A subset of neurons in the paraventricular nucleus of the hypothalamus directly project to liver-related premotor neurons in the ventrolateral medulla

Abstract

Sympathetic circuits including pre-sympathetic neurons in the ventrolateral medulla (VLM) and in the paraventricular nucleus (PVN) of the hypothalamus play an important role in the regulation of hepatic glucose metabolism. Despite the importance of central regulatory pathways, specific information regarding the circuits of liver-related neurons is limited. Here, we tested the hypothesis that PVN neurons are directly connected to spinally-projecting liver-related neurons in the VLM of mice. Pseudorabies virus (PRV) was used to identify liver-related neurons and time-dependent analyses revealed the location and distribution of neurons in the PVN and ventral brainstem. Four days following PRV injection, most liver-related neurons were found in the VLM and consist of both catecholaminergic (CA) and non-CA neurons. Furthermore, in addition to PRV inoculation, a monosynaptic viral tracer was used to identify VLM-projecting PVN neurons to specifically dissect PVN-VLM connections within the liver pathway. Five days following PRV inoculation, our anatomical findings revealed that a small population of liver-related PVN neurons projected to the VLM. In addition, photo-stimulation of axonal projections from SIM1-expressing PVN neurons resulted in evoked excitatory postsynaptic currents in a subset of spinally projecting liver-related neurons in the VLM. In summary, our data demonstrate the existence of monosynaptic, glutamatergic connections between PVN neurons and pre-sympathetic liver-related neurons in the VLM. These new findings regarding the central circuits involved in the sympathetic regulation of the liver provide further information necessary for developing new strategies to improve glucose homeostasis via modulation of the autonomic nerves.

Keywords: Liver; Paraventricular nucleus; Pseudorabies virus; Sympathetic nervous system; Ventral brainstem.

Fig. 1.

Liver-related neurons in the mouse brain four days after inoculation of the liver. (A): Representative images of liver-related neurons (PRV-152, green) in the Kolliker-Fuse nucleus (KF) (A1); noradrenaline cell group A5 (A2); locus coeruleus (LC) (A3); rostral ventrolateral medulla (RVLM), lateral paragigantocellular nucleus (LPG), raphe obscurus nucleus (ROb) and raphe pallidus nucleus (RPa) (A4); dorsal motor nucleus of the vagus (DMV) (A5) and lateral reticular nucleus (LRt) (A6). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (B): Distribution of liver-related neurons in the ventral medulla. Dash vertical lines positioned at bregma −6.84 mm and bregma −6.24 mm indicate the position of the VLM where the majority liver-related neurons (58.8 %) were identified. Data are expressed as % ± SEM of the total number of liver-related neurons counted in the ventral medulla of mice (n = 11 mice). (C): Images of confocal fluorescence microscopy showing co-expression of liver-related neurons (PRV-152, green) and DBH expressing neurons (tdTomato, red) in the RVLM of a DBH-tdT reporter mouse, at low (C1) and high (C2) magnification. Arrows indicate co-labeled neurons. Scale bar: 100 μm. (D): Representative images illustrating liver-related neurons (PRV-152, green) in the antero-posterior paraventricular nucleus of the hypothalamus (PVN) (D1-D6). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (E): Schematic drawing illustrates the brain regions that contain liver-related neurons four days after inoculation of the liver with pseudorabies virus 152. Abbreviations: 3 V: 3rd ventricle, 4 V: 4th ventricle, 7n: facial nerve, AH: anterior hypothalamic area, Aq: aqueduct, f: fornix, Gi: gigantocellular reticular nucleus, IO: inferior olive, NTS: nucleus of solitary tract, Pe: periventricular hypothalamic nucleus, PRV: pseudorabies virus, py: pyramidal tract, scp: superior cerebellar peduncle, SO: superior olive nucleus, VLM: ventrolateral medulla, VMH: ventromedial hypothalamic nucleus, VMM: ventromedial medulla.

Fig. 2.

