A role for dopamine in the peripheral sensory processing of a gastropod mollusc

Abstract

Histological evidence points to the presence of dopamine (DA) in the cephalic sensory organs of multiple gastropod molluscs, suggesting a possible sensory role for the neurotransmitter. We investigated the sensory function of DA in the nudipleuran Pleurobranchaea californica, in which the central neural correlates of sensation and foraging behavior have been well characterized. Tyrosine hydroxylase-like immunoreactivity (THli), a signature of the dopamine synthetic pathway, was similar to that found in two other opisthobranchs and two pulmonates previously studied: 1) relatively few (<100) THli neuronal somata were observed in the central ganglia, with those observed found in locations similar to those documented in the other snails but varying in number, and 2) the vast majority of THli somata were located in the peripheral nervous system, were associated with ciliated, putative primary sensory cells, and were highly concentrated in chemotactile sensory organs, giving rise to afferent axons projecting to the central nervous system. We extended these findings by observing that applying a selective D2/D3 receptor antagonist to the chemo- and mechanosensory oral veil-tentacle complex of behaving animals significantly delayed feeding behavior in response to an appetitive stimulus. A D1 blocker had no effect. Recordings of the two major cephalic sensory nerves, the tentacle and large oral veil nerves, in a deganglionated head preparation revealed a decrease of stimulus-evoked activity in the former nerve following application of the same D2/D3 antagonist. Broadly, our results implicate DA in sensation and engender speculation regarding the foraging-based decisions the neurotransmitter may serve in the nervous system of Pleurobranchaea and, by extension, other gastropods.

Fig 1. A specimen of Pleurobranchaea californica engaged in the food-localization task.

Fig 2. Summary diagram of the distribution of tyrosine hydroxylase-like immunoreactive neurons in the central nervous system.

Somata are indicated as black circles on the dorsal (A) and ventral (B) surfaces of the cerebropleural, pedal, and buccal ganglia (modified from [38]; cells not drawn to scale). The cerebrobuccal connectives (CBC) are shown cut here for clarity. Cerebropleural ganglion abbreviations: aCPC, anterior cerebropedal connective; BWN, body wall nerve; CBC, cerebrobuccal connective; CC, cerebral commissure; CL, cerebral lobe; CVC, cerebrovisceral connective; MN, mouth nerve; OVN, oral veil nerve; PL, pleural lobe; pCPC, posterior cerebropedal connective; RN, rhinophore nerve; sBWN, small body wall nerve; SCC, subcerebral commissure; TN, tentacle nerve. Pedal ganglia abbreviations: AccL, accessory lobe; LBWN, lateral body wall nerve; pPC, parapedal commissure; PC, pedal commissure; PLL, posterior lateral lobe; pPN, posterior pedal nerve; VML, ventromedial lobe. Buccal ganglion abbreviations: R1-R3, buccal roots 1–3; SGN, stomatogastric nerve.

Fig 3. Tyrosine hydroxylase-like immunoreactivity (THli) in the cerebropleural ganglion.

