Activity Regulates Neuronal Connectivity and Function in the C. elegans Motor Circuit: A Dissertation

Activity plays diverse roles in shaping neuronal development and function. These roles range from aiding in synaptic refinement to triggering cell death during traumatic brain injury. Though the importance of activity-dependent mechanisms is widely recognized, the genetic underpinnings of these processes have not been fully described. In this thesis, I use the motor circuit of Caenorhabditis elegans as a model system to explore the functional and morphological consequences of modulating neuronal activity. First, I used a gain-of-function ionotropic receptor to hyperactivate motor neurons and asked how increased excitation affects neuronal function. Through this work, I identified a cell death pathway triggered by excess activation of motor neurons. I also showed that suppression of cell body death failed to block motor axon destabilization, providing evidence that death of the cell body and of motor axons can be genetically separated. Secondly, I removed excitatory drive from a simple neural circuit and asked how loss of excitatory activity alters circuit development and function. I identified excitatory motor neurons as master regulators of inhibitory synaptic connectivity. Additionally, I was able to identify previously undescribed activity-dependent mechanisms for regulating inhibitory synapses in both developing and mature neural circuits. Finally, I show data to implicate the highly conserved genes neurexin and neuroligin in determining inhibitory synapse connectivity. Collectively this work has lent insight into activity-dependent mechanisms in place to regulate neuronal development and function, a core function of neurobiology that is relevant to the study of a wide range of neurological disorders

A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition

Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation

ACR-12 ionotropic acetylcholine receptor complexes regulate inhibitory motor neuron activity in Caenorhabditis elegans

Heterogeneity in the composition of neurotransmitter receptors is thought to provide functional diversity that may be important in patterning neural activity and shaping behavior (Dani and Bertrand, 2007; Sassoe-Pognetto, 2011). However, this idea has remained difficult to evaluate directly because of the complexity of neuronal connectivity patterns and uncertainty about the molecular composition of specific receptor types in vivo. Here we dissect how molecular diversity across receptor types contributes to the coordinated activity of excitatory and inhibitory motor neurons in the nematode Caenorhabditis elegans. We show that excitatory and inhibitory motor neurons express distinct populations of ionotropic acetylcholine receptors (iAChRs) requiring the ACR-12 subunit. The activity level of excitatory motor neurons is influenced through activation of nonsynaptic iAChRs (Jospin et al., 2009; Barbagallo et al., 2010). In contrast, synaptic coupling of excitatory and inhibitory motor neurons is achieved through a second population of iAChRs specifically localized at postsynaptic sites on inhibitory motor neurons. Loss of ACR-12 iAChRs from inhibitory motor neurons leads to reduced synaptic drive, decreased inhibitory neuromuscular signaling, and variability in the sinusoidal motor pattern. Our results provide new insights into mechanisms that establish appropriately balanced excitation and inhibition in the generation of a rhythmic motor behavior and reveal functionally diverse roles for iAChR-mediated signaling in this process

A dominant mutation in a neuronal acetylcholine receptor subunit leads to motor neuron degeneration in Caenorhabditis elegans

Inappropriate or excessive activation of ionotropic receptors can have dramatic consequences for neuronal function and, in many instances, leads to cell death. In Caenorhabditis elegans, nicotinic acetylcholine receptor (nAChR) subunits are highly expressed in a neural circuit that controls movement. Here, we show that heteromeric nAChRs containing the acr-2 subunit are diffusely localized in the processes of excitatory motor neurons and act to modulate motor neuron activity. Excessive signaling through these receptors leads to cell-autonomous degeneration of cholinergic motor neurons and paralysis. C. elegans double mutants lacking calreticulin and calnexin-two genes previously implicated in the cellular events leading to necrotic-like cell death (Xu et al. 2001)-are resistant to nAChR-mediated toxicity and possess normal numbers of motor neuron cell bodies. Nonetheless, excess nAChR activation leads to progressive destabilization of the motor neuron processes and, ultimately, paralysis in these animals. Our results provide new evidence that chronic activation of ionotropic receptors can have devastating degenerative effects in neurons and reveal that ion channel-mediated toxicity may have distinct consequences in neuronal cell bodies and processes

A conserved dopamine-cholecystokinin signaling pathway shapes context-dependent Caenorhabditis elegans behavior

