No abstract

Curarized cutaneous pectoris nerve-muscle preparations from frogs were stimulated at 10/s or at 2/s for periods ranging from 20 min to 4 h . End plate potential were recorded intracellularly and used to estimate the quantity of transmitter secreted during the period of stimulation . At the ends of the periods of stimulation the preparations were either fixed for electron microscopy or treated with black widow spider venom to determine the quantities of transmitter remainind in the terminal . Horseradish peroxidase or dextran was added to the bathing solution and used as a tracer to detect the formation of vesicles from the axolemma . During 4 h of stimulation at 2/s many new vesicles were formed from the axolemma and the quantity of transmitter secreted was several times greater than the quantity in the initial store . After this period of stimulation, the terminals were severely depleted of transmitter, but not of vesicles, and their general morphological organization was normal . During 20 min of stimulation at 10/s the nerve terminals swelled and were severely depleted both of vesicles and of transmitter . During a subsequent hour of rest the changes in morphology were largely reversed, many new vesicles were formed from the axolemma and the stores of transmitter were partially replenished, These results suggest (a) that synaptic vesicles fuse with, and re-form from, the membrane of the nerve terminal during and after stimulation and (b), that the re-formed vesicles can store and release transmitter .

The oscillatory behavior of the cephalopod giant axons in response to an applied current has been established by previous investigators. In the study reported here the relationship between the familiar "RC" electrotonic response and the oscillatory behavior is examined experimentally and shown to be dependent on the membrane potential. Computations based on the threecurrent system which was inferred from electrical measurements by Hodgkin and Huxley yield subthreshold responses in good agreement with experimental data. The point which is developed explicitly is that since the three currents, in general, have nonzero resting values and two currents, the "Na" system and the "K" system, are controlled by voltage-dependent time-variant conductances, the subthreshold behavior of the squid axon in the small-signal range can be looked upon as arising from phenomenological inductance or capacitance. The total phenomenological impedance as a function of membrane potential is derived by linearizing the empirically fitted equations which describe the time-variant conductances. At the resting potential the impedance consists of three structures in parallel, namely, two series RL elements and one series RC element. The true membrane capacitance acts in parallel with the phenomenological elements, to give a total impedance which is, in effect, a parallel R, L, C system with a "natural frequency" of oscillation. At relatively hyperpolarized levels the impedance "degenerates" to an RC system. The subthreshold behavior of the axonal membrane, exemplified by the classical "electrotonus" of the myelinated nerve (Hermann, 1905; Hodgkin and Rushton, 1946;Davis and Lorente de N6, 1947) and the "local response" and oscillatory behavior of the giant axon in the cephalopods, Loligo and Sepia (Hodgkin, 1938;Arvanitaki, 1939 Arvanitaki, , 1941Brink et al., 1946), have been studied by many investigators. In recent years, perhaps due to the advent of the microelectrode technique with its emphasis on large amplitude recordings, electrophysiologists have shown only moderate interest in the subthreshold be-497

