Differential effects of diazepam and MPEP on habituation and neuro

Phamedica.orgEffects of diazepam


7/17/2014
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Effects of diazepam
Differential effects of diazepam and MPEP on habituation and neuro

These results suggest that the strain differences observed in (non)adaptive anxiety behaviour are at least in part mediated by differences in gamma-aminobutyric acid- A and mGluR5 mediated transmission.

The electronic version of this article is the complete one and can be found online at: /content/8/1/30.

Amber R Salomons 1 2 *, Nathaly Espitia Pinzon 1, Hetty Boleij 1 2, Susanne Kirchhoff 1 2, Saskia S Arndt 1 2, Rebecca E Nordquist 2 3, Lothar Lindemann 4, Georg Jaeschke 4, Will Spooren 4 and Frauke Ohl 1 2.

This file can be viewed with: Adobe Acrobat Reader Table 1. Overview of behavioural parameters measured in the open field and the object recognition test Blood samples Basal blood samples ( basal) were collected 4 days before the start of the OF using tail vein incision for corticosterone (CORT) determination. Thirty minutes after behavioural testing a second blood sample was taken ( non-basal ). For blood sampling, mice were transported individually in their home cage to an adjacent laboratory (in order not to disturb circadian rhythm of the mice, the hallway and rooms were under red light conditions). By using tail vein incision a small blood sample was collected (±50 μl) and stored in prechilled Microvette tubes (CB300, Sarstedt, Numbrecht, Germany) containing lithium heparin. Blood samples were centrifuged (10 min at 12000 rpm, 4 °C) and stored at −20 °C until measurement. CORT levels were measured by radioimmunoassay (RIA) according to the protocol of the supplier with an ImmuChem Double Antibody Corticosterone kit for rats and mice (MPI Biochemicals, Amsterdam, The Netherlands).

All statistical analyses were carried out according to Field using the software program SPSS for Windows (version 16.0.1; SPSS Inc., IL, USA). Two-sided, exact i.e. for the non-parametric tests probabilities were estimated throughout. Continuous numerical data (CORT, c-Fos, latency and relative duration of behavioural parameters) were summarized as means with standard error of the mean (SEM), whereas discrete data on the ordinal scale (total number of behavioural parameters) were represented as medians with the interquartile range (IQR). The Kolmogorov-Smirnov one-sample test was used to check Gaussanity of the continuous numerical data. Several parameters that were not normally distributed were transformed to a Gaussian distribution by using a mathematical function or by rank transformation. Discrete numerical data (total numbers of the behavioural parameters of the open field) were fist rank-transformed. Behavioural numerical data from the open field and CORT values were tested for significant differences by multivariate repeated measures ANOVA. Tests of significance were derived using the Wilk’s lambda criterion (for the open field, time interval was taken as within-subject factor and strain and dose as between-subject factors). For CORT values the basal/non-basal CORT was taken as within-subject factor and strain and dose as between-subject factors. For latency and defecation data of the OF, ORT data and c-Fos results, a two-way ANOVA was used with strain and treatment (or dose for the open field) as main between-subject factors. If ANOVA detected significant effects, group means were further compared. Between-subject post hoc comparisons were done with either unpaired Student’s t tests for normally distributed data, or for non-normally distributed data and for discrete data, the same comparisons were performed using a Wilcoxon-Mann–Whitney test. For the ORT this was done to investigate whether the discrimination index differed significantly from zero i.e. no discrimination between novel and familiar object. To take the greater probability of a Type I error due to multiple hypotheses into account, we calculated for each behavioural category separate so-called Dunn-Šidák corrections: ANOVA’s: α = 1 – 1/q; q = number of parameters per behavioural category; post hoc Student’s t tests, the Wilcoxon-Mann–Whitney tests, and Wilcoxon matched-pairs signed ranks tests: α = 1 – 1/q; q = number of parameters per behavioural dimension multiplied by the number of times a group is used for a meaningful comparison. The corrected thresholds used for the ANOVA’s and post hoc comparisons can be found in the 1 : Table A1 (open field) and Additional file 1 : Table A2 (object recognition test).

However, it remains unclear whether the profound lack of habituation in 129P3/J mice is primarily based on anxiety-related characteristics. For example, avoidance behaviour can be confounded by other motivational systems, such as exploration or cognitive processes. We hypothesise that if the habituation profile in 129P3 mice was primarily based on anxiety-related characteristics, anxiolytic treatment should improve the habituation capacity in these mice.

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Diazepam (BUFA, The Netherlands) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP, Hoffmann-La Roche, Basel, Switzerland) were prepared in 0.1 % Tween 80 and saline in a volume of 10 ml/kg and injected i.p. in the experimental room 30 minutes prior to behavioural testing. Vehicle treatment consisted of 0.1 % Tween 80 and saline and was injected i.p. 30 minutes prior to behavioural testing. Anxiolytic effects after diazepam treatment in BALB/c mice have been found in a dose range of 1–5 mg/kg. For MPEP treatment, anxiolytic effects have been found in a dose range of 3–30 mg/kg. There is no knowledge about diazepam and MPEP effects in 129P3 mice specifically so a dose response curve was included in the open field test.

Recently, we found that 129P3/J mice are characterized by a profound lack of habituation to the modified hole board test as shown by highly increased avoidance behaviour over time while BALB/c mice, which have been reported to be highly anxious, show rapid habituation to the same test environment. In addition, in 129P3/J mice c-Fos expression was found to be lower after the habituation procedure in distinct brain areas (e.g. prelimbic cortex and lateral septum) in comparison to BALB/c mice. In a subsequent study we further demonstrated that exposure to chronic mild stress prior to repeated behavioural testing intensified the lack of habituation also in other behavioural parameters such as locomotion. Other studies have found specific behavioural characteristics in 129P3 mice, such as less locomotor activity and more anxiety-related behaviour compared to for example C57BL/6 mice. From these and our results we concluded that 129P3/J mice may represent an interesting animal model for non-adaptive anxiety.

Behavioural findings validated the initially high, but habituating phenotype of BALB/c mice, while 129P3 mice were characterized by impaired intrasession habituation. Diazepam had an anxiolytic effect in BALB/c mice, while in higher doses caused behavioural inactivity in 129P3 mice. MPEP revealed almost no anxiolytic effects on behaviour in both strains, but reduced stress-induced corticosterone responses only in 129P3 mice. These results were complemented by reduced expression of c-Fos after MPEP treatment in brain areas related to emotional processes, and increased c-Fos expression in higher integrating brain areas such as the prelimbic cortex compared to vehicle-treated 129P3 mice.

Basal blood samples ( basal) were collected 4 days before the start of the ORT using tail vein incision for corticosterone (CORT) determination. Thirty minutes after the ORT a second blood sample was taken (non-basal). The same procedure and analyses were used as described in the OF (section 2.3.2).

Naive male BALB/cJ (BALB/c, stock nr. 000651) and 129P3/J (129P3, stock nr. 000690) were obtained from the Jackson Laboratory (Bar Harbour, Maine, USA) and housed individually in Eurostandard Type II cages (size: 26.7 × 20.7 × 14 cm, Tecniplast, Buguggiate, Italy) provided with bedding material (Lignocel, J. Rettenmaier & Söhne GmbH, Germany), a tissue (KLEENEX Facial Tissue, Kimberly-Clark Professional BV, Ede, The Netherlands) and a shelter for cage enrichment. Mouse chow (CRM, Expanded, Special Diets Services Witham, England) and tap water were available ad libitum. For all experiments, the mice were acclimated to the experimental room for 17 days at the animal facilities of The Netherlands Vaccine Institute (Bilthoven, The Netherlands) under a reversed dark/light cycle (lights on between 18.00 h and 6.00 h). A radio played constantly as background noise (radio music interspersed with talk-shows, +/−60 dB). During this period the animals were handled three times a week by the person who also performed the behavioural tests. All behavioural testing took place between 9.00 and 13.00 h in the animal’s housing room and equipment (including the behavioural test set-ups) was installed before the animals arrived. Relative humidity was kept at a constant level of approximay 50%, room temperature was sustained at 22 °C ± 2 and ventilation rate was 15–20 air changes per hour.

The OF apparatus consisted of a circular grey PVC arena, 80 cm in diameter and 33 cm high grey PVC walls (Additional file 1 : Figure A1a). The arena was divided by red concentric circles in an outer zone, inner zone and centre area. Extra lines radiating out from the centre were placed on the floor as indicator for locomotion. One extra light bulb (red light) was fastened above the arena making the light intensity in the OF about 5–10 lux. Each animal was individually placed in the OF for 30 minutes, always starting from the same position at the edge of the OF. Behaviour was directly monitored and scored by a trained observer blind to treatment group using the program Observer 5.0 (Noldus Technology, The Netherlands). After each trial the OF was carefully cleaned with tap water and a towel. Behaviours scored in the OF included avoidance behaviour of the centre area, risk assessment, locomotor and exploratory behaviour and arousal. A description of all behaviours measured can be found in Table 1.

