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Effects of environmental enrichment and sexual dimorphism on the expression of cerebellar receptors in C57BL/6 and BTBR + Itpr3tf/J mice

Abstract

Objective

Environmental enrichment is used to treat social, communication, and behavioral deficits and is known to modify the expression of synaptic receptors. We compared the effects of environmental enrichment in the expression of glutamate and endocannabinoid receptors, which are widely expressed in the cerebellar cortex. These two receptors interact to regulate neuronal function and their dysregulation is associated with behavioral changes. We used BTBR + Itpr3tf/J mice, a strain that models behavioral disorders, and C57BL/6 mice for comparison. We studied the effects of genetic background, sex, environmental conditions, and layer of the cerebellar cortex on the expression of each receptor.

Results

The influence of genetic background and environmental enrichment had the same pattern on glutamate and endocannabinoid receptors in males. In contrast, in females, the effect of environmental enrichment and genetic background were different than the ones obtained for males and were also different between the glutamate and endocannabinoid receptors. Furthermore, an analysis of both receptors from tissue obtained from the same animals show that their expression is correlated in males, but not in females. Our results suggest that environmental enrichment has a receptor dependent and sexual dimorphic effect on the molecular expression of different receptors in the cerebellar cortex.

Introduction

The BTBR + Itpr3tf/J (BTBR) mouse model is widely used in studies related to social and communication deficits, and repetitive behaviors [1,2,3]. BTBR mice exposed to environmental enrichment show decreases in repetitive behaviors and anxiety [4, 5], and show an increase in social affiliation [6]. In separate studies at the molecular level, changes in the expression of NMDAR1s [7, 8] and CB1Rs [9] are modulated by environmental enrichment. Interactions between NMDAR1s and CB1Rs, contribute to regulate neuronal function [10, 11], including in the cerebellum [12] where they are widely expressed in the cerebellar cortex [13, 14]. For these reasons, we quantified the expression of NMDAR1s and CB1Rs in the cerebellar cortex. In particular, we measured these changes in lobule VII because changes in the structure and physiology of this area correlate with abnormal behaviors such as compulsive rituals, stereotypical performance, and difficulty to understand social cues [15], which are replicated in the abnormal behavioral phenotype of the BTBR strain [16, 17]. The BTBR and C57 groups of each sex were exposed to a standard or enriched environmental conditions. Our results suggest that environmental enrichment has a receptor dependent and sexual dimorphic effect in the cerebellar cortex.

Main text

Methods

Animal procedures

We bred a new colony for 4 months of C57 (stock #000664) and BTBR (stock #002282) mice (Jackson Laboratories, Farmington, CT) under the protocol MU113, approved by the UTSA Institutional Animal Care and Use Committee (IACUC). Animals were maintained on a 12 h light/dark-cycle with constant access to food and water. After weaning, animals were separated by sex and housed with their litter in standard caging (26 cm width × 16 cm long x 16 cm deep) until P75. Reaching this age, animals from the same litter were randomly selected and separated in those that stayed in the standard environment (standard caging all the time) and those that experienced the environmental enrichment protocol.

The environmental enrichment arena was 90 cm width × 40 cm long × 33 cm deep and contained dust free bedding (Sophresh Natural Aspen) and toys. The toys were balls, cubes, and pyramids of different sizes, shapes, colors, and textures (solid, hollow, furry); there was also a running wheel, and tunnels. The position of all the toys was changed every day. Animals were placed in the arena 1 h a day for 20 days during the second four hours of the light part of their light cycle. Animals were returned to their standard cages after each exposure to environmental enrichment. At the end of the 20 days period the standard and environmental enrichment animals were euthanized. We anesthetized mice with 4 mL isoflurane in an evaporation chamber and kept them in deep anesthesia using the nose cone method. Animals were transcardially perfused with saline followed by 4% paraformaldehyde (0.2 M phosphate buffer, pH 7.4).

