Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive

Abstract

Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either "birthdate" or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons.

Keywords: Pten; autism spectrum disorder; dendritic spine; neuron structure function; seizure; synaptogenesis.

Figure 1.

Retroviral deletion of Pten reveals early differences in neuronal morphology and activity. A, Retroviruses encoding a fluorescent protein only, or a distinct fluorescent protein and Cre via a T2A element, are coinjected into Pten-floxed mice at P7. At increasing DPI, control versus Pten KO dentate gyrus granule neurons are analyzed in acutely generated hippocampal slices (photomicrograph at 25 DPI). Scale bar, 100 μm. B, Immunolabeling Pten at 24.5 DPI shows that neurons expressing mcherry-T2A-Cre are Pten-depleted (*) versus control neurons expressing only eGFP. Scale bar, 20 μm. C, By analysis of tissue processed as in B, Pten KO increased soma size (p < 0.0001), and this effect was significant at each DPI. D, Compared with control neurons, p-S6 immunoreactivity is elevated in Pten KO neurons in an example image at 24.5 DPI. Scale bar, 10 μm. E, By analysis of tissue processed as in D, Pten KO elevates p-S6 immunoreactivity (p < 0.0001) compared with control neurons. This effect was significant after 7.5 DPI. F, Compared with a control neuron (*), a Pten KO neuron (**) has increased immunoreactivity for c-Fos (16.5 DPI). Scale bar, 10 μm. G, Quantitation of c-Fos immunoreactivity reveals that Pten KO neurons have higher c-Fos immunoreactivity (p = 0.0036) compared with control neurons, and this effect was significant at 16.5 DPI. Data are mean ± SEM; n = animals. Asterisks next to brackets between control and “Pten KO” indicate overall significance of two-way repeated-measures ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Figure 2.

Retroviral deletion of Pten alters intrinsic properties of developing neurons. A, Pten KO changed input resistance and cell capacitance; a Pten KO neuron exhibited an exaggerated response to a test pulse (20.5 DPI). Capacitance increased (B) and input resistance decreased (C) with neuronal age (p < 0.0001, p < 0.0001), but Pten KO exaggerated these changes (p < 0.0001, p < 0.0001), and these effects of Pten KO were significant at each DPI. D, Spike threshold for a 10 ms current injection was greater in a Pten KO than in a control neuron at 20.5 DPI. E, Current injection to elicit a spike rose as neurons matured (p < 0.0001), and Pten KO dramatically increased this trend (p < 0.0001); this effect was significant at DPI ≥ 12.5 DPI. F, Neurons fired at more hyperpolarized potentials as they matured (p < 0.0001), but this was exaggerated by Pten KO (p = 0.001), with the effect of Pten KO significant by 24.5 DPI. G, Spike trains from a 500 ms current injection show that a Pten KO neuron required more current to fire (20.5 DPI). H, As neurons matured, peak firing frequency required increased current injection (p < 0.0001), and this was exacerbated by Pten KO (p < 0.0001), with the effect of Pten KO reaching significance at DPI ≥ 12.5. I, Peak spike rate rose with age (p < 0.0001) but was higher with Pten KO (p = 0.0203). Data are mean ± SEM; n = neurons (indicated in bar bases). Asterisks next to brackets between control and “Pten KO” indicate overall significance of two-way ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. B–F, Gaussian fit lines. H, I, Quadratic fit lines. *p < 0.05. **p < 0.005. ***p < 0.001. ****p < 0.0001.

Figure 3.

Pten KO increases developmental sensitivity to afferent stimulation. A, Although a Pten KO cell required greater current injection to fire than did a control (815 vs 265 pA), the Pten KO cell fired at levels of presynaptic (perforant path) stimulation (0.1 mA), which did not elicit firing in the neighboring control cell (20.5 DPI). Arrowhead indicates the stimulus artifact. B, Spike probability rose with perforant path stimulation intensity (p < 0.0001), but Pten KO neurons were more likely to fire (p = 0.0004); this effect was significant at intensities ≥0.1 mA. Cumulative Gaussian fit lines. C, Design of retroviruses to infect newborn granule neurons with the Ca²⁺ sensor GcAMP6s and/or mCherry-T2A-Cre. D, Multiphoton image of live 20.5 DPI neurons illustrating basal fluorescence of Pten KO neurons, which are those dual-infected with mCherry-T2A-Cre and GCaMP6s retroviruses (Pten KO, **), and of a control cell, which is infected with GCaMP6s retrovirus alone (control,*). Scale bar, 30 μm. E, Top, A montage demonstrates GcAMP6s fluorescence, detected by high-speed widefield imaging (30 FPS), in control (C1–4) or Pten KO (KO1, 2) neurons before and after presynaptic perforant path stimulation at the indicated intensities. Bottom, Traces of 10× raw change in GcAMP6s intensity for the indicated cells. F, The intensity of somatically recorded GcAMP6s fluorescence (Peak ΔF_Real/F₀) rose with increasing afferent stimulation intensity (p < 0.0001), but Pten KO neurons had greater evoked GcAMP6s transients (p = 0.026); this effect was significant at stimulation intensities ≥0.2 mA. Data are mean ± SEM; n = neurons (indicated in bar bases). Asterisks next to brackets between control and Pten KO indicate overall significance of two-way repeated-measures ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. B, F, Quadratic fit lines. *p < 0.05. **p < 0.005. ***p < 0.001.

