Octopamine promotes rhythmicity but not synchrony in a bilateral pair of bursting motor neurons in the feeding circuit of Aplysia.

Octopamine-like immunoreactivity was localized to a limited number (<40) of neurons in the Aplysia central nervous system, including three neurons in the paired buccal ganglia (BG) that control feeding movements. Application of octopamine (OA) to the BG circuit produced concentration-dependent (10(-8)-10(-4) mol l(-1)) modulatory actions on the spontaneous burst activity of the bilaterally paired B67 pharyngeal motor neurons (MNs). OA increased B67's burst duration and the number of impulses per burst. These effects reflected actions of OA on the intrinsic tetrodotoxin-resistant driver potential (DP) that underlies B67 bursting. In addition to its effects on B67's burst parameters, OA also increased the rate and regularity of burst timing. Although the bilaterally paired B67 MNs both exhibited rhythmic bursting in the presence of OA, they did not become synchronized. In this respect, the response to OA differed from that of dopamine, another modulator of the feeding motor network, which produces both rhythmicity and synchrony of bursting in the paired B67 neurons. It is proposed that modulators can regulate burst synchrony of MNs by exerting a dual control over their intrinsic rhythmicity and their reciprocal capacity to generate membrane potential perturbations. In this simple system, dopaminergic and octopaminergic modulation could influence whether pharyngeal contractions occur in a bilaterally synchronous or asynchronous fashion.

Evidence for a novel chemotactic C1q domain-containing factor in the leech nerve cord.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.

Comparative anatomy of PACAP-immunoreactive structures in the ventral nerve cord ganglia of lumbricid oligochaetes.

By means of a whole mount immunocytochemical approach, the distribution patterns of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 were identified in the ventral nerve cord (VNC) ganglia of the earthworms Eisenia fetida and Lumbricus terrestris. Each PACAP form appears to occur in a distinct neuron population. Positions of these populations, as well as numbers and sizes of the constituting neurons do not essentially differ between the two species. The data suggest that in Lumbricid Oligochaetes, PACAP-27 and PACAP-38 neuron populations may mediate distinct physiological processes.

Serotonergic immunoreactivity in the pedal ganglia of the pulmonate snail Megalobulimus abbreviatus after thermal stimulus: A semi-quantitative analysis.

Using an immunohistochemical procedure and optical densitometry, the distribution of neurons containing serotonin (5-HT) was investigated in the pedal ganglia of Megalobulimus abbreviatus after thermal "non-functional stimulus" (22 degrees C) and stressful thermal conditions (50 degrees C). The animals were sacrificed at different times (3 h, 6 h and 24 h) following these stimuli. In control animals, the results showed the location of these serotonergic immunoreactive elements (5HT-ir) in this ganglion to be similar to those shown in other studies, where the anterior region of ventral sections showed the largest number of 5HT-ir neurons. In the anterior neurons, significant differences (p < 0.01) were observed between the groups of animals stimulated at 50 degrees C and 22 degrees C and sacrificed after 6 h. In the medial neurons, significant differences (p < 0.05) were observed between the control group and the groups of animals stimulated at 50 degrees C and sacrificed after 6 and 24 h. Neuropilar area 1 showed differences (p < 0.01) in 5HT-ir between the control group and the groups of animals stimulated at 50 degrees C and sacrificed after 3 and 24 h. Neuropilar area 2 showed a significant difference (p < 0.05) between the groups of animals stimulated at 22 degrees C and sacrificed after 3 and 24 h. These results suggest the involvement of 5-HT in the nociceptive circuit of M. abbreviatus, mainly that of the medial neurons and neuropilar area 1, which showed increases in 5HT-ir after thermal aversive stimuli. These results could be helpful in drawing cellular homologies with other gastropods.

Analysis of the effects of antibodies to gangliosides on the electrical activity of Retzius neurons in the leech and on the functional activity of influx sodium current channels.

