Dr Chris Connolly

Introduction Movie

The main focus of this laboratory is the study of the role of ligand-gated ion channel biology to the control of excitation/inhibition in neurons of the brain. Our studies concentrate on two family members of the cys-loop superfamily of ligand-gated ion channels (GABAA and 5-HT3 receptors) as well as the NMDA receptor subtype of glutamate receptors, to obtain an understanding of how such mechanisms contribute to pathological states such as epilepsy and ischaemia.

Cys-loop receptor assembly and trafficking

This research investigates the mechanisms of the Cys-loop (in particular 5-HT3 and GABAA) receptor biogenesis and how transport to the cell surface is controlled. We are using recombinant expression of novel cDNA constructs expressing chimaeric ‘living colour’ fusion proteins. We are using a combination of cell line, primary neuronal culture, organotypic culture and transgenic models to study these events.

Movie1: RIC-3a aggregation

Movie 2: RIC-3a aggregates consume ‘RIC-3 diffuse slicks’

Movie 3: RIC-3d in Golgi

Our interests include the role(s) of chaperone molecules such as RIC-3 on 5-HT3 receptor trafficking and targeting with a particular focus on how these processes are influenced by excessive exposure to serotonin. Such events may be relevant to psychosis, depression and irritable bowel syndrome. Similarly, we are interested in the responses (dynamic trafficking) of GABA(A) and glutamate (NMDA) receptors following excessive exposure to GABA and glutamate. Understanding how such a balance in excitatbility is altered may provide important information regarding alterations in behaviour and future vulnerability to neurotoxic insults.

Spreading neurotoxicity: Spreading poison versus spreading warning

We have shown previously that rapid (<10 min) neuronal morphological changes occur in response to neuronal insults as a result of NMDA receptor activation. It is not clear whether these changes are an attempt at neuronal protection or are early signs of neuronal toxicity. Interestingly, neuronal inactivation is also induced by excessive glutamate receptor activation. We are currently studying the events that follow a localised insult to monitor how these responses spread throughout neuronal networks and what impact this has to neuronal survival. We are investigating how neuronal inactivation is achieved and whether this process may be manipulated to provide better neuroprotection within the surrounding (penumbra) region of a lesion and thus limiting the spread of neurotoxicity.

Movie 4: Neuronal dendritic beading (blue), mitochondrial collapse (red) and mitochondrial depolarisation (green) in response to excess glutamate.

Movie 5: Sequential neuronal beading (blue), mitochondrial collapse (red) and mitochondrial depolarisation (green) in response to oxygen-glucose deprivation.

Dendritic beading of a hippoocampal neuron following an excitotoxic insult

The effects of miticides and pesticides on the nervous system of bees

Professor Neil Millar (UCL), Dr Nigel Raine (Royal Holloway), Dr Geraldine Wright (Newcastle), and Dr Christopher Connolly (Dundee). Photograph taken by Nancy Mendoza (BBSRC) at the Insect Pollinators Initiatve launch in June 2010.

This is a multi-disciplinary research programme (~£2M) studying the effects of miticides and pesticides on the nervous system on bees (honeybees and bumblebees). This involves research at the cellular and pathway level (University of Dundee and UCL), individual bees and whole colonies (Newcastle University and Royal Holloway University of London) and involves field studies (Scottish Beekeeper’s Association). This multi-disciplinary approach will allow us to integrate laboratory-based findings at multiple distinct levels into cohesive conclusions on the effects of these chemicals to bee health and correlate this information to the experiences of beekeepers. This high profile issue (Guardian article) and (AVAAZ.org) ) is highly controversial. To investigate this issue, this programme (2011-2015) has been funded by the Insect Pollinators Initiative as part of the Living with Environmental Change (LWEC) partnership. Further information.

