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DOPAMINERGIC PROJECTION TO THE CENTRAL AMYGDALA MEDIATES THE FACILITATORY EFFECT OF CCK-8US AND CAERULEIN ON MEMORY IN RATS
Pharmacological Research, Vol. 39, No. 6, 1999 Article No. phrs.1999.0496, available online at http:rrwww.idealibrary.com on
DOPAMINERGIC PROJECTION TO THE CENTRAL AMYGDALA MEDIATES THE FACILITATORY EFFECT OF CCK-8US AND CAERULEIN ON MEMORY IN RATS ´ MARIA M. WINNICKAU and KONSTANTY WISNIEWSKI Department of Pharmacology, Medical Uni¨ ersity, Mickiewicza 2c, 15-222 Białystok, Poland Accepted 11 February 1999
The involvement of dopaminergic projection to the central amygdala in the facilitatory effect of cholecystokinin unsulphated octapeptide ŽCCK-8US. and caerulein ŽCER. on memory motivated affectively was investigated in male rats. CCK-8US and CER were administered subcutaneously at the doses of 10 m g kgy1 and 0.5 m g kgy1 , respectively, immediately after a single learning trial in a passive avoidance situation, after bilateral 6-OHDA lesions to the central amygdala. Bilateral 6-OHDA lesions to the central amygdala totally abolished the facilitatory effect of CCK-8US and CER on retention of passive avoidance behaviour evaluated 24 h after the learning trial. These results may indicate that the facilitatory effect of CCK-8US and CER on memory motivated affectively is mediated by dopaminergic projection from ventral tegmental area to the central amygdala. Q 1999 Academic Press KEY
WORDS:
CCK-8US, CER, central amygdala, 6-OHDA lesions, passive avoidance, rats.
INTRODUCTION Cholecystokinin ŽCCK. was first described in the mammalian central nervous system as a gastrin-like immunoreactive material by Venderhaegen et al. w1x. At present, CCK is recognised as the most widely distributed neuropeptide in the brain. Immunohistochemical studies have demonstrated that CCK is present at high concentrations in the cerebral cortex, hippocampal formation, hypothalamus, several subcortical forebrain areas, ventral mesencephalon, nucleus tractus solitarius, and some neurones in the ventral medulla oblongata w1]5x. In the brain, CCK exists mainly as an octapeptide, both in sulphated and desulphated form w6x. The receptors for CCK have been divided into two types: the CCK A and CCK B subtypes. The CCK A receptor subtype was found in periphery and some brain regions, while CCK B receptor subtype is widely distributed in the brain. The highest density of CCK B receptor binding sites was found in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate nucleus, hippocampus, hypothalamus, amygdala, substantia nigra, ventral tegmental area, dorsal U
Corresponding author.
1043]6618r99r060445]06r$30.00r0
raphe, cerebellum, and vagus nerve w7]11x. CCK A receptors do occur in certain brain areas: the area postrema, nucleus of solitary tract, interpeduncular nucleus, nucleus accumbens, dorsal raphe, substantia nigra and ventral tegmental area w12, 13x. Several roles have been ascribed to CCK including: pain perception w14x feeding and satiety w15x, hypothermia w16x, and sedation w17x. Recent investigations have shown that CCK takes part in anxiety and panic attacks w18]20x. The increasing evidence supports the involvement of CCK on cognitive processes. Early studies have shown that peripheral injections of CCK-8 impaired acquisition and facilitated extinction of an active avoidance behaviour w21x. In a passive avoidance situation, CCK-8 improved retention when it was injected either after the single learning trial w21x or before the retention testing w22x. Moreover, CCK-8 given either intracerebroventricularly or subcutaneously prevented experimental amnesia induced by electroconvulsive shock, carbon dioxide inhalation, or cycloheximide injection w23, 24x, and reduced amnesia induced by scopolamine administration w25x. Central administration of CCK-8 antagonists, L-354 718, CR 1409 and proglumide, produced pronounced memory deficit in a passive and an active avoidance tasks w26, 27x and local injection of anti-CCK-8 antiQ 1999 Academic Press
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serum to the posterior cingulate cortex was shown to produce learning impairment in the discrimination task w28x. There is support for the notion that CCK may act as a neurotransmitter or neuromodulator, and appears to modulate the action of other neurotransmitters w29, 30x. Several reports indicate that CCK may exert the modulatory action of nigrostriatal and mesolimbic dopaminergic systems w29, 31, 32x. According to Kesner w33x a key structure for memory motivated affectively is the central amygdala. Dopaminergic projection from the ventral tegmental area to this structure appears to be closely related to motivational mechanism of reward w34x. Therefore, in the present study the involvement of dopaminergic projection to the central amygdala, in the facilitatory effect of cholecystokinin unsulphated octapeptide ŽCCK-8US, CCK B receptor agonist. and caerulein ŽCER, CCK A and CCK B receptor agonist. given after a single learning trial, on retention of information in a passive avoidance situation was evaluated.
