Biomedical subjects

M J During

Publications and source records attributed to M J During.

At least 55 records · Page 3Linked to original sources

PubMed1995

Combining laser Doppler flowmetry with microdialysis: a novel approach to investigate the coupling of regional cerebral blood flow to neuronal activity.

Regional cerebral blood flow (rCBF) is directly coupled to neuronal activity; however, the mediators of this coupling have not been established. The characterization of these vasoactive substances requires a technique which enables sampling of locally released mediators together with the simultaneous monitoring of rCBF. The goal of this study was to establish such a technique by combining microdialysis and laser doppler flowmetry. Laser doppler and microdialysis probes were inserted into the dorsal hippocampal, CA1-dentate hilus, of rats. Animals received sequentially increasing concentrations of N-methyl-D-aspartate (NMDA). rCBF responded in a dose-dependent manner, increasing to 106.8 +/- 2.3%, 119.7 +/- 6.3%, 148.0 +/- 16.6%, and 191.4 +/- 20.4% of baseline at 50 microM, 100 microM, 200 microM, and 500 microM NMDA, respectively. All doses of NMDA produced an increase in extracellular concentrations of adenosine and citrulline, an indirect measure of nitric oxide generation. These results indicate that the combination of microdialysis and laser doppler flowmetry is a valuable tool to investigate the coupling of rCBF to neuronal activity. Moreover our data suggest two possible mediators of this coupling, nitric oxide and adenosine, which require further investigation.

Adenosine↗

PubMed1995

An HSV-1 vector expressing tyrosine hydroxylase causes production and release of L-dopa from cultured rat striatal cells.

In this report we demonstrate that a defective herpes simplex virus type one (HSV-1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into L-DOPA-producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV-1 vector (pHSVth) and packaged into virus particles using an HSV-1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of L-DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of L-DOPA and low levels of dopamine were observed from cells in pHSVth-infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.

Animals↗

PubMed1995

Glucose modulates rat substantia nigra GABA release in vivo via ATP-sensitive potassium channels.

Glucose modulates beta cell insulin secretion via effects on ATP-sensitive potassium (KATP) channels. To test the hypothesis that glucose exerts a similar effect on neuronal function, local glucose availability was varied in awake rats using microdialysis in the substantia nigra, the brain region with the highest density of KATP channels. 10 mM glucose perfusion increased GABA release by 111 +/- 42%, whereas the sulfonylurea, glipizide, increased GABA release by 84 +/- 20%. In contrast, perfusion of the KATP channel activator, lemakalim, or depletion of ATP by perfusion of 2-deoxyglucose with oligomycin inhibited GABA release by 44 +/- 8 and 45 +/- 11%, respectively. Moreover, the inhibition of GABA release by 2-deoxyglucose and oligomycin was blocked by glipizide. During systemic insulin-induced hypoglycemia (1.8 +/- 0.3 mM), nigral dialysate GABA concentrations decreased by 49 +/- 4% whereas levels of dopamine in striatal dialysates increased by 119 +/- 18%. We conclude that both local and systemic glucose availability influences nigral GABA release via an effect on KATP channels and that inhibition of GABA release may in part mediate the hyperexcitability associated with hypoglycemia. These data support the hypothesis that glucose acts as a signaling molecule, and not simply as an energy-yielding fuel, for neurons.

Adenosine Triphosphate↗

PubMed1995

Interstitial fluid concentrations of glycerol, glucose, and amino acids in human quadricep muscle and adipose tissue. Evidence for significant lipolysis in skeletal muscle.

