Yale Psychiatry Grand Rounds: "Bidirectional Brain-Body Circuits in Stress and Depression"

Transcript

• 00:00Alright. Well, thank you very
• 00:01much. This is a real
• 00:03pleasure to be here. It's
• 00:04good to see a lot
• 00:05of old friends and a
• 00:06lot of new faces.
• 00:08Thanks especially to AZA and
• 00:09Marina for the invitation.
• 00:12So I'm gonna tell you
• 00:13about our work.
• 00:16I'm good? Okay.
• 00:21Good. Okay. I'm gonna tell
• 00:22you about our our work
• 00:24into,
• 00:25brain immune circuitry.
• 00:27Really taking kind of a
• 00:29whole body,
• 00:30more holistic integrative perspective into
• 00:32understanding,
• 00:34how our body responds to
• 00:35stress and ultimately
• 00:36what that means for psychiatric
• 00:38disease.
• 00:40So I think
• 00:41when I was a student,
• 00:43this picture was much less
• 00:45complex for me. We really
• 00:46thought about the brain and
• 00:47behavior in terms of kinda
• 00:49simple circuits in the brain.
• 00:51You have a nerve cell
• 00:52that's
• 00:53probably wrapped, with by an
• 00:55oligodendrocyte,
• 00:57and that then signals everything
• 00:58that's important to the brain,
• 01:01to guide our behavior.
• 01:03Well, it's much more complex
• 01:04than that. I'm sure there's
• 01:05a lot of, glial fanatics
• 01:07in the room. There's lots
• 01:08of other cell types beyond
• 01:09nerve cells that can do,
• 01:11things,
• 01:12as complex as regulate synaptic
• 01:13plasticity and circuit function.
• 01:16But but even beyond that,
• 01:17right, we kind of think
• 01:19that that the brain is
• 01:20somewhat cordoned off from the
• 01:22rest of the body when
• 01:23we do our studies, and
• 01:24we don't really think about
• 01:25these dynamic interactions
• 01:27in both directions between,
• 01:29how we feel
• 01:30centrally and and what that
• 01:32means for neural encoding
• 01:34and then how our body
• 01:35then senses those signals and
• 01:37and communicates that bidirectionally.
• 01:40This is particularly important, I
• 01:42think, for conditions like depression
• 01:43where we see a high
• 01:44degree of comorbidity.
• 01:46This is actually the these
• 01:47are the, datasets that really
• 01:49drove me towards this field
• 01:51was that depression is not
• 01:53a singular disease in most
• 01:54cases. You see depression associated
• 01:57with,
• 01:58several,
• 01:59general medical conditions as well
• 02:01as several other brain diseases,
• 02:03including both neurological and psychiatric
• 02:05diseases. And one of the
• 02:06common features that kind of
• 02:08that that connects all of
• 02:10these is the immune system.
• 02:12Many of these diseases
• 02:14are caused by either primary
• 02:16or secondary
• 02:17disturbances
• 02:17either in immunity,
• 02:19inflammation, or some immunological
• 02:22process.
• 02:24If you look at the
• 02:24prevalence rates, for example, for
• 02:26some of these kind of
• 02:27bonafide autoimmune or inflammatory diseases,
• 02:31depression risk goes way up.
• 02:33In in a given year,
• 02:34it might be,
• 02:36a risk of three point
• 02:36two percent in the general
• 02:38population. But look at asthma.
• 02:39Asthma
• 02:40is eighteen point one percent.
• 02:42This really does argue that
• 02:43there's something shared
• 02:45that's that's that's causing this
• 02:47really substantial
• 02:49increased risk of of developing
• 02:50a psychiatric syndrome. And the
• 02:52data for anxiety actually looks
• 02:54similar. So it's not
• 02:56just,
• 02:57depression or or or mood
• 02:59disorders. It does seem to
• 03:00be this broad swath of
• 03:01stress related conditions.
• 03:04We put together this table
• 03:06recently.
• 03:07Actually, postdoc in my lab,
• 03:08Kenny Chan, put this table
• 03:09together as as part of
• 03:10a
• 03:11review, trying to just,
• 03:13accumulate,
• 03:14knowledge around biomarkers,
• 03:16that are shared amongst these
• 03:18general conditions.
• 03:20Is there a a pointer?
• 03:23I have an extra one.
• 03:24You have an extra one?
• 03:25I'll use maybe I'll just
• 03:26use this. Yes. You guys
• 03:27can see that. Right? Oh,
• 03:29that is right. Yeah. That's
• 03:30a good idea.
• 03:31Perfect. So if you look,
• 03:33these are these are,
• 03:34cytokine profiles that came from
• 03:36a range of different studies
• 03:38that we see shared amongst
• 03:39patients with various illnesses from
• 03:41depression
• 03:42all the way down to
• 03:43coronary artery disease,
• 03:45and then there are equivalent
• 03:46mouse models. And you can
• 03:47see there's a lot of
• 03:48shared features. A lot of
• 03:49the same players are coming
• 03:50up in both.
• 03:52Back in the day, I
• 03:53think that we all looked
• 03:54at this data and thought,
• 03:55well, of course, you're you're
• 03:57you're inflamed. Of course, your
• 03:59body's responding.
• 04:00It's just a consequence of
• 04:01being stressed, but it's an
• 04:02unrelated biomarker
• 04:04because we know that the
• 04:05brain cordon off all of
• 04:06these factors and sequesters them
• 04:08in in in circulation and
• 04:10prevents them from actually acting
• 04:12upon brain circuits.
• 04:13So while it might be
• 04:14interesting for, you know, diagnostic
• 04:16purposes, I don't think there's
• 04:18a path forward for treatment.
• 04:21I didn't believe that.
• 04:23And so when I was
• 04:24starting my lab years ago,
• 04:25I posed, I think, three
• 04:27main questions that I set
• 04:28out to answer with respect
• 04:30to these. The first was,
• 04:31what are the cellular mechanisms
• 04:33that are underlying these effects?
• 04:34Because we had no concept
• 04:36of where these inflammatory molecules
• 04:38were coming from. They come
• 04:39they're produced and secreted by
• 04:41a diverse,
• 04:42number of different cell types
• 04:43throughout the brain and the
• 04:44body.
• 04:45And so we wanted to
• 04:46define what these cell,
• 04:48these cellular mechanisms were.
• 04:51Then we wanted to show
• 04:52that immune cells and their
• 04:53factors that they secrete could
• 04:55in fact interface with brain
• 04:57circuitry. I know it sounds
• 04:58kind of
• 04:59obvious now, but, you know,
• 05:01fifteen years ago,
• 05:02I remember my first post
• 05:04doc coming back from a
• 05:05meeting,
• 05:06where she said somebody came
• 05:07up to her and told
• 05:08her that her project was
• 05:09useless
• 05:10because I l six doesn't
• 05:12get into the brain. And
• 05:13so
• 05:14this was a concept that's
• 05:15really become more and more
• 05:17accepted, but wasn't as recently
• 05:18as fifteen years ago.
• 05:21And then lastly, this is
• 05:22maybe a a a a
• 05:23newer line of research, but,
• 05:26but I think it's starting
• 05:27to enable us to get
• 05:28at these comorbidities and, you
• 05:30know, go beyond psychosomatic medicine
• 05:32and actually define mechanisms that
• 05:34might drive these types of
• 05:35comorbidities because it's not just
• 05:36all in your brain. It's
• 05:37not just all in your
• 05:38mind.
