Peripuberty Is a Sensitive Period for Prefrontal Parvalbumin Interneuron Activity to Impact Adult Cognitive Flexibility

Abstract

Introduction: Developmental windows in which experiences can elicit long-lasting effects on brain circuitry and behavior are called "sensitive periods" and reflect a state of heightened plasticity. The classic example of a sensitive period comes from studies of sensory systems, like the visual system, where early visual experience is required for normal wiring of primary visual cortex and proper visual functioning. At a mechanistic level, loss of incoming visual input results in a decrease in activity in thalamocortical neurons representing the affected eye, resulting in an activity-dependent reduction in the representation of those inputs in the visual cortex and loss of visual perception in that eye. While associative cortical regions like the medial prefrontal cortex (mPFC) do not receive direct sensory input, recent findings demonstrate that changes in activity levels experienced by this region during defined windows in early development may also result in long-lasting changes in prefrontal cortical circuitry, network function, and behavior. For example, we recently demonstrated that decreasing the activity of mPFC parvalbumin-expressing (PV) interneurons during a period of time encompassing peripuberty (postnatal day P14) to adolescence (P50) led to a long-lasting decrease in their functional inhibition of pyramidal cells, as well as impairments in cognitive flexibility. While the effects of manipulating mPFC PV interneuron activity were selective to development, and not adulthood, the exact timing of the sensitive period for this manipulation remains unknown.

Methods: To refine the sensitive period in which inhibiting mPFC PV cell activity can lead to persistent effects on prefrontal functioning, we used a chemogenetic approach to restrict our inhibition of mPFC PV activity to two distinct windows: (1) peripuberty (P14-P32) and (2) early adolescence (P33-P50). We then investigated adult behavior after P90. In parallel, we performed histological analysis of molecular markers associated with sensitive period onset and offset in visual cortex, to define the onset and offset of peak-sensitive period plasticity in the mPFC.

Results: We found that inhibition of mPFC PV interneurons in peripuberty (P14-P32), but not adolescence (P33-P50), led to an impairment in set-shifting behavior in adulthood manifest as an increase in trials to reach criterion performance and errors. Consistent with a pubertal onset of sensitive period plasticity in the PFC, we found that histological markers of sensitive period onset and offset also demarcated P14 and P35, respectively. The time course of expression of these markers was similar in visual cortex.

Conclusion: Both lines of research converge on the peripubertal period (P14-P32) as one of heightened sensitive period plasticity in the mPFC. Further, our direct comparison of markers of sensitive period plasticity across the prefrontal and visual cortex suggests a similar time course of expression, challenging the notion that sensitive periods occur hierarchically. Together, these findings extend our knowledge about the nature and timing of sensitive period plasticity in the developing mPFC.

Introduction: Developmental windows in which experiences can elicit long-lasting effects on brain circuitry and behavior are called "sensitive periods" and reflect a state of heightened plasticity. The classic example of a sensitive period comes from studies of sensory systems, like the visual system, where early visual experience is required for normal wiring of primary visual cortex and proper visual functioning. At a mechanistic level, loss of incoming visual input results in a decrease in activity in thalamocortical neurons representing the affected eye, resulting in an activity-dependent reduction in the representation of those inputs in the visual cortex and loss of visual perception in that eye. While associative cortical regions like the medial prefrontal cortex (mPFC) do not receive direct sensory input, recent findings demonstrate that changes in activity levels experienced by this region during defined windows in early development may also result in long-lasting changes in prefrontal cortical circuitry, network function, and behavior. For example, we recently demonstrated that decreasing the activity of mPFC parvalbumin-expressing (PV) interneurons during a period of time encompassing peripuberty (postnatal day P14) to adolescence (P50) led to a long-lasting decrease in their functional inhibition of pyramidal cells, as well as impairments in cognitive flexibility. While the effects of manipulating mPFC PV interneuron activity were selective to development, and not adulthood, the exact timing of the sensitive period for this manipulation remains unknown.

Methods: To refine the sensitive period in which inhibiting mPFC PV cell activity can lead to persistent effects on prefrontal functioning, we used a chemogenetic approach to restrict our inhibition of mPFC PV activity to two distinct windows: (1) peripuberty (P14-P32) and (2) early adolescence (P33-P50). We then investigated adult behavior after P90. In parallel, we performed histological analysis of molecular markers associated with sensitive period onset and offset in visual cortex, to define the onset and offset of peak-sensitive period plasticity in the mPFC.

Results: We found that inhibition of mPFC PV interneurons in peripuberty (P14-P32), but not adolescence (P33-P50), led to an impairment in set-shifting behavior in adulthood manifest as an increase in trials to reach criterion performance and errors. Consistent with a pubertal onset of sensitive period plasticity in the PFC, we found that histological markers of sensitive period onset and offset also demarcated P14 and P35, respectively. The time course of expression of these markers was similar in visual cortex.

Conclusion: Both lines of research converge on the peripubertal period (P14-P32) as one of heightened sensitive period plasticity in the mPFC. Further, our direct comparison of markers of sensitive period plasticity across the prefrontal and visual cortex suggests a similar time course of expression, challenging the notion that sensitive periods occur hierarchically. Together, these findings extend our knowledge about the nature and timing of sensitive period plasticity in the developing mPFC.

Keywords: Adolescence; Parvalbumin; Peripuberty; Prefrontal cortex; Sensitive period.

Fig. 1.

Peripubertal inhibition of mPFC PV interneurons results in long-term effects in adult cognitive flexibility. a Experimental timeline. Animals expressing hM4DG_i or the control fluorophore mCherry in mPFC PV interneurons were given CNO during either peripuberty (P14–P32) or early adolescence (P33–P50). ED SS behavior was assessed in adulthood (P90+). b Immunofluorescence micrograph illustrates viral expression in the mPFC at P19 (red depicts mCherry and blue depicts DAPI). c Diagram of the SS task. Animals are initially required to learn that one dimension of a multidimensional stimulus (e.g., odor not texture) indicates the presence of a buried reward in a pot (IA). After performing 8 out 10 consecutive trials correctly, the rule is shifted, so now a different dimension (e.g., texture) indicates the presence of a buried reward (ED Set-Shift). O = odor; T = texture. The groups do not differ significantly on the number of trials it takes them to reach criterion d or the number of errors they make during IA e. During the ED Set-Shift phase of the task, the Peripuberty Inhibition group takes significantly more trials to reach criterion (one-way ANOVA, Sidak multiple comparison Control vs. Peripuberty Inhibition p = 0.0044) f and makes significantly more errors (one-way ANOVA, Sidak multiple comparison Control vs. Peripuberty Inhibition p = 0.0230) g than the control group. ^*p < 0.05; ^**p < 0.01.

Fig. 2.

Expression of markers of CP plasticity in primary visual cortex. a Experimental design. Sections from mice ranging in age from P14 to P90 that encompassed V1 were stained for PV and WFA and imaged on a confocal microscope. b Example images of PV and WFA staining in V1 across developmental ages. c The number of PV cells/section was averaged for all sections from a given animal and compared as a function of developmental age using a one-way ANOVA followed by a Sidak post hoc. The most dramatic increase in PV cells/section occurred between P14 and P21 (Sidak post hoc P14 vs. P21, p < 0.0001). PV cell number significantly decreased between P21 and P35 (p = 0.0072) and did not change between P35 and P50 and P50 and P90. d The intensity of WFA staining encapsulating identified PV cells was averaged for all cells for a given animal and compared for each developmental age against levels in the adult (P90) animal with a one-way ANOVA followed by a Sidak post hoc. Levels of WFA significantly differed between P14 and P90, P21 and P90 and P35 and P90 (Sidak post hoc: P14 vs. P90, p = 0.0019; P21 vs. P90, p = 0.0289; P35 vs. P90, p = 0.0374), but not between P50 and P90 (p > 0.9999). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Fig. 3.

Expression of markers of CP plasticity in prefrontal cortex. a Experimental design. Sections from mice ranging in age from P14 to P90 that encompassed the mPFC were stained for PV and WFA and imaged on a confocal microscope. b Example images of PV and WFA staining in mPFC across developmental ages. c The number of PV cells/section was averaged for all sections from a given animal and compared as a function of developmental age using a one-way ANOVA followed by a Sidak post hoc. The most dramatic increase in PV cells/section occurred between P14 and P21 (Sidak post hoc P14 vs. P21, p < 0.0001, P21 vs. P35, P35 vs. P50, P50 vs. P90, p > 0.05). d The intensity of WFA staining encapsulating identified PV cells was averaged for all cells for a given animal and compared for each developmental age against levels in the adult (P90) animal with a one-way ANOVA followed by a Sidak post hoc. Levels of WFA significantly differed between P14 and P90 and P21 and P90 (Sidak post hoc, P14 vs. P90, p < 0.0001; P21 vs.90, p = 0.0246). There was also a strong trend-level difference in WFA intensity between P35 and P90 (p = 0.0638) but no difference between P50 and P90 (p = 0.7594). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^#p < 0.1.