Neuroscience l.ellerx. 62 (1985) 231-235 23 I

Elsevier Scientific Publishers Ireland Ltd.

N S L 03663

y -AMINOBUTYRIC ACID A N D T A U R I N E RELEASE IN THE STRIATUM OF THE RAT DURING H Y P O G L Y C E M I C COMA, S T U D I E D BY MICRODIALYSIS

ULF TOSSMAN I *, T A D E U S Z WIELOCIf : and U R B A N U N G E R S T E D T I

i Dc,partment o! PharmacOlOLgy, Karolinska Institute. P.O. Box 60 400, S- 10401 Stockhohn, and :i+~lhor~llor l'

[i." t'.\perinwnla[ Brain Research, UniverL~ity O/Lund, Lend Hospital, S-221 85 Lend (Su'e&'n J

I Rcceived Aprit 25th. 1985: Revised version received September 4lh. 1985; Acceptcd September 5th. It#s<)

Key word~ taurme , '-aminobutyric acid microdialysis cxtracellular concentration hypoglyccmm rat

Extracellular levels of striatal 7-aminobutyric acid (GABA) and taurine were monitored during insulin-

reduced hypoglycemia using microdialysis. At the onset of isoelectricity m the electroencephalogram (EEC,), a transient 5-fold increase in the levels of G A B A occurred. Taurme levels increased 5 rain follox~-

ing the onset of isoelectricity and continued to increase during the entire isoelectric period. The result~

dcmonslratc that events associated with the onset of isoelectricity during hypoglycemia trigger an increase m extracellular concentrations of GAB A and taurine. The discrepancy in time-course of these changes may retlcct differences in compartmentat ion, function and metabolism of the two amino acids.

The microdialysis technique [17] offers the possibility to study the changes in the extracelluhtr concentrations of amino acids in the brain during various pathological situations, such as hepatic encephalopathy [16], kainic acid-induced seizures [6] or cerebral ischemia [3]. In the two later conditions, characterized among others by par- tial or complete membrane depolarization, a significant elevation of the extraccllular concentrations of amino acids were demonstrated.

The amino acids 7-aminobutyric acid (GABA) and taurine have demonstrated inhibitory and seizure-depressant properties [11, 14]. In the striatum, GABA has been identified as a transmitter of the striatonigral pathways but also in the recipro- cal nigrostriatal pathway and in interneurons [5, 12]. The function of taurine as a neurotransmitter is still controversial, although it is a major constituent of the free araino acid pool in the brain [I 1]. Based on lesion studies, taurine has been suggested to play a transmitter role in the striatum [10].

Insulin-induced hypoglycemia, when severe enough to extensively reduce blood glucose levels and dearrange brain energy metabolism, also induces membrane depo- larization [4, 13]. In the present investigation, we report on changes in the brain extracellular concentrations of GABA and taurine during hypoglycemia.

The study was performed on 8 male Wistar rats (30(~350 g). The animals were

*Author for correspondence.

<l~ ~l~ I~)R'~ FNevicr Scicnt i l ic Publ ishers I re land Ltd.

232

tracheostomized, immobilized with tubocurarine, and artificially ventilated on a res- pirator (for details see ref. 7). In the hypoglycemia group, the animals were injected i.p. with 40 IU of insulin (Actrapid; Novo Industries A/S). A femoral vein and artery were cannulated. Mean arterial blood pressure was continuously monitored, and blood pO2, pCO2, pH and glucose levels intermittently measured during the experi- ment. The animals were held in a stereotaxic instrument. The skull bone was exposed and the temporal muscles dissected out in order to expose the temporal bones, where bilateral craniotomies were made. A dialysis tube (Vita fiber, 3 x 50 Amicon, 0.3 mm diameter, molecular cut-off 50,000) was glued to a steel cannula and inserted hori- zontally (! mm anterior, 5.7 mm ventral from bregma) through the brain with the aid of the micromanipulator on the stereotaxic instrument. The dialysing part of the fiber (7 mm) was centered in the striatum, and the cannula was fixed to the skull bone with dental cement. The fiber was perfused with a Ringer solution (147 mM Na ~, 2.3 mM Ca 2+, 4 mM K + and 155.6 mM C1 , pH 6.0) with a microinfusion pump at a rate of 2/~l/min (for details see ref. 16). The perfusate was collected every 5 min. In the control group, the fractions were collected during a 2-h period. The fractions were immediately frozen and later analysed for GABA and taurine using a high-per- formance liquid chromatography system with fluorescence detection after a precol- umn o-phthaldialdehyde derivatization [8, 15]. The recovery values of the fibers were obtained by immersion of the fibers into solutions of known concentrations of tau- rine and GABA and perfusion with the dialysis solution. The calculated recovery values were 31~o for GABA and 51~o for taurine.

Statistical differences were calculated using a one-way analysis of variance (ANOVA) with Dunnett 's test.

