Neurovascular activation requires conduction through vessels

In experiments on rats, a hind leg was transected except for the femoral nerve, artery and vein. The femoral nerve was stimulated by electrical pulses, with one electrode attached to the nerve in the amputation gap and another placed in the inferior caval vein. At each pulse of stimulation, contraction could be recorded in femoral muscles. Ligation of the femoral vessels suspended the contractions within a fraction of a second; contractions resumed when ligation ended. Propagation of neural stimuli to femoral muscles therefore required an intact electrical communication through associated vessels. This is possible because conducting blood plasma and its capillary junction with the interstitial fluid form an "external" closed circuit branch of low resistance with the neuromuscular unit. In testing the interstitial tissue fluid as an alternative to the vascular "outer" communication, a higher voltage of stimulation was required for muscle contractions.     

Potential differences in the inferior vena cava and between cava and extravascular electrode at leg contraction in man.

When a voluntary adult male contracted his leg muscles, electric potential differences were recorded between platinum electrodes positioned inside the vena cava or between the vena cava and the grounded skin. Two experiments confirm that potential differences at leg contractions in man have a similar appearance as those in the rat at pain-evoked contractions of leg muscles. Platinum electrodes were used in man. The potential differences obtained are thereafter compared with recordings with Ag-AgCl electrodes in salt-bridges in the rat. Slow potential waves with superimposed irregular potential oscillations are recorded in man as previously found in the rat using platinum or Ag-AgCl electrodes. The findings indicate that vascular-interstitial routes participate in a vascular-interstitial-neuromuscular closed circuit, which is activated at contraction of the muscles.     

Synaptic connections between sensory afferents and the common inhibitory motoneuron in crayfish

The sensory inputs to the common inhibitory motoneuron that innervates every leg muscle of the crayfish Procambarus clarkii (Girard) were analyzed by performing intracellular recordings from its neurite within the neuropil of the 5th thoracic ganglion. Two types of sensory inputs involved in locomotion were studied, those from a movement coding proprioceptor (the coxobasal chordotonal organ) and those from sensory neu rons coding contact forces exerted at the tip of the leg on the substrate (the dactyl sensory afferents). Sinusoidal movements applied to the chordotonal organ strand induced a stable biphasic response in the common inhibitory motoneuron that consisted of bursts of spikes during release and stretch of the strand, corresponding to raising and lowering of the leg, respectively. Using ramp movements imposed on the chordotonal strand, we demonstrated that only movement-coding chordotonal afferents produce excitatory post-synaptic potentials in the common inhibitory motoneuron; these connections are monosynaptic. Mechanical or electrical stimulation of the dactyl sensory afferents resulted in an increase in the tonic discharge of the common inhibitory motoneuron through polysynaptic excitatory pathways. These two types of sensory cues reinforce the central command of the common inhibitory motoneuron and contribute to enhancing its activity during leg movements, and thus facilitate the relaxation of tonic muscle fibres during locomotion.Abbreviations ADR anterior distal root - A Lev anterior levator nerve - CB coxo-basipodite joint - CBCO coxo-basal chordotonal organ - CI common inhibitory motoneuron - Dep depressor nerve - DSA dactyl sensory afferents - EPSP excitatory post-synaptic potential - IN interneuron - MN motoneuron - PDR posterior distal root - P Lev posterior levator nerve - Pro promotor nerve - Rem remotor nerve     

VI. Synaptic Fuel Cell Reactions in Vascular-Interstitial-Neuromuscular Closed Circuit: Theory of Neuromuscular Activation

Spontaneous pulses of voltage occur in the caval vein at voluntary contraction of a leg muscle both in the rat and in humans. Morphology and vascular reactions indicate that vascular-interstitial-neuromuscular circuits (VINMC) exist. They require redox reactions, which are likely to occur at proteins in the cellular membranes of the synapse. Metabolic degradation of ATP in the nerve cell is known to generate a flow of current out of the cell, creating the resting potential. It corresponds to an electrochemical equilibrium potential. An overpotential leads to a closed circuit flow of current in the VINMC, producing redox products at the electrode equivalent redox proteins. The VINMC is thereby charged. Brain impulses open ionic channels of the nerve cell body, short-circuiting the VINMC. Electrochemical reactions by the redox products produce an action potential and reverse the current in the charged VINMC. The charging and discharging of the synaptic membranes are explained as electrochemical analogs. The action potential and its height, production, transport, and disappearance of various synaptic products, including vesicles and the voltage pulses in the associated vessels such as the caval vein and the aorta, can be explained.     

