Patch clamp study and kinetic modeling on the most abundant chloride channel on distal fibers of crayfish opener muscle

Whereas the inhibitory innervation of the deep extensor abdominal muscle in crayfish is mediated by a weakly acting common inhibitor, the opener muscle exhibits a stronger inhibition. In the present study the most abundant γ-aminobutyric acid-activated chloride channel on distal fibers of crayfish opener muscle was characterized by measuring the current responses after applying pulses of γ-aminobutyric acid to outside-out patches. The results were compared to those obtained earlier with the chloride channel on the deep extensor abdominal muscle of the same species. The double logarithmic plot of the dose-response relationship had a slope of n _H = 2.2 in contrast to n _H = 5.3 for the channel on the deep extensor abdominal muscle. The rise time of the current response declined to 1 ms at a γ-aminobutyric acid concentration of 50 mmol · l⁻¹. With lower concentrations the rise time increased to a maximal value of 280 ms. No peak of the rise time at low γ-aminobutyric acid concentrations, as observed for the channel on the deep extensor abdominal muscle, was obvious. The open and closed times were similar to those of the channel of the deep extensor abdominal muscle. Different reaction schemes were discussed to describe the kinetics of the chloride channel of the opener muscle. Accepted: 12 August 1996     

Rhythmicity of torpor in a marsupial hibernator, the mountain pygmy-possum (Burramysparvus), under natural and laboratory conditions

Circadian rhythms have been observed in most mammals, but their importance and function remain controversial with respect to daily cycles during hibernation. We investigated the timing of arousals from and entries into hibernation for both free-living and captive mountain pygmy-possums (Burramys parvus). Under both natural and laboratory conditions most arousals and entries were entrained with the light-dark cycle. Entries occurred mainly during the night and arousals preferably around dusk, which coincides with the onset of the normal activity phase for the nocturnal pygmy-possums. This entrainment prevailed throughout the hibernation season although only the laboratory animals were constantly subjected to photoperiodic stimuli, whereas under natural conditions hibernacula are shielded from photic cues and diurnal temperature fluctuations. Nevertheless, possums left their hibernacula frequently throughout winter and were occasionally trapped close to the snow surface suggesting that during the periods of post-arousal normothermia they can be exposed to environmental stimuli. It thus appears that the synchronisation with the photocycle was governed by a temperature-compensated circadian clock which was reset periodically during short activity periods. For the mountain pygmy-possum, entrainment with the photocycle probably has two functions: 1. Entrainment ensures that foraging bouts during the hibernation season remain synchronised with the dark phase. 2. Information about the prevailing climatic conditions sampled during short activity periods enables them to time final spring emergence from hibernation when snow melt begins and ensures that the breeding season can commence as early as possible. Accepted: 26 August 1998     

Fatty acid compositions of arthropod and cephalopod photoreceptors: interspecific,seasonal and developmental studies

Fatty acid compositions of the compound eyes of insects (soldier-bug, Hemiptera, and silk moth, Lepidoptera), crustaceans (crayfish and grapsid crab, Decapoda) and inner and outer segments of visual cells of a squid (Cephalopoda, Mollusca) were analyzed by gaschromatography for interspecific comparison. Fatty acid compositions showed great variation among species. In insect compound eyes, 16:0 and 18:0 were the main saturated fatty acids, and 18:1 was the dominant unsaturated fatty acid. Silk moth eyes contained, in addition, considerable amounts of 18:2 and 20:5. In crustacean compound eyes, the main saturated fatty acids were 16:0 and 18:0, and 14:0 (5.0%) was only detected in grapsid crabs; the main unsaturated fatty acids were 20:4, 20:5 and 22:6. Both whole eyes and rhabdom fraction of crayfish showed similar profiles of fatty acid compositions. Both inner and outer segments of squid retinae were characterized by high amounts of unsaturated fatty acids, especially 22:6. Compound eyes of grapsid crabs were used for the experiments on seasonal changes of fatty acid compositions. UFA/SFA ratios (weight in % of unsaturated fatty acids saturated fatty acids) were lowest (1.0) in July and highest (2.5) in March, and unsaturation indexes (average number of double bonds per molecule) were lowest (1.5) in July and highest (2.3) in March. Fatty acids 18:0 and 20:1 showed a significant correlation with the changes of seasonal temperature. Fatty acid analysis of the developing compound eyes of silk moths during the pupal stage revealed that eicosapentanoic acid (20:5) increased remarkably in parallel with the development of photoreceptive membranes, the rhabdoms. This suggests that eicosapentaenoic acid may play an important role in formation and function of rhabdoms.     

