PACAP-deficient Mice Research Articles

Effects of PACAP Deficiency on Immune Dysfunction and Peyer's Patch Integrity in Adult Mice.

PACAP (pituitary adenylate cyclase activating polypeptide) is a widespread neuropeptide with cytoprotective and anti-inflammatory effects. It plays a role in innate and adaptive immunity, but data are limited about gut-associated lymphoid tissue. We aimed to reveal differences in Peyer's patches between wild-type (WT) and PACAP-deficient (KO) mice. Peyer's patch morphology from young (3-months-old) and aging (12-15-months-old) mice was examined, along with flow cytometry to assess immune cell populations, expression of checkpoint molecules (PD-1, PD-L1, TIM-3, Gal-9) and functional markers (CD69, granzyme B, perforin) in CD3+, CD4+, and CD8+ T cells. We found slight differences between aging, but not in young, WT, and KO mice. In WT mice, aging reduced CD8+ T cell numbers frequency and altered checkpoint molecule expression (higher TIM-3, granzyme B; lower Gal-9, CD69). CD4+ T cell frequency was higher with similar checkpoint alterations, indicating a regulatory shift. In PACAP KO mice, aging did not change cell population frequencies but led to higher TIM-3, granzyme B and lower PD-1, PD-L1, Gal-9, and CD69 expression in CD4+ and CD8+ T cells, with reduced overall T cell activity. Thus, PACAP deficiency impacts immune dysfunction by altering checkpoint molecules and T cell functionality, particularly in CD8+ T cells, suggesting complex immune responses by PACAP, highlighting its role in intestinal homeostasis and potential implications for inflammatory bowel diseases.

Presence of Systemic Amyloidosis in Mice with Partial Deficiency in Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Aging

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with widespread expression and general cytoprotective effects, is also involved in aging. Previously, we observed accelerated systemic senile amyloidosis in PACAP knockout (KO) mice. As mice partially lacking PACAP (heterozygous-HZ) show variable symptoms, here we investigated whether HZ mice have accelerated aging, completed with observations in PAC1 receptor KO mice. As we have limited data on qualitative or quantitative changes in the blood of PACAP-deficient mice, we investigated whether these changes could be in the background of the amyloidosis. Routine histological staining was used to examine amyloid deposits, rated on a severity scale 0–3. Blood was collected from PACAP wild type/HZ mice for complete blood analysis. In contrast to receptor KO mice showing no amyloidosis, histopathological analysis revealed severe deposits in PACAP HZ mice, with kidney, spleen, skin, and intestines being most affected. Increased cholesterol, lipoprotein levels, and differences in several blood count parameters were found in HZ mice. In summary, amyloidosis also develops in partial absence of PACAP, in contrast to the lack of its PAC1 receptor. In addition to the earlier identified inflammatory and degenerative disturbances, the alteration in lipid metabolism and bone marrow activity can also be additional factors leading to systemic degenerative processes.

The Protective Effects of Endogenous PACAP in Oxygen-Induced Retinopathy

Pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues, including the retina. Previously, we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Wild-type and PACAP-knockout (KO) mouse pups at postnatal day (PD) 7 were maintained at 75% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15, animals underwent electroretinography (ERG) to assess visual function. On PD16, eyes were harvested for either immunohistochemistry to determine the percentage of the central avascular retinal area or molecular analysis to assess angiogenesis proteins by array kit and anti-apoptotic protein kinase B (Akt) change by western blot. Retinas of PACAP-deficient OIR mice showed a greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated due to OIR, and 11 different proteins markedly increased in PACAP-deficient mice, whereas western blot analysis revealed a reduction in Akt phosphorylation, suggesting an advanced cell death in the lack of PACAP. This is the first study to examine the endogenous effect of PACAP in the OIR model. Previously, we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings, we suggest that PACAP could be a novel retinoprotective agent in ROP.

