The role of cAMP in synaptic homeostasis in response to environmental temperature challenges and hyperexcitability mutations

Abstract

Homeostasis is the ability of physiological systems to regain functional balance following environment or experimental insults and synaptic homeostasis has been demonstrated in various species following genetic or pharmacological disruptions. Among environmental challenges, homeostatic responses to temperature extremes are critical to animal survival under natural conditions. We previously reported that axon terminal arborization in Drosophila larval neuromuscular junctions (NMJs) is enhanced at elevated temperatures; however, the amplitude of excitatory junctional potentials (EJPs) remains unaltered despite the increase in synaptic bouton numbers. Here we determine the cellular basis of this homeostatic adjustment in larvae reared at high temperature (HT, 29°C). We found that synaptic current focally recorded from individual synaptic boutons was unaffected by rearing temperature (<15°C to >30°C). However, HT rearing decreased the quantal size (amplitude of spontaneous miniature EJPs, or mEJPs), which compensates for the increased number of synaptic releasing sites to retain a normal EJP size. The quantal size decrease is accounted for by a decrease in input resistance of the postsynaptic muscle fiber, indicating an increase in membrane area that matches the synaptic growth at HT. Interestingly, a mutation in rutabaga (rut) encoding adenylyl cyclase (AC) exhibited no obvious changes in quantal size or input resistance of postsynaptic muscle cells after HT rearing, suggesting an important role for rut AC in temperature-induced synaptic homeostasis in Drosophila. This extends our previous finding of rut-dependent synaptic homeostasis in hyperexcitable mutants, e.g., slowpoke (slo). In slo larvae, the lack of BK channel function is partially ameliorated by upregulation of presynaptic Shaker (Sh) IA current to limit excessive transmitter release in addition to postsynaptic glutamate receptor recomposition that reduces the quantal size.

Keywords: input resistance; quantal content; quantal size; rutabaga adenylyl cyclase; synaptic growth.

Figure 1

A novel form of synaptic homeostasis in response to high temperature rearing. (A) Evoked EJP size was not affected by rearing temperatures. Upper (black) and lower (red) traces are from WT larvae reared at room temperature (23°C, RT) and high temperature (29°C, HT), respectively. (B) Miniature EJPs (mEJPs) became smaller when reared at HT. ***p < 0.001, t-test. (C) Quantal content (ratio of mean amplitudes of EJP to mEJP) in HT-reared larvae could be larger than that in RT-reared larvae, as indicated by a skewed distribution. + p < 0.05, F-test. (D) Frequency of mEJPs in HT-reared larvae was also more variable than that in RT-reared larvae. + p < 0.05, F-test. Scale bars: 5 mV and 20 ms (A) or 200 ms (B). Number of muscle fibers indicated in parenthesis. Data recorded in HL3.1 saline containing 0.2 mM (A,C) or 0.2 and 0.5 mM Ca²⁺ (B,D). (A) is constructed from data in Ueda and Wu (2012) with additional data.

Figure 2

Focal mEJCs and EJCs were not affected by rearing temperature. Focal EJCs (A) and mEJCs (B) remained stable despite ambient temperature fluctuations. Building temperature varied significantly: from as low as 15°C to beyond 30°C (mostly during May to July). Pooled data from August to April are compared to those from May to July. (C) Quantal content of the focal EJCs was not different between the two periods (p > 0.5, Wilcoxon rank-sum test). Number of recording sites indicated in parenthesis. (A) is reconstructed from data in Ueda and Wu (2012).

Figure 3

HT rearing decreased muscle input resistance in WT, but not rut larvae. (A) Muscle membrane potential traces in response to hyperpolarizing current injection in WT larvae reared at RT and HT. (B) Input resistance was decreased in HT-reared WT, but not rut, larvae. (C) Rearing temperature did not significantly affect half-decay time in WT and rut. *p < 0.05, One-way ANOVA. Number of muscle fibers indicated in parenthesis. Data from 0.2 and 0.5 mM Ca²⁺ were pooled. HL3.1 saline.

Figure 4

Focal EJC amplitudes affected by rearing temperature in rut. (A) Focal EJCs from the great majority of rut boutons were below 2 nA (dotted line) during the period of May to July, whereas those collected outside of this period were substantially larger. Compare to Figure 2 for constant EJC size for WT. (B) Focal mEJC amplitudes in rut did not vary with ambient temperature fluctuations. (C) Quantal content was significantly smaller at HT. Number of recording sites indicated in parenthesis. ***p < 0.001; **p < 0.01, Wilcoxon rank-sum test. (A) is reconstructed from data in Ueda and Wu (2012).

Figure 5

Altered transmitter release in HT-reared rut larvae. (A) Evoked EJP size became drastically smaller in rut when reared at HT. Upper (black) and lower (red) traces are from RT- and HT- reared larvae, respectively. In rut, mEJP size was not significantly altered by HT rearing (B), although quantal content was significantly reduced (C). Frequency of mEJP was reduced in rut when reared at HT (D). ***p < 0.001; *p < 0.05, t-test. Number of muscle fibers indicated in parenthesis. Data recorded in HL3.1 saline containing 0.2 mM (A,C) or 0.2 and 0.5 mM Ca²⁺ (B,D). (A) is constructed from data in Ueda and Wu (2012) with additional data.

Figure 6

Altered activity-dependent facilitation of EJPs in rut larvae. EJPs in rut larvae displayed enhanced facilitation when reared at HT. Ratios of EJP amplitudes evoked by 15-Hz–0.5 Hz provides an index for facilitation. Stimulus trains (10 s, 15 Hz) were applied and average EJP amplitudes were determined between 5–10 s. **p < 0.01, t-test with sequential Bonferroni adjustment for multiple comparisons of RT vs. HT. Number of muscle fibers indicated in parentheses. Error bars indicate SEM.

Figure 7

Comparisons between slo hyperexcitabilty mutation- and HT rearing-induced synaptic homeostasis. (A) slo mutation-induced pre- and post-synaptic homeostatic adjustment (Lee et al., , ; Lee and Wu, 2010): (1) upregulation of presynaptic Sh I_A K⁺ currents to compensate for the loss of slo BK currents, (2) growth of excessive, functional satellite boutons, and (3) altered GluRII receptor composition (subunit A vs. B ratio) resulting in decreased mEJP amplitude. The roles of cac and Dmca1D Ca²⁺ channels, as well as the rut AC-mediated adjustments are depicted. (B) HT rearing induced-homeostasis: (1) regulation of excitability by rut AC (Ueda and Wu, 2009), (2) increased number of boutons (Zhong and Wu, ; Berke et al., 2013), and (3) decreased muscle membrane resistance from increased effective muscle membrane area. The resultant modifications in amplitudes of mEJPs and EJPs are summarized. In both (A) and (B), rut AC plays an important role in homeostatic regulation of both synaptic function and growth.