Presynaptic Fmr1 Genotype Influences the Degree of Synaptic Connectivity in a Mosaic Mouse Model of Fragile X Syndrome

Abstract

Almost all female and some male fragile X syndrome (FXS) patients are mosaic for expression of the FMR1 gene, yet all research in models of FXS has been in animals uniformly lacking Fmr1 expression. Therefore, we developed a system allowing neuronal genotype to be visualized in vitro in mouse brain slices mosaic for Fmr1 expression. Whole-cell recordings from individual pairs of presynaptic and postsynaptic neurons in organotypic hippocampal slices were used to probe the cell-autonomous effects of Fmr1 genotype in mosaic networks. These recordings revealed that wild-type presynaptic neurons formed synaptic connections at a greater rate than presynaptic neurons lacking normal Fmr1 function in mosaic networks. At the same time, the postsynaptic Fmr1 genotype did not influence the probability that a neuron received synaptic connections. Asymmetric presynaptic function during development of the brain could result in a decreased participation in network function by the portion of neurons lacking FMR1 expression.

Introduction

Fragile X syndrome (FXS) is one of the most common inherited forms of mental retardation (Crawford et al., 2001). FXS is caused by trinucleotide expansion within the FMR1 gene on the X chromosome resulting in hypermethylation of the 5′ untranslated region and loss of fragile X mental retardation protein (FMRP) expression (Pieretti et al., 1991). FXS patients suffer mental retardation, macroorchidism, facial dysmorphologies, and behavioral problems (Hagerman, 2002) as well as a high incidence of epilepsy (Musumeci et al., 1999). Because a small but significant proportion of children with autism spectrum disorder (ASD) have a mutation in the FMR1 gene (Brown et al., 1986) and up to 33% of children with FXS fulfill a diagnosis of autism (Rogers et al., 2001), FXS is considered to be a genetic model of ASD (Hagerman et al., 2005).

Male patients carry the mutation on their single X chromosome and typically exhibit relatively severe mental impairment whereas female patients heterozygous for the mutation are generally less severely impaired (Reiss et al., 1995). In female patients, X-inactivation silences either the mutant or normal X chromosome in each cell, resulting in mosaic FMRP expression, with variable skewing of X inactivation leading to variability in the percentage of cells expressing FMRP (Martínez et al., 2005). In addition, variable silencing of the FMR1 gene in different cells, can results in mosaic expression of FMRP in some male patients (Pieretti et al., 1991; Nolin et al., 1994; Rousseau et al., 1994).

The mouse model of FXS is a knock-out (KO) of the Fmr1 gene and exhibits many phenotypic characteristics of FXS (The Dutch-Belgian Fragile X Consortium, 1994). This model has proven to be of great value in probing the alterations that result from loss of Fmr1 gene function (Kooy, 2003). For example, electrophysiological recordings have demonstrated both reduced long-term synaptic potentiation in the cortex and amygdala (Li et al., 2002; Larson et al., 2005; Zhao et al., 2005) and enhanced long-term synaptic depression in the hippocampus and cerebellum of Fmr1 KO mice (Huber et al., 2002; Koekkoek et al., 2005). Although the insight gained from such work has provided hope for identifying therapeutic targets (Bear et al., 2004), no studies have addressed the neuropathology specific to mosaic brains that contain neurons both with and without active Fmr1. Therefore, to gain insight into the types of neuronal alterations that could exist in FXS patients with mosaic FMR1 expression, we developed a system that models mosaic FXS, and is amenable to electrophysiological recordings from neurons of known genotype. Measurements of individual synaptic connections using this approach revealed a novel cell-autonomous presynaptic role for Fmr1 function in the establishment of functional synaptic connections.

Materials and Methods

Animals.

All experiments were performed using F1 females from the breeding of Fmr1 KO or Fmr1 wild-type (WT) controls on an FVB background (FVB.129P2-Fmr1^tm1Cgr/J, JAX catalog #4624, or FVB.129P2-Pde6b⁺Tyr^c-ch/AntJ, JAX catalog #4828; The Jackson Laboratory, Bar Harbor, ME) to mice carrying a green fluorescent protein (GFP) transgene on their X chromosome (Hadjantonakis et al., 1998) that were originally from a mixed 129/ICR background [stock transgenic(GFPX)4Nagy/J, JAX catalog #3116] and had been backcrossed onto the 129 background for six generations before use in this study.

