Apomorphine-induced Rotational Behavior Research Articles

Attenuation of Paraquat-Induced Dopaminergic Toxicity on the Substantia Nigra by (−)-Deprenyl in Vivo

(−)-Deprenyl (DEP) had been shown to slow of progression of Parkinson's disease (PD). The present study sought to determine whether DEP would attenuate the nigrostriatal system damage induced by intranigral administration of the herbicide paraquat (PQ) as a model of parkinsonism in vivo. Neurochemical and behavioral observations of Wistar rats were the focus of our study. In the neurochemical observation, the PQ injected in the rats caused dose-dependent depletion of dopamine (DA) in the ipsilateral striata. The coadministration of DEP with PQ partially increased the striatal DA level. The prediction of the striatal DA levels was calculated by regression coefficients obtained from multiple linear regression (r2 = 0.82): DA level (% of control) = 103.34 − 9.58 PQ (nmol) + 0.79 DEP (nmol). It was demonstrated that the high dose of 20 nmol DEP could significant attenuate the PQ (5 nmol)-elicited dopaminergic toxicity (p < 0.05). In the behavioral observation, the intranigral injection of PQ into the rats caused a rotation behavior contralateral to the lesioned side in response to apomorphine administration (0.5 mg/kg, sc). This apomorphine-induced rotational behavior could also be attenuated significantly by coadministration of DEP (20 nmol) and PQ (5 nmol) compared with PQ-treated (5 nmol) animals (p < 0.05). The above observations indicate that DEP could provide a protective effect on the moderate injury elicited by PQ toxicity of the nigrostrital dopaminergic system. DEP might be a useful therapeutic agent in treating patients with early-stage PD.

Microencapsulated Bovine Chromaffin Cell Xenografts into Hemiparkinsonian Rats: A Drug-Induced Rotational Behavior and Histological Changes Analysis

Bovine chromaffin cells were microencapsulated within alginate-polylysine-alginate (APA) membranes. Microencapsulated bovine chromaffin cells as well as unencapsulated cells and empty microcapsules were grafted into the brain of hemiparkinsonian rats with 6-hydroxydopamine (6-OHDA) lesions. Apomorphine-induced rotational behavior of the host animals and the survival of the grafted chromaffin cells were examined after transplantation. The animals receiving microencapsulated bovine chromaffin cells showed a significant decrease (17.6--35.6%) in apomorphine-induced rotation 1 week postimplantation that remained stable for the 10 month test period. Fluorescent histochemistry further revealed that microencapsulation increased the chromaffin cell survival with only a minimum host reaction for up to 10 months posttransplantation while the survival of free, unencapsulated chromaffin cells was only modest and was accompanied by a large inflammatory response. The reduction of apomorphine-induced rotations was correlated with the survival of bovine chromaffin cells in the host brain. The data indicate that encapsulation of bovine chromaffin cells in APA membranes reduces the host immune response to the xenograft and prolongs the viability of the grafted cells.

Intranigral transplantation of solid tissue ventral mesencephalon or striatal grafts induces behavioral recovery in 6-OHDA-lesioned rats

Parkinson’s disease (PD) is characterized by a degeneration of the dopamine (DA) pathway from the substantia nigra (SN) to the basal forebrain. Prior studies in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats have primarily concentrated on the implantation of fetal ventral mesencephalon (VM) into the striatum in attempts to restore DA function in the target. We implanted solid blocks of fetal VM or fetal striatal tissue into the SN to investigate whether intra-nigral grafts would restore motor function in unilaterally 6-OHDA-lesioned rats. Intra-nigral fetal striatal and VM grafts elicited a significant and long-lasting reduction in apomorphine-induced rotational behavior. Lesioned animals with ectopic grafts or sham surgery as well as animals that received intra-nigral grafts of fetal cerebellar cortex showed no recovery of motor symmetry. Subsequent immunohistochemical studies demonstrated that VM grafts, but not cerebellar grafted tissue expressed tyrosine hydroxylase (TH)-positive cell bodies and were associated with the innervation by TH-positive fibers into the lesioned SN as well as adjacent brain areas. Striatal grafts were also associated with the expression of TH-positive cell bodies and fibers extending into the lesioned SN and an induction of TH-immunolabeling in endogenous SN cell bodies. This finding suggests that trophic influences of transplanted fetal striatal tissue can stimulate the re-expression of dopaminergic phenotype in SN neurons following a 6-OHDA lesion. Our data support the hypothesis that a dopaminergic re-innervation of the SN and surrounding tissue by a single solid tissue graft is sufficient to improve motor asymmetry in unilateral 6-OHDA-lesioned rats.

