Paraneurons are receptosecretory cells producing substances of neurons. Like neurons, they are characterized by membrane-bound granules and vesicles which contain bioactive (and inactive) peptides, amines or other classic messengers, adenine nucleotides and acidic carrier proteins including chromogranins.
The cell-biological significance of the carrier proteins in the secretory granules and the utility of immunohistochemical detection of chromogranins as reliable markers of paraneurons are emphasized.
Cells “ectopically” producing bioactive peptides and/or amines, including ANP producing muscle cells of the heart, do not contain chromogranin immunoreactivity.
The boundary between the neuron and paraneuron and that of the paraneuron are unclear because the cells comprise gradational spectra from typical ones to atypical ones. It is arbitrary whether to include the mast cell in the paraneurons, although it seems reasonable to exclude it by defining a paraneuron as being epithelial in nature.

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This paper reviews the sequence of events in stimulus-secretion coupling in neurons and paraneurons which can be divided into three main steps: the reception of the stimuli, the conduction of excitation, and the extrusion of messenger substances. One physological criterion of the paraneuron is that its conductile region is believed to be short enough to transmit the net electrogenesis to the output portion. This contrasts with the neuron in which the depolarization in the input region can initiate spikes which in turn propagate via the axonal membrane to the output portion. The final step of the coupling, the extrusion of the secretory products by exocytosis, is common between neurons and paraneurons. The intracellular messengers acting rather directly in exocytosis are [Ca²⁺]_c and diacylglycerol; both of these seem to be under physiological control. An increase in [Ca²⁺]_c may also act on mitochondria to acceleate the synthesis of ATP, which may be used as the principal energy source and substrate for diacylglycerol-induced protein phosphorylation. In contrast to the common mechanism in the extrusion step of messenger substances, there is a wide diversity in the reception step in the variety of chemical and physical stimuli. This diversity is recognized not only among various neurons and paraneurons but also among the same type of cell in different species and at different stages of adaptation.

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This paper reviews the immunohistochemical distribution of four brain-derived proteins, NSE, NFP, spot 35 and S-100 protein, in neuronal and paraneuronal tissues, concentrating on the results of our own research group.
Neuron-specific enolase (NSE), a brain-specific isozyme of the glycolytic enzyme enolase, is characterized by its consistent occurrence in the cytoplasm of mature neurons. Immunoreactivity for NSE has been found in almost all paraneurons of both sensory and endocrine nature, suggesting that a unique system of intracellular energy metabolism may be shared by neurons and paraneurons.
Neurofilament protein (NFP), a neuronal cytoskeletal protein, is immunohistochemically recognized in only a part of neurons, apparantly due to the scarcity in neurofilaments in some neurons or to a decreased antigenicity for NFP in the cell. Similarly, only small populations of cells in restricted types of paraneurons, including gut endocrine cells and thyroid C cells, are immunoreactive for NFP. However, the potentiality for paraneurons to express NFP is given credence by the fact that its immunoreactivity is rather frequently found in the neoplasmas of paraneurons which normally do not show this immunoreactivity.
Spot 35 protein which is a cerebellar Purkinje cell-specific protein, has displayed immunohistochemical localization in sensory and endocrine paraneurons as well as some neurons including a specific type of intramural nerve cells in the gut. The selective localization of this calcium-binding protein may be correlated with the secretory function and calcium-dependent excitable membrane property in those cells.
S-100 protein, another brain-derived calcium-binding protein, is contained in sustentacular cells of paraneuronal organs as well as glial elements of nervous systems. Although it is now widely accepted that S-100 protein is not “glia-specific” but contained in a variety of non-glial cells, its β subunit protein is still a useful marker for a series of glial and sustentacular cells.

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Studies of proteins specific to a certain type of cell are of general interest because these proteins may be involved in the determination of morphological and functional characteristics of the cell. In the nervous tissue, many “neuron-specific” and “glia-specific” proteins have been identified by various biochemical and immunohistochemical means and their relationships to cell functions have been studied. This paper briefly reviews our studies on the chemical and cell-biological aspects of the neuron- and glia-specific proteins, with special reference to the 14-3-3 protein and the S100 protein. Our studies suggest that glial protein S100 may be involved in the regulation of cell growth and differentiation, while neuronal protein 14-3-3, in the regulation of serotonin and catecholamine biosynthesis in neurons and other monoamine-synthesizing cells.

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Among glucose-recognizing paraneurons, the gustatory cell is believed to have a glucoreceptor, while the pancreatic B cell is thought to metabolize glucose for signal production for insulin release. To investigate whether a common mechanism of glucose recognition exists among these cells, we have studied the interaction of the anomers of glucose or its derivative, known as a sugar taste inhibitor, with the pancreatic B cell of normal and diabetic rats. Inhibitors of the sugar taste response inhibited glucose-induced insulin release in various manners. A non-specific inhibitor, dibucaine, impaired not only the insulin response to glucose but also the ability to discriminate the anomers of glucose, without inhibiting glucose oxidation in the islets. Dibucaine also inhibited insulin release induced by a non-glucose secretagogue, tolbutamide. The α anomer, but not the β anomer, of p-nitrophenyl-D-glucopyranoside, a specific-competitive inhibitor of the sugar taste response, inhibited glucose-induced insulin release, but did not inhibit insulin release induced by non-glucose secretagogues or glucose oxidation. The pancreatic B cell of a non-insulin-dependent diabetes (NIDD) rat model, the NSZ rat, exhibited low insulin response to glucose and did not discriminate between the two anomers of glucose. The diabetic B cell responded to non-glucose secretagogues to the same extent as the control. Glucose oxidation in the diabetic islets was not impaired. These findings, together with previous ones, suggest that the gustatory and pancreatic B cells have a common glucose recognition site, which has a steric preference for the α anomer and is impaired in NIDD.

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The localization of glycine (GR) and adrenergic receptors (AdR) was examined in the rat brain using a monoclonal antibody against the affinity purified glycine receptor and a polyclonal antibody against purified beta₂-AdR. GR were concentrated in the lower brainstem, whereas no immunoreactivity was observed in the diencephalon and forebrain except in a few diencephalic nuclei. The highest density was found in the cranial motor nuclei, reticular formation, parabrachial area, dorsal and ventral cochlear nuclei, and dorsal and ventral tegmental nuclei. On the other hand, AdR were widely but unevenly distributed in the rat brain. The present study further showed the presence of AdR in catecholaminergic terminals in the hypothalamus.

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Although recent advances in gene technology have made it possible to produce peptides and proteins by the recombinant technique, chemical synthesis either by the solution or by the solid-phase technique is still only a method of choice for the syntheses of a brain-gut hormone and its superagonist and/or antagonist. In addition, synthetic replicates of braingut hormones, their analogus and fragments or their precursor-related peptides are important haptenic immunogens to produce region-specific antisera to the respective peptides.
In the present study, antisera against proglucagon and pro-LH-RH related pepties were proved to be useful not only for identifying hormone-producing cells, but also for demonstrating the post-translational biosynthetic processing in the cells.
For galanin, a novel brain-gut hormone, have structural similarity in its C-terminal region to tachykinins, a monoclonal antibody againt synthetic porcine galanin (1-15) was prepared for immunohistochemical study. This monoclonal antibody was found to recognize specifically galanin-producing cells and not to crossreact with any of tachykinins examined.
These observations indicate that monoclonal antibodies are highly useful for identifying cells possessing a brain-gut hormone containing structures common to those of different known peptides.

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Methods for non-radioactive in situ hybridization are rapidly progressing in refinement and application. In this overview, we present our experiences with the use of synthetic oligodeoxynucleotides, labeled with biotin- or digoxigenin-tagged nucleotides by the terminal transferase method.

