Marijuana smoke induces severe pulmonary hyperresponsiveness, inflammation, and emphysema in a predictive mouse model not via CB1 receptor activation.

Sporadic clinical reports suggested that marijuana smoking induces spontaneous pneumothorax, but no animal models were available to validate these observations and to study the underlying mechanisms. Therefore, we performed a systematic study in CD1 mice as a predictive animal model and assessed the pathophysiological alterations in response to 4-mo-long whole body marijuana smoke with integrative methodologies in comparison with tobacco smoke. Bronchial responsiveness was measured with unrestrained whole body plethysmography, cell profile in the bronchoalveolar lavage fluid with flow cytometry, myeloperoxidase activity with spectrophotometry, inflammatory cytokines with ELISA, and histopathological alterations with light microscopy. Daily marijuana inhalation evoked severe bronchial hyperreactivity after a week. Characteristic perivascular/peribronchial edema, atelectasis, apical emphysema, and neutrophil and macrophage infiltration developed after 1 mo of marijuana smoking; lymphocyte accumulation after 2 mo; macrophage-like giant cells, irregular or destroyed bronchial mucosa, goblet cell hyperplasia after 3 mo; and severe atelectasis, emphysema, obstructed or damaged bronchioles, and endothelial proliferation at 4 mo. Myeloperoxidase activity, inflammatory cell, and cytokine profile correlated with these changes. Airway hyperresponsiveness and inflammation were not altered in mice lacking the CB1 cannabinoid receptor. In comparison, tobacco smoke induced hyperresponsiveness after 2 mo and significantly later caused inflammatory cell infiltration/activation with only mild emphysema. We provide the first systematic and comparative experimental evidence that marijuana causes severe airway hyperresponsiveness, inflammation, tissue destruction, and emphysema, which are not mediated by the CB1 receptor.

The 5-HT immunoreactive innervation of the Helix procerebrum.

In the procerebrum of terrestrial snails, 5-HT is a key modulatory substance of the generation of synchronous oscillatory activity and odor learning capability. In this study, we have analyzed the characteristics of the 5-HT-immunoreactive (5-HT-IR) innervation of the distinct anatomical regions of the procerebrum of Helix pomatia, applying correlative light- and electron microscopic immunocytochemistry. A dense network of 5-HT-IR innervation was demonstrated in the cell body layer, meanwhile a varicose fiber system of different density occurred in the different neuropil regions. At the ultrastructural level, labeled varicosities were found to contact both procerebral cell bodies, and different unlabeled axon profiles in the neuropils. The labeled structures established mostly close non-specialized membrane contacts with the postsynaptic profiles. The overall dense distribution of 5-HT-IR innervation supports a general modulatory role of 5-HT in processing different olfactory events.

The neuroanatomical and ultrastructural organization of statocyst hair cells in the pond snail, Lymnaea stagnalis.

The ultrastructure, neuroanatomy and central projection patterns, including the intercellular connections of the statocyst hair cells of the pond snail, Lymnaea stagnalis, were studied, applying different intra- and extracellular cellular staining techniques combined with correlative light- and electron microscopy. Based on the ultrastructure different hair cells could be distinguished according to their vesicle and granule content, meanwhile the general organization of the sensory neurons was rather uniform, showing clearly separated perinuclear and "vesicular" cytoplasmic regions. Following intra- and extracellular labeling with fluorescence dyes or HRP a typical, local arborization of the hair cells was demonstrated in the cerebral ganglion neuropil, indicating a limited input-output system connected to the process of gravireception. Correlative light- and electron microscopy of HRP-labeled hair cells revealed both axo-somatic and axo-axonic output contacts of hair cell varicosities, and input on sensory axons located far from the terminal arborizations. Our findings suggest (i) a versatile ultrastructural background of hair cells corresponding possibly to processing different gravireceptive information, and (ii) the synaptic (or non-synaptic) influence of gravireception at different anatomical (terminal, axonal and cell body) levels when processed centrally. The results may also serve as a functional morphological background for previously obtained physiological and behavioral observations.