Liver-related neurons in the mouse brain five days after inoculation of the liver. (A): Representative images of liver-related neurons (PRV-152, green) in the antero-posterior paraventricular nucleus of the hypothalamus (PVN) (A1-A6). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (B): Images of confocal fluorescence microscopy showing co-expression of liver-related neurons (PRV-152, green) and oxytocin expressing neurons (red) in the PVN, at low (B1) and high (B2) magnification. Arrows indicate co-labeled neurons (B2). Scale bar: 100 μm. (C): Images of confocal fluorescence microscopy showing co-expression of liver-related neurons (PRV-152, green) and vasopressin expressing neurons (blue) in the PVN, at low (C1) and high (C2) magnification. Arrows indicate co-labeled neurons (C2). Scale bar: 100 μm. (D): Representative images of liver-related neurons (PRV-152, green) in the bed nucleus of the stria terminalis (BNST) (D1); paraventricular nucleus of the hypothalamus (PVN) (D2); central amygdaloid nucleus (CeA) (D3); lateral hypothalamic area (LH) (D4); parasubthalamic nucleus (PSTh) (D5); Kolliker-Fuse nucleus (KF) (D6); noradrenaline cell group A5 (D7); lateral paragigantocellular nucleus (LPG), gigantocellular reticular nucleus (Gi) and raphe pallidus nucleus (RPa) (D8); locus coeruleus (LC) (D9); rostral ventrolateral medulla (RVLM), LPG, raphe obscurus nucleus (ROb) and RPa (D10); dorsal motor nucleus of the vagus (DMV) (D11); lateral reticular nucleus (LRt) and ROb (D12). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (E): Schematic drawing illustrates the brain regions that contain liver-related neurons five days after inoculation of the liver with pseudorabies virus 152. Abbreviations: 3 V: 3rd ventricle, 4 V: 4th ventricle, aca: anterior commissure, AH: anterior hypothalamic area, cp: cerebral peduncle, f: fornix, ic: internal capsule, IO: inferior olive, LSO: lateral superior olive, mcp: middle cerebellar peduncle, ml: medial lemniscus, mt: mammillothalamic tract, NTS: nucleus of solitary tract, opt: optic tract, Pe: periventricular hypothalamic nucleus, Pr5: principal sensory trigeminal nucleus, PRV: pseudorabies virus, py: pyramidal tract, VLM: ventrolateral medulla, VMM: ventromedial medulla.

Fig. 3.

A subset of liver-related PVN neurons projects to the VLM. (A): Representative images of VLM-projecting neurons (AAVrg-GFP, green) in the vascular organ of the lamina terminalis (VOLT) and medial preoptic area (MPA) (A1); motor cortex (Mctx) (A2), insular cortex (Ictx) (A3, A7); ventrolateral preoptic nucleus (VLPO) (A4), bed nucleus of the stria terminalis (BNST) (A5); central amygdaloid nucleus (CeA) (A6); magnocellular nucleus of the lateral hypothalamus (MCLH) (A8); dorsomedial hypothalamic nucleus (DMH) (A9); rostral interstitial nucleus (RI) (A10); red nucleus (red) (A11); lateral lemniscus (LL) (A12, A16); dorsal raphe (DR) (A13); LL and middle cerebellar peduncle (mcp) (A14); tegmental nucleus (Tg) (A15); Kolliker-Fuse nucleus (KF) and DR (A17); locus coeruleus (LC) (A18, A19); nucleus of solitary tract (NTS) (A20). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (B): Representative images of VLM-projecting neurons (AAVrg-GFP, green) in the antero-posterior paraventricular nucleus of the hypothalamus (PVN) (B1-B6). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (C): Schematic drawing illustrates the brain regions that project to the VLM. (D): Images of confocal fluorescence microscopy showing co-expression of a VLM-projecting neuron (AAVrg-GFP, green) and an oxytocin expressing neuron (OX, red) in the PVN, at low (D1) and high (D2–4) magnification. The arrow indicates a co-labeled neuron (D2–4). Scale bar: 100 μm. (E): Images of confocal fluorescence microscopy showing co-expression of VLM-projecting neurons (AAVrg-GFP, green) and liver-related neurons (PRV-614, red) in the PVN, at low (E1) and high (E2–4) magnification. Arrows indicate co-labeled neurons (E2–4). Scale bar: 100 μm. Abbreviations: 12 N: hypoglossal nucleus, 3 V: 3rd ventricle, 4 V: 4th ventricle, AH: anterior hypothalamic area, Amb: nucleus ambigus, Aq: aqueduct, Cb: cerebellum, cp: cerebral peduncle, CPu: caudate putamen, Cu: cuneate nucleus, D3V: dorsal 3rd ventricle, DMV: dorsal motor nucleus of the vagus, ec: external capsule, f: fornix, fr: fasciculus retroflexus, HDB: nucleus of the horizontal limb of the diagonal band, ic: internal capsule, LV: lateral ventricle, mt: mammillothalamic tract, PAG: periaqueductal gray, PB: parabrachial nucleus, Pe: periventricular hypothalamic nucleus, RRF: retrorubral field, rs: rubrospinal tract, scp: superior cerebellar peduncle, Sctx: somatosensory cortex, VLM: ventrolateral medulla.

Fig. 4.

