Changes in murine anorectum signaling across the life course.

BACKGROUND: Increasing age is associated with an increase in the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to these conditions is not fully understood. This study examined the effects of increasing age on the function of the murine anorectum. METHODS: The effects of increasing age on cholinergic, nitrergic, and purinergic signaling pathways in the murine anorectum were examined using classical organ bath assays to examine tissue function and electrochemical sensing to determine age-related changes in nitric oxide and acetylcholine release. KEY RESULTS: Nitrergic relaxation increased between 3 and 6 months, peaked at 12 months and declined in the 18 and 24 months groups. These changes were in part explained by an age-related decrease in nitric oxide (NO) release. Cholinergic signaling was maintained with age by an increase in acetylcholine (ACh) release and a compensatory decrease in cholinesterase activity. Age-related changes in purinergic relaxation were qualitatively similar to nitrergic relaxation although the relaxations were much smaller. Increasing age did not alter the response of the anorectum smooth muscle to exogenously applied ACh, ATP, sodium nitroprusside or KCl. Similarly, there was no change in basal tension developed by the anorectum. CONCLUSIONS AND INFERENCES: The decrease in nitrergic signaling with increasing age may contribute to the age-related fecal impaction and constipation previously described in this model by partially obstructing defecation.

Digit ratio (2D:4D) and physical fitness (Eurofit test battery) in school children.

BACKGROUND: The relative lengths of the index finger to the ring finger (2D:4D) is sexually dimorphic and is thought to be a correlate of prenatal sex steroids (low 2D:4D=high prenatal testosterone and low prenatal oestrogen). In adults there have been reports that low 2D:4D is consistently associated with high sports performance. AIMS: To investigate correlations between 2D:4D and fitness levels in children. STUDY DESIGN: Right 2D:4D and body size were measured, in addition to flexibility, speed, endurance and strength (Eurofit tests). SUBJECTS: 922 boys and 835 girls (mean ages (years): 10.8 ± 1.01 and 10.07 ± 1.00 respectively). OUTCOME MEASURES: height, mass, BMI, triceps and subscapular skinfolds, 20 m shuttle run, sit and reach, standing broad jump, hand grip strength, 10 × 5m sprint, and the sit and reach test. RESULTS: Boys significantly outperformed the girls in the 10 × 5m sprints, the 20 m shuttle run, standing broad jump, and hand grip strength. In boys but not girls, 2D:4D was significantly negatively correlated with scores in all these tests except the standing broad jump. In girls but not boys, 2D:4D was significantly positively correlated to stature, mass, BMI and waist circumference. CONCLUSIONS: It is suggested that high prenatal testosterone and low prenatal oestrogen (low 2D:4D) is implicated in high sprinting speed, endurance and hand grip strength in boys. In girls low prenatal testosterone and high prenatal oestrogen is associated with large body size.

Changes in the innervation of the mouse internal anal sphincter during aging.

BACKGROUND: The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS: Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS: In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES: The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.

Reliability of a field based 2D:4D measurement technique in children.

BACKGROUND: There is limited literature on the relationship between second to fourth finger digit ratio (2D:4D) and health- and skill-related fitness in children. To examine this relationship it is important to establish a reliable method of assessing 2D:4D for use with large groups of children. AIM: The aim of the study was to examine the reliability of a field-based 2D:4D measure in children. METHODS/RESEARCH DESIGN: Fifty 8-11 year olds had 2D:4D of the right hand measured using a Perspex table top, a digital camera, and Adobe Photoshop software. Second to fourth finger digit ratio (and 2D and 4D) intra-observer and inter-observer reliabilities were assessed on the same day and intraobserver reliability was measured between days. Limits of agreement (LoA), coefficient of variation (CV) and Pearson's correlation coefficient were used for statistical analysis. RESULTS: High correlation coefficients (r=0.95-0.99) and low CV's (0.4-1.2%) were reported for intra- and inter-observer reliabilities on the same day and between days. LoA revealed negligible systematic bias with random error ranging from 0.02 to 0.12. CONCLUSION: These findings suggest that 2D:4D (and 2D and 4D) assessment in children using digital photography provides a reliable measure of 2D:4D that can be used during field-based testing.

