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. 2009 Mar 3;159(1):83-93.
doi: 10.1016/j.neuroscience.2008.12.003. Epub 2008 Dec 14.

5-HT inhibition of rat insulin 2 promoter Cre recombinase transgene and proopiomelanocortin neuron excitability in the mouse arcuate nucleus

K Hisadome  1 , M A Smith, A I Choudhury, M Claret, D J Withers, M L J Ashford
Affiliations

5-HT inhibition of rat insulin 2 promoter Cre recombinase transgene and proopiomelanocortin neuron excitability in the mouse arcuate nucleus

K Hisadome et al. Neuroscience. .

Abstract

A number of anti-obesity agents have been developed that enhance hypothalamic 5-HT transmission. Various studies have demonstrated that arcuate neurons, which express proopiomelanocortin peptides (POMC neurons), and neuropeptide Y with agouti-related protein (NPY/AgRP) neurons, are components of the hypothalamic circuits responsible for energy homeostasis. An additional arcuate neuron population, rat insulin 2 promoter Cre recombinase transgene (RIPCre) neurons, has recently been implicated in hypothalamic melanocortin circuits involved in energy balance. It is currently unclear how 5-HT modifies neuron excitability in these local arcuate neuronal circuits. We show that 5-HT alters the excitability of the majority of mouse arcuate RIPCre neurons, by either hyperpolarization and inhibition or depolarization and excitation. RIPCre neurons sensitive to 5-HT, predominantly exhibit hyperpolarization and pharmacological studies indicate that inhibition of neuronal firing is likely to be through 5-HT(1F) receptors increasing current through a voltage-dependent potassium conductance. Indeed, 5-HT(1F) receptor immunoreactivity co-localizes with RIPCre green fluorescent protein expression. A minority population of POMC neurons also respond to 5-HT by hyperpolarization, and this appears to be mediated by the same receptor-channel mechanism. As neither POMC nor RIPCre neuronal populations display a common electrical response to 5-HT, this may indicate that sub-divisions of POMC and RIPCre neurons exist, perhaps serving different outputs.

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Figures

Fig. 1
Fig. 1
5-HT alters the excitability of RIPCre neurons. Whole-cell current clamp recordings were made from RIPCre neurons in the absence and presence of 5-HT. (A) The predominant response to bath applied 5-HT (1 μM) was hyperpolarization and inhibition of firing. This action of 5-HT was readily reversible and a second application of 5-HT produced the same effect. (B) A smaller proportion of RIPCre neurons responded to 5-HT by depolarization and increased excitability. A diary plot of firing frequency for this neuron is shown, with bath-applied 5-HT demonstrating a clear excitation. (C) The remaining proportion of RIPCre neurons tested was insensitive to 5-HT, with no evidence of a ΔVm or of firing frequency. Expanded regions of this recording are displayed to show more clearly that bath-applied 5-HT had no effect on membrane potential. The diary plot of firing frequency is shown for this neuron. (D) Increasing doses of 5-HT were locally-applied to a RIPCre neuron, shown previously to respond to 5-HT by hyperpolarization. Increasing the duration of pressure ejection of 5-HT (5 s–25 s) increased the magnitude and duration of the 5-HT response. Note that increasing the dose of 5-HT did not induce receptor desensitization at the time intervals used for successive 5-HT challenges, and there was no evidence for heterogeneity of response to 5-HT.
Fig. 2
Fig. 2
5-HT2 receptors are not responsible for 5-HT-induced RIPCre neuron depolarization. (A) Bath-applied α-me 5-HT (1 μM) did not affect the membrane potential or firing frequency of a RIPCre neuron, which had been shown previously to respond to 5-HT with depolarization (upper trace). Expanded traces and the corresponding diary plot of firing frequency for this neuron are shown. (B) Increasing the concentration of α-me 5-HT to 10 μM mimicked the depolarizing effect of 5-HT on the same RIPCre neuron. The expanded traces (lower) show the depolarization more clearly. Note that the depolarization of this neuron by either agonist was sufficient to cause severe action potential truncation. The presence of (C) the selective 5-HT2C antagonist, SB242084 (100 nM) (D) ketanserin (10 nM) or (E) the 5-HT2B antagonist SB204741 (100 nM) did not prevent locally-applied 5-HT from depolarizing and increasing the firing frequency of RIPCre neurons. (F) Application of 5 μM BW72C86, a selective 5-HT2B agonist, had no effect on the excitability of RIPCre neurons that responded to 5-HT by depolarization.
Fig. 3
Fig. 3
5-HT2 receptors do not mediate RIPCre neuron hyperpolarization by 5-HT (local application). The presence of (A) SB242084 (100 nM), (B) ketanserin (100 nM) or (C) SB204741 (100 nM) did not prevent 5-HT from hyperpolarizing RIPCre neurons. (D) Local application of BW723C86 (5 μM) did not mimic the hyperpolarizing action of 5-HT. The time gap in the recording shown in (B) is 38 min.
Fig. 4
Fig. 4
5-HT1 receptors mediate RIPCre neuron hyperpolarization by 5-HT. (A) Zacopride (2 μM) could not mimic the hyperpolarization of RIPCre neurons by 5-HT. (B) Local application of 5-CT (1 μM) hyperpolarized and inhibited the firing of RIPCre neurons, in a manner identical to that of 5-HT. (C) 5-HT-mediated hyperpolarization of RIPCre neurons was insensitive to block by 200 nM methiothepin (upper trace), but could be prevented by 2 μM methiothepin (lower trace). Local application of (D) 8-OH DPAT (10 μM) or (E) CGS12066B (1 μM) was unable to mimic the hyperpolarizing action of 5-HT. (F) The presence of SB22489 (100 nM) did not prevent 5-HT from hyperpolarizing RIPCre neurons.
Fig. 5
Fig. 5
RIPCre neurons are hyperpolarized by BRL54443, a 5-HT1E/F agonist and express 5-HT1F receptors. (A) The 5-HT1D receptor agonist, L69247 (1 nM) did not alter RIPCre neuron membrane potential or firing frequency. The gap in the recording shown is 17 min. (B) BRL54443 (20 nM) hyperpolarized and inhibited the firing of RIPCre neurons that responded by hyperpolarization to 5-HT. The gap in the recording (upper trace) is 22 min. (C) RT-PCR detection of the 5-HT1F receptor transcript in (i) whole mouse brain and (ii), (iii) in separate mouse hypothalami. (D) Stacked (30 μm) confocal image of the ARC captured in 1 μm serial sections. Dual fluorescence for GFP (green) and 5-HT1F receptor (N-terminal domain antibody: red), 5-HT1F receptors can be observed on both GFP-positive (white circles) and GFP-negative (white squares) cells, although a minority of GFP-positive neurons display little or no immunoreactivity (e.g. white arrow). Note the C-terminal domain 5-HT1F receptor antibody produced similar images (data not shown). 3v, Third ventricle. Scale bar=20 μm in (D). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
Fig. 6
Fig. 6
BRL54443 increases IK, a delayed rectifier-type K+ current, in RIPCre neurons. (A) Voltage clamp pulse protocol for recording of potassium currents. The 5 ms pre-pulse to −170 mV was used to deactivate any residual voltage dependent potassium conductance. (B) Representative currents from a RIPCre neuron elicited by a −10 mV voltage step in the absence and presence of bath-applied TEA (40 mM). Inset traces show the relatively small effect of TEA on the transient, peak potassium current. (C) Representative current families in the absence and presence of BRL54443 (20 nM). (D) Expanded single test pulses to −10 mV, showing that BRL54443 increased the steady-state current amplitude, which was reversed on washout, but had only a small effect on the peak transient current amplitude (inset expanded traces).
Fig. 7
Fig. 7
POMC neurons exhibit heterogeneous electrical responses to 5-HT. Example of a POMC neuron hyperpolarized and inhibited (A) or depolarized and excited (B) by locally-applied 5-HT. Expanded traces and the corresponding diary plot of firing frequency for this neuron are shown. (C) The majority of POMC neurons, challenged with 5-HT (local application), exhibited no ΔVm or of firing frequency, as shown in the expanded traces. (D) Example of BRL54443 (20 nM) hyperpolarization of a POMC neuron. Expanded traces are shown below for each agonist application ((i)–(iii)). Note that a second application of BRL54443 (20 nM) ∼3 min after the first was unable to elicit a response, and even after ∼9 min an increased dose of BRL54443 was required to produce significant hyperpolarization and reduction in firing. (E, F) BRL54443 reversibly increased the amplitude of the steady-state potassium current in POMC neurons. Representative current families in the absence and presence of BRL54443 (20 nM) are shown (E) with expanded single test pulses to −10 mV (F) to demonstrate this action of BRL54443 is reversible and limited to the steady-state, not the peak, potassium current amplitude.
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