Early Disruption of Synaptic Function, Impairment of Plasticity, and Decreased of Cortical Circuit Connectivity in an Alzheimer’S Mouse Model of Amyloid Deposition

Mutations in genes encoding amyloid precursor proteins and presenilins lead to increased -amyloid (A) production and cause familial Alzheimer’s disease (AD), a neurodegenerative disorder often associates with aging and features synapse loss and impaired synaptic plasticity. A deposition is a pathological hallmark of AD. It is currently unknown whether and how AD risk alleles affects development of brain circuit function, and whether subtle synaptic pathology occurs prior to overt A deposition. Transgenic mutated APP/PS1 over-expression mice lines are key tools to study molecular mechanisms of AD pathogenesis. Among these mice lines, the 5XFAD mice rapidly recapitulate key features of AD pathology, and have proven utility in studying amyloid plaque formation and A-induced neurodegeneration. We reason that transgenic mutant APP/PS1 over-expression may lead to neurodevelopmental defects in early cortical neurons as a result of continuous APP/A expression from early life. We first explored agedependent plasticity changes in prefrontal cortical circuits in the 5XFAD mice, and found that at an early age (6-8 wks old) that does not produce overt impairment of hippocampus LTP, layer (L) 5 neuron circuit plasticity, measured by long term potentiation (LTP), is impaired. In addition, L5 neurons, which are reportedly vulnerable cortical neuron populations, show reduced mEPSC amplitude and frequency in early postweaning ages, indicating impaired synaptic transmission as a result of transgenic APP/A overloading during early postnatal development. These functional changes were corroborated by the morphological findings of smaller, sparser dendritic spines on L5 pyramidal neurons, indicative of impaired prefrontal circuit function. Lastly, laser scanning photostimulation (LSPS) combined with glutamate uncaging revealed that L2/3 > L5 cortical connectivity was decreased at this early age. Our results suggest 5XFAD transgenic mutant APP/PS1 over expression causes developmental defects of cortical circuits, which could contribute to the age-dependent synaptic pathology and neurodegeneration later in life.


INTRODUCTION
Alzheimer's disease (AD) represents the most frequent form of dementia. A hallmark pathology of the AD brain is the degenerating cortical neurons overloaded with neurofibrillary tangles and amyloid plagues 1 . At cellular and functional levels, early AD brain is characterized by impaired synaptic function and synapse loss, manifested as disrupted synaptic plasticity, learning, memory, and cognition 2 . Transgenic mice that overexpress the mutated human amyloid precursor protein (APP), presenilin (PS) and tau genes reproduce many aspects of AD pathology such as β-amyloid plaques (Aβ), neurofibrillary tangles, reactive gliosis, synaptic and neuronal loss, which are consistent with behavioral changes such as progressive age-dependent memory impairments 3 . As such, these mice line are useful to inform molecular and cellular changes associated with AD.
The 5XFAD mouse line, which co-overexpresses human APP and presenilin 1 (PS1) harboring five familial AD (FAD) mutations manifest very early-onset and aggressive amyloid deposition 4, 5 ; These mice start to develop amyloid plaques as early as ~2 months of age, at which they show dramatically accelerated intraneuronal Aβ42 production. Pathologically, A deposition emerges in the hippocampus subiculum and in layer 5 (L5) principal neurons of the neocortex, and rapidly increases in affected brain regions 4 . Synaptic failures reportedly taken place prior to overt plaque formation; for example, it has been shown that the hippocampal basal synaptic transmission and theta burst stimulus (TBS)-induced LTP is impaired at 6 months, but not prior to 4 months 6 . In addition, synaptic failure can manifest at multiple levels, ranging from functional alterations to structural disturbances 7 . Synaptic failure of L5 neurons in 5xFAD mice reportedly long precede their physical loss, which happens by 12 months of age 7 .
Although AD is a disease associated with aging, the mutant forms APP/PS1 is expected to be expressed throughout life span. As such, how transgenic over-expression of mutant forms APP/PS1 affects the developing neural circuit function may be an interesting question. The 5XFAD mouse line, which utilizes Thy1 promoter (Bradley, Ramirez, & Hagood, 2009) to drive transgenic APP/PS1 expression, offers a unique opportunity to study neurodevelopmental effects of APP/PS1 over-loading in the affected neuron populations. Despite a large literature exploring mechanisms of neurodegeneration in 5XFAD mice [8][9][10][11] , in this study we focused on early developmental synaptic deficits resulting from mutated forms of APP/PS1. Because Thy1promotor driven expression of mutated forms of APP/PS1 are expected to predominantly affect layer 5 neurons 7, 12, 13 , we reason that mutated transgenic APP/PS1 and the processed intracellular A may appear at an much early time point, thus impairing normal development of synapse function and connectivity at very early age. In this study, we provide electrophysiology evidence of impaired synaptic activity, plasticity, and decreased of cortical circuit connectivity in the L5 prefrontal cortex neurons at an early postweaning age (P25-35). Our data suggest that transgenic mutant APP/PS1 over-loading in the 5XFAD mouse model leads to impaired early cortical circuit development, an underappreciated neuropathology in this prevalent AD mouse model.

