Joseph Cichon, MD, PhD. February 2026

Transcript

• 00:00Joe gets his his time.
• 00:01So welcome everyone,
• 00:03to our February meeting of
• 00:04the, program for psychedelic science
• 00:06seminar series.
• 00:07Our speaker, Joe Chichon or
• 00:09I'm sorry. Chichon Sichon?
• 00:12Shishon.
• 00:13Sishon. I apologize.
• 00:15I'm sorry.
• 00:16Was,
• 00:17suggested and invited by, Pasha
• 00:19Davoodian, and I wanna invite
• 00:21Pasha to introduce the speaker.
• 00:24Thanks, Chris.
• 00:25Yeah. So we're very excited
• 00:26to have, Joe Sichon, MD
• 00:29PhD, assistant professor of anesthesia
• 00:31and neuroscience from UPenn here
• 00:33today.
• 00:34He earned his MD PhD
• 00:35at NYU working with one,
• 00:38doing some, like, really nice
• 00:39fundamental work on dendritic mechanisms
• 00:41of learning and memory.
• 00:43He then went on to
• 00:43complete an anesthesia
• 00:45residency at Penn, including, a
• 00:47research focus
• 00:48where he has since stayed
• 00:50on as an assistant professor.
• 00:52His main focus so far
• 00:53has been on rapid acting
• 00:55anesthetics as well as psychedelics
• 00:57and how they modulate neural
• 00:58circuits and health and disease
• 01:00with the goal of informing
• 01:01next generation,
• 01:02treatments for both anesthesia and
• 01:04antidepressants.
• 01:05In today's talk, he'll mostly
• 01:06talk about NMDA receptor antagonism
• 01:08work in aesthetics that he's
• 01:09done,
• 01:10in changing otherwise treatment resistant
• 01:12neuropsychiatric
• 01:13diseases.
• 01:14He uses several state of
• 01:15the art imaging approaches that
• 01:17are very unique and enable
• 01:18real time measurement of neural
• 01:19physiology during drug treatment at
• 01:21the level of single neurons,
• 01:22but also at the level
• 01:23of networks
• 01:24and synapses and, some really
• 01:26nice work hopefully illustrating how,
• 01:28mechanisms of ketamine and nature's
• 01:30oxide oxide engage in some
• 01:31distinct cellular and circuit level
• 01:33mechanisms to drive their respective
• 01:35rapid acting
• 01:37effects. So with that, I
• 01:38will not take any more
• 01:39time. The floor is yours,
• 01:40Joe.
• 01:41Thank you so much. Thank
• 01:43you to the Yale, Psychedelic
• 01:45Center,
• 01:45and Pasha for extending this
• 01:47invitation. This is truly a
• 01:49pleasure,
• 01:50and I am I am
• 01:51very
• 01:52overwhelmed by the invitation.
• 01:56So,
• 01:58with that, I, you know,
• 01:59I have no financial disclosures,
• 02:00but the one true disclosure
• 02:02is that I'm very much
• 02:03still in the lab doing,
• 02:05experiments on a daily basis,
• 02:07and I am briefly,
• 02:10completely engaged,
• 02:11in the data, and I've
• 02:13been told that I tend
• 02:14to present as if I'm
• 02:15a graduate student giving a
• 02:16data blitz.
• 02:17So, with that said, I
• 02:19will, try my best to
• 02:21not get, completely captivated by
• 02:23the data.
• 02:25So the lab is very
• 02:27much interested in how
• 02:29anesthetics with psychedelic properties and
• 02:31also psychedelics themselves
• 02:33induce,
• 02:35rapid and durable,
• 02:37corticoplasticity
• 02:39mechanisms.
• 02:40And I think many of
• 02:41you are also really interested
• 02:43in how certain drugs that
• 02:45are given for a very,
• 02:46very transient duration
• 02:48induce
• 02:49rather long lived responses in
• 02:51the brain, whether that's symptomatic
• 02:53improvements related to depression
• 02:55or
• 02:56behavioral changes that are beneficial,
• 02:59to the individual.
• 03:01How these drugs, such as
• 03:03ketamine,
• 03:05no, historically nitrous oxide, but,
• 03:08again, reemerging as an rapid
• 03:09anti
• 03:10antidepressant,
• 03:13and and psilocybin, LSD alike.
• 03:15How can they induce,
• 03:17such rapid changes in neural
• 03:18activity and changes in brain
• 03:20state and how this sets
• 03:21up,
• 03:22these these interesting,
• 03:25behavioral features and symptomatic,
• 03:27improvements.
• 03:29And so the the working
• 03:30hypothesis for how,
• 03:33this all happens,
• 03:36for my lab is related
• 03:38to activity
• 03:39dependent synaptic plasticity,
• 03:41and that changes in neural
• 03:43activity
• 03:44induce,
• 03:47distinct,
• 03:48receptors,
• 03:49engagement
• 03:50leading to, calcium entry, for
• 03:52example,
• 03:53leading to changes in gene
• 03:55expression,
• 03:56changes in kinase,
• 03:57leading to restructuring of cytoskeleton,
• 04:00and potentially the birth of
• 04:02new connections.
• 04:03And what's
• 04:04really fascinating is that these
• 04:06types of
• 04:07changes occur over different time
• 04:08scales,
• 04:09and how these drugs,
• 04:11that we find interesting
• 04:13induce these changes in these
• 04:14distinct steps is is is
• 04:16still rather unclear.
• 04:18And the way in which
• 04:19the lab has addressed, some
• 04:21of these,
• 04:24preclinical
• 04:25models using mouse,
• 04:27largely because we have
• 04:29access to unique cell types.
• 04:31We can understand connectivity,
• 04:33at the level of individual
• 04:35neurons and even local circuits,
• 04:37and then
• 04:38and we can also then,
• 04:40modulate,
• 04:41many of these distinct cells
• 04:43and and see their,
• 04:45consequences,
• 04:46to to circuit function and
• 04:48behavior.
• 04:52And my lab really got
• 04:54hooked,
• 04:56into this,
• 04:58outstanding question
• 05:01and and and and thinking
• 05:02about this was through work,
• 05:05done, you know, largely at
• 05:07Yale and at the NIH,
• 05:10where,
• 05:11you know, two separate groups
• 05:12independently,
• 05:14determined that subantanacetamin
• 05:16so this is not an
• 05:17anesthetic dose of ketamin. Subantanacetamin
• 05:19given over forty minutes to
• 05:20an hour induces a rapid
• 05:23change in, antidepressant
• 05:25symptoms
• 05:26that emerge within minutes upon
• 05:28completing, the infusion.
• 05:31And you can see here,
• 05:34that there's a distinct separation
• 05:35between the placebo group and
• 05:37the ketamine group that,
• 05:39spans,
• 05:40within the first day of
• 05:41receiving the treatment, and that
• 05:43lasts,
• 05:44for multiple days, if not
• 05:46weeks, in some patients.
• 05:49And this is, you know,
• 05:51amazing considering that this was,
• 05:53initially classified
• 05:55as an anesthetic.
• 05:57And what I found really
• 05:58unique, just given my,
• 06:01clinical training in anesthesia,
• 06:03is that,
• 06:05indeed, during this infusion, this
• 06:06sub hypnotic infusion, the patient's
• 06:08not anesthetized. They're able to
• 06:10respond.
• 06:12They experience a unique state,
• 06:14which we call disassociation.
• 06:15And it's, in fact, it's
• 06:17this property that gave ketamine
• 06:19the classification as a disassociated
• 06:21anesthetic
• 06:22because at these subanesthetic doses,
• 06:24it has this profound,
• 06:27induction of a different brain
• 06:29state. And what what I
• 06:30mean by disassociation
• 06:32is that it, it distorts,
• 06:35various different,
• 06:36percepts
• 06:37of of the world. It
• 06:39it distorts the your
• 06:41representation of body.
