Environmental Enrichment Mitigates the Long-Lasting Sequelae of Perinatal Fentanyl Exposure in Mice

Abstract

The opioid epidemic is a rapidly evolving societal issue driven, in part, by a surge in synthetic opioid use. A rise in fentanyl use among pregnant women has led to a 40-fold increase in the number of perinatally-exposed infants in the past decade. These children are more likely to develop mood-related and somatosensory-related conditions later in life, suggesting that fentanyl may permanently alter neural development. Here, we examined the behavioral and synaptic consequences of perinatal fentanyl exposure in adolescent male and female C57BL/6J mice and assessed the therapeutic potential of environmental enrichment to mitigate these effects. Dams were given ad libitum access to fentanyl (10 µg/ml, per os) across pregnancy and until weaning [postnatal day (PD)21]. Perinatally-exposed adolescent mice displayed hyperactivity (PD45), enhanced sensitivity to anxiogenic environments (PD46), and sensory maladaptation (PD47), sustained behavioral effects that were completely normalized by environmental enrichment (PD21-PD45). Additionally, environmental enrichment normalized the fentanyl-induced changes in the frequency of miniature EPSCs (mEPSCs) of layer 2/3 neurons in the primary somatosensory cortex (S1). We also demonstrate that fentanyl impairs short-term potentiation (STP) and long-term potentiation (LTP) in S1 layer 2/3 neurons, which, instead, exhibit a sustained depression of synaptic transmission that is restored by environmental enrichment. On its own, environmental enrichment suppressed long-term depression (LTD) of control S1 neurons from vehicle-treated mice subjected to standard housing conditions. These results demonstrate that the lasting effects of fentanyl can be ameliorated with a noninvasive intervention introduced during early development.SIGNIFICANCE STATEMENT Illicit use of fentanyl accounts for a large proportion of opioid-related overdose deaths. Children exposed to opioids during development have a higher risk of developing neuropsychiatric disorders later in life. Here, we employ a preclinical model of perinatal fentanyl exposure that recapitulates these long-term impairments and show, for the first time, that environmental enrichment can reverse deficits in somatosensory circuit function and behavior. These findings have the potential to directly inform and guide ongoing efforts to mitigate the consequences of perinatal opioid exposure.

Keywords: LTP; adolescence; in utero; somatosensory cortex; synaptic plasticity.

Figure 1.

Experimental timeline of exposure to perinatal fentanyl and postnatal housing conditions. A, Timeline depicting fentanyl exposure of mouse dams throughout pregnancy until weaning of offspring on PD21. B, Offspring were weaned into standard (top cage) or enriched (bottom cage) housing conditions until behavioral (PD45/47) and electrophysiological (PD48–PD55) analyses took place. Enrichment items are numbered (1–9) and listed in Table 1.

Figure 2.

Environmental enrichment reverses the sustained behavioral deficits that are induced by perinatal fentanyl exposure. A, Timeline depicting behavioral assays and a schematic of the open field test. n = 15–20 mice per group. B, C, Perinatal fentanyl exposure increases distance traveled across time. D, E, Perinatal fentanyl-exposed mice raised with environmental enrichment have comparable distance traveled with vehicle controls but spend less time in the center area of the open field. Fentanyl-exposed mice raised with environmental enrichment spend more time in the center area of the open field, comparable to vehicle controls. F, Graphic depicting von Frey tactile stimulation on the plantar surface of the hind paw. G, There were no differences across groups in the paw withdrawal threshold to tactile stimulation. n = 11–19 mice per group. H, Standard-housed fentanyl-exposed mice have a higher number of paw withdrawal responses to repeated stimuli than standard-housed vehicle mice. Fentanyl-exposed mice raised with environmental enrichment have a lower number of paw withdrawal responses, comparable to vehicle mice raised in standard and enriched environment. Data depict means, p values, and 95% confidence intervals.

Figure 3.

