Transition of pattern generation: the phenomenon of post-scratching locomotion.

A fundamental problem in neurophysiology is the understanding of neuronal mechanisms by which the central nervous system produces a sequence of voluntary or involuntary motor acts from a diverse repertory of movements. These kinds of transitions between motor acts are extremely complex; however, they could be analyzed in a more simple form in decerebrate animals in the context of spinal central pattern generation. Here, we present for the first time a physiological phenomenon of post-scratching locomotion in which decerebrate cats exhibit a compulsory locomotor activity after an episode of scratching. We found flexor, extensor and intermediate single interneurons rhythmically firing in the same phase during both scratching and the subsequent post-scratching locomotion. Because no changes in phase of these neurons from scratching to post-scratching locomotion were found, we suggest that in the lumbar spinal cord there are neurons associated with both motor tasks. Moreover, because of its high reproducibility we suggest that the study of post-scratching fictive locomotion, together with the unitary recording of neurons, could become a useful tool to study neuronal mechanisms underlying transitions from one rhythmic motor task to another, and to study in more detail the central pattern generator circuitry in the spinal cord.

Upregulation of voltage-gated calcium channel cav1.3 in bovine somatotropes treated with ghrelin.

Activation of the growth hormone (GH) secretagogue receptor (GHS-R) by synthetic GH releasing peptides (GHRP) or its endogenous ligand (Ghrelin) stimulates GH release. Though much is known about the signal transduction underlying short-term regulation, there is far less information on the mechanisms that produce long-term effects. In the current report, using an enzyme-linked immunosorbent assay for GH detection and whole-cell patch-clamp recordings, we assessed the long-term actions of such regulatory factors on voltage-activated Ca(2+) currents in bovine somatotropes (BS) separated on a Percoll gradient and detected by immunohistochemistry. After 24 h of treatment with Ghrelin (10 nM) or GHRP-6 (100 nM) enhanced BS secretory activity; GH secretion stimulated by GHS through the activation of GHS-R because treatment with the antagonist of GHS-R (D-Lys3-GHRP-6, 10 µM) blocked the GH secretion, and the effect was dose and time dependent (24, 48, and 72 h). GH secretion stimulated by GHRP-6 was abolished by nifedipine (0.5 µM), a blocker of L-type HVA Ca(2+) channels, and KN-62 (10 µM), an inhibitor of Ca(2+)/CaM-KII. After 72 h in culture, all recorded BS exhibited two main Ca(2+) currents: a low voltage-activated (LVA; T-type) and a high voltage-activated (HVA; mostly dihydropyridine-sensitive L-type) current. Interestingly, HVA and LVA channels were differentially upregulated by Ghrelin. Chronic treatment with the GHS induced a significant selective increase on the Ba(2+) current through HVA Ca(2+) channels, and caused only a small increase of currents through LVA channels. The stimulatory effect on HVA current density was accompanied by an augment in maximal conductance with no apparent changes in the kinetics and the voltage dependence of the Ca(2+) currents, suggesting an increase in the number of functional channels in the cell membrane. Lastly, in consistency with the functional data, quantitative real-time RT-PCR revealed transcripts encoding for the Cav1.2 and Cav1.3 pore-forming subunits of L-type channels. The treatment with Ghrelin significantly increased the Cav1.3 subunit expression, suggeting that the chronic stimulation of the GHS receptor with Ghrelin or GHRP-6 increases the number of voltage-gated Ca(2+) channels at the cell surface of BS.

Dopamine prevents muscarinic-induced decrease of glutamate release in the auditory cortex.

Acetylcholine and dopamine are simultaneously released in the cortex at the occurrence of novel stimuli. In addition to a series of excitatory effects, acetylcholine decreases the release of glutamate acting on presynaptic muscarinic receptors. By recording evoked excitatory postsynaptic currents in layers II/III neurons of the auditory cortex, we found that activation of muscarinic receptors by oxotremorine reduces the amplitude of glutamatergic current (A(oxo)/A(ctr) = 0.53 +/- 0.17) in the absence but not in the presence of dopamine (A(oxo)/A(ctr) = 0.89 +/- 0.12 in 20 microM dopamine). These data suggested that an excessive sensitivity to dopamine, such as postulated in schizophrenia, could prevent the decrease of glutamate release associated with the activation of cholinergic corticopetal nuclei. Thus, a possible mechanism of action of antipsychotic drugs could be through a depression of the glutamatergic signal in the auditory cortex. We tested the capability of haloperidol, clozapine and lamotrigine to affect glutamatergic synaptic currents and their muscarinic modulation. We found that antipsychotics not only work as dopamine receptor antagonists in re-establishing muscarinic modulation, but also directly depress glutamatergic currents. These results suggest that presynaptic modulation of glutamate release can account for a dual route of action of antipsychotic drugs.

