Pituitary adenylate cyclase-activating peptide (PACAP) was first isolated from ovine hypothalamic tissue and characterized as a peptide which stimulates cyclic AMP elevation in rat anterior pituitary cells in culture (Miyata et al., 1989). PACAP binding to its receptors Vipr1, Vipr2 and, predominantly, PAC1 initiates cell signaling through multiple intracellular pathways (Harmar, 2001). PACAP acting at PAC1 is generally considered to engage Gαs, activating adenylate cyclase, with some isoforms also activating phospholipase C via Gαq, leading to multiple cellular responses including increased neuronal excitability (Pisegna and Wank, 1993; Spengler et al., 1993; Kawasaki et al., 1998; Emery et al., 2013; Jiang et al., 2017; Johnson et al., 2019). The PACAP/PAC1 signaling pathway has consistently been related to psychogenic stress responding, and potentiation of this pathway has been linked to psychopathologies including anxiety and PTSD in human (Ressler et al., 2011; Wang et al., 2013a; Mustafa et al., 2015). PACAP gene knock-out in the mouse results in decreased hypothalamo-pituitary-adrenal (HPA) axis activation after physical or psychogenic stress (Stroth and Eiden, 2010; Tsukiyama et al., 2011), and a hypoarousal behavioral phenotype in response to psychogenic stress (Lehmann et al., 2013; Mustafa et al., 2015; Jiang and Eiden, 2016). However, interactions within and among populations of PACAP- and PAC1-expressing neurons in brain circuits mediating behavioral responses to environmental stimulation remain to be understood. This is a critical step in integrative understanding of the functional significance of PACAP-PAC1 neurotransmission.

Exploration of PACAP-containing circuits in rodent CNS has been based on reports of the distribution of PACAP peptide and mRNA, and on expression from reporter genes under the control of a PACAP promoter transgene (Hannibal, 2002; Condro et al., 2016; Koves, 2016) or knocked-in to the PACAP gene itself (Krashes et al., 2014). Hannibal reported the anatomical distribution of PACAP projection fields and cell groups in rat CNS employing immunohistochemistry (IHC) and in situ hybridization (ISH), using radiolabeled riboprobes, in a rigorous study. However, due to the paucity of PACAP in cell bodies, dendrites, and axons compared to nerve terminals, peptide IHC has not provided more definitive PACAP chemoanatomical circuit identification in rodent brain. Similarly, ISH with radiolabeled riboprobes, while identifying PACAP-positive cell bodies, lacks the resolution to identify the co-transmitter phenotypes and precise microanatomical features of these cell groups. Thus, heterogeneity of PACAP-containing neurons within and between brain regions, both with respect to cell type and accurate regional boundaries could not be discerned. Nevertheless, an essential function for PACAP as a neurotransmitter within one or more brain behavioral circuits, and consistent with the cellular and post-synaptic actions of PACAP, has not yet emerged. A systematic analysis with accuracy at the level of cellular co-phenotypes, and with anatomical resolution to the level of sub-nuclei within CNS, is essential to complete this task.

To address these issues, we conducted a systematic analysis placing the PACAP>PAC1 signaling into anatomical and basic sensorimotor circuit contexts. We first describe in detail the overall topographical organization of expression of mRNAs encoding PACAP (Adcyap1) and its predominant receptor PAC1 (Adcyap1r1), and their co-expression with the small-molecule transmitters, glutamate, and GABA, using probes for the expression of mRNAs encoding the vesicular transporters VGLUT1 (Slc17a7), VGLUT2 (Slc17a6), and VGAT (Slc32a1), in mouse brain. We then examined the distribution of PACAP/PAC1 hubs within well-established sensory input-to-motor output pathways passing through the cognitive centers, within the context of glutamate/GABA neurotransmission. This systematic analysis has revealed several possible PACAP-dependent networks involved in sensory integration allowing environmental cues to guide motor output.

We have studied Adcyap1 and Adcyap1r1 co-expression with Slc17a7, Slc17a6, and Slc32a1 in the mouse brain, with precise region and subfield identification, using a sensitive dual ISH (DISH) method. Figure 1; Figure 1—source data 1 (Adcyap1), Figure 2 (Adcyap1r1) show examples using this method, which unambiguously labels the co-expression of two mRNAs at the single-cell level for light microscopical examination. At the light microscopical level, facile low- and high- magnification switching allows detailed serial high-power images to be located in a global histological context for precise delineation of anatomical regions/subfields as well as their rapid photo-documentation. Single-cell co-expression of two mRNA targets can be clearly observed by light microscopy with both low and high magnification.

Figure 1

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Figure 1—source data 1

Comprehensive DISH mapping of PACAP co-expression with VGLUT1, VGLUT2, and VGAT throughout mouse brain reveals an extensive distribution and diversity of cell types.

Adcyap1, PACAP-mRNA mapping within glutamatergic and GABAergic subpopulations of mouse brain. Slc17a7, Slc17a6, and Slc32a1: mRNAs encoding the VGLUT1, VGLUT2, and VGAT with respective color coding for the chromogens labeling the corresponding mRNAs. Double arrows indicate examples of colocalization of two probe mRNAs and single arrows indicate no-co-expression in given cells. Panels A–O: coronal and sagittal sections taken from the indicated Bregma or medio-lateral coordinates. The combination of mRNA probes is indicated with respective colors corresponding to the DISH method end products. The high-magnification panels are examples with relevant molecular features, of the regions labeled within low-magnification photomicrographs (see the abbreviations vide infra). Note that the high-magnifications photos are not from the same experiment of the low magnification. 3 v: third ventricle; ACA: anterior cingulate area; ac: anterior commissure; AHN: anterior hypothalamic nucleus; AI: agranular insular area; AONpv: anterior olfactory nucleus, postero-ventral; AUD: auditory areas; AVPV: antero-ventral periventricular nucleus; BAC: bed nucleus of anterior commissure; BSTpl: Bed nucleus of the stria terminalis, posterolateral; CA3v: ventral hippocampal formation, CA3; CBpj: purkinje layer of the cerebellum; CENT: central lobule of the cerebellum; COA: cortical amygdala; CSm: superior central nucleus raphe, medial part; CSl: superior central nucleus raphe, lateral part; DCO: dorsal cochlear nucleus; DR: Dorsal raphe nucleus; DMHa: dorsomedial nucleus of the hypothalamus, anterior part; ECT: ectorrinal area; ENT: entorrinal area; FL: flocculus; NLOT: nucleus of the lateral olfactory tract; gcl: granule cell layer; GU: gustatory area; IC: inferior colliculus; ICe: inferior colliculus external nucleus; IF: interfascicular nucleus of the raphe; IL: infralimbic area; IPN: interpeduncular nucleus; ISN: inferior salivatory nucleus; KF: Koelliker-Fuse subnucleus; LH: lateral habenula; ISN: inferior salivatory nucleus; LPO: lateral preoptic area; MD: mediodorsal nucleus of the thalamus; MEA: medial amygdala; MEPO: median preoptic nucleus; MGm: medial geniculate complex, medial part; MH: medial habenula; MM: medial mammillary nucleus; MOB: main olfactory bulb; MOp: primary motor area; MOs: supplemental motor area; MPO: medial preoptic area; MRN: midbrain reticular nucleus; NTS: nucleus of the tractus solitarius; opt: optic tract; ORB: orbital area; OV: vascular organ of lamina terminalis; PA: posterior amygdalar nucleus; PAG: periaqueductal gray; PB: parabrachial nucleus; PCG: contine central gray; PG: pontine gray; PH: posterior hypothalamic nucleus; PIR: piriform area; PL: prelimbic area; POL: posterior limiting nucleus of the thalamus; PP: peripeduncular nucleus; PPN: pedunculo pontine nucleus; PRN: pontine reticular nucleus; PSTN: parasubthalamic nucleus; PSV: principal sensory nucleus of the trigeminal nerve; RR: midbrain reticular nucleus, retrorubral area; RSPd: retrosplenial area dorsal; RSPv: retrosplenial area dorsal; PVH: periventricular hypothalamic nucleus; PVi: periventricular hypothalamic nucleus, intermediate part; RM: raphe magnus; SC: superior colliculus; SCm: superior colliculus, motor related; SFO: subfornical organ; SPF: subparafascicular nucleus; STN: subthalamic nucleus; SUBd: subiculum dorsal part; SGN: suprageniculate nucleus; SOC: superior olivary complex; SUM: supramammillary nucleus; TEa: Temporal association area; TM: tuberomammillary nucleus; VCO: ventral coclear nucleus; VISam: anteromedial visual área; VISp: primary visual area; VISal: anterolateral visual area; VISl: lateral visual area; VISC: visceral area; vHi: ventral hippocampus; VMH: ventromedial hypothalamic nucleus; VMHc: ventromedial hypothalamic nucleus, central part; VMHdm: ventromedial hypothalamic nucleus, dorsomedial part; VMHvl: ventromedial hypothalamic nucleus, ventrolateral part; VTA: ventral tegmental area; ZI: zona incerta.

