>>   GOOD AFTERNOON EVERYBODY.

IT'S MY GREAT PRESSURE TO

INTRODUCE OUR SPEAKER JAIDEEP

BAINS THAT COMES FROM THE

UNIVERSITY OF CALGARY.

JAIDEEP GOT HIS PH.D. AT QUEEN'S

UNIVERSITY IN KINGSTON, ONTARIO

WHERE HE FIRST STARTED WORKING

ON HIG HYPO THALAMIC -- THEN HE

MOVED TO THE UNIVERSITY OF

COLORADO HEALTH SCIENCES CENTER

TO DO A POST DOC WHERE HE WAS IN

THE HIPPO CAMPO NETWORK AND DID

SOME CIRCUITRY WORK AND REALLY

SPECIAL EMPHASIS ON -- THEN HE

MOVED IN 2002 TO CALGARY WHERE

HE REMAINS TODAY AND HE'S MOVED

BACK TO I THINK HIS FIRST LOVE

PRIMARILY THE HYPO THALAMIC PUT

[INDISCERNIBLE] PAPUT -- PITUITARY.

HE'S REALLY MADE CONSIDERABLE

INSIGHT INTO A NUMBER OF REALLY

GREAT THOUGHT PROVOKING STEETS

THAT HAVE -- STUDIES THAT HAVE

RAMIFICATIONS ON THE NERVOUS

SYSTEM FUNCTION.

HE'S GOING TO PRESENT TOTAL

ENTITLED SNAP DISCUS SYNAPSES AND STRESS.

>> SNAP AND STRESS.

CHRIS, THANK YOU VERY MUCH FOR

THE VERY KIND INTRODUCTION AND

THE INVITATION TO SPEAK TODAY

ONE OF THE REASONS I LEFT THE

HIPPO CAMPUS IS THAT HAVING

DIPPED MY TOE IN I WAS WORRIED

SOMETHING WAS BOEING TO BITE IT

OFF.

-- WAS GOING TO BITE IT OFF.

WHAT WE TRY TO DO IN MY LAB IS

REALLY FOCUS ON TRYING TO

UNDERSTAND THE HYPOTHALAMUS FROM

IS INSIGHT OUT.

A LOT OF PEOPLE FOR DECADES HAVE

THOUGHT A LOT ABOUT THE

HYPOTHALAMUS.

HAVE THOUGHT ABOUT THE ALL-NOMIC

OUTPUT OF THE HYPOTHALAMUS, THE

ENDOCRINE OUT PUT OF THE

HYPOTHALAMUS, THE ROLE THE

HYPOTHALAMUS PLAYS IN A LOT OF

INNATE BEHAVIORS BUT ONE THING

THEY HAVEN'T DONE IS THOUGHT

VERY MUCH ABOUT THE SYNAPSES AND

THE NEURO CIRCUITS THAT CODES

ALL THIS INFORMATION AND DRIVES

THESE INNATE BEHAVIORS.

THIS IS WHERE MY LAB HAS COME

IN.

TO REALLY PUT THE SPOTLIGHT ON

THE SYNAPSES AND THE

HYPOTHALAMUS AND THINK ABOUT HOW

THESE SYNAPSES BEHAVE UNDER

NORMAL CONDITIONS.

HOW THESE SYNAPSES BEHAVE WHEN

THEY'RE CHALLENGED AND WHAT THIS

MIGHT MEAN FOR THE ORGANISMS.

SO THE WORK I'M GOING TO TALK

ABOUT TODAY WAS DONE BY TWO

GRADUATE STUDENTS IN MY LAB, ONE

OF THEM IS SARA HEWITT WHO

FINISHED A COUPLE YEARS AGO AND

THE OTHER IS JACQULIN WANSTEEKER

IS STILL A STUDENT IN MY LAB.

I'LL START WITH THE WORK SARA

ABOUT WITH HER PH.D. AND CLOSE

WITH JACKIE'S WORK WHICH IS

CURRENTLY UNPUBLISHED.

SO THE HYPOTHALAMUS IS THE

PRIMARY INTERFACE BETWEEN THE

BRAIN AND IMPORTANT INTERFACE

BETWEEN THE BRAIN AND THE BODY

THAT CONTROLS HOMEOSTASIS.

AS ALL OF YOU KNOW HOME STASIS

IS THE PROPERTY THAT REGULATES

THE INTERNAL ENVIRONMENTS THAT

MAINTAINS STABILITY AND

CONSTANCY.

SO THE HYPOTHALAMUS SITS AT THIS

INTERFACE AND IF WE TAKE A

CROSS-SECTION OF THE BRAIN, IT

SITS RIGHT AT THE BASE OF THE

WRAIN AND SENDS PROJECTIONS TO

AUTONOMIC NUCLEI AS WELL AS TO

THE PITUITARY.

IN THE RAT BRAIN, IN THE RAT

BRAIN, THIS IS A SECTION OF THE

RAT BRAIN SHOWING THE LOCATION

OF A NUMBER OF NUCLEI IN THE

HYPOTHALAMUS.

SO IT'S ONE DIFFUSELY CONNECTED

STRUCTURE THAT HAS A NUMBER OF

DIFFERENT FUNCTIONS.

AND SOME OF THOSE ARE JUST

HIGHLIGHTED HERE INCLUDING MOST

FUNCTION THAT ARE CRITICAL FOR

SURVIVAL, CIRCADIAN RHYTHMS,

ENERGY BALANCE, AS YOU MOW

REGULATION -- OS MOW REGULATION,

SEXUAL FUNCTION.

THE THING MY LAB IS INTERESTED

IN IS THE RESPONSE TO STRESS.

THESE ARE ESSENTIAL FOR SURVIVAL

SO THEY MUST BE HARD WIRE, THE

ABILITY TO RESPOND EXPEAN GAUGE.

THESE BEHAVIORS ARE HARD WIRED.

BUT INNATE BEHAVIORS HAVE A LOT

OF CHALLENGES.

SO AN ORGANISM EXISTS IN A VERY

DYNAMIC ENVIRONMENT.

THE CHALLENGES THAT IT PHASES

ARE UNPREDICTABLE SO THERE'S GOT

TO BE MECHANISMS THAT ARE BUILT

IN TO ALLOW THESE HONEY EIGHT

BEHAVIORS -- INNATE BEHAVIORS TO

WORK PROPERLY EVEN THOUGH THE

BASELINE IS CHANGING ALL THE

TIME.

SO SURVIVAL REQUIRES THAT THE

ORGANISM CONTINUE TO ADAPT.

AND THESE ADAPTATIONS REQUIRE

SOME FORM OF LEARNING AND

MEMORY.

IN OTHER WORDS, IF YOU'RE FACED

WITH ONE CHALLENGE AND FIGURE

OUT A WAY TO RESPOND

APPROPRIATELY TO THAT CHALLENGE,

IT WILL PROBABLY BE HELPFUL IF

YOU COULD USE THOSE SAME

MECHANISMS THE NEXT TIME AROUND.

SO WE THINK THERE'S GOT TO BE

SOME LEARNING AND MEMORY IN THIS

SYSTEM.

AND THERE ARE A LOT OF

EXPERIMENTS THAT HAVE GONE ON

THAT HAVE SHOWED THAT THESE

SYSTEMS ARE CAPABLE OF

RESPONDING EFFECTIVELY TO

CHALLENGES AND THEN ADAPTING TO

SUBSEQUENT CHALLENGES.

SO LEARNING IN MEMORY FROM A

SYNAPTIC POINT ARE ENCODED IN A

COUPLE WAYS.

ONE FORM THEY CAN TAKE ARE

IMMEDIATE OR EXPERIENCE

MODIFICATIONS IN SYNAPSES

ACTIVITY AND CIRCUITS.

AND THAT'S HOW WE CLASSALLY

PLASTICITY AND THE SECOND IS

LATENT EXPERIENCE DEPENDENT

MODIFICATIONS THAT ARE ONLY

EVIDENCE UPON SUBSEQUENT

RECRUITMENT OF SYSTEM OR

METAPLASTICITY.

THE BEHAVIOR CHALLENGE HAS DONE

SOMETHING TO THE SYSTEM BUT WHEN

YOU PROBE THE SYSTEM USING

PHYSIOLOGICAL MEANS LIKE WE DO,

THE SYSTEM DOESN'T REALLY SEEM

VERY DIFFERENT.