Electrical pulses appear in the inferior vena cava and abdominal aorta at contraction of leg muscles.

A vascular-interstitial neuromuscular closed circuit is described anatomically and electrically. The neuromuscular unit has a high resistance due to its axons. The "outer" vascular-interstitial branch has a low resistance due to a large cross section area. When a rat spontaneously contracts leg muscles after pinching a toe, electric potential pulses appear inside the inferior vena cava, between vena cava and peritoneum, between aorta and peritoneum, between vena cava and peritoneum and between aorta and subcutis. Minor pulses between electrodes in the peritoneal fluid are interpreted as induced by the intravascular potential pulses. The use of Ag-AgCl electrodes or platinum electrodes does not appreciably change the results.     

Immunohistochemical localization and vascular effects of vasoactive intestinal polypeptide in skeletal muscle of the cat

Summary Scattered vasoactive intestinal polypeptide (VIP) — immunoreactive nerves were found in the striated muscle of the hind limb of the cat, where they usually were associated with small blood vessels. VIP-immunoreactive nerves were also demonstrated in the sciatic nerve; after nerve ligation an abundance of intensely immunoreactive VIP fibres were seen proximal to the ligation. Intraarterial infusion of VIP into the isolated hind limb of the cat had dramatic effects on different sections of the vascular bed. Thus, VIP dilated the resistance vessels leading to a marked increment in muscle blood flow. VIP also relaxed the capacitance vessels causing regional pooling of blood; it increased the capillary surface area available for fluid exchange. Infusions of VIP at a dose of 8 g/min significantly inhibited the vasoconstriction induced by electrical stimulation of the regional sympathetic nerves. It is suggested that local nervous release of VIP may act as a modulator of vascular tone in skeletal muscle.     

Prefabricated sensate myocutaneous and osteomyocutaneous free flaps: an experimental model. Preliminary report

Principles of neovascularization have been reported for the successful creation of a variety of muscle and bone free flaps. This study demonstrates a simple and effective technique for construction of prefabricated sensate myocutaneous and osteomyocutaneous free flaps in a rat model. These experiments were carried out in 20 Sprague-Dawley male rats. In half the animals, a sensate myocutaneous flap was constructed by sandwiching the superficial inferior epigastric vessels between a laterally based external abdominal oblique muscle flap and a laterally based skin flap served by an identified cutaneous nerve. A similar preparation included a piece of iliac crest bone. Two to three weeks later, now neovascularized by the sandwiched vessels, the flaps were harvested and transferred as free flaps with high reliability. An increased number of potential donor sites, the versatility of design, and the ability to customize flaps to the specific recipient-site needs are proffered.     

Neuromuscular Junctions in Flight and Tymbal Muscles of the Cicada

The tymbal muscle fiber in the cicada closely resembles the indirect flight muscle fiber in its structural detail. We agree with other authors that the tymbal muscle is a modified indirect flight muscle. The peripheral nerve branches to the tymbal and flight muscle fibers are similar to those in the wasp leg. The axon is loosely mantled by irregular turns of the mesaxon, enclosing cytoplasm. The nerve is therefore a tunicated nerve. The neuromuscular junction in the high frequency muscle fibers shows direct apposition of plasma membranes of axon and muscle fiber, large numbers of mitochondria and synaptic vesicles in the axon, and concentrations of mitochondria, aposynaptic granules, and endoplasmic reticulum in the postsynaptic area of the muscle fiber. Of special interest is the multitude of intracellular, opposing membranes in the postsynaptic area. They form laminated stacks and whorls, vesicles, cysternae, and tubules. They occasionally show continuity with the plasma membrane, the outer nuclear envelope, and the circumfibrillar endoplasmic reticulum. The membrane system in this area is designated "rete synapticum." It is believed to add to the electrical capacity of the neuromuscular junction, to serve in transmission of potentials, and possibly is the site of the oscillating mechanism in high-frequency muscle fibers.     

