Post-Diapause DNA Replication during Oogenesis in a Capital-Breeding Copepod.

In high-latitude environments where seasonal changes include periods of harsh conditions, many arthropods enter diapause, a period of dormancy that is hormonally regulated. Diapause is characterized by very low metabolism, resistance to environmental stress, and developmental arrest. It allows an organism to optimize the timing of reproduction by synchronizing offspring growth and development with periods of high food availability. In species that enter dormancy as pre-adults or adults, termination of diapause is marked by the resumption of physiological processes, an increase in metabolic rates and once transitioned into adulthood for females, the initiation of oogenesis. In many cases, individuals start feeding again and newly acquired resources become available to fuel egg production. However, in the subarctic capital-breeding copepod Neocalanus flemingeri, feeding is decoupled from oogenesis. Thus, optimizing reproduction limited by fixed resources such that all eggs are of high quality and fully-provisioned, requires regulation of the number of oocytes. However, it is unknown if and how this copepod limits oocyte formation. In this study, the phase in oocyte production by post-diapause females that involved DNA replication in the ovary and oviducts was examined using incubation in 5-Ethynyl-2'-deoxyuridine (EdU). Both oogonia and oocytes incorporated EdU, with the number of EdU-labeled cells peaking at 72 hours following diapause termination. Cell labeling with EdU remained high for two weeks, decreasing thereafter with no labeling detected by four weeks post diapause, and three to four weeks before spawning of the first clutch of eggs. The results suggest that oogenesis is sequential in N. flemingeri with formation of new oocytes starting within 24 hours of diapause termination and limited to the first few weeks. Lipid consumption during diapause was minimal and relatively modest initially. This early phase in the reproductive program precedes mid-oogenesis and vitellogenesis 2, when oocytes increase in size and accumulate yolk and lipid reserves. By limiting DNA replication to the initial phase, the females effectively separate oocyte production from oocyte provisioning. A sequential oogenesis is unlike the income-breeder strategy of most copepods in which oocytes at all stages of maturation are found concurrently in the reproductive structures.

Em ambientes de alta latitude, onde as mudanças sazonais incluem períodos de condições adversas, muitos artrópodes entram em diapausa, um período de dormência regulado por hormônios. A diapausa é caracterizada por metabolismo muito baixo, resistência ao estresse ambiental e interrupção do desenvolvimento. Ele permite que um organismo otimize a reprodução sincronizando o crescimento e desenvolvimento da prole com períodos de alta disponibilidade de alimentos. Em espécies que entram em dormência como pré-adultos ou adultos, o término da diapausa é marcado pela retomada dos processos fisiológicos, um aumento nas taxas metabólicas e o início da oogênese. Em muitos casos, os indivíduos começam a se alimentar novamente e os recursos recém-adquiridos ficam disponíveis para abastecer a produção de ovos. No oceano subártico, a alimentação do copépode Neocalanus flemingeri é dissociada da oogênese e a reprodução é limitada por recursos fixos obtidos durante a pré-diapausa. No entanto, não se sabe como este copépode regula a formação de ovócitos para garantir que todos os óvulos sejam de alta qualidade e bem fornecidos. Neste estudo, a fase de produção de oócitos por fêmeas pós-diapausa foi examinada usando incubação em 5-Etinil-2'-desoxiuridina (EdU) para caraterizar a replicação do DNA no ovário e nos ovidutos. Tanto as oogônias quanto os oócitos incorporaram EdU, com o número de células marcadas atingindo o pico 72 horas após o término da diapausa. A marcação das células com EdU permaneceu alta por duas semanas, diminuindo na terceira e cessando na quarta semana. Desova da primeira ninhada de ovos ocorre três a quatro semanas depois. Os resultados sugerem que a oogênese é sequencial em N. flemingeri com a formação de novos oócitos começando dentro de 24 horas após o término da diapausa e limitado às primeiras semanas. O consumo de lipídios durante a diapausa foi mínima e relativamente modesto inicialmente. Essa fase inicial do programa reprodutivo precede a vitelogênese 2, quando os ovócitos aumentam de tamanho e acumulam reservas de vitelo e lipídios. Ao limitar a replicação do DNA à fase inicial, as fêmeas efetivamente separam a produção de oócitos do seu fornecimento. Uma oogênese sequencial é diferente da estratégia de reprodução da maioria dos copépodes que mantem oócitos em todos estágios de maturação nas estruturas reprodutivas.

Rapid conduction and the evolution of giant axons and myelinated fibers.

Nervous systems have evolved two basic mechanisms for increasing the conduction speed of the electrical impulse. The first is through axon gigantism: using axons several times larger in diameter than the norm for other large axons, as for example in the well-known case of the squid giant axon. The second is through encasing axons in helical or concentrically wrapped multilamellar sheets of insulating plasma membrane--the myelin sheath. Each mechanism, alone or in combination, is employed in nervous systems of many taxa, both vertebrate and invertebrate. Myelin is a unique way to increase conduction speeds along axons of relatively small caliber. It seems to have arisen independently in evolution several times in vertebrates, annelids and crustacea. Myelinated nerves, regardless of their source, have in common a multilamellar membrane wrapping, and long myelinated segments interspersed with 'nodal' loci where the myelin terminates and the nerve impulse propagates along the axon by 'saltatory' conduction. For all of the differences in detail among the morphologies and biochemistries of the sheath in the different myelinated animal classes, the function is remarkably universal.

Temperature compensation in the escape response of a marine copepod, Calanus finmarchicus (Crustacea).

Calanus finmarchicus, the dominant mesozooplankter of the North Atlantic, is an important food source for many fishes and other planktivores. This species, which has limited diel vertical migration, depends on its fast-start escape response to evade predators. It has myelinated neuronal axons, which contribute to its rapid and powerful escape response. The thermal environment that C. finmarchicus inhabits ranges from below 0 degrees C to 16 degrees C. Previous studies have shown that respiration, growth, and reproductive rates are strongly dependent on temperature, with Q10 > 2.5. A comparable dependence of the escape response could place the animal at higher risk for cold-compensated predators. Our work focused on the temperature dependence of the behavioral response to stimuli that mimic predatory attacks. We found that in contrast to other biological processes, all aspects of the escape response showed a low dependence on temperature, with Q10 values below 2. This low temperature dependence was consistent for escape parameters that involved neural as well as muscle components of the behavioral response. These findings are discussed in the contexts of the predator-prey relations of copepods and the thermal dependence of behavior in other taxa.

The need for speed. I. Fast reactions and myelinated axons in copepods.

A rapid and powerful escape response decreases predation risk in planktonic copepods. Calanoid copepods are sensitive to small and brief hydrodynamic disturbances: they respond with multiple nerve impulses to a vibrating sphere. Some species, such as Pleuromamma xiphias and Labidocera madurae, respond with very large spikes (1-4 mV), whereas maximum spike heights are an order of magnitude smaller in others, such as Undinula vulgaris and Neocalanus gracilis. A comparative study of the escape responses showed that all species reacted within 10 ms of the initiation of a hydrodynamic stimulus. However, U. vulgaris and N. gracilis had significantly shorter reaction times (minimum reaction times: 1.5 ms and 1.6 ms) than the other two, P. xiphias (6.6 ms) and L. madurae (3.1 ms). Examination of the first antenna and the central nervous system using transmission electron microscopy revealed extensive myelination of sensory and motor axons in the two species with the shorter reaction times. Axons of the other two species resembled typical crustacean unmyelinated fibers. A survey of 20 calanoids revealed that none of the species in two of the more ancient superfamilies possessed myelin, but myelination was present in the species from three more recently-evolved superfamilies.

The need for speed. II. Myelin in calanoid copepods.

Speed of nerve impulse conduction is greatly increased by myelin, a multi-layered membranous sheath surrounding axons. Myelinated axons are ubiquitous among the vertebrates, but relatively rare among invertebrates. Electron microscopy of calanoid copepods using rapid cryofixation techniques revealed the widespread presence of myelinated axons. Myelin sheaths of up to 60 layers were found around both sensory and motor axons of the first antenna and interneurons of the ventral nerve cord. Except at nodes, individual lamellae appeared to be continuous and circular, without seams, as opposed to the spiral structure of vertebrate and annelid myelin. The highly organized myelin was characterized by the complete exclusion of cytoplasm from the intracellular spaces of the cell generating it. In regions of compaction, extracytoplasmic space was also eliminated. Focal or fenestration nodes, rather than circumferential ones, were locally common. Myelin lamellae terminated in stepwise fashion at these nodes, appearing to fuse with the axolemma or adjacent myelin lamellae. As with vertebrate myelin, copepod sheaths are designed to minimize both resistive and capacitive current flow through the internodal membrane, greatly speeding nerve impulse conduction. Copepod myelin differs from that of any other group described, while sharing features of every group.

