Sample collection protocol for single-cell RNA sequencing of functionally identified neuronal populations in vivo.

Here, we present a sample collection protocol for single-cell RNA sequencing of functionally identified neuronal populations in vivo with a virally delivered activity-dependent labeling tool (CaMPARI2). We describe steps for photoconversion in mice during the onset of computationally relevant events in a virtual reality environment, followed by removal and dissociation of the photo-labeled tissue, and separation of differentially labeled groups with fluorescence-activated cell sorting (FACS). We then detail procedures for characterizing and examining functionally relevant groups using standard bioinformatic techniques. For complete details on the use and execution of this protocol, please refer to O'Toole et al.1.

Molecularly targetable cell types in mouse visual cortex have distinguishable prediction error responses.

Predictive processing postulates the existence of prediction error neurons in cortex. Neurons with both negative and positive prediction error response properties have been identified in layer 2/3 of visual cortex, but whether they correspond to transcriptionally defined subpopulations is unclear. Here we used the activity-dependent, photoconvertible marker CaMPARI2 to tag neurons in layer 2/3 of mouse visual cortex during stimuli and behaviors designed to evoke prediction errors. We performed single-cell RNA-sequencing on these populations and found that previously annotated Adamts2 and Rrad layer 2/3 transcriptional cell types were enriched when photolabeling during stimuli that drive negative or positive prediction error responses, respectively. Finally, we validated these results functionally by designing artificial promoters for use in AAV vectors to express genetically encoded calcium indicators. Thus, transcriptionally distinct cell types in layer 2/3 that can be targeted using AAV vectors exhibit distinguishable negative and positive prediction error responses.

A transcriptional constraint mechanism limits the homeostatic response to activity deprivation in mammalian neocortex.

Healthy neuronal networks rely on homeostatic plasticity to maintain stable firing rates despite changing synaptic drive. These mechanisms, however, can themselves be destabilizing if activated inappropriately or excessively. For example, prolonged activity deprivation can lead to rebound hyperactivity and seizures. While many forms of homeostasis have been described, whether and how the magnitude of homeostatic plasticity is constrained remains unknown. Here, we uncover negative regulation of cortical network homeostasis by the PARbZIP family of transcription factors. In cortical slice cultures made from knockout mice lacking all three of these factors, the network response to prolonged activity withdrawal measured with calcium imaging is much stronger, while baseline activity is unchanged. Whole-cell recordings reveal an exaggerated increase in the frequency of miniature excitatory synaptic currents reflecting enhanced upregulation of recurrent excitatory synaptic transmission. Genetic analyses reveal that two of the factors, Hlf and Tef, are critical for constraining plasticity and for preventing life-threatening seizures. These data indicate that transcriptional activation is not only required for many forms of homeostatic plasticity but is also involved in restraint of the response to activity deprivation.

The human brain is made up of billions of nerve cells called neurons which receive and send signals to one another. To avoid being over- or under-stimulated, neurons can adjust the strength of the inputs they receive by altering how connected they are to other nerve cells. This process, known as homeostatic plasticity, is thought to be necessary for normal brain activity as it helps keep the outgoing signals of neurons relatively constant. However, homeostatic plasticity can lead to seizures if it becomes too strong and overcompensates for weak input signals. Regulating this process is therefore central to brain health, but scientists do not understand if or how it is controlled. To address this, Valakh et al. analyzed the genes activated in neurons lacking incoming signals to find proteins that regulate homeostatic plasticity. This revealed a class of molecules called transcription factors (which switch genes on or off) that constrain the process. In brain samples from mice without these regulatory proteins, neurons received twice as much input, leading to an increase in brain activity resembling that observed during seizures. Valakh et al. confirmed this finding using live mice, which developed seizures in the absence of these transcription factors. These findings suggest that this type of regulation to keep homeostatic plasticity from becoming too strong may be important. This could be especially vital as the brain develops, when the strength of connections between neurons changes rapidly. The discovery of the transcription factors involved provides a potential target for activating or restraining homeostatic plasticity. This control could help researchers better understand how the process stabilizes brain signaling.

NMDA receptors in visual cortex are necessary for normal visuomotor integration and skill learning.

The experience of coupling between motor output and visual feedback is necessary for the development of visuomotor skills and shapes visuomotor integration in visual cortex. Whether these experience-dependent changes of responses in V1 depend on modifications of the local circuit or are the consequence of circuit changes outside of V1 remains unclear. Here, we probed the role of N-methyl-d-aspartate (NMDA) receptor-dependent signaling, which is known to be involved in neuronal plasticity, in mouse primary visual cortex (V1) during visuomotor development. We used a local knockout of NMDA receptors and a photoactivatable inhibition of CaMKII in V1 during the first visual experience to probe for changes in neuronal activity in V1 as well as the influence on performance in a visuomotor task. We found that a knockout of NMDA receptors before, but not after, first visuomotor experience reduced responses to unpredictable stimuli, diminished the suppression of predictable feedback in V1, and impaired visuomotor skill learning later in life. Our results demonstrate that NMDA receptor-dependent signaling in V1 is critical during the first visuomotor experience for shaping visuomotor integration and enabling visuomotor skill learning.

