Anna Holmberg

Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut

The autonomic nervous system develops following migration and differentiation of precursor cells originating in the neural crest. Using immunohistochemistry on intact zebrafish embryos and larvae we followed the development of the intrinsic enteric and extrinsic vagal innervation of the gut. At 3 days postfertilization (dpf), enteric nerve cell bodies and fibers were seen mainly in the middle and distal intestine, while the innervation of the proximal intestine was scarcer. The number of fibers and cell bodies gradually increased, although a large intraindividual variation was seen in the timing (but not the order) of development. At 11–13 dpf most of the proximal intestine received a similar degree of innervation as the rest of the gut. The main intestinal branches of the vagus were similarly often already well developed at 3 dpf, entering the gut at the transition between the proximal and middle intestine and projecting posteriorly along the length of the gut. Subsequently, fibers branching off the vagus innervated all regions of the gut. The presence of several putative enteric neurotransmitters was suggested by using markers for neurokinin A (NKA), pituitary adenylate cyclase‐activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide, serotonin (5‐hydroxytryptamine, 5‐HT), and calcitonin gene‐related peptide (CGRP). The present results corroborate the belief that the enteric innervation is well developed before the onset of feeding (normally occurring around 5–6 dpf). Further, the more detailed picture of how development proceeds at stages previously not examined suggests a correlation between increasing innervation and more regular and elaborated motility patterns. J. Comp. Neurol. 508:756–770, 2008.

Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae

SUMMARY Using digital motion analysis, the ontogeny of the cholinergic, tachykinin and pituitary adenylate cyclase-activating polypeptide (PACAP) control systems was studied in zebrafish Danio rerio larvae, in vivo. For the first time we show that the regular propagating anterograde waves that occur in the zebrafish larval gut before and around the onset [at 5–6 days post fertilization (d.p.f.)] of feeding are modulated by acetylcholine or atropine, PACAP and NKA (neurokinin A). At 3 d.p.f., when no spontaneous motility has developed, application of acetylcholine did not affect the gut. However, at 4 d.p.f., acetylcholine increased and atropine reduced the frequency of propagating anterograde waves. At 5 d.p.f., NKA increased and PACAP reduced the wave frequency. This suggests that both excitatory and inhibitory pathways develop at an early stage in the gut, independent of exogenous feeding. Immunohistochemistry established the presence of gut neurons expressing PACAP and NKA in the proximal part of the developing gut from the first stage investigated (2 d.p.f.) and before regular motility was observed. 1 d.p.f. (PACAP) or 2 d.p.f. (NKA) stages later the whole gut was innervated. This supports physiological results that gut motility is under neuronal control during the period when regular motility patterns develop.

TTX-sensitive and TTX-insensitive control of spontaneous gut motility in the developing zebrafish (Danio rerio) larvae.

SUMMARY Spontaneous regular gut motility in zebrafish begins around 4 days post fertilisation (d.p.f.) and is modulated by release of acetylcholine and nitric oxide. The role of intrinsic or extrinsic innervation for initiating and propagating the spontaneous contractions, however, is not well understood. By creating spatiotemporal maps, we could examine spontaneous motility patterns in zebrafish larvae in vivo at 4 and 7 d.p.f. in more detail. Tetrodotoxin (TTX) was added to elucidate the importance of nervous control. Anterograde and retrograde contraction waves originated in the same region, just posterior to the intestinal bulb. This area correlates well with the distribution of Hu (human neuronal protein C/D)-immunoreactive nerve cell bodies. Whereas numerous immunoreactive nerve cells were present in the mid and distal intestine at both 4 and 7 d.p.f., fewer cells were seen anterior to the origin of contractions. The overall frequency of contractions (1.16±0.15 cycles min–1, N=14 at 4 d.p.f.; 1.05±0.09 cycles min–1, N=13 at 7 d.p.f.) and the interval between individual anterograde contraction waves (54.8±7.9 s at 4 d.p.f., N=14; 56.9±4.4 s, N=13 at 7 d.p.f.) did not differ between the two stages but the properties of the contractions were altered. The distance travelled by each wave increased from 591.0±43.8 μm at 4 d.p.f. (N=14) to 719.9±33.2 μm at 7 d.p.f. (N=13). By contrast, the velocity decreased from 4 d.p.f. (49.5±5.5 μm s–1, N=12) to 7 d.p.f. (27.8±3.6 μm s–1, N=13). At 4 d.p.f., TTX did not affect any of the parameters whereas at 7 d.p.f. anterograde frequency (control 1.07±0.12 cycles min–1, N=8; TTX 0.55±0.13 cycles min–1, N=8) and distance travelled (control 685.1±45.9 μm, N=8; TTX 318.7±88.7 μm, N=6) were decreased. In conclusion, enteric or extrinsic innervation does not seem to be necessary to initiate spontaneous contractions of the gut in zebrafish larvae. However, later in development, nerves have an increasingly important role as modulators of intestinal activity.)

CD11c(high )dendritic cells are essential for activation of CD4+ T cells and generation of specific antibodies following mucosal immunization.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11chigh conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8+ T cells in vivo, but the role of cDCs in CD4+ T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4+ T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11chigh cDCs. Our results show that cDCs are absolutely required for activation of CD4+ T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4+ T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.