• 06:43It disconnects you from your
• 06:44environment, disconnects you from time.
• 06:47You become
• 06:49depersonalized,
• 06:50you realize, and you start
• 06:51to experience,
• 06:53even,
• 06:54illusions or hallucinations. And that
• 06:56this experience is often just
• 06:58tied to the drug exposure,
• 06:59maybe,
• 07:01trickles into periods after the,
• 07:04drug exposure,
• 07:05but it's eventually resolved,
• 07:07once the drug is supposedly,
• 07:10cleared and metabolized.
• 07:13And I'm gonna revisit this
• 07:14whole idea of this association.
• 07:16And so this was a
• 07:17remarkable finding because ketamine in
• 07:19this patient population did nearly
• 07:22twice as good as standard
• 07:24of care, which would be
• 07:25an SSRI.
• 07:26And in fact, it's really
• 07:28the
• 07:28single application of ketamine that
• 07:30was doing this,
• 07:34effect.
• 07:36And as a as a
• 07:36basic scientist, I I began
• 07:39to gauge literature and try
• 07:40to figure out exactly what
• 07:42people thought of how these
• 07:45rapid and sort of more
• 07:46durable effects
• 07:48are happening.
• 07:49And,
• 07:50I'm gonna make this very
• 07:52superficial, but the thinking is
• 07:53is that ketamine is an,
• 07:55NMDA blocker specifically targeting open,
• 07:58NMDA channels,
• 08:00and, this blockade preferentially happens
• 08:03on GABAergic interneurons
• 08:05leading to a suppression of
• 08:07interneuron activity
• 08:08that would subsequently
• 08:10drive
• 08:11excitatory
• 08:12activity.
• 08:13Excitatory activity dumping glutamate would
• 08:16then drive in subsequent cells,
• 08:19signaling changes,
• 08:20protein gene expression, protein expression,
• 08:23and potentially
• 08:24new synapse formation.
• 08:27So with these sort of
• 08:28activity changes underlying the initiating
• 08:30mechanisms
• 08:31followed by new formation of
• 08:33synapse
• 08:34underlying
• 08:35the sustaining mechanisms.
• 08:38If you follow this logic,
• 08:40then you might think, well,
• 08:42maybe we can do better
• 08:43than ketamine. Maybe we can
• 08:44design out the disassociation
• 08:46with very specific,
• 08:48NMD antagonists or NMD modulators.
• 08:50And indeed,
• 08:52you know, academics
• 08:53and and and pharma companies
• 08:55have thought deeply about this
• 08:57question and have, engineered,
• 09:00you know, amazing compounds
• 09:02that in preclinical models show
• 09:04efficacy, but in in in
• 09:06human
• 09:08trials, just fall short.
• 09:10And so,
• 09:11acknowledging all these efforts, through
• 09:14the years, I thought about
• 09:15this question slightly different.
• 09:18And this was also motivated
• 09:20by a bunch of papers
• 09:21coming, subsequently from the the
• 09:23Zarate
• 09:24group, at the NIH
• 09:26where they found an association
• 09:28between the dissociative
• 09:30state and the fact that
• 09:31if you experience this state,
• 09:32you might have a more
• 09:33robust sustained antidepressant
• 09:35response.
• 09:38And so I was curious,
• 09:40in a mouse, can we
• 09:41determine when that happens?
• 09:43And so I devised a
• 09:45series of behavioral experiments to
• 09:47see if I can pinpoint
• 09:48exactly where a mouse becomes
• 09:52disassociated.
• 09:53So so here you have
• 09:54a mouse
• 09:55in tail suspension. Tail suspension
• 09:57is often used as a,
• 09:59behavioral test to assess for
• 10:01learned helplessness.
• 10:02Here you have a mouse
• 10:03hanging by its tail,
• 10:05very commonly done, and you
• 10:06see that the mouse attempts
• 10:07to escape, and they do
• 10:08this for a period of
• 10:09time. This is considered,
• 10:11mobility, and then it interdispersed
• 10:13between,
• 10:14active escape events. You have
• 10:15these periods of immobility where
• 10:17the mice,
• 10:18sort of just give up,
• 10:19and and then they have
• 10:20a resurgence of wanting to
• 10:22escape and try to, curl
• 10:24and and and swing and
• 10:26and try to get out
• 10:26of this uncomfortable position.
• 10:29And what you find here
• 10:30at ketamine at a certain
• 10:32dose, it's very high relative
• 10:33to humans, and we can
• 10:34go into that later. You
• 10:35find that the mouse completely
• 10:37loses that escape behavior.
• 10:40There's no,
• 10:41attempt to escape.
• 10:43In fact, they sort of
• 10:44hang there,
• 10:46and you see this interesting
• 10:47head twitch. And this is
• 10:48very distinct from something that
• 10:50psilocybin or LSD would do,
• 10:52which would be a rotational
• 10:53head twitch, and a mouse
• 10:54was like like a dog
• 10:56shaking off its wet, wet
• 10:58fur
• 10:59to get dry. This, you
• 11:01see it's sort of vertical
• 11:02and that the how the
• 11:03the mouse's head sort of
• 11:04bobs.
• 11:06And interestingly, if you take
• 11:07that mouse out of tail
• 11:08suspension, they'll begin to move
• 11:10about.
• 11:12And so here's a dose
• 11:14response curve looking at,
• 11:16immobility time and tail suspension
• 11:18and also
• 11:19a head twitch response.
• 11:20And what you find is
• 11:21that once you get to
• 11:22a dose of fifty and
• 11:23a hundred mgs per kg,
• 11:25the mice,
• 11:26will become completely immobile, no
• 11:29escape behavior, and they'll also
• 11:30have this sort of sustained
• 11:32head twitch over this recording
• 11:34period.
• 11:35To convince myself that I
• 11:36was looking at,
• 11:38disassociation, I devised a few
• 11:40more behavioral tests. In this
• 11:41behavioral test, the mouse is
• 11:43head fixed, and I placed
• 11:44an adhesive on its nose,
• 11:46so it's sitting on its
• 11:47snout, and its whiskers can
• 11:49also feel the adhesive.
• 11:50And, mice normally find this
• 11:53very aversive and, knock it
• 11:55straight away within, like, a
• 11:56second or two,
• 11:57a very short period of
• 11:59time. But with ketamine at
• 12:00these doses, they become
• 12:02completely unaware of the sticker.
• 12:05Similarly, if you expose a
• 12:07mouse to a simple air
• 12:08puff,
• 12:09they have a
• 12:10withdrawal response, and ketamine at
• 12:12these, two particular doses, this
• 12:15modern high dose, failed to
• 12:16do that.
• 12:18Moreover,
• 12:19if you put a mouse
• 12:20in a rat's cage exposed
• 12:21to marbles, they have this
• 12:22intrinsic desire to bury these
• 12:24marbles,
• 12:25so they'll bury a fraction
• 12:27of them.
• 12:28And interestingly, if you give
• 12:30the mouse a a ketamine
• 12:32at these two different doses,
• 12:33they failed to bury a
• 12:34single marble. But if you
• 12:36record their movement throughout this
• 12:38rat's cage,
• 12:39they seem to be moving
• 12:41no different or
• 12:43some increase, some decrease
• 12:44relative to their, baseline.
• 12:47So it's not that the
• 12:48mouse is even sedated,
• 12:49at this particular
• 12:51at these two doses.
