Involvement of hippocampal CA3 KATP channels in contextual memory

Neuropharmacology 56 (2009) 615–625

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Involvement of hippocampal CA3 KATP channels in contextual memory Alexandre Betourne a, b, Ambre M. Bertholet a, c, Elodie Labroue a, b, He´le`ne Halley a, b, Hong Shuo Sun d, Anne Lorsignol a, c, Zhong-Ping Feng d, Robert J. French e, Luc Penicaud a, c, Jean-Michel Lassalle a, b, Bernard Frances a, b, * a

Universite´ de Toulouse, Centre de Recherches sur la Cognition Animale, France ˆt 4R3b3, 118 route de Narbonne, 31062 Toulouse, France CNRS-UMR 5169, Universite´ Paul Sabatier, UFR SVT Ba c ˆ t 4R3B1, 118 route de Narbonne, 31062 Toulouse Cedex, France Me´tabolisme, Plasticite´, Mitochondrie, CNRS-UMR 5241, Universite´ Paul Sabatier, UFR SVT, Ba d University of Toronto, Department of Physiology, Toronto, Ontario, Canada e University of Calgary, Department of Physiology and Biophysics, Calgary, Canada b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2008 Received in revised form 31 October 2008 Accepted 3 November 2008

This paper evaluates the involvement of hippocampal ATP-sensitive potassium channels (KATP) in learning and memory. After confirming expression of the Kir6.2 subunit in the CA3 region of C57BL/6J mice, we performed intra-hippocampal pharmacological injections of specific openers and blockers of KATP channels. The opener diazoxide, the blocker tolbutamide, or a mixture of both, were bilaterally injected in the CA3 region before we subjected the animals to a fear conditioning paradigm. Diazoxide strongly impaired contextual memory of mice at both doses tested. This impairment was specifically reversed by co-injecting the blocker tolbutamide. Moreover, we studied the mnemonic abilities of mice deleted for the Kir6.2 subunit. These mice were backcrossed to C57BL/6J mice and tested in two learning paradigms. We found a significant impairment of contextual and tone memories in the Kir6.2 knock-out mice when compared with heterozygous or wild-type animals. Furthermore, these animals were also slightly impaired in a spatial version of the Morris water maze task. Our data suggest a specific involvement of hippocampal KATP Kir6.2/SUR1 channels in memory processes. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Mouse Contextual memory Hippocampus CA3 KATP channels Diazoxide

1. Introduction ATP-sensitive potassium channels (KATP) belong to the inwardly rectifying super family of potassium channels. They are distributed in numerous tissues including pancreatic beta cells; smooth, skeletal and cardiac muscles; kidney and brain (Seino and Miki, 2003). The KATP channel acts as a metabolic sensor, coupling the energy status of the cell (represented by the [ATP]/[ADP] ratio) to its membrane potential and excitability. When the cell metabolism increases, these channels are able to sense the rise in intracellular ATP levels, consequently closing and depolarizing the cell. Conversely, in various stress conditions such as hypoglycemia or hypoxia, when the intracellular ATP levels fall, these channels open and hyperpolarize the cells, thus reducing their electrical activity, and in turn, activity-dependent release of hormones, mediators and neurotransmitters (Seino and Miki, 2003). Their physiological role

* Corresponding author. Universite´ de Toulouse, Centre de Recherches sur la Cognition Animale, CNRS UMR 5169, Baˆt 4R3 b3, Universite´ Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 4, France. Tel.: þ33 5 61 55 63 36; fax: þ33 5 61 55 61 54. E-mail address: [email protected] (B. Frances). 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.11.001

has been best characterized in pancreatic b-cells where they close in response to the elevation of blood glucose concentration, and trigger insulin secretion (Ashcroft, 2000). Structurally, KATP channels are hetero-octameric with four identical inwardly rectifying potassium channel subunits (either Kir6.1 or Kir6.2) forming the pore and four identical high-affinity sulphonylurea receptor subunits (either SUR1, SUR2A or SUR2B) (Shyng and Nichols, 1997). In the b-cell, the Kir6.2 subunit primarily confers inhibition of the channel by ATP whilst SUR1 subunits bind MgADP and ATP on two separate intracellular nucleotide binding folds. When the [ATP]/[ADP] ratio is decreased, the interaction of SUR1 with Kir6.2 reduces the affinity of Kir6.2 for ATP, thereby opening the KATP channel. SURx subunits also bind potassium channel openers (such as diazoxide) and blockers (such as sulfonylureas) with varying sensitivities (Seino et al., 2000). Therefore, tissue specific combination of different Kir6.x and SURx subunits will form a native channel with distinct electrophysiological and pharmacological properties (Seino and Miki, 2003). In the brain, as in the pancreatic b-cells, the Kir6.2/SUR1 channel seems to be the dominant KATP isoform (Bancila et al., 2005; Liss and Roeper, 2001; Miki et al., 2001; Sun et al., 2006; Zawar et al., 1999). Central Kir6.2/SUR1 KATP channels have been detected in many regions such as the hypothalamus, the substantia nigra (SNr)

