Direct projections from hypothalamic orexin neurons to brainstem cardiac vagal neurons

Abstract

Orexin neurons are known to augment the sympathetic control of cardiovascular function, however the role of orexin neurons in parasympathetic cardiac regulation remains unclear. To test the hypothesis that orexin neurons contribute to parasympathetic control we selectively expressed channelrhodopsin-2 (ChR2) in orexin neurons in orexin-Cre transgenic rats and examined postsynaptic currents in cardiac vagal neurons (CVNs) in the dorsal motor nucleus of the vagus (DMV). Simultaneous photostimulation and recording in ChR2-expressing orexin neurons in the lateral hypothalamus resulted in reliable action potential firing as well as large whole-cell currents suggesting a strong expression of ChR2 and reliable optogenetic excitation. Photostimulation of ChR2-expressing fibers in the DMV elicited short-latency (ranging from 3.2ms to 8.5ms) postsynaptic currents in 16 out of 44 CVNs tested. These responses were heterogeneous and included excitatory glutamatergic (63%) and inhibitory GABAergic (37%) postsynaptic currents. The results from this study suggest different sub-population of orexin neurons may exert diverse influences on brainstem CVNs and therefore may play distinct functional roles in parasympathetic control of the heart.

Keywords: brainstem; cardiac; neurons; optogenetic; orexin; parasympathetic.

Figure 1

Localization of orexin-A immunoreactivity (red, left panel) with ChR2-EYFP (green, middle panel) driven by orexin-Cre selective expression in the lateral hypothalamus. Co-localization is shown in the right panel. Representative example of n=4 animals. Scale bar, 100 μm.

Figure 2

Optogenetic stimulation of orexin cell bodies in the lateral hypothalamus evoked reliable action potentials (A) as well as large inward whole-cell currents (B) in all orexin neurons tested (n=8) in rats that received AAV1-ChR2-EYFP viral injections. In contrast, neither light-triggered action potential firing (C) nor large inward currents (D) were observed in animals that did not receive AAV1-ChR2-EYFP viral injections (n=5 orexin neurons). Black circles represent light pulses (3 ms, 1 Hz).

Figure 3

Glutamatergic EPSCs in CVNs. Representative example of optogenetically-evoked glutamatergic postsynaptic current in an individual CVN shown in A, left. Abolishment of this this synaptic response by application of CNQX (50 μM) is demonstrated in A, right. Properties of glutamatergic synaptic events in CVNs are illustrated in B. Black circles represent average from 60 sweeps in individual CVNs (3 ms stimulation at 1 Hz) while horizontal bars are population means ± SEM. Amplitude, latency and decay time constant of the glutamatergic responses are shown from left to right.

Figure 4

GABAergic IPSCs in CVNs. Representative example of optogenetically-evoked GABAergic postsynaptic current in an individual CVN shown in A, left. Abolishment of this this current by application of gabazine (25 μM) is demonstrated in A, right. Properties of GABAergic responses in CVNs are illustrated in B. Black circles represent average from 60 sweeps in individual CVNs (3 ms stimulation at 1 Hz) while horizontal bars are population means ± SEM. Amplitude, latency and decay time constant of the GABAergic responses are shown from left to right.