The influence of acute and chronic administration of 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline on the function of the nigrostriatal dopaminergic system in rats.

The contribution of (R)-enantiomer of N-methyl-salsolinol (1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; NMSal) to the degeneration of dopaminergic neurons in the course of Parkinson's disease (PD) has been predominantly suggested by in vitro experiments in cell culture and by an in vivo study in which this compound has been directly injected into the rat striatum. The aim of the present study was to examine the effects of racemic NMSal (50 and 100 mg/kg) administered systemically, acutely and chronically for 21 days to rats, on the neurochemical and behavioral markers of PD. Our results showed that racemic NMSal easily penetrated the blood-brain barrier. Its brain level was relatively high 2-6 h after a single injection than gradually decreased. NMSal was quickly eliminated from the rat brain, its concentration 24 h after withdrawal from chronic treatment was very low. NMSal at both examined doses did not affect striatal and nigral levels of dopamine (DA) 2 h after the first and last chronic injections, however, it markedly changed DA catabolism. In the striatum both its doses evoked a significant acceleration of the total and oxidative, monoamine oxidase (MAO)-dependent DA catabolism without affecting the catechol-O-methyltransferase (COMT)-dependent O-methylation. In the substantia nigra (SN), only the higher dose of NMSal produced such effect. DA catabolism in either structure was the same as in control 24 h after cessation of chronic treatment. The second characteristic marker of PD, the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SN, was not affected by chronic NMSal treatment as revealed by the stereological counting. In the behavioral study, it was found that racemic NMSal significantly suppressed spontaneous locomotor activity and effectively prevented that stimulated by apomorphine. Our results suggest that NMSal may play an important role in the regulation of dopaminergic activity rather than in inducing changes of parkinsonian type.

Degeneration of dopaminergic mesocortical neurons and activation of compensatory processes induced by a long-term paraquat administration in rats: implications for Parkinson's disease.

A deficiency of the dopaminergic transmission in the mesocortical system has been suggested to contribute to cognitive disturbances in Parkinson's disease. Therefore, the aim of the present study was to examine whether the long-term administration of a commonly used herbicide, paraquat, which has already been found to induce a slowly progressing degeneration of the nigrostriatal neurons, influences mesocortical dopaminergic neurons in rats. Paraquat at a dose of 10 mg/kg i.p. was injected either acutely or once a week for 4, 8, 12 and 24 weeks. Acute treatment with this pesticide increased the level of homovanillic acid (HVA) and HVA/dopamine ratio in the prefrontal cortex. After 8 weeks of administration paraquat increased the number of stereologically counted tyrosine hydroxylase-immunoreactive (TH-ir) neurons and their staining intensity in the ventral tegmental area (VTA), which is a source of the mesocortical dopaminergic projection. At the same time, few TH-ir neurons appeared in different regions of the cerebral cortex: in the frontal, cingulate, retrosplenial and parietal cortices. Chronic paraquat administration did not influence the level of dopamine in the prefrontal cortex but increased the levels of its metabolites: 3,4-dihydroxyphenylacetic acid (after 8-12 weeks), HVA (after 4 and 12 weeks) and HVA/dopamine ratio (4 weeks). After 24 weeks this pesticide reduced the number of TH-ir neurons in the VTA by 42% and of the Nissl-stained neurons by 26%, and induced shrinkage of this structure by ca. 25%. Moreover, TH-ir neurons in the cortex were no more visible after such a long period of administration and levels of dopamine metabolites returned to control values. The present results suggest that the long-term paraquat administration destroys dopaminergic neurons of the VTA. However, compensatory activation of the VTA neurons and cortex overcomes progressing degeneration and maintains cortical dopaminergic transmission.

Adaptive down-regulation of the serotonin transporter in the 6-hydroxydopamine-induced rat model of preclinical stages of Parkinson's disease and after chronic pramipexole treatment.

Our recent study has indicated that a moderate lesion induced by bilateral 6-hydroxydopamine (6-OHDA) injections into the ventrolateral region of the caudate-putamen (CP) in rats, modeling preclinical stages of Parkinson's disease, induces a "depressive-like" behavior which is reversed by chronic treatment with pramipexole (PRA). The aim of the present study was to examine the influence of the above lesion and chronic PRA treatment on binding to the serotonin transporter (SERT) in different brain regions. As before, 6-OHDA (15 µg/2.5 µl) was administered bilaterally into the CP. PRA (1mg/kg) was injected subcutaneously twice a day for 2 weeks. Serotonergic and dopaminergic neurons of the dorsal raphe (DR) were immunostained for tryptophan hydroxylase and tyrosine hydroxylase, respectively, and were counted stereologically. Binding of [(3)H]GBR 12,935 to the dopamine transporter (DAT) and [(3)H]citalopram to SERT was analyzed autoradiographically. Intrastriatal 6-OHDA injections decreased the number of dopaminergic, but not serotonergic neurons in the DR. 6-OHDA reduced the DAT binding in the CP, and SERT binding in the nigrostriatal system (CP, substantia nigra (SN)), limbic system (ventral tegmental area (VTA), nucleus accumbens (NAC), amygdala, prefrontal cortex (PFCX), habenula, hippocampus) and DR. A significant positive correlation was found between DAT and SERT binding in the CP. Chronic PRA did not influence DAT binding but reduced SERT binding in the above structures, and deepened the lesion-induced losses in the core region of the NAC, SN, VTA and PFCX. The present study indicates that both the lesion of dopaminergic neurons and chronic PRA administration induce adaptive down-regulation of SERT binding. Moreover, although involvement of stimulation of dopaminergic transmission by chronic PRA in its "antidepressant" effect seems to be prevalent, additional contribution of SERT inhibition cannot be excluded.

Alterations in striatal neuropeptide Y system activity of rats with haloperidol-induced behavioral supersensitivity.

The study was conducted to determine whether the expression of behavioral supersensitivity induced by haloperidol (HAL) administered once daily (2 mg/kg i.p.) for 14 days is associated with the alterations in activity of neuropeptide Y (NPY) system in the striatum (caudate-putamen) and nucleus accumbens. Dopamine supersensitivity was tested by measurement of locomotor activity and stereotyped behavior after administration of the dopamine D2/D3 receptor agonist quinpirole (1 mg/kg i.p.) on day 1, 3 and 7 after HAL withdrawal. Neuropeptide Y-like immunoreactivity (NPY-LI) was determined in the striatum and nucleus accumbens isolated 6 h after quinpirole injection on day 1, 3 and 7 after the end of HAL treatment. NPY mRNA was quantified in these structures on day 7 after HAL withdrawal. HAL increased spontaneous locomotor activity and prevalence of rearing, grooming and head-down sniffing. At the same time, striatal NPY-LI increased progressively from the reduced level found on day 1 of haloperidol withdrawal. NPY mRNA remained unchanged. In saline-treated rats, quinpirole enhanced locomotion, rearing, and induced intense head-down sniffing and oral activity. These behavioral effects were accompanied by a decrease in striatal NPY-LI. NPY mRNA was slightly increased. HAL treatment altered response to quinpirole, namely it increased locomotion, intensified oral activity and reduced rearing and head-down sniffing. The second and the third quinpirole injection decreased NPY-LI levels. NPY mRNA was unchanged. In the nucleus accumbens, apart from a decrease in NPY-LI on day 1 after the last haloperidol dose, the level of NPY-LI and NPY mRNA in any experimental group did not differ from the control value. The presented results suggest that the alterations in the activity of the striatal but not nucleus accumbens NPY system contribute to adaptive changes induced by long-term haloperidol treatment and may be of significance to the motor hyperactivity induced by intermittent stimulation of postsynaptic dopamine D2 receptors.

Effect of chronic imipramine or electroconvulsive shock on the expression of mGluR1a and mGluR5a immunoreactivity in rat brain hippocampus.

Previous studies showed that chronic electroconvulsive shock (ECS) or imipramine treatment induced a subsensitivity of group I metabotropic glutamate receptors (mGluR) in hippocampus. In the present study effects of antidepressant treatment on the expression of mGluR1a and mGluR5a, belonging to the group I mGluR, were investigated in rat brain hippocampus using immunohistochemical and Western blot methods, respectively. Male Wistar rats were treated singly or chronically for 21 days with imipramine, 10 mg/kg, twice daily; with ECS (90 mA, 50 Hz, 0.5 s) every second day; or with haloperidol, 1.2 mg/kg, once daily. Appropriate controls were injected with saline. Rats were sacrificed 24 h after the last treatment and their hippocampi were taken out for analysis. It was found that the mGluR1a-immunoreactivity expression increased significantly in Ammon's horn (CA) regions after chronic ECS. The most pronounced effect was observed in the CA3. No significant effects were found after single treatment or after haloperidol. The expression of mGluR5a increased significantly after chronic imipramine in the CA1 and after chronic ECS in the CA3 region. The results obtained indicate an influence of antidepressant treatment on group I mGluR. This increase in the receptor protein level may be a compensatory mechanism developing after chronic treatment.

Neuropeptide Y immunoreactivity in the locus coeruleus of the rat brain.

The detailed distribution of neuropeptide Y immunoreactive neurons and fibres is given for the rat locus coeruleus. The studies were carried out using indirect immunofluorescence and avidin-biotin-peroxidase techniques. It was shown that in colchicine pretreated rats, about 15-20% of locus coeruleus neurons contain neuropeptide Y immunoreactivity. Neuropeptide Y immunoreactive neurons form two populations: (1) medium-sized or large neurons, poorly immunostained, situated mainly in the dorsal and central locus coeruleus nucleus, and (2) small, strongly immunostained neurons in ventromedial parts of the nucleus. Neuropeptide Y immunoreactive fibres and terminals are scattered throughout the locus coeruleus, but are more numerous in its ventromedial and ventrorostral parts.

An inhibitory dopaminergic regulation of the neuropeptide Y immunoreactivity expression in the rat cerebral cortex neurons.

The effect of catecholamine depletion or blockade of dopaminergic or noradrenergic receptors on the neuropeptide Y immunoreactivity was studied in the rat brain cortex using immunohistochemical methods. Neuropeptide Y immunoreactive neurons were counted and the mean density of stained neurons per microscopic field was calculated. It was found that monoamine depletion by reserpine, the blockade of dopaminergic receptors by haloperidol or the specific D1 receptor blockade by SCH23390 caused a significant increase in the neuropeptide Y immunoreactivity in the cortex studied, after 24 h, evaluated as the density of immunoreactive neurons. No significant changes were observed after the blockade of alpha or beta adrenergic receptors (by phenoxybenzamine or propranolol, respectively). Specific D2 receptor blockade by sulpiride induced an insignificant increase only. The results suggest the existence of an inhibitory dopaminergic control of the neuropeptide Y content, mainly via D1 receptors, in neurons of the rat brain cortex.

Influence of imipramine on neuropeptide Y immunoreactivity in the rat brain.

The effects of treatment with the antidepressant drug imipramine on neuropeptide Y immunoreactivity were studied immunocytochemically in the rat brain cortex and hypothalamus. It was found that the level of neuropeptide Y immunoreactivity in the cortex was significantly lowered three and 24 h after the last dose of chronic (14 days) imipramine administration as well as 3 h after acute administration. A tendency to decrease neuropeptide Y immunoreactivity was also found in the hypothalamus. The results obtained suggest an important role of the cortical neuropeptide Y in the action of the drug.

The influence of some antidepressant drugs on the nuclear volume of rat cingular cortex cells in culture.

The changes in the volume of cell nuclei of the rat cingular cortex were investigated in culture after incubation with some antidepressant drugs. Two-week incubation of the cingular cortex culture with both tricyclic (desipramine, imipramine, amitryptyline) and non-tricyclic (mianserin) antidepressants in concentration of 3 X 10(-6) M resulted in a decrease of the volume of the cell nuclei. Because the size of the nucleus is regarded as a criterion of the cell metabolic activity, our results may point to a diminished activity of metabolic processes of the cells.

