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. 2021 Apr 13;7(4):e06730.
doi: 10.1016/j.heliyon.2021.e06730. eCollection 2021 Apr.

Effects of prenatal synthetic cannabinoid exposure on the cerebellum of adolescent rat offspring

Priyanka D Pinky  1   2 , Mohammed Majrashi  1 , Ayaka Fujihashi  1 , Jenna Bloemer  1   3 , Manoj Govindarajulu  1   2 , Sindhu Ramesh  1   2 , Miranda N Reed  1   2 , Timothy Moore  1   2 , Vishnu Suppiramaniam  1   2 , Muralikrishnan Dhanasekaran  1   2
Affiliations

Effects of prenatal synthetic cannabinoid exposure on the cerebellum of adolescent rat offspring

Priyanka D Pinky et al. Heliyon. .

Abstract

Cannabis is the most commonly used illicit drug worldwide. Recently, cannabis use among young pregnant women has greatly increased. However, prenatal cannabinoid exposure leads to long-lasting cognitive, motor, and behavioral deficits in the offspring and alterations in neural circuitry through various mechanisms. Although these effects have been studied in the hippocampus, the effects of prenatal cannabinoid exposure on the cerebellum are not well elucidated. The cerebellum plays an important role in balance and motor control, as well as cognitive functions such as attention, language, and procedural memories. The aim of this study was to investigate the effects of prenatal cannabinoid exposure on the cerebellum of adolescent offspring. Pregnant rats were treated with synthetic cannabinoid agonist WIN55,212-2, and the offspring were evaluated for various cerebellar markers of oxidative stress, mitochondrial function, and apoptosis. Additionally, signaling proteins associated with glutamate dependent synaptic plasticity were examined. Administration of WIN55,212-2 during pregnancy altered markers of oxidative stress by significantly reducing oxidative stress and nitrite content. Mitochondrial Complex I and Complex IV activities were also enhanced following prenatal cannabinoid exposure. With regard to apoptosis, pP38 levels were significantly increased, and proapoptotic factor caspase-3 activity, pERK, and pJNK levels were significantly decreased. CB1R and GluA1 levels remained unchanged; however, GluN2A was significantly reduced. There was a significant decrease in MAO activity although tyrosine hydroxylase activity was unaltered. Our study indicates that the effects of prenatal cannabinoid exposure on the cerebellum are unique compared to other brain regions by enhancing mitochondrial function and promoting neuronal survival. Further studies are required to evaluate the mechanisms by which prenatal cannabinoid exposure alters cerebellar processes and the impact of these alterations on behavior.

