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Understanding How the Brain Drives Hot Flashes
Ashley A. KrullNeuroscience Graduate Program
Annual RetreatSeptember 25, 2015
• Constellation of symptoms – Flushing, perspiration, heart rate, chills
• Troublesome, life-disrupting
• Triggered by acute sex steroid withdrawal
• Traditional treatments (e.g., hormone replacement therapy) risky with varied efficacy and often contraindicated
Hot Flashes
Need for better understanding of this phenomenon and
for novel, safer treatments
Hot flashes are brain-driven
phenomena
Disrupt HPG Axis Hot FlashesHypothalamus (Arcuate Nucleus - ARC)
GnRH neuron
KNDy neuron
Pituitary Ovary
GnRH
LH
Estradiol
Hot Flashes
KNDy Neurons
KNDy Neuron
KisspeptinNeurokinin B (NKB)Dynorphin
GlutamateGABA
MnPO
Working Hypothesis
GnRH neuron
KNDy neuron
Pituitary Ovary
GnRH
LH
Estradiol
Hot Flashes
Peripheral Effectors
NKB Receptors
Aim: Determine the effect of KNDy neuronal stimulation on thermoregulation
Technique: Use DREADD technology to selectively manipulate KNDy neuronal activity
in a murine model
Observations: Changes in thermoregulation
Experimental Plan
Kiss1Cre/+ Transgenic Mice
DREADD in Kiss1Cre/+ MiceAAV-hM3D(Gq)-mCherry
Kiss1-expressing neurons with
Cre-GFP
DREADD in Kiss1Cre/+ Mice
Kiss1-expressing neurons with
Cre-GFP
CNOCNO
CNOCNO
CNOCNO
Selective NeuronalActivation
AAV-hM3D(Gq)-mCherry
• Localized expression seen in ARC of gonadectomized Kiss1Cre/+ animals
Successful Receptor Expression
3V
ARC
3V
• GDX permanent KNDy neuronal activation• Hyperactive KNDy neurons will not react to CNO
• Need to transiently suppress steroids to leverage increased transcription of Cre by hyperactive KNDy neurons• Then, allow neurons to return to baseline activity
levels to enable measureable stimulation by CNO
• Solution: Acyline, a GnRH receptor antagonist
GDX vs. Transient Suppression
Acyline dosing trial results
From all we’ve learned…New Plan
Acyline
Steroids KNDy Activity
DREADDInjection
Steroids KNDy Activity
CNO/VEHX 4
Serum LHE2 / T
KNDy c-Fos
ThermoregulatoryStudies
• Aim: To observe behavioral thermoregulation following KNDy neuronal stimulation
Our Thermogradient
HypothesisAcyline
AAV-DREADD
29° C 22° C
CNO
Thermogradient
• Performing rounds of CNO/Vehicle injections in DREADD-expressing KissCre mice
• Running proof-of-concept experiments on the thermogradient
• Looking forward to exciting data in the near future!
Current Status
AcknowledgementsSteiner-Clifton LabSarah LarsenJessica LancasterAlejandra CabreraLee OrganickJaden BlazierJasmine CorreaDr. Don Clifton, Ph.D.Dr. Robert Steiner, Ph.D.
CollaboratorsDr. John Amory, MD, MPHDr. Susan Reed, MD, MPH
Thesis CommitteeDr. Charles Chavkin, Ph.D.Dr. G. Stanley McKnight, Ph.D.Dr. Richard Palmiter, Ph.D.Dr. Horacio de la Iglesia, Ph.D.
Graduate Program in NeuroscienceMolecular Medicine Training ProgramNeurobiology Training Grant
Plan B: Optogenetics
• Improved temporal precision
• Encouraging slice culture recordings
• Requires more engineering
Pilot Results
None of the mice exhibit co-localization of GFP and mCherry
(5) Male Kiss1Cre Mice (3) Female Kiss1Cre Mice
• Kiss1Cre mice do not express the DREADD – OR – some express the DREADD laterally
DREADD Not Localized in ARC
• Change AAV viral vector–Began using AAV5, shown to permeate brains better–Still did not express or expressed laterally–Mice are the issue
• Gonadectomize the animals to increase Cre-GFP– Increased viral expression but location issues persist
• Change animal strain to KissCre2–Continuous Cre expression restricted to Kiss1-expressing
neurons in ARC and AVPV
• Optimize perfusion, slicing, staining
Project Troubleshooting 101
• Kicker: LH was non-detectible• Acyline on board
at time of CNO (14-16 days after last dose)• Need single-shot
of acyline to transiently suppress sex steroids
Acyline + DREADD Stimulation