Haloperidol-induced increase in neuropeptide Y immunoreactivity in the locus coeruleus of the rat brain.

The effect of haloperidol, a dopamine (preferably D2) receptor blocking agent on neuropeptide Y immunoreactivity was studied immunohistochemically in neurons of the locus coeruleus and striatum of rat brain. It was found that haloperidol given four times (5 and 2.5 mg/kg, i.p.) induced, after 24 h, a significant increase in the level of neuropeptide Y immunoreactivity in the locus coeruleus but not in the striatum. No changes in neuropeptide Y immunoreactivity in studied structures were observed after alpha-adrenergic receptor blocking agent phenoxybenzamine or serotonin-synthesis inhibitor D,L-p-chlorophenylalanine. The results suggest that the content of neuropeptide Y-immunoreactive material in nerve cell bodies of the locus coeruleus is inhibitorally controlled by monoaminergic (may be dopaminergic D2) receptors.

The development of neuropeptide Y-immunoreactive neurons in a model of pure cortical culture.

The development of neuropeptide Y-immunoreactive neurons in the rat brain cerebral cortex was studied in a model of a pure cortical culture. In this model, development of neurons devoid of any afferents from other brain structures could be observed. Since mutual interactions between neuropeptide Y and catecholamines have been postulated, such a pure cortical culture offers a possibility of studying the development of neuropeptide Y neurons devoid of any brainstem monoaminergic afferents. A tissue dissected from 16-day-old rat fetuses and cultivated in a dissociated culture for 14 days was examined immunohistochemically for the presence of neuropeptide Y-immunoreactive neurons. Three main types of neuropeptide Y-immunoreactive neurons were found: unipolar, bipolar and multipolar. Cell processes and terminal varicose fibres were also observed. The results obtained indicate that neuropeptide Y-immunoreactive neurons and fibres may develop in a pure culture of the rat cerebral cortex without the influence of any other structures.

Influence of reserpine administration on neuropeptide Y immunoreactivity in the locus coeruleus and caudate-putamen nucleus of the rat brain.

The effects of treatment with reserpine (10 mg/kg, i.p.) a monoamine depleting agent, on neuropeptide Y immunoreactivity were studied immunohistochemically in neurons of two rat brain structures: locus coeruleus and caudate-putamen nucleus. It was found that reserpine after 24 h increased neuropeptide Y immunoreactivity level but no significant changes were observed 4 and 72 h or 5 days after the injection. The results indicate that despite the known co-existence of neuropeptide Y and noradrenaline in some neurons of the locus coeruleus no concomitant decrease in neuropeptide Y immunoreactivity level was found after reserpine when noradrenaline was depleted from nerve cell bodies and terminals. The increase in neuropeptide Y immunoreactivity observed 24 h after reserpine injection may suggest that the neuropeptide Y-containing neuronal systems of the locus coeruleus and caudate-putamen nucleus are controlled by monoaminergic afferents.

Chronic imipramine diminishes the nuclear size of neurons in the locus coeruleus and cingular cortex but not in the hippocampus of the rat brain.

The effect of an antidepressant drug--imipramine--on the nuclear volume of the rat brain neurons was studied. Imipramine was administered per os, 10 mg/kg acutely or chronically (twice a day, for 14 days). A reduction in the nuclear volume was observed after chronic treatment in neurons of the locus coeruleus and cingular cortex, but not in the hippocampus. The diminution in the nuclear volume of the affected cells suggests a decrease in their activity.

Influence of 6-hydroxydopamine lesion of the dopaminergic nigrostriatal pathway on the muscle tone and electromyographic activity measured during passive movements.

