Spontaneous nerve activity and sensitivity in catfish ampullary electroreceptor organs after tetanus toxin application.

The functioning of electroreceptor organs of Ictalurus sp. was investigated by inhibiting synaptic transmission by the administration of tetanus toxin in vitro. A piece of Ictalurus skin of about 20 mm diameter was mounted in an Ussing-type chamber. After establishing the normal functioning of the organ, tetanus toxin (TeTx) was applied basolaterally for 150 min in 66.7 pM and 400 pM concentrations, while the single unit nerve activity was recorded extracellularly. Spontaneous spike activity and the sensitivity of the electroreceptor organs were measured. The results show that TeTx reduces sensitivity to less then 20% of its original value, whereas the spontaneous activity is unaffected by the treatment. This indicates that the afferent nerve is capable of generating impulses independent of receptor cell neurotransmitter release. In the discussion we suggest two alternative mechanisms for the emergence of the spontaneous spike activity.

Simultaneous measurements of calcium mobilization and afferent nerve activity in electroreceptor organs of anesthetized Kryptopterus bicirrhis.

The transduction pathway of ampullary electroreceptor organs involves ionic currents. It has been shown that calcium, as well as sodium and potassium play important parts in this process. In this study we examine the stimulus-evoked changes in the Fura-2 ratio in electroreceptor cells. Furthermore, we recorded stimulus-evoked Fura-2 ratio changes while Na+ and K+ channels were blocked by amiloride and TEA. Simultaneously, extracellular recordings of the afferent spike activity were made. The results show the presence of stimulus evoked fluctuations in the Fura-2 ratio. These fluctuations can be abolished by the application of Cd2+, TEA, and amiloride. The stimulus-evoked activity of the afferent nerve was decreased due to application of these drugs. We conclude that the transduction current is carried by Na+, K+, and probably Ca2+. This fits the existing model on transduction in electroreceptors.

Effects of dietary fats on red blood cell membrane insulin receptor in normo- and hypercholesterolemic miniature swine.

It has been demonstrated that the type of dietary fat affects insulin receptors in various tissues in normal humans and animals by altering membrane fluidity. This study compares the effects of n-3 fatty acids from fish oil and n-6 fatty acids from corn oil on red blood cell membrane insulin receptors in normal and hypercholesterolemic minipigs. A group of minipigs were made hypercholesterolemic by feeding cholesterol and lard for 2 months; the other group served as controls and was fed stock diet. Both groups were then fed experimental diets containing either corn oil or menhaden oil or a mixture of the two for 23 additional weeks. Blood was collected at 0, 2, 12 and 23 weeks after the start of the experimental diets and membranes were prepared from the red blood cells. Insulin binding to red blood cell membranes was measured by radioreceptor assay. Plasma insulin was measured by radioimmunoassay. Insulin binding to red blood cell membrane was compared with the fluidity of the membrane measured and reported earlier. There was no significant effect of cholesterol feeding on plasma insulin concentrations. After 23 weeks on experimental diet plasma insulin was significantly higher in minipigs fed menhaden oil compared to those fed corn oil. No such effect was observed in hypercholesterolemic minipigs. No significant effect of either hypercholesterolemia or fish oil was observed on red blood cell insulin binding. A significant negative relationship was observed between insulin binding and anisotropy at 4°C for all probes but at 37°C significant negative relationship was observed only with polar probes. The data suggest that n-3 fatty acids from fish oil significantly increases plasma insulin in minipigs compared to n-6 fatty acids from corn oil. However, the unsaturation has no significant effect on insulin receptors on erythrocytes. Similarly, prior hypercholesterolemic state also has no effect on plasma insulin levels or the insulin binding to red blood cell membranes.

The electroreceptor organ of the catfish, Ictalurus melas, as a model for cisplatin-induced ototoxicity.

The ototoxic side-effects of the anti-cancer drug cisplatin (cis-diaminedichloroplatinum) have been widely investigated. However, the exact site of action remains unclear. In this study, the electroreceptor organ of the freshwater catfish Ictalurus melas is used as a model for examining the acute effects of cisplatin. The sensory cells in the electroreceptor organ are homologous to the inner hair cells in the cochlea of mammals. The effects of cisplatin administration can be investigated by in vivo recording of the spike trains from the electroreceptor organ primary afferents. Exposure of electroreceptor organs to 330 microM cisplatin for 1 h causes the spontaneous activity to drop, the overall sensitivity to diminish and the shape of the frequency characteristics to change. These effects persist in the week after administration. Control levels have returned at day 22. These results demonstrate an acute and, with considerable hysteresis, reversible cisplatin effect on the electroreceptor organs, which is to a large extent consistent with the cisplatin-induced effects in isolated hair cells in mammals. The time-course of the effect supports the hypotheses that ion channels are blocked immediately by cisplatin administration, and that cisplatin metabolites disturb enzymatic cellular processes.

