Institute of Health Sciences Education

Microelectrodes using single carbon fibres as the conducting element have been used since 1979 for electrophysiological measurements in vivo and in vitro. However, there is still considerable discussion about the manufacture of these electrodes, and no overall agreement as to the best way to minimise electrode noise. In this article we describe some revised methods for carbon fibre electrode manufacture, which we believe, gives the lowest noise performance for spike recording in vivo.

Electrical stimulation of the median forebrain bundle of the chloral hydrate anaesthetised rat evoked dopamine release in the ipsilateral striatum, which was monitored with fast cyclic voltammetry. On cessation of stimulation, the extracellular concentration of dopamine fell to sub-detectable levels over a period of about 15 s. This fall appeared to be due to a saturable, low affinity uptake system that could be inhibited by nomifensine (20 mg/kg i.p.). These experiments constitute the first characterisation of a dopamine uptake mechanism obtained in the intact animal.

Peripheral administration of 5-hydroxytryptophan (5-HTP) to rats causes 'wet dog' shakes and a parallel elevation of brain serotonin (5-HT) levels. The increase in 5°HT eoncentrauon does not, however, correlate with the endogenous 5-HT mnervat~on rinsing the possibility that some 5-HTP is decarboxylated in non-serotonergic cells. In the present study we used in vivo voltammetry to establish whether 5-HTP treatment led to formation of 5-HT as a 'false transmitter' in strlatal dopamine (DA) neurons. Fast cyclic voltammetry at carbon fibre microelectrodes (CFMs) was used to monitor stnatal monoamine release following electrical stimulation of the median forebrain bundle (MFB). In the absence of any pretreatment DA was the sole compound released by stimulation. However, when DA release was abolished with a-methyl-p-tyrosine (AMPT), 5-HTP administration (after peripheral decarboxylase inhibition) caused a dose-dependent release of 5-HT, confirmed by the voltammetric characteristics. Central decarboxylase inhibition prevented release indicating that 5-HTP itself was not released. By monitonng reduction peaks it was possible to record DA and 5-HT release simultaneously at a single CFM. Whale DA and 5-HT oxidised at the same potential their reduction peaks were separated by approximately 450 mV It was shown, using this means, that 5-HT was still detectable even when DA release was not abolished by AMPT DA and 5-HT release showed a sigmficant positive correlation suggesting that they were released from the same nerves. We conclude that, after 5-HTP treatment, 5-HT can be released as a false transmitter from striatal DA neurones.

Key words: striatum --dopamine --electrical stimulation --voltammetry --carbon fibre microelectrode High speed cyclic voltammetry was used to measure electrically stimulated striatal dopamine release in vivo with 25 ms time resolution. Dopamine release after 1 s stimulations was readily detectable and could be manipulated by drugs known to influence dopaminergic neurones. Using data-averaging dopamine release could be detected following stimulus trains as short as 100 ms. The use of carbon fibre microelectrodes gave a spatial resolution better than 5/~m.

Fast cyclic voltammetry at a carbon fibre microelectrode was used to monitor the time course of dopamine overflow in slices of rat corpus striatum incubated in a brain slice chamber. Dopamine release occurred in response to electrical stimulation. Electrochemical, physiological and pharmacological evidence indicates that release of endogenous dopamine can be measured reliably for up to 9 h and that fast cyclic voltammetry can be used in brain slices for quantitative studies of dopamine release in the CNS.

Key words: extracellular ascorbic acid --high speed cyclic voltammetry --carbon fibre microelectrode High speed cyclic voltammetry was used in combination with pressure ejection of ascorbate oxidase for the determination of extracellular ascorbic acid within the brain of the anaesthetized rat. Large variations in absolute levels of ascorbate were found between animals although distribution patterns showed a good degree of reproducibility. Ascorbate levels in the white matter of the corpus callosum were found to be higher than in adjacent areas of grey matter (striatum and cortex)•

This report describes a new form of fast cyclic voltammetry which samples electrochemical reactions of dopamine at one every 10 ms. We have called this technique 'continuous scan cyclic voltammetry' (CSCV). The technique uses a carbon fibre microelectrode which is connected to a continuously active sine-wave source. The applied voltage is a 100 Hz precision sine wave. Faradaic signals due to cycles of continuous oxidation and reduction of dopamine at the tip of the electrode can be observed superimposed on a fixed charging current. The technique is insensitive to moderate concentrations of ascorbate as a depletion zone for ascorbate is rapidly established around the electrode. In a brain slice preparation the technique can show the time course of dopamine release on a millisecond time scale. It should prove a valuable new tool for the investigation of dopamine transmission in the brain.

Key words: dopamine uptake --striatum --in vivo voltammetry --median forebrain bundle Electrical stimulation of the median forebrain bundle evoked dopamine release in the ipsilateral striatum which was monitored with high-speed cyclic voltammetry. After stimulation, the extracellular concentration of dopamine fell due to uptake in a biphasic manner, showing zero-order and first-order components. The zero-order phase corresponded to a Vma x of 42.8 +-1.8 nmol/min/g tissue. The uptake system could be blocked by D-amphetamine, methylphenidate and nomifensine, but not by benztropine, amfonelic acid or mazindol, The density of uptake sites showed a correlation with the density of striatal dopamine innervation.

