Exploring the response of a key Mediterranean gorgonian to heat stress across biological and spatial scales.

Understanding the factors and processes that shape intra-specific sensitivity to heat stress is fundamental to better predicting the vulnerability of benthic species to climate change. Here, we investigate the response of a habitat-forming Mediterranean octocoral, the red gorgonian Paramuricea clavata (Risso, 1826) to thermal stress at multiple biological and geographical scales. Samples from eleven P. clavata populations inhabiting four localities separated by hundreds to more than 1500 km of coast and with contrasting thermal histories were exposed to a critical temperature threshold (25 °C) in a common garden experiment in aquaria. Ten of the 11 populations lacked thermotolerance to the experimental conditions provided (25 days at 25 °C), with 100% or almost 100% colony mortality by the end of the experiment. Furthermore, we found no significant association between local average thermal regimes nor recent thermal history (i.e., local water temperatures in the 3 months prior to the experiment) and population thermotolerance. Overall, our results suggest that local adaptation and/or acclimation to warmer conditions have a limited role in the response of P. clavata to thermal stress. The study also confirms the sensitivity of this species to warm temperatures across its distributional range and questions its adaptive capacity under ocean warming conditions. However, important inter-individual variation in thermotolerance was found within populations, particularly those exposed to the most severe prior marine heatwaves. These observations suggest that P. clavata could harbor adaptive potential to future warming acting on standing genetic variation (i.e., divergent selection) and/or environmentally-induced phenotypic variation (i.e., intra- and/or intergenerational plasticity).

Population collapse of habitat-forming species in the Mediterranean: a long-term study of gorgonian populations affected by recurrent marine heatwaves.

Understanding the resilience of temperate reefs to climate change requires exploring the recovery capacity of their habitat-forming species from recurrent marine heatwaves (MHWs). Here, we show that, in a Mediterranean highly enforced marine protected area established more than 40 years ago, habitat-forming octocoral populations that were first affected by a severe MHW in 2003 have not recovered after 15 years. Contrarily, they have followed collapse trajectories that have brought them to the brink of local ecological extinction. Since 2003, impacted populations of the red gorgonian Paramuricea clavata (Risso, 1826) and the red coral Corallium rubrum (Linnaeus, 1758) have followed different trends in terms of size structure, but a similar progressive reduction in density and biomass. Concurrently, recurrent MHWs were observed in the area during the 2003-2018 study period, which may have hindered populations recovery. The studied octocorals play a unique habitat-forming role in the coralligenous assemblages (i.e. reefs endemic to the Mediterranean Sea home to approximately 10% of its species). Therefore, our results underpin the great risk that recurrent MHWs pose for the long-term integrity and functioning of these emblematic temperate reefs.

Strong linkages between depth, longevity and demographic stability across marine sessile species.

Understanding the role of the environment in shaping the evolution of life histories remains a major challenge in ecology and evolution. We synthesize longevity patterns of marine sessile species and find strong positive relationships between depth and maximum lifespan across multiple sessile marine taxa, including corals, bivalves, sponges and macroalgae. Using long-term demographic data on marine sessile and terrestrial plant species, we show that extreme longevity leads to strongly dampened population dynamics. We also used detailed analyses of Mediterranean red coral, with a maximum lifespan of 532 years, to explore the life-history patterns of long-lived taxa and the vulnerability to external mortality sources that these characteristics can create. Depth-related environmental gradients-including light, food availability, temperature and disturbance intensity-drive highly predictable distributions of life histories that, in turn, have predictable ecological consequences for the dynamics of natural populations.

Activation of a novel p70 S6 kinase 1-dependent intracellular cascade in the basolateral nucleus of the amygdala is required for the acquisition of extinction memory.

