Mullee, Daniel W., B.S.

, May 2016

Biochemistry

Glutamate transporter function at the neuromuscular junction of Drosophila Faculty Mentor: Dr. Michael Ka anau!h Abstract
Glutamate is the primary excitatory neurotransmitter at synapses in the mammalian central nervous system. Glutamate is released into the synapse by presynaptic neurons and activates postsynaptic receptors that propagate the excitatory signal. Glutamate transporters, also known as excitatory amino acid transporters (E !s", are responsible #or terminating the signal and maintaining the ambient glutamate concentration in the synapse by removing the neurotransmitter #ollowing presynaptic release. $n pathological states including amyotrophic lateral sclerosis and l%heimer&s disease, disruptions to transporter #unction have been shown to cause elevated synaptic glutamate levels, resulting in excitotoxicity and cell death. $n contrast to mammals, which use acetylcholine at the neuromuscular 'unction ((M)", insects o# the order *iptera also use glutamate #or signaling at this peripheral motor synapse. +omologous glutamate transporters in Drosophila, termed dE !, and dE !-, could provide valuable insight to the #unctions o# human E !s. +owever, the roles o# these transporters in modulating glutamate signaling at the Drosophila (M) has not been determined. Electrophysiological recording at the Drosophila (M) is well established and provides an ideal plat#orm #or studying the roles o# glutamate transporters in neurotransmission. !his proposal aims to #irst express the cloned Drosophila dE !, and dE !- transporters in "enopus oocytes. !he pharmacology o# the dE !s in oocytes will then be characteri%ed by applying E ! blockers with the goal o# identi#ying subtype selective inhibitors. .ynaptic transmission at the (M)s o# abdominal muscle o# Drosophila larvae will be recorded by voltage clamp with intracellular microelectrodes, and the e##ects o# selective dE ! blockers will then be tested. /y selectively inhibiting the glutamate transporter subtypes, it will be possible to test the hypothesis that dE !, #unction is re0uired #or proper control o# synaptic transmission at the (M) o# Drosophila.

Background
$n the mammalian central nervous system, neuronal and glial cells expressing excitatory amino acid transporters (E !s" regulate extracellular and synaptic glutamate concentrations. 1hen glutamate is released into the synapse by #usion o# presynaptic vesicles, it binds and activates postsynaptic receptors, triggering an excitatory postsynaptic potential (E2.2". !hese postsynaptic receptors allow the in#lux o# sodium ions which depolari%es the membrane potential. !his potential is then propagated along the dendrite o# the neuron to the soma where it is ultimately integrated. $# threshold is reached, another action potential is triggered causing release o# neurotransmitters at the synaptic cle#t o# a neighboring neuron. !his mechanism is replicated in mass networks that make up the nervous system. Excess glutamate in the synapse will cause excitotoxicity and is a #actor in many pathological conditions. !he neuromuscular 'unction ((M)" has been used to study synaptic transmission since the ,345&s when /ernard 6at% discovered the 0uantal nature o# synaptic transmission that we know to arise #rom vesicular release o# neurotransmitters (,". For our experiments, the larval Drosophila melano!aster (M) will be used as a model system because o# its large si%e, easy accessibility, and well7characteri%ed genetics. $n contrast to vertebrate muscle, the Drosophila muscle is isopotential, making it possible to record synaptic potentials anywhere in the muscle. !he Drosophila (M) is o# interest because it is a

glutamatergic synapse (as in other arthropods", in contrast to the mammalian (M) which is cholinergic (-". 8onse0uently, it has been proposed to be a use#ul potential model #or vertebrate central glutamatergic synapses. $n particular, we wish to study the roles o# glutamate transporters at this Drosophila synapse, which are not yet established. !he study o# homologous structure and #unction between species has helped scientists understand the #unction o# genes and proteins since *arwin #irst hypothesi%ed that phenotypic similarities and di##erences are variations to a #undamental structure derived #rom a common ancestor. s such, Drosophila (commonly known as the #ruit #ly" provides a homologous model to the human glutamate transporters. !he /asic 9ocal lignment .earch !ool (/9 .!" shows that dE !, and dE !- are more homologous to the human glutamate transporters (hE !,74" than any other known proteins. !he *( se0uence similarities can be seen in Figure ,. *espite the similarities, Drosophila glutamate transporters have been sub'ect to a limited number o# studies, and their roles at the (M) are virtually unknown. !he 6avanaugh lab has extensive experience with human glutamate transporter studies and $ propose to use well7established techni0ues to characteri%e the pharmacology o# the dE !s and their roles at the (M).

Figure 1. +omology o# the Drosophila dE

!,7- proteins with the human hE

!,74 proteins (:".

paper published in -5,5 by .tacey and Muraro et al., #ound that when non7#unctional dE !, mutants were expressed in larval Drosophila, the larvae were unable to per#orm the peristaltic contractions re0uired #or crawling (;". <ur research aims to examine the hypothesis that dE !s are critical to synaptic signaling at the (M) by using synthetic blockers to inhibit dE !, #unction and observing the e##ects on synaptic response. !he pro'ect will #irst involve characteri%ation o# the pharmacology o# several blockers that have previously been tested with the human E !s (4". n important goal o# this characteri%ation is to determine blockers that are speci#ic to each subtype, so that they may be selectively inhibited. n additional ob'ective is to analy%e the observations o# /irman and /esson et al., which show that dE !- is a low7a##inity glutamate transporter and a high7a##inity transporter #or aspartate and taurine (=". $t will also be o# scienti#ic value to con#irm that inhibition o#

dE !- transport #unction does not e##ect motor #unction o# Drosophila, as previously reported (>". !his can also be achieved by inhibiting dE !- transport #unction at the (M) and measuring the resulting synaptic transmission. !his is a novel experiment that may provide critical in#ormation about the role o# amino acid transport in synaptic transmission.

Research Methods
$ will be working in the laboratory o# *r. Michael 6avanaugh at the 8enter #or .tructural and Functional (euroscience (8.F(", located on the third #loor o# the .kaggs building. *r. 6avanaugh is an expert in glutamate transporters and has experience working with both Drosophila and "enopus. +e will provide all the necessary e0uipment re0uired to conduct molecular biology and electrophysiology. ll electrophysiological recordings will be ampli#ied, inter#aced through an ?* converter, and analy%ed by so#tware provided by *r. 6avanaugh. +e will provide the "enopus lae is, which are housed in the @niversity o# Montana animal care #acility in the basement o# the (orth .kaggs building. !he #acility is 9 87accredited and run by trained sta## with oversight o# a #ull time veterinarian. +e will also provide the Drosophila melano!aster stock and all the relevant e0uipment, which is located in the basement o# the .kaggs building. $ will be keeping a bound laboratory notebook #or recording observations and data. *ata will also be stored and analy%ed on Macintosh computers, and backed up using cloud storage. 9astly, *r. 6avanaugh is experienced in all o# the techni0ues that will be used in this experiment, and has generously o##ered to provide mentorship and oversight. Part A: Expression of the Drosophila dEAATs in Xenopus oocytes. !he #ollowing procedure is per#ormed to minimi%e su##ering and comply with ($+ and @niversity o# Montana $ 8@8 regulations. "enopus lae is #emales are used which have been prein'ected with human chorionic gonadotropin to induce superovulation. !he #rogs will then be anestheti%ed with ,A !ricaine in water by partial submersion in an aerated solution until unresponsive (47,5 minutes". small incision is then made to per#orm a partial ovarectomy, #ollowed by suturing using sterile techni0ue. Following surgery, #rogs are allowed to recover #or two hours be#ore being returned to the colony. Each #rog typically undergoes three to #our surgeries be#ore euthani%ation by decapitation under deep !ricaine?cold anesthesia. <ne advantage o# using oocytes is that they do not express many voltage7gated channels and the current that is produced by endogenous channels is relatively low. nother advantage is that voltage clamp current recordings allow #or a speci#ic type o# channel to be analy%ed in situ. !he *( plasmids encoding the wild7type dE !, and dE !- proteins are in the 6avanaugh lab. #n itro transcription o# the lineari%ed c*( with the !> promoter region will produce cB( #or in'ection o# the "enopus oocytes. +ealthy de#olliculated stage C7C$ oocytes will be selected and microin'ected with the cB( encoding the wild7type dE !, and dE !- proteins. !he in'ected oocytes will be kept in a Frog Binger&s solution at ,; o8 #or three to #ive days be#ore voltage clamp currents are recorded. Part B: Characterization of the pharmacology of loc!ers against the dEAATs. !ransporter analysis will be conducted by voltage clamp current recordings as shown by Figure -. !his method will allow #or the screening o# blockers against the subtypes. library o# blockers will be chosen based on prior success with human E !s (D". /lockers will be introduced and the resulting current measured using methods that have been established by *r. 6avanaugh (4". !he goal o# this assay is to characteri%e the blockers against each dE ! and to determine the concentrations re0uired #or inhibition o# transporter #unction. 1e will then attempt to determine a compound that is selective

#or each subtype. compound o# interest is the blocker threo7E7hydroxy7aspartic acid (!/< ", which binds all #ive human E !s with a##inity in the low micromolar range, but does not prevent glutamate #rom binding receptors. lso o# interest are the derivatives o# !/< such as !F/7!/< (D" and *97 !/< (;", which have larger groups attached to the E7hydroxyl group. <ther compounds we can characteri%e include -7F , 97!/ , and kainate. ll o# which have been characteri%ed with the human E !s in previous experiments by *r. 6avanaugh (3, ,5, ,,". 9astly, the characteri%ation o# blockers against glutamate transporters is o# biomedical signi#icance and may be o# value to research groups concerned with the treatment o# neurodegenerative pathologies.

Figure ". .chematic o# voltage clamp current recording (,-". Part C: #ynaptic transmission of dEAATs at the Drosophila neuromuscular $unction. !he .tacey and Muraro et al. research group that observed motor dys#unction in Drosophila larvae expressing non7#unctional dE !, mutants, also conducted electrophysiological assays in situ. !he group measured electrophysiological properties o# the dE !, mutant and wild7type Drosophila using whole7cell patch7clamp recordings o# the dorsal motor neurons. Becordings o# the dE !, mutants showed an increased amplitude, longer duration, and decreased #re0uency o# excitatory synaptic potentials. !hese results suggested that the crawling de#ects observed in dE !, mutants are due to synaptic signaling de#ects within the 8(. (;". !he group then used the same recording techni0ue to measure excitatory synaptic potentials in wild7type larvae a#ter the addition o# the E ! competitive antagonist *97!/< . !heir results showed an increased duration and decreased #re0uency o# excitatory synaptic potentials as in the dE !, mutants, but with no e##ect on amplitude. /ased on these results, the group proposed that the electrophysiological de#ects observed in dE !, mutants were consistent with a #ailure to transport glutamate (;". 1e plan to expand upon the research o# the .tacey and Muraro et al. group by measuring synaptic transmission at the (M) o# wild7 type larval Drosophila and selectively inhibiting the glutamate transporter subtypes, which has never been done. Figure %. *issection o# larval Drosophila to access the (M)s (,=".

!he glutamatergic evoked excitatory 'unction currents (E)8s" at the (M) will be measured using the intracellular two7electrode voltage clamp techni0ue. !his method is pre#erred because it allows #or an accurate readout o# synaptic transmission through glutamate receptors without contribution #rom voltage7gated ion channels or nonlinear summation o# synaptic activity during high #re0uency stimulation (,:". For the recording, third instar larvae will be dissected into a #illet to provide internal access to the (M)s. Figure :. provides an example o# the dissection. s shown in Figure ;., larval muscles are arranged in a stereotypical, repetitive manner, as are the motor neurons innervating them. !his allows #or the same individual neuromuscular 'unction to be identi#ied among di##erent individual larvae (,;". !he two7electrode voltage clamp techni0ue consists o# one intracellular electrode that will record voltage and a second intracellular electrode that will in'ect current. third electrode will partially suck up the #ree nerve ending o# the motor neuron #or electrical stimulation to induce action potentials. !he larvae will be kept in a saline solution at physiological p+ during dissections and assays. pplication o# pharmacological blockers will be accomplished by addition o# the blocker to the saline solution used during larval dissection (;". Finally, E)8s as well as miniature excitatory 'unction currents (mE)8s" will be recorded while varying the concentrations o# glutamate with the substrate inhibitors. $n addition to investigating the role o# dE !, in proper motor #unction, the contribution o# dE !- can also be 0uanti#ied by using subtype selective blockers. *r. 6avanaugh will provide guidance in data analysis, mathematical modeling, and interpretation o# the results. .hould time permit, additional experiments can be per#ormed using the same (M) model. <ne such experiment would employ the use o# optogenetics by expressing the light7activated ion channelrhodopsin7(8hB-" in Drosophila (,4". !he activation o# glutamatergic motor neurons at the (M) o# Drosophila could then be mediated by 8hB- light7activation while measuring the resulting E)8s. Figure &. (euromuscular connectivity and myotopic map o# larval Drosophila (,=".

Timeline
$ will be working at least -5 hours a week during the summer while taking one class. 1hen $ am not in summer school, $ will be available to work at least ;5 hours a week. *uring the utumn and .pring semesters, $ will be available to work ,57-5 hours a week, depending on my work load #rom classes. $ will continue to work on the proposed pro'ect until su##icient science has been done to publish an academic paper.

References
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