- Email: [email protected]
The effects of prefrontal intracortical microinjections of an alpha-2 agonist, alpha-2 antagonist and lidocaine on the delayed alternation performance of aged rats
BrainResearchBulletin,Vol.40, No. 2, pp. 117-119, 1996 Copyright© 1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0361-9230/96 $15.00 + .00 ELSEVIER
PII 0361-9230(96) 00026-3
The Effects of Prefrontal Intracortical Microinjections of an Alpha-2 Agonist, Alpha-2 Antagonist and Lidocaine on the Delayed Alternation Performance of Aged Rats HEIKKI TANILA, PIA R~,M~, AND SYNN£)VE CARLSON1
Institute of Biomedicine, Department of Physiology, P.O. Box 9, FIN-O0014, University of Helsinki, Helsinki, Finland [Received 11 October 1995; Revised 28 December 1995; Accepted 3 January 1996] ABSTRACT: A selective alpha-2 agonist medetomidine, a selective alpha-2 antagonist atipamezole and lidocaine were injected into the prefrontal cortex of aged rats that had been trained to perform a delayed alternation (DA) task in a T maze. Medetomidine at the dose of 0.01 /zg//zl improved the memory task performance in four of five rats. The fifth rat improved its performance at the dose of 1.0/~g//~l. Atipamezole did not have any effect on the task performance. The effect of a systemic dose of medetomidine (3/~g/kg), which was previously shown to improve the task performance in old rats, was reversed by an intracortical injection of atipamezole. Lidocaine impaired the DA performance significantly. The results suggest that the beneficial effects of medetomidine on the working memory of old rats are mediated at least partly through the prefrontal cortex.
wall of the frontal cortex in rats during the delayed performance on the U-shaped maze [6]. Ablations of the medial PFC impair retention of delayed performance in rats [11,33]. However, it is also suggested that the medial cortex of rats corresponds to the primate premotor and anterior cingulate cortices [ 25 ]. The aims of the present work were to study whether the beneficial effect of an alpha-2 agonist, medetomidine, is mediated through the PFC in the rat and whether an intracortical injection of an alpha2 antagonist, atipamezole, into the PFC could reverse the effect of systemically administered medetomidine. The alpha-2 agonist, medetomidine, and the alpha-2 antagonist, atipamezole, are highly selective to alpha-2 adrenoceptors. The alpha-2/alpha-1 receptor binding selectivity ratios of these compounds are much higher than those of the respective reference compounds, clonidine and yohimbine [30,31].
KEY WORDS: Alpha-2 adrenergic, Prefrontal cortex, Delayed alternation task, Aged rats.
METHODS INTRODUCTION The age-related decline in central catecholaminergic function [12,17,20] has been shown to be related to age-associated memory impairments in rats and mice [20,26] and monkeys [32]. Alpha-2 agonists have been shown to alleviate the age-related impairment in the delayed response (DR) [ 1,2 ] and delayed nonmatching to sample tasks of aged monkeys [ 19 ]. Earlier, we have shown that medetomidine, a highly selective alpha-2 agonist, improved at a dose of 3.0/zg/kg the performance of aged rats in the delayed alternation (DA) task, whereas it had no effect on the performance of young adult rats [9]. There are also contradictory results of the effects of alpha-2 agonists on working memory in monkeys [5,10]. The prefrontal cortex (PFC) has been shown to be involved in spatial working memory processes in monkeys [14,18]. There is evidence that the beneficial effects of alpha-2 agonists are due to their action on the PFC in the monkey [1]. Regional depletion of catecholamines by 6-hydroxydopamine in the PFC of rhesus monkeys produces inability to perform DR task [8,27]. This inability can be reversed by the alpha-2 agonist clonidine [1,8 ]. The prelimbic sector of the PFC has been shown to be essential for the DA performance in rats [7]. Spatially selective neuronal activity has been recorded in the medial
In the present study, we used the same eight male Wistar rats as in the previous longitudinal study [9]. However, two of the rats died during the anesthesia, and one had to be rejected because of an obstruction in the cannula. The age range of the rats was 20-24.5 months (mean 22.0 months) and they weighed 435-485 g. The rats had free access to water but were kept on a food restriction schedule to maintain body weight at 85% of the free feeding level. The rats were anesthetized with pentobarbital 4 0 - 5 0 mg/kg ip and positioned in the stereotaxic frame with the incisor bar 2.3 mm below the interaural line. A pair of guide cannulae were made from 22-gauge injection needles and cut to 12-mm-long pieces. They were positioned above the PFC at A + 4.0 mm anterior to the bregma, L ___0.9 mm lateral to the ~midline, V 0.0 mm ventral to the dura and fixed to the skull with ~jeweller's screws and dental cement. After the rats had fully recovered from the operation (after 3 - 5 days), they were given 1-3 daily sessions of 20 trials of DA training until they reached their preoperative performance level. The testing procedure has been described in detail in our previous paper [9]. In brief, the rat was placed in the starting compartment of the T maze where it waited 3 sec until the guillotine door to the maze was opened. The rat then entered the stem of
l To whom requests for reprints should be addressed. 117
118
TANILA, RAM~, AND CARLSON
FIG. 1. The injection sites based on glial reaction in the brain sections of five rats are plotted on standard sections of the PFC at the level of 4.70 mm anterior to the bregma [24]. the T maze and turned to either of the arms, after which the arm was blocked by the guillotine door. The rat was rewarded by a piece of a standard food pellet for entering the arm that was not chosen in the previous trial. During the delay period, the rat was placed into a round bucket without any view of the maze. Four alternating delays were used. The shortest one was 10 sec for all rats, and the longest one varied between 5 and 25 min and corresponded to just above chance level performance. The intermediate delays were adjusted so that the overall performance of the rats was about 65% correct choices in the 20 trials of the day. The effects of medetomidine (Domitor I m g / m l ) , atipamezole (Antisedan 5 mg/ml, both synthesized by the Farmos Group Ltd., Orion Pharmaceuticals Inc., Turku, Finland), and lidocaine (Lidocain 40 mg/ml, Medipolar, Orion Pharmaceuticals Inc., Oulu, Finland) were studied. The rats received bilateral microinjections of medetomidine (0.01 and 1.0 # g / # l ) , atipamezole (0.05 and 5.0 # g / # l ) , lidocaine (10 # g / # l ) or saline. Also, the effect of combined injections of atipamezole, 0.05 #g//A ic, and medetomidine, 3.0 # g / k g im, was tested. Before the intracortical injections, the rats were tested twice on saline to assess the effect of the intracortical injection itself on the task performance. The volume of each intracortical injection was 1 #1, which was injected with a Hamilton l-#l syringe through a 14-mm-long, 27gauge stainless steel cannula over 30 sec. A further 30 sec was allowed for diffusion before the injection cannula was withdrawn. The microinjections were given 10 rain and the intramuscular injections 15 min before the behavioral testing. The effects of the seven treatments were tested twice in each rat over 3 weeks. The experimenter was unaware of the nature of the injections except of the combined ic and im injections that were always given last in the sequence. At the completion of the experiment, the rats were killed by decapitation, the skulls were opened and the heads were immersed in formalin. The brains were removed, sectioned in 150-/.tin slices and stained with cresyl violet to verify the placement of the cannulae.
subject, drug treatment and the testing period. There were significant main effects of the treatment ( F ( 6 ) = 2.7, p < 0.04) and the testing period so that most rats performed better during the second than the first testing period ( F ( 1 ) = 16.7,p < 0.001 ). Intracortically injected medetomidine improved the DA performance in each of the five rats. The dose of 0.01 # g / # l improved the performance in four rats, whereas the fifth rat improved its performance at the dose of 1 /zg//A. The most beneficial dose of medetomidine for each rat improved the DA task performance significantly (t(4) = 4.24, p < 0.02; Fig. 2). Atipamezole did not have significant effects on the performance (0.05 # g / # l , t(4) = 0.19, p = 0.85; 5.0 # g / # l , t(4) = 1.73, p = 0.16). Lidocaine impaired the performance in all five rats ( t ( 4 ) = 2.99, p < 0.05; Fig. 2). The dose of 3 # g / k g of medetomidine was previously shown to improve the memory task performance of old rats [9]. Intracortical injection of a low dose of atipamezole (0.05 # g / # l ) , which itself did not impair the task performance, reversed the beneficial effect of a systemic administration of medetomidine at a dose of 3 # g / k g in all but one rat ( in which the performance remained improved despite of ic atipamezole) and therefore the performance of the rats did not differ significantly from that on saline ( t ( 4 ) = 0.678, p = 0.53). DISCUSSION In the present study, prefrontal intracortical injection of an alpha-2 adrenoceptor agonist, medetomidine, improved the DA task performance of aged rats, whereas an alpha-2 adrenoceptor antagonist, atipamezole, had no effect. The medial PFC in the rat is an essential structure for DA performance [7]. This was further verified in the present study by the deterioration of DA performance after intracortical administration of lidocaine. The improvement of performance in the working memory task by alpha-2 agonist, medetomidine, may be related to the declined levels of central monoaminergic and cholinergic neurotransmitters in aged rats [21,22]. The decline of noradrenaline levels has been demonstrated in basal forebrain nuclei and in frontal and entorhinal
16 ,,,,,i
12
o
8
"~
4
0)
//
//
O
N
-4
//
Et --12
RESULTS Histological sections indicated that the guide cannulae were well positioned for injections into the prelimbic sector of the PFC (Fig. 1 ). Intracortical injection of saline did not produce a significant change in the performance from the baseline levels ( t ( 4 ) = 2.25, p < 0.10). Multivariate repeated measures analysis of variance was used for testing the main effects of the factors:
-16
u
i
M
L
l
FIG. 2. The m e a n effects o f the beneficial d o s e s o f m e d e t o m i d i n e (M) (0.01 ~ g / # l t o r f o u r rats a n d 1.0 ~zg/#l for one rat) a n d o f lidocaine (L) (10 pg/~l). *p < 0.05, **p < 0.02.
