Decision letter: Conditional protein tagging methods reveal highly specific subcellular distribution of ion channels in motion-sensing neurons
2020-10-14

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Neurotransmitter receptors and ion channels shape the biophysical properties of neurons, from the sign of the response mediated by neurotransmitter receptors to the dynamics shaped by voltage-gated ion channels. Therefore, knowing the localizations and types of receptors and channels present in neurons is fundamental to our understanding of neural computation. Here, we developed two approaches to visualize the subcellular localization of specific proteins in Drosophila: The flippase-dependent expression of GFP-tagged receptor subunits in single neurons and ‘FlpTag’, a versatile new tool for the conditional labelling of endogenous proteins. Using these methods, we investigated the subcellular distribution of the receptors GluClα, Rdl, and Dα7 and the ion channels para and Ih in motion-sensing T4/T5 neurons of the Drosophila visual system. We discovered a strictly segregated subcellular distribution of these proteins and a sequential spatial arrangement of glutamate, acetylcholine, and GABA receptors along the dendrite that matched the previously reported EM-reconstructed synapse distributions. Introduction How neural circuits implement certain computations in order to process sensory information is a central question in systems neuroscience. In the visual system of Drosophila, much progress has been made in this direction: numerous studies examined the response properties of different cell-types in the fly brain and electron microscopy studies revealed the neuronal wiring between them. However, one element crucial to our understanding is still missing; these are the neurotransmitter receptors used by cells at the postsynaptic site. This knowledge is essential since neurotransmitters and corresponding receptors define the sign and the time-course of a connection, that is whether a synapse is inhibitory or excitatory and whether the signal transduction is fast or slow. The same neurotransmitter can act on different receptors with widely differing effects for the postsynaptic neuron. Glutamate for instance is mainly excitatory, however, in invertebrates it can also have inhibitory effects when it acts on a glutamate-gated chloride channel, known as GluClα (Cully et al., 1996; Liu and Wilson, 2013; Mauss et al., 2015). Recently, it has also been shown that acetylcholine, usually excitatory, might also be inhibitory in Drosophila, if it binds to the muscarinic mAChR-A receptor (Bielopolski et al., 2019). Hence, knowledge inferring the type of transmitter receptor at a synapse is essential for our understanding of the way neural circuits process information. Moreover, voltage-gated ion channels shape synaptic transmission and the integration of synaptic inputs by defining the membrane properties of every neural cell type. The voltage-gated calcium channel cacophony, for instance, mediates influx of calcium ions that drives synaptic vesicle fusion at presynaptic sites (Kawasaki et al., 2004; Fisher et al., 2017). Voltage-gated sodium channels like paralytic (para) are important for the cell’s excitability and the generation of sodium-dependent action potentials. The voltage-gated channel Ih influences the integration and kinetics of excitatory postsynaptic potentials (Magee, 1999; Littleton and Ganetzky, 2000; George et al., 2009). However, only little is known about how these channels are distributed in neurons and how this shapes the neural response properties. One of the most extensively studied neural circuits in Drosophila is the motion vision pathway in the optic lobe and the underlying computation for direction-selectivity. The optic lobe comprises four neuropils: lamina, medulla, lobula, and lobula plate (Figure 1A). As in the vertebrate retina, the fly optic lobe processes information in parallel ON and OFF pathways (Joesch et al., 2010; Borst and Helmstaedter, 2015). Along the visual processing chain, T4/T5 neurons are the first neurons that respond to visual motion in a direction selective way (Maisak et al., 2013; Behnia et al., 2014; Fisher et al., 2015a; Arenz et al., 2017; Strother et al., 2017). T4 dendrites reside in layer 10 of the medulla and compute the direction of moving bright edges (ON-pathway). T5 dendrites arborize in layer 1 of the lobula and compute the direction of moving dark edges (OFF-pathway) (Maisak et al., 2013). The four subtypes of T4/T5 neurons (a, b, c, d), project axon terminals