Paul G. Mermelstein

Background: Cocaine-induced changes in nucleus accumbens shell (NAcSh) medium spiny neurons (MSNs) differ based on dopamine receptor subtype expression, the sex of the animal, and for females, phase of the estrous cycle. These findings highlight the need to account for both sex and estrous cycle when studying drug-mediated alterations in neurophysiology. Whether MSNs of the nucleus accumbens core (NAcC), which serve different aspects of reward function, will exhibit similar sex and estrous cycle effects with cocaine administration was investigated. Methods: Mice underwent a 5-day locomotor sensitization paradigm via daily cocaine administration (15 mg/kg, s.c.) followed by a 1- to 4-day drug-free abstinence period. We examined NAcC MSN excitability by obtaining ex vivo whole-cell recordings from differentially labeled dopamine D1-receptor expressing MSNs (D1R-MSNs) and dopamine D2-receptor expressing MSNs (D2R-MSNs) obtained from male mice or female mice that were either in estrus or diestrus. Results: In this mouse strain, male and female mice sensitized to cocaine to a similar degree. In males, there were no cocaine-induced changes in NAcC D1R-MSN or D2R-MSN excitability. When comparing MSN subtypes, D2R-MSNs exhibited greater excitability. In saline-treated females, D1R-MSN excitability fluctuated across the estrous cycle with increased excitability during estrus. Following cocaine, estrous cycle-dependent D1R-MSN excitability was arrested, fixed at an intermediate value between estrus and diestrus when compared to saline controls. D2R-MSNs did not change across the estrous cycle or following cocaine. When comparing MSN subtypes, in diestrus, D2R-MSNs were more excitable under saline conditions, but indistinguishable from D1R-MSNs following cocaine. In contrast, during estrus, D1R- was indistinguishable from D2R-MSN excitability in saline treated animals, but with cocaine, D2R-MSNs displayed heightened excitability. Conclusions: There are fundamental sex differences in cocaine-induced changes to the excitability of D1R-MSNs in the NAcC. After cocaine exposure, female mice in diestrus saw a significant main effect change in MSN excitability, an inversion of what had previously been demonstrated in the NAcSh. These data suggest that there are fundamental sex differences in the neuropharmacological effect of cocaine in males versus females that are shell- and core-specific. Highlights: There are sex- and estrous-cycle dependent changes to D1R-MSNs in the NAcC that are sensitive to cocaine exposure. In males, cocaine has no effect on altering D1R- or D2R- MSNs excitability. During the estrous cycle, D1R-MSNs exhibit increased excitability during estrus. This fluctuation is halted by cocaine, such that D1R-MSNs recorded in diestrus show increased excitability following cocaine exposure whereas female D1R-MSNs recorded in estrus have decreased excitability. Plain language summary: The nucleus accumbens core (NAcC) is a brain region associated with regulating motivated behavior. The primary neuronal populations of the NAcC are dopamine D1 receptor expressing medium spiny neurons (D1R-MSNs) and dopamine D2 receptor expressing medium spiny neurons (D2R-MSNs). No studies exist which examine sex differences and estrous cycle effects in the NAcC following cocaine administration. Using ex vivo electrophysiology, we found inherent sex- and estrous-cycle differences in cocaine-induced MSN neuroplasticity. Following cocaine exposure, D1R-MSN excitability was unaffected in males, increased in females recorded during the diestrus phase, and decreased in females recorded during estrus. This ran counter to estrous cycle effects under drug-naive conditions where D1R-MSN excitability was higher in estrus versus diestrus. The estrous cycle effects on D1R-MSNs were eliminated following cocaine administration. For both sexes, D2R-MSN excitability was not impacted following cocaine. These results highlight fundamental sex differences in neurophysiology that might underpin differences in addiction. Graphical

Accumulating evidence has revealed the existence of functional astrocyte-neuron communication based on the ability of astrocytes to respond to neurotransmitters and release gliotransmitters. However, little is known about how other signaling molecules, such as hormones, impact astrocyte function. Estradiol (E2) is an important hormone known to regulate neuronal activity, synaptic transmission, plasticity, and animal behavior. However, whether E2 specifically signals to astrocytes in situ and the functional consequences on astrocyte-neuron communication remain unknown. Therefore, we investigated the impact of estradiol on astrocyte activity and astrocyte-neuron communication in the mouse hippocampus. Using an RNAscope approach, we determined that estrogen receptors (ERα and ERβ) are expressed in astrocytes in both female and male mice. In both sexes, confocal imaging of hippocampal slices determined that astrocytes respond to locally applied E2 with calcium elevations. In pyramidal neurons, slow inward currents (SICs) are mediated by the activation of extrasynaptic NMDA receptors and indicate gliotransmission. Electrophysiological recordings of hippocampal neurons determined that E2 increases the frequency, but not the amplitude, of SICs. We also recorded excitatory synaptic transmission evoked by Schaffer collateral stimulation. Here, only in females, did E2 produce a reduction in excitatory synaptic transmission. The E2-induced effects on the astrocyte calcium signal and gliotransmission were prevented by the broad estrogen receptor antagonist ICI 182,780. Taken together, these results demonstrate the existence of estradiol-mediated astrocyte-neuron communication in both female and male mice. They reveal that E2 can signal to astrocytes and, through this signaling, E2 may regulate neuronal activity and synaptic transmission.