Figure 4.

Developing Pten KO neurons have more dendritic protrusions and more dendritic arborization than do controls. A, Multiphoton microscope images of live distal dendrite segments of control or Pten KO granule neurons demonstrate that, although dendritic protrusion density increases with neuron age, Pten KO neurons consistently had more protrusions. Scale bar, 10 μm. B, Quantifying the protrusion density changes documented in A, protrusion density increased with age (p = 0.001), but Pten KO further increased protrusion density (p < 0.0001); this effect was significant at each DPI. C, From fixed tissue, Neurolucida reconstructions of control and Pten KO neurons at 24.5 DPI demonstrate a grossly altered dendritic architecture. D, Quantifying the morphological differences as in C. The number of dendrites, branching points (nodes), and dendrite ends were increased in Pten KO neurons at 20.5–24.5 DPI (p = 0.0008, p < 0.0001, and p = 0.0002, respectively). Likewise, total dendrite length, surface area, and volume were all also increased in Pten KO neurons (p < 0.0001, p = 0.0001, and p = 0.0016, respectively). Data are mean ± SEM; n = neurons (indicated in bar bases). B, Asterisks next to brackets between control and Pten KO indicate overall significance of two-way ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. D, **p < 0.005 (t test). ***p < 0.001 (t test). ****p < 0.0001 (t test).

Figure 5.

Dendritic protrusions have immunohistochemical markers of functional excitatory synapses. A, C, Dendrite segments of 24 DPI control or Pten KO neurons (retroviral expression of GFP only or expression of GFP and Cre, in Pten-floxed animals) visualized by confocal microscopy after immunohistochemical labeling of GFP (first panel). SH3 and multiple ankyrin repeat domain protein 2 (Shank2, second panel) was used to label the postsynaptic density of glutamatergic synapses. Presynaptic sites are labeled by the vesicle-associated membrane protein, Synaptobrevin 2 (SynB, third panel). B, D, GFP intensity is used as a threshold to define the territory of the infected neurons (first panel); in the territory of the infected neuron, Shank2 reactivity is found upon the dendritic shaft and dendritic protrusions (middle), whereas Syn2B is found in close apposition to or colocalized with Shank2 in these same domains (right), as evidenced in the mushroom spine inset images. Scale bar, 2 μm.

Figure 6.

Developing Pten KO neurons have more excitatory synapses. A, Evoked synaptic currents are greater in a Pten KO versus control neuron (20.5 DPI). Blocking GABA_A (SR95531) revealed a greater excitatory current (EPSC) in Pten KO, whereas the subtracted inhibitory current (IPSC) was similar. B, Neither age (p = 0.419) nor Pten KO (p = 0.115) changed IPSC amplitude. C, Pten KO (p = 0.0003) nor age (p = 0.167) increased EPSC amplitude. D, EPSC to IPSC amplitude ratio increased with age (p = 0.0043), but Pten KO further increased EPSC to IPSC amplitude ratio (p = 0.0018). E, Compared with Ca²⁺-containing solution, evoked EPSCs (eEPSCs) in Sr²⁺ were asynchronous and smaller. F, Quantal-like asynchronous EPSCs were detected in Sr²⁺ (aEPSCs, arrowheads; 10x scale from E). G, Averaged aEPSCs were larger in Pten KO cells. H, Peak-scaled averaged aEPSCs from control or Pten KO neurons had similar kinetics. I, Histogram: amplitude of aEPSC currents, which were greater in Pten KO versus control neurons. J, Mean eEPSC amplitude, recorded in Ca²⁺, was greater in Pten KO versus control neurons (p = 0.0259). K, Mean aEPSC amplitude, recorded in Sr²⁺, was slightly higher in Pten KO neurons (p = 0.0209). L, Pten KO neurons had more aEPSCs per eEPSC (p = 0.0322). M, The paired pulse ratio was similar between control and Pten KO neurons (p = 0.9193). Data are mean ± SEM; n = neurons (indicated in bar bases), except in I, where n = aEPSCs, indicated on graphs. B–D, Asterisks next to brackets between control and “Pten KO” indicate overall significance of two-way ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. I–L, Asterisks indicate significance by t test. *p < 0.05. **p < 0.005. ***p < 0.001.

