Species interactions and random dispersal rather than habitat filtering drive community assembly during early plant succession

Theory on plant succession predicts a temporal increase in the complexity of spatial community structure and of competitive interactions: initially random occurrences of early colonising species shift towards spatially and competitively structured plant associations in later successional stages. Here we use long-term data on early plant succession in a German post mining area to disentangle the importance of random colonisation, habitat filtering, and competition on the temporal and spatial development of plant community structure. We used species co-occurrence analysis and a recently developed method for assessing competitive strength and hierarchies (transitive versus intransitive competitive orders) in multispecies communities. We found that species turnover decreased through time within interaction neighbourhoods, but increased through time outside interaction neighbourhoods. Successional change did not lead to modular community structure. After accounting for species richness effects, the strength of competitive interactions and the proportion of transitive competitive hierarchies increased through time. Although effects of habitat filtering were weak, random colonization and subsequent competitive interactions had strong effects on community structure. Because competitive strength and transitivity were poorly correlated with soil characteristics, there was little evidence for context dependent competitive strength associated with intransitive competitive hierarchies.

The use of an electronic von Frey device for evaluation of sensory threshold in neurologically normal dogs and those with acute spinal cord injury

The utility and inter-session repeatability of sensory threshold measurements using an electronic von Frey anesthesiometer (VFA) were assessed in a group of six neurologically normal dogs. Sensory threshold values obtained in neurologically normal dogs were compared to those of dogs with acute spinal cord injury (SCI) caused by intervertebral disc extrusion (n=6) and to a group of neurologically normal dogs with cranial cruciate ligament rupture (CCLR; n=6). Sensory threshold values in neurologically normal dogs were 155.8 ± 37.7 g and 154.7 ± 67.2 g for the left and right pelvic limbs, respectively. The difference in mean sensory threshold values obtained for the group when two distinct testing sessions were compared was not statistically significant (P>0.05). Mean sensory threshold values for the group with SCI were significantly higher than those for neurologically normal dogs at 351.1 ± 116.5 g and 420.3 ± 157.7 g for the left and right pelvic limbs, respectively (P=0.01). A comparison of sensory threshold values for the group with CCLR and neurologically normal dogs was not statistically significant (P>0.05). The modified dorsal technique for VFA described here represents a reliable method to assess sensory threshold in neurologically normal dogs and in those with SCI.

A legacy of human-induced ecosystem changes: spatial processes drive the taxonomic and functional diversities of testate amoebae in Sphagnum peatlands of the Galápagos

Aim Our aims were to compare the composition of testate amoeba (TA) communities from Santa Cruz Island, Galápagos Archipelago, which are likely in existence only as a result of anthropogenic habitat transformation, with similar naturally occurring communities from northern and southern continental peatlands. Additionally, we aimed at assessing the importance of niche-based and dispersal-based processes in determining community composition and taxonomic and functional diversity. Location The humid highlands of the central island of Santa Cruz, Galápagos Archipelago. Methods We survey the alpha, beta and gamma taxonomic and functional diversities of TA, and the changes in functional traits along a gradient of wet to dry habitats. We compare the TA community composition, abundance and frequency recorded in the insular peatlands with that recorded in continental peatlands of Northern and Southern Hemispheres. We use generalized linear models to determine how environmental conditions influence taxonomic and functional diversity as well as the mean values of functional traits within communities. We finally apply variance partitioning to assess the relative importance of niche- and dispersal-based processes in determining community composition. Results TA communities in Santa Cruz Island were different from their Northern Hemisphere and South American counterparts with most genera considered as characteristic for Northern Hemisphere and South American Sphagnum peatlands missing or very rare in the Galápagos. Functional traits were most correlated with elevation and site topography and alpha functional diversity to the type of material sampled and site topography. Community composition was more strongly correlated with spatial variables than with environmental ones. Main conclusions TA communities of the Sphagnum peatlands of Santa Cruz Island and the mechanisms shaping these communities contrast with Northern Hemisphere and South American peatlands. Soil moisture was not a strong predictor of community composition most likely because rainfall and clouds provide sufficient moisture. Dispersal limitation was more important than environmental filtering because of the isolation of the insular peatlands from continental ones and the young ecological history of these ecosystems.

