Whole-Brain Mapping of Direct Inputs to and Axonal Projections from GABAergic Neurons in the Parafacial Zone

Abstract

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.

Keywords: GABAergic neurons; Parafacial zone; Parvocellular reticular formation; Trans-synaptic tracing.

Fig. 1

Experimental procedures for retrograde monosynaptic tracing to identify direct inputs to PZ GABAergic neurons. A Design of viral vectors for RV-mediated trans-synaptic retrograde tracing. B Schematic of the PZ injection procedure for AAV-CAG-DIO-TVA-EGFP, AAV-CAG-DIO-RvG, and RV-EnvA-ΔG-DsRed in VGAT-Cre mice. C Schematic showing unilateral injection site of virus in the PZ in VGAT-Cre mice. D Representative images showing RV-labeled neurons (red) in VGAT-Cre mice but not in wild-type mice (blue, nuclei stained with DAPI). Scale bar, 550 μm. E Representative immunostaining images of the PZ from VGAT-Cre mice (left-most) showing that virus co-expression was restricted to the PZ. Scale bar, 200 μm. Starter cells (yellow, expressing both EGFP and DsRed) indicated by arrows in enlarged view of the PZ (right). Scale bar, 20 μm. 7n, facial nerve or its root.

Fig. 2

Representative images of selected regions with monosynaptic inputs to PZ GABAergic neurons. RV-labeled cells identified by red dots in a series of coronal sections to show the whole-brain input pattern. Scale bar, 200 μm. DPGi, dorsal paragigantocellular nucleus; DpMe, deep mesencephalic nucleus; Int, interposed cerebellar nucleus; InWh, intermediate white layer of the superior colliculus; IPAC, interstitial nucleus of the posterior limb of the anterior commissure; IRt, intermediate reticular nucleus; LH, lateral hypothalamic area; Med, medial (fastigial) cerebellar nucleus; MVePC, medial vestibular nucleus, parvocellular part; PSTh, parasubthalamic nucleus; S2, secondary somatosensory cortex; SNR, substantia nigra, pars reticulata; SpVe, spinal vestibular nucleus; ZI, zona incerta. Regions were identified based on the mouse brain atlas [19].

Fig. 3

Whole-brain distribution of inputs to PZ GABAergic neurons. Percentages of retrogradely-labeled input neurons from 62 regions in VGAT-Cre mice. Brain areas are grouped into 8 general structures (cortex, pallidum, amygdala, hypothalamus, midbrain, pons, medulla, and cerebellum).

Fig. 4

Schematic of the major presynaptic inputs to PZ GABAergic neurons. Color scale represents the percentage of total inputs. BST, bed nucleus of the stria terminalis; DpMe, deep mesencephalic nucleus; IRt, intermediate reticular nucleus; LH, lateral hypothalamic area; LV, lateral ventricle; M1, primary motor cortex; M2, secondary motor cortex; Med, medial (fastigial) cerebellar nucleus; PnO, pontine reticular nucleus, oral part; SNR, substantia nigra, pars reticulata; ZI, zona incerta.

Fig. 5

Experimental procedures for tracing whole-brain efferents of PZ GABAergic neurons. A Design of the AAV construct expressing a loxP site and EGFP for Cre-dependent cell tracing. B Schematic of injection procedure of the output tracing virus, AAV-EF1α-DIO-EGFP, in VGAT-Cre mice. C Example image of virus expressed in the PZ (left). Scale bar, 100 μm. Arrows indicate virus-infected GABAergic neurons (middle). Green, EGFP; blue, DAPI. Scale bar, 30 μm. Axons exhibiting green fluorescence were traced by the virus (right). Scale bar, 100 μm. PZ, parafacial zone; 7n, facial nerve or its root.

Fig. 6

Representative coronal sections of PZ GABAergic neurons projecting to selected brain regions. Green, axon projections stained with EGFP; blue, nuclei stained with DAPI. Scale bar, 1 mm. BST, bed nucleus of the stria terminalis; CeA, central amygdaloid nucleus; Dk, nucleus of Darkschewitsch; DMTg, dorsomedial tegmental area; DpMe, deep mesencephalic nucleus; DR, dorsal raphe nucleus; DTT, dorsal tenia tecta; InC, interstitial nucleus of Cajal; LH, lateral hypothalamic area; LPB, lateral parabrachial nucleus; MnPO, median preoptic nucleus; MPB, medial parabrachial nucleus; mRt, mesencephalic reticular formation; MVePC, medial vestibular nucleus, parvocellular part; PCRtA, alpha part of the parvocellular reticular formation; PF, parafascicular thalamic nucleus; PnO, pontine reticular nucleus, oral part; PR, prerubral field; PSTh, parasubthalamic nucleus; SI, substantia innominata; VMPO, ventromedial preoptic nucleus; VPM, ventral posteromedial thalamic nucleus; VTA, ventral tegmental area; ZI, zona incerta. Brain regions were identified based on the mouse brain atlas [19].