Intraventricular Injection Of 6-hydroxydopamine Research Articles

Intraventricular injection of 6-hydroxydopamine results in an increased number of tyrosine hydroxylase immune-positive cells in the rat cortex

Previously we have demonstrated that intraventricular injection of 6-hydroxydopamine (6-OHDA) results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells 4days after lesion (Wachter et al., 2010).To find out if these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, we performed immunohistochemistry in the rat cortex following intraventricular injection of 6-OHDA. Four days after injection we demonstrated a strong emergence of TH-positive (TH+) somata in the cortices of 6-OHDA-lesioned animals. The number of TH+ cells in the cortex of 6-OHDA-lesioned animals was 15 times higher than in sham-operated animals, where virtually no TH+ somata occurred. Combining TH immunohistochemistry with classical Nissl stain yielded complete congruency, and ∼45% of the TH+ cells co-expressed calretinin, which indicates an interneuron affiliation. There was no co-staining of TH with other interneuron markers or with glial markers such as glial fibrillary acidic protein (GFAP) or the neural stem/progenitor marker Nestin, nor could we find co-localization with the proliferation marker Ki67. However, we found a co-localization of TH with glial progenitor cell markers (Sox2 and S100β) and with polysialylated-neural cell adhesion molecule (PSA-NCAM), which has been shown to be expressed in immature, but not recently generated cortical neurons.Taken together, this study seems to confirm our previous findings with respect to a 6-OHDA-induced expression of neuronal precursor markers in cells of the rat cortex, although the TH+ cells found in this study are not identical with the potentially de-differentiated astrocytes described recently (Wachter et al., 2010).The detection of cortical cells expressing the catecholaminergic key enzyme TH might indicate a possible compensatory role of these cells in a dopamine-(DA)-depleted system. Future studies are needed to determine whether the TH+ cells are capable of DA synthesis to confirm this hypothesis.

Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine toxicity for dopaminergic neurons

Angiotensin II activates (via type 1 receptors) NAD(P)H-dependent oxidases, which are a major source of superoxide, and is relevant in the pathogenesis of several cardiovascular diseases and certain degenerative changes associated with ageing. Given that there is a brain renin–angiotensin system and that oxidative stress is a key contributor to Parkinson's disease, we investigated the effects of angiotensin II and angiotensin type 1 (AT 1) receptor antagonists in the 6-hydroxydopamine model of Parkinson's disease. Rats subjected to intraventricular injection of 6-hydroxydopamine showed bilateral reduction in the number of dopaminergic neurons and terminals. Injection of angiotensin alone did not induce any significant effect. However, angiotensin increased the toxic effect of 6-hydroxydopamine. Rats treated with the AT 1 receptor antagonist ZD 7155 and then 6-hydroxydopamine (with or without exogenous administration of angiotensin) showed a significant reduction in 6-hydroxydopamine-induced oxidative stress (lipid peroxidation and protein oxidation) and dopaminergic degeneration. Dopaminergic degeneration was also reduced by the NAD(P)H inhibitor apocynin. Angiotensin may play a pivotal role, via AT 1 receptors, in increasing the oxidative damage of dopaminergic cells, and treatment with AT 1 antagonists may reduce the progression of Parkinson's disease.

Calcitriol Protection against Dopamine Loss Induced by Intracerebroventricular Administration of 6-Hydroxydopamine

Calcitriol has been implicated as an agent that has neuroprotective effects in various animal models of diseases, possibly by upregulating glial cell line-derived neurotrophic factor (GDNF). The present study examined the neuroprotective effects of calcitriol in a model of early Parkinson's disease. Rats were treated daily with calcitriol or saline for 7 days before an intraventricular injection of 6-hydroxydopamine (6-OHDA), and then for 1 day or daily for 3(1/2) to 4 weeks after lesioning. Evoked overflow and tissue content of dopamine (DA) were determined 3(1/2) to 4 weeks post lesion. The 8-day calcitriol treatment did not attenuate 6-OHDA-induced decreases in evoked overflow of DA, nor did it protect against 6-OHDA-induced reductions in tissue levels of DA in the striatum or substantia nigra. However, the long-term calcitriol treatment did significantly increase evoked overflow of DA, as well as the amount of DA in the striatum, compared to saline treated animals. GDNF was significantly increased in the substantia nigra, but not in the striatum, of non-lesioned, calcitriol treated rats. These results suggest that long-term treatment with calcitriol can provide partial protection for dopaminergic neurons against the effects of intraventricularly administered 6-OHDA.