every 2 min. The above procedure was non-traumatic since in sham-operated rats lesions appeared as a very slight vertical tract terminating in the central amygdala. Thirty-four randomly selected rats received 6OHDA, the remaining 30 sham-operated control rats underwent the same procedure except that they only received the vehicle solution. Thirty minutes before surgery rats were pre-treated with an intraperitoneal injection of 25 mg kgy1 of desmethylimipramine ŽSigma., an inhibitor of norepinephrine ŽNE. uptake, which has been shown to protect NE neurones from destruction by 6-OHDA w36x. Behavioural testing started after 10 days of recovery period. Unsulphated cholecystokinin octapeptide ŽCCK-8US, RBI. and caerulein ŽCER, Serva. dissolved in 0.9% NaCl were given subcutaneously Žs.c.. in the amount of 10 m g kgy1 and 0.5 m g kgy1 immediately after the learning trial, respectively. One group of lesioned as well as one group of sham-operated rats received 0.9% NaCl at the same time.
Beha¨ ioural testing
MATERIALS AND METHODS
Animals Male Wistar rats of laboratory strain, after 5 days of acclimatisation to the laboratory conditions were used. In the day of surgery their weight was 160]165 g and at the time of behavioural testing, 180]185 g. They were housed in plastic cages Ž50 = 40 = 20 cm., four animals per cage, in the temperature controlled room Ž228C. on a 12-h light]dark cycle beginning at 07:00 h. Food and water were freely available.
Surgery The rats were anaesthetised with chloral hydrate and were placed in a Kopf stereotaxic apparatus with tooth bar 5 mm above the interaural line. The skull was exposed, and burr holes, 1.5 mm in diameter were drilled above the appropriate coordinate targets. The coordinates Žin mm. for the central amygdala, anterior ŽA. from bregma, lateral ŽL. to the midline and below ŽV. the skull surface were selected with the aid of the atlas of Konig and ¨ Klippel w35x at A: y0.8, L: 3.7, and V: 8.0. Each site was infused with 1 m l of 0.9% NaCl containing 8 m g of 6-OHDA Žfree base, Sigma.. The neurotoxin was dissolved in a vehicle solution containing 5 mg mly1 of ascorbic acid, to prevent oxidation of 6-OHDA, and injected over 10 min through a stainless steel needle Žo.d.s 0.3 mm. of the syringe mounted to the sterotaxic apparatus manually at the rate of 0.1 m l miny1 . The needle was left in the place for an additional 5 min after the end of the infusion and subsequently slowly removed at the rate of 0.5 mm
Passi¨ e a¨ oidance Passive avoidance performance was studied in a one trial step-through passive avoidance situation w37x. The apparatus consisted of an illuminated Ž25 W bulb, 20 cm above. platform attached to a large dark compartment. After 2 min of habituation to the dark compartment the rats were placed on the platform and were allowed to enter the naturally preferred dark compartment. Two more approach trials with 2-min intervals were given on the following day. At the end of the second trial an inescapable, scrambled electric footshock Ž0.5 mA for 3 s. was delivered through the grid floor of the dark compartment Žlearning trial.. Retention of a passive avoidance behaviour was tested 24 h after the single learning trial by measuring the latency to re-enter the dark compartment up to a maximum of 300 s.