An organism\u27s ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output

Stem Cell Survey of Human Sebaceous Tumors

Mutations in stem cell signaling caused by alterations in the Lef1 beta-catenin pathway can lead to the development of sebaceous tumors in humans. A survey of known stem cell markers was conducted on a human sebaceous tumor line and on normal skin using immunoflourescence, PCR and Western Blots to verify the existence of stem cells in sebaceous tumors. The resulting data supports the existence of cancer stem cells in sebaceous tumors

The hair follicle barrier to involvement by malignant melanoma

BACKGROUND: Melanoma characteristically grows within the epidermis along the dermal-epidermal junction, sometimes extending outward up to several centimeters beyond the foci of invasive tumors. Although follicular involvement by malignant melanoma is widely recognized, to the authors\u27 knowledge no previously published data address this phenomenon. METHODS: To examine the growth characteristics of in situ melanomas in relation to the hair follicle microanatomy, the authors analyzed 100 cases of primary cutaneous melanomas (61 in situ and 39 invasive melanomas with significant in situ components) obtained from pathology clinical archives. RESULTS: Eighty-two (82%) cases of melanoma in situ demonstrated tumor cells within \u3eor=1 hair follicles. Of those, 57 (69.5%) cases demonstrated the tumor cells only within the infundibulum. Extension of the tumor cells down to the isthmus was observed in 24 cases (29.3%). In only 1 exceptional case (1%) were tumor cells detected beneath the level of the hair follicle bulge. CONCLUSIONS: The authors postulate that a physiologic barrier restricts the intraepithelial spread of melanoma tumor cells at or beyond the level of the stem cell niche in the hair follicle bulge. Although the nature of this barrier remains to be elucidated, the distinct biologic characteristics of the hair follicle bulge may provide clues to understanding this phenomenon

Excitatory neurons sculpt GABAergic neuronal connectivity in the C. elegans motor circuit

Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by a reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development-during the integration of post-embryonic born GABAergic neurons into the circuit-produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, likely through distinct mechanisms in the developing and mature nervous system

Linking businesses, employment services and neighbourhood learning centres (NLCs) in the Thames Valley.

This project, prepared for Groundwork Thames Valley, makes recommendations to help overcome the obstacles that prevent the citizens of the Slough, Reading, and Wycombe local authorities from receiving the necessary training and services to obtain local employment. The successful surveying of businesses, Neighbourhood Learning Centres (NLCs), and local employment services helped gain an understanding of the current interactions between said entities. Gathered data has allowed us to make recommendations to improve the links between all parties involved to better service jobseekers

Locomotory phenotypes associated with L-AChR<i>(gf)</i> expression require neuropeptide signaling.

<p>(<b>A</b>) Average body bends/min measured in liquid for the genotypes indicated. The strong locomotory defects of <i>egl-3</i> and <i>egl-21</i> mutants prevented analysis of L-AChR(<i>gf</i>) effects on agar (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584.s003" target="_blank">Fig. S3A, B</a>). Mutation of <i>pkc-1</i> normalized the locomotor effects of L-AChR(<i>gf</i>) in both liquid and on agar (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584.s003" target="_blank">Fig. S3C–E</a>). (<b>B</b>) Movement trajectories of wild type, L-AChR<i>(gf), nlp-12</i>;L-AChR<i>(gf)</i> animals as indicated. Each black line shows the trajectory of one animal monitored for 45 s on food. (<b>C</b>) Average body bend amplitude for the genotypes indicated. Each bar represents the mean (±SEM) of values calculated from recordings of at least 15 animals. For (A) and (C) ***, p<0.0001 by ANOVA with Sidak's post-hoc test. (<b>D</b>) Wide-field epifluorescent image of an adult animal expressing <i>nlp-12::SL2::mCherry</i>. The image is oriented with the head to the left. White rectangle: nerve ring. Arrow: DVA cell body. (<b>E</b>) Average body bend amplitude for the indicated genotypes and effects of DVA ablation (–DVA). Ablations were performed on L2 stage animals. Body bend amplitude was measured from recordings of young adult animals 2 days following laser ablation. Error bars indicate mean (±SEM) of at least 8 animals. ***, p<0.0001 student's t-test.</p