Rat Striatal Neurons Research Articles

Modulation by bicuculline and penicillin of the block by t-butyl-bicyclo-phosphorothionate (TBPS) of GABA(A)-receptor mediated Cl(-)-current responses in rat striatal neurones.

1. T-butyl-bicyclo-phosphorothionate (TBPS) is a prototypical representative of the cage-convulsants which act through a use-dependent block of the GABA(A)-receptor-ionophore complex. Using current recordings from cultured neurones of rat striatum the manner was investigated in which two antagonists, bicuculline and penicillin, presumably acting at the agonist binding site and in the ionic channel, respectively, modify the rate of block by TBPS. 2. Penicillin (5 or 10 mM) did not slow the rate of block by TBPS, but produced a significant enhancement of block rate, which, however, was inversely related to the degree of antagonism by penicillin of the GABA-induced current. 3. Bicuculline (10 microM) reduced the rate of block by TBPS. However, this effect was 3 fold weaker than its GABA-antagonistic action. The slowing of block rate and the current antagonism exhibited a biphasic, positive-negative relationship. Co-application of bicuculline (100 microM) in a concentration that produced nearly complete antagonism and TBPS (10 microM) resulted in a marked ( approximately 40%) reduction of subsequent GABA response amplitudes compatible with a direct, bicuculline-induced conformational change in the receptor required for the binding of and block by TBPS. 4. The lack of protection afforded by the channel blocker penicillin as well as the lack of correlation between bicuculline antagonism of the Cl(-)-current and its efficiency in protecting against TBPS block is evidence against an open channel blocking mechanism for TBPS. TBPS does, therefore, not appear to gain access to its binding site via the open pore but through alternative routes regulated from the agonist binding site.

ATP-sensitive potassium channels in freshly dissociated adult rat striatal neurons: activation by metabolic inhibitors and the dopaminergic receptor agonist quinpirole

ATP-sensitive potassium channels in freshly dissociated adult rat striatal neurons: activation by metabolic inhibitors and the dopaminergic receptor agonist quinpirole

Localization of alternatively spliced NMDAR1 glutamate receptor isoforms in rat striatal neurons

Alternative splicing of the mRNA encoding the N-methyl-D-aspartate (NMDA) receptor subunit NR1 changes the structural, physiologic, and pharmacologic properties of the resultant NMDA receptors. We used dual label immunocytochemistry and confocal microscopy to localize the four alternatively spliced segments of the NR1 subunit (N1, C1, C2, and C2') in rat striatal neurons. Striatofugal projection neurons and four populations of interneurons were studied. Projection neurons, which were identified by immunolabeling for calbindin and by retrograde tracing from the globus pallidus and the substantia nigra, were the only striatal neurons containing C1 segment immunoreactivity. Projection neurons were also C2 segment immunopositive, as were all other neuronal populations studied. Projection neurons were C2' segment immunonegative. In contrast, each of the interneuron types were labeled by the antibody to the C2' segment: nitric oxide synthase interneurons were labeled intensely, calretinin and parvalbumin neurons were labeled moderately strongly, and cholinergic neurons were also labeled but less strongly than the other types of interneurons. Parvalbumin interneurons showed distinct N1 segment immunolabeling, which was not found in other types of striatal neurons. Our results suggest that all striatal neurons studied synthesize NR1 subunit proteins, but the isoforms of the protein present in projection neurons and the several types of interneurons are distinct. This differential expression of NR1 isoforms may affect both neuronal function and selective vulnerability of neurons to injury.

