Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation.

The degenerative retinal disorders characterized by progressive cell death and exacerbating inflammation lead ultimately to blindness. The ubiquitous neuropeptide, PACAP38 is a promising therapeutic agent as its proliferative potential and suppressive effect on microglia might enable cell replacement and attenuate inflammation, respectively. Our previous finding that PACAP38 caused a marked increase of the amacrine cells in the adult (1-year-old) mouse retina, served as a rationale of the current study. We aimed to determine the proliferating elements and the inflammatory status of the PACAP38-treated retina. Three months old mice were intravitreally injected with 100 pmol PACAP38 at 3 months intervals (3X). Retinas of 1-year-old animals were dissected and effects on cell proliferation, and expression of inflammatory regulators were analyzed. Interestingly, both mitogenic and anti-mitogenic actions were detected after PACAP38-treatment. Further analysis of the mitogenic effect revealed that proliferating cells include microglia, endothelial cells, and neurons of the ganglion cell layer but not amacrine cells. Furthermore, PACAP38 stimulated retinal microglia to polarize dominantly into M2-phenotype but also might cause subsequent angiogenesis. According to our results, PACAP38 might dampen pro-inflammatory responses and help tissue repair by reprogramming microglia into an M2 phenotype, nonetheless, with angiogenesis as a warning side effect.

Secreted key regulators (Fgf1, Bmp4, Gdf3) are expressed by PAC1-immunopositive retinal ganglion cells in the postnatal rat retina.

Identified as a member of the secretin/glucagon/VIP superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP1-38) has been recognized as a hormone, neurohormone, transmitter, trophic factor, and known to be involved in diverse and multiple developmental processes. PACAP1-38 was reported to regulate the production of important morphogens (Fgf1, Bmp4, Gdf3) through PAC1-receptor in the newborn rat retina. To follow up, we aimed to reveal the identity of retinal cells responsible for the production and secretion of Fgf1, Bmp4, and Gdf3 in response to PACAP1-38 treatment. Newborn (P1) rats were treated with 100 pmol PACAP1-38 intravitreally. After 24 h, retinas were dissected and processed for immunohistochemistry performed either on flat-mounted retinas or cryosections. Brn3a and PAC1-R double labeling revealed that 90% of retinal ganglion cells (RGCs) expressed PAC1-receptor. We showed that RGCs were Fgf1, Bmp4, and Gdf3-immunopositive and PAC1-R was co-expressed with each protein. To elucidate if RGCs release these secreted regulators, the key components for vesicle release were examined. No labeling was detected for synaptophysin, Exo70, or NESP55 in RGCs but an intense Rab3a-immunoreactivity was detected in their cell bodies. We found that the vast majority of RGCs are responsive to PACAP, which in turn could have a significant impact on their development or/and physiology. Although Fgf1, Bmp4, and Gdf3 were abundantly expressed in PAC1-positive RGCs, the cells lack synaptophysin and Exo70 in the newborn retina, thus unable to release these proteins. These proteins could regulate postnatal RGC development acting through intracrine pathways.

Age-Related Alterations of Proteins in Albino Wistar Rat Retina.

Imbalance of homeostasis causes permanent changes in the body with time. The central nervous system is especially prone to these changes since it possesses limited regenerative capacity. In the retina, neurons are damaged during the aging process, and this eventually leads to deterioration of vision. In our 2-year-long study, we examined genetically closely related rat individuals to disclose the hidden retinal causes of age-associated visual dysfunction. Morphometric analysis showed significant reduction of the retina thickness with aging, particularly that of the inner plexiform layer. To reveal changes between the age groups, we used immunohistochemistry against vesicular glutamate transporter 1 protein for photoreceptor and bipolar cell terminals, Brn3a for ganglion cells, calbindin 28 kDa for horizontal cells, parvalbumin for AII amacrines, protein kinase Cα for rod bipolar cells, tyrosine hydroxylase for dopaminergic cells, glial fibrillary acidic protein for glial cells, and peanut-agglutinin labeling for cones. The most significant decrease was observed in the density of photoreceptor and the ganglion cells in the aging process. By using immunocytochemistry and western blot technique, we observed that calbindin and vesicular glutamate transporter 1 protein staining do not change much with aging; tyrosine hydroxylase, parvalbumin and calretinin showed the highest immunoreactivity during the midlife period. Most interestingly, the level of glial fibrillary acidic protein also changes similarly to the previously named markers. Our results provide further evidence that protein content is modified at least in some cell populations of the rat retina, and the number of retinal cells declined with aging. We conclude that senescence alone may cause structural and functional damage in the retinal tissue.

