Collagenous colitis as a possible cause of toxic megacolon.

Collagenous colitis is a microscopic colitis characterized by normal appearing colonic mucosa on endoscopy. It is regarded as a clinically benign disease which rarely results in serious complications. We report a case of toxic megacolon occurring in a patient with collagenous colitis. This is the first reported case of toxic megacolon occurring in this subset of patients.

Lack of awareness of oesophageal carcinoma among the public in Ireland.

BACKGROUND: Oesophageal cancer is advanced in the majority at presentation and its symptoms are usually present for many months suggesting poor awareness of its symptoms. Few studies have examined awareness of oesophageal cancer amongst the public. AIMS: This study aimed to identify the level of awareness among the general public of oesophageal cancer, of its symptoms, of its awareness campaigns and to compare it with other common cancers. METHODS: Face-to-face interviews were conducted with 279 members of the public. People were asked about their awareness of a range of cancers, and their knowledge of cancer symptoms and cancer awareness campaigns. RESULTS: Awareness of oesophageal cancer was low and knowledge of its symptoms was even lower. Despite the efforts of awareness campaigns, knowledge of these campaigns was poor amongst the public. CONCLUSION: Awareness of oesophageal cancer and its symptoms is low amongst the public. This needs to be addressed if disease is to be detected at an earlier and curable stage.

Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons.

We used a novel mammalian coculture system to study ACh receptor (AChR) redistribution and synaptic structure at nerve-muscle contacts. Ventral spinal cord (VSC) neurons were plated on cultures containing extensive myotubes but few fibroblasts. Neurite-induced redistribution of AChRs occurred within 6 hr after plating neurons and was maximal between 36-48 hr. This AChR redistribution appeared in two patterns: (1) AChR density at sites directly apposed to the neurite where neurites crossed preexisting AChR patches was sharply reduced, (2) Newly aggregated AChRs formed swaths lateral to the neurite path. VSC neurons induced more AChR aggregation than hippocampal, superior cervical ganglion and dorsal root ganglion neurons. The 43 and 58 kDa postsynaptic proteins were colocalized with AChR-enriched domains in all VSC neurite-induced aggregates whereas the colocalization of laminin was variable. Electron microscopy of regions with neurite-induced AChR aggregation showed postsynaptic membrane specializations characteristic of developing synapses and, in older cultures, features of more mature synaptic structure. Thus, the coculture system is useful for studying early stages of neuromuscular junction (NMJ) formation. Neurites in these cocultures were identified as axons or dendrites by morphological criteria and by their immunoreactivity for synaptophysin and phosphorylated heavy neurofilament subunits or for microtubule associated protein 2 (MAP2), respectively. Axons showed a 10-fold higher induction of AChR aggregation than did dendrites. Thus, at least one essential signaling molecule necessary for the induction of AChR aggregation at sites of interaction with muscle appears to be expressed in a polarized fashion in developing VSC neurons.

Excitatory amino acid regulation of the enkephalin phenotype in mouse embryonic spinal cord cultures.

Expression of the preproenkephalin gene in developing spinal cord-dorsal root ganglia (SC-DRG) cultures was determined by Northern analysis following treatments with different agonists and antagonists of the glutamate receptor. Cultures (10-12 days old) were treated with various concentrations (10(-7)-10(-3) M) of N-methyl-D-aspartate (NMDA), quisqualate, kainic acid (KA), 2-amino-5-phosphonovaleric acid (APV) and 5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine maleate (MK801) either with or without blocking spontaneous electrical activity with 1 microM tetrodotoxin (TTX). In electrically active cultures, treatments with NMDA and KA increased preproenkephalin transcripts (mRNAppENK), showing maximum effects at 1 microM (4-fold and 2-fold, respectively), while treatments with quisqualate and MK801 caused concentration-dependent down-regulation in mRNAppENK. The most effective concentrations of NMDA (1 microM) and quisqualate (10 microM) altered mRNAppENK levels within 4 h of treatment and peaked after 24 h for NMDA and 48 h for quisqualate treatment. Co-treatment with APV completely blocked the NMDA-induced rise of mRNAppENK. During electrical blockade, none of the concentrations of NMDA tested showed any effect on enkephalin expression, neither could NMDA pre-treatment prevent the TTX-induced down-regulation of mRNAppENK. Our results indicate that the activity-dependent establishment of the enkephalin phenotype is modulated through the selective activation of the NMDA-glutamate receptor.

In search of the central respiratory neurons: I. Dissociated cell cultures of respiratory areas from the upper medulla.

Dissociated cells from the areas of the nucleus ambiguus and the nucleus tractus solitarius obtained by tissue punch or block dissection from coronal slices of the medulla at the level of the obex were cultured from fetal rats at 18 to 21 days gestation. The dissociated neurons were plated either directly in vitrogen-coated 35 mm tissue culture dishes or in such dishes which had been seeded with subcultures of cortex- or medulla-derived astrocytes. After the astrocytes reached confluency and were treated with an antimitotic agent, dissociated nucleus ambiguus or nucleus tractus solitarius was plated at 0.5-1.0 x 10(6) cells per dish. Neurons grew well on monolayers of medullary or cortical astrocytes, but survived poorly on vitrogen-coated dishes without a cellular substrate. Rat medulla was preferred as the source of astrocytes. Tissue dissociation with papain rather than trypsin produced less cellular debris, and the neuronal yield from the tissue was higher. The neuronal population was heterogenous in morphology including small and large bipolar, pyramidal, and multipolar cells. Neurons sensitive to CO2 and/or low pH (Rigatto et al., J Neurosci Res 33:590-597, 1992) did not appear to have any definitive morphologic characteristics, but most were multipolar. These neurons stained well with antibodies to neuron-specific enolase and Fragment C of tetanus toxin, but not to choline acetyltransferase (ChAT). These findings suggest that neurons possibly responsible for the central regulation of respiration can be maintained for several weeks in dissociated cell culture, providing a system for neurotransmitter, electrophysiological, and morphological studies.

