Journal of Neuroscience Methods, 38 (1991 ) 25-33 c~ 1991 Elsevier Science Publishers B.V. 0165-0270/91/$03.50

25

NSM 01231

Fast cyclic voltammetry can be used to measure stimulated endogenous 5-hydroxytryptamine release in untreated rat brain slices J.J. O ' C o n n o r

and Z.L. Kruk

D~7~artment ~f Pharmacology, Queen Mary and Westfield College, Mile End Road, London El 4NS (U.K 1 (Received 12 October 1990) (Revised version received 15 February 1991) (Accepted 15 February., 1991)

Key words: Fast cyclic v o l t a m m e t r y ; 5 - H y d r o x y t r y p t a m i n e ; Release; Dorsal raphe n e u r o n e s ; Suprachiasmatic nuclei; In vitro Fast cyclic voltammetry at a carbon fibre microelectrode was used to monitor the time course of 5-hydroxytryptamine (5-HT) overflow in slices of rat dorsal raphe (DRN) and suprachiasmatic nuclei (SCN), incubated in a brain slice chamber for over 6 h. 5-HT overflow was detected in response to electrical brain stimulation in both regions. Voltammetric evidence showed that the released substance was identical to exogenously applied 5-HT. Overflow was reversibly abolished when Ca ~+ was removed from the incubating medium or when TTX was added. Ro4-1284, a reserpine like agent, irreversibly abolished 5-HT overflow from both nuclei. The 5-HT uptake blockers, citalopram, clomipramine, fenfluramine and fluvoxamine dose dependently increased overflow and slowed the rate of removal of 5-HT from the extracellular space in both regions. Benztropine had no effect on overflow in the DRN and SCN whereas it caused a significant increase in dopamine overflow in slices of caudate putamen (CPu). Xylamine had no effect on 5-HT overflow in the DRN and SCN. This evidence indicates that the release of endogenous 5-HT can be measured reliably for long periods and that FCV can be used in brain slices for quantitative studies of 5-HT release and uptake.

Introduction T h e use of fast cyclic v o l t a m m e t r y ( F C V ) to m e a s u r e real time d o p a m i n e overflow in vivo has b e e n well d o c u m e n t e d (Millar et al., 1985; Stamford et al., 1986a, b, c), a n d this t e c h n i q u e has b e e n a d a p t e d to the q u a n t i t a t i v e m e a s u r e m e n t of d o p a m i n e release in rat b r a i n slices (Bull et al., 1990; Palij et al., 1990) over relatively long periods of time. In vitro studies have shown that c a r b o n fibre m i c r o - e l e c t r o d e s ( C F M e ) used with F C V are

~orrespondence: Dr. J.J. O'Connor, Dept. of Pharmacology, Queen Mary and Westfield College, Mile End Road, London El 4NS, U.K. Phone: (71) 982 6351; Fax: (81) 983 0470.

more sensitive to 5 - H T t h a n d o p a m i n e a n d that 5 - H T can be d i s t i n g u i s h e d from d o p a m i n e v o l t a m m e t r i c a l l y (Stamford et al., 1990). In the same study, it was shown that s t i m u l a t e d 5 - H T overflow occurs from d o p a m i n e t e r m i n a l s in the c a u d a t e p u t a m e n only after 5 - H T P loading in vivo. Previous m e t h o d s to d e t e r m i n e the release of e n d o g e n o u s m o n o a m i n e s have b e e n insensitive in d e t e c t i n g release elicited by small trains of single pulses. In c o n t r a s t to the use of t r i t i u m release m e t h o d s for the m e a s u r e m e n t of m o n o a m i n e overflow w h e r e the entire b r a i n slice is stimulated, (activating a high p r o p o r t i o n of axons within the slice), fast cyclic v o l t a m m e t r y p e r m i t s stimulation of selected fibre b u n d l e s or nuclei. T h e precise a n a t o m i c a l location of C F M e in

26

regions known to have high densities of 5-HT cell bodies such as raphe neurones (Steinbusch, 1981) and 5-HT axon-terminal regions such as suprachiasmatic nucleus (Willoughby and Blessing, 1987; Wilson et al., 1989) is of benefit in the identification of 5-HT using FCV. Here we present electrochemical, biochemical and pharmacological evidence which we believe shows that it is possible to measure overflow and uptake of endogenous 5-HT in rat brain slices without either precursor loading, inhibition of enzymes of metabolism or inhibition of uptake processes.