• 05:39And so this this last
• 05:40part, I hope I can
• 05:41get to it. I'm being
• 05:42a little ambitious today, but
• 05:43this would, potentially tell you
• 05:45a little bit about, some
• 05:46of these upstream mechanisms where
• 05:48the brain is actually sensing
• 05:49stress and sending signals down
• 05:51to the periphery.
• 05:53So this is my outline.
• 05:55I'm gonna start with a
• 05:56bottom up perspective. This is
• 05:58where we know the most,
• 05:59where we're
• 06:01focusing on how,
• 06:02systemic immune compartments can interface
• 06:05with brain circuits to control
• 06:06behavior.
• 06:07And then in part two,
• 06:09I'm gonna talk about a
• 06:10top down mechanism in which
• 06:12the brain is regulating gut
• 06:13dysbiosis.
• 06:16So,
• 06:17you always have to have
• 06:18a good clinical partner if
• 06:19you're a preclinical basic scientist
• 06:21studying psychiatric disease because,
• 06:24mice are not depressed. Mice
• 06:25do not exhibit the classic
• 06:27psychiatric syndromes that you may,
• 06:30encounter in the clinic.
• 06:31And so,
• 06:33very early on at Mount
• 06:34Sinai, I found my clinical
• 06:35partner. That's James Murrell. Many
• 06:37of you may know him
• 06:38probably more from his work
• 06:39in ketamine,
• 06:41with Dennis Charney, but he's,
• 06:43now a convert. I would
• 06:44call him a burgeoning neuroimmunologist.
• 06:47And what we did was
• 06:48we built a bio,
• 06:49repository
• 06:51of, patients that were coming
• 06:52through his depression and anxiety
• 06:53clinic.
• 06:54They would come in,
• 06:56they get a clinical, workup,
• 06:58including,
• 07:00not just psychiatric workup, but
• 07:02basic labs like CBC with
• 07:04differential.
• 07:05We have lots of of
• 07:06rich data clinically,
• 07:09you know, of clinical scales.
• 07:10We have lots of imaging
• 07:11data on these. One of
• 07:12the things that became clear
• 07:14early on was that patients
• 07:15that suffered from depression, at
• 07:16least a subset of them,
• 07:18exhibited myelopoiesis.
• 07:19And this is an increase
• 07:21either in the production or
• 07:22the release of monocytes and
• 07:24neutrophils into the bloodstream.
• 07:26And you can see that
• 07:27shown over here. We don't
• 07:28see disturbances broadly across other,
• 07:31leukocyte subtypes, including lymphocytes. We've
• 07:33done more sophisticated analysis,
• 07:36in these, but it really
• 07:37does appear that myelopoiesis is
• 07:39an enduring,
• 07:41response of the body to
• 07:43being depressed or stressed. This
• 07:44has been shown by many
• 07:45other groups too, by the
• 07:46way. This just came up.
• 07:48This is not something that
• 07:49we've, proven ourselves only. So
• 07:51but what we did find
• 07:52because we have this rich
• 07:54clinical dataset was there are
• 07:56some clinical correlates of myelopoiesis
• 07:58that are interesting. For example,
• 08:00stress.
• 08:01This is one type of
• 08:02stress that we measure based
• 08:04on childhood trauma or the
• 08:05intensity of childhood trauma, but
• 08:07we see equivalent correlations with,
• 08:09for example, perceived stress. If
• 08:11you ask the patient how
• 08:11stressed are you, they'll endorse
• 08:13a number, and that's also
• 08:15correlated with myelopoiesis. So in
• 08:17humans, it does appear to
• 08:18be stress driven.
• 08:20So stress and trauma increases
• 08:22myeloid cells and potentially
• 08:24causes some disturbance in behavior.
• 08:26These cells are also more
• 08:27reactive. You can take them.
• 08:28You can harvest them from
• 08:29blood. You can put them
• 08:30into a dish. You can
• 08:31stimulate them with an agonist,
• 08:32and they release more pro
• 08:33inflammatory factors in response to
• 08:35it if they came from
• 08:36a depressed person versus a
• 08:38healthy control.
• 08:40But they can't they have
• 08:41to get into the brain.
• 08:42And around,
• 08:44I should also say, because
• 08:45I think I forgot to
• 08:46say this, but this was
• 08:47led by a very talented
• 08:48former postdoc, Florian Kathomas,
• 08:50a psychiatrist
• 08:51now back in Switzerland.
• 08:54An earlier postdoc in the
• 08:55lab wanted to understand whether
• 08:57or not there was a
• 08:58potential breach
• 08:59in the barrier systems that
• 09:01would have potentially prevented some
• 09:03of these,
• 09:05immune to brain interactions. And
• 09:06so she started by assessing
• 09:08this is Carolyn Menard, by
• 09:09the way. She started by
• 09:11assessing,
• 09:12various blood brain barrier health
• 09:13markers in brains of postmortem
• 09:15patients with depression.
• 09:18And she found a loss
• 09:19of one particular factor, cloudin
• 09:21five. Cloudin five is an
• 09:23epithelial
• 09:24specific tight junction protein. So
• 09:25it's really only expressed in
• 09:27the neurovasculature
• 09:28in brain. No other cell
• 09:29type. And it's a membrane
• 09:31spanning protein that links up.
• 09:33It kinda zips up to
• 09:34endothelial cells and blood vessels
• 09:36so that,
• 09:37it creates a a a
• 09:38an initial barrier. It's the
• 09:40first layer of the blood
• 09:41brain barrier so that,
• 09:43molecules can't passively diffuse through
• 09:45the spaces between those endothelial
• 09:47cells. And she found that
• 09:48it was lost in both
• 09:50cases.
• 09:52It wasn't reversed by antidepressant
• 09:54status, but I should caution
• 09:55you caution you. Most of
• 09:56these cases are suicide cases.
• 09:59We have
• 09:59basically no information on therapeutic
• 10:02efficacy. It's just at the
• 10:04time of death that they
• 10:05have an antidepressant in their
• 10:07system or not. If we
• 10:08do controlled rodent studies using,
• 10:10say, for example, social defeat
• 10:12stress and then treatment with
• 10:14thirty days of chronic omepramine,
• 10:16Animals that respond to that
• 10:18thirty day course of omepramine
• 10:20do show evidence of normalization
• 10:21of barrier proteins. So it's
• 10:23possible that even standard antidepressants,
• 10:25when therapeutically therapeutically
• 10:27efficacious,
• 10:28could have beneficial effects to
• 10:30your barrier.
• 10:31A
• 10:32more a more recent study
• 10:33came out confirming these data
• 10:34at a functional level at
• 10:35least in a subset of
• 10:36patients.
• 10:37For this, this group used
• 10:39something called d c DCE
• 10:41MRI.
• 10:42For the clinicians, you might
• 10:43use this,
• 10:45for example,
• 10:46in in neurology to identify
• 10:47a tumor or a stroke.
• 10:49What you do is you
• 10:50inject a contrast
• 10:52dye gadolinium.
• 10:53You inject it intravenously,
• 10:55and you let it perfuse
• 10:56into the into the brain,
• 10:57and you can then measure
• 10:58the fluorescence by MRI
• 11:00as a measure of of
• 11:01of, periphery to brain, infiltration.
• 11:04And they showed evidence that
• 11:05there was increased permeability at
• 11:06the functional level.
• 11:07We were disappointed a little
• 11:09bit because we were doing
• 11:10the same study with James's
• 11:11group. This is work led
• 11:12by, Sarah Costi,
• 11:14a resident, in James' group
• 11:16and, again, Florin Katamas.
