>>> WELCOME TO THE WEDNESDAY

AFTERNOON LECTURE SERIES.

MY NAME IS VERONICA ALVAREZ,

INVESTIGATOR AT NIAAA.

TODAY IS REALLY MY HONOR, AND I

HAVE LEARNED TO SAY THOSE WORDS

FROM WHEN THEY ARE REALLY

REQUIRED, TO INTRODUCE THE

SPEAKER OF THE LECTURE,

DR. BERNARDO SABATINI.

BERNARDO SABATINI OBTAINED A

Ph.D. FROM THE DEPARTMENT OF

NEUROBIOLOGY AT HARVARD MEDICAL

SCHOOL, AND HE IS MD DEGREE FROM

HARVARD AND MIT JOINT PROGRAM IN

HEALTH SCIENCES AND TECHNOLOGY.

NOT SO LONG AGO, IN 1999.

THEN FOLLOWING A FAMILY

TRADITION, BERNARDO HAS

DEDICATED HIS CAREER TO RESEARCH

AND WORKING IN THE LABORATORY.

HE DID A POSTDOCTORAL FELLOWSHIP

IN THE LABORATORY OF CARLOS AT

CARL SPRING HARBOR LABORATORIES

IN NEW YORK AND AFTER THAT, HE

JOINED THE FACULTY IN THE

DEPARTMENT OF NEUROBIOLOGY AT

HARVARD MEDICAL SCHOOL IN 2001,

AND HAS REMAINED FROM SINCE

THEN.

BERNARDO HAS RECEIVED NUMEROUS

AWARDS AND HONORS DURING HIS

CAREER AND I'M GOING TO START

FOR SOME OF THE LATEST.

IN 2008,Y HE WAS NAMED AN

INVESTIGATOR OF THE HOWARD

HUGHES MEDICAL INSTITUTE, IN

2010 HE WAS NAMED PROFESSOR OF

NEUROBIOLOGY AT HARVARD MEDICAL

SCHOOL.

BEFORE THAT, FOR INSTANCE, HE

HAD RECEIVED A YOUNG

INVESTIGATOR AWARD FOR SOCIETY

OF NEUROSCIENCE IN 2008.

MACK KNIGHT TECHNOLOGY

INNOVATION AWARD.

HE HAS BEEN A A McKNIGHT THAT

FUNDED HIS LAB FOR A LONG TIME

WHEN HE STARTED HIS LAB AT

HARVARD.

HIS LABORATORY FOCUSES ON

UNDERSTANDING THE FUNCTION AND

REVELATION OF SYNAPSES IN THE

MAMMALIAN BRAIN WITH PARTICULAR

INTEREST IN HOW THE FUNCTION OF

SYNAPSES IS PERTURBED DURING

DECEASED.

SOME OF THEM ARE AUTISM,

PARKINSON AND ALZHEIMERS THAT

RESEARCH PROJECTS HAVE BEEN

FOCUSING ON LATELY.

I HAVE TO SAY THAT I HAVE HAD

THE HONOR TO BE A POSTDOCTORAL

FELLOW IN BERNARDO'S LAB AND

RECEIVED TRAINING THERE.

I THINK THAT BEYOND HIS OR

BESIDES HIS VERY IMPRESSIVE CV,

IT ACTUALLY DOESN'T MAKE JUSTICE

TO THE REALLY AMAZING

INVESTIGATOR AND RESEARCHER HE

IS.

HE IS EXTREMELY CREATIVE, AND SO

WORKING WITH HIM IN SCIENCE IS

ACTUALLY A LOT OF FUN.

AND I THINK ANOTHER QUALITY OF

BERNARD OH, BEYOND THE USUAL,

SMART, HARDWORKING AND ALL THOSE

THINGS WE ALL KNOW, REQUIRED FOR

SCIENCE, I THINK BERNARDO IS

EXTREMELY BRAVE.

AND I LEARNED, I HAVE BEEN

INSPIRED BY THIS WHILE I WAS

WORKING WITH HIM.

I THINK YOU SOMETIMES NEED TO BE

BRAVE AND TAKE RISKS TO TACKLE

THESE IMPORTANT AND MEANINGFUL

QUESTIONS IN SCIENCE, AND I

THINK BERNARDO HAS THAT -- IS

DOING THAT REALLY WELL.

AND EXAMPLES OF THESE CREATIVITY

AND LACK OF FEAR IS THE AMOUNT

OF NEW TECHNOLOGIES, BOTH

OPTICAL AND CHEMICALS BEING

DEVELOPED IN HIS LAB IN ORDER TO

FURTHER STUDY THE FUNCTION OF

SYNAPSES.

SO HE IS GOING TO TELL US ABOUT

SELF STIMULATION, HOW THE BRAIN

WIRES ITSELF, AND SURPRISING NEW

WAYS IT TURNS ITSELF OFF.

WELCOME.

[ APPLAUSE ]

>> THANK YOU, VERONICA.

I'M HONORED TO HAVE BEEN THE

SUBJECT OF THAT INTRODUCTION AND

WHAT VERONICA DIDN'T TELL YOU IS

THAT SHE WAS ACTUALLY THE

FOUNDING MEMBER OF MY LAB.

SHE ARRIVED AT HARVARD ABOUT TWO

MONTHS BEFORE I DID AND WAS THE

FIRST PERSON THERE, OBVIOUSLY,

AND REALLY HELPED ME GET THINGS

GOING.

SO A LOT OF THE SUCCESS MY

LABORATORY HAS HAD IS REALLY DUE

TO PEOPLE LIKE VERONICA WHO ARE

PIONEERING AND BRAVE TO JOIN MY

LAB WHEN I WAS COMPLETELY

UNKNOWN.

SO I'M GOING TO TELL YOU

ABOUT -- I'M GOING TO PUSH THESE

AWAY.

I'M GOING TO TELL YOU ABOUT A

PROJECT THAT WAS JUST COMPLETED

IN THE LAB AND IS ABOUT TO

APPEAR ONLINE, HOPEFULLY TOO

MANY OF YOU HAVE NOT READ IT YET

SO IT WON'T BE BORING FOR YOU.

THE FIRST PART, I THINK YOU'LL

UNDERSTAND THE TITLE IN A MINUTE

F I HAVE TIME, I WILL GET SOME

SURPRISING NEW RESULTS THAT ARE

TOTALLY UNPUBLISHED AND REALLY

UNFINISHED AND THAT'S THE SECOND

PART HERE ABOUT HOW THE BRAIN

TURNS ITSELF OFF.

BUT I MAY NOT GET TO IT.

WE'LL SEE IF WE HAVE TIME.

SO THIS IS THE ANIMAL THAT WE

STUDY IN THE LAB.

THIS IS LABORATORY MOUSE ON THE

LEFT.

THIS IS OBVIOUSLY A NEW MOTHER.

SHE HAS A BUNCH OF PUPS THERE, A

LITTER OF PUPS.

AND ONE OF THE THINGS WE TRY TO

UNDERSTAND IN THE LABORATORY IS

THE REMARKABLE TRANSPORTATION

HAPPENS BETWEEN THIS STAGE OF

DEVELOPMENT AND THIS STAGE OF

DEVELOPMENT.

SO JUST AFTER BIRTH, THESE PUPS

ARE VERY LIMITED IN THEIR

BEHAVIORAL REPERTOIRE.

THEIR LACK OF MOTOR ABILITY AND

FINE MOTOR SKILLS ARE INCREDIBLY

POOR.

THERE IS VERY LITTLE THEY CAN

DO.

THEIR SENSORY SYSTEMS ARE NOT

YET FULLY DEVELOPED.

AND ESSENTIALLY THAT THE STAGE,

ALL THEY CAN REALLY DO IS FIND

THEIR MOTHER, SUCKEL AND GROW

AND CONTINUE TO DEVELOP.

WITHIN A FEW WEEKS AFTER BIRTH,

THESE ANIMALS BECOME FULLY

AUTONOMOUS CREATURES.

IN THE WILD THEY ARE ABLE TO

FORAGE OVER A SQUARE KILOMETER.

THEY ARE ABLE TO REMEMBER THEIR

WAY THROUGH THAT ENVIRONMENT.

THEY ARE ABLE TO ASSOCIATE

SENSORY CUES WITH THE PRESENCE

OF REWARDS LIKE FOOD, WATER OR

MATES OR THE PRESENCE OF

PREDATORS.

SO WE WANT TO UNDERSTAND WHAT

OCCURS IN THE BRAIN THAT ALLOW

THIS IS REMARKABLE DEVELOPMENTAL

MATURATION IN JUST A FEW WEEKS.

SO, IN PARTICULAR, WE ARE

INTERESTED IN THESE RAPID

PROGRESSIONS THAT ARE SHOWN

HERE, THE RAPID INCREASE IN

MOTOR FUNCTIONS AND IN

PARTICULAR THIS ABILITY TO FORM

ASSOCIATIONS BETWEEN STIMULI AND

REWARDS.

AND WHEN WE THINK ABOUT THESE

KINDS OF PROBLEMS, IT'S

IMPORTANT TO DIFFERENTIATE

BETWEEN TWO KINDS OF PLASTICITY

THAT CAN OCCUR IN THE BRAIN.

ONE IS THE THAT CHANGES IN THE

BRAIN ITSELF THAT ALLOW IT TO

BECOME AN ORGAN THAT CAN LEARN.

THAT'S REALLY A DEVELOPMENTAL

QUESTION.

WHAT CHANGES FROM HERE TO HERE

THAT THIS ANIMAL IS NOW CAPABLE

OF DOING QUITE SOPHISTICATED

LEARNING?

THAT'S ONE PROBLEM WE ARE

INTERESTED IN.

THAT'S MOSTLY WHAT I'LL TELL BUT

TODAY.

SECONDLY, WHAT ACTUALLY CHANGES

IN THE BRAIN ITSELF WHEN ANIMAL

ACUTELY LEARNS?

SO WHEN THIS ANIMAL LEARNS THIS

KIND OF LIFE TASTES GOODS AND

IT'S SOMETHING TO SEEK OUT, WHAT

HAPPENED IN HIS BRAIN?

THAT'S A MORE ACUTE PLASTICITY

THAT IS THOUGHT TO HAPPENED IN

HOW CELLS TALK TO EACH OTHER.

THE PART OF THE BRAIN I'M GOING

TO TELL YOU ABOUT IS THE BASAL

GANGLIA.

THAT'S BECAUSE IT IS NECESSARY

FOR MANY FUNDAMENTAL BEHAVIORS

SEEN IN MAMMALS AND HUMANS AS

WELL.

SO FOR EXAMPLE, THE PROPER

FUNCTION OF BASAL GANGLIA IS

NECESSARY TO GENERATE PURPOSEFUL

MOVEMENTS AND ALSO NECESSARY FOR

MANY FORMS OF REINFORCEMENT

LEARNING.

THIS IS MADE QUITE CLEAR BY

DISEASES OF THE BASAL GANGLIA IN

HUMANS.

SO FOR EXAMPLE, PARKINSON'S

DISORDER, HUNTINGTON'S DISORDER,

TWO DEVASTATING DISORDERS, ARE

BOTH DISEASES OF THE BASAL

GANGLIA.

FURTHERMORE, DRUG ADDICTION,

MANY FORMS OF DRUG ADDICTIONS

AND DRUGS OF ABUSE, THE

MECHANISM OF ACTION OF THESE

DRUGS IS BY CHANGING THE

ACTIVITY OF NEURONS WITHIN THE

BASAL GANGLIA.

MOST DRUGS OF ABUSE ACTS BY

INCREASING THE LEVEL OF DOPAMINE

IN ONE PART OF THE BASAL

GANGLIA, THE STRATUM.

THAT HAS PROFOUND EFFECTS ON

HUMAN BEHAVIOR AND CAN CORRUPT

THE BEHAVIORAL REP TIRE AND

DRIVE THEM INTO DRUG SEEKING

BEHAVIOR.

MANY OF THE DISEASES, INCLUDING

THE TERETS SYNDROME, ADHD AND

OTHER NEUROPSYCHIATRIC

DISORDERS, ALSO HAVE COMPONENTS

OF BASAL GANGLIA DYSFUNCTION.

SO JUST TO GET EVERYBODY ON THE

SAME PAGE, I'M GOING TO SPEND

SOME TIME OUTLINING THE

CIRCUITRY OF THE BASAL GANGLIA

AND FOR THOSE OF YOU IN THE

AUDIENCE THAT ARE EXPERTS IN THE

BASAL GANGLIA, PLEASE UNDERSTAND

THAT I'M GOING TO SHOW YOU VERY

SIMPLIFIED MOUSECENTRIC VIEW OF

THE BASAL GANGLIA.

