Francesca Stratta

Effects of dihydropyridine calcium antagonists on rat midbrain dopaminergic neurones

1 The effects of the dihydropyridine calcium channel antagonists, nifedipine and nimodipine (300 nm‐30 μm) were tested in vitro on intracellularly recorded dopaminergic neurones in the rat ventral mesencephalon. 2 Bath applied nifedipine and nimodipine inhibited in a concentration‐dependent manner the spontaneous firing discharge of the action potentials, whereas, the dihydropyridine calcium channel agonist, Bay K 8644 increased the firing rate. 3 Pacemaker oscillations and bursts of action potentials were produced by loading the cells with caesium. Nifedipine and nimodipine reduced the rate and the duration of the caesium‐induced membrane oscillations and decreased the number of action potentials in a burst. During the blockade of potassium currents the dopaminergic neurones often developed a prolonged (100–800 ms) afterdepolarization that was also inhibited by dihydropyridines. 4 The spontaneous discharge of calcium spikes was also inhibited by both dihydropyridine calcium antagonists. The apparent input resistance and the level of membrane potential were not affected by the dihydropyridine calcium antagonists. 5 If the action potential duration was less than 150 ms the shape of the spike was not clearly influenced by both calcium antagonists. However, when the duration of the action potential was longer than 150–200 ms due to the intracellular injection of caesium ions plus the extracellular application of tetraethylammonium (10–50 μm), both nifedipine and nimodipine reversibly shortened the plateau potential. 6 It is suggested that nifedipine and nimodipine depress the rhythmic and bursting activity of the dopaminergic cells and shorten the calcium action potential by blocking dihydropyridine‐sensitive high‐threshold calcium currents.

Activation of metabotropic glutamate receptors induces an inward current in rat dopamine mesencephalic neurons.

To investigate the electrophysiological effects of the stimulation of the metabotropic excitatory amino acid receptors, we applied trans-1-amino-cyclopentane-1,3-dicarboxylate, an agonist of this type of receptors, on presumed rat dopamine cells intracellularly recorded in vitro. Trans-1-amino-cyclopentane-1,3-dicarboxylate (3-30 microM, t-ACPD) caused a sustained increase of the spontaneous firing rate and a depolarization. When the membrane potential was held at about the resting level (-50, -60 mV), by the single-electrode voltage-clamp technique, t-ACPD induced an inward current. In 57% of the tested cells the inward current was associated with a decrease of the apparent input conductance. In the remaining cells no obvious changes in membrane conductance were observed. The active form of t-ACPD, (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylate [3-50 microM, (1S,3R)-ACPD] also produced a reversible inward current on the dopaminergic cells and this was antagonized by (S)-4-carboxy-3-hydroxyphenylglycine (300 microM), a selective antagonist of the (1S,3R)-ACPD-induced depolarization on central neurons. The (1S,3R)-ACPD-induced inward current was not antagonized by L-2-amino-3-phosphonopropionic acid (100 microM), an antagonist of the t-ACPD-induced activation of inositide synthesis. 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), an alfa-amino-3-hydroxy-5- methyl-isoxazole propionic acid/kainate antagonist, DL-amino-5-phosphonopentanoic acid (30 microM), an N-methyl-D-aspartate antagonist, and scopolamine (10 microM), a muscarinic antagonist, did not significantly affect the actions of t-ACPD. A block of synaptic transmission obtained by applying tetrodotoxin failed to prevent the action of t-ACPD.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotensin induces an inward current in rat mesencephalic dopaminergic neurons

Neurotensin (0.3-3 microM) depolarized the membrane and increased the firing discharge of dopaminergic cells in slices of the rat mesencephalon. Under voltage-clamp, at holding potentials from -50 to -60 mV (near the resting membrane potential), neurotensin produced a sustained inward shift in the holding current. This inward current was reduced with the hyperpolarization of the membrane to -125 mV. It was resistant to tetrodotoxin, but it was diminished following the perfusion with low sodium (choline chloride substitution) solution. It persisted in low calcium (0-0.5 mM). Changes in the intracellular concentration of chloride did not affect neurotensin-induced current. The neurotensin-induced inward current did not reverse at hyperpolarized potentials in 10.5 mM extracellular K+. It was also seen in the presence of the potassium channel blockers tetraethylammonium (10-20 mM), barium (1 mM), apamine (1 microM) and 4-aminopyridine (1-1.5 mM). Also the extracellular application of cesium (1-5 mM) had no effect on the cellular responsiveness to neurotensin. The action of neurotensin appears to be mediated, at least partially, by a TTX-insensitive but voltage-dependent inward current carried by sodium. The non-dopaminergic cells of the substantia nigra and ventral tegmental area were not affected by neurotensin.