• 11:19Ours is a has a
• 11:20little bit more rich clinical
• 11:21data, so we can kind
• 11:22of make associations better.
• 11:25But these are the different
• 11:26features that we collected across
• 11:28this cohort from whole brain
• 11:29permeability to,
• 11:31immune markers like this metalloproteinase,
• 11:34which I'll get into a
• 11:34little bit more later on,
• 11:36several clinical scales that tap
• 11:38into anxiety, depression,
• 11:41trauma, and then, we always
• 11:43get a CBC with differential
• 11:44for leukocyte subcount or subdistributions.
• 11:48We don't see broad group
• 11:49differences. And I think if
• 11:50you looked at the previous
• 11:51dataset, that was also the
• 11:53case. Right? It was a
• 11:54few subjects pulling them up.
• 11:55In fact, we think about
• 11:56twenty five percent of all
• 11:58patients with depression may exhibit
• 12:00this this type of immune
• 12:02biosignature.
• 12:04We don't think it's everybody,
• 12:05for example. But what we
• 12:06do see is some clinical
• 12:08predictors of brain health,
• 12:10and the biggest one being
• 12:11a history of childhood trauma.
• 12:13Much, much more predictive than
• 12:15even the individual's own perception
• 12:17of their stress, which is
• 12:18captured by the PSS score.
• 12:20As you can see strong
• 12:21correlations
• 12:22between MMP,
• 12:23brain permeability,
• 12:25and several myeloid,
• 12:27cell subtypes.
• 12:28If you look a little
• 12:29bit deeper into this data,
• 12:31you can see really,
• 12:32in terms of childhood trauma,
• 12:34it's a history of physical
• 12:35abuse and psychological abuse, so
• 12:37social trauma that's driving a
• 12:39lot of the brain permeability
• 12:40that we see. It's also
• 12:43of the two myeloid subtypes
• 12:44that we capture, it seems
• 12:46that monocytes seem to be
• 12:47more broadly in at least
• 12:49correlated with some of these
• 12:50factors,
• 12:51suggesting maybe that monocytes and
• 12:53a history of trauma might
• 12:55be the two features that
• 12:56we're looking at when we're
• 12:57thinking about the immune biotype
• 12:58of depression.
• 13:00All of this has led
• 13:01to this hypothesis. We think
• 13:02that stress somehow, maybe through
• 13:04direct circuits that go from
• 13:06brain to body and innervate
• 13:07bone marrow, are causing an
• 13:09increase in the differentiation release
• 13:10of these myeloid cells into
• 13:12circulation.
• 13:13Once in circulation,
• 13:15they're actively trans,
• 13:17locating to neurovascular
• 13:18spaces. This isn't just a
• 13:20passive diffusion effect. They're not
• 13:22getting into the brain, we
• 13:23don't think, at least, at
• 13:24this point.
• 13:25We think that they're they're
• 13:26adhering to the endothelial wall
• 13:28in local neurovascular
• 13:30niches and then secreting factors
• 13:32that can gain access to
• 13:33the parenchyma
• 13:34via holes, essentially, in the
• 13:36up endothelial,
• 13:38in in the endothelial lining.
• 13:41So before I get to
• 13:42my out mouse model, I
• 13:44mentioned we don't have models
• 13:45of depression. I think that's
• 13:46very clear. We probably don't
• 13:47have models of any psychiatric
• 13:48disease. But I do think
• 13:50that trauma is something that's
• 13:52evolutionarily conserved, and our bodies
• 13:54can respond to trauma in
• 13:55ways that are very similar.
• 13:57And we do have ethologically
• 13:58valid models of mouse trauma.
• 14:00We've been using it for
• 14:01years. We've been calling it
• 14:02a depression model.
• 14:04But, effectively, the social defeat
• 14:05stress model is a social
• 14:07trauma model.
• 14:08Animals are
• 14:10chronically and consistently exposed to
• 14:12short bouts of bullying and
• 14:14social subordination much like humans.
• 14:17And then
• 14:18on top of that physical
• 14:20abuse or that physical trauma,
• 14:22there's a a sensory period.
• 14:24There's this psychological trauma of
• 14:25being housed next to your
• 14:27aggressor
• 14:28knowing full well what's happening
• 14:29the following day.
• 14:31And so we like this
• 14:32model in terms of capturing
• 14:33aspects of social trauma that
• 14:35might be driving psychopathology.
• 14:37And I think one of
• 14:38the unique things that was
• 14:39was first characterized by Eric
• 14:41Nessler's group, and,
• 14:43I was lucky to be
• 14:44a postdoc. Ralph was a
• 14:45new assistant professor just getting
• 14:47going at the time. It
• 14:47was a really exciting time
• 14:48to be in the lab,
• 14:49but they showed that this
• 14:51genetically identical
• 14:53inbred line of mice showed
• 14:54vastly different responses to this
• 14:56trauma that could be captured
• 14:58in a simple readout of
• 14:59social interaction behavior, and that
• 15:01is simply asking the mouse
• 15:02to interact with an aggressor
• 15:04inside a cage or
• 15:06to interact with an empty
• 15:08cage.
• 15:09And it's been it's been
• 15:11really predictive of a lot
• 15:12of more complex behaviors, but
• 15:13we never really understood what
• 15:14it meant. We called it
• 15:16depression. We called it social
• 15:17reward. It's probably fear and
• 15:19trauma, if I'm honest.
• 15:21So a couple of years
• 15:23ago, just as a quick
• 15:24sidestep, and then I'll go
• 15:25back to the immunology bit.
• 15:27I had a postdoc in
• 15:28the lab that was interested
• 15:29in this to see if
• 15:29it's what this really is.
• 15:32And so this is we
• 15:33we think it's fear generalization,
• 15:35actually, is what we're capturing
• 15:36because here's a modified version
• 15:38of the model.
• 15:41So this is a resilient
• 15:42mouse. This is a mouse
• 15:43that doesn't show social avoidance
• 15:44to the to the aggressor
• 15:46after ten days of defeat.
• 15:47We drop a a same
• 15:48sex juvenile mouse that's nonthreatening
• 15:51and nonaggressive into its home
• 15:52cage, and we look at
• 15:54the interaction.
• 15:55Juveniles are rewarding. They actually
• 15:57press levers for these guys.
• 15:58It's really amazing. They develop
• 16:00place preference for them, and
• 16:02they're very interactive. They're very
• 16:03interested in them. Look what
• 16:05happens when we select a
• 16:06susceptible mouse. We drop a
• 16:08juvenile. Now, again, these juvenile
• 16:09mice are two to three
• 16:10weeks younger.
• 16:12They're they're they're not attacking.
• 16:13They're not showing any evidence
• 16:15of threat, and this susceptible
• 16:17mouse is clearly viewing that
• 16:18animal as a threat. He
• 16:20went on to show that,
• 16:21effectively, what's happening is this
• 16:22threat sensors in the brain
• 16:24are occluding the ability of
• 16:25these animals to develop social
• 16:26reward, and that's why we
• 16:28think it's something akin to
• 16:29fear generalization.
• 16:31Back to immunology.
