Peter Palij's research while affiliated with Royal Holloway, University of London and other places

Brain slices have been used for several years in the study of the factors controlling neurotransmitter release in animals. Despite this, most conventional studies have drawbacks. First, it is often necessary to label the neurotransmitter pool with tritium (Farnebo and Hamberger, 1971). This assumes, probably fallaciously, that release of the radiolabel accurately reflects the neurotransmitter. For example, when endogenous and radiolabeled catecholamine releases are simultaneously measured in response to various stimuli, there are significant discrepancies between the two (Herdon and Nahorski, 1987). Second, transmitter release is frequently evoked by elevating the potassium concentration. However, some transmitter is released in a calcium-independent fashion following potassium depolarization (Okuma and Osumi, 1986). Third, many superfusion methods need to block transmitter uptake in order for efflux to be measurable (Parker and Cubeddu, 1985). These are serious limitations.

Desipramine, like most antidepressants, does not exert clinical benefit until 2-3 weeks after the onset of treatment. It has been suggested that this delay might be due to enhanced autoreceptor activation, counteracting the acute uptake blockade. We therefore tested whether autoreceptor blockade might enhance the response to acute uptake blockade, using voltammetry at carbon fibre microelectrodes to monitor stimulated noradrenaline (NA) efflux in rat bed nucleus of stria terminalis brain slices. Desipramine significantly increased NA efflux and slowed NA uptake. The combination of rauwolscine and desipramine increased NA efflux significantly more than desipramine alone. We suggest that alpha 2 autoreceptor blockade functionally mimics alpha 2 autoreceptor down-regulation and thus may allow the full therapeutic effect of desipramine to be manifested more rapidly.

In functional terms, the brain is highly spatially and temporally organised. Its function may be studied by many types of implantable probe such as dialysis fibres, cannulae or microelectrodes. In vivo voltammety, in particular, has been extensively used to study neurochemistry in situ. However, spatial and temporal sampling constraints can profoundly affect not only the reproducibility but also the accuracy of all neurochemical data obtained by voltammetry and related sampling techniques. For instance, it is possible to get inaccurate yet highly reproducible temporal information about neurotransmission simply by injudicious choice of sampling parameters. This review discusses the importance of voltammetric sampling parameters to the quality of neurochemical data that we, as neuroscientists, obtain. Although these considerations apply equally to all sampling based methodologies we have chosen to focus on the case of fast cyclic voltammetry. The review is illustrated by data using fast cyclic voltammetry at carbon fibre microelectrodes in brain slices.

Fast cyclic voltammetry was used to investigate the effect of 7‐OH‐DPAT (7‐hydroxy‐ N,N ‐di‐n‐propyl‐2‐aminotetralin), a putative D 3 receptor agonist, on electrically stimulated endogenous dopamine release in slices of rat nucleus accumbens. 7‐OH‐DPAT inhibited single pulse stimulated dopamine release in a concentration‐dependent manner with a maximum inhibition of 95.5%. Analysis of concentration‐response curves to 7‐OH‐DPAT showed that they were biphasic, with the high affinity component contributing 18.0% to the total inhibition and the low affinity component 77.5%. 7‐OH‐DPAT exhibited a 560 fold selectivity between the high and low affinity components (0.015 nm compared to 8.4 n M ). Concentration‐response curves to the nonselective D 2 /D 3 agonist, apomorphine, were monophasic. The maximum inhibition was 93.1% and the EC 50 value 82 nM. The selective D 2 antagonist, haloperidol (30 n M ), antagonized the low affinity component of the concentration‐response cuve to 7‐OH‐DPAT whilst the high affinity component was essentially unaffected. The p K B values calculated for the high and low affinity components were 7.89 and 9.45 respectively. In conclusion, these results demonstrate that 7‐OH‐DPAT inhibits stimulated dopamine release by acting at two different sites. Furthermore, the results are consistent with the hypothesis that the high and low affinity components of the concentration‐response curve to 7‐OH‐DPAT may reflect activation of functional D 3 and D 2 release‐regulating autoreceptors respectively. However, the possibility that the biphasic nature of the curve may reflect different subtypes of the D 2 receptor cannot be excluded.

Fast cyclic voltammetry (FCV) at carbon fibre microelectrodes was used to monitor 'real time' endogenous noradrenaline (NA) efflux in superfused slices of rat locus coeruleus (LC) following local electrical stimulation. When stimulated with a standard train (30 pulses, 100 Hz, 0.2 ms, 10 mA, every 5 min), efflux of monoamine was constant over the experimental period (2.5 h): Amine efflux declined by only 16 +/- 5% while uptake half-life lengthened by only 9 +/- 8%. When calibrated in solutions of NA, peak amine efflux corresponded to 0.31 +/- 0.04 microM (mean +/- S.E.M., n = 28) and was removed by uptake with a half-life of 2.93 +/- 0.28 s (n = 16). The released compound was confirmed as NA on the basis of pharmacological and electrochemical criteria. Stimulated monoamine efflux was reversibly reduced by 78% by omission of Ca2+ from the superfusate for 30 min (P < 0.05). Ro 4-1284 (1 microM), a fast-acting reserpine-like drug, decreased amine efflux by 86% (P < 0.05). The monoamine oxidase inhibitor pargyline (2 microM) increased efflux by 30% (P < 0.05). Desipramine (0.05 microM), a selective NA uptake blocker, significantly increased amine efflux (by 96%, P < 0.05) and uptake half-life (by 314%, P < 0.05). Fluvoxamine (0.5 microM), the selective serotonin (5HT) uptake blocker, increased efflux by 59% (P < 0.05) and the uptake half-life by 122% (P < 0.05). Vanoxerine (GBR 12909: 0.3 microM), the dopamine (DA) uptake blocker, had no effect on amine efflux or uptake half-life. The voltammogram of the released amine had single oxidation and reduction peaks.(ABSTRACT TRUNCATED AT 250 WORDS)

Many previous studies have demonstrated the value of carbon-fibre microelectrodes (CFMs) for single-unit activity recording and for fast cyclic voltammetry. In this report we show that these two independent methodologies can be combined at a single CFM and used to study simultaneous electrochemical and electrophysiological events in brain slices. In superfused slices of rat locus coeruleus, dorsal raphe and substantia nigra, we were able to record stable electrophysiological signals and stimulated monoamine efflux for periods of at least 2 h, thereby allowing quantitative pharmacological interventions. The simultaneous recording of amine efflux and unit activity at the same locus facilitates comparison of drug effects at pre- and post-synaptic sites. Furthermore, the system described here uses commercially available instrumentation. The circuitry is described and examples of its application are shown.

Fast cyclic voltammetry (FCV) at carbon fibre microelectrodes was used to monitor stimulated noradrenaline (NA) efflux in slices of the ventral part of the rat bed nucleus of stria terminalis (BSTV) superfused with artificial cerebrospinal fluid at 32 degrees C. NA efflux was evoked by local electrical stimulation (trains of 10-50 pulses, 0.2 ms duration, 10 mA constant current at 10-500 Hz). The effects of four alpha 2 antagonists (yohimbine, rauwolscine, prazosin and WB 4101) and three alpha 2 agonists (clonidine, oxymetazoline and UK 14304) were examined. All drugs (1 microM) were added via the superfusate. Yohimbine and rauwolscine increased NA efflux on the lower but not the higher frequency trains: maximum increases (on 20 Hz, 50 pulse stimulation) were to 392 +/- 63% (yohimbine) and 243 +/- 7% (rauwolscine). There was a threshold train duration for demonstration of autoreceptor antagonism of 500-1000 ms. Prazosin and WB 4101 did not increase NA efflux but caused a modest decrease at the higher (100-500 Hz) frequencies. The effects of the alpha 2 agonists were also affected by stimulus train duration. Longer trains reduced agonist (clonidine) effects. When tested on pseudo-one pulse (POP) stimulations (less than 100 ms duration), the alpha 2 agonists decreased NA efflux. UK 14304 reduced NA efflux on 20 pulse/200 Hz stimulation to a greater degree (86 +/- 7%) than the partial agonists clonidine (39 +/- 3%) or oxymetazoline (40 +/- 8%). The present results demonstrate that alpha 2 autoreceptors are a major mechanism in the control of NA efflux in the BSTV.(ABSTRACT TRUNCATED AT 250 WORDS)