Lipocalin-type prostaglandin D synthase regulates light-induced phase advance of the central circadian rhythm in mice

We previously showed that mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) exhibit attenuated light-induced phase shift. To explore the underlying mechanisms, we performed gene expression analysis of laser capture microdissected suprachiasmatic nuclei (SCNs) and found that lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is involved in the impaired response to light stimulation in the late subjective night in PACAP-deficient mice. L-PGDS-deficient mice also showed impaired light-induced phase advance, but normal phase delay and nonvisual light responses. Then, we examined the receptors involved in the response and observed that mice deficient for type 2 PGD2 receptor DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) show impaired light-induced phase advance. Concordant results were observed using the selective DP2/CRTH2 antagonist CAY10471. These results indicate that L-PGDS is involved in a mechanism of light-induced phase advance via DP2/CRTH2 signaling.

Protective Effects of PACAP in Peripheral Organs.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide widely distributed in the nervous system, where it exerts strong neuroprotective effects. PACAP is also expressed in peripheral organs but its peripheral protective effects have not been summarized so far. Therefore, the aim of the present paper is to review the existing literature regarding the cytoprotective effects of PACAP in non-neuronal cell types, peripheral tissues, and organs. Among others, PACAP has widespread expression in the digestive system, where it shows protective effects in various intestinal pathologies, such as duodenal ulcer, small bowel ischemia, and intestinal inflammation. PACAP is present in both the exocrine and endocrine pancreas as well as liver where it reduces inflammation and steatosis by interfering with hepatic pathology related to obesity. It is found in several exocrine glands and also in urinary organs, where, with its protective effects being mainly published regarding renal pathologies, PACAP is protective in numerous conditions. PACAP displays anti-inflammatory effects in upper and lower airways of the respiratory system. In the skin, it is involved in the development of inflammatory pathology such as psoriasis and also has anti-allergic effects in a model of contact dermatitis. In the non-neuronal part of the visual system, PACAP showed protective effects in pathological conditions of the cornea and retinal pigment epithelial cells. The positive role of PACAP has been demonstrated on the formation and healing processes of cartilage and bone where it also prevents osteoarthritis and rheumatoid arthritis development. The protective role of PACAP was also demonstrated in the cardiovascular system in different pathological processes including hyperglycaemia-induced endothelial dysfunction and age-related vascular changes. In the heart, PACAP protects against ischemia, oxidative stress, and cardiomyopathies. PACAP is also involved in the protection against the development of pre-senile systemic amyloidosis, which is presented in various peripheral organs in PACAP-deficient mice. The studies summarized here provide strong evidence for the cytoprotective effects of the peptide. The survival-promoting effects of PACAP depend on a number of factors which are also shortly discussed in the present review.

Altered light induced EGR1 expression in the SCN of PACAP deficient mice.

The brain's biological clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus and generates circadian rhythms in physiology and behavior. The circadian clock needs daily adjustment by light to stay synchronized (entrained) with the astronomical 24 h light/dark cycle. Light entrainment occurs via melanopsin expressing retinal ganglion cells (mRGCs) and two neurotransmitters of the retinohypothalamic tract (RHT), PACAP and glutamate, which transmit light information to the SCN neurons. In SCN neurons, light signaling involves the immediate-early genes Fos, Egr1 and the clock genes Per1 and Per2. In this study, we used PACAP deficient mice to evaluate PACAP's role in light induced gene expression of EGR1 in SCN neurons during early (ZT17) and late (ZT23) subjective night at high (300 lux) and low (10 lux) white light exposure. We found significantly lower levels of both EGR1 mRNA and protein in the SCN in PACAP deficient mice compared to wild type mice at early subjective night (ZT17) exposed to low but not high light intensity. No difference was found between the two genotypes at late night (ZT23) at neither light intensities. In conclusion, light mediated EGR1 induction in SCN neurons at early night at low light intensities is dependent of PACAP signaling. A role of PACAP in shaping synaptic plasticity during light stimulation at night is discussed.

