Causes of progressive cerebellar ataxia: prospective evaluation of 1500 patients.

BACKGROUND: Cerebellar ataxias are the result of diverse disease processes that can be genetic or acquired. Establishing a diagnosis requires a methodical approach with expert clinical evaluation and investigations. We describe the causes of ataxia in 1500 patients with cerebellar ataxia. METHODS: All patients were referred to the Sheffield Ataxia Centre, UK, and underwent extensive investigations, including, where appropriate genetic testing using next-generation sequencing (NGS). Patients were followed up on a 6-monthly basis for reassessment and further investigations if indicated. RESULTS: A total of 1500 patients were assessed over 20 years. Twenty per cent had a family history, the remaining having sporadic ataxia. The commonest cause of sporadic ataxia was gluten ataxia (25%). A genetic cause was identified in 156 (13%) of sporadic cases with other causes being alcohol excess (12%) and cerebellar variant of multiple system atrophy (11%). Using NGS, positive results were obtained in 32% of 146 patients tested. The commonest ataxia identified was EA2. A genetic diagnosis was achieved in 57% of all familial ataxias. The commonest genetic ataxias were Friedreich's ataxia (22%), SCA6 (14%), EA2 (13%), SPG7 (10%) and mitochondrial disease (10%). The diagnostic yield following attendance at the Sheffield Ataxia Centre was 63%. CONCLUSIONS: Immune-mediated ataxias are common. Advances in genetic testing have significantly improved the diagnostic yield of patients suspected of having a genetic ataxia. Making a diagnosis of the cause of ataxia is essential due to potential therapeutic interventions for immune and some genetic ataxias.

Bisphenol A concentrations in maternal breast milk and infant urine.

PURPOSE: The present report describes the distribution of breast milk and urinary free and total bisphenol A (BPA) concentrations, from 27 postpartum women and their 31 infants, and explores the influence of age, sex, and nutritional source on infant BPA urinary concentration. METHODS: Both free (unconjugated) and total (free plus conjugated) BPA concentrations from women's breast milk samples and infants' urine samples were measured by online solid-phase extraction coupled to high-performance liquid chromatography-isotope dilution tandem mass spectrometry. Descriptive statistics and nonparametric tests of group comparisons were conducted. RESULTS: Total BPA was detected in 93 % of urine samples in this healthy infant population aged 3-15 months who were without known environmental exposure to BPA [interquartile range (IQR) = 1.2-4.4 µg/L)]. Similarly, 75 % of the mothers' breast milk samples had detectable concentrations of total BPA (IQR = 0.4-1.4 µg/L). The magnitude and frequency of detection of free BPA in the children's urine and the mothers' breast milk were much lower than the total concentrations. CONCLUSIONS: Total BPA was detected in 93 % of this healthy infant population aged 3-15 months who are without known environmental exposure to BPA. Neither free nor total BPA urinary concentrations differed significantly by infant's sex or by nutritional source (breast milk and/or formula) while age group was of borderline significance. There were no significant correlations between free or total BPA concentrations in mothers' breast milk and their infants' urine.

Antiparkinsonian potential of targeting group III metabotropic glutamate receptor subtypes in the rodent substantia nigra pars reticulata.

BACKGROUND AND PURPOSE: Increased firing of the glutamatergic pathway between the subthalamic nucleus and substantia nigra pars reticulata (SNpr) contributes to the abnormal firing of motor circuits and subsequent motor deficits seen in Parkinson's disease. Broad spectrum agonist-induced activation of presynaptic group III metabotropic glutamate (mGlu) receptors within the SNpr reduced glutamate release and reversed akinesia in the reserpine-treated rat model of Parkinson's disease. Here, we have sought to identify which subtypes of group III mGlu receptor in the SNpr were responsible for these beneficial effects. EXPERIMENTAL APPROACH: The ability of the mGlu(4) positive allosteric modulator, N-phenyl-7-(hydroxyminocyclopropa[b]chromen-1a-carboxamide) (PHCCC), the mGlu(7) allosteric agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) and the mGlu(8) -selective agonist (S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG] to inhibit KCl-evoked [(3) H]-D-aspartate release was examined in vitro in rat nigral prisms. Reversal of akinesia in reserpine-treated rats was also assessed following intranigral injection of these agents. KEY RESULTS: PHCCC and AMN082 inhibited [(3) H]-D-aspartate release by 42% and 53%, respectively when given alongside a sub-threshold concentration of the broad spectrum group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4; 1 µM). In contrast (S)-3,4-DCPG failed to inhibit [(3) H]-D-aspartate release. All three agents also reversed reserpine-induced akinesia although only the effects of PHCCC and AMN082 were inhibited by pre-treatment with the group III antagonist (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG). CONCLUSIONS AND IMPLICATIONS: These findings reveal that targeting SNpr mGlu(4) or mGlu(7) receptors, but not mGlu(8) receptors, provided relief from akinesia in the reserpine-treated rat model of Parkinson's disease, most likely reflecting inhibition of excess glutamate release in this region.

