, ‘ . , , A: ., . . .1. . . . . ‘y . j ,. 4 V j _ , , V .. . ,. . . _ . . . . , . W513 i .3, 1‘. :1 Mia; " , “like?” weaivami‘ty j This is to certify that the thesis entitled The Relation Between Epidermal Hydration State and the Activation of Neurons in the Primary Somatosensory Cortex presented by Michael Anthony Steinmetz has been accepted towards fulfillment of the requirements for Ph.D. Physiology degree in Major professor Dateg‘fl___,_a_05+ “ ‘0‘ 7- 0-7639 RETURNING MATERIALS: IV1531_] Place in book drop to LJBRARJES remove this checkout from .5252; your record. FINES will be charged if book is returned after the date stamped below. THE RELATION BETWEEN EPIDERMAL HYDRATION STATE AND THE ACTIVATION OF NEURONS IN THE PRIMARY SOMATOSENSORY CORTEX BY Michael Anthony Steinmetz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1982 ABSTRACT THE RELATION BETWEEN EPIDERMAL HYDRATION STATE AND THE ACTIVATON OF NEURONS IN THE PRIMARY SOMATOSENSORY CORTEX BY Michael Anthony Steinmetz A major function of the epidermis is to inhibit the loss of fluids from the body. Although the epidermis is an effective water diffusion barrier, it is not impermeable, and is capable of absorbing and storing water. .The epidermal hydration state is labile and determined by water diffusion from the dermis, sweat gland activity and ambient environmental conditions. Thermal, electrical and mechanical properties of intact skin depend on the epidermal hydration state. Moreover, it has been proposed that the activation of receptors at or below the dermo-epidermal junction depends on the energy transfer properties of the overlying epidermis. The purpose of this study was to test the effect of epidermal hydration state on the activation of cortical neurons which receive input from superficial glabrous skin mechanoreceptors. The electrical sign of cortical neuronal activity was recorded extracellularly using a glass-insulated tungsten microelectrode and conventional neurophysiological recording techniques, while punctate stimuli of controlled force were delivered to the glabrous skin surface of the cat's (Eglig Egggg) central footpad. Cats were used because of their well—mapped somatosensory cortex and the thick epidermal layers of their central footpad. Epidermalhydration state was varied by either exposing the footpad to air streams of different relative humidities, or by soaking the skin in deionized water. Statistical analysis of experimental results was performed using an analysis of variance. Data from these experiments show that the amount of force required to activate neurons in the primary somatosensory cortex varies with the epidermal hydration state of the glabrous skin. An inflection point occurs in this relationship, such that threshold forces are minimal for moist skin and are maximal for dry and water soaked skin. This study also shows that epidermal hydration has no significant effect on the spatial coupling of the stimulus to the response of single cortical neurons. To my wife, Susan - whose love made it possible ii ACKNOWLEDGEMENTS I am deeply grateful for the guidance, opportunity, support and constant enthusiasm which I received throughout my gradute training from my friend and advisor Dr. Thomas Adams. I am also forever indebted to Dr. S. Richard Heisey for his advice and encouragement, and to Jill Fisher and Craig Hartman for their collaborative and technical contributions to this research project. Much of the success of these experiments is owed to the excellent technical assistance provided by Amy Abrahamsen, Jeff Bruer and Kam Hunter. A special thanks is due to David Manner for his assistance with the electronics. iii LIST LIST II. III. IV. V. TABLE OF CONTENTS OF TABLESOOOOOOOOOOOOOOOOOOOOOOOOO OF FIGURESOOOOOOOOOOOOO0.0.0.0.... INTRODUCTIONOOOOOOOOOOOO...0.0... LITERATURE REVIEWOOOOOOOOOOOOOOO. A. B. C. D. E. F. G. The The The The Hydration effects on skin thermal epidemiSooooooooooooo ..... .0 epidermal strata....... demiSooooooooooooooooo 0.0.0.0... sweat glands.......................... Epidermal hYdrationOO0.0000000000000000000 properties................................ Hydration effects on skin electrical properties................................ Hydration effects on skin mechanical properties................................ Glabrous skin mechanoreceptors............. The mechanoreceptive pathway............... The primary somatosensory cortex........... MATERIALS AND METHODSOOOOOOOO0.0.0.0.0...00...... A. B. C. D. E. F. G. H. I. J. Surgery and experimental preparation....... Anesthesia supplementation................. Preparation for cortical recording......... Microelectrodes............................ Cortical recordings........................ Footpad preparation.......... Mechanical stimulation....... Skin hydration............... Experimental protocol and data reduction... Statistical analysis....................... RESULTSOOOOOOOOOOOO0.00.0.00...OOOOOOOOOOOOOOOOOO DISCUSSION.0.0...O...00.0.0.0...OOOOOOOOOOOOOOOO. A. B. Epidermal permeability..................... Epidermal water storage.................... iv PAGE vi vii 15 17 22 26 27 28 32 35 38 41 41 42 42 46 50 56 60 68 69 72 74 103 103 105 PAGE C. Epidermal hydration and skin thermal properties. .................. ............. 109 D. Epidermal hydration and skin electrical properties.............. ........... ......... 110 E. Epidermal hydration and skin mechanical properties. ..... .. .............. .... ..... ... 112 F. Cortical unit activity ....... .... .... 115 G. Stimulus intensity and cortical neuronal response.... .......................... H. Epidermal hydration effects on cortical neuronal response........................... 120 I. Stimulus location effects on cortical neuronal response........................... 125 ... 118 V. CONCLUSIONS......... .............................. 127 VI. BIBLIOGRAPHY.................................. LIST OF TABLES TABLE PAGE 1. Threshold Values for Cat PC06 ...... ..... ....... ... 90 2. Hydration Effects on Skin Pliability.............. 94 3. Anova Tables...................................... 96 4. Treatment Means and Statistical Comparisons....... 99 5. Skin Location Means and Statistical Comparisons... 101 vi FIGURE 1. 10. 11. 12. 13. 14. 15. 16. LIST OF FIGURES Diagram of Experimental Preparation.............. Copper-constantan Thermopiles.................... Microelectrode for Recording Unit Activity....... Block Diagram of Signal Amplification and recording system.O.......OOOO.........OOOOOOO0.0. Circuit Diagram of Second Stage Amplifier, Filter and Audio Monitor... ..... ....... .......... Recording System Calibration..................... Cortical Unit Response........................... Mechanical Stimulator and Hydration Capsule...... Cross Section of Mechanical Stimulator........... Circuit Diagram of the Mechanical Stimulator current source..0......OOOOOOOOOOOOOOOOOOOO0.0... Mechanical Stimulator Driver Calibration......... Circuit Diagram of the Stimulus Event Marker..... Cortical Neuronal Responses to Ten Successive MeChanical StimUIiOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Action Potential Amplitude Histogram — cat BCOlWA0084...0...........OOOOOOOOOOOOOO0.00.. Action Potential Amplitude Histogram - CAt BC07LW0125.0....0.0.0..........OOOOOOOOOOOOOO Representative Effect of Hydration State on Cortical Unit Response Probability - cat PC170241......OOOOOOOOOOOOOOO....OOOOOOOOOOOO vii PAGE 43 45 47 51 53 54 S7 59 61 62 65 66 67 77 78 80 FIGURE PAGE 17. Representative Effect of Hydration State on Cortical Unit Response Probability — Cat PC04.022................. ....... ............. 81 18. Representative Effect of Hydration State on Cortical Unit Response Probability — Cat PC01.084 ........ . .................. .......... 82 19. Representative Effect of Stimulus Location on Cortical Unit Response Probability - Cat BCO4RA............ acoo-cocoo-noooooooooooooso 84 20. Representative Effect of Hydration State on T50 Threshold - Cat PC17.241......................... 86 21. Representative Effect of Hydration State on T50 ThreShOld—Cat PC040022¢ooouoooooc-oooooo'tnoooa 88 22. Representative Effect of Hydration State on T50 Threshold - Cat PC01.084......................... 89 23. T50 Thresholds for Cat BC06RA.................... 93 24. Effect of Hydration Condition on T50 Threshold... 100 25. Effect of Stimulus Location on T50 Threshold..... 102 26. Proposed Stress-Strain Relationships............. 122 27. Summary of Hydration State Effects on Cortical Neuronal Force Thresholds........................ 124 viii INTRODUCTION Skin is a nonhomogeneous and anisotropic tissue composed of several distinct layers, each with unique physical and chemical properties. The outer skin layers (the epidermis) are devoid of blood vessels and contain only a few bare nerve endings. In contrast, the lowest skin layer (the dermis) has a well developed vascular system, and is richly innervated. This nerve supply includes sympathetic fibers to sweat glands, blood vessels and hair follicles, as well as afferent nerves subserving the sensory modalities of touch, pain and temperature. Although the specialized endings of these afferent nerves are found throughout the dermis, they are more concentrated near the junction of the dermis and epidermis. The secretory portion and proximal tubules of atrichial (eccrine) sweat glands are also located in the dermis. These glands are found throughout human skin and in the glabrous, tactile skin of some furred animals, such as in the central footpad of the cat. Because of the lack of a direct blood supply, the water content and hydration profile of the epidermis is extremely labile and dependent on at least three factors. These factors are: l) diffusion of water from the underlying dermis, 2) absorption or desorption of water from the l 2 environment, and 3) the lateral dboth salts and water from the helically coiled distal sweat gland tubules, which penetrate the epidermis. Although these factors have long been recognized to influence epidermal water content, little is known about their interactions, or of possible feedback mechanisms involved in controlling hydration in intact skin. It has been clearly demonstrated that thermal and electrical energy transfer properties of the epidermis are affected by the amount of water stored in the skin; For example, both the thermal and electrical conductivity of the epidermis vary directly with skin hydration. Mechanical properties of skin, which have been indexed by stress-strain relationships or by coefficients of friction at the skin surface, also depend on water content. Both "free" water, which occupies the intracellular and intercellular spaces, and "bound" water, which exists in chemical combination with proteins and other water binding molecules, have been implicated in these hydration effects. It seems reasonable that hydration induced effects on epidermal mechanical properties might affect the threeedimensional distribution of forces applied to the skin surface, and consequently affect the activation of mechanoreceptive nerve endings located at the dermal-epidermal border. At least three types of mechanoreceptive endings have been identified for glabrous skin. Two of these (Merkel Cells and Meissner Corpuscles) are located almost exclusively at the dermal-epidermal border, making them 3 particularly sensitive to epidermal hydration effects. The third type (Pacinian Corpuscles) are found deeper in the dermis and also in subdermal tissues. Primary afferent fibers arising from these receptors have been functionally classified as either rapidly or slowly adapting, according to their response to maintained skin surface indentations. Both of these receptor classes can be subdivided according to the relative size of their receptive fields. Individual rapidly adapting receptors show selectivity in the range of frequencies to which they respond when sinusoidal stimuli are given. It is generally assumed that the variation in primary afferent response is due to the topographical distribution of the fiber endings and to the viscoelastic and membrane electrical properties of the nerve ending. Some of the adaptation observed in the frequency of action potentials is likely due to the viscoelastic properties of the skin. Primary afferent fibers which enter the spinal cord relay information to the brain through two parallel pathways. The larger diameter nerve fibers project directly to the brainstem via the dorsal columns of the spinal cord and synapse in the dorsal column nuclei of the medulla. Second order fibers cross over to the contralateral side of the brainstem in the medial lemniscus, and their axons terminate in specific relay nuclei of the thalamus. In contrast, the smaller diameter mechanoreceptive afferents cross over and synapse in the spinal cord, and the axons of 4 the second order neurons ascend to thalamic nuclei by way of the spinothalamic tract. Although the importance of the large fiber system in fine tactile discrimination is unquestionable, the relative contribution of the smaller diameter fibers remains controversial. Axons of third order neurons which originate in the thalamus ascend through the internal capsule and terminate on the intrinsic neurons of the anterior parietal cortex. A high degree of organization and synaptic integrity is maintained throughout the mechanoreceptive projections, which results in a columnar, somatotopic organization in this cortical receiving area. Cells in a single cortical "column" have similar functional properties and similar receptive fields. Cells in adjacent "columns" represent either overlaping or adjacent peripheral receptive fields. Because of this somatotopic organization, a complete representation of the body surface exists in the somatosensory cortex. This representation or "homunculus" is somewhat distorted, because the area of cortical tissue devoted to input from a body region is directly related to the density of mechanoreceptive endings in that peripheral site. Accordingly, a relatively large proportion of the somatosensory cortex represents input from the hands and face, which have the greatest density of mechanoreceptive endings. Recent evidence suggests that at least two and perhaps as many as three or four overlapping representations of the 5 body can be located in this cortical area. Each representation corresponds roughly with previously identified architectonic zones, and each exhibits different functional properties. In the cortical depiction of the cat central footpad, at least three separate representations exist. First, a population of neurons which slowly adapt to small, maintained displacements of the skin surface; second, a population which rapidly adapts