Cognitive and anxiety-like impairments accompanied by serotonergic ultrastructural and immunohistochemical alterations in early stages of parkinsonism.

Parkinson's disease (PD) is mostly known as a dopamine deficiency syndrome due the structural and functional changes in striatal projection neurons. However, studies have considered this pathology as a multi-systemic disease in which the neurodegenerative process extends beyond the dopaminergic system. Therefore, the purpose of the present study was to investigate the morphological and immunohistochemical changes associated with behavioral and cognitive alterations in a model of parkinsonism induced by low dose of reserpine. Animals showed anxiety-like behavior and deficits in short-term recognition memory. Besides, Tyrosine Hydroxylase (TH) immunoreactive cells decreased in reserpine (RES) group in CA1 and serotonin (5-HT) immunoreactive cells decreased in RES group in CA1, CA3 and medial prefrontal cortex (mPFC). Moreover, an increase in the area (µm2) of 5 H T labeled ultrastructure (axon terminal) was observed in RES group only in CA1 and mPFC. The evidence of alterations in 5-HT immunoreactive in the premotor phase of model of parkinsonism highlights the importance of looking beyond the nigrostriatal system to elucidate the underling mechanisms and deficits in other neurotransmitters systems. This provides vital information regarding novel interventions for the management of non-motor symptoms. Additionally, the low-dose reserpine treatment has an early effect on axonal ultrastructure. As the axonopathy in PD has been increasingly recognized, the focus on axonal neurobiology is noteworthy for both neuroprotective and restorative therapeutics, and the progressive reserpine rat model can be a useful tool in this search.

Exercise in an animal model of Parkinson's disease: Motor recovery but not restoration of the nigrostriatal pathway.

Many clinical studies have reported on the benefits of exercise therapy in patients with Parkinson's disease (PD). Exercise cannot stop the progression of PD or facilitate the recovery of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) (Bega et al., 2014). To tease apart this paradox, we utilized a progressive MPTP (1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine) mouse model in which we initiated 4weeks of treadmill exercise after the completion of toxin administration (i.e., restoration). We found in our MPTP/exercise (MPTP+EX) group several measures of gait function that recovered compared to the MPTP only group. Although there was a small recovery of tyrosine hydroxylase (TH) positive DA neurons in the SNpc and terminals in the striatum, this increase was not statistically significant. These small changes in TH could not explain the improvement of motor function. The MPTP group had a significant 170% increase in the glycosylated/non-glycosylated dopamine transporter (DAT) and a 200% increase in microglial marker, IBA-1, in the striatum. The MPTP+EX group showed a nearly full recovery of these markers back to the vehicle levels. There was an increase in GLT-1 levels in the striatum due to exercise, with no change in striatal BDNF protein expression. Our data suggest that motor recovery was not prompted by any significant restoration of DA neurons or terminals, but rather the recovery of DAT and dampening the inflammatory response. Although exercise does not promote recovery of nigrostriatal DA, it should be used in conjunction with pharmaceutical methods for controlling PD symptoms.

Intervention with exercise restores motor deficits but not nigrostriatal loss in a progressive MPTP mouse model of Parkinson's disease.

