Hippocampal PACAP signaling activation triggers a rapid antidepressant response

Abstract

Background: The development of ketamine-like rapid antidepressants holds promise for enhancing the therapeutic efficacy of depression, but the underlying cellular and molecular mechanisms remain unclear. Implicated in depression regulation, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is investigated here to examine its role in mediating the rapid antidepressant response.

Methods: The onset of antidepressant response was assessed through depression-related behavioral paradigms. The signaling mechanism of PACAP in the hippocampal dentate gyrus (DG) was evaluated by utilizing site-directed gene knockdown, pharmacological interventions, or optogenetic manipulations. Overall, 446 mice were used for behavioral and molecular signaling testing. Mice were divided into control or experimental groups randomly in each experiment, and the experimental manipulations included: chronic paroxetine treatments (4, 9, 14 d) or a single treatment of ketamine; social defeat or lipopolysaccharides-injection induced depression models; different doses of PACAP (0.4, 2, 4 ng/site; microinjected into the hippocampal DG); pharmacological intra-DG interventions (CALM and PACAP6-38); intra-DG viral-mediated PACAP RNAi; and opotogenetics using channelrhodopsins 2 (ChR2) or endoplasmic natronomonas halorhodopsine 3.0 (eNpHR3.0). Behavioral paradigms included novelty suppressed feeding test, tail suspension test, forced swimming test, and sucrose preference test. Western blotting, ELISA, or quantitative real-time PCR (RT-PCR) analysis were used to detect the expressions of proteins/peptides or genes in the hippocampus.

Results: Chronic administration of the slow-onset antidepressant paroxetine resulted in an increase in hippocampal PACAP expression, and intra-DG blockade of PACAP attenuated the onset of the antidepressant response. The levels of hippocampal PACAP expression were reduced in both two distinct depression animal models and intra-DG knockdown of PACAP induced depression-like behaviors. Conversely, a single infusion of PACAP into the DG region produced a rapid and sustained antidepressant response in both normal and chronically stressed mice. Optogenetic intra-DG excitation of PACAP-expressing neurons instantly elicited antidepressant responses, while optogenetic inhibition induced depression-like behaviors. The longer optogenetic excitation/inhibition elicited the more sustained antidepressant/depression-like responses. Intra-DG PACAP infusion immediately facilitated the signaling for rapid antidepressant response by inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII)-eukaryotic elongation factor 2 (eEF2) and activating the mammalian target of rapamycin (mTOR). Pre-activation of CaMKII signaling within the DG blunted PACAP-induced rapid antidepressant response as well as eEF2-mTOR-brain-derived neurotrophic factor (BDNF) signaling. Finally, acute ketamine treatment upregulated hippocampal PACAP expression, whereas intra-DG blockade of PACAP signaling attenuated ketamine's rapid antidepressant response.

Conclusions: Activation of hippocampal PACAP signaling induces a rapid antidepressant response through the regulation of CaMKII inhibition-governed eEF2-mTOR-BDNF signaling.

Keywords: Antidepressant response; Ketamine; Novelty suppressed feeding (NSF); Optogenetic; Pituitary adenylate cyclase-activating polypeptide (PACAP).

