The role of chloride transport in postsynaptic inhibition of hippocampal neurons.

Hippocampal inhibitory postsynaptic potentials are depolarizing in granule cells but hyperpolarizing in CA3 neurons because the reversal potentials and membrane potentials of these cells differ. Here the hippocampal slice preparation was used to investigate the role of chloride transport in these inhibitory responses. In both cell types, increasing the intracellular chloride concentration by injection shifted the reversal potential of these responses in a positive direction, and blocking the outward transport of chloride with furosemide slowed their recovery from the injection. In addition, hyperpolarizing and depolarizing inhibitory responses and the hyperpolarizing and depolarizing responses to the inhibitory neurotransmitter gamma-aminobutyric acid decreased in the presence of furosemide. These effects of furosemide suggest that the internal chloride activity of an individual hippocampal neuron is regulated by two transport processes, one that accumulates chloride and one that extrudes chloride.

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.

A physiological role for GABAB receptors and the effects of baclofen in the mammalian central nervous system.

The inhibitory neurotransmitter GABA acts in the mammalian brain through two different receptor classes: GABAA and GABAB receptors. GABAB receptors differ fundamentally from GABAA receptors in that they require a G-protein. GABAB receptors are located pre- and/or post-synaptically, and are coupled to various K+ and Ca2+ channels presumably through both a membrane delimited pathway and a pathway involving second messengers. Baclofen, a selective GABAB receptor agonist, as well as GABA itself have pre- and post-synaptic effects. Pre-synaptic effects comprise the reduction of the release of excitatory and inhibitory transmitters. GABAergic receptors on GABAergic terminals may regulate GABA release, however, in most instances spontaneous inhibitory synaptic activity is not modulated by endogenous GABA. Post-synaptic GABAB receptor-mediated inhibition is likely to occur through a membrane delimited pathway activating K+ channels, while baclofen, in some neurons, may activate K+ channels through a second messenger pathway involving arachidonic acid. Some, but not all GABAB receptor-gated K+ channels have the typical properties of those G-protein-activated K+ channels which are also gated by other endogenous ligands of the brain. New, high affinity GABAB antagonists are now available, and some pharmacological evidence points to a receptor heterogeneity. The pharmacological distinction of receptor subtypes, however, has to await final support from a characterization of the molecular structure. The function importance of post-synaptic GABAB receptors is highlighted by a segregation of GABAA and GABAB synapses in the mammalian brain.

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.

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.

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.

Interaction of Noradrenergic and Cholinergic Agonists with Ligands Increasing K-conductance of Guinea Pig Hippocampal Neurons, in vitro.

Single electrode current clamp and voltage clamp recordings were employed to study the effects of noradrenergic agonists and a cholinergic agonist (carbachol, Cch) on the resting membrane potential of CA3 neurons in guinea pig hippocampal slices. Stimulation of muscarinic and beta-adrenergic receptors depolarized, and stimulation of alpha1-adrenergic receptor hyperpolarized, CA3 neurons but the membrane potential changes were small. Hyperpolarizations or outward currents induced by baclofen, adenosine or serotonin (5-HT) were strongly potentiated by alpha-noradrenergic agonists and suppressed by Cch at concentrations ten times lower than those having any direct effects on membrane potential. Both the enhancement of the baclofen-induced hyperpolarization by phenylephrine and its suppression by Cch were pronounced at low concentrations of baclofen, but diminished at higher concentrations. The modulatory effects persisted after blockade of sodium spikes by tetrodotoxin and after blockade of fast inhibitory and excitatory synaptic transmission by picrotoxin and 6-cyano-7-nitroquinoxaline-2,3-dione. Our data suggest that, through the postsynaptic interaction with ligands activating potassium conductance, noradrenergic and muscarinic receptor stimulation can exert a stronger inhibitory and excitatory effect on CA3 pyramidal neurons at their resting membrane potential than would be expected from the changes in membrane potential induced by these neuromodulators on their own.

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.

Adrenergic modulation of hilar neuron activity and granule cell inhibition in the guinea-pig hippocampal slice.

