Multiple effects of β-amyloid on single excitatory synaptic connections in the PFC

Abstract

Prefrontal cortex (PFC) is recognized as an AD-vulnerable region responsible for defects in cognitive functioning. Pyramidal cell (PC) connections are typically facilitating (F) or depressing (D) in PFC. Excitatory post-synaptic potentials (EPSPs) were recorded using patch-clamp from single connections in PFC slices of rats and ferrets in the presence of β-amyloid (Aβ). Synaptic transmission was significantly enhanced or reduced depending on their intrinsic type (facilitating or depressing), Aβ species (Aβ 40 or Aβ 42) and concentration (1-200 nM vs. 0.3-1 μ M). Nanomolar Aβ 40 and Aβ 42 had opposite effects on F-connections, resulting in fewer or increased EPSP failure rates, strengthening or weakening EPSPs and enhancing or inhibiting short-term potentiation [STP: synaptic augmentation (SA) and post-tetanic potentiation (PTP)], respectively. High Aβ 40 concentrations induced inhibition regardless of synaptic type. D-connections were inhibited regardless of Aβ species or concentration. The inhibition induced with bath application was hard to recover by washout, but a complete recovery was obtained with brief local application and prompt washout. Our data suggests that Aβ 40 acts on the prefrontal neuronal network by modulating facilitating and depressing synapses. At higher levels, both Aβ 40 and Aβ 42 inhibit synaptic activity and cause irreversible toxicity once diffusely accumulated in the synaptic environment.

Keywords: excitatory post-synaptic potential (EPSP); post-tetanic potentiation (PTP); short term potentiation (STP); synaptic augmentation (SA); synaptic connection; synaptic dynamics; β-amyloid (Aβ).

Figure 1

Excitatory synaptic connections in layer V of PFC. (A) A facilitating (F-type) PC-PC connection in layer V of the PFC of an 8-weeks old ferret. Left panel: 3D computer reconstruction of the connection: Both pre- (PC1 in black) and post-synaptic (PC2 in green) cells are complex PCs featured by an apical dendrite with multiple early-bifurcated major branches. A total of 20 putative synapses are marked with red stars onto the basal, apical, oblique and tuft dendrites of PC2. Right panel: Physiological responses of pre- (upper, in black) and post-synaptic (middle, in green) cells were induced by injections of depolarization currents into their somata. Excitatory postsynaptic potentials (EPSPs, down, in red) were recorded from PC2 by giving brief current injections to induced action potentials (APs, bottom, in black) in PC1. The postsynaptic response train is composed of 8 EPSPs at a 20 Hz frequency followed by a recover test response (RTR) with 500 ms delay. (B) A depressing (D-type) PC-IN connection in layer V of the PFC of a P30 rat. The color coding for the reconstructed pre- and post-synaptic cells and their physiological and synaptic responses are the same as the PC-PC connection in (A). A total of 7 putative synapses are marked with red stars onto basal dendrites of the postsynaptic interneuron. Note: The postsynaptic interneuron appears to be a fast-spiking basket cell according to its axonal and dendritic morphologies and fast AP firing induced by depolarization current injection to its soma.

