Combining ‘caged-dopamine’ photolysis with fast-scan cyclic voltammetry to assess dopamine clearance and release autoinhibition in vitro

ELSEVIER

Journal

of Neuroscience

Methods 67 ( 1996172 I-23 I

Combining ‘caged-dopamine’ photolysis with fast-scan cyclic voltammetry to assessdopamine clearance and release autoinhibition in vitro Tong H. Lee a3*, Kyle R. Gee b, Everett H. Ellinwood “ Dqwment ’ Dqmrrment Received

of Pswhiairyc.

Duke

Unicrrsi~

’ Molecular Probes. of Phnrmnrolog~ Duke Unil’UrJity 23 December

1995:

revised

MedicuI

Center.

Inc... Eugenr. Mediccd IO April

a.C,Frederic J. Seidler ’

Durhtrrn.

NC

OR. USA Ceutrr, Durham,

1996:

accepted

NC

16 April

27710, 27710.

llSA USA

1996

Abstract We have developed a methodology for inducing a rapid rise in extracellular dopamine concentrations. The clearanceof the applied dopamine.as well as its effect on the endogenous dopaminerelease(i.e., autoinhibition), was then examined using fast scan cyclic voltammetry.In a recordingchambermountedon a Nikon Optiphotepifluorescence microscope.corona4rat brainslicescontainingeither the caudate nucleus or prefrontal cortex were perfused with ACSF containing 400-200 IJ-M ‘caged-DA.’ UV iltumination (400-200 ms) focusedat the tip of the recordingelectrodeproduceda peak DA concentrationof l-2 FM within 400-200 ms of terminatingthe illumination. The caudate nucleus exhibited a faster clearance rate for photo-released DA compared to the prefrontal cortex. Cocaine

reduced the clearancerates in both the caudate nucleus and prefrontal cortex. In the prefrontal cortex a combination of desipramine/clomipramine

also reduced dopamine clearance, suggesting heterologous uptake of the applied DA by noradrenerpic

and/or

zerotonergicterminals.Photo-released dopamineinhibited releaseof endogenouscaudateDA releaseevoked by single electrical stimulation.The advantages of this methodologyare discussed. Kr!blnrrlstDopamine; Clearance: Autoinhibition; Caudate nucleus; Prefrontalcortex:Fast-scan cyclicvollammetry

1. Introduction Dopamine (DA) has been implicated in various neuropsychiatric conditions including Parkinson’s disease. schizophrenia. and drug addiction (see Weinberger et al. (1988) for overview). Consequently, regulation of DA pathways has been extensively studied. The regulation of DA releaseby autoreceptors and uptake by specific transporters are two important mechanismswhich regulate temporal and spatial distribution of the neurotransmitter in the synapses. During the last decade, various voltammetric techniques have been used to assessthe functional status of these two mechanisms(see Garris and Wightman, 1995: Gerhardt. 4995; Gonon, 1995; Rice and Nicholson, 1995; Stamford et al.. 1995; for reviews). Among these tech-

1 Corresponding author. Box 3870, Duke University Medical Center. Durham. E-mail:

NC 27710.

USA.

Tel.:

(I) 919-684-3032;

Fax:

(I) 919-681-8369:

[email protected]

0 I65-0270/96/S IS.00 PII SOI 65.0270(‘+6)00068-4

Published

by Else&r

Science

B.V.

niques, fast-scan cyclic voltammetry (FSCV) offers the advantages of millisecond sampling time (e.g., 8 ms) as well as the ability to identify chemical species based on their characteristic voltammograms. This technique. there-

fore, enables one to measure DA concentrations on a ‘real-time’ basis. thus allowing DA release and uptake processesto be discriminated (Ganis and Wightman, 1995: Jones et al., 1995: Kawagoe et al.. 1993; Stamford et al.. 1988). Using FSCV. it is possible to examine DA releaseand uptake associatedwith single-pulse electrical stimulation in slice preparations (Bull et al., 1990; Jones et al., 4995: Kennedy et al.. 1992). Thus, inhibition of DA releaseby agonists (e.g., quinpirole) and uptake by uptake blockers (e.g.. cocaine or nomifensine) have been demonstrated in the caudate nucleus and nucleus accumbensslices. Wightman and his colleagues, using a Michaelis-Menten based model, have quantitatively assessedDA release/uptake kinetics and their modulation by various pharmacological agents(Kawagoe et al.. 1993: Joneset al., 1995): rigorous

