Contacts between the endoplasmic reticulum and other membranes in neurons

The cytoplasm of eukaryotic cells is compartmentalized by intracellular membranes that define subcellular organelles. One of these organelles, the endoplasmic reticulum, forms a continuous network of tubules and cisternae that extends throughout all cell compartments, including neuronal dendrites and axons. This network communicates with most other organelles by vesicular transport, and also by contacts that do not lead to fusion but allow cross-talk between adjacent bilayers. Though these membrane contacts have previously been observed in neurons, their distribution and abundance has not been systematically analyzed. Here, we have carried out such analysis. Our studies reveal new aspects of the internal structure of neurons and provide a critical complement to information about interorganelle communication emerging from functional and biochemical studies

Hyperpolarization-Activated Currents and Subthreshold Resonance in Granule Cells of the Olfactory Bulb

An important contribution to neural circuit oscillatory dynamics is the ongoing activation and inactivation of hyperpolarization-activated currents (Ih). Network synchrony dynamics play an important role in the initial processing of odor signals by the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). In the mouse olfactory bulb, we show that Ih is present in granule cells (GCs), the most prominent inhibitory neuron in the olfactory bulb, and that Ih underlies subthreshold resonance in GCs. In accord with the properties of Ih, the currents exhibited sensitivity to changes in extracellular K(+) concentration and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride), a blocker of Ih. ZD7288 also caused GCs to hyperpolarize and increase their input resistance, suggesting that Ih is active at rest in GCs. The inclusion of cAMP in the intracellular solution shifted the activation of Ih to less negative potentials in the MOB, but not in the AOB, suggesting that channels with different subunit composition mediate Ih in these regions. Furthermore, we show that mature GCs exhibit Ih-dependent subthreshold resonance in the theta frequency range (4-12 Hz). Another inhibitory subtype in the MOB, the periglomerular cells, exhibited Ih-dependent subthreshold resonance in the delta range (1-4 Hz), while principal neurons, the mitral cells, do not exhibit Ih-dependent subthreshold resonance. Importantly, Ih size, as well as the strength and frequency of resonance in GCs, exhibited a postnatal developmental progression, suggesting that this development of Ih in GCs may differentially contribute to their integration of sensory input and contribution to oscillatory circuit dynamics

Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of mouse dorsal striatum (jrc_mus-dorsal-striatum)

Sample: Wild-type, 4 month old mouse, strain: C57BL6/129 from Jackson Lab Sample Description: Close appositions between the membrane of the endoplasmic reticulum (ER) and other intracellular membranes have important functions in cell physiology. These include lipid homeostasis, regulation of Ca2+ dynamics, and control of organelle biogenesis and dynamics. Although these membrane contacts have previously been observed in neurons, their distribution and abundance have not been systematically analyzed. Here, we have used focused ion beam-scanning electron microscopy to generate 3D reconstructions of intracellular organelles and their membrane appositions involving the ER (distance ≤30 nm) in different neuronal compartments. ER–plasma membrane (PM) contacts were particularly abundant in cell bodies, with large, flat ER cisternae apposed to the PM, sometimes with a notably narrow lumen (thin ER). Smaller ER–PM contacts occurred throughout dendrites, axons, and in axon terminals. ER contacts with mitochondria were abundant in all compartments, with the ER often forming a network that embraced mitochondria. Small focal contacts were also observed with tubulovesicular structures, likely to be endosomes, and with sparse multivesicular bodies and lysosomes found in our reconstructions. Our study provides an anatomical reference for interpreting information about interorganelle communication in neurons emerging from functional and biochemical studies. Protocol: Four month old female mice C57BL6/129 were anesthetized with a ketamine/xylazine anesthetic mixture before perfusion with 2% depolymerized paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed in 2% OsO4, 1.5% K4Fe(CN)6, 0.1 M sodium cacodylate buffer 1 h. Subsequently, specimens were stained in block with 2% aqueous uranyl acetate (1 h), dehydrated in increasing concentrations of ethanol, and embedded in Epon then re-embedded in durcupan. Contributions: Sample provided by Yumei Wu, Christina Whites, and Pietro De Camilli (Yale), prepared for imaging by Yumei Wu and Christina Whites (Yale), with imaging and post-processing by C. Shan Xu (Yale), Kenneth Hayworth (HHMI/Janelia), Gleb Shtengel, and Harald Hess(HHMI/Janelia). Acquisition ID: jrc_mus-dorsal-striatum Voxel size (nm): 4 x 4 x 4.44 (X, Y, Z) Data dimensions (µm): 10.2 x 10.3 x 6.0 (X, Y, Z) Imaging start date: 2016-09-09 Imaging duration (days): 3 Landing energy (eV): 700 Imaging current (nA): 0.20 Scanning speed (MHz): 0.400 Dataset URL (Redirect): https://data.janelia.org/XFBry Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-dorsal-striatum Publication: Wu et al., 2017; Xu et al., 2017; Xu et al., 2021</div

Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of mouse nucleus accumbens (jrc_mus-nacc-4)

Sample: Wild-type, adult male mouse, strain: C57/BL6J from Charles River Sample Description: Exemplary data used to demonstrate the capabilities of Enhanced FIB-SEM systems for large-volume 3D imaging. Protocol: Chemical fixation, reduced osmium processing, treated with 2% samarium trichloride and 1% uranyl acetate in maleate buffer, then embedded in Durcupan resin. Contributions: Sample provided by and prepared for imaging by Richard Weinberg (University of North Carolina), with imaging by C. Shan Xu (Yale), Kenneth Hayworth (HHMI/Janelia), and post-processing by Gleb Shtengel, and Harald Hess(HHMI/Janelia). Acquisition ID: jrc_mus-nacc-4 Voxel size (nm): 4 x 4 x 3.99 (X, Y, Z) Data dimensions (µm): 20.3 x 20.5 x 3.0 (X, Y, Z) Imaging start date: 2014-09-19 Imaging duration (days): 2 Landing energy (eV): 1100 Imaging current (nA): 0.21 Scanning speed (MHz): 0.400 Dataset URL (Redirect): https://data.janelia.org/ZssB7 Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-nacc-4 Publication: Wu et al., 2017; Xu et al., 2017; Xu et al., 2021</p

Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of mouse nucleus accumbens (jrc_mus-nacc-3)

Sample: Wild-type, adult male mouse, strain: C57/BL6J from Charles River Sample Description: Exemplary data used to demonstrate the capabilities of Enhanced FIB-SEM systems for large-volume 3D imaging. Protocol: Chemical fixation, reduced osmium processing, treated with 2% samarium trichloride and 1% uranyl acetate in maleate buffer, then embedded in Durcupan resin. Contributions: Sample provided by and prepared for imaging by Richard Weinberg (University of North Carolina), with imaging by C. Shan Xu (Yale), Kenneth Hayworth (HHMI/Janelia), and post-processing by Gleb Shtengel, and Harald Hess(HHMI/Janelia). Acquisition ID: jrc_mus-nacc-3 Voxel size (nm): 4 x 4 x 3.8 (X, Y, Z) Data dimensions (µm): 20.4 x 21.0 x 2.9 (X, Y, Z) Imaging start date: 2014-09-16 Imaging duration (days): 3 Landing energy (eV): 1100 Imaging current (nA): 0.21 Scanning speed (MHz): 0.403 Dataset URL (Redirect): https://data.janelia.org/eVzPC Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-nacc-3 Publication: Wu et al., 2017: Xu et al., 2017; Xu et al., 2021</div

Near-isotropic, reconstructed volume electron microscopy (FIB-SEM) of mouse nucleus accumbens (jrc_mus-nacc-2)

Sample: Wild-type, adult male mouse, strain: C57/BL6J from Charles River Sample Description: Exemplary data used to demonstrate the capabilities of Enhanced FIB-SEM systems for large-volume 3D imaging. Protocol: Chemical fixation, reduced osmium processing, treated with 2% samarium trichloride and 1% uranyl acetate in maleate buffer, then embedded in Durcupan resin. Contributions: Sample provided by and prepared for imaging by Richard Weinberg (University of North Carolina), with imaging by C. Shan Xu (Yale), Kenneth Hayworth (HHMI/Janelia), and post-processing by Gleb Shtengel, and Harald Hess(HHMI/Janelia). Acquisition ID: jrc_mus-nacc-2 Voxel size (nm): 4 x 4 x 2.96 (X, Y, Z) Data dimensions (µm): 10.4 x 10.0 x 1.7 (X, Y, Z) Imaging start date: 2015-03-09 Imaging duration (days): 2 Landing energy (eV): 200 Imaging current (nA): 0.22 Scanning speed (MHz): 0.200 Dataset URL (Redirect): https://data.janelia.org/PGuRe Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-nacc-2 Publication: Wu et al., 2017: Xu et al., 2017: Xu et al., 2021</p