• 16:32So from these mice, after
• 16:34this interaction, what we did
• 16:35was we did we collected
• 16:36blood, and we took a
• 16:37very high altitude
• 16:39assessment of their leukocyte subtype
• 16:42distribution throughout blood. Now this
• 16:44is approximately very close to
• 16:46the stress, so it's a
• 16:47little bit different than what
• 16:48we see in humans, but
• 16:49it gives us a sense
• 16:50of what's going on. This
• 16:51is, these are tSNE plots
• 16:53that came from a mass
• 16:54cytometry study. Mass cytometry is
• 16:57a lot like flow. It
• 16:57uses heavy metals conjugated to
• 17:00antibodies. It gives you a
• 17:01lot more flexibility in terms
• 17:03of the number of surface
• 17:04antigens you can apply. But,
• 17:06we we used approximately
• 17:08fifty different types of surface
• 17:09antigens to care broadly characterize
• 17:11these immune cell subtypes.
• 17:13And we found two interesting
• 17:14things. One is that stress
• 17:16really dramatically activates the innate
• 17:17immune cells. Right? So these
• 17:19are myeloid cells like neutrophils
• 17:21and monocytes.
• 17:22And it's broadly immunosuppressive.
• 17:24So these are,
• 17:26showing data from b cells,
• 17:28various subtypes of b cells
• 17:29involved in adaptive immunity, and
• 17:31then we get similar profiles
• 17:33with t cells, etcetera.
• 17:34And this is maybe also
• 17:35not surprising because we all
• 17:37know when we get sick,
• 17:39it's usually because we just
• 17:40came off of a very
• 17:41stressful time in our in
• 17:42our lives, and our our
• 17:43immune system's down,
• 17:45and and we're now more
• 17:46vulnerable. But the thing that
• 17:47we didn't see is any
• 17:49differences between our our our
• 17:50behaviorally distinct groups of mice,
• 17:52which was a little disappointing.
• 17:54But luckily, Florin was also
• 17:57piloting brain CyTOF at the
• 17:59time, so we were able
• 18:00to get immune cell profiles
• 18:01in the brain of these
• 18:02mice as well. And the
• 18:03first thing I want you
• 18:04to notice is there's obviously
• 18:05not as many leukocyte subtypes
• 18:07in the brain. Most of
• 18:08them are not resident, for
• 18:09example. But the most predominant
• 18:11one is, in fact, these
• 18:12microglia or brain resident macrophages,
• 18:14which isn't surprising.
• 18:16When we looked across all
• 18:18of these different subtypes that
• 18:19we were able to detect,
• 18:20really only monocytes gave us
• 18:22any inkling of a signature.
• 18:24And and and to be
• 18:26frank, this is a a
• 18:27pretty minor effect.
• 18:29And part of that is
• 18:30due to the fact that
• 18:30in these early days of
• 18:32CyTOF, in order to get
• 18:33enough cells, because there's not
• 18:34a lot of monocytes
• 18:36in the brain relative to
• 18:37other cell types, we had
• 18:38to crush down an entire
• 18:40brain per sample. And so
• 18:41we're getting nothing about kind
• 18:43of region specificity or selectivity.
• 18:46But we did see evidence
• 18:47that there might be trafficking
• 18:49increases in these susceptible mice
• 18:51where lies where these monocytes,
• 18:53though being the same in
• 18:54circulation,
• 18:55are somehow,
• 18:57entering or or or attaching
• 18:59to the brain neurovasculature
• 19:00at higher levels in susceptible
• 19:02mice. But to get more
• 19:04granular
• 19:05data,
• 19:05we decided to use,
• 19:07a brain clearing procedure called
• 19:09iDisco,
• 19:11combined with the this transgenic
• 19:13line that allows us to
• 19:15distinguish
• 19:16the, peripherally derived monocytes from,
• 19:18for example, central microglia.
• 19:21We use the CCR two
• 19:22promoter to drive, red fluorescent
• 19:24protein. And you can see
• 19:25there's actually a lot of
• 19:26monocytes
• 19:28in the brain, not as
• 19:29many as as, of, monocytes
• 19:31as there are, for example,
• 19:32of microglia. But as we
• 19:33start to kinda hone into
• 19:34these brains, you'll start to
• 19:36see these small round circles.
• 19:37Each one of those are
• 19:38a monocyte.
• 19:40So there's lots of these
• 19:41in the brain somewhere,
• 19:42and they seem to be
• 19:43expressed or or,
• 19:45attached in various places across
• 19:47the extent of it. So
• 19:49this time when we quantified
• 19:50again, we got the same
• 19:51type of effect,
• 19:53in susceptible mice across the
• 19:54entire brain,
• 19:56and it was it was
• 19:57correlated to our behavioral phenotype.
• 20:00But what became really interesting
• 20:01was when we started thinking
• 20:02about region specific
• 20:04accumulation of these monocytes,
• 20:06that's where we think
• 20:08the the data is is
• 20:09starting to uncover something, unique.
• 20:11So in incumbents, in the
• 20:12ventral striatum, we see an
• 20:14increase in susceptible mice, but
• 20:16we don't see anything in
• 20:17the cortex.
• 20:18So despite the fact that
• 20:19there's all these monocytes
• 20:21floating around in circulation,
• 20:23they're only attaching in certain
• 20:25parts of the brain.
• 20:27And
• 20:28based on a previous study
• 20:30that Carolyn had done back
• 20:31in the day where she
• 20:32used that same MRI,
• 20:34gadolinium method to measure brain
• 20:36wide permeability
• 20:37following social defeat stress.
• 20:39She found this this interesting
• 20:41pattern, which at least is
• 20:43aligned somewhat with these monocyte
• 20:45data.
• 20:46The accumbens, for example,
• 20:48shows increased permeability
• 20:50in susceptible mice,
• 20:52but not the prefrontal cortex.
• 20:54And so we've started to
• 20:55look at this more broadly,
• 20:56and it does appear that
• 20:57areas where monocytes are accumulating
• 20:59are the areas of the
• 21:00brain where we're starting to
• 21:01see these endothelial holes in
• 21:03this increase of brain permeability.
• 21:05We don't know if that
• 21:06they're causal yet, but we
• 21:07do think that they might
• 21:08be.
• 21:10However and, again, this gets
• 21:11back to this point of
• 21:13where these monocytes are. This
• 21:14is important because it changes
• 21:16how we think about the
• 21:17mechanism.
• 21:18They're not inside the brain.
• 21:19They're too big. They're not
• 21:21getting through these small little
• 21:22holes in the endothelium.
• 21:24What they're doing is they're
• 21:25adhering to the endothelial
• 21:27cells themselves, and we think
• 21:28we know why.
• 21:30We did a study recently
• 21:31where we, we did trap
• 21:33seek. This is using,
• 21:35the ribosomal
• 21:36affinity proto
• 21:38profiling protocol, not the cell
• 21:40capture protocol.
• 21:41And we're able to isolate,
• 21:43RNAs that are poised at
• 21:45the ribosome for translation.
• 21:47And when we seek those,
• 21:48a lot of adhesion molecules
• 21:50were upregulated
• 21:51in these vulnerable mice in
• 21:53endothelium
• 21:54in areas like accumbens, but
• 21:56not in cortex.
• 21:57So I think what's happening
• 21:58in the areas where we
• 21:59see this accumulation,
• 22:00these adhesion molecules, these junctional
• 22:03adhesion molecules molecules
• 22:05whose sole purpose is to
• 22:06reach into the luminal side
• 22:07of the blood vessel and
• 22:08grab on to myeloid cells
• 22:10are being upregulated in those
• 22:11regions where we're seeing blood
• 22:13brain barrier damage and monocyte
• 22:14accumulation.
• 22:17But what are these monocytes
• 22:18doing? And And can this
• 22:19give us any insight into,
• 22:21what what mechanisms might be
• 22:23driving this interaction? For this,
• 22:24we did,
• 22:25single cell RNA sequencing.