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and the neocortex. Briefly, hypothalamic KATP channels participate in central glucose sensing (Miki et al., 2001), while they prevent seizure propagation in the SNr (Yamada and Inagaki, 2005). In addition, accumulating evidence suggests that brain KATP channels influence the release of neurotransmitters. They may modulate glutamate release from fused nerve terminals of the rat motor cortex (Lee et al., 1996);: dopamine, acetylcholine (ACh), glutamate and GABA release in the striatum (Amoroso et al., 1990; Lee et al., 1997; Shi et al., 2008; Tanaka et al., 1995) and GABA release in the SNr (During et al., 1995). In rats, Zawar et al. (1999) suggested that the Kir6.2/SUR1 combination was the major isoform in CA1 pyramidal cells and interneurons. Likewise, the binding sites of radioactive glibenclamide, a KATP blocker with a higher affinity for SUR1 subunits, were found highly abundant in the granular layer of the dentate gyrus and in the stratum lucidum of the CA3 region where mossy fibers synapse with the pyramidal cells (Mourre et al., 1990). Furthermore, stratum lucidum channels were found mainly on presynaptic mossy fiber terminals, as indicated by a strong reduction of [3H]glibenclamide binding in the CA3 after specific lesion (Mourre et al., 1991; Tremblay et al., 1991). Besides, in both rats and mice, the Kir6.2 protein is abundantly expressed in neurons of all hippocampal regions including pyramidal neurons in the CA1 and CA3 fields, granule cells in the dentate gyrus and interneurons in the hilus area (Sun et al., 2006; Thomzig et al., 2005). Therefore, the hippocampus seems particularly enriched in Kir6.2/SUR1-based channels. The hippocampal KATP channels play a major role in neuroprotection. Their activation during cellular stress causes a transient membrane hyperpolarisation with a consequent reduction of energy demand, thus providing an effective protection to the metabolically compromised cell (Yamada and Inagaki, 2005). Transgenic mice overexpressing the SUR1 unit display a general reduced sensitivity to kainic-acid induced seizure (HernandezSanchez et al., 2001). Accordingly, Kir6.2 null mice are more prone to neuronal damage after ischemic insult than their wild-type littermates (Sun et al., 2006). Finally, several in vitro studies suggest a modulation of mossy fibers glutamate release by Kir6.2/SUR1 channels. Ben-Ari (1990) first demonstrated that glibenclamide was able to enhance glutamate release in rat hippocampal slices. Using rat mossy fibers synaptosomes, Bancila et al. (2004) showed that the potassium channel opener diazoxide was able to reduce glutamate release through the opening of presynaptic KATP Kir6.2/ SUR1 channels. In addition, Quinta-Ferreira and Matias (2005) reported that zinc, co-released with glutamate by the mossy fibers, was able to inhibit mossy fiber long-term potentiation (LTP) after presynaptic activation of KATP channels in rat hippocampal slices. Altogether, these results suggest that hippocampal KATP channels are involved in the modulation of glutamate (and zinc) release by the mossy fibers. Consequently, they may influence the processing of new information at the mossy fibers/CA3 synapses. Therefore, we investigated the putative involvement of CA3 KATP channels in learning and memory. First, we studied the effects of bilateral intra-CA3 infusions of specific KATP openers and/or blockers on contextual memory processing in C57BL/6J mice, tested in the fear conditioning paradigm (FC). We then investigated the learning abilities of Kir6.2 null mice in the FC and further characterized their behavior in the Morris water maze task (MWM). Altogether, our results suggest that CA3 KATP Kir6.2/SUR1 channels are involved in the creation of new memories. 2. Materials and methods 2.1. Animals A total of 92 C57BL/6J male mice (Iffa Credo, France), 9–12 weeks-old and weighing 23–28 g at the time of surgery were used for the pharmacological

experiments. Kir6.2 knock-out (KO) mice were kindly provided by Dr. S. Seino. The KATP knock-out mice (Kir6.2/) used in the present experiment were genetically engineered to carry a mutation in the Kir6.2 isoform of the inward rectifier Kþ channel member of the KATP channel (Miki et al., 1998). These mice, with a mixed genetic background of C57BL/6J and 129 Sv, were backcrossed to C57BL/6J mice. Therefore, Kir6.2/ mice were mated with wild-type C57BL/6J mice to produce F1 offspring and F2 homozygous, heterozygous, and wild-type offspring of F1  F1 intercrosses that were used for behavioral testing. The animals were allowed to familiarize with the animal facility for at least 5 days before the start of the experiment. The mice were housed in groups of 3–6 in standard plastic cages with access to food and water ad libitum in a temperature controlled room (22–25  C), with a 12 h light–dark cycle (light on at 8 a.m.). Animal surgery and experimentation are authorized by the French Direction of Veterinary Service to AB, BF and JML. The study was approved by The French Animal Care and Use Committee (Comite´ re´gional d’Ethique pour l’expe´rimentation animale Midi Pyre´ne´es, ref MP 02/02/02/ 06). Experiments were performed in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (NRC, 2003) and the European Communities Council Directive of November 24, 1986 (86/609/EEC).

2.2. Genotyping DNA for genotyping was extracted from tail punches utilizing Chelex 100 Resin (Bio-Rad Laboratories). Genotyping was performed via PCR and gel-electrophoresis (2% TBE-agarose gels) using the following set of primers: Forward: TAG GCC AAG CCA GTG TAG TG, Reverse-KO: GGA GGA GTA GAA GTG GCG C, Reverse-WT: GCC CTG CTC TCG AAT GTT CT. For homozygote Kir6.2 KO mice, a PCR band of 386 bp was amplified, for Kir6.2 WT, PCR product was 222 bp, for heterozygote mice both PCR amplificates were derived from genomic DNA. PCR was carried out in 25 ml reactions in a Crocodile Appligene thermocycler utilizing about 20 ng genomic DNA as template, 25 pmol each primer, 160 mM each dNTP, and Q-Biogene TaqPolymerase and buffers. PCR conditions: 94  C for 3 min, followed by 30 cycles: 94  C for 30 s, 60  C for 60 s, 72  C for 60 s, final extension 72  C for 7 min.

2.3. Immunocytochemistry Immunocytochemistry was performed as described before (Sun et al., 2006). C57BL/6J wild-type or Kir6.2 KO mice were deeply anesthetized by halothane inhalation, and then perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at pH 7.4. The brains were quickly removed and placed in the same fixative at 4  C overnight. They were then sectioned into 30mm-thick slices, blocked in 3% normal goat serum/0.3% Triton X-100/0.1% BSA in PBS (room temperature for 1 h), and followed by incubation with rabbit anti-Kir6.2 antibody, 1:500 (Suzuki et al., 1997), at 4  C overnight. Subsequently, the sections were incubated with the affinity-purified second antibody, goat anti-rabbit Alexa 568 antibody (1:200; Invitrogen). Sections were then rinsed, dried, and coverslipped with Dako (Carpinteria, CA) fluorescence mounting medium. Images were viewed with a confocal laser scanning microscope (Zeiss 510 META) and analyzed with a three-dimensional (3D) constructor (Image J). In each preparation, the slice was initially scanned to determine a depth halfway between the slice surfaces and the midslice section was used for imaging. We produced 3D digital reconstructions from a series of confocal images taken at 0.5-mm intervals through the region of interest, and optical stacks of 5–10 images were produced for the figures. Region specific immunofluorescence intensity was compared using a Student’s t-test.

2.4. Surgical procedure, drugs and microinfusions Mice were anaesthetized with a mixture of ketamine hydrochloride (100 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.) dissolved in isotonic saline sterile solution. Sources of the anesthetics were: ketamine hydrochloride (Virbac, France), and xylazine (RompunÒ 2%, Bayer Pharma, France). The mice were implanted bilaterally with permanent 26-gauge steel guide cannulae above the CA3 region of the dorsal hippocampus. The coordinates were adapted from the Franklin and Paxinos atlas for C57BL/6J mice (Franklin and Paxinos, 1997): AP 1.65 mm refer to bregma, 2.5 mm laterally to the midsagittal suture line and 1.5 mm ventral to the surface of the skull. Obturators were inserted into guide cannulae to avoid any intrusion by dust. After surgery, mice were allowed at least 7 days of recovery before starting the behavioral experiments. Diazoxide and tolbutamide were obtained from Sigma, France. These drugs were freshly dissolved using 60% dimethyl sulfoxide (DMSO; Sigma, France) in isotonic saline solution. On injection days, obturators were removed and an infusion cannula extending 1.1 mm beyond the end of the guide cannulae was inserted for CA3 infusions. The infusion cannula was connected by a polypropylene tube to a 1 ml Hamilton microsyringe that delivered the solution at the rate of 0.11 ml/min using an automated pump. A volume of 0.25 ml of drug solution, 60% DMSO (DMSO) or NaCl 0.9% (NaCl) was infused once into each dorsal hippocampus. The first injection took 2 min 30 s and the cannula was left in place for an additional 60 s before injecting the second CA3 region.

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2.5. Histological examination of infusions and cannulae location

2.7. Allocentric spatial learning in the Morris water maze (MWM)

At the end of behavioral experiments, all mice were killed by cervical dislocation and their brains removed. Brains were stored 24 h in a solution of 2.5% glutaraldehyde–30% sucrose (1:1), then placed in a 30% sucrose solution and stored at 4  C. Cannulae placements were checked by examination of serial coronal sections (40 mM) stained with thionine and identified by non-informative numbers to be blind to drug condition. Histological analysis of the cannulae placement was performed on all the animals injected, and only animals with correct cannulae placement were used for data analysis.

The MWM is a test of hippocampus-dependant spatial learning and memory, where mice are trained to find a hidden platform. Animals first have to learn the presence of an opportunity to escape and subsequently how to get there efficiently using primarily distal visual cues. This requires both spatial and procedural memory (Lassalle et al., 2000; Morris, 1984).

2.6. Contextual fear conditioning (FC) In the FC paradigm, mice have to associate a conditioned stimulus (CS) such as the context or a tone, to an unconditional stimulus (US) such as an electric footshock. Thereafter, when exposed to the CS, mice will show a conditioned fear response, corresponding to a species-specific defence reaction detectable as total immobility or freezing. The hippocampus is thought to govern primarily the processing of the contextual representation that will be associated with the US, whereas the amygdala has been shown to be mandatory for associating both the context and the tone to the US (Phillips and LeDoux, 1992). 2.6.1. Apparatus Fear conditioning took place in a rectangular polyvinyl chloride box (length 35 cm, width 20 cm and height 25 cm) with three light-brown sides and a Plexiglas front wall through which experimental subjects were videotaped. The floor was made of a grid with stainless steel rods (diameter 4 mm) spaced 1 cm apart and connected to a generator (Campden Instruments, UK) delivering shocks of defined duration (2 s) and intensity (0.7 mA) through a shock-scrambler unit. Light-brown disposable tissue paper covered the floor below the grid. The loud speaker producing the tone (85 dB, 30 s) was fixed on the top of the conditioning chamber. The experimental device was lit by a 60 W white bulb. Two black and white patterns were placed on surrounding curtains that faced the conditioning chamber at a distance of 1 m. Experiments were recorded by a video camera placed in front of the conditioning chamber, and connected to a TV monitor and a video tape recorder in the adjacent room, where the experimenter and all the electronic system were settled. The conditioning chamber was cleaned with 70% aqueous ethanol before each animal’s training session. Contextual learning was checked in the same experimental conditions as training, whereas tone learning was assessed 2 h later in a modified context where external visual cues were removed (tone test). The new chamber was triangular, with white Plexiglas walls and floor. The apparatus was washed with 1% acetic acid (instead of alcohol) and lit by a 40 W white bulb. 2.6.2. Behavioral procedure The different drugs were injected before the conditioning session to investigate their effects on the acquisition and/or on the consolidation of contextual memory. The animals received one injection per CA3 and each injection was performed separately in order to avoid any overpressure of the hippocampus. Conditioning began 10 min after the first drug infusion. Conditioning consisted of a single training session with two trials. After a 120 s exploration period, a sound was emitted for 30 s and a foot-shock was superimposed to the tone during the last 2 s. Then, this sequence was repeated once again. Thirty seconds after the second (and last) foot-shock, mice were gently removed from the chamber and returned to their home cage. During conditioning, mice stayed in the conditioning chamber for a total of 330 s. Twenty-four hours after conditioning, mice were individually checked for freezing to the context in the conditioning chamber for 4 min. Two hours later, they were tested for freezing to the tone in the modified context: 2 min after their introduction in the apparatus, mice received a 2 min tone presentation while freezing was measured. Freezing was defined as the lack of movement besides respiration. In order to ensure that drugs do not act directly on mobility, locomotor activity of mice, defined as the number of crossings of a virtual line dividing the chamber in two parts, was measured during the first 2 min of conditioning. 2.6.3. Data analysis and statistics Freezing was scored every 5 s during training and test sessions. The data were converted to the percentage of samples scored as freezing and calculated for conditioning, contextual and tone tests. For each animal, baseline freezing data during the first 2 min of conditioning were averaged and subtracted from the averaged freezing across the contextual test to yield normalized response for the context. To satisfy the requirements for the use of the ANOVA, the mean percentage of freezing scores (P) was transformed in Q ¼ arcsin(OP/100). Statistical analysis was performed on the Q variable, using a one-way analysis of variance (ANOVA), or a repeated measures ANOVA design for related samples (SYSTAT 9 for Windows). Post hoc comparisons were conducted using Fisher’s least significant difference (LSD) test. Alpha levels were set at P < 0.05 for all tests. However to facilitate graphs reading and homogenize freezing representations between studies, mean freezing time for each group (SEM) is presented in the figures, instead of transformed Q variable.

