Neonatal loss of FGFR2 in astroglial cells affects locomotion, sociability, working memory, and glia-neuron interactions in mice By Nate Welch Background – FGFR2 Fibroblast Growth Factor signaling has many developmental roles FGFR2 receptors are almost exclusively expressed in non-neurons in the postnatal forebrain Role of specific FGF receptors in astrocytes are still undefined Necessary to determine the separate roles at different critical stages of development How does astrocyte-dependent signaling through FGFR2 affect behavioral regulation across different domains? Overview and Research Goals Targeted FGF signaling in the brain at different times to determine the role FGFR2 plays at critical stages of development Three groups with cell targeted knockouts of FGFR2 were used: cKO: Knockout during embryogenesis in radial glial cells - hGFAP-cre nKO: Knockout during the rodent in early postnatal period “largely in proliferating astroglial precursors and glial progeny” GFAP-creERT2 iKO: Knockout during adulthood in post-mitotic astroglia GFAP-creERT2 Hypothesis: that early postnatal loss of FGFR2 in astrocytes would affect function and impact the regulation of behaviors that are disrupted at early postnatal time points in psychiatric disorders Mice – Conditional FGFR2 (KO) Conditional hGFAP-cre fgfr2 knockout mice conditional fgfr2 null allele harbors loxP recombination sites flanking regions encoding the Ig III binding and transmembrane domains of the fgfr2 gene (fgfr2 ) Homozygous fgfr2 mice were crossed with mice expressing the Cre recombinase transgene under the control of the human glial fibrillary acidic protein promoter (hGFAP) hGFAP-Cre transgene targets Cre recombination to radial glia progenitors of the dorsal telencephalon starting at E13.5 (cKO) Homozygous fgfr2 mice crossed with GFAP-CreERT2 (GCE) mice Administering tamoxifen in Cre+ mice induces KO Caveat - KO is assumed to be substantially more effective in astroglia than neural stem cells Inducible Postnatal KO Protocol First Neonatal Protocol (nKO) Mother received intraperitoneal injections of 1 mg/day of tamoxifen dissolved in sunflower seed oil for 5 consecutive days starting on P(1,2,3) during nursing Second Neonatal Protocol (iKO) Adult mice received injections of 0.5mg tamoxifen dissolved in sunflower seed oil twice daily for 5 consecutive days at 2-4 months Background – Behavior Testing Behavior testing started at least 9 days after the time of the last tamoxifen injection Performed during the light cycle in dedicated test room, mice were grouped according to age and strain Testing started at 2.5 months to 4.5 months, completed between 5.5 to 7.5 months of age Only male mice were tested Open Field Test • Operational definition: anxiety-like behaviors and locomotor activity • Square or rectangular plastic arena (1500cm^2) • First 5 min evaluated for “center time” • Distance traveled was evaluated in 5 min epochs Three Chamber Social Approach Operational definition: social behavior – social approach and social recognition Three Chamber apparatus using two “stranger” mice to test mouse (same strain and age) Zeroth Stage: Habitation First Stage: test mouse was habituated to center chamber with one stranger mouse and empty chamber Second Stage: New stranger mouse was added to empty chamber Social Approach vs Social Recognition Y and Radial Arm Water Maze Y maze Operational definition: working memory – “spontaneous alternations” Movement monitored noting arm entry order For each set of three entries, spontaneous alternations of those entries through all three arms were noted Assumes rodents prefer to investigate a new arm of the maze than return to a familiar area Radial Arm Water Maze Operational definition: spatial memory Tested with visual spatial cues over 2 days Day 1: working memory of platform location over 15 trials Day 2: testing consolidation of short-term memory using only hidden platforms over 15 trials Elevated Plus Maze Operational definition: anxiety like behavior “+” shaped maze elevated above the floor with two oppositely positioned closed arms, two oppositely positioned open arms and a center area Ratio of the time spent in open and closed zones indicate level of anxiety-like behavior Total entries into arms indicates locomotor activity Results - Analysis of KO efficacy Cre is expressed in radial glia beginning at E13.5 which affects neuronal and glial progeny in regions where hGFAP-Cre is expressed (Forebrain and Cerebellum) cKO - 80–91% loss of fgfr2 gene expression was previously found and a substantial reduction of FGFR2 protein level iKO - 80% reduction in fgfr2 gene expression Results - Analysis of KO efficacy Embryonic knock-out of FGFR2: Open Field, Social Approach, and