We studied the function, development and aging of the adult nervous system in the colonial tunicate Botryllus schlosseri. Adults, termed zooids, are filter-feeding individuals. Sister zooids group together to form modules, and modules, in turn, are linked by a shared vascular network to form a well-integrated colony. Zooids undergo a weekly cycle of regression and renewal during which mature zooids are replaced by developing buds. The zooid brain matures and degenerates on this 7-day cycle. We used focal extracellular recording and video imaging to explore brain activity in the context of development and degeneration and to examine the contributions of the nervous system and vascular network to behavior. Recordings from the brain revealed complex firing patterns arising both spontaneously and in response to stimulation. Neural activity increases as the brain matures and declines thereafter. Motor behavior follows the identical time course. The behavior of each zooid is guided predominantly by its individual brain, but sister zooids can also exhibit synchronous motor behavior. The vascular network also generates action potentials that are largely independent of neural activity. In addition, the entire vascular network undergoes slow rhythmic contractions that appear to arise from processes endogenous to vascular epithelial cells. We found that neurons in the brain and cells of the vascular network both express multiple genes for voltage-gated Na+ and Ca2+ ion channels homologous (based on sequence) to mammalian ion channel genes.

The absolute low-light sensitivity of four congeneric species of rockfish (genus Sebastes) was studied from analysis of electroretinograms measured in living fish. The purpose was: (1) to determine if temperature sensitive noise in rod photoreceptors affects the absolute limit to low-light sensitivity at environmentally realistic temperatures and light levels, and (2) to examine whether interspecific variations in habitat utilization within rockfish communities correlate with differences in visual sensitivity. It was found that the low-light sensitivity of individual retinae is inversely dependent on temperature, decreasing tenfold with a 10°C increase in temperature. While in all four species, temperature had a similar effect on sensitivity, the absolute sensitivity levels were different. The four species could be divided into two groups based on measured sensitivity. Kelp and olive rockfish form a high-sensitivity group capable of responding to light levels approximately 50-fold lower than blue and black rockfish. The sensitivity groups correlated with reported diel activity patterns; the high-sensitivity group forages nocturnally, whereas members of the low-sensitivity group are quiescent during twilight and night and forage during the day.

Bluefin Tuna ( Thunnus orientalis ) have the capacity to move from warm surface waters to cooler waters beneath the thermocline where they will be faced with the challenge of a cold heart delivering blood to metabolically warmed tissues. We used confocal microscopy to investigate the temporal and spatial characteristics of cellular Ca 2+ flux in bluefin tuna ventricular myocytes to further our understanding of the thermal tolerance of heart function in these animals. Tuna were acclimated to either 15°C or 23°C for a minimum of 3 weeks. Cardiac myocytes were isolated from the spongy myocardium and loaded with the Ca 2+ indicator dye Fluo‐4 and field‐stimulated to contract. Line scan images across the width of the myocyte revealed spatial inhomogeneities in the temporal properties of the Ca 2+ transients. The Ca 2+ wavefront initiated faster, rose faster and reached larger peak amplitude at the periphery of the cell compared with the centre. This pattern was exacerbated during acute cooling and when the sarcoplasmic reticulum was inhibited with ryanodine and thapsigargin.

SUMMARY Box jellyfish, or cubomedusae (class Cubozoa), are unique among the Cnidaria in possessing lens eyes similar in morphology to those of vertebrates and cephalopods. Although these eyes were described over 100 years ago, there has been no work done on their electrophysiological responses to light. We used an electroretinogram (ERG) technique to measure spectral sensitivity of the lens eyes of the Caribbean species Tripedalia cystophora. The cubomedusae have two kinds of lens eyes, the lower and upper lens eyes. We found that both lens eye types have similar spectral sensitivities, which likely result from the presence of a single receptor type containing a single opsin. The peak sensitivity is to blue-green light. Visual pigment template fits indicate a vitamin A-1 based opsin with peak sensitivity near 500 nm for both eye types.

