Electrophysiology of arcuate neurokinin B neurons in female Tac2-EGFP transgenic mice

Abstract

Neurons in the arcuate nucleus that coexpress kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) play an important role in the modulation of reproduction by estrogens. Here, we study the anatomical and electrophysiological properties of arcuate NKB neurons in heterozygous female transgenic mice with enhanced green fluorescent protein (EGFP) under the control of the Tac2 (NKB) promoter (Tac2-EGFP mice). The onset of puberty, estrous cyclicity, and serum LH were comparable between Tac2-EGFP and wild-type mice. The location of EGFP-immunoreactive neurons was consistent with previous descriptions of Tac2 mRNA-expressing neurons in the rodent. In the arcuate nucleus, nearly 80% of EGFP neurons expressed pro-NKB-immunoreactivity. Moreover, EGFP fluorescent intensity in arcuate neurons was increased by ovariectomy and reduced by 17β-estradiol (E2) treatment. Electrophysiology of single cells in tissue slices was used to examine the effects of chronic E2 treatment on Tac2-EGFP neurons in the arcuate nucleus of ovariectomized mice. Whole-cell recordings revealed arcuate NKB neurons to be either spontaneously active or silent in both groups. E2 had no significant effect on the basic electrophysiological properties or spontaneous firing frequencies. Arcuate NKB neurons exhibited either tonic or phasic firing patterns in response to a series of square-pulse current injections. Notably, E2 reduced the number of action potentials evoked by depolarizing current injections. This study demonstrates the utility of the Tac2-EGFP mouse for electrophysiological and morphological studies of KNDy neurons in tissue slices. In parallel to E2 negative feedback on LH secretion, E2 decreased the intensity of the EGFP signal and reduced the excitability of NKB neurons in the arcuate nucleus of ovariectomized Tac2-EGFP mice.

Figure 1.

Serum LH and estradiol in diestrous, OVX, and OVX + E₂-treated mice. Mice were OVX at 2 months of age and treated with sc capsules containing either E₂ or vehicle. Blood samples were taken at 3 months of age. A, Serum LH was significantly increased in OVX mice, compared with diestrous and E₂-treated OVX mice. Similar patterns of LH secretion were observed in wild-type and Tac2-EGFG mice. B, Treatment of OVX Tac2-EGFP mice with E₂ capsules resulted in diestrous levels of serum E₂. n = 5–10 mice/group. *, significantly different from diestrous and OVX + E₂ mice (within wild-type and Tac2-EGFP groups, ANOVA with Tukey's post hoc test).

Figure 2.

Photomicrograph (right) and computer-assisted map (left) of EGFP immunocytochemistry in a representative brain section from a Tac2-EFGP female mouse. Sections were visualized with nickel-intensified DAB. Each black dot on the left represents a single immunoreactive soma mapped with the Neurolucida image-combining computer microscope. See Supplemental Figures 1 and 2 for additional maps at multiple levels of the hypothalamus. 3V, third ventricle; ARC, arcuate nucleus; BLA, basolateral amygdaloid nucleus; CA1, field CA1 hippocampus; cc, corpus callosum; Cl, claustrum; cp, cerebral peduncle; CPu, caudate putamen (striatum); f, fornix; fr, fasciculus retroflexus; ic, internal capsule; LHA, lateral hypothalamic area; LHb, lateral habenular nucleus; ME, median eminence; MHb, med habenular nucleus; mt, mammilothalamic tract; ot, optic tract; PH, posterior hypothalamus; st, stria terminalis; TH, thalamus; VMN, ventromedial nucleus; ZI, zona incerta. Scale bar, 250 μm.

Figure 3.

Photomicrograph of EGFP immunocytochemistry in the arcuate nucleus of a Tac2-EGFP female mouse. Sections were visualized with nickel-intensified DAB. Intensely labeled cell bodies were obscured by numerous beaded axons and dendrites. Axons extended to the median eminence, periventricular zone, ependymal layer, and the pial surface. The adjacent ventromedial nucleus was devoid of EGFP-ir somata. See Supplemental Figures 1 and 2 for photomicrographs of EGFP immunohistochemistry of other brain regions. 3V, third ventricle; ME, median eminence; VMN, ventromedial nucleus. Scale bar, 50 μm.

Figure 4.

Representative photomicrographs of EGFP-fluorescence (A, green) and pro-NKB-immunofluorescence (B, red) in the arcuate nucleus of an OVX, Tac2-EGFP mouse. Nearly 80% of EGFP neurons in the arcuate nucleus were labeled by the pro-NKB antibody (C, yellow). ARC, arcuate nucleus; ME, median eminence; 3V, third ventricle. Scale bar in A, 100 μm (applies to A–C).

Figure 5.

EGFP fluorescence in the arcuate nucleus of Tac2-EGFP mice. Fluorescent arcuate neurons in the OVX mice (B) appeared qualitatively brighter than neurons in diestrous (A) and OVX + E₂ (C) mice. D, Semiquantitative image analysis revealed a significant increase in the average fluorescent intensity units per neuron in the arcuate nucleus of OVX mice. E, The mean number of fluorescent neurons counted in a unilateral arcuate nucleus section was not significantly different between groups. 3V, third ventricle; ARC, arcuate nucleus, ME, median eminence. Values are mean ± SEM, n = 5–6 mice/group. *, significantly different from diestrous and OVX + E₂ mice (one-way ANOVA with Tukey's post hoc test).

Figure 6.

Spontaneous activity of arcuate Tac2-EGFP neurons in OVX and OVX + E₂ mice. A, Representative recordings of neurons classified as either silent (mean firing frequency <0.1 Hz) or active. B, Proportions of silent or active or silent neurons in OVX mice (n = 22 neurons from 12 mice) and OVX + E₂ mice (n = 15 neurons from 11 mice). More active neurons were identified in OVX mice, but this difference was not statistically significant (Fisher's exact test). C, There was no significant difference between OVX and OVX + E₂ mice in the mean spontaneous firing frequency of neurons classified as either spontaneously active or silent. D, Similarly, no difference was detected in the EPSC events per minute between treatment groups; mean ± SEM; n, number of Tac2-EGFP neurons.

Figure 7.

Evoked activity of arcuate Tac2-EGFP neurons in OVX and OVX + E₂ mice. A, Representative patterns of activity evoked by a series of rectangular-pulse current injections. A tonic pattern was identified with little frequency adaptation (top), and a phasic pattern was characterized by a few spikes and rapid adaptation (middle). B, Proportions of neurons identified as tonic or phasic in OVX and OVX + E₂ mice. A greater percentage of tonic neurons was detected in the OVX mice, but these changes were not statistically significant (Fisher's exact test). C, In response to depolarizing current injections, OVX + E₂ mice exhibited reduced spike frequencies, compared with OVX mice (mean ± SEM; OVX, n = 16 neurons from 9 mice; OVX + E₂, n = 15 neurons from 11 mice). *, significantly different, OVX vs OVX + E₂ (two-way ANOVA with Tukey's post hoc tests).