Endocrinol Metab.  2023 Oct;38(5):545-556. 10.3803/EnM.2023.1725.

Insulin Preferentially Regulates the Activity of Parasympathetic Preganglionic Neurons over Sympathetic Preganglionic Neurons

• Hyun U
• Kweon YY
• Sohn JW

• KMID: 2546985
• DOI: http://doi.org/10.3803/EnM.2023.1725

Abstract

Background
Insulin is a peptide hormone that regulates post-prandial physiology, and it is well known that insulin controls homeostasis at least in part via the central nervous system. In particular, insulin alters the activity of neurons within the autonomic nervous system. However, currently available data are mostly from unidentified brainstem neurons of the dorsal motor nucleus of the vagus nerve (DMV).
Methods
In this study, we used several genetically engineered mouse models to label distinct populations of neurons within the brainstem and the spinal cord for whole-cell patch clamp recordings and to assess several in vivo metabolic functions.
Results
We first confirmed that insulin directly inhibited cholinergic (parasympathetic preganglionic) neurons in the DMV. We also found inhibitory effects of insulin on both the excitatory and inhibitory postsynaptic currents recorded in DMV cholinergic neurons. In addition, GABAergic neurons of the DMV and nucleus tractus solitarius were inhibited by insulin. However, insulin had no effects on the cholinergic sympathetic preganglionic neurons of the spinal cord. Finally, we obtained results suggesting that the insulininduced inhibition of parasympathetic preganglionic neurons may not play a critical role in the regulation of glucose homeostasis and gastrointestinal motility.
Conclusion
Our results demonstrate that insulin inhibits parasympathetic neuronal circuitry in the brainstem, while not affecting sympathetic neuronal activity in the spinal cord.

Keyword

Autonomic nervous system; Medulla oblongata; Spinal cord; Cholinergic neurons; Electrophysiology; Autonomic function

Figure

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  Fig. 1. Insulin directly inhibits parasympathetic preganglionic neurons. (A) Image demonstrating the location of the brainstem section (left) and a parasympathetic preganglionic neuron of the dorsal motor nucleus of the vagus nerve (DMV; right) targeted for whole-cell patch clamp recordings, obtained from ChATcre/+::tdTomato mice. (B) Bright-field, tetramethylrhodamine (TRITC), fluorescein isothiocyanate (FITC), and merged images (from left to right) of the targeted neuron (arrows) in the DMV of ChATcre/+::tdTomato mice. Scale bar=50 μm. (C) Membrane potential hyperpolarizes in response to insulin (50 nM) treatment. The dashed line indicates the baseline membrane potential. (D) Voltage responses to hyperpolarizing current steps from –50 to 0 pA, applied as indicated by arrows in (C). (E) The voltage-current relationship demonstrates insulin-induced decreases in input resistance. (F) Drawings of four rostrocaudal levels of the mouse brainstem summarize the location of the acute effects of insulin on parasympathetic preganglionic neurons. Blue dots indicate hyperpolarized cells, while black dots indicate cells with no effects. (G) The membrane potential hyperpolarized in response to insulin treatment in the presence of tetrodotoxin (TTX, 500 nM) and synaptic blockers (SBs, 1 mM kynurenic acid+50 μM picrotoxin). The dashed line indicates baseline membrane potential. (H) The membrane potential did not change after insulin treatment parasympathetic preganglionic neurons from ChATcre/+::InsRf/f::tdTomato mice. The dashed line indicates baseline membrane potential. (I) Plots summarizing insulin-induced changes in membrane potential. (J) Histogram summarizing the percentage of neurons showing insulin responses. ACSF, artificial cerebrospinal fluid; 4V, fourth ventricle; 12N, nucleus of the hypoglossal nerve; NTS, nucleus tractus solitaries; AP, area postrema; CC, central canal; Cu, nucleus cuneatus; Gr, nucleus gracilis; ChAT, choline acetyltransferase; InsRf/f, insulin receptor flox. aP<0.01, Fisher’s exact test for the occurrence of hyperpolarization and no effects.

