TY - JOUR
AU - Ballabh,, Praveen
AB - Abstract Preterm-born children suffer from neurological and behavioral disorders. Herein, we hypothesized that premature birth and non-maternal care of preterm newborns might disrupt neurobehavioral function, hippocampal dendritic arborization, and dendritic spine density. Additionally, we assessed whether 17β-estradiol (E2) replacement or the TrkB receptor agonist, 7,8-dihydroxyflavone (DHF), would reverse compromised dendritic development and cognitive function in preterm newborns. These hypotheses were tested by comparing preterm (E28.5) rabbit kits cared and gavage-fed by laboratory personnel and term-kits reared and breast-fed by their mother doe at an equivalent postconceptional age. Neurobehavioral tests showed that both premature-birth and formula-feeding with non-maternal care led to increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term-kits. Dendritic branching and number of total or mushroom dendritic spines were reduced in the CA1 field of preterm-kits compared with term controls. While CDC42 and Rac1/2/3 expression levels were lower, RhoA-activity was higher in preterm-kits compared with term controls. Both E2 and DHF treatment reversed prematurity-induced reduction in spine density, reduced total RhoA-GTPase levels, and enhanced cognitive function. Hence, prematurity and non-maternal care result in cognitive deficits, and reduced dendritic arbors and spines in CA1. E2 replacement or DHF treatment might reverse changes in dendritic spines and improve neurodevelopment in premature infants. dendritic spine, formula feeding, hippocampus, prematurity, Rho-GTPase, TrkBT agonist Introduction Approximately 10% of infants are born premature and 1.5% of neonates are born with very low birth weight (<1500 g) (Murphy et al. 2017). These infants are deprived of the safe intrauterine environment and supply of placental and maternal growth factors and hormones. They are frequently admitted to neonatal intensive care units, where they are cared for by neonatal nurses and fed with infant formula. Most of these infants survive and grow up with the help of sophisticated technologies, but face multiple health problems, including delayed brain development, cognitive deficits, and intellectual disabilities (Bhutta et al. 2002; Anderson et al. 2003; Aarnoudse-Moens et al. 2009). Conceivably, the abnormal structure and function of dendritic arbors and spines in these individuals may underlie information processing deficits and interfere with learning basic skills, such as reading and writing, as well as higher-level abilities, such as organization, memory formation, and attention. However, the effect of premature birth and non-maternal care as opposed to term birth and maternal care on dendrite development has not been evaluated. Therefore, we asked whether premature birth and non-maternal neonatal care would affect neurobehavior, dendritic integrity, and density of dendritic spines in preterm neonates, and, if so, whether this damage can be reversed. Preterm-born children often develop memory deficits and learning disabilities at school age. Quantitative MRI analyses have shown that hippocampal volumes are significantly reduced in preterm compared with full-term born children (Beauchamp et al. 2008). The hippocampus plays critical roles in memory, learning, flexible cognition, and social behavior. The hippocampal CA1 field is critical for detection of novelty, encoding overlapping information, and spatial and contextual learning (Ji and Maren 2008; Schlichting et al. 2014). Glucocorticoid receptors are especially highly expressed in the hippocampus, rendering it more vulnerable to stress than other brain areas (Conrad 2008; Joels 2008). Premature infants are exposed to a number of stressors including endotracheal intubation, intravenous line placement, blood draws, and other interventions, which can potentially affect structure and function of dendritic spines in the CA1. In fact, both human and animal studies have shown that stress related to maternal separation produces lasting changes in infant brain development and behavior (Feng et al. 2011; Flacking et al. 2012). A number of reports show that chronic stress affects dendritic branching, spine density, and synaptic plasticity in the hippocampus (Chen et al. 2008; Christian et al. 2011; Ke et al. 2016). Dendritic morphogenesis is regulated by extrinsic and intrinsic cues. Extrinsic factors include secreted neurotropic factors, cell adhesion molecules, and activity dependent calcium signaling, while intrinsic factors are transcription factors and cytoskeletal regulators (Ledda and Paratcha 2017). Rho-GTPase proteins (Cdc42, Rac1/2/3, RhoA) play vital roles in modulating dendrite arborization and spine plasticity (Duquette and Lamarche-Vane 2014). Because they modulate the cytoskeleton, Rho GTPases are critical for regulating spine plasticity at every stage. Premature birth may disturb dendritic actin cytoskeletal dynamics, consequently impairing dendritic arborization, and spine morphogenesis. In hippocampal pyramidal neurons, BDNF-TrkB receptor signaling increases the number of dendrites, their branching, and density of dendritic spines (Cheung et al. 2007). 17β-estradiol (E2) increases BDNF expression in the prefrontal cortex and the hippocampus (Su et al. 2016). Indeed, either E2 or BDNF administration results in enhanced dendritic arborization and increased spine density in adult animals (Frankfurt and Luine 2015; Tuscher et al. 2016). Although the E2 levels are known to drop 100-fold and serum BDNF levels are reduced in preterm infants (Trotter and Pohlandt 2000; Malamitsi-Puchner et al. 2004; Rao et al. 2009), the impact of correcting these levels in stressed preterm newborns on dendritic spines has not been studied. Based on these considerations, we hypothesized that premature birth and formula feeding of preterm newborns might disrupt neurobehavioral function, hippocampal dendritic arborization, and density of dendritic spines compared with breast-fed term newborns. In addition, we theorized that E2 administration or activation of TrkB signaling would restore dendritic arborization, dendritic spine density, and neurocognitive function in preterm and formula-fed kits. Materials and Methods Experimental Groups This study was approved by the Institutional Animal Care and Use Committee of New York Medical College, Valhalla, NY and Albert Einstein College of Medicine, Bronx, NY. Briefly, we purchased timed-pregnant New Zealand white rabbits from Charles River Laboratories (Wilmington, MA). Cesarean sections were performed to deliver premature kits at embryonic day 28.5 (E28.5; full-term 32 days), whereas term kits were delivered vaginally. After C-section and delivery of kits, the mother doe was euthanized. The animals were classified into 4 groups (n = 8–10/group): full-term breast-fed; full-term formula-fed; preterm breast-fed by a foster mother; and preterm formula-fed. The full-term breast-fed kits were nurtured by the dam from birth until being euthanized at 27 days. Full-term formula-fed kits were brought to the laboratory at birth, reared in an incubator, and gavage-fed by laboratory personnel. Preterm foster mother-fed kits were gavage-fed for the first 3 days in the laboratory and were placed under care of a different mother doe at postnatal day (D)4 until euthanized. Preterm formula-fed kits were brought to the laboratory immediately at birth, reared in an infant incubator with other kits at a temperature of 35 °C and gavage-fed by laboratory personnel. We used rabbit milk replacer (Wombaroo, Glen Osmond, South Australia) to gavage-feed the kits, a volume of ~2 mL every 12 h (100 ml/kg/d) for the first 2 days. The feeds were then advanced to 125, 150, 200, 250, and 280 mL/kg at postnatal D3, D5, D7, D10, and D14, respectively. After D21, all the animals were fed with lettuce, cauliflower, carrots, and broccoli. Preterm kits (E28.5) were euthanized at D30 and term kits (delivered at E32) at D27. Neurobehavioral Assessments Preterm and full-term rabbit kits underwent a battery of neurobehavioral assessments at an equivalent postconceptional age of D28–30 for preterm and D25–27 for term kits. Any-Maze Software (Stoelting Company, Wood Dale, IL) was used to collect and analyze all behavioral assessments by tracking movement and time spent in delineated zones using an overhead camera. 3-chambered Socialization evaluated general sociability and preference for social novelty by the tendency to spend more time with unfamiliar animals over familiar ones. Testing was performed in 3 sessions using a 3-chambered box (12 × 20 inches size for each chamber) with openings between the chambers. After habituation to the empty box, the subject encountered a never-before-met rabbit kit under one wired box and an empty wired box in the “sociability” session. The subject then met the first rabbit kit, as well as a second never-before-met rabbit kit under another wired box in the “social novelty” session. The kits were allowed to explore for 5 min in each of the 3 stages—habituation in empty box, sociability, and social novelty (total 15 min). The time spent sniffing the wired box, the time spent in each chamber, and the number of entries into each chamber were measured. Novel Object Recognition evaluates cognition and recognition memory by the spontaneous tendency to spend more time exploring a novel object over a familiar one. The rabbit was placed in a small arena (40 × 40 × 30 inches) with 2 identical objects for 3 min, followed by 1 h rest in home cage. This was repeated 3 times so that kits got accustomed to the 2 objects. The next day, the kit was placed in the arena for 3 min with one of the known objects replaced with a novel object in the same location. We analyzed the amount of time the rabbit spent with the novel versus known object. Elevated Plus Maze was used to assess anxiety-related behavior. The maze consisted of 4 arms (5 × 22-inches each) that are 30 inches above the ground. 2 arms had 20-inch high opaque sides. Kits were placed in the center and allowed to explore the maze for 5 min. The preference for open arms over closed arms is a measure for anxiety-like behavior. Open Field evaluates general activity levels, gross locomotor activity, and exploration habits. Animals were placed in a square arena (40 × 40 × 30 inches) and allowed to move freely for 10 min. Distance moved, velocity, and time spent in delineated zones were recorded. 17β-Estradiol and 7,8-Dihydroxyflavone Treatment The preterm rabbit kits (E28.5) were treated daily with either intramuscular E2 (Sigma-Aldrich, St. Louis, MO) or vehicle for 7 days starting at D1 until D7 and then euthanized at D14 (Tibrewal et al. 2018). The treatment was administered in a dose of 200 μg/kg. DMSO was used as vehicle. To stimulate TrkB, we treated E28.5 rabbit kits with DHF (Alfa Aesar, Tewksbury, MA) starting at D8, continued until D14, and then euthanized. DHF is a plant product that offers neuroprotection in animal models of depression, Alzheimer’s disease, Parkinson’s disease, demyelinating disease, and stroke (Liu et al. 2016). DHF was administered intramuscularly in a dose of 5 mg/kg 2 times daily (Cipolla et al. 2004). The comparison group received vehicle (15 μl of DMSO). Preterm kits treated with E2 or DHF were euthanized at D14 for evaluation of spine density. We also compared neurobehavioral function between with E2, DHF, and vehicle-treated kits at D28–30. Golgi Staining and Stereological Quantifications Golgi staining was performed on brain slices from preterm kits (E28.5) at D30 and term kits (E32) at D27 (n = 6/group). The FD Rapid GolgiStain Kit (FD NeuroTechnologies, Columbia, MD) was used. Brains were immersed in a 1:1 mixture of FD Solution A:B for 2 weeks at room temperature in the dark. The brains were then transferred to FD Solution C and kept in the dark for 48 h. We next froze the brains into optimum cutting temperature formulation. Cryosectioning was performed on a Leica CM 3050 S cryostat at −22 °C. Coronal sections of 150-μm thickness were cut and transferred to gelatin-coated slides onto small drops of FD Solution C. After allowing sections to dry at room temperature in the dark for at least 4 h (or overnight), slides were then stained exactly as described in the FD Rapid GolgiStain instructions. We quantified several stereological parameters using a Zeiss Axioskop 2 plus microscope equipped with Stereologer hardware and software (SRC Biosciences, Tampa, FL), including dendritic length, branching, and spines in the CA1 region. The reference space (CA1 region) was marked on the section under a 5x objective. The volume of the outlined area (reference space) was quantified using a point-counting probe (frame 25 × 25 μm, guard zone 2 μm, inter-frame interval 400 μm). Dendrite length was quantified using the Space Balls probe under 100× oil-immersion objective by a blinded investigator clicking on the focused dendrites that overlapped with the space ball perimeter. Dendrite branching (frame 25 × 25 μm, guard zone 2 μm, inter-frame interval 400 μm) and spines (frame 10 × 10 μm, guard zone 2 μm, inter-frame interval 400 μm) were quantified by a disector probe, in which a blinded investigator clicked on branches or spines that came into focus within the disector area (Mouton et al. 2009). A coefficient of error <0.10 was considered acceptable. This stererological quantification of dendritic length, branching, and number of spines resulted in absolute values. A blinded investigator next quantified spine density and 3 classes of dendritic spines (thin, stubby, mushroom) in Golgi-stained brains using Neurolucida 360 and AutoSpine software (MBF Biosciences, Williston, VT), as previously described (Dickstein et al. 2016). Briefly, 6 neurons were randomly selected in the CA1 of each brain by a blinded investigator. 3 basal dendrites and 3 apical secondary dendrites that were ~30–60 μm in length and originated at least 30 μm from the soma were traced in brightfield image stacks acquired at 100x using a Zeiss Axiophot 2 microscope. Dendritic spines were reconstructed and modeled using the spine autodetection tool on the traced dendrites. Quantification of spines in Neurolucida using this method led to an estimate of spine density, which was expressed as number of spines per μm. Diolistic Staining and Spine Quantification To assess the effect of E2 and DHF, we performed diolistic labeling (n = 5/group), which produces Golgi-like random labeling of full neuronal profiles using a lipophilic fluorescent dye. For diolistic labeling of neurites (Seabold et al. 2010; Foster Olive et al. 2018), the area between the ventral posterolateral nucleus and cerebellum of D14 brain sections were divided into left and right hemispheres and the diencephalon excised to isolate the hippocampus. The hippocampus was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; 0.1 M, pH 7.4) for 1 h, washed in PBS, and then cut into 300-μm thick sections using a vibratome (Leica Biosystems, Buffalo Grove, IL). Selected sections were then “shot” with DiI labeled tungsten beads using a Helios Gene Gun (Bio-Rad, Hercules, CA), allowed to incubate in PBS and sytox green nucleic acid stain (1:5000, ThermoFisher Scientific, Walthom, MA) for 6 h, then mounted onto glass slides