Corticotropin-releasing factor (CRF) that is released from the paraventricular nucleus (PVN) of the hypothalamus is essential for mediating stress response by activating the hypothalamic–pituitary–adrenal axis. CRF-releasing PVN neurons receive inputs from multiple brain regions that convey stressful events, but their neuronal dynamics on the timescale of behavior remain unknown. Here, our recordings of PVN CRF neuronal activity in freely behaving mice revealed that CRF neurons are activated immediately by a range of aversive stimuli. By contrast, CRF neuronal activity starts to drop within a second of exposure to appetitive stimuli. Optogenetic activation or inhibition of PVN CRF neurons was sufficient to induce a conditioned place aversion or preference, respectively. Furthermore, conditioned place aversion or preference induced by natural stimuli was significantly decreased by manipulating PVN CRF neuronal activity. Together, these findings suggest that the rapid, biphasic responses of PVN CRF neurons encode the positive and negative valences of stimuli.
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We are grateful to K. Narasimhan, L. Vendruscolo, and members of the Suh laboratory for critical comments on the manuscript. We also thank G. Schwartz, S. Han, J. Kim, and A. Watts for discussions of this work. We appreciate W. Jung for assisting with immunohistochemistry and data analysis, and the laboratory of the late W. Vale for providing an aliquot of anti-CRF antibody. This work is supported by the TJ Park Science Fellowship of the POSCO TJ Park Foundation and the KAIST Innovative Doctoral Research Fellowship to J.K., NIH grants (R01MH101377 to D.L. and R01DK106636 to G.S.B.S.), and KAIST Chancellor’s fund to G.S.B.S. and J.W.S.
The authors declare no competing interests.
Journal peer review information: Nature Neuroscience thanks Stephen Mahler and other anonymous reviewer(s) for their contribution to the peer review of this work.
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Integrated supplementary information
Supplementary Figure 1 PVN-CRFGCaMP6 signal is increased by the onset and is decreased by the offset of exposure to aversive stimuli.
a–c, PETH plot (upper) and heat map (bottom) from a representative animal shown in Fig. 1d to the onset of TRT (a), in Fig. 1f to the onset of overhead object (b), and in Fig. 1f’ to the onset of visual looming disk (c). e–g, PETH plot (upper) and heat map (bottom) for population activity across animals to the offset of TRT (e), the offset of overhead object (f), and the offset of visual looming disk (g). d,h, PETH plot (upper) and heat map (bottom) from a representative animal shown in Fig. 1f’, aligned to the onset of flight behavior (d) and population activity across animals (h). Data are presented as mean (solid line) ± s.e.m (shaded area). Related to Fig. 1.
Supplementary Figure 2 The changes in ∆F/F of PVN-CRFGFP signal in response to environmental stimuli were not due to a movement artifact.
a,b, Left: a representative trace illustrating no change in the normalized PVN-CRFGFP signal during FST (a) or TRT (b). Right: bar graph illustrating average ΔF/F of PVN-CRFGFP signals during FST (a) and TRT (b) (N = 5 mice). This control experiment was conducted to ascertain the validity of increased PVN CRF neuronal activity in response to stressors that caused movements. Data are presented as mean ± s.e.m. Related to Fig. 1.
Supplementary Figure 3 PVN-CRFGCaMP6 signal is increased by a flying object presented from above, but not by objects presented from either side or from below.
a, Schematic for a ‘flying’ object from above and side (left). Bar graph showing average ΔF/F of PVN-CRFGCaMP6 in mice that were exposed to the flying object from above (N = 7 mice) and side (N = 4 mice) (right). b, Schematic for a looming shadow disk (left). Bar graph showing average ΔF/F of PVN-CRFGCaMP6 in mice that were exposed to the disk from above, side, or bottom (right) (N = 6 mice). Mann–Whitney two-tailed U-test in a and paired two-tailed t-test in b. *P < 0.05, **P < 0.01. See Supplementary Table 1 for detailed description of statistics for this figure and subsequent figures. Data are presented as mean ± s.e.m. Related to Fig. 1.
Supplementary Figure 4 PVN-CRFGCaMP6 signals and c-Fos induction in PVN CRF neurons after periods of starvation.
a,b, Bar graphs summarizing the levels of the baseline intracellular calcium (a) and the frequencies of calcium transients in PVN-CRFGCaMP6 mice when fed, 22 h fasted, and refed with chow (b) (N = 7). c,d, A representative confocal image of PVN in mice fasted for 22 h (c) or for 9 h (d) immunostained with anti-CRF (red) and anti-c-Fos (green) antibodies. Similar results were independently obtained in three other mice for 22 h fasted and 3 mice for 9 h fasted. Scale bar, 50 μm. One-way ANOVA test with Holm–Sidak post hoc analysis. *P < 0.05, **P < 0.01. Data are presented as mean ± s.e.m. Related to Fig. 2.