Pediatric cortical dysplasia: correlations between neuroimaging, electrophysiology and location of cytomegalic neurons and balloon cells and glutamate/GABA synaptic circuits.

Seizures in cortical dysplasia (CD) could be from cytomegalic neurons and balloon cells acting as epileptic 'pacemakers', or abnormal neurotransmission. This study examined these hypotheses using in vitro electrophysiological techniques to determine intrinsic membrane properties and spontaneous glutamatergic and GABAergic synaptic activity for normal-pyramidal neurons, cytomegalic neurons and balloon cells from 67 neocortical sites originating from 43 CD patients (ages 0.2-14 years). Magnetic resonance imaging (MRI), (18)fluoro-2-deoxyglucose positron emission tomography (FDG-PET) and electrocorticography graded cortical sample sites from least to worst CD abnormality. Results found that cytomegalic neurons and balloon cells were observed more frequently in areas of severe CD compared with mild or normal CD regions as assessed by FDG-PET/MRI. Cytomegalic neurons (but not balloon cells) correlated with the worst electrocorticography scores. Electrophysiological recordings demonstrated that cytomegalic and normal-pyramidal neurons displayed similar firing properties without intrinsic bursting. By contrast, balloon cells were electrically silent. Normal-pyramidal and cytomegalic neurons displayed decreased spontaneous glutamatergic synaptic activity in areas of severe FDG-PET/MRI abnormalities compared with normal regions, while GABAergic activity was unaltered. In CD, these findings indicate that cytomegalic neurons (but not balloon cells) might contribute to epileptogenesis, but are not likely to be 'pacemaker' cells capable of spontaneous paroxysmal depolarizations. Furthermore, there was more GABA relative to glutamate synaptic neurotransmission in areas of severe CD. Thus, in CD tissue alternate mechanisms of epileptogenesis should be considered, and we suggest that GABAergic synaptic circuits interacting with cytomegalic and normal-pyramidal neurons with immature receptor properties might contribute to seizure generation.

NMDA receptor function in mouse models of Huntington disease.

Huntington disease (HD) is an autosomal dominant disorder in which degeneration of medium-sized spiny striatal neurons occurs. The HD gene and the protein it encodes, huntingtin, have been identified but their functions remain unknown. Transgenic mouse models for HD have been developed and we examined responses of medium-sized striatal neurons recorded in vitro to application of N-methyl-D-aspartate (NMDA) in two of these. The first model (R6/2) expresses exon 1 of the human HD gene with approximately 150 CAG repeats. In the R6/2 an enhancement of currents induced by selective activation of NMDA receptors as well as an enhancement of intracellular Ca(2+) flux occurred in both presymptomatic and symptomatic mice. These alterations appeared specific for the NMDA receptor because alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-mediated currents were reduced in symptomatic R6/2s. In R6/2 animals there were parallel increases in NMDA-R1 and decreases in NMDA-R2A/B subunit proteins as established by immunohistochemistry. The second model (YAC72) contains human genomic DNA spanning the full-length gene and all its regulatory elements with 72 CAG repeats. The phenotypical expression of the disorder develops more gradually than in the R6/2. In YAC72 mice we found similar but less marked increases in responses of medium-sized striatal neurons to NMDA. These findings indicate that alterations in NMDA receptor function may predispose striatal neurons to excitotoxic damage, leading to subsequent neuronal degeneration and underscore the functional importance of NMDA receptors in HD.

Pulmonary tuberculosis and associated factors in areas of high levels of poverty in Chiapas, Mexico.