2.7.1. Apparatus The water maze consisted of an ivory painted circular pool (110 cm diameter, 30 cm high) that was filled up to 15 cm from the base with water maintained at 23  1  C and made opaque by addition of non-toxic white opacifier. A circular goal platform painted white (9 cm of diameter) was positioned in the center of the target quadrant (i.e. North), 15 cm from the wall. A white curtain surrounded the swimming pool, delimiting the experimental environment. Several extra-maze visual cues, approximately 50–100 cm away from the pool, were attached to this curtain. Four start positions were located around the perimeter of the pool, dividing its surface into four equal quadrants: north (Target), south (Opposite), east (Adjacent 1, A1) and west (Adjacent 2, A2). The experimental room was illuminated with surrounding weak white lights, and the apparatus was surmounted by a video camera connected to a video recorder and a computerized tracking system (Ethovision1, Noldus). 2.7.2. Behavioral procedure We used a massed-procedure originally developed by Florian and Roullet (2004). Mice were placed in the experimental room under a red heating light for 10 min and were then individually submitted to a single familiarization session of three trials with the platform always located in the Target quadrant and protruding 0.5 cm over the surface of the water. The session started with the mouse standing on the platform for 60 s. At the beginning of each trial, the mice were released at one of the three possible starting points facing the wall, and allowed to swim freely until they reached the platform. Mice failing to find the platform within a fixed period of 60 s were gently guided by hand to the platform and a maximum escape latency of 60 s was recorded. After the animals had climbed onto the platform, they were allowed to stay on it for an additional 60 s, and subsequently replaced in the water from a different start position. The starting positions were determined in a pseudorandom order, such that each was used only once in a single session. The training phase started the next day. Training consisted of four consecutive sessions of three trials with an intersession delay of 15–20 min during which mice were returned to their home cage. The procedure was the same as for the familiarization, except that the platform was submerged 0.5 cm beneath the surface of the water. Mice were required to navigate to the invisible platform using the spatial cues available in the experimental environment. Twenty four hours after the last training session, the animals were submitted to a single-trial probe test to test their long term memory of spatial orientation. The platform was removed and mice, starting from the center of the pool and facing the Opposite wall (south), were allowed a 60 s search for the platform. The number of annulus crossings (number of times a mice crossed a small region around each of the four possible platform positions, diameter 9 cm) was obtained to characterize the search pattern used by the mice. 2.7.3. Statistics The results were expressed as mean (SEM) and analyzed using a one-way ANOVA, or repeated measures ANOVA when appropriate. Post hoc multiple comparisons were carried out when allowed, using Fisher’s LSD test.

3. Results 3.1. Kir6.2 protein expression in the hippocampus Using immunocytochemical staining, we studied Kir6.2 expression patterns in hippocampal sections from C57BL/6J wild-type mice (Fig. 1a). Numerous cell types were Kir6.2 positive, including pyramidal neurons in the CA1 and CA3 regions, granule cells in the dentate gyrus, and interneurons in the hilus. No fluorescence signal was observed in control slices treated only with secondary antibody (data not shown). In sharp contrast, the hippocampal region of Kir6.2 knock-out mice was devoid of any detectable labelling (Fig. 1b). Confocal imaging demonstrated that the fluorescencelabeled Kir6.2 protein was expressed throughout the hippocampal CA3 region of wild-type mice (Fig. 1c). In addition to the Kir6.2 positive pyramidal neurons, specific Kir6.2 expression was observed on the mossy fibers in the stratum lucidum. The estimation of immunofluorescence intensity in this region indicated that Kir6.2 expression on CA3 neurons was significantly higher (P < 0.001) and

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Fig. 1. Immunohistochemical imaging of Kir6.2 in the hippocampus of wild-type (WT) and knock-out (KO) mice. (a) Most of the cells from WT mice in CA1, CA3, dentate gyrus (DG), and hilus areas of hippocampus were stained, indicating expression of Kir6.2 protein. (b) The hippocampal region of Kir6.2 knock-out mice was devoid of any detectable labelling. (c) Representative confocal imaging in the CA3 region of hippocampus. Kir6.2 antibody staining was detected in CA3 neurons, and in the mossy fibers (MFs) region of C57BL/6J wildtype mice. (d) Comparison of estimated intensity of immunofluorescence (arbitrary units, AU) in stratum lucidum neurons (SLu) and the MFs region. The data (mean  s.e.m.) were obtained from 3 mice (n ¼ 3) and 10 neurons/regions were measured from each mouse. ***P < 0.001 (Student’s t-test).

approximately 2.5-fold that of the mossy fibers (Fig. 1d). In contrast to wild-type mice, the CA3 of Kir6.2 knock-out animals showed no detectable expression of the Kir6.2 protein. 3.2. Pharmacological activation of CA3 KATP channels impairs contextual fear memory KATP channel openers or blockers were microinjected in the CA3 regions via implanted cannulae before the beginning of the conditioning session. After histological examination of the brain slices, 27 mice were removed from the analysis because of misplacement of either the guide cannulae or the infusion tips. At the end of our study, group sizes ranged from 7 to 17 mice, all animals having cannulae tips located in the distal part of CA3 (Fig. 2). Six groups of mice were tested: NaCl (n ¼ 7), DMSO (¼8), diazoxide 1.5 (diaz 1.5; 1.5 nmol per CA3; n ¼ 9), diazoxide 2.5 (diaz 2.5; 2.5 nmol per CA3; n ¼ 17), tolbutamide 5 (tolb 5; 5 nmol per CA3; n ¼ 13) and diaz 2.5 þ tolb 5 (respectively 2.5 and 5 nmol per CA3; n ¼ 7). 3.3. Fear expression and locomotor activity We first checked for a possible effect of drugs (diazoxide and/or tolbutamide) on fear expression and learning performance levels by scanning freezing behavior during the conditioning session (Fig. 3a). A repeated ANOVA run on 30 s-blocks during the whole session revealed no significant difference between groups [F(5,55) ¼ 1.890; n.s., P > 0.111] and no significant time  drug interaction [F(50,550) ¼ 1.335; n.s., P > 0.068]. However there was a significant time effect [F(10,550) ¼ 88.490, P < 0.001], indicating

a normal increase in freezing after the electric shocks. Therefore, we compared the freezing levels group by group (i.e. NaCl vs. diaz 1.5, NaCl vs. diaz 2.5 and so on) during 30 s-blocks after the first shock occurrence. Two doses of the KATP channel opener diazoxide have been tested (1.5 and 2.5 nmol per CA3 region). Although freezing performances of the two diazoxide-injected groups appeared slightly decreased in comparison with their proper control (i.e. DMSO), no significant differences were detected. Therefore, diazoxide injections in the CA3 had no effects on fear expression. Locomotor activity was assessed by counting the number of chamber crossings during the first 2 min of conditioning; the ANOVA revealed no overall variation of crossings between groups [F(5,55) ¼ 0.689; n.s., P > 0.634; R2 ¼ 0.068], indicating a lack of effect of bilateral injections of DMSO, diaz 1.5, diaz 2.5 and/or tolb 5 on mouse locomotion (data not shown). 3.4. Acquisition and/or consolidation With regard to the contextual test, the ANOVA revealed a significant overall variation of freezing between the two control groups and the diazoxide groups [F(3,37) ¼ 11.258, P < 0.001, R2 ¼ 0.477]. As shown in Fig. 3b, post hoc analysis revealed no difference in freezing between NaCl and DMSO groups (n.s., P > 0.434). Furthermore, there was no significant differences between these two groups and sham (non-injected) animals (mean freezing  SEM: sham: 56.7%  6.1, n ¼ 10; NaCl: 50.3%  3.3, n ¼ 7; DMSO: 44.8%  3.8, n ¼ 8; data not shown). Thus, the intrahippocampal injection of a concentrated DMSO solution was without effects on memory, as previously reported in rats (Sharifzadeh et al., 2005). Two doses of the KATP channel opener

