biology

The Virus Zoo: A Quick Primer on Molecular Virology


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SARS-CoV-2

Genome and Structure:

The SARS-CoV-2 genome consists of about 30 kb of linear positive-sense ssRNA. There is a m7G-cap (specifically m7GpppA1) at the 5’ end of the genome and a 30-60 nucleotide poly-A tail at the 3’ end of the genome. These protective structures minimize exonuclease degradation.1 The genome also has a 5’ UTR and a 3’ UTR which contain sequences that aid in transcriptional regulation and in packaging. The SARS-CoV-2 genome directly translates two partially overlapping polyproteins, ORF1a and ORF1b. There is a –1 ribosomal frameshift in ORF1b relative to ORF1a. Within the polyproteins, two self-activating proteases (Papain-like protease PLpro and 3-chymotrypsin-like protease 3CLpro) perform cleavage events that lead to the generation of the virus’s 16 non-structural proteins (nsps). It should be noted that the 3CLpro is also known as the main protease or Mpro. The coronavirus also produces 4 structural proteins, but these are not translated until after the synthesis of corresponding subgenomic RNAs via the viral replication complex. To create these subgenomic RNAs, negative-sense RNA must first be made and then undergo conversion back to positive-sense RNA for translation. Genes encoding the structural proteins are located downstream of the ORF1b section.

SARS-CoV-2’s four structural proteins include the N, E, M, and S proteins. Many copies of the N (nucleocapsid) protein bind the RNA genome and organize it into a helical ribonucelocapsid complex. The complex undergoes packaging into the viral envelope during coronavirus budding. Interactions between the N protein and the other structural proteins may facilitate this packaging process. The N protein also inhibits host immune responses by antagonizing viral suppressor RNAi and by blocking the signaling of interferon production pathways.2

The transmembrane E (envelope) protein forms pentamers and plays a key but poorly understood role in the budding of viral envelopes into the endoplasmic reticulum Golgi intermediate compartment (ERGIC).3–5 Despite its importance in budding, mature viral particles do not incorporate very many E proteins into their envelopes. One of the posttranslational modifications of the E protein is palmitoylation. This aids subcellular trafficking and interactions with membranes. E protein pentamers also act as ion channels that alter membrane potential.6,7 This may lead to inflammasome activation, a contributing factor to cytokine storm induction.

The M (membrane) protein is the most abundant protein in the virion and drives global curvature in the ERGIC membrane to facilitate budding.5,8 It forms transmembrane dimers that likely oligomerize to induce this curvature.9 The M protein also has a cytosolic (and later intravirion) globular domain that likely interacts with the other structural proteins. M protein dimers also induce local curvature through preferential interactions with phosphatidylserine and phosphatidylinositol lipids.4,5 M proteins help sequester S proteins into the envelopes of budding viruses.10

The S (spike) protein of SARS-CoV-2 has been heavily studied due to its central roles in the infectivity and immunogenicity of the coronavirus. It forms a homotrimer that protrudes from the viral envelope and is heavily glycosylated. It binds the host’s ACE2 receptor (angiotensin-converting enzyme 2 receptor) and undergoes conformational changes to promote viral fusion.11 The S protein undergoes cleavage into S1 and S2 subunits by the host’s furin protease during viral maturation.12,13 This enhances SARS-CoV-2 entry into lung cells and may partially explain the virus’s high degree of transmissibility. The S1 fragment contains the receptor binding domain (RBD) and associated machinery while the S2 fragment facilitates fusion. Prior to cellular infection, most S proteins exist in a closed prefusion conformation where the RBDs of each monomer are hidden most of the time.14 After the S protein binds ACE2 during transient exposure of one of its RBDs, the other two RBDs quickly bind as well. This binding triggers a conformational change in the S protein that loosens the structure, unleashing the S2 fusion component and exposing another proteolytic cleavage site called S2’. Host transmembrane proteases such as TMPRSS2 cut at S2’, causing the full activation of the S2 fusion subunit and the dramatic elongation of the S protein into the postfusion conformation. This results in the viral envelope fusing with the host membrane and uptake of the coronavirus’s RNA into the cell.

The 16 nsps of SARS-CoV-2 play a variety of roles. For instance, nsp1 shuts down host cell translation by plugging the mRNA entry channel of the ribosome, inhibiting the host cell’s immune responses and maximizing viral production.15,16 Viral proteins still undergo translation because a conserved sequence in the coronavirus RNA helps circumvent the blockage through a poorly understood mechanism. The nsp5 protein is the protease 3CLpro.17 The nsp3 protein contains several subcomponents, including the protease PLpro. The nsp12, nsp7, and nsp8 proteins come together to form the RNA-dependent RNA polymerase (RdRp) that replicates the viral genome.17,18 The nsp2 protein is likely a topoisomerase which functions in RNA replication. The nsp4 and nsp6 proteins as well as certain subcomponents of nsp3 restructure intracellular host membranes into double-membrane vesicles (DMVs) which compartmentalize viral replication.19

Beyond the 4 structural proteins and 16 nsps of SARS-CoV-2, the coronaviral genome also encodes some poorly understood accessory proteins including ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF9b.20 These accessory proteins are non-essential for replication in vitro, but they are thought to be required for the virus’s full degree of virulence in vivo.

Life cycle:

As mentioned, SARS-CoV-2 infects cells by first binding a S protein RBD to the ACE2 receptor. This triggers a conformational change that elongates the S protein’s structure and reveals the S2 fusion fragment, facilitating fusion of the virion envelope with the host cell membrane.14 Cleavage of the S’ site by proteases like TMPRSS2 aid this change from the prefusion to postfusion configurations. Alternatively, SARS-CoV-2 can enter the cell by binding to ACE2, undergoing endocytosis, and fusing with the endosome to release its genome (as induced by endosomal cathepsin proteases).20 After release of the SARS-CoV-2 genome into the cytosol, the N protein disassociates and allows translation of ORF1a and ORF1b, producing polyproteins which are cleaved into mature proteins by the PLpro and 3CLpro proteases as discussed earlier. 

The RdRp complex synthesizes negative-sense full genomic RNAs as well as negative-sense subgenomic RNAs. In the latter case, discontinuous transcription is employed, a process by which the RdRp jumps over certain sections of the RNA and initiates transcription separately from the rest of the genome.21 The negative-sense RNAs are subsequently converted back into positive-sense full genomic RNAs and positive-sense subgenomic RNAs. The subgenomic RNAs are translated to make structural proteins and some accessory proteins.20

As described earlier, the nsp4, nsp6, and parts of nsp3 proteins remodel host endoplasmic reticulum (ER) to create DMVs.20 These DMVs are the site of the coronaviral genomic replication and serve to shield the viral RNA and RdRp complex from cellular innate immune factors. DMVs cluster together and are continuous with the ER mostly through small tubular connections. After replication, the newly synthesized coronavirus RNAs undergo export into the cytosol through molecular pore complexes that span both membranes of the DMVs.22 These molecular pore complexes are composed of nsp3 domains and possibly other viral and/or host proteins.

Newly replicated SARS-CoV-2 genomic RNAs complex with N proteins to form helical nucleocapsids. To enable packaging, the nucleocapsids interact with M protein cytosolic domains which protrude at the ERGIC.23 M proteins, E proteins, and S proteins are all localized to the ERGIC membrane. The highly abundant M proteins induce curvature of the membrane to facilitate budding. As mentioned, E proteins also play essential roles in budding, but the mechanisms are poorly understood. Once the virions have budded into the ERGIC, they are shuttled through the Golgi via a series of vesicles and eventually secreted out of the cell.

Adeno-associated virus (AAV)

Genome and Structure:

AAV genomes are about 4.7 kb in length and are composed of ssDNA. Inverted terminal repeats (ITRs) form hairpin structures at ends of the genome. These ITR structures are important for AAV genomic packaging and replication. Rep genes (encoded via overlapping reading frames) include Rep78, Rep68, Rep52, Rep40.24 These proteins facilitate replication of the viral genome. As a Dependoparvovirus, additional helper functions from adenovirus (or certain other viruses) are needed for AAVs to replicate.

AAV capsids are about 25 nm in diameter. Cap genes include VP1, VP2, VP3 and are transcribed from overlapping reading frames.25 The VP3 protein is the smallest capsid protein. The VP2 protein is the same as VP3 except that it includes an N-terminal extension with a nuclear localization sequence. The VP1 protein is the same as VP2 except that it includes a further N-terminal extension encoding a phospholipase A2 (PLA2) that facilitates endosomal escape during infection. In the AAV capsid, VP1, VP2, and VP3 are present at a ratio of roughly 1:1:10. It should be noted that this ratio is actually the average of a distribution, not a fixed number.

Frame-shifted start codons in the Cap gene region transcribe AAP (assembly activating protein) and MAAP (membrane associated accessory protein). These proteins help facilitate packaging and other aspects of the AAV life cycle.

Life cycle:

There are a variety of different AAV serotypes (AAV2, AAV6, AAV9, etc.) that selectively infect certain tissue types. AAVs bind to host cell receptors and are internalized by endocytosis. The particular receptors involved can vary depending on the AAV serotype, though some receptors are consistent across many serotypes. Internalization occurs most often via clathrin-coated pits, but some AAVs are internalized by other routes such as macropinocytosis or the CLIC/GEEC tubulovesicular pathway.26

After endocytosis, conformational changes in the AAV capsid lead to exposure of the PLA2 VP1 domain, which facilitates endosomal escape. The AAV is then transported to the nucleus mainly by motor proteins on cytoskeletal highways. It enters via nuclear pores and finishes uncoating its genome.

AAV genomes initiate replication using the ends of their ITR hairpins as primers. This leads to a series of complex steps involving strand displacement and nicking.24 In the end, new copies of the AAV genome are synthesized. The Rep proteins are key players in this process. It is important to realize that AAVs can only replicate in cells which have also been infected by adenovirus or similar helper viruses (this is why they are called “adeno-associated viruses”). Adenoviruses provide helper genes encoding proteins (e.g. E4, E2a, VA) that are vital for the successful completion of the AAV life cycle. After new AAV capsids have assembled from VP1, VP2, and VP3 and once AAV genomes have been replicated, the ssDNA genomes are threaded into the capsids via pores at their five-fold vertices.

