Behavioral and Molecular Responses to Exogenous Cannabinoids During Pentylenetetrazol-Induced Convulsions in Male and Female Rats

Abstract

Epilepsy is a disabling, chronic brain disease,affecting ~1% of the World's population, characterized by recurrent seizures (sudden, uncontrolled brain activity), which may manifest with motor symptoms (e.g., convulsions) or non-motor symptoms. Temporal lobe epilepsies (TLE) compromising the hippocampus are the most common form of focal epilepsies. Resistance in ~1/3 of epileptic patients to the first line of treatment, i.e., antiepileptic drugs (AEDs), has been an important motivation to seek alternative treatments. Among these, the plant Cannabis sativa (commonly known as marihuana) or compounds extracted from it (cannabinoids) have gained widespread popularity. Moreover, sex differences have been proposed in epilepsy syndromes and in cannabinoid action. In the hippocampus, cannabinoids interact with the CB1R receptor whose membrane levels are regulated by β-Arrestin2, a protein that promotes its endocytosis and causes its downregulation. In this article, we evaluate the modulatory role of WIN 55,212-2 (WIN), a synthetic exogenous cannabinoid on behavioral convulsions and on the levels of CB1R and β-Arrestin2 in female and male adolescent rats after a single injection of the proconvulsant pentylenetetrazol (PTZ). As epilepsies can have a considerable impact on synaptic proteins that regulate neuronal toxicity, plasticity, and cognition, we also measured the levels of key proteins markers of excitatory synapses, in order to examine whether exogenous cannabinoids may prevent such pathologic changes after acute seizures. We found that the exogenous administration of WIN prevented convulsions of medium severity in females and males and increased the levels of phosphorylated CaMKII in the hippocampus. Furthermore, we observed a higher degree of colocalization between CB1R and β-Arrestin2 in the granule cell layer.

Keywords: beta arrestin-2; cannabinoids; dentate gyrus; epilepsy; hippocampus; seizures.

Figure 1

Effect of WIN on PTZ-induced convulsions. (A–E) Seizure duration in females (PTZ: n = 16 for stages 1, 3, 4, 5; and n = 17 for stage 6. WIN + PTZ: n = 8, 10, 11, 12, 11 for stages 1, 3, 4, 5, 6, respectively). (F) Number of times that female rats reach stage 4 in the first 500 s (PTZ n = 9, WIN + PTZ, n = 8). (F–J) Seizure duration in males (PTZ: n = 7 for all stages. WIN + PTZ: n = 5 for stages 1, 3, 5, 6; n = 4 for stage 4). (K) Number of times that male rats reach stage 4 in the first 500 s (PTZ n = 7, WIN + PTZ, n = 5). ^*p < 0.05, t-test.

Figure 2

Latency to epileptic crisis at stages of increasing severity. (A–E) Latency in females (PTZ: n = 16 for stages 1 and 4, n = 5 for stage 3, n = 14 for stage 5, n = 13 for stage 6. WIN + PTZ: n = 12, 11, 9, and 8 for stages 1, 4, 5, 6, respectively, n = 3 for stage 3). (F–J) Latency to epileptic crisis at stages of increasing severity in males (PTZ: n = 7 stages 1 and 4; n = 4 for stage 3, n = 5 for stages 5 and 6. WIN + PTZ: n = 5 for stage 1, n = 2 for stage 3, n = 3 for stages 4, 5, and 6).

Figure 3

Expression levels of membrane-bound CB1R and AMPAR subunit GluA2 in females and males. (A) Representative Western blots of membrane-bound CB1R and GluA2, in females and (D) males. (B) Quantification of membrane-bound CB1R levels in females (C, n = 5; PTZ, n = 8; WIN, n = 5; WIN + PTZ, n = 6). (C) Quantification of membrane-bound GluA2 levels in females (C, n = 5; PTZ, n = 8; WIN, n = 5; WIN + PTZ, n = 7). (E) Quantification of membrane-bound CB1R levels in males (C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5). (F) Quantification of membrane-bound GluA2 levels in males (C, n = 4; PTZ, n = 6; WIN, n = 4; WIN + PTZ, n = 5). N-cadherin was used as protein loading control for all gels.

