Adolescent alcohol exposure reduces dopamine 1 receptor modulation of prelimbic neurons projecting to the nucleus accumbens and basolateral amygdala

Abstract

Binge drinking during adolescence is highly prevalent despite increasing evidence of its long-term impact on behaviors associated with modulation of behavioral flexibility by the medial prefrontal cortex (mPFC). In the present study, male and female rats underwent adolescent intermittent ethanol (AIE) exposure by vapor inhalation. After aging to adulthood, retrograde bead labelling and viral tagging were used to identify populations of neurons in the prelimbic region (PrL) of the mPFC that project to specific subcortical targets. Electrophysiological recording from bead-labelled neurons in PrL slices revealed that AIE did not alter the intrinsic excitability of PrL neurons that projected to either the NAc or the BLA. Similarly, recordings of spontaneous inhibitory and excitatory post-synaptic currents revealed no AIE-induced changes in synaptic drive onto either population of projection neurons. In contrast, AIE exposure was associated with a loss of dopamine receptor 1 (D1), but no change in dopamine receptor 2 (D2), modulation of evoked firing of both populations of projection neurons. Lastly, confocal imaging of proximal and apical dendritic tufts of viral-labelled PrL neurons that projected to the nucleus accumbens (NAc) revealed AIE did not alter the density of dendritic spines. Together, these observations provide evidence that AIE exposure results in disruption of D1 receptor modulation of PrL inputs to at least two major subcortical target regions that have been implicated in AIE-induced long-term changes in behavioral control.

Keywords: Adolescence; Alcohol; Development; Dopamine 1 receptor; Prelimbic cortex; Pyramidal neuron.

Fig. 1.

Retrobead labelling and injection location. (A) Schematic depicting the injection site of green fluorescent retrobeads in the NAc of the right hemisphere and the injection site of red fluorescent retrobeads in the NAc of the left hemisphere. (B) Representative images of neurons labeled with green retrobeads (left panel) and red retrobeads (right panel) in the PrL.

Fig. 2.

Intrinsic excitability of PrL neurons that project to the NAc (PrL^NAc). (A,B) Representative traces showing the input-output relationship between firing frequency and injected current in PrL^NAc cells in slices obtained from adult male and female Air control and AIE exposed rats. (C,D) Quantification of the evoked firing data revealed that AIE exposure did not alter the intrinsic excitability of PrL ^NAc neurons in either male or female rats. Data represent the mean ± SEM, n = 8 rats/group.

Fig. 3.

Intrinsic excitability of PrL neurons that project to the BLA (PrL ^BLA). (A,B) Representative traces showing the input-output relationship between firing frequency and injected current in PrL ^BLA cells in slices obtained from adult male and female Air control and AIE exposed rats. (C,D) Quantification of the evoked firing data revealed that AIE exposure did not alter the intrinsic excitability of PrL ^BLA neurons in either male or female rats. Data represent the mean ± SEM, n = 8 rats/group.

Fig. 4.

Dopamine receptor modulation of evoked firing of PrL neurons that project to the NAc (PrL ^NAc). (A) Representative traces showing the effects of the D1 agonist SKF 38393 (5 μM) on evoked firing of PrL ^NAc cells in slices obtained from adult male and female Air control and AIE exposed rats. (B) Representative traces showing the effects of the D2/D4 agonist quinpirole on evoked firing of PrL ^NAc cells in slices obtained from adult male and female Air control and AIE exposed rats. (C) Enhancement of evoked firing in response to bath application of SKF 38393 was significantly attenuated by AIE exposure in slices obtained from both male and female rats. (D) Inhibition of evoked firing in response to bath application of quinpirole (5 μM) was not significantly altered by AIE exposure in either male or female rats. Dashed lines indicate baseline firing. Data represent the mean ± SEM. * indicates significant difference compared to respective Air controls; p ≤ 0.05; n = 8 rats/group.

Fig. 5.