Repeated presentations of a previously conditioned stimulus lead to a new form of learning known as extinction, which temporarily alters the response to the original stimulus. Previous studies have shown that the consolidation of extinction memory requires de novo protein synthesis. However, the role of specific nodes of translational control in extinction is unknown. Using auditory threat conditioning in mice, we investigated the role of mechanistic target of rapamycin complex 1 (mTORC1) and its effector p70 S6 kinase 1 (S6K1) in the extinction of auditory threat conditioning. We found that rapamycin attenuated the consolidation of extinction memory. In contrast, genetic deletion and pharmacological inhibition of S6K1, a downstream effector of mTORC1, blocked within-session extinction, indicating a role for S6K1 independent of protein synthesis. Indeed, the activation of S6K1 during extinction required extracellular signal-regulated kinase (ERK) activation in the basolateral nucleus of the amygdala (BLA) and was necessary for increased phosphorylation of the GluA1 (Thr840) subunit of the AMPA receptor following extinction training. Mice exposed to brief uncontrollable stress showed impaired within-session extinction as well as a downregulation of ERK and S6K1 signaling in the amygdala. Finally, using fiber photometry we were able to record calcium signals in vivo, and we found that inhibition of S6K1 reduces extinction-induced changes in neuronal activity of the BLA. These results implicate a novel ERK-S6K1-GluA1 signaling cascade critically involved in extinction.

Regional and local environmental conditions do not shape the response to warming of a marine habitat-forming species.

The differential response of marine populations to climate change remains poorly understood. Here, we combine common garden thermotolerance experiments in aquaria and population genetics to disentangle the factors driving the population response to thermal stress in a temperate habitat-forming species: the octocoral Paramuricea clavata. Using eight populations separated from tens of meters to hundreds of kilometers, which were differentially impacted by recent mortality events, we identify 25 °C as a critical thermal threshold. After one week of exposure at this temperature, seven of the eight populations were affected by tissue necrosis and after 30 days of exposure at this temperature, the mean % of affected colonies increased gradually from 3 to 97%. We then demonstrate the weak relation between the observed differential phenotypic responses and the local temperature regimes experienced by each population. A significant correlation was observed between these responses and the extent of genetic drift impacting each population. Local adaptation may thus be hindered by genetic drift, which seems to be the main driver of the differential response. Accordingly, conservation measures should promote connectivity and control density erosion in order to limit the impact of genetic drift on marine populations facing climate change.

Re-shifting the ecological baseline for the overexploited Mediterranean red coral.

Overexploitation leads to the ecological extinction of many oceanic species. The depletion of historical abundances of large animals, such as whales and sea turtles, is well known. However, the magnitude of the historical overfishing of exploited invertebrates is unclear. The lack of rigorous baseline data limits the implementation of efficient management and conservation plans in the marine realm. The precious Mediterranean red coral Corallium rubrum has been intensively exploited since antiquity for its use in jewellery. It shows dramatic signs of overexploitation, with no untouched populations known in shallow waters. Here, we report the discovery of an exceptional red coral population from a previously unexplored shallow underwater cave in Corsica (France) harbouring the largest biomass (by more than 100-fold) reported to date in the Mediterranean. Our findings challenge current assumptions on the pristine state of this emblematic species. Our results suggest that, before intense exploitation, red coral lived in relatively high-density populations with a large proportion of centuries-old colonies, even at very shallow depths. We call for the re-evaluation of the baseline for red coral and question the sustainability of the exploitation of a species that is still common but ecologically (functionally) extinct and in a trajectory of further decline.

The birth, death and resurrection of avoidance: a reconceptualization of a troubled paradigm.

