 There's no Little foam thing came off Well, it seems I think you can clip it on with that, right? It's not easy Okay, okay. All right We're I Don't know what that means And And Thank you You know, I think there's two things you don't want in a meeting You don't want to have to get the after lunch talk and you don't want to have to talk after steward fires time Some are I got both I'd certainly like to thank the organizers for the opportunity to come here It's such a delight to be back in Trieste with such a wonderful group of people I look forward to learning a lot from not only the faculty that are here, but also all the students and postdocs that come So I've been interested for a long time in the development of the olfactory system And what we could learn about principles of development in the olfactory system so we could then apply to other Other parts of the central nervous system be it neocortex or brainstem or spinal cord and to that And we've carried out a number of studies taking a look at critical periods in development We've looked at that the role of afferent input and how it affects central organization how order exposure affects Organization the central nervous system and a number of other features But one thing that has remained largely less attended to at the very least I would say is the role of the embryo the role of embryology the earliest components of development beginning at the time of the neural tube and the neural epithelium and how determinants at that time May in fact serve as protomaps or models Directors for how the remainder of the olfactory system will continue to develop That's what I'd like to tell you a little bit about today But before I do that I want to continue with Stewart's introduction to the olfactory system And I'm gonna oops. Sorry. I'm gonna use the The same diagram that he started with this is from Santiago Ramonica Hall 19th century His original drawing of this is from 1897 it's considerably more primitive than this one In fact Hall said in one of his letters to one of his correspondence after his Histology of the nervous system had come out that it was fortunate He had learned how to make a composite drawing or an impressionistic drawing of what he had seen in the central nervous system Because if he had been required to make plates for each of the figures that he had included in that in that two volume text It would have required more than 3,000 separate plates So all the images that you see from Cajal like this one are impressionistic ones that he's gained or he's developed based upon his observations of individual neurons Reconstructed with the Golgi technique beginning in around 1891 So Stewart began out here in the olfactory epithelium I will as well with the populations of sensory neurons There are large numbers of them found in the mouse olfactory epithelium and a steward pointed out each one expresses only one odor receptor We did not point out to you that is that all of the sensory neurons expressing the same odor receptor go back to converge and Arborize in only two to three glomeruli within the olfactory bulb So there's a molecular specificity to a glomerulus that is determined by the nature of the properties of the odor receptor That's expressed by the population of olfactory sensory neurons that are projecting their axons into that particular glomerulus These axons are unbranched until they reach the glomerulus after which they branch 17 to 18 times Making a corresponding number of synapses not solely onto these large microcells But certainly heavily onto that primary population of projection neurons So the microcells are located in a single layer around the circumference of the olfactory bulb I'll show you more of that in just a few minutes Besides their large apical dendrite only one of which extends from the cell body and goes up to innovate a single glomerulus They also have populations of secondary dendrites seen here these arborize in this next deepest layer down called the external Plexiform layer where they establish synaptic interactions with populations of inner neurons called granular cells seen here Their cell bodies are in the deepest layer of the olfactory bulb They have an apical dendrite that extends up into the external Plexiform layer and something that I'll come back to you later on an interesting observation Sometimes controversial or at least under discussion is that populations of these granular cells may in fact segregate into two separate populations one of which arborizes deep within the external Plexiform layer and the other Arborizing superficially within the external Plexiform layer offering the opportunity to have a bilayer or bilamina Organization of local circuits within the external Plexiform layer So the axons of the mitral cells as Stuart mentioned do go out to pyriform cortex or olfactory Cortices including the olfactory tubercle and the pyriform cortex will Concentrate on for the latter half of the top in pyriform cortex We'll also find a laminar organization like the laminar organization We found here in the olfactory bulb it will be considerably different based upon the populations of cells there and certainly Relative to neocortex it will be different as well. It's a paleocortex It has three primer. It's really hot up here. I don't know what it's like out there, but up here. It's really hot Not that you're putting any pressure on me It's a paleocortex with three layers of organization in contrast to the six layers of organization seen in neocortex So now let's begin with the embryological development and we can begin with Ross Harrison He was a sterling professor of biology at Yale and he gave the crunian lecture to the Royal Society in London in 1933 and he made the following observation the distribution of dividing cells in the central nervous system of the embryos Inequal with a series of zones of more intense activity alternating with zones of less He went on to make the notation that was also a temporal determinant So he made the observation the seminal observation that within the neurogenic zone within the proliferative zone of the developing embryo There were populations of progenitor cells that were very busy at one time less busy at another time and those zones were were In his mind at least at the time kind of randomly distributed or arbitrarily distributed throughout the neural tube with a neurogenic zone Eight decades later beginning with this observation. We now understand that in fact there is a well-defined programmatic development beginning and beginning in the embryo for the development of the neocortex we can begin over here in the neuroepithelium with a ventricular zone populations of Intermediate progenitor early progenitors give rise to radial glial cells radio glial cells and turn divide asymmetrically giving