cAMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: alterations in the memory mutant dunce

Abstract

It is well known that cAMP signaling plays a role in regulating functional plasticity at central glutamatergic synapses. However, in the Drosophila CNS, where acetylcholine is thought to be a primary excitatory neurotransmitter, cellular changes in neuronal communication mediated by cAMP remain unexplored. In this study we examined the effects of elevated cAMP levels on fast excitatory cholinergic synaptic transmission in cultured embryonic Drosophila neurons. We report that chronic elevation in neuronal cAMP (in dunce neurons or wild-type neurons grown in db-cAMP) results in an increase in the frequency of cholinergic miniature EPSCs (mEPSCs). The absence of alterations in mEPSC amplitude or kinetics suggests that the locus of action is presynaptic. Furthermore, a brief exposure to db-cAMP induces two distinct changes in transmission at established cholinergic synapses in wild-type neurons: a short-term increase in the frequency of spontaneous action potential-dependent synaptic currents and a long-lasting, protein synthesis-dependent increase in the mEPSC frequency. A more persistent increase in cholinergic mEPSC frequency induced by repetitive, spaced db-cAMP exposure in wild-type neurons is absent in neurons from the memory mutant dunce. These data demonstrate that interneuronal excitatory cholinergic synapses in Drosophila, like central excitatory glutamatergic synapses in other species, are sites of cAMP-dependent plasticity. In addition, the alterations in dunce neurons suggest that cAMP-dependent plasticity at cholinergic synapses could mediate changes in neuronal communication that contribute to memory formation.

Fig. 1.

Elevated cholinergic sEPSC frequency indnc mutant neurons. A, Examples of typical recordings of rapid transient sEPSCs obtained from a single wt1 and a single dnc¹ neuron at 5 DIV. Each record represents four superimposed current traces. Currents in both genotypes are reversibly blocked by bath perfusion of 100 nm curare. B, The average sEPSC frequency is significantly higher in dnc¹neurons when compared with wt1 (Student's t test,p < 0.01). All recordings were obtained between 3 and 9 DIV. Error bars indicate SEM; number of neurons indicated inparentheses.

Fig. 2.

Elevated cholinergic mEPSC frequency indnc mutant neurons. A, mEPSC recordings obtained from indicated genotype in the presence of 1 μmTTX. Averaged mEPSC on a faster time scale from a single neuron is shown for each genotype (traces were normalized to the same peak current amplitude). B, The mEPSC frequencies indnc¹ and dnc²neurons are significantly higher (fourfold) than wt1 (ANOVA, *p < 0.05; **p < 0.005 Fisher's protected least significant difference). All recordings obtained between 3 and 9 DIV. C, Overlapping cumulative probability amplitude histograms at a holding potential of −75 mV constructed from wt1 (1895 individual mEPSCs recorded from 13 wt1 neurons) and dnc¹ (763 mEPSCs from 8dnc¹ neurons).

Fig. 3.

Differentiation of wild-type neurons in db-cAMP results in a dnc phenocopy. A, Ten superimposed sEPSC recordings obtained from two wt1 neurons (4 DIV) that were grown in either 1 μm (top) or 100 μm (bottom) db-cAMP. B,Dose-dependent increase in sEPSC frequency. Neurons were grown in the indicated concentration of db-cAMP and examined between 3 and 9 DIV. A sigmoidal fit to the data indicated an EC₅₀ of 17.7 ± 8.1 μm. C, mEPSCs recorded from four wt1 neurons grown in the absence of db-cAMP (first trace), presence of 100 μm db-cAMP (second trace), and 1 (third trace) or 2 (fourth trace) days after removal of db-cAMP at 3 DIV. D, The average mEPSC frequency in wt1 neurons grown in the presence of 100 μm db-cAMP (squares) was approximately sevenfold higher than that observed in the untreated wt1 neurons (circles) at all ages examined. In contrast, wt1 neurons that were grown in the presence of 100 μm db-cAMP for 3 DIV and then switched to db-cAMP-free media (triangles), showed an elevated mEPSC frequency at 1 d after removal of db-cAMP that returned to baseline levels by 2 d. All recordings were obtained in normal external saline at room temperature. Error bars indicate SEM. All data points represent values obtained from between 8 and 38 neurons examined in three or more separate platings.

Fig. 4.

Acute elevation in cAMP levels mediates a rapid onset, protein kinase-dependent increase in AP-dependent transmission at cholinergic synapses in wild-type neurons. A,Schematic of treatment protocol. sEPSCs were recorded in standard external saline 0–4 hr after termination of the db-cAMP pulse.B, Increasing the duration of cAMP exposure, between 0.5 and 4 hr, induced an increasing elevation in sEPSC frequency when compared with controls (*p < 0.01, **p < 0.01, ***p < 0.001, ANOVA, Scheffe's post hoc analysis). There was no further increase in sEPSC frequency when the db-cAMP exposure time was increased to 8 hr. When 50 nm staurosporine was present during the 4 hr db-cAMP exposure, the increase in sEPSC frequency was completely blocked, indicating a protein kinase-dependent mechanism. The mEPSC frequency was not significantly different in neurons immediately after 4 hr db-cAMP exposure compared with controls (value at db-cAMP concentration of 0). Error bars indicate SEM. All means represent data obtained from between 10 and 23 neurons examined in three or more separate platings.

Fig. 5.

Acute elevation in cAMP levels mediates a delayed onset, protein synthesis-dependent increase in AP-independent transmission at cholinergic synapses in wild-type neurons.A, Schematic of treatment protocol. mEPSCs were recorded in standard external saline at the indicated times (±2 hr) after termination of the db-cAMP pulse. B, There was a gradual increase in mEPSC frequency as a function of increasing time after termination of the db-cAMP treatment. The peak mEPSC frequency (a fourfold increase) was observed at 24 hr and subsequently declined over the next 8 hr. When 100 μm cycloheximide was present during the 4 hr db-cAMP exposure as well as the subsequent 20 hr, the increase in mEPSC frequency was completely blocked. Cycloheximide (CXM) alone for 24 hr resulted in mEPSC frequencies that were not different than the controls. Error bars indicate SEM. All data points represent values obtained from between 8 and 32 neurons examined in three or more separate platings.

Fig. 6.

A persistent increase in mEPSC frequency induced by repetitive, spaced db-cAMP in differentiated wild-type neurons is absent in neurons from the memory mutant dunce.A, db-cAMP treatment and recording paradigm.B, A single 4 hr exposure to db-cAMP results in a delayed onset increase in mEPSC frequency that peaks at 24 hr and returns to control levels 2 d after treatment in wt1 neurons. In contrast, repetitive space application of db-cAMP resulted in a slightly higher mEPSC frequency at 24 hr with a persistent elevation in frequency maintained for up to 3 d after treatment. (*p < 0.05, **p < 0.01, Student's t test, treated vs control). These data suggest that rest intervals are important in mediating persistent changes initiated by elevation in cAMP levels. C,dunce neurons have a higher basal mEPSC frequency than wt1. However, the mEPSC frequencies in dnc¹neurons treated for 4 hr with db-cAMP (single and repetitive spaced exposure) were only slightly elevated at 1 d after treatment when compared with the control dnc¹ neurons. The mean mEPSC frequencies in dnc¹ neurons at 2–3 d after either db-cAMP treatment paradigm were not different than the control neurons, indicating a reduction in cAMP-induced plasticity in dnc mutants. Error bars indicate SEM. All data points represent values obtained from between 9 and 32 neurons examined in three or more separate platings.