A new specific antibody reveals octopamine-like immunoreactivity in cockroach ventral nerve cord.

An antiserum was raised in rabbits immunized with octopamine conjugated to thyroglobulin. The specificity of this antiserum for octopamine is shown by dot blot immunoassay analysis. The antiserum does not crossreact with dopamine, noradrenaline, and serotonin, but slight crossreactivity with the amine tyramine at high concentrations was observed. The tyramine crossreactivity could be eliminated by preabsorption with a tyramine-glutaraldehyde-BSA conjugate. Using this antiserum, we describe the topographical distribution of octopamine-immunoreactive (ir) neuronal elements in wholemounts and paraffin sections of the ventral nerve cord of the American cockroach. The pattern of octopamine immunostaining is completely different from that obtained with an antidopamine serum, and can be blocked by preabsorbing the antioctopamine serum with BSA-conjugated octopamine. Cell bodies and dendritic processes of putatively octopaminergic dorsal (DUM) and ventral (VUM) unpaired median neurons were clearly octopamine-ir in all ganglia examined. The numbers of stained DUM somata in the mesothoracic, metathoracic, and terminal ganglion of females correspond to those of peripherally projecting DUM cells revealed previously by retrograde tracing (Gregory, Philos Trans R Soc Lond [Biol] 306:191, 1984; Tanaka and Washio, Comp Biochem Physiol 91A:37, 1988; Stoya et al., Zool Jb Physiol 93:75, 1989). In addition, various, previously unknown, paired cells with octopamine-like immunoreactivity were found in all ventral ganglia except abdominal ganglia 3-6. Some of these probably project intersegmentally.

Distribution of GABA-like immunoreactivity in the central nervous system of the cockroach, Periplaneta americana (L.).

Gamma-aminobutyric acid (GABA) is one of the most frequently occurring neurotransmitters in the central nervous system. Using an antiserum against a GABA-protein conjugate, this has also been demonstrated for insects. The distribution of GABA-like immunoreactive neuronal elements within the central nervous system of the insect Periplaneta americana has been investigated by means of immunocytochemistry. An overview of the distribution of neuronal elements with GABA-like immunoreactivity (GLIR) in the brain is given. The ventral nerve cord was extensively investigated. The highest number of neurons with GLIR was estimated in the metathoracic ganglion (ca. 1000). The function of the suboesophageal ganglion as an inhibitory centre for the central nervous system was further confirmed by the high number of GABAergic elements and descending GABAergic pathways within the ventral nerve cord. A GABAergic innervation of skeletal musculature by so-called common inhibitory neurons was revealed for the legs of cockroaches. Nevertheless, GABA mainly seems to be a neurotransmitter in central interneurons. Identical distribution patterns of neuronal elements after immunofluorescence double-staining with anti-GABA antibodies and antibodies against the GABA-synthesizing enzyme glutamic acid decarboxylase demonstrated the high specificity of the anti-GABA serum used in this paper.

Ca2+ currents in cockroach neurones: properties and modulation by neurohormone D.

In dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana two types of Ca2+ currents were found: a transient one activated by lower depolarizations (LVA) and a sustained one activated by higher voltage jumps (HVA). Both currents were strongly reduced by 100 microM Cd2+; Ni2+ was less effective. The HVA currents were insensitive to dihydropyridines, but sensitive to phenylalkylamines. The LVA component was sensitive to omega-conotoxin GVIA and SK&F96365 whereas the HVA component was insensitive to both blockers. The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), is known to increase the spiking frequence in these DUM cells. We demonstrate that NHD increases the LVA current component and decreases the HVA current component. These effects of NHD can explain the previously found NHD-induced stimulation of Ca2+ dependent K+ currents.

Periviscerokinin (Pea-PVK): a novel myotropic neuropeptide from the perisympathetic organs of the American cockroach.