Extracellular recordings from the motor nervous system of the nematode,Ascaris suum

1. The close association of muscle and neurons in Ascaris suum makes it difficult to determine whether spikes recorded from nerve cords originate in muscle or neurons. We have developed criteria that distinguish muscle and neuronal activity. There are two categories of extracellular spikes. 2. The first category consists of spikes with a wide range of amplitudes, marked by large spikes. These spikes, which can be recorded over lateral muscle and over the dorsal and ventral nerve cords, are abolished when muscle is disrupted or removed, or when curare is applied. Large spikes are relatively infrequent, are correlated with intracellularly recorded muscle events, and respond to polarizations of motor neurons, implying that they originate in muscle. 3. The second spike category, small amplitude spikes, is exclusive to the ventral nerve cord, occurs more frequently than large spikes and displays patterned firing. Small spikes are not affected by muscle removal or by curare, and are correlated with motor neuronal post-synaptic potentials, but not with intracellularly recorded muscle events. We infer that they originate in neurons. 4. Low level activity recorded extracellularly over nerve cords may represent muscle activity due to tonic motor neuronal synaptic transmission. It responds to motor neuronal polarization and is suppressed by curare or muscle removal.     

Cobalt injections into the pedunculopontine nuclei attenuate the reflex diaphragmatic responses to muscle contraction in rats.

Previous studies have suggested that neurons in the pedunculopontine nucleus (PPN) are activated during static muscle contraction. Furthermore, activation of the PPN, via electrical stimulation or chemical disinhibition, is associated with increases in respiratory activity observed via diaphragmatic electromyogram recordings. The present experiments address the potential for PPN involvement in the regulation of the reflex diaphragmatic responses to muscle contraction in chloralose-urethane anesthetized rats. Diaphragmatic responses to unilateral static hindlimb muscle contraction, evoked via electrical stimulation of the tibial nerve, were recorded before and subsequent to bilateral microinjections of a synaptic blockade agent (CoCl2) into the PPN. The peak reflex increases in respiratory frequency (9.0 +/- 1.0 breaths/min) and minute integrated diaphragmatic electromyogram activity (14.6 +/- 3.3 units/min) were attenuated after microinjection of CoCl2 into the PPN (2.6 +/- 0.9 breaths/min and 4.6 +/- 2.1 units/min, respectively). Consistent diaphragmatic responses were observed in the subset of animals that were barodenervated. Control experiments suggest no effects of PPN synaptic blockade on the cardiovascular responses to muscle contraction. The results are discussed in terms of a potential role for the PPN in modulation of the reflex respiratory adjustments that accompany muscular activity.     

On the generation of potential differences across the membranes of ellipsoidal cells in an alternating electrical field

Summary Particles with a nonconducting membrane, oriented in an alternating electrical field, will show the behaviour of electrical dipoles. Across the membranes there will be generated alternating electrical potential differences, which may be calculated for confocal ellipsoidal cells by solving Laplace's equation. We have evaluated a formula valid generally for single confocal ellipsoidal cells under physiological conditions, the cells being placed with one of their semi-axes parallel to an external electrical field. The values of the generated potential difference, considered at the position of their maximum values, are dependent on the shape and size of the cells, on their orientation to the electrical field and on the frequency and strength of the field. The relaxation frequency depends also on cell shape, size and orientation, but furthermore on the membrane properties and on the conductivities inside and outside the cells. For simple cases like spheres and cylinders perpendicular to the electrical field, our formula will correspond to known expressions. Values for the generated potential differences, form-factors and relaxation frequencies are given for different types of spheroids and at different orientations. Of some practical importance are long prolate spheroids with their long semi-axes parallel to the external field, because only small field strengths are necessary in order to generate large potential differences which may evoke action potentialse.g. in muscle or nerve cells. The significance of this mechanism concerning the determination of protection and safeguard standards for the exposure to low-frequency electrical fields is discussed.Dedicated to Prof. Dr. Dr. h. c. mult. B. Rajewsky on the occasion of his 80th birthday.     

