Effects of novelty and habituation on acetylcholine, GABA, and glutamate release from the frontal cortex and hippocampus of freely moving rats

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Neuroscience Vol. 106, No. 1, pp. 43^53, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00

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EFFECTS OF NOVELTY AND HABITUATION ON ACETYLCHOLINE, GABA, AND GLUTAMATE RELEASE FROM THE FRONTAL CORTEX AND HIPPOCAMPUS OF FREELY MOVING RATS M. G. GIOVANNINI,a A. RAKOVSKA,b R. S. BENTON,c M. PAZZAGLI,a L. BIANCHIa and G. PEPEUa * a

Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy b

Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Street, bl. 23, 1113 So¢a, Bulgaria c

University of Arizona, Division of Neural System, Memory and Aging, Arizona Research Laboratories, 384 Life Sciences North Building, Tucson, AZ 85724, USA

AbstractöThe involvement of the forebrain cholinergic system in arousal, learning and memory has been well established. Other neurotransmitters such as GABA and glutamate may be involved in the mechanisms of memory by modulating the forebrain cholinergic pathways. We studied the activity of cortical and hippocampal cholinergic, GABAergic and glutamatergic systems during novelty and habituation in the rat using microdialysis. After establishing basal release of the neurotransmitters, the animals were transferred to a novel environment and allowed to explore it twice consecutively for 30 min (60 min apart; exploration I and II). The motor activity was monitored. Samples were collected throughout the experiment and the release of acetylcholine (ACh), GABA and glutamate was measured. During the two consecutive explorations of the arena, cortical and hippocampal, ACh release showed a signi¢cant tetrodotoxin-dependent increase which was higher during exploration I than II. The e¡ect was more pronounced and longer-lasting in the hippocampus than in the cortex. Cortical GABA release increased signi¢cantly only during exploration II, while hippocampal GABA release did not increase during either exploration. Motor activity was higher during the ¢rst 10 min of exploration I and II and then gradually decreased during the further 20 min. Both cortical and hippocampal ACh release were positively correlated with motor activity during exploration II, but not during I. During exploration II, cortical GABA release was inversely correlated, while hippocampal GABA release was positively correlated to motor activity. No change in cortical and hippocampal glutamate release was observed. In summary, ACh released by the animal placed in a novel environment seems to have two components, one related to motor activity and one related to attention, anxiety and fear. This second component disappears in the familiar environment, where ACh release is directly related to motor activity. The negative relationship between cortical GABA levels and motor activity may indicate that cortical GABAergic activity is involved in habituation. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: exploration, microdialysis, motor activity.

tylcholine (ACh) and other neurotransmitters released from brain structures under physiological conditions and, by studying the out£ow changes in the behaving animal, to identify the behaviors and cognitive functions in which they may play a role. Using this approach, it has been demonstrated that ACh extracellular levels increase in the cerebral cortex and hippocampus of the rat after sensory stimulation (Nilsson et al., 1990; Inglis et al., 1994) in the presence of a novel environment (Aloisi et al., 1997; Ceccarelli et al., 1999, Inglis et al., 1994; Giovannini et al., 1998) during arousal associated with anticipation to feeding (Inglis and Fibiger, 1995), during the performance of a visual attentional task (Passetti et al., 2000), and during wakefulness (Mizuno et al., 1991). However, no simple relationship appears to exist between the increase in ACh levels and the di¡erent behaviors during which the increase can be detected. It is not clear, for instance, whether the increase in ACh release elicited by exposure to a novel environment is associated with, or depends on, locomotor activity or cognitive processes. Day and

There is a consensus that brain cholinergic neurotransmission plays a critical role in the processes underlying attention, learning, and memory (Everitt and Robbins, 1997; Sarter and Bruno, 1997, 2000). Two main cholinergic nuclei of the basal forebrain, the medial septum and the nucleus basalis magnocellularis (NBM) projecting to the hippocampus and neocortex subserve attentional functions and are considered of crucial importance in learning and memory processes (ZolaMorgan and Squire, 1993). In vivo microdialysis makes it possible to monitor ace-

*Corresponding author. Tel.: +39-55-4271-274; fax: +39-55-4271280. E-mail address: [email protected]¢.it (G. Pepeu). Abbreviations : Ach, acetylcholine; ANOVA, analysis of variance; AUC, area under the curve; EDTA, ethylenediaminetetraacetate; HPLC, high-performance liquid chromatography; NBM, nucleus basalis magnocellularis ; NMDA, N-methyl-D-aspartate ; OPA, o-phthalaldehyde; TTX, tetrodotoxin. 43

