Relationship of transepithelial electrical potential to membrane potentials and conductance ratios in frog skin

Summary Previous studies in anuran epithelia have shown that, after clamping the transepithelial voltage in symmetrical sequences for 4–6 min there is near-constancy of the rate of active Na transport and the associated oxidative metabolism, with a near-linear potential dependence of both. Here we have investigated in frog skin the cellular electrophysiological events associated with voltage clamping (V _t =inside-outside potential). Increase and decrease ofV _t produced converse effects, related directly to the magnitude ofV _t .Hyperpolarization resulted in prompt decrease in inward transepithelial currentI _t and increase in fractional outer membrane resistancefR ₀ (as evaluated from small transient voltage perturbations) and in outer membrane potentialV ₀. Overshoot ofV ₀ was followed by relaxation to a quasi-steady state in minutes. Changes infR ₀ were progressive, with half times of some 1–5 sec. Changes in transepithelial slope conductanceg _t were more variable, usually preventing precise evaluation of the outer and inner cell membrane conductancesg ₀ andg _i . Nevertheless, it was shown thatg ₀ is related inversely toV _t andV ₀. Presuming insensitivity ofV _i toV _t , the dependence ofg ₀ onV ₀ in the steady state much exceeds that predicted by the constant field equation. Apparent inconsistencies with earlier results of others may be attributable to differences in protocol and the complex dependence ofg ₀ onV ₀ and/or cellular current. In contrast to previous findings in tight epithelia at open circuit, differences inV _t were associated with substantial differences infR ₀ and inner membrane potentialV _i . Hyperpolarization ofV _t over ranges commonly employed in studies of active transport and metabolism appears to increase significantly the electrochemical work per Na ion transported.     

A method for the in situ measurement of the electrical quantities in frog skin

A method allowing the measurement of the electrical quantities related to the physiological functions of the frog skin in situ is presented. The method allows the performance of several experiments on the same pithed animal, which remains alive for a number of days. The preparation is very stable, and the electric potential difference and short-circuit current values are higher than in isolated skin. The theory of measurement and the possible systematic errors are discussed. The possibilities of the method are evaluated on comparing the pH and temperature dependence of the electrical quantities in situ with previous measurements on isolated skin.     

Quasi-periodic behaviour in a model for the lithium-induced,electrical oscillations of frog skin

The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g. relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). A realistic model was thus proposed under the form of a set of ordinary differential equations. In the past, numerical simulations using such a model reproduced the periodic experimental oscillations and was able to provide an explanation for the global synchronised oscillations of the whole skin. In that paper, new numerical simulations reproduce the non-periodic oscillations which were observed two decades ago, but not reproduced by the model. Moreover, the dynamical process under which all the local oscillators are synchronised is explained in terms of a tangent bifurcation.     

Evoked potentials in the frog medulla to electrical stimulation of the glossopharyngeal nerve

Evoked potentials in the frog medulla to stimulation of the glossopharyngeal nerve appear on the ipsilateral side in a zone of limited area. They are recorded at depths not exceeding 2000 µ. Depending on their form the surface evoked potentials are divided into two groups, negative and positive—negative, differing from each other in their parameters and properties. During insertion of the microelectrode the phase of the negative potential is changed. The principal slow components of the responses reflect postsynaptic processes. The first fast wave of the evoked potential is regarded as the presynaptic component. Differences in the properties of the evoked potential recorded at different points are determined by the neuromorphological heterogeneity of the structures of the primary center.     

Reversed potentials in isolated frog skin. II. Active transport of chloride

Net inward transport of Cl in the absence of an electrochemical potential difference was demonstrated in the skin of two species of frog, R. pipiens and R. esculenta under conditions of low (2 mM) Cl concentration in the bathing solutions. The electrical potential profile of skins of R. pipiens was examined with microelectrodes under conditions in which the inside solution was negative relative to the outside solution. This reversal of the normal potential difference was found to arise as a result of potential changes across the outward facing electrical barriers in the skin. The reversed potential difference appears to arise, at least in part, as a result of the inward Cl transport. The effect may be due either to electrogenic Cl transfer or to variations in internal composition of the epithelial cells arising as a result of Cl transport.     

Tissue electroporation. Observation of reversible electrical breakdown in viable frog skin.

Experiments by others have used isolated cell or bilayer membrane preparations to study the dramatic phenomena associated with electroporation. The present study observes electroporation behavior in an intact tissue. Viable samples of frog skin (Rana pipiens) were exposed to short electrical pulses of varying width and magnitude under "charge injection" conditions. After a pulse, the transtissue potential decayed with two distinct time constants, one short (tau approximately 0.3 ms) and the other longer (tau L approximately 2 ms). Above thresholds for the pulse magnitude and for the pulse width tau L decreased significantly, with progressively smaller tau L as the pulse magnitude and width increased. The postpulse potential, delta Utissue (t), and resistance, Rtissue, also decreased progressively. The tissue subsequently recovered to its original resistance and open circuit potential, delta U tissue,oc, within 2-3 min after a pulse. At that time another pulse experiment could be carried out, demonstrating repeatability and reversibility. No significant permanent changes in Rtissue and delta Utissue,oc were found. This is interpreted as avoidance of significant tissue damage. Taken together, these dramatic phenomena are characteristic of the reversible electrical breakdown previously observed in charge injection experiments with artificial planar bilayer membranes and with isolated cell membranes by similar very short pulses. The present experiments therefore demonstrate that electroporation can be repeatedly caused and observed in a viable tissue without apparent damage.     