Animals
The 5XFAD mice used in this study were obtained from Jackson laboratory (catalog #34840-JAX), and maintained on the B6SJLF1 background. This mouse line overexpresses both human amyloid precursor protein (APP) gene with K670N/M671L, V717I, and I716V mutations and the human PS1 M146L and L286V mutations under the Thy1
The brain slices were transferred to an interface chamber (AutoMate Scientific) to facilitate long-term slice viability, and superfused with ACSF saturated with 95% O2 / 5% CO2. Field excitatory postsynaptic potentials (fEPSPs) were recorded using a glass patch electrode in L5 pyramidal neuron layer (mPFC recording, in response to L2/3 stimulation) or in the CA1 stratum radiatum layer (hippocampus recording, in response to Schaffer collateral stimulation). The patch electrode had an electrical resistance of 1-2 MΩ at 1kHz when filled with ACSF. Electric stimuli were delivered by a bipolar tungsten electrode (FHC, Bowdoin, ME) that was placed ~200 μm away from the recording site, using biphasic stimuli (10-250 μA, 100 μs duration, 0.05 Hz for baseline recording). Stimulus was generated using a Digidata 1440A (Molecular Devices, San Jose, CA) device, and delivered through an optic isolator (Iso-flex, A.M.P.I). fEPSP signals were amplified using a differential amplifier (model 1800, A-M Systems, Carlsborg, WA), low-pass filtered at 2 kHz and digitized at 10 kHz.
For fEPSP recordings from both mPFC and HPC, a stimulus-response (input-output) curve was obtained by measuring fEPSP slope (first 1-ms response after fiber volley) as a function of the fiber volley amplitude, which is used to quantify basal synaptic transmission strength. We then chose a stimulus intensity produces a ∼40-50% maximum fEPSP amplitude throughout the experiments. Following a 10min stable baseline response of stimulus-evoked fEPSPs, we tested paired-pulse responses at inter-pulse intervals ranging from 20-200ms in order to probe potential changes in presynaptic transmission. An LTP induction stimulation protocol was then applied. Because stimulation pattern is critical for LTP/LTD induction and maintenance [15][16][17][18][19] , we adopted a tetanus stimulation protocol that consisted of two, 1-sec trains of stimulation at 100Hz 20 for both mPFC and CA1 LTP induction. Following LTP induction, fEPSP responses were recorded for an additional 1 hour.

Whole cell recording in brain slices
Whole cell recordings were conducted in L 5 pyramidal neurons in parasagittal mPFC slices, which are prepared similarly to slices used for fEPSP recordings, except they are perfused with 95%O2/5% CO2-saturated ACSF in a submerged chamber during recording. Slices were visualized under a 4X objective (Olympus UPlanApo, NA= 0.16) to locate the cytoarchitectural landmarks of layer 5. To minimize neurite cutoffs, only L5 pyramidal neurons with soma at least 50m below the slice surface were selected for whole cell recordings. Neuronal soma were identified and targeted using a 60X objective (NA = 0.9) under IR illumination (Olympus BX-51 WI).
Neuronal signals were amplified using a MultiClamp 700B amplifier (Molecular Devices, Forster City, CA), low-pass filtered at 1 kHz for voltage clamp recordings, and digitized at 20 kHz using a Digidata 1440A interface controlled by responses were obtained when neurons were sequentially voltage clamped at -70 mV (for AMPAR-mediated synaptic currents) and + 40 mV (AMPAR+NMDAR currents). A:N ratio was quantified using the peak of EPSC amplitude at -70 mV ( AMPAR current), and +40mV (NMDAR current, which is measured at 75ms after stimulus onset 21 ).