Immunodetection and localization of nitric oxide synthase in the olfactory center of the terrestrial snail, Helix pomatia.

The procerebrum of stylommatophoran snails produces nitric oxide (NO)-modulated oscillatory local field potentials which are considered the basis of olfactory information processing. Although the function of NO is well characterized in the PC, the identification and distribution of NO synthase (NOS) has not known completely. In the present study, applying a mammalian anti-NOS antibody, a 170 kDa molecular weight NOS-like protein was demonstrated in the procerebrum homogenate of Helix pomatia. NOS-like immunolabeling of the globuli cells, the internal and terminal neuropils displayed an identical distribution compared to that of NADPH-diaphorase reactive material, confirming the specificity of immunohistochemistry. The detailed characteristics of the immunostaining (different intensity of the neural perikarya, a gradual appearance in the terminal neuropil and in the axon bundles of the tentacular nerve, as well as an intense, homogeneous distribution of NOS-like immunoreactivity in the internal neuropil) suggest that NOS is expressed constitutively, maintaining a high level of the enzyme in neuropil areas. NOS accumulation in the internal neuropil suggests that NO plays an important role in delivering olfactory signals extrinsic to the procerebrum, and integrating them with other sensory modalities, respectively. Our results are the first, demonstrating unequivocally the presence of NOS and resolving its differential distribution in the Helix procerebrum.

Morphology, ultrastructure and contractile properties of muscles responsible for superior tentacle movements of the snail.

Bending, twitching and quivering are different types of tentacle movements observed during olfactory orientation of the snail. Three recently discovered special muscles, spanning along the length of superior tentacles from the tip to the base, seem to be responsible for the execution of these movements. In this study we have investigated the ultrastructure, contractile properties and protein composition of these muscles. Our ultrastructural studies show that smooth muscle fibers are loosely embedded in a collagen matrix and they are coupled with long sarcolemma protrusions. The muscle fibers apparently lack organized SR and transverse tubular system. Instead subsarcolemmal vesicles and mitochondria have been shown to be possible Ca2+ pools for contraction. It was shown that external Ca2+ is required for contraction elicited by high (40 mM) K+ or 10-4 M ACh. Caffeine (5 mM) induced contraction in Ca2+-free solution suggesting the presence of a substantial intracellular Ca2+ pool. High-resolution electrophoretic analysis of columellar and tentacular muscles did not reveal differences in major contractile proteins, such as actin, myosin and paramyosin. Differences were observed however in several bands representing presumably regulatory enzymes. It is concluded that, the ultrastructural, biochemical and contractile properties of the string muscles support their special physiological function.

Potassium channels in the Helix central nervous system: preliminary immunohistochemical studies. Short communication.

Distribution of the potassium channel of Kv4.3 type was investigated in the central nervous system (CNS) of Helix pomatia by immunohistochemistry. Immunopositive neurons were found widely distributed in the CNS, present mostly in smaller groups in the different central ganglia but not in the visceral ganglion. Labeled fibers were characteristic for not only the neuropils of all ganglia but also the connective tissue sheath around the CNS and the aorta wall were richly innervated. Western blot analysis revealed a clear identity with the mammalian Kv4.3 subunit, suggesting an evolutionary conserved structure of this channel type. Our preliminary results provide a steady basis for further experiments aiming partly at the identification of other potassium channel types and partly the ultrastructural localization of Kv4.3.

Inhibitory action of endomorphin-1 on sensory neuropeptide release and neurogenic inflammation in rats and mice.

