Recent parallel speciation in Antirrhinum involved complex haplotypes and multiple adaptive characters.

A role of ecological adaptation in speciation can be obscured by stochastic processes and differences that species accumulate after genetic isolation. One way to identify adaptive characters and their underlying genes is to study cases of speciation involving parallel adaptations. Recently resolved phylogenies reveal that alpine morphology has evolved in parallel in the genus Antirrhinum (snapdragons): first in an early split of an alpine from a lowland lineage and, more recently, from within the lowland lineage to produce closely related sympatric species with contrasting alpine and lowland forms. Here, we find that two of these later diverged sympatric species are differentiated by only around 2% of nuclear loci. Though showing evidence of recent gene flow, the species remain distinct for a suite of morphological characters typical of earlier-diverged alpine or lowland lineages and their morphologies correlate with features of the local landscape, as expected of ecological adaptations. Morphological differences between the two species involve multiple, unlinked genes so that parental character combinations are readily broken up by recombination in hybrids. We detect little evidence for post-pollination barriers to gene flow or recombination, suggesting that genetic isolation related to ecological adaptation is important in maintaining character combinations and might have contributed to parallel speciation. We also find evidence that genes involved in the earlier alpine-lowland split were reused in parallel evolution of alpine species, consistent with introgressive hybridisation, and speculate that many non-ecological barriers to gene flow might have been purged during the process.

A phylogeny of Antirrhinum reveals parallel evolution of alpine morphology.

Parallel evolution of similar morphologies in closely related lineages provides insight into the repeatability and predictability of evolution. In the genus Antirrhinum (snapdragons), as in other plants, a suite of morphological characters are associated with adaptation to alpine environments. We tested for parallel trait evolution in Antirrhinum by investigating phylogenetic relationships using restriction-site associated DNA (RAD) sequencing. We then associated phenotypic information to our phylogeny to reconstruct the patterns of morphological evolution and related this to evidence for hybridisation between emergent lineages. Phylogenetic analyses showed that the alpine character syndrome is present in multiple groups, suggesting that Antirrhinum has repeatedly colonised alpine habitats. Dispersal to novel environments happened in the presence of intraspecific and interspecific gene flow. We found support for a model of parallel evolution in Antirrhinum. Hybridisation in natural populations, and a complex genetic architecture underlying the alpine morphology syndrome, support an important role of natural selection in maintaining species divergence in the face of gene flow.

The ß-Secretase Substrate Seizure 6-Like Protein (SEZ6L) Controls Motor Functions in Mice.

The membrane protein seizure 6-like (SEZ6L) is a neuronal substrate of the Alzheimer's disease protease BACE1, and little is known about its physiological function in the nervous system. Here, we show that SEZ6L constitutive knockout mice display motor phenotypes in adulthood, including changes in gait and decreased motor coordination. Additionally, SEZ6L knockout mice displayed increased anxiety-like behaviour, although spatial learning and memory in the Morris water maze were normal. Analysis of the gross anatomy and proteome of the adult SEZ6L knockout cerebellum did not reveal any major differences compared to wild type, indicating that lack of SEZ6L in other regions of the nervous system may contribute to the phenotypes observed. In summary, our study establishes physiological functions for SEZ6L in regulating motor coordination and curbing anxiety-related behaviour, indicating that aberrant SEZ6L function in the human nervous system may contribute to movement disorders and neuropsychiatric diseases.

Shared Mutations in a Novel Glutaredoxin Repressor of Multicellular Trichome Fate Underlie Parallel Evolution of Antirrhinum Species.

