In vivo release of dopamine in the nucleus accumbens of the rat: modulation by cholecystokinin

We observed that the release of endogenous dopamine (DA), induced by perfusion of a 55 mM K⁺-containing buffer in the nucleus accumbens, was Ca²⁺-dependent and confined to a local region. We also demonstrated that the sulphated form of cholecystokinin octapeptide, but not the unsulphated form, suppressed this stimulated release of dopamine in a concentration-dependent manner. This suggests that cholecystokinin may act as a functional antagonist to dopamine within this structure.     

Effect of nomifensine on the toxicity of 6-hydroxydopamine for mesotelencephalic dopamine neurons

The ability of nomifensine to protect the dopaminergic cells of the substantia nigra and ventral tegmental areas against 6-hydroxydopamine-induced destruction was evaluated. Nomifensine at high doses (20 mg/kg, i.p.) protected the cells from the effects of low amounts of 6-hydroxydopamine (2 micrograms) injected intracerebrally. This protective effect was markedly decreased with an increased amount of 6-hydroxydopamine (8 micrograms), or by lower doses of nomifensine (6.7 mg/kg). These doses of nomifensine are higher than those required to protect dopaminergic nerve terminals.     

SCH 23390 enhances exogenous L-DOPA decarboxylation in nigrostriatal neurons

Summary. Exogenous L-DOPA enhances dopamine metabolism in the intact and denervated striatum, and is the treatment of choice for Parkinsonism. Aromatic L-amino acid decarboxylase (AAAD) converts L-DOPA to dopamine. Blockade of dopamine D₁-like receptors increases the activity of AAAD in both intact and denervated striatum. A single dose of SCH 23390, a dopamine D₁-like receptor antagonist, increases the activity of AAAD in the striatum and midbrain and induces small changes in dopamine metabolism. When L-DOPA is administered after SCH 23390, there is a significant increase in the formation of 3,4-dihydroxyphenylacetic acid and dopamine turnover in striatum and midbrain compared to L-DOPA alone, suggesting further enhancement of dopamine metabolism. When the studies are repeated in the MPTP mouse model of Parkinson's disease, there is significantly more dopamine metabolism in the striatum of lesioned mice pretreated with SCH 23390 than in a comparison group treated with L-DOPA alone. These studies suggest that it may be possible to enhance the conversion of L-DOPA to dopamine in Parkinson's disease patients by administering substances that augment brain AAAD. Received May 10, 1999; accepted September 14, 1999     

Lesions of the superior colliculus in the rat differentiate between nigrostriatal and mesolimbic dopamine systems

Bilateral electrolytic lesions of the superior colliculus in rats increased spontaneous locomotor activity, enhanced amphetamine-induced hyperactivity and attenuated apomorphine-induced biting. These lesions were associated with an increased rate of turnover of dopamine in the nucleus accumbens, but not in the striatum. Similarly concentrations of the dopamine metabolites homovanillic acid and 3,4-dihydroxyphenylacetic acid were elevated in accumbens tissue but not in striatum in rats with bilateral collicular lesions. The results indicate that lesions of the superior colliculus cause differentiation between hyperactivity and stereotypy, and that this may be related to blockade of a nigrostriatal outflow, and relief of inhibition on mesolimbic systems.     

Iontophoretically applied dopamine depolarizes and hyperpolarizes the membrane of cat caudate neurons

Dopamine (DA) was applied iontophoretically on intracellularly recorded cat caudate neurons. Ejected approximately 100 μm away from the cell soma, it caused slow depolarizations of the membrane while the ongoing firing rate was reduced. This last effect was not due to sodium inactivation. Cortically evoked EPSP-IPSP sequences were inhibited during the depolarizations. The latency of cortically evoked action potentials was consistently increased during DA-ejections. These effects were blocked by fluphenazine, a relatively selective blocker of the DA-sensitive adenylate cyclase. Nevertheless, there are serious doubts as to the specificity of these actions of DA as a number of other substances like naloxone, nicotine, acetylcholine or glutamate-diethylester occasionally had very similar effects on membrane potential, firing rate and cortically evoked EPSP-IPSP sequences.If DA was applied nearer to the soma, approximately 50 μm away, 70% of the recorded neurons continued to display the slow depolarizations above described, while 30% of the cells now reacted by a hyperpolarization accompanied also by a reduced firing rate. If DA was applied for prolonged periods on such cells, the initial hyperpolarization was followed by the slow depolarization.The observation that during the slow depolarization there is a decrease in firing rate and amplitude of the cortically evoked IPSP is explained by the assumption that the region of the axon hillock is hyperpolarized by DA, and that the slow depolarization is a phenomenon restricted to the distant recording site and possibly to the dendritic region.None of the 74 responsive neurons displayed an increased firing rate when DA was ejected either continously, i.e. for more than 5 sec, or in short pulses of 50–500 msec.     