Dopamine receptor modulation of evoked firing of PrL neurons that project to the BLA (PrL ^BLA). (A) Representative traces showing the effects of the D1 agonist SKF 38393 (5 μM) on evoked firing of PrL ^BLA cells in slices obtained from adult male and female Air control and AIE exposed rats. (B) Representative traces showing the effects of the D2/D4 agonist quinpirole on evoked firing of PrL ^BLA cells in slices obtained from adult male and female Air control and AIE exposed rats. (C) Enhancement of evoked firing in response to bath application of SKF 38393 observed in the Air controls was significantly attenuated by AIE exposure in slices obtained from both male and female rats. (D) Inhibition of evoked firing in response to bath application of quinpirole (5 μM) was not significantly altered by AIE exposure in either male or female rats. Dashed lines indicate baseline firing. Data represent the mean ± SEM. **, indicates significant group difference between males versus females, p ≤ 0.01; ***, indicates significant difference compared to respective Air controls; p ≤ 0.001; n = 7-8 rats/group.

Fig. 6.

Spontaneous excitatory and inhibitory inputs to PrL neurons that project to the NAc (PrL ^NAc). (A) Representative traces of sIPSCs recorded from PrL ^NAc neurons of Air and AIE exposed adult male and female rats. (B) Representative traces of sEPSCs recorded from PrL ^NAc neurons of Air and AIE exposed adult male and female rats. When compared across treatment and sex, there were no differences in either the frequency (C) or amplitude (D) of sIPSCs. Similarly, there were no differences in either the frequency (E) or amplitude (F) of sEPSCs. (G) Examination of the sEPSC/sIPSC ratios (as an index of the excitatory-inhibitory balance of the synaptic inputs) for frequency revealed a significant AIE-induced decrease in both male and female rats. (H) There were no differences in the ratio of the current amplitudes as a function of either treatment or sex. Data represent the mean ± SEM. *, indicates significant difference compared to respective Air controls; p ≤ 0.05; n = 8 rats/group.

Fig. 7.

Spontaneous excitatory and inhibitory inputs to PrL neurons that project to the BLA (PrL ^BLA). (A) Representative traces of sIPSCs recorded from PrL BLA neurons of Air and AIE exposed adult male and female rats. (B) Representative traces of sEPSCs recorded from PrL ^BLA neurons of Air and AIE exposed adult male and female rats. There were no differences in either the frequency (C) or amplitude (D) of sIPSCs across either treatment or sex. While AIE also had no effect on either the frequency (E) or amplitude (F) of the sEPSC in either male or female rats, the amplitudes of the sEPSC were significantly reduced in female rats compared to male rats. Examination of the sEPSC/sIPSC ratios as an index of excitatory-inhibitory balance of synaptic inputs revealed no differences in either frequency (G) or amplitude (H) of the currents. Data represent the mean ± SEM. *, indicates significant group difference between males and females; p ≤ 0.05; n = 8 rats/group.

Fig. 8.

Dendritic spine morphometric analyses in PrL neurons that project to the NAc (PrL ^NAc ). (A) Surgery schematic depicting localization of retrograde Cre virus and Cre-dependent mCherry viral vector infusion sites. A pyramidal neuron is also depicted with boxes around the areas of the apical dendrite where spine segments were imaged (B,C) Representative images of proximal apical (B) and apical tuft dendritic segments (C). Top panel shows viral labeling in red with the dendrite of interest highlighted in yellow. The center panel shows the isolated dendrite pseudo colored in green. The bottom panel provides the digital spine analyses images with the shaft region shown in green and the spine region shown in blue. Dendritic spine density and head diameter data for the proximal apical segments (D) and the apical tuft segments (E). No significant differences were observed in spine density or head diameter at either the proximal or tuft regions of the apical dendrite when comparing Air (yellow circles) and AIE (blue squares) groups (unpaired t-tests). Data represent the mean ± SEM. n = 5-6 rats/group.