Enhancement of [3H]dopamine release and its [3H]metabolites in rat striatum by nicotinic drugs

Recent evidence has identified directly muscarinic acetylcholine receptor (m-ACh R) and nicotinic acetylcholine receptor (n-ACh R) in the brain utilizing receptor binding assay. Several studies suggest that release of dopamine (DA) in the striatum is regulated by presynaptic receptors present on dopaminergic terminals. In the present study, the effects of cholinergic drugs on [3H]DA release were examined using micropunched tissue and synaptosomes obtained from rat striatum. ACh (5 x 10(-4) M) significantly increased spontaneous [3H]DA release, and the overflow was partially inhibited by D-tubocurarine (1 mM) but not atropine. Nicotine, lobeline, coniine and spartein, nicotinic agonists, significantly increased spontaneous and 25 mM K + evoked [3H]DA release almost in a dose-dependent manner. In contrast, oxotremorine (2 x 10(-4) M), muscarinic agonist, did not any change in [3H]DA efflux. Furthermore, the metabolites of [3H]DA were separated by column chromatography. The main metabolite of [3H]DA in the spontaneous release from rat striatal synaptosomes was [3H]DOPAC (3,4-dihydroxyphenylacetic acid). Lobeline (5 x 10(-5) M) accelerated the outflow of [3H]DOPAC and [3H]OMDA metabolites (O-methylated and deaminated metabolites). These results could give rise to the suggestion that there was n-ACh R on the dopaminergic nerve terminals in the striatum and n-ACh R might have related to a directly excitatory effect on the DA release.     

Desmethylimipramine potentiates NMDA responses in a mouse cortical slice preparation.

Desmethylimipramine (DMI) has been shown to interact with the N-methyl-D-aspartate (NMDA) receptor complex. Its probable action is through blockade of the cationic channel at the phencyclidine site and as a result it has potential anticonvulsant action. In this present study we have investigated the effects of DMI and ketamine on both NMDA-induced and spontaneous depolarizing shifts in cortical wedges prepared from genetically epilepsy-prone mice (DBA/2). Contrary to published reports, DMI potentiated the effects of NMDA and increased the frequency of spontaneous depolarizations. The actions of ketamine were inhibitory and these were reversed by DMI. Presynaptic mechanisms may be involved in the DMI-induced potentiation and this may explain the lowering of convulsive thresholds seen clinically with tricyclic antidepressants.     

Pyruvate protects against 3-nitropropionic acid neurotoxicity in corticostriatal slice cultures

Previously, we have shown that 3-nitropropionic acid (NPA) neurotoxicity in organotypic corticostriatal slice cultures is dependent on glucose and glutamate. Here we studied the neuroprotective potential of agents improving mitochondrial function, including creatine, malate, oxaloacetate, Pyruvate and L-lactate in NPA-treated slice cultures. pyruvate provided the best protection against the loss of glutamic acid decarboxylase activity, depletion of GABA levels, increased propidium iodide uptake and increased glial fibrillary acidic protein levels. ATP levels were significantly reduced by 100 microM NPA (but not by 50 microM) and restored by pyruvate (5 mM). Creatine and L-lactate had no significant protective effect. Protective mechanisms of pyruvate are probably multifold, including stimulation of the citric acid cycle, scavenging and reduction of excitotoxicity.     

Dopamine release from pharmacologically distinct storage pools in rat striatum following stimulation at frequency of neuronal bursting

The increase of dopamine overflow in the rat caudate and nucleus accumbens following repeated stimulation of the median forebrain bundle (MFB) at short intervals and the role of -methyl-p-tyrosine (AMPT) and reserpine sensitive storage pools in evoked dopamine overflow were investigated by in vivo voltammetry. In contrast to stimulation at 30-s intervals and longer, stimulation of the MFB at 5-s intervals led to increased dopamine release following each consecutive stimulation. This effect was much larger in the caudate than in the nucleus accumbens. We consider that the increase in dopamine release is due to translocation of dopamine from a reserpine-sensitive storage pool and this accounts for the increase of the rate of refilling of the readily releasable pool. The shorter the intervals between stimulation and the higher the frequency (with a plateau at 30 Hz), then the greater the fraction of dopamine which originates from the reserpine-sensitive storage pool. Exocytotic release of dopamine from the AMPT-sensitive storage pool does not seem to depend on the intervals between stimulation. We propose that the vesicles in presynaptic dopaminergic terminals near the outer membrane are more sensitive to AMPT. Distantly located vesicles have a relatively higher sensitivity to reserpine. Experiments using repeated stimulation at as low a frequency as 10–20 Hz, revealed that this phenomenon may take place under physiological conditions following bursting of dopamine neurones.     