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Cholecystokinin (CCK), the long known gut hormone has recently been found in the brain and suggested to be a neurotransmitter or neuromodulator. In order to clarify the structure of the CCK precursor, we cloned the cDNA to mRNA of rat brain. We determined the nucleotide sequence of the inserted cDNA and deduced the amino acid sequence and proteolytic cleavage sites of the CCK precursor. The availability of CCK cDNA probes allowed us to examine the levels of CCK mRNA in the brain and intestine. CCK mRNA was barely detectable in the fetal brain, but started to increase postnatally and attained a plateau after 20-30 days. The level of CCK mRNA was the highest in the frontal cortex, followed by those of the hippocampus and striatum. The cerebellum contained only negligible CCK mRNA. Furthermore, we succeeded in cloning the CCK gene and revealed its structure which contained three exons interrupted by two introns. The size of this gene spanned about 7kbp. The transcription initiation sites were determined by using S1 nuclease mapping and a primer extension procedure, showing evidence for the existence of the major and minor start sites. To analyze the mechanism for CCK gene expression in the neuron and paraneuron, we tried to examine the transcription of the DNA fragments from CCK gene by an in vitro cell free system. These experiments confirmed the existence of two transcription start sites. From experiments with the deleted mutants of DNA fragments, we could deduce the promoter sequence. Assuming the presence of the cell-specific regulation factor in the nuclear extract from the rat brain, we examined a brain-specific DNA binding protein in the rat brain nuclear extract by a gel shift assay and DNase footprinting assay with several DNA fragments from 5′-flanking region of the CCK gene. A brain nuclear protein could bind to a promoter region. The exact binding site was determined with several synthetic oligodeoxynucleotides and their mutants. Currently we are attempting to purify this brain nuclear factor and to examine the regulatory function of this factor in cell-free transcription experiments.

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Serotonin is one of the representative biogenic amines produced from tryptophan in the gastrointestinal tract as well as in the brain. The pineal gland also synthesizes serotonin, but as an intermediate in the biosynthesis of melatonin. The first enzymic step in the biosynthesis of serotonin is the hydroxylation of L-tryptophan catalyzed by tryptophan 5-monooxygenase. The 5-hydroxy-L-tryptophan (L-5HTP) formed is then decarboxylated to serotonin. Since the second step is catalyzed by non-specific aromatic L-amino acid decarboxylase, whether or not a given cell has the capacity to synthesize serotonin depends on the existence of tryptophan monooxygenase, and regulation of the serotonin biosynthesis is achieved mainly by modulating the activity of the first step enzyme.
The activity of tryptophan monooxygenase was detected in extracts of the mouse stomach and intestines, although this activity was as low as approximately 1/20 of that in similar extract of the mouse brainstem. The upper small intestine and colon had a higher level of activity than other parts, and in the upper small intestine the enzyme was found to reside primarily in enterochromaffin cells of the mucosa. The intestinal tryptophan monooxygenase shared a common antigenic character with the enzyme from murine mastocytoma and was immunologically different from the brain enzyme. The enzymic properties were also similar to those of mastocytoma tryptophan monooxygenase which requires loosely-bound functional iron (Fe²⁺) for the activity. It seemed likely that the activity of tryptophan monooxygenase in paraneurons such as mastocytoma and enterochromaffin cells depends on available ferrous iron, and is therefore regulated, at least in part, by the cytosolic level of chelatable iron.

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Several years ago we proposed a method for the localization of acetylcholine in the cholinergic nerve terminals. The method is based on a rapid precipitation of quarternary ammonium cations (such as acetylcholine or choline) by molybdic or tungstic heteropolyanions (such as phosphomolybdic acid, phosphotungstic acid and silicotungstic acid). The insoluble salts formed can be directly visualized under the electron microscope. In the synaptic vesicles, acetylcholine was localized as point-like precipitates, while the membranes were well preserved. Since these fixations are based on a rapid ionic interaction, the term “ionic fixation” was proposed. The ionic fixation performed on motor end-plates in various physiological conditions, provided different forms of cytochemical precipitates of acetylcholine (point-like, spot-like, diffuse or laminar).
In contrast with the numerous physiological and neurochemical investigations into the acetylcholine mechanism of neurons, very little is known about paraneurons. The historical evolution of paraneuron research, more or less bound to that of the sympathetic paraganglion and of the APUD cells may explain this lack of results. In certain physiological conditions, acetylcholine can be revealed in paraneurons and seems to reflect a plasticity of the cells, as already observed in sympathetic ganglion cells.
This paper further overviews the cholinergic mechanisms of exocrine secretory cells. The presence of a high cholinesterase activity exist in most of these exocrine cells, as far as we could verify. In addtion to cholinesterase activity, the presence of acetylcholine, probably related to exocrine cell metabolism, was investigated with our histochemical method.

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Mature secretory granules in paraneurons contain ATP amongst other small messenger molecules. In the islet organ such stores of adenine nucleotides readily can be demonstrated by means of the quinacrine fluorescence method. ATP is co-released together with other granule constituents when the major hormones are exocytosed. The distribution of ATP splitting enzymic activities was studied in the pancreas of the mouse and rat, in order to obtain information on the possible fate of this small messenger molecule.
ATPase, ADPase, and AMPase (5′-nucleotidase) were demonstrated with lead precipitation methods, L-tetramisole was used to inhibit unspecific alkaline phosphatase (alPase); alPase activities were shown with tetrazolium methods, using 5-bromo-4-chloro-3-indoxyl phosphate as substrate.
Most endothelial cells of the vascular bed, both in the exocrine and in the endocrine pancreas, are reactive for ATPase, ADPase, AMPase and alPase. Smooth muscle cells are strongly reactive for ATPase and AMPase, vascular adventitial fibroblasts (veil cells) stain for ATPase and alPase, as do some lamellar cells at the islets surface. Staining for ADPase serves as a selective method to demonstrate the vascular bed. Comparable results are obtained with the alPase reaction, though insular non-B-cells are also reactive. ATPase staining is less useful for demonstrating vascular connections because moderate reactivity of exocrine parenchyma and adventitial tissue obscures the picture. AMPase activity is strong in the venous segments of the capillary net and in collecting veins but the reaction obviously does not demonstrate significant portions of the residual capillary network. Weak AMPase activity is seen in the insular parenchyma.
These results indicate that in situ most of the secreted ATP readily is split down to AMP and adenosine before leaving the interstitial space. Vascular smooth muscle cells or equivalent contractile cells which are sensitive to these substances are rare in the insular vascular bed as well as in the connected capillary systems (insuloacinar and insulo-ductal connections). Therefore it is suggested that adenosine a) is taken up by the paraneurons to be re-used via the salvage pathway of purines, and b) may act as messenger for inhibitory cellular selfregulation (autocrinia).

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Chromogranins (Cg) are large acidic proteins, co-stored with amines or peptides in the secretory granules of a wide variety of paraneurons. By immuno-histochemistry performed on serial semithin sections, the distribution of Cg in the gastroenteropancreatic (GEP) system was analyzed in the endocrine pancreas of 10 mammalian species and in enteroendocrine cells of 6 species. Of the Cg families, CgA is primarily localized in GEP endocrine cells. CgA was regularly present in amine containing cells (EC and EC-like cells). In 11 other endocrine cell types, CgA-immunoreactivities showed inter-species and intra-species variations in their distribution or in the intensity of staining. However, no endocrine cell type lacked CgA-immunoreactivities in all species investigated. Findings from extended studies indicate that CgA is involved in the intracellular storage of resident amines, and in the storage of newly formed amines after exogenous application of the corresponding precursors. Finally, densitometric findings on possible reciprocal relationships between CgA- and peptide-immunoreactivities suggest that CgA also participates in the intragranular storage of peptide hormones in certain GEP endocrine cells.

