All animal protocols were approved by the Institutional Animal Care and Use Committee of The University of Chicago, in accordance with the National Institute of Health guidelines.

C57BL/6 mice of both sexes (P4 to P60) were used in this study. They were housed in AAALAC-approved facilities with a 12-h/12-h light-dark cycle and had ad libitum access to food and water. To ensure sufficient neonatal nutrition, litters were culled down to six pups prior to the start of experimental protocols. The dam and pups were kept together during all in vivo experimental protocols, allowing unrestricted access to nutrition for the offspring.

Five in vivo experimental protocols were used in this study.

Coronal hippocampal slices were prepared from mice either 24 h following the end of nIH (P14 to P15) or ~6 weeks after nIH (P55 to P60). Mice were anesthetized with isoflurane and euthanized by rapid decapitation. The brain was rapidly harvested and blocked, rinsed with cold artificial cerebrospinal fluid (aCSF), and mounted for vibratome sectioning. The mounted brain tissue was submerged in aCSF (4°C; equilibrated with 95% O₂, 5% CO₂), and coronal cortico-hippocampal brain slices (350 μm thick) were prepared. Slices were immediately transferred into a holding chamber containing aCSF equilibrated with 95% O₂ and 5% CO₂ (at 20.5 ± 1°C). Slices were allowed to recover a minimum of 1 h prior to the transfer into a recording chamber. Slices were used within 8 h following tissue harvest, with an aCSF composition (in) of 118 mM NaCl, 10 mM glucose, 20 mM sucrose, 25 mM NaHCO₃, 3.0 mM KCl, 1.5 mM CaCl₂, 1.0 mM NaH₂PO₄ and 1.0 mM MgCl₂. The osmolarity of aCSF was 305–315 mOsm/L, and when equilibrated with 95% O₂/5% CO₂, the pH was 7.42 ± 0.2.

The extracellular recording of the fEPSP was established in aCSF (31.0 ± 2°C, equilibrated with 95% O₂ 5% CO₂), which was superfused and recirculated over the preparation. A custom-constructed bipolar stimulation electrode composed of twisted Teflon-coated platinum wires (wire diameter: 127 μm, catalog number 778000, AM Systems, Washington, WA) was used to electrically evoke the fEPSP. The stimulation electrode was positioned in the Schaffer collaterals, and a recording electrode (< 2 MΩ) was positioned into the stratum radiatum of area CA1. The intensity of the electrical current (100–400 μA; 0.1–0.2 ms duration) was set to the minimum intensity required to generate 50% of the maximal fEPSP.

The fEPSP was evoked every 20 s. After 10 min of baseline recording, LTP was induced using Theta burst stimulation (TBS), which consisted of four trains of 10 bursts at 5 Hz, each burst was comprised of four pulses at 100 Hz. Recordings continued for up to 1 h. The fEPSP slope was normalized to baseline values. 50 μM D-AP5 or 100 μM D-L AP5 (Sigma-Aldrich, Saint Louis, MO; Cat#A5282) was used to block NMDAr, TCN-213 (5 μM, Tocris Bioscience, Bristol, UK; Cat#4163) was used to block GluN2A, and ifenprodil (5 μM, Tocris Bioscience, Bristol, UK; Cat#0545) was used to block GluN2B. The current stimulus was set at the minimum current value (150 to 250 μA) required to evoke the initial fEPSP at a maximal value of 50%. Recordings were made using either a MultiClamp 700B (Molecular Devices, San Jose, CA, USA) or using a differential amplifier (AM system, Washington, DC, USA).

