Carbon fibre micro-electrodes for concomitant in vivo electrophysiological and voltammetric measurements: no reciprocal influences

Abstrac

Differential pulse voltammetry and more recently cyclic voltammetry have been successfully used to monitor basal levels of
endogenous chemicals by means of treated carbon fibre microbiosensors inserted in specific brain regions. In this study, feasibility of
concomitant in vivo recordings of stable electrophysiological signals and basal ascorbate, catecholaminergic and indolaminergic
voltammetric peaks at the same cerebral site by means of a single electrically treated carbon fibre micro electrode (microbiosensor) is
presented. The results indicate that these two independent techniques can be combined in vivo at a single electrode, and that
voltammetric measurements of unstimulated levels of extracellular compounds do not alter concomitant basal cell firing for a period
long enough (more than 6 h) to allow pharmacological manipulations.

In the last decade, carbon based electrodes have been
extensively used with voltammetric techniques for in vitro and in vivo analysis of electroactive compounds such
as neurotransmitters and/or related metabolites [12]. Recently, low impedance carbon fibre electrodes (1-2 Mf~)
have been used to record evoked responses from brain
areas such as the somatosensory cortex [13]. Concomitant
voltammetric analysis of stimulated release of catechols
with electrophysiological measurements using carbon
fibre micro-electrodes (mCFE) has been recently proposed [3], based on the observation that an oxidation potential of +0.55 V (voltage applied with respect to a
silver/silver chloride electrode) used for chronoamperometrical analysis in vivo, does not interfere with
spontaneous neuronal activity in the brain as it appears to
be below the threshold required to alter neuronal excitability [11]. This is an interesting feature as it implies that
both pre-synaptic (release, metabolism) and post-synaptic
neuronal processes can potentially be studied simultaneously by means of a single sensor. This was first proposed
by Amstrong-James and Fox in 1983 [1], and more recently Su et al. [17] have reported the feasibility of performing combined electrochemical and electrophysiologi cal studies of monoamine overflow in rat slices with carbon based electrodes.


In addition to chronoamperometry (CA), other voltammetric methods for in vivo applications have been developed and have started to be used extensively to monitor central functions. Two major methodologies seem to
be most widely used at the present time, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) [16].
All three methods are concerned with measuring the current produced by oxidation or reduction of chemicals at
the surface of a working (carbon based) electrode, following the application of a suitable potential. In CA, the voltage is increased instantaneously to a potential sufficient to
oxidise the electroactive compound and held for a fixed
time (50 ms to 1 s). This results in an accurate evaluation
of the concentration of electroactive species but with poor
selectivity, since all chemicals that are oxidisable at or
below the applied potential, will contribute to the oxidation current measured. In contrast, with CV and DPV the
applied voltage is not maintained at a constant value but
is instead a waveform, such as a cyclic ramp (CV) or a
linear potential ramp with superimposed pulses (DPV).
The voltage input waveform determines the faradaic (due
to the oxidation of the electroactive species at the surface
of the electrode) to charging current ratio, which is in-

versely proportional to the scan rate [4]. This allows an
increased selectivity between the electroactive chemicals
present in solution (in vitro), as well as in the brain extracellular fluid. When oxidising at sufficiently different
potentials, these compounds can be selectively monitored
by means of slow scanning methods such as DPV (scan
speed 10-20 mV/s, versus faster techniques such as CA
or CV) applied in association with electrically treated
mCFE [6]. DPV can be applied between -0.2 V and
+0.9 V when one wants also to monitor peptidergic signals (which occur between +0.7 and +0.9 V) [7]. However, this slow scanning method is routinely used between
-0.2 V and +0.4 V scan range, which allows the simultaneous, selective analysis of ascorbate (peak 1 at approximately -100 mV), catechols (peak 2 at approximately
+60/90 mV) and indoles mixed with uric acid (UA) (peak
3 at approximately +250/290 mV) [6] (see Figs. 1 and 2).
Thus, it remains within the limits of the voltage which
does not alter neuronal functions [11]. Another widely
used voltammetric method is fast cyclic voltammetry
(FCV), which is normally applied in brain slices at the
scan speed of 300 V/s to measure electrically stimulated
release of neurotransmitters by means of untreated sensors [16]. In this method, the waveform extends from
-1 V up to +1.0 or to +1.4 V for the detection of catechol
or indole signals [5,14]. This is largely above 0.55 V,
nevertheless recent data indicates that FCV does not in

duce change in the cell firing rate measured in brain slices
with the same untreated voltammetric sensor [15]. Recently, we have suggested that CV at the scan speed of
500-1000 mV/s associated with electrically treated microbiosensors can simultaneously monitor in vivo basal
extracellular levels of catechol and indole-UA oxidation
signals with the same sensitivity as DPV but with an improved time resolution [10].
In this study, the feasibility of concomitant in vivo
electrophysiological (cell firing) and electrochemical
(voltammetric) recordings at the same rat brain area by
means of a single electrically treated mCFE is analysed.
In addition, the reciprocal influence of the in vivo voltammetric methods used (DPV, CV) and electrophysiological measurements performed with the same voltammetric microbiosensor, have been studied.
The mCFE (approximately 10/~m in diameter) were
prepared and electrically treated for voltammetric analysis
as described previously [6,9,16]. In order to obtain singleunit activity measurements with the same electrode, the
length of the active tip of the microbiosensor was approximately 30ktm according to Bickford-Wimer et al.
[3] and Stamford et al. [15]. (The commonly used length
for voltammetric analysis is approximately 500ktm, this
length allowing multiple-unit recordings only).
In vitro experiments were performed with the 500/~m
length and then with the 30/tm length mCFE in a solution


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