Polyandry increases offspring viability and mother productivity but does not decrease mother survival in Drosophila pseudoobscura.

Polyandrous mating is common, but the benefits for females of polyandry remain controversial. To test whether mating with multiple males affects female fitness, we compared lifetime components of fitness of three experimental sets of Drosophila pseudoobscura females: monogamous females allowed to copulate one time (MOC); monogamous females held with a male over her entire life and experiencing many copulations (MMC); and polyandrous females with a different male over each day of their lives and also experiencing many copulations (PMC). Consistent with previous studies in this species, females in treatments in which multiple copulations occurred, MMC and PMC, had offspring with significantly higher egg-to-adult survival (i.e., offspring viability) and higher numbers of adult offspring (i.e., productivity) than MOC females, showing that multiple inseminations enhance offspring and mother fitness. In addition, although MMC females laid significantly more eggs than polyandrous (PMC) females, percent egg-to-adult survival and number of adult offspring were higher for PMC than MMC females, showing that polyandrous mating enhances the fitness of females more than multiply mating with only one male. Inconsistent with the cost of reproduction, lifespan was not significantly longer for MOC females than for MMC or PMC females. To our knowledge, this is the first study to examine simultaneously in outbred WT Drosophila pseudoobscura the lifetime costs and benefits to females of polyandry, monogamy with a single copulation, and monogamy with repeat copulations.

NMDA antagonists differentiate epileptogenesis from seizure expression in an in vitro model.

In an electrographic model of seizures in the hippocampal slice, both of the N-methyl-D-aspartate (NMDA) antagonists 2-amino-5-phosphonovaleric acid and 5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801) prevented the progressive development of seizures but did not block previously induced seizures. Thus, a process dependent on the NMDA receptor-ionophore complex establishes a long-lasting, seizure-prone state; thereafter the seizures depend on non-NMDA receptor-ionophore mechanisms. This suggests that there is an important distinction between epileptogenesis and seizure expression and between antiepileptogenic and anticonvulsant pharmacological agents.

Diamond-anvil cell for radial x-ray diffraction.

We have designed a new diamond-anvil cell capable of radial x-ray diffraction to pressures of a few hundred GPa. The diffraction geometry allows access to multiple angles of Ψ, which is the angle between each reciprocal lattice vector g(hkl) and the compression axis of the cell. At the 'magic angle', Ψ≈54.7°, the effects of deviatoric stresses on the interplanar spacings, d(hkl), are significantly reduced. Because the systematic errors, which are different for each d(hkl), are significantly reduced, the crystal structures and the derived equations of state can be determined reliably. At other values of Ψ, the effects of deviatoric stresses on the diffraction pattern could eventually be used to determine elastic constants.

Evidence for non-neutrality of mitochondrial DNA haplotypes in Drosophila pseudoobscura.

Mitochondrial DNA (mtDNA) haplotypes usually are assumed to be neutral, unselected markers of evolving female lineages. This assumption was tested by monitoring haplotype frequencies in 12 experimental populations of Drosophila pseudoobscura which were polymorphic for mtDNA haplotypes. Populations were maintained for at least 10 generations, and in one case for 32 generations, while tests of mtDNA selective neutrality were conducted. In an initial population, formed from a mixture of two strains with different mitochondrial haplotypes, the frequency of the Bogota haplotype increased 46% in 3 generations, reaching an apparent equilibrium frequency of 82% after 32 generations. Perturbation of this equilibrium by addition of the less common haplotype resulted in a rapid, dramatic increase in frequency of the second haplotype, and a return to essentially the same equilibrium frequency as before perturbation. This behavior is not consistent with mtDNA neutrality, nor is the equilibrium consistent with a simple model of constant selection on the haploid mtDNAs. Replicate cage experiments with mtDNA haplotypes did not always generate the same result as the initial cage. Several lines of evidence, including manipulations of the nuclear genome, support the idea that both nuclear and mitochondrial genomes are involved in the dramatic mtDNA frequency changes. In another experiment, strong female viability selection was implicated via mtDNA frequency changes. Although the causes of the dramatic mtDNA frequency changes in our populations are not obvious, it is clear that Drosophila mitochondrial haplotypes are not always simply neutral markers. Our findings are relevant to the introduction of a novel mtDNA variant from one species or one population into another. Such introductions could be strongly favored by selection, even if it is sporadic.

