Relationship between postsynaptic NK(1) receptor distribution and nerve terminals innervating myenteric neurons in the guinea-pig ileum.

The amounts of neurokinin 1 (NK(1)) receptor immunolabelling on the membranes of myenteric cell bodies at appositions with tachykinin-immunoreactive nerve terminals, other nerve terminals, and glial cells were compared at the ultrastructural level using pre-embedding, double-label immunocytochemistry. NK(1) receptor immunoreactivity was revealed using silver-intensified, 1 nm gold, and tachykinin-immunoreactive nerve terminals were revealed using diaminobenzidine. The density of NK(1) receptor immunolabelling (silver particles per length of cell membrane) on the membrane at appositions with tachykinin-immunoreactive nerve terminals was not significantly different from that at appositions with other (nonimmunoreactive) nerve terminals or with glial cells. Synaptic specializations ("active zones") were present at a small proportion of the appositions between NK(1) receptor-immunoreactive cell bodies and tachykinin-immunoreactive or other nerve terminals. The density of NK(1) receptor immunolabelling at synaptic specializations was lower than that at regions of appositions where no synaptic specializations were present. The presence of NK(1) receptor on the cell surface in areas not directly apposed to tachykinin-containing nerve terminals suggests that tachykinins that diffuse away from their site of release may still exert an action via NK(1) receptors. Although NK(1) receptors do not appear to be targetted to particular sites on the surfaces of myenteric nerve cell bodies and proximal dendrites, they are reduced in density at regions of the membrane-forming synaptic specializations.     

Important roles of tachykinins in the development of allergic nasal hyperresponsiveness in guinea-pigs

Background Although it has been suggested that the use of tachykinin receptor antagonists might prove to be an effective treatment for allergic rhinitis (AR), they are not used clinically. Therefore, we decided to examine the effects of tachykinin receptor antagonists on AR symptoms in an appropriate experimental model. Objective To evaluate newly developed tachykinin receptor antagonists in a Japanese cedar pollen‐induced AR model and to determine their effect on allergen‐induced sneezing, nasal blockage, and nasal hyperresponsiveness (NHR). Methods Sensitized guinea‐pigs were challenged by forced inhalation of pollen once every week. Sneezing and nasal blockage were observed after pollen challenges. NHR (nasal blockage) to an intranasal application of leukotriene D₄ was assessed 2 days after an antigen challenge. We also evaluated whether intranasal dosing with a tachykinin causes NHR. NK₁ and NK₂ receptor antagonists were administered before an intranasal treatment with antigen or tachykinin. Amounts of tachykinins present in nasal cavity lavage fluid were measured by an enzyme immunoassay. Results Although an NK₁ and NK₂ receptor dual antagonist showed no effect on pollen‐induced sneezing and biphasic nasal blockage, it did completely suppress the development of NHR. Experiments using specific NK₁ or NK₂ receptor antagonists revealed that NK₂ receptor activation was preferentially involved in the development of hyperresponsiveness. Increases in the levels of substance P (SP) and neurokinin A (NKA) in the nasal tissue were noted 20 min–1 h after the challenge. Intranasal instillation of either SP or NKA‐induced NHR, which was almost completely inhibited by NK₂ receptor antagonists and partially inhibited by NK₁ receptor antagonists. Conclusions SP and NKA, which are released early after the challenge, mediate the development of NHR by preferentially activating NK₂ receptors. Therefore, NK₂ receptor antagonists might prove to be effective treatment of AR.     

