Distribution of Serotonin-Immunoreactive Neurons in the Brain and Gnathal Ganglion of Caterpillar Helicoverpa armigera

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) is an important biogenic amine that acts as a neural circuit modulator. It is widespread in the central nervous system of insects. However, little is known about the distribution of serotonin in the nervous system of the cotton bollworm Helicoverpa armigera. In the present study, we performed immunohistochemical experiments with anti-serotonin serum to examine the distribution of serotonin in the central nervous system of H. armigera larvae. We found about 40 serotonin-immunoreactive neurons in the brain and about 20 in the gnathal ganglion. Most of these neurons are wide-field neurons giving rise to processes throughout the neuropils of the brain and the gnathal ganglion. In the central brain, serotonin-immunoreactive processes are present bilaterally in the tritocerebrum, the deutocerebrum, and major regions of the protocerebrum, including the central body (CB), lateral accessory lobes (LALs), clamps, crepine, superior protocerebrum, and lateral protocerebrum. The CB, anterior ventrolateral protocerebrum (AVLP), and posterior optic tubercle (POTU) contain extensive serotonin-immunoreactive process terminals. However, the regions of mushroom bodies, the lateral horn, and protocerebral bridges (PBs) are devoid of serotonin-immunoreactivity. In the gnathal ganglion, the serotonin-immunoreactive processes are also widespread throughout the neuropil, and some process projections extend to the tritocerebrum. Our results provide the first comprehensive description of the serotonergic neuronal network in H. armigera larvae, and they reveal the neural architecture and the distribution of neural substances, allowing us to explore the neural mechanisms of behaviors by using electrophysiological and pharmacological approaches on the target regions.

Keywords: Helicoverpa armigera; brain; commissure; gnathal ganglion; immunoreactivity; neuropils; serotonin; wide-field neurons.

Figure 1

Confocal images and three-dimensional reconstruction of the brain and the gnathal ganglion of Helicoverpa armigera larvae. (A) Confocal image showing the neuropils of the brain and the gnathal ganglion. (B) Confocal image showing the serotonin-immunoreactive neurons in the brain and the gnathal ganglion. (C) Merged confocal image showing the neuropil (magenta) and the serotonin-immunoreactive neurons (green). (D) Three-dimensional reconstruction of the brain and the gnathal ganglion. Con, connective; GNG, gnathal ganglion. Scale bars, 100 μm.

Figure 2

Brain composition of H. armigera larvae. (A) Three-dimensional reconstuctions of one brain hemisphere in frontal view; (B) in posterior view; (C) in lateral view; (D) in sagittal view. α, alpha lobe; AL, antennal lobe; AMMC, antennal mechanosensory and motor center; AVLP, anterior ventrolateral protocerebrum; β, belta lobe; CA, calyx; CB, central body CL, clamp; CRE, crepine; GOR, gorget; IB, inferior bridge; ILP, inferior lateral protocerebrum; LAL, lateral accessory lobe; LH, lateral horn; LOB, mushroom body lobes; OL, optic lobe; PB, protocerebral bridge; PLP, posterior lateral protocerebrum; POTU, posterior optic tubercle; PS, posterior slope; PVLP, posterior ventrolateral protocereburm; SIP, superior intermediate protocerebrum; SLP, superior lateral protocerebrum; SMP, superior medial protocerebrum; TR, tritocerebrum. Directions: a, anterior; d, dorsal; l, lateral; m, medial; p, posterior, v, ventral. Scale bar, 100 μm.

