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Right-left discrimination in a biologically oriented model of the cockroach escape system.

We present a biologically oriented model that accounts for left-right discrimination in the cockroach's escape behavior. The model includes the main groups of neurons found to be involved in the escape response. Each one of the included neurons is described by the actual processes taking place in an individual neuron (formation of an action potential, transmitter release, conductance changes, etc.). Furthermore, realistic chemical synapses (excitatory or inhibitory and able to undergo different types of modulation) connect the various neurons. With this model, we ere able to achieve, for a wide range of inputs representing different wind directions, behavior which resembles that found experimentally. The model indicates that several synaptic properties, in particular postsynaptic inhibition and presynaptic facilitation, play a key role in the discrimination of wind direction.

Animals↗

Localization of leucomyosuppressin in the brain and circadian clock of the cockroach Leucophaea maderae.

The myosuppressins (X1DVX2HX3FLRFamide), which reduce the frequency of insect muscle contractions, constitute a subgroup of the FMRFamide-related peptides. In the cockroach Leucophaea maderae, we have examined whether leucomyosuppressin (pQDVDHVFLRFamide) is present in the accessory medulla, viz., the circadian clock, which governs circadian locomotor activity rhythms. Antisera that specifically recognize leucomyosuppressin stain one to three neurons near the accessory medulla. MALDI-TOF mass spectrometry has confirmed the presence of leucomyosuppressin in the isolated accessory medulla. Injections of 1.15 pmol leucomyosuppressin into the vicinity of the accessory medulla at various circadian times have revealed no statistically significant effects on the phase of circadian locomotor activity rhythms. This is consistent with the morphology of the myosuppressin-immunoreactive neurons, which restrict their arborizations to the circadian clock and other optic lobe neuropils. Thus, leucomyosuppressin might play a role in the circadian system other than in the control of locomotor activity rhythms.

Animals↗

Metabolism of propionate to acetate in the cockroach Periplaneta americana.

Carbon-13 NMR and radiotracer studies were used to determine the precursor to methylmalonate and to study the metabolism of propionate in the cockroach Periplaneta americana. [3,4,5-13C3]Valine labeled carbons 3, 4, and 26 of 3-methylpentacosane, indicating that valine was metabolized via propionyl-CoA to methylmalonyl-CoA and served as the methyl branch unit precursor. Potassium [2-13C]propionate labeled the odd-numbered carbons of hydrocarbons and potassium [3-13C]propionate labeled the even-numbered carbons of hydrocarbons in this insect. This labeling pattern indicates that propionate is metabolized to acetate, with carbon-2 of propionate becoming the methyl carbon of acetate and carbon-3 of propionate becoming the carboxyl carbon of acetate. In vivo studies in which products were separated by HPLC showed that [2-14C]propionate was readily metabolized to acetate. The radioactivity from sodium [1-14C]propionate was not incorporated into succinate nor into any other tricarboxylic acid cycle intermediate, indicating that propionate was not metabolized via methylmalonate to succinate. Similarly, [1-14C]propionate did not label acetate. An experiment designed to determine the subcellular localization of the enzymes involved in converting propionate to acetate showed that they were located in the mitochondrial fraction. Data from both in vivo and in vitro studies as a function of time indicated that propionate was converted directly to acetate and did not first go through tricarboxylic acid cycle intermediates. These data demonstrate a novel pathway of propionate metabolism in insects.

Acetates↗

Tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach.

