Regional distribution and effects of postmortal delay on endocannabinoid content of the human brain.

Tissue levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have been determined in 16 regions and nuclei from human brains, using liquid chromatography/in-line mass spectrometry. Measurements in brain samples stored at -80 degrees C for 2 months to 13 years indicated that endocannabinoids were stable under such conditions. In contrast, the postmortal delay had a strong effect on brain endocannabinoid levels, as documented in brain samples microdissected and frozen 1-6 h postmortem, and in neurosurgical samples 0, 5, 30, 60, 180 and 360 min after their removal from the brain. The tissue levels of AEA increased continuously and in a region-dependent manner from 1 h after death, increasing about sevenfold by 6 h postmortem. In contrast, concentrations of 2-AG, which were 10-100 times higher in human brain regions than those of AEA, rapidly declined: within the first hour, 2-AG levels dropped to 25-35% of the initial ('0 min') value, thereafter they remained relatively stable. As analyzed in samples removed 1-1.5 h postmortem, AEA levels ranged from a high of 96.3 fmol/mg tissue in the nucleus accumbens to a low of 25.0 fmol/mg in the cerebellum. 2-AG levels varied eightfold, from 8.6 pmol/mg in the lateral hypothalamus to 1.1 pmol/mg in the nucleus accumbens. Relative levels of AEA and 2-AG varied from region to region, with the 2-AG:AEA ratio being high in the sensory spinal trigeminal nucleus (140:1), the spinal dorsal horn (136:1) and the lateral hypothalamus (98:1) and low in the nucleus accumbens (16:1) and the striatum (31:1). The results highlight the pitfall of analyzing endocannabinoid content in brain samples of variable postmortal delay, and document differential distribution of the two main endocannabinoids in the human brain.

The role of the endocannabinoid system in the control of energy homeostasis.

The endocannabinoid system has recently emerged as an important regulator of energy homeostasis, involved in the control of both appetite and peripheral fat metabolism. We briefly review current understanding of the possible sites of action and cellular mechanisms involved in the central appetitive and peripheral metabolic effects of endocannabinoids. Studies in our laboratory, using leptin-deficient obese rodents and CB1 cannabinoid receptor (CB1)-deficient mice, have indicated that endocannabinoids acting via CB1 are involved in the hunger-induced increase in food intake and are negatively regulated by leptin in brain areas involved in appetite control, including the hypothalamus, limbic forebrain and amygdala. CB1-/- mice are lean and are resistant to diet-induced obesity (DIO) despite similar energy intake to wild-type mice with DIO, suggesting that CB1 regulation of body weight involves additional peripheral targets. Such targets appear to include both adipose tissue and the liver. CB1 expressed in adipocytes has been implicated in the control of adiponectin secretion and lipoprotein lipase activity. Recent findings indicate that both endocannabinoids and CB1 are present in the liver and are upregulated in DIO. CB1 stimulation increases de novo hepatic lipogenesis through activation of the fatty acid biosynthetic pathway. Components of this pathway are also expressed in the hypothalamus where they have been implicated in the regulation of appetite. The fatty acid biosynthetic pathway may thus represent a common molecular target for the central appetitive and peripheral metabolic effects of endocannabinoids.

Functional effect of adeno-associated virus mediated gene transfer of aromatic L-amino acid decarboxylase into the striatum of 6-OHDA-lesioned rats.

In animal models of Parkinson's disease, gene transfer of aromatic L-amino acid decarboxylase (AADC) leads to an increase in the capacity of the striatum to decarboxylate exogenous L-DOPA. However, the functional effects of enhanced L-DOPA to dopamine conversion have not been explored. Here, we show that following adeno-associated virus (AAV)-AADC transduction, the transgenic AADC is able to decarboxylate exogenous L-DOPA more efficiently so that a dose of L-DOPA ineffective before gene transfer elicits a motor asymmetry (rotational behavior) following gene transfer. Furthermore, rotation scores showed a strong correlation with AADC activity in the lesioned striatum, thus allowing for behavioral screening of successful gene transfer in the brain. In animals receiving AAV2-AADC, dopamine production was restored to 50% of normal levels 12 weeks after the infusion. Microdialysis experiments demonstrated an in vivo enhanced conversion of L-DOPA to dopamine, but no storage capacity as dopamine was released to the extracellular space in a continuous, nonregulated fashion. In addition to the potential clinical benefit of improving decarboxylation efficiency in Parkinson's disease, our approach may be relevant for the treatment of AADC deficiency, a rare, autosomal recessive disorder causing a severe movement disorder and progressive cognitive impairment.

