Fertility is regulated by the gonads, which are the primary reproductive organs and a part of the endocrine system. The gonads produce reproductive hormones such as progesterone and estrogen as well as gametes which are essential for reproductive cycle and fertility. The development of gonads is solely dependent on the gonadotopins (luteinizing hormone and follicle stimulating hormone) released from the pituitary gonadotrophs. The gonadotropin-releasing hormone (GnRH) neurons within the hypothalamus represent the final common pathway through which the brain regulates the release of gonadotropins from the pituitary. Thus the hypothalamus-pituitary-gonadal (HPG) axis forms the neuroendocrine system which is indispensable for reproduction and fertility (Figure 1).

Kisspeptin is a peptide encoded by the Kiss-1 gene and the interaction of kisspeptin and its receptor, G-protein coupled receptor 54 (GPR54) is essential for puberty onset and fertility. Functional mutations of GPR54 in both humans and mice cause severely impaired reproductive functions, such as failure to undergo puberty and lack of gonadotropin secretion (de Roux et al., 2003 and Seminara et al., 2003). A group of neurons in the hypothalamus that express the neuropeptide kisspeptin, known as the kisspeptin neurons, potently stimulate the GnRH neurons (Han et al., 2005) which express GPR54. In situ hybridization and immunohistochemistry studies have identified two populations of kisspeptin neurons located in the rostral periventricular (RP3V) and arcuate nucleus (ARN) of rodents, sheep and primates (Figure 2). Our group is greatly interested in ‘scrutinizing’ the kisspeptin neurons for their neuroanatomical distribution, morphological characteristics as well as their indispensable physiological roles in puberty onset and fertility.

Figure 1: Schematic diagram indicating how kisspeptin neurons regulate GnRH neurons, which are the critical component of the hypothalamic-pituitary-gonadal (HPG) axis for reproduction.

Figure 1: Schematic diagram indicating how kisspeptin neurons regulate GnRH neurons, which are the critical component of the hypothalamic-pituitary-gonadal (HPG) axis for reproduction.

 

Figure 2: Coronal brain sections indicating the immunostaining of RP3V (left) and ARN (right) kisspeptin neurons, kisspeptin-immunoreactive cell bodies appeared as black dots.

Figure 2: Coronal brain sections indicating the immunostaining of RP3V (left) and ARN (right) kisspeptin neurons, kisspeptin-immunoreactive cell bodies appeared as black dots.

Our research relies heavily upon the use of a variety of transgenic mouse models, including the global- and neuron-specific gene knockout mice as well as transgenic reporter mice. We have previously established the essential role of RP3V kisspeptin neurons in mediating the estrogen positive feedback on the gonadotropin-releasing hormone (GnRH) neurons which resulted in puberty onset and ovulation (Clarkson and Herbison, 2011; Liu et al., 2011; Clarkson et al., 2008; Clarkson and Herbison, 2006).  Whereas much of the RP3V kisspeptin neurons characteristics are well-established, the role of arcuate nucleus (ARN) kisspeptin neurons remains elusive. Hence, our current research focuses on understanding the functional roles of ARN kisspeptin neurons.  We aimed to define the neuroanatomical, morphological and physiological characteristics of the ARN kisspeptin neurons. Simon de Croft (PhD Student) has embarked on electrophysiological recordings of ARN kisspeptin neurons. His main research interest is in defining the electrical properties of ARN kisspeptin neurons. Shel-Hwa Yeo (PhD student) is interested in the neuroanatomical projections and physiological roles of ARN kisspeptin neurons. Much of her work involves in vivo models and immunohistochemistry.

 

Exploring electrophysiological aspects of kisspeptin neurons

Recently, using gene targeting technology, a mouse line has been created that allows kisspeptin neurons to be fluorescent (Mayer et al., 2010), making it possible to distinguish them from all other neurons. We have used dual-label immunohistochemistry to check the relationship between fluorescent neurons and those which are currently expressing kisspeptin. We found that in a sub-region of the ARN, 90-95% of fluorescent neurons express kisspeptin in adulthood. This validates the mouse model as being accurate at reporting kisspeptin neurons, and means that there is a high probability that electrical activity from a fluorescent neuron is that of a kisspeptin neuron.

Figure 3: Photomicrographs showing endogenous GFP fluorescence (green) in the ARN, kisspeptin immunostaining (red) and a merged image where yellow indicates co-expression of GFP and kisspeptin in a kisspeptin-reporter animal.

