Effects of intraseptally injected noradrenergic drugs on hippocampal sodium-dependent-high-affinity-choline-uptake in ‘resting’ and ‘trained’ mice

BRAIN RESEARCH ELSEVIER

Brain Research 652 (1994) 120-128

Research report

Effects of intraseptally injected noradrenergic drugs on hippocampal sodium-dependent-high-affinity-choline-uptake in 'resting' and 'trained' mice Aline Marighetto *, Robert Jaffard, Jacques Micheau Laboratoire de Neurosciences Comporternentales et Cognitil.'es, CNRS URA 339, Unit,ersitd de Bordeaux 1, A~,enue des Facultds, 33405 Talence C~dex, France Accepted 12 April 1994

Abstract

It has been shown in numerous studies that memory testing can alter presynaptic cholinergic activity within the hippocampus. In the present experiments, the role of the noradrenergic input to the septal cholinergic neurons in the immediate increase in cholinergic activity induced by the first training session of a spatial reference memory task in an 8-arm radial maze was investigated. The effects of bilateral intraseptal injections of noradrenergic drugs on hippocampal sodium-dependent-highaffinity-choline-uptake (SDHACU)were studied in 'resting' animals (basal level) or in 'trained' animals injected 20 rain before training and sacrificed immediately after the test. The results showed that: (1) the injection of maprotiline, a noradrenaline reuptake inhibitor (0.06 ng/site), induced an increase in hippocampal SDHACU in 'resting' animals, whereas the az-adrenoce ptor agonist UK 14304 (1.5 ng) significantly reduced the basal level of SDHACU; (2) none of the a-adrenoceptor antagonists used (phenoxybenzamine, 10 and 100 ng; BE 2254, 100 and 500 ng; yohimbine, 0.5 and 50 ng) significantly affected the basal level of hippocampal SDHACU, and only the al-adrenoceptor antagonist BE 2254 (500 ng) significantly reduced the testing-induced activation of SDHACU. Taken together, these findings suggest that noradrenaline may exert a bimodal regulatory influence on the activity of septo-hippocampal cholinergic neurons. The behavior-induced activation of hippocampal SDHACU could be partly mediated by the stimulation of aj-adrenoceptors, whereas postsynaptic a2-adrenoceptors may be important for the maintenance of a tonic inhibition of the steady-state cholinergic activity in the hippocampus. These results are further evidence for a regulatory noradrenergic influence on the activity of septal cholinergic neurons.

Key words: Cholinergic septo-hippocampal system; High-affinity choline uptake; Noradrenergic drugs; a-Adrenoceptor; Transsynaptic modulation; Mouse

1. Introduction

There is now a considerable body of evidence from both animal and human studies that central cholinergic systems play an important role in modulating the neural circuitry underlying the cognitive processes involved in memory. However, unravelling the exact nature of the involvement of central cholinergic synapses in learning and memory is no easy task [23,34], and it is becoming apparent that comprehension of the contribution of central cholinergic pathways to memory-related mechanisms largely hinges on progress made in

* Corresponding author. Fax: (33) 5684-8743. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 4 7 9 - V

more integrative approaches. For instance, Hasselmo and Bower [32] have recently presented a theoretical framework for describing the role of acetylcholine in cortical memory function based on a combination of electrophysiological, pharmacological data and computational modeling. In this theory, acetylcholine is thought to subserve learning of new information by modifying synaptic strengths in cortical networks, whereas recall processes are assumed to require a reduction in cholinergic modulation. This model highlights the dynamic aspect of the cholinergic modulation of memory function. According to this model, cholinergic activity would be expected to be altered during a n d / o r after training in memory tasks. By measuring the time

A. Marighetto et al. / Brain Research 652 (1994) 120-128

course of sodium-dependent-high-affinity-choline-uptake (SDHACU) in brain samples, or by microdialysis, several authors have demonstrated an acute increase in hippocampal cholinergic activity after a first exposure to different behavioral situations, including memory tasks [7,24,40,46,70]. However, these early changes may well depend on factors such as stress and locomotor activity that are unrelated to the specific response to be learned [13,18,35]. Nevertheless, there is evidence for a learning-specific commitment of cholinergic function as training progresses [18,46]. In our laboratory, we have shown that spatial memory testing induces changes in hippocampal SDHACU. Furthermore, the direction and magnitude of the alteration was found to depend on the post-testing time-interval, on the level of training and on the form of memory involved. At all levels of acquisition, we observed an immediate posttraining increase in SDHACU, followed by a stable plateau and a progressive decline as training continued [35,46]. In individual animals, these immediate and subsequent training-induced alterations of hippocampal S D H A C U were found to be directly related to the rate of acquisition [18,46]. Moreover, these changes were either not observed or much attenuated in older animals with impairments in a spatial memory task [18,25,41]. These findings point to a role for a cholinergic involvement in memory processes, although comprehension of the mechanisms underlying these training-induced variations will hinge on knowledge gained about the transsynaptic modulation of cholinergic pathways. The septal region receives numerous ascending and descending projections [43,50,66], and intraseptal injection of drugs has shown that a number of neurotransmitters exert net inhibitory or excitatory influences on cholinergic activity with either tonic or phasic actions [12,16]. With respect to the testing-induced activation and subsequent deactivation of hippocampal cholinergic activity, we postulated that cholinergic neurons in the septo-hippocampal pathway are under the control of at least two major transsynaptic components. Among the phasically active excitatory afferents to the septum that could mediate the observed daily acute testing-induced increase in hippocampal S D H A C U [12], the noradrenergic system, via a-receptors, appeared to be the best candidate in view of its implication in both learning and memory [59], attentional processes [22] and in age-related learning deficits [72]. In previous studies, we showed that the cholinergic system could be directly or indirectly deactivated by glutamatergic innervation of the lateral septum originating in the hippocampal formation [35,47]. The present study was designed to further characterize transsynaptic mechanisms underlying the testing-induced increase in hippocampal cholinergic activity. We report here our results on the effects of in-

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traseptal injections of noradrenergic agonists and aantagonists on the activity of cholinergic neurons of the septo-hippocampal pathway in mice kept under resting conditions or submitted to a first session of a mixed working reference memory task in an 8-arm radial maze.