A B S T R A C TCurarized cutaneous pectoris nerve muscle preparations from frogs were subjected to prolonged indirect stimulation at 2/see while recording" from end plate regions. At the ends of the periods of stimulation, the curare was removed and the preparations were fixed for electron microscopy or treated with black widow spider venom to determine the degree to which their stores of transmitter had been depleted. After 6--8 hr of stimulation the nerve terminals were almost completely depleted of their stores of transmitter and of their population of vesicles. Most of the transmitter release occurred during the first 4 hr of stimulation, and after this time most (about 80%) of the fibers were depleted of about 80% of tbeir transmitter. The organization of the nerve terminals in 4-hr preparations appeared normal and the terminals still contained many vesicles. When peroxidase was present in the bathing medium, terminals from stimulated preparations showed many vesicles that contained peroxidase, whereas the rested control p;eparations showed few such vesicles The fact that after 4 hr the total number of vesicles is not markedly changed while a large fraction (up to 45%) contained peroxidase suggests that in our experiments vesicles were continuously fusing with and reforming from the axolemma. I . N T R O D U C T I O NSeveral workers have tried to deplete neuromuscular junctions of their stores of transmitter and of their synaptic vesicles by tetanic stimulation of the nerve (1, 2, 3, 4). Depiction of transmitter has been obtained only when synthesis was inhibited by hemicholinium (2, 3) and, under this condition, a reduction in the number of vesicles occurred only in the regions of the axoplasm immediately adjacent to the axolemma (4). In these previous works the preparations were stimulated for from several minutes to a few hours at frequencies of 10/see or more. We have stimulated a neuromuscular preparation from the frog for 6-9 hr at a rate of 2/see in the absence of hemicholinium and have successfully depleted the terminals of their store of transmitter and of their population of vesicles. M A T E R I A L S A N D M E T H O D SThe cutaneous pectorls muscle of the frog, Rana p¢zens, was used. The muscles were mounted in the chamber described previously (5) and maintained at about 22°C in a Ringer's soIution that contained 116 rn~i NaC1, 2.0 mu KC1, 1.8 m:~ CaC12, 1 rn~ NaH2PO4, and 2 m~ Na2HPO4 (pH 7.0). End plate regions were impaled with mieropipettes filled with 3 • KC1. Conventional recording equipment was used and photographic records of the end plate potentials @.p.p.s) and miniature end plate potenrials (m.e.p.p.s) were obtained. The nerve was stimulated with square pulses 0.1 msec in duration and amplitude three to four times threshold. The muscle twitch was blocked by adding curare to the bath at a concentration of 3 X 10 -6 g/ml, an 30THE JOV~,tL O~" CELL BIOLOGr -VoLtnuE 54~ 197g • pages 30-38 on

The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50, pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of at least four protein components that are similar in their molecular weights (about 130,000) and isoclectric points (ranging from pH 5.2 to 5.5) and are immunologicaUy indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.The physiological effects of the extract of the venom glands of the black widow spider, Latrodectus mactans, particularly the variety tredecimguttatus, have been studied extensively and the primary effects seem to be exerted on the nervous system (2-5, 7, 8, 12, 13, 15-18, 20-23, 30, 31, 33, 36-40, 43). Some of the active agents are proteins (18), and they seem to act in at least two basic ways: (a) to depolarize the cell bodies of some neurons and (b) to cause the release of neurotransmitters from a variety of nerve endings. For example, the extract depolarizes the cell body of the crayfish stretch receptor (S. Obara and A. Mauro, unpublished data) and induces a discharge of impulses in the axon (26). This depolarizing action of the extract may be responsible for its ability to induce a discharge of action potentials in the abdominal ganglion of the cockroach (5, 17).In addition to depolarizing excitable cells, the extract causes a release of transmitter from cholinergic nerve endings in brain (21, 24), sympathetic ganglia (38, 39), and Torpedo electric tissue (25), and from adrenergic nerve endings in the iris and other tissues (20,22,24). At neuromuscular junctions, the extract increases the frequency of occurrence of miniature end plate potentials, and blocks neuromuscular transmission. The neuromuscular effects have been observed at the cholinergic junctions of frogs and mammals (31, 37), at adrenergic nerve endings (vas deferens) in mammals (27), at both the inhibitory (3,-aminobutyrate) 462

The Poisson-Boltzmann equation, which was derived by Shockley in his treatment of the p-n semiconductor junction at equilibrium, is applied to fixed charge ionic membranes. The fixed charges in ionic membranes play the same role as "doping" ions in semiconductors, the major difference between the two systems being that in the former the mobile particles are ions while in the latter the particles are electrons and phenomenological particles, "holes." An important consequence of spatial gradients of fixed charge is the presence of space charge regions which give rise to an intrinsic electric field and potential. These quantities are established first for the single "lattice" thus providing a continuous treatment of the Donnan equilibrium invoked by Teorell-Meyer-Sievers in their treatment of fixed charge membranes. It is shown further that when a positive and negative membrane are juxtaposed, the space charge region in the "junction" so formed provides a mechanism for the storage of electrical energy. Thus while the system is basically a "conductor" the presence of transition regions of fixed charge give rise to the additional property of capacitance. Experimental data are presented on ionic and p-n junctions. The implications of this mechanism for the physical basis of capacitance in biological cells are discussed.

The ventral photoreceptors of Limulus polyph,mus are unipolar cells with large, ellipsoidal somas located long both "lateral olfactory nerves." As a consequence of their size and location, the cells are easily impaled with microelectrodes. The cells have an average resting potential of --48 my. The resting potential is a function of the external concentration of K. When the cell is illuminated, it gives rise to the typical "receptor potential" seen in most invertebrate photoreceptors which consists of a transient phase followed by a maintained phase of depolarization. The amplitude of the transient phase depends on both the state of adaptation of the cell and the intensity of the illumination, while the amplitude of the maintained phase depends only on the intensity of the illumination. The over-all size of the receptor potential depends on the external concentration of Na, e.g. in sodium-free seawater the receptor potential is markedly reduced, but not abolished. On the other hand lowering the Ca concentration produces a marked enhancement of both components of the response, but predominantly of the steady-state component. Slow potential fluctuations are seen in the dark-adapted cell when it is illuminated with a low intensity light. A spike-like regenerative process can be evoked by either the receptor potential or a current applied via a microelectrode. No evidence of impulse activity has been found in the axons of these cells. The ventral photoreceptor cell has many properties in common with a variety of retinular cells and therefore should serve as a convenient model of the primary receptor cell in many invertebrate eyes. I N T R O D U C T I O NT h e ionic basis for receptor potentials in photoreceptors has been investigated in only a few preparations, namely, the cells in the o m m a t i d i a of the horseshoe c r a b (Kikuchi, Naito, and T a n a k a , 1962), the retinular cells of the crayfish (Eguchi, 1965), and the retinular cells of the h o n e y b e e d r o n e (Fulpius and B a u m a n n , 1969). T h e s e preparations all have the disadvantages of being quite small and e m b e d d e d in a meshwork of pigment-containing cells and glial cells. In the studies cited above it was shown that sodium is ira3Io

1. Characterizing thermal acclimation is a common goal of eco-physiological studies and has important implications for models of climate change and environmental adaptation. However, quantifying thermal acclimation in biological rate processes is not straightforward because many rates increase with temperature due to the acute effect of thermodynamics on molecular interactions. Disentangling such passive plastic responses from active acclimation responses is critical for describing patterns of thermal acclimation.2. Here, we reviewed published studies and distinguished between different study designs measuring the acute (i.e. passive) and acclimated (i.e. active) effects of temperature on metabolic rate. We then developed a method to quantify and classify acclimation responses by comparing acute and acclimated Q 10 values. Finally, we applied this method using meta-analysis to characterize thermal acclimation in metabolic rates of ectothermic animals.3. We reviewed 258 studies measuring thermal effects on metabolic rates, and found that a majority of these studies (74%) did not allow for quantifying the independent effects of acclimation. Such studies were more common when testing aquatic taxa and continue to be published even in recent years. 4.A meta-analysis of 96 studies where acclimation could be quantified (using 1,072 Q 10 values) revealed that 'partial compensation' was the most common acclimation response (i.e. acclimation tended to offset the passive change in metabolic rate due to acute temperature changes). However, 'no acclimation' and 'inverse compensation', in which acclimation further augmented the acute change in metabolic rate, were also common. 5. Acclimation responses differed among taxa, habitats and with experimental design. Amphibians and other terrestrial taxa tended to show weak acclimation responses, whereas fishes and other aquatic taxa tended to show stronger compensatory responses. Increasing how long the animal was allowed to adjust to a new test temperature increased the acclimation response, but body size did not.Acclimation responses were also stronger with longer acclimation durations.