Histology Placement of the needle was examined histologically. At the end of the behavioural testing, the rats were killed and the brains were removed and fixed in 10% formaldehyde for 7 days. Subsequently, coronal sections Ž20 m m thick. of the needle tract were cut using frozen sectioning method, saving every fifth section through the lesion, mounted on slides, and stained with cresyl violet. Rats in which the needle tip was located outside the centre of target structure were excluded from the experimental data. According to Agid et al. w38x the location of the tip of the needle or cannula is particularly critical for reproducibility of the decrease in dopamine levels in lesioned structures. After infusion of 1 m l of 6-
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gle learning trial, on the retention of information in a step-through passive avoidance situation ŽFig. 2.. The stimulating performance effect of CCK-8US and CER in the sham-operated groups of rats was fully preserved. ANOVA of three lesioned and three sham-operated group of rats, injected s.c. with CCK8US, CER or saline, yielded F1,54 s 139.63, P0.0001 and F2,54 s 34.83, P- 0.0001, when the influence of lesions and the influence of s.c. injections on rats’ performance in a passive avoidance task was evaluated, respectively. Further post hoc comparisons between these groups, made with the Newman]Keuls test, revealed significant increase of the mean step-through latency in the sham-operated groups of rats injected with CCK-8US and CER as compared with saline injected sham-operated and all lesioned groups of rats. Lesioned to the central amygdala rats, injected s.c. with saline, stayed on the illumination platform more than twofold shorter than injected with saline sham-operated animals, nevertheless this difference was insignificant. Only the sham-operated rats injected with CCK-8US and CER stayed on the illuminated platform significantly
Fig. 1. Typical localisation of the tip of the needle of 6-OHDA infusions into the central amygdala ŽCA., A}line drawing Žarrows., B}photomicrograph.
OHDA, the distribution of neurotoxin has spherical shape with a diameter of 3.0 mm. A typical localisation of the tip of the needle into the central amygdala is shown in Fig. 1ŽA,B..
Statistical analysis The results of experiments were evaluated by twoway analysis of variance ŽANOVA. followed by Newman]Keuls test. F-ratios, degrees of freedom, and P-values are reported only for significant differences. In all comparisons between the groups a probability of F 0.05 was considered significant.
RESULTS In the final analysis 31 lesioned and 29 sham-operated animals were included. Each group consisted of 9]11 rats.
Passi¨ e a¨ oidance Bilateral 6-OHDA induced lesions to the central amygdala totally abolished the facilitatory effect of CCK-8US and CER, given immediately after a sin-
Fig. 2. Effect of 10 m g kgy1 of CCK-8US and 0.5 m g kgy1 of CER, given s.c. immediately after the single learning trial, on the re-entry latencies in a passive avoidance situation in rats after bilateral 6-OHDA lesions to the central amygdala. Columns represent means " SEM of the values obtained from 9]11 rats. U P - 0.01 ¨ s sham-operated injected with saline and all lesioned groups of rats ŽANOVA and Newman]Keuls test..
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longer than all the other groups; almost four times longer than sham-operated animals injected with saline.