Keywords: Cannabinoid; Cerebellum; Developmental; Mitochondria; Oxidative stress; Prenatal exposure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of prenatal cannabinoid exposure on ROS, lipid peroxide, and nitrite content in the cerebellum: (A) Significant reduction in the oxidative stress level measured by ROS generation (p = .05) (B) significant reduction in cerebellar lipid peroxide content in prenatally cannabinoid exposed offspring (p = .04) (C) Nitrite content was significantly reduced in prenatally cannabinoid exposed group (p = .02). Results are expressed as Mean ± SEM, n = 4–5 rats per group. ∗ indicates a significant difference when p ≤ .05. Two tailed student's T test.
Figure 2
Figure 2
Effect of prenatal cannabinoid exposure on complex I and complex IV activity: (A) significant increase in complex I activity in prenatally WIN55,212-2 exposed group (p = .03). (B) Complex IV activity has also increased in WIN55,212-2 exposed group (p = .03). Results are expressed as Mean ± SEM, n = 4 rats per group. ∗ indicates a significant difference when p ≤ .05. Two tailed student's T test.
Figure 3
Figure 3
Effect of prenatal cannabinoid exposure on apoptotic markers: (A) Prenatal cannabinoid exposure did not cause any alteration in the caspase 1 activity (p > .05). (B) Caspase-3 activity was significantly reduced in the WIN55,212-2 exposed group. Representative immunoblots showing (C) pERK/ERK (p = .01), (D) pJNK/JNK (p = .02), (E) pP38/P38 (p = .05). relative densities. Results are expressed as Mean ± SEM, n = 3–4 rats per group. ∗ indicates a significant difference when p ≤ .05. Two tailed student's T test. Refer Supplementary material Figure 3.
Figure 4
Figure 4
Effect of prenatal cannabinoid exposure on cerebellar signaling molecules associated with cannabinoid and glutamatergic neurotransmission: Representative immunoblots showing (A) CB1/GAPDH (p > .05), (B) GluA1/GAPDH (p > .05) and (C) GluN2A/GAPDH relative expression (p = .04) Results are expressed as Mean ± SEM, n = 3–4 rats per group. ∗ indicates a significant difference when p ≤ 0.05. Two tailed student's T test. Refer Supplementary material Figure 4.
Figure 5
Figure 5
Effect of prenatal cannabinoid exposure on MAO and tyrosine hydroxylase activity: (A) MAO activity was significantly reduced in prenatally cannabinoid exposed group (p = 0.02). (B) No significant change in the tyrosine hydroxylase content in between the groups (p > .05). Results are expressed as (%) change as Mean ± SEM. n = 4 rats per group. ∗ indicates a significant difference when p ≤ .05. Two tailed student's T test.
Figure 6
Figure 6
Effect of prenatal cannabinoid exposure on cerebellar signaling molecules associated with markers of excitotoxicity and synaptic plasticity: Representative immunoblots showing pAKT/AKT, pGSK3β/GSK3β, ILK/Actin/GAPDH, relative expression. (A) No change in the phosphorylation of AKT in response to prenatal cannabinoid exposure (p > .05) (B) No change in the phosphorylation of GSK3β at Serine-9 between the two groups (p > .05). (C) No change in ILK expression in the cerebellum in response to prenatal cannabinoid exposure (p > .05). Results are expressed as Mean ± SEM. n = 3–4 rats per group. ∗ indicates a significant difference when p ≤ .05. Two tailed student's T test. Refer Supplementary material Figure 6.
Figure 7
Figure 7
Effect of prenatal cannabinoid exposure on the developing cerebellum: Prenatal cannabinoid exposure results in alteration of phosphorylation of several molecules i.e. ERK, JNK, P38. It can also increase complex I and complex IV activity accompanied with reduction in caspase 3 activity and lipid peroxidation content demonstrating altered mitochondrial function. This figure was produced using Servier Medical Art (https://smart.servier.com/) and Library of science and medical Illustrations (http://www.somersault1824.com/science-illustrations/).
GAPDH P38 Figure3E
GAPDH P38 Figure3E
GAPDH p P38 Figure3E
GAPDH p P38 Figure3E
JNK Figure3D
JNK Figure3D
P38 Figure 3E
P38 Figure 3E
pJNK Figure3D
pJNK Figure3D
pP38 Figure 3E
pP38 Figure 3E
GAPDH JNK Figure3D
GAPDH JNK Figure3D
GAPDH ERK Figure3C
GAPDH ERK Figure3C
GAPDH pERK Figure3C
GAPDH pERK Figure3C
GAPDH pJNK Figure3D
GAPDH pJNK Figure3D
pERK Figure3C
pERK Figure3C
ERK Figure3C
ERK Figure3C
CB1 figure 4A
CB1 figure 4A
GAPDH Figure 4A
GAPDH Figure 4A
GAPDH Figure 4C
GAPDH Figure 4C
GluA1 Figure 4B
GluA1 Figure 4B
GluN2A Figure 4C
GluN2A Figure 4C
AKT Figure 6A
AKT Figure 6A
GAPDH AKT Figure6A
GAPDH AKT Figure6A
GAPDH Figure 6C
GAPDH Figure 6C
GAPDH pAKT Figure 6A
GAPDH pAKT Figure 6A
ILK Figure 6C
ILK Figure 6C
GAPDH AKT Figure6A
GAPDH AKT Figure6A
p GSK3B Figure 6B
p GSK3B Figure 6B
pAKT Figure 6A
pAKT Figure 6A
GSK3B Figure 6B
GSK3B Figure 6B
beta acin GSK3B Figure 6B
beta acin GSK3B Figure 6B
beta acin pGSK3B Figure 6B
beta acin pGSK3B Figure 6B
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