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Electroconvulsive shock increases interstitial concentrations of uric acid in the rat brain
BRAIN RESEARCH ELSEVIER
Brain Research 660 (1994) 50-56
Research report
Electroconvulsive shock increases interstitial concentrations of uric acid in the rat brain G.G. Nomikos 1, A.P. Zis *, G. Damsma 2, H.C. Fibiger Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1W5, Canada Accepted 5 July 1994
Abstract
This study examined the effects of electroconvulsive shock (ECS) on striatal interstitial concentrations of the purine metabolite uric acid (UA) using microdialysis in freely moving rats. UA increased to about 200% of baseline following ECS. Intense seizure activity induced by the convulsant agent flurothyl also resulted in a two-fold increase of UA concentrations suggesting that the ECS-induced UA increase is related to the seizure activity per se. Local administration of tetrodotoxin or perfusion with a Ca2+-free solution failed to affect the basal or the ECS-induced increase in UA concentrations. These data indicate that both the basal and the stimulated interstitial concentrations of uric acid are not dependent upon neuronal activity and exocytotic release. The UA response to ECS appears to be refractory to a second ECS delivered 2 but not 24 h after the first. Intrastriatal infusion of aUopurinol (1 mM), an inhibitor of UA synthesis, decreased basal UA concentrations to 26% but did not influence the ECS-induced UA increase. Systemic injection of allopurinol (20 mg/kg, i.p.) decreased basal UA concentrations to 25% and prevented the ECS-induced UA elevation. ECS also increased serum concentrations of UA to almost 200% of baseline. Allopurinol (20 mg/kg, i.p.) markedly decreased serum UA concentrations to non-detectable levels and completely abolished the ECS-induced increase. The estimated concentration difference between blood and brain interstitial UA strongly suggests that ECS-induced increase in brain interstitial UA concentrations is of peripheral origin possibly due to disruption of the blood brain barrier during seizure activity.
Keywords: Electroconvulsive shock; Microdialysis; Striatum; Uric acid
1. Introduction
By employing the in vivo microdialysis technique recent studies have shown that electroconvulsive shock (ECS) produces distinct actions on brain monoaminergic neurotransmission. For example, acute ECS profoundly increases extracellular concentrations of dopamine (DA), serotonin and noradrenaline in the rat striatum, hippocampus and frontal cortex [7,15,18,35, 36]. In addition, acute ECS moderately but significantly
* Corresponding author. UBC Department of Psychiatry, 2255 Wesbrook Mall, Vancouver, B.C, V6T 2A1, Canada. Fax: (1) (604) 822-7756. i Present address: Karolinska Institute, Department of Pharmacology, Stockholm, Sweden. 2 Deceased. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 8 5 4 - X
elevates extracellular concentrations of the D A metabolites D O P A C and H V A in both the striatum and the nucleus accumbens [18,20,35]. One of the most consistent findings reported is a considerable increase in the outflow of the purine metabolite uric acid (UA) from the rat striatum after an ECS challenge [18]. The presence of U A in the brain has only recently been demonstrated [34]. Evidence has also been provided that U A is synthesized in the brain [3,9,16,21]. Although extracellular U A most likely reflects an endproduct in the catabolism of purines, the origin and the significance of changes in its levels remain largely unknown. Several natural and pharmacological stimuli as well as ischemic insults enhance extracellular brain concentrations of U A [10,13,16,22]. Thus, changes in U A concentrations may be associated with altered neuronal activity and energy use. The purpose of this study was to characterize further the ECS-elicited increase in the outflow of UA
G.G. Nomikos et aL /Brain Research 660 (1994) 50-56
from the striatum of freely moving rats. ECS was administered 24, 48 or 72 h after implantation of the microdialysis probe to determine whether the postimplantation interval affects the ECS-induced changes in U A concentrations. The effects of (a) a higher intensity electrical stimulus, (b) the convulsant drug flurothyl, and (c) a second ECS given 2 or 24 h after the first, on U A outflow were also assessed. The relationship of the UA responses to ECS to neuronal activity and exocytotic release was examined by administering locally tetrodotoxin or by perfusing with a calcium-flee solution. Systemic or central administration of allopurinol that inhibits the synthesis of UA has previously been found to decrease extracellular concentrations of UA [9,16]. In the present study the effects of ECS on UA outflow were examined in the presence of allopurinol that was given systemically or locally through the microdialysis probe. Finally, serum UA concentrations were measured in response to ECS given alone or after treatment with allopurinol.
2. Materials and methods 2.1. Animals, surgery and microdialysis Microdialysis experiments were performed on male Wistar rats (270-330 g) 48 h after implantation of a microdialysis probe unless otherwise indicated. Rats were stereotaxically implanted with a transverse microdialysis probe u n d e r sodium pentobarbital anesthesia (50-60 m g / k g , i.p.). The probe was placed in the dorsal striatum l A P : + 0 . 7 ram, DV: - 4 . 7 5 ram; relative to bregma [24]. Dialysis occurred through 6.4 m m (active surface) of a semipermeable hollow fiber (saponified cellulose ester, O.D. = 0.27 ram, 10,000 Da, Cordis Dow). After surgery the rats were housed individually in Plexiglass cages (35 x 35 x 40 cm), where they remained throughout the experimental period and had free access to food and water. All perfusion experiments were carried out in conscious animals during the light phase of the daily cycle (09.00-17.00 h). For all experiments, animals were handled according to the guidelines of the Animal Care Committee of the University of British Columbia, who approved our protocols. Microdialysis experiments were conducted using an on-line sample injection system [17]. The dialysis tubes were perfused with a physiological solution containing (in mM) NaCI 147, KC1 3.0, CaCI 2 1.3, MgCI 2 1.0, and sodium phosphate 1.0 (pH 7.4), at a rate of 5 / x l / m i n using a microperfusion p u m p (Harvard Apparatus). The dialysate was loaded directly into a 100 ~1 sample loop of the injector (Valco) and automatically injected to the analytical system every 10 min.
2.2. Biochemical assay of dialysate uric acid
51
vs. a A g / A g C I reference) and the chromatograms were registered on dual-pen chart recorder (Kipp and Zonnen). The identity of the uric acid peak was verified by differential potentiometry (LC4B, BAS), using the dual parallel mode setting with one electrode at +500 mV and the second at 700 mV. The mean ( + S . E . M . ) percent in vitro recovery of uric acid at 37°C was 17.8 (+2.5, n = 5).
2.3. Biochemical analysis of serum uric acid Animals were decapitated 10 or 20 min following administration of sham-ECS or ECS. Blood was collected in sterilized tubes and then was centrifuged at 3,000 rpm for 20 rain. The supernatant (serum) was kept in - 8 0 ° C , Uric acid was determined using an enzymatic method (Ektachem Clinical Chemistry Slide Uric, Kodak) combined with spectophotometric m e a s u r e m e n t s [11,27].
2.4. ECS- and flurothyl-induced seizures ECS was administered via earclip electrodes using a Medcraft B24 Ill clinical ECT apparatus. Sham treatments consisted only of handling and application of earclips. Stimulus parameters unless otherwise indicated were 15(1 V for 0.75 s (sinusoidal waveform, 60 Hz) which induced tonic extension and generalized seizures lasting about 15-20 s. A separate group received a higher electrical dosage (170 V for 1 s) that induced seizures lasting also 15-20 s. Separate group of animals received ECS at either 24 (day l) or 72 h (day 3) postimplantation intervals, while in some animals a second ECS was administered either 2 (day 2) or 24 h (day 3) following the first. For flurothyl treatment, animals were placed in an airtight glass jar measuring approximately 3 1. Flurothyl (0.2 ml, Anaquest) was introduced into the jar. Myoclonic jerks were apparent within 20 to 40 s with generalized seizures beginning within another 5 to 10 s at which point animals were removed from the jar and observed. Flurothyl-induced generalized seizures lasted a total of 3(1 to 40 s. Throughout this experimental phase the analysis of uric acid was kept on-line.
Uric acid]
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The quantification of uric acid was achieved by reverse phase liquid chromatography (150×4.6 ram, Nucleosil 5 ~ m , C 1 8 ) w i t h a mobile phase consisting of 0.06 M sodium acetate, 0.4-0.6 m M octanesulfonic acid, 0.01 mM, N a z E D T A , and 80 m l / l methanol (pH = 4.1, adjusted with glacial acetic acid). T h e mobile phase was delivered by an H P L C p u m p ( B I O - R A D ) at 1.5 m l / m i n . Electrochemical detection was accomplished using an LC4B amperometric detector (Bioanalytical Systems) with a glassy carbon electrode (0.7
ECS
Time (rain)
Fig. 1. Effects of electroconvulsive shock (ECS) on rat striatal dialysate concentrations of uric acid on day 1 (circle, n = 9), day 2 (square, n = 8) and day 3 (triangle, n 5) after surgery. Each point represents the m e a n ( ± S . E . M . ) percent change of baseline. Mean ( + S . E . M . ) baseline values of uric acid in p m o l / m i n are 9.1 _+ 1.0, 12.1+_1.1 and 10.6+1.8 for days l, 2 and 3, respectively.
52
( ; (;. Nornikos et al. / Brain Research 660 (1994) 50-56
2.5. Pharmacological treatments In some experiments a modified perfusion solution in which Ca 2+ had been replaced by equimolar concentration of Mg 2+ was used. ECS was administered 1 h after the initiation of perfusion with the modified solution. Tetrodotoxin (TTX, Sigma) and allopurinol (Sigma) were added to the perfusion solution at 1 # M and 1 m M concentrations, respectively. Allopurinol was also administered at a dose of 20 m g / k g i.p. ECS was administered 60, 120 and 90 rain after initiation of local infusion of TTX, local infusion of allopurinol, and peripheral injection of allopurinol, respectively.
concentrations of uric acid (Fig. 2A) to the same extent and with the same pattern as ECS (Fig. 11. As shown on Fig. 2B, administration of a second ECS (ECS2) 2 h following the first (ECSI) in the same rats produced an increase in uric acid concentrations which was significantly lower compared to the response of uric acid to the first ECS ( P < 0.05, paired t-test). However, when the second ECS (ECS2) was administered 24 h after the first, uric acid response was similar
2.6. Data analysis For purpose of graphic representation (Figs. 1 - 3 ) , the average of three baseline samples immediately preceeding treatment was defined as 100% and all measures were related to these values (percent changes). For the statistical evaluation of the data either percent changes (last baseline plus six posttreatment samples) or the absolute baseline values were used. Data were evaluated by analysis of variance ( A N O V A ) followed by N e w m a n - K e u l s multiple comparisons. The Student's t-test for independent and paired data was also used.
[Uric
acid]
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3. Results o
3.1. Effects of ECS and flurothyl on interstitial concentrations of uric acid Baseline (preECS) concentrations of uric acid 24 (day 1), 48 (day 2), and 72 (day 3) h following implantation of the microdialysis probe are reported on the legend of Fig. 1. One-way ANOVA did not reveal any statistically significant differences between the baseline concentrations of uric acid 24, 48, and 72 h following s u r g e r y (/'2,21 = 2.86, P = 0.081). Interstitial concentrations of uric acid substantially increased in response to ECS (Fig. 1). The effect was maximal (approximately 215%) within 20-30 min following ECS administration and then gradually decreased towards baseline. Twoway repeated measures ANOVA including the data from all 3 days revealed a significant time effect (F6,114 = 72.4, P < 0.001) but no significant day or day × time interaction. Post-hoc analysis revealed that ECS-induced increase in dialysate uric acid was statistically significant (P < 0.05) compared to the last baseline sample at all time points in all three groups. Sham-ECS did not affect dialysate concentrations of uric acid (data not shown). The use of a higher voltage and longer stimulus duration ECS (170 V, 1 s) resulted in a significant (P < 0.05, paired t-test between the last baseline and the peak sample) increase in uric acid concentrations (Fig. 2A). Both the magnitude of this effect and the time course were comparable to the uric acid response observed following the lower ECS dosage (150 V for 0.75 s Fig. 1). Flurothyl-induced convulsions significantly (P < 0.05, paired t-test) increased extracellular
×>
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ECS2-24hr
Fig. 2. A: effects of electrically (ECS) or chemically (flurothyl, double hatched bars, n = 6) induced seizures on rat striatal dialysate concentrations of uric acid. Stimulus parameters are t50 V for 0.75 s (open bars, n = 5). Bars indicate the mean ( + S . E . M . ) peak effect expressed as percent change of baseline. Mean ( + S . E . M . ) baseline values in p m o l / m i n are 12.1+1.1, 10.2+1.4, and 10.2+1.7 for the groups treated with ECS and flurothyl, respectively. B: effects of a second ECS (ECS2-2hr, hatched bars, n = 6) administered 2 h following the first (ECS1, open bars). Double hatched bars indicate the effects of a second ECS (ECS2-24hr, n = 5) administered 24 h after the first ECS (not depicted in the figure). Bars indicate the mean (-+S.E.M.) peak effect expressed as percent change of baseline. Mean ( ± S . E . M . ) baseline values in p m o l / m i n are 11.5+_1.6, 10.8-+ 1.5, and 10.7-+1.3 for the ECS1, ECS2-2hr, ECS 2-24hr groups, respectively.
G.G. Nomikos et al. / Brain Research 660 (1994) 50-56 to t h a t o b s e r v e d in a n i m a l s receiving E C S for t h e first time. Basal c o n c e n t r a t i o n s of uric acid w e r e not aff e c t e d 24 h following E C S t r e a t m e n t .
