Classificatin Rules (and Exceptins) Name: Cell type fllwed by either a clumn ID (determined by the visual lcatin f the cell) r a numeric identifier t separate ut different examples f a given cell type (when the lcatin is nt assciated with a single clumn) The lcatin is described by ne f the 7 clumn IDs: hme Central clumn A, B, C, D, E, F Always in capital text Letter in lwer case are used t represent subtypes f a given neurn. Example: T4a vs. T4b: Tw subtypes f the T4 neurn. The T4 neurn type als has an additinal descriptr related t its functin. T4 is knwn t functin in mtin detectin, and the directin selectivity f a given T4 can be determined frm its anatmical arbrizatin pattern Reference: Takemura, Bharike, et al. Nature (2013) In this case, ne f the fllwing 4 descriptrs is added t describe the directin selectivity: fb: frnt t back bf: back t frnt ud: up t dwn du: dwn t up Smetimes the relative psitin f the neurn, within the hexagnal array, is als added, in a case where it may be functinally relevant. We use ant (anterir) r pst (psteri) Example: Tm3 A ant This is always the final descriptr added t the name. Type: Determined thrugh cmparisn f cellular mrphlgy thrugh randm silver staining f different cell types r genetic targeting f flurescent prteins t different cell types Silver staining reference: Fischbach and Dittrich Cell and Tissue Research (1989) Genetic targeting reference: Unpublished wrk by Aljscha Nern (Janelia Farm) Class: Different cell types are gruped int classes by cmmn mrphlgical features (e.g. similar arbrizatin patterns). All classes: C: Centrifugal cells Cells thugh t send feedback signals frm the medulla back t the lamina L: Lamina Mnplar cells Cells sending input frm the lamina L1, L2, and L3 are strngly retintpic, with each receiving a large fractin f its input frm phtreceptrs transducing phtn inputs frm a single pint in visual space. LaWF: Large Wide field cells Neurns with wide arbrizatins in the lamina Dm: Distal medulla 1
Cells lcalized entirely t layers M1 t M6 Arbrize tangentially and are thught t prvide intercnnectins between different medulla clumns, within a regin f the medulla (circumscribed by depth) Pm: Prximal medulla Cells lcalized entirely t layers M8 M10 Like Dm cells; arbrize tangentially and are thught t prvide intercnnectins between different medulla clumns, within a regin f the medulla (circumscribed by depth) Mi: Intrinsic medulla cells Cells that traverse all layers f the medulla (and are generally clumnar) but d nt send a neurite prjectin ut f the medulla R: Phtreceptrs Tw clr phtreceptrs send inputs directly frm the retina t the medulla, bypassing the lamina. T1: T type neurn 1 T1 type cells have arbrizatins within the lamina and medulla. This, tgether with their cell bdy lcatin, at the edge f brder between the lamina and the medulla prvide them with a T shaped arbr. T2: T type neurn 2 Includes T2 and T2a neurns These neurns appear t be paired, and have very similar shapes and arbrizatins. Bth types arbrize within the medulla and lbula, and tgether with the prjectin t their cell bdies, als have a T shaped arbr. T3: T type neurn 3 Anther T type neurn that has arbrizatins in medulla and lbula T4: T type neurn 4 A T type neurn that has arbrizatins within the medulla and lbula plate. Tm: Transmedulla cells Cells that traverse all layers f the medulla (and are generally clumnar) but send a neurite prjecting ut f the medulla, generally t the lbula neurpil. TmY: Transmedulla Y cells Within the medulla, transmedulla Y cells appear generally similar t transmedulla cells. Hwever, their neurite prjectin splits int tw dwnstream neurites, and prjects t bth the lbula and lbula plate neurpils (with the skeletn thereby having the shape f an inverted Y) Y: Y cells Named fr the same prjectin t bth the lbula and lbula plate neurpils as the TmY cells (and hence an inverted Y arbrizatin), the difference between a Y cell and a TmY is that the arbrizatin within the medulla des nt crss all the medulla layers, and stays within the deeper layers. Superclass: Different cell classes can be gruped int a superclass, generally, by their cmmn arbrizatin patterns. Sme superclass are simply the full name f the class (withut including any additinal cell classes) All Superclasses: Lamina: Includes C, L, LaWF, and T1 cells Cells that have arbrizatins within the lamina Phtreceptrs Includes R cells 2
Distal Medulla Includes Dm Cells Prximal Medulla Includes Pm Cells Lbula T type Includes three classes f neurns: T2, T2a, T3 These are neurns with T shaped arbrs that arbrize within the medulla and the lbula Lbula Plate T type Includes T4 neurns These are neurns with T shaped arbrs that arbrize within the medulla and the lbula plate Transmedulla Cells Includes Tm neurns TmY type Cells Includes TmY cells Y Cells Includes Y cells Clumnar Spread: This key takes a value f "Single clumnar" r "Multi clumnar" Sme fractin f the ttal recnstructed arbr f a cell verlaps with the 7 clumn ROIs Of this fractin, we find the fractin that is in the clumn with the greatest verlap. If this fractin is >0.9, then we label the cell as "Single clumnar". This implies that >90% f the cell's