• 12:53When you look under the
• 12:54hood at the EEG, you
• 12:55find that ketamine at these
• 12:56two different doses, and these
• 12:57are not anesthetic doses, you
• 12:59couldn't perform a surgery
• 13:01on a mouse. You actually
• 13:02most likely need an adjunct
• 13:04like dexmedetomidine
• 13:06or, xylazine to reduce,
• 13:08an general anesthesia for a
• 13:10mouse. So at these subhypnoct
• 13:12doses I would describe,
• 13:14you could see these fast
• 13:15oscillations emerging,
• 13:18which is not terribly surprising.
• 13:20What is surprising
• 13:22is when you start doing
• 13:24two photon,
• 13:26imaging
• 13:28into the the the living
• 13:29mouse brain. And in these
• 13:31experiments, the mouse's head fixed.
• 13:33It's in the mouse that
• 13:34is expressing,
• 13:37thigh one.
• 13:39It's it's expressing GCaM under
• 13:41the thigh one promoter, which
• 13:42is labeling excitatory cells in
• 13:44the brain. And in these
• 13:46traces, you're looking at GCaMP
• 13:48fluorescence over time in a
• 13:50in a local cortical region.
• 13:51So this is a two
• 13:52dimensional slice, almost like a
• 13:53CT scan through the mouse's
• 13:55brain,
• 13:56and we're recording these calcium
• 13:58fluctuations,
• 13:59which are a proxy for
• 14:00neural activity. And so when
• 14:02you see a spike in
• 14:03calcium, that's most likely reflecting
• 14:05some type of action potential
• 14:11in
• 14:12you're recording this animal
• 14:13spontaneous activity,
• 14:15when you give this moderate
• 14:16and high dose of ketamine,
• 14:18you find that neurons that
• 14:19were previously active
• 14:22attenuate or even switch off,
• 14:23whereas cells that were previously
• 14:25silent,
• 14:26switch on. And this was
• 14:28surprising because when I first
• 14:29did the analysis, I I
• 14:31was looking at sort of
• 14:32the average activity across the
• 14:34two different states, and they
• 14:36were basically no different. But
• 14:38when you look at what
• 14:39are the neurons contributing to
• 14:40this activity,
• 14:42they're completely different.
• 14:47And this is the summary
• 14:48of
• 14:48of of neurons recorded in
• 14:50this particular region. You could
• 14:51see
• 14:52there's really not much in
• 14:53the way of a change
• 14:54with saline injection, but at
• 14:55these two different doses of
• 14:57ketamine,
• 14:57you see that cells that
• 14:58were previously,
• 15:00low and and a low
• 15:01activity state in wakefulness, they
• 15:03switch on under ketamine.
• 15:05Cells that were previously highly
• 15:07active in wakefulness switch off,
• 15:09and this was true at
• 15:10these two different doses.
• 15:12And what what ketamine is
• 15:14doing is completely in contrast
• 15:16to other anesthetics that don't
• 15:17have disassociated properties. Here, cecoflurane
• 15:20at two percent, midazolam,
• 15:22you could see that, neurons
• 15:24active in wakefulness completely turn
• 15:26off. You don't see this
• 15:28sort of,
• 15:29reconfiguration of its cells activating
• 15:31under these states.
• 15:33And to see if this
• 15:34is a more,
• 15:35more of a global effect,
• 15:37I look I can look
• 15:38across the mouse's brain. Because
• 15:40the mouse's brain is quite
• 15:41flat, you can impose windows
• 15:43in different areas that are,
• 15:45related to different functions.
• 15:47And looking at secondary motor
• 15:48cortex,
• 15:51forelimb motor cortex, visual cortex,
• 15:53retrosplenial cortex, you find a
• 15:55very similar motif emerging.
• 15:57So this is not really
• 15:58specific to somatosensory but maybe
• 16:00more of
• 16:01a global,
• 16:02cortical feature.
• 16:04And in this work, and
• 16:05I I don't wanna,
• 16:07go into all the mechanisms,
• 16:10with you here today,
• 16:11because I wanna get to
• 16:12some of my newer work.
• 16:13But I really thoroughly entertained
• 16:16the fact that ketamine is
• 16:17a dirty drug, and it
• 16:18has effects on neuromodulation.
• 16:21It has circuit effects through
• 16:22GABAergic interneurons.
• 16:23It also has effects, through
• 16:25various different types of,
• 16:27channels, ion channels.
• 16:29And what this work,
• 16:32many experiments later,
• 16:35began to tell me is
• 16:36that
• 16:36you would need at least
• 16:38modulation of GABAergic interneurons,
• 16:40and you need suppression of
• 16:42HCN and NMDA channels to
• 16:44recreate this switch.
• 16:47And
• 16:48when I finished this work
• 16:50and I I noticed the
• 16:51switch that I think is
• 16:53arising in under a dissociative
• 16:55like state, I be began
• 16:57to think about,
• 16:59how this might relate to,
• 17:01like, the typical forms of
• 17:03plasticity that a lot of
• 17:04people describe and,
• 17:06folks like Alex Vaughn has
• 17:07described where you might see
• 17:09the birth of new connections.
• 17:10And I was, beginning to
• 17:15oh, let let me let
• 17:16me just take you on
• 17:17this little sidetrack,
• 17:19to sort of connect that
• 17:20initial idea,
• 17:23with regards to disassociation
• 17:25to ketamine's antidepressant
• 17:27effect. And I I thought
• 17:28of a very, very,
• 17:31simple experiment,
• 17:32to get at this. And
• 17:34so in this experiment,
• 17:35I start with,
• 17:37naive mice, and I expose
• 17:39them to chronic stress.
• 17:41The prediction is is that
• 17:42you'll get a chronically stressed
• 17:44mouse.
• 17:45That mouse, if you expose
• 17:46it to ketamine at the
• 17:48subhypnotic dose,
• 17:50would, induce
• 17:52an antidepressant effect as measured
• 17:54by increase in immobility and
• 17:56tail suspension.
• 17:58But if you,
• 17:59then couple ketamine
• 18:01to isoflaurine,
• 18:03you might be able to
• 18:04quell,
• 18:05or suppress the dissociative
• 18:07effect and maybe render ketamine
• 18:10not useful to the mouse
• 18:11and you would,
• 18:13continue to show signs of
• 18:15a depression like state.
• 18:17And so,
• 18:19here you're looking at a
• 18:20very similar,
• 18:21experiment where a mouse is
• 18:22held in tail suspension and
• 18:24the mouse is exposed to
• 18:26this fifty mg per kg
• 18:27dose, and you could see
• 18:27that they failed to show
• 18:28that escape behavior and they
• 18:30have this sustained head twitch.
• 18:33When you couple ketamine with
• 18:34isoplaurine,
• 18:35you can completely eliminate,
• 18:38this vertical head twitch. It's
• 18:39again, I I I dare
• 18:41not to speculate what,
• 18:43the vertical head twitch means,
• 18:44but it is suggestive of
• 18:46perhaps maybe
• 18:47a psychedelic
• 18:48like response,
• 18:50but,
• 18:51hard to say.
• 18:52When you look at the
• 18:53neural activity,
• 18:55you could see that ketamine
• 18:57induces a reconfiguration
• 18:58of activity, but when you
• 19:00start to immediately couple that
• 19:01with isofluorine, the activity is
• 19:03lost.
• 19:04And that's true at a
• 19:05low moderate dose of isoflaurine
• 19:07or a higher dose of
• 19:08isoflaurine.
• 19:09And if you do the
• 19:10Converse experiment,
• 19:12we record activity under wakefulness
• 19:14followed by,
• 19:16isoflaurine.
• 19:18You could see the suppression
• 19:19of neural activity
• 19:20and followed by ketamine,
• 19:22you find that the activity
• 19:23is lost. So I think
• 19:24this is good evidence that
• 19:26when you couple the two,
• 19:27you start to eliminate or
• 19:29suppress neural activity.