to small, maintained skin displacements; and third, a population which responds to deep tissue stimulation by larger displacements of the skin or to movements of the limbs. The population of cortical neurons selected for study in this dissertation were located in the areas subserved by slowly and rapidly adapting cutaneous cells. These cells appear to receive input from the more superficial skin receptors, and are important in fine tactile discrimination. When activity is evoked by short duration (10 msec) square wave mechanical pulses, both types of cells respond similarly with single action potentials. Results of previous experiments indicate that thresholds for perception of mechanical stimuli of controlled force or displacement correlate well with thresholds for the activation of primary afferent nerve fibers. Two types of thresholds were identified in these studies of primary afferents, an absolute threshold for initiating activity. and a higher threshold, where neuronal responses correspond in a nearly 1:1 fashion with the stimulus. This study 6 examines thresholds for activating cortical neurons over activating cortical neurons. The hypothesis tested in this dissertation is: do the mechanical properties of the skin which depend upon epidermal hydration affect threshold response characteristics of rapidly or slowly adapting neurons in the primary somatosensory cortex, when punctate stimuli of controlled force are delivered to the glabrous skin surface? LITERATURE REVIEW Because numerous and voluminous reviews of skin and somatosensory anatomy and physiology have already been published, the scope of this literature review is limited to those aspects of skin anatomy, epidermal hydration, and mechanoreception judged crucial to this investigation. Textbooks by Rothman (1954), Montagna (1962), Tregar (1967), and Elden (1971), as well as review articles by Scheuplein and Blank (1971) and Newburgh and Johnston (1942) provide broad discussions of the structure and function of skin and its appendages. In addition, Holmes (1972) has reviewed the literature specific to the glabrous skin on the cat footpad. For comprehensive and detailed information on the somatosensory system, textbooks edited by Mountcastle (1980) and 1990 (1973), and review articles by Wall and Dubner (1972) and Dykes (1978) are recommended. THE EPIDERMIS The epidermis is a stratified tissue which ranges in thickness from 0.3mm (Conroy, 1964) to 0.9mm (Strickland, 1958). Early investigators identified two epidermal layers, the germinating layer (stratum germinativum) also known as 8 the Malpighian layer (Trautmann and Fiebiger, 1957), and a more superficial horny layer. Closer examination has revealed that there are at least five epidermal layers, which beginning with the deepest are: l) the stratum basale, 2) the stratum spinosum, 3) the stratum granulosum, 4) the stratum lucidum, and 5) the stratum corneum (Creed, 1958; Andrew, 1959; Montagna, 1962; Conroy, 1964). All of these layers appear to be present in the glabrous skin of the cat central footpad (Holmes, 1972). Although the relative thickness of each layer varies over the body surface, the proportionality is characteristic for any one location (Rushmer gt 31., 1966). Only cells in the deeper skin layers exhibit mitotic activity (Montagna, 1962). Cells formed in these layers migrate toward the skin surface while undergoing a gradual morphological transformation called cornification or keratinization. This process begins in the stratum germinativum and continues until cells typical of the stratum corneum are formed (Nicoll, 1972). Lavker and Matoltsy (1970) have identified two stages of keratinization called the "synthetic" and the "transformation" phases. During the "synthetic" phase, which occurs in the lower layers, three major intracellular constituents are formed. These constituents are: l) the filamentous protein bundles, 2) the membrane coating granules, and 3) the keratohyalin granules. The "transformation" phase which occurs in the upper skin strata is characterized by the digestion of 9 intracellular constituents by proteolytic enzymes, sparing the filamentous proteins and the keratohyalin granules. Jerrett gt El. (1965) proposed that these proteins are protected by extensive disulfide cross linkages and by phospholipid moieties with which they chemically interact. During the "transformation" phase, the membrane coating granules migrate toward the cell membrane and then extrude their contents into the interstitial space (Nicoll, 1972). THE EPI DERMAL STRATA The deepest epidermal layer, the stratum basale consists of a single layer of columnar cells which rests on the underlying dermis (Conroy, 1964). Microscopic cytoplasmic processes from the basal surface of these cells provide intimate contact with the dermis. Dick (1947) proposed that dermal collagen fibers attach to the cell membrane between these cytoplasmic processes, therby anchoring the epidermis. Medawar (1953) has shown a close relationship between the elastin fibers of the dermis and the basal layer cells. Trypsin, a proteolytic enzyme which readily attacks elastin, rapidly separates the dermis and epidermis (Medawar, 1953). Basal cells have a large basophilic nucleus and a "scanty amount" of cytoplasm (Conroy, 1964). These cells have an abundance of nucleoprotein and a large proportion of sulfhydryl groups (Creed, 1958). Electron micrographs reveal both mitochondria and ribosomes in these cells. 10 (Montagna, 1962; Dellmann, 1971). It is generally accepted that the basal layer has a high degree of mitotic activity, and that its main functions are to anchor the epidermis (Montagna, 1962) and to provide for the regeneration of skin through cell division (Nicoll, 1972). Scattered among the basal layer cells are melanocytes, which under neuroendocrine control produce and disperse the pigment melanin and give skin its characteristic color (Conroy, 1964; Strickland, 1958; Strickland and Calhoun, 1963). The stratum spinosum is superficial to the stratum basale; it is the thickest epidermal layer. This strata contains polygonal—shaped cells and has been reported to vary in thickness from 16 (Conroy, 1962) to 38 (Strickland, 1958) cell layers. As new cells are formed by the basal layer, the cells of the stratum spinosum migrate toward the skin surface and become progressivly more flattened (Andrew, 1959; Conroy, 1964; Dellmann, 1971). The cells of the stratum spinosum contain small protein fibrils (tonofibrils), some of which are organized into microscopic structures called tonofilaments (Montagna, 1962). Although the protein fibrils are distributed throughout the cytoplasm, the filaments are organized into bundles which are generally directed from the cell nucleus out toward specialized structures on the cell membrane, called desmosomes. The desmosomes serve as loci for attachment of adjacent cells (Montagna, 1962; Selby, 1957; Dellmann, 1971). Although early investigators believed the 11 cells had cytoplasmic continuity with each other at these sites (Chambers and Renyi, 1925), electron micrographs by Selby (1955, 1956), Odland (1958, 1960) and others do not support this belief (Montagna, 1962). The cell membranes in these regions of contact are thickened. Rather than cytoplasmic bridges, the intercellular cleft contains fine protein filaments which join and separate adjacent cells. It is these filamentous tufts which give this layer its name (Montagna, 1962). Desmosomes are most evident in the stratum spinosum. Some can be identified in the stratum basale and stratum germinativum, but they appear to be absent from the stratum corneum (Montagna, 1962). Data by Wolff and Schreiner (1968) and Mercer gt gt. (1968) show the presence of a mucopolysaccharide in the intracellular space, which is especially concentrated near the desmosomes. They have concluded that the carbohydrate material represents an "intracellular cement" which binds together adjacent cells and limits the movement of fluids in the interstitial space. Mitochondria can be found in the stratum spinosum, but they become fewer and completely disappear as cells migrate toward the skin surface (Montagna, 1962). The granular layer or stratum granularis lies superficial to the stratum spinosum and consists of flattened, diamond—shaped cells (Conroy, 1964). The granular cell membrane contains small villi, which give them a serrated appearance (Trautmann and Fiebiger, 1957). This layer has been reported to range in thickness from 3 12 (Conroy, 1964) to 8 (Strickland, 1958; Strickland and Calhoun, 1964) cell layers in the cat central footpad. In hairy skin, the cells of this layer are organized in vertical columns with the cells of adjacent columns interdigitating. Glabrous skin lacks this degree of columnar organization (Menton and Eisen, 1971). Both the size and number of keratohyalin granules increase in the stratum granularis (Trautmann and Fiebiger, 1957). These granules contain phospholipids which are closely associated with the tonofibril protein moieties (Spearman, 1970). Electron micrographs reveal a close association of these granules with cytoplasmic tonofilaments (Dellmann, 1971). Selby (1957) has demonstrated a pronounced consolidation of the protein fibers in this layer. The tonofilament proteins contain a large proportion of cysteine residues, and therefore have a large number of sulfhydryl groups. The sulfhydryl groups of adjacent polypeptide chains are oxidized as the cells migrate toward the surface forming the disulfide bonds of cystine (Giroud and Leblond, 1951; Spearman, 1970). The extensive disulfide cross-linkages between polypeptide chains is characteristic of keratin, the primary structural protein of the epidermis (Giroud and Leblond, 1951). It is also in the granular layer that the digestion of intracellular organelles begins which results in the gradual reduction in size and number of ribosomes, mitochondria, and cell nuclei (Jarrett gt 33., 1965). 13 The stratum lucidum is a very thin cell layer interposed between the stratum granulosum and the stratum corneum. This layer is not present in skin covering the general body surface, but is distinct in the glabrous skin on the tactile surfaces (Andrew, 1959; Creed, 1958; Strickland, 1958; Strickland and Calhoun, 1963). This layer has been reported to vary in thickness between 28 and 40 microns in the cat central footpad (Strickland, 1958). The cells of the stratum lucidum are flat and closely packed (Andrew, 1959; Dellmann, 1971; Trautmann and Fiebiger, 1957). They have been described as "anuclear" (Conroy, 1964; Trautmann and Fiebiger, 1957) and appear to be devoid of organelles (Dellmann, 1971). Distinct cell membranes and intracellular spaces are difficult to discern with light microscopy (Trautmann and Fiebiger, 1971; Spearman, 1970), but have been demonstrated in electron micrographs (Selby, 1957). Electron microscopy also revealed closely packed intracellular protein filaments (Dellmann, 1971). Spearman (1970) demonstrated the presence of disulfide linkages and protein bound phospholipids. Selby (1957) and Hashimoto (1969) noted an abrupt change in the structure of the desmosome in this layer which forms dense intercellular bands. Spearman (1970) speculated that the stratum lucidum may serve as the primary barrier to water diffusion across the epidermis, but Scheuplein and Blank (1971) state that there are few data to support this hypothesis. 14 The most superficial epidermal layer is the stratum corneum. The cells in this layer are usually flattened (Andrew, 1959; Dellmann, 1971; Trautmann and Fiebiger, 1957). On the tactile surfaces, these cells overlap and lack the architectural organizaton found on the general body surface (Christophers, 1971; Menton and Eisen, 1971). On the cat footpad, the corneum is thicker than at other skin locations, varying between 15 (Strickland, 1958; Strickland and Calhoun, 1963) and 320 (Conroy, 1964) microns. Cells of this layer are devoid of organelles (Dellmann, 1971) and contain a "tightly packed" matrix of tonofibrils (Matoltsy and Balsamo, 1955) in close association with cytoplasmic granules (Dellmann, 1971). Matoltsy and Balsamo (1955) reported that the dry mass of the stratum corneum is composed of 75% epidermal proteins (keratin), 10% amino acids, 7-9% lipids and 5% cell membranes. The sulfhydryl groups which are plentiful in the stratum granularis are fewer in the corneum, but a large number of disulfide bonds have been shown (Spearman, 1970). Giroud and Leblond (1951) demonstrated a pronounced decrease in the lipid and phospholipid content of the corneum. They proposed that the lipids that remained in the corneum were bound to proteins. Jerrett gt gt. (1965) give supportive evidence for this and attribute the reduction in lipid content to autolysis by epidermal hydrolytic enzymes. Spearman (1970) demonstrated the presence of protein—bound lipids in the stratum lucidum, but failed to find them in 15 the corneum. His observations also support the autolysis hypothesis. The thickened and enzyme resistant cell membranes of the corneum have long foliated villi on their surfaces (Menton and Eisen, 1971). Concomitant with a condensation of the "intracellular cement" at the desmosomes (Wolff and Schreiner, 1968) is a reduction of the interstitial space (Selby, 1957). Mercer (1968) concluded that the desmosomal sites were the "main device holding the (stratum corneum) formation together" (Holmes, 1972). THE DERMIS Unlike the epidermis which is of ectodermal origin, the dermis is derived embryonically from mesoderm. Its major components are collagen, elastin, reticular fibers, hyaluronic acid, and chondroitin sulfate. The first three of these are structural proteins which provide the viscoelastic properties of the dermis, and the latter two are mucopolysaccharides which form the "ground substance" of skin (Holmes, 1972). The dermis of glabrous skin contains blood vessels,.lymphatic vessels, both afferent and efferent nerves, and the secretory portions and proximal tubules of atrichial (eccrine) sweat glands. The dermis lies on top of the subcutaneous fat, but the border separating them is often indistinct (Andrew, 1959; Dellmann, 1971). 