3.2. Basal and postECS interstitial concentrations o f uric acid in response to infusion o f T T X or a Ca 2+ free perfusion solution
"6 E
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L o c a l a d m i n i s t r a t i o n of 1 /xM T T X or p e r f u s i o n with a m o d i f i e d solution in which Ca 2+ h a d b e e n r e p l a c e d with M g 2+ did not i n f l u e n c e b a s a l c o n c e n t r a tions o f uric acid. U n d e r t h e s e c o n d i t i o n s t h e E C S - i n d u c e d i n c r e a s e in e x t r a c e l l u l a r c o n c e n t r a t i o n s of uric acid r e m a i n e d also u n a f f e c t e d ( d a t a not shown).
53
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Fig. 4. Effect of electroconvulsive shock (ECS) on serum concentrations of uric acid. Sham-ECS or ECS was administered 10 or 20 min before sacrifice of the animals (n = 8 for all groups). Allopurinol (20 mg/kg) was injected i.p. 90 min before administration of Sham-ECS or ECS. The detection limit of the assay is 15 /zmol/l. *P < 0.001 compared to the respective Sham-ECS group. ND, non-detectable values.
80" 0
60-
~-
3.3. Effects o f striatal and peripheral administration o f allopurinol on basal and post-ECS concentrations of uric acid
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C o n t i n u o u s topical infusion of 1 m M of a l l o p u r i n o l r e s u l t e d in a significant ( P < 0.001, p a i r e d t-test b e t w e e n the last b a s e l i n e a n d the last p r e E C S s a m p l e ) r e d u c t i o n o f e x t r a c e l l u l a r c o n c e n t r a t i o n s o f uric acid to 2 6 % (Fig. 3A). S u b s e q u e n t l y , a d m i n i s t r a t i o n of E C S i n c r e a s e d e x t r a c e l l u l a r c o n c e n t r a t i o n s o f uric acid by 81% (relative to the p r e E C S c o n c e n t r a t i o n s ) . This i n c r e a s e was statistically significant ( P < 0.001, p a i r e d t-test b e t w e e n the last p r e E C S a n d t h e third p o s t E C S sample). Systemic injection of 20 m g / k g a l l o p u r i n o l also r e s u l t e d in a significant ( P < 0.001, p a i r e d t-test) d e c r e a s e o f uric acid levels to 25% (Fig. 3B). Subseq u e n t a d m i n i s t r a t i o n of E C S did not i n c r e a s e significantly uric acid c o n c e n t r a t i o n s .
3.4. Effect o f E C S on serum concentrations o f uric acid
Time [rain) F i g . 3. E f f e c t s
of electroconvulsive
shock
(ECS)
on dialysate
concen-
of uric acid from the striatum of rats treated with allopurinol. Allopurinol is either locally infused (A, 1 mM, n = 5) or systemically injected (B, 20 mg/kg i.p., n = 5). Each point represents the mean ( + S.E.M.) percent change of baseline. Mean ( + S.E.M.) baseline values of uric acid in pmol/min are 10.5 + 2.0 or 9.1 + 1.9 for the groups treated locally or systemically with allopurinol, respectively. trations
T h e d a t a a r e s u m m a r i z e d in Fig. 4. E C S i n c r e a s e d s e r u m c o n c e n t r a t i o n s o f uric acid within 1 0 - 2 0 min a f t e r a d m i n i s t r a t i o n . T h e s e i n c r e a s e s w e r e significant in c o m p a r i s o n to S h a m - E C S effects ( P < 0.001, Stud e n t ' s t-test). P e r i p h e r a l injection o f 20 m g / k g allopu r i n o l r e s u l t e d in a m a r k e d d e c r e a s e in s e r u m uric acid
54
(;.(i. Nomik~,s et al. ~Brain Research 660 (1994) 50-56
concentrations to less than 15 /.tmol/l which was the detection limit of the assay. Subsequently, administration of ECS did not produce a measurable effect on serum uric acid.
4. Discussion
Acute ECS treatment increases dialysate concentrations of U A from the rat striatum to approximately 200% of baseline. In accordance with the effects of ECS on extracellular concentrations of the dopamine metabolites D O P A C and HVA, but not of dopamine itself [35], the ECS-induced increase in UA did not differ across days following surgery. The basal dialysate UA concentrations also did not vary as a function of the postimplantation interval. Recently, O'Neill et al. [21] by using interpolated data from a rectangular hyperbolic curve fitted to recovery measurements over time, reported relatively stable extracellular UA concentrations between 1 and 5 days following probe implantation. Thus, it appears that dialysis measurements of basal and pharmacologically modified extracellular concentrations of U A are reliable within the 24-72 h postimplantation interval. A higher intensity electrical stimulus as well as seizure activity elicited by the convulsant agent flurothyl also resulted in a two-fold increase of baseline UA concentrations. In contrast to the ECS-induced effects on striatal dopamine concentrations, which appear to be mediated by the passage of current [15,35], the present results suggest that the ECT-induced UA increase is independent of electrical dosage and it is related to the seizure activity per se. Local administration of tetrodotoxin or perfusion with a calcium-free solution failed to affect the basal or the ECS elevated concentrations of UA. These data indicate that neither the basal nor the stimulated UA concentrations are dependent on neuronal activation and exocytotic release. Previous microdialysis studies have indicated that the basal concentrations of monoaminergic neurotransmitters but not of their metabolites are both tetrodotoxin and calcium dependent [19,31,32]. The UA response to an ECT appears to be decreased in response to a second stimulus administered 2 but not 24 h after the first. Although changes in seizure threshold after ECT have been documented, the decreased U A response in these series of experiments was not associated with a concomitant decrease in seizure duration in response to the second stimulus. It is noteworthy that a similar reduction of the dopamine response to a second E C T given 2 but not 24 h after the first has previously been reported [35]. In accordance with earlier in vivo studies using either voltammetry or dialysis, systemic administration of allopurinol resulted in a pronounced (to about 20%
of baseline) decrease of basal UA concentrations [9,16J. Also, local infusion of allopurinol through the microdialysis probe markedly decreased UA concentrations to about 20% of baseline. This finding provides additional support for the notion that UA is formed locally in the brain [15]. Pretreatment with allopurinol given systemically, but not locally, completely abolished the ECS-induced elevation of dialysate UA concentrations. These results indicate that inhibition of the synthesis of UA only at a central level is not sufficient to prevent the rise in extracellular brain concentrations of UA observed following ECS. In view of the fact that baseline concentrations of the UA decreased to the same extent following the systemic or local administration of allopurinol, the possibility that the ECS induced increase in UA dialysate derives from a brain area not covered by the local administration of allopurinot, although it cannot entirely be excluded, seems highly unlikely. Consequently, it is plausible to conclude that the ECS-induced UA increase is of peripheral origin rather than a result of a general increase in brain metabolism. The concentrations of UA in rat plasma reported by different investigators under slightly varying conditions, range from 20-100 p,M [26]. The extracellular concentrations of UA in the rat brain are estimated by means of in vivo voltammetry and microdialysis in the range of 11-50 g M [9,21]. However, as argued by O'Neill et al. [21] these estimated concentrations are anomalously high and are most likely due to tissue perturbation by the relatively large in vivo probes, such as carbon paste electrodes and dialysis tubes. It is noteworthy, that in humans the ratio between plasma and CSF (that most closely reflects brain extracellular fluid) UA is t0-25 [6,26]. By extrapolation and if the ratio of plasma to extracellular fluid UA concentrations in man and the rat is the same, then the extracellular fluid concentrations of UA in the rat brain may be in the range of 1-10 p,M. Thus, it is plausible to argue that there exists a concentration gradient between blood and brain UA. This concentration gradient becomes even higher following ECS since UA serum concentrations increase in response to the treatment. The ECS-induced rise in serum UA concentrations is most likely due to enhanced energy use and purine metabolism associated with intense muscular activity. In this regard, it is noteworthy that high levels of serum and urinary UA have been found in patients with seizure disorders [28], and that overproduction of purines is often accompanied by dramatic CNS symptoms including seizures [5,14]. Given that ECS disrupts blood brain barrier permeability [2,25,30], the ECS-induced elevation of dialysate UA concentrations may reflect enhanced transport of U A through the cerebral endothelium. Allopurinol treatment greatly decreased both basal and stimulated concentrations of UA in the blood to
G.G. Nomikos et al. /Brain Research 660 (1994) 50-56
non-detectable levels, thus preventing the influx of UA acid into the brain during the ECS induced disruption of blood brain barrier permeability. Of some relevance here are observations indicating that brain UA increases as a result of experimentally induced ischemia [12] and that this increase is prevented by the systemic administration of allopurinol which also reduces ischemic brain injury [8,23,33]. Although the mechanism of action of allopurinol in reducing ischemic brain injury remains controversial [1] and ECT does not appear to be associated with structural brain damage [4,29], it may nevertheless be of interest to examine the effect of allopurinol pre-treatment on ECS/or ECT induced memory disruption.
Acknowledgements This work was supported by the Medical Research Council of Canada (PG-23 and MT-10615). G.D. was supported in part by the Netherlands Organization for the Advancement of Research (Z.W.O.). We would like to thank C. Tham and C. Wilson for technical assistance and Professor Venturella, Anaquest, Murray Hill, NJ for supplying Flurothyl (Indoclon).
References [1] Betz, A.L., Randall, J. and Martz, D., Xanthine oxidase is not a major source of free radicals in focal cerebral ischemia, The American Physiological Society, 1991, pp. 563-568. [2] Bolwig, T.G., The influence of electrically induced seizures on deep brain structures. In B. Lerer, R.D. Weiner, R.H. Belmaker (Eds.), ECT: Basic Mechanisms, John Libbey, London, 1984, pp. 132-138. [3] Cespuglio, R., Sarda, N., Gharib, A., Faradji, H. and Chastrette, N., Differential pulse voltammetry in vivo with working carbon fibre electrodes: 5-hydroxyindole compounds or uric acid detection, Exp. Brain Res., 64 (1986) 589-595. [4] Coffey, C.E., Structural brain imaging and ECT. In C.E. Coffey (Ed.), The Clinical Science of Electroconvulsive Therapy, Chapter 5, Progress in Psychiatry, 38, American Psychiatric Press Inc., Washington, D.C., 1993, pp. 73-92. [5] Coleman, M., Landgrebe, M. and Landgrebe, A., Purine seizure disorders, Epilepsia, 27 (1986) 263-269. [6] Degrell, I. and Nagy, E., Concentration gradients for HVA, 5-HIAA, ascorbic, and uric acid in cerebrospinal fluid, Biol. Psychiatry, 27 (1990) 891-896. [7] Glue, P., Costello, M.J., Pert, A., Mele, A. and Nutt, D.J., Regional neurotransmission responses after acute and chronic electron-convulsive shock, Psychopharmacology, 100 (1990) 6065. [8] ltoh, T., Kawakami, M. and Yamauchi, B.S., Effect of allopurinol on ischemia and reperfusion-induced cerebral injury in spontaneously hypertensive rats, Stroke, 17 (1986) 1284-1287. [9] Joseph, M.H. and Young, A.M.J., Pharmacological evidence, using in vivo dialysis, that substances additional to ascorbic acid, uric acid and homovanillic acid contribute to the voltammetric signals obtained in unrestrained rats from chronically implanted carbon paste electrodes, J. Neurosci. Methods, 36 (1991) 209-218.
55
[10] Joseph, M.H., Hodges, H. and Gray, J.A., Lever pressing for food reward and in vivo voltammetry: evidence for increases in extracellular homovanillic acid, the dopamine metabolite, and uric acid in the rat caudate nucleus, Neuroscience, 32 (1989) 195-201. [11] Kageyama, N., A direct colorimetric determination of uric acid in serum and urine with uricase-catalase system, Clin. Chim. Acta, 31 (1971) 421-426. [12] Kanemitsu, H., Tamura, A., Kirino, T., Karasawa, S., Sano, K., lwamoto, T., Yoshiura, M. and Iriyama, K., Xanthine and uric acid levels in rat brain following focal ischemia, J. Neurochem., 51 (1988) 1882-1885. [13] Kendrick, K.M., Baldwin, B.A., Cooper, T.R. and Sharman, D.F., Uric acid is released in the zona incerta of the subthalamic region of the sheep during rumination and in response to feeding and drinking stimuli, Neurosci. Lett., 70 (1986) 272-277. [14] Lesch, M. and Nyhan, W.L., A familial disorder of uric acid metabolism and central nervous system function. Am..I. Med., 36 (1964) 361-370. [15] McGarvey, K.A., Zis, A.P., Brown, E.E,, Nomikos, G.G. and Fibiger, H.C., ECS-induced dopamine release: effects of electrode placement, anticonvulsant treatment and stimulus intensity, BioL Psychiatry, 34 (1993) 152-157. [16] Mueller, K., Palmour, R., Andrews, C.D. and Knott, P.J., In vivo voltammetric evidence of production of uric acid by rat caudate, Brain Res., 335 (1985) 231-235. [17] Nomikos, G.G., Damsma, G., Wenkstern, D. and Fibiger, H.C., Acute effects of bupropion on extracellular dopamine concentrations in rat striatum and nucleus accumbens studied by in vivo microdialysis, Neurop,sychopharmacology, 2 (1989) 273-27tL [18] Nomikos, G.G., Zis, A.P., Damsma, G. and Fibiger, H.C., Electroconvulsive shock produces large increases in interstitial concentrations of dopamine in the rat striatum: an in vivo midrodialysis study, Neuropsychopharmacolog~', 4 (1990) 65-69. [19] Nomikos, G.G., Damsma, G., Wenkstern, D. and Fibiger, H.C., In vivo characterization of locally applied dopamine uptake inhibitors by striatal microdialysis, Synapse, 6 (1990) 106- I 12. [20] Nomikos, G.G., Zis, A.P., Damsma, G. and Fibiger, H.C., Effects of chronic electroconvulsive shock on interstitial concentrations of dopamine in the nucleus accumbens, t~vvchopharmacology, 105 (1991) 230-238. [21] O'Neill, R.D., Gonzalez-Mora, J., Boutelle, M.G., Ormonde, D.E., Lowry, J.P., Duff, A., Fumero, B., Fillenz, M. and Mas, M., Anomalously high concentrations of brain extracellular uric acid detected with chronically implanted probes: implications for in vivo sampling techniques, J. Neurochem., 57 (1991) 22-29. [22] O'Neill, R.D., Fillenz, M., Grunewald, R.A., Bloomfield, M.R., Albery, W.J., Jamieson, C.M., Williams, J.H. and