Basis for Dual-Site ​in vivo

Recording of Neuronal
Activity
• ​Mackenzie Andrews​ •
PI : Charles Chavkin • Mentor : Antony Abraham
Co-Advisor : Alyssa Taylor

Abstract
Drug addiction is a highly prevalent disease with common potentiating risk factors including
stress-exposure, anxiety, and depression. A number of brain regions have been linked to
behaviors that drive drug seeking and abuse, however little is known about how these regions
communicate to regulate those behaviors. Currently, instruments can be used to record neuronal
activity ​in vivo​, however these devices are typically only able to record from one brain region at
a time and don’t allow for specificity of neuronal stimulation. In order to improve the
understanding of how brain regions communicate to drive behavior, there is a need to develop a
multiple-site ​in vivo ​recording device that can record broad excitation signals while allowing
optical stimulation of specific subtypes of neurons. In order to fill this need, I intend to design a
dual-site optic delivering electrode or optrode. This device would be small enough to mount on a
mouse’s skull to promote naturalistic behavior with the ability to record non-specific electrical
signals from the brain regions of interest, as well as optically stimulate specific subtypes of
neurons. I will also be designing a program to process and analyze the data recorded by the
device. If this device is successful, it will improve our understanding of how brain regions
communicate to drive addiction-associated behaviors which could lead to therapeutic solutions
for the treatment of addiction.
Background
Stress-exposure is a widely-recognized risk factor leading to compulsive drug abuse and
relapse during abstinence.​1 ​Drug abuse is highly prevalent in the United States with
approximately 25 million Americans (over 9 percent of the population) using an illicit drug in
any given month.​2​ Addiction potentiating stressors such as anxiety and major depressive
disorders are the most common mental illnesses in the United States with anxiety affecting about
18% of adults in any given year and major depression inflicting 6.7% of all U.S. adults.​3,4​ The
relationship between stress and addiction is reciprocally compounding where stress exposure
increases addiction risk and that drug exposure increases the vulnerability to stress.
Studies on the relationship between stress and addiction have shown that stress-exposure
causes the release of dynorphins, a class of endogenous opioid peptides. Dynorphins act
primarily through the κ-opioid receptor (KOR) to potentiate the rewarding effects of drugs of
abuse including cocaine, ethanol, and nicotine.​5​ Dynorphin is widely produced in the CNS and
regulates neurons that project to many different areas of the brain. Multiple synaptic inputs
converge on dopaminergic neurons in the ventral tegmental area (VTA) to regulate their
excitability. These neurons project broadly to critical targets in the brain to coordinate the drug
seeking behaviors initiated by addictive drugs (Figure 1).​7​ Understanding this circuit and the
effects of stress-mediators on neuronal function in the context of awake and behaving animals is
vital for the development of treatments for addiction.
Statement of Problem and Need
In order to develop new treatments for addiction and related mental illnesses, we must
understand how neuronal activity regulates specific behaviors such as drug seeking. Currently,
questions can be asked about neuronal populations by examining ​ex vivo ​preparations after a
behavior is observed, but this doesn’t show the activity of neurons in the alive and behaving
animal. In order to observe the behavior of neurons, ​in vivo ​recording techniques must be
employed. Imaging of neural activity using fluorescent calcium indicators has been
accomplished with ​in vivo​ two-photon microscopy; however, two-photon imaging must be
performed in head-fixed animals, greatly limiting the assessment of naturalistic behavior (Figure
2).​8,9,10​ Recording from a freely behaving animal, requires imaging devices that are small enough
to fit on an animal’s head and light enough to be carried by the animal.
There are a number of barriers against research using such imaging devices. Primarily,
few companies offer assembled set-ups for the specialized equipment needed for ​in vivo
recording techniques. These head mount assemblies include a microchip, female connector, fiber
bundles, and a probe (Figure 3). Additionally, many of the pre-assembled set-ups that are offered
are extremely expensive, on the range of $500 per instrument for use in a single animal.​11​ These
set-ups also offer limitations such as the type of recording available and the fact that only one
brain region can be recorded from at a time. Concurrently, the available recording techniques
produce a huge amount of data with no standardized processing techniques. Since ​in vivo
imaging is a new tool in the field, much of the available processing tools offer a very limited
number of analysis and still have many glitches in the software. This greatly reduces the ability
for a group to ask novel questions in an efficient and standardized way. In order to overcome
these barriers, there is a need to optimize a specialized ​in vivo​ recording tool and develop
software that allows for easy analysis of the data produced.