16 Unlike the avascular epidermis, the dermis has a well developed and unique vascular system. Blood flow to the skin greatly exceeds metabolic demand (Rushmer gt gt., 1966). Arterial blood brought to the skin in the arteriolar plexus located near the dermis—subcutaneous fat border is returned to the venous system through two routes (Newburgh, 1949). During cold stress, arterial blood can be shunted through communicating veins into venules which are closely opposed to the arterioles. This provides a counter—current heat exchange mechanism, which builds a steep thermal gradient across the skin and conserves body heat (Bazett, 1948). An alternate route for arterial blood is through long capillary loops, which extend into the dermal papillae at the dermal-epidermal junction. Blood flowing through these capillaries drains into venous plexuses located at two different levels of the dermis (Bazett, 1948). This circulatory scheme is an important thermoregulatory adaptation and provides the dermis with a direct source of water, while the avascular epidermis must rely on indirect sources. Nervous system control of skin blood flow appears to be mediated solely by the sympathetic division of the autonomic nervous system (Hertzman, 1959). Adrenergic nerve fibers are responsible for maintaining a vasoconstrictor tone, while sympathetic cholinergic fibers can indirectly cause vasodilation by stimulating bradykinin formation (Barcroft, 1960). Kontos gt gt. (1967) demonstrated a profound, local 17 vasodilator effect of C02, which is unaffected by either alpha or beta adrenergic blockade. The epidermis contains only a few, bare, afferent nerve endings which do not penetrate as far as the stratum corneum (Rushmer gt gt., 1966; Copenhaver, 1971). In contrast, the dermis has a rich supply of highly specialized afferent endings which are concentrated near the dermal-epidermal border, but are also found deeper in the dermis and in the subcutaneous fat (Rushmer gt gl., 1966; Janig, 1971; Malinovsky, 1966; Mountcastle, 1980). These afferent nerve terminals subserve the sensory modalities of touch-pressure, pain and temperature (Mountcastle, 1980) and will be discussed later in greater detail. THE SWEAT GLANDS The atrichial (eccrine) sweat gland is the only appendage of glabrous skin in both humans and cats (Conroy, 1964; Creed, 1958; Strickland, 1958; Strickland and Calhoun, 1963). Although the gross anatomy and histology of cat atrichial glands have been reported (Conroy, 1964; Munger and Brusilow, 1961; Sperling and Koppanyi, 1949), most of our knowledge of sweat gland blood supply, innervation and secretory processes comes from studies in primates including man (Holmes, 1972). Montagna gt gt. (1953) stated that the atrichial gland of the cat footpad is in no way comparable to the human eccrine gland. Using both light and electron 18 microscopy, Munger and Brusilow (1961) found the secretory portions of human and cat sweat glands to be identical. Electron micrographs by the same investigators, however: reveal the sweat gland tubule in cats to be “markedly different" from the human sweat duct. The osmolarity and composition of sweat collected at the skin surface is also different in cats and humans (Munger and Brusilow, 1961). The secretory portions of the atrichial sweat glands are located deep in the dermis, or in the subcutaneous fat (Munger and Brusilow, 1961). This portion of the gland is highly coiled and intertwined with capillaries and small arterioles (Ellis gt gt., 1958). The secretory apparatus is innervated by small unmyelinated, sympathetic nerves (Langley, 1891), which release acetylcholine as a neurotransmitter (Langley, 1923; Patton, 1948; Chalmers and Keele, 1951, 1952; Foster and Weiner, 1970). Fujisawa (1959) reported the presence of three cell layers in this segment which are: 1) the glandular cells, 2) the myoepithelial cell layer, and 3) the basement membrane. The glandular cells surround the lumen of the sweat gland secretory apparatus in a single cell layer (Montagna gt gl., 1953; Hibbs, 1958; Conroy, 1964). These cells have an abundant amount of cytoplasm, and can be classified as "dark" or "clear" according to their staining characteristics. The "dark" cells contain a prominent golgi apparatus, mitochondria, and secretory vacuoles containing mucopolysaccharides (Munger and Brusilow, 1961). These 19 cells also show a high degree of acid phosphatase activity (Kamamura, 1957). In contrast, the "clear" cells do not stain for mucopolysaccharides (Munger and Brusilow, 1961): and have little acid phosphatase activity (Kamamura, 1957). These cells contain glycogen granules (Hashimoto, 1971a), numerous mitochondria, and microvilli which project into the interstitial space (Munger and Brusilow, 1961). The interstitial spaces between adjacent tubular cells called intercellular canaliculi are greatly reduced in regions where adjacent cell membranes are closely opposed, where they are called tight junctions (Munger and Brusilow, 1961). A layer of myoepithelial cells is interposed between the glandular cells and the basement membrane. These elongated, fibrillar cells are oriented either along the tubular axis (Sperling.and Koppanyi, 1949), or spirally around the tubule (Hibbs, 1958). Sperling and Koppanyi (1949) reported that the myoepithelial cells are not contractile, but change their size by a reduction of their intracellular volume. This intracellular fluid contributes considerably to the initial secretion of the stimulated sweat gland. Using Hashimoto's anatomical data, Dobson and Sato (1972) postulated that sweat is formed by the osmotic flow of extracellular water into the canaliculi which are made hypertonic through the action of an energy dependent sodium pump on the "clear" cell membrane. Fluid from the interstitial space then flows from the canaliculi into the 20 lumen of the sweat gland and is brought to the skin surface by volume displacement. The dermal segment or proximal sweat gland tubule receives its blood supply from branches of the dermal capillary loops (Ellis gt_gt.,1958). In both humans and cats, the dermal segment is composed of a superficial cell layer, a basal cell layer, and a basement membrane (Ellis and Montagna, 1962; Munger and Brusilow, 1961). In cats, however, there are far fewer mitochondria in the superficial cells then are found in human sweat gland tubules (Montagna gt gl., 1953; Munger and Brusilow, 1961). The luminal side of the superficial cells has a dense border or cuticle which is thinner in cats than in humans (Munger and Brusilow, 1961; Hibbs, 1958; Holyoke and Lobitz, 1952; O'Brien, 1952). This cuticle is formed by a dense band of intracellular tonofilaments and granules which lie just below the cell membrane. The luminal membrane has microvilli which project into the lumen of the sweat duct (Hashimoto, 1971b; Munger, 1961; Munger and Brusilow, 1961). The cuticle has a large number of disulfide linkages which are similar to cells of the stratum corneum (Lobitz gt_gt., 1954). Adjacent superficial cells are joined by tight junctions which are at or near well developed desmosomes (Hashimoto, 1971b; Ellis and Montagna, 1961). Ellis and Montagna (1961) postulated that the cuticle and desmosomes provide a rigid ring which prevent collapse and occlusion of the sweat duct. 21 The cells of the basal layer contain glycogen granules and numerous mitochondria. These cells also have desmosomes, but they are fewer and less pronounced than in the superficial layer (Ellis and Montagna, 1961). Munger and Brusilow (1961) reported that like the superficial layer, the basal layer in cat tubules has fewer mitochondria than in human sweat glands. Human sweat collected at the skin surface is hypotonic; its osmolarity is directly related to the rate of sweat secretion (Lobitz and Dobson, 1961). Schwartz and Thaysen (1956) postulated that reabsorption of ions in the proximal sweat gland tubule might account for the hypotonicity. Brusilow and Munger (1962) noted that sweat collected from the skin surface of the cat footpad was hypertonic. They attributed this species difference to the shorter length and reduced number of mitochondria in the proximal tubule cells of the cat. Histological and enzymatic data from other investigators support this hypothesis (Hashimoto, 1971b; Sato and Dobson, 1970; Sato gt gt., 1971). The epidermal segment or distal sweat gland tubule is a helically coiled duct which opens to the skin surface in a pore (Sperling and Koppanyi, 1949; Takagi and Tagawa, 1955; Wolf, 1968b). The lumen of the duct is lined with one (Zelickson, 1961) or more (Hashimoto, 1971b; Pinkus, 1939; Takagi, 1952; Wolf, 1968a) cell layers which are anatomically distinct from the surrounding tissue. Blair (1968) has reported as many as 16 tightly wrapped cell 22 layers. The luminal cells of the distal tubule have a cuticle (Conroy, 1964) which gradually disintegrates as it approaches the skin surface (Ellis and Montagna, 1961). These tubular cells become keratinized sooner than the epidermal cells which surround the duct (Takagi, 1952; Lobitz gt gt., 1954). EPIDERMAL HYDRATION Sanctio Sanctorious demonstrated in 1614 that humans continuously lose weight even when sleeping or resting quietly (Benedict and Benedict, 1927). Until the early 1900's, this weight loss was attributed to evaporation from the respiratory surfaces and low rates of sweat secretion. Fleischer in 1877 concluded that the intact skin of man was totally impermeable to all substances (Scheuplein and Blank, 1971). More recent studies by Buettner (1959a,1959b,1959c), Adams (1966) and Scheuplein and Blank (1971) indicate that the skin is permeable to water, ions and other substances. Blank (1953) attempted to locate the "permeability barrier" of the epidermis by measuring evaporative water loss while successively removing thin layers of cells from the skin surface. He noted that the epidermis retained its semipermeable nature until the last few layers of stratum corneum cells were removed. This led him to the erroneous conclusion that a "thin permeability barrier" existed near the base of the stratum corneum (Scheuplein and Blank, 23 1971). Mackee gt gt. (1945) postulated that the stratum lucidum was the "thin barrier" to both ions and uncharged molecules. Spearman (1970), and Elias and Friend (1975) support the "thin barrier" hypothesis. Further studies by Blank and Gould (1962), Fredricksson (1962) and Matoltsy gt gt. (1962) present convincing arguments that the corneum is nearly uniformly permeable to water, and that its entire thickness provides the "permeability barrier". Adult human skin contains a large quantity (about 9 liters) of water (Skelton, 1927). Blank (1952) calculated that the epidermis alone was able to absorb up to six times its weight when fully hydrated. Buettner (1959a) also demonstrated that the skin was hydroscopic and could either take up or lose water depending on the environmental conditions. He also proposed the existence of an energy dependent process which could cause the movement of water from the epidermis into the general circulation. Adams (1966) presented data which suggest the reabsorption of sweat water from the epidermis. No direct evidence of an active transport process for water has been presented (Scheuplein and Blank, 1971). Laden and Spitzer (1967) summarized the factors which influence the hydration of the stratum corneum as follows: 1) the rate at which water reaches the stratum corneum from underlying tissues, 2) the rate at which water leaves the skin surface, 3) the ability of the corneum to hold water. 24 Water is brought to the corneum from underlying tissue by diffusion and through sweat gland activity. Diffusion into the corneum from the well hydrated dermis appears to be limited only by the permeability of the corneum itself (Laden and Spitzer, 1967). Sweat gland secretion brings a substantial amount of water to the upper skin strata. Experiments in cats (Adams, 1966) and raccoons (Steinmetz gt gt., 1977), in which sweat gland activity was precisely controlled by electrical stimulation of the sweat gland nerves and skin surface evaporative water loss rate was continuously measured, indicate a 5 to 10 fold increase in evaporation rate when the sweat glands were maximally stimulated. Sweat water can enter the epidermis either by absorption at the skin surface, or by lateral diffusion from the distal sweat gland tubule. Adams (1966) proposed a micro-circulation of water at low levels of sweat secretion as water diffusing laterally from the sweat ducts was reabsorbed across the dermal—epidermal border. The rate of water loss across the skin surface depends in part on environmental conditions. Buettner (1959a) demonstrated that skin absorbed water when exposed to solutions of low osmolarity or to air at relative humidities above 90%. Goodman and Wolf (1969) examined the effect of ambient relative humidity on water loss from the stratum corneum. They noted a paradoxical increase in evaporation rate when the relative humidity was raised from 2% to 50%. Further increases in ambient relative humidity caused the 25 expected decrease in evaporation as the gradient between air and skin was reduced. Grice gt gt. (1972) investigated this phenomena further and concluded that the permeability of the epidermis varied with its water content. There has been a great deal of discussion on the nature of substances in the skin responsible for its being able to hold water. Scheuplein and Morgan (1967) identified two "water pools" in the skin, which they identified as "free" and "bound" water. According to their data, "free" water accounts for about 20% of the total water stored. It is depleted in the first few minutes during exposure to a severely dehydrating environment. "Bound" water comprises 80% of skin water, and is removed slowly over a period of hours or days during dehydration. The ability of the corneum to hold water is impaired by its treatment with lipid solvents (Blank, 1953) or detergents (Blank and Shappirio, 1955). Blank (1953) showed that no change in water binding capacity occurred if the skin was exposed to lipid solvents or to water alone. If the skin was extracted with water after exposure to the lipid solvents, however, a significant reduction in its water binding capacity was noted. He concluded that the reduction in water binding capacity was due to the extraction of water soluble substances which are held in the skin by lipid-containing semipermeable membranes. Many of these hygroscopic substances have been identified. They include a mixture of amino acids, organic acids, urea and inorganic ions 26 (Bolliger and Gross, 1954; Laden, 1954). Sodium lactate (Fox gt gt., 1962) and 2—pyrrolidone-5—carboxylic acid (Laden and Spitzer, 1967) appear to be the most important of them (Middleton, 1968). Middleton (1968) showed that a disruption of cell membranes in the stratum corneum had the same effect as extraction with lipid solvents. His data are interpreted to show that either lipid extracted or membrane disrupted skin tissue retained about 59% of its water binding capacity. Park and Baddiel (1972b) suggested that intracellular and cell membrane proteins might account for the remaining water binding capacity. HYDRATION