The aim of the present study was to find out whether a 6-hydroxydopamine-induced lesion of the substantia nigra in rats would evoke muscular rigidity of the parkinsonian type. Simultaneous measurements of muscle resistance (mechanomyogram) of the hind foot to passive flexion and extension at the ankle joint, as well as of the electromyographic activity of the antagonistic muscles of the ankle joint--the gastrocnemius and tibialis anterior--in rats were carried out one, two and four weeks after bilateral injections of 6-hydroxydopamine (6.5 micrograms/microliter) into the substantia nigra. After immunohistochemical staining of brain sections for tyrosine hydroxylase, the rats were divided into two groups in which, on average, either 70% (63-80%) or 89% (81-96%) of nigral cells degenerated. Larger lesions increased the resistance (mechanomyogram) of the rat's hind leg to passive movements two weeks after 6-hydroxydopamine injection, whereas smaller lesions did not. Muscle rigidity was accompanied by an increase in the movement-induced reflex electromyographic activity in both muscles, mainly in long-latency components which are most probably influenced by supraspinal mechanisms. However, in spite of relatively large lesions of nigral dopamine cells, already four weeks after the lesion, muscle rigidity and the respective electromyographic activity diminished dramatically, which seems to result from very effective compensatory mechanisms operating in young lesioned rats. The results suggest that the muscle rigidity induced by the 6-hydroxydopamine nigral lesion seems to be a good model of parkinsonian rigidity.

Effect of acute and chronic administration of 1,2,3,4-tetrahydroisoquinoline on muscle tone, metabolism of dopamine in the striatum and tyrosine hydroxylase immunocytochemistry in the substantia nigra, in rats.

The effects of acute and chronic administration of 1,2,3,4-tetrahydroisoquinoline, an endogenous substance suspected of producing parkinsonism in humans, on the muscle tone and metabolism of dopamine in the striatum, and on the number of tyrosine hydroxylase-immunoreactive cells in the substantia nigra were investigated in rats. Muscle tone was examined using a combined mechanomyographic and electromyographic method which measured simultaneously the muscle resistance of the rat's hind foot to passive extension and flexion in the ankle joint and electromyographic activity of the antagonistic muscles of that joint: gastrocnemius and tibialis anterior. 1,2,3,4-Tetrahydroisoquinoline administered at doses of 50 and 100 mg/kg intraperitoneally for 19 days increased muscle resistance 1 h after the first injection (acute treatment), 1 h after the last injection (chronic treatment) and three days after compound withdrawal. Rigidity observed on the third day of 1,2,3,4-tetrahydroisoquinoline withdrawal was accompanied by an increased tonic (resting) electromyographic activity of the gastrocnemius and tibialis anterior muscles. At the same time, a significant reduction in the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a decrease in the dopamine level in the striatum were also found. A declining number of tyrosine hydroxylase-immunoreactive neurons in the whole substantia nigra showed a significant negative correlation with the enhanced muscle resistance, as well as with the tonic electromyographic activity recorded at rest, i.e. before the start of movements, from the gastrocnemius and tibialis anterior muscles. Our results suggest that 1,2,3,4-tetrahydroisoquinoline may be one of the endogenous substances involved in the progress of Parkinson's disease.

Effect of 6-hydroxydopamine on neuropeptide Y and corticotropin-releasing factor expression in rat amygdala.

The influence of dopaminergic denervation on neuropeptide Y and corticotropin-releasing factor-containing neurons in the amygdala was investigated in rats by examining the effects of a selective, unilateral 6-hydroxydopamine lesion of mesencephalic dopaminergic neurons in both the substantia nigra and the ventral tegmental area on these peptides and their messenger RNA expression, observed eight to 10 days after the lesion. The studies were conducted by immunocytochemical and in situ hybridization methods. Neuropeptide Y or corticotropin-releasing factor-immunoreactive neurons were counted in sections of the amygdala under a microscope, and the messenger RNA expression was measured as optical density units in autoradiograms. A significant increase in both neuropeptide Y and corticotropin-releasing factor messenger RNA expression was found in the amygdala on the lesioned side in comparison with the contralateral one, as well as with the ipsilateral side of vehicle-injected controls. Immunohistochemical studies showed that the number of neuropeptide Y-immunoreactive neurons increased in the whole amygdala on the lesioned side. At the same time, the number of corticotropin-releasing factor-immunoreactive neurons grouped in the central amygdaloid nucleus declined, and so did the staining intensity. The obtained results indicate that dopaminergic denervation stimulates the synthesis of neuropeptide Y and corticotropin-releasing factor in rat amygdala, but the peptide levels are differently regulated, which points to a diverse release of these peptides.

