Rapid and slow chemical synaptic interactions of cholinergic projection neurons and GABAergic local interneurons in the insect antennal lobe

Abstract

The antennal lobe (AL) of insects constitutes the first synaptic relay and processing center of olfactory information, received from olfactory sensory neurons located on the antennae. Complex synaptic connectivity between olfactory neurons of the AL ultimately determines the spatial and temporal tuning profile of (output) projection neurons to odors. Here we used paired whole-cell patch-clamp recordings in the cockroach Periplaneta americana to characterize synaptic interactions between cholinergic uniglomerular projection neurons (uPNs) and GABAergic local interneurons (LNs), both of which are key components of the insect olfactory system. We found rapid, strong excitatory synaptic connections between uPNs and LNs. This rapid excitatory transmission was blocked by the nicotinic acetylcholine receptor blocker mecamylamine. IPSPs, elicited by synaptic input from a presynaptic LN, were recorded in both uPNs and LNs. IPSPs were composed of both slow, sustained components and fast, transient components which were coincident with presynaptic action potentials. The fast IPSPs were blocked by the GABAA receptor chloride channel blocker picrotoxin, whereas the slow sustained IPSPs were blocked by the GABAB receptor blocker CGP-54626. This is the first study to directly show the predicted dual fast- and slow-inhibitory action of LNs, which was predicted to be key in shaping complex odor responses in the AL of insects. We also provide the first direct characterization of rapid postsynaptic potentials coincident with presynaptic spikes between olfactory processing neurons in the AL.

Keywords: GABA; acetylcholine; antennal lobe; local interneuron; olfaction; projection neuron.

Figure 1.

Type I LNs receive fast, excitatory input from uPNs. A, B, Single presynaptic action potentials in the uPN induced fast, transient short-latency EPSPs in the postsynaptic type I LN. C, Higher magnification of the frame in A showing fast synaptic input, with two additional fast EPSPs from the same neuron. The black dotted lines represent linear fits of the resting membrane potential and the rising phase of an EPSP. Their point of intersection was defined as the onset of the EPSP. D, Latency between the uPN spikes and EPSP in type I LNs. E, EPSP amplitudes in type I LNs that were elicited by single uPN action potentials. F, During high-frequency trains of action potentials the fast EPSPs can reach the action potential threshold (action potentials clipped). Gi, Morphology of the recorded uPN (green) and type I LN (magenta) revealed by staining of each neuron via the recording pipette. The green asterisk marks the position of the uPN soma that was lost during processing. Scale bar, 100 μm. ii–iv, Higher magnification of frame i showing neurites of both neurons in the same glomerulus. m, Medial; p, posterior. Scale bar, 50 μm. H, Recording from two type I LNs showing coincident EPSPs presumably due to dyadic input from a uPN. The black trace in the dotted box is enlarged underneath.

Figure 2.

Fast excitatory synaptic transmission between uPNs and type I LNs was reversibly blocked by the nicotinic acetylcholine receptor blocker mecamylamine. A, Example of a fast excitatory connection between a uPN and a type I LN that is reversibly blocked by 100 μm mecamylamine. B, Quantification of the effect of 100 μm mecamylamine on the normalized EPSP amplitude.

Figure 3.

uPNs receive fast action potential-evoked inhibitory input from type I LNs. A–C, Presynaptic depolarization in the type I LNs induced IPSPs in the uPN. The IPSPs consisted of a sustained component (A) and fast, transient components that were coincident with presynaptic action potentials (B, C). B is a higher magnification of the frame in A. C shows an overlay of three fast IPSPS in the same uPN that were evoked by single presynaptic action potentials. D, Latency between the presynaptic LN action potentials and the IPSPs in the uPNs. E, Amplitude of fast IPSPs in uPNs. F, The fast inhibitory synaptic transmission between type I LNs and uPNs was abolished by PTX, a blocker of the GABA_A receptor chloride channels (each trace is an average of 5). G, Hyperpolarization of type I LNs had no effect on the membrane potential of the postsynaptic uPN.

Figure 4.

The reversal potential of the sustained IPSCs in uPNs. A, Black trace, Whole-cell current of the presynaptic type I LN induced by a voltage step to +50 mV. Gray traces, Resulting currents in the postsynaptic uPN that was clamped at different holding potentials which are given in brackets. B, I–V plot showing the reversal potential of the sustained IPSC.

Figure 5.

The slow inhibitory synaptic transmission between type I LNs and uPNs was reversibly blocked by the GABA_B receptor blocker CGP-54626. A, Example of a sustained inhibitory connection between a type I LN and a uPN that was reversibly blocked by 5 μm CGP-54626. Note the slow depolarization that followed the sustained IPSPs, which increased during the wash. The wash trace is shown on the left in the same scale as the control and the CGP trace (large depolarization and action potential are clipped). To the right the same wash trace is shown in lower magnification to provide a better overview. B, Quantification of the CGP-54626 effect on the normalized sustained IPSP amplitude. C, PTX, a blocker of the GABA_A receptor chloride channels, had no effect on the sustained inhibitory transmission. D, Latency of the sustained IPSPs. E, Time from start of the voltage step to maximum IPSP, as depicted in A. F, The amplitude of the sustained IPSP (δv₁) and the slow depolarization (δv₂), as depicted in A.

Figure 6.

Inhibitory synaptic connections between pairs of type I LNs. A, B, Presynaptic trains of action potentials induced IPSPs in the postsynaptic type I LN. The IPSPs consisted of a sustained component (A) and fast, transient components that were coincident with presynaptic action potentials (B, C). B is a higher magnification of the frame in A. C, Three overlaid fast IPSPS that were evoked by presynaptic action potentials in the same postsynaptic LN. D, Latency between the presynaptic action potential and fast IPSPs. E, Amplitude of fast IPSPs in type I LNs elicited by single action potentials in the presynaptic type I LNs. F, Example of the reciprocal inhibitory connection between two type I LNs. Gi, Morphology of two simultaneously recorded type I LNs revealed by staining of each neuron via the recording pipette. Scale bar, 100 μm. ii–iv, Higher magnification of four glomeruli showing neurites of both LNs in the same glomeruli. l, lateral; p, posterior. Scale bar, 50 μm.

Figure 7.

GABA_A and GABA_B receptor-mediated inhibition can contribute to inhibitory transmission between type I LNs during strong presynaptic depolarization. A, B, The fast single action potential-evoked inhibitory synaptic transmission between type I LNs was reversibly blocked by 1 mm PTX, a blocker of the GABA_A receptor chloride channels. The fast IPSPs were not affected by 5 μm CGP-54626, a GABA_B receptor blocker. C–F, CGP-54626 (5 μm; C, D) and PTX (1 mm; E, F) decreased the sustained IPSPs that were evoked by strong presynaptic depolarization.