A voltage-clamp analysis of NMDA-induced responses on dopaminergic neurons of the rat subtantia nigra zona compacta and ventral tegmental area

The effects of NMDA-receptor activation on dopaminergic neurons of the rat substantia nigra zona compacta and ventral tegmental area were studied by using in vitro intracellular electrophysiological recordings (current and voltage-clamp). NMDA depolarized the membrane and increased the firing activity. A voltage-dependent inward current and a reduction of the apparent input conductance were observed in voltage-clamp experiments. Interestingly, the peak amplitude of the inward current occurred at approximately -60 mV. The NMDA-induced responses were reduced by the application of DL-2-amino-5-phosphonovaleric acid (APV). The NMDA-induced current was unaffected by potassium channel blockers, was present in low-sodium solutions or in solutions treated with TTX; but was reduced or blocked in low-calcium solutions containing cobalt. In addition, no reduction of the apparent input conductance was observed either in the solutions without magnesium or in those with low-sodium. Our data indicate that the activation of NMDA receptors produces a powerful excitatory stimulus on the dopaminergic neurons of the ventral mesencephalon and this may be primarily the result of a voltage-dependent influx of calcium ions. The degeneration of the dopaminergic cells after application of neurotoxins may be explained by their peculiar response to NMDA.

Responses of rat mesencephalic dopaminergic neurons to a prolonged period of oxygen deprivation.

We employed intracellular electrophysiological techniques to examine the effects of a prolonged anoxia (more than 7 min superfusion with artificial cerebrospinal fluid saturated with 95% N2-5% O2) on dopaminergic neurons of the rat ventral mesencephalon maintained in vitro. A prolonged anoxia caused an inhibition of the spontaneous firing and a sustained (mean 16 min) and slowing declining hyperpolarization of the membrane in 30 dopaminergic cells. This was associated with a decrease of the apparent input resistance at 5, 10, 15 and 20 min of O2 deprivation by 38% (n = 18), 42% (n = 8), 48% (n = 18) and 54% (n = 8) of control, respectively. The continuation of anoxia, 1-4 min after the hyperpolarizing period, induced an irreversible depolarization (n = 8). More than 50% of the cells (17 of 30) fully recovered their electrophysiological properties after 15 min of O2 deprivation. Since the intracellular diffusion of cesium (a potassium channel blocker) was able to block the hyperpolarization and to reveal a depolarization caused by anoxia, we tested whether the blockade of the hyperpolarization modified the resistance of the cells to O2 deprivation. We observed that the cells loaded with cesium were depolarized and damaged in a period of O2 deprivation less than 10 min. The apparent input resistance of these neurons was irreversibly reduced by 36% of the control at 5 min of anoxia (n = 6). Furthermore, in order to ascertain whether an impairment of the sodium/potassium pump due to energy failure is involved in the anoxia-induced depolarization, we blocked the Na+/K+ ATPase pump with the inhibitor ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Nomifensine but not amantadine increases dopamine-induced responses on rat substantia nigra zona compacta neurons

Responses of substantia nigra zona compacta neurons to nomifensine and amantadine were studied with intracellular recording techniques (current and voltage clamp) in in vitro slice preparation of rat mesencephalon. The application of nomifensine (1-10 microM) slightly hyperpolarized the cells and inhibited action potential discharge that occurs spontaneously. In voltage-clamp experiments (-50, -60 mV, holding potential) an outward current was observed. The membrane responses to exogenously-applied dopamine were potentiated by the concomitant superfusion of nomifensine. The effects of nomifensine were antagonized by (-)-sulpiride (1 microM), a D2 receptor antagonist. By contrast, the superfusion of amantadine (1-30 microM) on substantia nigra zona compacta cells was ineffective on firing rate, membrane potential or on sensitivity to exogenous dopamine. In the presence of high doses (300 microM to 1 mM) of amantadine a depolarization and an increase in firing activity was observed. While our results provide electrophysiological evidence for an inhibition of the dopamine uptake system by nomifensine, they do not support a dopaminergic mechanism for the actions of amantadine in the substantia nigra zona compacta.