Figure 7.

Excitatory quantal events are greater in size and frequency in Pten KO neurons. A, Example raw traces of mEPSCs recorded at 20.5 DPI from control and Pten KO neurons, illustrating the higher rate and peak amplitude of mEPSC events in Pten KO cells. B, The average waveform of mEPSCs recorded in Pten KO neurons has increased amplitude compared with mEPSCs from control neurons. C, The peak-scaled mEPSC demonstrates similar kinetics between control and Pten KO conditions. D, The frequency of mEPSCs is increased in Pten KO cells (p = 0.0265). E, mEPSCs from Pten KO cells have a greater peak amplitude (p = 0.0271). F, The mEPSC rise time is elevated by Pten KO (p = 0.0045). G, the mEPSC decay time is not significantly increased by Pten KO (p = 0. 147). Asterisks indicate significance by t test.*p < 0.05. **p < 0.005. N.S., Not significant.

Figure 8.

An increased density of dendritic filopodia precedes increased density of mushroom spines in developing Pten KO neurons. A, An exemplary multiphoton image of a live dendritic segment (12.5 DPI, Pten KO) is overlaid with selected protrusions labeled according to manually classified type. Scale bar, 5 μm. B, With age, thin/filopodial protrusion density decreased (p = 0.0027), but in Pten KO cells, overall thin/filopodial protrusion density was increased (p < 0.0001); this effect was significant at each DPI. C, With age, mushroom spine density increased (p < 0.0001), but in Pten KO cells, mushroom spine density was further elevated (p < 0.0001); this effect was significant at DPI > 12.5. D, *A thin/filopodial protrusion upon a dendritic segment of a 20.5 DPI control neuron undergoes a morphological transition to a mushroom-type spine over the 20 min imaging session. Scale bar, 5 μm. E, The total spatial density of maturation events (as in D) significantly increased with Pten KO (p = 0.0182). F, The overall proportion of protrusions that matured was not affected by Pten KO (p = 0.7303). Data are mean ± SEM; n = neurons (indicated in bar bases). B, C, Asterisks next to brackets between control and “Pten KO” indicate overall significance of two-way ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. E, F, Asterisks indicate significance by t test. *p < 0.05. **p < 0.005. ****p < 0.0001.

Figure 9.

Changes in morphology and excitability explain the increased activity of Pten KO neurons. A, Increased eEPSC amplitude from Pten KO (4.532 ± 1.144-fold, J) is accounted for by Pten KO-associated increases in dendrite length, spine density, and aEPSC amplitude (4.099 ± 0.425-fold). B, Experimentally measured aEPSCs (Fig. 6G) and aEPSCs from simulated neurons based on experimentally determined morphology had similar amplitudes and kinetics. C, Both modeled (p = 0.027) and experimentally recorded (p = 0.002) aEPSC amplitudes were increased by Pten KO, with no difference in measured versus modeled approaches (not significant, p = 0.297). D, Dendritic compartments were more proximal to the soma in Pten KO neurons. Inset, Reconstructions at 24.5 DPI. E, Increased arbor length, spine density, and synapse proximity account for >80% of the increased eEPSC in Pten KO neurons. F, Resting potential did not differ with Pten KO. G, Spike threshold was lower with Pten KO (p = 0.0064). H, The depolarization to bring neurons to threshold was decreased by Pten KO (p = 0.0325). I, aEPSP voltages were smaller with Pten KO (p < 0.0001). J, It took more aEPSPs (i.e., synaptic inputs) to make a Pten KO neuron fire (p < 0.0001). K, In the Pten KO condition, the increase in evoked synaptic inputs was greater than the increase in synaptic inputs required to depolarize Pten KO neurons. Thus, Pten KO neurons are predicted to be more likely to fire in response to afferent stimulation (p = 0.0143). Data are mean ± SEM; n = neurons (indicated in bar bases). B, Data are ±2 SE. C, K, Asterisks next to brackets between control and “Pten KO” indicate overall significance of two-way ANOVA. Asterisks above bars indicate significance by Bonferroni's post-test. F–J, Asterisks indicate significance by t test. *p < 0.05. **p < 0.005. ****p < 0.0001. NS, Not significant.