Cocaine-induced locomotor sensitization in rats correlates with nucleus accumbens activity on manganese-enhanced MRI

Abstract

A long-standing goal of substance abuse research has been to link drug-induced behavioral outcomes with the activity of specific brain regions to understand the neurobiology of addiction behaviors and to search for drug-able targets. Here, we tested the hypothesis that cocaine produces locomotor (behavioral) sensitization that correlates with increased calcium channel-mediated neuroactivity in brain regions linked with drug addiction, such as the nucleus accumbens (NAC), anterior striatum (AST) and hippocampus, as measured using manganese-enhanced MRI (MEMRI). Rats were treated with cocaine for 5 days, followed by a 2-day drug-free period. The following day, locomotor sensitization was quantified as a metric of cocaine-induced neuroplasticity in the presence of manganese. Immediately following behavioral testing, rats were examined for changes in calcium channel-mediated neuronal activity in the NAC, AST, hippocampus and temporalis muscle, which was associated with behavioral sensitization using MEMRI. Cocaine significantly increased locomotor activity and produced behavioral sensitization compared with saline treatment of control rats. A significant increase in MEMRI signal intensity was determined in the NAC, but not AST or hippocampus, of cocaine-treated rats compared with saline-treated control rats. Cocaine did not increase signal intensity in the temporalis muscle. Notably, in support of our hypothesis, behavior was significantly and positively correlated with MEMRI signal intensity in the NAC. As neuronal uptake of manganese is regulated by calcium channels, these results indicate that MEMRI is a powerful research tool to study neuronal activity in freely behaving animals and to guide new calcium channel-based therapies for the treatment of cocaine abuse and dependence.

Keywords: behavioral sensitization; calcium; cocaine; dopamine transporter; magnetic resonance imaging; manganese; nucleus accumbens; striatum.

Figure 1. The MEMRI data analysis process and brain regions of interests are shown

(A) For the image analysis process the MPRAGE (top) and PDGE (middle) images were registered on a voxel-by-voxel basis and then the signal intensities from MPRAGE by PDGE were divided to produce the resulting ratio MPRAGE/PDGE (bottom) image. The scale bar represents mean values for that image and the units are arbitrary. (B) A sample ratio image depicting the anterior striatum (AST), nucleus accumbens (NAC), and muscle (M) regions of interest outlined in yellow is shown. (C) A sample ratio image identifying the hippocampus (Hip) and muscle (M) regions of interest outlined in yellow is shown.

Figure 2. Cocaine increases locomotor activity and induces behavioral sensitization in rats

Behavioral (locomotor) activity was measured for 30 min followed by measurement over a 3 hour period after saline or cocaine injections daily for 5 days (only 3 h of activity after injection is shown). This was followed by a 2-day drug free period, and then a challenge day given on day 8, where Mn²⁺ was injected in all animals for later MEMRI analysis and locomotor activity was measured after saline or cocaine injections over a 6 hour period. (A) Cocaine (10 mg/kg, 3 injections daily) increased locomotor activity and induced robust behavioral sensitization over the first 5 days. (B) On the challenge day (day 8) after a 2-day drug free period, cocaine (3 mg/kg, 10 injections) induced robust locomotor activity compared to saline control. N=7 rats in the cocaine group and N=8 rats in the saline group; *p<0.05, **p<0.01, or ***p<0.001 for cocaine versus saline.

Figure 3. Neurofunctional activity only in the NAC is increased upon a cocaine challenge

Cocaine (3 mg/kg, repeated) or saline was given after a 2 day drug free period that followed a 5-day binge-pattern saline or cocaine injection paradigm, and following 6 h of Mn²⁺ uptake and measurement of locomotor activity on day 8, MEMRI was conducted. Group means show that on day 8 cocaine increases signal intensity relative to controls in the (A) NAC but not (B) AST. Sample ratio (MPRAGE/PDGE) images of a (C) saline-treated and (D) cocaine-treated rat illustrate the effect of cocaine on signal intensity and Mn²⁺ uptake (i.e. MEMRI) in the NAC and AST. A pseudo-color palette was used such that more signal intensity and Mn²⁺ uptake is represented by brighter colors (and higher values on the scale to the right). N=7 rats in the cocaine group and N=8 rats in the saline group; *p<0.05 for cocaine versus saline.

Figure 4. Neurofunctional activity in the hippocampus or average temporalis muscle is not affected by a cocaine challenge

Cocaine (3 mg/kg, repeated) or saline was given after a 2 day drug free period that followed a 5-day binge-pattern saline or cocaine injection paradigm, and following 6 h of Mn²⁺ uptake and measurement of locomotor activity on day 8, MEMRI was conducted. Group means show that cocaine challenge on day 8 does not affect signal intensity in the (A) hippocampus normalized to average muscle or (B) average temporalis muscle alone. Sample ratio (MPRAGE/PDGE) images of a (C) saline-treated and (D) cocaine-treated rat illustrate the effect of cocaine on signal intensity and Mn²⁺ uptake (i.e. MEMRI) in the hippocampus and average muscle. A pseudo-color palette was used such that more signal intensity and Mn²⁺ uptake is represented by brighter colors (and higher values on the scale to the right). N=7 rats in the cocaine group and N=8 rats in the saline group.

Figure 5. A significant correlation between locomotor activity on day 8 and signal intensity exists in the NAC, but not in the AST or hippocampus, of rats administered saline or cocaine

Spearman correlation and linear regression analyses revealed that a significant relationship between locomotor activity on day 8 and MEMRI signal intensity in (A) NAC but not (B) AST or (C) hippocampus. N=15 for each brain region analysis.

Figure 6. Administration of Mn²⁺ (88 mg/kg) does not alter DAT content of striatal synaptosomal membranes

A two-tailed t-test revealed no significant difference between Mn²⁺ administration and control on DAT protein in synaptosomal membranes isolated from whole striatum. Blots of the cytosolic fraction had no detectible DAT protein bands (data not shown). N=8 per group.