Organotypic slices.

Interface cultures of hippocampal slices were made from 5- to 6-d-old mice (n = 9 Fmr1 mosaic; 9 control mosaic). Hippocampi were dissected in minimum essential medium (MEM; Invitrogen, Eugene, OR) with 15 mm HEPES and 10 mm Tris buffer (Invitrogen). Four-hundred micrometer slices were cultured on Millicell CM culture plate inserts (Millipore, Temecula, CA). The culture medium consisted of 50% MEM, 25% HBSS, and 25% horse serum, with 12.5 mm HEPES buffer and penicillin (100 U/ml)/streptomycin (100 μg/ml) (all from Invitrogen). Cultures were maintained in 5% CO₂, at 37°C for 3 d and then at 34°C for the remaining culture period. Slices were recorded from after 6–10 d in culture.

Neuron visualization.

Pyramidal cells in area CA3 were visualized by infrared differential interference contrast microscopy (IR-DIC) and GFP labeling was visualized using epifluorescence. Photobleaching and phototoxicity were minimized by shuttering the UV light source so as to illuminate the slice for a duration of 50–150 ms at 1.5–2 s intervals using a Lambda SC Smart Shutter (Sutter, Novato, CA). Presynaptic recordings were obtained first and then potential postsynaptic partners were targeted so as to allow visualization of both neurons within the same field of view of the microscope. That control GFP mosaics showed no alterations to presynaptic or postsynaptic connectivity (see Fig. 4b) indicates that these measurements were free of any artifact that could have theoretically been caused by unintentional biasing of the distance between pairs of neurons as a function of GFP expression.

Figure 4.

Functional connection probability functions of genotype. A, The total proportion of neuron pairs with synaptic connections is shown as a function of either presynaptic or postsynaptic genotype for Fmr1 mosaics. Error bars show the 95% confidence intervals of the proportions. The asterisk indicates that the raw number of functional connections was significantly reduced in neuron pairs with Fmr1 KO presynaptic neurons relative to pairs with Fmr1 WT presynaptic neurons (p < 0.05, Pearson's χ² test) (see Results and Materials and Methods). At the same time, the proportion of functional connections was not significantly affected by the postsynaptic Fmr1 genotype. Note that because GFP neurons were selected for recordings at a much greater frequency than they occurred in the slices, the absolute connectivity rates calculated from these recordings do not necessarily reflect the overall mean connectivity of the slices. B, The total proportion of neuron pairs with synaptic connections is shown as a function of either presynaptic or postsynaptic GFP labeling in control mosaics (see Results) (total n = 58 pairs). There was no difference in the percentage of functional connections in control mosaics caused by either presynaptic or postsynaptic GFP labeling status.

After seal formation, but before going whole-cell, the presence or absence of GFP in each recorded neuron was confirmed as follows: 16–24 frames of epifluorescence were captured from an analog video camera and averaged using a LG-3 frame-grabber (Scion, Frederick, MD) and the contrast and brightness of the average image were optimized (Photoshop; Adobe, San Jose, CA). The image of the fluorescence was then aligned with an IR-DIC image from the same focal plane showing the position of the recording electrode and the cell body. As confirmation of GFP(+) status, at the end of each recording we examined another image of the recorded neuron to confirm that the soluble GFP had dialyzed out of the neuron and into the whole-cell electrode. As confirmation of GFP(−) status, at the end of the experiment the recorded neuron was imaged again to confirm the persistence of the background fluorescence (i.e., no evidence of dialysis of intracellular GFP).

Electrophysiological recordings.