Neuronal morphology
Analyses of the recorded L5 mFPC neuronal morphology, including dendritic arborization and spine morphometric measures, was performed as described previously 22,23 . After completion of whole cell recording, neurons were injected with 500pA current to facilitate biocytin diffusion into the neurite processes. The slices were then fixed in 4% PFA overnight, blocked with 1% BSA/0.01M PBS, permeabilized with 0.2% Triton X-100. Slices were further incubated with avidin-Alexa 488 for 24 h and mounted on glass slides with a pair of ~350 m spacers to prevent tissue crushing.
Neuronal dendritic arbors were reconstructed after collecting Z-stack images on a confocal microscope (Zeiss LSM 710). The maximal projection images were then imported into FIJI/ImageJ, and neurite arborization and Sholl analysis 24 were done using the Simple Neurite Tracer plugin 25 . This allows quantification of morphometric features such as dendritic arbor, length, and number of intersections as a function of distances from soma. To analyze spines, Z stacks of spines from the apical dendrites (200-450 μm away from soma, secondary branches) were collected with a 63x objectives (Plan-Apochromat, NA 1.4). Each Z stack was collected at 512 × 512 pixels, with 4 × digital zoom and 0.2 μm Z step size. Imaris software (V8.02, Bitplane, South Windsor, CT) was used to measure spine length, density, and head volumes, as we described previously 22 .

Laser scanning photostimulation for functional circuit mapping
To investigate how transgenic mutant APP/PS1 expression affects synaptic connectivity, we used laser scanning photostimulation (LSPS) combined with glutamate uncaging 26,27 to map synaptic connectivity made onto the L5 pyramidal neurons in the mPFC. 5XFAD and littermate control mice were sacrificed at P25-35, during which intracellular APP overload was confirmed by immunohistochemistry staining. Parasagittal mPFC slices were made and perfused in modified ACSF (4 mM Ca 2+ , 4 mM Mg 2+ ) that contains 0.2 mM MNI-caged glutamate and 5 μM R-CPP (block NMDA receptors and short-term plasticity). To minimize truncation of dendritic structures and preserve connectivity, only L5 neurons with pyramidal shaped soma that are >50 μm below the slice surface were selected for recording/mapping. LSPS mapping/glutamate uncaging was performed using a 4× objective lens (NA 0.16; Olympus) and a UV laser (355 nm; DPSS Lasers). 1-ms, 20-mW UV laser pulses were delivered onto slices through a pair of X-Y mirror to generate a 16X16 stimulation grid with 100μm spacing. The top of stimulation grid was aligned with the pia surface, and span the pia to white matter. Stimulation location was registered onto a digital image using a CCD camera (Retiga 2000DC, Qimaging). Laser power/timing was controlled by an optic shutter (Conoptics, model 3050), a mechanical shutter (Unibliz VCM-D1) and a neutral density filter (Edmund Optics), and constantly monitored using a photodiode (Edmund Optics) and current amplifier (Sanford Research Systems, model SR570) that feeds to the BNC-6259 boards (National Instruments, Austin, TX). Neuronal signals were amplified with a Multiclamp 700B amplifier, digitized at 10 kHz, and acquired using two BNC-6259 boards. Data synchronization, acquisition and analyses were implemented by Ephus software, a suite of customized MATLAB scripts 27 .

Immunohistochemistry/Immunofluorescence
Mice were anesthetized with 4% isoflurane, and transcardially perfused with 0.01M PBS, followed by cold 4% Images were collected on a LSM 710 confocal microscope (Zeiss) with appropriate laser lines.

Statistical analysis
All results were reported as mean ± s.e.m. (standard error of the mean). The experimenters were blinded to mouse genotypes/grouping during data collection and analyses. Sample sizes and number of independent experiments were estimated by power analyses using an R script that takes pre-specified effect size, type I and II errors as input arguments. Sex-segregated data were first analyzed for potential sex-specific effects, and pooled together for group analyses.