To study the effects of norepinephrine on synaptic inhibition in the dentate gyrus, intracellular recordings were made from hilar neurons in the guinea-pig hippocampal slice. The effects of norepinephrine on hilar neurons were compared with changes in the frequency of spontaneous inhibitory postsynaptic potentials recorded from granule cells. Hilar neurons comprised two electrophysiologically distinct groups: type I hilar neurons displayed a pronounced single spike afterhyperpolarization and little spike frequency accommodation, type II hilar neurons had small afterhyperpolarizations and pronounced spike frequency accommodation. The majority of recordings were from type I hilar neurons which are presumably inhibitory to granule cells. In most instances, effects of norepinephrine (2-10 microM) on hilar neurons could be mimicked by the beta-adrenergic agonist isoproterenol (0.1-1 microM). Isoproterenol induced a slight depolarization, blocked a slow afterhyperpolarization and, in type II neurons, reduced spike frequency accommodation. These effects were associated with an increase in the spontaneous discharge rate and an enhancement of spontaneous excitatory and inhibitory postsynaptic potentials. In accordance, isoproterenol and norepinephrine increased the frequency of inhibitory postsynaptic potentials in granule cells. In the presence of the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and the N-methyl-D-aspartate receptor antagonist CGP 37849, isoproterenol and norepinephrine also increased the frequency of Cl- -dependent inhibitory postsynaptic potentials in granule cells. Under this experimental condition, however, norepinephrine reduced the discharge rate of type I hilar neurons through an effect on alpha-receptors. In the presence of GABAA receptor blockers, norepinephrine increased the frequency of spontaneously occurring K(+)-dependent inhibitory postsynaptic potentials in granule cells. Accordingly, the frequency of burst discharges in type I hilar neurons was increased. We suggest that the discrepancy in the effect of norepinephrine on the discharge rate of presumed inhibitory hilar neurons and the frequency of Cl- -dependent inhibitory postsynaptic potentials in granule cells results from a direct effect of norepinephrine on GABAergic terminals because norepinephrine also enhanced the frequency of tetrodotoxin-resistant inhibitory postsynaptic potentials in granule cells. Thus, the net effect of synaptically released norepinephrine on synaptic inhibition in the dentate gyrus will be determined by opposing actions of alpha- versus beta-receptor stimulation at the synapse on hilar neurons.

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.

Postsynaptic-GABAergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus.

Intracellular recording and staining was applied to study non-pyramidal neurons in the guinea-pig hippocampus. To avoid accidental impalement of pyramidal or granule cells, two hippocampal regions known to be devoid of pyramidal or granule cells were chosen. In transverse and longitudinal slices, neurons of the deep hilar region (zone 4 of Amaral3), and in transverse slices, neurons of the stratum lacunosum-moleculare (CA3) were impaled. The intracellular staining with Lucifer Yellow revealed that of 20 neurons stained in these zones all were non-pyramidal neurons. Hilar neurons, situated just below the granular layer, differed from granule cells and CA3 neurons with respect to their action potential waveform and their current/voltage relationship. In contrast to granule cells, hilar neurons exhibited spontaneous bursts in the presence of bicuculline (25 microM). In all neurons impaled in the hilar region and the stratum lacunosum-moleculare (n = 42), inhibitory postsynaptic potentials could be elicited. These inhibitory postsynaptic potentials were blocked by bicuculline. In transverse slices, perforant path stimulation elicited inhibition preceding excitation in hilar neurons and excitation preceding inhibition in granule cells. Since non-pyramidal neurons are likely to be inhibitory neurons, our data suggest that GABAergic neurons in the hilus or in the stratum lacunosum-moleculare are controlled by inhibitory GABAergic synapses. This was verified by immunocytochemistry using antibodies against glutamate decarboxylase, the gamma-aminobutyric acid synthetizing enzyme. In both hippocampal regions studied, glutamate decarboxylase-positive synaptic terminals on glutamate decarboxylase-positive cells were observed. It is concluded that disinhibition is an important feature of information processing in the hippocampus, and that disinhibition is mediated by GABAergic synapses on GABAergic neurons.

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.

Intrinsic membrane properties of neostriatal neurons can account for their low level of spontaneous activity.