to one of the four layers in the lobula plate, each responding only to movement in one of the four cardinal directions, their preferred direction (Maisak et al., 2013). Figure 1 Download asset Open asset Overview of the fly optic lobe and anatomy of T4/T5 neurons with their presynaptic partners and distribution of input synapses. (A) Horizontal view of optic lobe with retina, lamina, medulla, lobula, and lobula plate. T4 dendrites (darker gray) reside in layer 10 of the medulla, T5 dendrites (lighter gray) in layer 1 of the lobula. T4/T5 axon terminals of all subtypes (a, b, c, d) project to the lobula plate in four layers. (B) Close-up, horizontal view of EM-reconstructed single T4 neuron with dendrite, axon, axon terminal, soma fiber and soma (image extracted from Seven medulla column connectome dataset, https://emdata.janelia.org/#/repo/medulla7column, #3b548, Janelia Research Campus). (C) Scheme of individual T4 dendrite and distribution of input synapses (frontal view). The dendrite depicted here is oriented pointing to the right side against its preferred direction from right to left (indicated by arrow). Input on proximal base of T4 dendrite: GABAergic CT1, Mi4 and C3. In the central area: GABAergic TmY15 and cholinergic Mi1 and Tm3. On the distal tips T4 receive input from cholinergic T4 from the same subtype and glutamatergic Mi9. Yellow circle labels first branching point of the dendritic arbor. Reproduced from Figure 4, Shinomiya et al., 2019, eLife, published under the Creative Commons Attribution 4.0 International Public License (CC BY 4.0; https://creativecommons.org/licenses/by/4.0/). (D) Scheme of individual T5 dendrite and distribution of input synapses (frontal view). The dendrite depicted here is oriented pointing to the right side against its preferred direction from right to left (indicated by arrow). The T5 dendrite receives GABAergic input from CT1 on the proximal base and from TmY15 in the central area. Cholinergic synapses are formed with Tm1, Tm2, and Tm4 in the central area and with Tm9 and T5 from the same subtype on the distal dendritic tips. Yellow circle labels first branching point of the dendritic arbor. Reproduced from Figure 4, Shinomiya et al., 2019, eLife, published under the Creative Commons Attribution 4.0 International Public License (CC BY 4.0; https://creativecommons.org/licenses/by/4.0/). How do T4/T5 neurons become direction-selective? Both T4 and T5 dendrites span around eight columns collecting signals from several presynaptic input neurons, each of which samples information from visual space in a retinotopic manner (Haag et al., 2016; Shinomiya et al., 2019). The functional response properties of the presynaptic partners of T4/T5 have been described in great detail (Behnia et al., 2014; Ammer et al., 2015; Fisher et al., 2015a; Fisher et al., 2015b; Serbe et al., 2016; Arenz et al., 2017; Strother et al., 2017; Strother et al., 2018; Drews et al., 2020) along with their neurotransmitter phenotypes (Takemura et al., 2017; Richter et al., 2018; Shinomiya et al., 2019; Davis et al., 2020). T4 dendrites receive glutamatergic, GABAergic and cholinergic input, whereas T5 dendrites receive GABAergic and cholinergic input only. These input synapses are arranged in a specific spatial order along T4/T5 dendrites (s. Figure 1C and D; for overview Takemura et al., 2017; Shinomiya et al., 2019). Which receptors receive this repertoire of different neurotransmitters at the level of T4/T5 dendrites? Recently, several RNA-sequencing studies described the gene expression pattern of nearly all cell-types in the optic lobe of the fruit fly including T4/T5 neurons (Pankova and Borst, 2016; Konstantinides et al., 2018; Davis et al., 2020; Hörmann et al., 2020). T4/T5 neurons were found to express numerous receptor subunits of different transmitter classes and voltage-gated ion channels at various expression strengths. However, RNA-sequencing studies do not unambiguously answer the above question for two reasons: mRNA and protein levels are regulated in complex ways via post-transcriptional, translational, and protein degradation mechanisms making it difficult to assign protein levels to RNA levels (Vogel and Marcotte, 2012). Secondly, standard RNA-sequencing techniques cannot provide spatial information about receptor localizations, hence, they are not sufficient to conclude which transmitter receptors receive which input signal. Both shortcomings could in principle be overcome by antibody staining since immunohistochemical techniques detect neurotransmitter receptors at the protein level