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coworkers (1991) found that cortical and hippocampal ACh release was correlated with locomotor activity, while other studies (Day and Fibiger, 1992; Moore et al., 1992; Thiel et al., 1998) did not con¢rm this correlation, rejecting the possibility that a simple relationship between ACh release and exploratory activity exists. Accordingly, novelty represented not by the environment, but by unconditioned stimuli, such as a tone or a light, caused a large increase in ACh extracellular levels in the frontal cortex and hippocampus, but little motor activity (Acquas et al., 1996). Moreover, stress activates the forebrain cholinergic pathways, presumably because it induces arousal (Ceccarelli et al., 1999). Behavioral habituation provides one of the most elementary forms of learning, both in animals and humans. In rodents, behavioral habituation is often analyzed in terms of exploratory behavior. The ¢rst exposure to an open ¢eld causes pronounced behavioral activation which is strongly attenuated by subsequent exposures that render the environment familiar (Cerbone and Sadile, 1994; Thiel et al. 1998). The decrease in exploratory activity as a function of repeated exposure to the same environment is taken as an index of memory. The response to novelty is a complex mechanism that involves several processes including arousal, attention, anxiety and fear, and stress-related factors. Habituation to a familiar environment requires its recognition and learning-related processes (Dai et al., 1995; Sadile, 1996; Platel and Porsolt, 1982), and results in a reduced response. Arousal and attention are necessary when the animal is exposed to a novel environment in order to get acquainted with it. Exploration of an open ¢eld and habituation involve the cholinergic innervation of the hippocampus (Gray and McNaughton, 1983; Izquierdo et al., 1992), and it has been shown that exploratory activity is associated with hippocampal theta-rhythm (Whishaw and Vanderwolf, 1973), which in turn depends, at least in part, on the septo-hippocampal cholinergic system (Stewart and Fox, 1990). Therefore, through their role in arousal and attention, the cortical and hippocampal cholinergic systems may allow for the detection of novelty and recognition during habituation. Regardless of whether the forebrain cholinergic neurons are activated by novelty, learning, sensory and stressor stimuli, the question arises as to which neuronal circuits and neurotransmitters trigger and/or accompany this activation. The interactions of forebrain cholinergic neurons with other neurotransmitter systems, such as the GABAergic and glutamatergic systems, during novelty, arousal and habituation have not yet been unraveled. The aim of the present study was to investigate the activity of cholinergic, GABAergic, and glutamatergic systems in the frontal cortex and dorsal hippocampus. This was done, using microdialysis, by measuring the extracellular ACh, GABA and glutamate levels during exploratory activity of a novel and then familiar environment where neither positive nor negative reinforcements were presented, and then correlating neurotransmitter release with the monitored motor activity. A preliminary report of this work has been presented (Giovannini et al., 2001).

EXPERIMENTAL PROCEDURES

Animal housing and surgery Male Wistar rats, weighing 260^280 g, were used (Harlan Nossan, Milan, Italy). The rats were individually housed in macrolon cages until surgery with ad libitum food and water and maintained on a 16/8 h light/dark cycle with lights on at 07.00 h. The room temperature was 23 þ 1³C. All rats were kept for at least 2 weeks in the animal house facility of the Department of Pharmacology at the University of Florence before beginning the experiments, and were frequently handled. All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive 86/609/ EEC). All e¡orts were made to minimize animal su¡ering and to use only the number of animals necessary to produce reliable scienti¢c data. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic apparatus (Stoelting, Stellar, Wood Dale, IL, USA). Microdialysis tubes were inserted transversally into the cortex and hippocampus, and vertically into the NBM, following the procedure described by Giovannini et al. (1994). The microdialysis tubing (AN 69 membrane, Dasco, Bologna, Italy) was covered with super-epoxy glue along its entire length except for the regions corresponding to the cortex (8 mm) and hippocampus (6 mm). The coordinates used for the implantation of the microdialysis tubing in the cortex and hippocampus were as follows (Paxinos and Watson, 1982): anterio-posterior +1.0 mm and 31.9 mm; lateral 33.3 mm and horizontal 33.3 mm, respectively. When I-shaped microdialysis probes (exposed membrane: 2 mm) were implanted vertically into the NBM the coordinates used were: anterio-posterior 30.8, lateral þ 2.8 and horizontal 38.5. All coordinates referred to the bregma, with the bregma and lambda on a horizontal plane. The day after surgery each rat was placed in a Plexiglas cage and transferred to the behavioral studies room, with free access to food and water. Microdialysis procedure The microdialysis probe was perfused with Ringer's solution (NaCl 147 mM, CaCl2 1.2 mM, KCl 4.0 mM, containing 7 WM physostigmine sulfate) at a constant £ow rate of 4 Wl/min using a microperfusion pump (Carnegie Medicine, Mod. CMA/100, Solna, Sweden). In all experiments the microdialysis membrane was allowed to stabilize for 1 h at the £ow rate of 4 Wl/min without collecting samples. At the end of the stabilization period, samples were collected at 10-min intervals at a £ow rate of 4 Wl/min. Five baseline samples were collected to evaluate baseline release of ACh, GABA, and glutamate. The animals were then gently transferred to a novel environment (see Exploratory behavior) and allowed to explore it twice consecutively for 30 min each, spaced by 60 min (exploration I and II). Between and after the explorations, the animals were returned to their home cage. The length of the inlet tubing (1.3 m) was long enough to allow free and unconstrained movements of the animal into the arena during behavioral testing. The microdialysis experiments were performed the day after surgery. All rats were tested during the light phase starting at 09.00 h. When needed, the samples were split into two to analyze, by high-performance liquid chromatography (HPLC), the content of ACh, GABA, and glutamate in the dialysate from the same animal. Assay of ACh in the dialysate ACh was directly assayed in the dialysate using a HPLC method with an electrochemical detector as previously described (Damsma et al., 1987; Giovannini et al., 1994). Brie£y, ACh was separated on a cation-exchange column, prepared by loading a reverse-phase column (Chromospher 5 C18, Chrompack, Middelburg, The Netherlands) with sodium lauryl sulfate (0.5 mg/ml). The mobile phase consisted of 0.2 M phosphate bu¡er (pH 8.0) containing 5 mM KCl, 1 mM tetramethylammonium