Figure 4

Expression levels of proteins obtained from a fraction enriched in cytoplasmic proteins/proteins bound to small organelles. (A) Representative Western blots of CB1R, GluA2R, βArr2, PSD95, p-CaMKII and total CaMKII in females and (I) males. (B–H) Quantification of (A) (Females. CB1R: C, n = 5; PTZ, n = 8; WIN, n = 4; WIN + PTZ, n = 6; GluA2: C, n = 5; PTZ, n = 9; WIN, n = 5; WIN + PTZ, n = 6; βArr2: C, n = 5; PTZ, n = 9; WIN, n = 5; WIN + PTZ, n = 6; PSD95: C, n = 5; PTZ, n = 8; WIN, n = 5; WIN + PTZ, n = 6, p-CaMKIIα: C, n = 5; PTZ, n = 8; WIN, n = 4; WIN + PTZ, n = 5; p-CaMKIIβ: C, n = 5; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5; total CaMKII: C, n = 3; PTZ, n = 4; WIN, n = 3; WIN + PTZ, n = 3). (J–P) Quantification of (D) (Males. CB1R: C, n = 4; PTZ, n = 6; WIN, n = 3; WIN + PTZ, n = 5; GluA2: C, n = 4; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5; βArr2: C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5; PSD95: C, n = 4; PTZ, n = 6; WIN, n = 4; WIN + PTZ, n = 5, p-CaMKIIα: C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5; p-CaMKIIβ: C, n = 4; PTZ, n = 5; WIN, n = 4; WIN + PTZ, n = 5; total CaMKII: C, n = 3; PTZ, n = 4; WIN, n = 3; WIN + PTZ, n = 3). Tubulin was used as protein loading control for all gels. ^*p < 0.05 and ^**p < 0.01, one-way ANOVA with Tukey’s multiple comparisons post hoc.

Figure 5

Hilar and granule celllayer immunohistochemistry in female rats. (A)Representative immunofluorescent images of the dentate gyrus showingthe granule cell layer (Gr) and the hilus (H) for each experimental group. Dashed square: areas used for colocalization analysis. Calibration bar: 40 μm. (B) Representative images used for analysis [amplified view of dashed square region from (A)]. Calibration bar: 25 μm. (C–L) quantification of (B). (C) CB1R + (C, n = 6; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5) and (D) βArr2+ (C, n = 5; PTZ, n = 7; WIN, n = 4; WIN + PTZ, n = 3) area in the hilus. (E) GluA2-CB1R (C, n = 5; PTZ, n = 5; WIN, n = 3; WIN + PTZ, n = 5), (F) GluA2-βArr2 (C, n = 5; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5) and (G) CB1R-βArr2 (C, n = 5; PTZ, n = 7; WIN, n = 4; WIN + PTZ, n = 4) colocalization in hilus. (H) CAS3+ neurons (C, n = 4; PTZ, n = 5; WIN, n = 3; WIN + PTZ, n = 4) in hilus. (I) GluA2-CB1R (C, n = 5; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5), (J) GluA2-βArr2 (C, n = 5; PTZ, n = 7; WIN, n = 5; WIN + PTZ, n = 5), and (K) CB1R-βArr2 (C, n = 5; PTZ, n = 6; WIN, n = 4; WIN + PTZ, n = 3) colocalization in the granular cell layer. (L) CAS3+ neurons (C, n = 4; PTZ, n = 5; WIN, n = 3; WIN + PTZ, n = 4) in granular cell layer. White arrows: examples of strong colocalization between CB1R and βArr2. Green arrows: example of CAS3+ cells. ^*p < 0.05 and ^**p < 0.01, one-way ANOVA with Tukey’s multiple comparisons post hoc.