Heparin coinfusion during convection-enhanced delivery (CED) increases the distribution of the glial-derived neurotrophic factor (GDNF) ligand family in rat striatum and enhances the pharmacological activity of neurturin.

Convection-enhanced delivery (CED) distributes macromolecules in the brain in a homogeneous, targeted fashion in clinically useful volumes. However, the binding of growth factors to heparin-binding sites in the extracellular matrix may limit the volume of distribution (V(d)). To overcome this limitation, we examined the effects of heparin coinfusion on V(d) of glial-derived neurotrophic factor (GDNF), neurturin (NTN), artemin, and a nonspecifically bound protein, albumin. Heparin coinfusion significantly enhanced the V(d) of GDNF and GDNF-homologous trophic factors, probably by binding and blocking heparin-binding sites in the extracellular matrix. Furthermore, coinfusion of heparin with NTN enhanced striatal dopamine metabolism, compared to trophic factor administered alone. The negligible benefit of GDNF in recent clinical trials of Parkinson's disease may result from limited tissue distribution. Heparin coinfusion during CED targeting the striatum may alleviate this important limitation. This study demonstrates the influence of receptor binding on the distribution of trophic factors in the CNS.

Estimation of striatal dopamine spillover and metabolism in vivo.

A microdialysis probe with an attached microinjection cannula was inserted into rat striatum. [3H]dopamine (or [14C]sucrose as a reference substance for diffusion) was infused via the cannula, with microdialysate sampled for concentrations of endogenous and [3H]-labeled dopamine and its metabolites. The calculated specific activities of the [3H]-labeled metabolites led to the conclusions that striatal extracellular dopamine undergoes inactivation mainly by extraneuronal but also by neuronal uptake and intracellular metabolism. Some of the dopamine taken up into nerve terminals slowly re-enters (spillover) the extracellular fluid unchanged. This spillover was calculated to be about 5 pmol/min. Destruction of dopaminergic terminals increases the turnover of vesicular stores in the surviving terminals, both by increased vesicular leakage and by increased release into the extracellular fluid.

Acidic dopamine metabolites are actively extruded from PC12 cells by a novel sulfonylurea-sensitive transporter.

Incubation of PC 12 cells with the sulfonylurea drug, glipizide (1-100 microM), increased intracellular levels of the acidic metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The levels of these acids in the medium were decreased, indicating the presence of a sulfonylurea-sensitive organic anion transporter. In the present study, we demonstrate that the sulfonylurea-sensitive transport of acidic dopamine metabolites is unidirectional, ATP dependent, unaffected by ouabain or by tetrodotoxin and blocked by drugs that interact with the multidrug-resistance protein-1 (MRP1). However, over-expression of MRP1 did not affect transport of the acid metabolites. The pharmacological profile and ion dependence of the transporter also differs from that of known ATP-binding cassette (ABC) family members. Using microdialysis, we also demonstrated a sulfonylurea-sensitive transport process in the striatum of freely moving rats. These results show that acidic dopamine metabolites are actively secreted from dopaminergic cells into surrounding extracellular fluid by a previously undescribed transporter.

Convection-enhanced delivery of AAV vector in parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach.

Using an approach that combines gene therapy with aromatic l-amino acid decarboxylase (AADC) gene and a pro-drug (l-dopa), dopamine, the neurotransmitter involved in Parkinson's disease, can be synthesized and regulated. Striatal neurons infected with the AADC gene by an adeno-associated viral vector can convert peripheral l-dopa to dopamine and may therefore provide a buffer for unmetabolized l-dopa. This approach to treating Parkinson's disease may reduce the need for l-dopa/carbidopa, thus providing a better clinical response with fewer side effects. In addition, the imbalance in dopamine production between the nigrostriatal and mesolimbic dopaminergic systems can be corrected by using AADC gene delivery to the striatum. We have also demonstrated that a fundamental obstacle in the gene therapy approach to the central nervous system, i.e., the ability to deliver viral vectors in sufficient quantities to the whole brain, can be overcome by using convection-enhanced delivery. Finally, this study demonstrates that positron emission tomography and the AADC tracer, 6-[(18)F]fluoro-l-m-tyrosine, can be used to monitor gene therapy in vivo. Our therapeutic approach has the potential to restore dopamine production, even late in the disease process, at levels that can be maintained during continued nigrostriatal degeneration.

3,4-Dihydroxyphenylacetaldehyde potentiates the toxic effects of metabolic stress in PC12 cells.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic metabolite formed by the oxidative deamination of dopamine. This aldehyde is mainly oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH), but is also partly reduced to 3, 4-dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase (ARs). In a previous study, we found that rotenone, a complex I inhibitor, induced a rapid accumulation of DOPAL and DOPET in the medium of cultured PC12 cells. Here, we examined the potential role of DOPAL in the toxicity induced by complex I inhibition in PC12 cells and compared the effects of rotenone on concentrations of DOPAL and DOPET to those of MPP(+). DOPAL and DOPET levels were increased by rotenone but decreased by MPP(+). Inhibition of ALDH by daidzein reduced the formation of DOPAC and increased the accumulation of DOPAL. Inhibition of ARs (with AL1576) diminished DOPET formation and elevated DOPAL concentrations. Combined inhibition of ALDH and ARs markedly elevated DOPAL concentrations while diminishing DOPET and DOPAC levels. The elevation of DOPAL levels induced by combined inhibition of ALDH and ARs had no effect on cell viability. However, combined inhibition of ALDH and ARs potentiated rotenone-induced toxicity. Both the potentiation of toxicity and the increase in DOPAL levels were blocked by inhibition of monoamine oxidase with clorgyline indicating that accumulation of DOPAL was responsible for the potentiated rotenone-induced toxicity following combined inhibition of ALDH and ARs. Since complex I dysfunction is reported to be involved in the pathogenesis of Parkinson's disease, DOPAL potentiation of the deleterious effects of complex I inhibition may contribute to the specific vulnerability of dopaminergic neurons to injury.

Metabolic stress in PC12 cells induces the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) has been reported to be a toxic metabolite formed by the oxidative-deamination of dopamine (DA) catalyzed by monoamine oxidase. This aldehyde is either oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase, an NAD-dependent enzyme or reduced to 3, 4-dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase. In the present study we examined whether levels of DOPAL are elevated by inhibition of the mitochondrial respiratory chain. Using inhibitors of mitochondrial complexes I, II, III and IV we found that inhibition of complex I and III increased levels of DOPAL and DOPET. Nerve growth factor-induced differentiation of PC12 cells markedly potentiated DOPAL and DOPET accumulation in response to metabolic stress. DOPAL was toxic to differentiated PC12 as well as to SK-N-SH cell lines. Because complex I dysfunction has been implicated in the pathogenesis of Parkinson's disease, the accumulation of DOPAL may explain the vulnerability of the dopaminergic system to complex I inhibition. The rapid appearance of DOPAL and DOPET after inhibition of complex I may be a useful early index of oxidative stress in DA-forming neurons.

Effect of glipizide on dopamine synthesis, release and metabolism in PC12 cells.

Sulfonylureas block ATP-dependent K(+) channels (K/ATP channels) in pancreatic beta cells and brain gamma-aminobutyric acid (GABA) containing neurons causing depolarization-evoked insulin or GABA release. In high concentrations, sulfonylureas also inhibit catecholamine release from bovine adrenal chromaffin cells and isolated guinea pig aorta. In this study, we examined the effect of glipizide, a sulfonylurea, on dopamine release from PC12 cells and found that neither basal nor K(+)-stimulated dopamine release was affected. Although PC12 cells expressed mRNA for the K/ATP channel, functional K/ATP channels could not be demonstrated electrophysiologically, consistent with the lack of effect of glipizide on dopamine release. Glipizide did, however, increase cytoplasmic retention of the acidic dopamine metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), indicating blockade of their outward transport. The cellular accumulation of DOPAC was accompanied by reduced tyrosine hydroxylase activity and reduced formation of dopamine and its metabolites presumably by a negative feedback effect of the increased cytoplasmic concentrations of DOPAC.

Increased cerebral blood flow but no reversal or prevention of vasospasm in response to L-arginine infusion after subarachnoid hemorrhage.

OBJECT: The reduction in the level of nitric oxide (NO) is a purported mechanism of delayed vasospasm after subarachnoid hemorrhage (SAH). Evidence in support of a causative role for NO includes the disappearance of nitric oxide synthase (NOS) from the adventitia of vessels in spasm, the destruction of NO by hemoglobin released from the clot into the subarachnoid space, and reversal of vasospasm by intracarotid NO. The authors sought to establish whether administration of L-arginine, the substrate of the NO-producing enzyme NOS, would reverse and/or prevent vasospasm in a primate model of SAH. METHODS: The study was composed of two sets of experiments: one in which L-arginine was infused over a brief period into the carotid artery of monkeys with vasospasm, and the other in which L-arginine was intravenously infused into monkeys over a longer period of time starting at onset of SAH. In the short-term infusion experiment, the effect of a 3-minute intracarotid infusion of L-arginine (intracarotid concentration 10(-6) M) on the degree of vasospasm of the right middle cerebral artery (MCA) and on regional cerebral blood flow (rCBF) was examined in five cynomolgus monkeys. In the long-term infusion experiment, the effect of a 14-day intravenous infusion of saline (control group, five animals) or L-arginine (10(-3) M; six animals) on the occurrence and degree of cerebral vasospasm was examined in monkeys. The degree of vasospasm in all experiments was assessed by cerebral arteriography, which was performed preoperatively and on postoperative Days 7 (short and long-term infusion experiments) and 14 (long-term infusion experiment). In the long-term infusion experiment, plasma levels of L-arginine were measured at these times in the monkeys to confirm L-arginine availability. Vasospasm was not affected by the intracarotid infusion of L-arginine (shown by the reduction in the right MCA area on an anteroposterior arteriogram compared with preoperative values). However, intracarotid L-arginine infusion increased rCBF by 21% (p < 0.015; PCO2 38-42 mm Hg) in all vasospastic monkeys compared with rCBF measured during the saline infusions. In the long-term infusion experiment, vasospasm of the right MCA occurred with similar intensity with or without continuous intravenous administration of L-arginine on Day 7 and had resolved by Day 14. The mean plasma L-arginine level increased during infusion from 12.7+/-4 microg/ml on Day 0 to 21.9+/-13.1 microg/ml on Day 7 and was 18.5+/-3.1 microg/ml on Day 14 (p < 0.05). CONCLUSIONS: Brief intracarotid and continuous intravenous infusion of L-arginine did not influence the incidence or degree of cerebral vasospasm. After SAH, intracarotid infusion of L-arginine markedly increased rCBF in a primate model of SAH. These findings discourage the use of L-arginine as a treatment for vasospasm after SAH.

Kir6.2 oligoantisense administered into the globus pallidus reduces apomorphine-induced turning in 6-OHDA hemiparkinsonian rats.

ATP-sensitive inwardly rectifying potassium channels (KATPs) couple cell metabolism with its membrane potential. The best characterized KATP is the pancreatic KATP which is an heteromultimer of Kir6.2 and SUR1 protein subunits. KATPs are found in a variety of excitable cells, including neurons of the central nervous system. Basal ganglia (BG), especially in the substantia nigra (SN) reticulata and the globus pallidus (GP), have a high density of KATPs. Pharmacological modulation of the KATPs within the BG alters GABAergic activity and produces behavioural changes. However, the relatively high concentrations of drugs used might not have been entirely selective for the KATPs and may have acted at presynaptic nerve terminals as well as on the post-synaptic neurons. As an alternative means of examining the role of KATPs in regulating motor behavior, we used oligoantisense technology to diminish selectively Kir6.2 formation in the GP neurons. We then examined the effect of reduction in Kir6.2 expression on apomorphine-induced turning behavior in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the SN. Two weeks after injection of 6-OHDA, contralateral circling in response to apomorphine (0.25 mg/kg sc) was recorded. Kir6.2 antisense oligodeoxyribonucleotide (ODN) was then administered daily for 6 days into the GP ipsilateral to the 6-OHDA injection. Responses to apomorphine were then tested again and the animals killed to determine the effect of the antisense ODN on Kir6. 2 mRNA. Administration of Kir6.2 antisense ODN significantly attenuated apomorphine-induced contralateral turning and specifically reduced Kir6.2 mRNA in the injected GP. These results are consistent with pharmacological experiments which suggest that KATP channels in the GP are involved in motor responses to apomorphine in 6-OHDA lesioned rats, localizing the effects to the GP neurons, probably through modulation of the GABAergic system.

Differential effects of chemical sympathectomy on expression and activity of tyrosine hydroxylase and levels of catecholamines and DOPA in peripheral tissues of rats.

Tyrosine hydroxylase (TH) mRNA and activity and concentrations of 3,4-dihydroxyphenylalanine (DOPA) and catecholamines were examined as markers of sympathetic innervation and catecholamine synthesis in peripheral tissues of sympathectomized and intact rats. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) markedly decreased norepinephrine and to a generally lesser extent TH activities and dopamine in most peripheral tissues (stomach, lung, testis, duodenum, pancreas, salivary gland, spleen, heart, kidney, thymus). Superior cervical ganglia, adrenals and descending aorta were unaffected and vas deferens showed a large 92% decrease in norepinephrine, but only a small 38% decrease in TH activity after 6-OHDA. Presence of chromaffin cells or neuronal cell bodies in these latter tissues, indicated by consistent expression of TH mRNA, explained the relative resistance of these tissues to 6-OHDA. Stomach also showed consistent expression of TH mRNA before, but not after 6-OHDA, suggesting that catecholamine synthesizing cells in gastric tissue are sensitive to the toxic effects of 6-OHDA. Tissue concentrations of DOPA were mainly unaffected by 6-OHDA, indicating that much of the DOPA in peripheral tissues is synthesized independently of local TH or sympathetic innervation. The differential effects of chemical sympathectomy on tissue catecholamines, DOPA, TH mRNA and TH activity demonstrate that these variables are not simple markers of sympathetic innervation or catecholamine synthesis. Other factors, including presence of neuronal cell bodies, parenchymal chromaffin cells, non-neuronal sites of catecholamine synthesis and alternative sources of tissue DOPA, must also be considered when tissue catecholamines, DOPA and TH are examined as markers of sympathetic innervation and local catecholamine synthesis.

Neurochemical alterations in the cerebellum of a murine model of Niemann-Pick type C disease.

Niemann-Pick disease Type C (NPC) is a progressive neurovisceral metabolic disorder that is caused in most patients by a defect in a recently found gene, NPC-1. Neurological damage includes visual disorders such as vertical supranuclear gaze palsy, movement disorders such as dystonia and ataxia, dementia, and seizures. So far the biochemical deficit, most likely manifested by delayed intracellular cholesterol transport, has not been correlated with the progressive neurological damage. A mutant Balb/C mouse with a defect in the same gene is used as a model to study NPC. Pathological examination of brain tissue obtained by autopsy from NPC patients or brains of affected NPC mice of different ages, revealed signs of extensive damage throughout the brain, including neurofibrillary tangles and intracellular storage of various compounds. Loss of cerebellar Purkinje cells was the most significant specific damage. The present study examined whether the neurochemical changes present in the NPC mouse brain were related to the pathological changes. The results show major alterations in the levels of serotonin and its main metabolite, 5-hydroxyindoleacetic acid, in the cerebellum and cortex of NPC mice. The levels of the inhibitory amino acid glycine were threefold higher in the cerebellum of NPC mice and those of glutamate and GABA decreased in the cortex. Tyrosine hydroxylase immunoreactivity was present in Purkinje cells, and the levels of L-DOPA increased specifically in the vermis of the cerebellum. These results are the first to indicate changes in neurotransmitters in NPC and that these could be correlated with some of the neuropathology of this disease.