2. Materials and methods 2.1. Animals O n e - h u n d r e d and fifty-one male mice of the C57B1/6 Jl co strain (IFFA-Credo, Lyon, France) were used. They were 6 - 8 weeks of age on arrival in the laboratory, and were housed in an animal house maintained at a constant temperature (22+2°C), on a 12/12 lightdark cycle (lights on at 07.00 h) with free access to food and water. After 3 - 6 weeks, the animals were placed in individual cages for at least one week prior to experimentation.

2.2. Surgery The mice were operated u n d e r general anesthesia (sodium thiopental, 70 m g / k g i.p.). Bilateral stainless steel guide-cannulae (30 gauge, 8 m m long) were stereotaxically implanted into the dorsolateral septum. Stereotaxic coordinates were 0.6 m m anterior to the bregma, _+0.3 m m lateral to the midline, and 1.9 m m below the dura. Guide-cannulae were fixed to the skull using acrylic dental cement and fine bone screws. The mice were allowed to recover from surgery for at least a fortnight before experimentation.

2.3. Injection protocol Bilateral intraseptal injections were performed in freely moving mice via injection cannulae (9.3 m m long) attached to 1 /~1 Hamilton syringes via polyethylene catheter tubing. The syringes were held in a constant rate infusion pump and fluids (0.2 /zl per injection site) were injected over a 3 min period. In all cases, correct injection flow rates were checked visually. The cannulae were left in place for a further 2 min before removal. Cannulae placements were verified visually during the brain dissection phase prior to neurochemical assay.

2.4. Drugs T h e a I antagonist BE 2254 was kindly provided by Dr. Eva Hofferber (Beiersdorf, Hamburg, FRG). The a antagonist phenoxybenzamine and the a 2 agonist U K 14304 (bromoxidine) were purchased from Research Biochemicals Inc. (Natick, MA, USA). The a 2 antagonist yohimbine and the noradrenaline uptake inhibitor maprotiline were obtained from Sigma. Solutions of drugs were made up freshly before use by dilution in sterile saline, with the exception of U K 14,304 which was dissolved in artificial CSF (glucose 5 mM, NaCl 125 mM, N a H C O 3 27 mM, KC1 2.5 mM, N a H 2 P O 4 0.5 mM, N a 2 H P O 4 1.2 mM, Na2SO 4 0.5 mM, MgCI 2 1.0 mM, CaCI 2 1.0 mM).

2.5. Behavioral testing The animals were submitted to a spatial discrimination task in an elevated 8-arm radial maze based on that of Olton et al. [58], and described in detail elsewhere [46]. Two days before the training session, the animals were food

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deprived to maintain their body weight at 88% of their free-feeding weight, and the behavioral testing began without any habituation to the apparatus. Each mouse was assigned a constant set of 3 baited arms. They were chosen such that the 3 angles separating them were always 90°, 135° and 135°. At the beginning of each trial, each of these arms was pre-baited with a single food pellet. A trial began by placing a mouse in the central platform, initially with all the doors closed. The doors were then simultaneously opened and remained so until the end of the trial. The mouse was allowed to choose freely among the 8 arms until the last reward was collected. The session ended after 6 trials or when 30 min had elapsed. The animals were killed 30 s after testing and brains were dissected for neurochemical analysis. The experimental design is summarized in Fig. 1.

2.6. Neurochemical analysis The animals were killed by a brief immersion in liquid nitrogen followed by immediate decapitation and dissection of the brain. The hippocampus was rapidly dissected on a glass plate placed over liquid nitrogen vapor. The in vivo cholinergic activity was quantified by measuring in vitro the rate of sodium-dependent-high-affinitycholine-uptake (SDHACU) in crude synaptosomal (P2) fractions from fresh samples of hippocampus using a modified technique of Atweh et al. [5]. Briefly, P2 samples (about 1 mg/ml protein) were incubated in normal Krebs-Ringer buffer and also in sodium-free medium (NaC1 was replaced by equimolar LiCI) at pH 7.4 for 4 min at 37°C, in the presence of 0.25 mM external [methyl-3H]choline chloride (NEN, France). Uptake was terminated by filtration on HA-type 0.45-/xm pore size filters (Millipore). Filters were dissolved and counted in a liquid scintillation counter (Beckman). The amount of choline taken up by the sodium-dependent high-affinity mecha-

nism was calculated from the difference between the sodium and sodium-free incubations.

3. Results

3.1. First experiment 3.1.1. Effects of intraseptal injection of the noradrenaline reuptake inhibitor maprotiline on 'basal' hippocampal cholinergic activity (SDHACU) measured in 'quiet' animals" E n h a n c e d r e l e a s e o f N A a n d its m e t a b o l i t e s h a s b e e n o b s e r v e d in v a r i o u s b r a i n s t r u c t u r e s i n c l u d i n g t h e h i p p o c a m p u s , in r e s p o n s e t o d i f f e r e n t s t i m u l i [1,38,57]. H o w e v e r , s y s t e m i c a d m i n i s t r a t i o n o f a m p h e t a m i n e in animals with lesions of the ventral noradrenergic bundie has led to contradictory results. Depending on the s t u d y , a n e x c i t a t o r y [62] o r a n i n h i b i t o r y [56] i n f l u e n c e of this noradrenergic input to the septo-hippocampal cholinergic system was observed. In the present experim e n t , m a p r o t i l i n e , a b l o c k e r o f N A r e u p t a k e [8], w a s injected bilateraly into the lateral septum of 'quiet' a n i m a l s in a n a t t e m p t t o m i m i c t h e b e h a v i o r - i n d u c e d i n c r e a s e in N A in t h e s y n a p t i c cleft. W e w e r e p a r t i c u larly i n t e r e s t e d in t h e e f f e c t o f s e p t a l i n j e c t i o n o f m a p r o t i l i n e o n h i p p o c a m p a l c h o l i n e r g i c activity.