Imaging and data analysis

A detailed description of the immunofluorescence protocol is in the Additional file . We visualized expression of NMDAR1, CB1R, and DAPI with a multi-photon microscope (Bruker Ultima, Madison, WI). All images were collected with a 20 × objective. The wavelength of the tunable laser (DeepSee 690–1300 nm, Spectr Physics) to excite at the same time DAPI-NMDAR1 was 1120 nm and for DAPI-CB1R was 1078 nm. Fluorescent signals were separated by a dichroic cube and detected by photo multipliers. In all cases the scanning laser spent 2.0 µs in each coordinate of the image. Images for Fig. 1 were background corrected, thresholded, contrast enhanced, and convolved with a Gaussian filter to enhance morphological features. All images used for statistical analyses were only background corrected.

Fig. 1
figure 1

Expression of NMDAR1 and CB1R in the cerebellar cortex of C57BL/6 (C57) and BTBR + Itpr3tf/J (BT) mice raised in standard environments. A Fluorescent signal of NMDAR1 (red). B Images corresponding to the squares in A. C Images obtained from averaging the fluorescence of a Z-stack of 10 images separated by 1 µm. D images corresponding to the squares in C. The contours emphasize the shape of Purkinje cell somas and dendrites. E–H As in A–F for the expression of CB1 receptors (green) H. The contours emphasize the CB1R presynaptic terminals around Purkinje cell soma. ML, Molecular cell layer, PCL, Purkinje cell layer; GCL, Granule cell layer. All slices were stained with DAPI and identically processed (blue)

We selected ROIs for the molecular, Purkinje, and granular layers for each receptor in every slice. In the molecular and granular layers, the ROIs were polygons with and average area of 13,072 μm2 with a rage from 1718 to 45,365 μm2 for the granular and 7098 μm2 with a range of 1273–22,125 μm2 for the molecular layer. The ROI for the Purkinje cells were squares of 19.7 μm side. We collected, in average, 9.5 Purkinje cell somas per slice with a range from 5 to 15 (see Additional file 1: Figure S1 for an example of ROIs). The ROIs and background correction areas were identical for the receptor and DAPI images. In each ROI we computed a mask of the pixels that contained fluorescent information and used it to calculate the average value of the signal. We then calculated the ratio of NMDAR1/DAPI and CB1R/DAPI. Other groups have successfully used this technique to image brain slices and organoids [18].

For each animal we collected 3 images. The slices were selected based on a clear display of DAPI staining. Each experimental group consisted of 3 animals. No data points were excluded. Confounders were not controlled (such as animal/cage location). Images were analyzed blindly. We used a sample size and power test (sampsizepwr function in Matlab) to calculate the minimum number of animals necessary to have a statistical power of 80%. Based on our experimental measurements, the average standard deviation in each experimental group was 0.54. The number of animals required to have at least a difference of 2 in the expression of the receptors with this standard deviation was 3. We applied a Lilliefors test for all the data and then performed ANOVA and post-hoc tests as described in the results. In all cases we set significance at p ˂ 0.05. We also estimated the effect size of the ANOVA test using the value of \({\eta }^{2}=\frac{Sum Squares Effect}{Sum Squares Effect+SumSquares Error}\); for post hoc analyses we used Cohens’ d, \(d=\frac{Mean group 1-Mean Group 2}{Population Standard Deviation}\) [19]. All the analyses were performed using custom MATLAB scripts (Natick, MA).

Results

We found NMDAR1s present throughout the cerebellar cortex. NMDAR1s in Purkinje cell somas, dendrites of the molecular layer, and granular cell layer (Fig. 1A–D). We found CB1Rs expression on pre-synaptic terminals around Purkinje cell somas, consistent with their presence in the pinceau (Fig. 1E–H). There was also diffuse expression in the molecular layer, corresponding to parallel fibers [20]. In the granule cell layer the expression corresponded to granule cells somas and dendrites [21].

We performed a multi-way ANOVA test on the expression patterns of each receptor. The test compared genetic background (C57 vs BTBR), sex (male vs female), environmental condition (standard vs enriched caging), and layer in the cerebellar cortex (molecular, Purkinje, and granular). In the case of NMDAR1/DAPI, this test showed that sex was the only significant effect (\(F(\mathrm{1,32}) = 56.95, p=1.71 \times {10}^{-10}\), and the effect size was \({(\eta }^{2}= 0.44\)). The same test for the expression of CB1R showed that sex (sex:\(F\left(\mathrm{1,32}\right)= 11.09, p=14.00 \times {10}^{-4}, {\eta }^{2}= 0.11\)) and environmental condition (\(F(\mathrm{1,32}) = 16.75, p=1.00 \times {10}^{-4}, {\eta }^{2}= 0.17\)) had a significant effect.

Since sex has a strong influence on the expression of both, NMDAR1 and CB1R, we performed another multi-way ANOVA separating the groups by sex. For NMDAR1 this shows that, for both males and females, the genetic background had a significant effect on the expression of this receptor (males: \(F(\mathrm{1,32}) = 13.73, p=8.00 \times {10}^{-4}, {\eta }^{2}=0.22\); females: \(F(\mathrm{1,32}) = 26.69, p = 1.33 \times {10}^{-5}, {\eta }^{2}=0.22\)). This was also the case for the environmental condition (males: \(F(\mathrm{1,32}) = 15.98, p = 3.00\times {10}^{-4}, {\eta }^{2}=0.25\); females: \(F(\mathrm{1,32}) = 56.94, p = 1.66 \times {10}^{-8}, {\eta }^{2}=0.48\)). We obtained the same result when performing the test for the CB1R images (Genetic background: males, \(F(\mathrm{1,32})=14.91, p= 5.00\times {10}^{-4},{\eta }^{2}=0.23\); females, \(F(\mathrm{1,32}) = 14.40, p = 6.00 \times {10}^{-4}, {\eta }^{2} = 0.27\); Environmental condition: males, \(F(\mathrm{1,32}) = 18.21, p = 1.00 \times {10}^{-4}, {\eta }^{2} = 0.28\); females, \(F\left(\mathrm{1,32}\right)= 6.27,p = 0.01, {\eta }^{2}=0.11\)).

Next, we performed a post-hoc multi-compare analysis of the multi-way ANOVA tests (groups separated by sex) to determine differences in the effect of environmental enrichment and genetic background in the expression of each receptor. In males, the expression of NMDAR1/DAPI in the BTBR enriched environment group was more than 5 times larger (and significantly different, mean 6.42) than for all the other experimental groups (C57 standard and enriched environment groups, and the BTBR standard environment group, Fig. 2A). The same analysis for expression of NMDAR1 in females shows that the effect of environmental enrichment is similar for the C57 and BTBR groups. In both cases, the enriched environment had a higher expression of the receptor than the corresponding standard environment group. Thus, suggesting that the genetic background does not modify the effect of environmental enrichment in females (Fig. 2B).

Fig. 2
figure 2

Effects of environmental enrichment on the expression of glutamate (NMDAR1) and endocannabinoid (CB1R) receptors in C57BL/6 (C57) and BTBR + Itpr3tf/J (BT) mice in the cerebellar cortex. A Values of NMDAR1/DAPI. The symbols correspond to the average expression of the receptor in each animal. Animal groups were from each genetic background (C57 or BT) exposed to standard (S) or enriched (E) environments. Each column shows mean and SEM. The horizontal lines indicate statistical significance (p < 0.05). B The same analysis as in A for female mice. C , D as in A and B for the expression of CB1R. Each data point is the average of 3 samples obtained from the same mice. We collected tissue from 3 animals for each experimental condition

We also performed a post-hoc analysis for the CB1R data. This shows that the expression of CB1R in males is identical to the pattern we found for NMDAR1 males (Fig. 2C). The expression in females was different from CB1R males and from NMDAR1 males or females. The BTBR groups showed a lower ratio of CB1R/DAPI than the C57 groups. Environmental enrichment had no effect in changing the expression of CB1R (Fig. 2D).

