Laura Farraway

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

Abstract The network of interstitial cells of Cajal associated with Auerbach’s (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach’s plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach’s plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities.

Development of Merkel Cell Populations with Contrasting Sensitivities to Neonatal Deafferentation in the Rat Whisker Pad

In this study, we used the quinacrine fluorescence technique to investigate the embryonic and early postnatal development of two distinct populations of Merkel cells in the rat whisker pad and the consequences of neonatal deafferentation on their subsequent development. Annular clusters of Merkel cells first appear in the epidermis near the caudal margin of the mystacial region between embryonic days E14 and E15 at dome sites located on horizontal ridges where the primordial vibrissal follicles develop. The development of these cells progresses in a caudorostral sequence across the whisker pad as does the development of the vibrissal follicles. Each cluster eventually forms a conical ridge or collar of about 130 Merkel cells that surrounds the vibrissal hair shaft as it penetrates the overlying pad epidermis. In the vibrissae, which develop as downgrowths from the horizontal ridges at the dome sites, Merkel cells first appear (caudally) between E16 and E17 and form a cylindrical cuff within the outer root sheath; cells are added progressively until about the end of the first postnatal week when a plateau level of about 750-800 cells is reached. Following unilateral transection of the infraorbital nerve at 24-36 hr after birth, these vibrissal Merkel cells continued to develop along a time course that was indistinguishable from normal, at least over the first 2 weeks of postnatal life. In contrast, all or most of the Merkel cells that normally develop within collars or annular clusters in the pad epidermis (around both the vibrissal and intervibrissal or pelage hairs) either disappeared within a few days or failed to develop. Other light and electron microscopic procedures supported the main findings and confirmed that the denervation was successful. Thus, the vibrissal Merkel cells, like those in the glabrous hindpaw, behaved as a distinct class which develops postnatally and is maintained (at least over a 2-week period) without the presence of sensory nerves. Since both the mystacial vibrissae and glabrous hindpaw have specialized cortical representations, a possible relationship between these findings and the organization of the somatosensory cortex during development is discussed.

Characterization of Merkel cells and mechanosensory axons of the rat by styryl pyridinium dyes

SummaryThe epidermal Merkel cells and their sensory innervation serve tactile sensation in vertebrates. In this study the fluorescent cationic mitochondrial dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), which has recently been used as a vital stain for motor and autonomic nerve terminals, was tested for its ability to stain Merkel cells and sensory fibers in the snout of the rat. Brightly-fluorescent structures resembling Merkel cells as well as nerve fibers and their terminations were evident in whole mounts of the vibrissal follicle. Unilateral denervation of the vibrissal follicles soon after birth resulted in a staining pattern remarkably similar to that obtained after labelling of the Merkel cells selectively with the fluorescent marker quinacrine, but all fiber staining was abolished. Likewise, in the separated epidermis of other skin regions, including the hairy and glabrous skin of the nose, the staining pattern revealed by 4-Di-2-ASP was indistinguishable from that obtained by quinacrine fluorescence. These results indicate that certain styryl pyridinium dyes may be used as vital stains for epidermal Merkel cells as well as cutaneous mechanosensory axons.

Pinaverium acts as L-type calcium channel blocker on smooth muscle of colon.

The effect of pinaverium was electrophysiologically characterized and compared with the established L-type calcium channel blockers diltiazem, D600, and nitrendipine on canine colonic circular smooth muscle. Effects were studied on the electrical activity of the smooth muscle cells, in particular the spontaneously occurring slow wave. In addition, effects were examined on spontaneous contraction patterns and contractile activities generated by stimulation of cholinergic nerves or directly by stimulating muscarinic receptors. Effects were also examined on excitation of NO-releasing intrinsic nerves. Pinaverium bromide affected the slow wave by selectively inhibiting the plateau potential that is associated with generation of contractile activity. Pinaverium, similar to diltiazem and D600, produced reductions in cholinergic responses as well as spontaneous contractions. The IC50 values for inhibition of cholinergic responses for pinaverium, diltiazem, and D600 were 1.0 x 10(-6), 4.1 x 10(-7), and 5.3 x 10(-7) M, respectively. The IC50 values for inhibition of spontaneous contractile activity for pinaverium, diltiazem, and D600 were 3.8 x 10(-6), 9.7 x 10(-7), and 8.0 x 10(-7) M, respectively. Increases in contractility by carbachol were abolished by pretreatment with either pinaverium or D600. In addition, neither pinaverium nor D600 had any effects on the inhibitory NO-mediated relaxations. These data provide a rationale for the use of pinaverium in the treatment of colonic motor disorders where excessive contraction has to be suppressed.

Do Developing Merkel Cells in Vibrissal Tactile Receptors Provide a Template for Pattern Formation in the Trigeminal System

The trigeminal pathway in rodents has served as an important model for studying pattern formation in the central nervous system. In this pathway there is a topographic point-to-point relation between the array of mystacial vibrissae on the whisker pad and their central projections in the barrel-field of the somatosensory cortex (Woolsey and Van der Loos, 1970; Welker, 1976; Belford and Killackey, 1980). Among the peripheral receptor sites the aggregates of Merkel cells, which are involved in tactile sensation in the vibrissal follicles, have been proposed as candidates for the master pattern or template for the central maps (Killackey, 1980). Support for this idea would be strengthened if the development of Merkel cells in the vibrissae should occur independently of retrograde influences within the whisker-to-barrel pathway. To test this we have investigated in the rat the effects of neonatal deafferentation of the whisker pad on Merkel cell development in the vibrissae using the quinacrine fluorescence technique (Nurse et al., 1983). Merkel cells are also present in the overlying whisker pad epidermis and these provided an additional population for comparing the effects of denervation. Since the method permits rigorous quantification of Merkel cell populations, this study was expected to address further the controversial issue of the role of sensory nerves on the development and maintenance of Merkel cells in different epithelial locations (Nurse et al., 1984).