EFFECTS ON SKIN THERMAL PROPERTIES It has been well established that the thermal energy transfer properties of skin are related to its water content. After reviewing the literature reporting thermal conductivity coefficients (k) for skin and other tissue, Tregar (1966) speculated that heat conduction in skin was primarily through water molecules. Using data from the footpads of arctic canines (Henshaw gt gt., 1972), Holmes (1972) calculated k values and concluded that the k of wet skin was more than double that for dry skin. Holmes (1972,1975) investigated the effect of hydration on thermal conductivity of intact cat footpad epidermis through the simultaneous measurement of unidirectional heat flow and temperature gradient. Epidermal hydration was varied either 27 by electrical stimulation of nerves innervating sweat glands, or by exposure to a high ambient relative humidity. These data show conclusively the direct relationship between epidermal water content and thermal conductivity. HYDRATION EFFECTS ON SKIN ELECTRICAL PHENOMENA The effect of hydration on skin electrical properties has been demonstrated. Electrical properties of the skin are usually indexed either as its endogenous, transcutaneous electrical potential, or as some measure of its resistance to electrical current flow. The latter may be expressed in terms of resistance, conductance or impedance depending on the measurement technique (Steinmetz and Adams, 1980). The methodology, terminology and physiological basis of these electrical phenomena have been reviewed by Edelberg (1971) and Venables and Christie (1973). An inverse relationship between sweat gland activity and skin electrical resistance was first demonstrated by Thomas and Korr (1957). A similar relationship was observed by Lloyd (1959a,l959b) between sweat gland activity and the electrical impedance of skin. Adams and Vaughan (1965) reported a strong correlation between skin electrical resistance and skin surface evaporative water loss when they were measured simultaneously in humans either resting quietly or engaged in provocative conversation. The implication of these data is that observed changes in 28 epidermal electrical characteristics are due either directly to the activity of sweat glands, or are a result of the consequent skin hydration. In studies on the cat foot pad model, Stombaugh and Adams (1971) noted an exponential decline in both skin electrical potential (SEP) and skin electrical conductance (SEC) during progressive dehydration of a previously wetted skin. These investigators also recorded the amplitude and time course of transient changes in SEP and SEC evoked by a single stimulus to the sweat gland nerves at various times during progressive dehydration. A similar relationship between these electrical phenomena and epidermal hydration was observed by Adams gt gt. (1980) during progressive skin wetting. In addition these investigators noted changes in SEP and SEC baselines and evoked responses when the ion content of the epidermis was altered either by allowing salt deposited in previous sweating bouts to accumulate, or by exposing the epidermis to solutions of varying osmolarity. The conclusion reached from these studies is that both the water and ion content of the epidermis are important determinants of steady state and transient skin electrical characteristics (Steinmetz and Adams, 1981). HYDRATION EFFECTS ON SKIN MECHANICAL PROPERTIES Measurements of coefficients of friction between various materials and the skin surface have been used as indices of 29 skin mechanical properties. Naylor (1955) determined coefficients of friction between the tip of a moving plastic rod and intact human skin. His studies show that skin moistened with "trace" amounts of water or by moderate sweating has a higher coefficient of friction than either dry or thoroughly wetted skin. Similar results were obtained by Adams and Hunter (1969), using a plexiglass plate drawn across the central footpad skin of the cat. In this latter study, epidermal hydration was varied by controlled electrical stimulation of sweat gland nerves. In sequential experiments in the same footpads, these investigators observed and measured sweat gland activity while activating the peripheral nerves with the same stimulation parameters. Their studies demonstrate that the increase in friction measured at the moist skin surface and the subsequent decrease with further hydration both occur before liquid sweat reaches the skin surface. Comaish and Bottoms (1971) and Highley gt gt. (1977) demonstrated hydration effects similar to those of Naylor (1955) and Adams and Hunter (1971). Epidermal hydration effects on skin mechanical properties have also been indexed by evaluation of stress-strain relationships. Blank (1952) first showed that the "pliability" of excised skin was directly related to its water content. By measuring the response of excised stratum corneum strips, Laden and Morrow (1970) determined that the relative "softness" and "flexibility" of the stratum corneum 30 were directly related to ambient relative humidity. Strips of corneum which were lipid solvent-water extracted exhibited a drastically reduced "flexibility", and were plasticized less by exposure to water vapor. Wildnauer gt gt. (1971) measured the strain (elongation) of excised stratum corneum strips uniaxially loaded, and the maximum stress (force) before fracture. They showed that the stress at any given strain is greater at high ambient relative humidities than at low ones. They observed that the stratum corneum cells were "fibrous protein bundles" covered with lipids. When the corneum was stretched to the breaking point, it fractured intercellularly rather than intracellularly. They concluded that water decreases interfibrillar protein interaction, lessening cohesion among corneal cells. Park and Baddiel (l972a,l972b) examined the rheology of excised stratum corneum strips in uniaxial loading experiments. They showed that the elastic modulus (slope of the stress—strain curve) is inversely related to the ambient relative humidity in a manner predicted by models based on data from other keratinous substrates and polymers. They proposed that the increase in elasticity which accompanies lipid solvent-water extraction is due to increased "protein—protein" interaction. Christensen gt gt. (1977) devised a method for measuring uniaxially loaded stress-strain relationships of the intact human stratum corneum. Using sinusoidal stimuli they demonstrated rapid 31 and pronounced changes when moist air was blown across the skin. They interpreted their data to indicate that hydration influences the mechanical properties of intact stratum corneum. There have been many other methods of indexing skin mechanical properties tg_gtttg. Lanier and Fung (l974a,1974b) developed a computer controlled device for measuring stress-strain relationships in biaxially loaded, excised skin samples. Their data have contributed to our understanding of the nonlinearity and directional anisotropicity of skin. They have demonstrated the importance of temperature and pre-loading conditions on experimental results. Many methods have been devised recently for measuring the mechanical properties of intact human skin. These techniques include methods in which the skin is loaded uniaxially (Burlin gt gt., 1977), torsionally (Barbenel and Evens, 1977), by vertical extension (Pierard and Lapiere, 1977), suction (Cook gt gt., 1977), ballistometry (Tosti gt gt., 1977) or compression (Daley and Odland, 1979). Although the hydration state of human skin is difficult to control in these human experiments, it is recognized by these investigators as an important consideration. 32 GLABROUS SKIN MECHANORECEPTORS Mechanoreceptors in the glabrous tactile skin of cats and primates appear to be similar in both structure and function among species (Burgess and Perl, 1973; Mountcastle, 1980). Early anatomists identified four types of receptors in the glabrous skin, which are: l) bare nerve endings, 2) Merkel cells, 3) Meissner corpuscles, and 4) Pacinian corpuscles. The functional characteristics of each of these receptors have been determined by recording nerve cell activity from primary afferent fibers, while delivering controlled stimuli to the skin surface. The bare nerve endings are distributed throughout the dermis and in the lower layers of the epidermis (Andres and During, 1973). These endings subserve the sensory modalities of pain and temperature, and are associated with small diameter myelinated (A-delta) and unmyelinated (C) primary afferent fibers (Mountcastle, 1980). Some nerve fibers of this size are associated with mechanoreceptors in hairy skin, but they do not appear to be important for mechanoreception in glabrous skin (Burgess and Perl, 1973; Mountcastle, 1980). The Merkel cell receptors were first described by F. Merkel in 1875, as an example of an "epithelial cell-neurite" complex (Munger, 1977; Gottschaldt and Vahle-Hinz, 1981). The structure and function of these 33 receptors has been characterized in the opossum snout by Munger (1965) and in the Cat by 1990 and Muir (1969) and Janig (1971). The Merkel cell is a specialized epithelial cell of the stratum basale, which is in intimate contact with other epidermal cells through tonofilament and desmosomal attachment (Andres and During, 1973: Munger, 1977). These cells contain secretory granules and are generally assumed to be the mechanoreceptive element (Munger, 1977). The Merkel cells make contact with specialized structures (Merkel Disks) on the primary afferent nerve ending. There is an abundance of secretory granules both in the Merkel cell and in the neuron at these sites which suggests bidirectional information transmittal at this synapse (Andres and During, 1973). Recent data by Gottschaldt and Vehle-Hinz (1981) suggest that the primary afferent ending may be the mechanoreceptor, while the Merkel cell serves as an abutment for the nerve ending. Although the transduction process is still uncertain, there is little disagreement concerning its functional properties. This receptor is generally considered to be a slowly adapting mechanoreceptor, whose function is to detect both velocity and position. (1990 and Muir, 1969; Burgess and Perl, 1973; Munger, 1977; Mountcastle, 1980; Gottschaldt and Vehle—Hinz, 1981). Stimulation of these receptors gives rise to the sensation of "touch-pressure" (Mountcastle, 1980). 34 Meissner corpuscles were identified in cat glabrous skin by Malinovsky (1966). These receptors like the Pacinian corpuscles are distinguished morphologically by concentric rings of lamellar cells which surround the nerve ending (Andres and During, 1973). lIn glabrous skin, these receptors are located in the dermis, just below the dermal—epidermal border. They are activated by small displacements of the epidermis which are transferred through the tonofilaments to dermal collagen fibers, which enter the upper portion of the receptor corpuscle. In contrast, displacements of the deeper dermis do not activate these receptors, because the collagen fibers of the lower dermal layers are not continuous with the lamellae of the receptor (Andres and During, 1973). Janig (1971) classified these receptors as rapidly adapting mechanoreceptors. They respond best to low frequency (30-40 Hz) sinusoids, and their stimulation evokes the sensation of contact and flutter (Mountcastle, 1980). Pacinian corpuscles are found in the deeper dermis and subcutaneous tissue in glabrous skin (Mountcastle, 1980). The structure and function of these mechanoreceptors has been described in detail by Loewenstein (1971). Like the Meissner corpuscle, these receptors consist of a nerve ending surrounded by concentric layers of lamellae (Andres and During, 1973). Pacinian corpuscles are also classified as rapidly adapting mechanoreceptors; they are most sensitive to high frequency (250-300 Hz) sinusoids. 35 Activation of these receptors evokes the sensation of contact and vibration (Mountcastle, 1980). Loewenstein (1971) has shown that it is the viscoelastic properties of the concentric lamellae which gives the corpuscular nerve endings their characteristic response. Because of their larger size and deeper location, the cutaneous receptive fields of the Pacinian corpuscles are much larger than those of the Meissner corpuscles and Merkel cells. All three of these receptor types are associated with large diameter (A—alpha and A-delta) nerve fibers (Eyzaguirre and Fidone, 1975; Mountcastle, 1980). THE MECHANORECEPTIVE PATHWAY The cell bodies of the primary afferents are located in the dorsal root ganglia (Eyzaguirre and Fidone, 1975). After entering the spinal cord, the axons of the primary afferents divide, sending collateral branches to several locations within the spinal cord. Some collateral branches of the primary afferent axons enter ascending spinal pathways, while others synapse with spinal neurons. Rethelyi and Szentagothai (1973) have reviewed the literature describing the spinal ramifications of primary afferents. Perl and his co-workers more recently have described the location and morphology of the spinal terminations of functionally identified primary afferents, with special emphasis on A-delta and C fiber 36 mechanoreceptors and nociceptors (Rethelyi gt gt., 1979; Light and Perl, 1979a, 1979b; Light gt gt., 1979). Rethelyi gt gt. (1979) demonstrated that some branches of the large diameter mechanoreceptive fibers terminated in the nucleus proprius (lamina III) of the spinal cord. Second order neurons which arise from this nucleus may either synapse with other neurons in the spinal cord, or project to the brain in any one of several parallel pathways. Other collateral branches of the primary afferents project directly to the brain in the dorsal columns of the spinal cord. Fibers are added to this tract in a highly organized fashion, so that a complete representation of the body surface can be mapped onto the dorsal columns at the base of the brain (Mountcastle, 1980). Brown (1973) reviewed the literature concerning the topographical organization and fiber content of the dorsal columnns. He concludes that: 1) the dorsal columns are the most direct and fastest ascending mechanoreception pathway, which relay information for fine tactile discrimination, 2) not all fibers which enter the dorsal columns terminate in the same location in the brain, and 3) some fibers other than the large diameter ones from mechanoreceptors also travel in this pathway. Wall and Dubner (1972) note that lesions of the dorsal columns do not always cause the sensory deficit that would be predicted on the basis of the fiber content of this tract. They suggest the possiblity of redundancy in the ascending projections which relay fine 37 tactile information. The small diameter mechanoreceptive fibers from skin project to the brain in several parallel pathways (anterolateral system), but few of these fibers originate in glabrous skin. The role of these small diameter fibers in fine tactile discrimination appears to be minimal (Burgess and Perl, 1973), but it may serve as the redundant pathway suggested by Wall and Dubner (1972). The fibers of the dorsal column pathway terminate in the dorsal column nuclei (nucleus gracilis and nucleus cuneatus) which are located in the caudal medulla. Johnson gt gt. (1968) demonstrated that the cells of the dorsal column nuclei are arranged in an accurate somatotopic fashion. The cells of these nuclei have a high degree of modality specificity which closely mimics the discharge patterns of the primary afferents (Eyzaguirre and Fidone, 1975). Although the modality specificity is preserved at this synapse, cells in the dorsal column nuclei have larger receptive fields than do primary afferents. It has also been observed that stimulation of a single primary afferent can result in the activation of several dorsal column nuclear cells. This suggests that some degree of both convergence and divergence of primary afferent nerve endings occur at this level (Eyzaguirre and Fidone, 1975). Mountcastle (1980) cautions that these nuclei should be considered as integrating centers, rather than simply as relay nuclei. 