A myotropic neuropeptide was isolated from extracts of 1000 abdominal perisympathetic organs of males of the cockroach, Periplaneta americana. This peptide, termed periviscerokinin, has excitatory actions on the hyperneural muscle of Periplaneta americana. After peptide sequence analysis and mass spectrometry, the structure of this peptide was confirmed by chemical synthesis and bioassay to be Gly-Ala-Ser-Gly-Leu-Ile-Pro-Val-Met-Arg-Asn-NH2. This sequence is different from the other known myotropic peptides in insects. The threshold concentration for stimulatory effects of the synthetic peptide on the isolated hyperneural muscle was about 10(-9) M, suggesting a physiological role as a neurohormone.

Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.

By monitoring the contractile activity of the hyperneural muscle of the American cockroach in vitro two peptides were isolated from the retrocerebral complex of the American cockroach. Three purification steps using reversed-phase high performance liquid chromatography on C-18 columns containing trifluoroacetic acid or heptafluorobutyric acid as organic modifiers were sufficient to achieve homogeneous peptide preparations. The structures of both peptides were elucidated by a combination of Edman degradation and mass spectrometry which yielded the following structures: His-Thr-Ala-Gly Phe-Ile-Pro-Arg-Leu-NH2 (Pea-PK-1) and Ser-Pro-Pro-Phe-Ala-Pro-Arg-Leu-NH2 (Pea-PK-2). The C-terminal sequence Phe-X-Pro-Arg-Leu-NH2 characterized the peptides as members of the insect pyrokinin family. The synthetic peptides were shown to have the same retention times as the natural peptides. The occurrence of both peptides in the retrocerebral complex suggests a physiological role as neurohormones. The effects of the synthetic pyrokinis were clearly distinguishable in their actions on the hyperneural muscle. Regarding the threshold concentrations, Pea-PK-2 was only 0.3% as active as Pea-PK-1.

Isolation of periviscerokinin-2 from the abdominal perisympathetic organs of the American cockroach, Periplaneta americana.

Using the isolated hyperneural muscle as bioassay, a novel myotropin was isolated from the abdominal perisympathetic organs of Periplaneta americana. This is the second neuropeptide identified from insect perisympathetic organs. Peptide sequence analysis and mass spectrometry yielded the following structure: Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH2. This peptide, named periviscerokinin-2, was confirmed to be amidated by chemical synthesis, bioassay, and comparison of retention times between native and synthetic peptides. A highly specific antiserum was used to determine sites of synthesis in the abdominal ganglia. Besides periviscerokinin-1, periviscerokinin-2 is the only putative myotropic neurohormone from the abdominal perisympathetic organs that is effective in the nanomolar range. This confirms the hypothesis that the neurohormonal system of the ventral nerve cord is remarkably different from that of the brain.

Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana.

By monitoring the contractile activity of the hindgut of the American cockroach in vitro eight myotropic neuropeptides were isolated from the retrocerebral complex of the American cockroach. Peptide sequence analysis and mass spectrometry yielded the following structures: Arg- Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-1), Asp-Ala-Ser-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-2), Asp-Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-3), Gly-Ala-Gln-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-4), Ser-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-5), Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lem-K-7), Gly-Ala-Asp-Phe-Tyr-Ser-Trp-Gly-NH2 (Lem-K-8) and Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lom-K). The C-terminal sequence Phe-X-Ser-Trp-Gly-NH2 characterized the peptides as members of the insect kinin family. All structures were confirmed by comparison of retention times between synthetic and natural peptides. The threshold concentration for stimulatory effects of the synthetic peptides on the isolated hindgut was about 10(-9) M and there was no significant difference measured between the different kinin forms. These neuropeptides are the first members of the insect kinin-family isolated from the American cockroach. Their occurrence in the retrocerebral complex suggests a physiological role as neurohormone.

A comparative study of the immunohistochemical localization of a presumptive proctolin-like peptide, thyrotropin-releasing hormone and 5-hydroxytryptamine in the rat central nervous system.