Electroresponsiveness of neostriatal neurons was studied by intracellular recording in a rat brain slice preparation maintained in standard solution or in solution containing K-channel blockers. In standard solution, the neurons fired repetitively at increasing frequencies with increasing amplitude of direct depolarization. The firing pattern was independent of the membrane potential from which firing was induced. In the presence of tetraethylammonium (20 mM), long-lasting (300-500 ms) plateau potentials could be elicited by the injection of short (5-10 ms) current pulses. Plateau potentials persisted in Na-free solution, in the presence of tetrodotoxin (1-3 microM) and if Ca in the perifusate was replaced by Ba. The plateau was blocked by Cd (500 microM). The plateaux were followed by depolarizing after-potentials. When the plateau potential failed due to fatigue, a small slow depolarization of short duration (10-30 ms) was elicited in Na-free or tetrodotoxin-containing solution, which increased in amplitude with membrane hyperpolarization. This slow depolarization was blocked by Cd, indicating that it was also mediated by Ca. By intrastriatal stimulation in the presence of 4-aminopyridine a long-lasting, voltage-dependent depolarization was triggered from the enhanced postsynaptic potential. In contrast, in the presence of tetraethylammonium, postsynaptic potentials were only slightly increased if they were compared at sizes subthreshold for the plateau potentials. It is concluded that neostriatal neurons, although being characterized as "silent" and "non-bursting", possess slow conductances for inward currents which they share with other mammalian central neurons. However, in contrast, to other central neurons, their Ca-spikes are suppressed by their K-conductances and, in contrast to oscillating neurons, low-threshold Ca-potentials are not prominent.

Effects of antagonists on quisqualate and nicotinic receptor-mediated currents of midbrain neurones in culture.

1. The action of non-N-methyl-D-aspartate (non-NMDA) and nicotinic antagonists on excitatory postsynaptic currents (e.p.s.cs) and on quisqualate (Quis)- and nicotine-gated currents was studied by use of whole-cell recording in dissociated culture of the rat midbrain. 2. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 0.1 microM) and kynurenic acid (0.1 mM) attenuated network-generated and miniature e.p.s.cs while mecamylamine (100 microM) and hexamethonium (400 microM) had no effect. Acetylcholine (ACh) enhanced or suppressed e.p.s.cs. The suppressing effect of ACh was blocked by atropine (0.1-10 microM). 3. ACh (50-1000 microM) and quisqualate (Quis, 0.1-20 microM) induced inward currents with the same reversal potential as e.p.s.cs. 4. Application of Quis and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) in a low concentration (0.5 and 5 microM, respectively) evoked a maintained current which was attenuated by CNQX (1 microM) and kynurenic acid (0.5 mM) but not by mecamylamine (100 microM). 5. Higher concentrations of Quis (5-20 microM) and AMPA (50-100 microM) evoked a transient and a maintained current component. Kynurenic acid (1 mM) reduced the transient but not the maintained component. CNQX (5-10 microM) increased the maintained component without reducing the transient one; 20 microM CNQX reduced both components. 6. ACh-induced transient current was mimicked by nicotine and reversibly and dose-dependently blocked by mecamylamine. Atropine (10 microM), hexamethonium (400 microM) as well as CNQX (100 microM) and kynurenic acid (1 mM) did not affect the current. 7. Hexamethonium (50-400 microM) voltage-dependently depressed the maintained current elicited by both Quis and ACh. 8. In conclusion, although the antagonists examined here seem to discriminate between non-NMDA and nicotinic receptor-mediated e.p.s.cs, they vary considerably in respect of their mode of action when tested on Quis, AMPA and ACh-induced currents.

Muscarinic slow EPSPs in neostriatal and hippocampal neurons in vitro.

Cholinergic slow excitatory postsynaptic potentials (slow EPSPs) can be elicited by presynaptic tetanic stimulation in brain slices obtained from rat neostriatum or guinea pig hippocampus. Slow EPSPs are generated by a reduction of a K-leak-conductance. In hippocampal neurons slow EPSPs are amplified by the reduction of an outward current termed IAHP through the activation of a second muscarinic receptor subtype. While hippocampal slow EPSPs might be involved in information processing across hippocampal pathways, muscarinic modulation in the neostriatum consists of a presynaptic tuning of nicotinic fast synaptic transmission.

Carbachol and pirenzepine discriminate effects mediated by two muscarinic receptor subtypes on hippocampal neurons in vitro.

Measurement of [Cch] in the bath and in slices demonstrated a considerable concentration discrepancy between the bath and the extracellular space. With fast (bolus) application of Cch this discrepancy is due to the speed of diffusion, while equilibration with continuous application is considerably impaired by cellular uptake of Cch (Creese and Taylor, 1967). Low concentrations (less than or equal to 1 microM) of Cch reduce the afterhyperpolarization following a train of action potentials and depolarize the membrane. Analysis of [Cch]0 (t) and the effects of pirenzepine allowed these effects to be assigned to two different muscarinic receptor subtypes.