Figure 3: Photomicrographs showing endogenous GFP fluorescence (green) in the ARN, kisspeptin immunostaining (red) and a merged image where yellow indicates co-expression of GFP and kisspeptin in a kisspeptin-reporter animal.

It is well-established that the mRNA of kisspeptin in the ARN is negatively regulated by estrogen, that is, when estrogen levels are high, kisspeptin mRNA levels are suppressed, and when estrogen levels are low, kisspeptin mRNA in the ARN is increased (Smith et al., 2005). However, neurons exert their influence by firing action potentials, and there is currently no data available on the effects of estrogen on the firing rate of ARN kisspeptin neurons. Therefore, we are examining the firing-rate of the ARN kisspeptin neurons, by using cell-attached electrophysiology, which is minimally invasive to the cell while recording their activity. Using this technique we record the spontaneous firing rate of ARN kisspeptin neurons during different stages of the estrous cycle since this represents accurately the physiological levels of circulating estrogen.

 

Figure 4: Electrophysiological recording of the spontaneous firing of a kisspeptin neuron in the ARN of a female mouse.

Figure 4: Electrophysiological recording of the spontaneous firing of a kisspeptin neuron in the ARN of a female mouse.

Recent evidence showed that ARN kisspeptin neurons co-express neurokinin B (NKB) and dynorphin (Figure 5), two other neuropeptides which are also essential for normal reproductive function (Goodman et al., 2004, Topaloglu et al., 2009 and Lehman et al., 2010). There is evidence to suggest that there are physical contacts between ARN kisspeptin neurons, giving rise to the suggestion of a neuronal network existing in the ARN which may be important in regulating GnRH release. The roles of dynorphin and NKB in this neuronal network are currently unknown.  Therefore, we are measuring the firing rate of ARN kisspeptin neurons in the presence of NKB and dynorphin, and also in the presence of specific antagonists to the receptors of NKB and dynorphin using cell-attached electrophysiology.

Figure 5: ARN kisspeptin neurons co-express both dynorphin and NKB. Adapted from Lehman et al., 2010.

Figure 5: ARN kisspeptin neurons co-express both dynorphin and NKB. Adapted from Lehman et al., 2010.

 

Exploring neuroanatomical aspects of ARN kisspeptin neurons

Neurons connect to each other by extending their axons into other areas of the brain to provide synaptic inputs to their efferent neurons. The distinctive projection patterns of each kisspeptin neuronal population is not known in any species yet and it is difficult to discern their discreet projection patterns using conventional immunohistochemistry. We undertook neuroanatomical tracing to map out the ARN kisspeptin neuronal projections as a means to envisage the possible physiological roles of these neurons.  To explore the projections of kisspeptin neurons, we performed stereotactic surgery to inject the anterograde tracer, Phaseolus vulgaris agglutinin (PHA-L) into the ARN of adult female mice. The anterograde tracer is used to trace axonal projections from neuronal cell body to their point of termination in the nerve terminal. Tracing the neuronal cell bodies labeled with the PHA-L (Figure 6) enabled us to visualize the axonal projections of ARN kisspeptin neurons. The retrograde tracer is complementary to the anterograde tracer where it is used to trace neural connections from their termination to the cell bodies. We injected the retrograde tracer, fluorogold into the rostral preoptic area of adult female mice to verify the projection of ARN kisspeptin neurons to the vicinity of GnRH neuronal cell bodies as indicated in our anterograde findings. Dual-label immunofluorescence and extensive confocal microscopy was employed to visualize the distribution of PHA-L-labeled ARN kisspeptin-immunoreactive fibers and kisspeptin-immunoreactive cell bodies labeled with fluorogold.

 Figure 6: Dual immunofluorescence showing one of the ARN kisspeptin neurons (green) labeled with PHA-L (yellow) after PHA-L injection. Adapted from Yeo and Herbison, Endocrinology, 2011.


Figure 6: Dual immunofluorescence showing one of the ARN kisspeptin neurons (green) labeled with PHA-L (yellow) after PHA-L injection. Adapted from Yeo and Herbison, Endocrinology, 2011.

Figure 7: Schematic, flat-map horizontal view of the female mouse brain showing the organization of kisspeptin neuronal projections. ARN kisspeptin neuron projections are represented by red dotted line on the right, whereas RP3V kisspeptin neuron projections are indicated on the left (black solid line). Adapted from Yeo and Herbison, Endocrinology, 2011.

Figure 7: Schematic, flat-map horizontal view of the female mouse brain showing the organization of kisspeptin neuronal projections. ARN kisspeptin neuron projections are represented by red dotted line on the right, whereas RP3V kisspeptin neuron projections are indicated on the left (black solid line). Adapted from Yeo and Herbison, Endocrinology, 2011.