arbr within the clumn ROIs is within a single clumn Nte that this des nt imply that the entire cell's vlume is within the clumn ROIs. We apply this analysis t an entire cell type We assume that each cell type must have the same classificatin (since cells f a given type are defined t be mrphlgically similar). Further, we fcus nly n cells within the central 7 clumns. These cells are mst cmpletely recnstructed. We ignre any cells that are within the uter sectins f the recnstructin (n matter the degree f cmpleteness) We chse the value (i.e. either single r multi clumnar) based n the fractin fund ver the majrity f cells within the type. Exceptins: Each f the neurn types that is an exceptin t this rule has a bilgical reasn fr the exceptin. We have attempted t detail them here. Dm6: The arbrizatins fr mst Dm6 recnstructins are within nly a single clumn. Hence, the quantitative metric crrectly labels them as single clumnar. Hwever, Dm6 is actually cmpsed f several bushy arbrs, each within a single clumn, cnnected by shrt, thin prcesses. These prcesses are ften missed during EM recnstructin, due t their small size. Nevertheless, the identificatin f a given (single clumnar) arbr as part f a Dm6 cell is unambiguus, due t the agreement with silver stained exemplars. The silver staining results, and genetic labelling f individual neurns clearly shw that all Dm4s encmpass arbrizatins is several clumns. If such a cmplete Dm6 was recnstructed, then it wuld be quantitatively classified as Multi clumnar. Therefre, despite the recnstructin f nly single clumn arbrs in mst cases, we have used an exceptin t classify Dm6 as Multi clumnar. 3
Dm1: The case f Dm1 is analgus t Dm6. The EM recnstructins cntain nly single clumnar fragments f the cells. Hwever, by cmparisn with light micrscpy, the cell type is clearly Multi clumnar, and we have used an exceptin t classify it as such. C2 There is a single C2 neurn arbrizing within each retintpic structure within the lamina. This des nt necessarily imply that it shuld be single clumnar. Hwever, C3 neurns have an arbrizatin very similar in structure t its sister neurn, C3. Further, C3 is a Single clumnar neurn. The main difference between the tw is that C2 has slightly bushier arbrizatins. Hence, a slightly greater fractin f its arbr is within ther clumn ROIs. Since the 90% threshld is simply an arbitrarily defined cut ff (chsen t select as many classificatins crrectly as pssible), it is pssible that C2 simply sits just shrt f the threshld. Therefre, by bservatin f its arbrizatin pattern, we decided that it was reasnable t utilize an exceptin and label it as Single clumnar. L4 T2 This lamina mnplar cell is lcated in the exterir f the clumn ROIs (see next sectin n Clumnar lcatin). Hence, its arbrizatins spread int three surrunding clumns. Hwever, the size f the arbr f L4 is similar t that f L1, L2, and L3 three ther lamina mnplar cells, which are clearly single clumnar (indeed, the extent f their arbrs is used as a wrking definitin f the extent f the clumn). Therefre, it is reasnable t label this cell as Single clumnar (and exterir) by analgy t the ther lamina mnplar cells. This cell type is quantitatively identified as single clumnar. Hwever, it is physically lcated adjacent (and ften intertwined) t tw ther analgus Lbula T type cells (T2a, and T3). Bth f these cells are Multi clumnar. Hence, we assume that this cell must simply sit just ver the arbitrary 90% threshld, and by analgy t the ther neurns f the same type we label this cell as Multi clumnar. Tm20 This cell appears visually t be Single clumnar. Hence, we have labeled it as such. We believe that the reasn fr the mis classificatin f this cell type may be the small number f examples within the central 7 clumns. Since this cell is nt fund within all clumns, we may simply have a small sample errr. Clumnar Lcatin: This key value takes a value f "Interir" r "Exterir". The metric used t define the value f this key is the fractin f the ttal recnstructed vlume f the cell (within the seven clumn regin) that is within the clumn ROIs. If this fractin is <0.3, then the cell is "Exterir". If the fractin is >=0.3, then the cell is "Interir". This threshld was chsen because 30% f the ttal vlume within the recnstructin is within the clumn ROIs. Therefre, a cmpletely randm cell which cvers the entire regin wuld necessarily have 30% f its vlume within the clumn ROIs. We wuld reasnably term such a cell as external (since it is nt internal t any clumn, and des nt have a cncentratin f its vlume within any f the clumns). This metric is nt identical t the Clumnar spread metric, thugh it is dependent n it. It is designed t measure the cncentratin f the vlume within the clumns vs. utside the clumns, whereas 4
