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Introduction: Our aim was to determine the psychological and educational impact of the 2017 Las Vegas mass shooting on the graduate medical education (GME) mission within two cohorts of resident physicians and attending faculty at two nearby academic trauma centers., Methods: A cross-sectional survey assessed 55 resident physicians and attending faculty involved in the acute care of the patients from the mass shooting. We measured the psychological impact of the event, post-traumatic growth, team cohesion, social support, and known risk factors for post-traumatic stress disorder (PTSD). Additionally, we assessed the impact of the event on GME-specific tasks., Results: Attending faculty and physicians in training in GME residencies evaluated over 300 penetrating trauma patients in less than 24 hours, and approximately 1 in 3 physicians had a patient die under their care. Despite this potential for psychological trauma, the majority of clinicians reported minimal distress and minimal impact on GME activities. However, 1 in 10 physicians screened positive for possible PTSD. Paradoxically, the minority of physicians who sought psychological counseling after the event (20%) were not those who reported the highest levels of distress. Residents generally assessed the event as having an overall negative impact on their educational goals, while attendings reported a positive impact. Psychological impact correlated inversely with social support and the amount of prior education relating to mass casualty incidents (MCI) but correlated directly with the degree of stress prior to the event., Conclusion: Despite the substantial level of exposure, most resident physicians did not report significant psychological trauma or an impact on their GME mission. Some reported post-traumatic growth. However, a minority reported a significant negative impact; institutions should consider broad screening efforts to detect and assist these individuals after a MCI. Social support, stress reduction, and education on MCIs may buffer the effects of future psychologically traumatic events on physicians in training.

CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.

A novel method is described for measuring the regional visual acuity in the vicinity of the fovea. A letter is flashed for 100 ms in one of nine positions. The size of the letters is increased or decreased systematically until the size of a letter that can be identified correctly in 75% of cases is obtained for each of the nine positions. The method has been applied to the determination of the time course of development of the acuity changes associated with a macular hole in a single case. The relationship of these changes to other changes occurring in the retina or in perception is also described. With appropriate modification the method could be applied to other parts of the retina. Some even simpler tests are also described.

We studied here the long-term maintenance of distinct populations of T helper type 1 (T(H)1)-lineage cells in vivo and found that effector T(H)1 cells, defined by their secretion of interferon-gamma (IFN-gamma), are short-lived and do not efficiently develop into long-term memory T(H)1 cells. In contrast, a population of activated T(H)1-lineage cells that did not secrete IFN-gamma after primary antigenic stimulation persisted for several months in vivo and developed the capacity to secrete IFN-gamma upon subsequent stimulation. These data suggest that a linear differentiation pathway, as defined by the transition from IFN-gamma-producing to resting memory cells, is relatively limited in vivo and support a revised model for T(H)1 memory differentiation.

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

We have investigated the responsiveness of adult guinea pig Schwann cells to a range of neuroligands, using ratiometric calcium imaging. The majority of cells responded to ATP (90 +/- 4%), adrenaline (57 +/- 5%), and noradrenaline (61 +/- 5%), as well as glutamate (60 +/- 5%). The number of cells responding to glutamate increased significantly (90 +/- 4%; p < 0.01) when the cells were grown in excitatory amino acid (EAA) free medium, indicating EAA-induced downregulation. Only a small number of cells (9 +/- 2%) responded to acetylcholine. Agonist and antagonist experiments show that these adult Schwann cells predominantly express ionotropic glutaminergic receptors (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isooxazolepropionic acid (AMPA), and kainate) as well as alpha1-, alpha2-, and beta-adrenoreceptors. We conclude that Schwann cells derived from adult guinea pigs express a variety of neuroligand receptors when established in culture and are particularly rich in glutamate receptors. This probably reflects a de-differentiated state important to development and regeneration.