Symptomatic and neuroprotective effects following activation of nigral group III metabotropic glutamate receptors in rodent models of Parkinson's disease.

BACKGROUND AND PURPOSE: Increased glutamatergic innervation of the substantia nigra pars reticulata (SNpr) and pars compacta (SNpc) may contribute to the motor deficits and neurodegeneration, respectively, in Parkinson's disease (PD). This study aimed to establish whether activation of pre-synaptic group III metabotropic glutamate (mGlu) receptors reduced glutamate release in the SN, and provided symptomatic or neuroprotective relief in animal models of PD. EXPERIMENTAL APPROACH: Broad-spectrum group III mGlu receptor agonists, O-phospho-l-serine (l-SOP) and l-2-amino-4-phosphonobutyrate (l-AP4), were assessed for their ability to inhibit KCl-evoked [(3)H]-d-aspartate release in rat nigral prisms or inhibit KCl-evoked endogenous glutamate release in the SNpr in vivo using microdialysis. Reversal of akinesia in reserpine-treated rats was assessed following intranigral injection of l-SOP and l-AP4. Finally, the neuroprotective effect of 7 days' supra-nigral treatment with l-AP4 was examined in 6-hydroxydopamine (6-OHDA)-lesioned rats. KEY RESULTS: l-SOP and l-AP4 inhibited [(3)H]-d-aspartate release by 33 and 44% respectively. These effects were blocked by the selective group III mGlu antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG). l-SOP also reduced glutamate release in the SNpr in vivo by 48%. Injection of l-SOP and l-AP4 into the SNpr reversed reserpine-induced akinesia. Following administration above the SNpc, l-AP4 provided neurochemical, histological and functional protection against 6-OHDA lesion of the nigrostriatal tract. Pretreatment with CPPG inhibited these effects. CONCLUSIONS AND IMPLICATIONS: These findings highlight group III mGlu receptors in the SN as potential targets for providing both symptomatic and neuroprotective relief in PD, and indicate that inhibition of glutamate release in the SN may underlie these effects.

DNA damage in human sperm is related to urinary levels of phthalate monoester and oxidative metabolites.

BACKGROUND: The ubiquitous use of phthalate esters in plastics, personal care products and food packaging materials results in widespread general population exposure. In this report, we extend our preliminary study on the relationship between urinary concentrations of phthalate metabolites and sperm DNA damage among a larger sample of men and include measurements of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), two oxidative metabolites of di-(2-ethylhexyl) phthalate (DEHP). METHODS: Among 379 men from an infertility clinic, urinary concentrations of phthalate metabolites were measured using isotope-dilution high-performance liquid chromatography-tandem mass spectrometry. Sperm DNA damage measurements, assessed with the neutral comet assay, included comet extent (CE), percentage of DNA in tail (Tail%) and tail distributed moment (TDM). RESULTS: Monoethyl phthalate (MEP), a metabolite of diethyl phthalate, was associated with increased DNA damage, confirming our previous findings. Mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of DEHP, was associated with DNA damage after adjustment for the oxidative DEHP metabolites. After adjustment for MEHHP, for an interquartile range increase in urinary MEHP, CE increased 17.3% [95% confidence interval (CI) = 8.7-25.7%], TDM increased 14.3% (95% CI = 6.8-21.7%) and Tail% increased 17.5% (95% CI = 3.5-31.5%). CONCLUSIONS: Sperm DNA damage was associated with MEP and with MEHP after adjusting for DEHP oxidative metabolites, which may serve as phenotypic markers of DEHP metabolism to 'less toxic' metabolites. The urinary levels of phthalate metabolites among these men were similar to those reported for the US general population, suggesting that exposure to some phthalates may affect the population distribution of sperm DNA damage.

