Drosophila connectome

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A Drosophila connectome is a list of neurons in the Drosophila melanogaster (fruit fly) nervous system, and the chemical synapses between them. The fly's nervous system consists of the brain plus the ventral nerve cord, and both are known to differ considerably between male and female. [1] [2] Dense connectomes have been completed for the female adult brain, [3] the male nerve cord, [4] and the female larval stage. [5] The available connectomes show only chemical synapses - other forms of inter-neuron communication such as gap junctions or neuromodulators are not represented. Drosophila is the most complex creature with a connectome, which had only been previously obtained for three other simpler organisms, first C. elegans .[ citation needed ] The connectomes have been obtained by the methods of neural circuit reconstruction, which over the course of many years worked up through various subsets of the fly brain to the almost full connectomes that exist today.[ citation needed ]

Contents

Why Drosophila

Connectome research (connectomics) has a number of competing objectives. On the one hand, investigators prefer an organism small enough that the connectome can be obtained in a reasonable amount of time. This argues for a small creature. On the other hand, one of the main uses of a connectome is to relate structure and behavior, so an animal with a large behavioral repertoire is desirable. It's also very helpful to use an animal with a large existing community of experimentalists, and many available genetic tools. Drosophila meets all of these requirements:

Structure of the fly connectome

The one fully-reconstructed adult female fruit fly brain contains about 128,000 neurons and roughly 50 million chemical synapses, and the single reconstructed male nerve cord has about 23,000 neurons and 70 million synapses. These numbers are not independent, since both the brain and the nerve cord contain portions of the several thousand ascending and descending neurons that run through the neck of the fly. The one female larval brain reconstructed contains roughly 3,000 neurons and 548 thousand chemical synapses. All of these numbers are known to vary between individuals. [8]

Adult brain

Drosophila connectomics started in 1991 with a description of the circuits of the lamina. [9] However the methods used were largely manual and further progress awaited more automated techniques.

In 2011, a high-level connectome, at the level of brain compartments and interconnecting tracts of neurons, for the full fly brain was published, [10] and is available online. [11] New techniques such as digital image processing began to be applied to detailed neural reconstruction. [12]

Reconstructions of larger regions soon followed, including a column of the medulla, [13] also in the visual system of the fruit fly, and the alpha lobe of the mushroom body. [14]

In 2017 a paper introduced an electron microscopy image stack of the whole adult female brain at synaptic resolution. The volume was available for sparse tracing of selected circuits. [15] [16]

In 2020, a dense connectome of half the central brain of Drosophila was released, [17] along with a web site that allows queries and exploration of this data. [18] The methods used in reconstruction and initial analysis of the 'hemibrain' connectome followed. [19]

In 2023, using the data from 2017 (above), the full brain connectome (for a female) was made available, containing roughly 5x10^7 chemical synapses between ~130,000 neurons. [3] A projectome, a map of projections between regions, can be derived from the connectome. In parallel, a consensus cell type atlas for the Drosophila brain was published, produced based on this 'FlyWire' connectome and the prior 'hemibrain'. [20] This resource includes 4,552 cell types: 3,094 as rigorous validations of those previously proposed in the hemibrain connectome; 1,458 new cell types, arising mostly from the fact that the FlyWire connectome spans the whole brain, whereas the hemibrain derives from a subvolume. Comparison of these distinct, adult Drosophila connectomes showed that cell type counts and strong connections were largely stable, but connection weights were surprisingly variable within and across animals.

Adult ventral nerve cord

In 2022, a group of scientists mapped the motor control circuits of the ventral nerve cord of a female fruit fly using electron microscopy. [21] In 2023, a dense reconstruction of the male fly ventral nerve chord was released. [22]

Larval brain

In 2023, Michael Winding et al. published a complete larval brain connectome. [23] [5] This connectome was mapped by annotating the previously collected electron microscopy volume. [24] They found that the larval brain was composed of 3,016 neurons and 548,000 synapses. 93% of brain neurons had a homolog in the opposite hemisphere. Of the synapses, 66.6% were axo-dendritic, 25.8% were axo-axonic, 5.8% were dendro-dendritic, and 1.8% were dendro-axonic.