This study tested the usefulness of Schwann cells in the repair of a severed nerve with a biosynthetic bridge or guide. Reinforced collagen nerve guides were used to bridge an 18 mm gap in the sciatic nerve of 21 young adult rats. The animals were divided into three groups and the guides were filled with: (i) more than 0.5 x 10(6) cultured syngeneic adult Schwann cells (group L, n = 12); (ii) less than 0.5 x 10(6) Schwann cells (Group S, n = 6); and (iii) phosphate buffered saline (control, n = 3). Schwann cells were pre-labelled with Hoechst dye. Regeneration was assessed functionally and histologically at 1, 2, 3 and 6 + months after surgery. Group L animals showed numerous regenerated axons surrounded by implanted Schwann cells within the first month. The total number of myelinated fibers (12.5 x 10(3)) remained above normal unoperated values (7 x 10(3)) in long-term animals. Regenerated axons were found in Group S in the third month, but no Hoechst labelled cells were found. The number of myelinated fibers (3.9 x 10(3)) remained below normal values in long-term animals. Control guides failed to support axonal regeneration. Functional recovery was evident at week 20 (Group L) and week 30 (Group S) after surgery, with no difference in function between the two groups by the end of the study. Supplementing guides with Schwann cells enhances regeneration of peripheral axons over a distance normally prohibitive. This effect is greatest in the early stages of regeneration (1-3 months) and is dependent on the number of cells implanted.

1. The effect on sarcomere organization of stretching intact single skeletal muscle fibres by 50% of their optimum length (Lo) during ten consecutive short tetani was investigated. Stretch reduced tetanic force to 36 +/- 4% of the pre-stretch condition. Sarcomere organization was analysed using both electron and confocal microscopy. For confocal microscopy the striation pattern was examined by fluorescently staining F-actin with rhodamine-phalloidin. 2. Electron microscopy revealed that fibres which had been stretched during contraction contained areas of severe sarcomere disorganization, as well as adjacent sarcomeres of normal appearance. 3. Confocal images of stretched fibres, which had been fixed and stained with rhodamine-phalloidin, showed focal regions of overstretched sarcomeres and regions where sarcomeres of adjacent myofibrils were out of alignment with each other. Analysis of all sarcomeres along the length of fibres showed regions of sarcomere inhomogeneity were distributed throughout the fibre length and cross-section. 4. Fibres were microinjected with the fluorescent [Ca2+]i indicator fura-2 before being stretched. Conventional wide-field fluorescence imaging microscopy showed that the tetanic [Ca2+]i was reduced after stretching but remained uniformly distributed. 5. This study confirms the finding that stretch-induced muscle injury has components caused by disorganization of the myofibrillar array and by failure of tetanic Ca2+ release. The structural damage is spatially heterogeneous whereas the changes in Ca2+ release appear to be spatially homogeneous.

We have previously reported that extracellular ATP causes a transient rise in intracellular calcium concentration ([Ca2+]i) in cultured Schwann cells derived from adult animals [Ansselin A. D. et al. (1994) Int. J. Neurosci. 74, 148]. In this study, the receptor mediating this response has been characterized. Established adult rat and rabbit Schwann cell cultures were loaded with fura-2 (acetoxymethyl ester, 10 micromol/l, 40 min, 37 degrees C). which indicated, by fluorescence imaging, a resting [Ca2+]i of 34.7 +/- 1.4 nmol/l (mean S.E., n=591). The cells were exposed to 100 micromol/l ATP, ADP, AMP, UTP and adenosine in defined medium for 1-2 min, and the change in [Ca2+]i was observed as a change in the Fura-2 ratio. Seventy-seven percent of adult rat Schwann cells (n=235) and 88% adult rabbit Schwann cells (n=356) responded to the presence of extracellular ATP (100 mmol/l) with a transient increase in [Ca2+]i (41 and 90 nmol/l from resting value, respectively), independent of the presence of [Ca2+]o. Calcium waves were observed in one experiment. The following order of agonist potency was observed: UTP= ATP>>ADP>AMP=adenosine. The agonists alpha,beta-methylene-ATP and 2-methylthio-ATP had a small effect on the cells, similar to AMP, and were mutually desensitizing. The ATP antagonist suramin blocked the response. We conclude that adult Schwann cells express a purinergic ATP receptor belonging to the G-protein-coupled P2u alpha subtype [O'Connor S. et al. (1991) Trends pharmac. Sci. 12, 137-141].

A study has been made of the distribution of nitric oxide synthase (NOS) in the developing avian ciliary ganglion. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity first appeared in ciliary neurones at embryonic day 10 (E10). The number of NADPH-d positive neurones appeared maximal at this age and thereafter declined; at post hatched day 4 (P4) these neurones were found predominately in the periphery of the ganglion. At the light microscope level the NADPH-d stain appeared throughout the cell soma of the ciliary neurones. This was confirmed using tissue culture techniques. Ultrastructural delineation of horseradish peroxidase-labelled NOS antibodies was also found in the calyx where it was bound to the membranes of the endoplasmic reticulum as well as to the outer membranes of mitochondria. This distribution of NOS in the soma and calyx is consistent with the physiological role of NO as a co-transmitter and retrograde messenger that regulates the quantal secretion of the principal transmitter, acetylcholine, from the calyx.