Dopamine release in ventral striatum of pathological gamblers losing money

Linnet J, Peterson E, Doudet DJ, Gjedde A, Møller A. Dopamine release in ventral striatum of pathological gamblers losing money. Objective: To investigate dopaminergic neurotransmission in relation to monetary reward and punishment in pathological gambling. Pathological gamblers (PG) often continue gambling despite losses, known as ‘chasing one’s losses’. We therefore hypothesized that losing money would be associated with increased dopamine release in the ventral striatum of PG compared with healthy controls (HC). Method: We used Positron Emission Tomography (PET) with [¹¹C]raclopride to measure dopamine release in the ventral striatum of 16 PG and 15 HC playing the Iowa Gambling Task (IGT). Results: PG who lost money had significantly increased dopamine release in the left ventral striatum compared with HC. PG and HC who won money did not differ in dopamine release. Conclusion: Our findings suggest a dopaminergic basis of monetary losses in pathological gambling, which might explain loss‐chasing behavior. The findings may have implications for the understanding of dopamine dysfunctions and impaired decision‐making in pathological gambling and substance‐related addictions.     

Dopamine release in human ventral striatum and expectation of reward

Using the ability of [11C]raclopride to compete with dopamine for D(2)/D(3) receptors, we investigated by positron emission tomography the effect of placebo (saline) injection on dopamine release in the ventral striatum of patients with Parkinson's disease. We found evidence for placebo-induced dopamine release of similar magnitude to that reported in healthy volunteers after amphetamine administration. However, in contrast to the dorsal striatum, there were no differences in [11C]raclopride binding potential changes between patients who experienced the reward (those who reported placebo-induced clinical benefit) and those who did not. We conclude that the release of dopamine in the ventral striatum (nucleus accumbens) is related to the expectation of reward and not to the reward itself. These observations have potential implications for the treatment of drug addiction.     

Intracellular study of rat entopeduncular nucleus neurons in an in vitro slice preparation: response to subthalamic stimulation

Responses of rat entopeduncular nucleus (EP) neurons after stimulation of the subthalamic nucleus (STh) and the morphology of the EP neurons were studied using brain slice preparations. EP neurons were classified into two types based on their electrophysiological properties as reported previously. Of 87 EP neurons, 72 were Type I and the rest were Type II. Synaptic responses to STh stimulation were different in these two cell types. STh stimulation evoked excitatory postsynaptic potentials (EPSPs) followed by strong inhibitory postsynaptic potentials (IPSPs) in Type I neurons and EPSPs without strong IPSPs in Type II neurons. The EPSPs were considered to be monosynaptic because no large change in the latency (1.7 ± 0.5ms) resulted by alteration of stimulus intensity. The EPSPs were reversibly suppressed by kynurenic acid in a dose-dependent manner. Bath application of (+)-tubocurarine (10–50 μM) had no effect on EPSPs or IPSPs. Bath application of bicuculline methiodide (50–100 μM) markedly suppressed IPSPs evoked by STh stimulation and at the same time increased the amplitude and duration of EPSPs without affecting the latency. In the presence of bicuculline methiodide, EPSPs could induce plateau potentials and slow action potentials. Some Type I and Type II neurons were intracellularly labeled by biocytin. Type I neurons were located throughout the EP but Type II neurons were located mainly in the dorsal portion of the EP. Medium sized somata of both Type I and Type II neurons were spine-free and fusiform or round in shape. They had 3–4 thick primary dendrites with diameters of 2–5 μm that branched into thin secondary dendrites. The secondary and tertiary dendrites of Type I neurons were sparsely covered with spines. Dendritic terminals of some Type I neurons had complex arborizations with abundant spines and appendages. The dendrites of Type II neurons were generally smooth and had no complex arborizations at their terminals.     