Nicotine, but neither the alpha4beta2 ligand RJR2403 nor an alpha7 nAChR subtype selective agonist, protects against a partial 6-hydroxydopamine lesion of the rat median forebrain bundle.

Although previous studies suggest nicotine protects against a 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal tract in rats, it is not known whether functional motor recovery occurs or which nicotinic acetylcholine receptor (nAChR) subtypes mediate this effect. These issues were investigated by comparing the effects of the subtype-specific nAChR agonists, RJR2403 (alpha4beta2 preferring) and (R)-N-(1-azabicyclo[2.2.2.]oct-3-yl)(5-(2-pyridyl)thiopene-2-carboxamide (Compound A; alpha7-selective) and nicotine given 30 min prior to and daily for 14 days after a partial 6-OHDA lesion. In vehicle treated animals, 6-OHDA (6 microg) produced a 65 +/- 1.8% loss of striatal tyrosine hydroxylase (TH) immunoreactivity in the lesion versus intact hemisphere. This loss was reduced in animals treated with nicotine (0.6 and 0.8 mg kg(-1)), reaching significance at the higher dose (36.6 +/- 3.7% loss; P < 0.01 versus vehicle). Treatment with nicotine (0.6 and 0.8 mg kg(-1)) also significantly reduced the number of amphetamine-induced rotations compared to vehicle treatment. In contrast, treatment with RJR2403 (0.2 and 0.4 mg kg(-1)) or Compound A (10 and 20 mg kg(-1)) reduced neither the degree of amphetamine-induced rotations nor the loss of striatal TH immunoreactivity. These data suggest that whilst nicotine is neuroprotective in this partial lesion model, activation of neither the alpha4beta2 nor alpha7 subtypes alone is sufficient to provide protection.

Chronic pre-treatment with nicotine enhances nicotine-evoked striatal dopamine release and alpha6 and beta3 nicotinic acetylcholine receptor subunit mRNA in the substantia nigra pars compacta of the rat.

Whilst local intrastriatal infusion of nicotine consistently elicits striatal dopamine release, systemic administration often fails to do so. Since chronic nicotine administration is known to result in desensitisation-induced upregulation of nicotinic acetylcholine receptors (nAChRs), the present study investigated whether chronic pre-treatment could enhance the response to systemic nicotine and, if so, whether increases in specific nAChR subunit mRNA levels in the substantia nigra pars compacta (SNc) may underlie this effect. In vivo microdialysis studies in male Sprague-Dawley rats revealed that following 4 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), local intrastriatal nicotine infusion (3 mM) elicited significantly higher dopamine efflux compared to vehicle pre-treated controls (peak release: 1273 +/- 199% basal versus 731 +/- 113% basal), whereas systemic nicotine challenge (0.8 mg kg(-1)s.c.) elicited no response. In contrast, following 8 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), systemic nicotine challenge (0.8 mg kg(-1)s.c.) now produced significantly higher dopamine efflux than that of vehicle pre-treated controls (147 +/- 30% basal versus 91 +/- 5% basal). Eight days pre-treatment with nicotine also significantly elevated the levels of alpha6 (approximately 55%) and beta3 (approximately 43%) nAChR subunit mRNA in the SNc, suggesting that up-regulation of these nAChR subunit genes in the nigrostriatal tract may contribute to the enhanced nicotine-evoked striatal dopamine release.

Changes in GABA(B) receptor mRNA expression in the rodent basal ganglia and thalamus following lesion of the nigrostriatal pathway.