The effect of proximo-distal orientation of peripheral nerve grafts upon axonal regeneration has been investigated using the sciatic nerve of the rat as a model. To test the hypothesis that the presence of nerve branches within a graft will cause misdirection of axons in normally oriented grafts but not in reversed grafts, all grafts studied contained branches. Qualitative electron microscopic examination of graft ultrastructure revealed no differences in nerve structure related to graft orientation. In most normally oriented grafts, branches persisted up to 12 months after surgery. These branches contained axons which terminated at the end of the branch. In all reverse oriented grafts, and in a small number of normally oriented ones, the branches could not be seen after two or more months of regeneration. Axons sprouting outside of the epineurium of the graft caused the branch to be incorporated into the nerve structure. Axon counts in the distal stump of grafted nerves after twelve months recovery revealed that normally oriented grafts with persistent branches led to poorer peripheral regeneration, especially of unmyelinated fibers. The results indicate that regeneration of axons to their peripheral targets may be facilitated by reversing the graft orientation.

Immunologically incompatible sciatic nerve grafts were inserted into the severed sciatic nerves of Wistar rats. In an attempt to induce graft tolerance, low-dose cyclosporin A (CsA) was administered to some animals for 20 weeks, then gradually withdrawn. Behavioural, electrophysiological and histological studies indicated that some degree of regeneration took place in all animals regardless of treatment. Neither a daily dose of 5 mg/kg nor 10 mg/kg was sufficient to prevent the rejection and subsequent disruption of allograft structure, and as a consequence reinnervation of the distal stump was limited. This was manifest both in the poor functional recovery of the denervated foot, and in the large number of regenerated axons found outside of the perineurial membranes of the transplanted fascicles. Therefore, tolerance was not induced at these doses. Furthermore, the significant decrease in the amplitude of electromyographs recorded from experimental and unoperated (control) animals suggests CsA may have a deleterious effect on unlesioned nerve even at these low doses. It would be prudent, therefore, to exercise caution in the combined use of nerve allografts and CsA immunosuppression, until the neurotoxicity of CsA has been investigated further. This is particularly important since CsA is sometimes used in the treatment of certain neuropathic autoimmune diseases.

Solid rhodamine B isothiocyanate microsurgically implanted into mammalian peripheral nerve labels a small number of axons within that nerve. These axons can be observed in whole mounts or slices following fixation of the tissue approximately 16 h after the rhodamine is applied. The labelling appears to involve axoplasmic transport in both directions, for axons can be followed over long distances in the whole mounts. Since only some axons are labelled, their structure can be observed within large populations of cells in a whole nerve. This is valuable in interpreting axon counts in developmental and regenerative studies, and in observing the pattern of sprouting at regenerating axon terminals.

A study has been made of the growth of segmental nerves 13 to 16 (SN13 to SN16) into the chick limb bud, from the time when they have just reached the ends to the brachial myotomes (stage 21: Hamburger and Hamilton, '51), until they enter the newly formed ventral (stage 24) and dorsal (stage 25) pre-muscle cell masses in the limb bud. At stage 22 axon bundles of SN13 to SN16 have grown off the ends of their respective myotomes, and converge towards the most densely packed mesenchyme in the limb bud at segmental level 15. As a consequence, the first axon bundles of SN14 and SN16 have almost joined those of SN15, whereas the further removed SN13 axon bundles have not yet reached the level of SN15. By stage 23 the first axon bundles from SN14 to SN16 have joined at segmental level 15 to form a nerve which grows toward the ventral pre-muscle cell mass. At stage 24 axon bundles from SN13 have joined those from SN14 to SN16 to form the brachialis longus inferior nerve, which enters the densest region of the ventral pre-muscle. Other axons from SN13 to SN15 grow along the pathways provided by the early arriving axon bundles towards the ventral pre-muscle, but diverge from those at segmental level 14 to grow to the dorsal pre-muscle. By stage 25 axon bundles from SN13 to SN15 have joined to form the brachialis longus superior nerve which enters the densest region of the dorsal pre-muscle. At stage 26 a plexus has formed due to this pattern of growth of the segmental nerves between stages 22 and 25. It is suggested that pre-muscle cells synthesize a nerve growth factor which directs the growth of axons into the limb bud.