To study the connectome, they treated it as a directed graph with the neurons forming nodes and the synapses forming the edges. Using this representation, Winding et al found that the larval brain neurons could be clustered into 93 different types, based on connectivity alone. These types aligned with the known neural groups including sensory neurons (visual, olfactory, gustatory, thermal, etc), descending neurons, and ascending neurons.

The authors ordered these neuron types based on proximity to brain inputs vs brain outputs. Using this ordering, they could quantify the proportion of recurrent connections, as the set of connections going from neurons closer to outputs towards inputs. They found that 41% of all brain neurons formed a recurrent connection. The neuron types with the most recurrent connections were the dopaminergic neurons (57%), mushroom body feedback neurons (51%), mushroom body output neurons (45%), and convergence neurons (42%) (receiving input from mushroom body and lateral horn regions). These neurons, implicated in learning, memory, and action-selection, form a set of recurrent loops.

Structure and behavior

One of the main uses of the Drosophila connectome is to understand the neural circuits and other brain structure that gives rise to behavior. This area is under very active investigation. [25] [26] For example, the fruit fly connectome has been used to identify an area of the fruit fly brain that is involved in odor detection and tracking. Flies choose a direction in turbulent conditions by combining information about the direction of air flow and the movement of odor packets. Based on the fly connectome, processing must occur in the “fan-shaped body” where wind-sensing neurons and olfactory direction-sensing neurons cross. [27] [28]

A natural question is whether the connectome will allow simulation of the fly's behavior. However, the connectome alone is not sufficient. Additional information needed includes gap junction varieties and locations, identities of neurotransmitters, receptor types and locations, neuromodulators and hormones (with sources and receptors), the role of glial cells, time evolution rules for synapses, and more. [29] [30]

See also

Related Research Articles

<span class="mw-page-title-main">Brain</span> Organ central to the nervous system

The brain is an organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It consists of nervous tissue and is typically located in the head (cephalization), usually near organs for special senses such as vision, hearing and olfaction. Being the most specialized organ, it is responsible for receiving information from the sensory nervous system, processing those information and the coordination of motor control.

<span class="mw-page-title-main">Neuron</span> Electrically excitable cell found in the nervous system of animals

A neuron, neurone, or nerve cell is an excitable cell that fires electric signals called action potentials across a neural network in the nervous system. Neurons communicate with other cells via synapses, which are specialized connections that commonly use minute amounts of chemical neurotransmitters to pass the electric signal from the presynaptic neuron to the target cell through the synaptic gap.

<span class="mw-page-title-main">Nervous system</span> Part of an animal that coordinates actions and senses

In biology, the nervous system is the highly complex part of an animal that coordinates its actions and sensory information by transmitting signals to and from different parts of its body. The nervous system detects environmental changes that impact the body, then works in tandem with the endocrine system to respond to such events. Nervous tissue first arose in wormlike organisms about 550 to 600 million years ago. In vertebrates, it consists of two main parts, the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS consists of the brain and spinal cord. The PNS consists mainly of nerves, which are enclosed bundles of the long fibers, or axons, that connect the CNS to every other part of the body. Nerves that transmit signals from the brain are called motor nerves (efferent), while those nerves that transmit information from the body to the CNS are called sensory nerves (afferent). The PNS is divided into two separate subsystems, the somatic and autonomic, nervous systems. The autonomic nervous system is further subdivided into the sympathetic, parasympathetic and enteric nervous systems. The sympathetic nervous system is activated in cases of emergencies to mobilize energy, while the parasympathetic nervous system is activated when organisms are in a relaxed state. The enteric nervous system functions to control the gastrointestinal system. Nerves that exit from the brain are called cranial nerves while those exiting from the spinal cord are called spinal nerves.

<span class="mw-page-title-main">Motor neuron</span> Nerve cell sending impulse to muscle

A motor neuron is a neuron whose cell body is located in the motor cortex, brainstem or the spinal cord, and whose axon (fiber) projects to the spinal cord or outside of the spinal cord to directly or indirectly control effector organs, mainly muscles and glands. There are two types of motor neuron – upper motor neurons and lower motor neurons. Axons from upper motor neurons synapse onto interneurons in the spinal cord and occasionally directly onto lower motor neurons. The axons from the lower motor neurons are efferent nerve fibers that carry signals from the spinal cord to the effectors. Types of lower motor neurons are alpha motor neurons, beta motor neurons, and gamma motor neurons.