Localization of alternatively spliced NMDAR1 glutamate receptor isoforms in rat striatal neurons

Alternative splicing of the mRNA encoding the N-methyl-D-aspartate (NMDA) receptor subunit NR1 changes the structural, physiologic, and pharmacologic properties of the resultant NMDA receptors. We used dual label immunocytochemistry and confocal microscopy to localize the four alternatively spliced segments of the NR1 subunit (N1, C1, C2, and C2′) in rat striatal neurons. Striatofugal projection neurons and four populations of interneurons were studied. Projection neurons, which were identified by immunolabeling for calbindin and by retrograde tracing from the globus pallidus and the substantia nigra, were the only striatal neurons containing C1 segment immunoreactivity. Projection neurons were also C2 segment immunopositive, as were all other neuronal populations studied. Projection neurons were C2′ segment immunonegative. In contrast, each of the interneuron types were labeled by the antibody to the C2′ segment: nitric oxide synthase interneurons were labeled intensely, calretinin and parvalbumin neurons were labeled moderately strongly, and cholinergic neurons were also labeled but less strongly than the other types of interneurons. Parvalbumin interneurons showed distinct N1 segment immunolabeling, which was not found in other types of striatal neurons. Our results suggest that all striatal neurons studied synthesize NR1 subunit proteins, but the isoforms of the protein present in projection neurons and the several types of interneurons are distinct. This differential expression of NR1 isoforms may affect both neuronal function and selective vulnerability of neurons to injury. J. Comp. Neurol. 415:204–217, 1999. © 1999 Wiley-Liss, Inc.

Neurochemical determination of the location of NMDA and GABA receptors on rat striatal cholinergic neurons

This paper reports on the protocol for neurochemical determination of the location of various receptors on cholinergic neurons in various brain regions. We applied this protocol to investigate whether NMDA and GABA receptors are located on rat striatal cholinergic neurons. When striatal cholinergic neurons were selectively destroyed by intrastriatal injection of cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A), the number of NMDA and GABA A receptors decreased. However, no significant changes were observed on the number of GABA B receptors. These results suggest that NMDA and GABA A, but not GABA B receptors are located on cholinergic neurons in the striatum. These results also indicate the usefulness and scientific applicability of the present protocol. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Regional localization of receptors and transmitters

Adrenergic α 2C-receptors reside in rat striatal GABAergic projection neurons: comparison of radioligand binding and immunohistochemistry

We have investigated the distribution of α 2C-adrenergic receptors in the rat striatum and characterized the striatal neuron types expressing these receptors. Sequential double-labelled immunocytochemistry was performed with a polyclonal antibody against rat α 2C-adrenoceptors and antibodies against GABA, Calbindin-D 28k, parvalbumin and calretinin. The subregional distribution of α 2C-adrenoceptor binding sites in the striatum was also quantitatively investigated using selective radioligands. Almost all lightly stained striatal GABAergic neurons, with the morphological characteristics of medium-sized spiny projection neurons (94% of GABAergic cells counted), contained α 2C-adrenoceptor-immunoreactive structures. Intensely labelled GABAergic interneurons (6%) were devoid of α 2C-adrenoceptor immunoreactivity. The co-localization of calbindin- and α 2C-adrenoceptor immunoreactivity in the majority of the cells confirmed the presence of α 2C-adrenoceptors in the population of medium-sized spiny neurons. Furthermore, the α 2C-adrenoceptor/calbindin double-labelling disclosed the existence of three neuronal subsets in the matrix compartment of the striatum: a large proportion (83%) of double-labelled neurons, a population of neurons (8%) that exhibited only α 2C-adrenoceptor immunoreactivity without calbindin immunoreactivity, and a population of neurons (9%) immunoreactive for calbindin, but lacking α 2C-adrenoceptors. In addition, α 2C-adrenoceptor immunolabelled neurons were observed in calbindin-free striatal patches. Parvalbumin- and calretinin-positive neurons never displayed α 2C-adrenoceptor immunoreactivity, confirming that striatal GABAergic interneurons are devoid of α 2C-adrenoceptors. The present findings indicate that α 2C-adrenoceptors are localized in GABAergic medium-sized spiny projection neurons but not in interneurons of the rat striatum, and that they may modulate both the direct and indirect pathways of the basal ganglia, as well as participate in the regulation of mesencephalic dopaminergic neurons.

Activation of nuclear factor kappaB by nitric oxide in rat striatal neurones: differential inhibition of the p50 and p65 subunits by dexamethasone.

