Hadley Wilson Horch

Destabilization of Cortical Dendrites and Spines by BDNF

Particle-mediated gene transfer and two-photon microscopy were used to monitor the behavior of dendrites of individual cortical pyramidal neurons coexpressing green fluorescent protein (GFP) and brain-derived neurotrophic factor (BDNF). While the dendrites and spines of neurons expressing GFP alone grew modestly over 24-48 hr, coexpressing BDNF elicited dramatic sprouting of basal dendrites, accompanied by a regression of dendritic spines. Compared to GFP-transfected controls, the newly formed dendrites and spines were highly unstable. Experiments utilizing Trk receptor bodies, K252a, and overexpression of nerve growth factor (NGF) demonstrated that these effects were mediated by secreted BDNF interacting with extracellular TrkB receptors. Thus, BDNF induces structural instability in dendrites and spines, which, when restricted to particular portions of a dendritic arbor, may help translate activity patterns into specific morphological changes.

Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

Local Effects of BDNF on Dendritic Growth

The development of dendrites is a crucial step in the formation of cortical circuitry. The morphogen brain-derived neurotrophic factor (BDNF) may mediate the effects of activity on dendritic morphology since its expression and release are thought to be activity-dependent. Using two-photon microscopy, the autocrine and paracrine effects of BDNF on dendritic morphology were assessed. Overexpression of BDNF profoundly altered the form and stability of basal dendritic arbors via an autocrine mechanism. Paracrine BDNF also altered dendritic branching, though in a highly local fashion. BDNF is capable of acting as an intercellular morphogen, and could hypothetically shape dendritic arbors to best fit the developing structure and function of the pre-synaptic circuit.

Developmental Gene Discovery in a Hemimetabolous Insect: De Novo Assembly and Annotation of a Transcriptome for the Cricket Gryllus bimaculatus

Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.

Synaptic and extrasynaptic distribution of two distinct populations of nicotinic acetylcholine receptor clusters in the frog cardiac ganglion

SummaryWe examined the distribution of neuronal nicotinic acetylcholine receptor clusters in relation to synaptic sites on autonomic neurons in the frog heart using immunofluorescence techniques and laser scanning confocal microscopy. Acetylcholine receptor clusters were visualized using the rat anti-Electrophorus acetylcholine receptor monoclonal antibody no. 22 and cyanine 3.18-labelled goat anti-rat secondary antibody. Synaptic boutons were labelled with the mouse anti-synaptic vesicle protein SV2, monoclonal antibody no. 10h and cyanine 5.18-labelled goat anti-mouse secondary antibody. Acetylcholine receptor clusters on the neuronal surface exist in two populations that vary in size, staining intensity, and surface distribution. The more prominent population consists of large, brightly stained clusters numbering 30±15 per cell, while the second class is smaller and less brightly stained and numbers over 100 per cell. The large clusters tend to be organized into groups of 2–6 members. This arrangement results from the fact that 80% of the large clusters colocalize at synaptic boutons and that single boutons can have several associated clusters. The remaining 20% of large/bright acetylcholine receptor clusters are extrasynaptic, but they, too, are clustered and are found in close proximity to synaptic boutons. The small/dim acetylcholine receptor clusters are randomly distributed over the cell surface. The large/bright synaptic acetylcholine receptor clusters presumably underlie fast excitatory synaptic transmission. The small/dim clusters and the large/bright extrasynaptic clusters may represent intermediates in the metabolism of large/bright synaptic clusters.

Bilateral consequences of chronic unilateral deafferentation in the auditory system of the cricket Gryllus bimaculatus.

The auditory system of the cricket has the unusual ability to respond to deafferentation by compensatory growth and synapse formation. Auditory interneurons such as ascending neuron 2 (AN-2) in the cricket Gryllus bimaculatus possess a dendritic arbor that normally grows up to, but not over, the midline of the prothoracic ganglion. After chronic deafferentation throughout larval development, however, the AN-2 dendritic arbor changes dramatically, and medial dendrites sprout across the midline where they form compensatory synapses with the auditory afferents from the contralateral ear. We quantified the extent of the effects of chronic, unilateral deafferentation by measuring several cellular parameters of 3 different neuronal components of the auditory system: the deafferented AN-2, the contralateral (or nondeafferented) AN-2 and the contralateral auditory afferents. Neuronal tracers and confocal microscopy were used to visualize neurons, and double-label experiments were performed to examine the cellular relationship between pairs of cells. Dendritic complexity was quantified using a modified Sholl analysis, and the length and volume of processes and presynaptic varicosities were assessed under control and deafferented conditions. Chronic deafferentation significantly influenced the morphology of all 3 neuronal components examined. The overall dendritic complexity of the deafferented AN-2 dendritic arbor was reduced, while both the contralateral AN-2 dendritic arbor and the remaining, intact, auditory afferents grew longer. We found no significant changes in the volume or density of varicosities after deafferentation. These complex cellular changes after deafferentation are interpreted in the light of the reported differential regulation of vesicle-associated membrane protein and semaphorin 2a.