Phenotypic characterization of testicular immune cells expressing immune checkpoint molecules in wild-type and pituitary adenylate cyclase-activating polypeptide-deficient mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several regulatory functions in the nervous system and in peripheral organs including those of the reproductive system. PACAP-deficient male mice have several morphological, biochemical, behavioral defects and show disturbed signaling in spermatogenesis affecting fertility in PACAP KO mice. Reproductive functions such as fertility, mating, and maternal behaviors have been widely investigated, but no immune analyses are available regarding the testicular immune-privileged environment in male PACAP-deficient mice. We performed detailed immunophenotyping of testicular immune cells and investigated the expression of TIM-3 and PD-1 Immune checkpoint molecules of immune cells together with the detection of galectin-9 and perforin. We investigated the percentage of numerous immune cell populations in the testis of wild-type and PACAP-deficient mice. We demonstrated a significant increase in the frequency of testicular CD8+ T cells together with the decrease in Treg cell number obtained from PACAP KO mice compared with wild-type mice. Investigating Immune checkpoint receptors, only PD-1 showed a significantly decreased expression in CD8+ T cells in PACAP KO mice compared with wild-type suggesting an impaired PD-1/PD-L1 pathway. Regarding TIM-3 expression, we did not find any significant difference between the investigated groups. We hypothesize that these local changes may result in an immune activation with disturbed testicular immunoregulation in PACAP KO mice; however, determining the exact function requires further investigations. Our data further support the view that besides a systemic immune tolerance, localized active immunosuppression is involved in the regulation of testicular immune privilege.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP)-Glutamate Co-transmission Drives Circadian Phase-Advancing Responses to Intrinsically Photosensitive Retinal Ganglion Cell Projections by Suprachiasmatic Nucleus

Results from a variety of sources indicate a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in light/glutamate-induced phase resetting of the circadian clock mediated by the retinohypothalamic tract (RHT). Attempts to block or remove PACAP’s contribution to clock-resetting have generated phenotypes that differ in their responses to light or glutamate. For example, previous studies of circadian behaviors found that period-maintenance and early-night phase delays are intact in PACAP-null mice, yet there is a consistent deficit in behavioral phase-resetting to light stimulation in the late night. Here we report rodent stimulus–response characteristics of PACAP release from the RHT, and map these to responses of the suprachiasmatic nucleus (SCN) in intact and PACAP-deficient mouse hypothalamus with regard to phase-resetting. SCN of PACAP-null mice exhibit normal circadian rhythms in neuronal activity, but are “blind” to glutamate stimulating phase-advance responses in late night, although not in early night, consistent with previously reported selective lack of late-night light behavioral responsiveness of these mice. Induction of CREB phosphorylation, a hallmark of the light/glutamate response of the SCN, also is absent in SCN-containing ex vivo slices from PACAP-deficient mouse hypothalamus. PACAP replacement to the SCN of PACAP-null mice restored wild-type phase-shifting of firing-rate patterns in response to glutamate applied to the SCN in late night. Likewise, ex vivo SCN of wild-type mice post-orbital enucleation are unresponsive to glutamate unless PACAP also is restored. Furthermore, we demonstrate that the period of efficacy of PACAP at SCN nerve terminals corresponds to waxing of PACAP mRNA expression in ipRGCs during the night, and waning during the day. These results validate the use of PACAP-deficient mice in defining the role and specificity of PACAP as a co-transmitter with glutamate in ipRGC-RHT projections to SCN in phase advancing the SCN circadian rhythm in late night.

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation.

