Receptors for L-glutamate and GABA in the nervous system of an insect (Periplaneta americana)

The nervous system of the cockroach Periplaneta americana is well suited to studies of invertebrate amino acid receptors. Using a combination of radioligand binding and electrophysiological techniques, several distinct receptors have now been identified. These include an l-glutamate-gated chloride channel which has no known counterpart in the vertebrate nervous system, and a putative kainate/quisqualate receptor with pharmacological properties different from those of the existing categories of vertebrate excitatory amino acid receptors. GABA receptors have also been characterized in the cockroach nervous system. Bicuculline, benzodiazepines and steroids have revealed important differences between certain insect GABA-gated chloride channels and vertebrate GABA receptors. Identifiable neurones may facilitate the allocation of specific functions to amino acid receptor subtypes. In view of the existence of subtypes of amino acid receptors in insects, it is of interest to examine how this is reflected at the molecular level in terms of receptor subunit composition and amino acid sequence. Preliminary molecular cloning studies on insect GABA receptors are described.     

Characterization of phenylalkylamine binding sites in insect (Periplaneta americana) nervous system and skeletal muscle membranes

This study has identified specific, stereoselective phenylalkylamine (PAA, (±)- [³H]verapamil) binding sites of low-affinity and high-density in cockroach (Periplaneta americana) nervous system and skeletal muscle membranes. Scatchard transformation of equilibrium binding data revealed a single population of binding sites in both tissues with dissociation constants (K_d) of 273 nM and 377 nM and binding capacities (B_max) of 23 pmol·mg protein^?1 and 37pmol·mg protein^?1 for cockroach nervous tissue and skeletal muscle membranes, respectively. The PAA binding site in cockroach nervous tissue membranes was found to be dihydropyridine (DHP)-insensitive, whereas the corresponding site in cockroach skeletal muscle membranes was DHP-sensitive. This property of a DHP-sensitive PAA receptor distinguishes the binding sites identified in cockroach skeletal muscle from those in cockroach nervous tissue and indicates that pharmacologically distinct putative Ca²⁺ channel subtypes are present in insect nerve and muscle. © 1993 Wiley-Liss, Inc.     

Distribution of GABA-like immunoreactive neurons in the optic lobes of Periplaneta americana

Summary Specific antisera against protein-conjugated -aminobutyric acid (GABA) were used in immunocytochemical staining procedures to study the distribution of the putative GABA-like immunoreactive neurons in the optic lobes of Periplaneta. GABA-like immunoreactive structures are evident in all three optic neuropil regions. Six different populations of GABAergic neurons, whose perikarya are grouped around the medulla, are found within the optic lobe. The number of these immunoreactive cells varies greatly and corresponds to the number of ommatidia of the eye. In the proximal part of the lamina, a coarse network of GABA-positive fibres is recognizable. These are the processes of large field tangential cells whose fibres pass through the distal surface of the medulla. A second fibre population of the lamina is made up of the processes of the centrifugal columnar neurons whose perikarya lie proximally to the medulla. The medulla contains 9 layers with GABAergic elements of variable immunoreactivity. Layers 1, 3, 5, 7 and 9 exhibit strong labelling, as a result of partial overlapping of the processes of centrifugal and centripetal columnar neurons, tangential fibres and/or lateral processes of perpendicular fibres and (possibly) processes of amacrines. A strong immunoreactivity is found in the proximal and distal layers of the lobula.     

Substance P-like immunoreactivity in the central nervous system of the blattarian insect Periplaneta americana L. revealed by a monoclonal antibody

Brains, retrocerebral complexes and frontal and suboesophageal ganglia of adult American cockroaches, Periplaneta americana, were immunohistochemically investigated with a specific monoclonal antibody (McAb) directed against a well characterized antigenic determinant, namely the COOH terminus of the endecapeptide substance P (SP). This resulted in the detection of several neurons and nerve fibres containing a substance antigenically closely related to this typically vertebrate neuropeptide. No difference in staining pattern could be observed between male and female insects. Related to the age of the adult specimens, however, a slight quantitative difference in SP immunoreactivity seems to occur, which probably might have functional implications. The SP-like peptide demonstrated in this study appears to be located in different neuronal structures than the ones that we earlier described as containing ACTH-, CRF-, OT-, AVP-, NP I-, NP II-, BPP-, FMRFamide-, AKH-, met-ENK-, FSH-, LH- and LHRF-like material (Verhaert et al. 1984a, b, 1985; Verhaert and De Loof 1985a, b).     

Substance P-like immunoreactivity in the central nervous system of the blattarian insect Periplaneta americana L. revealed by a monoclonal antibody

Summary Brains, retrocerebral complexes and frontal and suboesophageal ganglia of adult American cockroaches, Periplameta americana, were immunohistochemically investigated with a specific monoclonal antibody (McAb) directed against a well characterized antigenic determinant, namely the COOH terminus of the endecapeptide substance P (SP). This resulted in the detection of several neurons and nerve fibres containing a substance antigenically closely related to this typically vertebrate neuropeptide.No difference in staining pattern could be observed between male and female insects. Related to the age of the adult specimens, however, a slight quantitative difference in SP immunoreactivity seems to occur, which probably might have functional implications. The SP-like peptide demonstrated in this study appears to be located in different neuronal structures than the ones that we earlier described as containing ACTH-, CRF-, OT-, AVP-, NP I-, NP II-, BPP-, FMRFamide-, AKH-, met-ENK-, FSH-, LH- and LHRF-like material (Verhaert et al. 1984a, b, 1985; Verhaert and De Loof 1985a, b).     

Actions of deltamethrin and tralomethrin on cholinergic synaptic transmission in the central nervous system of the cockroach (Periplaneta americana)

Deltamethrin (RU 22974) and tralomethrin isomers (RU 24784 and RU 24785) block transmission at the cercal-afferent giant-interneuron synapses of the cockroach, when bath-applied to the desheathed ganglion at micromolar concentrations. The time-course of the events leading to the blockage suggests two possible target sites: one located presynaptically and the other situated on postsynaptic membranes.     

The uptake of L-glutamate by the central nervous system of the cockroach, Periplaneta americana.

A concentrative uptake mechanism for L-glutamate with the following characteristics has been identified in the abdominal nerve cord: 1. The uptake can be divided into Na+-sensitive and Na-plus-insensitive components. 2. The Na-plus-sensitive component showed the typical saturation kinetics of a carrier mediate process. It had a V of 15.9 x 10(6) times 10(6) muM/mg wet weight/min and a Km of 0-33 mm. Its magnitude was proportional to the first power of the Na-plus concentration of the medium. The uptake was specific for L-dicarboxylic amino acids and was sensitive to the presence of metabolic inhibitors. 3. The Na-plus-insensitive component was linearly related to the glutamate concentration of the medium. An isosmotic saline is described for use with the isolated intact abdominal nerve cord of P. americana.     

美洲大蠊中枢DUM神经元的分离和电压门控Na+电流的记录

【目的】建立美洲大蠊Periplaneta americana中枢神经系统背侧不成对中间神经元（dorsal unpaired median neurons, DUM neurons）的分离方法和DUM神经元电生理实验模型。【方法】IA型胶原酶法消化美洲大蠊末端腹神经节, 机械吹打得到DUM神经元细胞, 运用膜片钳技术记录DUM神经元细胞电压门控Na+电流。【结果】分离得到的DUM神经元细胞状态良好, 具有DUN神经元典型的梨状形态和表面特征。以膜片钳全细胞方式记录到的Na+电流符合钠通道电流特征。【结论】IA型胶原酶消化得到美洲大蠊DUM神经元细胞的方法可靠, 能稳定地记录到Na+电流。本文描述的方法为昆虫神经细胞的电生理机制研究提供一个可用的实验模型。     

Localization of corazonin in the nervous system of the cockroach Periplaneta americana