(A₁) Dorsal surface: THli fibers were abundant in the rhinophore nerve (RN). A cluster of 6–8 small cerebral neurons were stained proximal to the origin of each RN (arrows). An additional pair of cells was present lateral to each RN, one of which (arrowheads) projected a prominent fiber toward the midline. THli neurons were not observed in the pleural lobe. (A₂) Ventral surface: the majority of THli neurons in the cerebral ganglion were located near the confluence of the oral veil (OVN), tentacle (TN), and mouth nerves (MN). THli fibers were abundant in each of these nerves. One cluster of 15–20 small (10–20 μm) neurons was located near the origin of each mouth nerve (arrows). Calibration bar = 400 μm, applies to A₁ and A₂. (B₁) Dorsolateral quadrant of the right cerebral ganglion. At higher magnification, the cluster of THli neurons situated at the origin of the RN was observed to be composed of heterogeneous cell bodies, with larger (30–50 μm) somata located more posterior (arrow) and smaller (10–30 μm) cells in a more anterior position (arrowhead). (B₂, B₃) Ventrolateral quadrant of the right and left cerebral ganglia, respectively. The most intensely labeled cells were present in the cluster at the base of the MN (arrows) and exhibited less diversity in size, staining intensity, and segregation than the dorsal cluster cells. Bilateral homologs in this cluster were highly symmetrical with respect to number, location, and intensity. A pair of small (15–20 μm) neurons was located posteromedial to the confluence of the TN and OVN (arrowheads), while a less brightly staining cluster of 3–5 somata lay anterior to those (asterisks). Calibration bar = 100 μm, applies to b₁-b₃. Abbreviations: aCPC, anterior cerebropedal connective; BWN, body wall nerve; CL, cerebral lobe; CVC, cerebrovisceral connective; PL, pleural lobe; pCPC, posterior cerebropedal connective; sBWN, small body wall nerve; SCC, subcerebral commissure.

Fig 4. THli in the pedal ganglia.

(A₁, B₁) Dorsal surfaces of the left and right pedal ganglia, respectively. Immunoreactive fibers were present in each of the nerves and connectives, but no THli cell bodies were observed. (A₂, B₂) Ventral surfaces of left and right pedal ganglia, respectively. THli fibers were abundant in the anterior and medial pedal nerves (aPN, mPN). Labeling was also observed in 8–10 and 4–6 small (10–30 μm) neurons at the confluence of the aPN and mPN in the left and right ganglia, respectively. Several additional small THli neurons (10–15 μm) were situated just lateral to the anterior cerebropleural connective (aCPC; arrows). Calibration bar = 200 μm, applies to A₁, A₂, B₁, and B₂. (A₃, B₃) Higher magnification of the regions enclosed by dashed boxes in A₂ and B₂, respectively. The neurons embedded in the fiber tract exhibited diverse sizes and staining intensities. Calibration bar for A₃ = 100 μm; calibration bar for B₃ = 50 μm. Abbreviations: AccL, accessory lobe; DML, dorsomedial lobe; LBWN, lateral body wall nerve; pPC, parapedal commissure; PC, pedal commissure; PPL, posterior lateral lobe; pPN, posterior pedal nerve; VML, ventromedial lobe.

Fig 5. THli in the buccal nervous system.

(A₁) Ventral view of the right buccal hemiganglion. Outline of the ganglion is marked by the dashed blue line. THli fibers coursed through the ventral commissure (VC) and through the buccal neuropil. The majority crossed the ganglion toward buccal root (R3) but several fibers projected into the first buccal root (R1). Three neurons were stained on the anterior margin of the immunoreactive fiber tract projecting into R3. The two medial neurons were contiguous (arrow). (A₂) Ventral view of the left buccal hemiganglion. Four neurons were present in the medial region of the ganglion, including a medial pair (arrow) and a lateral pair (arrowhead). There was no immunoreactivity on the dorsal surface of the ganglion. Calibration bar = 100 μm, applies to A₁ and A₂. (B₁) Higher magnification of the medial pair of THli cells in the left buccal hemiganglion. Both cells had two fibers originating from the soma, one of which projected medially (arrows) while the other projected laterally (arrowheads). Calibration bar = 40 μm. (B₂) Anterolateral region of left buccal hemiganglion, ventral view. Two stout axons were present in the cerebrobuccal connective (CBC). Calibration bar = 100 μm. (B₃) A single THli neuron was located in the stomatogastric ganglion (STG), which is demarcated by the dashed blue line. Two fibers originated from the cell soma (arrowheads), one projecting toward the periphery via the lateral gastroesophegeal nerve (LGEN) and the other toward the CNS via the stomatogastric nerve (SGN). Calibration bar = 100 μm.

Fig 6. THli in the visceral ganglion.