Interactions of Serotoninergic,Cholinergic, and Tachykinin‐Containing Nerve Elements in the Rabbit Small Intestine

This report presents novel results on the effects of serotonin (5‐HT) on longitudinal muscle contractions in the rabbit ileum and the interactions of serotonin with some neuronal elements of the myenteric plexus. We showed previously that serotonin‐triggered contractions involved two mechanisms in the rabbit ileum: neuronal excitation (via 5‐HT₂ receptors in the neurons) and direct muscular stimulation (via 5‐HT₄ receptors in the muscle). Here, we focus on the neuronal 5‐HT₂ receptor pathway and report further pharmacological and immunocytochemical data clarifying the details of the mechanisms. We observed that antagonists for neurokinin (NK1 and NK2) receptors partially blocked the serotonin response, but NK3 receptor antagonists had no effect. Pretreatment by atropine (ATR) eliminated the NK1 receptor antagonist resistant contractions. In contrast, the NK1 antagonist did not depress the ATR‐resistant contraction when ATR was added first. 5‐HT₂ receptor agonist‐induced contractions were partially suppressed by ATR, hexamethonium, and NK1 or NK2 receptor antagonists. In conclusion, serotonin acting through 5‐HT₂ receptors could stimulate interneurons and excitatory motor neurons. Immunocytochemical staining revealed an extensive tachykinin‐immunoreactive (IR) network in the myenteric plexus. Approximately 52% of all myenteric neurons were labeled. 5‐HT‐IR fibers could be detected around both choline acetyltransferase‐ and tachykinin‐IR cells, suggesting functional relationships between them. Consistent with our pharmacological observations, we found that immunopositive nerve elements for 5‐HT_2A receptor and double‐labeled immunostaining revealed a remarkable overlap between tachykinin‐IR neurons and 5‐HT_2A‐IR elements. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Ultrastructure and synaptic relationships of calbindin-reactive, Dogiel type II neurons, in myenteric ganglia of guinea-pig small intestine

Summary Immunoreactivity for calbindin D 28K was localized ultrastructurally in nerve cell bodies and nerve fibres in myenteric ganglia of the guinea-pig small intestine. Reactive cell bodies had a characteristic ultrastructure: the cytoplasm contained many elongate, electron-dense mitochondria, numerous secondary lysosomes that were peripherally located, peripheral stacks of rough endoplasmic reticulum and dispersed Golgi apparatus. The cells were generally larger than other myenteric neurons and had mainly smooth outlines. The cytoplasmic features of these neurons were shared by a small group of immunonegative cells, but the majority of negative cells had clearly different ultrastructural appearances. Of 310 cells from 16 ganglia that were systematically examined, 38% were immunoreactive for calbindin, 10% were unreactive but similar in ultrastructure to the calbindin-reactive neurons and 51% were unreactive and dissimilar in the appearance of their cytoplasmic organelles. Immunoreactive varicosities with synaptic specializations were found on most unreactive neurons, but were markedly less frequent on the calbindin-immunoreactive cell bodies. Non-reactive presynaptic fibres were also more common on non-reactive neurons than on the calbindin-positive cell bodies. Numerous reactive varicosities, some showing synaptic specializations, were found adjacent to other fibres in the neuropil. Light microscopic studies show calbindin immunoreactive neurons to have Dogiel type-II morphology. Thus the present work links distinguishing ultrastructural features to a specific nerve cell type recognized by light microscopy in the enteric ganglia for the first time.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

The origins,pathways and terminations of neurons with VIP-like immunoreactivity in the guinea-pig small intestine

We have analyzed changes in the distributions of terminals with vasoactive intestinal polypeptide (VIP)-like immunoreactivity, and accumulations in severed processes, that occur after lesions of intrinsic and extrinsic nerve pathways of the guinea-pig small intestine. The observations indicate that enteric vasoactive intestinal polypeptide immunoreactive neurons have the following projections. Nerve cell bodies in the myenteric plexus provide varicose processes to the underlying circular muscle; the majority of these pathways, if they extend at all in the anal or oral directions, do so for distances of less than 1 mm. Nerve cell bodies of the myenteric plexus also project anally to provide terminals to other myenteric ganglia. The lengths of the majority of these projections are between 2 and 10 mm, with an average length of about 6 mm. Processes of myenteric neurons also run anally in the myenteric plexus and then penetrate the circular muscle to provide varicose processes in the submucous ganglia at distances of up to 15 mm, the average length being 9–12 mm. In addition, there is an intestinofugal projection of myenteric neurons whose processes end around nerve cell bodies of the coeliac ganglia. A similar projection from the colon supplies the inferior mesenteric ganglia. The nerve cell bodies in submucous ganglia give rise to a subepithelial network of fibres in the mucosa and also supply terminals to submucous arterioles.It is concluded that vasoactive intestinal polypeptide is contained in neurons of a number of intrinsic nerve pathways, influencing motility, blood flow and mucosal transport. The myenteric neurons that project to prevertebral sympathetic ganglia may be involved in intestino-intestinal reflexes.     

Synaptogenesis in the basilar papilla of the chick

Summary Synaptogenesis was studied in the basilar papilla of chicken embryos from days 7–21 of incubation. On the 9th day of incubation differentiating hair cells first appeared and a few growing nerve tips made contact with them, although no membrane specializations were apparent at this stage. Synaptic bodies associated with presynaptic membrane specializations were first observed on the 10th day. They lay opposite either supporting cells or afferent nerve processes; in the latter site slight membrane thickenings were occasionally found. During subsequent stages synaptic bodies and the surrounding vesicles increased in number. Synaptic bodies associated with presynaptic membrane specializations, but devoid of contact with afferent nerve endings, were often observed on the 14th day, whereas almost all the synaptic bodies associated with presynaptic specializations were in contact with afferent nerve processes by the 21st day. The efferent synapses were first recognized on the 14th day. These results suggest that in the hair cells of chicken basilar papilla the synaptic bodies and presynaptic membrane specializations appear first and after the synaptic sites are determined by the position of the synaptic bodies, the growing nerve tips seek out and establish synaptic contact at the pre-existing synaptic sites.     

Neurokinin-1 Receptor Immunoreactive Neuronal Elements in the Superficial Dorsal Horn of the Chicken Spinal Cord: With Special Reference to Their Relationship with the Tachykinin-containing Central Axon Terminals in Synaptic Glomeruli

Synaptic glomeruli that involve tachykinin-containing primary afferent central terminals are numerous in lamina II of the chicken spinal cord. Therefore, a certain amount of noxious information is likely to be modulated in these structures in chickens. In this study, we used immunohistochemistry with confocal and electron microscopy to investigate whether neurokinin-1 receptor (NK-1R)-expressing neuronal elements are in contact with the central primary afferent terminals in synaptic glomeruli of the chicken spinal cord. We also investigated which neuronal elements (axon terminals, dendrites, cell bodies) and which neurons in the spinal cord possess NK-1R, and are possibly influenced by tachykinin in the glomeruli. By confocal microscopy, NK-1R immunoreactivities were seen in a variety of neuronal cell bodies, their dendrites and smaller fibers of unknown origin. Some of the NK-1R immunoreactive profiles also expressed GABA immunoreactivities. A close association was observed between the NK-1R-immunoreactive neurons and tachykinin-immunoreactive axonal varicosities. By electron microscopy, NK-1R immunoreactivity was seen in cell bodies, conventional dendrites and vesicle-containing dendrites in laminae I and II. Among these elements, dendrites and vesicle-containing dendrites made contact with tachykinin-containing central terminals in the synaptic glomeruli. These results indicate that tachykinin-containing central terminals in the chicken spinal cord can modulate second-order neuronal elements in the synaptic glomeruli.     

The fine structure of serotonin and tyrosine hydroxylase immunoreactive terminals in the ventral posterior thalamic nucleus of cat and monkey

Summary Immunocytochemical methods have been combined with serial thin section analysis to study the synaptic organization of serotonin (5-HT) and tyrosine hydroxylase (TH) immunoreactive terminals in the ventral posterior nucleus of the cat and monkey thalamus. One hundred 5-HT immunoreactive terminals from the cat and approximately forty 5-HT and TH immunoreactive terminals from the monkey were selected for analysis in serial thin sections. Only 7–10% of the immunoreactive terminals could be revealed to form conventional synaptic contacts. Most of these could be identified as the asymmetrical type. Dendritic shafts belonging to relay neurons were the major targets of these monoamine immunoreactive terminals. The remainder made intimate membrane associations with relay cell dendrites and somata or with presynaptic dendrites of interneurons, but no overt membrane specializations could be detected. The present results suggest that the modulation of thalamocortical relay function by brainstem monoamine pathways in the somatosensory thalamus may occur by release of transmitters at atypical contact sites.     