The elongation of fatty acyl-CoAs, reactions involved in hydrocarbon biosynthesis, was examined in the cockroach, Periplaneta americana. Products were analyzed by radio-HPLC and radio-GLC. The majority of the elongation activity was observed in microsomes prepared from abdominal epidermal tissue. Linoleoyl-CoA (18:2-CoA) was elongated most efficiently followed by stearoyl-CoA (18:0-CoA), linolenoyl-CoA (18:3-CoA; n-3) and oleoyl-CoA (18:1-CoA). The products of 18:2-CoA elongation included all even numbered acyl groups up to 28 carbons, and the products of 18:0-CoA included all even numbered acyl groups to 26 carbons. The 18:3-CoA was elongated only to 20 and 22 carbons. Radioactivity from both 18:2-CoA (5.4%) and 18:0-CoA (1.2%) was recovered in the hydrocarbon fraction. Analysis of this hydrocarbon fraction showed that the radio-activity from 18:2-CoA was present in (Z,Z)-6,9-heptacosadiene and that the radioactivity from 18:0-CoA was present in n-pentacosane. These data demonstrate for the first time in an in vitro insect system that the fatty acid elongation reactions are coupled with the conversion of the elongated product to hydrocarbon. Thus, each of the expected intermediates in the conversion of 18:0 and 18:2 to 25 and 27 carbon hydrocarbons, respectively, was observed, and the results demonstrate high tissue, substrate, and product specificity.

Acyl Coenzyme A↗

A 13C-NMR study of the biosynthesis of 3-methylpentacosane in the American cockroach.

13C-NMR spectrometry was used to examine the in vivo incorporation of 13C-labeled precursors into 3-methylpentacosane in the cockroach Periplaneta americana. Natural abundance 13C-NMR of 3-methylpentacosane showed that carbons 1 through 6, 23 through 25 and the branching methyl carbon (C26) each gave distinct signals, with carbons 7 through 22 indistinguishable from each other. The label from dipotassium 2-[methyl-13C]methylmalonate was incorporated primarily into the methyl branch of 3-methylpentacosane, demonstrating the 2-methylmalonate is the precursor to the methyl branch unit. The carboxyl carbon from sodium [1-13C]propionate was incorporated exclusively into the 4-position. This indicates that propionate, as a 2-methylmalonyl derivative, is incorporated as the second unit during chain synthesis rather than toward the end of the elongation process. The labeled carbon from sodium [1-13C]acetate was incorporated into carbons 2, 6 and 24 and the labeled carbon from [2-13C]acetate was incorporated into carbons 1, 5, 23 and 25 of 3-methylpentacosane, respectively. These data are consistent with an elongation-decarboxylation pathway for 3-methylpentacosane biosynthesis.

Acetates↗

Characterization of the delta 12 desaturase in the American cockroach, Periplaneta americana: the nature of the substrate.

The cockroach, Periplaneta americana, can convert oleic acid (18:1(n - 9], to linoleic acid, (18:2(n - 6], by a microsomal delta 12 desaturase. Most of the desaturase activity was present in the fat body tissue, with lower activity in the epidermis and no detectable activity in the thorax or gut tissue. In incubations of microsomal preparations from fat body tissues with [1-14C]18:1-CoA, increased amounts of [1-14C]18:2 were found with increasing time and protein concentration. The form of the substrate for the delta 12 desaturase was determined to be 18:1-CoA by comparing activity towards [1-14C]18:1-CoA and [1-14C]18:1 transesterified to phospholipid. Ozonolysis of the 18:2 formed from [1-14C]oleoyl-CoA followed by radio-gas-liquid chromatography gave one labeled peak, 9-oxononanoate, which showed that the product of the delta 12 desaturase is the physiologically important isomer, 18:2(n - 6).

Acyl Coenzyme A↗

Vitellogenin: its structure, synthesis and processing in the cockroach Periplaneta americana.

The vitellogenin in the oocyte of the American cockroach contains three different polypeptides with approximate molecular weights of 123 000, 118 000 and 57 000. These are present in a molar ratio of 2 : 2 : 2; all three contain carbohydrate. The same subunit structure is observed in vitellogenin isolated from hemolymph and in that secreted by fat body in organ culture. The only protein found within fat body by precipitation with female-specific antiserum is a large polypeptide, with a molecular weight of approx. 250 000. No mature vitellogenin subunits are found in fat body, even after pulsing with labeled amino acids and chasing with unlabeled substrates for up to 4 h. These data suggest that vitellogenin is synthesized in the fat body as a large precursor, which is processed to smaller polypeptides upon secretion into the hemolymph.

Animals↗

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry.

Amino acid sequences have been assigned to two cockroach neuropeptides (greater than Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, M I, and greater than Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.

