Neuromuscular junctions and L-glutamate-sensitive sites in the fleshfly, Sarcophaga bullata.

Sites have been located on retractor unguis and trochantal depressor muscle fibres of Sarcophaga which respond to iontophoretic application of l-glutamate. No such sites could be found on flight muscle fibres. Ultrastructural examination of the three muscles reveals differences between the muscles in the positions of the neuromuscular junctions. A correlation can be made between the sites of the neuromuscular junctions and the iontophoretically sensitive sites. The possibility of l-glutamate fulfilling a transmitter rôle in these muscles is discussed.     

The elusive role of l-glutamate as an echinoderm neurotransmitter: evidence for its involvement in the control of crinoid arm muscles

Although l-glutamate is the most widespread excitatory neurotransmitter in vertebrate and invertebrate nervous systems, there is only sparse evidence that it has this role in echinoderms. Following our previous finding that l-glutamate is widely distributed in the arms of the featherstar (crinoid echinoderm) Antedon mediterranea and initiates arm autotomy (defensive detachment), we now provide evidence of glutamatergic involvement in the control of the arm muscles of the same species using immunocytochemical and physiological methods. Immunofluorescence and immunoenzymatic techniques, which employed the same polyclonal antibody against l-glutamate conjugated to glutaraldehyde, revealed a high level of glutamate-like reactivity in the brachial muscles. By recording the mechanical responses of isolated arm pieces, we found that l-glutamate, l-aspartate and elevated [K⁺]_o induced rhythmic muscle contractions, while glycine, γ-aminobutyric acid, adrenaline and acetylcholine had either no, or no consistent, effect. The frequency and duration of the dominant component of the rhythmic contractions indicated that these may be responsible for the rhythmic activity of the arms that occurs during swimming and after autotomy. We conclude that it is highly likely that l-glutamate has at least a neuromodulatory role in the neural pathways controlling the brachial muscles of A. mediterranea.     

The Quantal Nature of Synaptic Transmission at the Neuromuscular Junction of a Spider

Spontaneous and evoked release of transmitter at neuromuscular junctions in three different leg muscles of a tarantula (Dugesiella hentzi) was investigated. In most cases the spontaneous miniature potentials were released independently, although bursts from single synaptic junctions occasionally occurred. In contrast to recent findings in other arthropod muscles, focal extracellular recording from junctional areas revealed that the evoked release of transmitter quanta followed Poisson's theorem at low quantal content synaptic junctions in arachnid muscles.     

Degree of neuromuscular facilitation is correlated with contribution to walking in leg muscles of two species of crab

Despite decades of work on the neuromuscular physiology of crustacean leg muscles, little is known about how physiological differences between these muscles relate to their behavioral usage. We studied a sideways walking shore crab, Carcinus maenas, and a forward walking spider crab, Libinia emarginata, as part of our work to understand the neural control of locomotion. The two species differed significantly in facilitation at neuromuscular junctions for every muscle studied. Further, these differences are correlated exactly with the walking use of the muscles. The forward walking spider crab showed more facilitation in muscles which operate joints having larger ranges of motion in forward walking. Likewise, greater facilitation was seen in muscles more active during sideways walking in the predominantly sideways walking shore crab. These differences even occur between muscles innervated by the same motor neuron, and become more evident with higher stimulus frequency. The increased presynaptic facilitation might allow selective recruitment of fibers innervated by the same motor neuron and aid in temporal filtering. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Neuromuscular activation of vastus intermedius muscle during fatiguing exercise

The purpose of this study was to investigate neuromuscular activation of the vastus intermedius (VI) muscle during fatiguing contraction. Seven healthy men performed sustained isometric knee extension exercise at 50% of maximal voluntary contraction until exhaustion. During the fatiguing task, surface electromyograms (EMGs) were recorded from four muscle components of the quadriceps femoris muscle group: VI; vastus lateralis (VL); vastus medialis (VM); and rectus femoris (RF) muscles. For the VI muscle, our recently developed technique was used. Root mean square (RMS) and median frequency (MF) of the surface EMG signal were calculated and these variables were then normalized by the value at the beginning of the task. Normalized RMS of the VI muscle resembled those of the other three muscles at all given times. At 95% of exhaustion time, normalized MF of the VI muscle was significantly higher than that of the VL muscle (p < 0.05). These results suggested that neuromuscular activation is not consistent between the VI and VL muscles at the exhaustion for isometric submaximal contraction and this could reflect the dissimilar intramuscular metabolism between these muscles.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.)

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K_m using alanine as substrate and catalytic efficiency (k_cat/K_m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K_m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.     