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Fig. 1. Overview of the experimental design. The figure summarizes the experiments designed to evaluate the drug effects on ~basal' hippocampal sodium-dependent-high-affinity-choline-uptake (SDHACU) (Situation I) and on 'activated' SDHACU in trained animals (Situation II).

A. Marighetto et aL/ Brain Research 652 (1994) 120-128 Twenty-four animals with intra-septal c a n n u l a e were divided into three groups (vehicle ( 0 . 2 / z l / s i t e ) n = 10; maprotiline 0.2 pmol = 0.06 n g / s i t e n = 7; maprotiline 2 pmol = 0.6 n g / s i t e n = 7). Drugs were administered 20 min before the animals were killed. T h e results are summarized in Fig. 2A. A one-way A N O V A revealed a significant effect of t r e a t m e n t (Fe,2~ = 3.693, P = 0.042). Post-hoc pairwise comparisons showed a significant increase in hippocampal S D H A C U in the 0.06 ng maprotiline g r o u p c o m p a r e d to the vehicle group ( D u n n e t t t-test: P < 0.05), whereas no changes were observed at the higher dose (0.6 ng). These results suggest that elevation of N A in the synaptic cleft of the septal area induced by infusion of a N A r e u p t a k e inhibitor has an excitatory influence on cholinergic neurons. T h e lack of effect with the higher dose of maprotiline was attributed to the antimuscarinic activity of this drug [8], as it has been d e m o n s t r a t e d that injection of scopolamine or infusion of atropine into the septal region r e d u c e d release of acetylcholine (ACh) from the h i p p o c a m p u s [14,27]. Blockade of muscarinic a u t o r e c e p t o r s of the septal cholinergic n e u r o n s may also have antagonized the stimulating influence of NA. T h e results of this experim e n t are nevertheless consistent with the idea that the acute increase in hippocampal cholinergic activity observed immediately after training may be, at least partly, induced by the phasically active noradrenergic input to

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Fig. 3. Effects of intraseptal injections (0.2 p.l bilaterally) of different a-noradrenergic antagonists on hippocampal SDHACU measured in the 'quiet' condition (open bars) and at 30 s following the first training session in the radial-maze (shaded bars). For each experiment, values are expressed as percent variation of the corresponding 'quiet' vehicle control group (SDHACU (mean+S.E.M.) for the pooled vehicle animals (n = 17) was 13.75_+0.44 pmol/4 min/mg protein). * Significantly different from the 'quiet' condition (P < 0.05); ©P < 0.05; § significantly different from the dose of 0.5 ng (P < 0.05).

the septum. This transsynaptic stimulation of cholinergic n e u r o n s appears to be mediated by a - r e c e p t o r s [12,45]. Since these previous studies employed a nonselective a - n o r a d r e n e r g i c antagonist (phenoxybenzamine), the following set of experiments were c o n d u c t e d to determine the effects of intraseptal injection of different a - n o r a d r e n e r g i c antagonists ( a 1, a 2 and a t a 2) on the hippocampal cholinergic activity in both 'quiet' control mice and in animals submitted to a first session of m e m o r y testing.

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In preliminary experiments we evaluated the effects of doses of 10, 100 and 1000 ng of p h e n o x y b e n z a m i n e on 'basal' hippocampal S D H A C U observed in the 'quiet' condition. Since there was a significant treatment effect (one-way A N O V A : F3,27 = 4.62, P < 0.01) (see Fig. 2B), we used a dose of 100 n g / s i t e for the subsequent experiments on the influence of phenoxyb e n z a m i n e on the cholinergic activation induced by a first learning session in the radial-maze. T h e results are summarized in Fig. 3A.

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A two-way ANOVA (treatment × behavior) showed that radial-maze testing induced a significant increase in SDHACU (F~,2o = 42.64, P = 0.0001), whereas the drug treatment did not affect cholinergic activity ( F < 1). The drug tended to decrease 'basal' SDHACU in the 'quiet' mice without modifying the absolute level of SDHACU in the 'active' mice, and thus somewhat enhanced activation of SDHACU in these animals (see Fig. 3A), although this effect failed to reach significance (treatment × behavior interaction) (F~,20 = 2.382, P <0.14).

3.2.2. Effects of intraseptal injection of the a l antagonist BE 2254 on the activation of hippocampal cholinergic activity produced by behavioral testing Two doses of BE 2254 (100 and 500 ng) were used in this experiment. The results are illustrated in Fig. 3B. Immediately after training, hippocampal SDHACU was increased in the 'active' animals as compared to the 'quiet' controls (two-way ANOVA: Fn,xs = 72.63, P = 0.0001). There was a significant treatment effect (F2.2s = 8.79, P = 0.0011), that was dependent on the behavioral situation (treatment x behavior interaction) (F2,2s = 7.12, P=0.0032). Although treatment had a slight effect on SDHACU in the 'quiet' animals ( F < I), it reduced (100 ng) or blocked (500 ng) the increase in SDHACU normally induced by the learning session. As indicated in Fig. 3B, post-hoc analysis showed that training induced a significant increase in SDHACU in the vehicle group (+31.9%; Scheffe F-test: P < 0.01) as in the group BE-100 ng (+20.4%; P < 0.01), but this increase was almost completely blocked by the 500 ng dose of BE 2254 ( + 8.9%; F < 1). Moreover, hippocampal SDHACU in the 'active' animals was significantly lower in the BE 500 ng group than in the other two groups ( P < 0.02). 3.2.3. Effects of intraseptal injection of the a 2-antagonist yohimbine on the actiz,ation of hippocarnpal cholinergic actit,ity produced by memory testing The effects of two doses of yohimbine (0.5 and 50 ng) were investigated in this experiment. The results are summarized in Fig. 3C. The two-way ANOVA revealed a main effect of behavior (Fn.30=49.85, P=0.0001), and no overall treatment effect (F2,30 = 2.21, P = 0.13), although there was a treatment-behavior interaction (F2.3o = 4.84, P = 0.015). As illustrated in Fig. 3C, the training-induced activation was statistically significant in both vehicle and yoh.0.5 ng groups (+29.89% and +43.32%, respectively; P < 0.01), but not in the yoh.50 ng group ( + 12.56%; F < 1). Moreover, the two doses of yohimbine had significantly different effects on SDHACU in both 'quiet' and 'active' animals (treatment (yoh.0.5/ yoh.50) × behavior interaction) (FL20 = 16.46, P =

