Dopamine- and Tyrosine Hydroxylase-Immunoreactive Neurons in the Brain of the American Cockroach, Periplaneta americana.

The catecholamine dopamine plays several vital roles in the central nervous system of many species, but its neural mechanisms remain elusive. Detailed neuroanatomical characterization of dopamine neurons is a prerequisite for elucidating dopamine's actions in the brain. In the present study, we investigated the distribution of dopaminergic neurons in the brain of the American cockroach, Periplaneta americana, using two antisera: 1) an antiserum against dopamine, and 2) an antiserum against tyrosine hydroxylase (TH, an enzyme required for dopamine synthesis), and identified about 250 putatively dopaminergic neurons. The patterns of dopamine- and TH-immunoreactive neurons were strikingly similar, suggesting that both antisera recognize the same sets of "dopaminergic" neurons. The dopamine and TH antibodies intensively or moderately immunolabeled prominent brain neuropils, e.g. the mushroom body (memory center), antennal lobe (first-order olfactory center) and central complex (motor coordination center). All subdivisions of the mushroom body exhibit both dopamine and TH immunoreactivity. Comparison of immunolabeled neurons with those filled by dye injection revealed that a group of immunolabeled neurons with cell bodies near the calyx projects into a distal region of the vertical lobe, which is a plausible site for olfactory memory formation in insects. In the antennal lobe, ordinary glomeruli as well as macroglomeruli exhibit both dopamine and TH immunoreactivity. It is noteworthy that the dopamine antiserum labeled tiny granular structures inside the glomeruli whereas the TH antiserum labeled processes in the marginal regions of the glomeruli, suggesting a different origin. In the central complex, all subdivisions excluding part of the noduli and protocerebral bridge exhibit both dopamine and TH immunoreactivity. These anatomical findings will accelerate our understanding of dopaminergic systems, specifically in neural circuits underlying aversive memory formation and arousal, in insects

Location of dopaminergic cell groups in the brain of <i>Periplaneta Americana</i>.

<p>Location of dopaminergic cell groups in the brain of <i>Periplaneta Americana</i>.</p

Dopamine immunoreactivity in the deutocerebrum and medial antennal lobe tract.

<p>Dopamine immunoreactivity is shown in magenta (A, D, G, J) and synapsin immunoreactivity in green (B, E, H, K). Corresponding merged images (C, F, I, L). <b>A-C</b>: Frontal sections though the antennal lobe (AL). Antennal lobe glomeruli (G in inset of B) are innervated by moderately immunolabeled fine fibers, which arise from local interneurons (LNs in inset of A) of the DAL cell cluster. <b>D-F</b>: Horizontal sections through the antennal lobe. Dopamine-immunoreactive local interneurons arborize to bear fine fibers (arrow). <b>G-I</b>: Frontal sections through the medial antennal lobe tract (MALT) containing a pair of dopamine-positive fibers (arrowheads). <b>J-L</b>: Frontal sections though the lobus glomerulatus (LG) and antennal mechanosensory and motor center (AMMC). A dorso-lateral part of the AMMC, and ventral and dorso-medial region of the lobus glomerulatus receive dopamine-positive fibers. D, dorsal; M, medial; P, posterior. Scale bars = 100 μm.</p

Dopamine immunoreactivity in the central complex and lateral accessory lobe.

<p>A-L: Frontal sections. M-O: Horizontal sections. Dopamine immunoreactivity shown in magenta (A, D, G, J, M) and synapsin immunoreactivity in green (B, E, H, K, N). Corresponding merged images in C, F, I, L, O. <b>A-C</b>: Frontal sections through the central body (CB) and lateral accessory lobe (LAL). The upper and lower divisions of the central body (CBU and CBL, respectively) display intense dopamine immunoreactivity. Immunoreactive fibers innervate the dorsal and ventral shell of the lateral accessory lobe (DS and VS, respectively) and parts of the pedunculus (PED; arrowheads in A,C). A bundle of fibers connecting the central body to the lateral accessory lobe also exhibits dopamine immunoreactivity (arrows in A,C). <b>D-F</b>: Frontal sections through the anterior lip (ANL) innervated by dense dopamine-immunoreactive fibers. Labeled fibers are also seen in the superior medial protocerebral neuropil (SMP). <b>G-I</b>: Frontal sections through the central body and medial lobe (ML). Immunoreactive fibers are widely distributed in both structures, except for part of the medial lobe indicated by brackets in G. The immuno-negative region is flanked by a dense meshwork of dopamine-immunoreactive fibers. Double arrow in G indicates a plexus of immunoreactive processes in the posterior groove, originating from DP2 neurons. <b>J-L</b>: Frontal sections through the posterior portion of the central body. DP1 cell bodies (arrowheads) extend their axons ventrally, and a part of the noduli (NO) display dopamine immunoreactivity. <b>M-O</b>: Horizontal sections through the protocerebral bridge (PB). DP2 clusters (indicated by triangles) extend their axons anteriorly. Immunoreactive fibers bypass the protocerebral bridge, which lacks dopamine immunolabeling. D, dorsal; ON, ocelluar nerve; P, posterior. Scale bars = 100 μm.</p

Dopamine-immunoreactivity in the mushroom body.