3H]GBR-12935 binding to dopamine uptake sites in rat striatum

The binding of the selective dopamine uptake inhibitor [3H]GBR-12935 to rat striatum was studied. Competition by mazindol and dopamine against [3H]GBR-12935 binding revealed monophasic binding curves. The addition of 100 microM dopamine to the mazindol competition inhibited only 80% of the binding, indicating more than one [3H]GBR-12935 binding site in rat striatum. When a binding fraction that could be discriminated by 1 microM mazindol or 1 mM dopamine was defined as specific binding, a single site binding model was obtained. The [3H]GBR-12935 binding was of protein nature, since it was abolished after protease treatment. Drug inhibition studies with the addition of low concentrations of mazindol and dopamine resulted in alterations in apparent Kd values only, suggesting competitive inhibition by these compounds against [3H]GBR-12935 binding. It is concluded that the [3H]GBR-12935 binding to rat striatum discriminated by 1 microM mazindol reflects binding to the substrate recognition site for the dopamine uptake.     

Enhancement of [H]dopamine release and its [H]metabolites in rat striatum by nicotinic drugs

Recent evidence has identified directly muscarinic acetylcholine receptor (m-ACh R) and nicotinic acetylcholine receptor (n-ACh R) in the brain utilizing receptor binding assay. Several studies suggest that release of dopamine (DA) in the striatum is regulated by presynaptic receptors present on dopaminergic terminals. In the present study, the effects of cholinergic drugs on [³H]DA release were examined using micropunched tissue and synaptosomes obtained from rat striatum. ACh (5 × 10⁻⁴M) significantly increased spontaneous [³H]DA release, and the overflow was partially inhibited by d-tubocurarine (1 mM) but not atropine. Nicotine, lobeline, coniine and spartein, nicotinic agonists, significantly increased spontaneous and 25 mM K⁺ evoked [³H]DA release almost in a dose-dependent manner. In contrast, oxotremorine (2 × 10⁻⁴M), muscarinic agonist, did not any change in [³H]DA efflux. Furthermore, the metabolites of [³H]DA were separated by column chromatography. The main metabolite of [³H]DA in the spontaneous release from rat striatal synaptosomes was [³H]DOPAC (3,4-dihydroxyphenylacetic acid). Lobeline (5 × 10⁻⁵M) accelerated the outflow of [³H]DOPAC and [³H]OMDA metabolites (O-methylated and deaminated metabolites).These results could give rise to the suggestion that there was n-ACh R on the dopaminergic nerve terminals in the striatum and n-ACh R might have related to a directly excitatory effect on the DA release.     

D2 autoreceptor switches CB2 receptor effects on [3H]-dopamine release in the striatum

CB₂ receptors (CB₂R) are expressed in midbrain neurons. To evidence the control of dopamine release in dorsal striatum by CB₂R, we performed experiments of [³H]-dopamine release in dorsal striatal slices. We found a paradoxical increase in K⁺-induced [³H]-dopamine release by CB₂R activation with GW 833972A and JWH 133 two selective agonist. To understand the mechanism involved, we tested for a role of the D₂ autoreceptor in this effect; because in pallidal structures, the inhibitory effect of CB₁ receptors (CB₁R) on GABA release is switched to a stimulatory effect by D₂ receptors (D₂R). We found that the blockade of D₂ autoreceptors with sulpiride prevented the stimulatory effect of CB₂R activation; in fact, under this condition, CB₂R decreased dopamine release, indicating the role of the D₂ autoreceptor in the paradoxical increase. We also found that the effect occurs in nigrostriatal terminals, since lesions with 6-OH dopamine in the middle forebrain bundle prevented CB₂R effects on release. In addition, D₂–CB₂R interaction promoted cAMP accumulation, and the increase in [³H]-dopamine release was prevented by PKA blockade. D₂–CB₂R coprecipitation and proximity ligation assay studies indicated a close interaction of receptors that could participate in the observed effects. Finally, intrastriatal injection of CB₂R agonist induced contralateral turning in amphetamine-treated rats, which was prevented by sulpiride, indicating the role of the interaction in motor behavior. Thus, these data indicate that the D₂ autoreceptor switches, from inhibitory to stimulatory, the CB₂R effects on dopamine release, involving the cAMP → PKA pathway in nigrostriatal terminals.     