AAVs are nonpathogenic, though a large fraction of people possess antibodies against at least some serotypes, so exposure to them is fairly common.

Adenovirus

Genome and Structure:

Adenovirus genomes are about 36 kb in size and are composed of linear dsDNA. They possess inverted terminal repeats (ITRs) which help facilitate replication and other functions. These genomes contain a variety of transcriptional units which are expressed at different times during the virus’s life cycle.27 E1A, E1B, E2A, E2B, E3, and E4 transcriptional units are expressed early during cellular infection. Their proteins are involved in DNA replication, transcriptional regulation, and suppression of host immune responses. The L1, L2, L3, L4, and L5 transcriptional units are expressed later in the life cycle. Their products include most of the capsid proteins as well as other proteins involved in packaging and assembly. Each transcriptional unit can produce multiple mRNAs through the host’s alternative splicing machinery.

The capsid of the adenovirus is about 90 nm in diameter and consists of three major proteins (hexon, penton, and fiber proteins) as well as a variety of minor proteins and core proteins. Hexon trimer is the most abundant protein in the capsid, the pentameric pentons occur at the vertices, and trimeric fibers are positioned on top of the pentons.28 The fibers point outwards from the capsid and end in knob domains which bind to cellular receptors. In Ad5, a commonly studied type of adenovirus, the fiber knob primarily binds to the coxsackievirus and adenovirus receptor (CAR). That said, it should be noted that Ad5’s fiber knob can also bind to alternative receptors such as vascular cell adhesion molecule 1 and heparan sulfate proteoglycans.

Minor capsid proteins include pIX, pIIIa, pVI, and pVIII. The pIX protein interlaces between hexons and helps stabilize the capsid. Though pIX is positioned in the crevices between the hexons, it is still exposed to the outside environment. By contrast, the pIIIa, pVI, and pVIII proteins bind to the inside of the capsid and contribute further structural stabilization. When the adenovirus is inside of the acidic endosome during infection, conformational changes in the capsid release the pVI protein, which facilitates endosomal escape through membrane lytic activity.

Adenovirus core proteins include pV, pVII, protein μ (also known as pX), adenovirus proteinase (AVP), pIVa2, and terminal protein (TP).29 The pVII protein has many positively-charged arginine residues and so functions to condense the viral DNA. The pV protein bridges the core with the capsid through interactions with pVII and with pVI. AVP cleaves various adenoviral proteins (pIIIa, TP, pVI, pVII, pVIII, pX) to convert them to their mature forms.30 The pIVa2 and pX proteins interact with the viral DNA and may play roles in packaging or replication. TP binds to the ends of the genome and is essential for localizing the viral DNA in the nucleus and for viral replication.

Life Cycle:

Adenovirus infects cells by binding its fiber knob to cellular receptors such as CAR (in the case of Ad5). The penton then binds certain αv integrins, positioning the viral capsid for endocytosis.31 When the endosome acidifies, the adenovirus capsid partially disassembles, fibers and pentons fall away, and pVI is released.32 The pVI protein’s membrane lytic activity facilitates endosomal escape. Partially disassembled capsids then undergo dynein-mediated transport along microtubules and dock at the entrance to nuclear pores. The capsids further disassemble and releases DNA through the nuclear pore. This DNA remains complexed with pVII after it enters the nucleus.

Adenoviral transcription is initiated by the E1A protein, inducing expression of early genes.33 This subsequently leads to expression of the E2, E3, and E4 transcriptional units, which help the virus escape immune responses. This cascade leads to expression of the L1, L2, L3, L4, and L5 transcriptional units, which mainly synthesize viral structural proteins and facilitate capsid assembly.

In the nucleus, adenovirus genomes replicate within dense complexes of protein that can be seen as spots via fluorescence microscopy. Replication begins at the ITRs and is primed by TP.34 Several more viral proteins and host proteins also aid the initiation of replication. Nontemplate strands are displaced during replication but may reanneal and act as template strands later. Adenovirus DNA binding protein and adenovirus DNA polymerase play important roles in replication. Once the genome has been replicated, TP undergoes cleavage into its mature form, signaling for packaging of new genomes.

The adenoviral capsid assembly and maturation process occurs in the nucleus.33 Once enough assembled adenoviruses have accumulated, they rupture the nuclear membrane using adenoviral death protein and subsequently lyse the cell, releasing adenoviral particles.

Herpes Simplex Virus 1 (HSV-1)

Genome and Structure:

HSV-1 genomes are about 150 kb in size and are composed of linear dsDNA. These genomes include a unique long (UL) region and a unique short (US) region.35 The UL and US regions are both flanked by their own inverted repeats. The terminal inverted repeats are called TRL and TRS while the internal inverted repeats are called IRL and IRS. HSV-1 contains approximately 80 genes, though the complexity of its genomic organization makes an exact number of genes difficult to obtain. As with many other viruses, HSV-1 genomes encode early, middle, and late genes. The early genes activate and regulate transcription of the middle and late genes. Middle genes facilitate genome replication and late genes mostly encode structural proteins.

The diameter of HSV-1 ranges around 155 nm to 240 nm.36 Its virions include an inner icosahedral capsid (with a 125 nm diameter) surrounded by tegument proteins which are in turn enveloped by a lipid membrane containing glycoproteins.

HSV-1’s icosahedral capsid consists of a variety of proteins. Some of the most important capsid proteins are encoded by the UL19, UL18, UL38, UL6, UL17, and UL25 genes.37 The UL19 gene encodes the major capsid protein VP5, which forms pentamers and hexamers for the capsid. These VP5 pentamers and hexamers are glued together by triplexes consisting of two copies of VP23 (encoded by UL18) and one copy of VP19C (encoded by UL38).38 The UL6 gene encodes the protein that makes up the portal complex, a structure used by HSV-1 to release its DNA during infection. Each HSV-1 capsid has a single portal (composed of 12 copies of the portal protein) located at one of the vertices. UL17 and UL25 encode additional structural proteins that stabilize the capsid by binding on top of the other vertices. These two proteins also serve as a bridge between the capsid core and the tegument proteins.

The tegument of HSV-1 contains dozens of distinct proteins. Some examples include pUL36, pUL37, pUL7, and pUL51 proteins. The major tegument proteins are pUL36 and pUL37. The pUL36 protein binds on top of the UL17-UL25 complexes at the capsid’s vertices.39 The pUL37 protein subsequently associates with pUL36. The pUL51 protein associates with cytoplasmic membranes in infected cells and recruits the pUL7 protein.40 This pUL51-pUL7 interaction is important for HSV-1 assembly. HSV-1 has many more tegument proteins which play various functional roles.

HSV-1’s envelope contains up to 16 unique glycoproteins. Four of these glycoproteins (gB, gD, gH, and gL) are essential for viral entry into cells.41 The gD glycoprotein first binds to one of its cellular receptors (nectin-1, herpesvirus entry mediator or HVEM, or 3-O-sulfated heparan sulfate). This binding event triggers a conformational change in gD that allows it to activate the gH/gL heterodimer. Next, gH/gL activate gB which induces fusion of HSV-1’s envelope with the cell membrane. Though the remaining 12 envelope glycoproteins are poorly understood, it is thought that they also play roles that influence cellular tropism and entry.

Life cycle:

After binding to cellular receptors via its glycoproteins, HSV-1 induces fusion of its envelope with the host cell membrane.42 The capsid is trafficked to nuclear pores via microtubules. Since the capsid is too large to pass through a nuclear pore directly, the virus instead ejects its DNA through the pore via the portal complex.43

HSV-1 replicates its genome and assembles its capsids in the nucleus. But the assembled capsids are again too large to exit the nucleus through nuclear pores. To overcome this issue, HSV-1 first buds via the inner nuclear membrane into the perinuclear cleft (the space between nuclear membranes), acquiring a primary envelope.42 This process is driven by a pair of proteins (pUL34 and pUL31) which together form the nuclear egress complex. Next, the primary envelope fuses with the outer nuclear membrane, releasing the assembled capsids into the cytosol.

To acquire its final envelope, the HSV-1 capsid likely buds into the trans-Golgi network or into certain tubular vesicular organelles.44 These membrane sources contain the envelope proteins of the virus as produced by transcription and various secretory pathways. One player is the pUL51 tegument protein that starts associated with the membrane into which the virus buds. The interaction between pUL51 and pUL7 helps facilitate recruitment of the capsid to the membrane. (Capsid envelopment is also coupled in many other ways to formation of the outer tegument). The enveloped virion eventually undergoes trafficking through the secretory system and eventually is packaged into exosomes that fuse with the cell membrane and release completed virions into the extracellular environment.

In humans, HSV-1 infects the epithelial cells first and produces viral particles.45 It subsequently enters the termini of sensory neurons, undergoes retrograde transport into the brain, and remains in the central nervous system in a dormant state. During periods of stress in the host, the virus is reactivated and undergoes anterograde transport to infect epithelial cells once again.

References

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Resource: List of Biotechnology Companies to Watch


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PDF version: List of Biotechnology Companies to Watch – by Logan Thrasher Collins

I created this list to serve as a resource to help people learn about and keep track of key biotechnology companies. Some of these are emerging startups, some are established giants, and some provide useful services. Though this list is far from comprehensive, I have tried to cover as many of the key players as possible. It is also important to realize that this landscape is constantly changing, so some of the information on this list will eventually transition into antiquity. This version was written over the course of 2021 and updated during the summer of 2022. I hope you enjoy delving into the exciting world of biotechnology! 