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Figure 2

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Mapping of Adcyap1r1 co-expression with Adcyap1, Slc17a7, Slc17a6, and Slc32a1 suggests the PACAP-PAC1 system can function in autocrine and paracrine modes

Adcyap1r1 expression was studied in 152 mouse brain regions. In Figure 3, panels A–F, we show the semi-quantitative expression levels of Adcyap1r1, based on microscopic observation as different intensities of blue shading. Adcyap1 expression was also symbolized with either red or green dots (VGAT vs VGLUT mRNA co-expression) in corresponding regions. Contrasting with the discrete expression of Adcyap1, Adcyap1r1 expression was diffuse and widespread. Adcyap1r1-positive cells co-expressed Slc17a7 in the temporal hippocampus (Figure 2 panels A and A'), anterior cingulate area (ACA, panel B) and bed nucleus of anterior commissure (BAC, panel C) and Scl32a1 in pallidum and striatum structures (Figure 2D–G and Table 2). Almost all the Adcyap1-expressing neurons we studied co-expressed Adcyap1r1 (Figure 3G, from ACA and H from medial preoptic nucleus, MEPO). Besides, most neurons neighboring Adcyap1-positive cells also expressed Adcyap1r1 (single arrows). These observations suggest that the PACAP/PAC1 pathway may use autocrine and paracrine mechanisms in addition to classical neurotransmission through axon innervation and transmitter co-release.

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Figure 3—source data 1

DISH mapping of Vipr1 co-expression with VGAT in selective brain regions.

Each panel from A to J show a low-magnification image of the coronal section analyzed, indicating with arrows the regions where the corresponding high-magnification photomicrographs were taken. The red signal corresponds to SLC32a1 (the mRNA for VGAT) and the green signal correspond to Vipr1 (the mRNA for VPAC1). The abbreviatures correspond to the Allen Brain Map and are indicated in the Figure 3—source data 5, where a comparison with the expression observed in the Vipr1 ISH experiments from Allen (73927619 and 77924538) and a semiquantitative analysis of the co-expression with VGAT mRNA was done. Scale bar: 2 mm for low-amplification and 50 µm for high-amplification photomicrographs.

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Figure 3—source data 2

DISH mapping of Vipr2 co-expression with VGAT in selective brain regions.

Each panel from A to J show a low-magnification image of the coronal section analyzed, indicating with arrows the regions where the corresponding high-magnification photomicrographs were taken. The red signal corresponds to Slc32a1 (the mRNA for VGAT) and the green signal correspond to Vipr2 (the mRNA for VPAC2). The abbreviations correspond to the Allen Brain Map and are indicated in the Figure 3—source data 5, where a comparison with the expression observed in the Vipr2 ISH experiments from Allen (1104 and 1105) and a semiquantitative analysis of the co-expression with VGAT mRNA was done. Scale bar: 2 mm for low-amplification and 50 µm for high-amplification photomicrographs.

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Figure 3—source data 3

Density of VipR1 mRNA expressing cells in the mouse brain: analysis of VGAT mRNA co-expression and comparison with data from Allen Brain Atlas.

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Figure 3—source data 4

Density of VipR2 mRNA expressing cells in the mouse brain: analysis of VGAT mRNA co-expression and comparison with data from Allen Brain Atlas.

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Figure 3—source data 5

Density distribution of PAC1 expressing cells in selective cortical regions.

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PACAP also binds to two other G protein–coupled receptors highly related to PAC1, called VPAC1 (Vipr1), and VPAC2 (Vipr2) (Harmar, 2001). In all the regions where Adcyap1r1 was expressed, the expression of mRNA for either or both VIP receptors (Vipr1 and Vipr2) was also found. To simplify this already extensive report, we present the data for these two receptors in Figure 3—source data 1–4 .

Distribution and glutamatergic/GABAergic vesicular transporter mRNA co-expression with Adcyap1 and Adcyap1r1 suggests a broad function for PACAP signaling in sensorimotor processing system(s)

Retina

Retinal ganglion cells (RGC) have been reported to express PACAP at various levels of abundance previously in the literature. In rat, Adcyap1 was reported at a low level (‘+') (Hannibal, 2002) within the RGC population. CD1 mice were reported to express PACAP in retina (Kawaguchi et al., 2010), and in Adcyap1 promoter-EGFP reporter mice, EGFP expression was reported to be low (‘+') (Condro et al., 2016). With the DISH method employed here, we found a higher percentage of RGCs co-expressing Adcyap1 and Slc17a6 than previously reported (Figure 1—source data 1A1). Expression levels of Adcyap1 oscillate daily from 50% to 80% with highest levels during subjective night (see Lindberg et al., 2019 for details).

Cerebral cortex: structures derived from cortical plate

Olfactory area

High levels of PACAP expression in the olfactory area have been previously reported (Hansel et al., 2001). Here, we report the subfields of olfactory area Adcyap1-expressing neurons in detail. In the main olfactory bulb (MOB, Figure 3B, area 1), Adcyap1 was intensely expressed in outer plexiform (OPL) and mitral layers. In OPL we observed the co-expression with Slc17a7, Slc17a6 and Slc32a1. (Table 1 and Figure 1—source data 1A2 and insets). Other cell types in the internal plexiform cell layers expressed Adcyap1 at low levels with mixed glutamate/GABA molecular signatures (see Table 1). In contrast, in the accessory olfactory bulb (AOB, Figure 3C, area 83), Adcyap1 was mainly expressed in the mitral cell layer with co-expression of Slc17a7, Slc17a6 (intense), and Slc32a1 (weak) mRNAs (Table 1). Other olfactory areas intensely expressing Adcyap1 and Slc17a7 were AON (layer 1, Figure 3C, area 82), TT (Figure 3C, area 34b), DPA (Figure 3C, area 34a), Pir (layer 3, Figure 3D and E, area 129), NLOT (layer, Figure 3D, area 108; SI Figure 1F). The COA (layer, Figure 3D, areas 107a and 107b) co-expressed Adcyap1 and Slc17a6 (Figure 1—source data 1F and F5).

