Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ

Abstract

Behavioral and genetic studies in Drosophila have contributed to our understanding of molecular mechanisms that underlie the complex processes of learning and memory. Use of this model organism for exploration of the cellular mechanisms of memory formation requires the ability to monitor synaptic activity in the underlying neural networks, a challenging task in the tiny adult fly. Here, we describe an isolated whole-brain preparation in which it is possible to obtain in situ whole-cell recordings from adult Kenyon cells, key members of a neural circuit essential for olfactory associative learning in Drosophila. The presence of sodium action potential (AP)-dependent synaptic potentials and synaptic currents in >50% of the Kenyon cells shows that these neurons are members of a spontaneously active neural circuit in the isolated brain. The majority of sodium AP-dependent synaptic transmission is blocked by curare and by alpha-bungarotoxin (alpha-BTX). This demonstrates that nicotinic acetylcholine receptors (nAChRs) are responsible for most of the spontaneous excitatory drive in this circuit in the absence of normal sensory input. Furthermore, analysis of sodium AP-independent synaptic currents provides the first direct demonstration that alpha-BTX-sensitive nAChRs mediate fast excitatory synaptic transmission in Kenyon cells in the adult Drosophila brain. This new preparation, in which whole-cell recordings and pharmacology can be combined with genetic approaches, will be critical in understanding the contribution of nAChR-mediated fast synaptic transmission to cellular plasticity in the neural circuits underlying olfactory associative learning.

Figure 1.

Holder used to stabilize isolated whole brain in the recording chamber. The U-shaped frame was fashioned, by hand, from platinum wire (0.5 mm diameter; World Precision Instruments, Sarasota, FL). Nylon fibers (0.07 mm diameter) were glued to the frame, separated by ∼0.5 mm, as shown. The holder was placed on top of the brain to stabilize the tissue during the recording. Brains were oriented perpendicular to cross hairs, with the fibers contacting the tissue at the borders between the central brain region and the optic lobes.

Figure 2.

Spontaneous activity in Kenyon cells in an isolated whole-brain preparation. A, GFP+ Kenyon cell-body clusters are visible in the mushroom-body region (right hemisphere cluster delineated by large white square), on the posterior surface of an entire brain isolated from a 2-d-old OK107 adult fly. Kenyon cell axons appear as L-shaped dorsomedial projections. A fluorescent mask projected on the Nomarski image. B, Individual GFP+ Kenyon cell bodies can be distinguished at higher magnification (top right region of square in A) and targeted for electrophysiological recording in the living isolated brain preparation. C, Whole-cell, currentclamp recording of spontaneous depolarizations from a single Kenyon cell. RP, Resting potential. D, Voltage-clamp recording of the underlying spontaneous currents from the same Kenyon cell. HP, Holding potential.

Figure 3.

Spontaneous currents in Kenyon cells. A, Whole-cell, voltage-clamp recording from a GFP+ OK107 Kenyon cell exhibiting the most common activity pattern (49 of 66 neurons) characterized by the presence of discrete sPSCs. Inset, High time resolution of the indicated region illustrates the rapid rise and slow decay phase of the sPSCs. Holding potential, –75 mV. B, Activity pattern in a second GFP+ OK107 Kenyon cell, classified as bursting sPSCs, was observed less often (8 of 66). This class was defined by clusters of fast synaptic currents. Inset, High time resolution of single burst. C, Slow-wave inward current activity, seen in 6 of 66 GFP+ OK107 Kenyon cells, was dominated by large slowly rising and decaying inward currents. D, Distribution of activity patterns, as a percentage of active GFP+ Kenyon cells in OK107. E, Kenyon cells in wild-type brains were not significantly different (χ² analysis). The number of Kenyon cells with an activity pattern in each class was expressed as a percentage of the total number of active Kenyon cells in each genotype.

Figure 4.

nAChRs mediate most of the spontaneous sodium AP-dependent excitatory drive in the Kenyon cell circuit in situ. A, sPSCs recorded from a Kenyon cell in control saline. Bath application of TTX (1 μm) blocked many of the synaptic currents. Events remaining in the presence of TTX were classified as mPSCs. Each record represents two superimposed current traces. B, Bath perfusion of TTX caused a significant reduction in PSC frequency (***p < 0.001, Wilcoxon signed rank test; n = 16). C, Bath perfusion of TTX did not significantly reduce PSC amplitude. All recordings were made at a holding potential of –75 mV. D, sPSCs recorded from a Kenyon cell in control saline were blocked by bath application of curare. Partial recovery of sPSCs after extensive washing is shown. Each record represents two superimposed current traces. E, The sPSC frequency was significantly reduced by bath application of curare (20 μm; n = 6) or αBTX (5 μm; n = 4). **p < 0.01, Wilcoxon signed rank test (n = 10). Error bars indicate SEM.

Figure 5.

Sodium AP-independent synaptic currents mediated by α-BTX-sensitive nAChRs in Kenyon cells in situ. A, The frequency of mPSCs recorded in the presence of TTX was significantly reduced by the addition of curare (20 μm; n = 6) or α-BTX (5 μm; n = 4). **p < 0.01, Wilcoxon signed rank test. B, Sodium AP-independent synaptic currents recorded from a Kenyon cell in saline containing TTX, PTX, APV, and CNQX are completely blocked by bath perfusion of 5 μm α-BTX, indicating they are mediated by nAChRs. Only partial recovery was observed after extensive washing. C, mEPSC amplitude decreases with increasing depolarization. Ensemble average mEPSCs constructed from 7–15 events at each holding voltage. HP, Holding potential. D, The mEPSC amplitude histogram from a single cell reveals a typical, relatively narrow size distribution (mean, 4.5 pA). E, The ensemble average mEPSC from the same cell, compiled from 225 individual events, exhibits a typical shape with a relatively fast rise and slow decay. The decay was fit with a single exponential (dotted line; τ = 4 ms). Error bars indicate SEM.