Regeneration of a central synapse restores nonassociative learning

Abstract

Sensitization is a form of nonassociative learning in which a strong or noxious stimulus persistently enhances the response produced by a weaker stimulus. In the leech Hirudo medicinalis, the S-interneuron is required for sensitization of the shortening response. A single S-cell axon was surgically separated from its sole synaptic partner, the neighboring S-cell. This consistently eliminated sensitization without impairing reflexive shortening itself, as measured in semi-intact specimens. Sensitization of the shortening reflex returned after 3 weeks when the severed axon grew and regenerated its specific electrical synapse within the nerve cord, as shown by restored conduction of impulses between S-cells. This confirms the essential role of one neuron, the S-cell, in sensitization, and it demonstrates that regeneration of the synapse between S-cells restores this example of nonassociative learning.

Fig. 1.

Elements of the sensory motor reflex, including sensory and motor connections with the S-cell. ⊣, Excitatory synaptic connections, both chemical and rectifying electrical; •, strong, nonrectifying electrical connections. The touch (T) cells excite the S-cell through a disynaptic pathway, with coupling interneurons (C) interposed. The S-cell in each ganglion is linked to the S-cells in the neighboring ganglia (shown without shading, distances not drawn to scale). The synapses are electrical. The S-cell and the T, pressure (P), and nociceptive (N) sensory neurons each excite the L motor neuron, innervating longitudinal musculature, thereby shortening the leech. In addition, there are apparently connections of P and N cells with the S-cell (not shown) (Shaw and Kristan, 1995), perhaps indirectly through C cells.Subscripts refer to the number of cells of each type within the ganglion.

Fig. 2.

Schematic diagram of the semi-intact leech preparation, dorsal view, showing tension transducer attached to the anterior (top) and placement of stimulating electrodes on skin. The test and habituating stimuli were delivered to the anterior region of the animal, whereas sensitizing stimuli were delivered to the posterior region, as shown. Placements of pins, including one in the posterior sucker, are also indicated. The S-cell axon was cut anterior to ganglion 7, between 6 and 7, which in the figure is at the anterior margin of the posterior portion of the animal.

Fig. 3.

Extracellular stimulation and recording demonstrate disconnection and reconnection of S-cells.A, Diagram of preparation for recording, depicting locations of suction electrodes at the ends of the connectives, an intracellular microelectrode in S₈, and the lesion site in the medial connective (open arrow) anterior to ganglion 7. B, For short-term specimens, 5 d after surgery, the impulses elicited in the S-cell by stimulating the connectives extracellularly with the posterior suction electrode did not propagate across the lesion to the anterior suction electrode. Stimulus artifact is marked (•), and the usual location of the propagated extracellular action potential, missing here, is marked (★). C, For long-term specimens, here 78 d, the S-cell impulses propagated across the lesion. In D, depolarization of S₈ produced a pair of impulses that were also recorded extracellularly. The recording sites on the diagram inA are linked by dashed lines to the corresponding voltage traces in B–D. Calibration bars at right for B and C, 1 mV, extracellular trace; 10 mV, intracellular trace; and 10 msec.

Fig. 4.

Impairment of sensitization of the shortening reflex by S-cell axon lesions. Shown is the mean percentage of initial contraction for leeches in the experimental (lesion), sham (control), and habituation–control groups across habituation training 6–8 d after cutting the S-cell axon. Leeches in the experimental and control groups experienced a sensitizing stimulus before the onset of repeated presentations of the weak, habituating stimulus, whereas leeches in the habituation–control group did not experience a sensitizing stimulus. Error bars indicate SEM.

Fig. 5.

Restoration of behavioral sensitization after regeneration of the S-cell axon. Mean percentage of initial contraction for leeches in experimental (lesion), sham (control) and habituation–control groups across habituation training 3–6 weeks after lesions. The performance of leeches in the experimental group is not different from that in the sham group. They show significant sensitization compared with the habituation–control group, which did not experience the sensitizing stimulus.