The action potential: an effect of fuel cell reactions in the synapse

The neuron and its vascular-interstitial communications form, in vivo, an electrophoretic closed circuit. It is charged by ionic pumps in the nerve cell membrane at rest. Electrophoretic products of reaction collect at biologic electrodes, represented by redox proteins. These are located in the pre- and postsynaptic membranes and also in associated capillary membranes in the vascular part of the closed circuit. Efferent brain impulses start a series of events preceding muscle contraction. They open ionic channels in the membrane of the nerve cell body. A short-circuiting is thereby created, and cations flow into the cell. The membrane pumps cannot withstand this ionic inflow and maintain the transmembranous potential difference. The circuit is no longer driven but starts selfdriving reactions by previously formed products of reaction at the biological electrodes. Fuel cell reactions start at these and create in the axon the peak of the action potential. In vivo, the action potentials preceding the contracting of a muscle are transmitted through the circuit. In the vascular pathway of the closed circuit, the action potentials appear, by summation, as the previously described slow potential waves. The function of nerve cell matrices, as well as the nodes of Ranvier, are discussed. The proposed theory is in accordance with the vascular-interstitial-neuromuscular closed circuit. It provides new possibilities to explain the development of the action potential, transport and disappearance of various synaptic structures and the neurotransmitter. Technical analogues are presented to illustrate a new possible background mechanism for understanding structure and function in neuromuscular transmission.     

Neural control of the expression of a Ca2+-activated K+ channel involved in the induction of myotonic-like characteristics

Summary 1. Expression of the apamin-sensitive K⁺ channel (SK⁺) in rat skeletal muscle is neurally regulated. The regulatory effect of the nerve over the expression of some muscle ion channels has been attributed to the electrical activity triggered by the nerve and/or to a trophic effect of some molecules transported from the soma to the axonal endings. 2. SK⁺ channels apparently are involved in myotonic dystrophy (MD), therefore understanding the factors that regulate their expression may ultimately have important clinical relevance. 3. To establish if axoplasmic transport is involved in this process, we used two experimental approaches in adult rats: (a) Both sciatic nerves were severed, leaving a short or a long nerve stump attached to the anterior tibialis (AT). (b) Colchicine or vinblastine (VBL), two axonal transport blockers of different potencies, was applied on one leg to the sciatic nerve. To determine whether electrical activity affects the expression of SK⁺ channels, denervated AT were directly stimulated. The corresponding contralateral muscles were used as controls. 4. With these experimental conditions we measured (a) apamin binding to muscle membranes, (b) muscle contractile characteristics, and (c) electromyographic activity. 5. In the short- and long-nerve stump experiments, 5 days after denervation¹²⁵I-apamin binding to AT membranes was 2.0 times higher in the short-stump side. This difference disappeared at longer times. The delayed expression of SK⁺ channels in the muscle left with a longer nerve stump can be attributed to the extra axoplasm contained in the longer stump, which maintains a normally repressive signal for a longer period of time. Ten to 15 days after application of axonal transport blockers we found that the muscle half-relaxation time increased in the drug-treated side and apamin partially reverted the prolonged relaxation. Myotonic-like discharges specifically blockable by apamin were always present in the drug-treated leg.¹²⁵I-Apamin binding, which is undetectable in a microsomal preparation from hind leg control muscles, was increased in the drug-treated preparations. Apamin binding to denervated and stimulated AT muscles was lower than in the contralateral unstimulated muscles [3.3±1.0 vs 6.8±0.8 (n=4) fmol/mg protein]. 6. Our results demonstrate that electrical activity and axoplasmic transport are involved in the control of expression of SK⁺ in rat skeletal muscle. However, the increased expression of this channel induces myotonic-like characteristics that are reversed by apamin. This myotonic activity could be a model for MD.     