SO THE BASAL GANGLIA CONSISTS OF

TWO INTERLOCKING LOOPS.

SO THERE IS A PROJECTION FROM

CORTEX FROM MANY AREAS OF CORTEX

INTO THE STRATUM.

THIS IS AN EXCITATORY

PROJECTION, CELLS THAT RELEASE

GUTTE MATE AND CAN STIMULATE THE

STRATUM.

THERE IS A PROJECTION OF

INHIBITORY NEURONS THAT GO TO

THE -- INHIBITORY PROJECTION

THAT RELEASES GABA.

IN TURN, THERE IS INHIBITORY

PROJECTION BACK TO THE THALAMUS

AND THEN AN EXCITATORY

PROJECTION BACK TO CORTEX AND

TRIATUM.

IF YOU LOOK AT THIS LOOP, THERE

ARE SEVERAL EXCITATORY

PROJECTIONS AND TWO INHIBITORY

PROJECTIONS.

THIS IS WOULD CATHE DIRECT

PATHWAY.

IT IS THOUGHT THAT ACTIVITY OF

THE CORTEX ACTS THROUGH THIS

LOOP TO REINFORCE ACTIVITY.

IT'S A POSITIVE FEEDBACK LOOP.

IN PARALLEL, THERE IS A SECOND

PATHWAY, THE INDIRECT PATHWAY,

IN WHICH IS THERE AN EXTRA

SYNAPSE, SO WE HAVE INHIBITORY

NEURONS THAT MAKES NEURONS INTO

THE GLOBE US PAL DOZEN AND THEN

IT SENDS A SECOND NEURON.

IN THIS CASE, WE HAVE THREE

INHIBITORY SYNAPSES WITHIN THE

ARE LOOP AND IT IS THOUGHT THAT

THE ACTIVITY OF INDIRECT PATHWAY

SUPPRESSES CORTEX.

AND THIS DIVISION OF DIRECT AND

INDIRECT PATHWAY IS SOMETHING WE

WILL PAY ATTENTION TO THROUGHOUT

THE WHOLE TALK.

SO HERE IS ANOTHER VIEW OF THE

BASAL GANGLIA CORTICAL

CIRCUITRY.

WHAT WE THOUGHT WAS INTERESTING

ABOUT THE DEVELOPMENT OF THIS

CIRCUIT IS THAT HERE YOU HAVE A

CIRCUIT WITH MANY COMPONENTS IN

IT.

SOME OF WHICH ARE EXCITE TORY

AND SOME OF WHICH ARE

INHIBITORY.

IT WAS UNKNOWN IN THE MAMMALIAN

BRAIN HOW SUCH A CIRCUIT WOULD

DEVELOP AND WIRE ITSELF.

WHAT WAS THE ROLE OF ACTIVITY IN

THESE KINDS OF RECURRENT LOOPS

IN STABILIZING THE CIRCUITRY AND

HELPING IT FORM?

WE KNOW A LOT OF MATURATION

HAPPENS AFTER BIRTH.

FOR EXAMPLE, IN A MOUSE IN THE

SECOND WEEK OF LIFE.

SO IT'S QUITE LIKELY TO BELIEVE

THAT THE EXPERIENCE OF THE

ANIMAL SENSORY AND ENVIRONMENT

AND PATTERNS OF ACTIVITY IN THE

BRAIN IN THE POSTNATAL PERIOD

WOULD PLAY A ROLE IN GOVERNING

THE FORMATION OF THIS CIRCUIT.

SO NOW LET'S SPEND MORE TIME ON

THESE TWO PATHWAYS, DIRECT AND

INDIRECT.

BECAUSE THE DIVISION IS VERY

IMPORTANT.

SO IT TURNS OUT THAT BOTH THE

DIRECT AND INDIRECT PATHWAY,

THESE TWO PATHWAYS OF

INFORMATION FLOWING THROUGH THE

BASAL GANGLIA ARE FORMED BY A

CLASS OF NEURON CALLED THE

MEDIUM SPINY NEURONS.

SOME CALL THEM PROJECTION

NEURONS.

THESE ARE THE PRINCIPLE CELLS OF

TRIATUM AND THEY ARE A CURIOUS

CELL TYPE.

THEY ARE INHIBITORY.

THAT BREAKS ONE OF THE GENERAL

RULES IN THE BRAIN WHICH IS THAT

THE LONG-RANGE PROJECTIONS ARE

USUALLY FORMED BY EXCITATORY

NEURONS.

THESE CELLS ARE ALSO COVERED

WITH A HIGH DENSITY OF DENDRITIC

SPINES.

THESE ARE THESE PECULIAR

MUSHROOM-SHAPED SPECIALIZATIONS

AT WHICH EXCITATORY SYNAPSES ARE

FORMED.

IT'S A TYPICAL FOR INHIBITORY

NEURTONS HAVE DENDRITIC SPINES.

THIS IS ANOTHER RULES THAT IS

BROKEN BY THIS TYPE OF CELL IN

THE BRAIN.

THERE ARE 3 OR 4 CELLS EXAMPLES.

THE DIRECT AND INDIRECT PATHWAY

PRETTY MUCH LOOK THE SAME.

YOU CAN'T REALLY TELL THEM APART

BUT THERE ARE DIFFERENCES

BETWEEN THEM.

ONE OF WHICH IS AN ANATOMICAL

DIFFERENCE I MENTIONED, THE

DIRECT PATHWAY NEURONS PROJECT

THE -- WHEREAS THE INDIRECT

PROJECT THE GLOBE US PAL TUS.

THESE CELLS EXPRESS THE TYPE ONE

DOPAMINE RECEPTOR WHEREAS THESE

CELLS EXPRESS THE TYPE TWO

DOPAMINE RECEPTOR.

THAT IS FUNDAMENTALLY IMPORTANT

BECAUSE THESE TWO TYPES OF

RECEPTORS DIFFERENTIALLY SIGNAL

TO INTRACELLULAR PATHWAYS.

ONE OF THEM ACTIVATES PKA AND

THE OTHER INHIBITS.

THIS IS A PUSH-PULL TYPE OF

ARRANGEMENT.

THESE GUYS EXPRESS THE PEPTIDE

SUBSTANCE P AS WELL AS THE

OPIOID IN CAP 1.

WHAT THAT MEANS IS HOW IT

CHANGES THE FUNCTION OF THE

CIRCUIT THAT IS UNCLEAR AND

SOMETHING WE ARE INTERESTED IN.

EXFURTHERMORE, AS PROPOSED IN

VERY CLASSIC MODELS OF FUNCTION,

THIS DIRECT PATHWAY, WHEN IT IS

ACTIVE, PROMOTES MOVEMENT IN THE

ANIMAL, PROMOTES LOCOMOTION,

WHEREAS THIS INHIBITS MOVEMENT.

IT'S THOUGHT PARKINSON'S

DISEASE, ONE OF THE MAIN CAUSES

IS AN IMBALANCE BETWEEN ACTIVITY

OF THESE TWO PATHWAYS SUCH THAT

INDIRECT PATHWAY ACTIVITY IS

FAVORED IN PARKINSONS AND THAT

EXPLAINS SOME OF THE DIFFICULTY

OF PEOPLE WITH PARKINSON'S

INITIATING MOVEMENTS.

NOW ANOTHER FEATURE OF THE BASAL

GANGLIA CIRCUITRY, WE HAVEN'T

TOUCHED ON YET IS THAT THIS KIND

OF BOX DIAGRAM IS NOT THAT ALL

OF CORTEX PROJECTS TO ALL OF

TRIATUM.

THERE ARE SEVERAL INTERLOCKING

SUBLOOPS SUCH THAT ONE PART OF

CORTEX WILL PROJECT DOWNSTREAM

AREAS AND ANOTHER WILL PROJECT

TO OTHER DOWNSTREAM AREAS.

WE NEED TO THINK OF THIS AS A

SERIES OF LOOPS BETWEEN ALL

THESE STRUCTURES.

SO THAT IS THE QUESTION THAT

REALLY IS VERY INTERESTING TO

US.

BECAUSE HOW CAN THIS SYSTEM WIRE

ITSELF?

HOW CAN WE HAVE CONNECTIONS THAT

ARE MAINTAINED ACROSS FOUR OR

FIVE NUCLEI, SOME EXCITE TORY

AND SOME INHIBITORY?

HOW DOES THE CELL KNOW WHERE TO

PROJECT IN THE DOWNSTREAM AREA

IN ORDER TO CLOSE THIS LOOP?

AND THAT'S THE SUBJECT OF

TODAY'S TOPIC.

WE THOUGHT THIS KIND OF LOOP IS

MORE EASILY FORMED VIA ACTIVITY

WIRING.

THAT SOMEHOW THE LOOP CAN DETECT

ONE IS CORRECTLY WIRED BASED ON

THE PATTERNS OF ACTIVITY THEY

LIE WITHIN IT.

THAT'S WHAT I HOPE TO CONVINCE

YOU OF TODAY.

NOW I WANT TO STRESS THIS IS

FUNDAMENTALLY DIFFERENT THAN THE

BEST STUDIES CIRCUITS WITHIN THE

BRAIN IN TERMS OF DEVELOPMENT,

WHICH ARE SENSORY SYSTEMS.

SO FOR EXAMPLE, IN THE EYE, WE

KNOW THAT OF COURSE THE

ORGANIZATION OF THE RETINA IS

TOPOGRAPHIC.

IT REFLECTS THE ORGANIZATION OF

THE WORLD OUTSIDE US.

SO FOR EXAMPLE, NEAREST

NEIGHBORING CELLS WITHIN THE

RETINA ARE RECEIVING INFORMATION

FROM NEAREST NEIGHBOR PORTIONS

OF THE VISUAL FIELD AND THAT TOP

GRAPHIC ARRANGEMENT IS

MAINTAINED TO VARIOUS STAGES

WITHIN THE PROCESSING SYSTEM FOR

THE VISUAL SYSTEM.

FOR EXAMPLE, IN THE THALAMUS AND

NUCLEUS AND CORTEX.

FURTHERMORE, EARLY IN

DEVELOPMENT, EVEN BEFORE ANIMAL

IS CAPABLE OF SEEING, EVEN IN

THE MOUSE, EVEN BEFORE THE EYE

OPENED, IT TURNS OUT THERE ARE

WAVES OF ACTIVITY THEY ARE

SPONTANEOUSLY GENERATED WITHIN

THESE STRUCTURES THAT CAN

PROJECT INFORMATION ABOUT

NEAREST NEIGHBOR RELATIONSHIPS

FORWARD.

SO FOR EXAMPLE, THERE ARE WAVES

OF ACTIVITY THAT OCCUR IN THE

EYE THAT ARE TRANSMITTED FORWARD

INTO THE LGN AND IN THE CORTEX

THAT ARE PASSING INFORMATION

ABOUT WHAT CELLS ARE NEAR EACH

OTHER AND THEREFORE LISTENING TO

OR SEEING NEIGHBORING PARTS OF

THE VISUAL FIELD.

SO THERE IS A LOT OF THEORIES

ABOUT THE DEVELOPMENT OF THIS

VISUAL SYSTEM IN WHICH IT IS

CLEAR THESE KINDS OF WAVES,

COUPLED WITH SOME LEARNING RULES

FOR STABILIZING AND GROWING

SYNAPSES, IS SUFFICIENT TO

EXPLAIN A LOT OF THE ARRANGEMENT

OF THE SENSORY SYSTEM.

AND THIS IS WORK THAT IS

APPEARED OVER MANY DECADES FROM

PEOPLE LIKE CARLA SHAT, RACHEL

WONG AND MANY OTHERS.

NOW QUITE INTERESTINGLY, THERE

HAS BEEN RECENT WORK BY NICK AND

WRITE, SO SHOWING A SIMILAR

PHENOMENON IN THE AUDITORY

SYSTEM.

IN THE INNER EAR WHERE THERE IS

A TONE TOPIC ARRANGEMENT SO

NEAREST FREQUENCY RESPONDING

CELLS ARE CLOSE TO EACH OTHER,

THEY ARE ALSO PATTERNED

ACTIVITY, WAVES OF ACTIVITY THEY

PROP GADE THROUGH THAT STRUCTURE

THAT ALSO TRANSMIT INFORMATION

ABOUT NEAREST NEIGHBORHOOD

RELATIONSHIPS TO DOWNSTREAM

STRUCTURES.

