Thermal Dependence of Passive Electrical Properties of Lizard Muscle Fibres

Passive electrical properties are important in determining the excitability of muscle fibres (Bryant & Morales-Aguilera, 1971; Adrian & Bryant, 1974; Jack, Noble & Tsein, 1975). Previous studies have demonstrated significant effects of temperature on the passive electrical properties of muscle fibres from a wide variety of ectothermic animals: membrane resistance (R_m) decreases with increasing temperature in fibres from crab (Fatt & Katz, 1953), crayfish (White, 1983), fish (Klein & Prosser, 1985), frog (del Castillo & Machne, 1953; Hodgkin & Nakajima, 1972), lobster (Coulton & Freeman, 1975) and locust muscle (Kornhuber & Walther, 1987). In contrast, the effects of temperature upon passive electrical properties of mammalian muscle fibres are less uniform: R_m decreases with increasing temperature in rat diaphragm (Palade & Barchi, 1977), but increases with increasing temperature in cat tenuissimus (Boyd & Martin, 1959) and goat intercostal muscle (Lipicky & Bryant, 1972).

Little information is available regarding the passive electrical properties of reptilian muscle fibres (Levine, 1966; Proske & Vaughan, 1968; Ridge, 1971), and the thermal dependence of those properties is unknown. As ectotherms, reptiles must contend with variable environmental and body temperatures and may experience a 20 °C range of body temperature during the course of one day (Brattstrom, 1965). The experienced range of body temperatures may include, at its lower end, the activity temperatures of fish and amphibians and, at its upper end, the regulated temperature of mammals. Temperature excursions of this magnitude may significantly affect the passive electrical properties of muscle fibres, and thereby influence muscle excitability and functional capacity.

This study investigated the thermal dependence of passive electrical properties of muscle fibres from three lizard species: Anolis cristatellus, Sceloporus occidentalis and Dipsosaurus dorsalis. These species differ in preferred body temperature (PBT) and upper critical temperature (CT_max): Anolis has a PBT of 30°C and a CT_max of 37°C (Huey, 1983), Sceloporus has a PBT of 35°C (Brattstrom, 1965) and a CT_max of 44°C (Larson, 1961), and Dipsosaurus has a PBT of 40°C and a CT_max of 48°C (Norris, 1953; Brattstrom, 1965). The results are compared with those obtained from other studies, and the implications for muscle function are discussed.

Anoles (Anolis cristatellus, four females, six males; mean body mass 7·9 ± 0·39g, range 6·0–9·6g) were collected in Puerto Rico by Dr Paul Hertz (permit no. DRN-84-67). Western fence lizards (Sceloporus occidentalis, eight females, seven males; mean body mass 10·2±0·64g, range 6·3–15·1g) and desert iguanas (Dipsosaurus dorsalis, 14 females, 16 males; mean body mass 46·1 ±1·90g, range 27·7–73·6g) were collected locally in Southern California under California State Fish and Game permits no. 2071 and no. 2141. Lizards were held in glass aquaria, and provided with a photothermal gradient that allowed behavioural thermoregulation. Anolis were maintained for several weeks on a diet of crickets. Sceloporus were used within several days of capture. Dipsosaurus were maintained for several months on a diet of chopped lettuce that was coated with pulverized Purina Puppy Chow and Special-H high vitamin cereal. Access to water was unrestricted for all species.

The iliofibularis (IF) muscle was removed with a portion of the ilium attached and pinned at 120% resting length (defined as the in situ length with the knee at a 90° angle and the femur perpendicular to the ilium) in a Sylgard-lined chamber. The ranges of in situ muscle lengths were 12–15 mm (Anolis), 9–12 mm (Sceloporus) and 13–20 mm (Dipsosaurus). The preparations were superfused with lizard saline (145mmoll⁻¹ NaCl, 4mmoll⁻¹ KC1, 2·5mmoll⁻¹ CaCh, 20mmoll⁻¹ imidazole buffer, 10 mmoll⁻¹ glucose, pH7·50 at 23°C) throughout the experiments. Imidazole was chosen as the buffer because its pH changes with temperature in a manner very similar to the plasma of a variety of ectothermic vertebrates (Reeves, 1977), including Dipsosaurus (Bickler, 1981). Temperature was varied by heating or cooling the saline before it entered the chamber and was recorded with a small thermocouple placed within 1 mm of the muscle. The sequence of temperature exposures was random, except for the highest temperature, which was done last. The preparation was allowed to equilibrate at each newly selected temperature for at least 5 min before measurements were taken.

The iliofibularis of lizards is a parallel-fibred muscle composed of discreet ‘white’ and ‘red’ regions. In Dipsosaurus the two regions are particularly distinct; the red region contains 69 % fast oxidative glycolytic (FOG) fibres and 28 % tonic fibres, and the white region contains 2% FOG and 98% fast glycolytic (FG) fibres (Gleeson, Putnam & Bennett, 1980). Data presented here were collected separately from twitch fibres of both the red and white regions of the IF of Dipsosaurus-, those fibres examined in the white region of the IF are taken to be FG fibres, and those from the red region are considered to be FOG fibres. Fibres having relatively high input resistance (>1MΩ) and relatively long membrane time constant (>30ms) were occasionally encountered in the red region of the IF from Dipsosaurus-, such fibres were presumed to be tonic fibres, and were not included in the analysis.

In the iliofibularis of Anolis and Sceloporus the red region is not as distinct as in Dipsosaurus. Hence, only twitch fibres from the unambiguously ‘white’ region of the iliofibularis of these species were examined; these fibres had large diameters (80–150 μm), were very transparent, and generated propagated action potentials when stimulated directly via an intracellular electrode.

The upper experimental temperature to which muscles from each species were exposed was determined in preliminary experiments to be the highest temperature the preparations could tolerate without obvious deterioration (depolarization of resting potential and decreased transparency). These maximum temperatures were always several degrees cooler than the species’ upper critical temperature (CT_max).

The passive electrical properties of muscle fibres were determined using hyperpolarizing square pulses, following the procedure of Josephson & Stokes (1982). The preparation was viewed at 50× magnification with transverse illumination; an ocular micrometer was used to measure distances to the nearest 10 μm. The current-passing electrode (10–20 MΩ, tip filled with 4% Lucifer Yellow solution, back-filled with 3 mol l⁻¹ LiCl₂) was inserted near the middle of a fibre, and the recording electrode (10·20 M Ω, 3 mol l⁻¹ KC1) was inserted in the same fibre 1–2 mm away. For determination of length constant (L), rectangular hyperpolarizing pulses (500 ms duration) of constant magnitude were injected for 3–5 separations of the two electrodes. L was taken as the reciprocal slope of the plot of electrode separation versus the natural logarithm of the change in membrane potential.

Data from a given muscle fibre were included in the analysis only if the following criteria were met : ( 1 ) the initial resting potential, measured upon first impalement of a fibre with the recording electrode, was at least −70 mV and declined no more than 10 mV during the analysis; (2) voltage offsets in response to constant current pulses were successfully measured at 3–5 electrode separations and (3) current-voltage relationships (I–V plots) were linear and had correlation coefficients of at least 0-95. For any given muscle preparation, several fibres were analysed, each at a different temperature. Fibres from at least five different individuals of each species were examined at each temperature.

Data for each species were analysed using one-way analysis of variance (ANOVA) followed by a multiple range test (Student-Neuman-Keuls; SNK). In some cases the data were log-transformed prior to statistical analysis to correct for unequa variance among groups. R₁₀ (analogous to Q₁₀; Bennett, 1984) values were calculated for consecutive 5 °C intervals using the mean values for each passive electrical property. Values presented in the text are mean ± S.E.M.

There were clear differences in fibre diameter among species, with Anolis having the largest and Dipsosaurus the smallest diameter fibres (Table 1). In Dipsosanrus, FG fibres from the white region of the IF were larger than FOG fibres from the red region. However, within species and within the red and white regions of the IF from Dipsosaurus, diameter did not vary significantly among the groups of fibres examined at different temperatures (P>0·2; ANOVA). Therefore, all fibres from a species (or region) were combined to calculate average fibre diameters, which were: Anolis white IF, 126 ± 4·5 μm (N =42 fibres); Sceloporus white IF, 101 ±3·0 μm (N = 46 fibres) ; Dipsosaurus white IF, 86 ± 1·8 μm (N = 62 fibres) ; Dipsosaurus red IF, 57 ± 1·3 gm (N = 61 fibres).

All the passive electrical properties displayed significant thermal dependence (Table 1 ; Figs 1,2). The thermal ratios (R₁₀ values), calculated for each consecutive temperature interval are shown for each passive electrical property in Table 2.

Input resistance (R_in) decreased with increasing temperature (Fig. 1A), and the change was significant in fibres from all three species (Table 1). The absolute value of R_in increased in order of decreasing fibre diameter; at any given temperature, R_in was lowest in fibres from Anolis, intermediate in fibres from Sceloporus, and highest in Dipsosaums red IF fibres (Table 1).

Length constant (L) decreased with increasing temperature, except in Anolis fibres (Fig. 1B). At 15 and 25°C, length constant was inversely correlated with fibre diameter, being shortest in Anolis fibres and longest in Dipsosaurus red IF fibres (Table 1).

Time constant (τ) decreased with increasing temperature (Fig. 1C). Time constant tended to be inversely proportional to fibre diameter; it was shortest in Anolis fibres and longest in Dipsosaurus red IF fibres (Table 1).

Sarcoplasmic resistivity (R_s) decreased with increasing temperature (Fig. 2A). There were no consistent differences in R_s among species, and the value of R_s was not correlated with fibre diameter (Table 1).

Apparent membrane resistance (R_m) decreased with increasing temperature (Fig. 2B). Apparent R_m was inversely related to fibre diameter; fibres from Anolis had the lowest apparent R_m and red IF fibres from Dipsosaurus had the highest apparent R_m (Table 1).

Apparent membrane capacitance (C_m) increased with increasing temperature (Fig. 2C). Apparent C_m was not strictly correlated with fibre diameter; C_m was greatest in fibres from Sceloporus (101 μm), next highest in fibres from Anolis (126gm), and lowest in fibres from the red IF of Dipsosaurus (57μm; Table 1).

There were significant differences in average fibre diameter among species, and between the red and white IF of Dipsosaurus (Table 1). These differences are important because the passive electrical properties depend upon fibre cross-sectional area and the total membrane area available for current flow, and these areas are functions of fibre diameter. Consequently, many of the interspecific and interregional differences in passive electrical properties might simply reflect differences in fibre diameter. However, within each species or muscle region, the changes observed in the passive electrical properties are due to temperature variation.

Input resistance

As temperature increased, R_in decreased significantly in muscle fibres from all three species (Fig. 1A; Table 1). This result is qualitatively similar to that obtained in prior studies of muscle fibres from other ectothermic animals, but qualitatively different from results obtained with some mammalian fibres (see section on Membrane resistance below for further discussion).

The thermal dependence of R_in (average R₁₀ = 0·7) is similar to that for the aqueous diffusion of ions (Rio = 0·67–0·83 ; Hille, 1984). Thus the decline in R_in with increasing temperature can be attributed to a purely passive decrease in membrane resistance, due to enhanced conductance of ions through open membrane channels.

Of all the passive electrical properties, R_in is perhaps the most important, because it strongly influences the response of a fibre to synaptic current. As temperature increases, R_in decreases, and the depolarization in response to a given synaptic current will be less. Temperature-dependent changes in R_in have significant effects on the excitability of muscle; the fibres become less excitable as temperature is increased, and require larger depolarizing currents to reach threshold (B. A. Adams, in preparation).

As temperature increased, τ decreased significantly (Fig. 1C). The thermal dependence of τ was fairly similar in fibres from the different species (range of R₁₀ = 0·7–1·0; Table 2). Since τ is equal to the product of R_m and C_m, the shortening of r with increasing temperature must be entirely due to the decrease in R_m, because C_m is either constant or increases with increasing temperature (Table 1 ; Fig. 2C); increasing C_m would increase, not decrease, τ.

The time constants reported here are within the range reported for lizard scalenus fibres (6–40ms at 20–25°C; Proske & Vaughan, 1968) and snake costocutaneus fibres (8–14ms at 20–23°C; Ridge, 1971), but are shorter than those reported for turtle retractor capitis fibres (20–40 ms at 15–24°C; Levine, 1966).

