Role of adenosine in drug-induced catatonia in mice

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1 Indian Journal of Experimental Biology Vol. 40, August 2002, pp Role of adenosine in drug-induced catatonia in mice Amanpreet Singh & S K Kulkarni* Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, , India Received 11 June 2001 ; revised 21 August 2001 Parkinson's disease is one of the most common neurodegenerative disorders affecting large majority of population ho are older than age of 65. Apart from dopamine, acetylcholine and glutamate, adenosine has also been identified in the basal ganglia. Adenosine modulates the release of a variety of neurotransmitters including dopamine. In order to establish adenosine-dopamine interactions in drug-induced catatonia e studied the effect of adenosine in drug-induced catatonia in mice. [n the present study adenosine dose dependently produced catatonia hen assessed on rota-rod and bar tests in mice. Adenosine also potentiated the catatonic effect of perphenazine. L-dopa plus carbidopa or OR-486 (a potent centrally acting COMT inhibitor) completely reversed adenosine-induced catatonia. Since reversal by scopolamine of adenosine-induced catatonia as not to the same extent as ith I-dopa and OR-486 it appears that catecholamines particularly dopamine rather than cholinergic modulation is more important in adenosine induced catatonia. The motor dysfunction (catatonia) could be easily assessed using rota-rod test apparatus in mice. Adenosine is knon to be present in the CNS. Adenosine receptors namely A2A are present in high concentration in striatum, nucleus accumbens and olfactory tubercule (regions hich are rich in dopamine) '. A2A and dopamine D2 receptors, hich are co-localized in subpopulation of striatal projection neurons, the GABAergic output neurons of striatopallidal pathay have shon to be interacting antagonistically on different levels. The A2A receptor agonists are reported to reduce the affinity for D2 receptors thus modulate D2 function 2.3. Adenosine modulates the release of variety of neurotransmitters both in vivo and in vitro. Adenosine has been shon to affect the release of norepinephrine, GABA, dopamine serotonin, acetylcholine, histamine, aspartate and glutamate. Although the mechanism by hich adenosine affects neurotransmission is not established it has been generally accepted that adenosine acts through presynaptic modulation of neurotransmitter release 4 5. The striatopallidal neuronal function seems to be mainly regulated not only by Au, receptors at the postsynaptic levels by direct antagonistic A2A-D2 and D2-A2A receptor-receptor interactions) but also at the presynaptic levels by an A2A receptor mediated regulation of acetylcholine release and, possibly, of GABA release. The evidence that stimulation of A2A receptors most probably localized in the striatal cho- *Correspondent author : Skpu@yahoo.com Fax : linergic intemeurons has been reported to induce acetylcholine release in striatal synaptosomal preparations 6 7 supports this. With this background the present ork as carried out to investigate adenosine-dopamine and adenosinecholinergic interactions. Various models to test the motor function, including the rota-rod apparatus as used as an approach for the evaluation of catatonic moments in animals and the fall-off time in this test as compared ith the other existing model of catatonia namely the bar immobility tests. Materials and Methods Animals-Laka mice of either sex (20-30g), bred in central animal house (CAR) of Panjab University, Chandigarh, maintained on a 12-h light and dark cycles ere used in the study. Animals ere housed under standard laboratory conditions, ith free access to food and ater. All behavioural experiments ere carried out beteen 0900 and 1400 hrs. The experimental protocol as approved by Institutional Animals Ethics Committee. Assessment of catatonia-using the folloing three-test procedures assessment of catatonia as done in animals. Rota-rod test-mice ere subjected to motor function evaluation by placing them individually on rota-rod 9, hich as adjusted to the speed of 21 r.p.m. The fall off time as recorded for each mouse and the longest time any animal as kept on the rod as 300 sec.

2 SINGH & KULKARNI: ADENOSINE IN DRUG-INDUCED CATATONIA 883 -In the bar test, front pa of the mice ere gently placed on a horizontal metal bar ith 2 mm diameter and placed 4 cm above ground level and the length of time the mouse maintained this abnormal posture ith at least one pa as measured 8. The test as terminated hen the pa of animal touched the ground or 180 sec had passed. If the animal did not hold on to the bar after three attempts, it received the score of 0 seconds. Drug treatment-the folloing drugs ere used in the present study. Adenosine (Loba Chemicals, Mumbai, India) as dissolved ith the aid of minimum quantity of hydrochloric acid, the volume as adjusted ith distilled ater, and finally ph as adjusted to neutral. Perphenazine (Schering Co., Kenilorth, NJ, USA) as dissolved ith the aid of minimum quantity of hydrochloric acid, the volume as adjusted ith distilled ater, and finally ph as adjusted to neutral. Scopolamine HBr (Merck & Co., Inc., NJ, USA) as dissolved in distilled ater. L dopa (Hi Media, Mumbai, India), carbidopa (Sun Pharmaceuticals, Mumbai, India), OR-486, (gift sample from Prof. P.T Mannisto Finland) ere suspended in 0.3% sodium CMC. Treatment schedule-all the drugs ere administered intraperitonially ip, in a constant volume of 1 ml per 100g of body eight of mice. Adenosine as administered 30 min prior to the behavioural assessment. Scopolamine, perphenazine, OR-486 as coadministered ith adenosine respectively. Carbidopa as co-administered ith adenosine as ell as perphenazine 15 min prior to the administration of L dopa, behavioural assessment as done 1 hr and 2 hr of L-dopa administration, respectively. Statistical analysis-the fall-off time in rota-rod test, the time spent in bar test ere expressed as mean± SE. The data as analyzed using analysis of variance (ANOYA) folloed by Dunnett's test by a statistical package STAT. In the test, the criterion for statistically significance as P<0.05. Results Effect of perphenazine on fall-off time from the rota-rod test and time spent on the bar in bar test Perphenazine (1-5 mg/kg, ip) decreased the fall-off time and increased the time spent by the animal on the bar in a dose dependent manner. Significant differences ere observed at all the doses as compared to the non-treated control group (Figs 1 A & B). Effect of adenosine on fall-off time and time spent on the bar in bar tests-adenosine ( mg/kg, ip) decreased the fall-off time and increased the time spent by the animal on the bar in a dose dependent manner. Significant differences ere observed at dose of (50-mg/kg, ip) and (100 mg/kg, ip) as compared to the non-treated control group (Figs 2 A & B). Adenosine (l0-100 mg/kg, ip) hen administered in combination ith perphenazine (5 mg/kg, ip) decreased the fall-off time and increased the time spent by the animal on bar in a dose dependent manner as compared to the perphenazine (5mg/kg, ip) alone treated group. Significant differences ere observed at various dose of (25, 50 and 100 mg/kg, ip) of adenosine (Figs 3 A & B). Effect of I-dopa and carbidopa combination on adenosine induced catatonia-l-dopa (100 mglkg, ip and 200 mg/kg, ip) and carbidopa (10 mg/kg, ip 20 mg/kg, ip) combination dose dependently increased the fall-off time hen given in combination ith adenosine (100 mg/kg, ip). Significant differences ere found after 1 hr of I-dopa administration as compared to the control (adenosine 100 mg/kg, ip) - I/) 250! 150 -II I/) -II 100!:z 80 Q. 60 I/) ::; i= (A) Rota-rod test CONTROL PPZ(1) PPZ(2.5) PPZ(5) a TREATMENT (mg /kg) (8) CONTROL PPZ(1) PPZ(2.5) PPZ(5).. TREA TMENT(mgIkg) Fig. 1-Effect of perphenazine (1-5 mg/kg) on (A) fall-off time (8) time spent 011 bar by mice. *P<0.05 as compared untreated conrrol group. ** P < 0.05 as compared perphelluzine (I mg/kg) treated group. (ANOvA folloed by Dunnett's test) a Animals did not hold on to the bar in three attempts. PPZ = perphenazine n=5

3 884 INDIAN J EX? BIOL, AUGUST 2002 alone treated group. Although there as no dose dependency after 2 hr but difference as stastically significant (Fig. 4). In the bar test this combination hen administered in combination ith adenosine (loa 350 ill' (A) Rota- rod test CONTROL AOE(10) ADE (25) ADE(50) ADE (100) TREATMENT (mgikg) (B) 100 f- 80 z (/) :;; 40 i= z 20 ;1i 0 a a a CONTROL ADE (10) ADE(25) ADE(50) AOE(100) Fig. 2 - TREATMENT(mglkg) Effect of adenosine ( mg/kg) on (A) fall off time, (B) time spent on bar by mice. * P < 0.05 as compared to the control group. **p < 0.05 as compared to the adenosine 50 mg/kg (ANOY A folloed by Dunnett's test). n=5 a Animal did not hold on to the bar in three attempts ADE = Adenosine mg/kg, ip) completely reversed adenosine-induced catatonia after 1 and 2 hr of I-dopa administration. The animals did not hold on to the bar in three attempts indicating a complete reversal (Table 1). Effect of l-dopa and carbidopa combination on adenosine plus perphenazine induced catatonia L-dopa (loa mg/kg, ip and 200 mg/kg, ip) and carbidopa (10 mg/kg, ip and 20 mg/kg, ip) combination increased the fall-off time hen administered along ith the combination of adenosine (loa mg/kg, ip) and perphenazine (5 mg/kg, ip) treated group. Although no dose dependency as observed but the effect as statistically significant after 1 and 2 hi' of I-dopa administration as compared to the control group, treated ith combination of adenosine (loa mg/kg,ip) plus perphenazine (5 mg/kg, ip) (Fig. 5). In the bar test I-dopa (loa mg/kg, ip) and carbidopa (10 mg/kg, ip) combination significantly decreased the time spent by the animal on the bar hen given along ith the combination of adenosine (loa mg/kg, ip) plus perphenazine (5 mg/kg, ip) after I hr of I-dopa administration as compared ith the control group treated ith the combination of adenosine (loa mg/kg, ip) plus perphenazine (5 mg/kg, ip). There as a complete reversal seen after 2 hi' of I-dopa administration ith this dose. L-dopa (200 mg/kg, ip) and carbidopa (20 mg/kg, ip) combination hen administered along ith the combination of adenosine (loa mg/kg, ip) plus perphenazine (5 mg/kg, ip) completely reversed adenosine (loa mg/kg, ip) plus perphenazine (5 mg/kg, ip). Induced catatonia after 1 and 2 hr of I-dopa administration as the animal ere not able to hold on to the rod in three attempts (Table 2). PPZ(5) (A)Rota-rod test (B)Bartest E i=' Z 0:::... ADE(IOO) PPZ(S)ADE(IO) PPZ(5}+ADE(2S) Ppz(S}+ADE(SO) * * Ppz(S}+ADE(IOO) * MEAN FALL- OFF TIME (sec tse) MEAN TIME SPENT (sec tsem) Fig. 3-Effect of adenosine ( mg/kg) on perphenazine (5 mg/kg) induced catatonia in (A) rota rod test (B) bar test. *P<0.05 as compared to control (perphenazine)(anoy A folloed by Dunnett's test). n = 5-10 P?Z = perphenazine ADE=adenosine

4 SINGH & KULKARNI: ADENOSINE IN DRUG-INDUCED CATATONIA 885 OAdeno.lne(I00mg/kg) 350.Adeno.Ine(IOOmg1kg}+Ldopa(lOCln'1Wkg)+C8I1lkIop8 (10 tnwkg) 300 W BAdeno.Ine(I00mIVkll}+L-d0pa(200)+ II) +I 250 carbidopa (20 rnwku)! 200 i= oj oj 50 z 0 2 Test time (hr) Fig. 4-Effect of combination of L-dopa (100 mg/kg, 200 mglkg) and carbidopa (10 mg/kg, 20 mglkg) on adenosine induced catatonia in rota-rod test. *P<0.05 as compared to the control adenosine (100 mg/kg) at respective time intervals.