Peter G. Osborne

An in vivo microdialysis characterization of extracellular dopamine and GABA in dorsolateral striatum of awake freely moving and halothane anaesthetised rats

This study describes the results of a systematic characterization of extracellular dopamine (DA) and gamma-aminobutyric acid (GABA) recovered from dorsolateral striatum using in vivo microdialysis in rats following acute (2.5 h) and chronic (1 day, 2 day and 4 day) implantation of the probe. The voltage and calcium dependence of DA and GABA overflow was characterised by perfusion with the sodium channel blocker tetrodotoxin (TTX 10-6M) and with Ca2(+)-free Ringers perfusion medium. In addition, the effect of halothane anaesthesia on the responsiveness of these neurotransmitter substances to TTX and Ca2(+)-free perfusion medium was investigated. Perfusion with TTX decreased basal DA levels by at least 60% in all groups. The TTX-induced decrease was most profound in halothane-anaesthetised rats, 24 h after implantation of the probe. Responsiveness of GABA to TTX infusion was different between the groups. In acutely implanted halothane-anaesthetised rats basal GABA levels were unaltered by perfusion with TTX while in the remaining groups at least a 35% reduction was observed. In awake rats 2 days following implantation of the probe removal and replacement of the Ca2+ from the perfusion medium resulted in a reversible reduction of basal DA by 87%. In addition, basal GABA levels were decreased by 52%. This decrease was delayed and was not reversed 1.5 h after the Ca2(+)-free perfusion medium was replaced with normal perfusion medium although basal GABA levels returned to pre-experimental levels by the following day.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo characterisation of extracellular dopamine, GABA and acetylcholine from the dorsolateral striatum of awake freely moving rats by chronic microdialysis.

Basal extracellular (EC) DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), gamma aminobutyric acid (GABA) and acetylcholine (ACh) were measured in dialysates from the dorsolateral striatum (DLS) of awake rats, every 30 min for 4.5 h each day over a 4-day period. The responsiveness of basal EC DA, DOPAC, HVA and GABA to local perfusion with tetrodotoxin (1 micron) was measured 1 and 4 days after implantation. In addition EC ACh was also measured 4 days after probe implantation. The results of this study indicate that EC levels of DA, DOPAC, HVA, GABA and ACh can be reliably monitored for up to 4 days after probe implantation. In addition, we show that striatal EC levels of DA, GABA and ACh may be regarded as a reflection of ongoing neuronal activity for up to 4 days after implantation of a microdialysis probe.

Ascorbate and glutathione regulation in hibernating ground squirrels

Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousals from torpor. Metabolic suppression likely plays a primary adaptive role which allows hibernating species to tolerate such phenomena. However, several other aspects of hibernation physiology are also consistent with tolerance to dramatic fluctuations in cerebral blood flow, suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work was to study the dynamics of the low molecular weight antioxidants, ascorbate and glutathione (GSH), during hibernation. Alterations in concentrations of ascorbate during hibernation and arousal in two species of hibernating ground squirrels suggest that it could play a protective role during hibernation or arousal. Samples were collected during the hibernation season from arctic ground squirrels (AGS; Spermophilus parryii) and 13-lined ground squirrels (TLS; S. tridecemlineatus) during prolonged torpor and in squirrels that did not hibernate or had not been hibernating for several weeks. We determined antioxidant levels in plasma, cerebrospinal fluid (CSF), and in frontal cortex, hippocampus and cerebellum using high-performance liquid chromatography (HPLC). Plasma ascorbate concentrations increased dramatically (3-4-fold) in both species during hibernation and rapidly returned to prehibernation levels upon arousal. By contrast, plasma GSH concentrations fell slightly or remained stable during hibernation. Ascorbate levels in the CSF doubled in hibernating AGS (not determined in TLS), while brain ascorbate content fell slightly (10-15%) in both species. Substantial increases in plasma and CSF ascorbate concentrations suggest that this antioxidant could play a protective role during hibernation and reperfusion upon arousal from hibernation.