IT'S ONLY WHEN YOU THEN AGAIN

CHALLENGE IT WITH SOME SORT OF

PARADIGM THAT YOU NOTICE THAT

THESE SYNAPSES STORE INFORMATION

A LITTLE BIT DIFFERENTLY.

SO THE NUCLEUS WE'LL FOCUS ON

FOR ALL THIS TALK IS

PARAVENTRICULAR NUCLEUS.

IT'S A BILATERAL STRUCTURE THAT

SITS AROUND THE THIRD VENTRICLE.

AND THESE PBN NEURONS RECEIVE

SIGNALS FOR ALL SORTS OF

STRESSFUL CHALLENGES, INCLUDING

PHYSICAL THREATS, INFLAMMATION,

BLOOD LOSS, HUNGER AS WELL AS

PERCEIVED OR PSYCHOLOGICAL

THREATS.

FOR EXAMPLE, SOMETHING THAT

CHALLENGES AN ORGANISM'S

SURVIVAL.

ALL OF THESE SYNAPSES CONVERGE

ON TO THE NEURONS AND THE PBN ON

THE -- SECRETORY CELLS THAT SIT

IN THE MEDIAL ASPECT OF THE PBN

AROUND THE VENTRICLES.

THESE CELLS MAKE A NUMBER OF

NEUROPEPTIDES INCLUDING --

RELEASING HORMONES -- STATIN AND

CORTICO APPROPRIATEN RELEASING

HORMONES WHICH IS THE KEY

INITIATOR OF THE -- AXIS.

I JUST SHOW YOU THIS VERY

QUICKLY.

THIS IS A SCENE FOR C FAUST

FOLLOWING 30 MINUTES OF STRESS.

AND YOU CAN SEE LARGE MAJORITY

OF CELLS IN THE PVN SEEMS TO

LIGHT UP.

THERE IS THIS KIND OF BROAD

ACTIVATION OF THIS NUCLEUS IN

RESPONSE TO A VERY BRIEF 30

MINUTE RESTRAINED STRESS.

SO THIS RESTRAINT STRESS OR AN

IMMEDIATE THREAT LAUNCHES AN

IMMEDIATE INCREASE IN CIVIL

THETIC ACTIVITY -- SYMPATHETIC

ACTIVITY WHAT WE ARE AWARE OF A

FLIGHT OR FIGHT RESPONSE BUT IT

RELEASES HORMONES.

CRH IS RELEASED AND FROM THERE

IT'S TAKEN INTO THE CIRCULATION

TO THE ANTERIOR PITUITARY

CAUSING THE RELEASE OF ACTH

WHICH CIRCULATES IN THE BODY AND

CAUSES THE RELEASE OF

CORTICOSTEROIDS.

OKAY.

SO WHEN WE GOT INTO THIS, THERE

WERE A FEW OBSERVATIONS THAT

WERE PREVIOUS LUCT IN THE

LITERATURE, AND I THOUGHT --

PREVALENT IN THE LITERATURE AND

I THOUGHT I WOULD SUMMARIZE

BEFORE I GET INTO THE MEAT OF

THE TALK.

STRESS INCREASES NEURO ACTIVITY

IN PVN AND COUPLED IN INCREASING

CORTICOSTEROIDS.

THESE CORTICOSTEROIDS, THEY

TERMINATE THE NEUROENDOCRINE

RESPONSE TO STRESS.

THE THIRD THING WE KNEW IS THAT

REPETITIVE RECRUITMENT OF THE

STRESS ACCESS MUD FIES THE

OUTPUT AT THE AXIS.

THE MORE YOU RECRUIT IT, IT

DOESN'T BEHAVE THE SAME WAY FOR

EVERY SUBSEQUENT RECRUITMENT.

SO EVERY KIND OF STRESS

GENERATES A STEREOTYPED HORMONE

RESPONSE.

I'LL JUST BREAK DOWN THE TIME

FRAME KINETICS OF THE HORMONE

RESPONSE.

SO THIS IS A CRH NEURON.

WHEN THE NEURON FIRES IT

RELEASES HORMONE INTO THE

VESSELS OF THE -- AND THIS

HORMONE IS CRH SOMETIMES ALSO

VASO PRES PRESTON -- THIS TRIGGERS

THE RELEASE OF -- INTO BLOOD

WHICH CIRCULATES IT INTO THE

PITUITARY AND HITS THE ADRENAL

CORTEX THAT CAUSE THE RELEASE OF

CORTICOSTEROIDS INTO PLASMA.

AND THEN OVER TIME

CORTICOSTEROIDS CIRCULATE AND

THE CORTICO STEROID

CONCENTRATION IN THE BRAIN ALSO

STARTS TO BUILD BUT YOU'LL

NOTICE IT'S MANY MINUTES AFTER

THE INITIAL INDUCTION OF THE

STRESS RESPONSE.

AND THESE CORTICOSTEROIDS ARE

THOUGHT AND HAVE BEEN SHOWN TO

FEEDBACK AND ALTER THE MACHINERY

IN THESE CRH NEURONS.

SO THIS PLAYS ANOTHER WHAT WE

THINK IS A VERY IMPORTANT ROLE

AND THAT IS THAT CORTICOSTEROIDS

ENCODE A FORM OF MEMORY IN THE

SYSTEM AND THEY CHANGE THE WAY

THE SYSTEM WORKS TO SUBSEQUENT

STRESSES.

SO I'M JUST GOING TO DESCRIBE AN

EXPERIMENT THAT WAS DONE BY

WISEALL LAST YEAR.

THEY INFUSE THE CORTICOSTEROIDS

INTO THE PVN PRIOR TO AND DURING

15 MINUTES OF STRAINED STRESS.

WHEN YOU INFUSE -- IT GIVES YOU

A LARGE INCREASE IN THE RELEASE

OF THIS HORMONE, HCTH.

IF CORTISONE BOARD IN THE PVN IT

COMPLETELY BLUNTS THIS RESPONSE

TO STRESS.

THIS HAS A PROFOUND ABILITY TO

MODIFY RESPONSES TO STRESS.

SO IT'S NOT NECESSARILY WHAT YOU

WOULD THINK OF A NEGATIVE

FEEDBACK BREAK IN A SYSTEM ONCE

IT'S LAUNCH, IT PREVENTING THE

LAUNCH OF THE SYSTEM IN THE IF

YOU ARE PLACE.

THEY PROPOSE THAT STRESS OR

CORED CO-STEROIDS IMPOSE THIS

REFRACTORY PERIOD ON THE SYSTEM

SO THAT IMMEDIATELY FOLLOWING A

SINGLE STRESS THE SYSTEM IS NOT

VERY RESPONSIVE TO SUBSEQUENT

STRESSES.

SO BASED ON THESE DATA I THINK

WE CAN MODIFY A COUPLE OF THESE

STATEMENTS.

SO RATHER THAN THINK IT

TERMINATES THE RESPONSE IT MUD

FIES THE END KROIN RESPONSE AND

MODIFIES THE RESPONSE TO

SUBSEQUENT STRESS.

SO WHAT I'M GOING TO TELL YOU

ABOUT TODAY IS ONE EXAMPLE OF

HOW WE THINK THIS MIGHT HAPPEN

AT THE SYNAPTIC LEVEL.

WHEN WE BEGAN THIS WORK WE TRIED

TO DISTILL THE QUESTIONS DOWN

INTO VERY SIMPLE QUESTIONS.

THE TWO QUESTIONS I'M GOING TO

ANSWER TODAY WHICH WERE

UNANSWERED WHEN WE GOT INTO THIS

IS WHAT TURNS THE HPA, THE

HYPOTHALAMUS PITUITARY ADRENAL

AXIS ON AND HOW DOES THIS HPA

ADAPT.

OKAY.

SO THERE ARE TWO FACTORS THAT

DETERMINE THE ACTIVITY OF THESE

NEURONS.

THE FIRST IS INPUT FROM ACCURATE

SYNAPSES AND THE SECOND ARE THE

CIRCULATING LEVELS OF CORT.