Synaptic potentials in the rat neostriatum in dissociated embryonic cell culture.

Neostriatal cells of embryonic days 19-21 were grown in dissociated cell culture. To test whether the cultures contained predominantly neostriatal cells, a glyoxylic acid staining procedure was used which, after dopamine loading, stained neostriatal cells but not neurons of embryonic neocortical tissue. Whole cell current clamp recording was performed in the neurons after 1-2 weeks in cell culture. Although cells could be driven to discharge by direct depolarization, spontaneous activity was low. All cells responded to gamma-aminobutyric acid (GABA) (0.1-0.5 mM), and the majority of them responded to glutamate (Glu) (0.1 mM). Only about 50% were depolarized by acetylcholine (ACh) (0.1-0.5 mM). Atropine (1-10 microM) did not block this depolarization. Barrages of postsynaptic potentials (PSPs) were induced by applications of Glu or ACh, even if the neuron under observation was not depolarized. All PSPs were depressed by bicuculline (50 microM), indicating their mediation by GABAergic receptors. Exclusively GABAergic PSPs were also observed in cultures raised in the presence of nerve growth factor. The study indicates that neostriatal cells form GABAergic, but not excitatory cholinergic synapses when cultured at this embryonic age under our conditions, resembling the pattern of development observed in slices obtained from neonatal rats.

Identification of projecting neurons in rat neostriatal slices.

Spiny neurons projecting to substantia nigra (SN) were identified by combining retrograde transport of tracers in vivo with intracellular staining in vitro. First, neostriatal projection neurons were retrogradely labeled by the injection of fluorescent tracers into the ipsilateral SN. Then, slices were taken from the neostriata containing the retrogradely labeled cells. Cells were impaled for intracellular recording and stained by intracellular dye injection. All cells were of the spiny type and 75% contained both dyes, the intracellularly injected dye as well as the retrogradely transported tracer. Thus, most intracellular recordings in neostriatal slices are obtained from spiny neurons projecting to SN.

Effects of serotonin on hilar neurons and granule cell inhibition in the guinea pig hippocampal slice.

Intracellular recordings in guinea pig hippocampal slices were used to study the effects of serotonin (5-HT) on presumed inhibitory hilar neurons and on postsynaptic inhibition of granule cells. 5-HT applied by the bath hyperpolarized only 50% of the hilar neurons tested but all CA3 neurons and granule cells, presumably by activating a K-conductance. The bath application of 4-aminopyridine (4-AP, 50 microM) induced burst discharge activity in hilar neurons and giant inhibitory postsynaptic potentials (IPSPs) in granule cells consisting of a Cl- and K-component. 5-HT (5-10 microM) reversibly blocked the K-component of giant IPSPs in granule cells, but not their Cl-component. In the majority of hilar neurons 5-HT increased the frequency of 4-AP induced burst discharges even when hilar neurons were hyperpolarized. Only in a few hilar neurons 5-HT blocked 4-AP induced burst discharges. We conclude that the changes in burst discharge pattern of hilar neurons correspond with the differential effect of 5-HT on Cl- and K-mediated inhibition of granule cells.

Late development of intrinsic excitation in the rat neostriatum: an in vitro study.

Functional maturation of intrinsic circuitry in the neostriatum was studied by intracellular recording and intracellular staining with Lucifer yellow in slices obtained from rat pups at postnatal days (P)1-20 and from adult rats. The most striking observation was that intrastriatal stimulation elicited predominantly inhibitory responses in slices obtained from animals of P1-6. In contrast, intrastriatally evoked responses in slices obtained after P10 were predominantly excitatory. The inhibitory postsynaptic potentials (IPSPs) recorded in slices obtained from pups were blocked by bicuculline (50 microM) and exhibited a reversal potential at about -60 mV which shifted in depolarizing direction when intracellular Cl- activity increased. Thus, these IPSPs correspond to IPSPs observed in adult animals. It is concluded that maturation of excitatory synapses is the main change during postnatal development. The changes of postsynaptic potentials were paralleled by the appearance of spines on dendrites around P7 as revealed by intracellular staining. The apparent input resistances and time constants of young neurons were very high and responses to large current injections were often distorted by humps which could not be observed in adult neurons. Only young neurons responded with bursts to synaptic activation in the presence of bicuculline (50 microM). It appears that dendritic conductances have a stronger influence on somatic discharge in the electrotonically compact young neurons than in adult neurons.