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mathematical analysis of this model has recently been presented (Nicholson, 1995). Furthermore, FSCV has been used to examine alterations in autoreceptor sensitivity and uptake inhibition by cocaine following withdrawal from chronic cocaine treatment (Jones et al., 1996; Lee et al., 1996). Two of the difficulties in measuring the kinetics of endogenous DA release/uptake in slices are that the amount of release shows a considerable regional and subregional heterogeneity, and that stimuli used to evoke the release also induce uncontrolled release of other neurotransmitters. The latter neurotransmitters can exert unpredictable effects on the DA kinetics. Furthermore, for experiments designed to probe the uptake mechanism, a method that allows for control of the initial DA concentrations would be desirable. Moreover, it has been noted that repeated electrical stimulations lead to decreases in the amount of DA release under some experimental conditions (e.g., Hafizi et al., 1992; Jones et al., 1995). For example, infusing increasing concentrations of cocaine over a long period of time (e.g., 2 h) can lead to decreasing amounts of DA released by single pulses (Jones et al., 1995). Variables responsible for this cocaine-associated decrease probably include DA uptake inhibition leading to loss of endogenous stores and synthesis inhibition via the synthesis-regulating autoreceptors. Participation by these synthesis-modulating autoreceptors is supported by the observation that sulpiride can partially prevent the decrease in the amount of released DA during long cocaine perfusion (unpublished observation). Another variable that limits the utility of the current FSCV methodology in studying DA regulation is that some of important DA terminal areas (e.g., prefrontal cortex) contain low concentrations of endogenous DA (Bull et al., 1991; Ganis and Wightman, 1995; Jones et al., 1994; Rice et al., 1994). In slice preparations, this limitation has necessitated the use of long, high-frequency, non-physiological stimuli to induce measurable DA release. In addition to theoretical considerations, this necessity for intense stimulation presents a practical problem in that these stimuli interfere with voltammetric measurement of DA by changing the local extracellular environment (e.g., changes in pH, [Ca’+ I, or the extracellular ‘volume fraction’; Jones et al., 1994; Rice and Nicholson, 1995; Rice et al., 1994). To circumvent the problems associated with nonphysiological induction of endogenous DA release, a number of investigators have examined clearance of exogenous DA applied locally by either micro-iontophoresis or pressure application (Cass et al., 1993; Nicholson, 1995; Rice and Nicholson, 1995). For example, Nicholson and his colleagues have presented elegant quantitative analyses of DA clearance from a well-defined source deposited microiontophoretically in the caudate nucleus (see Rice and Nicholson, 1995). Gerhardt and his colleagues have also utilized local pressure application to examine regulation of

Methods

67 (19961221-231

RorR’=H, 01;

C02H

Caged-Dopamine

NO2 CNB Oroup

(6)

B

XA = dopamlne

Aei-nltro intermediate

o-NPG

Fig. 1. (A) Chemical structure of CNB caged-DA, trifluoroacetate salt (compound 6). (B) Synthesis of CNB caged-DA. (C) Proposed photolysis mechanism for CNB caged-DA. See text for details. rds. rate-determining step.

DA clearance in various DA terminal areas (see Gerhardt, 1995). The present experiments develop the methodology that allows a selective, local application of DA without using micropipettes and a simultaneous measurement of its physiological effects and clearance. We have synthesized a ‘caged’ form of DA by linking the parent compound covalently to a ‘caging’ moiety (a-carboxy-2-nitrobenzyl-; see Fig. 1). This ‘inactivated’ form can be bath-applied without producing physiological/pharmacological effects, until a focal UV illumination breaks the covalent bond (‘photolysis’). The technique of UV-induced photolysis has been previously used to characterize physiological/pharmacological responses to other effecters such as ATP, Ca’+, agonists and antagonists (Katz and Dalva, 1994; Walker et al., 1993; see Adams and Tsien, 1993 for review).

2. Materials

and methods

2.1. Arlinzals Male Sprague-Dawley rats (250-300 g, Charles Rivers. Raleigh. NC) were cared for in accordance with the Guide for Care and Use of Laboratory Animals (NIH publication 865-23. Bethesda. MD). Food and water were provided ad libitum. 2.2. Swtlwsis

ert’.