IN THIS CASE, WE HAVE AT LEAST A

MODEL FOR HOW TOPOGRAPHY CAN BE

ORGANIZED.

AND I THINK THAT'S FUNDAMENTALLY

DIFFERENT THAN THIS CIRCUIT.

THIS CIRCUIT IS NOT A DIRECT

SENSORY CIRCUIT.

IT'S EMBEDDED WITHIN

SUBSTRUCTURE OF THE BRAIN AND

SUB-CORTICALS.

IT CONSISTS OF MIXED EXCITATORY

AND INHIBITORY PROJECTIONS.

SO HOW CAN WE HAVE THIS SYSTEM

WIRE ITSELF?

AND THAT WAS THE SUBJECT OF

THESE TWO PEOPLE HERE.

THEY WORKED TOGETHER ON THIS

PROJECT.

THEY DECIDED IN ORDER TO

UNDERSTAND, IF ACTIVITY IN THE

CIRCUIT PLAYS A ROLE IN THIS

DEVELOPMENT, THE BEST STRATEGY

WAS TO DO WHAT IT SAYS HERE,

THAT SILENCE EITHER THE DIRECTOR

INDIRECT PATHWAY AND UNDERSTAND

HOW THESE IMBALANCES ALTER THE

CIRCUIT.

NOW THESE GUYS DID THE WORK.

BUT I WANTED TO TAKE SOME TIME

TO THANK CHIP, WHO IS HERE.

SO CHIP'S EFFORTS, AS ONE OF THE

CO-PIs IN THE PROJECT ALONG

WITH MAT HINES, HAS REALLY

ENABLED ALL OF MODERN BASAL

GANGLIA RESEARCH IN THE MOUSE.

SO CHIP WAS ONE OF THE PEOPLE

THAT REALLY PUSHED FORWARD THE

DEVELOPMENT OF BACK TRANSGENIC

MICE THAT ALLOW GFP TO BE

EXPRESSED IN PARTICULAR

SUBPOPULATIONS.

THANKS TO HIS EFFORTS THAT WE

NOT ONLY HAVE THE MICE BUT WE

HAVE WELL CHARACTERIZED MICE

THAT ALLOW US TO PUT PROTEINS

INTO VERY SPECIFIC ELEMENTS OF

THE BASAL GANGLIA.

WE'LL SEE HOW WE USE THAT

TECHNOLOGY.

SO, WHAT I'M SHOWING YOU HERE

ARE IMAGES OF FLUORESCENCE

PATTERNS IN A REPORTER ANIMAL

THAT EXPRESSES A FLORESCENT

PROTEIN.

ON THE LEFT, WE HAVE CROSSED

THAT REPORTER ANIMAL WITH

ANOTHER MOUSE THAT EXPRESSED

PRE

RECOMBINASE UNDER CONTROL OF THE

TYPE 1 DOPAMINE RECEPTOR.

I TOLD YOU AT THE BEGINNING,

THAT THE TYPE 1 DOPAMINE

RESECTOR IS SELECTIVE.

FLUORESCENCE WITHIN THE STRATUM

AND THEN THIS COALESCENCE OF

AXONS INTO THE VERTIC LIE.

THIS IS THE TOOL THAT WILL BE

USED TO TURN ON EXPRESSION OF

PROTEINS WITHIN THE DIRECT

PATHWAY.

ON THE RIGHT, IS A SIMILAR IMAGE

OBTAINED BY CROSSING THE

REPORTER ANIMAL WITH THE SECOND

ANIMAL THAT EXPRESSES

RECOMBINASE UNDER THE TYPE 2

DOPAMINE RECEPTOR.

THIS LABELS THE INDIRECT

PATHWAY.

YOU CAN SEE THE SHORT AXONS

HERE.

SO WE TOOK THESE ANIMALS AND

CROSSED THEM WITH ANOTHER

ANIMAL.

THIS IS ANIMAL MADE BY BRAD

LOWEL AT THE BETH ISRAEL, A

CONDITIONAL ALLELE OF THIS JEAN,

SOC32A1.

THIS GENE ENCODES A PROTEIN

KNOWN AS VGAT.

AND THIS IS THE ONLY PROTEIN

KNOWN TO TAKE GABA FROM THE

CYTOPLASM AND PACKAGE IT.

SO THE IDEA IS THAT THIS MOUSE

NOW WILL LOSE EXPRESSION OF VGAT

IN CELLS THAT EXPRESS COUNTRY

RECOMBINASE.

SO THE D1 -- CRE RECOMBINASE.

IT'S IMPORTANT TO KEEP IN MIND

THAT WE DON'T EXPECT THIS TO BE

TOXIC TO THE CELLS IN ANY WAY.

THE CELLS SHOULD BE ABLE TO

PRODUCE SYNAPTIC VESICLES,

SIMPLY WITH NO GABA.

THEY WON'T BE ABLE TO OUTPUT TO

THE DOWNSTREAM.

SO HERE IS AN IMAGE OF THESE

MICE.

THESE MICE ARE QUITE SICK AND

THEY ALL DIE BY ABOUT 20 DAYS.

WE CAN KEEP THEM ALIVE LONGER BY

POLING THE LITTERS SUCH THAT

ONLY THE MUTANT MICE ARE PRESENT

WITH THE MOTHER AND THAT ALLOWS

THEM TO GET BETTER ACCESS TO

MOTHER AND SURVIVE LONGER.

HERE IS AN IMAGE OF A MOUSE THAT

HAS ONE COPY OF VGAT COMPARED TO

A LITTERINATE HAS 0 COPIES.

THIS ANIMAL IS OFTENY AND QUITE

SICK.

THE INDIRECT PATHWAY SILENCED

ANIMALS ARE BETTER OFF.

THEY SURVIVE LONGER.

THEY ARE A LITTLE BIT RUNTY AS

WELL.

SMALLNER WEIGHT BUT NOT AS

SERIOUSLY EFFECTED.

NOW WHAT IS INTERESTING IS THAT

THESE ANIMALS, DESPITE BEING

SICK, ALREADY SHOW THE LOCO

MOTOR PHENOTYPES ONE EXPECT FOR

PERTUBATION OF DIRECT AND

INDIRECT PATHWAY.

WHAT I'M SHOWING YOU HERE IS

VIDEO TRACKING OF THE MOVEMENT

OF THESE ANIMALS IN AN ARENA

OVER AN HOUR.

YOU CAN SEE THAT AT 14 DAYS OF

AGE, THIS ANIMAL HERE LOCO

MOATS.

THIS IS A CONTROL.

IT HAS ONE COPY OF VGAT.

WHEREAS ITS SIBLING WITH NO

COPIES OF VGAT SHOWS MUCH

REDUCED LOCO MOTOR ABILITY.

CONSISTENT WITH MODELS OF BASAL

GANGLIA FUNCTION PROPOSED MANY

YEARS AGO AND WERE VERY RECENTLY

PROVEN TO BE TRUE BY ACUTE

MANIPULATIONS OF DIRECT AND

INDIRECT PATHWAYS BY PEOPLE WHO

ARE COMING HERE TO THE

INTRAMURAL PROGRAM.

SO THE DIRECT PATHWAY SILENCED

ANIMAL IS QUITE -- WHEREAS THE

INDIRECT PATHWAY SILENCED ANIMAL

DESPITE BEING SICK AND RUNTY

SHOWS GREATLY EXAGGERATED LOCO

MOTOR ABILITY.

SO CONSISTENT.

THE IMPORTANCE HERE IS THAT EVEN

AT THESE YOUNG AGES, P14-P17,

ALREADY THE OPPOSING

ORGANIZATION OF THE BASAL

GANGLIA IS IN PLACE SUCH AS

DIRECT PATHWAY HAS ONE

BEHAVIORAL PHENOTYPE WHEREAS THE

INDIRECT PATHWAY HAS THE

OPPOSITE.

SO WE HAVE DONE A LOT OF

ANALYSIS TO THESE ANIMALS.

I'M GOING SKIP MANY DETAILS.

A COUPLE OF CONTROLS I WANT TO

SHOW YOU ARE THAT THE NUMBER OF

CELLS OF THE DIRECT AND INDIRECT

PATHWAY AND EACH ONE OF THESE

ANIMALS IS UNPERTURBED.

IT'S NOT AS IF SILENCING THE

DIRECT PATHWAY MAKES THESE CELLS

DIE.

THERE ARE JUST AS MANY THERE AS

IN THE CONTROL ANIMAL.

FURTHERMORE, THE AXONS OF THESE

CELLS AND THIS WAS SURPRISING,

ARE PERFECTLY FINE IN REACHING

THEIR TARGETS.

SO YOU CAN SEE HERE FLUORESCENCE

FILLING THE VERTIC LATA IN A

NULL ANIMAL COMPARED TO CONTROL

OR THE GLOBE US PAL TUS IN A

NULL ANIMAL AND CONTROL.

WE HAVE DONE MORE REFINED

ANALYSIS TO CONVINCE OURSELVES

OF THE FINE STRUCTURE IS OKAY AS

WELL.

FOR THE POINT OF THIS TALK, JUST

KEEP IN MIND THAT CELL SURVIVAL

IS FINE AND DOWNSTREAM TARGETING

OF NUKE SLY FINE AS WELL.

SO, OBVIOUSLY ANIMALS ARE SICK.

THERE IS SOME MAJOR PERTUBATION

IN THE CIRCUITRY.

SO SINCE THE OUTPUT OF THE

SYSTEM SEEM TO BE CORRECT, WE

DECIDED TO FOCUS INSTEAD ON

LOOKING AT THE INPUT OF THE

MEDIUM SPINY NEURONS.

THIS IS AN IMAGE OF A MEDIUM

SPINY NEURON FILLED WITH

FLORESCENT MOLECULE.

SO WE CAN RECORD ELECTROCURRENTS

THROUGH THE PI PET AND IMAGE THE

STRUCTURE BASE OF THE

FLUORESCENCE.

IT'S ABOUT 200 MICRON LONG DEN

DRIED COVERED IN HIGH DENSITY OF

DENDRITIC SPINE.

AND SO HERE IS A BLOW UP VIEW OF

A DENDRITE.

YOU CAN SEE THESE SPINES ON

THEM, LOLLIPOP-SHAPED

PROTRUSIONS.

IN THIS CELL, THERE IS

ESSENTIALLY A VERY CLOSE

CORRESPONDENCE BETWEEN THE

NUMBER OF SYNAPSES IN THE

DENSITY OF SPINES.

MOST OF THESE SPINES HOUSE ONE

GLUTAMATERGIC SYNAPSE.

SO COUNT SPINES AS AN INDIRECT

PROXY TO COUNT THE NUMBER OF

GLUTAMATERGIC SYNAPSES.

WE WERE SURPRISED TO FIND THAT

SILENCING THE OUTPUT OF THIS

CELL HAS TREMENDOUS EFFECTS ON

ITS INPUT ON A NUMBER OF SNAPS

THAT IS IT RECEIVES.

SO HERE IS AN IMAGE OF A DIRECT

PATHWAY SPINY NEURON.

HERE IS A BLOW UP OF A SPINY

SECTION FROM A CONTROL ANIMAL

AND HERE IS FROM A SILENCED

ANIMAL IN WHICH TWO COPIES OF

VGAT HAVE BEEN LOST.

WHAT I HOPE YOU CAN APPRECIATE

IS THAT THIS NORMAL DENSITY OF

DENDRITE DRIEDIC SPINES IS

GREATLY REDUCED IN THIS ANIMAL T

LOST HALF OF ITS DENDRITIC

SPINE.

FURTHERMORE, IF WE LOOK AT THE

ELECTRICAL RECORDING OF THE

CELL, WE CAN SEE THESE VERY

SHORT BURSTS OF CURRENT FLOW

WHICH CORRESPOND TO THE

SPONTANEOUS ACTIVATION OF A

SINGLE SINNANS AND BY COUNTING

HOW OFTEN THESE OCCUR, WE CAN

GET ANOTHER MEASURE OF HOW MANY

GLUTAMATERGIC SYNAPSES ARE ON

THE CELL.

AND ALSO THE NUMBER OF

GLUTAMATERGIC SYNAPSES MEASURED

BY THIS ASSAY IS GREATLY

REDUCED.

SO FROM THIS KIND OF DATA, WHAT

WE LEARNED THAT SILENCING THE

DIRECT PATHWAY DECREASES THE

DENSITY OF DENDRITIC SPINES AND

THE FREQUENCY OF MINIATURE

EPSCs, ELECTRICAL ANALYSIS

OCCURRENCE, ON TO THESE CELLS.