Selection by fertility in Drosophila pseudoobscura.

Fertility, the component of selection due to female fecundity and male mating success, differed significantly among the ST/ST, ST/AR, and AR/AR karyotypes in experimental populations and varied with karyotypic frequency. In relation to ST/AR, ST/ST females and males had higher fertilities at low frequency; AR/AR males and females were at a significant fertility disadvantage at intermediate frequency, while at low and at high frequencies their fertilities matched or exceeded that of the heterokaryotype. These fertility differences were comparable in size to viability differences previously reported for D. pseudoobscura karyotypes. Differential fertility seems likely to be an important element, perhaps just as important as differential viability, in the balancing selection that maintains the chromosomal polymorphism in this species.

A Test for Rare Male Mating Advantage with DROSOPHILA PSEUDOOBSCURA Karyotypes.

Recent work has called into question the reality of the rare male mating advantage, pointing out that it could be a statistical artifact of marking flies for behavioral observation or of experimental bias in collecting males. We designed an experiment to test for rare male mating advantage that avoids these sources of bias. Large numbers of males of three Drosophila pseudoobscura karyotypes were allowed to mate with females of one karyotype in population cages. The females were then isolated before multiple mating occurred and their progeny used to diagnose the males that mated them. Populations were studied at five sets of male karyotypic frequencies. The mating success of the male homokaryotypes ST/ST and CH/CH, relative to that of the heterokaryotype ST/CH, was frequency dependent. Both ST/ST and CH/CH males displayed a statistically significant mating advantage at low frequency by comparision with their mating success in the midrange of karyotypic frequencies. Both male homokaryotypes also showed a significantly greater mating success at high homokaryotypic frequency than at intermediate frequencies, which is the same as saying that the heterokaryotype not only failed to show a rare male advantage but actually suffered a mating disadvantage at low frequency. We conclude that rare male mating advantage is not always an experimental or methodological artifact but does occur in laboratory populations of D. pseudoobscura. It may occur for some genotypes and not for others, however, and it may be only one of several forms of frequency-dependent mating behavior operating in a population.

Drift or Selection: A Statistical Test of Gene Frequency Variation over Generations.

The method used by Fisher and Ford (1947) to study the spread of a gene in a natural population has been modified to analyze the variation in allele frequencies from generation to generation in a common experimental procedure. A further analysis has been developed that is more sensitive to directional trends in the allele frequency over generations, and its use in detecting the action of directional selection on gene frequency at a locus is discussed. The power of each of these statistical tests is calculated for a number of cases, and the tests are applied to sets of isozyme data from Drosophila pseudoobscura and Zea mays.

Gene Frequency Changes at the alpha-Amylase Locus in Experimental Populations of DROSOPHILA PSEUDOOBSCURA.

The frequencies of alleles at the alpha-Amylase locus of D. pseudoobscura were followed in both large and small experimental populations. No evidence for balancing or directional selection was found, although our ability to detect weak selection is limited. The gene frequency changes in our experimental populations were consistent with the hypothesis of selective neutrality and genetic drift due to sampling error.

DNA sequence evolution of the amylase multigene family in Drosophila pseudoobscura.

The alpha-Amylase locus in Drosophila pseudoobscura is a multigene family of one, two or three copies on the third chromosome. The nucleotide sequences of the three Amylase genes from a single chromosome of D. pseudoobscura are presented. The three Amylase genes differ at about 0.5% of their nucleotides. Each gene has a putative intron of 71 (Amy1) or 81 (Amy2 and Amy3) bp. In contrast, Drosophila melanogaster Amylase genes do not have an intron. The functional Amy1 gene of D. pseudoobscura differs from the Amy-p1 gene of D. melanogaster at an estimated 13.3% of the 1482 nucleotides in the coding region. The estimated rate of synonymous substitutions is 0.398 +/- 0.043, and the estimated rate of nonsynonymous substitutions is 0.068 +/- 0.008. From the sequence data we infer that Amy2 and Amy3 are more closely related to each other than either is to Amy1. From the pattern of nucleotide substitutions we reason that there is selection against synonymous substitutions within the Amy1 sequence; that there is selection against nonsynonymous substitutions within the Amy2 sequence, or that Amy2 has recently undergone a gene conversion with Amy1; and that Amy3 is nonfunctional and subject to random genetic drift.

