Two types of local interneurons are distinguished by morphology, intrinsic membrane properties, and functional connectivity in the moth antennal lobe

Abstract

Neurons in the silkmoth antennal lobe (AL) are well characterized in terms of their morphology and odor-evoked firing activity. However, their intrinsic electrical properties including voltage-gated ionic currents and synaptic connectivity remain unclear. To address this, whole cell current- and voltage-clamp recordings were made from second-order projection neurons (PNs) and two morphological types of local interneurons (LNs) in the silkmoth AL. The two morphological types of LNs exhibited distinct physiological properties. One morphological type of LN showed a spiking response with a voltage-gated sodium channel gene expression, whereas the other type of LN was nonspiking without a voltage-gated sodium channel gene expression. Voltage-clamp experiments also revealed that both of two types of LNs as well as PNs possessed two types of voltage-gated potassium channels and calcium channels. In dual whole cell recordings of spiking LNs and PNs, activation of the PN elicited depolarization responses in the paired spiking LN, whereas activation of the spiking LN induced no substantial responses in the paired PN. However, simultaneous recording of a nonspiking LN and a PN showed that activation of the nonspiking LN induced hyperpolarization responses in the PN. We also observed bidirectional synaptic transmission via both chemical and electrical coupling in the pairs of spiking LNs. Thus our results indicate that there were two distinct types of LNs in the silkmoth AL, and their functional connectivity to PNs was substantially different. We propose distinct functional roles for these two different types of LNs in shaping odor-evoked firing activity in PNs.

Keywords: antennal lobe; insect brain; patch-clamp recording.

Fig. 1.

Distinct physiological properties with two morphologically distinct local interneurons (LNs) and projection neuron (PN). A: whole cell current-clamp recordings of PNs showing their typical membrane voltage responses to current steps (left) and the morphology of a recorded neuron, which is visualized by filling with biocytin (right). D, dorsal; M, medial. Scale bar, 100 μm. B: whole cell current-clamp recordings of spiking LNs showing their typical membrane voltage responses to current steps (left) with MGC-allGs-sparse morphology (global multiglomerular type of LN that arborizes in both the MGC and most ordinary glomeruli; see text), which is visualized by filling with biocytin (right). Scale bar, 100 μm. C: mean firing rate vs. injected current for PNs (left) and for spiking LNs (right). The mean firing frequency of action potentials during the current injection was calculated (n = 13 for PNs; n = 56 for LNs). D: whole cell current-clamp recordings of nonspiking LNs and their MGC-allGs-dense morphology, which is visualized by filling with biocytin. D1: “rectified” response of nonspiking LNs showing strong rectification in response to a depolarizing current injection (left) and the MGC-allGs-dense morphology of the recorded neuron (right). D2: “spikelet” response of nonspiking LNs showing a tetrodotoxin (TTX)-insensitive mini-spike waveform in response to a depolarizing current injection (left) and the MGC-allGs-dense morphology of the recorded neuron (right). Scale bars, 100 μm. E: single-cell RT-PCR analysis of the voltage-gated sodium channel gene (BmNa_V). Single-cell RT-PCR was conducted with total RNA from PNs and spiking and nonspiking LNs. The minus sign indicates a negative control without reverse transcriptase. The ribosomal protein gene (Bmrp49) was used as a positive control.

Fig. 2.

Comparison of transient and sustained K⁺ currents (I_transient and I_sustained) in individual antennal lobe (AL) neurons in whole cell voltage-clamp configuration. The conductance (G) is determined by assuming E_K = −98.5 mV. A: I_transient and I_sustained. A1: current traces showing voltage-dependent activation of I_transient and I_sustained, which were measured during the step depolarization in cells loaded with a K^-gluconate patch pipette internal solution and bathed in a solution containing 10⁻⁷ M TTX and 10⁻³ M CdCl₂. A2: current traces showing the pharmacologically isolated I_transient with additional application of 2 × 10⁻² M tetraethylammonium chloride (TEA). A3: current traces showing the pharmacologically isolated I_sustained with additional application of 5 × 10⁻³ M 4-aminopyridine (4-AP). Traces in A1, A2, and A3 were obtained from different cells. B: conductance-voltage (G-V) relationship of I_transient. C: G-V relationship of I_sustained. D: comparison of absolute maximum current amplitude of I_transient. E: comparison of absolute maximum current amplitude of I_sustained. F: comparison of peak current density of I_transient. G: comparison of peak current density of I_sustained. *P < 0.05; n.s., not significant.

Fig. 3.

Comparison of Na⁺ currents (I_Na) in PNs and spiking LNs in whole cell voltage-clamp configuration. A: current traces showing voltage-dependent activation of I_Na, measured during the step depolarization in cells loaded with a CsCl patch pipette internal solution and bathed in a solution containing 10⁻³ M CdCl₂, 5 × 10⁻³ M 4-AP, and 2 × 10⁻² M TEA. The representative traces shown are from spiking LNs. B: current traces showing voltage-dependent inactivation of I_Na, measured during the constant test pulse of 0 mV before the step depolarization in cells loaded with a CsCl patch pipette internal solution and bathed in a solution containing 10⁻³ M CdCl₂, 5 × 10⁻³ M 4-AP, and 2 × 10⁻² M TEA. The representative traces shown are from spiking LNs. C: I-V relationship of voltage-dependent activation of I_Na. D: I-V relationship of voltage-dependent inactivation of I_Na. The curves in C and D were fit to a first-order Boltzmann equation. E: comparison of absolute maximum current amplitude of I_Na. F: comparison of peak current density of I_Na.