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Fig. 2. Schema of the individual tip of infusion cannulae position on dorsal hippocampus for all pre-conditioning injected mice according to the corresponding coronal sections from the mouse brain atlas (number on the left indicating the distance from bregma). Black crosses are for NaCl treated mice; black squares for DMSO; black triangles for diazoxide [1.5 nmol]; white triangles for diazoxide [2.5 nmol]; black circles for tolbutamide [5 nmol] and white circles for diazoxide [2.5 nmol] þ tolbutamide [5 nmol] treated mice.

diazoxide have been tested (1.5 and 2.5 nmol per CA3) and compared to DMSO-injected mice (Fig. 3b). During the contextual test, freezing was strongly impaired in mice injected with diazoxide as revealed by the post hoc analysis (ANOVA, post hoc analysis, P < 0.001, diaz 1.5 vs. DMSO; P < 0.01, diaz 2.5 vs. DMSO), and diaz 1.5 had a stronger impact (P < 0.05, diaz 1.5 vs. diaz 2.5). Thus,

diazoxide impaired the acquisition and/or the early consolidation of contextual memory. Finally, we injected the KATP channel blocker tolbutamide (5 nmol per CA3) and a mixture of diazoxide and tolbutamide (respectively, 2.5 nmol and 5 nmol per hippocampus). The ANOVA for the contextual test (Fig. 3c) showed an overall variation

Fig. 3. Effects of intra-hippocampal injections of the KATP channel opener diazoxide, the KATP channel blocker tolbutamide, or both, before the acquisition of contextual fear memory. (a) Fear expression of mice during conditioning. Fear expression has been assessed during contextual fear conditioning by scanning freezing behavior by 30 s-blocks. Black arrows on the X-axis indicate time of foot-shocks. (b) Effects of the opener diazoxide, (c) the blocker tolbutamide and a mixture of both, on contextual (b and c) and tone fear memory acquisition (d). Results are presented as percentage of time spent on freezing during the contextual test or the tone presentation (mean  SEM). Baseline freezing during pre-conditioning was averaged and subtracted from the mean freezing during the contextual test. Injection of diazoxide before conditioning impaired the contextual freezing at both doses tested (1.5 and 2.5 nmol per CA3, b), whereas freezing was not disturbed by tolb 5 injection (c). Treatment with a mixture of diazoxide 2.5 nmol and tolbutamide 5 nmol completely reversed the learning impairment caused by the opener alone (c). There was no difference in freezing to the tone between all groups tested (d). ***P < 0.001, diaz 1.5 or diaz 2.5 vs. NaCl, DMSO, tolb 5 or diaz 2.5 þ tolb 5. þP < 0.05, diaz 1.5 vs. diaz 2.5.

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between groups [F(3,41) ¼ 8.789, P < 0.001, R2 ¼ 0.391]. The specific KATP channel blocker was without effects (n.s., P > 0.536 vs. DMSO) but reversed the impairment in contextual freezing due to diaz 2.5 (P < 0.001, diaz 2.5 þ tolb 5 vs. diaz 2.5 alone). This indicates that the effects of the opener diazoxide were specific for KATP channels. 3.5. Tone test Freezing to the tone in the modified contextual chamber was similar in all tested groups, as indicated by the ANOVA [F(5,53) ¼ 0.946; n.s., P > 0.459; R2 ¼ 0.082]. This result presented in Fig. 3d confirms the specific effect of diazoxide on contextual memory, and the expected absence of effect in hippocampalinjected mice on the amygdala-dependant association between the tone and the electric shock. 3.6. Genetic approach showing that KATP channels are involved in contextual memory formation The Kir6.2/SUR1 KATP channels seem to be the dominant neuronal and hippocampal combination (Sun et al., 2006). Furthermore the Kir6.2/SUR1 KATP channels are highly sensitive to diazoxide and tolbutamide (Liss and Roeper, 2001), but these two molecules may bind other isoforms (Nagashima et al., 2004; Seino and Miki, 2003). Therefore, we used Kir6.2 knock-out mice to further investigate the specific involvement of Kir6.2-based channels in memory. We looked for a possible effect of the gene deletion in the fear conditioning and the Morris water maze tests. Wild-type (Kir6.2þ/þ), heterozygous (Kir6.2þ/) or homozygous (Kir6.2/) animals were tested in the fear conditioning paradigm (n ¼ 41: Kir6.2þ/þ, 10; Kir6.2þ/, 22 and Kir6.2/, 9) or in the Morris water maze (n ¼ 40: Kir6.2þ/þ, 10; Kir6.2þ/, 21 and Kir6.2/, 9).

3.7. Fear conditioning 3.7.1. Fear expression and locomotor activity First we checked for a possible effect of the Kir6.2 gene deletion on fear expression and learning performance by scanning freezing behavior during the conditioning session (Fig. 4a). A repeated ANOVA run on 30 s-blocks after the first shock occurrence revealed a time effect [F(6228) ¼ 23.829, P < 0.001] but neither genotype nor time  genotype interaction effect [respectively: F(2,38) ¼ 0.107; n.s., P > 0.899/F(12,228) ¼ 0.483; n.s., P > 0.924], suggesting the lack of genotype effect on fear expression. Locomotor activity was assessed by counting the number of chamber crossings during the first 2 min of conditioning; the ANOVA revealed no overall variation of crossings between groups [F(2,38) ¼ 8.916; n.s., P > 0.502; R2 ¼ 0.036], indicating a lack of genotype effect on mice locomotion (data not shown) although Kir6.2 KO mice have been originally described as hypoactive and impaired in motor coordination (Deacon et al., 2006). 3.7.2. Acquisition and/or consolidation The ANOVA revealed a significant variation of freezing between groups during the contextual test [F(2,37) ¼ 3.986, P < 0.05, R2 ¼ 0.177]. As presented in Fig. 4b, Kir6.2/ contextual memory was reduced when compared with Kir6.2þ/þ mice and significantly impaired in comparison with the Kir6.2þ/ group (respectively n.s.: P > 0.062, and P < 0.01). Although Kir6.2þ/þ animals presented a rather low memory of the context, they were not statistically different from the heterozygous group (n.s., P > 0.554). This result suggests a contextual memory deficit in the Kir6.2/ mice. 3.7.3. Tone test As shown in Fig. 4c, the Kir6.2/ mice also presented a small reduction in tone-shock memory association, as freezing to the