Background

Honeybee decline has been identified as a major world problem and is estimated to contribute in excess of £440M to the UK economy every year. A similar decline in other native pollinators is also a major concern for our biodiversity and food security. The major identified natural threats to UK honeybees are Varroa mites and the viruses they transmit, Foul Brood diseases, Nosema and Small Hive Beetle. The control of Varroa infestation often involves the chronic exposure of a colony to miticides. These miticides target the Varroa nervous system, in particular, cholinergic neurotransmission. The central basis of our hypothesis is that chronic miticide treatment may put honeybees under significant chemical stress to the extent that they become vulnerable to otherwise sub-toxic doses of other pesticides. Honeybee pollination is important for the fertilization of a large amount of our fruit, nuts, vegetables and livestock feed. The other major pollinator of crops and native plants are bumblebees as they fly earlier in the season and are increasingly important for the pollination of UK crops such as potatoes, berries, red clover, alfalfa and greenhouse crops. The loss of bees would also have a major impact on the production of native food for wildlife, with unknown knock-on effects at the ecosystem level. While it is widely acknowledged that honeybee populations are in global decline, how the different factors are interacting to produce this decline is poorly understood. In addition to the natural threats and the chronic use of miticides, bees are also exposed to sub-toxic levels of pesticides (including herbicides and fungicides) that are vital to maintenance of crop quality and yield and so, food security. When bees are exposed to multiple pesticides, these may synergize to cause enhanced toxicity to bee populations. In order to understand the potential for such synergistic threats, we need to consider the major neuronal targets in bees for the agents used (miticides and insecticides). These targets include action potential firing (sodium channels) and the balance between excitatory (cholinergic signalling) and inhibitory (GABA and glutamate-gated chloride channels) neurotransmission (Figure 1). Perturbation of these pathways disrupts brain function in all animals, leading to locomotor, behavioural or social problems. The insect brain structure involved in bee learning (olfactory and visual) is the mushroom body. Cholinergic neurotransmission is the major excitatory pathway in this brain structure. The responses are terminated by the rapid inactivation of the released acetylcholine (ACh), by acetylcholinesterase (AChE). In insects, this excitatory activity is balanced by the inhibitory actions of chloride-gated GABA or glutamate receptors. The pharmacological manipulation of any these individual steps may have a profound effect on honeybee learning.

Given the central importance of ACh signalling in the insect brain, it has become a major target for the development of insecticides. The AChE inhibitors (eg. coumaphos [Checkmite]), have been shown to alter neuronal morphology and bee foraging behaviour. Interestingly, AChE activity decreases naturally in adult foragers and this decrease correlates with enhanced olfactory learning. Pyrethroids, (eg. fluvinate in Apistan) target neurotransmission by potentiating the voltage-gated sodium channels (Nav’s) and so prolongs action potential firing. Neonicotinoid pesticides act as partial agonists at honeybee nAChRs, but are not inactivated by AChE and may lead to prolonged receptor activation. Some neonicotinoids are more resistant to cytochrome P450 detoxicification, or generate toxic metabolites. That neonicotinoids decrease bee foraging behaviour [9] is counter-intuitive, suggesting that the role of these pesticides is inconsistent. However, the long-term consequences of the chronic exposure to neonicotinoids, such as receptor desensitization and agonist-induced receptor internalization, are unknown. To add to the confusion, imidacloprid (a neonicotinoid) exhibits “off target” activity by operating as a GABA receptor antagonist. Other pesticides, including cyclodienes and phenylpyrazoles, also inhibit GABA receptors, whereas, the ‘natural’ pesticide, thymol (the active ingredient of the miticide, Apiguard) is a GABA receptor agonist. Avermectins target the other major inhibitory receptor, the glutamate-gated chloride channel. Domestic insecticides also contain ‘synergist’ agents, e.g. piperonyl butoxide, to reduce the pyrethroid detoxification by cyctochrome P450. Despite this potential for pesticide toxicity to bees, analyses of actual doses found in pollen, bees or their honey are below toxic levels. We will investigate whether pesticides, at levels that are sub-toxic by themselves, may synergise with in-hive miticides to alter the brain activity of bees, disrupt their locomotion, ‘higher cognitive function’ (eg. learning and memory), bee communication or social interaction.