Many studies have investigated exercise therapy in Parkinson's disease (PD) and have shown benefits in improving motor deficits. However, exercise does not slow down the progression of the disease or induce the revival of lost nigrostriatal neurons. To examine the dichotomy of behavioral improvement without the slowing or recovery of dopaminergic cell or terminal loss, we tested exercise therapy in an intervention paradigm where voluntary running wheels were installed half-way through our progressive PD mouse model. In our model, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is administered over 4 weeks with increased doses each week (8, 16, 24, 32-kg/mg). We found that after 4 weeks of MPTP treatment, mice that volunteered to exercise had behavioral recovery in several measures despite the loss of 73% and 53% tyrosine hydroxylase (TH) within the dorsolateral (DL) striatum and the substantia nigra (SN), respectively which was equivalent to the loss seen in the mice that did not exercise but were also administered MPTP for 4 weeks. Mice treated with 4 weeks of MPTP showed a 41% loss of vesicular monoamine transporter II (VMAT2), a 71% increase in the ratio of glycosylated/non-glycosylated dopamine transporter (DAT), and significant increases in glutamate transporters including VGLUT1, GLT-1, and excitatory amino acid carrier 1. MPTP mice that exercised showed recovery of all these biomarkers back to the levels seen in the vehicle group and showed less inflammation compared to the mice treated with MPTP for 4 weeks. Even though we did not measure tissue dopamine (DA) concentration, our data suggest that exercise does not alleviate motor deficits by sparing nigrostriatal neurons, but perhaps by stabilizing the extraneuronal neurotransmitters, as evident by a recovery of DA and glutamate transporters. However, suppressing inflammation could be another mechanism of this locomotor recovery. Although exercise will not be a successful treatment alone, it could supplement other pharmaceutical approaches to PD therapy.

Intervention with 7,8-dihydroxyflavone blocks further striatal terminal loss and restores motor deficits in a progressive mouse model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurological disorder and current therapies help alleviate symptoms, but are not disease modifying. In the flavonoid class of compounds, 7,8-dihydroxyflavone (7,8-DHF) has been reported to elicit tyrosine kinase receptor B (TrkB) dimerization and autophosphorylation that further stimulates signaling cascades to promote cell survival/growth, differentiation, and plasticity. In this study we investigated if 7,8-DHF could prevent further loss of dopaminergic cells and terminals if introduced at the midpoint (i.e. intervention) of our progressive mouse model of PD. In our model, 1-methyl-4phenyl-1,2,3,6-tetrahyrdopyridine (MPTP) is administered with increased doses each week (8, 16, 24, 32-kg/mg) over a 4-week period. We found that despite 4 weeks of MPTP treatment, animals administered 7,8-DHF starting at the 2-week time period maintained 54% of the tyrosine hydroxylase (TH) levels within the dorsolateral (DL) striatum compared to the vehicle group, which was comparable to animals treated with MPTP for 2 weeks and was significantly greater compared to animals treated with MPTP for the full 4 weeks. Animals treated with MPTP and 7,8-DHF also demonstrated increased levels of, a sprouting-associated protein, superior cervical ganglion-10 (SCG10), phosphorylated TrkB (pTrkB), and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) within the DL striatum and substantia nigra (SN) compared to the 4-week MPTP-treated animals. In addition, motor deficits seen in the 2- and 4-week MPTP-treated animals were restored following administration of 7,8-DHF. We are reporting here for the first time that intervention with 7,8-DHF blocks further loss of dopaminergic terminals and restores motor deficits in our progressive MPTP mouse model. Our data suggest that 7,8-DHF has the potential to be a translational therapy in PD.

Effect of intermittent washout periods on progressive lesioning of the nigrostriatal pathway with 1-methyl-2-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

We have previously reported that a progressively increased dose of MPTP over the course of 4 weeks induces the gradual impairment of the nigrostriatal dopamine (DA) pathway and several behaviors [Goldberg et al. (in press) Neuroscience]. To our knowledge, this is the first report of specific behavioral deficits correlated with discrete thresholds of DA loss in this pathway. In that study, MPTP was administered 5 d/wk, with behavioral and tissue analysis being carried out 3 days following the final injection at each dose. However, in order to better represent long-term progressive neurodegeneration the present study introduced a washout period of 10 days between each increased dose of MPTP. This implementation also controlled for any transient de-activation of tyrosine hydroxylase (TH), the enzyme that catalyzes synthesis of DA, caused by MPTP-induced oxidative stress which has been suggested following acute administration of the toxin [Smeyne and Jackson-Lewis (2005) Brian Res Mol Brain Res 134:57-66]. Additionally, by the end of the previous study, there was an ultimate decrease of 62% in the mean number of TH-labeled neurons/section in the substantia nigra pars compacta (SNpc) and a 74% decrease in caudate putamen (CPu) TH optical density with continuous MPTP. In the present study, we find that the washout periods lead to a final 79% decrease in the mean number of TH-labeled SNpc neurons/section, and a similar 74% decrease in CPu TH following the 32 mg/kg MPTP dose. Additionally, a dose-dependent decrease was observed in the mean number of SNpc TH-ir neurons/section in the current study which was not seen in the continuous MPTP protocol. These results suggest that a washout period following each increased MPTP dose allows for observation of continued cell death that might occur during the week following MPTP administration, and for therapeutic interventions to be applied at any of several stages during progressive neurodegeneration.