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Two novel monoclonal antibodies, called B11 and B13, directed exclusively against human chromogranin B (CgB) and another antibody, All, specific for human chromogranin A (CgA), were obtained by immunization of mice with chromaffin granules, the fission of their splenocytes, the screening of hybridomas supernatants by ELISA and immunohistochemistry, and characterization of the antibodies by two-dimensional immunoblotting.
The antibodies were used in immunohistochemical tests to investigate the distribution of CgA and CgB in hormonally-identified cells of the human endocrine system. The All antibody confirmed the occurrence of CgA in gut EC, ECL, gastrin, secretin and neurotensin cells, pancreatic A and PP cells, parathyroid chief cells, pituitary TSH and gonadotrope cells and adrenal medullary cells. Only a fraction of CgA-immunoreactive cells in the human gut and pancreas showed C-terminus arginineglycinamide immunoreactivity, suggesting pancreastatin storage. Both CgB antibodies showed immunoreactivity in gastrin cells, intestinal (but not gastric) EC cells, pancreatic A and PP cells, pituitary TSH and gonadotrope cells and adrenal medullary cells. In addition, the B11 antibody stained thyroid C cells and the B13 antibody stained the Golgi area of pituitary GH cells. It is concluded that most CgB is stored in the same cells showing CgA, although some CgA-rich cells, like gastric EC and ECL cells, lacked B11 and B13 immunoreactivities and some CgA-poor cells, like human thyroid C cells, showed intense B11 immunostaining.

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The concept of a neurosecretory system consisting of a secretory center (production of bioactive substances in neuronal soma), a transport pathway (transport by axonal flow) and a neurohemal organ (storage and release at nerve endings) has been established through a series of histological studies on the hypothalamic magnocellular nuclei by two pioneers, SCHARRER and BARGMANN, together with their coworkers.
In the early stages of the investigation, these actively secreting neurons were considered as exceptional neurons, separated from ordinary ones. However, since the application of modern techniques, such as electron microscopy, immunohistochemistry, and in situ hybridization, have made progress in tracing the cytological course from the gene expression to the release of bioactive product, it has become obvious that the classical definition of neurosecretion must be extended to include a variety of neuroepithelial derivatives, such as aminergic and peptidergic neurons and also paraneurons. At present, all neurotransmitters and neuromodulators can be regarded as secretory substances produced in neurons.
Newly developed precise techniques, e. g., a tracing method with a computer graphic system for the demonstration of axonal arborizations, have led to a dramatic change in our understanding of the fine details of cytological features, offering much more complicated structures than images presented by the classical impregnation technique. Immunohistochemically demonstrated serotoninergic neurons in the brain have revealed enormous reticular extensions and anastomoses of beaded processes which were previously unknown. Neurons composing the nervous system show greatly varied shapes and structures. This morphological heterogeneity makes possible the diversity of the brain function.
This review stresses that advances in studies in the field will be promoted by a dual strategy: one to investigate neurons in their general features especially the Secretory aspects, and the other to pay attention to the special features of each variety of neurons.

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Anatomical and functional aspects of serotonin (5HT) neurons in the central nervous system are described with special reference to their neurohumoral function. The central 5HT neurons show morphological features characteristic of the modulating nervous system: a small number of cell bodies and extremely broad innervation. These may play some part in EEG arousal through the divergent projection. Being most active during the waking state, the 5HT neurons are responsible for the onset of sleep. 5HT released during waking may initiate the humoral, as well as neuronal mechanisms leading to sleep. Although the 5HT dependent hypogenic structure is highly localized in the anterior hypothalamus, the effect seems to be due to neurohumoral action of 5HT. The neurohumoral function of the 5HT neuron is suggested by the ultrastructure of 5HT terminals almost devoid of a synaptic structure (less than 10%). Intraventricular 5HT fibers also represent a substrate supporting the neurohumoral function. A strong activity of monoamine oxidase (MAO) is found in intercellular spaces in the arcuate nucleus region of the hypothalamus, where a high 5HT immunoreactivity is seen in the young rat before MAO activity appears. It is ljkely that 5HT liberated in the cerebrospinal fluid is drained through these intercellular channels.

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The effects of angiotensin II (A II) and ANP on spontaneously active neurons in the subfornical organ (SFO), anteroventral third ventricle (AV3V) and supraoptic (SON) and paraventricular nucleus (PVN) were investigated using slice preparations and extracellular recordings. Application of A II (10^-7M) excited the neural activity of 66% of the SFO neurons, 28% of the AV3V neurons and 44% of the SON neurons. The threshold concentration to produce responses in SFO and AV3V neuron was less than 10^-10M, while that in SON neurons was 10^-9M. The excitatory effects of A II were reversibly antagonized by saralasin and persisted after synaptic blockade in a low Ca²⁺ and high Mg²⁺ medium. Application of ANP (10^-7M) inhibited the neural activity of 41% of the AV3V neurons, 22% of the PVN neurons and only 14% of the SFO neurons but had no effect on SON neurons. The threshold concentration for ANP in the AV3V was 10^-11M. Interestingly, ANP inhibited A II induced excitation in most of the SFO neurons (87%), while ANP had little effects on their spontaneous firing rates. These results show that both peptides of A II and ANP have direct central actions on hypothalamic neurons although ANP can not directly influence magnocellular neurons, suggesting that these blood borne peptides are detected in the SFO and AV3V and that they are acting as a neurotransmitter or a neuromodulator in the central nervous system to regulate water homeostasis.

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The neuropeptides vasopressin and oxytocin were first characterized as hormones, i. e., signalling molecules which are synthesized in hypothalamic neurones, transported toward the neurohypophysis and from there secreted into the general circulation. Although extrahypophysial Gomori-positive pathways were described in the brain as early as the mid-1950s, it is only during the last decade that the neurotransmitter role of vasopressin and oxytocin has begun to be investigated. Recent electrophysiological and morphological studies from our laboratory are summarized. Using extracellular and intracellular recordings from in vitro brain slices, a direct excitatory action of vasopressin was demonstrated in the lateral septum and in the nucleus of the solitary tract. This action of vasopressin was mediated by V₁-type receptors. An excitatory effect of oxytocin, mediated by receptors similar to those present in uterus, has also been found in the dorsal motor nucleus of the vagus nerve. In accordance with these results, light microscopic autoradiography showed the presence in these brain areas of high affinity binding sites for vasopressin and for oxytocin, respectively. While these data corroborate the notion that vasopressin and oxytocin are probably involved in interneuronal communications, several questions, however, remain. The membrane mechanism by which vasopressin and oxytocin cause neuronal excitation is still unknown and evidence that endogenous vasopressin and oxytocin act at central synapses deserves further investigation.

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We have found a large portion of the neurons in the abdominal ganglia of the crayfish, Procambarus clarkii (Girard) to be local nonspiking neurons. These neurons are anaxonic, have extensive arborizations of neurites and have their entire structure confined within a single ganglion. They show relatively low membrane potentials of 40-50mV and under no circumstance generate action potentials, yet their membrane potential changes can effectively modulate the activity of postsynaptic neurons which are generally motoneurons. The transmission is chemical and transmitter release in many, if not all, of them is continuous even at their “resting” level. Electron microscope examination showed an extensive intermingled distribution of both input and output synaptic structures. This synaptic distribution together with the passive electric property suggests that individual neurites of this type of neurons function rather independently, constituting numerous local circuits. Similar nonspiking communication is also found among the ordinary “spiking” motoneurons, indicating that this mode of communication is far more widely used in the crayfish central nervous system than generally believed. These findings appear to necessitate a radical revision of our understanding of how the central nervous system operates in arthropods.