Solution and Prior Art
The Chavkin Lab seeks to construct a replicable, low-cost version of the hardware needed
for ​in vivo​ recording. In order to achieve the conditions of this solution, the Chavkin Lab will
develop a 3-D printable hardware or “optrode” assembly (Figure 4). The optrode will include the
microdrive that will be placed on each animal’s head and fiber bundles including optical and/or
electrical cables placed into the specific brain region for ​in vivo​ recording. The hardware will be
recording GCaMP, a genetically encoded calcium indicator, and/or extracellular
electrophysiological signals from the brain to determine neuronal activity. The signal picked up
by the fiber will be transmitted to the microdrive for analog processing before being set to the
digital processing system external to the head mount. The microdrives we are intending to use
are $200 a piece and can are intended to be reused multiple times.
In addition, the Chavkin Lab seeks to develop programs to (1) link specific timepoints of
behavior to time points in recorded data and (2) analyze the data for trends in behavior-linked
neuronal activity. MATLAB (matrix laboratory) will be used to organize, process, and analyze
the data collected by the optrode recordings. This software is intended to ask a variety of
questions about the recorded neuronal activity with a simple user interface. The final program
will be packaged and made available for open source use.
The ​in vivo​ recording technology will be tested in dopaminergic neurons due to their role
in stress and addiction as well as the extensive data available about the behavior of dopaminergic
populations which will allow for easy validation of results. A simple KOR-dependant behavioral
task, such as a T-maze assay, will be chosen as the basis for ​in vivo​ recording. A T-maze assay
allows an animal to travel down a single arm before coming to a split path in which the animal
must make a decision as to which arm to travel down (Figure 5). The decision is often
accompanied with a memory and reward component such as a food-paired arm.
The solution will be building off of the current optrode assemblies available in the field
such as those described by Gillaume Dugué (Figure 3).​12​ These assemblies are typically 16
channel recording devices with either electrical and/or optical controls. The hardware is
cemented onto the animal’s skull with fibers projecting to the brain region of interest. For
electrophysiological recording, insulated tungsten or platinum–iridium wires are typically used.
For GCaMP recording, fused silica fiber optic cables are often employed. In order to optimize
the current assemblies, I will be making modifications to reduce the weight of the device as well
as design an optrode capable of dual site recording.
The software solution will be an adaption of open source MATLAB code available from
the Zweifel Lab.​13 ​The software available allows for analysis of burst firing of neurons. Using a
program such as LabChart to analyze clustering of spikes in order to identify specific neuronal
populations, the cluster information can be uploaded to MATLAB to analyze how different
populations of neurons are responding to recorded behaviors of the animal. In order to ask more
complex questions about the neuronal activity, the addition of other MATLAB processing
techniques will be implemented.
Preliminary Data
This work will be building off of previous research in the Chavkin Lab exploring the
effects of drug seeking behaviors mediated by the release of prodynorphin-derived opioid
peptides and the subsequent activation of KOR (Figure 6).​14​ This work outlined the basis of the
underlying mechanism of drug seeking behavior at a mechanistic level with a pharmacological
context. The Chavkin Lab has previously used optogenetic stimulation techniques on cannulated
animals as a method for ​in vivo ​exploration of neuronal population dependant behavior.
Optogenetics uses fiber optic bundles to stimulate or inhibit specific neuron populations. The
methods for optogenetic control of neurons including surgerizing, cable tethering and the
behavioral assays employed are very similar to those used for optrode recording.
In order to step into the field of ​in vivo​ imaging, I will be collaborating with the Zweifel
Lab who have constructed an electrophysiology optrode set-up (Figure 4). Their current setup is
manufactured by a 3D printing company and the optrode assembly is done by the Zweifel Lab. I
have learned the assembly process and assembled 3 optrodes under the supervision of a Zweifel
Lab member. The Zweifel Lab has also used dual site stimulation and recording techniques
which could be adapted to the dual site optrode imaging.​15​ I will be adapting the current set-up
from the Zweifel Lab to examine the specific aims of the Chavkin Lab.