A proctolin (PROC)-like peptide was studied immunohistochemically in the hypothalamus, lower brainstem and spinal cord of the rat using an antiserum against PROC conjugated to thyroglobulin. Neuronal cell bodies containing PROC-like immunoreactivity (PROC-LI) were observed in the dorsomedial, paraventricular and supraoptic nuclei of the hypothalamus and in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus and nucleus interfascicularis nervi hypoglossi in the medulla oblongata. Fibers containing PROC-LI were seen in the median eminence and in other hypothalamic nuclei, and in the lower brainstem in cranial motor nuclei including the dorsal motor nucleus of the vagus nerve, the motor trigeminal nucleus, the facial nucleus and nucleus ambiguous, and in lower numbers in the nucleus of the solitary tract and locus coeruleus. Fibers containing PROC-LI were also located in the spinal cord, in the intermediolateral cell column at thoracic levels and in the ventral horns at all levels of the spinal cord. After transection of the spinal cord, all PROC-immunoreactive fibers below the lesion disappeared. Following injection of Fast blue into the thoracic spinal cord, retrogradely labeled cells in the nuclei raphe pallidus, obscurus and magnus and nucleus interfasciculari nervi hypoglossi were seen to contain PROC-LI. PROC-LI had a similar distribution as thyrotropin-releasing hormone (TRH)-LI in the above-mentioned areas and coexistence of TRH-LI and PROC-LI was shown in cell bodies in the hypothalamus and medulla oblongata. PROC-LI could also be shown to coexist with 5-hydroxytryptamine (5-HT)-LI in neuronal cell bodies in the lower brainstem. The results demonstrate the occurrence of a PROC-like peptide in the mammalian nervous system, and these neurons seem to be at least largely identical to previously described TRH systems. A possible involvement of the PROC-like peptide in spinal motor control is discussed in relation to the well-established role of PROC in control of motor behavior in insects and invertebrates.

Proctolin-related material in the mouse brain as revealed by immunohistochemistry.

By use of a specific antiserum against the insect peptide proctolin we were able to identify proctolin-immunoreactive neurons in the mouse brain. These nerve cells belong to the nuc. mesencephalicus n. trigemini. Furthermore, the antiserum stained very few nerve fibers with varicosities in the immediate neighborhood of the roof of the third ventricle. The chemical identity of the immunoreactive material with genuine proctolin remains to be elucidated.

Effect of morphine and naloxone on shock avoidance learning in headless cockroaches (Periplaneta americana L.).

Systemic administration of high doses of morphine (56 micrograms morphine/g body weight) or of naloxone (54 micrograms naloxone/g body weight) results in a significant improvement of shock avoidance behavior in headless cockroaches. In both cases the learning parameter stimulation time (time during which an animal receives shocks) is significantly decreased. The behavioral parameters, stimulation rate (activity) and mean stimulation duration (shock responsiveness), as the two factors of the stimulation time considered individually, do not significantly change with morphine. Only the responses of both parameters together give the significant decrease of stimulation time (improvement of shock avoidance behavior) as mentioned above. However, the administration of naloxone causes a significant shortening of the mean stimulation duration indicating that these animals avoid the shocks by learning to lift their legs more quickly out of electrified saline in order to terminate the shocks (escape learning). Combined administration of both drugs together also causes a significant improvement of the leg-lifting response even at lower doses (morphine: 0.56 microgram plus naloxone: 0.54 microgram/g; morphine: 0.0056 microgram/g plus naloxone: 0.0054 microgram/g). Although the various effective doses of these drugs administered in combination have similar effects on the stimulation time, they affect the behavioral parameters in different ways. These different actions on activity and shock responsiveness as well as the efficacy of doses smaller than those known from vertebrates are discussed as behavioral evidence of opiate receptors in the cockroach.

A comparison of shock avoidance learning in headless cockroaches in leg-lifting and lowering task.

Headless cockroaches were trained either to lift or to lower the prothoracic leg to avoid electric shocks. The learning process in both cases was very distinct. However, the learning performance in the lifting was better than in the lowering task. The reason for this poorer learning in the lowering task is due to the unconditioned leg raising movement of the animals to electric shocks. This unconditioned response affects the headless cockroaches avoid shocks in the lifting task by escape learning, whereas they avoid shocks in the lowering task by true avoidance learning. Injection of 30 microliter of Ringer's solution has no significant effect on the learning process in the lifting task. In the lowering task, however, Ringer's solution impairs the learning ability considerably. Crude extract of corpora cardiaca strongly inhibits the learning of both tasks.