Nitric oxide (NO), an intercellular messenger in the brain, has been implicated in both neuronal plasticity and neurotoxicity. It has been suggested that NO can activate the DNA binding activity of nuclear factor kappaB (NF-kappaB) family proteins in some cell types while having an inhibitory effect in others. In this study we have investigated the effect of acute NO in primary neuronal cultures of rat striatum using immunohistochemistry. Exposure of neurones to the NO-mimetic S-nitroso-n-acetylpenicillamine (SNAP; 200 microM) and to bacterial lipopolysaccharide (LPS; 10 microg/ml) for 30 min increased nuclear protein expression of the p50 subunit of NF-kappaB. SNAP also enhanced nuclear protein expression of the p65 subunit of NF-kappaB. Simultaneously, the cytoplasmic expression of phosphorylated inhibitory protein IkappaB alpha was dramatically increased by SNAP (200 microM), LPS (10 microg/ml), and kainate (50 microM) treatment. In the adult rat, stimulation with NOR-3 (2 mg/kg), a NO donor, increased NF-kappaB DNA binding activity in the striatum after 45 min. Because glucocorticoids inhibit NF-kappaB activity, primary cultures were pretreated with dexamethasone (50 microM) before SNAP, LPS, and kainate treatment, and the effect on the protein expression level of the individual subunits p50 and p65 present in the classical form of the transcription factor NF-kappaB was assessed. Dexamethasone pretreatment resulted in a marked reduction of p65 protein in striatal neurones after SNAP, LPS, and kainate, whereas p50 expression was reduced by dexamethasone pretreatment only after an LPS stimulus. This study indicates that NO-releasing compounds can directly induce nuclear NF-kappaB subunit expression in rat striatum and that glucocorticoids selectively inhibit p65 subunit expression following exposure to NO.

Native human neocortex release-regulating dopamine D2 type autoreceptors are dopamine D2 subtype.

Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1 nM-10 microM) inhibited tritium release with a calculated EC50 of 17 nM and a maximal inhibition of approximately 75% reached at 1 microM. In the presence of the D2/D3 receptor antagonist (-)-sulpiride (0.1 and 1 microM), the concentration-response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14-8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor-selective antagonist [7-N,N-dipropylamino-5,6,7, 8-tetrahydro-naphtho(2,3b) dihydro,2,3-furane] (S 14297) and the D4 receptor-selective antagonist 3-(4-[4-chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine (L-745,870) (0.01-1 microM in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype.

Sodium influx plays a major role in the membrane depolarization induced by oxygen and glucose deprivation in rat striatal spiny neurons.

Striatal spiny neurons are selectively vulnerable to ischemia, but the ionic mechanisms underlying this selective vulnerability are unclear. Although a possible involvement of sodium and calcium ions has been postulated in the ischemia-induced damage of rat striatal neurons, the ischemia-induced ionic changes have never been analyzed in this neuronal subtype. We studied the effects of in vitro ischemia (oxygen and glucose deprivation) at the cellular level using intracellular recordings and microfluorometric measurements in a slice preparation. We also used various channel blockers and pharmacological compounds to characterize the ischemia-induced ionic conductances. Spiny neurons responded to ischemia with a membrane depolarization/inward current that reversed at approximately -40 mV. This event was coupled with an increased membrane conductance. The simultaneous analysis of membrane potential changes and of variations in [Na+]i and [Ca2+]i levels showed that the ischemia-induced membrane depolarization was associated with an increase of [Na+]i and [Ca2+]i. The ischemia-induced membrane depolarization was not affected by tetrodotoxin or by glutamate receptor antagonists. Neither intracellular BAPTA, a Ca2+ chelator, nor incubation of the slices in low-Ca2+-containing solutions affected the ischemia-induced depolarization, whereas it was reduced by lowering the external Na+ concentration. High doses of blockers of ATP-dependent K+ channels increased the membrane depolarization observed in spiny neurons during ischemia. Our findings show that, although the ischemia-induced membrane depolarization is coupled with a rise of [Na+]i and [Ca2+]i, only the Na+ influx plays a prominent role in this early electrophysiological event, whereas the increase of [Ca2+]i might be relevant for the delayed neuronal death. We also suggest that the activation of ATP-dependent K+ channels might counteract the ischemia-induced membrane depolarization.

