A journey through the field of fish hearinga).

My interest in fish bioacoustics was ignited more than 50 years ago and resulted in a zigzag time travel between various interesting problems that were unsettled at the time. The present paper gives a brief overview of the main topics I have worked on in the field of fish hearing, i.e., auditory function of the swim bladder, directional hearing, function of the lateral line system, and infrasound sensitivity. Rather than being a comprehensive review of these issues, the paper is autobiographical and limited. The aim is to show young scientists that experimental science can be exciting, diverse, and rewarding-and open doors to a rich collegial network, collaboration, and friendships.

Electrophysiological differences between fshb- and lhb-expressing gonadotropes in primary culture.

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²âº responses were similar. Expression of Ca²âº-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²âº influx.

Signal transduction involved in GnRH2-stimulation of identified LH-producing gonadotropes from lhb-GFP transgenic medaka (Oryzias latipes).

We have characterized the response to gonadotropin-releasing hormone 2 (GnRH2) in luteinizing hormone producing cells from gfp-transgenic medaka. Teleosts have separate cells producing the two types of gonadotropins, enabling us for the first time to study the intracellular signaling that controls secretion of each gonadotropin separately. Pituitary cell cultures were prepared, and lhb-producing cells were selected by their GFP expression. Cytosolic Ca(2+) imaging revealed three response patterns to GnRH2, one monophasic and two types of biphasic patterns. The Ca(2+) sources were examined by depleting intracellular Ca(2+) stores and preventing influx of extracellular Ca(2+). Both treatments reduced response amplitude, and affected latency and time to peak. Blocking L-type Ca(2+) channels reduced amplitude and time to peak, but did not remove extracellular Ca(2+) contribution. Patch-clamp recordings showed spontaneous action potentials in several cells, and GnRH2 increased the firing frequency. Presence of Ca(2+)-activated K(+) channels was revealed, BK channels being the most prominent.

Electrophysiological properties of the microstome and macrostome morph of the polymorphic ciliate Tetrahymena vorax.

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately -30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca(2+) spikes with a maximum rate of rise of 7.5 Vs(-1) in microstome and 4.7 Vs(-1) in macrostome cells. Depolarizing voltage steps from a holding potential of -40 mV induced an inward Ca(2+) -current (I(ca) ) peaking at -10 mV, reaching approximately the same value in microstome (-1.4 nA) and macrostome cells (-1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I(Ca) in these morphs supports the notion that the voltage-gated Ca(2+) channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na(+) -free solution and was only partially inhibited by extracellular Cs(+) . The inward rectification was completely blocked by replacing Ca(2+) with Co(2+) or Ba(2+) in the recording solution, and is probably due to Ca(2+) -activated inward K(+) current secondary to Ca(2+) influx through channels activated by hyperpolarization.

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts.

Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO2, and pH.

Protocols for primary cultures of teleost cells are commonly only moderately adjusted from similar protocols for mammalian cells, the main adjustment often being of temperature. Because aquatic habitats are in general colder than mammalian body temperatures and teleosts have gills in direct contact with water, pH and buffer capacity of blood and extracellular fluid are different in fish and mammals. Plasma osmolality is generally higher in marine teleosts than in mammals. Using Atlantic cod (Gadus morhua) as a model, we have optimized these physiological parameters to maintain primary pituitary cells in culture for an extended period without loosing key properties. L-15 medium with adjusted osmolality, adapted to low pCO(2) (3.8mm Hg) and temperature (12°C), and with pH 7.85, maintained the cells in a physiologically sounder state than traditional culture medium, significantly improving cell viability compared to the initial protocol. In the optimized culture medium, resting membrane potential and response to releasing hormone were stable for at least two weeks, and the proportion of cells firing action potentials during spawning season was about seven times higher than in the original culture medium. The cells were moderately more viable when the modified medium was supplemented with newborn calf serum or artificial serum substitute. Compared to serum-free L-15 medium, expression of key genes (lhb, fshb, and gnrhr2a) was better maintained in medium containing SSR, whereas NCS tended to decrease the expression level. Although serum-free medium is adequate for many applications, serum supplement may be preferable for experiments dependent on membrane integrity.

Microstome--macrostome transformation in the polymorphic ciliate Tetrahymena vorax leads to mechanosensitivity associated with prey-capture behaviour.

