Procognitive impact of ciproxifan (a histaminergic H3 receptor antagonist) on contextual memory retrieval after acute stress

Summary Aim Although cognitive deficits commonly co‐occur with stress‐related emotional disorders, effect of procognitive drugs such as histaminergic H3 receptor antagonists are scarcely studied on memory retrieval in stress condition. Methods Experiment 1. Memory of two successive spatial discriminations (D1 then D2) 24 hours after learning was studied in a four‐hole board in mice. H3 receptor antagonist ciproxifan (ip 3 mg/kg) and acute stress (three electric footshocks; 0.9 mA; 15 ms) were administered 30 and 15 minutes respectively before memory retrieval test. Fos immunostaining was performed to evaluate the neural activity of several brain areas. Experiment 2. Effects of ciproxifan and acute stress were evaluated on anxiety‐like behavior in the elevated plus maze and glucocorticoid activity using plasma corticosterone assay. Results Experiment 1. Ciproxifan increased memory retrieval of D2 in nonstress condition and of D1 in stress one. Ciproxifan mitigated the stress‐induced increase of Fos expression in the prelimbic and infralimbic cortex, the central and basolateral amygdala and the CA1 of dorsal hippocampus. Experiment 2. Ciproxifan dampened the stress‐induced anxiety‐like behavior and plasma corticosterone increase. Conclusion Ciproxifan improved contextual memory retrieval both in stress and nonstress conditions without exacerbating behavioral and endocrine responses to stress. Overall, these data suggest potential usefulness of H3 receptor antagonists as cognitive enhancer both in nonstress and stress conditions.


| INTRODUC TI ON
The histamine H 3 receptor is a central inhibitory autoreceptor located on histaminergic nerve terminals that are found mainly on cholinergic and dopaminergic neurons. H3 receptor activation resulted in a reduction of the release of histamine in the brain, whereas its inhibition (by inverse agonist or antagonist) increased the release of histamine [1][2][3] . Ciproxifan (cyclopropyl 4-(3-(1H-imidazol- 4-yl) propyloxy) phenyl ketone) is an extremely potent histamine H 3 receptor (H 3 R) inverse agonist/antagonist which enhanced the release of histamine and increases sustained attention and alertness states. [4][5][6] Both thioperamide (a potent HRH3 antagonist) and ciproxifan enhance working memory 4,7-10 and long-term memory [11][12][13][14][15][16][17] and counteract scopolamine-induced amnesia. 11,12,[18][19][20][21] The therapeutic use of procognitive compounds might be integrated into a stressful context. Indeed, patients suffering cognitive deficits often show anxiety disorders 22 and stress impairs hippocampus-dependent memory retrieval. 23 The deleterious effect of stress on cognitive functions is observed in stress-related disorders such as anxiety and depression. [24][25][26] In such context, histaminergic system is a relevant target because histamine is an indicator of stress response [27][28][29][30][31][32] since stress is a potent activator of histamine neurons in the tuberomammillary nucleus of the hypothalamus. 33 A consensus view is that histamine antagonists have more impact in tasks having an anxiety component. 34 For example, it has been found that ciproxifan prevented the deleterious effects of chronic stress exposure in spatial memory. 35 In contrast, central administration of histamine can also increase plasma corticosterone via its action on hypothalamic neuropeptides 36 and promotes anxiety-like behavior. [37][38][39][40] Research efforts are now needed to determine emotional impact of H 3 R antagonists at procognitive dose.
Although numerous studies concerned procognitive action of 3 mg/kg ciproxifan in control (nonstressed) conditions, 4,[8][9][10][11] its cognitive action in hippocampus-dependent memory in stress condition has not been yet studied and more particularly at the retrieval phase. 41 To that aim, we investigated the effects of an acute stress (three electric footshocks 0.9 mA) on memory in a contextual serial spatial discrimination task (CSD) where stress is not directly associated with the memory task. In the CSD task, mice learned two successive discriminations and are tested 24 hours later for memory of the first or second discrimination using distinct internal context in mice. 42 We previously showed that memory of the first discrimination involved the dorsal hippocampus in nonstress condition, whereas memory of the second discrimination involved the prefrontal cortex and the amygdala in stress condition. 43,44 Using this behavioral model, we investigated in a first experiment the effects of pre-test injection of 3 mg/kg ciproxifan on memory of D1 and D2 in nonstress or stress conditions. Fos immunohistochemistry has been found to be a powerful tool for identifying the modifications of neural activity in brain areas particularly after stress. For example, the number of immunostained cells was increased in the amygdala of animals submitted to stressful situations. 45,46 Therefore, in a first experiment, the impact of ciproxifan on Fos expression was performed in brain areas involved in the CSD behavioral task such as the prefrontal cortex (PrL for prelimbic and Il for infralimbic cortex), the dorsal hippocampus (CA1, CA3 for Cornu Ammonis areas 1 and 3 and DG for dentate gyrus) and lateral (LA), basolateral (BLA) and central (CeA) nucleus of amygdala. A second experiment was designed to study the effect of ciproxifan 3 mg/kg on emotional reactivity in the elevated plus maze (EPM) behavior and plasma corticosterone levels. 47 This second experiment was designed to determine if the 3 mg/kg procognitive dose of ciproxifan could be dissociated from its emotional impact.

| Animals
144 subjects were 3-month-old male mice of the C57Bl/6J inbred strain obtained from Charles River and assigned to Experiment 1 (eight groups of 13; n = 104) and Experiment 2 (four groups; n = 40).
All mice were maintained in a ventilated colony room at 22 ± 1°C, under a 12:12 light-dark cycle (lights on at 7:00 am). They were pro- F I G U R E 1 Contextual Serial spatial Discriminations (CSD) protocol. Mice were ip injected (vehicle solution for all groups) 30 min before acquisition phase then were exposed to the first discrimination D1 (only one hole on 4 is randomly baited by ten 20-mg saccharose pellets) on a specific floor (white and smooth; random use for each mouse). Note that each treatment is done in a specific room. In the second discrimination (D2), the baited hole is in the opposite corner than discrimination 1 and the color and texture of the floor is changed (black and rough). On the following day, mice were randomly treated (ip injection and acute stress, 30 and 15 min before behavioral test respectively). During the retrieval test phase, no hole is baited and mice were randomly exposed to either D1 or D2 floor (independent groups) after treatment.

| Experiment 1: effects of ciproxifan on memory and neural activities
Contextual serial discrimination (CSD) task After 3 days of food restriction to maintain 90% of initial body weight, mice learn two spatial discriminations (D1 then D2) in a four-holeboard apparatus (45 × 45 × 30 cm; Room A; Figure 1). On the floor, four holes opening on a food cup (three diameter × 2.5 cm in depth) were located 6 cm away from the sidewalls. During the acquisition session, the two serial discriminations differed by the color (black versus white) and texture (rough versus smooth) of the floor. For D1, ten 20-mg saccharose pellets (BIOSERV, France) were available only in one randomly chosen hole. For D2, ten pellets were located in the opposite symmetrical hole. The environmental spatial cues (outside the board) were made of colored and striped paper sheets positioned at 1.00 m above the four-hole board. These allocentric cues remained at the same place for both D1 and D2 discriminations and also for the memory retrieval test. Thus, both discriminations D1 and D2 differed only by way of the internal (floor) contextual cues.
24 hours after acquisition, memory retrieval was tested either on D1 or on D2 using the specific floor of each acquisition. Mice were allowed to freely explore the apparatus and performance was assessed by measuring the number of head-dips in each hole during 6 minutes without any pellets in the apparatus. All mice tested in the retrieval phase were included in statistics. The following parameters were calculated: (a) % of correct responses (number of head-dips into the hole previously baited on the same floor-context/total number of headdips × 100), (b) % of spatial responses: interfering responses (number of head-dips into the hole previously baited on the other floor-context/total number of head-dips × 100) + correct responses. Spatial responses refer specifically to head-dips into the two previously baited holes, regardless of the floor used at the acquisition phase. Thus, within the framework of our analysis, spatial responses depended exclusively on knowledge of the external allocentric cues which remained stable over the learning of D1 and D2 and during the test phase. Conversely, "correct" responses emerges as an index of contextual memory which can be considered as reflecting a unique event-related memory. 48

Drug administration
All animals were injected intraperitoneally with vehicle 30 minutes before the acquisition phase (0.9% saline solution; 0.1 ml/10 g body weight; Figure 1). During the test session, the animals randomly

Stress administration
Fifteen minutes before behavioral test (CSD and EPM tests), mice were randomly chosen and placed in the stress delivery cage for 1 minute. Three consecutive inescapable electric footshocks (0.9 mA; 15 ms) were delivered every 20 seconds.

| Experiment 2: effects of ciproxifan and stress on behavioral and endocrinal reactivity
General protocol Drug injections (vehicle or ciproxifan 3 mg/kg) and stress (three footshocks 0.9 mA) were performed respectively 30 minutes and 15 minutes before EPM. Mice were randomly assigned to four experimental groups (NS Veh; NS Cipro3, Str Veh; Str Cipro3).