Figure 2

Synaptic failure rates vary according to synaptic type and upon Aβ species and concentration. (A) Superimposed 15 single EPSP traces recorded at 0.5 Hz from an F-type connection in pre-application, application and washout phases of 1 μ M Aβ 40. EPSPs were generally reduced and the number of failures increased during application of Aβ for 20 min, which tended to recover on washout for 10 min. (B) Average failure rates of F- and D-connections in pre-application, application and washout phases in the presence of Aβ were charted according to Aβ species and concentration, and connection type. The synaptic failure rate tended to decrease in F-connections after low-dose Aβ 40 applied. In contrast, the failure rates appear to increase in all other cases. Low and high doses of Aβ 40 applied to D-connections reached significance (P = 0.01 and P = 0.024, respectively). A trend to enhance failure rates is shown in the case of high-dose Aβ 40 to F-connection (n = 4) and low-dose Aβ 42 to F- (n = 3) and D-(n = 4) connections. After washout for 10–30 min., the failure rate are further exacerbated in applications of low-dose Aβ 42 to F- and D-connections (compared with pre-application, P = 0.04; compared with Aβ application, P = 0.05). Note: ^*compared with pre-application, ^Δcompared with Aβ application; ^* or ^Δ P < 0.05; ^** P < 0.01. (C) Net changes in average failure rates following exposure to Aβ (the failure rate in Aβ application - the failure rate in pre-application). The net rate change in low-dose Aβ 40 to F-connections was opposite in direction to that of the other cases. The difference between the net rate changes corresponding to low-dose Aβ 40 vs. high-dose Aβ 40 to F-connections did not quite reach statistically significance possibly due to the low n (n = 3) in the latter case. (D) Net changes to average failure rates by washout of Aβ (the failure rate in washout of Aβ-the failure rate in pre-application). The differential change in failure rates remained virtually similar to that in (C). Notably, the net rate change became smaller (from 41 to 14%) in high-dose Aβ 40 to F-connection, but became bigger (from 21 to 49%) in the cased of low-dose Aβ 42 to F-and D-connections.

Figure 3

EPSP trains of F- and D- connections change differentially depending upon the synapse type and the Aβ species and concentration. In each case, representative traces (each was an average of 15–30 individual traces) from pre-application, Aβ application and washout phases are presented at the top of each graph. The chart in the middle gives average EPSP amplitudes that were normalized to the mean of pre-application EPSPs for the comparison between pre-application and Aβ application and washout. The chart at the bottom alternatively shows average EPSP amplitudes that were normalized instead to the 1st EPSP of their intrinsic run in order to access changes in EPSP patterns (for clarity, traces of washout were not plotted). (A) Low-dose Aβ 40 enhanced F-connections. The overall increase in the EPSP train was followed by a comparably larger increment in the RTR. The enhancement tended toward recovery after washout. (B) Low-dose Aβ 40 inhibited D-connections. The EPSP amplitudes were all significantly diminished while the EPSP pattern remained virtually similar to that in pre-application. (C) High-dose Aβ 40 inhibited F-connections. EPSPs were unevenly reduced, in which the decrement of the 1st EPSP was greater. In the chart at the bottom, the empty circles between two arrows showed the real pattern of subsequent EPSPs as they relate to the 1st EPSP. The red dots between two arrows that were disassociated with the 1st EPSPs, show a match up of patterns of subsequent EPSPs and RTR between pre-application and Aβ application conditions. (D) High-dose Aβ 40 inhibited D-connections. The 1st EPSP was notably reduced while the amplitudes of steady state EPSPs (4th through 8th EPSPs) remained unchanged, followed by a reduced RTR. Between two arrows in the chart at the bottom, the empty circles show the pattern of 3rd through 8th EPSPs + RTR as they relate to the 1st and 2nd EPSPs, and the red dots between two arrows were disassociated with the 1st and 2nd EPSPs, giving the matching patterns of 3rd–8th EPSPs and RTR between pre-application and Aβ applications. (E) Low-dose Aβ 42 inhibited F-connections. EPSPs were unevenly reduced, also indicating that the decrement of the 1st EPSP was greater (The same chart presentation was made as in C). (F) Low-dose Aβ 42 inhibited D-connections. EPSPs were all significantly diminished while the EPSP pattern remained almost the same as in pre-application. Note: Upon washout of Aβ (see the middle charts, also see Table 1), the enhancement of low-dose Aβ 40 to F-connections (in A) appeared to recover to the pre-application level while EPSPs inhibited by Aβ did not recover in B–D or even further diminished in E and F.

Figure 4

Full recovery of EPSPs from inhibition by brief local application of Aβ. 1 nM Aβ 42 was briefly applied near the somata of the connected neurons (diagram) for 2 min. Recordings were carried out before (pre-application) and at the end of application of Aβ, and lastly after washout of Aβ for 10 min. The average EPSP traces display full recovery from the inhibition of Aβ (left panel). In the single traces recorded at the end of Aβ application (right panel), the EPSPs almost completely disappeared. These started to recover at ~1 min. after terminating the Aβ “puff.”