The impedance of carbon fibre-containing microelectrodes was measured at a range of frequencies. From this and other data an equivalent circuit model for the microelectrodes was constructed. Various parameters of the model that contribute to the overall noise performance of the microelectrodes when recording extracellular nerve action potentials were discussed.

Microelectrodes using single carbon fibres as the conducting element have been used since 1979 for electrophysiological measurements in vivo and in vitro. However, there is still considerable discussion about the manufacture of these electrodes, and no overall agreement as to the best way to minimise electrode noise. In this article we describe some revised methods for carbon fibre electrode manufacture, which we believe, gives the lowest noise performance for spike recording in vivo.

The present experiments were designed to test further the idea that 7-OH-DPAT (7-hydroxy-N,N-di-n-propyl-2-aminotetralin), a putative dopamine (DA) D 3 agonist, has effects at DA autoreceptors to reduce intracranial DA levels and to reduce behaviours that are DA-dependent. Rats were trained to respond on a self-stimulation protocol for electrical stimulation to the ventral tegmental area (VTA). Each press of a lever delivered a 0.5 s train of square wave, 1.5 ms duration, 100 Hz, 90-120 mA stimulation. Systemic administration of 7-OH-DPAT at 0.01-0.3 mg/kg i.p., quickly dose-dependently reduced responding. Electrical stimulation using similar parameters to those that supported self-stimulation were then applied to the VTA of anaesthetized rats. Fast cyclic voltammetry (FCV) revealed that this stimulation released DA in the nucleus accumbens (NAC). 7-OH-DPAT i.p. (0.1-3.0 mg/kg) quickly and potently reduced the size of the DA-generated voltammetric signal. This effect of 0.3 mg/kg 7-OH-DPAT was not blocked by sulpiride (60 mg/kg, i.p.) a D2-specific antagonist that may preferentially block D 2 autoreceptors. These data are discussed with reference to the possibility that 7-OH-DPAT reduces the release of dopamine in the NAC, at D3, but not at D 2, autoreceptors and that this in turn may reduce the rewarding effect of VTA stimulation.

Despite their presumed importance in kinesthesia 11 (the sense of limb position and movement), joint afferent nerve fibres have been investigated by relatively few authors 2-4,6,%14. A particularly interesting observation in one of the early studies 14 was that the knee joint afferents appeared to be influenced by the tension in the muscles acting at the joint, when the joint was held immobile by clamps. This observation gains particular interest in relation to recent studies of kinesthesia in man 8, and it seemed worthwhile to investigate the responsiveness of other joint afferent nerves to muscle activity. This report shows that fibres in the elbow joint nerve of the forelimb are selectively sensitive to several types of muscle activity, and suggests that one of the functions of muscles in kinesthesia may be to modulate joint afferent discharge.

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 pM) 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 pM) 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.

The somatotopic map of the gracile nucleus was determined using tungsten electrodes in anesthetized adult cats. Following deafferentation of the hind leg by cutting all dorsal roots caudal to L3, the nucleus was remapped. The total number of points in the map where abdomen responses were recorded doubled immediately following deafferentation. The latency of responses of cells responding to brush of the abdomen was not different after deafferentation. Some of the cells which began responding to abdominal stimulation had one or more of the following characteristics : (i) a rapid flick of the hair was required to fire the cell, (ii) habituation, and (iii) widespread inhibitory fields. To confirm that individual cells had switched the afferents which excite them, we examined the respone of single cells before, during, and after a reversible cold block of all the afferents from the hind leg. Of 40 single cells examined, 11 which had receptive fields on the leg switched their receptive fields to the abdomen as soon as the block of leg afferents was complete. Mapping the gracile nucleus 8 months following deafferentation still showed an increase in abdominal representation. Examination of the dorsal horn in the L6-7 segment of these chronically deafferented cats showed that abdominal afferents excited cells in a region normally responding to leg afferents. These results suggest that deafferentation results in the unmasking of normally ineffective connections. Unmasking could be a mechanism which explains many of the observed changes in the responses of central nervous system neurons following lesions.

Fast cyclic voltammetry was used to measure NO and dopamine (DA) simultaneously in rat caudate putamen (CPu) slices. Analysis of electrochemical signals obtained from mixtures of DA and NO showed that subtraction of either the DA or the NO component revealed the contribution of the other component. Application of such data manipulation to signals obtained in CPu slices indicated that DA and NO components contributed to electrochemical signals. Following electrical stimulation (>1 s), site-dependent NO-like signals were observed. Pressure ejection of NMDA yielded a biphasic electrochemical signal. The first phase (lasting 10-20 s) had electrochemical characteristics of DA and was observed only during the first application of NMDA. The second phase developed more slowly, was of longer duration (1-3 mm), and had electrochemical characteristics of a mixture of DA and NO. Generation of the NO-like signal by NMDA was antagonised by L-N-monomethylarginine. Pressure ejection of NO into CPu slices caused dose-and site-dependent DA release. More DA was released in the dorsolateral than in the dorsomedial CPu. Pressure ejection of DA did not generate NO. It is concluded that electrically stimulated DA release is not mediated by prior release of NO. Key Words: Brain slice-Caudate putamen-Cerebellum-Dopamine-Electrochemistry-Electrical stimulation-Nitric oxide-N-Methyl-D-aspartate -Pressure ejection-Voltammetry.