SUMMARY The nudibranch mollusc Melibe leonina swims by bending from side to side. We have identified a network of neurons that appears to constitute the central pattern generator (CPG) for this locomotor behavior, one of only a few such networks to be described in cellular detail. The network consists of two pairs of interneurons, termed `swim interneuron 1' (sint1) and `swim interneuron 2' (sint2), arranged around a plane of bilateral symmetry. Interneurons on one side of the brain, which includes the paired cerebral, pleural and pedal ganglia, coordinate bending movements toward the same side and communicate via non-rectifying electrical synapses. Interneurons on opposite sides of the brain coordinate antagonistic movements and communicate over mutually inhibitory synaptic pathways. Several criteria were used to identify members of the swim CPG, the most important being the ability to shift the phase of swimming behavior in a quantitative fashion by briefly altering the firing pattern of an individual neuron. Strong depolarization of any of the interneurons produces an ipsilateral swimming movement during which the several components of the motor act occur in sequence. Strong hyperpolarization causes swimming to stop and leaves the animal contracted to the opposite side for the duration of the hyperpolarization. The four swim interneurons make appropriate synaptic connections with motoneurons, exciting synergists and inhibiting antagonists. Finally, these are the only neurons that were found to have this set of properties in spite of concerted efforts to sample widely in the Melibe CNS. This led us to conclude that these four cells constitute the CPG for swimming. While sint1 and sint2 work together during swimming, they play different roles in the generation of other behaviors. Sint1 is normally silent when the animal is crawling on a surface but it depolarizes and begins to fire in strong bursts once the foot is dislodged and the animal begins to swim. Sint2 also fires in bursts during swimming, but it is not silent in non-swimming animals. Instead activity in sint2 is correlated with turning movements as the animal crawls on a surface. This suggests that the Melibe motor system is organized in a hierarchy and that the alternating movements characteristic of swimming emerge when activity in sint1 and sint2 is bound together.

A B S TRACT The relationship between the deplet ion of IP3-releasable intracellular Ca z+ stores and the activation of Ca2+-selective membrane current was determined dur ing the st imulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca 2+ is required for refilling Ca 2+ stores and the voltage-independent , receptor-regulated Ca 2+ current represents a significant Ca 2+ source for refilling. The time course of Ca z+ store deplet ion was measured with fura-2 fluorescence imaging, and it was compared with the time course of Ca 2+ current activation measured with nystatin patch voltage clamp. At the time of max imum current density (0.18 + .03 pA/pF; n = 48), the Ca ~+ conten t of the IP3-releasable Ca z+ pool is reduced to 39 + 3 % (n = 10) of its resting value. Calcium stores deplete rapidly, reaching a m i n i m u m Ca 2+ conten t in 15-30 s. The activation of Ca 2+ current is delayed by 10-15 s after the beg inn ing of Ca z+ release and continues to gradually increase for nearly 60 s, long after Ca 2+ release has peaked and subsided. The delay in the appearance of the current is consistent with the idea that the product ion and accumulat ion of a second messenger is the rate-limiting step in current activation. The time course of Ca 2+ store deplet ion was also measured after adding thapsigargin to block intracellular Ca 2+ ATPase. After 15 min in thapsigargin, IP3-releasable Ca z+ stores are depleted by >90% and the Ca z+ current is maximal (0.19 + 0.05 pA/pF; n = 6). Intracellular loading with the Ca 2+ buffer EGTA/AM (10 p~M; 30 min) depletes 1P3-releasable Ca 2+ stores by between 25 and 50%, and it activates a vol tage-independent inward current with properties similar to the cur ren t activated by agonist or thapsigargin. The current density after EGTA/AM loading (0.61 + 0.32 pA/pF; n = 4) is three times greater than the current density in response to agonist or thapsigargin. This could result from partial removal of Ca2+-dependent inactivation.

Chelators and associated computer programs are commonly used to buffer metal ions in biological experiments. This communication discusses common misunderstandings and pitfalls in use of these buffers and provides information on choosing the best metal buffer for different experimental situations.

Postinhibitory rebound (PIR) is an intrinsic property of many neurons but the underlying mechanism is not well understood. We studied PIR and its relationship to spike adaptation in B-cells isolated from the buccal ganglia of Aplysia. These neurons exhibit PIR following inhibitory synaptic input and following direct membrane hyperpolarization. Hyperpolarizing and depolarizing voltage clamp pulses from the resting potential evoke slow changes in membrane current that persist in the form of tail currents following the pulses. A subtraction method was used to isolate slow tail currents for study. Current-voltage measurements indicate that slow outward tail currents following depolarizing pulses result from increases in membrane conductance, while inward tail currents following hyperpolarizations to −50 and −60 mV result from conductance decreases. The reversal potential of both outward and inward tail current is between −60 and −70 mV. Tail currents activated by pulses more positive than −60 mV are sensitive to the external K+ concentration and blocked by injection of Cs+ and TEA. When Ca2+ influx is prevented by bathing cells in Ca2+ free saline or by adding Co2+ or Ni2+, the tail currents are reduced but a significant fraction of the current is insensitive to these treatments. More negative conditioning pulses activate a second component of inward tail current that is weakly sensitive to K+ but more strongly effected by substitution of N-methyl glucamine or Li+ for external Na+. We conclude that both PIR and adaptation result from slow changes in a voltage dependent, non-inactivating K+ conductance that is active at voltages near the resting potential and is not tightly coupled to Ca2+ influx. In addition, a second inward current is activated by large hyperpolarizing pulses that results from an increase in Na+ and K+ conductance. This second process is likely to contribute to PIR under particular circumstances.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest—particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species1,2. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Stochastic analysis was applied to observations of spontaneous behavior in the carnivorous mollusc Melibe leonina. Six behaviors were defined that could be easily recognized on inspection and it was found that transitions between each of these behaviors could be fully described by a first-order random process without memory of past behavioral choices, The behaviors are organized by frequency of transition into two modes, a feeding mode and a resting mode. Transitions within modes are more likely than transitions between modes, and the feeding and resting modes are linked by a preferred pair of behavioral transitions. The amount of time spent in the feeding mode is positively correlated with body size, but the average length of a feeding episode is independent of size, This suggests that body size regulates the probability of entry into feeding behavior but does not influence the basic pattern of feeding, In the presence of food the animals express nearly continuous feeding behavior, suggesting that food reduces the probability of exiting the feeding mode. This model of spontaneous behavior in Melibe is used to form hypotheses amenable to further exploration through neurophysiological experiments.