Isocortex

PACAP’s role in isocortex has in general been little studied (Zhang and Eiden, 2019). Moderate expression of Adcyap1 was initially reported in the cingulate and frontal cortices, with lower concentrations found in other neocortical areas using radiolabeled riboprobe ISH (Mikkelsen et al., 1994). Hannibal subsequently reported that Adcyap1-expressing cells were observed mainly in layers 1–3 and layers 5–6, and PACAP‐IR nerve fibers in all layers of the cerebral cortex; however, no detailed information about the differential expression levels across cortical regions was presented (Hannibal, 2002).

In our study, we found intense expression of Adcyap1 in isocortex to be in the frontal pole of the telencephalon, including prelimbic, infralimbic and anterior cingulate and orbital area (Figure 3, A and B, areas 43, 42, 32a and 32b, and 31, respectively). Approximately 80% of the neuronal population of the layer 2 and layer 5 co-expressed Adcyap1 and Slc17a7. A significant population of Adcyap1-expressing cells in the layer 5 of prefrontal cortices co-expressed Slc17a6 or Slc32a1 (Table 1 and Figure 1—source data 1B and B1).

In the primary and secondary motor cortices (MOp and MOs, Figure 3B, area 44), Adcyap1 was found expressed in layer 2/3 and layer 5. This pattern was also observed in somatosensory, gustatory, auditory, visual, visceral, temporal association, ectorhinal, perirhinal, retrosplenial, and post-parietal association areas (see Table 1 for more cortical area expression and strength and Figure 1—source data 1A–K, low-magnification panels).

Adcyap1r1 expression in neocortex was widespread with more homogenous aspects concerning the different cortical areas, except that in the ACA and the entorhinal cortex layers 2–3 and layers 5–6 showed very intense expression levels (Figure 3, panels B and F, areas 32 and 32a, 139 and 140) and https://gerfenc.biolucida.net/images?selectionType=collection&selectionId=98. We sampled eight neocortex regions at two coronal levels, Bregma 0.14 mm and Bregma 1.7 mm, where we observed that more than 80% of neurons in layers 2–3 and layer 5 expressed Adcyap1r1 (Figure 3—source data 5). As approximately 20% of cortical neurons are GABAergic (Ascoli et al., 2008), we tested three of the main GABAergic cell types in these cortical regions, finding that in the selected cortical areas we sampled, all of somatostatin (Sst), parvalbumin (PV) and corticotropin releasing hormone (CRH) neurons co-expressed Adcyap1r1 (Figure 3—figure supplement 1 F,G,H).

Hippocampal formation

In the mouse dorsal (septal pole) hippocampal formation, in contrast to data obtained in rat (Figure 3—figure supplement 2) and from the PACAP-EGFP transgenic reporter mouse (Condro et al., 2016), we did not find Adcyap1-expressing cells in cell body layers of CA1, CA3, and DG, as previously reported (Hannibal, 2002; Condro et al., 2016). However, we report here the marked and selective expression of Adcyap1 in pyramidal neurons of the CA2 region (Table 1 and Figure 4C, left inset). Adcyap1r1 expression was observed to be low in CA subfields and Adcyap1r1 was selectively expressed in Slc32a1-expressing cells (Figure 3—figure supplement 1, A and B). In contrast, the DG-GCL had very intense expression level of Adcyap1r1, among all brain regions, in both Slc17a7- and Slc32a1-expressing cells (Figure 2A and Figure 3—figure supplement 1, A and C). In DG hilar region (polymorphic layer), we observed few cells co-expressed Adcyap1 and Slc17a7 (Figure 4A and D). These were mossy cells co-expressing calretinin mRNA (Calb2) (Figure 4B). The Adcyap1-expressing mossy cell quantity increased in the caudo-temporal direction. This population was also described in the previous reports (Hannibal, 2002; Condro et al., 2016), however, without identification of cell type, as reported here.

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In the ventral (temporal pole) hippocampal formation, we identified two cell populations with Adcyap1 expression. One was the Slc17a7-expressing mossy cell population in the hilar region mentioned above, which was distributed from septo-dorsal to temporo-ventral hilus with increasing quantity (Figure 4C and Figure 1—source data 1J) Mossy cells are major local circuit integrators and they exert modulation of the excitability of DG granule cells (Scharfman and Myers, 2012; Sun et al., 2017). The DG granule cells strongly expressed Adcyap1r1 (Figure 3—figure supplement 1A and C). Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether they project directly to granule cells or to local inhibitory interneurons (Scharfman and Myers, 2012). However, the net effect of mossy cell loss on granule cell activity is not clear. Interestingly, dentate gyrus has a unique feature: there are two principal populations of glutamatergic cell type, the granule cells and the mossy cells. The former intensely expressed Adcyap1r1 and the latter intensely expressed Adcyap1, indicating that PACAP/PAC1 signaling may play a pivotal role for granule cell excitability.

A second population of Adcyap1-expressing neurons in ventral hippocampus was a subset of CA3 pyramidal neurons in the ventral tip, and have been addressed in the literature as CA3vv pyramidal neurons expressing the gene coch (Thompson et al., 2008; Fanselow and Dong, 2010). This population was previously photo-documented without comment in Figure 11J of the referenced report (Hannibal, 2002). This represents a distinct and novel group of pyramidal neurons in ventral CA3 (Figure 4A, C and D). These neurons strongly expressed Adcyap1, and co-expressed Slc17a7 (Figure 4, inset of B and D) as well as Calb2 (Figure 4B), with the rest of pyramidal neurons expressing Slc17a7 but neither Calb2 nor Adcyap1.

Retrohippocampal regions expressing Adcyap1 and either Slc17a7 or Slc17a6 were entorhinal area, prominently in the layer 5, parasubiculum, postsubiculum, presubiculum, and subiculum (Figure 3E and F, areas 139 and 140, 128, 124, 104). This latter region, subiculum, together with the pyramidal layer of dorsal CA1, CA2 and CA3, exhibited large differences in Adcyap1 expression strength between rat and mouse (Figure 3—figure supplement 2). Developmental studies of these regions, as well as extended amygdala, may indicate a recapitulation of phylogeny by development that is relevant to the evolution of PACAP neurotransmission across mammalian species (Zhang and Eiden, 2019).