Evidence for a dopaminergic innervation of the cat lateral habenula: its role in controlling serotonin transmission in the Basal ganglia

The presence of a dopaminergic innervation of the cat lateral habenula and its possible role in modulating serotonin transmission within the basal ganglia were investigated using both in vitro and in vivo approaches. A high density of [³H]spiroperidol binding sites with similar affinities for domperidone and apomorphine as those present in the cat striatum were found in the habenula. By means of the push-pull cannula technique, a substantial release of [³H]dopamine continuously formed from [³H]tyrosine was detected in the lateral habenula of halothane-anesthetized cats since the amount of [³H]catecholamines was enhanced in the presence of benztropine, an inhibitor of dopamine uptake into dopaminergic nerve terminals. Furthermore, in anesthetized animals with a push-pull cannula implanted in each caudate nucleus and substantia nigra habenular applications of dopamine (10⁻⁷ M) reduced nigral but not striatal release of [³H]serotonin continuously formed from [³H]tryptophan. This change was prevented either by the delivery of domperidone to the lateral habenula or by the blockade of GABAergic transmission (picrotoxin 10⁻⁵ M) in the dorsal raphe. These data support the involvement of habenula-raphe pathways in the regulation of serotonin transmission in the cat basal ganglia and indicate that dopaminergic inputs to the lateral habenula participate in such a control.     

Cholecystokinin octapeptide excites dorsal horn neurons both in vivo and in vitro

Cholecystokinin octapeptide (CCK-8) applied microiontophoretically causes a moderate to strong excitation of about half of all tested dorsal horn neurons located in laminae I-VII of both the cat intact spinal cord and the rat in vitro spinal cord slice preparation. In the cat intact spinal cord the excitation is not limited to a single population of neurons but is observed in all categories of units recognized in spinal preparations of cats in this area on the basis of their excitability by different kinds of cutaneous afferent input. In the spinal cord slice preparation the excitatory action of CCK-8 persists even when the spinal cord slices are perfused with a Ca2+-free, Mg2+-high Krebs solution. The latter finding indicates that the action of CCK-8 might be a direct one exerted on the postsynaptic sites of dorsal horn units. These results are consistent with the possibility that CCK-8 acts on postsynaptic sites in the dorsal horn of the spinal cord as a neurotransmitter or modulator.     

Anatomical and functional reconstruction of the nigrostriatal system in vitro: selective innervation of the striatum by dopaminergic neurons.

To study development of the nigrostriatal pathway in an in vitro model system, organotypic slices obtained from rat pups (P4) and containing the striatum and the cortex were grown together with apposed embryonic (E13.5) mesencephalic blocks according to the static slice culture method of Stoppini et al. (1991; J. Neurosci. Methods 37:173-182). Under these conditions, mesencephalic dopaminergic (DA) fibers rapidly grow through the slice, preferentially its striatal portion. This innervation provides a true synaptic innervation to the striatum, as shown by the presence of DA terminals on striatal neurons. DA fibers are able to exert a functional influence, as seen by their ability to modulate c-Fos expression in striatal neurons in the same way as in vivo. Thus, blockade, under basal conditions, of the effect of spontaneously released dopamine by the D2 receptor antagonist haloperidol leads to the activation of c-Fos expression in the striatum. Furthermore, stimulation of DA release by amphetamine induces striatal c-Fos expression in a D1 receptor-dependent manner. Next, the mechanisms of the selective striatal innervation were examined. Indeed, DA fibers innervated specifically the striatum, avoiding the cortical portion of the slice. This selectivity seems to be specific for DA neurons; no selectivity could be observed when noradrenergic neurons were substituted for DA neurons. Short-term cocultures in a collagen gel of mesencephalic blocks with striatal blocks failed to reveal any oriented outgrowth of DA fibers from the mesencephalon, suggesting that the selective innervation observed in the organotypic slices results from some contact-dependent, presumably adhesive interactions rather than from the presence of some diffusible substance orienting the growth of DA fibers towards the striatum. On the other hand, DA neurons seeded onto striatal slices did not attach selectively onto the striatal portion of the slice, indicating that the putative specific adhesive interactions governing the selective striatal innervation are not the same as those determining the adhesion of the DA neurons. These results show that cocultures of cortex-striatum and mesencephalic slices result in a system that displays a number of the morphological and functional traits of the normal nigrostriatal system and that can be relied on as a good in vitro model of in vivo development.     

