Low-threshold L-type calcium channels in rat dopamine neurons.

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.

Muscarine reduces calcium-dependent electrical activity in substantia nigra dopaminergic neurons.

The effect of muscarine on Ca2+ dependent electrical activity was studied in dopamine (DA) neurons located in the substantia nigra pars compacta (SNc) in brain slices from young rats, using sharp electrodes. In most DA neurons tested, muscarine (50 microM) reduced the amplitude of spontaneous oscillatory potentials and evoked Ca2+-dependent potentials recorded in the presence of TTX. Muscarine also reduced the amplitude of the slow afterhyperpolarization (sAHP) following action potentials in most DA neurons. These data suggest that muscarine reduces Ca2+ entry in SNc DA neurons. The reduction of the amplitude of the sAHP by muscarine in DA neurons may facilitate bursting initiated by glutamatergic input by increasing the frequency at which DA neurons can fire. The reduction of the sAHP via activation of muscarinic receptors in vivo may provide a mechanism whereby cholinergic inputs to DA neurons from the tegmental peduncular pontine nucleus could modulate dopamine release at dopaminergic targets in the brain.

Unilateral striatal dopamine depletion: time-dependent effects on cortical function and behavioural correlates.

Previously, we showed that unilateral blockade of D1 dopamine receptors in the striatum inhibits immediate-early gene expression bilaterally throughout large parts of the cortex, including sensory-evoked expression in the barrel cortex. To further investigate this dopamine regulation of cortical function, we examined the effects of dopamine depletion on cortical gene regulation and behavioural correlates. Two days after unilateral infusion of 6-hydroxydopamine into the midbrain, rats displayed a (to some degree) bilateral reduction in cortical zif 268 expression that was more pronounced on the lesioned side. This decrease was found across motor, somatosensory, insular and piriform, but not cingulate, cortex, similar to the effects of blockade of striatal D1 receptors. Furthermore, whisker stimulation-evoked c-fos and zif 268 expression in the barrel cortex ipsilateral to the lesion was also attenuated by acute dopamine depletion. These cortical deficits were accompanied by a breakdown of spontaneous behaviours in an open-field test. In contrast, 21 days after dopamine depletion, both basal and sensory-evoked gene expression in the cortex were near-normal. This cortical recovery was paralleled by recovery in locomotion and in sensory-guided behaviour (scanning) related to the hemisphere contralateral to the lesion, but not in scanning by the dopamine-depleted hemisphere. Our results suggest that striatal dopamine exerts a widespread facilitatory influence on cortical function that is necessary, but not sufficient, for normal behaviour. Moreover, the mechanisms mediating this cortical facilitation appear to be subject to substantial neuroplasticity after dopamine perturbation.

Morphological study of the tegmental pedunculopontine nucleus, substantia nigra and subthalamic nucleus, and their interconnections in rat organotypic culture.

The morphological organization of the tegmental pedunculopontine nucleus, midbrain extrapyramidal area, substantia nigra and subthalamic nucleus and their interrelationships were studied in rat organotypic culture using immunohistochemistry and NADPH-diaphorase histochemistry. Three coronal sections, one containing the tegmental pedunculopontine nucleus/midbrain extrapyramidal area, another with the substantia nigra and the third with the subthalamic nucleus, were obtained from postnatal 1-2-day-old rats. These sections were co-cultured for 3-4 weeks using the roller-tube technique. In the tegmental pedunculopontine nucleus/midbrain extrapyramidal area, the distribution pattern of cholinergic neurons was similar to that found in the in vivo study. We could, therefore, identify the subdivisions of the tegmental pedunculopontine nucleus (i.e., pars compacta and pars dissipata) and the midbrain extrapyramidal area. As in the in vivo situation, glutamate immunoreactive neurons were also located in these areas. Approximately 10% of NADPH-diaphorase positive neurons in the tegmental pedunculopontine nucleus, were glutamate immunoreactive. In the substantia nigra, as in the in vivo, tyrosine hydroxylase immunoreactive (putative dopaminergic) neurons were identified predominantly in the substantia nigra pars compacta, and parvalbumin immunoreactive neurons (putative GABAergic) mainly in the substantia nigra pars reticulata. The subthalamic nucleus was ladened with glutamate immunoreactive neurons. NADPH-diaphorase stained axons originating from the tegmental pedunculopontine nucleus were traced into the substantia nigra and subthalamic nucleus. They were often in close apposition to tyrosine hydroxylase immunoreactive neurons in the substantia nigra. Parvalbumin immunoreactive fibers from the substantia nigra projected heavily to the midbrain extrapyramidal area, but only sparsely to the tegmental pedunculopontine nucleus and the subthalamic nucleus. These findings indicate that the tegmental pedunculopontine nucleus/midbrain extrapyramidal area, substantia nigra and subthalamic nucleus in the organotypic culture have retained a basic morphological organization and connectivity similar to those seen in the in vivo situation. Therefore, this preparation could be a useful model to conduct further studies to investigate functional circuits among the structures represented.