(A) Dorsal view of the visceral ganglion. No immunoreactive somata were observed. (B) Ventral view of the visceral ganglion. THli fibers with an anteroposterior orientation were found passing through the ventral surface of the ganglion, colocated between one of two cerebrovisceral connectives (lCVC, rCVC) and the visceralgenital connective (VGC). Calibration bar = 80 μm, applies to A and B.

Fig 7. THli in peripheral cephalic tissues.

(A₁) Immunoreactive fibers were present in each of the tentacle nerve (TN) branches. Two to three small (15–20 μm) neurons were resolved at one of the bifurcations (arrow). Calibration bar = 200 μm. (A₂) Small (5–20 μm) neurons were present in the most distal region of the tentacle ganglion (TG). Calibration bar = 100 μm. (A₃) The largest somata in the TG gave rise to fibers that projected toward the CNS via the TN. Calibration bar = 30 μm. (B₁) Numerous immunoreactive fibers were present in the peripheral branches of the lateral oral veil nerve (LOVN). (B₂) THli fibers branched repeatedly throughout the oral veil epithelium, sometimes terminating in papillae. Calibration bar = 100 μm. (B₃) Higher magnification of the area enclosed by the dashed white rectangle in panel B₂. Groups of specialized, cilia-like terminations within the papillae projecting from small somata (< 5 μm) penetrated the oral veil epithelium. Calibration bar = 30 μm.

Fig 8. The D₂/D₃ dopamine antagonist sulpiride increased latency to bite in a food-localization task.

(A) Unilateral application of sulpiride to the OVTC of Pleurobranchaea (N = 24) significantly increased the latency to bite at shrimp (two-way repeated measures ANOVA, F(1,23) = 13.28, p = 0.0014). Sulpiride increased latency to bite at stimuli placed on the experimental tentacle by 18.8 ± 3.1 s (mean ± SEM, p < 0.0001), with post-treatment latencies measured on the experimental tentacle 15.7 ± 3.1 s longer than those on the control tentacle (p = 0.0002). (B) Bite latencies did not significantly change on either side of the OVTC when food was presented to animals (N = 6) treated with the D₁ antagonist SCH-23390 (two-way repeated measures ANOVA, F(1,5) = 1.21, p = 0.321). Individual bars depict means with SEMs. ****, p < 0.0001; ***, p < 0.001; N.S. = not significant.

Fig 9. The D₂/D₃ dopaminergic antagonist sulpiride significantly reduced sensory responses to tactile stimuli measured in the tentacle nerve (TN).

(A) Stimulus-evoked activity in the TN was attenuated by treating the OVTC with sulpiride, both through immersion (weighted-means one-way repeated measures ANOVA, F(2,18) = 5.75, p = 0.0117) and painting (F(2,9) = 4.98, p = 0.0350). When measured at 5 minutes following sulpiride treatment, TN activity had decreased by 18.81 ± 16.81 (mean ± SEM; Tukey’s test, p = 0.0130) in immersed preparations and by 22.85 ± 15.68 spikes (p = 0.0288) in preparations that had been painted. Evoked activity in the TN at 60 minutes following treatment was not significantly different than pre-treatment levels but exhibited no significant recovery relative to activity elicited through stimulation at 5 minutes post-treatment. (B) Evoked activity in the LOVN was not significantly changed following sulpiride treatment in either immersed (weighted-means one-way repeated measures ANOVA, F(2,18) = 0.07, p = 0.937) or painted preparations (F(2,6) = 0.74, p = 0.517). Individual bars depict weighted means with SEMs. *, p < 0.05; N.S. = not significant. TN: N = 11 for immersion, N = 7 for painting; LOVN, N = 11 for immersion, N = 5 for painting. (C) Sets of representative electrophysiological records obtained from a single preparation demonstrate responses in the TN and LOVN to tactile stimulation before, 5 minutes after, and 60 minutes after sulpiride treatment. Shading represents the 2 s over which tactile stimulation was applied.