using Prolong Diamond Antifade Mountant (ThermoFisher Scientific). Fluorescence image Z-stacks (75 images at 0.2 μm interval) of dendrites at 120x magnification were taken by confocal laser scanning microscopy (Nikon Instruments, Melville, NY). Image stacks were analyzed by a blinded investigator using NeuronStudio to quantify spine density and type (thin, stubby, mushroom) using previously established protocols (Rodriguez et al. 2008) for the same dendritic segment types as described for Golgi-stained specimens. The analyses led to spine density expressed as number of spines per μm. Western Blot Analyses Western blot analyses were performed as described previously (Ballabh et al. 2007) (n = 5/group). Briefly, homogenates made from brain slices taken at the level of the ventral posterolateral nucleus of thalamus were quantified for protein using a BCA protein assay kit (ThermoFisher Scientific, Waltham, MA). Equal amounts of protein (10–20 μg) were loaded onto 4–15% or 4–20% gradient precast gels (Bio-Rad, Hercules, CA), depending on the molecular weight of the target protein. Separated proteins were transferred onto polyvinylidene difluoride membranes by electrotransfer. Membranes were incubated overnight with primary antibodies (1:2000 rabbit anti-Rac1/2/3, CDC42, and RhoA antibodies, Cell Signaling Technology, Danvers, MA; 1:20 000 mouse anti-Actin antibody, Sigma Aldrich, St. Louis, MO) and target proteins were detected with an ECL system by using secondary antibodies conjugated with horseradish peroxidase (1:2000 HRP antibody, Jackson Immunoresearch, West Grove, PA). The blots from each experiment were run 2 to 3 times and were densitometrically analyzed using ImageJ (NIH, Bethesda, MD). Optical density values were normalized to β-actin. RhoA pull-down activation assay (Cytoskeleton, Denver, CO) was performed, as described (Yan et al. 2014). Briefly, rhotekin conjugated beads, which have a high affinity for GTP-bound RhoA, were incubated for 1 h at 4°C with lysates containing 300–800 μg total protein in which protein content was adjusted to acquire a relatively equal amount of total RhoA as determined by Western blot, at a concentration of 1 mg/mL to extract activated RhoA, then used to perform a Western blot using primary RhoA antibody. Statistical Analyses Data are expressed as means and SD. All the behavioral tests were analyzed by 2-way ANOVA to assess the interaction between the 2 independent categorical variables (breast vs. formula feeds and term vs. preterm) on one dependent variables, including time spent with stranger in the 3-chamber socialization test, time spent with novel object in the novel object recognition test, and others. All post hoc comparisons to test for differences between means were done using the Tukey multiple comparison test at the 0.05 significance level. Dendritic length, branching, and density were compared between term and preterm groups as well as between E2/DHF treatment and vehicle controls by unpaired t-test. The effect of E2 and DHF on neurobehavioral tests were analysis using 1-way ANOVA. Western blot analyses were also analyzed by a t-test. The data on effect of 11-Statistical significance was set using an α level of 0.05. Results Formula-Feeding and Prematurity Results in Poor Social Interaction, Less Risk-Taking Behavior, and Low Motor Activity As premature infants cared in neonatal units often suffer from cognitive deficits and neurobehavioral impairments, we evaluated the effect of prematurity and type of feeding on neurobehavior in rabbit newborns. We compared 4 groups of rabbit kits: (1) preterm kits (E28.5): gavage-fed and taken care by laboratory personnel, (2) preterm kits: breast-fed and foster-reared by mother rabbits (foster mother), (3) term kits (E32): gavage-fed and taken care by laboratory personnel, and (4) term kits: breast-fed and reared by the mother rabbits (biological mother). The 3-chambered socialization test revealed that the term-born kits (gavage- and breast-fed combined) spent more time with the stranger animal compared with preterm-born kits (P = 0.001, 2-way ANOVA, F = 12.9). Likewise, all the breast-fed kits (both term and preterm) were for a longer time with stranger compared with formula-fed kits (P = 0.001, 2-way ANOVA, F = 8.19; Fig. 1A). Within term kits, breast-fed ones spent more time with stranger relative to formula-fed kits (P = 0.009, post hoc Tukey test). Similarly, among breast-fed kits, term-born kits were for a longer period with stranger relative to preterm ones (P = 0.003, post hoc Tukey test). However, among preterm kits, there was no difference between breast- and formula-fed rabbits; and also within formula-fed animals, there was no difference between term and preterm kits for this parameter. Formula-fed preterm kits also spent shorter time with stranger compared with breast-fed term kits (P < 0.001, post hoc analyses). Together, both the type of birth (term vs. preterm) and rearing (breast vs. formula) affects social recognition in rabbit newborns. We next performed novel object recognition test, which showed that term kits (gavage- and breast-fed combined) spent longer time with novel object relative to preterm kits (P = 0.039, F = 5.1, 2-way ANOVA, Fig. 1B). However, breast-fed and formula-fed kits (term and preterm combined) spent similar time with novel object (P = 0.13, F = 1.2, 2-way ANOVA). Again among term and preterm kits, there was no significant difference between breast- and formula-fed kits for this metric. Data suggest that there is reduced preference for novelty as well as poorer recognition and memory in preterm relative to term kits. Figure 1. Open in new tabDownload slide Formula-fed premature-kits display increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term kits. (A) Representative track plots of full-term breast-fed and preterm formula-fed kits in the 3-chambered socialization test. Bar charts show means ± SD (n = 8–10 each group). Among term kits, breast-fed ones spent more time with stranger relative to formula-fed kits. Within breast-fed kits, term-born rabbits spent longer period with stranger relative to preterm kits. (B) Representative traces of full-term breast-fed and preterm formula-fed kits in the novel object recognition test. Bar charts show means ± SD (n = 8–10). Term kits, when gavage- and breast-fed combined, spent longer time with novel object relative to preterm kits. Again among term and preterm kits, there was no significant difference between breast- and formula-fed kits for this metrics. (C) Representative track plots of full-term breast-fed and preterm formula-fed kits in the closed vs. open arm of elevated plus maze. Bar charts show means ± SD (n = 8–10 each group). Among formula-fed kits, preterm rabbits spent less time in open arm compared with breast-fed kits. Within preterm rabbits, formula-fed kits spent shorter time in the open arm compared to breast-fed kits. (D–F) Representative track plots of locomotor activity for full-term breast-fed and preterm formula-fed kits in open field box. Data show means ± SD (n = 8–10 each group). Within preterm kits, breast-fed ones traveled longer distance and displayed higher speed relative to formula-fed kits. Among formula-fed kits, term rabbits traveled longer distance at higher speed compared with preterm kits. * indicate P < 0.05 and ** indicate P < 0.01. Figure 1. Open in new tabDownload slide Formula-fed premature-kits display increased anxiety behavior, poor social interaction, and lack of novelty preference compared with term kits. (A) Representative track plots of full-term breast-fed and preterm formula-fed kits in the 3-chambered socialization test. Bar charts show means ± SD (n = 8–10 each group). Among term kits, breast-fed ones spent more time with stranger relative to formula-fed kits. Within breast-fed kits, term-born rabbits spent longer period with stranger relative to preterm kits. (B) Representative traces of full-term breast-fed and preterm formula-fed kits in the novel object recognition test. Bar charts show means ± SD (n = 8–10). Term kits, when gavage- and breast-fed combined, spent longer time with novel object relative to preterm kits. Again among term and preterm kits, there was no significant difference between breast- and formula-fed kits for this metrics. (C) Representative track plots of full-term breast-fed and preterm formula-fed kits in the closed vs. open arm of elevated plus maze. Bar charts show means ± SD (n = 8–10 each group). Among formula-fed kits, preterm rabbits spent less time in open arm compared with breast-fed kits. Within preterm rabbits, formula-fed kits spent shorter time in the open arm compared to breast-fed kits. (D–F) Representative track plots of locomotor activity for full-term breast-fed and preterm formula-fed kits in open field box. Data show means ± SD (n = 8–10 each group). Within preterm kits, breast-fed ones traveled longer distance and displayed higher speed relative to formula-fed kits. Among formula-fed kits, term rabbits traveled longer distance at higher speed compared with preterm kits. * indicate P < 0.05 and ** indicate P < 0.01. We next performed elevated plus maze test to assess anxiety and fear of open space and height in the above 4 sets of kits. We found that formula-fed kits (term and preterm combined) spent less time in the open arms relative to breast-fed kits (P = 0.023, F = 5.8, 2-way ANOVA, Fig. 1C). Among formula-fed kits, preterm rabbits spent less time in open arm compared with breast-fed kits (P = 0.02, Tukey test). Preterm kits (both formula- and breast-fed combined) also showed an insignificant trend toward spending less time in open relative to all term kits (P = 0.07, F = 3.36, 2-way ANOVA,). Within preterm rabbits, formula-fed kits were for a shorter time in the open arm compared with breast-fed kits (P = 0.009, Tukey test). Taken together, these data suggest both prematurity and formula feeding results in heightened anxiety, increased risk aversion, and diminished exploratory behavior relative to term birth and breast feeding. We next performed open field test and assessed 3 parameters, including distance traveled, period of activity, and average speed of the animal. All the 3 variables (distance traveled, duration of activity, and speed) were higher in term kits (breast- and formula-fed kits combined) relative to all preterm kits (P = 0.016, 0.01, 0.015; F = 6.6, 7.7, 6.7, respectively, 2-way ANOVA; Fig. 1D–F). However, there was no difference in between formula- and breast-fed kits (both term and preterm combined) for any of the 3 parameters (P = 0.13, 0.3, 0.13, F = 2.4, 0.9, 2.4, respectively; 2-way ANOVA). Within preterm kits, breast-fed ones covered longer distance and exhibited higher speed relative to formula-fed kits (P = 0.04 both, Tukey test); and among formula-fed kits, term rabbits traveled longer distance at higher speed compared with preterm kits (P = 0.01 both, Tukey test). There was no difference between term and preterm subgroups of breast-fed animals and between breast- and formula-fed subgroups of term kits for any of the 3 parameters. Together, the behavioral study revealed a loss of social novelty in 3-chambered-socialization test, reduced preference for object novelty in novel object recognition test, increased anxiety behavior in elevated plus maze test and reduced activity in open field test in preterm and formula-fed kits relative to term and breast-fed ones. Overall, the data suggest that prematurity and type of feeding independently affect cognitive and motor behavior of animals. Hippocampal CA1 Dendritic Branching and Spines are Reduced in Premature Kits There is compelling evidence on the role of dorsal hippocampus in social memory, recognition of object novelty, and anxiety responses, therefore we chose to study dorsal hippocampus (Antunes and Biala 2012; Cominski et al. 2014; Montagrin et al. 2017). The hippocampal CA1 regions plays key role in novelty recognition, social memory and learning (Ohtsuki et al. 2007; Fernandez-Lopez et al. 2012). Because the largest behavioral differences were observed between the full-term breast-fed and preterm formula-fed animals, we evaluated dendritic spine morphology and underlying biochemical differences in the CA1 field of hippocampus between these 2 groups. To this end, dendrite length, branching, and spines were quantified using stereological methods. No difference was observed in dendrite length between full-term and preterm animals (Fig. 2A, B). However, dendrite branching and total number of spines were significantly decreased in the CA1 of preterm kits relative to term kits (P = 0.002 and 0.003, respectively, t-test; Fig. 2C–E). Together, reduced dendritic arborization and spine density in the CA1 of formula-fed premature kits might be the structural basis of their cognitive and other neurobehavioral deficits. Figure 2. Open in new tabDownload slide Dendritic branching and spines are reduced in CA1 of premature kits. (A) Representative images of Golgi-stained hippocampal CA1 neurons from preterm- and term-born kits. Note reduced dendritic branching in preterm formula-fed compared with the full-term breast-fed kits. Scale bar = 50 μm. (B–C) Data are presented as means ± SD (n = 6 each). Dendrite length was comparable between the preterm formula-fed group and full-term breast-fed group in the CA1 region. However, dendrite branching was significantly reduced in the preterm formula-fed group compared with the full-term breast-fed group. (D, E) Representative images of Golgi-stained CA1 hippocampal dendritic spines from preterm and term born kits. Data are presented as means ± SD (n = 6 each). The number of spines were significantly reduced in the preterm formula-fed group compared with the full-term breast-fed group. Scale bar = 10 μm. * indicate P < 0.05 preterm formula-fed versus full-term breast-fed kits. Figure 2. Open in new tabDownload slide Dendritic branching and spines are reduced in CA1 of premature kits. (A) Representative images of Golgi-stained hippocampal CA1 neurons from preterm- and term-born kits. Note reduced dendritic branching in preterm formula-fed compared with the full-term breast-fed kits. Scale bar = 50 μm. (B–C) Data are presented as means ± SD (n = 6 each). Dendrite length was comparable between the preterm formula-fed group and full-term breast-fed group in the CA1 region. However, dendrite branching was significantly reduced in the preterm formula-fed group compared with the full-term breast-fed group. (D, E) Representative images of Golgi-stained CA1 hippocampal dendritic spines from preterm and term born kits. Data are presented as means ± SD (n = 6 each). The number of spines were significantly reduced in the preterm formula-fed group compared with the full-term breast-fed group. Scale bar = 10 μm. * indicate P < 0.05 preterm formula-fed versus full-term breast-fed kits. Premature Kits Exhibit Reduced Number of Mushroom and Higher Number of Stubby Spines Relative to Full-term Kits Spines display high levels of plasticity and have a cycle that starts with filopodia/thin spines, then either grow into mushroom spines to form mature synapses or collapse into stubby spines (Nag et al. 2007). To determine differences in dendritic spine plasticity between term and preterm kits, we quantified and classified CA1 dendritic spines