• 22:27And by doing this, we
• 22:28were able to, characterize four
• 22:30unique subtypes based on
• 22:33on transcriptional profiles,
• 22:36and one of them seemed
• 22:37to be enriched in susceptible
• 22:39mice. You can see this
• 22:40cluster zero right here.
• 22:42What is cluster zero? We
• 22:43did a gene ontology analysis,
• 22:46and, actually, some unexpected thing
• 22:47some expected things came up
• 22:48and some unexpected things came
• 22:50up. And, of course, we
• 22:51went after the unexpected things
• 22:52because we thought they were
• 22:53more interesting.
• 22:54And that is,
• 22:56these genes that were making
• 22:57up up terms such as
• 22:59the extracellular matrix and the
• 23:00extracellular
• 23:01space. We thought why the
• 23:02hell would an immune cell
• 23:04have a transcription profile in
• 23:06brain to regulate the extracellular
• 23:07matrix. Prominent among these was
• 23:09MMP eight. This is matrix
• 23:11metalloproteinase.
• 23:12We've done some some investigation
• 23:14into it. It happens to
• 23:16be upregulated in depression.
• 23:18Three studies have now validated
• 23:20this finding since we published
• 23:22our paper, which is really
• 23:23cool.
• 23:25So it it's bonafide. It's
• 23:26a subset of patients.
• 23:28It's shown at multiple levels,
• 23:30and it
• 23:31seems to be coming from
• 23:32a myeloid cell. And, again,
• 23:34it correlates with that trauma
• 23:35score that I talked about
• 23:36earlier.
• 23:38But what is MMP eight?
• 23:40It's not there's not a
• 23:40lot known, and there's almost
• 23:41nothing in brain because it's
• 23:43not produced in the brain.
• 23:44Right? There's other MMPs that
• 23:45are definitely produced in the
• 23:47brain, and we've characterized them.
• 23:48This is produced by myeloid
• 23:50cells. It's only expressed by
• 23:51neutrophils
• 23:52and monocytes.
• 23:54And I think the best
• 23:56characterized role for it
• 23:58and and if if you
• 23:59guys are any MMP eight
• 24:00aficionados and have another explanation,
• 24:02you might be right. But
• 24:04I like the story about
• 24:05this role in cardiovascular
• 24:06disease.
• 24:07So because it helps me
• 24:08to understand what it might
• 24:09be doing in the brain.
• 24:11So I'm gonna try to
• 24:12explain this, but, you know,
• 24:14basically, this is an,
• 24:15an, an aortic plaque, and
• 24:17you can see that it's
• 24:18got this really dense lipid
• 24:20core.
• 24:21The stability of this plaque
• 24:22is critically important because if
• 24:24it ruptures and that lipid
• 24:26core diffuses,
• 24:27that can be really bad,
• 24:29medically speaking. Right? So you
• 24:31wanna stable if you're gonna
• 24:32have plaques, you want a
• 24:33stable plaque. And a stable
• 24:34plaque is derived from this
• 24:36fibrous cap right here, which
• 24:37is made up of collagen.
• 24:39Now in the case of
• 24:40of inflammation
• 24:42and cardiovascular
• 24:43disease,
• 24:44macrophages
• 24:45infiltrate the fibrous cap. You
• 24:47can see them shown right
• 24:48here and here, and they
• 24:49produce MMP eight. MMP eight
• 24:52is secreted,
• 24:53and it breaks down the
• 24:54collagen network
• 24:55that makes up that fibrous
• 24:57cap. Now you get plaque
• 24:58instability, and you get a
• 25:00cardiovascular
• 25:00event.
• 25:02And in fact, I'm gonna
• 25:03take another little detour.
• 25:05Susceptible mice do show exactly
• 25:06this profile. So if you
• 25:07take,
• 25:09and look at aortic plaques
• 25:10in these different subgroups of
• 25:12animals, susceptible mice have the
• 25:14largest aortic plaques,
• 25:15and they have the highest
• 25:16distribution of macrophages.
• 25:18The CD eight marker labels
• 25:20macrophages. So it does appear
• 25:23that this mechanism
• 25:24could be involved in increasing
• 25:26one's risk for cardiovascular
• 25:28disease. It's a whole lecture.
• 25:29I'm not gonna talk about
• 25:30it. I'm I'm gonna talk
• 25:31about the brain today, but
• 25:32I did wanna take this
• 25:33little detour because I want
• 25:35you to be thinking whole
• 25:36body now. You know, these
• 25:37are not mechanisms that were
• 25:38derived to affect the brain.
• 25:40These are things that are
• 25:41disrupted in our body, and
• 25:42they're gonna impact lots of
• 25:44different systems in our body.
• 25:46And this is the type
• 25:46of work that may explain
• 25:48why
• 25:49cardiovascular
• 25:50mortality
• 25:51is is a hundredfold higher
• 25:52in a patient with depression,
• 25:53for example, if they have
• 25:54a heart attack.
• 25:56At any rate, what does
• 25:57it do in the brain?
• 25:58Well, there's not a lot
• 25:59known in the brain. There's
• 26:00some evidence actually that MMP
• 26:01eight cleaves
• 26:03junctional proteins, so that might
• 26:04be why monocytes
• 26:06that accumulate that express this
• 26:08can are associated with open
• 26:10regions of the blood brain
• 26:11barrier possibly. We haven't tested
• 26:12it yet. We were more
• 26:14interested in its potential role
• 26:15for regulating synaptic physiology because
• 26:18there is a big literature
• 26:20in neuroscience that's
• 26:22characterized
• 26:22the impact of the brain's
• 26:24extracellular matrix on plasticity
• 26:26and synaptic physiology.
• 26:29But it's not MMP eight.
• 26:30It's about other MMPs. For
• 26:32example, MMP nine, which is
• 26:33secreted by glial cells locally.
• 26:35And what this does is
• 26:36it breaks down
• 26:38the extracellular
• 26:39proteins that are made up
• 26:40of collagens and proteoglycans
• 26:42that provide sort of structure
• 26:44and support for the brain.
• 26:45It breaks them down, and
• 26:46it opens up windows of
• 26:47plasticity where new synapse can
• 26:49be added,
• 26:50existing synapses can be strengthened,
• 26:52and, ultimately, what you end
• 26:53up with is increased synaptic
• 26:55transmission.
• 26:56We have a similar phenotype
• 26:58in our stressed mice. Using
• 26:59EM, we found that, in
• 27:01fact, there is an increase
• 27:02in the extracellular
• 27:03space. So there is an
• 27:04opening or a loosening
• 27:06of the space between that,
• 27:07which suggests maybe
• 27:09that some of these gly
• 27:11proteoglycans
• 27:12that make up the extracellular
• 27:14matrix are in fact decreased,
• 27:15and we do see that.
• 27:17I do wanna point out,
• 27:19in these mice, we measured
• 27:20circulating levels of MMP eight,
• 27:22and the correlation between circulating
• 27:23levels of MMP eight in
• 27:25extracellular space in just a
• 27:27small cohort like this, I
• 27:28think, is pretty impressive.
• 27:29Really
• 27:30suggesting maybe that there is
• 27:32some causal
• 27:33links to MMP eight and
• 27:34circulation and these effects.
• 27:38Coming back I mean, come
• 27:40going back to some of
• 27:41our earliest work, when I
• 27:42first started my lab, we
• 27:43had been characterizing
• 27:45some of the synaptic adaptations
• 27:46that occur that are very
• 27:48much in line with the
• 27:49idea of this permissive extracellular
• 27:51matrix state adding new synapses.
