Probes for INS

Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expressionnetworks that regulate synapse development and plasticity. These networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.

Fluorescent detection of transcripts using RNAscope has quickly become a standard in situ hybridization (ISH) approach in neuroscience with over 400 publications since its introduction in 2012. RNAscope's sensitivity and specificity allow the simultaneously detection of up to three low abundance mRNAs in single cells (i.e., multiplexing) and, in contrast to other ISH techniques, RNAscope is performed in 1 day. BaseScope, a newer ultrasensitive platform, uses improved amplification chemistry of single oligonucleotide probe pairs (∼50 bases). This technique allows discrimination of single nucleotide polymorphisms or splice variants that differ by short exons. A present limitation of BaseScope is that expression analysis is limited to a single gene (i.e., single-plexing). This article outlines detailed protocols for both RNAscope and BaseScope in neuronal tissue. We discuss how to perform ISH experiments using either fresh-frozen or formalin-fixed paraffin-embedded sections, as well as dissociated cultured neurons. We also outline how to obtain quantitative data from hybridized tissue sections.

Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease, is lacking. To investigate the breadth of transcriptional changes that occur across cell types in the mouse visual cortex after exposure to light, we applied high-throughput single-cell RNA sequencing. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, thus revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibited inter- and intralaminar heterogeneity in the induction of stimulus-responsive genes. Non-neuronal cells showed clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of the stimulus-dependent transcriptional changes occurring across cell types in the visual cortex; these changes are probably critical for cortical function and may be sites of deregulation in developmental brain disorders.

Acetylcholine is a potent neuromodulator capable of modifying patterns of acoustic information flow. In auditory cortex, cholinergic systems have been shown to increase salience/gain while suppressing extraneous information. However, the mechanism by which cholinergic circuits shape signal processing in the auditory thalamus (medial geniculate body, MGB) is poorly understood. The present study, in male Fischer Brown Norway rats, seeks to determine the location and function of presynaptic neuronal nicotinic acetylcholine receptors (nAChRs) at the major inputs to MGB and characterize how nAChRs change during aging. In vitro electrophysiological/optogenetic methods were used to examine responses of MGB neurons following activation of nAChRs during a paired-pulse paradigm. Presynaptic nAChR activation increased responses evoked by stimulation of excitatory corticothalamic and inhibitory tectothalamic terminals. Conversely, nAChR activation appeared to have little effect on evoked responses from inhibitory thalamic reticular nucleus and excitatory tectothalamic terminals. In situ hybridization data showed nAChR subunit transcripts in GABAergic inferior colliculus neurons and glutamatergic auditory cortical neurons supporting the present slice findings. Responses to nAChR activation at excitatory corticothalamic and inhibitory tectothalamic inputs were diminished by aging. These findings suggest that cholinergic input to the MGB increases the strength of tectothalamic inhibitory projections, potentially improving signal-to-noise ratio and signal detection while increasing corticothalamic gain, which may facilitate top-down identification of stimulus identity. These mechanisms appear negatively affected by aging, potentially diminishing speech perception in noisy environments. Cholinergic inputs to the MGB appear to maximize sensory processing by adjusting both top-down and bottom-up mechanisms in conditions of attention and arousal.Significance StatementThe pedunculopontine tegmental nucleus (PPTg) is the source of cholinergic innervation for sensory thalamus and is a critical part of an ascending arousal system which controls the firing mode of thalamic cells based on attentional demand. The present study describes the location and impact of aging on presynaptic neuronal nicotinic receptors (nAChRs) within the circuitry of the auditory thalamus (medial geniculate body; MGB). We show that nAChRs are located on ascending inhibitory and descending excitatory presynaptic inputs onto MGB neurons, likely selectively increasing gain and improving temporal clarity. In addition, we show that aging has a deleterious effect on nAChR efficacy. Cholinergic dysfunction at the level of MGB may negatively impact speech understanding in the elderly population.

Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.