Research on avoidance conditioning began in the late 1930s as a way to use laboratory experiments to better understand uncontrollable fear and anxiety. Avoidance was initially conceived of as a two-factor learning process in which fear is first acquired through Pavlovian aversive conditioning (so-called fear conditioning), and then behaviors that reduce the fear aroused by the Pavlovian conditioned stimulus are reinforced through instrumental conditioning. Over the years, criticisms of both the avoidance paradigm and the two-factor fear theory arose. By the mid-1980s, avoidance had fallen out of favor as an experimental model relevant to fear and anxiety. However, recent progress in understanding the neural basis of Pavlovian conditioning has stimulated a new wave of research on avoidance. This new work has fostered new insights into contributions of not only Pavlovian and instrumental learning but also habit learning, to avoidance, and has suggested that the reinforcing event underlying the instrumental phase should be conceived in terms of cellular and molecular events in specific circuits rather than in terms of vague notions of fear reduction. In our approach, defensive reactions (freezing), actions (avoidance) and habits (habitual avoidance) are viewed as being controlled by unique circuits that operate nonconsciously in the control of behavior, and that are distinct from the circuits that give rise to conscious feelings of fear and anxiety. These refinements, we suggest, overcome older criticisms, justifying the value of the new wave of research on avoidance, and offering a fresh perspective on the clinical implications of this work.

Imaging of the non-traumatic brachial plexus.

The first line imaging of the non-traumatic brachial plexus is by MRI. Knowledge of the anatomy and commonest variants is essential. Three Tesla imaging offers the possibility of 3D isotropic sequences with excellent spatial and contrast enhancement resolutions, which leads to time saving and quality boosting. The most commonly seen conditions are benign tumor lesions and radiation damage. Gadolinium is required to assess inflammatory or tumour plexopathy. MRI data should be correlated with FDG-PET if tumor recurrence is suspected.

Phylogeography of the red coral (Corallium rubrum): inferences on the evolutionary history of a temperate gorgonian.

The red coral Corallium rubrum (Cnidaria, Octocorallia) is an exploited, long-lived sessile species from the Mediterranean Sea and the adjacent coastline in the Atlantic Ocean. Surveys of genetic variation using microsatellites have shown that populations of C. rubrum are characterized by strong differentiation at the local scale but a study of the phylogeography of this species was still lacking. Here, we used seven polymorphic microsatellite loci, together with sequence data from an intron of the elongation factor 1 (EF1) gene, to investigate the genetic structure of C. rubrum across its geographical range in the western Mediterranean Sea and in the Adriatic Sea. The EF1 sequences were also used to analyse the consequences of demographic fluctuations linked with past environmental change. Clustering analysis with microsatellite loci highlighted three to seven genetic groups with the distinction of North African and Adriatic populations; this distinction appeared significant with AMOVA and differentiation tests. Microsatellite and EF1 data extended the isolation by distance pattern previously observed for this species at the western Mediterranean scale. EF1 sequences confirmed the genetic differentiation observed between most samples with microsatellites. A statistical parsimony network of EF1 haplotypes provided no evidence of high sequence divergence among regions, suggesting no long-term isolation. Selective neutrality tests on microsatellites and EF1 were not significant but should be interpreted with caution in the case of EF1 because of the low sample sizes for this locus. Our results suggest that recent Quaternary environmental fluctuations had a limited impact on the genetic structure of C. rubrum.

From global to local genetic structuring in the red gorgonian Paramuricea clavata: the interplay between oceanographic conditions and limited larval dispersal.

Defining the scale of connectivity among marine populations and identifying the barriers to gene flow are tasks of fundamental importance for understanding the genetic structure of populations and for the design of marine reserves. Here, we investigated the population genetic structure at three spatial scales of the red gorgonian Paramuricea clavata (Cnidaria, Octocorallia), a key species dwelling in the coralligenous assemblages of the Mediterranean Sea. Colonies of P. clavata were collected from 39 locations across the Mediterranean Sea from Morocco to Turkey and analysed using microsatellite loci. Within three regions (Medes, Marseille and North Corsica), sampling was obtained from multiple locations and at different depths. Three different approaches (measures of genetic differentiation, Bayesian clustering and spatially explicit maximum-difference algorithm) were used to determine the pattern of genetic structure. We identified genetic breaks in the spatial distribution of genetic diversity, which were concordant with oceanographic conditions in the Mediterranean Sea. We revealed a high level of genetic differentiation among populations and a pattern of isolation by distance across the studied area and within the three regions, underlining short effective larval dispersal in this species. We observed genetic differentiation among populations in the same locality dwelling at different depths, which may be explained by local oceanographic conditions and which may allow a process of local adaptation of the populations to their environment. We discuss the implications of our results for the conservation of the species, which is exposed to various threats.

Ultrastructural characterization of noradrenergic axons and Beta-adrenergic receptors in the lateral nucleus of the amygdala.

Norepinephrine (NE) is thought to play a key role in fear and anxiety, but its role in amygdala-dependent Pavlovian fear conditioning, a major model for understanding the neural basis of fear, is poorly understood. The lateral nucleus of the amygdala (LA) is a critical brain region for fear learning and regulating the effects of stress on memory. To understand better the cellular mechanisms of NE and its adrenergic receptors in the LA, we used antibodies directed against dopamine beta-hydroxylase (DßH), the synthetic enzyme for NE, or against two different isoforms of the beta-adrenergic receptors (ßARs), one that predominately recognizes neurons (ßAR 248) and the other astrocytes (ßAR 404), to characterize the microenvironments of DßH and ßAR. By electron microscopy, most DßH terminals did not make synapses, but when they did, they formed both asymmetric and symmetric synapses. By light microscopy, ßARs were present in both neurons and astrocytes. Confocal microscopy revealed that both excitatory and inhibitory neurons express ßAR248. By electron microscopy, ßAR 248 was present in neuronal cell bodies, dendritic shafts and spines, and some axon terminals and astrocytes. When in dendrites and spines, ßAR 248 was frequently concentrated along plasma membranes and at post-synaptic densities of asymmetric (excitatory) synapses. ßAR 404 was expressed predominately in astrocytic cell bodies and processes. These astrocytic processes were frequently interposed between unlabeled terminals or ensheathed asymmetric synapses. Our findings provide a morphological basis for understanding ways in which NE may modulate transmission by acting via synaptic or non-synaptic mechanisms in the LA.

Fine-scale genetic structure and inferences on population biology in the threatened Mediterranean red coral, Corallium rubrum.

Identifying microevolutionary processes acting in populations of marine species with larval dispersal is a challenging but crucial task because of its conservation implications. In this context, recent improvements in the study of spatial genetic structure (SGS) are particularly promising because they allow accurate insights into the demographic and evolutionary processes at stake. Using an exhaustive sampling and a combination of image processing and population genetics, we highlighted significant SGS between colonies of Corallium rubrum over an area of half a square metre, which sheds light on a number of aspects of its population biology. Based on this SGS, we found the mean dispersal range within sites to be between 22.6 and 32.1 cm, suggesting that the surveyed area approximately corresponded to a breeding unit. We then conducted a kinship analysis, which revealed a complex half-sib family structure and allowed us to quantify the level of self-recruitment and to characterize aspects of the mating system of this species. Furthermore, significant temporal variations in allele frequencies were observed, suggesting low genetic drift. These results have important conservation implications for the red coral and further our understanding of the microevolutionary processes acting within populations of sessile marine species with a larval phase.

Genetic survey of shallow populations of the Mediterranean red coral [Corallium rubrum (Linnaeus, 1758)]: new insights into evolutionary processes shaping nuclear diversity and implications for conservation.

Combined action from over-harvesting and recent mass mortality events potentially linked to ongoing climate changes has led to new concerns for the conservation of shallow populations (5-60 m) of Corallium rubrum, an octocorallian that is mainly found in the Mediterranean Sea. The present study was designed to analyse population structure and relationships at different spatial scales (from 10s of meters to 100s of kilometres) with a focus on dispersal pattern. We also performed the first analysis of the distribution of genetic diversity using a comparative approach between regional-clusters and samples. Forty populations dwelling in four distinct regions between 14 and 60 m in depth were genotyped using 10 microsatellites. Our main results indicate (i) a generalized pair-sample differentiation combined with a weak structure between regional-clusters; (ii) the occurrence of isolation by distance at the global scale, but also within two of the three analysed regional-clusters; (iii) a high level of genetic diversity over the surveyed area with a heterogeneous distribution from regional-cluster to sample levels. The evolutionary consequences of these results are discussed and their management implications are provided.