Free radical scavenger OPC-14117 attenuates quinolinic acid-induced NF-κB activation and apoptosis in rat striatum

Oxidative stress has long been implicated in the pathogenesis of both the acute and chronic neurotoxic effects of glutamate acting through ionotrophic receptors of the N-methyl- d-aspartate (NMDA) subtype. To evaluate the contribution of oxidative stress to the NMDA receptor-mediated apoptotic death of rat striatal neurons in vivo, the effects of a novel, orally administered free radical scavenger, OPC-14117, was studied following intrastriatal infusion of the NMDA receptor agonist quinolinic acid (QA). Receptor autoradiography and in situ hybridization histochemistry showed that pretreatment with OPC-14117 (600 mg/kg) reduced the QA (120 nmol)-induced loss of striatal D 1 dopamine receptors by about 20% ( p<0.01) and NMDA receptors by 15% ( p<0.01) as well as 67 kDa glutamic acid decarboxylase mRNA (34%; p<0.01) and proenkephalin mRNA (36%; p<0.01). OPC-14117 also decreased the apomorphine-induced ipsilateral rotational response in unilaterally QA-lesioned animals by about 70% ( p<0.05). In addition, OPC-14117 pretreatment inhibited QA-induced internucleosomal DNA fragmentation. Western blot analysis and electrophoresis mobility shift assay further revealed that the free radical scavenger (300 and 600 mg/kg) blunted the QA-induced degradation of IκBα (increased IκBα levels from about 15% to 33 and 62% of control, respectively; p<0.01) as well as the ensuing activation of NF-κB by 25 to 34%, respectively ( p<0.01) and the augmentation in c-Myc (35 to 70%, respectively) and p53 expression by 50–80%, respectively (both p<0.01). In contrast, OPC-14117 had no significant effect on the QA-induced increase in AP-1 binding activity. These results suggest that the NMDA receptor-mediated generation of reactive oxygen species contributes to the QA-induced activation of NF-κB and further that orally administered OPC-14117 partially protects against excitotoxin-induced apoptosis of striatal neurons through inhibition of the NF-κB apoptotic cascade.

U-37883A potently inhibits dopamine-modulated K + channels on rat striatal neurons

An 85 pS K + channel of rat caudate–putamen neurons, which is activated by dopamine D 2 receptors and inhibited by sulfonylurea drugs, was studied using cell-attached patch-clamp electrophysiology. This channel was inhibited by externally-applied U-37883A (4-morpholinecarboximidine- N-1-adamantyl- N′-cyclohexyl hydrochloride), a blocker of vascular ATP-sensitive K + channels, with a half-maximal effect at a concentration of approximately 0.1 μM. Channel inhibition occurred in a time-dependent fashion when U-37883A was applied to the membrane from a back-filled patch pipette. Inhibition was associated with a decrease in fractional open time, but was voltage-insensitive and did not alter channel conductance, suggesting an effect on channel gating at a site largely insensitive to the electrical field of the channel. U-37883A was about 50 times more potent at inhibiting this channel than was the sulfonylurea drug glibenclamide. This relative potency, opposite to that found in pancreatic tissue, indicates that U-37883A is a useful tool to distinguish amongst different subtypes of sulfonylurea-sensitive K + channels.

Selective blockade of a slowly inactivating potassium current in striatal neurons by (±) 6‐chloro‐APB hydrobromide (SKF82958)