NOW THE SURPRISE CAME WHEN WE

ANALYZED THE EFFECTS OF

SILENCING THE INDIRECT PATHWAY.

SO THIS IS AN INDIRECT PATHWAY

MEDIUM SPINY NEURON ANALYZED IN

THE SAME WAY AND NOW YOU CAN

APPRECIATE THE SILENCED NEURON

HAS MANY MORE SPINES WITH A

CONTROL NEURON.

SO REMEMBER THAT -- SO SILENCING

THE INDIRECT PATHWAY NOW

ENHANCES THE DENSITY OF

DENDRITIC SPINES AND

ELECTROPHYSICALLY, WE SEE THE

ENHANCE EVENTS BOTH LEADING TO

THE CONCLUSION THAT SILENCING

THE PATHWAY INCREASES THE

DENSITY OF SPINES AND IN

ACTIVATION OF SYNAPSES.

SO THE OPPOSITE OF BEFORE.

HERE IS THE QUANTINDICATION OF

THAT DATA.

IF THE SILENCE OF THE DIRECT

PATHWAY AND LOOK AT DIRECT

PATHWAY NEURONS, WE SEE LESS

ELECTROPHYSIOLOGICAL OF SYNAPSES

AND LESS MORPHOLOGICAL.

IF WE SILENT THE INDIRECT

PATHWAY WE GAIN

ELECTROPHYSIOLOGICAL AND GAIN

MORPHOLOGICAL CORRELATION OF

SYNAPSES.

SO THERE ARE TWO POSSIBILITIES

THAT WE NEED TO CONSIDER.

ONE IS THAT MAYBE THIS GENE THAT

WE HAVE KNOCKED OUT AND THE

PROTEIN PRODUCT, HAS OPPOSING

FUNCTIONS IN THESE 2 CELL

POPULATIONS.

SOMEHOW, THIS GENE, EVEN THO IT

PACKAGE GATHERS IN THE VESICLES,

ACTS TO REINFORCE SYNAPSES AND

WHEN WE LOSE THE GENE, WE LOSE

THE SYNAPSES AND IN THIS CASE,

ACTS TO DESTABILIZE THE SYNAPSES

SO WHEN WE LOSE THE GENE WE

INCREASES SYNAPSES.

THAT'S ONE POSSIBILITY.

THE SECOND IS THAT BY SILENCING

THESE TWO PATHWAYS, WE HAVE

OPPOSING EFFECTS ON THE

CIRCUITRY AND WE ARE REALLY

REVEALING SOME KIND OF ACTIVITY

DEPENDENT LEARNING RULE WITHIN

THE SYSTEM.

AND I HOPE TO CONVINCE YOU THE

SECOND ONE IS TRUE.

NOW THE FIRST PIECE OF DATA WE

HAVE THAT THE SECOND MODEL WAS

LIKELY TRUE IS OBTAINED BY

LOOKING AT THE OPPOSITE PATHWAY.

SO WHEN WE SILENCE A DIRECT

PATHWAY, WHAT HAPPENS TO THE

INDIRECT PATHWAY?

GENOTYPICALLY NORMAL?

WE HAVEN'T DONE ANYTHING TO IT.

YOU WHAT SEE IS THE INDIRECT

PATHWAY ALSO LOSES

ELECTROPHYSIOLOGICAL CORRELATES

OF SYNAPSES AND MORPHOLOGICAL

CORRELATES OF SYNAPSES.

WHEN WE SILENCE THE INDIRECT

PATHWAY, WE SAW THE OPPOSITE

EFFECT.

SILENCE OF THE INDIRECT PATHWAY

INCREASES NUMBER OF SYNAPSEOS TO

DIRECT PATH AND MEDIUM SPINY

NEURONS.

EVEN THOUGH THOSE CELLS ARE

NORMAL.

SO I THINK THIS IS BEST

EXPLAINED BY A CIRCUIT EFFECT AS

OPPOSED TO A CELL TUNE MUSEFFECT

OF THAT GENE.

AND SO WHAT WE ARE SEEING IS

THAT WHEN WE SILENCE A DIRECT

PATHWAY, WE LOSE THE INHIBITORY

PROJECTION BECAUSE OF THAT, THIS

STRUCTURE IS OVERACTIVE.

BECAUSE OF THAT, THIS IS

OVERINHIBITTED AND THEREFORE

DRIVE IN THE SURGAT REDUCED.

WE PREDICT THAT SILENCING THIS

WOULD REDUCE ACTIVITY IN THE

WHOLE CIRCUIT AND IF THIS

CIRCUIT PAYS ATTENTION TO ITS

OWN ACTIVITY TO DRIVE SNAPS

GENESIS, IT WOULD LOSE SYNAPSES.

CONVERSELY, SILENCING THE

INDIRECT PATHWAY WOULD HAVE THE

OPPOSITE EFFECT.

IT WOULD BOOST ACTIVITY IN THE

CIRCUIT AND POSSIBLY PROMOTE THE

FORMATION OF SYNAPSES.

WE WANTED TO THEFT MODEL

DIRECTLY, WHICH MEANS WE NEEDED

TO -- WE ALSO WANTED TO DO IT IN

A TEMPORALLY RESTRICTED MANNER

AS OPPOSED TO RESTRICTING THE

ACTIVITY OF THESE CELLS FROM

BIRTH AS I SHOWED YOU IN A

PREVIOUS SLIDE.

TO DO THAT, WE NEED TO HAVE A

WAY OF ALTERING THE ACTIVITY OF

GENETICALLY DEFINED NEURONS

DURING A DEFINED TEMPORAL TIME

WINDOW AFTER BIRTH.

AND SO WE TURN TO A TOOL CALLED

HM4D.

THIS WAS A G PROTEIN COUPLED

RECEPTOR DEVELOPED BY BRIAN

ROTH.

WHAT HE DID IS TOOK THE MUSTER

INIC COLONNER GICK RECEPTOR AND

HE MUTATED THAT RECEPTOR SUCH

THAT IT NO LONGER BINDS ASEATEL

COALINE BUT CAN BE ACTIVATED BY

A SMALL MOLECULE.

SO THE POINT NOW IS THAT IF WE

CAN TAKE THIS PROTEIN AND

EXPRESS IN A PARTICULAR CELL IN

THE BRAIN AND THEN ADMINISTER

CNO TO THE ANIMAL, WE CAN

SELECTIVELY REDUCE THE ACTIVITY

OF THOSE TAGGED NEURONS.

SO WE TOOK THIS FROM BRIAN ROTH

AND WE PACKAGED IT INTO A CRE

DEPENDENT RECOMBINANT ADDS NO

ASSOCIATED VIRUS WHICH WE CAN

AFFECT THE BRAIN AND DIRECT

EXPRESSION IN CELLS THAT EXPRESS

CRE.

SO LET ME SHOW YOU SOME DATA HOW

THAT WORKS.

THIS IS AN IMAGE OF A TRIATUM OF

ANIMAL THAT HAS BEEN INFECTED

WITH THIS HM4D CONTAINING VIRUS.

IN THIS CASE, IT IS FUSED TO RED

FLORA FORAND YOU CAN SEE

EXPRESSION OF THE PROTEIN WITHIN

THE CELL.

AND JUST IN THE ANECDOTE HERE,

YOU CAN SEE WE CAN TAKE A CELL

THAT SPIKES AND INHIBITS THE

SPIKING BY APPLICATION OF CNL.

THE WAY WE QUANTIFIED IF THE

SYSTEM REALLY WORKS IS TO USE A

COMBINATION TWO OF VIRUSES.

AND SO WHAT WE DID IS INJECT

INTO THE BRAIN OF DIRECT PATHWAY

LABELED ANIMAL, TWO VIRUSES, ONE

THAT ENCODES HM4D THEY JUST TOLD

YOU ABOUT AND THE SECOND ONE

THAT ENCODES ALSO IN A CRE

DEPENDENT MANNER, SO THIS IS A

ION CHANNEL WHICH IS GATED BY

BLUE LIGHT THAT CARL HAS

INTRODUCED TO NEUROSCIENCE AND

MADE A USEFUL TOOL.

SO, WHAT WE SEE IS THAT NEURONS

THAT EXPRESS CHANNELS, WE CAN

TURN ON WITH LIGHT.

SO ESSENTIALLY HERE BELOW, YOU

SEE IF WE RECORD FROM MEDIUM

SPINY NEURON, WHEN WE GIVE A

FLASH OF BLUE LIGHT, WE INCREASE

ACTIVITY OF THE CELL.

THEN WE CAN WASH IN AND REDUCE

THE ACTIVITY OF THE CELL AND NOW

WE HAVE SHOWN ESSENTIALLY THAT

CONSTANT AMOUNT OF DRIVE TO THE

CELL PROVIDED, IS LESS EFFECTED

AT SPIKING THE CELL.

SO CNO IS ABLE TO INHIBIT

ACTIVITY OF EXPELS WE CAN

RECOVER THAT WITH MORE LASER

POWER.

WE HAVE DONE THIS KIND OF

ANALYSIS FOR THE D ONE CRE

ANIMAL T WORKS THERE.

FOR THE D2 IT WORKS THERE AS

WELL AND AS WELL AS AN RPB4-CRET

WORKS THERE AS WELL.

SEE CAN REDUCE THE SPIKING OF

CELLS BY 30-50% USING THIS TRICK

OF HM40 PLUS CNL.

WE LOOKED AT TWO MANIPULATIONS.

FIRST WE INJECTED A SMALL OF

ADNO-VIRUS INTO ONE SIDE OF THE

BRAIN IN THE STRATUM.

SO WE GOT VERY SPARSE INFECTIONS

SO THERE WERE VERY FEW OF THESE

RED HM4D CELLS IN THE STRATUM.

AND THEN WE DELIVERED CN.

TO THERE ANIMAL FROM P8 TO P14

DURING THIS TIME OF MATURATION

AND ASKED WHAT HAPPENS.

ESSENTIALLY WHAT HAPPENED IS

NOTHING.

SO INDIVIDUAL CELLS WHOSE

ACTIVITY HAVE BEEN KNOCKED OUT

DURING THIS TIME OF P8-P15, HAD

NO PERTUBATION OF DENDRITIC

SPINES OR MINIATURE UPSCs.

I'M NOT GOING TO SHOW YOU THAT

DATA.

IT DIDN'T MATTER IF WE INHIBITED

DIRECTLY.

SO THIS KIND OF DATA SAYS THAT

SPARSELY INHIBITING NEURONS HAS

NO EFFECT ON SYNAPSE NUMBER.

IT'S NOT REALLY THE ACTIVITY OF

A SINGLE CELL THAT MATTERS IN

DETERMINING INTERIVATION.

BUT LIKELY THE ACTIVITY OF THE

CIRCUIT.

SO TO PROVE IT IS THE ACTIVITY

OF THE CIRCUIT THAT MATTERS, WE

DID THE CONVERSE EXPERIMENT IN

WHICH WE BILATERALLY INJECTED

LARGE AMOUNTS SO WE COULD INFECT

MOST OF THE DIRECT PATHWAY OR

INDIRECT PATHWAY OF SPINY

NEURONS AND THEN ONCE AGAIN

DELIVERED CN PROMPT THIS PERIOD

TO P15.

YOU CAN SEE THAT NOW WITH THIS

WIDESPREAD INFECTION, WIDESPREAD

PERTUBATION OF CIRCUIT ACTIVITY,

WE CAN NOW CAUSE DIRECT PATHWAY

MEDIUM SPINY NEURONS TO LOSE

HALF THEIR SPINES AND SYNAPSES

AND CAUSE THE PATHWAY NEURONS TO

DOUBLE THEIR SPINES AND DOUBLE

THEIR SYNAPSES.

THIS IS TELLING US THAT ACTIVITY

IN THIS NOW MORE RESTRICTED

DEVELOPMENTAL WINDOW IN A SECOND

POSTNATAL WEEK IS WHAT ACTS TO

GOVERN THE FORMATION OF THE

STRATUM CIRCUIT.

SO HERE IS THE QUANTIFICATION OF

THAT DATA.

WE LOSE ABOUT HALF THE SPINES IN

THE DIRECT PATHWAY WHEN WE

INHIBIT ACTIVITY DURING THIS

TIME WINDOW AND WE GAIN A LOT OF

SPINES IN THE INDIRECT PATHWAY.

A LOT OF NOISE IS IN THIS DATA

BECAUSE THE BASAL DENSITY OF

SPINES AND SYNAPSES IN THE CELLS

IS LOW.

