Oxytocin activity in the paraventricular and supramammillary nuclei of the hypothalamus is essential for social recognition memory in rats.

Oxytocin plays an important role in modulating social recognition memory. However, the direct implication of oxytocin neurons of the paraventricular nucleus of the hypothalamus (PVH) and their downstream hypothalamic targets in regulating short- and long-term forms of social recognition memory has not been fully investigated. In this study, we employed a chemogenetic approach to target the activity of PVH oxytocin neurons in male rats and found that specific silencing of this neuronal population led to an impairment in short- and long-term social recognition memory. We combined viral-mediated fluorescent labeling of oxytocin neurons with immunohistochemical techniques and identified the supramammillary nucleus (SuM) of the hypothalamus as a target of PVH oxytocinergic axonal projections in rats. We used multiplex fluorescence in situ hybridization to label oxytocin receptors in the SuM and determined that they are predominantly expressed in glutamatergic neurons, including those that project to the CA2 region of the hippocampus. Finally, we used a highly selective oxytocin receptor antagonist in the SuM to examine the involvement of oxytocin signaling in modulating short- and long-term social recognition memory and found that it is necessary for the formation of both. This study discovered a previously undescribed role for the SuM in regulating social recognition memory via oxytocin signaling and reinforced the specific role of PVH oxytocin neurons in regulating this form of memory.

Light and electron microscopic imaging of synaptic vesicle endocytosis at mouse hippocampal cultures.

Following the release of neurotransmitters at synaptic vesicles via exocytosis, endocytosis is initiated to retrieve vesicles that have fused with the plasma membrane of nerve terminals and recycle them, thus sustaining synaptic transmission. Here, we describe imaging-based protocols for quantitative measurements of endocytosis at cultured synapses. These protocols include (1) primary culture of mouse hippocampal neurons, (2) studying endocytosis at neurons transfected with a pH-sensitive synaptophysin-pHluorin2× using fluorescent microscopy, and (3) imaging endocytosis at fixed neurons with electron microscopy. For complete details on the use and execution of this protocol, please refer to Wu et al. (2016) and Wu et al. (2021).

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells.

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated Ω-shape profile until it merges completely at the plasma membrane. To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C δ (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.