To further scrutinize this unusual reverse EAG responses, we used gas chromatography with electroantennographic detection . In GC-EAD analyses, injected mixtures are separated by GC and subjected to antennal preparations under the same condition thus ruling out any possibility of mechanical interference and minor sample contamination. Here, methyl salicylate responded with regular EAG responses, i.e., with the first phase , which is referred to as rise of the receptor potential, and the second phase starting at the end of the stimulus, commonly referred to as the decline of the receptor potential . This is analogous to the depolarization, repolarization, and hyperpolarization of a nervous impulse. As opposed to methyl salicylate, eucalyptol consistently gave inverse EAD responses  thus corroborating what we observed in EAG analyses . Next, we recorded EAG responses when flies were challenged with odorants and an inhibitor. First, we compared the response of w1118 and Orco-Gal4/UAS-CqOR32 flies to -2-hexenal when it was delivered alone or in combination with eucalyptol. EAG responses from w1118 flies to 0.1% -2-hexenal alone or in combination with 10% eucalyptol did not differ significantly . By contrast, EAG responses from Orco-Gal4/UAS-CqOR32 flies to 0.1% -2-hexenal plus 10% eucalyptol were significantly lower than those elicited by 0.1% -2-hexenal alone . We then examined the dose-dependent effect of this inhibition by using Orco-Gal4/UAS-CqOR32 flies. Robust responses to 0.1% methyl salicylate were reduced in a dose-dependent manner with the addition of eucalyptol but remained unchanged at the end of the tests. Likewise, EAG responses to 0.01% -2-hexenal were reduced when coapplied with eucalyptol . Of note, -2-hexenal does not activate CquiOR32 . Such inhibition presumably results from CquiOR32 indirectly inhibiting responses of the fly endogenous receptors to -2-hexenal. In these continuous experiments, a small difference between EAG responses before and after costimulus tests may be due to loss of this volatile semiochemical from the cartridge rather than adaptation. Similar inhibition was observed when 2-heptanone was applied alone or coapplied with eucalyptol . Taken together, these results further suggest that intrareceptor inhibition occurs in vivo as indicated by the inhibitory effect of eucalyptol on methyl salicylate responses. Additionally,drainage planter pot the effect of eucalyptol on the response to -2-hexenal suggests that intraneuronal inhibition occurred.

A few lines of evidence support this hypothesis. First and foremost, eucalyptol does not cause inhibition in control flies and -2-hexenal does not activate CquiOR32 . The simplest explanation is that, in Orco-Gal4/UAS-CqOR32 flies, all endogenous receptors are coexpressedwith CquiOR32. Thus, CquiOR32 response to eucalyptol interferes with the response of DmelOR7a to -2-hexenal. In short, inhibitor and agonist are likely to be acting on different receptors in the same neurons, thus an intraneuron inhibition. To further test the notion of intraneuronal inhibition, we turned to single sensillum recordings .The best ligand for ab4A, the neuron in ab4 sensilla with a large spike amplitude, is -2-hexenal , although ab4A is also very sensitive to other ligands, including hexanal . Contrary to ab4B, ab4A houses only one OR, namely, DmelOr7a . Because expression of CquiOR32 was driven by DmelOrco, ab4A neurons in our transgenic flies house both DmelOr7a and CquiOR32. Coexpression was confirmed by a significantly stronger response to methyl salicylate recorded from Orco-Gal4/UAS-CquiOR32 than from WT flies , while retaining response to hexanal . It is known that methyl salicylate is the best ligand for DmelOr10a in ab1D but elicits only very low response in ab4A . The low response of WT flies to methyl salicylate did not differ significantly when the odorant was delivered alone or codelivered with eucalyptol . By contrast, responses recorded from Orco-Gal4/UAS-CquiOR32 flies were significantly lower when the two stimuli were delivered simultaneously from two different cartridges . Next, we tested whether CquiOR32 response to eucalyptol would affect DmelOR7a response to a cognate ligand, hexanal. Responses of WT flies to hexanal did not differ significantly when comparing hexanal alone with hexanal plus eucalyptol . Recordings from ab4 sensilla in the Orco-Gal4/UAS-CquiOR32 flies showed a slight, albeit not significant, increase in response to hexanal. This is unlikely to be due to hexanal activation of CquiOR32 . When hexanal and eucalyptol were delivered simultaneously firing of DmelOR7a was completely abolished . We also recorded from ab7 sensilla, which expresses DmelOR98a, in ab7A and for which butyl acetate is one of the best ligands . Eucalyptol elicited inhibitory response in ab7A neurons of Orco-Gal4/UASCquiOR32 flies . In the transgenic flies both methyl salicylate and butyl acetate generated excitatory responses , which were inhibited by eucalyptol . Because methyl salicylate and eucalyptol elicit inward and reverse currents in CquiOR32, this in vivo inhibition is not surprising.

However, the consistent observation that eucalyptol inhibits the response of an endogenous receptor to a cognate ligand supports the notion that intraneuronal inhibition occurs when receptors are colocated in a neuron. Specifically, the inhibitory responses of CquiOR32 interferes with the activation of a collocated receptor by a cognate ligand. For example, activation of DmelOR7a in ab4A neuron by hexanal and activation of DmelOR98a in ab7A neuron by butyl acetate were both inhibited by eucalyptol upon interaction with CquiOR32. Contrary to the fruit fly, which expresses only one receptor per neuron , mosquitoes can coexpress multiple ORs in the same neuron .CquiOR32 has an orthologue in the genome of the yellow fever mosquito, AaegOR71 , with 55.5% identity. We sequenced 20 clones and obtained 19 AaegOR71 sequences. Five clones showed sequences identical to the sequence in VectorBase and were, therefore, considered the WT. We expressedAaegOR71-WT in the Xenopus oocyte recording system and challenged the oocytes with compounds that elicited inward and inhibitory currents in CquiOR32. Cyclohexanone elicited inward currents, but the compounds generating the largest inward currents were 4,5-dimethylthiazole and 2-methyl-2-thiazoline ; no response was observed with methyl salicylate. Although eucalyptol and fenchone did not elicit measurable inhibitory currents, these two compounds reduced AaegOR71 responses to cyclohexanone, DMT, and 2MT . Four clones differed from WT in 7 amino acid residues, and 3 clones differed from WT in 11 amino acid residues. They both showed weak responses to odorants when tested in the Xenopus oocyte recording system. The other 7 clones differed from the WTin 6–12 amino acid residues. AaegOR71-V5 , AaegOR71-V14 , and AaegOR71-V15 differed in 12, 9, and 11 amino acid residues, respectively, and none of them responded to odorants. AaegOR71-V8 differed in 10 amino acid residues and showed very weak response only to the Orco agonist VUAA-1. AaegOR71-V4 , AaegOR71-V9 , AaegOR71- V17 differed in 8, 10, and 6 amino acid residues but gave weak to moderate responses to odorants. Next, we tested whether intrareceptor inhibition might be manifested in vivo in the antennae of the yellow fever mosquito. With 350 contacts, 69 recordings were made from SST-2 sensilla.

SSR showed that cyclohexanone, 2-methyl-2-thiazoline, and 2,4-dimethylthiazole elicited dose-dependent excitatory responses in neuron-A in SST-2, whereas eucalyptol and fenchone showed inhibitory responses . When costimulated with 2-methyl-2-thiazoline and eucalyptol, the response to the odorant decreased markedly . We then analyzed the effect of inhibitors on the responses to the three odorants that caused excitatory responses. Both eucalyptol and fenchone inhibited the responses of ORN-A in SST-2 to cyclohexanone , 2-methyl-2-thiazoline , and 4,5-dimethylthiazole in a dose-dependent manner.In 1970, Philip Bjork described a small fossil bear from the Pliocene Glenn’s Ferry Formation of southwestern Idaho. Based on a single m1 as the holotype, he was understandably perplexed and named it Ursus abstrusus. Additional material has not been forthcoming since its initial description and this bear has remained an enigma. Hence the discovery in the 1990s of a similar bear from more complete fossils in the Pliocene of the Canadian High Arctic throws much needed light onto the mystery . In addition to resolving the riddle of Ursus abstrusus, with a moderately complete skull and lower jaws with associated post cranials,plant pot with drainage the new materials present a rare opportunity to fill a large gap in our knowledge of North American High Arctic at a time in the early Pliocene when mean annual temperatures in the High Arctic were ~22 °C warmer than the present polar temperatures. Such a warm climate supported an extensive boreal-type forest biome, radically different from today’s arid polar tundr. Thus the evidence of this primitive bear in an extinct polar forest offers valuable information about the diet and habitat of this basal ursine. Te fossil records of basal ursines has improved with recent discoveries of three relatively complete specimens of basal ursines from China – a very advanced Ursavus and a very primitive Protarctos . We are now in a position to more tightly bracket the North American Pliocene bears as well as providing a wealth of information about cranial anatomy of basal ursines previously unavailable. Te present description of P. abstrusus and a phylogenetic analysis combining molecular and morphological data of most fossil and living ursines for the first time allows a much more detailed view of the history of bears at the critical juncture of their initial diversifcation. In addition, the presence of dental caries provides insight into the evolutionary history of diet of ursines.Protarctos abstrusus is a basal ursine the size of a small Asian black bear. It has a fat forehead covering an uninfated frontal sinus; very high sagittal crest that projects backward to overhang the occipital condyle ; P4 with a small, distinct protocone situated at the level of carnassial notch; M2 talon modestly developed but not very elongated ; no pre-metaconid on m1, smooth posterior surface of m1 trigonid without zigzagpattern, presence of a distinct pre-entoconid; m2 shorter than m1 . It is about the same size as P. boeckhi and difers from it in the relatively smaller p4, presence of a tiny cuspule on lingual side of posterior crest in p4, and presence of a pre-entoconid on m1. P. abstrusus is also similar in size to P. yinanensis and can be distinguished from the latter in a fattened forehead, posteriorly projected sagittal crest, p4 posterior accessory cuspule on lingual side of posterior crest, an m1 pre-entoconid, and less elongated M1 and M2. P. abstrusus difers from P. ruscinensis by its lack of unique features of the latter such as a deep angular process, reduction of P4 protocone, and a single entoconid on m1.There is much disagreement over the generic taxonomy of ursines. Most mammalogists and some paleontologists include all living black bears , brown bears, and polar bears in the genus Ursus but allow separate generic status for the sloth bear, Melursus, and sun bear, Helarctos, although some include all of above in Ursus and others use Talarctos for the polar bear. With a deep time perspective, vertebrate paleontologists either adopt some subgeneric names, such as Ursus for sloth bear, Ursus for Asian black bear, Ursus for American black bear, Ursus for some extinct bears or elevate some of them to generic status. In his remarks about carnivoran classifcation, Kretzoi erected a new genus, Protarctos, for Ursus boeckhi Schlosser, 1899. Kretzoi’s name has been adopted either at full generic rank or as a subgenus, although many authors still prefer a more inclusive usage of Ursus. In our cladistic framework in this study, some generic reassignment becomes necessary to maintain monophyly, especially in light of the general preference to giving sloth and sun bears distinct generic status.Te Beaver Pond site, 78° 33′N 82° 22′W, is a >20m succession of fne to coarse cross-bedded fuvial sands conformably overlain by cobble gravels interpreted to be glacial outwash and capped by 2m of till on the northeastern edge of an interfuvial plateau southeast of Strathcona Fiord on Ellesmere Island, Nunavut . A peat deposit near the base of the sequence, up to 2.4 m thick, produced exceptionally well-preserved plant, invertebrate and vertebrate remains , and is disconformably overlaying light-colored, tilted Eocene sediments. Abundant beaver-cut branches and cut saplings of larch trees suggest that the peat growth may have been promoted by beaver activity. Further supporting this view are the skeletal remains of multiple beaver individuals, and two clusters of beaver-cut branches found within the peat unit, at least one of which was interpreted to be the core of a dam. Using terrestrial cosmogenic nuclide burial dating, four samples of quartz-rich coarse sand from above the peat unit yielded a weighted mean date of >3.4+0.6/−0.4Ma, suggesting the peat accumulation was formed during a mid-Pliocene warm phase.At 78°N, the Beaver Pond site on Ellesmere Island is presently extremely cold and arid, with ice sheets, permafrost, and sparse vegetation.