The effects of anti-ganglioside antibodies on the functional states of two types of influx Na+ current channels were studied. Experiments used 20% anti-ganglioside antiserum prepared by standard methods by immunizing rabbits with total bovine brain gangliosides. These experiments showed that incubation of neurons in physiological saline containing antiserum induced discordance in the operation of the two types of influx current Na+ channels responsible for spike generation. This reaction was found to be associated with the slowed rate of activation of TTX-sensitive Na+ channels. Synaptic stimulation of cells in the presence of antiserum induced blockade of TTX-insensitive influx Na+ current channels. High-frequency synaptic activation of cells (10 Hz) showed that apart from blockade of TTX-insensitive Na+ channels, anti-ganglioside antibodies prevented plastic rearrangements in the gate system of TTX-sensitive Na+ channels. This resulted in impairment of the development of the acclimation process - the response of the neuron to high-frequency stimulation seen in normal conditions.

[Effect of the antibodies to gangliosides on the Retzius neuron electrical activity and the functional activity of sodium channels for incoming current in leech]. / Analiz vliianiia antitel k gangliozidam na élektricheskuiu aktivnost' neirona Rettsiusa piiavki i na funktsional'nuiu aktivnost' natrievykh kanalov vkhodiashchego toka.

In the present study, the effect of antibodies to gangliosides on the Retzius neurons of the leech was investigated to study the spike activity and the functional activity of the Na-channels which generate the spike. A forty-minute incubation of the Retzius neurons in a 20% solution of antiganglioside serum in a Ringer solution provoked appearance of a double spike (a spike with two parts) connected with a decrease of the speed of the activation of the tetrodotoxin (TTX)-sensitive Nachannels. The high frequency synaptic activation of the neuron (10 Hz during 10 minutes) under the plasticity exchange of the gate system of the TTX-sensitive Na-channels. As a result of this, there was a disturbance of the habituation of the Retzius neuron to the high-frequency stimulation.

Development of neuronal ganglion xenografts from gastropoda in rat brain.

Survival of neuronal ganglia from newborn snail (Helix aspera L.) in the brain of adult rats was studied. Snail ganglion survived in the brain of warm-blooded animals for 6 months without inducing immune conflict. At early stages (5 days) after transplantation, xenografts increased in size and were several times larger than native ganglia from 10-day-old snails, thereafter (on days 28 and 180) they became smaller still surpassing the sizes of ganglia from snail of the corresponding age. Rapid enlargement of the xenograft was due to cell reactive processes in the ganglion. Deep penetration of large vessels from xenografts to rat brain was observed.

Projection neurons with shared cotransmitters elicit different motor patterns from the same neural circuit.

Specificity in the actions of different modulatory neurons is often attributed to their having distinct cotransmitter complements. We are assessing the validity of this hypothesis with the stomatogastric nervous system of the crab Cancer borealis. In this nervous system, the stomatogastric ganglion (STG) contains a multifunctional network that generates the gastric mill and pyloric rhythms. Two identified projection neurons [modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1)] that innervate the STG and modulate these rhythms contain GABA and the pentapeptide proctolin, but only MCN1 contains Cancer borealis tachykinin-related peptide (CabTRP Ia). Selective activation of each projection neuron elicits different rhythms from the STG. MPN elicits only a pyloric rhythm, whereas MCN1 elicits a distinct pyloric rhythm as well as a gastric mill rhythm. We tested the degree to which CabTRP Ia distinguishes the actions of MCN1 and MPN. To this end, we used the tachykinin receptor antagonist Spantide I to eliminate the actions of CabTRP Ia. With Spantide I present, MCN1 no longer elicited the gastric mill rhythm and the resulting pyloric rhythm was changed. Although this rhythm was more similar to the MPN-elicited pyloric rhythm, these rhythms remained different. Thus, CabTRP Ia partially confers the differences in rhythm generation resulting from MPN versus MCN1 activation. This result suggests that different projection neurons may use the same cotransmitters differently to elicit distinct pyloric rhythms. It also supports the hypothesis that different projection neurons use a combination of strategies, including using distinct cotransmitter complements, to elicit different outputs from the same neuronal network.

A new method for double immunolabelling with primary antibodies from identical species.