the Clumnar spread metric is designed t measure the cncentratin f vlume within a single clumn vs. the rest. The metric is dependent n the Clumnar spread metric in the fllwing way: If the Clumnar spread is Multi clumnar, the Clumnar lcatin is assumed t be "Exterir". This is nt due t any deeper bilgical understanding. It simply allws the smallest set f exceptin cases. Exceptins: Each f the neurn types that is an exceptin t this rule has a bilgical reasn fr the exceptin. We have attempted t detail them here. L4: L4 is a single clumnar neurn (see abve fr the exceptin) that thrugh visual inspectin appears displaced frm the single clumnar interir neurns assciated with the surrunding clumns. Hence it appears reasnable t define it as exterir. The reasn it is nt autmatically classified as exterir may have t d with its small ttal arbr size. Any small differences in the extent f its arbr, n a specific side, wuld greatly affect the fractin f its vlume that is within a specific ROI (when the main bdy extent is between three different clumns). This means that ften a significant fractin f its arbr is within ne specific clumn, althugh visually, it appears t have its main bdy axis lcated between clumns. This randmness is supprted by the fact that the identity f the clumn where it has its maximum extent appears randm, and uncrrelated with the retintpic pint in space frm which it receives mst f its inputs (in the lamina). (This is determined by identifying the L1, L2, and L3 neurns crrespnding t the same pint in space, and identifying the clumn t which these single clumnar interir neurns send their prjectins.) Dm8, Tm9, and Tm4: These three cell types are the nly examples f multi clumnar interir neurns. Althugh they send prcesses t many clumns (and hence have a large fractin f their vlume utside the clumns), they still have a majrity f their primary arbrizatin riented alng a single clumn. Hence, anatmists have classified them as interir (and we include them as an exceptin t the rule). Tm23/24: We define Tm23/24 as an exterir cell. This neurn has a particularly unique mrphlgy with nly a thin neurite that traverses the entire medulla withut having any branching arbrizatins. Given its structure, it can crrespnd t tw different identified neurns within the light micrscpy datasets either Tm23 r Tm24. Hwever, these cells cannt be distinguished in the limited recnstructin within the medulla (hence the naming cnventin f the cell type). These thin neurites appear t pass thrugh the medulla at randm. This des mean that, in sme cases, they travel thrugh a clumn ROI. Indeed, numerically mre axns within the 7 clumn regin traverse clumn ROIs than nt Hwever, the general anatmical impressin including axns utside f the 7 clumn regin suggests that this is simply a sampling bias by the randm chice f which 7 clumns t recnstruct. Therefre, it is reasnable t say that Tm23/24 is nt rganized with respect t the medulla clumns and, hence, is nt interir t the clumns (i.e. in this tw valued key, it is labeled as exterir). 5
One imprtant caveat is that this ppulatin f neurns culd actually be cmpsed f bth Tm23 and Tm24 neurns, and hence ne might be interir, while the ther might be exterir (with the cmbinatin appearing t be randm). Hwever, at present, we cannt distinguish this. Clumn ID: The clumn ID is the clumn in which a specific single clumnar, interir neurn is lcated in. Fr multi clumnar r exterir neurns, by definitin, it is nt pssible t define such a clumn, and hence we have left this value empty. We als d nt prvide an ID fr any neurns which are nt in the central 7 clumns. This is because there are nly clumn ROIs fr the central clumns. Hence the quantitative verlaps fr the uter clumns are nt available, in ur dataset. Clumn Vlume Fractin: The fractin f the ttal recnstructed vlume within the 7 central clumns: Hme, A, B, C, D, E,F. The nrmalizatin is made f the ttal vlume within the 7 clumn recnstructed regin (in the DVID database, these vlumes can be fund by querying the graph_ri_full labelgraph bject). Indeed, all the vlumes within "clumn I" can be fund by querying the apprpriate labelgraph bject in the DVID database (i.e. graph_ri_i). T cmpute the fractins used t btain the Clumnar spread, it is necessary t renrmalize these values t include nly the fractin f the vlume within the ROIs. Hwever, t cmpute Clumnar lcatin, it is nly necessary t sum ver all the fractins (and cmpare against the ttal that is nt included, i.e. that is exterir t the clumns). Synaptic Fractins: There are fur sets f synaptic fractins that have been precmputed. Each can be cmputed frm the synapse.jsn, by querying all the clumnar / layer ROIs t identify which ne (if any) cntains each synapse. The synapses are then assciated by bdy, s as t identify the fractin f synapses in a specific ROI, fr a given bdy. The lcatin f each synapse can be divided int a presynaptic specializatin, a Tbar, and a pstsynaptic specializatin, a PSD. Clumn Tbar Fractin: Fractin f Tbars fr a given cell, in each f the 7 clumns. Details where the cell prvides utput t ther cells. Layer Tbar Fractin: Fractin f Tbars fr a given cell, in each f the 10 layers. Details where the cell prvides utput t ther cells. Clumn PSD Fractin: Fractin f PSDs fr a given cell, in each f the 7 clumns. Details where the cell receives input frm ther cells. Layer PSD Fractin: Fractin f Tbars fr a given cell, in each f the 10 layers. Details where the cell receives input frm ther cells. In all these vectrs, the ttal may nt sum t 1, because f the presence f synapses utside f the ROIs. Hwever, they shuld always be <= 1. 6