Novel Approaches to Monitor and Manipulate Single Neurons In Vivo

The complexity of the vertebrate brain poses an enormous challenge to experimental neuroscience. One way of dealing with this complexity has been to investigate different aspects of brain function in widely different preparations, each best suited to address a particular question. Accordingly, cellular questions are typically addressed with intracellular recordings in in vitro preparations such as brain slices or neuronal cultures, whereas network behavior and sensory or motor response properties are analyzed in vivo, often with extracellular recordings. This division of labor has proved to be an experimentally effective strategy. However, although there seems to be no limit to the wealth of data that can be generated in this way, integrating results derived in different preparations comes with its own set of challenges. The enormous difficulties encountered when one attempts to link cellular phenomena such as synaptic plasticity to systems properties such as spatial memory (Martin et al., 2000) have shown us that close collaboration between molecular−cellular and systems neuroscience is required (Tonegawa et al., 2003) and that we need more convergence of experimental techniques to analyze the cellular basis of neural function under more natural conditions. Studying neurons under naturalistic conditions is, however, easier said than done. A return to in vivo preparations will only be successful if we are able to solve the technical problems that led previous researchers to abandon the study of intact brains in the first place. Thus, studying neurons at the cellular level in vertebrate brains is today first and foremost a technological challenge. Here we highlight recent efforts to improve our ability to analyze functions of single neurons in vivo. Given th

Brain-wide mapping of c-fos expression in the single prolonged stress model and the effects of pretreatment with ACH-000029 or prazosin.

Post-traumatic stress disorder (PTSD) is a mental health condition that is triggered by a stressful event, with symptoms including exaggerated startle response, intrusive traumatic memories and nightmares. The single prolonged stress (SPS) is a multimodal stress protocol that comprises a sequential exposure to physical restraint, forced swimming, predator scent and ether anesthesia. This procedure generates behavioral and neurobiological alterations that resemble clinical findings of PTSD, and thus it is commonly used to model the disease in rodents. Here, we applied c-fos mapping to produce a comprehensive view of stress-activated brain regions in mice exposed to SPS alone or to SPS after oral pretreatment with the serotonin-noradrenaline receptor dual modulator ACH-000029 or the α1-adrenergic blocker prazosin. The SPS protocol evoked c-fos expression in several brain regions that control the stress-anxiety response, including the central and medial amygdala, the bed nucleus of the stria terminalis, the pallidum, the paraventricular hypothalamus, the intermediodorsal, paraventricular and central medial thalamic nuclei, the periaqueductal gray, the lateral habenula and the cuneiform nucleus. These effects were partially blocked by pretreatment with prazosin but completely prevented by ACH-000029. Collectively, these findings contribute to the brain-wide characterization of neural circuits involved in PTSD-related stress responses. Furthermore, the identification of brain areas regulated by ACH-000029 and prazosin revealed regions in which SPS-induced activation may depend on the combined or isolated action of the noradrenergic and serotonergic systems. Finally, the dual regulation of serotonin and α1 receptors by ACH-000029 might represent a potential pharmacotherapy that can be applied in the peri-trauma or early post-trauma period to mitigate the development of symptoms in PTSD patients

Select Overexpression of Homer1a in Dorsal Hippocampus Impairs Spatial Working Memory

Long Homer proteins forge assemblies of signaling components involved in glutamate receptor signaling in postsynaptic excitatory neurons, including those underlying synaptic transmission and plasticity. The short immediate-early gene (IEG) Homer1a can dynamically uncouple these physical associations by functional competition with long Homer isoforms. To examine the consequences of Homer1a-mediated “uncoupling” for synaptic plasticity and behavior, we generated forebrain-specific tetracycline (tet) controlled expression of Venus-tagged Homer1a (H1aV) in mice. We report that sustained overexpression of H1aV impaired spatial working but not reference memory. Most notably, a similar impairment was observed when H1aV expression was restricted to the dorsal hippocampus (HP), which identifies this structure as the principal cortical area for spatial working memory. Interestingly, H1aV overexpression also abolished maintenance of CA3-CA1 long-term potentiation (LTP). These impairments, generated by sustained high Homer1a levels, identify a requirement for long Homer forms in synaptic plasticity and temporal encoding of spatial memory

Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.P 28338 - Austrian Science Fund FWF; U01 MH105971 - NIMH NIH HHS; U01 MH114824 - NIMH NIH HHS; Howard Hughes Medical InstituteAccepted manuscrip

Dopamine neurons projecting to the posterior striatum form an anatomically distinct subclass

Combining rabies-virus tracing, optical clearing (CLARITY), and whole-brain light-sheet imaging, we mapped the monosynaptic inputs to midbrain dopamine neurons projecting to different targets (different parts of the striatum, cortex, amygdala, etc) in mice. We found that most populations of dopamine neurons receive a similar set of inputs rather than forming strong reciprocal connections with their target areas. A common feature among most populations of dopamine neurons was the existence of dense ‘clusters’ of inputs within the ventral striatum. However, we found that dopamine neurons projecting to the posterior striatum were outliers, receiving relatively few inputs from the ventral striatum and instead receiving more inputs from the globus pallidus, subthalamic nucleus, and zona incerta. These results lay a foundation for understanding the input/output structure of the midbrain dopamine circuit and demonstrate that dopamine neurons projecting to the posterior striatum constitute a unique class of dopamine neurons regulated by different inputs. DOI: http://dx.doi.org/10.7554/eLife.10032.00

Incomplete and Inaccurate Vocal Imitation after Knockdown of FoxP2 in Songbird Basal Ganglia Nucleus Area X

The gene encoding the forkhead box transcription factor, FOXP2, is essential for developing the full articulatory power of human language. Mutations of FOXP2 cause developmental verbal dyspraxia (DVD), a speech and language disorder that compromises the fluent production of words and the correct use and comprehension of grammar. FOXP2 patients have structural and functional abnormalities in the striatum of the basal ganglia, which also express high levels of FOXP2. Since human speech and learned vocalizations in songbirds bear behavioral and neural parallels, songbirds provide a genuine model for investigating the basic principles of speech and its pathologies. In zebra finch Area X, a basal ganglia structure necessary for song learning, FoxP2 expression increases during the time when song learning occurs. Here, we used lentivirus-mediated RNA interference (RNAi) to reduce FoxP2 levels in Area X during song development. Knockdown of FoxP2 resulted in an incomplete and inaccurate imitation of tutor song. Inaccurate vocal imitation was already evident early during song ontogeny and persisted into adulthood. The acoustic structure and the duration of adult song syllables were abnormally variable, similar to word production in children with DVD. Our findings provide the first example of a functional gene analysis in songbirds and suggest that normal auditory-guided vocal motor learning requires FoxP2

Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels

Silencing and Un-silencing of Tetracycline-Controlled Genes in Neurons

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (Ptets). We have discovered that stably integrated Ptet becomes functionally silenced in the majority of neurons when it is inactive during development. Ptet silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced Ptet gene silencing, possibly by inducing promoter accessibility

Serial two-photon tomography for automated ex vivo mouse brain imaging

Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders

Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence

Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors