Quantification and neurochemical coding of the myenteric plexus in humans: No regional variation between the distal colon and rectum.

BACKGROUND: It remains unclear whether regional variation exists in the human enteric nervous system (ENS) ie, whether intrinsic innervation varies along the gut. Recent classification of gastrointestinal neuropathies has highlighted inadequacies in the quantification of the human ENS. This study used paired wholemounts to accurately quantify and neurochemically code the hindgut myenteric plexus, comparing human distal colon and rectum. METHODS: Paired human descending colonic/rectal specimens were procured from 15 patients undergoing anterior resection. Wholemounts of myenteric plexi were triple-immunostained with anti-Hu/NOS/ChAT antibodies. Images were acquired by motorized epifluorescence microscopy, allowing assessment of ganglionic density/size, ganglionic area density, and neuronal density. 'Stretch-corrected' values were calculated using stretched/relaxed tissue dimensions. KEY RESULTS: Tile-stitching created a collage with average area 99 300 000 µm2 . Stretch-corrected ganglionic densities were similar (colon: median 510 ganglia/100 mm2 [range 386-1170], rectum: 585 [307-923]; P = .99), as were average ganglionic sizes (colon: 57 593 µm2 [40 301-126 579], rectum: 54 901 [38 701-90 211], P = .36). Ganglionic area density (colon: 11.92 mm2 per 100 mm2 [7.53-18.64], rectum: 9.84 [5.80-17.19], P = .10) and stretch-corrected neuronal densities (colon: 189 neurons/mm2 [117-388], rectum: 182 [89-361], P = .31) were also similar, as were the neurochemical profiles of myenteric ganglia, with comparable proportions of NOS+ and ChAT+ neurons (P > .10). CONCLUSIONS AND INFERENCES: This study has revealed similar neuronal and ganglionic densities and neurochemical profiles in human distal colon and rectum. Further investigation of other components of the ENS, incorporating additional immunohistochemical markers are required to confirm that there is no regional variation in the human hindgut ENS.

Impairment of human proprioception by high-frequency cutaneous vibration.

These experiments assessed whether the impairment in proprioceptive acuity in the hand during 'interfering' cutaneous stimulation could be caused by inputs from Pacinian corpuscles. The ability to detect passive movements at the proximal interphalangeal joint of the index finger was measured when vibrotactile stimuli were applied to the adjacent middle finger and thenar eminence at frequencies and amplitudes that favour activation of rapidly adapting cutaneous afferents. Inputs from Pacinian corpuscles are favoured with high-frequency vibration (300 Hz), while those from Meissner corpuscles are favoured by lower frequencies (30 Hz). Detection of movement was significantly impaired when 300 Hz (20 microm peak-to-peak amplitude) complex vibration or 300 Hz (50 microm) sinusoidal vibration was applied to the middle finger and thenar eminence. In contrast, detection of movements was not altered by low-frequency sinusoidal vibration at 30 Hz with an amplitude of 50 microm or with a larger amplitude matched in subjective intensity to the 300 Hz sinusoidal stimulus. Thus it is unlikely that the impairment in detection was due to attention being diverted by vibration of an adjacent digit. In addition, an increase in amplitude of 300 Hz vibration led to a greater impairment of movement detection, so that the impairment was graded with the input. The time taken to nominate the direction of applied movement also increased during 300 Hz but not during 30 Hz sinusoidal vibration. These findings suggest that stimuli which preferentially activate Pacinian, but not Meissner corpuscles, impair proprioceptive acuity in a movement detection task.

Processing of vibrotactile inputs from hairy skin by neurons of the dorsal column nuclei in the cat.

The capacity of single neurons of the dorsal column nuclei (DCN) for coding vibrotactile information from the hairy skin has been investigated in anesthetized cats to permit quantitative comparison first with the capacities of DCN neurons responding to glabrous skin vibrotactile inputs and second with those of spinocervical tract neurons responding to vibrotactile inputs from hairy skin. Dynamically sensitive tactile neurons of the DCN the input of which came from hairy skin could be divided into two classes, one associated with hair follicle afferent (HFA) input, the other with Pacinian corpuscle (PC) input. The HFA-related class was most sensitive to low-frequency (<50 Hz) vibration and had a graded response output as a function of vibrotactile intensity changes. PC-related neurons had a broader vibrotactile sensitivity, extending to > or =300 Hz and appeared to derive their input from the margins of hairy skin, near the footpads, or from deeper PC sources such as the interosseous membranes or joints. HFA-related neurons had phaselocked responses to vibration frequencies up to approximately 75 Hz, whereas PC neurons retained this capacity up to frequencies of approximately 300 Hz with tightest phaselocking between 50 and 200 Hz. Quantitative measures of phaselocking revealed that the HFA-related neurons provide the better signal of vibrotactile frequency up to approximately 50 Hz with a switch-over to the PC-related neurons above that value. In conclusion, the functional capacities of these two classes of cuneate neuron appear to account for behavioral vibrotactile frequency discriminative performance in hairy skin, in contrast to the limited capacities of vibrotactile-sensitive neurons within the spinocervical tract system.

Vibrotactile coding capacities of spinocervical tract neurons in the cat.

The response characteristics and tactile coding capacities of individual dorsal horn neurons, in particular, those of the spinocervical tract (SCT), have been examined in the anesthetized cat. Twenty one of 38 neurons studied were confirmed SCT neurons based on antidromic activation procedures. All had tactile receptive fields on the hairy skin of the hindlimb. Most (29/38) could also be activated transynaptically by electrical stimulation of the cervical dorsal columns, suggesting that a common set of tactile primary afferent fibers may provide the input for both the dorsal column-lemniscal pathway and for parallel ascending pathways, such as the SCT. All but 3 of the 38 neurons studied displayed a pure dynamic sensitivity to controlled tactile stimuli but were unable to sustain their responsiveness throughout 1s trains of vibration at vibration frequencies exceeding 5-10 Hz. Stimulus-response relations revealed a very limited capacity of individual SCT neurons to signal, in a graded way, the intensity parameter of the vibrotactile stimulus. Furthermore, because of their inability to respond on a cycle-by-cycle pattern at vibration frequencies >5-10 Hz, these neurons were unable to provide any useful signal of vibration frequency beyond the very narrow bandwidth of approximately 5-10 Hz. Similar limitations were observed in the responsiveness of these neurons to repetitive forms of antidromic and transynaptic inputs generated by electrical stimulation of the spinal cord. In summary, the observed limitations on the vibrotactile bandwidth of SCT neurons and on the precision and fidelity of their temporal signaling, suggest that SCT neurons could serve as little more than coarse event detectors in tactile sensibility, in contrast to DCN neurons the bandwidth of vibrotactile responsiveness of which may extend beyond 400 Hz and is therefore broader by approximately 40-50 times than that of SCT neurons.

Vibrotactile frequency discrimination in human hairy skin.

The human capacity for vibrotactile frequency discrimination has been compared directly for glabrous and hairy skin regions by means of a two-alternative, forced-choice psychophysical procedure in five subjects. Sinusoidal vibratory stimuli, delivered by means of a 4-mm-diam probe, were first used to obtain detection threshold values for the two skin sites, the finger tip and the dorsal forearm, at four standard frequencies, 20, 50, 100, and 200 Hz. Values confirmed previous results showing detection thresholds were markedly higher on hairy skin than on glabrous skin. For the discrimination task, each standard frequency, at an amplitude four times detection threshold, was paired with a series of comparison frequencies, and discrimination capacity then was quantified by deriving from psychometric function curves, measures of the discriminable frequency increment (Deltaf) and the Weber Fraction (Deltaf/f), which, when plotted as a function of the four standard frequencies, revealed similar capacities for frequency discrimination at the two skin sites at the standard frequencies of 20, 100, and 200 Hz but an equivocal difference at 50 Hz. Cutaneous local anesthesia produced a marked impairment in vibrotactile detection and discrimination at the low standard frequencies of 20 and 50 Hz but little effect at higher frequencies. In summary, the results reveal, first, a striking similarity in vibrotactile discriminative performance in hairy and glabrous skin despite marked differences in detection thresholds for the two sites, and, second, the results confirm that vibrotactile detection and discrimination in hairy skin depend on superficial receptors at low frequencies but depend on deep, probably Pacinian corpuscle, receptors for high frequencies.

Mechanosensory perception: are there contributions from bone-associated receptors?

