Disruption of Timing: NeuroHIV Progression in the Post-cART Era

The marked increase in life expectancy for HIV-1 seropositive individuals, following the great success of combination antiretroviral therapy (cART), heralds an examination of the progression of HIV-1 associated neurocognitive disorders (HAND). However, since the seminal call for animal models of HIV-1/AIDS in 1988, there has been no extant in vivo animal model system available to provide a truly longitudinal study of HAND. Here, we demonstrate that the HIV-1 transgenic (Tg) rat, resembling HIV-1 seropositive individuals on lifelong cART, exhibits age-related, progressive neurocognitive impairments (NCI), including alterations in learning, sustained attention, flexibility, and inhibition; deficits commonly observed in HIV-1 seropositive individuals. Pyramidal neurons from layers II-III of the medial prefrontal cortex (mPFC) displayed profound synaptic dysfunction in HIV-1 Tg animals relative to controls; dysfunction that was characterized by alterations in dendritic branching complexity, synaptic connectivity, and dendritic spine morphology. NCI and synaptic dysfunction in pyramidal neurons from layers II-III of the mPFC independently identified the presence of the HIV-1 transgene with at least 78.5% accuracy. Thus, even in the absence of sensory or motor system deficits and comorbidities, HAND is a neurodegenerative disease characterized by age-related disease progression; impairments which may be due, at least partly, to synaptic dysfunction in the mPFC. Further, the progression of HAND with age in the HIV-1 Tg rat and associated synaptic dysfunction affords an instrumental model system for the development of therapeutics and functional cure strategies.

litters) and control (N = 17 litters) rats were received at the animal vivarium, between PD 7 and PD 9, housed with their biological dam. At approximately PD 21, animals were sampled from each litter (HIV-1 Tg: male, n = 37, female, n = 33; Control: male n = 34; female, n = 33), weaned and pair-or group-housed with animals of the same sex for the duration of experimentation. During the original acquisition of signal detection, animals were placed on food restriction, beginning at approximately PD 60, to maintain 85% body weight. After animals successfully acquired the signal detection task (PD 100-PD 277), rodent food was again provided ad libitum, including during all retest assessments. HIV-1 Tg and control animals had ad libitum access to water throughout the duration of the study.
HIV-1 Tg and control animals were sacrificed following the successful completion of the reversal task at approximately 20 months of age to assess synaptic dysfunction in pyramidal neurons from layers II-III of the mPFC and putative markers of neuroinflammation in the hippocampus. Due to health issues, including significant weight loss (n = 23), tumors (n = 6), natural death (n = 3) or other (n = 4), some animals were euthanized prior to 20 months of age.
Recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health were used for the maintenance of HIV-1 Tg and control animals in AAALAC-accredited facilities. The targeted environmental conditions for the animal vivarium were 21° ± 2°C, 50% ± 10% relative humidity and a 12-h light:12-h dark cycle with lights on at 0700 h (EST). The Institutional Animal Care and Use Committee (IACUC) at the University of South Carolina approved the project protocol (Federal Assurance # D16-00028).
Phase 1: Original Acquisition. Apparatus. Sixteen operant chambers, located inside sound-attenuating chambers (Med Associates, Inc., Fairfax, VT), were used to train animals in a signal detection task tapping sustained attention. A pellet dispenser (45 mg), located between two retractable levers, and three panel lights were located on the front wall of the operant chambers. Only the panel light located above the pellet dispenser was used in the present experiment. A house light was located at the top of the rear wall of the operant chamber. The house light, used for shaping, and central panel light, used for signal presentation during the original acquisition of signal detection and retest assessments, were incandescent (22 lux). PC and Med-PC for Windows software (V 4.1.3; Med Associates, Inc., Fairfax, VT) controlled signal presentation, lever operation, reinforcement delivery, and data collection. Shaping. A standard shaping response protocol was used to train animals to lever-press at approximately 2 months of age, described in detail by McLaurin et al. 18 . All animals met criteria in shaping (i.e., 60 reinforcers for 3 consecutive or 5 non-consecutive days), indicating learning the operant response, prior to beginning the signal detection task.
