Model Systems for Studying Kisspeptin Signalling: Mice and Cells - Kisspeptin Signaling in Reproductive Biology - page 481

their chance of fi ring. If this sub-population of GnRH neurons act as pacemakers to
co-ordinate the fi ring rate of the non-oscillating GnRH neurons, then this could
provide a mechanism by which kisspeptin can regulate GnRH pulsatility.
Transgenic Mice with Fluorescently Marked Kiss1 Neurons
Transgenic mice in which Kiss1 neurons are marked with a fl uorescent protein such
as GFP allow unequivocal identifi cation of these neurons in brain slice preparations
to interrogate their function and responses to neuromodulators. Patch clamp record-
ings of GFP-labelled neurons in the ARC of female mice have shown that most
Kiss1 neurons express both a hyperpolarization-induced cation current (h-current)
and a T-type Ca 2+ current, which may produce intrinsic pacemaker activity in Kiss1
neurons [ 42 ]. Expression of the relevant genes (HCN1-4 and Ca v 3.1) to produce
these pacemaker channels was confi rmed by RT-PCR analysis from individual
Kiss1 neurons. Kiss1 neurons also respond to NMDA or glutamate by increasing
their fi ring rates and to GABA by inhibiting fi ring rates [ 42 ]. In male mice, the
majority of the Kiss1 neurons in the ARC are depolarized by NKB and show an
increased fi ring rate [ 21 ]. This observation is consistent with the hypothesis that
NKB stimulates GnRH release via activation of Kiss1 neurons in the ARC.
Cell Lines
Immortalized cell lines provide a less complex model system to study kisspeptin
signalling pathways and how these infl uence cell biology and behaviour. Cell lines
have the advantage that they represent a homogeneous cell population without the
complex neuronal inputs found in vivo, greatly simplifying the experimental analy-
ses. It is also easier to control experimental variables using cell lines rather than
whole animals.
Cell lines were initially used to establish the intracellular signalling pathways
associated with kisspeptin binding to its receptor. Originally, immortalized cell lines
transfected with Kiss1r were used for these studies (CHO, HEK293, NIH 3T3 and
Cos7 cells), but similar results have been found in cell lines with endogenous kiss-
peptin receptor expression (for review, see [ 62 ]). Kisspeptin receptor activation was
found to couple through G q/11 to activate phospholipase C and increase intracellular
IP 3 and Ca 2+ levels [ 3 , 4 , 63 ]. Kisspeptin was also found to activate mitogen-
activated-protein-kinase (MAPK) pathways with increased phosphorylation of
ERK1/2 and p38MAPK [ 3 ]. Activation of the MAPK pathways is thought to occur
by Src-dependent transactivation of the EGF receptor [ 64 , 65 ].
The Kiss1 gene was originally identifi ed as a suppressor of metastasis [ 66 ] and
cell lines have been used to study the mechanism by which kisspeptins inhibit cell
migration. Migratory inhibition may be caused by increased cell adhesion to the
extracellular matrix at focal adhesion points or increased cell-cell binding at adhe-
rens junctions. Kisspeptin increases the phosphorylation of focal adhesion kinase