Anatomy of neurons crossing the tritocerebral commissures of the cockroach Periplaneta americana (Blattaria).

The neuronal connections of the tritocerebral commissures of Periplaneta americana were studied in the brain-suboesophageal ganglion complex and the stomatogastric nervous system by means of heavy metal iontophoresis through cut nerve ends followed by silver intensification. The tritocerebral commissure 1 (Tc1) contains mainly the processes of the subpharyngeal nerve (Spn) whose neurons are located in both tritocerebral lobes and in the frontal ganglion. Some neurons of the frontal ganglion project through the Tc1 to the contralateral tritocerebrum. A few fibers in this commissure were observed projecting to the protocerebrum and the suboesophageal ganglion. There are tritocerebral neurons which pass through the Tc1 or the tritocerebral commissure 2 (Tc2) and extend on into the stomatogastric nervous system. One axon of a descending giant neuron appears in the Tc2. This neuron lies in the tritocerebrum and connects the brain to the contralateral side of the ventral nerve cord. In addition, sensory fibers of the labral nerve (Ln) traverse both commissures to the opposite tritocerebrum. The anatomical and physiological relevance of the identified neuronal pathways is discussed.

Proctolin immunoreactive neurons in the human brain stem.

Using the peroxidase-antiperoxidase (PAP) technique it could be established that a variety of nerve cells of human Pons and Medulla oblongata contain proctolin-like material. These neurons belong to the Nuc. olivaris caudalis, Nuc. originis n. hypoglossi, Nuc. raphes dorsalis and the Nuc. ambiguus. Furthermore, proctolin immunoreactive peptide was found to be contained in certain fiber systems (Lemniscus medialis and fiber tracts near the Raphe).

Function and modulation of the antennal heart of Periplaneta americana (L.).

The antennal heart of Periplaneta americana, a small accessory circulatory pump in the head, shows a rhythmicity with myogenic automatism. The muscle fibres extending throughout of the dilator muscle are electrically coupled. Among the peptides, proctolin causes a dose-dependent strong excitation, but allatostatin does not affect heart rhythm. However, allatostatin applied before proctolin antagonized the proctolin effect. In contrast to this, an immediate heart block produced by octopamine is similar to that produced by electrical stimulation of the nervus cardioantennalis. This inhibition is caused by a K(+)-dependent hyperpolarization. The second effect of octopamine is a delayed increase of the cAMP level. Because octopamine is present at the antennal heart, a physiological role is assumed.

Stress-induced release of octopamine in the American cockroach Periplaneta americana L.

After stress, changes of octopamine content were observed in the antennal heart and the retrocerebral complex of cockroaches. Only individually handled animals exposed to short and extreme stress showed an alteration in the haemolymph octopamine levels. The removal of the retrocerebral complex resulted in an elevation of the octopamine content in the haemolymph. We suppose that the corpora cardiaca are not the only source for the octopamine released into haemolymph in stress situations.

The octopaminergic system within the ventral nerve cord of the American cockroach.

Octopamine-immunoreactive neurons within the ventral nerve cord of the cockroach, Periplaneta americana, were mapped with a new anti-octopamine serum. The specificity of this antiserum was demonstrated by dot blot immunoassay and by comparing the immunocytochemical staining patterns obtained after incubation with anti-dopamine and anti-octopamine serum. Putative octopaminergic dorsal and ventral unpaired median (DUM resp. VUM) neurons showed octopamine-like immunoreactivity in all ventral ganglia. The numbers of DUM cells in the mesothoracic, metathoracic and terminal ganglia of females correspond to those previously characterized by retrograde staining 19, 33, 34. It could be shown that besides segmentally projecting there are also intersegmentally projecting DUM neurons within the thoracic ganglia. In addition various, previously unknown, paired octopamine-ir cells were revealed in all ventral ganglia except the abdominal ganglia 2-5.