1. The identity of the receptors and afferent nerve fibres that mediate the sense of touch varies somewhat with body location. Those that have been most intensively characterized are associated with the distal glabrous skin of the limbs and, in primates, mediate the sense of touch in the fingertips and palms. In this glabrous skin region, there appear to be three or four principal classes of tactile sensory nerves that fall into two broad groups. One group, the so-called slowly adapting (SA) receptors and afferent fibres, is responsive to static mechanical displacement of skin tissues and is made up of two classes, the type I (SAI) fibres that innervate Merkel receptors and the type II (SAII) fibres that innervate Ruffini endings. The second broad group displays a pure dynamic sensitivity to tactile stimuli and also falls into two principal classes, the rapidly adapting (RA) tactile fibres that are associated with Meissner corpuscle receptors and the Pacinian corpuscle (PC)-associated class of tactile afferent fibres. 2. In other regions of the skin, such as the hairy skin of the arms, legs and trunk, there are similar functional classes of tactile sensory nerves, although the receptor endings differ somewhat from those of the glabrous skin. 3. Receptors in close association with the long bones of the limbs include groups of Pacinian corpuscles distributed along the interosseous membranes. These are highly sensitive to dynamic forms of mechanical stimuli, in particular vibrotactile disturbances. However, despite their close association with bone, these receptors probably cannot be legitimately considered 'osseoreceptors'. 4. Both the periosteum and the bone marrow are richly supplied by nerve fibres. However, much evidence indicates that these are largely or entirely in the fine-diameter category of nerve fibres, whose roles may be confined to either nociception or to the efferent autonomic regulation of bone-associated blood vessels. 5. In conclusion, it remains uncertain whether any aspects of our innocuous touch or kinaesthetic senses, in either the limbs or in orofacial regions, can be ascribed to 'osseoreceptors' located in the periosteum or within the bone marrow itself.

Tactile neural mechanisms in monotremes.

Monotremes, perhaps more than any other order of mammals, display an enormous behavioural reliance upon the tactile senses. In the platypus, Ornithorhynchus anatinus, this is manifest most strikingly in the special importance of the bill as a peripheral sensory organ, an importance confirmed by electrophysiological mapping that reveals a vast area of the cerebral cortex allocated to the processing of tactile inputs from the bill. Although behavioural evidence in the echidna, Tachyglossus aculeatus, suggests a similar prominence for tactile inputs from the snout, there is also a great reliance upon the distal limbs for digging and burrowing activity, pointing to the importance of tactile information from these regions for the echidna. In recent studies, we have investigated the peripheral tactile neural mechanisms in the forepaw of the echidna to establish the extent of correspondence or divergence that has emerged over the widely different evolutionary paths taken by monotreme and placental mammals. Electrophysiological recordings were made from single tactile sensory nerve fibres isolated in fine strands of the median or ulnar nerves of the forearm. Controlled tactile stimuli applied to the forepaw glabrous skin permitted an initial classification of tactile sensory fibres into two broad divisions, according to their responses to static skin displacement. One displayed slowly adapting (SA) response properties, while the other showed a selective sensitivity to the dynamic components of the skin displacement. These purely dynamically-sensitive tactile fibres could be subdivided according to vibrotactile sensitivity and receptive field characteristics into a rapidly adapting (RA) class, sensitive to low frequency (

Impulse propagation over tactile and kinaesthetic sensory axons to central target neurones of the cuneate nucleus in cat.

Paired, simultaneous recordings were made in anaesthetized cats from the peripheral and central axons of individual tactile and kinaesthetic sensory fibres. The aim was to determine whether failure of spike propagation occurred at any of the three major axonal branch points in the path to their cuneate target neurones, and whether propagation failure may contribute, along with synaptic transmission failures, to limitations in transmission security observed for the cuneate synaptic relay. No evidence for propagation failure was found at the two major axonal branch points prior to the cuneate nucleus, namely, the T-junction at the dorsal root ganglion, and the major branch point near the cord entry point, even for the highest impulse rates (approximately 400 impulses s(-1)) at which these fibres could be driven. However, at the highest impulse rates there was evidence at the central, intra-cuneate recording site of switching between two states in the terminal axonal spike configuration. This appears to reflect a sporadic propagation failure into one of the terminal branches of the sensory axon. In conclusion, it appears that central impulse propagation over group II sensory axons occurs with complete security through branch points within the dorsal root ganglion and at the spinal cord entry zone. However, at high rates of afferent drive, terminal axonal propagation failure may contribute to the observed decline in transmission security within the cuneate synaptic relay.

Transmission security for single, hair follicle-related tactile afferent fibers and their target cuneate neurons in cat.