A population of SIM1 expressing PVN neurons project to the VLM as part of the hypothalamus-liver pathway. (A): Representative images of the anteroposterior paraventricular nucleus of the hypothalamus (PVN) (A1-A6) illustrating the location of SIM1 expressing neurons with projections to the ventrolateral medulla (AAVrg-GFP, green). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (B): Images of confocal fluorescence microscopy showing co-expression of liver-related neurons (PRV-152, green) and SIM1 expressing neurons (tdTomato, red) in the PVN of a SIM1-tdT reporter mouse, at low (B1) and high (B2–4) magnification. Arrows indicate co-labeled neurons (B2–4). Scale bar: 100 μm. Abbreviations: 3 V: 3rd ventricle, AH: anterior hypothalamic area, Pe: periventricular hypothalamic nucleus, SIM1: Single-minded homolog 1.

Fig. 5.

Projection sites of SIM1 expressing PVN neurons. (A): Representative images illustrating the presence of axonal projections from SIM1 expressing PVN neurons (ChR2-mCherry, red) throughout the brain. (A1) Asterisks indicate bilateral injection of AAV5-EF1a-DIO-mCherry into the paraventricular nucleus of the hypothalamus (PVN) of SIM1-Cre mice. Axonal projections from SIM1 expressing PVN neurons were found in the supraoptic decussation (sox) (A2); median eminence (Me) (A3); paraventricular thalamic nucleus (PV) (A4); posterior hypothalamus (PH) (A5); posterior subthalamic nucleus (PSTh) (A6); arcuate nucleus (ARC) (A7); periaqueductal gray (PAG) (A8, A11); ventral tegmental area (VTA) (A9); substantia nigra (SN) (A10); retrorubral field (RRF) (A12); parabrachial nucleus (PB) (A13); raphe magnus nucleus (RMg) and raphe pallidus nucleus (RPa) (A14); noradrenaline cell group A5 (A15); locus coeruleus (LC) and PB (A16); rostral ventrolateral medulla (RVLM) and lateral paragigantocellular nucleus (LPG) (A17); dorsal motor nucleus of the vagus (DMV) and nucleus of the solitary tract (NTS) (A18, A22); inferior olive (IO) and raphe obscurus nucleus (ROb) (A19); LPG (A20); rostral ventral respiratory group (RVRG) (A21); DMV, NTS and area postrema (AP); NTS (A24). Distance from bregma (β) is indicated at the bottom left. Scale bar: 100 μm. (B): Schematic drawing illustrates the brain regions that contain fibers from SIM1 expressing PVN neurons. Abbreviations: 3 V: 3rd ventricle, 4 V: 4th ventricle, 7n: facial nerve, Amb: nucleus ambigus, Aq: aqueduct, cp: cerebral peduncle, Cu: cuneate nucleus, D3V: dorsal 3rd ventricle, DMH: dorsomedial hypothalamic nucleus, f: fornix, fr: fasciculus retroflexus, Hb: habenular nucleus, LL: lateral lemniscus, ml: medial lemniscus, MT: medial terminal nucleus, mtg: mammillotegmental tract, opt: optic tract, pc: posterior commissure, Py: pyramidal tract, RM: retromammillary nucleus, scp: superior cerebellar peduncle, SO: superior olive, VLM: ventrolateral medulla, VMH: ventromedial hypothalamic nucleus, VMM: ventromedial medulla.

Fig. 6.

Functional connections between SIM1 expressing PVN neurons and liver-related VLM neurons. (A): Image illustrating ChR2 expression in the paraventricular nucleus of the hypothalamus (PVN) of a SIM1-Cre mouse injected with AAV5-Ef1a-DIO-hChR2-h134R-mCherry (A1). Representative traces demonstrate that light stimulation (3 ms) evoked inward current (A2) and action potential (A3) in a ChR2-expressing PVN-SIM1 neuron. (B): Images illustrate that liver-related VLM neurons (PRV-152, green) are surrounded with axonal projections (ChR2-mCherry, red) from SIM1 expressing PVN neurons (B1). The RVLM (boxed area) is shown on the higher magnification images where an arrow indicates a liver-related VLM neuron (B2-B4). Scale bar: 100 μm. (C): Arrow points to a recorded liver-related VLM neuron. Enlarged images illustrate that the recording (neurobiotin 350, blue) was conducted from a liver-related (PRV-152, green) neuron that responded to light stimulation of ChR2 expressing fibers (ChR2-mCherry, red). Scale bar: 100 μm (low magnification), 25 μm (high magnification). (D): Representative traces following light stimulation of ChR2-expressing projections from SIM1 PVN neurons. In some cases, excitatory post-synaptic currents (holding −60 mV, left traces) were evoked after 100 ms light stimulation. Abbreviations: 3 V: 3rd ventricle, LPG: lateral paragigantocellular nucleus, RVLM: rostral ventrolateral medulla.