In the Drosophila antenna, different subtypes of olfactory receptor neurons (ORNs) housed in the same sensory hair (sensillum) can inhibit each other non-synaptically. However, the mechanisms underlying this underexplored form of lateral inhibition remain unclear. Here we use recordings from pairs of sensilla impaled by the same tungsten electrode to demonstrate that direct electrical (“ephaptic”) interactions mediate lateral inhibition between ORNs. Intriguingly, within individual sensilla, we find that ephaptic lateral inhibition is asymmetric such that one ORN exerts greater influence onto its neighbor. Serial block-face scanning electron microscopy of genetically identified ORNs and circuit modeling indicate that asymmetric lateral inhibition reflects a surprisingly simple mechanism: the physically larger ORN in a pair corresponds to the dominant neuron in ephaptic interactions. Thus, morphometric differences between compartmentalized ORNs account for highly specialized inhibitory interactions that govern information processing at the earliest stages of olfactory coding.

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications.

In the Drosophila antenna, different subtypes of olfactory receptor neurons (ORNs) housed in the same sensory hair (sensillum) can inhibit each other non-synaptically. However, the mechanisms underlying this unusual form of lateral inhibition remain unclear. Here we use recordings from pairs of sensilla impaled by the same tungsten electrode to prove that direct electrical ("ephaptic") interactions mediate lateral inhibition between ORNs. Intriguingly, within individual sensilla, we find that ephaptic lateral inhibition is asymmetric such that one ORN exerts greater influence onto its neighbor. Serial block-face scanning electron microscopy of genetically identified ORNs and circuit modeling indicate that asymmetric lateral inhibition reflects a surprisingly simple mechanism: the physically larger ORN in a pair corresponds to the dominant neuron in ephaptic interactions. Thus, morphometric differences between compartmentalized ORNs account for highly specialized inhibitory interactions that govern information processing at the earliest stages of olfactory coding.

26Electron microscopy (EM) offers unparalleled power to study cell substructures at the 27 nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as 28 it captures cellular structures instantaneously in their near-native states. However, the 29 applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using 30 genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning 31 electron microscopy (SBEM). In addition, it is challenging to perform correlated light and 32 electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods 33 cannot be applied to address questions requiring optimal morphological preservation and high 34 temporal resolution. Here we developed an approach that overcomes these limitations; it 35 enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. 36Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory 37 organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality 38 ultrastructural preservation for diverse EM applications. 39Cryofixation is especially critical, and often necessary, for properly fixing tissues with cell 55 walls or cuticles that are impermeable to chemical fixatives, such as samples from yeast, plant, 56 Winey et al., 58 1995). As cryofixation instantaneously halts all cellular processes, it also provides the temporal 59 control needed to capture fleeting biological events in a dynamic process (Hess et al., 2000; 60 Watanabe et al., 2013; Watanabe et al., 2013;Watanabe et al., 2014). 61Despite the clear benefits of cryofixation, it is incompatible with diaminobenzidine (DAB) 62 labeling reactions by genetic EM tags. For example, APEX2 (enhanced ascorbate peroxidase) 63is an engineered peroxidase that catalyzes DAB reaction to render target structures electron 64 dense (Lam et al., 2015; Martell et al., 2012). Despite the successful applications of APEX2 to three-dimensional (3D) EM (Joesch et al., 2016), there has been no demonstration that APEX2 66 or other genetic EM tags can be activated following cryofixation. Conventionally, cryofixation is 67 followed by freeze-substitution (Steinbrecht and Müller, 1987), during which water in the sample 68 is replaced by organic solvents. However, the resulting dehydrated environment is incompatible 69 with the aqueous enzymatic reactions required for DAB labeling by genetic EM tags. 70 EM structures can also be genetically labeled with fluorescent markers through 71 correlated light and electron microscopy (CLEM). Yet, performing CLEM with cryofixed samples 72 also presents challenges. Fluorescence microscopy commonly takes place either before 73 cryofixation (Brown et al., 2009; Kolotuev et al., 2010;McDonald, 2009) or after the sample is 74 embedded (Kukulski et al., 2011;Nixon et al., 2009;Schwarz and Humbel, 2009). However, if 75 the specimen is dissected from live animals, the ti...

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.