SO IN CONTRAST WHAT I SHOWED YOU

BEFORE, WIDESPREAD INHIBITION OF

SPIKING IN MEDIUM SPINY NEURONS

WITH THIS PHARMACOGENETIC TOOL,

HM40D PLUS CNO, REVEALS PATHWAY

SPECIFICS ON SYNAPSES.

LET ME SUMMARIZE THE DATA I HAVE

SHOWN YOU HERE BEFORE I MOVE ON

TO THE FURTHER ANALYSIS OF THIS

MODEL.

SO I HAVE THRONE YOU GENETIC

INHIBITION OF DIRECT PATHWAY

MINY NEURONS CAUSES

HYPOINVASION.

ON THE OTHER HAND, GENETIC

INHIBITION OF THE INDIRECT

PATHWAY - TYPO THERE.

CAUSES A HYPERINNERIVATION OF

STREETAL MEDIUM SPINY NEURONS.

PHARMACOLOGICAL INHIBITION OF

DIRECTOR INDIRECT PATHWAYS

CAUSES THE SAME EFFECT.

WE ARE PROPOSING THAT GLUTAMATE

DRIVES ISN'T GENESIS AND THEN

FURTHERMORE IT'S THE ACTIVITY OF

THESE INNERIVATING AXONS CAUSING

THE SYNAPSE FORMATION PROCESS TO

GET TRIGGERED AND OF COURSE,

SINCE WE ARE SILENCING THE

OUTPUT OF CELLS BUT SEEING

EFFECTS ON THE INPUTS THIS

SUGGESTS THAT ACTIVITY IS ACTING

THROUGH THE RECURRENT LOOP IN

ORDER TO DRIVE THE FORMATION OF

THIS CIRCUIT.

SO THERE ARE TWO QUESTIONS EM

BEDDED IN THAT PROPOSAL.

ONE IS, IS IT REALLY THE ONGOING

ACTIVITY OF CORTICAL AXONS IN

THE STRATUM THAT DRIVES

FORMATION?

IS IT THE ACTIVITY OF THESE

CORTICAL STREETAL PROJECTION

NEURONS THAT IS DRIVING SYNAPSE

FORMATION?

SO LUCKILY HERE, WE ARE ABLE TO

USE ANOTHER TOOL THAT CHIP GAVE

TO US, THIS RPB4CRE LINE.

THIS SAY BEAUTIFUL MOUSE THAT

EXPRESSES CRE RECOMBINASE IN A

WIDE OR HIGH PERCENTAGE OF

CORTICOSTREETAL PROJECTION

NEURONS.

YOU YOU CAN SEE EXPRESSION OF

THE RED IN THE DEEP PLAYER

CORTICAL STRIATAL NEURONS.

IF WE LOOK IN THE TRIATUM, YOU

CAN SEE DENSE INNERIVATION OF

THE FIBERS.

WE NOW HAVE A TOOL TO MA NIP THE

CELLS THAT PROJECT FROM CORTEX

INTO THE TRIATUM.

IT'S IMPORTANT.

THIS LINE DOESN'T EFFECT

CORTICAL PROJECTION.

SO HERE IS AN IMAGE OF ANOTHER

MOUSE.

THIS IS AN RPB4-CRE MOUSE

CROSSED WITH A SECOND BACK

TRANSGENIC ALLELE.

THIS IS GFP UNDER THE TYPE 2

DOPAMINE PROMOTOR.

SO THIS ALLOWS US TO VISUAL ICE

INDIRECT PATHWAY MEDIUMS AND TRY

TRIATUM WHILE MANIPULATING.

SO IN THIS CASE, WE INJECTED

INTO CORTEX THE SAME ADD NO

ASSOCIATED VIRUS THAT CARRIES

THE HM4 FUSION PROTEIN.

SO IF WE BLOW UP AN IMAGE OF

TRIATUM AND TURN OFF THE GREEN

CHANNEL, WE CAN SEE THIS

BEAUTIFUL PATTERN OF CORTICAL

STRIATAL PROJECTION AXONS.

AND AS CAN SEE, WE CAN HIT A

HUGE NUMBER OF THESE AXONS.

SO NOW WE HAVE A TOOL TO

MANIPULATE THE ACTIVITY OF THESE

CELLS DURING DEVELOPMENT AND ASK

WHAT THE EFFECTS ARE ON INDIRECT

PATHWAYS.

AND SO NOW WHAT HAPPENED

CONSISTENT WITH OUR MODEL IS

WHEN WE REDUCED THE ACTIVITY OF

CORTICAL NEURONS AGAIN DURING

THIS NARROW TIME WINDOW, WE WERE

ABLE TO REDUCE THE INNERIVATION

OF BOTH INDIRECT PATHWAY AND

DIRECT PATHWAY IN MEDIUM SPINY

NEURONS.

CUTTING IT DOWN IN ABOUT HALF.

FURTHERMORE, AS EXPECTED, WE

ALSO LOST DENDRITIC SPINES.

THIS SHOWS THAT THE ONGOING

ACTIVITY OF CORTICAL NEURONS

NORMALLY DRIVES THE PROCESS OF

SYNAPSE FORMATION SUCH THAT WHEN

WE TURN THAT DOWN, WE GET LESS

SYNAPSES.

NOW I WANT TO TAKE A SLIGHT

DETOUR.

THE SECOND PART OF OUR MODEL IS

GLUTAMATE RELEASE IS SUFFICIENT

TO CREATE NEW SIN ASS.

IS IT TRUE THAT EXPOSE TOWER

GLUTAMATE CAN MAKE IT FORM A

SYNAPSE?

THIS IS A QUESTION THAT A

POSTDOC IN MY LAB PREVIOUSLY

ADDRESSED IN CORTEX IN WHICH HE

HAD SHOWN THAT GLUTAMATE WAS

SUFFICIENT TO FORM NEW SYNAPSES.

I WANT TO SHOW YOU THAT DATA

PUBLISHED LAST YEAR.

SO HE HAD BEEN STUDYING THE

PROCESS OF POSTNATAL SYNAPSE

DEVELOPMENT IN THE CORTEX WHICH

IS EQUALLY IMPRESSIVE AS IN THE

STRATUM, IN DAY 6-DAY 21, THERE

IS A MASSIVE INCREASE NOT ONLY

FROM THE COMPLEXITY OF THE

DENDRITE BUT ALSO IN THE

DENNITIES OF THESE DENDRITIC

SPINES AND NUMBER OF GLUTE MA

NERGIC SYNAPSES.

WHAT HE WAS ABLE TO SHOW IS THAT

GLUTAMATE CAN DRIVE THIS

PROCESS.

SO THE TOOL THAT WE USED FOR

THAT IS THIS MOLECULE SLONE ON

THE LEFT.

THIS IS CALLED MNI GLUTAMATE.

SO GLUTE ESTIMATE THIS PORTION

HERE IN WHICH THERE IS ACID HERE

AND THIS IS DERIFF TIDES WITH

THIS AROMATIC LIGHT ABSORBING

GROUP.

WHEN THIS ABSORBS LIGHT, IT

TRIGGERS HYDROLYSIS REACTION

THAT THE BOND RELEASING

GLUTAMATE.

SO USING THIS APPROACH, WE CAN

NOW USURY PULSES OF LIGHT TO

RELEASE GLUTAMATE WHENEVER WE

WANT.

THIS ALLOWS US TO IMAGE A CELL,

SEE A SPINE, AND DELIVER A PULSE

OF GLUTAMATE TO THAT SPINE THAT

CAN ELUCIT REACTION OF GLUTAMATE

RECEPTORS.

SO HE DID THAT.

BUT INSTEAD OF TARGETING SPINES,

HE TARGETED REGIONS OF DENDRITE

NEURONS THAT HAD NO SPINES.

WHAT HE SAW WAS REMARKABLE.

WITH REPEATED STIMULATION OF OF

A SINGLE SITE, OVER 20 SECONDS,

HE WAS ABLE TO VERY QUICKLY

INDUCE THE GROWTH OF A NEW

SPINE.

AND HE DID A LOT OF WORK, WHICH

I'M NOT GOING TO SHOW YOU NOW TO

PROVE THAT THESE NEW SPINES ARE

ASSOCIATED WITH NEW FUNCTIONAL

SYNAPSES.

THEY HAVE BOTH OF THE DOMINANT

TYPES OF GLUTAMATE AND

RECEPTORS.

AND THIS PROCESS OF NEW GROWTH

REQUIRES THE ACTIVATION AND NMDA

RECEPTORS, PKA AND MAP KINASE.

AND THIS IS DEVELOPMENTALLY

REGULATED PROCESS THAT OCCURS IN

YOUNG ANIMALS.

SO HERE IS A SCHEMATIC OF THAT

SIGNALING PATHWAY CRUDELY

OUTLINED IN WHICH GLUTAMATE

ACTIVATES MDA RECEPTORS AND

INCLUDING A COMPONENT OF CALCIUM

RELEASED IN THE VERTIC LUM,

ACTIVATES CALCIUM ACTIVATED

DENDRITE SIGH CLASSES LEADING TO

ACTIVATION OF PKA AND MAP KINASE

AND THE GROWTH OF A NEW SPINE.

NOW WHAT INTRIGUED SUSTHAT

MODULATORS OF PKA ARE PROMINENT

IN THE STRATUM.

SO DOPAMINE IS A MODULATOR OF

PKA.

IT TURNS ON PDA EXPRESSION AND

SHUTS THEM DOWN.

SO THERE IS LOTS OF HITS IN

PATHWAYS OR PARTS OF THIS

PATHWAY THAT MAY BE INVOLVED

WHEN MANIPULATING SYNAPSES.

SO HE HAD SHOWN THAT IN THE

CORTEX DOES. THIS APPLY IN THE

STRATUM?

SO WE RETURNED TO THE STRATUM.

AND LOOKED THAT THE PROTOCOL.

HERE ON THE LEFT, YOU'RE SEEING

A SECTION OF THE STRATUM OF A

D2GFP ANIMAL EXPRESSED IN

INDIRECT PATHWAY NEURONS.

THIS ANIMAL CARRIES ONE OR TWO

COPIES OF THIS PREDEPENDENT RED

REPORTER.

AND THEN VERY SPARSELY INFECTED

THE STRATUM WITH AN ADD NO

ASSOCIATED VIRUS THAT ENCODES

CRE RECOMBINASE.

SO NOW THEY WILL CRESS CR.

AND TURN ON THE RED AND ALLOW US

TO SEE THEIR DENDRITES.

IN THIS WAY, HE WAS ABLE TO TAKE

STRETCHES OF DENDRITE IN WHICH

THERE WERE HOLES, NO SPINES, AND

NOW DELIVER GLUTAMATE AND GROW A

NEW SPINE.

AND THE PROTOCOL IN THE STRATUM

WORKS JUST AS WELL AS IT DID IN

CORTEX AND ABLE TO WITH ABOUT

50% EFFICIENCY, SO 50% OF THE

TRIALS RENDERED A NEW SPINE.

SHE COULD GROW A SPINE IN EITHER

DIRECT PATHWAY OR INDIRECT

PATHWAY.

SO THIS KIND OF DATA SHOWS THAT

GLUTAMATE RELEASED IN THE

STRATUM IS SUFFICIENT TO DRIVE

SYNAPSE FORMATION AND FAVORS A

MODEL WE HAVE BEEN BUILDING UP

OVER THE LAST FEW YEARS IN WHICH

IT IS THE ACTIVITY OF THE AXONS

AS A SOURCE OF GLUTAMATE THAT

FUNDAMENTALLY REMODELS THE

CIRCUIT BY DRIVING SUCH ACTIVITY

DEPENDENCE.

SO I HAVE BEEN TALKING TO BUT

THIS LOOP AND I SHOWED YOU DATA

HOW THE DIRECT PATHWAY AND

INDIRECT PATHWAY CAN INFLUENCE

THE ISN'T GENESIS IN THE STRATUM

AND ACTIVITY-DEPENDENT MANNER.

ONE THING I LEFT OFF THIS

CONVERSATION SO FAR, THE SUBJECT

OF THIS LAST PART OF THE TALK,

IS DOPAMINE ITSELF.

SO I MENTIONED THAT DOPAMINE CAN

MODULATE PKA.

NOW OF COURSE THERE IS A MAJOR

SOURCE OF DOPAMINE WITHIN THE

BASAL GANGLIA.

THIS IS SUBSTANTIAL NIAGRA

COMPACTA WHICH SENDS

DOPAMINERGIC PROJECTIONS TO ALL

NUCLEI WITHIN THE STRATUM AND A

LOT OF THE BRAIN.

BUT VERY DENSELY INNERIVATES THE

STRATUM.

WE KNOW DOPAMINE IS A

FUNDAMENTAL PART OF REWARD

REINFORCEMENT.

THIS IS HOW DRUGS OF ABUSE ACT.

STOW WOULD BE NATURAL TO THINK

THAT DOPAMINE ITSELF MIGHT PLAY

A ROLE IN REGULATING ISN'T

GENESIS IN THIS STRUCTURE.

IN MORE KLIM NARY DATA, THIS IS

WHAT WE FIND.

SO HERE IS AN EXAMPLE OF THE

TRACING OF THE MOVEMENT OF

ANIMAL.

IN THIS CASE, A VERY YOUNG

ANIMAL, A P12 MOUSE.

YOU CAN SEE IT BARELY MOVES

AROUND IN THE CAGE IN AN HOUR.

AFTER WE GIVE IT AN AGONIST OF

THE D1 RECEPTOR, IT TURNS ON

DIRECTLY THE RECEPTOR AND NOW

THIS ANIMAL RUNS AROUND LIKE

CRAZY.

AND THIS IS A, WE THINK PKA

DEPENDENT PROCESS.

BECAUSE IF WE ADMIN STRAIGHT A

BLOCKER OF PKA, ALBEIT NOT A

GREAT BLOCKER, BUT A BLOCKER, WE

CAN PREVENT THIS SKF INDUCED

RUNNING OF THE ANIMAL.

NOW, HERE IS THE DATA.

ON AVERAGE WE GET ABOUT A

TWO-FOLD DIFFERENCE IN THE

LOCOMOTION AF SKF.

WHAT WAS REALLY DRAMATIC IS THAT

IN THAT SINGLE HOUR OF

TREATMENT, THESE CELLS MASSIVELY

GREW NEW FINES AND I'M NOT GOING

TO SHOW YOU THE DATAA -- NEW

SPINES.

THIS IS QUITE CLEAR AND A SALINE

TREATED ANIMAL VERSUS SKF

TREATED ANIMAL AND ON AVERAGE WE

ABOUT DOUBLED THE DENSITY OF

DENDRITIC SPINE IN JUST ONE

HOUR.

THIS IS ALSO OCCURRED WITH THE

NUMBER OF FUNCTIONAL SYNAPSES.

SO TO SUMMARIZE THIS DATA, WHAT

I HAVE SHOWN YOU IS THAT

DECREASING SPIKE AND ELIMINATION

OF GABA RELEASE IN DIRECT

PATHWAY SPINY NEURONS DECREASES

SPINE DENSITY IN SYNAPSES OF

BOTH DIRECT AND INDIRECT

PATHWAYS.

AND THEY HAVE THE OPPOSITE

EFFECT.

FURTHERMORE, DECREASED IN THE

ACTIVITY OF CORTICAL STRATUM

PROJECTION NEURONS INCREASES

SPINE DENSITY HERE AND GLUTAMATE

RELEASES SUFFICIENT TO DRIVE

ISN'T GENESIS IN THE STRATUM.

FURTHERMORE, THE D1 RECEPTOR

ACTIVATION PROMOTES SPINE

DEACTIVATION.

BASED ON THAT, WE PROPOSE THAT

ACTIVITY GUIDES SYNAPSE

FORMATION OF THE BASAL GANGLIA

AND ACTS IN A POSITIVE FEEDBACK

LOOP SO MANIPULATION THAT IS

TURN ON THE DIRECT PATHWAY AND

HENCE TURN ON THE ACTIVITY OF

CORTEX ARE SELF REINFORCING BY

DRIVING MORE ISN'T GENESIS IN

THE STRATUM.

WE THINK THESE MECHANISMS ALLOW

FOR SELF-GUIDED WIRING OF THIS

LOOP THROUGH MULTIPLE STAGES,

ESSENTIALLY AS THE LOOP RANDOMLY

TRIES DIFFERENT CONNECTIONS,

WHEN IT FINDS ONE, IT REINFORCES

THE PROPAGATION OF ACTIVITY

THROUGH THE WHOLE LOOP, THAT

DRIVES MORE SYNAPSE FORMATION.

DORM MEAN CAN ACCEL 38 PROCESS.

POTENTIALLY ALLOWING FOR A

CONTEXT-DEPENDENT COMPONENT TO

THIS REGULATION OF THE LOOP SUCH

THAT FOR EXAMPLE, WHEN ANIMAL

PERFORMANCE LOCO MOTOR ACTION

THAT LETS IT FIND THE MOTHER AND

SUCKEL, THIS WOULD RELEASE

DOPAMINE AND THEREFORE REINFORCE

THAT LOCO MOTOR ACTION.

AND SO WE PROPOSE THAT EARLY IN

DEVELOPMENT THE DIRECT PATHWAY

INNERIVATION OCCURS FIRST IS

ENHANCED WHEN REWARD IS ACHIEVED

AND THAT LATER THE INDIRECT

PATHWAY INNERIVATIONLESS AND

PROVIDE A BRAKE FOR THE SYSTEM.

NOW ON THE LAST FEW MACHINES

THAT REMAIN, I WANT TO -- FEW

MINUTES -- I WANT TO SWITCH

TOPICS.

WE KNOW THAT DOPAMINE ACTS AS A

LONG-TIME SCALE NEUROMODULATOR

BOTH THE STRAIGHTAL FUNCTION AND

BEHAVIOR.

BUT THERE IS STILL A LOT OF

QUESTIONS AS TO WHAT DOPAMINE

DOES IN THE STRATUM.

WE ARE INTERESTED IN THIS

QUESTION.

IS DOPAMINE PURELY A REGULATOR

OF PLASTICITY?

DID IT JUST CHANGE THE STATE OF

THE STRATUM TO ALLOW GROWTH?

OR IS IT ALSO A DYNAMIC

REGULATOR STREETAL ACTIVITY?

NOW IT'S WELL DESCRIBED BY WORK

OF THE GROUPS AND ALSO HAS BEEN

PROPOSED BY DAVID AT COLUMBIA,

THAT DOPAMINE NEURONS ALSO

PRODUCE AND RELEASE GLUTAMATE.

SO BY RELEASING GLUTAMATE, WE

ALREADY HAVE ONE MECHANISM BY

WHICH DOPAMINERGIC NEURONS

QUICKLY ALTER STRAIGHTAL

ACTIVITY.

NIC AND JUN DECIDED TO ADDRESS

THIS PROBLEM.

AND THE WAY THAT ADDRESSED IT IS

AGAIN USING THESE MODERN TOOLS.

THEY TOOK A DOPAMINE TRANSPORTER

CRE DRIVER ANIMALS AND THIS

EXPRESSES CRE AND THEY INFECTED

THAT ANIMAL AGAIN WITH AN ADD NO

ASSOCIATE THE VIRUS BUT NOW THIS

VIRUS CARRIED CHANNEL -- THIS

BLUE LIGHT ACTIVATED CHANNEL.

THEY LET THIS ANIMAL RETURN TO

ITS CAGE FOR 30 TO 100 DAYS AND

LATER CUT AN ACUTE BRAIN SLICE

AND USED BLUE LIGHT TO STIMULATE

THESE FIBERS THAT NORMALLY

RELEASED DOPAMINE AND ARE COMING

FROM SUBSTANCE EYEINGRA.

WHILE RECORDING THE

CONSEQUENCES.

SO HERE IS AN IMAGE OF THE

BRAINS.

HERE YOU CAN SEE A SLICE SHOWING

COMPACT BTAN THIS CASE, HE

INJECTED A VIRUS THAT EXPRESSED

GFP IN A CONDITIONAL NORTHERN

SEE THE SPECIFICITY OF LABELING.

AND YOU CAN SEE ON THE RIGHT,

THE BLOW UP OF THAT IMAGE, A

STAIN OF THE BIOSYNTHETIC

ENZYMES IN THE DOPAMINE PATHWAY

AND THE EXPRESSION OF GFP.

YOU CAN TARGET AND COMPACT A

SECTION OF THIS AREA AND THERE

IS REALLY ALL OF THE GFPs

EXPRESSED IN THE POSITIVE

NEURONS.

I'M NOT SHOWING QUANTIFICATION.

IF WE LOOK IN THE STRATUM, WE

CAN SEE THE FIBERS OF THESE

AXONS SHOWN HERE IN GREEN FROM

THE GFP DENSELY INNERIVATING

LARGE PORTIONS OF THE DORSAL

STRATUM.

SO WHAT NIC DID IS CARBON

FIBER -- TO MEASURE THE BLUE

FLASH.

HERE YOU CAN SEE SCHEMEATIZED

SHORT FLASH OF BLUE LIGHT, A

MILLISECOND LONG, FOLLOWED BY A

TRANSIENT IN DOPAMINE MEASURED

BY CARBON FIBER.

AND IT TURNS OUT THIS TRANSIENT

HAS ALL OF THE PROPERTIES THAT

ONE WOULD EXPECT FOR TRUE

REFLEECE DOPAMINE.

SO IT'S BLOCKED BY A VOLTAGE

GATED SODIUM CHANNEL ANTAGONIST

NECESSARY TO FIRE ACTION

POTENTIALS AND IS BLOCKED

IMPORTANTLY BY TWO DRUGS WE'LL

RETURN TO WHICH ARE ANTAGONISTS.

MONOAMINE TRANSPORTERS.

THIS IS NECESSARY FOR PUTTING

DEEP MEAN INTO VESICLES.

IF YOU BLOCK THAT TRANSPORTER,

AS EXPECTED YOU LOSE DOPAMINE.

THIS MIMICS THE STATE IN WHICH

DOPAMINERGIC NEURONS ARE LOST.

FURTHERMORE, IF WE USE AN

ANTAGONIST OF TYROSINE HYDROXYL.

SO THAT ENZYME FOR DOPAMINE, WE

GREATLY DIMINISH THE RELEASE OF

DOPAMINE QUINCY.

SO HERE IS THE QUANTIFICATION OF

THAT DATA.

WE DON'T NEED TO GO THROUGH IT

ALL BUT THE BOTTOM LINE IS THIS

LOOKS LIKE GOOD VESTICULAR

DOPAMINERGICKIC FIBERS.

IT'S INHIBITED BY TYROSINE

HYDROXYLASE AND VOLTAGE GATED

SODIUM CHANNELS.

BUT MOST IMPORTANTLY IS WHAT IS

SHOWN ON THE RIGHT.

THIS IS KNOCKED DOWN BY ABOUT

TWO-THIRDS BY AN AGONIST.

IT'S KNOWN THAT DOPAMINERGIC

RECEPTORS ACTS AS AN AUTO

RECEPTOR TO LOWER DOPAMINE

RELEASE.

THIS IS CONTROLS SAY ARE GETTING

GOOD RELEASE.

SO NIC INJECTED CURRENTS TOEE

LEASEIT FIRING POTENTIALS AND

WHAT CAN YOU SEE IS WHEN HE

FLASH ITS THE BLUE LIGHT, WHAT

WE SEE IS A PAUSE IN THE

ACTIVITY.

IT WAS SURPRISING BECAUSE IT WAS

NOT EXPECTED IN THE ACTIONS OF

DOPAMINE.

NOR FROM GLUTAMATE WHICH SHOULD

INCREASE EXCITABILITY.

HERE IS A QUANTIFICATION.

IF WE LOOK AT THE INTERSPIKE

INTERVAL BEFORE THE BLUE LIGHT

FLASH, RIGHT AROUND THE BLUE

LIGHT FLASH OR AFTER, YOU CAN

SEE WHEN THE BLUE LIGHT FLASH

COMES, THERE IS A SLOWING OF THE

SPIKE RATE CONSISTENT WITH

INCREASE IN THE INTERSPIKE

INTERVAL AND SURPRISINGLY, THIS

CHANGE WAS INDEPENDENT OF THE

ACTION OF DOPAMINE RECEPTORS.

IF YOU BLOCK RECEPTORS WE STILL

GOT THIS.

INSTEAD, THIS PAUSE WAS

MEDEIATED BY ACTIVATION OF GABA

RECEPTORS SHOWN IN PURPLE BY THE

LACK OF EFFECT AND THE PRESENCE

OF GABA RECEPTOR ANTAGONIST.

THIS EFFECT IS SEEN IN BOTH

DIRECT PATH AND MEDIUM SPINY

NEURONS AND INDIRECT PATHWAYS.