Consequences of Success
If the Chavkin Lab successfully produces a low-cost, replicable optrode with
standardized data processing software, the accessibility of this powerful and adaptable recording
technique will greatly increase. On a topic specific level, this could lead to novel discoveries
about the neural pathways and mechanisms involved in stress and drug seeking behavior in
animals which could ultimately lead to a new treatment for addiction in humans.
On a broader scale, increasing the accessibility to ​in vivo ​recording tools would allow the
field of neuroscience to advance as a whole as the technology being standardized is adaptable to
any brain region/neuronal population. In addition, if the Chavkin Lab is able to successfully
manufacture a dual site recording optrode, questions about the interactions of separate brain
regions can be examined in novel and informative ways. Dual site GCaMP imaging would be a
new tool for the field of neuroscience and could vastly increase the understanding of neuronal
communication and cooperation.
Real-World Constraints
The major foreseeable constraint for this project is funding limitations contingent on the
grants available to the lab and the cost of the other projects being conducted by the lab. The
major funding sources for the lab come from the National Institute of Drug Abuse (NIDA) and
the National Institute of Mental Health (NIMH) which are both branches of the National Institute
of Health (NIH). The current grants available to the Chavkin Lab have specific research aims
associated with the funding and thus my project must uphold to those aims. There are no
foreseeable translation constraints because this project is intended to stay in a research setting.
From a regulatory standpoint, the project involves animal research, so all of my work
must follow and be approved by The Institutional Animal Care and Use Committee (IACUC)
regulations. The project also involves the use of Schedule I drugs such as cocaine and morphine
so all procedures must adhere to the Drug Enforcement Administration (DEA) guidelines.
Regular inspections by both of these regulatory agencies are mandated.
From a social standpoint, the project is potentially controversial on two fronts. First, the
involvement of animal research makes the project prone to backlash from animal rights activist
groups such as People for the Ethical Treatment of Animals (PETA). Second, since the topic of
research is drug abuse, the social stigma around addiction as a medical and societal disease
makes research in the field a sensitive and controversial subject.
Figures, Schematics, Tables

Figure 1: Schematic showing KOR-mediated GABAergic and glutamatergic input to the VTA.
The VTA has dopaminergic and glutamatergic projections to the nucleus accumbens (NAc) also
mediated by KOR. These projections signal ‘incentive salience’ to the NAc, a primary driver of
addictive behavior. The NAc also sends dynorphin containing afferents to the VTA presumably
contributing to the reciprocal stress-addiction relationship.
Figure 2: Two examples of head fixing for operant behavior showing drastic reductions in
naturalistic behavior of animals. A) Mouse is fixed in space but allowed to walk in two
dimensions on a styrofoam ball.​9​ B) Rat is fixed in space but allowed to push a lever in response
to a cue.​10
Figure 3: Example of a 16 channel optoelectrode available on the market through Neuronexus.
This device is cemented to the animal’s skull with a silicon probe and optical fibers inserted into
the animal’s brain.
Figure 4: Current optrode assembly from Zweifel Lab. A) The hardware set-up allows for
adjustable depth of fiber bundle. B) The microchip and female connector for electrophysiology
recording. C) Example of optrode placement on live mouse, the fiber bundle is connected to the
female connector for recording.
Figure 5: Schematic of a T-maze where the animal is shown in the start arm. Once traveling up
the start arm, the animal must make a choice to travel into arm A or arm B. Often there is a
reward associated with one of the arms and a memory task to learn which arm will yield the
reward.
Figure 6: Prodynorphin knockout mice show a significantly decreased preference for the
cocaine-paired side of the chamber during a conditioned placed preference (CPP) task following
social defeat stress (SDS). Data collected and published by the Chavkin Lab.
