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Brain Research 882 (2000) 217–220

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Short communication

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Activation of ATP receptor increases the cytosolic Ca

concentration

in nucleus accumbens neurons of rat brain

*

Masaru Sorimachi , Takashi Moritoyo, Kazuhiko Yamagami

Department of Physiology, Kagoshima University, Faculty of Medicine, Kagoshima 890-8520, Japan

Accepted 8 August 2000

Abstract

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ATP increased the cytosolic Ca concentration ([Ca] ) in nucleus accumbens neurons acutely dissociated from rat brain. The ATPi

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response was dependent on external Ca and Na , and was blocked by voltage-dependent Ca channel blockers. The results suggest

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that the ATP-induced depolarization increases Ca influx resulting in the increase in [Ca] .i  2000 Elsevier Science B.V. All rights

reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Other neurotransmitters

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Keywords: ATP; Fura-2 microfluorometry; Ca channel; Nucleus accumbens; Purinoceptor

The nucleus accumbens (NAc) has been considered to neurons express P2 type receptor, which is less permeable 21

function in promoting locomotion, in motivation, be- to Ca .

havioral drive, and reward [10,16,24] including the rein- The neurons of NAc were dissociated from 15- to forcing effects of addictive drugs [18], and also in the 23-day-old Wistar rats, which were decapitated under ether pathogenesis of schizophrenia [1,4,21]. The NAc neurons anesthesia after the study protocol was approved by the receive excitatory glutamatergic inputs, which may be Committee on Animal Experimentation, Faculty of Medi-modulated by a wide variety of endogenous neuro- cine, Kagoshima University. The brain was quickly re-modulators [2,11], from the cortical and subcortical limbic moved from the skull and placed in ice-cold saline area [7,8]. For example, dopamine depresses excitatory containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl , 1 mM2 synaptic transmission [13,14,22,23] and thus has been MgCl , 10 mM HEPES and 5.5 mM glucose, the pH of the2 assumed to contribute to the behavioral and rewarding solution was adjusted to 7.4 by adding tris(hydroxy-effects of psychostimulants. The principal medium spiny methyl)aminomethane (Tris-base). The brain was sliced at GABAergic NAc neurons are generally quiescent with a thickness of 400 mm with a microslicer (DTK-1000, highly negative resting potentials [15,28,29], and are Dosaka, Japan), and the core and shell regions of NAc therefore strongly dependent on excitatory input to gener- were micropunched using a sample corer (FST; internal ate output. However, little is known about other excitatory diameter of 0.5 or 1 mm). The tissue pieces were treated inputs. We now report that the excitatory transmitter with saline containing dispase (1600–2500 pU / ml;

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candidate ATP [3,25] increased the cytosolic Ca con- Goudoushusei, Japan) and DNase (0.02%; Sigma, type 1) centration ([Ca] ) in a subset of acutely isolated NAci for 45 min at 348C under continuous O2 bubbling, neurons, using fura-2 microfluorometry, and that these followed by further incubation in saline at room tempera-ture for 1 h. Finally, the tissues were gently triturated in saline containing 1% bovine serum albumin (BSA) using finely polished glass pipettes in a range of sizes, and the *Corresponding author. Tel.: 181-99-275-5227; fax: 1

81-99-275-dissociated cells were allowed to adhere to poly-L

-lysine-5231.

coated glass coverslips. Cells on the glass coverslip were E-mail address: ciliary@med2.kufm.kagoshima-u.ac.jp (M.

Sorimachi). loaded with 1 mM fura-2 acetoxymethyl ester (fura-2 /

218 M. Sorimachi et al. / Brain Research 882 (2000) 217 –220

AM), 0.1% dimethyl sulfoxide, and 0.2% BSA for 30 min synthetic enzyme for GABA, using the primary antibody at 378C. The coverslips were then mounted in a superfu- (Chemicon, USA).