Gray, J.A., Voltammetric carbon paste electrodes monitor uric acid and not 5HIAA at the 5-hydroxyindole potential in the rat brain, Neurosci. Lett., 45 (1984) 39-46. [23] Palmer, C., Towfighi, J., Roberts, R.L. and Heitjan, D.F., AIIopurinol administered after inducing hypoxia-isehemia reduces brain injury in 7-day-old rats, Pediat. Res., 33 (1993) 405-411 [24] Paxinos, G. and Watson, P.G., The Rat Brain in Stereotaxic Coordinates, Academic Press, London, 1986. [25] Petito, C.K., Schaefer, J.A. and Plum, F., Ultrastructural characteristics of the brain and blood-brain barrier in experimental seizures, Brain Res., 127 (1977) 251 267. [26] Roch-Ramel, F. and Peters, G., Urinary excretion of uric acid in nonhuman mammalian species. In W.N. Kelley, I.M. Weiner (Eds.), Handbook of Experimental Pharmacology, 51, Uric Acid, Springer-Verlag, Berlin, 1978, pp. 211 255. [27] Trivedi, R.C., Rebar, L., Desai, K. and Stong, L.J., New ultraviolet (340 nm) method for assay of uric acid in serum or plasma, Clin. Chem., 24 (1978) 562-566. [28] Warren, D.J., Leitch, A.G. and Leggett, R.J.E., Hyperuricaemic
56
[29] [30]
[31]
[32]
G.G. Nornikos et al. /Brain Research 660 (1994) 50-56 acute renal failure after epileptic seizures Lancet, 2 (1975) 385-387. Weiner, R.D., Does electroconvulsive therapy cause brain damage?, BehaL,. Brain Sci., 7 (1984) 1-53. Westergaard, M., Hertz, M.M. and Bolwig, T.G, Increased permeability to horseradish peroxidase across cerebral vessels, evoked by electrically induced seizures in the rat, Acta Neuropath., 41 (1978) 73-80. Westerink, B.H.C., Hofsteede, R.M., Tuntler, J. and de Vries, J.B., Use of calcium antagonism for the characterization of drug-evoked dopamine release from the brain of conscious rats determined by microdialysis, J. Neurochem., 52 (1989) 722-729. Westerink, B.H.C., Tuntler, J., Damsma, J., Rollema, H. and de Vries, J.B., The use of tetrodotoxin for the characterization of drug-enhanced dopamine release in conscious rats studied by brain dialysis, Naunyn Schmiedebergs Arch. Pharmacol., 336 11987) 502-507.
[33] Williams, G.D., Palmer, C., Heitjan, D.F. and Smith, M.B., Allopurinol preserves cerebral energy metabolism during perinatal hypoxia-ischemia: 31p NMR study in unanesthetized immature rats, Neurosci. Lett., 144 (1992) 103-1~. [34] Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, U., In vivo measurement of dopamine and its metabolites by intrac, erebral dialysis: changes after t~-amphetamine. Z Neurochem., (1983) 1769. [35] Zis, A.P., Nomikos, G.G., Damsma, G. and Fibiger, H.C., In vivo neurochemical effects of electroconvulsive shock studied by microdialysis in the rat striatum, Psychopharmacology, t03 (1991) 343-350. [36] Zis, A.P., Nomikos, G.G., Brown, E.E., Damsma, G. and Fibiger, H.C., Neurochemical effects of electrically and chemically induced seizures: an in vivo microdialysis study in the rat hippocampus, Neuropsychopharrnacology, 7 (1992) 189-195.
Brain Research 660 (1994) 50-56
Research report
Electroconvulsive shock increases interstitial concentrations of uric acid in the rat brain G.G. Nomikos 1, A.P. Zis *, G. Damsma 2, H.C. Fibiger Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1W5, Canada Accepted 5 July 1994
Abstract
This study examined the effects of electroconvulsive shock (ECS) on striatal interstitial concentrations of the purine metabolite uric acid (UA) using microdialysis in freely moving rats. UA increased to about 200% of baseline following ECS. Intense seizure activity induced by the convulsant agent flurothyl also resulted in a two-fold increase of UA concentrations suggesting that the ECS-induced UA increase is related to the seizure activity per se. Local administration of tetrodotoxin or perfusion with a Ca2+-free solution failed to affect the basal or the ECS-induced increase in UA concentrations. These data indicate that both the basal and the stimulated interstitial concentrations of uric acid are not dependent upon neuronal activity and exocytotic release. The UA response to ECS appears to be refractory to a second ECS delivered 2 but not 24 h after the first. Intrastriatal infusion of aUopurinol (1 mM), an inhibitor of UA synthesis, decreased basal UA concentrations to 26% but did not influence the ECS-induced UA increase. Systemic injection of allopurinol (20 mg/kg, i.p.) decreased basal UA concentrations to 25% and prevented the ECS-induced UA elevation. ECS also increased serum concentrations of UA to almost 200% of baseline. Allopurinol (20 mg/kg, i.p.) markedly decreased serum UA concentrations to non-detectable levels and completely abolished the ECS-induced increase. The estimated concentration difference between blood and brain interstitial UA strongly suggests that ECS-induced increase in brain interstitial UA concentrations is of peripheral origin possibly due to disruption of the blood brain barrier during seizure activity.
Keywords: Electroconvulsive shock; Microdialysis; Striatum; Uric acid
1. Introduction
By employing the in vivo microdialysis technique recent studies have shown that electroconvulsive shock (ECS) produces distinct actions on brain monoaminergic neurotransmission. For example, acute ECS profoundly increases extracellular concentrations of dopamine (DA), serotonin and noradrenaline in the rat striatum, hippocampus and frontal cortex [7,15,18,35, 36]. In addition, acute ECS moderately but significantly
* Corresponding author. UBC Department of Psychiatry, 2255 Wesbrook Mall, Vancouver, B.C, V6T 2A1, Canada. Fax: (1) (604) 822-7756. i Present address: Karolinska Institute, Department of Pharmacology, Stockholm, Sweden. 2 Deceased. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 8 5 4 - X
elevates extracellular concentrations of the D A metabolites D O P A C and H V A in both the striatum and the nucleus accumbens [18,20,35]. One of the most consistent findings reported is a considerable increase in the outflow of the purine metabolite uric acid (UA) from the rat striatum after an ECS challenge [18]. The presence of U A in the brain has only recently been demonstrated [34]. Evidence has also been provided that U A is synthesized in the brain [3,9,16,21]. Although extracellular U A most likely reflects an endproduct in the catabolism of purines, the origin and the significance of changes in its levels remain largely unknown. Several natural and pharmacological stimuli as well as ischemic insults enhance extracellular brain concentrations of U A [10,13,16,22]. Thus, changes in U A concentrations may be associated with altered neuronal activity and energy use. The purpose of this study was to characterize further the ECS-elicited increase in the outflow of UA
G.G. Nomikos et aL /Brain Research 660 (1994) 50-56
from the striatum of freely moving rats. ECS was administered 24, 48 or 72 h after implantation of the microdialysis probe to determine whether the postimplantation interval affects the ECS-induced changes in U A concentrations. The effects of (a) a higher intensity electrical stimulus, (b) the convulsant drug flurothyl, and (c) a second ECS given 2 or 24 h after the first, on U A outflow were also assessed. The relationship of the UA responses to ECS to neuronal activity and exocytotic release was examined by administering locally tetrodotoxin or by perfusing with a calcium-flee solution. Systemic or central administration of allopurinol that inhibits the synthesis of UA has previously been found to decrease extracellular concentrations of UA [9,16]. In the present study the effects of ECS on UA outflow were examined in the presence of allopurinol that was given systemically or locally through the microdialysis probe. Finally, serum UA concentrations were measured in response to ECS given alone or after treatment with allopurinol.
2. Materials and methods 2.1. Animals, surgery and microdialysis Microdialysis experiments were performed on male Wistar rats (270-330 g) 48 h after implantation of a microdialysis probe unless otherwise indicated. Rats were stereotaxically implanted with a transverse microdialysis probe u n d e r sodium pentobarbital anesthesia (50-60 m g / k g , i.p.). The probe was placed in the dorsal striatum l A P : + 0 . 7 ram, DV: - 4 . 7 5 ram; relative to bregma [24]. Dialysis occurred through 6.4 m m (active surface) of a semipermeable hollow fiber (saponified cellulose ester, O.D. = 0.27 ram, 10,000 Da, Cordis Dow). After surgery the rats were housed individually in Plexiglass cages (35 x 35 x 40 cm), where they remained throughout the experimental period and had free access to food and water. All perfusion experiments were carried out in conscious animals during the light phase of the daily cycle (09.00-17.00 h). For all experiments, animals were handled according to the guidelines of the Animal Care Committee of the University of British Columbia, who approved our protocols. Microdialysis experiments were conducted using an on-line sample injection system [17]. The dialysis tubes were perfused with a physiological solution containing (in mM) NaCI 147, KC1 3.0, CaCI 2 1.3, MgCI 2 1.0, and sodium phosphate 1.0 (pH 7.4), at a rate of 5 / x l / m i n using a microperfusion p u m p (Harvard Apparatus). The dialysate was loaded directly into a 100 ~1 sample loop of the injector (Valco) and automatically injected to the analytical system every 10 min.
2.2. Biochemical assay of dialysate uric acid
51
vs. a A g / A g C I reference) and the chromatograms were registered on dual-pen chart recorder (Kipp and Zonnen). The identity of the uric acid peak was verified by differential potentiometry (LC4B, BAS), using the dual parallel mode setting with one electrode at +500 mV and the second at 700 mV. The mean ( + S . E . M . ) percent in vitro recovery of uric acid at 37°C was 17.8 (+2.5, n = 5).
2.3. Biochemical analysis of serum uric acid Animals were decapitated 10 or 20 min following administration of sham-ECS or ECS. Blood was collected in sterilized tubes and then was centrifuged at 3,000 rpm for 20 rain. The supernatant (serum) was kept in - 8 0 ° C , Uric acid was determined using an enzymatic method (Ektachem Clinical Chemistry Slide Uric, Kodak) combined with spectophotometric m e a s u r e m e n t s [11,27].
2.4. ECS- and flurothyl-induced seizures ECS was administered via earclip electrodes using a Medcraft B24 Ill clinical ECT apparatus. Sham treatments consisted only of handling and application of earclips. Stimulus parameters unless otherwise indicated were 15(1 V for 0.75 s (sinusoidal waveform, 60 Hz) which induced tonic extension and generalized seizures lasting about 15-20 s. A separate group received a higher electrical dosage (170 V for 1 s) that induced seizures lasting also 15-20 s. Separate group of animals received ECS at either 24 (day l) or 72 h (day 3) postimplantation intervals, while in some animals a second ECS was administered either 2 (day 2) or 24 h (day 3) following the first. For flurothyl treatment, animals were placed in an airtight glass jar measuring approximately 3 1. Flurothyl (0.2 ml, Anaquest) was introduced into the jar. Myoclonic jerks were apparent within 20 to 40 s with generalized seizures beginning within another 5 to 10 s at which point animals were removed from the jar and observed. Flurothyl-induced generalized seizures lasted a total of 3(1 to 40 s. Throughout this experimental phase the analysis of uric acid was kept on-line.
Uric acid]
200-
150-
o
100[] day 2 day 3 1'0
2'0
'T
4'0
5'0
6'0
7'0
8'0
9'0
/
The quantification of uric acid was achieved by reverse phase liquid chromatography (150×4.6 ram, Nucleosil 5 ~ m , C 1 8 ) w i t h a mobile phase consisting of 0.06 M sodium acetate, 0.4-0.6 m M octanesulfonic acid, 0.01 mM, N a z E D T A , and 80 m l / l methanol (pH = 4.1, adjusted with glacial acetic acid). T h e mobile phase was delivered by an H P L C p u m p ( B I O - R A D ) at 1.5 m l / m i n . Electrochemical detection was accomplished using an LC4B amperometric detector (Bioanalytical Systems) with a glassy carbon electrode (0.7
ECS
Time (rain)
Fig. 1. Effects of electroconvulsive shock (ECS) on rat striatal dialysate concentrations of uric acid on day 1 (circle, n = 9), day 2 (square, n = 8) and day 3 (triangle, n 5) after surgery. Each point represents the m e a n ( ± S . E . M . ) percent change of baseline. Mean ( + S . E . M . ) baseline values of uric acid in p m o l / m i n are 9.1 _+ 1.0, 12.1+_1.1 and 10.6+1.8 for days l, 2 and 3, respectively.