<i>Drosophila melanogaster</i> Species of fruit fly

Drosophila melanogaster is a species of fly in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly", "pomace fly", or "banana fly". In the wild, D. melanogaster are attracted to rotting fruit and fermenting beverages, and are often found in orchards, kitchens and pubs.

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<span class="mw-page-title-main">Neuroanatomy</span> Branch of neuroscience

Neuroanatomy is the study of the structure and organization of the nervous system. In contrast to animals with radial symmetry, whose nervous system consists of a distributed network of cells, animals with bilateral symmetry have segregated, defined nervous systems. Their neuroanatomy is therefore better understood. In vertebrates, the nervous system is segregated into the internal structure of the brain and spinal cord and the series of nerves that connect the CNS to the rest of the body. Breaking down and identifying specific parts of the nervous system has been crucial for figuring out how it operates. For example, much of what neuroscientists have learned comes from observing how damage or "lesions" to specific brain areas affects behavior or other neural functions.

<span class="mw-page-title-main">Glia</span> Support cells in the nervous system

Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system and the peripheral nervous system that do not produce electrical impulses. The neuroglia make up more than one half the volume of neural tissue in the human body. They maintain homeostasis, form myelin in the peripheral nervous system, and provide support and protection for neurons. In the central nervous system, glial cells include oligodendrocytes, astrocytes, ependymal cells and microglia, and in the peripheral nervous system they include Schwann cells and satellite cells.

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<span class="mw-page-title-main">Neural circuit</span> Network or circuit of neurons

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<span class="mw-page-title-main">Ventral nerve cord</span>

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<span class="mw-page-title-main">Connectome</span> Comprehensive map of neural connections in the brain

A connectome is a comprehensive map of neural connections in the brain, and may be thought of as its "wiring diagram". An organism's nervous system is made up of neurons which communicate through synapses. A connectome is constructed by tracing the neuron in a nervous system and mapping where neurons are connected through synapses.

Connectomics is the production and study of connectomes: comprehensive maps of connections within an organism's nervous system. More generally, it can be thought of as the study of neuronal wiring diagrams with a focus on how structural connectivity, individual synapses, cellular morphology, and cellular ultrastructure contribute to the make up of a network. The nervous system is a network made of billions of connections and these connections are responsible for our thoughts, emotions, actions, memories, function and dysfunction. Therefore, the study of connectomics aims to advance our understanding of mental health and cognition by understanding how cells in the nervous system are connected and communicate. Because these structures are extremely complex, methods within this field use a high-throughput application of functional and structural neural imaging, most commonly magnetic resonance imaging (MRI), electron microscopy, and histological techniques in order to increase the speed, efficiency, and resolution of these nervous system maps. To date, tens of large scale datasets have been collected spanning the nervous system including the various areas of cortex, cerebellum, the retina, the peripheral nervous system and neuromuscular junctions.

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Gregory Stephen Xavier Edward Jefferis is a British neuroscientist known for his work on the circuit basis of olfactory perception in the vinegar fly, Drosophila melanogaster. He is a tenured Programme Leader at the MRC Laboratory of Molecular Biology in Cambridge (UK) and associated with the Department of Zoology at the University of Cambridge.

A descending neuron is a neuron that conveys signals from the brain to neural circuits in the spinal cord (vertebrates) or ventral nerve cord (invertebrates). As the sole conduits of information between the brain and the body, descending neurons play a key role in behavior. Their activity can initiate, maintain, modulate, and terminate behaviors such as locomotion. Because the number of descending neurons is several orders of magnitude smaller than the number of neurons in either the brain or spinal cord/ventral nerve cord, this class of cells represents a critical bottleneck in the flow of information from sensory systems to motor circuits.

References

  1. ↑ Cachero, Sebastian, Aaron D. Ostrovsky, Y. Yu Jai, Barry J. Dickson, and Gregory SXE Jefferis (2010). "Sexual dimorphism in the fly brain" (PDF). Current Biology. 20 (18): 1589–1601. doi:10.1016/j.cub.2010.07.045. PMC   2957842 . PMID   20832311. S2CID   14207042.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  2. ↑ Kelley, Darcy B.; Bayer, Emily A. (March 22, 2021). "Sexual dimorphism: Neural circuit switches in the Drosophila brain". Current Biology. 31 (6): R297–R298. doi: 10.1016/j.cub.2021.02.026 . PMID   33756143. S2CID   232314832.
  3. 1 2 "CODEX: Connectome Data Explorer". Princeton Neuroscience Institute., as described in non-peer-reviewed preprint Dorkenwald S, Matsliah A, Sterling AR, Schlegel P, Yu SC, McKellar CE, et al. (June 2023). "Neuronal wiring diagram of an adult brain". bioRxiv   10.1101/2023.06.27.546656 .
  4. ↑ "Analysis tools for Connectomics". Janelia Research Campus, HHMI., as described in non-peer-reviewed preprint Takemura SY, Hayworth KJ, Huang GB, Januszewski M, Lu Z, Marin EC, et al. (June 2023). "A Connectome of the Male Drosophila Ventral Nerve Cord". bioRxiv   10.1101/2023.06.05.543757 .
  5. 1 2 Winding M, Pedigo BD, Barnes CL, Patsolic HG, Park Y, Kazimiers T, et al. (March 2023). "The connectome of an insect brain". Science. 379 (6636): eadd9330. doi:10.1126/science.add9330. PMC   7614541 . PMID   36893230.
  6. ↑ Alivisatos AP, Chun M, Church GM, Greenspan RJ, Roukes ML, Yuste R (June 2012). "The brain activity map project and the challenge of functional connectomics". Neuron. 74 (6): 970–974. doi:10.1016/j.neuron.2012.06.006. PMC   3597383 . PMID   22726828.
  7. ↑ DeWeerdt S (July 2019). "How to map the brain". Nature. 571 (7766): S6–S8. Bibcode:2019Natur.571S...6D. doi: 10.1038/d41586-019-02208-0 . PMID   31341309.
  8. ↑ Rihani, Karen, and Silke Sachse (2022). "Shedding light on inter-individual variability of olfactory circuits in Drosophila". Frontiers in Behavioral Neuroscience. 16 (16): 835680. doi: 10.3389/fnbeh.2022.835680 . PMC   9084309 . PMID   35548690.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  9. ↑ Meinertzhagen IA, O'Neil SD (March 1991). "Synaptic organization of columnar elements in the lamina of the wild type in Drosophila melanogaster". The Journal of Comparative Neurology. 305 (2): 232–263. doi:10.1002/cne.903050206. PMID   1902848. S2CID   35301798.
  10. ↑ Chiang AS, Lin CY, Chuang CC, Chang HM, Hsieh CH, Yeh CW, et al. (January 2011). "Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution". Current Biology. 21 (1): 1–11. doi: 10.1016/j.cub.2010.11.056 . PMID   21129968. S2CID   17155338.
  11. ↑ "FlyCircuit - A Database of Drosophila Brain Neurons". National Center for High-Performance Computing (NCHC). Retrieved 30 Aug 2013.
  12. ↑ Rivera-Alba M, Vitaladevuni SN, Mishchenko Y, Lu Z, Takemura SY, Scheffer L, et al. (December 2011). "Wiring economy and volume exclusion determine neuronal placement in the Drosophila brain". Current Biology. 21 (23): 2000–2005. doi:10.1016/j.cub.2011.10.022. PMC   3244492 . PMID   22119527.
  13. ↑ Takemura SY, Bharioke A, Lu Z, Nern A, Vitaladevuni S, Rivlin PK, et al. (August 2013). "A visual motion detection circuit suggested by Drosophila connectomics". Nature. 500 (7461): 175–181. Bibcode:2013Natur.500..175T. doi:10.1038/nature12450. PMC   3799980 . PMID   23925240.
  14. ↑ Takemura SY, Aso Y, Hige T, Wong A, Lu Z, Xu CS, et al. (July 2017). "A connectome of a learning and memory center in the adult Drosophila brain". eLife. 6: e26975. doi: 10.7554/eLife.26975 . PMC   5550281 . PMID   28718765.
  15. ↑ Yeager A (31 May 2017). "Entire Fruit Fly Brain Imaged with Electron Microscopy". The Scientist Magazine. Retrieved 2018-07-15.