Dopaminergic and behavioral correlates of progressive lesioning of the nigrostriatal pathway with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

A number of neurotoxin- and gene-based rodent models of acute neurodegeneration of nigrostriatal dopamine (DA) neurons are used to study Parkinson's disease (PD). The rapid degeneration achieved by many of these current models limits the capacity of the model to develop pathogenic mechanisms and display the various stages of motor degradation representative of the human Parkinsonian condition. Chronic rodent models have been the only ones to reproduce these characteristics, yet do not show correlated progress of DA loss with multiple stepwise behavioral deficits as seen in humans. In the present study, we have developed a progressive model of increasing DA loss and motor dysfunction via progressively increased administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), in the C57Bl/6J mouse. Mice were administered a daily (5 d/wk) dose of MPTP that increased weekly over the course of 4 weeks (4 mg/kg, 8 mg/kg, 16 mg/kg and 32 mg/kg). Each treatment group was tested for exploratory and motor behavioral changes after every week leading up to their final dose, as well as changes in tyrosine hydroxylase immunoreactivity (TH-ir) of the substantia nigra pars compacta (SNpc) and caudate putamen (CPu). We detected a 24% decrease in the mean number of TH-ir SNpc neurons/section after 1 week, and a 62% decrease after 4 weeks as compared to the vehicle group. CPu TH-ir began at a 35% loss after 1 week and increased to a 74% loss after 4 weeks compared to the vehicle group. CPu DA content showed an initial decrease of 20% after 1 week, and a final decrease of 70% following week 4 versus the vehicle group. Free-standing rears (versus wall-assisted rears, in a cylinder), decreased from 35% to 8% of total rears as the dose of MPTP increased from 4 mg/kg to 32 mg/kg, respectively. However, motor impairment as measured by a Parallel Rod Activity Chamber test was not significant until week 4 at 32 mg/kg compared to the vehicle group. The present study is the first to show stepwise progression of behavioral deficits which correlate with gradual dopaminergic decline in the nigrostriatal pathway. This progressive lesioning regiment may be appropriate for future investigation of pathogenic mechanisms and various intervention therapies in PD.

Enriched environment promotes similar neuronal and behavioral recovery in a young and aged mouse model of Parkinson's disease.

Environmental enrichment has been shown to be neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Because PD patients are not typically diagnosed until later neuropathological stages, the current study investigated the capacity of an enriched environment (EE) to stimulate restoration of neurons in the substantia nigra pars compacta (SNpc) and locomotor recovery after lesioning, as opposed to before. A low-dose chronic MPTP regimen was used to achieve a partial, less severe lesion of the nigrostriatal pathway not seen in acute MPTP models. Both young adult (10 weeks) and aged (12 months) C57BL/6J male mice were used to assess the effects of aging on recovery with EE intervention. After the first week of either MPTP (7 mg/kg/d in young; 5 mg/kg/d in aged) or saline injection, animals from both groups were housed in a standard environment (SE) or an EE for 3 weeks, with continued daily administration of MPTP. We are the first to report that following 3 weeks exposure to an EE, young and aged MPTP-lesioned mice showed a significant 53% and 52% restoration of tyrosine hydroxylase (TH)-labeled neurons in the SNpc, respectively. This increase in TH-labeled cells in the MPTP+EE group was correlated with recovery of free-standing rear (FSR) behavior in both age groups; however, improved locomotor control as measured by foot faults (FF) per total activity was only seen in the aged MPTP+EE group. Our data demonstrate that an EE promotes neurorestoration in TH protein expression in SNpc neurons as well as some locomotor recovery in both young and aged animals in this mouse model of PD.