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Small foci of photocoagulation necrosis (diameter about 0.5mm) were produced in the wall of the guinea-pig small intestine by argon laser irradiation applied to the serosal surface. Features of the nerve elements around the necrotic masses were examined by immunocytochemistry, scanning electron microscopy, and transmission electron microscopy. The severed ends of nerve strands were examined at various intervals from 10 to 60h after the laser irradiation, and the acute cytological responses of the nerve terminals, glial cells and connective tissue cells were studied.
The laser irradiated foci in the deep muscular plexus (plexus muscularis profundus: CAJAL, 1911) had concentric configurations: 1) central necrotic area, 2) inner transitional zone, 3) outer transitional zone, and 4) normal area. In the necrotic area, there were inflammatory cells such as neutrophile leukocytes and macrophages. Contours of the smooth muscle cells and nerve strands were well preserved in spite of the damage to their subcellular structures. In the inner transitional area, individual nerve fibers gathered to form bundles. Some of these nerve bundles were devoid of a glial cell framework. In the outer transitional zone, there were swollen nerve terminals with a strong immunoreactivity for methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸. In transmission electron microscopy, some of these swollen nerve fibers contained many large cored vesicles. Glial cells in the outer transitional zone exhibited an S-100b proteinlike immunoreactivity. Scanning electron microscopy revealed gatherings of fibroblast-like (FBL) cells in the outer transitional zone. Two subtypes of these were distinguished: the first subtype (FBL I) was characterized by short, blunt cytoplasmic processes; the second subtype (FBL II), possessing long, slender dendritic processes, assembled to form a dense network which intermeshed with that of the nerve strands.
The enteric nerve plexuses severed by laser-irradiation affords a unique experimental model in the cytological investigation of the plasticity of the peripheral autonomic nervous system.

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Views about the roles and nature of the enteric nervous system have changed dramatically in the last ten years. This system of neurons is recognized to control, through reflex pathways intrinsic to the gut wall, motility, transport of water and electrolytes, and blood flow in the small and large intestines. There are thus a range of neuron types in the enteric nervous system, including sensory neurons, motor neurons, and interneurons, involved in the control of each of these functions. The present paper deals with the recent efforts to provide an integrated functional and structural description of the nerve circuits. One of the challenges in this quest has been to identify primary sensory neurons and final motor neurons involved in motility control. Evidence is presented that in the guinea-pig small intestine the primary sensory neurons have Dogiel type II morphology and are, electrophysiologically, AH neurons. They send circumferential processes to adjacent myenteric ganglia and some of them, at least, have processes leading from the mucosa. Motor neurons are S neurons, with cell bodies in the myenteric plexus. Those that supply the circular muscle are Dogiel type I neurons and provide processes that run circumferentially for about one third of the circumference of the intestine.
The circuits for secretomotor reflexes have been partly worked out. The secretomotor neurons have cell bodies in the submucous ganglia and are activated to return water and electrolytes to the lumen during digestion. The secretomotor reflexes are regulated, in accord with whole body water and electrolyte homeostasis, via sympathetic neurons which lower the excitability of secretomotor neurons.

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This study focuses on establishing the nature and extent of the changes that occur in gastroenteric innervation, specifically the myenteric plexus of the rat ileum, following an injury generated by experimental obstruction. A partial obstruction was accomplished by placing a cuff around the terminal portion of the ileum of the rat. Substantial hypertrophy of the enteric muscle wall occurred after 3-5 weeks.
Light microscopic examination of the myenteric plexus revealed changes in the numbers of neuronal perikarya, ganglia and perikarya per ganglia; sizes and shapes of perikarya; and thicknesses of nerve fiber bundles.
Using vasoactive intestinal polypeptide (VIP) and substance P light microscopic immunohistochemistry, we observed indications of transmitter accumulation in cell bodies and nerve fibers and reactive, degenerative and regenerative changes in axonal endings.
Electron microscopic studies provided evidence for neuroplastic changes, as demonstrated by the appearance of reactive and regenerative, or growth, cones in the myenteric plexus.

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In order to understand better the neuronal circuitry involved in the regulation of gut functions, we have studied the distribution and projections of those enteric neurons in the rat intestines that contain vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), galanin, substance P (SP), calcitonin gene-related peptide (CGRP), gastrin releasing peptide (GRP), somatostatin and enkephalin. The origin of the peptide-containing nerve fibers was examined by immunocytochemistry after extrinsic denervation. Most of them were found to be intramural in origin, each population displaying its own characteristic topographic distribution. The projections of each neuronal population were studied immunocytochemically by examining the subsequent axonal degeneration after local severing of nervous pathways. This study revealed that myenteric neurons issue predominantly descending projections to other myenteric ganglia and to the muscle layers. Submucous neurons project to other submucous ganglia and to the mucosa and submucosa. Most of these neurons issue both ascending and descending projections. The projection distances varied considerably between the different neuronal populations, the majority being in the range of 4-10mm. Myenteric GRP and galanin neurons in the small intestine issued the longest projections, 20 and 15mm, respectively. The shortest projections were those issued from myenteric VIP/NPY neurons and submucosal galanin and GRP neurons in the small intestine and from submucosal VIP neurons in the large intestine (2mm in length). On the whole our results on the projections of enteric neurons in the rat agree with observations in the guinea pig and dog. However, there are species differences that remain to be explained.

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We systematically studied the intramural nervous system of the alimentary tract in patients with Parkinson's disease and found that Lewy bodies were distributed widely in the Auerbach's and Meissner's plexuses. In the central nervous system, we recognized a striking similarity between the distribution of Lewy bodies and that of monoaminergic neurons. More recently, we have demonstrated that neuronal somata immunoreactive for tyrosine hydroxylase (TH) exist in the Auerbach's and Meissner's plexuses of normal humans. We consider a possible relation between these TH-immunoreactive catecholaminergic neurons to the occurrence of Lewy bodies in the enteric nervous system in Parkinson's disease. The affinity of Lewy bodies to the central and enteric neurons seems to be attributable to an unknown cell-biological characteristic apparently shared by both neurons.

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Cerebrospinal fluid (CSF)-contacting neurons are located periventricularly or inside the brain ventricles; they contact the CSF via their dendrites, perikarya or axons. Most of these neurons form ciliated dendrite terminals in the internal CSF as do retinal and pineal photoreceptors in the optic ventricle and pineal recess. The peculiar localization, polarization and synaptic connections of the CSF-contacting neuronal elements suggest receptor and integrative functions.
The present review pays special attention to vitamin A (retinoids) immunoreactivity in CSF-contacting neurons as compared with that present in retinal and pineal photoreceptor cells, common neurons, glial and adenohypophysial cells. The immunoreactivity of the darkadapted photoreceptor outer segments was strong, but decreased after illumination, suggesting the functioning of vitamin A as the chromophore of the retinal and pineal photopigments. Retinoid immunoreaction was also found in the endoplasmic reticulum, nuclei, nucleoli and mitochondria of the cell types studied. This cytological localization suggests that vitamin A compounds may be involved in the function of these organelles.
The CSF-contacting neurons contain varying amounts of bioactive materials. The intracellular distribution of immunoreactive serotonin (5-HT), substance P (SP) and gamma-aminobutyric acid (GABA) is compared with that of immunoreactive vitamin A. Immunogold labeling for SP was demonstrated in dense-core vesicles of preoptic neurons; 5-HT marking was found on the dense-core vesicles of subependymal CSF-contacting neurons of the paraventricular organ, while GABA immunoreaction was localized in the cytoplasm of distal infundibular CSF-contacting neurons.
The CSF-contacting neurons are considered to synthesize and release their bioactive substances at transmitter synapses, and/or at neurohormonal terminals into the external CSF in accord with information received by their dendrites from the internal CSF and by afferent fiber connections from various brain areas.