rise to populations of neurons that go up to occupy the upper layers of cortex They also divide symmetrically to generate more radial glial cells Which also then go on to continue to divide in some cases producing intermediate progenitor cells also with asymmetric Provisions eventually leading to the generation of glial cells very late in development We can take this program and we can look at it not only from a from a spatial perspective But we also also can look at it from a temporal perspective so the deepest layer Population of cells and we're only highlighting here or these authors I should say only highlighted here the pyramoral neurons the deep layer parameter on Paramount neurons layer six primal neurons are the first to be generated. They have a unique molecular signature Sorry about that Unique molecular signature the development of their dendritic processes is unique or Characteristic for that particular layer and they also have projections that go into the corpus callosum and go outside of the cortex in contrast to the superficial layer pyramoral cells also Unique in the distribution of the both their cell bodies and dendritic processes and now with axons that state Intracortically now for a long time. It was thought many years It was thought that progenitor cells depending upon the timing of neurogenesis that you see down here going from E11 to E16 Any one progenitor cell could be pluripotential and give rise to both superficial Paraminal neurons as well as deep layer pyramoral neurons Non-cystonic Yale and many many others throughout the world that in fact most progenitor cells are fate limited Limited meaning simply that they're going to give rise to a narrower perspective or a narrower population of cells They may give rise to more than one type of cell But that the number of cells that they give rise to will be limited now as we go on with development We can recognize again from the work of summa connell and now also poshka Rikish something called the radial unit Radial glial cell unit hypothesis and what this refers to is the spatial Organization of the neurogenic zone so we can think up here at the top and in the cortex of an embryo You can just think of this in fact is that as the neuro epithelial zone and each of these zones is going to be giving rise Particular to a particular type of specialization in neocortex So the reddish area here will give rise to visual cortex the yellow area will give rise to auditory cortex The blue area to somatosensory and so on and so forth So as we look at development in cortex in particular what we can recognize is there there is a spatial determinant to the Laminar organization therefore the circuit organization of the cells found in peripheral cortex And there's also a determine for the kind of information that's going to be processed a spatial organization How that applies to the olfactory system is not well understood and what I'd like to talk to you about today are two aspects of the olfactory system Beginning with work we've done previously on on on the organization of microcells in the olfactory bulb and how the timing of neuro genesis of those cells Influences where they're distributed and their connectivity and then move on to some new unpublished data on peripheral cortex and the role of timing and Neurogenic and migratory Migratory behavior of these populations of cells as they move out into peripheral cortex to occupy their final positions one of the thing that I forgot to mention I mean, yeah, so Questions are welcome at any time. Please feel free to interrupt me. You have a question Stuart will be happy to answer it. So jump in So we'll begin with the olfactory bulb and and and microcells and the role of microcell fate is determined by time of progenitor cell division This is a coronal sections through the olfactory bulb We've arbitrarily divided into a dorsal medial zone and a ventral lateral zone based upon the expression of OCam olfactory cell adhesion Molecule shown in green which is expressed by the axons of olfactory sensory neurons. It's not completely arbitrary So when the work from Kinsako Mori would suggest that this more dorsal medial aspect of the olfactory bulb may be involved in Processing intrinsic odors associated with fear and avoidance Where is this more ventral ventral lateral aspect of the olfactory bulb? Maybe more associated with competitive behaviors and food seeking behavior But with this is beginning now we can start to ask about the distribution of mitral cells So we've labeled mitral cells here again going around the circumference of the olfactory bulb with a transcription factor called TVX 21 which is fairly not completely but reasonably exclusive to mitral cells within the olfactory bulb Giving us the opportunity to look at individual mitral cells and ask where their cell bodies are located in the mitral cell layer Around this circumference dorsal medial or ventral lateral also the distribution of their secondary dendrites within the external plexiform layer for any clues about the local circuit Local circuit interactions with granule cells that may be occurring We began with a relatively simple experiment Using thymidine analogs any number of them Bromodioxyuridine CLDU or IDU we injected at these five different agents Just one injection per age and then sacrificed all the animals that post natal day 20 and looked at the distribution of labeled cells around the circumference of the olfactory bulb and both the both the Mediolateral aspect as well as dorsal ventral aspect and anterior posterior aspect We use the following criteria to identify labeled cells that have been labeled at the time of our injection So green is the is the uptake of bromodioxyuridine indicating that this cell Divided at the time the analog was administered red is a a TVX 21 positive cell But in the absence of any green staining it means it was born or it was its neurogenic period was outside the time frame in which We had administered the bromodioxyuridine and any of the cells that we recognize that were double labeled with both Red and green often appearing yellow were cells that were both Born within the time frame that we were interested in as well as expressing TVX 21 meaning they were in fact mitral cells Yeah, it does but tough to cells don't even begin to be born until around embryonic day 14 So we had looked at that at an earlier point in time So we didn't worry too much about that that confounding the data analysis. That's a good point I mean we could we could in fact carry out a similar analysis beginning with the administration of Thymidine analogs that say embryonic day 14.5 or 15 and look exclusively at toughed cells to see if they follow a similar neurogenic pattern so the data Showed us early on or immediately that the bulk of neurogenic activity for