Neurokinin B and Dynorphin A in Kisspeptin Neurons of the Arcuate Nucleus Participate in Generation of Periodic Oscillation of Neural Activity Driving Pulsatile Gonadotropin-Releasing Hormone Secretion in the Goat

“The pulsatile release of gonadotropin-releasing hormone (GnRH) is a prerequisite for sustaining normal gonadotropin secretion in mammals (Knobil, 1980; Karsch, 1984); however, the cellular and molecular mechanisms that generate the rhythmic discharge of GnRH are unknown.”

“LH pulses were identified by the PULSAR computer program (Merriam and Wachter, 1982).”

“Finally, kisspeptin/ NKB/Dyn neurons in the ARC have been proposed to be a primary site of negative feedback action of E2 and P (Smith et al., 2005a,b, 2007; Burke et al., 2006; Foradori et al., 2006; Goodman et al., 2007), and we demonstrated here that E2 and P suppress the occurrence of MUA…”

“duces a prolonged activation of GnRH neurons in the mouse (Han et al., 2005; Pielecka-Fortuna et al., 2008).”

“Indeed, Topaloglu et al. (2009) have recently shown that mutations of either Trc3 or Tacr3, which encode NKB and NK3, respectively, produce gonadotropin deficiency and pubertal failure in humans.”

“Kisspeptin neurons in the hypothalamus play a key role in the regulation of GnRH neurons (Oakley et al., 2009), but the precise nature of the interaction between kisspeptin and GnRH neurons is just emerging.”

“Finally, we tested the hypothesis that NKB and Dyn play crucial roles in driving GnRH pulse generator activity by analyzing the effects of centrally administered NKB, Dyn, and a KOR antagonist on pulsatile MUA and LH secretion.”

“Finally, kisspeptin/ NKB/Dyn neurons in the ARC have been proposed to be a primary site of negative feedback action of E2 and P (Smith et al., 2005a,b, 2007; Burke et al., 2006; Foradori et al., 2006; Goodman et al., 2007), and we demonstrated here that E2 and P suppress the occurrence of MUA…”

“A vehicle injection was performed in each of the three treatment groups: OVX (n 7), OVX-plus-E2-treated (n 4), and OVX-plus-E2plus-P-treated (n 4), and these data served as the reference control for both the NKB and Dyn experiments.”

“Moreover, Roseweir et al. (2009) have demonstrated in several species that a kisspeptin antagonist blocks pulsatile GnRH/luteinizinghormone(LH)secretion.”

“duces a prolonged activation of GnRH neurons in the mouse (Han et al., 2005; Pielecka-Fortuna et al., 2008).”

“A similar dense plexus of NKB/Dyn fibers has been reported in the ARC of the rat and sheep (Burke et al., 2006; Foradori et al., 2006), and an electron microscopic study in the sheep revealed that Dyn neurons in the ARC receive synaptic contact with Dyn fibers (Foradori et al.”

“DOI:10.1523/JNEUROSCI.5848-09.2010 Copyright © 2010 the authors 0270-6474/10/303124-09$15.00/0 the ME (Ramaswamy et al., 2008).”

“…of either an NKB receptor (NK3) agonist or a Dyn receptor [the -opiate receptor (KOR)] antagonist profoundly influences GnRH/LH secretion (Goodman et al., 2004; SandovalGuzmán and Rance, 2004); moreover, mutations in either Trc3 or Tacr3 (which encode NKB and NK3, respectively) cause…”

“These anatomical observations suggest that kisspeptin/NKB/Dyn neurons in the ARC comprise an oscillatory feedback loop interconnected through collaterals that synchronize their activity, as previously proposed (Burke et al., 2006; Foradori et al., 2006; Rance, 2009; Navarro et al., 2009).”

“Indeed, it has been reported that continuous administration of either kisspeptin (Seminara et al., 2006) or GnRH (Belchetz et al., 1978) results in reduced LH secretion.”

“Double labeling of mRNA and proteins on the same section was performed as previously described (Wakabayashi et al., 2007; Ohkura et al., 2009).”

“Another possibility is that, in addition to activating kisspeptin neurons, NKB also acts on NKB receptors on GnRH nerve terminals in the ME (Krajewski et al., 2005) to inhibit GnRH secretion, perhaps through a Gi-coupled mechanism (Laniyonu et al., 1988).”

“Third, the administration of NKB to the OVX goat increased MUA but decreased LH secretion, which is consistent with previous observations that an NK3 agonist inhibits LH release in rats and mice (Sandoval-Guzmán and Rance, 2004; Navarro et al., 2009).”

“, 2006), and an electron microscopic study in the sheep revealed that Dyn neurons in the ARC receive synaptic contact with Dyn fibers (Foradori et al., 2002).”

“LH concentrations in single aliquots of 50 l plasma sample were measured by a double-antibody radioimmunoassay (Mori and Kano, 1984) with rabbit anti-ovine LH serum (Ohkura et al., 2004) and expressed in terms of ovine LH standard (NIDDK-oLH-I-4).”