Myenteric neuron numbers are maintained in aging mouse distal colon.

BACKGROUND Age-associated myenteric neuronal loss has been described in several species. In some studies,cholinergic neurons have been reported to be selectively vulnerable, whereas nitrergic neurons are spared. Aging of the mouse enteric nervous system(ENS) and the subtypes of mouse myenteric neurons that may be lost have been little studied. We therefore investigated changes in the numbers of total neurons and two neuronal subpopulations in the mouse distal colon during aging. METHODS Wholemount preparations from 3­4-, 12­13-, 18­19-, and 24­25-month-old C57BL/6 mice were double immunolabeled with HuC/D antibody to identify the total neuronal population and antisera to either calbindin or neuronal nitric oxide synthase (nNOS) to identify myenteric neuronal subpopulations. Samples were analyzed by confocal microscopy. New procedures were employed to ensure unbiased counting and to correct for changes in gut dimensions with age and stretch during sample preparation. The density of nerve fibers in the tertiary plexus was also studied. KEY RESULTS No significant change in numbers of total neurons or of either subpopulation with age was measured, but because of gut growth, the density of myenteric neurons decreased between 3­4 and 12­13 months. The density of nNOS-immunoreactive nerve fibers in the tertiary plexus increased significantly with age, up to 18­19 months. Numerous swollen processes of CB and nNOS-immunoreactive neurons were observed in 18­19- and 24­25-month-old animals. Conclusions &Inferences These results indicate that aging does not result in a loss of myenteric neurons in mouse distal colon at the ages studied, although neurodegenerative changes, which may impact on neuronal function, do occur.

Ageing reduces the number of vesicular glutamate transporter 2 containing immunoreactive inputs to identified rat pelvic motoneurons.

The immunocytochemical localisation of vesicular glutamate transporters, VGLUT1 and VGLUT2, was employed to identify putative glutamatergic axon terminals innervating pelvic motoneurons. VGLUT1 terminals were sparsely distributed within lumbosacral spinal motoneuron pools, including the dorsolateral nucleus, retrodorsolateral nucleus and spinal nucleus of the bulbospongiosus. This was in marked contrast to VGLUT2 which was expressed in a robust innervation of these areas. Retrograde tracing was used to reveal motoneurons innervating the levator ani (LA) muscle. On these neurons, associations with VGLUT2 immunoreactive terminals were abundant while those with VGLUT1 were rare. Ultrastructural investigations revealed that VGLUT2 immunoreactive terminals made asymmetric synaptic contacts with dendrites of retrogradely labelled LA motoneurons. Quantification of VGLUT2 immunoreactive boutons in close association with these dendrites was carried out in young and aged animals using light microscopy. This revealed a significant decline in the numbers of VGLUT2 immunoreactive boutons on the more distal dendrites of motoneurons in aged rats. VGLUT2 boutons were reduced by approximately 21% from 11.25+/-0.5 per 35-mum length of dendrite in young rats to 8.89+/-0.5 in aged animals. This decline in glutamatergic input may reduce the excitability of LA motoneurons and consequently decrease the capacity of the rat to induce reflexive erections.

The effects of ageing on the distribution of vesicular acetylcholine transporter immunoreactive inputs to pelvic motoneurons of male Wistar rats.

Age-related changes in the number and size of large cholinergic terminals immunoreactive for vesicular acetylcholine transporter (VAChT), were documented for the dorsolateral nucleus (DLN), retrodorsolateral nucleus (RDLN) and spinal nucleus of the bulbospongiosus (SNB) of the lumbosacral spinal cord of male rats. The most significant changes were a large increase in the number and size of cholinergic terminals within the DLN of aged animals, together with a small decrease in terminal number within the RDLN. No significant age-associated differences in VAChT labeling were seen within the SNB. In both age groups, SNB motoneurons projecting to the levator ani muscle received about 9 to 10 contacts from large cholinergic terminals. Ultrastructural examination of the terminals revealed structures likely to be postsynaptic subsurface cisterns that are characteristic of type C terminal boutons. Since both the DLN and SNB contain motoneurons innervating pelvic muscles and sphincters, these findings provide further evidence for a central cholinergic influence on micturition and sexual reflexes and suggest that this may remain robust in the face of ageing.