Most angiosperms produce trichomes-epidermal hairs that have protective or more specialized roles. Trichomes are multicellular in almost all species and, in the majority, secretory. Despite the importance of multicellular trichomes for plant protection and as a source of high-value products, the mechanisms that control their development are only poorly understood. Here, we investigate the control of multicellular trichome patterns using natural variation within the genus Antirrhinum (snapdragons), which has evolved hairy alpine-adapted species or lowland species with a restricted trichome pattern multiple times in parallel. We find that a single gene, Hairy (H), which is needed to repress trichome fate, underlies variation in trichome patterns between all Antirrhinum species except one. We show that H encodes a novel epidermis-specific glutaredoxin and that the pattern of trichome distribution within individuals reflects the location of H expression. Phylogenetic and functional tests suggest that H gained its trichome-repressing role late in the history of eudicots and that the ancestral Antirrhinum had an active H gene and restricted trichome distribution. Loss of H function was involved in an early divergence of alpine and lowland Antirrhinum lineages, and the alleles underlying this split were later reused in parallel evolution of alpines from lowland ancestors, and vice versa. We also find evidence for an evolutionary reversal from a widespread to restricted trichome distribution involving a suppressor mutation and for a pleiotropic effect of H on plant growth that might constrain the evolution of trichome pattern.

Evidence that a defined population of neurons in lateral amygdala is directly involved in auditory fear learning and memory.

Memory is thought to be encoded within networks of neurons within the brain, but the identity of the neurons involved and circuits they form have not been described for any memory. Previously, we used fos-tau-lacZ (FTL) transgenic mice to identify discrete populations of neurons in different regions of the brain which were specifically activated following fear conditioning. This suggested that these populations of neurons form nodes in a network that encodes fear memory. In particular, one population of learning activated neurons was found within a discrete region of the lateral amygdala (LA), a key nucleus required for fear conditioning. In order to provide evidence that this population is directly involved in fear conditioning, we have analysed the expression of a key molecular requirement for fear conditioning in LA, phosphorylated Extracellular Signal Regulated Kinase 1 and 2 (pERK1/2). The only neurons in LA that specifically expressed pERK1/2 following auditory fear conditioning were in the ventrolateral nucleus of the LA (LAvl), in the same discrete region where we found learning specific FTL+ neurons. Double labelling experiments in FTL mice showed that a substantial proportion of the learning activated neurons expressed both pERK1/2 and FTL. These experiments provide clear evidence that the learning specific neurons we identified within LAvl are directly involved in auditory fear conditioning. In addition, learning specific expression of pERK1/2 was found in a dense network of dendrites contained within the border region of the LAvl. This network of dendrites may represent an activated dendritic field involved in fear conditioning in LA.

The malleable brain: plasticity of neural circuits and behavior - a review from students to students.

One of the most intriguing features of the brain is its ability to be malleable, allowing it to adapt continually to changes in the environment. Specific neuronal activity patterns drive long-lasting increases or decreases in the strength of synaptic connections, referred to as long-term potentiation and long-term depression, respectively. Such phenomena have been described in a variety of model organisms, which are used to study molecular, structural, and functional aspects of synaptic plasticity. This review originated from the first International Society for Neurochemistry (ISN) and Journal of Neurochemistry (JNC) Flagship School held in Alpbach, Austria (Sep 2016), and will use its curriculum and discussions as a framework to review some of the current knowledge in the field of synaptic plasticity. First, we describe the role of plasticity during development and the persistent changes of neural circuitry occurring when sensory input is altered during critical developmental stages. We then outline the signaling cascades resulting in the synthesis of new plasticity-related proteins, which ultimately enable sustained changes in synaptic strength. Going beyond the traditional understanding of synaptic plasticity conceptualized by long-term potentiation and long-term depression, we discuss system-wide modifications and recently unveiled homeostatic mechanisms, such as synaptic scaling. Finally, we describe the neural circuits and synaptic plasticity mechanisms driving associative memory and motor learning. Evidence summarized in this review provides a current view of synaptic plasticity in its various forms, offers new insights into the underlying mechanisms and behavioral relevance, and provides directions for future research in the field of synaptic plasticity. Read the Editorial Highlight for this article on page 788. Cover Image for this issue: doi: 10.1111/jnc.13815.