Rapid Jumps and Bioluminescence Elicited by Controlled Hydrodynamic Stimuli in a Mesopelagic Copepod, Pleuromamma xiphias.

Actively vertically migrating mesopelagic copepods are preyed upon by a wide variety of fishes and invertebrates. Their responses to predatory attacks include vigorous escape jumps and discharge of bioluminescent material. Escape jumps and bioluminescent discharges in the calanoid copepod Pleuromamma xiphias were elicited by quantified hydrodynamic disturbances. Brief weak stimuli (peak water velocity 64 +/- 21 {mu}m s-1) elicited weak (peak force 6.5 dynes) propulsive responses ("jumps") and no bioluminescence. Moderate stimuli (1580 +/- 780 {mu}m s-1) produced strong propulsive responses consisting of long trains of coordinated power strokes by the four pairs of swimming legs ("kicks"). Peak forces averaged 42 dynes. Strong stimuli (5520 +/- 3420 {mu}m s-1) were required to elicit both a jump and a bioluminescent discharge. In several cases, multiple stimuli were needed to evoke bioluminescence, given the limits on stimulus magnitude imposed by the apparatus. Repeated bioluminescent discharges could be evoked, but this responsiveness waned rapidly. Latencies for the jump response (14 +/- 4 ms) were shorter than for the accompanying bioluminescent discharge (49 +/- 26 ms). The higher threshold for eliciting bioluminescent discharge compared to escape jumps suggests that the copepods save this defense mechanism for what is perceived to be a stronger threat.

Effects of soma isolation on outward currents measured under voltage clamp in spiny lobster stomatogastric motor neurons.

1. Outward currents in identified cell types from the pyloric system of the stomatogastric ganglion (STG) of the spiny lobster, Panulirus marginatus, were studied under two-microelectrode voltage clamp. A comparison was made between data from intact cells and somata isolated by ligation of the primary neurite of these monopolar neurons. 2. Despite the elimination of current contributions from the extensive arborizations of STG neurons, few significant differences were found in the mean values of parameters for outward currents between populations of isolated somata and intact cells of a given type. Measurements that showed little difference included magnitude and activation threshold of a calcium-dependent outward current (IJ) and magnitude, activation threshold, voltage dependence, and inactivation time course of A current (IA). Although previous work has suggested that IJ might reside predominantly in the soma, IA is known to be distributed in poorly space-clamped neurite processes. The absence of obvious effects of isolation was thus unexpected. 3. To better understand the mechanisms involved, we used compartmental models derived from reconstructed neurons to simulate the effects of isolation. It was concluded that, for the particular conditions present in stomatogastric neurons, with a large, uniformly distributed outward current conductance activated, even though neurites and axon remain attached, most measured current flows through well-clamped soma membrane. 4. Factors contributing to this result included the outward sign of the current, the large specific conductance activated in these neurons (among the larger reported in somata), and the presence of only a single major process leaving the soma. The potential for serious errors in voltage-clamp measurements from intact cells remains if these conditions are not met.

Voltage clamp analysis of intact stomatogastric neurons.

Two-electrode voltage clamp of intact, identified pyloric neurons of the spiny lobster stomatogastric ganglion reveals two major outward currents. A rapidly inactivating, tetraethylammonium- (TEA) insensitive, 4-aminopyridine- (4AP) sensitive, outward current resembles IA of molluscan neurons; it activates rapidly on depolarizations above rest (e.g. -45 mV), delaying both the axonal-sodium and the neuropil-calcium spikes which escape voltage-clamp control. We infer that A-current is distributed both in a space clamped region (on or near the soma) and in a non-space clamped region with access to the generators for sodium and calcium spikes. A calcium-dependent outward current, IO(Ca), activates rapidly at clamp steps above -25 mV and inactivates at depolarizing holding voltages. Increasing depolarization results in an increase in both IO(Ca) and firing rate but a reduction in the amplitude of the sodium spike current. Blockage of IO(Ca) with Cd2+ causes little change in spike firing pattern. These observations are consistent with IO(Ca) being activated primarily in the soma and nearby regions which are under good control with a soma voltage clamp (and distant from the Na(+)-spike trigger zone). While the lack of space clamp limits resolution of charging transients and tail currents, the identification of the major current subgroups can still be readily accomplished, and inferences about the location and function of currents can be made which would not be possible if the cells were space clamped or truncated.