The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.     

Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines

Tyrosine hydroxylase-immunoreactive fibres in the rat neostriatum were studied in the electron microscope in order to determine the nature of the contacts they make with other neural elements. The larger varicose parts of such fibres contained relatively few vesicles and rarely displayed synaptic membrane specializations; however, thinner parts of axons (0.1-0.4 micron) contained many vesicles and had symmetrical membrane specializations, indicative of en passant type synapses. By far the most common postsynaptic targets of tyrosine hydroxylase-immunoreactive boutons were dendritic spines and shafts, although neuronal cell bodies and axon initial segments also received such input. Six striatonigral neurons in the ventral striatum were identified by retrograde labelling with horseradish peroxidase and their dendritic processes were revealed by Golgi impregnation using the section-Golgi procedure. The same sections were also developed to reveal tyrosine hydroxylase immunoreactivity and so we were able to study immunoreactive boutons in contact with the Golgi-impregnated striatonigral neurons. Each of the 280 immunoreactive boutons examined in the electron microscope displayed symmetrical synaptic membrane specializations: 59% of the boutons were in synaptic contact with the dendritic spines, 35% with the dendritic shafts and 6% with the cell bodies of striatonigral neurons. The dendritic spines of striatonigral neurons that received input from immunoreactive boutons invariably also received input, usually more distally, from unstained boutons that formed asymmetrical synaptic specializations. A study of 87 spines along the dendrites of an identified striatonigral neuron showed that the most common type of synaptic input was from an individual unstained bouton making asymmetrical synaptic contact (53%), while 39% of the spines received one asymmetrical synapse and one symmetrical immunoreactive synapse. It is proposed that the spatial distribution of presumed dopaminergic terminals in synaptic contact with different parts of striatonigral neurons has important functional implications. Those synapses on the cell body and proximal dendritic shafts might mediate a relatively non-selective inhibition. In contrast, the major dopaminergic input that occurs on the necks of dendritic spines is likely to be highly selective since it could prevent the excitatory input to the same spines from reaching the dendritic shaft. One of the main functions of dopamine released from nigrostriatal fibres might thus be to alter the pattern of firing of striatal output neurons by regulating their input.     

An ultrastructural study of nerve and glial cells by freeze-substitution

Summary The ultrastructure of nerve and glial cells in the cerebral and cerebellar cortices of mice was studied after rapid freezing followed by substitution fixation. The cerebral and cerebellar cortices were frozen by bringing them into contact with a polished pure copper block cooled at a temperature of about –196 ° C. The tissues were fixed and substituted in acetone containing 2–4% OsO₄ at –78 ° C for 2–3 days and then prepared for electron microscopy. Tissue fixed by this method displayed the following characteristics. (1) The contour of cells, processes and intracellular membrane systems was smooth. (2) The extracellular spaces were of variable widths. (3) Microtubules were well preserved and were often observed to extend into nerve terminals and to run close to presynaptic membranes. (4) The matrix of cytoplasm and mitochondria was electron dense. Dense granules, possibly binding sites of divalent cations, were often found in the mitochondrial matrix. (5) The plasma membrane of neuronal processes was thicker than that of glial processes. (6) The plasma membranes of nerve fibres and glial processes appeared asymmetrical, the inner leaflet being slightly thicker than the outer leaflet, whereas membranes of cell organelles such as smooth endoplasmic reticulum, Golgi bodies, lysosomes, multivesicular bodies, mitochondria and synaptic vesicles, were symmetrical.     