Acute versus chronic haloperidol : relationship between tolerance to catalepsy and striatal and accumbens dopamine, GABA and acetylcholine release

Using in vivo microdialysis, changes in extracellular dorsolateral striatum and nucleus accumbens dopamine, GABA and acetylcholine following acute and chronic haloperidol (0.25 mg/kg, s.c.) were evaluated in rats concurrent with the measurement of catalepsy. When administered to drug-naive and chronically treated rats, haloperidol was associated with a consistent and prolonged (> 150 min) increase in dorsolateral striatum and nucleus accumbens DA release and a transient (60 min) increase in dorsolateral striatum GABA release. Haloperidol was also associated with a transient (30 min) increase in dorsolateral striatum acetylcholine release in the chronically treated rats. Basal dopamine and acetylcholine levels were similar in both brain regions; however, basal dorsolateral striatum GABA levels were two-fold higher in the chronically treated rats. Administration of haloperidol was associated with a prolonged (> 150 min) catalepsy in the drug-naive rats which was greatly diminished or absent in chronically treated rats. Additionally, serum haloperidol levels were shown to be similar 120 min following administration of haloperidol in both groups. These results indicate a marked behavioral difference in the effects of haloperidol in drug-naive and chronically treated rats which is not related to an altered bioavailability of the drug and which is dissociated from both basal and haloperidol induced effects on dopamine and acetylcholine release in both brain regions. However, the selective elevation of basal dorsolateral striatum GABA release following chronic administration of haloperidol may contribute to the development of tolerance to catalepsy as well as providing an in vivo neurochemical marker of the long-term effects of haloperidol.

Effect of Varying the Ionic Concentration of a Microdialysis Perfusate on Basal Striatal Dopamine Levels in Awake Rats

Abstract: In vivo microdialysis was used to study the effects of Ca2+, Mg2+, and K+ ion concentrations on basal extracellular (EC) levels of striatal DA and metabolites in awake rats on the second day (48 h) after implantation of a microdialysis probe. Basal EC striatal dopamine (DA) levels were markedly (90%) and reversibly reduced by removal and subsequent replacement of Ca2+ ions from the microdialysis perfusate. This implies that the EC DA in this preparation is primarily of synaptic origin. The addition and subsequent removal of 1.7 mM MgCl2 to the Mg2+‐free perfusate produced a reversible decrease (20%) in basal EC DA levels. This decrease may reflect a competitive interaction between Ca2+ and Mg2+ in the process of vesicular release. Basal EC DA levels were also reduced (27%) by decreasing the K+ concentration of the perfusate from 4 mM to 3 mM. However, after restoring the K+ concentration to 4 mM, EC DA levels were slow to return to pretreatment levels. Basal EC 3,4‐dihydroxyphenylacetic acid and homovanillic acid levels exhibited a parallel but diminished response to each manipulation of the ionic concentration of the perfusate. This study demonstrates that small variations in the concentrations of Ca2+, Mg2+, and K+ in the perfusate employed in microdialysis preparations will affect basal EC striatal DA and metabolite levels.

State-dependent regulation of cortical blood flow and respiration in hamsters: response to hypercapnia during arousal from hibernation

Hibernation is characterised by a global reduction of metabolism, body temperature and blood flow, while arousal from hibernation is achieved by the reversal of these processes. Our experiments were performed on Syrian hamsters that had been chronically implanted with a cortical thermocouple and an optical fibre over the contralateral cortex, and acutely implanted with thermocouples in the rectal, cheek pouch and interscapular brown adipose tissue (BAT). Measurements revealed large thermal gradients in the body of the arousing animals. Maximum whole‐body metabolic rate, which was 2.4 times normal cenothermic resting metabolic rate, coincided not with rectal temperature but more closely with respiratory rate (RR) or BAT temperature. Regional cortical blood flow (rCBF), as measured by laser‐Doppler flowmetry, changed in parallel with whole‐body metabolic rate, peaking at 3.8 times the normal cenothermic resting levels, when rectal temperature was 15 °C. When BAT temperature was less than 25 °C, RR, rCBF and heart rate (HR) were decreased by breathing hypercapnic gas, but these parameters were unresponsive to hyperoxic gases. At cenothermia the RR and rCBF of anaesthetised hamsters was increased by exposure to hypercapnic gases. Exposure to hyperoxic gas decreased RR but had no effect on rCBF. The mechanisms regulating rCBF, HR and RR exhibit state‐dependent sensitivities to hypercapnic and hyperoxic stimuli. The large increase in rCBF observed during arousal implies that cerebral autoregulation is temporarily suspended and suggests that hamsters effectively use endogenous mechanisms to minimise the pathology normally associated with dramatic increases in rCBF.

