Alfons B.A. Kroese

Frequency response of the lateral-line organ of xenopus laevis

The stimulus response relation of the epidermal lateral-line organ of Xenopus laevis was studied by recording activity of single afferent nerve fibres in isolated preparations. Linear frequency response analysis over a frequency range of 0.1–100 Hz was performed under steady-state conditions, using small amplitude, plitude, sinusoidal water displacements produced by a glass sphere at a short distance from the skin.Period histograms of afferent nerve activity were computed, and amplitude, phase and mean activity of the response were determined by means of Fourier analysis. A standardization procedure at the start of each experiment made scaling of the frequency responses of different preparations unnecessary.The results show that for small stimulus amplitudes the response of the lateral-line organ over the whole range of frequencies studied can adequately be described as a modulation of the spontaneous activity. The amplitude of the response is proportional to the stimulus amplitude, and the phase of the response is independent of stimulus amplitude. The lateral-line organ of Xenopus laevis can thus be regarded as a linear system for stimuli which produce modulation of the spontaneous activity.The frequency response demonstrates unequivocally that the lateral-line organ of Xenopus laevis functions as a water velocity detector. For frequencies of stimulation from 0.1–20 Hz the gain increases with a slope of 7.5 dB/oct, and up to 5 Hz the response is almost in phase with the water velocity. The extent to which the different transmission steps between stimulus and response will contribute to the frequency response is discussed.

Consequences of intestinal inflammation on the enteric nervous system: Neuronal activation induced by inflammatory mediators

The ENS is responsible for the regulation and control of all gastrointestinal functions. Because of this critical role, and probably as a consequence of its remarkable plasticity, the ENS is often relatively well preserved in conditions where the architecture of the intestine is seriously disrupted, such as in IBD. There are structural and functional changes in the enteric innervation in animal models of experimental intestinal inflammation and in IBD. These include both up and down regulation of transmitter expression and the induction of new genes in enteric neurons. Using Fos expression as a surrogate marker of neuronal activation it is now well established that enteric neurons (and also enteric glia) respond to inflammation. Whether this “activation” is limited to a short‐term functional response, such as increased neuronal excitability, or reflects a long‐term change in some aspect of the neuronal phenotype (or both) has yet to be firmly established, but it appears that enteric neurons are highly plastic in their response to inflammation. Anat Rec 262:79–90, 2001.

CCKA and CCKB receptor subtypes both mediate the effects of CCK-8 on myenteric neurons in the guinea-pig ileum

The effects of cholecystokinin (CCK-8) on myenteric S neurons were investigated by intracellular recording techniques, with the aim to determine the CCK receptor subtypes involved. CCK-8 (1-1000 nM) evoked concentration-dependent long-lasting excitatory responses in 45 of 54 neurons. CCK receptor antagonists were applied to 15 neurons in which CCK-8 evoked an excitatory response. In 5 of these neurons, application of the CCKA antagonist L-364,718 (100-500 nM) antagonized the action of CCK-8 and the CCKB antagonist L-365,260 (500 nM) had no effect. L-365,260 (100-500 nM) antagonized the CCK-8 induced response in 5 neurons, on which L-364,718 had no effect. In the other 5 neurons each antagonist (500 nM) partly inhibited the CCK-8 evoked excitation and application of both antagonists (500 nM) caused a complete blockade of the response to CCK-8. The selective CCKB receptor agonist CCK-8NS had similar excitatory effects as CCK-8, but only on the neurons in which CCK-8 evoked effects were antagonized by L-365,260. The results demonstrate that the excitatory effects of CCK-8 are mediated by both CCKA and CCKB receptor subtypes. Further, the results indicate that some neurons possess exclusively the CCKA or the CCKB receptor subtype, but others possess both subtypes.

Effects of ototoxic antibiotics on sensory hair cell functioning

The isolated lateral-line organ of Xenopus laevis was used to study reversible effects of low concentrations of aminoglycoside antibiotics on sensory hair cell functioning. Extracellular receptor potentials and single fibre afferent nerve activity were recorded during stimulation of the lateral-line organ with sinusoidal water movements. The results show that dihydrostreptomycin in concentrations between 2.5 and 17.5 micrograms/ml caused an increase in spontaneous afferent nerve activity, probably by increasing the spontaneous transmitter release at the afferent synapse. In addition, the same concentrations of dihydrostreptomycin markedly affected the mechano-electric transduction process, resulting in a phase lag in th receptor potential, the magnitude of which increased with increasing concentration up to a value of 0.4 pi radians. The effect of dihydrostreptomycin on the amplitude of the receptor potentials depended on the stimulus frequency. Other aminoglycoside antibiotics, namely streptomycin, gentamycin and kanamycin, produced similar effects but at different concentrations. It is concluded that low concentrations of aminoglycoside antibiotics have a dual action upon the sensory hair cells. They increase the spontaneous activity by affecting the hair cell membrane and at the same time they impair the mechano-electric transduction process resulting in a large phase lag in the receptor potentials. The latter effect, which is antagonized by Ca2+, may be due to interference by the antibiotics with the mode of motion of the sensory hairs.

Evidence for a role of cholecystokinin as neurotransmitter in the guinea-pig enteric nervous system

Intracellular recordings were made of neurons in the myenteric plexus of the guinea-pig distal ileum. Slow excitatory postsynaptic potentials (sEPSPs) were evoked by electrical stimulation of an interganglionic fibre tract. The effect of cholecystokinin (CCK) receptor antagonists on the sEPSPs was investigated in 11 neurons. Application of the CCK receptor antagonists L-364,718 and L-365,260 (each 250 nM) markedly attenuated the sEPSPs in five of 11 neurons. The amplitude of the sEPSP reduced from 15 +/- 3 to 7 +/- 2 mV and the change in membrane resistance during the sEPSP was reduced from 28 +/- 9 to 11 +/- 8 MS. In six of 11 neurons the CCK antagonists had no effect on the sEPSPs. The results provide evidence that neurally released CCK is involved in the mediation of sEPSPs in some enteric neurons.

Pretreatment but not treatment with probiotics abolishes mouse intestinal barrier dysfunction in acute pancreatitis

BACKGROUND Intestinal barrier failure during acute pancreatitis (AP) is associated with translocation of luminal bacteria, resulting in infectious complications. We examined the effects of multispecies probiotics on the intestinal barrier impairment in a murine model of AP. METHODS Mice were injected with cerulein to induce AP and were sacrificed 11 (early AP) or 72 hours (late AP) after start of induction. AP and associated systemic effects were confirmed by histology of pancreas and lung. Animals received daily probiotics starting 2 days prior to AP induction (pretreatment) or at the moment of AP induction (treatment). Mucosal barrier function of the distal ileum was assessed in Ussing chambers by measurement of the epithelial electrical resistance and the permeability to Na-fluorescein. RESULTS Histological analysis revealed pancreatic injury in both phases of AP, and lung damage in the early phase. Epithelial resistance of the ileum was reduced and permeability increased in both phases of AP, indicating impairment of the intestinal barrier. Pretreatment had no effect on resistance or permeability in the early phase of AP. In the late phase of AP, pretreatment but not treatment abolished the AP induced resistance decrease and permeability increase. Administration of probiotics as such (ie, without induction of AP) had no effect on intestinal barrier function. CONCLUSION Pretreatment with multispecies probiotics for 2 days abolishes intestinal barrier dysfunction in the late phase of AP, while treatment does not. The effectiveness of probiotics in this model depends on the timing of administration. Clinical trials with probiotics should seek conditions where treatment can be started prior to onset of disease or elective surgical intervention.

