Evidence that the swim afferent neurons of tritonia diomedea are glutamatergic

Abstract

The escape swim response of the marine mollusc Tritonia diomedea is a well-established model system for studies of the neural basis of behavior. Although the swim neural network is reasonably well understood, little is known about the transmitters used by its constituent neurons. In the present study, we provide immunocytochemical and electrophysiological evidence that the S-cells, the afferent neurons that detect aversive skin stimuli and in turn trigger Tritonia's escape swim response, use glutamate as their transmitter. First, immunolabeling revealed that S-cell somata contain elevated levels of glutamate compared to most other neurons in the Tritonia brain, consistent with findings from glutamatergic neurons in many species. Second, pressure-applied puffs of glutamate produced the same excitatory response in the target neurons of the S-cells as the naturally released S-cell transmitter itself. Third, the glutamate receptor antagonist CNQX completely blocked S-cell synaptic connections. These findings support glutamate as a transmitter used by the S-cells, and will facilitate studies using this model system to explore a variety of issues related to the neural basis of behavior.

Figure 1

The Tritonia isolated brain preparation and the escape swim network. (A) The escape swim circuit, which includes the afferent neurons (S-cells), the pre- central pattern generator excitatory interneurons (Tr1 and DRI), central pattern generator interneurons (C2, DSI and VSI-B), and the flexion neurons (VFN and DFN). Pleural cells 5–8, located outside the swim network, are also indicated. Excitatory synapses are indicated as bars, inhibitory as circles. (B) Schematic illustration of the isolated brain preparation, depicting the positions of the S-cells, the Pleural cells 5–8 and the suction electrode placement onto Pedal Nerve 3 for the peripheral nerve stimulation used to trigger the swim motor program.

Figure 2

Glutamate immunoreactivity in S-cells. (A) Tritonia diomedea brain ganglia in the living state, pinned out before fixation. Lettered boxes indicate the specific areas depicted in the corresponding figure panels. (B) Light micrograph image of a non-immunostained 2 µm epoxy section of the left dorsal cerebro-pleural ganglion through the S-cell cluster (circled). (C) Confocal image of a 50 µm horizontal section through the left dorsal pleural ganglion. The S-cells (circled) showed strong glutamate immunolabeling in their cytoplasm. (D) Sections incubated with a solution in which the primary antibody was pre-absorbed to a glutamate-glutaraldehyde conjugate failed to show immunoreactivity (section though the S-cell cluster). (E) C1 (star), a known serotonergic cell, showed no specific immunolabeling and served as a control for antibody specificity. All calibration bars 100µm.

Figure 3

Lucifer Yellow and glutamate co-labeling of S-cells. (A) Two S-cells (yellow cells) were injected with Lucifer Yellow prior to fixation. They are shown here in the live isolated brain preparation. (B) One of the dye-filled S-cells appears in this confocal image of a 50 µm section taken through the glutamate immunolabeled dorsal pleural cluster (circle), independently confirming it as the S-cell cluster. Calibration bar 100 µm.

Figure 4

S-cell postsynaptic neurons are excited by pressure-applied glutamate to the soma. (A) Schematic illustration of the monosynaptic connection between S-cells and Pleural cells 5–8 and of the recording (left) and puffing (right) microelectrode placement. (B) Response of Pleural Cells 5–8 in normal saline. B1. A puff of 100mM glutamate elicited action potentials. B2. A puff of the saline vehicle produced no depolarizing response. (C) Pleural Cells 5–8 depolarized and fired in response to a 100 mM glutamate puff in 0 mM calcium, 10 mM cobalt saline. (D) Schematic illustration of the monosynaptic connection between S-cells and of the microelectrode placement. (E) Response of S-Cells in normal saline. E1. A puff of 100 mM glutamate elicited action potentials. E2. A puff of the saline vehicle produced no depolarizing response. (F) S-cells depolarized and fired in response to a 100 mM glutamate puff in 0 mM calcium, 10 mM cobalt saline. In all panels, the horizontal bar beneath the response indicates the duration of the puff.

Figure 5

The glutamate antagonist CNQX blocked the ability of sensory input to elicit the swim motor program. (A) Upper trace: Pedal Nerve 3 stimulation elicited a 3-cycle swim motor program, recorded in a pedal ganglion flexion neuron in normal saline. Middle trace: The motor program response was blocked during bath application of 300 µM CNQX. Lower trace: The motor program was starting to reoccur after 45 min in wash. (B) Group data for the experiment (n = 5 preparations).

Figure 6

CNQX blocked an S-cell synapse. (A) Schematic illustration of the monosynaptic S-cell to Pleural cell 5–8 connection, together with the stimulating (left) and recording (right) microelectrode placement. (B) Intracellular recording showing elimination of the S-cell-to-Pl 5–8 EPSP before (Saline), and again after 8 min in CNQX. In the drug, the EPSPs are eliminated, with only the stimulus artifacts remaining. In each 2 minute test, an impaled S-cell was stimulated with intracellular current to fire for 1 s at 10 Hz. Just the first three EPSPs of the test trains are shown. (C) Mean amplitude of the first EPSP in each train, normalized to its amplitude in the first pretest train, for CNQX (black circles, n = 5 preparations) and control experiments (white circles, n = 5 preparations). In the CNQX group, the mean normalized EPSP was blocked during the 10 min bath application of 100 µM CNQX as compared to the control group. Figure plots are expressed as means ± S.E.M.