Timothy G. Kingan

Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta

SummaryWe have prepared and characterized specific rabbit antisera against γ-aminobutyric acid (GABA) coupled covalently to bovine serum albumin and keyhole-limpet hemocyanin. Using these antisera in immunocytochemical staining procedures, we have probed the antennal lobes and their afferent and efferent fiber tracts in the sphinx moth Manduca sexta for GABA-like immunoreactivity in order to map putatively GABAergic central neurons in the central antennal-sensory pathway. About 30% of the neuronal somata in the large lateral group of cell bodies in the antennal lobe are GABA-immunoreactive; cells in the medial and anterior groups of antennal-lobe cells did not exhibit GABA-like immunoreactivity. GABA-immunoreactive neurites had arborizations in all of the glomeruli in the antennal lobe. Double-labeling experiments involving tandem intracellular staining with Lucifer Yellow and immunocytochemical staining for GABA-like immunoreactivity demonstrated that at least some of the GABA-immunoreactive cells in the antennal lobe are amacrine local interneurons. Several fiber tracts that carry axons of antennal-lobe projection neurons exhibited GABA-immunoreactive fibers. Among the possibly GABA-containing projection neurons are several cells, with somata in the lateral group of the antennal lobe, that send their axons directly to the lateral protocerebmm.

Immunocytochemistry of GABA in the brain and suboesophageal ganglion ofManduca sexta

SummaryWe have used specific antisera against protein-conjugatedγ-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. About 20000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers.

A competitive enzyme-linked immunosorbent assay: applications in the assay of peptides, steroids, and cyclic nucleotides

Indirect competitive enzyme-linked immunosorbent assays (ELISAs) that can be used to quantify several types of small, bioactive molecules, including peptides, steroids, and cyclic nucleotides, are described. The assays require no special expertise to perform, and the sensitivities are very high, equally or exceeding what is commonly achieved in radioimmunoassay (RIA). The molecule to be assayed or a synthetic derivative is coupled to a protein carrier (= conjugate). The conjugate is adsorbed to the wells of a microtiter plate where it is bound by antibody in inverse proportion to free hapten in a sample or standard. Bound antibody is then quantified with enzyme-labeled anti-immunoglobulin and appropriate substrate. The assay of peptides is illustrated for the sulfated cholecystokinin octapeptide, in which an ED50 of 20 fmol (2 x 10(-10) M in 100 microliters assay volume) is attained. The ED50s and slopes of the dose-response curves in the steroid and cyclic nucleotide ELISAs are compared with those parameters obtained earlier by RIA using the same antisera. This comparison indicates that a steroid, ecdysone, can be quantified with no apparent participation of the bridging group of the conjugate in the competitive assay. Furthermore, the ED50s in the ecdysone assays (ecdysone 2 beta, 3 beta, 14 alpha, 22R, 25-pentahydroxy-5 beta-cholest-7-en-6-one, 7.7 fmol; 20-hydroxyecdysone, 16 fmol) are 19- to 38-fold lower for ELISA than for RIA. In the cyclic nucleotide assay, the bridge of a cAMP conjugate (homologous with the bridge of the immunogen) decreases the slope of the dose-response curve. This effect is minimized by the use of short incubations with anti-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Mushroom body feedback interneurones in the honeybee show GABA-like immunoreactivity

The distribution of the transmitter substance GABA was investigated in the mushroom body neuropile of the honeybee by means of immunohistochemistry. The protocerebro-calycal tract (PCT) links a mushroom body output area with the calycal input sites. Interneurones contained within the PCT exhibit GABA-like immunoreactivity and may function as negative feedback loop.

Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity.

SummaryUsing an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. More than 2000 neurons per hemisphere exhibit FMRFamide-like immunoreactivity. Most of these cells reside within the optic lobe. Particular types of FMRFamide-immunoreactive neurons can be identified. Among these are neurosecretory cells, putatively centrifugal neurons of the optic lobe, local interneurons of the antennal lobe, mushroom-body Kenyon cells, and small-field neurons of the central complex. In the suboesophageal ganglion, groups of ventral midline neurons exhibit FMRFamide-like immunoreactivity. Some of these cells have axons in the maxillary nerves and apparently give rise to FMRFamide-immunoreactive terminals in the sheath of the suboesophageal ganglion and the maxillary nerves. In local interneurons of the antennal lobe and a particular group of protocerebral neurons, FMRFamide-like immunoreactivity is colocalized with GABA-like immunoreactivity. This suggests that FMRFamide-like peptides may be cotransmitters of these putatively GABAergic interneurons. All FMRFamide-immunoreactive neurons are, furthermore, immunoreactive with an antiserum against bovine pancreatic polypeptide, and the vast majority is also immunoreactive with an antibody against the molluscan small cardioactive peptide SCPB. Therefore, it is possible that more than one peptide is localized within many FMRFamide-immunoreactive neurons. The results suggest that FMRFamide-related peptides are widespread within the nervous system ofM. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types.

γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta

We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27–50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate ( <0.2 mM) and higher levels of certain other amino acids such as serine, glutamine and proline. The concentration of proline in hemolymph increases up to 2-fold after eclosion. Glutamate, glutamine and proline are interconvertible in the CNS, and each can serve as precursor for GABA synthesis both in vivo and in vitro. The efficiency of the precursor role in vitro is similar for each amino acid, as estimated from the ratio of the specific radioactivities of GABA and glutamic acid in the ganglion derived from each precursor. Exogenous proline and glutamine can equilibrate rapidly with the ganglionic pools of the same amino acids while glutamic acid is relatively excluded. Taken together, the findings of this study show that proline and glutamine may contribute substantially to synthesis of GABA in the CNS of M. sexta.

Radioimmunologic detection of proctolin in arthropods

Abstract 1. Antisera to the insect pentapeptide proctolin (Arg-Tyr-Leu-Pro-Thr) have been prepared and used to quantify proctolin radioimmunoassay (RIA) activity in the central nervous system (CNS) of three orthopterous insects and one crustacean. 2. RIA activity was found throughout the CNS of the orthopterous insects with the 6th abdominal. thoracic and supraesophageal (brain) ganglia being enriched in the peptide relative to the remaining abdominal ganglia. 3. No activity was found in the larval or adult CNS of a Lepidopteran ( Manduca sexta ). 4. RIA activity was also found throughout the crustacean CNS.

Transport and metabolism of l-glutamic acid by abdominal ganglia of the hawk moth, Manduca sexta

Abstract 1. The transport of exogenous L -glutamate was characterized in an insect ganglion with an in vitro preparation consisting of whole ganglia in short-term organ culture. The characteristics studied include the ionic dependence and specificity of transport, as well as its rate of operation. 2. Rates for the Na+-dependent and Na+-independent components were measured, revealing lowaffinity carriers; the apparent KMs are 0.48 and 1.27 mM, respectively. 3. Accumulation of exogenous glutamate is saturable, sensitive to metabolic inhibitors, and specific for certain dicarboxylic acids, thus exhibiting features of a carrier-mediated process. Transport is partially dependent on Na+ in the medium, and it is reduced in the presence of the competitor amino acids, L-and D-aspartate. 4. Exogenous glutamate is metabolized by abdominal ganglia; the major amino acid metabolites were measured in extracts of the ganglia and in the culture medium. Major amino acid metabolites derived from glutamate and recovered in the ganglia are glutamine, proline, GABA, and small amounts of aspartate and alanine. 5. Proline and glutamine are spontaneously released by the ganglia. A portion of this material is newly synthesized from exogenous glutamate, and its release follows a different time course for proline and glutamine. 6. Metabolism of exogenous glutamate may be a biochemical aspect of the insect blood-brain barrier.

Neurotransmitters and Neuropeptides in the Olfactory Pathway of the Sphinx Moth Manduca Sexta

Like other animals, insects have many and diverse chemical “messengers” in their nervous systems. A growing list of synaptic neurotransmitters, neuromodulators, neuropeptides, and neurohormones -- collectively “transmitters” -- prompts efforts to seek physiological roles and mechanisms of action for these substances. An improved understanding of these chemical messengers in the insect nervous system promises to reveal key regulatory mechanisms, novel and accessible targets for pharmacological agents, and phyletic differences that can be exploited in new approaches to the manipulation of insect behavior and the selective destruction of harmful populations of insect pests and disease vectors. Toward such goals, we study the biochemistry, cellular distribution, and physiological actions of transmitter candidates in an experimentally favorable insect “model”, the sphinx moth Manduca sexta. In contrast with significant advances that have been made in many laboratories investigating peripheral neural and neuromuscular systems, relatively much less is known about chemical signalling in the insect central nervous system (CNS). With this in mind, and building upon substantial previous and ongoing studies of the anatomy, physiology, and development of the insect CNS in many laboratories including our own, we focus on the cellular neurochemistry of the CNS in Manduca. In particular we are exploring the olfactory pathway in the brain, for which we have accumulated much information about the types of neurons and their functional organization and development (e.g. see recent reviews: Hildebrand, 1985; Hildebrand and Montague, 1986; Christensen and Hildebrand, 1987).