PACAP for Retinal Health: Model for Cellular Aging and Rescue.

Retinal aging is the result of accumulating molecular and cellular damage with a manifest decline in visual functions. Somatostatin (SST) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in neuroprotection through regulating disparate aspects of neuronal activity (survival, proliferation and renewal). The aim of the present study was to validate a transgenic model for SST-expressing amacrine cells and to investigate the chronic effect of PACAP on the aging of SSTergic and dopaminergic cells of the retina. SST-tdTomato transgenic mice that were 6, 12 and 18 months old were treated intravitreally with 100 pmol of PACAP every 3 months. The density of SST and dopaminergic amacrine cells was assessed in whole-mounted retinas. Cells displaying the transgenic red fluorescence were identified as SST-immunopositive amacrine cells. By comparing the three age groups. PACAP treatment was shown to induce a moderate elevation of cell densities in both the SST and dopaminergic cell populations in the 12- and 18-month-old animals. By contrast, the control untreated and saline-treated retinas showed a minor cell loss. In conclusion, we report a reliable transgenic model for examining SSTergic amacrine cells. The fundamental novelty of this study is that PACAP could increase the cell density in matured retinal tissue, anticipating new therapeutic potential in age-related pathological processes.

Neuroprotective Potential of Pituitary Adenylate Cyclase Activating Polypeptide in Retinal Degenerations of Metabolic Origin.

Pituitary adenylate cyclase-activating polypeptide (PACAP1-38) is a highly conserved member of the secretin/glucagon/VIP family. The repressive effect of PACAP1-38 on the apoptotic machinery has been an area of active research conferring a significant neuroprotective potential onto this peptide. A remarkable number of studies suggest its importance in the etiology of neurodegenerative disorders, particularly in relation to retinal metabolic disorders. In our review, we provide short descriptions of various pathological conditions (diabetic retinopathy, excitotoxic retinal injury and ischemic retinal lesion) in which the remedial effect of PACAP has been well demonstrated in various animal models. Of all the pathological conditions, diabetic retinopathy seems to be the most intriguing as it develops in 75% of patients with type 1 and 50% of patients with type 2 diabetes, with concomitant progression to legal blindness in about 5%. Several animal models have been developed in recent years to study retinal degenerations and out of these glaucoma and age-related retina degeneration models bear human recapitulations. PACAP neuroprotection is thought to operate through enhanced cAMP production upon binding to PAC1-R. However, the underlying signaling network that leads to neuroprotection is not fully understood. We observed that (i) PACAP is not equally efficient in the above conditions; (ii) in some cases more than one signaling pathways are activated; (iii) the coupling of PAC1-R and signaling is stage dependent; and (iv) PAC1-R is not the only receptor that must be considered to interpret the effects in our experiments. These observations point to a complex signaling mechanism, that involves alternative routes besides the classical cAMP/protein kinase A pathway to evoke the outstanding neuroprotective action. Consequently, the possible contribution of the other two main receptors (VPAC1-R and VPAC2-R) will also be discussed. Finally, the potential medical use of PACAP in some retinal and ocular disorders will also be reviewed. By taking advantage of, low-cost synthesis technologies today, PACAP may serve as an alternative to the expensive treatment modelities currently available in ocular or retinal conditions.

Pituitary Adenylate Cyclase-Activating Polypeptide: 30 Years in Research Spotlight and 600 Million Years in Service.