Regulation of rat cortex function by D1 dopamine receptors in the striatum.

Interactions between the basal ganglia and the cerebral cortex are critical for normal goal-directed behavior. In the present study, we used immediate-early genes (c-fos, zif 268) as functional markers to investigated how basal ganglia output altered by stimulation/blockade of D1 dopamine receptors in the striatum affects cortical function. Systemic administration of the mixed D1/D2 receptor agonist apomorphine (3 mg/kg) increased immediate-early gene expression in the striatum and throughout most of the cortex. Unilateral intrastriatal infusion of the selective D1 receptor antagonist SCH-23390 (0.5-10 microg) blocked this response bilaterally in striatum and cortex in a dose-dependent manner. Even apparently regionally restricted blockade of striatal D1 receptors attenuated gene expression throughout striatum and cortex in both hemispheres. Intrastriatal administration of the D1 antagonist inhibited apomorphine-induced sniffing/whisking, whereas other motor behaviors were unaffected. To determine whether such changes in cortical gene expression could reflect altered cortical function, we examined the effects of blocking striatal D1 receptors on whisker stimulation-evoked immediate-early gene expression in the sensorimotor cortex. Apomorphine increased sensory stimulation-evoked gene expression in the barrel cortex, and intrastriatal infusion of SCH-23390 attenuated this effect. These results suggest that stimulation of D1 dopamine receptors in the striatum exerts a widespread facilitatory effect on cortical function.

Electrophysiological characteristics of substantia nigra neurons in organotypic cultures: spontaneous and evoked activities.

Morphological and electrophysiological characteristics of dopaminergic and non-dopaminergic neurons in the substantia nigra and their postsynaptic responses to stimulation of the tegmental pedunculopontine nucleus were studied in rat organotypic triple cultures. These cultures consisted of the subthalamic nucleus explant, ventral mesencephalic explant, inclusive of the substantia nigra and the mesopontine tegmentum explant, inclusive of the tegmental pedunculopontine nucleus, prepared from one- to two-day-old rats. Intracellular sharp and whole-cell recordings were obtained from three- to eight-week-old organotypic cultures. Recorded neurons were identified as dopaminergic and non-dopaminergic neurons with tyrosine hydroxylase immunohistochemistry. Dopaminergic neurons had long duration action potentials, prominent afterhyperpolarization, time-dependent inward and outward rectification and strong frequency adaptation. Spontaneous firing patterns varied from regular, irregular to burst firing. Non-dopaminergic neurons had short duration action potentials, in general no rectifying currents, and maintained high firing frequencies. Spontaneous firing patterns in these neurons were irregular or burst firing. Morphological analysis of the recorded neurons labeled with neurobiotin revealed that non-dopaminergic neurons had more extensive arborization of higher-order dendrites than dopaminergic neurons. Dopaminergic and non-dopaminergic neurons receive glutamatergic and cholinergic excitatory inputs from the tegmental pedunculopontine nucleus. These results indicate that morphological and electrophysiological characteristics of substantia nigra neurons in the organotypic culture are generally similar to those reported in in vitro slice and in vivo studies. However, spontaneous activities of dopamine neurons observed in the organotypic culture preparation more closely resemble those in in vivo preparation compared to in vitro preparation.

Afferent modulation of dopamine neuron firing patterns.