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors

In crayfish photoreceptor cells, Gq-type G-protein plays a central role in the phototransduction pathway, and the translocation of Gqα has been proposed as one of the molecular mechanisms to control photoreceptor sensitivity. We here investigated β subunit of Gq and its localization profiles under various light conditions in the crayfish photoreceptor cells to understand the functional characteristic of visual Gq in the phototransduction pathway. An immunoprecipitation experiment was performed using an anti-Gqα antibody and a thiol-cleavable crosslinker. A 39 kDa protein was co-immunoprecipitated with Gqα, but not by irradiation, in the presence of GTPγS. The partial amino acid sequence of the 39 kDa protein was similar to Gβe in Drosophila photoreceptors, indicating that the crayfish Gβ which combines with Gqα is a Gβe homologue. Immunohistochemical and immunoblot analyses revealed that the amount of the Gβ decreased in the rhabdomeric membranes and increased in the cytoplasm in the light, compared with that in the dark. The profile of the translocation was similar to that reported for Gqα. Since both α and βγ subunits are necessary for G-proteins to be activated by rhodopsin in the rhabdom, the light-modulated translocation of a Gβe homologue possibly controls the amount of Gq which can be activated by light-stimulated rhodopsin. Accepted: 27 June 1998     

Non-spiking local interneurones mediate abdominal extension related descending control of uropod motor neurones in the crayfish terminal abdominal ganglion

The role of non-spiking local interneurones in the synaptic interactions between abdominal extension-evoking descending interneurones and uropod motor neurones in the terminal abdominal ganglion of the crayfish Procambarus clarkii (Girard) was investigated electrophysiologically. Continuous electrical stimulation of the lateral region of the 3rd-4th abdominal connective that included abdominal extension evoking interneurones excited the opener motor neurones and inhibited the closer, reductor motor neurone. Spikes from a single descending interneurone evoked consistent and short latency (0.8–0.9 ms) excitatory postsynaptic potentials (e.p.s.ps) in the opener motor neurones, and evoked rather long-latency (1.5–2.7 ms) inhibitory postsynaptic potentials (i.p.s.ps) in the reductor motor neurone. Many non-spiking interneurones also received depolarizing p.s.ps (0.8–2.5 ms in latency) that were usually faster than i.p.s.ps of the reductor motor neurone if both neurones were recorded sequentially in the same preparation. Non-spiking interneurones received convergent inputs from several descending interneurones and made inverting connection with the reductor motor neurone. Elimination of descending inputs to a particular non-spiking interneurone could reduce the inhibitory response of the reductor motor neurone. These observations strongly suggested that descending inhibitory inputs to the closer, reductor motor neurone were mediated by non-spiking interneurones. Furthermore, some non-spiking interneurones made output connections with the opener motor neurones. The disynaptic pathway through non-spiking interneurones is significant to control and modulate the opening pattern of the uropod during abdominal extension. Accepted: 27 December 1996     

Effect of the removal of cleaner fish on the abundance and species composition of reef fish

The ecological significance of cleaner fish on coral reefs was investigated. I removed all cleaner fish, Labroides dimidiatus, from eight small reefs, measured the subsequent effect on the abundance and species composition of all reef fish after 3 and 6 months, and compared it with eight control reefs with cleaner fish. The removal of cleaner fish had no detectable effect on the total abundance of fish on reefs and the total number of fish species at both times. Multivariate analysis by non-metric multidimensional scaling and ANOSIM pairwise tests based on 191 fish species revealed no effect of cleaners on the community structure of fish. Similar results were obtained using principal components analysis on subsets of the data using the 33 most common fish species and the 15 most abundant species (≥5 individuals per reef ) with both log10 (x + 1) transformed data and with fish numbers standardized for abundance. This study demonstrates that the removal of cleaner fish for 6 months did not result in fish suffering increased mortality nor in fish leaving reefs to seek cleaning elsewhere. Received: 28 October 1996 / Accepted: 7 February 1997     

