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Neurological diseases such as ischemic stroke can be debilitating and have limited treatments available. The progestin Nestorone® (segesterone acetate) has been evaluated for use in birth control and hormone replacement therapy due to its potency and high affinity for the progesterone receptor. Interestingly, Nestorone also exerts neuroprotection in animals afflicted with various central nervous system diseases, including stroke, which implicates its potential for treating these maladies in clinical settings. In fact, a recent Brain Research paper by Tanaka and colleagues demonstrates Nestorone's ability to reduce infarct sizes and preclude functional impairments in rats subjected to ischemic stroke. This commentary highlights Nestorone's properties as a progestin, its neuroprotective capabilities in animal studies, and how the Tanaka team's findings and previous clinical trials contribute to Nestorone's translation into a therapeutic agent for stroke and other neurological diseases.

Amyotrophic lateral sclerosis (ALS) was recently recognized as a neurovascular disease. Accumulating evidence demonstrated blood-spinal-cord barrier (BSCB) impairment mainly via endothelial cell (EC) degeneration in ALS patients and animal models. BSCB repair may be a therapeutic approach for ALS. We showed benefits of human bone marrow endothelial progenitor cell (hBMEPC) transplantation into symptomatic ALS mice on barrier restoration; however, cellular mechanisms remain unclear. The study aimed to characterize hBMEPCs in vitro under normogenic conditions. hBMEPCs were cultured at different time points. Enzyme-linked immunosorbent assay (ELISA) was used to detect concentrations of angiogenic factors (VEGF-A, angiogenin-1, and endoglin) and angiogenic inhibitor endostatin in conditioned media. Double immunocytochemical staining for CD105, ZO-1, and occludin with F-actin was performed. Results showed predominantly gradual significant post-culture increases of VEGF-A and angiogenin-1 levels. Cultured cells displayed distinct rounded or elongated cellular morphologies and positively immunoexpressed for CD105, indicating EC phenotype. Cytoskeletal F-actin filaments were re-arranged according to cell morphologies. Immunopositive expressions for ZO-1 were detected near inner cell membrane and for occludin on cell membrane surface of adjacent hBMEPCs. Together, secretion of angiogenic factors by cultured cells provides evidence for a potential mechanism underlying endogenous EC repair in ALS through hBMEPC transplantation, leading to restored barrier integrity. Also, ZO-1 and occludin immunoexpressions, confirming hBMEPC interactions in vitro, may reflect post-transplant cell actions in vivo.

Neurological diseases such as ischemic stroke can be debilitating and have limited treatments available. The progestin Nestorone® (segesterone acetate) has been evaluated for use in birth control and hormone replacement therapy due to its potency and high affinity for the progesterone receptor. Interestingly, Nestorone also exerts neuroprotection in animals afflicted with various central nervous system diseases, including stroke, which implicates its potential for treating these maladies in clinical settings. In fact, a recent Brain Research paper by Tanaka and colleagues demonstrates Nestorone's ability to reduce infarct sizes and preclude functional impairments in rats subjected to ischemic stroke. This commentary highlights Nestorone's properties as a progestin, its neuroprotective capabilities in animal studies, and how the Tanaka team's findings and previous clinical trials contribute to Nestorone's translation into a therapeutic agent for stroke and other neurological diseases.

