Name: tracking drosophila larval behavior in response to optogenetic stimulation of olfactory neurons
Uploaded: 2018-03-21T14:30:00
Description: Watch this Scientific Journal Video about Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons at JoVE.com

This work was supported by startup funds from the University of Nevada, Reno and by NIGMS of the National Institute of Health under grant number P20 GM103650.

1. Building a Behavior Arena and Preparing Hardware to Enable Optogenetic Stimulation in the Behavior Arena
<ol>
	<li>To build a light-deprived behavior arena, construct a box with a dimension of 89 x 61 x 66 cm3 (35&#34; L x 24&#34; W x 26&#34; H) made of black colored plexiglass acrylic sheets (3 mm thick) (see Table of Materials). Materials to build such a box should be available at local hardware stores. Place this box on a table-top in the behavior room (Figure 1</s...

<ol>
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Here, we described a method that allows for the measurement of Drosophila larval behavior in response to simultaneous optogenetic activation of olfactory neurons. Previously described larval tracking methods1,8 use different odor delivery technology to activate ORNs. However, these methods cannot control for either the specificity or temporal patterns of ORN activation. Our method overcomes these deficits by using light stimuli instead of odor stimuli fo...

Olfactory information in a Drosophila larva&#39;s environment is sensed by only 21 functionally distinct ORNs, the activities of which ultimately determine larval behavior1,2,3,4. Yet, relatively little is known about the logic by which sensory information is encoded in the activities of these 21 ORNs. There is thus a need to experimentally measure the functional contributions of each larval ORN to behavior.
Although the sensory response profile of the entire repertoire of Drosophila larval ORNs has been studied in detail1,4,5, the contributions of individual ORNs to the olfactory circuit and thereby to navigational behavior remain largely unknown. Difficulties in larval behavior studies, so far, arise due to the inability to spatially and temporally activate single ORNs. A panel of odorants that specifically activate 19 of the 21 Drosophila larval ORNs was recently described1. Each odorant in the panel, at low concentrations, elicits a physiological response only from its cognate ORN. However, at higher concentrations that are normally used for conventional behavior assays, each odorant elicits physiological responses from multiple ORNs1,5,6. Further, odorants in this panel have varied volatilities that complicate interpretation of behavior studies that depend on formation of stable odor gradients7,8. Finally, naturally occurring odor stimuli have a temporal component that is difficult to replicate under laboratory conditions. It is therefore important to develop a method that can measure larval behavior while simultaneously activating individual ORNs in a spatial and temporal manner.
Here, we demonstrate a method that has advantages over previously described larval tracking assays1,8. The tracking assay described in Gershow et al. uses electronically controlled valves to maintain a stable gradient of odor in the behavior arena8. However, due to the level of complex engineering involved to build the odor stimulus setup, this method is difficult to replicate in other laboratories. Further, the issues related to using odorants to specifically activate single ORNs remain unresolved. The tracking assay described in Mathew et al. uses a simpler odor delivery system, but the resulting odor gradient is dependent on the volatility of test odorant and is unstable for long durations of the assay1. Thus, by replacing odor stimuli with light stimuli, our method has the advantages of specificity and precise temporal control of ORN activation and is not dependent on formation of odor gradients of different strengths.
Our method is easy to set up and is appropriate for researchers interested in measuring aspects of Drosophila larval navigation. This technique could be adapted to other model systems provided that the researcher is able to drive the expression of CsChrimson in their favorite system&#39;s neuron(s) of choice. CsChrimson is a red-shifted version of channel rhodopsin. It is activated at wavelengths that are invisible to the larva&#39;s phototaxis system. We are therefore able to manipulate the activity of neurons with specificity, reliability, and reproducibility9. By modifying the custom written software to account for size changes of the subjects, this method could easily be adapted for crawling larvae of other insect species.

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			<td height="33" style="height:33px;">2&#34; Square Threaded Filter Holder for Imaging Lenses&#160;</td>
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			<td>amazon.com</td>
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			<td height="33" style="height:33px;">630nm Quad-row LED strip lights</td>
			<td>environmentallights.com</td>
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			<td height="33" style="height:33px;width:359px;">850nm LED strips</td>
			<td>environmentallights.com</td>
			<td>wp-4000K-CC5050-60x2-kit</td>
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			<td height="33" style="height:33px;width:359px;">Software&#160;</td>
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			<td height="33" style="height:33px;width:359px;">Matlab</td>
			<td>Mathworks Inc.</td>
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			<td height="33" style="height:33px;width:359px;">Ubuntu MATE v16.04</td>
			<td>Nubuntu</td>
			<td>https://github.com/yslo/nubuntu</td>
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			<td>Home Depot</td>
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			<td>66-112</td>
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			<td height="33" style="height:33px;">22cm X 22cm square petri-dish</td>
			<td style="width:296px;">VWR Inc.</td>
			<td>25382-327</td>
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			<td>Sigma-Aldrich</td>
			<td>D2650</td>
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			<td>Sigma-Aldrich</td>
			<td>84097</td>
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			<td height="33" style="height:33px;">All trans-retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R2500</td>
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			<td height="33" style="height:33px;">Flies</td>
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			<td height="33" style="height:33px;">UAS-IVS-CsChrimson&#160;</td>
			<td>Bloomington Drosophila Stock Center</td>
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			<td>Bloomington Drosophila Stock Center</td>
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			<td>Bloomington Drosophila Stock Center</td>
			<td>23907</td>
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tracking drosophila larval behavior in response to optogenetic stimulation of olfactory neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

