Loss of protein tyrosine phosphatase receptor delta PTPRD increases the number of cortical neurons, impairs synaptic function and induces autistic-like behaviors in adult mice

Abstract

Background: The brain cortex is responsible for many higher-level cognitive functions. Disruptions during cortical development have long-lasting consequences on brain function and are associated with the etiology of brain disorders. We previously found that the protein tyrosine phosphatase receptor delta Ptprd, which is genetically associated with several human neurodevelopmental disorders, is essential to cortical brain development. Loss of Ptprd expression induced an aberrant increase of excitatory neurons in embryonic and neonatal mice by hyper-activating the pro-neurogenic receptors TrkB and PDGFRβ in neural precursor cells. However, whether these alterations have long-lasting consequences in adulthood remains unknown.

Results: Here, we found that in Ptprd+/- or Ptprd-/- mice, the developmental increase of excitatory neurons persists through adulthood, affecting excitatory synaptic function in the medial prefrontal cortex. Likewise, heterozygosity or homozygosity for Ptprd also induced an increase of inhibitory cortical GABAergic neurons and impaired inhibitory synaptic transmission. Lastly, Ptprd+/- or Ptprd-/- mice displayed autistic-like behaviors and no learning and memory impairments or anxiety.

Conclusions: These results indicate that loss of Ptprd has long-lasting effects on cortical neuron number and synaptic function that may aberrantly impact ASD-like behaviors.

Keywords: ASD; Anxiety; Learning; Medial prefrontal cortex; Memory; Protein tyrosine phosphatase receptor delta; Synaptic transmission.

Fig. 1

Adult Ptprd+/- or Ptprd-/- mice show an increase of glutamatergic neurons in the mPFC and somatosensory cortex. (A-H) Coronal sections of mPFC (A, C) or somatosensory cortex (E, G) of Ptprd+/+ (PTPRD-WT) and Ptprd-/- (PTPRD-KO) 3-month-old animals were immunostained against Tbr1 (A, E) or Satb2 (C, G). Relative total Tbr1-positive cells in the mPFC (for B, PTPRD-WT, n = 3; PTPRD-HET, n = 4; PTPRD-KO, n = 4) or somatosensory cortex (for F, PTPRD-WT, n = 3; PTPRD-HET, n = 5; PTPRD-KO, n = 5) of Ptprd+/+, Ptprd+/- (PTPRD-HET), and Ptprd-/- 3-month-old mice were quantified. Relative total Satb2-positive cells in the mPFC (for D, PTPRD-WT, n = 3; PTPRD-HET, n = 3; PTPRD-KO, n = 4) or somatosensory cortex (for H, PTPRD-WT, n = 3; PTPRD-HET, n = 4; PTPRD-KO, n = 5) of Ptprd+/+, Ptprd+/-, and Ptprd-/- 3-month-old mice were quantified. In (A) and (C), the dotted line shows mPFC and specific cortical layers. (I, J) Tbr1 (I) or Satb2 (J) positive neurons were quantified per cortical layers II/III, V and VI in the mPFC. (K, L) Tbr1 (K) or Satb2 (L) positive neurons were quantified per cortical layers II/III, IV, V and VI in the somatosensory cortex. mPFC = medial prefrontal cortex. SSC = somatosensory cortex. WM = white matter. *p < 0.05; **p < 0.01; ***p < 0.001. In all the images, scale bars represent 200 μm. In all graphs, the error bars denote SEMs

Fig. 2

Excitatory synaptic transmission in layer 2/3 of the mPFC is disrupted in the Ptprd-/- mice. (A) Sample traces (left) and quantitative analysis (right) of sEPSC activity recorded from layer 2/3 pyramidal neurons in the mPFC in Ptprd+/+ (PTPRD-WT), Ptprd+/- (PTPRD-HET), and Ptprd-/- (PTPRD-KO) mice. A significant increase in the frequency but not in the amplitude of sEPSC in PTPRD-KO was detected. PTPRD-WT, n = 4 animals, 10 cells; PTPRD-HET, n = 5 animals, 11 cells; PTPRD-KO, n = 8 animals, 15 cells. **p < 0.01. (B) Miniature excitatory synaptic current (mEPSC) also shows an increase in the frequency but not in the amplitude in Ptprd-/- compared to Ptprd+/+ and Ptprd+/- mice. PTPRD-WT, n = 5 animals, 8 cells; PTPRD-HET, n = 5 animals, 8 cells; PTPRD-KO, n = 6 animals, 11 cells. *p < 0.05. (C) Input/output function measured as the EPSP amplitude as a function of stimulus intensity also is increased in Ptprd-/- compared to Ptprd+/+ and Ptprd+/- mice. PTPRD-WT, n = 3 animals, 6 cells; PTPRD-HET, n = 5 animals, 9 cells; PTPRD-KO, n = 5 animals, 7 cells. (D) Paired-pulse responses superimposed after subtraction of the first pulse at 10, 30, 70, 100, and 300 ms interstimulus intervals (ISI) are similar between genotyping. PTPRD-WT, n = 4 animals, 6 cells; PTPRD-HET, n = 6 animals, 8 cells; PTPRD-KO, n = 5 animals, 8 cells. (E) Short-term synaptic responses (top) and normalized summary data (bottom) evoked by a burst of 25 stimuli at 14 Hz are also similar between genotyping. PTPRD-WT, n = 3 animals, 6 cells; PTPRD-HET, n = 4 animals, 8 cells; PTPRD-KO, n = 4 animals, 9 cells. (F) AMPA/NMDA ratio shows no significant differences between genotyping. PTPRD-WT, n = 3 animals, 6 cells; PTPRD-HET, n = 3 animals, 8 cells; PTPRD-KO, n = 5 animals, 14 cells. In all graphs, the error bars denote SEMs