Ciliates feed by phagocytosis. Some ciliate species, such as Tetrahymena vorax, are polymorphic, a strategy that provides more flexible food utilization. Cells of the microstomal morph of T. vorax feed on bacteria, organic particles and organic solutes in a non-selective manner, whereas macrostome cells are predators that consume specific prey ciliates. In the present study, we investigated a possible correlation between phagocytosis and mechanosensitivity in macrostome T. vorax. Microstome cells seem to be insensitive to mechanical stimulation whereas macrostome cells depolarise in response to mechanical stimulation of the anterior part of the cell. The amplitude of the receptor potential induced by either a prey ciliate or a 5 µm push by a glass needle was sufficient to elicit a regenerative Ca²âº spike. The difference in mechanosensitivity of the two forms correlates with the swimming behaviour when hitting an obstacle; microstome cells swim alongside the obstacle whereas macrostome cells swim backwards, turn and resume forward swimming. Macrostome cells prevented from backward swimming and the subsequent turn failed to capture prey cells in their pouch. Macrostome cells consumed heterospecific prey ciliates preferentially over conspecific microstome cells. This selectivity is not due to electrical membrane responses elicited by physical contact. Both microstome and macrostome cells accumulated in an area containing putative substances released from heterospecific prey ciliates, but the substances did not elicit any electrophysiological membrane responses. We conclude that the mechanosensitivity of macrostome cells is associated with the prey-capture behaviour, whereas the selective phagocytosis is probably due to chemo-attraction to heterospecific prey ciliates.

Chemo-accumulation without changes in membrane potential in the microstome form of the ciliate Tetrahymena vorax.

The swimming behaviour of ciliates is mainly determined by membrane potential and transmembrane fluxes. In a chemical gradient, swimming ciliates may approach or move away from the source. Based on experiments on Paramecium, it is generally assumed that chemical attractants and repellents affect the swimming behaviour of ciliates by specific changes in the membrane potential. We have examined whether there is a causal relationship between membrane potential and chemo-accumulation in the microstome form of the polymorphic ciliate Tetrahymena vorax. Effects of chemo-attractants on the membrane potential of Tetrahymena have not been previously reported. Microstome T. vorax cells aggregated close to a point source of l-cysteine and the complex meat hydrolysate proteose peptone. Chemo-accumulated cells displayed a significantly higher turning frequency than control cells at a similar cell density. A concentration of 20 mmol l(-1) l-cysteine did not evoke any detectable change in the membrane potential whereas 1% proteose peptone depolarised the cells by ∼12 mV. This is contrary to the current model, which predicts agents that induce a moderate depolarisation to be repellents. A solution of 1% proteose peptone contains 21 mmol(-1) Na(+). A solution of 21 mmol(-1) NaCl without organic compounds also caused ∼12 mV depolarisation but had no aggregating effect on the cells. Collectively, the electrophysiological and behavioural data indicate that chemo-accumulation in the microstome form of T. vorax is not governed obligatorily by the membrane potential. We thus suggest that the simple membrane potential model for chemokinesis in Paramecium may not be valid for T. vorax.

Depolarization-induced Ca2+ entry preferentially evokes release of large quanta in the developing Xenopus neuromuscular junction.

The amplitude histogram of spontaneously occurring miniature synaptic currents (mSCs) is skewed positively at developing Xenopus neuromuscular synapses formed in culture. To test whether the quantal size of nerve-evoked quanta (eSCs) distributes similarly, we compared the amplitude histogram of single quantum eSCs in low external Ca(2+) with that of mSCs and found that nerve stimulation preferentially released large quanta. Depolarization of presynaptic terminals by elevating [K(+)] in the external solution or by direct injection of current through a patch pipette increased the mSC frequency and preferentially, but not exclusively, evoked the release of large quanta, resulting in a second broad peak in the amplitude histogram. Formation of the second peak under these conditions was blocked by the N-type Ca(2+) channel blocker, ω-conotoxin GVIA. In contrast, when the mSC frequency was elevated by thapsigargin- or caffeine-induced mobilization of internal Ca(2+), formation of the second peak did not occur. We conclude that the second peak in the amplitude histogram is generated by Ca(2+) influx through N-type Ca(2+) channels, causing a local elevation of internal Ca(2+). The mSC amplitude in the positively skewed portion of the histogram varied over a wide range. A competitive blocker of acetylcholine (ACh) receptors, d-tubocurarine, reduced the amplitude of smaller mSCs in this range relatively more than that of larger mSCs, suggesting that this variation in the mSC amplitude is due to variable amounts of ACh released from synaptic vesicles. We suggest that Ca(2+) influx through N-type Ca(2+) channels preferentially induces release of vesicles with large ACh content.