Creatine protects against 3-nitropropionic acid-induced cell death in murine corticostriatal slice cultures

In murine corticostriatal slice cultures, we studied the protective effects of the bioenergetic compound creatine on neuronal cell death induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). 3-NP caused a dose-dependent neuronal degeneration accompanied by an increased lactate dehydrogenase (LDH) activity in the cell culture medium. An increased ratio of lactate to pyruvate concentration in the medium suggested that metabolic activity shifted to anaerobic energy metabolism. These effects were predominantly observed in the 24-h recovery period after 3-NP exposure. Creatine protected against 3-NP neurotoxicity: LDH activity was reduced and aerobic respiration of pyruvate was stimulated, which resulted in lower lactate levels and less cell death. In both striatum and cortex, apoptosis in 3-NP-exposed slices was demonstrated by increased activation of the pro-apoptotic protein caspase-3 and by numerous cells exhibiting DNA fragmentation detected by the terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL) technique. Creatine administration to the 3-NP-exposed corticostriatal slices resulted in a reduced number of TUNEL-positive cells in the recovery period. However, in the striatum, an unexpected increase of both TUNEL-positive cells and caspase-3-immunostained cells was observed in the exposure phase in the presence of creatine. In the recovery phase, caspase-3-immunostaining decreased to basal levels in both striatum and cortex. These findings suggest that 3-NP-induced neuronal degeneration in corticostriatal slices results from apoptosis that in the cortex can be prevented by creatine, while in the more vulnerable striatal cells it may lead to an accelerated and increased execution of apoptotic cell death, preventing further necrosis-related damage in this region.     

Effect of sensory stimulation in rat barrel cortex, dorsolateral striatum and on corticostriatal functional connectivity

The striatum integrates sensory information to enable action selection and behavioural reinforcement. In the rat, a large topographical projection from the rat barrel cortex to widely distributed areas of the striatum is assumed to be an important structural component supporting these processes. The striatal sensory response is, however, not comprehensively understood at a network level. We used a 10-Hz, 100-ms air puff, allowing undamped movement of multiple whiskers, to look at functional connectivity in contralateral cortex and striatum in response to sensory stimulation. Simultaneous recordings of cortical and striatal local field potentials (LFPs) were made under isoflurane anaesthesia in 15 male Brown Norway rats. Four electrodes were placed in the barrel cortex while the dorsolateral striatum was mapped with a 500-μm resolution, resulting in a maximum of 315 recording positions per animal. Significant event-related responses were unevenly distributed throughout the striatum in 34.8% of positions recorded within this area. Only 10.3% of recorded positions displayed significant total power increases in the LFPs during the stimulation period at the stimulus frequency. This suggests that the responses seen in the LFPs are due to phase rearrangement rather than an amplitude increase in the signal. Analysis of corticostriatal imaginary coherence revealed stimulus-induced changes in the functional connectivity of 12% of corticostriatal pairs, the sensory response of sparsely distributed neuronal ensembles within the dorsolateral striatum is reflected in the phase relationship between the cortical and striatal local fields.     

Dopamine depletion causes fragmented clustering of neurons in the sensorimotor striatum: evidence of lasting reorganization of corticostriatal input