EFFECTS OF P R E F R O N T A L I N T R A C O R T I C A L MICROINJECTIONS
cortical areas [ 22 ] and shown to be related to age-associated memory impairement [23,26]. The results of the present study as well as our earlier finding that medetomidine improved the DA task performance of aged rats but not that of young adult rats [ 9 ] are in line with the abovementioned studies. In our studies, systemically administered atipamezole had no significant effect on the DA task performance in rats [9 ] or on the DR task performance in monkeys [28]. Intraperitoneally injected afipamezole has been shown to increase the central turnover of noradrenaline (NA) at doses 3.0 mg/ kg and higher by blocking presynaptic release-inhibiting receptors [29]. Intracortically injected atipamezole in the present study had no effect on the task performance of aged rats. In monkeys, it has been shown that systemically injected alpha-2 antagonist, yohimbine, increases age-associated memory impairement [1]. However, recently, Arnsten and Cai [4] showed that yohimbine at low doses improved working memory task performance in a subset of aged monkeys, and it was suggested that the improvement might be due to increased NA release on to postsynaptic alpha-2 receptors and might depend on an intact NA system. The role of the prefrontal cortex of monkeys in spatial working memory functions is well documented in electrophysiological and lesion studies [13,15,16]. It has been suggested that the effects of alpha-2 agonists on memory performance of aged monkeys is mediated through postsynaptic alpha-2 adrenoceptors in the frontal cortex [1,8]. The inability to perform the DR task after regional depletion of catecholamines by 6-hydroxydopamine in the PFC can be reversed by clonidine [ 8 ]. The present study provides direct evidence of a prefrontally mediated action of an alpha-2 agonist in rats. Intracortically administered medetomidine improved the DA task performance and intracortical injection of an alpha-2 antagonist, atipamezole, reversed the beneficial effect of the systemically administered medetomidine. The present results support the idea that the beneficial effect of alpha2 agonists on the working memory performance of aged animals is mediated at least partly through the PFC.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
REFERENCES 1. Arnsten, A. F. T.; Goldman-Rakic, P. S. Alpha-2 adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates. Science 230:1273-1276; 1985. 2. Arnsten, A. F. T.; Cai, J. X.; Goldman-Rakic, P. S. The alpha-2 adrenergic agonist guanfacine improves memory in aged monkeys without sedative or hypotensive side effects: Evidence for alpha-2 receptor subtypes. J. Neurosci. 8:4287-4298; 1988. 3. Amsten, A. F. T.; Leslie, F. M. Behavioral and receptor binding analysis of the alpha2-adrenergic agonist, 5-bromo-6 [2-imidazoline-2-yl amino] quinoxaline (UK-14304): Evidence for cognitive enhancement at an alpha2-adrenoceptor subtype. Neuropharmacology 30:1279-1289; 1991. 4. Arnsten, A. F. T.; Cai, J. X. Postsynaptic alpha-2 receptor stimulation improves memory in aged monkeys: Indirect effects of yohimbine versus direct effects of clonidine. Neurobiol. Aging 14:597603; 1993. 5. Bartus, R. T.; Dean, R. L. Lack of efficacy of clonidine on memory in aged Cebus monkeys. Neurobiol. Aging 9:409-411; 1988. 6. Batuev, A. S.; Kursina, N. P.; Shutov, A. P. Unit activity of the medial wall of the frontal cortex during delayed performance in rats. Behav. Brain Res. 41:95-102; 1990. 7. Brito, G. N. O.; Thomas, G. J.; Davis, B. J.; Gingold, S. I. Prelimbic cortex, mediodorsal thalamus, septum, and delayed alternation in rats. Exp. Brain Res. 46:52-58; 1982. 8. Brozoski, T. J.; Brown, R. M.; Rosvold, H. E.; Goldman, P. S. Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929-932; 1979. 9. Carlson, S.; Tanila, H.; R~na~i,P.; Mecke, E.; Pertovaara, A. Effects of medetomidine, an alpha-2 adrenoceptor agonist, and atipamezole,
24. 25. 26. 27.
28. 29. 30. 31. 32. 33.
119
an alpha-2 antagonist, on spatial memory performance in adult and aged rats. Behav. Neural Biol. 58:113-119; 1992. Davis, R. E.; Callahan, M. J.; Downs, D. A. Clonidine disrupts agedmonkey delayed response performance. Drug Dev. Res. 12:279286; 1988. Dunnett, S. B. Role of prefrontal cortex and striatal output systems in short-term memory deficits associated with ageing, basal forebrain lesions, and cholinergic-rich grafts. Can. J. Psychol. 44:210-232; 1990. Estes, K. S.; Simpkins, J. W. Age-related alterations in catecholamine concentrations in discrete preoptic area and hypothalamic regions in the male rat. Brain Res. 194:556-560; 1980. Funahashi, S.; Bruce, C. J.; Goldman-Rakic, P. S. Mnemonic coding of visual space in the monkey's dorsolateral prefrontal cortex. J. Neurophysiol. 61:331-349; 1989. Funahashi, S.; Kubota, K. Working memory and prefrontal cortex. Neurosci. Res. 21:1-11; 1994. Fuster, J. M. Unit activity in prefrontal cortex during delayed-response performance: Neuronal correlates of transient memory. J. Neurophysiol. 36:61-78; 1973. Goldman, P. S.; Rosvold, H. E. Localization of function within the dorsolateral prefrontal cortex of the rhesus monkey. Exp. Neurol. 27:291-304; 1970. Goldman-Rakic, P. S.; Brown, R. M. Regional changes of monoamines in cerebral cortex and subcortical structures of aging rhesus monkeys. Neuroscience 6:177-187; 1981. Goldman-Rakic, P. S. Cellular basis of working memory. Neuron 14:477-485; 1995. Jackson, W. J.; Buccafusco, J. J. Clonidine enhances delayed matching-to-sample performance by young and aged monkeys. Pharmacol. Biochem. Behav. 39:79-84; 1991. Leslie, F. M.; Loughin, S. E.; Stenberg, D. B.; McGaugh, J. L.; Young, L. E.; Zornetzer, S. F. Noradrenergic changes and memory loss in aged mice. Brain Res. 359:292-299; 1985. Luine, V.; Hearns, M. Spatial memory deficits in aged rats: Contributions of the cholinergic system assessed by CHAT. Brain Res. 523:321-324; 1990. Luine, V.; Bowling, D.; Hearns, M. Spatial memory deficits in aged rats: Contributions of monoaminergic systems. Brain Res. 537:271-278; 1990. Markowska, A. L.; Stone, W. S.; Ingram, D. K.; Reynolds, J.; Gold, P. E.; Conti, L. H.; Ponteccorvo, M. J.; Wenk, G. L.; Olton, D. S. Individual differences in aging: Behavioral and neurobiological correlates. Neurobiol. Ageing 10:31-43; 1989. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Sydney, London and New York: Academic Press. Preuss, T. M. Do rats have prefrontal cortex? The Rose-WoolseyAkert Program reconsidered. J. Cogn. Neurosci. 7:1-24; 1995. Rapp, P. R.; Rosenberg, R.; GaUagher, M. An evaluation of spatial information processing in the aged rats. Behav. Neurosci. 101:3-12; 1987. Roberts, A. C.; De Salvia, M. A.; Wilkinson, L. S.; Collins, P.; Muir, J. L.; Everitt, B. J.; Robbins, T. W. 6-Hydroxydopamine lesions of the prefrontal cortex in monkeys enhance performance on an analog of the Wisconsin card sort test: Possible interactions with subcortical dopamine. J. Neurosci. 14:2531-2544; 1994. R~imii, P.; Linnankoski, I.; Tanila, H.; Pertovaara, A.; Carlson, S. Medetomidine, atipamezole and guanfacine in delayed response performance of aged monkeys. Submitted. Scheinin, H.; MacDonald, E.; Scheinin, M. Behavioural and neurochemical effects of atipamezole, a novel alpha-2 adrenoceptor antagonist. Eur. J. Pharmacol. 151:35-42; 1988. Virtanen, R.; Savola, J. M.; Saano, V.; Nyman, L. Characterization of the selectivity, specificity and potency of medetomidine as an alpha-2 adrenoceptor agonist. Eur. J. Pharmacol. 150:9-14; 1988. Virtanen, R.; Savola, J. M.; Saano, V. Highly selective and specific antagonism of central and peripheral alpha-2 adrenoceptors by atipamezole. Arch. Int. Pharmacodyn. Ther. 297:190-204; 1989. Walker, L. C.; Kitt, C. A.; Struble, R. G.; Wagster, M. V.; Price, D. L ; Cork, L. C. The neural basis of memory decline in aged monkeys. Neurobiol. Ageing 9:657-666; 1988. Wikmark, R. G. E.; Divac, I.; Weiss, R. Retention of spatial delayed alternation in rats with lesions in the frontal lobes. Brain Behav. Evol. 8:329-339; 1973.
