Fragile X mental retardation protein knockdown in the developing Xenopus tadpole optic tectum results in enhanced feedforward inhibition and behavioral deficits
© The Author(s). 2016
Received: 19 May 2016
Accepted: 3 August 2016
Published: 8 August 2016
Fragile X Syndrome is the leading monogenetic cause of autism and most common form of intellectual disability. Previous studies have implicated changes in dendritic spine architecture as the primary result of loss of Fragile X Mental Retardation Protein (FMRP), but recent work has shown that neural proliferation is decreased and cell death is increased with either loss of FMRP or overexpression of FMRP. The purpose of this study was to investigate the effects of loss of FMRP on behavior and cellular activity.
We knocked down FMRP expression using morpholino oligos in the optic tectum of Xenopus laevis tadpoles and performed a series of behavioral and electrophysiological assays. We investigated visually guided collision avoidance, schooling, and seizure propensity. Using single cell electrophysiology, we assessed intrinsic excitability and synaptic connectivity of tectal neurons.
We found that FMRP knockdown results in decreased swimming speed, reduced schooling behavior and decreased seizure severity. In single cells, we found increased inhibition relative to excitation in response to sensory input.
Our results indicate that the electrophysiological development of single cells in the absence of FMRP is largely unaffected despite the large neural proliferation defect. The changes in behavior are consistent with an increase in inhibition, which could be due to either changes in cell number or altered inhibitory drive, and indicate that FMRP can play a significant role in neural development much earlier than previously thought.
Fragile X Syndrome (FXS) is the leading monogenetic cause of autism and most common form of inherited intellectual disability [1–3]. FXS is typically caused by the expansion of a trinucleotide (CGG) repeat in the 5′ untranslated region of the Fragile X mental retardation 1 (FMR1) gene [4, 5]. The mutation prevents expression of Fragile X Mental Retardation Protein (FMRP) throughout development. The most well understood neuroanatomical marker in FXS is the presence of immature dendritic spines in the cortex [6, 7]. This is thought to occur because FMRP is a RNA binding protein that inhibits protein synthesis downstream of group 1 metabotropic glutamate receptor activation , and therefore prevents normal plasticity and synaptic maturation. However, FMRP disruption prior to synapse formation results in abnormalities that may lead to neurodevelopmental deficits , indicating a possible role for FMRP much earlier than initially known.
Previous work from Faulkner et al.  identified a cell proliferation defect associated with excessive and decreased levels of FMRP. In this study, Xenopus laevis tadpoles were used to assess the early effects of FMRP upregulation and knockdown in the optic tectum. Tadpoles express FMRP throughout central nervous system (CNS) development [11, 12], and thus are ideally placed for studying the effects of FMR1 gene disruption during early development. Since tadpole development occurs in the absence of a womb, this experimental animal provides easy access to developmental stages that occur in utero in mammals . The tadpole optic tectum is homologous to the mammalian superior colliculus and is the main sensory processing area in the tadpole . The optic tectum receives direct visual input via retinal ganglion cells and generates outputs that directly inform behavior, and thus can be used to assay the emergence of functional properties of neural circuits during development.
Faulkner et al.  showed that FMR1 is expressed in neural progenitor cells that line the brain ventricle and neurons located lateral to the progenitor cells, as well as in puncta throughout the optic tectum. Both knockdown and overexpression of FMRP reduced cell proliferation in the tectum and increased cell death, providing evidence that FMRP is required at tightly controlled levels. Furthermore, Faulkner et al.  showed that FMRP regulates neuronal differentiation and dendritic morphology, with both overexpression and knockdown of FMRP levels resulting in abnormal numbers of neural progenitor cells and reduced dendritic arborization of tectal neurons. These results indicate a critical role for FMRP in early development, both in the generation of new neurons and in the wiring of the proper neural circuit. These data also suggest a clear role for FMRP prior to synapse formation. However, this study did not investigate the consequences of abnormal proliferation and arborization on the functional properties of tectal circuits. Furthermore, it is not clear how the cells that do survive are affected by knockdown of FMRP.