R₃ decreased with increasing temperature (Fig. 2A), as has been observed in other muscle fibres (del Castillo & Machne, 1953; Fatt & Katz, 1953; Hodgkin & Nakajima, 1972; Klein & Prosser, 1985; Kornhuber & Walther, 1987). The average value of R_s at 25°C was approximately 180Ωcm (Table 1), which is in good agreement with the value of 170Ωcm (20°C) reported by Hodgkin & Nakajima (1972) for frog fibres, and the value of 150 Ωcm (25 °C) reported by Klein & Prosser (1985) for fish fibres. In previous studies of reptilian fibres, It, was assumed and not measured, so no comparisons can be made with the present results. The thermal dependence of R_s (average R₁₀ = 0·8) also agrees fairly well with that calculated by earlier authors (R₁₀ = 0·72–0·73; Hodgkin & Nakajima, 1972; Klein & Prosser, 1985).

There were no consistent differences in either the absolute value or the thermal dependence of R_s among species (Tables 1, 2; Fig. 2A). Therefore, the interspecific differences in passive electrical properties must be solely attributable to differences in the total membrane surface area, and/or the specific membrane resistances of the fibres.

As temperature increased, apparent R_m declined significantly (Fig. 2B). This result is qualitatively similar to that reported for muscle fibres from other poikilothermic animals and appears to result from passive changes in membrane conductances. However, the change in R_m is opposite to that observed in some mammalian muscle fibres; in cat tenuissimus (Boyd & Martin, 1959) and goat intercostal muscle (Lipicky & Bryant, 1972) R_m increases with increasing temperature. This difference in the behaviour of R_m between poikilothermic and mammalian muscle suggests that the resting conductances of mammalian muscle membranes may be actively increased (via channel opening) at low temperature and actively decreased (via channel closure) at high temperature. Such a response, if it produced changes in Na⁺ or K⁺ conductances, could influence the activity of Na⁺,K⁺-ATPase and hence be involved in cellular thermogenesis.

The thermal dependence of apparent R_m (average R₁₀ = 0·7) was indistinguishable from that for the aqueous diffusion of ions (R₁₀ = 0·67–0·83; Hille, 1984). This similarity suggests that the observed decrease in R_m can be explained by increased single-channel conductance at higher temperatures.

The inverse correlation between fibre diameter and apparent R_m (see Table 1) has also been observed for different sized fibres from a single species (Rana temporana;Hodgkin & Nakajima, 1972), and is based upon the underestimation of true membrane surface area caused by assuming that muscle fibres are simple cylinders; such an assumption ignores the contribution of t-tubular membrane to total fibre surface area.

The clear interspecific differences in apparent R_m (Table 1) could result from differences in true R_m or from differences in total membrane surface area. Unfortunately, true R_m cannot be calculated accurately without measuring the precise membrane area of the fibres, which was not attempted in this study.

In general, C_m did not change significantly over the lower range of temperatures, but then increased at the highest temperatures (Fig. 2C) with R₁₀ values as high as 2 ·1 (Sceloporus, Table 2). The average thermal ratio was 1·3. Previous authors (del Castillo & Machne, 1953; Klein & Prosser, 1985; Hodgkin & Nakajima, 1972) have reported smaller increases in apparent C_m as temperature is increased (range of R10= 1·02–1·10), but in these studies apparent C_m was not measured at the high temperatures used here. Membrane capacitance should be a function of the area, thickness and dielectric constant of the membrane; temperature must alter one or more of these variables to affect apparent C_m. Possibly the membrane becomes thinner as temperature increases, or perhaps membrane processes such as pinocytosis and exocytosis are greatly increased at the higher temperatures, and more membrane surface area is actually present then.

Apparent membrane capacitance was highest in fibres from Sceloporus (mean diameter = 101 μm), and next highest in fibres from Anolis (mean diameter = 126 μm). This result suggests a more extensive development of the t-tubular system, and/or a higher specific membrane capacitance in Sceloporus than in Anolis fibres. Quantitative morphological data, which would provide an estimate of relative t-tubule development, are unfortunately not available for these muscle fibres. Apparent C_m was lowest in the smallest diameter fibres (red IF of Dipsosaurus, mean diameter 57 μm). A positive correlation between apparent C_m and fibre diameter has been previously demonstrated in Rana sartorius and semitendinosus fibres (Hodgkin & Nakajima, 1972), and in Schistocerca leg muscle fibres (Kornhuber & Walther, 1987).