Transmission from single, identified hair follicle afferent (HFA) nerve fibers to their target neurons of the cuneate nucleus was examined in anesthetized cats by means of paired recording from individual cuneate neurons and from fine, intact fascicles of the lateral branch of the superficial radial nerve in which it is possible to identify and monitor the activity of each group II fiber. Selective activation of individual HFA fibers was achieved by means of focal vibrotactile skin stimulation. Forearm denervation precluded inputs from sources other than the monitored HFA sensory fiber. Transmission characteristics were analyzed for 21 HFA fiber-cuneate neuron pairs in which activity in the single HFA fiber of each pair reliably evoked spike output from the target neuron at a fixed latency. As the cuneate responses to each HFA impulse often consisted of 2 or 3 spikes, in particular at HFA input rates up to approximately 20 imp/s, the synaptic linkage displayed potent amplification and high-gain transmission, characteristics that were confirmed quantitatively in measures of transmission security and cuneate spike output measures. In response to vibrotactile stimuli, the tight phase locking in the responses of single HFA fibers was well retained in the cuneate responses for vibration frequencies up to approximately 200 Hz. On measures of vector strength, the phase locking declined across the synaptic linkage by no more than approximately 10% at frequencies up to 100 Hz. However, limitations on the impulse rates generated in both the HFA fibers their associated cuneate neurons meant that the impulse patterns could not directly signal information about the vibration frequency above 50-100 Hz. Although single HFA fibers are also known to have secure synaptic linkages with spinocervical tract neurons, it is probable that this linkage lacks the capacity of the HFA-cuneate synapse for conveying precise temporal information, in an impulse pattern code, about the frequency parameter of vibrotactile stimuli.

NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31]NPY24-36, reduces renal vasoconstrictor activity in anaesthetised dogs.

The actions of neuropeptide Y (NPY) at the autonomic neuroeffector junction have been attributed to two main receptor subtypes. At NPY Y1 receptors, located postsynaptically, NPY has been shown to produce vasoconstriction, or to potentiate the action of other vasoconstrictor agents. At NPY Y2 receptors, located presynaptically on nerve terminals, NPY inhibits the release of neurotransmitter from autonomic nerve terminals. In these experiments we have used the specific NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31]NPY, which lacks local constrictor activity, and have demonstrated inhibition of nerve-evoked vasoconstriction in the renal circulation of anaesthetised dogs in a way that suggests an intra-renal regional specificity. Under control conditions stimulation of the renal sympathetic nerves over a range of frequencies (1-5 Hz) reduced renal vascular conductance and glomerular filtration rate (GFR). Following the injection of the selective NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31]NPY24-36, nerve-evoked reductions in renal conductance were reduced by over 45%. At the lowest stimulation frequencies, reduced vasoconstrictor activity was associated with a marked increase in GFR in the presence N-acetyl [Leu28,Leu31]NPY24-36. At both higher levels of stimulation N-acetyl [Leu28,Leu31]NPY24-36 significantly inhibited vasoconstrictor activity and attenuated the nerve-evoked reductions in GFR. Full recovery of both variables was observed 20 min after N-acetyl [Leu28,Leu31]NPY24-36 injection. N-acetyl [Leu28,Leu31]NPY24-36 produced a similar inhibition of renal vasoconstrictor activity when the renal nerves were left intact and activated reflexly. These results suggest that NPY can act via NPY Y2 receptors to inhibit sympathetic vasoconstrictor activity in the renal circulation of dogs. On the basis of the demonstrated dissociation of effects on vascular conductance and GFR, we suggest that this might result from a preferential action of the NPY Y2 agonist on sympathetic nerves supplying the afferent arteriole of the kidney.

Inhibition of vagal vasodilatation by a selective neuropeptide Y Y2 receptor agonist in the bronchial circulation of anaesthetised dogs.

Neuropeptide Y (NPY) is both co-stored and co-released with noradrenaline from sympathetic nerve terminals. In the cardiovascular system, NPY acts on two main receptor subtypes. At postjunctional, or Y1 receptors, NPY can cause both direct vasoconstriction and the potentiation of various constrictor agents. NPY acting at the presynaptic, or Y2 receptor, inhibits the release of neurotransmitter from autonomic nerves. In the present paper, we have used both sympathetic stimulation and the selective NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31] NPY24-36, to examine the role of NPY in the inhibition of vagally mediated vasodilatation in the bronchial circulation of the anaesthetised dog. Stimulation of the cardiac end of the cervical vagus nerve at 1 Hz for 15 s (1 ms, 70 V) increased bronchial vascular conductance by 45%. This increase in flow was abolished by atropine. Sympathetic stimulation for 2.5 min at 16 Hz (1 ms, 20 V) produced a significant (P < 0.05) and prolonged (9 min) inhibition of the subsequent parasympathetically evoked vasodilatation. Similarly, the NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31] NPY24-36, produced a significant (P < 0.05) and prolonged (15 min) inhibition of parasympathetically evoked vasodilatation. When vagus was stimulated at 2.5 Hz for 30 s (1 ms, 70 V), an atropine-resistant, but capsaicin-sensitive vasodilatation was observed. Neither sympathetic stimulation nor the NPY Y2 receptor agonist could be demonstrated to inhibit this vasodilatation. These results suggest that NPY can inhibit cholinergic parasympathetic vasodilatation in the bronchial circulation by an action on NPY Y2 receptors.