Inhibitory postsynaptic membrane specializations are formed in gephyrin-deficient mice

Gephyrin is a major postsynaptic scaffolding protein at GABAergic and glycinergic inhibitory synapses. Gephyrin-deficient (geph^−/−) mice die after birth due to disinhibition of motor and sensory pathways resulting from a lack of postsynaptic glycine receptor and GABA_A receptor clusters. Here, immunoelectron and confocal microscopy revealed that postsynaptic membrane specializations are formed in the absence of gephyrin. First, in brainstem sections obtained from newborn geph^−/− mice inhibitory nerve terminals identified by immunogold labeling of either the vesicular inhibitory amino acid transporter (VIAAT) or GABA were found to be apposed to postsynaptic membrane areas decorated by electron-dense material. Second, neuroligin-2, a membrane protein of inhibitory postsynapses, was clustered beneath glutamate decarboxylase 65 (GAD-65) positive nerve terminals in geph^−/− hippocampal cultures. These results indicate that proteins other than gephyrin define the ultrastructure of inhibitory postsynaptic membrane specializations.     

GABAB and group I metabotropic glutamate receptors in the striatopallidal complex in primates

Glutamate and GABA neurotransmission is mediated through various types of ionotropic and metabotropic receptors. In this review, we summarise some of our recent findings on the subcellular and subsynaptic localisation of GABA_B and group I metabotropic glutamate receptors in the striatopallidal complex of monkeys. Polyclonal antibodies that specifically recognise GABA_BR1, mGluR1a and mGluR5 receptor subtypes were used for immunoperoxidase and pre‐embedding immunogold techniques at the light and electron microscope levels. Both subtypes of group I mGluRs were expressed postsynaptically in striatal projection neurons and interneurons where they aggregate perisynaptically at asymmetric glutamatergic synapses and symmetric dopaminergic synaptic junctions. Moreover, they are also strongly expressed in the main body of symmetric synapses established by putative intrastriatal GABAergic terminals. In the globus pallidus, both receptor subtypes are found postsynaptically in the core of striatopallidal GABAergic synapses and perisynaptically at subthalamopallidal glutamatergic synapses. Finally, extrasynaptic labelling was commonly seen in the globus pallidus and the striatum. Moderate to intense GABA_BR1 immunoreactivity was observed in the striatopallidal complex. At the electron microscope level, GABA_BR1 immunostaining was commonly found in neuronal cell bodies and dendrites. Many striatal dendritic spines also displayed GABA_BR1 immunoreactivity. Moreover, GABA_BR1‐immunoreactive axons and axon terminals were frequently encountered. In the striatum, GABA_BR1‐immunoreactive boutons resembled terminals of cortical origin, while in the globus pallidus, subthalamic‐like terminals were labelled. Pre‐embedding immunogold data showed that postsynaptic GABA_BR1 receptors are concentrated at extrasynaptic sites on dendrites, spines and somata in the striatopallidal complex, perisynaptically at asymmetric synapses and in the main body of symmetric striatopallidal synapses in the GPe and GPi. Consistent with the immunoperoxidase data, immunoparticles were found in the presynaptic grid of asymmetric synapses established by cortical‐ and subthalamic‐like glutamatergic terminals. These findings indicate that both GABA and glutamate metabotropic receptors are located to subserve various modulatory functions of the synaptic transmission in the primate striatopallidal complex. Furthermore, their pattern of localisation raises issues about their roles and mechanisms of activation in normal and pathological conditions. Because of their ‘modulatory’ functions, these receptors are ideal targets for chronic drug therapies in neurodegenerative diseases such as Parkinson's disease.     