• 27:53We showed that susceptible mice
• 27:55have more of these, dendritic
• 27:57spine structures, which are excitatory
• 27:59synapses.
• 28:00We showed at a functional
• 28:01level, they have more functional
• 28:03synapses. These are,
• 28:04EPSC recordings in the presence
• 28:06of tetrodotoxin.
• 28:08And then to test whether
• 28:10MMP eight was involved in
• 28:11any of these things, we
• 28:12generated bone marrow chimeric mice.
• 28:14And the way you do
• 28:15this is think about a
• 28:16patient that has leukemia. What
• 28:17you wanna do is you
• 28:18wanna give them series of
• 28:20radiation therapy to get rid
• 28:22of all of the cancerous
• 28:23immune cells,
• 28:26and
• 28:26and and all of the
• 28:27hematopoietic,
• 28:29stem cells that they're derived
• 28:30from. Well, we do the
• 28:31same thing to mice. So
• 28:32you can irradiate a mouse.
• 28:33You can deplete their peripheral
• 28:35immune system, and then you
• 28:36can,
• 28:37reconstitute that mouse with immune
• 28:39cells from anything.
• 28:41Stress susceptible mouse. It confers
• 28:43stress susceptibility.
• 28:45In this case, we actually
• 28:47did it with MMP eight
• 28:48knockout mouse mice. So we
• 28:49have now a wild type
• 28:51adult,
• 28:52normally developed c fifty seven
• 28:53mouse that completely lacks MMP
• 28:56eight in their in their
• 28:57bone marrow, which means that
• 28:58none of their immune cells
• 28:59express MMP eight, and it
• 29:01completely blocks that effect on
• 29:03synaptic physiology that I just
• 29:04showed you, which is amazing.
• 29:06Right? That is that a
• 29:07circulating factor
• 29:08can completely block or abolish,
• 29:11the effects of stress on
• 29:12the physiology of our nucleus
• 29:14accumbens medium spinae neurons.
• 29:16We also saw that it
• 29:17prevented that increase in extracellular
• 29:19space, again, suggesting that there's
• 29:21a direct causal link between
• 29:23systemic
• 29:24circulating MMP eight.
• 29:25And then lastly, it normalized,
• 29:28social interaction behavior. It also
• 29:30normalized several other depression associated
• 29:33or trauma associated behaviors.
• 29:35But this is all that
• 29:35I'm gonna show you.
• 29:37And as I recoup myself,
• 29:39this is probably unnecessary, but
• 29:41I spent a lot of
• 29:42time
• 29:43making this slide, so you're
• 29:44all so you're all gonna
• 29:45watch it. Okay?
• 29:49So this is our model.
• 29:51What we think is happening
• 29:52is that we're sensing stress
• 29:54in our environment,
• 29:55and our body is reacting
• 29:56by mobilizing immune cells. And
• 29:58those immune cells happen to
• 29:59be myeloid derived
• 30:01of the innate immune,
• 30:02subtype
• 30:04include
• 30:05more specifically, probably,
• 30:07sorry, monocytes, but also potentially
• 30:09neutrophils.
• 30:11Those cells actively transport. We've
• 30:13got data that I haven't
• 30:14shown you here,
• 30:15that that molecular
• 30:17signatures of active,
• 30:19leukocyte migration are upregulated in
• 30:21these cells, and they can
• 30:22be prevented by pharmacological
• 30:24blockade.
• 30:25When they when they attach
• 30:26or adhere to regions,
• 30:28there seems to be barrier
• 30:29damage and secreted factors of
• 30:31which MMP eight is one,
• 30:33but there's probably many other
• 30:34can now access the parenchyma,
• 30:36break down that brain extracellular
• 30:38matrix, open up a window
• 30:40of plasticity that leads to
• 30:41changes
• 30:42ultimately in the in the
• 30:43firing properties of these nerve
• 30:45cells. And these are the
• 30:46same types of electrophysiological
• 30:47adaptations
• 30:48that we've shown Mary k
• 30:50Lobo at at University of
• 30:51Maryland has shown and and
• 30:53many others.
• 30:54So I'm gonna pivot now
• 30:56to talk about
• 30:58colons,
• 30:58if everybody's okay with that.
• 31:02So,
• 31:04what are the what are
• 31:05the possible reasons why myeloid
• 31:08cells are activated by stress?
• 31:09This is a question that
• 31:10we've asked ourselves a lot.
• 31:12And we don't know yet
• 31:13know the answer, but I
• 31:14think some of these studies
• 31:15might get, a little bit
• 31:16closer to that.
• 31:18So why is this interesting?
• 31:20Like other comorbidities,
• 31:22bowel symptoms are a a
• 31:24very common symptom of patients
• 31:26that have depression. And in
• 31:27fact, the the frontline treatment
• 31:29for IBS is actually
• 31:31I think it is still.
• 31:32It's it's one of the
• 31:32common. Is it frontline for,
• 31:34like, the old school, amitriptyline,
• 31:37for for IBS still? Or
• 31:39do they have better drugs?
• 31:40Anyways, it's used a lot.
• 31:42We'll say it that way.
• 31:44And so what's really thought
• 31:45is that stress
• 31:47exacerbates. So stress conditions like
• 31:49depression exacerbate
• 31:50bowel physiology
• 31:51problems
• 31:52and disrupt disrupt normal intestinal
• 31:55processes.
• 31:56It's thought to increase the,
• 31:57to, alter the, motility and
• 32:00transit from the gut. It's
• 32:02thought to change mucus production
• 32:04from goblet cells. It's thought
• 32:05to increase intestinal permeability similar
• 32:08to brain endothelial permeability,
• 32:10and then it's thought to
• 32:11do so through an enteric
• 32:12neuronal population, which is heavily
• 32:14innervating,
• 32:15gut tissue. And all of
• 32:16this leads to dysbiosis at
• 32:18the level of the microbiome
• 32:19and low grade systemic inflammation.
• 32:21That's kind of the working
• 32:23model from a clinical perspective.
• 32:26Keddy Chan,
• 32:27also postdoc in my lab,
• 32:29currently on the job market,
• 32:31by the way. He's spectacular.
• 32:32Decided to to to dig
• 32:34into this phenomenon a little
• 32:35bit more. And he hypothesized
• 32:36that chronic stress was gonna
• 32:38be causing a shift in
• 32:40the inflammatory,
• 32:42profile
• 32:43of of the gut, specifically
• 32:44within the lamina propria. And
• 32:46this would be marked by
• 32:47a shift from
• 32:48anti inflammatory t cells to
• 32:50pro inflammatory t cells. That
• 32:52would then lead to a
• 32:53breakdown of of the epithelium
• 32:55through a loss of tight
• 32:56junctions,
• 32:57and an increase in the
• 32:59release of of of bacterial
• 33:01end products,
• 33:02I e endotoxins
• 33:03like lipopolysaccharide,
• 33:05from the gut into circulation.
• 33:07Myeloid cells contain or are
• 33:09enriched actually in an LPS
• 33:12receptor called the TLR four
• 33:14receptor. So LPS
• 33:15endotoxemia
• 33:17activates innate immune cells
• 33:19in circulation
• 33:20to then be pro inflammatory
• 33:22and release inflammatory molecules, and
• 33:24this was his hypothesis.
• 33:25So he first started with,
• 33:27flow cytometry,
• 33:28investigation of the gut immune
• 33:30environment,
• 33:31and he found that chronic
• 33:32social defeat stress increases
• 33:35these, t h one inflammatory
• 33:37t cells. They're marked by
• 33:39high levels expression of interferon
• 33:41and, CD four.