OBJECTIVES: To estimate the prevalence of pulmonary tuberculosis (PTB) and factors associated with PTB in areas of high levels of poverty in Chiapas, Mexico. METHODS: In 1998 active case-finding was carried out among those aged over 14 years who had a cough of > or =15 days duration, in a convenience sample of 1894 households in 32 communities selected at random based on the level of poverty and on the level of access to health services, measured by travelling time (<1 hour, > or =1 hour) from the community to the nearest health care unit. Of the 277 identified with a productive cough, we obtained sputum samples from 228 for the purposes of detecting PTB through acid-fast smears and cultures. Mycobacteria characterization was carried out using the BACTEC method. The identification of factors associated with PTB was performed using bivariate analysis and via logistic regression models. RESULTS: A PTB rate of 276.9 per 100 000 persons aged > or =15 years was found (95% CI : 161-443). Blood in sputum was the only factor associated with PTB (none of the demographic or socioeconomic characteristics were). Of 16 positive cultures, 14 became contaminated. The two cultures characterized were Mycobacterium tuberculosis (one being multiresistant). CONCLUSION: The high prevalence of PTB detected indicates the need, both in the area studied and in others with similar conditions, to develop PTB control programmes which give priority to early diagnosis and to the provision of adequate treatment.

Dopamine D4 receptor-deficient mice display cortical hyperexcitability.

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.

Coordinated expression of muscarinic receptor messenger RNAs in striatal medium spiny neurons.

The postsynaptic effects of acetylcholine in the striatum are largely mediated by muscarinic receptors. Two of the five cloned muscarinic receptors (M1 and M4) are expressed at high levels by the medium spiny neurons-the principal projection neurons of the striatum. Previous studies have suggested that M4 muscarinic receptors are found primarily in medium spiny neurons that express substance P and participate in the "direct" striatonigral pathway. This view is difficult to reconcile with electrophysiological studies suggesting that nearly all medium spiny neurons exhibit responses characteristic of M4 receptors. To explore this apparent discrepancy, the coordinated expression of M1-M5 receptor messenger RNAs in identified medium spiny neurons was assayed using single-cell reverse transcription-polymerase chain reaction techniques. Nearly all medium spiny neurons had detectable levels of M1 receptor messenger RNA. Although M4 receptor messenger RNA was detected more frequently in substance P-expressing neurons (70%), it was readily seen in a substantial population of enkephalin-expressing neurons (50%). To provide a quantitative estimate of transcript abundance, quantitative reverse transcription-polymerase chain reaction experiments were performed. These studies revealed that M4 messenger RNA was expressed by both substance P and enkephalin neurons, but was roughly five-fold higher in abundance in substance P-expressing neurons. This quantitative difference provides a means of reconciling previous estimates of M4 receptor distribution and function.

Facilitated glutamatergic transmission in the striatum of D2 dopamine receptor-deficient mice.

Dopamine (DA) receptors play an important role in the modulation of excitability and the responsiveness of neurons to activation of excitatory amino acid receptors in the striatum. In the present study, we utilized mice with genetic deletion of D2 or D4 DA receptors and their wild-type (WT) controls to examine if the absence of either receptor subtype affects striatal excitatory synaptic activity. Immunocytochemical analysis verified the absence of D2 or D4 protein expression in the striatum of receptor-deficient mutant animals. Sharp electrode current- and whole cell patch voltage-clamp recordings were obtained from slices of receptor-deficient and WT mice. Basic membrane properties were similar in D2 and D4 receptor-deficient mutants and their respective WT controls. In current-clamp recordings in WT animals, very little low-amplitude spontaneous synaptic activity was observed. The frequency of these spontaneous events was increased slightly in D2 receptor-deficient mice. In addition, large-amplitude depolarizations were observed in a subset of neurons from only the D2 receptor-deficient mutants. Bath application of the K+ channel blocker 4-aminopyridine (100 microM) and bicuculline methiodide (10 microM, to block synaptic activity due to activation of GABA(A) receptors) markedly increased spontaneous synaptic activity in receptor-deficient mutants and WTs. Under these conditions, D2 receptor-deficient mice displayed significantly more excitatory synaptic activity than their WT controls, while there was no difference between D4 receptor-deficient mice and their controls. In voltage-clamp recordings, there was an increase in frequency of spontaneous glutamate receptor-mediated inward currents without a change in mean amplitude in D2 receptor-deficient mutants. In WT mice, activation of D2 family receptors with quinpirole decreased spontaneous excitatory events and conversely sulpiride, a D2 receptor antagonist, increased activity. In D2 receptor-deficient mice, sulpiride had very little net effect. Morphologically, a subpopulation of medium-sized spiny neurons from D2 receptor-deficient mice displayed decreased dendritic spines compared with cells from WT mice. These results provide evidence that D2 receptors play an important role in the regulation of glutamate receptor-mediated activity in the corticostriatal or thalamostriatal pathway. These receptors may function as gatekeepers of glutamate release or of its subsequent effects and thus may protect striatal neurons from excessive excitation.

Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the rat.

Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by application of N-methyl-d-aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large-sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in medium-sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium- and large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders.

Neurons recorded from pediatric epilepsy surgery patients with cortical dysplasia.

PURPOSE: Cortical dysplasia (CD) is a common pathological substrate in patients with early-onset childhood epilepsy. In CD tissue, little is known about the mechanisms responsible for cellular hyperexcitability. In this study, we report initial electrophysiological and morphological observations from normal and dysmorphic cells in pediatric CD patients. METHODS: Neocortical "most" and "least" epileptogenic areas were sampled based on neuroimaging and electrocorticography from 15 CD patients (ages 0.3 to 14 years). Whole-cell voltage clamp recordings combined with infrared videomicroscopy sampled abnormal cells (cytomegalic neurons, cells with bifurcated dendrites, disoriented pyramidal cells, etc.) compared with normal-appearing neurons from the same patient. Cells were filled with biocytin, and adjacent tissue blocks were stained for neuronal and glial markers. RESULTS: About 15% of the 161 recorded cells were abnormal in appearance. Abnormal cells showed electrophysiological irregularities ranging from intrinsic cellular hyperexcitability to hyposensitivity after application of ionotropic receptor agonists. Other findings included increased excitatory postsynaptic currents and alterations in gamma-aminobutyric acid reversal potentials. CONCLUSIONS: In pediatric CD tissue, these preliminary results indicate that abnormal-appearing cells showed abnormalities in electrophysiological measures compared with normal-appearing neurons. The abnormalities varied from hyperexcitability to hypoexcitability. More detailed results and conclusions will be forthcoming as additional patient material is analyzed.

D(1) dopamine receptor activation reduces GABA(A) receptor currents in neostriatal neurons through a PKA/DARPP-32/PP1 signaling cascade.

Dopamine is a critical determinant of neostriatal function, but its impact on intrastriatal GABAergic signaling is poorly understood. The role of D(1) dopamine receptors in the regulation of postsynaptic GABA(A) receptors was characterized using whole cell voltage-clamp recordings in acutely isolated, rat neostriatal medium spiny neurons. Exogenous application of GABA evoked a rapidly desensitizing current that was blocked by bicuculline. Application of the D(1) dopamine receptor agonist SKF 81297 reduced GABA-evoked currents in most medium spiny neurons. The D(1) dopamine receptor antagonist SCH 23390 blocked the effect of SKF 81297. Membrane-permeant cAMP analogues mimicked the effect of D(1) dopamine receptor stimulation, whereas an inhibitor of protein kinase A (PKA; Rp-8-chloroadenosine 3',5' cyclic monophosphothioate) attenuated the response to D(1) dopamine receptor stimulation or cAMP analogues. Inhibitors of protein phosphatase 1/2A potentiated the modulation by cAMP analogues. Single-cell RT-PCR profiling revealed consistent expression of mRNA for the beta1 subunit of the GABA(A) receptor-a known substrate of PKA-in medium spiny neurons. Immunoprecipitation assays of radiolabeled proteins revealed that D(1) dopamine receptor stimulation increased phosphorylation of GABA(A) receptor beta1/beta3 subunits. The D(1) dopamine receptor-induced phosphorylation of beta1/beta3 subunits was attenuated significantly in neostriata from DARPP-32 mutants. Voltage-clamp recordings corroborated these results, revealing that the efficacy of the D(1) dopamine receptor modulation of GABA(A) currents was reduced in DARPP-32-deficient medium spiny neurons. These results argue that D(1) dopamine receptor stimulation in neostriatal medium spiny neurons reduces postsynaptic GABA(A) receptor currents by activating a PKA/DARPP-32/protein phosphatase 1 signaling cascade targeting GABA(A) receptor beta1 subunits.

Physiological and molecular properties of AMPA/Kainate receptors expressed by striatal medium spiny neurons.