Here we investigate the behavioral and cellular changes induced by FMRP knockdown in the optic tectum. We use translation-blocking antisense morpholino oligonucleotides to decrease FMRP expression in the optic tectum during a key developmental period. We measured behavior using several assays that measure swimming speed, escape responses, social aggregation, and seizure susceptibility. We also investigated the electrophysiological properties of cells in the optic tectum. Our results show that FMRP knockdown results in decreased swimming speed, reduced schooling behavior and decreased seizure severity. However, FMRP knockdown does not perturb intrinsic properties of tectal neurons, but rather results in enhanced synaptic inhibition. This circuit abnormality is consistent with the behavioral results and shows that the early effects seen for FMRP knockdown have important functional consequences.
All animal experiments were performed in accordance with and approved by Brown University Institutional Animal Care and Use Committee standards.
Tadpoles were raised in Steinberg’s rearing media on a 12 h light/dark cycle at 18–21 °C for 7–8 d, until they reached developmental stage 46 [10, 14]. They were then electroporated with the FMR1 or scrambled control morpholino and reared until they reached developmental stages of either 47 or 49, depending on the experiment (see below). Developmental stages of tadpoles were determined according to . The rearing medium was renewed every 3 d. Tadpoles that were used for acoustic startle, schooling, or seizure protocols were not used again for other experiments, whereas after visual avoidance experiments, tadpoles were in some cases also used in startle and schooling experiments after having 24 h of rest in the rearing solution. At least two different clutches of tadpoles from different husbandry were used for every set of behavioral experiments. Animals of either sex were used because at these developmental stages tadpoles of either sex are phenotypically indistinguishable.
As described and validated in a prior study , a Xenopus laevis homolog of FMR1, fmr1a, was knocked down using a 3′ lissamine-tagged translation-blocking antisense morpholino oligonucleotide (GeneTools) with the sequence 5′- AGCTCCTCCATGTTGCGTCCGCACA-3′ (start codon underlined), referred to as fmr1a MO to generate FMRP knockdown (FMRP KD). Control lissamine-tagged oligonucleotides had the sequences 5′-TAACTCGCATCGTAGATTGACTAAA-3′ or 5′-CCTCTTACCTCAGTTACAATTTATA-3′, referred to as control. Morpholinos were dissolved in water. Morpholinos were injected into the brain ventricle, then platinum electrodes were placed on each side of the midbrain and voltage pulses were applied across the midbrain to electroporate optic tectum cells in stage 46 tadpoles.
For seizure experiments, stage 47 tadpoles were transferred into individual wells in a six-well plate (Corning), each filled with 7 ml of 5 mM pentylenetetrazol (PTZ) solution in Steinberg’s rearing media. The plate was diffusely illuminated from below and imaged from above with a SCB 2001 color camera (Samsung) at 30 frames/s. Tadpole positions were tracked in Noldus EthoVision XT (Noldus Information Technology) and processed offline in a custom MATLAB program (MathWorks). Onset of regular seizures happened on average 3.9 ± 1.3 min into the recording; seizure events were defined as periods of rapid and irregular movement, interrupted by periods of immobility , and were detected automatically using swimming speed thresholding at a level of half of the maximal swimming speed. Frequency of seizures and length of seizure events were measured across 5 min intervals of a 20-min-long recording.
Stage 49 tadpoles were placed in a clear plastic Petri dish (8.5 cm in diameter) filled to an approximate depth of 1 cm with Steinberg’s solution at 18 °C. The dish was put on top of a CRT monitor screen (maximum luminance, 57 cd/m2 and minimum luminance, 0.3 cd/m2; Dell Ultrascan 1600 SH Series; Dell Computer Company) and screened from all sides with an opaque black cloth. Stimuli were generated by a custom-written MATLAB program using the Psychophysics Toolbox [16, 17]. A black circle of a radius of 0.3 cm was projected in the center of the dish. Every 30 s, this circle was sent toward the tadpole at a speed of 1.4 cm/s. Only collisions in which the animal was swimming within 1 s before the encounter with the circle were included in the dataset. Experiments were performed in the morning (from 9:00 A.M. to 1:00 P.M.), because animals seemed to be less responsive in the afternoon; each testing session lasted for 5 min. Videos were acquired in EthoVision; both the tadpole and the stimulus were manually tracked offline, and trajectories were exported for additional automated analysis in MATLAB. Avoidance response initiation points were identified as points of peak acceleration immediately after an encounter with a visual stimulus; escape speed was averaged over a 17 ms window (five frames) around the swimming velocity peak.