Samples for immunoblot assays were prepared from the isolated hippocampus that was homogenized using N-PER (Thermo Fisher Scientific, Cat#87792; Whaltam, MA) in ice following manufacturer instructions. Briefly, tissues were homogenized in the cytoplasmic extraction buffer. After the isolation of the cytoplasmic fragment, the insoluble pellet containing nuclear proteins was suspended in a nuclear extraction buffer and separated by centrifugation (16,000 rcf). Halt™ Protease Inhibitor Cocktail (Thermo Fisher, Whaltam, MA; Cat#78429) was added into cytoplasmic and nuclear extraction buffers to prevent protein degradation. Samples were boiled for 15 min in a loading buffer (Bio-Rad, Hercules, CA, USA), denaturated by β-mercaptoethanol at 75°C before loading 50–60 μg protein onto 4–20% Mini-PROTEAN TGX Stain-FreeTM Protein Gels (Bio-Rad, Hercules, CA, USA), and electrophoresed at 120 V for 120 min. Then, gels were transferred to PVDF membrane (Bio-Rad) using the Transfer-Blot Turbo System (Bio-Rad, Hercules, CA). Membranes were subsequently blocked for 2 h at room temperature with 5% bovine serum albumin (BSA) (Sigma-Aldrich, MN, USA) in Tris-buffered saline (TBS) (Bio-Rad, Hercules, CA, USA). Membranes were then incubated under constant shaking with primary antibodies for HIF1a (1:500; Abcam, Bristol, UK; Cat# ab1, RRID:AB_296474) and TBP (1:2000; Danvers, MA; Cat# 44059, RRID:AB_2799258). After washing three times with 0.3% TBS-Tween for 20 min, the membranes were incubated for 1.5 h at room temperature with appropriate secondary antibodies. Finally, the membranes were washed three times with 0.3% TBS-Tween for 20 min, and immunoreactive proteins were detected with Super Signal^TM West Femto reagents according to the manufacturer's instructions (Thermo Fisher Scientific, Whaltam, MA; Cat#34095). Signals were captured with the ChemiDoc Imaging system (Bio-Rad, Hercules, CA). The ImageJ program (National Institutes of Health, USA) was used to quantify optical band intensity.

Samples for immunoblot assays were prepared from the isolated hippocampus that was homogenized using M-PER^TM (Thermo Fisher Scientific, Whaltam, MA; Cat#78501) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Whaltam, MA; Cat#78429) kept on ice. Samples were centrifuged at 12,000 rpm for 15 min at 4°C, and the pellet was discarded. Samples were boiled for 15 min in a loading buffer (Bio-Rad, Hercules, CA), denaturated by β-mercaptoethanol at 75°C before loading 20–30 μg protein onto 4–20% Mini-PROTEAN TGX Stain-FreeTM Protein Gels (Bio-Rad, Hercules, CA), and electrophoresed at 120 V for 120 min. Then, gels were transferred to PVDF membrane (Bio-Rad) using the Transfer-Blot Turbo System (Bio-Rad, Hercules, CA). Later, membranes were subsequently blocked for 2 h at room temperature with 5% non-fat milk (Bio-Rad, Hercules, CA) or 5% bovine serum albumin (BSA) (Sigma-Aldrich, MN, USA) in Tris-buffered saline (TBS) (Bio-Rad, Hercules, CA). Membranes were incubated under constant shaking with primary antibodies for GluN1 (1:2000; Abcam, Bristol, UK; Cat# ab109182, RRID:AB_10862307), GluN2A (1:2000; Danvers, MA; Cat# 4205, RRID:AB_2112295), GluN2B (1:2000; Danvers, MA; Cat# 14544, RRID:AB_2798506), NOX2 (1:2000; Abcam, Bristol, UK; Cat# ab129068, RRID:AB_11144496), NOX4 (1:500; Saint Louis, MO; Cat# NB110-58841B, RRID:AB_1217375), and GAPDH (1:10000; Abcam, Bristol, UK; Cat# ab8245, RRID:AB_2107448). Incubations were performed at 4°C overnight in 5% non-fat milk or BSA. After washing three times with 0.2% TBS-Tween for 15 min, the membranes were incubated for 1.5 h at room temperature with appropriate secondary antibodies. Finally, the membranes were washed three times with TBS-Tween 0.2% for 15 min and immunoreactive proteins were detected with enhanced chemiluminescence reagents according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Signals were captured with the ChemiDoc system (Bio-Rad, Hercules, CA). The ImageJ program (National Institutes of Health, USA) was used to quantify optical band intensity.

Whole-cell protein lysates were isolated from the entire hippocampal tissues using the RIPA buffer (Thermo Fisher Scientific, Catalog No. R0278) in the presence of protease and phosphatase inhibitors (Thermo Fisher Scientific, Catalog No. 78429) kept on ice. Protein lysates were immediately processed and stored at −80°C until use. The amount of lipid-peroxidation was determined using a TBARS Assay Kit (Cayman Chemical, Cat#10009055) as per manufacturer's instructions. Absorbance was measured between 530 and 540 nm using a plate reader. The analysis for determining MDA values was conducted in accordance with the manufacturer's instructions.

