Charles K. Meshul

Exercise-induced behavioral recovery and neuroplasticity in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse basal ganglia.

Physical activity has been shown to be neuroprotective in lesions affecting the basal ganglia. Using a treadmill exercise paradigm, we investigated the effect of exercise on neurorestoration. The 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐lesioned mouse model provides a means to investigate the effect of exercise on neurorestoration because 30–40% of nigrostriatal dopaminergic neurons survive MPTP lesioning and may provide a template for neurorestoration to occur. MPTP‐lesioned C57 BL/6J mice were administered MPTP (four injections of 20 mg/kg free‐base, 2 hr apart) or saline and divided into the following groups: (1) saline; (2) saline + exercise; (3) MPTP; and (4) MPTP + exercise. Mice in exercise groups were run on a motorized treadmill for 30 days starting 4 days after MPTP lesioning (a period after which MPTP‐induced cell death is complete). Initially, MPTP‐lesioned + exercise mice ran at slower speeds for a shorter amount of time compared to saline + exercise mice. Both velocity and endurance improved in the MPTP + exercise group to near normal levels over the 30‐day exercise period. The expression of proteins and genes involved in basal ganglia function including the dopamine transporter (DAT), tyrosine hydroxylase (TH), and the dopamine D1 and D2 receptors, as well as alterations on glutamate immunolabeling were determined. Exercise resulted in a significant downregulation of striatal DAT in the MPTP + exercise compared to MPTP nonexercised mice and to a lesser extent in the saline + exercised mice compared to their no‐exercise counterparts. There was no significant difference in TH protein levels between MPTP and MPTP + exercise groups at the end of the study. The expression of striatal dopamine D1 and D2 receptor mRNA transcript was suppressed in the saline + exercise group; however, dopamine D2 transcript expression was increased in the MPTP + exercise mice. Immunoelectron microscopy indicated that treadmill exercise reversed the lesioned‐induced increase in nerve terminal glutamate immunolabeling seen after MPTP administration. Our data demonstrates that exercise promotes behavioral recovery in the injured brain by modulating genes and proteins important to basal ganglia function.

Dopamine Modulates Release from Corticostriatal Terminals

Normal striatal function is dependent on the availability of synaptic dopamine to modulate neurotransmission. Within the striatum, excitatory inputs from cortical glutamatergic neurons and modulatory inputs from midbrain dopamine neurons converge onto dendritic spines of medium spiny neurons. In addition to dopamine receptors on medium spiny neurons, D2 receptors are also present on corticostriatal terminals, where they act to dampen striatal excitation. To determine the effect of dopamine depletion on corticostriatal activity, we used the styryl dye FM1-43 in combination with multiphoton confocal microscopy in slice preparations from dopamine-deficient (DD) and reserpine-treated mice. The activity-dependent release of FM1-43 out of corticostriatal terminals allows a measure of kinetics quantified by the halftime decay of fluorescence intensity. In DD, reserpine-treated, and control mice, exposure to the D2-like receptor agonist quinpirole revealed modulation of corticostriatal kinetics with depression of FM1-43 destaining. In DD and reserpine-treated mice, quinpirole decreased destaining to a greater extent, and at a lower dose, consistent with hypersensitive corticostriatal D2 receptors. Compared with controls, slices from DD mice did not react to amphetamine or to cocaine with dopamine-releasing striatal stimulation unless the animals were pretreated with l-3,4-dihydroxyphenylalanine (l-dopa). Electron microscopy and immunogold labeling for glutamate terminals within the striatum demonstrated that the observed differences in kinetics of corticostriatal terminals in DD mice were not attributable to aberrant cytoarchitecture or glutamate density. Microdialysis revealed that basal extracellular striatal glutamate was normal in DD mice. These data indicate that dopamine deficiency results in morphologically normal corticostriatal terminals with hypersensitive D2 receptors.

Time-dependent changes in striatal glutamate synapses following a 6-hydroxydopamine lesion

The goal of this study was to investigate changes in glutamatergic synapses in the striatum of rats at two different time-points following a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. One month following this lesion of the nigrostriatal pathway, there was an increase (70%) in the mean percentage of asymmetrical synapses within the dorsolateral striatum containing a discontinuous, or perforated, postsynaptic density, possibly suggesting an increase in glutamatergic activity. This was correlated, in the same brain region, with a decrease (44%) in the density of glutamate immunoreactivity within nerve terminals associated with all asymmetrical synapses and also with those terminals associated with a perforated postsynaptic density. These morphological changes were consistent with an increase (>two-fold) in the basal extracellular level of striatal glutamate, as measured by in vivo microdialysis. The density of GABA immunolabeling within symmetrical nerve terminals was increased (25%) at this one month time-period. Dopamine levels within the lesioned striatum were >99% depleted. However, at three months, while an increase in the mean percentage of striatal perforated synapses was maintained, a significant increase (50%) in the density of striatal nerve terminal glutamate immunolabeling within all asymmetrical synapses and those associated with a perforated postsynaptic density was observed. This was correlated with a small, but significant, decrease (32%) in the basal extracellular level of striatal glutamate. The density of GABA immunolabeling within nerve terminals associated with a symmetrical contact remained elevated at this three month time-period, while striatal dopamine levels remained depleted. While the density of nerve terminal GABA immunolabeling remained elevated at both the one and three month time-periods, there appeared to be a differential effect on glutamatergic synapses. The in vivo microdialysis data suggest that glutamate synapses were more active at a basal level at one month and become less active compared to the control group at the three month time-period. These data suggest that there are compensatory changes in glutamatergic synapses within the striatum following a 6-hydroxydopamine lesion that appear to be independent of the level of striatal dopamine or GABA. We propose that changes in the activity of the thalamo-cortico-striatal pathway may help to explain the differential time-course change in striatal glutamatergic synaptic activity.