Since we used tissue from the same animals to stain for CB1R or NMDAR1 we studied the relative expression of these receptors. We calculated the correlation coefficient between these values across all animals. We found that the correlation coefficient for all males was large and significant (\(r = 0.89, p = 1.10\times {10}^{-4}\)). In contrast, the correlation coefficient for females was not significant (\(r = 0.10, p = 0.77\)). This analysis suggests a differential regulation in the co-expression of NMDAR1 and CB1R between males and females.

Discussion

We demonstrated that environmental enrichment affects the expression of NMDAR1s and CB1Rs in vermal lobule VII of the cerebellar cortex. Our results show differences that depended on sex and receptor type, suggesting a complex effect of environmental enrichment in receptor expression in the cerebellar cortex. These findings contribute to the use of the BTBR strain to study sexual dimorphism in neurological disorders in humans [22,23,24].

While there is no single environmental enrichment paradigm, in all protocols as in this study, the arena is larger than the standard cage and there is a constant reorganization of “toys” [25,26,27,28,29]. In our protocol, the session duration for environmental enrichment exposure was based on therapies used in human patients with ASD [30, 31]. These environmental therapies change the individual’s experience by sensorimotor stimulation, self-directed patterns of attention, and social learning [32, 33]. Our protocol reproduced these features by the daily change in textures, shapes, sizes, and location of objects, and the mouse-mouse interaction inside the arena. As such, our arena provided an enhanced sensory, motor, social, and cognitive stimulation which meets the criteria in the implementation of this paradigm [29].

Glutamate and endocannabinoid receptors and their pathways can be therapeutic targets to treat abnormal behaviors [2, 34,35,36,37]. In the BTBR mouse NMDAR1 agonist and antagonist have been used to improve sociability and spontaneous grooming [1, 38]. The activation of endocannabinoid production by acetaminophen enhances social behavior in BTBR mice [39]. BTBR animals have a mutation in the Kmo and Ext1 genes, involved in the production of a glutamate receptor antagonist and excitatory synaptic transmission [40, 41]. It is possible that the increased expression of NMDAR1 in male BTBR mice found in our work, is a compensation for the dysregulation in the function of the proteins encoded by these genes, which could have sex differences [24].

Limitations

Our research would benefit from quantification of changes in behavior to correlate with our molecular expression measurements. A limitation in the interpretation of our results for translational applications in ASD is that while the BTBR mouse model has a strong construct validity it may not reproduce behavioral and cellular symptoms reported in human patients. In addition, data from ASD adult human postmortem brains is not available, and neither data from ASD adult after environmental enrichment, so our data cannot be contrasted.

Our study would benefit from a larger sample size. Particularly, to study small differences between the standard housing groups. However, our current results have a statistical power of 80% and the effect size shows that the magnitude of the experimental effect is medium to large [42] for the receptor expression.

We concentrated our measurements in Lobule VII because of the known effects of ASD in this area; future work should compare changes in expression across the cerebellum. As an additional limitation the two genotypes used in this work were not littermates, therefore there could be further genetic differences introduced de novo. As a future direction, other molecular techniques such as RT-qPCR should be use to quantify the NMDAR1 and CB1R expression at mRNA level.

Availability of data and materials

All data and analysis code are available at github.com/Santamarialab/BTBR and upon request.

Abbreviations

ASDs:

Autism spectrum disorders.

BTBR:

BTBR + Itpr3tf/J strain mice.

NMDR1s:

N-methyl-d-aspartate receptors subunit1.

CB1Rs:

Endocannabinoid receptors type1

C57:

C57BL/6 strain mice group

BT:

BTBR + Itpr3tf/J strain mice group

S:

Standard

E:

Enriched

ROIs:

Regions of interest

LARC:

Laboratory animal resources center

UTSA:

University of texas at san antonio

PBS:

Phosphate buffer saline

TBS:

Tris-buffered saline

DAPI:

4′, 6-Diamidino-2-phenylindole

ANOVA:

Analysis of variance

Env:

Environment

Gen:

Genetic background

M:

Male

F:

Female

P:

Postnatal day

References

  1. Burket JA, Benson AD, Tang AH, Deutsch SI. D-Cycloserine improves sociability in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorders with altered Ras/Raf/ERK1/2 signaling. Brain Res Bull. 2013;96:62–70.