Tracking the fear memory engram: discrete populations of neurons within amygdala, hypothalamus, and lateral septum are specifically activated by auditory fear conditioning.

Memory formation is thought to occur via enhanced synaptic connectivity between populations of neurons in the brain. However, it has been difficult to localize and identify the neurons that are directly involved in the formation of any specific memory. We have previously used fos-tau-lacZ (FTL) transgenic mice to identify discrete populations of neurons in amygdala and hypothalamus, which were specifically activated by fear conditioning to a context. Here we have examined neuronal activation due to fear conditioning to a more specific auditory cue. Discrete populations of learning-specific neurons were identified in only a small number of locations in the brain, including those previously found to be activated in amygdala and hypothalamus by context fear conditioning. These populations, each containing only a relatively small number of neurons, may be directly involved in fear learning and memory.

Reduction of p75 neurotrophin receptor ameliorates the cognitive deficits in a model of Alzheimer's disease.

Alzheimer's disease (AD) is an extremely prevalent cause of dementia. It is characterized by progressive memory loss, confusion, and other behavioral and physiological problems. The amyloid-ß (Aß) protein is thought to be involved in the pathogenesis of AD, and there is evidence that Aß may act through the p75 neurotrophin receptor (p75) to mediate its pathogenic effects. This raises the possibility that reducing levels of p75 could be a treatment for AD by preventing the effects of Aß. In this study, we have crossed the transgenic AD model mice, Tg2576, with p75(-/-) mice to generate Tg2576/p75(+/-) mice with reduced levels of p75. These mice are rescued from the deficits in learning and memory and hippocampal function which were found in the Tg2576 mice. These findings suggest that reduction of p75 can ameliorate some of the primary symptoms of AD.

The bile acid receptor TGR5 activates the TRPA1 channel to induce itch in mice.

BACKGROUND & AIMS: Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G-protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice. METHODS: Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice was analyzed by immunofluorescence, in situ hybridization, and single-cell polymerase chain reaction. TGR5-induced activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary sensory neurons by measuring Ca(2+) signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1(-/-) mice. RESULTS: TGR5 and TRPA1 protein and messenger RNA were expressed by cutaneous afferent neurons. In HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent activation and sensitization of TRPA1 by mechanisms that required Gßγ, protein kinase C, and Ca(2+). Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice. CONCLUSIONS: BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.

The genetic basis for natural variation in heteroblasty in Antirrhinum.

Heteroblasty refers to the changes in leaf shape and size (allometry) along stems. Although evolutionary changes involving heteroblasty might contribute to leaf diversity, little is known of the extent to which heteroblasty differs between species or how it might relate to other aspects of allometry or other developmental transitions. Here, we develop a computational model that can quantify differences in leaf allometry between Antirrhinum (snapdragon) species, including variation in heteroblasty. It allows the underlying genes to be mapped in inter-species hybrids, and their effects to be studied in similar genetic backgrounds. Heteroblasty correlates with overall variation in leaf allometry, so species with smaller, rounder leaves produce their largest leaves earlier in development. This involves genes that affect both characters together and is exaggerated by additional genes with multiplicative effects on leaf size. A further heteroblasty gene also alters leaf spacing, but none affect other developmental transitions, including flowering. We suggest that differences in heteroblasty have co-evolved with overall leaf shape and size in Antirrhinum because these characters are constrained by common underlying genes. By contrast, heteroblasty is not correlated with other developmental transitions, with the exception of internode length, suggesting independent genetic control and evolution.

Identification of neurons specifically activated after recall of context fear conditioning.