0.0006). This interaction was due mainly to the drug effect in the 'quiet' animals (yoh.0.5 vs yoh.50; P < 0.05). Several findings emerged from this set of experiments which will be discussed in greater detail in a later section. (1) The basal level of hippocampal cholinergic activity was not significantly affected by intraseptal injection of different doses of o~-adrenoceptor antagonists (except the 1000 ng dose of phenoxybenzamine, which increased SDHACU). (2) Only one treatment (BE 2254; 500 ng) significantly reduced the training-induced activation of SDHACU. (3) A closer examination of the yohimbine experiment revealed a significant increase of basal SDHACU by the 50 ng dose compared to the 0.5 ng dose. Furthermore, the testing-induced increase of SDHACU was relatively little potentiated by the yohimbine treatment. These observations have led us to propose the existence of two types of noradrenergic influence on the cholinergic neurons: (i) the findings of the BE 2254 experiment suggest that the behavior-induced activation of hippocampal SDHACU could be mediated by stimulation of al-adrenoceptors; (ii) on the basis of the dose effects of yohimbine on 'basal' cholinergic activity and to a lesser extent on 'activated' cholinergic activity, noradrenergic afferents were thought to exert an inhibitory influence via c~2-adrenoceptors on septal cholinergic neurons. There is evidence that cQ-antagonists such as rauwolscine, idazoxan and yohimbine have a higher absolute affinity for the presynaptic than for the postsynaptic c~2-adrenoceptor [33]. Consequently, the lack of effect with the small dose of yohimbine may be a net result of blockade of prc- and postsynaptic a,adrenoceptors, whereas the effects of the higher dose of yohimbine may be the result of total blockade of the post-synaptic receptors thereby relaxing the inhibitory control of the a2-adrenoceptors on the cholinergic neurons. In line with this interpretation, yohimbine would be expected to potentiate the testing-induced increase of SDHACU since the al-mediated stimulation would no longer be opposed by the a2-mediated inhibition. A possible explanation for this discrepancy may lie in the c~-adrenoceptor selectivity of yohimbine. A selectivity ratio (OL2/Og 1 ) of 5.7 was found in rat cerebral cortex by Doxey et al. [21]. This could explain the effect of the higher dose of yohimbine in the 'active' condition, in which the behavioral activation of SDHACU may in fact be slightly reduced by partial blockade of c~-receptors rather than potentiated. In order to assess the possibility of a 2 mediated tonic inhibition, the next experiment was designed to examine the effects of an intraseptal injection of the cr2-agonist UK 14,304 on 'basal' hippocampal choliner-

A. Marighetto et al. / Brain Research 652 (1994) 120-128

gic activity. The expected result was a reduction in SDHACU.

3.3. Third experiment 3.3.1. Effects of intraseptal injection of the full ot2-agonist UK 14,304 on 'basal' hippocampal cholinergic activity In this experiment we measured SDHACU in hippocampal synaptosomes 20 min after drug administration in animals under the 'quiet' condition. Fourteen mice were divided into two groups (n = 7 in each group); a vehicle group (0.2 /zl/site) and an experimental group (UK 14,304, 1.5 ng/site = 5 pmol/site). The results are summarized in Fig. 2C. A one-way ANOVA revealed that the intraseptal injection of UK 14,304 induced a significant decrease in hippocampal SDHACU (FI.12 = 14.65, P < 0.003). This result is consistent with the assumption that the noradrenergic system exerts a tonic inhibitory influence on the septal cholinergic neurons via a2-adrenoceptors.

4. Discussion

The present findings show that choline uptake in the hippocampus can be influenced by intraseptal injection of noradrenergic drugs, and they are consistent with the idea that the noradrenergic innervation of the septal area has a bimodal regulatory influence on the activity of the septo-hippocampal cholinergic neurons. We suggest that the behavioral-induced activation of hippocampal SDHACU is partly mediated by stimulation of al-adrenoceptors, whereas az-adrenoceptors are involved in maintaining tonic inhibition of steadystate cholinergic activity in the hippocampus. These results will be discussed before considering the functional implications of the tonic and phasic influences of the noradrenergic system on the septo-hippocampal cholinergic neurons. It is now well documented that both the septal area and the hippocampus receive a dense innervation of noradrenergic fibers arising mostly from the locus coeruleus [43,44]. Radioligand binding and autoradiographic studies have shown a high density of the different subtypes of a-adrenoceptors in the septo-hippocampal area [36,53,60]. The number and the localization of the different oq- or az-adrenoceptor subtypes appear to vary widely between the different brain regions. Concerning the distribution of az-subtypes in the septo-hippocampal area, the ratio between [3H] idazoxan and [3H]rauwolscine binding was found to be 2:1 in the stratum radiatum and the lacunosum moleculare of the hippocampus, whereas it was 20 : 1 in the lateral septum [9]. This regional diversity tends to complicate pharmacological investigation of the respec-