PII 0361-9230(96) 00026-3
The Effects of Prefrontal Intracortical Microinjections of an Alpha-2 Agonist, Alpha-2 Antagonist and Lidocaine on the Delayed Alternation Performance of Aged Rats HEIKKI TANILA, PIA R~,M~, AND SYNN£)VE CARLSON1
Institute of Biomedicine, Department of Physiology, P.O. Box 9, FIN-O0014, University of Helsinki, Helsinki, Finland [Received 11 October 1995; Revised 28 December 1995; Accepted 3 January 1996] ABSTRACT: A selective alpha-2 agonist medetomidine, a selective alpha-2 antagonist atipamezole and lidocaine were injected into the prefrontal cortex of aged rats that had been trained to perform a delayed alternation (DA) task in a T maze. Medetomidine at the dose of 0.01 /zg//zl improved the memory task performance in four of five rats. The fifth rat improved its performance at the dose of 1.0/~g//~l. Atipamezole did not have any effect on the task performance. The effect of a systemic dose of medetomidine (3/~g/kg), which was previously shown to improve the task performance in old rats, was reversed by an intracortical injection of atipamezole. Lidocaine impaired the DA performance significantly. The results suggest that the beneficial effects of medetomidine on the working memory of old rats are mediated at least partly through the prefrontal cortex.
wall of the frontal cortex in rats during the delayed performance on the U-shaped maze [6]. Ablations of the medial PFC impair retention of delayed performance in rats [11,33]. However, it is also suggested that the medial cortex of rats corresponds to the primate premotor and anterior cingulate cortices [ 25 ]. The aims of the present work were to study whether the beneficial effect of an alpha-2 agonist, medetomidine, is mediated through the PFC in the rat and whether an intracortical injection of an alpha2 antagonist, atipamezole, into the PFC could reverse the effect of systemically administered medetomidine. The alpha-2 agonist, medetomidine, and the alpha-2 antagonist, atipamezole, are highly selective to alpha-2 adrenoceptors. The alpha-2/alpha-1 receptor binding selectivity ratios of these compounds are much higher than those of the respective reference compounds, clonidine and yohimbine [30,31].
KEY WORDS: Alpha-2 adrenergic, Prefrontal cortex, Delayed alternation task, Aged rats.
METHODS INTRODUCTION The age-related decline in central catecholaminergic function [12,17,20] has been shown to be related to age-associated memory impairments in rats and mice [20,26] and monkeys [32]. Alpha-2 agonists have been shown to alleviate the age-related impairment in the delayed response (DR) [ 1,2 ] and delayed nonmatching to sample tasks of aged monkeys [ 19 ]. Earlier, we have shown that medetomidine, a highly selective alpha-2 agonist, improved at a dose of 3.0/zg/kg the performance of aged rats in the delayed alternation (DA) task, whereas it had no effect on the performance of young adult rats [9]. There are also contradictory results of the effects of alpha-2 agonists on working memory in monkeys [5,10]. The prefrontal cortex (PFC) has been shown to be involved in spatial working memory processes in monkeys [14,18]. There is evidence that the beneficial effects of alpha-2 agonists are due to their action on the PFC in the monkey [1]. Regional depletion of catecholamines by 6-hydroxydopamine in the PFC of rhesus monkeys produces inability to perform DR task [8,27]. This inability can be reversed by the alpha-2 agonist clonidine [1,8 ]. The prelimbic sector of the PFC has been shown to be essential for the DA performance in rats [7]. Spatially selective neuronal activity has been recorded in the medial
In the present study, we used the same eight male Wistar rats as in the previous longitudinal study [9]. However, two of the rats died during the anesthesia, and one had to be rejected because of an obstruction in the cannula. The age range of the rats was 20-24.5 months (mean 22.0 months) and they weighed 435-485 g. The rats had free access to water but were kept on a food restriction schedule to maintain body weight at 85% of the free feeding level. The rats were anesthetized with pentobarbital 4 0 - 5 0 mg/kg ip and positioned in the stereotaxic frame with the incisor bar 2.3 mm below the interaural line. A pair of guide cannulae were made from 22-gauge injection needles and cut to 12-mm-long pieces. They were positioned above the PFC at A + 4.0 mm anterior to the bregma, L ___0.9 mm lateral to the ~midline, V 0.0 mm ventral to the dura and fixed to the skull with ~jeweller's screws and dental cement. After the rats had fully recovered from the operation (after 3 - 5 days), they were given 1-3 daily sessions of 20 trials of DA training until they reached their preoperative performance level. The testing procedure has been described in detail in our previous paper [9]. In brief, the rat was placed in the starting compartment of the T maze where it waited 3 sec until the guillotine door to the maze was opened. The rat then entered the stem of
l To whom requests for reprints should be addressed. 117
118
TANILA, RAM~, AND CARLSON