and preserve spatial information. However, high-quality antibodies are not available for every protein of interest and may have variable affinity due to epitope recognition (Fritschy, 2008). Furthermore, labeling ion channels via antibodies and ascribing expression of a given channel to a cell-type in dense neuronal tissue remains challenging. The disadvantages of the above techniques highlight the need for new strategies for labeling neurotransmitter receptors in cell types of interest. In this study, we employed existing and generated new genetic methods to label and visualize ion channels in Drosophila. For endogenous, cell-type-specific labeling of proteins, we developed a generalizable method called FlpTag which expresses a GFP-tag conditionally. Using these tools, we explored the subcellular distribution of the glutamate receptor subunit GluClα, the acetylcholine receptor subunit Dα7, and the GABA receptor subunit Rdl in motion-sensing T4/T5 neurons. We found these receptor subunits to be differentially localized between dendrites and axon terminals. Along the dendrites of individual T4/T5 cells, the receptor subunits GluClα, Rdl, and Dα7 reveal a distinct distribution profile that can be assigned to specific input neurons forming synapses in this area. Furthermore, we demonstrated the generalizability of the FlpTag approach by generating lines for the metabotropic GABA receptor subunit Gaba-b-r1 and the voltage-gated ion channels para and Ih. The strategies described here can be applied to other cells as well as other proteins to reveal the full inventory and spatial distribution of the various ion channels within individual neurons. Results Subcellular localization of the inhibitory glutamate receptor GluClα in T4/T5 neurons As suggested by the connectome (Takemura et al., 2017; Shinomiya et al., 2019) and antibody staining against the vesicular glutamate transporter VGluT (Richter et al., 2018), T4 cells receive input on their dendrites from the glutamatergic medulla neuron Mi9. Since a multitude of glutamate receptors exist, both excitatory and inhibitory, we explored which glutamate receptor forms the synapse between the glutamatergic Mi9 input and T4 dendrites. According to a RNA-sequencing study, GluClα is the most highly expressed glutamate receptor in T4 neurons (Davis et al., 2020). To investigate the distribution of this glutamate receptor in T4 and T5 neurons, we developed a transgenic fly line that allowed us to express a GFP-tagged GluClα in a cell-type specific way. We created a UAS-GluClα::GFP line bearing the cDNA of GluClα with a GFP-insertion (Supplementary file 1). This construct can be combined with any Gal4-line to study the receptor’s expression and its subcellular localization. We combined the UAS-GluClα::GFP line with a membrane-bound UAS-myr::tdTomato and expressed both constructs under the control of a T4/T5-specific Gal4-driver line. We found GluClα in T4 dendrites of the medulla, where it is distributed in discrete puncta (Figure 2A; horizontal section, first two panels). A top view of the medulla of these flies reveals that these puncta are arranged in circular clusters, each corresponding to one column (Figure 2A, right panel). Since Mi9 is the only glutamatergic presynaptic partner of T4 cells in the medulla (Takemura et al., 2017; Richter et al., 2018; Shinomiya et al., 2019), this columnar arrangement likely reflects the columnar array of Mi9 cell inputs. Conversely, T5 dendrites are completely devoid of GluClα signal (Figure 2A, first two panels). This result is in agreement with T5 dendrites not receiving glutamatergic input (Richter et al., 2018). In addition to the medulla layer 10, GFP signal of GluClα::GFP is also visible in the axon terminals of T4/T5 in the lobula plate (Figure 2A, first two panels). However, both T4 and T5 cells send their axons into the lobula plate, therefore, this staining cannot be assigned to one of the cell types specifically. To differentiate between the two cell types, we used two different driver lines, one specific for either T4 or T5 cells. We confirmed the presence of GluClα in the dendritic layer of T4 cells (Figure 2B) and the lack thereof in the dendritic layer of T5 cells (Figure 2C). Interestingly, with these specific driver lines, both T4 and T5 neurons express the glutamate receptor in their axon terminals in the lobula plate (Figure 2B and Figure 2C). The presence of GluClα in the axon terminals of T5 neurons explains the high GluClα-mRNA levels in T5 (Davis et al., 2020) even though T5 dendrites are missing a glutamatergic