Motor depression and head twitches induced by IP injection of GABA.

Influence of GABA onlocomotor activity and gross behavior of mice, rats, and rabbits was studied. In mice and rats, IP GABA injection produced decreased locomotor activity, but in rats and rabbits head twitches and disturbances in body balance were seen. GABA-induced head twitches were inhibited by picrotoxin, clonidine, morphine, or cyproheptadine. Our results suggest a serotonergic component in GABA-induced head twitches, but it is not the only mechanism involved in this behavior.

Inhibitory effect of intrahippocampal NPY injection on amphetamine-induced behavioural activity.

The aim of the present study was to investigate the influence of intrahippocampal neuropeptide Y (NPY) administration on the rats' behaviour. The CA1 field or dentate area (GD) of the dorsal hippocampus was chronically implanted with intracerebral cannulae. NPY (or redistilled water in the control group) was injected bilaterally or unilaterally in a volume of 1 microliter to each hippocampus, in a dose of 1 or 2 micrograms per rat. A separate group of rats was pretreated with amphetamine (1 mg/kg, s.c.), 15 min before a bilateral microinjection of NPY (2 micrograms) into the CA1 region. Immediately after the intrahippocampal injection, the rats' behaviour was tested in an open field box. It was found that NPY did not change the locomotor and the exploratory activity after either CA1 or GD administration to non-pretreated animals. In amphetamine pretreated rats, NPY injected into the CA1 field inhibited the amphetamine induced increase in sniffing and rearing and, to a lesser extent, the number of line-crossings. The obtained results may suggest an inhibitory action of NPY in the CA1 hippocampal field on the behavioural hyperactivity.

Morphine and cocaine influence on CRF biosynthesis in the rat central nucleus of amygdala.

The central nucleus of the amygdala is a CRF-containing limbic brain site which mediates both fear-like and avoidance behaviors; moreover it has been hypothesized that atypical stress responses may contribute to compulsive drug use. Therefore, we studied in rat amygdala the level of CRF mRNA by in situ hybrydization, and the level of the peptide using immunocytochemistry after acute and chronic administration of morphine and cocaine and after their withdrawal. Acute injection of morphine (20 mg/kg i.p.) increased CRF mRNA level, but did not change significantly CRF immunoreactivity in the central nucleus of the amygdala. Chronic morphine administration significantly increased the level of CRF mRNA 3, 24 and 48 h after the last dose. Both, acute and chronic cocaine administration increased CRF mRNA, but the peptide level was decreased only after acute cocaine administration. However, in the late withdrawal (48 h after the last dose of cocaine) both mRNA and the peptide levels tended to decrease. The above data suggest that amygdalar CRF system activity is potently activated after administration of morphine and cocaine, and that activation of this system observed at the time of withdrawal from morphine may be responsible for aversion and anxiety related to these states; therefore a CRF1 receptor may be a target for prospective pharmacotherapies of the withdrawal from abused drugs.

The behavioural effects of neuropeptide Y (NPY) injection into the rat brain frontal cortex.

The aim of this study was to investigate the influence of intracortical administration of neuropeptide-Y on the behaviour of rats. One week before the behavioural test the frontal cortex of rats was chronically implanted with intracerebral cannulae. Neuropeptide-Y (or saline in the control group) was injected in a volume of 1 microliter. After treatment the behaviour of the rats was tested in an open field apparatus and a light-beam actometer. The number of line crossings, episodes of rearing, sniffing, washing and head poking were counted. The results indicate that administration of neuropeptide-Y to the frontal cortex of rats slightly increased exploratory and locomotor activity.