The mechanisms by which glutamate shapes the activity of striatal medium spiny neurons are of fundamental importance to our understanding of normative and pathological striatal physiology. Non-N-Methyl-D-aspartate (non-NMDA) glutamate receptor expression and function were studied in medium spiny neurons with a combination of single cell RT-PCR, immunocytochemistry and whole-cell voltage-clamp techniques. Reverse transcription polymerase chain reaction analysis found that GluR2 mRNA appeared to be the most abundant and widely distributed AMPA receptor mRNA. GluR1 was also commonly detected. However, GluR3 mRNA was preferentially expressed by neurons coexpressing substance P and enkephalin and GluR4 mRNA was not detected in identified medium spiny neurons. All neuronal classes appeared to express GluR5 or GluR6 and/or GluR7 mRNA in addition to kainate (KA) subunit mRNA. Immunocytochemical studies confirmed the mRNA distributions and also revealed that GluR1 protein was largely restricted to dendritic spines. Although the mRNA and protein for both alpha-amino-3-hydroxy-5-methyl-ioxyzole-4-proprionic acid (AMPA) and KA class subunits was detected, the physiological response to glutamatergic ligands and the benzothiadizine cyclothiazide was characteristic of AMPA, not KA receptors. The AMPA receptor antagonist GYKI 52466 blocked the response to AMPA and all but a small transient component of the response to KA. The current-voltage relationship of the AMPA-evoked currents was relatively linear but Ca2+ fluorometry revealed that substantial changes in intracellular Ca2+ concentration accompanied exposure to either agonist. These results argue that somatodendritic non-NMDA glutamate receptors in medium spiny neurons are primarily GluR2-containing receptors of the AMPA class but that activation of these receptors as a group nevertheless results in a significant Ca2+ influx.

Passive properties of neostriatal neurons during potassium conductance blockade.

Voltage recordings from neostriatal projection neurons were obtained using in vitro intracellular techniques before and during K+-conductance blockade. Neurons were stained with the biocytin technique. Somatic surface area (AS) was determined by both whole-cell recordings in isolated somata and by measuring stained somata recorded in slices. Dendritic measurements were done in reconstructed neurons. Average determinations of dendritic (AD) and neuronal (AN) surface areas coincided with previously reported anatomical data. Thus: As approximately 6.5 x 10(-6) cm2; AD approximately 1.9 x 10(-4) cm2; AN approximately AD + AS approximately 2 x 10(-4) cm2; AD/AS approximately 30. Measurements were done before and after superfusion with K+-conductance blockers (K+-blockers). Cells whose neuronal morphology was not obviously distorted by K+-blockade were chosen for the present study. Electrotonic transients were matched to a somatic shunt equivalent cylinder model adjusted with the generalized correction factor (Fdga) that constrains the parameters for neuronal anatomy. Neuronal input resistance (RN; mean +/- SEM) increased when it was corrected for somatic shunt, from 49 +/- 2 Momega (n = 80) to 179 +/- 7 Momega (n = 32). A difference was also obtained between the slowest time constant, tau0 = 16 +/- 0.9 ms (n = 49), and the dendritic membrane time constant, taumD = 33 +/- 1.6 ms (n = 36). When these electrophysiological measurements were used to calculate AN, the value obtained was similar to the anatomical measurements. Combining anatomical and electrophysiological data, somatic and dendritic input resistances were determined: RD = 182 +/- 7 Momega; Rs (with shunt) = 74 +/- 4 Momega (n = 32). The generalized correction factor, Fdga = 0.91 +/- 0.007 (n = 10), implied a short effective electrotonic length for dendrites: LD = 0.46 +/- 0.014 (n = 32). Saturating concentrations of the K+-blockers tetraethylammonium, Cs+, and Ba2+ increased RN and induced charging curves well fitted by single exponential functions in 56% of neostriatal neurons. Ba2+ greatly decreased the somatic shunt (n = 5): (RN = 216 +/- 21 Momega, tau0 = 46 +/- 2 ms, RD = 239 +/- 25 Momega, and RS = 3.2 +/- 0.5 Gomega), rendering values similar to those obtained with whole-cell recordings (e.g., RN approximately 198 Momega, RS approximately 2.62 Gomega) (n = 52). Cs+ (n = 5) had less effect on the somatic shunt (RN = 115 +/- 19 Momega, tau0 = 49 +/- 13 ms, RS = 161 +/- 8 Momega), although dendritic conductance was equally blocked (RD = 261 +/- 16 Momega). The Cs+-sensitive conductance exhibited inward rectifying properties not displayed by the Ba2+-sensitive conductance, suggesting that Cs+ preferentially acted upon inward rectifier conductances. In contrast, Ba2+ significantly acted upon linear conductances making up the somatic shunt. This suggests a differential action of different K+-blockers on the somato-dendritic membrane, implying a differential distribution of membrane conductances. Another action of K+-blockers, in about 40% of the cells, was to induce dye and probably electrical coupling between neighboring neurons.

mRNAs for clozapine-sensitive receptors co-localize in rat prefrontal cortex neurons.