DISCUSSION
Bilateral lesions to the dopaminergic endings in the central amygdala totally abolished the improvement of memory in a passive avoidance situation after s.c. injection of CCK-8US and CER in rats. The positive effect of CCK-8US and CER, given s.c. immediately after the learning trial, on retrieval process was fully preserved only in sham-operated groups of rats. Injected with the peptides sham-operated animals stayed on the illuminated platform almost four times longer than sham-operated injected with saline controls. After both peptides, the improvement of performance was demonstrable when the treatment was administered after the learning trial, what is, according to McGaugh and Dawson w39x, an indication that the effect was memory-specific, since the substances were administered after the learning experience, unspecific effects resulting from altered perception, motivation and emotion can be ruled on. Bilateral 6-OHDA lesions to the central amygdala on their own attenuated retrieval process insignificantly, however, lesioned to the central amygdala rats injected with saline stayed on the illuminated platform more than twofold shorter than injected with saline sham-operated ones. There is a lot of data supporting the involvement of the amygdala in the emotional memory. In the majority of studies, amygdalatomised rats were impaired in acquiring or relearning a two-way avoidance problem w40]42x. Similarly, amygdalatomised rats were found to be impaired in a passive avoidance test w43]45x. Also electrical stimulation of the amygdala produced profound memory deficits in the variety of tasks, in which reinforcement contingencies of sufficiently high magnitude were used w46, 47x. There are some reports showing that lesions to the basolateral or central amygdaloid nuclei consistently produced an impaired passive avoidance behaviour w48]50x, whereas corticomedial amygdaloid lesions induced only minor deficit or had no effects w48, 51x. On the basis of the above studies, which support a role for the amygdala in mediating emotional experiences, Kesner w33x, in the multidimensional attribute model of memory proposed, that amygdala are critically involved in the encoding of emotional Žpositive and negative. attributes of memory providing that reinforcement input is of sufficient intensity to elicit a relatively strong emotional reaction. According to this theory the amygdala are probably also influenced by sensory-perceptual attribute information
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and can influence selective operation of the autonomic component of the response attribute. In mentioned reports only total amygdaloid lesions or amygdalectomy produced significant impairment of learning and memory processes observed in a different behavioural tasks. However, in the present and also our previous studies w52]55x after removal of dopaminergic projection from the central amygdala only insignificant attenuation of a passive avoidance behaviour was observed, so it may point to an important role of dopaminergic projection arriving at this structure in the mediation of cognitive processes. Jellestad et al. w56x comparing behavioural effects after ibotenic acid, 6-OHDA and electrolytic lesions in the central amygdala of the rat have shown that in contrary to the electrolytic lesions resulted in pronounced passive avoidance impairments, the 6-OHDA lesions similar to our observations resulted in no significant changes in a passive avoidance task. Moreover, one could consider, whether 6-OHDA lesion to the central amygdala abolished only CCK-8US and CER improvement of memory, or made facilitation of cognitive processes by any other memory stimulating substance impossible. Since it was found that the same lesion did not affect the positive influence of vasopressin peptide on retrieval of passive avoidance behaviour w52x, this may indicate that intact dopaminergic projection to the central amygdala is necessary for the memory facilitation exerted by CCK-8US and CER. Co-localisation of CCK and dopamine ŽDA. in the neurones of ventral tegmental area and the ascending mesolimbic pathways w2]4x suggests that CCK could act as a neuromodulator of dopaminergic transmission. These dopaminergic pathways have been closely related to motivational mechanisms of reward w34x, and, thus, CCK would have a place in the regulation of affectively motivated behaviour. Ventral tegmental neurones which contain both DA and CCK project to the caudal and medial portions of the nucleus accumbens, the olfactory tubercles, the anterior cortex, and the central nucleus of the amygdala w2, 3, 57x. Crawley et al. w58x have demonstrated that in the portions of the nucleus accumbens innervated by mixed CCK-DA neurones CCK potentiated the behavioural response to DA. Biochemical studies with DA turnover reported that CCK reduced DA turnover in the striatum and anterior nucleus accumbens, but not in posterior nucleus accumbens and olfactory tubercles where DA terminals contain CCK w59x. These several studies supported the hypothesis that the regions in which CCK is co-localised with DA may delineate unique actions of CCK in modulating dopaminergic function. Also our results are consistent with above hypothesis. Bilateral lesions of dopaminergic endings
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in the central amygdala totally abolished the facilitatory effect of CCK-8US and CER on memory evaluated in a passive avoidance task. This may point to the involvement of DA projection from ventral tegmental area to the central amygdala in modulation of CCK effect on memory motivated affectively. Since similar results were observed after s.c. injection of CCK-8US and CER it may indicate that the above effect is mediated by CCKB receptor subtype.