There are several double immunolabelling methods but each has its drawbacks. More often than not, antibodies with the required specificities are available in only one species and their use normally produces false labels due to cross-reactivity. We describe a new and reliable technique for staining with primary antibodies from the same species, that can even be employed on tissues of the donor species. The protocol avoids cross-reactivities without loss in sensitivity, uses commercially available reagents and takes advantage of enzymatic detection, although it can be adapted for fluorescent labelling. Briefly, tissue is incubated with one primary antibody, followed by a peroxidase-coupled secondary antibody which is detected using amino ethyl carbazol to give a red reaction product. Meanwhile, the next primary antibody is coupled in vitro to a biotinylated secondary antibody and excess binding sites quenched with normal immune serum from the same species as the primary antibody. This complex is applied to tissue and detected by the avidin-biotin/alkaline phosphatase technique using naphthol-AS-MX-phosphate/Fast Blue BB to produce a blue label. In addition to extensive controls, the reliability and broad applicability of this method has been confirmed in (1) murine skin cryostat sections to co-visualize antigen-presenting cells (MHC class II-immunoreactive; "-ir') with either antigen detecting T lymphocytes (CD4-ir) or Langerhans cells (NLDC-145-ir) and (2) locust (Insecta) abdominal ganglion paraffin sections, where it is known that immunoreactivities for octopamine and a FMRFamide-related peptide are colocalized in only one, uniquely identifiable neuron.

Dopamine in the lobster Homarus gammarus. I. Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the nervous system of the juvenile.

As a catecholamine, dopamine belongs to a class of molecules that have multiple transmitter and hormonal functions in vertebrate and invertebrate nervous systems. However, in the lobster, where many central neurons have been identified and the peripheral innervation pattern is well known, the distribution of dopamine-containing neurons has not been examined in detail. Therefore, immunocytochemical methods were used to identify neurons likely to contain dopamine and tyrosine hydroxylase in the central nervous system of the juvenile lobster Homarus gammarus. Approximately 100 neuronal somata stain for the catecholamine and/or its synthetic enzyme in the brain and ventral nerve cord. The systems of neurons labeled with dopamine and tyrosine hydroxylase antibodies have the following characteristics: 1) the two systems are nearly identical; 2) every segmental ganglion contains at least one pair of labeled neurons; 3) the positions and numbers of cell bodies labeled with each antiserum are similar in the various segmental ganglia; 4) six labeled neurons are anatomically identified; two interneurons from the brain project within the ventral cord to reach the last abdominal ganglion, two neurons from the commissural ganglia are presumably neurosecretory neurons, and two anterior unpaired medial abdominal neurons project to the hindgut muscles; and 5) no cell bodies are labeled in the stomatogastric ganglion, but fibers and terminals in the neuropil are stained. The remarkably small numbers of labeled neurons and the presence of very large labeled somata with far-reaching projections are distinctive features consistent with other modulatory aminergic systems in both vertebrates and invertebrates.

Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea.

The presence and distribution of nitric oxide synthase (NOS) in the CNS and peripheral organs (buccal muscles, oesophagus, salivary glands, foot, mantle and pneumostome) of the pulmonate mollusc, Lymnaea stagnalis were studied using an antiserum developed against rat cerebellar NOS. NOS-immunopositive neurones in Lymnaea were localized predominantly in the buccal ganglia as well as in distinct areas of the cerebral and suboesophageal ganglia. NOS-immunoreactive terminals were also found on the somata of some central neurones. In the periphery, NOS-immunostaining was detected only in a few neurones in the pneumostome area and in the osphradial ganglion. In addition, approximately 100 NOS-immunopositive cells have been found in the salivary glands. Our data supports other recent reports indicating that NO may be a signal molecule in the CNS of molluscs.

Ontogeny of serotonin-immunoreactive neurons in juvenile Aplysia california: implications for the development of learning.