Vascular pathology, including blood-brain/spinal cord barrier (BBB/BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage, identifying a neurovascular disease signature for ALS. However, BBB/BSCB competence in sporadic ALS (SALS) is still undetermined. In this study, BBB/BSCB integrity in postmortem gray and white matter of medulla and spinal cord tissue from SALS patients and controls was investigated. Major findings include (1) endothelial cell damage and pericyte degeneration, (2) severe intra- and extracellular edema, (3) reduced CD31 and CD105 expressions in endothelium, (4) significant accumulation of perivascular collagen IV, and fibrin deposits (5) significantly increased microvascular density in lumbar spinal cord, (6) IgG microvascular leakage, (7) reduced tight junction and adhesion protein expressions. Microvascular barrier abnormalities determined in gray and white matter of the medulla, cervical, and lumbar spinal cord of SALS patients are novel findings. Pervasive barrier damage discovered in ALS may have implications for disease pathogenesis and progression, as well as for uncovering novel therapeutic targets.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with a complicated pathogenesis. Compelling evidence indicates impairment of all neurovascular unit components including the blood-brain and blood-spinal cord barriers (BBB/BSCB) in both patients and animal models, leading to classification of ALS as a neurovascular disease. The present review provides an updated analysis of the normal and impaired BBB/BSCB, focusing on the ALS-altered barrier. Here we describe the roles of cellular components, tight junctions, transport systems, cell interactions, cytokines, matrix metalloproteinases, and free radicals in the BBB/BSCB disruption, along with recent evidence from experimental and clinical ALS studies. The BBB/BSCB is a promising research area in ALS and this review will reveal some aspects of microvascular pathology in ALS and hopefully provide ideas for the development of new therapeutic strategies.

We explored the effects of exogenous and endogenous erythropoietin (EPO) in a seizure model of rat. Adult male Fischer 344 rats received continuous intraventricular infusion of EPO dissolved in saline containing 1 mg/ml of rat serum albumin, anti-EPO antibody, saline containing 1 mg/ml of rat serum albumin or combined EPO and neuropeptide Y (NPY) Y2-receptor antagonist. Animals were behaviorally evaluated for seizure development over 6 h after kainic acid injection followed by immunohistochemical assays. Mortality rate, seizure severity, apoptotic cell death and abnormal cell proliferation in the hippocampus of EPO-treated epileptic rats were significantly attenuated, compared to control rats. Anti-EPO antibody in non-EPO-treated animals worsened seizures and CA1 neuronal cell death, while NPY Y2-receptor antagonist cancelled the therapeutic effects of exogenous EPO. Both exogenous and endogenous EPO might modulate seizure severity and protect the hippocampal neurons in epileptic rats, via novel mechanistic pathways involving blockade of epileptogenic cell formation coupled with NPY receptor modulation in the hippocampus.

Metabotropic glutamate receptors (mGluRs) have been recently implicated as robust therapeutic targets for Parkinson's disease (PD). Here, we explored how activation of mGluRs in globus pallidus (GP) affected the amphetamine-induced rotational behavior in the unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. The amphetamine-induced rotations were completely suppressed by the ipsilateral intrapallidal injection of the non-selective mGluR agonist, 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD) and the selective group I mGluR agonist, (R,S)-3,5-dihydroxyphenylglycine (DHPG), but not the selective group III mGluR agonist, l-2-amino-4-phosphonobutyric acid (l-AP4). The suppressive effects were detected at 2, 4, 6, 8, and 12 h after ACPD injection, but returned to the control level at 24 h. A remarkable c-fos expression was found in the lesioned side of GP, subthalamic nucleus (STN), and substantia nigra pars reticulata (SNr) of rats that received the ACPD or DHPG injection, compared to rats treated with L-AP-4 or phosphate buffer-injection. The results indicate that the blockade of amphetamine-induced rotations might be at least partially mediated by group I mGluR activation. This study advances the use of selective group I mGluRs directed toward the GP for PD treatment.

Hippocampal cholinergic neurostimulating peptide (HCNP), originally isolated from soluble fraction of young rat hippocampus and released from hippocampus by the stimulation of N -methyl- d -aspartate (NMDA) receptors, enhances the cholinergic phenotype development in vitro . HCNP precursor protein (HCNP-pp) has multiple functions, not only acting as the precursor of HCNP but also serving as an inhibitor of phosphorylation of Erk and contributing to neuronal growth and memory formation. In this study, the accumulation of HCNP and/or HCNP precursor in hippocampus was found to progress from 2 to 5 months of age in senescence-accelerated mouse-prone 8 (SAM P8). This HCNP surge in the hippocampus appears to correspond to the age of onset of memory deterioration, reduction of amount of NMDA-type receptor, and morphological aberration in this dementia model mouse, SAM P8. The present findings, together with our previously published results, suggest that the HCNP and/or HCNP precursor is involved in the dysfunction of the cholinergic neuronal system and memory deterioration in this model mouse via NMDA-type receptor signaling and the activation of the MAP cascade.