Measurement of sympathetic nerve activity in the unanesthetized rat

A new system was developed in our laboratory to continuously monitor intra-arterial pressure, heart rate, and sympathetic nerve activity in unanesthetized rats. The animals were prepared 24 h before the start of the experiments. Sympathoneural traffic was measured at the level of splanchnic nerve. The amplitude of the spikes recorded at this level was utilized to express sympathetic nerve activity. The amplitude of the residual electroneurogram signal present 30 min after the rats were killed was 32 +/- 2 mV (mean +/- SE; n = 11). For analysis, these background values were subtracted from values determined in vivo. The nerve we studied contains postganglionic fibers, since electrical activity decreased in response to ganglionic blockade with pentolinium (1.25 mg/min iv for 4 min). The amplitude of spikes fell by 43 +/- 4% (n = 4). Sympathetic nerve activity was highly reproducible at a 24-h interval (104 +/- 26 vs. 111 +/- 27 mV for the amplitude of spikes; n = 11). Dose-response curves to the alpha 1-stimulant methoxamine and to bradykinin were established in four rats. The increase in blood pressure induced by methoxamine caused a dose-dependent fall in sympathetic nerve activity, whereas the blood pressure reduction resulting from bradykinin was associated with a dose-dependent activation of sympathetic drive. These data therefore indicate that it is possible with out system to accurately measure sympathetic nerve activity in the awake rat, together with intra-arterial pressure and heart rate.     

Model study of time‐dependent muscle response to pulsed electrical stimulation

A systems‐level model analysis of neuromuscular response to external electrical stimulation is presented. Action potential (AP) generation, dynamics of voltage‐based calcium release at the motor endplates controlled by the arrival of APs, and muscle force production are all comprehensively included. Numerical predictions exhibit trends that are qualitatively similar to measurements of muscle response in rats from a burst of cortical stimulation and a nanosecond impulse. Modulation of neural membrane conductances (including possible electroporation) that alters the neural impulse generation frequency is hypothesized as a possible mechanism leading to observed changes in muscle force production. Other possibilities such as calcium release at nerve end endings also exist. It is also proposed that multipulsing strategies and changing the electric field direction by using multielectrode systems would be useful. Bioelectromagnetics 31:361–370, 2010. © 2010 Wiley‐Liss, Inc.     

The cardiac muscle in the pulmonary vein of the rat: A morphological and electrophysiological study

The pulmonary veins of albino Wistar rats were studied by means of light and electron microscopy. The media of larger veins consists of cardiac muscle fibers which extend until the vessels attain about 100 μ in diameter. This coat consists of external longitudinal fibers and internal circular fibers. The vasa vasorum are well developed and the capillaries show pseudofenestrations. The numerous adrenergic and cholinergic nerve endings do not form typical motor end-plates as seen in skeletal muscles. The ultrastructure of these media muscle fibers is similar to that of rat hearts. The smooth muscle layer of larger pulmonary veins is not continuous as it is in smaller veins where it forms cushions. Comparisons of albino rats and other rodents reveal striking differences. Action potential shape and propagation velocity (0.5–1.2 m/s) along the myocardial coat of the pulmonary vein were similar to those observed in the left atrium and so was their sensitivity to locally applied acetylcholine. The physiological direction of propagation in rat pulmonary veins is toward the lung. This finding lends support to the hypothesis of a rhythmic, valve-like action of the striated musculature of the pulmonary venous wall during the systole and a possible role in the capacitance of the pulmonary circulation.     