Emerging from the depths of evolution, pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (i.e., PAC1, VPAC1, VPAC2) are present in multicellular organisms from Tunicates to humans and govern a remarkable number of physiological processes. Consequently, the clinical relevance of PACAP systems spans a multifaceted palette that includes more than 40 disorders. We aimed to present the versatility of PACAP1-38 actions with a focus on three aspects: (1) when PACAP1-38 could be a cause of a malfunction, (2) when PACAP1-38 could be the cure for a malfunction, and (3) when PACAP1-38 could either improve or impair biology. PACAP1-38 is implicated in the pathophysiology of migraine and post-traumatic stress disorder whereas an outstanding protective potential has been established in ischemia and in Alzheimer's disease. Lastly, PACAP receptors could mediate opposing effects both in cancers and in inflammation. In the light of the above, the duration and concentrations of PACAP agents must be carefully set at any application to avoid unwanted consequences. An enormous amount of data accumulated since its discovery (1989) and the first clinical trials are dated in 2017. Thus in the field of PACAP research: "this is not the end, not even the beginning of the end, but maybe the end of the beginning."

The Neuroprotective Peptide PACAP1-38 Contributes to Horizontal Cell Development in Postnatal Rat Retina.

Purpose: PACAP1-38, a member of the secretin/glucagon superfamily, is expressed in the developing retina with documented neuroprotective effects. However, its function in retinal cell differentiation has yet to be elucidated. Our goals, therefore, were to identify PAC1 expressing cells morphologically, investigate the PACAP1-38 action functionally, and establish PACAP1-38 regulated events developmentally during the first postnatal week in rat retina. Methods: P1 retinal sections or whole mounts of Wistar rats were used to reveal PAC1 and calbindin immunoreactive structures. P1, P3, or P7 pups were injected intravitreally with 100 pmol PACAP1-38. Tissues were harvested 24 hours post-treatment, then processed for calbindin immunohistochemistry to determine horizontal cell number, or 6, 12, 24 hours post-treatment for real-time PCR and immunoblots to detect PCNA expression. To localize proliferating cells, anti-PCNA antibody was applied. Results: We showed various PAC1 expressing cells in RPE, NBL, and GCL in P1 retina including calbindin positive horizontal cells. We found that PACAP1-38 induced a marked cell number increase at P3 and P7 and showed upregulated cell proliferation as its mechanism; however, it was ineffective at P1. PACAP1-38 induced proliferative cells localized in the NBL, and double-marker studies demonstrated that the induced proliferative cells were horizontal cells. Conclusions: PACAP1-38 appears to act in retinal differentiation by inducing mitosis selectively in a time and cell specific manner through PAC1. The control of horizontal cell proliferation raises the novel possibilities that (1) PACAP1-38 may be a major player in retinal patterning and (2) PACAP signaling may be critical in retinoblastoma.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP1-38) Exerts Both Pro and Anti-Apoptotic Effects on Postnatal Retinal Development in Rat.

PACAP1-38, a ubiquitous and multifunctional regulator has been in the focus of neurotoxicity research due to its impressive neuroprotective potential. Although the literature extensively demonstrated its repressive effect on the apoptotic machinery in neurodegenerative models, there is a striking absence of analysis on its role in normal development. We performed quantitative analyses on caspase activity in developing retina upon 100, 50, 25 or 1 pmol intravitreal PACAP1-38 injection from postnatal day 1 (P1) through P7 in Wistar rats. Retinas were harvested at 6, 12, 18, 24 or 48 h post-injection. Apoptotic activity was revealed using fluorescent caspase 3/7 enzyme assay, western blots and TUNEL assay. Unexpectedly, we found that 100 pmol PACAP1-38 increased the activity of caspase 3/7 at P1 and P5 whereas it had no effect at P7. At P3, as a biphasic effect, PACAP1-38 repressed active caspase 3/7 at 18 h post-injection while increased their activity in 24 h post-injection. Amounts, smaller than 100 pmol, could not inhibit apoptosis whereas 50, 25 or 1 pmol PACAP1-38 could evoke significant elevation in caspase 3/7 activity. TUNEL-positive cells appeared in the proximal part of inner nuclear as well as ganglion cell layers in response to PACAP1-38 treatment. The fundamental novelty of these results is that PACAP1-38 induces apoptosis during early postnatal retinogenesis. The dose as well as stage-dependent response suggests that PACAP1-38 has a Janus face in apoptosis regulation. It not only inhibits development-related apoptosis, but as a long-term effect, facilitates it.