Electrophysiological effects of amineptine on neurones of the rat substantia nigra pars compacta: evidence for an inhibition of the dopamine uptake system

1 Intracellular recordings were made from substantia nigra pars compacta neurones of the rat maintained in vitro in order to study the effects of the tricyclic antidepressant drug, amineptine. 2 Amineptine hydrochloride (1–30 μm) decreased spontaneous firing and slightly hyperpolarized the membrane potential. In neurones voltage‐clamped at − 50 or − 60 mV, amineptine produced an outward membrane current. These actions were concentration‐dependent and were completely antagonized by (−)‐sulpiride. 3 The amineptine‐induced hyperpolarization was resistant to tetrodotoxin (1 μm) but it was abolished in 0 mm Ca2+ (plus 13 mm MgCl2) solutions. 4 Amineptine (300 nm‐30 μm) and cocaine (10–30 μm) increased the amplitude and duration of responses to exogenously applied dopamine. The effects of dopamine were potentiated about 5 fold by 10 μm amineptine; this potentiation persisted in calcium‐free solutions. 5 Cocaine (10 μm) had no additional effect on the dopamine‐induced responses in the presence of amineptine (30 μm). Amineptine (10 μm) produced no detectable effects in the presence of cocaine (30 μm). 6 It is concluded that amineptine acts as a dopamine uptake blocker in slices of rat substantia nigra.

Electrophysiology of dopamine D-1 receptors in the basal ganglia: Old facts and new perspectives

1. The dopamine (DA) D1-receptor family is highly represented in the mammalian brain and particularly in the nigrostriatal system, whose integrity is crucial for the execution of motor performances. 2. In the last decade, our understanding of the electrophysiology of D1 receptors on caudate-putamen neurons has greatly improved. The effects of the activation of striatal D1 receptors were studied by extracellular single unit recordings in the intact animal as well as by intracellular recordings in rat brain slice preparation. More recently, whole-cell recordings on isolated striatal neurons have further addressed this issue and confirmed the inhibitory modulatory role of D1 receptor on the electrical activity of striatal neurons. 3. Several important questions, however, concerning the functional effects of D1 receptor activation in the basal ganglia are still debated: the cellular segregation of the distribution of D1-D2-like receptors, their synergistic or opposite functional roles at the second messenger level, the effects of D1 receptor activation on the transmitter release and the modifications of D1 receptor pharmacology in dopamine-denervated striata. 4. A different perspective will also be discussed: the involvement of D1 receptors in long-term changes of synaptic efficacy in the striatum as a possible correlate of motor learning.

Basic Electrophysiology and Possible New Therapeutic Approaches to Movement Disorders

What the basal ganglia do, is it the on-going question? New models have reevaluated the input/output ratio of single structures as inserted in parallel, functional systems (Alexander and Crutcher, 1990). These models have reinforced the assumption that the basal ganglia are a key station for the execution of organized movements (DeLong, 1990; Goldman-Rakic and Selemon, 1990). At the molecular level, new families of receptors are explored. The cloning of glutamate metabotropic receptors is heading the surprising multiplicity of the neurobiology of excitatory transmission (Gasic, 1992). The definition of new subclasses of dopamine receptors is an invitation to reconsider the pharmacology of the amine (Surmeier et al., 1992). Radical changes, however, in the therapy of movement disorders have barely taken place, being the introduction of levo-dopa still a “cornerstone” of the therapy of the parkinsonian patient (Hornykiewicz, 1966). Whatever are the fundamental functions of the basal ganglia, a striking dichotomy risks to develop between basic research acquisition and the daily urgency of patient’s quality of life. In presenting our recent findings, we aim to highlight those aspects of mesencephalic, neostriatal and pallidal physiology whose clinical impact could be relevant.