Cytoplasmic incompatibility and mating preference in Colombian Drosophila pseudoobscura.

MACRAE and ANDERSON observed a large frequency change of mitochondrial DNA (mtDNA) haplotypes in a population initiated with two allopatric strains of Drosophila pseudoobscura, BogER from Colombia and AH162 from California. They concluded that mtDNA haplotypes in D. pseudoobscura are not always selectively neutral. NIGRO and PROUT suggested, however, that a maternally transmitted incompatibility system, similar to the one they observed in two strains of D. simulans from Italy, could account for the observed mtDNA frequency changes. SINGH and HALE postulated that a mating preference between the strains BogER and AH162 in MACRAF and ANDERSON's experiment, in the form of negative assortative mating, could also account for the mtDNA frequency changes. We report two experiments designed to test the hypotheses: that a maternally transmitted cytoplasmic incompatibility system exists between D. pseudoobscura strains BogER and AH162; and, that BogER females mate preferentially with AH162 males. Our results do not support either hypothesis.

The drug MK-801 attenuates the development, but not the expression, of long-term potentiation and stimulus train-induced bursting in hippocampal slices.

Recent studies have demonstrated that (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), a use-dependent blocker of N-methyl-D-aspartate (NMDA)-activated membrane channels, attenuates the development of long-term potentiation in vitro and kindling in vivo. Both of these phenomena are manifestations of physiological plasticity related to behavioural states and the results of these studies add to the gathering evidence for the involvement of the NMDA receptor/channel system in such processes. In the present experiment, slices of hippocampus, prepared from rats, were electrically stimulated to produce either long-term potentiation of the CA1 population spike or stimulus train-induced epileptiform bursting in area CA3. At 10 microM, MK-801 attenuated the development of long-term potentiation, but had no attenuating effect upon the previously-potentiated population spike. Similarly, 10 microM MK-801 attenuated the development of epileptiform activity in area CA3, but had little or no effect on the previously-established bursting in area CA3. These data support the suggestion that MK-801 exerts an antiepileptogenic, but not an anticonvulsant effect, at concentrations which also inhibit long-term potentiation.

An investigation of the expression mechanism of LTP of AMPA receptor-mediated synaptic transmission at hippocampal CA1 synapses using failures analysis and dendritic recordings.

There is considerable controversy surrounding the mechanism of expression of long-term potentiation of AMPA receptor-mediated synaptic transmission in the CA1 region of the hippocampus, a process thought to be important for learning and memory in the mammalian CNS. We have re-examined the expression mechanism of this form of synaptic plasticity using whole-cell dendritic recordings, minimal stimulation to activate one or a few synapses, and failures analysis. Dendritic recordings provide improved resolution of small synaptic events, as compared to previous studies using somatic recordings, because there is less dendritic filtering of signals. We find that long-term potentiation (LTP) is associated with changes in the size of synaptic responses when they occur (potency) in all cells and this is accompanied by significant decreases in failure rate in approximately 60% of the experiments. This suggests that in some cells an increase in quantal amplitude is the sole expression mechanism for LTP and, in the cells where failure rate decreased, there is an additional mechanism causing a change in quantal content.

The LTP Program: a data acquisition program for on-line analysis of long-term potentiation and other synaptic events.

The LTP Program is a stimulation, acquisition and on-line analysis program for studying long-term potentiation (LTP), long-term depression (LTD), and stimulus-evoked synaptic responses in general. The program is freely available from the website: www.ltp-program.com. It is a 32-bit DOS program that runs on Windows 3/95/98 computers having a Pico Technologies ADC-42, Axon Instruments' Digidata 1200, or Scientific Solution's Labmaster acquisition board. The program records two channels of activity in extracellular, current- or voltage clamp modes. It acquires < or =1,000,000 samples per sweep, and has extracellular dual pathway stimulation and epoch-like intracellular stimulation. Basic protocols include slow alternating dual pathway stimulation. LTP is induced by single train, theta burst, or primed burst stimulation. LTD is induced using fast repetitive 1 pulse sweeps (< or =2 Hz). The program analyzes all stimulus-evoked synaptic responses in both acquisition channels. Analyzes include: slope, peak amplitude/latency, population spike amplitude/latency, average amplitude, duration, area, rise time, decay time, coastline, cell resistance and patch electrode series resistance. Sweeps can be averaged and digitally filtered. Trains can be analyzed by measuring the responses of all pulses relative to the baseline of the first pulse. Stimulus artifacts can be automatically removed for accurate determination of synaptic areas and peaks during a train.