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  Fig. 2. Insulin reduces excitatory and inhibitory postsynaptic currents onto parasympathetic preganglionic neurons. (A) Traces demonstrating spontaneous excitatory postsynaptic currents (sEPSCs) before (upper) and during (lower) insulin (50 nM) treatment. Holding potential=–60 mV. (B, C) Insulin reduced the frequency (B) and mean amplitude (C) of sEPSCs. (D) Traces demonstrating spontaneous inhibitory postsynaptic currents (sIPSCs) before (upper) and during (lower) insulin treatment. Holding potential=–10 mV. (E, F) Insulin reduced the frequency (E) and mean amplitude (F) of sIPSCs. (G) Traces demonstrating miniature EPSCs (mEPSCs) before (upper) and during (lower) insulin treatment. Holding potential=–60 mV. (H, I) Insulin reduced the frequency (H) and mean amplitude (I) of mEPSCs. (J) Traces demonstrating miniature IPSCs (mIPSCs) before (upper) and during (lower) insulin treatment. Holding potential=–10 mV. (K, L) Insulin reduced the frequency (K), but not the mean amplitude (L) of mIPSCs. ACSF, artificial cerebrospinal fluid; TTX, tetrodotoxin; ns, not significant (P>0.05). aP<0.05; bP<0.01; cP<0.001 by the Wilcoxon matched-pairs signed rank test.

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  Fig. 3. Insulin hyperpolarizes GABAergic neurons of the nucleus tractus solitaries (NTS) and dorsal motor nucleus of the vagus nerve (DMV). (A) Bright-field, tetramethylrhodamine (TRITC), fluorescein isothiocyanate (FITC), and merged images (from left to right) of the targeted neuron (arrows) in the NTS of Phox2bcre/+::tdTomato mice. Scale bar=50 μm. (B) The membrane potential hyperpolarized in response to insulin (50 nM) treatment. The dashed line indicates the baseline membrane potential. (C) Plots summarizing insulin-induced changes in membrane potential. (D) Bright-field, TRITC, FITC, and merged images (from left to right) of the targeted neuron (arrows) in the NTS of Vgatcre/+::tdTomato mice. Scale bar=50 μm. (E) The membrane potential hyperpolarized in response to insulin treatment. The dashed line indicates the baseline membrane potential. (F) Plots summarize insulin-induced changes in the membrane potential. (G) Bright-field, TRITC, FITC, and merged images (from left to right) of the targeted neuron (arrows) in the DMV of Vgatcre/+::tdTomato mice. Scale bar=50 μm. (H) The membrane potential hyperpolarized in response to insulin treatment. The dashed line indicates the baseline membrane potential. (I) Plots summarizing insulin-induced changes in the membrane potential. (J) Illustration summarizing modulation of parasympathetic preganglionic neurons and their upstream neurons by insulin and insulin receptors. Phox2b, paired-like homeobox 2b; Vgat, vesicular GABA transporter; ACSF, artificial cerebrospinal fluid; AP, area postrema; ChAT, choline acetyltransferase; InsR, insulin receptor; GABA, γ-aminobutyric acid.

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  Fig. 4. Insulin does not affect the activity of sympathetic preganglionic neurons. (A) Image demonstrating the location of the spinal cord section (left) and the sympathetic preganglionic neuron of intermediolateral column (IML, right) targeted for whole-cell patch clamp recordings, obtained from ChATcre/+::tdTomato mice. (B) Bright-field, tetramethylrhodamine (TRITC), fluorescein isothiocyanate (FITC), and merged images (from left to right) of targeted neuron (arrows) in the IML of ChATcre/+::tdTomato mice. Scale bar=50 μm. (C) The membrane potential did not change in response to insulin (50 nM) treatment. The dashed line indicates the baseline membrane potential. (D, E) Plots summarizing insulin-induced changes in the membrane potential (D) and input resistance (E). Not significant (ns) (P>0.05) by Wilcoxon matched-pairs signed rank test. ChAT, choline acetyltransferase; ACSF, artificial cerebrospinal fluid.

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