Loss of striatal dopaminergic innervation in Parkinson's disease (PD) is accompanied by widespread alterations in GABAergic activity within the basal ganglia and thalamus. Accompanying changes in GABA(B) receptor binding have been noted in some basal ganglia regions in parkinsonian primates, suggesting that plasticity of this receptor may also occur in PD. However, the molecular mechanisms underlying the changes in receptor binding and the manner and extent to which different GABA(B) receptor mRNA subunits and splice-variants are affected remain unknown. This study used in situ hybridisation to examine the full profile of changes in expression of the known rat GABA(B) receptor genes and gene variants in the basal ganglia and thalamus of rats, brought about by degeneration of the nigrostriatal tract. All of the GABA(B) mRNA species examined showed unique expression patterns throughout the basal ganglia and thalamus. In addition, all exhibited a marked loss of expression (between 46 and 80%) in the substantia nigra pars compacta of animals bearing a complete 6-hydroxydopamine-induced lesion of the nigrostriatal tract, confirming the presence of these variants in dopaminergic neurones in this region. Further analysis of autoradioagrams revealed additional changes only in GABA(B(1a)) mRNA in discrete anatomical regions. Expression of the GABA(B(1a)) variant was significantly increased in the substantia nigra pars reticulata (33+/-2%), entopeduncular nucleus (26+/-1%) and the subthalamic nucleus (16+/-1%). Since these regions all receive reduced GABAergic innervation following nigrostriatal tract lesioning, it is possible that the increased expression occurs as a compensatory measure. In conclusion, these data demonstrate that GABA(B) receptor genes exhibit regional- and subunit/variant-specific plasticity at the molecular level under parkinsonian conditions.

Changes in metabotropic glutamate receptor 1-8 gene expression in the rodent basal ganglia motor loop following lesion of the nigrostriatal tract.

Metabotropic glutamate (mGlu) receptors in the basal ganglia motor loop may increase cell excitability (Group I) or modulate neurotransmitter release (Group I, II and III). Nigrostriatal tract degeneration in Parkinson's disease (PD) produces downstream pathological disturbances in glutamate and GABA transmission. The present study examined whether changes in mGlu receptor gene expression may either contribute to, or compensate for these pathological changes in transmission. In situ hybridisation studies examined the levels of mGlu receptor mRNA in motor loop regions in rats bearing a 6-hydroxydopamine-induced unilateral nigrostriatal tract lesion. Gene expression was reduced in the lesion compared to intact hemispheres for mGlu(1) in the substantia nigra pars compacta (SNc; 51.8+/-11.5%), mGlu(3) in the striatum and globus pallidus (11.7+/-2.8% and 18.9+/-1.4%, respectively) and mGlu(4) in the striatum and premotor cortex (13.8+/-2.7% and 15.8+/-5.5%, respectively). Loss of mGlu(1) mRNA in the SNc confirms that mGlu(1) is highly expressed on dopaminergic neurones where it may contribute to their vulnerability in PD. The down-regulation of mGlu(3) and mGlu(4) mRNA may reflect reduced transcriptional activity in response to increased levels of extracellular glutamate in these regions under parkinsonian conditions. These changes are likely to exacerbate the pathophysiological glutamate and GABA transmission within these regions in PD.

Reliability of the comet assay in cryopreserved human sperm.

BACKGROUND: Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. METHODS: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. RESULTS: The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. CONCLUSIONS: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.

The role of neuronal nicotinic acetylcholine receptors in acute and chronic neurodegeneration.

In the last five years there has been a rapid explosion of publications reporting that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in neurodegenerative disorders. Furthermore, there is a well-established loss of nAChRs in post-mortem brains from patients with Alzheimer's disease, Parkinson's disease and a range of other disorders. In the present review we discuss the evidence that nicotine and subtype selective nAChR ligands can provide neuroprotection in in vitro cell culture systems and in in vivo studies in animal models of such disorders. Whilst in vitro data pertaining to a protective effect of nicotine against nigral neurotoxins like MPTP is less robust, most studies agree that nicotine is protective against glutamate and beta-amyloid toxicity in various culture systems. This effect appears to be mediated by alpha7 subtype nAChRs since the protection is blocked by alpha-bungarotoxin and is mimicked by alpha7 selective agonists. In vivo studies indicate that alpha7 receptors play a critical role in protection from cholinergic lesions and enhancing cognitive function. The exact subtype involved in the neuroprotectant effects seen in animal models of Parkinson's disease is not clear, but in general broad spectrum nAChR agonists appear to provide protection, while alpha4beta2 receptors appear to mediate symptomatic improvements. Evidence favouring a protectant effect of nicotine against acute degenerative conditions is less strong, though some protection has been observed with nicotine pre-treatment in global ischaemia models. A variety of cellular mechanisms ranging from the production of growth factors through to inactivation of toxins and antioxidant actions of nicotine have been proposed to underlie the nAChR-mediated neuroprotection in vitro and in vivo. In summary, although the lack of subtype selective ligands has hampered progress, it is clear that in the future neuronal nAChR agonists could provide functional improvements and slow or halt the progress of several crippling degenerative diseases.

