Cranial Irradiation Impairs Juvenile Social Memory and Modulates Hippocampal Physiology

Introduction

Radiation therapy is one of the most utilized and effective treatments for cancer, though certain tissues, such as those in the central nervous system, are particularly vulnerable to being damaged in the process. For some types of cancer, such as acute lymphoblastic leukemia, whole-brain or craniospinal radiation therapy of 10 −30 Gy is used destroy and prevent the spread of tumors throughout the central nervous system. Pediatric patients are particularly susceptible to acquiring long-lasting side effects as the radiation induces damage during a critical window of brain development. Nearly half of pediatric recipients of whole-brain radiotherapy have neurocognitive problems throughout their life, thus creating a list of concerns for patients once the cancer has been effectively treated. ^1,2 These problems include decreases in cognitive proficiency, processing speed, and working memory. Quality of life and overall functionality are also compromised, as these patients have been shown to have increased use of special education services, decreased high school graduation rate, and greater likelihood of developing symptoms of depression.

Children treated at a younger age and/or with higher doses of radiation showed greater incidences and degrees of cognitive impairments ^3–5. The process of neurogenesis happens into adulthood when new concepts are learned. The formation of new neurons in the subgranular zone of the hippocampus is believed to be responsible for learning and the formation of memories. Once these new neurons are incorporated into the circuit of the hippocampus, a new memory is formed ⁶. In a study by Cheng et al, they used male mice that were exposed to 5 Gy of irradiation at either 2, 4, 6, 8, 12, or 18 months of age. After irradiation, they conducted immunostaining where they measured the levels of doublecortin in the hippocampi of these animals. They found significant decreases in doublecortin positive cells after irradiation. Interestingly, mice irradiated at older time points did not exhibit the same level of damage as did younger mice. Such that, younger mice were less radioresistant than the older mice. This suggests that age plays a major role in the susceptibility of patients to cognitive decline following cranial radiotherapy⁷

The hippocampus consists of several subregions— the dentate gyrus, CA1, CA2, CA3, and CA4— each of which contributes to different aspects or types of memory. For example, while CA1 and CA3 are particularly important for the processing and encoding of spatial memories, CA2 region is thought to govern object recognition and social memory ^8,9. The proper function of the hippocampus relies on the rapid remodeling of postsynaptic processes known as spines. The dynamic reorganization of the number, density, and location of hippocampal dendritic spines is crucial to the processes of learning, memory, and cognition¹⁰.

Chakraborti et al, used 2 month old male mice to measure negative effects of 10 Gy of cranial radiation on hippocampal spine density and morphology in the hippocampus. They found that the spine morphology and density of the hippocampus was greatly affected by cranial radiation¹¹. Cranial radiation has also been shown to significantly compromise neuronal morphology in the hippocampus. After 10 Gy of cranial irradiation, Parihar et al observed significant decreases in dendritic complexity. Specifically, they found significant changes in dendritic branching, area and length in irradiated mice¹². Gamma-ray and proton doses spanning from 0.1 Gy to 10.0 Gy have been found to cause significant, dose-responsive reductions in dendritic complexity and spine density in the hippocampus lasting at least 1 month post-irradiation ^11–13. However, the majority of these studies have focused on adult murine models. We aimed to study mouse brains subjected to irradiation on postnatal day 21, an age approximately equivalent to a juvenile child ¹⁴. Here we extend our investigation to the effects of juvenile cranial radiation on social memory and CA2 dendritic spine morphology in mice.

Results

Three-Chamber Sociability

Social behavior was assessed via the three-chamber test. All mice displayed normal habituation by spending approximately equal time exploring both lateral chambers (F _{(5, 64)} = 0.82; P = 0.54; Fig 1). All experimental groups also socialized normally, by spending significantly more time exploring a stranger (stimulus 1) than an identical chamber with an empty cage during stage 2 (F _{(3, 28)} = 16.41; P < 0.001; Holm-Sidak multiple comparisons Stimulus vs Empty; Sham P < 0.001; 10 Gy P < 0.0001: Fig 1). Radiation affected social discrimination during the third stage where radiation eliciting an inability to discriminate between the familiar and stranger mouse, while sham animals successfully spent more time exploring the novel stranger (F (_{3, 28}) = 4.18; P < 0.05; Holm-Sidak multiple comparisons Stimulus 1 vs Novel Stimulus; Sham P < 0.05 Fig 1).