Adaptive anxiety in mice may be characterised by changes in behavioural responses over time, for example habituation to a novel environment. In contrast, non-adaptive anxiety might be mirrored by a lack of such a habituation, a phenomenon which may severely interfere with the normal interaction of the animal with its physical and social environment.

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* Corresponding author: Amber R Salomons Author Affiliations.

Brains from treatment groups: vehicle, 1 mg/kg diazepam and 10 mg/kg MPEP were removed for c-Fos immunohistochemistry. After removal, brains were frozen in liquid (−80 °C) 2-methylbutane which was cooled with dry ice and stored at −80 °C. Coronal sections were cut (20 μm) and mounted on Menzel SuperFrost Plus slides (Menzel GmbH & Co, Braunschweig, Germany) and stored at −20 °C. The sections were processed for c-Fos immunohistochemistry as described previously, dilution with a polyclonal primary antibody (1:1000, SC-52 Santa Cruz Biotechnology, Santa Cruz, USA), and a donkey-anti-rabbit IgG Biotin SP conjugated secondary antibody (1:400, Jackson ImmunoResearch Laboratories, Inc USA). Cells containing a nuclear brown-black reaction product were considered as c-Fos positive cells and counted in several brain areas which are known to be involved in anxiety : medial prefrontal cortex (prelimbic, PrL ), lateral septum (dorsal, LSD ; intermediary, LSI ; ventral, LSV ), bed nucleus of the stria terminalis (medial anterior, BSTMA ; lateral posterior, BSTLP ; medial ventral, BSTMV ), hippocampus (granular layer dentate gyrus, DG ), hypothalamus (paraventricular nucleus, PVN; dorsal medial hypothalamus, DMH ), amygdala (basolateral nucleus, BLA ; central nucleus cAmy ) and the periaqueductal gray (dorsolateral, dlPAG ; dorsomedial, dmPAG ; lateral, lPAG ; ventrolateral, vlPAG ). The anatomical localization was aided by use of adjacent Nissl stained sections and the illustrations in a stereotaxic atlas. For each region at least two overt landmarks were used. For quantitative analysis of c-Fos positive cells, the program Leica Qwin (image processing and analysis software, Cambridge, United Kingdom) was used. Left and right hemispheres were analyzed for stained neurons per mm2 and calculated for one section separay and averaged for each animal.

Additional file 1. Schematic representations of the open field and the object recognition test.Corrected P value thresholds of significance for the open field and object recognition test.Overview of all behavioural results in the open field after MPEP or diazepam treatment.Overview of CORT levels before and after open field or object recognition testing.Overview of c-Fos results for all brain areas investigated.

Previous studies have demonstrated a profound lack of habituation in 129P3 mice compared to the habituating, but initially more anxious, BALB/c mice. The present study investigated whether this non-adaptive phenotype of 129P3 mice is primarily based on anxiety-related characteristics.

Results from the open field led to the conclusion that 1 mg/kg diazepam and 10 mg/kg MPEP could be used as the minimal effective dose range, without causing side effects in both strains. We used these doses to investigate object memory. A total of 27 mice per strain was used for the ORT (n = 9 per treatment group). On day 1, the familiar object was placed in the home cage. On day 2, the animals were i.p. injected with diazepam (1 mg/kg), MPEP (10 mg/kg) or vehicle 30 minutes before behavioural testing. Thirty minutes after the ORT, a blood sample was taken and 120 minutes after testing the animals were euthanized by decapitation.

In the present study, the two mouse strains BALB/c and 129P3/J, were tested after acute treatment with either diazepam or MPEP for 30 minutes in the open field (intra-session habituation), and an object recognition test (cognitive performance). In addition to behavioural parameters, levels of plasma corticosterone (CORT) were determined before and after behavioural testing. Finally, the expression of c-Fos, a marker for neural activity, was investigated after behavioural testing in brain areas involved in emotional and cognitive processing. Based on the hypothesis that the non-adaptive phenotype of 129P3 mice is primarily anxiety-related, we expected anxiolytic treatment to facilitate habituation and decrease post-testing corticosterone levels. In addition we expected enhanced c-Fos expression in brain areas involved in the integration of emotional and cognitive processes.