+1.4V

A

-10V

B o

Methods

Preparation of brain slices The brain was removed from male Wistar rats (150-200 g) and prepared for sectioning in icecold artificial cerebrospinal fluid (ACSF) of the following composition (mM): NaC1 125, KC1 2, K H z P O 4 1.25, MgSO 4. 7 H 2 0 2, N a H C O 3 25, D-glucose 11, CaCI 2 2, saturated with 95% 0 2 / 5 % CO 2. Brain slices were prepared as previously described by Bull et al. (1990). Briefly, blocks of tissue containing either the CPu, D R N or SCN 9.2-9.9 ram, 7.5-8.2 mm and 1.0-1.7 mm anterior to the interaural line respectively, were dissected out according to the atlas of Paxinos and Watson (1982). A single slice (350/xm thick) was cut from the block of tissue using a vibrotome and placed in an full immersion incubation chamber (Richards and Tegg, 1977) on a stainless steel grid. The slice was maintained at 32 ° C, and superfused (1.1 m l / m i n ) with ACSF for 1 h before recordings were started.

Fast cyclic t,oltammetry Fast cyclic voltammetry was carried out using a Millar voltammeter (PD Systems Ltd., Surrey, U.K.) using a working CFMe (Armstrong-James and Millar, 1979), Pt auxiliary electrode and A g / A g C l reference electrode. The applied waveform was 1.5 cycles of a 100 Hz triangular ramp, scanning between - 1 . 0 and + 1.4 V relative to the reference electrode at a voltage scan rate of 480 V / s (Fig. 1A). This potential scan was applied to the CFMe at 2 Hz; zero potential was

60nA rl

C 6nA

o

D 5nA r

5ms

Fig. 1. Fast cyclic voltammetry. A: triphasic waveform applied to the carbon fibre microelectrode (CFMe). B: current waveforms flowing in the CFMe in response to the applied waveform before (lower trace) and after placing the electrode into a solution of ACSF containing 10 -7 M 5-HT (upper trace). The points indicated by the letters show the oxidation (O: +610 mV) and reduction potentials (rl: - 1 0 mV and r2: -600 mV) characteristic for 5-HT. C: current waveform, (subtractogram) for 5-HT produced by subtraction of t h e two waveforms shown in B. The height of the oxidation potential (O) is used to quantify the amount of 5-HT present at the CFMe. D: subtractogram produced by taking the current waveform produced in the presence of 3 x 10 7 M dopamine from the background current. An oxidation (O: +585 mV) and a single reduction (r: - 2 2 0 mV) potential can be seen. Vertical bar for A: V and B, C and D: nA. Horizontal bar: 5 ms.

27 maintained between scans. The signal was amplified and fed into a CED 1401 interface (Cambridge Electronic Design, Cambridge, U.K.) for waveform capture, storage and analysis using CED Sigavg software. The background (charging) current in the CFMe obtained in response to the input voltage is shown in Fig. 1B, and the Faradaic current obtained in the presence of 10 . 7 M 5-HT is superimposed on the charging current. When the charging current signal is subtracted from that obtained in the presence of 5-HT, a faradaic waveform ('subtractogram') shown in Fig. 1C results. There is an upward directed oxidation peak at +610 mV (O), and two reduction peaks at - l(I mV and - 6 5 0 mV (r~ and r=, respectively). The subtractogram obtained in the presence of 3 × 10 7 M DA is shown in Fig. 1D. The oxidation potential for dopamine occurs at + 590 mV and a single reduction potential occurs at - 2 2 0 inV.

Position of stimulating and recording electrodes In all experiments, the CFMe working electrode was placed 8(1/xm below the surface of the slice. In the CPu, the CFMe was placed centrally in a light band of grey matter as seen with a binocular dissecting microscope. A bipolar stimulating electrode (tip separation 150 bern) was placed so as to straddle the same light band, 200 # m ventromedial to the CFMe. In the DRN the CFMe was placed centrally, ventral to the aquaduct, and in the SCN centrally in the left nucleus. The bipolar stimulating electrode was placed 200 # m ventral to the CFMe in these regions. Constant voltage electrical stimulation and timing was carried out using a Neurolog system and a DS2 constant voltage stimulator. Attempts in the D R N and SCN to stimulate 5-HT release using single-square wave pulses (0.1 ms; 1-40 V) met with varied success; to produce a consistent detectable amount of oxidisable material it was necessary to use trains of pulses (25 pulses, 50 Hz, 0.1 ms width, 20 V) in both the D R N and SCN.

20

15

v

10

5

0

.

.

.

.

.

.

.

.

,

.

.

.

.

.

.

.

.

,

10 ~

10 7 1()~ 5-HT Concentration (M) Fig. 2. Calibration curve for 5-HT. Electrodes were calibrated in vitro after each experiment in solutions of known 5-HT molarity (10 x to 10 ~' M) since stimulated 5-HT overflow was typically within this range. The average (nA+SEM) ol four observations is shown. voltammeter, set at the oxidation peak for 5-HT ( + 6 1 0 mV), and from a calibration curve the concentration of 5-HT overflow in a slice was estimated (Fig. 2). The output from the sample and hold was fed into a Y / t chart recorder (Servogor 220) for on-line monitoring of changes in extracellular 5-HT concentrations between and during electrical stimulation. The rate of uptake of 5-HT was also measured from the sample and hold traces, and defined as the time for voltammetric signal to decline to half of its maximal value following electrical stimulation (T 1/2).