• 33:44There was also a reduction
• 33:46in, t h two anti
• 33:47inflammatory cells marked by interleukin
• 33:50four and CD four. So
• 33:51the ratio of this these
• 33:53two markers is really important,
• 33:55and it seemed to be
• 33:55skewed to a highly inflammatory
• 33:57state.
• 33:59We're no longer we've been
• 34:00reprimanded statistically for years about
• 34:03using the term susceptibility and
• 34:04resilience.
• 34:06So we're now using it
• 34:07as a continuous variable,
• 34:08but you could still see
• 34:09those populations
• 34:10right here. So
• 34:13Okay. So permeability.
• 34:15To get permeability
• 34:16assessments, Kenny
• 34:18took,
• 34:18physidextrin. It's a dye,
• 34:21and he gavaged mice following
• 34:22social defeat.
• 34:24And then if you take
• 34:25blood from these mice, you
• 34:26can,
• 34:28perform an an analysis of
• 34:29of, the how much FITC
• 34:31dextrin is now in circulation.
• 34:33Under normal conditions, FITC doesn't
• 34:34get into the it doesn't
• 34:36bypass the epithelial barrier.
• 34:38And so anything you detect
• 34:39in blood is typically
• 34:41caused by an increase in
• 34:42permeability, and we saw an
• 34:43increase in in brain per
• 34:45brain permeability and gut permeability.
• 34:48Can't take the neuroscientist
• 34:49out of me.
• 34:50And this again correlated with
• 34:52some of these behavioral features.
• 34:56The the the whole idea
• 34:57of this was that
• 34:59this was leading to a
• 34:59more permissive state so that
• 35:01things in the gut that
• 35:03shouldn't get into the bloodstream
• 35:04were now getting in. And
• 35:05so we performed a shotgun
• 35:07meta genomic analysis to look
• 35:09at the different,
• 35:10types of pathways,
• 35:11of the microbes in our
• 35:13gut that were disrupted.
• 35:14And one of the top
• 35:15pathways was lipopolysaccharide
• 35:18biosynthesis.
• 35:19So, again, this is this
• 35:20idea that there's an increase
• 35:22in the bacterial
• 35:23composition in our guts that's
• 35:25that shifted towards endotoxemia.
• 35:29And last but not least,
• 35:30of course, if you look
• 35:31at those endotoxins in the
• 35:32bloodstream, you see,
• 35:34an elevation of it.
• 35:38And so last but not
• 35:39least, with with respect to
• 35:40this kind of basic characterization
• 35:41of the physiology of the
• 35:42gut,
• 35:44Kenny wanted to establish
• 35:46that the the myeloid cell
• 35:48itself was responsible for this.
• 35:50So he generated a bone
• 35:51marrow chimeric animal that lacked
• 35:53TLR four in these bone
• 35:55marrow derived myeloid cells and,
• 35:57of course, that partially normalized
• 35:59social defeat. I think it
• 36:00would have fully normalized social
• 36:02defeat, but, you know, over
• 36:03the years I've been doing
• 36:04that, this was a pretty
• 36:04brutal defeat. So I think
• 36:06that this is why we're
• 36:07seeing just a partial reversal,
• 36:08but certainly
• 36:10suggesting causally that that these,
• 36:12endotoxins
• 36:13are possibly activating myeloid cells.
• 36:15And now what we're doing
• 36:16is is we're going back
• 36:17to see if this is
• 36:18also involved in this process
• 36:20of trafficking.
• 36:21I think once they're activated
• 36:22by LPS,
• 36:23you're gonna see this this
• 36:24transcriptional profile
• 36:26that that that, drives leukocyte
• 36:28migration, and that's what's bringing
• 36:29the cells up to the
• 36:30neurovasculature.
• 36:31And when the neurovasculature
• 36:33expresses
• 36:34the lock for that key
• 36:35to go into, I e
• 36:36these junctional proteins, that's where
• 36:37you see accumulation.
• 36:38But that's, something that we're
• 36:40still coming back to. And
• 36:42in the last few minutes
• 36:43this was really ambitious, but
• 36:44I hope I can get
• 36:45through this.
• 36:48I'm good? Okay. So how
• 36:49does how does the brain
• 36:50do all of this stuff?
• 36:51Is there is our hypothesis
• 36:53correct that the brain is
• 36:54actually sensing this process of
• 36:56of of being stressed and
• 36:57sending signals outward through circuits
• 37:00that ultimately impinge upon,
• 37:02the colon and and and
• 37:03impact,
• 37:04gut dysbiosis.
• 37:09Oh, boy. This I had
• 37:10to take from a textbook.
• 37:12So I was never good
• 37:13at anatomy, but, basically,
• 37:16the the pathways for these
• 37:17things have been really well
• 37:19worked out.
• 37:20For the intestines, for example,
• 37:22I don't know if you
• 37:23can see that very well.
• 37:24I should get a bigger
• 37:25version of this.
• 37:27But they are innervated
• 37:29by cells that emanate from
• 37:31the celiac ganglion.
• 37:34That then goes through,
• 37:36the spinal cord up to
• 37:37the brain stem
• 37:39and beyond. And that's the
• 37:41pathway that we think
• 37:42could be controlling this process.
• 37:45So we, of course, had
• 37:47to validate
• 37:48it with modern methods because
• 37:50the textbook was not right.
• 37:52We put a a psorohrabies
• 37:53virus into the into the
• 37:55colon,
• 37:56and then did a time
• 37:57sack. Every twenty four hours,
• 37:59they jump about a synapse
• 38:01or so, so you kinda
• 38:03loosely label a monosynaptic
• 38:04synapse every twenty four to
• 38:06forty eight hours.
• 38:07So So we started seeing
• 38:08brainstem expression from colon injections
• 38:11at about ninety six hours.
• 38:13So about three synapses away,
• 38:15which makes sense. You can
• 38:16see it really nicely at
• 38:17a hundred and twenty hours.
• 38:18And here's the overall way
• 38:20in which the circuit is
• 38:21laid out exactly like the
• 38:23textbook, so we confirmed it,
• 38:24thankfully.
• 38:26But it it goes the
• 38:27one, I think, interesting than
• 38:28the thing that we did
• 38:29learn is the the the
• 38:30spinal cord,
• 38:33domain that it goes through.
• 38:34It goes through the thoracic,
• 38:36not the cervical level. So
• 38:37but, again, it it's a
• 38:39it's a well defined circuit,
• 38:40and,
• 38:41it's it's, certainly still there
• 38:43in the mouse.
• 38:46So after stress, we wanted
• 38:47to look at each node
• 38:48to see whether or not
• 38:49there was evidence of differences
• 38:51of activation within this kind
• 38:52of multisynaptic
• 38:53circuit.
• 38:54So we first looked at
• 38:55the celiac gaglion, and you
• 38:57can see, I think I
• 38:58don't know. Was that two?
• 38:59You can see,
• 39:00we use CFOS as a
• 39:01as a marker of activity,
• 39:02and you can see that
• 39:03there's an increase in activity
• 39:05and,
• 39:06marked by increases in CFOS
• 39:07in the celiac ganglion.
• 39:10We found also the same
• 39:11type of increase
• 39:12in,
• 39:13the colon itself.
• 39:16Except for this, we actually
• 39:17counter stained with DBH
• 39:19to show that it's actually
• 39:20is these enteric neurons. These
• 39:22are dopamine beta hydroxylase containing
• 39:24TH cells,
• 39:26which are which are,
• 39:28sympathetic in nature, for example.