The 5HT(1B) receptor agonist, CP-93129, inhibits [(3)H]-GABA release from rat globus pallidus slices and reverses akinesia following intrapallidal injection in the reserpine-treated rat.

This study examined whether activation of 5HT(1B) receptors in the rodent globus pallidus (GP) could reduce GABA release in vitro and reverse reserpine-induced akinesia in vivo. Microdissected slices of GP from male Sprague Dawley rats (300-350 g) were preloaded with [(3)H]-GABA. During subsequent superfusion, 4 min fractions were collected for analysis of release. The effects of the 5HT(1B) receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3, 2-b]pyrid-5-one (CP-93129), on 25 mM KCl-evoked release were examined using a standard dual stimulation paradigm. Male Sprague Dawley rats (270 - 290 g), stereotaxically cannulated above the GP, were rendered akinetic by injection of reserpine (5 mg kg(-1) s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of CP-93129 was examined. CP-93129 (0.6-16.2 microM) produced a concentration-dependent inhibition of 25 mM KCl-evoked [(3)H]-GABA release reaching a maximum inhibition of 52.5+/-4.5%. The effect of a submaximal concentration of CP-93129 (5.4 microM) was fully inhibited by the 5HT(1B) receptor antagonist, isamoltane (10 microM). Following intrapallidal injection, CP-93129 (30-330 nmol in 0.5 microl) produced a dose-dependent increase in net contraversive rotations reaching a maximum of 197+/-32 rotations in 240 min at 330 nmol. Pre-treatment with isamoltane (10 nmol in 1 microl) inhibited the effects of a submaximal dose of CP-93129 (220 nmol) by 84+/-6%. These data suggest that at least some 5HT(1B) receptor function as heteroreceptors in the GP, reducing the release of GABA. Moreover, CP-93129-mediated activation of these receptors in the GP provides relief of akinesia in the reserpine-treated rat model of PD.

Changes in [3H]zolpidem and [3H]Ro 15-1788 binding in rat globus pallidus and substantia nigra pars reticulata following a nigrostriatal tract lesion.

Changes in GABA(A) receptor alpha(1) subunit gene expression occur in the globus pallidus and substantia nigra pars reticulata following lesions of the nigrostriatal tract. To determine whether these changes are translated at the protein level, we performed quantitative autoradiography with the alpha(1) selective ligand, [3H]zolpidem, and the non-selective benzodiazepine site ligand, [3H]Ro 15-1788. Binding of both [3H]zolpidem and [3H]Ro 15-1788 was significantly increased in the substantia nigra pars reticulata (13. 5+/-4.1 and 26.3+/-2.9%, respectively) and significantly reduced in the globus pallidus (20.9+/-0.8 and 18.3+/-1.3%, respectively). These changes in alpha(1) subunit protein expression may help to compensate for the pathological changes in GABAergic activity that occur after striatal dopamine depletion.

The group II metabotropic glutamate receptor agonist, DCG-IV, alleviates akinesia following intranigral or intraventricular administration in the reserpine-treated rat.

1. This study examined whether activation of group II metabotropic glutamate (mGlu) receptors in the substantia nigra pars reticulata (SNr) could reverse akinesia in a rodent model of Parkinson's disease (PD). 2. Male Sprague Dawley rats, stereotaxically cannulated above either the SNr or third ventricle, were rendered akinetic by injection of reserpine (5 mg kg-1 s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of the group II mGlu receptor agonist, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), was examined. 3. Following intranigral injection, DCG-IV (0.125-0.75 nmol in 0.1 microliter) produced a dose-dependent increase in net contraversive rotations (n = 6-8 animals per dose), reaching a maximum of 395 +/- 51 rotations 60 min-1 after 0.75 nmol. The effects of DCG-IV (0.5 nmol) were inhibited by 63.0 +/- 9.0% following 30 min pre-treatment with the group II mGlu receptor antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 100 nmol in 0.2 microliter; n = 6). 4. Following intraventricular injection, DCG-IV (0.125-1.5 nmol in 2 microliters) produced a dose-dependent increase in bilateral locomotor activity (n = 6-7 animals per dose), reaching a maximum of 180 +/- 21 locomotor units 30 min-1 after 0.5 nmol. Pre-treatment with EGLU (200 nmol in 2 microliters) inhibited the effects of DCG-IV (0.5 nmol) by 68.2 +/- 12.3% (n = 5). 5. These data show that activation of group II mGlu receptors in the SNr provides relief of akinesia in the reserpinized rat model of PD. The reversal seen following intraventricular administration supports the likely therapeutic benefit of systemically-active group II mGlu receptor agonists in PD.

