Sex differences in mouse infralimbic cortex projections to the nucleus accumbens shell

Abstract

Background: The nucleus accumbens (NAc) is an important region in motivation and reward. Glutamatergic inputs from the infralimbic cortex (ILC) to the shell region of the NAc (NAcSh) have been implicated in driving the motivation to seek reward through repeated action-based behavior. While this has primarily been studied in males, observed sex differences in motivational circuitry and behavior suggest that females may be more sensitive to rewarding stimuli. These differences have been implicated for the observed vulnerability in women to substance use disorders.

Methods: We used an optogenetic self-stimulation task in addition to ex vivo electrophysiological recordings of NAcSh neurons in mouse brain slices to investigate potential sex differences in ILC-NAcSh circuitry in reward-seeking behavior. Glutamatergic neurons in the ILC were infected with an AAV delivering DNA encoding for channelrhodopsin. Entering the designated active corner of an open field arena resulted in photostimulation of the ILC terminals in the NAcSh. Self-stimulation occurred during two consecutive days of testing over three consecutive weeks: first for 10 Hz, then 20 Hz, then 30 Hz. Whole-cell recordings of medium spiny neurons in the NAcSh assessed both optogenetically evoked local field potentials and intrinsic excitability.

Results: Although both sexes learned to seek the active zone, within the first day, females entered the zone more than males, resulting in a greater amount of photostimulation. Increasing the frequency of optogenetic stimulation amplified female reward-seeking behavior. Males were less sensitive to ILC stimulation, with higher frequencies and repeated days required to increase male reward-seeking behavior. Unexpectedly, ex vivo optogenetic local field potentials in the NAcSh were greater in slices from male animals. In contrast, female medium-spiny neurons (MSNs) displayed significantly greater intrinsic neuronal excitability.

Conclusions: Taken together, these data indicate that there are sex differences in the motivated behavior driven by glutamate within the ILC-NAcSh circuit. Though glutamatergic signaling was greater in males, heightened intrinsic excitability in females appears to drive this sex difference.

Keywords: Glutamate; Infralimbic cortex; Intracranial self-stimulation; Intrinsic excitability; Motivation; Nucleus accumbens shell; Optogenetics; Reward; Sex differences; Synaptic strength.

Fig. 1

Timeline of the behavioral experiment. Seven days after arrival, mice were received viral delivery of channelrhodopsin. The fiber optic cannulae were placed on day 21. Behavioral testing began on approximately day 28 with the baseline test, followed by 10 Hz acquisition then reversal trials. 20 Hz acquisition and reversal trials, then 30 Hz acquisition and reversal trials followed spaced approximately one week apart, and for females, in accordance with the appropriate phases of the estrous cycle. Mice were perfused following the final behavioral trial

Fig. 2

AAV and fiber optic placement. A Representative image of targeted AAV injection, with cell bodies in the infralimbic cortex (ILC) expressing eYFP-ChR2. B Representative image of fiber optic cannula placement above the nucleus accumbens shell (NAcSh). Projections from the ILC are in green. Fiber optic placement (fo) indicated by the solid lines. ac, anterior commissure; ACC, anterior cingulate cortex; dPA, dorsal peduncular area; fo, fiber optic cannula; ILC, infralimbic cortex; NAcC, nucleus accumbens core; NAcSh, nucleus accumbens shell; PLC, prelimbic cortex. Scale bars indicate 500 μm

Fig. 3

Sex differences in entries made and time spent in the acquisition zone during acquisition. A At all stimulation frequencies, the total number of entries into the acquisition zone were significantly greater than the average number of entries into the inactive zones (indicated by asterisks). There were also significant differences in the number of entries made into the active zone between 10 and 20 Hz, 10 Hz and 30 Hz, and 20 Hz and 30 Hz (denoted by different letters above each bar). At 30 Hz, there were significant differences between the sexes, shown as the colored bar. B Males entered the acquisition zone significantly more than the inactive zones only at 30 Hz. Males displayed no significant differences across frequencies in the number of entries into the acquisition zone. C In females, the total time spent in the acquisition zone was greater than the time spent in the inactive zones across all frequencies. There were also significant differences in the time spent in the acquisition zone between 10 and 20 Hz, and between 10 and 30 Hz. There were sex differences in the amount of time spent in the acquisition zone at 20 Hz and 30 Hz. D Males spent more total time in the acquisition zone versus the inactive zones at all frequencies. However, there were no significant differences in the time spent in the acquisition zone between any of the stimulation frequencies. Representative track plots illustrate travel throughout the duration of the 30-Hz acquisition trial for E females and F males. The acquisition zone is indicated by the blue square. Data presented as mean ± SEM