In recent studies examining the modulation of dopamine (DA) cell firing patterns, particular emphasis has been placed on excitatory afferents from the prefrontal cortex and the subthalamic nucleus. A number of inconsistencies in recently published reports, however, do not support the contention that tonic activation of NMDA receptors is the sole determinate of DA neuronal firing patterns. The results of work on the basic mechanism of DA firing and the action of apamin suggest that excitatory projections to DA neurons from cholinergic and glutamatergic neurons in the tegmental pedunculopontine nucleus, and/or inhibitory GABAergic projections, are also involved in modulating DA neuron firing behavior.

Differential expression of ErbB3 and ErbB4 neuregulin receptors in dopamine neurons and forebrain areas of the adult rat.

Neuregulins have been shown to play an important role in the development of the central nervous system, but their function in adult tissues is still unclear. We investigated the expression of the neuregulin receptors erbB3 and erbB4 in the adult rat brain by in situ hybridization histochemistry. Areas with considerable expression of erbB4 receptor mRNA include cortex, amygdala, hippocampus, medial habenula, reticular thalamic nucleus, several hypothalamic nuclei, subthalamic nucleus, substantia nigra pars compacta, and ventral tegmental area. Immunostaining for tyrosine hydroxylase and dopamine depletion by 6-hydroxydopamine indicate that erbB4 is expressed in dopamine neurons in the latter two nuclei. Substantial erbB4 expression is also present in clusters of cells along the ventral and medial border of the striatum/nucleus accumbens and in the subependymal zone along the lateral and olfactory ventricles (rostral migratory stream), suggesting a role for neuregulins in adult cell proliferation. In contrast, erbB3 mRNA is mostly expressed in white matter throughout the brain and in the ependyma of the ventral half of the third ventricle (tanycytes). These results demonstrate that expression of erbB3 and erbB4 receptors is widespread in the adult rat brain and suggest a function for neuregulins into adulthood.

Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice.

The basal ganglia, a brain structure critical for sensorimotor and motivational aspects of behavior, contain very high levels of CB1 cannabinoid receptors. These receptors are activated by endogenous lipophilic ligands, and they are thought to mediate behavioral effects of cannabinoid drugs. To evaluate the role of the endogenous cannabinoid system in the regulation of basal ganglia pathways, we have investigated the effects of targeted deletion of CB1 receptors on gene expression of various neuropeptides and transmitter-related enzymes in basal ganglia neurons. Mice without CB1 receptors are extremely hypoactive in a test for exploratory behavior (open-field test), showing markedly reduced locomotion and rearing. These CB1 mutants display significantly increased levels of substance P, dynorphin, enkephalin, and GAD 67 mRNAs in neurons of the two output pathways of the striatum that project to the substantia nigra and the globus pallidus. Our findings demonstrate that elimination of CB1 receptors results in behavioral abnormalities and functional reorganization of the basal ganglia.

Postsynaptic nicotinic receptors on dopaminergic neurons in the substantia nigra pars compacta of the rat.

Previous studies have shown that application of nicotinic agonists in the substantia nigra pars compacta increases the firing rate of dopaminergic neurons. We have used intracellular recordings to show that the response of these neurons to nicotine is postsynaptic, since it persists in the presence of low-calcium buffer containing tetrodotoxin. Burst firing in the presence of nicotine was not observed. The presence of postsynaptic nicotinic receptors was confirmed by immunohistochemical localization of the alpha4 nicotinic receptor subunit on dendrites in the substantia nigra pars compacta. The majority of tyrosine hydroxylase-immunopositive neurons in the substantia nigra pars compacta were also immunopositive for the alpha4 subunit. Immunohistochemical localization of the alpha4 and beta2 subunits in adjacent brain sections produced similar patterns of staining. Electron micrographs clearly indicated the presence of alpha4 subunit at postsynaptic densities. The predominant role of nicotinic receptors in the central nervous system has been suggested to be the presynaptic modulation of neurotransmitter release [McGehee D. S. and Role L. W. (1995) A. Rev. Physiol. 57, 521-546]. Although several postsynaptic nicotinic responses have also been reported in the literature, it is unclear as to whether the postsynaptic nicotinic receptors mediating responses to exogenously applied agonists are involved in synaptic transmission. From our electrophysiological and immunohistochemical results, we conclude that alpha4-containing nicotinic receptors are found at synapses on dopaminergic neurons. These synapses are similar to the cholinergic synapses described at these neurons, suggesting that nicotinic receptors are important in modulating the excitability of dopaminergic neurons by direct synaptic transmission.

Morphological organization of the globus pallidus-subthalamic nucleus system studied in organotypic cultures.

The morphological organization of the globus pallidus (GP), the subthalamic nucleus (STN), and the pallidosubthalamic projection was studied in organotypic cultures. Coronal slices from the GP, the STN, the striatum (CPu), and the cortex (Cx) were taken from the rat after postnatal days 0-2 and grown for 2 or 5-6 weeks. For analysis, immunocytochemistry against glutamate (GLU), parvalbumin (PV), and calretinin (CR) was combined with confocal microscopy. After 2 weeks in vitro, the STN showed a densely packed, homogeneous GLU-immunoreactive (ir) cell population. Pallidal GLU-ir neurons were heterogeneous, consisting of large-sized weakly GLU-ir neurons and small-sized intensively GLU-ir neurons. After 5-6 weeks in vitro, pallidal axons had radiated from numerous large-sized PV-ir cells and selectively innervated the STN, where they heavily ramified. Cultured STN neurons were not stained for PV; however, multipolar intensely PV-ir neurons were located at the border of the STN with their dendrites oriented towards the STN. Double labeling for PV and CR in both mature cultures and in the adult rat revealed that the culture CR-ir neurons from the GP, the Cpu, and from areas adjacent to the STN were different from cultured PV-ir neurons and their morphologies and distribution corresponded to that in vivo. These results demonstrate that 1) cultured CP and STN neurons display similar morphologies found in in vivo, 2) PV-ir pallidal neurons heavily and selectively innervate the STN; 3) there is a specific class of STN border neurons; and 4) in contrast to the in vivo situation, most cultured STN neurons are PV-negative.

Regulation of the nigrostriatal pathway by metabotropic glutamate receptors during development.

Dopamine neurons in the substantia nigra heavily innervate the striatum, making it the nucleus with the highest levels of dopamine in the adult brain. The present study analyzes the time course and the density of striatal innervation by nigral dopamine neurons and characterizes the role of the neurotransmitter glutamate during the development of the nigrostriatal pathway. For this purpose, organotypic cultures containing the cortex, the striatum, and the substantia nigra (triple cultures) were prepared from rat brains at postnatal day (PND) 0-2 and were cultured for up to 60 d in vitro (DIV). Dopamine fibers and neurons were labeled using tyrosine hydroxylase (TH) immunohistochemistry. Striatal TH-ir fiber density was quantitatively analyzed using confocal laser scanning microscopy (CLSM). In long-term triple cultures (44 +/- 3 DIV), the striatal dopamine fiber density was high and was weakly correlated with the number of nigral dopamine neurons. The high striatal dopamine fiber density mainly resulted from an enhanced ingrowth and ramification of dopamine fibers from nigral neurons during 8-17 DIV. The metabotropic glutamate receptor (mGluR) antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP-3) selectively inhibited this dopaminergic innervation of the striatum, whereas ionotropic GluR antagonists had no effect. The L-AP-3-mediated inhibition was prevented by the mGluR agonist 1S, 3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). The inhibition of the striatal dopaminergic innervation by L-AP-3 was further confirmed by anterograde tracing of the nigrostriatal projection with Phaseolus vulgaris leucoagglutinin. These results indicate that glutamate, by acting on group I mGluRs, plays an important "trophic" role for the development of the nigrostriatal dopamine pathway.

Somatodendritic depolarization-activated potassium currents in rat neostriatal cholinergic interneurons are predominantly of the A type and attributable to coexpression of Kv4.2 and Kv4.1 subunits.