The relationship between serotonin, dopamine beta hydroxylase and GABA immunoreactive inputs and spinal preganglionic neurones projecting to the major pelvic ganglion of Wistar rats.

Preganglionic neurones in the lumbosacral spinal cord give rise to nerves providing the parasympathetic and sympathetic innervation of pelvic organs. These neurones are modulated by neurotransmitters released both from descending supra-spinal pathways and spinal interneurones. Though serotonin has been identified as exerting a significant influence on these neurones, few studies have investigated the circuitry through which it achieves this particularly in relation to sympathetic preganglionic neurones. Using a combination of neuronal tracing and multiple immunolabeling procedures, the current study has shown that pelvic preganglionic neurones receive a sparse, and probably non-synaptic, axosomatic/proximal dendritic input from serotonin-immunoreactive terminals. This was in marked contrast to dopamine beta hydroxylase-immunoreactive terminals, which made multiple contacts. However, the demonstration of both serotonin, and dopamine beta hydroxylase immunoreactive terminals on both parasympathetic and sympathetic preganglionic neurones provides evidence for direct modulation of these cells by both serotonin and norepinephrine. Serotonin-containing terminals displaying conventional synaptic morphology were often seen to contact unlabeled somata and dendritic processes in regions surrounding the labeled preganglionic cells. It is possible that these unlabeled structures represent interneurones that might allow the serotonin containing axons to exert an indirect influence on pelvic preganglionic neurones. Since many spinal interneurones employ GABA as a primary fast acting neurotransmitter we examined the relationship between terminals that were immunoreactive for serotonin or GABA and labeled pelvic preganglionic neurones. These studies were unable to demonstrate any direct connections between serotonin and GABA terminals within the intermediolateral or sacral parasympathetic nuclei. Colocalization of serotonin and GABA was very rare but terminals immunoreactive for each were occasionally seen to contact the same unlabeled processes in close proximity. These results suggest that in the rat, the serotonin modulation of pelvic preganglionic neurones may primarily involve indirect connections via local interneurones.

Terminals of paraventricular spinal neurones are closely associated with adrenal medullary sympathetic preganglionic neurones: immunocytochemical evidence for vasopressin as a possible neurotransmitter in this pathway.

A recent study using transsynaptically transported pseudorabies virus, injected into the adrenal gland, showed labelled neurones in the paraventricular nucleus (PVN) of the hypothalamus, indicating that these neurones send projections to sympathoadrenal preganglionic neurones (SPNs). However, this technique cannot conclusively demonstrate that the pathway is monosynaptic. In order to investigate the possibility of a direct projection from the PVN to SPNs, the present study used the anterograde tracer biotin dextran amine to label paraventricular spinal projections and the retrograde tracer cholera toxin B conjugated to horseradish peroxidase to label SPNs. In addition, because electrophysiological evidence suggests vasopressin to be a neurotransmitter candidate in this pathway, immunocytochemical identification of the peptide and retrograde labelling of SPNs to the adrenal medulla were used to investigate this. The results of these studies show spinally projecting paraventricular axons with terminal varicosities closely associated with SPNs. Therefore some of these associations may represent boutons forming synaptic contact on SPNs. Similarly, vasopressin fibres were found close to the dendrites and soma of SPNs. It is suggested that spinal axons originating from paraventricular neurones can provide a direct influence on adrenal medullary function, that vasopressin is a possible neurotransmitter involved in some of these connections and this is one means by which the paraventricular nucleus can generate a defence to stressful stimuli.