Explanatory role of sociodemographic, clinical, behavioral, and social factors on cognitive decline in older adults with diabetes.

BACKGROUND: The aim of the study was to examine the explanatory role of sociodemographic, clinical, behavioral, and social factors on racial/ethnic differences in cognitive decline among adults with diabetes. METHODS: Adults aged 50+ years with diabetes from the Health and Retirement Survey were assessed for cognitive function (normal, mild cognitive impairment [MCI], and dementia). Generalized estimating equation (GEE) logistic regression models were used to account for repeating measures over time. Models were adjusted for sociodemographic (gender, age, education, household income and assets), behavioral (smoking), clinical (ie. comorbidities, body mass index), and social (social support, loneliness, social participation, perceived constraints and perceived mastery on personal control) factors. RESULTS: Unadjusted models showed non-Hispanic Blacks (NHB) and Hispanics were significantly more likely to progress from normal cognition to dementia (NHB OR: 2.99, 95%CI 2.35-3.81; Hispanic OR: 3.55, 95%CI 2.77-4.56), and normal cognition to MCI (NHB OR = 2.45, 95%CI 2.14-2.82; Hispanic OR = 2.49, 95%CI 2.13-2.90) compared to non-Hispanic Whites (NHW). Unadjusted models for the transition from mild cognitive decline to dementia showed Hispanics were more likely than NHW to progress (OR = 1.43, 95%CI 1.11-1.84). After adjusting for sociodemographic, clinical/behavioral, and social measures, NHB were 3.75 times more likely (95%CI 2.52-5.56) than NHW to reach dementia from normal cognition. NHB were 2.87 times more likely (95%CI 2.37-3.48) than NHW to reach MCI from normal. Hispanics were 1.72 times more likely (95%CI 1.17-2.52) than NHW to reach dementia from MCI. CONCLUSION: Clinical/behavioral and social factors did not explain racial/ethnic disparities. Racial/ethnic disparities are less evident from MCI to dementia, emphasizing preventative measures/interventions before cognitive impairment onset are important.

Dicer maintains the identity and function of proprioceptive sensory neurons.

Neuronal cell identity is established during development and must be maintained throughout an animal's life (Fishell G, Heintz N. Neuron 80: 602-612, 2013). Transcription factors critical for establishing neuronal identity can be required for maintaining it (Deneris ES, Hobert O. Nat Neurosci 17: 899-907, 2014). Posttranscriptional regulation also plays an important role in neuronal differentiation (Bian S, Sun T. Mol Neurobiol 44: 359-373, 2011), but its role in maintaining cell identity is less established. To better understand how posttranscriptional regulation might contribute to cell identity, we examined the proprioceptive neurons in the dorsal root ganglion (DRG), a highly specialized sensory neuron class, with well-established properties that distinguish them from other neurons in the ganglion. By conditionally ablating Dicer in mice, using parvalbumin (Pvalb)-driven Cre recombinase, we impaired posttranscriptional regulation in the proprioceptive sensory neuron population. Knockout (KO) animals display a progressive form of ataxia at the beginning of the fourth postnatal week that is accompanied by a cell death within the DRG. Before cell loss, expression profiling shows a reduction of proprioceptor specific genes and an increased expression of nonproprioceptive genes normally enriched in other ganglion neurons. Furthermore, although central connections of these neurons are intact, the peripheral connections to the muscle are functionally impaired. Posttranscriptional regulation is therefore necessary to retain the transcriptional identity and support functional specialization of the proprioceptive sensory neurons.NEW & NOTEWORTHY We have demonstrated that selectively impairing Dicer in parvalbumin-positive neurons, which include the proprioceptors, triggers behavioral changes, a lack of muscle connectivity, and a loss of transcriptional identity as observed through RNA sequencing. These results suggest that Dicer and, most likely by extension, microRNAs are crucially important for maintaining proprioception. Additionally, this study hints at the larger question of how neurons maintain their functional and molecular specificity.

Regulation of ECM degradation and axon guidance by growth cone invadosomes.

Invadopodia and podosomes, collectively referred to as invadosomes, are F-actin-rich basal protrusions of cells that provide sites of attachment to and degradation of the extracellular matrix. Invadosomes promote the invasion of cells, ranging from metastatic cancer cells to immune cells, into tissue. Here, we show that neuronal growth cones form protrusions that share molecular, structural and functional characteristics of invadosomes. Growth cones from all neuron types and species examined, including a variety of human neurons, form invadosomes both in vitro and in vivo. Growth cone invadosomes contain dynamic F-actin and several actin regulatory proteins, as well as Tks5 and matrix metalloproteinases, which locally degrade the matrix. When viewed using three-dimensional super-resolution microscopy, F-actin foci often extended together with microtubules within orthogonal protrusions emanating from the growth cone central domain. Finally, inhibiting the function of Tks5 both reduced matrix degradation in vitro and disrupted motoneuron axons from exiting the spinal cord and extending into the periphery. Taken together, our results suggest that growth cones use invadosomes to target protease activity during axon guidance through tissues.

Abelson interactor-1 (ABI-1) interacts with MRL adaptor protein MIG-10 and is required in guided cell migrations and process outgrowth in C. elegans.

Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two-hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-immunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, our results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth.