The apical tuft is the most remote area of the dendritic tree of neocortical pyramidal neurons. Despite its distal location, the apical dendritic tuft of layer 5 pyramidal neurons receives substantial excitatory synaptic drive and actively processes corticocortical input during behavior. The properties of the voltage-activated ion channels that regulate synaptic integration in tuft dendrites have, however, not been thoroughly investigated. Here, we use electrophysiological and optical approaches to examine the subcellular distribution and function of hyperpolarization-activated cyclic nucleotide-gated nonselective cation (HCN) channels in rat layer 5B pyramidal neurons. Outside-out patch recordings demonstrated that the amplitude and properties of ensemble HCN channel activity were uniform in patches excised from distal apical dendritic trunk and tuft sites. Simultaneous apical dendritic tuft and trunk whole-cell current-clamp recordings revealed that the pharmacological blockade of HCN channels decreased voltage compartmentalization and enhanced the generation and spread of apical dendritic tuft and trunk regenerative activity. Furthermore, multisite two-photon glutamate uncaging demonstrated that HCN channels control the amplitude and duration of synaptically evoked regenerative activity in the distal apical dendritic tuft. In contrast, at proximal apical dendritic trunk and somatic recording sites, the blockade of HCN channels decreased excitability. Dynamic-clamp experiments revealed that these compartment-specific actions of HCN channels were heavily influenced by the local and distributed impact of the high density of HCN channels in the distal apical dendritic arbor. The properties and subcellular distribution pattern of HCN channels are therefore tuned to regulate the interaction between integration compartments in layer 5B pyramidal neurons.

We performed patch-clamp recordings from morphologically identified and anatomically mapped pyramidal neurons of the ventral hippocampus to test the hypothesis that bursting neurons are distributed on a gradient from the CA2/CA1 border (proximal) through the subiculum (distal), with more bursting observed at distal locations. We find that the well-defined morphological boundaries between the hippocampal subregions CA1 and subiculum do not correspond to abrupt changes in electrophysiological properties. Rather, we observed that the percentage of bursting neurons is linearly correlated with position in the proximal-distal axis across the CA1 and the subiculum, the percentages of bursting neurons being 10% near the CA1-CA2 border, 24% at the CA1-subiculum border, and higher than 50% in the distal subiculum. The distribution of bursting neurons was paralleled by a gradient in afterdepolarization (ADP) amplitude. We also tested the hypothesis that there was an association between bursting and two previously described morphologically distinct groups of pyramidal neurons (twin and single apical dendrites) in the CA1 region. We found no difference in output mode between single and twin apical dendrite morphologies, which was consistent with the observation that the two morphologies were equally distributed across the transverse axis of the CA1 region. Taken together with the known organization of connections from CA3 to CA1 and CA1 to subiculum, our results indicate that bursting neurons are most likely to be connected to regular spiking neurons and vice versa.

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. Here, we investigate how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. We identify a class of segmentally repeated second-order somatosensory interneurons, that we named Wave, whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, we identify premotor circuits including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.

The Drosophila central brain develops from a fixed number of neuroblasts. Each neuroblast makes a clone of neurons that exhibit common trajectories. Here we identified 15 distinct clones that carry larval-born neurons innervating the Drosophila central complex (CX), which consists of four midline structures including the protocerebral bridge (PB), fan-shaped body (FB), ellipsoid body (EB), and noduli (NO). Clonal analysis revealed that the small-field CX neurons, which establish intricate projections across different CX substructures, exist in four isomorphic groups that respectively derive from four complex posterior asense-negative lineages. In terms of the region-characteristic large-field CX neurons, we found that two lineages make PB neurons, 10 lineages produce FB neurons, three lineages generate EB neurons, and two lineages yield NO neurons. The diverse FB developmental origins reflect the discrete input pathways for different FB subcompartments. Clonal analysis enlightens both development and anatomy of the insect locomotor control center.

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNA interference (RNAi) varies dramatically within the Caenorhabditis elegans natural population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others, the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains but dampens it further in others. Among wild strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans and suggest that RNAi processes are evolving rapidly and dynamically in nature.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

The C9orf72 hexanucleotide repeat expansion (HRE) is the most frequent genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we describe the pathogenic cascades that are initiated by the C9orf72 HRE DNA. The HRE DNA binds to its protein partner DAXX and promotes its liquid-liquid phase separation, which is capable of reorganizing genomic structures. An HRE-dependent nuclear accumulation of DAXX drives chromatin remodeling and epigenetic changes such as histone hypermethylation and hypoacetylation in patient cells. While regulating global gene expression, DAXX plays a key role in the suppression of basal and stress-inducible expression of C9orf72 via chromatin remodeling and epigenetic modifications of the promoter of the major C9orf72 transcript. Downregulation of DAXX or rebalancing the epigenetic modifications mitigates the stress-induced sensitivity of C9orf72-patient-derived motor neurons. These studies reveal a C9orf72 HRE DNA-dependent regulatory mechanism for both local and genomic architectural changes in the relevant diseases.

Progressive depletion of midbrain dopamine neurons (PDD) is associated with deficits in the initiation, speed, and fluidity of voluntary movement. Models of basal ganglia function focus on initiation deficits; however, it is unclear how they account for deficits in the speed or amplitude of movement (vigor). Using an effort-based operant conditioning task for head-fixed mice, we discovered distinct functional classes of neurons in the dorsal striatum that represent movement vigor. Mice with PDD exhibited a progressive reduction in vigor, along with a selective impairment of its neural representation in striatum. Restoration of dopaminergic tone with a synthetic precursor ameliorated deficits in movement vigor and its neural representation, while suppression of striatal activity during movement was sufficient to reduce vigor. Thus, dopaminergic input to the dorsal striatum is indispensable for the emergence of striatal activity that mediates adaptive changes in movement vigor. These results suggest refined intervention strategies for Parkinson’s disease.