Fig. 4

Sex differences in optogenetic stimulation during acquisition. A In females, there were significant differences in the number of optogenetic stimulation trains received during each of the three frequencies. Sex differences in the number of stimulation trains occurred at both 20 Hz and 30 Hz. B There were no differences in the number of optogenetic stimulation trains received by males across frequencies. C Across all stimulus conditions, there were significant differences in the amount of time female received optogenetic stimulation. At both 20 Hz and 30 Hz, there were also sex differences in optogenetic stimulation time. D In males, increasing stimulation frequency had no effect on the amount of time optogenetic stimulation was received

Fig. 5

No sex differences in distance traveled or travel speed during acquisition. Heightening the stimulation frequency increased the mean distance traveled in A females and B males, though there were no sex differences at any frequency. Similarly, increasing stimulation frequency also increased the travel speed in both C females and D males, again without differences between the sexes

Fig. 6

Entries made and sex differences in time spent in the reversal zone. A Females entered the reversal zone versus the previously active zone a greater number of times at 20 Hz and 30 Hz. The number of entries into the reversal zone was also greater at 30 Hz than 10 Hz. B Males entered the reversal zone significantly more only at 10 Hz and showed no differences in entries across stimulation frequencies. C Females spent more time in the reversal zone versus the acquisition zone at all three stimulus frequencies. They also spent more time in the reversal zone at 20 Hz and 30 Hz in comparison to 10 Hz. At 30 Hz, there was a sex difference in the time spent in the reversal zone. D In males, there were significant differences in the time spent in the reversal zone as compared with time spent in the acquisition at all stimulus frequencies. However, there were no differences across frequency. Representative track plots illustrate travel throughout the duration of the 30-Hz reversal trial for both E females and F males. The reversal zone is indicated by the solid blue square, and the previous acquisition zone is indicated by the dashed blue square

Fig. 7

Optogenetic stimulation during reversal trials. A In females, there were significant differences in the number of optogenetic stimulation trains received during reversal trials between 10 Hz versus 20 Hz and 30 Hz. B In males, increasing the optogenetic stimulation frequency did not increase the number of stimulations. C Increasing stimulation frequency in females resulted in differences in the mean amount of time optogenetic stimulation was received, again between 10 Hz versus 20 Hz and 30 Hz. At 30 Hz stimulation, females received significantly more optogenetic stimulation than did males. D In males, there were no significant differences in mean time optogenetic stimulation

Fig. 8

Distance traveled and travel speed during reversal trials. As with the acquisition trials, heightening the stimulation frequency increased the mean distance traveled in both A females and B males, without any differences between the sexes. Similarly, increasing frequency stimulation also increased the travel speed in C females and D males, without sex differences

Fig. 9

Males exhibit stronger ILC-NAcSh glutamatergic neurotransmission, while females display increased MSN intrinsic excitability. A Neurons in the infralimbic cortex (ILC) in both female and male mice were transfected with an eYFP-labeled AAV expressing channelrhodopsin, and the terminals in the shell of the nucleus accumbens (NAcSh) were optogenetically stimulated ex vivo. The shaded area in the enlarged box indicates recording area. Representative traces of the measured optogenetic local field potentials (oLFPs) in both female (B) and male (C) mice are presented at light stimulation durations of 1 ms (ms) (B: female, light red; C: male, light blue) and 4 ms (B female, dark red; C male, dark blue). D At all stimulus frequencies, males (blue) exhibited a significantly greater glutamatergic response (p = 0.002) than females (red). E Whole-cell current clamp recordings were measured in medium-spiny neurons (MSNs) in NAcSh. The shaded area in the enlarged box indicates recording area. Representative current clamp recordings of female (F) and male G NAcSh MSNs at 160 pA (top) and 200 pA (bottom) depolarizations are presented. Input current elicited significantly more action potentials in females than in males at 160 pA (F top) and 200 pA (F bottom), indicated by the asterisk. H Females (red) exhibited significantly greater intrinsic firing frequencies than males (blue) in response to current injection (p = 0.0489), and post hoc analysis indicated significant differences at 160 pA through 200 pA (p < 0.05). I Females (red) had significantly increased NAcSh MSN action potential firing frequency at maximum current injection (+ 220 pA) compared to males (blue) (p = 0.0435). NAcSh, nucleus accumbens shell; ac, anterior commissure; AAV, AAV adeno-associated virus; ILC infralimbic cortex

Fig. 10

Females have a lower rheobase and shorter time to initiate an action potential compared to males. A Representative ramp current injection traces for females (red, top) and males (bottom, blue). B Females (red) have a significantly lower rheobase compared to males (blue) (p = 0.005). C Females (red) have a significantly shorter time to initiate an action potential compared to males (blue) (p = 0.005). D There is no apparent sex difference in voltage threshold to firing an AP between females (red) and males (blue) (p = 0.1064)