microcells occurs here between embryonic day 10 11 and 12 what was pleasing Was that there was not a there was not a symmetry in the distribution of labeled cells across across that time frame when we administered one Thymidine analog and embryonic day 10 and then in the same animal administered a second thymidine analog at embryonic day 12 We in fact found that they had a complimentary distribution the early born microcells those born at E 10 actually E 9 as well And then on through E 10 are distributed predominantly not exclusively but predominantly in the dorsal medial Aspect of the olfactory bulb while those administered at embryonic day 12 and also to some extent embryonic day 13 Distribute predominantly in the ventral medial abspect of the olfactory bulb So we interpreted that to say that the distribution of early and late generated microcells Form a basic temporal or protomap within the olfactory bulb with early born cells occupying one region of the olfactory bulb and Later born occupying later born microcells occupying different regions Given the distribution of primary aphorins coming in from the nose This obviously has implications for how different odors may be coded as we work through the olfactory system Stuart So this was not a this is not a planted question. Yes. No, it's not so So Peter gonna is gonna say something about this is being SOS same old shit It's intended mostly as an introduction So we know from from both work that Linda and Susan Sullivan did as well as work that that we did later on in the embryo beginning at as early as embryonic day 8.5 that there is a temporal Coordinate to the appearance of populations of cells expressing specific odor receptors They don't all come on at the same time and their peak appearance appears to vary across time as well We did But but my point my point is that you and Susan also recognized that there were populations of odor receptors that you could detect very early in embryonic Yeah, and then we went on to suggest that it was variable So I think the answer to your question Stuart is that there probably is variability and there was a recent paper Kind of ashamed. I don't remember his name a former associate of Sikhanos That recently took a look at the development of the olfactory epitheum and found a dorsal to ventral gradient Suggesting that the most dorsal aspect of the epitheum which in fact is the part of the epitheum That would be going here to the dorsal medial aspect of the olfactory bulb develops prior to then the Develops earlier several days earlier than the more ventral aspect of the olfactory. Yeah, so it looks like there's correspondence on that level So so the bromide oxyuregine is only taken up a time of cell division So that's that's that's one of the parameters that we have and we know that that thymidine analogs when they're injected as they were here Into the mother intrepair it nearly are only present for about six hours as a viable marker of dividing cells They're not taken up at later time points and that's been established Not by my lab, but by other labs doing double labeling studies So we know at least within a six-hour interval that we're labeling a limited population of cells No, is it possible that we would have a progenitor cell that undergoes a symmetric division? Generating now a second progenitor cell which then undergoes an asymmetric division to generate a mitral cell. It's possible Seems unlikely based on these prior studies that I just mentioned with double labeling, but it's certainly possible so That was fine But you know it only gives us information about the localization or the expression of a thymidine analog in the nucleus of the cell It doesn't tell us much about the about the structure of the cell and where its processes may be distributed We wanted to use that information to gain some better insight into into how they may be integrated into local circuits So we turn to electroporation the proliferative zone for the for the micro cells in the olfactory bulb is located all of the way at The rostral pole of the telencephalic vesicle and you can begin to label cells there as early as embryonic day 9 But certainly embryonic day, you know a 10 11 and 12 There's a there's a greater frequency of cell division and it's easier to get cells at that point So we began as early as embryonic day 10 to use electroporation to label populations of dividing cells So we introduced our plasmid GFP in this case into the ventricle and then positioned our Electrodes in order to drive that plasmid rostrally into this very tip of the telencephalic pole Where the micro cells are being generated and then we sacrificed at at various points afterwards mostly Postnatal day 20 to find out where these cells were located in the olfactory bulb and where they're dendritic processes and eventually where their axons were Distributing and an example of that is shown here So at the top row we Electroporated at embryonic day 10 at the bottom row. We electroporated at embryonic day 12 We sacrificed at postnatal day 20 and all of the green cells are cells that we labeled with our electroporation at embryonic day 10 Versus embryonic day 12. These were obviously not different groups of animals as opposed to the previous experiment And what I'd like you to recognize and I'll back it up with a with a subsequent image as well Is those cells electroporated at embryonic day 10? Microcells tended to have their secondary dendrites distributed in the deepest portion of the external plexiform layer That is that portion of the external plexiform layer Proximal to the cell body so the cell bodies are here in the dendrites We're coming out and spreading out quite immediately and that's in contrast to what you see here at embryonic day 12 where the Electroporated cells now have a cell body here and an apical secondary dendrite rather than extends up Like the arms of an umbrella and then spreads out now in the more superficial Partion of the external plexiform layer that's highlighted here where we've taken a couple of these embryonic day 12 cells and Also labeled them with a red dye to help contrast them from the others that were also labeled at embryonic day 12 And again, you see their secondary dendrites moving up into this most superficial portion of the external plexiform layer Now together with the data that has has suggested that Granule cells have apical dendrites that also segregate between the superficial and the deep portions of the external plexiform layer We've been interpreting this and it's open to debate But we've been interpreting this as as as evidence that early-born mitral cells are interacting with a population of Granule cells local circuits that are segregated segregated or different from the population of local circuits or granule cells But the late-born mitral cells are interacting with not unlike the kind of segregation to see in In pure from court to I mean neocortex if you're looking at the superficial versus the deep pyramidal cells So a segregation of local circuit interactions that seems to be I think is a determinant of or is Determined in part by the time of neurogenesis Now we've gone on to take the yeah So so again the tufted cells are born later, so it's unlikely that we're getting those So a tufted cells form form there are actually three populations There's a most superficial tufted form population of tufted cells that stay intra bulb are there's a middle portion of Projection neurons tufted cell projection neurons that go out to peer from anterior peer from cortex and olfactory cubicle, and then there's a deep Consoc to call the mitral cell type to That seems to follow this the same behavior as other mitral cells But their cell body is maybe slightly displaced out of the main main layer of mitral cells It would it would you don't you don't see much evidence at all of tufted cells going to posterior peripheral cortex They don't go to the amygdala they go heavily to the tubercle and to the anterior peer from cortex But not all the way to posterior and not to antirhinal cortex And speaking of going to cortex So we also use use use the data from electroporation to take a look at the distribution of axons and the LOT going from anterior peer from cortex all the way back to posterior peer from cortex as well as to the olfactory tubercle and And Yeah, so the deep tufted came. Yeah. Well, I don't think there is one the nomenclature has varied through the years from deep tufted to mitral cell type 2 or or your variation on on superficial mitral cells But I don't think there's a difference. I think they're I think they're I personally think they're mitral cells We're sadly, yeah We're sadly lacking reliable markers of projection neurons tufted cells and mitral that distinguish tufted Cells and mitral cells that project out of the olfactory bone So we wanted to understand a little more about where populations and cells were going and we thought we might be able to do it with This electroporation strategy, which was clearly inadequate because we couldn't follow populations of individual axons in any manner whatsoever So instead we turned to a strategy in which we labeled cells and Bromagioxy urine thymidine analogs and then put a drop of dye I into either the olfactory tubercle or into pure from cortex And then looked at the distribution of the label cells in the olfactory bone In the following way because the dye I would go retrograde way back to the cell bodies and label everything in there In its entirety we picked a relatively young age just because as the cortex and the brain grows It takes longer and longer for the dye I to diffuse and we want it to be reasonably effective in our use of time And what we found is that there is an asymmetrical distribution When we place the dye in the olfactory tubercle We label predominantly cells that are late born and are located here in the ventral lateral aspect of the olfactory So you see all the red here that red is the labeling of the secondary dendrites of mitral cells that are located here in the ventral lateral aspect of the mitral cell layer and now have axons projecting out to the olfactory tubercle We did the complementary experiment by placing a dye crystal in pure from cortex as well And in that case we found the predominant labeling here in the dorsal medial aspect of the olfactory bulb This is not a completely dichotomous distribution I don't want you to go away with the impression that all dorsal medial cells go to pure from cortex and all ventral lateral cells go To olfactory tubercle, but there certainly is a preponderance of a projection from the ventral lateral aspect of late born cells Going to the tubercle and the dorsal medial aspect Dorsal medial cells going to pure from cortex for the most from the earliest born primal cells Don't think I mean certain. There's certainly a paper from from Joel Price's lab Beth Friedman and Joel Price in 1970 something early 70s Using using a double label on nuclear yellow and died nuclear blue and diatomino yellow To look at at populations of projection neurons and they found that there is a segregation for mitral cells and tufted cells For tubercle and and pure from cortex, but I don't believe they talked about this kind of segregation So we put the story together and and what we have is a situation in which we have populations of projection neurons That are born early with secondary dendrites that segregate in the external plexiform layer from populations of mitral cells That are born later in development with the former being distributed predominant former dendrites being distributed Predominantly in the deep layers of extra the deep portions of external plexiform layer and the latter having their secondary dendrites Predominantly in the most superficial aspect of the external plexiform layer exactly what this means with regard to odor information processing Is still open to speculation? The axons of these cells are certainly extending out to the cortices with a very heavy projection From the late born cells going to the olfactory tubercle also some going out to tear from cortex and the heaviest fraction of Early born microcells going predominantly the pure from cortex What leaves open here and what we don't understand yet is about the organization of pure from cortex itself And the degree to which these kinds of events are similar events may shape the way in which cells are distributed in pure from cortex Or the kinds of connections they make I don't understand exactly Yeah, yeah, this is not it this is not intended to suggest duplicate glomeruli in any way that's a great question It wasn't it wasn't drawn with an intent to show particular access or an intent to explore the two symmetrical glomerula or the Symmetrical glomerular on our other side. I say the truth. I've not thought about that, but it's a great question It could suggest the possibility that odor information originating in subpopulations of Sensory neurons could in fact be going to both areas would buy a separate pathways. It's an interesting idea Um So what we don't know about a lot is is the organization of cortex and some of you know that I have this this this tenancy to be fond of history Above Norland Library in at the University of Colorado is a statement that says he who knows only his own generation remains forever ignorant And and you know using that as a guideline I wanted to go back and find out what the history was or at least some of the history for understanding the organization of pure form cortex a colicor Prokinje all the early anonymous from from beginning in the 1800s through the end of the end of the 