Impaired glutamate homeostasis and programmed cell death in a chronic MPTP mouse model of Parkinson's disease.

The pathogenesis of Parkinson's disease is not fully understood, but there is evidence that excitotoxic mechanisms contribute to the pathology. However, data supporting a role for excitotoxicity in the pathophysiology of the disease are controversial and sparse. The goal of this study was to determine whether changes in glutamate signaling and uptake contribute to the demise of dopaminergic neurons in the substantia nigra. Mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid or vehicle (probenecid or saline alone). Extracellular levels of glutamate in the substantia nigra were substantially increased, and there was an increase in the affinity, but no change in the velocity, of glutamate transport after MPTP/probenecid treatment compared to vehicle controls. In addition, the substantia nigra showed two types of programmed death, apoptosis (type I) and autophagic (type II) cell death. These data suggest that increased glutamate signaling could be an important mechanism for the death of dopaminergic neurons and trigger the induction of programmed cell death in the chronic MPTP/probenecid model.

l-dopa-induced reversal in striatal glutamate following partial depletion of nigrostriatal dopamine with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

We have reported that 1 month following acute (20mg/kg x 4) or subchronic (30 mg/kg/day x 7d) administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, there is an increase or decrease, respectively, in the extracellular level of striatal glutamate as determined by in vivo microdialysis [Robinson S, Freeman P, Moore C, Touchon JC, Krentz L, Meshul CK (2003) Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function. Exp Neurol 180:73-86]. The goal of this study was to determine the effects of treatment with l-dopa (15 mg/kg) for 21 days on striatal glutamate starting on day 8 after the first dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was administered to mice. Following acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the increase in extracellular striatal glutamate due to lesion of the nigrostriatal pathway was completely reversed to a level below that found in the vehicle-treated group after l-dopa treatment. Subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment resulted in a decrease in striatal extracellular glutamate that was reversed to the level close to that observed in the vehicle-treated group. There was no change in the density of nerve terminal glutamate immunolabeling associated with the synaptic vesicle pool, suggesting that the alterations in extracellular glutamate most likely originated from the calcium-independent pool. There was a similar decrease in the relative density of tyrosine hydroxylase immunolabeling, a marker for dopamine terminals, within the dorsolateral striatum in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups that had been administered l-dopa. There was a decrease in the relative density of immunolabeling within the dorsolateral striatum for the glutamate transporter, GLT-1, following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in the groups administered either vehicle or l-dopa. There was no change in GLT-1 immunolabeling following subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The results demonstrate that the reversal in the extracellular level of striatal glutamate following l-dopa treatment in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups is not due to changes in either striatal dopamine nerve terminals or in the density of the glutamate transporter, GLT-1.

Differential regulation of the growth-associated proteins GAP-43 and superior cervical ganglion 10 in response to lesions of the cortex and substantia nigra in the adult rat.

Investigation of the elements underlying synapse replacement after brain injury is essential for predicting the neural compensation that can be achieved after various types of damage. The growth-associated proteins superior cervical ganglion-10 and growth-associated protein-43 have previously been linked with structural changes in the corticostriatal system in response to unilateral deafferentation. To examine the regulation of this response, unilateral cortical aspiration lesion was carried out in combination with ipsilateral 6-hydroxydopamine lesion of the substantia nigra, and the time course of the contralateral cortical molecular response was followed. Unilateral cortical aspiration lesion in rats corresponds with an upregulation of superior cervical ganglion-10 mRNA at 3 and 10 days post-lesion, and protein, sustained from three to at least 27 days following lesion. With the addition of substantia nigra lesion, the response shifts to an upregulation of growth-associated protein-43 mRNA at 3 and 10 days post-lesion, and protein after 10 days. Nigral lesion alone does not alter contralateral expression of either gene. Likewise, motor function assessment using the rotorod test revealed no significant long-term deficits in animals that sustained only nigrostriatal damage, but cortical lesion was associated with a temporary deficit which was sustained when nigrostriatal input was also removed. Growth-associated protein-43 and superior cervical ganglion-10, two presynaptic genes that are postulated to play roles in lesion-induced sprouting, are differentially upregulated in corticostriatal neurons after cortical versus combined cortical/nigral lesions. The shift in contralateral gene response from superior cervical ganglion-10 to growth-associated protein-43 upregulation and associated behavioral deficit following combined cortical and nigral denervation suggest that nigrostriatal afferents regulate cortical lesion-induced gene expression and ultimate functional outcome.

Time-dependent changes in extracellular glutamate in the rat dorsolateral striatum following a single cocaine injection.

Acute cocaine administration has been shown to alter dorsal striatal plasticity [Proc Natl Acad Sci USA 87 (1990) 6912; Brain Res Bull 30 (1993) 173] and produce long-term neurochemical changes [Pharmacol Biochem Behav 27 (1987) 533]. To date, the effects of acute cocaine on extracellular glutamate and nerve terminal glutamate immunolabeling in the rat dorsolateral striatum have not been reported. To investigate cocaine-induced changes in extracellular glutamate, in vivo microdialysis was carried out in the dorsolateral striatum of rats 1-14 days after receiving a single injection of either vehicle or 15 mg/kg cocaine. There was an increase in the group injected with cocaine 1 day prior to measuring extracellular glutamate as compared with the control group. The group injected with cocaine 3 days prior to the microdialysis session had decreased extracellular glutamate levels. Furthermore, extracellular glutamate remained attenuated 14 days after acute cocaine treatment. Striatal glutamate decreased in the cocaine-treated rats after calcium removal, suggesting that cocaine-induced changes in extracellular glutamate were partially calcium-dependent. The density of nerve terminal glutamate immunolabeling was measured using immunogold electron microscopy in the contralateral striatum of the same rats that had been acutely treated with cocaine or vehicle. There were no changes in the density of glutamate immunolabeling within identified nerve terminals making an asymmetrical (excitatory) synaptic contact 1, 2, 3, or 14 days after acute cocaine exposure as compared with the control groups. Hence, these alterations in extracellular glutamate did not result from changes in glutamate immunolabeling within the synaptic vesicle pool. In addition, no changes in glutamate immunolabeling were found in rats that received cocaine 2 h previously or were withdrawn after 1 week of cocaine administration. The results demonstrate that a single injection of cocaine produces biphasic, time-dependent changes in extracellular glutamate in the rat dorsolateral striatum.

Nerve terminal glutamate immunoreactivity in the rat nucleus accumbens and ventral tegmental area after a short withdrawal from cocaine.

Cocaine administration has been shown to alter glutamate transmission in numerous studies. Using quantitative electron microscopic immunogold labeling, our laboratory has previously reported that nerve terminal glutamate immunoreactivity is transiently altered following cocaine administration. The present study was undertaken to examine presynaptic nerve terminal glutamate immunoreactivity at shorter time points after withdrawal from cocaine. Animals received saline or cocaine for 7 days followed 3 days later by a cocaine or saline challenge. Most (>75%) cocaine-challenged animals had a heightened locomotor response to cocaine compared to the first day of cocaine and were considered behaviorally sensitized. One day after the challenge, glutamate immunogold-labeling was quantified in nerve terminals making asymmetrical synaptic contacts within the core and shell of the nucleus accumbens and ventral tegmental area. A single dose of cocaine did not alter the density of presynaptic nerve terminal glutamate immunoreactivity in the nucleus accumbens (NAc) or ventral tegmental area (VTA). The density of nerve terminal glutamate immunoreactivity in the shell, but not the core, was significantly increased in the animals receiving repeated cocaine. In the VTA the density of nerve terminal glutamate immunoreactivity did not change in the cocaine-sensitized group, but was significantly increased in the nonsensitized group. The finding that repeated cocaine treatment increased glutamate nerve terminal immunolabeling within the nucleus accumbens shell, but not the core, supports the hypothesis that glutamate synapses in the core and shell are differentially sensitive to repeated cocaine administration. Overall, our study does not support a role for changes in presynaptic glutamate in the development of behavioral sensitization.

