Two modulatory inputs exert reciprocal reinforcing effects on synaptic input of premotor interneurons for withdrawal in terrestrial snails

Abstract

A cluster of serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in modulation of withdrawal behavior, and to be necessary for elaboration of aversive withdrawal conditioning in intact snails. In the present experiments local extracellular stimulation of the serotonergic cells elicited a pairing-specific increase (difference between paired and explicitly unpaired sessions was significant, P<0.01) of synaptic responses in the premotor interneurons involved in withdrawal to paired nerve stimulation. Intracellular stimulation of only one Pd4 cell from the pedal group of serotonergic neurons increased (P<0.05) synaptic responses to contingent test nerve stimulation significantly in the same premotor interneurons for 2-3 hr. Mesocerebral cells are known to participate in male sexual behavior, and their extracellular stimulation was shown previously to suppress the amplitude of synaptic responses in withdrawal interneurons. Local extracellular stimulation of the mesocerebral cells elicited a pairing-specific decrease (P<0.01) of synaptic responses to contingent test nerve stimulation in the premotor interneurons involved in withdrawal for 2-3 hr. Paired application of met-enkephaline (10(-6) M, some mesocerebral cells are enkephaline-like immunoreactive) also selectively decreased synaptic responses to contingent nerve stimulation in the premotor interneurons for hours. Thus, two modulatory inputs exert pairing-specific effects that influence the same synaptic connection in opposite directions, which may underlie the long-term up- and down-regulation of behavioral responses.

Figure 1

Properties of synaptic input to the premotor parietal interneurons. (A) Schematic representation of protocol of the experiment; (B) averaged amplitude of responses to test stimuli via intestinal nerve (n.int.) or second cutaneous nerve (n.cut.) before and 2 min after extracellular stimulation of mesocerebrum; open bars represent the EPSP before mesocerebral cell stimulation; hatched bars represent the EPSP after stimulation. (C) Dynamics of habituation to the independent test stimuli to two nerves without the contingent stimulation.

Figure 2

(A) Averaged changes (mean ± s.e.m.) in amplitude of complex EPSPs evoked in parietal giant neurons by stimulation of intestinal nerve (n.int.) paired and second cutaneous nerve (n.cut) explicitly unpaired with extracellular activation (arrows) of mesocerebral cells. (B) Same protocol of experiment, but n.cut. was paired. Initial response was taken in all experiments as 100%. Arrows mark paired trials. (C) Examples of complex EPSPs in right parietal neuron 2 in response to test stimuli to n.int. (left, unpaired) and n.cut. (right, paired procedure) before and after pairing (solid and dotted lines, respectively); the example is taken from one of experiments averaged in B. Stimulus artifact is seen. Here and in Figs. 3–5: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, Mann–Whitney rank sum test.

Figure 3

Averaged changes (mean ± s.e.m.) in amplitude of complex EPSPs evoked in parietal giant neurons by paired stimulation of intestinal nerve (n.int.) with bath application of metenkephalin. Stimulation of second cutaneous nerve (n.cut.) was explicitly unpaired with application of met-enkephalin.

Figure 4

Averaged changes (mean ± s.e.m.) in amplitude of complex EPSPs evoked in parietal giant neurons by paired stimulation of intestinal nerve (n.int.) and stimulation of second cutaneous nerve (n.cut.) explicitly unpaired with extracellular activation (arrows) of serotonergic pedal cells. Initial response was taken in all experiments as 100%. (*) P < 0.05; (**) P < 0.01, Mann–Whitney rank sum test.

Figure 5

Changes in amplitude of complex EPSPs evoked in parietal giant neurons by stimulation of intestinal nerve (n.int.) paired and explicitly unpaired with intracellular activation of cell Pd4. (A) Diagram of experiment; (B) scheme of recording and experimental setup; (C) averaged results (mean ± s.e.m.) (*) P < 0.05; (**) P < 0.01, Mann–Whitney rank sum test. (▾) Paired; (▿) unpaired. Initial response was taken in all experiments as 100%. The training session is marked by breaks in axes and an arrow.