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ACh, GABA and glutamate release during novelty and habituation

and 0.3 mM EDTA. The £ow rate was 0.75 ml/min. ACh was hydrolyzed by acetylcholinesterase to acetate and choline in a post-column enzyme reactor; choline was oxidized by choline oxidase to produce betaine and hydrogen peroxide. Hydrogen peroxide was electrochemically detected by a platinum-working electrode at +500 mV with a Ag/AgCl reference electrode. For the quantitative analysis of ACh, we constructed a calibration curve by spiking the Ringer's solution with standard ACh in the concentration range we expected to ¢nd in the dialysates. Three or four concentrations for each calibration curve were then injected at the beginning and end of the analysis and the heights of the recorded peaks were then plotted against the concentrations. A regression line was calculated and quanti¢cation of unknown samples was carried out by the method of inverse prediction. Under these experimental conditions the sensitivity limit (s/n ratio s 3/1) was 100 fmol for ACh. Assay of GABA and glutamate in the dialysate Glutamate and GABA analyses were carried out as previously described (Bianchi et al., 1999) by HPLC with £uorimetric detection (at an excitation and emission wavelength of 340 and 455 nm, respectively) after o-phthalaldehyde (OPA) derivatization of the amino acids. A 5-Wm nucleosil C18 column (200U4 mm i.d., Macherey-Nagel, Duren, Germany) was used. The mobile phase consisted of methanol^potassium acetate (0.1 M) adjusted to pH 5.52 with glacial acetic acid. A gradient (£ow rate 0.9 ml/min) of three linear steps: from 25 to 43% methanol (1 min), from 43 to 70% methanol (10 min) and from 70 to 90% (1 min), followed by an isocratic hold at 90% methanol (1 min) and then back from 90 to 25% methanol (1 min). One volume (10 Wl) of dialysate was mixed with 1 Wl of the OPA derivatization reagent (Bianchi et al., 1999) in a glass capillary tube and injected after 1.5 min. Standard curves were linear over the concentration range of 25^1000 fmol/Wl. The minimum detectable concentration was 2 fmol/Wl. Exploratory behavior Apparatus. The exploratory behavior was investigated in an arena formed by a white-colored polyvinyl chloride box (70U60U30 cm) with a grid £oor which could be easily cleaned. The arena was illuminated by two 75-W lamps suspended 50 cm above the box. A microwave sensor was placed 70 cm above the arena to measure the rats' motor activity. Handling and exploration. Each animal was taken from its home cage and transferred to the adjacent arena, to which it had never been exposed before. To do this, the animal was manually picked up from its home cage, lifted approximately 50 cm and placed gently into the open ¢eld. This procedure lasted less than 5 s. We had previously demonstrated that this handling procedure, to which the animal was used to, did not modify ACh release (Giovannini et al., 1998). After a period of 30 min in the arena (exploration I), the animal was again picked up and returned to its home cage. One hour later, the rat was placed for another 30 min in the open ¢eld (exploration II), repeating the above procedure in an identical manner, and then was put back in its home cage for a further 60 min. During the entire procedure, dialysis samples were collected at 10-min intervals. A total of 21 samples were collected. Motor activity. Total motor activity in the arena during exploration was recorded every 10 min by means of a microwave sensor placed 70 cm above the cage, in order to cover the entire arena. The impulses created by the movements were recorded every 0.1 s by a counter, which added and expressed them in s/10 min. No analysis of the movement type was made. Statistical analysis The changes in neurotransmitter levels in the 10-min samples were expressed as percent variation over basal level, which in each group of experiments was the mean þ S.E.M. of all deter-

