Chronic voluntary oral methamphetamine induces deficits in spatial learning and hippocampal protein kinase Mzeta with enhanced astrogliosis and cyclooxygenase-2 levels

Abstract

Methamphetamine (MA) is an addictive drug with neurotoxic effects on the brain producing cognitive impairment and increasing the risk for neurodegenerative disease. Research has focused largely on examining the neurochemical and behavioral deficits induced by injecting relatively high doses of MA [30 mg/kg of body weight (bw)] identifying the upper limits of MA-induced neurotoxicity. Accordingly, we have developed an appetitive mouse model of voluntary oral MA administration (VOMA) based on the consumption of a palatable sweetened oatmeal mash containing a known amount of MA. This VOMA model is useful for determining the lower limits necessary to produce neurotoxicity in the short-term and long-term as it progresses over time. We show that mice consumed on average 1.743 mg/kg bw/hour during 3 hours, and an average of 5.23 mg/kg bw/day over 28 consecutive days on a VOMA schedule. Since this consumption rate is much lower than the neurotoxic doses typically injected, we assessed the effects of long-term chronic VOMA on both spatial memory performance and on the levels of neurotoxicity in the hippocampus. Following 28 days of VOMA, mice exhibited a significant deficit in short-term spatial working memory and spatial reference learning on the radial 8-arm maze (RAM) compared to controls. This was accompanied by a significant decrease in memory markers protein kinase Mzeta (PKMζ), calcium impermeable AMPA receptor subunit GluA2, and the post-synaptic density 95 (PSD-95) protein in the hippocampus. Compared to controls, the VOMA paradigm also induced decreases in hippocampal levels of dopamine transporter (DAT) and tyrosine hydroxylase (TH), as well as increases in dopamine 1 receptor (D1R), glial fibrillary acidic protein (GFAP) and cyclooxygenase-2 (COX-2), with a decrease in prostaglandins E2 (PGE2) and D2 (PGD2). These results demonstrate that chronic VOMA reaching 146 mg/kg bw/28d induces significant hippocampal neurotoxicity. Future studies will evaluate the progression of this neurotoxic state.

Keywords: Neuroscience.

Fig. 1

Timeline for the 50d study, Average Methamphetamine (MA) Consumption and Body Weights. (A) Days 1–2: behavioral shaping on RAM. Days 5–32: VOMA for 28d. Day 37: working memory assessment. Days 40–49: cognitive flexibility assessment (spatial learning). Day 50: RAM test and tissue harvest. (B) The total amount of MA consumed over the 28 day VOMA period, grouped into 4-week averages for the Methamphetamine (Meth) group. (C) Mice were weighed during the consumption period of VOMA. No significant differences were found between the two groups when analyzed by two-way ANOVA [F_(1,32) = 1.55, p > 0.05; η²_G = 0.023, non-significant effect size].

Fig. 2

VOMA impairs spatial working memory. (A) Two-way Repeated ANOVA showed an overall effect of bait group (*** p < 0.001). Post hoc analyses reveal that both control and Meth mice performed significantly worse to retrieve baits 5–8 compared to baits 1–4 (# indicates different from control bait 1–4, p < 0.05; ! indicates different from Meth bait 1–4, p < 0.001). Baits 5–8 reveal a significant difference between groups (^ indicates different from control in same bait condition p < 0.01). (B) There was no significant difference in latency to complete the trial between the groups.

Fig. 3

VOMA impairs spatial learning. All statistical analyses involved two-way ANOVA across 10 consecutive training days (experiment days 40–49 shown on graphs) and treatment conditions. Differences between treatment conditions during asymptotic performance (training days 6–10, experiment days 45–49) were analyzed separately. (A) % Correct shows a significant effect of training days (^ p < 0.0001) and treatment conditions (# p < 0.0001). Bonferroni-corrected post hoc analyses revealed significant differences between treatment conditions on training days 8 and 9, (experiment days 47–48) (** p < 0.01; *** p < 0.001). During asymptotic performance (training days 6–10) there is a significant treatment effect (! p < 0.05). (B) Reference memory errors show a significant effect on training days (^ p < 0.01), a significant interaction (# p < 0.01), and a significant treatment effect during asymptotic performance (training days 6–10, ! p < 0.01). (C) Working memory errors show a significant effect of training days (^ p < 0.01), without a significant difference between treatment conditions except during asymptotic performance (training days 6–10, ! p < .05). (D) Latency to complete the trials show a significant effect of training days (^ p < 0.01), and a significant interaction between treatment conditions (# p < 0.01). Bonferroni post-hoc tests show a significant difference between treatment conditions on training day 1 (p<0.05).

Fig. 4

Chronic VOMA affects dopaminergic marker expression. Compared to controls, VOMA decreases cytosolic TH (*p < 0.05; A). In the synaptic fraction, D1R significantly increased (**p < 0.01; B) and DAT significantly decreased (*p < 0.05; C) compared to controls. Representative blots show control (left band) and Meth (right band) treatments. See supplementary figures for original images of blots.

Fig. 5

Chronic VOMA increases neuroinflammation marker expression. In the synaptic (A) and cytosolic (B) fractions COX-2 levels significantly increased compared to controls (^*p < 0.05). (C) Cytosolic GFAP increased compared to controls (**p < 0.01). See supplementary figures for original images of blots. (D,E). PGE2 and PGD2 significantly decreased with VOMA compared to controls (*p < 0.05; picograms/milligram). (F). PGJ2 expression did not change significantly between treatment conditions. Representative blots show control (left band) and Meth (right band) treatments.

Fig. 6

Chronic VOMA decreases memory associated marker expression. In the synaptic fraction, VOMA decreased (A) GluA2 (^**p < 0.01) and (B) PSD-95 compared to controls (^**p < 0.01). (C) In the cytosolic fraction, VOMA decreased PKMζ compared to controls (*p < 0.05). (D) VOMA did not significantly affect cytosolic PKCι/λ expression. Representative blots show control (left band) and Meth (right band) treatments. See supplementary figures for original images of blots.