Alterations in pituitary adenylate cyclase-activating polypeptide (PACAP), a multifunctional neuropeptide, and its receptors have been identified as risk factors for certain psychiatric disorders, including schizophrenia. Increasing evidence from human genetic and animal model studies suggest an association between various psychiatric disorders and altered dendritic spine morphology. In the present study, we investigated the role of exogenous and endogenous PACAP in spine formation and maturation. PACAP modified the density and morphology of PSD-95-positive spines in primary cultured hippocampal neurons. Notably, PACAP increased the levels of microRNA (miR)-132 and decreased expression of corresponding miR-132 target genes and protein expression of p250GAP, a miR-132 effector known to be involved in spine morphology regulation. In corroboration, PSD-95-positive spines were reduced in PACAP-deficient (PACAP-/-) mice versus WT mice. Golgi staining of hippocampal CA1 neurons revealed a reduced spine densities and atypical morphologies in the male PACAP-/- mice. Furthermore, viral miR-132 overexpression reversed the reduction in hippocampal spinal density in the male PACAP-/- mice. These results indicate that PACAP signaling plays a critical role in spine morphogenesis possibly via miR-132. We suggest that dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through its effects on spine formation.SIGNIFICANCE STATEMENT Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling dysfunction and dendritic spine morphology alterations have recently been suggested as important pathophysiological mechanisms underlying several psychiatric and neurological disorders. In this study, we investigated whether PACAP regulates dendritic spine morphogenesis. In a combination of pharmacological and viral gain- and loss-of-function approaches in vitro and in vivo experiments, we found PACAP to increase the size and density of dendritic spines via miR-132 upregulation. Together, our data suggest that a dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through abnormal spine formation.

VPAC1 receptors play a dominant role in PACAP-induced vasorelaxation in female mice.

BackgroundPACAP and VIP are closely related neuropeptides with wide distribution and potent effect in the vasculature. We previously reported vasomotor activity in peripheral vasculature of male wild type (WT) and PACAP-deficient (KO) mice. However, female vascular responses are still unexplored. We hypothesized that PACAP-like activity is maintained in female PACAP KO mice and the mechanism through which it is regulated differs from that of male PACAP KO animals.MethodsWe investigated the vasomotor effects of VIP and PACAP isoforms and their selective blockers in WT and PACAP KO female mice in carotid and femoral arteries. The expression and level of different PACAP receptors in the vessels were measured by RT-PCR and Western blot.ResultsIn both carotid and femoral arteries of WT mice, PACAP1-38, PACAP1-27 or VIP induced relaxation, without pronounced differences between them. Reduced relaxation was recorded only in the carotid arteries of KO mice as compared to their WT controls. The specific VPAC1R antagonist completely blocked the PACAP/VIP-induced relaxation in both arteries of all mice, while PAC1R antagonist affected relaxation only in their femoral arteries.ConclusionIn female WT mice, VPAC1 receptors appear to play a dominant role in PACAP-induced vasorelaxation both in carotid and in femoral arteries. In the PACAP KO group PAC1R activation exerts vasorelaxation in the femoral arteries but in carotid arteries there is no significant effect of the activation of this receptor. In the background of this regional difference, decreased PAC1R and increased VPAC1R availability in the carotid arteries was found.

Protective Role of Endogenous PACAP in Inflammation-induced Retinal Degeneration.

Pituitary adenylate Cyclase-Activating Polypeptide (PACAP) is a neuroprotective peptide that has been shown to exert protective effects in different models of neurodegenerative diseases, including retinal degenerations. Data obtained from PACAP-deficient (PACAP KO) mice provide evidence that endogenous PACAP has a neuroprotective role in different pathologies. PACAP KO mice show enhanced sensitivity to different insults, such as oxidative stress, hypoxia and inflammation. The aim of the present study was to investigate the protective effects of endogenous PACAP in retinal inflammation. Endotoxin-induced eye inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) in PACAP KO and wild-type (Wt) mice. After LPS treatment, retinas were processed for histological examination. To detect the alterations of different proteins and cytokines, immunohistochemical, western blot and cytokine array were used. We also performed dark-adapted electroretinography (ERG) to detect the functional differences. The thickness of nearly all layers was significantly less in LPS-injected PACAP KO mice compared to Wt animals. Increased expression of Glial Fibrillary Acidic Protein (GFAP) was induced in Müller glial cells after LPS treatment, which was more intense in PACAP KO mice. The levels of pAkt and pGSK were decreased in PACAP KO group during inflammation. LPS treatment significantly increased cytokines (sICAM-1, JE, TIMP-1) in both treated groups, but it was more expressed in PACAP KO animals. Furthermore, ERG responses were disturbed after LPS injection in PACAP KO mice. Our results showed that endogenous PACAP has a protective role in LPS-caused retinal inflammation.