Figure 1: Sociability.

(A) Animals of all cohorts displayed no significant chamber-exploration bias. (B) All cohorts displayed normal sociability by spending significantly more time with a novel mouse (stranger). (C) Sham-irradiated and mice were able to discriminate between a novel stranger, and a now-familiar stranger, whereas mice treated with 10 Gy failed to distinguish strangers. Average ± SEM (n = 10); **P < 0.01.

Spine Morphology

To investigate the effects of radiation on hippocampal physiology, we performed spine-density analyses between cohorts (Table 1). CA2 was a found to have reduced mushroom-spine density in the apical CA2 at (Sham vs. 10 Gy, P < 0.05 Fig 2) and in the basal CA2 (Sham vs. 10 Gy, P < 0.05; Fig 2).

Table 1.

Morphological Analysis of Apical and Basal Dendrites in CA2

Cell types and measurements	O Gya	10 Gya	P value
CA2 apical
Thin spines (no.)	56.07 ± 1.62	54.08 ±2.09	P = 0.58
Stubby spines (no.)	29.54 ± 1.39	30.74 ± 1.47	P = 0.67
Mushroom spines (no.)	14.40 ± 0.69	11.31 ± 1.03	P < 0.05
Overall density (no.)	18.15 ± 0.60	18.18± 0.51	P = 0.96
CA2 basal
Thin spines (no.)	52.39 ± 1.60	55.89 ±1.74	P = 0.25
Stubby spines (no.)	33.36 ±1.75	33.39 ± 1.26	P = 0.99
Mushroom spines (no.)	14.26 ±0.59	10.72 ± 0.93	P < 0.05
Overall density (no.)	16.89 ±0.58	18.16 ± 0.39	P < 0.05

^aMean ± SEM.

^bValues in boldface are significant.

Figure 2: 10 Gy reduces mushroom spine density in the CA 2 hippocampus.

(A)The Apical CA2 underwent reduced mushroom spine density (B) The basal CA2 mushroom spine density was significantly decreased by Irradiation. (C-D) Representative Images. Average ± SEM (n = 5); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Proteomics

Identification of proteins differentially expressed relative to neurocognitive deficits

Identification of specific proteins and pathways associated with neurocognitive dysfunction in 10 Gy-treated C57BL/6 male mice as well as putative candidates that may underlie the behavioral and neuronal changes observed in cranial irradiation treatment, 10 Gy-treated, hippocampal sample cell lysates were analyzed using proteomic analysis relative to a control. A set of 2,076 proteins out of a total of 4,560 identified proteins were differentially expressed between the 10 Gy-treated animals and control animals with a p-value of < 0.05 and a fold change >2 (Table 3, Supplementary 1). Of these 14/2076 were highlighted for their association oxidative stress, neuronal function, and a significant log ratio fold change greater or equal to +/− 2.

Table 3.

Top five IPA protein networks associated with 10 Gy treatment

Network 1	Associated network functions: Cell Morphology, Cellular Assembly and Organization, Cell-To-Cell Signaling and Interaction Number of “focus molecules” contained in the network: 31 IPA p-score: 44 Network proteins: CACNB4,CCDC136,CCDC47,CDIPT,CEP170B,CKAP4,CLASP1,CNP,CTNND2,CTTN BP2,DSG1,EMC1,EMC2,GHITM,GRIA2,LRRC59,MAT2B,MTFR1L,NT5C3A,PGP,PI4 KA,PTPase,Ppp2c,SHANK1,SHANK2,SHISA7,SMAP2,SRC (family),SRCIN1,STRN,STRN4,Sod,TECR,TNIK,TSPAN7
Network 2	Associated network functions: Cellular Assembly and Organization, Cellular Function and Maintenance, Molecular Transport Number of “focus molecules” contained in the network: 30 IPA p-score: 42 Network proteins: AP- 3,AP3D1,AP3S1,ARL8B,BLVRB,Bvr,C1QTNF4,CLIP2,DAD1,DDOST,DIRAS1,DIRAS 2,DMXL2,LMAN1,MCAT,NFkB
Network 3	Associated network functions: Developmental Disorder, Neurological Disease, Organismal Injury and Abnormalities Number of “focus molecules” contained in the network: 30 IPA p-score: 42 Network proteins: 14-3-3(α, ε, ζ) ,ACTB,ATAT1,C2orf72,CD81,DCPS,EEF1A1,EMD,Gm10358,HIST1H1C,HTRA2,IFN
Network 4	Associated network functions: Protein Synthesis, Cellular Compromise, Inflammatory Response Number of “focus molecules” contained in the network: 29 IPA p-score: 39 Network proteins: ARSB,CPPED1,CROCC,CST3,CTSA,CTSZ,Cathepsin,ENDOD1,ERGIC1,Ephb,FIS1,GL B1,Glycoprotein
Network 5	Associated network functions: Cellular Movement, Molecular Transport, Developmental Disorder Number of “focus molecules” contained in the network: 29 IPA p-score: 39 Network proteins: 14-3-3 (α,β,η, δ, ζ, 14-3-3(δ, η, ζ),ANXA3,ATAD3A,CA2,CA4,CADM3,CALB1,CLNS1A,CYFIP2,EPB41L2,EPB41L3,F N3KRP,IDI1,IGSF21,IMPA1,Integrin alpha 3 beta 1,KCNMA1,LIN7A,MARK1,MPC1,MPC2,MPP1,MPP2,NPL,PLLP,PLPP3,SARNP,SER PINB6,SLC4A4,TLR7/8,Vegf,WDR45,YWHAH,c-Src

^*Using a proprietary algorithm, IPA generates these networks and adds proteins not contained within the original dataset. Additionally, IPA calculates a p-score, which indicates the probability of finding a given number of focus molecules in a given network selected randomly from IPA’s Global Molecular Network. The p-score (−log10 (p-value)) is calculated by the Fisher’s exact test.

Proteins, pathways, and protein networks affected by radiation treatment

To gain insight on the potential pathways and networks involved in mediating irradiation-induced neurocognitive dysfunction IPA analysis was conducted on all 2,076 proteins that were identified to be differentially expressed between the 10 Gy-treated and control animals. IPA is a web-based software application that is used to extract from biological meaningful information from our list of differentially expressed proteins. This analysis was able to determine the top 5 canonical pathways (Synaptogenesis Signaling Pathway, Sirtuin Signaling Pathway, EIF2 Signaling, Mitochondrial Dysfunction, and Rac Signaling), which are predictions of pathways that are changing based on gene expression due to 10 Gy radiation treatment (Table 2). The number of proteins within each of these pathways thought to be disrupted is represented in (Fig 3). Interestingly, each of these pathways have associations with neurocognitive disorders^15–18. Additionally, IPA provided non-directional gene interaction maps in the form of networks with associated functions (Table 3). These IPA networks provide information on how proteins with similar functions interact with each other as well as predictions on which direction each of the proteins within the network are dysregulated. For example, in this analysis network 1 has associated functions with cell morphology, cell assembly and organization, and cell to cell signaling and interaction (Fig 4). Similarly, network 3 which has associated networks functions of developmental disorders, neurological disease, and organismal injury and abnormalities (Fig 5). Additional canonical pathways and protein networks disrupted due to 10 Gy radiation treatment can be found in the supplementary material.

Table 2.

Top five pathways associated with memory deficits in 10 Gy treated mice. Overlapping proteins that met statistical significance for differential expression in 10 Gy treated animals (10 Gy vs. 0Gy) comparisons were uploaded into Ingenuity Pathway Analysis (IPA) to identify enriched pathways.

Top Canonical Pathways	P-value
Synaptogenesis Signaling Pathway	1.23e-15
Sirtuin Signaling Pathway	1.11e-14
EIF2 Signaling	2.12e-14
Mitochondrial Dysfunction	7.15e-12
Rac Signaling	1.08e-10

Figure 3.

Number of proteins determined to be dysregulated within each of top 5 pathways associated with memory deficits in 10 Gy-treated mice.

Figure 4. Graphic representation of mouse hippocampus protein network 1, identified by IPA as being affected by 10 Gy-treatment.

The color of the node depicts differential expression. Red represents upregulated proteins. Green represents downregulated proteins. The intensity of the color denotes the degree of regulation where brighter colors are more regulated. Gray node color reflects proteins that were found in the data set but were insignificant expression wise. Uncolored nodes were not identified as differentially expressed in our data, but were incorporated into the computational network based on evidence stored in the Ingenuity Knowledge Base. Known direct and indirect interactions between network proteins, as well as the direction of the interaction, are indicated by arrows or blocked lines.

Figure 5. Graphic representation of mouse hippocampus protein network 3, identified by IPA as being affected by 10 Gy-treatment.

Green indicates downregulation and red indicates. The color of the node depicts differential expression. Red represents upregulated proteins. Green represents downregulated proteins. The intensity of the color denotes the degree of regulation where brighter colors are more regulated. Gray node color reflects proteins that were found in the data set but were insignificant expression wise. Uncolored nodes were not identified as differentially expressed in our data, but were incorporated into the computational network based on evidence stored in the Ingenuity Knowledge Base. Known direct and indirect interactions between network proteins, as well as the direction of the interaction, are indicated by arrows or blocked lines

Discussion

Though cranial radiation is a necessary therapy for some types of cancer, the treatment itself can induce long-term neurocognitive deficits, particularly in children. In this study we investigated how cranial radiation in juvenile mice affected a specific aspect of neurocognition—social recognition. We then explore the radiation-induced changes in neuroanatomy and cellular function of the CA2 of the hippocampus, a brain region known to be important for social memory. Social recognition, which includes the ability to recognize familiar versus novel individuals of the same species, is a major component of social memory. In our study we assay for differences in social cognitive abilities in sham versus irradiated mice by using the three-chamber sociability paradigm, previously established by Dr. Jaqueline Crawley, which has been employed to study social affiliation and social memory ¹⁹. In order to habituate the mice to the experimental apparatus, mice are first placed in the middle chamber, then are allowed to freely explore the flanking compartments. Given that both the left and right compartments are devoid of a stimulus, mice tend to spend equal amounts of time in each. In the next stage of experiments, sociability is tested by giving the subject a choice between an unfamiliar mouse in one chamber and a novel object in the other. As social mammals, the subjects spend more time with the mouse than the object ¹⁹, a trend that was observed in both our sham and our irradiated mice. Sociability includes the choice to engage in social behaviors, but is not an implicit component of social memory. Social novelty, however, can be used an indicator of social recognition. When given a choice between a familiar mouse versus a novel one, mice prefer to explore the novel individual^19,20. We found that 10 Gy-irradiated mice failed to show a preference between a familiar individual and a stranger, perhaps due to an inability to actually recognize the familiar mouse.

The Cornu Ammonis 2 (CA2) of the hippocampus has emerged as an important region for social memory formation. Social recognition memory in animals is essential for social hierarchy, territorial defense, interspecies recognition²¹. Silencing of the CA2, leads to a pronounced deficit in social memory, without impairment in sociability or other hippocampal-dependent behaviors, such as contextual or spatial memory^22,23. In this study, we identified a decrease in overall spine mushroom spine density within CA 2 apical and basal dendrites, but there were no changes in thin spine and stubby spines apical dendrites. Spines are activity-dependent, as high-frequency stimulation induces spine development in hippocampal neurons ²⁴. Thin spines, or “learning” spines, are transient and mobile; mushroom spines, or “memory” spines, are more stable and persist for months ^25,26. Changes in spine density or length are thought to represent a morphological correlate of altered brain functions associated with learning and memory ²⁷. Alterations in spine morphology can disrupt synapse formation and/or stability, which ultimately can lead to neurological and cognitive disorders, such as autism spectrum disorders, Alzheimer’s disease, schizophrenia, anxiety, and depression ²⁸.

In order to understand the molecular mechanisms contributing to hippocampal dependent cognitive impairment after 10 Gy irradiation treatment a comparative proteomic analysis was conducted. Currently there are few studies that have used proteomic technology to explore the molecular underpinnings behind radiation-induced cognitive impairment. One study found that juvenile mice 7 months after receiving low doses of radiation (≤1.0 Gy) treatment there were significant disruptions in key signaling pathways, such as Ephrin B, Rho Family GTPases, RhoGDI, Axonal Guidance, and Ephrin Receptor signaling ²⁹. A similar study was conducted on both 10 week and 10 day old mice 6 months after receiving low/moderate (0.1 and 2 Gy) cranial radiation. In their proteomic analysis, they saw significant dysregulation in pathways such as superoxide radicals degradation, oxidative phosphorylation, and mitochondrial dysfunction for both 10-week-old and 10 day old mice³⁰.

In our study, we implemented a comparative proteomic analysis on 21 day old mice hippocampal tissue 30 days after receiving radiation treatment (10 Gy). Interestingly, in our study which demonstrated short term effects at higher dose of radiation revealed some key similarities to the prior studies discussed. Like previous studies our IPA revealed that one of the top five canonical mechanisms by which 10 Gy cranial irradiation contributes to hippocampal dysfunction is mitochondrial dysfunction (Table 3). These pathways each contain a number of proteins that have been dysregulated as a result of 10 Gy treatment (Figure 4). It has been determined that radiation can damage mitochondria resulting in mitochondrial-induce cellular damage particularly within the central nervous system. The extent of mitochondrial-induced damage on the central nervous system has been characterized as dosage dependent with low doses inducing via antioxidant defenses and higher dosages inducing damage through oxidative stress and neuroinflammation³¹. Also, in this analysis several significantly differentially disrupted proteins were found in 10-Gy hippocampal samples associated with mitochondrial dysfunction (Table 4). For example, subunits of both the cytochrome b (Cytochrome b-c1 complex subunit 6, mitochondrial) and c complex (Cytochrome c oxidase subunit 5A, mitochondrial) were determined to be significantly upregulated with fold changes of 6.74 and 4.90 respectively.

Table 4.

Top up regulated proteins differentially expressed associated with cognitive decline. A comparison of 10 Gy-treated mice versus control mice identified 2076 proteins as meeting statistical significance for differential expression.

Protein	Description	Location	Type	Fold Change (log ratio)
P99028	Cytochrome b-c1 complex subunit 6, mitochondrial	Mitochondrion	Other	6.747313809
B1ARW4	NADH dehydrogenase [ubiquinone] iron-sulfur protein 5;NADH dehydrogenase [ubiquinone] iron-sulfur protein 5, N-terminally processed	Mitochondrion	Other	4.914809526
P12787	Cytochrome c oxidase subunit 5A, mitochondrial	Mitochondrion	Other	4.909787013
Q56A15	Cytochrome c, somatic	Mitochondrion	Other	5.001447813
Q9CQB4	Cytochrome b-c1 complex subunit 7	Mitochondrion	Other	4.529687357
A2RSV8	Cytochrome c oxidase subunit 4 isoform 1, mitochondrial	Mitochondrion	Other	3.750787159
Q06185	ATP synthase subunit e, mitochondrial	Mitochondrion	Other	5.050258482
Q9WVA2	Mitochondrial import inner membrane translocase subunit Tim8 A	Mitochondrion	Other	4.163148008
Q9CQ54	NADH dehydrogenase [ubiquinone] 1 subunit C2	Mitochondrion	Other	3.629549791
Q9CQZ5	NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6	Mitochondrion	Other	3.500211383
Q9ERS2	NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13	Mitochondrion	Other	3.288135065
Q9D6J6	NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial	Mitochondrion	Other	3.940287473
P20108	Thioredoxin-dependent peroxide reductase, mitochondrial	Mitochondrion	Enzyme	3.217668799

Upregulation of mitochondrial proteins after radiation have been characterized in other tissues as well. One study found that irradiation of lung carcinoma cells resulted in an increase of mitochondria abundance and an increase of electron transport chain activity after 3.9 Gy irradiation³². What we can gather from these findings is that low to moderate doses of irradiation seems to consistently disrupt mitochondrial function both in long term and in the short-term post irradiation. This suggests evidence for a radiation response in hippocampal tissue that results in mitochondrial damage and subsequently an increase in reactive oxygen species tissue damage. Thus, oxidative stress is a likely candidate mechanism for the social behavioral deficits we see in these juvenile 10 Gy-irradiated mice (Fig 1). Additionally, IPA identified other disrupted canonical pathways such as EIF2, Rac, Sirutin, and Synaptogenesis signaling pathways, which have all have been implicated in neurocognitive disorders ^33–36.

Another feature of the proteomic analysis was the identification of top disrupted protein networks by the IPA-network functions tool (Table 3). These networks provide non-directional predictions of how significantly differentially expressed proteins identified in the initial analysis interact with each other as well as their relative expression levels. For example, network 1 from this analysis has associated functions with cell morphology, cellular assembly and organization, and cell to cell signaling and interaction (Table 3). This network contains dysregulated central node proteins such as GRIA2 (Glutamate ionotropic receptor AMPA type subunit 2), TNIK (TRAF2-and NCK interacting kinase), and STRN (Striatin) all generally related to dendritic organization, synaptic activity, and spine morphology (Fig 4). GRIA2 is a glutamate ionotropic receptor often expressed on the surface of spines and is responsible for mediating synaptic transmission in the central nervous system thus having a key role in processes like learning and memory³⁷. TNIK has a role in organizing dendritic spines, critical for glutamatergic signaling, and regulation of synaptic strength³⁸. STRN is part of a family of proteins that is largely localized in dendritic spines and has been implicated in dendritic outgrowth and remodeling ⁴². Network 3 from this analysis also contains proteins like Tuba1a (Tubulin alpha-1a chain), which is a major constituent of microtubules and is associated with regulating synapse organization as well as spine development (Fig 5) ^35,39. These results suggest that disruption of these regulators of dendritic spine and synaptic morphology could be molecular candidates to explain the decreases in mushroom spines density observed after 10 Gy treatment (Fig 2). Interestingly, mitochondria have also been implicated in having a role in dendritic spine morphology and synaptic function.

Mitochondria in dendrites are often localized in dendritic shafts and enhance the activity of both spines and synapses. Reduction of mitochondrial content in dendrites has been shown to directly decrease both spine and synaptic abundance⁴⁰. Thus, it is possible that dysregulation of these regulators of spine and synaptic morphology may work mechanistically in tandem with mitochondrial dysfunction to induce the changes in hippocampal physiology seen after 10 Gy-irradiation treatment. Altogether, we conclude that these cognitive deficits and changes in hippocampal physiology after 10 Gy treatment are indicative of both mitochondrial damage and disruption molecular regulators of dendritic morphology. Further validation of these highlighted proteins in this analysis will be needed to further support these findings.

MATERIALS AND METHODS

Animals and Irradiation

Animals

Male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME), 21 days old (n = 20), were used in this study. Mice were irradiated (Schematic). Behavior testing was conducted when mice were 2 months old. The mice were housed under a constant 12-h light/12-h dark cycle and were cared for in compliance with the UAMS Institutional Animal Care and Use Committee.

Schematic diagram showing experimental design.

21 day old C57BL/6J male mice received head only irradiation with the SARRP at UAMS. 30 Days post irradiation behavioral testing was performed.

Small-Animal Radiation Research Platform

The male mice were irradiated with the use of the SARRP. They were anaesthetized with 1% isoflurane inhalation for the duration of the radiation exposure at 0 Gy (n = 10) 10 Gy (n = 10). Each mouse was placed prone on the horizontal bed in the SARRP. A cone beam computed tomographic image of each mouse was obtained to deliver image-guided radiation targeted to the brain at 60 kVp and 0.8 mA; reconstruction was accomplished with the use of 720 projections. From Merislice, image precision targeting of the brain was determined to avoid the eyes and frontal lobe. One cohort received 1 round of 2 × 5-Gy x-ray irradiation from 90° and −90° gantry angles with an isocenter tissue depth of 5 mm. The brain-targeted radiation doses were delivered with a 0.5-mm copper filter with a 5 × 5 mm collimator using 220 kVp and 13 mA. All sham-irradiated mice were anaesthetized and placed in the SARRP for an equivalent amount time as the irradiated mice.

Three-Chamber Sociability

Next, mice underwent testing for social memory via the three-chamber sociability paradigm, in which mice freely explore three adjacent chambers for 10 minutes in three consecutive trials. The first trial consists of habituation, allowing mice to explore the 3 empty chambers. Trial 2 involves social familiarization, where a previously unknown animal (stimulus 1) of the same sex, strain, and age is placed in a small vertical cage on one of the lateral chambers, and the other lateral chamber contains an identical, but empty cage. For trial 3, another previously unknown animal (stimulus 2) of the same sex, strain, and age is placed in a small vertical cage on the opposite lateral chamber. Chamber selections for trials 2 and 3 are randomized to ensure there is no location bias. Strangers were randomly selected on a non-consecutive basis for each subject. Strangers were naïve animals housed in separate facilities and had no contact with test animals prior to behavioral testing, and were not aggressive. The apparatus is made of an aluminum floor, transparent acrylic walls, and an open ceiling. Each adjacent chamber has a dimension of (40 × 20 × 23 cm). Cages are made of aluminum and plastic. The tracking software was programmed to track animal nose-points during all trials.

Golgi Staining

The day after behavioral testing, animals were anesthetized with isoflurane, their brains were subsequently collected and dissected along the midsagittal plane. The Golgi method of staining has long proven to be a reliable method for assessing dendrite and dendritic spine dynamics due to various conditions, because of its resistance to fading or photobleaching over time.^41,42 We adapted a staining protocol and used the reagents contained in the superGolgi kit (Bioenno Tech).⁴³ Right hemispheres were immediately impregnated in a potassium dichromate solution for two weeks (n = 5). Next, sections were immersed for at least 48 hours in a post-impregnation buffer. Samples were sectioned at 200 μm in 1X PBS along the coronal plane. Samples were then transferred into wells and washed with 0.01 M PBS buffer (pH 7.4) with Triton X-100 (0.3%) (PBS-T). Immediately after washing, samples were stained with ammonium hydroxide and then immersed in a post-staining buffer. Sections were again washed in PBS-T, mounted on 1% gelatin-coated slides, and allowed to dry. Sections were finally dehydrated with ethanol solutions, followed by cleaning in xylene, and coverslipped with Permount™ (Thermo Fisher Scientific).

Dendritic Morphology Quantification

Dendritic spines were also analyzed using Golgi-stained brain sections featuring the hippocampus. The criteria for neurons chosen for analysis was the following: 1. non-truncated dendrites; 2. consistent Golgi staining along dendrites; 3. relative isolation from other neurons to avoid interference ⁴⁴. The neurons were traced using Neurolucida software version 11. Six to seven neurons per brain with 5 dendritic segments (at least 20 nm long) per neuron were analyzed ⁴⁵.

Tissue Preparation

The hippocampus was removed and placed in 400 μl of RIPA lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5 M EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl). The tissue was homogenized on ice, incubated for 30 min on ice, and then centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was transferred to a new microcentrifuge tube and stored at −80 °C until processing. The Compat-Able™ Protein Assay Preparation Reagent Kit (Thermo Scientific ™) was used to eliminate EGTA as an interfering substance for the BCA Pierce TM BCA Protein Assay Kit (Thermo Scientific ™). Protein was separated by SDS-PAGE using the 4–15% Criterion™ TGX™ Precast Midi Protein Gel, 18-well, 30 uL (Bio-Rad) ran at 120V for 75 min. The gel was stained using Coomassie Blue staining (Bio-Rad). The samples were then sent to the UAMS Proteomics Core for further processing for mass spectrometry.

GeLC-MS/MS Analysis

Each SDS-PAGE gel lane was sectioned into 12 segments of equal volume. Each segment was subjected to in-gel trypsin digestion as follows. Gel slices were destained in 50% methanol (Fisher), 100 mM ammonium bicarbonate (Sigma-Aldrich), followed by reduction in 10 mM Tris[2-carboxyethyl]phosphine (Pierce) and alkylation in 50 mM iodoacetamide (Sigma-Aldrich). Gel slices were then dehydrated in acetonitrile (Fisher), followed by addition of 100 ng porcine sequencing grade modified trypsin (Promega) in 100 mM ammonium bicarbonate (Sigma-Aldrich) and incubation at 37^oC for 12–16 hours. Peptide products were then acidified in 0.1% formic acid (Pierce). Tryptic peptides were separated by reverse phase XSelect CSH C18 2.5 um resin (Waters) on an in-line 150 × 0.075 mm column using a nanoAcquity UPLC system (Waters). Peptides were eluted using a 30 min gradient from 97:3 to 67:33 buffer A: B ratio. [Buffer A = 0.1% formic acid, 0.5% acetonitrile; buffer B = 0.1% formic acid, 99.9% acetonitrile.] Eluted peptides were ionized by electrospray (2.15 kV) followed by MS/MS analysis using higher-energy collisional dissociation (HCD) on an Orbitrap Fusion Tribrid mass spectrometer (Thermo) in top-speed data-dependent mode. MS data were acquired using the FTMS analyzer in profile mode at a resolution of 240,000 over a range of 375 to 1500 m/z. Following HCD activation, MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with precursor mass-dependent normalized collision energy between 28.0 and 31.0.

Data Analysis

Proteins were identified and quantified by searching the UniprotKB database restricted to Mus musculus using MaxQuant (version 1.6.5.0, Max Planck Institute). The database search parameters included selecting the MS1 reporter type, trypsin digestion with up to two missed cleavages, fixed modifications for carbamidomethyl of cysteine, variable modifications for oxidation on methionine and acetyl on N-terminus, the precursor ion tolerance of 5 ppm for the first search and 3 ppm for the main search, and label-free quantitation with iBAQ normalized intensities. Peptide and protein identifications were accepted using the 1.0% false discovery rate identification threshold. Protein probabilities were assigned by the Protein Prophet algorithm ⁴⁶.

MaxQuant iBAQ intensities for each sample were median-normalized so the medians were equal to the sample with the maximum median. Median-normalized iBAQ intensities were then imported into Perseus (version 1.6.1.3, Max Planck Institute) to perform log2 transformation and impute the missing values using a normal distribution with a width of 0.3 and a downshift of 2 standard deviations. The Linear Models for Microarray Data (limma) Bioconductor package was used to calculate differential expression among the experimental conditions using the lmFit and eBayes functions ^46,47. Proteins were considered to be significantly different with a fold change > 2 and an FDR adjusted p-value < 0.05. Differentially expressed proteins were analyzed using Ingenuity Pathway Analysis (IPA, QIAGEN Redwood City, www.qiagen.com/ingenuity) to identify pathways and networks.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD017596 and 10.6019/PXD017596”.