The learning of new information and recall of that information presumably involves modification of and access to shared circuitry in the brain. However, learning and recall may involve the activation of distinct parts of that circuitry, according to the quite distinct functional differences between these two processes. Previously we examined neuronal activation following learning of context fear conditioning. Using the Fos-Tau-LacZ (FTL) transgenic mouse to label activated neurons, we identified a number of distinct populations of neurons in amygdala and hypothalamus which showed learning specific activation. These populations of neurons showed much less activation following recall. Here we ask what populations of neurons might be specifically activated following recall. We trained mice in context fear conditioning, and then looked at FTL activation following recall of context fear. We identified a number of populations of neurons which showed recall specific activation in nucleus accumbens shell, the anterio-medial bed nucleus of stria terminalis, the anterior commissural nucleus and the periventricular hypothalamic nucleus. These were all different populations of neurons compared with those activated following context fear learning. These different functional activation patterns occurring between learning and recall may reflect the different brain functions occurring between these two memory related processes.

Context fear learning specifically activates distinct populations of neurons in amygdala and hypothalamus.

The identity and distribution of neurons that are involved in any learning or memory event is not known. In previous studies, we identified a discrete population of neurons in the lateral amygdala that show learning-specific activation of a c-fos-regulated transgene following context fear conditioning. Here, we have extended these studies to look throughout the amygdala for learning-specific activation. We identified two further neuronal populations, in the amygdalo-striatal transition area and medial amygdala, that show learning-specific activation. We also identified a population of hypothalamic neurons that show strong learning-specific activation. In addition, we asked whether these neurons are activated following recall of fear-conditioning memory. None of the populations of neurons we identified showed significant memory-recall-related activation. These findings suggest that a series of discrete populations of neurons are involved in fear learning in amygdala and hypothalamus. The lack of reactivation during memory recall suggests that these neurons either do not undergo substantial change following recall, or that c-fos is not involved in any such activation and change.

Audiogenic seizure proneness requires the contribution of two susceptibility loci in mice.

Juvenile mice of the DBA/2J strain undergo generalised seizures when exposed to a high-intensity auditory stimulus. Genetic analysis identified three different loci underlying this audiogenic seizure proneness (ASP)-Asp1, Asp2 and Asp3 on chromosomes 12, 4 and 7, respectively. Asp1 is thought to have the strongest influence, and mice with only Asp1 derived from the DBA/2J strain are reported to exhibit ASP. The aim of this study was to characterise more accurately the contributions of the Asp1 and Asp3 loci in ASP using congenic strains. Each congenic strain contains a DBA/2J-derived interval encompassing either Asp1 or Asp3 on a C57BL/6J genetic background. A double congenic C57BL/6J strain containing both Asp loci derived from DBA/2J was also generated. Here, we report that DBA/2J alleles at both of these Asp loci are required to confer ASP because congenic C57BL/6 mice harbouring DBA/2J alleles at only Asp1 or Asp3 do not exhibit ASP, whereas DBA/2J alleles at both loci resulted in increased susceptibility for audiogenic seizure in double congenic C57BL/6 mice.

The evolutionary history of Antirrhinum suggests that ancestral phenotype combinations survived repeated hybridizations.

The model species Antirrhinum majus (the garden snapdragon) has over 20 close wild relatives that are morphologically diverse and adapted to different Mediterranean environments. Hybrids between Antirrhinum species have been used successfully to identify genes underlying their phenotypic differences, and to infer how selection acts on them. To better understand the genetic basis for this diversity, we have examined the evolutionary relationships between Antirrhinum species and how these relate to geography and patterns of phenotypic variation in the genus as a whole. Large population samples and both plastid and multilocus nuclear genotypes resolved the relationships between many species and provided some support for the traditional taxonomic division of the genus into morphological subsections. Morphometric analysis of plants grown in controlled conditions supported the phenotypic distinction of the two largest subsections, and the involvement of multiple underlying genes. Incongruence between nuclear and plastid genotypes further suggested that several species have arisen after hybridization between subsections, and that some species continue to hybridize. However, all potential hybrids appear to have retained a phenotype similar to one of their ancestors, suggesting that ancestral combinations of characters are maintained by selection at many different loci.