Differential gene expression during compensatory sprouting of dendrites in the auditory system of the cricket Gryllus bimaculatus.

Neurones that lose their presynaptic partners because of injury usually retract or die. However, when the auditory interneurones of the cricket Gryllus bimaculatus are denervated, dendrites respond by growing across the midline and forming novel synapses with the opposite auditory afferents. Suppression subtractive hybridization was used to detect transcriptional changes 3 days after denervation. This is a stage at which we demonstrate robust compensatory dendritic sprouting. Whereas 49 unique candidates were down‐regulated, no sufficiently up‐regulated candidates were identified at this time point. Several candidates identified in this study are known to influence the translation and degradation of proteins in other systems. The potential role of these factors in the compensatory sprouting of cricket auditory interneurones in response to denervation is discussed.

Developmental and adult expression of semaphorin 2a in the cricket Gryllus bimaculatus.

Developmental guidance cues act to direct growth cones to their correct targets in the nervous system. Recent experiments also demonstrate that developmental cues are expressed in the adult mammalian nervous system, although their function in the brain is not yet clear. The semaphorin gene family has been implicated in the growth of dendrites and axons in a number of different species. While the expression of semaphorin and its influence on tibial pioneer neurons in the developing limb bud have been well characterized in the grasshopper, the expression of semaphorin 2a (sema2a) has not been explored in the adult insect. In this study we used polymerase chain reaction (PCR) with degenerate and gene‐specific primers to clone part of the secreted form of sema2a from Gryllus bimaculatus. Using in situ hybridization and immunohistochemistry, we confirmed that sema2a mRNA and protein expression patterns in the embryonic cricket were similar to that seen in the grasshopper. We also showed that tibial neuron development in crickets was comparable to that described in grasshopper. An examination of both developing and adult cricket brains showed that sema2a mRNA and protein were expressed in the Kenyon cells in mushroom bodies, an area involved in learning and memory. Sema2a expression was most obvious near the apex of the mushroom body in a region surrounding the neurogenic tip, which produces neurons throughout the life of the cricket. We discuss the role of neurogenesis in learning and memory and the potential involvement of semaphorin in this process. J. Comp. Neurol. 503:169–181, 2007.

Quantification of dendritic and axonal growth after injury to the auditory system of the adult cricket Gryllus bimaculatus

Dendrite and axon growth and branching during development are regulated by a complex set of intracellular and external signals. However, the cues that maintain or influence adult neuronal morphology are less well understood. Injury and deafferentation tend to have negative effects on adult nervous systems. An interesting example of injury-induced compensatory growth is seen in the cricket, Gryllus bimaculatus. After unilateral loss of an ear in the adult cricket, auditory neurons within the central nervous system (CNS) sprout to compensate for the injury. Specifically, after being deafferented, ascending neurons (AN-1 and AN-2) send dendrites across the midline of the prothoracic ganglion where they receive input from auditory afferents that project through the contralateral auditory nerve (N5). Deafferentation also triggers contralateral N5 axonal growth. In this study, we quantified AN dendritic and N5 axonal growth at 30 h, as well as at 3, 5, 7, 14, and 20 days after deafferentation in adult crickets. Significant differences in the rates of dendritic growth between males and females were noted. In females, dendritic growth rates were non-linear; a rapid burst of dendritic extension in the first few days was followed by a plateau reached at 3 days after deafferentation. In males, however, dendritic growth rates were linear, with dendrites growing steadily over time and reaching lengths, on average, twice as long as in females. On the other hand, rates of N5 axonal growth showed no significant sexual dimorphism and were linear. Within each animal, the growth rates of dendrites and axons were not correlated, indicating that independent factors likely influence dendritic and axonal growth in response to injury in this system. Our findings provide a basis for future study of the cellular features that allow differing dendrite and axon growth patterns as well as sexually dimorphic dendritic growth in response to deafferentation.

De novo assembly of a transcriptome for the cricket Gryllus bimaculatus prothoracic ganglion: An invertebrate model for investigating adult central nervous system compensatory plasticity

The auditory system of the cricket, Gryllus bimaculatus, demonstrates an unusual amount of anatomical plasticity in response to injury, even in adults. Unilateral removal of the ear causes deafferented auditory neurons in the prothoracic ganglion to sprout dendrites across the midline, a boundary they typically respect, and become synaptically connected to the auditory afferents of the contralateral ear. The molecular basis of this sprouting and novel synaptogenesis in the adult is not understood. We hypothesize that well-conserved developmental guidance cues may recapitulate their guidance functions in the adult in order to facilitate this compensatory growth. As a first step in testing this hypothesis, we have generated a de novo assembly of a prothoracic ganglion transcriptome derived from control and deafferented adult individuals. We have mined this transcriptome for orthologues of guidance molecules from four well-conserved signaling families: Slit, Netrin, Ephrin, and Semaphorin. Here we report that transcripts encoding putative orthologues of most of the candidate developmental ligands and receptors from these signaling families were present in the assembly, indicating expression in the adult G. bimaculatus prothoracic ganglion.