Figure 5

Differential effects of low nanomolar Aβ 40 and Aβ 42 on the SA and PTP. (A1) Low-dose Aβ 40 enhanced synaptic responses (i.e., EPSPs) under all the recording phases (pre-tetanus baseline, SA induction and PTP induction) in F-connections (all P < 0.01, n = 4 pairs). After washout for 10–30 min, the EPSPs during the pre-tetanus phase recovered to the pre-application level (P = 0.557), but still remained significantly higher during the SA and PTP induction phases (both P < 0.01, inset table). EPSP amplitudes were normalized to the mean of pre-tetanus EPSPs in pre-application. Paired t-test with multiple outcome values per connection was performed between pre-application and Aβ application, and between pre-application and washout phases. (A2) Comparison of increments during pre-tetanus, SA and PTP induction in the case of low-dose Aβ 40 application. Compared with the baseline level (0 ± 3%) of increment during pre-tetanus phase of pre-application condition, low-dose Aβ 40 enhanced the average baseline EPSP by 23 ± 6% (P = 0.01), recovering after a 10–30 min washout (4 ± 4%, P = 0.556). Compared with 16 ± 9% in pre-application, the SA appeared to be enhanced by low-dose Aβ40 to 30% ± 13% (P = 0.141), and remained enhanced at an average level of 58 ± 16% after a 10–30 min. washout (P = 0.05). Similarly, compared with 2 ± 2% in pre-application condition, the PTP appeared to be enhanced by low-dose Aβ40 to 5 ± 3% (P = 0.284), and remained enhanced to a statistically significant level after a 10–30 min. washout (10 ± 2%, P = 0.01). (B1) Low-dose Aβ 42 depressed synaptic responses at the pre-tetanus baseline, and significantly at the SA and PTP inductions (both P < 0.01, n = 6 pairs). After a 10–30 min. washout, the EPSPs under all the recording phases (pre-tetanus, SA induction and PTP induction) became significantly depressed (P < 0.01, inset table). (B2) Comparison of increments during pre-tetanus, SA and PTP inductions in low-dose Aβ 42 applications. Compared with the baseline level (0 ± 2%) of increment during the pre-tetanus phase of pre-application condition, low-dose Aβ 42 depressed the average baseline EPSP by −6 ± 5% (P = 0.08). This became statistically significant after washout (−28 ± 4%, P < 0.01). Compared with 33 ± 5% in pre-application, the SA was significantly depressed by low-dose Aβ 42 to 22 ± 5% (P = 0.01), recovering after washout (27 ± 7%, P = 0.530). Compared with 6 ± 1% of the increment in pre-application, the PTP was significantly depressed by low-dose Aβ 42 to −4 ± 1% (P = 0.01), again recovering after washout (6 ± 2%, P = 0.154). ^*P < 0.05; ^**P < 0.01.

Figure A1

Western blot monitoring of Aβ stock solutions. Monomer predominant preps as shown in lane 1 were used for the recording of single synaptic connections. Synthetic Aβ peptides were solubilized in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), aliquoted, dried and stored at −80°C. A working stock solution (0.1 mM) was then prepared and stored at −80°C for dilution immediately before use. Aged or oligomerized preps as appear in lane 5 were not used in the current work.

Figure A2

Computer simulation of synaptic responses. The 10 Hz EPSP train followed by a RTR with 500 ms delay was recorded from a PC-IN connection (A). Using a model of dynamic synaptic transmission, the synaptic dynamics were assessed by fitting the EPSPs (B). The modeling extracts four key parameters of the connection: DFUA (D, the time constant of recovery from depression; F, the time constant of recovery from facilitation; U, utilization of synaptic resources used analogously to release probability, p; A, the absolute synaptic strength). If samplings were big enough, these parameters could be quantitatively analyzed enabling a quantitative comparison of basal synaptic dynamic changes induced by Aβ. Trends of changes induced by Aβ were revealed despite low samplings in most data sets (see Table A1).