Cerebral cortex: structures derived from cortical subplate

The subplate is a largely transient cortical structure that contains some of the earliest generated neurons of the cerebral cortex and has important developmental functions to establish intra- and extra-cortical connections (Bruguier et al., 2020). The concept of the subplate zone as a transient, dynamically changing and functional compartment arose from the combined application of functional and structural criteria and approaches (for a historical review see Judaš et al., 2010). Here, we adapt our classification from that of the Allen Brain Map (https://portal.brain-map.org/). Two noteworthy structures derived from the cortical subplate that expressed Adcyap1 and Adcyap1r1 are the claustrum (CLA) and the lateral amygdalar nucleus (LA).

The claustrum (CLA)

Owing to its elongated shape and proximity to white matter structures, the claustrum (CLA, Figure 3, panels D, E, F, area 118) is an anatomically well-defined yet functionally poorly described structure, once speculated to be the ‘seat of consciousness’ due to its extensive interconnections (Crick and Koch, 2005). CLA is located between the insular cortex and the striatum: it is a thin sheet of gray matter considered as a major hub of widespread neocortical connections (Bruguier et al., 2020). The CLA is recently reported to be required for optimal behavioral performance under high cognitive demand in the mouse (White et al., 2020). Consistent with recent work (White et al., 2018), rat CLA receives a dense innervation from the anterior cingulate cortex (ACA), one of the most prominent PACAP mRNA-expressing regions in frontal cortex, co-expressing Slc17a7 and Slc17a6 (Table 1 and Figure 3A, areas 32a and 32b, and G) and is implicated in top-down attention (Zhang et al., 2016b). The CLA interconnects the motor cortical areas in both hemispheres through corpus callosum (Smith and Alloway, 2010), where EGFP+ projections were reported in PACAP promoter-EGFP reporter mice (Condro et al., 2016). Expression of the neuropeptides somatostatin (SOM), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP) has been reported in the rat CLA (Eiden et al., 1990). In mouse CLA, more than 80% of the neurons were Slc17a7- and Slc17a6-coexpressing and less that 20% of the neurons expressed Slc32a1. PACAP content in rodent CLA has not been reported. In our study, we observed 10–15% of glutamatergic cells of the CLA co-expressed Adcyap1 and almost 100% of cells expressed Adcyap1r1 (Figure 3D, area 118).

Endopiriform nucleus and amygdalar complex

The endopiriform nucleus (Figure 3E and F, area 117) and divisions of lateral (Figure 3F, area 144), basolateral (Figure 3F, area 143), basal medial (Figure 3E, area 133), and posterior amygdalar (Figure 3F, area 106, Figure 4C) nuclei are, from a phylogenetic point of view, olfactory structures (Groor, 1976) derived from cortical subplate which the main cell population is glutamatergic, co-expressing Slc17a7 and Slc17a6 (see Table 1). Adcyap1 was intensely expressed in the lateral (dorsal) amygdala (Figure 3F, area 144), anterior basomedial amygdala (Figure 3E, area 133), posterior amygdalar nucleus (area 106), and with low expression in the endopiriform nucleus (area 117) and basomedial amygdala posterior subnucleus (Figure 1—source data 1G) and weak expression in the basolateral amygdala (Figure 3F, area 143).

Structures derived from cerebral nuclei

The main structures expressing Adcyap1 in striatum were the lateral septum (LS, Figure 3B, areas 36 and 39) and medial amygdala (MEA, Figure 3D, areas 111 and 112) (SI Figure 1G). Most of these neurons coexpressed both Slc17a7 and Slc17a6 (see Table 1). In contrast to rat brain, where expression of Adcyap1 is prominent in central amygdala and intercalated cells (Figure 3—figure supplement 2, C), in the mouse Adcyap1-expressing cells were quite sparse in these structures (Figure 3—figure supplements 2C'). Adcyap1r1 was intensely expressed in these mainly Slc32a1-expressing structures (Figure 2D,E,F and G, Table 2).

In structures within pallidum, Adcyap1 was expressed, in order of abundance, in posterior, and anterior divisions of BST, and very weakly, in the oval nucleus (Table 1).

The very intense expression of Adcyap1r1 was observed on mainly GABAergic structures derived from cerebral nuclei. Figure 2D and G show examples illustrating Adcyap1r1- and Slc32a1-coexpressing neurons in some subcortical regions. Most of cells in the BST complex co-expressed Adcyap1r1 (Figure 2D and E). In BSTov, we tested the three main GABAergic cell types that co-express somatostatin (Sst), parvalbumin (Pvalb), and corticotropin releasing hormone (Crh) and found all the three types of neurons co-expressed Adcyap1r1 (Figure 3—figure supplement 1, I,J and K). Table 2 summarizes the distribution, cell types, and strength of expression of the main Adcyap1r1-expressing group in mouse cerebral nuclei.

Table 2

Cell group / sub-field	Slc17a7 (VGLUT1)	Slc17a6 (VGLUT2)	Slc17a8 (VGLUT3)	Slc32a1 (VGAT)
Striatum
Caudoputamen
Nucleus accumbens	-	-	-	+++
Fundus of striadum	-	-	-	++
Olfactory tubercle	-	-	-	+
Lateral septum complex	-	-	-	++++
Medial amygdala (MeA)
MeAav	-	++++	-	+
MeApd	-	+	-	++++
Central amygdala (CeA)
CeA medial	-	-	-	+++
CeA lateral	-	-	-	+++
CeA capsular	-	-	-	++++
Anterior amygdala area	-	-	-	+++
Intercalated nucleus	-	-	-	++++
Cell group / sub-field	Slc17a7 (VGLUT1)	Slc17a6 (VGLUT2)	Slc17a8 (VGLUT3)	Slc32a1 (VGAT)
Pallidum
Globus pallidum internal	-	-	-	++
Globus pallidum external	-	-	-	++
Globus pallidum ventral (VP)
Substantia innominata	-	-	-	++
Magnocellular nucleus	-	-	-	+++
Medial septal complex	-	-	-	+++
Bed nuclei stria terminalis (BNST)
BNSToval	-	-	-	++++
BNSTam	-	-	-	+++
BNSTdm	-	-	-	+++
BNSTpr	-	-	-	+++
Nucleus of Diagonal Band	-	-	-	++++
Bed nucleus of anterior commissure	+	+	-	-

1. Semiquantitative annotations are used here the percentage of expressing cell/total Nissl stained nuclei: ‘-”, not detectable; '+”' weak (<20%); '++', low (40–20%); '+++', moderate (60–40%); '++++', intense (80–60%); '+++++', very intense (>80).

The bed nucleus of anterior commissure (BAC) is defined here, for the first time, as a major Adcyap1-expressing nucleus (Figure 1C, Figure 3B, area 47, Figure 5A, F, Si Figure 1E). BACs are bilateral triangular cell groups located on the dorsal corners of the chiasm of anterior commissure (ac) between the anterior part and posterior part (Papp et al., 2014). Most BAC neurons (90–95%) express Slc17a6 intensely (Figure 5B) and Slc17a7 weakly (Figure 5C) and around 5% express Slc32a1 (Figure 5D and inset). Calb2 is intensely expressed in BAC (Figure 5E), as in the case of the subset of CA3vv pyramidal neurons mentioned earlier (Figure 4A, C). The Adcyap1-expressing neurons were densely packed (Figure 1, C and Figure 5A). Almost all the Adcyap1-positive neurons co-expressed Slc17a6 mRNA (Figure 5F) and Slc17a7. Co-expression of Adcyap1 within the Slc32a1-positive neurons was not found in BAC (Figure 5G).