Cholinergic interneurons (ChIs) act as master regulators of striatal output, finely tuning neurotransmission to control motivated behaviors. ChIs are a cellular target of many peptide and hormonal neuromodulators, including corticotropin releasing factor, opioids, insulin and leptin, which can influence an animal’s behavior by signaling stress, pleasure, pain and nutritional status. However, little is known about how sex hormones via estrogen receptors influence the function of these other neuromodulators. Here, we performed in situ hybridization on mouse striatal tissue to characterize the effect of sex and sex hormones on choline acetyltransferase ( Chat ), estrogen receptor alpha ( Esr1 ), and corticotropin releasing factor type 1 receptor ( Crhr1 ) expression. Although we did not detect sex differences in ChAT protein levels in the striatum, we found that female mice have more Chat mRNA-expressing neurons than males. At the population level, we observed a sexually dimorphic distribution of Esr1 - and Crhr1 -expressing ChIs in the ventral striatum that demonstrates an antagonistic correlational relationship, which is abolished by ovariectomy. Only in the NAc did we find a significant population of ChIs that co-express Crhr1 and Esr1 . At the cellular level, Crhr1 and Esr1 transcript levels were negatively correlated only during estrus, indicating that changes in sex hormones levels can modulate the interaction between Crhr1 and Esr1 mRNA levels. Together, these data provide evidence for the unique expression and interaction of Esr1 and Crhr1 in ventral striatal ChIs, warranting further investigation into how these transcriptomic patterns might underlie important functions for ChIs at the intersection of stress and reproductive behaviors.

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.

Recent studies have implicated the ethanol metabolite, acetic acid, as neuroactive, perhaps even more so than ethanol itself. In this study, we investigated sex-specific metabolism of ethanol (1, 2, and 4 g/kg) to acetic acid in vivo to guide electrophysiology experiments in the accumbens shell (NAcSh), a key node in the mammalian reward circuit. There was a sex-dependent difference in serum acetate production, quantified via ion chromatography only at the lowest dose of ethanol (males > females). Ex vivo electrophysiology recordings of NAcSh medium spiny neurons (MSN) in brain slices demonstrated that physiological concentrations of acetic acid (2 mM and 4 mM) increased NAcSh MSN excitability in both sexes. N-methyl-D-aspartate receptor (NMDAR) antagonists, AP5 and memantine, robustly attenuated the acetic acid-induced increase in excitability. Acetic acid-induced NMDAR-dependent inward currents were greater in females compared to males and were not estrous cycle dependent. These findings suggest a novel NMDAR-dependent mechanism by which the ethanol metabolite, acetic acid, may influence neurophysiological effects in a key reward circuit in the brain from ethanol consumption. Furthermore, these findings also highlight a specific sex-dependent sensitivity in females to acetic acid-NMDAR interactions. This may underlie their more rapid advancement to alcohol use disorder and increased risk of alcohol related neurodegeneration compared to males.

Cocaine-induced plasticity in the nucleus accumbens shell of males occurs primarily in D1 dopamine receptor expressing neurons (D1-MSNs), with little if any impact on D2 dopamine receptor neurons (D2-MSNs). Using ex vivo whole cell recordings in male and female mice, we observe alterations in D1-MSN excitability across the estrous cycle similar in magnitude to the actions of cocaine in males. Furthermore, cocaine shifts estrous cycle-dependent plasticity from intrinsic excitability changes in D1-MSNs to D2-MSNs. Overall, while there are similar cocaine-induced disparities regarding the relative excitability of D1-MSN versus D2-MSN between the sexes, in males this is mediated through reduced D1-MSN excitability, whereas in females it is due to heightened D2-MSN excitability.

Recent studies have implicated the ethanol metabolite, acetic acid, as neuroactive, perhaps even more so than ethanol itself. In this study, we investigated sex-specific metabolism of ethanol (1, 2, and 4g/kg) to acetic acid in vivo to guide electrophysiology experiments in the accumbens shell (NAcSh), a key node in the mammalian reward circuit. There was a sex-dependent difference in serum acetate production, quantified via ion chromatography only at the lowest dose of ethanol (males>females). Ex vivo electrophysiology recordings of NAcSh neurons in brain slices demonstrated that physiological concentrations of acetic acid (2 mM and 4 mM) increased NAcSh neuronal excitability in both sexes. N -methyl- D -aspartate receptor (NMDAR) antagonists, AP5, and memantine robustly attenuated the acetic acid-induced increase in excitability. Acetic acid-induced NMDAR-dependent inward currents were greater in females compared to males. These findings suggest a novel NMDAR-dependent mechanism by which the ethanol metabolite, acetic acid, may influence neurophysiological effects in a key reward circuit in the brain.

Like many social behaviors, aggression can be rewarding, leading to behavioral plasticity. One outcome of reward-induced aggression is the long-term increase in the speed in which future aggression-based encounters is initiated. This form of aggression impacts dendritic structure and excitatory synaptic neurotransmission in the nucleus accumbens, a brain region well known to regulate motivated behaviors. Yet, little is known about the intracellular signaling mechanisms that drive these structural/functional changes and long-term changes in aggressive behavior. This study set out to further elucidate the intracellular signaling mechanisms regulating the plasticity in neurophysiology and behavior that underlie the rewarding consequences of aggressive interactions. Female Syrian hamsters experienced zero, two or five aggressive interactions and the phosphorylation of proteins in reward-associated regions was analyzed. We report that aggressive interactions result in a transient increase in the phosphorylation of extracellular-signal related kinase 1/2 (ERK1/2) in the nucleus accumbens. We also report that aggressive interactions result in a transient decrease in the phosphorylation of mammalian target of rapamycin (mTOR) in the medial prefrontal cortex, a major input structure to the nucleus accumbens. Thus, this study identifies ERK1/2 and mTOR as potential signaling pathways for regulating the long-term rewarding consequences of aggressive interactions. Furthermore, the recruitment profile of the ERK1/2 and the mTOR pathways are distinct in different brain regions.