Overview and glomeruli.

A. Left: Low-resolution EM image showing a cross-section through the adult zebrafish OB. Box approximates the area where the high-resolution EM volume was acquired. Right: Single high-resolution section in the center of the volume (composite of 40 tiles). Boundaries of the GCL, PL, and GL are outlined. B. Left: Examples of PNs. Right: Quantification of skeletal length, soma size, and the ratio of terminal branches to total dendritic length. Box plots show the distribution of morphological features, where each data point represents an individual neuron within the respective subclass. C. Overlay of PNs showing organization into 12 discrete glomeruli (colors). Left: Anterior view of all reconstructed PNs. Glomeruli are defined by multiple PNs (same color) with overlapping dendritic arbors. Inset: Example showing a precise boundary between two glomeruli (GL6, dark blue, and GL7). Right: All 12 individual glomeruli.

Mitral and ruffed cells.

A. Morphology of a large MC. Insets: 1. The MC dendrite exhibits (1) Example of frequent, irregular varicosities containing mitochondria. These enlargements are sites of inbound and outbound synapses with local INs (arrows highlight synapses; asterisks indicate reciprocal synapses). (2) Myelinated MC axons forming small, loose bundles. (3) Irregular surface of a MC soma. The perikaryon contains an abundance of small mitochondria, vacuoles, and endoplasmic reticulum (ER); the Golgi apparatus frequently extends far into the primary dendrite. (4) MC dendrite receiving dense sensory input (arrows) on terminal dendritic branches. B. Morphology of a RC. Insets: (1) RC soma with labyrinth-like ER structures around the nucleus and a multitude of organelles within the endomembrane system. (2,3) Cross sections through the ruff showing RC branches embedded in neuropil that are sites of prominent synaptic input and output, including reciprocal synapses (arrows). The axon (*) emerges from the soma, contains large vesicular structures and lacks obvious outbound synapses. (4) Input synapse from a sensory axon (arrows), which are consistently observed but less frequent than in MCs. (5) Typical thin RC dendrite with a bulbar enlargement containing a few small, round mitochondria with a light appearance. (6) Two primary cilia projecting parallel to the soma surface. Similar structures were observed in approximately half of all RCs. (7) GL6 with three highlighted RCs, showing the location of the somata and ruffs relative to the glomerular neuropil.

Association between RCs and MCs.

A. Center: RC within GL6 and four associated MCs. Arrows highlight synapses. The thin RC dendrites follow and wrap around the MC dendrites (1, 3, 6, 8, 10). Synapses from MCs onto RCs are rare, typically small, and characterized by low anatomical confidence (2, 11), though larger synapses are occasionally observed (9). Terminal branches of the RC often protrude into the space adjacent to synapses between MCs and INs (4, 7, 11), sometimes receiving direct input from the respective IN (7). MCs and RCs receive shared sensory input (5). B. Intertwined dendrites of a RC and two MCs. Inset shows enlargement of boxed area.

GLIN1.

A. Four GLIN1s (arrows: axons). B. Representative example of a GLIN1 with a branched axon (black arrow). Axonal branches contain amorphous varicosities with mitochondria, outbound synapses, and occasional inbound synapses, including reciprocal arrangements (1, arrows). The bushy dendrite, located close to the soma, has frequent bulbar enlargements with mitochondria (2). The soma (3) is small and surrounded by a thin layer of cytosol characterized by small, round mitochondria with a light matrix (see also 2). C. Seven GLIN1s associated with an RC of GL7. GLIN1 dendrites are entirely immersed within the ruff neuropil (1). GLIN1 dendrites tightly wrap around (2) and protrude into (4) RC neurites. Synapses from the ruff onto GLIN1s are often small (arrow in 4). GLIN1s receive input from synaptic partners of the RC (3). Half of the reconstructed GLIN1s interacted with multiple RCs (5).

GLIN2.

A. Five GLIN2s (arrows: axons) highlighting variable morphology reflecting the shape of the innervated glomerulus (right). B. Example of a GLIN2s with a beaded axon (arrow) extending far into the PL and forming synapses with local INs. Somata are smooth with only occasional small protrusions. (1) Synapse onto a PLIN2 of a neighboring glomerulus (arrow). (2) Nucleus embedded in a prominent layer of cytosol containing relatively little ER and compact, darkly stained mitochondria. (3,4) Minor dendritic segments projecting outside the glomerular neuropil (black arrows in 4; observed in 83% of GLIN2s). These segments form large amorphous structures targeted by axons originating from outside the imaged volume (3). (5) Close-up of the dendrite showing typical spine-like protrusions with a very thin and long neck and a large head with multiple filiform extensions. Bottom left: Association of a GLIN2 with a MC. (6,7,8) Synaptic input from the MC to the GLIN2 via multiple synaptic contacts (arrows) targeting the filiform spine heads.