Firing during sensorimotor exam was used to categorize single neurons in the lateral striatum of awake, unrestrained rats. Five rats received unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle to deplete striatal dopamine (DA; >98% depletion, postmortem assay). Three months after treatment, rats exhibited exaggerated rotational behavior induced by L-dihydroxyphenylalanine (L-DOPA) and contralateral sensory neglect. Electrode track "depth profiles" on the DA-depleted side showed fragmented clustering of neurons related to sensorimotor activity of single body parts (SBP neurons). Clusters were smaller than normal, and more SBP neurons were observed in isolation, outside of clusters. More body parts were represented per unit volume. No recovery in these measures was observed up to one year post lesion. Overall distributions of neurons related to different body parts were not altered. The fragmentation of SBP clusters after DA depletion indicates that a percentage of striatal SBP neurons switched responsiveness from one body part to one or more different body parts. Because the specific firing that characterizes striatal SBP neurons is mediated by corticostriatal inputs (Liles and Updyke [1985] Brain Res. 339:245-255), the data indicate that DA depletion resulted in a reorganization of corticostriatal connections, perhaps via unmasking or sprouting of connections to adjacent clusters of striatal neurons. After reorganization, sensory activity in a localized body part activates striatal neurons that have switched to that body part. In turn, switched signals sent from basal ganglia to premotor and motor neurons, which likely retain their original connections, would create mismatches in these normally precise topographic connections. Switched signals could partially explain parkinsonian deficits in motor functions involving somatosensory guidance and their intractability to L-DOPA therapy-particularly if the switching involves sprouting.     

Broad cortical activation in response to tactile stimulation in newborns

Tactile sensation, which is one of the earliest developing sensory systems, is very important in the perception of an individual's body and the surrounding physical environment, especially in newborns. However, currently, only little is known about the response of a newborn's brain to tactile sensation. The objective of the present study was to determine the response of a newborn's brain to tactile sensation and to compare the brain responses to various sensory stimuli. Ten healthy newborns, 2-9 days after birth, were enrolled. A multichannel near-infrared spectroscopy system was used to measure brain responses. The probe array covered broad cortical areas, including the parietal, temporal, and occipital areas. We measured cortical hemodynamic changes in response to three different types of stimuli: tactile, auditory, and visual. Activated areas were analyzed by t-tests, and the number of activated channels among the three different stimuli was compared by χ2-tests. The results showed that when the brain responded to each type of stimulation, the corresponding primary sensory area was activated, and tactile stimuli induced broader areas of brain activation than the other two types of stimuli (auditory or visual). Thus, broad brain areas, including the temporal and parietal areas, were activated by tactile stimuli in early newborn periods. These results suggest that there are differences in newborns' reactions to various types of sensory stimuli, which may reflect the importance of tactile sensation in the early newborn period.     

Dopamine receptor-modulated [S]GTPγS binding in striatum of 6-hydroxydopamine-lesioned rats

The role of dopamine receptor–G protein coupling in the development of striatal dopamine receptor supersensitivity was studied in rats with a 6-hydroxydopamine (6-OHDA)-induced unilateral lesion of the nigrostriatal pathway. This coupling was assessed by the measurement of dopamine agonist-induced guanosine 5′-O-(γ[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in striatal membranes, at different periods of time (1–5 weeks) following the microinjection of the neurotoxin. From the first to the fifth week following the lesion, basal and dopamine-stimulated [³⁵S]GTPγS-specific binding were found to be enhanced in the denervated striata as compared to their control counterpart. D₂ dopamine receptors were clearly demonstrated to be involved in this supersensitivity, as assessed by measuring N-propylnorapomorphine (NPA)-, quinpirole- and bromocriptine-induced [³⁵S]GTPγS-specific binding. The involvement of D₁ dopamine receptors was indirectly studied by the combination of dopamine with a saturating concentration of the selective and potent D₂ antagonist domperidone. In these conditions, the remaining response to dopamine was also found to be significantly increased following the lesion. These results are consistent with the hypothesis that, in addition to D₂ dopamine receptor upregulation, modulation of dopamine receptor–G protein interaction is involved in the hypersensitivity accompanying striatal dopamine depletion.     

Responses of suprachiasmatic nucleus neurons to optic nerve stimulation in rat hypothalamic slice preparation

Stimulation of the optic nerve evoked two positive waves (P₁ and P₂) with short latencies and a following large negative wave (N) in the suprachiasmatic nucleus (SCN) of hypothalamic slice preparations. The conduction velocities of the P₁, P₂ and N waves were assumed to be 2.9 m/s, 0.5 m/s and 0.4 m/s, respectively. The N wave disappeared in low Ca²⁺ medium and all responses disappeared when TTX was added to the medium at a concentration of 10⁻⁶ M. The results indicate that the P₁ and P₂ waves were responses of the optic nerve in the SCN and the N wave was that of SCN neurons. Following optic nerve stimulation 15 out of 212 single SCN neurons were excited; 10 were excited then inhibited, 14 were excited followed by rhythmic oscillation, and 4 were inhibited. The results demonstrate neuronal projections from the optic nerve to the SCN, and suggest that the major role of the excitatory influence on SCN neurons is responsible for the elevation of neuronal activity in the SCN during the daytime.     