Seizure-like events in brain slices: suppression by interictal activity.

A major concern in epilepsy research is the relationship between ictal (seizure) electrophysiological activity and interictal (between seizure) activity. Much research is carried out in vitro using brain slice models. Although they allow detailed electrophysiology, the events recorded are generally more similar to interictal than ictal activity. We have described an in vitro model of epileptiform activity in the hippocampal slice (exposure to artificial cerebrospinal fluid containing no added magnesium) in which the events closely resemble those seen in vivo during seizures. However, this model is limited by the brief period during which this ictaform activity occurs before it is replaced by interictal-like activity. We now report that as the frequency of the interictal activity is suppressed by the GABAB agonist baclofen, the ictal activity returns. Moreover, when frequent interictal activity is reinduced, the ictal activity again is suppressed. These results suggest that interictal activity may decrease the probability of a seizure. Furthermore, they suggest that substances which may be shown to inhibit interictal activity in various models of epilepsy may not necessarily inhibit ictal activity.

Magnesium-free medium activates seizure-like events in the rat hippocampal slice.

The effect of magnesium-free medium on the electrical activity in CA3 of the rat hippocampal slice was examined. Magnesium removal resulted in the development of spontaneous and triggered interictal-like bursting, followed by spontaneous ictal-like events and finally periodic clustered bursts. The ictal-like events consisted of a tonic firing phase and a phase of clustered burst discharges resembling the tonic and clonic phases of seizures. The return to normal medium resulted in spontaneous and triggered interictal-like bursts.

Regenerative, all-or-none electrographic seizures in the rat hippocampal slice in Mg-free and physiological medium.

All-or-none electrographic seizures (EGSs) were studied in hippocampal slices from young (21- to 38-day-old) rats in medium containing low (0 mM) or physiological (0.9 mM) levels of magnesium, with and without the GABAB agonist baclofen. Extracellular recording and stimulation were performed in stratum pyramidale and stratum radiatum of CA3, respectively. EGS activity was induced by exposure to low-Mg medium or by delivering repetitive stimulus trains in physiological Mg medium. After EGS activity had stabilized, the EGSs were tested for all-or-none behavior by varying the number of pulses in a train. An EGS was considered all-or-none if subthreshold stimulation produced no afterdischarge bursts, and if the EGS duration was largely independent of the number of suprathreshold stimulus pulses. According to this measure, EGSs in Mg-free + baclofen medium were all-or-none. EGSs evoked in physiological Mg medium were also all-or-none, although the threshold was higher, and the EGS duration lower, than in Mg-free medium. This all-or-none characteristic was observed whether the EGSs were induced by prior exposure to Mg-free medium or by repetitive stimulation, and in the presence and absence of baclofen. The all-or-none characteristic suggests that while the triggering mechanism for EGSs is strongly dependent on stimulus intensity, regenerative mechanisms--independent of stimulus intensity--are responsible for the maintenance of EGSs. EGSs are also terminated by mechanisms not dependent on stimulus intensity.

Clinical features of pruritus among patients undergoing maintenance hemodialysis.

Patients undergoing maintenance hemodialysis were asked to complete a questionnaire to define further the nature of uremic pruritus. Of the 237 respondents, 87 (37%) reported "prolonged bothersome itchiness" at the time surveyed, and an additional 97 (41%), not affected at that time, had experienced this problem in the past. Of the 184 patients who reported pruritus in either the past or present, discomfort occurred only during or soon after dialysis in 46 (25%) patients and was most severe at those times in an additional 78 (42%) patients. Topical emollients and orally administered antipruritic agents provided relief in only 33 (18%) and 31 (17%) patients, respectively. These data provide the first statistical basis for certain clinical impressions concerning uremic pruritus and suggest it is not as common among patients who are undergoing dialysis as has been previously implied.