Jack-of-all-trades effects drive biodiversity–ecosystem multifunctionality relationships in European forests

There is considerable evidence that biodiversity promotes multiple ecosystem functions (multifunctionality), thus ensuring the delivery of ecosystem services important for human well-being. However, the mechanisms underlying this relationship are poorly understood, especially in natural ecosystems. We develop a novel approach to partition biodiversity effects on multifunctionality into three mechanisms and apply this to European forest data. We show that throughout Europe, tree diversity is positively related with multifunctionality when moderate levels of functioning are required, but negatively when very high function levels are desired. For two well-known mechanisms, ‘complementarity’ and ‘selection’, we detect only minor effects on multifunctionality. Instead a third, so far overlooked mechanism, the ‘jack-of-all-trades’ effect, caused by the averaging of individual species effects on function, drives observed patterns. Simulations demonstrate that jack-of-all-trades effects occur whenever species effects on different functions are not perfectly correlated, meaning they may contribute to diversity–multifunctionality relationships in many of the world’s ecosystems.

Mechanisms of TGF-beta1-induced neuronal plasticity in Aplysia

Transforming growth factor beta-1 (TGF-β1) is a cytokine and neurotrophic factor whose neuromodulatory effects in Aplysia californica were recently described. Previous results demonstrated that TGF-β1 induces long-term increases in the efficacy of sensorimotor synapses, a neural correlate of sensitization of the defensive tail withdrawal reflex. These results provided the first evidence that a neurotrophic factor regulates neuronal plasticity associated with a simple form of learning in Aplysia, and raised many questions regarding the nature of the modulation. No homologs of TGF-β had previously been identified in Aplysia, and thus, it was not known whether components of TGF-β1 signaling pathways were present in Aplysia. Furthermore, the signaling mechanisms engaged by TGF-β1 had not been identified, and it was not known whether TGF-β1 regulated other aspects of neuronal function.^ The present investigation into the actions of TGF-β1 was initiated by examining the distribution of the type II TGF-β1 receptor, the ligand binding receptor. The receptor was widely distributed in the CNS and most neurons exhibited somatic and neuritic immunoreactivity. In addition, the ability of TGF-β1 to activate the cAMP/PKA and MAPK pathways, known to regulate several important aspects of neuronal function, was examined. TGF-β1 acutely decreased cAMP levels in sensory neurons, activated MAPK and triggered translocation of MAPK to the nucleus. MAPK activation was critical for both short- and long-term regulation of neuronal function by TGF-β1. TGF-β1 acutely decreased synaptic depression induced by low frequency stimuli in a MAPK-dependent manner. This regulation may result, at least in part, from the modulation of synapsin, a major peripheral synaptic vesicle protein. TGF-β1 stimulated MAPK-dependent phosphorylation of synapsin, a process believed to regulate synaptic vesicle mobilization from reserve to readily-releasable pools of neurotransmitter. In addition to its acute effect on synaptic efficacy, TGF-β1 also induced long-term increases in sensory neuron excitability. Whereas transient exposure to TGF-β1 was not sufficient to drive short-or long-term changes in excitability, prolonged exposure to TGF-β1 induced long-term changes in excitability that depended on MAPK. The results of these studies represent significant progress toward an understanding of the role of TGF-β1 in neuronal plasticity. ^

The role of sensory neuron outgrowth in long-term sensitization of the tail-elicited tail-siphon withdrawal reflex of Aplysia

Neuronal outgrowth has been proposed in many systems as a mechanism underlying memory storage. For example, sensory neuron outgrowth is widely accepted as an underlying mechanism of long-term sensitization of defensive withdrawal reflexes in Aplysia. The hypothesis is that learning leads to outgrowth and consequently to the formation of new synapses, which in turn strengthen the neural circuit underlying the behavior. However, key experiments to test this hypothesis have never been performed. ^ Four days of sensitization training leads to outgrowth of siphon sensory neurons mediating the siphon-gill withdrawal response in Aplysia . We found that a similar training protocol produced robust outgrowth in tail sensory neurons mediating the tail siphon withdrawal reflex. In contrast, 1 day of training, which effectively induces long-term behavioral sensitization and synaptic facilitation, was not associated with neuronal outgrowth. Further examination of the effect of behavioral training protocols on sensory neuron outgrowth indicated that this structural modification is associated only with the most persistent forms of sensitization, and that the induction of these changes is dependent on the spacing of the training trials over multiple days. Therefore, we suggest that neuronal outgrowth is not a universal mechanism underlying long-term sensitization, but is involved only in the most persistent forms of the memory. ^ Sensory neuron outgrowth presumably contributes to long-term sensitization through formation of new synapses with follower motor neurons, but this hypothesis has never been directly tested. The contribution of outgrowth to long-term sensitization was assessed using confocal microscopy to examine sites of contact between physiologically connected pairs of sensory and motor neurons. Following 4 days of training, the strength of both the behavior and sensorimotor synapse and the number of appositions with follower neurons was enhanced only on the trained side of the animal. In contrast, outgrowth was induced on both sides of the animal, indicating that although sensory neuron outgrowth does appear to contribute to sensitization through the formation of new synapses, outgrowth alone is not sufficient to account for the effects of sensitization. This indicates that key regulatory steps are downstream from outgrowth, possibly in the targeting of new processes and activation of new synapses. ^