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minations made in the home cage. Since the exploration-associated increase in neurotransmitter release always returned to basal levels once the animal was put back in its home cage, we expressed the `evoked' neurotransmitter release by calculating the areas under the curves (AUCs, expressed in arbitrary units). AUCs were also calculated for control animals within the same time interval. Signi¢cance of the e¡ect of exploration on neurotransmitter release was therefore evaluated on the AUCs by means of one- or two-way analysis of variance (ANOVA), followed by Newman^Keuls multiple post-hoc test. P values of less than 0.05 were considered signi¢cant. Motor activity, evaluated at 10-min intervals during exploration of the arena (exploration I and II), was expressed as s/10 min of recording. Correlation between motor activity and neurotransmitter release (expressed as pmol or fmol/10 min) was calculated by means of the linear regression analysis. All statistical analyses were performed using GraphPad Prism 3.0 or NCSS 6.0.

RESULTS

Basal release of ACh, GABA, and glutamate from the cortex and hippocampus of freely moving rats, obtained by averaging the ¢rst ¢ve samples collected before the animal was transferred from its home cage to the arena, is shown in Table 1. In control animals, kept in their home cage during the entire experiment and manipulated similarly to the arena-exposed animals, the basal out£ow of ACh, GABA, and glutamate remained relatively constant throughout the entire experiment (see also Figs. 2, 3 and 4). According to the map of Zilles and Wree (1985), the membrane placed in the cortex is inserted through the parietal region of one hemisphere and reaches the opposite hemisphere by running under the frontal and through the pre-frontal cortices, areas involved in motor-sensory and cognitive functions. Since no signi¢cant di¡erences were observed in the rats' motor activity when the membranes were placed either in the cortices or in the hippocampi (exploration I, ¢rst 10 min, Student's

Fig. 1. Motor activity of rats in the arena during exploration I and II. Bars represent pooled values of motor activity (expressed as s/10 min) from all rats tested in the experiments (n = 22). Twoway ANOVA, followed by Newman^Keuls multiple-comparison test, showed that exploration I is signi¢cantly di¡erent from exploration II. **P 6 0.05 versus all other groups and *P 6 0.05 versus 20 and 30 min, exploration II.

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M. G. Giovannini et al. Table 1. Basal release of ACh, GABA and glutamate from the parietal cortex and dorsal hippocampus of freely moving rats

Neurotransmitter

Parietal cortex

Dorsal hippocampus

ACh (pmol/10 min) GABA (fmol/10 min) Glutamate (pmol/10 min)

2.42 þ 0.26 (n = 13) 51.48 þ 9.07 (n = 12) 3.31 þ 0.41 (n = 7)

1.19 þ 0.23 (n = 9) 56.23 þ 19.76 (n = 7) 3.08 þ 1.07 (n = 6)

Dialysate outputs are expressed as the mean þ S.E.M. of the ¢rst ¢ve basal samples before exploration I. Number of animals in parentheses.

t-test, P = 0.25, not signi¢cant), it appears that no signi¢cant damage was done to the motor-sensory areas by the membrane spanning through them. Therefore, in order to have a better understanding of the animals' behavior during exploration and habituation, the motor activity data obtained from animals that were operated in the cortex and hippocampus were pooled together. Placing

the animals in the arena elicited a thorough exploration of the environment, which was more prominent during exploration I, when the environment was new, than during exploration II, when the animals were already acquainted with the arena and, therefore, had undergone habituation (Fig. 1). During exploration I, the rats' motor activity decreased steadily from the ¢rst 10 min

Fig. 2. E¡ect of exploration on cortical and hippocampal ACh release. (A) Time-course of cortical ACh release (curves, left y-axis) and motor activity (bar graph, right y-axis) from animals exposed to the arena during exploration I and II. ACh release was expressed as percent changes of basal release. Black squares: treated animals (placed in the arena); open circles: control animals (kept in their home cages throughout the experiment). The samples collected while the animals were in the arena during exploration I and II and the exploratory activity during the same periods are framed by the thin rectangles. (B) AUC calculated between samples 5 and 12 (exploration I) and between samples 14 and 17 (exploration II), both from treated and control animals. One-way ANOVA (F3;23 = 6.48; P 6 0.005), followed by Newman^Keuls multiple-comparison test, showed that exploration I was signi¢cantly di¡erent from its control and from exploration II (**P 6 0.01) and exploration II was signi¢cantly di¡erent from its control (*P 6 0.05). (C) Time-course of hippocampal ACh release (curves, left y-axis) and motor activity (bar graph, right y-axis) from animals exposed to the arena during exploration I and II. ACh release was expressed as percent changes of basal release. Black squares: treated animals (placed in the arena); open circles: control animals (kept in their home cages throughout the experiment). The samples collected while the animals were in the arena during exploration I and II and the exploratory activity during the same periods are framed by the thin rectangles. (D) AUC calculated between samples 5 and 13 (exploration I) and between samples 14 and 20 (exploration II), both from treated and control animals. One-way ANOVA (F3;14 = 12.1; P 6 0.0001), followed by Newman^Keuls multiple-comparison test, showed that exploration I was signi¢cantly di¡erent from its control and from exploration II (**P 6 0.01) and exploration II was signi¢cantly di¡erent from its control (*P 6 0.05).

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(318 s motor activity/10 min) to the second 10 min (109 s/10 min) and reached a minimum during the third 10 min, when the animals showed almost no motor activity (37 s/10 min). During exploration II, only the ¢rst 10 min showed some motor activity (140 s/10 min). Two-way ANOVA (explorationU10-min period) revealed a signi¢cant di¡erence between exploration I and II (F2;113 = 53.66; P 6 0.0001), between 10-min periods (F1;113 = 25.99; P 6 0.0001) and in the interaction between the two (F5;113 = 7.87; P 6 0.0005). No analysis of the type of motor activity was performed. ACh release from the fronto-parietal cortex and dorsal hippocampus of the behaving animal When the animals were removed from their home cage and placed in the arena (exploration I), a signi¢cant increase, both in cortical (maximal increase +64%) and hippocampal (maximal increase +200%) ACh release,

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was observed (Fig. 2A, C). When the animals were placed back in their home cage, ACh release slowly returned to basal levels. The exploration of the same arena during exploration II caused an increase, although less intense and shorter lasting than during exploration I, both in cortical and hippocampal ACh release (maximal increase +37% in the cortex and +51% in the hippocampus). Total ACh release evoked by exploration was calculated by measuring the AUCs during exploration I and II. Corresponding AUCs were also calculated for control animals within the same time intervals. Results are shown in Fig. 2B, D. Statistical analysis performed using one-way ANOVA on the AUCs showed that during the two explorations of the arena, ACh release signi¢cantly increased both in the cortex (Fig. 2B, F3;23 = 6.482; P 6 0.005) and dorsal hippocampus (Fig. 2D, F3;14 = 12.10; P 6 0.0001), as compared to the respective areas calculated in control animals. However, in both areas, the e¡ect was higher during exploration I

Fig. 3. E¡ect of exploration on cortical and hippocampal GABA release. (A) Time-course of cortical GABA release (curves, left y-axis) and motor activity (bar graph, right y-axis) from animals exposed to the arena during exploration I and II. GABA release was expressed as percent changes of basal release. Black squares: treated animals (placed in the arena); open circles: control animals (kept in their home cages throughout the experiment). The samples collected while the animals were in the arena during exploration I and II and the exploratory activity during the same periods are framed by the thin rectangles. (B) AUC calculated between samples 5 and 9 (exploration I) and between samples 14 and 18 (exploration II), both from treated and control animals. One-way ANOVA (F3;15 = 3.65; P 6 0.05), followed by Newman^Keuls multiple-comparison test, showed that exploration II was signi¢cantly di¡erent from its control (*P 6 0.05). (C) Time-course of hippocampal GABA release (curves, left y-axis) and motor activity (bar graph, right y-axis) from animals exposed to the arena during exploration I and II. GABA release was expressed as percent changes of basal release. Black squares: treated animals (placed in the arena); open circles: control animals (kept in their home cages throughout the experiment). The samples collected while the animals were in the arena during exploration I and II and the exploratory activity during the same periods are framed by the thin rectangles. AUC calculated between samples 5 and 9 (exploration I) and between samples 14 and 18 (exploration II), both from treated and control animals. One-way ANOVA showed that neither exploration I nor II was signi¢cantly di¡erent from controls.

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M. G. Giovannini et al.