The ion channels of rat striatal neurons are known to be modulated by stimulation of D1 dopamine receptors. The susceptibility of depolarization-activated K+ currents to be modulated by the D1 agonist, 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzazepine (APB) was investigated using whole-cell voltage-clamp recording techniques from acutely isolated neurons. APB (0.01–100 μM) produced a concentration-dependent reduction in the total K+ current. At intermediate concentrations (ca. 10 μM), APB selectively depressed the slowly inactivating A-current (IAs). A similar effect was produced by application of the D1 agonist, 7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1-H-2-benzazepine (SKF38393, 10 μM). APB reduced IAs rapidly, having onset and recovery time constants of 1.2 sec and 1.6 sec, respectively. Unexpectedly, the effect of APB could not be mimicked by application of Sp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (Sp-cAMPS, 100–200 μM), a membrane-permeable analog of cyclic AMP (cAMP), or by pretreatment with forskolin (25 μM), an activator of adenylyl cyclase. The reduction in IAs also was not blocked by pretreatment with the D1 receptor antagonist, R(+)-SCH23390 hydrochloride (SCH23390, 10–20 μM). In addition, intracellular dialysis with guanosine-5′-O-(2-thiodiphosphate (GDP-β-S, 200 μM) did not preclude the APB-induced inhibition of IAs, nor did dialysis with guanosine-5′-O-(3-thiotriphosphate (GTP-γ-S, 400 μM) prevent reversal of the effect. The effect of APB was produced by a reduction in the maximal conductance of IAs without changing the voltage-dependence of the current. Collectively, these results argue that APB does not inhibit IAs through D1 receptors coupled to stimulation of adenylyl cyclase, but rather by allosterically regulating or blocking the channels giving rise to this current. Synapse 29:213–224, 1998. © 1998 Wiley-Liss, Inc.

Selective blockade of a slowly inactivating potassium current in striatal neurons by (+/-) 6-chloro-APB hydrobromide (SKF82958).

The ion channels of rat striatal neurons are known to be modulated by stimulation of D1 dopamine receptors. The susceptibility of depolarization-activated K+ currents to be modulated by the D1 agonist, 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzaze pine (APB) was investigated using whole-cell voltage-clamp recording techniques from acutely isolated neurons. APB (0.01-100 microM) produced a concentration-dependent reduction in the total K+ current. At intermediate concentrations (ca. 10 microM), APB selectively depressed the slowly inactivating A-current (I(As)). A similar effect was produced by application of the D1 agonist, 7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1-H-2-benzazepine (SKF38393, 10 microM). APB reduced I(As) rapidly, having onset and recovery time constants of 1.2 sec and 1.6 sec, respectively. Unexpectedly, the effect of APB could not be mimicked by application of Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Sp-cAMPS, 100-200 microM), a membrane-permeable analog of cyclic AMP (cAMP), or by pretreatment with forskolin (25 microM), an activator of adenylyl cyclase. The reduction in I(As) also was not blocked by pretreatment with the D1 receptor antagonist, R(+)-SCH23390 hydrochloride (SCH23390, 10-20 microM). In addition, intracellular dialysis with guanosine-5'-O-(2-thiodiphosphate (GDP-beta-S, 200 microM) did not preclude the APB-induced inhibition of I(As), nor did dialysis with guanosine-5'-O-(3-thiotriphosphate (GTP-gamma-S, 400 microM) prevent reversal of the effect. The effect of APB was produced by a reduction in the maximal conductance of I(As) without changing the voltage-dependence of the current. Collectively, these results argue that APB does not inhibit I(As) through D1 receptors coupled to stimulation of adenylyl cyclase, but rather by allosterically regulating or blocking the channels giving rise to this current.

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. l-glutamate and the selective group I agonist, ( S)-dihydroxyphenylglycine ( S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5′- N-ethylcarboxamidoadenosine (NECA), with EC 50 values of 3.41±0.39 and 5.69±1.64 μM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 μM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, l-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC 50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19±1.18 and 3.78±1.42 μM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu 5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2 S,2′ R,3′ R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.

Modification of nitric oxide synthase activity and neuronal response in rat striatum by melatonin and kynurenine derivatives.