To determine the relationship between circulating metabolic fuels and their local concentrations in peripheral tissues we measured glycerol, glucose, and amino acids by microdialysis in muscle and adipose interstitium of 10 fasted, nonobese human subjects during (a) baseline, (b) euglycemic hyperinsulinemia (3 mU/kg per min for 3 h) and, (c) local norepinephrine reuptake blockade (NOR). At baseline, interstitial glycerol was strikingly higher (P < 0.0001) in muscle (3710 microM) and adipose tissue (2760 microM) compared with plasma (87 microM), whereas interstitial glucose (muscle 3.3, fat 3.6 mM) was lower (P < 0.01) than plasma levels (4.8 mM). Taurine, glutamine, and alanine levels were higher in muscle than in adipose or plasma (P < 0.05). Euglycemic hyperinsulinemia did not affect interstitial glucose, but induced a fall in plasma glycerol and amino acids paralleled by similar changes in the interstitium of both tissues. Local NOR provoked a fivefold increase in glycerol (P < 0.001) and twofold increase in norepinephrine (P < 0.01) in both muscle and adipose tissues. To conclude, interstitial substrate levels in human skeletal muscle and adipose tissue differ substantially from those in the circulation and this disparity is most pronounced for glycerol which is raised in muscle as well as adipose tissue. In muscle, insulin suppressed and NOR increased interstitial glycerol concentrations. Our data suggest unexpectedly high rates of intramuscular lipolysis in humans that may play an important role in fuel metabolism.

Adipose Tissue↗

PubMed1995

Local ventromedial hypothalamus glucopenia triggers counterregulatory hormone release.

To test the hypothesis that nuclei of the ventromedial hypothalamus (VMH) play a key role in the detection of counterregulatory responses to hypoglycemia, we delivered the glucopenic agent 2-deoxyglucose via bilaterally placed microdialysis probes into the VMH of conscious, chronically catheterized rats. The goal was to produce cellular glucopenia localized to the VMH. The volume of brain tissue exposed to 2-deoxyglucose was determined by adding [3H]2-deoxyglucose to the dialysate; its distribution in cerebral tissue was almost exclusively limited to the VMH. Rats with microdialysis probes placed into the frontal lobes served as a control group. Local perfusion of 2-deoxyglucose (but not glucose) into the VMH caused a prompt twofold increase in plasma glucose in association with a striking elevation of plasma glucagon (3.5-fold), epinephrine (30-fold), and norepinephrine (3.5-fold). No effect was seen when 2-deoxyglucose was delivered into the frontal lobes. We conclude that glucopenia localized to the VMH triggers the release of counterregulatory hormones that defend against hypoglycemia. Thus, the neurons that sense glucopenia may be situated in the VMH.

Animals↗

PubMed1995

Cellular and molecular characterization of a brain-enriched protein tyrosine phosphatase.

Regional variations in the expression of a striatal enriched protein tyrosine phosphatase called STEP were studied in the adult rat brain by a combination of immunocytochemistry, lesion studies, Western blotting, and in situ hybridization. Monoclonal antibodies generated against STEP identified multiple polypeptides of M(r) 46, 37, 33 and a doublet of M(r) 64-66 kDa on Western blots. Although the three STEP immunoreactive bands with lower molecular weights were enriched in cytosolic fractions, the 64-66 kDa doublet was enriched in membrane fractions. All of the immunoreactive forms were abundant in the caudate-putamen and were present in lower amounts or were undetectable in other brain regions. In substantia nigra, the M(r) 64-66 kDa doublet was not detected but bands with M(r) 46, 37, and 33 kDa were present. Immunocytochemical and lesion experiments demonstrated that the cytosolic STEP isoforms present in the substantia nigra are in presynaptic axons originating from the projection neurons of the caudate putamen, which innervate this structure. Additional in situ hybridization studies showed that STEP mRNA expression patterns correlate with the patterns of immunocytochemical staining. These findings indicate that there are multiple polypeptide isoforms of STEP enriched in the basal ganglia and related structures which differ in terms of their intracellular locations and functional roles.

Animals↗

PubMed1994

Long-term behavioral recovery in parkinsonian rats by an HSV vector expressing tyrosine hydroxylase.