The temperature dependence of the passive electrical properties has important implications for muscle function. Because input resistance largely determines the synaptic currents required to depolarize muscle fibres-to threshold, decreases in R_in will directly increase the excitatory current requirement. Thus, at higher temperatures, larger synaptic currents are necessary to excite the fibres (B. A. Adams, in preparation). Unless the nerve terminal compensates for the decrease in R_in by increasing transmitter release, the safety factor for neuromuscular transmission may decline as temperature increases. If safety factor declines enough, or is small to begin with, some fibres may fail to be excited at high temperature; such failure would reduce muscular power output, and would affect locomotor capacity negatively.

At the lower temperatures, R_in is relatively high, and the requirement for synaptic current should be reduced. Increased R_in would tend to compensate for the decreased transmitter release that may occur at low temperatures (Li & Gouras, 1958; Takeuchi, 1958; Hubbard, Jones & Landau, 1971; Jensen, 1972).

Although iliofibularis fibres from the three lizard species react similarly to changing temperature, the absolute values of the passive electrical properties differ markedly (Table 1). These differences may be provisionally explained by interspecific differences in fibre diameter. However, it should be noted that the absolute value of the fibre properties is correlated with the thermal ecology of the species. Input resistance, length constant, time constant and apparent membrane resistance were consistently lowest in fibres from Anolis (PBT = 30°C, CT_max = 37°C), intermediate in fibres from Sceloporus (PBT = 35 °C, CT_max = 44°C) and highest in fibres from Dipsosaurus (PBT = 40°C, CT_max = 48°C; Table 1). This correlation may indicate a functional basis for the interspecific differences in passive electrical properties.

Since R_in is temperature-dependent, and is important in determining whether a given muscle fibre becomes excited, it may be functionally advantageous for species that operate at high temperature (such as Dipsosaurus) to maintain Ri_n at relatively higher levels than less thermophilic species (e.g. Anolis). High R_in would tend to lower the synaptic current required to excite the fibres at all temperatures, and would help to maintain synaptic effectiveness at the unusually high activity temperatures (38–46°C; Norris, 1953) of Dipsosaurus. In an evolutionary’ sense, high R_in could be achieved by reducing fibre diameter, decreasing the extent of the t-tubule system, or by increasing specific membrane resistance.

In contrast, species that operate at lower temperatures (such as Anolis) may not need to maintain R_in at high levels, because they do not normally experienced temperatures where muscle fibre excitability or neuromuscular transmission might fail due to low R_in. Low values of R,_n are not necessarily advantageous at any temperature, but may simply result from the large membrane area and extensive t-tubule development usually found in large-diameter twitch fibres. Consequently, species such as Anolis may have more freedom to increase the maximum diameter of their muscle fibres compared with thermophilic species such as Dipsosaurus. Large muscle fibres may be more efficient than small muscle fibres during high-speed burst locomotion, because the tension and power generated per nerve impulse would be maximized.

The red region of the Dipsosaurus IF contains approximately 28% tonic fibres (Gleeson et al. 1980). However, in the present study only twitch fibres capable of propagating action potentials were examined. Within the IF of Dipsosaurus, twitch fibres from the red region had higher R_in, L, τ and R_m values than fibres from the white region. These differences in passive electrical properties suggest that twitch fibres from the red region may maintain postsynaptic excitability and effective neuromuscular transmission at higher temperatures than fibres from the white region. This suggestion is in accord with preliminary observations (B. Adams, unpublished data) indicating that red region fibres are more resistant to block by curare than are white region fibres, and appear to possess a greater safety factor for neuromuscular transmission.

This work was supported by NSF grants PCM 81-02331 and DCB 85-02218 to Dr Albert F. Bennett, a Grant-in-Aid of Research from Sigma Xi, The Scientific Research Society to BA, and by a University of California Chancellor’s Patent Fund Award to BA. I thank Drs A. F. Bennett and R. Josephson for their guidance and comments on the manuscript, Drs R. Miledi and I. Parker for numerous helpful discussions, and Drs T. Bradley, H. Koopowitz and R. Josephson for the generous loan of equipment.