presynaptic partner (Takemura et al., 2017; Richter et al., 2018; Shinomiya et al., 2019). Figure 2 with 1 supplement see all Download asset Open asset Subcellular localization of the inhibitory glutamate receptor GluClα in T4/T5 neurons. (A) Optic lobe with T4/T5 neurons labeled with myr::tdTomato and GluClα::GFP. Left panel: horizontal view on the optic lobe overview (scale bar: 20 μm). Central panel: close-up of medulla layer M10, lobula layer Lo1 and lobula plate layers 1–4 (scale bar: 5 μm). Right panel: Frontal view on medulla layer M10 with T4 dendrites (scale bar: 20 μm); inset: close-up of columnar GluClα::GFP structure in layer 10 of the medulla. (B) Close-up of T4 dendrites in layer 10 of the medulla and axon terminals in lobula plate labeled with myr::tdTomato and GluClα::GFP (scale bar: 5 μm). (C) Close-up of T5 dendrites in layer 1 of the lobula and axon terminals in lobula plate labeled with myr::tdTomato and GluClα::GFP (scale bar: 5 μm). (D) Individual T4 dendrites labeled with tdTomtato and GluClα::GFP; subtypes a-d pointing in their natural orientation in visual space coordinates (A = anterior, p=posterior, D = dorsal, V = ventral). White arrows indicate preferred directions for every subtype and the dendrites’ proximal (Prox.), central (Cent.) and distal (Dist.) areas are labeled (scale bar: 2 μm). Yellow circle labels first branching point of the dendrite. (E) Quantification of GluClα distribution over the whole dendritic length (normalized distance) averaged across several T4 dendrites from all subtypes (n = 8). All dendrites were aligned pointing to the right with the most proximal point at 0.0 and the most distal point at 1.0. (F) Quantification of GluClα puncta averaged across several T4 dendrites from all subtypes (mean ± SD = 20.5, 4.98 [n = 8]) (same cells used in E) compared to number of glutamatergic input synapses from Mi9 (mean ± SD = 23.0, 9.34 [n = 20]) (EM numbers: personal communication, K. Shinomiya, May 2020). n.s., not significant p>0.05 (p=0.37, t-test). Figure 2—source data 1 Table with numbers of GluClα puncta quantified for T4 dendrites. https://cdn.elifesciences.org/articles/62953/elife-62953-fig2-data1-v3.xlsx Download elife-62953-fig2-data1-v3.xlsx One caveat associated with overexpression-lines is a potential mis-localization of proteins. To control for this effect, we used a pan-neuronal Gal4-line to express the UAS-GluClα::GFP construct and compared this expression pattern to an existing MiMIC protein trap line with GFP insertion (MiMIC GFSTF) in the endogenous locus of GluClα (Mi02890) (Nagarkar-Jaiswal et al., 2015a). We observed broad expression of GluClα throughout all neuropils of the optic lobe in both genotypes (Figure 2—figure supplement 1A and B). We quantified the mean fluorescence intensity of manually drawn ROIs around the medulla and found both values to be similar for the pan-neuronal UAS-GluClα::GFP and the MiMIC line (Figure 2—figure supplement 1D). Furthermore, we expressed the UAS-GluClα::GFP line with a driver line for T1, a cell-type which lacks GluClα mRNA (Davis et al., 2020). Our UAS-line confirmed this result as we could not detect significant levels of GluClα::GFP protein in T1 (Figure 2—figure supplement 1E). Hence, overexpression of GFP-tagged GluClα, introduced as a transgene, leads to a subcellular localization pattern that seems to be identical to the endogenous GluClα protein. Given that Mi9 is the only glutamatergic input neuron to T4 dendrites and GluClα is the corresponding glutamate receptor, we hypothesized that GluClα should localize on the individual T4 dendrite exclusively where Mi9 makes glutamatergic synapses with the latter. Therefore, we wanted to visualize the distribution of GluClα at the single-cell level along individual T4 dendrites. The dendrites of each T4/T5 subtype are oriented pointing against their preferred direction (Takemura et al., 2017; Shinomiya et al., 2019). With respect to the point of axonal attachment to the dendrite, T4/T5 dendrites can be divided into a proximal, central and distal region (summarized in Figure 1B–D). Electron microscopy studies have shown that Mi9 forms synaptic contacts with T4 on the distal tips of its dendrite (Figure 1C; Takemura et al., 2017; Shinomiya et al., 2019). Since T4/T5 dendrites are strongly intermingled in their respective layers, it is not possible to resolve receptor localizations at the single-cell level by labeling the whole population. We used a flippase-based mosaic approach (Gordon and Scott, 2009) to sparsely label single T4/T5 neurons with tdTomato together with