Antisera raised to the cardioactive peptide corazonin were used to localize immunoreactive cells in the nervous system of the American cockroach. Sera obtained after the seventh booster injection were sufficiently specific to be used for immunocytology. They recognized a subset of 10 lateral neurosecretory cells in the protocerebrum that project to, and arborize and terminate in the ipsilateral corpus cardiacum. They also reacted with bilateral neurons in each of the thoracic and abdominal neuromeres, a single dorsal unpaired median neuron in the suboesophageal ganglion, an interneuron in each optic lobe, and other neurons at the base of the optic lobe, in the tritocerebrum and deutocerebrum. The presence of corazonin in the abdominal neurons and the lateral neurosecretory cells was confirmed by HPLC fractionation of extracts of the abdominal ganglia, brains and retrocerebral complexes, followed by determination of corazonin by ELISA, which revealed in each tissue a single immunoreactive peak co-eluting with corazonin in two different HPLC systems. Antisera obtained after the first three booster injections recognized a large number of neuroendocrine cells and neurons in the brain and the abdominal nerve cord. However, the sera from the two rabbits reacted largely with different cells, indicating that the majority of this immunoreactivity was due to cross-reactivity. These results indicate that the production of highly specific antisera to some neuropeptides may require a considerable number of booster injections.     

Identification of immunoreactive neurophysin-like proteins in the central nervous system of an insect; Locusta Migratoria

Proteins exhibiting the immunoreactivity of vertebrate neurophysin were detected in acido-ethanolic extracts of suboesophagial ganglia of the locust. These components possess an apparent molecular weight approximating 10,000, acidic isoelectric points (4.5 and 4.75) and bind to antibovine neurophysin antibodies immobilized on CH-Sepharose 4B. These results suggest that the immunoreactive material observed in the Locusta Migratoria is composed of proteins structurally related to vertebrate neurophysin.     

Mass spectrometric analysis of FMRFamide-like immunoreactive neurons in the prothoracic and subesophageal ganglion of Periplaneta americana

MALDI-TOF mass spectrometry combined with immunocytochemistry and retrograde labeling, was used to study the expression pattern and morphology of Pea-FMRFamide-related peptides in single neurons of the prothoracic ganglion and the subesophageal ganglion (SEG) of the American cockroach Periplaneta americana. In contrast to the postero-lateral cells (PLCs) of the meta- and mesothoracic ganglion, the prothoracic FMRFamide-related peptides expressing neurons not only extend in the posterior median nerve but also in an anterior median nerve, which is described herein. The peptidome of the prothoracic PLCs is identical with that of the meso- and metathoracic neurons, respectively. In this study, we identified a truncated form of Pea-FMRFa-24 which was found to be more abundant than the peptide originally designated as Pea-FMRF-24. FMRFamide-related peptides expressing postero-lateral cells were also detected in the labial neuromere of the SEG. Although their projection could not be solved, mass spectrometric analyses revealed the same peptide complement in these neurons as found in the thoracic postero-lateral cells. In all neurons which we studied no co-localized peptides of other peptide families were observed.     

[N-Methyl-3H]Scopolamine binding sites in the central nervous system of the cockroach Periplaneta americana

The binding of [N-methyl-³H]scopolamine to a cockroach nerve cord preparation has been investigated. Specific [N-methyl-³H]scopolamine binding was found to be saturable and of high affinity (K_d = 13.9 nM). Muscarinic ligands were found to displace [N-methyl-³H]scopolamine binding more effectively than nicotinic ligands. The distribution of these [N-methyl-³H]scopolamine binding sites was examined in the metathoracic ganglion at the light microscope level by autoradiographical techniques. Specific binding was found to be localized to distinct regions of the neuropile. This pattern showed certain similarities to that seen when the ganglion was stained for acetylcholinesterase, suggesting a functional role for these insect muscarinic acetylcholine receptors.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

FMRFamide immunoreactive neurons in the central nervous system of the snail,Achatin a fulica

• 1.1. FMRFamide immunoreactive neurons were detected in the central nervous system of the snail, Achatina fulica.
• 2.2. FMRFamide immunoreactive neurons were found in all the ganglia comprising the central nervous system. In particular, the immunoreactivity was recognized in both the ordinary and giant neurons of the visceral and right parietal ganglia.
• 3.3. In the cerebral and pleural ganglia, FMRFamide immunoreactive neurons were found only in the ordinary neurons. The immunoreactivity was shown to have a tendency to form a group in the cerebral and pedal ganglia.

     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10^-3, 10^-4, 10^-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.