperactivity, the cotransporter accumulates Cl- in neurons, thereby further enhancing neuronal excitability.

Pore mutation in a G-protein-gated inwardly rectifying K+ channel subunit causes loss of K+-dependent inhibition in weaver hippocampus.

Weaver (wv) mice carry a point mutation in the pore region of a G-protein-gated inwardly rectifying K+ channel subunit (Kir3.2). wvKir3.2 conducts inward currents that may cause the loss of neurons in the cerebellum and substantia nigra. Although Kir3.2 is widely expressed in the CNS, significant morphological or physiological changes have not been reported for other brain areas. We studied the role of wvKir3.2 in hippocampal slices of young [postnatal day (P) 4-18] and adult wv/wv (>/=P24) mice, because protein levels of Kir 3. 1 and Kir3.2 appear to be normal in the first 3 postnatal weeks and only decrease thereafter. In disinhibited slices, the GABAB receptor agonist R-baclofen reduced burst activity in wv/wv mice but was much more potent in wild-type mice. Mean resting membrane potential, slope input resistance, and membrane time constant of CA3 neurons of adult wv/wv and wild-type mice were indistinguishable. However, R-baclofen or chloroadenosine did not induce K+ currents or any other conductance change in wv/wv mice. Moreover, electrical or chemical stimulation of inhibitory neurons did not evoke slow IPSPs in adult wv/wv mice. Only in a few cells of young wv/wv mice did GABAB receptor activation by R-baclofen or presynaptic stimulation induce small inward currents, which were likely caused by a Na+ ion influx through wvKir3.2 channels. The data show that the pore mutation in wvKir3.2 channels results in a hippocampal phenotype resembling Kir3.2-deficient mutants, although it is not associated with the occurrence of seizures.

Synaptic modulation of oscillatory activity of hypothalamic neuronal networks in vitro.

1. Rhythmic bursts of action potentials in neurosecretory cells are a key factor in hypothalamic neurosecretion. Rhythmicity and synchronization may be accomplished by pacemaker cells synaptically driving follower cells or by a network oscillator. 2. In this review we describe a hypothalamic cell culture which may serve as a model for a hypothalamic network oscillator. An overview is given of neurochemical phenotypes, synaptic mechanisms and their development, properties of receptors for fast synaptic transmission, and membrane properties of cells in dissociated rat embryonic hypothalamic culture. 3. Rhythmic activity spreads in the cultured network through synapses that release glutamate, activating a heteromultimeric AMPA-type receptor containing a GluR2 subunit which is associated with a high-conductance channel for Na+ and K+. Rhythmic activity is controlled by synapses that release GABA to activate GABAA receptors. The presumed function of the two receptor types is facilitated by their respective location, GABAA receptors predominating near the soma and AMPA receptors being abundant in dendrites. 4. Network oscillators may be more reliable for the presumed function than single-cell oscillators. They are controlled through synaptic modulation, which may prove to represent a process important for the release of hormones.

Dopamine D1 receptors facilitate GABAA synaptic currents in the rat substantia nigra pars reticulata.

GABA neurons in the substantia nigra pars reticulata receive input from GABAergic fibers originating in the forebrain. The role of dopaminergic D1 receptors located on these fibers was investigated using tight-seal whole-cell recordings from visually identified pars reticulata neurons of rat substantia nigra slices. Nondopaminergic pars reticulata neurons were characterized by their electrophysiological properties. Postsynaptic currents evoked by minimal stimulation in the presence of ionotropic glutamate receptor antagonists were blocked by bicuculline, indicating that they were GABAA IPSCs. Evoked GABAA IPSCs were potentiated by D1 receptor agonists. After application of D1 receptor agonists, miniature IPSCs [recorded in the presence of tetrodotoxin (TTX) and the Ca2+ channel blocker Cd2+] increased in frequency but not in amplitude. Effects of D1 receptor stimulation were mimicked by forskolin, as expected, if a cAMP-dependent mechanism was involved. The D1 antagonist SCH23390 blocked the effects of the agonists, and perfusion with SCH23390 resulted in a reduction of evoked IPSCs. In TTX and Cd2+, which prevented dopamine release, the D1 antagonist had no effect on miniature IPSCs. Blocking of monoamine uptake by imipramine increased the amplitude of evoked IPSCs. We conclude that dopamine released from dendrites of dopaminergic neurons enhances GABA release in the pars reticulata of the substantia nigra through D1 receptors presumably located on striatonigral afferents. These D1 receptors, thereby, can reinforce D1 receptor-mediated activation of striatal projection neurons that inhibit the inhibitory output neurons of the basal ganglia in substantia nigra.

