Molecular profiling of activated neurons by phosphorylated ribosome capture - PubMed (original) (raw)

Molecular profiling of activated neurons by phosphorylated ribosome capture

Zachary A Knight et al. Cell. 2012.

Abstract

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Phosphorylated Ribosome Profiling

(A) Neurotransmitters and neuromodulators activate a core set of signaling pathways in neurons. Rps6 is a common target of these pathways and is phos-phorylated on five C-terminal residues. (B) Asparse subpopulationofneuronsis activated in response a stimulus (red cells). Activated neurons show enhanced pS6, and thus, capture of phosphorylated ribosomes enriches for the mRNA expressed in the activated cells. Quantifying the enrichment (IP/input) of a large panel of cell-type-specific marker genes reveals the genes uniquely expressed in the neurons that were activated.

Figure 2

Figure 2. Colocalization between c-Fos and pS6 following Diverse Stimuli

(A) Response to the drugs kainate (pS6 235/236), cocaine (pS6 244), clozapine (pS6 235/236), and olanzapine (pS6 235/236). (B) Response to stimuli that induce defensive behavior or aggression, including introduction of an intruder (pS6 244) or exposure to a worn cat collar (pS6 244). (C) Response to nutritional stimuli, including dehydration (pS6 244), salt challenge (pS6 235/236), overnight fast (pS6 244), and ghrelin (pS6 235/236). (D) Response inthe suprachiasmatic nucleus (SCN) following light stimulation at the end of the dark phase (pS6 235/236). Inset region is shown in the second row. All scale bars, 50 mm except kainate (100 µm). See also Figure S1.

Figure 3

Figure 3. Selective Capture of Phosphorylated Ribosomes In Vitro and In Vivo

(A) Western blot for ribosomal proteins from wild-type or S6S5A MEFs. Input lysate (left) and the pS6 240/244 immunoprecipitate (right) are shown. (B) RNA yield from pS6 240/244 immunoprecipitates from wild-type and S6S5A MEFs. (C) Bioanalyzer analysis of immunoprecipitated RNA from wild-type and S6S5A MEFs. 18S and 28S ribosomal RNA are labeled. FU, fluorescence units. (D) Colocalization of MCH and pS6 in the hypo-thalamus of Tsc1fl/fl and MCHCre Tsc1fl/fl mice. Scale bar, 50 mm. (E) Left: pS6 244 immunostaining density in MCH neurons following Tsc1 deletion. Right: volume of MCH neurons following Tsc1 deletion. (F) Five major S6 phosphorylation sites and the diphospho-motifs recognized by commonly used pS6 antibodies. (G) Enrichment of a panel of cell-type-specific genes following immunoprecipitation with antibodies recognizing pS6 240/244 or only pS6 244. Black bars represent immunoprecipitates from hypothalamic homogenates of MCHCre Tsc1fl/fl mice, whereas white bars represent Tsc1fl/fl controls. The enrichment of cell-type-specific genes in pS6 immunoprecipitates was determined by Taqman. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using two-tailed unpaired t test. All error bars are mean ±SEM. See also Figure S2.

Figure 4

Figure 4. Identification of Neurons Activated by Salt Challenge

(A) Hypothalamic staining for pS6 244 from mice given an injection of vehicle (PBS) or 2 M NaCl. (B) Differential enrichment of cell-type-specific genes in pS6 immunoprecipitates determined by Taqman. Data are expressed as the ratio of fold enrichment (IP/input) for salt-treated animals divided by the fold enrichment (IP/input) for controls and plotted on a log scale. Genes with a fold enrichment > 3.0 and p < 0.05 are labeled. (C) Fold enrichment (IP/input) for a panel of activity-dependent genes following osmotic stimulation. Actin is shown for reference. (D) RNA-seq analysis of differential enrichment of cell-type-specific genes in pS6 immunoprecipitates. Inset, magnification of region (blue) showing >4-fold enrichment. Genes labeled in (B) are highlighted. (E) Colocalization between Avp, Oxt, and Crh with pS6 244 in salt-treated and control animals. Crh neurons were analyzed as two separate populations in the rostral and caudal PVN. (F) pS6 intensity within individual Avp, Oxt, and Crh neurons from salt-treated and control animals. (G) Colocalization between FosB, Cxcl1, and pS6 in salt-treated and control animals. (H) Percentage of FosB-positive cells in the PVN and SON that are also pS6 positive. (I) Percentage of pS6-positive cells in the PVN and SON that are also FosB positive. All scale bars, 50 mm except (A) (200 mm). All error bars are mean ±SEM. See also Figure S3.