Excitatory neurotransmitters in the tentacle flexor muscles responsible for space positioning of the snail olfactory organ

Recently, three novel flexor muscles (M1, M2 and M3) in the posterior tentacles of the snail have been described, which are responsible for the patterned movements of the tentacles of the snail, Helix pomatia. In this study, we have demonstrated that the muscles received a complex innervation pattern via the peritentacular and olfactory nerves originating from different clusters of motoneurons of the cerebral ganglia. The innervating axons displayed a number of varicosities and established neuromuscular contacts of different ultrastructural forms. Contractions evoked by nerve stimulation could be mimicked by external acetylcholine (ACh) and glutamate (Glu), suggesting that ACh and Glu are excitatory transmitters at the neuromuscular contacts. Choline acetyltransferase and vesicular glutamate transporter immunolabeled axons innervating flexor muscles were demonstrated by immunohistochemistry and in Western blot experiments. Nerve- and transmitter-evoked contractions were similarly attenuated by cholinergic and glutamatergic antagonists supporting the dual excitatory innervation. Dopamine (DA, 10^?5 M) oppositely modulated thin (M1/M2) and thick (M3) muscle responses evoked by stimulation of the olfactory nerve, decreasing the contractions of the M1/M2 and increasing those of M3. In both cases, the modulation site was presynaptic. Serotonin (5-HT) at high concentration (10^?5 M) increased the amplitude of both the nerve- and the ACh-evoked contractions in all muscles. The relaxation rate was facilitated suggesting pre- and postsynaptic site of action. Our data provided evidence for a DAergic and 5-HTergic modulation of cholinergic nerves innervating flexor muscles of the tentacles as well as the muscles itself. These effects of DA and 5-HT may contribute to the regulation of sophisticated movements of tentacle muscles lacking inhibitory innervation.     

Neuromuscular control of prey capture in frogs.

While retaining a feeding apparatus that is surprisingly conservative morphologically, frogs as a group exhibit great variability in the biomechanics of tongue protraction during prey capture, which in turn is related to differences in neuromuscular control. In this paper, I address the following three questions. (1) How do frog tongues differ biomechanically? (2) What anatomical and physiological differences are responsible? (3) How is biomechanics related to mechanisms of neuromuscular control? Frog species use three non-exclusive mechanisms to protract their tongues during feeding: (i) mechanical pulling, in which the tongue shortens as its muscles contract during protraction; (ii) inertial elongation, in which the tongue lengthens under inertial and muscular loading; and (iii) hydrostatic elongation, in which the tongue lengthens under constraints imposed by the constant volume of a muscular hydrostat. Major differences among these functional types include (i) the amount and orientation of collagen fibres associated with the tongue muscles and the mechanical properties that this connective tissue confers to the tongue as a whole; and (ii) the transfer of intertia from the opening jaws to the tongue, which probably involves a catch mechanism that increases the acceleration achieved during mouth opening. The mechanisms of tongue protraction differ in the types of neural mechanisms that are used to control tongue movements, particularly in the relative importance of feed-forward versus feedback control, in requirements for precise interjoint coordination, in the size and number of motor units, and in the afferent pathways that are involved in coordinating tongue and jaw movements. Evolution of biomechanics and neuromuscular control of frog tongues provides an example in which neuromuscular control is finely tuned to the biomechanical constraints and opportunities provided by differences in morphological design among species.     

Myogenesis in the thoracic limbs of the American lobster

Newly hatched lobster larvae have biramous thoracic limbs composed of an endopodite, which is used for walking in the adult, and an exopodite used for swimming. Several behavioural and physiological aspects of larval locomotion as well the ontogeny of the neuromuscular system have been examined in developing decapod crustaceans. Nevertheless, the cellular basis of embryonic muscle formation in these animals is poorly understood. Therefore, the present report analyses muscle formation in embryos of the American lobster Homarus americanus Milne Edwards, 1837 (Malacostraca, Eucarida, Decapoda, Homarida) using the monoclonal antibody 016C6 that recognizes an isoform of myosin heavy chain. 016C6 labelling at 25% of embryonic development (E25%) revealed that syncytial muscle precursor cells establish the muscles in the endopodites. During subsequent embryogenesis, these muscle precursors subdivide into several distinct units thereby giving rise to pairs of antagonistic primordial muscles in each of the successive podomeres, the layout of which at E45% already resembles the arrangement in the adult thoracopods. The pattern of primordial muscles was also mapped in the exopodites of thoracic limbs three to eight. Immunohistochemistry against acetylated α-tubulin and against presynaptic vesicle-associated phosphoproteins at E45% demonstrated the existence of characteristic neural tracts within the developing limbs as well as putative neuromuscular synapses in both the embryonic exo- and endopodites. The results are compared to muscle development in other Crustacea.     