Effect of unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway on GABA(A) receptor subunit gene expression in the rodent basal ganglia and thalamus.

In Parkinson's disease, changes in GABAergic activity occurring downstream of the striatal dopamine loss are accompanied by reciprocal changes in GABA(A) receptor binding, the underlying molecular mechanisms for which are unknown. This study examined whether changes in expression of the genes encoding known GABA(A) receptor subunits (alpha(1-4), beta(1-3), gamma(1-3) and delta) could account for this receptor plasticity using a rodent model of Parkinson's disease with a 6-hydroxydopamine-induced nigrostriatal lesion. Analysis of autoradiograms of the basal ganglia and thalamus revealed changes in expression of only four of the 11 subunits studied. Expression of alpha1 and beta2 subunit genes was altered in a parallel manner following a 6-hydroxydopamine lesion; messenger RNA levels for both were significantly increased in the substantia nigra pars reticulata (11 +/- 4% and 17 +/- 1%, respectively), and significantly reduced in the globus pallidus (18 +/- 3% and 16 +/- 3%, respectively) and parafascicular nucleus (19 +/- 3% and 16 +/- 5%, respectively). Smaller changes in the messenger RNA levels encoding the alpha1 subunit in the lateral amygdala (8 +/- 1% decrease) and the alpha4 and gamma2 subunits in the striatum (10 +/- 2% and 6 +/- 1% increase, respectively) were also observed. No changes in expression were noted for any other subunits in any region studied. Clearly, both region- and subunit-specific regulation of GABA(A) receptor subunit gene expression occurs following a nigrostriatal tract lesion. The changes in expression of the alpha1 and beta2 subunit genes probably contribute to the documented changes in GABA(A) receptor binding following striatal dopamine depletion. Moreover, they provide a molecular basis by which the pathological changes in GABAergic activity in Parkinson's disease may be partially compensated.

Topographical organization of opioid peptide precursor gene expression following repeated apomorphine treatment in the 6-hydroxydopamine-lesioned rat.

Many studies have previously described changes in preproenkephalin-A (PPE-A) and preproenkephalin-B (PPE-B) gene expression in the striatum of the 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (both with or without dopamine replacement treatment). To date, these studies have either taken the striatum as a whole or have focused on a single subregion of the striatum. However, the striatum is organized into anatomically discrete parallel circuits serving different functions (motor, associative, and limbic). We have therefore employed in situ hybridization to examine the detailed topography of changes in opioid precursor expression following dopamine depletion and subsequent treatment with apomorphine (5 mg/kg twice daily for 10 days). In the untreated 6-OHDA-lesioned striatum PPE-A expression was elevated only in the dorsal (sensorimotor) caudate-putamen. Following apomorphine treatment PPE-A mRNA levels were further raised in the sensorimotor striatum (

Enhancement of the behavioral response to apomorphine administration following repeated treatment in the 6-hydroxydopamine-lesioned rat is temporally correlated with a rise in striatal preproenkephalin-B, but not preproenkephalin-A, gene expression.

Repeated dopamine receptor stimulation in the 6-hydroxydopamine (6-OHDA)-lesioned rat produces a marked enhancement in the behavioral response to a given dose of dopamine agonist. Such treatment also increases the synthesis of preproenkephalin-B (PPE-B) throughout the striatum and preproenkephalin-A (PPE-A) rostrally. To examine the relationship between these changes, 6-OHDA-lesioned rats were treated with apomorphine (5 mg/kg) twice-daily. Between 0.5 and 7 days treatment, behavior was monitored and the levels of PPE-A and PPE-B mRNA were determined by in situ hybridization. A single injection of apomorphine induced a well-known rotational response contraversive to the lesion (314 +/- 22 rotations h-1). However, with repeated injection, the response to apomorphine was markedly enhanced, being significantly higher on Days 3 (704 +/- 60 rotations h-1), 5 (1354 +/- 104 rotations h-1), and 7 (1680 +/- 117 rotations h-1). In the lesioned caudate-putamen, but not in the nucleus accumbens, PPE-B expression rose in a manner that was temporally correlated with the enhanced locomotor response to apomorphine. PPE-A expression in the lesioned striatum, significantly increased only after 7 days treatment, did not correlate with the behavioral response. In conclusion, PPE-B may contribute to the development of behavioral supersensitivity following repeated dopamine-replacement therapy in animal models of Parkinson's disease.

