Losing Control: Excessive Alcohol Seeking after Selective Inactivation of Cue-Responsive Neurons in the Infralimbic Cortex

Abstract

Loss of control over drinking is a key deficit in alcoholism causally associated with malfunction of the medial prefrontal cortex (mPFC), but underlying molecular and cellular mechanisms remain unclear. Cue-induced reinstatement of alcohol seeking activates a subset of mPFC neurons in rats, identified by their common expression of the activity marker cFos and comprised of both principal and interneurons. Here, we used cFos-lacZ and pCAG-lacZ transgenic rats for activity-dependent or nonselective inactivation of neurons, respectively, which by their lacZ encoded β-galactosidase activity convert the inactive prodrug Daun02 into the neurotoxin daunorubicin. We report that activity-dependent ablation of a neuronal ensemble in the infralimbic but not the prelimbic subregion induced excessive alcohol seeking. The targeted neuronal ensemble was specific for the cue-induced response because stress-induced reinstatement was not affected in these animals. Importantly, nonselective inactivation of infralimbic neurons, using pCAG-lacZ rats, was without functional consequence on the cue-induced reinstatement task. Thus, inhibitory control over alcohol seeking is exerted by distinct functional ensembles within the infralimbic cortex rather than by a general inhibitory tone of this region on the behavioral output. This indicates a high level of functional compartmentation within the rat mPFC whereat many functional ensembles could coexist and interact within the same subregion.

Significance statement: Hebb's (1949) idea of memories as being represented in local neuronal networks is supported by identification of transiently stable activity patterns within subgroups of neurons. However, it is difficult to link individual networks to specific memory tasks, for example a learned behavior. By a novel approach of activity-dependent ablation, here we identify a specific neuronal ensemble located in the infralimbic subregion of the medial prefrontal cortex that controls a seeking response for alcohol in rats. Our data demonstrate that functional output depends on specific neuronal ensembles within a given brain region rather than on the global activity of that region, which raises important questions about the interpretation of numerous earlier experiments using site-directed silencing or stimulation for elucidating brain function.

Keywords: Daun02 inactivation method; alcohol self-administration; conditioned cues; medial prefrontal cortex; neuronal ensembles; reinstatement.

Figure 1.

Experimental design and Daun02 inactivation method. A, Time line of cue-induced alcohol self-administration, extinction, and repeated reinstatement (RE1, RE2) tests. B, Principle designs of activity-dependent and nonselective neuronal inactivation by Daun02 in cFos-lacZ (top) and pCAG-lacZ (bottom) transgenic rats. Blue triangles represent lacZ-positive and activated cells, gray triangles represent nonactivated cells, and red triangles represent inactivated cells after Daun02 treatment.

Figure 2.

Activity-dependent Daun02 inactivation in cFos-lacZ rats. A, Exposure to alcohol-associated cues results in coexpression of cFos and lacZ in cFos-lacZ rats. Scale bar, 20 μm. B, Representative X-Gal staining of lacZ-positive cells in the IL region of cFos-lacZ rats after RE2. Scale bar, 300 μm. Drawing of coronal section adapted from Paxinos and Watson (1998). Quantification of lacZ-positive nuclei after RE2 showed a significant reduction in cFos-lacZ rats after Daun02 treatment compared with vehicle. **p < 0.01.

Figure 3.

Characterization of Daun02 inactivation in pCAG-lacZ rats. A, Representative image of Daun02 and Daun02 + Z-VAD-FMK injections into the mPFC of pCAG-lacZ rats. Scale bar, 300 μm. Fluorojade-B signal is visible by green fluorescence. Daun02 injections induced a high level of neurodegeneration, whereas there was less Fluorojade-B signal in the Daun02 + Z-VAD-FMK injection site. Integrated density measurement of Fluorojade-B signal in mPFC (mean ± SEM, n = 30 sections/group). The combination of Daun02 + Z-VAD-FMK significantly reduced the Fluorojade-B signal. ***p < 0.001. For detailed statistics, see Results. B, Representative images of Flurojade-B stainings are shown for cFos-LacZ rats after cue-induced reinstatement and for pCAG-LacZ rats. 2 μg Daun02 injections into the PL of pCAG-LacZ rats induced massive neurodegeneration. Daun02 infusions into cFos-lacZ rats after cue-induced reinstatement caused less, therefore specific neurodegeneration. Vehicle injections into cFos-lacZ rats caused no neurodegeneration. Scale bar, 25 μm.

Figure 4.

Effects of mPFC Daun02 inactivation on alcohol-seeking behavior. Responses at the active lever (mean + SEM) are shown during extinction (EXT) and reinstatement before (RE1) and after (RE2) Daun02 injection. Principle schemes as in Figure 1B. D2, Daun02. A, Daun02 microinjection into IL of cFos-lacZ rats after RE1 resulted in a significant increase in alcohol seeking in RE2 (n = 10–11/group). B, Nonselective IL inactivation by Daun02 in pCAG-lacZ rats (n = 7/group) was without effect on reinstatement behavior. C, The increase in alcohol seeking in the cFos-lacZ rats from A after Daun02 treatment persists for at least 2 weeks (RE 2 same as in A), RE3 at 7, and RE4 at 14 d after RE1 (n = 8–9/group). Red triangles represent cells inactivated in A. D, The cFos-lacZ animals from A were tested on stress-induced reinstatement of alcohol seeking. There was no significant difference between the groups (n = 6–8/group). Red triangles represent cells inactivated in A. Blue triangles represent newly activated cells by stress. E, Daun02 microinjection into PL of cFos-lacZ rats after RE1 has no effect on alcohol-seeking behavior. F, Microinjections of vehicle and Daun02 into IL of wild-type littermates had no effect on cue-induced reinstatement. Gray triangles represent lacZ-negative cells. *p < 0.05; **p < 0.01; ***p < 0.001. For detailed statistics, see Results.

Figure 5.

Inactive lever responses from Daun02 inactivation experiments and cannula placements. For explanation of triangles see Figures 2–4. A, Responses at the inactive lever (mean ± SEM) during extinction (EXT) and reinstatement before (RE1) and after (RE2) Daun02 injection into IL of cFos-LacZ rats. There were no significant differences between the groups during EXT, RE1, and RE2 (n = 10–11/group). B, There was also no significant difference in inactive lever presses after nonselective IL inactivation by Daun02 in pCAG-LacZ rats (n = 7/group). C, The animals from A were further tested on their reinstatement behavior. There was again no significant difference in inactive lever presses between the groups (n = 8–9/group). D, The cFos-LacZ animals from A and C were tested on a stress-induced reinstatement of alcohol-seeking task. There was no significant difference in inactive lever presses between the groups (n = 6–8/group). E, There was no significant difference in inactive lever presses between the groups in a third cohort of animals, which received PL Daun02 injections. F, There was no significant difference in inactive lever presses in cue-induced reinstatements of wild-type animals after IL vehicle and Daun02 injections. Approximate locations of the 28 gauge injection-cannula tips are indicated by small black triangles. Cannula placements were verified within the infralimbic cortex from +3.2 to +2.7 anterior to bregma and within the prelimbic cortex from +4.2 to +2.7 anterior to bregma (Paxinos and Watson, 1998).

Figure 6.

Characterization of cue-responsive neurons in the prelimbic and infralimbic cortex of cFos-lacZ rats after cue-induced reinstatement of alcohol seeking. A, Representative images of cFos and NeuN (top row), cFos and GAD67 (middle row), cFos and CaMKII (bottom row) double-labeling are shown. Scale bar, 50 μm. Arrows indicate colocalization. B, Quantification of double-labelings of the PL neurons, 12.7% were cFos-positive; 6% of the cFos-positive neurons were GAD 67-positive, and 66.6% of all cFos-positive neurons were CaMKII-positive; 11.2% of all IL neurons were cFos-positive; 8.8% of the cFos-positive neurons were GAD67-positive, and 70.6% of the cFos-positive neurons were CaMKII-positive.