Amino Acid Sequence↗

Insect hypertrehalosemic hormone: isolation and primary structure from Blaberus discoidalis cockroaches.

A new neurohormone was isolated and structurally characterized that increased hemolymph carbohydrate (trehalose) levels in the cockroach, Blaberus discoidalis. The hormone was isolated in high yield by a rapid HPLC procedure. The sequence, pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr-NH2, was suggested from gas-phase Edman degradation of a peptide fragment of the natural peptide after deblocking with pyroglutamate aminopeptidase. The structure was confirmed by synthesis of the suggested sequence. The synthetic peptide had identical chromatographic and biological properties as the natural peptide.

Amino Acid Sequence↗

Energy metabolism and nerve function in cockroaches (Periplaneta americana).

Levels of ATP are near normal in the cockroach nerve cord when hypoxia is sufficient to cause coma as determined by behavioral observations and nerve cord electrical activity. During anoxia the major change in ATP occurs after loss of measurable nerve activity. High glycogen levels and reduced energy demands due to coma may contribute to the resistance to anoxic death.

Adenosine Triphosphate↗

Protein synthesis in regenerating motor neurons in the cockroach.

Protein synthesis was studied in the thoracic ganglia of the cockroach. All the motor neurons leaving the metathoracic ganglion were axotomized by cutting the nerve roots. In vivo experiments demonstrated that within two days of the operation those ganglia with cut nerve roots had an increased [3H]leucine incorporation into protein resulting from an increased uptake and labeling of the leucine pool. These effects were non-specific responses to the damage and trauma produced by the operation. In vitro labeling experiments failed to demonstrate any change in incorporation or uptake of [3H]leucine up to 16 days after the operation. Autoradiographic experiments were done on ganglia in which only nerve root 5 on one side was cut. Grain densities and cytoplasmic areas were measured for the ipsilateral, axotomized, identified, fast motor neuron, Df and the homologous, contralateral, intact cell of the same ganglion. The first statistically significant increase in grain density in the axotomized Df was observed 12 days after the operation. This may be compared with the 13 day lag period after the operation before the nerves start growing from the proximal stump. The significance of this relatively long lag period before the increased [3H]leucine incorporation and the start of nerve growth is discussed in terms of the ability of regenerating neurons to specifically reinnervate the cells to which they were originally connected.

Animals↗

Extracellular potassium activity during frequency-dependent conduction block of giant axons in the metathoracic ganglion of the cockroach.

In the metathoracic ganglion (T3) of the cockroach, extracellular potassium activity (aK) was measured with ion-sensitive microelectrodes and intracellular recordings were simultaneously made from giant axons (GAs) during high frequency stimulation of the connectives. Blockade of spike conduction through T3 was associated with intraganglionic aK rises of 0.2-0.5 mM, which were only 10% of the periaxonal aK rises suggested from GA depolarizations. When aK in the bath was increased 10-fold, GA conduction block during 1 Hz stimulation did not occur until much higher levels of aK and GA depolarization were achieved. The results suggest that glial sheaths surrounding GAs significantly impede K+ movement, and may thus prevent non-specific axonal interactions, and that stimulus-induced conduction block is not primarily due to K+-induced depolarization and consequent Na+-inactivation.

Action Potentials↗

Neural basis of wind-receptive fields of cockroach giant interneurons.

The pattern of connectivity between the nine most prominent columns of sensory hairs of one cercus and ipsilateral giant interneurons (GIs) 1 and 2 of the cockroach Periplaneta americana was studied using intracellular methods. GI 1, a somewhat directionally selective interneuron receives excitatory synaptic input from all hair columns of the ipsilateral cercus. GI 2, an omni-directional cell, also receives synaptic input from all ipsilateral hair columns. However, for 2 of the 9 columns of hairs the response in GI 2 is greater than in GI 1. This difference in response magnitude represents a real difference in the functional strength of connection and not the number of synapsing afferents since the response in both GI 1 and 2 declines stepwise as the number of hairs tested was reduced one-by-one. Thus, the difference in the shapes of the wind-receptive fields of GI 1 and 2 can be explained by differences in the functional strength of connection of certain identified afferents.