38 The axons of the second order neurons decussate in the medulla and project in the medial lemniscus to the ventrobasal complex of the thalamus. This complex consists of two groups of cell bodies, the ventroposterolateral and ventroposteromedial nuclei. Welker and Johnson (1965) demonstrated that the third order neurons of these nuclei exhibit a complete somatotopic representation of the body. These third order neurons also retain the same functional properties as the primary afferents (Mountcastle, 1980). In addition to receiving input from the medial lemniscus, the thalamic nuclei receive projections from the anterolateral system and possibly from the contralateral lemniscal system (Ruch gt_gt., 1965; Eyzaguirre and Fidone, 1975; Mountcastle, 1980). Axons from the third order neurons in the thalamus project to the cortex through the internal capsule. As in the other portions of this ascending system, the axons of the third order neurons remain highly organized, so the somatotopic representation remains preserved (Mountcastle, 1980). THE PRIMARY SOMATOSENSORY CORTEX The somatotopic organization of the somatosensory cortex was first suggested by Bard (1938). Complete somatotopic maps of the body were first described in the 1940's by Woolsey, and have since been described for many species (Dykes, 1980). These representations consist of 39 two-dimensional "sheets" of cortical neurons which are activated by small deformations of the skin or deeper body tissues (Jones and Powell, 1973). The cortical maps are distorted because the area devoted to input from a body region is proportional to the tactile acuity (Weinstein, 1968) or the number of fibers innervating that region (Mountcastle, 1980), rather than the surface area. Measurements of the response latency suggest that cutaneous mechanoreceptors activate the cortical neurons through a fast conducting pathway with little synaptic delay (Dykes, 1980). This and other anatomical data implicate the dorsal column-medial lemniscus as the major input pathway (Jones and Powell, 1973). In addition to the primary somatosensory cortex (SI), a complete representation of the body has been located in a secondary (SII) cortical receiving area in many species. In general, the SII area is located posterior and ventral to SI in the parietal lobe (Werner and Whitsel, 1973; Dykes, 1978; Mountcastle, 1980). The somatotopic maps in SII differ from SI, in that either ipsilateral or contralateral stimuli will evoke SII cortical responses (Mountcastle, 1980). Mountcastle (1957) showed that an electrode penetration normal to the cortical surface would encounter nerve cells of the same functional type (ie. rapidly or slowly adapting) and with the same peripheral receptive field. He described this organization as "columnar". Mountcastle (1957) also noted that different functional columns were arranged in a 40 mosaic, implying a mixing of functionally different columns within the same body representation. Recent data by Dykes gt gt. (1980) suggest that at least three separate representations of the body occur in SI, each with different functional properties. One set of neurons receives inputs exclusively from "deep" receptors (joint and muscle), another set receives input from slowly adapting ”cutaneous" receptors, and the third set is driven by rapidly adapting "cutaneous“ receptors. The cytoarchitecture of SI is not uniform. Four different architectonic zones have been identified. Dykes ‘gt gt. (1980) have shown that the "deep" body representation corresponds with one of these zones, while the two ”cutaneous" representations overlap somewhat within one of the other zones. He proposes that closer examination will reveal at least two more complete representations within SI, each with unique functional properties. Dykes concludes that, "the concept of modality segregation for separate but parallel processing of afferent input is in fact a major principle of cortical organization" (Dykes gt gt., 1980). MATERIALS AND METHODS SURGERY AND EXPERIMENTAL PREPARATION Adult cats (Felis catus) of either sex, weighing between 2.4 and 6.5 kg were anesthetized by intravenous injection of sodium pentobarbital (25 mg.kg-1; Abbott Laboratories, Chicago, IL) into the cephalic vein and intubated with a tracheal cannula (4.0-5.5 mm ID.; American Hospital Supply, McGraw Park, IL). Cannulae (PE-90; Clay-Adams, Parsippany, NY) were inserted into the femoral artery and vein and advanced approximately 7 cm into the abdominal aorta and inferior vena cava, respectively. Systemic arterial pressure was monitored by means of a blood pressure transducer (Statham model P23Dc; Grass Instruments, Quincy, MA) and a low level DC preamplifier (model 5P1; Grass Instruments, Quincy, MA) and displayed continuously on a calibrated strip chart recorder (model 7100B; H—P Mosley, Pasadena, CA). Body fluids were supplemented with a slow saline (5% dextrose in lactated Ringer's solution; Cutter Medical Laboratories, Berkley, CA) drip infusion into the venous cannula. Body temperature was monitored by means of a rectal thermistor probe inserted approximatly 10 cm into the lower bowel and connected to a calibrated resistance 41 42 bridge and temperature scale (model 44TD; Yellow Springs Instruments, Yellow Springs, OH). A heating pad (lZOVAC/6A; General Electric, Cleveland, OH) placed under the animal was used to maintain body temperature between 38.5 and 39.5°C. A diagram of the experimental preparation is shown in figure 1. ANESTHESIA SUPPLEMENTATION Anesthesia was supplemented by intravenous injections of sodium pentobarbital (1.25-2.50 mgokg-l) at approximately one hour intervals. The depth of anesthesia was monitored through periodic evaluations of spontaneous cortical nerve cell activity, respiratory frequency, systemic arterial pressure, and palpebral, corneal and paw pinch reflexes. In order to standardize the effect of pentobarbital on evoked nerve cell activity, each animal was given an intravenous injection of 1.25 mg-kg-1 ten minutes prior to each footpad stimulation sequence, but not supplemented during the subsequent 45 min. testing period. PREPARATION FOR CORTICAL RECORDING The cat was placed in a laterally recumbent position on its right side, with its head fixed in a stereotaxic frame (model 1204; David Kopf Instruments, Tujunga, CA). A 5 to 8 cm midline incision was made in the scalp and the wound 43 .xwuuoo Hounmumo m.HmEficm on» souu wows mums mocflcuoomu HmowooHoflm>no|0uuumHm can coumHsEfium mm: :me msounmam on» oaflcz uuommsm anewcmcooe o no momuusm cocoon on» unawmmm cmcMmuumou mm: 3mm unmwn mum .Acsonm uocv wsoum odxmuomuoum 6 ca cam: mm: coon m.Hmecm one .ousumuomEou moon mu“ samucflms o» Assocm uoc. pom madame; poaaouucoo o co cmumou umo pmswuonummcm one .cOAumummoum HmucmEfiuomxm uo Emummfln .H musmfim .732» W3 1:531.>; .xswmz< motzo: \ dam» 5m .322.»m .2<10m2 motzo: ism» om ozEcoomm 3-rE-$:-$:3:%-5o:€1$5c§:;:-43-35:-:-5=:3§!E£E$§;:3:435:637'525 flzfiéf,’ 6:33;” '53 .-.~.-:-.-:~:'-.°~:-:-.:-. '- :~.'=:=:,,-’I§§§:'§5$: " \- . ....- Figure 3. This sketch of the microelectrode for recording unit activity is not drawn to scale. Electrodes varied between S and 8 cm in length with uninsulated tips 10-50u in length, and tapering from 10—25u in diameter. 48 crimped at the butt end tight enough to insure good electrical connection. About 0.5 cm of the exposed tungsten wire was etched electrolytically in a saturated sodium nitrite (Mallinkrodt Inc., St Louis, MO) solution at about 20VAC (Powerstat; Superior Electric, Bristol, CT) to produce a stubby but sharp tip approximately 50u in diameter. Electrode tips were tapered further either by etching at lOVAC for 50 sec. or by repeatedly dipping the electrode in and out of the solution at 30VAC until the desired shape was obtained (figure 3). The possibility of sodium nitrite contamination of the electrode tip was eliminated by successive washings with deionized water, acetone, ethanol and ether. The metal microelectrode was electrically insulated by drawing molten glass tubing over it using a vertical micropipet puller (model 7008; David Kopf Instruments, Tujunga, CA). This was accomplished in the following series of steps. First the electrode was inserted butt end down into a 6.0 cm length of 2 mm OD glass tubing which had been heat sealed at its lower end. The electode was postioned so that the middle of the stainless steel tubing was in the center of the heating coil. Allowing the lower clamp to drop about 1 cm while heating the glass tubing (24A) crimped the glass tightly around the steel tubing. The electrode was then inverted and positioned in the clamp with the tip 1 cm below the heating coil and then pulled again at a heater current of 24A, until the diameter of the glass tubing was 49 reduced about 50%. The electrode tip was lowered to a position slightly below the heating coil, and then the lower clamp was allowed to drop very slowly with a 20A heater current, drawing the molten glass over the electrode tip. In properly insulated electrodes, the glass was bound to the metal electrode along its entire length and covered the tip. The glass insulation was then removed from the very tip of the electrode, by etching in concentrated hydrofluoric acid (Mallinckrodt Inc., ST Louis, MO) under mineral oil (Fisher Scientific, Pittsburgh, PA). The acid-oil interface was viewed through a dissecting microscope (Stereozoom 7; Bauch and Lomb, Rochester, NY) in order to monitor the etching process. Electrodes produced in this fashion for extracellular recording of activity of small clusters of nerve cells, characteristically had conical tips 14.0 to 16.5u in diameter and 35.0 to 45.0u in height. A flexible wire lead with a gold pin connector was soldered to the butt end of the electrode after breaking off about 5 mm of the glass insulation. Electrodes chosen for use were radio-frequency shielded by securing them inside 12 gauge stainless steel tubing (Laboratory Accessories Inc.; Millburn, NJ) with heat shrinkable tubing (Newark Electronics, Milwaukee, WI). 50 CORTICAL RECORDINGS A block diagram of the recording system is shown in figure 4. Microelectrode signals in reference to an indifferent electrode clipped to the skin were amplified with a differential input, AC preamplifer (type 122; Tektronix Inc., Beaverton, OR) having an approximate gain of 1000 and lower and upper filter cut off frequencies set at 0.8 Hz. and 1.0 KHz respectively. The preamplifier output was filtered with a 60 Hz. notch filter and further amplified by the circuit diagramed in figure 5. The gain of this amplifier was adjusted to give a maximum output of about 2.5 volts. Voltage spikes were monitored on a dual beam storage oscilloscope (model 564; Tektronix Inc., Beaverton, OR) or recorded on magnetic tape using a 7 track, FM tape recorder (model 2000; Sanborn-Ampex, Waltham, MA). A tape speed of 3 3/4 inosec-l was used for both recording and playback, with a resulting band width of 50Hz. to 6.25KHz (13dB) and a 40dB signal to noise ratio. In addition, the amplifier output was used to drive an audio monitor (figure 5). The recording system was calibrated with lOOuV square waves generated with a DC stimulator (model 88; Grass Instruments, Quincy, MA) and a voltage divider. A representative calibration signal is shown in figure 6. 51 .Eoummm mcficuooou can coaumuHuMHmEm Hmcmwm mo Emummfic xuon .q musmfim EmomOOmm mm<._. mwx0um uouHEMH oflosm one .fi=@\u=v usmuoo omou ofluocmme no A=c05=v ugmuso uofluflamsm uODHcos Cu umcno CH conouflsm on casoo scuflcos ofiosm one .o>m3 ocflm um om 6 mo mcfiuouaflw Hmefiumo ocfl>oum ou muouoeofiucwuom =cfimm= cam =c0= .=03= on» spas cmumsmpm mums nouaflu um ow manmcouflsm ecu wo cflmm can wuflamsq mcflumuaflu .nucflspcmm .uouHCOE oflcsm can nouafiu .uoamHHmEm ommum ocooow mo Emummfic ufisouflu .m musmflm 2 53 mO.~_ZOE O_DD< mm_u__qa_2< Z_<0 m..m<_m<> .m gunman J)“ h :05 III .“ an -mos.zfi_ .oc co»<:zms»< .ao c. «N I h. Wm. xo. xoow «d 3m>w5 ox. can con o.Jm< m I I so... u I“. 7 v.0 1%: _ I I m so. T1 MH + TIY. I owou oou _ poo N- - xo.l OI. a\. II. z_ma