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Many strains of rats develop adrenal medullary hyperplasia and/or neoplasia after chronic administration of a wide variety of pharmacologic agents. The same changes may occur spontaneously in the course of aging. Current evidence suggests that chromaffin cell proliferation in adult rats is regulated by a combination of hormonal and neurogenic signals. Drugs associated with adrenal medullary tumors might act directly or indirectly on the hypothalamic-endocrine axis or the autonomic nervous system to stimulate chromaffin cell proliferation through mechanisms which normally adjust cell number to meet physiological needs. The increased cell turnover rate might be a prelude to other events leading to neoplastic transformation.

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The development of hypothalamic neurons was examined in vivo and in transplanted grafts in rats. The neurons appeared in vivo in distinctive chronotopical schedules showing such morphological characteristics as synaptocrine or hemocrine neurons. These phenotypical properties seemed to be primed already by day 12.5 of gestation in rats, because the grafted hypothalamic primordia from 12.5-day-old embryonal rats differentiated neurons, which express these neuronal properties in the third ventricle of adult female rats. The synaptocrine neurons projected to other neurons, suggesting the establishment of synaptic contacts, and the hemocrine neurons projected to vasculatures developed in the grafts, suggesting the accomplishment of neurovascular associations.

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From results of phylogenetical investigations of the neuron-paraneuron or neuroendocrine system it is known that cells producing hormonal peptides appear first in the nervous system, then also as disseminated cells of an open or closed type in the mucosa of the alimentary tract, and, lastly, as the parenchyma of the classical endocrine glands. In all these three parts of the neuroendocrine system the parenchymal cells can undergo neoplastic transformation, expressing “eutopic” and/or “ectopic” peptide hormones and/or biogenic amines. The neuro-paraneuronal tumours arising in the central or peripheral nervous system in man are rare. In contrast, they are more common when they originate from the disseminated neuroendocrine cells in the gut mucosa, and when they arise in the endocrine glands. Here, a broad spectrum of histopathologically benign or malignant neoplasms, hyperplastic nodules, and other tumour-like lesions occur, some of which are associated with well-known clinicopathological entities. Typical examples are the gastrointestinal carcinoids, pancreatic insulomas, pituitary adenomas, medullary thyroid carcinomas, and phaeochromocytomas.
The fact that neoplastically transformed cells of some traditionally non-endocrine tumours, e. g., mammary and prostatic carcinomas, can undergo neuroendocrine differentiation has recently become well established. The “malignancy potential” of neuroendocrine carcinomas and that of the paraneuronally differentiated cells of prostatic and mammary carcinomas have both been studied by means of cytochemical DNA assessments of the nuclei of the tumour cells, applying a recently developed deparaffinisation-disintegration procedure using archival material. So far, it seems as if about 1/4 of common gut carcinoids show a DNA distribution pattern of the “aneuploid” type, whereas the other 3/4 are “euploid” i. e., show a “diploid” or “diploid/tetraploid” DNA distribution pattern. The cell nuclei in areas in prostatic and mammary carcinomas with high amounts of paraneuronally differentiated cells displayed to a greater extent “euploid” DNA patterns than those in areas of the same carcinomas without such paraneuronal cells. Thus, the “malignancy potential” of the neoplasms of neuro-paraneuronal cells seems to be rather low.

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Human endocrine tumours were studied in in vitro systems (cell suspensions and tissue cultures) and in in vivo systems (tumour transplants to the anterior eye-chamber of immunosuppressed rats). In the experimental systems the tumour cells were demonstrated to synthesize and secrete the same hormonal products as in the patient. Intraocular transplants of a gastrinoma secreted gastrin-17 into the chamber fluid. This molecule, normally not secreted in the rat, was also detected in the peripheral plasma of tumour-bearing rats. Intraocular transplants of midgut carcinoid tumours released serotonin (5-HT) at adrenoceptor stimulation, of a similar type as demonstrated in acute tumour cell suspensions. However, in tissue cultures genuine β-adrenoceptors seemed to be modified, since pretreatment with β-adrenoceptor antagonists or calcium deprivation did not prevent stimulated 5-HT release. Tachykinins were not liberated by adrenoceptor stimulation.
In certain cultures of midgut carcinoid tumour cells, two different phenotypes developed: small rounded endocrine tumour cells with positive immunoreactions against 5-HT and tachykinins (TK), and large elongated neuron-like cells, which gradually lost 5-HT immunoreactivity, while TK immunoreactivity remained unchanged. These cultured tumour cells may produce an endogenous factor inducing transformation into a neuron-like phenotype. One candidate factor is nerve growth factor (NGF), since NGF-like immunoreactivity was demonstrated in cells of the endocrine phenotype.

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Egg laying in the hermaphrodite freshwater snail Lymnaea stagnalis is a highly complex activity, including a series of internal activities (ovulation, egg and egg mass formation) which are closely correlated to a pattern of behaviours (alteration of locomotion and feeding, specific postures, oviposition). In this snail egg laying is induced by the neuroendocrine caudodorsal cells (CDCs), consisting of two homogeneous clusters at a total of 100 neurons. At egg laying these neurons release their products during a 60min period of firing. The genes coding for these products have been cloned and characterized. There are two genes, CDCH-I and -II. Each gene codes for 12 peptides; one of these is the ovulation hormone (CDCH). The genes display over 90% homology. The most striking difference is a 17 bp deletion near the carboxy-terminal region.
With immunocytochemistry and in situ hybridization both CDCH genes appeared to be expressed in the CDC and in paired groups of ectopic CDC-like neurons in the pleural ganglia, while a group of small neurons in the cerebral ganglia expresses the CDCH-I gene only. In addition, a widespread expression of the CDCH genes has been demonstrated in peripheral tissues. In the female part of the reproductive tract neurons were found to express the CDCH-I gene. In the male part of the tract exocrine secretory cells express the CDCH-I or -II gene. The gene products are secreted into the male tract and transferred to the female partner during copulation. Finally, sensory neurons in the head skin and mantle edge were found to express the CDCH-I gene.
The presence of insulin-related peptides has been demonstrated in the brain as well as the digestive system of Lymnaea. The brain insulin-related peptides are produced in 4 groups of 50 giant neurons each (Light green cells, LGC). These neurons are involved in various physiological activities, related to body growth and glycogen metabolism. The major gene products expressed in the LGC have been cloned and characterized. It appeared that the predicted proteins represent three types of insulin-related molecules (MIP, molluscan insulin-related peptide). In these MIPs, those elements important in the determination of the tertiary structure, have been conserved. The MIP of the digestive system has been characterized up to now only at the peptide level. The snail gut MIP is more hydrophobic compared to bovine insulin. Cells containing MIP have been identified immunocytochemically in the gut epithelium.

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The integrative mechanisms in phylum Platyhelminthes are of considerable phylogenetic interest since ancestral flatworms are believed to be progenitors to all “higher” metazoans. The phylum consists of free-living and parasitic worms. In this group of early bilateral animals, the nervous system is condensed and consists of a bilobed brain and longitudinal nerve cords. Endocrine glands and a circulatory system are still lacking.
The present review deals mainly with microturbellarians and parasitic worms. Three main types of neurons, all containing vesicles, occur in the worms: neurons containing dense-core vesicles, peptidergic neurons and sensory neurons. All types of neurons react positively to antisera towards invertebrate and vertebrate neuropeptides. Neurons containing dense-core vesicles are the most frequent type. The dense-core vesicles occur both in the cell soma and in their long processes. Neurons of this type form synaptocrine and paracrine release sites. We ask: Is the neuron containing dcv an archaic type of neuron in which the characteristics of the stem cells for neurons have been preserved?