Figure 3

Serial confocal images from anterior to posterior showing the serotonin- immunoreactive neurons (green) and the brain neuropils (magenta) of Helicoverpa armigera larvae. (A) Confocal image of the brain section at a depth of 21 μm of total 138 μm; (B) at 42 μm; (C) at 57 μm; (D) at 72 μm. Double arrows indicate the processes from the cells in the cluster of PR-M extending to the AVLP; (E) at 78 μm. Arrowheads indicate the processes of the cell in the cluster of DE-L; (F) at 84 μm; (G) at 96 μm. Arrowhead indicates the processes of cells in the cluster of PR-L; (H) at 111 μm. α, alpha lobe; AL, antennal lobe; AMMC, antennal mechanosensory and motor center; AVLP, anterior ventrolateral protocerebrum; β, belta lobe; CA, calyx; CB, central body; CL, clamp; CRE, crepine; GOR, gorget; IB, inferior bridge; ILP, inferior lateral protocerebrum; LAL, lateral accessory lobe; LOB, mushroom body lobes; OL, optic lobe; PB, protocerebral bridge; PLP, posterior lateral protocerebrum; POTU, posterior optic tubercle; PS, posterior slope; PVLP, posterior ventrolateral protocereburm; SIP, superior intermediate protocerebrum; SLP, superior lateral protocerebrum; SMP, superior medial protocerebrum; TR, tritocerebrum. DE-L, PR-A, PR-L, PR-LD, PR-M, and TR-A are cell clusters. 1, the posterior protocerebral commissure linking the POTU; 2, the median protocerebral commissure linking the CB and bilateral SIP; 3, the posterior great commissure linking the bilateral AVLP and the CL; 4, the LAL commissure; 5, the dorsal protocerebral commissure linking the bilateral AL and the SLP; 6, the anterior protocerebral commissure linking the bilateral tritocerebrum. Scale bar, 100 μm.

Figure 4

Confocal image and three-dimensional reconstructions showing the distribution of the serotonin-immunoreactive neuronal processes and cell bodies in the brain. (A) Projection view of the confocal stack images. (B) Three-dimensional reconstructions of the brain including the labeled cell body clusters in frontal view. (C) Three-dimensional reconstructions of the brain including labeled cell body clusters in sagittal view. (D) Reconstructed skeleton trees of the thick neuronal processes showing their projection patterns in frontal view. (E) Reconstructed skeleton trees of 1–6 commissures in frontal view. (F) Reconstructed skeleton trees of 1–6 commissures in dorsal view. AL, antennal lobe; AVLP, anterior ventrolateral protocerebrum; CA, calyx; LAL, lateral accessory lobe; TR, tritocerebrum. DE-L, PR-A, PR-L, PR-LD, PR-M, and TR-A are cell clusters. 1, the posterior protocerebral commissure linking the POTU; 2, the median protocerebral commissure linking the CB and bilateral SIP; 3, the posterior great commissure linking the bilateral AVLP and the CL; 4, the LAL commissure; 5, the dorsal protocerebral commissure linking the bilateral AL and the SLP; 6, the anterior protocerebral commissure linking the bilateral tritocerebrum. Directions: a, anterior; d, dorsal; l, lateral; p, posterior; v, ventral. Scale bars, 100 μm.

Figure 5

Confocal images and three-dimensional reconstructions showing the distribution of the serotonin-immunoreactive neuronal processes and cell bodies in the protocerebrum. (A) Reconstructed skeleton trees of PR-M neurons in frontal view. (B) Reconstructed skeleton trees of PR-M neurons in dorsal view. (C) Confocal image showing the serotonin-immunoreactive processes (green) in the POTU. (D) Three-dimensional reconstructions of serotonin-immunoreactive neurons in the cluster of PR-M1 and related neuropils in frontal view. (E) Three-dimensional reconstructions of the serotonin-immunoreactive neurons of PR-M1 and related neuropils in lateral view. (F–H) Confocal images showing the serotonin-immunoreactive processes of neurons PR-M2 (green). (I) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-M2 and related neuropils in frontal view. (J) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-M2 and related neuropils in lateral view. (K–M) Confocal images showing the serotonin-immunoreactive processes of neurons PR-M3 (green). (N) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-M3 and related neuropils in frontal view. (O) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-M3 and related neuropils in lateral view. α, alpha lobe; AVLP, anterior ventrolateral protocerebrum; β, belta lobe; CA, calyx; CB, central body; CL, clamp; CRE, crepine; LAL, lateral accessory lobe; PB, protocerebral bridge; PED, pedunculus; POTU, posterior optic tubercle; PVLP, posterior ventrolateral protocereburm; SIP, superior intermediate protocerebrum; SMP, superior medial protocerebrum. PR-M1-3 are cell clusters. 2, the median protocerebral commissure linking the CB and bilateral SIP; 6, the anterior protocerebral commissure linking the bilateral tritocerebrum. Directions: a, anterior; d, dorsal; l, lateral; m, medial; p, posterior; v, ventral. Scale bars, 50 μm.

