Amphetamine distorts stimulation-dependent dopamine overflow: effects on D2 autoreceptors, transporters, and synaptic vesicle stores.

Amphetamine (AMPH) is known to raise extracellular dopamine (DA) levels by inducing stimulation-independent DA efflux via reverse transport through the DA transporter and by inhibiting DA re-uptake. In contrast, recent studies indicate that AMPH decreases stimulation-dependent vesicular DA release. One candidate mechanism for this effect is the AMPH-mediated redistribution of DA from vesicles to the cytosol. In addition, the inhibition of stimulation-dependent release may occur because of D2 autoreceptor activation by DA that is released via reverse transport. We used the D2 receptor antagonist sulpiride and mice lacking the D2 receptor to address this issue. To evaluate carefully AMPH effects on release and uptake, we recorded stimulated DA overflow in striatal slices by using continuous amperometry and cyclic voltammetry. Recordings were fit by a random walk simulation of DA diffusion, including uptake with Michaelis-Menten kinetics, that provided estimates of DA concentration and uptake parameters. AMPH (10 microm) promoted the overflow of synaptically released DA by decreasing the apparent affinity for DA uptake (K(m) increase from 0.8 to 32 microm). The amount of DA released per pulse, however, was decreased by 82%. This release inhibition was prevented partly by superfusion with sulpiride (47% inhibition) and was reduced in D2 mutant mice (23% inhibition). When D2 autoreceptor activation was minimal, the combined effects of AMPH on DA release and uptake resulted in an enhanced overflow of exocytically released DA. Such enhancement of stimulation-dependent DA overflow may occur under conditions of low D2 receptor activity or expression, for example as a result of AMPH sensitization.

Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system.

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson's disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.

Caffeine-sensitive calcium stores regulate synaptic transmission from retinal rod photoreceptors.

We investigated the role of caffeine-sensitive intracellular stores in regulating intracellular calcium ([Ca(2+)](i)) and glutamatergic synaptic transmission from rod photoreceptors. Caffeine transiently elevated and then markedly depressed [Ca(2+)](i) to below prestimulus levels in rod inner segments and synaptic terminals. Concomitant with the depression was a reduction of glutamate release and a hyperpolarization of horizontal cells, neurons postsynaptic to rods. Caffeine did not affect the rods' membrane potentials indicating that caffeine likely acted via some mechanism(s) other than a voltage-dependent deactivation of the calcium channels. Most of caffeine's depressive action on [Ca(2+)](i), on glutamate release, and on I(Ca) in rods can be attributed to calcium release from stores: (1) caffeine's actions on [Ca(2+)](i) and I(Ca) were reduced by intracellular BAPTA and barium substitution for calcium, (2) other nonxanthine store-releasing compounds, such as thymol and chlorocresol, also depressed [Ca(2+)](i), and (3) the magnitude of [Ca(2+)](i) depression depended on basal [Ca(2+)](i) before caffeine. We propose that caffeine-released calcium reduces I(Ca) in rods by an as yet unidentified intracellular signaling mechanism. To account for the depression of [Ca(2+)](i) below rest levels and the increased fall rate of [Ca(2+)](i) with higher basal calcium, we also propose that caffeine-evoked calcium release from stores activates a calcium transporter that, via sequestration into stores or extrusion, lowers [Ca(2+)](i) and suppresses glutamate release. The effects of store-released calcium reported here operate at physiological calcium concentrations, supporting a role in regulating synaptic signaling in vivo.

D2-Like dopamine autoreceptor activation reduces quantal size in PC12 cells.

D2-like dopamine autoreceptors regulate dopamine release and are implicated in important actions of antipsychotic drugs and rewarding behaviors. To directly observe the effects of D2 autoreceptors on exocytic neurotransmitter release, we measured quantal release of dopamine from pheochromocytoma PC12 cells that express D2 and D4 autoreceptors. High potassium-evoked secretion in PC12 cells produced a unimodal population of quantal sizes. We found that exposures to the D2-like agonist quinpirole that inhibited tyrosine hydroxylase activity by approximately 50% also reduced quantal size by approximately 50%. The reduced quantal size was blocked by the D2 antagonist sulpiride and reversed by L-DOPA. Quinpirole also decreased the frequency of stimulation-evoked quantal release. Together, these findings indicate effects on quantal neurotransmission by D2-like dopamine autoreceptors previously distinguished as synthesis-modulating autoreceptors that regulate tyrosine hydroxylase activity versus impulse-regulating autoreceptors that modulate membrane potential. The results also provide an initial demonstration of a receptor-mediated mechanism that alters quantal size.