Signal Detection. Following successful acquisition of shaping, all HIV-1 Tg (N = 20 litters; male, n = 37, female, n = 33) and control animals (N = 17 litters; male n = 34; female, n = 33) were trained in a signal detection task, tapping sustained attention. Three vigilance programs, initially described by McGaughy and Sarter 19 , were employed. In brief, animals were trained to discriminate between signal (i.e., central panel light illumination) and non-signal (i.e., no illumination) trials. In the first vigilance program, consisting of 160 trials per session, termination of the stimulus light was contingent upon a response. In the second vigilance program, consisting of 160 trials per session, the length of the stimulus light was 1 sec. Correction trials, including up to three repetitions of the trial, and force-choice trials were an integral component of the first two vigilance programs, and occurred when an animal responded incorrectly. In the third vigilance program, consisting of 162 trials per session, the length of the stimulus was manipulated (i.e., 1000, 500, 100 msec) across trials using a block randomized experimental design. Correction trials and force-choice trials were removed in the third vigilance program. During the initial acquisition, no data was censored i.e., each and every animal successfully acquired the signal detection task. A preliminary report of original acquisition, on approximately half of the animals, was presented in McLaurin et al. 18 . Additional methodological details are presented in the Supplementary Information Text available online. Phase 2: Retest Assessments. Apparatus. Throughout the retest assessments, HIV-1 Tg and control animals were assessed in the operant chambers described above.
Procedure. After the successful completion of original acquisition, HIV-1 Tg and control animals were assessed in the third vigilance program with varying signal durations every 60 days through approximately 18 months of age. Animals were given up to 60 days to successfully complete each retest assessment and were assessed in up to 6 retest assessments. Phase 3: 18 Month Assessment. Apparatus. At the 18 Month Assessment, HIV-1 Tg and control animals were assessed in the operant chambers described above with a minor modification. An LED light bulb (11 lux), instead of an incandescent light bulb, was used for signal presentation during the 18 month acquisition and reversal assessment due to the short (i.e., 10 msec) signal duration times.
Procedure. After the successful completion of the 6 th retest assessment or at 18 months of age, HIV-1 Tg and control animals were challenged with two tasks. First, HIV-1 Tg (N = 20 litters; male, n = 29, female, n = 29) and control animals (N = 17 litters; male n = 28; female, n = 31) were challenged for 5-consecutive days with shorter signal durations (i.e., 1000, 100, 10 msec). Second, HIV-1 Tg (N = 20 litters; male, n = 18, female, n = 29) and control animals (N = 17 litters; male n = 27; female, n = 28) were assessed in a reversal task, tapping flexibility and inhibition. Response contingencies were reversed from those originally learned in the signal detection task. Animals were given up to 60 days to successfully complete the reversal assessment or were sacrificed at approximately 20 months of age after 45 days in the task. Phase 4: Neuroanatomical Assessments. Synaptic Dysfunction. Preparation of Tissue: Animals were deeply anesthetized using sevoflurane (Abbot Laboratories, North Chicago, IL) and transcardially perfused using methodology adapted from Roscoe et al. 12 .
DiOlistic Labeling: A DiOlisitc labeling technique, originally described by Seabold et al. 20 , was used to visualize pyramidal neurons from layers II-III of the medial prefrontal cortex (mPFC). Methodology for the preparation of DiOlistic cartridges, preparation of Tefzel tubing, and DiOlistic labeling is described in detailed by McLaurin et al. 21 .
The AutoNeuron and AutoSpine extension modules, available in Neurolucida 360 (MicroBrightfield, Williston, VT), were used for the analysis of spine parameters. Selection criteria, including continuous dendritic staining, low background/dye clusters, and minimal diffusion of the DiI dye into the extracellular space, were used for spine analysis. Based on the selection criteria, one neuron from each animal was chosen for the analysis of spine parameters. Neurons not meeting the selection criteria were not included in the analysis, yielding Control, N = 16 litters, male, n = 26, female, n = 20, and HIV-1 Tg N = 19 litters, male, n = 28, female, n = 27.