Unlike other neostriatal neurons, cholinergic interneurons exhibit spontaneous, low-frequency, repetitive firing. To gain an understanding of the K+ channels regulating this behavior, acutely isolated adult rat cholinergic interneurons were studied using whole-cell voltage-clamp and single-cell reverse transcription-PCR techniques. Cholinergic interneurons were identified by the presence of choline acetyltransferase (ChAT) mRNA. Depolarization-activated potassium currents in cholinergic interneurons were dominated by a rapidly inactivating, K+-selective A current that became active at subthreshold potentials. Depolarizing prepulses inactivated this component of the current, leaving a delayed, rectifier-like current. Micromolar concentrations of Cd2+ dramatically shifted the voltage dependence of the A current without significantly affecting the delayed rectifier. The A-channel antagonist 4-aminopyridine (4-AP) produced a voltage-dependent block (IC50, approximately 1 mM) with a prominent crossover at millimolar concentrations. On the other hand, TEA preferentially blocked the sustained current component at concentrations <10 mM. Single-cell mRNA profiling of subunits known to give rise to rapidly inactivating K+ currents revealed the coexpression of Kv4.1, Kv4.2, and Kv1.4 mRNAs but low or undetectable levels of Kv4.3 and Kv3.4 mRNAs. Kv1.1, beta1, and beta2 subunit mRNAs, but not beta3, were also commonly detected. The inactivation recovery kinetics of the A-type current were found to match those of Kv4.2 and 4.1 channels and not those of Kv1.4 or Kv1. 1 and beta1 channels. Immunocytochemical analysis confirmed the presence of Kv4.2 but not Kv1.4 subunits in the somatodendritic membrane of ChAT-immunoreactive neurons. These results argue that the depolarization-activated somatodendritic K+ currents in cholinergic interneurons are dominated by Kv4.2- and Kv4. 1-containing channels. The properties of these channels are consistent with their playing a prominent role in governing the slow, repetitive discharge of interneurons seen in vivo.

Up and down states in striatal medium spiny neurons simultaneously recorded with spontaneous activity in fast-spiking interneurons studied in cortex-striatum-substantia nigra organotypic cultures.

In vivo intracellular spontaneous activity in striatal medium spiny (MS) projection neurons is characterized by "up" and "down" states. How this type of activity relates to the neuronal activity of striatal fast-spiking (FS) interneurons was examined in the presence of nigral and cortical inputs using cortex-striatum-substantia nigra organotypic cultures grown for 45 +/- 4 d. The nigrostriatal projection was confirmed by tyrosine hydroxylase immunoreactivity. Corticostriatal (CS) projection neurons, striatal MS neurons, and FS neurons were intracellularly recorded and morphologically and electrophysiologically characterized. Intracellular spontaneous activity in the cultures consisted of intermittent depolarized periods of 0.5-1 sec duration. Spontaneous depolarizations in MS neurons were restricted to a narrow membrane potential range (up state) during which they occasionally fired single spikes. These up states were completely blocked by the glutamate antagonist CNQX. In FS interneurons, depolarized periods were characterized by large membrane potential fluctuations that occupied a wide range between rest and spike threshold. Also, FS interneurons spontaneously fired at much higher rates than did MS neurons. Simultaneous intracellular recordings established that during spontaneous depolarizations MS neurons and FS interneurons displayed correlated subthreshold neuronal activity in the low frequency range. These results indicate that (1) the CS projection neurons, striatal MS neurons, and FS interneurons grown in cortex-striatum-substantia nigra organotypic cultures show morphological and electrophysiological characteristics similar to those seen in vivo; (2) striatal MS neurons but not FS interneurons show an up state; (3) striatal MS neurons and FS interneurons receive common, presumably cortical inputs in the low frequency range. Our results support the view that the cortex provides a feedforward inhibition of MS neuron activity during the up state via FS interneurons.

Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat.

Neurons in the substantia nigra pars reticulata and pars compacta of the rat were studied using a combination of intracellular electrophysiological recording in in vitro and subsequent immunocytochemical double and triple labelling techniques. The neurons recorded in the pars reticulata were identified as either GABA or dopamine neurons: neurons were considered to be GABA neurons if they were immunopositive for glutamate decarboxylase, whereas those neurons which were immunopositive for tyrosine hydroxylase were considered to be dopaminergic. The GABA neurons had short duration action potentials (0.45+/-0.03 ms halfwidth), no apparent rectifying currents, no low threshold calcium spikes, were spontaneously active (7.4+/-3.7 Hz), and could maintain high firing rates. The dopamine neurons had long duration action potentials (1.49+/-0.10 ms), displayed both anomalous inward and transient outward rectifying currents, and more than half (12/17 neurons) displayed a low threshold calcium spike. Their spontaneous firing rate was lower than that of the GABA neurons (2.3+/-1.0 Hz), and they displayed strong frequency adaptation. Morphological reconstruction of neurobiotin-filled neurons revealed that the pars reticulata GABA neurons had more extensive local dendritic arborization than the dopamine neurons from either the pars reticulata or the pars compacta. All of the neurons recorded from the pars compacta were dopamine neurons; they were found not to be different either electrophysiologically or morphologically from pars reticulata dopamine neurons. The electrophysiology of the GABA neurons suggests that input activity is translated linearly to spike frequency. These GABA neurons probably represent the projection neurons of the pars reticulata, and it is thus likely that this basal ganglia output is frequency coded. The close similarity between the dopamine neurons in the pars compacta, which give rise to the nigrostriatal pathway, and those in the pars reticulata supports the notion that the dopamine neurons in these two regions are part of the same neuronal population.

