Verification of a new model of the time course of RNA synthesis. Measurement of the rates of initiation and elongation.

The time course of RNA synthesis in vitro commonly starts with a lag followed by a linear phase. Differing from the earlier interpretation we have previously proposed that, under conditions where the initiation rate is low, the lag represents the time taken for the first RNA polymerase molecule to reach a termination site. During the linear phase, initiation is balanced by termination (Mahon, G.A.T., McWilliam, P., Gordon, R.L. and McConnell, D.J. (1980) J. Theor. Biol. 87, 483-515). We report the use of rifampicin as a further test of this new model. We show that it does apply under conditions of high ionic strength (0.3 M KCl), and under these conditions time courses may be analyzed to yield unbiased estimates of the initiation (Vi) and chain elongation (Vp) rates. We illustrate the application of the method of time course analysis and confirm some of its features by examining the effect of variation in the concentrations of RNA polymerase and nucleoside triphosphate on the estimates of Vi and Vp. The alternative interpretation of the time course applies under conditions of low ionic strength, where the initiation rate is high. (Chamberlin, M.J., Nierman, W.C., Wiggs, J. and Neff, N. (1979) J. Biol. Chem. 254, 10061-10069.) The advantages of each model in measuring Vi and Vp (the major parameters of the transcription reaction) are discussed.

Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: projections from the heart preferentially express vesicular glutamate transporter 1.

The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as peptide markers of nociceptive afferent neurones. These data suggest that the population of cardiac vagal afferents labeled by pericardial cholera toxin B-subunit injection is neurochemically varied, which may be related to a functional heterogeneity of baroreceptive, chemoreceptive and nociceptive afferent fibers. A high proportion of cardiac neurones appear to be glutamatergic, but differ from other vagal afferents in expressing vesicular glutamate transporter 1.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the nucleus ambiguus of the cat.

The distribution of calcitonin gene-related peptide (CGRP) in the cat nucleus ambiguus was examined by means of a combination of horseradish peroxidase (HRP) tracing and immunohistochemical techniques. Vagal motoneurones in the nucleus ambiguus were identified by applying HRP to either the thoracic vagus or the superior laryngeal nerve or the cervical vagus. Motoneurones in the nucleus ambiguus labelled with HRP from the thoracic vagus did not contain CGRP-like immunoreactivity although CGRP-like immunoreactive cells were present in this nucleus on the same sections. In contrast, a large proportion of the motoneurones labelled from the superior laryngeal nerve and a smaller proportion of cells labelled from the cervical vagus did contain CGRP-like immunoreactivity. It is concluded that CGRP-like immunoreactivity is absent from vagal preganglionic motoneurones projecting to structures in the thorax and abdomen but is present in vagal motoneurones projecting to striated muscle of the larynx and pharynx.

Distribution of dopamine-containing neurons and fibres in the feline medulla oblongata: a comparative study using catecholamine-synthesizing enzyme and dopamine immunohistochemistry.

The distribution of dopamine-immunoreactive neurons and fibres in the feline medulla oblongata was examined by immunocytochemistry with antisera to the catecholamine-synthesizing enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase, and with antisera to the catecholamines dopamine and L-dihydroxyphenylalanine. Neurons immunoreactive for the catecholamine-synthesizing enzymes were found in two regions of the medulla, the ventrolateral A1 region and the dorsomedial A2 region. Double-staining studies with antisera to the enzymes indicated that a population of neurons within both regions were immunoreactive for tyrosine hydroxylase but not dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase, implying that they synthesize dopamine. Studies using the dopamine antisera demonstrated the presence of dopamine-immunoreactive neurons in both the ventrolateral and dorsomedial regions of the medulla; in the dorsomedial region, they were found in the area postrema, nucleus tractus solitarius and dorsal motor vagal nucleus, mainly at levels caudal to the obex. Dopamine-immunoreactive fibres were found in several areas of the medulla including the nucleus tractus solitarius, inferior olive, dorsal motor vagal, spinal trigeminal, hypoglossal, cuneate, gracile, and raphe nuclei. Double-staining studies with antisera to dopamine and dopamine-beta-hydroxylase revealed a population of cells immunoreactive for dopamine alone. The presence of some double-stained neurons, however, implies some cross-reactivity of the dopamine antiserum with noradrenaline or adrenaline and/or recognition of dopamine present as a metabolic intermediary in some noradrenergic neurons. No L-dihydroxyphenylalanine-immunoreactive neurons were found in the medulla, although fibres were seen. These data provide evidence for the existence of catecholamine neurons which utilize dopamine as a final synthetic product within the medulla oblongata.