Supplementary Figure 5 PVN-CRFGCaMP6 signals decrease when mice investigate and consume food for the first time.
a–f, PETH plot of population activity across animals, for the first bout and all other bouts to the onset of investigation of a mesh-covered cup with object (a), chow pellet (c), or peanut butter (e); investigation of object (b); and consumption of chow pellet (d) and peanut butter (f) in fed (left) or fasted (right) mice (N = 11). Data are presented as mean ± s.e.m. Related to Fig. 2.
Supplementary Figure 6 The frequency of calcium transients decreases during the presentation of food or a pup.
a,c, Representative traces with denoted calcium transients (red dots) of raw PVN-CRFGCaMP6 signals in a mouse that was fed (left) or fasted (right) when exposed to object or chow (a); and in a male (left) or a female (right) when exposed to a pup (c). Blue line denotes the moment at which chow (a) or a pup (c) was introduced. b,d, Bar graph summarizing the frequency of calcium transients of PVN-CRFGCaMP6 signals during the presentation of object and chow (N = 11 mice) (b); before and after pups were introduced to male (N = 12 mice) or female mice (N = 8 mice) (d). One-way ANOVA test with Holm–Sidak post hoc analysis in b and paired two-tailed t-test in d. *P < 0.05, **P < 0.01. Data are presented as mean ± s.e.m. Related to Figs. 1 and 3.
Supplementary Figure 7 PVN-CRFGCaMP6 signals decrease when female mice approach and interact with pups for the first time.
a,b, PETH plots for population activity across animals to the onset of approach to pups (a) and interaction with pups (b) by male (left, N = 12) or female (right, N = 8) mice. First bout and all other bouts are overlaid. c, Representative traces illustrating no change in the normalized PVN-CRFGCaMP6 signals in a male (left) or a female (right) during the presence of a fake animal. Shaded bars depict the epochs during which the recorded mouse approached toward the fake animal (light gray) and interaction with the fake animal (light purple). d,e, PETH plots for population activity across animals to the onset of approach to fake animals (d) and interaction with fake animals (e) by male (left, N = 12) and female (right, N = 7) mice. Data are presented as mean ± s.e.m. Related to Fig. 3.
Supplementary Figure 8 PVN-CRFGCaMP6 signals of recorded female mice while being aggressed on by CD1 female aggressor.
a, A representative trace illustrating acute surge in PVN-CRFGCaMP6 signal in a female mouse to the onsets of being investigated (light gray) or being attacked (red) by a female aggressor, and being introduced or removed from the arena (gray). b, PETH plot across trials of being attacked from the representative trace (n = 10 trials). Similar results were independently obtained by four other female mice. Data are presented as mean ± s.e.m. Related to Fig. 3.
A population PETH plot from recorded mice during investigation with aggressive intruders that does not involve attack (left) and bar graph that measures the changes in ∆F/F of PVN-CRFGCaMP6 signal (right) (N = 5 mice). Paired two-tailed t-test. P = 0.11. Data are presented as mean ± s.e.m. Related to Fig. 3.
Supplementary Figure 10 Optogenetic activation of PVN CRF neurons of food-restricted mice reduces preference for food and for food-paired chamber.
a–e, Time spent in eating normalized to time spent in the food-paired chamber (%) (a), the number of eating bouts (b), the duration of eating bout (c), probability of initiating eating when mice are in the food-paired chamber (%) (d), and cumulative bouts from days 3, 4, and 5 (e) in PVN-CRFChR2 mice (blue, N = 12) and control PVN-CRFeYFP (gray, N = 9) mice. Two-way ANOVA test with Holm–Sidak post hoc analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± s.e.m. Related to Fig. 6.
Supplementary Video 1 GCaMP6 signal by visual looming disk. A recorded mouse is exposed to the visual looming shadow disk presented from the top. When exposed to the visual disk, the mouse quickly move to a nesting area. The synchronized PVN-CRFGCaMP6 trace is shown below. The real-time activity of PVN-CRFGCaMP6 is shown on the right. Related to Fig. 1.
Supplementary Video 2 GCaMP6 signal by consuming peanut butter in a fed mouse. A recorded fed mouse approaches and consumes peanut butter. The synchronized PVN-CRFGCaMP6 trace is shown below. The real-time activity of PVN-CRFGCaMP6 is shown on the right. Related to Fig. 2.
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Kim, J., Lee, S., Fang, YY. et al. Rapid, biphasic CRF neuronal responses encode positive and negative valence. Nat Neurosci 22, 576–585 (2019). https://doi.org/10.1038/s41593-019-0342-2
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