Overexpression of 5-HT(1B) mRNA in nucleus accumbens shell projection neurons differentially affects microarchitecture of initiation and maintenance of ethanol consumption

Abstract

Serotonin 1B (5-HT(1B)) heteroreceptors on nucleus accumbens shell (NAcSh) projection neurons have been shown to enhance the voluntary consumption of alcohol by rats, presumably by modulating the activity of the mesolimbic reward pathway. The present study examined whether increasing 5-HT(1B) receptors expressed on NAcSh projection neurons by means of virus-mediated gene transfer enhances ethanol consumption during the initiation or maintenance phase of drinking and alters the temporal pattern of drinking behavior. Animals received stereotaxic injections of viral vectors expressing either 5-HT(1B) receptor and green fluorescent protein (GFP) or GFP alone. Home cages equipped with a three-bottle (water and 6 and 12% ethanol) lickometer system recorded animals' drinking behaviors continuously, capturing either initiation or maintenance of drinking behavior patterns. Overexpression of 5-HT(1B) receptors during initiation increased consumption of 12% ethanol during both forced-access and free-choice consumption. There was a shift in drinking pattern for 6% ethanol with an increase in number of drinking bouts per day, although the total number of drinking bouts for 12% ethanol was not different. Finally, increased 5-HT(1B) expression induced more bouts with very high-frequency licking from the ethanol bottle sippers. During the maintenance phase of drinking, there were no differences between groups in total volume of ethanol consumed; however, there was a shift toward drinking bouts of longer duration, especially for 12% ethanol. This suggests that during maintenance drinking, increased 5-HT(1B) receptors facilitate longer drinking bouts of more modest volumes. Taken together, these results indicate that 5-HT(1B) receptors expressed on NAcSh projection neurons facilitate ethanol drinking, with different effects during initiation and maintenance of ethanol-drinking behavior.

Figure 1

Schematic of experimental designs. Boxes indicate surgical injection days; bars above indicate training days; shaded areas indicate testing days. (A) This experiment assessed the effects of 5-HT_1B receptor overexpression on the initiation of ethanol drinking. Viral vectors were injected on day 1. Animals had forced ethanol exposure (6% and 12%) on days 2–4 and free-choice drinking (water, 6%, and 12%) on days 5–11. (B) This experiment determined the effect of 5-HT_1B receptor overexpression on maintenance of ethanol drinking. Animals had forced ethanol exposure (6% and 12%) on days 1–3. Viral vectors were injected on day 4. Days 5–10 were free choice drinking (water, 6%, and 12%). (C) This experiment assessed the effects of 5-HT_1B receptor overexpression on sucrose solution intake. Animals were drink trained on days 1–3. Viral vectors were injected on day 4. Drinking preference was tested on days 5–9. (D) This experiment assessed the effects of 5-HT_1B receptor overexpression on saccharin solution intake. Animals were trained on days 1 and 2. Virus was injected on day 3. Animals rested on days 4–6. Drinking preference was tested on day 7. (E) This experiment assessed 5-HT_1B receptor overexpression on baseline locomotion. Viral vectors were injected on day 1 and locomotor behavior was recorded on days 2–5.

Figure 2

NAcSh injection site. (A) A schematic diagram that includes the NAcSh target is shown in figure 2A. Figures 2B and C are a representative section showing GFP fluorescence in the NAcSh; 2B is magnified by a factor of 4X, while 2C is magnified by a factor of 10X. pHSV/GFP signal is present in both cell bodies and processes.

Figure 3

Consumption of ethanol and drinking patterns during initiation drinking. (A) Drinking volume data from generalized estimating equations were back transformed to yield ethanol intake ratios (See analysis and statistics section), which revealed that pHSV-HA1B/GFP animals drank almost twice as much 12% ethanol during both forced and free access drinking, compared to pHSV-GFP animals. There were no significant differences between groups in total average licks per day on ethanol sippers (B). The number of bouts per day, as well as overall average bouts for 6% are depicted in C. pHSV-HA1B/GFP animals had significantly increased bouts for 6% ethanol, compared to pHSV-GFP animals. (D) There were no differences in bouts for 12% ethanol. (E) Representative 48-hour period of circadian activity shows the number of bouts on 6% and 12% ethanol sippers (combined) per 2 hours. Dark bars denote lights-off period. Data are presented as mean ± SEM. * = p<0.05, between groups.

Figure 4

Consumption of ethanol and drinking patterns during maintenance drinking. (A) Drinking volume data from generalized estimating equations were back transformed to yield ethanol intake ratios (See analysis and statistics section), which revealed no significant differences between groups during either forced or free access to ethanol. (B) There was a main effect of treatment on number of licks, with pHSV-HA1B/GFP animals having increased total licks on the 12% ethanol sipper. (C) There were no differences in number of bouts per day or overall average bouts for 6% ethanol. (D) pHSV-HA1B/GFP animals significantly increased bouts on 12% ethanol sippers, compared to pHSV-GFP animals. (E) Representative 48-hour period of circadian activity shows the number of bouts on 6% and 12% ethanol sippers (combined) per 2 hours. Dark bars denote lights-off period. Data are presented as mean ± SEM. * = p<0.05, between groups.

Figure 5

Sucrose and saccharin preference after pHSV-HA1B/GFP or pHSV-GFP injection. There were no significant differences between pHSV-HA1B/GFP and pHSV-GFP groups in preference for either sucrose or saccharin solutions. Data are presented as mean ± SEM.

Figure 6

Locomotor behavior after pHSV-HA1B/GFP or pHSV-GFP injection. There were no significant differences between pHSV-HA1B/GFP and pHSV-GFP groups in fine motor movements, ambulation, or total locomotion. Data are presented as mean ± SEM.