Failure of Neuroprotection by Embryonic Striatal Grafts in a Double Lesion Rat Model of Striatonigral Degeneration (Multiple System Atrophy)

In the present experiment we studied the ability of embryonic striatal grafts to protect against striatal quinolinic acid (QA)-induced excitotoxicity in a previously established double lesion rat model of striatonigral degeneration (SND), the neuropathological substrate of parkinsonism associated with multiple system atrophy (MSA). Male Wistar rats received under halothane inhalation anesthesia a 6-hydroxydopamine 6-OHDA injection into the left medial forebrain bundle. Four to 5 weeks later apomorphine-induced rotation behavior was tested. Rats were divided into two treatment groups receiving either embryonic striatal cell suspensions or sham injections. Apomorphine-induced rotation behavior was retested 2 and 4 weeks after the grafting procedure. Following the rotation test animals of the striatal and sham graft group received a stereotaxic injection of 150 nmol QA. Again rotation behavior was assessed 2 and 4 weeks after lesioning. Brains were then processed to dopamine reuptake ([3H]mazindol), dopamine D1 ([3H]SCH23390), and D2 ([3H]spiperone) receptor autoradiography. Gliosis was detected using [3H]PK11195, a marker for peripheral benzodiazepine binding sites. Behavioral and autoradiographic analysis failed to show striatal protection in 6-OHDA prelesioned animals receiving embryonic striatal grafts. These findings indicate that beneficial protective effects of striatal grafts implanted into host striatum prior to excitotoxic insults are abolished in the presence of severe dopaminergic denervation. Our present results are relevant to future applications of neural grafting in MSA-SND.

Development of an adenoviral vector for intracerebral delivery of the dopamine D 2 receptor

The age-related loss of striatal dopamine D 2 receptors (D 2R) has been observed in numerous species, including rodents, monkeys, and man, and is partly responsible for impaired motor function in aged mammals. We have developed an adenoviral vector designed for intracerebral transfer of cDNA for D 2R. Results of in vitro studies demonstrated that the vector produced abundant message for D 2R and that the vector was membrane bound and capable of binding appropriate ligand. Results of in vivo studies provided clear evidence of D 2R production when injected into the striatum of rats. The D 2R produced were capable of binding appropriate ligand. In addition, evidence of functional receptors was produced by demonstrating apomorphine-induced rotational behavior in rats receiving a unilateral injection of the vector. Despite these successes, we have been unable to demonstrate improvement in the motor behavior of aged rats receiving bilateral injections of the vector. A major problem with this vector as with similar adenoviral vectors is the loss of expression beginning 3–5 days after injection to undetectable levels at 21 days. Because of the lack of motor functional effects in aged rats and the loss of expression of the vector, other strategies for development of the vector are being pursued. Regarding functional effects, we have examined the feasibility of manipulating hippocampal acetylcholine (ACh) release through D 2R manipulation to improve memory performance. Using microdialysis, we have demonstrated in vivo in rats that treatment with a D 2R agonist increases hippocampal ACh release while treatment with a D 2R antagonist attentuates this effect as well as impairs performance in a complex maze task. In addition, a D 2R null mutant mouse is being used to examine possible therapeutic effects of the vector. These mice show specific motor deficits. Recent studies using positron emission tomography have also demonstrated the feasibility of in vivo imaging of the vector. Thus, use of adenoviral vectors specific for neurotransmitter receptors can provide a highly useful research tool for examining age-related alterations in behavioral function and a possible strategy for therapeutic intervention.