Full-wave rectification from a mixed electrical-chemical synapse.

Electrical and chemical synapses usually reinforce one another, but the pyloric late-to-lateral pyloric (PL-to-LP) neuronal connections in lobster stomatogastric ganglia create an inverted U-shaped transfer function between the two neurons: regardless of whether the PL membrane voltage swings positive or negative, the postsynaptic LP voltage will go negative. When the presynaptic cell voltage goes negative, the effect on the LP voltage is due to electrical coupling. During positive presynaptic voltages, the strong contribution of graded chemical inhibition from the PL to the LP neuron overrides the positive electrical coupling to produce net negativity.

A microprocessor-controlled stimulator for generating voltage clamp command sequences.

A device for generating programmed sequences of voltage pulses is described. Initial pulse parameters are entered from a key pad. Parameters can be varied in systematic fashion to generate a range of test pulse sequences automatically. An application for a two-microelectrode voltage clamp is used to illustrate the capabilities.

Endogenous burst capability in a neuron of the gastric mill pattern generator of the spiny lobster Panulirus interruptus.

The gastric system of the lobster stomatogastric ganglion has previously been thought to include no neurons capable of endogenous bursting. We describe conditions under which one of the motorneurons, the CP cell, can burst endogenously in a free-running manner in the absence of other phasic network activity. Isolated preparations of the foregut nervous system were used, and the CP bursting was either spontaneous or was activated by continuous stimulation of an input nerve. Three criteria were applied to establish the endogenous nature of such burst generation in CP: absence of phasic input, reset of the bursting pattern by pulses of current in a characteristic phase-dependent manner, and modulation of burst rate by sustained injected current. (1) The firing of other cells which are known to be related synaptically to CP was monitored in nerve records. These other cells were either silent or fired only tonically. Cross-correlograms showed that CP bursting was not ascribable to phasic activity in these other network cells. (2) A depolarizing current pulse of sufficient strength injected intracellularly between bursts triggered a burst prematurely and reset the subsequent rhythm. A hyperpolarizing pulse during a burst terminated it and reset the subsequent rhythm. Reset behavior was similar to that described for other endogenous bursters. (3) Application of a positive-going ramp current initially caused an increase in burst rate, as described for other endogenous bursters. However, further depolarization caused a slower burst rate due to lengthening of the individual bursts, although mean firing frequency continued to increase throughout the range tested. Such free-running endogenous repetitive bursting appeared to result from the CP's ability to produce slow regenerative depolarizations ("plateau potentials"). When bursting was present, so was the plateau property, as determined by I-V analysis and by the ability of brief current pulses to trigger and terminate bursts. The previous inability to observe endogenous bursting in preparations with central input removed may be due to the usual absence of the plateau property in such preparations. CP bursting during normal gastric mill rhythms, while underlain by plateau potentials, is strongly controlled by network interactions. CP appears not to be well placed in the network to be considered a source of normal gastric rhythmicity. Nevertheless, endogenous bursting in CP may explain some of the partial gastric rhythms seen in behavioral studies, and illustrates one way that cellular properties might contribute to rhythmic behaviors.

Synaptic regulation of cellular properties and burst oscillations of neurons in gastric mill system of spiny lobsters, Panulirus interruptus.