GLIN3.

A. Four large and two small GLIN3s. B. Representative GLIN3 with an uneven, craggy surface bearing excrescences and protrusions, as observed in a subset of GLIN3s (e.g., orange and purple GLIN3s in A). The nucleus is surrounded by sparse cytosol. The Golgi apparatus is frequently located within bulges of the primary dendrite at a distance from the soma (1). Proximal dendrites are hairy, covered with thin branches and long spine-like protrusions that are exclusively postsynaptic (4). The primary dendrite exhibits regular enlargements (3; black arrows). (2) Close-up of the distal dendrite showing spine-like varicosities forming reciprocal synapses with MCs (arrows). C. GLIN3 that is reciprocally connected to a MC by multiple synapses in GL6. Inset: Synapse from the GLIN3 onto the MC (arrow).

GLIN4.

A. Five GLIN4s (arrows: axons). B. Representative GLIN4 with an ovoid nucleus embedded in sparse cytosol, organelles at the base of the primary dendrite, and two short primary cilia (1). The primary dendrite branches into a bushy arbor and axons remain local, branching extensively. (2) Distal dendrite with an input synapse. (3) Axon with amorphous varicosities and synaptic connections onto dendrites two other DLINs (green, magenta). Bottom: cross sections showing synapses (arrows). C. Representative anatomical association of a GLIN4 with a RC. Insets show intertwined dendrites (top) and shared input from another IN (bottom).

A. Five PLIN1s. B. Another representative PLIN1 with and irregular soma and sparse cytoplasm (1). Proximal dendrites exhibit spine-like protrusions that are exclusively postsynaptic (2; arrows). Distal dendrites have amorphous varicosities with outbound synapses (3, 4; arrows). C. A PLIN1 with distal dendrites immersed into the ruff neuropil of four RCs. Innervation of ruffs can be dense (1) or sparse (2, 3) and contain both inbound and outbound synapses (1, bottom, arrows).

PLIN2.

A. Four PLIN2s. B. Two representative PLIN2s with bulbar somata, a thin layer of cytosol, and organelles of the endomembrane system at the base of the primary dendrite (1). Protrusions are sparse along the proximal dendrite and receive synaptic input (2, 3; arrows). Dendrites project toward the GL where they branches into a small, bushy arbor with inbound and outbound synapses on small varicosities (4). C. Example of a PLIN2 connected through a large number of synapses to a MC dendrite in GL5. Insets: top: 12 synapses (arrows) along approximately 20 μm of the MC dendrite. Bottom: Reciprocal (left, right) and unidirectional (center) synapses (red arrows in 1).

PLIN3.

A. Two varicose (green, violet) and two ribbon-like PLIN3s (orange, blue). B. Detailed morphology of a ribbon- like (green) and a varicose (pink) PLIN3. Somata are ovoid, with organelles at the base of the primary dendrite and no protrusions or excrescences. The surface of the ribbon-like PLIN3 is smooth, whereas the varicose PLIN3 is more irregular (1). (2). Proximal branch of the ribbon-like PLIN3 with stubby and long-necked, round-headed spines receiving synaptic input (arrows). (3) Proximal\ branch of the varicose PLIN3 with synaptic input (arrow) onto more irregular, stubby spines. (4, 5) Distal dendrites with varicosities containing inbound and outbound synapses (arrows). Varicosities are flattened in the ribbon-like PLIN3 (4) and bulgy in the varicose PLIN3 (5). (6) Flattened dendrites of the ribbon-like PLIN3 that form ribbon-like curls. C. Connectivity to PNs. PLIN3s are multiglomerular. Top left: association of a representative varicose PLIN3 (red) with GL9 (violet), GL7 (blue), and GL11 (turquoise). Bottom left: A varicose (black arrows) and a ribbon-like (purple arrows) PLIN3 connected to a MC of GL11 via three synapses each. Both PLIN3s form reciprocal synapses (1, 2, arrows). Right: A ribbon-like PLIN3 providing input to the thin dendrites of RCs in GL11 (3, 4, arrows).

PLIN4.

A. Five PLIN4s. B. Representative PLIN4 with an ovoid, uneven soma lacking spine-like protrusions and thin, tortuous dendrites. (1) Nucleus surrounded by sparse mitochondria and ER; most organelles accumulate at the base of the primary dendrite. (2) Long and sparse on the proximal dendrite with a head-like protrusion that contains vesicles and inbound but no outbound synapses (arrow). (3) Bulbous distal varicosities with multiple outbound synapses (red arrows), one of which (black arrow) is shown in cross-section (top). C. Example of a PLIN4 connected to mitral cells and RCs of different glomeruli. Insets show (1) a reciprocal synapse with a MC (arrows), (2) a unidirectional synapse from the PLIN4 onto a MC (arrow), and (3) a synapse from the PLIN4 onto a RC dendrite (arrow).

DLIN spines.

Spine-like appendages are important features used for the classification of DLINs. Spines receive at least one and often multiple synaptic inputs (indicated by arrows in A.1, B.6, C.2, C.3). A. Spines of DLIN1s. Heads are predominantly rounded. Spines can be short-necked with complex heads (1), cupped (3), or voluminous (5) heads, or long-necked with flattened (2) or voluminous heads (7). Sessile spines can occur either as flattened membrane protrusions (4) or voluminous stumps (6). B. Spines of DLIN2. Spines are typically bulgy and frayed. Necks are of moderate length and typically terminate in bulky, amorphous heads with one or more frayed or pointed protrusions. Some branched (7) and sessile spines (3) are observed. C. Spines of DLIN3s. Spines are typically thin, elongated protrusions several micrometers in length that occur at high frequency. Filopodial as well as mushroom-like branches can arise either along the middle of a spine neck or at the distal end (4, 5). Many spines also exhibit amorphous dilations along the neck that are typically targeted by synapses and continue into a filopodium (2, 3).

DLIN1 subclasses.

A. Three rough DLIN1s (rDLIN1s) with large primary dendrites projecting to the ventrolateral GL. Two rDLIN1s (green, pink) project a secondary large dendrite medio-dorsally, presumably towards glomeruli outside the imaged volume. Within the GL, the widespread distal dendrites bear varicosities at a low to moderate frequency. (1) Somata are ovoid with an egg-shaped nucleus surrounded by moderate amounts of cytosol. (2) Somata and proximal dendrites are densely covered with flattened membrane excrescences and stubby, short-necked, rounded spines. (3) Proximal dendrites are thin and studded with short-necked, round-headed spines at moderate frequency. Inset: Spines receive synapses from thin axons densely packed with small, dark vesicles that originate outside the imaged volume (arrow). (4) Distal dendrite with ribbon-like curls (top). The elongated expansions of DLIN1 dendrites envelop neighboring neurites and contain outbound (bottom, arrows) and inbound synapses (not shown). B. A long-spiny DLIN1 that is characterized by a moderate density of long-necked spines with rounded heads. (1) The soma bears occasional spines but is otherwise smooth. Long-spiny DLIN1s have numerous thin basal dendrites with long- and short-necked spines that are exclusively postsynaptic (inset). (2) Small varicosities of distal dendrites—either bulky or cup-shaped and enveloping adjacent neurites—are sites of synaptic output (top, arrow) and input (not shown). C. A sparsely-spiny DLIN1. (1) The soma bears membrane excrescences and occasional spines. Basal dendrites (arrows), seen in a subset of sDLIN1s, bear predominantly stubby and short-necked spines at low to moderate frequency. Inset: Synapse onto a stubby spine. (2) Varicosities of distal dendrites are mostly flattened, amorphous, or coin-shaped. They are densely filled with synaptic vesicles and contain inbound and outbound synapses (inset, arrows).

Connectivity between DLIN1s and PNs.

A. A rough DLIN1 connecting to MCs and RCs from six different glomeruli. It is reciprocally connected to intermediate (1) and large MCs (2, 4, 5) and one RC (3). In addition, it provides unidirectional input to a small MC (6) and two RCs (7, 8). B. Large synapses between a rough DLIN1 and MCs. The distal dendrite of the rough DLIN1 forms extensive sheaths that wrap around MC dendrites. Synapses onto the MCs can comprise multiple active zones or single zones extending over several hundred nanometers.

DLIN2 subclasses.

A. Two representative common DLIN2s with somata in the superficial and deep GCL. Somata are globular and smooth; spines are rare. The superficial DLIN2 has a smaller soma with less cytosol (1, 4). The primary cilia are among the longest within this class (2, 4, arrows). (2) Proximal dendrites are smooth and exhibit regular bulbar dilations that ear frayed spines. (3) Characteristic beaded dendrites extend through the PL and GCL neuropil with frequent bulbar dilations separated by thin dendritic segments, resulting in a pearl-necklace appearance. These varicosities lack mitochondria but are rich in ER (inset). Pre- and postsynaptic contacts are found at a low frequency (not shown). (5) Varicosities of the distal dendrite are typically voluminous (left) but can also be coin-shaped and contain inbound and outbound synapses (arrows in insets). B. A representative stubby DLIN2s (1) The soma and proximal dendrites are densely covered with irregular membrane excrescences and stubby, short- necked, frayed spines. (2) A varicosity or stubby spine within the PL establishing an outbound synapse onto a soma (arrow). (3) Varicosities of distal dendrites occur at a moderate frequency and are predominantly flattened. Insets, top: Varicosities can form multiple outbound synapses. Bottom: Some varicosities receive input from sensory axons (SeA). C. Two representative fringy DLIN2s. Somata are globular and typically covered with numerous short- and long-necked frayed spines, resulting in a rough appearance (1, 2). Inset: The Golgi apparatus extends deep into the primary dendrite. (3) Spines on proximal dendrites receive synaptic inputs from varicosities of thin neurites packed with small vesicles. (4) Inbound and outbound synapses on a beaded dendrite passing through the PL (insets, arrows). (5) Voluminous varicosities of a distal dendrite in the GL establish synapses onto local neurons (insets).