The effects of endogenous and exogenous nitric oxide on gut motility in zebrafish Danio rerio embryos and larvae.

SUMMARY Using motion analysis, the ontogeny of the nitrergic control system in the gut was studied in vivo in zebrafish Danio rerio embryos and larvae. For the first time we show the presence of a nitrergic tonus, modulating both anterograde and retrograde contraction waves in the intestine of developing zebrafish. At 4 d.p.f. (days post fertilisation), the nitric oxide synthase (NOS) inhibitor l-NAME (three boluses of 50–100 nl, 10–3 mol l–1) increased the anterograde contraction wave frequency by 0.50±0.10 cycles min–1. Subsequent application of the NO donor sodium nitroprusside (SNP; three boluses of 50–100 nl, 10–4 mol l–1) reduced the frequency of propagating anterograde waves (–0.71±0.20 cycles min–1). This coincided with the first appearance of an excitatory cholinergic tonus, observed in an earlier study. One day later, at 5 d.p.f., in addition to the effect on anterograde contraction waves, application of l-NAME increased (0.39±0.15 cycles min–1) and following SNP application reduced (–1.61±0.36 cycles min–1) the retrograde contraction wave frequency. In contrast, at 3 d.p.f., when no spontaneous motility is observed, application of l-NAME did not induce contraction waves in either part of the gut, indicating the lack of a functional inhibitory tonus at this early stage. Gut neurons expressing NOS-like immunoreactivity were present in the distal and middle intestine as early as 2 d.p.f., and at 1 day later in the proximal intestine. In conclusion, the present study suggests that a nitrergic inhibitory tonus develops shortly before or at the time for onset of exogenous feeding.

Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses

Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyers patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7+ B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.

A Unique Role of the Cholera Toxin A1-DD Adjuvant for Long-Term Plasma and Memory B Cell Development

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD–adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80+, but not CD80−, B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

Does waterborne citalopram affect the aggressive and sexual behaviour of rainbow trout and guppy

Citalopram is one of several selective serotonin reuptake inhibitors (SSRIs) commonly found in treated sewage effluents. Accordingly, there are concerns about possible adverse effects of SSRIs on aquatic organisms, particularly behavioural effects similar to those associated with SSRI use in humans. Rainbow trout fry and adult male guppies were therefore exposed to waterborne citalopram, ranging from environmentally relevant to high concentrations (1, 10, 100 μg/L) for 3-7 days. Under these experimental conditions citalopram does not appear to cause significant effects on aggression in rainbow trout fry or on sexual behaviour in male guppies. This may be explained by a relatively low uptake of citalopram from water to fish.

Effects of digestive status on the reptilian gut

Reptiles, including the Burmese python, Python molurus bivittatus, that feed at infrequent intervals show a prominent increase in gastrointestinal mass, metabolism and brush border transport rates after feeding. Current knowledge and theories around these phenomena, as well as studies on the innervation of the reptilian gut, are summarised in this review. Little is known about the putative changes in the nervous and humoral control systems of the gut, and it is not known whether feeding affects innervation and motility of the stomach and intestine. Using immunohistochemistry, we have investigated possible up/down regulation of several neurotransmitters in specimens that had been fasted for a minimum of 3 weeks and specimens that had ingested a large meal 2 days before the experiments were conducted. There were no major changes in the innervation by nerves containing calcitonin gene-related peptide (CGRP), galanin, nitric oxide synthase (NOS), pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM), substance P/neurokinin A (SP/NKA), or vasoactive intestinal polypeptide (VIP)-like immunoreactivity. Nor did we find any differences in the effect of substance P (stomach and intestine), galanin (intestine), or bradykinin (intestine) on motility in strip preparations from the gut wall. A significant increase in dry weight of the intestine was obtained 48 h after feeding. We conclude that although there are considerable changes in gut thickness and absorptive properties after feeding, the smooth muscle and its control appear little affected.

Occurrence of neurotrophin receptors and transmitters in the developing Xenopus gut

Abstract. The ontogeny of gut innervation in the anuran amphibian Xenopus laevis was studied using immunohistochemistry on sections of whole larvae from NF stages 38–52. Immunoreactivity to acetylated tubulin confirmed the presence of nerve fibres as early as stages 38–39. Actin immunoreactivity was found at stage 41, indicating the presence of smooth muscle cells. Trk-like neurotrophin receptors were occasionally found in nerve fibres as soon as stages 38–39. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) immunoreactivities coexisted in nerves innervating the gut wall from stages 40–41, and nitric oxide synthase (NOS) from stage 42. Substance P/neurokinin A (SP/NKA) occurred at stage 42. In all these cases, the first fibres were observed in the oesophagus. Calcitonin gene-related peptide (CGRP) was first observed in nerves at stage 48. In general, VIP/PACAP and NOS innervation was denser than the tachykinin innervation. In conclusion, the development of nerve fibres in the Xenopus gut is probably dependent on neurotrophins that may act via Trk-like receptors and occur before the gut wall is fully organised morphologically. Feeding in Xenopus larvae starts at NF stage 45. The study demonstrates that several of the transmitters investigated are expressed in the gut innervation (and in endocrine cells) prior to this stage.