• 19:31When you look at a
• 19:32more,
• 19:35classic,
• 19:39experiment
• 19:40that,
• 19:41would be indicative of a
• 19:42plasticity event such as CFOS
• 19:44expression,
• 19:46you find that ketamine induces
• 19:48CFOS expression in the prefrontal
• 19:50cortex. But when you couple
• 19:51this,
• 19:53with isoflaurine,
• 19:55the CFOS expression is reduced.
• 19:57And lastly, if you look
• 19:58at behavior
• 19:59where ketamine induces an antidepressant
• 20:01like effect, when you start
• 20:03to couple this with isoflurane,
• 20:05you fail to induce,
• 20:07an antidepressant
• 20:08effect.
• 20:09And I guess what that
• 20:10means, to me is that
• 20:12if you're gonna use ketamine,
• 20:15for the treatment
• 20:16of of, depression,
• 20:19you're gonna wanna probably give
• 20:20ketamine to an awake patient,
• 20:24that can experience,
• 20:26activity dependent plasticity,
• 20:29and and can,
• 20:31have complete benefit from from
• 20:32the drug exposure.
• 20:34I think if you begin
• 20:35to couple ketamine
• 20:36with, various different drugs that
• 20:38are sort of GABAergic in
• 20:39nature, you will begin to,
• 20:42suppress these, various forms of
• 20:44activity dependent plasticity.
• 20:46You'll and,
• 20:47and you'll,
• 20:50block the therapeutic effect.
• 20:56And, you might then also
• 20:58think in in the setting
• 20:59of ketamine with isoflaurine,
• 21:02you might not also be
• 21:03able to, trigger the disassociative
• 21:06state, but that's, again, hard
• 21:07to determine because the patient,
• 21:10is unconscious.
• 21:11And so in this first
• 21:13portion of the talk, I
• 21:14I really wanted to stress
• 21:15that,
• 21:17ketamine,
• 21:18can be modeled in a
• 21:19rodent and to induce a
• 21:20disassociated like state, and not
• 21:23one behavioral test will really
• 21:24capture that. But I think
• 21:25the the, summation of several
• 21:28different behavioral tests,
• 21:30can show that. Ketamine induces
• 21:32a behavioral,
• 21:33induces a rapid switch in
• 21:35neural activity
• 21:36and that this switch is
• 21:37widespread across the brain,
• 21:39and it seems that,
• 21:41ketamine,
• 21:42its antidepressant effects are sensitive
• 21:44to, general anesthetic. And I
• 21:46will touch again on this
• 21:48in a in a little
• 21:48bit later in the talk.
• 21:50So
• 21:51a few slides ago, I
• 21:52was sort of drawing in
• 21:53this concept
• 21:54that
• 21:55if you can induce disassociation,
• 21:57you can induce this rapid
• 21:59switch in activity,
• 22:00and then the prediction is
• 22:01is that,
• 22:03different forms of plasticity
• 22:05might be
• 22:06shown in cells that activate
• 22:09or cells conversely if they're
• 22:11suppressed.
• 22:12And so I developed recently
• 22:13a technique that enables you
• 22:15to label cells in a
• 22:16very sparse fashion, and you
• 22:18can begin to record,
• 22:20not only the neural activity
• 22:21of that individual cell, but
• 22:23then you can also go
• 22:25more superficially or deeper into
• 22:27its dendritic regions,
• 22:29and you can look at
• 22:30the synapses and investigate how
• 22:32these synapses,
• 22:33acutely change with regards to
• 22:35preexisting synapses.
• 22:36But then, also, you can
• 22:37look at the formation of
• 22:39new synapses and see how
• 22:40that contributes
• 22:41to internal activity. And so
• 22:43in this example, you can
• 22:44see two cells
• 22:45very active in wakefulness
• 22:47following ketamine at this moderate
• 22:49dose. You could see its
• 22:51rapid suppression.
• 22:52And
• 22:54as I was doing these
• 22:55experiments,
• 22:56I was approached,
• 22:58by an old colleague that
• 23:00I met during my PhD
• 23:02and has been a close
• 23:03collaborator,
• 23:04and,
• 23:05he said, hey, Joe. Would
• 23:06you this is Lauren Luger
• 23:08at at UCSD now, but
• 23:09previously at,
• 23:11Janelia,
• 23:13said, hey, Joe. We've we've
• 23:14sort of reengineered
• 23:16a, ketamine sniffer a ketamine
• 23:18sensor, which they also call
• 23:19a sniffer.
• 23:20Would you be interested in
• 23:22sort of exploring this because
• 23:23of your interest in ketamine?
• 23:25I said absolutely.
• 23:27And,
• 23:28what this molecule is is
• 23:30is basically a a bacterial
• 23:33protein that has been,
• 23:35exquisitely engineered to sense specifically
• 23:38ketamine. And in fact, in
• 23:39this new unpublished version of
• 23:40the sensor, we can actually
• 23:42begin to detect
• 23:43different enantiomers of ketamine in
• 23:45real time. And so if
• 23:47you're looking at this a
• 23:48panel, this is how the,
• 23:49sensor expresses,
• 23:51and what you can see
• 23:52here, this is fluorescence over
• 23:53time. And following ketamine injection,
• 23:55you could see within almost
• 23:57ten seconds, ketamine is already
• 23:59entering the brain, meaning it's
• 24:00circulating from the intraperitoneal
• 24:03cavity
• 24:04into the circulatory system, getting
• 24:06into the brain through the
• 24:07blood brain barrier, and being
• 24:08detected locally,
• 24:10in in various different brain
• 24:12regions within,
• 24:14basically, a ten second
• 24:16period of time.
• 24:17And, interestingly, if you look
• 24:19at some of these kinetic
• 24:20measurements,
• 24:21you find that, the time
• 24:23to,
• 24:24peak signal of ketamine is
• 24:25around ten to fifteen minutes,
• 24:27until it gets to its
• 24:29final fluorescence,
• 24:31its max, fluorescence intensity.
• 24:33And that's interesting, but you
• 24:35said you might say, Joel,
• 24:37I can probably measure that
• 24:38in blood, and you wouldn't
• 24:39be wrong. What I think
• 24:40is interesting about some of
• 24:41this unpublished data is that
• 24:43you can begin to use
• 24:44clever genetic tricks to target
• 24:46these sensors to various different
• 24:48locations within a neuron. You
• 24:49could target this,
• 24:51sensor specifically the cytoplasm
• 24:53versus the nucleus
• 24:55and plasma membrane.
• 24:57And what I'm showing you
• 24:58here is sort of how
• 24:59these sensors express,
• 25:01And here on the right
• 25:02looking at these traces is
• 25:03what you find
• 25:05is something really amazing.
• 25:07When you sense ketamine at
• 25:08the plasma membrane, it's rapid
• 25:10in its detection, and it's
• 25:12relatively short lived, and this
• 25:13sort of mirrors plasma men
• 25:15plasma
• 25:16measurements of ketamine.
• 25:18When you look within the
• 25:19cell, what you begin to
• 25:21find is that there's a
• 25:22rapid detection within ketamine. So
• 25:23ketamine is just going right
• 25:25through the plasma membrane, getting
• 25:27into the cytoplasm,
• 25:28and even getting into the
• 25:29nucleus.
• 25:31And what's outstanding is the
• 25:33fact that this,
• 25:35recording
• 25:36signals that in this particular
• 25:37mouse given a ten mg
• 25:38per kg injection, which is
• 25:40the common injection to induce
• 25:41an antidepressant like effect of
• 25:43a mouse,
• 25:44these signals are sustained for
• 25:46over hours.
• 25:48And if you look at
• 25:48ninety minutes, you see sustained
• 25:50signal.