From frog integument to human skin: dermatological perspectives from frog skin biology

For over a century, frogs have been studied across various scientific fields, including physiology, embryology, neuroscience, (neuro)endocrinology, ecology, genetics, behavioural science, evolution, drug development, and conservation biology. In some cases, frog skin has proven very successful as a research model, for example aiding in the study of ion transport through tight epithelia, where it has served as a model for the vertebrate distal renal tubule and mammalian epithelia. However, it has rarely been considered in comparative studies involving human skin. Yet, despite certain notable adaptations that have enabled frogs to survive in both aquatic and terrestrial environments, frog skin has many features in common with human skin. Here we present a comprehensive overview of frog (and toad) skin ontogeny, anatomy, cytology, neuroendocrinology and immunology, with special attention to its unique adaptations as well as to its similarities with the mammalian integument, including human skin. We hope to provide a valuable reference point and a source of inspiration for both amphibian investigators and mammalian researchers studying the structural and functional properties of the largest organ of the vertebrate body.     

The heart-brain connection: mechanistic insights and models

While both cardiac dysfunction and progressive loss of cognitive function are prominent features of an ageing population, surprisingly few studies have addressed the link between the function of the heart and brain. Recent literature indicates that autoregulation of cerebral flow is not able to protect the brain from hypoperfusion when cardiac output is reduced or atherosclerosis is prominent. This suggests a close link between cardiac function and large vessel atherosclerosis on the one hand and brain perfusion and cognitive functioning on the other. Mechanistically, the presence of vascular pathology leads to chronic cerebral hypoperfusion, blood brain barrier breakdown and inflammation that most likely precede neuronal death and neurodegeneration. Animal models to study the effects of chronic cerebral hypoperfusion are available, but they have not yet been combined with cardiovascular models.     

Dermal tracts in frog skin: fibronectin pathways for cell migration

The dermis of the frog skin (Rana esculenta) displayed a remarkable organization of vertical and horizontal tracts. Vertical thick tracts connected the dermal Stratum spongiosum with the subcutaneous tissue. Horizontal thin tracts were found alongside and contiguous to them. The thick tracts were sheathed by collagen fibrils of the Stratum compactum which were vertically oriented (i.e. parallel to the axes of the tracts) according to the horizontal and orthogonal arrangement of the collagen bundles of the Stratum compactum. The thin tracts devoid of collagenous sheath were formed by clear spaces between superimposed collagen bundles of the dermal Stratum compactum. On vertical sections, the thick tracts were seen to contain fibronectin (FN), detected by indirect immunoperoxidase. Continuous vertical FN lines were centred in these tracts. On horizontal sections, a clear zone around these FN-centred lines was also sheathed by FN. The thick tracts contained flattened pigmentary cells and fibroblasts; these cells were FN-outlined. The thin tracts contained patches of FN and FN-outlined fibroblasts. In culture, in vertical thick tracts, both pigmentary cells and fibroblasts disappeared when antiserum to FN was added to the culture medium. This suggested that thick tracts were pathways allowing pigmentary cells to move upward or downward between their usual upper dermal and lower subcutaneous localizations. Fewer fibroblasts were found in the thin tracts in the presence of antiserum to FN.     

The nerves in frog skin

In the epidermis of frog skin, most nerves are situated at the top of the basal layer. More superficial nerve fibres are usually adjacent to flask cells; it is concluded that this is not a functional association, but a consequence of the pattern of moulting. There are nerve fibres in the walls of the granular glands; mucous glands appear to have no intrinsic innervation although nerves pass within a short distance of their walls. The smooth muscle bundles of the dermis are innervated, and have a physical attachment to the overlying epidermis.     

Mosquito repellents in frog skin

The search for novel insect repellents has been driven by health concerns over established synthetic compounds such as diethyl-m-toluamide (DEET). Given the diversity of compounds known from frog skin and records of mosquito bite and ectoparasite infestation, the presence of mosquito repellents in frogs seemed plausible. We investigated frog skin secretions to confirm the existence of mosquito repellent properties. Litoria caerulea secretions were assessed for mosquito repellency by topical application on mice. The secretions provided protection against host-seeking Culex annulirostris mosquitoes. Olfactometer tests using aqueous washes of skin secretions from L. caerulea and four other frog species were conducted to determine whether volatile components were responsible for repellency. Volatiles from Litoria rubella and Uperoleia mjobergi secretions were repellent to C. annulirostris, albeit not as repellent as a DEET control. The demonstration of endogenous insect repellents in amphibians is novel, and demonstrates that many aspects of frog chemical ecology remain unexplored.     

Effect of prolactin on short circuit current, potential and electrical resistance across isolated frog skin

The effect of a range of ovine prolactin doses (10(-9)-10(-6)M) on the short circuit current (Isc), potential difference (E) and electrical resistance (R) of isolated frog skin has been studied. Prolactin produced a dose dependent stimulation of Isc and generally a fall in R, although the latter was only significant after 10(-9) and 10(-6)M prolactin. The effect of prolactin on E was found to be more dependent upon the initial E (at the time of hormone addition) than on the dose of hormone. 10(-9)M prolactin, in contrast to higher doses, produced a sustained fall in R without stimulating Isc. Thus the effect of prolactin on frog skin appears to be predominantly on passive permeability at low doses, and on active ion transport at higher doses.