APP/A overload in mPFC L5 neuron during early postnatal neurodevelopment
It has been previously shown that 5XFAD mice exhibit a robust A pathology with an onset of extracellular plaque pathology at approximately 2 months of age, and manifest abundant intraneuronal and plaqueassociated pathology as early as 3 months of age 4 . Because of Thy1 promoter-driven transgenic APP/PS1 expression is enriched in layer 5 neurons and is across life span 12, 13 , we asked whether intraneuronal APP/A load can be detected in cortical L5 neurons at very early developmental stages. We conducted IHC staining to detect APP/A (6E10 antibody), and found that in most cortical regions including the prefrontal cortex, retrosplenial cortex, L5 neurons show strong APP/Ab immunoreactivity at postnatal day (P) 21, which appear stronger than CA1 neuron immunolabeling (Fig 1A). In addition, we observed strong APP/A immunoreactivity in the subiculum region in the temporal levels of hippocampus, consistent with reports that the subiculum is one of earliest structure that shows APP/A overloading 4 . We also found that APP/A overloading in L5 neuron at this early age is associated with few Iba1-positive activated microglia (Fig. 1B) that is comparable to the density of Iba1-labeled microglia non-transgenic controls (not shown). Importantly, at this early age, no extracellular amyloid plaque or Thio-S+ insoluble fibrillar dense core A deposits was observed. In contrast, mPFC layer 5 neurons in 5 months old 5XFAD mice show extensive APP/A immunoreactivity, increased A plaque deposition, clustered Iba-1+ reactive microglia surrounding the plaques, and the Thio-S -labeled dense core fibrillar amyloid deposits (Fig 1B).
Notably, the APP/A intracellular overloading in L5 mPFC neurons was also seen at earlier ages (P11 and P14, data not shown), indicating transgenic expression of mutant APP/PS1 is a continuous process spanning early neurodevelopment.

Impaired L5 PFC neuron synaptic plasticity early in development
It has been well established that impaired plasticity, such as LTP, is a core synaptic deficit in AD brain 28,29 .

Decreased dendritic spine size and density in L5 mPFC neurons in early postweaning 5XFAD mice
We next focused on potential anatomical changes of mPFC L5 neurons during early development by analyzing the dendritic structure and spine morphometry at P22-30, during which we found impaired synaptic activity. L5 neuron morphology was revealed by biocytin injection (Fig. 4A), followed by confocal imaging of the dendritic structure and Z-stack images of spines. Sholl analysis revealed that 5XFAD did not affect the number of dendritic intersections as a function of distance from soma (Fig. 4B. Main group effect: F(1, 15) = 2.93, p = 0.11, two-way ANOVA), nor did it affect dendritic length distribution (Fig. 4C. main group effect: F(1, 15) = 0.033, p = 0.86, two-way ANOVA). However, dendritic spine from 5XFAD neurons exhibited a reduced density (Fig. 4D, 4E. Number of spines/10m: WT, 12.52 ± 0.45; 5XFAD, 10.8 ± 0.37, t(21) = 2.89, p = 0.008). In comparison, spine length from 5XFAD L5 neurons did not differ (Fig. 4F. Averaged length in m: WT, 2.06 ± 0.13; 5XFAD, 2.20 ± 0.17, t(21) = 0.69, p = 0.49). We next quantified the spine head volume, and found that 5XFAD neurons show significantly reduced spine head volume distribution ( Fig. 4G. WT, n = 274 spines/6 neurons/6 mice; 5XFAD, n = 258 spines/7 neurons/5 mice. D = 0.286, p< 0.0001, K-S test). Considering that spine size and geometry are well established to correlate with glutamate receptor content and functional maturity 30 , these data are consistent with decreased mEPSC amplitude and frequency, and further suggest impaired synaptic function at very early development age in the PFC.

mPFC L5 neurons from 5XFAD mice show reduced intracortical circuit connectivity at 4-5 wks age
We hypothesize that the early transgenic mutant APP/PS1 expression may alter intracortical functional connectivity in L5 mPFC neurons, either as a result of impaired establishment of developmental circuits, or as a result of early degradation of synaptic connectivity. mPFC cortical circuits show conserved connectivity patterns, with balanced excitation and inhibition distributed across both columnar and laminar dimensions 26,31,32 . We used LSPS mapping 14,26,27 to investigate synaptic connectivity made onto L5 pyramidal neurons in mPFC in parasagittal brain slices (Fig. 5A). L5 neurons from P25-35 5XFAD mice and littermate controls were voltage clamped, and glutamate uncaging at different cortical locations produces excitatory currents that reflect either soma/dendrite-mediated direct EPSC responses or synaptic EPSC responses (see methods) (Fig. 5B). In addition, by holding at 0mV, postsynaptic inhibitory currents (IPSCs) can be also obtained. This allows us to construct a 'map' of local circuit connectivity (both excitatory and inhibitory. Fig. 5C).
These data suggest reduced excitatory synaptic connectivity onto L5 neurons in mPFC at very early (4-5 wks) age, and are consistent with reduced mEPSC amplitude/frequency and dendritic spine size and density found on these neurons.

