Effects of Hydrocodone Overdose and Ceftriaxone on Astrocytic Glutamate Transporters and Glutamate Receptors, and Associated Signaling in Nucleus Accumbens as well as Locomotor Activity in C57/BL Mice

Abstract

Chronic opioid treatments dysregulate the glutamatergic system, inducing a hyperglutamatergic state in mesocorticolimbic brain regions. This study investigated the effects of exposure to hydrocodone overdose on locomotor activity, expression of target proteins related to the glutamatergic system, signaling kinases, and neuroinflammatory factors in the nucleus accumbens. The locomotor activity of mice was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS). CLAMS data showed that exposure to hydrocodone overdose increased locomotion activity in mice. This study tested ceftriaxone, known to upregulate major glutamate transporter 1 (GLT-1), in mice exposed to an overdose of hydrocodone. Thus, ceftriaxone normalized hydrocodone-induced hyperlocomotion activity in mice. Furthermore, exposure to hydrocodone overdose downregulated GLT-1, cystine/glutamate antiporter (xCT), and extracellular signal-regulated kinase activity (p-ERK/ERK) expression in the nucleus accumbens. However, exposure to an overdose of hydrocodone increased metabotropic glutamate receptor 5 (mGluR5), neuronal nitric oxide synthase activity (p-nNOS/nNOS), and receptor for advanced glycation end products (RAGE) expression in the nucleus accumbens. Importantly, ceftriaxone treatment attenuated hydrocodone-induced upregulation of mGluR5, p-nNOS/nNOS, and RAGE, as well as hydrocodone-induced downregulation of GLT-1, xCT, and p-ERK/ERK expression. These data demonstrated that exposure to hydrocodone overdose can cause dysregulation of the glutamatergic system, neuroinflammation, hyperlocomotion activity, and the potential therapeutic role of ceftriaxone in attenuating these effects.

Keywords: GLT-1; ceftriaxone; glutamate; opioids; xCT.

Figure 1

Timeline of the experimental procedure. CLAMS, comprehensive laboratory animal monitoring system.

Figure 2

Effects of exposure to hydrocodone overdose on locomotion activity in mice. (A) Statistical analysis revealed that x activity increased in the hydrocodone-treated group compared to the control group, while treatment with ceftriaxone (200 mg/kg, i.p.) reduced x activity compared to the hydrocodone-treated group. (B) Statistical analysis demonstrated that x ambulatory increased in the hydrocodone-treated group compared to the control group, and there was no significant difference in x ambulatory in the hydrocodone–ceftriaxone group compared to the hydrocodone-treated group. (C) Statistical analysis revealed that z activity increased in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg/kg, i.p.) reduced z activity compared to the hydrocodone-treated group. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8/group), (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). Hyd, hydrocodone.

Figure 3

Effects of exposure to hydrocodone overdose on GLT-1, xCT, and mGluR5 expression in the NAc. (A) Western blots for GLT-1 and β-tubulin in the NAc. One-way ANOVA followed by the Newman–Keuls multiple comparisons test showed downregulation of GLT-1 expression in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg/kg, i.p.) normalized GLT-1 expression in the NAc compared to the hydrocodone-treated group. (B) One-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed downregulation of xCT expression in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg/kg, i.p.) normalized xCT expression in the NAc compared to the hydrocodone-treated group. (C) One-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed upregulation of mGluR5 expression in the hydrocodone-treated group compared to the control group, and ceftriaxone treatment (200 mg/kg, i.p.) attenuated this effect. There was also a significant difference between the control and hydrocodone–ceftriaxone-treated groups in the expression of mGluR5 in the NAc. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8/group), (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). GLT-1, glutamate transporter 1; xCT, cystine/glutamate antiporter; mGluR5, metabotropic glutamate receptor subtype 5; Hyd, hydrocodone.

Figure 4

Effects of exposure to hydrocodone overdose on the expression of nNOS and ERK in the NAc. (A) Western blots for p-nNOS and NOS in the NAc. One-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed that hydrocodone exposure increased nNOS expression compared to the control group, and ceftriaxone treatment (200 mg/kg, i.p.) decreased nNOS expression in the NAc compared to the hydrocodone group. (B) Western blots for p-ERK and ERK in the NAc. One-way ANOVA followed by the Newman–Keuls multiple comparisons test revealed that hydrocodone exposure downregulated ERK expression compared to the control group, and ceftriaxone treatment (200 mg/kg, i.p.) upregulated ERK expression in the NAc compared to the hydrocodone group. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8/group), (* p < 0.05, *** p < 0.001 and **** p < 0.0001). nNOS, neuronal nitric oxide synthase; ERK, extracellular signal-regulated kinases; Hyd, hydrocodone.

Figure 5

Effects of exposure to hydrocodone overdose on RAGE expression in the NAc. One-way ANOVA followed by the Newman–Keuls multiple comparisons test showed that RAGE expression was upregulated in the hydrocodone group compared to the control group, while ceftriaxone (200 mg/kg) downregulated RAGE expression in the NAc compared to the hydrocodone group. Data from the control group are represented as 100%. Each column is expressed as mean ± S.E.M (n = 7–8/group), (* p < 0.05). RAGE, receptor for advanced glycation end products; Hyd, hydrocodone.

Figure 6

Schematic representation summarizing the effects of exposure to hydrocodone overdose on the mGluR5-nNOS-ERK pathway and GLT-1, xCT, and RAGE expression in the NAc. Exposure to hydrocodone overdose may increase synaptic glutamate release, resulting in an increase in extracellular glutamate concentrations. Under a hyper-glutamatergic state, mGluR5 and NMDAR are overstimulated, thereby increasing intracellular calcium and subsequently upregulating nNOS activity. Activation of nNOS activity can lead to inhibition of the downstream ERK signaling pathway. Additionally, exposure to hydrocodone overdose is associated with an increase in the inflammatory response, such as upregulation of RAGE. Ceftriaxone treatment attenuates hydrocodone-induced mGluR5-nNOS-ERK pathway activation, glutamatergic system dysregulation, and RAGE upregulation. (Blue arrows indicate the downstream pathways; Red arrows indicate upregulation or downregulation of all target proteins or markers).