SO THERE'S BEEN A LOT OF WORK

LOOKING AT AND DEFINING CARE

EMPLO --CAREFULLY DESCRIBING THE INPUTS.

THE INPUTS ORIGINATE FROM A

NUMBER OF DIFFERENT BRAIN

STRUCTURES, INCLUDING THE BRAIN

STEM, THE NUCLEUS -- THE C1, A1

ENERGETIC CELL GROUPS AS WELL AS

OTHER OTHER HYPO THALAMIC

STRUCTURES AND LIKE THE MEDIUM

PREOP -- OPTIC.

OF THESE INPUTS ARE GABA LARGIC.

THEY MAKE UP THE SYNAPSES ON THE

ENDOCRINE CELL.

THIS IS DIFFERENT ACCORDING TO

THIS PAPER.

THEY CLASSIFY THE -- NEURONS

HAVING ABOUT TWO AND-A-HALF, 3%

OF THEIR SYNAPSES.

IN OUR CASE 50% OF THE SYNAPSES

ARE GABA ENERGETIC.

THAT MEANS IT HAS A PROFOUND

DAMPENING EFFECT ON THE SYSTEM.

SO WE BASICALLY STARTED WITH

THIS STATEMENT.

SO GIVEN THAT SYNAPTIC

INHIBITION ACTIVELY RESTRAINS HP

OUTPUT IT'S BEEN PROPOSED THAT

RELEASE FROM INHIBITION IS

NECESSARY FOR THE INITIATION OF

THE NEUROENDOCRINE RESPONSE TO

STRESS.

SO THIS MAKES SENSE WHEN HALF OF

YOUR SYNAPSES ARE INHIBITORY YOU

REMOVE THE INHIBITION, YOU CAN

LAUNCH THIS SYSTEM VERY QUICKLY.

WE WANTED TO TAKE A LITTLE

CLOSER LOOK AT THIS AND ASK WHAT

WAS HAPPENING AT THE LEVEL OF

GABA SNAP SUGGESTS.

SO WE DID -- SYNAPSES.

WE DID IN VIVO EXPERIENCES WHICH

WE INJECTED DIRECTLY INTO THE

PVN AND JUST MEASURE CIRCULATING

CORTICOSTEROIDS.

WHEN YOU INJECT YOU SEE THIS

TIME DEPENDENT INCREASE IN

CIRCULATING CORTICOSTEROIDS

WHICH IS REPORTED BY DOZEN AND

DOZEN OF LABS.

THIS IS NOT NEW.

WHAT'S INTERESTING, THOUGH, IS

THAT WHEN YOU DO THE SAME

EXPERIMENT AFTER YOU'VE STRESSED

AN ANIMAL FOR 30 MINUTES, YOU DO

NOT SEE THE SAME INCREASE IN

CIRCULATING CORTICOSTEROIDS,

OKAY.

SO THE SYSTEM HAS CHANGED IN

SOME WAY FOLLOWING STRESS.

WHEN WE LOOKED A LITTLE BIT MORE

CAREFULLY AT THE FIRING OF THESE

CELLS SO WE TOOK SLICES

CONTAINING THE PVN EITHER FROM

NAIVE ANIMALS OR FROM ANIMAL

THAT HAVE BEEN STRESSED AND

ATTACHED RECORDINGS, DOESN'T

PERTURB THE ENVIRONMENT AND JUST

RECORD ACTION POTENTIAL IN THESE

CELLS.

WHAT YOU FIND IS THAT WHEN YOU

LOCK GABA RECEPTORS, THE CELLS

INCREASE, THEY ARE FIRING GREAT.

THAT SHOULDN'T BE SURPRISING.

WHEN YOU BLOCK THE RECEPTORS

FROM STRESSED ANIMALS, THOUGH,

WE SEE NO CHANGE IN FIRING,

OKAY.

SO THIS SYSTEM IS DISINHIBITED

AND GABA APPEARS NOT TO BE

INHIBITORY ANYMORE.

SO THIS IS JUST SUMMARY DATA

FROM A NUMBER OF CELLS SHOWING

CONTROL INCREASE IN FIRING AFTER

STRESS BY FAILED TO INCREASE THE

ACTIVITY OF THESE CELLS.

SO WHAT TURNS THE HP ON.

SO OUR APPROACH IS TO TAKE

CORONAL SLICES OF THE

HYPOTHALAMUS THAT CONTAIN THE

PARAVENTRICULAR NUCLEUS.

THIS IS A SECTION CONTAINING THE

PVN AND WE MAKE RECORDINGS FROM

NEURONS, LOOK AT THE NEURONS AND

LOOK AT THE MORPHOLOGY, THE

RELATIVELY BORING CELLS THEY

HAVE TWO DENY RATE DENDRITES AND NOT

VEHEMENT SYNAPSES.

ELECTRICALLY COMPACT CELLS AND

HUNDREDS OF MEGAOMS MAKING

SYNAPTIC PARAMETERS RELATIVELY

EASY, OKAY.

AND THESE GREEN CELLS BY NEURONS

THAT HAVE BEEN IMMUNOSTAINED FOR

CRH.

SO WE CAN IDENTIFY THESE

NEURONS, EITHER POST HAWK WITH

THE CHEMISTRY OR PRIOR TO OR

DURING THE RECORDING JUST BY THE

ELECT FULL FINGERPRINT OF THESE

NEURON.

THEY HAVE A STEREO ELECTRICAL

FINGERPRINT WITH A FIRE ACTION

POTENTIAL WITHOUT ANY DELAY.

THERE'S LOW POTENTIALISTIC WHICH

IS NO EVIDENT IN OTHER FORMS OF THE

NEURONS.

WE USE EXTRACELLULAR

ELECTRODES -- IN THE PRESENCE OF

CNQX TO BLOCK GLUTAMATE

TRANSMISSION AND THESE CURRENTS

ARE ENTIRELY BLOCKED BY GABA A

RECEPTOR ANTAGONISTS.

OKAY.

SO ONE OF THE FIRST THINGS WE

DID WAS WE JUST LOOKED AT THE

BASAL PARAMETERS FOLLOWING

STRESS.

WHAT WE FOUND IS THAT 30 MINUTE

RESTRAINT STRESS DIDN'T DO VERY

MUCH TO EITHER THE INPUT

RESISTANCE, THE PULSE RATIO, THE

FREQUENCY OF SPONTANEOUS IPST'S

OR THE AMPLITUDE OF SPONTANEOUS

ACTIVITY.

STRESS ITSELF DOESN'T APPEAR TO

BE CHANGING USING WHOLE CELL

RECORDING THE BASAL PROPERTIES

OF THE SYNAPSES ON TO THESE

CELLS.

BUT STRESS DOES ALTER HOW GABA

WORKS.

AND WE STARTED TO THINK ABOUT

GABA A LITTLE BIT DIFFERENTLY.

WE THNT THINK ABOUT RELEASE

PROBABILITY OR THE NUMBER OF

SYNAPSES OR HOW MANY GABA

RECEPTORS WERE THERE.

WE STARTED THINKING ABOUT GABA

JUST FROM FIRST PRINCIPLES OF

WHAT GABA ACTUALLY DOES.

WE KNOW THAT GABA'S INHIBITORY

BECAUSE IN THE ANIMAL,

CONCENTRATIONS IN THE CELLS ARE

VERY LOW.

WHEN GABA BINDS TO ITS GABA A

RECEPTORS, CHLORIDE TENDS TO

FLOW DOWN THE GRADIENT WHEN THE

CELL IS AT REST AND HYPER

POLARIZES ITSELF.

THE REASON THE GREAT YUBT IS LOW

BECAUSE MOST NEURONS IS A

TRANSPORTER CALLED KC2 WHICH IS

A POTASSIUM CHLORIDE

COTRANSPORTER WHICH PUMPS

CHLORIDE OUT OF THE CELL.

AND SO WE ASKED A SIMPLE

QUESTION WE SAID WELL WHAT IF

STRESS ALTERS THE FUNCTION OF

THIS TRANSPORTER.

EITHER CHANGES THE EXPRESSION OF

THE TRANSPORTER OR CHANGES THE

WAY THE TRANSPORTER WORKS.