of CNB-raged DA (Fig. I I

Dopamine hydrochloride (RBI, Natick, MA) was converted to its N-P-(trimethylsilyl)ethoxycarbonyl derivative (1) by treatment with the chloroformate of 2-trimethylsilylethanol in acetonitrile (Fig. 1B; Carpino and Lsao, 1978). For 1: ’ H-NMR (CD,OD) 6 6.62 (m, 2H), 6.51 (d, IH). 4.13 (t, 2H). 3.20 (t. 2H). 2.49 (t. 2H), 0.93 (t, 2H), 0.05 (5, 9H). The resulting carbamate 1 was alkylated on either of the phenolic hydroxyl groups with the 2-trimethylsilyl ester (2) of 2-bromo-o-nitrophenylacetic acid (BNPA) in acetonitrile. mediated by potassium carbonate. The ester 2 (‘H-NMR (CDCI,) 6 8.04 (m. 2H). 7.70 (t. IH), 7.55 (t. IH), 6.08 (s. 1H). 4.30 (m, 2H). 1.05 (m, 2H). 0.04 (s, 9H)) was prepared by dicyclohexylcarbodiimide (DCC)mediated coupling of BNPA (Chang et al., 1995) and 2-trimethylsilylethanol in acetonitrile. The alkylation reaction gave two products. The first product (3) was a mixture of monoaikylated bis-protected phenolic ethers formed in 41 Q yield: ’ H-NMR showed that the ratio of the two regioisomeric mono-ethers in 3 to be 1: 1, as determined by the relative integrations of the dopamine methylene signials at 3.4 and 2.7 ppm. For 3: ‘H-NMR (CDCI,) 6 8.1 (d. lH), 7.88 (d. IH). 7.74 (m. IH), 7.60 (t. IH), 6.95 (d. IH), 6.8 (m. 2H). 6.30 (two s. IH). 4.6 (br s, IH), 4.24 (m. 2H). 4. IS (m. 2H), 3.4 (two m. 2H, I:1 integration ratio). 2.7 (two t, 2H. 1: 1 integration ratio), 0.96 (m, 4H), 0.08 (s. 1XH). The second product from the alkylation reaction was formed in 36% yield. and ’ H-NMR analysis showed it to be the mono-protected carbamate (4) in which the 2-trimethylsilyl ester protecting group had been cleaved. Again. NMR analysis showed a 1: 1 mixture of regioisomers. For 4: ‘H-NMR (CD~~D) 6 7.90 (d. iH). 7.70 (t. iH), 7.5 (m. 2H). 7.8 (m. 3H). 6.5 (two s. IH), 4.08 (m, 2H), 3.20, 3.10 (two t. 2H. 1: 1 integration ratio), 2.63, 2.52 (two t. 2H, 1: 1 integration ratio). 0.92 (m, 2H). 0.07 (s, 9H). Thin-layer chromatographic (TLC) analysis showed that 4 was not the cyciized lactone 5: i.e.. treatment of 4 (R, 0.15, chloroform/methanol/acetic acid 50:5:1) with DCC in dichloromethane generated the much less polar 5 (R, O.84,chloroforn~/methanol/acetic acid 50:5:1) and dicyclohexylurea. Treatment of either 3 or 4 with trifluoroacetic acid (TFA) in dichloromethane at room temperature rapidly afforded CNB-caged DA (6) as its TFA salt in 6O-70% yield. after the compound was purified by chromatography on Sephadex LH-20 using water as eluant.

Again, ’ H-NMR analysis indicated a 1: I mixture of regioiSomers. and this mixture was subsequently used in biological experiments. The photochemical properties of the two isomers are expected to be identical. For 6: ’ H-NMR (D,O) 6 8.10 (d. IH). 7.79 (m, ZH). 7.65 (m. IH). 7.85 (m, 3H). 6.69 (two s. 1H). 3.2 (two m. 2H. I: 1 integration ratio). 2.8 (m. 2H): “F-NMR (D,O) @ 73.3: IR (KBr) 1627 cm ‘. Anal. Caicd for C,,H ,hNZOh . 1/2CF,CO,H: C. 52.91; H. 4.33: N. 7.30. Found: C. 52.98; H. 4.73; N. 7.53. The in vitro photochemical properties of 6 were not quantified. However, TLC experiments showed that photolysis of 6 (R, 0.85. methanol/chloroform/water/acetic acid 13: 10:4:0.2) with a UV handlamp at 254 nm for 30 min cleanly generated free DA (R, 0.70. methanol/chloroform/water/acetic acid 13: 10:4:0.2) and a non-polar side product, presumably o-nitrosophenylglyoxylate (oNPG: vide infra). The photolysis rate and quantum yield for 6 are expected to be similar to an analogous compound, o-CNB-caged phenylephrine, a biogenic amine caged on its phenolic hydroxyl group with the CNB photolabile group (Walker et al., 1993). Its quantum yield was reported as 0.28 upon irradiation at 300-350 nm. and it\ photolysis rate was 1980 ss’ (Walker et al., 1993). The photolysis mechanism of compounds caged as onitrobenzyl derivatives is generally accepted as proceeding through an aci-nitro intermediate, which is generated on the order of nanoseconds upon UV photolysis (Fig. IC; Walker et al.. 1988). The rate of agonist production is directly proportional to the rate at which the aci-nitro intermediate decomposes. in a dark reaction. to release free agonist and an o-nitrosobenzoyl side product. The CNB variant of o-nitrobenzyl caging groups not only affords generally the fastest photolysis rates and highest quantum yields, but it also affords the binlogicaliv inert photobyproduct o-NPG (Fig. IC).