Air Movements↗

Elimination of inappropriate axonal branches of regenerating cockroach motor neurons as detected by the retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin.

A procedure is described for identifying the motor neurons innervating individual cockroach muscles by use of the retrograde transport of extracellularly injected horseradish peroxidase and the staining of cells in whole mount preparations. A modification of this procedure using wheat germ agglutinin covalently conjugated to horseradish peroxidase is required in order to identify regenerating motor neurons that have an axonal process that had regrown into a particular leg muscle at various times after axotomy. At early stages of regeneration 23 out of 31 axotomized motor neurons with axons leaving the metathoracic ganglion through nerve root 5 send an axonal process into muscle 178. In intact animals this muscle receives innervation from only one motor neuron, Df. At later stages of regeneration these inappropriate axonal branches become eliminated until this muscle is again innervated only by Df. These results indicate that the specificity required for the reformation of the original innervation pattern of the leg muscles does not come from factors which are components of paths along the nerve root or which are being released from denervated muscles which could selectively control the direction of growth of axons of particular motor neurons.

Animals↗

Cholinergic receptors on cultured neurones from the central nervous system of embryonic cockroaches.

Cultured neurones from the cockroach, Periplaneta americana, have been used to investigate putative acetylcholine receptors. Ligand-binding experiments revealed that these neurones possessed an alpha-bungarotoxin binding site that was saturable, had an apparent affinity constant of 3.51 nM and was predominantly nicotinic in nature. An individual culture of 50,000 neurones had a maximum of 4200 pmol. binding sites per gram of protein. [I125]alpha-BTX autoradiography showed the binding sites to be distributed over both the neuronal cell bodies and their associated axonal processes. Both acetylcholine and nicotine applied by pressure ejection to the neuronal soma induced depolarizing responses and in the majority of cells tested the response was blocked by alpha-BTX at a concentration of 25 nM in a time dependent manner. Some of the neurones, however, were depolarized by acetylcholine and nicotine after 3 h incubation in alpha-BTX. These experiments suggest that two populations of cells possessing extrajunctional nicotinic receptors were present in these cultures. In the majority of cells these receptors were sensitive to alpha-BTX but in a subpopulation the receptors were unaffected by this toxin.

Acetylcholine↗

Tryptophan hydroxylase and 5-HTP-decarboxylase activity in cockroach brain and the effects of p-chlorophenylalanine and 3-hydroxybenzylhydrazine (NSD-1015).

The activities of two enzymes of the serotonin biosynthetic pathway, namely tryptophan hydroxylase (TPH) and 5-hydroxytryptophan-decarboxylase (5-HTP-D), were determined in the cockroach, Periplaneta americana, brain using radiometric techniques, significant levels of both enzymes were found. p-Chlorophenylalanine (PCPA) inhibited brain TPH activity strongly both in vitro and in vivo. In vitro, 10 mM PCPA reduced the TPH activity by 76% and in vivo either 15 micrograms/g or 30 micrograms/g of PCPA inhibited brain TPH activity by 80-85%. The activity of 5-HTP-D enzyme was inhibited significantly in vivo by 3-hydroxybenzylhydrazine, commonly known as NSD-1015. A recovery in the activity of 5-HTP-D enzyme was observed 12 h after 50 micrograms/g of NSD-1015 but 100 micrograms/g caused an inhibition lasting for the 24 h period studied.

Animals↗

Reduction of sensory activity produced by cercal displacement modifies response of wind-sensitive interneurons in the cockroach.

The cockroach can reduce the amount of sensory activity received by its CNS from the cerci, paired wind-responsive appendages. This reduction is produced by medial displacement of the cerci, a movement the animal performs naturally during flying. We demonstrate here that this sensory reduction significantly reduces activity of the postsynaptic wind-sensitive interneurons in the abdominal nerve cord, cells which carry the wind information to higher centers of the nervous system. In addition, we have found that the wind-evoked activity of two identified giant interneurons that are involved in escape and locomotion behaviors, is significantly reduced by cercal displacement.

Action Potentials↗