19th century Or almost the end of the 19th century largely dismissed pure form cortex They were interested in aspects of development of the olfactory bulb In fact, they were interested in the glomeruli in the olfactory bulb and what influenced the way in which they were Distributed and how they came to receive primary sensory information, but as far as cortex was concerned There's a there's a quote out of out of brazen anatomy from around 1850 It says it's of little importance and therefore will not be considered further at this point It went on to comment as well that it was clearly in a state of progressive atrophy Referring to the human and therefore was not worthy of further study, and it was not until 1893 that the first serious paper came out this came out from Carlos Kaya who was a PhD student with With the Santiago Ramon Ramonica Hall here in this PhD from the University of Madrid in 1895 And he published this paper the regional factorial in the in the cortex in 1893. This is a pamphlet You know wasn't in a journal per se And it is in Spanish, which was a challenge at the time and you might notice up at the top that there's an inscription Signature, which is is C. S. Sherrington Sherrington was was one of the leading Neurophysiologists in the early 20th century was at Oxford It was actually one of the first to show that that the synaptic organization of the central nervous system was a critical Determinant not just the spatial organization But specifically the way in which the synapses functioned within circuits was a critical determinant to how information was being processed So as I as I dug on this to find out more if you flip back the front cover on this Which you find is a letter from Sherrington inside Directed to the the librarian at Oxford and it says it's a little hard to read First it stated October 19th, 1931 I came across another celebrated early paper from the Cajal lab laboratory and send it without delay because it should be alongside the others Already sent you these early papers of the Madrid school are not very difficult to cure I also have the original edition of Cajals el Sestimen de Riosso del Hombre y de los recta product Madrid 1897 two volumes if the college library has not this and would wish it I shall be pleased to offer it It is not bound. It appeared in paper covers only originally as it has long been out of print So Sherrington, you know recognize the importance of this work in 1931, but between 1893 and 1931 almost nothing was done on paraffin cortex Despite the observations that Kaia was able to make so this is again one of the composite drawings It's a horizontal section of paraffin cortex as he recognized it based upon Golgi impregnations You see the nerve layer of the olfactory bulb down here the glomeruli populations of mitral cells Their axons going out to join the lateral facet tract here They are arborizing heavily and what we'll come to describe as layer 1a of paraffin cortex You see populations of horizontally oriented cells and populations of more more radially oriented cells that will come to recognize It's layer 2 Parametal neurons and then finally deeper parametal neurons that are at the deepest aspect In this more simplified drawing on the right hand side also from the same text We again see the lateral facet tract out here, but now they've removed a lot of the a lot of the Detail so you can see the outlines of populations of parametal cells here deep parametal cells down here the heavy Arborization of parametal cell dendrites here and layers what we'll describe as layers 1a and 1b And then finally the lot above they did recognize four layers rather than the three layers We recognize now that a b and c by including the lateral facet tract most contemporary folks described as three layers Beginning with this plexiform layer as layer 1 the Parametal cells as layer 2 and then the deep parametal cells as layer 3 Now the next significant advance in understanding how piriform cortex work didn't have occur in till 1942 When Lord Adrian did the first recordings the first electrical recordings from piriform cortex use the hedgehog Because Lorenta they know a few years earlier had published a paper Describing the organization of piriform cortex and the hedgehog and made it clear that it would be accessible for electrophysiological Analysis I learned I did know by the way it was was a Cajal's last student So this is like a multi-unit recording up the top is just breathing normal air the animals anesthetized Then they administered or exposed the animal to as a fatida, which is a fetid smell It's a it's a spice used in Indian cooking and then finally the odor of decaying worms And what you recognize if you analyze the data is there are a series of relatively Low-frequency peaks that occur here in the absence of odors and in the presence of odors There's a there's a decreased amplitude but a significant increase in the frequency of spikes that are occurring leading them to conclude That in fact piriform cortex was the recipient of direct olfactory information coming in and then it resulted in alterations in functional activity Now that seems trivial now, but in fact, it was the first time it was ever demonstrated in the years that followed this There have been a number of publications Walter Freeman in the 50s improved in early 60s improved upon these recordings Lou Haberly was the first one to do single-cell recordings in the 1960s Joel Price's lab looked at aspects of postnatal development and plasticity Avery and Lorenta didn't know were the first ones to use in 1947 Silver stains to demonstrate definitively where the primary axons of the mitral cells were distributing in the piriform cortex These and so on and so forth until today almost recently We have a wonderful paper from Alex Fleischman's lab that you'll see I think you know later this week Yes, Alex will see yes describing aspects of odor representation in piriform cortex cortex that were Unrecognized, you know just a few short months ago So progress has continued in a really exciting fashion and all of that has led us to a view of piriform cortex It's just summarized here. So this is largely work from from review article by Sakano and Mori in 2011 but it shows effectively the broad distribution of primary aprons coming from the olfactory bulb and tear piriform cortex Well factory-tubical and additional areas as well and this in a review from from John Becker's lab is a nice summary of the distribution of what we now recognize as the different types of neurons that are found in Piriform Cortex some insight into their laminar distribution and also the kinds of synaptic organization what we're lacking sorry, but we're What we're lacking here is the kind of insight about how Embryogenesis influences the way in which these cells are