Dietary menhaden and corn oils and the red blood cell membrane lipid composition and fluidity in hyper- and normocholesterolemic miniature swine.

Fatty acids in the diet are readily incorporated into lipids in various tissues. However, it is not clear whether all tissues have the same level of incorporation. Second, (n-6) unsaturated fatty acids increase the fluidity of membranes, but this has not been shown for (n-3) fatty acids. In this study, we measured the incorporation of (n-6) and (n-3) fatty acids into erythrocyte membrane lipids and studied their effects on the fluidity of erythrocyte membranes. One group of female miniature swine was made hypercholesterolemic by feeding the swine cholesterol and lard for 2 mo; the other group served as controls and was fed a stock diet. Both groups were then fed either corn oil or menhaden oil or a mixture of the two for 23 additional weeks. Blood was collected at 0, 2, 4, 12 and 23 wk after initialization of the experimental diets, and fatty acid composition of phospholipids was assessed. Membrane phospholipids of pigs fed menhaden oil had elevated (n-3) fatty acids (20:5 and 22:6), and lower 18:2 than those fed corn oil. There was no difference in 20:4 content. The fatty acid changes occurred as early as 2 wk after consumption of the corn oil or menhaden oil in pigs previously fed a stock diet, but it took longer in pigs previously fed lard + cholesterol, indicating residual effects of pretreatment. Menhaden oil increased anisotropy (indicating decreased fluidity) more than corn oil for the nonpolar probe diphenylhexatriene (DPH) at earlier time points, but not at 23 wk. Erythrocyte membrane fluidity was significantly related to membrane polyunsaturate content, with (n-6) fatty acids having a greater influence than (n-3) fatty acids. A comparison of the present red blood cell fatty acid compositions with brain synaptosome fatty acid compositions for the same animals showed poor correlations for some of the fatty acids. There was no significant direct relationship between docosahexaenoate (DHA) concentrations in erythrocyte membranes with DHA concentrations in brain synaptosomes from cerebellum, forebrain and caudate nucleus.

Converging electroreceptor cells improve sensitivity and tuning.

We studied the effect of convergent clustering of ampullary electroreceptor organs on stimulus transduction and transmission in the catfish Icalurus melas by electrophysiologically recording primary afferent activity of single ampullae (singlets) and pairs (doublets) innervated by the same afferent. Doublets were twice as sensitive as singlets, and showed sharper tuning around the best frequency. The slope of the phase curve in the doublets was slightly steeper than in the singlets. The spontaneous activity and scatter in interspike interval were not correlated with clustering. The implications of these findings for signal averaging in sensory neurons and their relevance for behaviour are discussed.

Electrophysiological demonstration of N-methyl-D-aspartate receptors at the afferent synapse of catfish electroreceptor organs.

An excitatory amino acid, most probably L-glutamate, acts as a neurotransmitter at the receptor cell--afferent fibre synapses in the ampullary electroreceptor organs of the freshwater catfish Ictalurus nebulosus. In the present study, we have used an electrophysiological approach to investigate the presence of N-methyl-D-aspartate receptors at this level. N-Methyl-D-aspartate, dissolved in an Mg(2+)-containing (normal) solution, had no effect on afferent activity, not even at 5 mM. However, addition of 5 mM N-methyl-D-aspartate to an Mg(2+)-free solution evoked an enduring increase in firing rate. The application of N-methyl-D-aspartate combined with electrical sine wave stimulation produced a firing increase in the primary afferents, even in the presence of Mg2+ (1.5 mM). Glycine (0.01-0.001 mM) significantly potentiated the N-methyl-D-aspartate responses. Addition of antagonists of the actions of N-methyl-D-aspartate, 7-chlorokynurenic acid, DL-2-amino-5-phosphonovaleric acid and ketamine in concentrations of 0.5-2.0 mM led to a decrease in resting and stimulus-evoked activity. 7-Chlorokynurenic acid also blocked the responses to application of N-methyl-D-aspartate. The glycine agonist D-serine (0.01 mM) prevented the 7-chlorokynurenic inhibitory effect. These results suggest the involvement of N-methyl-D-aspartate receptors in mediating the actions of L-glutamate at the afferent synapses of the electroreceptor organs of the catfish.