125

tive role of a 1- and a2-adrenoceptors. This difficulty is increased by the observation that az-adrenoceptors in the brain are located both pre- and post-synaptically [20,67], so that opposite pharmacological effects may be observed according to the extent of activation of each population of adrenoceptors [49]. The increase in hippocampal SDHACU produced by intraseptal infusion of maprotiline suggests that elevation of NA in the synaptic cleft may stimulate septo-hippocampal cholinergic activity. The present finding is consistent with the increase in septo-hippocampal cholinergic activity observed after systemic administration of amphetamine [12,56,62]. Since this excitatory effect was prevented by intraseptal phenoxybenzamine, the action of amphetamine was assumed to be mediated by a-adrenoceptors [12]. Because BE 2254 was the only compound that blocked the testing-induced activation of hippocampal SDHACU without altering the basal level, we concluded that this stimulatory influence was mediated by a~-adrenoceptors. Since biochemical and electrophysiological studies indicate that cholinergic and noradrenergic systems have complex interactions [16], we suggest that, apart from a phasic excitatory modulation, the noradrenergic innervation also exerts a tonic inhibitory control on the septo-hippocampal cholinergic pathway via postsynaptic az-adrenoceptors. In line with these findings, Nilsson et al. have observed an increase in hippocampal ACh release after either systemic injection of amphetamine or lesion of the dorsal and ventral noradrenergic bundle, which is indicative of both excitatory and inhibitory noradrenergic influences on the septohippocampal cholinergic system [56]. It has been suggested that noradrenergic fibers originating in the locus coeruleus exert a tonic inhibitory effect on cortical cholinergic neurons [68] via az-adrenoceptors located on cholinergic terminals [6,52]. The functional distinction between pre- and post-synaptic az-adrenoceptors assessed in the present study is in line with electrophysiological data showing that activation of az-adrenergic autoreceptors with low doses of clonidine results in a reduction in the tonic inhibitory effect of endogenous noradrenaline on serotonin (5-HT) neurotransmission, whereas higher doses of clonidine are thought to decrease 5-HT neurotransmission via a direct activation of a2-adrenergic heteroreceptors on 5-HT terminals [49]. Although these complex cholinergic-noradrenergic interactions are undoubtedly of functional significance, we are far from understanding their role in modulating cognitive processes. In most studies, systemic noradrenergic manipulations or chronic NA depletion induced by neurotoxins have little impact on memory [11,17,28,63]. There is evidence, however, that noradrenergic dysfunction is implicated in age-related memory deficits [42,71]. This is supported by studies

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reporting the effects of a combination of cholinergic and noradrenergic disruption on learning and memory performance in similar tasks. Most studies have reported additive or synergistic negative effects [15,30], whereas other authors have reported compensatory effects [51,65]. To resolve these discrepancies, Sara et al. [65] have suggested that the balance between cholinergic and noradrenergic activity is critical and that under some circumstances memory deficits induced by cholinergic degeneration may be alleviated by a reduction in noradrenergic function. Although these results highlight the importance of reciprocal interactions between the noradrenergic and cholinergic systems in cognitive function, little is known about the involvement of the adrenoceptors mediating noradrenergic input on the cholinergic neurons during learning and memory processes. Electrophysiological studies provide evidence that endogenously released NA may facilitate excitatory cholinergic action [39,54, 69] via a~-adrenoceptors [54,69]. Despite the physiological importance of these interactions, to our knowledge, no studies have investigated the role of a~-adrenoceptors in learning and memory. Some experiments using non-selective a-adrenoceptor antagonists have shown that systemic or intraseptal injections of phentolamine or phenoxybenzamine have detrimental effects on spatial learning [31] or spatial working memory [45]. Although phenoxybenzamine has greater affinity for o~1 than for a2-adrenoceptors [48], we cannot rule out the possible involvement of az-adrenoceptors in the memory deficit observed in the phenoxybenzamine-injected animals. Conversely, several studies have reported positive effects on learning and memory tasks of systemic administration of a2-adrenoceptors agonists in either lesioned or aged rats [2,10,30] and in aged monkeys [3,4] but not in normal subjects. In apparent contradiction, other studies have showed a facilitation of either memory retrieval [64] or attentional performance [19,61] after administration of a 2 antagonists. In the first series of experiments, c~2 agonists were used to reverse the memory deficits subsequent to either brain damage or aging, whereas a2-antagonists were administered to normal subjects to enhance release of NA. In these studies, the pharmacological manipulation was aimed at increasing noradrenergic transmission either at the postsynaptic level with a2-agonists in an attempt to compensate the possible decrease in ~2-adrenoceptor sites [26,37] (but see [29]), or at a presynaptic level by blocking autoreceptor inhibition of NA release with c~ antagonists. Most of these studies have only considered the impact of a2-1igands on learning and memory processes without investigating the interaction with the cholinergic system. However, it has been recently demonstrated that forebrain cholinergic plus noradrenergic lesion-induced deficits can be reversed by a treatment combining the cholinergic and noradrenergic ag-

onists physostigmine and clonidine [30]. Although physostigmine treatment was found to enhance the efficacy of clonidine the nature of the mechanisms involved is still unclear. These studies emphasize the modulatory influence of the noradrenergic system on septo-hippocampal cholinergic activity. Apart from the various roles played by c~-adrenoceptor subtypes, the intervention of /3-adrenoceptors should not be overlooked since their influence on the cholinergic septo-hippocampal system may differ from that of the c~-adrenoceptors [31,55,69]. The nature of these noradrenergic cholinergic interactions and their functional role represent exciting problems for future studies.

Acknowledgements This research was supported by the 'Centre National de la Recherche Scientifique' and by the 'Conseil R6gional d'Aquitaine'.