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The first nervous system probably evolved in coelenterates. Many neurones in coelenterates have morphological characteristics of both sensory and motor neurones, and appear to be multifunctional. Using immunocytochemistry with antisera to the sequence Arg-Phe-NH₂ (RFamide), RFamide-like peptides were demonstrated in the nervous systems of all classes of coelenterates. Using a radioimmunoassay for RFamide, three such peptides were isolated from the sea anemone Anthopleura elegantissisma and sequenced: <Glu-Gly-Arg-Phe-NH₂ (Antho-RFamide), <Glu-Ser-Leu-Arg-Trp-NH₂ (Antho-RWamide I) and <Glu-Gly-Leu-Arg-Trp-NH₂ (Antho-RWamide II). The general structure of these peptides can be described as <Glu…Arg-X-NH₂, where X is an aromatic amino acid. From the hydromedusa Polyorchis penicillatus, the peptide <Glu-Leu-Leu-Gly-Gly-Arg-Phe-NH₂ (Pol-RFamide I) was isolated, which also belongs to the <Glu…Arg-X-NH₂ family. Using specific antisera it was shown that all four peptides were located in neurones. Application of low doses of Antho-RFamide, or Antho-RWamide I or II induced contractions of endodermal muscles of sea anemones. This indicates that these neuropeptides play a role in neurotransmission.

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The characteristics of neurosecretory granules include the presence of an acidic interior, a hyperosmolar concentration of granule solutes, the presence of chromogranin (CG) or CG-like soluble acidic proteins and a high content of nucleotides, predominantly ATP. The identification of “nucleotides” within the neuroendocrine “stem cells” of coelenterates (e. g. Hydra) has raised some interesting evolutionary questions as to the function of intragranular nucleotides.
The chromaffin granules of adrenal medullary cells have been studied extensively, and are representative of the neurohormone/neurotransmitter packaging problems encountered in neurosecretory granules, in general. At the acid pH (5.7) of the interior of the chromaffin granule, ATP has three negative charges based on the pK value of the γ-phosphate group. ATP can therefore interact with positively charged amines, acetylcholine and divalent cations, forming binary and ternary complexes. The results of nuclear magnetic resonance (NMR) spectroscopy indicate that the hyperosmolar solutes within the chromaffin granule exist in a viscous, but fluid state; one function of ATP could be to help lower the osmotic pressure of the granule contents through extensive, but weak, intermolecular bonding. In addition, ATP is an excellent buffer to help maintain a pH of 5.7 within the interior of the chromaffin granule. An acidic milieu contributes to neurohormone/neurotransmitter packaging and granule stability. The presence of nucleotides within neurosecretory granules cannot, however, be explained on the basis of the ability of ATP to simply reduce osmotic pressure, since insulin molecules exist in a crystalline phase, a condition which, by itself, could substantially reduce osmotic pressure; nucleotides, nevertheless, co-exist in these insulin cores. ATP and ATP metabolites such as ADP, AMP and adenosine, formed as a result of the action of ectonucleotidases, can have extensive extracellular trophic and feedback effects after secretion. Extracellular nucleotides and adenosine can function as neuromodulators, agonists and antagonists to inflammatory cells, and regulators of blood flow, etc.
It is possible that intragranular nucleotides were retained through a billion or more years of evolution because of the importance of these trophic and feedback effects. Parts of the neurosecretory granule, such as the F₁ subunit of the proton-translocating ATPase, can be traced back to the aerobic bacteria, vacuolar amine transport to yeast and a CG-like acidic protein to protozoan secretory granules (i. e., the trichocysts of Paramecia).

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This review indicates that the heart is a polypeptide-producing organ which should be classified among the traditional endocrine tissues. Cardiac hormones have only been known for a few years, the discovery of their endocrine functions, however, occurred in the 1950ies when GAUER, HENRY and KISCH observed specific physiological and morphological features of the heart atria indicative of an endocrine activity. Because of their basic effects many target organs involved in the regulation of body fluid pressure and composition are related to this endocrine organ located in the atrial appendages of the heart. The compact endocrine portion of the heart is built up by myoendocrine cells which form the functional endocrine units and produce a variety of polypeptide hormones called cardiodilatin (CDD) or atrial natriuretic polypeptide (ANP), which belong to one family. Also, costorage of a partially homologous regulatory polypeptide called brain natriuretic polypeptide (BNP) occurs, as has been determined by immunohistochemistry and radioimmunoassay. CDD and/or BNP are found in numerous organs where they exert paracrine and neurocrine functions, e. g., in the brain, peripheral nervous system, kidney, and adrenal medulla. In these organs, a differential post-translational processing of cardiac polypeptides is observed, resulting in different functional activities according to discriminating receptor interactions and degrading metabolism. Some of the extra-auricular sites of synthesis and storage of CDD-like peptides are briefly mentioned. In summary the heart constitutes the center of a multilocal and multifunctional system of specific cardiac polypeptides of endocrine, paraneuronal, and neuronal character.

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The authors present an overview of the main amphibian peptide families mainly derived from the skin and mostly discovered by ERSPAMER and his associates. The studies of the peptides do not only promote the understanding of their chemical, metabolical and physiological features of those molecules in amphibians, but also contribute to progress in our knowledge of the corresponding mammalian counterparts. Particular reference is made on sauvagine, tachykinin, bombesin and dermorphin families, offering new data mostly from personal contributions to this field.

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It is well known that the Atrial Natriuretic Peptide (ANP) plays a major role in vasorelaxation and in lowering systolic blood pressure. However, little information is available concerning its effect on the autonomic outflows.
The present study deals with the effect of rat ANP (rANP) on the efferent activity of the sympathetic and vagal outflows.
Under anesthesia (urethane 700mg/kg and chrolarose 50mg/kg, intraperitoneal), the effect of intravenous administration of rANP was observed in rats. A suppression of the efferent activity was recognized after administration of rANP in adrenal, renal and splenic sympathetic nerve fibers, and also in vagal gastric, pancreatic and hepatic nerve fibers. The smallest effective dose was 100pg.
In decerebrated preparations no change in efferent activity after administration of rANP was recognizable.
The results indicate that rANP released from the atrium to systemic circulation modulates autonomic outflows through the hypothalamic neurons surrounded by an insufficient blood brain barrier.

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Aspects of the functional anatomy of the chromaffin cell since 1950 are reviewed—beginning with the identification of noradrenaline in the adrenal gland and the first descriptions of distinct adrenaline- and noradrenaline-storing cells.
Reference is made to the identification of specific proteins and neuropeptides in chromaffin cells, of the storage of endogenous and exogenous amines including 5HT.
The influence of corticosteroids and nerve growth factor on cell phenotypes is discussed and a plea made for the specific use of descriptive terms with due regard to historical and functional context: this relating particularly to the appropriate labelling of elements as SIF cells.
The review includes reference to recent quantitative studies on the rat adrenal medulla and adrenal chromaffin cells and to the innervation of the adrenal medulla by pre- and postganglionic sympathetic neurons, by spinal afferent neurons and by sensory and motor vagal fibers.

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Additional evidence for the existence of subclasses of SIF cells is described. Type II SIF cells in the inferior mesenteric ganglia contain enkephalin, vasoactive intestinal polypeptide (VIP), somatostatine (SOM), neuropeptide tyrosine (NPY), or dynorphin (DYN) in variable combinations in addition to noradrenalin. These transmitters or modulators can affect the autoreceptors of the SIF cell themselves or modify the synatpic transmission and the activity of sympathetic ganglionic neurons through portal blood vessels. Tyrosine hydroxylase/NPY immunoreactive nerve terminals from sympathetic ganglia innervate blood vessels and both submucous and myenteric ganglia. DYN/VIP/cholecystokinin neurons in the nerve plexus send axon collaterals to the inferior mesenteric ganglia and form a feedback loop. Substance P (SP) and calcitonin gene related peptide may exist in sensory nerve terminals. SP neurons in the myenteric ganglia innervate smooth muscles. SOM neurons inhibitory interneurons in the ganglia. SIF cells act as secretory paraneurons, the effect being long-lasting and nonspecific, while sympathetic neurons innervate both nerve plexus and intestinal tissues, and the effects in this case are specific, fast and of short duration.