Fig. 1

Effects of augment or attenuation of hippocampal pituitary adenylate cyclase-activating polypeptide (PACAP) signaling on the onset of antidepressant response or depression-like behaviors. Requirement of hippocampal PACAP expression upregulation for the onset of the antidepressant response following paroxetine treatment. a Latency to eat and food consumption in novelty-suppressed feeding (NSF) at 4, 9, 14 d by paroxetine treatment (n = 10). b The expression of PACAP in hippocampus 4, 9, 14 d by paroxetine treatment by Western blotting analysis. c The expression of PACAP in the hippocampus 14 d by paroxetine treatment by ELISA analysis (n = 6). t-test, ^***P < 0.001. Intra-dentate gyrus (DG) PACAP infusion elicited a rapid and long-lasting antidepressant response. d The procedure of experimental design, drug treatment and behavioral test (upper), and the tract of the cannulation for intra-DG infusion of PACAP (below, scale bar = 100 μm). e Latency to eat and food consumption in NSF at 30 min post-PACAP [F (3, 28) = 5.940, P = 0.0029; F (3, 28) = 14.16, P < 0.0001]. f Time in the center and total distance traveling in open filed test (OFT) at 1 d post-PACAP [F (3, 28) = 7.349, P = 0.0009; F (3, 28) = 0.8864, P = 0.4602]. g Immobility time in forced swimming test (FST) at 1 d post-PACAP [F (3, 28) = 16.99, P < 0.0001]. h The experiment was designed for tests of long-term antidepressant effects by different doses of PACAP. i Immobility time in tail suspension test (TST) at 30 min post-PACAP [F (3, 28) = 10.82, P < 0.0001]. j Latency to eat and food consumption in NSF at 2 d post-PACAP [F (3, 28) = 16.35, P < 0.0001; F (3, 28) = 41.92, P < 0.0001]. k Immobility time in TST at 3 d post-PACAP [F (3, 28) = 5.658, P = 0.0037]. l Immobility time in FST at 7 d post-PACAP [F (3, 28) = 3.415, P = 0.0309] (n = 8). One-way ANOVA, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. The immediate and lasting antidepressant activity with the infusion of 0.4 ng/site of hippocampal PACAP in mice subjected to chronic mild stress (CMS). m Behavioral comparisons across different groups, including immobility time in the TST at 30 min [F (2, 21) = 39.74, P < 0.0001]. n Post-PACAP-infusion, immobility time in the FST [F (2, 21) = 96.85, P < 0.0001]. o Sucrose preference in the SPT [F (2, 21) = 63.94, P < 0.0001]. p Time in the center and total distance in OFT [F (2, 21) = 0.1119, P = 0.8947; F (2, 21) = 0.08789, P = 0.9162] at day 1 post-PACAP [F (2, 21) = 0.1119, P = 0.8947; F (2, 21) = 0.08789, P = 0.9162]. q Latency to eat and food consumption in NSF [F (2, 21) = 61.25, P < 0.0001; F (2, 21) = 45.74, P < 0.0001] at day 2 post-PACAP [F (2, 21) = 61.25, P < 0.0001; F (2, 21) = 45.74, P < 0.0001] (n = 7 – 8). One-way ANOVA, ^***P < 0.001

Fig. 2

Social defeat (SD) stress or lipopolysaccharide (LPS) injections attenuated the hippocampal pituitary adenylate cyclase-activating polypeptide (PACAP) levels, and knockdown of the dentate gyrus (DG) PACAP induced depression-like behaviors. Following SD exposure (a, b), LPS injections (c, d), or control (Con) condition, animals received behavioral tests on the same day in order after the termination of the stress protocol. The brains were harvested to test the expression of PACAP in the hippocampus. a Behavioral test following SD for sucrose preference in the sucrose preference test (SPT); the ratio of social interaction times in the social interaction test (SIT); and immobility time in the tail suspension test (TST) (n = 6). b The expression of PACAP in the hippocampus following SD (n = 5). c Behavioral tests following LPS for sucrose preference in SPT, immobility time in TST, and immobility time in forced swimming test (FST) (n = 8). d The expression of PACAP in the hippocampus following LPS injections (n = 5). t-test, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. e Experimental timeline for tests after bilateral hippocampal dentate gyrus (DG) transfected with virus containing PACAP RNAi or scramble control sequence (SCR) and the design of PACAP RNAi construct and a representative figure for the bilateral hippocampal DG of viral transfection (scale bar = 100 μm). f The behavioral comparisons between PACAP knockdown and SCR mice, including immobility time in the TST, immobility time in FST, and latency to eat and food consumption in the novelty-suppressed feeding (NSF) test [F (1, 28) = 12.78, P = 0.0013; F (1, 28) = 9.638, P = 0.0043; F (1, 28) = 20.25, P = 0.0001; F (1, 28) = 39.69, P < 0.0001] (n = 8). Two-way ANOVA, ^***P < 0.001