Accelerated retinal aging in PACAP knock-out mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide. PACAP and its receptors are widely distributed in the retina. A number of reports provided evidence that PACAP is neuroprotective in retinal degenerations. The current study compared retina cell type-specific differences in young (3-4months) and aged adults (14-16months), of wild-type (WT) mice and knock-out (KO) mice lacking endogenous PACAP production during the course of aging. Histological, immunocytochemical and Western blot examinations were performed. The staining for standard neurochemical markers (tyrosine hydroxylase for dopaminergic cells, calbindin 28 kDa for horizontal cells, protein kinase Cα for rod bipolar cells) of young adult PACAP KO retinas showed no substantial alterations compared to young adult WT retinas, except for the specific PACAP receptor (PAC1-R) staining. We could not detect PAC1-R immunoreactivity in bipolar and horizontal cells in young adult PACAP KO animals. Some other age-related changes were observed only in the PACAP KO mice only. These alterations included horizontal and rod bipolar cell dendritic sprouting into the photoreceptor layer and decreased ganglion cell number. Also, Müller glial cells showed elevated GFAP expression compared to the aging WT retinas. Furthermore, Western blot analyses revealed significant differences between the phosphorylation state of ERK1/2 and JNK in KO mice, indicating alterations in the MAPK signaling pathway. These results support the conclusion that endogenous PACAP contributes to protection against aging of the nervous system.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP), a Novel Secretagogue, Regulates Secreted Morphogens in Newborn Rat Retina.

Purpose: Pituitary adenylate cyclase-activating peptide (PACAP)1-38 has been reported to be responsible for regulation of a disparate array of developmental processes in the central nervous system, and its antiapoptotic effect has been revealed in numerous models, pointing to its relevance in the etiology of neurodegenerative disorders. However, its function in retinal development remains unclear. Here, we aimed to point out that versatility can be achieved through interaction with other regulators, in which PACAP can act indirectly on the retinal microenvironment. Methods: Wistar rats at age postnatal day 1 were injected intravitreally with PACAP or PAC1 receptor antagonist (PACAP6-38, M65) or VPAC1 antagonist (PG97-269) alone or in combination. Retinas were removed at 3, 6, 12, or 24 hours after injection. Changes in mRNA level were assessed using quantitative PCR, whereas changes in protein levels were measured by Western blot. Results: Intravitreal injection of PACAP or PAC1 receptor antagonists or the VPAC1 antagonist showed that PACAP receptors regulate the expression of five key secreted molecules (i.e., Fgf1, Bmp4, Wnt1, Gdf3, and Ihh), wherease other crucial morphogens (i.e., Fgf2, Fgf4, Fgf8, Fgf9, Shh, and Bmp9) were not affected. Pharmacologic dissection revealed that both PAC1 and VPAC1 induced downstream signaling and could cause upregulation of Fgf1, Bmp4, and Wnt1, whereas expression of Gdf3 might be mediated through the VPAC2 receptor. Conclusions: Our data are the first to shed light on PACAP as a secretagogue regulating a sustained production of morphogens, which in turn could enable PACAP to serve as a mitogen for retinal cells, to induce ganglion cell differentiation, and to contribute to RPE development.

Pituitary Adenylate Cyclase-Activating Polypeptide Receptors Signal via Phospholipase C Pathway to Block Apoptosis in Newborn Rat Retina.