 

Overall, our neuroanatomical tracing revealed that ARN kisspeptin neurons project widely in the forebrain into multiple hypothalamic nuclei and associated limbic structures (Figure 7). Projections of ARN kisspeptin neurons to many hypothalamic areas highlight the diverse neuronal circuits that are likely to be regulated by kisspeptin in addition to the GnRH neurons. Our tracing results indicated that ARN kisspeptin neurons project to the vicinity of GnRH neurons in the rostral preoptic area suggesting that ARN kisspeptin neurons might innervate GnRH neuron cell body.  However, the fundamental evidence for presynaptic inputs awaits further morphological characterization of these neurons. To examine the detailed morphology of ARN kisspeptin neurons, we are currently employing juxtacellular cell-filling of living kisspeptin neurons with small molecular weight dyes in the acute brain slice preparation. Using triple immunofluorescence labeling for kisspeptin, GnRH and cell-filling dye immunoreactivities, we aim to track the fibres projecting out from the filled kisspeptin neuron, which could possibly lead to the identification of synaptic inputs onto GnRH neurons. With relation to the electrical activity of the ARN kisspeptin neurons, these morphological characteristics are essential to clarify the possible role of ARN kisspeptin neurons in regulating GnRH neuronal activities to maintain tonic GnRH secretion.

Our investigations so far have shown that ARN kisspeptin neurons exert spontaneous firing activity and possibly provide efferent inputs to GnRH neurons. However, we are still unsure about i) how ARN kisspeptin neurons regulate GnRH neuronal activity ii) how estrogen affects the firing activity of ARN kisspeptin neurons iii) what the roles of the co- transmitters (dynorphin and neurokinin B) in these neurons are and iv) what the physiological importance of these neurons are. Hence, our quest for elucidating the role of ARN kisspeptin neurons is still ongoing.

 

Written by Shel-Hwa Yeo and Simon de Croft 

 

Relevant Citations:

Clarkson J and Herbison AE (2011). Dual phenotype kisspeptin-dopamine neurones of the rostral periventricular area of the third ventricle project to gonadotrophin-

releasing hormone neurones. J Neuroendocrinol 23: 293-301. 

Clarkson J, d’Anglemont de Tassigny X, Moreno AS, Colledge WH, Herbison AE (2008). Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge. J Neurosci 28:8691-8697.

Clarkson J, Herbison AE (2006). Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons.Endocrinology 147:5817-5825.

de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E (2003). Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972-10976.

Goodman RL, Coolen LM, Anderson GM, Hardy SL, Valent M, Connors JM, Fitzgerald ME, Lehman MN (2004). Evidence that dynorphin plays a major role in mediating progesterone negative feedback on gonadotropin-releasing hormone neurons in sheep. Endocrinology 145:2959-2967.

Han SK, Gottsch ML, Lee KJ, Popa SM, Smith JT, Jakawich SK, Clifton DK, Steiner RA, Herbison AE (2005). Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. J Neurosci 25:11349-11356.

Lehman MN, Merkley CM, Coolen LM, Goodman RL (2010). Anatomy of the kisspeptin

neural network in mammals. Brain Res 1364: 90-102.

Liu X, Porteous R, d’Anglemont de Tassigny X, Colledge WH, Millar R, Petersen SL, Herbison AE(2011). Frequency-dependent recruitment of fast amino acid and slow neuropeptide neurotransmitter release controls gonadotropin-releasing hormone neuron excitability. J Neurosci31: 2421-2430.

Mayer C, Acosta-Martinez M, Mayer C, Acosta-Martinez M, Dubois SL, Wolfe A, Radovick S, Boehm U, Levine JE (2010). Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons. Proc Natl Acad Sci USA 107: 22693-22698.

Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Jr., Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley WF, Jr., Aparicio SA, Colledge WH (2003). The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614-1627.

Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA (2005). Regulation of Kiss1

gene expression in the brain of the female mouse. Endocrinology 146:3686-3692.

Topaloglu AK, Reimann F, Guclu M, Yalin AS, Kotan LD, Porter KM, Serin A, Mungan NO, Cook JR,Ozbek MN, Imamoglu S, Akalin NS, Yuksel B, O’Rahilly S, Semple RK (2009). TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction. Nat Genet 41:354-358.

Yeo SH, Herbison AE (2011). Projections of arcuate nucleus and rostral periventricular

kisspeptin neurons in the adult female mouse brain. Endocrinology 152: 2387-2399.

 

 

 

 

 

 

 

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