8-Hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, may be a good biomarker for monitoring the progression of Parkinson's disease (PD). Unfortunately, there are no basic laboratory data examining 8-OHdG levels in animal models of PD. In this study, we demonstrate that rats lesioned with 6-hydroxydopamine (6-OHDA) in the medial forebrain bundle display significantly elevated 8-OHdG levels in urine, serum, and substantia nigra, but not cerebrospinal fluid and striatum, compared to sham controls. These increments in 8-OHdG levels were detected at 2 days, but not at 7 days after the lesion suggesting that oxidative stress is restricted to the acute phase of 6-OHDA neurotoxicity. The present results support 8-OHdG as a biomarker that may aid both in the diagnosis and in the documentation of progression in PD.

Vascular endothelial growth factor (VEGF) has been shown to display neuroprotective effects on dopaminergic (DA) neurons. Here, we investigated the neurorescue effects of VEGF on 6-hydroxydopamine (6-OHDA)-treated DA neurons in vitro and in vivo. Initially, we examined in vitro whether 1, 10, or 100 ng/ml of VEGF administration at 2 or 4 h after 6-OHDA treatment rescued DA neurons derived from E14 murine ventral mesencephalon. The earlier treatment of VEGF suppressed 6-OHDA-induced loss of DA neurons more than the delayed treatment. Next, we examined whether the continuous infusion of VEGF had neurorescue effects in a rat model of Parkinson's disease. We established a human VEGF secreting cell line (BHK-VEGF) and encapsulated the cells into hollow fibers. The encapsulated cells were unilaterally transplanted into the striatum of adult rats at 1 or 2 weeks after 6-OHDA lesions, and animals subsequently underwent behavioral and immunohistochemical evaluations. Compared to lesioned rats that received BHK-Control capsules, lesioned rats transplanted with BHK-VEGF capsules showed a significant reduction in the number of amphetamine-induced rotations, a significant preservation of TH-positive neurons in the substantia nigra pars compacta, and a remarkable glial proliferation in the striatum, with the earlier transplantation exerting much more benefits than the delayed transplantation. Parallel studies revealed that the observed in vitro and in vivo neurorescue effects were likely mediated by VEGF's angiogenic and glial proliferative effects, as well as its direct effects on the neurons. Our results suggest that VEGF is a highly potent neurorescue molecule for Parkinson's disease therapy.

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D(3) (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague-Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H(2)O(2) alone caused significant cell death. Pretreatment with D3 (10(-10) M) protected VM neurons against 6-OHDA- or H(2)O(2)-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H(2)O(2) and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.

Huntington's disease is a progressive neurodegenerative disorder associated with severe degeneration of basal ganglia neurons, especially the intrinsic neurons of the striatum, and characterized by involuntary abnormal choreiform movements and progressive dementia. With the discovery of the gene underlying HD, genetic therapy may be the next logical step towards finding a cure, but no such treatment is currently available. Animal models that closely mimic the neurobiological and clinical symptoms of the disease may offer an alternative approach for the development of new therapies. We report that systemic administration of 3-nitropropionic acid, an inhibitor of the mitochondrial citric acid cycle, results in a progressive locomotor deterioration resembling that of HD. We further demonstrate that manipulating the time course of 3-nitropropionic acid injections leads to sustained hyperactivity (early HD) or hypoactivity (advanced HD). These data suggest that this animal model can be used to test experimental treatments for HD across different stages of the disease.