2 Rudolf Magnus Institute of Neuroscience, Universiteitsweg 100, Utrecht, CG, 3584, The Netherlands.

Since our previous studies evaluated between trial or intersession habituation, we were now interested in extending our knowledge about the behavioural profile in 129P3/J mice by investigating within-trial intrasession habituation as well. It has been suggested that intersession habituation reflects memory or retention of the previous exposures, while intrasession habituation might indicate adaptive capacity.

The experimental protocols were approved by the Animal Experiments Committee of the Academic Biomedical Centre Utrecht, The Netherlands. Furthermore, all animal experiments followed the ‘Principles of Laboratory Animal Care’ and refer to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioural Research (National Research Council 2003).

3 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, Utrecht, CL, 3584, The Netherlands.

To test this hypothesis, two anxiolytic compounds were used in the present study: the benzodiazepine Diazepam as a standard anxiolytic, and the metabotropic glutamate receptor 5 antagonist (mGlu5R) MPEP (2-methyl-6-(phenylethynyl)pyridine). Diazepam has shown anxiolytic efficiency over decades. However, this compound is known to induce side effects at the cognitive level i.e. amnesia as well as in activity sedation. Therefore, the putative anxiolytic MPEP was included in the present study. A number of studies have shown anxiolytic properties of MPEP. For example, MPEP significantly reduced fear potentiated startle and increased the number of open arm entries in the elevated plus maze, similar as treatment with diazepam Furthermore, treatment with MPEP attenuated stress-induced hyperthermia and decreased the number of buried marbles in a marble burying test, whereas no effect of MPEP treatment was observed on spontaneous locomotor activity.

1 Department of Animals in Science and Society, Division of Animal Welfare and Laboratory Animal Science, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584, Utrecht, CM, The Netherlands.

2012 Salomons et al.; licensee BioMed Central Ltd.

The test apparatus was a Eurostandard Type II L cage (size: 36.5 x 21 x 14 cm; Tecniplast, Buguggiate, Italy) without any bedding and equally divided in 6 squares by black lines on the floor (Additional file 1 : Figure A1b). Light conditions were the same as in the experimental room, red light with an illumination intensity of approximay 5 lux. Two different objects were used that differed in colour, material and shape (screw nut and die). Both objects were considered too heavy to be displaced by the animals. All animals were allowed to familiarize with one of the two objects for 24 h in their home cage (randomized for each strain and treatment i.e. half the animals of each testing group received a nut, the other half received a die) one day before the ORT. Twenty-four hours later, the animals were tested in a one-trial ORT, without habituation to the test arena. This was done to study the effect of anxiolytic treatment on object memory per se. Both objects (for the familiar object a duplicate was used) were always placed in the same corner of the apparatus, each was positioned at the same distance from the wall. For testing, the animals were individually placed in the apparatus always in the same corner opposite of the objects and scored by a trained observer blind to treatment group for 10 minutes. After each trial the ORT was carefully cleaned with tap water and a towel. Behaviours scored in the ORT included object memory, risk assessment, locomotor and exploratory behaviour and arousal. A description of all behaviours measured can be found in Table 1.

To test this hypothesis and extend our knowledge on the behavioural profile of 129P3 mice, the effects of the anxiolyticdiazepam (1, 3 and 5 mg/kg) and the putative anxiolytic metabotropic glutamate receptor 5 (mGlu5R) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP, 3, 10 and 30 mg/kg) treatment on within-trial (intrasession) habituation, object recognition (diazepam: 1 mg/kg; MPEP 10 mg/kg) and on the central-nervous expression of the immediate early gene c-Fos (diazepam: 1 mg/kg; MPEP 10 mg/kg) were investigated.

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Behavioral and Brain Functions 2012, 8 :30 doi:10.1186/

4 F. Hoffmann-La Roche Ltd. Pharmaceuticals Division, Discovery Neuroscience, Basel, Switzerland.

A total of 56 mice per strain were used and randomly assigned to 3 different dose groups per compound and one vehicle group (n = 8 per treatment group). Animals were tested with either diazepam (1, 3 or 5 mg/kg), MPEP (3, 10 or 30 mg/kg) or vehicle. While we used one vehicle group only in order to decrease the number of animals needed, the diazepam or MPEP treated mice were considered as parallel experiments and were thus analyzed per compound. Although, all treated mice were tested conjointly in a given test session in order to prevent variability of baseline behaviours among experiments. For all animals, diazepam MPEP or vehicle was i.p. injected 30 minutes before behavioural testing. Thirty minutes after behavioural testing a blood sample was taken and 120 minutes after testing the animals were euthanized by decapitation and brains were removed.


Effects of diazepam