In search of the central respiratory neurons: II. Electrophysiologic studies of medullary fetal cells inherently sensitive to CO2 and low pH.

Although extensively pursued, the central respiratory neurons have remained elusive. We departed from the more conventional physiologic and morphologic methods of system and tissue examination and cultured dissociated fetal rat cells (Fitzgerald et al., J Neurosci Res 33:579-589, 1992) from the area of the nucleus ambiguus and the nucleus tractus solitarius located within the 2 mm rostral to the obex. Pacemaker-like cells, with a regular single or bursting activity, studied at 3-5 weeks of age, responded to very small pulses of CO2 (50 ms) and low pH with an increase in spike frequency and a decrease in spike amplitude. Other irregularly beating or silent cells did not respond or else required very large pulses (> 200 ms) to do so. The pacemaker cells also responded to hypoxia induced by administration of sodium hydrosulfite with an increase in spike frequency and amplitude; high oxygen (> 600 torr) and adenosine produced a decrease in electrical activity. Most of these cells were multipolar after staining with antibodies to neuron-specific enolase (NSE) and Fragment C of tetanus toxin. They did not stain for choline acetyltransferase (ChAT). The results suggest that these cultured cells, expressing a phenotype inherently responsive to CO2 and low pH, have the characteristics of central respiratory chemoreceptors, and may be involved in the generation of the respiratory rhythm.

Differential effects of tetanus toxin on inhibitory and excitatory neurotransmitter release from mammalian spinal cord cells in culture.

The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.

Cholinergic function in cultures of mouse spinal cord neurons.

Cholinergic synapses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetyltransferase (ChAT). Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recordings showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparations indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co-cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non-neuronal cells from cerebral cortex, than in co-cultures with VH alone or with SC and dorsal root ganglion cells. Consistency between cholinergic physiology and staining for ChAT-positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG. Application of acetylcholine, muscarine, and/or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synaptic interactions between SC neurons in only one-third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures. The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.

Differential neurochemical effects of chronic exposure of cerebral cortical cell culture to valproic acid, diazepam, or ethosuximide.

We have assessed the relative neurochemical effects of valproic acid, ethosuximide, and diazepam on dissociated cultures of mouse cerebral cortex. Cultures were exposed chronically (11 days) to each antiepileptic drug and assayed for number of neurons, total protein, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, choline acetyltransferase activity, and specific and clonazepam-displaceable benzodiazepine binding. Ethosuximide-exposed cultures did not evidence neuronal toxicity; exposure to valproic acid and diazepam resulted in modest neuronal toxicity. However, exposure to each of these drugs resulted in a marked reduction in benzodiazepine binding. This effect may relate to a common mechanism of action of drugs used to treat absence seizures.

Differential toxicity of chronic exposure to phenytoin, phenobarbital, or carbamazepine in cerebral cortical cell cultures.

The effects of phenytoin (30 micrograms/ml), phenobarbital (64 micrograms/ml), and carbamazepine (24 micrograms/ml) were assessed in cerebral cortical cell cultures. After antiepileptic drug exposure for eleven days, cultures were assayed for total protein, number of neurons, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, activity of choline acetyltransferase, and benzodiazepine binding. Carbamazepine-exposed cultures demonstrated minimal effects, whereas highly significant deficits related to generalized toxicity were observed in cultures exposed to phenytoin or phenobarbital.

A method for large-scale production of mouse brain cortical cultures.

Fetal cerebral cortex can be grown for several weeks in dissociated cell culture using a relatively simple protocol for culture preparation. It is possible to establish large numbers of very similar cultures which serve as an effective test system for studies of toxicity and mechanism of action of neuroactive compounds. Light microscopic and ultrastructural studies document the neuronal component of the cultures as well as the developmental sequence. Cell counts, protein concentration and choline acetyltransferase activity demonstrate the reproducibility of the cultures from dish to dish.

Neuronal maturation in mammalian cell culture is dependent on spontaneous electrical activity.

Fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) neurons undergo a process of maturation in cell culture lasting a month or more. We have investigated the role of electrical activity in this maturational process with the use of tetrodotoxin (TTX), the specific blocker of the voltage-sensitive sodium channel responsible for action potential generation. This agent completely eliminates the spikes and related synaptic activity which occur abundantly in untreated cultures. Such blockade of electrical activity in the cultures, when begun early (day 1 or day 8 in vitro), results in a 85-95% reduction in the number of large SC neurons, without affecting DRG neuron numbers. TTX treatment initiated when cultures are mature (day 70) has no significant effect on either DRG or SC neurons. Intermediate effects are obtained when treatment is initiated at day 35 in vitro. The activity of the nerve-specific enzyme choline acetyltransferase, is significantly decreased by early TTX treatment, while DNA and protein content of the cultures (primarily contributed by glial and fibroblastic cells) is not affected.

Utilization of a further miniaturized serological microtechnique.

A new 0.01-ml serological microtechnique system produced accurate and reproducible complement-fixation and indirect hemagglutination tests. This equipment involves the use of smaller amounts of antigens and sera, and thus increases the value of the method when employed with expensive reagents or limited qualities of sera.