Specific Aims
This project aims to design a device to record the activity of neurons in the awake and
behaving animal with the ultimate goal of unraveling the neuronal circuitry involved in drug
seeking and abuse. In order to achieve this goal, I will be designing a device that can be attached
to a mouse’s skull with a fiber bundle that will project into the brain region of interest. The fiber
bundle will include optic fibers for fluorescent Calcium (GCaMP) imaging and/or electrical
fibers for electrophysiology recording. I will also be developing programs to (1) link specific
timepoints of behavior to time points in recorded data and (2) analyze the data for trends in
behavior-linked neuronal activity.
Aim 1 - Optimize Current Optrode Design
The first aim of this project is to optimize the current optrode design in use and
development by the Zweifel Lab. Optimization parameters include weight, size, construction
time, and cost. A device with minimized weight and size would reduce the strain on the animal’s
neck, reduce discomfort and stress on the animal, and provide for more naturalistic behavior
from the animal. A device with minimized construction time and cost would allow less resources
to be spent on the device itself so that more resources could be spent on asking additional
research questions.
Aim 2 - Design Dual-Site Optrode
Once the current design has been optimized, I aim to design a dual-site optrode capable
of recording from two brain regions simultaneously. A dual-site optrode would allow researchers
the ability to ask questions about the role of two brain regions on specific behaviors with the
unique ability to observe firing patterns between regions to get a sense of synchronicity,
cooperativity, and communication between regions.
Aim 3 - Develop Software Package
Finally, I aim to develop a software package to process and analyze the data produced by
the recording device. This software package would have the ability to link behavioral time-points
with time-points in the data, perform firing analysis to identify neuron clusters, and run a variety
of analysis to answer specific questions about the neuronal activity in the animal.
Design Strategy
Aim 1
Approach
I will begin the optimization of the current design by replicating the single-site optrode in
a computer-aided design (CAD) platform such as Autodesk Inventor or Creo Parametric. I will
make modifications to the CAD model to reduce the overall size and weight and run structural
analysis to determine the integrity of the modified design. Part of this design process will include
determining a lightweight, 3D printable plastic that is durable, biocompatible, and degradable by
acetone. Once I’ve achieved an optimized model, I will 3D print and assemble the design.The
design assembly will include the 3D printed device, metal rods for support and fiber threading,
screws to mount the microdrive and to drive the fiber placement, optical (glass) and/or electrical
(tungsten) fibers.
In order to determine the viability of the design, an aseptic surgery to implant the
recording fibers will be performed. Once the optrode is mounted to the animal’s skull and the
animal has been given time to recover from surgery, the overall behavior of the animal will be
assessed for variables such as animal fatigue, head drooping, and food intake to determine if the
optimized device promotes more naturalistic behavior. Data will then be collected from the
device during behavioral assays to validate that the optimized design does not cause reductions
in recording ability or data quality. The data will be analyzed using the current processing
software available in the Zweifel Lab and compared to control recordings gathered by the current
optrode design.
Deliverables
The deliverables for this aim include (1) the CAD design for the optimized optrode, (2)
the final optimized device, and (3) preliminary recording data for device validation. The CAD
design can be used as reference material or as a basis for further iterations of the device design.
The final device can be used answer research questions in a more naturalistically behaving
animal. The preliminary data can be used as a control for future assays.
Anticipated Outcomes
In the design process, a number of decisions must be made to determine the most valid
and feasible optimizations. In order to not entirely redesign the device, small modifications will
be made to reduce the size and weight of the device such as rounding corners, reducing platform
thickness, and minimizing stabilization with metal posts.
Potential problems with this aim include lack of accessibility to lightweight,
biocompatible 3D printer material. If this is the case, the design will be prototyped and reiterated
with the accessible printing materials, then the final design will be outsourced to a printing
company with appropriate materials. Another risk is that the optimized design shows no
significant increases in animal recovery time or behavior. If this is the case, the design can either
be reiterated or I can move on to Aim 2.