Immunohistochemical profiles of spinal lamina I neurones retrogradely labelled from the nucleus tractus solitarii in rat suggest excitatory projections.

Three morphologically distinct types of lamina I neurones, fusiform, flattened and pyramidal, project from the spinal cord to the caudal part of the nucleus tractus solitarii in the rat, and may represent a pathway whereby peripheral stimuli can modify autonomic functions. The neurochemistry of these three types of projection neurones was investigated using retrograde neuronal tracing with cholera toxin B-subunit combined with dual and triple immunofluorescence labelling for different neuroactive substances. None of the lamina I neurones with immunoreactivity for GABA or glycine were found to project to the nucleus tractus solitarii, whereas high levels of glutamate immunoreactivity, which may indicate a glutamatergic phenotype, were found in 18.4% of fusiform, 9.6% of pyramidal and 2.1% of flattened projection neurones. Immunoreactivity for calbindin-D28K was present in 34.9% of fusiform cells, 18.3% of pyramidal cells and 10.5% of flattened cells, and nitric oxide synthase immunoreactivity was detected in 13.8% of fusiform cells, 1.1% of pyramidal cells and 4.2% of flattened cells that had projections to the nucleus tractus solitarii. Calbindin immunoreactivity was co-localised in major subpopulations of projection neurones of each morphological type that contained glutamate immunoreactivity, whereas co-localisation of nitric oxide synthase immunoreactivity in these neurones was relatively uncommon. The pyramidal cell was the only retrogradely labelled cell type found to be immunoreactive for substance P, but few (<5%) of these neurones were immunolabelled. These data are consistent with the hypothesis that lamina I neurones projecting to the dorsal vagal complex are not inhibitory, and that some of them, belonging mostly to the fusiform and pyramidal types, may exert excitatory, glutamate- or substance P-mediated effects upon inhibitory interneurones in the nucleus tractus solitarii. Such excitatory pathways could be involved in the attenuation of the reflex control of blood pressure by both painful and innocuous peripheral stimuli, such as those arising in injury and exercise.

Co-localization of c-Fos and neurotransmitter immunoreactivities in the cat brain stem after carotid sinus nerve stimulation.

To reveal neurones in the cat medulla oblongata involved in carotid baroreceptor/chemoreceptor reflexes, the distribution of c-Fos oncoprotein immunoreactivity was studied following electrical stimulation of the right carotid sinus nerve. The neurochemistry of the activated neurones was investigated using antisera to tyrosine hydroxylase, neuropeptide Y, somatostatin, and glutamate. Nitric oxide containing neurones were identified using antiserum to nitric oxide synthase (NOS) and by the histochemical localization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Following sinus nerve stimulation numerous c-Fos-IR cells were detected both ipsilaterally and contralaterally in the nucleus tractus solitarii, the area postrema and throughout the ventrolateral medulla. Dual labelling studies revealed that 3.3% of c-Fos-immunoreactive cells in the nucleus tractus solitarii were also immunoreactive for tyrosine hydroxylase. The double labelled cells were scattered within the medial and ventrolateral subnuclei, predominantly rostral to obex. A higher proportion (10.3%) of c-Fos-IR cells in the ventrolateral medulla also showed tyrosine hydroxylase immunoreactivity. Caudal to obex, these were scattered in the reticular formation between the spinal trigeminal nucleus and the lateral reticular nucleus, while more rostrally they were found within the lateral reticular nucleus, the nucleus ambiguus and the lateral tegmental field. Cells expressing c-fos and reactive for glutamate, neuropeptide Y or NADPH-diaphorase (or NOS) were only rarely seen, and co-localization of c-Fos and somatostatin immunoreactivities was not seen. These results suggest that of the neurones forming pathways within the medulla activated on carotid sinus nerve stimulation, presumably mediating baro- and chemoreceptor reflexes, relatively few utilize catecholamines, glutamate, neuropeptide Y or nitric oxide as their transmitter substance.

Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem.

The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH-diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH-diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed.

Nitric oxide producing neurones in the rat medulla oblongata that project to nucleus tractus solitarii.

The production of nitric oxide in neurones of the rat medulla oblongata that project to the nucleus tractus solitarii (NTS) was examined by simultaneous immunohistochemical detection of nitric oxide synthase (NOS) and of cholera toxin B-subunit (CTb), which was injected into the caudal zone of the NTS. Neurones immunoreactive for CTb and neurones immunoreactive for NOS were widely co-distributed and found in almost all the anatomical divisions of the medulla. Dual-labelled cells, containing both CTb and NOS immunoreactivities were more numerous ipsilaterally to the injection sites. They were concentrated principally in the more rostral zone of the NTS, raphé nuclei, dorsal, intermediate and lateral reticular areas, spinal trigeminal and paratrigeminal nuclei and the external cuneate and medial vestibular nuclei. Isolated dual-labelled neurones were also scattered throughout most of the divisions of the reticular formation. These observations indicate that many areas of the medulla that are known to relay somatosensory and viscerosensory inputs contain NOS immunoreactive neurones that project to the NTS, and may, therefore, contribute to the dense NOS-immunoreactive innervation of the NTS. The release of nitric oxide from the axon terminals of these neurones may modulate autonomic responses generated by NTS neurones in relation to peripheral sensory stimuli, and thus ultimately regulate sympathetic and/or parasympathetic outflow.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the medulla oblongata of the cat, in relation to choline acetyltransferase-immunoreactive motoneurones and substance P-immunoreactive fibres.

The immunohistochemical distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (ir) in the cat medulla oblongata was examined using an antiserum to rat alpha-CGRP. Comparative distributions of substance P (SP)-like and choline acetyltransferase (ChAT)-like ir were also studied on sections adjacent to those stained for CGRP, and on sections simultaneously stained for CGRP by double staining techniques. The vast majority of ChAT-ir motoneurones in somatomotor or branchiomotor cranial nuclei (of VI, VII and XII nerves) and their accessory nuclei also displayed a coarsely granular CGRP-ir, shown by electron microscopic examination to represent immunoreactive Golgi bodies. The nucleus ambiguus (IX and X nerves), a mixed branchiomotor and visceromotor nucleus, showed CGRP-ir in a lower proportion of its motoneurones, whereas the purely viseromotor dorsal motor vagal nucleus (X nerve) contained no CGRP-ir cells. A few CGRP-ir but ChAT-negative cells were seen in the ventromedial reticular formation, lateral cuneate nucleus, infratrigeminal nucleus and nucleus of the solitary tract. Coarse, often varicose CGRP-ir fibres were most prominent in the X and IX cranial nerve rootlets, the spinal tract of the V nerve and the solitary tract, and also in the V spinal nucleus and nucleus of the solitary tract. Many of these also appeared to contain SP-ir. The central patterns of CGRP and SP-ir fibres thus reflect the previously reported coexistence of these peptides in sensory afferent cells of the trigeminal and nodose ganglia. These results are consistent with a role for CGRP as a transmitter or modulator in efferents to striated muscle, sensory afferents and intrinsic neurones in the cat brain stem.

Labelling of rat vagal preganglionic neurones by carbocyanine dye DiI applied to the heart.

We describe a method for applying the carbocyanine dye DiI to the rat heart that takes advantage of the dye's lipophilic properties and its ability to diffuse easily into tissues, and results in specific retrograde labelling of cardiac vagal preganglionic neurones in the medulla oblongata. Most of the labelled neurones were found bilaterally in the nucleus ambiguus (81%), with a few sparsely distributed in the dorsal motor vagal nucleus (6.5%), and in an intermediate area located between these two nuclei (12.5%). We contend that the method of applying DiI crystals to the surface of the heart is a more efficient, accurate and reproducible method of retrograde labelling than the injection of tracers into this very delicate tissue.

