TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex.

Thiouracil (TU)-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4TU. This technique has been applied in mouse using a Cre-dependent UPRT transgene, CA>GFPstop>HA-UPRT, to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood-brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here, we crossed the CA>GFPstop>HA-UPRT transgenic mouse to a Sepw1-cre line to express UPRT in layer 2/3 of visual cortex or to an Nr5a1-cre line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day (P)12, as well as from wild-type (WT) mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here, we show that Cre-dependent UPRT expression can be used to purify cell type-specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the BBB to label RNA in vivo.

Perceptual gap detection is mediated by gap termination responses in auditory cortex.

BACKGROUND: Understanding speech in the presence of background noise often becomes increasingly difficult with age. These age-related speech processing deficits reflect impairments in temporal acuity. Gap detection is a model for temporal acuity in speech processing in which a gap inserted in white noise acts as a cue that attenuates subsequent startle responses. Lesion studies have shown that auditory cortex is necessary for the detection of brief gaps, and auditory cortical neurons respond to the end of the gap with a characteristic burst of spikes called the gap termination response (GTR). However, it remains unknown whether and how the GTR plays a causal role in gap detection. We tested this by optogenetically suppressing the activity of somatostatin- or parvalbumin-expressing inhibitory interneurons, or CaMKII-expressing excitatory neurons, in auditory cortex of behaving mice during specific epochs of a gap detection protocol. RESULTS: Suppressing interneuron activity during the postgap interval enhanced gap detection. Suppressing excitatory cells during this interval attenuated gap detection. Suppressing activity preceding the gap had the opposite behavioral effects, whereas prolonged suppression across both intervals had no effect on gap detection. CONCLUSIONS: In addition to confirming cortical involvement, we demonstrate here for the first time a causal relationship between postgap neural activity and perceptual gap detection. Furthermore, our results suggest that gap detection involves an ongoing comparison of pre- and postgap spiking activity. Finally, we propose a simple yet biologically plausible neural circuit that reproduces each of these neural and behavioral results.

Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons.

Understanding how neural circuits work requires a detailed knowledge of cellular-level connectivity. Our current understanding of neural circuitry is limited by the constraints of existing tools for transsynaptic tracing. Some of the most intractable problems are a lack of cellular specificity of uptake, transport across multiple synaptic steps conflating direct and indirect inputs, and poor labeling of minor inputs. We used a novel combination of transgenic mouse technology and a recently developed tracing system based on rabies virus (Wickersham et al., 2007a,b) to overcome all three constraints. Because the virus requires transgene expression for both initial infection and subsequent retrograde transsynaptic infection, we created several lines of mice that express these genes in defined cell types using the tetracycline-dependent transactivator system (Mansuy and Bujard, 2000). Fluorescent labeling from viral replication is thereby restricted to defined neuronal cell types and their direct monosynaptic inputs. Because viral replication does not depend on transgene expression, it provides robust amplification of signal in presynaptic neurons regardless of input strength. We injected virus into transgenic crosses expressing the viral transgenes in specific cell types of the hippocampus formation to demonstrate cell-specific infection and monosynaptic retrograde transport of virus, which strongly labels even minor inputs. Such neuron-specific transgenic complementation of recombinant rabies virus holds great promise for obtaining cellular-resolution wiring diagrams of the mammalian CNS.