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.Download video file.^{(61M, mov)}     

Peptide-containing nerve fibers in the respiratory tract of the ferret

Summary The ferret is widely used in functional and neuromorphological studies on the respiratory tract. We have examined the occurrence and distribution of peptide-containing and adrenergic nerve fibers (using dopamine--hydroxylase as a marker). Adrenergic nerve fibers and fibers storing vasoactive intestinal peptide have a widespread distribution along the entire respiratory tract. Adrenergic nerve fibers were found in the lamina propria, as well as around blood vessels and glands and in smooth muscle. Nerve fibers storing vasoactive intestinal peptide occurred in the epithelium, the lamina propria, around blood vessels and glands, and among muscle bundles. Substance P-, neurokinin A- and calcitonin gene-related peptide-containing nerve fibers predominated beneath and within the epithelium along the entire respiratory tract. Neuropeptide Y-containing nerve fibers were prominent among smooth muscle bundles and around glands. The blood vessels in the wall of the airways were richly supplied with peptidecontaining nerve fibers and adrenergic fibers. Ganglia located over the outer or dorsal surface of the tracheal wall harbored vasoactive intestinal peptide-containing nerve cell bodies. Substance P and neurokinin A invariably coexisted in the same nerve fibers. Further, coexistence of substance P/neurokinin A and calcitonin gene-related peptide was observed in the nerve fibers associated with the epithelium. Vasoactive intestinal peptide, neuropeptide Y and occasionally also substance P coexisted in the population of nerve fibers associated with blood vessels and smooth muscle. Many adrenergic nerve fibers contained neuropeptide Y.     

Cardioregulatory nerves in the spider Eurypelma marxi Simon

The objective of this study was to locate nerves arising from the CNS that have a cardioregulatory function in the tarantula, Eurypelma marxi Simon. Ramifications of the paired abdominal nerve VIIIb merge with the cardiac ganglion within the first heart segment. Electrical stimulation of the branches of nerve VIIIb that connect with the cardiac ganglion produce changes in heartbeat rate and amplitude. Nerve cutting experiments indicate that no other cardioregulatory nerves are present. Both increases and decreases in heart activity can be produced upon electrical stimulation of nerve VIIIb on each side of the heart. Only one action potential associated with the response of each type could be recorded in each member of the nerve pair. Therefore, we conclude that there are two inhibitory and two acceleratory neurons that arise in the central nervous system to modulate heartbeat activity. The inhibitory effect becomes maximal at a stimulation frequency of 20-30 Hz and the accelerator effect at 30-40 Hz. The aftereffect of acceleratory nerve activity exceeds that of inhibitory nerve activity. When the inhibitor and accelerator are activated simultaneously, the inhibitor dominates. The regulatory nerves interact with neurons in the cardiac ganglion. During inhibition, the number of externally recorded spikes in each ganglionic burst is decreased. The rate and magnitude of the heartbeat are decreased concomitantly. Stimulation of the accelerator enhances electrical activity in the cardiac ganglion at the same time that the heartbeat rate and amplitude are increased.     

Ionic properties of the acetylcholine receptor in cultured rat myotubes

The acetylcholine reversal potential (Er) of cultured rat myotubes is -3mV. When activated, the receptor is permeable to K+ and Na+, but not to Cl- ions. Measurement of Er in Tris+-substituted, Na-free medium also indicated a permeability to Tris+ ions. Unlike adult frog muscle the magnitude of Er was insensitive to change in external Ca++ (up to 30 mM) or to changes in external pH (between 6.4 and 8.9). The equivalent circuit equation describing the electrical circuit composed of two parallel ionic batteries (EK and ENa) and their respective conductances (gK and gNa), which has been generally useful in describing the Er of adult rat and frog muscle, could also be applied to rat myotubes when Er was measured over a wide range of external Na+ concentrations. The equivalent circuit equation could not be applied to myotubes bathed in media of different external K+ concentrations. In this case, the Er was more closely described by the Goldman constant field equation. Under certain circumstances, it is known that the receptor in adult rat and frog muscle can be induced to reversibly shift from behavior described by the equivalent circuit equation to that described by the Goldman equation. Attempts to similarly manipulate the responses of cultured rat myotubes were unsussessful. These trials included a reduction in temperature (15 degress C), partial alpha-bungarotoxin blodkade, and activation of responses with the cholinergic agonist, decamethonium.