than II. Furthermore, two-way ANOVA (brain structureUexploration) showed that the e¡ect in the hippocampus was signi¢cantly higher than in the cortex (F1;21 = 10.11; P 6 0.005). Figure 2A, C shows motor activity during exploration I and II for this group of animals. Perfusing 0.5 WM tetrodotoxin (TTX) through the cortex or hippocampus via the dialysis membrane, after collecting the ¢ve basal samples and throughout exploration I, completely blocked the release of ACh from either structure, which fell to undetectable levels within 40 min from the beginning of perfusion (data not shown). These results indicate that the increase observed during exploration was due to an increased ¢ring of cholinergic neurons projecting to the cortex and hippocampus. TTX administration did not signi¢cantly change the rats' motor activity during exploration (not shown). Animals implanted with a vertical dialysis probe in the NBM (n = 3) did not show any variation in ACh extracellular levels from the NBM during exploration (data not shown) GABA release from the fronto-parietal cortex and dorsal hippocampus of the behaving animal In contrast to what was observed with ACh, cortical GABA release did not signi¢cantly change during exploration I, even though the animals showed regular exploration of the novel environment, as shown by motor activity measured for this group of animals (Fig. 3A, C). However, cortical GABA release showed a marked increase during exploration II, which was slow in onset and delayed, with maximal increase (+99%) during the third sample collected during the rats' exposure to the arena. The calculation of the AUCs indicated that the increase in GABA release was signi¢cant during exploration II only, compared to control animals (Fig. 3B, one-way ANOVA, F3;15 = 3.654; P 6 0.05). Hippocampal GABA release increased during exploration I and II in two out of seven rats only. When these two rats were excluded from the analysis, hippocampal GABA release did not signi¢cantly increase during the two exploratory periods (Fig. 3C, D). Glutamate release from the fronto-parietal cortex and dorsal hippocampus of the behaving animal Neither cortical nor hippocampal glutamate release changed signi¢cantly during the animals' two exposures to the arena, even though the animals showed regular exploration of the novel environment, as shown by motor activity measured for this group of animals (Fig. 4A, B). Therefore the AUCs were not calculated for these animals. Correlation between neurotransmitter release and motor activity In order to better understand if a relationship between the increase in motor activity and neurotransmitter release during exploration existed, the amount of motor

Fig. 4. E¡ect of exploration on cortical and hippocampal glutamate release. (A) Time-course of cortical glutamate release (curves, left y-axis) and motor activity (bar graph, right y-axis) from animals exposed to the arena during exploration I and II. Glutamate release was expressed as percent changes of basal release. Black squares: treated animals (placed in the arena); open circles: control animals (kept in their home cages throughout the experiment). The samples collected while the animals were in the arena during exploration I and II are framed by the thin rectangles. (B) Timecourse of hippocampal glutamate release (curves, left y-axis) and motor activity (bar graph, right y-axis) from animals exposed to the arena during exploration I and II. Glutamate release was expressed as percent of basal release. Black squares: treated animals (placed in the arena); open circles: control animals (kept in their home cages throughout the experiment). The samples collected while the animals were in the arena during exploration I and II are framed by the thin rectangles.

activity during the three 10-min periods of each exploration were correlated with the respective neurotransmitter release levels (expressed as pmol/10 min for ACh or fmol/10 min for GABA) measured during the same three 10-min periods. The results are shown in Fig. 5 for the cortex and Fig. 6 for the hippocampus. During exploration I, neither cortical (Fig. 5A) nor hippocampal (Fig. 6A) ACh and GABA releases were correlated to motor activity. However, during exploration II (Fig. 5B), cortical ACh release was directly correlated to motor activity (r2 = 0.3987; P 6 0.005), while cortical GABA release was inversely correlated to motor activity (r2 = 0.2517; P 6 0.05). In contrast, during exploration II (Fig. 6B), both ACh and GABA release in the hippocampus were directly correlated to motor activity (r2 = 0.8234; P 6 0.0001 and r2 = 0.3572; P 6 0.05, respectively). No correlation was found in the cortex and hippocampus between motor activity and glutamate release.

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Fig. 5. Correlation between motor activity and ACh and GABA release from the cortex. Linear regression analysis was performed on each neurotransmitter release and motor activity measured during exploration I and II, separately. Open symbols: exploration I; ¢lled symbols : exploration II; squares: ACh; triangles: GABA.

DISCUSSION

In the present study, we investigated the activity of the cholinergic, GABAergic, and glutamatergic systems in the frontal cortex and dorsal hippocampus of the rat in response to novelty and habituation by placing the rats twice consecutively in the same arena. The activity of the cholinergic, GABAergic and glutamatergic neurons was monitored by measuring cortical and hippocampal extracellular levels of ACh, GABA and glutamate by transversal microdialysis during the performance of the exploratory tasks. A relationship between the change in neurotransmitter levels and motor activity was sought. Cortical and hippocampal ACh release during novelty and habituation In our experiments, the rats, accustomed to being handled, were twice exposed to the same arena with 60-min intervals between each exposure. Thus, the arena was new at ¢rst exposure and had become familiar by the second. Handling had previously been shown to activate the cholinergic system in the frontal cortex, hippocampus and nucleus accumbens (Acquas et al., 1996; Thiel et al., 1998; Moor et al., 1998). However, in the present experiments, as in previous ones (Giovannini et al., 1998), no changes in neurotransmitter out£ow was