SO WE CONCLUDE FROM THAT

STIMULATION OF DOPAMINERGIC

FIBERS INHIBITS THE FIRING OF

STREETAL PROJECTION NEURONS.

THERE ARE TWO MODELS WE NEED TO

DISTINGUISH HERE.

ONE IS THESE CELLS RELEASE THEE

NEUROTRANSMITTERS.

WE KNOW THEY RELEASE DOPAMINE.

THEY RELEASE GLUTAMATE.

MAYBE THEY RELEASE GABA TWO.

WE CAN CONTRAST THAT INSTEAD

WITH A INDIRECT MODEL IN WHICH

WHAT THIS CELL DOES IS ONLY

RELEASED DOPAMINE AND GLUTAMATE

BUT THESE GUYS ACTIVATE AN

INTERMEDIATE NEURON WHICH GENTLY

RELEASES GABA.

SO WE TURNED TO WHOLESALE

CLAMPINGS TO ISOLATE EITHER THE

GLUTAMATERGIC CURRENT OR THE

GABAERGIC CURRENT IN RED AND

THEN USED PHARMACOLOGY.

WHEN WE APPLIED THIS COMPOUND

AND BLOCKER OF GABA A RECEPTORS,

WE LOSE THE GABA A RECEPTOR

MEDIATED INHIBITORY CURRENT IN

RED WHILE PRESERVING THE

GLUTAMATERGIC SHOWN HERE IN RED.

NOW IF WE ADD LOCKERS OF

GLUTAMATE RECEPTORS, WE

ELIMINATE THE GLUTAMATERGIC.

NOW YOU CAN DO THE EXPERIMENT IN

THE OPPOSITE WAY.

IF THIS WERE IN DISYNAPTIC

MODEL, THEN THE RELEASE OF GABA

WOULD REQUIRE THE RELEASE OF

GLUTAMATE TO STIM 38

INTERMEDIATE CELL.

THAT'S NOT WHAT WE SEE HERE.

WHEN WE ADD ANTAGONISTS, WE LOSE

THE GLUTAMATERGIC RESPONSE BUT

GABA IS PRESERVED.

SO THIS SUGGESTS THAT THE

RELEASE OF GABA IS INDEPENDENT

OF THE ACTION OF GLUTAMATE.

I SHOWED YOU THOSE INDEPENDENT

REACTION OF DOPAMINE.

THIS FAVORS THE DIRECT MODEL.

ANOTHER PIECE OF EVIDENCE IN

FAVOR OF DIRECT MODEL IS IF

RELOOK AT THE LATENCE OF THE

GABA AND GLUTAMATERGIC CURRENT,

THEY ARE IDENTICAL.

THE TIME TO HAVE MAXIMAL

ACTIVATION AND THE TIME TO PEAK,

WE CAN SEE ANYTHING THE IPSC

PRECEDES THE EPS.

THE GABA ACTIVATION COMES BEFORE

THE MA TERGIC.

THIS IS AN ARTIFACT OF THESE

CURRENTS AND SO MUCH BIGGER THAN

THESE.

IT'S NOT TRUE THAT THE

GLUTAMATERGIC COMES FIRST.

FURTHERMORE, WE CAN REALLY SHOWS

THESE ARE INDEPENDENT BY DOING A

LOT OF EXPERIMENTS.

NO REASON TO GO THROUGH ALL OF

THESE.

BUT THE EPSZ BLOCKED BY

GLUTAMATE ANTAGONISTS AND

UNTOUCHED BY GABA RECEPTORS AND

REDUCED BY THIS AGONIST

CONSISTENT WITH DOPAMINERGIC

FIBERS.

IPSZ BLOCKED BY TWO SEPARATE

ANTAGONISTS AND INDEPENDENT GABA

C RECEPTORS AND INDEPENDENT

GLUTAMATE RECEPTORS AND

SENSITIVE D2 RECEPTORS.

I'M NOT SHOWING YOU ALL THE

THINGS WE HAVE DONE BUT WE CAN

DISCOUNT THIS MODEL AND FAVOR

THE DIRECT RELEASE.

NOW THIS WAS GOOD NEWS.

BECAUSE WE ALREADY KNEW HOW TO

ELIMINATE GABA RELEASE FROM A

NEURON.

I SHOWED THAT YOU BEFORE.

WE HAVE THIS GABA TRANSPORTER

CONDITION ALLELE.

WE CAN LOSE THAT PROTEIN AND

LOSE GABA RELEASE.

SO, WE SAY OKAY, VGAS WHAT

PATCHES GABA INTO VESKELS.

LET'S CROSS THE DOPAMINE

TRANSPORTER CRE ANIMAL AND

GENERATE A MOUSE THAT CANNOT

RELEASE GABA FROM DOPAMINERGIC

NEURONS.

SO NIC WENT IN TO CONFIRM THIS

WAS TRUE AND IN CONTROL WE CAN

GET THE GABAERGIC CURRENT IN RED

AND THE WE FOUND THE VGAT

CONDITIONAL ANIMAL HAS PERFECTLY

GOOD GABA RELEASE.

AND THIS IS REALLY GABA A

RECEPTOR ACTIVATION BLOCKED BY

THE GABA A RECEPTOR ANTAGONIST.

THIS WAS PUZZLING.

WE WENT AHEAD AND MATE MADE THE

VGLUT2 ANIMAL BECAUSE WE CAME UP

WITH THEORIES.

WE MADE THIS ANIMAL AND WE FOUND

THAT OF COURSE WE ABOLISHED THE

GLUTAMATE RESPONSE.

WE DOCK DO THIS KIND OF

EXPERIMENT.

BUT THE GABA RESPONSE IS STILL

THERE.

WE HAVE A SITUATION IN WHICH THE

RELEASE OF GABA IS INDEPENDENT

OF THE GABA TRANSMITTER.

AND SO, WE HYPOTHESIZED THERE IS

ANOTHER TRANSPORTER THERE.

THE MONOAMINE TRANSPORTER IS

PRESENT.

IT NORMAL PATCHES DOPAMINE.

THIS IS V MAT 2 EXPRESSED.

WE SAID MAYBE THAT PATCHES GAB A

THAT'S WHAT WE FOUND.

WE USED THREE DIFFERENT

ANTAGONISTS OF THE MONOAMINE

TRANSPORTER AND ABOLISHED THE

RESPONSES.

SO HERE IT IS REVERSIBLE AND WE

TREATED THE ANIMAL BEFORE GABA

RESPONSES ARE GONE.

USING TETRABEANZINE, REVERSAL

ANTAGONIST, WE CAN BLOCK IT AND

A THIRD ANTAGONIST.

SO THREE DIFFERENT COMPOUNDS ALL

WHICH TARGET VMAT2.

NOW I'LL QUICKLY MOVE THROUGH

THIS BECAUSE I'M RUNNING LONG.

IF DOPAMINERGIC NEURONS MAKE

GABA AND USE V MAT 2 TO PACKAGE

IT, WE SHOULD BE ABLE TO BLOCK

VMAT2 AND REPLACE ACTIONS BY

PUTTING VGAT INTO THESE CELLS.

SO WE MADE A VIRUS THAT

EXPRESSED VGAN A PRECONDITIONAL

MANNER, INFECTED THESE CELLS AND

THEN TREATED THEM.

AND THEN WE COULD GET MAMMOTH

GABA RELEASE IN THESE CELLS.

SO THIS SUGGESTS THAT

DOABA -- DOPAMINERGIC NEURONS

HAVE EVERYTHING THEY NEED TO

RELEASE GABA.

FURTHERMORE, THE LATENCY OF

THESE RESPONSES WERE CONSISTENT

WITH DIRECTORY LEASES.

SO VMAT2 IS NECESSARY FOR GABA

RELEASE BUT IT CAN BE REBLADES

BY VGAT.

LASTLY, IF VMA2 IS A GABA

TRANSPORTER THEN IT SHOULD BE

ABLE TO REPLACE VGAT.

SO WE TOOK MEDIUM SPINY NEURONS

AND GENERATED A ANIMAL THAT

LACKED VGAT AND INFECTED THESE

CELLS WITH BOTH A VIRUS OR

INFECTED THESE CELLS AND IF WE

EXPRESS OR RECORD FROM A

NEIGHBORING NEURON, WE CAN NOW

GET LIGHT OF OAK GABA RELEASE

BLOCKED BY GABA RECEPTOR

ANTAGGISTANIST.

IF WE RECOMBINE VGAT AS I SHOWED

YOU IN THE FIRST HALF OF THE

TALK, WE CAN ABOLISH IT AND NOT

FULLY ELIMINATE.

THIS IS GABA RELEASE DEPENDENT

ON VGAT.

NOW IF WE GO IN AND INFECT THE

SAME AS WELL AS A VIRUS THAT

ENCODES, WE CAN PARTIALLY RESCUE

THIS GABA RELEASE.

MANY REASONS WHY THERE SHOULD BE

A PARTIAL RESCUE BECAUSE WE HAVE

TO FAVOR NOT PULLING ACTIVITY OF

THE CELLS FLORIDA TO GET THIS ON

TO WORK.

WE SEE THE VMA2 CAN PARTIALLY

RESCUE GABA RELEASE IN A NEURON.

NOW BASED ON ALL OF THAT, WE

COME TO THE CONCLUSION FROM A

LOT OF GENETICS AND

PHARMACOLOGIC EXPERIMENTS, WE

HAVE SHOWN THAT STIMULATION OF

DOPAMINERGIC FIBERS ACTIVATES

GABA RECEPTORS AND INHIBITION IS

VIA DIRECTORY LEASE OF GABA AND

INDEPENDENT OF VGAT BUT REQUIRES

VMA2 AND IT CAN RESCUE GABA

RELEASE.

THIS LEADS US TO CONCLUDE THAT

VMA2 IS NECESSARY AND SUFFICIENT

FOR VESICULAR GABA RELEASE.

AND THAT VMA2 IS A VESICULAR

GABA TRANSPORTER.

SO WHAT?

IS IT A CURIOSITY?

IT'S MORE MORE THAN THAT.

DOPAMINERGIC NEURONS MEDIATE

INHIBITION OF THE STRATUM.

THIS NEEDS TO TAKE INTO ACCOUNT

AND ALTER OUR MODELS OF

REINFORCEMENT LEARNING.

SECONDLY, MANY MONOMA NERGIC

NEURONS INCLUDING HIS TA MEAN,

NORADRENALINE AND 5HT ALL RELY

ON GABA.

WE BELIEVE MANY OF THESE ARE

LIKELY TO RELEASE GABA AS WELL.

EVEN IF THEY DON'T EXPRESS VGAT.

IT'S NOT NECESSARY FOR THIS

PROCESS.

I THINK THIS MAY BE QUITE

IMPORTANT ALSO FOR HUMAN

DISEASE.

THIS SUGGESTS OR INDICATES THAT

THE PARK IN STONIAN STATE

INCLUDES A LOSS OF GABA RELEASE.

SO MAY BE SOME OF THE LONG TERM

HYPEREXCITABILITY SEEN IN THE

STRATUM IN THE PARK INSONIAN

STATE, REALLY REFLECTS THE

CHRONIC LOSS OF GABA IN THAT

CIRCUIT AND THEREFORE THE

HYPEREXCITABILITY.

LASTLY, NEUROLEAPTIC DRUGS USED

TO TREAT SCHIZOPHRENIA AND MANY

OTHER PSYCHIATRIC DISEASES ARE

AGONISTS.

AND THEY WILL REDUCE DOPAMINE

RELEASE BUT ALSO TO INHIBIT GABA

RELEASE.

THIS MAY HAVE CONSEQUENCES FOR

THE MECHANISM OF ACTION FOR THE

ACTIVITY.

SO THAT'S IT.

LET ME GIVE THANKS.

WE HAVE TECHNICAL SUPPORT IN THE

LAB TA MAKES THESE PROJECTS

POSSIBLE.

THESE ARE DOUBLE AND TRIPLE

TRANSGENIC ANIMALS AND MODULATED

WITH VIRUSES AND WHATEVER ELSE.

SO TECHNICIANS, CAROLINE AND

JESS HELPED TO GET THIS GOING.

BRAD GAVE US VGAT CONDITIONAL

ANIMAL AND IMPORTANT

COLLABORATOR IN THIS PROJECT.

CHIP AND NAT FOR ALL THE BACK

TRANSGENICS THAT MAKE THIS KIND

OF STUDY POSSIBLE.

BRIAN GAVE US THE HM4D AND CARL

THE CHR2 AND THE BACKBONE WE USE

TO MAKE THINGS LIKE THE VGA.

AND RESCUE VIRUSES AND VIRUSES

WERE MADE MOSTLY AT UNC AND THEN

FUNDING.

SO THIS IS THE LAB.

I ALREADY POINTED OUT NIC AND

JUN WHO DID DOPAMINE INHIBITION

WORK.

RP SANDERS AND JENIA WHO DID

THIS CIRCUITING AND MODELING

WORK AND QUAN WHO DID THE

CORTICAL WORK LAST YEAR.

THANK YOU.

[ APPLAUSE ]

>> THIS MAY BE A LITTLE OFF THE

TOPIC BUT IT WAS A GREAT TALK.

I HAVE TWO IDENTICAL TWIN

GRANDCHILDREN, BOYS.

AND THEY BOTH ARE QUITE BRIGHT,

THEY COMMUNICATE WELL.

THEY UNDERSTAND A LOT.

BUT THEY WERE VERY SLOW AT

LEARNING TO TALK TO PEOPLE THAT

ONLY UNDERSTAND ENGLISH.

SO, MY DAUGHTER, THEIR MOM,

DECIDED TO SEPARATE THEM MORE

OFTEN SO THEIR DAD WOULD TAKE

ONE OFF TO ONE ACTIVITY AND THE

OTHER TO ANOTHER OR THEY DID THE

SAME WHEN THEY WERE PLAYING WITH

OTHER CHILDREN.

THEN THEIR ABILITY TO SPEAK

ENGLISH STARTED TO PICK UP

QUICKLY.

SO WHAT I WONDERED IS, ONE, IS

THERE ANY UNDERSTANDING ON A

MOLECULAR LEVEL?

AND TWO, IS IT TOO MUCH OF A

LEAP TO ASK HOW YOUR WORK MIGHT

INFLUENCE EARLY LEARNING?

>> WELL, OF COURSE IT'S PURE

SPECULATION ON MY PART BECAUSE I

HAVE NO DATA TOWARDS THAT.

I THINK THAT THE WORKING HAVE A

REAL INFLUENCE ON EARLY LEARNING

BECAUSE WE THINK THAT VIA THESE

MECHANISMS, PATTERNS OF ACTIVITY

THEY TURN ON PARTICULAR PARTS OF

THE BRAIN MAY REINFORCE THE

ACTIVITY IN THAT PART OF THE

BRAIN.

SO PARTICULARLY WHEN YOU'RE

THINKING ABOUT TWO KINDS OF

DISEASES, ONE IS ADHD, WHERE ONE

COULD SPECULATE THAT SOME EARLY

MANIPULATIONS EITHER

PHARMACOLOGICAL OR ENVIRONMENTAL

OR STRESS, MIGHT LEAD TO

ACTIVATION OF DIRECT PATHWAY IN

A WAY THAT REINFORCES ITSELF.

I DIDN'T SHOW THAW THESE

PERTUBATIONS ARE MAINTAINED

TOWARDS LATER ADULTHOOD.

THAT'S ONE POSSIBILITY.

THE ROLE OF THE BASAL GANGLIA

AND LANGUAGE AND THESE KINDS OF

THINGS ARE STILL UNCLEAR.

ALTHOUGH THERE IS EVIDENCE

EMERGING IT DOES PLAY A ROLE.

SO THERE COULD BE BUT I DON'T

HAVE ANY DATA.

ON THE RIGHT.

MY RIGHT.

>> THANK YOU FOR THIS VERY

BEAUTIFUL TALK.

I REALLY ENJOYED IT.

TO SEPARATE INTERSTATEAL

EFFECTS -UE SUGGESTED A CHANGE

IN FIRING RATES OF THE OUTPUT.

DID YOU RECORD ACTIVITY IN THOSE

MICE?

>> RIGHT.

>> DID YOU FIND THAT PREDICTION?

>> SO THAT'S A VERY IMPORTANT

THING.

I PRESENTED THIS LOOP AS IF

THESE CHANGES IN ACTIVITY

OCCURRED.

I HAVEN'T SHOWN YOU THEY OCCUR.

AND IN FACT I CAN'T.

YOU HAVE TO REMEMBER THESE ARE

ANIMALS ABOUT P8-P12.

WHEN ANIMALS ARE PLACED UNDER

ANESTHESIA, THE MAJORITY OF

RECURRENT ACTIVITY IN THE BASAL

GANGLIA SHUT DOWN AND THE

STRATUM BECOMES QUIET.

SO IN ORDER TO DO THE EXPERIMENT

WE SUGGEST, WE WOULD HAVE TO

RECORD FROM THE P8-P12 ANIMAL

UNANESTHETIZED AND PRESUMABLY

ENGAGE IN A TASK TO TURN ON THE

STRATUM.

AND I THINK THAT IS QUITE AN

IMPOSSIBLE THING TO DO.

SO WE DON'T KNOW THAT.

WE DO KNOW THAT IN ADULTS, WE

HAVE DONE MANY OF THESE

EXPERIMENTS THE LOOP ACTS IN A

WAY THAT IT SHOULD ACT AND AS

PROPOSED BY THESE MODELS.

BUT WE DON'T KNOW THAT IN YOUNG

ANIMALS.

THE LOCO MOTOR EFFECTS MAKE ME

BELIEVE THAT IS TRUE.

I HAVE NO WAY OF ADDRESSING

THAT.

THE SECOND QUESTION THAT YOU

ASKED WAS ABOUT INTERSTATEAL

CONNECTIVITY.

AND YOU'RE RIGHT, MEDIUM SPINY

NEURONS SEND AXONS WITHIN THE

STRATUM TO INHIBIT.

THE DEGREE OF INHIBITION IS

ESSENTIALLY THE SAME.

SO IT WOULD BE HARD TO EXPLAIN

HOW THAT COULD EXPLAIN OUR

RESULTS.

FURTHERMORE, WE WANT ON TO

DIRECTLY MANIPULATE TORE KICKS

AND SAW THE SAME THING.

WE THINK IT IS CURRENT ACTIVITY

IN THE LOOP.

THESE ARE VERY IMPORTANT CAVEATS

AND WE DO ADDRESS THESE MORE

THROUGHLY LATER.

>> AGAIN, MAYBE IT'S DIFFICULT

TO PERFORM THE EXPERIMENT BUT I

WAS WONDER IF YOU GO HAVE SOME

IDEA IF THE EXTRACELLULAR AMOUNT

OF DOPAMINE IS DIFFERENT BETWEEN

THOSE TWO DIFFERENT MICE GIVEN

THAT'S PART OF THE LOOP AND

SECOND, THERE IS A LOT OF

LITERATURE THAT SUGGESTS THAT

INTERACTION BETWEEN THE D1 AND

DA RECEPTORS SYSTEM THAT COULD

BASICALLY EXPLAIN A LOT WHAT

HAVE YOU SEE WITH REGARD TO

GLUTAMATERGIC TRANSMISSION.

>> THE FIRST PART, I ACCEPT

THERE MAY BE CHANGINGS IN

DOPAMINE.

VERY HARD TO DO THAT KIND ENVY

OH.

THE SECOND PART I DON'T QUITE

GET.

I DON'T UNDERSTAND WHAT EXACTLY

THAT ALTERNATIVE WOULD BE.

>> IF SPINY MEDIUM NEURONS HAS

PARTICULAR EXTRA -- POST

SYNAPTIC D1, YOU MAY THINK THAT

ACTIVATION COULD LEAD TO THE

INCORPORATION OF NDA RECEPTORS

FOR THE SURFACE WHILE THE ONES

THAT ARE ENRICHED IN D2, IF

ACTIVATED BY DOPAMINE, WOULD

HAVE NEGATIVELY COUPLED AND NOT

PROMOTE --

>> RIGHT.

SO I THINK WHAT ARGUES AGAINST

THAT MODEL IS THAT WHEN WE

INHIBIT THE DIRECT PATHWAY, WE

SEE CHANGES IN BOTH THE DIRECT

AND INDIRECT PATHWAY.

WHICH ARE OCCURRING IN THE SAME

ANIMAL AND SAME CONTEXT AND

EXPERIENCED IN THE SAME AMOUNT

OF DOPAMINE.

I DON'T THINK IT HAS ANYTHING TO

DO WITH POST SYNAPTIC RESPONSE

TO THE CONTEXT BUT RATHER THE

PRESYNAPTIC MANIPULATION.

>> SO, YOU MENTIONED SOME 50s

OF AGONISTS -- EFFECTS -- OF THE

D1 RECEPTOR ON SPINE DENSITY AND

SO FORTH.

I WANTED TO KNOW WHETHER THERE

IS DEFINED WINDOW OF PLASTICITY

WHERE THIS OCCURS OR WHETHER YOU

EXPECT THESE EFFECTS TO BE TRUE

ALSO IN ADULTS?

>> YES, I THINK THAT'S A GREAT

QUESTION.

IN CORTEX, WHAT WE HAVE SHOWN IS

THAT THE ABILITY OF GLUTAMATE TO

INDUCE THE GROWTH OF THE SPINE

FALLS OFF DRAMATICALLY.

THAT'S IN THE SENSORY CORTEX IN

WHICH YOU IMAGINE THERE SHOULD

BE A WINDOW OF POST SUSTAINABLE

DEVELOPMENT THAT REFINES THE

ABILITY OF THE ANIMAL TO DETECT

THE SENSORY DEVELOPMENT.

THE BASAL GANGLIA IS DIFFERENT.

I FAVOR YOU WHAT SUGGESTED WHICH

IS THAT PLASTICITY WILL REMAIN

IN THAT STRUCTURE UNTIL LATER.

WE HAVEN'T TEST TODAY YET.

BUT THE GAZEAL GANGLIA IS WHAT

ALLOWS US TO FORM NEW

ASSOCIATIONS BETWEEN SENSORY

ENVIRONMENT AND REWARD

THROUGHOUT LIFE.

AND THAT SUGGESTS THAT THIS IS A

PLASTIC STRUCTURE THAT SHOULD BE

RESPONDING TO DOPAMINE BY

MANIPULATING THROUGHOUT LIFE.

WE NEED TO EXAMINE THAT.

WE FAVOR THAT MODEL BUT HAVEN'T

SHOWN IT YET.

>> I HAD A QUESTION RELATED TO

THE NATURE OF THE LOOPS AND

INTRODUCTION SAID THAT THOUGH

YOU SHOW A SINGLE LOOP, IT'S IN

RELATE MULTIPLE PARALLEL LOOPS.

SO WHAT I WAS CURIOUS ABOUT IS

WHEN YOU DO THESE BROAD

MANIPULATIONS, WHY IS THE

REINFORCEMENT SO SPECIFIC IN THE

OUTCOME ON CERTAIN MOTOR

BEHAVIOR AND CAN YOU SELECTIVELY

STIMULATE PARTS OF THAT THING TO

GET MORE SELECTIVE

REINFORCEMENT?

>> SO THESE ARE VERY GOOD

QUESTIONS TO WHICH I DON'T KNOW

THE ANSWER.

THE TOPOGRAPHY THE LOOPS AND

STRATUM IS FUZZY.

IT'S NOT LIKE IN SENSORY CORTEX

AND THE VISUAL SYSTEM WHERE

THERE IS REALLY POINT TO POINT

PROJECTIONS.

SO THERE IS A GENERAL TOPOGRAPHY

THERE THAT IS NOT AS TIGHT AND I

THINK THAT THAT NATURALLY LEADS

TO DIVERGENCE IN THE CIRCUIT.

OF COURSE WE STARTED WITH

RELATIVELY GROSS MANIPULATIONS

TO SEE AN EFFECT.

THESE ARE EFFECTS THAT WERE

LIMITED TO DORSAL AND LARGELY

DORSAL MEDIAL STRATUM.

SO WE DIDN'T EFFECT THE VENTAL

STRATUM OR ANY OF THE STRUCTURES

AND WE HAVEN'T ANALYZED THEM AT

ALL.

THIS IS SPECIFICITY TO IT.

WE ARE NOW TRYING TO DO YOU WHAT

SUGGEST.

AND I THINK THAT EXPERIMENT IS

BEST DONE BY STARTING IN CORTEX.

VERY SMALL FOCAL MANIPULATIONS

AND TRACING THE PATHWAY THROUGH

AND WE HOPE TO BOTH DEFINE THE

SCALE OF THE LOOP AND TOPOGRAPHY

AS WELL AS SEE IF THESE EFFECTS

ARE PRESERVED.

THANK YOU FOR COMING.

[ APPLAUSE ]