Behavioral effects of intra-nigral microinjections of manganese chloride: Interaction with nitric oxide

1. 1. Microinjection of manganese chloride (MnCl 2) into the rat substantia nigra pars compacta (SNc) induces a neurodegenerative process manifested by apomorphine-induced rotational behavior. Manganese intoxication produces a parkinsonism-like phenotype in humans. 2. 2. In addition to motor control the substantia nigra has also been proposed to be related to epilepsy and emotional behavior. 3. 3. Although nitric oxide (NO) participation in neurodegenerative processes is still questioned, neurons stained for NAPDH-diaphorase, a marker of NO-producing cells, are spared in several experimental neuronal lesions. Additionally, NO has also been suggested to participate in motor control. 4. 4. The objective of this study was to analyze the effects of MnCl 2 -induced nigral degeneration in audiogenic seizure susceptibility, anxiety and motor activity. We also analyzed if NO synthesis inhibition (N G-nitro-L-arginine 25 mg/Kg twice a day for 4 days) modifies MnCl 2 — induced neurodegenerative process. 5. 5. MnCl 2 (50 μg) microinjection into the SNc caused a statistical significant higher number of apomorphine (0.75 mg/kg s.c.)-induced rotations. No sensitization to audiogenic seizure was found but the lesion induced an increase of open arm exploration in the elevated plus maze, suggesting an anxiolytic effect. 6. 6. The MnCl 2-nigral lesion was accompanied by an increased number of NADPH-d positive neurons in the ipsilateral SNc and striatum (both sides). NO synthesis inhibition potentiated the MnCl 2-nigral lesion and reversed the NADPH-d cell number increase. 7. 7. The present results show that MnCl 2-nigral lesion can influence emotional behavior and suggest that NO may modify the progression of manganese-induced degenerative process.

Effect of Acute [formula omitted]-Dopa Pretreatment on Apomorphine-Induced Rotational Behavior in a Rat Model of Parkinson's Disease

Currently, reduction of apomorphine-induced rotational behavior in the 6-hydroxydopamine (6-OHDA) lesioned rat is the most utilized drug-induced paradigm for assessing functional efficacy in a rat model of Parkinson's disease (PD). Any clinically predictive animal model of PD should include a positive response to l-dopa, the standard pharmacotherapy for PD. However, the acute interaction between l-dopa and apomorphine has never been studied to determine if l-dopa pretreatment could reduce apomorphine-induced rotational behavior in a 6-OHDA lesioned rat. The present study was designed to explore whether, indeed, pretreatment with subrotational doses of l-dopa could inhibit apomorphine-induced rotations. The data indicate that l-dopa significantly reduced apomorphine-induced rotational behavior only at one dose (5.0 mg/kg) for 12 min. Based on these and other data, it is concluded that although the apomorphine-induced rotational paradigm may continue to be utilized as one additional indicator of efficacy in the 6-OHDA rat model of PD, it is not in itself a completely valid functional assay.

Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hydroxydopamine lesion.

The neurotoxin 6-hydroxydopamine (6-OHDA) has been used extensively in animal models of Parkinson's disease. Typically, rodents develop severe unilateral movement deficiencies coupled with apomorphine-induced rotation behavior at least 1 week after an ipsilateral 6-OHDA lesion of the nigrostriatal dopamine (DA) system. The short-term morphological effects of 6-OHDA have not been determined in detail, however, and the exact process by which neurons die has not been elucidated. Thus, novel degenerative markers were used to determine the temporal pattern of acute phenotypic and degenerative alterations following a unilateral 6-OHDA injection into the medial forebrain bundle of adult rats. 6-Hydroxydopamine administration resulted in an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining as early as 6 hours postlesion. Staining for FluoroJade, a marker of neuronal degeneration, was evident at all time points examined but was maximal at 48 hours. Loss of tyrosine hydroxylase (TH) immunoreactivity began in axons at 6 hours, and progressed to cell bodies at later time points postlesion. Morphological examination of these neurons supported the conclusion of their death via apoptosis. Thus, whereas behavioral manifestations typically become evident 1 week or more following a 6-OHDA lesion, it is evident that nigral cell degeneration begins much earlier. This suggests multiple therapeutic possibilities, including the prevention of apoptosis, in affected neurons.