Serotonergic transmission in the periaqueductal gray matter in relation to aversive behaviour: morphological evidence for direct modulatory effects on identified output neurons

Intracellular recordings were made from 21 cells in the dorsolateral periaqueductal gray matter in coronal midbrain slices. In the majority (n=20) bath application of 5-hydroxytryptamine (30 or 150 mM) evoked either hyperpolarizing (n=11) or depolarizing (n=9) responses. Reconstructions of 11 neurons in the dorsolateral periaqueductal gray matter after filling with biocytin revealed a population of output neurons whose axons followed a dorsolateral trajectory towards the perimeter of the ipsilateral periaqueductal gray matter. In seven cells, the axon could be followed into the adjacent mesencephalic reticular formation. At the light microscopic level, immunostaining for 5-hydroxytryptamine revealed immunoreactive processes throughout the dorsolateral periaqueductal gray matter but no labelled somata or dendrites. Close associations (i.e. no discernible gap) were observed between serotonergic profiles and the somata and dendrites of biocytin-filled cells. At the ultrastructural level, serial sections through 21 appositions on to biocytin-filled dendrites in three slices revealed 19 true appositions (i.e. having closely parallel plasma membranes with no intervening glial cell profiles) with the biocytin-filled dendrite. Only four of the appositions (21%) showed evidence of synaptic specializations which included aggregations of synaptic vesicles, and some thickening of the apposing membrane. The dense reaction product in the biocytin-filled cells precluded identification of the ultrastructure of postsynaptic elements. However, examination of contacts between 5-hydroxytryptamine-immunoreactive profiles and unlabelled elements in material taken from the contralateral side of the periaqueductal gray matter (i.e. no biocytin present) or in material taken from perfusion-fixed whole brain, in which ultrastructural preservation was superior compared with slices, revealed a similar incidence (21% and 23%, respectively) of synaptic specializations.The data indicate that serotonergic transmission on to output neurons in the dorsolateral periaqueductal gray matter is largely mediated by non-junctional contacts, suggesting that the actions of 5-hydroxytryptamine on these cells are mediated predominantly by volume rather than wiring transmission.     

Correlation Between the Distribution of SP and CGRP Immunopositive Neurons in Dorsal Root Ganglia and the Afferent Sensation of Preputial Frenulum

The aim of this study was to explore the distribution of substance P (SP) and calcitonin gene‐related peptide (CGRP) immunoreactive nerve terminals in the penis prepuce and the preputial frenulum. The possible correlation between SP‐ and CGRP‐immunopositive neurons in dorsal root ganglia (DRG) and the afferent sensation of the penile preputial frenulum is also discussed. Immunohistochemistry showed SP‐ and CGRP‐positive nerve terminals in the epidermal basal layer of the prepuce and frenulum in adult human males. The majority of the nerve terminals presented as bundles of different lengths and a few as enlarged nodosities. The density of SP‐ and CGRP‐immunopositive nerve terminals in the preputial frenulum was significantly higher than those in the penis prepuce (P<0.01). Fluoro‐Gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in Sprague‐Dawley rats. SP and CGRP immunofluorescence labeling was employed to detect the distribution of SP‐ and CGRP‐immunoreactive neurons in DRG. FG retro‐labeled neurons were localized in L₆‐DRG and S₁‐DRG. All the FG/SP and FG/CGRP double‐labeled neurons were medium or small‐sized. One‐third of the FG‐labeled neurons were SP‐immunoreactive, and a half of them CGRP‐immunoreactive in L₆‐DRG and S₁‐DRG, respectively. The FG/SP/CGRP‐labeled neurons accounted for one fifth of the FG retro‐labeled neurons. Taken together, these data suggest that the SP‐ and CGRP‐immunopositive nerve fibers may participate in the transmission of afferent sensation in the preputial frenulum. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Ultrastructural relationships between serotonin and dopamine neurons in the rat arcuate nucleus and medial zona incerta: a combined radioautographic and immunocytochemical study

Combined radioautographic and immunocytochemical detection of [3H]serotonin-labeled axon terminals and tyrosine hydroxylase-immunoreactive processes in the same thin sections allowed for electron microscopic demonstration of direct appositions between serotoninergic axonal varicosities and dopaminergic nerve cell bodies and/or dendrites in the anterior part of the arcuate nucleus and in the medial zona incerta. Although no junctional specializations were apparent at the sites of contacts, it is proposed that the observed appositions may represent a serotonin input onto tubero-infundibular and incerto-hypothalamic dopaminergic neurons. This innervation could account for some of the central neuroendocrine effects of serotonin, particularly its regulatory role on prolactin and gonadotropin secretion.     