Ablynx
Nanobodies as therapeutics and as laboratory reagents.
AgeX Therapeutics
Treating aging using stem cell therapies, induced tissue regeneration, related methods.
Allonnia
Engineering microorganisms and enzymes to degrade environmental pollutants.
Funded by the Ferment Consortium of Ginkgo Bioworks.
Alora
Engineering salt-tolerant rice via CRISPR for ocean agriculture to feed the world.
Formerly known as Agrisea.
Early stage: raised a $1.4M seed round as of September 2022.
Asimov
Developing computer aided design tools for synthetic biology, making host cell lines for viral vector and biologics manufacturing, constructing genetic parts database.
One of the co-founders is Christopher Voigt.
James Collins is on the scientific advisory board.
Beam Therapeutics
Developing base editor technologies towards therapeutic applications.
David Liu and Feng Zhang are among the co-founders.
Bioasis
Has developed a peptide called xB3 that facilitates transcytosis across the blood-brain barrier.
Working towards applications in glioblastomas, brain metastases, and neurodegenerative diseases.
Biogen
Large pharmaceutical company focusing on developing treatments for neurological diseases.
Has made moves towards developing gene therapy pipelines for treating neurological diseases, though the company has experienced some setbacks in this space (i.e. failed clinical trials).
BioMarin Pharmaceutical
Enzyme replacement therapies for rare diseases.
During April 2021, announced a collaboration with the Allen Institute to develop AAV gene therapies for rare diseases of the brain.
Bionaut Labs
Microrobotics as a new paradigm for drug delivery.
BioViva
Developing gene therapies to treat aging, offers tests for determining biological age.
Elizabeth Parrish (the company’s CEO) tested an experimental gene therapy on herself and reports positive results, though she did not intend for this information to go public.
George Church and Aubrey de Grey are on the scientific advisory board.
Anders Sandberg is the company’s ethics advisor.
Capsida Biotherapeutics
Developing targeted AAV gene therapies for a variety of brain diseases.
Has made blood-brain barrier crossing AAVs that are liver untargeted and brain targeted.
Founded by Viviana Gradinaru.
Capsigen
Engineering superior AAV gene therapy vectors through a proprietary method called Transcription-Dependent Directed Evolution (TRADETM).
Have developed greatly improved neurotrophic AAVs.
Entered into a partnership with Biogen during May of 2021 to develop AAV gene therapies that treat various brain and neuromuscular disorders.
CATALOG
Building a DNA-based platform for massive digital data storage and computation.
Colossal
Centered on moonshot projects that are using advanced CRISPR methods to bring back the Wooly Mammoth, the Thylacine (Tazmanian Tiger), and other extinct animals.
Aims to reintroduce lost biodiversity and thus repair ecosystems.
Will develop biomedical technologies such as artificial wombs in conjunction with its de-extinction research, providing additional benefits to humanity and acting as a way to bring in funding.
Cofounded by George Church, Ben Lamm, and Andrew Busey.
Cortical Labs
Developing hybrid bioelectronic devices which incorporate cultured biological neurons to perform computational tasks. These devices are power efficient, scalable, robust to physical damage, and have the potential for fluid adaptation to many different computational problems.
Creative Biolabs
Custom services for antibody engineering, membrane protein production and characterization, bioconjugation, gene therapy development, viral vector engineering, cell therapy development, molecular dynamics simulations, drug development consulting, and more.
Cultivarium
Developing molecular techniques, hardware platforms, and software tools to accelerate adoption of non-model microorganisms for biotechnology.
Cultivarium is a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
Dyno Therapeutics
Using deep learning to improve properties of AAV capsids as a platform technology for gene therapy.
George Church is one of the co-founders.
E11 Bio
Building moonshot technologies involving superior molecular barcoding, spatial -omics, and viral circuit tracing to help neuroscientists map the brain. Has a long-term goal of mapping brains at the one-hundred billion neuron scale.
E11 Bio is a focused research organization (FRO), so it possesses a distinct funding approach and different goals compared to traditional startups. For more information, see this open access article describing FROs in Nature.
Editas Medicine
CRISPR-based gene therapy.
George Church, David Liu, Jennifer Doudna, Feng Zhang, and J. Keith Joung are the co-founders.
Eikon Therapeutics
Superior drug discovery platform which leverages high-throughput automated super-resolution microscopy for tracking single protein movements in living cells.
Eric Betzig is one of the advisors.
Emerald Cloud Lab
Remote automated laboratory as a service for researchers.
Has a large array of automated equipment for synthetic biology and genetic engineering, physical and biophysical chemistry, structural biology, biochemistry, analytical chemistry, etc.
Provides a software interface for users to instruct the automated equipment.
GATTAquant
DNA origami imaging probes, fluorescence microscopy reagents.
First commercial application of DNA origami.
GenScript
Services in artificial DNA synthesis, synthetic biology, antibodies, cell therapies, enzyme engineering, etc.
Ginkgo Bioworks
Synthetic biology, biomanufacturing, microorganism design, enzyme engineering, etc.
Acquired Gen9 in 2017.
HelixNano
Developing an mRNA-based SARS-CoV-2 vaccine which might protect from all possible variants of the virus.
Pivoted from original plan of developing cancer vaccines using the same technology.
Co-founded by Hannu Rajaniemi, who is also a successful science fiction author.
George Church is an advisor.
Immunai
Combining multi-omic single cell profiling technologies and machine learning to comprehensively map the immune system and thereby enable greatly improved immunotherapies as well as accelerate clinical trials and avoid costly failures.
Impossible Foods
Uses synthetic biology and biochemical engineering to develop plant-based substitutes for meat products.
Their signature product is the Impossible Burger. They also make a product which mimics sausages.
One notable strategy employed by Impossible Foods is production of leghemoglobin in yeast. This compound gives a meaty flavor when added to their food products. They also add other plant-based compounds to mimic the fats found in animal meat.
Intellia Therapeutics
Developing therapies which employ CRISPR gene editing technology.
Has conducted some successful clinical trials using CRISPR gene therapy to treat transthyretin amyloidosis (as of February 2022, this is not yet FDA approved though).
Also working on CRISPR therapeutics for engineering T cells towards targeting acute myeloid leukemia.
Partnered with Regeneron, Novartis, and others.
Jennifer Doudna was one of the co-founders.
Kernel
Neurotechnology, noninvasive brain-computer interfaces, invasive neural prostheses.
Some noninvasive products anticipated to be released during 2021.
Founded by Bryan Johnson who personally invested $54 million.
Raised an additional $53 million from outside investors.
Early goal is to help treat brain disease, has ambitions to enable human enhancement.
Laronde
Developing therapies which utilize circular RNAs (Laronde calls these “endless RNAs”) as expression vehicles for proteins. Such circular RNAs are much more stable and less immunogenic than linear RNAs.
Ligandal
Peptide nanoparticles for targeted CRISPR-Cas gene therapy delivery, immunotherapy, hematological gene therapy, aging treatments.
Founded by Andre Watson.
LyGenesis
Allogenic cell therapy that uses host lymph nodes as bioereactors to grow ectopic replacement organs.
Has developed a method for generating ectopic livers via patient lymph nodes that is in early clinical trials as of September 2022.
Mammoth Biosciences
CRISPR-based diagnostics.
Jennifer Doudna is one of the co-founders.
ManifoldBio
System for barcoding protein therapeutics to enable high-throughput design and testing in complex environments, machine learning to optimize drug design.
George Church is one of the co-founders.
Moderna
Biomedical technologies which utilize mRNA inside of lipid nanoparticles; application areas include drug discovery, drug development, and vaccines.
Major player in COVID-19 pandemic since it was one of the first companies which developed and distributed SARS-CoV-2 vaccines to the world.
Nautilus Biotechnology
Developing a high-throughput single-molecule proteomics platform which integrates many novel techniques to decipher protein networks and thereby help accelerate basic science, new therapeutics, and new diagnostics.
Neurable
Developing a non-invasive brain-computer interface based on headphones that use electroencephalography to record brain signals, allowing people to control devices like phones with their minds.
As of September 2022, the company appears fairly far along in its product development process and is likely to release their headphones within a year or so.
Neuralink
High-bandwidth brain-machine interfaces, surgical robots which implant the interfaces in a manner resembling a sewing machine.
Early goal is to help treat brain disease, has ambitions to enable human enhancement.
Founded by Elon Musk and others, highly publicized by Elon Musk.
Has done testing on rats, pigs, monkeys, and other animals as of April 2021.
Openwater
Portable medical imaging technologies which employ novel optoelectronics, lasers, and holographic systems.
Wearable imaging technologies which could be 1,000x cheaper than MRI and achieve similar or better results.
Has speculated that their technology might eventually allow telepathic communication.
Organovo
3D tissue bioprinting for in vivo clinical applications, in vitro tissue models for disease modeling and toxicology.
Long-term goal is to print entire human organs for transplants.
Oxford Nanopore Technologies
Portable nanopore sequencing devices, high-throughput desktop nanopore sequencing devices, sample preparation kits.
The company states that they have the first and only nanopore DNA and RNA sequencing platform as of May 2021.
Oxitec
Genetically modified male insects which curb the reproduction of populations of their species in the wild, acting as a precise and environmentally friendly way of controlling dangerous pests that spread disease or destroy crops.
After years of battles with activists and regulatory bodies, the company will release 750 million genetically modified mosquitos in the Florida Keys (the first time this has been done in the U.S.) with the goal of reducing rates of illnesses such as yellow fever and dengue. 
Panacea Longevity
Enhancing longevity and health using a fasting-mimetic metabolite supplementation.
Early stage as of May 2021.
Prime Medicine
Developing CRISPR Prime editing technology as a novel therapeutic modality.
David Liu and Andrew Anzalone are co-founders.
Proteinea
Mass-produced insect larvae as an affordable way of manufacturing recombinant proteins.
Early stage as of May 2021.
Repair Biotechnologies
Developing a cholesterol degrading platform therapy which can reverse atherosclerosis.
The CEO, who is known as Reason, is outspoken about the need to combat aging.
Has preclinical proof-of-concept as of May 2021.
Resilience
New manufacturing platforms to service partners for development and scaling of gene therapies, cell therapies, vaccines, protein therapies, and more.
Received $800 million in funding during 2020.
Sherlock Biosciences
CRISPR-based diagnostics.
Feng Zhang is one of the co-founders.
Somalogic
Proteomics platform called SomaScan for protein biomarker discovery which aids researchers in the development of new diagnostics.
SomaScan is an aptamer-based platform which can simultaneously measure 7,000 protein biomarkers.
Founded by Larry Gold, who is the inventor of SELEX.
Strateos
Offers R&D services through remotely controlled automated laboratories.
Has extensive automated equipment for research in drug discovery, synthetic biology, imaging, cell and gene therapy, etc.
Synchron
Endovascular brain-computer interfaces as a minimally invasive approach for neural prosthetics, neuromodulation, and neurodiagnostics.
Has developed the strentrode, an endovascular electrode array that can record or stimulate neurons from within blood vessels.
As of September 2022, a technology called brain.io (that employs stentrodes) is in early clinical trials and gives paralyzed patients the ability to control digital devices.
Synthego
CRISPR genome engineering services, custom cell lines, custom screening libraries, CRISPR reagents and kits, aiding both academic researchers and clinical drug developers.
Syzygy Plasmonics
Developing a photocatalytic reactor system which leverages a nanoparticle-based plasmonic photocatalyst. The photocatalyst consists of a larger light-harvesting plasmonic nanoparticle decorated with smaller catalytic nanoparticles. Their first product will be a clean hydrogen fuel production system which does not rely on petroleum.
More of a chemical engineering company than a biotechnology company, but their technology may eventually have applications in biology.
Tilibit Nanosystems
Service which gives researchers predesigned and custom DNA origami nanostructures, including ones with chemical modifications.
Founded by Hendrik Dietz, who was CEO from 2012-2014. He is now a scientific advisor.
Twist Bioscience
Artificial DNA synthesis services. Synthetic biology towards insulin manufacturing in yeast, scalable spider silk manufacturing, combating malaria, and DNA data storage.
Emily Leproust is a co-founder.
Vault Pharma
Protein vault nanocompartments as a drug delivery platform to treat cancers and other diseases, protein vaults as a vaccine platform.
Co-founded by Leonard Rome.
VectorBuilder
Services in vector cloning, virus packaging, library construction, cell lines, etc.
Verve Therapeutics
Developing CRISPR base editing therapies to turn off key genes (e.g. PCSK9 and ANGPTL3) involved in atherosclerotic plaque formation and thus to combat cardiovascular disease.
The delivery mechanism involves lipid nanoparticles carrying gRNA and mRNA encoding a base editor protein.
Has potential to save tens of millions of lives due to the status of heart disease as one of the most common causes of death.
Early clinical trials began in July 2022.
Zymergen
Synthetic biology, metabolic engineering, biomanufacturing of materials and compounds as a substitute for chemical engineering practices.
4D Molecular Therapeutics
Using high-throughput screening and recombination methods to develop novel AAV serotypes that evade immune responses and that target and transduce specific organs.
Clinical trials for several new AAV vectors that treat pulmonary, cardiac, and eye diseases are ongoing as of September 2022
10x Genomics
Spatial transcriptomics, genomics, proteomics, immune cell profiling, etc.
Acquired ReadCoor and Cartana in 2020.
64x Bio
High-throughput screening and computational design of new mammalian cell lines for manufacturing gene and cell therapies.
George Church and Pamela Silver are among the co-founders.