Release of endogenous dopamine and cholecystokinin from rat striatal slices: effects of amphetamine and dopamine antagonists

The release of immunoreactive cholecystokinin (CCK) and dopamine was monitored simultaneously from superfused rat striatal slices. Exposure of the tissue to medium containing elevated of dopamine (10⁻⁷ and 10⁻⁶)M), the dopamine agonist pergolide (10⁻⁷, M), the D₂-antagonist sulpride (1 μM) or the D₁-antagonist (SCH 23390) had no significant effect on basal overflow or on evoked release of CCK. On the other hand, preincubation of striatal slices withd-amphetamine (10⁻⁵ M) enhanced basal and veratrine-stimulated dopamine release but markedly suppressed evoked CCK release. Sulpiride blocked this action of amphetamine whereas SCH 23390 was ineffective. The data suggests that whereas it is difficult to observe any effects of exogenous dopamine agonists or antagonists on evoked CCK release, endogenously released dopamine appears to interact with D₂-receptors to suppress evoked CCK release from rat striatal slices.     

Effects of fencamfamine on single unit activity of mesencephalic dopaminergic neurons in rats

Summary Systemic fencamfamine (0.5–16 mg/kg, i.v.) significantly but incompletely inhibited spontaneous activity of nigrostriatal and mesolimbic/mesocortical dopamine (DA) neurons. Inhibition was reversed by haloperidol (0.1 mg/kg, i.v.) and prevented by pretreatment with -methyltyrosine (50 mg/ kg, i.v.) plus reserpine (5 mg/kg, i.p.). Pretreatment with -methyltyrosine alone attenuated inhibition at high but not low doses of fencamfamine. Microiontophoresed fencamfamine had little direct effect on DA neurons and did not consistently modulate the effects of co-microiontophoresed DA. In contrast, systemic fencamfamine blocked the inhibitory effects of low doses of apomorphine (10–40 g/kg, i.v.). Fencamfamine appears to be an indirect DA agonist which interacts with both vesicular and newly synthesized DA storage pools. Fencamfamine may also cause a rapid desensitization to the effects of DA autoreceptor stimulation.     

Electrophysiological study on the corticoreticular projection neurons of the cat

Thirty-two motor cortical neurons of the cat which project on medullary reticular formation (RF) were electrophysiologically classified into 3 groups, i.e., 22 slow PT-RF neurons (collateral projections to RF from slow pyramidal tract (PT) neurons), 6 fast PT-RF neurons (those from fast PT neurons) and 4 RF (non-PT) neurons without any relation to PT neurons. Firings of slow PT-RF and RF (non-PT) neurons elicited by thalamic stimulation showed close correlation to the surface negative component of cortical evoked potentials. It was suggested that activities of these neurons are controlled mainly by excitatory thalamic inputs onto the distal and superficial part of their apical dendrites.     

Dopamine synthesis in inbred mouse strains which differ in numbers of dopamine neurons

BALB/cJ and CBA/J mice have been shown to have different numbers of dopamine (DA) neurons in the central nervous system, with BALB/cJ mice having 20–50% more DA neurons in each dopaminergic cell group which is reflected in a difference in tyrosine hydroxylase activity in these cell groups. The present study compared the levels of DA and the rate of DA synthesis between these two inbred mouse strains. Three measures were used to reflect the rate of DA synthesis: the levels of DA metabolites (DOPAC and HVA) in the striatum, the rate of disappearance of DA following inhibition of tyrosine hydroxylase withα-methyl-P-tyrosine, and the rate of accumulation of DOPA following inhibition of aromatic amino acid decar☐ylase with NSD-1015. Striatal DA levels were slightly higher in CBA/J mice than BALB/cJ mice. The rate of DA synthesis in the striatum, as estimated from the accumulation of DOPA following NSD-1015 injection or from the decline of DA levels followingα-methyl-p-tyrosineinjection, was from 30–50% greater in the BALB/cJ mice compared to the CBA/J mice. In striatum, DOPAC levels were higher, HVA levels lower, and DOPAC plus HVA levels equal in CBA/J mice compared to BALB/cJ mice. The results show that BALB/cJ mice, with more DA neurons than CBA mice, also synthesize more DA. In addition, the data suggest that DA levels do not necessarily reflect numbers of DA neurons, and that catecholamine metabolite levels are not a good measure for comparing catecholamine synthesis between inbred animal strains.     