Effects of phencyclidine on schedule-controlled responding following neurotoxic lesions of the striatum

The effects of phencyclidine on an operant task were evaluated prior to and after neurotoxic lesions of the striatum in rats. Subjects were trained to respond on a fixed-interval 90-second schedule for water presentation. The degree to which phencyclidine disrupted responding was first evaluated (dose range 1.0–4.0 mg/kg). The subjects were then divided into three matched groups and received bilateral intraventricular injections of 6-hydroxydopamine (6-OHDA) (100 μg), kainic acid (0.25 μg), or vehicle delivered stereotaxically. 6-OHDA was used to destroy the presynaptic neurons of the nigro-striatal pathway and kainic acid was employed to destroy the postsynaptic neurons whose cell bodies are located in the striatum. Following recovery, the phencyclidine dose-response curve was repeated in the fixed-interval paradigm. It was observed that 6-OHDA-induced damage resulted in a rightward shift of the dose-response curve indicating tolerance to phencyclidine and caused a significant depletion of striatal dopamine and gamma-aminobutyric acid (GABA). Kainic acid-induced damage resulted in a leftward shift in the dose-response curve indicating sensitivity to the schedule-disruptive effects of phencyclidine and produced a significant GABA depletion. The vehicle-treated rats exhibited no shift in their sensitivity to phencyclidine. These observations indicate that the effects of phencyclidine are mediated, at least in part, by striatal dopaminergic neurons.

Bromocriptine protects mice against 6-hydroxydopamine and scavenges hydroxyl free radicals in vitro

Pretreatment with bromocriptine (5 mg/kg, i.p., 7 days) completely protected against the decrease in mouse striatal dopamine and its metabolites induced by intraventricular injection of 6-hydroxydopamine after intraperitoneal administration of desipramine, but similar pretreatment with l-DOPA/carbidopa (75/7.5 mg/kg, i.p., 7 days) showed only partial protective effect. Furthermore, in an in vitro system that generated ·OH from FeSO 4-H 2O 2, bromocriptine dose-dependently reduced the number of ·OH radicals. These findings indicate that bromocriptine has a neuroprotective effect against neurotoxins such as 6-hydroxydopamine, probably due, in part, to its hydroxyl radical scavenging activity and inhibiting effect on dopamine turnover rate. This suggests that early introduction of bromocriptine in the therapy of Parkinson's disease may be superior to treatment with l-DOPA alone.

Enhanced Efficacy of Nigral-Striatal Cotransplants in Bilaterally Dopamine-Depleted Rats: An Anatomical and Behavioral Analysis

When embryonic ventral mesencephalic tissue containing nigral dopamine (DA) neurons is transplanted into adult DA-depleted striatum, synaptic connections form and behavioral effects are observed. This study investigated the cotransplantation of embryonic striatal tissue as a means of enhancing the innervation, survival, and functional effects of nigral transplants. Rats neonatally DA-depleted, via bilateral intraventricular injections of 6-hydroxydopamine, developed turning in response to amphetamine and stress following unilateral transplantation of either nigral or combination nigral-striatal cell suspensions. Animals with cotransplants developed higher levels of turning to both stimuli and maintained these responses for a longer period of time post-transplantation, when compared with animals receiving transplants of nigral cells alone. In addition, these combination transplants required fewer dopaminergic cells to produce a strong behavioral effect on the host. Dense patches of tyrosine hydroxylase (TH)-immunoreactive fibers were observed within the cotransplants, yet no greater outgrowth of DA fibers into host striatum was detected. Amphetamine produced widespread induction of the immediate-early gene c-fos in cells of host striatum that extended beyond the transplant-derived DA innervation. After both amphetamine and stress, Fos protein was found within both types of transplants, but these F os-immunoreactive cells did not colocalize with TH-immunoreactive cells nor dense TH-immunoreactive patches within the grafts. Thus, cotransplanted embryonic striatal tissue augments the effects of ventral mesencephalic transplants, possibly by providing a trophic influence that enhances the function of the DA cells without increasing cell survival.

The role of the striatum in organizing sequences of play fighting in neonatally dopamine-depleted rats

Juvenile rats sustaining dopamine depletions by intraventricular injections of 6-hydroxydopamine (6-OHDA) as neonates were used to study the role of the striatum in controlling play fighting. As juveniles, the rats exhibited all the behavior elements typical of play fighting. However, they were more likely to use defensive tactics that shortened the playful contact between partners; and when contacting the partner, they were more likely to switch to other behaviors, such as allogrooming and sexual mounting, rather than continue with the play sequence. It is suggested here that the striatum is important for maintaining sequential organization of play fighting.

6-Hydroxydopamine induced impairment of Pavlovian conditioning in the rabbit.

This study employed bilateral, intraventricular injections of 6-hydroxydopamine (6-HDA) to examine the effects of monoamine depletion on Pavlovian conditioning of the rabbit's nictitating membrane response. 6-HDA produced dose-dependent and highly correlated decreases in the rate of acquisition of conditioned responses and in the telencephalic content of 5-HT, DA, and NE. At the highest dose of 6-HDA (1340 micrograms), 5-HT, DA, and NE were reduced by 42, 48, and 89%, respectively, and the number of trials required to achieve criterion acquisition was increased by 123%. Control experiments established that the highest dose of 6-HDA: 1) had no effect on the unconditioned nictitating membrane reflex; 2) had no effect on the threshold of the conditioned stimulus for eliciting conditioned responses; and 3) produced only a small, less than 5%, decrease in nonassociative responding. It was concluded that decreases in 5-HT, DA, and NE can impair associative learning without altering sensory or motor function.

Environmental stress increases extracellular dopamine in striatum of 6-hydroxydopamine-treated rats: in vivo microdialysis studies

Bilateral intraventricular injections of 6-hydroxydopamine depleted dopamine (DA) in striatal tissue of rats by 88%. Tail shock increased DA in striatal extracellular fluid of these rats from 10 to 17 pg/20 μl as measured by microdialysis, values less than half those seen in normal striatum. The increase was observed even in rats that were akinetic. DA in extracellular fluid after stress was negatively correlated with behavioral performance. Thus, residual nigrostriatal DA neurons are responsive to stress but do not produce normal DA levels in striatal extracellular fluid.

Dopamine released from mesencephalic transplants restores modulation of striatal acetylcholine release after neonatal 6-hydroxydopamine: An in vitro analysis

After chemical lesions which destroy the nigrostriatal dopamine pathway, transplants rich in dopamine neurons innervate the striatum and, with appropriate stimulation, drive host motor behaviors normally mediated by dopamine. We wished to determine whether dopamine released from the transplant also reinstated dopaminergic inhibition of striatal acetylcholine release. Three-day-old rat pups received bilateral intraventricular injections of 6-hydroxydopamine. Three days later cell suspensions prepared from embryonic ventral mesencephalon were injected unilaterally into the striatum. Tail pinch and amphetamine were able to elicit contralateral turning in many of these animals. Only those animals which rotated ≥5 turns/min were included for further analysis. Subsequent assays indicated that 6-hydroxydopamine had depleted striatal dopamine to 4% of control and that the transplant had increased dopamine levels to 11% of control. Superfused striatal slices were stimulated (8 Hz, 1 min) and then exposed to amphetamine (10 μ M, 3 min). The slice released dopamine, as measured by HPLC, and acetylcholine, as measured by tritium efflux after preincubation with [ 3H]choline. Moreover, the release of acetylcholine was inhibited by endogenous dopamine as indicated by the ability of sulpiride (1 μ M) to increase tritium efflux. Striatal slices prepared from lesioned animals showed a reduction in dopamine overflow in response to both electrical stimulation (0.6% of control) and amphetamine (1% of control), and a decrease in the ability of sulpiride to increase electrically evoked acetylcholine overflow (12% of control). Transplantation partially restored the dopaminergic response to electrical stimulation (21% of control), and amphetamine (15% of control) and fully restored the sulpiride-induced increase in acetylcholine overflow (98% of control). We conclude that transplanted dopaminergic neurons are able to release dopamine and to restore inhibitory control over the release of striatal acetylcholine.

Tyrosine Hydroxylase Content of Residual Striatal Dopamine Nerve Terminals Following 6‐Hydroxydopamine Administration: A Flow Cytometric Study

Fluorescence-activated cell sorting based on immunolabeling with a monoclonal antibody to tyrosine hydroxylase and a fluorescein-conjugated secondary antibody was used to identify striatal synaptosomes derived from nigrostriatal dopamine nerve terminals. The amount of tyrosine hydroxylase immunoreactivity in dopaminergic striatal synaptosomes prepared from control rats was compared to the amount in dopaminergic synaptosomes prepared from rats that had received intraventricular injections of 6-hydroxydopamine. Although the absolute number of dopaminergic synaptosomes was decreased in lesioned animals, those residual dopamine terminals present contained more tyrosine hydroxylase than did dopamine terminals from control rats. Both the decrease in the absolute number of dopamine terminals and the increase in tyrosine hydroxylase immunoreactivity in residual terminals were proportional to the extent of the lesion, as determined by measurement of striatal dopamine levels. These results suggest that an increase in the amount of tyrosine hydroxylase protein in residual terminals may represent one compensatory mechanism by which residual dopamine neurons maintain normal striatal function after partial destruction of the nigrostriatal dopamine projection.