Enhancement of synaptic excitation by GABAA receptor antagonists in rat embryonic midbrain culture.

Alterations of synaptic excitation induced by exposure to gamma-aminobutyric acid-A (GABAA) receptor antagonists were investigated employing tight-seal whole cell recording from single neurons or pairs of neurons in rat embryonic midbrain culture. Application of GABAA receptor antagonists led to sustained depolarizations followed by synchronous paroxysmal depolarization shifts (PDSs). PDSs induced a transient increase in miniature excitatory postsynaptic currents in the presence as well as in the absence of a N-methyl-aspartate receptor antagonist. The increase in glutamate release supports the excitatory drive required to reinitiate PDSs from the quiescent interburst intervals. After washout of GABAA receptor antagonists, synaptic activity remained grouped, regardless of the presence or absence of PDS blockade by tetrodotoxin (TTX). Impediment of action potential-triggered transmitter release by Cd2+ or TTX also induced grouped activity. We conclude that changes in synaptic excitation are produced by the impaired GABAA inhibition per se and by the initiation of PDSs.

Effects of serotonin through serotonin1A and serotonin4 receptors on inhibition in the guinea-pig dentate gyrus in vitro.

The role of serotonin1A and serotonin4 receptors in the modulation of synaptic inhibition in the dentate gyrus of guinea-pig hippocampal slices was studied. The effects of serotonin (5-hydroxytryptamine) on hilar neurons and on inhibitory postsynaptic potentials in granule cells were compared using intracellular recording in the presence of glutamatergic receptor antagonists. On the basis of electrophysiological properties hilar neurons were classified as type I neurons (presumably inhibitory) and type II neurons (presumably excitatory). Serotonin hyperpolarized a proportion of type I hilar neurons (60%) and decreased their input resistance through activation of a K+-conductance. This effect was mediated by serotonin1A receptors since it was mimicked by the selective serotonin1A receptor agonist (+/-)-8-hydroxy-dipropylaminotetralin hydrobromide and blocked by the selective serotonin1A receptor antagonist (+) WAY 100135. In some type I hilar neurons (40%) neither serotonin nor (+/-)-8-hydroxydipropylaminotetralin hydrobromide induced a membrane hyperpolarization. Instead, serotonin induced an excitatory response, depolarizing the cells and blocking the slow afterhyperpolarization. Similar effects were seen in all hilar neurons after blockade of serotonin1A receptors. They were mimicked by the serotonin4 receptor agonist zacopride. Serotonin induced either decreases or increases in the frequency of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic potentials in granule cells via activation of serotonin1A and of serotonin4 receptors, respectively. 4-aminopyridine-evoked GABA(B) receptor-mediated inhibitory postsynaptic potentials were inhibited by serotonin via activation of serotonin1A receptors. However, after blockade of serotonin1A receptors, serotonin increased the frequency of GABA(B)-inhibitory postsynaptic potentials through the activation of serotonin4 receptors. We conclude that a proportion of inhibitory neurons in the dentate area does not express serotonin1A receptors and is excited by serotonin. Other inhibitory neurons express serotonin1A receptors and are inhibited by serotonin.

GABAB receptor-mediated inhibition of spontaneous inhibitory synaptic currents in rat midbrain culture.

1. Tight-seal, whole-cell recording was used to study GABAB receptor-mediated inhibition of spontaneous inhibitory synaptic currents in cultured rat midbrain neurones. 2. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were recorded in tetrodotoxin (TTX), Cd2+ and Ba2+. (R)-(-)-baclofen reduced the frequency of mIPSCs through a presynaptic mechanism. The EC50 for this effect was 7 microM. It was antagonized by the GABAB receptor antagonist CGP55845A (0.5 microM). 3. In pertussis toxin (PTX)-treated cultures, some GABAB receptor-mediated reduction of the frequency of mIPSCs persisted. In contrast, PTX treatment totally abolished inhibition of miniature excitatory postsynaptic currents (mEPSCs). 4. In PTX-treated cultures, a saturating concentration of (R)-(-)-baclofen inhibited action potential-generated IPSCs but no EPSCs. 5. PTX treatment abolished the (R)-(-)-baclofen-mediated inhibition of high voltage-activated somatic Ca2+ currents and of spontaneous IPSCs depending on presynaptic Ca2+ entry. 6. We conclude that cellular mechanisms underlying GABAB receptor-mediated inhibition of mIPSCs contribute to auto-inhibition of GABA release.

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.

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.

(-)-Baclofen-induced and constitutively active inwardly rectifying potassium conductances in cultured rat midbrain neurons.

Biophysical and pharmacological properties, and development of the (-)-baclofen-induced potassium (KBac) conductance and the constitutively active inwardly rectifying potassium (KIR) conductance were characterised using the patch-clamp technique in cultured embryonic rat midbrain neurons. KBac conductance was induced by (-)-baclofen acting on gamma-aminobutyric acid B (GABAB) receptors, and displayed a high degree of selectivity for potassium ions, an approximate square-root dependence of conductance on extracellular potassium concentration and strongly voltage-dependent activation. Ba2+ blocked the conductance in a voltage-independent manner, whereas Cs+ produced a voltage-dependent block. In the same preparation, the KIR conductance displayed biophysical properties indistinguishable from those of the KBac conductance. Block of KIR currents by Ba2+ was voltage independent (KI, 4 microM), whereas Cs+ produced a voltage-dependent block (KI, 370 microM at -100 mV, equivalent valence, z', 1.67). The KBac and KIR conductances additionally displayed a strikingly similar pattern of development in culture; the specific conductance (nS/pF) of both conductances increased two- to three-fold between the first and second week in vitro, and remained constant thereafter.

Role of chloride-homeostasis in the inhibitory control of neuronal network oscillators.

1. Spontaneous synaptic activity in networks formed by dissociated neurons from embryonic rat midbrain was analyzed in tight seal whole cell recordings. 2. Application of furosemide (0.5 mM) to the cell and its surrounding area increased the frequency of spontaneous synaptic currents. Incubation of the culture with furosemide resulted in "rhythmic" burst activity. 3. Furosemide (0.1-0.5 mM) changed equilibrium potentials of inhibitory postsynaptic currents, gamma-aminobutyric acid-A (GABAA) or glycine receptor-mediated Cl- currents by a blockade of Cl(-)-outward transport. Furosemide did not alter the slope conductance of GABAA receptor-mediated currents. Membrane conductance and cell excitability were also unaffected. 4. We conclude that furosemide locked the activity of the network in "burst activity" mode through impairment of inhibition resulting from the disturbance of Cl- homeostasis.

Suppression by GABAB receptors of 4-aminopyridine-induced hyperactivity in guinea-pig dentate neurons.

Double intracellular recording from granule cells and hilar neurons was performed in hippocampal slices to study the effect of gamma-aminobutyric acid (GABA) receptor antagonists on the activity induced by the convulsant 4-aminopyridine (4-AP) in the dentate gyrus. 4-AP evoked GABA-mediated responses in granule cells and burst discharges in hilar neurons. In the presence of GABAB but not GABAA receptor antagonists, 4-AP evoked discharge activity in dentate granule cells. When both GABAA and GABAB receptors were blocked 4-AP induced synchronous 'paroxysmal depolarizing shift'-like activity in granule cells and hilar neurons. Our data indicate that GABAB receptor-mediated mechanisms protect dentate cells against the convulsant effects of 4-AP.

5,7-Dihydroxytryptamine uptake discriminates living serotonergic cells from dopaminergic cells in rat midbrain culture.

Dissociated cells from embryonic rat midbrain develop in dissociated culture into glutamatergic, GABAergic and aminergic cells. The autofluorescent serotonin analogue, 5,7-dihydroxytryptamine (5,7-DHT), is taken up by a small population of cells that is immunoreactive to 5-hydroxytryptamine. Tyrosine hydroxylase-immunoreactive cells do not accumulate 5,7-DHT. 5,7-DHT uptake, therefore, is well suited for the identification of living serotonergic cells and their discrimination from dopaminergic cells.