SKF 38393 alters the rate-dependent D2-mediated inhibition of nigrostriatal but not mesoaccumbens dopamine neurons

Previous electrophysiological studies have failed to identify significant effects of the D1 dopamine (DA) agonist SKF 38393, either alone or in combination with the D2 agonist quinpirole (LY 171555), on the spontaneous firing rate of midbrain DA neurons. We have utilized extracellular single-unit recording techniques to examine whether SKF 38393 can alter D2-mediated inhibition of DA cell activity. Quinpirole-induced inhibition of the spontaneous activity of midbrain DA neurons was observed to be positively correlated with the basal firing rate of the neuron being examined (i.e., faster cells required higher doses to achieve 50% and maximal inhibition). Pretreatment with SKF 38393 (1.0 mg/kg, i.v.; 4 minutes) eliminated the rate dependency of quinpirole-induced inhibition of nigrostriatal but not mesoaccumbens DA neurons. This effect of SKF 38393 was blocked both by the D1 antagonist SCH 23390 and by hemitransections of the forebrain. In summary, SKF 38393 appears to exert Dl-specific, feedback pathway-dependent effects on the profile of responsiveness of nigrostriatal DA neurons to D2-mediated inhibition of cell firing rate.     

D1 receptor activation enhances sciatic nerve stimulation-induced inhibition of nigrostriatal dopamine neurons

Nigrostriatal dopamine (NSDA) neurons have been hypothesized to play an important regulatory role in neostriatal sensorimotor integration. In order to provide further information on the nature of sensory modulation of NSDA cells, we have examined the pharmacology of the responsiveness of these neurons to peripheral nerve stimulation. The selective D1 dopamine receptor agonist SKF 38393 enhanced the normal inhibition of NSDA neurons produced by electrical stimulation of the sciatic nerve. The SKF 38393-induced enhancement, but not the basal stimulation-induced inhibition itself, was blocked by prior hemitransection of the forebrain and was reversed by the selective D1 antagonist SCH 23390 but not by the selective D2 antagonist 1-sulpiride. SCH 23390 alone, however, exerted no effect on this inhibition. The selective D1 receptor agonist fenoldopam, which does not cross the blood-brain barrier, also failed to alter the response to sciatic nerve stimulation (i.v. administration). Thus, central D1 receptors (rostral to the midbrain) appear to be involved in a system which mediates phasic control over sensory modulation of NSDA neuronal activity.     

Neuroleptic-like disruption of the conditioned avoidance response requires destruction of both the mesolimbic and nigrostriatal dopamine systems

An examination of the ability to learn an active avoidance response was made in rats subjected to 6-hydroxydopamine (6-OHDA) lesions of the individual terminal areas of the midbrain dopamine (DA) system or a lesion to all these terminal regions in one group. Lesions were made by infusing 8 micrograms (base) of 6-OHDA in 2 microliter of vehicle into the following forebrain regions (each region representing a separate group of rats); frontal cortex, nucleus accumbens, corpus striatum and a double lesion of nucleus accumbens and corpus striatum. A separate group of rats received a smaller 6-OHDA lesion of the ventral substantia nigra. Only those rats with the combined double lesion of both the nucleus accumbens and corpus striatum (90% total depletion of dopamine) showed a severe deficit in acquisition of active avoidance. However, the rats with the separate 6-OHDA lesions to the mesolimbic or nigrostriatal DA systems did show the appropriate blockade of the amphetamine-induced locomotion or stereotyped behavior, respectively. In contrast, the rats with the double lesion showed no response to a low or high dose of amphetamine, remained cataleptic for the duration of the experiment but rapidly recovered from transient aphagia and adipsia (less than 10 days post lesion). Results suggest that a severe deficit in acquisition of an active avoidance response, similar to that observed with high doses of neuroleptics, requires destruction of all of the dopamine innervation of nucleus accumbens and corpus striatum. Results also suggest that both the mesolimbic and nigrostriatal dopamine systems act in concert to produce response enabling to important environmental events, and that the severe response enabling deficits observed in Parkinson's disease involves not only degeneration of the nigrostriatal dopamine system, but of the mesolimbic dopamine system as well.     