Spine Parameters: An observer-assisted automatic classification of dendritic spines (i.e., thin, mushroom, stubby) was conducted using an algorithm in Neurolucida 360 23 . The number of segments at each branch order, an assessment of dendritic branching complexity, was examined for branch orders 1 through 10. A Sholl analysis was conducted to evaluate neuronal complexity (i.e., the number of intersections at each successive radii) and dendritic spine connectivity (i.e., the number of dendritic spines between each successive radii). Some animals were excluded due to processing errors, yielding Control, N = 16 litters, male, n = 21, female, n = 18, and HIV-1 Tg N = 17 litters, male, n = 26, female, n = 23. Subsequently, dendritic spine morphology was assessed using three parameters, including backbone length (µm), head diameter (µm), and volume (µm 3  defined using well-accepted previously published results [i.e., backbone length, 0.1 to 4.1 µm 24 ; head diameter, 0 to 0.825 µm 25 ; volume, 0.05 to 0.5 µm 3 26 ]. Neuroinflammatory Markers. The selection of the hippocampus for the assessment of neuroinflammation was based on the publication of Fitting et al. 27,28 , which displayed frank cell loss in the hippocampus from viral protein exposure, maximizing the likelihood of detecting neuroinflammation. Total RNA isolation and cDNA synthesis: Total RNA was isolated from 30 mg of hippocampal tissue using RNeasy FFPE kit (QIAGEN) according to manufacturer's protocol (Control: N = 17 litters, male, n = 31 female, n = 30; HIV-1 Tg: N = 20 litters, male n = 32, female n = 30). The total RNA quality and quantity were assessed by a Nano drop spectrophotometer (Thermo Scientific, USA). One µg of total RNA from each sample was converted into cDNA by Cloned AMV first-strand cDNA synthesis kit (Invitrogen) for real-time PCR. The 20 μl first-strand cDNA synthesis reaction mixture contained 1 μl random-hexamer primer, 1 µg total RNA sample, 2 μl of 10 mM dNTP mix, 5x cDNA synthesis buffer, 1 μl of 0.1 M DTT, 1 μl RNaseOUT, 1 µl cloned AMV RT and 1 µl DEPC-treated water. The following conditions were used: 65 °C for 5 min, 25 °C for 10 min, 50 °C for 50 min and terminated at 85 °C for 5 min. The cDNA products were used immediately for further analysis.
Real-time PCR: The neuroinflammation related cytokines (TNF-α, IL-1β, IL-6) were quantified using a real-time PCR detection system and SsoAdvanced Universal SYBR Green Supermix kit (BIO-RAD). In brief, the 20 μl reaction mixture contained 10 μl 2x SsoAdvanced universal SYBR Green supermix, 1 μl forward primer and reverse primers (250 nM each), 1 μl template (100 ng) and 7 μl of DEPC-treated water. Reactions were performed with the DNA Engine Opticon 2 system (M J Research, USA) using the following cycling conditions: 30 sec at 95 °C and 40 cycles of 15 sec at 95 °C and 30 sec at 58 °C. Animals failing to reach threshold after 40 cycles were considered to have undetectable gene expression and accordingly these censored data were not included in the figures or statistical analysis, yielding Control, N = 8-15 litters, male, n = 7-18, female, n = 8-12, and HIV-1 Tg N = 11-13 litters, male, n = 6-14, female, n = 9-15, dependent upon gene. The data were analyzed using Intuitive Opticon Monitor TM software. Each primer, including the corresponding GenBank numbers, are listed in Supplementary Table 1, and β-Actin was used as an internal control. The relative gene expression of each neuroinflammatory marker (i.e., TNF-α, IL-1β, IL-6) was analyzed using the 2 −ΔΔCt method to examine relative changes in expression dependent upon genotype and biological sex 30 . statistical Analysis. Given the nested experimental design (i.e., pups within litter), figures, created using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA), represent litter means and standard errors, dependent upon biological sex 31,32 .
Analysis of variance (ANOVA) and regression techniques were used for the analysis of all data (SAS/STAT Software 9.4, SAS Institute, Inc., Cary, NC; SPSS Statistics 24, IBM Corp., Somer, NY; GraphPad Software, Inc., La Jolla, CA). For all statistical analyses, litter was used as the unit of analysis to preclude the violation of the assumption of independence, leading to an inflation of Type 1 error rate 33 . Orthogonal decomposition of the repeated-measures factors or the conservative Greenhouse-Geisser df correction factor [p GG 34 ] were used to preclude potential violations of sphericity. Effect size was assessed using partial eta squared (η p 2 ). An alpha value of p ≤ 0.05 was considered significant for all statistical tests. Additional details on the statistical analyses are available in the Supplementary Information Text.