The clinical efficacy of clozapine in treating schizophrenia may stem from its lack of receptor selectivity. If true, several clozapine-sensitive receptors may be co-expressed by neurons dysfunctional in schizophrenia. To test this hypothesis, neurons from the rat medial prefrontal cortex were acutely isolated and subjected to single cell RT-PCR analysis. The co-ordinated expression of five clozapine-sensitive receptors (D4, m1, 5-HT2a, 5-HT2c, 5-HT7) was examined in interneurons and pyramidal neurons. Profiling of GABAergic interneurons commonly revealed the co-expression of two or more clozapine-sensitive receptor mRNAs. Although co-expression of these receptors was less extensive in pyramidal neurons, it was also commonly found. These results suggest that clozapine's therapeutic effects may be mediated by antagonism of dopaminergic, cholinergic and serotoninergic signaling pathways at the single cell level.

Dopamine selects glutamatergic inputs to neostriatal neurons.

Glutamatergic synaptic potentials induced by micromolar concentrations of the potassium conductance blocker 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in the presence of 10 microM bicuculline (BIC). These synaptic potentials originate from neostriatal cortical and thalamic afferents and were completely blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 100 microM D-2-amino-5-phosphonovaleric acid (2-APV). Their inter-event time intervals could be fitted to exponential distributions, suggesting that they are induced randomly. Their amplitude distributions had most counts around 1 mV and fewer counts with values up to 5 mV. Since input resistance of the recorded neurons is about 40 M omega, the amplitudes agree to quantal size measurements in mammalian central neurons. The action of a D2 agonist, quinpirole, was studied on the frequency of these events. Mean amplitude of synaptic potentials was preserved in the presence of 2-10 microM quinpirole, but the frequency of 4-AP-induced glutamatergic synaptic potentials was reduced in 35% of cases. The effect was blocked by the D2 antagonist sulpiride (10 microM). Input resistance, membrane potential, or firing threshold did not change during quinpirole effect, suggesting a presynaptic site of action for quinpirole in some but not all glutamatergic afferents that make contact on a single cell. The present experiments show that dopaminergic presynaptic modulation of glutamatergic transmission in the neostriatum does not affect all stimulated afferents, suggesting that it is selective towards some of them. This may control the quality and quantity of afferent flow upon neostriatal neurons.

Firing frequency modulation of substantia nigra reticulata neurons by 5-hydroxytryptamine.

Unitary extracellular recordings were made in in vitro rat brain slices to explore the effects of serotoninergic analogues on the spontaneous activity of substantia nigra reticulata (SNr) neurons. Most SNr neurons exhibited regular spontaneous firing (23.4 +/- 8.9 Hz, mean +/- S.E.M., n = 30) similar to that found in vivo. The most reproducible effect of serotonin (5-HT) was an increase in firing frequency found in 53% of the cells. The effect was concentration dependent and blocked by the 5-HT1/2 antagonist methysergide (1-10 microM) but unaffected by the 5-HT4- and 5-HT1-preferring antagonists DAU 6285 (5 microM) and metiothepin (5 microM), respectively. However, 5-HT also decreased the firing frequency in several neurons. In 19% of the neurons an inhibition was found alone but a biphasic response (inhibition and excitation) was found in another 28% of the neurons. Interestingly, the effect of the 5-HT-uptake inhibitor, duloxetine (100-400 nM), was frequency inhibition. Agonists that mimicked the 5-HT-induced inhibition were of the 5-HT1B-class (25 microM CP 93129 and 25 microM TFMPP). Neither the 5-HT2-antagonist ritanserin (5 microM) nor the GABA(A)-antagonist, bicuculline (30 microM) were able to block the inhibition suggesting that some SNr neurons may be directly inhibited by 5-HT.

M3 muscarinic receptors mediate cholinergic excitation of the spontaneous activity of subthalamic neurons in the rat.