Mechanism of electrographic seizure generation in the hippocampal slice in Mg2+-free medium: the role of GABAa inhibition.

We recently have described a new model of ictal-like electrographic activity in the hippocampal slice. When magnesium is eliminated from the medium bathing the hippocampal slice, spontaneously occurring electrical events which closely resemble electrographic seizures can be recorded extracellularly from area CA3. In order to begin to understand the mechanisms of initiation and termination of these seizures, the present study investigated the role of GABAa-mediated inhibition in these processes. Prior to the onset of a seizure recorded from stratum pyramidale of CA3, a twin-pulse stimulus to stratum radiatum of CA3 evoked twin EPSPs. Following the seizure, the same stimulation triggered a strong epileptiform burst. This is consistent with the known reduction of GABAa inhibition after seizures in vivo. Addition of bicuculline, picrotoxin, or penicillin, which reduce the efficacy of GABAa-mediated inhibition in this system, caused triggered epileptiform bursting to occur prior to the seizure as well. They also lowered the threshold for the production of seizures, facilitating their spontaneous occurrence or elicitation by fewer stimulus pulses. This suggests that the GABAa inhibition present in magnesium-free (0-Mg) plus baclofen medium tends to suppress the onset of seizures and raises the seizure threshold. However, these drugs did not prolong the ictal events. This suggests that in this model, GABAa-mediated inhibition is not responsible for termination of the seizure-like activity. Although there is some potentiation of the tonic firing phase of the seizures under these conditions, there is no gross change in the morphology of the events when inhibition is suppressed. In this model, therefore, GABAa-mediated inhibition plays a limited role in determining the structure and duration of the ictal events, but may contribute to the seizure threshold.

Exposure of hippocampal slices to magnesium-free medium produces epileptiform activity and simultaneously decreases calcium and calmodulin-dependent protein kinase II activity.

The effect of magnesium-free medium on electrical and CaM kinase II activity in the rat hippocampal slice was examined. Experimental slices were incubated in 2 mM Mg, then exposed to magnesium-free medium for 1 h. Control slices were concurrently run in 2 mM Mg. Slices were then frozen and CaM kinase II activity was measured in homogenates. Exposure of hippocampal slices to magnesium-free medium resulted in spontaneous epileptiform activity and a concurrent 38 +/- 5.47% decrease in CaM kinase II activity (range 38.8-75.4% of control; n = 7, P less than 0.001, paired Student's t test). The decrease in CaM kinase II activity was not reversible by treatment with protein phosphatases 1 and 2A (58.8 +/- 4.77% of control activity; range 28.6-69.7, P less than 0.01, paired Student's t-test), indicating that the decrease in CaM kinase II activity cannot be accounted for exclusively by autophosphorylation. The results demonstrate that magnesium-free medium treatment can induce spontaneous epileptiform activity and simultaneous changes in CaM kinase II activity.

Seizure activity in vitro: a dual focus model.

Recently, we have reported that the exposure of hippocampal slices in vitro to artificial cerebrospinal fluid (ACSF) containing no added magnesium results in ictal-like (ictaform) activity in area CA3 of the hippocampal formation. Other reports describe such activity in slices of entorhinal cortex (EC) under similar conditions. Because of the close interrelationship between the entorhinal area and the hippocampal formation, we have begun to study, in vitro, brain slices which contain both the entorhinal cortex and the hippocampal formation. In these slices, we have found that, in the magnesium-free (0-Mg2+) model, there is good electrical communication between area CA3 and the EC. Simultaneous recordings of the activity in the EC and CA3 showed that, when the circuitry linking the two areas was intact, the EC tended to initiate the ictaform activity and lead CA3. However, late in the event, CA3 could lead EC. Furthermore, interictal-like spontaneous bursting in CA3 led to a disorganized pattern of ictaform activity in EC. Finally, when the EC was separated from the hippocampal formation, both areas were capable of ictaform activity which was temporally unrelated. This model provides the opportunity to explore the relationship between two epileptogenic areas in vitro, and to compare and contrast the morphology of the ictaform activity present in both structures. As such, it may prove valuable in both pharmacological and physiological studies of seizure disorders.