PACAP deficiency as a model of aging.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. In the long list of neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) represents a highly effective cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. PACAP has neuro- and general cytoprotective effects due to anti-apoptotic, anti-inflammatory, and antioxidant actions. As PACAP is also a part of the endogenous protective machinery, it can be hypothesized that the decreased protective effects in lack of endogenous PACAP would accelerate age-related degeneration and PACAP knockout mice would display age-related degenerative signs earlier. Recent results support this hypothesis showing that PACAP deficiency mimics aspects of age-related pathophysiological changes including increased neuronal vulnerability and systemic degeneration accompanied by increased apoptosis, oxidative stress, and inflammation. Decrease in PACAP expression has been shown in different species from invertebrates to humans. PACAP-deficient mice display numerous pathological alterations mimicking early aging, such as retinal changes, corneal keratinization and blurring, and systemic amyloidosis. In the present review, we summarize these findings and propose that PACAP deficiency could be a good model of premature aging.

Altered Notch Signaling in Developing Molar Teeth of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Deficient Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with neuroprotective and neurotrophic effects. This suggests its influence on the development of teeth, which are, similarly to the nervous system, ectoderm and neural crest derivatives. Our earlier studies have shown morphological differences between wild-type (WT) and PACAP-deficient mice, with upregulated sonic hedgehog (SHH) signaling in the lack of PACAP. Notch signaling is a key element of proper tooth development by regulating apoptosis and cell proliferation. In this study, our main goal was to evaluate the possible effects of PACAP on Notch signaling pathway. Immunohistochemical staining was performed of Notch receptors (Notch1, 2, 3, 4), their ligands [delta-like protein (DLL)1, 3, 4, Jagged1, 2], and intracellular target molecules [CSL (CBF1 humans/Su (H) Drosophila/LAG1 Caenorhabditis elegans transcription factor); TACE (TNF-α converting enzyme), NUMB] in molar teeth of 5-day-old WT, and homozygous and heterozygous PACAP-deficient mice. We measured immunopositivity in the enamel-producing ameloblasts and dentin-producing odontoblasts. Notch2 receptor and DLL1 expression were elevated in ameloblasts of PACAP-deficient mice compared to those in WT ones. The expression of CSL showed similar results both in the ameloblasts and odontoblasts. Jagged1 ligand expression was elevated in the odontoblasts of homozygous PACAP-deficient mice compared to WT mice. Other Notch pathway elements did not show significant differences between the genotype groups. The lack of PACAP leads to upregulation of Notch pathway elements in the odontoblast and ameloblast cells. The underlying molecular mechanisms are yet to be elucidated; however, we propose SHH-dependent and independent processes. We hypothesize that this compensatory upregulation of Notch signaling by the lack of PACAP could represent a salvage pathway in PACAP-deficient animals.

Comparative analysis of decidual and peripheral immune cells and immune-checkpoint molecules during pregnancy in wild-type and PACAP-deficient mice.

PACAP is a neuropeptide having a major relevance in the nervous system and in several peripheral organs including those of the reproductive system. PACAP-deficient mice have several morphological, biochemical, behavioral defects, and show reduced fertility. Female reproductive functions such as fertility, mating behavior, maternal behaviors, and implantation alterations have been widely investigated, but no comparative immune analyses are available in pregnant wild-type (WT) and PACAP knockout (KO) mice. Therefore, we performed a detailed immunophenotyping of decidual and peripheral immune cells and investigated the expression of two immune-checkpoint molecules by immune cells together with immunohistochemistry detecting Galectin-9 in placental tissues. We investigated the percentage of numerous immune cell populations in the periphery and in the decidua of pregnant mice. We demonstrated a significant increase in the frequency of decidual Gal-9+ Th cells obtained from PACAP KO mice compared to the decidua of WT mice. We could not determine statistical differences in TIM-3 and programmed cell death-1 expression by different immune cells in the decidua and in the periphery between WT and KO mice. In conclusion, we could not find any significant alteration either in the distribution or in the cytotoxicity of the investigated decidual immune cells which could elucidate any reproductive alterations in PACAP KO mice. The only remarkable finding is the recruitment of Gal-9+ Th cells to the decidua promoting local immune homeostasis in PACAP KO mice, which nevertheless cannot explain the reduced fertility observed in these mice.

Role of light and the circadian clock in the rhythmic oscillation of intraocular pressure: Studies in VPAC2 receptor and PACAP deficient mice

The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24 h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24 h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24 h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values.In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness.

Disturbed spermatogenic signaling in pituitary adenylate cyclase activating polypeptide-deficient mice

PACAP is a neuropeptide with diverse functions in various organs, including reproductive system. It is present in the testis in high concentrations, and in addition to the stage-specific expression within the seminiferous tubules, PACAP affects spermatogenesis and the functions of Leydig and Sertoli cells. Mice lacking endogenous PACAP show reduced fertility, but the possibility of abnormalities in spermatogenic signaling has not yet been investigated. Therefore, we performed a detailed morphological analysis of spermatozoa, sperm motility and investigated signaling pathways that play a role during spermatogenesis in knockout mice. No significant alterations were found in testicular morphology or motility of sperm in homozygous and heterozygous PACAP-deficient mice in spite of the moderately increased number of severely damaged sperms. However, we found robust changes in mRNA and/or protein expression of several factors that play an important role in spermatogenesis. Protein kinase A expression was markedly reduced, while downstream phospho-ERK and p38 were elevated in knockout animals. Expression of major transcription factors, such as Sox9 and phospho-Sox9, was decreased, while that of Sox10, as a redundant factor, was increased in PACAP-deficient mice. The reduced phospho-Sox9 expression was partly due to increased expression and activity of phosphatase PP2A in knockout mice. Targets of Sox transcription factors, such as collagen type IV, were reduced in knockout mice. In summary, our results show that lack of PACAP leads to disturbed signaling in spermatogenesis, which could be a factor responsible for reduced fertility in PACAP knockout mice, and further support the role of PACAP in reproduction.

Backup Mechanisms Maintain PACAP/VIP-Induced Arterial Relaxations in Pituitary Adenylate Cyclase-Activating Polypeptide-Deficient Mice

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide in the VIP/secretin/glucagon peptide superfamily. Two active forms, PACAP1-38 and PACAP1-27, act through G protein-coupled receptors, the PAC1 and VPAC1/2 receptors. Effects of PACAP include potent vasomotor activity. Vasomotor activity and organ-specific vasomotor effects of PACAP-deficient mice have not yet been investigated; thus, the assessment of its physiological importance in vasomotor functions is still missing. We hypothesized that backup mechanisms exist to maintain PACAP pathway activity in PACAP knockout (KO) mice. Thus, we investigated the vasomotor effects of exogenous vasoactive intestinal peptide (VIP) and PACAP polypeptides in PACAP wild-type (WT) and PACAP-deficient (KO) male mice. Methods: Carotid and femoral arteries were isolated from 8- to 12-week-old male WT and PACAP-KO mice. Vasomotor responses were measured with isometric myography. Results: In the arteries of WT mice the peptides induced relaxations, which were significantly greater to PACAP1-38 than to PACAP1-27 and VIP. In KO mice, PACAP1-38 did not elicit relaxation, whereas PACAP1-27 and VIP elicited significantly greater relaxation in KO mice than in WT mice. The specific PAC1R and VPAC1R antagonist completely blocked the PACAP-induced relaxations. Conclusion: Our data suggest that in PACAP deficiency, backup mechanisms maintain arterial relaxations to polypeptides, indicating an important physiological role for the PACAP pathway in the regulation of vascular tone.

