Catharina Olsson

The control of gut motility.

Gut motility in non-mammalian vertebrates as in mammals is controlled by the presence of food, by autonomic nerves and by hormones. Feeding and the presence of food initiates contractions of the stomach wall and subsequently gastric emptying, peristalsis, migrating motor complexes and other patterns of motility follow. This overview will give examples of similarities and differences in control systems between species. Gastric receptive relaxation occurs in fish and is an enteric reflex. Cholecystokinin reduces the rate of gastric emptying in fish as in mammals. Inhibitory control of peristalsis is exerted, e.g. by VIP, PACAP, NO in fish and amphibians, while excitatory stimuli arise from nerves releasing tachykinins, acetylcholine or serotonin (5-HT). In crocodiles, we have found the presence of the same nerve types, although the effects on peristalsis have not been studied. Recent studies on signal transduction in the gut smooth muscle of fish and amphibians suggest that external Ca2+ is of great importance, but not the only source of Ca2+ recruitment in tachykinin-, acetylcholine- or serotonin-induced contractions of rainbow trout and Xenopus gastrointestinal smooth muscle. The effect of acetylcholine involves reduction of cAMP-levels in the smooth muscle cells. It is concluded that, in general, the control systems in non-mammalian vertebrates are amazingly similar between species and animal groups and in comparison with mammals.

Aerobic scope fails to explain the detrimental effects on growth resulting from warming and elevated CO2 in Atlantic halibut

As a consequence of increasing atmospheric CO2, the worlds oceans are becoming warmer and more acidic. Whilst the ecological effects of these changes are poorly understood, it has been suggested that fish performance including growth will be reduced mainly as a result of limitations in oxygen transport capacity. Contrary to the predictions given by the oxygen- and capacity-limited thermal tolerance hypothesis, we show that aerobic scope and cardiac performance of Atlantic halibut (Hippoglossus hippoglossus) increase following 14–16 weeks exposure to elevated temperatures and even more so in combination with CO2-acidified seawater. However, the increase does not translate into improved growth, demonstrating that oxygen uptake is not the limiting factor for growth performance at high temperatures. Instead, long-term exposure to CO2-acidified seawater reduces growth at temperatures that are frequently encountered by this species in nature, indicating that elevated atmospheric CO2 levels may have serious implications on fish populations in the future.

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.

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.)

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.

Neurochemical characterization of extrinsic innervation of the guinea pig rectum

The presence of markers for parasympathetic, sympathetic, and glutamatergic or peptidergic sensory innervation was investigated by using in vitro tracing with biotinamide, combined with immunohistochemistry, to characterise quantitatively extrinsic axons to myenteric ganglia of the guinea pig rectum. Of biotinamide‐filled varicose axons, 3.6 ± 1.3% were immunoreactive for tyrosine hydroxylase (TH) and 16.0 ± 4.8% for vesicular acetylcholine transporter (VAChT). TH and vesicular monoamine transporter (VMAT1) showed high coexistence (83–100%), indicating that varicosities lacking TH immunoreactivity also lacked VMAT1. VAChT was detectable in 77% of choline acetyltransferase (ChAT)‐immunoreactive varicosities. Calcitonin gene‐related peptide (CGRP) was detected in 5.3 ± 1.6% of biotinamide‐labeled varicosities, the vesicular glutamate transporter (VGluT) 1 in 2.8 ± 0.8%, and VGluT2 in 11.3 ± 4.2% of varicosities of extrinsic origin. Varicosities from the same axon showed consistent immunoreactivity. A novel type of nerve ending was identified, with branching, flattened lamellar endings, similar to the intraganglionic laminar endings (IGLEs) of the proximal gut. Rectal IGLEs were frequently immunoreactive for VGluT1 and VGluT2. Thus most varicose axons of extrinsic origin, which innervate rectal myenteric ganglia, lack detectable levels of immunoreactivity for TH, VMAT1, VAChT, ChAT, VGluT1/2, or CGRP, under conditions in which these markers are readily detectable in other axons. Although some unlabeled varicosities may belong to afferent axons that lack detectable CGRP or VGluT1/2 in the periphery, this suggests that a large proportion of axons do not release any of the major autonomic or sensory transmitters. We speculate that this may vary under particular circumstances, for example, inflammation or obstruction of the gut. J. Comp. Neurol. 470:357–371, 2004.

Autonomic control of gut motility: A comparative view

Gut motility is regulated to optimize food transport and processing. The autonomic innervation of the gut generally includes extrinsic cranial and spinal autonomic nerves. It also comprises the nerves contained entirely within the gut wall, i.e. the enteric nervous system. The extrinsic and enteric nervous control follows a similar pattern throughout the vertebrate groups. However, differences are common and may occur between groups and families as well as between closely related species. In this review, we give an overview of the distribution and effects of common neurotransmitters in the vertebrate gut. While the focus is on birds, reptiles, amphibians and fish, mammalian data are included to form the background for comparisons. While some transmitters, like acetylcholine and nitric oxide, show similar distribution patterns and effects in most species investigated, the role of others is more varying. The significance for these differences is not yet fully understood, emphasizing the need for continued comparative studies of autonomic control.

Coexistence of NADPH-diaphorase and vasoactive intestinal polypeptide in the enteric nervous system of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias)

The distribution of NADPH (nicotinamide adenine dinucleotide phosphate)-diaphorase in nerve cells in the gastrointestinal tract has been investigated and compared in three fish species representing different evolutionary branches. In mammals, NADPH-diphorase is identical to nitric oxide synthase (NOS) and can, in the presence of NADPH, reduce the dye nitroblue tetrazolium, resulting in a blue product. Using this method, we have found numerous NADPH-diaphorase-containing nerve cells in the myenteric plexus of the Atlantic cod (Gadus morhua) and the spiny dogfish (Squalus acanthias) but none in the hagfish (Myxine glutinosa). In the cod, nerve fibres were sparsely stained, whereas in the dogfish, they formed a dense pattern of fibre bundles. Double-staining for NADPH-diaphorase and the neuropolypeptides VIP (vasoactive intestinal polypeptide) and PACAP (pituitary adenylate cyclase activating peptide) revealed three separate populations designated VIP/NADPH, VIP/- and NADPH/-. The majority but not all of the NADPH-diaphorase-positive cells also showed VIP or PACAP immunoreactivity and vice versa. The presence of NADPH-diaphorase in neurons and the distribution of these neurons in the gastrointestinal tract of the two species indicate a physiological role for nitric oxide in the control of gut motility.

Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.

Comparison of extrinsic efferent innervation of guinea pig distal colon and rectum

The extrinsic efferent innervation of the distal colon and rectum of the guinea pig was compared, by using retrograde tracing combined with immunohistochemistry. Application of the carbocyanine tracer DiI to the rectum filled significantly greater numbers of extrinsic neurons than similar injections into the distal colon. Approximately three‐fourths of all filled neurons from either location were either sympathetic or parasympathetic; the rest were spinal sensory neurons. Nerve cell bodies in sympathetic prevertebral ganglia labelled from the two regions were similar in number. Both regions were innervated by sympathetic neurons in paravertebral ganglia; however, the rectum received much more input from this source than the colon. The rectum received significantly more input from pelvic ganglia than the colon. The rectum also received direct innervation from two groups of neurons in the spinal cord. Neurons located in the spinal parasympathetic nucleus in segment S2 and S3 were labelled by DiI injected into the rectal wall. Similar numbers of neurons, located in intermediolateral cell column and dorsal commissural nucleus of lumbar segments, also projected directly to rectum, but not colon. The great majority (>80%) of retrogradely labelled nerve cell bodies in sympathetic ganglia were immunoreactive for tyrosine hydroxylase. In pelvic ganglia, retrogradely labelled neurons contained choline acetyltransferase and/or nitric oxide synthase or tyrosine hydroxylase. Although the rectum and colon in this species are continuous and macroscopically indistinguishable, they have significantly different patterns of extrinsic efferent innervation, presumably reflecting their different functions. J. Comp. Neurol. 496:787–801, 2006.