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14.
15. 16. 17.
ACKNOWLEDGEMENTS This work was supported by AMB project Nr 3-10686.
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DOPAMINERGIC PROJECTION TO THE CENTRAL AMYGDALA MEDIATES THE FACILITATORY EFFECT OF CCK-8US AND CAERULEIN ON MEMORY IN RATS ´ MARIA M. WINNICKAU and KONSTANTY WISNIEWSKI Department of Pharmacology, Medical Uni¨ ersity, Mickiewicza 2c, 15-222 Białystok, Poland Accepted 11 February 1999
The involvement of dopaminergic projection to the central amygdala in the facilitatory effect of cholecystokinin unsulphated octapeptide ŽCCK-8US. and caerulein ŽCER. on memory motivated affectively was investigated in male rats. CCK-8US and CER were administered subcutaneously at the doses of 10 m g kgy1 and 0.5 m g kgy1 , respectively, immediately after a single learning trial in a passive avoidance situation, after bilateral 6-OHDA lesions to the central amygdala. Bilateral 6-OHDA lesions to the central amygdala totally abolished the facilitatory effect of CCK-8US and CER on retention of passive avoidance behaviour evaluated 24 h after the learning trial. These results may indicate that the facilitatory effect of CCK-8US and CER on memory motivated affectively is mediated by dopaminergic projection from ventral tegmental area to the central amygdala. Q 1999 Academic Press KEY
WORDS:
CCK-8US, CER, central amygdala, 6-OHDA lesions, passive avoidance, rats.
INTRODUCTION Cholecystokinin ŽCCK. was first described in the mammalian central nervous system as a gastrin-like immunoreactive material by Venderhaegen et al. w1x. At present, CCK is recognised as the most widely distributed neuropeptide in the brain. Immunohistochemical studies have demonstrated that CCK is present at high concentrations in the cerebral cortex, hippocampal formation, hypothalamus, several subcortical forebrain areas, ventral mesencephalon, nucleus tractus solitarius, and some neurones in the ventral medulla oblongata w1]5x. In the brain, CCK exists mainly as an octapeptide, both in sulphated and desulphated form w6x. The receptors for CCK have been divided into two types: the CCK A and CCK B subtypes. The CCK A receptor subtype was found in periphery and some brain regions, while CCK B receptor subtype is widely distributed in the brain. The highest density of CCK B receptor binding sites was found in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate nucleus, hippocampus, hypothalamus, amygdala, substantia nigra, ventral tegmental area, dorsal U
Corresponding author.
1043]6618r99r060445]06r$30.00r0
raphe, cerebellum, and vagus nerve w7]11x. CCK A receptors do occur in certain brain areas: the area postrema, nucleus of solitary tract, interpeduncular nucleus, nucleus accumbens, dorsal raphe, substantia nigra and ventral tegmental area w12, 13x. Several roles have been ascribed to CCK including: pain perception w14x feeding and satiety w15x, hypothermia w16x, and sedation w17x. Recent investigations have shown that CCK takes part in anxiety and panic attacks w18]20x. The increasing evidence supports the involvement of CCK on cognitive processes. Early studies have shown that peripheral injections of CCK-8 impaired acquisition and facilitated extinction of an active avoidance behaviour w21x. In a passive avoidance situation, CCK-8 improved retention when it was injected either after the single learning trial w21x or before the retention testing w22x. Moreover, CCK-8 given either intracerebroventricularly or subcutaneously prevented experimental amnesia induced by electroconvulsive shock, carbon dioxide inhalation, or cycloheximide injection w23, 24x, and reduced amnesia induced by scopolamine administration w25x. Central administration of CCK-8 antagonists, L-354 718, CR 1409 and proglumide, produced pronounced memory deficit in a passive and an active avoidance tasks w26, 27x and local injection of anti-CCK-8 antiQ 1999 Academic Press
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serum to the posterior cingulate cortex was shown to produce learning impairment in the discrimination task w28x. There is support for the notion that CCK may act as a neurotransmitter or neuromodulator, and appears to modulate the action of other neurotransmitters w29, 30x. Several reports indicate that CCK may exert the modulatory action of nigrostriatal and mesolimbic dopaminergic systems w29, 31, 32x. According to Kesner w33x a key structure for memory motivated affectively is the central amygdala. Dopaminergic projection from the ventral tegmental area to this structure appears to be closely related to motivational mechanism of reward w34x. Therefore, in the present study the involvement of dopaminergic projection to the central amygdala, in the facilitatory effect of cholecystokinin unsulphated octapeptide ŽCCK-8US, CCK B receptor agonist. and caerulein ŽCER, CCK A and CCK B receptor agonist. given after a single learning trial, on retention of information in a passive avoidance situation was evaluated.