Autoradiographic study of striatal dopamine re-uptake sites and dopamine D 1 and D 2 receptors in a 6-hydroxydopamine and quinolinic acid double-lesion rat model of striatonigral degeneration (multiple system atrophy) and effects of embryonic ventral mesencephalic, striatal or co-grafts

The influence of embryonic mesencephalic, striatal and mesencephalic/striatal co-grafts on amphetamine- and apomorphine-induced rotation behaviour was assessed in a rat model of multiple system atrophy/striatonigral degeneration type using dopamine D 1 ([ 3H]SCH23390) and D 2 ([ 3H]spiperone) receptor and dopamine re-uptake ([ 3H]mazindol) autoradiography. Male Wistar rats subjected to a sequential unilateral 6-hydroxydopamine lesion of the medial forebrain bundle followed by a quinolinic acid lesion of the ipsilateral striatum were divided into four treatment groups, receiving either mesencephalic, striatal, mesencephalic/striatal co-grafts or sham grafts. Amphetamine- and apomorphine-induced rotation behaviour was recorded prior to and up to 10 weeks following transplantation. 6-Hydroxydopamine-lesioned animals showed ipsiversive amphetamine-induced and contraversive apomorphine-induced rotation behaviour. Amphetamine-induced rotation rates persisted after the subsequent quinolinic acid lesion, whereas rotation induced by apomorphine was decreased. In 11 of 14 animals receiving mesencephalic or mesencephalic/striatal co-grafts, amphetamine-induced rotation scores were decreased by >50% at the 10-week post-grafting time-point. In contrast, only one of 12 animals receiving non-mesencephalic (striatal or sham) grafts exhibited diminished rotation rates at this time-point. Apomorphine-induced rotation rates were significantly increased following transplantation of mesencephalic, striatal or sham grafts. The largest increase of apomorphine-induced rotation rates approaching post-6-hydroxydopamine levels were observed in animals with striatal grafts. In contrast, in the co-graft group, there was no significant increase of apomorphine-induced rotation compared to the post-quinolinic acid time-point. Morphometric analysis revealed a 63–74% reduction of striatal surface areas across the treatment groups. Striatal [ 3H]mazindol binding on the lesioned side (excluding the demarcated graft area) revealed a marked loss of dopamine re-uptake sites across all treatment groups, indicating missing graft-induced dopaminergic re-innervation of the host. In eight (73%) of the 11 animals with mesencephalic grafts and reduced amphetamine-induced circling, discrete areas of [ 3H]mazindol binding (“hot spots”) were observed, indicating graft survival. Dopamine D 1 and D 2 receptor binding was preserved in the remaining lesioned striatum irrespective of treatment assignment, except for a significant reduction of D 2 receptor binding in animals receiving mesencephalic grafts. “Hot spots” of dopamine D 1 and D 2 receptor binding were observed in 10 (83%) and nine (75%) of 12 animals receiving striatal grafts or co-grafts, consistent with survival of embryonic primordial striatum grafted into a severely denervated and lesioned striatum. Our study confirms that functional improvement may be obtained from embryonic neuronal grafts in a double-lesion rat model of multiple system atrophy/striatonigral degeneration type. Co-grafts appear to be required for reversal of both amphetamine- and apomorphine-induced rotation behaviour in this model. We propose that the partial reversal of amphetamine-induced rotation asymmetry in double-lesioned rats receiving mesencephalic or mesencephalic/striatal co-grafts reflects non-synaptic graft-derived dopamine release. The changes of apomorphine-induced rotation following transplantation are likely to reflect a complex interaction of graft- and host-derived striatal projection pathways and basal ganglia output nuclei. Further studies in a larger number of animals are required to determine whether morphological parameters and behavioural improvement in the neurotransplantation multiple system atrophy rat model correlate.

Synthesis and dopamine receptor modulating activity of substituted bicyclic thiazolidine lactam peptidomimetics of L-prolyl-L-leucyl-glycinamide.