Elevated plus maze
The EPM consisted of two open-arms (30 cm long, 7 cm wide) and

Plasma corticosterone
At the end of the EPM task, animal were decapitated, trunk blood was immediately centrifuged at 4°C and plasma was stored at −20°C until corticosterone assay. Corticosterone concentrations were quantified using a commercially Enzyme Immunoassay kit (DetectX, Arbor Assays).
The limit of detection of this assay was 1.7 µg/dL that is 4-fold lower than the minimal value (8.05 µg/dL) obtained in the present study.

| Statistical analysis
The normality of the distribution was evaluated using Shapiro-Wilk test.
Data are displayed with bar graph representing mean ± SEM (standard error mean). One or two-way factorial analysis of variance (ANOVA) of drug and stress factors were performed for behavioral analyses (experiment 1 and 2) and plasma corticosterone assay followed by post hoc comparisons (Scheffe multiple comparison test; two-sided method adjustment). Insofar that stress increase Fos immunoreactivity, 50 the interaction between drug and stress factors was not performed. Therefore, drug effect on Fos staining in nonstressed animals was analyzed independently of stressed animals by the mean of unpaired t test. The significance level was set at P < 0.05, nonsignificant results are reported as NS. Statistical analysis was performed using Statistica ® 7.0. software.

| Contextual serial discrimination (CSD)
Acquisition. The number of animals per group is mentioned in Table 1.
No significant between-groups difference was observed both for the total number and the percentage of head-dips in the baited hole, both for discrimination 1 and Discrimination 2) (P > 0.10 in all analyses).

Correct responses in stressed animals
Ciproxifan effect In stress condition, ANOVA also revealed a significant interaction between drug and discrimination (F(2, 46) = 5.56; P < 0.05): ciproxifan increased the % of correct responses at D1 (3.6% ± 3.7% for ciproxifan group versus 25.3% ± 3.3% TA B L E 1 Behavioral performances during acquisition phase in CSD task
Performance of spatial responses were all above chance level (50%;

| Fos expression in hippocampus, prefrontal cortex and amygdala
In all groups, CSD behavioral test increased significantly Fos immunostaining as compared to naive (nonbehaving) mice (P < 0.0001 in all comparisons and for all brain areas).

| Elevated plus maze
Distance travelled in open-arms ( Figure 4A)

Stress effect
Stress did not significantly alter plasma corticosterone levels in cip-

| D ISCUSS I ON
Main results are as follows. In experiment 1, ciproxifan (3 mg/kg) enhanced contextual memory retrieval both in nonstress and stress con-

| Stress, ciproxifan, and memory retrieval
It has been reported that compounds activating histamine receptors H1 and/or H2 or increasing histamine release (via H3 histamine receptor blockade) substantially improve memory processes. 4,[7][8][9][11][12][13][14][15][16] In our study, ciproxifan did not modify spatial memory performance that were already high (around 70%) in control animals, suggesting likely a ceiling effect. In contrast, a procognitive impact of ciproxifan is observed on contextual memory (see also 15,21 ). The sparing of spatial but not contextual memory retrieval after stress in the CSD task agrees with studies showing that flexible forms of memory are more vulnerable to the deleterious effects of stress as compared to stable ones. 51 Spatial memory in CSD procedure evaluates a reference memory component. In contrast, "correct" responses depended on the retrieval of a unique internal context (floor) which is associated to a specific spatial location; thus, contextual memory could be an index of the flexible memory processes involved in declarative-like memory in animals. 48 A specific contribution of our study is to show that this improvement is observed on the retrieval phase of memory processes, and both in nonstress and stress conditions. Indeed, it has been reported that histamine may act differently on memory consolidation according to the emotional component of the task. 52 To explain this discrepancy, it could be hypothesized that ciproxifan (injected 35 minutes before blood sampling) could activate hippocampus and/or prefrontal cortex neurons 9 that exert a negative feedback on the HPA axis activity, leading to dampened corticosterone levels. 56 In the present experimental paradigm, ciproxifan did not increase corticosterone levels also in nonstressed animals probably via a masking effect because the behavioral task itself induces corticosterone release. 44 It is known that the release of histamine induced anxiety-like reactivity through the activation of H1 receptors. 39

| CON CLUS ION
Ciproxifan 3 mg/kg enhanced contextual memory retrieval, both in stress and nonstress conditions and dampened emotional reactivity and glucocorticoid responses to an acute stress. Overall, this study emphasized the usefulness of H3 receptor antagonist to enhance cognitive functions both in stress and nonstress conditions.

ACK N OWLED G M ENTS
We thank Mathieu Coutan from IRBA for expert technical assistance. This work has benefited from the facilities and expertize of the imagery platform from IRBA. We also like to thank Dr. Frances Ash for language proofreading (contact: ashberac@free.fr).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.