every 2 min. The above procedure was non-traumatic since in sham-operated rats lesions appeared as a very slight vertical tract terminating in the central amygdala. Thirty-four randomly selected rats received 6OHDA, the remaining 30 sham-operated control rats underwent the same procedure except that they only received the vehicle solution. Thirty minutes before surgery rats were pre-treated with an intraperitoneal injection of 25 mg kgy1 of desmethylimipramine ŽSigma., an inhibitor of norepinephrine ŽNE. uptake, which has been shown to protect NE neurones from destruction by 6-OHDA w36x. Behavioural testing started after 10 days of recovery period. Unsulphated cholecystokinin octapeptide ŽCCK-8US, RBI. and caerulein ŽCER, Serva. dissolved in 0.9% NaCl were given subcutaneously Žs.c.. in the amount of 10 m g kgy1 and 0.5 m g kgy1 immediately after the learning trial, respectively. One group of lesioned as well as one group of sham-operated rats received 0.9% NaCl at the same time.
Beha¨ ioural testing
MATERIALS AND METHODS
Animals Male Wistar rats of laboratory strain, after 5 days of acclimatisation to the laboratory conditions were used. In the day of surgery their weight was 160]165 g and at the time of behavioural testing, 180]185 g. They were housed in plastic cages Ž50 = 40 = 20 cm., four animals per cage, in the temperature controlled room Ž228C. on a 12-h light]dark cycle beginning at 07:00 h. Food and water were freely available.
Surgery The rats were anaesthetised with chloral hydrate and were placed in a Kopf stereotaxic apparatus with tooth bar 5 mm above the interaural line. The skull was exposed, and burr holes, 1.5 mm in diameter were drilled above the appropriate coordinate targets. The coordinates Žin mm. for the central amygdala, anterior ŽA. from bregma, lateral ŽL. to the midline and below ŽV. the skull surface were selected with the aid of the atlas of Konig and ¨ Klippel w35x at A: y0.8, L: 3.7, and V: 8.0. Each site was infused with 1 m l of 0.9% NaCl containing 8 m g of 6-OHDA Žfree base, Sigma.. The neurotoxin was dissolved in a vehicle solution containing 5 mg mly1 of ascorbic acid, to prevent oxidation of 6-OHDA, and injected over 10 min through a stainless steel needle Žo.d.s 0.3 mm. of the syringe mounted to the sterotaxic apparatus manually at the rate of 0.1 m l miny1 . The needle was left in the place for an additional 5 min after the end of the infusion and subsequently slowly removed at the rate of 0.5 mm
Passi¨ e a¨ oidance Passive avoidance performance was studied in a one trial step-through passive avoidance situation w37x. The apparatus consisted of an illuminated Ž25 W bulb, 20 cm above. platform attached to a large dark compartment. After 2 min of habituation to the dark compartment the rats were placed on the platform and were allowed to enter the naturally preferred dark compartment. Two more approach trials with 2-min intervals were given on the following day. At the end of the second trial an inescapable, scrambled electric footshock Ž0.5 mA for 3 s. was delivered through the grid floor of the dark compartment Žlearning trial.. Retention of a passive avoidance behaviour was tested 24 h after the single learning trial by measuring the latency to re-enter the dark compartment up to a maximum of 300 s.