6-Substituted bicyclic thiazolidine lactam peptidomimetics of Pro-Leu-Gly-NH(2) (1) were synthesized to test the hypothesis that incorporation of a hydrophobic side chain into the bicyclic thiazolidine lactam scaffold would further enhance the dopamine receptor modulating activity of such peptidomimetics. The substituents employed were the isobutyl, butyl, and benzyl groups to give peptidomimetics 3-5, respectively. These peptidomimetics were evaluated in vivo as modulators of apomorphine-induced rotational behavior in the 6-hydroxydopamine-lesioned rat model of hemiparkinsonism and were compared with the unsubstituted bicyclic thiazolidine lactam Pro-Leu-Gly-NH(2) peptidomimetic 2. Peptidomimetics 3-5 each affected rotational behavior in a bell-shaped dose-response relationship producing maximal increases of 44% (1 microgram/kg,ip), 56% (0.1 microgram/kg,ip), and 30% (1 microgram/kg, ip), respectively. In comparison, unsubstituted peptidomimetic 2 increased rotational behavior by only 23% at a dose of 0.1 microgram/kg, ip.

Design, synthesis, and dopamine receptor modulating activity of spiro bicyclic peptidomimetics of L-prolyl-L-leucyl-glycinamide.

In the present study, the synthesis of the 5.5.6. and 5.6.5. spiro bicyclic lactam PLG peptidomimetics, compounds 3 and 4, respectively, was undertaken. These peptidomimetics were designed to examine the following: (1) the effect that changing the size of the thiazolidine and lactam ring systems would have on the ability of these systems to mimic the type-II beta-turn and (2) the effect that these structural perturbations would have on the ability of the peptidomimetics to modulate dopamine receptors. Through the use of the [3H]spiroperidol/N-propylnorapomorphine (NPA) dopamine D2 receptor competitive binding assay, 3 and 4, at a concentration of 100 nM, decreased the dissociation constant of the high-affinity state of the dopamine receptor for the agonist. These effects were observed when either Gpp(NH)p was absent or present and they were comparable to those produced by PLG at a concentration of 1 microM. Peptidomimetics 3 and 4 also increased the percentage of D2 receptors that existed in the high-affinity state. Even with Gpp(NH)p present, 3 and 4 were able to return the RH/RL ratios to values observed in the respective controls where Gpp(NH)p was absent. Furthermore, both peptidomimetics were able to attenuate the Gpp(NH)p-induced shift to the low-affinity state to a greater extent than PLG. Peptidomimetics 3 and 4 were evaluated in vivo as modulators of apomorphine-induced rotational behavior in the 6-hydroxydopamine-lesioned rat model of hemiparkinsonism, and each affected the rotational behavior in a bell-shaped dose-response relationship producing increases of 95 +/- 31% (0.01 mg/kg, ip) and 88 +/- 14% (0.001 mg/kg, ip), respectively. In comparison, the previously reported 5.5.5. spiro bicyclic lactam 2 increased rotational behavior by 25 +/- 11% (0.01 mg/kg, ip).

Modulation of Rat Rotational Behavior by Direct Gene Transfer of Constitutively Active Protein Kinase C into Nigrostriatal Neurons

The modulation of motor behavior by protein kinase C (PKC) signaling pathways in nigrostriatal neurons was examined by using a genetic intervention approach. Herpes simplex virus type 1 (HSV-1) vectors that encode a catalytic domain of rat PKCbetaII (PkcDelta) were developed. PkcDelta exhibited a constitutively active protein kinase activity with a substrate specificity similar to that of rat brain PKC. As demonstrated in cultured sympathetic neurons, PkcDelta caused a long-lasting, activation-dependent increase in neurotransmitter release. In the rat brain, microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of pkcDelta in a small percentage of nigrostriatal neurons (approximately 0.1-2%) was sufficient to produce a long-term (>/=1 month) change in apomorphine-induced rotational behavior. Nigrostriatal neurons were the only catecholaminergic neurons that contained PkcDelta, and the amount of rotational behavior was correlated with the number of affected nigrostriatal neurons. The change in apomorphine-induced rotational behavior was blocked by a dopamine receptor antagonist (fluphenazine). D2-like dopamine receptor density was increased in those regions of the striatum innervated by the affected nigrostriatal neurons. Therefore, this strategy enabled the demonstration that a PKC pathway or PKC pathways in nigrostriatal neurons modulate apomorphine-induced rotational behavior, and altered dopaminergic transmission from nigrostriatal neurons appears to be the affected neuronal physiology responsible for the change in rotational behavior.