Potential Role of Adenosine Signaling in Acetic Acid Activation of Murine Sensory Neurons

Chronic respiratory illnesses are a significant cause of morbidity and mortality, and acute changes in respiratory function often lead to hospitalization. Air pollution is known to exacerbate asthma, but the molecular mechanisms of this are poorly understood. The current studies were aimed at clarifying the roles of nerve subtypes and purinergic receptors in respiratory reflex responses following exposure to irritants. In C57Bl/6J female mice, inspired adenosine produced sensory irritation, shown to be mediated mostly by A-delta fibers. Secondly, the response to inhaled acetic acid was discovered to be dually influenced by C and A-delta fibers, as indicated by the observed effects of capsaicin pretreatment, which selectively destroys TRPV1-expressing fibers (mostly C fibers) and pretreatment with theophylline, a nonselective adenosine receptor antagonist. The responses to both adenosine and acetic acid were enhanced in the ovalbumin-allergic airway disease model, although the particular pathway altered is still unknown.

Intrinsic pH-Sensitivity of Cells in the Retrotrapezoid Nucleus: Possible Role of Glia in Respiratory Drive

An increase in carbon dioxide (CO2) and protons (H+) are the primary signals for breathing. Cells that sense changes in CO2/H+ levels and increase breathing accordingly are located in a region of the caudal medulla oblongata called the retrotrapezoid nucleus (RTN). Specifically, select RTN neurons are intrinsically pH sensitive and send excitatory projections to the respiratory rhythm generator to drive breathing. Glial cells in the RTN are thought to contribute to this respiratory drive, possibly by releasing ATP in response to increases in CO2/H+ levels. However, pH sensitivity of RTN glial cells has yet to be determined. Therefore, the goal of my thesis is to determine if acutely dissociated RTN cells can respond to changes in pH in isolation. To make this determination I used ratiometric fluorescent microscopy to measure intracellular calcium in dissociated RTN cells during changes in bath pH. I found that a small percentage of RTN cells (16%) respond to bath acidification from pH 7.3 to pH 6.9 with an increase in fluorescence indicating an increase in intracellular calcium. Preliminary electrophysiological findings suggest that responsive cells are unable to make action potentials, thus suggesting their identity to be glia. These results indicate that a subset of pH sensitive cells in the RTN are intrinsically pH sensitive and that glia cells may possibly play a role in central chemoreception.

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Long-term hyperexcitability of sensory and motor axons in Aplysia californica: Induction by axotomy, serotonin, and transient depolarization

Neuropathic pain is a debilitating neurological disorder that may appear after peripheral nerve trauma and is characterized by persistent, intractable pain. The well-studied phenomenon of long-term hyperexcitability (LTH), in which sensory somata become hyperexcitable following peripheral nerve injury may be important for both chronic pain and long-lasting memory formation, since similar cellular alterations take place after both injury and learning. Though axons have previously been considered simple conducting cables, spontaneous afferent signals develop from some neuromas that form at severed nerve tips, indicating intrinsic changes in sensory axonal excitability may contribute to this intractable pain. Here we show that nerve transection, exposure to serotonin, and transient depolarization induce long-lasting sensory axonal hyperexcitability that is localized to the treated nerve segment and requires local translation of new proteins. Long-lasting functional plasticity may be a general property of axons, since both injured and transiently depolarized motor axons display LTH as well. Axonal hyperexcitability may represent an adaptive mechanism to overcome conduction failure after peripheral injury, but also displays key features shared with cellular analogues of memory including: site-specific changes in neuronal function, dependence on transient, focal depolarization for induction, and requirement for synthesis of new proteins for expression of long-lasting effects. The finding of axonal hyperexcitability after nerve injury sheds new light on the clinical problem of chronic neuropathic pain, and provides more support for the hypothesis that mechanisms of long-term memory storage evolved from primitive adaptive responses to injury. ^