sion chamber and placed on the stage of an inverted Although the NAc neurons were reported to be electri-microscope (Diaphot-TMD, Nikon, Japan). Cells were cally quiescent [15,28,29], we sometimes observed sponta-continuously superfused at a rate of 1 ml / min with neous fluctuations in [Ca] , which were markedly inhibitedi standard saline at 25–268C through a polyethylene tube, by adding 10 mM GABA (N57; data not shown). We the tip of which was placed 1–2 mm away from the cells. tentatively assumed that neurons in slice preparations are The changes in fluorescence ratios at 340 and 380 nm tonically inhibited by recurrent axon collaterals of other excitation wavelengths (F340 / F380) were measured using GABAergic neurons [5,17]. The addition of 10 mM a CAM-200 spectrometer (Jasco, Japan). The absolute glutamate increased [Ca] in most cells examined, buti value of [Ca] was calculated using the formula describedi about 34% of these cells responded to 100mM ATP (109 by Grynkiewicz et al. [12]: [Ca]i5b3K (Rd 2Rmin) / of 320 cells). The diameter of ATP-responsive cells ranged (Rmax2R). Calibration constants were determined in between 10 and 12.5mm, and these cells exhibited GAD-separate experiments with the same experimental set-up, as like immunoreactivity (32 of 32 cells; Fig. 1C), showing described previously [27]. Several points on the F340 / that purinoceptors are expressed on the GABA-containing F380 ratio tracing were selected, and the values of [Ca]i neurons.

are shown on the ordinates in the figure. After measuring As shown in Fig. 1A, the response to 10–500mM ATP the ATP-induced [Ca] increase, cells on a coverslip werei was compared with 100mM ATP, and the dose–response fixed with 4% (v / v) paraformaldehyde in 0.1 M phosphate relationship was obtained (Fig. 1B). Both 2-methyl-thio-buffer for 15–30 min at 48C, followed by the conventional ATP (2MeS-ATP) and ATPgS were effective in increasing immunostaining of glutamate decarboxylase (GAD), a [Ca]i (Fig. 1B), but neither a, b-methylene-ATP nor

Fig. 1. [Ca] increases induced by various concentrations of ATP and its analogues. (A) [Ca] increases induced by 10, 20, and 50i i mM ATP were compared with that by 100mM ATP. (B) Dose–response curves of ATP (s_{), 2-methyl-thio-ATP (MeS-ATP;}d_{), or ATP}gS (n_{). The mean of two responses to 100}

M. Sorimachi et al. / Brain Research 882 (2000) 217 –220 219

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b,g-methylene-ATP had any effect on [Ca] at a con-i Mn entry into chick ciliary ganglion cells [26] when centration of 200–500mM (N57 or 6, respectively). membrane depolarization was minimized by removal of

1

The ATP-induced [Ca] increase was dose-dependentlyi external Na , and considered that the purinoceptor chan-21

inhibited by P2 purinoceptor antagonists, suramin [9] and nels expressed are permeable to Ca . We therefore pyridoxal-phosphate-6-azophenyl-29,49-disulphonic acid employed the same manipulation to investigate the per-21 (PPADS; [20]) (Table 1). The ATP-induced [Ca] increasei meability of purinoceptor associated channels to Ca .

1 1

in the absence of external Na (replaced by NMDG ) was When the cells were stimulated for 1 min with 100 mM

1 1

only 562% (N55) of the mean of two controls at 150 mM ATP in a Na -free (NMDG ) solution containing 10 mM

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Na and completely nullified in the absence of external Ca , the response was only 1063% (N511) of the mean 21

Ca (N56). These results suggest that ATP increased of two controls obtained with 15 s ATP stimulation at 150

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Ca influx by depolarizing the membrane potential via mM Na . These results suggest that nonselective cationic 21

activation of P2 receptor type. channels on NAc neurons are less permeable to Ca .

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To determine the types of voltage-dependent Ca Previous studies have demonstrated that P2 receptors are channels involved, we investigated the effects of specific situated at both the dopaminergic terminals of ventral antagonists on the ATP response. Individual antagonists tegmental area (VTA) neurons and the glutamatergic for L-type (nitrendipine), N-type (v-conotoxin-M VIIA), terminals of corticofugal pyramidal neurons, and that these

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and P/ Q-type (v-agatoxin IVA) Ca channels caused receptors play an essential role in modifying the release of slight inhibitions (Table 1), the magnitudes of which were dopamine [19]. The present results add a new finding that comparable to that found in electrophysiological studies ATP acts on P2 receptors present on a subset of NAc [6]. The combined addition of three antagonists involving neuron as an excitatory neurotransmitter. The origin of

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10 M nitrendipine reduced the ATP responses to ATP has not been investigated in the present study, but it 21

2263% of controls (N510). Cd at 0.2 mM alone or in is possible that ATP is released from axon terminals of 21

combination with three Ca channel antagonists reduced VTA neurons, where it is co-stored with dopamine [19]. the responses to 1463% (N59) or 962% (N55),

respec-tively. The responses in the presence of 10 and 50 mM

D-600 (methoxyverapamil) were also reduced to 3665% _References (N56) and 564% (N53), respectively. On the other hand,

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