Download Histologic Analysis of Chronos in an

Histologic Analysis of Chronos in an Optogenetics-Based
Auditory Brainstem Implant Model
1,2
MD,
Massachusetts
Eye and Ear
1,2
MD,
2,3
MS,
Ashton E. Lehmann,
Elliott D. Kozin,
A. Ed Hight,
Keith Darrow,
5
1,2
1,2
Ed Boyden, PhD, M. Chris Brown, PhD, Daniel J. Lee, MD
2,4
PhD,
1 Department
of Otology and Laryngology, Harvard Medical School, Boston, MA. 2 Eaton-Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston, MA; 3Program in
Speech and Hearing Bioscience and Technology, Harvard Medical School, Boston, MA. 4 Department of Communication Sciences and Disorders, Worcester State University,
Worcester, MA. 5 Departments of Brain and Cognitive Sciences and Biological Engineering, MIT Media Lab and McGovern Institute, MIT, Cambridge, MA.
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
Objectives: Optogenetics affords the potential for
improved spatial resolution compared to electric
stimulation in future auditory neuroprostheses.
No histologic studies have yet examined the
virally-mediated gene transfer of Chronos, a new
opsin, to the murine cochlear nucleus (CN).
Herein, we aim to 1) identify CN regions and
neurons receptive to gene transfer of Chronos
and 2) describe the morphology of putative cell
types that express Chronos.
• Current auditory brainstem implants rely on electrical
stimulation to provide sound awareness to patients
who are not candidates for cochlear implants.
• Electric stimulation of neurons results in current
spread, compromising signal specificity.1
• As optical stimulation may be able to excite discrete
sets of neurons more selectively, optogenetics affords
neuronal systems the potential for improved spatial
and temporal resolution.2,3
• To date, no histologic studies have characterized the
CN-targeted gene transfer of Chronos, a new
generation auditory opsin.
• We aim to 1) identify CN regions receptive to gene
transfer of Chronos and 2) describe the morphology
of putative cell types that express Chronos.
1. Opsin-linked GFP fluorescence demonstrates Chronos
expression throughout the injection-targeted dorsal
CN with labeling in all 3 CN subdivisions. Chronos
expression contiguously extends routinely into the
ventral CN and variably on into the auditory nerve.
Extension medially into the inferior cerebellar
peduncle is also occasionally noted.
• The present results demonstrate expression of
Chronos in somata and terminals within all layers of
the CN, paralleling previous findings of
Channelrhodopsin-2 fused to GFP.4
Methods: CBA/CaJ mice underwent CN-targeted
injection of Chronos. The Chronos construct
consisted of an adeno-associated viral vector
(AA2/8), CAG promoter, and a fluorescent marker.
Following a 4-week incubation period, mice were
sacrificed and intravascularly fixed with
paraformaldehyde, and brains were extracted,
sucrose cryoprotected, and cryostatically
sectioned. Sections of 35-micron thickness were
co-labeled with neuron-specific markers
microtubule associated protein-2 and antitubulin, beta III isoform and DAPI-fluoromounted.
Sections of 60-micron thickness were DAPIfluoromounted, and confocal microscopy
revealed cellular morphologies.
Results: Opsin-linked fluorescence demonstrates
Chronos expression throughout the dorsal CN
with contiguous extension routinely into the
ventral CN and variably into the auditory nerve
and inferior cerebellar peduncle. Chronos
localizes to neuronal-specific and non-neuronal
populations. Confocal microscopy suggests
involvement of a wide array of CN cell types,
including morphologies consistent with
pyramidal/fusiform, giant, and cartwheel cells.
Conclusion: Our histologic analyses confirm
widespread infection of multiple neuronal
populations throughout the CN. This work sets
the stage for correlation with ongoing
neurophysiology experiments. Future work with
CN-specific promoters to target neuronal
subpopulations may further improve clinical
potential for an optogenetics-based auditory
neuroprosthesis.
CONTACT
Ashton Lehmann, MD
Email: [email protected]
Daniel J. Lee, MD, FACS
Department of Otolaryngology
Massachusetts Eye and Ear Infirmary
Email: [email protected]
• Chronos is also poised to influence activity in cells
crucial to modulating auditory input as inhibitory
cartwheel cells shape the output of the dorsal CN in
auditory processing.7,9
METHODS & MATERIALS
GENE TRANSFER & OPTICAL STIMULATION
CBA/CaJ mice (n=4) underwent CN-targeted injection of
Chronos via an adeno-associated viral vector (AA2/8)
with a CAG promoter and a green fluorescent protein
(GFP) marker. Following a 4-week incubation period,
blue light (473nm) was delivered to the CN and
responses were recorded in upstream regions of the
auditory pathway.
TISSUE PREPARATION
Mice were sacrificed, intravascularly fixed with
paraformaldehyde; brains were extracted, sucrose
cryoprotected, and cryostatically sectioned. Sections of
35-micron thickness were co-labeled with neuronspecific markers microtubule associated protein-2
(MAP2) and anti-tubulin, beta III isoform (TUJ1) and
DAPI-fluoromounted. Sections of 60-micron thickness
were DAPI-fluoromounted, and confocal microscopy
revealed cellular morphologies.
IMMUNOHISTOCHEMISTRY
Day1:
• Thaw & rehydrate with rinse (PBS x5min x3)
• Block/Permeabilize: 1% Triton-X in 5% normal horse
serum (NHS) x1hr; then rinse
• Primary antibody incubation: 1:500 mouse
monoclonal MAP2, TUJ1 with 0.3% Triton-X in 1%
NHS, overnight (omit primary for negative controls)
Day 2:
• Rinse then secondary antibody incubation: 1:1000
goat-anti-mouse Alexafluor 568 with 0.3% Triton-X in
1% NHS x1hr30min; then rinse
• Tertiary antibody incubation: 1:1000 donkey-antigoat Alexafluor 568 with 0.3% Triton-X in 1% NHS
x1hr30min; then rinse
• DAPI-fluoromount, coverslip
Inferior Cerebellar
Peduncle
• Following virally-mediated opsin gene transfer, the
locations and numbers of infected cells differ
between cases with likely implications for signal
quality achieved optogenetically.10,11
1. Colabeling with neuron-specific markers MAP2 and
TUJ1 demonstrates localization of Chronos to both
neuronal and non-neuronal populations.
• Future experimentation with different promoters may
enable targeting of specific tissues and neuronal
subpopulations to refine the expression of opsins in
the auditory system.
DAPI
Chronos-GFP MAP2-TUJ1 (Neuron)
1. Confocal microscopy enables visualization of complex
cellular morphology. Chronos expression involves a
wide array of CN cell types, including morphologies
consistent with pyramidal/fusiform cells, giant cells,
and cartwheel cells.
VIII
VII nerve root
300 μm
CONCLUSION
• Histologic characterization of Chronos gene transfer
reveals extensive involvement of multiple CN
subregions and cell types, laying the groundwork for a
new generation of improved auditory
neuroprostheses based on optogenetics.
REFERENCES
IV Ventricle
Neutral Red
Nissl Stain
FUTURE STUDIES
• This work sets the stage for correlation with ongoing
neurophysiology experiments.
Dorsal
CN
Ventral
CN
• Chronos localizes to both principal excitatory cell
types of the CN: the numerous pyramidal/fusiform
cells and the sparse multipolar giant cells receive
multimodal input from auditory nerve afferents and
parallel fibers and both give off axons which join to
form the dorsal acoustic stria, projecting directly
upstream to the inferior colliculus.5,6,7,8
Arrow indicates pyramidal/fusiform cell
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