Increased striatal dopamine production from L-DOPA following selective inhibition of monoamine oxidase B by R(+)-N-propargyl-1-aminoindan (rasagiline) in the monkey.

Striatal extracellular fluid concentrations of dopamine and metabolites in response to direct striatal administration of two L-DOPA boluses administered sequentially were determined in three rhesus monkeys during halothane anesthesia. Whereas in an initial microdialysis run, generation of dopamine was less following the second L-DOPA bolus than the first, in a subsequent run, in which the selective MAO-B inhibitor R(+)-N-propargyl-1-aminoindan (rasagiline) was administered systemically (0.2 mg/kg s.c.) between the two L-DOPA boluses, generation of dopamine was greater following the second bolus.

Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis.

A sensitive method for quantitatively determining submicromolar levels of neurotransmitter amino acids (e.g. Asp, Glu and gamma-aminobutyric acid) in microdialysates from brain and cerebrospinal fluids is reported. 6-Aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC) was employed as the derivatization reagent, followed by HPLC separation and fluorescence detection of the derivatives. The derivatization was conducted simply by mixing the AQC directly with the microdialysis samples. The reaction was complete within seconds after mixing at room temperature. Separation development optimizing the gradient profile, eluent pH and column temperature resulted in an excellent separation of the required amino acids in less than 30 min. Other resolved amino acids in the same profile include Gly, taurine, and Pro. Recoveries for the amino acids of interest spiked into high salt containing perfusion buffers were greater than 97%. The sensitivity of the method was increased by employing a 16-microliter flow cell in the detector and analyzing 20-microliter aliquots of the derivatization mixtures. With the optimized conditions, the detection limits were 3-7 nM (fmol/microliter). Typical reproducibility (%R.S.D.) for quantitation of these amino acids at submicromolar levels was approximately 2%. Excellent linearity (r2 > 0.999) was achieved over the range 0.2-20 microM. The low detection limits permitted the analysis of a number of different microdialysate samples including those from cerebrospinal fluid, as well as substantia nigra and hypothalamus from brain samples, even at basal levels where gamma-aminobutyric acid concentration may be < 50 nM. The excellent sensitivity made it easy to distinguish basal from stimulated levels of neurotransmitter amino acids, even from sample sizes as small as 10 microliters.

Potentiation of NMDA receptor-mediated responses by dynorphin at low extracellular glycine concentrations.

The effect of dynorphin A(1-13) on N-methyl-D-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1-13) (10 microM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: epsilon1/zeta1, 3,377 +/- 1,416% (mean +/- SE); epsilon2/zeta1, 1,897 +/- 893%; epsilon3/zeta1, 4,356 +/- 846%; and epsilon4/zeta1, 1,783 +/- 503%. Potentiation of NMDA-activated current by dynorphin A(1-13) was concentration dependent between 0.1 and 10 microM dynorphin A(1-13), with a half-maximal concentration value of 2.77 microM and an apparent Hill coefficient of 2.53, for epsilon2/zeta1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1-13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 microM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations > 0.1 microM for epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 subunits, or at > 1 microM for epsilon3/zeta1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1-13) was not inhibited by 1 microM of the kappa-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 microM of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1-13) was inhibited by the glycine antagonist kynurenic acid (50 microM). NMDA-activated current was also potentiated at low glycine concentrations by 10 microM dynorphin A(2-13) or (3-13), both of which have a glycine as the first amino acid, but not by 10 microM dynorphin A(4-13), which does not have glycine as an amino acid. In hippocampal neurons, 10 microM dynorphin A(1-13) or (2-13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 microM dynorphin A(1-13), (2-13), (3-13), or (4-13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1-13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.

Brain MRI, lumbar CSF monoamine concentrations, and clinical descriptors of patients with spinocerebellar ataxia mutations.

OBJECTIVES: To serially assess changes in lumbar CSF biogenic amines, radiographic characteristics, and neurological signs in 34 patients with dominantly inherited ataxia. METHODS: Mutational analysis was used to identify genetic subgroups. Annual assessment of lumbar CSF monoamine metabolites using a gas chromatographic/mass spectrometric method and morphometric measurements of the cerebellum, pons, and the cervical spinal cord on MRI were analysed for each patient and compared with normal controls. RESULTS: Patients with CAG trinucleotide repeat expansions on chromosome 6p (mutSCA1) and chromosome 14q (mutSCA3) had only about one half the normal concentrations of lumbar CSF homovanillic acid (HVA) whereas, 5-hydroxyindoleacetic acid (5-HIAA) concentrations were similar to those in age matched normal subjects. The HVA and 5-HIAA concentrations in clinically similar patients without mutSCA1 or mutSCA3 were normal. One year after the first study, HVA concentrations were reduced by a mean of 22% regardless of the patient's SCA mutation. Abnormalities on MRI were consistent with a spinopontine atrophy in patients with mutSCA3, spinopontocerebellar atrophy in patients with mutSCA1, and "pure" cerebellar atrophy in patients without these mutations. CONCLUSIONS: Quantitative MRI measurements were not useful in monitoring progression of disease but lumbar CSF HVA concentrations and total scores on a revised version of the ataxia clinical rating scale seemed to progress in parallel.

Differential inhibition of neuronal and extraneuronal monoamine oxidase.

This study examined whether the neuronal and extraneuronal sites of action of two monoamine oxidase (MAO) inhibitors, l-deprenyl and debrisoquin, could be distinguished by their effects on plasma concentrations of catecholamine metabolites. Plasma concentrations of the intraneuronal deaminated metabolite of norepinephrine, dihydroxyphenylglycol (DHPG), were decreased by 77% after debrisoquin and by 64% after l-deprenyl administration. Plasma concentrations of the extraneuronal O-methylated metabolite of norepinephrine, normetanephrine, were increased substantially more during treatment with l-deprenyl than with debrisoquin (255% compared to a 27% increase). The comparable decreases in plasma concentrations of DHPG indicate a similar inhibition of intraneuronal MAO by both drugs. Much larger increases in normetanephrine after l-deprenyl than after debrisoquin are consistent with a site of action of the latter drug directed at the neuronal rather than the extraneuronal compartment. Thus, differential changes in deaminated and O-methylated amine metabolites allows identification of neuronal and extraneuronal sites of action of MAO inhibitors.

Monoaminergic effects of folinic acid, L-DOPA, and 5-hydroxytryptophan in dihydropteridine reductase deficiency.

Plasma and CSF concentrations of endogenous L-DOPA, catecholamines, and metabolites of monoamines were assayed in a patient with atypical phenylketonuria due to absent dihydropteridine reductase (DHPR), before and during treatment with folinic acid, Sinemet, and 5-hydroxytryptophan. The patient had low but detectable levels of L-DOPA, 3,4-dihydroxyphenylacetic acid (DOPAC), and 3,4-dihydroxyphenylglycol (DHPG) in plasma and low but detectable levels of these compounds and of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in CSF, with approximately normal plasma and CSF levels of norepinephrine [noradrenaline (NA)]. Folinic acid treatment approximately doubled plasma levels of L-DOPA, NA, DOPAC, and DHPG, compared with values during dietary phenylalanine restriction alone. Detection of L-DOPA, catecholamines, and monoamine metabolites in this patient indicates that monoamine synthesis in humans does not absolutely require DHPR. The results are consistent with the existence of an alternative biochemical pathway, with folinic acid treatment augmenting activity along this pathway. Low plasma levels of L-DOPA, DOPAC, and DHPG may reflect decreased catecholamine synthesis and turnover in sympathetic nerves, with compensatory increases in exocytotic release normalizing plasma NA levels.