References [l] Abercrombie, E.D., Keller, R.W. and Zigmond, M.J., Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: pharmacological and behavioral studies, Neuroseience. 27 (1988) 897-904. [2] Ammassari-Teule, M., Maho, C. and Sara, S.J., Clonidine reverses spatial learning deficits and reinstates 0 frequencies in rats with partial fornix section, Behat. Brain Res., 45 (19911 1-8. [3] Arnsten. A.F.T. and Leslie, F.M., Behavioral and receptor binding analysis of the ~2 adrenergic agonist, UK-14304 (5-bromo6[2-imidazoline-2-yl amino]quinoxaline): evidence for cognitive enhancement at an alpha-2 adrenoreceptor subtype, Neuropharmacology, 3(1 (1991) 1279 1289. [4] Arnsten, A.F.T., Cai, J.X. anti Goldman-Rakic, P.S., The ~t~ adrenergic agonist guanfacine improves memory in aged monkeys without sedative or hypotensive side effects: evidence for ¢~ receptor subtypes..L Neurosci., 8 (1988) 4297 4298. [5] Atweh, S.F.. Simon, J.R. and Kuhar, M.J., Utilization of sodium-dependent high-affinity choline uptake in vitro as measure of the activity of cholinergic neurons in vivo, Li[k, Sei., 17 (1975) 1535-1544. [6] Beani, L., Tanganelli, S., Antonelli, T. and Bianchi, C., Noradrenergic modulation of cortical acetylcholine release is both direct and y-aminobutyric acid-mediated, J. Pharmacol. Exp. Ther.. 236 (19861 2311 236. [7] B6racoch&a, D., Micheau, J. and Jaffard, R., Memory deficits following chronic alcohol consumption in mice: relationships with hippocampal and cortical cholinergic activities, Pharmacol. Biochem. Behat'., 42 (1992) 749-753. [8] Blackwell, B., Newer antidepressant drugs. In Meltzer tt.Y. (Ed.), Psycho-Pharmacolo~,n,: the Third Generation of Progress. Raven Press, New York, 1987, pp. 1(/41-1049. [9] Boyajian, C.L., Loughlin, S.E. and Leslie, F.M., Anatomical evidence for a : heterogeneity: differential autoradiographic distributions of [-~H]-rauwolscine and [~H]-idazoxan in rat brain, J. PharrnacoL Exp. Ther.. 241 (1987) 1079-1091. [10] Carlson, S.. Tanila, H., Riimfi, P,, Mecke, E. and Pertovaara, A..

A. Marighetto et aL /Brain Research 652 (1994) 120-128 Effects of medetomidine, an c~z-adrenoceptor agonist, and atipamezole, an c~2-antagonist, on spatial memory performance in adult and aged rats, Behat'. Neural Biol., 58 (1992) 113-119. [11] Chrobak, J.C., DeHaven, D.L. and Walsh, T.J., Depletion of brain norepinephrine with DSP-4 fails to alter acquisition or performance of a radial arm maze task, BehaL,. Neural Biol., 44 (1985) 144-150. [12] Costa, E., Panula, P., Thompson, H.K. and Cheney, D.L., The transsynaptic regulation of the septal hippocampal cholinergic neurons, Life Sci., 32 (1983) 165-179. [13] Day, J., Damsma, G. and Fibiger, H.C., Cholinergic activity in the rat hippocampus, cortex and striatum correlates with locomotor activity: an in vivo microdialysis study, PharmacoL Biochem. Behat'., 38 (1991) 723-729. [14] De Boer, P., Moor, E., Zaagsma, J. and Westering, B.H.C., Consequences of septal manipulation on the release of acetylcholine from the ventral hippocampus: an in vivo microdialysis study. In Rollema, H., Westering, B. and Drijfhout, W.J. (Eds.), Monitoring Molecules" in Neurosciences, Proceedings of the 5th International Conference on in vivo methods, Elsevier, Amsterdam, 1991, pp. 279-282. [15] Decker, M.W. and Gallagher, M., Scopolamine-disruption of radial arm maze performance: modification by noradrenergic depletion, Brain Res., 417 (1987) 59-69. [16] Decker, M.W. and McGaugh, J.L., The role of interactions between the cholinergic system and other neuromodulatory systems in learning and memory, Synapse, 7 (1991) 151-168. [17] Decker, M.W., Cholinergic/noradrenergic interactions and memory. In E.D. Levin, M.W. Decker and L.L. Butcher (Eds.), Neurotransmitter Interactions and Cognitil'e Function, Birhk~iuser, Boston, 1992, pp. 78-90. [18] Decker, M.W., Pelleymounter, M.A. and Gallagher, M., Effects of training on a spatial memory task on high affinity choline uptake in hippocampus and cortex in young adult and aged rats, J. Neurosci., 8 (1988) 90-99. [19] Devauges, V. and Sara, S.J., Activation of the noradrenergic system facilitates an attentional shift in the rat, Behat,. Brain Res.. 39 (1990) 19-28. [20] Dooley, D.J., Bittiger, H., Hauser, K.L., Bischoff, S.F. and Waldmeier, P.C., Alteration of central d e- and /3-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin, Neuroscience, 9 (1983)889-898. [21] Doxey, J.C., Lane, A.C., Roach, A.G. and Virdee, N.K., Comparison of the a-adrenoceptor antagonist profiles of idazoxan (RX 781094), yohimbine, rauwolscine and corynanthine, Naunyn-Schmiedeberg's Arch. Pharmacol., 325 (1984) 136-144. [22] Everitt, B.J., Robbins, T.W. and Selden, N.R.W., Functions of the locus coeruleus noradrenergic system: a neurobiological and behavioural synthesis. In Heal, D.J. and Marsden, C.A., (Eds.), The Pharmacology of Noradrenaline in the Central Nert,ous System, Oxford University Press, Oxford, 1990, pp. 349-378. [23] Fibiger, H.C., Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence, Trends Neurosci., 14 (1991) 220-223. [24] Fordyce, D.E. and Farrar, R.P., Physical activity effects on hippocampal and parietal cortical cholinergic function and spatial learning in F344 rats, Behat,. Brain Res., 43 (1991) 115-123. [25] Gallagher, M. and Pelleymounter, M.A., An age-related spatial learning deficit: choline uptake distinguishes 'impaired' and 'unimpaired' rats, Neurobiol. Aging, 9 (1988) 363-369. [26] Gelgmann, C.M. and Mfiller, W.E., Specific decrease of high-affinity agonist states of a2-adrenoceptors in the aging mouse brain, J. Neural Transm., 79 (1990) 131-136. [27] Gorman, L.K., Pang, K., Givens, B., Kwon, C. and Olton, D.S., lntraseptal infusion alters acetylcholine release in the hippocampus of the rat, Soc. Neurosci. Abstr., 17 Part 1 (1991) 55.8.