Severe degeneration of basal ganglia neurons, particularly the intrinsic neurons of the striatum, is the major underlying neuropathology implicated in clinical attributes of Huntington's disease (HD). The excitotoxin-lesioned striatum provides a useful model for evaluating behavioral parameters of HD. Animals with unilateral excitotoxic lesions exhibit asymmetrical rotational behavior in response to dopamine agonists, such as apomorphine. However, the observed behavior is a pharmacological reaction, and subject to sensitization effects. A behavioral test using undrugged animals may demonstrate a more natural response of the animals to the lesion effects. Recently, we have developed the ‘drug-free’ elevated body swing test (EBST), and demonstrated that hemiparkinsonian rats exhibited significant biased swing activity. In the present study, we observed that animals with unilateral intrastriatal 3-nitropropionic acid or quinolinic acid lesions displayed a significant biased swing activity with the direction ipsilateral to the lesioned side of the brain. This ipsilateral swing corresponded to the ipsilateral rotational behavior exhibited by the lesioned animals when challenged with apomorphine. The present results demonstrated that the EBST is a sensitive measure for characterizing asymmetrical behavior in animals with striatal lesions.

Hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, has been known to promote the differentiation of septo-hippocampal cholinergic neurons. Recently, the precursor protein of HCNP (HCNP-pp) has also received attention as a multifunctional protein with roles, in addition to serving as the HCNP precursor, such as acting as an ATP-binding protein, a Raf kinase inhibitor protein (RKIP), and phosphatidylethanolamine-binding protein (PEBP). In particular, the function of RKIP has attracted attention over several years for its role in controlling cellular proliferation and metastasis in cancer cells. HCNP-pp is also thought to be important in regulating the proliferation and differentiation of neuronal cells in vitro and in vivo by modification of the MAPK cascade. In the present study, we used cultured adult rat hippocampal progenitor cells (AHPs), which are thought to be important for memory formation, and focused on the role of HCNP-pp in adult neurogenesis, namely, the production of new neurons from neural stem/progenitor cells. We found that HCNP-pp expression in AHPs was closely associated with differentiation into MAP2ab-positive neurons and RIP-positive oligodendrocytes, but not into GFAP-positive astrocytes. By contrast, a down-regulated HCNP-pp expression in AHPs accompanied differentiation into GFAP-positive astrocytes. Direct manipulations of HCNP-pp via viral over-expression or siRNA downregulation further confirmed the HCNP-pp contribution to specific neural lineage commitment of AHPs. Our results show that the expression level of HCNP-pp acts as a key regulator for differentiation of cultured AHPs into specific neural lineages, indicating that the control of neural stem cell fate can be achieved via the HCNP-pp pathway.

We investigated a non-human primate (NHP) transient global ischemia (TGI) model which was induced by clipping the arteries originating from the aortic arch. Previously we demonstrated that our TGI model in adult Rhesus macaques (Macaca mulatta) results in marked neuronal cell loss in the hippocampal region, specifically the cornu Ammonis (CA1) region. However, we observed varying degrees of hippocampal cell loss among animals. Here, we report for the first time an anomaly of the aortic arch in some Rhesus macaques that appears as a key surgical factor in ensuring the success of the TGI model in this particular NHP. Eleven adult Rhesus macaques underwent the TGI surgery, which involved 10-15-minute clipping of both innominate and subclavian arteries. Animals were allowed to survive between 1 day and 28 days after TGI. Because of our experience and knowledge that Japanese macaques exhibited only innominate and subclavian arteries arising from the aortic arch, macroscopic visualization of these two arteries alone in the Rhesus macaques initially assured us that clipping both arteries was sufficient to produce TGI. During the course of one TGI operation, however, we detected 3 arterial branches arising from the aortic arch, which prompted us to subsequently search for 3 branches in succeeding TGI surgeries. In addition, we performed post-mortem examination of the heart to confirm the number of arterial branches in the aortic arch. Finally, in order to reveal the pathological effect of the aortic arch anomaly, we compared the hippocampal cell loss between animals found to have 3 arterial branches but had all or only two branches clipped during TGI operation. Post-mortem examination revealed that eight NHPs had the typical two arterial aortic branches, but three NHPs displayed an extra arterial aortic branch, indicating that about 30% of Rhesus macaques had 3 arterial branches arising from the aorta. Histological analyses using Nissl staining showed that in NHPs with the aortic arch anomaly clipping only two of three arterial branches led to a partial cell loss and minimal alteration in number of cell layers in the hippocampal region when compared with clipping all three branches, with the hippocampal cell death in the latter resembling the pathological outcome achieved by clipping the two arterial branches in NHPs displaying the typical two-artery aortic arch. The finding that 3 of 11 NHPs exhibited an extra arterial aortic branch recognizes this aortic arch anomaly in Rhesus macaques that warrants a critical surgical maneuver in order to successfully produce consistent TGI-induced hippocampal cell loss.

Ropinirole, which is a non-ergot dopamine agonist derivative, exerts therapeutic benefits in Parkinson's disease (PD). Based on recent studies implicating dopamine receptors 2 and 3 (D2R and D3R) as possible targets of ropinirole, we over-expressed these dopamine receptor genes in the dopamine-denervated striatum of rodents to reveal whether their over-expression modulated ropinirole activity. Adult Sprague-Dawley rats initially received unilateral 6-hydroxydopamine lesion of the medial forebrain bundle. At 1 month after surgery, successfully lesioned animals (3 or less forelimb akinesia score, and 8 or more apomorphine-induced rotations/min over 1 h) were randomly assigned to intrastriatal injection (ipsilateral to the lesion) of blank lentiviral vector, D2R, D3R or both genes. At about 5 months post-lesion, ropinirole (0.2 mg/kg, i.p.) was administered daily for 9 consecutive days. The subtherapeutic dose of ropinirole improved the use of previously akinetic forelimb and produced robust circling behavior in lesioned animals with striatal over-expression of both D2R and D3R compared to lesioned animals that received blank vector. In contrast, the subtherapeutic dose of ropinirole generated only modest motor effects in lesioned animals with sole over-expression of D2R or D3R. Western immunoblot and autoradiographic assays showed enhanced D2R and D3R protein levels coupled with normalized D2R and D3R binding in the ventral striatum of lesioned animals with lentiviral over-expression of both D2R and D3R relative to vehicle-treated lesioned animals. Immunohistochemical analyses showed that D2R and D3R GFP fluorescent cells colocalized with enkephalin and substance P immunoreactive medium spiny neurons. These data support the use of the subtherapeutic dose of ropinirole in a chronic model of PD.

We investigated the neuroprotective effects of immunosuppressant cyclosporine-A (CsA) and the anti-inflammatory methylprednisolone (MP) in a stroke model. Adult Sprague-Dawley rats underwent middle cerebral artery (MCA) occlusion then were randomly treated with either: low dose CsA, MP, low dose CsA plus MP, high dose CsA, or vehicle. Ischemic animals that received low dose CsA, MP or vehicle displayed profound motor and neurological impairments at days 1-3 after stroke. In contrast, ischemic animals that received high dose CsA exhibited near normal motor and neurological functions throughout the test period. Of note, ischemic animals that received low dose CsA plus MP showed significantly less motor and neurological deficits at day 1, but thereafter displayed behavioral impairments. Histological analysis at 3 days post-stroke revealed that only those ischemic animals treated with high dose CsA had significantly reduced cerebral infarcts. This study is the first report to demonstrate partial and transient neuroprotection against stroke by low dose CsA when combined with MP.

Methanesulfonyl fluoride (MSF), a highly selective CNS inhibitor of acetylcholinesterase, has been recently demonstrated to promote improvement in cognitive performance in patients with senile dementia of Alzheimer type. Because a similar cognitive impairment may accompany stroke, we investigated in the present study whether treatment with MSF could produce beneficial effects in adult rats subjected to an experimental stroke model. Sprague-Dawley rats received transient 60 min intraluminal occlusion of the right middle cerebral artery (MCAo) and were given i.p. injections of either MSF (1 mg/kg at 24 and 48 h post-MCAo and 0.3 mg/kg thereafter every other day) or the vehicle, peanut oil, for 4 weeks. Behavioral tests and biochemical assays were performed at 28 days post-surgery. MSF treatment produced about 90% inhibition of acetylcholinesterase in the brain. Ischemic animals that received the vehicle displayed significant elevated body swing biased activity (84.8 +/- 10%) and significantly prolonged acquisition (398 +/- 62 s) and shortened retention (79 +/- 26 s) of the passive avoidance task. Interestingly, while the ischemic animals that received the MSF exhibited elevated body swing biased activity (87.7 +/- 8%), they performed significantly better in the passive avoidance task (255 +/- 36 s and 145 +/- 18 s in acquisition and retention) than the vehicle-treated animals. Moreover, whereas brains from both groups of animals revealed similar extent and degree of cerebral infarction, the MSF-treated ischemic animals showed more intense immunoreactivity, as well as a significantly higher number (10-15% increase) of septal choline acetyltransferase-positive cells than the vehicle-treated ischemic animals. These results show that MSF, possibly by preserving a functional cholinergic system, attenuated stroke-induced deficits in a simple learning and memory task.

We examined in the present study alterations in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability following intrastriatal transplantation of mouse bone marrow stromal cells (BMSCs) or saline infusion in adult Sprague-Dawley rats. Laser Doppler revealed that transplanted animals exhibited near normal cerebral blood flow (CBF, 150 perfusion units) at a much earlier period post-transplantation (day 4) compared to animals that received saline infusion (day 12) (p's

We monitored alterations in cerebral blood flow (CBF) and blood-brain barrier (BBB) permeability following middle cerebral artery occlusion (MCAo) and intrastriatal transplantation of mouse bone marrow stromal cells (BMSCs) or saline infusion in adult Sprague-Dawley rats. Laser Doppler and Evans Blue assay revealed that BMSC grafts dose-dependently restored CBF and BBB to near normal levels at a much earlier period (Days 4-5 post-MCAo) in transplanted stroke animals compared to stroke animals that received saline infusion (Days 11-14 post-MCAo). Xenografted BMSCs survived in the absence of immunosuppression, and elevated levels of transforming growth factor-beta superfamily of neurotrophic factors were detected in transplanted stroke animals. These data suggest that early restoration of CBF and BBB following transplantation of BMSCs could mediate the reported functional outcomes in stroke animals.

Several kidney cell lines were investigated for their ability to produce glial cell line-derived neurotrophic factor (GDNF). Cell line-conditioned medium was analyzed using ELISA and two cell lines were identified which produce GDNF in physiologically active concentrations. ELISA analyses revealed that conditioned medium from these two cell lines also contained PDGF, bFGF, TGFβ1 and TGFβ2. Both of these cell lines were then transplanted into the striatal penumbra of rats, 1 h following middle cerebral artery occlusion. Behavioral testing revealed that both cell lines reduced the deficit associated with cerebral ischemia and reduced the infarct volume relative to controls. Reduction of infarct volume was likely achieved by the action of GDNF and/or other growth factors produced by the cells.

Systemic administration of 3-nitropropionic acid (3-NP) results in striatal atrophy by irreversibly inhibiting the citric acid cycle, and thereby resulting in cellular ATP depletion. The neuropathological outcome following 3-NP injections is thought to resemble that seen in Huntington's disease (HD) [1]. The current study administered systemic injections in 6- and 10-week-old rats of low-dose 3-NP every other 4 days for a period of 28 days in order to investigate the effects on locomotor behavior and striatal D1 dopamine receptor binding. Experimental and control animals received intraperitoneal injections of 3-NP (10 mg/kg in 0.9% saline) and 0.9% saline, respectively. Animals were tested behaviorally prior to the first and after the last 3-NP administration. Brains were then removed and striatal tissue samples were analyzed for D1 dopamine receptor binding using [3H]SCH23390. Behaviorally, 6-week-old injected animals developed bradykinesia with no signs of stiffness or rigidity, while 10-week-old injected animals displayed an uncoordinated gait, stiffness and ventral recumbency with hind limbs extended in a rigid or fixed position. These visual observations of hypoactivity were supported by a significant decline in both experimental groups' locomotor activity as measured by Digiscan monitors. Furthermore, [3H]SCH23390 specific binding to D1 dopamine receptors revealed a small but significant decrease in 10-week-old injected animals compared to controls. These results demonstrate that both 6- and 10-week-old rats do exhibit behavioral alterations after long-term 3-NP administration, however the former may not show accompanying gross D1 receptor changes.