Cover image is a photograph of a part of Emerald Cloud Lab modified from https://www.linkedin.com/company/emerald-therapeutics/ .

Global Highlights in Neuroengineering 2005-2018


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PDF version: global highlights in neuroengineering 2005-2018 – logan thrasher collins

Optogenetic stimulation using ChR2

(Boyden, Zhang, Bamberg, Nagel, & Deisseroth, 2005)

  • Ed Boyden, Karl Deisseroth, and colleagues developed optogenetics, a revolutionary technique for stimulating neural activity.
  • Optogenetics involves engineering neurons to express light-gated ion channels. The first channel used for this purpose was ChR2 (a protein originally found in bacteria which responds to blue light). In this way, a neuron exposed to an appropriate wavelength of light will be stimulated.
  • Over time, optogenetics has gained a place as an essential experimental tool for neuroscientists across the world. It has been expanded upon and improved in numerous ways and has even allowed control of animal behavior via implanted fiber optics and other light sources. Optogenetics may eventually be used in the development of improved brain-computer interfaces.

optogenetics

Blue Brain Project cortical column simulation

(Markram, 2006)

  • In the early stages of the Blue Brain Project, neuronal cell types from the layers of the rat neocortex were reconstructed. Furthermore, their electrophysiology was experimentally characterized.
  • Next, a virtual neocortical column with about 10,000 multicompartmental Hodgkin-Huxley-type neurons and over ten million synapses was built. Its connectivity was defined according the patterns of connectivity found in biological rats, (though this involved the numbers of inputs and outputs quantified for given cell types rather than explicit wiring). In addition, the spatial distributions of boutons forming synaptic terminals upon target cells reflected biological data.
  • The cortical column was emulated using the Blue Gene/L supercomputer and the dynamics of the emulation reflected its biological counterpart.

cortical column

Optogenetic silencing using halorhodopsin

(Han & Boyden, 2007)

  • Ed Boyden continued developing optogenetic tools to manipulate neural activity. Along with Xue Han, he expressed a codon-optimized version of a bacterial halorhodopsin (along with the ChR2 protein) in neurons.
  • Upon exposure to yellow light, halorhodopsin pumps chloride ions into the cell, hyperpolarizing the membrane and inhibiting neural activity.
  • Using halorhodopsin and ChR2, neurons could be easily activated and inhibited using yellow and blue light respectively.

halorhodopsin and chr2 wavelengths

Brainbow

(Livet et al., 2007)

  • Lichtman and colleagues used Cre/Lox recombination tools to create genes which express a randomized set of three or more differently-colored fluorescent proteins (XFPs) in a given neuron, labeling the neuron with a unique combination of colors. About ninety distinct colors were emitted across a population of genetically modified neurons.
  • The detailed structures within neural tissue equipped with the Brainbow system can be imaged much more easily since neurons can be distinguished via color contrast.
  • As a proof-of-concept, hundreds of synaptic contacts and axonal processes were reconstructed in a selected volume of the cerebellum. Several other neural structures were also imaged using Brainbow.
  • The fluorescent proteins expressed by the Brainbow system are usable in vivo.

brainbow

High temporal precision optogenetics

(Gunaydin et al., 2010)

  • Karl Deisseroth, Peter Hegemann, and colleagues used protein engineering to improve the temporal resolution of optogenetic stimulation.
  • Glutamic acid at position 123 in ChR2 was mutated to threonine, producing a new ion channel protein (dubbed ChETA).
  • The ChETA protein allows for induction of spike trains with frequencies up to 200 Hz and greatly decreases the incidence of unintended spikes. Furthermore, ChETA eliminates plateau potentials (a phenomenon which interferes with precise control of neural activity).

ultrafast optogenetics

Hippocampal prosthesis in rats

(Berger et al., 2012)

  • Theodore Berger and his team developed an artificial replacement for neurons which transmit information from the CA3 region to the CA1 region of the hippocampus.
  • This cognitive prosthesis employs recording and stimulation electrodes along with a multi-input multi-output (MIMO) model to encode the information in CA3 and transfer it to CA1.
  • The hippocampal prosthesis was shown to restore and enhance memory in rats as evaluated by behavioral testing and brain imaging.

In vivo superresolution microscopy for neuroimaging

(Berning, Willig, Steffens, Dibaj, & Hell, 2012)

  • Stefan Hell (2014 Nobel laureate in chemistry) developed stimulated emission depletion microscopy (STED), a type of superresolution fluorescence microscopy which allows imaging of synapses and dendritic spines.
  • STED microscopy uses a torus-shaped de-excitation laser that interferes with the excitation laser to deplete fluorescence except in a very small spot. In this way, the diffraction limit is surpassed since the resulting light illuminates extremely small regions of the sample.
  • Neurons in transgenic mice (equipped with glass-sealed holes in their skulls) were imaged using STED. Synapses and dendritic spines were observed up to fifteen nanometers below the surface of the brain tissue.

superresolution microscopy in vivo

In vivo three-photon microscopy

(Horton et al., 2013)

  • Multi-photon excitation uses pulsed lasers to excite fluorophores with two or more photons of light with long wavelengths. During the excitation, the photons undergo a nonlinear recombination process, yielding a single emitted photon with a much shorter wavelength. Because the excitation photons possess long wavelengths, they can penetrate tissue much more deeply than traditional microscopy allows.
  • Horton and colleagues developed a three-photon excitation method to facilitate even deeper tissue penetration than the commonly used two-photon microscopic techniques.
  • Since three photons were involved per excitation event, even longer excitation wavelengths (about 1,700 nm) were usable, allowing the construction of a 3-dimensional image stack that reached a depth of up to 1.4 mm within the living mouse brain.
  • Blood vessels and RFP-labeled neurons were imaged using this approach. Furthermore, the depth was sufficient to enable imaging of neurons within the mouse hippocampus.

3-photon microscopy

Whole-brain functional recording from larval zebrafish

(Ahrens, Orger, Robson, Li, & Keller, 2013)

  • Laser-scanning light-sheet microscopy was used to volumetrically image the entire brains of larval zebrafish (an optically transparent organism).
  • The genetically encoded calcium sensor GCaMP5G facilitated functional recording at single-cell resolution from about 80% of the total neurons in the larval zebrafish brains. Computational methods were used to distinguish between individual neurons.
  • Populations of neurons that underwent correlated activity patterns were identifiedto show the technique’s utility for uncovering the dynamics of neural circuits. These populations included hindbrain neurons that were functionally linked to neural activity in the spinal cord and a population of neurons which showed coupled oscillations on the left and right halves.

whole-brain recording from larval zebrafish

Eyewire: crowdsourcing method for retina mapping

(Marx, 2013)

  • The Eyewire project was created by Sebastian Seung’s research group. It is a crowdsourcing initiative for connectomic mapping within the retina towards uncovering neural circuits involved in visual processing.
  • Laboratories first collect data via serial electron microscopy as well as functional data from two-photon microscopy.
  • In the Eyewire game, images of tissue slices are provided to players who then help reconstruct neural morphologies and circuits by “coloring in” the parts of the images which correspond to cells and stacking many images on top of each other to generate 3D maps. Artificial intelligence tools help provide initial “best guesses” and guide the players, but the people ultimately perform the task of reconstruction.
  • By November 2013, around 82,000 participants had played the game. Its popularity continues to grow.

eyewire

The BRAIN Initiative

(“Fact Sheet: BRAIN Initiative,” 2013)

  • The BRAIN Initiative (Brain Research through Advancing Innovative Technologies) provided neuroscientists with $110 million in governmental funding and $122 million in funding from private sources such as the Howard Hughes Medical Institute and the Allen Institute for Brain Science.
  • The BRAIN Initiative focused on funding research which develops and utilizes new technologies for functional connectomics. It helped to accelerate research on tools for decoding the mechanisms of neural circuits in order to understand and treat mental illness, neurodegenerative diseases, and traumatic brain injury.
  • The BRAIN Initiative emphasized collaboration between neuroscientists and physicists. It also pushed forward nanotechnology-based methods to image neural tissue, record from neurons, and otherwise collect neurobiological data.