One therapeutic approach to treating Parkinson's disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (L-dopa)-producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine-lesioned rats, used as a model of Parkinson's disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity.

Animals↗

PubMed1994

Phenytoin concentrations in the human brain: an in vivo microdialysis study.

We report the first human study of phenytoin concentration using in vivo microdialysis, which permits sampling the extracellular environment of the brain. This technique has been applied to patients undergoing intracranial electrode investigation for intractable epilepsy. By varying the rate of perfusion (from 2.5 to 0.25 microliters/min), it is possible to quantify the concentration of drug in the extracellular fluid (ECF), which reflects the concentration on the outer neuronal cell membrane. Samples were obtained from four catheters in two patients, in whom serum phenytoin (PHT) concentrations were held constant. Unbound serum concentrations were measured following ultrafiltration at 37 degrees C. In one patient, with left and right hippocampal probes, steady state ECF/unbound serum ratios were 87 and 84% respectively. In the second patient, with hippocampal and frontal probes, ECF/unbound serum ratios were 87 and 85% respectively. Flow rate for 50% maximal recovery averaged 1.65 microliters/min (1.5-1.7 microliters/min). We found that steady state ECF PHT concentrations corresponded closely to unbound serum concentrations. No differences are observed between different sites within the brain. Flow rates needed for equilibration of dialysate with the extracellular space were slower than reported for carbamazepine, but faster than those we found for carbamazepine-epoxide and valproate.

Brain↗

PubMed1994

Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.

Adeno-associated viral (AAV) vectors are non-pathogenic, integrating DNA vectors in which all viral genes are removed and helper virus is completely eliminated. To evaluate this system in the post-mitotic cells of the brain, we found that an AAV vector containing the lacZ gene (AAVlac) resulted in expression of beta-galactosidase up to three months post-injection in vivo. A second vector expressing human tyrosine hydroxylase (AAVth) was injected into the denervated striatum of unilateral 6-hydroxydopamine-lesioned rats. Tyrosine hydroxylase (TH) immunoreactivity was detectable in striatal neurons and glia for up to four months and we also found significant behavioural recovery in lesioned rats treated with AAVth versus AAVlac controls. Safe and stable TH gene transfer into the denervated striatum may have potential for the genetic therapy of Parkinson's disease.

Adenoviruses, Human↗

PubMed1994

Direct measurement of extracellular lactate in the human hippocampus during spontaneous seizures.

The effect of clinical, spontaneous-onset seizures on extracellular fluid lactate was investigated by the method of lactography, the in vivo on-line measurement of lactate levels using microdialysis. Studies of experimental animals have suggested that generation of extracellular lactate as measured by microdialysis is an index of local glucose utilization and is dependent on the activity of neurons under physiological conditions. Patients with medically refractory complex partial epilepsy underwent stereotactic implantation of combination depth electrode/microdialysis probes into both hippocampi for 7-16 days. During spontaneous complex partial seizures with secondary generalization, extracellular lactate levels rose by 91 +/- 32%. Moreover, this increase persisted for 60-90 min. During a unilateral hippocampal seizure that did not propagate to the contralateral hippocampus, the increase in lactate content was restricted to the side of seizure activity. Between seizures, extracellular lactate levels correlated with the frequency of interictal spikes. In summary, these data suggest that brief clinical seizures increase nonoxidative glucose metabolism significantly as measured by the generation of extracellular lactate. Furthermore, the increase in extracellular lactate levels is limited to the site of seizure activity. Lactate is transported extracellularly via a lactate/proton cotransporter, therefore, the rise in extracellular lactate level may mediate the drop in pH0 associated with seizure activity. As acidification of the extracellular compartment has an inhibitory effect on neuronal excitability, the rise in extracellular lactate content may be a mechanism of seizure arrest and postictal refractoriness. Moreover, extracellular lactate may also mediate the decreased seizure susceptibility associated with frequent interictal spikes.