The Barnes maze test was performed using a custom-made opaque white circular acrylic platform (92.4 cm in diameter) with 20 equidistant holes (5.08 cm in diameter and 2.54 cm from the edge). The platform was elevated (30 cm from the floor) from ground and surrounded by four identical walls (27.94 cm high). By default, each hole was closed with a fixed piece of opaque acrylic that could be removed to lead to a dark exit box. Lighting was achieved through diffuse overhead fluorescent lighting such that all holes were equally lit. An overhead camera was suspended above the maze. Data collection and post-hoc analysis were performed using CinePlex Digital Video Recording/Tracking System (Plexon, Dallas, TX).

As described in a previous study (Arias-Cavieres et al., 2020), the task was performed using a 4-day protocol consisting of one training trial per day for 3 consecutive days and a PROBE trial on the fourth day. The Barnes maze protocol began after 6-week exposure to IH with respective controls run at the same time. For the training trials, all but one of the holes (i.e., the exit zone) were closed. An exit box with a small ramp was placed directly underneath the exit zone. Animals were given a maximum of 6 min to locate the exit, and if unable to locate the exit, they were gently guided to the exit. If the mouse found and entered the exit before the given duration of 6 min were over, the trial ended at the time that the mouse entered the exit and the mouse was promptly returned to its home cage. During the PROBE trial, all holes were closed, and the animal was given 6 min to explore the maze. To reduce potential confounders due to odor, the arena was sanitized with 75% ethanol between trials.

Entry probability for each hole during the PROBE trail was calculated by the following:

where EP_nis the entry probability for hole n; x_nis the number of entries into hole n; and x_total is the sum of entries across all holes during the PROBE trial.

The Object Location task was performed using a modified protocol described by Wimmer et al. (2012). The procedure included three phases: open field, familiarization, and object location. Each phase was performed in the acrylic open arena (W: 33 cm, L: 36 cm, H: 33 cm). During the open field phase, the mouse was habituated and allowed to freely explore the arena for 10 min. The familiarization session consisted of three consecutive trials (session duration: 5 min; intersession duration: 5 min), where the mouse was placed in the arena and allowed to explore two different objects (i.e., object A and object B). The time for exploring both objects was recorded. The object location phase occurred 24 h following familiarization, where one object presented in the familiarization session was repositioned (B). The time taken for exploring the repositioned object (object B) was compared between experimental groups. Exploration time was expressed as a percentage describing the proportion of time the subject explored object B in relation to total exploration time.

To eliminate odor cues between trials, the experimental apparatus and all objects were cleaned with 75% ethanol after each trial. The behavior of mice was recorded with a video camera positioned over the behavioral apparatus, and the collected videos were analyzed with the ANY-MAZE software (Stoelting Co., Wood Dale, IL, USA).

Statistical analysis was performed using Prism 6 (GraphPad Software, Inc.; RRID:SCR_015807), and the results were plotted in either Prism 6 or Origin 2018b (OriginLab, RRID:SCR_014212). Comparisons between the two groups were conducted using unpaired two-tailed t-test with Welch's correction or paired comparisons, where appropriate. The equality of variances between the two groups was determined with an F test. Analyses that involved comparisons beyond more than two groups were assessed using a one-way ANOVA of means followed by a post-hoc Bonferroni's multi-comparison test. Data are presented as mean ± S.E.M, and where appropriate, individual responses are overlaid over box plots. The upper and lower limits of each box represented the 25th and 75th percentile of the cohort, respectively, while the inter-limit line within the box represented as median. The box plot error bars represented the maximum and minimum values in the data set. Significance was defined as a P-value of < 0.05.

Measurements of the malondialdehyde (MDA) content in neonatal hippocampal homogenates revealed that the MDA levels were increased with nIH (Figure 2A, control: 7.2 ± 0.82 nmol · mg⁻¹ protein and nIH: 18.59 ± 1.65 nmol · mg⁻¹ protein, P = 0.004; N = 6 preparations per condition). This was accompanied by greater nuclear content of the prooxidant transcription factor, HIF1a (Figure 2B, control = 1.06 ± 0.04 and nIH = 1.59 ± 0.12, P = 0.010; N = 5 per condition) and increased expression of two isoforms of the prooxidant enzyme NADPH oxidase (NOX), NOX-2 (Figure 2C control = 1.02 ± 0.02 and nIH = 1.31 ± 0.12; P = 0.04; N = 6 per condition), and NOX-4 (Figure 2D, NOX-4: control = 0.96 ± 0.03 and nIH = 1.30 ± 0.07; P = 0.003; N = 5 per condition).