detected in the rats which had been gently handled for several days before the experiment. It may, therefore, be assumed that the increase in ACh release observed in both the cortex and hippocampus during the two exposures to the arena was associated, at least in part, with the exploratory activity, as already shown by Giovannini et al. (1998). Studies in rats and monkeys involving the lesioning of the basal forebrain cholinergic neurons (Dunnett et al., 1991; Olton and Markowska, 1994) indicate that the corticopetal and hippocampopetal cholinergic pathways are involved primarily in attentional functions (Buzsaki and Gage, 1989). In particular, one role of the hippocampus in associative learning seems to be the reduction in attention to stimuli that are irrelevant or for which the behavioral consequences are known (Baxter and Chiba, 1999). Furthermore, Inglis and Fibiger (1995) suggested that an increase in cortical and hippocampal ACh release plays a role in arousal and/or attention. Therefore, the marked and long-lasting increase in cortical and hippocampal ACh release present when the rats were placed in the new environment can be, at least in part, attributed to the increase in attention and arousal triggered by novelty, and may be relevant for information processing. Fear, elicited by novelty, may also play a role in the increased ACh release observed (Acquas et al., 1996). During the second exposure to the arena, the increase in cortical and hippocam-

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M. G. Giovannini et al.

Fig. 6. Correlation between motor activity and ACh and GABA release from the hippocampus. Linear regression analysis was performed on each neurotransmitter release and motor activity measured during exploration I and II, separately. Open symbols: exploration I; ¢lled symbols: exploration II; squares: ACh; triangles: GABA.

pal ACh was signi¢cantly smaller than during the ¢rst exposure. Similarly, Acquas et al. (1996) observed that an unconditioned sensory stimulus does not increase cortical and hippocampal ACh release in habituated rats. In our experiments, habituation is also demonstrated by the decrease in motor activity during exploration II, in comparison with exploration I. Interestingly, a signi¢cant correlation between the increase in cortical and hippocampal ACh out£ow and motor activity was detected only during the second exposure to the arena. These results indicate that no simple correlation exists between cortical or hippocampal extracellular ACh levels and locomotor activity. Measurements of ACh release in awake animals during motor activity have provided contradictory results. Day et al. (1991) found a correlation between motor activity and ACh release in the cerebral cortex, hippocampus and striatum, and a similar correlation was found in the hippocampus by Mizuno et al. (1991). However, Day and Fibiger (1992), Moore et al. (1992) and Thiel et al. (1998) did not con¢rm this correlation. Thiel and coworkers (1998) found no di¡erences in the increase in hippocampal ACh out£ow in rats exposed twice to an open ¢eld with an interval of 24 h. In a previous paper (Giovannini et al., 1998), we also found no di¡erences in cortical ACh release when the animals were re-exposed to the same environment 24 h after ¢rst exposure. These ¢ndings suggest that habituation evoked by this paradigm results in decreased explor-

atory activity, which is not paralleled by decreased cortical and hippocampal cholinergic activation (Thiel et al., 1998). In contrast, the present data show that habituation results in a lesser increase in both exploratory activity and ACh release. The di¡erence between the present, our previous and Thiel's group data may arise from the di¡erences in the time elapsed between the two exposures to the arena, i.e. 1 h versus 24 h. It should be mentioned that in the object-recognition test, rats no longer discriminate between familiar and novel objects 24 h after the ¢rst presentation (Bartolini et al., 1996). Presumably, after 24 h away from the arena, the environment has once again become unfamiliar to the animals, thus inducing arousal and fear. The lack of correlation between ACh release and motor activity in the ¢rst exposure to a novel environment in our experiments and in the second exposure after a 24-h interval in Thiel et al.'s (1998) experiments, indicates that cortical and hippocampal cholinergic system activation has several components, one of which is motor activity. Other components could be attention, arousal and stress. Attentional tasks performed, in order to obtain food or water, are associated with an increase in ACh release (Orsetti et al., 1996; Himmelheber et al., 2000; Passetti et al., 2000), but a correlation between attentional e¡ort and ACh release has not always been found (Passetti et al., 2000). Stressor stimuli, such as prolonged handling (Nilsson et al., 1990;

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ACh, GABA and glutamate release during novelty and habituation