Histology Placement of the needle was examined histologically. At the end of the behavioural testing, the rats were killed and the brains were removed and fixed in 10% formaldehyde for 7 days. Subsequently, coronal sections Ž20 m m thick. of the needle tract were cut using frozen sectioning method, saving every fifth section through the lesion, mounted on slides, and stained with cresyl violet. Rats in which the needle tip was located outside the centre of target structure were excluded from the experimental data. According to Agid et al. w38x the location of the tip of the needle or cannula is particularly critical for reproducibility of the decrease in dopamine levels in lesioned structures. After infusion of 1 m l of 6-
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gle learning trial, on the retention of information in a step-through passive avoidance situation ŽFig. 2.. The stimulating performance effect of CCK-8US and CER in the sham-operated groups of rats was fully preserved. ANOVA of three lesioned and three sham-operated group of rats, injected s.c. with CCK8US, CER or saline, yielded F1,54 s 139.63, P0.0001 and F2,54 s 34.83, P- 0.0001, when the influence of lesions and the influence of s.c. injections on rats’ performance in a passive avoidance task was evaluated, respectively. Further post hoc comparisons between these groups, made with the Newman]Keuls test, revealed significant increase of the mean step-through latency in the sham-operated groups of rats injected with CCK-8US and CER as compared with saline injected sham-operated and all lesioned groups of rats. Lesioned to the central amygdala rats, injected s.c. with saline, stayed on the illumination platform more than twofold shorter than injected with saline sham-operated animals, nevertheless this difference was insignificant. Only the sham-operated rats injected with CCK-8US and CER stayed on the illuminated platform significantly
Fig. 1. Typical localisation of the tip of the needle of 6-OHDA infusions into the central amygdala ŽCA., A}line drawing Žarrows., B}photomicrograph.
OHDA, the distribution of neurotoxin has spherical shape with a diameter of 3.0 mm. A typical localisation of the tip of the needle into the central amygdala is shown in Fig. 1ŽA,B..
Statistical analysis The results of experiments were evaluated by twoway analysis of variance ŽANOVA. followed by Newman]Keuls test. F-ratios, degrees of freedom, and P-values are reported only for significant differences. In all comparisons between the groups a probability of F 0.05 was considered significant.
RESULTS In the final analysis 31 lesioned and 29 sham-operated animals were included. Each group consisted of 9]11 rats.
Passi¨ e a¨ oidance Bilateral 6-OHDA induced lesions to the central amygdala totally abolished the facilitatory effect of CCK-8US and CER, given immediately after a sin-
Fig. 2. Effect of 10 m g kgy1 of CCK-8US and 0.5 m g kgy1 of CER, given s.c. immediately after the single learning trial, on the re-entry latencies in a passive avoidance situation in rats after bilateral 6-OHDA lesions to the central amygdala. Columns represent means " SEM of the values obtained from 9]11 rats. U P - 0.01 ¨ s sham-operated injected with saline and all lesioned groups of rats ŽANOVA and Newman]Keuls test..
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longer than all the other groups; almost four times longer than sham-operated animals injected with saline.