In adult rodents, IH dependency increased nuclear HIF1a, and a shift toward a prooxidant state is associated with impaired NMDAr-dependent synaptic plasticity (Arias-Cavieres et al., 2020, 2021). Therefore, we next characterized how nIH impacted LTP, evoked by theta-burst stimulation (LTP_TBS) in neonatal hippocampal slices from control and nIH mice. LTP_TBS was evoked in area CA1 in both control (Figure 3A, black, n = 7 slices; N = 6 mice) and nIH (Figure 3A, red, n = 6; N = 5) mice, and blockade of NMDAr, using AP5, prevented LTP_TBS in both groups (Figure 3A, control (green): 99.74 ± 2.88, n = 4, N = 4; nIH (blue): 99.07 ± 3.38, n = 4, N = 4). However, the magnitude of LTP_TBS was smaller in nIH slices as compared to control mice (Figure 3A, control: 177.02 ± 5.9% vs. nIH: 135.10 ± 6.8% over baseline, P = 0.009). Such a difference may have resulted from an overall downregulation in NMDAr expression or a change in receptor subunit composition.

While the expression of GluN1, the obligatory subunit of the NMDAr, was similar between control and nIH (Figure 3B, control: 1.06 ± 0.01, N = 5; nIH: 1.11 ± 0.09, N = 5; P = 0.55), GluN2 subunit composition was different between the groups. GluN2A expression was reduced following nIH (Figure 3C, control: 1.07 ± 0.04, N = 5; nIH: 0.88 ± 0.02, N = 5; P = 0.006), whereas GluN2B expression following nIH was greater than control (Figure 3D, control: 1.06 ± 0.03; N = 5; nIH: 1.38 ± 0.09; N = 5, P = 0.014). These differences coincided with a larger GluN2B:GluN2A ratio following IH (Figure 3E, P = 0.0019). Since GluN2 subunit composition is a significant factor in dictating NMDAr-dependent physiology (Kumar and Huguenard, 2003; Paoletti et al., 2013; Kumar et al., 2019), we sought to determine whether changed LTP_TBS following nIH was related to changes in GluN2 subunit composition. In control slices, the magnitude of LTP_TBS was reduced when blocking either the GluN2A containing NMDAr with TCN-213 (Figure 3F, orange, 129.87 ± 3.07% over baseline; n = 7 slices, N = 6) or the GluN2B containing NMDAr with ifenprodil (Figure 3F, cyan, 146.67 ± 3. 29% over baseline; n = 6 slices, N = 6). When both agents were applied, LTP_TBS was not effectively evoked (Figure 3F, olive, 96.37 ± 3.36% over baseline; n = 4 slices, N = 4; P < 0.0001).

Following nIH, LTP_TBS appeared to be unaffected by TCN-213 (Figure 3G; light yellow, 137.58 ± 2.24% over baseline; n = 4 slices, N = 4 mice) when compared to treated nIH slices (from Figure 3A); however, ifenprodil appeared to prevent LTP_TBS following nIH (Figure 3G; light blue, 106.13 ± 2.16% over baseline, n = 5 slices, N = 5; P = 0.0016). Collectively, these results indicated that NMDAr-dependent plasticity following nIH is exclusively driven by GluN2B-containing receptors, which is consistent with the nIH-mediated remodeling of GluN2 subunit composition to favor GluN2B expression over GluN2A.

