The effects of temperature on vesicular supply and release in autaptic cultures of rat and mouse hippocampal neurons

Abstract

Membrane fusion plays a central role in the synaptic vesicle cycle. While many of the pre- and postfusion events have been investigated at room temperature, few researchers have investigated these processes at more physiologically relevant temperatures. We have used autaptic cultures of hippocampal neurons to investigate changes in the size and refilling rate of the readily releasable pool (RRP) of synaptic vesicles brought about by an increase in temperature from 25 to 35 degrees C. We have also examined temperature-dependent changes in spontaneous and action potential (AP)-evoked release as well as the fraction of the RRP that is released during an AP. Although we found a threefold increase in the refilling rate of the RRP at the higher temperature, there was no apparent change in the size of the RRP with increased temperature. Moreover, we observed a slight but significant decrease in the quanta released during an AP. This increased refilling rate and decreased release probability resulted in a reduction of both the degree and time course of synaptic depression during high frequency stimulation at the higher temperature. This reduction in synaptic depression was accompanied by an increased maintenance of the synchronous component of release during high frequency stimulation. These findings indicate that the dynamics of vesicular supply and release in hippocampal neurons at room temperature are significantly different at near physiological temperatures and could affect our present understanding of the way in which individual neurons and networks of neurons process information.

Figure 1. Refilling of the RRP is faster at the higher temperature

A, example trace of paired hypertonically mediated responses at 25 and 35 °C. Bars represent 3 s applications of 500 mm sucrose. Interpulse intervals are 1, 4, 7, 10, 13 and 16 s. Additionally, the total time of one episode (paired pulse) was between 30 and 60 s in order to allow adequate time for refilling of the RRP. Since refilling was significantly faster at the higher temperature, additional interpulse interval times (0.5, 1.5, 2, 2.5, 3 and 3.5 s) were also examined at 35 °C in order to obtain better resolution of the fast component of refilling (raw traces not shown). B, the degree to which the charge of the second response (A2) had recovered relative to the charge of the first response (A1) was plotted [1 - (A1 - A2)/A1] as a function of the interpulse interval time for both 35 (○) and 25 °C (•). The data were best fitted by biexponential curves, indicating that refilling of the RRP involves both a fast and a slow component (n = 9–24 for each temperature point).

Figure 2. Changes in the shape and frequency of mEPSCs occur with increasing temperature

A, example traces of mEPSCs recorded in the presence of 200 nm TTX from the same cell at both 25 and 35 °C. B, superimposed traces of the average mEPSC from the same cell as shown in A at 25 (continuous line) and 35 °C (dotted line). C, statistical analysis indicated that the amplitude, charge and frequency of release increased significantly at the higher temperature (n = 13; Table 1). Furthermore, the decay time decreased significantly at the higher temperature (n = 13; Table 1). Asterisks indicate changes that were statistically significant (P < 0.05).

Figure 3. The size of the RRP remains constant at both temperatures

A, example traces of the hypertonically mediated release of quanta upon application of 500 mm sucrose from the same cell at both 25 and 35 °C. The response rises monoexponentially to a peak rate of release and then decays monoexponentially to a steady-state level of release that represents release of vesicles newly refilled to the RRP. B, by dividing the charge of the transient response by the mean quantal charge at the appropriate temperature, we obtained the size of the RRP of vesicles (n = 25). We found no significant difference in the size of the RRP between the two temperatures. C, by examining the steady-state component of the response, we were able to determine the fraction of the pool refilled per second (n = 8). As expected from Fig. 1C, this value increased significantly (* P < 0.05) at the higher temperature.

Figure 4. The release probability decreases at the higher temperature

A, example traces of the EPSC from the same cell at both 25 (continuous line) and 35 °C (dotted line). B, plot of the instantaneous change in the EPSC amplitude (○), charge (•) and decay time (□) (n = 10). The amplitude increased significantly with increasing temperature, while the charge and decay time decreased significantly with increasing temperature (Table 1). C, by dividing the charge of the EPSC by the mean quantal charge at each temperature in the absence (n = 10) and presence (n = 11) of CTZ, we quantified the quanta released per EPSC. For both conditions, we observed a significant (* P < 0.05) decrease in the release probability at the higher temperature. We also observed a significant increase in the total number of quanta released at both temperatures in the presence of CTZ.

Figure 5. Depression of the synaptic response during high frequency stimulation is reduced at the higher temperature

The amplitude of the EPSC normalized to the amplitude of the first response is plotted as a function of time (n = 10–17). Stimulations at a frequency of 10 (A), 20 (B) and 40 Hz (C) were performed for 5 s at both 25 and 35 °C. Biexponential curves with an added time constant could be fitted to graphs plotting the normalized EPSC as a function of time for each temperature and stimulation frequencies of 20 and 40 Hz (Table 2). A biexponential curve without an added constant was used to fit the data obtained during 10 Hz stimulation, presumably because depression had not reached a level that allowed fitting of an added constant (Table 2).

Figure 6. Maintenance of synchronous release during high frequency stimulation is improved at the higher temperature

A, comparison of the response of the same cell at the start (continuous line) and end (after 5 s; dotted line) of 20 Hz stimulation at both temperatures. Interestingly, the increase in decay time of the EPSC and lowering of the baseline were not as pronounced at the higher temperature. The components of synchronous (B) and asynchronous (C) release were quantified as described in the Methods and plotted as a function of time for the first 2 s of stimulation at 20 Hz at both 25 and 35 °C (n = 10).