Characterization of Intrastriatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer of Human Tyrosine Hydroxylase and Human GTP-Cyclohydrolase I in a Rat Model of Parkinson’s Disease

To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.

Significant behavioral recovery in Parkinson's disease model by direct intracerebral gene transfer using continuous injection of a plasmid DNA-liposome complex.

As an alternative to virus-mediated gene transfer, we previously demonstrated a simple, safe, and efficient transfer of foreign gene into the central nervous system using continuous injection of a plasmid DNA-cationic liposome complex. To explore whether this approach can be applied to the treatment of certain neurological disorders, we used an experimental model of Parkinson's disease (PD) in the present study. Following continuous injection for 7 days, tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) genes carried by a bovine papilloma virus-based plasmid vector were efficiently introduced into glial cells in the striatum of 6-hydroxydopamine-lesioned rats. Significant recovery in apomorphine-induced rotational behavior of PD models was obtained by transfection of TH gene and this effect continued for up to 5 weeks after injection. Moreover, cotransfection of TH with AADC genes was readily accomplished by this procedure and resulted in a greater and longer-lasting improvement of apomorphine-induced rotational behavior than was achieved by transfection of TH gene alone. We suggest that this approach is a controllable and manageable alternative to other methods of gene therapy for the treatment of PD.

Intracerebral transplantation of testis-derived sertoli cells promotes functional recovery in female rats with 6-hydroxydopamine-induced hemiparkinsonism.

Recently, we demonstrated amelioration of behavioral deficits associated with 6-hydroxydopamine-induced hemiparkinsonism by transplanting rat testis-derived Sertoli cells into adult male rat brains. In the present study, we used adult female hemiparkinsonian rats to investigate whether the beneficial effects of transplantation of Sertoli cells may be differentially affected by gender of the animal transplant recipient. At 1 month posttransplantation, animals transplanted with Sertoli cells showed functional recovery as revealed by significant reductions in apomorphine-induced rotational behavior and asymmetrical elevated body swing behavior. Control animals that received medium alone did not display any visible behavioral recovery. These results suggest that transplantation of Sertoli cells is not male hormone-dependent and further support the use of these cells as a graft source for Parkinson's disease and other neurological disorders.

Design, synthesis, and dopamine receptor modulating activity of diketopiperazine peptidomimetics of L-prolyl-L-leucylglycinamide.

The diketopiperazine "C5" conformational mimic has been incorporated into the L-prolyl-L-leucylglycinamide (PLG, 1) structure and into the bicyclic lactam PLG peptidomimetic structure 3 to give compounds 5 and 6, respectively. These analogues were designed to explore the idea that the N-terminal "C5" conformation, which was found in the crystal structure of 2 and which was mimicked in 4 by the diketopiperazine function, was a factor in the high potency of these two agents. Through the use of the [3H]spiroperidol/N-propylnorapomorphine (NPA) D2 receptor competitive binding assay, both 5 and 6 were found to increase the affinity of the dopamine receptor for agonists and both were found to increase the percentage of D2 receptors which existed in the high-affinity state. These effects were observed when Gpp(NH)p was either absent or present, and they were analogous to the effects observed previously for PLG and the PLG peptidomimetics 2 and 4. However, the potency seen with 5 and 6 was less than that seen for 2 and 4, suggesting that while the N-terminal "C5" conformation may play a role in the potency of the gamma-lactam peptidomimetics of PLG, it does not appear to be the primary factor. In the 6-hydroxydopamine-lesioned animal model of Parkinson's disease, 5 altered apomorphine-induced rotational behavior in a dose-dependent manner. The maximum effect occurred at a dose of 0.01 mg/kg i.p. and resulted in a 52.27 +/- 13.96% (p < 0.001, n = 7) increase in rotations compared to apomorphine administered alone.

Long-term enhanced chromaffin cell survival and behavioral recovery in hemiparkinsonian rats with co-grafted polymer-encapsulated human NGF-secreting cells.