Fig. 4. Effect of the Kir6.2 deletion in backcrossed mice on fear expression during conditioning (a), contextual memory acquisition (b) and tone–shock association memory (c). (a) Fear expression of backcrossed Kir6.2þ/þ, Kir6.2þ/ and Kir6.2/ mice during conditioning. Fear expression has been assessed during contextual fear conditioning by scanning freezing behavior by 30 s-blocks after the first shock occurrence. Black arrows on the X-axis indicate time of foot-shocks. There was no difference in fear expression between all groups tested. (b) Kir6.2/ contextual memory was impaired in contrast with the other two groups. Results are presented as percentage of time spent on freezing during the contextual test (mean  SEM). **P < 0.01 Kir6.2/ vs. Kir6.2þ/. (c) Impairment of tone fear memory in Kir6.2/ animals. Results are presented as percentage of time spent on freezing during the tone presentation (mean  SEM). ***P < 0.001 for the Kir6.2/ mice vs. Kir6.2þ/ animals, þþP < 0.01, Kir6.2/ vs. Kir6.2þ/þ.

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tone in the modified context was significantly impaired in comparison with the other two groups (ANOVA [F(2,34) ¼ 8.008, P < 0.01, R2 ¼ 0.320]; P < 0.01 vs. Kir6.2þ/þ, P < 0.001 vs. Kir6.2þ/). This intriguing result suggests a putative involvement of KATP channels in the elementary association between the tone and the electric shock, but, regarding the high freezing response displayed by the KO animals, could as well be an artefact. These results strongly support a contextual memory impairment of the Kir6.2 knock-out mice. In order to clarify if this disability was specific of contextual memory in the FC or a more generalized deficit, we decided to test hippocampo-dependent spatial memory in a Morris water maze paradigm. 3.8. Morris water maze 3.8.1. Effect of the Kir6.2 genotype on training in the Morris water maze Fig. 5a illustrates the mean latencies before escape onto the hidden platform across the four training sessions. A repeated ANOVA revealed a significant group effect [F(2,37) ¼ 6.745, P < 0.01], a time effect [F(3111) ¼ 16.226, P < 0.001] but no

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time  group interaction [F(6,111) ¼ 0.976; n.s., P > 0.445]. This difference is explained by longer escape latencies in the Kir6.2/ group as indicated by a separate one-way ANOVA performed on each of the last three sessions (for example on session 4: [F(2,37) ¼ 5.098, P < 0.05, R2 ¼ 0.216]). As indicated by Fig. 5b, a repeated ANOVA performed on the distance moved during the 4 training sessions revealed that there is no significant difference between groups [F(2,37) ¼ 2.441, n.s., P > 0.101] but a significant time effect [F(3,111) ¼ 18.547, P < 0.001] and more importantly a time  group interaction [F(6,111) ¼ 2.671, P < 0.05]. This latter is explained by the longer distance travelled by the Kir6.2 Knock-out animals as indicated by a one-way ANOVA performed on the distance moved by the three groups on Session 2 only [F(2,37) ¼ 6.191, P < 0.01, R2 ¼ 0.251] and on Session 4 only [F(2,37) ¼ 4.020, P < 0.05, R2 ¼ 0.179]. Although the Kir6.2/ mice were slightly less swift and spent more time in the border of the pool than the other groups, the swimming speed between the three groups tested was not statistically different [F(2,37) ¼ 0.874; n.s., P > 0.426; R2 ¼ 0.045], neither was the time spent in a virtual ring 8 cm from the border of the pool which is an indicator of thigmotactism [F(2,37) ¼ 1.683; n.s., P > 0.200, R2 ¼ 0.083] (Table 1). 3.8.2. Effect of the Kir6.2 genotype on spatial memory in the Morris water maze Spatial memory was evaluated by a probe test conducted 24 h after the last training session. A repeated ANOVA performed on the time spent in the different quadrants revealed no group effect [F(2,35) ¼ 0.000; n.s., P > 1.000] but a quadrant effect [F(3,105) ¼ 20.273, P < 0.001] and a group  quadrant interaction [F(6,105) ¼ 2.684, P < 0.05]. A one-way ANOVA performed on the target quadrant unveiled a difference between the three groups [F(2,35) ¼ 3.938, P < 0.05, R2 ¼ 0.184]. As presented in Fig. 6a, homozygous animals spent significantly less time in the target quadrant than the other groups (P < 0.01 vs. Kir6.2þ/; n.s., P > 0.066 vs. Kir6.2þ/þ). An intra-group analysis revealed that Kir6.2/ mice spent as much time searching in the adjacent quadrants (east and west) as in the target location (ANOVA, F(2,16) ¼ 0.589; n.s., P > 0.566). In terms of annuli crossings (Fig. 6b), there was only a small group effect [F(2,37) ¼ 3.287, P < 0.05, actual P-value was 0.049], a quadrant effect [F(3,111) ¼ 34.094, P < 0.001] but no group  quadrant interaction [F(6,111) ¼ 1.567; n.s., P > 0.163]. Moreover, there was no significant difference in the number of crossings of the target annulus [ANOVA F(2,37) ¼ 2.149; n.s., P > 0.131; R2 ¼ 0.104]. During the first 30 s of the probe test trial (Fig. 6c), the ANOVA showed an overall variation between groups [F(2,36) ¼ 3.737, P < 0.05], quadrants [F(3,108) ¼ 34.250, P < 0.001] and a group  quadrant interaction [F(6,108) ¼ 2.768, P < 0.05]. This difference is explained by a reduced number of target annuli crossings in the Kir6.2/ group as indicated by a one-way ANOVA [F(2,36) ¼ 3.764, P < 0.05, R2 ¼ 0.173]. Altogether, these results support a slight spatial memory impairment for the Kir6.2/ animals. These mice searched the

Fig. 5. Effect of the Kir6.2 deletion in backcrossed mice on acquisition of spatial memory in the Morris water maze. (a) Mean latencies to find the hidden platform across the four training sessions (three trials per session, mean  SEM). Training was impaired in the Kir6.2/ group as indicated by a separate one-way ANOVA performed on the escape latency in each of the last three sessions, *P < 0.05, **P < 0.01. (b) Mean distance travelled to find the hidden platform (mean  SEM). The Kir6.2 knock-out animals swam a longer distance as indicated by a one-way ANOVA performed on the distance moved by the three groups on Session 2 only [F(2,37) ¼ 6.191, P < 0.01, R2 ¼ 0.251] and on Session 4 only [F(2,37) ¼ 4.020, P < 0.05, R2 ¼ 0.179]. *P < 0.05, **P < 0.01.