Early Neurobehavioral Development of Mice Lacking Endogenous PACAP.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.

Activation of the HPA axis and depression of feeding behavior induced by restraint stress are separately regulated by PACAPergic neurotransmission in the mouse

We measured serum CORT elevation in wild-type and PACAP-deficient C57BL/6N male mice after acute (1 h) or prolonged (2–3 h) daily restraint stress for 7 d. The PACAP dependence of CORT elevation was compared to that of stress-induced hypophagia. Daily restraint induced unhabituated peak CORT elevation, and hypophagia/weight loss, of similar magnitude for 1, 2, and 3 h of daily restraint, in wild-type mice. Peak CORT elevation, and hypophagia, were both attenuated in PACAP-deficient mice for 2 and 3 h daily restraint. Hypophagia induced by 1-h daily restraint was also greatly reduced in PACAP-deficient mice, however CORT elevation, both peak and during recovery from stress, was unaffected. Thus, hypothalamic PACAPergic neurotransmission appears to affect CRH gene transcription and peptide production, but not CRH release, in response to psychogenic stress. A single exposure to restraint sufficed to trigger hypophagia over the following 24 h. PACAP deficiency attenuated HPA axis response (CORT elevation) to prolonged (3 h) but not acute (1 h) single-exposure restraint stress, while hypophagia induced by either a single 1 h or a single 3 h restraint were both abolished in PACAP-deficient mice. These results suggest that PACAP’s actions to promote suppression of food intake following an episode of psychogenic stress is unrelated to the release of CRH into the portal circulation to activate the pituitary-adrenal axis. Furthermore, demonstration of suppressed food intake after a single 1-h restraint stress provides a convenient assay for investigating the location of the synapses and circuits mediating the effects of PACAP on the behavioral sequelae of psychogenic stress.

Structural and Morphometric Comparison of Lower Incisors in PACAP-Deficient and Wild-Type Mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread distribution. PACAP plays an important role in the development of the nervous system, it has a trophic and protective effect, and it is also implicated in the regulation of various physiological functions. Teeth are originated from the mesenchyme of the neural crest and the ectoderm of the first branchial arch, suggesting similarities with the development of the nervous system. Earlier PACAP-immunoreactive fibers have been found in the odontoblastic and subodontoblastic layers of the dental pulp. Our previous examinations have shown that PACAP deficiency causes alterations in the morphology and structure of the developing molars of 7-day-old mice. In our present study, morphometric and structural comparison was performed on the incisors of 1-year-old wild-type and PACAP-deficient mice. Hard tissue density measurements and morphometric comparison were carried out on the mandibles and the lower incisors with micro-CT. For structural examination, Raman microscopy was applied on frontal thin sections of the mandible. With micro-CT morphometrical measurements, the size of the incisors and the relative volume of the pulp to dentin were significantly smaller in the PACAP-deficient group compared to the wild-type animals. The density of calcium hydroxyapatite in the dentin was reduced in the PACAP-deficient mice. No structural differences could be observed in the enamel with Raman microscopy. Significant differences were found in the dentin of PACAP-deficient mice with Raman microscopy, where increased carbonate/phosphate ratio indicates higher intracrystalline disordering. The evaluation of amide III bands in the dentin revealed higher structural diversity in wild-type mice. Based upon our present and previous results, it is obvious that PACAP plays an important role in tooth development with the regulation of morphogenesis, dentin, and enamel mineralization. Further studies are required to clarify the molecular background of the effects of PACAP on tooth development.