One to three independent pairs of neurons were recorded per slice and when multiple pairs were recorded in the same slice, pairs with differing genotype profiles were targeted. On average, approximately four slices were used from each animal. Slices were immersed in oxygenated artificial CSF at room temperature, containing (in mm) 119 NaCl, 2.5 KCl, 1.3 MgSO₄, 2.5 CaCl2, 1 Na₂HPO₄, 26.2 NaHCO3, and 11 glucose, perfused at a rate of 2 ml/min. The internal electrode solution consisted of the following (in mm): 120 K gluconate (presynaptic cell) or Cs gluconate (postsynaptic cell), 40 HEPES, 5 MgCl2, 0.3 MgGTP, 2 NaATP, and 5 QX314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride; postsynaptic cell only], pH 7.2, with KOH or CsOH. Presynaptic neurons were held in current-clamp mode and postsynaptic neurons were voltage clamped using a MultiClamp 700A amplifier (Molecular Devices, Foster City, CA). Input resistance was measured in postsynaptic neurons and was not significantly different between WT and KO neurons in mosaic slices (mean ± SD, 267 ± 153 MΩ for WT vs 265 ± 126 for KO; n = 26 and 32, respectively; p > 0.05, t test). Presynaptic cells were kept at −70 mV by injection of bias current and action potentials were induced by 20 ms current pulses (typically 20 pA) and were elicited at 0.1 Hz. Action potential threshold measured during the current pulses did not significantly differ between WT and KO neurons (−37.8 ± 3.6 mV for WT vs −36.5 ± 4.4 for KO; p > 0.05 t test). Postsynaptic currents in response to presynaptic action potential firing were recorded while the postsynaptic cell was held at −70 mV. All neuron pairs not showing a functional connection were tested by pairing 1 Hz presynaptic with depolarization of the postsynaptic neuron to 0 mV. In contrast to work in different mouse strains (Hanson et al., 2007), silent synapses (synapses without initial responses at −70 mV, but which could be awakened by this stimuli) were very infrequently observed (∼5% occurrence). Because the occurrence was too low, this class of synapse could not be independently analyzed. However, this indicates that strong synaptic activation did not significantly cause new connections to form.

Statistics.

The proportions of pairs of neurons that were connected versus unconnected were compared using Pearson's χ² test to analyze the raw number of observations of each category as a function of genotype.

Results

To create a tractable model of mosaic expression, homozygous Fmr1 KO mice on a FVB background were bred to mice with a GFP transgene on their X chromosome on a predominantly 129 background (Fig. 1A). The F1 female offspring of this cross have one X chromosome that is Fmr1 KO and one X chromosome that is Fmr1 WT and carries the GFP transgene. During embryonic development of these F1 females, the process of X inactivation leaves each neuron in the brain either Fmr1 KO and GFP(−) or Fmr1 WT and GFP(+). Likely because the X chromosome with the GFP transgene is from the 129 strain and therefore carries the weakest X controlling element (Xce) allele, Xce^a (Simmler et al., 1993), skewed X inactivation was observed in the F1 mosaic females, with a minority of cells expressing GFP (∼10%). Because the pyramidal cells body layer spreads out during flattening of the slices in culture, clumps of GFP(+) neurons that might be expected in vivo because of the clonal organization of the hippocampus were not observed. Instead, a salt and pepper pattern was observed that facilitated visual identification of the GFP(+) neurons, which were distributed among the more abundant GFP(−) neurons (Fig. 1B). The same GFP expression pattern was seen when Fmr1 WT controls were bred to the GFP transgenic mice, indicating that the skewing was not related to Fmr1 function.

Figure 1.

Generation of labeled mosaics for electrophysiological recordings. A, Generation of mosaic F1 female mice for generation of hippocampal slices (see Results). B, Example IR-DIC and epifluorescence images of recorded presynaptic and postsynaptic neurons. In this example, the presynaptic neuron is GFP(+) and therefore Fmr1 WT, whereas the postsynaptic neuron is GFP(−) and therefore Fmr1 KO. Scale bar, 10 μm.

The GFP labeling of wild-type cells allowed us to target whole-cell recordings to CA3 pyramidal neurons of known genotype during measurements from potentially synaptically coupled pairs of neurons in organotypic hippocampal slices (Fig. 1B). The organotypic preparation was chosen because synaptic regrowth after slicing makes feasible the measurement of individual synaptic connections between natural synaptic partners. We did not detect any differences in synaptic efficacy attributable to either presynaptic or postsynaptic Fmr1 genotype in synaptically coupled pairs of neurons (Fig. 2), which is consistent with field recordings from hemizygous Fmr1 KO mice showing no abnormalities in stimulus–response relationships or paired-pulse ratios (Godfraind et al., 1996; Huber et al., 2002).