Table 1 Results of analysis of two parameters tested in Morris water maze during training: swimming speed and thigmotactism. Although the Kir6.2/ mice were slightly less active and spent more time in the border of the pool than the other groups, the swimming speed between the three groups tested was not statistically different [F(2,37) ¼ 0.874, P ¼ 0.426, R2 ¼ 0.045], neither was the time spent in a virtual ring 8 cm from the border of the pool [F(2,37) ¼ 1.683, P ¼ 0.200, R2 ¼ 0.083], an indicator of thigmotactism. Data in the table represent the mean values  SEM.

Swimming speed (cm/s) Time in border zone (s)

Kir6.2þ/þ

Kir6.2þ/

Kir6.2/

17.8  0.7 16.2  2.6

17.0  0.54 15.9  1.6

16.4  0.7 21.3  2.4

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Fig. 6. Effect of the Kir6.2 deletion in backcrossed mice on long term retention of spatial memory in the Morris water maze during a 24 h post-training probe test. (a) Histograms represent the time (seconds) spent in the four quadrants of the pool. Kir6.2/ mice spent less time in the North (where the platform was located during acquisition), *P < 0.05 Kir6.2/ vs. Kir6.2þ/. (b) This figure represents the number of annulus crossings during the entire 60-s of the probe test. There was no difference between the three genotypes. (c) Number of annulus crossings during the first 30 s of the probe test. Kir6.2/ mice performances were decreased in comparison with the other groups. *P < 0.05, Kir6.2/ vs. Kir6.2þ/. (d) Representative paths taken by the three groups of mice during the probe test (left to right, Kir6.2þ/þ, Kir6.2þ/ and Kir6.2/).

platform in the correct location of the pool, but were less accurate in their search than the other groups. 4. Discussion We present here the first pharmacological study arguing for a specific implication of hippocampal CA3 KATP channels in memory processes in the fear conditioning test. When injected before conditioning, the KATP opener diazoxide strongly impaired mice contextual memory, an effect reversed by tolbutamide. The mnemonic consequences of central KATP channels modulation under physiological conditions have been poorly studied. Mainly, a previous study by Ghelardini et al. (1998) found a memory impairment in mice tested in the passive avoidance test after intracerebroventricular injections of several KATP channel openers. Importantly, the molecules used (minoxidil, pinacidil, and cromakalim) were far more selective for SUR2-based channels, having little or no effect on SUR1-based channels. In contrast, we studied the effects of diazoxide, a molecule preferentially active on potassium channels incorporating the SUR1 subunit, but with a relative affinity only slightly above other commonly used openers (Ashcroft and Gribble, 2000; Schwanstecher et al., 1998). Indeed, studies performed on reconstituted KATP channels showed clearly that diazoxide is a non-selective KATP opener (Schwanstecher et al., 1998). Diazoxide was shown to activate both SUR1- and SUR2Bbased channels (Inagaki et al., 1995a; Isomoto et al., 1996). However, while SUR2A incorporating channels may also be sensitive to diazoxide, in mice, the forebrain is apparently devoid of SUR2A subunits (Ashcroft and Gribble, 2000; Chutkow et al., 1996). On the opposite, tolbutamide is clearly specific of SUR1 subunits with a binding affinity 10-fold higher than for SUR2A or SUR2B subunits (Davis-Taber et al., 2003; Dickinson et al., 1997; Dorschner et al., 1999). Thus, the impairment observed in our pharmacological

experiments is more likely attributable to the opening of SUR1based channels. As Kir6.1/SUR1 and Kir6.2/SUR1 channels are both sensitive to diazoxide (Ammala et al., 1996), the KATP isoform naturally occurring in the hippocampus needs further discussion. Kir6.1 and Kir6.2 mRNAs are both expressed in the brain (Dunn-Meynell et al., 1998; Inagaki et al., 1995b; Zawar et al., 1999). In rats, the Kir6.1 protein is expressed throughout the hippocampus with a restricted mitochondrial localisation (Zhou et al., 1999). Furthermore, functional Kir6.1/SUR1 channels have been described in striatal cholinergic interneurons and glial cells, and recently at Schaffer collateral-CA1 synapses (Soundarapandian et al., 2007). Kir6.2 mRNA is widely distributed in the brain and a striking overlap with the SUR1 mRNA puts forward the Kir6.2/SUR1 complex as the best candidate for the brain functional KATP channel (Karschin et al., 1997; Zawar et al., 1999). Indeed, this combination has been readily observed in the SNr (Liss et al., 2005) and the ventromedial hypothalamus (Miki et al., 2001), but only suggested on the mossy fibers (Bancila et al., 2005). The Kir6.2 protein had already been evidenced in neurons throughout the hippocampus of both rats and mice (Sun et al., 2006; Zhou et al., 2002). Yet, in rats, the mossy fibers were found devoid of any Kir6.2 immunohistological staining (Thomzig et al., 2005) and a puzzling study in mice by Soundarapandian et al. (2007) presented the Kir6.1/SUR1 channel as the dominant plasmalemmal combination at presynaptic terminals in the hippocampus. In contrast, our work confirms the presence of the Kir6.2 subunit along the mossy fibers and gives the first quantification of the protein expression in the stratum lucidum of the CA3 region (Sun et al., 2006). Among other possible alternatives, the discrepancies with our studies might be best explained by the presence of both Kir6.1 and Kir6.2 proteins on mossy fibers terminals. In addition, whilst SUR1 is thought to be the predominant sulphonylurea receptor isoform expressed in the CNS (Karschin et al., 1997), SUR2B might as

