Florence P. Varodayan

Background: Norepinephrine (noradrenaline; NE) is a stress signal released from the locus coeruleus (LC) into the prefrontal cortex (PFC) to govern arousal, attention, and cognition. The LC is sexually dimorphic, and PFC NE dysfunction contributes to alcohol use disorder and several stress-related neuropsychiatric disorders that manifest differently in men and women. However, most preclinical studies of the medial PFC (mPFC) NE system have only used male subjects. Additionally, even though each mPFC subregion and layer forms unique circuits that mediate different aspects of cognitive behavior, their specific neuromodulation by NE is not understood. Methods: We comprehensively probed potential sex differences in the mouse mPFC NE system, starting with fluorescent tracing of the LC→mPFC circuit. Basal mPFC NE tissue content and adrenergic receptor mRNA were measured using high performance liquid chromatography and real-time quantitative polymerase chain reaction. Ex vivo electrophysiology assessed NE modulation of glutamate synapses in layers 2/3 and 5 of the prelimbic and infralimbic subregions of the mPFC. Finally, we used an α2 adrenergic receptor antagonist to increase NE release and tested for mPFC-associated reversal learning and episodic memory. Results: Females had a greater percentage of LC NE neurons→mPFC than males, with no differences in basal mPFC NE concentration or adrenergic receptor mRNA. NE increased mPFC glutamate release broadly in males, but its effects in females were restricted to prelimbic layer 5 and infralimbic layer 2/3. Finally, while there were dose-dependent effects of the α2 receptor antagonist on cognitive behavior, they did not vary between sexes. Conclusions: We uncovered complex sex differences in LC→mPFC structure and mPFC NE function, and future studies should examine NE activation in the context of greater cognitive load, such as during alcohol withdrawal or periods of stress. Clinically, women exhibit greater stress-induced activation of the NE system, are more likely to be diagnosed with affective disorders, and are more likely to drink alcohol to regulate negative affect and stress reactivity than men. Therefore, our study highlights the importance of considering specific subpopulations (e.g. women, or people with comorbid stress and alcohol use disorders) during the development of new NE-based treatments. Plain english summary: Norepinephrine (also known as noradrenaline) is a stress signal that regulates activity in the brain region the medial prefrontal cortex (mPFC) to optimize decision making, emotional processing, inhibitory control, and learning and memory. Its dysfunction contributes to both alcohol use disorder and stress-related neuropsychiatric disorders, though its role may differ between men and women. It is well known that the brain region that makes norepinephrine (i.e. locus coeruleus; LC) is larger and more complex in women and female rodents than their male counterparts. However, most preclinical studies have only used male subjects so the impact of these sex differences remains unclear. In this study, we used male and female mice to probe the LC→mPFC brain circuit and understand how norepinephrine shapes mPFC neuronal communication. We also tested how increasing norepinephrine levels alters cognitive behaviors that are mediated by the mPFC. We identified complex sex differences; female mice had a larger LC→mPFC circuit but their mPFC neurons were less sensitive to norepinephrine compared to male mice. There were no sex differences in norepinephrine’s effects on reversal learning and episodic memory under baseline conditions, but future studies should examine whether sex differences emerge during alcohol withdrawal or periods of stress. This work expands our understanding of mPFC norepinephrine signaling in both sexes and highlights the importance of considering specific subpopulations (e.g. women, or people with comorbid stress and alcohol use disorders) during the development of new norepinephrine-based treatments. Highlights: Females have a larger locus coeruleus → medial prefrontal cortex circuit than males. Basal norepinephrine levels and adrenergic receptor gene expression levels are similar in the medial prefrontal cortex of male and female mice. Norepinephrine broadly increases glutamate release onto medial prefrontal cortex pyramidal neurons in male mice. In female mice, norepinephrine-induced glutamate release is restricted to prelimbic cortex layer 5 and infralimbic cortex layer 2/3 pyramidal neurons. Norepinephrine altered medial prefrontal cortex-dependent cognitive behaviors (reversal learning and episodic memory) in mice, but there were no sex differences in its effects.

Alzheimer's disease (AD) is the most common age‐related dementia. Lifestyle factors, including alcohol use, can increase the risk of AD. While human studies demonstrate that alcohol use can negatively impact AD risk and disease progression, the underlying ethanol‐dependent mechanisms that increase Aβ burden and impair cognition remain elusive. We have recently shown that ethanol can acutely affect microglial dynamics, which are critical to microglial function, and many other studies have reported inflammatory activation of microglia after long‐term ethanol exposure in both humans and animal models. Here, we administered 4 weeks of ethanol at dosages that mimic human binge drinking to 2.5‐month‐old male 5xFAD mice, a common mouse model of AD. After a 2‐week abstinence period, we performed behavioral assays and analyzed amyloid pathology and microglial morphology in the subiculum where amyloid pathology develops earlier than in other brain regions. We also analyzed the microglial transcriptome in the hippocampus. We found that ethanol exposure facilitated amyloid pathology and worsened cognitive function in 5xFAD mice, while microglial expression patterns, arborization, and phagocytosis appeared unchanged. Overall, our results suggest that pronounced ethanol exposure, when started early in the disease before amyloid pathology is established, can worsen AD progression in an amyloidosis model.

Individuals with alcohol use disorder (AUD) struggle with inhibitory control, decision making, and emotional processing. These cognitive symptoms reduce treatment adherence, worsen clinical outcomes, and promote relapse. Neuroimmune activation is a key factor in the pathophysiology of AUD, and targeting this modulatory system is less likely to produce unwanted side effects compared to directly targeting neurotransmitter dysfunction. Notably, the cytokine interleukin-1β (IL-1β) has been broadly associated with the cognitive symptoms of AUD, though the underlying mechanisms are not well understood. Here we investigated how chronic intermittent 24-hour access two bottle choice ethanol drinking affects medial prefrontal cortex (mPFC)-related cognitive function and IL-1 synaptic signaling in male and female C57BL/6J mice. In both sexes, ethanol drinking decreased reference memory and increased mPFC IL-1 receptor 1 (IL-1R1) mRNA levels. In neurons, IL-1β can activate either pro-inflammatory or neuroprotective intracellular pathways depending on the isoform of the accessory protein (IL-1RAcP) recruited to the IL-1R1 complex. Moreover, ethanol drinking sex-dependently shifted mPFC IL-1RAcP isoform gene expression and IL-1β regulation of mPFC GABA synapses, both of which may contribute to female mPFC resiliency and male mPFC susceptibility. This type of signaling bias has become a recent focus of rational drug development. Therefore, in addition to increasing our understanding of how IL-1β sex-dependently contributes to mPFC dysfunction in AUD, our current findings also support the development of a new class of pharmacotherapeutics based on biased IL-1 signaling. Highlights: - Female mice consumed more ethanol, but were less cognitively impaired than males - Ethanol altered IL-1β effects at mPFC GABA synapses, with females less sensitive than males - Ethanol altered mPFC Il1rap mRNA to promote female neuroprotection and male neuroinflammation

Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by the recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin-releasing factor (CRF)-CRF₁ receptor and Gamma-Aminobutyric Acid (GABA)-ergic signaling dysregulation occurring in alcohol-dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch-clamp electrophysiology to examine the effects of alcohol dependence on the CRF modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared to males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had fewer CeA neurons containing mRNA for the CRF₁ receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike in males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.