Glutamate induced cell death mechanisms gained considerable attention lately as excessive release of extracellular glutamate was reported to cause neurodegeneration in brain areas including the retina. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP) was shown to provide neuroprotection through anti-apoptotic effects in the glutamate-model and also in other degeneration assays. Although PACAP is known to orchestrate complex intracellular signaling primarily through cAMP production, the mechanism that mediates the anti-apoptotic effect in glutamate excitotoxicity remains to be clarified. To study this mechanism we induced retinal neurodegeneration in newborn Wistar rats by subcutaneous monosodium-glutamate injection. 100 pmol PACAP and enzyme inhibitors were administered intravitreally. Levels of caspase 3, 9, and phospho-protein kinase A were assessed by Western blots. Changes in cAMP levels were detected employing a competitive immunoassay. We found that cAMP blockade by an adenylyl-cyclase inhibitor (2',4'-dideoxy-adenosine) did not abrogate the neuroprotective effect of PACAP1-38. We show that following intravitreal PACAP1-38 treatment cAMP was unaltered, consistent with the inhibitor results and phospho-protein kinase A, an effector of the cAMP pathway was also unaffected. On the other hand, blockade of the alternative phosphatidylcholine-specific PLC pathway using an inhibitor (D609CAS) abrogated the neuroprotective effects of PACAP1-38. Our results highlight PACAP1-38 ability in protecting retinal cells against apoptosis through diverse signaling cascades. It seems that at picomolar concentrations, PACAP does not trigger cAMP production, but nonetheless, exerts a significant anti-apoptotic effect through PLC activation. In conclusion, PACAP1-38 may signal via both AC and PLC activation producing the same protective outcome.

Metastasis blood test by flow cytometry: in vivo cancer spheroids and the role of hypoxia.

Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis-compared to the high circulating tumor-cell numbers (CTCs)-appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid-size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light-scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC-association with in vivo spheroids. We propose that the mechanism involves a portable "micro-niche" in spheroids that may support CSC-survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression.

High male chimerism in the female breast shows quantitative links with cancer.

Clinical observations suggest that pregnancy provides protection against cancer. The mechanisms involved, however, remain unclear. Fetal cells are known to enter the mother's circulation during pregnancy and establish microchimerism. We investigated if pregnancy-related embryonic/fetal stem cell integration plays a role in breast cancer. A high-sensitivity Y-chromosome assay was developed to trace male allogeneic cells (from male fetus) in females. Fixed-embedded samples (n = 206) from both normal and breast cancer patients were screened for microchimerism. The results were combined with matching clinicopathological and histological parameters and processed statistically. The results show that in our samples (182 informative) more than half of healthy women (56%) carried male cells in their breast tissue for decades (n = 68), while only one out of five in the cancer sample pool (21%) (n = 114) (odds ratio = 4.75, CI at 95% 2.34-9.69; p = 0.0001). The data support the notion that a biological link may exist between chimerism and tissue-integrity. The correlation, however, is non-linear, since male microchimerism in excess ("hyperchimerism") is also involved in cancer. The data suggest a link between hyperchimerism and HER2-type cancers, while decreased chimerism ("hypochimerism") associates with ER/PR-positive (luminal-type) breast cancers. Chimerism levels that correlate with protection appear to be non-random and share densities with the mammary progenitor components of the stem cell lineage in the breast. The results suggest that protection may involve stem/progenitor level interactions and implicate novel quantitative mechanisms in chimerism biology.

Development-related splicing regulates pituitary adenylate cyclase-activating polypeptide (PACAP) receptors in the retina.