Adolescent↗

PubMed1994

Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia.

The central nervous system has been implicated in the activation of counterregulatory hormone release during hypoglycemia. However, the precise loci involved are not established. To determine the role of the ventromedial hypoglycemia, we performed hypoglycemic clamp studies in conscious Sprague-Dawley rats with bilateral VMH lesions produced by local ibotenic acid injection 2 wk earlier. Rats with lesions in the lateral hypothalamic area, frontal lobe, sham operated (stereotaxic needle placement into hypothalamus without injection), and naive animals served as control groups. The clamp study had two phases. For the first hour plasma glucose was fixed by a variable glucose infusion at euglycemia (approximately 5.9 mM). Thereafter, for an additional 90 min, glucose was either allowed to fall to (a) mild hypoglycemia (approximately 3.0 mM) or (b) more severe hypoglycemia (approximately 2.5 mM). Glucagon and catecholamine responses of lateral hypothalamic area-, frontal lobe-lesioned, sham operated, and naive animals were virtually identical at each hypoglycemic plateau. In contrast, glucagon, epinephrine, and norepinephrine responses in the VMH-lesioned rats were markedly inhibited; hormones were diminished by 50-60% during mild and by 75-80% during severe hypoglycemia as compared with the other groups. We conclude that the VMH plays a crucial role in triggering the release of glucagon and catecholamines during hypoglycemia.

Animals↗

PubMed1994

Measurement of carbamazepine and carbamazepine epoxide in the human brain using in vivo microdialysis.

We report the first study of carbamazepine and carbamazepine-10,11-epoxide concentrations determined by using intracerebral microdialysis in three patients undergoing depth electrode studies for the evaluation of medically intractable epilepsy. Very small microdialysis catheters, affixed to and inserted with the depth electrodes, sampled drug concentration in the extracellular environment. We perfused artificial extracellular fluid continuously, and varied the perfusion rate to permit estimation of the absolute drug concentration in the extracellular space. Serum samples were obtained simultaneously. The relation between dialysate and extracellular concentration (recovery fraction) depended, in vivo but not in vitro, on the relative lipophilicity of the compounds, suggesting that diffusion of the drug within the brain is a major determinant of microdialysate drug concentration. When this is taken into account, the steady-state extracellular concentrations of these compounds closely mirror their unbound serum concentrations.

Brain Chemistry↗

PubMed1993

Effect of caffeine on the recognition of and responses to hypoglycemia in humans.

OBJECTIVE: To determine whether two effects of acute caffeine ingestion--decrease in cerebral blood flow and increase in brain glucose use--alter the recognition of and physiologic responses to hypoglycemia. DESIGN: On two occasions, a hyperinsulinemic glucose clamp technique (2 mU/kg body weight per minute) was used to maintain plasma glucose at 5 mmol/L for 90 minutes, followed by 60 minutes at 3.8 mmol/L, and then 2.8 mmol/L. After 30 minutes at 5 mmol/L, participants consumed, using a randomized, double-blind design, caffeine-free cola with or without caffeine (400 mg) added. SETTING: Yale Clinical Research Center. PARTICIPANTS: Eight healthy, nonobese volunteers (5 men; age range, 20 to 33 years). MEASUREMENTS: Middle cerebral artery velocity (V MCA), counter-regulatory hormone levels, hypoglycemic symptoms, and cognitive function (P300 evoked potentials). RESULTS: Caffeine caused an immediate and sustained 23% decrease in VMCA from 64 to 49 cm/s (point estimate of difference, +15 cm/s [95% CI, 10 to 21 cm/s], P < 0.001). At a glucose level of 3.8 mmol/L, only the participants given caffeine had warning symptoms and "felt hypoglycemic." Moreover, the level of epinephrine was 118% ([CI of point difference, 76% to 158%] [CI, P < 0.001]) higher after caffeine consumption compared with placebo. Similarly, levels of norepinephrine (41% [CI, 26% to 60%], P < 0.002), cortisol (65% [CI, 26% to 78%], P < 0.008), and growth hormone (60% [CI, 16% to 143%], P < 0.05) were higher after caffeine consumption compared with placebo. At 2.8 mmol/L, epinephrine (40% [point estimate of the percentage difference], P < 0.05), norepinephrine (27%, P < 0.05), and cortisol (24%, P < 0.05) levels were higher, participants were more aware (P < 0.02) of hypoglycemia, and P300 latency was prolonged in the group that consumed caffeine (7.2%, P < 0.05). CONCLUSIONS: Acute ingestion of caffeine is associated with sympathoadrenal activation and awareness of hypoglycemia at a glucose level not usually considered hypoglycemic. Our data suggest that individuals who ingest moderate amounts of caffeine may develop hypoglycemic symptoms if plasma glucose levels fall into the "low-normal" range, as might occur in the late postprandial period after ingestion of a large carbohydrate load.