127

[28] Hagan, J.J., Alpert, J.E., Morris, R.G.M. and Iversen, S.D., The effects of central catecholamine depletions on spatial learning in rats, Behat'. Brain Res., 9 (1983) 83-104. [29] Harik, S.I., Sromek, S.M. and Kalaria, R.N., or- and /3-adrenergic receptors of the rat cerebral cortex and cerebral microvessels in aging, and their response to denervation, NeurobioL Aging, 12 (1991) 567-573. [30] Haroutunian, V., Kanof, P.D., Tsuboyama, G. and Davis, K.L., Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats, Brain Res., 507 (1990) 261-266. [31] Harrel, L.E., Peagler, A. and Parsons, D.S., Adrenoreceptor antagonist treatment influences recovery of learning following medial septal lesions and hippocampal sympathetic ingrowth, PharmacoL Biochem. Behat,., 35 (1990) 21-28. [32] Hasselmo, M.E. and Bower, J.M., Acetylcholine and memory, Trends Neurosci., 16 (1993) 218-222. [33] Hieble, J.P., Sulpizio, A.C., Nichols, A.J., Willette, R.N. and Ruffolo, R.R., Pharmacologic characterization of SK&F104078, a novel ae-adrenoceptor antagonist which discriminates between pre- and postjunctional c~2-adrenoceptors, J. PharmacoL Exp. Ther, 247 (1988) 645-652. [34] Jaffard, R. and Micheau, J., Central cholinergic systems, learning and memory. In J. Delacour (Ed.), Neural Basis of Learning and Memory, World Scientific Press, in press. [35] Jaffard, R., Marighetto, A. and Micheau, J., Septal noradrenergic and glutamatergic influences on hippocampal cholinergic activity in relation to spatial learning and memory in mice. In E.D. Levin, M.W. Decker and L.L. Butcher (Eds.), Neurotransmitter Interactions and CognitiL~e Function, Birhk~iuser, Boston, 1992, pp. 103-117. [36] Jones, C.R., Hoyer, D. and Palacios, J.M., Adrenoceptor autoradiography. In Heal, D.J. and Marsden, C.A. (Eds.), The Pharmacology of Noradrenaline in the Central Nert'ous System, Oxford University Press, Oxford, 1990, pp. 41-75. [37] Kalaria, R.N. and Andorn, A.C., Adrenergic receptors in aging and Alzheimer's disease: decrease a2-receptors demonstrated by [3H]p-aminoclonidine binding in prefrontal cortex, NeurobioL Aging, 12 (1991) 131-136. [38] Kalen, P., Rosegren, E., Lindvall, O. and Bj6rklund, A., Hippocampal noradrenaline and serotonin release over 24 h as measured by the dialysis technique in freely moving rats: correlation to behavioural activity state, effect of handling and tail-pinch, Eur. J. Neurosci., 1 (1989) 181-188. [39] Kruglikov, R.I., On the interaction of neurotransmitter systems in processes of learning and memory. In Matthies, H. and Marsan, C.A. (Eds.), Neuronal Plasticity and Memory Formation, Raven Press, New York, 1982, pp. 339-351. [40] La'i, H., Zabwska, J. and Horita, A., Sodium-dependent high-affinity choline uptake in hippocampus and frontal cortex of the rat affected by acute restraint stress, Brain Res., 372 (1986) 366-369. [41] Lebrun, C., Durkin, T.P., Marighetto, A. and Jaffard, R., A comparison of the working memory performances of young and aged mice combined with parallel measures of testing and drug-induced activations of septohippocampal and nbm-cortical cholinergic neurones, NeurobioL Aging, 11 (1990) 515-521. [42] Leslie, F.M., Loughlin, S.E., Sternberg, D.B., McGaugh, J.L., Young, L.E. and Zornetzer, S.F., Noradrenergic changes and memory loss in aged mice, Brian Res., 359 (1985) 292-299. [43] Lindvall, O. and Steveni, U., Dopamine and noradrenaline neurons projecting to the septal area in the rat, Cell Tiss. Res., 190 (1978) 383-405. [44] Loy, R., Koziell, D.A., Lindsay, J.D. and Moore, R.Y., Noradrenergic innervation of the adult rat hippocampal formation, J. Comp. NeuroL, 189 (1980) 699-710.