Caffeine reduces the efficacy of electroreceptor cell synapses: an electrophysiological single-unit in vivo study.

Ampullary electroreceptor organs of catfish, Ictalurus melas, were exposed apically to caffeine solutions at concentrations of 0, 5, 7.5, 10, and 15 mM. Recording sinusoidally-modulated activity of single-unit afferents reveals a dose-dependent decrease in mean afferent activity and sensitivity. A rebound effect of average activity occurs after caffeine is washed out. After 25 min exposure to 15 mM caffeine the peak of the gain curve shifts from 8 Hz to 4 Hz. The corresponding phase characteristic shows an increased phase lag with a maximum shift of 35 degrees at 20 Hz. The latency between stimulus and response increases from 12 to 19 ms; the recovery time after onset of the pulse decreases with 60 ms. The most probable explanation for the recorded effects is that caffeine reduces the availability of intracellular Ca2+ by blocking of the inositol triphosphate receptors in the endoplasmic reticulum. This in turn would affect many intracellular properties and processes. The unavailability of Ca2+ could reduce the synaptic efficacy and increase latency by suppressing fusion of synaptic vesicles with the presynaptic membrane and by depressing vesicle transport. The change in frequency response corresponds in part to reduction of the apical membrane surface area of the receptor cells, and in part to the increased latency. Accumulation of glutamate-containing vesicles could account for the higher mean activity and modulation amplitude in the lower frequency range after caffeine is washed out. Caffeine might act postsynaptically by inducing hyperpolarization of the terminals of the primary afferents.

Vitamin E levels and susceptibility to lipid peroxidation increase with aging in heart plasma membrane from miniature swine.

Age-related changes in heart plasma membrane fatty acid composition, vitamin E content, membrane fluidity, susceptibility to lipid peroxidation, and the subcellular distribution of vitamin E were observed in male and female Hormel-Hanford miniswine over a wide range of ages: prepubertal, < 0.5 years; young, 0.5-2.5 years; middle-aged, 5.9-10 years; and old, 11.5-13.9 years. Pigs were continuously fed the same low-fat, cholesterol-free, vitamin E-adequate stock diet at restricted maintenance levels. Membrane lipid peroxidation tended to increase in middle-aged and elderly pigs, but not significantly, perhaps being somewhat ameliorated by the significantly increased membrane vitamin E in middle-aged and old pigs. Mid-bilayer membrane fluidity was significantly increased in old pigs, but fluidity of the polar headgroup domains decreased with age. Thus, lipid peroxidation tended to increase over the long life span of miniswine even when they are food restricted.

Fatty acid modification of membrane fluidity in Chinese hamster ovary (TR715-19) cells.

Dietary saturated fatty acids, especially lauric (12:0), myristic (14:0) and palmitic (16:0) acids, which are hypercholesterolemic, influence cell membrane fatty acid composition and affect LDL receptor function. When membrane phospholipid fatty acids in Chinese hamster ovary cells, containing the human LDL receptor, were modified (Hannah J. S. et al., 1995 Metabolism 44, 1428-1434), LDL receptor function was affected, but correlations with DPH-determined membrane fluidity were weak. The role of fluidity in various membrane domains with respect to the LDL receptor is examined here. Membrane fluidity was assessed by measuring steady-state fluorescence polarization of diphenylhexatriene (DPH) and its polar propionic acid (DPH-PA) and trimethylammonium (TMA-DPH) derivatives from 38 to 4 degrees C in fatty acid modified Chinese hamster ovary cells. Fatty acid changes modulated mid-bilayer fluidity as determined with DPH, but fluidity in phospholipid headgroup domains, assessed with DPH-PA and TMA-DPH, was independent of fatty acyl composition. The DPH fluidity was related to membrane unsaturation (P < 0.02), oleate contents (P < 0.009) in particular, but inversely related (P < 0.0002) to the longer chain (> or = 20 C atoms) unsaturated fatty acids with from four to six double bonds. The LDL binding was independent of fluidity, but there were weak relations between LDL internalization and DPH-PA anisotropy and between LDL degradation and TMA-DPH anisotropy. It was concluded that LDL binding was not related to mid-bilayer fluidity, but the results with the polar probes suggest a role of fluidity in modulating vertical displacement of the LDL/LDL receptor complex across the plasma membrane.

The effect of hyperosmotic treatment on the functioning of ampullary electroreceptor organs.

The condition of the microvilli of the apical membrane in ampullary electroreceptor organs of the freshwater catfish, Ictalurus nebulosus, was manipulated by administration of hyperosmotic solutions (500 mM) in order to investigate to what extent the apical surface area determines the sensitivity and the shape of the frequency curves. Electrophysiological recordings demonstrated that hyperosmotic sucrose and glucose solutions reduce the sensitivity by 80%, and alter the shape of the frequency curves. The synaptic delay and the average spontaneous activity remained almost unchanged. Calculations with an equivalent electric circuit of an electroreceptor cell indicate that a reduction in apical surface area can account for these effects. Apparently, the sensitivity and the frequency curves in ampullary organs in freshwater catfish depend on the apical surface area. A hyperosmotic urea solution proved to be less suitable to investigate the effect of manipulations of the apical surface area. The almost total loss in sensitivity caused by administration of urea is caused by both shunting of the stimulus by leaky tight junctions and a reduced synaptic efficacy. The repetitive activity observed after exposure to glucose or sucrose must be attributed to malfunctioning of postsynaptic parts of the receptor organ.

Vincristine disturbs spontaneous firing of the afferent nerve fibre in ampullary electroreceptor organs.

Ampullary electroreceptor organs of the catfish were apically exposed to 0.3 mM vincristine in order to investigate the part played by the microtubular system in stimulus transduction. The main effects were repetitive firing of the afferent fibre, a reduction of the mean spontaneous activity and a reduction of the spike amplitude two to four days after exposure to vincristine. The mean sensitivity was less susceptible to vincristine than the spontaneous activity. Since the shape of the frequency curves remained unchanged and similar effects as described above were also observed after denervation, we conclude that vincristine most likely does not affect electroreceptor cell functioning, but causes degeneration of the afferent fibre.

Periodic firing pattern in afferent discharges from electroreceptor organs of catfish.

Spontaneous afferent activity was recorded from 26 single ampullary electroreceptive organs of freshwater catfish (Ictalurus nebulosus LeS) at various temperatures. Regular grouping of action potentials was apparent in this secondary sensory system at 35 degrees C and occasionally at 30 degrees C. Impulse groups consisted of up to seven impulses. The precise timing of impulse generation and the temporal sequence of impulses indicated that oscillating processes are involved. Expectation density functions were calculated for records of afferent activity obtained at various temperatures below 35 degrees C. In the majority of records the function was periodic. Impulse grouping and expectation density functions became more distinct in units exhibiting extremely high thresholds (i.e. being insensitive) to electrical stimuli. The results suggest that the oscillations originate from the postsynaptic membrane. The temporal pattern of impulse generation within impulse groups of ampullary electroreceptor organs and of specific warm and cold receptors was compared and found to be similar. Application of cadmium and menthol, which both reduce calcium entry, suppressed spontaneous activity in normal and insensitive electroreceptor systems, attenuated the sensitivity of normal receptors and modified the periodic pattern. This indicates that calcium is implicated in sensory transduction and in postsynaptic mechanisms. The data suggest that an oscillating process is one component of signal transmission in ampullary electroreceptor organs of teleost fish.

Heart and liver fatty acid composition and vitamin E content in miniature swine fed diets containing corn and menhaden oils.

Female miniature swine, 4-11 yr, were fed 15% fat diets containing n-3 and/or n-6 polyunsaturated fat for 6 months, at 1.95 g fat/kg body weight. Liver lipids from menhaden oil-fed minipigs were elevated in the n-3 fatty acids: 20:5, 22:5 and 22:6, but heart lipids only in 20:5 and 22:6. Liver cell plasma membrane was elevated in 20:5, 22:5 and 22:6 and lowered in the n-6 acids 18:2 and 20:4 in menhaden oil-fed animals, to a greater extent than in the total tissue lipids. Liver alpha-tocopherol tended to decrease upon feeding menhaden oil, but heart alpha-tocopherol concentrations were not affected.