Formation of very large conductance channels by Bacillus cereus Nhe in Vero and GH(4) cells identifies NheA + B as the inherent pore-forming structure.

The nonhemolytic enterotoxin (Nhe) produced by Bacillus cereus is a pore-forming toxin consisting of three components, NheA, -B and -C. We have studied effects of Nhe on primate epithelial cells (Vero) and rodent pituitary cells (GH(4)) by measuring release of lactate dehydrogenase (LDH), K(+) efflux and the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Plasma membrane channel events were monitored by patch-clamp recordings. Using strains of B. cereus lacking either NheA or -C, we examined the functional role of the various components. In both cell types, NheA + B + C induced release of LDH and K(+) as well as Ca(2+) influx. A specific monoclonal antibody against NheB abolished LDH release and elevation of [Ca(2+)](i). Exposure to NheA + B caused a similar K(+) efflux and elevation of [Ca(2+)](i) as NheA + B + C in GH(4) cells, whereas in Vero cells the rate of K(+) efflux was reduced by 50% and [Ca(2+)](i) was unaffected. NheB + C had no effect on either cell type. Exposure to NheA + B + C induced large-conductance steps in both cell types, and similar channel insertions were observed in GH(4) cells exposed to NheA + B. In Vero cells, NheA + B induced channels of much smaller conductance. NheB + C failed to insert membrane channels. The conductance of the large channels in GH(4) cells was about 10 nS. This is the largest channel conductance reported in cell membranes under quasi-physiological conditions. In conclusion, NheA and NheB are necessary and sufficient for formation of large-conductance channels in GH(4) cells, whereas in Vero cells such large-conductance channels are in addition dependent on NheC.

Peptide pheromone plantaricin a produced by Lactobacillus plantarum permeabilizes liver and kidney cells.

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca(2+) ([Ca(2+)](i)) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10-100 microM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca(2+)](i), and diffusional loss of fluorophore from the cytosol. At a concentration of 5 microM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 microM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.

Plantaricin A, a peptide pheromone produced by Lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells.

Antimicrobial peptides produced by multicellular organisms protect against pathogenic microorganisms, whereas such peptides produced by bacteria provide an ecological advantage over competitors. Certain antimicrobial peptides of metazoan origin are also toxic to eukaryotic cells, with preference for a variety of cancerous cells. Plantaricin A (PlnA) is a peptide pheromone with membrane permeabilizing strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. Recently, we have reported that PlnA also permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant. To investigate if preferential effect on cancerous cells is a general feature of PlnA, we have studied effects of the peptide on normal and cancerous lymphocytes and neuronal cells. Normal human B and T cells, Reh cells (from human B cell leukemia), and Jurkat cells (from human T cell leukemia) were studied by flow cytometry to detect morphological changes (scatter) and viability (propidium iodide uptake), and by patch clamp recordings to monitor membrane conductance. Ca(2+) imaging based on a combination of fluo-4 and fura-red was used to monitor PlnA-induced membrane permeabilization in normal rat cortical neurons and glial cells, PC12 cells (from a rat adrenal chromaffin tumor), and murine N2A cells (from a spinal cord tumor). All the tested cell types were affected by 10-100 microM PlnA, whereas concentrations below 10 microM had no significant effect. We conclude that normal and cancerous lymphocytes and neuronal cells show similar sensitivity to PlnA.

Comment on "silent research vessels are not quiet" [J. Acoust. Soc. Am. 121, EL145-EL150].

The recent paper by Ona et al. [J. Acoust. Soc. Am. 121, EL145-EL150] compared avoidance reactions by herring (Clupea harengus) to a traditional and a "silent" research vessel. Surprisingly, the latter evoked the strongest avoidance, leading to the conclusion that "candidate stimuli for vessel avoidance remain obscure." In this Comment, it is emphasized that the otolith organs in fish are linear acceleration detectors with extreme sensitivity to infrasonic particle acceleration. Near-field particle motions generated by a moving hull are mainly in the infrasonic range, and infrasound is particularly potent in evoking directional avoidance responses in several species of fish. The stimuli initiating vessel avoidance may thus include infrasonic particle acceleration.