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PACAP→PAC1 signaling within sensory and behavioral circuits

Here, we analyze the chemo-anatomical aspects of PACAP/PAC1 mRNA expression using the results described above, but putting them into basic sensory circuit wiring maps, as well as in behavioral state and survival instinctive brain longitudinal structures, especially the hypothalamic hubs, based on existing classification schema (Swanson, 2012; Sternson, 2013; Swanson et al., 2016; Zimmerman et al., 2017; Swanson, 2018). Based on PACAP mRNA expression in these proposed sensory/behavioral circuits, we have addressed consequences of PACAP deficiency on neuronal activation and behavioral output in a mouse model of predator odor exposure and defensive behavior.

PACAP-PAC1 co-expression in forebrain sensory system

Thirst circuit for osmotic regulation

As shown above, Adcyap1 was intensely expressed in all peri/para ventricular structures directly related to thirst and osmotic regulation (Figure 7). These structures include SFO, OVLT, MEPO, and PVH (vide supra). Other hypothalamic nuclei intrinsically related to osmotic control and anticipatory drinking are SO and SCH, which were intensely Adcyap1r1-expressing.

The SFO is an embryonic differentiation of the forebrain roof plate, in a dorsal region between the diencephalon (interbrain, thalamus) and the telencephalon (endbrain) (Swanson and Cowan, 1979; Anderson et al., 2001). This nucleus lacks a normal blood-brain barrier, and so its neurons are exposed directly to peptide hormones in the blood. One such hormone is angiotensin II, whose blood levels are elevated upon loss of body fluid due to dehydration or hemorrhage. Hence, the SFO is a humorosensory organ that detects hormone levels in the circulation to control drinking behavior and body water homeostasis. The SFO is situated immediately dorsal to the third ventricle and contains intermingled populations of glutamatergic (VGLUT2-PACAP-PAC1) and GABAergic (VGAT-PAC1) neurons with opposing effects on drinking behavior. Optogenetic activation of SFO-GLUT neurons stimulates intensive drinking in hydrated mice, whereas optogenetic silencing of SFO-GLUT neurons suppresses drinking in dehydrated mice (Zimmerman et al., 2017). By contrast, optogenetic activation of SFO-GABA neurons suppresses drinking in dehydrated mice (Bichet, 2018). SFO-GLUT projections to the median preoptic nucleus (MEPO) and OVLT drive thirst, whereas SFO-GLUT projections to the ventrolateral part of the bed nucleus of the stria terminalis (BSTvl) promote sodium consumption (Zimmerman et al., 2017). SFO-GLUT projections to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus Anderson et al., 2001 have not yet been functionally annotated with cell-type specificity, but classic models suggest that these projections mediate secretion of arginine vasopressin (AVP) and, in rodents, oxytocin (OXT) into the circulation by posterior pituitary (PP)-projecting magnocellular neurosecretory cells (MNNs). Recent studies also demonstrated that these MNNs possess ascending projections innervating limbic structures such as amygdala, hippocampus, lateral habenula, and lateral hypothalamus (Hernández et al., 2015; Hernández et al., 2016; Zhang et al., 2021) in a cell-type specific manner (Zhang and Hernández, 2013; Zhang et al., 2016a; Zhang et al., 2018). When these are activated, their central collaterals can exert motivational effect on exploration and drinking behavior.

Thirst and AVP release are regulated not only by the classical homeostatic, interosensory plasma osmolality negative feedback (through SFO as a humorosensory organ), but also by novel, exterosensory, anticipatory signals (Gizowski et al., 2016). These anticipatory signals for thirst and vasopressin release converge on the same homeostatic neurons of circumventricular organs that monitor the composition of the blood. Acid-sensing taste receptor cells (which express polycystic kidney disease 2-like one protein) on the tongue that were previously suggested as the sour taste sensors also mediate taste responses to water. Recent findings obtained in humans using blood oxygen level-dependent (BOLD) signals demonstrating that the increase in the lamina terminalis (LT) BOLD signal observed during an infusion of hypertonic saline is rapidly decreased after water intake well before any water absorption in blood. This is relevant in the context of this paper since the MEPO of the hypothalamus has been shown to mediate this interesting phenomenon, integrating multiple thirst-generating stimuli (Allen et al., 2017; Gizowski and Bourque, 2017); however, functionally annotated cell-type specific circuitry has not been clarified. Very intense expression of Adcyap1 was observed in MEPO. Together, these observations open new possibilities to further understand the role of PACAP-PAC1 signaling within this nucleus for homeostatic and allostatic control.

Information about plasma sodium concentration enters the circuit through specialized aldosterone-sensitive neurons in the NTS, an intense PACAP-expressing nucleus in the medulla, that expresses 11β-hydroxysteroid dehydrogenase type 2 (NTS-HSD2 neurons) (Zimmerman et al., 2017; Bichet, 2018), which promote salt appetite and project to the LC and PBN, both intense Adcyap1-expressing and BSTvl, moderate Adcyap1-expressing and intense Adcyap1r1-expressing (Figure 6A, basic circuit presented based on above literature).

Figure 6

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Olfactory pathway

In the main olfactory system, input from olfactory sensory neurons reaches the main olfactory bulb (MOB), and the axons of the projection neurons in MOB travel to the anterior olfactory nucleus (AON), piriform cortex (Pir), and amygdala for additional processing (Wacker et al., 2011). This multi-step pathway brings olfactory information to be processed in multiple areas of the cerebral cortex, including the anterior AON and Pir. The AON, a cortical area adjacent to the olfactory bulb, is part of the main olfactory pathway. A parallel system, the accessory olfactory system, brings information, for instance that conveyed by pheromones, from the vomeronasal organ into the accessory olfactory bulb, which innervates the MEA, BST, and cortical amygdala (Figure 10). Although it was originally assumed that only the accessory olfactory system processed pheromonal and other socially relevant odors, more recent evidence suggests that social information is processed by both pathways (Wacker and Ludwig, 2012).

The olfactory system appears specially to use PACAP/PAC1 as one of its main modes of co-transmission (see section The claustrum), especially within the cell population in the outer plexiform layer of the MOB and in the mitral cell layer of the AOB, in which some cells were observed to co-express Adcyap1 and Slc17a7, Slc17a6, or Slc32a1 (Figure 1—source data 1A2) and Figure 1A–F.