    CAS  Article  Google Scholar 

  2. Meyza KZ, Blanchard DC. The BTBR mouse model of idiopathic autism—Current view on mechanisms. Neurosci Biobehav Rev. 2017;76(Pt A):99–110.

    CAS  Article  Google Scholar 

  3. Pearson BL, Pobbe RL, Defensor EB, Oasay L, Bolivar VJ, Blanchard DC, et al. Motor and cognitive stereotypies in the BTBR T+tf/J mouse model of autism. Genes Brain Behav. 2011;10(2):228–35.

    CAS  Article  Google Scholar 

  4. Reynolds S, Urruela M, Devine DP. Effects of environmental enrichment on repetitive behaviors in the BTBR T+tf/J mouse model of autism. Autism Res. 2013;6(5):337–43.

    Article  Google Scholar 

  5. Woo CC, Donnelly JH, Steinberg-Epstein R, Leon M. Environmental enrichment as a therapy for autism: a clinical trial replication and extension. Behav Neurosci. 2015;129(4):412–22.

    Article  Google Scholar 

  6. Diamond, B. A. (2019). Environmental enrichment improves sociability in BTBR mice, a rodent model for autism (Order No. 22583654). Available from ProQuest One Academic. (2311757505). https://www.proquest.com/dissertations-theses/environmental-enrichment-improves-sociability/docview/2311757505/se-2?accountid=7122

  7. Huang Y, Jiang H, Zheng Q, Fok AHK, Li X, Lau CG, et al. Environmental enrichment or selective activation of parvalbumin-expressing interneurons ameliorates synaptic and behavioral deficits in animal models with schizophrenia-like behaviors during adolescence. Molecular Psychiatry. 2021. https://doi.org/10.1038/s41380-020-01005-w.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Wang X, Meng Z-X, Chen Y-Z, Li Y-P, Zhou H-Y, Yang M, et al. Enriched environment enhances histone acetylation of NMDA receptor in the hippocampus and improves cognitive dysfunction in aged mice. Neural Regen Res. 2020;15(12):2327.

    Article  Google Scholar 

  9. El Rawas R, Thiriet N, Nader J, Lardeux V, Jaber M, Solinas M. Early exposure to environmental enrichment alters the expression of genes of the endocannabinoid system. Brain Res. 2011;1390:80–9.

    Article  Google Scholar 

  10. Rodríguez-Muñoz M, Sánchez-Blázquez P, Merlos M, Garzón-Niño J. Endocannabinoid control of glutamate NMDA receptors: the therapeutic potential and consequences of dysfunction. Oncotarget. 2016;7(34):55840–62.

    Article  Google Scholar 

  11. Sánchez-Blázquez P, Rodríguez-Muñoz M, Garzón J. The cannabinoid receptor 1 associates with NMDA receptors to produce glutamatergic hypofunction: implications in psychosis and schizophrenia. Front Pharmacol. 2014. https://doi.org/10.3389/fphar.2013.00169.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Netzeband JG, Conroy SM, Parsons KL, Gruol DL. Cannabinoids enhance NMDA-elicited Ca2+ signals in cerebellar granule neurons in culture. J Neurosci. 1999;19(20):8765–77.

    CAS  Article  Google Scholar 

  13. Kawamura Y, Fukaya M, Maejima T, Yoshida T, Miura E, Watanabe M, et al. The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum. J Neurosci. 2006;26(11):2991–3001.

    CAS  Article  Google Scholar 

  14. Lonchamp E, Gambino F, Dupont JL, Doussau F, Valera A, Poulain B, et al. Pre and post synaptic NMDA effects targeting Purkinje cells in the mouse cerebellar cortex. PLoS ONE. 2012;7(1): e30180.