Programme

This research brings together a group of scientists with diverse but complimentary expertise in cellular and molecular neuroscience, neuroethology and behavioural ecology. Using a systems biology approach, we are performing a series of integrated interdisciplinary experiments to address these key issues. We are exploring the responses of bees to sub-lethal exposure to miticide/pesticide combinations at multiple levels of organisation: We (Connolly lab) will investigate individual neuronal responses to determine neurotoxicity, sub-lethal neuronal responses such as dendritic beading (movie 1), mitochondrial structural collapse (movie 2) and mitochondrial depolarisation (movie 3) and long term changes to receptor expression. The results of these studies will inform investigations into neurotransmission using fluorescence assays (Connolly lab) and electrophysiology, including plasticity (Harvey lab)

Movie 1
Dendritic beading of a neuron treated with a sub-lethal chemical insult

Movie 2
Mitochondrial collapse and halt to dendritic transport in a neuron treated with a sub-lethal chemical insult

Movie 3
Dendritic beading (blue), mitochondrial collapse (red) and mitochondrial depolarisation (loss of green staining) in a neuron following oxygen-glucose deprivation

Movie 4
Bumblebees (Bombus terrestris) in their nest. Courtesy of Dr Nigel Raine, RHUL

Movie 5
Bumblebees (Bombus terrestris) in a flight arena following training. Courtesy of Dr Nigel Raine, RHUL

(As part of this work Dr Raine will use Radio Frequency Identification tags to monitor bee foraging by record individual bees as they leave and re-enter the nest (Figure 2). A comparison of the difference in the weight of the bee when it returns to the nest, to when it leaves on a subsequent foraging trip, will provide information on the delivery of new resources for the colony.

Figure 2. RFID tagging of bumblebees. Courtesy of Dr Nigel Raine (RHUL)

An important tool for the screening of future agricultural chemicals is the availability of pest species and honeybee cell lines. This aspect of the programme is being conducted at University College London in the lab of Professor Neil Millar

The overall conclusions of this project will be related to the results of the Scottish Beekeepers Association (SBA members’ survey. This will be obtained over a 3-year period to investigate the significance of our findings to the real environment. In particular, SBA will survey, using a large number of honeybee colonies across Scotland, whether particular miticide treatment regimes are detrimental to honeybee health and performance or cause honeybees to become more vulnerable to exposure to other pesticides encountered more sporadically. From this information we hope to correlate the health, productivity and overwintering survivorship of honeybee bee colonies with respect to miticide load and potential pesticide exposure.

Walstab, J., Hammer, C., Lasitschka, F., Moller, D., Connolly, C.N., Rappold, G., Bruss, M., Bonisch, H., Niesler, B. (2010). RIC-3 exclusively enhances the surface expression of human homomeric 5-hydroxytryptamine type 3A (5-HT3A) receptors, despite direct interactions with 5-HT3A, C, D and E subunits. J. Biol. Chem. 285, 26956-26965.

Moult, P., Cross, A., Santos, S., Carvalho, AL., Lindsay, Y., Connolly, CN., Irving, A., Leslie, N., Harvey, J. (2010). Leptin regulates AMPA receptor trafficking via PTEN inhibition. J. Neurosci. 30, 4088-4101.

Mizielinska, S.M., Greenwood, S.M., Tummala, H., Connolly, C.N. (2009) Rapid dendritic and axonal responses to neuronal insults. Biochem. Soc. Trans. 37, 1389-1393.

Bollan, K.A., Baur, R., Hales, T.G., Sigel, E. & Connolly, C.N. (2008) The promiscuous role of the epsilon subunit in GABAA receptor biogenesis. Mol. Cell. Neuro. 37, 610-621.

Connolly, C.N. (2008) Trafficking of 5-HT3 and GABAA receptors. Mol. Memb. Biol. 25, 293-301..

Greenwood, S.M. & Connolly, C.N. (2007) Dendritic and mitochondrial changes during glutamate excitotoxicity. Neuropharmacology 53, 891-898.