The paraventricular nucleus of the hypothalamus sends efferents to the spinal cord of the rat that closely appose sympathetic preganglionic neurones projecting to the stellate ganglion.

Using a combination of anterograde and retrograde neuronal tract-tracing techniques, the descending projections from the paraventricular nucleus of the hypothalamus (PVN) to the brain/spinal cord and in particular those axonal projections that appear to be contiguous with sympathetic preganglionic neurones (SPN) projecting to the stellate ganglion have been studied. Descending PVN pathways were located by the anterograde transport of biotinylated dextran amine (BDA), whilst SPN were retrogradely labelled with cholera B toxin subunit conjugated to horseradish peroxidase (CB-HRP). BDA-labelled PVN axons terminated in both hypothalamic and extrahypothalamic (including the midbrain, medulla and spinal cord) brain nuclei, with dense terminal labelling observed particularly in the arcuate hypothalamic nucleus and adjacent median eminence, in the solitary tract, vagal nuclei and in the intermediolateral region of the spinal cord (IML). Varicose descending PVN fibres in the IML were often observed to closely appose both the cell soma and dendrites of retrogradely labelled SPN (projecting to the stellate ganglion) in the spinal cord. In addition, it was shown that PVN descending axons crossing to the contralateral side of the spinal cord were closely associated with retrogradely labelled SPN projecting to the superior cervical ganglion. Such findings suggest that descending pathways from the PVN may exhibit a direct influence on cardiac sympathetic outflow and may also influence the behaviour of the contralateral population of SPN projecting to the superior cervical ganglion.

Studies on nerves of the upper respiratory tract in the ferret and the mink.

The central localization of the superior laryngeal nerve (SLN), recurrent laryngeal nerve (RLN) and pharyngeal nerve (PHAR) in the ferret (Mustela putorius furo) and mink (Mustela vision) was determined by neuronal tract tracing techniques. The anterograde transport of horseradish peroxidase (HRP)/wheat germ aggluttinin conjugated HRP mixtures (WGA-HRP) revealed afferent fibres of the SLN projecting ipsilaterally in the tractus solitarius (TS) before terminating in the ipsilateral nucleus of the tractus solitarius (nTS). In both mustelids large concentrations of terminal reaction product were observed in the dorsal/dorsolateral and medial subnuclei of the nTS; however, at levels near obex significant projections of the SLN to the interstitial subnucleus were also observed. Caudal to obex, sparse terminal labelling was identified bilaterally in the nucleus commissuralis (n com). There were no labelled afferent projections of the SLN to the spinal trigeminal complex in either mustelid; neither was afferent terminal reaction product detected in the medulla oblongata following labelling of the RLN (in either the ferret or the mink) or PHAR (in the ferret). Projection of the SLN to areas of the nTS associated with upper airway functions, like swallowing and respiration, suggest a substantial role for this nerve in the initiation and control of airway reflexes. Extensive labelling of the nucleus ambiguus (nA) was revealed following HRP/WGA-HRP application to the PHAR, RLN and SLN and there was some evidence of a topographical arrangement of neuronal cell bodies in the nA, relative to the different nerves, in the ferret. In the case of the SLN, retrogradely labelled cells were also identified in the dorsal motor nucleus of the vagus (DmnX), nTS and reticular formation (rf). Electrical stimulation of the SLN produced periods of apnoea/ataxic breathing in both mustelids. As well as respiratory effects, stimulation of the SLN often caused bradycardia in the mink, but not in the ferret, in which heart rate generally remained unchanged. This difference did not reflect anatomical differences in the central localization of the SLN.

The central localization of the vagus nerve in the ferret (Mustela putorius furo) and the mink (Mustela vison).