Y-Maze • FGFR2 cKO demonstrated locomotor hyperactivity (55% greater) (Fig.1A) • Exhibited reduced anxiety-like behavior (Fig.1B) • Demonstrated higher preference for interacting socially with a novel stranger than a novel object, regardless of proximity (Fig.1C) • Social recognition relatively unchanged (Fig.1D) • Small impairment to working memory (Fig.1E) Embryonic knock-out of FGFR2: cKO Elevated Plus Maze FGFR2 cKO demonstrated reduced anxiety-like behavior (Fig.1 F-G) Showed increased locomotor acvitiy(Fig.1H) Neonatal knock-out of FGFR2: nKO Open Field, Social Approach, Y-Maze, and R.A.W. Maze FGFR2 nKO exhibited a small increase in locomotor activity (Fig.2A) Showed no difference in anxiety-like behaviors from time in center-open field(Fig.2B) Higher social preference with no deficit in social recognition (Fig.2C-D) Minor deficit in working and spatial memory (Fig.2E-F) Neonatal knock-out of FGFR2: nKO Elevated Plus Maze nKO exhibited a small decrease in anxiety-like behaviors comparable to the cKO mice (Fig.2G-H) Increased locomotor activity (Fig.2I) Adult knock-out of FGFR2: iKO Cre+ and Cresimilarities iKO exhibited no alterations in locomotor activity, working memory, or social preference (Fig.3A,C-F) Adult knock-out of FGFR2: Decreased Anxiety behaviors in iKO iKO demonstrated a decrease in anxiety like behaviors Increased time spent at center of open field (Fig.3B) Less time spent in closed arms of elevated plus maze, with a higher ratio of open to closed (Fig.3G-I) Neurobiological Findings: nKO GFAP + Astrocyte Density in Hippocampus • No difference in density: (n = 3,3; p = 0.36, control = 8.45 ± 0.97 × 10−6, FGFR2 nKO=10.14 ± 1.37 × 10−6 cells/μm^3) • Unchanged volume: (n = 3,3, p = 0.86, control = 3.1 ± 0.6 mm3, FGFR2 nKO = 3.2 ± 0.6 mm3) • cKO also showed no difference in cell density indicating astrocyte numbers were unaffected by early loss of FGFR2 Neurobiological Findings: Astrocyte Morphology Analysis of the astrocytic coverage of the cell membrane of neurons showed decreased coverage (Fig.4A-C) Note: Astrocyte-neuron contact was coupled to increased number of synapses on the same cells (Fig.4D) Neurobiological Findings: Glutamine Synthetase Expression • nKO/cKO showed increased density in the hippocampus • nKO exhibited increased density in medial frontal cortex • iKO little change in expression Neurobiological Findings: nKO Cortical Density of Synaptic Proteins Increased density of vGAT and vGLUT1 (Fig.4E-H) Gephyrin and PSD-95 puncta analysis indicated no change in density (Fig.4E-H) vGAT and vGLUT1 increases were localized to upper cortical layers (Fig4I, J) Gene expression analysis – vGAT and vGlut1 increased in juvenile hippocampus (n = 8,5; vGat: p < 0.005, control = 1.00 ± 0.07, FGFR2 nKO = 3.62 ± 0.91; vGlut1: p = 0.09 control = 1.00 ± 0.12, FGFR2 nKO = 3.70 ± 1.86) Discussion – Results Summary Embryonic/Neonatal Mice lacking FGFR2 expression in astroglia (cKO/nKO) exhibited locomotor hyperactivity, working memory deficits, and increased sociability nKO mice demonstrated decreased astroglia-neuron membrane contacts, increased neuronal synapses, and increased signaling indicated by EM and puncta density of their presynaptic vesicular proteins Suggests increased glutamine synthetase expression in cKO/nKO is a functional aftereffect of increased neuronal signaling All assessed development periods exhibited a small decrease in anxiety-like behaviors Discussion They suggest FGFR2 nKO/cKO behavioral findings indicate that fibroblast growth factor signaling in the early postnatal brain may affect the risk for behavioral disorders Results shows that varying the FGFR2 pathway during sensitive periods of development create different behavior patterns FGF is well documented in regulating glial proliferation and fate – astrocyte count was maintained with differing behavior outcomes cKO/nKO affected astrocytes changed neuronal development which manifested as the observed deviant behavioral triad Increased presynaptic vesicular proteins and GS expression in nKO glial cells suggests increased neuronal signaling Comments Astrocyte Morphology relatively unchanged? co-localized pre- and postsynaptic protein densities were unchanged - data not shown Calcium Imaging? Interpretation of animal testing - generally inform the understanding Sample Exam Question "How do the observed changes in astrocyte-neuron interactions, like reduced coverage and altered expression of synaptic vesicular proteins in the Neonatal FGFR2 knockout mice contribute to the observed behavioral changes, and how might these findings inform our understanding of the potential role of astrocytes in the etiology and treatment of neuropsychiatric disorders like ADHD?" Questions?