• 25:51So
• 25:52the classic sort of description
• 25:54of ketamine interacting with the
• 25:55receptor at the plasma membrane
• 25:57is is is rather superficial,
• 26:00and, ketamine is completely permeating
• 26:03a neuron from its,
• 26:05receptor surfaces
• 26:06to all the way the
• 26:07the the within the nucleus.
• 26:11And we don't quite understand
• 26:12the,
• 26:14implications
• 26:15of these sort of, observations.
• 26:19And so you could see
• 26:20I easily get sidetracked,
• 26:23and which which brings me
• 26:24to this, question of nitrous
• 26:26oxide. And when I was
• 26:28doing this very,
• 26:32very experiment, I was approached
• 26:33by a colleague in the
• 26:34field of anesthesia named Peter
• 26:36Nagley, who,
• 26:38is working at the University
• 26:39of Chicago, and he was
• 26:41basically,
• 26:43thinking about nitrous oxide and
• 26:44its role in
• 26:46in in the treatment of
• 26:47resistant to
• 26:49treatment resistant depression.
• 26:51And he said, Joe, you
• 26:52know, it's great that you're
• 26:52working with ketamine,
• 26:54but ketamine,
• 26:55as you know, is a
• 26:56dirty drug. And,
• 26:58you know, moreover, you know,
• 27:00ketamine has a lot of
• 27:01metabolites that are thought to
• 27:02be neuroactive. They could be
• 27:03dissociative. They could also be
• 27:05antidepressant.
• 27:06And you really don't know,
• 27:08how that's all gonna shake
• 27:10out,
• 27:10so why not work with
• 27:12a cleaner drug? And I
• 27:13said, well, that's, you know,
• 27:14an interesting,
• 27:16you know, observation.
• 27:18And
• 27:19and he's like, well, let
• 27:20me just show I'll show
• 27:21you my data, and,
• 27:23I can maybe convince you
• 27:25to to explore this in
• 27:26in your preclinical
• 27:28models.
• 27:29And,
• 27:30so so so he did.
• 27:32And,
• 27:33long story short, he convinced
• 27:34me this was worthy of
• 27:35pursuit.
• 27:37And one thing that I
• 27:38thought was really interesting is
• 27:39that, you know, when you
• 27:40look back at some of
• 27:41the older,
• 27:42in vitro data with regards
• 27:43to nitrous oxide molecular mechanisms,
• 27:46nitrous oxide is again thought
• 27:47to be primarily an NMDA
• 27:49antagonist,
• 27:50but that was really never
• 27:51tested in the mammalian brain.
• 27:53It was all done in
• 27:54sort of,
• 27:55altered neurons
• 27:57or or brain slice.
• 27:59And what really got me
• 28:00hooked and really engaged in
• 28:02in this particular study is
• 28:04the fact that nitrous oxide
• 28:05is a gas. It's only,
• 28:07three atoms.
• 28:12And, nitrous oxide has to
• 28:14be inhaled.
• 28:15It so it it,
• 28:17it will be inhaled, and
• 28:18it'll, this gas will,
• 28:21partition into the blood and
• 28:22then get to the brain
• 28:23and diffuse across the blood
• 28:25brain barrier and then,
• 28:27you know, interact and do
• 28:28its thing, and then it
• 28:29has to be blown off.
• 28:31And what's even more surprising
• 28:33is that there is no
• 28:34metabolite of nitrous oxide. It
• 28:36is breathed in as nitrous
• 28:37oxide. It is inhaled as
• 28:38nitrous
• 28:39oxide. So if anything, I
• 28:41uncover with regards to a
• 28:42mechanism
• 28:43has to be related to
• 28:45the drug exposure itself.
• 28:48And so, you know, this
• 28:50is one example that he
• 28:51showed me of a patient
• 28:52who is treatment resistant depressed,
• 28:55who who came in for
• 28:56their trial,
• 28:57received one hour duration of
• 28:58fifty percent nitrous oxide. So
• 29:00this is not an anesthetic
• 29:01dose of nitrous oxide. This
• 29:02is something that you would
• 29:03get in a dentist's office
• 29:05if you're getting,
• 29:06your wisdom teeth pulled.
• 29:08You could see that there's
• 29:09a rapid reduction in the
• 29:10symptoms.
• 29:12This patient,
• 29:14eventually was,
• 29:16crossed over to the placebo
• 29:17group in February
• 29:19and then in March had
• 29:20some a worsening of symptoms
• 29:22and then subsequently received a
• 29:23twenty five percent,
• 29:26inhalation of nitrous oxide, and
• 29:28their symptoms
• 29:29were, further improved.
• 29:32And so in this study,
• 29:34I transitioned
• 29:36to studying three cohorts of
• 29:38mice, a control group, and
• 29:40two chronic stress groups,
• 29:42one
• 29:43which,
• 29:44receives
• 29:45chronic social defeat,
• 29:47by an aggressor mouse, and
• 29:48that lasts for ten days.
• 29:50And the other is a
• 29:51more classic model where you
• 29:52infuse choristerone
• 29:53and the drinking order for
• 29:55twenty one days, and that
• 29:56is a pharmacological
• 29:58way to
• 29:59induce a depression like state,
• 30:01if I may. And then
• 30:03so these mice are gonna
• 30:04be chronically stressed, and then
• 30:06in and then,
• 30:09following that will be, imaged
• 30:11under the two photon microscope.
• 30:13And so, I devised a
• 30:15way to mix,
• 30:17nitrous oxide, and that's with
• 30:18oxygen and and,
• 30:20a hundred percent nitrous oxide
• 30:22through a blender. And I
• 30:23can create a fifty percent
• 30:26concentration. And I can monitor
• 30:27also how that, is being
• 30:29delivered to a mouse by
• 30:30using a gas analyzer, something
• 30:32that we commonly use in
• 30:33the operating room,
• 30:35and then the remaining gas
• 30:36is,
• 30:37scavenged.
• 30:39And,
• 30:39in this,
• 30:41experiment, I record across a
• 30:43prefrontal area called c g
• 30:44one, which will be equivalent
• 30:45to the anterior cingulate cortex
• 30:47in the human.
• 30:48And we can image
• 30:50basically across this cortical,
• 30:52across this cortical column through
• 30:53layer two, three, and layer
• 30:54five.
• 30:58Before I show you some
• 30:59of the imaging data, I
• 31:00just wanna convince you that
• 31:02we can detect, an antidepressant
• 31:04like effect
• 31:05following nitrous oxide in the
• 31:07chronic stress group. So here
• 31:08you're looking
• 31:09at, immobility time and tail
• 31:11suspension. Here you're looking at
• 31:17open arm time and elevated
• 31:18plus assess whether a mouse
• 31:21displays a depression like state.
• 31:22It's not like one test.
• 31:23It's a combination of tests,
• 31:25but you can see that
• 31:27that the oxygen group, which
• 31:29is in green,
• 31:30begins to separate from, the
• 31:32blue group, which is nitrous
• 31:33oxide following treatment.
• 31:35And so there's, some behavioral
• 31:37evidence that nitrous oxide
• 31:39induces a behavioral change.
• 31:41And my hypothesis leading into
• 31:43this experiment, unfortunately, wasn't too
• 31:45thoughtful or creative. I thought
• 31:47there might be,
• 31:49sort of a reconfiguration
• 31:51like I saw with ketamine
• 31:52because I think
• 31:53perhaps this, NDA antagonist effect
• 31:56would suppress these active cells
• 31:58that were active following chronic
• 32:00stress,
• 32:02and then maybe the shift
• 32:03in network activity might drive
• 32:05a different population.
• 32:07It turns out I couldn't
• 32:08be, further, from the truth.
• 32:10And so here's a a
• 32:12raw data. So this is
• 32:13a a mouse, following chronic
• 32:15stress.