?. . < m Irillo xou goo. . xoo ////u,-.§$fi4l+l¢llo xoopuoaow c. . ll . m xoo _ xom ouod Q _ xoo. “ o)... I .r.. t. mmuusm Z_<0 mood \ X.» 20 mwk.:u IOPOZ NI ow >._._ZD 54 In this recording system calibration the upper trace shows tape recorder output, and the lower trace shows -100uV input signal. Calibrations: Upper trace= 0.5V/cm Lower trace= lOOuV/cm div.; Sweep: lOmsec/cm div. Figure 6. div.; 55 A shielded microelectrode, mounted in a micromanipulator (Narishige model 2138; Eric Sobotka Co., Farmingdale, NY) was positioned over probable sites for recording extracellular activity from small clusters of cortical neurons having receptive fields on the central footpad. Experience gained from preliminary experiments and data from experiments by Mountcastle (1957) and Dykes gt gt. (1980) were used to locate central footpad units. Cortical sulci and postcentral gyrus topography served as visual landmarks. Prior to covering the brain surface with the translucent agar layer, stereotaxic coordinates were determined for probable recording locations, taking care to avoid surface blood vessels. Once the agar had solidified over the brain surface, the microelectrode was slowly advanced through the gelatin layer until contact with the brain surface was verified by distinctive reductions in the noise levels displayed on the oscilloscope and broadcasted by the audio monitor. The microelectrode was then advanced in 50 to 200u steps, to a maximum depth of 1.0-2.0 cm from the brain surface, or where evoked responses were no longer recordable. After pausing at each site long enough for spontaneous activity induced by electrode movement to subside, the boundaries of the peripheral receptive field for that unit were determined by lightly stimulating the skin with a sharp tipped wooden rod, a fine paintbrush or with the mechanical stimulator set for a midscale force. After determining the locations of the receptive field and 56 considering receptive fields encountered during previous penetrations, the electrode was withdrawn and repositioned for a new penetration. A small area of the primary somatosensory cortex was systematically explored in this manner until a cortical unit was located which responded to light tactile stimulation of the glabrous skin on the central footpad. A representative neuronal response is shown in figure 7. Once a unit of this type was located, no further movements of the microelectrode were made, and the preparation was allowed to stabilize for 30 to 45 min. before data were recorded. FOOTPAD PREPARATION Fur was removed from the right foreleg and paw by clipping (model A2; Oster, Milwaukee, WI) and from the central foot pad area with a depilatory cream (Neet; Whitehall Laboratories, New York, NY). Caution was exercised so that the depilatory agent did not come in contact with the glabrous skin. The forepaw was positioned and gently restrained against a padded platform, with the plantar surface upward and parallel to the table surface as shown in figures 1 and 8. Rubber dental dam (Light-Thin; Hygienic Corporation, Akron, OH) with holes cut to expose the central glabrous footpad and toepad skin was used to restrain the paw. The limb was carefully positioned at or slightly below the level of the heart in order to keep 57 , _ ......— y-———-.-—--_--.- '7~""-'——"”"‘ Figure 7. In this recording of a cortical unit response, the upper trace shows a single biphasic cortical unit action potential with negative polarity in an upward direction, and the lower trace shows the stimulus event marker. Calibrations: Upper trace= lOOuV/cm div.; Lower trace= 1.0V/cm div.; Sweep= 20msec/cm div. 58 .000: oflummam ummao may Lo mmmco on» pcsouw uso can mos» node“ may 20:0ucu cmsoau Lam cocofiuflpcou .pouwwu on o» mcoflumooH comu00m may m>onm pocofiuflmom on Co Ewcu cmsoaam soaps Acsocm uOcv uOumHschmEOLUME m ch paw: who: wasmmmo cofiumupmc cam uOomHSEwum Hmoflcmcomz .m wusmflm ELECTRICAL INPUT MECHANICAL STIMULATOR WIRE SUPPORT 59 CLEAR PLASTIC HOOD Figure 8. 60 footpad blood flow unobstructed. Footpad temperature was monitored with a thermopile (figure 2) constructed from 36 gauge copper constantan thermocouple wire (Omega Engineering; Stamford, CT) and displayed on a calibrated strip chart recorder (model 7100B; H—P Mosley, Pasadena, CA) and served as an index of an uncompromised circulation. A footpad temperature of 34-380C was accepted as indicating that the extremity had an unimpeded circulation. MECHANICAL STIMULATION A device capable of delivering small displacement stimuli of varying force was assembled (figure 9), using the design of Brown (1974). This stimulator was constructed by cutting away the cone and support bracket from an 8 ohm: 2.25 in. transistor radio speaker (Philmore Manufacturing, Inwood, NY) leaving intact only its permanent magnet, central cylinder and support baffle. Punctate stimuli were delivered to the skin through a brass rod cemented with epoxy to the center of the speaker cylinder. The stimulating rod was tapered using a jeweler's lathe to a tip diameter of 0.8 mm The mechanical stimulator was driven with a DC stimulator (model S9; Grass Instruments, Quincy, MA) capable of producing 1.0 to 7.0VDC square waves pulses, 10 msec. in duration and at a frequency of 1 Hz. These pulses were used as input to the circuit diagramed in figure 10, which provided the current necessary to drive the 61 SUPPORT BRACKET W W/ PERMANENT MAGNET / SPEAKER CON. / SUPPORTIHAPHRAGM ELECTRICAL INPUT STIMULATING ROD Figure 9. This drawing is a cross section of the mechanical stimulator. Displacements of the stimulating rod were obtained by applying a current pulse to the electrical inputs of the speaker coil. .mCOMDMHQLHmo u0u Ha musmflu wow .=:fl uw>fiuc: um coucomoum womasm ommuao> o» mucommmu CH guso uo>fiuc= um cwcfimuno mums uOumHDEHum Hmoflcmsooe on» uOL momaso acouusu .wousow ucwuuso uOumassflum Hmoficmcoms on» mo Emummfic uwsouwo on» mzonm vauosonum mane .oH musmwm I or- ..- < .3 + swoauo h. xop m . l I or+ «o I Foo mm>_mo + II . < H 2 o xop I z.mm>_mo 6 .o_\7 I. go. so. oP+ mccrnv_z_ wmnv2¢frm_wm:u mOhw_wZo usmuso ecu uo monumocMH on» mwumuumcosmc cco .musmcfl wmouao> cwaaouucoo o» uncommon CH CH wusmfim :H cosmummflp uflsouwo on» mo usmuso ucmuuso on» wzocm musmflw mace .coflumunflaoo uo>Huc uOumassflum Hmoflcmnomz .HH ousmfim 65 .HH magmas o.o o.o oé o.o o.o 9 q q u u u q a d L o A: mo<.:o> :EE . J 1 or L .. ow . oo 2:: szmmmao ; ov 66 .A=m\ugv mam» ofiumcmme sou“ a“ wouwcoe new cocoon ou no Ascoszv mauowuflc aw uOuHcoe o» umcuo cfi cmnoufizm on canoe adsouwo was» soon usmuso .uwsouflo uoxuoe uco>o on» 0» yoga“ cmcfi>oum ca wusmflu cw czonm ufisouwo on» mcfi>fiuc wou50m wmmuao> on» no usmuso Hmaamumm .ousmww wasp cfi cwEEmumch ufisouwo ecu an popfl>0um mum: ma can b mmusmfim CH csocm mxums ucm>o msaseflum .uoxums uco>m modsEHum ocu mo Emummflc ufisouwo .NH wusmfim Q:IJI— .COE #30 NQ m.o III m.m s.v o.o o.o m.m v.m a.m v.N v.m m.N s.N N.o s.v awe o.m v.m N.m o.o III o.o w.m o.o v.m s.o N.m m.m s.m s.m m.v m.H v.m m.H o.H m.N N.N awe a.m w.o N.m o.o III v.m m.m s.h w.v m.m N.m N.m m.o s.m o.m m.H s.m w.H m.H o.N w.H awe s.m o.o N.m o.v III v.m o.m o.o s.o o.N s.m N.m m.m s.m m.N m.H s.m m.H m.H N.N m.H ems ®.m v.v v.N w.v III &.v m.N v.v v.m N.m o.o N.m w.m s.m m.N m.H m.N w.H m.H m.H w.H ave s.m o.o v.N m.v III s.w m.N o.o v.m N.m v.v N.m w.m m.N o.N m.H v.N w.H v.H ©.H m.H sme v.N v.0 c.H ®.m &.m m.m m.N s.v v.m m.N m.m N.N m.H m.N ©.N N.H N.N w.H N.H v.H v.H 8N9 &.H m.m N.H v.H N.m N.H w.H N.m &.H w.H N.m s.N m.H v.H w.H &.H s.N &.H N.H N.H ¢.H SHE m N H m N H m N H m N H m N H m N H m N H mmHmmm o m m a m N H meHm .cocommu uoc 0003 cOHcs mOHocmOLLD mumoHccH mosHm> mcmeHz .msum 000 com mmsHm> OHocmOCce .H oHnmE 91 are plotted as a function of the skin location in figure 23. The variability in response associated with the stimulus location appears to be consistent among these 3 series of stimulations, and no consistent variation in the response associated with the stimulation sequence is observed. The lack of a response from skin location 6 during the second series of stimulations was most likely the result of poor contact of the stimulator probe with the skin surface. In cats in which the epidermal hydration state was experimentally varied, the initial mechanical properties of the skin during exposure to room air, and the effect of the wet air, dry air and liquid water exposures on these mechanical properties were quite variable. Subjective evaluations of the pliability of the skin were made during each of the hydration states. Table 2 summarizes these subjective evaluations for the 6 animals which are examined in the analysis of variance. In general, skin exposed to the wet air stream appeared to be either slightly softer or not different from the room air exposed skin. In all cases, the skin exposed to dry air appeared to be less pliable than either the room air or wet air exposed skin. The effect of liquid water soaking on skin pliability was the most variable. Although in some animals the liquid water soaked skin appeared to be softer than the dry air exposed skin, the most common effect was a pronounced swelling and stiffening of the skin. 92 mo mOHuow u .AomeOmOnv mcoHunOOH cmmuoo m 00 mcHocmmucu macaw whomflu once .amooum boo 0WucmwwmnmmuhmuwwechHumHBEHum .MN munmwm 93 A w v N F q q a H J ZO_P mo Nomoom Noe owe MN.ooN mm.moN oHH mameoe III mo.o NN.oe III so.o av.ss om mommm amoonmm mo.H oo.o SN.HH oo.H No.H HN.NH NH soommom x 92mze mo Nomoom Nae ome so.HmH mm.mm mmw. mameoe III om.o mm.mo III om.o oN.oo om mommm amoonmm Hm.o oo.o eo.m Hm.o oN.o mo.m NH ooammom x ezmzommme .oa.m mo.a oo.ma NN.N NH.H oo.o a Aonemooac meoammom III me.H NH.HN III mo.o NH.SH ma mommm ozmzommme .om.o Nm.o mm.mH .mm.o om.a mm.e~ m Aonemmowms 92mzemmme mN.N oN.m Hm.oa oo.a Ho.a Ho.m m “memos mmooum m m: mm m mm mm mm onemHm<> mo momoom oNe ode .lma.ovmv moabmu m unmozoaoosm News 1.3 mxmauobmm .mchmB m>oc¢ .m OHOBB 97 me.omm om.soa mwm. maaeoe III NH.H mm.oo III em.H Ho.ooH om mommm Hmoonmm mm.o mo.H oo.NH oe.o mm.o HH.HH NH ooummom x ezmzemmme .mo.o mm.s om.om .mm.o Nm.o oH.am o Honemoosv moosmmom III mo.o Hm.oo III Hm.m Hm.mm mH mommm ezmzemmme .ss.m Ho.om SN.NHH .om.NH oo.ma om.omH m Honemmowms 92mzemmme «Hm.m oH.oH mm.os .oo.m mo.mH oN.mo m Hmemos mmooam m mm mm m m: mm mm onomHm<> mo momoom ooHe ome NN.oNo om.NNo whw mameoe III MN.H mm.mo III HH.H oo.mm om mommm HmoaHmmm so.o Nm.o om.o om.o oo.o os.s NH eoammom x ezmzemmme .os.o mm.m Hm.mm .mo.m om.m mo.sm o Honemooav meoammom III mm.m NH.om III Ho.m NH.em mH mommm 92mzemmme .om.NH oo.oa Ho.moH «Ho.oH No.om om.HmH m Honemmowmv 92mzo mo momoom owe one .H.o.0ooos m mHnme 98 treatment-location interaction, treatment means at individual skin locations or location means for individual hydration states were not compared. Table 4 summarizes treatment means, standard errors and statistical comparisons for the four hydration conditions. The histogram in figure 24 of the T50 treatment means is representative of the other threshold levels. Data in table 4 and figure 24 indicate that there is no statistical difference between the responses of room air and wet air exposed skin. Thresholds for dry air exposed and liquid water soaked skin are significantly greater than the both wet air and room air exposed skin. Thresholds for liquid water soaked and dry air exposed skin are statistically different from each other at the T30, T40, T50, and T60 levels, but higher (T70-T100) or lower (T10—T20) threshold values are not different. Skin location means, standard errors and statistical comparisons are shown in table 5. Although there is some variability in the statistical comparisons at the different threshold levels, the histogram of T50 values shown in figure 25 is representative of the general pattern. Data in table 5 and figure 25 indicate that the threshold is significantly lower in skin location 1 than in the other skin locations. Threshold values at skin locations 2-4 are not significantly different from each other. The threshold at skin location 5 is significantly greater than the thresholds at the other skin locations. 99 HCHmwv. w: co «3 mm HoN.smm.m Hom.VNm.m ANN.vom.o HoN.voH.o ooHe m3 mo 43 mm HHm.vmm.m ANN.sHm.m HAN.cqo.m loN.va.m ome m3 «a <3 mm HNm.va.a HMN.voo.m ASN.omH.m HNm.VHo.m owe m3 mo <3 mm ANm.vmo.o HHN.VHH.N HmN.Vom.N Hmm.vmm.m one m3 mo <3 mm ANN.Vo>.m ANN.me.N ANN.VHo.N HNm.vom.o owe m3 <0 <3 mm HmN.vmm.m HoH.voH.N ANN.VHN.N Hmm.vso.o ome m3 mo 43 mm AHN.me.N HHH.VNo.N HoH.seH.N HNm.me.m Noe m3 an 43 mm HoH.Voo.N AmH.Vom.H HoN.voo.N Hom.vom.m ome m: mo «3 mm HNH.VHN.N HHH.VNo.H HHH.VNH.H ANN.VHm.N oNB m: an 43 mm HoH.smm.H Hmo.voH.H Hoo.vmm.H umN.voo.N oHo ......m.... ......H.... MM“ .mwm “My .wummm ”mnwmmmmm .Hms.svmv Conuo some 800“ ucouomuHc wHucmonHcme uoc mum ocHH 060w mcu mp cwuoomuocc: memos wmone .umwanc Ou uwoon EOuw coxcmu cum memos .comHquEoo 00m .mcomuummsoo HmOHumHumum cad mcmoz ucoEuBOHB .v mHnt 99 ESE. m3 <0 <3 HucmonHcmHm uoc mum mcHH Osmm ocu >0 cmuoomuocc: memos 00029 .umoann Ou umoon EOuu coxcmu Ono memos .comHummEoo Com .mcomHummEOU HmoHumHumum pad mcomz ucoEumoue .v oHnme 100 7-0 " THRESHOLD T50 6.0 - HYDRATION CONDITION 5.0 - * 4.0 - 3.0 - T 7 2.0 - % 1.0 L- //%/ HQUW WET ROOM DRY WATER AIR AIR AIR Figure 24. Effect of hydration condition on T50 threshold. Mean values and standard errors for 4 hydration conditions are from table 4. Bars marked with an asterisk are significantly different (p<0.05) from all other hydration condition means. 101 4E .®.w Va. mm o N m H Hmm.soo.m 1mm.som.m Hom.oma.a Ham.vao.m HHo.soH.a ooHe m a N m H 1mm.va.m Hom.ch.N Hom.saa.o Ham.sma.o Hao.vmo.m Nae m a m N H Hom.vmo.a Hoa.sem.a Hom.smH.a 1mm.vso.m Hoa.ssm.m owe m a m N H Hem.vos.a Hos.smH.H Ham.voo.m 1mm.sos.m 1mm.vmo.m Nee m a m N H ASN.3NN.H Hem.som.m Hem.vom.m le.vma.m HAN.st.N Noe m a N m H Hmm.som.m Ham.smH.m Hom.VmH.m HNm.va.m HmN.VNH.N ems m m a N H Ham.st.m HoN.soH.N HNm.soN.N HmN.soo.N ANN.vao.N Nae m m N o H ASN.3mo.m ASN.3oN.N HAN.VHH.N HHN.VNN.N HNH.VNN.H one m N a m H HNm.vNN.N HEN.VNH.H HmH.sNN.H HHN.VHN.H HmH.vmo.H oNe m N a m H ASN.cmo.H HoH.VNm.H HoH.me.H HoN.som.H “No.3aN.H SHE mmozmmmmmHo H<0HomHeHucmOHchmHm uoc mum mcHH 080m msu >0 concomuocc: mcoms whose .uwmann ou ummon scum coxcmu mum mcmme .comHquEOO Com .mcomHummEOU HmoHumHumum cad mcmmz coHumooq cwa .m anmB 720 SI) SI) 41) 31) 2i) 1.0 102 F THRESHOLD T50 SKIN LOCATION \\‘ 1 2 3 4 5 Figure 25. Effect of stimulus location on T50 threshold. Mean values and standard errors for 5 skin locations are from table 5. Bars marked with an asterisk are significantly different (p<0.05) from all other skin location means. DISCUSSION EPIDERMAL PERMEABILITY A major function of skin is to minimize the loss of body water. The marked increase in evaporation rate which occurs after either partial or complete removal of skin clearly demonstrates its water barrier function. Many early studies showed that a diffusion barrier was located within skin epidermal layers. This is not surprising considering the desmosomal attachments and tight interstitial spaces among cells in these layers. The identification of a single epidermal layer serving as a water diffusion barrier, however, is unresolved. Studies in which epidermal layers were serially removed by stripping them at the skin surface demonstrated that skin surface evaporation rate changes little‘until a thin layer of cells near the base of the stratum corneum is removed (Blank, 1952). This was initially interpreted by these investigators to indicate that a "thin barrier" at the base of the stratum corneum functions as the primary impediment to water diffusion, although it was concluded later that the stratum corneum is a nearly uniform "thick barrier" to transcutaneous water loss. 103 104 Other studies show, however, that the rate of transcutaneous water loss from "nonsweating" skin is greater on the tactile