FIG. 1. The injection sites based on glial reaction in the brain sections of five rats are plotted on standard sections of the PFC at the level of 4.70 mm anterior to the bregma [24]. the T maze and turned to either of the arms, after which the arm was blocked by the guillotine door. The rat was rewarded by a piece of a standard food pellet for entering the arm that was not chosen in the previous trial. During the delay period, the rat was placed into a round bucket without any view of the maze. Four alternating delays were used. The shortest one was 10 sec for all rats, and the longest one varied between 5 and 25 min and corresponded to just above chance level performance. The intermediate delays were adjusted so that the overall performance of the rats was about 65% correct choices in the 20 trials of the day. The effects of medetomidine (Domitor I m g / m l ) , atipamezole (Antisedan 5 mg/ml, both synthesized by the Farmos Group Ltd., Orion Pharmaceuticals Inc., Turku, Finland), and lidocaine (Lidocain 40 mg/ml, Medipolar, Orion Pharmaceuticals Inc., Oulu, Finland) were studied. The rats received bilateral microinjections of medetomidine (0.01 and 1.0 # g / # l ) , atipamezole (0.05 and 5.0 # g / # l ) , lidocaine (10 # g / # l ) or saline. Also, the effect of combined injections of atipamezole, 0.05 #g//A ic, and medetomidine, 3.0 # g / k g im, was tested. Before the intracortical injections, the rats were tested twice on saline to assess the effect of the intracortical injection itself on the task performance. The volume of each intracortical injection was 1 #1, which was injected with a Hamilton l-#l syringe through a 14-mm-long, 27gauge stainless steel cannula over 30 sec. A further 30 sec was allowed for diffusion before the injection cannula was withdrawn. The microinjections were given 10 rain and the intramuscular injections 15 min before the behavioral testing. The effects of the seven treatments were tested twice in each rat over 3 weeks. The experimenter was unaware of the nature of the injections except of the combined ic and im injections that were always given last in the sequence. At the completion of the experiment, the rats were killed by decapitation, the skulls were opened and the heads were immersed in formalin. The brains were removed, sectioned in 150-/.tin slices and stained with cresyl violet to verify the placement of the cannulae.
subject, drug treatment and the testing period. There were significant main effects of the treatment ( F ( 6 ) = 2.7, p < 0.04) and the testing period so that most rats performed better during the second than the first testing period ( F ( 1 ) = 16.7,p < 0.001 ). Intracortically injected medetomidine improved the DA performance in each of the five rats. The dose of 0.01 # g / # l improved the performance in four rats, whereas the fifth rat improved its performance at the dose of 1 /zg//A. The most beneficial dose of medetomidine for each rat improved the DA task performance significantly (t(4) = 4.24, p < 0.02; Fig. 2). Atipamezole did not have significant effects on the performance (0.05 # g / # l , t(4) = 0.19, p = 0.85; 5.0 # g / # l , t(4) = 1.73, p = 0.16). Lidocaine impaired the performance in all five rats ( t ( 4 ) = 2.99, p < 0.05; Fig. 2). The dose of 3 # g / k g of medetomidine was previously shown to improve the memory task performance of old rats [9]. Intracortical injection of a low dose of atipamezole (0.05 # g / # l ) , which itself did not impair the task performance, reversed the beneficial effect of a systemic administration of medetomidine at a dose of 3 # g / k g in all but one rat ( in which the performance remained improved despite of ic atipamezole) and therefore the performance of the rats did not differ significantly from that on saline ( t ( 4 ) = 0.678, p = 0.53). DISCUSSION In the present study, prefrontal intracortical injection of an alpha-2 adrenoceptor agonist, medetomidine, improved the DA task performance of aged rats, whereas an alpha-2 adrenoceptor antagonist, atipamezole, had no effect. The medial PFC in the rat is an essential structure for DA performance [7]. This was further verified in the present study by the deterioration of DA performance after intracortical administration of lidocaine. The improvement of performance in the working memory task by alpha-2 agonist, medetomidine, may be related to the declined levels of central monoaminergic and cholinergic neurotransmitters in aged rats [21,22]. The decline of noradrenaline levels has been demonstrated in basal forebrain nuclei and in frontal and entorhinal
16 ,,,,,i
12
o
8
"~
4
0)
//
//
O
N
-4
//
Et --12
RESULTS Histological sections indicated that the guide cannulae were well positioned for injections into the prelimbic sector of the PFC (Fig. 1 ). Intracortical injection of saline did not produce a significant change in the performance from the baseline levels ( t ( 4 ) = 2.25, p < 0.10). Multivariate repeated measures analysis of variance was used for testing the main effects of the factors:
-16
u
i
M
L
l
FIG. 2. The m e a n effects o f the beneficial d o s e s o f m e d e t o m i d i n e (M) (0.01 ~ g / # l t o r f o u r rats a n d 1.0 ~zg/#l for one rat) a n d o f lidocaine (L) (10 pg/~l). *p < 0.05, **p < 0.02.