The effect of the muscarinic antagonist on carbachol-induced increase in spontaneous activity of neurons of the subthalamic nucleus was examined by recording the extracellular unitary activity in an in vitro slice preparation. Carbachol produced (98% of the 263 neurons tested) an increase (twofold of the basal at 500 nM) of the discharge frequency. The EC50 for the carbachol-induced effect was 375 +/- 8.7 nM (mean +/- SEM). The response was blocked by muscarinic antagonists in a dose dependent manner. However, the IC50 (94 +/- 3 nM) for the M3 antagonist 4-diphenyl acetoxy-N-methyl piperidine methobromide (4-DAMP) was considerably less than the other muscarinic antagonists (M1 antagonist pirenzepine, IC50 1340 +/- 110 nM; M2 antagonist AF-DX-116, IC50 6780 +/- 690 nM). These results suggest that the cholinergic input to the rat subthalamic nucleus exerts a postsynaptic excitatory action and this effect is likely mediated via muscarinic receptor type 3.

Muscarinic receptors modulate the afterhyperpolarizing potential in neostriatal neurons.

The actions of carbachol were studied on the firing response of neostriatal neurons recorded intracellularly from in vitro slice preparations of the rat brain. Carbachol (1-10 microM) reversibly reduced the afterhyperpolarization in neostriatal neurons. This effect was accompanied by an increase in both firing frequency and input resistance in the subthreshold voltage range. Atropine (1-10 microM) reversibly blocked carbachol effects, suggesting muscarinic receptor modulation. Pirenzepine (up to 1 microM), but not AF-DX 384 (10 microM) or gallamine (30 microM), blocked the effects of carbachol on the afterhyperpolarization. The protein kinase C activator, phorbol 12,13 dibutyrate, but not the inactive phorbol ester, 4 alpha-phorbol 12-myristate 13-acetate, mimicked carbachol effects. The results suggest that muscarinic receptors, probably of the M1 type, regulate neostriatal excitability by modulating afterhyperpolarization.

Patterns of excitatory and inhibitory synaptic transmission in the rat neostriatum as revealed by 4-AP.

1. Synaptic potentials induced by 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in an in vitro slice preparation. EC50 for this 4-AP action was approximately 120 microM. The threshold for activation of synaptic potentials was 5 microM. 2. 4-AP-induced synaptic potentials appeared stochastically. Most were blocked by 1 microM tetrodotoxin or 400 microM Cd2+. Therefore they reflect a release of neurotransmitters dependent on both Ca2+ entry to the terminals and action potential firing. 3. Bicuculline (BIC) (< or = 10 microM), a gamma-aminobuturic acid-A (GABAA) antagonist, blocked about half of the 4-AP-induced synaptic potentials. This suggests that intrinsic inhibitory connections within the neostriatum are activated by 4-AP administration. 4. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; < or = 10 microM) plus D-2-amino-5-phosphonovaleric acid (D-APV; < or = 100 microM) blocked most of the BIC-resistant 4-AP-induced synaptic potentials. This suggests that 4-AP induced release of glutamate (GLU) from extrinsic glutamatergic afferents. As most glutamatergic afferents are extrinsic, these afferents then would be able to fire spikes and release transmitter for several hours after they are cut from their somata. 5. If CNQX plus D-APV were administered before BIC, neostriatal neurons responded in different ways. In one half of the neurons, all induced synaptic potentials were blocked. This suggests that most GABAergic intrinsic connections between neostriatal neurons are activated indirectly by 4-AP. 4-AP would first activate extrinsic glutamatergic afferents and these in turn would activate GABAergic intrinsic neurons and connections. 6. In the remaining half of the recorded neurons, administration of CNQX plus D-APV blocked most, but not all of the 4-AP-induced synaptic potentials. The synaptic potentials that remained had a characteristic pattern: they were high amplitude, rhythmic, bursts of synaptic potentials. They were blocked by BIC (5 microM) but not by mecamylamine (> 10 microM). This suggests that these bursts of synaptic potentials were GABAergic and generated by intrinsic neurons. Therefore these neurons would not innervate all neostriatal neurons equally but just a subset of them. 7. Records from an identified aspiny neostriatal interneuron, obtained from the same preparation, are shown. This interneuron fired in bursts and its morphologically and physiologically similar to the recently described, fast spiking, parvalbumin immunoreactive, GABAergic, aspiny interneuron is functional in the slice preparation.(ABSTRACT TRUNCATED AT 400 WORDS)