Congenic mouse strains enable discrimination of genetic determinants contributing to fear and fear memory.

The ability to learn and remember is variable within a population of a given species, including humans. This is due in part to genetic variation between individuals. However, only few genes have been identified that contribute to variation in learning and memory. Two inbred mouse strains, C57Bl/6J (B6) and DBA/2J (D2), show significant variation both in fear conditioning memory as well as primary responsiveness to fear. Several studies have identified quantitative trait loci (QTL) on chromosomes (Chr) 1 and 12 associated with performance in fear conditioning, but it is unclear if these QTL were associated with fear memory or innate fear responsiveness. To determine if these QTL are associated with fear memory or fear responsiveness, we studied congenic mouse strains harbouring D2-derived DNA from Chr1 or Chr12 on a B6 genetic background. Cohorts of D2, B6 and the congenic mice were tested throughout the process of fear conditioning by measuring a series of fear-related parameters. The Chr1 congenic mice showed clear deficits in fear memory compared to B6 mice, which established the presence of a QTL on Chr1 directly influencing fear memory. The Chr12 congenic mice also showed alterations in fear conditioning, but this was more associated with alterations in fear responsiveness. These findings thus provide evidence for the localisation of independent genetic determinants for fear memory and fear responsiveness.

Evolution of allometry in antirrhinum.

Correlated variation in shape and size (allometry) is a major component of natural diversity. We examined the evolutionary and genetic basis for allometry using leaves and flower petals of snapdragon species (Antirrhinum). A computational method was developed to capture shape and size variation in both types of organ within the Antirrhinum species group. The results show that the major component of variation between species involves positively correlated changes in leaf and petal size. The correlation was maintained in an F2 population derived from crossing two species with organs of different sizes, suggesting that developmental constraints were involved. Identification of the underlying genes as quantitative trait loci revealed that the larger species carried alleles that increased organ size at all loci. Although this was initially taken as evidence that directional selection has driven diversity in both leaf and petal size, simulations revealed that evolution without consistent directional selection, an undirected walk, could also account for the parental distribution of organ size alleles.

A discrete population of neurons in the lateral amygdala is specifically activated by contextual fear conditioning.

There is no clear identification of the neurons involved in fear conditioning in the amygdala. To search for these neurons, we have used a genetic approach, the fos-tau-lacZ (FTL) mouse, to map functionally activated expression in neurons following contextual fear conditioning. We have identified a discrete population of neurons in the lateral amygdala that are activated specifically following learning. These neurons have the morphology of principal neurons of the amygdala, and are immunoreactive for glutamate. The highly specific localization of these neurons within the lateral amygdala suggests that these neurons may be a discrete population of neurons involved in fear learning.

A single exposure to an enriched environment stimulates the activation of discrete neuronal populations in the brain of the fos-tau-lacZ mouse.

Storage of experience, including learning and memory, is thought to involve plasticity within pre-existing brain circuits. One model for looking at experience-dependent changes is environmental enrichment (EE), which involves exposing animals to a complex novel environment. Animals exposed to EE have previously been shown to exhibit a variety of behavioural and structural alterations in the brain, including decreased stress, improved learning and memory, altered levels of immediate early genes and synaptic change in the visual cortex. We were interested in understanding what regions of the brain are activated during the initial stages of EE. We used fos-tau-lacZ (FTL) transgenic mice to examine changes in functional activation throughout the brain after a single exposure to EE. We found that there was a significant increase in FTL expression within particular morphologically identified neurons in a series of brain regions in the enriched group compared to control groups, indicating that multiple circuits were activated. These regions include the claustrum, infralimbic cortex, hippocampus, amygdala and the hypothalamus. The data suggest that EE stimulates an initial strong increase in activation of multiple functional circuits. These circuits are presumably involved in the initial response of the animal to the enriched environment.

A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections.

We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of beta-galactosidase (betagal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of betagal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.