Connectivity between DLIN2s and PNs.

A common DLIN2 (cDLIN2) connecting to 19 MCs and nine RCs across nine glomeruli. Fifteen of the connections with MCs (e.g., 2, 8, 9) and two of the connections with RCs are reciprocal (not shown). Unidirectional connections comprised outbound connections to three MCs (e.g., 5, 6, 10) and five RCs (e.g., 1, 3) and inbound connections from one MC (4) and two RCs (e.g., 7). Most connections consist of a single or occasionally two synapses.

DLIN3.

A. Three DLIN3s with typical ovoid to globular, smooth somata occasionally bearing long spines. (1) Hairy spines on proximal dendrites. Some spines contain large vesicular structures resembling synaptic specializations (left; arrow). Right: A spine receiving synaptic input (arrow). (2) Soma with a thin layer of cytosol surrounding the nucleus and a concentration of ER, Golgi apparatus, and mitochondria at the base of the primary dendrite. (3–5) Details of spines within the GCL and PL with outbound synapses (arrows) targeting neurites of presumed other DLINs. (6) Compact, voluminous varicosities in the distal dendrite with pointed tips (black arrows). The inset (top) shows multiple outbound synapses (arrows). B. A DLIN3s connected to seven MCs and one RC across three glomeruli. Connections comprise five reciprocal DLIN3-MC connection, one unidirectional DLIN3-to-MC connection, and one unidirectional DLIN3-to-RC connection. (1) The connection onto the RC consists of a single synapse onto the RC dendrite (arrow). (2) The DLIN3 forms 11 synapses across the dendrite of a single MC (arrows). Center: Three dendritic varicosities closely enveloping the MC dendritic shaft. Bottom: Reciprocal (*, ***) and unidirectional (**) IN-to-MC synapses (arrows). (3) Three reciprocal synapses between another DLIN3 and a RC at the ruff (inset, arrows).

Classification of neuron types: validation and summary.

A. Validation of classifications. The assignment of cells into distinct classes by five independent experts is represented as an adjacency matrix of the joint classification graph for PNs (left) and INs (right). B. Association of neuron types with a glomerulus, schematically illustrated by overlays of reconstructed neurons representing different types (color-coded). Dark dashed line approximates the outline of a glomerulus defined by the dendritic arbors of MCs and RCs. Light dashed lines represent adjacent glomeruli. The GL contains the monoglomerular GLIN1–GLIN4. The PL features four predominantly multiglomerular IN types (PLIN1–PLIN4). The GCL contains three main classes of INs (DLIN1–DLIN3) that are further divided into subclasses.

Connectivity between INs and sensory axons.

A. Synapses of sensory axons onto a PLIN3 (left), a DLIN3 (center) and an fDLIN2 (right). B. Synapses (arrows) onto sensory axons (SeA) from a GLIN3 (top) and synaptic contacts between multiple other INs and SeAs (bottom). All highlighted neurites provide input to the SeAs (some synapses are out of the focal plane). Synapses onto the SeA frequently target varicosities and can be reciprocal. Bottom center: Six fully reconstructed neurons providing input onto SeAs. Right: Synapses between INs and a SeA; a reciprocal synapse is visible on the left.

Intraglomerular connectivity: summary.

A. Summary of the connectivity between IN and PN classes in the glomerular neuropil. Top: IN-MC connectivity. Bottom: IN-RC connectivity. B. Qualitative summary of observed connection strengths between IN classes and MCs or RCs, respectively. This qualitative assessment of connection strength is based on the intraglomerular connectivity shown in (A) and additional non-systematic observations of connections outside the glomerular neuropil. C. Two\ synaptic microcircuits anchored to MC and RCs. Microcircuits share a similar architecture but are anatomically distinct. Monoglomerular GLINs (GLIN2s and GLIN1s) receive unidirectional input from MCs and RCs, respectively, and project to extraglomerular targets. Multiglomerular PLINs (varicose PLIN3s and PLIN1s) are reciprocally connected to MCs and RCs, respectively. These microcircuits may mediate lateral inhibition and recurrent inhibition (gain control) in subnetworks involving different types of output hneurons.