the UAS-GluClα::GFP construct. By using a FRT-Gal80-FRT with an hs-FLP, both UAS-myr::tdTomato and UAS-GluClα::GFP expression are dependent on the same stochastic FLP-event. A heat-shock-activated flippase removes the FRT-flanked Gal4-repressor Gal80, which disinhibits Gal4, promoting transcription of both UAS-reporters simultaneously resulting in expression of membrane-bound tdTomato and GFP-tagged GluClα in only a few cells of interest. In individual T4 dendrites, we observed that GluClα was predominantly localized to the distal tips, which holds true for all four T4 subtypes (Figure 2D). We quantified the intensity distribution of the GluClα::GFP-signal over dendritic distance in individual T4 dendrites. To combine and average this distribution for all four subtypes, we rotated dendrites from each subtype such that the proximal side was on the left side of the image and the distal tips were pointing to the right. Averaged intensities across all subtypes confirmed our observations on individual cells, showing that GluClα is indeed localized toward the distal dendritic tips of T4 dendrites (Figure 2E). In addition, we quantified the numbers of GluClα puncta for all subtypes and compared them to the synapse numbers of glutamatergic Mi9 inputs onto T4 determined by the previous EM study et al., 2019). The number of T4 cell dendrite the number of glutamatergic input synapses made by Mi9 onto one T4 cell synaptic personal communication, K. Shinomiya, May 2020) (Figure This that every by microscopy in labeled T4 dendrites one postsynaptic GluClα receptor corresponding to one In GluClα to the dendrites of T4 cells and to the axon terminals of both T4 and T5 cells. the single-cell GluClα is distributed toward the distal tips of the dendrites in all T4 the number of GluClα puncta the number of input synapses by the only glutamatergic input neuron to T4 dendrites. Rdl to T4/T5 dendritic receiving GABAergic input glutamatergic we employed similar methods to visualize GABAergic synapses in T4/T5 neurons. T4 dendrites receive input from several GABAergic cell-types in the on the proximal base of the dendrite, these are the columnar cells the cell CT1 in the and distal of the dendrite as well as TmY15 (Figure In T5 dendrites receive GABAergic input from only two CT1 on the proximal base and TmY15 throughout the central and distal area of the dendrite (Figure 1D). In T4 and T5 dendrites receive the same number of GABAergic input synapses (Takemura et al., 2017; Shinomiya et al., 2019). GABA receptor subunits are described in the Drosophila Rdl, and et al., We on the GABA receptor subunit Rdl, since RNA-sequencing studies Rdl as the most highly expressed GABA receptor subunit in T4 and T5 neurons (Pankova and Borst, 2016; Davis et al., 2020). Rdl subunits can a chloride channel which leads to a receptor et al., studies created and used a line to investigate the distribution of this GABA receptor subunit in Drosophila and et al., et al., and 2013). In our the staining of this line was for not hence, we created a of the of Rdl and a GFP-tag (Supplementary file this line with a T4/T5 specific Gal4-line and a membrane-bound tdTomato revealed Rdl expression in both T4/T5 dendrites, not in the axon terminals (Figure both T4 and T5 neurons receive GABAergic via Rdl receptors on their dendrites. Figure with 1 supplement see all Download asset Open asset Subcellular localization of the GABA receptor Rdl in T4/T5 neurons. (A) Optic lobe with T4/T5 neurons labeled with myr::tdTomato and Left panel: horizontal view on the optic lobe overview (scale bar: 20 μm). Right panel: close-up of medulla layer M10, lobula layer Lo1 and lobula plate layers 1–4 (scale bar: 5 μm). (B) Individual T4 dendrites labeled with tdTomtato and subtypes a-d pointing in their natural orientation in visual space coordinates (A = anterior, p=posterior, D = dorsal, V = ventral). White arrows indicate preferred directions for every subtype and the dendrites’ proximal (Prox.), central (Cent.) and distal (Dist.) areas are labeled (scale bar: 2 μm). circle labels first branching point of the dendrite. (C) Individual T5 dendrites labeled with tdTomtato and subtypes a-d pointing in their natural orientation in visual space coordinates (A = anterior, p=posterior, D = dorsal, V = ventral). White arrows indicate preferred directions for every subtype and the dendrites’ proximal (Prox.), central (Cent.) and distal (Dist.) areas are labeled (scale bar: 2 μm). circle labels first branching point of the dendrite. (D) Quantification of Rdl distribution over the whole dendritic