EFFECTS OF P R E F R O N T A L I N T R A C O R T I C A L MICROINJECTIONS
cortical areas [ 22 ] and shown to be related to age-associated memory impairement [23,26]. The results of the present study as well as our earlier finding that medetomidine improved the DA task performance of aged rats but not that of young adult rats [ 9 ] are in line with the abovementioned studies. In our studies, systemically administered atipamezole had no significant effect on the DA task performance in rats [9 ] or on the DR task performance in monkeys [28]. Intraperitoneally injected afipamezole has been shown to increase the central turnover of noradrenaline (NA) at doses 3.0 mg/ kg and higher by blocking presynaptic release-inhibiting receptors [29]. Intracortically injected atipamezole in the present study had no effect on the task performance of aged rats. In monkeys, it has been shown that systemically injected alpha-2 antagonist, yohimbine, increases age-associated memory impairement [1]. However, recently, Arnsten and Cai [4] showed that yohimbine at low doses improved working memory task performance in a subset of aged monkeys, and it was suggested that the improvement might be due to increased NA release on to postsynaptic alpha-2 receptors and might depend on an intact NA system. The role of the prefrontal cortex of monkeys in spatial working memory functions is well documented in electrophysiological and lesion studies [13,15,16]. It has been suggested that the effects of alpha-2 agonists on memory performance of aged monkeys is mediated through postsynaptic alpha-2 adrenoceptors in the frontal cortex [1,8]. The inability to perform the DR task after regional depletion of catecholamines by 6-hydroxydopamine in the PFC can be reversed by clonidine [ 8 ]. The present study provides direct evidence of a prefrontally mediated action of an alpha-2 agonist in rats. Intracortically administered medetomidine improved the DA task performance and intracortical injection of an alpha-2 antagonist, atipamezole, reversed the beneficial effect of the systemically administered medetomidine. The present results support the idea that the beneficial effect of alpha2 agonists on the working memory performance of aged animals is mediated at least partly through the PFC.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
REFERENCES 1. Arnsten, A. F. T.; Goldman-Rakic, P. S. Alpha-2 adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates. Science 230:1273-1276; 1985. 2. Arnsten, A. F. T.; Cai, J. X.; Goldman-Rakic, P. S. The alpha-2 adrenergic agonist guanfacine improves memory in aged monkeys without sedative or hypotensive side effects: Evidence for alpha-2 receptor subtypes. J. Neurosci. 8:4287-4298; 1988. 3. Amsten, A. F. T.; Leslie, F. M. Behavioral and receptor binding analysis of the alpha2-adrenergic agonist, 5-bromo-6 [2-imidazoline-2-yl amino] quinoxaline (UK-14304): Evidence for cognitive enhancement at an alpha2-adrenoceptor subtype. Neuropharmacology 30:1279-1289; 1991. 4. Arnsten, A. F. T.; Cai, J. X. Postsynaptic alpha-2 receptor stimulation improves memory in aged monkeys: Indirect effects of yohimbine versus direct effects of clonidine. Neurobiol. Aging 14:597603; 1993. 5. Bartus, R. T.; Dean, R. L. Lack of efficacy of clonidine on memory in aged Cebus monkeys. Neurobiol. Aging 9:409-411; 1988. 6. Batuev, A. S.; Kursina, N. P.; Shutov, A. P. Unit activity of the medial wall of the frontal cortex during delayed performance in rats. Behav. Brain Res. 41:95-102; 1990. 7. Brito, G. N. O.; Thomas, G. J.; Davis, B. J.; Gingold, S. I. Prelimbic cortex, mediodorsal thalamus, septum, and delayed alternation in rats. Exp. Brain Res. 46:52-58; 1982. 8. Brozoski, T. J.; Brown, R. M.; Rosvold, H. E.; Goldman, P. S. Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 205:929-932; 1979. 9. Carlson, S.; Tanila, H.; R~na~i,P.; Mecke, E.; Pertovaara, A. Effects of medetomidine, an alpha-2 adrenoceptor agonist, and atipamezole,
24. 25. 26. 27.
28. 29. 30. 31. 32. 33.