Fura-2 fluorescence imaging was used to measure changes in intracellular Ca2+ concentration in individual N1E-115 neuroblastoma cells during repeated activation of M1 muscarinic receptors with carbachol. Ca2+ transients could be elicited repeatedly at 4 min intervals with little decrement as long as external Ca2+ was present. When the cells were bathed in Ca(2+)-free saline, however, the response amplitude decreased rapidly in a use-dependent fashion, indicating that external Ca2+, and presumably Ca2+ influx, is required for refilling Ca2+ stores during the interval between trials. The response amplitude also decreased during repeated stimulation in cells treated with the NO-synthase inhibitor L-NMMA or with the guanylyl cyclase inhibitor LY-83583 even when Ca2+ was present. Application of the membrane permeable cGMP analog 8-Br-cGMP reversed the effect of L-NMMA and promoted refilling in the continued presence of NO-synthase inhibitor. These results indicate that activation of the NO/cGMP pathway is necessary for refilling Ca2+ stores during muscarinic signaling. Evidence is also presented suggesting that the NO/cGMP pathway is involved in long term modulation of the content of Ca2+ stores.

The aminoglycoside G418 inhibited the release of calcium (Ca2+) from internal stores coupled to muscarinic receptors in murine N1E-115 neuroblastoma cells carrying the aminoglycoside resistance gene neomycin phosphotransferase (NPT). No significant effect was observed on responses coupled to histamine or bradykinin receptors. Cells were transfected using the eukaryotic expression vector pH beta APr-1-neo and selected using G418. Two groups were differentiated either in the continued presence of G418 or in the absence of G418. Carbachol (1 mM), histamine (200 microM) and bradykinin (100 nM) were administered to cells for thirty seconds and changes in [Ca2+]i were measured with fluorescence video microscopy of single cells loaded with the Ca2+ indicator fura-2. The effects of G418 on carbachol evoked Ca2+ release included a 73% reduction in the number of cells responding, a two fold increase in the time to reach half-maximal response, a 35% reduction of the peak [Ca2+]i in response to agonist and an elevation of resting [Ca2+]i from 99 +/- 14 nM (mean +/- S.E.M.) to 155 +/- 27 nM. Acute application (20 min) of G418 to transfected cells differentiated without G418 also reduced the percentage of cells responding to carbachol. This effect was less pronounced in non-transfected parent cells. Thus, the mechanism might involve a metabolite of G418 produced in cells expressing NPT. These results indicate that G418 attenuates Ca2+ release coupled to muscarinic receptors.

�Recovery from K current inactivation was studied in molluscan neurons using two-microelectrode and internal perfusion voltage clamps . Experiments were designed to study the voltage-dependent delayed outward current (IK) without contamination from other K currents . The amount of recovery from inactivation and the rate ofrecovery increase dramatically when the membrane potential is made more negative . The time course of recovery at the resting potential, -40 mV, is well fit by a single exponential with a time constant of 24.5 s (n = 7). At more negative voltages, the time course is best fit by the sum of two exponentials with time constants at -90mV of 1 .7 and 9.8 s (n = 7) . In unclamped cells, a short hyperpolarization can cause rapidrecovery from inactivation that results in a shortening of the action potential duration . We conclude that there are two inactivated states of the channel and that the time constants for recovery from both states are voltagedependent . The results are discussed in terms of the multistate model for K channel gating that was developed by R. N. Aldrich (1981, Biophys. J., 36:519-532) .

The current-voltage curves of repetitively firing neurons show non-linearities in the subthreshold region. Microsurgically isolated molluscan neuron somata were studied under voltage clamp using ramp voltage command signals. During the depolarizing 1/2 cycle a region of negative slope conductance was observed. Ion substitution experiments suggest that this results from non-inactivating or slowly inactivating Na+ and Ca2+ currents. The hyperpolarizing 1/2 cycle reveals a hysteresis effect which results at least in part from a Ca2+ activated K+ current. Similar characteristics have been described in bursting neurons. Their occurrence in the non-bursting neurons studied here shows that they are not unique to this class of neurons and suggests that their primary contribution is to create electrical instability necessary for repetitive firing.