Cheng, A., Bollan, K.A., Greenwood, S.M., Irving, A.J. & Connolly, C.N. (2007) Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition. J. Biol. Chem. 282, 26158-66.

Greenwood, S.M., Mizielinska, S.M., Frenguelli, B.G., Harvey, J. & Connolly, C.N. (2007) Mitochondrial dysfunction and dendritic beading during neuronal toxicity. J. Biol. Chem. 282, 26235-44.

O’Mally, D., MacDonald, N., Mizielinska, S., Connolly, C.N., Irving, A.J. & Harvey, J. (2007) Leptin promotes rapid dynamic changes in hippocampal dendritic morphology. Mol. Cell. Neuro. 35, 559-572.

McDonald, N.A., Henstridge, C., Connolly, C.N. & Irving, A.J. (2007) Generation and functional characterisation of fluorescent, N-terminally tagged CB1 receptor chimeras for live-cell imaging. Mol. Cell. Neuro. 35, 237-248.

McDonald, N.A., Connolly, C.N. & Irving, A. (2007) An essential role for constitutive endocytosis, but not activity, in the axonal targeting of the CB1 cannabinoid receptor. Mol. Pharmacol. 71, 976-984.

Krzywkowski, K., Jensen, A.A., Connolly, C.N. & Brauner-Osborne, H. (2007) Naturally occurring variations in the human 5-HT3A gene profoundly impact 5-HT3 receptor function and expression. Pharmacogenetics & Genomics 17, 255-266.

Mizielinska, S., Greenwood, S. & Connolly, C.N. (2006) The role of GABA(A) receptor biogenesis, structure and function in epilepsy. Biochem. Soc. Trans. 34, 863-867.

Hales, T.G., Deeb, T.Z., Tang, H., Bollan, K.A., King, D.P., Johnson, S.J., & Connolly, C.N. (2006) An asymmetric contribution to GABA(A) receptor function of a conserved lysine within TM2-3 of alpha 1, beta 2 and gamma 2 subunits. J. Biol. Chem. 281, 17034-17043.

Cheng, A., McDonald, N.A., & Connolly, C.N. (2005) Cell surface expression of 5-HT3 receptors is promoted by RIC-3. J.Biol.Chem. 280(23), 22502-22507.

Hales, T.G., Tang, H., Johnson, S.J., Bollan, K.A., King, D.P. McDonald, N.A., Cheng, A., & Connolly, C.N. (2005) The epilepsy mutation, gamma2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABA(A) receptors. Molecular & Cellular Neuroscience 29(1), 120-127.

Ward, M.W., Kushnareva, Y., Greenwood, S., Connolly, C.N. (2005) Cellular and Sub-cellular Calcium Accumulation During Glutamate Induced Injury in Cerebellar Granule Neurons. J.Neurochem. 92(5), 1081-1090.

Cole, A.R., Knebel, A., Morrice, N.A., Robertson, L.S., Irving, A.J., Connolly, C.N., Sutherland, C. (2004) GSK-3 phosphorylation of the Alzheimers epitope within collapsin response mediator proteins regulates axon elongation in primary neurons. J. Biol. Chem. 276: 50176-50180.

Connolly CN. Protein Trafficking in neurons. In Molecular Biology of the neuron (second edition). Oxford University Press (2004).

Connolly, C,N., & Wafford, K.A. (2004). The cys-loop superfamily of ligand-gated ion channels: The impact of receptor structure on function. Biochem. Soc. Trans. 32 (3), 529-534.

Boyd, G.W., Doward, A.I., Kirkness, E.F., Millar, N.S. & Connolly, C.N. (2003) Cell surface expression of 5-HT3 receptors is controlled by an endoplasmic reticulum retention signal. J.Biol.Chem. 278: 27681-27687.

Bollan, K., Robertson, L.A., Tang, H., & Connolly, C.N. (2003). Multiple assembly signals in g-aminobutyric acid (type A) receptor subunits combine to drive receptor construction and composition. Biochem. Soc. Trans. 31(4): 875-879.