Substance P (SP) and calcitonin gene-related peptide (CGRP) released from capsaicin-sensitive sensory nerves induce local neurogenic inflammation in the innervated area. The aim of the present study was to investigate the effects of an endogenous opioid peptide, endomorphin-1, on sensory neuropeptide release in vitro and acute neurogenic and non-neurogenic inflammatory reactions in vivo. Electrical field stimulation (EFS; 40 V, 0.1 ms, 10 Hz, 120 s; 1200 impulses) was performed to evoke SP and CGRP release from peptidergic afferents of the isolated rat tracheae which was determined from the incubation medium with radioimmunoassay. Neurogenic inflammation in the skin of the acutely denervated rat hind paw was induced by topical application of 1% mustard oil and detected by Evans Blue leakage. Mustard oil-induced ear swelling of the mouse was determined with a micrometer during 3 h and myeloperoxidase activity as an indicator of granulocyte accumulation was measured with spectrophotometry at 6 h. EFS evoked about a twofold elevation in the release of both pro-inflammatory sensory neuropeptides. Endomorphin-1 (5 nM-2 microM) diminished the release of SP and CGRP in a concentration-dependent manner, the EC50 values were 39.45 nM and 10.84 nM, respectively. The maximal inhibitory action was about 80% in both cases. Administration of endomorphin-1 (1-100 microg/kg i.p.) dose-dependently inhibited mustard oil-evoked neurogenic plasma protein extravasation in the rat skin as determined by microg Evans Blue per g wet tissue. Repeated i.p. injections of the 10 microg/kg dose three times per day for 10 days did not induce desensitization in this model. Neurogenic swelling of the mouse ear was also dose-dependently diminished by 1-100 microg/kg i.p. endomorphin-1, but non-neurogenic neutrophil accumulation was not influenced. These results suggest that endomorphin-1 is able to inhibit the outflow of pro-inflammatory sensory neuropeptides. Based on this mechanism of action it is also able to effectively diminish neurogenic inflammatory responses in vivo.

Functional neuroanatomy of the 5-HTergic system in the developing and adult buccal complex of the pond snail, Lymnaea stagnalis.

Organization of the innervation of the buccal region by 5-HT-immunoreactive (IR) elements was investigated in the pond snail, Lymnaea stagnalis, with special attention to developmental aspects. A gradual maturation is characteristic for the 5-HT-IR muscle innervation, appearing first by late (E80-90%) embryogenesis. It runs parallel with the muscle development and the maturation of the 5-HTergic innervation in the buccal ganglia, peaking by the mid-postembryogenesis (P3) with the presence of a 5-HT-IR network in the buccal mass and rich innervation in the buccal ganglia, including axo-somatic contacts. The whole process seems to match with the appearance of the adult-like feeding (radula protrusion).

GABAergic synaptic connections in mushroom bodies of insect brains.

Distribution and synaptic connections of GABA fibres in neuropil parts of the mushroom bodies in brains of crickets (Gryllus bimaculatus) and bees (Apis mellifera) were investigated by immuno-light and electron microscopy. In the inner calyx neuropil of cricket mushroom bodies, GABA fibres are pre- and post-synaptically connected with proximal Kenyon cell dendrites, indicating synaptic contacts differing from those of the Kenyon cell dendritic tips in the peripheral microglomeruli of the calyces. A more complex interaction of GABAergic fibres and Kenyon cell dendrites than assumed before is shown. In the mushroom bodies of bees, dendritic like strata of GABA fibre projections contribute to the subcompartmental layers of the vertical lobe. The GABA-immunostained fibre profiles exhibit pre- and postsynaptic sites as well and can therefore not be considered purely postsynaptic dendritic neuron parts. The micromorphology and synaptic contacts of the dendrites and dendritic like arborizations are seen as parts of local circuits within mushroom bodies.

Inhibitory effect of PACAP-38 on acute neurogenic and non-neurogenic inflammatory processes in the rat.

