Postnatal day 2 to 11 constitutes a 5-HT-sensitive period impacting adult mPFC function

Abstract

Early-life serotonin [5-hydroxytryptamine (5-HT)] signaling modulates brain development, which impacts adult behavior, but 5-HT-sensitive periods, neural substrates, and behavioral consequences remain poorly understood. Here we identify the period ranging from postnatal day 2 (P2) to P11 as 5-HT sensitive, with 5-HT transporter (5-HTT) blockade increasing anxiety- and depression-like behavior, and impairing fear extinction learning and memory in adult mice. Concomitantly, P2-P11 5-HTT blockade causes dendritic hypotrophy and reduced excitability of infralimbic (IL) cortex pyramidal neurons that normally promote fear extinction. By contrast, the neighboring prelimbic (PL) pyramidal neurons, which normally inhibit fear extinction, become more excitable. Excitotoxic IL but not PL lesions in adult control mice reproduce the anxiety-related phenotypes. These findings suggest that increased 5-HT signaling during P2-P11 alters adult mPFC function to increase anxiety and impair fear extinction, and imply a differential role for IL and PL neurons in regulating affective behaviors. Together, our results support a developmental mechanism for the etiology and pathophysiology of affective disorders and fear-related behaviors.

Keywords: SSRI; anxiety; development; mouse; prefrontal cortex; serotonin.

Figure 1.

PN-FLX treatment from either P2 to P21 or P2 to P11 enhances anxiety and depression-like behaviors in adult mice. A–C, In the novel OF test, FLX treatment from P2 to P21 or P2 to P11 results in significantly reduced time ambulating (A) and rearing (B); FLX treatment from P2 to P11 also results in significantly reduced time in the center region of the chamber (C). D–F, In the NSF test, FLX treatment from P2 to P21 or P2 to P11 significantly increases the latency to feed (D), while the percentage weight loss after 24 h food deprivation (E) and home-cage food consumption (F) are not affected by treatment. G–I, In the SESC test, FLX treatment from P2 to P21 or P2 to P11 results in significantly increased latency to escape (G) and suppressed activity between shocks (H), but does not affect activity during the pretest habituation phase (I). N values are indicated in the figure. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

PN-FLX from P2 to P11 increases depression-like behavior in the SPT and FST. A, In the SPT, FLX treatment from P2 to P11 significantly reduced sucrose consumption. B, In the FST, FLX treatment from P2 to P11 results in significantly increased time spent immobile. N values are indicated in the figure. *p < 0.05.

Figure 3.

PN-FLX treatment alters dendritic morphology of IL pyramidal neurons. A–D, IL neurons from PN-FLX mice have significantly less complex apical dendritic trees, relative to controls, as evidenced by a decrease in the number of intersections (A), number of nodes (B), and dendritic material (C), with no overall decrease in average apical arbor length (D). E, Decreased complexity was most pronounced in the proximal to medial region of the soma. F, A schematic of altered dendritic morphology. N = 3 mice/group; N values for cells per condition are indicated in the figure. *p < 0.05; **p < 0.01.

Figure 4.

PN-FLX treatment results in concurrent and opposite changes in IL and PL neuronal excitability. Input–output curves displaying frequency (in Hz) of spikes (action potentials) generated in response to increasing current injections (increased by 50 pA per step), from resting membrane potential. A, IL pyramidal neurons from PN-FLX mice display a significantly lower frequency of spikes in response current steps relative to PN-VEH controls. B, Conversely, prelimbic neurons from PN-FLX mice display significantly increased frequency of spikes in response to current injections. N = 3–6 mice/treatment; N values for cells per condition are indicated in the figure. **p < 0.01; ***p < 0.001.

Figure 5.

Normal sEPSCs in IL and PL pyramidal neurons of PN-FLX mice. A, B, PN-FLX treatment does not alter the frequency (A) or amplitude (B) of sEPSCs in IL (left) or PL (right) pyramidal neurons. N = 3–6 mice/treatment; N values for cells per condition are indicated in the figure.

Figure 6.

PN-FLX treatment results in fear extinction learning and recall deficits. A, PN-FLX mice exhibit comparable levels of freezing at baseline (pre), in response to the first and last tones of the training session, and in the post-training period (post), when compared with PN-VEH controls. B, During the extinction learning session, no baseline differences in pretone freezing in a novel extinction training environment is observable (pre). B, After the onset of the tones (without shocks), PN-VEH mice exhibit a progressive decrease in freezing to tone over the extinction session, while PN-FLX mice display significantly blunted decreases in freezing over the session (horizontal line indicates significant treatment × time interaction). PN-FLX mice also exhibit significantly higher post-training freezing levels, relative to controls (B, post). C, To investigate freezing behavior over multiple days, freezing levels of PN-FLX and PN-VEH mice were normalized to their respective starting freezing levels at D1. As observed in the non-normalized data of B, PN-FLX-treated mice displayed a deficit in extinction learning, when compared with PN-VEH controls on D1 (C, horizontal line indicates significant treatment × time interaction, stars without horizontal line indicate treatment effect on D1 end). Between D1 and D2, neither PN-VEH nor PN-FLX mice displayed extinction recall (D1 start vs D2 start, not statistically significantly different for either PN-VEH or PN-FLX). On D2, PN-VEH and PN-FLX mice displayed extinction learning (main effect of effect of time, no time × treatment interaction). Between D2 and D3, only PN-VEH displayed extinction recall, and PN-FLX mice froze significantly more than PN-VEH mice on D3. N values are indicated in the figure. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7.

IL lesions in control mice phenocopy the effects of PN-FLX treatment on anxiety-like behavior. Mice received bilateral injections in the IL using ibotenic acid (IL lesion) or saline solution (SHAM). A, B, Lesion extent was assessed histologically after behavioral experiments were concluded, with light gray demarcating the additive area lesioned, and dark gray demarcating the largest common area lesioned. C–E, Although IL lesions do not alter ambulatory time in the OF test (C), they significantly reduce the time spent rearing (D) and the time spent in the more anxiogenic center region (E) relative to SHAM lesioned controls. F–H, In the NSF test, IL lesioned mice take significantly longer to approach the food pellet relative to PN-VEH mice (F), while the percentage of weight loss after 24 h of food deprivation (G) and home-cage food consumption (H) are comparable. I–K, IL lesions did not significantly affect latency to escape (I), intratrial activity (J), or baseline pretrial activity (K) in the shock escape test. N values are indicated in the figure. *p < 0.05.

Figure 8.

PL lesions in control mice do not affect anxiety-like behavior. Mice received bilateral injections in the PL using ibotenic acid (PL lesion) or saline solution (SHAM). A, B, Lesion extent was assessed histologically after behavioral experiments were concluded, with light gray demarcating the additive area lesioned, and dark gray demarcating the largest common area lesioned. C–E, In the OF test, PL lesions do not alter ambulatory time (C), time spent rearing (D), or time spent in the center region (E) relative to SHAM lesioned controls. F–H, In the NSF test, PL lesions do not affect the time to feed (F), percentage of weight loss after 24 h of food deprivation (G), or home-cage food consumption (H). I, K, PL lesions do not impact the latency to escape (I) or baseline pretrial activity (K) in the SESC test. J, PL lesions significantly increased the intratrial activity. N values are indicated in the figure. *p < 0.05.