distributed and their timing of their distribution and Maybe some additional insights into what finally shapes their functional properties based upon when they come into being So that's one of the problems that we've tried to tackle We know a little bit about the progenitor zones that give rise to Crameral neurons in particular in piriform cortex. We know that they're coming largely from the paleo sub paleo border There's a very heavy projection from both the ventral pallium as well as the lateral pallium Shown here in blue and also from the dorsal late dorsal I'm sorry the lat dorsal aspect of the lateral ganglionic eminence going into piriform cortex as well There is a lesser distribution that comes from the medial telence phallic wall and also a small contribution from it from the From the septum, but these these these are minor relative to the projections coming from the paleo sub paleo border The the intern neurons that are found in piriform cortex are coming exclusively from the medial geniculate nucleus Which is important because in the studies. I'm about to describe We're going to target the paleo sub paleo border border It will be looking at cells derived from the progenitor is found largely or if not predominant If not exclusively in this particular area now they make their way To their final positions in the cortex well by following ventral migratory streams. It's called the lateral cortical strain And that stream is established by populations of glial cells radial glial cells that leave this paleo sub paleo border and Adopt kind of an S shape as they move ventrally from the paleo sub paleo border out into the particular region of piriform cortex That they're going to that they're going to target. This is not posterior factory cortex It's meant to it's meant to designate presumptive olfactory cortex So we've been trying to look at this process in a little more detail First we wanted to expand a little bit on what we had done earlier about the definition of the layers found in piriform cortex And we use several labels to make that happen Cal Retinin is found exclusively in olfactory sensory non axons Map 2 is in the dendritic processes of both deep and superficial pyramidal neurons and drac 5 is is a nuclear market Just to provide perspective and real and I'll mention in just a minute if we look at the anterior and posterior Piriform cortex for the expression of these you see that there are profound differences in the the thickness of the layers based upon differentiation of the number of axons that are there so the lateral of factory track, which is prominent anteriorly Is not gone but seemingly disappears in posterior piriform cortex as it actually merges into layer 1a of piriform cortex At the same time in posterior piriform cortex layer 1b becomes much thicker These are important distinctions because they reflect different populations of local circuits That are involved in processing information layer 1a here and here is the sole Recipient of primary afferent information coming in via the lot versus layer 1b Which is the recipient of local circuit interactions occurring both feet forward and feedback interactions Within piriform cortex and outside of piriform cortex We've also defined the layers of Layer 2 layer 2 can be distinguished with this line that you probably can't see here But it distinguishes between two populations of neurons found within layer 2 a lot found within layer 2 superficially in in in layer 1a a population of Semi lunar cells that most people call pyramidal neurons that are small in diameter Versus deeper in layer 2b populations of pyramidal cells that are larger in diameter Posteriorly we can use reeling to identify layer 2a of piriform cortex reeling is not expressed in Layer 2a of anterior piriform cortex Developmentally, we also see reeling expressed heavily in the area of the lateral factory track and to some extent extending into Layer 1a of piriform cortex as well where the primary afference of the lateral of factory tract are terminating We carried out a series of thymidine analog studies like we did in the olfactory bulb And again, we use the same kind of criteria for identifying the cells So this is a this is a neuron that is positive for TBR 1 So we know it's a pyramidal neuron from piriform cortex This one is TBR 1 and new and which means that it's its developmental progression is greater or Farther along than that shown here expressing only TBR 1 BDR you BDR you is shown in green So we have three criteria or three populations of cells we could identify BDR you cells that were both TBR 1 and new and negative were not interested in they were not pyramidal neurons cells that were both TBR 1 and new and pregnant New and positive as well as being positive for the thymidine analog We identified as mature pyramidal neurons and finally cells with the thymidine analog But expressing just PR PDR TBR 1 and not new and we identified as immature pyramidal cells We did a series of ages a broader series now than than what had been looked at previously Ranging from embryonic day 10 out through embryonic day 18 and the story is right here at embryonic day 11 through embryonic day 13 So you see that the heavy distribution of the green the VR du labeled cells distributing in the deeper. I'm sorry. You looked at 21 days after we administered the Thymidine analogs that each of the ages shown here and you can see at embryonic day 10 Most of the labeled cells are quite deep at embryonic day 11 They continue to be quite deep in layer three of paraform cortex by embryonic day 12 They're moving up into layer 2 embryonic 13 They're still in layer 2 but with an asymmetric distribution and then from 14 and 15 to 16 onward the number of labeled cells decreased decreased profoundly We quantify this in a number of different ways so we could we could look at labeling density across ages irregardless of across Layers irregardless of age. We found out the cells are distributing predominantly in layers 1a 1b and layer 3 of paracentra paracentra cortex We could also disregard layers We find out that the heaviest labeling or the most extensive neurogenesis is occurring here between embryonic day 10 and about embryonic day 13 or perhaps 14 then with a significant decrease if you look at it more closely I think there more there's more interesting data than emergence first is that the LOT layer 1a and 1b have little going on at the earliest ages There's some going on at the later ages probably reflecting the appearance of populations of interneuromes, but if we look at layer Layer 2a 2b and 3 a couple of interesting observations emerge one is that the preponderance of cells are the heaviest labeling the heaviest Targeting of cells born at the earlier ages is going to layer 3 and you see that here at embryonic day 10. That's maintained through embryonic day 11 and even into 12 and then drops off layer 1a has a relatively comparatively fewer cells generated at embryonic day 10 but increases significantly at embryonic day 11 drops off a bit of 12 and then drops off more significantly at 13 and 14 an area Lamina a which again contains predominantly the semi lunar cells that I mentioned earlier is again a little different from from from 2a Here we see that that again there are relatively few cells being generated in embryonic day 10 It ramps up significantly at 12, but it stays up at 12 13 and 14 and even to even to an extent I'm sorry at embryonic day 14 So it has a much more extended and longer period of neurogenesis relative to relative to layer 2a Offering the opportunity to consider potential differences between the two layers. We did that In part by looking at where cells were distributed in piriform cortex following injections at either I'm react 11 12 13 and 14 and each of these examples that you see in the rows the cells have been The time we need analogs have been administered twice. So I can't see quite well from over there So on the far left there were administer either that embryonic day 11 and 13 12 and 14 11 and 13 or 12 and 14 And we could then plot the distribution of cells across the different layers of piriform cortex to get a better understanding Of how they would be migrating into their final positions and both anterior piriform cortex and posterior piriform cortex at embryonic Postal day zero. We saw this largely stochastic distribution of cells across the three layers There may be have been a tendency for the cells to be somewhat in the deeper layers consistent with their migration in from the Lateral cortical stream, but broadly their distribution seems seems to be more stochastic But by posting the day 21 when the organization of the laminae within piriform cortex is stable we see a quite different picture and that's that the oldest cells the yellow ones seen here are Located predominantly in layer 2b while the earliest born cells only by a day or so But still earlier born cells shown in red tend to be located in layer 2a in both anterior piriform cortex and posterior piriform cortex Suggesting that the timing of neurogenesis for the pyramidal neurons found in within layer 2 was a determinant for where they were Positioning themselves within within within layer 2 of piriform cortex again driving the notion forward that these are different Populations of cells and perhaps even being derived from different populations of progenitors We wanted to look at that in one additional perspective and the way we did that was to ask about the maturation of cells So the sequence of molecular maturation in in piriform cortex is tbr1 it comes on prior to new n And and then drac5 just as a general descriptor So as I mentioned earlier when we see a cell that's labeled with new n and tbr1 We can classify it as a mature pyramidal neuron if we see it labeled only with tbr1 and not new n We can classify it as an immature cell and we see it labeled with new n but not tbr1 It's not a pyramidal neuron and it's not of interest to us So in broad terms very rapidly the number of immature cells in piriform cortex drops off quite quickly across postnatal development but if we look more closely at the earlier time periods of development at postnatal day zero following the administration of New n and drac5 we see that there are not a lot of differences Across the layers of of piriform cortex at postnatal day zero these deepest three bins here are layer 2b of piriform cortex Where the smart where the pyramidal neurons are found layer of 5 is going to be The more superficial layer 2a where the semi lunar cells are found the more interesting observation occurs here at postnatal day 7 With a number of immature Paramidal neurons that is those expressing tbr1 But not new n is much higher than it is in the more superficial aspect Suggesting to us that the neurons in layer 2b mature more slowly than the neurons in 2a So they're born later and they also mature at a much slower rate than the cells found in the more superficial aspect Like you know again, we don't understand the functional implications of this But I think insights like this will provide us with the kinds of tools We need to ask some very pointed questions about how these populations of cells may differ in the way they process Information and even their capacity for plasticity in the early developmental periods, particularly the early postnatal periods So we weren't entirely sad. Yeah. Oh, please That's a great question. There've been two papers one came from stewards laboratory and the most the more recent one came from Alex's laboratory. So in both cases, they found that that that regions of Piriform cortex they described as dorsal and ventral aspects of piriform cortex It's not quite clear how that relates to layer 2 and then different regions of piriform cortex had different projection patterns Particularly going to frontal areas, I believe And you know, I mean Alex should be addressing this better than I at this point because he also found not only were the projection patterns Different but the expression of transcription factors varied as a function of where they were projecting to So, you know, I think there's I think there's a continuing story there that still needs to be told Maybe we'll hear more about it. Okay So we wanted to know a little more about about the development of piriform cortex and what we turned to here was a strategy of electroporation Not unlike what we used in the olfactory bubble what differs here is we used a piggyback Placement to introduce different fluorophores into the Ventric along with a piggyback transplace ace which meant that these fluorophores would Stochastically or randomly get integrated into populations of progenitor neurons where they would continue to be expressed because they would be Incorporated in the genome so that meant that a progenitor cell that that incorporated any one of these all of the daughter cells that came from it would continue to express these fluorescent markers these fluorescent probes at at a at a measurable level and in contrast to the strategy that we used to label the olfactory bulb in this case We inject it again into the ventricle But now positioned our electroporation paddle so that we drove it specifically into the lateral pallium and a little bit of the Pallial sub-pallial border as well to label specifically the progenitors of cells that were going going to target the olfactory bulb and and you know It worked. It was amazing We get these populations of cells labeled with our three markers EGFP TD tomato and far red that we coated here as blue and we could characterize populations of cells as neurons as well as glia and then we said