Carbon-fiber microelectrodes ( t- = 5 p,m. Thornel P-55. Amoco, Greenville. SC) were prepared as described before (Kawagoe et al.. 1993). The electrodes were dip-coated with Nafion (2.5% w/v in isopropanol) to exclude anions from the electrode surface (Gerhardt et al.. 1984). FSCV employed either an EI 400 potentiostat in the ‘two-electrode’ mode (Ensman Instrumentation. Bloomington, IN) or an Axoclamp 2-B amplifier in the ‘single-electrode continuous voltage clamp’ mode (Axon Instrument. Foster City. CA): the Axoclamp was modified to increase the external voltage command compliance ( 1 120 mV/V external command. instead of k20 mV/V). An Ag/AgCi electrode was used as a reference. Either the Internal triangle command generator (El 400) or external D/A voltage input (Axoclamp) was used to scan the electrode potential linearly from -400 to + 1000 mV at 350 V/s every 100 ms (i.e.. sampling rate = IO Hz) To determine

224

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DA concentration versus time plots, the current at the peak oxidation potential in each voltammogram (+600 mV, current ‘integrated’ between 500 and 700 mV) was converted to concentration based on post-calibration of the electrode with DA in PBS. Background-subtracted vohammograms were constructed by subtracting the baseline current. Electrochemical treatment of the working electrodes sharpened the DA oxidative peak (probably by increasing the heterogenous electron transfer rate and/or promoting DA adsorption to the electrode surface; McCreery, 1995). This electrochemical modification was accomplished by superimposing an externally supplied 500 mV DC voltage step with continued application of the usual triangular voltage command (i.e., cycling at 10 Hz between + 100 and + 1500 mV instead of the usual - 400 and + 1000 mV); the duration of the treatment was 60 s. A distinctive increase in the electrode capacitance was noted as evidenced by increased potential-switching current. More intense electrochemical treatments such as that utilized by Gonon et al. (1981) were not used becauseof the tendency to promote excessive DA adsorption (see Gonon, 1995). While very useful for ‘slow’ (e.g., minutes) voltammetric techniques such as differential pulse voltammetry, DA adsorption markedly slows down the electrode response time, rendering the electrodes unusable for ‘fast’ (e.g., seconds) voltammetric techniques such as FSCV. Our electrochemical treatment protocol thus representsa compromise between enhancementof the sensitivity/selectivity of the electrodes and excessive decrease in their ‘frequency response’due to DA adsorption. The DA current measuredby our electrodes reached its peak within l-2 voltammetric measurementsof electrical stimulation (i.e., 100-200 ms); these values compare favorably with those reported in the literature (e.g., Kawagoe et al., 1993; seeSection 3 for further discussion). 2.4. In L.&-Orecording Coronal slices (400 pm thick) containing either the anterior caudate nucleus (approx. 2 mm anterior to the bregma) or prefrontal cortex (w 2.7 mm anterior, Paxinos and Watson, 1986) were prepared in ice-cold artificial cerebrospinal fluid (ACSF) using a vibratome (Technical Products International, Inc., St. Louis, MO). The composition of ACSF was (in mM): 124 NaCl, 1.0 KCl, 1.24 KHzPO,, 1.3 MgSO,, 26 NaHCO,, 2.4 CaCl,, and 10 glucose, saturatedwith 95/5% OZ/COZ. Following a I-2 h. recovery period in ACSF at room temperature, single slices were transferred to a submersion-type recording chamber mounted on a fixed-stage Nikon Optiphot microscopeand perfusedwith the control medium (33.5 + 0.5”C) at 1.5-2 ml/min. A minimum of 20 min of control perfusion was allowed before beginning voltammetry assessment. Caged-DA (150-200 PM) was dissolved in ACSF the same day of the experiments except for 2-3

Methods

67 (1996)

221-231

experiments, for which l-day-old solution that had been stored at 4°C was used with identical results. Because of the limited quantity of caged-DA, the solution was recirculated through the system using two peristaltic pumps; typical experiments lasted l-2 h. The carbon-fiber working microelectrode was inserted 75 pm below the surface of the slice; electrode placements were made with the aid of the Nikon microscope. In some experiments, a bi-polar stainless-steelstimulating electrode (Plastic Products, Roanoke, VA) was position on the slice surface 100-200 km from the carbon working electrode. Electrical stimulation consisted of biphasic, constant current (350 pA) pulses, 2 ms each phase; the stimuli were computer-generatedand output via a linear-responsestimulus isolation unit (Isolator- 10, Axon Instrument, Foster City, CA). Only single pulseswere used throughout these experiments. The DA clearance was analyzed using the exponential equation - d[DA]/dt = k[DA], as describedpreviously by Garris and Wightman (1995). Stamford and his colleagues have also utilized ‘half-life’ of DA clearance(estimated by 0.693/k) to characterize endogenous DA kinetics (see Stamford et al., 1995). A more analytical approach, based on Michaelis-Menten kinetics (e.g., Jones et al., 1995; Nicholson, 1995; Lee et al., 19961,was not attempted in this initial study, especially considering that the ‘mild’ electrochemical treatment of the electrodes, while enhancing their sensitivity/selectivity, also leads to a discemable reduction in their frequency responses(see Section 3). 2.5. Photolysis Caged-DA was photolyzed using an epifluorescence attachment on the Nikon microscope. A 100 W mercury lamp was used as the light source; the following modifications to standard attachment were made (Fig. 2). First, an Uniblitz@ shutter (Vincent Associates, Rochester, NY) was mounted between the lamp housingand the main body

Excitation Filter (Removed)

Field Diaphragm ,-

-

\

\/

‘; .’ \

/ ‘!