Results
Phase 1: Original Acquisition. The factor of biological sex was the driving factor for observed differences in the temporal process of acquisition and sustained attention during the initial acquisition of signal detection. Temporal Process of Acquisition: All HIV-1 Tg and control animals were able to successfully acquire the task, achieving 70% accuracy for 5 consecutive or 7 non-consecutive days in each of the three vigilance programs. Profound sex differences were observed in the temporal process of task acquisition, best fit using a sigmoidal dose-response curve ( Fig. 2A; R 2 s ≥ 0.98). Female animals, independent of genotype, acquired the task significantly slower than male animals (Main Effect: Sex [F(1,61) = 101.1, p ≤ 0.001]). Presence of the HIV-1 transgene failed to significantly alter the temporal process of initial acquisition of signal detection ( Fig. 2B; p > 0.05).
Signal Detection: The factor of biological sex (Fig. 2C) was the driving factor for observed differences in sustained attention during the initial acquisition of signal detection. Female animals, collapsed across genotype, displayed a decreased response rate, as well as a prominent rightward shift in the loss of signal detection (i.e., where the number of hits and misses intersect), relative to male animals (approximately 535 msec vs. 436 msec, respectively; Sex × Response Type or 7 non-consecutive days. To assess the progression of task acquisition across retest assessments, the number of days required for 80% of the population sampled to meet criteria was quantified (Fig. 3). Control animals displayed a continuous improvement in task acquisition across retest assessments, reaching asymptotic performance at approximately 7 days. In sharp contrast, HIV-1 Tg animals exhibited an improvement in task acquisition through retest session 2, followed by a progressive decline. Assessment of the temporal process of acquisition at each individual retest assessment (see Supplementary Fig. 1) revealed a significant main effect of genotype (p ≤ 0.05) at all retest assessments.
Despite the "savings" afforded by repeated testing, HIV-1 Tg animals displayed a progressive, relative impairment in the detection of shorter signal durations. Signal Detection: Across retest assessments, genotype became the driving factor for observed differences in sustained attention. Independent of genotype, HIV-1 Tg and control animals exhibited a significant improvement in the detection of shorter signal durations across retest assessments (Fig. 4). However, HIV-1 Tg animals displayed a progressive, relative impairment in the detection of shorter signal durations relative to control animals (Genotype × Response Type × Duration × Retest Assessment Interaction [F(8,488) = 2.2, p GG ≤ 0.05, η p 2 = 0.034, Power = 0.763] with a prominent quadratic-quadratic component [F(1,61) = 7.3, p ≤ 0.009, η p 2 = 0.106, Power = 0.756]). Specifically, HIV-1 Tg animals took significantly longer to reach the same level of performance (e.g., Loss of Signal Detection at 10 msec: Control animals (Retest 1); HIV-1 Tg animals (Retest 5)) and failed to improve to the level of control animals at any retest assessment.
Relative to genotype, the factor of biological sex played a less prominent role in the progression of sustained attention (Sex × Response Type × Duration × Retest Assessment Interaction with a prominent linear-linear Complementary analyses were conducted for each sex to determine the locus of the interaction. In male animals (Fig. 5C), presence of the HIV-1 transgene significantly altered the temporal process of acquisition with male HIV-1 Tg animals acquiring the task significantly slower than male control animals (Main Effect, Genotype: [F(1,26) = 9.5, p ≤ 0.005]). In sharp contrast, presence of the HIV-1 transgene failed to significantly alter the temporal process of acquisition in female animals ( Fig. 5D; p > 0.05).
Signal Detection: Assessment of signal detection data revealed a significant Task × Response Type × Duration Male HIV-1 Tg animals revealed marked impairment in signal detection relative to male control animals evidenced by a significant rightward shift in the loss of signal detection during the 18 month acquisition (Fig. 6A,C). During the reversal assessment, male control animals displayed a significant increase in both the number of hits and misses, suggesting an overtraining reversal effect. No significant alterations in the number of hits and/ or misses was observed in the male HIV-1 Tg animals, suggesting that, despite extensive training, there was no improvement in their performance during the reversal task.
In sharp contrast, at 18 months of age, female HIV-1 Tg animals displayed no impairment in the detection of shorter signal durations relative to female control animals (Fig. 6B,D). However, female HIV-1 Tg animals, relative to female control animals, exhibited marked impairment in the reversal task evidenced by an inability to reliably detect the signal at any duration assessed.

