Multiple roles for mammalian target of rapamycin signaling in both glutamatergic and GABAergic synaptic transmission

Abstract

The mammalian target of rapamycin (mTOR) signaling pathway in neurons integrates a variety of extracellular signals to produce appropriate translational responses. mTOR signaling is hyperactive in neurological syndromes in both humans and mouse models that are characterized by epilepsy, autism, and cognitive disturbances. In addition, rapamycin, a clinically important immunosuppressant, is a specific and potent inhibitor of mTOR signaling. While mTOR is known to regulate growth and synaptic plasticity of glutamatergic neurons, its effects on basic parameters of synaptic transmission are less well studied, and its role in regulating GABAergic transmission is unexplored. We therefore performed an electrophysiological and morphological comparison of glutamatergic and GABAergic neurons in which mTOR signaling was either increased by loss of the repressor Pten or decreased by treatment with rapamycin. We found that hyperactive mTOR signaling increased evoked synaptic responses in both glutamatergic and GABAergic neurons by ∼50%, due to an increase in the number of synaptic vesicles available for release, the number of synapses formed, and the miniature event size. Prolonged (72 h) rapamycin treatment prevented these abnormalities and also decreased synaptic transmission in wild-type glutamatergic, but not GABAergic, neurons. Further analyses suggested that hyperactivation of the mTOR pathway also impairs presynaptic function, possibly by interfering with vesicle fusion. Despite this presynaptic impairment, the net effect of Pten loss is enhanced synaptic transmission in both GABAergic and glutamatergic neurons, which has numerous implications, depending on where in the brain mutations of an mTOR suppressor gene occur.

Figure 1.

Loss of Pten increases synaptic transmission in glutamatergic and GABAergic neurons. A, Representative images from wild-type (left) and Pten^loxP/loxP (right) neurons infected with a cre-RFP-expressing lentivirus showing the fluorescence from the RFP fusion protein (red) and the immunoreactivity against phospho-S6 (green). B, Representative traces showing 2 ms depolarization evoked synaptic responses from control (black traces) and Pten-cre (red traces) glutamatergic (left traces) and GABAergic (right traces) neurons. GABAergic responses are inward currents due to the Cl⁻ concentration of the internal solution. C, Peak amplitudes of evoked EPSCs from glutamatergic (left) and evoked IPSCs from GABAergic (right) neurons. D, The charge contained in the evoked EPSCs of glutamatergic (left) and evoked IPSCs of GABAergic (right) neurons. All values are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01.

Figure 2.

Loss of Pten increases the amplitude but not the frequency of glutamatergic and GABAergic miniature events. A, Representative traces showing the average of mEPSCs collected from one control (black trace) and one Pten-cre (red trace) glutamatergic neuron. B, Representative traces showing the average of mIPSCs collected from one control (black trace) and one Pten-cre (red trace) GABAergic neuron. C, Peak amplitudes of mEPSCs from glutamatergic (left) and mIPSCs from GABAergic (right) neurons. D, The charge contained in the mEPSCs of glutamatergic (left bars) and mIPSCs of GABAergic (right bars) neurons. E, F, Representative traces showing control (black traces) and Pten-cre (red traces) miniature postsynaptic current activity from glutamatergic (E) and GABAergic (F) neurons. G, Bar graph showing no significant difference in the miniature event frequency between control and Pten-cre neurons in glutamatergic (left bars) and GABAergic (left bars) neurons. All values are presented as mean ± SEM. **p ≤ 0.01.

Figure 3.

Loss of Pten increases the number of synaptic vesicles in the RRP. A, Representative traces of the response of glutamatergic control (black trace) and Pten-cre (red trace) neurons to a 4 s pulse of 500 mm sucrose. B, Representative traces of the response of GABAergic control (black trace) and Pten-cre (red trace) neurons to a 4 s pulse of 500 mm sucrose. C, The charge contained in the transient current induced by sucrose application in glutamatergic (left) and GABAergic (right) neurons. D, The number of synaptic vesicles contained in the RRP of glutamatergic (left) and GABAergic (right) neurons. E, The rate constant of spontaneous release of synaptic vesicles from glutamatergic (left) and GABAergic (right) neurons. All values are presented as mean ± SEM. *p ≤ 0.05; ***p ≤ 0.001.