DISCUSSION
In this study, we report very early synaptic deficits in 5XFAD mice, which include impaired synaptic plasticity (LTP) in the mPFC L5 neurons, deficits in synaptic activity and intracortical connectivity. These functional changes are correlated with morphological alternations at dendritic spine levels and reflect the age-dependent intracellular transgenic APP/A overloading. Our data support that 5XFAD transgenic expression of mutant APP/PS1 leads to impaired development of neuronal function and connectivity at very early post-weaning age. The main focus of this paper is not to exhaustively pursue age-dependent pathological or electrophysiological changes, but rather to focus on L5 mPFC neurons at a very early age and probe circuit impairment using sensitive physiological and morphological measures. To the best of our knowledge, this study reports the earliest synaptic function and connectivity changes in 5XFAD mice in a major mPFC projection neuron population. Our data demonstrate that mutant forms of APP/PS1 and associated A production could pose a neurodevelopmental syndrome featuring early synaptic loss and dysconnectivity. While it is possible that by early post-weaning ages, synaptic loss and degradation already occurs as a result of APP/A overloading, an alternative explanation for these observations is that the synaptic connectivity, maturation, and circuit function in L5 mPFC neurons in 5XFAD mice are impaired developmentally and never achieved the same functionality as the control neurons.
Using immunohistochemistry staining, we have shown that L5 mPFC neurons exhibit increased intracellular APP/A loading at P21 and earlier. Similarly, another vulnerable cortical population in terms of APP/A overloading is the subiculum neurons of the temporal hippocampus, whose functional changes remain to be investigated. Because the 6E10 antibody for A does not distinguish APP and A 33, 34 , we cannot attribute these observed synaptic and circuit connectivity changes to APP or to intracellular A production. Yet, our IHC staining revealed that minimum microglia activation at P21, and there was no extracellular amyloid deposition or dense core fibrillar A plaques in extracellular space. It is therefore less likely L5 neuronal loss occurs at this early age. It has been reported that cortical L5 neurons are among the first neuronal populations that develop synaptic pathology 4, 7 , including decreased spontaneous mEPSCs frequency and amplitude, higher threshold for firing of action potentials, which happened before any overt structural dystrophy 7 . Yet, these pathological changes occur at much later stages that those observed by this study.
It is unclear how mutant APP over-expression affects synaptic function in the developing brain and how these detrimental effects evolve with age. Neurophysiological data exploring the effects of transgenic APP/PS1 on the prefrontal cortex circuit function are rather limited. Yet, mPFC is a primary and early target that develops AD pathology [35][36][37] . Transgenic overloading of APP/A in the developing cortical circuits may disrupt a myriad physiological function of neurons, including defective endo-lysosomal trafficking 38 39 , impaired intracellular cargo transport 40 , altered intracellular signaling 41,42 , or defective neurotransmitter release 43 that collectively contribute to failure of synaptic connectivity or synaptic degradation. That the impaired synaptic plasticity/LTP was observed at 6-8 wks, but not at early postweaning (P22-30), during which synaptic connectivity was reduced, suggest disruption of plasticityrelated mechanisms by APP/A may not be as sensitive as connectivity of developing cortical circuits. In some studies impaired LTP has been detected in AD mice models before 4 months of age 44,45 but typically appears at later age when intracellular A load in higher 6,[46][47][48][49][50][51] . It was also reported that LTP in layer 5 of somatosensory cortex was more severely impaired than LTP triggered in CA1 area in 6 months 5xFAD mice, which could be attributed to increased somatosensory cortex amyloid deposition 52 . It has also been reported that theta burst stimulation-induced hippocampus LTP is impaired at 6 months but not prior to 4 months 6,52 . In contrast to these reports, using a stronger LTP induction protocol (tetanus stimulation), we found that LTP in L5 mPFC neurons was impaired as early as 6-8 wks, during which the CA1 LTP seems normal. This may be due to overall higher L5 mPFC neuron intracellular APP/ A production or increased vulnerability of this neuronal population.
In summary, our study revealed that synaptic function and plasticity changes occur at very early postweaning age in the 5XFAD AD mouse model, and provided the first demonstration of reduced circuit connectivity in a major prefrontal projection neuron type as a result of APP/A overloading. These functional disruptions suggest that transgenic mutant APP/PS1 over-expression has a profound effect on developing cortical circuit, and as such changes in neuronal function may be a life-long process that impact neuronal degeneration in later stages. This thought-provoking hypothesis justifies further experimentation.