(anoy A folloed by Dunnett's test). n = 6 Effect of OR-486 on adenosine-induced catatonia OR-486 (30 mglkg, ip) significantly increased the fall-off time and hen it as administered in combination ith adenosine (100 mglkg, ip) as compared to adenosine (100 mg/kg, ip) alone treated group (Fig. 6). In the bar test OR-486 (30 mglkg, ip) completely reversed adenosine (100 mg/kg, i.p)- induced catatonia hen it as administered in combination ith adenosine (100 mglkg, ip) as compared ith adenosine (100 mglkg, ip) treated group. The animals ere not able to hold on to the rod in three attempts (Table 3). Effect of scopolamine on adenosine-induced catatonia-scopolamine (1 mglkg, ip and 2 mglkg, ip) increased the fall-off time in a dose dependent manner hen administered in combination ith adenosine (100 mglkg, ip). Significant difference as observed at dose of 2 mglkg, ip as compared to the adenosine (100 mglkg, ip) alone treated group (Fig. 7). In the bar test scopolamine (1 mglkg, ip) did not decrease the time spent by the animal on the bar hen adminis- Table I-Effect of combined treatment of L-dopa and carbidopa on adenosine induced catatonia in mice as tested on bar test Treatment (mg/kg) Adenosine (100) Adenosine (100) after 1 hr Adenosine (100) +L-dopa (100) +carbidopa (10) after Ihr Adenosine (100) +L-dopa (200) +carbidopa (20) after I hr Adenosine (100) after 2 hr Adenosine (100) +L-dopa (100) +carbidopa (10) after 2 hr Adenosine (100) +L-dopa (200) +carbidopa (20) after 2 hr 85.2 ± ± ± 5.2 ** Animal did not hold the bar in three attempts. Table 2-Effect of combined treatment of L-dopa and carbidopa on adenosine plus perphenazine induced catatonia in bar test Treatment (mglkg) Adenosine (100) + PPZ ( 5) Adenosine (100) + PPZ (5) after 1 hr Adenosine (100)+ PPZ (5) +L-dopa (100) +carbidopa (10) after Ihr Adenosine (100)+ PPZ (5) +L-dopa (200) +carbidopa (20) after Ihr Adenosine (100) + PPZ (5) after 2 hr Adenosine (100) + PPZ (5) +L-dopa (100) +carbidopa (10) after 2hr Adenosine (100)+ PPZ (5) +L-dopa (200) +carbidopa (20) after 2hr 175.8± ±4.56 Reversed ** Reversed ** 154.4±9.64 Reversed ** 92.6±2.6* *P<0.05 as compared to the control group (adenosine 100 mg/kg + perphenazine 5mglkg) (ANOYA folloed by Dunnett's test) **Animal did not hold onto the bar in three attempts.

5 886 INDIAN J EXP BIOL, AUGUST II) -+125! 20 l- ll.. ll.. o...j 15..J ;t z i5 10 ::le 5 Rota-rod te.t C Adenosine(1 OOmg/kg)+per phenazine(5rn!vkil) Adenosine(1 OOmg/kIl)+per phenazine(5rn!vkil)+ldopa(100mg/kg)+carbidop a (10 mg!kll) "'Adenosine(100mg/k1l)+per phenazine(5mgikg)+ldopa(200)+catbidopa( 20 mglkll) O+-J Test time(hr) Fig. S.-Effect of combination of L-dopa (100 mglkg, 200 mglkg) and carbidopa (10 mg/kg, 20 mglkg) on adenosine (100 mglkg) + perphenazine (Smg/kg) induced catatonia in rota-rod test. *p < 0.05 as compared to the control (adenosine +perphenazine l at respective time intervals.(anoya folloed by Dunnett's test) n = S 250 CIJ -ti g 200 Rota-rod test * ::E 150 t= c;> J ;J, 50 ::E 0 Adenosine (100) Adenosine (100)+ OR466(30) TREA TMENT(mglkg) Fig. 6-Effect of OR-486 on adenosine induced catatonia in rotarod test. * P < O.OS as compared to the control (adenosine). (ANOY A folloed by Dunnett's) n = S tered in combination ith adenosine (100 mg/kg, ip). Scopolamine (2 mg/kg, ip) hen administered in combination ith adenosine (l00 mg/kg, ip) significantly reversed adenosine (100 mg/kg, ip) induced catatonia. The animals did not hold on to the rod in three attempts (Table 4). When scopolamine (2 mg/kg, ip) as administered to the animals receiving various doses of adenosine (10, 25, 50 mg/kg, ip) and perphenazine (5 mg/kg, i.p), there as an increase in the fall-off time significant effect being observed at doses of 10 and 25 mg of adenosine (Fig. 8 A). Although there as a decrease in the time spent by the animal on the bar, the 90 W 80 II) -+I u <II W 60 :!: i= J -' 30 ll.. Z 20 i5 :!: 10 0 Rota rod test ADENOSINE (100) ADENOSINE ADENOSINE (100) (1oo).seo (1).seo (2) TREATMENT(mglkg) Fig. 7-Effect of scopolamine (1-2 mglkg) on adenosine induced catatonia in rota rod test. *P<0.05 as compared to control (adenosine) (ANOY A folloed by Dunnett's test. seo = scopolamine n=s Table 3-Effect of OR-486 on adenosine induced catatonia in bar test in mice Treatment (mglkg) Adenosine (100) Adenosine (100) +OR 486 (30) **Animal did not hold the bar in three attempts 8S.2±11.07 Table 4-Effect of scopolamine on adenosine induced catatonia in bar test in mice Treatment (mglkg) Adenosine (100) Adenosine (100) +Scopolamine (I) Adenosine (100) +Scopolamine (2) ** Animal did not hold onto the bar in three attempts 8S ± response as not statistically significant as compared to their respective combinations per se (Fig 8 B). Discussion Since adenosine A2A and dopamine D2 receptors hich are co-localized in a sub-population of striatalprojection neurons, the GABAergic output neurons of the striatopallidial pathay have been shon to interact antagonistically on different levels' A2A receptor agonists are reported to reduce the affinity of dopamine agonist for D2 receptors and thus modulate D2 function 2,3. The result of our study indicated that adenosine dose dependently (l0-100 mg/kg, ip) produced catatonia as indicated by rota-rod! bar tests. Adenosine

6 SINGH & KULKARNI: ADENOSINE IN DRUG-INDUCED CATATONIA 887 (A) Rota-rod test (B) ppz (5) +ade (10) 1--_... ppz (5) +ade (10)+ sco (2) * ppz(5)+ade(2s) Z W :E ppz (5) +ade (25) + sco (2) S)+adeSO.- ppz (5) +ade( 50) + sco( 2) t--... o o MEAN F ALL- OFF TIME (sec± SE) MEAN TIME SPENT Fig. 8-Effect of scopolamine (2 mg/kg) on perphenazine (5 mglkg)+adenosine (10-50 mglkg) induced catatonia in (A) rota-rod test (8) bar test. P <0.05 as compared to control (perphenazine plus adenosine). (ANOYA folloed by Dunnett's test). ppz = perphenazine, ADE = adenosine sco= scopolamine n = 5 also potentiated the perphenazine-induced catatonia in a dose dependent manner. In accordance ith the above explanation adenosine might have acted on the adenosine A2A receptors in the striatum co-localized ith D2 receptors and thus causing a decreased affinity of dopamine toards the dopamine D2 receptors leading to motor dysfunction of the dopamine D2 receptors. This ould again lead to disinhibition of negative control of dopamine D2 receptors toards the indirect pathay leading to the increased activity of indirect pathay, and hence a decreased movement by indirect modulation of cortex as observed in the animals in the form of catatonia. When adenosine is co-administered along ith perphenazine, decreased function of the dopamine D2 receptors already existing due to the blockade by perphenazine, adenosine potentiated this decreased function by possibly reducing the affinity of dopamine toards the D2 receptors and causing severe catatonic effect. The striatopallidal neuronal function seems to be mainly regulated not only by A2A receptors at the postsynaptic levels (by direct antagonistic A2A-D2 receptor-receptor interactions) but also at the pre synaptic level by an A2A receptor mediated regulation of acetylcholine release. The evidence that stimulation of A2A receptors, most probably localized on the striatal cholinergic interneurons, has been reported to induce acetylcholine release in striatal synaptosomal preparations supports this 6 : In accordance ith the above explanation scopolamine might have acted on postsynaptic muscaranic receptors, co-localized along ith dopamine O2 and adenosine A2 receptors in the striatopallidal pathay in basal ganglia. The blockade of postsynaptic muscaranic receptors ould have attenuated the effects due to increased pre-synaptic acetylcholine