surfaces of the hands and feet than it is from skin on the general body surface. This occurs despite the greater thickness and intercellular cohesion of the stratum corneum on the tactile surfaces (King gt gt., 1981). One explanation for this apparent contradiction to the "thick barrier" hypothesis is related to humans serving as experimental subjects for these studies. The distribution of eccrine sweat glands in humans is not uniform over the body surface. Kuno (1956) and others have shown a greater density of these sweat glands in skin on the tactile surfaces than in other regions of the body. Lateral diffusion of water from distal sweat gland tubules provides a mechanism for "short circuiting" the epidermal water barrier. Studies in animal models other than humans (Adams, 1966; Adams and Hunter, 1969; Steinmetz gt gt., 1977) in which sweat gland activity was precisely controlled indicate that water vapor loss from the skin surfaces is increased by electrical stimulation of sweat gland nerves. This increase in skin surface evaporation occured at levels of sweat gland stimulation not strong enough to bring liquid sweat to the skin surface, so that low levels of sweat gland activity undetectable as such at the skin surface may, nonetheless, contribute to the relatively higher skin surface evaporation rate from the tactile skin. 105 There are at least three additional considerations which make intact human skin a difficult experimental model. First, the sympathetic nervous system differentially activates sweat glands in different body regions. For example, hyperhidrosis is characterized by profuse sweating that is generally restricted to the tactile surfaces of the hands and feet. Secondly, sweat secretion is activated reflexly by both ipsilateral and contralateral stimuli. Thirdly, because sweat gland activity is difficult to control in humans, the cat footpad or some other experimental model in which eccrine sweating is more precisely regulated is more appropriate for quantitative studies of water movement and storage in skin. EPIDERMAL WATER STORAGE Because the stratum corneum is an effective barrier to transcutaneous water movement, some investigators have concluded that it absorbs little if any water. Precise measurements from skin when sweat glands are denervated, show a persistent low rate of transcutaneous water movement (Adams, 1966; Steinmetz gt gt., 1977). Buettner (1959a, 1959b, 1959c) showed that not only is the epidermis permeable to water, but also that it absorbs water readily when exposed to either liquid water or to air with a high -ambient relative humidity. Laden and Spitzer (1967) also demonstrated that the stratum corneum is hygroscopic and 106 that it can absorb both water and ions. These studies indicate that although the epidermis is an effective barrier to the loss of body water, it is not only permeable, but it is also capable of absorbing and storing significant amounts of water. The amount of water stored in the epidermis is variable and depends on environmental conditions, sweat gland activity and the rate of diffusion of water from lower skin layers. Adams proposed a hydration gradient across the epidermis, the profile of which depends on these factors and the horizontal and vertical permeability coefficients within the skin (Adams gt_gt., 1981; Steinmetz and Adams, 1981). Photoacoustic spectroscopy provides direct evidence for this hypothesis. Using this technique, Pines and Cunningham (1981) showed a variable water content across the thickness of the intact stratum corneum. They also showed that the amount of water stored in the skin is a nonlinear function of the distance from the skin surface, and that the shape of this curve depends on the total amount of water in the epidermis. These investigators concluded that normal and delipidized epidermis are "vastly different" in their ability to store water. They also concluded that "the uppermost skin layers are more sensitive to changes in water content" than are the lower skin layers, and therefore are "very important in the clinical conditions characterized by dry skin." 107 It is a common observation that when hands are immersed in warm water containing a detergent, there is a pronounced wrinkling of the skin, particularly of that of the fingers. This phenomenon is the result of cutaneous water absorption and a concomitant swelling of the epidermis (Bull and Henry, 1977). Skin wrinkling under these conditions appears to be related to the activity of sympathetic nerves innervating the hands (Lewis and Pickering, 1935). Patients suffering from cystic fibrosis or hyperhidrosis show a propensity for such skin wrinkling (Braham gt gt., 1979; Bull and Henry, 1977; Moynahan, 1974). Both of these conditions are also characterized by production of large volumes of hypertonic sweat. Peripheral nerve lesions, sympathectomy and peripheral nerve blockade with local anesthetics prevent wrinkling in patients suffering from these disorders which may provide a simple test of peripheral nerve integrity (Braham gt gt., 1979, Bull and Henry, 1977). One explanation for this phenomenon is that the increased turgor of the dermis owing to the peripheral vasodilation which accompanies sympathetic nervous system lesions may prevent swelling of the epidermis. An alternate explanation is that ions deposited in the epidermis as a result of normal or hyperactive sweat secretion increase water absorbtion by the epidermis. This wrinkling is prevented by either sympathectomy or peripheral nerve lesions which preclude sweat gland function and thereby 108 decrease skin osmolarity. These explanations are not mutually exclusive and both mechanisms may be involved. There is substantial evidence that the epidermis is capable of varying its hydration. The factors which influence the epidermal hydration state have been identified as being related to environmental conditions, diffusion of water from the dermis and sweat gland activity. Adams (1966) proposed a mechanism whereby water and salts deposited in the epidermis may be reabsorbed across the dermo—epidermal junction. Although several lines of indirect evidence support such a mechanism, there have been no attempts to demonstrate directly such an active process in mammalian skin. The epidermal hydration state of normal skin remains relatively constant except during extreme changes in environmental conditions or sympathetic nervous system activity. Sweat gland secretion rates and the ionic content of sweat are undoubtedly important factors in the control of epidermal hydration. Because of a paucity of related information, feedback mechanisms remain speculative. Several investigators (Janig and Spilok, 1978; Janig and Rath, 1980) have demonstrated reflex activation of sweat glands when mechanoreceptors, thermoreceptors or nociceptors are stimulated which may be important in the normal control of skin hydration. Specific receptors for sensing skin hydration have not been described. Bare nerve endings which 109 penetrate the epidermis and for which no known function has been ascribed might be involved. EPIDERMAL HYDRATION AND SKIN THERMAL PROPERTIES Holmes (Holmes, 1972; Holmes and Adams, 1975) showed that the thermal conductivity of the epidermis increases 2-3 fold with an increase in epidermal water content due either to sweat gland activity, or exposure to a high ambient relative humidity. These authors suggested that the temperature at the dermo-epidermal junction where skin thermoreceptors are located is closer to environmental temperature when the skin is hydrated. Holmes proposed also that this may account at least in part for differences in thermal perception in environments with different relative humidities. Although trained human observers are incapable of detecting changes in temperature of 6-7 0C if they occur slowly over a 20-30 min period, they are able to identify correctly the larger of two temperature changes which differ by as little as 0.05 °C if they occur within a few seconds (Darian-Smith and Johnson, 1977). These rapid changes are similar to those one would expect were the skin to come in contact with a warm metallic block, for example. It appears that epidermal hydration state is an important variable in thermal sensibility. Stoll and co-workers (Stoll, 1977; Stoll 35 31., 1981) used the heat transfer properties of the epidermis as 110 an indirect method for measuring epidermal thickness. They showed that the time to reach a thermal pain threshold after the skin touched a hot object is related to the thickness of the epidermis, as well as to the temperature and heat transfer properties of the object itself. Considering the influence of hydration on epidermal thermal conduction, the water content of the skin may be an important consideration in these and other measures which depend on skin thermal transfer properties. Perceived differences in skin temperature can be used as an important clinical sign in palpatory physical diagnosis. Both absolute and bilateral differences in regional skin temperature are used by some physicians as clinical indices. Adams 35 El° (1982) recently discussed the interaction of variables which might influence a physician's temperature perception in such an examination. Although these tests are important and useful diagnostic aids, the epidermal hydration states of both the patient and the examiner are important variables, and the results from such examinations require cautious interpretation. EPIDERMAL HYDRATION AND SKIN ELECTRICAL PROPERTIES The endogenous transcutaneous DC electrical potential and measures of resistance to electrical current flow in the skin are commonly used as indices of sweat gland activity 111 and sympathetic nervous system tone (Janig and Spilok, 1978; Janig and Rath, 1980). For example, a change in skin electrical resistance is the basis of the commonly used so called "lie detector" examination. Despite their common use, the underlying physiological mechanisms for these electrical phenomena are poorly understood. Epidermal hydration and the ionic content of the skin have been shown by Adams and co-workers to be important variables in all measures of skin electrical properties (Adams and Vaughan, 1965; Stombaugh and Adams, 1971; Adams 35 31., 1980; Steinmetz and Adams, 1981). Presumably these factors influence skin electical properties through alterations in the electrical volume conductor characteristics of the epidermis. The measurement of biopotentials from the skin surface is important in examination of the electrical activity of both the heart (EKG) and brain (EEG). In both of these skin surface measures of internal electrical events, artifacts associated with the attachment and movement of electrodes, as well as changes in skin electrical properties may restrict the usefulness of these techniques, although some of these problems currently are minimized through the use of special electrodes, electrode pastes and electrode attachment techniques. A better understanding of the physical and physiological basis for skin electrical phenomena may provide new diagnostic techniques for evaluating sweat gland activity, skin blood flow and sympathetic nervous system activity. In addition, a better 112 understanding could result in the reduction of artifacts associated with the measurement of biopotentials from the skin surface, and might extend the usefulness of these techniques. The cat footpad model may be a more appropriate model for quantitative studies of skin electrical properties than human skin in which sweat gland activity and peripheral circulation are difficult to control. EPIDERMAL HYDRATION AND SKIN MECHANICAL PROPERTIES The importance of hydration in the evaluation of the mechanical properties of whole skin (dermis and epidermis) is well documented. The "pliability" of excised whole skin samples is directly related to its water content (Blank, 1952; Lanier and Fung, 1974a, 1974b). Most of the effect of hydration on skin mechanical properties has been attributed to changes in the dermis. The importance of the epidermis in this regard has been disputed. Laden and Morrow (1970) and Park and Baddiel (1972a, 1972b) showed that the "softness" and "flexibility" of samples of excised stratum corneum are also directly related to their water content. Christensen §t_al. (1977) demonstrated rapid and significant changes in the mechanical properties of intact human skin when moist air was blown across its surface. Because of the methods used in these tests and the rapid time course of the hydration effects, these investigators attributed the observed effects to an 113 alteration of epidermal mechanical properties especially involving the stratum corneum. The relative contributions of the dermis and the epidermis to the overall mechanical properties of intact skin is unclear. This is largely because of the wide range of methodological differences used to evaluate skin mechanical properties. For this reason, there is now a major effort to establish standard nomenclature, techniques and procedures for evaluating the mechanical properties of intact skin (Payne 33 31., 1981). The hydration state of the epidermis appears to be more labile than that of the dermis. Hydration has been shown to have significant effects on the mechanical properties of both excised and intact epidermis. For these reasons, several investigators have recently concluded that epidermal mechanical properties are important to consider when evaluating either overall skin mechanical properties, or pathological conditions which are characterized by dry skin (Christensen 33 31., 1977; Pines and Cunningham, 1981; Steinmetz and Adams, 1981). The mechanism for hydration—induced effects on skin mechanical properties appears to be related to hydrogen bonding within and among protein filaments. Park and Baddiel (l972a, 1972b) showed that the decrease in elasticity which occurs in hydrated samples of excised stratum corneum follows patterns predicted by experiments in other keratinous substrates and polymer models. They concluded 114 that the decrease in elasticity which accompanies increased water content is owed to decreased protein-protein interaction. In dry skin, intrafibrillar hydrogen bonding produces tightly coiled three-dimensional protein configurations and extensive interfibrillar cross-linking. As the corneum is hydrated, the three-dimensional structure of proteins relaxes and interaction between adjacent protein fibers decreases which allows stretching and sliding of protein filaments (decreased elasticity) within the skin. Takahashi 3E 31. (1981) demonstrated that in human skin, the most drastic decreases in elasticity occur when the skin in equilibrated at relative humidities