Figure 2.

Synaptic amplitudes as a function of presynaptic or postsynaptic genotype. A, Example current-clamp recording of an action potential elicited by a brief injection of current in a presynaptic pyramidal neuron (bottom) and the resulting EPSC recorded from a postsynaptic pyramidal neuron voltage clamped at −70 mV (top). B, There was no significant difference in the average amplitudes of EPSCs recorded in synaptically connected pairs of neurons based on the presynaptic Fmr1 genotype (mean ± SD pA is shown, 15.7 ± 19.5 Fmr1 WT vs 21.5 ± 24.3 Fmr1 KO; n = 13 and 14, respectively; p > 0.05, t test). C, There was also no significant difference based on the postsynaptic Fmr1 genotype (23.6 ± 24.9 Fmr1 WT vs 14.8 ± 19.1 Fmr1 KO; n = 12 and 15, respectively; p > 0.05).

In contrast to analysis of the efficacy of synapses that were encountered during recordings from pairs of neurons, analysis of the probability of encountering a functional synaptic connection revealed a striking presynaptic effect of Fmr1 genotype (Fig. 3). Specifically, Fmr1 KO presynaptic neurons were found to make functional synapses onto WT and KO postsynaptic neurons in only 44 and 39% of recordings, respectively, whereas Fmr1- WT presynaptic neurons made functional synapses onto WT and KO neurons in 70 and 71% of recordings, respectively. The converse of these observations is that Fmr1 genotype had no relationship to the probability that postsynaptic neurons received functional synapses. To determine the statistical significance of these observations, the raw numbers of recordings showing a connection or no connection were compared as either a function of presynaptic genotype or postsynaptic genotype using the Pearson's χ² test (Fig. 4A). This analysis indicated that although presynaptic genotype had a significant effect on the number of connections formed [17 connected and 7 unconnected (WT) vs 14 and 20 (KO), respectively; p = 0.026], there was no significant effect caused by postsynaptic genotype [14 and 12 (WT) vs 17 and 15 (KO), respectively; p = 0.956]. Thus, it appears that Fmr1 KO presynaptic neurons are impaired at establishing or maintaining synapses compared with Fmr1 WT presynaptic neurons, whereas the ability of postsynaptic neurons to receive connections is independent of Fmr1 genotype.

Figure 3.

Synaptic connection probability in mosaic slices. A, Whereas some pairs of neurons shared functional synaptic connections as defined by the presence of an EPSC at −70 mV, other pairs did not (see Materials and Methods). B, Pie charts show the proportions of pairs with functional synapses (black) as a function of both presynaptic and postsynaptic Fmr1 genotypes (n = 58; 10 WT → WT, 16 KO → WT, 14 WT → KO, 18 KO → KO). Note that connection probability varies with presynaptic genotype but not postsynaptic genotype.

As a control, we repeated the measurements of connection probability in GFP mosaic mice generated from the breeding of GFP transgene carrying mice to Fmr1 WT mice from the same genetic background as the Fmr1 KO mice (Fig. 4B). Recordings from these control mosaics revealed no significant differences in the numbers of connections between GFP(+) or GFP(−) neurons as a function of either presynaptic [10 connected, 14 unconnected (WT) vs 15 and 19 (KO), respectively; p = 0.853], or postsynaptic genotype [11 and 15 (WT) vs 14 and 18 (KO); p = 0.912). These results rule out any potential contributions of the GFP transgene or strain differences in X chromosome genes to the finding of altered presynaptic connectivity.

Comparison of the Fmr1 mosaics (Fig. 4B) to the uniformly Fmr1 WT control mosaics (Fig. 4B) suggests that the differential presynaptic connectivity is largely caused by WT presynaptic neurons out-competing the KO neurons in a mosaic environment. Analysis of connection proportions shows that WT neurons in mosaics are significantly more connected than in controls [17 connected, 7 unconnected (Fmr1 mosaic) vs 10 and 14 (control), respectively; p = 0.042]. Together with the skewed abundance of WT versus KO neurons in the mosaic animals, this finding is consistent with the interpretation that synaptic competitiveness is dependent of Fmr1 genotype in the mosaic environment. Because ∼10% of the neurons are WT and ∼90% are KO, each of the WT presynaptic neurons should be very privileged in forming connections at the slight decrement of many KO neurons. Whereas the resulting increased connectivity in WT neurons in mosaic animals is large and statistically significant, the predicted decreased connectivity of KO neurons, which is shared by ∼9 KO neurons per privileged WT neuron, is predicted to be of a small magnitude, explaining the lack of a statistically significant decrease.