Tryptophan is mainly metabolized in the brain through methoxyindole and kynurenine pathways. The methoxyindole pathway produces (among other compounds) melatonin, which displays inhibitory effects on human and animal central nervous systems, including a significant attenuation of excitatory, glutamate-mediated responses. The kynurenine pathway produces kynurenines that interact with brain glutamate-mediated responses. Nitric oxide (NO) increases glutamate release, and melatonin and kynurenines may act via modification of NO synthesis. In the present study, the effects of melatonin and four synthetic kynurenines were studied on the activity of rat striatal nitric oxide synthase (NOS) and on the response of rat striatal neurons to sensorimotor cortex (SMCx) stimulation, a glutamate-mediated response. Melatonin inhibited both NOS activity and the striatal glutamate response, and these effects were dose-related. Compound A (2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric acid) did not inhibit NOS activity but inhibited the striatal response similarly to melatonin. Compound B (2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid) was more potent than melatonin in inhibiting both NOS activity and the striatal response. Compound C (2-butyramide-4-(3-methoxyphenyl)-4-oxobutyric acid) did not change NOS activity, but increased the striatal response. Compound D (2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid) showed potent inhibitory effects on both NOS activity and striatal glutamate-mediated response. A structure-related effect of the kynurenine derivatives was observed, and those with an amino group in position 2 of the benzenic ring had more potent effects than melatonin itself in inhibiting striatal NOS activity and the response of striatal neurons to SMCx.

3-Nitropropionic acid exacerbates N-methyl- d-aspartate toxicity in striatal culture by multiple mechanisms

We examined the effects of 3-nitropropionic acid-induced succinate dehydrogenase inhibition on neuronal ATP content, N-methyl- d-aspartate-induced neuronal death, resting membrane potential, and N-methyl- d-aspartate-induced changes in cytosolic calcium concentration ([Ca 2+] c) in cultured rat striatal neurons. Exposure of cultures to 3 mM 3-nitropropionic acid for 3 h did not cause overt toxicity, but reduced ATP content by 35%. Treatment with 3-nitropropionic, or removal of Mg 2+ from the medium, enhanced subsequent N-methyl- d-aspartate toxicity, reducing the LC 50 from 250 μM to 12 μM or 30 μM, respectively. Even after Mg 2+ removal, enhancement of N-methyl- d-aspartate toxicity by 3-nitropropionic acid remained pronounced, with the LC 50 further decreasing to 3 μM. The mean resting membrane potential of neurons treated with 3-nitropropionic acid was −37 mV, while that in control neurons was −61 mV. Treatment with 3-nitropropionic did not affect baseline [Ca 2+] c as determined by fura-2 microfluorimetry. N-methyl- d-aspartate (30 μM) caused a rapid rise in [Ca 2+] c, the initial magnitude of which was not affected by 3-nitropropionic acid. However, after a 1-h treatment, [Ca 2+] c was dramatically higher in 3-nitropropionic acid-treated neurons. This increased calcium load was washed out slowly and only partially, although calcium in control neurons washed out rapidly and almost completely. These results suggest that in striatal neurons, the enhancement of N-methyl- d-aspartate toxicity caused by succinate dehydrogenase inhibition may be due to synergism between partial relief of the Mg 2+ blockade of the N-methyl- d-aspartate receptor and other mechanisms, including disruption of neuronal calcium regulation. This synergism may be relevant to the neuronal death observed in neurodegenerative disorders.

Evidence for the preferential localization of Glutamate Receptor-1 subunits of AMPA receptors to the dendritic spines of medium spiny neurons in rat striatum

Although immunohistochemical studies have typically found the perikarya of striatal projection neurons to be devoid of immunohistochemical labelling for the GluR1 AMPA type glutamate receptor subunit, the striatal neuropil is rich in GluR1 immunolabelling and in situ hybridization histochemistry has indicated the presence of GluR1 message in many striatal neurons. To explore the possibility that GluR1 subunits may be synthesized by many striatal projection neurons, but selectively localized to their dendrites, we have used light-microscopic and electron-microscopic immunohistochemistry in combination with single-cell reverse transcription–polymerase chain reaction. Light-microscopic immunohistochemical studies confirmed the presence of abundant GluR1 immunoreactivity in the striatal neuropil in rats. Perikaryal labelling was restricted to neurons previously identified as parvalbuminergic neurons. Single-cell reverse transcription–polymerase chain reaction for individual striatal neurons in rats confirmed that most striatal projection neurons (i.e. containing either or both substance P message or enkephalin message) make GluR1 message. For example, 94% of enkephalin-containing neurons, 75% of substance P-containing neurons, and 87% of enkephalin and substance P co-containing neurons expressed GluR1 messenger RNA. Electron-microscopic immunohistochemistry revealed that GluR1 immunolabelling was prominent in 61% of dendritic spines and 53% of dendritic shafts. While prominent perikaryal GluR1 immunolabelling was observed only in a small population of interneurons, sparse perikaryal GluR1 immunolabelling was found associated with the rough endoplasmic reticulum, the Golgi apparatus, the outer membranes of the mitochondria, and the outer envelope of the nucleus of about 30% of striatal projection neurons (identified by their non-indented nuclei). These results indicate that striatal projection neurons selectively target GluR1 subunits to their spines and dendritic shafts. Our finding has implications for the functioning of striatal projection neurons and for the general issue of whether neurons can control the subcellular localization of glutamate receptors.