    CAS  Article  Google Scholar 

  15. Riva D, Annunziata S, Contarino V, Erbetta A, Aquino D, Bulgheroni S. Gray matter reduction in the vermis and CRUS-II is associated with social and interaction deficits in low-functioning children with autistic spectrum disorders: a VBM-DARTEL Study. Cerebellum. 2013;12(5):676–85.

    Article  Google Scholar 

  16. Chao OY, Fernandez Marron, de Velasco E, Pathak SS, Maitra S, Zhang H, Duvick L, et al. Targeting inhibitory cerebellar circuitry to alleviate behavioral deficits in a mouse model for studying idiopathic autism. Neuropsychopharmacol. 2020;45(7):1159–70.

    Article  Google Scholar 

  17. Xiao R, Zhong H, Li X, Ma Y, Zhang R, Wang L, et al. Abnormal cerebellar development is involved in dystonia-like behaviors and motor dysfunction of autistic BTBR mice. Front Cell Deve Biol. 2020;8:231.

    Article  Google Scholar 

  18. Smyrek I, Stelzer EH. Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy. Biomed Opt Express. 2017;8(2):484–99.

    CAS  Article  Google Scholar 

  19. Lakens D. Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVAs. Front Psychol. 2013;4:863.

    Article  Google Scholar 

  20. Safo PK, Regehr WG. Endocannabinoids control the induction of cerebellar LTD. Neuron. 2005;48(4):647–59.

    CAS  Article  Google Scholar 

  21. Egertová M, Cravatt BF, Elphick MR. Comparative analysis of fatty acid amide hydrolase and cb(1) cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling. Neuroscience. 2003;119(2):481–96.

    Article  Google Scholar 

  22. Baron-Cohen S, Knickmeyer RC, Belmonte MK. Sex differences in the brain: implications for explaining autism. Science. 2005;310(5749):819–23.

    CAS  Article  Google Scholar 

  23. Schumann CM, Sharp FR, Ander BP, Stamova B. Possible sexually dimorphic role of miRNA and other sncRNA in ASD brain. Molecular autism. 2017;8:4.

    Article  Google Scholar 

  24. Werling DM, Parikshak NN, Geschwind DH. Gene expression in human brain implicates sexually dimorphic pathways in autism spectrum disorders. Nat Commun. 2016;7:10717.

    CAS  Article  Google Scholar 

  25. Hulbert SW, Bey AL, Jiang YH. Environmental enrichment has minimal effects on behavior in the Shank3 complete knockout model of autism spectrum disorder. Brain Behav. 2018;8(11): e01107.

    Article  Google Scholar 

  26. Janssen H, Bernhardt J, Collier JM, Sena ES, McElduff P, Attia J, et al. An enriched environment improves sensorimotor function post-ischemic stroke. Neurorehabil Neural Repair. 2010;24(9):802–13.

    Article  Google Scholar 

  27. Stamenkovic V, Stamenkovic S, Jaworski T, Gawlak M, Jovanovic M, Jakovcevski I, et al. The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment. Brain Struct Funct. 2017;222(1):393–415.

    CAS  Article  Google Scholar 

  28. Xie H, Wu Y, Jia J, Liu G, Zhang Q, Yu K, et al. Enrichment-induced exercise to quantify the effect of different housing conditions: a tool to standardize enriched environment protocols. Behav Brain Res. 2013;249:81–9.

    Article  Google Scholar 

  29. Kempermann G. Environmental enrichment, new neurons and the neurobiology of individuality. Nat Rev Neurosci. 2019;20(4):235–45.

    CAS  Article  Google Scholar 

  30. Boso M, Emanuele E, Minazzi V, Abbamonte M, Politi P. Effect of long-term interactive music therapy on behavior profile and musical skills in young adults with severe autism. J Alternat Complement Med. 2007;13(7):709–12.

    Article  Google Scholar 

  31. Kandalaft MR, Didehbani N, Krawczyk DC, Allen TT, Chapman SB. Virtual reality social cognition training for young adults with high-functioning autism. J Autism Dev Disord. 2013;43(1):34–44.