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The development of the noradrenaline (NA)- and adrenaline (A)-storing cells was examined in the adrenal gland of pre- and postnatal rats and pigs. Cryostat sections were immunostained with antibodies to NA and A. Amine levels were estimated in homogenates of adrenals by ion-pair reversed-phase liquid chromatography with electrochemical detection.
1. In adult animals separate NA- and A-storing cells were found. In the rat A-cells comprised about 80% of the parenchyma of the adrenal medulla. NA-cells, the remaining 20%, were randomly arranged in clusters. In the pig, in contrast, the A- and NA-storing cells were equally distributed, with spherical clusters of NA-cells were surrounded by A-cells.
2. In the earliest developmental stages examined (16th day of gestation in the rat, and 42nd day in the pig) the adrenals only contained NA-immunoreactive cells. In the rat separate NA- and A-storing cells were first noticed 2 or 3 days after birth, whereas in the pig separated cells were already present at the 56th prenatal day (full term at 114th day).
3. In both rat and pig adrenals dopamine (DA), NA and A increased in amount rapidly during development. In the rat this change mainly took place after birth (an increase of 13 times from the 17th day of gestation, and a 100-fold increase from birth to adult age). In porcine adrenal increase of the total amount of amines mainly occurred before birth (200 times between the 42nd day of gestation and birth, whereas only 10 times after birth).
4. In both species DA-levels remained low during preand postnatal development. On the contrary, the relative concentrations of NA decreased while that of A increased correspondingly. Again, species differences were noticed: in the rat NA decreased from 90% (17th day of gestation) via 35% (just before birth) to 20% at adult age, while the porcine adrenal showed a more gradual decrease, i. e., from 90% (42nd day of gestation) to 70% (birth) ending up with 50% in the adult stage.
5. The immunohistochemical and biochemical data indicate that, in rat adrenal medulla three phases of development can be distinguished. First, up to the 18th day of gestation, medullary cells synthesize and store only NA. Second, from the 18th day to 2 or 3 days after birth, NA and A are synthesized and stored in a single cell type (“mixed cell type”), and third, NA and A are localized in separate cell types. It remains to be proved whether similar events also occur in the porcine adrenal.
6. Decapitation in pig foetuses (foetal hypophysectomy) at the 42nd, 56th and 92nd day of gestation was found to cause atrophy of the cortex, a strong reduction in the number of A-immunoreactive cells and a marked decrease in the relative concentration of A. On the contrary, foetal decapitation affected neither the number of NA-immunoreactive cells nor the total amount of amines contained in the adrenal. These observations indicate a functional relationship between the foetal adrenal cortex and medulla.

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The peptidergic innervation of arterial chemoreceptor organs (the rat carotid body and vagal paraganglia; guinea pig carotid body) was studied immunohistochemically. Five different populations of nerve fibres in the guinea pig carotid body could be discriminated according to their origin and their chemical coding. The innervation pattern of the rat carotid body differed in some aspects. Comparison of the rat carotid body and vagal paraganglia suggested that autonomic neuropeptide Y-like immunoreactive fibres act primarily via vascular mechanisms rather than directly on the chemoreceptor tissue. Sensory fibres were shown to contain immunoreactivities for substance P, calcitonin gene-related peptide (rat and guinea pig) and somatostatin (guinea pig). The functional role of the identified peptide-containing sensory fibres remains to be established.

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The neuronal and epithelial parts of the hypothalamo-hypophyseal complex (proximal and distal neurohypophysis, adenohypophysis) have in common the task of message transfer. According to the paraneuron concept, neurons and endocrine cells as paraneurons produce similar or identical substances, store them in vesicles and release them in response to specific stimuli. The released substances reach their targets via the intercellular spaces in a paracrine mode or via the blood in a more or less far-reaching hemocrine way. Special contacts for message transfer are not mandatory, even in the case of neurons. The biological activity of substances released by neurons and paraneurons depends on their affinities to binding sites, most probably localized on the outer target cell membrane. The neurons and paraneurons under discussion are mostly localized close to the mid-sagittal plane. The common ontogenetic roots of some neurons and paraneurons might be a further important point of the paraneuron concept.

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The immunohistochemical localization of nervous system-specific proteins and cytokeratin in the olfactory mucosa is described mainly in humans and guinea pigs. Immunoreactivity for neuron-specific enolase (NSE) was demonstrated in olfactory receptor cells including their dendrite and axon. Immunoreactivity for neurofilament protein (NFP) was contained in the dendrite and perikaryon of the olfactory cells, but not in the axon. Immunoreactivity for spot-35 protein, a kind of neuron-specific proteins was selectively localized in several flask-shaped cells of the olfactory epithelium only in the guinea pig. These spot-35 protein-positive cells were suspected of being microvillar cells, considered the second type of receptor cells dispersed in the olfactory epithelium. Immunoreactivity for gliaspecific S-100 protein was demonstrated in the Bowman's glands as well as Schwann cells associated with the olfactory nerves in the lamina propria. Supporting cells were not immunolabelled with any antisera against nervous system-specific proteins. Cytokeratin, a useful marker for epithelial cells, was expressed exclusively in basal cells.
Axotomy of olfactory nerves induced the disappearance of the olfactory receptor cells with NSE immunoreactivity 3 days after the operation. Regenerating cells showing NSE immunoreactivity were first recongnized in the lower portion of the epithelium within 7 days. The epithelium was completely repaired 3-4 weeks later and came to contain many NSE-reactive mature receptor cells. Bromodeoxyuridine (BrdU), administered during regeneration of the olfactory epithelium, was immunohistochemically detected in the cells of the layer above the basal cell layer, but not in the latter itself. This result does not support the general contention that the receptor cells originate from the basal cells.

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Chemoreceptive and mechanoreceptive paraneurons in the tongue include gustatory cells and basal cells of the taste buds, Merkel cells in the lingual epithelium and Merkel or Grandry corpuscles in avian subepithelial connective tissue. Neuron-specific enolase immunoreactivity is recognized in the basal cells, a type of Merkel cells, in the taste buds of teleost fishes and amphibia, certain taste bud cells of mammals and the Merkel cells in birds. The present study also concerns itself with the ultrastructural features of the gustatory cells of the rabbit taste buds, and the Merkel cells of the finch tongue. The functional significance of the densecored granules in the gustatory cell is still enigmatic. On the other hand, the dense-cored granules in the Merkel cells may have dual functions storing both neurotransmitters and local hormones. Shortly after giving a mechanical stimulus to the finch tongue, we could demonstrate massive exocytotic changes of the Merkel cell granules.

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The taste buds and associated nerves in the guinea pig, rat, cat, and mouse were investigated by immunocytochemistry and formaldehyde-induced fluorescence histochemistry. The antisera used were against spot 35 protein, neuron-specific enolase (NSE), neurofilament protein (NFP), and substance P. The spot 35 protein immunoreactivity was confined to taste bud cells in the guinea pig and rat; the immunoreactive cells, slender in shape, comprised half the number of the total taste bud cells in the guinea pig but were fewer in the rat. For NSE, on the other hand, taste bud cells as well as neural elements localized in both the taste bud and the subepithelial connective tissue were immunoreactive in all the species investigated. Furthermore, all of the spot 35 protein-immunoreactive cells proved to be NSE-immunoreactive in the guinea pig and rat. For NFP, neither the bud cells nor the nerves in the taste bud were reactive, whereas a part of nerves in the connective tissue was immunostained in all the species. The antiserum against substance P exclusively detected some parts of nerves in and out of the taste buds in the cat, rat, and mouse. The aminergic innervation was rather meager and appeared in the nerve fibers localized in the taste buds and connective tissue of the cat and mouse.