AND IF IT DID THAT, CHLORIDE

WOULD BUILD UP IN THE CELL AND

MAO WHEN THE GABA A RECEPTOR IS

ACTIVATED UNDER SOME CONDITIONS,

CHLORIDE WOULD FLOW OUT OF THE

CELL AND PERHAPS LEAD TO

EXCITATION.

SO THIS IDEA HAS BEEN EXPLORED

EXTENSIVELY IN THE EPILEPSY

LITERATURE, IN THE DEVELOPMENTAL

LITERATURE AND IN THE ADULT

NERVOUS SYSTEM, IT'S AN IDEA

THAT'S BEEN DEVELOPED BY MIKE

SALTER LOOKING AT CHANGES IN KC2

FOLLOWING PERIPHERAL INDUCTION

OF NEUROPATHIC PAIN.

AND SO WE ASK WELL ARE THERE

PHYSIOLOGICAL SITUATIONS WHERE

THIS MIGHT BECOME IMPORTANT WITH

AN OBVIOUS THOUGHT ABOUT

DISTRESS AXIS.

SO THIS IS THE HYPOTHESES

THAT -- WILL CONTRIBUTE TO THIS

INHIBITION AND INCREASED

EXCITABILITY OF THESE NERVESZ

SECRETORY CELLS FOLLOWING ACUTE

STRESS.

ONE OF THE FIRST THINGS WE DID

WAS DID A SIMPLE EXPERIMENT

WHERE WE JUST HELD THE CELL AT

DIFFERENT MEMBRANE PO TESTIFIED

AND E -- POTENTIALS SO THESE ARE

IPSC'S THE REVERSE POTENTIAL IS

ABOUT HERE IN THESE CELLS.

ABOUT MINUS 75 -- OR SO.

WE STRESS THE ANIMAL, DID THE

SAME EXPERIENCE AGAIN AND WHAT

WE FOUND IS THE DEPOLARIZING

SHIFT.

SO THESE ARE EXPERIMENTS THAT

ARE DONE WITH GRAM AWACIDE -- SO

THAT NOW AT REST GABA IS NOT

INHIBITORY UNDER SOME CONDITIONS

IT'S EXCITATORY.

WHAT ARE THE CONSEQUENCES OF

THIS POOR CHLORIDE.

WE DID THIS EXPERIMENT

DIFFERENTLY.

WE SAID OKAY IF KC2 THIS

TRANSPORTER IS WHAT'S REGULATING

CHLORIDE, WHAT HAPPENS IF WE

JUST FARM COLLAGELY INHIBIT KC2.

WHEN WE DO THAT, WE SEE A

DEPOLARIZING SHIFT IN E GABA.

THESE ARE WHOLE CELL RECORDINGS

AND IT WORKS JUST AS WELL IN

WHOLE CELL ALTHOUGH YOU'LL

NOTICE THE SHIFT IS NOT AS

ROBUST.

AND THEN WE DID AN EXPERIMENT IN

WHICH WE INJECTED IT INTO THE

PVN AND ASKED IS THAT SUFFICIENT

TO LAUNCH THIS REST RESPONSE.

JUST WALKING KC2 IN VIVO

INCREASES THE RELEASE OF

CORTICOSTEROIDS IN THE

PERIPHERAL FIBRILLATION.

SO IT LOOKS LIKE THIS

TRANSPORTER IS PRETTY IMPORTANT.

WE DID A CURSORY EXAMINATION OF

WHAT'S GOING ON AT THE PROTEIN

LEVEL.

WE FOUND THAT THERE'S NO CHANGE

IN OVERALL KC2 PROTEIN SO THERE

ARE TWO BANDS BECAUSE PROTEIN IS

REQUIRED TO BE DIMERIZED,

TRANSPORTED AND ININTEURTD THE

MEMBRANE.

WE FOUND NO CHANGE IN TOTAL

PROTEIN OR THE DIMER RATIO OF

KC2.

SO ONE OF THE OTHER THINGS WE

DID WAS WE DID SYNAPTIC

STIMULATION OF GABA INPUTS TO

THESE CELLS.

WHILE WE'RE RECORDING THE CELLS

AND THE CELL ATTACHED CONFIGURE

CONFIGURATION.

WHAT WE FOUND IS UNDER LOW

FREQUENCY WITHIN HERTZ OR FIVE

HERTZ STIMULATION WE FOUND NO

CHANGE IN THE FIRING RANGE OF

THESE CELLS.

WHEN YOU START TO PUSH THE

SYSTEM GO TO 10 HERTZ OR 20

HERTZ STIMULATION YOU START TO

UNMAUNMASK INCREASES WHICH IS A

CONTRAST WHAT YOU SEE UNDER

CONTROLLED CONDITIONS WHICH IS

AN INHIBITION OF FIRING OF THESE

CELLS.

SO OF COURSE WHEN YOU PUBLISH

WORK FROM YOUR LAB, YOU'RE LIKE

WELL NOBODY ELSE IS REALLY

REPORTING THIS, WHAT'S GOING ON.

SO WE WERE VERY PLEASED A COUPLE

YEARS LATER TO SEE THIS PAPER

OUT OF JAMIE MAC WIRE'S LAB

WHERE THEY SHOWED IF YOU

ACTIVATE EXTRA SYNAPTIC GABA A

RECEPTORS FOLLOWING -- YOU GET A

DECREASE IN FIRING BUT IN

STRESSED ANIMALS YOU SEE AN INIS

CREASE IN FIRING OF THESE SAME

CELLS.

SO SUGGESTING THAT ACTUALLY

THERE'S A ROBUST CHANGE LIKE

GABA WHICH IS DRIVING INCREASED

ACTIVITY OF THESE CELLS.

OKAY.

SO JUST TO BRIEFLY SUMMARIZE THE

FIRST PART OF THE TALK.

SO STRESS IS INDUCED TO DOWN

REGULATION OF KC2 WHICH RESULTED

IN THE COLLAPSE OF THE

TRANSMEMBRANE CHLORIDE GREAT

YENT AND UNMAP THESE GATHERING

INDUCED EXCITATIONS.

JAMIE LAB HAS SHOWN THE --

DEPHOSPHORYLATION OF THE PROTEIN

AND THESE RESULTS ARE ENTIRELY

CONSISTENT WITH OUR ALWAYS

INFORMATION OF POST TRANSLATION

MODIFICATIONS IN THE

TRANSPORTER.

SO FOR THE SECOND PART OF THE

TALK I'LL SWITCH GEARS AND TALK

ABOUT THE WORK JACKIE UPTION --

JACKIE'S BEEN DOING.

HOW DOES THIS SYSTEM ADAPT.

THOSE OF YOU THINKING ABOUT

SYNAPSES IS LEARNING ADAPTATION

IN MOST CIRCUITS BOILS DOWN TO

UNDERSTANDING THESE PHENOMENA,

LONG TERM POTENTIATION.

THEY ARE WIDELY TO BE IMPORTANT

SUBSTRATES FOR EXPERIENCE

DEPENDENT LEARNING IN

NEUROCIRCUIT.

AT GABA SYNAPSES RETROGRADE

SIGNALS ARE THOUGHT TO BE PRETTY

IMPORTANT PLASTICITY MOLECULES,

THE NUMBER OF LABS, HAS SHOWN

THAT THINGS LIKE NITRIC OXIDE

ARE IMPORTANT FOR LTP AT GABA

SYNAPSES.

AND LABS LIKE -- HAVE SHOWN THAT

ENDO CAB NOIDS ARE IMPORTANT.

THIS IS A FIGURE OUT OF --

SHOWING IN THE HIP CA HIPPOCAMPUS YOU

CAN GET INHIBITORY LTD -- WHICH

CAN BE ENTIRELY BLOCKED IF YOU

PRETREAT SLICES WITH --

ANTAGONIST OF RECEPTORS.

THEY PROPOSED A MODEL WHICH

LOOKS SOMETHING LIKE THIS THAT

YOU HAVE THE HETERO SYNAPTIC

RECRUITMENT OF ENDO CAB NOIDS

WHICH PRODUCES -- TO DECREASE

THE RELEASE OF GABA.

THIS IS RELATIVELY WELL

ESTABLISHED PHENOMENA ABOUT HOW

ENDO CAB NOIDS SIGNAL.

SO WE STARTED TO THINK ABOUT

RETRO GREAT SIGNALS AS

PLASTICITY SIGNALS AT THESE GABA

SYNAPSES.

AND WE DID SOME, JUST SOME

PRETTY SIMPLE EXPERIMENTS

SHOWING THAT IF WE USE PROTOCOLS

THAT INDOING ENDO CAB NOID

RELEASE LIKE THE POST SIGNAL

PARTICULAR CELL WE CAN GET

WHAT'S DESCRIBED PREVIOUSLY AT

MANY SYNAPSES THE

DEPOLARRATION -- YOU CAN DO I

OVER AND OVER IN THIS CELL AND

YOU CAN DO IT IN MANY CELLS DSI

IS PRESENT.

IT'S ROBUST.

SO THIS IS JUST THAT ENDO CAB

NOIDS ARE MADE IN THE SYSTEM AND

THEY CAN ACT AS RETRO GATED

SIGNALS AT LEAST TRANSIENTLY IN

THESE CASES.

ENDO CAB NOIDS AS WE ALL KNOW IS

NOT ONLY THE RETROGRADE SIGNALS.

THERE'S A OATH OF OTHER SIGNALS.

-- SUMMARIZED THESE NICELY IN A

REVIEW IN NEURONS SHOWING NOT

ONLY LIPID DERIVED SIGNALS THE

NITRIC OXIDES I JUST SHOWED A

SECOND AGO, GASES LIKE NITRIC

OXIDE BUT A ALSO PEPTIDES LIKE

OPIOID PEPTIDES, OTHER

NEUROPEPOIDS CAN ALSO PLAN AN

IMPORTANT ROLE AS RETROGRADE

SIGNALS.

I WANT YOU TO HOLD ON TO THIS

THOUGHT BECAUSE WE'LL COME BACK

TO IT.

OKAY.

SO WE TRIED TO THINK ABOUT HOW

WE MIGHT INDUCE PLASTICITY IN

THIS SYSTEM.

AND WE USED A NUMBER OF

DIFFERENT PROTOCOLS TO TRY TO

INDUCE PLASTICITY AND I'M JUST

GOING TO SHOW THE RAW DATA BELOW

HERE.

YOU CAN INDUCE ROBUST IPSC'S AND

WE TRIED MANY MANY PROTOCOLS --

REGARDLESS OF WHAT WE TRIED IN

THIS SYSTEM, WE FAILED TO INDUCE

ANY FORM OF TOXICITY IN THIS

SYSTEM, OKAY.

SO THEN WE ASKED WELL WHAT

HAPPENS WHEN WE BEGIN TO STRESS

ANIMALS AND START TO ASK THESE

SIMILAR TYPES OF QUESTIONS.

IF WE INTERROGATE THE SYNAPSES

USING A PROTOCOL LIKE THIS AFTER

STRESSING AN ANIMAL, DOES THE

SYSTEM BEHAVE DIFFERENTLY.

OKAY.

SO WE KNOW THAT A COUPLE THINGS

DETERMINE ACTIVITY IN THE

SYSTEM.

I TALKED ABOUT THE SYNAPSES AND

ALSO TALKED ABOUT CIRCULATING

LEVELS OF CORTICOSTEROIDS, OKAY.

AND SO WE ASKED AN ASSOCIATIVE

SIGNAL DURING OWE AFTER STRESS

THAT IMPACTS SUBSEQUENT

PLASTICITY ON THE GABA SYNAPSES.

OF COURSE IT'S THOUGHT TO BE A

VERY IMPORTANT SIGNAL FOR THE

INDUCTION OF PLASTICITY AT

GLUTAMATE SYNAPSES IN THE

HIPPOCAMPUS IN THE TR STRIATUM AND

THOUGHT TO PLAY A ROLE IN THE --

INCREASING THE RELEASE OF ENDO

CAB NOIDS IN THE HIPPOCAMPUS AS

WELL.

SO WE ASKED WHETHER IT COULD

PLAY SIMILAR ROLES HERE AND

ARTICLE THE INDUCTION OF

PLASTICITY FOLLOWING STRESS.

OKAY.

AND SO I'LL JUST WALK YOU

THROUGH THE PROTOCOL.

THIS TIME LINE SHOWS UP IN ONE

FORM OR ANOTHER IN A NUMBER OF

SLIDES.

SO WE PERFORMED EXPERIMENTS FROM

NAIVE ANIMALS.

SO SLICES OF NAIVE ANIMALS OR WE

STRESS THE ANIMALS FOR 30

MINUTES USING IMMOBILIZATION

THREAT, IMMOBILIZATION STRESS

AND THEN SACRIFICE AT DIFFERENT

TIME POINTS, EITHER IMMEDIATELY

AFTER OR 30 MINUTES AFTER THE

STRESS HAD BEEN TERMINATED OR 60

MINUTES OR 90 MINUTES, OKAY.

SO I SHOULD SAY THESE ARE ALL

MALES, RATS, THEY'RE AGED UP TO

21 TO ABOUT T40.

OKAY.

SO WE DID THE SAME PAIRING

PROTOCOLS IN NAIVE ANIMALS THAT

I SHOWED YOU THIS ALREADY.

IF YOU STRESS AND YOU WAIT 30

MINUTES, YOU START TO SEE THIS

DEPRESSION.

IF YOU WAIT 60 MINUTES, THE

DEPRESSION STARTS TO EVOLVE AND

GETS MORE ROBUST.

AND IF YOU WAIT 90 MINUTES, YOU

GET WHAT IS A BONE FIDE LONG

LASTING ROBUST LTD SYNAPSES,

OKAY.

SO WHAT IS THE, HOW DOES THE LTD

MANIFEST, WHAT ARE THE

PROPERTIES OF THE LTD.

WE LOOK AT THIS A LITTLE BIT

MORE CAREFULLY AND ONE OF THE

FIRST THINGS WE NOTED WAS THAT

THIS LTD IS ASSOCIATED WITH AN

INCREASE IN THE PARAPULSE RATIO

THAT IS SHOWN HERE IN THE LTD IN

STRESS SLICES THEY CHANGE IN THE

NAIVE SLICES AND ALSO A CHANGE

IN THE CB SQUARED CONSISTENT

WITH THE INCREASE IN THE RELEASE

PROBABILITY OF GABA.

SO UNLIKE ENDO CAB NOID LTD HAS

BEEN DESCRIBED IT WAS ASSOCIATED

WITH THE DEGREASE IN SPONTANEOUS

GABA IPSCS.

THESE ARE TRIESES FROM NAIVE

ANIMAL BEFORE PAIRING AND 30

MINUTES AFTER PAIRING.

THERE'S NO CHANGE IN THE

FREQUENCY OF AMPLITUDE.

IN STRESSED ANIMALS HOWEVER IF

YOU STRESS AND WAIT 90 MINUTES

AND YOU PAIR, THERE'S A ROBUST

INCREASE.

IT LOOKS LIKE THERE'S A DECREASE

IN THE RELEASE PROBABILITY OF

MANY GABA SYNAPSES ON TO THESE

NEURONS.

SO THAT DATA IS JUST SUMMARIZED

RIGHT HERE SHOWING A DECREASE IN

FREQUENCY OF THE 135U7 SPONTANEOUS BUT

NO CHANGE IN THE AMPLITUDE.

SO WE ASKED WELL, ARE

CORTICOSTEROIDS THE SIGNALS FOR

THE INDUCTION OF THE CELL PD.

IS GLUCOCORTICOID ACTIVATION

NECESSARY.

WE DID THIS EXPERIMENT IN WHICH

WE EITHER INJECTED -- OR THE

ANTAGONIST OF CYTOSOLIC COURT

COAL RECEPTORS PRIOR TO THE

STRESS.

SUBJECTED THE ANIMAL TO STRESS

AND THEN PREPARED SLICES 90

MINUTES LATER THAT YOU SEE IN

THE LTD.

SO IN THE VEHICLE CONTROLS WE

SEE VERY ROBUST LTD IN THIS

SYSTEM.

FOLLOWING -- BLOCKED

GLUCOCORTICOID RECEPTORS, WE

COMPLETELY WIPED OUT THE LCD'S.

SO IT APPEARS THAT

GLUCOCORTICOID RECEPTOR

ACTIVATION IS NECESSARY IN VIVO

FOR THE INDUCTION OF THIS LTD.

SO WE ASKED WHETHER WE COULD

JUST INDUCE THE LTD JUST BY

TAKING THE SLICES FROM NAIVE

ANIMALS AND INCUBATING THEM WITH

CORTICOSTEROIDS.

SO BASICALLY DOES THIS MIMIC THE

EFFECTS OF CIRCULATING

GLUCOCORTICOID STEROIDS.

HERE'S A TIME LINE, REKURVETION

INCUBATE -- RECOVER, ARE

INCUBATE AND THEN REGARD 30

MINUTES LATER.

AND UNDER THESE CASES ALSO WE

SEE VERY ROBUST LTD'S.

VERY VERY SIMILAR TO WHAT WE SAW

90 MINUTES AFTER STRESS.

OKAY.

AND SO WE STARTED TO THINK ABOUT

THE IDEA THAT CORTICOSTEROIDS

WERE ACTING ON THESE NEUROSECRET

TREE -- NEUROSECRETORY CELLS

PRODUCING GABA RELEASE.

WHAT'S THE TRIGGER FOR LTD.

FOR END OWE CAB NOIDS ARE VERY

VERY IMPORTANT SO THE SCHEME

LOOKS SOMETHING LIKE THIS.

PERHAPS COUPLED WITH CALCIUM

INFLUX TO CALCIUM CHANNELS

CAUSES THE PRODUCTION OF

SOMETHING THE CAB NOID, SOME

OTHER TYPE OF PEPTIDE THAT CAN

ACT AS A RETROGRADE SIGNAL.

WE TESTED THIS AND USED

ANTAGONISTS -- RECEPTORS AND

USING THE MGLUR5 WE NOTED WE

COULD COMPLETELY WIPE OUT LTD'S

IN OUR SYSTEM.

IT LOOKS LIKE MGLUR IS VERY

IMPORTANT.

SO THE MGLUR ACTIVATION IS NOT

SUFFICIENT BY ITSELF TO GENERATE

LTD, OKAY.

SO NORMALLY IF YOU JUST APPLY

MGLUR AGONIST, DHPG THE BROAD

SPECTRUM AGONIST WE GET A LITTLE

BIT OF POTENTIATION, A VERY HIGH

DOSE.

IF WE HAVE EVIDENCE IN SLICES

THAT HAVE INCUBATED WITH

CORTICOID STEROID WE SEE A

LITTLE BIT OF DEPRESSION BUT NOT

VERY MUCH DEPRESSION LIKE WE SAW

WHEN WE INDUCED LTD WITH A

PAIRING PROTOCOL OARK.

IF WE DEPOLARIZE THE CELL AND IF

WE TREAT THE SLICES OF CORTICOID

STEROID AND DEPOLARIZE THE

CELLS, WE SEE THIS VERY ROBUST

DEPRESSION, WHICH IS ACCOMPANIED

BY AN INCREASE IN PARAPULSE

RATIO JUST LIKE WE OBSERVED

DURING THE INDUCTION OF ACTIVITY

DEPENDENT LTD.

SO IT LOOKS LIKE DEPOLARIZATION,

CORTICOID STEROID MGLUR

ACTIVATION ALTOGETHER CONVERGE

TO GIVE US THIS HE RHO BUS LONG

TERM DEPRESSION AT THESE

SYNAPSES.

WE HAVE GDP DATA S AND FOUND WE

COULD COMPLETELY BLOCK THE LTD.

IF IT'S CALCIUM DEPENDENT WE

COULD LOAD OURSELF WITH BAPTA

CALCIUM AND SHOWED IT COULD

COMPLETELY BLOCK LTD.

WE HAVE A SCHEME THAT BEGINS TO

DEVELOP SOMETHING LIKE THIS.

YOU NEED ACTIVATION OF POST SNAB

SYNAPTIC MGLUR5 -- WHICH

LIBERATES THE RETROGRADE SIGNAL.

SO WE ASKED IS THE RETROGRADE

SIGNAL INVOLVED SO WE DID THE

EXPERIMENT THAT EVERYBODY WHO IS

LOOKING AT IO -- AND THAT IS

INCUBATE OUR SLICES WITH THREE

MICRMY MOLAR AND SO THIS IS NO

DIFFERENT THAN LTD WE OBSERVED

IN SLICES THAT HAVE BEEN TREATED

LIKE THIS.

IT APPEARS NOT TO BE CD1

RECEPTOR DEPENDENT.

A COUPLE LABS HAVE RECENTLY

SHOWN ENDO CAB NOIDS CAN ACT

THROUGH -- RECEPTORS ALTHOUGH WE

DIDN'T THINK THIS WAS A LIKELY

CANDIDATE.

WITH A HE DECIDED TO TEST IT

ANYWAY JUST TO MAKE SURE.

AND SO IF YOU INCUBATE WITH 10

MICRO MOLAR TO BLOCK THESE

RECEPTORS LIKE OTHER PEOPLE HAVE

SHOWN, WE SAW ABSOLUTELY NO

EFFECT IN THE INDUCTION OF LTD.

SO IT LOOKS LIKE IF WE HAVE A

RETROGRADE SIGNAL WHICH IT LOOKS

LIKE WE DID WHICH IS CALCIUM

DEPENDENT, IT APPEARS NOT TO BE

AN ENDO CAB NOID THAT'S ACTING

THROUGH A CB1 RECEPTOR.

WHEN WE GOT TO THIS POINT I WAS

REALLY PUZZLED.

I REALLY WANTED JACKIE TO TRY

CB2 ANTAGONISTS, TRY THIS AND

TRY THAT.

SHE WAS REALLY INSISTENT THAT IT

WAS THE GOING TO BE END OW ENDO CAB

NOID.

I DON'T KNOW WHY.

SHE DECIDED TO DO AN EXPERIMENT

TO PROVE ME WRONG.

THEY RELISH THE IDEA TO PROVE

THEIR SUPERVISORS WRONG.

SO JACKIE LOOKED INTO LITERATURE

A LITTLE BIT AND WE KNEW THIS

WAS PRESENT ALREADY IS THAT

THESE NEURONS THE CRH NEURONS AS

WELL AS THE BASAL -- OX OXYTOCIN

NEURON -- DOWN THE AX SU AXON AND

RELEASE THE TERMINALS IN THE

DENY DIETLE -- PERHAPS AUTO

CRINE SIGNALS TO SCULPTED THE

SYNAPTIC INPUTS TO THESE CELLS.

WE KNOW THESE ARE RELEASED IN A

MANNER THAT ARE SIMILAR TO

SYNAPTIC, IN OTHER WORDS THEY --

RETEENPROTEINS FOR EXOCYTOSIS.

JACKIE DID THE EXPERIMENT WHERE

SHE INCLUDED THE -- TOXIN C,

TRIED THE PAIRING PROTOCOL AND

SHOWED IT COULD COMPLETELY WIPE

OUT LTD, OKAY MUCH SO IT LOOKS

LIKE SOME FUSION EVENT IS

REQUIRED FOR THE INDUCTION OF

LTD.

WE'RE NOW LOOKING AT MORE

CLASSICAL TRANSMITTER SIGNALS

THAT ARE NOT LIPIDS OR GASES,

OKAY.

SO WHAT IS THAT SIGNAL.

SO THE LITERATURE OVER THE LAST

20 TO 30 YEARS STARTING WITH

WORK BY THOMAS HOT FIELD, A

SWEDISH ANATOMIST SHOWED THESE

ALSO CONTAIN OTHER SIGNALS THAT

SHOW UP IN A STRESS DEPENDENT

FASHION.

SO ONE OF THOSE SIGNALS IS THE

OPIOID PEPTIDE CAP LYNN.

WE DID -- SO THESE ARE CRH

NEURONS AND THESE ARE KEF LYNN

NEURONS LABELED IN RED.

THESE ARE NAIVE SLICES WITH YOU

THE ANIMALS HAVE BEEN TREATED

WITH -- TO ALLOW US TO VISUALIZE

THE PEPTIDE CONTENT A LITTLE BIT

MORE READILY.

WHEN WE DO THIS EXPERIENCE WHAT

WE FOUND IS THAT THERE INDEAD

IS -- NEUL NOTIC YOU'LL NOTICE -- WE'RE

DOING EXPERIMENTS RIGHT NOW WITH

STRESSED ANIMALS TO SEE IF

THERE'S AN INCREASE IN THE

EXPRESSION OF -- SOME OF THEM--

IF YOU THINK BACK TO THE SLIDE I

SHOWED YOU EARLIER, ALMOST ALL

OF THE NEURONS ARE ACTIVATED

THROUGH STRESS.

IT LOOKS LIKE THERE'S ENKEPHALIN

EXPRESSION IN THIS SYSTEM.

A LOT OF PEOPLE HAVE LOOKED WHAT

ENDOGENOUS DOES -- DIFFERENT

TYPES OF BEHAVIOR.

THERE ARE A FEW EXAMPLES OF

ACTIONS OF ENDOGENOUS OPIOIDS ON

GLUTAMATE TRANSMISSIONS BUT THEY

ARE REMARKABLY FEW EXAMPLES OF

EFFECTIVE ENDOGENOUS OPIOIDS ON

GABA TRANSITIONS.

SO WE DID AN EXPERIMENT AND WE

JUST INFUSED 500 -- WHICH IS A

NEW DELTA AGONIST ON TO THESE

SLICES AND SHOWED THAT WE COULD

SEE DEPRESSION.

THIS HAS BEEN REPORTED BY OTHER

PEOPLE IN DIFFERENT SYSTEMS

WHICH IS ACCOMPANIED BY AN

INCREASE IN THE PARAPULSE RATIO.

WHAT WE FOUND IS THAT WE DID AN

EXPERIMENT WHICH WE TRIED TO

BLOCK THE LCD.

A FEW INQUEUE BAIT SLICES

WITH -- COULD COMPLETELY WIPE

OUT THE LTD'S SUGGESTING IT'S AN

OPIOID SIGNAL.

IF WE DO IT MORE CAREFULLY WITH

A NEW RECEPTOR ANTAGONIST, CTAP,

WE CAN ALSO COMPLETELY WIPE OUT

THE SIGNALS.

SO IT LOOKS LIKE WE'RE GETTING

RELEASE OF AN OPIOID PEPTIDE AT

THIS POINT WE'D SAY IT'S MOST

LIKELY ENKEPHALIN WE HAVEN'T

NAILED THIS DOWN 100% I THINK

WHICH IS ACTING ON A PRESYNAPTIC

OPIOID RECEPTOR TO DECREASE THE

GABA.

IF THIS IS THE CASE, IF IS

ENKEPHALIN RELEASE ACTING ON --

WE SHOULD BE ABLE TO ACTIVATE

THE RECEPTOR WITH AN ENDOGENOUS

LYINGENELIGAND AND WE CAN OCCLUDE THE

ACTIVITY.

THAT'S THE EXPERIMENT WE DID

NEXT SO WE WE APPLIED ONE MICRO

MOLAR -- SUGGESTING THAT THESE

TWO PROTOCOLS SHARE A SIMILAR

MECHANISM THAT INDEED WE ARE

GETTING RELEASE OF ENKEPHALIN

WHICH IS ACTING ON A PRESYNAPTIC

OPIOID RECEPTOR.

SO WHAT I HOPE I'VE BEEN ABLE TO

SHOW YOU QUICKLY I THINK IS THAT

THESE STRESS CIRCUITS LAUNCH AND

ADAPT IANDADAPT INANDADAPT IN UNIQUE WAYS.

I TALKED ABOUT CORTICOIDS THAT

CAN ACT ON MGLUR5 TO CAUSE THE

RELEASE OF AN ENKEPHALIN OPIOID

PEPTIDE WHICH ACT AS A

PRESYNAPTIC OPIOID RECEPTOR TO

DECREAS THE PROBABILITY OF

RELEASE AT GABA SYNAPSES.

UNLIKE ENDO CAB NOIDS WHICH SEEM

TO TARGET -- THAT ARE ACTIVE,

THESE OPIOIDS SEEM TO TARGET

MOST OF THE GABA SYNAPSES ON

THESE NEURONS.

SO THERE'S A LITTLE DIFFERENT

MECHANISM OF ACTION HERE.

SO IT DOESN'T REQUIRE

PRESYNAPTIC DEPOLARIZATION TO

TARGET THE SNAPS.

AT THE BEGINNING OF THE TALK I

DIDN'T TALK ABOUT THE ALPHA --

DRIVEN DOWN REGULATION OF KC2

WHICH RENDERS THIS GABA

INHIBITION EFFECTIVELY MUTE,

OKAY.

SO IT ALLOWS THIS SYSTEM TO

LAUNCH ITSELF TO BE DISINHIBITED

AND THEN ONCE THE SYSTEM IS

4R5U67D AND YOU TRY TO RECRUIT

THESE GABA SYNAPSES AT HIGHER

FREQUENCIES, THESE CELLS

ACTUALLY BECOME EXCITED.

SO WE WOULD SAY THAT AFTER

STRESS THE SYSTEM BECOMES

CONDITIONALLY EXCITATORY AND

THAT PLASTICITY OF GABA SNAPS IS

ACTUALLY HE EVER -- SYNAPSE IS

NOT DISSIMILAR TO THE FUNCTION

SUREFUNCTION -- I ALSO THINK THIS

SYSTEM KIND OF I THINK THESE

OBSERVATIONS BEGIN TO ANSWER

THESE TWO QUESTIONS WHAT TURNS

IT ON AND HOW DOES THE HP ADAPT.

I SHOULD SAY THESE ARE VERY,

THESE ARE ELECTROPHYSIOLOGICAL

OBSERVATIONS SO WHEN WE THINK

WHAT IT TURNS IT ON, WE THINK

ABOUT THE IMMEDIATE SWITCH THAT

TURNS IT ON.

MOST PEOPLE WHO HAVE THOUGHT

ABOUT THESE SEIZE TUMS HAVE

THOUGHT ABOUT G TRANSCRIPTION

AND THE EXPRESSION OF PROTEINS

AND WE HAVEN'T THOUGHT VERY MUCH

ABOUT THIS.

SO WE THINK, WE'RE JUST DEALING

WITH INITIAL IGNITION SWITCHES

AND VERY RAPID SYNAPTIC

ADAPTATIONS TO STRESS.

I THINK THESE WILL GO HAND IN

HAND WITH A LOT OF THE

PREVIOUSLY DESCRIBED CHANGES IN

MRNA PROTEIN THAT ALLOW THE

SYSTEM TO BE MODIFIED AND TO

ADAPT TO SUBSEQUENT STRESSORS.

SO I THINK THAT WHAT THESE

OBSERVATIONS BEGIN TO TELL US IS

PAINTED A LITTLE BIGGER PICTURE

ABOUT WHAT HAPPENS IN THE BRAIN

AS IT HAS TO MAKE THIS JOURNEY

FROM BIRTH TO DEATH.

AND SO AT ANY GIVEN POINT IN

THIS TRAJECTORY THE BRAIN CAN

EXIST IN A NUMBER OF DIFFERENT

STATES AND SO I THINK WE'VE USED

STRESS TO MODEL A PARTICULAR

STATE IN THE BRAIN ESPECIALLY

FOR THE HYPOTHALAMUS.

AND THAT THE LAUNCH OF THIS

STRESS STATE, WHAT GOES ON

WITHIN THE STRESS STATE AND THE

FIRM NATION OTERMINATION OF THE STRESS STA
TE

ALLOW THE SYSTEM TO LEARN VERY

COLLOQUIALLY.

SO LEARN IN A WAY -- VERY

UNIQUELY.

SO TO LEARN IN WAY -- THIS

LEARNING THEN ALLOWS THE SYSTEM

LATER ON WHEN IT'S PRESENTED

WITH THE SAME PROBLEM, THE SAME

CHALLENGE TO REMEMBER WHAT

HAPPENED AND LAUNCH A RESPONSE A

LITTLE BIT MORE EFFECTIVELY.

SO THERE ARE YOU NEED TO FIND WAYS

TO ERASE THIS TOXICITY BECAUSE

YOU DOANT WITH A THE SYSTEM TO

BE ABLE TO GET BACK TO BASELINE

EVENTUALLY TO REPAIR ITSELF FOR

THE NEXT STRESS.

YOU DON'T WANT THIS TO BE

PERMANENTLY ENCODEDMENT WHAT ARE

THE BEHAVIORAL MECHANISMS WHICH

ALLOW THIS SYSTEM TO RESET AND

PREPARE FOR THE NEXT CHALLENGE.

DOES IT REMEMBER THIS STIMULUS

FOR THE NEXT CHALLENGE.

DOES IT RESPOND MORE QUICKLY,

MORE ROBUSTLY, WHAT IS THE

NATURE OF THAT CHALLENGE.

THESE ARE THE QUESTIONS THAT MY

LAB IS JUST BEGINNING TO PERSEAF

BUT I THINK NOW -- PERCEIVE BUT

NOW WE HAVE THE TOOLS AND THE

RULES, THE SYNAPTIC RULES THAT

WILL ALLOW US TO GET TO THESE

CHALLENGES IN A MORE PRECISE

FASHION.

LET ME CLOSE BY THANKING OF

COURSE SARA AND JACKIE FOR

REALLY A TREMENDOUS AMOUNT OF

WORK AND REALLY PROVIDING SOME

INSIGHT HOW THIS SYSTEM WORKS.

AND LET ME JUST THANK CIA CHAR

WHICH FUNDS OUR SYSTEM AND FUNDS

MY SALARY.

THANKS TO CHRIS FOR THE

INVITATION.

THANK YOU.

[APPLAUSE]

>> THAT'S A REALLY GREAT TALK

AND ANSWERS A LOT OF QUESTIONS

I'VE HAD ABOUT HOW STRESS WORKS

SORT OF THE MOLECULAR CELLULAR

LEVEL.

AND I'VE ALWAYS WONDERED ABOUT

THIS SORT OF SWAPPING OUT OF

STRESS FOR GLUCOCOUR GLUCOCORTICOID WHICH

YOU CLAIM CAN BE DONE YET IN ALL

OF YOUR DRAWINGS YOU'VE HAD NO

PICTURES OF THE GLUCOCORTICOID

RECEPTOR AND THROUGH THAT THESE

CHANGES MIGHT TAKE PLACE.

SO DO WE NEED THAT IN THE

PICTURE.

AND THEN ALSO AFTER REPEATED

STRESSORS, ARE THE CHEENGS IN

THE GLUCOCORTICOID RECEPTOR

SHOULD BE TAKEN INTO RECEPTION.

>> I GUESS I'M GUILTY OF

DOING -- TREAT THE SYNAPSES LIKE

A BLACK BOX.

I REFER TIGHT AS  TO IT AS A BLACK BALK.

WE -- BLACK BOX.

WE HAVE NOT TALKED ABOUT WHAT

HAPPENS DOWN STREAM FROM THAT

RECEPTOR.

YOU'RE ABSOLUTELY RIGHT, THE

RECEPTOR NEEDS TO BE IN THAT

PICTURE.

OTHERS IN THIS, OTHERS WORKING

IN THIS FIELD HAVE SHOWN THAT

THERE MAY BE A POTENTIAL SECOND

GLUCOCORTICOID RECEPTOR WHICH IS

MEMBRANE BOUND AND INTENSIVE TO

RU4 AND 6.

MAYBE.

BUT THAT'S NOT, THOSE AREN'T THE

EFFECTS THAT WE'RE LOOKING AT.

THESE ARE DEFINITELY THE CLASSIC

CYTOSOLIC GLUCOCORTICOID

RECEPTOR.

WE HAVEN'T THOUGHT VERY MUCH

ABOUT WHAT THIS RECEPTOR DOES

AND WHY IT TAKES THAT LONG,

RIGHT.

SO I MEAN THE STRESS IN 90

MINUTES OR HAVE YOU TO INCUBATE

WITH COURT CORED STEROIDS FOR AT

LEAST 60 MINUTES.

CLEARLY THERE ARE OTHER THINGS

GOING ON.

I DON'T KNOW IF IT'S CAUSING A

TRAFFICKING MGLUR5 FOR THE

MEMBRANE OR SWIVING OFF

SECONDARY ELEMENTS WHICH MAY

TERMINATE IT MORE QUICKLY LIKE

RGS PROTEINS.

THESE ARE ALL KIND OF QUESTIONS

THAT ARE BOUNCING AROUND IN HER

HEADS.

THOSE ARE REALLY REALLY GREAT

QUESTIONS.

I THINK THAT IS A, FOR THIS

FIELD, FOR THIS STRESS FIELD

UNDERSTANDING WHAT THAT RECEPTOR

REALLY DOES IN THIS RELATIVELY

SHORT MINUTE TIME FRAME IS

REALLY UNEXPLORED.

PEOPLE HAVE LOOKED A LOT DOWN

STREAM AT GENE TRANSCRIPTION AND

THINGS LIKE THAT BUT I THINK

THERE ARE A LOT OF CYTOSOLIC

EFFECTS WE HAVEN'T TOUCHED YET.

I'LL PUT IT IN MY DRAWING,

THANKS.

>> [INDISCERNIBLE]

>> SORRY, I'M NOT UNDERSTANDING

YOU.

>> [INDISCERNIBLE]

>> THE SIZE OF THE BASELINE

RESPONSE IS NOT SOMETHING THAT

WE CAN CONTROL REALLY WELL.

SO THAT'S WHY WHEN WE START TO

POOL DATA, WE HAVE TO NORMALIZE

IT.

WE DON'T ACTUALLY COMPARE

BASELINE IN ONE CONDITION NAIVE

VERSUS BASELINE ABOVE THE OTHER

CONDITION STRESS.

WE HAVE NOTHING LIKE THE RATIO

FOR THESE SYNAPSES THAT WE CAN

REALLY GET AT THAT.

SO THE SIZE OF THAT IS JUST A

FUNCTION OF HOW MANY FIBERS YOU

STIMULATE IN THE RELEASE

PROBABILITY OF THOSE GIVEN

FIBERS.

WE CAN'T NECESSARILY VERY

ACTIVELY CONTROL THE NUMBER OF

FIBERS UNLESS WE GO TO A

PARARECORDING -- TYPE OF

APPROACH TO ACTIVATE SPECIFIC

INPUT.

I THINK THAT'S JUST A

PRESENTATION ARTIFACT.

>> [INDISCERNIBLE]

>> IT'S INTERESTING.

PEOPLE HAVE LOOKED AT IT A

NUMBER OF WAYSMENT ONE IS MORE

FIN AND ONE IS TO KNOCK OUT

ENKEPHALIN OR TO KNOCK OUT --

EXACTLY WHAT YOU PREDICT

HAPPENS.

WHEN YOU KNOCK IT OUT, YOU

ACTUALLY, SO YOU GET LESS GABA.

THE SYSTEM ACTUALLY LAUNCHES.

SO YOU GET AN EXACERBATED STRESS

RESPONSE.

WHAT'S REALLY REALLY INTERESTING

IS THAT WHEN YOU THEN FOLLOW THE

STRESS RESPONSE, THE STRESS

RESPONSE ACTUALLY ALSO LASTS

LONGER.

SO IT'S ALMOST LIKE, SO, DID I

SAY THAT RIGHT.

I GOT THAT BACKWARDS.

SO WHEN YOU HAVE NO OPIOID, YOU

HAVE NO GABA INHIBITION, RIGHT.

SO YOU'RE NOT INHIBITING GABA.

I'M TALKING MYSELF INTO A

CIRCULAR HERE.

YES.

YOU AMPHI THE RESPONS AMPLIFY THE RESPONSE
 OF

STRESS.

WE DON'T KNOW WHERE THAT EFFECT

IS HAPPENING.

WE HAVEN'T DONE A TARGETED

APPROACH.

SO WE'RE JUST THINKING ABOUT,

WE'VE JUST GOT SOME, WE'RE

THINKING ABOUT SPECIFICALLY

INJECTING TRYING TO KNOCK DOWN

OPIOID RECEPTORS.

WE'LL SEE WHAT HAPPENS TO

STRESS.

>> THANK YOU VERY MUCH.

>> THANKS.