Serotonin has been implicated in both nonassociative learning (sensitization and dishabituation) as well as associative learning (classical conditioning) in Aplysia californica. Dishabituation and sensitization, and their underlying physiological analogs, emerge according to different developmental timetables--sensitization develops 4 to 6 weeks after dishabituation (Rankin & Carew, 1988; Nolen & Carew, 1988; Wright, McCance, Lu, & Carew, 1991). Since the late emergence of sensitization could result from the delayed expression of facilitatory neurotransmitters, we have examined the ontogeny of serotonin immunoreactivity in juvenile A. californica by means of indirect immunohistofluorescence. The purpose of these experiments was to describe the developmental timetable for the expression of serotonin immunoreactivity and to correlate the emergence of immunoreactive neurons with the ontogenetic expression of different forms of learning. While the addition of serotonin-immunoreactive cells tracked the growth of the central nervous system, juveniles contained a relatively higher proportion of immunoreactive cells than adults. Immunoreactive cell bodies were present in the abdominal, cerebral, and pedal ganglia as early as juvenile Stage 9, prior to the emergence of dishabituation in Stage 10. The posterior cerebral cluster (PCC) contained four pairs of immunoreactive cells by Stage 9, including the facilitator CB1, which, as shown in adults, heterosynaptically facilitates siphon sensory neurons. The PCC reached the adult complement of five pairs of cells, by Stage 10, long before the development of sensitization, but at the time corresponding to the emergence of dishabituation. These results suggest that the late emergence of sensitization is not simply a consequence of the late expression of serotonergic facilitatory interneurons.

Neurotensin-like immunoreactivity in locust supraesophageal ganglion and optic lobes.

A substance immunoreactive to antibodies directed against bovine neurotensin (NT) was localized in neurons in the supraesophageal ganglion (SEG) and optic lobes of larval and adult Locusta migratoria L. Two large somata were located in the caudal cortex, ventral to the calyces and symmetrical to the median of the SEG. Four smaller somata also in the caudal cortex were located as two symmetrical pairs at the level of the central body. These somata formed a diffuse network of varicose fibers from the superior lateral to the ventro-lateral protocerebrum between the pedunculi and frontal cortical region. Some fibers crossed the median to the contralateral sides of the SEG. Another pair of immunoreactive somata whose terminating processes remained unclear was found at the level of the antennal lobes. Intrinsic networks of fibers were labeled in the accessory medulla and in layer 4/5 of the medulla. These fibers originated from 8-10 small somata near the dorso-frontal rim of the medulla. All larval stages contained these NT-like immunoreactive structures. Results from isoelectric focusing and press-blot analysis of SEG homogenates, synthetic neurotensin and neurotensin fragments indicate that this substance is similar to bovine neurotensin(1-13).

Taurine-like immunoreactivity in octopaminergic neurones of the cockroach, Periplaneta americana (L.).

Taurine (2-aminoethanesulphonic acid) is reported to interact with the octopaminergic system. The distribution of taurine-like immunoreactivity (-LIR) in relation to octopamine-like immunoreactive dorsal unpaired median (DUM) neurones was investigated with the aim of revealing possible colocalization of these two neuromediators. The specificity of the anti-taurine serum used was demonstrated by dot blot immunoassay and by use of preabsorption controls. There was no crossreactivity with octopamine. The specificity of the octopamine antiserum employed has been described elsewhere. Taurine-LIR could be demonstrated in large dorso-median cells in the suboesophageal and the mesothoracic ganglion as well as in the abdominal ganglia. In addition taurine-LIR is distributed in numerous other regions of the ganglia. A comparison of the immunostaining for taurine and octopamine indicates that several of the taurine-like immunoreactive (-LI) neurones are probably members of the octopamine-immunoreactive DUM cell population. These taurine-LI neurones resemble octopamine-LI DUM cells in soma position and size as well as in the projections of their primary neurites. Colocalization of octopamine-LIR and taurine-LIR within the same neuronal element could be shown by alternate immunostaining of consecutive sections. It is probable that all octopamine-LI DUM neurones also exhibit taurine-LIR, and the possible physiological significance of this coexistence is discussed.

Distribution of serotonin-like immunoreactive neurones in the embryonic nervous system of lymnaeid and planorbid snails.

The distribution of 5HT-like immunoreactive (5HTLI) neurones was investigated in lymnaeid and planorbid snail embryos. The time of appearance and the pattern of development of homologous 5HTLI cells in the CNS were found to be similar in all species investigated. However, 5HTLI cells characteristic only of planorbid embryos were also found. These specific neurones which were detected outside the future CNS at stage E30 of embryogenesis and which disappeared close to hatching, were named transient serotoninergic 1 cells. A correlation is suggested between the embryogenetic development of 5HTLI neurones and the development of 5HT-dependent motor behaviour in these animals.