Visual pathway and the circadian circuits for brain states

There is a vast literature on the visual system and we only touch on selected hubs here to emphasize the PACAP-PAC1 signaling role for visual information processing (Figure 6C). PACAP-PAC1 in the visual system was first discovered via identification of the PACAP immunopositive RGCs, soon after the discovery of PACAP itself (Hannibal et al., 1997), which project to the SCH. Here, using DISH method, we report Adcyap1 co-expressed with Slc17a6 in retina ganglion cells. Adcyap1 expression intensity followed a circadian oscillation mode (see section Retina). The RGC sends visual information through its axons forming the optic nerve, chiasm and optical tract and accessory optical tract, with its first offshoot to SCH for brain state modulation. The SCH was found intensely expressing Adcyap1r1 in both Slc32a1- and Slc17a6-expressing neurons widely and homogenously distributed, at variance with the observation in rat that PAC1 is expressed mainly in the ventral part of the SCH (Hannibal et al., 1997). Leaving the optic chiasm, the optic tract courses latero-caudally and emits a second offshoot, the accessory optical tract, splits off and courses to the midbrain, where it ends in three terminal nuclei, that is medial, lateral and dorsal terminal nuclei. They play an important role in controlling eye movements and are thus parts of the motor system and were all Adcyap1-expressing. The main optic tract continues on to end in the lateral geniculate complex (LG) of the thalamus, after giving off collaterals to the superior colliculus (SC) of the midbrain and to the olivary pretectal nucleus (OP). The dorsal part of the LG then projects to primary visual cortex (Vis), whereas the OP is involved in visual reflex, and the superior colliculus has two main roles: projecting to the motor system, and projecting to secondary visual cortical area via thalamus (Swanson, 2012). All above optic nerve/tract/accessory tract targeted structures were observed to have intense expression of Adcyap1r1. The visual cortex, parietal associated area, and retrosplenial area intensely expressed Adcyap1 (Table 1 and Figure 1—source data 1I, J, K and Figure 1A–F).

Adcyap1r1 was strongly expressed in SCH (Figure 6C and Figure 7), which projects in turn to several Adcyap1r1-expressing regions, which control brain state (sleep-wake cycle), such as midbrain behavioral state related structures (MBSR: pendunculopontine, midbrain raphe nuclei, all Adcyap1-expressing) and pons behavioral state related (PBSR: locus coeruleus, superior central nucleus of raphe, pontine reticular nucleus, all Adcyap1-expressing) (Figure 1A–F).

Figure 7

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PACAP-PAC1 co-expression in the central processing of ganglion cell sensory systems

The sensory ganglion cells were observed to express PACAP shortly after its discovery (Sundler et al., 1996). PACAP mRNA and peptide levels are increased in sensory ganglion cells within 1 day following axotomy, suggesting possible roles in neuronal protection, differentiation, nerve fiber outgrowth, and/or restoration of perineuronal tissue upon neuronal damage. The sensory ganglion cells send their axons to primary sensory nuclei in the dorsal medulla, which include: (a) auditory system, which ends in the cochlear nuclei (Figure 6D); (b) vestibular system, which ends in the vestibular nuclei (not included in this analysis); (c) gustatory system which ends in the rostral nucleus of tractus solitarius (NTS, Figure 6E); and (d) vagal/glossopharyngeal visceroceptive system, which ends in the caudal nucleus of the NTS (not included in this analysis). The special sensory nuclei in the medulla are all derived in the embryo from a highly differentiated, dorsal region of the primary hindbrain vesicle, the rombic lip (Figure 5.14 of Swanson, 2012) – they were all observed to co-express Adcyap1 and Slc17a6 from very intense (NTS) to moderate levels (DCN and VCN) (see Table 1, Figure 1A–F, Figure 1—source data 1M5-M6 and website).

Here, we analyze the PACAP/PAC1 participation in the auditory and gustatory central processing circuits.

Auditory pathway

Auditory information, for example the spectrum, timing, and location of sound, is analyzed in parallel in the lower brainstem nuclei, that is, dorsal and ventral cochlear nuclei (DCN and VCN), medial nucleus of trapezoid body (MNTB), superior olivary complex (SO), and nuclei of the lateral lemniscus (NLL). These hindbrain structures (NLL and SO) project to the inferior colliculus (IC, in midbrain) through both the excitatory and inhibitory inputs to IC. From IC, the information travels toward thalamus via relay in medial geniculate complex (MG), to reach the auditory cortex, associate auditory cortex, temporal association area; hippocampus, which subsequently projects to prefrontal cortices (Ono and Ito, 2015). Main neuronal structures that are relevant to the auditory pathway are shown in the schematic diagram of Figure 6D. The glutamatergic population of these structures were all found to co-express Adcyap1, Slc17a6 and/or Slc176a7, as well as Adcyap1r1 (Figure 3A–F).

The mainly Sc321a1-expressing structures located in subcortical regions which strongly co-expressed Adcyap1r1 (Figure 2D–G and Table 2), include, for instance, BST and the CeA with its three subdivisions (Figure 6D, structures symbolized in pink-GABAergic, and blue outline–PAC expressing) participate in cognitive and emotional auditory information, obtaining auditory sensory input from brain stem>midbrain>thalamic MG, which project to lateral amygdala (LA, a main PACAP-containing nucleus derived from cortical plate) which send input to the cognitive centers through basal lateral amygdala (BLA, Figure 2F and G).

Gustatory pathway

Chemicals (taste substances) in foods detected by sensory cells in taste buds distributed in the oropharyngeal epithelia are recognized as tastes in the gustatory cortex (GC), including granular insular cortex (GI) and agranular insular cortex (AI), also called dysgranular insular cortex (DIC), which is the primary gustatory cortex (Figure 6E). Between the peripheral sensory tissue and the GC, many neuronal hubs participate in the gustatory information processing, beginning with the geniculate ganglion (GG) of the facial nerve, cranial nerve CN-VII which receive taste stimuli from the anterior 2/3 of the tongue; the petrosal ganglion (PG) of the glossopharyngeal nerve, CN-XI, which receives the taste stimuli from the posterior third of the tongue; and nodose ganglion (NG), also called the inferior ganglion of the vagus nerve (CN-X, which innervates the taste buds of the epiglottis). The lingual nerve, a branch of mandibular nerve, from trigeminal nerve (CN-V), also innervates the anterior 2/3 portion of tongue. All these ganglia express PACAP as their co-transmitter and this expression is potentiated during injury (Sundler et al., 1996). Taste information from sensory ganglia of CN V, VII, IX, and X project to the rostral NTS of the medulla, which intensely expressed Adcyap1 and Adcyap1r1. NTS neurons send taste information to parabrachial nucleus (PB, which also intensely expressed Adcyap1 and Adcyap1r1) and then to the parvocellular division of the ventral posteromedial nucleus VPM of the thalamus, which projects to GC, as well as to the cognitive centers, central amygdala (CEA), bed nucleus of stria terminalis (BNST) and lateral hypothalamus (LH) which also reciprocally innervate the PB (Halsell, 1992). In Figure 6 panel E, the basic wiring of taste circuit was based on the literature (Halsell, 1992; Carleton et al., 2010), and modified with PACAP-PAC1 signaling annotated (Figure 1A–F).

PACAP-PAC1 signaling in major cell groups associated with behavioral state control system and hypothalamic instinctive survival system

In the course of a day, brain states fluctuate, from conscious awake information-acquiring states to sleep states, during which previously acquired information is further processed and stored as memories (Tukker et al., 2020). Anatomically and chemically distinct neuronal cell groups stretching from medulla, pons, midbrain, through the hypothalamus to the cerebral nuclei all participate in modulation of behavioral state (Swanson, 2012). We have briefly referred to sleep and waking behavioral states in analysis of the hypothalamic SCH in the visual/photoceptive pathways that project to midbrain and hindbrain behavioral state structures broadly defined. In Figure 7, left column we complemented the model of behavioral state cell groups and chemical signatures previously presented (Swanson, 2012), adding PACAP-PAC1 as informed by the current study (Figure 1A–F). Three behavioral state-related glutamatergic structures in medulla, that is raphé magnus, raphé pallidus, and raphé obscurus are not represented in the left column in recognition of the original figure design of the author (Figure 9. 5, Swanson, 2012).