Blockade of NMDA receptors by MK-801 reverses the changes in striatal glutamate immunolabeling in 6-OHDA-lesioned rats.

A lesion of the dopamine (DA)-containing nigrostriatal pathway with 6-hydroxydopamine (6-OHDA) results in an increase in the density of nerve terminal glutamate immunolabeling and in the mean percentage of asymmetrical synapses containing a discontinuous postsynaptic density [Meshul et al. (1999) Neuroscience 88:1-16]. Similar alterations in striatal glutamate synapses have been reported following blockade of striatal DA D-2 receptors with subchronic haloperidol treatment [Meshul et al. (1994) Brain Res 648:181-195]. The haloperidol-induced change in glutamate synapses was blocked by coadministration of the N-methyl-D-aspartate (NMDA) noncompetitive receptor antagonist MK-801. In order to determine if blockade of NMDA receptors could alter the density of nerve terminal glutamate immunolabeling following a 6-OHDA lesion of the nigrostriatal pathway, MK-801 was administered to lesioned animals for 14 days. In addition, the number of apomorphine-induced contralateral rotations was determined prior to and following the administration of MK-801. MK-801 administration reversed the increase in the density of nerve terminal glutamate immunolabeling due to a 6-OHDA lesion. There was a small but significant decrease in the number of apomorphine-induced contralateral rotations following administration of MK-801 compared to the number of rotations prior to treatment with the NMDA antagonist. These results demonstrate that blockade of postsynaptic NMDA receptors affects the density of presynaptic glutamate immunolabeling and that this change in nerve terminal glutamate density is associated with a decreased behavioral response to direct DA receptor stimulation. Whether the effect of MK-801 is directly on the striatum or acts through other excitatory pathways of the basal ganglia remains unclear.

The effects of acute or repeated cocaine administration on nerve terminal glutamate within the rat mesolimbic system.

Cocaine administration alters glutamate function within several brain regions. Using quantitative electron microscopic immunocytochemistry, the present study investigated the effect of repeated intermittent cocaine (resulting in behavioral sensitization) or acute cocaine administration on the density of glutamate immunogold labeling within nerve terminals. Rats were treated daily with saline or cocaine for 7 days. Following a 14-day withdrawal animals were challenged with saline or cocaine. On the challenge day, most (75%) animals that received cocaine repeatedly showed a heightened locomotor response to cocaine compared to the first day of cocaine administration, and were considered behaviorally sensitized.Three days after the challenge, glutamate immunogold labeling was quantified in nerve terminals making asymmetrical synaptic contacts within the core and shell of the nucleus accumbens, ventral tegmental area and medial prefrontal cortex. There was a decrease in such labeling in the nucleus accumbens in the group receiving acute cocaine. Locomotor activity was positively correlated with glutamate immunolabeling within nerve terminals in the nucleus accumbens core only for the cocaine-sensitized group. Nerve terminal glutamate immunolabeling in the nucleus accumbens core, but not the shell, was increased in the non-sensitized compared to the cocaine-sensitized group. In the ventral tegmental area, glutamate immunolabeling was significantly higher in the cocaine-sensitized compared to the acute cocaine group. In the prefrontal cortex, there were no significant differences in glutamate immunogold labeling between treatment groups. This study indicates that acute cocaine administration significantly decreases nerve terminal glutamate immunoreactivity in the nucleus accumbens. We suggest that sensitization results in differential changes in the nucleus accumbens core versus the shell, and may alter presynaptic mechanisms regulating glutamate release or re-uptake in the core.