Two types of cholinergic neurons in the rat tegmental pedunculopontine nucleus: electrophysiological and morphological characterization.

Two types of tegmental pedunculopontine nucleus neurons have been reported previously based on their electrophysiological characteristics: type I neurons were characterized by low-threshold Ca spikes and type II neurons displayed a transient outward current. This report describes the membrane properties, synaptic inputs, morphologies and axonal projections of two subgroups of type II neurons examined in an in vitro slice preparation. Type II neurons were divided into two groups based on their spike durations: short-duration neurons with an action potential duration of 0.7-1.5 ms and long-duration neurons with an action potential duration of 1.6- 2.9 ms. Choline acetyltransferase immunohistochemistry combined with biocytin labeling indicated that 56% of short-duration neurons and 61% of long-duration neurons were immunopositive for choline acetyltransferase. Short-duration neurons had a high input resistance and the capacity to discharge with high frequency. By contrast, long-duration neurons had a low input resistance and low firing frequency and upon current injection displayed an accommodation (spike-frequency adaptation) before reaching a steady firing frequency. Microstimulation of the substantia nigra pars compacta evoked antidromic responses in both short-duration neurons (n=5/14, 36%) and long-duration neurons (n=20/39. 51%). Stimulations of the subthalamic nucleus and the substantia nigra pars reticulata induced in these neurons excitatory and inhibitory postsynaptic potentials, respectively. Short-duration neurons were dispersed equally throughout the extent of the tegmental pedunculopontine nucleus area, while long-duration neurons were located more in the rostral tegmental pedunculopontine nucleus. Short-duration neurons were small with two to four thin primary dendrites. Long-duration neurons were medium to large with three to six thick primary dendrites. Cell size was positively correlated with spike duration and axonal conduction velocity, but negatively with input resistance and spontaneous firing frequency. Both groups of neurons had ascending (toward thalamus, pretectal areas and tectum) and descending (toward pontomedullary reticular formation) axons in addition to nigropetal axons. Ascending axons were observed in 75% (6/8) of short-duration neurons and in 45% (15/33) of long-duration neurons, while nigropetal axons were observed in 50% (4/8) of short-duration neurons and in 76% (25/33) of long-duration neurons. These results suggest that the tegmental pedunculopontine nucleus cholinergic projection system is composed of heterogeneous populations of neurons in terms of electrophysiological and morphological characteristics as well as their distribution patterns in the nucleus.

Ionic mechanisms involved in the spontaneous firing of tegmental pedunculopontine nucleus neurons of the rat.