Exploring parents' memories of their child's death related sensory experiences as a dimension of grieving

Little is known about how dying children and their parents experience death. Dying children have reported death related sensory experiences (DRSEs), defined as seeing or hearing someone or something not visible or audible to others, associated with dying. Although parents report that they and the dying child benefit from these experiences, healthcare providers often unknowingly dismiss them. The aims of this phenomenological inquiry were to describe children's DRSEs and their meaning from the parents' perspectives. Four fathers and six mothers of African American, Caucasian, or Hispanic ethnicity, all Christian, ranging in age from 35 to 59 years, whose child died 23 to 52 months prior and was treated at a children's cancer center, were interviewed in the home or hospital setting of their choice. Children's ages at the time of their death ranged from 4 to 13 years. A modification of van Kaarn's phenomenological method of analysis was used to analyze data. Themes emerging from the data for the first aim were: perceiving someone or something from a spiritual realm others could not, expressing awareness tempered by parental reactions, and embracing transcendence. Themes emerging from the data for the second aim were: spiritual beings prepared child; child revealed reality, preparing parents; and child transcended wholly, easing parents' grief. Post-interview surveys revealed that parents found participating in this study a "very positive" or "positive" experience, particularly being able to tell the story of their child. Children's DRSEs have clinical implications for all who provide care near the end of life. Informing parents of DRSEs, cautioning that not all dying children express them, may help parents to anticipate this phenomenon, which may decrease anxiety when their child expresses them, increasing the opportunity for open dialogue between parent and child about dying and death, and decrease regrets associated with being unreceptive to their child's expressions of death awareness. Validating a child's DRSE can have profound effects on bereaved parents. Examining DRSEs from the child's perspective and the influence of informing parents of DRSEs on the dying experience of the child and the parental grieving process are recommended. ^

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

The role of the intracellular second messenger cAMP in the induction of long-term morphological changes in sensory neurons of {\it Aplysia\/}

The present work examines the role of cAMP in the induction of the type of long-term morphological changes that have been shown to be correlated with long-term sensitization in Aplysia.^ To examine this issue, cAMP was injected into individual tail sensory neurons in the pleural ganglion to mimic, at the single cell level, the effects of behavioral training. After a 22 hr incubation period, the same cells were filled with horseradish peroxidase and 2 hours later the tissue was fixed and processed. Morphological analysis revealed that cAMP induced an increase in two morphological features of the neurons, varicosities and branch points. These structural alterations, which are similar to those seen in siphon sensory neurons of the abdominal ganglion following long-term sensitization training of the siphon-gill withdrawal reflex, could subserve the altered behavioral response of the animal. These results expose another role played by cAMP in the induction of learning, the initiation of a structural substrate, which, in concert with other correlates, underlies learning.^ cAMP was injected into sensory neurons in the presence of the reversible protein synthesis inhibitor, anisomycin. The presence of anisomycin during and immediately following the nucleotide injection completely blocked the structural remodeling. These results indicate that the induction of morphological changes by cAMP is a process dependent on protein synthesis.^ To further examine the temporal requirement for protein synthesis in the induction of these changes, the time of anisomycin exposure was varied. The results indicate that the cellular processes triggered by cAMP are sensitive to the inhibition of protein synthesis for at least 7 hours after the nucleotide injection. This is a longer period of sensitivity than that for the induction of another correlate of long-term sensitization, facilitation of the sensory to motor neuron synaptic connection. Thus, these findings demonstrate that the period of sensitivity to protein synthesis inhibition is not identical for all correlates of learning. In addition, since the induction of the morphological changes can be blocked by anisomycin pulses administered at different times during and following the cAMP injection, this suggests that cAMP is triggering a cascade of protein synthesis, with successive rounds of synthesis being dependent on successful completion of preceding rounds. Inhibition at any time during this cascade can block the entire process and so prevent the development of the structural changes.^ The extent to which cAMP can mimic the structural remodeling induced by long-term training was also examined. Animals were subjected to unilateral sensitization training and the morphology of the sensory neurons was examined twenty-four hours later. Both cAMP injection and long-term training produced a twofold increase in varicosities and approximately a fifty percent increase in the number of branch points in the sensory neuron arborization within the pleural ganglion. (Abstract shortened by UMI.) ^