Oral dyskinesias and morphological changes in rat striatum during long-term haloperidol administration.

RATIONALE: Neuroleptic-induced oral dyskinesias in rats, a putative analogue to human tardive dyskinesia, may be due to increased glutamate release within the striatum. This may lead to excitotoxic degeneration and, as a consequence, persistent motor side effects. OBJECTIVES: To investigate whether alterations in glutamatergic synapses within the striatum are associated with the development of neuroleptic-induced oral dyskinesia. METHODS: Haloperidol was administered for 20 weeks, and rats with high and low levels of vacuous chewing movements (VCM) were analyzed for morphological changes with electron microscopy at three time points. RESULTS: At week 8, the high VCM rats had a larger nerve terminal area and lower density of nerve terminal glutamate immunoreactivity than the other groups. After 18 weeks of treatment, the nerve terminal area was increased relative to controls in both the high and low VCM groups. After discontinuation of treatment, there were no significant morphological differences between the groups, but the level of VCM was still significantly increased in the high VCM group. CONCLUSIONS: These results show that striatal glutamatergic transmission is affected during haloperidol treatment and the nerve terminal area and the density of nerve terminal glutamate immunoreactivity are important in determining the VCM response to haloperidol treatment. This indicates that increased glutamatergic synaptic activity in the striatum contributes to the development of human tardive dyskinesia.

Dopamine D4 receptor-deficient mice display cortical hyperexcitability.

The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.

Alterations in rat striatal glutamate synapses following a lesion of the cortico- and/or nigrostriatal pathway.

Ultrastructural changes within the ipsilateral dorsolateral striatum were investigated 1 month following a unilateral ablation of the rat frontal cortex (CTX), removing corticostriatal input, or injection of the neurotoxin, 6-hydroxydopamine (6-OHDA), into the substantia nigra pars compacta, removing nigrostriatal input. In addition, a combined ipsilateral cortical and 6-OHDA lesion (CTX/6-OHDA) was carried out. We find that following a CTX, 6-OHDA, or CTX/6-OHDA lesion, there was a significant decrease in the density of striatal nerve terminal glutamate immunoreactivity compared to the control group. There was also a significant increase in all three lesion groups in the mean percentage of asymmetrical synapses associated with a perforated postsynaptic density. There was a large increase within the CTX/6-OHDA-lesioned group and a smaller but still significant increase in the CTX-lesioned group in the percentage of terminals or boutons with multiple synaptic contacts (i.e., multiple synaptic boutons, MSBs), compared to either the 6-OHDA or the control group. There was no change in any of these measurements within the contralateral striatum. There was a significant decrease in the number of apomorphine-induced contralateral rotations in the CTX/6-OHDA versus the 6-OHDA-lesioned group. Animals receiving just the single CTX or 6-OHDA lesion recovered in motor function compared to the control group as measured by the Rotorod test, while the CTX/6-ODA-lesioned group recovered to less than 50% of the control level. The data suggest that following a CTX and/or 6-OHDA lesion, there is an increase in striatal glutamatergic function. The large increase in the percentage of MSBs in the combined lesion group suggests that dopamine or other factors released by the dopamine terminals assist in regulating synapse formation.

Haloperidol reverses the changes in striatal glutamatergic immunolabeling following a 6-OHDA lesion.