Fifteen to twenty tadpoles at developmental stage 49 were transferred to a glass bowl 17 cm in diameter (for each batch, control tadpoles matched FMRP KD tadpoles in number). A still image of tadpole distribution in the bowl was made every 5 min using Yawcam software (Magnus Lundvall, Yawcam) for 1 h (13 images per experiment). A strong acoustic stimulus was delivered 2.5 min after each photo was taken to elicit a startle response and force tadpoles to redistribute . Coordinates of tadpole heads and tails were tracked manually in NIH ImageJ and exported for additional processing in MATLAB. We defined neighboring tadpoles through point set triangulation and used a Kolmogorov–Smirnov test to compare distributions of inter-tadpole distances between FMRP KD tadpoles and matched controls. For all pairs of “neighboring tadpoles” that were located closer than 5.7 cm to each other (two-thirds of the bowl radius), we also estimated the angle between their orientations in the bowl [19, 20].
Statistics and presentation of behavior data
For behavioral data, averages and SDs are presented. When the Mann–Whitney test was used to compare values between the groups, significance values were reported as PMW, whereas for Kolmogorov–Smirnov test, p values are reported as PKS. Sample sizes are reported as n = x, N = y, where lowercase n stands for the number of measurements and capital N stands for the number of animals.
For whole-brain recordings, tadpole brains were prepared as described by  and . In brief, tadpoles were anesthetized in 0.02 % tricainemethane sulfonate (MS-222). To access the ventral surface of the tectum, brains were filleted along the dorsal midline and dissected in HEPES-buffered extracellular saline [in mM: 115 NaCl, 2 KCl, 3 Cacl2, 3 MgCl2, 5 HEPES, 10 glucose, and 0.1 picrotoxin, pH 7.2 (osmolarity 255 mOsm)]. Brains were then pinned to a submerged block of Sylgard in a recording chamber and maintained at room temperature (24 °C). To access tectal cells, the ventricular membrane surrounding the tectum was carefully removed using a broken glass pipette. For evoked synaptic response experiments, a bipolar stimulating electrode (FHC) was placed on the optic chiasm to activate retinal ganglion cell (RGC) axons.
Whole-cell voltage-clamp and current-clamp recordings were performed using glass micropipettes (8–12 MΩ) filled with K-gluconate intracellular saline [in mM: 100 K-gluconate, 8 KCl, 5 NaCl, 1.5 MgCl2, 20 HEPES, 10 EGTA, 2 ATP, and 0.3 GTP, pH 7.2 (osmolarity 255 mOsm)]. Recordings were restricted consistently to retinorecipient neurons in the middle one-third of the tectum, thus avoiding any developmental variability existing along the rostrocaudal axis [21, 23, 24]. Electrical signals were measured with a Multiclamp 700B amplifier (Molecular Devices), digitized at 10 kHz using a Digidata 1440A analog-to-digital board, and acquired using pClamp 10 software. Leak subtraction was done in real time using the acquisition software. Membrane potential in the figures was not adjusted to compensate for a predicted 12 mV liquid junction potential. Data were analyzed using AxographX software. The GABAA antagonist picrotoxin (100 μM) was added to the external saline in a subset of experiments. Spontaneous synaptic events were collected and quantified using a variable amplitude template . Spontaneous excitatory post-synaptic currents (sEPSCs) were recorded at −60 mV in the presence of picrotoxin, whereas spontaneous inhibitory post-synaptic currents (sIPSCs) were collected in control media at 5 mV (the reversal for glutamatergic currents). For each cell, 60 s of spontaneous activity was recorded. For evoked synaptic response experiments, a bipolar stimulating electrode (FHC) was placed on the optic chiasm to activate RGC axons. Synaptic stimulation experiments were conducted by collecting EPSCs evoked by stimulating the optic chiasm at a stimulus intensity that consistently evoked maximal amplitude EPSCs. Evoked responses at −45 mV (excitation) and 5 mV (inhibition) were used to calculate the excitation/inhibition (E/I) ratio. Excitation and inhibition were calculated as a measure of area under the curve for a 250 ms time