The present manuscript by Lass G and colleagues explores the role of MePD Kiss1 neurons in the control of GnRH pulses through a series of opto and pharmacological approaches in the mouse. The authors found that the photo-stimulating MePD neurons increases the LH pulse frequency while this, in combination with GABA or glutamate blockers, slows down or completely prevents LH pulses. These findings add important mechanistic information to the role of these neurons in the control of the GnRH pulse generator.

Comments:
- It is unclear why the authors used sustained stimulation for 90 min rather than pulsatile when trying to decipher the role of these neurons in the control of LH pulses. Still, it is interesting that despite this sustained protocol, LH pulses were observed. This, however, has not been addressed in the discussion as it could have major implications in the slowing down and blockade of LH pulses observed in some of the experiments. Understanding whether MePD Kiss1 neurons can induce a pulse of LH would be informative.
- The working model is complex and quite speculative; and needs further clarification. It seems to imply that photo-stimulation of MePD Kiss1 neurons leads to kisspeptin action within the MeA that releases GABA and or Glut to modify the GnRH pulse generator. However, low frequency stimulation favors the release of neurotransmitters, thus, it would be more likely that MePD Kiss1 neurons would be releasing GABA themselves (or Glut in a fraction of them) that is playing this role. Thus, testing a higher frequency would have offered valuable information on this regard to compare neuropeptide vs neurotransmitter release. Nonetheless, 5Hz could be high enough to induce some peptidic release. For this reason, an experiment with GABA + Glut antagonists could have addressed the question of whether inhibitory peptidic factors (e.g. dynorphin, which is highly expressed in the MeA), are contributing to this effect if the inhibition of the LH pulses is still present rather than affecting the glutamate/GABA balance.
- The canula system delivers the GABA and glutamate antagonists on the MePD Kiss1 neurons, thus it seems likely that they are either inhibiting presynaptic inputs or acting postsynaptically in neurons located close to them (somatodendritic interaction). However, it is unclear where the authors think the effect of MePD Kiss1 neurons is happening as the antagonists may not have reached the target areas of these neurons.
- The authors should include relevant references supporting the inhibitory effect of over-stimulating the GnRH pulse generator (Han SU et al 2020).
- Figure 1 shows a considerable number of cells expressing EYFP but no tdTomato. This is concerning giving that the AAV-ChR2 is cre-dependent and may indicate unspecific (non-Kiss1 neuron) effect in the results obtained.

The authors have successfully addressed all of my comments, mostly the one relating to the complexity of continuous stimulation to induce pulsatile release of GnRH. As we are learning more about the mechanisms underlying this phenomenon, the current manuscript will significantly add to this endeavor.