Synergism between two BLA-to-BNST pathways for appropriate expression of anxiety-like behaviors in male mice

BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 functionally connects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

Optogenetic stimulation of posterior BLA terminals strongly increased firing of oval BNST neurons and evoked robust excitatory (and inhibitory) postsynaptic currents there, consistent with the dense terminal field.

Fig. 2e–g; Supplementary Fig. 4a–d

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BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 functionally connects to BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 · Mouse

Optogenetic activation of adBNST-projecting anterior BLA principal neurons increased the spike rate of GABAergic interneurons recorded in the lateral posterior BLA.

Fig. 8f–h

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AUD (Auditory areas) MBA:247 synapses onto BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 · Mouse

Projection-specific monosynaptic rabies tracing from adBNST-projecting anterior BLA neurons labelled presynaptic cells in auditory cortical areas; the share of labelled cells there differed from that for ovBNST-projecting posterior BLA neurons.

Fig. 9c, d

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 functionally connects to BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 · Mouse

Photostimulating ovBNST-projecting lateral posterior BLA principal neurons robustly suppressed firing of adBNST-projecting anterior BLA principal neurons, an effect dependent on GABA-A receptors.

Fig. 8j–n, r

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 functionally connects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

Although posterior BLA terminals produced monosynaptic excitatory currents in adBNST neurons, the disynaptic GABAergic currents they recruited were larger, giving a high inhibition/excitation ratio and a net suppression of adBNST firing that was reversed by GABA-A receptor blockade; chemogenetic activation of pBLA to dBNST neurons likewise lowered adBNST calcium signals in vivo.

Fig. 2h–j; Fig. 3c, e, f; Supplementary Fig. 3, 5

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AUD (Auditory areas) MBA:247 synapses onto BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 · Mouse

Monosynaptic rabies tracing from ovBNST-projecting lateral posterior BLA neurons labelled presynaptic neurons in auditory areas, at a proportion significantly different from that of the aBLA-to-adBNST population.

Fig. 9c, d

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BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 functionally connects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

Light activation of ChR2-expressing anterior BLA terminals in dorsal BNST raised the tonic firing rate of adBNST neurons, and evoked monosynaptic excitatory currents (TTX-blocked, 4-AP-restored) whose amplitude exceeded the accompanying disynaptic inhibitory currents; chemogenetic activation of aBLA to dBNST neurons likewise increased adBNST calcium signals in vivo.

Fig. 2b–d; Fig. 3a, b, d; Supplementary Fig. 3b, c

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 functionally connects to BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 · Mouse

Activation of ovBNST-projecting lateral posterior BLA principal neurons increased the firing of GABAergic interneurons recorded in the anterior BLA.

Fig. 8q

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BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

AAV-vGluT1-EYFP in the anterior BLA labelled axon terminals that were found almost only in the anterodorsal part of the dorsal BNST, quantified as relative fluorescence intensity in four mice.

Fig. 1a–d

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

Retro-AAV-mCherry injected into the anterodorsal BNST labelled principal neurons in the posterior BLA as well as the anterior BLA, showing adBNST-projecting cells are spread along the antero-posterior axis.

Fig. 1e–h

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BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

After oval BNST retro-AAV injection only a few labelled neurons were seen in the anterior BLA, in contrast with the many labelled cells in posterior BLA.

Fig. 1i–l

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CA1 (Field CA1) MBA:382 synapses onto BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 · Mouse

Monosynaptic rabies tracing showed ventral CA1 neurons among the direct presynaptic inputs to adBNST-projecting anterior BLA principal neurons.

Fig. 9c, d

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

Retro-AAV-mCherry restricted to the oval BNST labelled principal neurons located mainly in the posterior BLA (3 mice).

Fig. 1i–l

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AI (Agranular insular area) MBA:95 synapses onto BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 · Mouse

Rabies-based input mapping of ovBNST-projecting lateral posterior BLA neurons revealed labelled presynaptic cells in the agranular insular cortex, with a proportion differing from the anterior BLA population.

Fig. 9c, d

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ENT (Entorhinal area) MBA:909 synapses onto BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 · Mouse

Entorhinal cortex neurons were retrogradely labelled by projection-specific monosynaptic rabies tracing from adBNST-projecting anterior BLA neurons.

Fig. 9c, d

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ENT (Entorhinal area) MBA:909 synapses onto BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 · Mouse

Entorhinal cortex neurons were retrogradely labelled by monosynaptic rabies tracing from ovBNST-projecting lateral posterior BLA neurons, with a labelling share significantly different from that of the aBLA-to-adBNST population.

Fig. 9c, d

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

Besides the dense oval BNST labelling, posterior BLA anterograde tracing also produced scattered, moderate terminal labelling in the anterodorsal BNST.

Fig. 1c, d

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BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

A tiny (15 nl) retro-AAV-mCherry deposit confined to the anterodorsal BNST retrogradely labelled principal neurons distributed through the anterior BLA subdivision (4 mice).

Fig. 1e–h

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

A small AAV-vGluT1-EYFP injection into the medial part of posterior BLA labelled fibers that terminated chiefly in the anterodorsal BNST, spatially separate from the lateral pBLA to ovBNST projection.

Supplementary Fig. 8d–i

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CA1 (Field CA1) MBA:382 synapses onto BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 · Mouse

Monosynaptic rabies tracing showed ventral CA1 neurons among the direct presynaptic inputs to ovBNST-projecting lateral posterior BLA principal neurons, at a different proportion than for the anterior population.

Fig. 9c, d

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BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 projects to BST (Bed nuclei of the stria terminalis) MBA:351 · Mouse

AAV-vGluT1-mCherry in the posterior BLA produced densely packed terminal labelling in the oval subregion of dorsal BNST, measured as relative fluorescence intensity in four mice.

Fig. 1a–d

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AI (Agranular insular area) MBA:95 synapses onto BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 · Mouse

Rabies-based input mapping of adBNST-projecting anterior BLA neurons revealed labelled presynaptic cells in the agranular insular cortex.

Fig. 9c, d

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BLAa (Basolateral amygdalar nucleus, anterior part) MBA:303 functionally connects to BLAp (Basolateral amygdalar nucleus, posterior part) MBA:311 · Mouse

Photostimulating ChR2-expressing adBNST-projecting anterior BLA principal neurons reduced the firing of ovBNST-projecting lateral posterior BLA principal neurons; the suppression was abolished by the GABA-A antagonist picrotoxin, showing it is mediated by local interneurons.

Fig. 8c–e, i

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