Localization of [3H]cyclohexyladenosine and [3H]nitrobenzylthioinosine binding sites in rat striatum and superior colliculus

The localization of adenosine receptors labelled with [3H]cyclohexyladenosine ([3H]CHA) and adenosine transport sites labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined in striatum and superior colliculus (SC) using radioligand binding and lesioning methods. Striatal kainic acid lesions significantly reduced the number (Bmax) of a single class of high affinity binding sites for [3H]CHA by 50% and that for [3H]NBI by 15% without altering Kd values for either ligand. In SC, enucleations significantly reduced both high and low affinity [3H]CHA binding sites by about 60% while levels of [3H]NBI binding were unaffected. Thus, adenosine receptors are present on striatal interneurons and retinal projections to the SC and some [3H]NBI binding sites are located on striatal interneurons.     

Low-frequency stimulation of the direct cortical input to area CA1 induces homosynaptic LTD and heterosynaptic LTP in the rat hippocampal-entorhinal cortex slice preparation

The entorhinal cortex (EC) innervates area CA1 and the subiculum directly via the portion of the perforant path, which originates from EC layer III cells referred to as direct cortical input (dCI), and indirectly via the trisynaptic loop through the dentate gyrus and area CA3. The dCI is of great importance as it mediates positional information for activation of place cells that is not prevented after interrupting information flow from area CA3 to CA1. In this study, we investigated the effects of low-frequency stimulation of the dCI on homo- and heterosynaptic plasticity in area CA1 and tested for the contribution of NMDA, GABA(A), GABA(B), kainate and group I mGlu receptors. We demonstrate that 1 Hz stimulation of the dCI induces homosynaptic long-term depression (LTD) and that 1 Hz stimulation of the dCI induces a long-lasting augmentation of stratum radiatum-induced population spikes in area CA1 (heterosynaptic long-term potentiation). Additionally we show that homosynaptic effects depend on activation of GABA(B) and kainate receptors, whereas the heterosynaptic effects are GABA(A) and mGlu receptor dependent.     

Involvement of corticostriatal glutamatergic terminals in striatal dopamine release elicited by stimulation of delta-opioid receptors

We have previously shown that striatal dopamine release induced locally by a delta-opioid receptor agonist was totally inhibited by a glutamate N-methyl-D-aspartate receptor antagonist, indicating the involvement of glutamatergic receptors in this effect. The aim of the present study was to specify this mechanism. Firstly, we investigated the effect of [D-Pen2,D-Pen5]-enkephalin (DPDPE) on glutamate release in rats by intrastriatal microdialysis. The infusion of DPDPE (10 microm) enhanced the glutamate content in dialysate by approximately 34%, an effect which did not appear to result from inhibition of glutamate uptake. We then considered the consequences of a unilateral thermocoagulation of the frontal cortex on either glutamate or dopamine release induced by stimulation of delta-opioid receptors 2 days later. This lesion, which decreased the glutamate content in ipsilateral striatum by approximately 30%, totally prevented the increase in dialysate levels of glutamate induced by DPDPE. Moreover, whereas DPDPE (10 microm) was found to increase the striatal dopamine release in intact animals by approximately 59%, this effect was also completely suppressed by the cortical lesion. Finally, we studied the effect of the lesion on the [3H]-DPDPE binding to striatal membranes prepared from the whole striatum. In the ipsilateral striatum a significant decrease in this [3H]-DPDPE binding (by approximately 18%) was found 2 days after the lesion. Our results indicate that the increase in striatal dopamine release induced by DPDPE probably depends on glutamate release from corticostriatal glutamatergic afferents in response to the stimulation of delta-opioid receptors located on terminals of these neurons.