The properties of neurons in the stomatogastric ganglion (STG) participating in the pattern generator for the gastric mill rhythm were studied by intracellular current injection under several conditions: during ongoing gastric rhythms, in the nonrhythmic isolated STG, after stimulation of the nerve carrying central nervous system (CNS) inputs to the STG, or under Ba2+ or Sr2+. Slow regenerative depolarizations during ongoing rhythms were demonstrated in the anterior median, cardiopyloric, lateral cardiac, gastropyloric, and continuous inhibitor (AM, CP, LC, GP, and CI) neurons according to criteria such as voltage dependency, burst triggering, and termination by brief current pulses, etc. Experiments showed that regenerative-like behavior was not due to synaptic network interactions. The slow regenerative responses were abolished by isolating the stomatogastric ganglion but could be reestablished by stimulating the input nerve. This indicates that certain CNS inputs synaptically induce the regenerative property in specific gastric neurons. Slow regenerative depolarizations were not demonstrable in gastric mill (GM) motor neurons. Their burst oscillations and firing rate were instead proportional to injected current. CNS inputs evoked a prolonged depolarization in GM motor neurons, apparently by a nonregenerative mechanism. All the gastric cells showed prolonged regenerative potentials under 0.5-1.5 mM Ba2+. We conclude that the gastric neurons of the STG can be divided into three types according to their properties: those with a regenerative capability, a repetitively firing type, and a nonregenerative "proportional" type. The cells are strongly influenced by several types of CNS inputs, including "gastric command fibers."

Graded synaptic transmission between identified spiking neurons.

Graded synaptic transmission between spiking motoneurons of the pyloric group was studied in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. Intracellular microelectrodes were placed in the cell bodies of both pre- and postsynaptic neurons. Graded synaptic transmission was found between all tested cell pairs that were known to display spike-evoked synaptic transmission, including PD to LP, PD to PE, PD to PL, PL to LP, and LP to PD. Graded synaptic transmission was effective below the threshold for spikes. Thus, it was possible to study the influence of graded synaptic transmission in normally active ganglia without blockage of spikes by tetrodotoxin. PD and LP neurons that were known to produce spike-evoked inhibitory postsynaptic potentials (IPSPs) were also capable of producing inhibitory effects on postsynaptic cells below the threshold for spikes. When tetrodotoxin (TTX) was used to eliminate both spikes and endogenous membrane oscillations, depolarization of presynaptic neurons produced hyperpolarization of postsynaptic cells. The presynaptic response to a current step usually showed a small early peak and a maintained, slightly lower plateau. The postsynaptic response had a delay, then a rise to a pronounced peak, and a roughly exponential decline to a maintained plateau. There was a presynaptic voltage threshold for any postsynaptic response; beyond the threshold, both pre- and postsynaptic peak and plateau responses increased with increasing current. PD neurons normally are depolarized beyond their release threshold in tetrodotoxin and, thus, released transmitter tonically for the many-hour duration of these experiments. Chemical, tonic synaptic transmission, here called graded synaptic transmission, was demonstrated by the presence of the following criteria: 1) reversal in sign of the postsynaptic response, 2) synaptic delay, 3) reversal potential, 4) postsynaptic conductance increase, 5) graded and reversible block by reduction of external Ca2+, and 6) specific graded block of the LP-to-PD synapse without effect on the PD-to-LP synapse by less than 10 microM picrotoxin added to the bathing medium.

Slow active potentials and bursting motor patterns in pyloric network of the lobster, Panulirus interruptus.

1. Neurons in the central pattern generator for the "pyloric" motor rhythm of the lobster stomatogastric ganglion were investigated for the possible involvement of regenerative membrane properties in their membrane-potential oscillations and bursting output patterns. 2. Evidence was found that each class of pyloric-system neurons can possess a capability for generating prolonged regenerative depolarizations by a voltage-dependent membrane mechanism. Such responses have been termed plateau potentials. 3. Several tests were applied to determine whether a given cell possessed a plateau capability. First among these was the ability to trigger all-or-none bursts of nerve impulses by brief depolarizing current pulses and to terminate bursts in an all-or-none fashion with brief hyperpolarizing current pulses. Tests were made under conditions of a high level of activity in the pyloric generator, often in conjunction with the use of hyperpolarizing offsets to the cell under test to suppress ongoing bursting. 4. For each class, the network of synaptic interconnections among the pyloric-system neurons was shown to not be the cause of the regenerative responses observed. 5. Plateau potentials are viewed as a driving force for axon spiking during bursts and as interacting with the synaptic network in the formation of the pyloric motor pattern.

A multiaction synapse evoking both EPSPs and enhancement of endogenous bursting.