Excitatory synaptic inputs on myenteric Dogiel type II neurones of the pig ileum

The synaptic input on myenteric Dogiel type II neurones (n = 63) obtained from the ileum of 17 pigs was studied by intracellular recording. In 77% of the neurones, electrical stimulation of a fibre tract evoked fast excitatory postsynaptic potentials (fEPSPs) with an amplitude of 6 ± 5 mV (mean ± S.D.) and lasting 49 ± 29 ms. The nicotinic nature of the fEPSPs was demonstrated by superfusing hexamethonium (20 μM). High‐frequency stimulation (up to 20 Hz, 3 seconds) did not result in a rundown of the fEPSPs, and did not evoke slow excitatory or inhibitory postsynaptic potentials. The effects of neurotransmitters, possibly involved in these excitatory responses, were investigated. Pressure microejection of acetylcholine (10 mM in pipette) resulted in a fast nicotinic depolarisation in 67%(18/27) of the neurones (13 ± 9 mV, duration 7.0 ± 7.2 seconds) as did 1,1‐dimethyl‐4‐phenylpiperazinium iodide (DMPP) application (10 mM; 14 ± 10 mV, duration 4.1 ± 2.8 seconds) in 76%of the cells. The fast nicotinic response to acetylcholine was sometimes (6/27) followed by a slow muscarinic depolarisation (8 ± 4 mV; duration 38.7 ± 10.8 seconds). Immunostaining revealed 5‐hydroxytryptamine hydrochloride (5‐HT)‐ and calcitonin gene‐related peptide (CGRP)‐positive neuronal baskets distributed around and in close vicinity to Dogiel type II neuronal cell bodies. Microejection of 5‐HT (10 mM) resulted in a fast nicotinic‐like depolarisation (12 ± 6 mV, duration 3.0 ± 1.3 seconds) in 4 of 8 neurones tested, whereas microejection of CGRP (20 mM) gave rise to a slow muscarinic‐like depolarisation (6 ± 2 mV, duration 56.0 ± 27.5 seconds) in 8 of 12 neurones tested. In conclusion, myenteric Dogiel type II neurones in the porcine ileum receive diverse synaptic input. Mainly with regard to the prominent presence of nicotinic responses, these neurones behave contrary to their guinea pig counterparts. J. Comp. Neurol. 432:137–154, 2001.

Prostaglandin E2 activation of VIP secretomotor neurons in the guinea pig ileum

The effect of prostaglandin E2 (PGE2) on the activity-related expression of the proto-oncogene c-fos in specific populations of enteric neurons was investigated. Segments of guinea-pig ileum were incubated in vitro in the presence or absence of PGE2, and whole mounts of the myenteric and submucosal plexus were prepared for immunocytochemical localization of Fos, VIP and NPY. Control tissues exhibited a low number of Fos-immunoreactive (Fos-IR) neurons (7 +/- 2% of total). Incubation of the tissues with 10-1000 nM PGE2 for 30 min caused a concentration-dependent increase in Fos-IR submucosal neurons (maximum at 100 nM; 39 +/- 6%), which was not inhibited by TTX. PGE2 did not evoke an increase in Fos-IR myenteric neurons. In double labeling experiments, Fos colocalized exclusively with VIP in the submucosal plexus, and not with NPY. Exposure of stripped segments of guinea pig ileum in Ussing chambers to 100 nM PGE2 evoked an increase in short circuit current (20 +/- 7 microA/cm2), of which the initial rapid phase could be abolished by TTX, and not by atropine and hexamethonium. It is concluded that PGE2 can activate VIP non-cholinergic secretomotor neurons.

Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells.

The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA(A) and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca(2+) concentration ([Ca(2+)](i)). As Ca(2+) triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca(2+) homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca(2+)](i) imaging experiments with fura-2 demonstrated that lindane (≥ 10 μM) rapidly increases basal exocytosis and basal [Ca(2+)](i). Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca(2+) channels (VGCC). On the other hand, lindane (≥ 3 μM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca(2+)](i) and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca(2+) influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic. These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca(2+)](i) and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca(2+)](i) and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.

CGRP1 receptor activation induces piecemeal release of protease‐1 from mouse bone marrow‐derived mucosal mast cells

Background  The parasitized or inflamed gastrointestinal mucosa shows an increase in the number of mucosal mast cells (MMC) and the density of extrinsic primary afferent nerve fibers containing the neuropeptide, calcitonin gene‐related peptide (CGRP). Currently, the mode of action of CGRP on MMC is unknown.