Collagen gels populated with rat neonatal heart cells can be used for optical recording of rhythmic contractions which also show ECG-like potentials.

Neonatal heart cells populated collagen gels show rhythmic contractions which can be recorded optically and electrically. Optical recordings revealed two populations of rhythmically contracting gels: 1) highly coherent contracting collagen gels with normally distributed contraction interval times and contraction amplitudes, and 2) irregularly contracting gels with a multi-modal distribution of contraction interval times and amplitudes. The irregularly contracting gels were shown to be 'semi regular', which means that a short contraction interval was preferentially followed by another short interval. The volume of the collagen gel during the contraction decreased, and our calculations indicate that the myocytes expel 3-10 times their own volume from the gel. Changes in electrical potential were observed depending on the location of the electrodes. These electrical, ECG-like changes in potential were maximal when one electrode was placed in the centre and the other at the edge of the gel. The results of this study indicate that myocyte-populated collagen gels are a very promising system for studies of electrophysiology and coherent contractions.

Identification of AMPA receptors in catfish electroreceptor organs.

Single afferent unit recording in microampullae of the catfish revealed that bath-applied AMPA increases both resting discharge frequency and electrically evoked responses. The potency of AMPA is of the order of 10 microM. DNQX strongly inhibits the excitatory effects of AMPA. The results suggest the presence of AMPA receptors at the synaptic membrane of ampullary electroreceptor organs in the catfish.

In vitro electroreceptor organs for pharmacological studies.

Electroreception is a well-established sensory faculty in aquatic vertebrates. The general physiology of the receptor organs is comprehensively documented. The transduction mechanism of the receptor cells and the synaptic transmission mechanism are less well understood. Research has been hampered by the inaccessibility of the synaptic site. This paper describes how to prepare an in vitro preparation of ampullary electroreceptor organs which allows exposure of both the mucosal and the serosal sides of the receptor cells to superfusion of test solutions. The preparation is quite robust and has been shown to function reliably for more than 8 h. Furthermore, the use of in vitro electroreceptors organs as a model for pharmacological studies is evaluated.

Quantitative aspects of transduction in an electroreceptor organ studied by means of experimental manipulation of the interspike interval.

The interspike interval histogram of spontaneously firing electroreceptor organs of freshwater catfish (Ictalurus nebulosus LeS.) fits well with the probability density function of a gamma distribution. The shape parameter r of this probability density function can be used as a measure for the firing threshold of the spike generator, and the scale parameter lambda provides a measure for the input rate of synaptic quanta. Here, we have studied the physiological meaning of the two parameters of the fitted gamma probability density function by manipulating the mean firing rate. This was done in two ways: "frequency clamp" stimulation and a change in temperature. Frequency clamp stimulation, compensating for adaptation of the response, keeps the afferent firing rate at a fixed value for a limited period. We show that within such a period not only the mean spike rate remains constant, but these spike trains can be regarded as renewal series: the spike generator is functionally uncoupled from the frequency-dependent parts of the transduction path. Concerning the gamma parameters, two types of responses are found: at strong excitation lambda increases, r does not change; with small stimuli r is inversely proportional to the stimulus strength, lambda remains unchanged. This is difficult to explain when stimulus transduction acts via the synaptic input rate only. Stimulation seems to influence the firing threshold of the spike generator directly. At strong inhibition a regular firing pattern suddenly takes over; the major part of the variability suddenly disappears from the spike train. A change in temperature causes a shift in lambda (Q10 approximately 2.3), but not in r. This points to a temperature effect on the input rate only.

Synaptic noise in spike trains of normal and denervated electroreceptor organs.

The sequence of interspike intervals of the spontaneous activity in denervated electroreceptor organs of the catfish is analysed with several statistical techniques: visual inspection of dot displays, interval histograms, serial correlograms, tests for stationary and trends, and tests for renewal properties, based on the spectrum of intervals. The interspike interval train of primary afferents can usually be treated as a renewal process. Following denervation, a number of non-renewal properties emerge. The interval histogram of the renewal spike trains can be fitted well with a gamma probability density function; non-renewal cases need a more complex approach. We propose that the stochastic fluctuations in interval duration arise from randomly occurring quantal depolarizations (Stein's model of stochastic neuronal excitations. Two important properties with regard to synaptic transduction are: afferent firing results from capture of a limited number of depolarizing quanta; and the quantal input rate and the threshold for firing appear to be correlated.