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