To determine the contribution of the nIH-dependent prooxidant state to the observed changes in the neonatal hippocampus, we administered the superoxide anion scavenger MnTMPyP to a cohort of mice during nIH. While the MDA content in neonatal hippocampi from the control and the cohort receiving MnTMPyP during nIH (nIH_Mn) were similar, the MDA content in the nIH cohort treated with a saline vehicle (nIH_saline) was greater than control (Figure 4A; control: 9.31 ± 0.98 nmol per mg protein; nIH_saline: 17.75 ± 1.78 nmol per mg protein; nIH_Mn: 11.33 ± 0.93 nmol per mg protein; P = 0.0011; n = 5 per group). Nuclear HIF1a content (Figure 4B, control: 0.99 ± 0.07; nIH_saline: 1.32 ± 0.10 and nIH_Mn: 1.03 ± 0.05, P = 0.017; n = 5 per group), NOX2 (Figure 4C, control: 1.03 ± 0.03, nIH_saline: 1.32 ± 0.051 and nIH_Mn: 0.99 ± 0.046, P = 0.0024; n = 6 per group), and NOX4 were increased in nIH_saline (Figure 4D, control: 1.02 ± 0.03; nIH_saline: 1.35 ± 0.08 and nIH_Mn: 1.10 ± 0.01, P = 0.0034; n = 4 per group) and remaned unchanged in nIH_Mn. GluN2A expression was also reduced in nIH_saline while the expression was unchanged in nIH_Mn (Figure 4E, control: 1.08 ± 0.07; nIH_Saline: 0.68 ± 0.06; nIH_Mn: 0.92 ± 0.06; P = 0.0086; N = 6 per group). Furthermore, while GluN2B expression was increased in nIH_saline, GluN2B expression remained unchanged in nIH_Mn (Figure 4F, control: 1.03 ± 0.030; nIH_saline: 1.38 ± 0.12; nIH_Mn: 0.92 ± 0.059, P = 0.010; n = 5 per group). Similarly, the magnitude of NMDAr-dependent LTP in nIH_Mn was greater than nIH (Figure 4G, nIH_Mn: 166.65 ± 6.20% over the baseline; n = 7 slices, N = 5 mice), and the sensitivity of LTP to ifenprodil was evident when compared to nIH (Figure 4H, 133.26 ± 5.03% over the baseline; n = 7, N = 5).

To asses the consequences of nIH, we characterized LTP in adult mice that were allowed to recover from nIH for 6 weeks in room air (Adult_nIH). In the hippocampal slices from Adult_nIH, the magnitude of LTP was smaller compared to that in Adult_control preparations [Figure 5A; Adult_control (black): 167.58 ± 4.08% over baseline; n = 6 slices, N = 6 mice; Adult_nIH (red): 139.47 ± 2.76% over baseline; n = 7 slices, N = 7 mice; P = 0.003]. We next determined whether differences in LTP magnitude observed in Adult_nIH hippocampal slices were related to GluN2 subunit composition. In Adult_control slices, NMDAr-dependent LTP appeared to be largely dependent on the GluN2A containing NMDAr and independent of GluN2B-containing NMDAr as TCN-213 suppressed LTP (Figure 5B, orange, 119.53 ± 1.97% over baseline; n = 4 slices, N = 4 mice), while ifenprodil minimally affected LTP (Figure 5B, blue, 150.04 ± 3.59% over baseline; n = 4 slices, N = 4 mice; P = 0.0009). In contrast, LTP from Adult_nIH slices appeared to depend on GluN2B-containing NMDAr and be independent of GluN2A-containing NMDAr as ifenprodil blocked LTP (Figure 5C, blue: 108.1 ± 1.55 over baseline; n = 6, N = 5), while TCN-213 failed to block LTP (Figure 5C, orange, 132.08 ± 5.1 over baseline; n = 5, N = 3; P < 0.001).

As MnTMPyP prevented the immediate effects of nIH on LTP, we assessed how nIH_Mn and MnTMPyP administration following nIH (Adult_{REC − Mn}) impacted LTP in adult hippocampal slices. In adult mice, the magnitude of LTP from the Adult_{REC − Mn} was smaller than the magnitude of LTP from Adult_{nIH − Mn} (Figure 5D, Adult_{nIH − Mn}: green, 161.35 ± 8.39% over baseline; n = 6 slices, N = 6 mice; Adult_{REC − Mn}: blue, 124.50 ± 4.87% over baseline; n= 7 slices, N = 7 mice; P = 0.0030).

We next examined biochemical and protein expression in the adult hippocampus. Neither HIF1a nor NOX isoforms were different from Adult_control in Adult_nIH, Adult_{nIH − Mn}, or Adult_{REC − Mn} (Supplementary Figure 1). Similarly, no differences in GluN1 subunit expression was found across experimental groups (Figure 5E, control = 1.00 ± 0.07; Adult_nIH = 0.99 ± 0.07; Adult_{nIH − Mn} = 1 ± 0.05 and Adult_{REC − Mn} = 0.95 ± 0.03, N = 5; P = 0.93). However, differences in both the subunit expression of GluN2A and GluN2B were evident. In Adult_nIH and Adult_{REC − Mn}, GluN2A expression was suppressed when compared to Adult_control; however, no difference was evident in Adult_{nIH − Mn} (Figure 5F, Adult_control: 1 ± 0.05; Adult_nIH: 0.56 ± 0.72; Adult_{nIH − Mn}: 0.92 ± 0.07 and Adult_{REC − Mn}: 0.71 ± 0.07, N = 4; P = 0.0029). While GluN2B expression was increased in Adult_nIH, no differences were observed in Adult_{nIH − Mn} and Adult_{REC − Mn} (Figure 5G, Adult_control: 1.02 ± 0.13; Adult_nIH: 1.47 ± 0.11, Adult_{nIH − Mn}: 1.14 ± 0.08, and Adult_{REC − Mn}: 0.98 ± 0.13, N = 4; P =0.018). These changes coincided with a larger GluN2B:GluN2A ratio in adult mice exposed to nIH (Figure 5H, P = 0.0083).