above 60 percent. Their results also indicate that it is at this relative humidity that "free“ or "loosely bound" water increases in the skin. These same investigators have shown that substances such as urea and lithium bromide which are known to interfere with hydrogen bonding in proteins, produce a plasticizing effect in skin similar to that produced by water. In the present study, both the initial mechanical properties of the cat central footpad skin and those induced by hydration were quite variable (table 2). Animals were preselected to have relatively soft, pliable skin free from abrasions and lacerations. Exposure of the footpad skin to an air stream saturated with water vapor had little or no effect on skin mechanical properties. One explanation for this may be that skin exposed to room air retained water 115 from sweat gland activity caused by restraint of the animal prior to anesthetization, and also from exposure of the skin to water during the footpad preparation procedures. Exposure of the footpad to the dry air stream caused a significant decrease in skin pliability (table 2). The effect of soaking the footpad in deionized water, however, was quite variable. In some animals, this had a plasticizing effect on the skin which made it softer than the skin exposed to dry air. More commonly, it resulted in a significant swelling and stiffening of the skin (table 2). A similar result has been described in human skin (Dikstein and Hartzshtark, 1981), where there was a decrease in compressibility of the forehead skin when it was soaked in water. One explanation for this effect is that absorption of a large amount of water fills the intracellular and interstitial spaces of the skin with an incompressible liquid, which overwhelms the plasticizing effect that accompanies the breaking of protein hydrogen bonds. Soaking skin in deionized water may potentiate this effect, especially at those sites where the stratum corneum is thin, as it is on the forehead. CORTICAL UNIT ACTIVITY Much of the variability in action potential amplitudes recorded in these experiments (fig l3, l4 and 15) is related to the size of the microelectrode tip, brain pulsations and 116 baseline noise of the recording system. The effect of external noise sources was minimized by RF shielding of the electrodes and the experimental preparation. Brain surface pulsations were effectively damped by covering the cortical surface with a layer of agar. Only cortical units which were characterized by sharp initially negative biphasic waveforms were chosen for study. Neurons with these characteristics are more distant from the microelectrode tip and are less damaged than those which have initially positive waveforms. Experiments were immediately terminated if sudden changes in spike amplitudes or waveforms were detected. Using these criteria and amplitude window discrimination gives reasonable assurance that the responses detected represent changes in firing characteristics of single cortical units. Variations in cortical neuron responses may also have been related to the effect of anesthesia since pentobarbital has been shown to depress their activity (Richards, 1972; Tsuchiya and Kitagawa, 1976; Harding 35 31., 1979; Collins and Roppolo, 1980). Collins and Roppolo (1980) demonstrated there were decreases in both spontaneous and evoked firing rates of neurons in the primary somatosensory cortex in awake monkeys given increasing doses of pentobarbital. In this study, 81 neurons were driven to high rates of activity (150 impulses per second) by a stimulus moving across the skin surface. Their results indicated an exponential decrease in both spontaneous and evoked activity which approached an asymptote 20 to 30 percent below control 117 levels at an anesthetic concentration of 16 to 20 mg per kg of body weight. This maximum dose of pentobarbital was less than that required for surgical anesthesia because animals could still be aroused by noxious stimuli. Animals used in the present study were anesthetized to a surgical plane, and then maintained at that level as described in section III. Although the cortical cells of deeply anesthetized animals are certainly less responsive than they are in the waking state, small variations in circulating anesthetic are likely to have only a minimal effect on excitability, once a surgical plane of anesthesia has been reached. Richards (1972) examined the mechanism for the depression of cortical activity by barbiturates and found that pentobarbital depresses the amplitude of EPSP's, but does not alter either axonal conduction or postsynaptic responsiveness to neurotransmitters. He concluded that pentobarbital depresses neurotransmitter release without affecting postsynaptic excitability. His data show an exponential decrease in EPSP amplitudes as a function of increasing anesthetic concentration which reaches an asymptote at subanesthetic concentrations. This result was similar to that demonstrated by Collins and Roppolo. Richards (1972) also showed that EPSP amplitudes in repetitively firing neurons were indistinguishable from control values at firing rates less than 2 to 5 impulses per second. 118 Small changes in circulating anesthetic are therefore unlikely to have a major effect on the responsiveness of . cortical neurons in already deeply anesthetized animals, especially if their firing rate is less than 2-5 impulses per second, as they were in this study. In order to test this hypothesis, 3 series of mechanical stimulations on seven footpad locations were conducted over four hours, without varying the skin hydration (table 1 and figure 22). The time course for data collection and anesthesia supplementation was the same as in other experiments. The results from these tests show that the changes in thresholds as a function of time were minimal and without any consistent trends. STIMULUS INTENSITY AND CORTICAL NEURONAL RESPONSE Freeman and Johnson (1982a, 1982b) have recently developed a mathematical model which accurately predicts discharge patterns in primary mechanoreceptive afferents when sinusoidal stimuli are delivered to the skin surface. Data recorded from cutaneous afferents show there is a sigmoidal relationship between the response probability and the stimulus amplitude with low frequencies of mechanical stimulation (1982b). Their model predicts such a relationship only if the effect of randomly distributed membrane noise is introduced into their calculations. One explanation for this is that at low stimulus frequencies, 119 receptor depolarization is superimposed on random temporal variations in the receptor resting membrane potential. At low stimulus frequencies, the probability of evoking a neuronal response in the primary afferent depends on both the stimulus amplitude and the temporal variation in the resting membrane potential. As the frequency of stimulation is increased, the stimulus-response relationship is further complicated by the time constant for membrane repolarization following the preceding stimulus. There was a curvilinear relationship between the probability of cortical neuronal response and the strength of the stimulus in the present study (figures 16-19). One explanation for this is that punctate stimuli were delivered at a low frequency (1 Hz), and the curvilinear response of the cortical neurons reflects the sigmoidal relationship expected in primary afferents when so stimulated. This explanation is supported by the results of previous studies which have shown that the subjective evaluation of stimulus intensity, as well as activity in neurons of the primary somatosensory cortex and ventrobasal nucleus of the thalamus, closely parallels the activity in primary mechanoreceptive afferents and are linear with respect to the stimulus amplitude (Mountcastle, 1980). This implies that the transfer functions at lemniscal system synapses are linear operators, and that cortical activity will be an accurate reflection of the activity in primary afferents. 120 Another explanation may be that the curvilinear relationship is the result of nonlinear skin mechanical properties. The stimulus characteristic controlled in this study was the amount of force (stress) delivered to the skin surface. It is well known that mechanoreceptors in skin are activated by the amount of displacement (strain) rather than the amount of stress (Lindblom, 1966). It is possible that the curvilinear relationships are due to nonlinear stress-strain characteristics of the skin. Although many studies have shown that skin mechanical properties are nonlinear, this appears to be true only for relatively large stresses and strains. Over the range of displacements which were used in this study, and for skin which is separated from underlying bone structure by a thick layer of subcutaneous fat such as that found in the cat footpad, the stress-strain relationship is linear as predicted by Hook's law (Phillips and Johnson, 1982). EPIDERMAL HYDRATION EFFECTS ON CORTICAL NEURONAL RESPONSE Epidermal hydration state has a significant effect on the amount of force required to activate neurons in the primary somatosensory cortex when punctate stimuli are delivered to the skin surface (figures 16-18, 20-23, tables 3,4). One explanation is that the slope of the stress-strain relationship in the epidermis has been affected so that more 121 force is required to achieve any given amount of skin indentation in the dry skin. There are at least two observations which support this explanation. First, data from studies of skin mechanical properties indicate that the elastic modulus (slope of the stress-strain relationship) is decreased in moistened skin. Second, comparison of threshold values for skin exposed to wet and dry air (table 4) show no statistical difference between the thresholds for low amounts of force (T10-T40). The difference between mean values becomes greater and significantly different, however, as the stress increases (T50-T100). The proposed stress-strain relationship in figure 26 is consistent with both of these observations. The effect of water soaking the skin appears to be much more complicated. Data reported in table 4 show that thresholds for water soaked skin are statistically different from those for wet air exposed skin over the whole range of applied stresses. The thresholds from water soaked skin are statistically different from those obtained from dry skin at low (T10-T20) and high (T70-T100) stresses, but are not different within the intermediate range (T30-T60). One explanation for this effect is that swelling of the epidermis with water soaking drastically alters skin mechanical properties which results in a curvilinear stress-strain relationship (figure 26). The stress-strain relationships shown in figure 26 are presented as a working STR ESS 122 WATER SOAKED SKIN DRY SKIN MOIST SKIN STRAIN + Figure 26. Proposed stress-strain relationships in the epidermis. 123 hypothesis to explain the results of the present study and can be tested directly in the cat footpad skin. Results from skin tested during room air exposure indicate that thresholds are not different from those in wet air—exposed skin over the entire range of applied stresses (table 4). For low stresses (T10-T30), threshold values from room air-exposed skin are not statistically different from those obtained from dry skin, but are significantly different for higher stresses (T40-T100). These results are consistent with the hypothesis that under the conditions of this experiment, the mechanical properties of normal skin are very similar to those of the wet air-exposed skin. Data shown in table 4 and figure 27 suggest a general relationship for thresholds of cortical mechanoreceptive neurons. It is proposed that there is an inflection point in the relationship between cortical neuronal thresholds and epidermal water content at or near the point of the normal hydration state (figure 27). Either absorption or desorption of large amounts of water from the epidermis causes an increase in the amount of force required to activate cortical neurons. 13‘1133 measurements of the frictional porperties of skin (Adams, 1969) show a similar inflection point at intermediate hydration states. A better understanding of a control system for maintaining epidermal hydration within the range of the lowest threshold levels may yield information critical to the understanding of FORCE THRESHOLD 124 DEHYDRAHON Sgrififl; MOIST SKIN SKIN HYDRATION 4)» Figure 27. Summary of hydration state effects on cortical neuronal force thresholds. 125 pathological conditions characterized by extremes in the cutaneous hydration state. STIMULUS LOCATION EFFECTS ON CORTICAL NEURONAL RESPONSES The location of the mechanical stimulus on the footpad surface has a significant effect on the threshold for activating cortical neurons (figures 19, 25, tables 3, 5). Although there are some slight differences in the statistical comparisons for very low and very high stresses, data presented in figure 25 are representative of the observed relationships. These data show that there is a region (locations 2-4) between 4 and 12 mm from the most sensitive skin site (location 1) where the amount of stress required to activate a single cortical neuron does not change as a function of distance. Previous studies have shown that the threshold for single mechanoreceptive afferent fibers increases as a function of the square of the distance from the center of the receptive field (Johnson, 1974). This suggests that the region of equal thresholds in the present study is not due to the characteristics of the receptive field of a single primary afferent, but is more likely due to processing within the ascending mechanoreceptor pathway or the cerebral cortex. This is supported by the lack of a significant treatment-location interaction (table 3) which would be expected if spatial coupling were through the skin. 126 A general pattern of synaptic connections whereby a single cell receives its major input from one cell, and minor inputs from many cells with surrounding and overlapping peripheral receptive fields occurs at all levels of the lemniscal system (Mountcastle, 1981). The results of the present study are consistent with this known pattern of convergence within the ascending pathway. A study of the response of single primary afferents to closely spaced stimuli would be more appropriate to describe accurately the effect of epidermal hydration on spatial coupling of stimulus and response in the skin. CONCLUSIONS Epidermal hydration state affects the amount of force required to activate Sl cortical neurons when punctate stimuli are delivered to the skin surface. 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