Morphometrical analysis of the distribution of corticotrophin releasing factor, glucocorticoid receptor and phenylethanolamine-N-methyltransferase immunoreactive structures in the paraventricular hypothalamic nucleus of the rat

By means of the indirect immunoperoxidase technique the corticotrophin releasing factor (CRF) and glucocorticoid receptor (GR) immunoreactive nerve cell bodies and the phenylethanolamine-N-methyltransferase (PNMT) immunoreactive nerve terminals in the paraventricular hypothalamic nucleus of the rat have been mapped out in adjacent vibratome sections (30 micron thick). By means of morphometrical analysis using a semiautomatic image analyser, it was possible to obtain density maps of CRF, GR and PNMT immunoreactive structures within the paraventricular hypothalamic nucleus. The statistical analysis by the use of correlation coefficients gives evidence that the PNMT immunoreactive nerve terminals innervate the majority of the CRF immunoreactive nerve cell bodies and that GR are located in the majority of the CRF immunoreactive neurons.     

G2-acetylcholinesterase is presynaptically localized in Torpedo electric organ.

Summary InTorpedo electric organ, much of the acetylcholinesterase (AChE) is a globular dimer (G₂), anchored to the plasma membrane via covalently attached phosphatidylinositol and selectively solubilized by a bacterial phosphatidylinositol-specific phospholipase C. While the structure of this form of the enzyme is well-established, the ultrastructural localization of G₂-AChE is still unclear. Selective solubilization with phosphatidylinositol-specific phospholipase C was, therefore, combined with immunocytochemistry at the electron microscope level, in order to localize G₂-AChE in electric organ ofTorpedo ocellata. Thin sections of electric organ were labelled with antibodies raised againstTorpedo AChE, followed by gold-conjugated second antibodies, before or after exposure to the phospholipase. For comparison, the location of AChE was examined using histochemical methods. We show that (1) immunolabelling is concentrated in the synaptic clefts between nerve terminals and the innervated face of the electrocyte; (2) this labelling co-localizes with AChE histochemical reaction products; and (3) prior exposure to the phospholipase causes a decrease in AChE-associated labelling. Quantitative analysis of immunolabelling in the synaptic clefts shows that the phospholipase treatment had reduced primary labelling at or adjacent to the presynaptic membrane. Together with our earlier biochemical and immunofluorescent evidence, these results support our previous assignment of a neuronal and synaptic localization for G₂-AChE inTorpedo electric organ.     

The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: Electron microscopy

We have studied the posterior division of the anteroventral cochlear nucleus, where the cochlear nerve root enters the brain, in the cat. In Nissl preparations, this region contains two types of neuronal cell bodies: globular and multipolar. The two types can be identified in the electron-microscope by comparing Nissl substance and rough endoplasmic reticulum. Globular cell bodies receive many synaptic terminals, which cover 85% of the surface. In contrast, multipolar cell bodies are almost entirely wrapped by thin glial sheets—synaptic terminals contact less than 15% of the surface and tend to cluster at the bases of dendrites. Synaptic terminals are of three kinds, types 1, 2, and 3, which contain large round, small round-to-oval, and small flattened synaptic vesicles, respectively. Terminals of all three kinds synapse on both types of cell bodies. However, only globular cell bodies receive the largest type 1 terminals, which correspond to end-bulbs, seen in Golgi impregnations to arise from cochlear nerve axons. Cochlear ablation leads to degeneration of type 1, but not type 2 or 3 terminals.We conclude that neurons with globular cell bodies receive heavy somatic input from the cochlear nerve, as well as from other sources. Neurons with multipolar cell bodies receive very little input to their perikarya—giving their dendrites a more important role in determining their response properties. We suggest a morphological basis for correlating individual kinds of neurons with certain electrophysiological response types.