Rosenblad and Nilsson, 1993), restraint stress (Imperato et al., 1991) and fear (Acquas et al., 1996), also strongly activate the cholinergic system. It may be assumed that when a rat is exposed to a novel environment, activation of the forebrain cholinergic system is triggered by fear, stress and motor activity. This activation is required for the attention needed for a ¢nalized exploration that may lead to obtaining food (Orsetti et al., 1996; Passetti et al., 2000). After habituation to the environment, attention is not needed since there is no fear, therefore, activation of the cortical and hippocampal cholinergic system is related to motor activity only, as shown by the correlation between motor activity and ACh release in exploration II. However, the neural connections between motor activity and activation of the cholinergic system in the cortex and hippocampus are far from clear, and the possibility that the increase in ACh release elicited by sensory input causes an increase in motor activity has not been ruled out. Cortical and hippocampal glutamate and GABA release during novelty and habituation No information is yet available on the changes in brain extracellular levels of glutamate and GABA during exploration and habituation. In our experiments, no changes in glutamate extracellular levels were found in the cerebral cortex or hippocampus of the rats during either exploration I or II. Apparently, cortical and hippocampal glutamatergic systems are not involved in exploration of a novel environment and habituation. However, glutamatergic pathways modulate cortical and hippocampal cholinergic activity both directly and indirectly (Pepeu and Blandina, 1998): directly, since antagonists of the N-methyl-Daspartate (NMDA) receptors injected into the nucleus basalis inhibit spontaneous and stimulated ACh release (Rasmusson et al., 1996; Giovannini et al., 1997). Administration of NMDA and K-amino-3-hydroxy5-methyl-4-isoxazole propionate/kainate antagonists in the medial septum inhibits handling-evoked ACh release in the hippocampus (Moor et al., 1998); indirectly, since glutamatergic pathways regulate the activity of GABAergic neurons impinging on cholinergic neurons in the medial septum (Giovannini et al., 1994). Presumably, changes in glutamate release could be detected if the probes were placed in the nucleus basalis or medial septum. Nevertheless, Timmerman and Westerink (1997) question the possibility of detecting changes in the small glutamate pool of neuronal origin, which is only a fraction of the larger non-synaptic glutamate pool. The determination of GABA extracellular levels revealed that cortical GABA release increased signi¢cantly only during exploration II, while hippocampal GABA release did not increase during either exploration periods. A correlation between motor activity and GABA release was detected, again, only during exploration II. However, the correlation was negative in the cortex and positive in the hippocampus. The cortical cholinergic network is regulated by cortical GABAergic interneurons inhibiting ACh release

51

at a pre-synaptic level (Giorgetti et al., 2000) and by GABAergic neurons originating from septal nuclei (Giovannini et al., 1997) and the nucleus basalis (Freund and Gulya©s, 1991). Moreover, GABAergic neurons, presumably originating from the nucleus accumbens, regulate the corticopetal cholinergic neurons in the nucleus basalis (see reference in Sarter and Bruno, 2000). No information is available on the relationship between spontaneous behavior, learning and memory and cortical GABA release. According to Timmerman and Westerink (1997), handling did not increase GABA e¥ux in the rat cerebral cortex. Enhancing the GABAergic tone through the administration of GABA receptor agonists reduced cortical ACh release (Casamenti et al., 1986; Moore et al., 1995), impaired several cognitive tasks (see references in Decker and McGaugh, 1991) and reduced attentional levels (Holley et al., 1995). It may be suggested that the inverse relationship between cortical GABA release and motor activity indicates that the activation of GABAergic neurons plays a role in habituation by dampening neuronal activity. The GABAergic basal forebrain projection innervating cortical inhibitory interneurons may determine whether the response of cortical principal neurons is potentiated or depressed (Freund and Meskenaite, 1992). However, the question regarding which stimulus activates the cortical GABAergic network in a familiar environment remains. Moreover, our ¢ndings in the hippocampus seem to contradict the hypothesis. In this region, although no clear-cut increase in GABA release occurred during exploration II, a direct relationship between GABA release and motor activity was detected. A relationship between GABAergic activation and motor activity was demonstrated by the ¢nding that perfusion of the medial septum with the GABAA receptor agonist, muscimol, increased spontaneous locomotor activity in rats without a¡ecting memory (Osborne, 1994).

CONCLUSIONS

No correlation was found between ACh release from the cerebral cortex and hippocampus and motor activity during exploration of a novel environment, while the correlation existed in the second exposure to the same environment when habituation had occurred. This ¢nding demonstrates that ACh release in a novel environment has two components, one related to motor activity and one related to attention, anxiety and fear. The second component is not present in the familiar environment, where ACh release appears to be directly related to motor activity. Our ¢ndings are among the ¢rst attempting to correlate glutamate and GABA release with a spontaneous behavior. The cortical and hippocampal extracellular levels of glutamate do not seem to be a¡ected by exploration and habituation. In contrast, cortical GABA release increased during the second exposure when habituation had developed. On the basis of the negative relationship between GABA levels and motor activity, the hypothesis may be put forward that cortical GABAergic activity is

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52

M. G. Giovannini et al.

involved in habituation. However, a similar relationship was not found in the hippocampus. Therefore, in order to understand the meaning of the relationship between GABA cortical and hippocampal out£ow and spontaneous behavior, further investigation is needed.

AcknowledgementsöWe thank M.A. Colivicchi for performing GABA and glutamate HPLC analyses. This work was supported by a Grant from MURST (Co¢n number 9805108207). R.S.B. was a recipient of a BRAVO program fellowship from the University of Arizona. A.R. was supported by a grant from CNR.

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