Sex Differences in Neural Networks Recruited by Frontloaded Binge Alcohol Drinking

Abstract

Frontloading is an alcohol drinking pattern where intake is skewed toward the onset of access. The goal of the current study was to identify brain regions involved in frontloading. Whole brain imaging was performed in 63 C57Bl/6J (32 female and 31 male) mice that underwent 8 days of binge drinking using the drinking-in-the-dark (DID) model. On days 1-7, three hours into the dark cycle, mice received 20% (v/v) alcohol or water for two hours. Intake was measured in 1-minute bins using volumetric sippers, which facilitated analyses of drinking patterns. On day 8 mice were perfused 80 minutes into the DID session and brains were extracted. Brains were then processed to stain for Fos protein using iDISCO+. Following light sheet imaging, ClearMap2.1 was used to register brains to the Allen Brain Atlas and detect Fos+ cells. For brain network analyses, day 8 drinking patterns were used to characterize mice as frontloaders or non-frontloaders using a recently developed change-point analysis. Based on this analysis the groups were female frontloaders (n = 20), female non-frontloaders (n = 2), male frontloaders (n = 13) and male non-frontloaders (n = 8). There were no differences in total alcohol intake in animals that frontloaded versus those that did not. Only two female mice were characterized as non-frontloaders, thus preventing brain network analysis of this group. Functional correlation matrices were calculated for each group from log₁₀ Fos values. Euclidean distances were calculated from these R values and hierarchical clustering was used to determine modules (highly connected groups of brain regions). In males, alcohol access decreased modularity (3 modules in both frontloaders and non-frontloaders) as compared to water drinkers (7 modules). In females, an opposite effect was observed. Alcohol access (9 modules for frontloaders) increased modularity as compared to water drinkers (5 modules). These results suggest sex differences in how alcohol consumption reorganizes the functional architecture of neural networks. Next, key brain regions in each network were identified. Connector hubs, which primarily facilitate communication between modules, and provincial hubs, which facilitate communication within modules, were of specific interest for their important and differing roles. In males, 4 connector hubs and 17 provincial hubs were uniquely identified in frontloaders (i.e., were brain regions that did not have this status in male non-frontloaders or water drinkers). These represented a group of hindbrain regions (e.g., locus coeruleus and the pontine gray) functionally connected to striatal/cortical regions (e.g., cortical amygdalar area) by the paraventricular nucleus of the thalamus. In females, 16 connector and 17 provincial hubs were uniquely identified which were distributed across 8 of the 9 modules in the female frontloader alcohol drinker network. Only one brain region (the nucleus raphe pontis) was a connector hub in both sexes, suggesting that frontloading in males and females may be driven by different brain regions. In conclusion, alcohol consumption led to fewer, but more densely connected, groups of brain regions in males but not females, and recruited different hub brain regions between the sexes. These results suggest that alcohol frontloading leads to a reduction in network efficiency in male mice.

Figure 1.

Intakes in the first 20 minutes indicate that male (A) and female (E) frontloaders drink more in the early part of the session. Change point over days indicates earlier change points for both male (B) and female (F) frontloaders, suggesting they consume at a quicker rate than the other groups. Intake patterns on day 8 for males (C) and females (G) are displayed. There were no differences between male (D) and female (H) frontloaders and non-frontloaders in BEC when mice were sacrificed at 80 minutes on day 8. The dashed line on figures D and H represents 80 mg/dL, which is the NIAAA-defined threshold for binge drinking.

Figure 2.

Functional correlation matrices organized using the Allen Brain Atlas. Males (A-C) and females (D, E) are represented. Note that there is no non-frontloading group represented for females as there were only 2 female mice who did not frontload when brains were extracted on day 8, which inhibits the ability to make meaningful conclusions about female non-frontloaders in the current study.

Figure 3.

Correlation strength (R value) is compared across groups and sex within anatomical subdivisions. In most anatomical divisions, the strength of correlation is decreased in female frontloaders as compared to water drinkers. This was true in all divisions except for the cortical subplate, thalamus (no differences), and midbrain (where frontloading increased co-activation). Further, there were main effects of sex in all divisions except for the pallidum, with interactions of sex indicated in many divisions. These results suggest that correlation strength is altered differently between sexes, with male frontloaders showing higher R values in all anatomical divisions than female frontloaders.

Figure 4.

Hierarchical clustering of Euclidean distance matrices for each group. Modules for all groups were determined using a tree-cut height of 50%. In male water drinkers, 7 modules were identified (A). In male non-frontloaders, 3 modules were identified (B). In male frontloaders, 3 modules were identified (C). The decreased modularity in male alcohol drinking groups (i.e. non-frontloaders and frontloaders) is observed regardless of tree cut percentage height chosen (with the exception of extreme cut off values), (D). In female water drinkers, 5 modules were identified (D). In female frontloaders, 9 modules were identified (E). The increased modularity in frontloaders is observed regardless of tree cut percentage height chosen (with the exception of extreme cut off values), (D).

Figure 5.

A visualized network of functional connectivity in male water drinkers. Each brain region is a circle. The size of the circle represents the participation coefficient. The color inside the circle represents the WMDz. The color on the outside of the circle represents the module. Seven distinct modules were identified using hierarchical clustering (Figure 4A) and each is represented by a different color.

Figure 6.

A visualized network of functional connectivity in male non-frontloaders.

Figure 7.

A visualized network of functional connectivity in male frontloaders.

Figure 8.

A visualized network of functional connectivity in female water drinkers.

Figure 9.

A visualized network of functional connectivity in female frontloaders.

Figure 10.

Network cartography. Male water drinkers (A) had more identified connector hubs and non-hub connector nodes - and fewer ultra-peripheral nodes - as compared to male non-frontloaders (B) and male frontloaders (C). These results suggest that water drinking mice have a more globally connected brain network. Female water drinkers (D) and female frontloaders (E) displayed a similar breakdown of types of hubs and nodes within their respective network.

Figure 11.

Connector hubs, provincial hubs, and non-hub connector nodes which were unique to male frontloaders (A) and female frontloaders (B). Each brain region is a circle. The color inside the circle represents the role of the brain region in the male frontloader network. The color on the outside of the circle represents the module. As these brain regions were not key brain regions in male non-frontloaders or male water drinkers, they may play a unique role in alcohol frontloading.