A metabotropic l-Glutamate receptor agonist: Pharmacological difference between rat central neurones and crayfish neuromuscular junctions

1. 2S,3S,4S-2-(carboxycyclopropyl)glycine (l-CCG-I), a conformationally restricted glutamate analogue, is a potent metabotropic l-glutamate receptor agonist in the mammalian central nervous system.2. Depolarizing actions of l-CCG-I and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) in the newborn rat spinal motoneurone are temperature-sensitive, and are not depressed by 3-[(±)-2-carboxypiperazin-4-yl] propyl-1-phosphonic acid (CPP) and/or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).3. l-CCG-I and trans-ACPD induced oscillatory responses in Xenopus oocytes injected with rat brain mRNA. Oocytes with oscillatory responses to l-CCG-I and trans-ACPD showed reversal potential of about −20 mV, which was very close to the equilibrium potential of chloride ions.4. In rat hippocampal synaptoneurosomes, l-CCG-I stimulated phosphoinositide hydrolysis in a concentration dependent manner. l-CCG-I was less potent than quisqualate but more potent than trans-ACPD.5. At low concentrations, l-CCG-I did not cause any depolarization of newborn rat spinal motoneurones, but reduced substantially amplitudes of monosynaptic reflexes.6. At the crayfish neuromuscular junction l-CCG-I, acting presynaptically, reduced the amplitude of excitatory junctional potentials. This action was prevented by application of picrotoxin but not pertussis toxin. The actions of trans-ACPD differ from those of either l-CCG-I or ibotenate at the crayfish neuromuscular junction.7. l-CCG-I has a potential to provide further useful information on metabotropic l-glutamate receptor function.     

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.     

The influence of bar diameter on neuromuscular strength and activation: inferences from an isometric unilateral bench press

The purpose of this study was to examine the influence of two different bar diameters on neuromuscular activation and strength. The bar diameters used reflected a standard Olympic bar (28 mm (1.1 inch); THIN) and a larger fat bar (51 mm [2 inch]; THICK). Eighteen healthy men (age 25.0 +/- 1 years) were assessed for their maximal voluntary contraction (MVC) during a unilateral isometric bench press exercise with the 2 bar types at 2 different joint angles (angle 1 and angle 2; elbow joint at approximately 45 and 90 degrees , respectively). Additionally, on a separate day, subjects performed three 10-second isometric repetitions at an intensity of 80% MVC using the 2 different bars at angle 1 and angle 2. Electromyographic recordings were collected in the pectoralis major and the muscles of the forearm flexor region at a sampling rate of 1000 Hz during the second day of testing. Analysis of variance was used to examine differences in MVC between bars and also examine between bar differences in electromyographic activity for each muscle group at each joint angle. A significance level of 0.05 was used for all tests. MVC was not different between bar types, although there was a main effect of joint angle on MVC such that it was greater at angle 2. There was a main effect of bar at both angles for the forearm muscles and at angle 1 for the pectoralis such that electromyographic activity was greater with THIN. Our data do not support the hypothesis that bar diameter influences performance during an isometric bench press exercise. However, higher electromyographic activity with THIN suggests greater neuromuscular activation with a standard Olympic bar as opposed to a larger diameter "fat" bar. Although our data do not support the use of a fat bar for increasing neuromuscular activation, these findings should be confirmed in other resistance training exercises.     

Tensiomyography of selected lower-limb muscles in professional soccer players

Tensiomyography is a non-invasive method of neuromuscular assessment used to measure muscle action characteristics, muscle tone, and muscle fiber type, and provides information on acute and chronic responses of muscle to different training loads. The aims of the present study were: to analyse differences in muscle response and mechanical characteristics of two major muscles of the lower extremity in a large group of Spanish soccer players according to playing position, and to provide group norms against which clinical findings may be compared. Data were collected from 78 professional soccer players (age 26.6 ± 4.4 years; height: 179.2 ± 5.3 cm; body mass: 75.8 ± 5.3 kg). Tensiomyography was recorded from the rectus femoris (RF) and biceps femoris (BF) muscles after 2 days without take part in any strenuous exercise or training. Five tensiomyographic parameters were analyzed: maximal displacement (D_m), contraction time (T_c), sustain time (T_s), delay time (T_d), and half-relaxation time (T_r). A good to excellent intra-session reliability was found for all contractile parameters (ICC ranged from 0.78 to 0.95). No significant differences between players of any position were observed in absolute values of BF. However, significant differences were observed for T_c, T_r and T_s between the different playing positions on RF (P < 0.05, effect size ranged from 1.3 to 1.6). Professional soccer players showed muscles with ability to rapidly generate force during contractions. The neuromuscular profile provided could help in identifying the normative data that are important for the different positions in order to optimize the training and recovery process of each individual player.     