• 39:31And then we wanted to
• 39:32say, okay. What what's the
• 39:33next level? So how is
• 39:34it then that the brain
• 39:35is connected up? We know
• 39:36that the brain stem is
• 39:38somewhere in there, but what
• 39:39are the higher order
• 39:40brain regions that might be
• 39:42activated by stress that are
• 39:43also innervating gut tissue? And
• 39:45so for this, we used
• 39:46a kind of a multifactorial
• 39:48approach. We injected Pseudorhabes into
• 39:50the colon,
• 39:52mapped out the brain wide
• 39:54expression of PRV by using
• 39:56a whole brain clearing method
• 39:58and then staining for and
• 39:59visualizing PRV expression.
• 40:01And we overlaid this onto
• 40:03another whole brain,
• 40:05clearing,
• 40:07stain with, with CFOS so
• 40:09we could get kind of,
• 40:10like, an activity,
• 40:11map of these.
• 40:13And we found a lot
• 40:14of regions that were enriched.
• 40:15So this means regions that
• 40:16were activated by social defeat
• 40:19that also showed strong innervation,
• 40:22by PRV.
• 40:24And, of course, the PVH
• 40:25was one of the top
• 40:26regions. This is the paraventricular
• 40:28hypothalamus.
• 40:29The PVH is really interesting.
• 40:31It contains
• 40:33a lot of
• 40:34of stress responsive nerve cells,
• 40:36for example. It it contains
• 40:38a really,
• 40:39significant population of corticotropin releasing
• 40:41hormone expressing cells. It's definitely
• 40:43involved in stress. It regulates
• 40:44stress at a behavioral level.
• 40:48We used, again, this kind
• 40:50of intersectional approach to get
• 40:51a sense of what the
• 40:53the the innervation patterns look
• 40:55like. And the predominant cell
• 40:57type that is innervating the
• 40:58gut from the PVH does
• 40:59seem to be CRH positive,
• 41:01Makes up about fifty two
• 41:02point nine percent. It's not
• 41:03all of them, but it
• 41:04certainly is a significant proportion
• 41:06of them.
• 41:07So the first study that
• 41:08we did was to was
• 41:09really simple and nonspecific. We
• 41:11put an inhibitory dread into
• 41:13the CRH presales,
• 41:15or into the c r
• 41:16the PVH of a CRH
• 41:18cream mouse,
• 41:20waited for expression, put them
• 41:21through ten days of defeat,
• 41:22and then gave them CNO
• 41:23every day just before the
• 41:25defeat. So we silenced that
• 41:26circuit, and then we measured
• 41:27gut physiology.
• 41:29We were able to prevent
• 41:30that change in gut permeability,
• 41:32and we were able to,
• 41:33again, partially,
• 41:35reverse the social avoidance deficits.
• 41:38Now this is a very
• 41:39gross,
• 41:41manipulation, and I'm I'm hoping
• 41:43that all of you guys
• 41:44are are are thinking that
• 41:45I'm full of crap right
• 41:46now, that really all I'm
• 41:48doing is activating the HPA
• 41:49axis. Right? It sounds like
• 41:51it's Really, like, just so
• 41:52I I just showed all
• 41:54of you that the HPA
• 41:55axis is now involved in
• 41:56stress responses. It's it's profound.
• 41:59So
• 42:00we did a few studies
• 42:01to kinda rule this out.
• 42:03The first one, I think,
• 42:04is straightforward. We got rid
• 42:06of the the the HPA
• 42:07response with metirophone.
• 42:09You can use adrenalectomy
• 42:10as well, but, this did
• 42:12not affect any of these
• 42:13parameters. So,
• 42:15even in the in the
• 42:16absence of corticosterone,
• 42:17for example,
• 42:19CNO is still capable of
• 42:20doing all of these things.
• 42:21So that's one thing. It
• 42:22doesn't seem to be HPA
• 42:23driven, but we weren't really
• 42:25convinced. And so, we partnered
• 42:26up with a with one
• 42:27of your former colleagues, Ivan
• 42:28de Arrajo,
• 42:30and did local denervation
• 42:31of the gut. Right? So
• 42:33this bypasses
• 42:34all of those endocrine mechanisms
• 42:35that I just mentioned. We
• 42:37used, an antibody against DBH,
• 42:39and we we conjugated it
• 42:41to a toxin called saponin.
• 42:43So whenever this antibody
• 42:44binds to DBH positive cells,
• 42:46it kills them off, and
• 42:48we get a local denervation
• 42:50of sympathetic inputs to the
• 42:52gut. And you can see
• 42:53that right there. And under
• 42:54these circumstances, we were still
• 42:55able to prevent gut permeability
• 42:57changes
• 42:58and normalize
• 42:59social avoidance behavior. So we
• 43:01do think it's local. We
• 43:02think it's neural driven. It's
• 43:03not, through the the HPA.
• 43:06Not that the HPA is
• 43:07not important for stress responses,
• 43:08but it might not be
• 43:09important for this particular
• 43:11stress response.
• 43:14I did it. Alright. This
• 43:16is my final slide.
• 43:17So I hope that I
• 43:19have convinced you that we
• 43:20should be thinking about brain
• 43:22diseases in a more holistic
• 43:24way.
• 43:25Brain diseases are complicated,
• 43:28and now we've thrown another
• 43:29layer of complication into an
• 43:31already complicated system. But I
• 43:33think it's really critical that
• 43:34we investigate
• 43:36the way in which these
• 43:37brain and body circuits
• 43:38and and processes interact in
• 43:40health and disease. I think
• 43:42we're always gonna be up
• 43:43against the glass ceiling in
• 43:45in treatment if we don't
• 43:47get at these comorbidities
• 43:49and address them. You know,
• 43:51I have I will I
• 43:53was telling Azi, I have
• 43:54a bit of experience in
• 43:55in psychological,
• 43:57as a clinical psychology intern.
• 43:58And every patient that I
• 43:59saw during that time
• 44:01had something else other than
• 44:03depression, for example. And I
• 44:04think that most of these
• 44:05clinical cases are incredibly complex,
• 44:08and we need to start
• 44:09to think more broadly across
• 44:10the possibilities of of these
• 44:12brain body connections.
• 44:14So with that, I'm gonna
• 44:15thank the people in my
• 44:16lab that did it. Of
• 44:17course, Foran and Kenny.
• 44:19I featured a lot of
• 44:20their work prominently. Carrie Lynn,
• 44:22played a major role in
• 44:23this. Georgia Hodes was my
• 44:25first postdoc.
• 44:26Started the whole thing. Without
• 44:28her, I wouldn't be here.
• 44:29Carolyn and Maddie, it's the
• 44:31same. You know? They've just
• 44:32all gone on to do
• 44:33amazing work. Our collaborators I'd
• 44:34like to give a, a
• 44:35big thanks
• 44:37to Yvonne,
• 44:37Wenfei,
• 44:38Sarah, and Hongzhen who've really
• 44:40allowed us to be able
• 44:41to dissect out these peripheral
• 44:44systems for which I have,
• 44:46you know, no knowledge base
• 44:48to pull from. So you
• 44:49gotta find yourself good collaborators
• 44:50if you wanna if you
• 44:51wanna kinda think outside of
• 44:52your comfort zone. Of course,
• 44:54I'd like to thank my
• 44:55funding sources and all of
• 44:57you guys for actually coming
• 44:58in person and listening to
• 44:59me talk. So thank you
• 45:00very much.