• 32:16You're looking at these, neurons
• 32:17expressing
• 32:18g
• 32:19chem. You can see them
• 32:20flicker, which means that they're
• 32:28active,
• 32:30and this is in wake
• 32:31light at fifty percent. I'm
• 32:32gonna give it a good
• 32:33ten minutes to fifteen minutes
• 32:34to get to equilibrium in
• 32:36the brain. It probably happens
• 32:38much faster than that, but
• 32:39I don't quite know the
• 32:41concentration of nitrous oxide in
• 32:43a mouse brain.
• 32:45And so this is following
• 32:53is following equilibrium
• 32:55of nitrous oxide. You could
• 32:56see there's a sudden active
• 32:58right and sustained,
• 33:00and,
• 33:01this was obviously very, very
• 33:02surprising. You could see, like,
• 33:03these little dots here,
• 33:05also flickering, and those are
• 33:07their apical dendrites. As these
• 33:08cells fire, they also,
• 33:11can fire their dendrites.
• 33:13And so this was,
• 33:15quite shocking.
• 33:22If you pseudo
• 33:24color them
• 33:27and show wakefulness activity in
• 33:29yellow and nitrous oxide in
• 33:30and blue, you could see
• 33:32this really widespread activation
• 33:34as opposed to what's seen
• 33:35in wakefulness.
• 33:38And here,
• 33:40I've given you a pretty
• 33:41good,
• 33:44display of activity in layer
• 33:45two three here and layer
• 33:46five. You could see comparing
• 33:48controls to chronic stress groups,
• 33:50you could see that chronic
• 33:51stress induces a hypoactive
• 33:53state in these neurons in
• 33:54layer two three and layer
• 33:55five, and that's not terribly
• 33:57surprising. That's,
• 33:59been shown over and over
• 34:00again as, chronic stress induces
• 34:02synaptic loss.
• 34:04And what's really interesting is
• 34:06that when you give nitrous
• 34:07oxide, there's robust activation of
• 34:09layer five. You could see
• 34:11this in even the control
• 34:12group
• 34:13as well as the chronic
• 34:14stress groups.
• 34:15But you don't really see
• 34:16much in layer two three,
• 34:17and that was very, very
• 34:18curious to me, because that,
• 34:20I couldn't imagine how a
• 34:22small gas,
• 34:24n two o,
• 34:26could show specificity.
• 34:29I thought it would, be
• 34:30a,
• 34:31an equal opportunity activator.
• 34:35And, also, I was wasn't
• 34:36sure what fifty percent nitrous
• 34:37oxide meant to a rodent.
• 34:39I knew I couldn't induce
• 34:40anesthesia with fifty percent nitrous
• 34:42oxide because that because that
• 34:43can't be done with a
• 34:44human. But here, I devised
• 34:46a simple experiment where I
• 34:47exposed,
• 34:48either oxygen or nitrous oxide
• 34:50at fifty percent in a
• 34:51closed
• 34:52chamber and monitored the animal's
• 34:54overall gross movement. And what
• 34:56you find is that
• 34:58at fifty percent nitrous oxide,
• 34:59the mice move a lot.
• 35:01There's a lot more distance
• 35:02traveled. Their speed is increased,
• 35:05but not necessarily their max
• 35:06speed. And these sort of
• 35:07upper deflections and these traces
• 35:09are exploratory
• 35:10events. So it seems that
• 35:11the mouse is engaged in
• 35:12its environment and exploring more,
• 35:15but they're definitely not sedated
• 35:17in any way under this
• 35:18dose.
• 35:20And what I wanna really,
• 35:22emphasize here is this particular
• 35:23slide, which I think is
• 35:25rather amazing.
• 35:26And so if you look
• 35:27at these layer five neurons
• 35:28and looking at their calcium
• 35:30activity over time, you could
• 35:31see that nitrous oxide activates
• 35:33them.
• 35:34And then I'm gonna blow
• 35:35off all this gas, and
• 35:36then I'm gonna revisit these
• 35:38same neurons an hour later.
• 35:40And so there's no drug
• 35:41in the system, but when
• 35:42you look at their activity,
• 35:43they're profoundly
• 35:45active.
• 35:46And that's true in the
• 35:47control.
• 35:48It's also true in the,
• 35:50chronic stress,
• 35:53cohorts.
• 35:54And interestingly,
• 35:55if you now go back
• 35:56and reimage layer two three,
• 35:59what you find is all
• 36:00of a sudden, re layer
• 36:01two three,
• 36:03is is now active, which
• 36:05when they previously weren't active
• 36:07under the drug treatment. So
• 36:09it's as if layer five,
• 36:11was recruited during the drug
• 36:13treatment,
• 36:14displayed persistent activity following the
• 36:16drug treatment.
• 36:17That persistent activity following the
• 36:19drug treatment,
• 36:21reawakens
• 36:22layer two three. And so
• 36:23now the
• 36:24both
• 36:25sort of the output layer,
• 36:26which is layer five, and
• 36:27also
• 36:30integrator layer, layer two three,
• 36:32is now,
• 36:33recruited,
• 36:34following the treatment. This is
• 36:36the analysis.
• 36:37And to really get at
• 36:39and I and I really
• 36:40became fascinated is how these
• 36:42neurons are activating and why
• 36:43are they specific to layer
• 36:45five.
• 36:46And so I turned to
• 36:47some of the,
• 36:49classic work done by the
• 36:50Zwaromski lab,
• 36:55at
• 36:58that
• 37:01that
• 37:03showed
• 37:05that,
• 37:06again,
• 37:08this is in, cultured neurons,
• 37:11like, if nitrous oxide is
• 37:12working through the NMDA receptor.
• 37:16And here you're looking at
• 37:17activity,
• 37:18in wakefulness,
• 37:19and then I'm gonna
• 37:21locally
• 37:24use an MDA antagonist, which
• 37:26was MKO. You find
• 37:28the typical of MKO one,
• 37:30you're gonna actually suppress calcium
• 37:32activity.
• 37:33And then if you then
• 37:34record these neurons under nitrous
• 37:36oxide, you can activate them.
• 37:37My prediction was is that
• 37:39they should stay silent, if
• 37:40not silence more.
• 37:43And then furthermore, if you
• 37:44perform a, like, a rather
• 37:46full synaptic block and you
• 37:47inhibit
• 37:48AMPA receptors, you could see
• 37:50the effect of, of the
• 37:52this pharmacological
• 37:54treatment.
• 37:55And then if
• 37:59you give nitrous oxide, you
• 38:00could still act it, sort
• 38:01of almost like synaptic independent
• 38:03mechanism
• 38:05here. Here's a movie to
• 38:07sort of, keep you interested.
• 38:09I'm recording spontaneous
• 38:11activity wakefulness.
• 38:12This is again layer five,
• 38:14sort of deep within layer
• 38:15five.
• 38:16You could see these neurons
• 38:17activating.
• 38:18Then I'm gonna expose nitrous
• 38:19oxide at fifty percent. The
• 38:21prediction is they're gonna be
• 38:22activated, and when they're activated,
• 38:24they're in a very
• 38:25robust
• 38:32regimen of firing, maybe burst
• 38:34firing. And then the ten,
• 38:36you could see it's actually,
• 38:37quite different.
• 38:39And the activity is rather
• 38:41sustained, but in a more,
• 38:43modest
• 38:44way.
• 38:45And so in this work,
• 38:47I really tried to figure
• 38:49out how nitrous oxide could
• 38:51recruit these layer five neurons.
• 38:53And as you might imagine,
• 38:55all being neuroscientists,
• 38:57that there are many possibilities.
• 38:58There There could be changes
• 38:59in synaptic input. There could
• 39:00be changes in intergenic integration.
• 39:02Inhibition could change. You could
• 39:03have various different types of
• 39:05activating channels.