Adult↗

PubMed1993

Long-term increases in neurotransmitter release from neuronal cells expressing a constitutively active adenylate cyclase from a herpes simplex virus type 1 vector.

Signal-transduction pathways mediate a wide range of short-term changes in the physiology of neuronal systems from invertebrates to mammals. However, examples of long-term changes in neuronal physiology mediated by these pathways have been limited to invertebrate systems. In this report, long-term changes in the physiology of mammalian neurons were studied by using genetic intervention to cause a long-lasting activation of the cAMP pathway. The catalytic domain of yeast adenylate cyclase (cyr), encoding a constitutive enzyme activity, was expressed in neuronal cells infected with a defective herpes simplex virus vector (pHSVcyr). In PC-12 cells infected with pHSVcyr, increases were seen in cAMP levels, protein kinase A activity, protein phosphorylation, phosphorylation of the tyrosine hydroxylase protein kinase A site (Ser40), and catecholamine release. Infection of sympathetic neurons with pHSVcyr increased cAMP levels, protein phosphorylation, and catecholamine release. Yeast adenylate cyclase immunoreactivity and elevated cAMP levels were localized to the cell bodies of sympathetic neurons. The increase in neurotransmitter release was both Ca(2+)- and activity-dependent and persisted for at least 1 week after infection of the sympathetic neurons, suggesting that sustained physiological activation of the cAMP pathway may mediate long-term changes in the neuronal physiology of mammalian systems.

Adenylyl Cyclases↗

PubMed1993

Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.

An alteration in excitatory and inhibitory influences may underlie epilepsy. We used bilateral intrahippocampal microdialysis to test the hypothesis that an increase in extracellular glutamate may trigger spontaneous seizures. The concentrations of glutamate and gamma-aminobutyric acid (GABA), the brain's major inhibitory neutrotransmitter, were measured in microdialysates before and during seizures in 6 patients with complex partial epilepsy investigated before surgery. Before seizures, concentrations of glutamate were higher in the epileptogenic hippocampus, whereas GABA concentrations were lower. During seizures, there was a sustained increase in extracellular glutamate to potentially neurotoxic concentrations in the epileptogenic hippocampus. Moreover, the increase preceded seizure. GABA concentrations were unchanged before seizures, but increased during them, with a greater rise in the non-epileptogenic hippocampus, suggesting that a rise in extracellular glutamate may precipitate seizures and that the concentrations reached may cause cell death.

Adolescent↗

PubMed1993

A gamma-aminobutyric acid transporter driven by a proton pump is present in synaptic-like microvesicles of pancreatic beta cells.