length (normalized distance) averaged across several T4 (n = and T5 dendrites (n = from all All dendrites were aligned pointing to the right with the most proximal point at 0.0 and the most distal point at 1.0. (E) Quantification of Rdl puncta averaged across several T4 (mean ± SD = [n = and T5 dendrites (mean ± SD = [n = (same cells used in from all subtypes compared to number of GABAergic input synapses from T4 (mean ± SD = [n = 20]) and T5 (mean ± SD = [n = 20]) (EM numbers: personal communication, K. Shinomiya, May 2020). n.s., not significant p>0.05 and t-test). Figure data 1 Table with numbers of Rdl puncta quantified for T4/T5 dendrites. Download In a control we for potential overexpression of the line. According to Rdl is not expressed in the neuron (Davis et al., 2020). we by of a driver Rdl signal is not in dendrites (Figure supplement 1). The protein was only visible in the cell due to protein This that Rdl only to endogenous GABA synapses that are of the Rdl Hence, this line can be used to study the subcellular localization of Rdl in any given cell of interest. we at the distribution of the GABA receptor Rdl on individual T4 and T5 dendrites. Using the labeling described we examined the distribution in individual T4/T5 dendrites. We found Rdl on the proximal base and in the central area of both T4 and T5 dendrites across all four subtypes (Figure and Figure On the proximal base most of the was arranged in discrete clusters, whereas puncta localized to the central area and toward the distal tips. The on the proximal base of the dendrite likely to the high number of GABAergic inputs by the CT1, Mi4 and for T4 and CT1 for T5 communication, K. Shinomiya, May 2020). The sparsely distributed in the and tips likely to TmY15 inputs for both T4 and T5 dendrites. This distribution is in the intensity across all T4/T5 subtypes, with high Rdl intensity on the proximal side and signal in the central and distal area (Figure We quantified the numbers of Rdl receptor in T4 and T5 dendrites and compared them to the of all GABAergic input synapses CT1, TmY15 for T4 and CT1, TmY15 for to T4/T5 by EM We found similar numbers of receptor for both T4 and T5 which the of all GABAergic input synapses to T4 and T5 (Figure (EM numbers: personal communication, K. Shinomiya, May 2020). Rdl receptor subunits localize to the proximal and to a in the central area of the dendritic of T4 and T5 neurons, their GABAergic inputs revealed by EM et al., 2019). Dα7 to T4/T5 dendritic receiving cholinergic input According to connectome T4 dendrites receive most of their input synapses from cholinergic Mi1 and cells at the of their dendrite (Takemura et al., 2017; Shinomiya et al., 2019). Furthermore, T4 neurons of the same subtype synapses with each other at the distal tips of their dendrites (Figure As T4 neurons are cholinergic et al., 2014; Davis et al., these synapses are to be cholinergic as With the of GABAergic CT1, T5 dendrites receive cholinergic input from Tm1, Tm2, and Tm4 in the central area of the dendrite. Tm9 and T5 provide cholinergic input mainly the distal tips of the dendrite (Figure Takemura et al., 2017; Shinomiya et al., 2019). T5 dendrites receive as cholinergic inputs as and et al., 2019). We used an existing GFP-tagged line to the subcellular distribution of these cholinergic synapses et al., 2009). Dα7 is one of 10 different receptor subunits and found in the Drosophila All these subunits can receptors of two or In addition, and Dα7 can also receptors and 2004; et al., 2012). According to RNA-sequencing both T4 and T5 neurons express every receptor for and (Davis et al., 2020). of with a revealed the distribution of Dα7 to both T4 and T5 dendrites their axon terminals devoid (Figure Figure with 1 supplement see all Download asset Open asset Subcellular localization of the receptor subunit Dα7 in T4/T5 neurons. (A) Optic lobe with T4/T5 neurons labeled with myr::tdTomato and Left panel: horizontal view on the optic lobe overview (scale bar: 20 μm). Right panel: close-up of medulla layer M10, lobula layer Lo1 and lobula plate layers 1–4 (scale bar: 5 μm). (B) Individual T4 dendrites labeled with tdTomtato and subtypes a and pointing in their natural orientation in visual space coordinates (A = anterior, p=posterior, D = dorsal, V = ventral). White arrows indicate preferred directions for every subtype and the dendrites’ proximal (Prox.), central (Cent.) and distal (Dist.) areas are labeled (scale bar: 2 μm). Yellow circle labels first branching point of the dendrite. (C) Individual T5 dendrites labeled with tdTomtato and