During the whole experiment, the blood pO2 was kept at 100--120 mmHg, pCO2 at 35-40 mmHg and pH 7.41. Blood pressure was 120-140 mmHg. The normal levels of blood glucose in control animals are 7 #moi/g. At the onset of isoelectricity, the blood glucose was 0.4_+0.2 pmol/g. The electroencephalogram (EEG) was contin- uously monitored via gold-plated screws positioned in the skull on each hemisphere. During hypoglycemia, the EEG showed a progressive slowing followed by a burst pattern with intermittent isoelectric periods preceding total electrical silence (Fig. 1). The EEG pattern and the progression of its changes did not differ from earlier reported findings [7].

The level of GABA in the perfusate of the control group (n =4 ) was !.06_+0.12 pmol/10 ktl dialysate. In the hypoglycemic group (n =4), the levels of GABA during the burst-suppresion period (Fig. 1 B) were not significantly different from control levels (Fig. 2A). In the fraction collected during 5 min following the onset of isoelec- tricity, a significant (P<0.01) increase in GABA levels was noted which amounted to 5.7__+1.8 pmol. In the following fractions, a slow decrease in concentration was noted, and after 15 rain the levels were not significantly different from control values.

The level of taurine in the perfusate of the control group was 40.6+ 2.7 pmol. In the hypoglycemic group, the levels were not significantly different from control levels during the polyspike period. Interestingly, the levels did not change during the first 5 min following the onset of isoelectricity (Fig. 2B). Thereafter, a progressive increase

233

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B

"1 C | 5 0 u V

m

I I

1 0 S

Fig. 1. Three different EEG recorded during insulin-induced hypoglycemia. A: control. B: burst pattern

with intermittent periods of isoelectricity. C: isoelectricity.

10.0

8 . 0

< m 6 . 0 <

o E 4 . 0

2.0

A I s o

L ~e

- 2 0 - 1 5 - 1 0 - 5 0 5 10 15 20

M i n u t e s f r o m i s o e l e c t r l c l t y

150

"E 1 0 0 = u

o E

50

B

I s o

1 ± J-

:!:i:i:i :?:!: :!:i:i:! i:i:i:

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M i n u t e s f r o m I s o o l e c t r l c i t y

Fig. 2. The changes of the extracellular levels of GABA (panel A) and taurine (panel B) during insulin-

induced hypoglycemia. The concentrations of GABA and taurine are given as pmol/10/A perfusate. The

dotted bar denotes the control levels of GABA and taurine, respectively, obtained from a control group.

Iso marks the fraction collected from the onset of isoelectric EEG. Values are mean_+ S.E.M. *P< 0.05,

** P < 0.01 denote statistical differences from the control group; Dunnett 's test.

234

in the levels occurred , reaching 160 + 15 pmol a l te r 20 min o f isoelectricity. With the-

known recovery values for the fibers, the s teady-s ta te levels o f G A B A and taur ine

in the s t r ia tum of the cont ro l g roup were ca lcula ted to be 0.2 and 9 ILM, respectively.

Hypog lycemia is charac te r ized by progress ive decrease in b lood glucose concentra-

tions [7]. With the onset o f isoelectrici ty, a rap id de te r io ra t ion o f the s teady-s ta te l e~

els o f labile phospha te c o m p o u n d s ensues, and ca tabol ic react ions are act ivated [ 13].

O f pa r t i cu la r interest f rom the present inves t igat ion is that isoelectr ici ty is associa ted

with m e m b r a n e depo la r iza t ion , i.e. an increase in ext racel lu lar po tass ium concentra-

t ions and a decrease in calcium concen t ra t ions [1, 4]. The m e m b r a n e depo la r iza t ion

associa ted with the observed electrical silence could thus trigger the reteasc of the

t r ansmi t t e r pool o f G A B A , observed as an increase in the G A B A concent ra t ion in

the perfusate. This is in agreement with the observa t ions that increased ext racel lu lar

levels o f po tass ium s t imula te the release o f both G A B A and taur ine [2, 9]. Dur ing

isoelectricity, the adenos ine t r iphospha te levels are still 30?0 o f cont ro l values [13],

suggest ing that reup take o f G A B A could prevail and thus explain the decrease in

G A B A levels in the later per iods o f isoelectricity. This seems par t i cu la r ly relevant

under condi t ions o f subs t ra te deficiency.

In contras t , the increase in the taur ine concen t ra t ion was first observed fol lowing

5 rain o f isoelectricity. This indicates that taur ine and G A B A are released from differ-

cnt synapses. Fu r the rmore , the levels of taur ine con t inuous ly increase dur ing the

isoelectric period. This could ei ther be due to an increased uptake from the blood,

a con t inuous release from presynapt ic sites, or a decrease in cel lular uptake. How-

ever, since the tissue level o f taur ine in the s t r ia tum does not change dur ing 30 min

of hypoglycemic coma [18], the lat ter two exp lana t ions seem more plausible.

Energy failure due to hypoglycemia occurs in o ther brain areas as well [13]. It is

thus p robab le that the increase in the ext racel lu lar concen t ra t ions o f taur ine and

G A B A dur ing hypoglycemic coma occurs in o ther brain areas as well.

This s tudy was suppor t ed by the Swedish Medical Research Counci l , (}rant 14X-

263, B85-14X-03574-14B, the N I U S P H S G r a n t 5 N01 NS07838. The au thors wish

to thank Mrs. Agne ta Eliasson and A n n e m a r i e Andersson for their skillful technical

assistance.

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