To determine whether the persistent changes in synaptic plasticity and NMDAr subunit expression due to neonatal IH corresponded with behavioral deficits related to spatial learning and memory, we next examined the performance of control adult mice (Adult_control) and Adult_nIH and in the Barnes maze and Object Location task.

In the Barnes maze test, both velocity and distance traveled during the training sessions were similar in Adult_control (N = 14) and Adult_nIH (N = 20) (Table 2). Additionally, both Adult_control and Adult_nIH, exhibited a progressive decrease in total latency to exit the maze across the training sessions (Supplementary Figure 2). During the PROBE trial, the initial distance (Figure 6A; control: 0.13 ± 0.017 m vs. Adult_nIH: 0.33 ± 0.05 m, P = 0.0019) and latency to initial entry (Figure 6A; control: 43.04 ± 10.44 s vs. Adult_nIH: 83.13 ± 15.99 s, P = 0.04) into the exit zone were greater in Adult_nIH. These differences were accompanied by a smaller entry probability into the exit zone in Adult_nIH (Figure 6B; control: 12.63 ± 1.42% vs. Adult_nIH: 7.14 ± 1.21%, P = 0.0067).

During the open field session in the object location task, Adult_control (N = 14) and Adult_nIH (N = 14) exhibited similar velocities, distance traveled, time in the periphery, and time in the center (Table 3), suggesting no locomotor differences between groups. During the familiarization session, control and Adult_nIH both exhibited a progressive decrease in exploration times and similar exploration times with both objects (Supplementary Figure 3). These data suggested that both groups learned the location of both objects and did not have a preference for either of the objects tested. During the PROBE trial of the Object Location task, control mice exhibited greater exploration time compared to nIH (Figure 6C: control = 28.81 ± 3.38% and nIH = 19.42 ± 2.63%; P = 0.038).

We sought to determine whether the adult behavioral performance was impacted by MnTMPyP administration during nIH (i.e., Adult_{nIH − Mn}) or by MnTMPyP administration following nIH (i.e., Adult_{REC − Mn}). Both Adult_{nIH − Mn} (N = 16) and Adult_{REC − Mn} (N = 13) exhibited progressive reductions in the distance traveled across training sessions without changed velocities (Table 3). These reductions corresponded with progressive improvement in exiting the Barnes maze during the three training sessions (Table 2). During the PROBE trial, the initial distance (Figure 6D; Adult_{nIH − Mn}: 0.13 ± 0.016 m vs. Adult_{REC − Mn}: 0.27 ± 0.05 m, P = 0.039) and latency to initial entry (Figure 6D; Adult_{nIH − Mn}: 43.83 ± 9.03 s vs. Adult_{REC − Mn}: 84.56 ± 15.79s, P = 0.037) into the exit zone were lower in Adult_{nIH − Mn} when compared to Adult_{REC − Mn}. Adult_{nIH − Mn} also showed a greater entry probability into the exit zone when compared to Adult_{REC − Mn} (Figure 6E; Adult_{nIH − Mn}: 11.45 ± 1.52% vs. Adult_{REC − Mn}: 7.43 ± 5.18%, P = 0.040).

During the open field session of the Object Location task, Adult_{nIH − Mn} (N = 16) and Adult_{REC − Mn} (N = 13) exhibited similar velocities, distance traveled, time in the periphery, and time in the center (Table 3), suggesting no locomotor differences between groups. During the familiarization session, Adult_{nIH − Mn} and Adult_{REC − Mn} both also exhibited a progressive decrease in exploration times (Table 4) and similar times exploring both objects (Table 5). However, during the PROBE trial of the Object Location task, exploration time for the moved object (i.e., object B) was greater in Adult_{nIH − Mn} when compared to Adult_{REC − Mn} (Figure 6F; Adult_{nIH − Mn}: 27.21 ± 2.51 % vs. Adult_{REC − Mn}: 20.44 ± 1.31 %, P = 0.024)