Fear conditioning induces distinct patterns of gene expression in lateral amygdala.

The lateral nucleus of the amygdala (LA) has been implicated in the formation of long-term associative memory (LTM) of stimuli associated with danger through fear conditioning. The current study aims to detect genes that are expressed in LA following associative fear conditioning. Using oligonucleotide microarrays, we monitored gene expression in rats subjected to paired training where a tone co-terminates with a footshock, or unpaired training where the tone and footshock are presented in a non-overlapping manner. The paired protocol consistently leads to auditory fear conditioning memory formation, whereas the unpaired protocol does not. When the paired group was compared with the unpaired group 5 h after training, the expression of genes coding for the limbic system-associated membrane protein (Lsamp), kinesin heavy chain member 2 (Kif2), N-ethylmaleimide-sensitive fusion protein (NSF) and Hippocalcin-like 4 protein (Hpcal4) was higher in the paired group. These genes encode proteins that regulate neuronal axonal morphology (Lsamp, Kif2), presynaptic vesicle cycling and release (Hpcal4 and NSF), and AMPA receptor maintenance in synapses (NSF). Quantitative real-time PCR (qPCR) showed that Kif2 and Lsamp are expressed hours following fear conditioning but minutes after unpaired training. Hpcal4 is induced by paired stimulation only 5 h after the training. These results show that fear conditioning induces a unique temporal activation of molecular pathways involved in regulating synaptic transmission and axonal morphology in LA, which is different from non-associative stimulation.

Observation of eta' decays to pi+pi-pi0 and pi+pi-e+e-.

Using psi(2S)-->pi;{+}pi;{-}J/psi, J/psi-->gammaeta;{'} events acquired with the CLEO-c detector at the CESR e;{+}e;{-} collider, we make the first observations of the decays eta;{'}-->pi;{+}pi;{-}pi;{0} and eta;{'}-->pi;{+}pi;{-}e;{+}e;{-}, measuring absolute branching fractions (37_{-9};{+11}+/-4)x10;{-4} and (25_{-9};{+12}+/-5)x10;{-4}, respectively. For eta;{'}-->pi;{+}pi;{-}pi;{0}, this result probes the mechanism of isospin violation and the roles of pi;{0}/eta/eta;{'}-mixing and final state rescattering in strong decays. We also set upper limits on branching fractions for eta;{'} decays to pi;{+}pi;{-}micro;{+}micro;{-}, 2(pi;{+}pi;{-}), pi;{+}pi;{-}2pi;{0}, 2(pi;{+}pi;{-})pi;{0}, 3(pi;{+}pi;{-}), and invisible final states.

Observation of Upsilon(2S)-->etaUpsilon(1S) and search for related transitions.

We report the first observation of Upsilon(2S)-->etaUpsilon(1S), with a branching fraction B=(2.1(-0.6)+0.7(stat)+/-0.3(syst)) x 10(-4) and a statistical significance 5.3sigma. Data were acquired with the CLEO III detector at the CESR e+e(-) symmetric collider. This is the first process observed involving a b-quark spin flip. For related transitions, 90% confidence limits in units of 10(-4) are B(Upsilon(2S)-->pi0Upsilon(1S)) < 1.8, B(Upsilon(3S)-->etaUpsilon(1S)) < 1.8, B(Upsilon(3S)-->pi0Upsilon(1S)) < 0.7, and B(Upsilon(3S)-->pi0Upsilon(2S)) < 5.1.