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well be expressed in the rodent CA3 (Chutkow et al., 1996; Pelletier et al., 2000; Wang et al., 2004; Zawar et al., 1999). Hence, even if our results point toward a specific involvement of CA3 Kir6.2/SUR1 KATP channels, we cannot exclude that diazoxide opened Kir6.1/SUR1, Kir6.2/SUR2B or Kir6.1/SUR2B channels. Importantly, one can question the specific site of action of the molecules injected in the CA3 region. Our immunohistochemical labelling of the hippocampal region indicates that diazoxide most probably impaired memory by opening Kir6.2-based channels at the postsynaptic level thereby hyperpolarizing CA3 pyramidal cells. Also, as the Kir6.2 protein was also detected on mossy fiber terminals, diazoxide could have reduced glutamate release at this level. This would be in agreement with a putative retro-inhibition exerted by zinc on glutamate release through the activation of presynaptic Kir6.2/SUR1 KATP channels, a mechanism plausibly involved in memory processing by reducing mossy fibers presynaptic LTP (Bancila et al., 2004, 2005; Minami et al., 2006; Quinta-Ferreira and Matias, 2005). Obviously, diazoxide may have modulated the release of other neurotransmitters such as GABA (Ohno-Shosaku et al., 1993) or ACh (Stefani and Gold, 2001). Such a modulation may have participated in the mnemonic deficit observed in our study and will deserve further examination. The use of Kir6.2/ mice provided a specific and discriminating strategy to explore the mnemonic role of Kir6.2-based channels. Deacon et al. (2006) first described the behavioral phenotype of Kir6.2 KO mice. These animals are nearly identical to wild-type animals but have very slight motor coordination impairments and a complex emotional reactivity. Their working memory is also slightly impaired in the spontaneous alternation test (Choeiri et al., 2006; Deacon et al., 2006). We further investigated the cognitive impact of the Kir6.2 deletion in these animals. In the fear conditioning paradigm, as immediate post-shock freezing was not impaired, our result suggests a conspicuous impact on the early steps of memory acquisition and/or on memory consolidation. Contextual memory processing requires the functional integrity of the hippocampus, and particularly of the pivotal auto-associative CA3 region (Daumas et al., 2004, 2007). Therefore, it is tempting to speculate that the loss of CA3 Kir6.2 subunits is responsible for the contextual memory impairment in transgenic animals. Both the pore forming and sulfonylurea receptor subunits must be expressed and co-assemble in order to form a functional Kir6.2-based KATP channel (Clement et al., 1997; Seino et al., 2000). Moreover, previous results with Kir6.1 null mice or kir6.2 overexpressing mice suggests that there is generally no compensation at the transcriptional level of the remaining subunits in KATP knockout models (Heron-Milhavet et al., 2004; Miki et al., 2002). Thus, as expected, Kir6.2 knock-out mice express various cell phenotypes relevant of a loss of KATP channels. Specifically, neurons from Kir6.2 KO animals are highly sensitive to brief episodes of metabolic stress in all hippocampal subregions, CA3 included. In the CA1, this phenotype was ascribed to a loss of protective neuronal hyperpolarization normally induced by the opening of Kir6.2/SUR1 channels (Sun et al., 2006). Of course, since we analyzed a general Kir6.2 KO mouse with Kir6.2-containing KATP channels absent throughout all brain (and specifically in the CA1, DG and hilus) and peripheral tissues, it is not possible to identify the cellular substrates of the changed behavioral phenotype. Moreover, we cannot exclude a role for remaining (or upregulated) Kir6.1-based channels. Further experiments such as restricted viral delivery of specific siRNAs targeting both Kir6.x and SURx subunits in wild-type mice will be useful to clarify the hypothesis of a specific implication of CA3 KATP Kir6.2/SUR1 channels. Audition of Kir6.2/ animals has never been described but appeared normal as indicated by a high level of freezing in response to the tone. Yet, the amygdala-dependant tone–shock association was significantly impaired in comparison with the other groups.

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While this result might be unrelated, Kir6.2/SUR1 mRNAs are weakly expressed in mice and rats amygdala (Karschin et al., 1997), and at least in rats, in the amygdalo-hippocampal area (Chen et al., 2001). Therefore, a cognitive role for KATP channels in this region cannot be excluded. During training in the Morris water maze test, the Kir6.2/ group escape latencies’ performances were altered in comparison with the other two groups. This difference is consistent with the working memory impairment previously described (Choeiri et al., 2006), but may also indicate a higher emotional reaction to the stressful pool environment as the animals progressively learned the task. Conversely, the distance swam by the Kir6.2/ animals was above the other two groups but their velocity was nearly identical, indicating that the previously reported motor impairments were merely without effects in this task (Deacon et al., 2006). As indicated by a probe test conducted 24 h after training, long term spatial memory was also slightly impaired in this group. However, the Kir6.2 deleted animals correctly explored the previous platform location despite a less intense and accurate search. Thence, whether this small spatial memory impairment is a result of the impaired training or a true deficit will need further confirmation. Why does Kir6.2 deletion would impair contextual memory? As pharmacological activation of the KATP channels impairs contextual memory, one would not expect a cognitive impairment in Kir6.2 KO animals. Importantly, KATP channels may couple neurosecretion to neuronal glucose metabolism (Amoroso et al., 1990). As originally postulated by Bancila et al. (2004), during learning, as the energy demand increases, mossy fibers KATP channels may sense a rise in intracellular ATP, thereby closing and enhancing glutamate release. Conversely, KATP channels opening immediately after intense electrical activity may also protect the CA3 cells from an excessive glutamate shower. In both hypotheses, loss of functional plasmalemmal KATP Kir6.2-based channels may have a deleterious effect on memory, either by decreased or excessive glutamate release. Furthermore, as a metabolic sensor the KATP channel modulates neuronal excitability and ventromedial hypothalamus neurons of Kir6.2/ mice exhibit an increase in spontaneous activity frequency (Miki et al., 2001). In the same view, increased excitability of mossy fibers or CA3 pyramidal cells may induce learning impairments and potentially alter memory processes such as LTP. Hence, the consequences of Kir6.2 deletion emphasize the importance of the KATP Kir6.2-based channels in contextual memory, in agreement with our pharmacological data. This study confirms the importance of hippocampal KATP channels in learning and long-term memory processes. So far, conflicting results have been found regarding a beneficial effect of improved glycemic control on cognitive function in patients suffering from type 2 diabetes (Meneilly et al., 1993; Mussell et al., 2004; Ryan et al., 2006). In addition to improved glycemic control through peripheral KATP modulation, sulfonylurea may cross the blood brain barrier and exert a specific action on cognition (Slingerland et al., 2008). Therefore, a better understanding of the regulation of brain glucose consumption in function of the learning pressure on the hippocampus will surely improve the treatment of disease with cognitive repercussions. Acknowledgements This work was supported by grant #03340 from ACI ‘‘Neurosciences inte´gratives et computationnelles’’, the Paul Sabatier University and the Centre National de la Recherche Scientifique. The work of Drs. Sun, Feng and French was supported by the Canadian Institutes of Health Research. We would like to thank Dr. Laure Verret for helpful comments on the manuscript. We are grateful to Dr. S. Seino for providing Kir6.2 null mice.

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