about Quantifying them in either anterior-pure from cortex Summarized very briefly here or posterior-pure from cortex shown here anterior-pure from cortex We defined largely as when the anterior commissure was present in posterior-pure from cortex We defined as the onset and about the middle of the hippocampal formation didn't go all the way back to enter rhino cortex The important question that we had if we were going to try to understand lineage a little bit was the degree to which any one of The progenitors would be incorporating just one two or all three of these of these of these markers that we introduced And what we found not surprisingly was that the probability of all three markers being incorporated either in the neurons or in the glial cells Was quite low relative to the number of cells that would incorporate one marker or two markers and based upon that Plus some earlier work that had been done in Lorela Pesma scar case lab. We felt that at least we could make some reasonable assumptions about the relationship between cells Between cells all of which we're expressing all three markers simultaneously We then look at the distribution of markers across the layers of Here from cortex for both neurons shown in the dark colors and glia shown in the in the more transparent colors not surprisingly the predominance Predominantly cells were labeled here in layers 2a 2b and layer 2 3 where we do and find back in fine Most of the neurons under any condition. So perhaps it's not surprising that we found most of our markers there so we then set about doing an experiment in which we again took took The plasmids and introduced them and now double labeled with different markers to determine what kinds of cells were picking them up The higher magnifications are shown on the right The area in which it's coming from of layer 2 is shown on the left And we labeled double labeled with new n readily doubled labeled with TBR 1 readily We found no double labeling with Gad 67 and he guesses why Gad 67 labels glutamatergic cells interneurons They're derived from the medial geniculate ganglion medial ganglionic eminence And we targeted the lateral ganglion eminence and the paleo sub paleo border So we weren't getting any of our label going into the medial ganglionic eminence and therefore we didn't expect in fact If we had found labeling of of Gad 67 cells, it would have been disappointing We've also found labeling of populations of cells in layer 2a of With reeling in posterior pyrrhic cortex not anterior pyrrhic cortex and then finally as I mentioned We also find found labeling of populations of glial cells in both anterior and posterior pyrrhic cortex Labeled with GFAP as well as s100 beta and I'll come back to that in just a minute because we're almost done So the next question which is a harder question is we wanted to ask what is the probability That cells that express all three colors have a narrow distribution in pyrrhic cortex and particularly wanted to understand What's the probability of cells expressing all three colors? Being found in either either layer one either layer to a to be or or layer three And this is a bit of a heat map at the bottom We've done these analyses first in anterior pyrrhic cortex Looking at individual animals and we've needed to look at individual animals because in any one animal Virginia cells that we were likely labeling is going to be different and what you see here is the warmer colors the red Indicate the highest probability of triple labeling and in all three of these aim animals The highest probability was in layer 1b an animal for the highest probability of labeling was in layer 3 So there's an asymmetric distribution a non uniform non random Distribution of cells expressing all three colors in any one Cells expressing all three colors in any one animal leading us to to to Tentatively conclude that there is a progenitor relationship that progenitors give rise to Populations of cells that are going to be targeted or are targeted to specific layers of the olfactory bone We found similar results with glial cells that are perhaps a little less interesting Looked in posterior pyrrhic cortex as well In this case animals 2 3 and 5 also heaviest labeling in layer where in layer 1a well layer 1b is also in animals in animal 3 and then also also an animal 5 But for any individual animal again There is a propensity for the triple labeled cells to be found in only one layer of the olfactory bone And again, we've only analyzed here cells that are expressing all three colors We didn't do anything with cells expressing one color or just two colors So our next question and our last question I must be about done am I overdone overdone Okay, so let me let me just give you the take-home message here then This is the migratory behavior of cells that we've labeled by electroporating the The Halial sub-halial border. This is posterior pyrrhic cortex. This is anterior pyrrhic cortex posterior pyrrhic cortex matures About 24 hours prior to the maturation of anterior pyrrhic cortex here at embryonic day 14 You see readily the distribution of layer 2 cells in posterior pyrrhic cortex If we look at anterior pyrrhic cortex, there's a lack of that by day 15 Posterior pyrrhic cortex has emerged and then on day 16 and 17 We just see a continued maturation of the cells found within each of those areas and then really briefly I showed you the summary of of Laminogenesis in neocortex, and this is now a new summary of laminar genesis in pyrrhic cortex beginning in the neuro epithelium At embryonic day 10 embryonic day 11. We have progenitor cells giving rise to radial glial cells from which Projection neurons will migrate up to the upper layers Will follow the lateral cortical stream to move out to their final positions in pyrrhic cortex that continues at embryonic day 13 with the emergence of the medial and lateral ganglionic Eminences giving rise to interneurons in red and projection neurons in blue if you if you move out to embryonic day 1415 that continues to be elaborated as more populations of cells are found Populations of interneurons do not follow a radial migration They follow a tangential migration from the medial ganglionic eminence, which is quite different and I haven't had time to talk about that by embryonic day 1618 almost all of the neuro genesis of Grammar neurons projection neurons is completed, but they continue to migrate along this densely populated lateral cortical stream until they reach the respective areas of cortex with layer 2 a cells getting there Yeah, layer 2 a cells getting there prior to the arrival of layer 2b cells until finally we have the emergence of a Typical adult organization with 2b with 2a 2b cells and the distribution of Dendritic processes in 1a and 1b, and I'm sorry it went too long I'll stop