The bacterial peptide pheromone plantaricin A permeabilizes cancerous, but not normal, rat pituitary cells and differentiates between the outer and inner membrane leaflet.

Plantaricin A (PlnA) is a 26-mer peptide pheromone with membrane-permeabilizing, strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. We investigated the membrane-permeabilizing effects of PlnA on cultured cancerous and normal rat anterior pituitary cells using patch-clamp techniques and microfluorometry (fura-2). Cancerous cells displayed massive permeabilization within 5 s after exposure to 10-100 microM PlnA. The membrane depolarized to nearly 0 mV, and the membrane resistance decreased to a mere fraction of the initial value after less than 1 min. In outside-out membrane patches, 10 microM PlnA induced membrane currents reversing at 0 mV, which is compatible with an unspecific conductance increase. The D and L forms of the peptide had similar potency, indicating a nonchiral mechanism for the membrane-permeabilizing effect. Surprisingly, inside-out patches were insensitive to 1 mM PlnA. Primary cultures of normal rat anterior pituitary cells were also insensitive to the peptide. Thus, PlnA differentiates between plasma membranes and membrane leaflets. Microfluorometric recordings of [Ca(2+)](i) and cytosolic concentration of fluorochrome verified the rapid permeabilizing effect of PlnA on cancerous cells and the insensitivity of normal pituitary cells.

Electrophysiological properties of pituitary cells in primary culture from Atlantic cod (Gadus morhua).

The aim of the present study was to explore the electrophysiological properties of pituitary cells from Atlantic cod (Gadus morhua), as a basis for future studies of the signaling pathways involved in the control of pituitary secretion in this species. Primary cultures of pituitary cells from maturing Atlantic cod were prepared by trypsin treatment and mechanical dispersion. Electrophysiological recordings were performed using the perforated patch clamp method. A subpopulation of large cells were selected for recordings. Spontaneous action potentials were observed in about 30% of the cells. The action potentials displayed a fast initial spike followed by a prolonged plateau. Correspondingly, the inward current elicited by depolarizing steps consisted of both a transient, tetrodotoxin-sensitive Na(+) component and a nifedipine-sensitive Ca(2+) component that was sustained when Ba(2+) replaced Ca(2+) as current carrier. The outward current was partially blocked both by 5 mM tetraethylammonium and 10 mM 4-aminopyridine. The voltage-activated ion channels present in these cells largely correspond to the ion channels of pituitary cells in other teleosts (goldfish, Carassius auratus, and tilapia, Oreochromis mossambicus) and mammals, although differences exist regarding the shape and duration of action potentials.

Infrasound initiates directional fast-start escape responses in juvenile roach Rutilus rutilus.

Acoustic stimuli within the sonic range are effective triggers of C-type escape behaviours in fish. We have previously shown that fish have an acute sensitivity to infrasound also, with acceleration thresholds in the range of 10(-5) m s(-2). In addition, infrasound at high intensities around 10(-2) m s(-2) elicits strong and sustained avoidance responses in several fish species. In the present study, the possible triggering of C-escapes by infrasonic single-cycle vibrations was examined in juvenile roach Rutilus rutilus. The fish were accelerated in a controlled and quantifiable manner using a swing system. The applied stimuli simulated essential components of the accelerations that a small fish would encounter in the hydrodynamic flow field produced by a predatory fish. Typical C- and S-type escape responses were induced by accelerations within the infrasonic range with a threshold of 0.023 m s(-2) for an initial acceleration at 6.7 Hz. Response trajectories were on average in the same direction as the initial acceleration. Unexpectedly, startle behaviours mainly occurred in the trailing half of the test chamber, in which the fish were subjected to linear acceleration in combination with compression, i.e. the expected stimuli produced by an approaching predator. Very few responses were observed in the leading half of the test chamber, where the fish were subjected to acceleration and rarefaction, i.e. the stimuli expected from a suction type of predator. We conclude that particle acceleration is essential for the directionality of the startle response to infrasound, and that the response is triggered by the synergistic effects of acceleration and compression.