  16. ↑ Zheng Z, Lauritzen JS, Perlman E, Robinson CG, Nichols M, Milkie D, et al. (July 2018). "A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster". Cell. 174 (3): 730–743.e22. doi:10.1016/j.cell.2018.06.019. PMC   6063995 . PMID   30033368.
  17. ↑ Xu CS, Januszewski M, Lu Z, Takemura SY, Hayworth KJ, Huang G, et al. (2020). "A connectome of the adult Drosophila central brain". bioRxiv   10.1101/2020.01.21.911859 .
  18. ↑ "Analysis tools for connectomics". Howard Hughes Medical Institute (HHMI).
  19. ↑ Scheffer LK, Xu CS, Januszewski M, Lu Z, Takemura SY, Hayworth KJ, et al. (September 2020). "A connectome and analysis of the adult Drosophila central brain". eLife. 9. doi: 10.7554/eLife.57443 . PMC   7546738 . PMID   32880371.
  20. ↑ Philipp Schlegel, Yijie Yin, Alexander S. Bates, Sven Dorkenwald, Katharina Eichler, Paul Brooks, Daniel S. Han, Marina Gkantia, Marcia dos Santos, Eva J. Munnelly, Griffin Badalamente, Laia Serratosa Capdevila, Varun A. Sane, Markus W. Pleijzier, Imaan F.M. Tamimi, Christopher R. Dunne, Irene Salgarella, Alexandre Javier, Siqi Fang, Eric Perlman, Tom Kazimiers, Sridhar R. Jagannathan, Arie Matsliah, Amy R. Sterling, Szi-chieh Yu, Claire E. McKellar, FlyWire Consortium, Marta Costa, H. Sebastian Seung, Mala Murthy, Volker Hartenstein, Davi D. Bock, Gregory S.X.E. Jefferis (2023). "Whole-brain annotation and multi-connectome cell typing quantifies circuit stereotypy in Drosophila". pp. 2023–06. bioRxiv   10.1101/2023.06.27.546055 .{{cite bioRxiv}}: CS1 maint: multiple names: authors list (link)
  21. ↑ Phelps JS, Hildebrand DG, Graham BJ, Kuan AT, Thomas LA, Nguyen TM, et al. (February 2021). "Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy". Cell. 184 (3): 759–774.e18. doi:10.1016/j.cell.2020.12.013. PMC   8312698 . PMID   33400916.
  22. ↑ Takemura, Shin-ya; et al. (5 Jun 2023). "A Connectome of the Male Drosophila Ventral Nerve Cord". bioRxiv. doi:10.1101/2023.06.05.543757. S2CID   259120564.{{cite journal}}: Cite journal requires |journal= (help)
  23. ↑ Leffer L (9 March 2023). "First Complete Map of a Fly Brain Has Uncanny Similarities to AI Neural Networks". Gizmodo. Retrieved 10 March 2023.
  24. ↑ Ohyama T, Schneider-Mizell CM, Fetter RD, Aleman JV, Franconville R, Rivera-Alba M, et al. (April 2015). "A multilevel multimodal circuit enhances action selection in Drosophila". Nature. 520 (7549): 633–639. Bibcode:2015Natur.520..633O. doi:10.1038/nature14297. PMID   25896325. S2CID   4464547.
  25. ↑ "Kavli Workshop on Neural Circuits and Behavior of Drosophila". Howard Hughes Medical Institute. 2019.
  26. ↑ "Crete Workshop on Neural Circuits and Behaviour of Drosophila". Queensland Brain Institute. 2023.
  27. ↑ Mackenzie D (6 March 2023). "How animals follow their nose". Knowable Magazine. Annual Reviews. doi: 10.1146/knowable-030623-4 . Retrieved 13 March 2023.
  28. ↑ Matheson AM, Lanz AJ, Medina AM, Licata AM, Currier TA, Syed MH, Nagel KI (August 2022). "A neural circuit for wind-guided olfactory navigation". Nature Communications. 13 (1): 4613. Bibcode:2022NatCo..13.4613M. doi:10.1038/s41467-022-32247-7. PMC   9360402 . PMID   35941114.
  29. ↑ "Columbia Workshop on Brain Circuits, Memory and Computation". Center for Neural Engineering and Computation. New York, NY: Columbia University. March 2019.
  30. ↑ Scheffer LK, Meinertzhagen IA (November 2021). "A connectome is not enough - what is still needed to understand the brain of Drosophila?". The Journal of Experimental Biology. 224 (21): jeb242740. doi: 10.1242/jeb.242740 . PMID   34695211. S2CID   239887246.

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