Effects of training on functional variables of muscles in reared Atlantic salmon Salmo salar smolts: connection to downstream migration pattern.

The relative amount of muscle contraction regulating dihydropyridine and ryanodine receptors in the swimming muscles of trained reared Atlantic salmon Salmo salar smolts was compared with those of untrained and wild smolts. After an optimized 2 week training period, i.e. swimming with a velocity of 1·5 body lengths per second for 6 h per day, the level of both receptors was significantly higher in the muscles of trained S. salar than in the untrained ones before they were released into the natural environment. This difference persisted after downstream migration in the river. The highest level of receptors was observed in wild S. salar. Swimming performance was also higher in trained fish compared to untrained ones. Furthermore, swimming performance was positively associated with the level of receptors in both red and white muscle types. Downstream migration after release into the wild was significantly slower in trained smolts than in untrained fish. This indicates that trained smolts were most probably swimming harder against the current in the river than untrained smolts. The possible advantages for a slower migration in the river are discussed. This study shows that the prerequisites for effective contraction of the swimming muscles are better met in trained S. salar compared to untrained fish, and the muscles of trained smolts more closely resemble those of wild smolts. The results also imply that the capacity of untrained, reared smolts to swim against the current is not equal to that of their trained or wild counterparts which affects the downstream migration pattern of S. salar smolts.

Niacin in the prevention of atherosclerosis: Significance of vasodilatation.

There is a rising interest towards the old drug, nicotinic acid (niacin, vitamin B(3)), because at pharmacological concentrations it has a beneficial effect on HDL cholesterol. Its use, however, was limited due to its adverse effect, flushing. When the mechanism of flushing was solved, a combination of niacin and DP1 receptor antagonist or prostaglandin inhibitor is used, there has been a comeback of niacin with extensive clinical trials. This paper argues that the new strategy with niacin for the prevention of atherosclerosis should be re-evaluated, because vasodilatation of the peripheral vessels might be crucially important in the early primary prevention according to our "vasa vasorum hypoxia" hypothesis.

Testosterone and Ca2+ regulation in skeletal muscle.

The aim of this study was to examine the effect of testosterone treatment on the expression of dihydropyridine and ryanodine receptors in skeletal muscle of mouse. Furthermore, the effects of training, a method also known to elevate the plasma testosterone level, were studied and compared to the effects of pure testosterone administration. Male mice were either administered with testosterone or trained with treadmill. After 6 weeks, hindlimb muscles were excised and the expression of receptors was measured by Western blotting. Furthermore, the alterations in myosin heavy chain phenotypes were studied. In general, both training and testosterone administration induced changes in the expression of both receptors and in myosin heavy chain composition. In testosterone treated mice the expression of dihydropyridine receptor in extensor digitorum longus was higher compared to the control ones (38.9 %, p = 0.026). In soleus the expression was quite the contrary (- 27.3 %, p = 0.044), as was the case with ryanodine receptor (- 51.4 %, p = 0.012). The amount of ryanodine receptors was higher in rectus femoris (144.0 %, p = 0.044) and plantaris (48.1 %, p = 0.037) in testosterone treated mice. In trained mice, the expression of ryanodine receptor was significantly higher in gastrocnemius (27.6 %, p = 0.018), soleus (57.2 %, p = 0.025), plantaris (28.5 %, p = 0.009) and extensor digitorum longus (94.8 %, p = 0.009) than in the control ones. No differences were observed in the dihydropyridine receptor level. To conclude, training has a more important role in skeletal muscle adaptation compared to increased plasma testosterone level. However, in postural muscles both treatments have comparable effects.

Effects of low-intensity training on dihydropyridine and ryanodine receptor content in skeletal muscle of mouse

To evaluate low-intensity exercise training induced changes in the expression of dihydropyridine (DHP) and ryanodine (Ry) receptors both mRNA and protein levels were determined by quantitative RT-PCR and immunoblot analysis from gastrocnemius (GAS) and rectus femoris (RF) muscles of mice subjected to a 15-week aerobic exercise program. The level of muscular work was assayed by changes in myosin heavy chain (MHC) content, myoglobin (Mb) expression and muscle size. The mRNA expression and optical density of DHP receptor increased significantly in GAS by 66.8 and 39.5%, respectively. The expression of Ry receptor, on the other hand, was not up-regulated. In RF, there was a significant increase of 38.4% in the mRNA expression of DHP receptor, although the protein level remained the same. No changes in Ry receptor expression was observed. The training resulted in a 1.58% increase in the amount of MHC IIa and a 2.34% decrease in that of IIb and IId in GAS. A significant 8.3% increase in the Mb content was observed. In RF, no significant changes in MHC or in Mb content were noted. Our results show that an evident increase in the mRNA and protein expression of DHP receptor was induced in GAS even by a relatively low-intensity exercise. Surprisingly, contrast to DHP receptor expression, no changes in Ry receptor mRNA, or protein levels were found, indicating more abundant demand for DHP receptor after increased muscle activity (AU)

