No abstract

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect brain.

Accumulating evidence suggests that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla of the insect brain are involved in circadian pacemaking functions. We have used immunocytochemical techniques to investigate the neurochemical organization of the accessory medulla in the locust Schistocerca gregaria. Local neurons with arborizations largely restricted to the accessory medulla are immunoreactive with antisera against serotonin, Manduca sexta allatotropin, and Diploptera punctata allatostatin 7. Projection neurons with arborizations in the accessory medulla and fibers to the lamina and/or several areas in the midbrain including the posterior optic tubercles, the inferior and the superior protocerebrum show Phe-Met-Arg-Phe (FMRF)amide-, gastrin/cholecystokinin-, crustacean cardioactive peptide-, and substance P immunoreactivities. A unique neuron with tangential ramifications in the medulla and lamina and varicose terminals in the accessory medulla contains a peptide related to locustatachykinin I/II. Double-label experiments show colocalization of pigment-dispersing hormone-immunoreactivity with substances related to gastrin/cholecystokinin, FMRFamide, substance P, or crustacean cardioactive peptide in certain projection neurons of the accessory medulla. The results suggest that neuropeptides and biogenic amines play major neuroactive roles in the accessory medulla of the locust. The abundance and extensive colocalization of neuropeptides in the locust accessory medulla is discussed with respect to the possible involvement of this brain area in circadian pacemaking functions.

Several lines of evidence suggest that the accessory medulla of orthopteroid insects is implicated in the control of circadian rhythms. To investigate the role of this brain area in more detail, anatomical and physiological properties of accessory-medulla neurons of the locust were studied by intracellular recordings combined with Lucifer dye injections. The responses of these neurons to visual stimuli were compared with visual responses of adjacent tangential neurons of the medulla. Principal neurons of the accessory medulla showed weak tonic excitations to stationary light stimuli, but they were not sensitive to movement stimuli or to different e-vector orientations of polarized light. These neurons connected the accessory medulla to the lamina, the anterior medulla, and to several areas in the midbrain including the superior protocerebrum and the posterior optic tubercle. A second class of neurons had tangential arborizations in the medulla, a few sidebranches in the accessory medulla, and projections to the lamina or to the contralateral optic lobe. Several of these neurons were sensitive to polarized light. Finally, a third class of neurons had tangential arborizations in the medulla and axonal projections to the lobula and to the lateral protocerebrum. These neurons showed phasic responses to light and nondirectional selective responses to motion stimuli. The results show that neurons of the accessory medulla receive photic input and support an involvement of this neuropil in circadian timekeeping functions. The possible role of the accessory medulla in polarization vision is discussed.

We have developed a new double immunofluorescence technique by which two neuroactive substances in the same tissue section can be labeled with primary antisera raised in the same species. The optic lobes of the locust Schisrocerca gregaria were used as a model system to develop the staining procedure. FMRFamide-immunoreactive neurons were detected by rabbit antisera against FMRFamide and FIXconjugated secondary antibodies. Antibodies against the second peptide, pigment-dispersmg hormone (PDH), also raised in rabbit, were biotinylated and detected via streptavidinTexas Red. Crossreactivity of the PDH immunoglobulins IntroductionImmunocytochemical techniques are widely used to investigate the distribution of neuroactive substances in the nervous system, and many antisera against neuropeptides and neurotransmitters are now available. Such studies have shown that peptides are often colocalized with classical neurotransmitters and other peptides (7,(9)(10)(11)(12) tographed after the first staining sequence and, after elution of the antiserum, a second staining is performed. Under certain conditions, the elution step can be omitted (24). Sequential techniques have several disadvantages and depend on the success of the elution. Co-localization can be demonstrated on the same section, but usually not at the same time, because in immunoenzyme techniques the mixed colors are often difficult to distinguish from the individual signals, For simultaneous demonstration of two antigens with antisera from the same species, we have developed a simple double i " unofluorescence staining technique after biotinylation of one of the antisera. Parts of this study have been reported in abstract form (28). Materials and MethodsAntisera. The anti-PDH serum (3B3) was raised in rabbits against conjugates of synthetic Uca pugiLatodCancer magtiter P-PDH and bovine thyroglobulin as described by Dircksen et al. Biotinylation of the PDH Antiserum. To obtain an immunoglobulin fraction of the PDH antiserum, serum proteins were removed by ammonium sulfate precipitation (8). The anti-PDH immunoglobulins were biotinylated as described by Bayer and Wilchek (l), using biotinamidocaproate N-hydroxy succinimide ester (Sigma; Deisendorf, Germany). The biotinylated PDH immunoglobulins were used at a dilution of 1:7500. 627 -EN, HOMBERGImmunocytochemistry. Immunocytochemistry was performed on adult male and female Schistocercu greguriu obtained from laboratory cultures at the University of Konstanz. After dissection, brains were fixed at room temperature for 4 hr or overnight in 4% paraformaldehyde/7.5% picric acid in phosphate buffer (0.1 M, pH 7.4). Brains were embedded in a gelatinlalbumin mixture and cut at 30 pm with a Vibratome (Technical Products; St Louis, MO). To reduce nonspecific background staining, the sections were incubated at room temperature for 1 hr in 0.1 M Tris HCI-0.3 M NaCl (pH 7.4) containing 2% normal goat serum (NGS) (Gibco Laboratories; Chagrin Falls, OH) and 0.5% Triton X-100. The sections were next incubated with anti-FMRFamide...