Engineering Design Standards
To ensure the safety and efficacy of this device, a number of regulatory practices must be
employed. First, all regulations put in place by The Institutional Animal Care and Use
Committee (IACUC) must be followed.​16​ Since my lab has not done any recording with
optrodes, an amendment to our current protocol must be made, likely in accordance with the
Zweifel Lab’s current protocol. In addition, all materials used in the device that are in contact
with the animal’s tissues must be evaluated for biocompatibility in accordance with standard
Biological Evaluation of Medical Devices which addresses cytotoxicity, genotoxicity, delayed
hypersensitivity, and USP plastic Class VI, which includes the test for irritation, acute systemic
toxicity and implantation.​17
Aim 2
Approach
I will begin the development of a dual-site optrode by determining which brain regions I
aim to be recording from. Currently, I am planning to design a device to record concurrently
from the VTA and prefrontal cortex (PFC). The surgery coordinates for these brain regions will
determine the dimensions for the device including appropriate displacement between fibers, fiber
depth from the skull mount, and angle of fiber driving legs. Once the dimensions have been
determined, I will produce a CAD model in Inventor and optimize as described in Aim 1 for
weight and size and durability. Once optimized, the design will be 3D printed and assembled
with the same materials as Aim 1 (metal rods for support and fiber threading, screws to mount
the microdrive and to drive the fiber placement, optical (glass) and/or electrical (tungsten)
fibers). The design will be validated in the same way as the optimized optrode from Aim 1 by
implanting the fibers and fixing the device to the animal’s skull, performing behavioral analysis
of the animal post-surgery, and running preliminary data-collection trials.
Deliverables
The deliverables for this aim include (1) the CAD design for the dual-site optrode, (2) the
final device, and (3) preliminary recording data for device validation. The CAD design can be
used as reference material or as a basis for further iterations of the device design including
adaptations to record from other brain regions. The final device can be used answer research
questions about the synchronicity, cooperativity, and communication between behavior driving
regions. The preliminary data can be used as a control for future assays.
Anticipated Outcomes
In addition to the material barriers described in Aim 1, a major risk during this process is
the fact that adding a second recording site will likely make the device much heavier so
designing a dual-site optrode within the weight requirements may be difficult. In order to
overcome this barrier, I could modify my design to be limited to an electrode because optical
fibers are heavier than electrical wires. I will also aim to reduce the need for metal supports in
my design. Finally, I could perform a cost-benefit analysis on using a lighter weight plastic
against loss of durability.
Engineering Design Standards
All engineering design standards from Aim 1 must be followed in the manufacturing and
implementation of the dual-site optrode. In addition, this device will allow for the ability to ask
more complex questions about depression, anxiety, drug seeking, and motivation which will
entail the use of Schedule I and II drugs such as cocaine and morphine. In order to use scheduled
drugs, all protocols must follow the regulations in place by the Drug Enforcement
Administration (DEA).​18
Aim 3
Approach
I will develop the processing and analysis software in MATLAB (matrix laboratory). I
will begin by developing an eternal system to store behavioral time points (recorded from a
video camera) into an array of values to link timepoints in behavior to timepoints in the recorded
data. Once recorded data has been run through a clustering software such as Cluster 3.0, it will
be loaded into MATLAB. I will build off of the pre-existing programs developed by the Zweifel
Lab for burst firing analysis to develop more advanced analysis tools to answer research
questions. Once the processing and analysis software has been developed, I will develop a simple
user interface to allow the user to enter data, chose an analysis to run, and set figure options.
Deliverables
The deliverables for this aim include (1) the packaged software capable of data
processing, analysis, and figure generation and (2) the analysis outcomes from the data collected
from the recording devices. The packaged software will be made open source so that any lab
using a similar data collection platform can streamline their data analysis and figure generation
with the aim of making optrode recording more accessible to the neuroscience community. The
analysis outcomes from the data collected by my devices will answer questions about depression,
anxiety, drug seeking, and motivation.
Anticipated Outcomes
I will likely be able to successfully develop a program to ask basic questions however
developing a working user interface that includes a broad range of analysis options may be more
difficult. I will focus my time on developing the analysis tools for the questions being asked by
my lab before working on additional analysis options or a user interface.
Engineering Design Standards
To ensure the validity of my software, I will perform block verifications to ensure that
each section of my code is running properly. I will then validate analysis outcomes against
published results to ensure that the results generated by my program are consistent with expected
outcomes.
Key Personnel
I will be working in the lab of Dr. Charles Chavkin under the direct mentorship of Dr.
Antony Abraham to determine research aims and design parameters. I will also be collaborating
with Dr. Larry Zweifel and members of the Zweifel Lab to learn the optrode manufacturing
process and recording procedures.