Figure 4.

Presynaptic neurotransmitter release efficiency in Pten-cre neurons. A, The probability that an individual synaptic vesicle fuses in response to a 2 ms depolarization (P_vr) in control (black) and Pten-cre (red) neurons. B, Representative traces of the response of control (black trace) and Pten-cre (red trace) glutamatergic neurons to paired 2 ms depolarizations separated by 20 ms. C, Representative traces of the response of control (black trace) and Pten-cre (red trace) GABAergic neurons to paired 2 ms depolarizations separated by 100 ms. D, Paired pulse ratios in control (black) and Pten-cre (red) neurons. E, Line plot of the responses of control (black line) and Pten-cre (red line) glutamatergic neurons to 10 Hz stimulation. Values are normalized to the peak amplitude of the first response in the train. F, Line plot of the responses of control (black line) and Pten-cre (red line) GABAergic neurons to 5 Hz stimulation. Values are normalized to the peak amplitude of the first response in the train. All values are presented as mean ± SEM. *p ≤ 0.05.

Figure 5.

Loss of Pten increases the length of dendrites and number of glutamatergic and GABAergic synapses. A, Representative images from control and Pten-cre glutamatergic (left images) and GABAergic (right images) neurons showing immunoreactivity for MAP2 (green) and either VGLUT1 or VGAT (red). B, Representative tracings of the control (top) and Pten-cre (bottom) GABAergic images shown in A. C, The number of VGLUT1 (left) and VGAT (right) punctae per each control (black) and Pten-cre (red) neuron. D, The total length of dendrites from control (black bars) and Pten-cre (red bars) glutamatergic (left) and GABAergic (right) neurons. E, The number of VGLUT1 (left) and VGAT (right) punctae per micron of dendritic length in each control (black) and Pten-cre (red) neuron. All values are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01.

Figure 6.

Seventy-two hour rapamycin treatment prevents the increase in RRP size and miniamplitude caused by Pten loss. A, Representative traces of the response of glutamatergic Pten-cre vehicle-treated (red trace) and 100 nm rapamycin-treated (gray trace) neurons to a 4 s pulse of 500 mm sucrose. B, Representative traces of the response of GABAergic Pten-cre vehicle-treated (red trace) and 100 nm rapamycin-treated (gray trace) neurons to a 4 s pulse of 500 mm sucrose. C, The number of synaptic vesicles contained in the RRP of glutamatergic vehicle-treated control, vehicle-treated Pten-cre, and rapamycin-treated Pten-cre neurons. D, The number of synaptic vesicles contained in the RRP of GABAergic vehicle-treated control, vehicle-treated Pten-cre, and rapamycin-treated Pten-cre neurons. E, Representative traces showing the average of mEPSCs collected from one vehicle-treated Pten-cre (red trace) and one rapamycin-treated Pten-cre (gray trace) glutamatergic neuron. F, Representative traces showing the average of mIPSCs collected from one vehicle-treated Pten-cre (red trace) and one rapamycin-treated Pten-cre (gray trace) GABAergic neuron. G, mEPSC peak amplitudes from vehicle-treated control, vehicle-treated Pten-cre, and rapamycin-treated Pten-cre glutamatergic neurons. H, mIPSC peak amplitudes from vehicle-treated control, vehicle-treated Pten-cre, and rapamycin-treated Pten-cre GABAergic neurons. All statistical tests are one-way ANOVA with a Student–Newman–Keuls post-test. All values are presented as mean ± SEM. *p ≤ 0.05; **p ≤ 0.01.

Figure 7.