The effects of norepinephrine depletion on cerebral blood flow in the rat

Cerebral blood flow (CBF) was measured by [ 14C]butanol indicator fractionation in 10 rats given intraventricular injections of 6-hydroxydopamine (6-OHDA) compared to 8 saline-injected controls. Rats treated with 6-OHDA displayed an 83% reduction in cortical norepinephrine (NE) levels. CBF was significantly increased in 6-OHDA-treated rats compared to controls (average whole brain blood flow of 126.0 ± 8.3 and 97.1 ± 10.6 ml·min −1·10 −2g −1 respectively, P<0.05). These studies suggest that noradrenergic innervation of the brain and cerebral microvasculature exerts a moderating effect on resting CBF.

Flow cytometric analysis and isolation of permeabilized dopamine nerve terminals from rat striatum

Fluorescence-activated cell sorting (FACS) was used to identify and isolate permeabilized dopaminergic nerve terminals from rat striatum based on immunofluorescent labeling of tyrosine hydroxylase (TH). Striatal synaptosomes were permeabilized by fixation with modified Zamboni fluid. A highly fluorescent subpopulation of particles was detected by FACS following sequential incubation with a monoclonal antibody to TH (LNC 1) and a fluorescein-conjugated secondary antibody. After correcting for nonsynaptosomal particles present in the synaptosomal fraction, analysis of these data suggested that approximately 12-15% of striatal synaptosomes were dopaminergic, consistent with previous estimates. Specific labelling by LNC 1 was decreased if synaptosomes were prepared from rats that had received intraventricular injections of 6-hydroxydopamine. The decrease in labeling was highly correlated with the extent of the lesion as determined by measurement of striatal dopamine levels, suggesting that LNC 1-labeled synaptosomes were derived from nigrostriatal dopamine terminals. In order to verify that LNC 1-labeled synaptosomes were enriched for TH, equal numbers of labeled and control synaptosomes were isolated by FACS and analyzed by SDS-PAGE. LNC 1-labeled synaptosomes were shown by Western blot techniques to be enriched 6-fold for TH compared with control synaptosomes, suggesting that the labeled population consisted almost entirely of dopaminergic synaptosomes.

Central noradrenergic neurons and vascular non-collagen protein in the initial phase of two-kidney, one-clip renovascular hypertension.

Rats had been given intraventricular injection of 6-hydroxydopamine (6-OHDA) before clipping the unilateral renal artery (2K-1C) that caused selective ablation of the central noradrenergic neurons. Central catecholamines and the in vivo incorporation of 3H-proline into vascular non-collagen protein were determined in 2K-1C rats in the acute hypertensive stage. It is suggested that increased non-collagen protein synthesis in the mesenteric artery and the low level of hypothalamic norepinephrine concentration may participate in the development of 2K-1C hypertension in rats.

Noradrenaline and neuropeptide Y innervation of the rat hypothalamus are differentially affected by 6-hydroxydopamine.

The neuropeptide Y (NPY) innervation of the hypothalamus is thought to arise largely from noradrenaline (NA) neurons of the medullary tegmentum in the rat. This view was tested in this study by analyzing the effects of intraventricular injections of 6-hydroxydopamine (6-OHDA) on hypothalamic NA and NPY innervation. 6-OHDA markedly depletes or eliminates the NA innervation of the hypothalamus, as demonstrated by dopamine-beta-hydroxylase immunohistochemistry, but does not affect the NPY innervation of the hypothalamus. These results indicate that the hypothalamic NPY innervation arises in large part from intrinsic NPY-producing neurons rather than from medullary neurons in which NA and NPY coexist.

Absence of sparing of spatial navigation, skilled forelimb and tongue use and limb posture in the rat after neonatal dopamine depletion

Depletion of caudate-putamen dopamine (DA) was produced by intraventricular injection of 6-hydroxydopamine (6-OHDA) in three day old rats. When adult, the rats were given cognitive and motor tasks sensitive to adult dopamine depletion, including: tests of cue and place spatial navigation in a swimming pool, a test of skilled forelimb use, requiring reaching for small pellets of food, a test of tongue protrusion, requiring tongue extension to lick mash from a spatula, a test of limb posture, sensorimotor tests of orienting to tactile stimulation and catalepsy. Despite apparent normal physical appearance and locomotor behavior, the rats were impaired on all tasks except orienting to tactile stimulation and catalepsy. There was a significant positive correlation between the degree of impairment on the behavioral tasks and the extent of caudate-putamen dopamine depletion. The results show that sparing of function following neonatal dopamine depletion is selective and incomplete. The results are discussed with respect to the questions of sparing of function following neonatal lesions, the heterogeneous nature of the neonatal depletions and the contributions of dopamine to complex spatial and motor behavior in the rat.