PURPOSE: The ubiquitous pituitary adenylate cyclase-activating peptide (PACAP) has a disparate array of functions in development (e.g., proliferation and apoptosis). Among three types of PACAP receptor (VPAC1, VPAC2, and PAC1), PAC1 is subject to alternative splicing that generates isoforms. Although the literature documenting the presence of PACAP receptors in the central nervous system is vast, their expression during development has not been established yet. Here, we performed quantitative analyses on the expression of PACAP receptors during the postnatal development of the rat retina. METHODS: Retinas were harvested from postnatal days 0 to 20 (P0-P20). Using a comprehensive primer system, expression changes were followed employing quantitative real-time PCR. Changes at the protein level were detected by immunoblotting using anti-VPAC1, -VPAC2, and -PAC1 receptor antibodies. RESULTS: The expression of VPAC1 showed increases at P10 and P15. Peaks in VPAC2 expression were observed at P5 and P15. Using splicing variant-specific primers for PAC1 receptor, splicing regulation of Null, Hip, Hop1, and Hiphop1 variants was revealed in correlation with postnatal development. Transcript levels of the Null and Hip variants showed a decline, while Hop1 became the major PACAP receptor by P20. Hiphop1 transcript levels did not display remarkable changes except for a transient increase at P10. Immunoblotting confirmed the presence and expression level changes of the receptors. CONCLUSIONS: We conclude that both VPAC1 and VPAC2 could have roles at all stages of retinal development, that PACAP acts through a specific set of PAC1 isoforms, and that Hip and Hop1 are predominantly involved in the postnatal development of rat retina.

Glial cells in the central nervous system of earthworm, Eisenia fetida.

Glial elements in the central nervous system of Eisenia fetida were studied at light- and electron microscopic level. Cells were characterized with the aid of toluidine blue, Glial Fibrillary Acidic Protein (GFAP), S100 staining. We identified neurilemmal-, subneurilemmal-, supporting-nutrifying- and myelinsheath forming glial cells. Both neuronal and non-neuronal elements are S100-immunoreactive in the CNS. Among glial cells neurilemmal and subneurilemmal cells are S100-immunopositive. With the antibody against the S100 protein one band is visible at 15 kDa. GFA P-immunopositive supporting-nutrifying glial cells are localized around neurons and they often appear as cells with many vacuoles. GFA P-positive cell bodies of elongated neurilemmal glial cells are also visible. Western blot analysis shows a single 57 kDa GFA P immunoreactive band in the Eisenia sample. At ultrastructural level contacts between neuronal and glial cells are recognizable. Glial cell bodies and their filopodia contain a granular and vesicular system. Close contacts between neuronal cell membranes and glial filopodia create a special environment for material transport. Vesicles budding off glial cell granules move towards the cell membranes, probably emptying their content with kiss and run exocytosis. The secreted compounds in return may help neuronal survival, provide nutrition, and filopodia may also support neuronal terminals.

A precise temporal dissection of monosodium glutamate-induced apoptotic events in newborn rat retina in vivo.

Although L-glutamate is the main excitatory neurotransmitter in the retina, excess glutamate level triggers severe neuronal damages. Therefore, monosodium glutamate has been used to probe neurodegenerative mechanisms but precise toxicity schedule is not available in vivo. We report, for the first time, a temporal analysis of apoptotic processes induced by subcutaneously applied monosodium glutamate. We investigated the glutamate triggered subcellular processes over a time scale of 48 h in neonatal retina. We employed immunoblots to measure the level of activated apoptotic factors and immunocytochemistry to reveal the dying cells. Upregulation of active caspase-9 started at 3 h and peaked at 6 h post-injection. Activations of caspase-3, caspase-6 and caspase-7 consistent with their late-phase roles increased at 6 h post-injection. The apoptotic processes were terminated by 24 h post-injection. Caspase 12 and calpain-2 seemed unaffected by subcutaneous monosodium glutamate administration. Uniquely, we found that the ubiquitous calpain-1 is not expressed in newborn rat retina.

Interactions of serotoninergic, cholinergic, and tachykinin-containing nerve elements in the rabbit small intestine.

