Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus

Abstract

Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis.

Keywords: Development; Embryonic chicken; Energy homeostasis; Hypothalamus; Kv1; Potassium channel.

Fig. 1.. Embryonic avian hypothalamic slice cultures for electrophysiological recordings.

Representative photomicrograph of an E12 chicken coronal brain slice affixed to the electrophysiological recording chamber (left). Corresponding regions of interest as adapted from an avian neuroanatomy atlas of a 2-week old chicken brain (right) [34]. Opt, optic tract; 3v, third ventricle; ARC, arcuate nucleus; VMH, ventromedial hypothalamus; ME, median eminence; Elec, electrode. (Scale bar = 200µm)

Fig. 2.. Developmental changes in outward current recorded from avian hypothalamic neurons.

Average voltage-clamp recordings from neurons of E8 (A; n = 6) and E12 (B; n = 8) hypothalamic brain slices. Membrane capacitance was estimated by calculating the integral of the initial capacitance transient in response to a 10 mV hyperpolarizing pulse (C). Capacitance compensated steady state current-voltage relationships (D) demonstrate a significant developmental increase in outward currents recorded from hypothalamic neurons (* p < 0.05).

Fig. 3.. Kv1 channel mRNA expression in the embryonic avian hypothalamus.

Genomic DNA analysis (A) reveals predicted fragment sizes for the following Kv1 channels: Kv1.2 (448 bp); Kv1.3 (474 bp); Kv1.5 (347 bp). RT-PCR products for Kv1 channels expressed at E8 (B) and E12 (C) were separated via electrophoresis. All cDNA synthesis reactions were run side by side with a no reverse transcriptase (-RT) condition as a negative control. β-actin primers were used as a positive control for a reference gene.

Fig. 4.. Developmental increase in Kv1 channel gene expression in the avian hypothalamus.

A) Real-time quantitative PCR primers were designed to amplify predicted fragment sizes for Kv1.2 (98bp), Kv1.3 (100bp), Kv1.5 (102bp), and β-actin (100bp) expressed in hypothalamic tissue. B) Hypothalamic Kv1.2 and Kv1.5 gene expression increases from E8 to E12 (n = 3; p < 0.05); no significant change in gene expression was detected for Kv1.3 (n = 3). Real-time quantitative PCR data was calculated using the standard curve method and all Kv1 channel expression data was normalized to the β-actin reference gene for each condition.

Fig. 5.. Developmental changes in action potential characteristics in avian hypothalamic neurons.

Representative current-clamp recordings from arcuate neurons of E8 (A) and E12 (B) hypothalamic brain slices. Current was administered for either 400 ms (A) or 200 ms (B). While no difference in resting membrane potential (C) was detected between E8 (n = 3) and E12 (n = 7) time points, a significant increase in the action potential overshoot (D) was observed (* p < 0.05).