Quantitation of contacts among sensory, motor, and serotonergic neurons in the pedal ganglion of aplysia

Abstract

Present models of long-term sensitization in Aplysia californica indicate that the enhanced behavioral response is due, at least in part, to outgrowth of sensory neurons mediating defensive withdrawal reflexes. Presumably, this outgrowth strengthens pre-existing connections by formation of new synapses with follower neurons. However, the relationship between the number of sensorimotor contacts and the physiological strength of the connection has never been examined in intact ganglia. As a first step in addressing this issue, we used confocal microscopy to examine sites of contact between sensory and motor neurons in naive animals. Our results revealed relatively few contacts between physiologically connected cells. In addition, the number of contact sites was proportional to the amplitude of the EPSP elicited in the follower motor neuron by direct stimulation of the sensory neuron. This is the first time such a correlation has been observed in the central nervous system. Serotonin is the neurotransmitter most closely examined for its role in modulating synaptic strength at the sensorimotor synapse. However, the structural relationship of serotonergic processes and sensorimotor synapses has never been examined. Surprisingly, serotonergic processes usually made contact with sensory and motor neurons at sites located relatively distant from the sensorimotor synapse. This result implies that heterosynaptic regulation is due to nondirected release of serotonin into the neuropil.

Figure 1

Schematic representation of tail sensory and motor neurons in the central nervous system of Aplysia. The somata of tail sensory neurons lie in the ventrocaudal cluster of the pleural ganglion. Sensory neurons, one of which is illustrated here (red), project through the pleural–pedal connective into the pedal ganglion. Monosynaptic contacts are formed with one or more tail motor neurons (green), whose cell bodies are located in the pedal ganglion. Both neurons project to the tail through peripheral nerve P9. The pleural abdominal (Pl-Ab), pleural–cerebral (Pl-C), and pedal–cerebral (Ped-C) connectives are shown for orientation.

Figure 2

Identification of potential sites of contact between tail sensory and motor neurons. (A) Confocal image through a region of the pedal ganglion containing branches of both the sensory neuron (red) and the motor neuron (green). The image was calculated by projecting through a stack of 43 optical sections. Although there are numerous branches from both neurons, relatively few potential contacts (yellow) were observed. One of these (arrowhead) was examined more closely in subsequent panels. Scale bar, 20 μm. (B) Contacts are more reliably observed by analyzing a subset of the data. This image was calculated by projecting through six optical sections surrounding the site of a potential contact (arrowhead). The scale is the same as A. (C) A small volume surrounding the potential contact from the image stack in B (six optical sections) was then rotated around the vertical axis. Examples from 0°, 30°, 60°, 90°, 120°, and 150° angles are shown. The yellow zone of contact is observed in all panels, confirming proximity. Scale bar in the 0 degree projection, 4 μm. Because the voxels are rectangular (see Materials and Methods), the scale bar corresponds to 10 μmfor the 90° projection. (D) A single optical section from the stack illustrated in A. In this case, the site of contact (arrowhead) is still observed as yellow pixels. Scale bar, 4 μm.

Figure 3

Quantification of contacts between tail sensory neurons and follower motor neurons. (A) The total number of contact sites were counted from serial sections through the extent of the pedal ganglion. More contacts were found between pairs of physiologically connected neurons (+) than between neurons that were not connected (-; 24 ± 4.2 vs. 0.7 ± 0.4; Mean ± SEM). This difference was statistically significant (t₁₄ = 4.94, p < 0.001). (B) In physiologically connected neurons, the number of contact sites was proportional to the amplitude of the EPSP (Spearman r = 0.79, p < 0.05, n = 7). This result indicates that the number of contacts is a reasonable estimate of the number of synapses, even if the presence of an active zone cannot be confirmed at each contact. The regression line was constrained to pass through (0,0).

Figure 4

Localization of serotonergic contacts with sensory and motor neurons. (A) Sections with labeled sensory and motor neurons were stained with antibody directed against 5-HT, the presumed modulatory transmitter of the sensorimotor synapse. This image is a projection through 16 optical sections. As in Figure 2, contacts between sensory (red) and motor (green) neurons appear yellow (large arrowhead). Serotonergic fibers appear blue. In the projection, apparent contacts between sensory neurons and 5-HT-immunoreactive processes appear magenta (arrow). Contacts between motor neurons and 5-HT immunoreactive processes appear cyan. In this print, magenta and cyan may be difficult to distinguish from red and green, respectively. The straight-line distance between the sensorimotor contact and the nearest serotonergic–sensory contact is indicated by the dashed line, and measures 25 μm in this case. Scale bar, 10 μm. (B) A subset of the image stack shown in A (16 optical sections) was examined by rotation around the vertical axis. The contact between the sensory and motor neuron (arrowhead) was indicated by presence of overlap (yellow pixels, arrowhead) from all directions, confirming that the two points are, indeed, in close proximity. Examples from images rotated 0°, 30°, 60°, 90°, 120°, and 150° are shown. Scale bar in the 0 degree projection, 5 μm. Because the voxels are rectangular (see Materials and Methods), the scale bar corresponds to 12.5 μm for the 90° projection. (C) The contact between serotonergic and sensory neurites (arrow) in A was also examined following rotation. Note that in this field, there is another apparent contact (dashed line), but on rotation the neurites were clearly not in proximity. Same scale as B.

Figure 5

Serotonergic inputs were not in close proximity to SN–MN contacts. (A) The straight-line distance between a sensorimotor contact and the closest serotonergic–sensory neuron (5-HT–SN) contact. (B) The straight-line distance between a sensorimotor contact and the closest serotonergic–motor neuron (5-HT–MN) contact. (C) The straight-line distance between a sensorimotor contact and the closest serotonergic process.