I

L&.4

’

100 W Hg Lamp

i’ c/

1 g L Electronic Shutter

L 40x Objective

Fig. 2. Utilization DA.

of epifluorescence

microscope

for photolysis

of caged-

to control the UV exposure timing and duration via a computer-controlled shutter driver/timer (Model Tl32, Vincent Associates). Second. the excitation filter in the tluorescence filter cube (UV-2A. Nikon) was removed in most of the experiments to maximize the amount of light reaching the slice. A long working distance objective (40 X . N.A. = 0.4. Nikon) was usedto focus the light and IO place electrodes in the center of the illumination. A 5 X objective was used to confirm the placement within specific terminal areas (i.e., caudate nucleus or prefrontal cortex). The field diaphragm for the epifluorescence attachment was closed to produce an illumination focus size of 500 pm. In some experiments. the effects of the UV illumination itself on recorded current were compared to higher wavelength illumination by using either the UV-2A excitation filter or G- 1B rhodamine filter cubes.

illumh-ration. That is. the general shapeof’ the background shift was that of the applied voltage (AZ= t//AR, ‘triangular-shaped’; Fig. 4A. ‘ACSF UV on’). The overall shape of the background shift was similar to that encountered during background cycling of ‘bad’ working electrodes, which typically show a pronounced resistive component (e.g., compare Fig. 4A. ‘ACSF with UV on’ to Fig. 4 in Stamford et al., 1995). Importantly. no distinctive current ‘humps, . which could interfere with DA oxidanve peak current (i.e.. u 400-800 mV) were noted. Summation of the UV-induced background shift in ACSF with the signal due to endogenousDA release(Fig. 3A, ‘EndogenousDA. UV off’) yielded a voltammogram, which was very similar to that after photolysis of caged-DA (Fig. 4A. ‘Caged-DA’

A

/

3. Results and discussion

Caged-DA gave rise to a distinctive shift in the baseline voltammetric signal. Fig. 3A shows a background-subtracted voltammogram (i.e.. ‘ACSF + caged-DA’ ‘ACSF’) for 200 p,M solution; a voltammogram for 5 FM DA is overlapped for a direct comparison. Single-pulse electrical stimulation of caudate slices in ACSF or cagedDA solution induced endogenous DA release (Fig. 3B). The peak concentrations of i--Z pM were achieved within 150-250 ms of the stimulation with a rapid clearance(l-2 s) of DA from the extracellular space. The backgroundsubtracted voltammogram at the peak DA concentration is shown above the temporal trace. Photolysis of caged-DA t 100-200 ms UV illumination) also produced rapid changes in measuredDA current at t- 600 mV (Fig. 3C); voltammogramsfor the time interval indicated by the arrows (caged-DA. ‘UV on’ - ‘UV off) are al$o shown above the temporal trace. Similar to the time required for peak detection following electrical stimulation (i.e.. within 150-250 ms of the single stimulus). the W-induced current change reached its peak within IOO200 ms of turning off UV illumination. In addition to DA current. the voltammograms above the trace also reveal background shifts: this superimposing shift was responsible for the oxidation peak current not returning to the baseline. Following adjustment for the baseline shift, the DA oxidative current decreased to O-IO%, of the peak current (see belovv). The background shifts associatedwith photolysis were examined in PBS or ACSF (either outside or inside slices when using ACSF) to assessthe contribution from the high-energy UV illumination itself. This evaluation suggested that a major contributor to the overall background shift during caged-DA photolysis was an apparent decrease in the resistanceof the working electrodes induced by UV

200 pM Caged-DA vs 5 pM DA

6

Endogenous DA

Caged-DA Fig.

3. (A)

Background

(AUF)-subtracted

voltammogram

caged-DA. Voltammopram for 5 &M DA is overlapped (B) Endogenous caudate DA release evoked by electrical the time represented by the square. (C) Photalysis-induced

for

-+ -400 mV at 350 V/s) above the temporal traces.

for

times

indicated

FM

stimulation ‘release’

DA from caped-DA (UV illumination between the two squares). prolonged elevation of DA -concentrations‘ caused by background in B. Background-subtracted voltammograms (voltage command: -+ + 1000 are shown

XU

for comparison.

hy the

at of

Note

the shift5 - JO0

arrows

226

TX.

Lee et al. /Journal

of Neuroscience

following UV illumination). More importantly, the resulting voltammogram could be reproduced experimentally by turning on UV illumination while electrically stimulating the slice for endogenous DA release (Fig. 4A, ‘Endogenous DA, UV on’). Considering the UV-induced background shift displayed a pronounced resistive component, it was compensated by subtracting the linear trend in the background-subtracted voltammogram for each determination (Fig. 4B). This is similar to the background adjustment technique used to resolve overlapping peaks in chromatograms. This simple linear correction method compensated for most of the prolonged elevation in oxidative current associated with photolysis. Thus, identical results for endogenous DA release/uptake were obtained, when the same locations in the caudate slices were stimulated before and after (within l-2 s of) UV exposure (see Fig. 6A). Physiologically, the unaltered release and clearance of endogenous DA over repeated photolysis is consistent with the interpretation that exposure to UV per se has no effect on the kinetics of release/clearance of DA and, moreover, on the viability of slice preparations. It is unlikely that UV illumination would simultaneously alter the electrode sensitivity and endogenous DA release so as to yield similar current magnitudes during UV exposure. The utility of our linear correction method was further verified by finding that it could correct for the baseline shift during UV illumination of solution containing a known concentration of DA and 2-nitromandelic acid (Fig.