window beginning at the onset of the synaptic response. Evoked monosynaptic events (at a stimulus intensity that does not evoke polysynaptic activity, typically 30–60 % of the maximum) were used to collect AMPA/NMDA ratios. Peak current amplitude at −65 mV (1 ms window at peak; 10–15 trials per cell) was used to calculate AMPAR-mediated currents, and average current amplitude collected at 55 mV (10 ms window 20 ms after peak AMPA; 5–15 trials per cell) was used to calculate NMDAR-mediated currents. Experiments to measure polysynaptic network activity were performed by collecting EPSCs evoked by stimulating the optic chiasm at a stimulus intensity that evoked the maximal amplitude EPSC. Quantification of polysynaptic activity was calculated by measuring the total change in current over 100 ms time bins beginning at the onset of the evoked response. A spontaneous barrage was defined as a change in holding current of 10 or 20 pA intervals for a period of >200 ms. To quantify intrinsic cell excitability, cells were presented with a series of depolarizing steps (20 pA intervals) in current clamp, starting from −65 mV. The number of spikes elicited by current injection was quantified using the following criteria: to qualify as a spike, the height of the spike had to be at least half the height of its preceding spike and no wider than three times the width of the first original spike . Voltage-gated Na+ and K+ current–voltage (I–V) curves were calculated as in the study by , by measuring the early Na+ peak current and the steady-state K+ current. All data were tested for normality; parametric statistical tests were completed on normally distributed data and nonparametric Mann–Whitney U tests were completed on non-normal data. Graphs show mean and standard deviation as error bars, and data in the text show means and standard deviations, unless otherwise indicated.
Behavioral and electrophysiological experiments were performed on stage 49 tadpoles in which FMRP expression was knocked down using a morpholino-antisense oligomer  during critical neural proliferation and circuit wiring time periods, referred to as FMRP KD tadpoles throughout. Tadpoles were compared with a control group which was transfected with a scrambled version of the morpholino. Our results show behavioral and electrophysiological deficits that implicate impaired inhibitory circuitry as the primary change resulting from knockdown of FMRP.
Without major effects to the basic escape behavior, we wanted to identify if behaviors particularly relevant to FXS were affected in our FMRP KD tadpoles. A common behavioral marker of FXS and autism spectrum disorders (ASDs) is social interaction deficits. Tadpoles normally engage in a social aggregation behavior, called schooling . Schooling is defined as structured aquatic animal aggregation marked by coordinated unidirectional group swimming behavior . Recent work in our lab found that control tadpoles that are in schools normally swim parallel to each other (with an inter-tadpole body-axis angle less than 45°) and within at least two-thirds of the bowl’s radius to each other . This schooling behavior requires integration of various sensory cues, including visual, auditory and olfactory. Here, we observed abnormal schooling patterns in FMRP KD tadpoles (Fig. 1d–e). Comparing the distributions of angles and distances of 2035 control and 2054 FMRP KD sample measurements in 51 experimental runs, we found that FMRP KD tadpoles showed a significantly different distribution of inter-tadpole distances (Fig. 1d, PKS < 0.05; N = 51 for FMRP and control tadpoles; see Fig. 1e inset), with decreased short and long inter-tadpole distances and increased intermediate distances, indicating more disperse swimming and less aggregation in FMRP KD tadpoles. Consistently, FMRP KD tadpoles also had fewer neighboring tadpoles that swam in the same direction (angle < 45°) and more tadpoles that swam perpendicular and opposite (90° and 180°, respectively) to their neighboring tadpoles (Fig. 1e, PKS < 10−20; N = 51 for both FMRP KD and control tadpoles). Both of these measures indicate that FMRP KD tadpoles show decreased schooling behavior.