Differences between substrate specificities of l-glutamate uptake by neurons and glia,studied in cell lines and primary cultures

High affinity uptake of [³H]l-glutamate was studied in cultures of continuous cell lines, originating either from mouse neuroblastoma or rat glioma, and in two types of primary cultures containing cerebellar granule cells and astrocytes from cerebral cortex, respectively. In the continuous lines, d- and l-aspartate-4-hydroxamate were found to interact preferentially with the uptake of [³H]l-glutamate in glioma cells while l-glutamate-5-hydroxamate and 2-aminoadipate interacted more strongly with [³H]l-glutamate uptake in neuroblastoma cells, d-Aspartate-4-hydroxyamate, l-glutamate-5-hydroxamate and 2-aminoadipate were inactive as inhibitors of [³H]l-glutamate uptake by either granule cells or astrocytes, grown in primary culture, but several other glutamate analogues, which did not differentiate between neuroblastomal and gliomal uptake of [³H]l-glutamate, were somewhat stronger inhibitors of [³H]l-glutamate uptake in astrocytes as compared to that in granule cells. However, all of these compounds (N-acetyl-l-glutamate, formimino-l-aspartate, d-homocysteate, l-homocysteate and dl-2-methylglutamate) were only very weak inhibitors and, consequently, it is unlikely that any of them could be useful in experiments with central nervous tissue in vivo or, at least, in brain slices in vitro, attempting to resolve the uptake of l-glutamate into glia- and neuron-localized components.     

Common and specific inhibition in leg muscles of decapods: sharpened distinctions

Crustaceans are characteristically parsimonious in their neuromuscular innervation. In extreme instances, a single efferent axon, excitatory or inhibitory, may innervate two or more muscles that have totally different actions. In particular, the inhibitory axons of the reptantian decapod leg have been reported, in various studies within four different infraorders, to innervate anywhere from one to all seven of the leg's distal muscles and to vary in number from two to four. These axons' often inexplicable combinations of target muscles have in many cases precluded interpretation of their behavioral significance. Recent findings reviewed in this paper suggest that in fact all reptants share the same three inhibitory axons: one is a universal common inhibitor, making synaptic connections within all leg muscles; the other two are specific (single-target) inhibitors of the opener and stretcher muscles, respectively (muscles which share a single excitatory axon as their sole source of activation even though they act on different joints). The literature suggests two distinct roles in the control of limb movement for these two classes of inhibitors.     

Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Neuromuscular alterations during walking in persons with moderate knee osteoarthritis.

This paper compared the neuromuscular responses during walking between those with early-stage knee osteoarthritis (OA) to asymptomatic controls. The rationale for studying those with mild to moderate knee OA was to determine the alterations in response to dynamic loading that might be expected before severe pain, joint space narrowing and joint surface changes occur. We used pattern recognition techniques to explore both amplitude and shape changes of the surface electromyograms recorded from seven muscles crossing the knee joint of 40 subjects with knee OA and 38 asymptomatic controls during a walking task. The principal patterns for each muscle grouping explained over 83% of the variance in the waveforms. This result supported the notion that the main neuromuscular patterns were similar between asymptomatic controls and those with OA, reflecting the specific roles of the major muscles during walking. ANOVA revealed significant (p<0.05) differences in the principal pattern scores reflecting both amplitude and shape alterations in the OA group and among muscles. These differences captured subtle changes in the neuromuscular responses of the subjects with OA throughout different phases of the gait cycle and most likely reflected changes in the mechanical environment (joint loading, instability) and pain. The subjects with OA attempted to increase activity of the lateral sites and reduce activity in the medial sites, having minimal but prolonged activity during late stance. Therefore, alterations in neuromuscular responses were found even in this high functioning group with moderate knee OA.     

The relationship between the lethal freezing temperatures and the amounts of ice formed in the foot muscle of marine snails (Mollusca: Gastropoda).

The differences in the lethal freezing temperatures of the foot muscles of the marine snails used in this study were related to the vertical distributions of the snails on the shore. The muscles of the subtidal species Thais lapillus and Nassarius obsoletus were injured at temperatures that were significantly higher than those of the muscles of the intertidal species Littorina obtusata, Littorina littorea, and Littorina saxatilis. The lethal freezing temperatures also varied among the intertidal species. The foot muscle of the high-intertidal species, L. saxatilis, was injured at a significantly lower temperature than the foot muscles of the low-intertidal species L. obtusata.Calorimetry was used to show that the differences in the lethal freezing temperatures between the subtidal and intertidal snails were related to the amounts of tissue ice formed. The ability of the muscles of the intertidal snails to tolerate lower subfreezing temperatures was associated with an increased tolerance to greater quantities of tissue ice. In contrast, the differences in the lethal freezing temperatures among the intertidal species were independent of the amounts of tissue ice formed. The percentage of water frozen in the muscles of these snails at their respective lethal freezing temperatures were not significantly different and were equal to 82%. Thus, the physiological mechanism responsible for the differences in the lethal freezing temperatures of the muscles of the intertidal snails is associated with an increased tolerance to a factor other than the amounts of tissue ice formed.