Unconditioned stimulus pathways to the amygdala: effects of lesions of the posterior intralaminar thalamus on foot-shock-induced c-Fos expression in the subdivisions of the lateral amygdala.

The lateral nucleus of the amygdala (LA) is a site of convergence for auditory (conditioned stimulus) and foot-shock (unconditioned stimulus) inputs during fear conditioning. The auditory pathways to LA are well characterized, but less is known about the pathways through which foot shock is transmitted. Anatomical tracing and physiological recording studies suggest that the posterior intralaminar thalamic nucleus, which projects to LA, receives both auditory and somatosensory inputs. In the present study we examined the expression of the immediate-early gene c-fos in the LA in rats in response to foot-shock stimulation. We then determined the effects of posterior intralaminar thalamic lesions on foot-shock-induced c-Fos expression in the LA. Foot-shock stimulation led to an increase in the density of c-Fos-positive cells in all LA subnuclei in comparison to controls exposed to the conditioning box but not shocked. However, some differences among the dorsolateral, ventrolateral and ventromedial subnuclei were observed. The ventrolateral subnucleus showed a homogeneous activation throughout its antero-posterior extension. In contrast, only the rostral aspect of the ventromedial subnucleus and the central aspect of the dorsolateral subnucleus showed a significant increment in c-Fos expression. The density of c-Fos-labeled cells in all LA subnuclei was also increased in animals placed in the box in comparison to untreated animals. Unilateral electrolytic lesions of the posterior intralaminar thalamic nucleus and the medial division of the medial geniculate body reduced foot-shock-induced c-Fos activation in the LA ipsilateral to the lesion. The number of c-Fos labeled cells on the lesioned side was reduced to the levels observed in the animals exposed only to the box. These results indicate that the LA is involved in processing information about the foot-shock unconditioned stimulus and receives this kind of somatosensory information from the posterior intralaminar thalamic nucleus and the medial division of the medial geniculate body.

Human fear-related motor neurocircuitry.

Using functional magnetic resonance imaging and an experimental paradigm of instructed fear, we observed a striking pattern of decreased activity in primary motor cortex with increased activity in dorsal basal ganglia during anticipation of aversive electrodermal stimulation in 42 healthy participants. We interpret this pattern of activity in motor neurocircuitry in response to cognitively-induced fear in relation to evolutionarily-conserved responses to threat that may be relevant to understanding normal and pathological fear in humans.

Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala.

Learning and memory depend on signaling molecules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the training stimuli were presented in a non-associative manner. Anatomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically implicated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nucleus of the amygdala. When ML-7 was applied without associative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the circuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala.

Glucocorticoids, released in high concentrations from the adrenal cortex during stressful experiences, bind to glucocorticoid receptors in nuclear and peri-nuclear sites in neuronal somata. Their classically known mode of action is to induce gene promoter receptors to alter gene transcription. Nuclear glucocorticoid receptors are particularly dense in brain regions crucial for memory, including memory of stressful experiences, such as the hippocampus and amygdala. While it has been proposed that glucocorticoids may also act via membrane bound receptors, the existence of the latter remains controversial. Using electron microscopy, we found glucocorticoid receptors localized to non-genomic sites in rat lateral amygdala, glia processes, presynaptic terminals, neuronal dendrites, and dendritic spines including spine organelles and postsynaptic membrane densities. The lateral nucleus of the amygdala is a region specifically implicated in the formation of memories for stressful experiences. These newly observed glucocorticoid receptor immunoreactive sites were in addition to glucocorticoid receptor immunoreactive signals observed using electron and confocal microscopy in lateral amygdala principal neuron and GABA neuron soma and nuclei, cellular domains traditionally associated with glucocorticoid immunoreactivity. In lateral amygdala, glucocorticoid receptors are thus also localized to non-nuclear-membrane translocation sites, particularly dendritic spines, where they show an affinity for postsynaptic membrane densities, and may have a specialized role in modulating synaptic transmission plasticity related to fear and emotional memory.