“MUA recording and blood sampling MUA was recorded as previously described (Mori et al., 1991; Ichimaru et al., 2001; Ohkura et al., 2009) with a slight modification.”

“A similar dense plexus of NKB/Dyn fibers has been reported in the ARC of the rat and sheep (Burke et al., 2006; Foradori et al., 2006), and an electron microscopic study in the sheep revealed that Dyn neurons in the ARC receive synaptic contact with Dyn fibers (Foradori et al.”

“…for 1 h. Sections were then incubated with a solution containing a monoclonal antibody to kisspeptin (1:4000; Takeda; no. 254) (Ohkura et al., 2009; Takase et al., 2009) and either a polyclonal antibody to NKB (1:4000; Peninsula Laboratories) or Dyn (1:4000; Phoenix Pharmaceuticals) in PBST-BSA…”

“Rising levels of E2 would reduce the stimulatory drive of NKB/NK3 signaling in ARC kisspeptin/NKB/Dyn neurons by suppressing the expression of NKB (Rance and Bruce, 1994; Dellovade and Merchenthaler, 2004; Navarro et al., 2009) as well as NK3 (Navarro et al., 2009), which would reduce…”

“Furthermore, it has been suggested that Dyn/KOR expressed in vasopressin neurons modulates the phasic firing of those neurons and the release of vasopressin by an autosynaptic loop (Brown et al., 1998; Iremonger and Bains, 2009).”

“Furthermore, it has been suggested that Dyn/KOR expressed in vasopressin neurons modulates the phasic firing of those neurons and the release of vasopressin by an autosynaptic loop (Brown et al., 1998; Iremonger and Bains, 2009).”

“…reduce the stimulatory drive of NKB/NK3 signaling in ARC kisspeptin/NKB/Dyn neurons by suppressing the expression of NKB (Rance and Bruce, 1994; Dellovade and Merchenthaler, 2004; Navarro et al., 2009) as well as NK3 (Navarro et al., 2009), which would reduce kisspeptin/NKB/Dyn (MUA) burst…”

“For example, activation of NK3 increases firing in noradrenergic and dopaminergic neurons (Jung et al., 1996; Nalivaiko et al., 1997), whereas NK3 antagonists attenuate pharmacologically induced firing of dopaminergic neurons (Gueudet et al., 1999).”

“LH concentrations in single aliquots of 50 l plasma sample were measured by a double-antibody radioimmunoassay (Mori and Kano, 1984) with rabbit anti-ovine LH serum (Ohkura et al., 2004) and expressed in terms of ovine LH standard (NIDDK-oLH-I-4).”

“Another possibility is that, in addition to activating kisspeptin neurons, NKB also acts on NKB receptors on GnRH nerve terminals in the ME (Krajewski et al., 2005) to inhibit GnRH secretion, perhaps through a Gi-coupled mechanism (Laniyonu et al., 1988).”

“The pulsatile release of gonadotropin-releasing hormone (GnRH) is a prerequisite for sustaining normal gonadotropin secretion in mammals (Knobil, 1980; Karsch, 1984); however, the cellular and molecular mechanisms that generate the rhythmic discharge of GnRH are unknown.”

“For example, activation of NK3 increases firing in noradrenergic and dopaminergic neurons (Jung et al., 1996; Nalivaiko et al., 1997), whereas NK3 antagonists attenuate pharmacologically induced firing of dopaminergic neurons (Gueudet et al., 1999).”

“Since Dyn inhibits MUA volleys and central administration of nor-BNI reverses the inhibitory effect of P on pulsatile LH secretion (Gallo, 1990; Goodman et al., 2004), Dyn/KOR signaling could also mediate the negativefeedback action of P.”

“MUA was recorded as previously described (Mori et al., 1991; Ichimaru et al., 2001; Ohkura et al., 2009) with a slight modification.”

“Double labeling of mRNA and proteins on the same section was performed as previously described (Wakabayashi et al., 2007; Ohkura et al., 2009).”

“Dynorphin and neurokinin B colocalize with kisspeptin in many mammals (Goodman et al, 2007); dynorphin is proposed to act back on the releasing kisspeptin neurons to synchronize and shape pulsatile release patterns of kisspeptin (Navarro et al, 2009; Wakabayashi et al, 2010).”

“Thereafter, the presence of such KNDy neurons has been documented by a combination of in situ hybridization and IHC techniques in the mouse, goat, and monkey, although in the latter only NKB coexpression was analyzed (304, 305, 363, 491).”

“Although each of these three peptides is found in separate sets of neurons in other brain regions, recent evidence suggests that the colocalization of KNDy peptides seen in the ARC is unique among brain regions and is conserved across multiple mammalian species that include the rat (17), mouse (18), sheep (13), and go…”

“Although GnRH and LH pulses generally exhibit a 1:1 ratio, preclinical data exist suggesting that a faster GnRH pulse frequency may be associated with decreased LH secretion [52].”