Fig. 3

Ptprd+/- or Ptprd-/- mice show an increase of GABAergic neurons in the mPFC and somatosensory cortex at 3 months. (A-H) Coronal sections of the mPFC or somatosensory cortex of Ptprd+/+ (PTPRD-WT) and Ptprd-/- (PTPRD-KO) 3-month-old animals were immunostained against parvalbumin (A, E) or somatostatin (C, G). Relative total parvalbumin-positive cells in the mPFC (for B, PTPRD-WT, n = 4; PTPRD-HET, n = 3; PTPRD-KO, n = 5) or somatosensory cortex (for F, PTPRD-WT, n = 4; PTPRD-HET, n = 5; PTPRD-KO, n = 3) of Ptprd+/+, Ptprd+/- (PTPRD-HET), and Ptprd-/- 3-month-old mice. Relative total of somatostatin-positive cells (arrows head) in the mPFC (for D, PTPRD-WT, n = 4; PTPRD-HET, n = 3; PTPRD-KO, n = 4) or somatosensory cortex (for H, PTPRD-WT, n = 3; PTPRD-HET, n = 4; PTPRD-KO, n = 4) of Ptprd+/+, Ptprd+/- and Ptprd-/- 3-month-old mice. In (A) and (C), the dotted line shows mPFC and specific cortical layers. (I, J) Parvalbumin (I) or Somatostatin (J) positive neurons were quantified per cortical layers II/III, V and VI in the mPFC. (K, L) Parvalbumin (K) or Somatostatin (L) positive neurons were quantified per cortical layers II/III, IV, V and VI in the somatosensory cortex. mPFC = medial prefrontal cortex. SSC = somatosensory cortex. PV = parvalbumin. SOM = somatostatin. WM = white matter. In all the images, scale bars represent 200 μm, and arrows show positive cells. *p < 0.05; **p < 0.01; ***p < 0.001. In all graphs, the error bars denote SEMs

Fig. 4

Inhibitory synaptic transmission in layer 2/3 of the mPFC is impaired in the Ptprd-/- mice. (A) Representative traces (left) and quantitative analysis (right) of sIPSC activity recorded from layer 2/3 pyramidal neurons in the mPFC in Ptprd+/+ (PTPRD-WT), Ptprd+/- (PTPRD-HET), and Ptprd-/- (PTPRD-KO). PTPRD-WT, n = 5 animals, 9 cells; PTPRD-HET, n = 6 animals, 8 cells; PTPRD-KO, n = 4 animals, 9 cells. *p < 0.05. (B) Representative traces (left) and quantitative analysis (right) of mIPSC activity recorded from layer 2/3 pyramidal neurons in the mPFC in Ptprd+/+ (PTPRD-WT), Ptprd+/- (PTPRD-HET), and Ptprd-/- (PTPRD-KO). PTPRD-WT, n = 4 animals, 9 cells; PTPRD-HET, n = 4 animals, 8 cells; PTPRD-KO, n = 4 animals, 8 cells. *p < 0.05. (C) Evoked IPSC amplitudes as a function of stimulus intensity plotted as input/output curves show an increase in Ptprd-/- compared to Ptprd+/+ and Ptprd+/- mice. Significant changes between Ptprd+/+ and Ptprd-/- groups are as “*”. *p < 0.05, **p < 0.01, and ***p < 0.001. PTPRD-WT, n = 4 animals, 7 cells; PTPRD-HET, n = 5 animals, 9 cells; PTPRD-KO, n = 4 animals, 8 cells. (D) Paired-pulse responses at 10, 30, 70, 100, and 300 ms interstimulus intervals (ISI) are similar between genotyping. PTPRD-WT, n = 4 animals, 6 cells; PTPRD-HET, n = 4 animals, 6 cells; PTPRD-KO, n = 5 animals, 8 cells. (E) Short-term synaptic responses (top) and normalized summary data (bottom) evoked by a burst of 20 stimuli at 10 Hz are also similar between genotyping. PTPRD-WT, n = 4 animals, 7 cells; PTPRD-HET, n = 6 animals, 10 cells; PTPRD-KO, n = 4 animals, 7 cells. (F) Excitatory and inhibitory balances show no significant differences between genotyping. PTPRD-WT, n = 4 animals, 8 cells; PTPRD-HET, n = 4 animals, 10 cells; PTPRD-KO, n = 5 animals, 12 cells. In all graphs, the error bars denote SEMs