of breast-fed term versus formula-fed preterm kits. We evaluated 3 basal and 3 apical secondary dendrites in 6 neurons to determine the dendritic spine density (per μm) in Golgi-stained sections. We found that apical, basal, and total (apical plus basal) spine densities were significantly reduced in the preterm kits relative to term kits (P < 0.001, P = 0.007, and P = 0.002, respectively; t-test, Fig. 3B). The proportion of total and basal mushroom spines was significantly lower in preterm compared with full-term kits (P = 0.014 and P = 0.002, respectively; t-test), whereas, total and basal stubby spines was significantly higher in proportion in preterm relative to term controls (P = 0.024 and P = 0.017, respectively; t-test; Fig. 3C–E). No significant difference was observed in thin spine proportion between full-term and preterm group. Given the reduced density of total, apical as well as basal spines and increased proportion of stubby spines in the dendrites of preterm relative to term kits, the data suggest a diminished spine morphogenesis and augmented propensity for spine collapse in preterm animals relative to term controls. Figure 3. Open in new tabDownload slide Reduced number of mushroom and higher number of stubby spines in preterm kits relative to full-term kits. (A) Representative images of CA1 dendritic spines in Golgi-stained sections. Note reduced number of spines in preterm kits compared to full-term kits. Scale bar = 10 μm. (B–E) Data are presented as means ± SD (n = 6 each). Total, apical, and basal spine density was significantly decreased in the preterm formula-fed group compared to the full-term breast-fed group. Thin spines were comparable between groups. Total and basal stubby spines were significantly increased in the preterm formula-fed group compared to the full-term breast-fed group, while total and basal mushroom spines were significantly decreased in the preterm formula-fed group compared to the full-term breast-fed group. * indicate P < 0.05 for preterm formula-fed versus full-term breast-fed kits. Figure 3. Open in new tabDownload slide Reduced number of mushroom and higher number of stubby spines in preterm kits relative to full-term kits. (A) Representative images of CA1 dendritic spines in Golgi-stained sections. Note reduced number of spines in preterm kits compared to full-term kits. Scale bar = 10 μm. (B–E) Data are presented as means ± SD (n = 6 each). Total, apical, and basal spine density was significantly decreased in the preterm formula-fed group compared to the full-term breast-fed group. Thin spines were comparable between groups. Total and basal stubby spines were significantly increased in the preterm formula-fed group compared to the full-term breast-fed group, while total and basal mushroom spines were significantly decreased in the preterm formula-fed group compared to the full-term breast-fed group. * indicate P < 0.05 for preterm formula-fed versus full-term breast-fed kits. RhoA-GTPase Activity is Elevated in Premature Formula-fed Kits Compared with Full-term Breast-fed Kits Small GTPase of Rho/Rac/CDC42 family has emerged as a key regulator of morphology and function of dendritic spines (Ledda and Paratcha 2017). We assessed expression levels of CDC42, Rac1/2/3, and RhoA in preterm (formula-fed) and term (breast-fed) kits by western blot analyses. CDC42 and Rac1/2/3 expression levels were significantly reduced in the preterm compared with the full-term kits (P = 0.004 and P = 0.005; respectively; t-test; Fig. 4B, C). However, RhoA levels were comparable between term and preterm kits (Fig. 4D). Figure 4. Open in new tabDownload slide CDC42 and Rac1/2/3 reduced, but RhoA-GTPase activity elevated in premature formula-fed kits compared with full-term breast-fed kits. (A) Representative western blot analyses for CDC42, Rac1/2/3, and RhoA on homogenates made from coronal brain slices taken at the level of the ventral posterolateral nucleus of thalamus in full-term and -preterm kits. Values were normalized to β actin levels. The lower panel shows results of RhoA pull-down assay. (B–D) The bar charts show means ± SD (n = 5 each). CDC42 and Rac1/2/3 protein expression was significantly decreased the preterm kits relative to term kits. Total RhoA was similar between 2 groups, (E) RhoA activity, determined by RhoA pull-down assay, was significantly increased in the preterm kits relative to term controls. * indicate P < 0.05 for preterm formula-fed versus full-term breast-fed kits. Figure 4. Open in new tabDownload slide CDC42 and Rac1/2/3 reduced, but RhoA-GTPase activity elevated in premature formula-fed kits compared with full-term breast-fed kits. (A) Representative western blot analyses for CDC42, Rac1/2/3, and RhoA on homogenates made from coronal brain slices taken at the level of the ventral posterolateral nucleus of thalamus in full-term and -preterm kits. Values were normalized to β actin levels. The lower panel shows results of RhoA pull-down assay. (B–D) The bar charts show means ± SD (n = 5 each). CDC42 and Rac1/2/3 protein expression was significantly decreased the preterm kits relative to term kits. Total RhoA was similar between 2 groups, (E) RhoA activity, determined by RhoA pull-down assay, was significantly increased in the preterm kits relative to term controls. * indicate P < 0.05 for preterm formula-fed versus full-term breast-fed kits. We next measured RhoA activity for term and preterm kits by employing RhoA pull-down assay. We found that RhoA activity was significantly elevated in the preterm kits relative to term kits (P = 0.039; Fig. 4E). Together, reduced density of dendritic spines in preterm kits relative to term controls was associated with diminished CDC42 and Rac1/2/3 levels, and elevated RhoA activity. These findings reinforce the notion that reduced CDC42 and Rac1/2/3 levels and elevated RhoA activity reduce dendritic arborization and cause spine collapse (Lee et al. 2000; Scott et al. 2003). E2 Supplementation Reverses Spine Loss in Preterm Rabbit Kits Estrogen receptors are highly expressed in the brain, and prior evidence suggests E2 administration increases spine density in the CA1 (Smith et al. 2009). Thus, we supplemented preterm rabbits with E2 in an attempt to reverse spine reduction. Spine density and classification were determined in both the basal and apical dendrite regions of CA1 pyramidal neurons in diolistically stained D14 brain sections. Basal spine density was significantly increased (P = 0.027; t-test) and total spine density showed a strong trend towards increasing (P = 0.075; Fig. 5B) in E2-supplemented preterm kits compared with vehicle controls. Basal thin spines were also significantly increased (P = 0.047; Fig. 5C), but apical mushroom spines were reduced in E2-supplemented preterm kits relative to controls (P = 0.039; t-test; Fig. 5E). There was no difference in stubby spines between groups (Fig. 5D). E2 supplementation, therefore, appeared to mitigate spine reduction in preterm formula-fed animals. We further assessed the effects of E2 administration on Rho-GTPase expression levels and RhoA activity. E2-treated preterm kits had significantly lower expression of CDC42, Rac1/2/3, and RhoA (P = 0.001, 0.001, 0.006, respectively; t-test; Fig. 6B–D), while RhoA activity showed a trend toward decline (P = 0.082; Fig. 6E). Together, E2 treatment increased spine density and decreased RhoA-GTPase expression, but not RhoA activity. As downregulation RhoA activity promotes spine density, lack of reduction in RhoA activity upon E2 treatment suggests that mechanism(s) other than RhoA-GTPase might be responsible for reversing spine loss in E2-treated animals. Figure 5. Open in new tabDownload slide E2 supplementation restored spine loss in basal CA1 dendrites of preterm rabbit kits. (A) Representative images of dendritic spines in DiI-stained hippocampal CA1 dendrites of full-term mon-fed and preterm formula-fed kits. Scale bar = 10 μm. (B–E) Data show means ± SD (n = 5 each). Basal spine density was significantly higher in the E2-supplemented preterm kits compared to controls, but not the total and apical spine density. Basal thin spines were significantly increased in E2-supplemented preterm kits. No differences were observed in stubby spines. Apical mushroom spines were decreased in E2-treated preterm kits relative to vehicle controls, but not total mushroom spines. * indicate P < 0.05 for E2- versus vehicle-treated preterm kits. Figure 5. Open in new tabDownload slide E2 supplementation restored spine loss in basal CA1 dendrites of preterm rabbit kits. (A) Representative images of dendritic spines in DiI-stained hippocampal CA1 dendrites of full-term mon-fed and preterm formula-fed kits. Scale bar = 10 μm. (B–E) Data show means ± SD (n = 5 each). Basal spine density was significantly higher in the E2-supplemented preterm kits compared to controls, but not the total and apical spine density. Basal thin spines were significantly increased in E2-supplemented preterm kits. No differences were observed in stubby spines. Apical mushroom spines were decreased in E2-treated preterm kits relative to vehicle controls, but not total mushroom spines. * indicate P < 0.05 for E2- versus vehicle-treated preterm kits. Figure 6. Open in new tabDownload slide E2 supplementation reduced CDC42, Rac1/2/3, and RhoA levels in preterm kits. (A) Representative western blot analyses for CDC42, Rac1/2/3, and RhoA on homogenates made from coronal brain slices taken at the level of the ventral posterolateral nucleus of thalamus in E2 and vehicle-treated preterm kits. Values were normalized to β actin levels. Lower panel shows blot of RhoA pull-down assay. (B–D) The bar charts show means ± SD (n = 5 each). CDC42, Rac1/2/3, and RhoA expression was significantly decreased in the E2-supplemented preterm kits compared with vehicle controls. (E) RhoA activity, determined by RhoA pull-down assay, was comparable between groups. * indicate P < 0.05 for E2- versus vehicle-treated preterm kits. Figure 6. Open in new tabDownload slide E2 supplementation reduced CDC42, Rac1/2/3, and RhoA levels in preterm kits. (A) Representative western blot analyses for CDC42, Rac1/2/3, and RhoA on homogenates made from coronal brain slices taken at the level of the ventral posterolateral nucleus of thalamus in E2 and vehicle-treated preterm kits. Values were normalized to β actin levels. Lower panel shows blot of RhoA pull-down assay. (B–D) The bar charts show means ± SD (n = 5 each). CDC42, Rac1/2/3, and RhoA expression was significantly decreased in the E2-supplemented preterm kits compared with vehicle controls. (E) RhoA activity, determined by RhoA pull-down assay, was comparable between groups. * indicate P < 0.05 for E2- versus vehicle-treated preterm kits. TrkB Agonist 7,8-Dihydroxyflavone Treatment Restores Spine Loss in Preterm Rabbit Kits BDNF-TrkB receptor signaling increases the number of dendrites, their branching, and density of dendritic spines in hippocampal pyramidal neurons (Cheung et al. 2007). Hence, we postulated that TrkB agonist might promote density of dendritic spines in CA1 pyramidal neurons. To these end, we treated preterm formula-fed kits with a TrkB agonist, DHF, and assessed dendritic spine density and types in CA1 pyramidal neurons of diolistically stained D14 brain sections. Total and apical spine density were significantly increased (P = 0.029 and P = 0.013, respectively), while basal spine density showed an upward trend (P = 0.068; Fig. 7A) in DHF-treated preterm kits compared to vehicle controls. No significant difference was observed in proportions of thin spines, stubby spines, or mushroom spines between 2 groups (Fig. 7B–D). TrkB stimulation, therefore, appeared to promote recovery of spine density in preterm formula-fed animals. We next evaluated the effects of DHF treatment on Rho-GTPase expression and RhoA activity. We found that CDC42, Rac1/2/3, and RhoA levels were significantly lower in DHF-treated preterm kits relative to vehicle controls (P < 0.001 all; t-test; Fig. 8B–D). Relative RhoA activity was also significantly reduced in DHF-treated animals compared to controls (Fig. 8E). Hence, DHF treatment restored total and apical spine density, which appears to be mediated by reduction in RhoA-GTPase activity. Figure 7. Open in new tabDownload slide DHF treatment restored spine loss in preterm rabbit kits. (A) Representative images of dendritic spines in DiI-stained hippocampal CA1 dendrites of DHF- and vehicle-treated kits. Note increased spine density in DHF-treated kits relative to vehicle controls. Scale bar = 10 μm. (B–E) Data are presented as means ± SD (n = 5 each). Total and apical spine densities were significantly increased in the DHF-supplemented preterm kits compared with vehicle controls, but not the basal spine density. No differences were observed in thin spines, stubby spines, and mushroom spines between the groups. * indicate P < 0.05 for DHF- versus vehicle-treated preterm kits. Figure 7. Open in new tabDownload slide DHF treatment restored spine loss in preterm rabbit kits. (A) Representative images of dendritic spines in DiI-stained hippocampal CA1 dendrites of DHF- and vehicle-treated kits. Note increased spine density in DHF-treated kits relative to vehicle controls. Scale bar = 10 μm. (B–E) Data are presented as means ± SD (n = 5 each). Total and apical spine densities were significantly increased in the DHF-supplemented preterm kits compared with vehicle controls, but not the basal spine density. No differences were observed in thin spines, stubby spines, and mushroom spines between the groups. * indicate P < 0.05 for DHF- versus vehicle-treated preterm kits. Figure 8. Open in new tabDownload slide DHF treatment reduced CDC42, Rac1/2/3, and total as well as active RhoA levels in preterm kits. (A) Representative western blot analyses for CDC42, Rac1/2/3, and RhoA on homogenates made from coronal brain slices taken at the level of the ventral posterolateral nucleus of thalamus in DHF- and vehicle-treated preterm kits. Values were normalized to β actin levels. The lower panel shows blot of RhoA pull-down assay. (B–D) The bar charts show means ± SD (n = 5 each). CDC42, Rac1/2/3, and RhoA expression was significantly decreased in the DHF-treated preterm kits compared to vehicle-treated preterm controls. (E) RhoA activity, determined by RhoA pull-down assay, was significantly decreased in DHF-treated kits relative to vehicle controls. * indicate P < 0.05 for DHF- versus vehicle-treated preterm kits. Figure 8. Open in new tabDownload slide DHF treatment reduced CDC42, Rac1/2/3, and total as well as active RhoA levels in preterm kits. (A) Representative western blot analyses for CDC42, Rac1/2/3, and RhoA on homogenates made from coronal brain slices taken at the level of the ventral posterolateral nucleus of thalamus in DHF- and vehicle-treated preterm kits. Values were normalized to β actin levels. The lower panel shows blot of RhoA pull-down assay. (B–D) The bar charts show means ± SD (n = 5 each). CDC42, Rac1/2/3, and RhoA expression was significantly decreased in the DHF-treated preterm kits compared to vehicle-treated preterm controls. (E) RhoA activity, determined by RhoA pull-down assay, was significantly decreased in DHF-treated kits relative to vehicle controls. * indicate P < 0.05 for DHF- versus vehicle-treated preterm kits. E2 and DHF Treatment Supplementation Enhances Cognitive Function in Preterm Rabbit Kits We next compared neurobehavioral function among E2-, DHF-, and vehicle-treated kits (Fig. 9). The 3-chambered socialization test demonstrated that the both E2- and DHF-treated kits spent more time with the stranger animal relative to vehicle-treated kits (P = 0.04 and 0.016, respectively, 1-way ANOVA, Tukey post hoc test). We next compared the time spent in the central chamber between the 3 groups (158.3 ± 24.4, 85 ± 25.2, and 111.3 ± 19.4 s for control, E2, and DHF treatment, respectively). We found that control kits were for longer time in the central chamber compared to E2-treated kits (P = 0.042, Tukey test), but not to DHF-treated kits (P = 0.14). Together, this indicates that both E2 and DHF treatment enhances general sociability and interest in the social novelty. We next performed novel object recognition test, which showed that there was no significant difference in the time spent with novel or familiar object between E2-, DHF-, and vehicle-treated kits. We then accomplished elevated plus maze test to assess anxiety and fear of open space and height in the above 3 sets of kits. We found that both E2- and DHF-treated kits spent less time in the closed arms relative to vehicle-treated kits (P = 0.023 and 0.049, respectively, 1-way ANOVA, Tukey post hoc test). DHF-treated kits spent also spent longer time in open arm relative to controls pups (P = 0.006). However, this difference between E2- and vehicle-treated kits was not significant (P = 0.15). Open field test showed that the 3 sets of pups traveled similar distance, exhibited comparable speed, and spent similar time in center and periphery of arena. The data suggest that E2 or DHF treatment improves social interaction and reduces anxiety for open space and height in premature rabbit newborns. Figure 9. Open in new tabDownload slide E2 replacement and DHF treatment reduces anxiety behavior and enhances social interaction in preterm kits. (A) Representative track plots of E2-, DHF-, and vehicle-treated kits for the 3-chambered socialization test. Bar charts show means ± SD (n = 9–10 each group). E2- and DHF-treated kits spent longer time with the stranger compared with vehicle-treated controls. (B) Representative traces of E2-, DHF-, and vehicle-treated kits in the novel object recognition test. Bar charts show means ± SD (n = 9–10). Time spent with a novel or familiar object was comparable across the 3 groups of kits. (C) Representative track plots for E2-, DHF-, and vehicle-treated kits in the closed versus open arm of elevated plus maze. Bar charts show means ± SD (n = 9–10). DHF-treated kits spent more time in open arm relative to controls. Both E2- and DHF-treated kits spent less time in the closed arms relative to vehicle-treated kits. (D) Data show means ± SD (n = 9–10). All the 3 groups of kits traveled similar distance in the open field. * indicate P < 0.05 and ** indicate P < 0.01. Figure 9. Open in new tabDownload slide E2 replacement and DHF treatment reduces anxiety behavior and enhances social interaction in preterm kits. (A) Representative track plots of E2-, DHF-, and vehicle-treated kits for the 3-chambered socialization test. Bar charts show means ± SD (n = 9–10 each group). E2- and DHF-treated kits spent longer time with the stranger compared with vehicle-treated controls. (B) Representative traces of E2-, DHF-, and vehicle-treated kits in the novel object recognition test. Bar charts show means ± SD (n = 9–10). Time spent with a novel or familiar object was comparable across the 3 groups of kits. (C) Representative track plots for E2-, DHF-, and vehicle-treated kits in the closed versus open arm of elevated plus maze. Bar charts show means ± SD (n = 9–10). DHF-treated kits spent more time in open arm relative to controls. Both E2- and DHF-treated kits spent less time in the closed arms relative to vehicle-treated kits. (D) Data show means ± SD (n = 9–10). All the 3 groups of kits traveled similar distance in the open field. * indicate P < 0.05 and ** indicate P < 0.01. Discussion Preterm infants suffer from cognitive deficits and neurobehavioral disorders. As survival of premature infants has strikingly improved with advancements in neonatal intensive care, prematurity-related complications have emerged as a major public health problem. In terms of financial impact, the burden related to the care of preterm infants exceeds $26 billion dollars per year (Gilbert et al. 2003). Herein, we report that preterm rabbits exhibit poor learning and memory, reduced dendritic arborization, and less dendritic spines compared with full-term kits. Importantly, this study shows that 2 interventions—E2 supplementation or treatment with the TrkB agonist (DHF)—restored hippocampal dendritic spine density, decreased Rho-GTPase expression levels, and improved cognitive function in preterm kits. Hence, this study indicates that the observed impairments in risk-taking behavior, social interaction, and low novelty preference in premature kits may be the result of abnormal dendritic structure and spines and suggests a mechanism-based strategy to reverse these deficits. Our data firmly establish the occurrence of reduced dendritic arborization and spine density in an animal model of prematurity. Our behavioral data suggest that both premature birth and formula feeding adversely influence neurobehavioral outcomes. The major functional implications of these deficits may be attributed to a number of conditions, including cessation in the maternal supply of estrogen and growth factors, suboptimal nutrition, and stress related to maternal separation and laboratory rearing. Previous studies have shown that E2 levels in preterm infants drop by 100-fold (Trotter and Pohlandt 2000). BDNF levels are lower in preterm infants compared with term infants, and that reduced levels of both E2 and BDNF directly affect morphology of dendritic spines (Frankfurt and Luine 2015). Our preterm formula-fed rabbits weighed relatively less than the term rabbits reared by their doe. Studies in rodents have shown that protein calorie malnutrition results in lack of synchrony of age-related changes and an increased spine density in the malnourished rats at D30 and a widening deficit in this parameter on D90 and D220 (Cintra et al. 1997). In contrast, the present study showed reduced number of dendritic spines in the underweight preterm rabbits relative to term controls. Both acute and chronic stress in adult animals results in memory deficit and diminished dendritic arborization and spines (Magarinos and McEwen 1995; Magarinos et al. 1996). Short-term stress lasting for hours has been reported to reduce dendritic spine density in the CA1 and CA3 fields (Chen et al. 2008). Chronic immobilization stress in mice also results in robust retraction of dendrites in CA1 pyramidal neurons (Donohue et al. 2006; Christian et al. 2011), and social stress in adolescent mice (P35) reduces stubby spines and increases long thin spines in the hippocampal CA1 region (Iniguez et al. 2016). Together, the reduction in dendritic spines in preterm rabbits might be stress-related, caused by maternal separation and/or withdrawal of estrogen or growth factors after premature delivery. Rabbits mature sexually by 3–6 months of age and kits of 30 days age are considered to be in their childhood (Frame et al. 1994). We found reduced hippocampal dendritic arborization and spine density in preterm-born kits at postnatal day 30, which indicates a structural mechanism of the underlying developmental behavioral deficits in prematurely born rabbits. Extremely premature human infants also display significant reduction in cortical gray matter volume and exhibit impaired cortical growth even in childhood and adolescence (de Kieviet et al. 2012). Moreover, they often suffer from neurobehavioral disorders during childhood and adolescence, including inattention, hyperactivity, anxiety disorders, autism, epilepsy, and others (Botting et al. 1997; Anderson et al. 2003). Hence, a histological evaluation of spine density and their behavioral assessment at 6 month of age will be of interest to determine whether the neurological impairment is transient or permanent. Both BDNF and E2 are critical neuromodulators of synaptic plasticity. They activate similar cascade pathways and second messenger systems, and have genomic effects in the hippocampus that promote dendritic growth (Luine and Frankfurt 2013). An E2 response element has been identified on the BDNF gene, which may directly trigger BDNF expression when bound to E2 (Scharfman and MacLusky 2006). Studies have shown that both E2 and BDNF play a role in the maintenance of dendritic spines and increase the spine density in adult models of disease (Zhao et al. 2015; Sifat et al. 2017). Consistent with this notion, we found that both E2 and DHF, a BDNF mimetic, increased dendritic spine density, and enhanced cognitive function in the premature kits. We chose to evaluate the effect of E2 supplementation because it is a physiological approach to restoring normal E2 levels in premature infants. Second, DHF was selected for treatment of preterm rabbits because it is a small molecule BDNF mimetic, orally bioavailable, blood–brain barrier penetrant, and thus, can compensate for reduced serum BDNF levels in premature infants and animals (Malamitsi-Puchner et al. 2004; Rao et al. 2009). Moreover, E2 treatment has shown improved cognitive function including learning and memory in animal models of Alzheimer’s disease, diabetes mellitus, and cerebral ischemia (Almli et al. 2000; Aarnoudse-Moens et al. 2009; Pereira et al. 2014; Zhang et al. 2018; Tang et al. 2019). Similarly, BDNF-mimetic therapy has enhanced learning, memory and other cognitive functions in animal models of Down syndrome, Alzheimer’s disease, and neonatal hypoxic-ischemic brain injury (Han et al. 2016; Kazim and Iqbal 2016; Parrini et al. 2017). Hence, these studies reinforce the notion that estrogen and BDNF mimetics can enhance cognitive function in both newborn and adult neuropathologies. The present study shows reductions in CDC42 and Rac1/2/3 levels and elevation in RhoA activity in preterm kits compared with term controls, which is consistent with fewer dendritic spines in premature kits. Rho, Rac, and CDC42 are the most extensively studied small GTPases and are in both inactive GDP-bound and active GTP-bound forms. CDC42 and Rac1/2/3 stabilize actin to promote dendrite branching and spine morphogenesis (Scott et al. 2003), while RhoA destabilizes actin causing dendrite extension and spine collapse (Lee et al. 2000; Tashiro et al. 2000). Overexpression of dominant negative Rac1 reduces spine density in hippocampal slices in culture (Nakayama et al. 2000), indicating GTPase regulation of dendrite arborization and spine density is complex and warrants further investigation. Rac1 triggers F-actin depolarization to stabilize dendritic spine through activation of P21-activated kinase (PAK), LIM-kinase-1 (LIMK-1), and actin-binding protein cofilin (Zhang et al. 2005). RhoA activation affects F-actin polymerization through Rho-associated kinase (ROCK) and LIMK-1 (Maekawa et al. 1999). To determine the levels of active RhoA-GTPase, we used the RhoA pull-down activation assay and found that those levels were increased in preterm rabbits compared with term controls. DHF treatment not only augmented spine density, but also reduced the levels of both total and active RhoA-GTPase proteins, suggesting that a decline in RhoA-GTPase levels by DHF treatment may underlie the observed stabilization of dendritic spines. Moreover, E2 treatment reduced levels of total RhoA-GTPase but not active RhoA-GTPase, although previous studies have shown that stimulating estrogen-related receptor α reduces RhoA stability and its expression to promote spine stability (Sailland et al. 2014). This indicates that E2 treatment might be mediating spinogenesis by mechanisms other than RhoA-GTPase. Interestingly, E2 supplementation also increased the density of thin and basal spines, and showed a trend toward increase for total and apical spine density. However, the density of mushroom spine was reduced in E2-treated kits. E2 treatment quickly increases thin spine formation in hippocampal slice cultures, which is thought to prime dendrites for forming new synapses (Wylot et al. 2015). As thin spines are considered an immature and transient spine species, E2 supplementation may not effectively promote formation of synapse and functional neuronal circuits, unlike DHF supplementation. BDNF-TrkB signaling plays a role in synapse maturation as well as spine maintenance and survival (Fernandez-Lopez et al. 2012). Hence, E2 and DHF treatment appears to promote spinogenesis by different mechanisms. Further investigation is warranted in elucidating the mechanisms of action in dendritic spine development and maintenance in vivo. Taken together, recovery in the density of dendritic spines and downregulation in both total and active Rho-GTPse levels on DHF treatment suggest that restoration of dendritic spines with treatment may be mediated by reduction in Rho-GTPase signaling. Preterm infants often exhibit cognitive and memory deficits and learning disabilities that may be attributed to hippocampal or cortical dysmaturation. We modeled these behavioral deficits in preterm rabbit kits and these premature rabbits displayed loss of social novelty, lack of preference for novel object, heightened anxiety, and diminished exploratory behavior reflecting poor memory and learning. In view of compelling evidence that the CA1 region of the dorsal hippocampus plays a key role in social memory, recognition of object novelty, and anxiety responses, we chose to study dendritic spines in dorsal hippocampus (Antunes and Biala 2012; Cominski et al. 2014). Consistent with our hypotheses we found reduced dendritic spine and arborization in premature rabbits relative to term rabbits. Together, reduced dendritic arborization and spine density in the CA1 could contribute to behavioral deficits in premature kits. Although we did not evaluate prefrontal cortex and other hippocampal areas, it is possible that dendritic morphology would be affected in these brain regions as well. In conclusion, the present study revealed that premature birth leads to cognitive deficits, reduced dendritic arborization, and fewer dendritic spines in the CA1 field. Of significant interest is the finding that E2 and a TrkB agonist reverses prematurity-induced reduction in dendritic density and cognitive function in premature kits. We speculate that E2 supplementation or TrkB agonists might restore changes in dendritic spine and enhance neurodevelopment outcome in premature infants. Funding Supported by NIH grants R01 NS083947-01 (PB) and R01 NS092786 (JV). 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TI - Reduced Hippocampal Dendrite Branching, Spine Density and Neurocognitive Function in Premature Rabbits, and Reversal with Estrogen or TrkB Agonist Treatment
JF - Cerebral Cortex
DO - 10.1093/cercor/bhz033
DA - 2019-12-17
UR - https://www.deepdyve.com/lp/oxford-university-press/reduced-hippocampal-dendrite-branching-spine-density-and-hInG69EQ5W
SP - 4932
VL - 29
IS - 12
DP - DeepDyve
ER -