Inhibitory actions of pituitary adenylate cyclase activating polypeptide (PACAP) have been described on cellular/vascular inflammatory components, but there are few data concerning its role in neurogenic inflammation. In this study we measured PACAP-like immunoreactivity with radioimmunoassay in the rat plasma and showed a two-fold elevation in response to systemic stimulation of capsaicin-sensitive sensory nerves by resiniferatoxin, but not after local excitation of cutaneous afferents. Neurogenic plasma extravasation in the plantar skin induced by intraplantar capsaicin or resiniferatoxin, as well as carrageenan-induced paw edema were significantly diminished by intraperitoneal PACAP-38. In summary, these results demonstrate that PACAP is released from activated capsaicin-sensitive afferents into the systemic circulation. It diminishes acute pure neurogenic and mixed-type inflammatory reactions via inhibiting pro-inflammatory mediator release and/or by acting at post-junctional targets on the vascular endothelium.

Effect of pituitary adenylate cyclase activating polypeptide-38 on sensory neuropeptide release and neurogenic inflammation in rats and mice.

Substance P (SP) and calcitonin gene-related peptide (CGRP), released from capsaicin-sensitive sensory nerves induce local neurogenic inflammation, while somatostatin exerts systemic anti-inflammatory actions. The aim of the present study was to investigate the release of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) and its effects on sensory neuropeptide release in vitro and acute neurogenic ear swelling in vivo. Capsaicin (10(-6) M) or electrical field stimulation (EFS; 40 V, 0.1 ms, 10 Hz, 120 s; 1200 impulses)-induced release of PACAP-38, SP, CGRP and somatostatin from isolated rat tracheae was measured with radioimmunoassay. Mustard oil-induced neurogenic inflammation in the mouse ear was determined with a micrometer and in the rat hind paw skin by the Evans Blue leakage technique. Capsaicin and EFS evoked 27% and more than twofold elevation of PACAP-38 release respectively, compared with the prestimulated basal values from isolated trachea preparation. Exogenously administered PACAP-38 (20-2000 nM) diminished both capsaicin- and EFS-evoked sensory neuropeptide release in a concentration-dependent manner. The maximal inhibitory effects of PACAP on capsaicin-induced substance P, CGRP and somatostatin release amounted to 75.4%, 73.3% and 90.0%, while EFS-evoked release of these peptides was 80.03%, 87.7% and 67.7%. In case of capsaicin stimulation the EC50 values for substance P, CGRP and somatostatin were 82.9 nM, 60.1 nM and 66.9 nM, respectively. When EFS was performed, these corresponding EC50 data were 92.1 nM, 67.8 nM and 20.9 nM. PACAP-38 (10, 100 and 1000 microg/kg i.p. in 200 microl volume) inhibited neurogenic ear swelling in the mouse. Furthermore, 100 microg/kg i.p. PACAP also significantly diminished mustard oil-evoked plasma protein extravasation in the rat skin. These results suggest that PACAP-38 is released from the stimulated peripheral terminals of capsaicin-sensitive afferents and it is able to inhibit the outflow of sensory neuropeptides. Based on this mechanism of action PACAP is also able to effectively diminish/abolish neurogenic inflammatory response in vivo after systemic administration.

Effects of the somatostatin receptor subtype 4 selective agonist J-2156 on sensory neuropeptide release and inflammatory reactions in rodents.

