Research indicates that responses to stress are sexually dimorphic, particularly in regard to learning and memory processes: while males display impaired cognitive performance and hippocampal CA3 pyramidal cell dendritic remodeling following chronic stress, females exhibit enhanced performance and no remodeling. Leu-enkephalin, an endogenous opioid peptide found in the hippocampal mossy fiber pathway, plays a critical role in mediating synaptic plasticity at the mossy fiber-CA3 pyramidal cell synapse. Estrogen is known to influence the expression of leu-enkephalin in the mossy fibers of females, with leu-enkephalin levels being highest at proestrus and estrus, when estrogen levels are elevated. Since stress is also known to alter the expression of leu-enkephalin in various brain regions, this study was designed to determine whether acute or chronic stress had an effect on mossy fiber leu-enkephalin levels in females or males, through the application of correlated quantitative light and electron microscopic immunocytochemistry. Both acute and chronic stress eliminated the estrogen-dependence of leu-enkephalin levels across the estrous cycle in females, but had no effect on male levels. However, following acute stress leu-enkephalin levels in females were consistently lowered to values comparable to the lowest control values, while following chronic stress they were consistently elevated to values comparable to the highest control values. Ultrastructural changes in leu-enkephalin labeled dense core vesicles paralleled light microscopic observations, with acute stress inducing a decrease in leu-enkephalin labeled dense core vesicles, and chronic stress inducing an increase in leu-enkephalin labeled dense-core vesicles in females. These findings suggest that alterations in leu-enkephalin levels following stress could play an important role in the sex-specific responses that females display in learning processes, including those important in addiction.

Opioids play a critical role in hippocampally dependent behavior and plasticity. In the hippocampal formation, mu opioid receptors (MOR) are prominent in parvalbumin (PARV) containing interneurons. Previously we found that gonadal hormones modulate the trafficking of MORs in PARV interneurons. Although sex differences in response to stress are well documented, the point at which opioids, sex and stress interact to influence hippocampal function remains elusive. Thus, we used quantitative immunocytochemistry in combination with light and electron microscopy for the phosphorylated MOR at the SER375 carboxy-terminal residue (pMOR) in male and female rats to assess these interactions. In both sexes, pMOR-immunoreactivity (ir) was prominent in axons and terminals and in a few neuronal somata and dendrites, some of which contained PARV in the mossy fiber pathway region of the dentate gyrus (DG) hilus and CA3 stratum lucidum. In unstressed rats, the levels of pMOR-ir in the DG or CA3 were not affected by sex or estrous cycle stage. However, immediately following 30 minutes of acute immobilization stress (AIS), males had higher levels of pMOR-ir whereas females at proestrus and estrus (high estrogen stages) had lower levels of pMOR-ir within the DG. In contrast, the number and types of neuronal profiles with pMOR-ir were not altered by AIS in either males or proestrus females. These data demonstrate that although gonadal steroids do not affect pMOR levels at resting conditions, they are differentially activated both pre- and post-synaptic MORs following stress. These interactions may contribute to the reported sex differences in hippocampally dependent behaviors in stressed animals.