HIV-1 Tg animals displayed a profound shift in the distribution of dendritic spines in pyramidal neurons from layers
II-III of the medial prefrontal cortex, supporting an alteration in synaptic connectivity. Sholl analyses were subsequently utilized to determine where dendritic spines were located on the neuron, assessed using spine type (i.e., Thin Spines, Stubby Spines, Mushroom Spines), determined using an observer-assisted automatic classification system in Neurolucida 360, genotype (i.e., HIV-1 Tg, Control), biological sex (i.e., Male, Female) and radii as factors (Fig. 8). HIV-1 Tg animals exhibited a prominent shift in the distribution of dendritic spines dependent upon the factor of biological sex and spine type (Genotype × Sex × Spine Type × Radii Interaction [F(2, 6265) = 21.5, p ≤ 0.001]). The effect or lack of effect of biological sex on spine type is illustrated in Supplementary Fig. 2.
Complementary analyses were conducted on each spine type to determine the locus of these interactions. HIV-1 Tg animals displayed a prominent shift, with an increased relative frequency of thin dendritic spines on more distal branches relative to control animals (Genotype  During the 18 month acquisition task, HIV-1 Tg and control animals were challenged for 5 consecutive days with shorter signal durations (i.e., 1000, 100, 10 msec). Male HIV-1 Tg animals (A) displayed a shift towards a decreased number of days meeting criteria (i.e., 70% accuracy) relative to male control animals (p ≤ 0.001); a distribution shift not observed in female HIV-1 Tg animals (B) p > 0.05). The temporal process of acquisition in the reversal task was assessed for HIV-1 Tg animals that had two months (i.e., 60 days) to acquire the task. Male HIV-1 Tg animals took significantly longer to acquire the reversal task relative to male control animals (C) p ≤ 0.005); an impairment not observed in female HIV-1 Tg animals (D) p > 0.05). Data are presented as cumulative frequencies with 95% confidence intervals fit to the curve. (Genotype × Radii Interaction: p > 0.05). No significant differences in the distribution of mushroom dendritic spines were observed (p > 0.05). There was no significant difference in neuroinflammation in the hippocampus between HIV-1 Tg and control animals. Three putative neuroinflammatory markers, including IL-1β, IL-6, and TNF-α, were assessed in the hippocampus of HIV-1 Tg and control animals (Fig. 9). Overall, HIV-1 Tg and control animals, independent of biological sex, reaching threshold after 40 cycles, displayed low levels of gene expression. No significant genotype (p > 0.05) or sex (p > 0.05) differences in gene expression, examined using the 2 −ΔΔCt method, were observed.  Synaptic dysfunction, assessed in pyramidal neurons from layers II-III of the medial prefrontal cortex, explains significant genotypic variance. A DFA was utilized to determine whether synaptic dysfunction in pyramidal neurons from layers II-III of the mPFC could classify animals based on genotype (Fig. 10B). Variables included in the stepwise DFA represented alterations in dendritic branching complexity, dendritic spine connectivity, and dendritic spine morphology.