BACKGROUND AND PURPOSE: Substance P (SP) and calcitonin gene-related peptide (CGRP) released from capsaicin-sensitive sensory nerves induce local neurogenic inflammation; somatostatin exerts systemic anti-inflammatory actions presumably via sst4/sst1 receptors. This study investigates the effects of a high affinity, sst4-selective, synthetic agonist, J-2156, on sensory neuropeptide release in vitro and inflammatory processes in vivo. EXPERIMENTAL APPROACH: Electrically-induced SP, CGRP and somatostatin release from isolated rat tracheae was measured with radioimmunoassay. Mustard oil-induced neurogenic inflammation in rat hindpaw skin was determined by Evans blue leakage and in the mouse ear with micrometry. Dextran-, carrageenan- or bradykinin-induced non-neurogenic inflammation was examined with plethysmometry or Evans blue, respectively. Adjuvant-induced chronic arthritis was assessed by plethysmometry and histological scoring. Granulocyte accumulation was determined with myeloperoxidase assay and IL-1beta with ELISA. KEY RESULTS: J-2156 (10-2000 nM) diminished electrically-evoked neuropeptide release in a concentration-dependent manner. EC50 for the inhibition of substance P, CGRP and somatostatin release were 11.6 nM, 14.3 nM and 110.7 nM, respectively. J-2156 (1-100 microg kg(-1) i.p.) significantly, but not dose-dependently, inhibited neurogenic and non-neurogenic acute inflammatory processes and adjuvant-induced chronic oedema and arthritic changes. Endotoxin-evoked myeloperoxidase activity and IL-1beta production in the lung, but not IL-1beta- or zymosan-induced leukocyte accumulation in the skin were significantly diminished by J-2156. CONCLUSIONS AND IMPLICATIONS: J-2156 acting on sst4 receptors inhibits neuropeptide release, vascular components of acute inflammatory processes, endotoxin-induced granulocyte accumulation and IL-1beta synthesis in the lung and synovial and inflammatory cells in chronic arthritis. Therefore it might be a promising lead for the development of novel anti-inflammatory drugs.

Topographic organization of serotonergic and dopaminergic neurons in the cerebral ganglia and their peripheral projection patterns in the head areas of the snail Helix pomatia.

The distribution of monoaminergic neurons within the cerebral ganglia was investigated in the pulmonate snail Helix pomatia. Simultaneous serotonin and tyrosine hydroxylase double immunostaining revealed that the immunoreactive cell groups are concentrated in a putative monoaminergic center on the ventral surface of the cerebral ganglia. Simultaneous cobalt (Co)- and nickel (Ni)-lysine backfills of cerebral nerves were combined with 5, 6-dihydroxytryptamine pigment-labelling of serotonergic neurons, or with fluorescence immunocytochemistry of dopaminergic neurons. This showed that the serotonergic and dopaminergic cell groups can be divided into smaller subgroups on the basis of their axonal projections into different cerebral nerves. These subgroups show a topographic organization within the serotonergic and dopaminergic neuronal clusters. In the serotonergic system, the different regions of the head are represented in a rostrocaudal direction, whereas a caudorostral organization is characteristic for the dopaminergic system. No serotonin- or dopamine-immunoreative cell bodies but numerous fibers were observed in the head areas, indicating that these are innervated by cerebral monoaminergic neurons and show different innervation patterns. Serotonin-immunoreactive fibers mostly innervate muscle fibers, whereas dopamine-immunoreactive processes do not innervate effector cells, but terminate within the nerve branches of the head areas. On the basis of their innervation pattern, we suggest that dopaminergic neurons may take part in en route modulation of sensory afferent and efferent processes in an as yet unknown manner. The serotonergic neurons, on the other hand, may play a direct role in the modulation of muscle function.

Dopamine-immunoreactive neurones in the central nervous system of the pond snail Lymnaea stagnalis.

The distribution of dopamine and dopamine-immunoreactive neurones was studied in the central nervous system of the snail Lymnaea stagnalis. The results from immunocytochemical labelling were compared with those from the application of the glyoxylic acid fluorescence method and 6-hydroxydopamine-induced pigment labelling. Comparisons were also made between the number of dopamine immunoreactive neurones and the dopamine content of the ganglia, measured by high-performance liquid chromatography. Dopamine immunocytochemistry proved to be superior to the other two histochemical techniques in terms of specificity and sensitivity. The 6-hydroxydopamine-induced pigment labelling failed to prove a useful tool for the in vivo identification of all dopamine-containing neurones. The distribution and number of dopamine-immunoreactive neurones and levels of biochemically measured dopamine in specific ganglia showed a close correspondence. By using the results of the dopamine immunocytochemistry and glyoxylic acid technique, a detailed map of dopamine-containing neurones was constructed. Dopamine-containing inter- and intra-ganglionic axon tracts were also demonstrated. The mapping of dopamine-containing neurones will facilitate further neurophysiological analysis of dopaminergic neural mechanisms in Lymnaea.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors.