Bollan, K., King, D., Robertson, L.A., Brown, K., Taylor, P.M., Moss, S.J., and Connolly, C.N., (2003). GABAA receptor composition is determined by distinct assembly signals within a and b subunits. J.Biol.Chem. 278: 4747-4755.

Boyd, GW, Low P, Dunlop JI, Ward M, Vardy AW, Lambert JJ, Peters JA & Connolly CN. (2002). Assembly and cell surface expression of homomeric and heteromeric 5-HT3 receptors: The role of oligomerisation and chaperone proteins. Molecular & Cellular Neuroscience 21: 38-50.

Kittler, J.T., Wang, J., Connolly, C.N., Vicini, S., Smart, T.G. and Moss, S.J. (2000). Analysis of GABA(A) receptor assembly in mammalian cell lines and hippocampal neurons using g2 Subunit Green Fluorescent Protein Chimeras. Molecular & Cellular Neuroscience 16: 440-452.

Taylor, P., Connolly, C.N., Kittler, J.F., Gorrie, G., Hosie, A., Smart, T.G. and Moss, S.J. (2000). Identification of Residues within GABAA Receptor aï€ Subunits that Mediate Specific assembly with Receptor b Subunits. J.Neuroscience 20: 1297-1306.

Connolly, C.N., Kittler, J.T., Thomas, P., Uren, J.M., Brandon, N.J., Smart, T.G. and Moss, S.J. (1999). Cell surface stability of g-aminobutyric acid type A receptors. J. Biol. Chem. 274; 36565-36572.

Taylor, P.M., Thomas, P, Gorrie, G.H., Connolly, C.N., Smart, T.G. and Moss, S.J. (1999). Identification of amino acid residues within GABAA receptor b sububnits which mediate both homomeric and heteromeric receptor expression. J. Neuroscience 19: 6360-71.

Brandon, N.J., Bedford, F.K., Connolly, C.N., Couve, A., Kittler, J.T., Hanley, J.G., Jovanovic, J.N., Uren, J., Taylor, P., Thomas, P., Smart, T.G. & Moss, S.J. (1999). Synaptic targeting and regulation of GABA(A) receptors. Biochem. Soc.Trans. 27 (4): 527-530.

Connolly, C.N., Thomas, P., Gorrie, G.H., Gibson, A., Smart, T.G. and Moss, S.J. (1999). Subcellular localization and endocytosis of homomeric g2 subunit splice variants of g-aminobutyric acid type A receptors. Molecular & Cellular Neuroscience 13: 259-71.

Amato, A., Connolly, C.N., Moss, S.J. and Smart, T.G. (1999). Modulation of neuronal and recombinant GABAA receptors by redox reagents. J.Physiology 517: 35-50.

McDonald, B.J., Amato, A., Connolly, C.N., Benke, D., Moss, S.J. and Smart, T.G. (1998). Adjacent phosphorylation sites on GABAA receptor b subunits determine regulation by cAMP-dependent protein kinase. Nature Neuroscience 1: 23-28.

Connolly, C.N., Wooltorton, J.R.A., Smart, T.G. and Moss, S.J. (1996). Subcellular localisation of GABAA receptors is determined by receptor b subunits. Proc. Natl. Acad. Sci. USA 93: 9899-9904.

Connolly, C.N., Krishek, B.J., McDonald, B., Smart, T.G., and Moss, S.J. (1996). Assembly and cell surface expression of heteromeric and homomeric g-aminobutyric acid type A receptors. J. Biol. Chem. 271: 89-96.

Krishek, B.J., Amato, A., Connolly, C.N., Moss, S.J., and Smart, T.G. (1995). Proton sensitivity of the GABAA receptor is associated with the receptor subunit composition. J.Physiol. 492: 431-443.

Futter, C.E., Connolly, C.N., Cutler, D. & Hopkins, C.R. (1995). Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface. J. Biol. Chem. 270: 10999-11003.

Connolly, C.N., Futter, C.E., Hopkins, C.R. & Cutler, D. (1994). Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase. J. Cell Biol. 127, 641-652.