• 39:07You could begin to open
• 39:09intracellular
• 39:09stores that'll leak calcium,
• 39:11or you could
• 39:12have more classic changes in
• 39:14neuro excitability through, say, potassium
• 39:16channels.
• 39:18And so I won't be
• 39:20able to show you all
• 39:21of these possibilities, but I
• 39:22thoroughly thought about them
• 39:26as I went through this
• 39:27work. And so here, you're
• 39:29looking at,
• 39:30synapses here in the apical
• 39:33tuft of a layer five
• 39:34neuron,
• 39:35and we can detect these
• 39:36synaptic events,
• 39:39by using calcium indicators like
• 39:40I showed you before.
• 39:42And when you,
• 39:43turn on nitrous oxide or
• 39:44record under nitrous oxide, you
• 39:45don't really see much in
• 39:46the way of, recruitment of
• 39:49synapses, which is, again, very
• 39:51curious because you need synaptic
• 39:52input to drive cells.
• 39:54When you also record these
• 39:56apical dendrites, they produce these
• 39:58fantastic calcium nonlinear
• 40:00nonlinearities.
• 40:02And, under nitrous oxide, you
• 40:04don't see them, occurring. And
• 40:06the presence of these types
• 40:08of dendritic
• 40:09spikes, so called dendritic spikes,
• 40:11would also give rise to,
• 40:13the activation
• 40:14of the of the cell
• 40:16body in layer five, but
• 40:17we don't really quite see
• 40:18that. What you see, again,
• 40:20if you're imaging across this
• 40:21individual neuron, you see that
• 40:23the soma is activated in
• 40:25sort of this burst configuration.
• 40:27And they actually see some
• 40:28calcium activity in the trunk,
• 40:30deep down, and this might
• 40:31be really the two backpropagating
• 40:33action potentials in these particular
• 40:35neurons.
• 40:37And then,
• 40:38I became,
• 40:40sort of,
• 40:42not convinced, but I wanted
• 40:43to prove to myself with
• 40:44a little bit more certainty
• 40:46that these apical tough dendrites
• 40:47don't really contribute to these
• 40:49somatic,
• 40:50calcium events.
• 40:51And so in this experiment,
• 40:52I can record at two
• 40:53different layers,
• 40:55superficial layer and a deep
• 40:56layer,
• 40:57and I can see them
• 40:58activate under nitrous oxide.
• 41:01But what I then did
• 41:02is I then used the
• 41:03two photon laser to cut
• 41:05the dendrite and completely sever
• 41:07these,
• 41:08these apical tufts from their
• 41:10parent dendrite.
• 41:11And so in this particular
• 41:12example, I performed two cuts
• 41:14across this dendrite,
• 41:16completely, and then you could
• 41:18see the beating of the
• 41:19dendrite here suggesting that it's
• 41:20a clean break,
• 41:22and dendritic signals of loss,
• 41:24but yet the somatic activity
• 41:26persists. So this again suggests
• 41:28that synapses
• 41:29are probably
• 41:30not the underlying mechanism
• 41:32driving the activation
• 41:33of these layer five neurons
• 41:35under nitrous oxide.
• 41:37In in the quest to
• 41:38figure out how can I,
• 41:41modulate this layer five activity,
• 41:44I then turned to one
• 41:45of those earlier experiments where
• 41:46I coupled,
• 41:47isofluorine,
• 41:49with nitrous oxide?
• 41:50And what you see here
• 41:52is that nitrous oxide recruits
• 41:53these layer five neurons,
• 41:55but even a small dose
• 41:57of isofluorine.
• 41:58So this would not be
• 41:59considered general anesthesia. This wouldn't
• 42:01even be considered sedation. This
• 42:02is
• 42:03this is, sub,
• 42:06very subnautic. I don't even
• 42:07know what you would wanna
• 42:08call this. This is a
• 42:08very low dose. This would
• 42:10be, considered,
• 42:11sub hypnotic isoflaurine.
• 42:14One point two percent is
• 42:15considered hypnotic isoflaurine.
• 42:17You could see that activity
• 42:18is completely lost.
• 42:20And then if you look
• 42:21at the behavior, its antidepressant
• 42:23effect is lost when you
• 42:24couple this with isofluorane. So,
• 42:26again, this is separate evidence
• 42:28with a different type of
• 42:29anesthetic that has psychedelic properties
• 42:31that if you couple nitrous
• 42:33oxide or even ketamine to
• 42:35sort of a GABAergic
• 42:36anesthetic,
• 42:37you begin to eliminate some
• 42:39of its activity profile
• 42:41and also behavioral effects.
• 42:44And so in this,
• 42:46part of the talk, I've
• 42:47really told you that nitrous
• 42:48oxide recruits layer five, and
• 42:50this is quite surprising consider
• 42:52this gas is everywhere
• 42:54at a high concentration, which
• 42:55is fifty percent,
• 42:56and that this activity induced
• 42:58by nitrous oxide persists once
• 43:00the drug is eliminated,
• 43:02and layer five is required
• 43:03for its antidepressant
• 43:04like effects.
• 43:06And I've also showed you
• 43:07that nitrous oxide doesn't seem
• 43:09to behave like an NMD
• 43:10antagonist
• 43:11as, I've defined an NMD
• 43:13antagonist in others, and it's
• 43:15quite sensitive to anesthesia.
• 43:17And so I wanna conclude
• 43:19with this. I I and
• 43:20I evaluated,
• 43:21some of these other components
• 43:23that I won't be able
• 43:23to tell you about and,
• 43:27to to sort of,
• 43:29really,
• 43:31to to figure out how
• 43:32nitrous oxide might recruit layer
• 43:34five, I turned to looking
• 43:35at
• 43:36sodium channels,
• 43:38calcium channels,
• 43:39serotonin uptake
• 43:41properties,
• 43:43opioid receptors, intracellular calcium release
• 43:46events,
• 43:47by using different type of,
• 43:49pharmacological
• 43:50agents. And all these agents
• 43:51induce changes in layer five
• 43:52activity, which is probably not
• 43:54surprising,
• 43:55but nitrous oxide was still
• 43:57capable of activating,
• 43:59these types of cells, indicating
• 44:01that it's just not working
• 44:02through this particular mechanism.
• 44:06And then I did a
• 44:06deep dive into the Allen
• 44:08Brain
• 44:09sequencing,
• 44:10open source sequencing,
• 44:12database,
• 44:13and used one of their
• 44:15databases to sort of, maybe
• 44:17more
• 44:18systematically
• 44:19screen,
• 44:21what might be,
• 44:23upregulated
• 44:24in two particular cell types.
• 44:26And I was looking particularly
• 44:27at layer five, and I
• 44:28was also looking at,
• 44:29a interneuron
• 44:30called VIP. And data I
• 44:32didn't show you is that
• 44:33nitrous oxide also turns on
• 44:34a VIP interneuron,
• 44:36which is thought to underlie
• 44:38disinhibition.
• 44:41And so I identified this,
• 44:43potassium channel. It's a small
• 44:44conductance,
• 44:45calcium activated potassium channel,
• 44:48which has been explored for
• 44:49years and its role in
• 44:51excitability,
• 44:51synaptic transmission, and plasticity.
• 44:54And this, particular
• 44:55potassium channel has, increased transcripts
• 44:58in both these types of
• 44:59cells. So I said, okay.
• 45:00This might be a a
• 45:01a good candidate to explore.
• 45:03And then Allen Brain also
• 45:04has some phenomenal in situ,
• 45:07hybridization data,
• 45:08again, showing you that these,
• 45:10mRNA,
• 45:11is located in layer five.
• 45:13And then if you look,
• 45:14like, a little bit closely
• 45:15at these sort of coronal
• 45:16slices, you could see that
• 45:17there's, these sparse
• 45:19cells in layer two three,
• 45:21which would most likely be
• 45:22these,
• 45:24interneurons.