We have previously defined three types of tegmental pedunculopontine nuclei neurons based on their electrophysiological characteristics: Type I neurons characterized by low-threshold Ca2+ spikes, Type II neurons which displayed a transient outward current (A-current), and Type III neurons having neither low-threshold spikes nor A-current [Kang Y. and Kitai S. T. (1990) Brain Res. 535, 79-95]. In this report, ionic mechanisms underlying repetitive firing of Type I (n=15) and Type II (n=69) neurons were studied in in vitro slice preparations. Type I neurons did not fire rhythmically but their spontaneous firing frequency ranged from 0 to 19.5 spikes/s (mean 9.7 spikes/s). The spontaneous firing of Type II neurons was rhythmic, with a mean frequency of 9.6 spikes/s (range 3.5-16.0 spikes/s). Choline acetyltransferase immunohistochemistry combined with biocytin labeling indicated that none of the Type I neurons were immunopositive to choline acetyltransferase, while 60% (42 of 69) of Type II neurons were immunopositive. There was no apparent difference in the electrophysiological membrane properties of immunopositive and immunonegative Type II neurons. At membrane potentials subthreshold for Na+ spikes (-50 mV), spontaneous membrane oscillations (11.6 Hz) were observed: these underlie the spontaneous repetitive firing of Type I neurons. The subthreshold membrane oscillation was tetrodotoxin sensitive but was not affected by Ca2+-free medium. A similar tetrodotoxin-sensitive subthreshold membrane oscillation (10.5 Hz) was also observed in Type II neurons. However, in Type II neurons a membrane oscillation was also observed at higher membrane potentials (-50 mV). This high-threshold oscillation was insensitive to tetrodotoxin and Na+-free medium, but was eliminated in Ca2+-free conditions. The amplitude and frequency of the high-threshold oscillation was increased upon membrane depolarization. At the most prominent oscillatory level (around -40 mV), the high-threshold oscillation had a mean frequency of 8.8 Hz. The high-threshold Ca2+ spike was triggered from the peak potential (-35 to -30mV) of the high-threshold oscillation. Application of tetraethylammonium chloride (< 5 mM) increased the amplitude of the high-threshold oscillation, while nifedipine greatly attenuated the high-threshold oscillation without changing the shape of the high-threshold Ca2+ spike. Application of Cd2+ eliminated both the high-threshold oscillation and the high-threshold Ca2+ spike, and omega-conotoxin reduced the size of the high-threshold Ca2+ spike without affecting the frequency of the high-threshold oscillation. Nickel did not have any effect on either the high-threshold oscillation or the high-threshold Ca2+ spike. These data suggest an involvement of N- and L-type Ca2+ channels in the generation of the high-threshold oscillation and the high-threshold Ca2+ spike, respectively. The results indicate that a persistent Na+ conductance plays a crucial role in the subthreshold membrane oscillation, which underlies spontaneous repetitive firing in Type I neurons. On the other hand, in addition to a persistent Na+ conductance for subthreshold membrane oscillation, a voltage-dependent Ca2+ conductance with Ca2+-dependent K+ conductance (for the high-threshold oscillation) may be responsible for rhythmic firing of Type II neurons.

State-dependent regulation of neuronal excitability by dopamine.

Since the discovery that the loss of the dopaminergic innervation of the striatum resulted in Parkinson's disease, physiologists have attempted to understand the role of dopamine on striatal activity. Hypotheses relying upon concepts derived from studies of fast synaptic transmission have consistently failed to explain the actions of dopamine or other receptors coupled to G-proteins which modulate the properties of voltage-dependent ionic conductances responsible for synaptic integration and spike activity. Recently, patch clamp studies have revealed that in medium spiny striatal neurons dopamine D1-class receptors modulate voltage-dependent Na+, K+ and Ca2+ channels. From a consideration of the biophysical properties of these channels and the state transitions that medium spiny neurons undergo while responding to cortical input, a novel picture of dopamine's actions is beginning to emerge. Our results and those of others suggest that D2-class receptors serve to make the transition to the depolarized 'upstate' from the hyperpolarized 'downstate' more probable in response to cortical input. But, once the transition has occurred, the alteration in excitability should be short-lived unless the neuron has recently been active. This state-dependent modulation provides a mechanism by which dopamine could shape global striatal activity governing the execution of motor behaviors.

Generation of high-frequency oscillations in local circuits of rat somatosensory cortex cultures.

1. Rhythmic cortical activity was investigated with intracellular recordings in cortex-striatum-mesencephalon organotypic cultures grown for 42 +/- 3 (SE) days in vitro. 2. Electrical stimulation of supragranular layers induced a self-sustained high-frequency oscillation (HFO) in pyramidal neurons and interneurons. 3. The HFO started 197 +/- 39 ms after stimulation and had a mean duration of 1.0 +/- 0.2 s and an initial frequency of 38 +/- 2 Hz. A decrease in frequency at a rate of 11.5 +/- 2.7 Hz/s started on average 547 +/- 109 ms after the onset of the HFO. 4. During the HFO, local interneurons and pyramidal neurons synchronized their activities. The synaptic origin of the HFO was confirmed by its reversal potential at -57 +/- 4 mV. 5. These results suggest that a self-maintained HFO can be induced in local cortical circuits by excitation of supragranular layers. This HFO would facilitate synchronization between distant cortical and thalamic regions.