A study has been made of the growth of cervical nerves C3-C6 to the rat diaphragm. At 11 days of embryonic age these cervical nerves first project out of the spinal cord toward the cardinal veins and later form the left and right phrenic nerve trunks. During the next 2 days, the phrenic nerves grow caudally in close association with the cardinal veins toward the diaphragm. At the growing tips of these nerve trunks the growth cones of axons were observed every 1-2 micrometers. The last axon did not project more than 2 micrometers ahead of any neighbouring axons. At 14 days the phrenic nerves reach the level of the developing diaphragm and converge into pools of premuscle cells. Previous studies have suggested that the phrenic nerve enters the premuscle masses of the diaphragm at an early developmental stage when the premuscle masses are at approximately the segmental levels C3-C6. This study shows that the phrenic nerves must grow to more caudal levels in order to reach the premuscle cells of the diaphragm. Furthermore, the leading axons of the phrenic nerve trunk do not project in a pioneering fashion, far in advance of the trailing axons.

The extraocular muscles of the carp Carassius contain two types of muscle fibre. Large white fibres have ribbon-shaped peripheral myofibrils and triads located at the Z line. Small red fibres, rich in mitochondria, have polygonal-shaped myofibrils and triads at the A-I junction. Silver- and cholinesterase-stained preparations show that the large fibres are innervated by axons which spiral around them and exhibit intense cholinesterase activity over long distances. Axons supplying small muscle fibres run across bundles of fibres, making one contact with each fibre. By electron microscopy the nerve endings on each fibre type appear identical, both having a smooth post-junctional muscle membrane. The differences in structure and innervation pattern of the two fibre types are discussed in relation to their possible functional roles.

A description is given of the growth of segmental nerves 13 to 16 (SN13 to SN16) from the brachial myotomes into the ventral and dorsal premuscle cell masses of the chick forelimb. At stage 25 (Hamburger and Hamilton, '51), segmental nerves have converged to form two nerve trunks which enter the ventral and dorsal premuscle cell masses. These nerves grow into the limb, parallel to its proximodistal axis. The nerve trunk in the ventral compartment, the brachialis longus inferior (bli n), grows on the brachial artery as far as the metacarpals. The nerve trunk in the dorsal compartment, the brachialis longus superior (bls n), grows on the precartilage adjacent to a dorsal premuscle density as far as the metacarpals. The brachialis longus inferior and superior nerve trunks give rise to a number of nerves during their growth to the metacarpals. Each of these nerves appears at the time of formation of a new premuscle cell density. At late stage 25, a posterior and ventral premuscle density forms in the middle of the stylopodium; posterior fascicles of the bli n diverge at this level and grow toward this new density to form the ulnar nerve. By stage 26 a posterior and ventral premuscle density is prominent over the bli n, at the junction of the stylopodium and the zeugopodium; ventral fascicles of the bli n grow into this density to form the flexor digitorum profundus nerve. At this stage a posterior and dorsal premuscle density forms just beyond the junction of the stylopodium and zeugopodium. Posterior fascicles of the bls n diverge at this level and grow toward this density to form a nerve from which the extensor metacarpi ulnaris, extensor digitorum communis, and radialis laterialis nerves are destined to form. At stage 29 the premuscle cell masses of the autopodium have formed. The remaining nerve fascicles comprising the bls n (the radialis profundus nerve) grow into the autopodium and branch to innervate each of the dorsal premuscles. The remaining nerve fascicles comprising the bli n (the middle nerve) grow into the autopodium and branch to innervate each of the ventral premuscles.

At the time of hindlimb development in the axolotl there is a well-established but still developing trunk innervation. The trunk innervation is primarily composed of the segmental nerves, each of which consists of a dorsal and a ventral ramus and its branches. At a few segmental levels in the region of the hindlimb a large number of additional axons arise to innervate the limb. To reach the limb, they must grow via the ventral rather than the dorsal rami. The precision with which this pathway is selected was determined by counting axons in the dorsal and ventral rami at limb segmental levels, prior to and during the period of maximum axon proliferation. If outgrowth is highly directed rather than random, then the ratio of ventral/dorsal ramus axons should increase significantly during the period when large numbers of additional axons are produced. In addition, since the dorsal trunk varies little in size between limb segments and immediately caudal 'non-limb' segments, the number of axons in the dorsal rami can be compared at the two levels. Mistaken projections should result in inordinately large axon numbers in dorsal rami at limb compared to non-limb levels. The results show that there is approximately a tenfold increase in the ratio of ventral/dorsal ramus axons at the time of maximum outgrowth to the limb, thus the mode of distribution at the ventral-dorsal branch point is significantly altered in favour of growth toward the limb, and outgrowth appears to be highly directed rather than random. Moreover at this time there is no discernible increase in the number of dorsal ramus axons at limb levels while those at non-limb levels increase fourfold. The apparent growth of all axons into the ventral ramus suggests the presence of a strong, non-specific attraction.