release, resulting from the stimulation of A2 receptors by adenosine. The mechanism by hich adenosine affects neurotransmission is not fully established although it is generally accepted that adenosine acts through presynaptic modulation of neurotransmitter release. Al receptors are located pre and post synaptically on cell bodies, and on axons here they mediate inhibition of the neurotransmission, by the inhibition of the neurotransmitter release, hyperpolarizing neuronal membranes, reducing excitability and firing rate and altering axonal neurotransmission 4 When the combination of I-dopa and a peripheral DOPA decarboxylase inhibitor, carbidopa as administered ith adenosine, a complete reversal of adenosine as observed. The reversal as far better as compared to scopolamine as ell as OR-486 (central COMT inhibitor), as indicated by both bar and rota-rod test. L-dopa hen given in combination ith peripheral DOPA decarboxylase inhibitor carbidopa gets decarboxylated into dopamine, thus increasing the levels of dopamine. These increased levels of dopamine ould have restored the depleted levels of dopamine due to adenosine. OR-486, a centrally acting COMT inhibitor hen administered along ith the adenosine completely reversed adenosine-induced catatonia. OR-486

7 888 INDIAN J EXP BIOL, AUGUST 2002 might have inhibited the metabolism of catecholamines thus further increasing the levels of dopamine. When it as administered along ith adenosine it completely reversed the adenosine effect. Thus this study confirms the potent adenosine-catecholamine, particularly adenosine-dopamine interactions. This study further gives clues to the use of adenosine A2A receptor antagonist as neer therapeutic agents in drug therapy of Parkinson's disease 13. Acknoledgement The authors appreciate Prof. P.T.Mannisto of University of Kupio, Kupio, Finland for generously providing COMT inhibitors for the study. References I Ongini E & Fredholm B B, Pharmacology of adenosine A2A receptors. Trends Pharmacol Sci, 17 (1996) Richarson P J, Kase H & Jenner P J, Adenosine A2A receptor antagonist as ne agents for the treatment of Parkinson's disease. Trends Pharmacol Sci, 18 (1997) Ferre S, Fredholm B B, Morelli M, Popoli P& Fuxe K, Adenosine-dopamine receptor- receptor interactions as an integrative mechanism in the basal ganglia. Trends Neurosci, 20 (1997) Kulkarni S K & Thorat S N, Purinergic transmission: An update. Drugs Of Today, 26 (1990) Fredholm B B & Duniddie T V, Ho does adenosine inhibit transmitter release? Trends Pharmacol Sci, 5 (1988) Ralevic V & Burnstock G, Receptors for Purines and Pyrimidines. Pharmacol Rev, 50 (1998) Jin S, Johansson B & Fredholm B B, Effects of adenosine AI and A2 receptor activation on electrically evoked dopamine and acetylcholine release from rat striatal slices. J Pharmacol ExpTher, 267 (1993) Costall B & Naylor R J, On catalepsy and catatonia and the predictability of the catalepsy test for neuroleptic activity. Psychopharmacology, 34 (1974b) Dunham N W & Miya T S, A note on a simple apparatus for detecting neurological deficits in rats and mice. JAm Pharm Ass, 46 (1957) Nikodijevic 0, Sarges R, Daly J W & Jacobson K A, Behavioural effects of AI and A2A- selective agonist and antagonists: Evidence for synergism and antagonism. J Pharmacol Exp Ther, 259 (1991) 286. II Jain N, Kemp N, Adeyemo 0, Buchanan P & Stone T W, Anxiolytic activity of adenosine receptor activation in mice. Br J Pharmacol, 116 (1995) Mahlotra J & Gupta Y K, Effect of adenosine receptor modulation on pentylenetetrazole-induced seizures in rats. Br J Pharmacol, 120 (1997) Muller C E, A2A adenosine receptor antagonist-future drugs for Parkinson's disease? Drugs Of The Future, 25(10) (2000) 1043.