• 45:25And so I thought this
• 45:26was a a a reasonable,
• 45:29molecule to test a little
• 45:30bit more robustly.
• 45:31And so what this channel
• 45:33does is that this channel
• 45:35really controls the after hyperpolarization.
• 45:38And so when a neuron
• 45:39fires
• 45:40and has calcium entry,
• 45:42this potassium channel interestingly has
• 45:44a calcium sensitive domain.
• 45:46And when it's sensed, it's
• 45:48gonna,
• 45:49activate this channel to efflux
• 45:50potassium
• 45:51and, create
• 45:53a,
• 45:54hyperpolarization
• 45:55and keep that cell from
• 45:56firing further,
• 45:58potentially.
• 45:59If you were to close
• 46:00this channel via a poor
• 46:01mechanism,
• 46:04you would reduce this after
• 46:05hyperpolarization,
• 46:06keeping the mem keeping the
• 46:08neuron closer to its,
• 46:10threshold for firing, and you
• 46:11might even see, increased firing.
• 46:14And so a decreased s
• 46:16k two, function might lead
• 46:18to, enhanced excitability
• 46:20and firing.
• 46:22And so I I,
• 46:24reached out to Chuck Soromsky,
• 46:25the the the the the,
• 46:27you know, very esteemed Chuck
• 46:29Soromski who did some of
• 46:30the
• 46:30classic work on nitrous oxide
• 46:32and said, you know, I
• 46:33presented this data to him,
• 46:35and he was, very much
• 46:36convinced and willing to explore
• 46:38this mechanism,
• 46:40in his preparation. So here,
• 46:41you're looking at,
• 46:44not only the expression that
• 46:46we identified
• 46:47of the angiosus s k
• 46:48two channel here in a
• 46:49a brain slice consisting mostly
• 46:51in layer five and scattered
• 46:53cells in layer two three,
• 46:54But here,
• 46:56when he looks at the
• 46:57after,
• 46:58hyperpolarization
• 46:59potential,
• 47:00under baseline and under nitrous
• 47:02oxide, you could see, like
• 47:03I showed in that previous
• 47:04schematic, it's reduced.
• 47:07And this, was a challenging
• 47:08experiment because it's hard to
• 47:10hold a gas,
• 47:12efficiently in a in a
• 47:13brain slice preparation
• 47:15where this,
• 47:16medium has to be infused
• 47:18with a, continuous gas. And
• 47:20if you look at, the
• 47:21control group here, it is
• 47:23is no different from its
• 47:24baseline.
• 47:26So this is a pretty
• 47:27good indicator that nitrous oxide
• 47:28can, inhibit this potential
• 47:31mediated by,
• 47:33s k two.
• 47:34And to sort of put
• 47:36some meat on the bones
• 47:36and to figure out if
• 47:37this is happening in vivo,
• 47:39I started
• 47:40looking at whether
• 47:42SK two inhibition,
• 47:44via pharmacological approach could drive
• 47:46layer five. And so here,
• 47:48you're looking at layer five
• 47:49neurons and also these VIP
• 47:50interneurons,
• 47:51before the drug and after
• 47:53the drug, and you can
• 47:54see that the drug itself
• 47:55can recruit the spontaneous activity
• 47:57of these neurons,
• 47:59and doesn't seem to have
• 48:00an effect in cells that
• 48:01don't have this receptor, such
• 48:03as lamin two three, PV
• 48:04interneurons, or SST interneurons.
• 48:07Moreover,
• 48:08if you put this s
• 48:10k two channel into neurons
• 48:12that don't have the receptor,
• 48:13so now they overexpress this
• 48:15receptor that's not natural to
• 48:16them, you can make them
• 48:17responsive to nitrous oxide.
• 48:19Conversely, if you knock down
• 48:21s k two
• 48:23protein,
• 48:24inside layer fiber VIP,
• 48:26you render them,
• 48:28mute to nitrous oxide or
• 48:29even apamint, this sort of
• 48:31specific
• 48:32antagonist of s k two
• 48:33channels.
• 48:35And then,
• 48:37because,
• 48:37you know, I'm in a
• 48:38very interesting department that contains
• 48:40neuroscientists
• 48:41but also computational,
• 48:43neurobiologists,
• 48:44I proposed this.
• 48:45I, again, presented this data
• 48:47to to a fellow colleague
• 48:48of mine who does
• 48:50various different types of simulations.
• 48:52And I said, you know,
• 48:53I have this effect of
• 48:54nitrous oxide on this particular
• 48:56type of potassium channel.
• 48:59Is it possible to run
• 49:01some type of
• 49:02simulations
• 49:03of this
• 49:04this small drug inside the
• 49:06pore region of this, particular
• 49:08protein.
• 49:09And he did, various different
• 49:11types of calculations that are
• 49:13by far above my pay
• 49:14grade and found that there's
• 49:16indeed,
• 49:17substantial interaction,
• 49:19and,
• 49:20energy changes that would potentially
• 49:22hold nitrous oxide sort of
• 49:24deep within the pore,
• 49:25potentially,
• 49:26creating an inhibition type effect.
• 49:31And so I will,
• 49:33skip this summary slide just
• 49:35so that I have time
• 49:35for questions.
• 49:37And I want to acknowledge,
• 49:39some of the my lab
• 49:40is now a little bit
• 49:41more,
• 49:43it's growing at a in
• 49:44a, at a pace,
• 49:46where I have a a
• 49:48graduate student, a postdoc, I
• 49:49have a few undergrads,
• 49:51which is all very exciting
• 49:52and sort of expanding the
• 49:53scope of our science. I'd
• 49:55like to thank a few
• 49:55of the people that have
• 49:56helped me,
• 49:57perform some of this work,
• 49:58Alex Proick, Max Tells,
• 50:00Tom Joseph,
• 50:03and Andy at Penn. And
• 50:04a special thanks to Lauren
• 50:05Lugar who's been a a
• 50:06close
• 50:07colleague and mentor,
• 50:09since my PhD,
• 50:11and and Kahlal,
• 50:14for for, his extraordinary work
• 50:16with that ketamine
• 50:17sensor,
• 50:19fantastic science,
• 50:20scientist who's, I believe, on
• 50:22the job market now,
• 50:24and Chuck Saromski for helping
• 50:25out with some of the,
• 50:26brain slice recordings and to
• 50:28Peter Nagley who drew me
• 50:30to nitrous oxide,
• 50:32when I was previously infatuated
• 50:33with ketamine.
• 50:35And and lastly,
• 50:37since I have your attention,
• 50:39I just wanna,
• 50:40do a a a plug
• 50:41here for a colleague,
• 50:43who's running,
• 50:45or chairing
• 50:46a fantastic Gordon conference that's
• 50:48gonna be held in in
• 50:49Texas this year, previously in
• 50:50Italy, that is really sort
• 50:52of bridging the gap between
• 50:54conscious neuroscience,
• 50:56anesthesia,
• 50:57psychedelic science,
• 51:00I mean, sleep science.
• 51:02So it's bringing together a
• 51:03lot of extraordinary people who
• 51:05are interested in in sort
• 51:06of similar questions,
• 51:08maybe using different approaches.
• 51:10It's a great venue. So
• 51:12if you have any interest,
• 51:14in any of this, I
• 51:14would highly suggest you attend
• 51:16just given you can trial
• 51:17it because it's gonna be
• 51:18in the States and a
• 51:19little bit more approachable than
• 51:20going to Italy,
• 51:21which I think is gonna
• 51:22be the same.
• 51:24So thank you so much
• 51:25for your attention. I'm I'm
• 51:26happy to address any,
• 51:28questions.