The CLARITY method for making brains translucent

(Chung & Deisseroth, 2013)

  • Karl Deisseroth and colleagues developed a method called CLARITY to make samples of neural tissue optically translucent without damaging the fine cellular structures in the tissue. Using CLARITY, entire mouse brains have been turned transparent.
  • Mouse brains were infused with hydrogel monomers (acrylamide and bisacrylamide) as well as formaldehyde and some other compounds for facilitating crosslinking. Next, the hydrogel monomers were crosslinked by incubating the brains at 37°C. Lipids in the hydrogel-stabilized mouse brains were extracted using hydrophobic organic solvents and electrophoresis.
  • CLARITY allows antibody labeling, fluorescence microscopy, and other optically-dependent techniques to be used for imaging entire brains. In addition, it renders the tissue permeable to macromolecules, which broadens the types of experimental techniques that these samples can undergo (i.e. macromolecule-based stains, etc.)

clarity imaging technique

X-ray microtomography used to reconstruct Drosophila brain hemisphere

(Mizutani, Saiga, Takeuchi, Uesugi, & Suzuki, 2013)

  • Mizutani and colleagues stained Drosophila brains with silver nitrate and tetrachloroaurate (a gold-containing compound), facilitating 3-dimensional imaging using X-ray microtomography at a voxel size of 220 × 328 × 314 nm.
  • To generate the X-rays, a synchrotron source was used. It should be noted that synchrotron sources require large facilities to operate.
  • Neuronal tracing was performed manually on the 3-dimensional X-ray images of the fly brain, a process which took about 1,700 person-hours. Some neuronal processes were too dense to be resolved, so they were “fused” into unified structures. Furthermore, some neuronal traces were fragmented and most of the cell bodies were not considered. This decreased the number of traces to one third of the estimated number of actual processes in the hemisphere.
  • Mizutani’s investigation represents an early effort at large-scale connectomics that sets the stage for further initiatives as neuronal tracing, sample preparation, and X-ray microtomography technologies continue to improve.

traced drosophila brain hemisphere

Telepathic rats engineered using hippocampal prosthesis

(S. Deadwyler et al., 2013)

  • Berger’s hippocampal prosthesis was implanted in pairs of rats. When “donor” rats were trained to perform a task, they developed neural representations (memories) which were recorded by their hippocampal prostheses.
  • The donor rat memories were run through the MIMO model and transmitted to the stimulation electrodes of the hippocampal prostheses implanted in untrained “recipient” rats. After receiving the memories, the recipient rats showed significant improvements on the task that they had not been trained to perform.

rat telepathy

Integrated Information Theory 3.0

(Oizumi, Albantakis, & Tononi, 2014)

  • Integrated information theory (IIT) was originally proposed by Giulio Tononi in 2004. IIT is a quantitative theory of consciousness which may help explain the hard problem of consciousness.
  • IIT begins by assuming the following phenomenological axioms; each experience is characterized by how it differs from other experiences, an experience cannot be reduced to interdependent parts, and the boundaries which distinguish individual experiences are describable as having defined “spatiotemporal grains.”
  • From these phenomenological axioms and the assumption of causality, IIT identifies maximally irreducible conceptual structures (MICS) associated with individual experiences. MICS represent particular patterns of qualia that form unified percepts.
  • IIT also outlines a mathematical measure of an experience’s quantity. This measure is called integrated information or ϕ.

Openworm

(Szigeti et al., 2014)

  • The anatomical elegans connectome was originally mapped in 1976 by Albertson and Thomson. More data has since been collected on neurotransmitters, electrophysiology, cell morphology, and other characteristics.
  • Szigeti, Larson, and their colleagues made an online platform for crowdsourcing research on elegans computational neuroscience, with the goal of completing an entire “simulated worm.”
  • The group also released software called Geppetto, a program that allows users to manipulate both multicompartmental Hodgkin-Huxley models and highly efficient soft-body physics simulations (for modeling the worm’s electrophysiology and anatomy).

c. elegans connectome

Expansion microscopy

(F. Chen, Tillberg, & Boyden, 2015)

  • The Boyden group developed expansion microscopy, a method which enlarges neural tissue samples (including entire brains) with minimal structural distortions and so facilitates superior optical visualization of the scaled-up neural microanatomy. Furthermore, expansion microscopy greatly increases the optical translucency of treated samples.
  • Expansion microscopy operates by infusing a swellable polymer network into brain tissue samples along with several chemical treatments to facilitate polymerization and crosslinking and then triggering expansion via dialysis in water. With 4.5-fold enlargement, expansion microscopy only distorts the tissue by about 1% (computed using a comparison between control superresolution microscopy of easily-resolvable cellular features and the expanded version).
  • Before expansion, samples can express various fluorescent proteins to facilitate superresolution microscopy of the enlarged tissue once the process is complete. Furthermore, expanded tissue is highly amenable to fluorescent stains and antibody-based labels.

expansion microscopy

Japan’s Brain/MINDS project

(Okano, Miyawaki, & Kasai, 2015)

  • In 2014, the Brain/MINDS (Brain Mapping by Integrated Neurotechnologies for Disease Studies) project was initiated to further neuroscientific understanding of the brain. This project received nearly $30 million in funding for its first year alone.
  • Brain/MINDS focuses on studying the brain of the common marmoset (a non-human primate abundant in Japan), developing new technologies for brain mapping, and understanding the human brain with the goal of finding new treatments for brain diseases.

The TrueNorth chip from DARPA and IBM

(Akopyan et al., 2015)

  • The TrueNorth neuromorphic computing chip was constructed and validated by DARPA and IBM. TrueNorth uses circuit modules which mimic neurons. Inputs to these fundamental circuit modules must overcome a threshold in order to trigger “firing.”
  • The chip can emulate up to a million neurons with over 250 million synapses while requiring far less power than traditional computing devices.

Human Brain Project cortical mesocircuit reconstruction and simulation

(Markram et al., 2015)

  • The Human Brain Project reconstructed a 0.29 mm3 region of rat cortical tissue including about 31,000 neurons and 37 million synapses based on morphological data, statistical connectivity rules (rather than exact connectivity), and other datasets. The cortical mesocircuit was emulated using the Blue Gene/Q supercomputer.
  • This emulation was sufficiently accurate to reproduce emergent neurological processes and yield insights on the mechanisms of their computations.

cortical mesocircuit

Recording from C. elegans neurons reveals motor operations

(Kato et al., 2015)

  • Live elegans worms were immobilized in microfluidic devices and the neurons in their head ganglia as well as some of their motor systems were imaged and recorded from using the calcium indicator GCaMP. As the C. elegans connectome is well-characterized, Kato and colleagues were able to determine the identities of most of the cells that underwent imaging (with the help of computational segmentation techniques).
  • Principal component analysis was used to reduce the dimensionality of the neural activity datasets since over 100 neurons per worm were recorded from simultaneously.
  • Next, phase space analysis was utilized to visualize the patterns formed by the recording data. Motor behaviors including dorsal turns, ventral turns, forward movements, and backward movements were found to correspond to specific sequences of neural events as uncovered by examining the patterns found in the phase plots. Further analyses revealed various insights about these brain dynamics and their relationship to motor actions.

c. elegans brain dynamics

Neural lace

(Liu et al., 2015)

  • Charles Lieber’s group developed a syringe-injectable electronic mesh made of submicrometer-thick wiring for neural interfacing.
  • The meshes were constructed using novel soft electronics for biocompatibility. Upon injection, the neural lace expands to cover and record from centimeter-scale regions of tissue.
  • Neural lace may allow for “invasive” brain-computer interfaces to circumvent the need for surgical implantation. Lieber has continued to develop this technology towards clinical application.

neural lace

BigNeuron initiative towards standardized neuronal morphology acquisition

(Peng et al., 2015)

  • Because of the inconsistencies between neuronal reconstruction methods and lack of standardization found in neuronal morphology databases, BigNeuron was established as a community effort to improve the situation.
  • BigNeuron tests as many automated neuronal reconstruction algorithms as possible using large-scale microscopy datasets (from several types of light microscopy). It uses the Vaa3D neuronal reconstruction software as a central platform. Reconstruction algorithms are added to Vaa3D as plugins. These computational tests are performed on supercomputers.
  • BigNeuron aims to create a superior community-oriented neuronal morphology database, a set of greatly improved tools for neuronal reconstruction, a standardized protocol for future neuronal reconstructions, and a library of morphological feature definitions to facilitate classification.

Human telepathy during a 20 questions game

(Stocco et al., 2015)

  • Using an interactive question-and-answer setup, Stocco and colleagues demonstrated real-time telepathic communication between pairs of individuals via EEG and transcranial magnetic stimulation. Five pairs of participants played games of 20 questions and attempted to identify unknown objects.
  • EEG data were recorded from the respondent, computationally processed, and transmitted as transcranial magnetic stimulation signals into the mind (occipital lobe stimulation) of a respondent. The respondent’s answers were translated into higher-intensity transcranial magnetic stimulation pulses corresponding to “yes” answers or lower-intensity transcranial magnetic stimulation pulses corresponding to “no” answers.
  • When compared to control trials in which sham interfaces were used, the people using the brain-brain interfaces were significantly more successful at playing 20 questions games.