Immediate and long-term effects of 5,7-dihydroxytryptamine on rat striatal serotonergic neurons measured using in vivo voltammetry.

The immediate and long-term effects of the selective serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) on rat striatal serotonergic neurons were examined after its intracerebroventricular administration using in vivo voltammetry. Extracellular concentration of 5-hydroxyindoles increased immediately following intracerebroventricular 5,7-DHT injection (200 microg in 24 microl, 18 min), peaked at 1.5-2 h, and returned to normal by 4 h. 5,7-DHT diffused to the contralateral striatum in detectable amounts 9 to 12 min after the start of injection and returned to basal levels by 1.5 h. Three to 6 days after 5,7-DHT lesions, 5-hydroxytryptophan administration produced an increase in striatal 5-hydroxyindoles that was greater than that produced in pre-lesioned rats. This effect was maximal at 14 to 17 days post-lesion, and remained even after 50 days. The short-term effect of 5,7-DHT may be attributable to increased serotonin release, inhibition of uptake, or monoamine oxidase inhibition. The long-term effect of 5,7-DHT lesions may attributable to increased synthesis of serotonin or decreased reuptake in remaining serotonergic neurons.

Immunohistochemical localization of GABA bR1 in the rat CNS

Intracellular recordings were obtained from rat neostriatal slices. Bath-applied dopamine (1–10 μM) produced a reversible inhibition of the action potentials evoked by direct stimulation and a decrease in the amplitude of the intrastriatally evoked depolarizing postsynaptic potentials. No change in membrane potential was detected during the application of 1–10 μM dopamine. Dopamine application also produced a decrease in anomalous rectification in the depolarizing direction. This subthreshold inward rectification was abolished by tetrodotoxin, but not by calcium-free and cadmium (0.1–1 mM)-containing solutions. The dopamine-induced decrease in excitatory postsynaptic potential amplitude was evident at resting membrane potential or at more positive levels, but was absent at hyperpolarized values of the membrane potential. Addition of bicuculline (50–500 μM) to the medium did not affect the inhibitory action of dopamine. The inhibitory action of dopamina also persisted in calcium-free and cadmium-containing solutions. The adenosine 3′,5′-cyclic monophosphate analogue, 8-bromo-adenosine 3′,5′-cyclic monophosphate (0.1–1 mM), mimicked the effects produced by D1 receptor activation. Bath application of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) (1–10 μM), a selective D1 dopaminergic agonist, mimicked the effects of micromolar concentrations of dopamine. The D2 dopaminergic agonists,4,4a,5,6,7,8,8a,9-octahydro-5-n-propyl-2H-pyrazolo-3,4-g-quinoline (LY 171555) and bromocriptine (both at 10 nM-10 μM), had no effects on neostriatal cells. The inhibition induced by micromolar doses of dopamine or SKF 38393 was antagonized by bath applications of R-( + )-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (SCH 23390; 0.1–10 μM), a D1-selective antagonist, but not by sulpiride (10nM–10μM), a D2 antagonist.We conclude that the inhibitory effect of dopamine on rat striatal neurons is postsynaptically mediated by the activation of D1 dopaminergic receptors via the reduction of a voltage-dependent tetrodotoxin sensitive inward conductance.

O-191 - Exicitatory effects of nicotine on rat striatal large neurons

O-191 - Exicitatory effects of nicotine on rat striatal large neurons