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Neuroepithelial bodies (NEB) are small groups of cells bound together by desmosomes and which form part of the epithelium lining the bronchopulmonary ducts.
The cells have an irregular, columnar shape often with laterally placed microvilli and unspecialized cytoplasmic protrusions into the intercellular spaces. Their most distinctive cytoplasmic feature is the numerous dense cored vesicles (DCV) located towards the basal region of each cell. A variety of peptides and the monoamine serotonin are distributed within these DCV. Evidence has been shown for the occurrence of more than one type of DCV for the co-existence of various peptides and serotonin in the same DCV and for morphological and functional variations in cells forming a NEB. In some animals the apical pole is in direct contact with the lumen of the duct, whereas in others the NEB is isolated from the lumen by either ciliated epithelial or Clara-like cells. When the apical pole contacts the lumen, the surface of each cell bears microvilli and occasionally a single cilium.
The NEB of eutherian mammals are sensitive to a number of experimental procedures including hypoxia, hypercapnia and vagal stimulation. Experimental lesions above and below the nodose ganglion have shown that the major part of the innervation is afferent with collateral branches off the afferent forming efferent, cholinergic endings. A small efferent input is also derived directly from the central nervous system. These studies suggest that a minor component of the efferent innervation is derived contralaterally or from intrapulmonary ganglia. Other forms of innervation identified by transmission electron microscopy in some mammals and lower vertebrates include cholinergic reciprocal synapses, adrenergic and peptidergic nerve junctions.
If due allowance is made for different electronmicroscopic and experimental procedures, two tentative evolutionary interpretations can be made. First, there has been a quantitative decrease in the adrenergic, efferent innervation from amphibian to mammal. Second, the cholinergic reciprocal synapse of the amphibian has been modified towards the afferent terminal with the efferent collateral branch present in eutherian mammals.
In most species examined, a capillary plexus is present either beneath or surrounding the NEB. In view of their intraepithelial location, cytochemical features, innervation and vascular supply, the NEB are considered to be endocrine/paracrine pulmonary receptors.
The essential features of NEB are similar in a variety of vertebrates encompassing amphibia, reptiles, birds and mammals. The essential histological features of these organoids is therefore presumed to have evolved within the ancestral Crossopterygian fish possibly as part of a vascular shunting mechanism in response to conditions of stress during the transition from gill to obligatory pulmonary respiration.

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Amines and/or peptide-producing cells, deserving to be called paraneurons, were demonstrated in the urethro-prostatic complex of the man, rabbit, buffalo and sheep and in the uterine horns of the pig, horse and mouse, by means of histochemical, immunohistochemical, immunofluorescent and double labeling immunofluorescent techniques. In particular, the urethro-prostatic complex of the sheep contains cells producing serotonin, chromogranin A and somatostatin. Often the amine and the “marker”-protein were colocalized in the same cells. Chromogranin A- and somatostatin-containing cells were found in the uterine horns of the pig.
Serotonin probably plays a role in the contraction/relaxation of the musculature of the male urogenital tract, thus regulating the urinary and spermatic flow.
The function of somatostatin cells in the urogenital tract is unknown. The presence and direction of their cytoplasmic processes lead us to hypothesize a double endocrine/paracrine modality of action.

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In lower vertebrates, Merkel cells are widely distributed in the epidermis. Dense-cored specific granules similar to those in mammalian Merkel cells are found in amphibians, dipnoans and lampreys, but only in some species of teleosts, others having few and smallcored vesicles. The characteristic microvilli are found in all these groups, but their number and disposition vary.
In amphibians a Merkel cell is associated with a single nerve fibre, known to be mechanoreceptive, which forms reciprocal synapses at a varicosity against the cell and continues on in the epidermis. In lampreys most of the specific granules are grouped around spur-like processes of a nerve fibre which has other branches in the epidermis. In teleosts, one or more nerve fibres wind around a Merkel cell; synaptic modifications are variable.
Fluorescence histochemistry shows the presence of quinacrine, neuron-specific enolase, met-enkephalin and serotonin in Merkel cells of some species that have been investigated, but experimental results from amphibians suggest that the receptive element may be the nerve fibre.
The Merkel cell can act as a target for growing nerve fibres, but other functions, especially connected with the synapses, most of which are morphologically aferent, remain unknown. Merkel cells do not require trophic maintenance from the nerve. The basal cells of taste buds in teleosts and amphibians have been compared to Merkel cells, but are not identical, although in frogs the basal cells have dense-cored vesicles. In teleosts, both cell types qualify as paraneurons, but there are considerable differences in their cytology especially in the form of the synaptic specializations.

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By the use of light microscopic immunohistochemistry, epidermal Merkel cells have been examined for the coexistence of some neuropeptides and chromogranin A (CGA). Peptide and CGA-immunophenotypes were similar in adult Merkel cells but variable in fetal skin, where CGA preceded the expression of peptides which were partly expressed only in a subpopulation of Merkel cells from hair follicles. Thus, only Substance P (SP) and calcitonin gene-related peptide (CGRP) were expressed in a subpopulation of Merkel cells from hair follicles. There were similar Merkel cell densities visualized on consecutive paraffin sections by the use of antisera against peptides, CGA and cytokeratin offering useful tools for a future systematical complementary mapping of Merkel cell populations in various species, locations and developmental stages. Electron microscopic immunohistochemistry has shown that CGA-immunoreaction is localized in the secretory granules which, again, supports the view that the Merkel cell is a paraneuron, i. e., neurosecretory in function.

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The pinealocytes—the main cellular elements of the pineal organ—are polarized, displaying a (photo) receptor and an axonic effector cell pole. The receptor endings are of two main types: they bear rod-type or cone-type outer segments characterized by the presence of immunoreactive opsin-, S-antigen- and vitamin A-binding sites. The effector pole may form ribbon-containing synapses on the secondary pineal neurons, and/or neurohormonal terminals on the basal lamina of the pineal nervous tissue.
Applying potassium pyroantimonate (PPA) to electron-microscopic histochemistry, we found in the frog that both effector terminals and photoreceptor outer segments contained a large amount of Capyroantimonate deposit similar to retinal cones and rods. Rods and rod-like pinealocytes contained more deposits than cones. The higher concentration of calcium on the cell membranes of dark pinealocytes in the rat may be connected with their rod-like character. In the frog, a high amount of calcium seemed to be concentrated in the photoreceptor effector terminals, especially around their synaptic ribbons, and in myeloid bodies of the pineal ependyma and retinal pigment epithelium. Calcium was richly found in or around corpora arenacea in the human and rat pineal. It is suggested that the formation of concrements may be connected with the high demand of Ca-exchange of pinealocytes for their receptor and effector membrane functions.
In the rat, lymphocytes were found to migrate through the wall of the vena magna of Galen and to closely contact pinealocytes, presumably to receive immunological information as an additional pineal output.

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Data on the cell biology of pineal transducers (chief cells: typical and modified photoreceptors, pinealocytes) which belong to the paraneuron family, are reviewed in the vertebrate series. In spite of major changes throughout phylogeny, it is proposed that pineal chief cells share a common feature: they somehow transform the information derived from the light/ dark cycle into daily rhythms of neural (an excitatory neurotransmitter) and/or hormonal (melatoninergic) output and appear invariably involved in the temporal organization of physiological and behavioral processes.

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