Neuroleptic-like distruption of the conditioned avoidance response requires destruction of both the mesolimbic and nigrostriatal dopamine systems

An examination of the ability to learn an active avoidance response was made in rats subjected to 6-hydroxydopamine (6-OHDA) lesions of the individual terminal areas of the midbrain dopamine (DA) system or a lesion to all these terminal regions in one group. Lesions were made by infusing 8 μg (base) of 6-OHDA in 2 μl of vehicle into the following forebrain regions (each region representing a separate group of rats); frontal cortex, nucleus accumbens, corpus striatum and a double lesion of nucleus accumbensand corpus striatum. A separate group of rats received a smaller 6-OHDA lesion of the ventral substantia nigra. Only those rats with the combined double lesion of both the nucleus accumbens and corpus striatum (90% total depletion of dopamine) showed a severe deficit in acquisition of active avoidance. However, the rats with the separate 6-OHDA lesions to the mesolimbic or nigrostriatal DA systems did show the appropriate blockade of the amphetamine-induced locomotion or stereotyped behavior, respectively. In contrast, the rats with the double lesion showed no response to a low or high dose of amphetamine, remained cataleptic for the duration of the experiment but rapidly recovered from transient aphagia and adipsia (< 10days post lesion). Results suggest that a severe deficit in acquisition of an active avoidance response, similar to that observed with high doses of neuroleptics, requires destruction of all of the dopamine innervation of nucleus accumbens and corpus striatum. Results also suggest that both the mesolimbic and nigrostriatal dopamine systems act in concert to produce response enabling to important environmental events, and that the severe response enabling deficits observed in Parkinson's disease involves not only degeneration of the nigrostriatal dopamine system, but of the mesolimbic dopamine system as well.     

Coexistence of neuropeptides in projection neurons of the thalamus in the cat

Coexistence of neuropeptides was suggested by double-staining immunohistochemistry in projection neurons in the thalamus of the cat; cholecystokinin (CCK)-like immunoreactivity (LI) and vasoactive intestinal polypeptide (VIP)-LI in the rostral group of the intralaminar nuclei, CCK-LI and neurotensin (NT)-LI in the anterodorsal nucleus and NT-LI and VIP-LI in the laterodorsal nucleus.     

Origin of extracellular dopamine from dopamine and noradrenaline neurons in the medial prefrontal and occipital cortex

Our recent studies suggest that extracellular dopamine (DA) in the cerebral cortex not only originates from dopaminergic terminals but is also coreleased with noradrenaline (NA) from noradrenergic terminals [Devoto et al. (2001) Mol Psychiatry 6:657-664]. To further clarify this issue, the concentrations of extracellular DA, its deaminated metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), and NA were compared by microdialysis in the medial prefrontal cortex (mPFC), an area densely innervated by NA and DA neurons, and in the occipital cortex (OCC), equally innervated by NA but receiving scarce DA projections. Moreover, the effect of the alpha(2)-adrenoceptor agonist clonidine locally perfused into the locus coeruleus (LC) on extracellular NA, DA, and DOPAC in the mPFC, OCC, and ventral striatum was investigated. Consistent with the homogeneous NA innervation, extracellular NA concentration was similar in both cortices, while extracellular DA in the OCC, in spite of the scarce DA afference in this area, was only 37% lower than in the mPFC; extracellular DOPAC in the OCC was 81% lower than in the mPFC. Consistent with its ability to inhibit NA neurons, clonidine (10 microM) reduced extracellular NA by about 65 and 80% in the OCC and the mPFC, respectively, but also reduced extracellular DA by 70 and 50% in the OCC and the mPFC, respectively. Clonidine reduced DOPAC in the OCC (by about 40%) but not in the mPFC. In the ventral striatum clonidine reduced NA (by 30%) but not DA and DOPAC. After inhibition of the DA and NA transporter, by perfusing 100 microM desmethyl-imipramine into the mPFC, clonidine perfusion into the LC reduced extracellular NA and DA in the mPFC by about 50%. The results indicate that most of extracellular DA in the OCC and a significant portion in the mPFC reflect the activity of NA neurons and support the hypothesis that extracellular DA in the cerebral cortex may originate not only from DA but also from NA neurons.     

Presynaptic regulation of the release of catecholamines in the cat hypothalamus

The posterior hypothalamus of cats was superfused through a push-pull cannula and the release of endogenous catecholamines was determined in the superfusate. Superfusion with yohimbine, isoprenaline, salbutamol or tazolol increased, while superfusion with propanolol decreased, the release of all three catecholamines. Transection of the brain caudal to the hypothalamus inhibited ‘resting’ and drug-induced release. It is concluded that α- and β-adrenoceptors of the hypothalamus are involved in the regulation of the release of catecholamines.