Selective stimulation of two identified input neurons called the 'IV neurons' has a dual influence on the endogenous bursting activity of certain 'PD' motorneurons in the stomatogastric ganglion of the spiny lobster. The effects include: (i) large, conventional and apparently monosynaptic EPSPs; and (ii) enhancement of the endogenous bursting of the pyloric dilator (PD) cells, seen as an increased amplitude of PD oscillations and a higher spiking rate during bursts. The burst enhancement decayed relatively slowly after stimulation ceased, over seconds or tens-of-seconds, depending on stimulus parameters. Modification of the voltage-dependent membrane properties of the PD cells appeared to underlie this effect. The dual-action nature of the IV-to-PD connection was confirmed by selectively blocking the brief EPSP component with 5 x 10(-4) M curare, under which conditions the burst enhancement still persisted. Data from low-Ca2+ experiments were consistent with a conventional mode of synaptic transmitter release underlying the burst enhancement. Enhancement was found to differ significantly from actions of injected current. The IV inputs appear to act on at least two types of synaptic receptors on PD neurons: a curare-sensitive receptor for the brief conventional EPSP, and a curare-resistant receptor for burst enhancement. Analogies may be drawn to the nicotinic and muscarinic cholinergic receptors of vertebrates. These findings may be considered within the contexts of multiaction synapses, modification of cellular properties, and mechanisms for the CNS activation of motor pattern generators.

Graded synaptic transmission between spiking neurons.

Graded synaptic transmission occurs between spiking neurons of the lobster stomatogastric ganglion. In addition to eliciting spike-evoked inhibitory potentials in postsynaptic cells, these neurons also release functionally significant amounts of transmitter below the threshold for action potentials. The spikeless postsynaptic potentials grade in amplitude with presynaptic voltage and can be maintained for long periods. Graded synaptic transmission can be modulated by synaptic input to the presynaptic neuron.

Pattern generation in the lobster (Panulirus) stomatogastric ganglion. II. Pyloric network simulation.

1. Results from the companion paper were incorporated into a physiologically realistic computer model of the three principal cell types (PD/AB, LP, PY) of the pyloric network in the stomatogastric ganglion. Parameters for the model were mostly calculated (sometimes estimated) from experimental data rather than fitting the model to observed output patterns. 2. The initial run was successful in predicting several features of the pyloric pattern: the observed gap between PD and LP bursts, the appropriate sequence of the activity periods (PD, LP, PY), and a substantial PY burst not properly simulated by an earlier model. 3. The major discrepancy between model and observed patterns was the too-early occurrence of the PY burst, which resulted in a much shortened LP burst. Motivated by this discrepancy, additional investigations were made of PY properties. A hyperpolarization-enabled depolarization-activated hyperpolarizing conductance change was discovered which may make an important contribution to the late phase of PY activity in the normal burst cycle. Addition of this effect to the model brought its predictions more in line with observed patterns. 4. Other discrepancies between model and observation were instructive and are discussed. The findings force a substantial revision in previously held ideas on pattern production in the pyloric system. More weight must be given to functional properties of individual neurons and less to properties arising purely from network interactions. This shift in emphasis may be necessary in more complicated systems as well. 5. An example has been provided of the value quantitative modeling can be to network physiology. Only through rigorous quantitative testing can qualitative theories of how the nervous system operates be substantiated.

Pattern generation in the lobster (Panulirus) stomatogastric ganglion. I. Pyloric neuron kinetics and synaptic interactions.

There are a number of perspectives gained from a quantitative analysis of the pyloric system which may be applicable to other simple pattern generators: 1. The system is organized around a dominant, endogenously-bursting neuron group, and its properties are tailored to that dominance. In particular, synaptic strengths and firing frequencies of that group appear just sufficient to suppress postsynaptic "follower" cells if the latter are not too highly excited. 2. Repetitive firing properties of follower neurons are such as to facilitate their switch-like mode of activity. This includes pacemaker response nonlinearities, rebound properties, and "burstiness" properties. 3. Proper sequencing of follower cells may be controlled by particular synaptic strengths and time-courses, feedback on the oscillator cells, and functional cellular properties of follower neurons (e.g., rebound; see also next paper). All such properties interact and must be tuned to each other for proper patterns to result.