Seventy-two hour rapamycin treatment normalizes dendritic length and synapse number. A, Representative images of a vehicle-treated Pten-cre (left) and rapamycin-treated Pten-cre (right) neuron infected with the cre-RFP-expressing lentivirus showing the fluorescence from the RFP fusion protein (red) and the immunoreactivity against phospho-S6 (green). B, Representative images from vehicle-treated (left) and rapamycin-treated (right) Pten-cre glutamatergic neurons showing immunoreactivity for MAP2 (green) and VGLUT1 (red). C, Normalized values (mean ± SEM) for the soma area, VGLUT1 punctae per neuron, and dendritic length of vehicle-treated (red bars) and rapamycin-treated (gray bars) glutamatergic Pten-cre neurons. The values are normalized to the corresponding values from vehicle-treated control neurons, which are represented by the dashed black line and surrounding error bars. D, Representative images from vehicle-treated (left) and rapamycin-treated (right) Pten-cre GABAergic neurons showing immunoreactivity for MAP2 (green) and VGAT (red). E, Normalized values (mean ± SEM) for the soma area, VGAT punctae per neuron, and dendritic length of vehicle-treated (red bars) and rapamycin-treated (gray bars) GABAergic Pten-cre neurons. The values are normalized to the corresponding values from vehicle-treated control neurons, which are represented by the dashed black line and surrounding error bars. All statistical tests are one-way ANOVA with a Student–Newman–Keuls post-test. *p ≤ 0.05; **p ≤ 0.01 versus control.

Figure 8.

Twelve hour rapamycin treat does not prevent the increase in synaptic transmission, but increases the minifrequency. A, Representative traces showing evoked synaptic responses from vehicle-treated Pten-cre (red traces), 12 h rapamycin-treated Pten-cre (blue traces), and 72 h rapamycin-treated Pten-cre (gray traces) neurons. B, Representative traces showing sucrose application responses from vehicle-treated Pten-cre (red traces), 12 h rapamycin-treated Pten-cre (blue traces), and 72 h rapamycin-treated Pten-cre (gray traces) neurons. C, Representative traces showing miniature EPSC activity from vehicle-treated Pten-cre (red traces), 12 h rapamycin-treated Pten-cre (blue traces), and 72 h rapamycin-treated Pten-cre (gray traces) neurons. D, Miniature event frequencies (mean ± SEM) of vehicle-treated control (black) and Pten-cre (red) neurons, 12 h rapamycin-treated Pten-cre neurons (blue), and 72 h rapamycin-treated Pten-cre neurons (gray). *p ≤ 0.05 versus all other groups (one-way ANOVA with a Student–Newman–Keuls post-test).

Figure 9.

Seventy-two hour rapamycin treatment decreases synaptic transmission in glutamatergic but not GABAergic neurons. A, Peak amplitudes of evoked EPSCs from glutamatergic (left bars) and evoked IPSCs from GABAergic (right bars) neurons. Black bars are vehicle-treated wild-type neurons, and green bars are 72 h 100 nm rapamycin-treated neurons. B, Representative traces showing sucrose application responses from glutamatergic (left traces) and GABAergic (right traces) wild-type neurons treated with vehicle (black traces) or rapamycin (green traces). C, The number of synaptic vesicles (mean ± SEM) contained in the RRP of glutamatergic (left bars) and GABAergic (right bars) neurons treated with vehicle (black) or rapamycin (green). D, Representative traces showing average mEPSCs from glutamatergic (left traces) or average mIPSCs from GABAergic (right traces) wild-type neurons treated with vehicle (black traces) or rapamycin (green traces). E, mEPSC peak amplitudes (left bars) and mIPSC peak amplitudes from (right bars) neurons treated with vehicle (black) or rapamycin (green). F, Representative traces showing miniature IPSC activity from vehicle-treated wild-type (black trace) or 72 h rapamycin-treated wild-type (green trace) GABAergic neurons. G, The frequency of miniature events collected from vehicle-treated and rapamycin-treated wild-type neurons. All values are presented as mean ± SEM. *p ≤ 0.05.