The transplantation of genetically modified cells represents one potential means of delivering trophic factors to the brain to support the survival of host neurons and to increase the survival of co-grafted cells. The present study examined the ability of encapsulated baby hamster kidney (BHK) fibroblasts, which were genetically modified to produce human nerve growth factor (hNGF), to provide long-term trophic support to co-grafted adrenal chromaffin cells. Following polymer encapsulation, BHK-hNGF cells were grafted into the striatum of hemiparkinsonian rats together with unencapsulated adrenal medullary chromaffin cells. Secretion of hNGF from the encapsulated cells, morphology of these cells, apomorphine-induced rotational behavior of the host animals, and survival of the co-grafted chromaffin cells were examined 1, 6, and 12 months after transplantation. Analysis of retrieved capsules revealed that the BHK cells survived and continued to release hNGF at a level of 2-3 ng/day even 12 months after transplantation. Although the animals receiving adrenal medulla alone did not show recovery of apomorphine-induced rotational behavior, the animals receiving adrenal medulla intrastriatal hNGF-secreting cells showed a significant decrease (40-50%) in apomorphine-induced rotation within 1 month postimplantation that remained stable for the 12-month test period. Tyrosine hydroxylase immunocytochemistry further revealed that while survival of chromaffin cells without hNGF support was poor, co-grafting of adrenal medulla and BHK-hNGF cells dramatically 926- to 32-fold) increased chromaffin cell survival 1, 6, and 12 months after transplantation. These results demonstrate that (1) encapsulated BHK cells survive for extended periods of time in vivo while continuing to secrete hNGF, (2) the continued secretion of hNGF provides trophic support for co-grafted adrenal chromaffin cells, and (3) the increased chromaffin cell survival is associated with long-term, stable behavioral recovery. These data further support the potential use of this approach for treating Parkinson's disease.

In vivo microdialysis studies on somatodendritic dopamine release in the rat substantia nigra: effects of unilateral 6-OHDA lesions and GDNF.

Dopamine (DA) release and metabolism within the substantia nigra (SN) were studied in normal rats, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions, and 6-OHDA-lesioned rats treated with glial cells line-derived neurotrophic factor (GDNF). Animals with > 99% DA depletions, as determined by apomorphine-induced circling behavior, also showed significant deficits in several measures of spontaneous motor activity. In vivo microdialysis recordings in the SN were carried out in normal and unilaterally 6-OHDA-lesioned rats. Basal levels of DNA were detectable only in the dialysates of normal animals, and basal levels of t he primary DA metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were found to be significantly reduced in the SN of 6-OHDA-lesioned animals. In the presence of d-amphetamine, either alone or in combination with potassium, significant reductions in DA release were observed in the SN of 6-OHDA-lesioned animals compared to normal animals. Potassium-evoked DA release alone was not significantly different between the groups. A single intranigral administration of GDNF into 6-OHDA-lesioned animals elicited a significant reduction in apomorphine-induced rotation behavior and a significant increase in spontaneous motor activities. These behavioral changes were apparent at 1 week and persisted through 4 weeks following treatment. In vivo microdialysis showed that, although DA metabolism was altered 1 week following GDNF treatment, DA release was not significantly affected until 4 weeks following treatment.

Glial cell line-derived neurotrophic factor attenuates behavioural deficits and regulates nigrostriatal dopaminergic and peptidergic markers in 6-hydroxydopamine-lesioned adult rats: comparison of intraventricular and intranigral delivery