128

A. Marighetto et al. / Brain Research 652 (1994) 120-128

[45] Marighetto, A., Durkin, T., Toumane, A., Lebrun, C. and Jaffard, R., Septal a-noradrenergic antagonism in vivo blocks the testing-induced activation of septohippocampal cholinergic neurones and produces a concomitant deficit in working memory performance of mice, Pharmacol. Biochem. Behac., 34 (1989) 553-558. [46] Marighetto, A., Micheau, J. and Jaffard, R., Relationships between testing-induced alterations of hippocampal cholinergic activity and memory performance on two spatial tasks in mice, Behat. Brain Res., 56 (1993) 133-144. [47] Micheau, J., Marighetto, A. and Jaffard, R., Influence of the septal glutamatergic input on the training-induced changes in hippocampal cholinergic activity in mice, J. Psychopharmacol., Abstract book (1992) A87: 345. [48] Minneman, K.P., Phenoxybenzamine is more potent in inactivating a 1- than a2-adrenergic receptor binding sites, Eur. J. Pharmacol., 94 (1983) 171-174. [49] Mongeau, R., Blier, P. and de Montigny, C., In vivo electrophysiological evidence for tonic activation by endogenous noradrenaline of a~-adrenoceptors on 5-hydroxytryptamine terminals in the rat hippocampus, Naunyn-Schmiedeberg's Arch. Pharmacol., 347 (1993) 266-272. [50] Moore, R.Y., Catecholamine innervation of the basal forebrain. I. The septal area, J. Comp. Neurol., 177 (1978) 665-684. [51] Moran, P.M., LeMa]tre, M.H., Philouze, V., Reymann, J.M., Allain, H. and Leonard, B.E., Reversal of learning and memory impairments following lesion of the nucleus basalis magnocellularis (NBM) by concurrent noradrenergic depletion using DSP4 in the rat, Bra& Res., 595 (1992) 327-333. [52] Moroni, F., Tanganelli, S., Antouelli, T., Carla, V., Bianchi. C. and Beani, L., Modulation of cortical acetylcholine and yaminobutyric acid release in freely moving guinea pigs: effects of clonidine and other adrenergic drugs, J. Pharmacol. Exp. Ther., 227 (1983) 435-440. [53] Morrow, A.L. and Creese, I., Characterization of al-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]-WB4104 and [3H]-prazosin binding, Mol. Pharmacol., 29 (1986) 321-330. [54] Mouradian, R.D., Sessler, F.M. and Waterhouse, B.D., Noradrenergic potentiation of excitatory transmitter action in cerebrocortical slices: evidence for mediation by an a t receptorlinked second messenger pathway, Brain Res., 546 (1991) 83-95. [55] Miieller, A.L., Hoffer, B.J. and Dunwiddie, T.V., Noradrenergic responses in rat hippocampus: evidence for mediation by a and /3 receptors in the in vitro slice, Brain Res., 214 (1981) 113-126. [56] Nilsson, O.G, Leanza, G. and Bj6rklund, A., Acetylcholine release in the hippocampus: regulation by monoaminergic afferents as assessed by in vivo microdialysis, Brain Res., 584 (1992) 132-140. [57] Nisenbaum, L.K., Zigmond, M.J., Sved, A.F. and Abercrombie. E.D., Prior exposure to chronic stress results in enhanced synthesis and release of hippocampal norepinephrine in response to a novel stressor, J. Neurosci., 11 (1991) 1478-1484. [58] Olton, D.S., Becker, J.T. and Handelmann, G.E., Hippocampus, space and memory, Behat,. Brain Sci., 2 (1979) 313-365. [59] Quatermain, D., The role of catecholamines in memory process-

[60]

[61]

[62]

[63]

[64]

[65]

[66] [67]

[68]

[69]

[70]

[71]

[72]

ing. In Deutsch, J.A. (Ed.), Physiological Basis of Memory, Academic Press, New York, 1983, pp. 387-423. Raymon, H.K., Smith, T.D. and Leslie, F.M., Further pharmacological characterization of [3H]-idazoxan binding sites in rat brain: evidence for predominant labeling of a2-adrenergic receptors, Brain Res., 582 (1992) 261-267. Riekkinen, P.J., Sirvio, J., Riekkinen, P., Valjakka, A., Jakala, A., Koivisto, E. and Lammintausta, R., The role of the noradrenergic system in higher cerebral functions: experimental studies about the effects of noradrenergic modulation on electrophysiology and behavior. In E.D. Levin, M.W. Decker and L.L. Butcher (Eds.), Neurotransmitter Interactions and Cognitice Function, Birkh~iuser, Boston, 1992, pp. 91-102. Robinson, S., 6-Hydroxydopamine lesion of the ventral noradrenergic bundle blocks the effect of amphetamine on hippocampal acetylcholine, Brain Res., 397 (1986) 181-184. Sara, S., Noradrenergic modulation of selective attention: its role in memory retrieval. In D. Olton, E. Gamzu and S. Corkin (Eds.), Memory Dysfunctions: an Integration of Animal and Human Research from Clinical and Preclinical Perspectices, Ann. NY Acad. Sci., Vol. 444, 1985, pp. 178-193. Sara, S.J. and Devauges, V., ldazoxan, an a-2 antagonist, facilitates memory retrieval in the rat, Behac. Neural Biol., 51 (1989) 4(/1-411. Sara, S.J., Dyon-Laurent, B., Guibert, B. and Leviel, V., Noradrenergic hyperactivity after partial fornix section: role in cholinergic dependent memory performance, Exp. Brain Res., 89 (1992) 125-132. Swanson, L.W. and Cowan, W.M., The connections of the septal region in the rat, J. Comp. Neurol., 186 (1979) 621-656. U'Prichard, D.C., Reisine, T.D., Mason, S.T., Fibiger, H.C. and Yamamura, H.I., Modulation of rat brain c~- and /3-adrenergic receptor populations by lesion of the dorsal noradrenergic bundle, Brain Res., 187 (1980) 143-154. Vizi, E.S., Modulation of cortical release of acetylcholine by noradrenaline released from nerves arising from the rat locus coeruleus, Neuroscience, 5 (1980) 2139-2144. Waterhouse, B.D., Moises, H.C. and Woodward, D.J., a-receptor-mediated facilitation of somatosensory cortical neuronal responses to excitatory synaptic inputs and iontophoretically applied acetylcholine, Neuropharmacology, 20 (1981) 907-920. Wenk, G., Hepler, D. and Olton, D., Behavior alters the uptake of [3H]choline into acetylcholinergic neurons of the nucleus basalis magnocellularis and medial septal area, Behal'. Brain Res., 13 (1984) 129-138. Zornetzer, S.F., Catecholamine system involvement in age-related memory dysfunction. In D. Olton, E. Gamzu and S. Corkin (Eds.), Memory Dysfunctions: an Integration of Animal and Human Research .from Clinical and Preclinical Perspectit'es, Ann. NY Acad. Sci., Vol. 444, 1985, pp. 242-254. Zornetzer, S.F., The noradrenergic locus coeruleus and senescent memory dysfunction. In Crook, T., Bartus, R., Ferris, S. and Gershon, S. (Eds.), Treatment Decelopment Strategies for Alzheimer~ Disease, Mark Powley Assoc., Madison, 1986, pp. 337-360.