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120-140 fmol per animal by around E40. Dopamine content remained stable during mid-embryogenesis (E40-65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde- and glyoxylate-induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32-35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: II. Postembryonic development of central and peripheral cells.

Catecholamines have long been thought to play important roles in different mollusc neural functions. The present study used glyoxylate- and aldehyde-induced histofluorescence to identify central and peripheral catecholaminergic neurons in the snail Lymnaea stagnalis. The majority of these cells were also found to react to antibodies raised against tyrosine hydroxylase. A minority of the catecholaminergic neurons, however, exhibited no such immunoreactivity. The number of central catecholaminergic neurons nearly doubled (from about 45 to about 80 cells) during the first 2-3 days of postembryonic development. Thereafter, catecholaminergic neurons again doubled in number and generally grew by about 100-200% in soma diameter as the snails grew by 1,000% in overall linear measurements. In contrast to the relatively meager addition of central catecholaminergic neurons, several thousand catecholaminergic somata were added to different peripheral tissues during postembryonic development. These small, centrally projecting neurons were particularly concentrated in the lips, esophagus, anterior margin of the foot, and different regions of the male and female reproductive tracts. Chromatographic analyses indicated that dopamine was the major catecholamine present in the central ganglia, foot, and esophagus, although detectable levels of norepinephrine (approximately 20% of dopamine levels) were also found in the ganglia. The total content but not the concentration of dopamine increased within the tissue samples during postembryonic development. The companion study (Voronezhskaya et al. [1999] J. Comp. Neurol. 404:285-296) and the present study furnish a complete description of central and peripheral catecholaminergic neurons from their first appearance in early embryonic development to adulthood.

Peptidergic neurons in the snail Helix pomatia: distribution of neurons in the central and peripheral nervous systems that react with an antibody raised to the insect neuropeptide, leucokinin I.

In this study, an antiserum raised against an insect myotropic peptide, leucokinin I (DPAFNSWGamide), was used for mapping leucokinin-like immunoreactive (LK-LI) neurons in the gastropod mollusc, Helix pomatia. Immunocytochemistry performed on both whole-mounts and cryostat sections demonstrated LK-LI neurons in all ganglia of the central nervous system (CNS), except the visceral ganglion. Altogether about 700 immunolabelled neurons have been found, with nearly one-half (46%) in the cerebral ganglia. A large proportion of the LK-LI neurons have small cell bodies and are likely to be interneurons. The most prominent LK-LI cell group is represented by the entire neuron population of the mesocerebri, which is the major source of a thick fiber bundle system, encircling and innervating the whole CNS. One single LK-LI giant neuron was found, which is located in the left pedal ganglion and is termed GLPdLKC (giant left pedal leucokinin immunoreactive cell). This cell has not been identified previously. The ganglion neuropils are heavily innervated by varicose LK-LI fiber arborizations. Some integrative centers, such as the medullary neuropil of the procerebri, reveal an extreme density of LK-LI innervation. All major peripheral nerves contain a large number of LK-LI axons, and LK-LI innervation is found in the musculature of different peripheral organs (buccal mass, lip, tentacles, oviduct, intestine). Among the peripheral organs investigated, the intestine contains a rich varicose LK-LI network, composed of both intrinsic and extrinsic elements. Radioimmunoassay (RIA) demonstrates a very high content of LK-LI material in Helix ganglion extracts (about 50 pmol/CNS). This is the first report on the occurrence of a substance resembling the myotropic neuropeptide leucokinin I in a phylum outside arthropods. Based on our immunocytochemical observations, a role for leucokinin-like peptides in both central and peripheral regulatory processes in Helix is suggested. According to double-labelling experiments, only a small number of the LK-LI neurons are labelled with an antibody to the vertebrate tachykinin substance P.

Serotonin-immunoreactive varicosities in the cell body region and neural sheath of the snail, Helix pomatia, ganglia: an electron microscopic immunocytochemical study.

