Large-volume EM and connectomics - An (incomplete) history of neuronal connectomics

ZooCELL 1st Practical Course | 03-14 February 2025, COS and EMBL, Heidelberg

Gáspár Jékely

Centre for Organismal Studies, Heidelberg University

@jekely@biologists.social

An (incomplete) history of neuronal connectomics

“Its technology - who are we kidding?”

Winfried Denk

Towards the first connectome

• Caenorhabditis elegans nematode worm
• 1 mm long
• hermaphrodites and males

The mind of the worm

• the hermaphrodite wiring diagram
• 5,000 chemical synapses, 2,000 neuromuscular junctions and 600 gap junctions
• from John White’s start in 1969 to completion, the project took 15 years
• a 340 page magnum opus by Sydney Brenner, John White and others

Manual annotation and naming of all cell types

• transverse section through the ventral ganglion
• each cell type individually identified and named
• left-right stereotypy

The computational challenge

• John White standing next to the Modular I computer
• the box in front is the storage disk drive (22 MB)
• room-sized computer with only 64 K of memory
• input was punched paper tape
• capacity was not enough!

Back to manual

• large, 12 × 16 inch glossy prints
• marked with Rotring Rapidograph coloured pens
• trace neurons through the stack of images
• movie film reel of serial EM images in the background

The first circuit diagrams

• C. elegans pharynx nervous system
• 20 neurons
• the connectivity of these neurons has been described at the level of synapses

The first circuit diagrams

• hermaphrodite nervous system
• a total complement of 302 neurons
• invariant structure
• neurons with similar morphologies and connectivities have been grouped together into 118 classes
• ~5000 chemical synapses, ~2000 neuromuscular junctions and ~600 gap junctions
• several detailed circuit diagrams

Serial Block-Face Scanning Electron Microscopy

• automated block-face imaging
• combined with serial sectioning inside the chamber of a SEM
• backscattering contrast to visualise heavy-metal staining

Axon tracing is possible

• tracking axons across large distances in volumes

Method improvements

• Rotring Rapidograph coloured pens for tracing!
• automated sectioning and imaging (SBEM, FIB-SEM)
• better cameras with larger FoV (e.g. 4k x 4k pixels)
• autoloader and camera array for TEM
• improved SBEM embedding (conducting block)
• enhanced FIB-SEM
• multi-beam SEM
• automated TEM with beam deflection
• ATUM-TOME
• improvements in alignments, segmentation, proofreading
• …

Improved TEM methods

• combination of in vivo physiology and network anatomy
• mouse cortex
• custom TEM Camera Array
• 1,215 thin sections (40–45 nm), 450 μm x 350 μm sections

Re-analysis of legacy C. elegans data

• posterior nervous system of adult male
• sexually dimorphic circuits for mating
• short synaptic pathways directly connecting sensory neurons to end organs
• reciprocal connectivity among sensory neurons
• interneurons acting in feedforward loops

Mouse inner plexiform layer

• mouse inner plexiform layer
• reconstruction of 950 neurons and their mutual contacts
• crowd-sourced manual annotation and machine-learning-based volume segmentation

Visual connectomes - fly medulla

• fly medulla connectome as matrix and as graph

• reconstruction of the Drosophila optic medulla
• 379 neurons and 8,637 chemical synapses
• reconstruction of a motion detection circuit

The first complete visual circuit

• neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis dumerilii
• 71-neuron circuit from eyes to muscles
• circuit motif for contrast enhancement and left-right comparison of light
• spatial light detection during visual phototaxis

A volume of the Drosophila larval brain

• an EM volume that spans the entire fly larval nervous system
• collaborative annotation by CATMAID
• many circuits mapped over the years on the same volume

The connectome of the Ciona tadpole larva

• Ciona intestinalis (ascidian) larva
• symmetrical undulations of the non-segmented tail
• simple nervous system

The connectome of the Ciona tadpole larva

• total network of synaptic pathways
• grouped by cell type
• line width indicates the total synaptic number
• arrows indicate direction of synaptic connection
• 177 CNS neurons, 6618 synapses including 1772 neuromuscular junctions, 1206 gap junctions
• asymmetries (e.g. right ocellus, left coronet cells)

Connectome of both sexes of C. elegans

• both male and hermaphrodite whole-animal connectomes
• the nervous system differs between sexes at multiple levels
• several sex-shared neurons are sexually dimorphic

Developmental connectomics

• full brain of eight Caenorhabditis elegans individuals across postnatal stages
• investigate how wiring changes with age
• connections become stronger, new connections appear

Enhanced FIB-SEM

• Focused Ion Beam Scanning Electron Microscopy (FIB-SEM)
• re-engineered commercial FIB-SEMs
• imaging speed improvement and error detection
• generating continuously imaged volumes > 10^6 µm^3

The fly hemibrain connectome

• link to data in neuroglancer

• ‘hot knife approach’ (slice the brain into 20-μm-thick slabs)

• central brain FIB-SEM dataset
• ~25,000 neurons and ~20 million connection
• half of the cell types newly identified

FAFB - a whole fly brain volume

• custom high-throughput EM platform

• serial sectioning and TEM imaging of a fly brain
• brain-spanning mapping of circuits at synaptic resolution
• 100,000-neuron adult brain
• https://catmaid-fafb.virtualflybrain.org/

Partial reconstructions in the FAFB dataset - olfactory system

• complete inventory of olfactory projections connecting the antennal lobe to higher-order brain regions
• the lateral horn is the main target for olfactory information
• many new PN cell types identified

Fly larva whole-brain connectome

• Drosophila larval brain
• 3,016 neurons and 548,000 synapses
• 93 neuron types
• extensive multisensory integration

Whole-body three-segmented Platynereis larva

• Platynereis dumerilii three-segmented larva
• whole-body connectome
• ~2,000 neurons
• 202 neuronal and 92 non-neuronal cell types

Complete fly brain connectome

• 139,255 neurons
• hierarchical annotations
• 8,453 annotated cell types (3,643 were previously proposed in the partial hemibrain connectome)
• available in CATMAID (https://fafb-flywire.catmaid.org/)

Complete fly brain connectome

• adult female Drosophila melanogaster
• full reconstruction of the FAFB dataset
• FlyWire Consortium, over 200 scientists contributed
• principle of openness: “similar approaches based on an open ecosystem will enable connectomics to scale more efficiently, economically and equitably”

Octopus connectomics

• Octopus vulgaris vertical lobe (memory center)
• small volume of 892 serial sections
• unique circuit architecture for associative learning based on feedforward information flow

The brain of a mini-wasp

• microinsect Megaphragma viggianii
• highly simplified visual system
• denucleated neurons

The Mind of a Mouse

• Each 1,000 cubic microns of brain volume is schematically represented by a 1 cm linear distance.
• ~1 cm³ (125,000x larger than a Platynereis larva)
• ~1 million terabytes of data will need to be acquired and analyzed to provide a complete mouse brain connectome

The importance of openness

• ZooCELL volumes should be released as soon as they are acquired and aligned (short data papers)
• collaboration via online tools
• better annotations, faster progress
• rely on communities of experts
• fly larva and FlyWire are good examples