Equipment and Facilities
The funding for this project will come from the National Institute of Drug Abuse (NIDA)
and the National Institute of Mental Health (NIMH). I will be using the optrode assembly
equipment available in the Zweifel Lab (dremel, soldering iron, optical fiber polisher). I will also
be using the 3D printers available in the UW Bioengineering Department for prototyping
designs. Software packages I will be using include Cluster 3.0, MATLAB, and Inventor. I will
also be using the animal inventor, housing supplies, and animal facilities of the Chavkin Lab in
the UW Health Sciences Building.

Figures/Schematics

Figure 1: Design Concept - ​Prototype design of dual-site optrode.

Figure 2: Functional Decomposition - ​Numbers refer to Need # from needs table.
Figure 3: Flowchart of Design Strategy ​(Approach - dashed gray circles on top, Deliverables -
Solid gray circles on bottom, iterative process - circle arrow, dependant deliverables - swoop
arrow, dependant approach - dotted lines)

Milestone Title Milestone Description

Measure Current Measure and record the dimensions of the current optrode design in order to produce
Optrode a CAD model
CAD Current
Optrode Produce a CAD model of the current optrode design in Inventor and verify weight
Determine appropriate modifications to reduce weight and size of device while
Optimize CAD maintaining structural integrity
3D Print CAD 3D print the optimized CAD model and verify design (measure weight/dimensions,
test durability, etc.)
Assemble Optrode Assemble the 3D printed parts with the other device parts to produce a complete
optrode
Implant Optrode Perform surgery on the animal to implant the fiber bundles in the brain and mount the
optrode on the skull
Behavioral Assess postoperative animal for variables such as animal fatigue, head drooping, and
Analysis food intake
Collect Data Run behavioral assays to collect data to validate the device and answer research
questions
Validate Data Validate preliminary data against published values and experimental data against
hypothesis predictions
Determine Determine the fiber spacing and driving dimensions of the dual-site optrode based on
Dimensions brain region coordinates
CAD Model
Produce and optimize a CAD model of the dual-site optrode in Inventor
3D Print CAD 3D print the dual-site CAD model and verify design (measure weight/dimensions, test
durability, etc.)
Assemble Optrode Assemble the 3D printed parts with the other device parts to produce a complete
dual-site optrode
Implant Optrode Perform surgery on the animal to implant the fiber bundles in the brain and mount the
optrode on the skull
Behavioral Assess postoperative animal for variables such as animal fatigue, head drooping, and
Analysis food intake
Collect Data Run behavioral assays to collect data to validate the device and answer research
questions
Validate Data Validate preliminary data against published values and experimental data against
hypothesis predictions
Time Point Develop a program to link specific timepoints of behavior to time points in recorded
Processor data
Analysis for Lab Develop a program with analysis tools to answer the specific research aims of the
Chavkin Lab
Additional Analysis Add additional analysis options to the program to widen the variety of questions that
can be asked about the data
User Interface Design a user interface to allow the user to enter data, chose an analysis to run, and
set figure options
Table 1: Research Milestones ​(Aim 1 - light gray, Aim 2 - medium gray, Aim 3 - dark gray)

Table 2: Timeline/Process Tracking Tool ​(Aim 1 - light gray, Aim 2 - medium gray, Aim 3 -
dark gray)
Chart 1: Gantt Chart ​(Aim 1 - light gray, Aim 2 - medium gray, Aim 3 - dark gray)
 Stakeholder I - Chavkin Lab
Rank
# Need Sig.
1 Answers Research Aim 3
2 Low Cost 4
3 Reproducible 5
4 Promotes Naturalistic Behavior 3
Stakeholder II - Mice
Need Rank
5 Minimally Harmful 5
4 Promotes Naturalistic Behavior 4
Stakeholder III - IACUC
Need Rank
5 Minimally Harmful 4
6 Follows Protocol 5
Stakeholder IV - NIMH/NIDA
Need Rank
1 Answers Research Aim 5
2 Low Cost 2
7 Novel 2
8 Time Efficient 1
Table 3: Needs Table
Table 4: Needs-Metric Table
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