Environmental enrichment restores potentiation in S1 layer 2/3 neurons after perinatal fentanyl exposure. A–D, Time course (left) of STP and LTP (STP/LTP) of EPSCs following paired induction (80 electrical stimulation pulses at 2 Hz paired with postsynaptic depolarization to +30 mV). Bar graphs (right) show group data comparing EPSC amplitudes at baseline −5–0 min (1), STP 0–5 min (2), and LTP 25–30 min (3) after LTP induction. Insets depict sample traces from times indicated on time course graph. A, Within-group comparisons indicate STP and LTP were induced in S1 layer 2/3 neurons from vehicle-exposed, standard-housed mice. B, LTP paired induction parameters induced LTD of the EPSC in fentanyl-exposed, standard-housed mice. C, LTP paired induction protocol failed to further potentiate EPSC amplitude from vehicle-exposed, enriched-housed mice. D, The paired induction protocol induced STP, but not LTP, in fentanyl-exposed, enriched-housed mice. E, Between-group comparisons of the EPSC amplitudes post-LTP induction. Fentanyl-exposed, standard-housed mice had lower EPSC amplitudes than vehicle-exposed, standard-housed mice at 0- to 5- and 25- to 30-min time points. Environmental enrichment increased the EPSC amplitude of fentanyl-exposed, enriched-housed mice, relative to their fentanyl-exposed, standard-housed counterparts also at 0- to 5- and 25- to 30-min time points. F, Between-group comparisons indicate fentanyl-exposed, standard-housed mice had a lower occurrence of neurons that exhibit STP and LTP than vehicle-exposed, standard-housed mice. Raising fentanyl-exposed mice in environmental enrichment increased the proportion of neurons that potentiate. n = 6–8 mice/group, 1 neuron/mouse. Data depict means and 95% confidence intervals.

Figure 4.

Environmental enrichment suppresses LTD. A–D, Time course (left) of LTD of EPSCs following low frequency stimulation (900 electrical stimulation pulses at 1 Hz). Bar graphs (right) show group data comparing EPSC amplitudes at baseline (1) and at 25–30 min after LTD induction (2). Insets depict sample traces from times indicated on time course graph. A, Within-group comparisons indicate LTD was induced in S1 layer 2/3 neurons from vehicle-exposed, standard-housed mice and in (B) fentanyl-exposed, standard-housed mice. C, LTD was not induced in vehicle-exposed, enriched-housed mice, but was in (D) fentanyl-exposed, enriched-housed mice. E, Between-group comparisons of the EPSC amplitudes post-LTD induction indicate that vehicle-exposed, enriched-housed mice failed to induce LTD compared with their vehicle-exposed, standard-housed counterparts. F, Between-group comparisons of the occurrence of LTD indicate vehicle-exposed, enriched-housed mice had a lower occurrence of neurons that exhibit LTD than vehicle-exposed, standard-housed mice. n = 5–6 mice/group, 1 neuron/mouse. Data depict means and 95% confidence intervals.

Figure 5.

Evoked glutamate release probability is not influence by perinatal fentanyl exposure or environmental enrichment. A, Representative traces of EPSC responses to paired pulse stimulation. B, There were no differences in the PPR between groups before plasticity induction (n = 9–12 mice/group, 1 neuron/mouse). C, D, Within each group, there were no differences in baseline PPR compared with PPR post-LTP or LTD induction (n = 4–6 mice/group, 1 neuron/mouse). Data depict means and 95% confidence intervals.

Figure 6.

Environmental enrichment restores changes in mEPSC frequency induced by perinatal fentanyl exposure. A, Representative traces of mEPSCs. B, Grouped data of the averaged events from each neuron and animal indicating that mEPSC frequency is higher after environmental enrichment, compared with controls. C, Cumulative probability curves of the frequency suggest perinatal fentanyl exposure results in increased mEPSC frequency, and that raising fentanyl-exposed mice with environmental enrichment reverses this increase. Enrichment also increased mEPSC frequency of vehicle control mice independent of perinatal fentanyl exposure. D, E, There were no differences in mEPSC amplitudes. n = 6–8 mice/group, 1–2 neurons/mouse. Data depict means, and 95% confidence intervals.