A

r-4-‘L

b-J

Methods

67 11996) 221-231

4A). The latter solution yields the same end products as photolyzed caged-DA (i.e., o-nitrosophenylglyoxylate and DA; see Fig. lB, ‘X’ = ‘OH’ on 2-nitromandelic acid). Repeated UV illumination over a 1 h period in either one of the solutions did not alter the measurement of DA current. These findings are consistent with the hypothesis that the amount of DA released from caged-DA can be estimated using this background subtraction method. In contrast, shifts observed with changing pH or [Ca*+ I are associated with distinctive peak ‘humps’ that interfere with detection of DA (Jones et al., 1994; Rice and Nicholson, 1995). Although a more rigorous qualitative assessment would be needed to characterize fully the background shift induced by UV per se, a likely mechanism underlying the background change is the photoelectric effect induced by the high-energy illumination. In support of this view, less energetic light (green light) failed to induce a similar current shift, while filtering illumination with a UV excitation filter still produced the characteristic shift, albeit smaller in magnitude (data not shown). 3.2. Assessment of DA clearance in the catidate nucleus and prefrontal cortex

The clearance of photo-released DA and uptake inhibition were compared between the caudate nucleus and prefrontal cortex; these regions exhibit different uptake kinetics (Garris and Wightman, 1995). The number of determinations from slices prepared from 4-5 animals is

Caged-DA

following

UV-illumination

\/ ACSF

CNB

+ DA (3 IJM) UV on

with UV on

CNB

$4~3

Endogenous

pM)

B.G.

DA

“““u;

Endogenous UV on

oD,A-‘~3$4

DA

CNB

UV on

correction C

Fig. 4. (A) UV illumination per se induces a voltammogram background shift similar to that observed during photolysis of caged-DA. This shifts accounts for much of the changes observed during measurement of DA after caged-DA photolysis. See text for details. (B) Following a linear subtraction of the W-induced background shift, DA concentrations after caged-DA photolysis decrease to 0- 10% of the peak concentration.

T.H.

Lee er al./Jnurnaf

~fi\ieuroscienre

Metlmd,~

presented in parentheses for each group. As shown in Fig. SA for a representative experiment, the baseline clearance rate was slower in the prefrontal cortex (Fig. 5A; k = 1.3 + 0.2 (231 vs. 0.70 + 0.14 SK’ (121, mean f SD, p < 0.0001). In addition to these two regions, clearance was measured in the corpus callosum which should be devoid of any DA uptake sites. Indeed, this white matter exhibited the slowest clearance rates among the three areas examined in this study (data not shown); diffusion probably plays the primary role in the removal. The sensitivity of DA uptake to its inhibitors were assessedusing cocaine. This drug at 5 p.M clearly decreasedthe DA clearance rate in both the caudate nucleus (I .3 + 0.2 (23) vs. 0.88 + 0.17 (8) s-l, p < 0.0001, r-test> and prefrontal cortex (0.70 + 0.14 (12) vs. 0.54 & 0.08 (7) SC’, p < 0.02, r-test). The extent of caudate clearance inhibition by cocaine is similar to the voltammetric data obtained in caudate slices using endogenous DA release (e.g.. Jones et al.. 1995, 1996). A full concentration/responsedetermination is planned for a quantitative analysis. Since cocaine is also a potent uptake inhibitor for other monoamines.the reduction in the DA uptake rate observed

67 CIYY6)

-._ “7

_7_71-31

with cocaine might be partly mediated by its effects on noradrenergic and/or 5-HT terminals (i.e., reduced ‘heterologous’ uptake). This confound could be expected to be exacerbatedin the prefrontal cortex, which possesses a less efficient DA uptake mechanismas well as greater innervation by the other monoaminergic terminals (see Weiss et al., 1995). The extent to which 5-HT and noradrenergic terminals contribute to DA clearance was examined at 3 sites(each terminal area) by comparing DA clearancerates before and after perfusion with clomipramine and desimipramine (5HT and noradrenergic uptake inhibitors, respectively). Clomipramine and desipramine(5 PM each) had a minimal effect on DA clearance in the caudate nucleus; on the other hand. they decreasedthe clearance rate in the prefrontal cortex (0.90, 0.68, 0.85 (pre-) vs. 0.67. 0.53. 0.70 sm.’(post-inhibition)). Fig. SB shows data for single experiments for the two terminal areas: as shown, a subsequentaddition of cocaine (5 FM) to the circulating solution clearly reduced DA clearance rates both in the caudate nucleus and prefrontal cortex. The sensitivity to clomipramine and desipramine in the prefrontal cortex is consistent with the idea that S-HT and

A 2-

1

2

3

4

5 TIME

6

7

8

PREFRONTAL

->-f -+

1

1 TIME

10

(SEC.)

CAUDATE

0

9

2 (SEC.)

3

0

CORTEX

BASELINE DESIP + CLOM DESIP + CLOM + COC’ .-.

1 TIME

2 (SEC

3 )

Fig. 5. DA clearance and its inhibition in the caudate nucleus and prefrontal cortex for single representative experiments. (A) The two DA terminal areas exhibit differential clearance rates for photo-released DA. (B) Effects of uptake inhibitors on the DA clearance rates. Combination of desipramine and clomipramine (5 CM each) selectively reduces the clearance in the prefrontal cortex. Subsequent addition of cocaine (5 p,M) markedly reduces the clearance in both the caudate nucleus and prefrontal cortex (all DA concentrations return to similar levels. i.e., 0- 10% of the peak).

T. H. Lee et al. /JoumaI

228

of Neuroscience

noradrenergic terminals map play significant roles in clearing extracellular DA in this region (see Weiss et al., 1995). The difference in the clearance rates between the caudate nucleus and prefrontal cortex was smaller than those reported by Garris and Wightman (19951, who also utilized nafion-coated electrodes. We hypothesize that this smaller clearance difference is due to a reduced frequency response of the working electrodes, which resulted from their electrochemical treatment; for example, an increased DA adsorption to the electrode surface would reduce the signal offset rates. The slowed response of the electrodes is expected to have a relatively greater effect on the faster kinetic events, i.e., uptake in the caudate nucleus. In support, it is noted that the prefrontal clearance rate measured in the current study is close to that reported by

Methods

67 119961221-231

Garris and Wightman (1995) in vivo. We are currently in the process of quantifying the frequency response of our electrochemically treated electrodes using a ‘flow injection’ system (Kristensen et al., 1986), as well as assessing a possibility of eliminating the electrochemical treatment step in our methodology. 3.3. Inhibition of endogenous photo-released DA

caudate DA

release by

The physiological effect of photo-released DA was assessed by its ability to modulate stimulated DA release from the caudate nucleus (5 slices from 4 animals; Fig. 6B shows data from a representative experiment). Increase in DA oxidation current induced by single-pulse electrical

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Time (set) Fig. 6. Inhibition of endogenous DA release by photo-released DA (data from single experiments). (A) UV illumination during perfusion with regular ACSF produces no effect on endogenous DA release. (B) Photolysis of caged-DA inhibits endogenous DA release. The inset illustrates how DA release * voltammogram immediately before electrical was estimated. * * Peak voltammogram after endogenous release by a single electrical stimulation: DA voltammogram, from which the amount of endogenous release is estimated (‘Endogenous stimulation. Subtracting ’ from ’ * yields the characteristic DA’, solid line). The peak voltammogram observed in the absence of photo-released DA is overlapped in dotted lines to demonstrate a - 30% inhibition.

stimulation (i.e.. endogenous DA release) was first measured with UV illumination off (Fig. 6B, ‘UV off’); 2-3 min later, the slice was again stimulated, this time l-2 s after UV-induced photolysis (Fig. 6B. ‘UV on’). The amount of endogenous release was estimated by subtracting the DA current immediately before the stimulation from the subsequent measurements (see inset, Fig. 6B). Photo-released DA (0.9-2.4 pM peak concentrations) reduced the peak height of the endogenous DA release by 12-30% (19 &- 6% (8). mean f SD); recovery back to pre-photolysis release (> 9.5%) was observed within IO min. Thus. inhibition of endogenous DA release by a rapidly applied (100-200 ms) exogenous DA could be readily determined by FSCV. Importantly, because the application was highly localized, inhibition of endogenous dopamine release by extraceliular DA could be measured at multiple sites in single slices. This ability would improve the confidence in the measurements. Sulpiride at 2 p,M prevented the inhibition by photolyzed cage-DA, indicating that the inhibition was indeed due to stimulation of terminal autoreceptors modulating terminal DA release (n = 2; 92 and 106% of ‘UV off’ measurements). The extent of autoreceptor-mediated inhibition by photo-applied DA (12-30s) compares well with a previous result that, in a 2-pulse electrical stimulation paradigm (pulses 100 ms apart), DA release evoked by the second pulse is inhibited - 15% via autoreceptor stimulation by 1-2 pM DA released by the first (Kennedy et al., 1992). The weak release inhibition in both studies is not surprising as levels of 50-100 p.M are typically required for full autoinhibition of impulse flow (Pinnock, 1983; Lee et al., 1993). A more powerful illumination source such as a laser would likely overcome this limitation (Katz and Dalva, 1994). 3.4. Main adcanrages

of the phatolysis method&q?:

A number of investigators have previously examined clearance of exogenous DA applied locally by either micro-iontophoresis or pressure application (Cass et al., 1993; Rice and Nicholson, 1995). Compared to these techniques, photolytic release of caged-DA provides improvement in the time for application and detection of exogenous DA. With our methodology, extracellular DA concentrations greater than 1 FM can be reached within 100-200 ms, compared to tens of seconds or more for typical microiontophoretic application. The time resolution is further enhanced by focusing the UV illumination at the center of the working electrode, thus. achieving the highest DA concentration at its immediate vicinity (i.e., spatial resolution). This strategy minimizes the problem inherent in micropipette techniques (both micro-iontophoresis and pressure application), namely a time delay between DA application and peak achievement at the recording sites due to the diffusional barrier between the micropipette and voltammetric electrode, which are typically ICKJ-200 km apart. Under the latter conditions, DA diffusion into the

recording site (down the concentration gradient) becomes a significant, if not the rate-limiting, factor in detecting concentration changes; non-linear equations containing both diffusional and uptake parameters are required for a quantitative assessment (Nicholson, 1995; Rice and Nicholson, 1995). On the other hand, the abihty to achieve an ‘instantaneous’ concentration peak (i.e.. time resolution) at the center of the working electrode (i.e.. hpatial resolution) allows one to use simpler equations. containing only clearance parameters. It is noted that the delay of 100-200 ms between UV offset and detection of the peak DA oxidation current in our methodology is likely due to the nafion coating (Kawagoe et al., 1993). With regard to spatial resolution of our methodology, we adjusted the illumination size to approx. 500 Pm in diameter by closing down the field diaphragm; this seemingly large size was chosen in order to expose the working elliptical electrode (- 20 Frn, major diameter) to a DA bolus with an ‘infinite’ diameter (i.e., > 20 km). This strategy is another means of ensuring that the electrode is not exposed to a significant DA concentration gradient nearby, so as to complicate the clearance analysis (see above). In our mercury-lamp system. consistent photolysis could be obtained with a focus size of + 250 pm. With an appropriate illumination system and optics (e.g.. argon laser with a high-power/NA objective), reliable illumination of slice areas smaller than 20 pm in diameter (even after considering light scattering by the tissue) has been reported (Katz and Dalva. 1994). Another important advantage of caged-DA utilization is that reproducible concentrations of extracellular DA can be achieved regardless of the endogenous content within given areas. Thus, we can directly compare DA uptake rates between the prefrontal cortex and caudate nucleus with similar initial extracellular concentrations; the same technique could be used to examine DA uptake mechanisms in other areas such as the basolaterai amygdala. substantia nigra and ventral tegmental area. These areas all present practical problems because of their low DA content. Previously, these areas required using either non-physiological depolarizing stimuli (e.g., elevated [K’], veratridine. or multiple high-frequency electrical stimulations; Jones et al.. 1994; Rice et al.. 1994) or local pressure/micro-iontophoretic DA application (Cass et al.. 1993: Rice and Nicholson. 1995). As discussed above, background current shifts during non-physiological stimulation interfere with DA peak current measurement. Although background shifts were also observed with our technique, the critical difference is that these shifts produce minimal interference with DA peak measurement. The ability to achieve consistent DA concentrations with minimal disturbances in the endogenous DA stores or interference with DA measurement offers significant improvements over previous techniques using endogenous DA release. The advantages over micro-iontophoretic or pressure-application techniques have been discussed (see above).

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One of the disadvantages of the photolytic methodology described here is that it cannot be used for in vivo studies; thus, it suffers from the usual short-comings found with slice preparations (e.g., results not necessarily reflective of the intact brain). However, to the extent that these preparations have been successively used forvoltammetric examination of DA release and clearance regulation (Bull et al., 1990, 1991; Jones et al., 1995, 1996; Lee et al., 1996; Kennedy et al., 1992), the photolytic methodology should enhance our ability to study the DA system and its regulations. In the present study, FSCV was chosen not only for the increased sampling rates (e.g., every 100 ms) but also to provide characteristic redox currents profiles. Thus, it was possible to examine and compensate for non-specific changes in the background current. A previous study has demonstrated the advantage of FSCV when there are possible non-specific alterations in the measure current (e.g., Jones et al., 1994).

4. Summary In the present paper, we have described the use of caged-DA to examine DA release and clearance in rat brain slice preparations. Compared to previously available techniques, the main advantages include: (1) a rapid rise in extracellular DA concentrations, which is secondary to the inherent photolysis kinetics and minimization of extracellular diffusion; (2) a reproducible level of initial DA regardless of the endogenous DA content; and (3) a combination of excellent temporal and spatial resolutions, allowing for multiple measurements in single preparations. This methodology allows rapid and consistent assessment of DA clearance uptake mechanisms as well as autoreceptorregulation of endogenous DA release (using FSCV) or DA impulse-flow (using single-unit recording).

Acknowledgements This research was supported by the National Institute on Drug Abuse (DA-06519). We would like to thank Drs. L. C. Katz and R. Mark Wightman for helpful comments and Ms. C. Calvi and W.-Y. Gao for excellent technical assistance.

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