The effects of intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor were tested on low dose (0.05 mg/kg) apomorphine-induced rotations and tyrosine hydroxylase activity in the substantia nigra and striatum of stable 6-hydroxydopamine-lesioned rats. In addition, we determined if 6-hydroxydopamine lesions in the absence or presence of treatment affected neuropeptide (substance P, met-enkephalin, dynorphin) content in the striatum. Glial cell line-derived neurotrophic factor, when administered intranigrally, prevented apomorphine-induced rotational behaviour for 11 weeks following a single injection. In comparison, intraventricularly-administered glial cell line-derived neurotrophic factor produced a transient reduction in rotational behaviour that lasted for two to three weeks following a single injection. We also show that rotational behaviour is reduced following each subsequent intraventricular injection of glial cell line-derived neurotrophic factor given every six weeks, a time-point when baseline rotation deficits were re-established. Intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor significantly reduced weight gain in all 6-hydroxydopamine-lesioned rats in this study. Following behavioural analysis where a confirmed improvement of behaviour was established, tissues were dissected for neurochemical analysis. In lesioned rats with intranigral injections of glial cell line-derived neurotrophic factor, significant increases of nigral, but not striatal tyrosine hydroxylase activity were measured. Additionally, 6-hydroxydopamine lesions significantly increased striatal dynorphin (61–139%) and met-enkephalin (81–139%), but not substance P levels. In these rats, intranigrally-administered glial cell line-derived neurotrophic factor injections reversed lesion-induced increases in nigral dynorphin A levels and increased nigral dopamine levels, but did not alter nigral met-enkephalin or substance P levels nor striatal dopamine levels. In lesioned rats with intraventricular injections of administered glial cell line-derived neurotrophic factor, tyrosine hydroxylase ispilateral to the lesion was increased in the substantia nigra, but not in the striatum. Intraventricularly-administered glial cell line-derived neurotrophic factor did not reverse lesion-induced increases in nigral dynorphin A or met-enkephalin levels nor did glial cell line-derived neurotrophic factor affect substance P levels in the striatum. These results suggest that in an animal model of Parkinson's disease, the neurotrophic factor glial cell line-derived neurotrophic factor reverses behavioural consequences of 6-hydroxydopamine administration, an effect that may involve both dopaminergic and peptidergic neurotransmission.

Effects of α-phenyl-tert-butyl nitrone on neuronal survival and motor function following intrastriatal injections of quinolinate or 3-nitropropionate

We have investigated the neuroprotective effects of the the spin-trapping agent α-phenyl-tert-butyl nitrone on striatal lesions produced by local injections of the excitotoxin quinolinate or the mitochondrial toxin 3-nitropropionate. We have assessed both the behavioural and morphological consequences of the lesion. Thus, we tested paw-reaching ability and amphetamine- and apomorphine-induced rotational behaviour in lesioned rats with or without α-phenyl-tert-butyl nitrone treatment, and also explored the relationship between the outcome of the behavioural studies and the extent of the lesion. In the morphological analysis, we chose dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32,000 as a specific marker for striatal neurons. The paw-reaching ability of rats with the quinolinate and 3-nitropropionate lesions was significantly impaired compared to normal control animals. Treatment with α-phenyl-tert-butyl nitrone significantly ameliorated the paw-reaching deficits produced by the quinolinate lesion, whereas the 3-nitropropionate-induced deficits were unaffected by α-phenyl-tert-butyl nitrone. Both quinolinate and 3-nitropropionate lesions resulted in a rotation asymmetry towards the lesioned side in response to both amphetamine and apomorphine. In the quinolinate lesion model, the α-phenyl-tert-butyl nitrone treatment resulted in a less marked motor asymmetry in response to both drugs. By contrast, α-phenyl-tert-butyl nitrone did not significantly reduce the drug-induced rotation asymmetry in rats with a 3-nitropropionate lesion. Morphological analyses disclosed that α-phenyl-tert-butyl nitrone significantly increased the size of the spared striatum in the quinolinate lesions, but only caused a non-significant trend towards an attenuation of the 3-nitropropionate lesions. The behavioural deficits were inversely correlated to the size of the spared residual striatum. The intrastriatal injection of 3-nitropropionate, unlike the injection of quinolinate, caused degeneration of the nigrostriatal dopamine system as well as of transverse fibre bundles of the internal capsule in the striatum, in addition to the striatal lesion. The behavioural studies revealed that the combination of multiple lesions seen in 3-nitropropionate-lesioned rats significantly exacerbated paw-reaching deficits and amphetamine-induced rotation asymmetry.In conclusion, α-phenyl-tert-butyl nitrone attenuated behavioural and morphological consequences of striatal lesions induced by local injections of quinolinate, but not of 3-nitropropionate. Deficits in behavioural tests of striatal function reflected well the extent of striatal lesion. The intrastriatal injection of 3-nitropropionate led to degeneration of both intrinsic striatal neurons and the nigrostriatal dopaminergic system, suggesting that this lesion may provide an animal model of a form of multiple system atrophy rather than Huntington's disease.