The secretion of a quantum from groups of release sites (me) declines along the length of terminal branches at the amphibian neuromuscular junction. The morphological basis of this decline in me has been studied at neuromuscular junctions in juvenile muscles (fibre length 4 mm) and adult muscles (fibre length 22 mm). Serial sections cut through the length of the junctions have been examined with both light and electron microscopy. Juvenile junctions consist of two short (less than 50 micron) terminal branches; adult junctions often consist of 4 long (100-500 micron) terminal branches. Synaptic contacts are largest near the origins of terminal branches and decline in size towards the end of branches. The number of horseradish peroxidase-labelled synaptic vesicles at release sites, following stimulation in the presence of the enzyme, is largest for sites closest to the origin of the terminal branches. The results suggest that the decline in me along the length of terminal branches is due to decline in the size of release sites.

Peripheral nerve transplants are used for the surgical repair of nerve loss. If normally oriented grafts contain branches, regenerating axons may be lost into them, limiting re-innervation and restoration of function in the denervated area. Since axons entering a reversed graft may not be lost in this way, we have investigated whether reverse implantation could enhance the number of axons that reach the distal stump. In young adult rats, a section of sciatic nerve, including one major branch, was removed and reinserted with either the normal or reversed proximo-distal orientation, using microsurgical techniques. After signs of recovery of function, the operated and contralateral unoperated nerves of each animal were fixed and processed for electron microscopy. Regenerated axons were seen to enter branches in normally orientated grafts, and there was substantial loss in the cross-sectional area of the graft distal to the branch termination. Reverse grafts are as well reinnervated by regenerating axons as normally oriented grafts and show a smaller loss in the cross-sectional area, even though the branches of these implants seemed to disappear.

A study was made of the decline in the number of motor neurons and axons of the brachial spinal cord of the rat during postnatal development. The injection of horseradish peroxidase (HRP) into the biceps muscle showed that it was innervated by motor neurons located in the dorsolateral position of the lateral motor column in segments C5 and C6; HRP injections into the triceps muscle showed that it was innervated by motor neurons located in the ventrolateral position of the lateral motor column in segments C7 and C8. There was no change in the position of these motor neuron pools between birth and maturity. However, there was a decline in the number of neurons in each pool during the postnatal period; over 35% of the neurons present at birth had disappeared by maturity. This loss of neurons was uniform throughout the rostrocaudal extent of each pool. It was accompanied by a similar percentage loss in the number of axons in a ventral root at the branchial level (C8). Electrophysiological measurements showed that the disappearance of motor neurons was accompanied by a loss in the polyneuronal innervation of synaptic sites in the biceps muscle. The possibility that a decrease in the number of neurons contributes to the loss of polyneuronal innervation is discussed.

(1) The effects of glycerol-treatment on the ultrastructure, tension, and electrical properties of rat sternomastoid muscle fibers are described. (2) The effect upon the ultrastructure of fibers differed from that previously reported for amphibian fibers, in that the sarcoplasmic reticulum, as well as the T-system, was disrupted. (3) Tension (tension (tetanus and K-contracture) was abolished when preparations were returned to normal Krebs after exposure to a glycerol-Krebs solution (exposure periods were 1 hr in 200--350 mM-glycerol or 10--60 min in 350 mM-glycerol), although fibers had normal resting membrane potentials and action potentials. (4) Fibers treated for 1 hr with 350 mM-glycerol were detubulated when returned to normal Krebs. Specific membrane capacity was reduced and exogenous horseradish peroxidase (HRP) did not penetrate the T-system. (5) Fibers were not detubulated after treatment for 1 hr with 200 to 300 mM-glycerol or after treatment for 10 to 30 min with 350 mM glycerol. Specific membrane capacity and resistance were normal and HRP entered the T-system. (6) Ultrastructural disruption of the triad junction became progressively more extensive with increasing glycerol concentration used and may be responsible for uncoupling.

A study has been made of the factors which determine that the terminals of a foreign flexor nerve at synaptic sites in a unrodele extensor muscle regress on return of the original extensor nerve. The quantal content (m) of the endplate potential (EPP) at flexor nerve terminal synapses, during innervation of a previously denervated extensor muscle, increased in about 8 weeks to reach the same size as at normal extensor nerve terminals; the same time was taken for m to reach normal size at extensor nerve terminals when these reinnervated their own muscle. At flexor nerve terminals, m decreased eventually to zero if the extensor nerve terminals returned within about 6 weeks of synapse formation by the flexor nerve terminals to the same or an immediately adjacent synaptic site to that occupied by these terminals. During this decrease in m at flexor nerve terminals, stimulation of the flexor nerves in the presence of horseradish peroxidase (HRP) showed HRP-labelled flexor nerve terminals present only in those regions of the extensor muscle in which the electrical signs of flexor nerve terminals were observed, indicating that the decrease in m at regressing flexor terminals was accompanied by their vacating synaptic sites. However, flexor nerve terminals failed to regress from the extensor muscle on return of the original nerve supply if the flexor nerve allowed to form synapses for more than about 10 weeks before the return of the extensor nerve.

1. The fine structure of rabbit rectococcygeus muscle has been studied with the electron microscope. 2. The mechanical performance and the heat production of this muscle has been investigated during tetanic contractions at 27 degrees C. 3. In isometric contractions a force of 164 +/- 27 mN/mm2 (mean +/- S.D., n = 17) is developed and the heat production is linearly related to the force. 4. There is a relationship between the duration of stimulation (t) and the total heat production (H) of the type H = A plus bt, where A and b are constants. 5. After-loaded isotonic experiments show that the relationship between force and velocity can be fitted by the 'characteristic equation' of Hill (1938). 6. The value of a/P0 (0-302 +/- 0-093, mean +/- S.D.) is slightly higher than in frog skeletal muscle but the constant b is about 50 times smaller. 7. The ratio of work/total energy production, for the stimulus conditions employed, was maximally 0-185. 8. The ratio of total enthalpy to initial enthalpy is difficult to measure accurately but is probably about 2.

1. Changes in the dimensions of the sarcoplasmic reticulum in frog sartorius muscles exposed to hypertonic and hypotonic solutions have been studied with the electron microscope.2. The volume of the sarcoplasmic reticulum has been found to be linearly related with a negative slope to the reciprocal of the osmotic pressure. Over the range 0.75 to 3.5 x normal osmotic pressure the reticulum volume has been calculated to change from 11.5 to 18.5% of normal cell volume.3. These changes in sarcoplasmic reticulum volume correspond to the calculated changes in the volume of the intra-fibre sucrose compartment, postulated by earlier workers on the basis of studies on changes in cell volume with changes in osmotic pressure in living muscles.4. To explain these and other related findings on the distribution of electrolytes in muscle, it is proposed that the sarcoplasmic reticulum of skeletal muscle is an extracellular compartment.5. The significance of this hypothesis for the mechanism of excitation-contraction coupling is discussed.

The volume responses of the T-system of frog skeletal muscle to isotonic solutions of altered ionic composition, as studied by electron microscopic examination of osmium-fixed tissue, have been re-investigated using aldehyde fixatives.1. In confirmation of the earlier work we find that the T-tubes swell in frog sartorius muscles which have been soaked in Ringer solution in which the sodium chloride concentration has been reduced to 40 mM and tonicity maintained with sucrose before fixation in osmium.2. The swelling does not occur in muscles similarly treated, but fixed with acrolein or glutaraldehyde.3. Swelling of the T-tubes, which has been reported for osmium-fixed muscle following exposure to Ringer in which chloride is replaced by acetate, or reduced and sulphate substituted, does not occur when fixation is by acrolein.4. Analysis of the data from the earlier work with sucrose substitution shows that the degree of T-tube swelling is proportional to the sucrose concentration of the soaking medium, whether the solution be isotonic or anisotonic.5. It is concluded that the increase in T-tube volume arises in muscles which have been exposed to certain impermeant solutes during fixation by osmium, and a possible mechanism to account for the effect is described.6. The relevance of these observations to the hypothesis that the sarcoplasmic reticulum is an extracellular compartment in muscle and to the proposal that the T-tubes may represent an intermediate compartment for potassium fluxes is discussed.