Projection sites of superficial and deep spinal dorsal horn cells in the nucleus tractus solitarii of the rat.

By using anterograde transport of biotin dextran amine injected into the cervical spinal dorsal horn, we have shown that fibres from superficial and deep dorsal horn project to the nucleus tractus solitarii via two distinct pathways. Afferent fibres from the superficial lamina (I-III) were found to course in the dorsal funiculus and terminate bilaterally in the caudal zone of the nucleus tractus solitarii (NTS), mainly within the commissural subnucleus. In contrast, afferents from the deeper dorsal horn laminae (IV-V) were found to course in the dorsolateral fasciculus and terminate ipsilaterally, mostly in the lateral areas of the caudal nucleus tractus solitarii. Similar, but more extensive patterns of labelled fibres were produced by injections into the white matter of the dorsal funiculus and dorsolateral fasciculus, respectively. These observations suggest that the caudal NTS not only serves as a location of visceral afferent convergence and integration, but may also be a receptive area for monosynaptic projections from dorsal horn neurons receiving sensory afferent inputs. Such projections may represent pathways through which NTS neurons are influenced by nociceptive and non-nociceptive information from the dorsal horn and thereby can co-ordinate the appropriate autonomic response, including adjustments in cardiorespiratory reflex output.

A GABA-mediated inhibition of neurones in the nucleus tractus solitarius of the cat that respond to electrical stimulation of the carotid sinus nerve.

Extracellular recordings were made from 39 single neurones in the nucleus tractus solitarius which were activated following electrical stimulation of the carotid sinus nerve in chloralose anaesthetised cats. Iontophoretic application of GABA abolished or markedly reduced the evoked activity of all neurones. The inhibitory effect of GABA could be antagonised by the simultaneous iontophoretic application of bicuculline. The iontophoretic application of bicuculline alone resulted in a significant increase in the evoked activity of eleven out of seventeen neurones. These results support the notion that a GABAergic inhibitory system, with the potential to modulate sinus nerve reflexes, acts within the nucleus tractus solitarius.

Trichostrongyle larval populations on herbage from grazed and ungrazed cattle pastures in south west Scotland.

In the course of studies on the control of bovine parasitic gastroenteritis in south west Scotland using a morantel sustained release bolus the populations of trichostrongyle infective larvae on herbage were monitored over a two-year period. From the results of the herbage analysis and the worm burdens in parasite naive tracer calves introduced at the end of 18 months, it was clear that considerable numbers of infective larvae of Ostertagia ostertagi and Cooperia oncophora survived for at least 18 months on pastures not grazed at all. The results suggest that in south west Scotland after one, or even two, seasons with no further contamination of grazing, permanent cattle pastures cannot be assumed to be helminthologically 'safe' and that this should be taken into consideration when determining prophylactic strategies.

Response to transplanted adult Ostertagia ostertagi in calves.

Plasma pepsinogen levels became elevated in groups of recipient calves immediately after transplant with adult Ostertagia ostertagi. These rises occurred in both previously parasite-naive calves and in calves which had experienced prior infection terminated with an anthelmintic either seven or 21 days before transplant. From the results it appears that adult O ostertagi play a significant role in the elevated plasma pepsinogen levels associated with bovine ostertagiasis.

Effect of sustained release and pulse release anthelmintic intraruminal devices on development of pathophysiological changes and parasite populations in calves infected with Ostertagia ostertagi and Cooperia oncophora.

An experiment was conducted in calves to investigate the effect of sustained release and pulse release anthelmintic intraruminal boli on the development of pathophysiological changes following daily infection with Ostertagia ostertagi and Cooperia oncophora for six weeks. After infection various pathophysiological changes were detected including increases in serum pepsinogen concentration, enteric plasma protein losses and in the catabolic rate of albumin. Such changes developed rapidly in the unprotected calves following patency after 17 days and persisted until the termination of the study. There were indications that the sustained anthelmintic release device was more efficacious than the pulse anthelmintic release device in reducing the worm burdens and early pathophysiological changes associated with infection. It was found at necropsy that the release of anthelmintic by the oxfendazole pulse release bolus had been delayed in several calves.