Para evaluar los cambios inducidos en la expresión de los receptores de dihidropiridina (DHPR) y rianodina (RyR) por el entrenamiento con ejercicio de baja intensidad, se determinan los niveles de mRNA y de proteína mediante el análisis de RT-PCR cuantitativa e inmunoblot de los músculos gastrocnemius (GAS) y rectus femoris (RF) de ratón sometido a un programa de ejercicio aeróbico durante 15 semanas. El nivel de trabajo muscular fue determinado por los cambios en contenido de cadena pesada de miosina (MHC), expresión de mioglobina (Mb) y tamaño del músculo. La cantidad de mRNA y de proteína de DHPR aumentó significativamente en un 66,8 y 39,5% respectivamente. La expresión de RyR, por otro lado, no se vio incrementada. En RF hubo un aumento significativo del 22,7% en la expresión del mRNA de DHPR, aunque los niveles de proteína permanecieron inalterados. Tampoco se observaron cambios en la expresión de RyR en RF. El entrenamiento dio lugar a un aumento del 1,58% en la cantidad de MHC IIa y disminución del 2,34% en MHC IIb y IId en GAS, con incremento significativo del 8,3% en el contenido de Mb. En RF no se detectaron cambios significativos en el contenido en MHC ni en Mb. Nuestros resultados muestran que se induce un evidente aumento en el nivel de RNAm y de proteína DHPR en GAS mediante un ejercicio de relativamente baja intensidad. Sorprendentemente, en contraste con la expresión de DHPR, no se encontraron cambios en los niveles de mRNA ni de proteína de RyR, indicando mayor demanda de DHPR al incrementar la actividad muscular (AU)

Effects of low-intensity training on dihydropyridine and ryanodine receptor content in skeletal muscle of mouse.

To evaluate low-intensity exercise training induced changes in the expression of dihydropyridine (DHP) and ryanodine (Ry) receptors both mRNA and protein levels were determined by quantitative RT-PCR and immunoblot analysis from gastrocnemius (GAS) and rectus femoris (RF) muscles of mice subjected to a 15-week aerobic exercise program. The level of muscular work was assayed by changes in myosin heavy chain (MHC) content, myoglobin (Mb) expression and muscle size. The mRNA expression and optical density of DHP receptor increased significantly in GAS by 66.8 and 39.5%, respectively. The expression of Ry receptor, on the other hand, was not up-regulated. In RF, there was a significant increase of 38.4% in the mRNA expression of DHP receptor, although the protein level remained the same. No changes in Ry receptor expression was observed. The training resulted in a 1.58% increase in the amount of MHC IIa and a 2.34% decrease in that of IIb and IId in GAS. A significant 8.3% increase in the Mb content was observed. In RF, no significant changes in MHC or in Mb content were noted. Our results show that an evident increase in the mRNA and protein expression of DHP receptor was induced in GAS even by a relatively low-intensity exercise. Surprisingly, contrast to DHP receptor expression, no changes in Ry receptor mRNA, or protein levels were found, indicating more abundant demand for DHP receptor after increased muscle activity.

Expression of L-type calcium channels associated with postnatal development of skeletal muscle function in mouse.

Several factors have an influence on the improvement of muscle activity and motor co-ordination of mammals during post-natal development. One of them is voltage sensitive L-type calcium channel function. In striated muscles of adult mammals these channels are located in T-tubule membranes thus linking the on-coming action potential to the molecular process of muscle contraction. The postnatal development of L-type calcium channels is therefore critical not only for contraction but also for all subsequent motor learning. We used high affinity enantiomer of dihydropyridine labelled with a fluorophore in order to show the relative amount of L-type calcium channels by histofluorescence in tissue. We found by qualitative microscopical analysis that the amount of L-type calcium channels increased during the postnatal development in the mouse skeletal muscle (m. rectus femoris and m. gastrocnemius). We also noted variation between different fibre types in the increase of the amount of L-type calcium channels, as judged by the intensity of histofluorescence. We showed by histochemical staining and statistical analysis that the high density of L-type calcium channels in adult muscles is correlated with fast oxidative glycolytic fibre type of striated muscles rather than slow oxidative or fast glycolytic fibres. Based on this finding we propose that the development of L-type calcium channels can be considered as one of the factors determining the different physiological properties of fibre types.

Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway.

We used the secretion of the novel salmon cardiac peptide (sCP) as a model to examine the mechanisms of ventricular hormone release. Mechanical load increased dose dependently the secretion of immunoreactive sCP from isolated perfused salmon ventricle, with 3. 3-fold increase when a load of 13 cmH(2)O was applied. Endothelin-1 (5 nmol/l) was also able to rapidly increase the secretion of sCP. The released peptide corresponded to the biologically active sCP-29, whereas the large ventricular storage consisted of pro-sCP-sized material. With the use of immunoelectron microscopy, a large number of granules containing immunoreactive sCP could be detected in salmon ventricle. As judged by RNA blot analysis, there was very active basal expression of the sCP gene in the ventricle, which was not increased by mechanical load of up to 2-h duration. Our results show that the ventricle actively expresses the gene of sCP, stores the prohormone in secretory granules, and releases the peptide in response to mechanical load and endothelin-1. Thus the salmon ventricle uses the regulated pathway to produce and release a hormone structurally related to the mammalian natriuretic peptides.

Tuning of photoreceptor spectral sensitivity in fireflies (Coleoptera: Lampyridae).

Sexual communication between male and female fireflies involves the visual detection of species-specific bioluminescent signals. Firefly species vary spectrally in both their emitted light and in the sensitivity of the eye, depending on the time when each is active. Tuning of spectral sensitivity in three firefly species that occupy different photic niches was investigated using light and electron microscopy, microspectrophotometry, and intracellular recording to characterize the location and spectral absorption of the screening pigments that filter incoming light, the visual pigments that receive this filtered light, and the visual spectral sensitivity. Twilight-active species had similar pink screening pigments, but the visual pigment of Photinus pyralis peaked near 545 nm, while that of P. scintillans had a lambdamax near 557 nm. The night-active Photuris versicolo, had a yellow screening pigment that was uniquely localized, while its visual pigment was similar to that of P. pyralis. These results show that both screening and visual pigments vary among species. Modeling of spectral tuning indicates that the combination of screening and visual pigments found in the retina of each species provides the best possible match of sensitivity to bioluminescent emission. This combination also produced model sensitivity spectra that closely resemble sensitivities measured either with electroretinographic or intracellular techniques. Vision in both species of Photinus appears to be evolutionarily tuned for maximum discrimination of conspecific signals from spectrally broader backgrounds. Ph. versicolor, on the other hand, appears to have a visual system that offers a compromise between maximum sensitivity to, and maximum discrimination of, their signals.

Thapsigargin has similar effect on p53 protein response to benzo[a]pyrene-DNA adducts as TPA in mouse skin.

The level of p53 tumor suppressor protein increases in response to DNA damage caused by benzo[a]pyrene (B[a]P). The most used tumor promoter in the two step mouse skin carcinogenesis model, 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases this response in mouse skin. In this study the effect of another promoter, thapsigargin was tested on B[a]P-induced p53 response using immunohistochemistry, western blotting and immunoelectron microscopy. We also studied the localization of p53 protein after treatments with BP and TPA or thapsigargin. Thapsigargin had a TPA-like effect on the acute induction of p53 protein related to benzo[a]pyrene-7, 8-diol-9,10-epoxide-DNA adducts in the skin of C57BL/6 mouse. After B[a]P treatment, there was slightly more putatively wild-type p53 protein in nuclei than in cytoplasm of the cells. Neither TPA nor thapsigargin affected the localization of p53 protein. Since both compounds increase the level of intracellular calcium, the inhibition of the p53 response may depend on the level of intracellular calcium. Inhibition of the putatively genome-protecting increase in p53 protein may be one of the critical effects of tumor promoters.

A digital feedback controller application for studying photoreceptor adaptation by 'voltage clamp by light'.

We present a new digital feedback application for the study of the sensitivity characteristics of photoreceptors. The amplitude of the recorded membrane voltage of a cell is steered by changing the incoming light intensity with a motor-driven circular, linear neutral-density wedge (CFW). The voltage response is sampled and fed to a software position controller of the CFW. The controller determines the position of the wedge according to the desired (command) value of the response. The light intensity changes during steady-state represent the sensitivity change, the time-course of adaptation.