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons in the crayfish Procambarus clarkii Girard

Dendritic properties of uropod motoneurons and premotor nonspiking interneurons of crayfish have been studied using intradendritic recording and current injection. The input resistance of phasic motoneurons (5.20 ± 0.5 M; mean ± standard error) measured by injecting constant hyperpolarizing current was significantly lower than that of tonic motoneurons (10.3 ± 2.6 M; 0.02 < P < 0.05). The membrane time constant of phasic motoneurons (7.3 ± 0.9 ms) was also significantly shorter than that of tonic motoneurons (24.3 ± 2.5 ms; P < 0.001). Both types of motoneurons behaved linearly during hyperpolarization and sub-threshold depolarization. Nonspiking interneurons showed outward rectification upon depolarization. During hyperpolarization, their membrane behaved linearly and showed significantly higher input resistance (19.5 ± 2.5 M) than phasic and tonic motoneurons (P < 0.001). Their membrane time constant (38.0 ± 5.7 ms) was significantly longer than that of phasic motoneurons (P < 0.001) but not than that of tonic motoneurons (P > 0.05). In response to intracellular injection of sinusoidally oscillating current, phasic motoneurons showed one or two spikes per depolarization period irrespective of oscillating frequency ranging from 1 to 16 Hz. Tonic motoneurons showed larger numbers of spikes per stimulus period at lower frequencies. Nonspiking interneurons also showed phase-locked effects on the motoneuron spike activity. The effective frequency range over which injected oscillating current could modulate motoneuron spike activity was similar for tonic motoneurons and nonspiking interneurons.     

Regulation of methyl farnesoate production by mandibular organs in the crayfish, Procambarus clarkii: a possible role for allatostatins

Decapod crustaceans do not appear to produce juvenile hormone, but rather its immediate precursor, methyl farnesoate (MF). Both MF and its immediate precursor, farnesoic acid (FA) are produced by the mandibular organs (MO) in crustaceans. The MO are homologous to the insect corpora allata (CA), the site of juvenile hormone biosynthesis. However, the FGLamide allatostatin (ASTs) peptides, of which there are about 60 distinct forms reported from crustaceans, have previously been found to have no effect on MO activity in crustaceans. We have identified by immunocytochemistry the presence of FGLamide-like AST immunoreactivity in neurosecretory cells throughout the CNS as well as in neurohaemal structures such as the sinus gland and pericardial organs. The ASTs are likely delivered to the MO hormonally and/or by local neurohaemal release. Using MO from adult males, we have found wide variability between animals in the in vitro rates of MF and FA biosynthesis. Treatment with Dippu-ASTs has a statistically significant stimulatory effect on MF synthesis, but only in MO that are initially producing MF at lower rates. No effect on FA production was observed, suggesting that the FGLamide ASTs exert their effect on the o-methyl transferase, the enzyme responsible for the conversion of FA to MF.     

Sexual dimorphism and species differences in the neurophysiology and morphology of the acoustic communication system of two neotropical hylids

We examined auditory tuning and the morphology of the anatomical structures underlying acoustic communication in female Hyla microcephala and H. ebraccata and compared our findings to data from a previous study (Wilczynski et al. 1993) in which we showed species differences in the traits that in males relate to differences in the species-typical calls. Female species differences in the best excitatory frequency (BEF) of the basilar papilla (BP) were similar to the differences seen in males, and females had a significantly lower BEF in H. ebraccata, but not H. microcephala. In both species, females had lower BP thresholds. Snout-vent length, head width, and tympanic membrane diameters were sexually dimorphic in both species and larger in females, whereas laryngeal components were sexually dimorphic and larger in males. Middle and inner ear volumes were not sexually dimorphic. Despite the significant species differences in laryngeal morphology seen in males, female larynges are not significantly different. Furthermore, the interaction of species and sex differences resulted in significantly different degrees of sex dimorphism in the species, particularly for the larynx, which is more sexually dimorphic in H. microcephala, and measures of body size, which are more dimorphic in H. ebraccata. Accepted: 6 December 1996     