Regional, reversible ultrastructural changes in rat brain with chronic neuroleptic treatment

Administration of the dopamine receptor antagonist (neuroleptic, antipsychotic), haloperidol, resulting in an increase in the number of dopamine binding sites in the striatum and nucleus accumbens, has been well established. These increases disappear following withdrawal of treatment. Ultrastructurally, we found an increase in the number of synapses containing perforated postsynaptic densities (PSDs) following haloperidol administration within the caudate nucleus but not within the nucleus accumbens. The effect in the caudate reversed following cessation of treatment. We speculate that the terminals undergoing the change are not dopaminergic but may originate from the cerebral cortex. This reversible morphological increase associated with dopamine antagonist drug therapy may be reflective of the tolerance developed to neuroleptic drug-induced extrapyramidal syndromes and/or may be associated with abnormal motor movements of tardive dyskinesia that occur following long-term treatment.

Astrocytes play a role in regulation of synaptic density

Exposure of neonatal cerebellar explants to cytosine arabinoside destroys granule cells and arrests surviving glia in an early stage of maturation. Purkinje cells lack astroglial ensheathment and are hyperinnervated by sprouted Purkinje cell recurrent axon collateral terminals. Such granuloprival cultures were transplanted with optic nerve in order to supply mature glial cells. It was observed that not only were Purkinje cells almost completely ensheathed by astroglia, but there was a greater than 60% reduction in the number of somatic synapses compared to the non-transplanted granuloprival cultures. This astroglial ensheathment, which may be neuronally directed, could be the physical element provoking the reduction in the number of synapses.

Hypoxia–ischemia preferentially triggers glutamate depletion from oligodendroglia and axons in perinatal cerebral white matter

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13±0.25 counts/μm2), and declined significantly during hypoxia-ischemia (0.93±0.15 counts/μm2: P < 0.001). Astrocytes had lower glutamate levels (0.7±0.07 counts/μm2), and showed a relatively small decline during hypoxiaischemia. Axonal regions contained high levels of glutamate (1.84±0.20 counts/μm2), much of which was lost during hypoxia-ischemia (0.72±0.20 counts/μm2: P >0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90±0.20 counts/μm2), and ependymal cells (2.20±0.28 counts/μm2), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97±0.08 counts/μm2: P >0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

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

The pathogenesis of Parkinsons 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.

Loss of Leucine-rich Repeat Kinase 2 (LRRK2) in Rats Leads to Progressive Abnormal Phenotypes in Peripheral Organs

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson’s disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype. In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats [1] and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson’s disease.

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.

Effects of subchronic clozapine and haloperidol on striatal glutamatergic synapses

Abstract: Subchronic treatment with haloperidol increases the number of asymmetric glutamate synapses associated with a perforated postsynaptic density in the striatum. To characterize these synaptic changes further, the effects of subchronic (28 days) administration of an atypical antipsychotic, clozapine (30 mg/kg, s.c.), or a typical antipsychotic, haloperidol (0.5 mg/kg, s.c.), on the binding of [3H]MK‐801 to the NMDA receptor‐linked ion channel complex and on the in situ hybridization of riboprobes for NMDAR2A and 2B subunits and splice variants of the NMDAR1 subunit were examined in striatal preparations from rats. The density of striatal glutamate immunogold labeling associated with nerve terminals of all asymmetric synapses and the immunoreactivity of those asymmetric synapses associated with a perforated postsynaptic density were also examined by electron microscopy. Subchronic neuroleptic administration had no effect on [3H]MK‐801 binding to striatal membrane preparations. Both drugs increased glutamate immunogold labeling in nerve terminals of all asymmetric synapses, but only haloperidol increased the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated postsynaptic density. Whereas subchronic administration of clozapine, but not haloperidol, resulted in a significant increase in the hybridization of a riboprobe that labels all splice variants of the NMDAR1 subunit, both drugs significantly decreased the abundance of NMDAR1 subunit mRNA containing a 63‐base insert. Neither drug altered mRNA for the 2A subunit, but clozapine significantly increased hybridization of a probe for the 2B subunit. The data suggest that some neuroleptic effects may be mediated by glutamatergic systems and that typical and atypical antipsychotics can have varying effects on the density of glutamate in presynaptic terminals and on the expression of specific NMDA receptor splice variant mRNAs. Alternatively, NMDAR1 subunit splice variants may differentially respond to interactions with glutamate.