Contrast gain, signal-to-noise ratio, and linearity in light-adapted blowfly photoreceptors.

Response properties of short-type (R1-6) photoreceptors of the blowfly (Calliphora vicina) were investigated with intracellular recordings using repeated sequences of pseudorandomly modulated light contrast stimuli at adapting backgrounds covering 5 log intensity units. The resulting voltage responses were used to determine the effects of adaptational regulation on signal-to-noise ratios (SNR), signal induced noise, contrast gain, linearity and the dead time in phototransduction. In light adaptation the SNR of the photoreceptors improved more than 100-fold due to (a) increased photoreceptor voltage responses to a contrast stimulus and (b) reduction of voltage noise at high intensity backgrounds. In the frequency domain the SNR was attenuated in low frequencies with an increase in the middle and high frequency ranges. A pseudorandom contrast stimulus by itself did not produce any additional noise. The contrast gain of the photoreceptor frequency responses increased with mean illumination and the gain was best fitted with a model consisting of two second order and one double pole of first order. The coherence function (a normalized measure of linearity and SNR) of the frequency responses demonstrated that the photoreceptors responded linearly (from 1 to 150 Hz) to the contrast stimuli even under fairly dim conditions. The theoretically derived and the recorded phase functions were used to calculate phototransduction dead time, which decreased in light adaptation from approximately 5-2.5 ms. This analysis suggests that the ability of fly photoreceptors to maintain linear performance under dynamic stimulation conditions results from the high early gain followed by delayed compressive feed-back mechanisms.

The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye.

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

The dynamic nonlinear behavior of fly photoreceptors evoked by a wide range of light intensities.

Fly photoreceptor cells were stimulated with steps of light over a wide intensity range. First- and second-order Volterra kernels were then computed from sequences of combined step responses. Diagonal values of the second-order Volterra kernels were much greater than the off-diagonal values, and the diagonal values were roughly proportional to the corresponding first-order kernels, suggesting that the response could be approximated by a static nonlinearity followed by a dynamic linear component (Hammerstein model). The amplitudes of the second-order kernels were much smaller in light-adapted than in dark-adapted photoreceptors. Hammerstein models constructed from the step input/output measurements gave reasonable approximations to the actual photoreceptor responses, with light-adapted responses being relatively better fitted. However, Hammerstein models could not account for several features of the photoreceptor behavior, including the dependence of the step response shape on step amplitude. A model containing an additional static nonlinearity after the dynamic linear component gave significantly better fits to the data. These results indicate that blowfly photoreceptors have a strong early gain control nonlinearity acting before the processes that create the characteristic time course of the response, in addition to the nonlinearities caused by membrane conductances.

A digital feedback controller application: a light response control system to reveal photoreceptor adaptation.

We present a new digital feedback application for the study of sensitivity characteristics of biological photoreceptors. The voltage response of a cell is controlled by feeding back to the stimulating LED--via the controller--the recorded membrane voltage. The light intensity changes represent the change in the sensitivity of the photoreceptors, revealing the time course of adaptation.

Spike-like potentials in the axons of nonspiking photoreceptors.

1. The voltage responses to light of dark-adapted cockroach photoreceptors were recorded from the somata in the retina and the axons below the two basement membranes. 2. One or more spike-like fast depolarizations superimposed on the graded receptor potential were recorded in photoreceptor axons identified by Lucifer yellow injections. These spikes are voltage dependent in as much as they could be elicited with depolarizing current pulses as well as with light stimuli. In photoreceptor somata only graded receptor potentials were recorded. 3. The physiological function of these axonal spikes may be to serve as an amplification mechanism that counteracts the unfavorable combination of photoreceptor geometry and electrical properties.

Non-linearities in response properties of insect visual cells: an analysis in time and frequency domain.