Post-dispersal seed predation and the establishment of vertebrate dispersed plants in Mediterranean scrublands

The post-dispersal fate of seeds and fruit (diaspores) of three vertebrate-dispersed trees, Crataegus monogyna, Prunus mahaleb and Taxus baccata, was studied in the Andalusian highlands, south-eastern Spain. Exclosures were used to quantify separately the impact of vertebrates and invertebrates on seed removal in relation to diaspore density and microhabitat. The three plant species showed marked differences in the percentage of diaspores removed, ranging from only 5% for C. monogyna to 87% for T. baccata. Although chaffinches (Fringilla coelebs) fed on diaspores, rodents (Apodemus sylvaticus) were the main vertebrate removers of seed and fruit. Two species of ant (Cataglyphis velox and Aphaenogaster iberica) were the only invertebrates observed to remove diaspores. However, the impact of ants was strongly seasonal and they only removed P. mahaleb fruit to any significant extent. While removal of seed by rodents was equivalent to predation, ants were responsible for secondary dispersal. However, their role was limited to infrequent, small-scale redistribution of fruit in the vicinity of parent trees. Rodents and ants differed in their use of different microhabitats. Rodents foraged mostly beneath trees and low shrubs and avoided open areas while the reverse was true of ants. Thus, patterns of post-dispersal seed removal will be contigent on the relative abundance and distribution of ants and rodents. Studies which neglect to quantify separately the impacts of these two guilds of seed removers may fail to elucidate the mechanisms underlying patterns of post-dispersal seed removal. The coincidence of both increased seed deposition by the main avian dispersers (Turdus spp.) and increased seed predation with increasing vegetation height suggested that selection pressures other than post-dispersal seed predation shape the spatial pattern of seed dispersal. Rather than providing a means of escaping post-dispersal seed predators, dispersal appears to direct seeds to microhabitats most suitable for seedling survival. Nevertheless, the reliance of most vertebrate-dispersed trees on regeneration by seed and the absence of persistent soil seed banks imply that post-dispersal seed predators may exert a strong influence on the demography of the plants whose seeds they consume. Even where microsites are limited, the coincidence of the most suitable microhabitats for seedling establishment with those where seed predation is highest provide a means by which selective seed predators can influence community composition. Received: 19 August 1996 / Accepted: 25 January 1997     

The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant

In order to test whether an increased export of carbohydrates by leaves and starch mobilization are critical for floral transition in Arabidopsis thaliana, the Columbia ecotype as well as its starchless mutant pgm and starch-in-excess mutant sex1 were investigated. Induction of flowering was achieved by exposure of plants to either one long day (LD) or one displaced short day (DSD). The following conclusions were drawn: (i) Both the pgm and sex1 mutants have a late-flowering phenotype in days shorter than 16 h. (ii) When inductive treatments cause a large percentage of induced plants, there is always a large, early and transient increase in carbohydrate export from leaves. By contrast, when an inductive treatment results in only a low percentage of induced plants (pgm plants exposed to one DSD), the export of carbohydrates from leaves is not increased, supporting the idea that phloem carbohydrates have a critical function in floral transition. (iii) Starch mobilization is not required to obtain an increased carbohydrate export when induction is by one LD (extended period of photosynthesis), but is absolutely essential when induction is by one DSD (period of photosynthesis unaffected). (iv) Floral induction apparently increases the capability of the leaf phloem-loading system. Received: 27 August 1997 / Accepted: 6 March 1998     

Fatty acid composition of brown adipose tissue in genetically heat-tolerant FOK rats

The phospholipid fatty acid composition of brown adipose tissue (BAT) was examined in inbred heat-tolerant FOK rats and compared with that in conventional Wistar rats not previously exposed to heat. The FOK rats showed higher unsaturation states, as indicated by higher levels of polyunsaturated fatty acids and a higher unsaturation index and polyunsaturated fatty acids/saturated fatty acids ratio. This higher level of unsaturation was characterized by the higher amount of polyunsaturated fatty acids such as linoleic acid, arachidonic acid and docosahexaenoic acid. It may be concluded that the increased docosahexaenoic acid level in BAT phospholipids brings about the hyperplasia of BAT, causing an enhancement of its in vivo thermogernic activity as well as the systemic non-shivering thermogenesis observed in heat-tolerant FOK rats. Received: 17 May 2000 / Revised: 21 August 2000 / Accepted: 21 September 2000     

Application of a 15N tracer to simulate and track the fate of atmospherically deposited N in the coastal forests of the Waquoit Bay Watershed, Cape Cod, Massachusetts

The central grassland region of the United States encompasses major gradients in temperature and precipitation that determine the distribution of plant life forms, which in turn may influence key ecosystem processes such as nutrient cycling and soil organic matter dynamics. One such gradient is the threefold increase in precipitation from the eastern Colorado shortgrass-steppe, in the rain shadow of the Rocky Mountains, to the tallgrass prairie in eastern Kansas. We investigated the relative roles of plant species and plant cover in influencing soil C and N cycling in three sites along this gradient. Plant cover (i.e., the presence or absence of an individual plant) was relatively more important than plant species in explaining variability in soil properties at the dry site, the Central Plains Experimental Range in␣northeastern Colorado. However, plant species explained relatively more of the variability in soil properties than did plant cover at the two wetter sites, Hays and Konza, in central and eastern Kansas. The wetter sites had more continuous plant cover, resulting in less plant-cover-induced variation in soil C and N, than did the dry site, which had distinct patches of bare ground. Plant species at the wetter sites had higher and more variable levels of tissue C:N than plant species at the dry site, due to both within species changes and changes in species composition. Aboveground tissue C:N was better correlated with net nitrogen mineralization rates at the wet sites than the dry site. Thus, tissue chemistry appears to exert more control on nitrogen dynamics at the wet than the dry sites. The results suggest that plant species traits that are relevant to nutrient cycling (e.g., tissue C:N ratios, spatial patterns, productivity) reflect environmental limitations as well as species' physiological potentials. Furthermore, a dominant environmental driver such as precipitation may ameliorate or exaggerate the importance of individual species traits for nutrient cycling. Received: 11 July 1996 / Accepted: 5 December 1996     

Alignment of the genetic and physical maps in the dpy-5 bli-4 (I) region of C. elegans by the serial cosmid rescue of lethal mutations

Lethal mutations in the 0.5 map unit region between dpy-5 and bli-4 on chromosome I in Caenorhabditis elegans were serially rescued using cosmid-containing transgenic strains. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described previously. Germline transformation with cosmid DNA was used to create 25 transgenic strains bearing heritable extrachromosomal arrays. These arrays were used as small duplications for the fine-scale mapping of essential genes, via the rescue of lethal mutations. Lethal mutations in 13 essential genes have been phenotypically rescued, allowing the alignment of the genetic and physical maps in this region. Extrachromosomal arrays were found to be transmitted 2- to 7-fold less frequently in oocytes than in hermaphrodite sperm for 12 of the 16 arrays that were examined. Three of these strains showed a subsequent 4- to 13-fold increase in array stability in oocytes. This phenomenon may be influenced by cosmid sequences. Early mitotic loss of the arrays was observed in all 17 transgenic strains examined, suggesting that loss of the array can occur at any time during development when cell divisions are occurring. As a result of this work, 13 of the essential loci positioned between dpy-5 and bli-4 are anchored to the physical map, thereby providing links between the physical and genetic maps on average every 85 kb. Received: 8 May 1996 / Accepted: 27 January 1997     

Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans

The unstable linear chromosome of Streptomyces lividans was circularized by homologous recombination and its terminal inverted repeats deleted. Strains with circularized chromosomes showed no obvious phenotypic disadvantages compared to the wild type. However, they segregated about 20 times more chloramphenicol-sensitive mutants than the wild type (24.3% vs. 1.4%), due to a higher incidence of large deletions. In addition, in all circularized chromosomes amplification of 30–60 kb fragments was observed at the new chromosomal junction, to levels of approximately 10 copies per chromosome. Arginine auxotrophs that arose spontaneously among the progeny of strains with a circularized chromosome showed high-copy-number amplification of the DNA element AUD1, as also seen in mutants of the wild type. These observations demonstrate that the circular form of the Streptomyces chromosome is more unstable than the linear one. Received: 28 July 1996 / Accepted: 18 November 1996     

An Aspergillus nidulans nuclear protein, AnCP, involved in enhancement of Taka-amylase A gene expression, binds to the CCAAT-containing taaG2 , amdS , and gatA promoters

Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A.␣nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells. Received: 28 June 1996 / Accepted: 7 October 1996     

Effects of hydration state on hormonal and renal responses during moderate exercise in the heat

The effects of hydromineral hormones and catecholamines on renal concentrating ability at different hydration states were examined in five male volunteers while they performed three trials. Each of these trials comprised a 60-min exercise bout on a treadmill (at 50% of maximal oxygen uptake) in a warm environment (dry bulb temperature, 35°C; relative humidity, 20–30%). In one session, subjects were euhydrated before exercise (C). In the two other sessions, after thermal dehydration (loss of 3% body mass) which markedly reduced plasma volume (PV) and increased plasma osmolality (osm_pl), the subjects exercised either not rehydrated (Dh) or rehydrated (Rh) by drinking 600 ml of mineral water before and 40 min after the onset of exercise. During exercise in the Dh compared to C state, plasma renin, aldosterone, arginine vasopressin (AVP), noradrenaline and adrenaline concentrations were increased (P < 0.05). A reduction in creatinine clearance and urine flow was also observed (P < 0.05) together with a decrease in urine osmolality, osmolar clearance and sodium excretion, while free water clearance increased (P < 0.05). However, compared to Dh, Rh partially restored PV and osm_pl and induced a marked reduction in the time courses of both the plasma AVP and catecholamine responses (P < 0.05). Values for renal water and electrolyte excretion were intermediate between those of Dh and C. Plasma atrial natriuretic peptide presented similar changes whatever the hydration state. These results demonstrate that during moderate exercise in the heat, renal concentrating ability is paradoxically reduced by prior dehydration in spite of high plasma AVP levels, and might be the result of marked activation of the sympatho-adrenal system. Rehydration, by reducing this activation, could partially restore the renal concentrating ability despite the lowered plasma AVP. Accepted: 23 April 1997     

Effects of experimental manipulation of inflorescence size on pollination and pre-dispersal seed predation in the hummingbird-pollinated plant Ipomopsis aggregata

Large floral displays should theoretically provide advantages to plants through increased pollinator visitation and resulting fruit and seed set. However empirical tests of the response of pollinators to floral display size have been limited by a lack of direct experimentation, and the results of such studies have been equivocal. In addition, other selective agents such as pre-dispersal seed predators might modulate effects of floral display on pollination. By artificially altering flower number, we examined the direct effects of floral display in the monocarpic herb, Ipomopsis aggregata (Polemoniaceae), on visitation rates by broad-tailed and rufous hummingbird pollinators, as well destruction of fruits by a pre-dispersal seed predator (Hylemya: Anthomyiidae). In addition, we quantified the ultimate effects of flower number on female reproductive success. Plants with larger floral displays were most likely to be visited first in any given foraging bout (P < 0.01). As expected, plants with more flowers received more total flower visits. However, we found no gain in the proportion of flowers visited for many- versus few-flowered plants, or the total number of approaches/hour. In fact, a significantly greater percentage of flowers were visited on few-flowered plants. Plants did not compensate for our reduction in flowers by increasing investment in the number or proportion of flowers that set fruit, the number of seeds/fruit, or seed weight. Pre-dispersal seed predation was greater for many- than for few-flowered plants (P < 0.001), but this did not offset the potential fitness gains of producing large displays. Our data support the hypothesis that large floral displays function primarily in long-distance attraction of pollinators, and enhance maternal success. Received: 21 March 1996 / Accepted: 24 October 1996