In the right column of Figure 7, we present the main hypothalamic survival and locomotor control hubs with PACAP/PAC1 signaling (Figure 1A–F) noted. These hubs are SFO, OVLT, MEPO, AHN, PVN, VMH, LH, MBO, and periventricular neuroendocrine motor zone. Recent evidence suggests that these cell groups control the expression of motivated or goal-oriented behaviors, such as drinking, feeding, sex, aggression, fear, foraging behaviors (Figure 7, color-filled rectangles represent the correlative behaviors with anatomical structure based on the literature [Sternson, 2013]). The rostral segment of this behavior control column has controllers for the basic classes of goal-oriented ingestive, reproductive and defensive behaviors, common to all animals, where the caudal segment has the controllers for exploratory behavior used to obtain any goal object (Swanson, 2012).

Other relevant structures more caudal in this longitudinal brain stem column are the reticular part of SN (not labeled in Figure 7), which is involved in the control of orientating movements of the eyes and head via projecting to SC, the VTA, Adcyap1-expressing, which together with ACB (intensely Acyap1r1-expressing) and STN (intensely Adcyap1-expressing), form the hypothalamic locomotor region.

After analyzing the above presence of PACAP and its receptor PAC1 in sensory-cognitive to motor output, a question emerged: what would be the behavioral and neuronal activation consequences if PACAP signaling were deficient?

PACAP knockout impairs predator odor salience processing via reducing neuronal activation and vesicular transporter expression in key PACAP-PAC1 nuclei

To assess the behavioral implications of PACAP action within one of these circuits, we examined the effect of knockout of PACAP expression in brain on defensive behavior initiated via olfaction, using a predator odor paradigm (Figure 8A and B). Using a modified open-field box with a lidded container with cat urine litter, and wild type (WT) and PACAP-deficient (KO) C57Bl/6N mice (Hamelink et al., 2002), we assessed the defensive locomotion patterns during cat odor exposure, which include displacement pattern in the four quadrants of the box; approach to the stimulus (urine-containing cat litter) with complete retreats and incomplete retreats; and periods of immobility (freezing behavior). DISH for fos, Slc17a7, Slc17a6, and Slc32a1 expression in the two experimental groups was performed and analyzed post hoc.

Figure 8

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Raw data for panels C, D and E.

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Cat urine triggered purposeful movement such as sniff>retreat in WT subjects (Figure 8Ai, green dashed lines indicate complete sniff>retreat cycles). In contrast, the movement patterns exhibited by KO subjects consisted in sniff>retreat cycles of short distances and the mice often returned to the container location area (Figure 8Bi, red dashed lines indicate incomplete/short sniff>retreat cycles). PACAP-deficient mice had significant reduction of complete retreats (p<0.001) and significant augmentation of incomplete retreats (p<0.001) (Figure 8D). Figure 8Aii and Bii show the X-Y dimension movement heat-maps for each group. Movement analysis by quadrants showed a significant increase in pixel values of the KO group in the quadrant where the odorant container was located (Figure 8C, p<0.05, C1). It is worth mentioning that movement in Z dimension (repetitive jumping), characteristic of PACAP-deficient mice (Hashimoto et al., 2001) was not represented in the X-Y dimension heat maps, and investigating the phenomenon is beyond the scope of the study. KO mice also exhibited significant reduction in freezing behavior (Figure 8D, p<0.001). To test whether or not the behavior of PACAP-deficient mice could be explained in whole or part by hypo- or anosmia, a standard test for olfaction was carried out. Wild-type and PACAPko mice were tested for the ability to retrieve a buried cookie as described in Materials and methods. Retrieval times for wild-type and PACAP-deficient mice were not significantly different (wild-type retrieval latency 38.0 ± 10.4 s; PACAPko retrieval latency 44.5 ± 12 s, p=0.7058, t-test, n = 6/group, each group comprising three male and three female mice approximately 8 weeks of age). We conclude that PACAP-deficient and wild-type C57Bl6/N mice were equally able to locate a food source using olfaction.

The Fos expression associated with the predator odor exposure behavioral test was significantly reduced, in PACAP-deficient mice, in olfactory areas, the mitral layer and glomerular layers, as well as higher predator-odor processing centers, for instance, the amygdaloid complex MEA and CEA, the LS, the endbrain ACA, PL and IL cortical areas, the BSTov; the hindbrain PB complex, which provides the main upstream PACAP signaling source especially for BSTov and CEA (http://connectivity.brain-map.org/) for the above cognitive centers, and the hypothalamic VMH, which control the aggressive behavior (Figure 8E–G), as well as the cerebellar cortex in which we found reduced fos expression in the granule cell layer of the ansiform lobule, which is reported to influence limb movement control (Manni and Petrosini, 2004; Zhu et al., 2006). In contrast, KO mice had increased Fos expression, compared to wild type, in the motor cortex layer VI and V (Figure 8E,H and I).

We assessed fos expression in the PACAP/PAC1 system including both glutamatergic and GABAergic neurons in response to odorant exposure, using DISH technique and Slc17a7, Slc17a6, and Slc32a1 probes to identify the glutamatergic and GABAergic neurons. Surprisingly, we observed a sharp reduction in the abundance of three vesicular transporters in the main PACAP-containing nuclei we described vide supra (Figure 9). This reduction was observed both as reduced abundance of the ISH staining puncta at the single-cell level and the density of expressing cells in the given region (Figure 9E).

Figure 9

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Here, we describe in detail the overall topographical organization of expression of mRNA encoding PACAP, and that of its predominant receptor PAC1, and their co-expression with the small-molecule transmitters, glutamate and GABA, using probes for mRNA encoding the vesicular transporters VGLUT1/2 and VGAT, as obligate markers for glutamatergic and GABAergic phenotypes, respectively, in mouse brain. A highly sensitive dual in situ hybridization method (RNAscope 2.5HD duplex detection) was used and corroborated with Allen Brain Atlas ISH data throughout brain. We report glutamatergic or GABAergic identity of 181 Adcyap1-expressing cell groups as indicated by co-expression of corresponding vesicular transporters. All Adcyap1-expressing neurons studied, and most of their neighboring cells, co-express mRNA encoding PAC1 (Adcyap1r1), indicating that the PACAP/PAC1 pathway, besides using classical neurotransmission through axon innervation and transmitter release, likely employs autocrine and paracrine mechanisms for signal transduction as well.

With the information obtained by PACAP/PAC1 expression and co-localization with small-molecule transmitters, we explored the hypothesis that the broad brain distribution of PACAP may reflect a more specific physiological function at a systems level. We examined the distribution of PACAP/PAC1 expression within specific sensory input-to-motor output pathways passing through the cognitive centers mentioned in these studies, in which most of the hubs analyzed used PACAP>PAC1 signaling within the context of glutamate/GABA neurotransmission. This systematic analysis has revealed several possible PACAP-dependent networks involved in the highest levels of motor control,that is the hypothalamic pattern initiator and controller system, intermediate (or intervening) levels of somatosensory information processing, and the complex cognitive and behavioral state control for behavior.