This report presents novel results on the effects of serotonin (5-HT) on longitudinal muscle contractions in the rabbit ileum and the interactions of serotonin with some neuronal elements of the myenteric plexus. We showed previously that serotonin-triggered contractions involved two mechanisms in the rabbit ileum: neuronal excitation (via 5-HT(2) receptors in the neurons) and direct muscular stimulation (via 5-HT(4) receptors in the muscle). Here, we focus on the neuronal 5-HT(2) receptor pathway and report further pharmacological and immunocytochemical data clarifying the details of the mechanisms. We observed that antagonists for neurokinin (NK1 and NK2) receptors partially blocked the serotonin response, but NK3 receptor antagonists had no effect. Pretreatment by atropine (ATR) eliminated the NK1 receptor antagonist resistant contractions. In contrast, the NK1 antagonist did not depress the ATR-resistant contraction when ATR was added first. 5-HT(2) receptor agonist-induced contractions were partially suppressed by ATR, hexamethonium, and NK1 or NK2 receptor antagonists. In conclusion, serotonin acting through 5-HT(2) receptors could stimulate interneurons and excitatory motor neurons. Immunocytochemical staining revealed an extensive tachykinin-immunoreactive (IR) network in the myenteric plexus. Approximately 52% of all myenteric neurons were labeled. 5-HT-IR fibers could be detected around both choline acetyltransferase- and tachykinin-IR cells, suggesting functional relationships between them. Consistent with our pharmacological observations, we found that immunopositive nerve elements for 5-HT(2A) receptor and double-labeled immunostaining revealed a remarkable overlap between tachykinin-IR neurons and 5-HT(2A)-IR elements.

APRIN is a unique Pds5 paralog with features of a chromatin regulator in hormonal differentiation.

Activation of steroid receptors results in global changes of gene expression patterns. Recent studies showed that steroid receptors control only a portion of their target genes directly, by promoter binding. The majority of the changes are indirect, through chromatin rearrangements. The mediators that relay the hormonal signals to large-scale chromatin changes are, however, unknown. We report here that APRIN, a novel hormone-induced nuclear phosphoprotein has the characteristics of a chromatin regulator and may link endocrine pathways to chromatin. We showed earlier that APRIN is involved in the hormonal regulation of proliferative arrest in cancer cells. To investigate its function we cloned and characterized APRIN orthologs and performed homology and expression studies. APRIN is a paralog of the cohesin-associated Pds5 gene lineage and arose by gene-duplication in early vertebrates. The conservation and domain differences we found suggest, however, that APRIN acquired novel chromatin-related functions (e.g. the HMG-like domains in APRIN, the hallmarks of chromatin regulators, are absent in the Pds5 family). Our results suggest that in interphase nuclei APRIN localizes in the euchromatin/heterochromatin interface and we also identified its DNA-binding and nuclear import signal domains. The results indicate that APRIN, in addition to its Pds5 similarity, has the features and localization of a hormone-induced chromatin regulator.

Laminin deficits induce alterations in the development of dopaminergic neurons in the mouse retina.

Genetically modified mice lacking the beta2 laminin chain (beta2null), the gamma3 laminin chain (gamma3 null), or both beta2/gamma3 chains (compound null) were produced. The development of tyrosine hydroxylase (TH) immunoreactive neurons in these mouse lines was studied between birth and postnatal day (P) 20. Compared to wild type mice, no alterations were seen in gamma3 null mice. In beta2 null mice, however, the large, type I TH neurons appeared later in development, were at a lower density and had reduced TH immunoreactivity, although TH process number and size were not altered. In the compound null mouse, the same changes were observed together with reduced TH process outgrowth. Surprisingly, in the smaller, type II TH neurons, TH immunoreactivity was increased in laminin-deficient compared to wild type mice. Other retinal defects we observed were a patchy disruption of the inner limiting retinal basement membrane and a disoriented growth of Müller glial cells. Starburst and AII type amacrine cells were not apparently altered in laminin-deficient relative to wild type mice. We postulate that laminin-dependent developmental signals are conveyed to TH amacrine neurons through intermediate cell types, perhaps the Müller glial cell and/or the retinal ganglion cell.

Neurochemical characterization of nervous elements innervating the body wall of earthworms (Lumbricus, Eisenia): immunohistochemical and pharmacological studies.

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological-physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus sub-muscularis is composed of nerve fibres only, whereas the plexus sub-epithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT(3) and GABA(B) receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.