Kisspeptin photometry recordings during pregnancy and lactation

The specific role that prolactin plays in lactational infertility, as distinct from other suckling or metabolic cues, remains unresolved. Here, deletion of the prolactin receptor (Prlr) from forebrain neurons or arcuate kisspeptin neurons resulted infailure to maintain normal lactation-induced suppression of estrous cycles. Kisspeptin immunoreactivity and pulsatile LH secretion were increased in these mice, even in the presence of ongoing suckling stimulation and lactation. GCaMP fibre photometry of arcuate kisspeptin neurons revealed that the normal episodic activity of these neurons is rapidly suppressed in pregnancy and this was maintained throughout early lactation. Deletion of Prlr from arcuate kisspeptin neurons resulted in early reactivation of episodic activity of kisspeptin neurons prior to a premature return of reproductive cycles in early lactation. These observations show dynamic variation in arcuate kisspeptin neuronal activity associated with the hormonal changes of pregnancy and lactation, and provide direct evidence that prolactin action on arcuate kisspeptin neurons is necessary for suppressing fertility during lactation in mice.

Methods
GCaMP fibre photometry recordings of arcuate nucleus kisspeptin neurons throughout pregnancy and lactation. 

Fluorescence signals were acquired using a custom-built fibre photometry system made primarily from Doric components. Violet (405nm) and blue (490nm) fibre-coupled LEDs were sinusoidally modulated at 531 and 211 Hz, respectively, and focused into a 400μm, 0.48 numerical aperture fibre optic patch cord connected to the mouse. Emitted fluorescence was collected by the same fibre and focused onto a femtowatt photoreceiver (2151, Newport). The two GCaMP6s emission signals were collected at 10 Hz in a scheduled 5s on/15s off mode by demodulating the 405nm (non-calcium dependent) and 490nm (calcium-dependent) signals. The power output at the tip of the fibre was set at 50μW. Fluorescent signals were acquired using a custom software acquisition system (Tussock Innovation, Dunedin, New Zealand) and analyzed using custom templates created by Dr Joon Kim (University of Otago, Dunedin, New Zealand) based on mathematics and calculations similar to those previously described (see references in manuscript). Briefly, the fluorescent signal obtained after stimulation with 405nm light was used to correct for movement artefacts as follows: first, the 405nm signal was filtered using a savitzky-golay filter and fitted to the 490nm signal using least linear square regression. The fitted 405nm signal was then subtracted and divided from the 490nm signal to obtain the movement and bleaching corrected signal. The output of these templates is 490-adjusted405/adjusted405, which was multiplied to get the final ΔF/F as a percentage increase. All photometry data is reported as ΔF/F(%) which is given as ΔF/F(%) in the data files.