Gain of rod to horizontal cell synaptic transfer: relation to glutamate release and a dihydropyridine-sensitive calcium current.

We related rod to horizontal cell synaptic transfer to glutamate release by rods. Simultaneous intracellular records were obtained from dark-adapted rod-horizontal cell pairs. Steady-state synaptic gain (defined as the ratio of horizontal cell voltage to rod voltage evoked by the same light stimulus) was 3.35 +/- 0.60 for dim flashes and 1.50 +/- 0.03 for bright flashes. Under conditions of maintained illumination, there was a measurable increment of horizontal cell hyperpolarization for each light-induced increment of rod hyperpolarization over the full range of rod voltages. In separate experiments we studied glutamate release from an intact, light-responsive photoreceptor layer, from which inner retinal layers were removed. Steady light reduced glutamate release as a monotonic function of intensity; spectral sensitivity measures indicated that we monitored glutamate release from rods. The dependence of glutamate release on rod voltage was well fit by the activation function for a high-voltage-activated, dihydropyridine-sensitive L-type calcium current, suggesting a linear dependence of glutamate release on [Ca]i in the synaptic terminal. A simple model incorporating this assumption accounts for the steady-state gain of the rod to horizontal cell synapse.

Dependence of photoreceptor glutamate release on a dihydropyridine-sensitive calcium channel.

A "reduced retina" preparation, consisting of the photoreceptor layer attached to the pigment epithelium in the eyecup, was used to study the pharmacology of the calcium channels controlling glutamate release by photoreceptors in Xenopus. Glutamate release was evoked either by dark adaptation or by superfusion with elevated (20 mM) potassium medium. Both darkness- and potassium-induced release were blocked by cadmium (200 microM). The N-type calcium channel blocker, omega-conotoxin GVIA (500 nM), the P-type calcium channel blocker, omega-agatoxin IVA (20 nM), and the P- and Q-type channel blocker omega-conotoxin MVIIC (1 microM) had no effect on glutamate release. In contrast, the dihydropyridines, nifedipine (10 microM) and nitrendipine (10 microM), which affect L-type calcium channels, blocked both darkness- and potassium-induced release. Bay K 8644 (10 microM), which promotes the open state of L-type calcium channels, enhanced glutamate release. These results indicate that photoreceptor glutamate release is controlled mainly by dihydropyridine-sensitive calcium channels. A dependence of glutamate release on L-type calcium channels also has been reported for depolarizing bipolar cells of a fish retina. Thus, it appears that non-inactivating L-type calcium channels are appropriate to mediate transmitter release in neurons whose physiological responses are sustained, graded potentials.

Projection from the cerebellar lateral nucleus to precerebellar nuclei in the mossy fiber pathway is glutamatergic: a study combining anterograde tracing with immunogold labeling in the rat.

The pontine nuclei (PN) and the nucleus reticularis tegmenti pontis (NRTP) are sources of an excitatory projection to the cerebellar cortex via mossy fibers and a direct excitatory projection to the cerebellar nuclei. These precerebellar nuclei, in turn, receive a feedback projection from the cerebellar nuclei, which mostly originate in the lateral nucleus (LN). It has been suggested that the feedback projection from the LN partially uses gamma-aminobutyric acid (GABA) as a transmitter. We tested this hypothesis by using a combination of anterograde tracing (biotinylated dextran amine injection into the LN) and postembedding GABA and glutamate immunogold histochemistry. The pattern of labeling in the PN and the NRTP was compared with that of cerebellonuclear terminals in two other target structures, the parvocellular part of the nucleus ruber (RNp) and the ventromedial and ventrolateral thalamus (VM/VL). The projection to the inferior olive (IO), which is known to be predominantly GABAergic, served as a control. A quantitative analysis of the synaptic terminals labeled by the tracer within the PN, the NRTP, and the VL/VM revealed no GABA immunoreactivity. Only one clearly labeled terminal was found in the RNp. In contrast, 72% of the terminals in the IO were clearly GABA immunoreactive, confirming the reliability of our staining protocol. Correspondingly, glutamate immunohistochemistry labeled the majority of the cerebellonuclear terminals in the PN (88%), the NRTP (90%), the RNp (93%), and the VM/VL (63%) but labeled only 5% in the IO. These data do not support a role for GABAergic inhibition either in the feedback systems from the LN to the PN and the NRTP or within the projections to the RNp and the VM/VL.

