At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought-imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium-dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February-March) when these toads are active, to toads obtained from dry season (October-November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro-555 conjugated α-bungarotoxin. Confocal microscopy and three-dimensional reconstructions were then used to examine the pre-and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.

1. [Ca2+] was recorded in voltage-clamped gastric myocytes from Bufo marinus. Repolarization to -110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast-slow-fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 +/- 6% (mean +/- S.E.M., n = 21) when compared with unconditioned transients. 2. [Ca2+]i decay was biphasic in cells injected with the calmodulin-binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n = 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 +/- 19% in conditioned transients (n = 6). 3. Cell injection with CK3AA, a pseudo-substrate inhibitor of calmodulin-dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n = 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 +/- 17% increase after the train (n = 5). 4. Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay. 5. These results demonstrate that [Ca2+]i decay is accelerated by Ca(2+)-calmodulin and calmodulin-dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.

Synaptic connections of amacrine cells with substance P-like or neuropeptide Y-like immunoreactivity (SP-LI or NPY-LI) in the retina of the cane toad, Bufo marinus, were investigated using ultrastructural immunocytochemistry. The perikarya of SP-LI or NPY-LI amacrine cells were located in the innermost row of the inner nuclear layer. The synapses associated with SP-LI amacrine cells were distributed mainly in sublaminae 3 and 4 with about 10% in sublamina 1 of the inner plexiform layer. The synapses formed by NPY-LI amacrine cells were found in sublaminae 1, 2, and 4 with approximately equal frequency. Of a total of 175 SP-LI profiles, 56% were in presynaptic positions and 44% in postsynaptic positions. The synaptic inputs to SP-LI profiles predominantly derived from other unlabeled amacrine cell dendrites, and to a lesser extent, from bipolar cell terminals. The majority of synaptic outputs from SP-LI amacrine cell dendrites were directed onto unlabeled amacrine cell processes. The SP-LI profiles also made synapses onto bipolar cell terminals and formed synapses onto presumed ganglion cell dendrites. Of a total of 200 NPY-LI profiles, 48% were in presynaptic positions and 52% in postsynaptic positions. The profiles of NPY-LI amacrine cells mainly received their synaptic inputs from other unlabeled amacrine cell processes, and to a lesser extent, from bipolar cell terminals. The majority of NPY-LI amacrine cell profiles gave their synaptic outputs onto unlabeled amacrine cell dendrites, and others formed synapses onto presumed ganglion cell processes. These results suggest that these two populations of neuropeptide-containing amacrine cells in the Bufo retina are involved in different synaptic circuits.

The synaptic connections of amacrine cells synthesizing or accumulating serotonin in the retina of the cane toad, Bufo marinus, were studied by using preembedding double-labeling electron-microscopic immunocytochemistry. The binding sites of an anti-serotonin antibody were revealed by the diaminobenzidine reaction, whilst a colloidal gold-conjugated secondary antibody was used to detect an antibody to phenylalanine hydroxylase. Since the latter antibody recognizes tryptophan 5-hydroxylase, one of the synthesizing enzymes for serotonin, as well as tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, the double labeling of the present study enabled us to identify three groups of labeled profiles at the ultrastructural level. The profiles of serotonin-synthesizing amacrine cells contained both diaminobenzidine reaction product and colloidal gold particles, whilst those of serotonin-accumulating and dopaminergic amacrine cells contained only diaminobenzidine reaction product or colloidal gold particles, respectively. The synapses of serotonin-synthesizing or serotonin-accumulating amacrine cells were distributed all through the inner plexiform layer of the retina. The profiles of serotonin-synthesizing amacrine cells predominantly received synapses from, and made synapses onto, unlabeled amacrine cell dendrites. They also received synapses from, and made synapses onto, bipolar cell terminals. They also made synapses onto presumed ganglion cell dendrites. However, the profiles of serotonin-accumulating cells made synapses only with unlabeled amacrine cell processes. There were close contacts between the profiles of serotonin-synthesizing and serotonin-accumulating amacrine cells. No synaptic relationships were observed between dopaminergic and serotonin-synthesizing or serotonin-accumulating amacrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.

A nano-flow high-resolution screening platform, featuring a parallel chip-based microfluidic bioassay and mass spectrometry coupled to nano-liquid chromatography, was applied to screen animal venoms for nicotinic acetylcholine receptor like (nAChR) affinity by using the acetylcholine binding protein, a mimic of the nAChR. The potential of this microfluidic platform is demonstrated by profiling the Conus textile venom proteome, consisting of over 1,000 peptides. Within one analysis (<90 min, 500 ng venom injected), ligands are detected and identified. To show applicability for non-peptides, small molecular ligands such as steroidal ligands were identified in skin secretions from two toad species (Bufo alvarius and Bufo marinus). Bioactives from the toad samples were subsequently isolated by MS-guided fractionation. The fractions analyzed by NMR and a radioligand binding assay with α7-nAChR confirmed the identity and bioactivity of several new ligands.

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.

Angiotensin II (Ang II) stimulates oral water intake by causing thirst in all terrestrial vertebrates except anurans. Anuran amphibians do not drink orally but absorb water osmotically through ventral skin. In this study, we examined the role of Ang II on the regulation of water-absorption behavior in the Japanese tree frog (Hyla japonica). In fully hydrated frogs, intracerebroventricular (ICV) and intralymphatic sac (ILS) injection of Ang II significantly extended the residence time of water in a dose-dependent manner. Ang II-dependent water uptake was inhibited by ICV pretreatment with an angiotensin II type-1 (AT(1)) receptor antagonist but not a type-2 (AT(2)) receptor antagonist. These results suggest that Ang II stimulates water-absorption behavior in the tree frog via an AT(1)-like but not AT(2)-like receptor. We then cloned and characterized cDNA of the tree frog AT(1) receptor from the brain. The tree frog AT(1) receptor cDNA encodes a 361 amino acid residue protein, which is 87% identical to the toad (Bufo marinus) AT(1) receptor and exhibits the functional characteristics of an Ang II receptor. AT(1) receptor mRNAs were found to be present in a number of tissues including brain (especially in the diencephalon), lung, large intestine, kidney and ventral pelvic skin. When tree frogs were exposed to dehydrating conditions, AT(1) receptor mRNA significantly increased in the diencephalon and the rhombencephalon. These data suggest that central Ang II may control water intake behavior via an AT(1) receptor on the diencephalon and rhombencephalon in anuran amphibians and may have implications for water consumption in vertebrates.

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence.

Dissections of >1,200 wild-caught cane toads (Rhinella marina) in tropical Australia confirm a laboratory report that anurans can expel foreign objects from the coelom by incorporating them into the urinary bladder. The foreign objects that we found inside bladders included a diverse array of items (e.g., grass seeds, twigs, insect prey, parasites), many of which may have entered the coelom via rupture of the gut wall. In some cases, the urinary bladder was fused to other organs including liver, fat bodies, ovaries, Bidder's organs, lungs, mesentery, stomach wall, gall bladder, and the abdominal wall. Acanthocephalan parasites (of a range of developmental stages) were identified from the walls of the urinary bladders of three cane toads. This organ may play a significant role in destroying or excreting metazoan parasites, as well as inanimate objects.