Partial reversal of asymmetry in microvessel neurochemical changes after ischemia by corpus callosum section.

Common carotid occlusion in the rat significantly decreases the density of beta-adrenergic receptors in preparations of microvessels obtained from ipsilateral and contralateral cerebral cortices. The disruption of nerve pathways connecting the hemispheres (callosal transection) partially reverses the effect of common carotid occlusion on beta-adrenergic receptor density in capillaries of the contralateral cortex. In addition, the destruction of the central noradrenergic system by intraventricular injection of 6-hydroxydopamine abolishes the effect of ischemia on capillary beta-adrenergic receptor function in both hemispheres. The results suggest that beta-adrenergic receptors located on microvessels are partially regulated by neuronal pathways and that focal ischemia induces neurochemical and functional changes in remote areas of the brain.

Sprouting of serotoninergic afferents into striatum after dopamine‐depleting lesions in infant rats: A retrograde transport and immunocytochemical study

Intraventricular injections of 6-hydroxydopamine in 3-day-old rats resulted in the near-total loss of tyrosine-hydroxylase-immunoreactive processes in the striatum when examined 2-6 months later. This destruction of dopamine (DA) afferents was accompanied by an increase in the density of serotonin (5-HT)-immunoreactive fibers in the striatum. The hyperinnervation was most striking in the rostral striatum, an area containing few 5-HT-immunoreactive fibers in control rats. Retrograde tracing, with either horse-radish peroxidase or rhodamine-labelled microspheres, indicated a significant increase in the number of neurons projecting to the rostral striatum from the dorsal raphe nucleus of lesioned animals. The increase was largely confined to the rostral extent of the dorsal raphe, and overlapped the distribution of cells labelled after injections of HRP into caudal striatum of control and lesioned animals. In sections additionally processed for immunocytochemistry, 80-90% of retrogradely labelled raphe neurons in both groups of animals were found to be 5-HT-immunoreactive. None of changes encountered in infant-lesioned rats were observed 2-4 weeks after 6-HDA was given to adult animals. These findings demonstrate that removal of DA afferents during development leads to an enlargement of the serotoninergic projection from the raphe nucleus to the striatum.

Effects of pargyline, reserpine and neurotoxin lesions on [ 3H]tryptamine binding sites in rat brain

[ 3H]Tryptamine binding sites were measured in 4 areas of rat brain following treatment with either pargyline or reserpine for 12 days, or 5 days and 30 days following intraventricular injections of 6-hydroxydopamine or 5,7-dihydroxytryptamine. Pargyline treatment decreased [ 3H]tryptamine binding in cerebral cortex, hippocampus, striatum and hypothalamus. Reserpine treatment increased binding in the cerebral cortex and hippocampus, but not in the striatum or hypothalamus. Neither 6-hydroxydopamine nor 5,7-dihydroxytryptamine altered [ 3H]tryptamine binding in any of the 4 brain areas. These results indicate that [ 3H]tryptamine binding sites in brain may be modified by drugs that can potentially affect tryptamine metabolism, and that the sites are not located on catecholamine or serotonin axons.

Implications of dopamine agonist-induced hypothermia following increased density of dopamine receptors in the mouse

An investigation was made of hypothermia induced by dopamine (DA) agonists as a model of the effect of various treatments or conditions on the sensitivity of central postsynaptic DA receptors. Selective supersensitivity of these receptors (defined as an increase in B max) was induced by means of intraventricular injections of 6-hydroxydopamine (6-OHDA) in animals pretreated with desmethylimipromine (DMI). Supersensitivity was also produced by the chronic administration of haloperidol. The supersensitivity of DA receptors induced by 6-OHDA was found to be associated with a reduced hypothermic response to apomorphine. Supersensitivity elicited by the chronic administration of haloperidol, which very probably did not produce a specific effect on the density of DA receptors but also affected serotonergic receptors, did not elicit any change in hypothermia induced by apomorphine. The results of the present study are not consistent with the view that DA receptors mediate hypothermia per se, but rather suggest that hypothermia induced by DA agonists is more complex, probably involving serotonergic receptors primarily, though other factors may also be contributory. Furthermore, the results of the present study suggest that the functional significance of supersensitivity of DA receptors induced by 6-OHDA versus chronic treatment with haloperidol may be quite different, depending upon the effector system examined.