<p>All panels show a right brain hemisphere (dorsal to the top, medial to the right), except M-O (posterior to the top). Dopamine immunoreactivity is shown in magenta (A, D, G, J, M) and synapsin immunoreactivity in green (B, E, H, K, N). Corresponding merged images in C, F, I, L, O. <b>A-C</b>: Frontal sections through the lateral protocerebrum. DCa1 and 2 clusters are visible. <b>D-F</b>: Frontal sections through the vertical lobe. The distal part of the vertical lobe (VL) as well as the superior protocerebral neuropil are innervated by numerous dopamine-immunoreactive fibers. <b>G-I</b>: Horizontal sections through the tip of the vertical lobe, where dopamine-positive fibers are widely distributed. Dopamine-positive fibers in the calyx were faintly labeled by fluorescent immunolabeling technique (arrowheads in G) while those fibers were visualized by the HRP-mediated technique (G’), possibly because of the low content of dopamine. <b>J-L</b>: Horizontal sections through the calyx, ventral to panels G-I. DCa1 cell bodies project axons toward the anterior face of the vertical lobe bypassing the medial margin (arrowheads). These axons bear small fibers that invade layers of the vertical lobes that are immunolabeled by a synapsin antibody. DSP1 cell bodies are clustered in a region lateral to the vertical lobe. The anteriormost layer (γ layer) is predominantely innervated by immunoreactive fibers. <b>M-O</b>: Horizontal sections through the medial lobes (MLs). A band of dopamine-immunoreactive fibers invades the tip of the medial lobe parpendicularly to synapsin-positive layers (arrows). In a very marginal region around the midline, dopamine-positive fibers follow synapsin-positive layers. The DIP1 cell clusters are located in the inferior medial protocerebrum (IMP) anterior to the medial lobe (triangles). D, dorsal; KFL, Kenyon fiber layer; LCA, lateral calyx; LO, lobula; M, medial; MCA, medial calyx; NL, neuropil layer; P, posterior; SMP, superior medial protocerebrum; γ, γ layer; VLP, ventro-lateral protocerebrum. Scale bars = 100 μm.</p

Diagram depicting cell body locations and the principal fiber trajectory of dopaminergic neurons in the American cockroach.

<p>Solid lines indicate confirmed pathways, and dotted lines in color are putative pathways. All abbreviations for neuropil structures are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160531#pone.0160531.g002" target="_blank">Fig 2</a>.</p

Diagram in a frontal plane showing the distribution of dopamine-immunoreactive cell bodies in the brain of the cockroach, <i>Periplaneta americana</i>.

<p>Cell bodies in the anterior cell body rind in the brain are shown in black while those in the posterior are in outline. Most are clustered: DP0-2, three groups in the pars intercerebralis (PI); DCa1-2; two groups lateral to the calyx; DSP1-2, two groups in the superior lateral protocerebrum; DIP1-3, three groups in the inferior protocerebrum; DAL, a group near the antennal lobe (AL); DD1-2, two groups in the deutocerebrum; DPOT, a group near the posterior optic tract (POT); DPOTu, a group near the posterior optic tubercle. CBL, central body lower division; CBU, central body upper division; LCA, lateral calyx; MCA, medial calyx; ML, medial lobe; PED, pedunculus; TR, tritocerebrum; VL, vertical lobe.</p

Antibodies used for immunocytochemistry.

<p>Antibodies used for immunocytochemistry.</p

Structure of the central complex and associated neuropils.

<p><b>A</b>: Diagram of a frontal section of the central complex and lateral accessory lobe (LAL), the latter further subdivided into the dorsal shell (DS) and ventral shell (VS). Dotted lines indicate a pair of globular noduli (NO) posterior to the central body (CB). The central body comprises the upper division (CBU) and the lower division (CBL). Each nodulus consists of four subunits (I-IV, see panel B). <b>B</b>: Diagram of a sagittal section of the central complex. The protocerebral bridge (PB) is situated above the central body. The anterior lip (ANL) is located ventro-anteriorly to the lower division of the central body. C-G: Bodian-stained brain sections. <b>C</b>: Frontal section through the upper division of the central body and the lateral accessory lobes. Arrows indicate fibers connecting the central body and the lateral accessory lobe. <b>D</b>: Frontal section through the anterior lip and the medial lobe (ML). <b>E</b>: Frontal sections through the protocerebral bridge. Longitudinal fibers span the midline. <b>F</b>: Frontal section through the central body. The upper division is divided into eight subunits called slices, four per hemisphere. <b>G, H</b>: Sagittal sections through the central body. A bundle of fibers connects a subunit IV of the nodulus (triangle) to lower division of the central body (arrowheads in H). PCH, posterior chiasma; PG, posterior groove; SMP, superior medial protocerebrum. Scale bars = 100 μm in C, D, F; 50 μm in E, G, H.</p

Tyrosine hydroxylase immunoreactivity in the deutocerebrum and medial antennal lobe tract.

<p>Tyrosine hydroxylase (TH) immunoreactivity is shown in magenta (A, D, G, J) and synapsin immunoreactivity in green (B, E, H, K). Corresponding merged images (C, F, I, L). <b>A-C</b>: Frontal sections through the antennal lobe (AL). Insets indicate single optical slice (< 2 μm thickness) of the three glomeruli, which are marked by asterisks in main panel B. TH-positive fibers innervate antennal lobe glomeruli, predominantly the peripheral areas (see arrowheads in inset of A). In the macroglomerular complex (MGC), TH-positive fibers innervate exclusively the medial side (arrows in A). <b>D-F</b>: Horizontal sections through the antennal lobe. A single TH-positive multiglomerular projection neuron (PN) is labeled, which projects its axon toward the medial antennal lobe tract (MALT, see also panel G). <b>G-I</b>: Frontal sections through the medial antennal lobe tract containing several TH-positive axons (arrowheads). <b>J-L</b>: Frontal sections through the lobus glomerulatus (LG) and the antennal mechanosensory and motor center (AMMC). TH-immunoreactive fibers are widely distributed in the AMMC. In the lobus glomerulatus, immunoreactive fibers predominantly innervate the ventral portion. D, dorsal; M, medial; ML, medial lobe; NO, noduli; P, posterior; PB, protocerebral bridge. Scale bars = 50 μm in inset of C; 100 μm in C, F, I, L.</p