The location of vagal preganglionic neurones (VPN) has been determined in nine ferrets (Mustela putorius furo) and seven mink (M. vison) using neuronal tract-tracing techniques employing horseradish peroxidase (HRP) and wheat-germ agglutinin conjugated HRP (WGA-HRP) mixtures injected into the nodose ganglion of the vagus nerve. Labelled VPN were located ipsilaterally in the dorsal motor nucleus of the vagus (DmnX), nucleus ambiguus (nA), and reticular formation (rf) of the medulla oblongata. In four of the ferrets, labelled VPN were also identified in the nucleus dorsomedialis (ndm) and the nucleus of the spinal accessory nerve (nspa). In a single mink a few labelled cells were observed in the ndm but no labelled VPN were found in the nspa. Labelling of afferent components of the vagus nerve was seen in two ferrets and two mink with the best labelling obtained following an injection of an HRP/WGA-HRP mixture into the nodose ganglion. Labelled afferents were observed to cross the ipsilateral spinal trigeminal tract (SpV) before entering the tractus solitarius (TS) in regions separate from the motor axons which exit the medulla in separate fasicles. Sensory terminal fields were identified bilaterally in the nucleus of the tractus solitarius (nTS) in both species and bilaterally in the area postrema (ap) of the ferret; however, the contralateral labelling was sparse in comparison to the densely labelled ipsilateral nTS/ap. Maximal terminal labelling was seen in regions just rostral and caudal to obex in both species.

Handling of 5-hydroxytryptamine by platelets in migraine.

There is little dispute that a link exists between 5-hydroxytryptamine (5HT) and migraine but the exact mechanism of an attack has yet to be established. The handling of 5HT by the platelet is regarded as a simple model of the handling of 5HT by nerve terminals. If differences are seen in how the platelets from migraineurs handle 5HT compared to those from a control population, it is possible that a similar difference exists in the nerve terminal. The Haemostatometer allows the rapid and simultaneous in vitro assessment of platelet function (shear-induced haemostasis), coagulation and thrombolysis from non anticoagulated blood samples. In this study, a baseline comparison of haemostasis was made on 20 migraineurs between attacks and 20 controls. No differences were found in the results from each of the two groups. 5 microM of 5HT was then added to blood taken from 10 migraineurs and 10 controls and the recordings were repeated. Again, no differences were found between the results from the two groups. In blood taken from both migraineurs and controls, the effect of 5HT was to significantly enhance clotting time and clot lysis. No effect was seen on primary aggregation. The possible reasons for and significance of these findings is discussed.

The similarities and differences of migraine with aura and migraine without aura: a preliminary study.

A preliminary study was undertaken to provide clinical evidence to support the hypothesis that: "Migraine with aura, migraine without aura and aura alone are the same condition, which differ in degree rather than pathophysiology." At the City of London Migraine Clinic, 50 patients consecutively attending the clinic with a past or present history of migraine with aura were questioned. Of the 50 patients questioned 36 (70%) had a combination of migraine with aura, migraine without aura and/or aura alone; i.e. 70% had had more than one type of migraine attack. The duration, severity and frequency of attacks did not differ between migraine with and migraine without aura. Conclusion--the results support the hypothesis that migraine with and migraine without aura, and aura alone are not separate conditions, because: (1) most patients suffer from more than one type of migraine attack; (2) there are no significant differences in the characteristics of the migraine attacks in the different groups; (3) there are no significant differences in the characteristics of the subjects.

A kinetic study of the pH-dependent properties of the ferric undecapeptide of cytochrome c (microperoxidase).

The ferric form of the haem undecapeptide, derived from horse cytochrome c by peptic digestion, undergoes at least three pH-induced transitions with pK values of 3.4, 5.8 and 7.6. Temperature-jump experiments suggest that the first of these is due to the binding of a deprotonated imidazole group to the feric iron while the second and third arise from the binding of the two available amino groups present (the alpha-NH2 of valine and the epsilon-NH2 of lysine). Molecular models indicate that steric retraints on the peptide dictate that these amino groups may only coordinate to iron atoms via intermolecular bonds, thus leading to the polymerization of the peptide. Cyanide binding studies are in agreement with these conclusions and also yield a value of 3.6 X 10(6) M-1 s-1 for the intrinsic combination constant of CN- anion with the haem. A model is proposed which describes the pH-dependent properties of the ferric undecapeptide.