Fig. 5

Heterozygous or homozygous Ptprd mice showed no learning and memory deficits. (A-E) 3-month-old Ptprd+/+ (PTPRD-WT), Ptprd+/- (PTPRD-HET), and Ptprd-/- (PTPRD-KO) mice were tested in the Morris water maze (MWM) test. (A) Latency escape during the 4 training days. (B) Time spent by the animals swimming in the target quadrant area on test day (day 5). (C) Distance traveled in the target quadrant zone on test day. (D) Time spent by the animals swimming in the platform area on test day. (E) Distance traveled in the platform zone on test day. PTPRD-WT, n = 8; PTPRD-HET, n = 16; PTPRD-KO, n = 9. (F) 3-month-old Ptprd+/+, Ptprd+/-, and Ptprd-/- mice were tested in the Y-maze test. Percentage of preference for spontaneous alternations (SAP). PTPRD-WT, n = 6; PTPRD-HET, n = 11; PTPRD-KO, n = 8. In all graphs, the error bars denote SEMs

Fig. 6

Loss of one or both Ptprd alleles does not induce anxious behavior. (A-C) 3-month-old Ptprd+/+ (PTPRD-WT), Ptprd+/- (PTPRD-HET), and Ptprd-/- (PTPRD-KO) mice were tested in open-field anxiety test. (A) Percentage of the total time that the mice spent in the central zone. (B) Number of times that the animals enter the central zone. (C) Total distance traveled. PTPRD-WT, n = 10; PTPRD-HET, n = 16; PTPRD-KO, n = 11. (D-G) 3-month-old Ptprd+/+, Ptprd+/-, and Ptprd-/- mice were tested in the elevated plus maze test (EPM). (D) Percentage of the total time spent in open arms. (E) Percentage of the total time spent in closed arms. (F) Number of times that the animals enter the open arms. (G) Number of times that the animals enter the closed arms. PTPRD-WT, n = 10; PTPRD-HET, n = 12; PTPRD-KO, n = 11. In all graphs, the error bars denote SEMs

Fig. 7

Ptprd+/- or Ptprd-/-mice showed autistic-like behaviors. (A-D) 3-month-old Ptprd+/+ (PTPRD-WT), Ptprd+/- (PTPRD-HET), and Ptprd-/- (PTPRD-KO) mice were tested for sociability (A) and social novelty (B), self-grooming (C) or marble burying (D). (A) Social Interaction Test was measured in the three-chamber test. The graph shows the cumulative duration of Ptprd+/+, Ptprd+/-, and Ptprd-/- mice in the mouse or object chambers. PTPRD-WT, n = 12; PTPRD-HET, n = 13; PTPRD-KO, n = 9. (B) Social Novelty Test was assessed in the three-chamber test. The graph shows the cumulative duration of Ptprd+/+, Ptprd+/-, and Ptprd-/- mice in the old mouse and new mouse chambers. PTPRD-WT, n = 12; PTPRD-HET, n = 13; PTPRD-KO, n = 9. *p < 0.05; **p < 0.01. (C, D) Repetitive behavior was evaluated by the time spent in spontaneous self-grooming (C) or marble-burying behaviors (D). (C) Cumulative time in which Ptprd+/+, Ptprd+/-, and Ptprd-/- mice did self-grooming behavior in a total of 10 min. PTPRD-WT, n = 7; PTPRD-HET, n = 7; PTPRD-KO, n = 8. (D) Percentage of marbles buried by Ptprd+/+, Ptprd+/-, and Ptprd-/- mice after 30 min. PTPRD-WT, n = 7; PTPRD-HET, n = 14; PTPRD-KO, n = 7. *p < 0.05; **p < 0.01. In all graphs, the error bars denote SEMs