Cyclohexane Inhalation Produces Long-Lasting Alterations in the Hippocampal Integrity and Reward-Seeking Behavior in the Adult Mouse

Abstract

Cyclohexane (CHX) is an organic solvent commonly used as a drug-of-abuse. This drug increases the oxidative stress and glial reactivity in the hippocampus, which suggests that this brain region is vulnerable to CHX effects. This study aimed to establish the behavioral changes and the pathological alterations that occur in the Cornu Ammonis 3 (CA3) and Dentate Gyrus (DG) after a long-lasting exposure to CHX. We exposed CD1 mice to a recreational-like dose of CHX (~ 30,000 ppm) for 30 days and explored its consequences in motor skills, reward-seeking behavior, and the CA3 and DG hippocampal subfields. Twenty-four hours after the last administration of CHX, we found a significant decrease in the number of c-Fos+ cells in the hippocampal CA3 and DG regions. This event coincided with an increased in NMDAR1 expression and apoptotic cells in the CA3 region. At day 13th without CHX, we found a persistent reduction in the number of c-Fos+ and TUNEL+ cells in DG. At both time points, the CHX-exposed mice showed a strong overexpression of neuropeptide Y (NPY) in the CA3 stratum lucidum and the hippocampal hilus. In parallel, we used an operant-based task to assess motor performance and operant conditioning learning. The behavioral analysis indicated that CHX did not modify the acquisition of operant conditioning tasks, but affected some motor skills and increased the reward-seeking behavior. Altogether, this evidence reveals that CHX exposure provokes long-lasting changes in the hippocampal subfields, induces motor impairments and increases the motivation-guided behavior. These findings can help understand the deleterious effect of CHX into the adult hippocampus and unveil its potential to trigger addiction-like behaviors.

Keywords: Apoptosis; Excitotoxicity; Hippocampus; Inhalant abuse; Motor skills; NMDAR1; Neuropeptide Y; Reward motivation; Solvent.

Fig. 1

a Representative body postures observed during seizures. b Analysis of seizure-like behavior in the group exposed to CHX. The X-axis shows each animal exposed to CHX and the Y-axis shows the incidence of seizures in each one of the 60 sessions. One bar represents a single seizure per subject, and the color of the bar indicates the latency in which the seizure occurred. We did not observed seizures before the 10th min

Fig. 2

CHX inhalation reduces the number of c-Fos-expressing cells in CA3 and DG. 24 h after the last CHX exposure, the number of c-Fos+ cells decreased significantly in the CA3 and DG regions of the CHX group (a–f). Thirteen days after the last CHX exposure, the number of c-Fos+ cells decreased in the DG granule neurons of CHX-exposed animals as compared to controls (j–l). In the CA3 region, we did not find statistically significant differences (g–i). All data are expressed as the median and interquartile range (IQR Q3−Q1); n = 5 mice per group. CA3 Cornu Ammonis, DG Dentate gyrus, CHX CHX. Circles represent outliers. Asterisks P ≤ 0.05; Mann–Whitney “U” test. Objective = × 40 (field area = 0.15 mm²). Bars = 20 µm

Fig. 3

Analysis of TUNEL-positive cells in CA3 pyramidal neurons and DG granule cells. a Confocal image of a representative TUNEL+ cell in the CA3 at 24 h. b After 24 h without the drug, the CHX group showed a significant increase in the number of TUNEL-positive cells in CA3 neurons as compared to controls. c Confocal image of a representative TUNEL+ cell in the DG at day 13th without CHX. d At day 13th without the drug, the experimental group showed a significant increase in the number of TUNEL-positive cell in the DG as compared to controls. Data are expressed as the median and interquartile range (IQR Q3−Q1); n = 5–6 mice per group. CHX CHX. Circles = outliers. Asterisks P < 0.05; Mann–Whitney “U” test. Objective × 40 (field area = 0.15 mm²). Bar = 20 µm

Fig. 4

NPY expression in the CA3 stratum lucidum and hilus. a–c 24 h after the last exposure to CHX, we found a significant increase in NPY expression in the CA3 stratum lucidum and hilus as compared to controls. d–f This NPY expression persisted in the same brain regions of the CHX group at day 13th. Data are expressed as the median and interquartile range (IQR Q3–Q1); n = 5–6 mice per group. CA3 Cornu Ammonis 3, SL stratum lucidum, CHX CHX. Asterisks P < 0.05; Mann–Whitney “U” test. Objective = × 40 (field area = 0.15 mm²). Bars = 20 µm

Fig. 5

NMDAR1 immunohistochemistry of the hippocampal CA3 region. NMDAR1 expression in the CA3 pyramidal neurons of controls (a) and the CHX group (b). Inset: higher magnifications of NMDAR1 receptors that surround neuronal bodies. (c) 24 h after CHX exposure, we observed high NMDAR1 expression in the CA3 region, which significantly decreased on day 13 (d). Data are expressed as the median and interquartile range (IQR Q3−Q1); n = 4–5 mice per group. CHX CHX. Circles = outliers. Asterisks P < 0.05; Mann–Whitney “U” test. Objective × 40 (field area = 0.15 mm²). Bars = 20 µm

Fig. 6

a Experimental design of the operant-based task used for assessing motor performance and motivation-guided behavior. The plots show the frequency of lever presses recorded during the acquisition (b) and maintenance session (c), and the maximum number of lever presses that mice were willing to do for the reward (animals’ breakpoint) (d). These three analyses did not show statistically significant differences between both groups. e, f Analysis of behavioral performance. The control group displayed a high activity level that decays rapidly as compared with CHX-exposed mice (e). However, control animals were more proficient during the first 3/4 of the assay (f). These results indicate that the CHX-exposed animals were slower but more persistent than controls in obtaining the reward. g In the analysis of the minimum inter-response time, the control group showed shorter intervals than the CHX group, suggesting that CHX induces motor impairment. Interestingly, the analysis of the activation time (h) indicates that control mice showed less activity than the drug-exposed group, suggesting that CHX increases motivation-guided behavior. PR progressive ratio, CRF continuous schedules of reinforcement, FT fixed time

Fig. 7

Hippocampal drawings that summarize the main findings observed after CHX withdrawal