    Article  Google Scholar 

  32. Mukherjee SB. Autism spectrum disorders - diagnosis and management. Indian J Pediatr. 2017;84(4):307–14.

    Article  Google Scholar 

  33. Woo CC, Leon M. Environmental enrichment as an effective treatment for autism: a randomized controlled trial. Behav Neurosci. 2013;127(4):487–97.

    Article  Google Scholar 

  34. Carlson GC. Glutamate receptor dysfunction and drug targets across models of autism spectrum disorders. Pharmacol Biochem Behav. 2012;100(4):850–4.

    CAS  Article  Google Scholar 

  35. Chakrabarti B, Persico A, Battista N, Maccarrone M. Endocannabinoid signaling in autism. Neurotherapeutics. 2015;12(4):837–47.

    CAS  Article  Google Scholar 

  36. Kerr DM, Downey L, Conboy M, Finn DP, Roche M. Alterations in the endocannabinoid system in the rat valproic acid model of autism. Behav Brain Res. 2013;249:124–32.

    CAS  Article  Google Scholar 

  37. Rojas DC. The role of glutamate and its receptors in autism and the use of glutamate receptor antagonists in treatment. J Neural transm. 2014;121(8):891–905.

    CAS  Article  Google Scholar 

  38. Silverman JL, Tolu SS, Barkan CL, Crawley JN. Repetitive self-grooming behavior in the BTBR mouse model of autism is blocked by the mGluR5 antagonist MPEP. Neuropsychopharmacology. 2010;35(4):976–89.

    CAS  Article  Google Scholar 

  39. Gould GG, Seillier A, Weiss G, Giuffrida A, Burke TF, Hensler JG, et al. Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice. Prog Neuropsychopharmacol Biol Psychiatry. 2012;38(2):260–9.

    CAS  Article  Google Scholar 

  40. McFarlane HG, Kusek GK, Yang M, Phoenix JL, Bolivar VJ, Crawley JN. Autism-like behavioral phenotypes in BTBR T+tf/J mice. Genes Brain Behav. 2008;7(2):152–63.

    CAS  Article  Google Scholar 

  41. Meyza KZ, Defensor EB, Jensen AL, Corley MJ, Pearson BL, Pobbe RL, et al. The BTBR T+ tf/J mouse model for autism spectrum disorders-in search of biomarkers. Behav Brain Res. 2013;251:25–34.

    CAS  Article  Google Scholar 

  42. Vandekar S, Tao R, Blume J. A robust effect size index. Psychometrika. 2020;85(1):232–46.

    Article  Google Scholar 

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Acknowledgements

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Funding

Contex (UT System-CONACYT) and NIMH-NIBIB 1R01EB026939.

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Authors

Contributions

DM: performed experiments; analyzed data; wrote manuscript. JM: conceptualization and funding. FS: conceptualization; data analysis; wrote manuscript; funding. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Fidel Santamaria.

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All animal procedures were in compliance with the UTSA IACUC and LARC (protocol MU113).

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Supplementary Information

Additional file 1

: Figure S1. Regions of interest (ROI) to calculate average fluorescent signal from a receptor marker and DAPI in the cerebellar cortex. Example image from a cerebellar Lobule VII slice expressing CB1R (green). The nuclei are marked with DAPI (blue). The image was obtained simultaneously. The molecular and granular layers were polygons drawn by hand. The Purkinje layer ROIs consisted in squares 19.8 μm on each side.

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Monje-Reyna, D., Manzo Denes, J. & Santamaria, F. Effects of environmental enrichment and sexual dimorphism on the expression of cerebellar receptors in C57BL/6 and BTBR + Itpr3tf/J mice. BMC Res Notes 15, 175 (2022). https://doi.org/10.1186/s13104-022-06062-8

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Keywords

  • Synaptic receptors
  • Excitability
  • Behavior
  • Plasticity
  • Multi-photon imaging
  • Autism spectrum disorders