Expansion FISH

(F. Chen et al., 2016)

  • Boyden, Chen, Marblestone, Church, and colleagues combined fluorescent in situ hybridization (FISH) with expansion microscopy to image the spatial localization of RNA in neural tissue.
  • The group developed a chemical linker to covalently attach intracellular RNA to the infused polymer network used in expansion microscopy. This allowed for RNAs to maintain their relative spatial locations within each cell post-expansion.
  • After the tissue was enlarged, FISH was used to fluorescently label targeted RNA molecules. In this way, RNA localization was more effectively resolved.
  • As a proof-of-concept, expansion FISH was used to reveal the nanoscale distribution of long noncoding RNAs in nuclei as well as the locations of RNAs within dendritic spines.

expansion fish

Neural dust

(Seo et al., 2016)

  • Michel Maharbiz’s group invented implantable, ~ 1 mm biosensors for wireless neural recording and tested them in rats.
  • This neural dust could be miniaturized to less than 0.5 mm or even to microscale dimensions using customized electronic components.
  • Neural dust motes consist of two recording electrodes, a transistor, and a piezoelectric crystal.
  • The neural dust received external power from ultrasound. Neural signals were recorded by measuring disruptions to the piezoelectric crystal’s reflection of the ultrasound waves. Signal processing mathematics allowed precise detection of activity.

neural dust

The China Brain Project

(Poo et al., 2016)

  • The China Brain Project was launched to help understand the neural mechanisms of cognition, develop brain research technology platforms, develop preventative and diagnostic interventions for brain disorders, and to improve brain-inspired artificial intelligence technologies.
  • This project will be take place from 2016 until 2030 with the goal of completing mesoscopic brain circuit maps.
  • China’s population of non-human primates and preexisting non-human primate research facilities give the China Brain Project an advantage. The project will focus on studying rhesus macaques.

Somatosensory cortex stimulation for spinal cord injuries

(Flesher et al., 2016)

  • Gaunt, Flesher, and colleagues found that microstimulation of the primary somatosensory cortex (S1) partially restored tactile sensations to a patient with a spinal cord injury.
  • Electrode arrays were implanted into the S1 regions of a patient with a spinal cord injury. The array performed intracortical microstimulation over a period of six months.
  • The patient reported locations and perceptual qualities of the sensations elicited by microstimulation. The patient did not experience pain or “pins and needles” from any of the stimulus trains. Overall, 93% of the stimulus trains were reported as “possibly natural.”
  • Results from this study might be used to engineer upper-limb neuroprostheses which provide somatosensory feedback.

somatosensory stimulation

Simulation of rat CA1 region

(Bezaire, Raikov, Burk, Vyas, & Soltesz, 2016)

  • Detailed computational models of 338,740 neurons (including pyramidal cells and various types of interneurons) were equipped with connectivity patterns based on data from the biological CA1 region. External inputs were also estimated using biological data and incorporated into the simulation. It is important to note that these connectivity patterns described the typical convergence and divergence of neurites to and from particular cell types rather than explicitly representing the exact connections found in the biological rat.
  • Each neuron was simulated using a multicompartmental Hodgkin-Huxley-type model with its morphological structure based on biological data from the given cell type. Furthermore, different cell types received different numbers of presynaptic terminals at specified distances from the soma. In total, over five billion synapses were present within the CA1 model.
  • The simulation was implemented on several different supercomputers. Due to the model’s complexity, a four second simulation took about four hours to complete.
  • As with the biological CA1 region, the simulation gave rise to gamma oscillations and theta oscillations as well as other biologically consistent phenomena. In addition, parvalbumin-expressing interneurons and neurogliaform cells were identified as drivers of the theta oscillations, demonstrating the utility of detailed neuronal simulations for uncovering biological insights.

ca1 simulation

UltraTracer enhances existing neuronal tracing software

(Peng et al., 2017)

  • UltraTracer is an algorithm that can improve the efficiency of existing neuronal tracing software for handling large datasets while maintaining accuracy.
  • Datasets with hundreds of billions of voxels were utilized to test UltraTracer. Ten existing tracing algorithms were augmented.
  • For most of the existing algorithms, the performance improvements were around 3-6 times, though a few showed improvements of 10-30 times. Even when using computers with smaller memory, UltraTracer was consistently able to enhance conventional software.
  • UltraTracer was made opensource and is available as a plugin for the Vaa3D tracing software suite.

Whole-brain electron microscopy in larval zebrafish

(Hildebrand et al., 2017)

  • Serial electron microscopy facilitated imaging of the entire brain of a larval zebrafish at 5.5 days post-fertilization.
  • Neuronal tracing software (a modified version of the CATMAID software) was used to reconstruct all the myelinated axons found in the larval zebrafish brain.
  • The reconstructed dataset included 2,589 myelinated axon segments along with some of the associated soma and dendrites. It should be noted that only 834 of the myelinated axons were successfully traced back to their cell bodies.

ssem of larval zebrafish brain

Hippocampal prosthesis in monkeys

(S. A. Deadwyler et al., 2017)

  • Theodore Berger continued developing his cognitive prosthesis and tested it in Rhesus Macaques.
  • As with the rats, monkeys with the implant showed substantially improved performance on memory tasks.

The $100 billion Softbank Vision Fund

(Lomas, 2017)

  • Masayoshi Son, the CEO of Softbank (a Japanese telecommunications corporation), announced a plan to raise $100 billion in venture capital to invest in artificial intelligence. This plan involved partnering with multiple large companies in order to raise this enormous amount of capital.
  • By the end of 2017, the Vision Fund successfully reached its $100 billion goal. Masayoshi Son has since announced further plans to continue raising money with a new goal of over $800 billion.
  • Masayoshi Son’s reason for these massive investments is the Technological Singularity. He agrees with Kurzweil that the Singularity will likely occur at around 2045 and he hopes to help bring the Singularity to fruition. Though Son is aware of the risks posed by artificial superintelligence, he feels that superintelligent AI’s potential to tackle some of humanity’s greatest challenges (such as climate change and the threat of nuclear war) outweighs those risks.

Bryan Johnson launches Kernel

(Regalado, 2017)

  • Entrepreneur Bryan Johnson invested $100 million to start Kernel, a neurotechnology company.
  • Kernel plans to develop implants that allow for recording and stimulation of large numbers of neurons at once. The company’s initial goal is to develop treatments for mental illnesses and neurodegenerative diseases. Its long-term goal is to enhance human intelligence.
  • Kernel originally partnered with Theodore Berger and intended to utilize his hippocampal prosthesis. Unfortunately, Berger and Kernel parted ways after about six months because Berger’s vision was reportedly too long-range to support a financially viable company (at least for now).
  • Kernel was originally a company called Kendall Research Systems. This company was started by a former member of the Boyden lab. In total, four members of Kernel’s team are former Boyden lab members.

Elon Musk launches NeuraLink

(Etherington, 2017)

  • Elon Musk (CEO of Tesla, SpaceX, and a number of other successful companies) initiated a neuroengineering venture called NeuraLink.
  • NeuraLink will begin by developing brain-computer interfaces (BCIs) for clinical applications, but the ultimate goal of the company is to enhance human cognitive abilities in order to keep up with artificial intelligence.
  • Though many of the details around NeuraLink’s research are not yet open to the public, it has been rumored that injectable electronics similar to Lieber’s neural lace might be involved.

Facebook announces effort to build brain-computer interfaces

(Constine, 2017)

  • Facebook revealed research on constructing non-invasive brain-computer interfaces (BCIs) at a company-run conference in 2017. The initiative is run by Regina Dugan, Facebook’s head of R&D at division building 8.
  • Facebook’s researchers are working on a non-invasive BCI which may eventually enable users to type one hundred words per minute with their thoughts alone. This effort builds on past investigations which have been used to help paralyzed patients.
  • The building 8 group is also developing a wearable device for “skin hearing.” Using just a series of vibrating actuators which mimic the cochlea, test subjects have so far been able to recognize up to nine words. Facebook intends to vastly expand this device’s capabilities.

DARPA funds research to develop improved brain-computer interfaces

(Hatmaker, 2017)

  • The U.S. government agency DARPA awarded $65 million in total funding to six research groups.
  • The recipients of this grant included five academic laboratories (headed by Arto Nurmikko, Ken Shepard, Jose-Alain Sahel and Serge Picaud, Vicent Pieribone, and Ehud Isacoff) and one small company called Paradromics Inc.
  • DARPA’s goal for this initiative is to develop a nickel-sized bidirectional brain-computer interface (BCI) which can record from and stimulate up to one million individual neurons at once.

Human Brain Project analyzes brain computations using algebraic topology

(Reimann et al., 2017)

  • Investigators at the Human Brain Project utilized algebraic topology to analyze the reconstructed ~ 31,000 neuron cortical microcircuit from their earlier work.
  • The analysis involved representing the cortical network as a digraph, finding directed cliques (complete directed subgraphs belonging to a digraph), and determining the net directionality of information flow (by computing the sum of the squares of the differences between in-degree and out-degree for all the neurons in a clique). In algebraic topology, directed cliques of n neurons are called directed simplices of dimension n-1.
  • Vast numbers of high-dimensional directed cliques were found in the cortical microcircuit (as compared to null models and other controls). Spike correlations between pairs of neurons within a clique were found to increase with the clique’s dimension and with the proximity of the neurons to the clique’s sink. Furthermore, topological metrics allowed insights into the flow of neural information among multiple cliques.
  • Experimental patch-clamp data supported the significance of the findings. In addition, similar patterns were found within the elegans connectome, suggesting that the results may generalize to nervous systems across species.

hbp algebraic topology

Early testing of hippocampal prosthesis algorithm in humans

(Song, She, Hampson, Deadwyler, & Berger, 2017)

  • Dong Song (who was working alongside Berger) tested the MIMO algorithm on human epilepsy patients using implanted recording and stimulation electrodes. The full hippocampal prosthesis was not implanted, but the electrodes acted similarly, though in a temporary capacity. Although only two patients were tested in this study, many trials were performed to compensate for the small sample size.
  • Hippocampal spike trains from individual cells in CA1 and CA3 were recorded from the patients during a delayed match-to-sample task. The patients were shown various images while neural activity data were recorded by the electrodes and processed by the MIMO model. The patients were then asked to recall which image they had been shown previously by picking it from a group of “distractor” images. Memories encoded by the MIMO model were used to stimulate hippocampal cells during the recall phase.
  • In comparison to controls in which the same two epilepsy patients were not assisted by the algorithm and stimulation, the experimental trials demonstrated a significant increase in successful pattern matching.