Figure 6

Confocal images and three-dimensional reconstructions showing the distribution of the serotonin-immunoreactive neuronal processes and cell bodies in the cluster of PR-L and PR-A in the protocerebrum. (A–C) Confocal images showing the serotonin-immunoreactive processes of neurons PR-L (green). (D) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-L and related neuropils in frontal view. (E) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-L and related neuropils in lateral view. (F) Confocal image showing the serotonin-immunoreactive processes of neurons PR-A (green). (G) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-A and related neuropils in frontal view. (H) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of PR-A and related neuropils in lateral view. α, alpha lobe; AL, antennal lobe; AVLP, anterior ventrolateral protocerebrum; β, belta lobe; CA, calyx; CB, central body; LAL, lateral accessory lobe; PED, pedunculus; PLP, posterior lateral protocerebrum; POTU, posterior optic tubercle; PVLP, posterior ventrolateral protocereburm; SLP, superior lateral protocerebrum. PR-A and PR-L are cell clusters. 4, the LAL commissure. Directions: a, anterior; d, dorsal; l, lateral; m, medial; p, posterior; v, ventral. Scale bars, 50 μm.

Figure 7

Confocal images and three-dimensional reconstructions showing the distribution of serotonin-immunoreactive neuronal processes in the protocerebrum, deutocerebrum and tritocerebrum. (A–D) Confocal images showing the serotonin-immunoreactive processes of neurons DE-L (green). (E) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of DE-L and related neuropils in frontal view. (F) Three-dimensional reconstructions of the serotonin-immunoreactive neurons in the cluster of DE-L and related neuropils in lateral view. (G,H) Confocal images showing the serotonin-immunoreactive processes in the tritocerebrum (green). (I) Confocal image showing the serotonin-immunoreactive processes in the tritocerebrum and the frontral ganglion (green). (J) Three-dimensional reconstructions of the serotonin-immunoreactive processes in the tritocerebrum and related neuropils in frontal view. (K) Three-dimensional reconstructions of serotonin-immunoreactive processes in the triotocerebrum in lateral view. α, alpha lobe; AL, antennal lobe; β, belta lobe; CA, calyx; FC, frontal connective; FG. Frontal ganglion; PED, pedunculus; SLP, superior lateral protocerebrum; SMP, superior medial protocerebrum; TR, tritocerebrum. DE-L are cell bodies. 5, the dorsal protocerebral commissure linking the bilateral AL and the SLP. Directions: a, anterior; d, dorsal; l, lateral; m, medial; p, posterior; v, ventral. Scale bars, 50 μm.

Figure 8

Confocal images and three-dimensional reconstructions showing the distribution of the serotonin-immunoreactive neuronal processes and cell bodies in the gnathal ganglion. (A) Projection view of confocal stack images of the gnathal ganglion. Serotonin-immunoreactive neurons in green and the surrounding neuropil in magenta. (B–D) Serial confocal images of the gnathal ganglion at different depths. (E) Three-dimensional reconstructions of the gnathal ganglion including labeled cell body clusters in frontal view. Arrow in (C) indicated the dorsal commissure formed by neurons of GNG-L2. (F) Three-dimensional reconstructions of the gnathal ganglion including the labeled cell body clusters in lateral view. (G) Reconstructed skeleton trees of the thick neuronal processes showing their projection patterns in frontal view. Arrow indicates the dorsal commissure formed by neurons of GNG-L2. (H) Reconstructed skeleton trees of the thick neuronal processes showing their projection patterns in lateral view. Con, connective; GNG-L1–L3, GNG-AD, GNG-AV, and GNG-M are cell clusters. 1, the commissure formed by neurons of GNG1; 2, the commissure formed by neurons of GNG-L2; 3, the commissure formed by neurons of GNG-L3. Directions: a, anterior; d, dorsal; l, lateral; p, posterior; v, ventral. Scale bars, 100 μm.