119
an alpha-2 antagonist, on spatial memory performance in adult and aged rats. Behav. Neural Biol. 58:113-119; 1992. Davis, R. E.; Callahan, M. J.; Downs, D. A. Clonidine disrupts agedmonkey delayed response performance. Drug Dev. Res. 12:279286; 1988. Dunnett, S. B. Role of prefrontal cortex and striatal output systems in short-term memory deficits associated with ageing, basal forebrain lesions, and cholinergic-rich grafts. Can. J. Psychol. 44:210-232; 1990. Estes, K. S.; Simpkins, J. W. Age-related alterations in catecholamine concentrations in discrete preoptic area and hypothalamic regions in the male rat. Brain Res. 194:556-560; 1980. Funahashi, S.; Bruce, C. J.; Goldman-Rakic, P. S. Mnemonic coding of visual space in the monkey's dorsolateral prefrontal cortex. J. Neurophysiol. 61:331-349; 1989. Funahashi, S.; Kubota, K. Working memory and prefrontal cortex. Neurosci. Res. 21:1-11; 1994. Fuster, J. M. Unit activity in prefrontal cortex during delayed-response performance: Neuronal correlates of transient memory. J. Neurophysiol. 36:61-78; 1973. Goldman, P. S.; Rosvold, H. E. Localization of function within the dorsolateral prefrontal cortex of the rhesus monkey. Exp. Neurol. 27:291-304; 1970. Goldman-Rakic, P. S.; Brown, R. M. Regional changes of monoamines in cerebral cortex and subcortical structures of aging rhesus monkeys. Neuroscience 6:177-187; 1981. Goldman-Rakic, P. S. Cellular basis of working memory. Neuron 14:477-485; 1995. Jackson, W. J.; Buccafusco, J. J. Clonidine enhances delayed matching-to-sample performance by young and aged monkeys. Pharmacol. Biochem. Behav. 39:79-84; 1991. Leslie, F. M.; Loughin, S. E.; Stenberg, D. B.; McGaugh, J. L.; Young, L. E.; Zornetzer, S. F. Noradrenergic changes and memory loss in aged mice. Brain Res. 359:292-299; 1985. Luine, V.; Hearns, M. Spatial memory deficits in aged rats: Contributions of the cholinergic system assessed by CHAT. Brain Res. 523:321-324; 1990. Luine, V.; Bowling, D.; Hearns, M. Spatial memory deficits in aged rats: Contributions of monoaminergic systems. Brain Res. 537:271-278; 1990. Markowska, A. L.; Stone, W. S.; Ingram, D. K.; Reynolds, J.; Gold, P. E.; Conti, L. H.; Ponteccorvo, M. J.; Wenk, G. L.; Olton, D. S. Individual differences in aging: Behavioral and neurobiological correlates. Neurobiol. Ageing 10:31-43; 1989. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Sydney, London and New York: Academic Press. Preuss, T. M. Do rats have prefrontal cortex? The Rose-WoolseyAkert Program reconsidered. J. Cogn. Neurosci. 7:1-24; 1995. Rapp, P. R.; Rosenberg, R.; GaUagher, M. An evaluation of spatial information processing in the aged rats. Behav. Neurosci. 101:3-12; 1987. Roberts, A. C.; De Salvia, M. A.; Wilkinson, L. S.; Collins, P.; Muir, J. L.; Everitt, B. J.; Robbins, T. W. 6-Hydroxydopamine lesions of the prefrontal cortex in monkeys enhance performance on an analog of the Wisconsin card sort test: Possible interactions with subcortical dopamine. J. Neurosci. 14:2531-2544; 1994. R~imii, P.; Linnankoski, I.; Tanila, H.; Pertovaara, A.; Carlson, S. Medetomidine, atipamezole and guanfacine in delayed response performance of aged monkeys. Submitted. Scheinin, H.; MacDonald, E.; Scheinin, M. Behavioural and neurochemical effects of atipamezole, a novel alpha-2 adrenoceptor antagonist. Eur. J. Pharmacol. 151:35-42; 1988. Virtanen, R.; Savola, J. M.; Saano, V.; Nyman, L. Characterization of the selectivity, specificity and potency of medetomidine as an alpha-2 adrenoceptor agonist. Eur. J. Pharmacol. 150:9-14; 1988. Virtanen, R.; Savola, J. M.; Saano, V. Highly selective and specific antagonism of central and peripheral alpha-2 adrenoceptors by atipamezole. Arch. Int. Pharmacodyn. Ther. 297:190-204; 1989. Walker, L. C.; Kitt, C. A.; Struble, R. G.; Wagster, M. V.; Price, D. L ; Cork, L. C. The neural basis of memory decline in aged monkeys. Neurobiol. Ageing 9:657-666; 1988. Wikmark, R. G. E.; Divac, I.; Weiss, R. Retention of spatial delayed alternation in rats with lesions in the frontal lobes. Brain Behav. Evol. 8:329-339; 1973.