A selective population shift in dendritic spine morphology was observed in layers II-III pyramidal neurons of the medial prefrontal cortex dependent upon presence of the
HIV-1 Tg and control animals were maximally separated (canonical correlation of 0.72) by selecting seven variables corresponding to dendritic spine connectivity (i.e., Relative Frequency of Thin Dendritic Spines at 150 µm; Relative Frequency of Stubby Dendritic Spines at 40 µm, 110 µm, and 170 µm) and dendritic spine head diameter

Discussion
Progressive NCI, including alterations in learning, sustained attention, flexibility, and inhibition, were observed in the population of HIV-1 Tg rats sampled. During initial acquisition, biological sex was the driving factor for observed differences in learning and sustained attention. Across retest assessments, presence of the HIV-1 transgene became the prominent factor underlying alterations in the temporal process of task acquisition, as well as sustained attention. Nevertheless, at 18 months of age, sex-dependent expression of NCI were observed in HIV-1 Tg animals, relative to controls. Pyramidal neurons from layers II-III of the mPFC revealed profound synaptic dysfunction in HIV-1 Tg animals relative to controls; dysfunction that was characterized by alterations in dendritic branching complexity, synaptic connectivity, and dendritic spine morphology. NCI across multiple domains (i.e., learning, sustained attention) and synaptic dysfunction in the mPFC independently identified the presence of the HIV-1 transgene with at least 78.5% accuracy, accounting for at least 61.6% of the genotypic variance. Thus, even in the absence of sensory or motor system deficits 14 and comorbidities, HAND is a neurodegenerative disease characterized by age-related disease progression; impairments which may be due, at least partly, to synaptic dysfunction in the mPFC. Located at the anterior pole of the mammalian brain, the prefrontal cortex (PFC), recognized as an anatomically complex brain region 35 , is organized in a laminar fashion and comprised of three major subdivisions (i.e., mPFC, orbital PFC (oPFC) and lateral PFC). Executive functions, including attention, flexibility, and inhibition, are the primary, most basic function of the PFC 36 . Although executive functions cannot be localized to any single subdivision of the PFC, the mPFC, oPFC, and lateral PFC each exhibit some degree of functional specialization 35,36 . Specifically, the mPFC has been implicated as having a primary role in attention, including pre-attentive processes [e.g. 37 ] and sustained attention [e.g. 38 ], whereas the oPFC is involved in stimulus-reinforcement learning [e.g. 39 ] and reversal [i.e., flexibility and inhibition 40 ].
The utilization of a series of neurocognitive tasks across the functional lifespan tapped multiple subdivisions of the PFC, revealing progressive NCI in the HIV-1 Tg rat. At the genotypic level, HIV-1 Tg animals displayed a progressive, relative impairment in tasks tapping both the mPFC (i.e., sustained attention) and the oPFC (i.e., stimulus-reinforcement learning). Most notably, HIV-1 Tg animals also exhibited alterations in the progression of prepulse inhibition, a pre-attentive process tapping, in part, the mPFC 14 . However, sex differences in the presentation of NCI at 18 months of age were dependent upon the brain region tapped via neurocognitive assessments (i.e., mPFC vs. oPFC). Specifically, male HIV-1 Tg animals exhibited significantly greater impairment, relative to male control animals, in sustained attention and stimulus-reinforcement learning in the reversal assessment, tasks tapping both the mPFC and oPFC. Female HIV-1 Tg animals, however, displayed profound impairment, relative to female control animals, in flexibility and inhibition, tasks predominantly associated with the oPFC. Elucidating the mechanisms underlying sex-dependent expression of NCI is vital for the development of therapeutic treatments and cure strategies. The rich cortical and subcortical connections make the PFC ideally positioned to serve as a central hub, integrating and relaying information from multiple afferent sources 41 . Broadly, the PFC receives afferents from thalamic nuclei, the limbic system, the frontal cortex, and other brain regions [e.g., hypothalamus, cerebellum, mesencephalon 36 ]. Dense innervation from multiple neurotransmitter systems to the PFC has also been well-established 36 ; afferents which play a prominent regulatory role across the PFC 36 and are functionally involved in the control of executive functions [for review 42 ]. Specifically, noradrenergic projections from the locus coeruleus (LC), dopaminergic afferents from the ventral tegmental area (VTA), and serotonergic projections from the raphe nuclei innervate the PFC.
Excitatory pyramidal neurons, characterized by a single apical dendrite, multiple shorter basal dendrites, and thousands of dendritic spines are abundant throughout the PFC 43 . A synaptic relationship between pyramidal neurons and the major afferent systems is established via postsynaptic sites on dendritic shafts and spines; the vast majority of which occur on dendritic spines [e.g. 44 ]. Morphologically, dendritic spines are classically characterized into three primary categories [i.e., thin, stubby, mushroom 45 ]. Thin spines, the predominant spine type in both HIV-1 Tg and control animals, are characterized by a long, thin neck and a small bulbous head, whereas the head volume to neck volume ratio is higher in mushroom spines. In sharp contrast, stubby spines are devoid of a spine neck, exhibiting an approximately equal head and neck volume ratio 45 . Most notably, asymmetric excitatory synapses are primarily formed between dendritic spine heads and presynaptic axons [e.g. 46 ], however, some dendritic spines receive additional input on their neck 47 . Thus, morphological characteristics of dendritic spines, which are reflective of functionality and capacity for structural change 48 , and their distribution along the dendrite, may be associated with synaptic dysfunction in the HIV-1 Tg rat.
Neuronal morphology, assessed using a branch order analysis, tapping dendritic branching complexity, as well as the classical Sholl analysis, tapping neuronal arbor complexity, revealed prominent alterations in HIV-1 Tg animals relative to controls (Fig. 11). In pyramidal neurons from layers II-III of the mPFC, assessed in the present study, HIV-1 Tg animals exhibited decreased dendritic branching complexity; an effect independent of biological sex. No statistically significant genotypic differences were observed in dendritic length, consistent with previous reports in younger HIV-1 Tg animals 49 or neuronal arbor complexity. In sharp contrast, in medium spiny neurons (MSNs) from the nucleus accumbens core subregion (NAcc), published results suggest sex-dependent alterations in neuronal morphology 50 . Specifically, female HIV-1 Tg animals, but not male HIV-1 Tg animals, displayed profound alterations in dendritic branching complexity and neuronal arbor complexity 50 . Morphological differences between pyramidal neurons, characterized as polar neurons, and MSNs, characterized by a centrifugal Figure 11. Dendritic spine connectivity in pyramidal neurons of the medial prefrontal cortex is illustrated as a function of genotype (HIV-1 Tg vs. control). Pyramidal neurons in HIV-1 Tg animals (B) displayed decreased dendritic branching complexity, with a decreased relative frequency of higher order branches relative to control animals (A) with no observed alterations in total dendrite length. Control animals exhibited a preponderance of thin spines on more proximal branches, receiving dopaminergic afferents from the ventral tegmental area (VTA) and noradrenergic innervation from the locus coeruleus (LC). In sharp contrast, HIV-1 Tg animals displayed a preponderance of stubby spines on more proximal branches. Morphological characteristics of stubby spines, including the absence of a spine neck 45  morphology, may underlie differences in branch order and Sholl analyses. Overall, results support alterations in neuronal morphology in HIV-1 Tg rats independent of brain region assessed. However, the comparison of results from branch order analyses with those from Sholl analyses suggests that the two analytic measures may reflect different measures of neuronal morphology. HIV-1 Tg animals, independent of biological sex, exhibited a profound shift in the distribution of dendritic spines along the apical dendrite, supporting a prominent alteration in synaptic connectivity. Specifically, a preponderance of thin spines were observed on more distal dendrites, extending into layer I of the mPFC, in HIV-1 Tg animals, relative to controls. However, HIV-1 Tg animals, relative to controls, displayed an increased relative frequency of stubby spines on more proximal dendrites; an effect which was most prominent in female HIV-1 Tg rats. Notably, pyramidal neurons within layers II-III of the mPFC express multiple monoamine receptors, including noradrenergic receptors (i.e., primarily α 1A , α 1D ), dopaminergic receptors (i.e., primarily D 1 ) and serotonergic receptors (i.e., primarily 5-HT 1A , 5-HT 2A ); minimal, if any, receptor expression within layer I of the mPFC 51 . The preponderance of stubby spines, morphologically characterized by the absence of a dendritic spine neck 45 and a smaller postsynaptic density 52 , on more proximal dendrites suggests that HIV-1 Tg animals fail to receive afferent projections, and thus neurotransmitter innervation, from the VTA, LC, and raphe nuclei (Fig. 11); consistent with neurotransmitter system alterations, specifically dopaminergic system dysfunction, commonly reported in HIV-1 [e.g. [53][54][55][56] ]. One previous study 49 and one conference abstract 57 have also suggested alterations in dendritic spine density in the mPFC in younger HIV-1 Tg animals; an effect which may also influence synaptic connectivity. Thus, profound synaptic dysfunction was observed in HIV-1 Tg animals, characterized by alterations in dendritic branching complexity, synaptic connectivity (i.e., VTA-PFC-LC), and dendritic spine morphology, supporting a key neural mechanism for expression of NCI in HAND.
Multiple animal systems are available to model components of neuroHIV, each with their own advantages and constraints. Several major considerations of the prominent animal species and their applicability to provide a biological system to model neuroHIV in the post-cART era are highlighted in Fig. 12. Central nervous system (CNS) infection occurs within two weeks of HIV-1 infectivity in humans 58,59 , leading to NCI [e.g. 6,7 ] and behavioral alterations [e.g., Apathy 60 ; Depression 61 ]. Selective NCI [e.g., selective attention appears relatively spared 15 ] have been observed across multiple domains in prominent animals systems [e.g., HIV-1 Tg rat, pre-attentive processes 13 ; learning and memory 16,17 ; executive function 15 ; SIV, spatial working memory 62 ; Humanized Mice, memory 63 ; Tat Transgenic Mice, spatial learning 64,65 , reversal learning 64 ]. Examples of behavioral alterations, similar to those observed in HIV-1 seropositive individuals, include depressive-like behaviors [e.g., HIV-1 Tg rat 66 ; Tat transgenic mice 67,68 ] and motivational alterations [e.g., HIV-1 Tg rat 56 ]. Furthermore, although generally understudied, prominent sex differences in the expression of NCI have been observed in HIV-1 seropositive individuals, with HIV-1 seropositive females displaying more severe deficits relative to HIV-1 seropositive males 69,70 . Sex differences in the expression of NCI have been translationally modeled in the HIV-1 Tg rat 18,71,72 , but inconsistent results have been reported in other animal systems [e.g., Tat transgenic mice 73 ]. Given the increased life expectancy of HIV-1 seropositive individuals in the post-cART era, as well as the persistence of NCI, an in vivo biological system able to provide a longitudinal study of HIV-1 is critical. HIV-1 seropositive individuals display progressive NCI, with individuals exhibiting asymptomatic NCI at baseline having an increased risk (i.e., two to six times) of developing symptomatic HAND 74 . The HIV-1 Tg rat, as demonstrated in the present study, displays age-related disease progression in multiple neurocognitive domains across the functional lifespan. Thus, the present study further elucidates the advantages and constraints of the HIV-1 Tg rat biological system to model neuroHIV in the post-cART era.
Despite the aforementioned strengths of the present study, a few caveats are acknowledged. First, the neuronal analysis focused exclusively on pyramidal neurons from layers II-III of the mPFC. Although the generalizability of synaptic dysfunction cannot be directly assessed within the present study, observations across multiple brain regions [i.e., nucleus accumbens, mPFC 12,21,49,50 ], ages [i.e., 4 months; 14-17 months; 20 months 12,21,50 ] and in HIV-1 Tg rats following psychostimulant exposure [i.e., methylphenidate 21 ] suggests the importance of further studies investigating synaptic dysfunction as a neural mechanism underlying NCI in HAND. Second, neuroinflammatory markers were assessed exclusively in the hippocampus and using only three cytokines (i.e., IL-1β, IL-6, and TNF-α). Despite the utilization of two experimental methodologies and sufficient statistical power, no significant differences in neuroinflammation in the hippocampus were suggested between HIV-1 Tg and control animals; results consistent with those observed using [(18)F]-DPA714 PET imaging and a profile of 24 neuroinflammatory markers in (3 and 9 month) adult HIV-1 Tg animals 75 and following PD 1 stereotaxic injections of HIV-1 viral proteins [i.e., Tat 1-86 , gp120 29 ]. However, this does not preclude neuroinflammation in the adolescent brain, in other brain regions, or using other methodology [e.g., ELISA 76 ]. For example, results of the present study are in contrast to those reported by Royal et al. 76 , which observed significant increases in the expression of INF-γ, TNF-α, and IL-β, assessed using ELISA, in brain lysates prepared from the frontal cortex and subcortical white matter of the HIV-1 Tg rat. Even in the absence of sensory or motor system deficits 14 and comorbidities, HAND is a neurodegenerative disease. NCI are characterized by age-related disease progression across multiple neurocognitive domains, tapping two primary subdivisions of the PFC. Most notably, alterations in sustained attention and learning across the functional lifespan, account for approximately 61.6% of the genotypic variance in NCI, independent of biological sex. Synaptic dysfunction in pyramidal neurons from layers II-III of the mPFC, characterized by alterations in dendritic branching complexity, synaptic connectivity, and dendritic spine morphology, supports a key, albeit not exclusive, neural mechanism underlying HAND, accounting for approximately 64.6% of the genotypic variance. Thus, the progression of HAND with age in the HIV-1 Tg rat and the associated synaptic dysfunction affords an instrumental model system for the development of therapeutics and functional cure strategies.

Data Availability
All relevant data are within the paper.