Experiments with uptake blockers In experiments using monoamine reuptake blockers, control values were obtained for 30 min prior to drug infusion and post drug values monitored in the same slice for up to 2 h. Pre- and post-drug 5-HT overflow and uptake were compared for each slice, and data from different slices was pooled for statistical analysis.

Measurement o[ stimulated overflow

Drugs and solutions

The height of the oxidation peak was measured with a sample and hold device in the Millar

Drugs were obtained from the following sources: xylamine HCI (Research Biochemical

2~

Inc); fluvoxamine and citalopram (kindly donated by Dr. David Bull); clomipramine (Geigy lnc); benztropine (Merck, Sharp & Dohme); nialamide (donated by Dr. Stamford); fenfluramine HCt, uric acid, 5-hydroxyindole-3-acetic acid, 5-hydroxytryptamine creatinine sulphate, and dopamine HC! (all Sigma). All drugs were dissolved in distilled water.

Statistical analysis The statistical significance of the difference between means (+ SEM) was assessed using the Student's t-test (paired or unpaired as appropriate).

after electrical stimulation in the CPu. A single oxidation potential occurs at +595 mV and a single reduction potential at -230 inV. This is indicative of the presence of dopamine which has a characteristic oxidation potential of + 590 mV and a reduction potential of -220 mV (see Fig. 1D). Fig. 3C shows the subtractogram obtained after exposure of 10 5 M 5-HIAA onto the CFMe. A concentration 100 times that of 5-HT was required to give similar oxidation potential peak

I

Results

Voltammetric identification of substance The current waveform after electrical stimulation (0.1 ms width; 20 V; 500 ms duration) in the DRN is shown in Fig. 3A. An oxidation potential at + 620 mV and two reduction potentials at - 20 mV and - 6 8 0 mV can be seen. This waveform is indistinguishable from that see in Fig. 1C for perfused 5-HT; a similar current waveform was obtained after electrical stimulation in the SCN (not shown). Fig. 3B shows the current waveform

Fig. 3. Current waveforms produced after electrical stimulation in rat brain slices (A, B) and in vitro (C, D). A: electrical stimulation (25 pulses at 50 Hz, 0.1 ms pulse width, 20 V) in the DRN. Note the single oxidation (+ 620 mV) and double reduction peaks ( - 2 0 mV and - 6 8 0 mV). These positions are identical to that obtained with perfused 5-HT (Fig. 1C). B: similar electrical stimulation carried oat in the CPu. The single oxidation (+ 595 mV) and reduction (-230mV) peaks for dopamine are seen. C: subtractogram obtained with the same CFMe as used in A and B, placed in a solution of ACSF containing 10 -5 M 5-HIAA. A single oxidation (+660 mV) and double reduction ( - 6 0 and - 5 2 0 mV) peaks can be seen. The CFMe was 50-100 times less sensitive to 5-HIAA than 5-HT. D: subtractogram obtained in ACSF containing 3×10 -5 M uric acid. A single oxidation (+700 mV) and reduction ( - 1 4 0 mV) potential can be seen. The subtractograms shown are typical traces obtained either in a brain slice (A and B) or in vitro (in pure solution; C and D), and they have neither been averaged or filtered, and all were obtained using the same eletrodes. The noise is a combination of digital, charging and electronic inputs.

B 7nA

10nA

6nA

5ms

29

A 0 m M Ca++

50

A

45 40 35 30

25

30nM [

20

5-HT

15 10 5

rain

5 0

I

20

40

S0

100

80

B

B

,°nMI

90

5-HT

70 60

20 sec Fig. 4. Time course for stimulated 5-HT release in the D R N obtained by monitoring the oxidation potential ( + 6 0 0 mV) for 5-HT with a sample and hold circuit. A: Time course obtained after electrical stimulation (25 pulses at 50 Hz, 0.1 ms pulse width, 20 V) in the D R N . The trace shows the rate of increase and decline of the voltammetric signal scanned twice a sccond and plotted onto a Y/t chart recorder stimulated every 5 rain and 40 rain time lapse is shown. B: Expanded time scale for one stimulation indicated by asterisk in A.

TFX (10-7M)

80

50 I-"1"

40 30 20 10 0

1

20

40

60

80

100

120

140

C 9080

4

RO4 1284 (10-7M)

70 •

heights. The oxidation potential for 5 H I A A occurred at + 660 mV with double reduction potentials at - 6 0 mV and - 5 2 0 mV. The subtractogram obtained in the presence of 3 x 10 -5 M uric acid is shown in Fig. 3D. There is a single oxidation potential at + 7 0 0 mV and a single reduction at - 140 inV.

60" 50" 4030

0

°'~

3-

1

20

i

i

40

Go

,

i

8o

•

~

loo

-

i

12o

Time (mins)

Time course for 5-HT ouerflow Trains of 25 pulses at 50 Hz (0.1 ms width; 20 V; 500 ms duration) elicited reproducible electrochemical signals in both D R N and SCN. Fig. 4A shows the time course of overflow obtained from the sample and hold output, after electrical stimulation in the D R N plotted on a Y/t chart recorder. A rise time of 1 - 2 s and a time of 3 - 4 s for the overflow of substance to decline to 50% m a x i m u m (Tl/2) can be seen (Fig. 4B). This was found to be equivalent to 60 nM 5-HT after

Fig. 5. Effect of Calcium, T T X and Ko4-1284 on electrically evoked overflow in the D R N (e) and SCN(©). A: effect of removal of Ca 2+ and replacement with Mg 2+ on stimulated release (25 pulses at 50 Hz, 0.1 ms pulse width, 20 V). B: effect of addition of TTX (10 v M) to the superfusion medium. TTX abolished release in both regions within 30-40 rain and 75% recovery had occurred within 30 rain of washout for both regions. C: effect of addition of R o 4 - 1 2 8 4 (10 - 7 M) to the superfusing medium. Ro4-1284 caused 90% irreversible inhibition of release after 80 rain in both regions. Each point in A, B and C represents the mean 5-HT overflow (nM _+SEM; n = 4 - 5 ) except for the SCN in D where the average of 2 experiments is shown,

3(}

calibrating the CFMe. Similar recordings were obtained after electrical stimulation in the SCN (not shown), where overflow was equal to 40 nM 5-HT. When stimulated every 5 rain, this signal was reliably evoked for over 6 h. Evoked release of substance was highly sensitive to the location of the CFMe in the slice. Movement of the electrode outside of the DRN by 2 0 - 5 0 / x m for example, resulted in an 80-90% drop in the size of the signal.

Effect of Ca 2 ~, TTX, Ro4 1284 or nialamMe on stimulated release in the DRN and SCN Removal of Ca z" from the ACSF and replacement with equimolar Mg 2+ completely abolished the electrically evoked voltammetric signal within 15-20 min in both the DRN and SCN (Fig. 5A). The response showed full recovery 15-20 rain after restoration of calcium to the ACSF. T T X (10 7 M) abolished the signal within 40 min (Fig. 5B). On washout the response returned to 90% control value after 30 min for both regions. Ro-4 1284, the reserpine-like agent (10 -7 M) irreversible abolished the electrochemical signal in both regions (Fig. 5C). The monoamine oxidase (MAO) inhibitor nialamide (1 p.M), did not affect stimulated overflow in the D R N or SCN (n = 3 for both regions; results not shown). Effect of dopamine and noradrenaline reuptake blockers on evoked release Benztropine, a dopamine reuptake blocker (Coyle and Snyder, 1969; 10 - 7 M ) , when added

"FABLI2 1 EFFECT OF X Y L A M I N E O V E R F L O W IN T H E SCN

ON

STIMULATED

5-1FI

n = 3 for all points. Values are means + SEM.

Control Xylamine I0 s M 1(1 7 M 10 ~' M

5-HT overflow (nM)

7"!j,

37.3 +_3.9

4. I 47(1.8

36.7+2.3 35.6_+3.4 34.7_+3.2

4.7+0.7 6.0± 1.0 7.1 :+: I.l

(s)

to a bath containing slices of CPu and DRN, caused a significant increase in the electrochemical signal in the CPu (360 + 57% control; P < 0.05, n = 6) while there was no effect on the signal in the D R N (Fig. 6). Xylamine, a noradrenergic reuptake blocker (Kammerer et al., 1979) was found at concentrations of 1 0 - s - 1 0 -~ M to have no effect on overflow of substances from the DRN (n = 3) and SCN (Table 1).

Effect of 5-HT reuptake blockers on ecoked release in the DRN and SCN Citalopram (1 ~m), the 5-HT re-uptake blocker increased the measured signal for 5-HT in the DRN (Fig. 7A). In addition, the rate of removal of 5-HT from the extracellular space (t~/2), as indicated by the rate of decline of the voltammetric signal, was reduced in its presence (4.5 _+ 0,9 to 22 + 7 s, P < 0.05; Fig. 7B). Citalopram at this concentration had no significant effect on stimulated dopamine overflow in slices of CPu in the

CPu

DRN

60

1.6

50 1.2 O

O

40 3o

0.8 0