Discussion

That wild-type neurons are more successful than Fmr1 KO neurons in forming functional synapses in a mosaic network demonstrates a cell autonomous, presynaptic role for Fmr1 in synaptic development. Several lines of evidence support the explanation that differential competitiveness results from direct impairments in presynaptic axonal development of Fmr1 KO neurons. (1) The Drosophila orthologs of FMRP (dFXR) and its interacting proteins play essential roles in axon growth and neuronal connectivity (Morales et al., 2002; Schenck et al., 2003; Pan et al., 2004). (2) FMRP is present in axons and growth cones of hippocampal neurons, and the regulation of axonal growth is altered in hippocampal neurons from Fmr1 KO mice (Antar et al., 2005, 2006). (3) Alterations to presynaptic terminal staining have been observed in the hippocampi of Fmr1 KO mice (Ivanco and Greenough, 2002; Mineur et al., 2002).

Potential molecular substrates for presynaptic alterations are provided by biochemical measurements that reveal abnormal regulation of proteins that play key roles in axonal development. For example, the translation of microtubule-associated protein 1B, a protein important for growth cone dynamics (Bouquet et al., 2004), and its Drosophila ortholog, Futsch, are negatively regulated by FMRP and dFXR, respectively (Zhang et al., 2001; Lu et al., 2004). In addition, studies in both fly and mouse models of FXS show abnormal expression of proteins involved in Rho GTPase signaling (Schenck et al., 2003; Gantois et al., 2006), a pathway that plays key roles in axonal development (Luo, 2000). Whereas the majority of studies in FXS models have focused on postsynaptic phenotypes, our findings along with the studies discussed above clearly identify presynaptic alterations. While it is possible that presynaptic and postsynaptic phenotypes are independent manifestations of disrupted Fmr1 function, it is also possible that alterations on one side of the synapse are causative of the abnormalities on the other side of the synapse. Therefore, future work probing the cell-autonomous roles of Fmr1 using physiological and anatomical measurements in mosaic models will provide key insight into the ontogeny of FXS.

The dependence of synapse formation on presynaptic Fmr1 genotype in mouse organotypic slices suggests the possibility that a similar phenomenon occurs during synaptic development or remodeling in the brains of mosaic FXS patients. Even given overall normal numbers of neurons and synapses, if the subset of wild-type neurons form a disproportionately larger number of synapses in a mosaic network, the result could be a decrease effective neural network size, complexity, and information carrying capacity. Thus, an impairment unique to mosaic patients may come from the inability of mutant axons to out-compete wild-type axons in the making of synapses during development.

Therefore there are two distinct theoretical implications of mosaic FMRP expression. (1) Mosaic patients should have fewer neurons exhibiting previously described deficits such as aberrant synaptic plasticity compared with patients uniformly lacking FMRP, and would be predicted to have some phenotypes ameliorated compared with nonmosaic patients. This is supported by studies showing a correlation of FMRP levels with intellectual function (Reiss et al., 1995; Tassone et al., 1999). (2) Our findings predict that mosaic patients may be uniquely predisposed to phenotypes that could arise from imbalanced presynaptic connectivity. A possible example of this is provided by a previous report suggesting that autistic patients who have disrupted FMR1 function disproportionately involve mosaic FMRP expression (Reddy, 2005). That both altered functional connectivity and autistic phenotypes could result from mosaic FMR1 expression is intriguing given that abnormal functional connectivity and disrupted information carrying capacity of neural networks play central roles in theories about the neural substrates of autism (Belmonte and Bourgeron, 2006). This underscores the importance of testing for links between mosaic FMRP expression and autistic phenotypes in future studies of large cohorts of ASD and FXS patients.