The distribution and connections of serotonin-immunoreactive fibers in the cell body region and neural sheath of the central ganglia of the snail, Helix pomatia, have been examined. The cell body region of the ganglia is supplied by an extremely dense network of varicose serotonin-immunoreactive fibers which surround neuronal perikarya in the ganglia. Immunoreactive processes also run to the neural sheath of both the ganglia and the peripheral nerve roots, forming a dense network. Electron microscopy revealed five different connections of serotonin-immunoreactive varicosities, according to their target: (i) non-specialized contacts with neuronal perikarya; (ii) non-specialized contacts with axon processes on the surface of the peripheral nerve roots; (iii) non-specialized neuromuscular connections with smooth muscle fibers in the neural sheath; (iv) varicosities engulfed by glial processes in both the cell body region and neural sheath; (v) varicosities embedded in the connective tissue elements of the sheath either partly or completely free of glial processes. In all cases of appositions no membrane specializations could be observed on either site of the contacts. These observations provide morphological evidence for non-synaptic regulatory actions of serotonin-containing neurons in Helix central nervous system: (i) modulation of the activity of neuronal perikarya; (ii) involvement in neuromuscular regulation; (iii) neurohormonal modulation of peripheral processes by release through the neural sheath.

The mormyrid brainstem--III. Ultrastructure and synaptic organization of the medullary "pacemaker" nucleus.

The ultrastructure and synaptic organization of the presumed medullary pacemaker nucleus, nucleus c of the weakly electric mormyrid fish, Gnathonemus petersii has been investigated. Nucleus c consists of about 12-15 small (20-25 micron) neurones (P-cells), which form a group situated ventrally to the medullary relay nucleus and embedded in a neuropil of myelinated fibres and dendritic processes. The P-cells often exhibit an enhanced electron density of their cytoplasm and dendroplasm. They possess several dendrites of different diameter, a short, thin axon initial segment and a thickly myelinated axon running in dorsal direction. The pacemaker neurons are interconnected by complex electronic coupling, established by somatosomatic, dendrosomatic and dendrodendritic gap junctions. Perikarya and dendrites are frequently interconnected serially by gap junctions; dendrites showed sometimes triadic gap-junction arrangement. It is suggested that this high degree of electrotonic coupling amongst the pacemaker cells represents the first level of the highly ordered synchronization processes which characterize the electric discharge command system of Gnathonemus. Pacemaker cells receive synaptic input from club endings with mixed synapses and from bouton-like terminals with chemical synapses, both of them originating from medium-sized myelinated fibres and contacting mainly neuronal perikarya and dendritic processes. The axon initial segment receives only few synaptic inputs. Bouton-like terminals were found to be of two types according to their vesicle content, namely, boutons with ovoid, clear synaptic vesicles forming Gray type-1 synapses and boutons with pleomorphic clear synaptic vesicles forming Gray type-2 synapses. Different functional roles for the two types of boutons in modulating pacemaker cell activity are suggested.

Immunocytochemical evidence for the GABAergic innervation of the stretch receptor neurons in crayfish.

The GABAergic innervation of the stretch receptor neurons of the crayfish Orconectes limosus has been investigated by means of light- and electron microscope immunocytochemistry using an antibody to GABA. Both whole-mount preparations and post-embedding semithin sections revealed a massive GABAergic innervation of both the slowly and the fast adapting receptor neurons. The stretch receptor organ is supplied by one principle GABA-immunoreactive axon, which gives off several branches that innervate the receptor neurons. Cell body, initial axon segment and dendritic region of the sensory neurons are covered by numerous GABA-immunoreactive varicose fibers. Electron microscopy revealed that the GABA-immunoreactive varicosities establish specialized synaptic contacts with the sensory neurons. The functional significance of the occurrence of GABA-immunoreactive varicosities on the different parts of the sensory neurons is discussed. The results support the physiological and pharmacological evidence that GABA is a transmitter substance of the efferent inhibitory neurons which innervate the crayfish stretch receptor neurons.