Inhibition of sympathetic cholinergic vasodilatation by a selective NPY Y2 receptor agonist in the gracilis muscle of anaesthetised dogs.

Neuropeptide Y (NPY) is known to be co-stored and co-released from sympathetic nerve terminals. In the cardiovascular system NPY acts on two main receptor subtypes. At the postjunctional or Y1 receptor NPY causes constriction directly in addition to potentiating other vasoconstrictor agents. NPY acting at the prejunctional, or Y2 receptor, inhibits the release of neurotransmitter from autonomic nerve terminals. In these experiments we used the selective Y2 receptor agonist N-acetyl[Leu28,Leu31]NPY24-36 to examine the role of NPY in the modulation of sympathetic vascular control in skeletal muscle in anaesthetised dogs. No systemic pressor or local constrictor activity was observed in response to N-acetyl[Leu28, Leu31]NPY24-36 administration, therefore allowing us to examine the neuroinhibitory actions of NPY in the absence of direct vascular effects on blood flow. Stimulation of the sympathetic nerves to the gracilis muscle engages both sympathetic cholinergic and sympathetic adrenergic fibres and produces an initial vasodilatation followed by a slower vasoconstriction. Nerve evoked vasodilatation was inhibited by over 50% in the presence of the selective NPY Y2 agonist N-acetyl[Leu28,Leu31]NPY24-36. This dilatation was abolished by atropine, confirming its cholinergic nature. N-Acetyl[Leu28,Leu31]NPY24-36 was found to have no effect on nerve evoked vasoconstriction. The results demonstrate a NPY Y2-receptor mediated inhibition of nerve evoked sympathetic cholinergic vasodilatation but not of sympathetic vasoconstriction.

The effect of galanin and galanin fragments on blood pressure in the Cane toad, Bufo marinus.

Galanin has previously been shown to have a slight vasodepressor or no effect on blood pressure in placental mammals, but causes potent increases in blood pressure in several other vertebrate species. In this paper, the part of the galanin molecule responsible for the pressor activity was investigated in the Cane road, Bufo marinus by administration of fragments of galanin into anaesthetised toads and isolated arterial segments in an organ bath. In anaesthetised toads, the order of efficacy was human galanin > porcine galanin > Gal 1-16 > Gal 1-15 > Gal 3-15 > Gal 21-29 = 0. In isolated vessels, the first three peptides were equally effective. When four fragments of human galanin were tested in anaesthetised toads, the order of efficacy was human galanin > Gal 1-15 > Gal 5-20 > Gal 10-25 > Gal 15-30. The substitution of alanine for tryptophan at position 2 or for tyrosine at position 9 abolished the pressor response to the human galanin fragment 1-18 in anaesthetised toads. These results suggest that vascular activity in the toad is retained within the N-terminus and that positions 2 (tryptophan) and 9 (tyrosine) are key amino acids in retention of the vascular activity of galanin.

Effect of three galanin antagonists on the pressor response to galanin in the Cane toad, Bufo marinus.

Galanin is a neuropeptide that causes a marked pressor response in several non-mammalian vertebrate species, and some marsupials. In this study, the effect of three galanin antagonists were tested on the pressor response to an intravenous dose (6.3 nmol/kg) of porcine galanin in anaesthetised Cane toads, Bufo marinus. Antagonists were injected at either 20 or 50 times the molar dose (x MD) of galanin. The antagonist, C7 (Galanin 1-13-spantide) reduced the pressor effect of galanin by 32.2 +/- 6.0% when delivered at 20 x MD (n = 4) and by 42.9 +/- 15.7% when delivered at 50 x MD (n = 4) of galanin, the response recovering within 30 min. A second antagonist, M32a (Galanin 1-13-NPY 24-36) had no effect on the pressor response to galanin at 20 x MD (n = 4), but significantly reduced the pressor effect by 54.8 +/- 6.4% at 50 x MD (n = 5), which also recovered within 30 min. Administration of a third antagonist, galantide or M15 (Galanin 1-13-Substance P5-11), resulted in a profound drop in blood pressure, and did not affect the response to galanin at either dose. In conclusion, C7 and M32a are effective, short-term antagonists of the blood pressure effects of galanin in the toad.