Figure 5

Figure 5. Identification of Neurons Activated by Fasting

(A) Hypothalamic staining for pS6 244 from fasted and fed mice. Top panels show the arcuate nucleus and DMH, and bottom panels show the preoptic area (highlighted). (B) Relative enrichment of cell-type-specific genes in pS6 immunoprecipitates from fasted and fed animals. Data are expressed as the ratio of fold enrichment (IP/input) for fasted animals divided by the fold enrichment (IP/input) for fed controls and are plotted on a log scale. Genes with fold enrichment >2.5 and p < 0.05 are labeled. (C) Left: colocalization between AgRP and pS6 244 in fed and fasted mice. Right: pS6 intensity in AgRP neurons. (D) Left: colocalization between POMC and pS6 in fed and fasted mice. Right: pS6 intensity in POMC neurons. (E) Colocalization between Gal and pS6 in fed and fasted mice in the MPA and DMH. (F) Colocalization between Gal and c-Fos in fed and fasted mice in the MPA and DMH. All scale bars, 50 mm except (A) (100 mm). All error bars are mean ±SEM. See also Figure S4.

Figure 6

Figure 6. Identification of Neurons Activated by Ghrelin and Scheduled Feeding

(A) Hypothalamic staining for pS6 244 in response to ghrelin (intraperitoneal injection, 1 hr) or scheduled feeding (2 hr following food presentation). (B) pS6 staining in the DMH in mice acclimated to a protocol of scheduled feeding between circadian time (CT) 4–7. Mice were either fed (top) or not fed (bottom) on the day of the experiment. (C) Number of pS6-positive cells in the DMH (left) and Arc (right) in mice on a scheduled feeding protocol. Black, fed on the day of the experiment. Red, not fed. (D) Differential enrichment of cell-type-specific transcripts in pS6 IPs from mice that were given ghrelin (y axis) or subjected to scheduled feeding (x axis). Data are expressed as the ratio of fold enrichment (IP/input) from ghrelin or scheduled feeding animals relative to the fold enrichment of their controls and are plotted on a log scale. Key genes are labeled. (E) Colocalization between NPY and pS6 in ad libitum, ghrelin-treated, and scheduled feeding mice. (F) Expression of Pdyn in the hypothalamus and its colocalization with pS6 in mice subjected to scheduled feeding and sacrificed at CT6. The Arc, DMH, and LH are labeled for reference. (G) Colocalization between Pdyn and pS6 in various hypothalamic nuclei of mice fed ad libitum or subjected to scheduled feeding and sacrificed at CT6. (H) Colocalization between Pdyn and pS6 in the DMH of ad libitum and scheduled feeding. (I) Colocalization between Pdyn and c-fos at CT6 in mice subjected to scheduled feeding. Scale bars, 50 mm except (A) and (F) (100 µm). All error bars are mean ±SEM. See also Figure S5 and Movies S1, S2, and S3.

Figure 7

Figure 7. KOR Signaling Restrains Food Intake during Scheduled Feeding

Mice were treated with KOR inhibitors (red) or vehicle (black) by central or peripheral injection, and their food intake and body weight were recorded during ad libitum or scheduled feeding (gray). (A) Mice givenanintraperitoneal injection ofthe KOR antagonistJDTic (red) orvehicle (black) and switched from adlibitum toscheduled feedingon day 0.p=0.01 for the difference in cumulative food intake on days 2–7. p = 0.055 for the body weight difference for days 4–7. (B) Mice given an intraperitoneal injection of JDTic (red) or vehicle (black) and maintained on an ad libitum diet. (C) Mice given an icv injection ofthe KOR antagonist norbinaltorphimine (red)orvehicle (black). Mice were switched from adlibitum toscheduled feedingonday 0. p < 0.01 for the difference in food intake for days 2–5 by t test. p < 0.02 for difference in body weight for days 5–8. (D) Mice given an icv injection of the norbinaltorphimine (red) or vehicle (black) and fed ad libitum. All error bars are mean ±SEM; p values calculated by two-tailed unpaired t test.

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