Dating human occupation and adaptation in the southern European last glacial refuge : The chronostratigraphy of Grotta del Romito (Italy)

Grotta del Romito has been the subject of numerous archaeological, chronological and palaeoenvironmental investigations for more than a decade. During the Upper Palaeolithic period the site contains evidence of human occupation through the Gravettian and Epigravettian periods, multiple human burials, changes in the pattern of human occupation, and faunal, isotopic and sedimentological evidence for local environmental change. In spite of this rich record, the chronological control is insufficient to resolve shifts in subsistence and mobility patterns at sufficiently high resolution to match the abrupt climate fluctuations at this time. To resolve this we present new radiocarbon and tephrostratigraphic dates in combination with existing radiocarbon dates, and develop a Bayesian age model framework for the site. This improved chronology reveals that local environmental conditions reflect abrupt and long-term changes in climate, and that these also directly influence changing patterns of human occupation of the site. In particular, we show that the environmental record for the site, based on small mammal habitat preferences, is chronologically in phase with the main changes in climate and environment seen in key regional archives from Italy and Greenland. We also calculate the timing of the transitions between different cultural phases and their spans. We also show that the intensification in occupation of the site is chronologically coincident with a rapid rise in Mesic Woody taxa seen in key regional pollen records and is associated with the Late Epigravettian occupation of the site. This change in the record of Grotta del Romito is also closely associated stratigraphically with a new tephra (the ROM-D30 tephra), which may act as a critical marker in environmental records of the region

Ion Channel Expression in the Developing Enteric Nervous System

<div><p>The enteric nervous system arises from neural crest-derived cells (ENCCs) that migrate caudally along the embryonic gut. The expression of ion channels by ENCCs in embryonic mice was investigated using a PCR-based array, RT-PCR and immunohistochemistry. Many ion channels, including chloride, calcium, potassium and sodium channels were already expressed by ENCCs at E11.5. There was an increase in the expression of numerous ion channel genes between E11.5 and E14.5, which coincides with ENCC migration and the first extension of neurites by enteric neurons. Previous studies have shown that a variety of ion channels regulates neurite extension and migration of many cell types. Pharmacological inhibition of a range of chloride or calcium channels had no effect on ENCC migration in cultured explants or neuritogenesis <i>in vitro</i>. The non-selective potassium channel inhibitors, TEA and 4-AP, retarded ENCC migration and neuritogenesis, but only at concentrations that also resulted in cell death. In summary, a large range of ion channels is expressed while ENCCs are colonizing the gut, but we found no evidence that ENCC migration or neuritogenesis requires chloride, calcium or potassium channel activity. Many of the ion channels are likely to be involved in the development of electrical excitability of enteric neurons.</p></div

Ion channel genes differentially expressed by purified (FACS-sorted) E14.5 ENCCs compared to E11.5 ENCCs.

<p>Only genes showing an average cycle length less than 30 at either E11.5 or E14.5 are shown. Expression of some genes was confirmed at E14.5 by RT-PCR (bold)</p><p>§Expression was previously detected in the E11.5 gut [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123436#pone.0123436.ref035" target="_blank">35</a>]</p><p>* Expression was not confirmed by RT-PCR at E14.5. Expression of other channels was not examined</p><p>Ion channel genes differentially expressed by purified (FACS-sorted) E14.5 ENCCs compared to E11.5 ENCCs.</p

Ion channels expressed at similar levels in E11.5 and E14.5 ENCC.

<p>Only genes showing an average cycle length less than 30 at either E11.5 or E14.5 are shown. Expression of some genes was confirmed at E14.5 by RT-PCR (bold); expression of KCa3.1 (also called IK1 or IK_Ca), which is encoded by <i>Kcnn4</i>, by ENCCs was also previously reported using immunohistochemistry [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123436#pone.0123436.ref050" target="_blank">50</a>].</p><p>* Expression was not confirmed by RT-PCR at E14.5. Expression of other channels was not examined.</p><p>Ion channels expressed at similar levels in E11.5 and E14.5 ENCC.</p

Expression of chloride channels and the effects of chloride channel blockers on ENCC migration and neurite outgrowth.

<p><b>A.</b> RT-PCR confirming expression of transcripts encoding 6 chloride channels in FACS-sorted ENCCs from E14.5 gut. Adult mouse brain (Br) was used as a positive control, and-RT was a negative control (-). <b>B.</b> Migration assay to assess effects of blocking chloride channels on ENNC migration. Explants of gut were removed from E11.5 mice, when the wavefront of ENCCs (grey) is just beyond the caecum, and grown in culture for 48 hours, during which time the ENCCs migrate into the distal hindgut. The preparations were fixed and the distance from the ileo-caecal junction to the most distal ENCC was then measured (dotted line). There was no significant difference in the distance migrated by ENCCs in explants grown in the presence of bumetanide (n = 9) or NPPB (n = 8) compared to controls (n = 10) (mean ± SEM; one way ANOVA). <b>C.</b> Effects of chloride channel blockers on neurite outgrowth. The small and large intestines from E14.5 mice were dissociated, allowed to adhere to coverslips for 3 hours and then exposed to bumetanide or NPPB for 9 hours. The cells were then fixed and processed for immunohistochemistry using an antibody to Tuj1. There was no significant difference in the percentage of Tuj1+ cells that extended neurites between control and drug-treated cultures (one way ANOVA; a minimum of 1750 Tuj1+ cells was examined from 6 coverslips from 2 experiments).</p