Fig. 3

Optogenetic stimulation or inhibition of dentate gyrus (DG) pituitary adenylate cyclase-activating polypeptide (PACAP)-expressing neurons bidirectionally regulated depression-related behaviors immediately and lastingly. a The scheme of optogenetic manipulation of DG PACAP-expressing neuronal subtypes, the histological verification of the AAV transfection sites (scale bar = 50 μm), and electrophysiological verification of the neuronal activity. b The experiment was designed for a transient (4 min) activation of PACAP neurons. c Depression-related behavioral effects by 4 min opto-stimulation in the ChR2 and scramble control (Con) mice, including immobility time in the tail suspension test (TST) before (light off) and during (light on) 4 min opto-stimulation [F (1, 20) = 21.45, P = 0.0002]. Two-way ANOVA, ^***P < 0.001. d Immobility time in the forced swimming test (FST) tested at 1 d after the opto-stimulation (n = 6). e The experiment was designed for a long-time (30 min) optogenetic activation of PACAP-expressing neurons. f Immobility time in the TST at 30 min after the opto-stimulation. g Immobility time in FST at 3 d after the opto-stimulation. h Latency to feed and food consumption in the novelty-suppressed feeding (NSF) test at 5 d post-opto-stimulation (n = 6). t-test, ^**P < 0.01, ^***P < 0.001. i Immobility time in TST was measured before (light off) and during 4 min opto-inhibition (light on) [F (1, 20) = 31.73, P < 0.0001]. Two-way ANOVA, ^***P < 0.001. j Immobility time in FST measured at 1 d post 4 min opto-inhibition. k Immobility time in TST following 30 min opto-inhibition. l Latency to feed and food consumption in NSF test. m sucrose preference test (SPT) at 3 d post-opto-inhibition (n = 6). t-test, ^***P < 0.001

Fig. 4

Intra-dentate gyrus (DG) pituitary adenylate cyclase-activating polypeptide (PACAP) infusion regulated neuroplasticity signaling of CaMKII/mTOR/eEF2. a Expressions of p-CaMKII/CaMKII, p-eEF2/eEF2, p-mTOR/mTOR, p-4EBP1, PKA, BDNF, PSD95, and PACAP at 30 min following intra-DG PACAP or control (Con) infusion (n = 5). b Cell signaling at 30 min post a 4-minute optogenetic activation of PACAP-ergic neurons in the DG between the ChR2 group and scramble control (Con) group, including expressions of PACAP, p-CaMKII/CaMKII, p-mTOR/mTOR, BDNF (n = 5). t-test, ^**P < 0.01, ^***P < 0.001. CaMKII calcium/calmodulin-dependent protein kinase II, mTOR mammalian target of rapamycin, eEF2 eukaryotic elongation factor 2, 4EBP1 4E-binding protein 1, PKA protein kinase A, BDNF brain-derived neurotrophic factor, PSD95 postsynaptic density protein 95

Fig. 5

Effects of intra-dentate gyrus (DG) blockade of CaMKII signaling inhibition on the rapid onset of antidepressant response and protein synthesis-related signaling induced by pituitary adenylate cyclase-activating polypeptide (PACAP). a Experimental procedure for intra-DG microinfusion of CaMKII agonist (CALM) or cerebrospinal fluid (CSF) followed by PACAP or control (Con) microinfusion. b Latency to feed and food consumption in the novelty-suppressed feeding (NSF) test at 30 min [F (1, 28) = 31.91, P < 0.0001; F (1, 28) = 20.43, P = 0.001]. c Immobility time in tail suspension test (TST) at day 1 [F (1, 28) = 0.144, P = 0.707] (n = 8). d The effects of pre-activation of CaMKII on intra-DG PACAP-induced cell signaling response (n = 6). Expressions of p-mTOR/mTOR, p-4EBP1, p-eEF2/eEF2, and BDNF at 30 min after intra-DG PACAP infusion [p-mTOR/mTOR: F (1, 20) = 53.35, P < 0.0001; p-4EBP1: F (1, 20) = 33.65, P < 0.0001; p-eEF2/eEF2: F (1, 20) = 26.09, P < 0.0001; BDNF: F (1, 20) = 79.54, P < 0.0001]. Two-way ANOVA, ^***P < 0.001. CaMKII calcium/calmodulin-dependent protein kinase II, mTOR mammalian target of rapamycin, eEF2 eukaryotic elongation factor 2, 4EBP1 4E-binding protein 1, BDNF brain-derived neurotrophic factor

Fig. 6

The influence of dentate gyrus (DG) pituitary adenylate cyclase-activating polypeptide (PACAP) signaling on the rapid onset of the antidepressant response of ketamine. a Co-immunostaining of PACAP (red color) and NMDAR subunit NR1 (green color) in the DG. The cell nuclei were stained with DAPI (blue color). Yellow arrows point to representative cells co-labeled with PACAP and NR1, and white arrows point to those singly labeled (scale bars = 50 μm and 20 μm, respectively). b The peptide expression of PACAP in the hippocampus at 30 min post a single administration of ketamine or control (Con) by Western blotting analysis (n = 5). c The peptide expression of PACAP in the hippocampus at 30 min post a single administration of ketamine or Con by ELISA analysis (n = 6). t-test, ^***P < 0.001. d The experiment design for intra-DG microinfusion of PACAP 6-38 or cerebrospinal fluid (CSF). e Latency to eat and food consumption in novelty-suppressed feeding (NSF) at 30 min post ketamine [F (1, 28) = 28.93, P < 0.0001; F (1, 28) = 16.20, P = 0.0004]. Two-way ANOVA, ^***P < 0.001. f Immobility time in the forced swimming test (FST) at 1 d post ketamine [F (1, 28) = 0.038, P = 0.847] (n = 8). g The expression of hippocampal BDNF at 30 min post-intra-DG PACAP 6-38 microinfusion and ketamine [F (1, 16) = 18.23, P < 0.001] (n = 5). h The experiment was designed for transfected with a virus containing PACAP RNAi or scramble control sequence (SCR). i Immobility time in the tail suspension test (TST) at 30 min post ketamine [F (1, 20) = 4.475, P = 0.047]. j Immobility time in the FST at 2 h post ketamine [F (1, 20) = 7.197, P = 0.0143]. k Latency to eat and food consumption in the NSF at 2 d post ketamine [F (1, 20) = 49.86, P < 0.0001; F (1, 20) = 6.557, P = 0.0186]. l The percentage of the drinking of sucrose water in the sucrose preference test (SPT) at 2 d post ketamine [F (1, 20) = 3.828, P = 0.045] (n = 6). Two-way ANOVA, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 7

The schematic diagram for hippocampal dentate gyrus (DG) pituitary adenylate cyclase-activating polypeptide (PACAP) regulation of the rapid and persistent antidepressant response. Activating hippocampal DG PACAP signaling produces the onset of antidepressant response, following a repeated treatment of the delayed-onset antidepressant paroxetine to increase the PACAP transcription, or acute treatment of ketamine to instantly enhance PACAP peptide expression. PACAP signaling activation initially inhibits CaMKII signaling, which controls the subsequent eEF2-mTOR-BDNF signaling for eliciting the rapid antidepressant response. PACAP activation of PKA-CREB signaling was involved in the following persistent antidepressant response. CaMKII calcium/calmodulin-dependent protein kinase II, mTOR mammalian target of rapamycin, eEF2 eukaryotic elongation factor 2, PKA protein kinase A, BDNF brain-derived neurotrophic factor, LPS lipopolysaccharide, CREB cAMP-response element binding protein