Brain imaging factory in China

(Cyranoski, 2017)

  • Qingming Luo started the HUST-Suzhou Institute for Brainsmatics, a brain imaging “factory.” Each of the numerous machines in Luo’s facility performs automated processing and imaging of tissue samples. The devices make ultrathin slices of brain tissue using diamond blades, treat the samples with fluorescent stains or other contrast-enhancing chemicals, and image then using fluorescence microscopy.
  • The institute has already demonstrated its potential by mapping the morphology of a previously unknown neuron which “wraps around” the entire mouse brain.

china brain mapping image

Automated patch-clamp robot for in vivo neural recording

(Suk et al., 2017)

  • Ed Boyden and colleagues developed a robotic system to automate patch-clamp recordings from individual neurons. The robot was tested in vivo using mice and achieved a data collection yield similar to that of skilled human experimenters.
  • By continuously imaging neural tissue using two-photon microscopy, the robot can adapt to a target cell’s movement and shift the pipette to compensate. This adaptation is facilitated by a novel algorithm called an imagepatching algorithm. As the pipette approaches its target, the algorithm adjusts the pipette’s trajectory based on the real-time two-photon microscopy.
  • The robot can be used in vivo so long as the target cells express a fluorescent marker or otherwise fluoresce corresponding to their size and position.

automated patch clamp system

Genome editing in the mammalian brain

(Nishiyama, Mikuni, & Yasuda, 2017)

  • Precise genome editing in the brain has historically been challenging because most neurons are postmitotic (non-dividing) and the postmitotic state prevents homology-directed repair (HDR) from occurring. HDR is a mechanism of DNA repair which allows for targeted insertions of DNA fragments with overhangs homologous to the region of interest (by contrast, non-homologous end-joining is highly unpredictable).
  • Nishiyama, Mikuni, and Yasuda developed a technique which allows genome editing in postmitotic mammalian neurons using adeno-associated viruses (AAVs) and CRISPR-Cas9.
  • The AAVs delivered ssDNA sequences encoding a single guide RNA (sgRNA) and an insert. Inserts encoding a hemagglutinin tag (HA) and inserts encoding EGFP were both tested. Cas9 was encoded endogenously by transgenic host cells and in transgenic host animals.
  • The technique achieved precise genome editing in vitro and in vivo with a low rate of off-target effects. Inserts did not cause deletion of nearby endogenous sequences for 98.1% of infected neurons.

genome editing neuronsNeuropixels probe

(Jun et al., 2017)

  • Jun and colleagues created the Neuropixels probe to facilitate simultaneous recording from hundreds of individual neurons with high spatiotemporal resolution. Previous extracellular probes were only able to record from a few dozen individual neurons.
  • The Neuropixels recording shank is one centimeter long and includes 384 recording channels. Due to the small size of the accompanying apparatus (a 6×9 mm base and a data transmission cable), it enables high-throughput recording in freely moving animals. Because the shank is quite long, Neuropixels can record from multiple brain regions at once.
  • Voltage signals are processed directly on the base of the Neuropixels apparatus, allowing for noise-free data transmission along the cable for further analysis.

neuropixels

EEG-based facial image reconstruction

(Nemrodov, Niemeier, Patel, & Nestor, 2018)

  • EEG data associated with viewing images of faces was collected and used to determine the neural correlates of facial processing. In this way, the images were computationally reconstructed in a fashion resembling “mind reading.”
  • It should be noted that the images reconstructed using data taken from multiple people were more accurate than the images reconstructed using single individuals. Nonetheless, the single individual data still yielded statistically significant accuracy.
  • In addition to reconstructing the images themselves, the process gave insights on the cognitive steps involved in perceiving faces.

eeg reconstructions of faces

Near-infrared light and upconversion nanoparticles for optogenetic stimulation

(S. Chen et al., 2018)

  • Upconversion nanoparticles absorb two or more low-energy photons and emit a higher energy photon. For instance, multiple near-infrared photons can be converted into a single visible spectrum photon.
  • Shuo Chen and colleagues injected upconversion nanoparticles into the brains of mice and used them to convert externally applied near-infrared (NIR) light into visible light within the brain tissue. In this way, optogenetic stimulation was performed without the need for surgical implantation of fiber optics or similarly invasive procedures.
  • The authors demonstrated stimulation via upconversion of NIR to blue light (to activate ChR2) and inhibition via upconversion of NIR to green light (to activate a rhodopsin called Arch).
  • As a proof-of-concept, this technology was used to alter the behavior of the mice by activating hippocampally-encoded fear memories.

upconversion nanoparticles and nir

Map of all neuronal cell bodies within mouse brain

(Murakami et al., 2018)

  • Ueda, Murakami, and colleagues combined methods from expansion microscopy and CLARITY to develop a protocol called CUBIC-X which both expands and clears entire brains. Light-sheet fluorescence microscopy was used to image the treated brains and a novel algorithm was developed to detect individual nuclei.
  • Although expansion microscopy causes some increased tissue transparency on its own, CUBIC-X greatly improved this property in the enlarged tissues, facilitating more detailed whole-brain imaging.
  • Using CUBIC-X, the spatial locations of all the cell bodies (but not dendrites, axons, or synapses) within the mouse brain were mapped. This process was performed upon several adult mouse brains as well as several developing mouse brains to allow for comparative analysis.
  • The authors made the spatial atlas publicly available in order to facilitate global cooperation towards annotating connectivity among the neural cell bodies within the atlas.

cubic-x

Clinical testing of hippocampal prosthesis algorithm in humans

(Hampson et al., 2018)

  • Further clinical tests of Berger’s hippocampal prosthesis were performed. Twenty-one patients took part in the experiments. Seventeen patients underwent CA3 recording so as to facilitate training and optimization of the MIMO model. Eight patients received CA1 stimulation so as to improve their memories.
  • Electrodes with the ability to record from single neurons (10-24 single-neuron recording sites) and via EEG (4-6 EEG recording sites) were implanted such that recording and stimulation could occur at CA3 and CA1 respectively.
  • Patients performed behavioral memory tasks. Both short-term and long-term memory showed an average improvement of 35% across the patients who underwent stimulation.

Precise optogenetic manipulation of fifty neurons

(Mardinly et al., 2018)

  • Mardinly and colleagues engineered a novel excitatory optogenetic ion channel called ST-ChroME and a novel inhibitory optogenetic ion channel called IRES-ST-eGtACR1. The channels were localized to the somas of host neurons and generated stronger photocurrents over shorter timescales than previously existing opsins, allowing for powerful and precise optogenetic stimulation and inhibition.
  • 3D-SHOT is an optical technique in which light is tuned by a device called a spatial light modulator along with several other optical components. Using 3D-SHOT, light was precisely projected upon targeted neurons within a volume of 550×550×100 μm3.
  • By combining novel optogenetic ion channels and the 3D-SHOT technique, complex patterns of neural activity were created in vivo with high spatial and temporal precision.
  • Simultaneously, calcium imaging allowed measurement of the induced neural activity. More custom optoelectronic components helped avoid optical crosstalk of the fluorescent calcium markers with the photostimulating laser.

optogenetic control of fifty neurons

Whole-brain Drosophila connectome data acquired via serial electron microscopy

(Zheng et al., 2018)

  • Zheng, Bock, and colleagues collected serial electron microscopy data on the entire adult Drosophila connectome, providing the data necessary to reconstruct a complete structural map of the fly’s brain at the resolution of individual synapses, dendritic spines, and axonal processes.
  • The data are in the form of 7050 transmission electron microscopy images (187500 x 87500 pixels and 16 GB per image), each representing a 40nm-thin slice of the fly’s brain. In total the dataset requires 106 TB of storage.
  • Although much of the the data still must be processed to reconstruct a 3-dimensional map of the Drosophila brain, the authors did create 3-dimensional reconstructions of selected areas in the olfactory pathway of the fly. In doing so, they discovered a new cell type as well as several other previously unrealized insights about the organization of Drosophila’s olfactory biology.

drosophila connectome with sem

Human telepathy using BrainNet

(Jiang et al., 2018)

  • EEG recordings were taken from two individuals (termed senders) while they played a Tetris-like game. Next, the recordings were converted into transcranial magnetic stimulation signals that acted to provide a third individual (called a receiver) with the necessary information to make decisions in the game without seeing the screen. The occipital cortex was stimulated. Fifteen people (five groups of three) took part in the study.
  • To convey their information, the senders were told to focus upon either a higher or a lower intensity light corresponding to commands within the game (the two lights were placed on different sides of the computer screen). In the receiver’s mind, this translated to perceiving a flash of light. The receiver was able to distinguish the intensities and implement the correct command within the game.
  • Using only the telepathically provided stimulation, the receiver made the correct game-playing decisions 81% of the time.

brainnet

Transcriptomic cell type classification across mouse neocortex

(Tasic et al., 2018)

  • Single-cell RNA sequencing was used to characterize gene expression across 23,822 cells from the primary visual cortex and the anterior lateral motor cortex of mice.
  • Using dimensionality reduction and clustering methods, the resulting data were used to classify the neurons into 133 transcriptomic cell types.
  • Injections of adeno associated viruses (engineered to express fluorescent markers) facilitated retrograde tracing of neuronal projections within a subset of the sequenced cells. In this way, correspondences between projection patterns and transcriptomic identities were established.

 

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Notes on Honeybee Sensory Neurobiology


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PDF version: Notes on Honeybee Sensory Neurobiology – Logan Thrasher Collins

Olfaction

Antennal lobes

  • Honeybee antennal lobes (ALs) are composed of about 160 regions called glomeruli in which olfactory receptor neurons from the antennae make synapses on projection neuron cell bodies as well as inhibitory local neurons.
  • The projection neurons send cholinergic axons to the mushroom bodies and to the lateral horn (LH) while the GABAergic local neurons facilitate olfactory computations within the antennal lobes.

Mushroom bodies

  • The mushroom bodies are paired structures located on either side of the central brain (CB). They are known to facilitate higher sensory integration as well as associative learning processes.Fig. 1
  • In honeybees, the mushroom bodies use cup-shaped medial calyces (MCAs) and lateral calyces (LCAs) as their major sensory input regions while using the pedunculi (PEDs) as their major sensory output regions.
  • The calyces contain Kenyon cells which receive cholinergic axons from the projection neurons of the antennal lobes and the pedunculi contain the efferent axons of the Kenyon cells.

