Characterisation of an Oxytocin‐Ires‐Cre Mouse Model for Recording Oxytocin Neuron Bursts in Behaving Mice
Authors: Perkinson Michael R. , Kim Joon S. , Brown Colin H. , Iremonger Karl J. 
Published: April 2020

The hormone oxytocin initiates uterine contractions required for parturition and mammary duct contraction for milk‐ejection during lactation. Oxytocin is secreted directly into the circulation at the posterior pituitary gland by magnocellular neurons of the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus. During parturition and lactation, oxytocin neurons fire high frequency bursts that are coordinated across the population, to generate pulses of high circulating oxytocin concentrations required for rhythmic contractions of the uterus and mammary ducts. However, researching oxytocin neuron bursts has stalled because oxytocin neuron bursts are blocked by surgical anaesthesia and are not seen in brain slice preparations. Furthermore, oxytocin neuron bursts have not been studied in freely behaving animals. Hence, new techniques are required to elucidate the mechanisms that generate oxytocin neuron bursts. Fiber photometry uses neuron specific expression of GCaMP, a modified green fluorescent protein (GFP) that senses calcium. Implantation of an optic fiber allows monitoring of changes in GCaMP fluorescence (fluorescence increases during spiking activity) as a proxy measurement of neuronal activity in freely behaving animals. Oxytocin neurons can be transfected using a virus carrying a Cre‐dependant GCaMP. We have crossed oxyotcin‐Ires‐Cre mice (oxytocin neurons express Cre) with tdTomato (red fluorescent protein) mice. The tdTomato expression is Cre‐dependant which allows visual identification of Cre‐positive neurons. We first determined the degree of colocalisation between Cre expression and oxytocin within the PVN. Five oxytocin‐Cre x tdTomato mice were transcardially perfused, their brains removed and sliced coronally at 30 μm. Immunohistochemistry (IHC) for oxytocin was used to determine the colocalisation of oxytocin and endogenous tdTomato (Cre‐positive neurons). Oxytocin positive neurons were found extensively throughout the entire length of the PVN and oxytocin colocalisation with tdTomato was 78.3% (n = 5). We then determined the transfection of Cre‐dependent AAV1.CAG.Flex.GCaMP6s (1.9 x 1013 GC/ml) in oxytocin neurons following an injection. GCaMP6s virus (1 μl) was injected unilaterally immediately dorsal to the PVN using a Hamilton syringe (0.08 cm posterior and 0.022 cm lateral from bregma at a depth of 0.45 cm from the brain surface). Three weeks after the injection, mice were perfused, their brains removed and sliced coronally at 30 μm. Single label IHC for GCaMP6s was used to quantify transfection. Following injections, GCaMP6s is expressed in the top two thirds of the PVN in tdTomato positive neurons (n = 3). To determine the relationship between changes in GCaMP6s fluorescence and spiking activity in oxytocin neurons, we are currently completing in‐vitro from oxytocin neurons while simultaneously recording GCaMP6s fluorescence in brain slices from oxytocin‐Cre mice. Finally, in another group of oxytocin‐Cre mice, an optic fiber will be implanted above the PVN to record oxytocin neurons bursts during lactation in freely behaving mice.