Synaptic plasticity alterations associated with memory impairment induced by deletion of CB2 cannabinoid receptors

Highlights

• CB2KO mice present a cognitive impairment in the SDIA test.

• The morphological alterations observed in the HIP of CB2KO mice.

• The reduced BDNF and GR gene expression observed in the HIP of CB2KO mice.

• The alterations of mTOR/p70S6K signaling cascade observed in PFC and HIP of CB2KO.

• The cognitive enhancement induced by JWH133.

Abstract

In this study, the role of CB₂r on aversive memory consolidation was further evaluated. Mice lacking CB₂r (CB2KO) and their corresponding littermates (WT) were exposed to the step-down inhibitory avoidance test (SDIA). MAP2, NF200 and synaptophysin (SYN)-immunoreactive fibers were studied in the hippocampus (HIP) of both genotypes. The number of synapses, postsynaptic density thickness and the relation between the synaptic length across the synaptic cleft and the distance between the synaptic ends were evaluated in the HIP (dentate gyrus (DG) and CA1 fields) by electron microscopy. Brain-derived neurotrophic factor (BDNF), glucocorticoid receptor (NR3C1) gene expressions and mTOR/p70S6K signaling cascade were evaluated in the HIP and prefrontal cortex (PFC). Finally, the effects of acute administration of CB₂r-agonist JWH133 or CB₂r-antagonist AM630 on memory consolidation were evaluated in WT mice by using the SDIA.

The lack of CB₂r impaired aversive memory consolidation, reduced MAP2, NF200 and SYN-immunoreactive fibers and also reduced the number of synapses in DG of CB2KO mice. BDNF and NR3C1 gene expression were reduced in the HIP of CB2KO mice. An increase of p-p70S6K (T389 and S424) and p-AKT protein expression was observed in the HIP and PFC of CB2KO mice. Interestingly, administration of AM630 impaired aversive memory consolidation, whereas JWH133 enhanced it. Further functional and molecular assessments would have been helpful to further support our conclusions.

These results revealed that CB₂r are involved in memory consolidation, suggesting that this receptor could be a promising target for developing novel treatments for different cognitive impairment-related disorders.

Introduction

Over the last few years, the endocannabinoid system (ECS) has emerged as a neuromodulatory system involved in different processes such as stress, anxiety, depression, drug addiction and memory (Urigüen et al., 2004, Puighermanal et al., 2009). Most of these studies focused on the role of cannabinoid CB1 receptors (CB₁r) due to their wide distribution in human and rodent brains (Herkenham et al., 1991). The presence of a high density of CB₁r in the brain areas which regulate learning and memory, such as the hippocampus (HIP), suggested the potential role of this receptor in cognitive behavior. Different animal models have been used extensively to evaluate the function of CB₁r in various states of learning and memory, including acquisition, consolidation and retrieval (Riedel and Davies, 2005). CB₁r-agonists impair working memory and the acquisition of long-term memory (Costanzi et al., 2004, Mishima et al., 2001). The fact that these effects were blocked by the administration of CB₁r-antagonist, SR141716A, supports CB₁r as being the target involved in the acute effects of cannabinoids on memory impairments (Da and Takahashi, 2002, Pamplona and Takahashi, 2006). Interestingly, pharmacological or genetic disruption of CB₁r signaling enhances learning and memory in a variety of spatial and operant paradigms (Thiemann et al., 2007). Further studies revealed the modulation of GABA neurotransmission through the activation of CB₁r located on GABA interneurons, the indirect activation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) signaling cascade and NMDA receptors as the main mechanisms underlying the cognitive impairments produced by cannabinoids (Puighermanal et al., 2009). In contrast, the fact that a number of effects mediated by cannabinoids persisted in mice lacking the CB₁r (CB1KO) pointed to the involvement of additional cannabinoid receptors (Breivogel et al., 2001, Hajos et al., 2001, Kofalvi et al., 2003).

Cannabinoid CB₂ receptors (CB₂r) is expressed in several brain regions including the HIP, cerebral cortex, amygdala, striatum, thalamus and cerebellum (Gong et al., 2006, García-Gutiérrez et al., 2010). In contrast, some studies found a lack of specific CB₂ binding and lack of CB₂ mRNA in the rat brain (Griffin et al., 1999). One possible explanation for this controversy is based on the lack of specificity of the available CB₂r antibodies (Atwood and Mackie, 2010, Baek et al., 2013).

Nevertheless, several evidences supported the potential functional effects of CB₂r in several brain regions (Van Sickle et al., 2005, Onaivi et al., 2008, Morgan et al., 2009, García-Gutiérrez et al., 2010, Xi et al., 2011, García-Gutiérrez and Manzanares, 2011, Ortega-Álvaro et al., 2011, Aracil-Fernández et al., 2012;). Transgenic mice overexpressing CB₂r in the central nervous system (CB2xP) exhibited a clear endophenotype resistance to anxiogenic (García-Gutiérrez and Manzanares, 2011) and depressogenic-like (García-Gutiérrez et al., 2010) stimuli associated with molecular adaptations in different key elements, such as the hypothalamic–pituitary–adrenal (HPA) axis (García-Gutiérrez and Manzanares, 2011), neurotrophic factors (García-Gutiérrez et al., 2010) and the GABAergic system (García-Gutiérrez and Manzanares, 2011). Interestingly, deletion of the CB₂r gene produced behavioral alterations that are commonly expressed in preclinical animal models of schizophrenia, namely altered locomotor activity, anxiety-like and depressive-like behaviors and cognitive deficits, including impaired sensorimotor gating (Ortega-Álvaro et al., 2011).

The high expression of CB₂r in areas involved in learning and memory, such as the HIP, the close interaction between CB₂r and the GABAergic system and the cognitive alterations observed in CB2KO mice, suggests that CB₂r might be an important neurobiological substrate for cognitive processes. In the present study, we further investigated the specific function of CB₂r in learning and memory. CB2KO mice were exposed to the step-down inhibitory avoidance test (SDIA). Immunohistological analyses of MAP2, NF200 and synaptophysin (SYN) were studied in the dentate gyrus (DG) and CA1 fields of the HIP of CB2KO and WT mice. In addition, brain-derived neurotrophic factor (BDNF) and glucocorticoid receptor (NR3C1) gene expression were analyzed in the HIP of both genotypes. The protein expression of the phosphorylated p-p70S6K, the phosphorylated protein kinase B (pAKT) and the phosphorylated ribosomal protein S6 (P-S6) in the HIP and prefrontal cortex (PFC) of CB2KO were studied to establish the possible involvement of the phosphatidylinositol-3 kinase (PI3K)/mTOR pathway in the cognitive impairment produced by the deletion of CB₂r. Finally, the effects of the cannabinoid CB₂r-agonist JWH133 or cannabinoid CB₂r-antagonist AM630 on memory consolidation were evaluated in WT mice by using the SDIA.