Double labeling of contralateral histamine-like immunoreactive neurons projecting toward the cerebral ganglionin <i>B</i>. <i>glabrata</i>.

<p><b>A:</b> Biocytin backfill of the right CBc labeled fibers in the buccal commissure (Bc) and numerous cells on the dorsal surface of the left buccal ganglion (magenta). Dashed box encloses area shown at higher magnification in panel D. <b>B</b>: Many of the histaminergic neurons on the dorsal surface of the left ganglion (green) exhibited similar sizes and locations to those labeled by the backfill. Dashed box encloses area shown at higher magnification in panel E. <b>C:</b> Merging of panels A and B demonstrates that several histaminergic neurons project to the CBc (double labeled neurons appear white). Dashed box encloses area shown at higher magnification in panel F. Calibration bar = 50 μm, applies to panels A-C. <b>D:</b> Higher magnification of central region of the left dorsal buccal ganglion. Image contains neurons that were labeled only by the backfill (diamonds) and cells that also contained histamine immunoreactive material (asterisks). <b>E:</b> Same region of the ganglion contains neurons that were labeled only by the immunohistochemistry protocol (triangles) and others that were double-labeled (asterisks). <b>F:</b> Overlay of panels D and E confirms the presence of backfilled neurons (magenta, diamonds), histaminergic neurons (green, triangles), and double-labeled cells (white, asterisks). Symbols mark representative neurons, but not all cells in each class. Calibration bar = 10 μm applies to panels D-F.</p

HA-Like immunoreactivity in the left parietal and visceral ganglia of <i>Biomphalaria alexandrina</i>.

<p><b>A:</b> Left parietal ganglion contains a single large, intensely immunoreactive neuron (large solid white arrow) on the dorsal surface near the center of the ganglion with a thick axon projecting into the underlying neuropil (small solid white arrowhead). This cell near two pairs of lightly stained, large neurons (brackets). A large dim oval cell (inside rectangle) is located anterior to the parietal-visceral connective (LPaVc) projecting an axon toward the visceral ganglion (insert). A cluster of large, lightly stained cells (asterisk) can be seen on the posterior and lateral edge of the dorsal surface. Numerous fibers run in the parietal-pleural connective (PaPlc) including some prominent, thick axons (contrasted black arrow). <b>B:</b> Visceral ganglion. A prominent intense immunoreactive, spherical, bipolar neuron (rectangle) with two main axonal projections (right insert) giving rise to small numerous sub-branches. At different focal plane, another intense monopolar cell can be noticed superficial to the previously described neuron, with axonal projection toward the parietal-visceral connective (left insert). Note along the posterior lateral edge of the visceral ganglion situated dorsally a group of small separated intense neurons (small, solid white arrowheads) in addition to a large heavily stained neuron (arrow) with noticeable axon projecting toward the ganglionic body (large, solid white arrowhead). <b>C:</b> Ventral surface of the visceral ganglion (Vg). Histamine-like immunoreactive material is present in two clusters of 4–5 large neurons, including a group of cells (30–40 μm diameter, arrow) at the anterolateral margin of the ganglion near the right parietal-visceral connective (RPaVc) and a second medial group (25–30 μm diameter, arrowhead) near the left parietal-visceral connective (LPaVc). <b>D:</b> Higher magnification of medial histminergic cluster on the ventral surface of the visceral ganglion. Stout axons (arrows) project from these cells to the LPaVc. The fibers system connecting the left parietal and visceral ganglia together through the connective and connects the two ganglia with their follower ones (<b>A, B</b> contrasted black arrowheads). The z-stack spanned the thickness of the ganglion and both dorsal and ventral cells are shown in the projection. Calibration bar = 50 μm, all panels.</p

Histamine-like immunoreactivity in the buccal ganglia of <i>Biomphalaria</i>.

<p><b>A:</b> Approximately 30 histamine-like immunoreactive neurons were present on the caudal surface of each buccal ganglion of <i>B</i>. <i>glabrata</i>. One population of neurons (stars) lay medially, and generally posterior to the buccal commissure. A single pair of larger (20–30 μm) more intensely labeled cells (arrows) flank the buccal commissure (Bc). A second population (single asterisks) formed an oblique band across each ganglion, while a third cluster (brackets) was located along the lateral margin. A few cells of a group located primarily on the ventral surface of the right ganglion are also indicated (double asterisks). The parabuccal nerves (PBn) and the esophageal trunks (Et) are rich in HA-immunoreactive fibers. Calibration bar = 50 μm. <b>B:</b> Cells comprising a medial cluster (star), central oblique band (single asterisk) and lateral population (bracket) can also be seen on the dorsal surface in a posteriorly rotated left buccal ganglion of <i>B</i>. <i>alexandrina</i>. Calibration = 50 μm. <b>C:</b> An encapsulated cluster (arrow) of immunoreactive neurons adhered to each esophageal trunk, near the ganglion. <b>D:</b> The parabuccal nerves project to the mouth region, where they branch to produce a rich innervation. Immunoreactive fibers also cover the salivary gland (arrow). Calibration bar = 100 μm.</p

HA-like immunoreactivity in the cerebral ganglia.

<p><b>A:</b> Dorsal view of the left cerebral ganglion of <i>B</i>. <i>glabrata</i>. The white arrow and double asterisks denote clusters along the lateral margin of the dorsal surface while the white arrowhead indicates an identified individual neuron at the base of the tentacular nerve (Tn). The star, single asterisk and bracket indicate ventral clusters that are not clearly in focus. <b>B:</b> Higher magnification view of area in part A indicated by the rectangle with the arrow showing an axon projecting from a cell in the lateral cluster. <b>C:</b> A deeper focus showing additional cells along the ventral surface in a similar region of another specimen of <i>B</i>. <i>glabrata</i>. <b>D:</b> Medial and, <b>E</b>: lateral regions of the right cerebral ganglia of <i>B</i>. <i>alexandrina</i>, as seen from a ventral vantage. The confocal z-stack spanned the thickness of the ganglion so that both dorsal and ventral cells are shown in the projection and many of the same cells and cell clusters indicated by stars, asterisks and double asterisks can be recognized here as described above for <i>B</i>. <i>glabrata</i>. One cluster of neurons (D, asterisk) is situated near the origin of the cerebral commissure (Cc) and another (E, star) near the cerebral-pleural connective (CPlc). The anterolateral cluster of neurons observed on the dorsal surface (A, double asterisk) continues to the anterior ventral surface (E, double asterisk) near the origin of the cerebral-buccal connective and lip nerves (out of view). Additional individual cells are located anterolaterally on the dorsal surface (E, large solid white arrows), and near the center of the ganglion on the ventral surface (D, large solid white arrows), and one solitary cell (E, small solid white arrow) at the base of the cerebral-buccal connective (diagonally above and out of view of this image). Numerous immunoreactive fibers can be observed in the cerebral commissures and connectives (large, solid white arrowheads). <b>F:</b> Ventral view of the left cerebral ganglia of <i>B</i>. <i>alexandrina</i>. Two clusters of neurons are observed, one (star) located at the origin of cerebral pleural connective (CPlc) and the other (asterisk) near the cerebral commissure (out of view to the left of this image). The subesophageal cerebral commissure contains a pair of fine immunoreactive filaments exiting the anterior ventral surface of the cerebral ganglia (contrasted black arrow). Scale bars = 50 μm, all panels.</p