Electrochemical carbon fiber-based technique for simultaneous recordings of brain tissue PO2, pH, and extracellular field potentials.

A method for simultaneous electrochemical detection of brain tissue PO2 (PtO2) and pH changes together with neuronal activity using a modified form of fast cyclic voltammetry with carbon fiber electrodes is described. This technique has been developed for in vivo applications and recordings from discrete brain nuclei in experimental animals. The small size of the carbon fiber electrode (⌀7 µm, length <100 µm) ensures minimal disruption of the brain tissue and allows recordings from small brain areas. Sample rate (up to 4 Hz) is sufficient to resolve rapid changes in PtO2 and pH that follow changes in neuronal activity and metabolism. Rapid switching between current and voltage recordings allows combined electrochemical detection and monitoring of extracellular action potentials. For simultaneous electrochemical detection of PtO2 and pH, two consecutive trapezoidal voltage ramps are applied with double differential-subtraction of the background current. This enables changes in current caused by protons and oxygen to be detected separately with minimal interference between the two. The profile of PtO2 changes evoked by increases in local neuronal activity recorded using the described technique was very similar to that of blood-oxygen-level-dependent responses recorded using fMRI. This voltammetric technique can be combined with fMRI and brain vessel imaging to study the metabolic mechanisms underlying neurovascular coupling response with much greater spatial and temporal resolution than is currently possible.

Abnormal oxygen homeostasis in the nucleus tractus solitarii of the spontaneously hypertensive rat.

NEW FINDINGS: What is the central question of this study? Arterial hypertension is associated with impaired neurovascular coupling in the somatosensory cortex. Abnormalities in activity-dependent oxygen consumption in brainstem regions involved in the control of cardiovascular reflexes have not been explored previously. What is the main finding and its importance? Using fast-cyclic voltammetry, we found that changes in local tissue PO2 in the nucleus tractus solitarii induced by electrical stimulation of the vagus nerve are significantly impaired in spontaneously hypertensive rats. This is consistent with previous observations showing that brainstem hypoxia plays an important role in the pathogenesis of arterial hypertension. The effects of arterial hypertension on cerebral blood flow remain poorly understood. Haemodynamic responses within the somatosensory cortex have been shown to be impaired in the spontaneously hypertensive rat (SHR) model. However, it is unknown whether arterial hypertension affects oxygen homeostasis in vital brainstem areas that control cardiovascular reflexes. In this study, we assessed vagus nerve stimulation-induced changes in local tissue PO2 (PtO2) in the caudal nucleus tractus solitarii (cNTS) of SHRs and normotensive Wistar rats. Measurements of PtO2 were performed using a novel application of fast-cyclic voltammetry, which allows higher temporal resolution of O2 changes than traditional optical fluorescence techniques. Electrical stimulation of the central cut end of the vagus nerve (ESVN) caused profound reductions in arterial blood pressure along with biphasic changes in PtO2 in the cNTS, characterized by a rapid decrease in PtO2 ('initial dip') followed by a post-stimulus overshoot above baseline. The initial dip was found to be significantly smaller in SHRs compared with normotensive Wistar rats even after ganglionic blockade. The post-ESVN overshoot was similar in both groups but was reduced in Wistar rats after ganglionic blockade. In conclusion, neural activity-dependent changes in tissue oxygen in brainstem cardiovascular autonomic centres are significantly impaired in animals with arterial hypertension.

Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT).

The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12-50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg(-1) ). However, decynium-22 (600 µg kg(-1) ), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not noradrenaline, whereas desipramine had the reverse action. The OCT3 inhibitor corticosterone (10 mg kg(-1) ) had no effect. Blockade of glutamate receptors with topical kynurenate (10-50 nm) reduced the vagally evoked 5-HT signal by 50%, indicating that this release was from at least two sources. It is concluded that vagally evoked 5-HT release is under the regulation of the high-capacity, low-affinity transporter PMAT, not the low-capacity, high-affinity transporter SERT. This is the first demonstration that PMAT may be playing a physiological role in the regulation of 5-HT transmission and this could indicate that 5-HT is acting, in part, as a volume transmitter within the NTS.

Calibration of NO sensors for in-vivo voltammetry: laboratory synthesis of NO and the use of UV-visible spectroscopy for determining stock concentrations.

The increasing scientific interest in nitric oxide (NO) necessitates the development of novel and simple methods of synthesising NO on a laboratory scale. In this study we have refined and developed a method of NO synthesis, using the neutral Griess reagent, which is inexpensive, simple to perform, and provides a reliable method of generating NO gas for in-vivo sensor calibration. The concentration of the generated NO stock solution was determined using UV-visible spectroscopy to be 0.28+/-0.01 mmol L(-1). The level of NO(2) (-) contaminant, also determined using spectroscopy, was found to be 0.67+/-0.21 mmol L(-1). However, this is not sufficient to cause any considerable increase in oxidation current when the NO stock solution is used for electrochemical sensor calibration over physiologically relevant concentrations; the NO sensitivity of bare Pt-disk electrodes operating at +900 mV (vs. SCE) was 1.08 nA micromol(-1) L, while that for NO(2) (-) was 5.9 x 10(-3) nA micromol(-1) L. The stability of the NO stock solution was also monitored for up to 2 h after synthesis and 30 min was found to be the time limit within which calibrations should be performed.

Simultaneous real-time amperometric measurement of catecholamines and serotonin at carbon fibre 'dident' microelectrodes.

Amperometry allows real-time measurement of in vivo electrochemical signals, albeit with no capacity to identify the constituents of the signal. In the present study, differential amperometry was used to monitor catecholamine and serotonin (5-HT) simultaneously at the same location. 'Dident' carbon fibre microelectrodes (microelectrodes with two working electrodes in a single assembly) were constructed and coated with Nafion to prevent poisoning on contact with brain tissue. One electrode (channel A) was held at +200 mV versus Ag/AgCl to monitor catecholamines selectively. This potential is too low to oxidise 5-HT. The second electrode (channel B), recording faradaic current at +500 mV versus Ag/AgCl, measured the sum of catecholamine and 5-HT oxidation. The 5-HT current component was the difference of channel B minus channel A. With appropriate balancing of the two channels, it is possible to record catecholamines and 5-HT simultaneously at the same dident microelectrode. Examples of measurements in striatum, cortex and locus coeruleus are shown.

Differential Actions of Endogenous and Iontophoretic Dopamine in Rat Striatum.

We have voltammetrically monitored dopamine release in the rat striatum evoked either by stimulation of the median forebrain bundle or by iontophoresis. Neuronal spike activity was recorded with the same microelectrode alternately with voltammetric scans. Low levels of stimulation-evoked dopamine (< 1 microM) excited all striatal units tested. However, this effect could be converted into an inhibition at higher levels of release. In contrast to the results with neuronally released dopamine, excitation was never seen when dopamine was applied by iontophoresis. Inhibition was, however, observed at high concentrations (> 1 - 5 microM) of exogenous dopamine. These data show that endogenous dopamine at low levels may be predominantly an excitatory transmitter in the striatum, but exogenous (iontophoretic) dopamine does not have the same effect on striatal unit activity as the endogenous material.