DISCUSSION
Bilateral lesions to the dopaminergic endings in the central amygdala totally abolished the improvement of memory in a passive avoidance situation after s.c. injection of CCK-8US and CER in rats. The positive effect of CCK-8US and CER, given s.c. immediately after the learning trial, on retrieval process was fully preserved only in sham-operated groups of rats. Injected with the peptides sham-operated animals stayed on the illuminated platform almost four times longer than sham-operated injected with saline controls. After both peptides, the improvement of performance was demonstrable when the treatment was administered after the learning trial, what is, according to McGaugh and Dawson w39x, an indication that the effect was memory-specific, since the substances were administered after the learning experience, unspecific effects resulting from altered perception, motivation and emotion can be ruled on. Bilateral 6-OHDA lesions to the central amygdala on their own attenuated retrieval process insignificantly, however, lesioned to the central amygdala rats injected with saline stayed on the illuminated platform more than twofold shorter than injected with saline sham-operated ones. There is a lot of data supporting the involvement of the amygdala in the emotional memory. In the majority of studies, amygdalatomised rats were impaired in acquiring or relearning a two-way avoidance problem w40]42x. Similarly, amygdalatomised rats were found to be impaired in a passive avoidance test w43]45x. Also electrical stimulation of the amygdala produced profound memory deficits in the variety of tasks, in which reinforcement contingencies of sufficiently high magnitude were used w46, 47x. There are some reports showing that lesions to the basolateral or central amygdaloid nuclei consistently produced an impaired passive avoidance behaviour w48]50x, whereas corticomedial amygdaloid lesions induced only minor deficit or had no effects w48, 51x. On the basis of the above studies, which support a role for the amygdala in mediating emotional experiences, Kesner w33x, in the multidimensional attribute model of memory proposed, that amygdala are critically involved in the encoding of emotional Žpositive and negative. attributes of memory providing that reinforcement input is of sufficient intensity to elicit a relatively strong emotional reaction. According to this theory the amygdala are probably also influenced by sensory-perceptual attribute information
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and can influence selective operation of the autonomic component of the response attribute. In mentioned reports only total amygdaloid lesions or amygdalectomy produced significant impairment of learning and memory processes observed in a different behavioural tasks. However, in the present and also our previous studies w52]55x after removal of dopaminergic projection from the central amygdala only insignificant attenuation of a passive avoidance behaviour was observed, so it may point to an important role of dopaminergic projection arriving at this structure in the mediation of cognitive processes. Jellestad et al. w56x comparing behavioural effects after ibotenic acid, 6-OHDA and electrolytic lesions in the central amygdala of the rat have shown that in contrary to the electrolytic lesions resulted in pronounced passive avoidance impairments, the 6-OHDA lesions similar to our observations resulted in no significant changes in a passive avoidance task. Moreover, one could consider, whether 6-OHDA lesion to the central amygdala abolished only CCK-8US and CER improvement of memory, or made facilitation of cognitive processes by any other memory stimulating substance impossible. Since it was found that the same lesion did not affect the positive influence of vasopressin peptide on retrieval of passive avoidance behaviour w52x, this may indicate that intact dopaminergic projection to the central amygdala is necessary for the memory facilitation exerted by CCK-8US and CER. Co-localisation of CCK and dopamine ŽDA. in the neurones of ventral tegmental area and the ascending mesolimbic pathways w2]4x suggests that CCK could act as a neuromodulator of dopaminergic transmission. These dopaminergic pathways have been closely related to motivational mechanisms of reward w34x, and, thus, CCK would have a place in the regulation of affectively motivated behaviour. Ventral tegmental neurones which contain both DA and CCK project to the caudal and medial portions of the nucleus accumbens, the olfactory tubercles, the anterior cortex, and the central nucleus of the amygdala w2, 3, 57x. Crawley et al. w58x have demonstrated that in the portions of the nucleus accumbens innervated by mixed CCK-DA neurones CCK potentiated the behavioural response to DA. Biochemical studies with DA turnover reported that CCK reduced DA turnover in the striatum and anterior nucleus accumbens, but not in posterior nucleus accumbens and olfactory tubercles where DA terminals contain CCK w59x. These several studies supported the hypothesis that the regions in which CCK is co-localised with DA may delineate unique actions of CCK in modulating dopaminergic function. Also our results are consistent with above hypothesis. Bilateral lesions of dopaminergic endings
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in the central amygdala totally abolished the facilitatory effect of CCK-8US and CER on memory evaluated in a passive avoidance task. This may point to the involvement of DA projection from ventral tegmental area to the central amygdala in modulation of CCK effect on memory motivated affectively. Since similar results were observed after s.c. injection of CCK-8US and CER it may indicate that the above effect is mediated by CCKB receptor subtype.
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14.
15. 16. 17.
ACKNOWLEDGEMENTS This work was supported by AMB project Nr 3-10686.
18. 19.
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