Hippocampal Insulin Exists! In Vivo Sampling Methods, Basal Concentrations, Recurrent Hypoglycemia, and Behavioral Extracellular Insulin Responses

Insulin has widespread actions within the brain, including energy homeostasis, modulation of feeding, and as a key component of memory processes. Further, brain insulin dysfunction correlates with Alzheimer's disease and other neurodegenerative disorders. To this point, measurement of hippocampal insulin has been limited. Determining in vivo hippocampal insulin levels both at a healthy baseline and after, e.g., induction of a disease state or in response to cognitive challenge, is essential to advance our understanding of both insulin's procognitive actions and to guide potential therapeutic interventions. We first sought to establish a sampling technique for insulin within awake and moving rats that would minimize tissue damage and maximize the accuracy of measurements by comparing microdialysis and cerebral open flow microperfusion in dual-cannulated 15-week-old male and female Sprague Dawley rats, taking simultaneous measurements counterbalanced across the hippocampi. Insulin sampling was comparable between probe types. Gliosis was measured using immunohistochemical markers for GFAP, TMEM-119, and CD68, and neither probe consistently resulted in more gliosis. Hence, we used MD for subsequent experiments. Basal extracellular hippocampal insulin was 0.3 ng/mL +/- 0.02 ng/mL. We showed that both a disease state (recurrent hypoglycemia) and a cognitive task (spontaneous alternation) altered hippocampal insulin in male and female 15-week-old Sprague Dawley rats. Further, these changes were associated with changes in Akt and its phosphorylation. For the first time, we confirmed that hippocampal insulin levels fluctuate acutely and chronically across conditions. Our data contains mechanisms by which insulin can modulate hippocampal function and offer guidance for future clinical approaches.