The autocrine/paracrine and neuroendocrine nature of PACAP>PAC1 signaling and glutamate and GABA vesicular transporter expression

The analysis of our data showed an impressive amount of Adcyap1 and Adcyap1r1 coexpression at both single-cell and regional levels (Figure 3), suggesting that the PACAP/PAC1 pathway uses autocrine and paracrine mechanisms in addition to classical neurotransmission through axon innervation and transmitter co-release (Hökfelt et al., 1984; Zhang and Eiden, 2019). That is, besides sending PACAP-containing axons to innervate regions where PAC1 is strongly expressed, PACAP may be released through soma and dendrites, to bind PAC1 expressed on the same and neighboring cells, to prime the neuron for optimum function (Leng, 2018). We still know relatively little about the possible functional interactions and control of release of PACAP>PAC1 signaling in this aspect. However, the observation reported in this study about down-regulation of vesicular transporter mRNAs in PACAP knockout mice, in regions/cell populations which were normally Adcyap1-expressing in wild-type mice, provides one possible example of such a function for PACAP. Activity-dependent regulation of both glutamate and GABA vesicular transporter synthesis and membrane insertion has been reported (De Gois et al., 2005; Erickson et al., 2006; Doyle et al., 2010). PACAP signaling most commonly leads to a net increase in neuronal excitability through modulation of intrinsic membrane currents and transiently increasing intracellular calcium concentration (Johnson et al., 2019). Decreased vesicular transporter mRNA expression would be expected at the cellular level to decrease transmitter quantal size, thus decreasing excitatory/inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) (Billups, 2005), and is consistent with our behavior data. PACAP absence in KO mice resulted in a behavioral hypoarousal response to moderate predator-odor stimulus. The vesicular transporter mRNA loss observed at RNA level upon complete PACAP deficiency, revealed in predator odor-exposed mice, may reflect a modulatory role for PACAP, in wild-type animals, on vesicular transporter mRNA (and protein) abundance within a more restricted, but functionally relevant, range depending on the level of PACAP release and autocrine signaling.

PACAP-deficient mice show hyperlocomotion and abnormal gait, as well as bouts of repetitive jumping (data not shown, but see Hashimoto et al., 2001, Gaszner et al., 2012, Hattori et al., 2012). In 1930, Hinsey, Ranson and McNattin reported a visionary experimental result, done in cats and rabbits about the rôle of the hypothalamus in locomotion (Hinsey, 1930). Quoting Swanson’s interpretation of this early experiment: “when the central nervous system is transected roughly between the mesencephalon and diencephalon, the animals displayed no spontaneous locomotor behavior. They remain immobile until stimulated. On the other hand, animals with transection roughly between diencephalon and telencephalon (or upon complete removal of the cerebral hemispheres, and the thalamus) display considerable spontaneous behavior. In fact, they can be hyperactive when the transection is a bit caudal, but cannot spontaneously eat, mate, or defend themselves. Notably, these last three functions are preserved at a primitive level when the transection is just slightly more rostral". This evidence, combined with the selective lesions or stimulations of the hypothalamus, suggests that the ventral half of the diencephalon (hypothalamus) contains neural mechanisms regulating setpoints for locomotor and other classes of motivated behavior’ (Figure 8.9, Swanson, 2012). As reported here, the major cell groups in the ventral diencephalon, associated with behavioral state (Figure 7, left column) and behavioral control (Figure 7, right column) all use PACAP/PAC1 signaling for co-transmission. Hence, movement pattern/initiation disorders would be expected in PACAP-deficient animals. Moreover, we found marked reductions in Slc32a1 in Purkinje cells (PC) of the ansiform lobule of the cerebellum, as well as significant reduction in Fos expression in the granule cells (Figure 9 panels Ds), the main excitatory input of the PC (Manni and Petrosini, 2004). The reduced VGAT availability in PC in the lateral lobules of the cerebellum, involved mainly in coordinating the movement patterns of the limbs (Manni and Petrosini, 2004, Sakayori et al., 2019), necessarily weakens the inhibitory influence on the excitatory output of the deep cerebellar nuclei which make ascending projections to several forebrain and midbrain structures, including hypothalamus (Zhu et al., 2006). In Figure 10, we propose a model based on the analysis of this study to provide a rationale for hyperlocomotion and hypoarousal behavior observed in PACAP- deficient mice. Under normal conditions, the hypothalamic motor initiator/controller centers (Figure 7, longitudinal PACAP cell groups in hypothalamus) are modulated, for instance, by direct excitatory (e.g. from deep cerebellar nuclei, DCN), indirect excitatory (through disinhibition of GABAergic projections from BNST, CEA to GABAergic neuronal circuits of hypothalamus) and inhibitory (from lateral septum) influences (Swanson, 2012; Figure 10A). We showed in this study that PACAP deficiency caused sharp downregulation of both vesicular glutamate and GABA transporters in the key nuclei that in wild-type mice Adcyap1 is expressed, such as cerebellar Purkinje cells that send inhibitory input to DCN; the prefrontal cortex, that with reduced VGLUT1 expression (Figure 9), could result in a deficient activation of striatum and pallidum cognitive structures, that send GABAergic input to the hypothalamic centers, through direct inhibition or indirect excitation through disinhibition mechanisms, for locomotor patterning and initiation. Unbalanced excitation/inhibition occurring in this hypothalamic region may result in hyperexcitation to motor pathways ending in primary motor cortex (Figure 8, higher fos expression in primary motor cortex in KO subjects), which command the spinal motor neurons for muscle activities (Figure 10B).

Figure 10

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All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures.

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Author details

1. Limei Zhang

   1. Department of Physiology, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico
   2. Section on Molecular Neuroscience, National Institute of Mental Health, Intramural Research Program, Bethesda, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Contributed equally with

   Vito S Hernandez

   For correspondence

   limei@unam.mx

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7422-5136

2. Vito S Hernandez

   Department of Physiology, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing

   Contributed equally with

   Limei Zhang

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1486-1659

3. Charles R Gerfen

   Laboratory of Systems Neuroscience, National Institute of Mental Health, Intramural Research Program, Bethesda, United States

   Contribution

   Resources, Data curation, Software, Writing - review and editing

   Competing interests

   No competing interests declared

4. Sunny Z Jiang

   Section on Molecular Neuroscience, National Institute of Mental Health, Intramural Research Program, Bethesda, United States

   Contribution

   Data curation, Investigation, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

5. Lilian Zavala

   Department of Physiology, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico

   Contribution

   Software, Formal analysis, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

6. Rafael A Barrio

   1. Section on Molecular Neuroscience, National Institute of Mental Health, Intramural Research Program, Bethesda, United States
   2. Department of Complex Systems, Institute of Physics, National Autonomous University of Mexico (UNAM), Mexico, Mexico

   Contribution

   Formal analysis, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0987-0785

7. Lee E Eiden

   Section on Molecular Neuroscience, National Institute of Mental Health, Intramural Research Program, Bethesda, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Project administration, Writing - review and editing

   For correspondence

   eidenl@nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7524-944X

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