We reported previously that 3 months following a unilateral lesion of the nigrostriatal pathway with 6-hydroxydopamine (6-OHDA), there was a decrease in the extracellular level of striatal glutamate as determined by in vivo microdialysis. This resulted in an accumulation or increase in the density of nerve terminal glutamate immunolabeling (Meshul et al., 1999). We also reported on blockade of dopamine D-2 receptors with haloperidol resulting in ultrastructural changes within the striatum consistent with increased functioning of the glutamatergic corticostriatal pathway (Meshul and Tan 1994). We hypothesized that administration of haloperidol to 6-OHDA-lesioned rats may be capable of activating the corticostriatal pathway and thereby counteracting the effects of the unilateral nigrostriatal lesion. Striatal glutamatergic function was evaluated using electron microscopy and quantitative glutamate immunocytochemistry. Starting 1 month after a unilateral lesion of the nigrostriatal pathway with 6-OHDA, haloperidol (0.5 mg/kg/d) was administered for the next 2 months. Within the dorsolateral caudate nucleus, the main area of innervation from the motor cortex, haloperidol blocked the 6-OHDA-induced increase in the density of nerve terminal glutamate immunolabeling. Within all three experimental groups (6-OHDA, haloperidol, 6-OHDA/haloperidol) there was an increase in the mean percentage of striatal asymmetrical synapses containing a perforated postsynaptic density. In addition, haloperidol treatment resulted in a reduction in the number of apomorphine-induced contralateral rotations in unilaterally 6-OHDA lesioned rats. The data suggests that the decrease in striatal glutamatergic function 3 months following a unilateral 6-OHDA lesion can be reversed by daily haloperidol treatment. This finding is discussed in terms of current therapy for Parkinson's disease. Synapse 36:129-142, 2000. Published 2000 Wiley-Liss, Inc.

Kappa opioid receptor immunoreactivity in the nucleus accumbens and caudate-putamen is primarily associated with synaptic vesicles in axons.

A rabbit polyclonal antiserum, raised against a C-terminal oligopeptide of the mouse kappa opioid receptor, was used to localize the cellular distribution of kappa receptors in the dorsal and ventral striatum of rats with light and electron microscopic immunocytochemistry. Prominent, diffuse kappa receptor immunoreactivity was present in the nucleus accumbens, particularly in the shell, ventral caudate-putamen and olfactory tubercle. The density of receptor immunoreactivity decreased in more dorsal areas of the caudate-putamen. In contrast, neuronal cell bodies stained clearly in the dorsal endopiriform nucleus, claustrum and layer VI of the adjacent cerebral cortex. Observations at the electron microscopic level in the dorsomedial shell of the nucleus accumbens and caudate-putamen revealed that the kappa receptor immunoreactivity was predominantly located in axons, often associated with synaptic vesicles, remote from the terminal or preterminal area. The few terminals which were labeled made slightly more asymmetrical than symmetrical contacts and the percentage of asymmetrical contacts observed was greater in the caudate than in the accumbens. A small number of postsynaptic spines was labeled; most of them were contacted by asymmetrical terminals. No labeling was observed in dendritic shafts.Thus, the predominant localization of kappa receptor immunoreactivity in axons is consistent with its role as a major inhibitor of glutamate and dopamine release in the dorsal and ventral striatum.

Prolonged survival of adult rat pancreatic islets transfected with E1A-12S adenovirus.

It has been reported that various mutants of the E1A-adenovirus can activate quiescent differentiated cells to start proliferating. The aim of this study was to determine whether transfection with E1A-12S could extend the life span and functionality of pancreatic islets in culture. Rat pancreatic islets were isolated and transfected with retrovirus containing the adenovirus E1A-12S, E1A-13S, or control vectors. Transfection with the retroviral E1A-13S mutant produced extensive islet necrosis compared with nontransfected islets. Islets transfected with the control E1A mutant Ad5-dl312 vector (containing no E1A-12S or E1A-13S segments) were similar to nontransfected islets in their characteristics. We found that the E1A-12S transfected islets maintained greater viability, insulin granule structure, and glucose-induced insulin responsiveness over a 6-week period compared with mock or control islets. At 6 weeks of culture, the E1A-12S transfected islets also had fewer apoptotic cells compared with nontransfected islets. These data suggest that adenovirus E1A-12S can extend the functional life span of cultured rat pancreatic islets.