Distinct subnetworks of the thalamic reticular nucleus.

The thalamic reticular nucleus (TRN), the major source of thalamic inhibition, regulates thalamocortical interactions that are critical for sensory processing, attention and cognition1-5. TRN dysfunction has been linked to sensory abnormality, attention deficit and sleep disturbance across multiple neurodevelopmental disorders6-9. However, little is known about the organizational principles that underlie its divergent functions. Here we performed an integrative study linking single-cell molecular and electrophysiological features of the mouse TRN to connectivity and systems-level function. We found that cellular heterogeneity in the TRN is characterized by a transcriptomic gradient of two negatively correlated gene-expression profiles, each containing hundreds of genes. Neurons in the extremes of this transcriptomic gradient express mutually exclusive markers, exhibit core or shell-like anatomical structure and have distinct electrophysiological properties. The two TRN subpopulations make differential connections with the functionally distinct first-order and higher-order thalamic nuclei to form molecularly defined TRN-thalamus subnetworks. Selective perturbation of the two subnetworks in vivo revealed their differential role in regulating sleep. In sum, our study provides a comprehensive atlas of TRN neurons at single-cell resolution and links molecularly defined subnetworks to the functional organization of thalamocortical circuits.

Differential excitatory vs inhibitory SCN expression at single cell level regulates brain sodium channel function in neurodevelopmental disorders.

The four voltage-gated sodium channels SCN1/2/3/8A have been associated with heterogeneous types of developmental disorders, each presenting with disease specific temporal and cell type specific gene expression. Using single-cell RNA sequencing transcriptomic data from humans and mice, we observe that SCN1A is predominantly expressed in inhibitory neurons. In contrast, SCN2/3/8A are profoundly expressed in excitatory neurons with SCN2/3A starting prenatally, followed by SCN1/8A neonatally. In contrast to previous observations from low resolution RNA screens, we observe that all four genes are expressed in both excitatory and inhibitory neurons, however, exhibit differential expression strength. These findings provide molecular evidence, at single-cell resolution, to support the hypothesis that the excitatory/inhibitory (E/I) neuronal expression ratios of sodium channels are important regulatory mechanisms underlying brain homeostasis and neurological diseases. Modulating the E/I expression balance within cell types of sodium channels could serve as a potential strategy to develop targeted treatment for NaV-associated neuronal developmental disorders.

Synthetic lethal screening in the mammalian central nervous system identifies Gpx6 as a modulator of Huntington's disease.

Huntington's disease, the most common inherited neurodegenerative disease, is characterized by a dramatic loss of deep-layer cortical and striatal neurons, as well as morbidity in midlife. Human genetic studies led to the identification of the causative gene, huntingtin. Recent genomic advances have also led to the identification of hundreds of potential interacting partners for huntingtin protein and many hypotheses as to the molecular mechanisms whereby mutant huntingtin leads to cellular dysfunction and death. However, the multitude of possible interacting partners and cellular pathways affected by mutant huntingtin has complicated efforts to understand the etiology of this disease, and to date no curative therapeutic exists. To address the general problem of identifying the disease-phenotype contributing genes from a large number of correlative studies, here we develop a synthetic lethal screening methodology for the mammalian central nervous system, called SLIC, for synthetic lethal in the central nervous system. Applying SLIC to the study of Huntington's disease, we identify the age-regulated glutathione peroxidase 6 (Gpx6) gene as a modulator of mutant huntingtin toxicity and show that overexpression of Gpx6 can dramatically alleviate both behavioral and molecular phenotypes associated with a mouse model of Huntington's disease. SLIC can, in principle, be used in the study of any neurodegenerative disease for which a mouse model exists, promising to reveal modulators of neurodegenerative disease in an unbiased fashion, akin to screens in simpler model organisms.

Enhancement of consolidated long-term memory by overexpression of protein kinase Mzeta in the neocortex.

Memories are more easily disrupted than improved. Many agents can impair memories during encoding and consolidation. In contrast, the armamentarium of potential memory enhancers is so far rather modest. Moreover, the effect of the latter appears to be limited to enhancing new memories during encoding and the initial period of cellular consolidation, which can last from a few minutes to hours after learning. Here, we report that overexpression in the rat neocortex of the protein kinase C isozyme protein kinase Mζ (PKMζ) enhances long-term memory, whereas a dominant negative PKMζ disrupts memory, even long after memory has been formed.

Boundary conditions for the maintenance of memory by PKMzeta in neocortex.

We report here that ZIP, a selective inhibitor of the atypical protein kinase C isoform PKMzeta, abolishes very long-term conditioned taste aversion (CTA) associations in the insular cortex of the behaving rat, at least 3 mo after encoding. The effect of ZIP is not replicated by a general serine/threonine protein kinase inhibitor that is relatively ineffective toward PKMzeta, is independent of the intensity of training and the perceptual quality of the taste saccharin (conditioned stimulus, CS), and does not affect the ability of the insular cortex to re-encode the same specific CTA association again. The memory trace is, however, insensitive to ZIP during or immediately after training. This implies that the experience-dependent cellular plasticity mechanism targeted by ZIP is established following a brief time window after encoding, consistent with the standard period of cellular consolidation, but then, once established, does not consolidate further to gain immunity to the amnesic agent. Hence, we conclude that PKMzeta is not involved in short-term CTA memory, but is a critical component of the cortical machinery that stores long- and very long-term CTA memories.

A Saccharomyces cerevisiae cell-based quantitative beta-galactosidase assay compatible with robotic handling and high-throughput screening.

Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-beta-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 microl yeast cell culture and with < 100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.

Rapid erasure of long-term memory associations in the cortex by an inhibitor of PKM zeta.

Little is known about the neuronal mechanisms that subserve long-term memory persistence in the brain. The components of the remodeled synaptic machinery, and how they sustain the new synaptic or cellwide configuration over time, are yet to be elucidated. In the rat cortex, long-term associative memories vanished rapidly after local application of an inhibitor of the protein kinase C isoform, protein kinase M zeta (PKMzeta). The effect was observed for at least several weeks after encoding and may be irreversible. In the neocortex, which is assumed to be the repository of multiple types of long-term memory, persistence of memory is thus dependent on ongoing activity of a protein kinase long after that memory is considered to have consolidated into a long-term stable form.