Circuit and synaptic organization of forebrain-to-midbrain pathways that promote and suppress vocalization

CEA (Central amygdalar nucleus) MBA:536 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from both PAG-USV neurons and GABAergic PAG neurons labelled neurons in the central amygdala, which were almost entirely GABAergic by in situ hybridization.

Fig. 1A–B; Fig. 1—figure supplement 1C

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sAMY (Striatum-like amygdalar nuclei) MBA:278 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Monosynaptic rabies tracing from PAG-USV neurons and from GABAergic PAG neurons labelled neurons at the boundary between central and medial amygdala; nearly all of these amygdalar input cells (~98%) were VGAT-positive.

Fig. 1A–B; Fig. 1—figure supplement 1B

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sAMY (Striatum-like amygdalar nuclei) MBA:278 projects to LPO (Lateral preoptic area) MBA:226 · Mouse

tdTomato-labelled axons of AmgC/M-PAG neurons were observed innervating the lateral preoptic area.

Fig. 2—figure supplement 3 (top left)

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AI (Agranular insular area) MBA:95 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from the PAG vocal gating circuit labelled upstream neurons in insular cortex.

Fig. 1—figure supplement 2B

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LH (Lateral habenula) MBA:186 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from the PAG vocal gating circuit labelled neurons in the lateral habenula.

Fig. 1—figure supplement 4D

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MPO (Medial preoptic area) MBA:523 projects to sAMY (Striatum-like amygdalar nuclei) MBA:278 · Mouse

GFP-labelled axons from PAG-projecting preoptic neurons were found in the amygdalar central-medial boundary zone, overlapping the cell bodies of AmgC/M-PAG neurons.

Fig. 2—figure supplement 3 (middle left)

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BST (Bed nuclei of the stria terminalis) MBA:351 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Transsynaptically labelled neurons in the bed nucleus of the stria terminalis provided monosynaptic input to PAG-USV and GABAergic PAG neurons.

Fig. 1—figure supplement 4C

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ILA (Infralimbic area) MBA:44 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Monosynaptic rabies tracing from PAG-USV neurons and from GABAergic PAG neurons labelled neurons in infralimbic cortex.

Fig. 1—figure supplement 2A

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sAMY (Striatum-like amygdalar nuclei) MBA:278 projects to PAG (Periaqueductal gray) MBA:795 · Mouse

AAV-retro-Cre injected into the caudolateral PAG together with a Cre-dependent fluorophore in the amygdala labelled cell bodies in the central-medial boundary zone, showing these neurons project to the PAG; about 92% of them expressed VGAT.

Fig. 4A; Fig. 4—figure supplements 1–2

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PALv (Pallidum, ventral region) MBA:835 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Monosynaptic rabies tracing from PAG-USV and GABAergic PAG neurons labelled neurons in the ventral pallidum.

Fig. 1—figure supplement 4A

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MPO (Medial preoptic area) MBA:523 projects to SNc (Substantia nigra, compact part) MBA:374 · Mouse

GFP-labelled axons of PAG-projecting preoptic neurons densely innervated the substantia nigra pars compacta.

Fig. 2—figure supplement 3; Results, axonal projections

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PVT (Paraventricular nucleus of the thalamus) MBA:149 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Transsynaptic tracing from PAG-USV and GABAergic PAG neurons labelled neurons in the paraventricular thalamic nucleus.

Fig. 1—figure supplement 4E

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MOp (Primary motor area) MBA:985 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Transsynaptically labelled neurons providing monosynaptic input to PAG-USV and GABAergic PAG neurons were found in primary motor cortex.

Fig. 1—figure supplement 2C–D

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AHN (Anterior hypothalamic nucleus) MBA:88 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Monosynaptic rabies tracing from the PAG vocal gating circuit labelled neurons in the anterior hypothalamus.

Fig. 1—figure supplement 3A

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MPO (Medial preoptic area) MBA:523 projects to PAG (Periaqueductal gray) MBA:795 · Mouse

Injection of a retrogradely infecting AAV driving Cre into the caudolateral PAG, combined with a Cre-dependent reporter/ChR2 in the preoptic area, labelled POA cell bodies, demonstrating a preoptic projection to the PAG. All 319 labelled POA-to-PAG cells expressed VGAT and 278 of 319 co-expressed Esr1.

Fig. 2A, 2C; Fig. 4—figure supplement 2

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VMH (Ventromedial hypothalamic nucleus) MBA:693 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from the PAG vocal gating circuit labelled upstream neurons in the ventromedial hypothalamus.

Fig. 1—figure supplement 3C

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MPO (Medial preoptic area) MBA:523 projects to VTA (Ventral tegmental area) MBA:749 · Mouse

Anterograde labelling of PAG-projecting preoptic neurons with GFP showed dense axonal projections to the ventral tegmental area.

Fig. 2—figure supplement 3; Results, axonal projections

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MPO (Medial preoptic area) MBA:523 projects to PAG (Periaqueductal gray) MBA:795 · Mouse

GFP labelling of PAG-projecting preoptic neurons revealed dense terminal fields in the caudolateral PAG that overlapped with those of AmgC/M-PAG neurons.

Fig. 2—figure supplement 3 (bottom right)

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MPO (Medial preoptic area) MBA:523 functionally connects to PAG (Periaqueductal gray) MBA:795 · Mouse

Optogenetic activation of ChR2-expressing preoptic axons within the PAG evoked IPSCs in 26 of 36 fluorescently identified VGAT+ PAG neurons (mean ~329 pA at 0 mV); the currents persisted in TTX/4AP and were blocked by gabazine, indicating direct GABAergic synapses.

Fig. 6A–D

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LS (Lateral septal nucleus) MBA:242 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from the PAG vocal gating circuit labelled lateral septal neurons as direct inputs.

Fig. 1—figure supplement 4B

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sAMY (Striatum-like amygdalar nuclei) MBA:278 projects to PAG (Periaqueductal gray) MBA:795 · Mouse

tdTomato/ChR2 labelling of PAG-projecting amygdalar central-medial boundary neurons revealed dense axon terminal fields in the caudolateral PAG overlapping those of POAPAG neurons.

Fig. 2—figure supplement 3 (bottom right); Fig. 5B

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MPO (Medial preoptic area) MBA:523 functionally connects to PAG (Periaqueductal gray) MBA:795 · Mouse

When preoptic axons in the PAG were optogenetically stimulated, IPSCs were detected in only 1 of 23 recorded PAG-USV neurons, even though IPSCs could be evoked in neighbouring unlabelled cells, suggesting few or weak direct preoptic synapses onto PAG-USV neurons.

Fig. 6—figure supplement 1

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MEZ (Hypothalamic medial zone) MBA:467 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies-labelled input neurons to the PAG vocal gating circuit were found in the premammillary nucleus of the medial hypothalamus.

Fig. 1—figure supplement 3E

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AUD (Auditory areas) MBA:247 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from PAG-USV neurons and GABAergic PAG neurons labelled neurons in auditory cortex.

Fig. 1—figure supplement 2E

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ACA (Anterior cingulate area) MBA:31 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Monosynaptic rabies tracing from PAG-USV and GABAergic PAG neurons labelled cingulate cortical neurons.

Fig. 1—figure supplement 2C

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PVH (Paraventricular hypothalamic nucleus) MBA:38 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Transsynaptically labelled input neurons to PAG-USV and GABAergic PAG neurons were observed in the hypothalamic paraventricular nucleus.

Fig. 1—figure supplement 3B

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MPO (Medial preoptic area) MBA:523 projects to RR (Midbrain reticular nucleus, retrorubral area) MBA:246 · Mouse

Axons of PAG-projecting preoptic neurons densely innervated the retrorubral (A8) dopaminergic cell group.

Fig. 2—figure supplement 3 (top right); Results, axonal projections

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SSp (Primary somatosensory area) MBA:322 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Transsynaptic labelling from the PAG vocal gating circuit revealed input neurons in primary somatosensory cortex.

Fig. 1—figure supplement 2D

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MOs (Secondary motor area) MBA:993 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from the PAG vocal gating circuit labelled upstream neurons in secondary motor cortex.

Fig. 1—figure supplement 2C–D

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PH (Posterior hypothalamic nucleus) MBA:946 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Transsynaptic tracing from PAG-USV and GABAergic PAG neurons labelled neurons in the posterior hypothalamus.

Fig. 1—figure supplement 3D

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LHA (Lateral hypothalamic area) MBA:194 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Rabies tracing from the PAG vocal gating circuit labelled neurons in the lateral hypothalamic area.

Fig. 1—figure supplement 3B–C

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sAMY (Striatum-like amygdalar nuclei) MBA:278 projects to RR (Midbrain reticular nucleus, retrorubral area) MBA:246 · Mouse

Dense axonal labelling from AmgC/M-PAG neurons was seen in the retrorubral (A8) area.

Fig. 2—figure supplement 3; Results, axonal projections

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ZI (Zona incerta) MBA:797 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Neurons in the zona incerta were transsynaptically labelled as monosynaptic inputs to PAG-USV and GABAergic PAG neurons.

Fig. 1—figure supplement 3B

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sAMY (Striatum-like amygdalar nuclei) MBA:278 projects to SNc (Substantia nigra, compact part) MBA:374 · Mouse

Axons of AmgC/M-PAG neurons densely innervated the substantia nigra pars compacta.

Fig. 2—figure supplement 3; Results, axonal projections

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sAMY (Striatum-like amygdalar nuclei) MBA:278 projects to VTA (Ventral tegmental area) MBA:749 · Mouse

Anterograde tracing showed dense axonal projections from PAG-projecting amygdalar central-medial boundary neurons to the ventral tegmental area.

Fig. 2—figure supplement 3; Results, axonal projections

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sAMY (Striatum-like amygdalar nuclei) MBA:278 functionally connects to PAG (Periaqueductal gray) MBA:795 · Mouse

In brain slices, optogenetic stimulation of ChR2-expressing GABAergic AmgC/M axons in the PAG evoked IPSCs in 16 of 29 CANE-tagged PAG-USV neurons recorded in TTX/4AP (mean ~180 pA at 0 mV); these currents reversed near the chloride equilibrium potential and were eliminated by gabazine, and no EPSCs were seen at -70 mV.

Fig. 5C–F

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MPO (Medial preoptic area) MBA:523 synapses onto PAG (Periaqueductal gray) MBA:795 · Mouse

Pseudotyped rabies tracing from CANE-tagged PAG-USV neurons and from VGAT+ caudo/ventrolateral PAG neurons labelled cell bodies in the medial preoptic area, indicating monosynaptic preoptic input to both PAG cell types. In situ hybridization showed about 84% of these labelled preoptic cells express VGAT.

Fig. 1A–B; Fig. 1—figure supplement 1A

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CEA (Central amygdalar nucleus) MBA:536 projects to PAG (Periaqueductal gray) MBA:795 · Mouse

AAV-retro-Cre injected into the caudolateral PAG of a Cre-dependent tdTomato reporter mouse produced no cell body labelling in the central amygdala, but the authors attribute this to restricted tropism of the retrograde AAV rather than to an absent projection, since rabies tracing did label CeA neurons.

Fig. 4—figure supplement 2

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