Intracellularly recorded voltage responses of the visual cells of the blowfly (Calliphora erythrocephala) were analysed in the time and frequency domains. The photoreceptors were stimulated with pulse (impulse), sine, sine-sweep and pseudorandomly (white noise) modulated green light. The blowfly photoreceptor responses, as analysed from the linear transfer functions, seem to arise from a system similar to that of cascaded low-pass filters, with a corner frequency at about 63 Hz (SD +/- 12 Hz). The system is likely to have at least five poles, including one linear second order term, and a pure delay element. Arising from the non-linearities a second harmonic can be seen in the power spectra of responses elicited by sine modulated light. This non-linearity is at least partly explained by the self-shunting property of the membrane voltage response. Light adaptation increases the non-linearities in frequencies lower than 20 Hz, as seen in the decrease of the coherence function with the signal-to-noise ratio remaining constant. Light adaptation also accelerates the transduction process and it appears in the linear transfer function in a form typical to negative feedback. With low stimulus frequencies it causes a 'phase lead'-type non-linearity. In addition, the sine-sweep responses show quite different frequency characteristics in respect of depolarization and repolarization. Lateral inhibition between photoreceptor responses recorded from retinular cell axons in the lamina appears as a drop in gain and as an increasing phase-lag in frequencies from 30 Hz upwards in linear transfer functions. The source of this capacitive-like coupling can be considered to be in the high resistance barriers compartmentalizing the second optic ganglion into discrete anatomical units.

Anti-adhesive coating for glass microelectrodes.

A simple procedure is described for coating the tips of glass micropipette electrodes with a durable thin film of a commercially available anti-adhesive agent (Antispread). This lowers the surface tension of the electrode glass, and helps reduce damage and distortion to tissue, by preventing the microelectrode from sticking permanently to cell membranes. In controlled trials, the coating actually increased the probability that nearby cells could be recorded from successively by about 30%. Electrode resistance increased only very slightly (on average by 8%) as a result of the coating. Coated electrodes yielded excellent long-term penetrations of small visual cells, and in addition allowed these to be dye-filled in the normal way. The coating may have applications in all situations where prevention of wetting by tissue is desirable.

Photoreceptor latency: analysis and definition.

Latency as a time difference between the stimulating light-pulse and the recorded potential response was studied with intracellular recording techniques on blowfly (Calliphora) visual receptors. Latency was shown to be a meaningful parameter in analysing the information content of the recorded receptor potential responses by comparing latency vs. amplitude functions using different intensities of stimulating light, stimulating angles of the incident light, ambient temperatures, and flies of different developmental stages. The maximum amplitude of the response is not sufficient to describe stimulus-response functions. With the help of the linear correlation analysis the most useful latency criterion among three widely used latency parameters (viz. the amplitude threshold (1 mV), the visual estimation (detectable voltage) and the maximum slope intersection with the base-line) was investigated. These were studied by correlating them with other commonly used response parameters. An arbitrarily chosen latency parameter that shows the lowest correlation coefficient is considered to contain more additional information from the photoreceptor voltage response than a latency parameter with high correlation. According to our analysis the most useful latency determination is proposed to be the time from the stimulus onset to the intersection of the maximum tangent at the base line, a criterion which also allows one to use concepts derived from systems analysis. The concept of latency is discussed and a definition proposed in the light of this and other recent findings.

Spectral sensitivity in insect photoreceptors at a range of temperatures.

The function of the visual receptors of blowflies (Calliphora erythrocephala) was studied using intracellular recording techniques where by the eye was kept at various temperatures below room temperature. Cooling was found to reduce the response amplitude, decrease the time course, and increase the latency. The Q10 as calculated from different recordings and from various temperature steps was found to be dependent both on the temperature range and the stimulation intensity, although more on the latter. From different kinds of spectral sensitivities of the receptor cells those with a double peak (UV and green) were studied with regard to the effect of the temperature change. We found a difference in the relative sensitivity change of the two peaks if the temperature was lowered. The sensitivity in UV remained enhanced in relation to green after cooling. This finding can be considered as supporting the sensitizing pigment theory, according to which the double peaked spectral sensitivity is explained by assuming a photostable pigment transferring the energy to the photopigment. This support is valid if the UV sensitivity at room temperature is assumed to be higher, and is limited by the reduced energy transfer to the photopigment caused by the increased mobility of the sensitizing pigment.

Spinal evoked potentials evaluated with two relevant electrode types.

The spinal evoked potentials (SEPs) were recorded at the level of the 7th lumbar segment in five cats by stimulating the popliteal nerve separately on both sides. The recordings were performed both with silver-silver chloride cup electrodes and stainless steel needle electrodes. The shapes and latencies of the responses were highly similar, when comparing the responses recorded with the two electrode types as shown by means of transfer function: attenuation and phase curves for both electrode types are highly similar in the bandpass used in the present study. It is concluded that the properties of stainless steel needle electrodes are highly correspondent with the conventional silver-silver chloride electrodes when somatosensory evoked responses are recorded.