A variety of peptide-secreting endocrine cells contain a population of recycling microvesicles that share several major membrane polypeptides with neuronal synaptic vesicles (SVs). The function of these synaptic-like microvesicles (SLMVs) remains to be elucidated. It was previously suggested that SLMVs of pancreatic beta cells may store and secrete gamma-aminobutyric acid (GABA). GABA, the major nonpeptide inhibitory neurotransmitter of the central nervous system, is stored in and secreted from SVs. GABA uptake into SVs is mediated by a transporter that is driven by a vacuolar proton ATPase. GABA is also present at high concentration in the endocrine pancreas where it is selectively localized in insulin-secreting beta cells, the core cells of pancreatic islets. GABA is not present in peripheral islet cells (mantle cells), represented primarily by glucagon-secreting alpha cells. In this study, an immunoisolation procedure was used to purify SLMVs from cell lines derived from mouse beta cells and alpha cells. SLMVs obtained from the beta-cell line, but not those obtained from the alpha-cell line, displayed a GABA-transport activity dependent upon a proton electrochemical gradient generated by a vacuolar proton ATPase. These data support the hypotheses that (i) SLMVs have a secretory function similar to that of SVs and (ii) beta-cell SLMVs are involved in the secretion of GABA, which in turn may have a paracrine function on mantle cells of the islet.

Adenosine Triphosphate↗

PubMed1993

Transient presence of GABA in astrocytes of the developing optic nerve.

Immunostaining and high-pressure liquid chromatography (HPLC) were used to study the developmental time course of astrocytic gamma-aminobutyric acid (GABA) expression in rat optic nerve. GABA immunostaining was carried out on cultured astrocytes, and on whole optic nerve. Confocal scanning laser microscopy was used to obtain optical sections in excised whole tissue in order to localize the cellular origins of GABA within the relatively intact optic nerve. GABA immunoreactivity was localized in astrocytes identified by GFAP staining; GABA staining was most intense in early neonatal optic nerve and attenuated over 3 weeks of postnatal development. The staining was pronounced in the astrocyte cell bodies and processes but not in the nucleus. There was a paucity of GABA immunoreactivity by postnatal day 20, both in culture and in whole optic nerve. A biochemical assay for optic nerve GABA using HPLC indicated a relatively high concentration of GABA in the neonate, which rapidly attenuated over the first 3 postnatal weeks. Immunoreactivity for the GABA synthesis enzyme glutamic acid decarboxylase (GAD) was pronounced in neonates but also attenuated with development. These results indicate that GABA and the GABA synthesis enzyme GAD are localized in astrocytes of optic nerve, and that their expression is transient during postnatal development.

Animals↗

PubMed1993

Effect of anoxia on excitatory amino acids in brain slices of rats and turtles: in vitro microdialysis.

Using in vitro microdialysis, we tested the hypothesis that anoxia-induced release of excitatory amino acids is greater in adult rat brain than in turtle brain. Ten minutes of anoxia produced significant elevation of glutamate (from 0.39 +/- 0.03 to 0.90 +/- 0.18 microM dialysate, means +/- SE, P < 0.05), aspartate (from 0.28 +/- 0.12 to 1.20 +/- 0.49 microM, P < 0.05), glycine, and alanine in the rat brain slice. During reoxygenation, alanine and glycine returned toward baseline values, whereas aspartate and glutamate remained elevated. In contrast, prolonged anoxia (60 min) in the turtle brain slice resulted in only minimal increase in aspartate (from 0.06 +/- 0.01 to 0.09 +/- 0.02 microM, P < 0.05) and, interestingly, a decrease in glutamate (from 0.50 +/- 0.11 to 0.33 +/- 0.09 microM, P < 0.05). Levels of glycine, alanine, and taurine were unchanged. We conclude that oxygen deprivation causes marked increase in excitatory amino acids in the anoxia-sensitive rat brain slice, while oxygen deprivation for an even longer period of time in the turtle brain slice produces substantially less change. We speculate that the difference in sensitivity to anoxia between rat and turtle is at least partly attributable to the major difference in interstitial levels of excitotoxic amino acids during oxygen deprivation.

Alanine↗