Brain antioxidant levels in hamsters during hibernation, arousal and cenothermia

Warming from hibernation to cenothermia involves intense metabolic activity coincident with large fluxes in blood flow and is considered to be a period of oxidative stress during which utilization of endogenous antioxidants prevents pathology. Very slow flow brain microdialysis enabled temperature independent sampling of the brain striatal extracellular fluid (ECF) during hibernation, arousal and cenothermia in Syrian hamsters (Mesocricetus auratus). Brain tissue and dialysates were analyzed to provide the first profile of changes in ECF levels of ascorbate (AA), glutathione (GSH) and urate during hibernation and the transition to cenothermia. Brain tissue content of AA and GSH was unchanged between hibernation and cenothermia; however, arousal was associated with substantial oxidation of AA from the brain ECF and plasma compartments. ECF GSH increased during arousal. Brain tissue urate content was decreased 50% during hibernation. ECF urate levels were unchanged in hibernation and cenothermia but transiently increased 100% during arousal. These experiments demonstrate that arousal from hibernation is a suitable experimental model for examination of the mechanisms by which non-pathological tissue integrity is maintained in the face of the generation of free radicals during increasing metabolism, temperature and cerebral reperfusion.

Electrocatalytic reduction of dioxygen on hemin based carbon paste electrode

A hemin-modified carbon paste electrode was constructed by a simple, rapid and effective method. The electrochemical behaviour of the modified electrode was characterized by cyclic voltammetry. The modified electrode obtained was very stable and exhibited electrocatalytic response for the reduction of oxygen. The possible mechanism for the catalytic reduction of dioxygen is discussed. The dioxygen is reduced via a one-step reduction accompanying four electrons and four protons transfer at pH 7–11.

Determination of striatal extracellular γ-aminobutyric acid in non-hibernating and hibernating Arctic ground squirrels using quantitative microdialysis

This study determined extracellular concentrations of gamma-aminobutyric acid ([GABA](ecf)) in striatum of non-hibernating and hibernating arctic ground squirrels to test the hypothesis that an increase in [GABA](ecf) was associated with profound CNS depression during hibernation. Quantitative microdialysis procedures were employed to circumvent the effects of low temperature on the relative recovery of the analyte across the dialysis membrane and yielded for the first time quantitative in vivo estimates of [GABA](ecf) in any brain region or any species. Laboratory housed, wild caught Arctic ground squirrels (Spermophilus parryii) were implanted intraperitoneally with radio transmitters that enabled the telemetric monitoring of activity and core body temperature (T(b)) and bilaterally implanted with cranial guide tubes that enabled the implantation of microdialysis probes into the striatum. Striatal [GABA](ecf) was determined in unrestrained, non-hibernating ground squirrels (T(b) range 34.7-38.9 degrees C) and hibernating ground squirrels (T(b) range 2.9-3.9 degrees C) using extrapolation to zero flow and very slow flow microdialysis techniques. The results show that [GABA](ecf) in non-hibernating squirrels was 73 nM and this level was decreased by approximately 50% during hibernation thereby suggesting that an increase in [GABA](ecf) does not play a major role in CNS depression during hibernation. The reduction of [GABA](ecf) parallels a decrease in plasma and CSF [glucose] and may be related to a decrease in GABA synthesis or reduced voltage dependent release. This paper demonstrates that measurement of extracellular concentrations of neurotransmitters in animals with vastly different body temperatures is possible using microdialysis techniques of extrapolation to zero flow or very slow flow rates that enable 100% recovery. Such quantitative techniques may prove valuable in the study of the neurochemistry of the cerebral mechanisms of hibernation and tolerance to cerebral ischemia exhibited by hibernating animals.

Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high-performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor

To determine the basal acetylcholine level in the dialysate of rat frontal cortex, a horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) was employed instead of the conventional platinum electrode used in high-performance liquid chromatography-electrochemical detection (HPLC-ED). In initial experiments, an oxidizable unknown compound interfered with the detection of basal acetylcholine release on HPLC-HRP-GCE. An immobilized peroxidase-choline oxidase precolumn (pre-reactor) was included in the HPLC system, to eliminate the interference from the unknown compound. This combination could detect less than 10 fmol of standard acetylcholine and basal acetylcholine levels in the dialysate from a conventional concentric design microdialysis probe, without the use of cholinesterase inhibitor, and may facilitate physiological investigation of cholinergic neuronal activity in the central nervous system.