To demonstrate the specificity of ORN activation, our method was successfully applied to determine the impact of two different ORN (ORN::7a &#38; ORN::42a) (ORNs expressing either Or7a or Or42a) activation on larval behavior (Figure 3). Consistent with recent studies that individual larval ORNs are functionally distinct1,10,13, our representative data demonstrates th...

Watch this Scientific Journal Video about Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons at JoVE.com

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

The overall goal of this methodology is to analyze navigational behavior of drosophila larva in response to simultaneously optogenetic stimulation of its olfactory neurons. This method enables the comprehensive dissection of olfactory circuit function and complements studies on how olfactory circuit function translates into behavior response. The main advantage of this technique is its specificity and temporal control by using a light instead of traditional odor gradients to activate individual olfactory neurons.To begin the experiment, build a light deprived behavior arena. Construct a box that is 89 by 61 by 66 centimeters, made of black-colored plexiglass acrylic sheets, three millimeters thick. Place the box on a tabletop in the behavior room.Next, mount a monochrome USB 3.0 CCD camera fitted with an IR Longpass 830 nanometer filter and an eight millimeter F1.4 C mount lens on the center of the ceiling of the black box. Place two infrared LED strips on the tabletop to illuminate the larvae in the dark arena. Then, obtain a 22 centimeter by 22 centimeter square aluminum plate to build the LED platform.In the center of the plate, use a metal cutter to cut a hole that is large enough to fit around the CCD camera. Cover the metal plate with red LED strip lights. Solder the LED light strip wires in series, and feed the strip wires into an optocoupler relay controlled by a Raspberry Pi 2B microprocessor.Next, mount the LED platform around the CCD camera. Then install and configure an Ubuntu MATE Raspbian Jessie Linux-based operating system on the Raspberry Pi processor before connecting the optocoupler relay to the LED strips. Attach a power supply to power the LED strips and the optocoupler.Ensure there is homogenous irradiance at various points around the CCD camera in the behavior arena. Ensure the absolute irradiance at the surface of the arena with the help of a spectrometer and determine it to be approximately 1.3 watts per meter squared throughout the surface of the arena. Maintain the flies on standard fly food at 25 degrees Celsius, 50 to 60%relative humidity in a 12-hour, 12-hour light/dark cycle.In order to express CsChrimson in a single pair of ORNs, cross the virgin females from a UAS-IVS-CsChrimson line to males from an ORX-GAL4 line. After male and female flies in a cross have been allowed to mate and lay eggs for 48 hours, transfer the adults to a fresh vial. On the surface of the food vial containing eggs, add 400 microliters of a mixture containing 400 micromolar all-trans-retinal, or ATR, dissolved in dimethyl sulphoxide, or DMSO, in 89 millimolar sucrose dissolved in distilled water.Once ATR is added to the food vials containing eggs, incubate the vials in the dark for an additional 72 hours. After incubation, extract third instar larvae from the surface of fly food by floating them using a high-density 15%sucrose solution. Using a P1000 micro-pipette, separate the larvae floating on the surface of the sucrose solution into a 1, 000 milliliter glass beaker.Wash larvae four times by exchanging 800 milliliters of fresh distilled water in the glass beaker each time. After the final wash, allow the larvae to rest for 10 minutes before subjecting them to the behavior assay. For the behavior assay, maintain a consistent temperature and humidity in the behavior room.Prepare larval crawling medium by pouring 150 milliliters of melted 1.5%agarose into a 22 centimeter by 22 centimeter square Petri dish. Allow the agarose to solidify and cool for one to two hours in the Petri dishes before using them in the behavior assay. After the agarose has solidified, transfer no more than 20 of the prepped third instar larvae to the center of the Petri dish and cover the Petri dish with its lid.Place the Petri dish in the behavior arena under the CCD camera. Turn on the 850 nanometer infrared LED light source to visualize larvae in the video. Start the CCD camera to record larval movement.Finally, move on to data processing and analysis. When ORN 7a expressing CsChrimson was stimulated by light, there was a significant decrease in run length compared to control animals. Conversely, when ORN 42a expressing CsChrimson was stimulated by light, there was a significant increase in run length compared to control animals.We found that for ORN 42a, different temporal patterns of light stimulation elicited different behavioral responses. While we have demonstrated this method for the first order olfactory receptor neurons, our method also permits a variety of future applications, such as measuring the impact of downstream neurons such as projection neurons and local neurons on larval behavior. We expect this technique to pave the way for a comprehensive dissection of larval olfactory circuit function.

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Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

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Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...