Associative olfactory learning

  • Honeybee associative olfactory learning can occur where the olfactory pathway converges with other pathways.Fig. 2
  • Specific odors can serve as conditioned stimuli when they are associated with unconditioned stimuli of appetitive or aversive character.
  • Experimental evidence shows that the VUMmx1 neuron is sufficient for olfactory reward learning in bees. Its cell body is located within a region called the subesophageal ganglion and it synapses upon cells in the calyces, the lateral horn, and the antennal lobe.

Vision

Honeybee eyes

  • Honeybees possess two frontal compound eyes and three ocelli (simple eyes) located on the top of the head.
  • The retinas of honeybee compound eyes are composed of ommatidia, each with nine photoreceptor cells. The types of bee photoreceptor cells include S, M, and L photoreceptors corresponding to UV, blue, and green wavelengths respectively.
  • Ocellar retinas are composed of rod cells (note that they do not have ommatidia) and are covered by a lens. However, the focal plane of this lens is behind the actual retina, leading to much lower resolving power than that of the compound eyes. Although the function of ocelli is not entirely understood, they may operate as widefield detectors of illumination changes. In addition, ocellar retinas can be subdivided into dorsal and ventral regions which view the horizon and the sky respectively. Distinct neuronal pathways are associated with these subdivisions.

Optic lobe

  • Honeybee vision (associated with the compound eyes) starts with the optic lobe’s three regions; the lamina (La), medulla (Me), and lobula (Lo).
  • The lamina is positioned directly under the compound eye’s photoreceptors. It receives inputs mainly from the L photoreceptors, which are involved in the achromatic pathway and exhibit fast response times. However, some very rough color processing may still occur in the lamina. Fig. 3
  • In the medulla, neurons are organized in a columnar retinotopic fashion with eight layers. The columns also possess horizontal connections (unlike the lamina) which likely facilitate color opponency. The medulla’s outer layers contain neurons that respond to specific wavelengths and neurons that respond to a broad range of wavelengths while the medulla’s inner layers contain color-opponent neurons that compare colors at center and surround regions of receptive fields.
  • The lobula consists of six layers. Its outer layers (1-4) are part of the achromatic pathway and exhibit motion sensitivity. Its inner layers (5-6) continue the color processing pathway. Some projections from the inner layers go to the mushroom bodies, possibly facilitating sensory crosstalk and learning.
  • Beyond the optic lobe, further visual processing of the achromatic and color pathways occurs in the protocerebrum and central brain.

Audition and antennal somatosensation

Johnston’s organ

  • Honeybees use Johnston’s organ as their sensory organ for audition. In bees, audition also acts as a form of somatosensation. Johnston’s organ is located on the antennae. It detects vibrations during the waggle dance and air currents during flight. Fig. 4
  • Johnston’s organ contains about 240 scolopidia, mechanosensory complexes which include bristles that deform and trigger action potentials along efferent axons.
  • The soma of neurons within Johnston’s organ are divided into dorsal (dJO), ventral (vJO), and anterior groups (aJO).

Projections from Johnston’s organ

  • The main axons from the soma within Johnston’s organ trifurcate into the fascicles called T6I, T6II, and T6III. The T6I axons terminate at the ventro-medial superior posterior slope (vmSPS), the T6II axons terminate at the antennal mechanosensory and motor center (AMMC), and the T6III axons terminate at the ventro-central superior posterior slope (vcSPS). Fig. 5
  • In the vmSPS, the axons show some degree of somatotopy arising from the dorsal, ventral, and anterior Johnston’s organ regions. Somatotopy is not observed in the AMMC or vcSPS.
  • All the sensory axons from Johnston’s organ also send small collateral branches to the bee’s dorsal lobe (DL).

The AMMC

  • The AMMC contains two classes of interneuron, AMMC-Int-1 and AMMC-Int-2. AMMC-Int-1 neurons have somas located in the honeybee’s primary auditory center, which is near the central brain. They densely arborize at the AMMC and thinly arborize in the ventral protocerebrum (the protocerebrum is a region of the insect brain that includes the mushroom bodies and central brain as well as several other structures). Their dense arborization in the AMMC runs close to the T6 collaterals at the dorsal lobe.
  • AMMC-Int-1 neurons demonstrate spontaneous spiking without sensory input.Fig. 6 During exposure to a vibratory stimulus, the spike rate slows slightly. After the stimulus is removed, the spike rate increases to a higher rate than that of the spontaneous spiking, but eventually returns to the basal rate. However, it should be noted that olfactory stimuli and other modulating factors can drastically alter the response properties of AMMC-Int-1 neurons.
  • AMMC-Int-2 neurons have somas located in the dorsal lobe. Their dendrites split into three main branches called x, y, and z. Branch y is the axon while branches x and z are dendritic. It sends a long process to the lateral protocerebrum (LP) and makes synapses. The x arborization represents the densest of the three branches and is located in the AMMC. Branch z passes through the dorsal lobe and into the lateral superior posterior slope (lateral SPS). Fig. 7
  • AMMC-Int-2 neurons respond to relatively high vibratory amplitudes, especially those which cause 30 μm (or greater) shifts in antennal position. Their sensitivity reaches a maximum at 265 Hz (a frequency that occurs during the waggle dance), though they also respond to other frequencies.

The SPS

  • The SPS contains an interneuron known as SPS-D-1 which projects to the ipsilateral and contralateral SPS.
  • SPS-D-1 does not respond to 265 Hz alone. However, it responds to long-lasting 265 Hz vibratory stimulation with simultaneous olfactory stimulation at the contralateral antenna.

Gustation

Gustatory sensilla

  • Gustatory receptor cells are found in sensilla, structures which resemble hairs or pegs. Sensilla are located on the glossa, antennae, labial palps, and several other parts of the bee’s body. Fig. 8
  • Each sensillum contains 3-5 gustatory receptor neurons that send dendrites up the shaft towards a pore at the sensillum’s tip. The somas of the receptor cells (along with a mechanoreceptor cell) are encapsulated by auxiliary cells and bathed in a receptor hemolymph. The gustatory receptor neurons likely use GPCRs to detect various food molecules while the mechanoreceptor facilitates evaluation of the food’s position and density.
  • Antennal sensilla respond in a dose-dependent and highly sensitive manner to sucrose solutions. In addition, antennal sensilla respond to aqueous NaCl. As the antennal sensilla do not respond to very low concentrations of KCl, they probably do not contain a receptor that responds to water alone (unlike in many other insects). Sensilla on the mouthparts respond to aqueous sucrose, glucose, fructose, LiCl, KCl, and NaCl. They do not respond to CaCl2 or MgCl2. Foreleg sensilla respond to sucrose as well as very low concentrations of KCl, suggesting that these sensilla may contain a receptor that responds to water alone (unlike the bee’s other sensilla).

Honeybee central gustatory processing

  • Honeybee central gustatory processing takes place primarily in their subesophageal ganglion (SEG). Axons of gustatory neurons and the mechanosensory neurons found in the sensilla project to the SEG’s mandibular, maxillary, and labial neuromeres via the mandibular, maxillary, and labial nerves respectively.
  • As mentioned, the SEG contains the VUMmx1 neuron, which facilitates pairing of olfactory and gustatory stimuli for reward learning. Other VUM neurons have been identified in the SEG, but their function remains unclear.
  • Beyond the SEG, other neurons might be involved in the honeybee’s gustatory processing. In the mushroom bodies, the PE1 neuron exhibits increased spiking in response to sucrose gustation. However, PE1 also responds to mechanical and olfactory inputs. Also located in the mushroom bodies are cells dubbed as “feedback neurons” which respond to odors and sucrose as well. In these cases, multisensory integration likely occurs.

With the exception of image created for the section “projections from Johnston’s organ,” images were modified from: (Steijven, Spaethe, Steffan-Dewenter, & Härtel, 2017), (R. Menzel, 2012), (Kiya & Kubo, 2011),  and (Galizia, Eisenhardt, & Giurfa, 2011).

References

Dyer, A. G., Paulk, A. C., & Reser, D. H. (2011). Colour processing in complex environments: insights from the visual system of bees. Proceedings of the Royal Society B: Biological Sciences, 278(1707), 952 LP-959. Retrieved from http://rspb.royalsocietypublishing.org/content/278/1707/952.abstract

Galizia, C. G., Eisenhardt, D., & Giurfa, M. (2011). Honeybee Neurobiology and Behavior: A Tribute to Randolf Menzel. Springer Netherlands.

Heisenberg, M. (2003). Mushroom body memoir: from maps to models. Nature Reviews Neuroscience, 4, 266. Retrieved from https://doi.org/10.1038/nrn1074

Hung, Y.-S., & Ibbotson, M. (2014). Ocellar structure and neural innervation in the honeybee. Frontiers in Neuroanatomy. Retrieved from https://www.frontiersin.org/article/10.3389/fnana.2014.00006

Kiya, T., & Kubo, T. (2011). Dance Type and Flight Parameters Are Associated with Different Mushroom Body Neural Activities in Worker Honeybee Brains. PLOS ONE, 6(4), e19301. Retrieved from https://doi.org/10.1371/journal.pone.0019301

Menzel, R. (2012). The honeybee as a model for understanding the basis of cognition. Nature Reviews Neuroscience, 13, 758. Retrieved from http://dx.doi.org/10.1038/nrn3357

Mota, T., Yamagata, N., Giurfa, M., Gronenberg, W., & Sandoz, J.-C. (2011). Neural Organization and Visual Processing in the Anterior Optic Tubercle of the Honeybee Brain. The Journal of Neuroscience, 31(32), 11443 LP-11456. Retrieved from http://www.jneurosci.org/content/31/32/11443.abstract

Sandoz, J.-C. (2013). Chapter 30 – Neural Correlates of Olfactory Learning in the Primary Olfactory Center of the Honeybee Brain: The Antennal Lobe. In R. Menzel & P. R. B. T.-H. of B. N. Benjamin (Eds.), Invertebrate Learning and Memory (Vol. 22, pp. 416–432). Elsevier. https://doi.org/https://doi.org/10.1016/B978-0-12-415823-8.00030-7

Steijven, K., Spaethe, J., Steffan-Dewenter, I., & Härtel, S. (2017). Learning performance and brain structure of artificially-reared honey bees fed with different quantities of food. PeerJ, 5, e3858. https://doi.org/10.7717/peerj.3858