Next we tested for seizure susceptibility. When presented with a convulsant, tadpoles develop seizures within 20 min . Prior work has shown that abnormalities in excitatory connectivity, or in local inhibition can strongly affect the severity and length of these seizures. Seizures were induced pharmacologically with 5 mM pentylenetetrazol (PTZ) applied to the rearing media . Under control conditions, increasing the concentration of PTZ results in an increase in seizure frequency and a decrease in seizure length. We found that FMRP KD tadpoles had less frequent (Fig. 1f, Pt = 0.038 1.06 ± 0.16 events per minute for controls and 0.94 ± 0.23 events per minute for FMRP; N = 22 for controls and FMRP KD tadpoles) and significantly longer seizures (Fig. 1g, Pt < 0.005, length of seizure 19.24 ± 7.37 s for controls and 25.28 ± 5.86 s for FMRP). The decreased seizure frequency and increased seizure length (Fig. 1h) are consistent with decreased seizure severity, indicating that FMRP KD tadpoles show decreased seizure susceptibility.
Disrupted schooling behavior indicates abnormal integration of multisensory input potentially resulting from abnormal neural circuit development, and decreased seizure susceptibility and lower baseline swimming could indicate decreased overall excitation or enhanced inhibition in the brain. Together with the prior observation that FMRP KD tadpoles show changes in tectal neuron proliferation and arborization , these findings lead us to perform electrophysiological recordings to assess whether there was a corresponding alterations in tectal excitability or network function. We explored three potential mechanisms to account for decreased tectal activity, including lowered intrinsic excitability of tectal neurons, abnormal development of excitatory synaptic transmission and increased synaptic inhibition.
Cell size, action potential threshold, and membrane resistance in control and FMR1 KD tadpoles
10.82 ± 4.90 pF, n = 31
11.28 ± 2.90 pF, n = 33
2.419 ± 1.93 mΩ, n = 31
1.387 ± 0.638 mΩ, n = 33
Action potential threshold
−22.90 ± 4.55 mV, n = 29
−20.93 ± 3.80 mV, n = 29
Our findings indicate that FMRP knockdown by FMR1 morpholino in the developing optic tectum has behavioral and electrophysiological consequences. Behaviorally, FMRP KD animals showed slightly reduced baseline swimming activity, decreased schooling behavior and decreased seizure susceptibility, but normal visual avoidance. These behavioral findings are consistent with decreased excitation within the tectum. Electrophysiologically we confirmed that neuronal intrinsic excitability and development of synaptic connectivity appear to be normal, however we found a significantly larger amount and longer lasting evoked network inhibition within the tectum, suggesting that this imbalance in the excitation to inhibition ratio may be responsible for the behavioral phenotypes.
These findings are unexpected since Faulkner et al.  identified a clear proliferative defect and abnormal tectal cell dendritic branching. Our experiments here show that despite these deficits individual cells develop normal excitability profiles and that excitatory and inhibitory synapses show normal maturation in the absence of FMRP. Nevertheless our findings also confirm that there is abnormal network connectivity since there is an increase in evoked inhibitory currents. Since we did not observe any differences in spontaneous inhibitory transmission, our findings suggest that the effects observed are likely occurring in cells other than the principal deep layer cells that we recorded from in this study. For example one could speculate that a change in intrinsic excitability of inhibitory interneurons, or increased excitatory drive to interneurons may explain our findings. It could also be that there are a greater number of inhibitory cells present in the tecta of FMRP KD tadpoles, or elevated numbers of excitatory interneurons driving inhibition. These changes could link our findings to the abnormal neuronal proliferation described previously using the same experimental manipulation. Furthermore, our findings that excitatory synapse maturation is unaffected, together with previous findings that neuronal proliferation is disrupted, are consistent with the view that FMRP deficits can have effects much earlier in development than previously thought. Development of normal brain connectivity requires a careful interplay between cell proliferation, migration and differentiation, and altering this interplay could result in abnormal neural circuit formation and behavioral deficits, even if individual neurons still appear to develop normally .
Disruptions in the normal balance of excitation and inhibition during development have been implicated in a number of neurodevelopmental disorders, ranging from schizophrenia to autism [33, 35–37]. Thus our findings are consistent with this view, and consistent with findings in other models of autism in which increased levels of inhibition are observed . Other studies, in contrast, have also associated autism and Fragile X syndrome with alterations that result in decreased inhibition over excitation, resulting in increased seizure susceptibility [39–42]. For example in the rodent cortex, knockout of the FMR1 gene results in decreased synaptic drive to inhibitory interneurons and increased intrinsic excitability of excitatory cortical neurons, which result in prolonged evoked “up states” [43, 44]. In the amygdala, FMR1 knockouts have overall decreased tonic inhibition, leading to altered E/I balance and hyper excitability (Martin 2014). Interestingly, in the amygdala during development (p14) the FMR1 knockouts show a transient period of enhanced inhibition, which they ascribe to a homeostatic adaptation that ultimately fails in adult animals . It is also worth noting that the seizures associated with Fragile X syndrome tend to be relatively mild and occur in only 10–20 % of individuals. This suggests that there could be a compensatory mechanism to counteract increased excitability, and perhaps this mechanism is more strongly evident in our brain structure and model organism. The effects of FMRP knockout also seem to be brain region specific and may manifest differently in the midbrain. However, the basic principle that small disruptions in network connectivity can lead to imbalances in information processing, which can then cascade into visible behavioral phenotypes, seems to be a common factor conserved across different brain regions, species and disorders.
To follow up on this study, it will be important to investigate the network properties of the optic tectum. The interaction of the decreased neural proliferation found by Faulkner et al.  and the increased evoked inhibition at the single cell level may manifest itself as a change in how the neural network interacts and interprets visual information to generate behavioral output. These studies could be carried out via in vivo Ca++ imaging of ensemble neuronal activity within the tectum . It will also be important to identify whether the increased evoked inhibition is due to a larger proportion of GABAergic interneurons, or to alterations in interneuron physiology.
Fragile X Syndrome and other neurodevelopmental disorders affect people in many ways, but have proven difficult to treat clinically. With our work, we have shown an explanation of why that may occur. It is clear that one particular insult does not result in a single, robust phenotype. Our research shows the opposite: small changes at the cellular level, combined with a neural proliferation defect, gives rise to the behavioral phenotype.
ASD, autism spectrum disorders; CNS, central nervous system; FMR1, fragile X mental retardation 1 gene; FMRP, fragile X mental retardation protein; FXS, Fragile X Syndrome; K, Potassium; KD, knock down; MO, morpholino; Na, sodium; Pks, P value for Kolmogorov-Smirnov test; Pmw, P value for Man-Whitney u-test; Pt, P value for t-test; PTZ, pentylenetetrazol; RGC, retinal ganglion cell; RNA, ribonucleic acid; sEPSC, spontaneous excitatory post-synaptic current; sIPSC, spontaneous inhibitory post-synaptic current
We thank Mimi Oupravanh for animal care and experimental support and Carolina Ramirez Vizcarrondo for critical help and training in the behavioral protocols.
T.L.S.T is supported by NIH F31 NS09379001. E.J.J. is supported by NSF GFRP, T.J.W. by NIH T34GM087193. M.H. supported by Brown University UTRA Fellowship. This research was supported by funds from Brown University and NSF IOS (to C.D.A), NIH grants EY011261 to H.T.C.), an endowment from the Hahn Family Foundation (to H.T.C.), the Department of Defense (Grant W81XWH-12-1-0207 to H.T.C.) and NIH P30-GM-32128 (to K.G.P. and Z.L.). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets supporting the conclusions of this article will be available in the Brown Digital Repository, [doi pending].
TLST and CDA wrote the manuscript. TLST, EJJ, ZL and KGP conducted, analyzed and interpreted the electrophysiology experiments. MH and TW conducted, analyzed and interpreted behavior experiments. The project was conceived and designed by CDA and HTC. All authors had a role in revising and approving the final version of the manuscript and made substantial contributions to the conception and experimental design of the study.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All experimental manipulations and care of Xenopus laevis frogs and tadpoles has been approved by the Brown University Institutional Care and Use Committee.
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