Fig. 4.

Comparison of Ca²⁺ currents (I_Ca) in individual AL neurons in whole cell voltage-clamp configuration. A: current traces showing voltage-dependent activation of I_Ca, measured during the step depolarization in cells loaded with a CsCl patch pipette internal solution and bathed in a solution containing 10⁻⁷ M TTX, 5 × 10⁻³ M 4-AP, and 2 × 10⁻² M TEA. B: current traces showing tail I_Ca, measured during the step depolarization in cells loaded with a CsCl patch pipette internal solution and bathed in a solution containing 10⁻⁷ M TTX, 5 × 10⁻³ M 4-AP, and 2 × 10⁻² M TEA. C: I-V relationship of I_Ca. D: I-V relationship of tail I_Ca. The curves in C and D were fit to a first-order Boltzmann equation. E: comparison of absolute maximum current amplitude of I_Ca. F: comparison of peak current density of I_Ca. *P < 0.05.

Fig. 5.

Functional connectivity of PNs and spiking LNs. A: paired recording of a spiking LN and a PN (n = 10) showing that activation of the PN elicited depolarization responses in the paired spiking LN, whereas activation of the spiking LN induced no substantial responses in the paired PN. Ten individual trials (gray traces) were superimposed and averaged (black trace). A1: presynaptic membrane voltage of the spiking LN in response to a depolarizing current injection (top), simultaneously recorded postsynaptic membrane voltage of the PN (middle), and simultaneously recorded postsynaptic membrane voltage of the PN with additional application of 5 × 10⁻³ M Cd²⁺ (bottom). A2: presynaptic membrane voltage of the PN in response to a depolarizing current injection (top), simultaneously recorded postsynaptic membrane voltage of the spiking LN (middle), and simultaneously recorded postsynaptic membrane voltage of the spiking LN with additional application of 5 × 10⁻³ M Cd²⁺ (bottom). The data set was obtained in the same cells shown in A1. B: quantification of the change in postsynaptic membrane voltage in the paired recording provided in A. *P < 0.05.

Fig. 6.

Functional connectivity of PNs and nonspiking LNs. A: paired recording of a PN and a nonspiking LN (n = 8) showing that activation of the PN elicited depolarization responses in the paired nonspiking LN, and activation of the nonspiking LN induced long-lasting hyperpolarization responses in the PN. Eight individual trials (gray traces) were superimposed and averaged (black trace). A1: presynaptic membrane voltage of the nonspiking LN in response to a depolarizing current injection (top), simultaneously recorded postsynaptic membrane voltage of the PN (middle), and simultaneously recorded postsynaptic membrane voltage of the PN with additional application of 5 × 10⁻³ M Cd²⁺ (bottom). A2: presynaptic membrane voltage of the PN in response to a depolarizing current injection (top), simultaneously recorded postsynaptic membrane voltage of the nonspiking LN (middle), and simultaneously recorded postsynaptic membrane voltage of the nonspiking LN with additional application of 5 × 10⁻³ M Cd²⁺ (bottom). The data set was obtained in the same cells shown in A1. B: quantification of the change in postsynaptic membrane voltage in the paired recording provided in A. *P < 0.05.

Fig. 7.

Functional connectivity of spiking LNs. A: paired recording of spiking LNs (n = 13) showing synaptic transmission via chemical synapses. Thirteen individual trials (gray traces) were superimposed and averaged (black trace). A1: presynaptic membrane voltage of the spiking LN in response to a depolarizing current injection (top), simultaneously recorded postsynaptic membrane voltage of the spiking LN (middle), and simultaneously recorded postsynaptic membrane voltage of the spiking LN with additional application of 5 × 10⁻³ M Cd²⁺ (bottom). A2: the same results were obtained when presynaptic and postsynaptic sites were inverted. The data set was obtained in the same cells shown in A1. B: quantification of the change in postsynaptic membrane voltage in the paired recording provided in A. *P < 0.05.

Fig. 8.

Electrical coupling. A: electrical coupling between spiking LNs (n = 13). A1: membrane potential changes of the same polarity were induced in postsynaptic cells (bottom) by hyperpolarization and subthreshold depolarization in presynaptic cells (top). A2: the same results were obtained when presynaptic and postsynaptic sites were inverted. B: no electrical coupling between PNs and spiking LNs (n = 10). B1: Membrane potential in postsynaptic cells (bottom) by hyperpolarization and subthreshold depolarization in presynaptic cells (top). B2: the same results were obtained when presynaptic and postsynaptic sites were inverted. C: no electrical coupling between PNs and nonspiking LNs (n = 8). C1: membrane potential in postsynaptic cells (bottom) by hyperpolarization and subthreshold depolarization in presynaptic cells (top). C2: the same results were obtained when presynaptic and postsynaptic sites were inverted. D: quantification showing the coupling coefficient in paired recordings from A–C, which was determined as the ratio of the voltage response in the noninjected cell divided by the voltage response in the injected cell. The results for the hyperpolarization current in the presynaptic cell are shown at top, and the results for the depolarization current in the presynaptic cell are shown at bottom.