Further studies on the response to transplanted adult Ostertagia ostertagi in calves.

Calves infected by surgical transplantation of adult Ostertagia ostertagi had raised levels of plasma pepsinogen and those in which the largest number of worms established also had elevated plasma gastrin concentrations. Despite the elevated plasma pepsinogen values, the abomasal pH of the animals did not change significantly, and there was no significant difference in the percentage establishment of adult parasites in calves previously infected with O ostertagi third stage larvae and those which had been maintained parasite-naive before transplant.

Metabolism and growth in housed calves given a morantel sustained release bolus and exposed to natural trichostrongyle infection.

Three groups of calves, previously grazed on permanent pastures contaminated with bovine trichostrongyle (Ostertagia and Cooperia species) infective larvae, were housed from October to May. During the grazing season one group had received fenbendazole at fortnightly intervals to suppress trichostrongyle infections, one received a morantel sustained release bolus before grazing to limit trichostrongyle contamination of the pasture, and the control group was only medicated when heavy infections caused clinical type 1 ostertagiasis. Digestibility of the whole diet was poorer in the control cattle, particularly for dry matter, crude protein and energy fractions. Balance studies conducted throughout the winter housed period showed that both increased faecal and urinary nitrogen outputs in the control animals contributed to a significantly reduced overall nitrogen retention. The effects were most apparent during clinical type 2 ostertagiasis, which occurred in March in the control group.

Production, parasitological and carcase evaluation studies in steers exposed to trichostrongyle infection and treated with a morantel bolus or fenbendazole in two consecutive grazing seasons.

Friesian steers were exposed to natural trichostrongyle infection and left untreated (controls) or treated with either a morantel sustained release bolus or fenbendazole at two week intervals (clean) in each of two consecutive grazing seasons. In the second grazing season the mean liveweight gains of the clean, morantel treated and control groups of cattle over 152 days were 105, 131 and 109 kg respectively (morantel treated greater than clean, control P less than 0.02). The cattle were slaughtered following an indoor fattening period. The dressed carcase weight, killing out percentage and related carcase measurements were superior in the treated groups. Eighty per cent of the clean and morantel treated cattle graded satisfactorily and qualified for subsidy payment whereas less than 30 per cent of the control cattle were successful. Rib joint analyses showed significantly lower total weight, eye muscle weight and area together with a higher bone content in control cattle.

Sequential in vivo measurements of body composition of calves exposed to natural infection with gastrointestinal nematodes.

Sequential in vivo measurements of total body water, exchangeable sodium and exchangeable potassium, made by radioisotopic dilution techniques, were used to determine changes in body composition in calves exposed to natural infections with gastrointestinal nematodes during their first grazing season. Two groups of calves were studied, one of which received a sustained release anthelmintic bolus at turn out. Over the grazing season the bolus-treated calves showed a significantly improved pattern of bodyweight gain, compared with the untreated control calves. There were also significant increases in both total body water and exchangeable potassium, as a percentage of the bodyweight, in the bolus calves compared with the controls. These findings indicated that body protein as a percentage of bodyweight was increased in the bolus animals. The bolus-treated calves also had significantly lower serum pepsinogen concentrations, faecal egg counts and worm burdens.

Pathophysiological and parasitological studies on Ostertagia ostertagi infections in calves.

Friesian calves given a low level infection of the abomasal parasite Ostertagia ostertagi over a six week period displayed a mild diarrhoea with high faecal egg counts and elevated plasma pepsinogen values. At necropsy on day 23 abomasal lesions characteristic of ostertagiasis were widespread. At 42 and 84 days oedema and congestion were also prominent. Total worm burdens on days 23 and 42 were similar but a marked decrease had occurred by day 84. Feed digestibility and nitrogen economy were not markedly affected but radioisotopic measurements demonstrated an increase in albumin disappearance and catabolic rates, and plasma faecal clearance during the course of the infection. Prior administration of a morantel sustained release bolus to a group of similarly infected calves reduced the total worm burdens to less than 50 per cent of those recorded in the infected calves on days 23 and 42 and this fell to 3 per cent on day 84. Abomasal damage and the adverse pathophysiological changes associated with infection were prevented in this group.