Modulation of GABA transmission by diazoxide and cromakalim in the globus pallidus: implications for the treatment of Parkinson's disease.

An ATP-sensitive potassium channel (KATP) is known to modulate insulin release from pancreatic beta cells. It has been proposed that potassium channels related to KATP in the nervous system might similarly modulate neurotransmitter release. We have therefore investigated the effects of KATP opening agents on GABA release in the globus pallidus. Diazoxide and cromakalim decreased the K(+)-evoked release of [3H]GABA from pallidal slices. The maximum inhibition observed for diazoxide (59%) and cromakalim (66%) was achieved at a concentration of 100 microM. The effects of both cromakalim and diazoxide were significantly antagonized by the concurrent application of the sulfonylurea glibenclamide (100 microM). Intrapallidal injections of diazoxide in the reserpine-treated rat model of Parkinson's disease reduced akinesia in a dose-dependent manner. These data suggest that manipulation of neuronal potassium channels with pharmacological properties similar to KATP may prove useful in the treatment of Parkinson's disease.

The effects of glibenclamide on tetanic force and intracellular calcium in normal and fatigued mouse skeletal muscle.

In this study the effects of ATP-sensitive K+ channel modulators were studied in intact single fibres dissected from mouse skeletal muscle. Indo-1 was used to measure [Ca2+]i simultaneously with force during normal and fatiguing stimulation. In control fibres, opening of ATP-sensitive K+ channels with BRL 38227 produced a small reduction in tetanic force and [Ca2+]i. In contrast, glibenclamide, a selective blocker of the ATP-sensitive K+ channel, slightly increased tetanic force and [Ca2+]i in these fibres and also increased Ca2+ sensitivity. Glibenclamide produced a more marked increase in tetanic force and [Ca2+]i during the later stages of fatiguing stimulation, although this effect was observed in only 50% of fibres examined. We conclude from this study that glibenclamide produces a partial reversal of the later stages of fatigue in a subpopulation of muscle fibres. Opening of ATP-sensitive K+ channels may therefore contribute to the decline in tetanic force and [Ca2+]i characteristic of skeletal muscle fatigue.

The distribution of intracellular calcium concentration in isolated single fibres of mouse skeletal muscle during fatiguing stimulation.

Fluorescence microscopy has been used to examine the distribution of intracellular calcium concentration ([Ca2+]i) in isolated single fibres from the mouse flexor brevis muscle during fatiguing stimulation. Under control conditions there was a virtually uniform distribution of [Ca2+]i in fura-2 loaded fibres either at rest or during short (0.35 s, 100 Hz) tetani. Fatigue produced by repeated short tetani was accompanied by an early rise, followed by a marked fall, in tetanic [Ca2+]i. Throughout the period of fatiguing stimulation the distribution of [Ca2+]i remained uniform with no detectable gradients observed. In contrast, when fatigue was produced by continuous 100 Hz stimulation, a small gradient of [Ca2+]i developed across the fibre with the [Ca2+]i in the centre of the fibre lower than that at the edge of the fibre. This gradient was apparent after 1.7 s, persisted for at least 11 s and was superimposed on a rise followed by a fall in spatially averaged [Ca2+]i. Reduction of the extracellular Na+ to 50% caused reduced force production and a reduced [Ca2+]i in the centre of the fibre. To assess the contribution of reduced response of the myofibrillar proteins to [Ca2+]i during continuous tetani, the relation between [Ca2+]i and force throughout the long tetanus was compared with that obtained in short, unfatigued tetani. These results show that in long tetani, reduced tetanic [Ca2+]i and reduced responsiveness of the myofibrillar proteins to [Ca2+]i each make important contributions to the decline of force, whereas the gradients of [Ca2+]i make only a small contribution.