Glutamate release by the intact light-responsive photoreceptor layer of the Xenopus retina.

In order to study glutamate release from light responsive photoreceptors, we used an eyecup preparation treated with detergent and distilled water, which permitted removal of the inner retina. The remaining 'reduced' retina consists mainly of photoreceptors attached to the pigment epithelium. The viability of the preparation was established by exclusion of trypan blue, light and electron microscopic examination of the photoreceptor layer and by intracellular recordings from rods. The 'reduced' retina was superfused at 1 ml/h and overflow samples were analyzed for their glutamate content by a fluorimetric enzyme assay. We tested the response to dark and light adaptation and to treatment with 100 microM CdCl2. We found a baseline glutamate level in light-adapted preparation which was not affected by cadmium. Dark adaptation induced a 2-fold increase of glutamate release, which was completely blocked by cadmium.

Evidence for calcium/calmodulin dependence of spinule retraction in retinal horizontal cells.

Horizontal cells of the carp retina alter their synaptic connections with cones during dark and light adaptation. At light onset, dendrites of horizontal cells, which are positioned laterally at the ribbon synapse, form "spinules," little processes with membrane densities. Spinules are retracted again during dark adaptation. Spinule retraction is also elicited upon glutamate application to the retina. In the present study, we address the question whether calcium/calmodulin-dependent pathways are involved in dark- and glutamate-evoked spinule retraction. Light-adapted retinas were isolated and subsequently dark adapted during incubation in media of different calcium concentrations. Spinule retraction was clearly blocked in low-calcium solutions (5 microM and 50 nM CaCl2). Incubation in medium containing cobalt chloride (2 mM) had the same effect. Both treatments blocked the glutamate-induced spinule retraction as well. These results indicate that spinule retraction is induced by a calcium influx into horizontal cells. To investigate whether calmodulin, the primary calcium receptor in eukaryotic cells, is present at the site of spinule formation, light- and dark-adapted retinas, embedded in LR White resin, were labelled with an antibody against calmodulin and gold-conjugated secondary antibodies. Horizontal cell dendrites at the ribbon synapse revealed strong calmodulin immunoreactivity, which was more than twice as high in light- as in dark-adapted retinas. The incubation of isolated retinas with the calmodulin antagonists W5 and W13 inhibited spinule retraction. In summary, these results suggest that spinule retraction may be regulated by calcium influx into horizontal cells and subsequent calcium/calmodulin-dependent pathways.

Spinule formulation in the fish retina: is there an involvement of actin and tubulin? An electronmicroscopic immunogold study.

During light-adaptation dendrites of teleost horizontal cells form finger-like processes, called 'spinules', which are characterized by synaptic membrane densities. To investigate the involvement of cytoskeletal elements in the formation and retraction of spinules, effects of the microtubule and actin inhibitors colchicine and cytochalasin D were examined by injection into the vitreous. Both substances inhibited the light-induced spinule formation. The ultrastructural immunolocalization of tubulin revealed labelling of dendrites only in their proximal parts. The distal parts of dendrites which invaginate into cone pedicles were free of label. Treatment with anti-actin revealed immunoreactivity along the entire length of dendrites up to the dendritic terminals. The spinules, however, showed no labelling. This finding does not support the hypothesis that spinules are protruded by actin polymerization. After cytochalasin D treatment the density of label in the dendritic terminals was enhanced by a factor of three, which suggests an accumulation of actin. Thus, spinule inhibition by cytochalasin D is probably caused by distortion of a functional actin network in the dendritic terminals.

Gap junction morphology of retinal horizontal cells is sensitive to pH alterations in vitro.

Isolated goldfish retinae were incubated in NaHCO3-reduced solutions, a treatment known to lower intracellular pH and to decrease gap-junction-mediated coupling between cells. The morphology of the gap junctions of horizontal cells examined by means of freeze-fracture replicas and ultrathin sections displays alterations after such treatment. The gap-junctional particles aggregate into dense clusters or crystalline arrays, whereas controls (pH 7.5) display a loose arrangement of particles. Incubation in NaHCO3-reduced solution leads to the appearance, in ultrathin sections, of prominent, electron-dense material beneath the gap-junctional membranes. Both effects, the increasing density of particles and the appearance of electron-dense material, are reversible. The application of dopamine, which uncouples horizontal cells, and its antagonist haloperidol produce less clear-cut effects on particle density in vitro.