52
( ; (;. Nornikos et al. / Brain Research 660 (1994) 50-56
2.5. Pharmacological treatments In some experiments a modified perfusion solution in which Ca 2+ had been replaced by equimolar concentration of Mg 2+ was used. ECS was administered 1 h after the initiation of perfusion with the modified solution. Tetrodotoxin (TTX, Sigma) and allopurinol (Sigma) were added to the perfusion solution at 1 # M and 1 m M concentrations, respectively. Allopurinol was also administered at a dose of 20 m g / k g i.p. ECS was administered 60, 120 and 90 rain after initiation of local infusion of TTX, local infusion of allopurinol, and peripheral injection of allopurinol, respectively.
concentrations of uric acid (Fig. 2A) to the same extent and with the same pattern as ECS (Fig. 11. As shown on Fig. 2B, administration of a second ECS (ECS2) 2 h following the first (ECSI) in the same rats produced an increase in uric acid concentrations which was significantly lower compared to the response of uric acid to the first ECS ( P < 0.05, paired t-test). However, when the second ECS (ECS2) was administered 24 h after the first, uric acid response was similar
2.6. Data analysis For purpose of graphic representation (Figs. 1 - 3 ) , the average of three baseline samples immediately preceeding treatment was defined as 100% and all measures were related to these values (percent changes). For the statistical evaluation of the data either percent changes (last baseline plus six posttreatment samples) or the absolute baseline values were used. Data were evaluated by analysis of variance ( A N O V A ) followed by N e w m a n - K e u l s multiple comparisons. The Student's t-test for independent and paired data was also used.
[Uric
acid]
A
3O0
@
7=
200-
J:l
100-
3. Results o
3.1. Effects of ECS and flurothyl on interstitial concentrations of uric acid Baseline (preECS) concentrations of uric acid 24 (day 1), 48 (day 2), and 72 (day 3) h following implantation of the microdialysis probe are reported on the legend of Fig. 1. One-way ANOVA did not reveal any statistically significant differences between the baseline concentrations of uric acid 24, 48, and 72 h following s u r g e r y (/'2,21 = 2.86, P = 0.081). Interstitial concentrations of uric acid substantially increased in response to ECS (Fig. 1). The effect was maximal (approximately 215%) within 20-30 min following ECS administration and then gradually decreased towards baseline. Twoway repeated measures ANOVA including the data from all 3 days revealed a significant time effect (F6,114 = 72.4, P < 0.001) but no significant day or day × time interaction. Post-hoc analysis revealed that ECS-induced increase in dialysate uric acid was statistically significant (P < 0.05) compared to the last baseline sample at all time points in all three groups. Sham-ECS did not affect dialysate concentrations of uric acid (data not shown). The use of a higher voltage and longer stimulus duration ECS (170 V, 1 s) resulted in a significant (P < 0.05, paired t-test between the last baseline and the peak sample) increase in uric acid concentrations (Fig. 2A). Both the magnitude of this effect and the time course were comparable to the uric acid response observed following the lower ECS dosage (150 V for 0.75 s Fig. 1). Flurothyl-induced convulsions significantly (P < 0.05, paired t-test) increased extracellular
×>
ECS 150 V 0 . 7 5 see
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Flurothyl
170 V 1 sec
U_ri c acidJ 300-
== al .o
200"
o //
o.
100-
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ECS2-2hr
ECS2-24hr
Fig. 2. A: effects of electrically (ECS) or chemically (flurothyl, double hatched bars, n = 6) induced seizures on rat striatal dialysate concentrations of uric acid. Stimulus parameters are t50 V for 0.75 s (open bars, n = 5). Bars indicate the mean ( + S . E . M . ) peak effect expressed as percent change of baseline. Mean ( + S . E . M . ) baseline values in p m o l / m i n are 12.1+1.1, 10.2+1.4, and 10.2+1.7 for the groups treated with ECS and flurothyl, respectively. B: effects of a second ECS (ECS2-2hr, hatched bars, n = 6) administered 2 h following the first (ECS1, open bars). Double hatched bars indicate the effects of a second ECS (ECS2-24hr, n = 5) administered 24 h after the first ECS (not depicted in the figure). Bars indicate the mean (-+S.E.M.) peak effect expressed as percent change of baseline. Mean ( ± S . E . M . ) baseline values in p m o l / m i n are 11.5+_1.6, 10.8-+ 1.5, and 10.7-+1.3 for the ECS1, ECS2-2hr, ECS 2-24hr groups, respectively.
G.G. Nomikos et al. / Brain Research 660 (1994) 50-56 to t h a t o b s e r v e d in a n i m a l s receiving E C S for t h e first time. Basal c o n c e n t r a t i o n s of uric acid w e r e not aff e c t e d 24 h following E C S t r e a t m e n t .
3.2. Basal and postECS interstitial concentrations o f uric acid in response to infusion o f T T X or a Ca 2+ free perfusion solution
"6 E
200
rO 150-
x
i.-
X ¢J eO
L o c a l a d m i n i s t r a t i o n of 1 /xM T T X or p e r f u s i o n with a m o d i f i e d solution in which Ca 2+ h a d b e e n r e p l a c e d with M g 2+ did not i n f l u e n c e b a s a l c o n c e n t r a tions o f uric acid. U n d e r t h e s e c o n d i t i o n s t h e E C S - i n d u c e d i n c r e a s e in e x t r a c e l l u l a r c o n c e n t r a t i o n s of uric acid r e m a i n e d also u n a f f e c t e d ( d a t a not shown).
53
o E
100"
x
50-
~
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x
x~ x~ ×>
x
k.
x~
Or) i tU
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tU
ND
ND
i
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"~. tU
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E~ +
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acid
A Q
~¢/I
1201oo.
Fig. 4. Effect of electroconvulsive shock (ECS) on serum concentrations of uric acid. Sham-ECS or ECS was administered 10 or 20 min before sacrifice of the animals (n = 8 for all groups). Allopurinol (20 mg/kg) was injected i.p. 90 min before administration of Sham-ECS or ECS. The detection limit of the assay is 15 /zmol/l. *P < 0.001 compared to the respective Sham-ECS group. ND, non-detectable values.
80" 0
60-
~-
3.3. Effects o f striatal and peripheral administration o f allopurinol on basal and post-ECS concentrations of uric acid
4020-
' ' ' 6 ' '3'0' '6'0' '9'0' ' 'I' ' '15() '180 ECS
B (D C
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0
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~ i T /
~ i 60
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~
i 9JO . . . .
AIIopurinol 120 mg/kg, IP)
I /
i ~0 I 1
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ECS
C o n t i n u o u s topical infusion of 1 m M of a l l o p u r i n o l r e s u l t e d in a significant ( P < 0.001, p a i r e d t-test b e t w e e n the last b a s e l i n e a n d the last p r e E C S s a m p l e ) r e d u c t i o n o f e x t r a c e l l u l a r c o n c e n t r a t i o n s o f uric acid to 2 6 % (Fig. 3A). S u b s e q u e n t l y , a d m i n i s t r a t i o n of E C S i n c r e a s e d e x t r a c e l l u l a r c o n c e n t r a t i o n s o f uric acid by 81% (relative to the p r e E C S c o n c e n t r a t i o n s ) . This i n c r e a s e was statistically significant ( P < 0.001, p a i r e d t-test b e t w e e n the last p r e E C S a n d t h e third p o s t E C S sample). Systemic injection of 20 m g / k g a l l o p u r i n o l also r e s u l t e d in a significant ( P < 0.001, p a i r e d t-test) d e c r e a s e o f uric acid levels to 25% (Fig. 3B). Subseq u e n t a d m i n i s t r a t i o n of E C S did not i n c r e a s e significantly uric acid c o n c e n t r a t i o n s .
3.4. Effect o f E C S on serum concentrations o f uric acid
Time [rain) F i g . 3. E f f e c t s
of electroconvulsive
shock
(ECS)
on dialysate
concen-
of uric acid from the striatum of rats treated with allopurinol. Allopurinol is either locally infused (A, 1 mM, n = 5) or systemically injected (B, 20 mg/kg i.p., n = 5). Each point represents the mean ( + S.E.M.) percent change of baseline. Mean ( + S.E.M.) baseline values of uric acid in pmol/min are 10.5 + 2.0 or 9.1 + 1.9 for the groups treated locally or systemically with allopurinol, respectively. trations
T h e d a t a a r e s u m m a r i z e d in Fig. 4. E C S i n c r e a s e d s e r u m c o n c e n t r a t i o n s o f uric acid within 1 0 - 2 0 min a f t e r a d m i n i s t r a t i o n . T h e s e i n c r e a s e s w e r e significant in c o m p a r i s o n to S h a m - E C S effects ( P < 0.001, Stud e n t ' s t-test). P e r i p h e r a l injection o f 20 m g / k g allopu r i n o l r e s u l t e d in a m a r k e d d e c r e a s e in s e r u m uric acid
54
(;.(i. Nomik~,s et al. ~Brain Research 660 (1994) 50-56
concentrations to less than 15 /.tmol/l which was the detection limit of the assay. Subsequently, administration of ECS did not produce a measurable effect on serum uric acid.
4. Discussion
Acute ECS treatment increases dialysate concentrations of U A from the rat striatum to approximately 200% of baseline. In accordance with the effects of ECS on extracellular concentrations of the dopamine metabolites D O P A C and HVA, but not of dopamine itself [35], the ECS-induced increase in UA did not differ across days following surgery. The basal dialysate UA concentrations also did not vary as a function of the postimplantation interval. Recently, O'Neill et al. [21] by using interpolated data from a rectangular hyperbolic curve fitted to recovery measurements over time, reported relatively stable extracellular UA concentrations between 1 and 5 days following probe implantation. Thus, it appears that dialysis measurements of basal and pharmacologically modified extracellular concentrations of U A are reliable within the 24-72 h postimplantation interval. A higher intensity electrical stimulus as well as seizure activity elicited by the convulsant agent flurothyl also resulted in a two-fold increase of baseline UA concentrations. In contrast to the ECS-induced effects on striatal dopamine concentrations, which appear to be mediated by the passage of current [15,35], the present results suggest that the ECT-induced UA increase is independent of electrical dosage and it is related to the seizure activity per se. Local administration of tetrodotoxin or perfusion with a calcium-free solution failed to affect the basal or the ECS elevated concentrations of UA. These data indicate that neither the basal nor the stimulated UA concentrations are dependent on neuronal activation and exocytotic release. Previous microdialysis studies have indicated that the basal concentrations of monoaminergic neurotransmitters but not of their metabolites are both tetrodotoxin and calcium dependent [19,31,32]. The UA response to an ECT appears to be decreased in response to a second stimulus administered 2 but not 24 h after the first. Although changes in seizure threshold after ECT have been documented, the decreased U A response in these series of experiments was not associated with a concomitant decrease in seizure duration in response to the second stimulus. It is noteworthy that a similar reduction of the dopamine response to a second E C T given 2 but not 24 h after the first has previously been reported [35]. In accordance with earlier in vivo studies using either voltammetry or dialysis, systemic administration of allopurinol resulted in a pronounced (to about 20%
of baseline) decrease of basal UA concentrations [9,16J. Also, local infusion of allopurinol through the microdialysis probe markedly decreased UA concentrations to about 20% of baseline. This finding provides additional support for the notion that UA is formed locally in the brain [15]. Pretreatment with allopurinol given systemically, but not locally, completely abolished the ECS-induced elevation of dialysate UA concentrations. These results indicate that inhibition of the synthesis of UA only at a central level is not sufficient to prevent the rise in extracellular brain concentrations of UA observed following ECS. In view of the fact that baseline concentrations of the UA decreased to the same extent following the systemic or local administration of allopurinol, the possibility that the ECS induced increase in UA dialysate derives from a brain area not covered by the local administration of allopurinot, although it cannot entirely be excluded, seems highly unlikely. Consequently, it is plausible to conclude that the ECS-induced UA increase is of peripheral origin rather than a result of a general increase in brain metabolism. The concentrations of UA in rat plasma reported by different investigators under slightly varying conditions, range from 20-100 p,M [26]. The extracellular concentrations of UA in the rat brain are estimated by means of in vivo voltammetry and microdialysis in the range of 11-50 g M [9,21]. However, as argued by O'Neill et al. [21] these estimated concentrations are anomalously high and are most likely due to tissue perturbation by the relatively large in vivo probes, such as carbon paste electrodes and dialysis tubes. It is noteworthy, that in humans the ratio between plasma and CSF (that most closely reflects brain extracellular fluid) UA is t0-25 [6,26]. By extrapolation and if the ratio of plasma to extracellular fluid UA concentrations in man and the rat is the same, then the extracellular fluid concentrations of UA in the rat brain may be in the range of 1-10 p,M. Thus, it is plausible to argue that there exists a concentration gradient between blood and brain UA. This concentration gradient becomes even higher following ECS since UA serum concentrations increase in response to the treatment. The ECS-induced rise in serum UA concentrations is most likely due to enhanced energy use and purine metabolism associated with intense muscular activity. In this regard, it is noteworthy that high levels of serum and urinary UA have been found in patients with seizure disorders [28], and that overproduction of purines is often accompanied by dramatic CNS symptoms including seizures [5,14]. Given that ECS disrupts blood brain barrier permeability [2,25,30], the ECS-induced elevation of dialysate UA concentrations may reflect enhanced transport of U A through the cerebral endothelium. Allopurinol treatment greatly decreased both basal and stimulated concentrations of UA in the blood to
G.G. Nomikos et al. /Brain Research 660 (1994) 50-56
non-detectable levels, thus preventing the influx of UA acid into the brain during the ECS induced disruption of blood brain barrier permeability. Of some relevance here are observations indicating that brain UA increases as a result of experimentally induced ischemia [12] and that this increase is prevented by the systemic administration of allopurinol which also reduces ischemic brain injury [8,23,33]. Although the mechanism of action of allopurinol in reducing ischemic brain injury remains controversial [1] and ECT does not appear to be associated with structural brain damage [4,29], it may nevertheless be of interest to examine the effect of allopurinol pre-treatment on ECS/or ECT induced memory disruption.
Acknowledgements This work was supported by the Medical Research Council of Canada (PG-23 and MT-10615). G.D. was supported in part by the Netherlands Organization for the Advancement of Research (Z.W.O.). We would like to thank C. Tham and C. Wilson for technical assistance and Professor Venturella, Anaquest, Murray Hill, NJ for supplying Flurothyl (Indoclon).
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