The role of proctolin in the antenna-heart beat acceleration of Periplaneta americana (L.).

So-called accessorial pulsatile circulatory organs in insects have developed during evolutionary reduction of the vascular system. As such a peripheral organ, the antenna-heart supplies the antennae with haemolymph. In the antenna-heart, the beat rhythm is generated by a myogenic automatism and is controlled neuronally. The electrical stimulation of the antenna-heart nerve produces an immediate heart beat inhibition brought about by octopamine release. Following this heart inhibition, an accelerating effect can be observed, which is based on a peptidergic mechanism. Proctolin-like material was found in both the dilator muscle and the antenna-heart nerve by means of immunocytochemistry. After HPLC-separation of antenna-heart tissue, proctolin was identified by sequencing the bioactive material and subsequent mass spectrometry. Proctolin proved to be extraordinarily effective producing stimulation of the antenna-heart beat rate up to 450%. The threshold concentration is 10(-10) mol.1(-1) and the value of the dissociation constant was fixed to KO = 1.3 x 10(-8) mol.1(-1). The chrono-trophic effect is caused by an increase in the steepness of the rising phase of pacemaker activity. The enhancement of the membrane resistance Rinput indicates a reduction of K(+)-conductance by proctolin. The PI-second messenger system is involved as well. Proctolin was identified and isolated from a real target organ. This fact and the physiological results furnished proof of peptidergic transmitter function of proctolin in the antenna-heart.

omega-Toxins affect Na+ currents in neurosecretory insect neurons.

omega-toxins specifically block certain Ca2+ channels in mammalian neurons as well as in dorsal unpaired median neurons isolated from the cockroach Periplaneta americana. In these cockroach neurons both the P/Q-type blockers omega-agatoxin IVA and omega-conotoxin MVIIC but not the N-type Ca2+ channel blocker omega-conotoxin GVIA affected fast Na+ currents sensitive to tetrodotoxin and veratridine. Both omega-toxins enhanced Na+ current decay and thus decreased the amplitudes of the peak currents. They also led to a slower recovery from inactivation. Toxin effects developing within a few min were ot removed upon washing. They were not use-dependent. The description of the effect of omega-conotoxin MVIIC on current kinetics in terms of the Hodgkin-Huxley model revealed that steady-state parameters were not affected whereas the time constant of inactivation was considerably reduced. Under control conditions, the inactivation time constant is similar to the time constant of recovery from inactivation. The toxin-induced increase of the latter time constant and the decrease of the inactivation time constant indicate that inactivation can no longer be described by first-order kinetics.

Identification of neuronal pathways between the stomatogastric nervous system and the retrocerebral complex of the cockroach Periplaneta americana (L.).

The neuronal pathways connecting the stomatogastric nervous system with the retrocerebral complex of the cockroach, Periplaneta americana, were investigated by means of axonal cobalt chloride iontophoresis. Somata in the hypocerebral ganglion and in the nervus recurrens sending their axons to different parts of the stomatogastric nervous system were traced. Some axons in the oesophageal nerve arise from large perikarya in the anterior part of the pars intercerebralis and pass via the NCCI to the corpora cardiaca and the oesophageal nerve. The form a profuse dendritic tree in the protocerebrum. Fibers of the NCC I and NCC II as well as the NCA I and NCA II enter the stomatogastric nervous system via the hypocerebral ganglion.

The neuronal connections of the frontal ganglion of the cockroach Periplaneta americana. A histological and iontophoretical study.

The frontal ganglion of the cockroach Periplaneta americana was studied histologically and its neuronal pathways were mapped by use of axonal cobalt iontophoresis. Neurons and fiber tracts of the frontal ganglion are directly linked with different regions of the central nervous system (tritocerebrum, protocerebrum, subesophageal ganglion) and with the more caudal parts of the stomatogastric nervous system (hypocerebral ganglion, nervus oesophagei).