Fine structure of neurons synthesizing vasoactive intestinal peptide in the human colon from patients with Hirschsprung's disease

Summary The fine structure of neuronal perikarya and processes containing VIP-like immunoreactive material in the colon of patients with Hirschsprung's disease was investigated by immunoelectron microscopy. No VIP-like immunoreactive terminals were found in Auerbach's plexus of the ganglionic segment. However, VIP-like immunoreactive preterminal axons were frequently found to make synaptic contact with both immunoreactive and non-immunoreactive elements within Meissner's plexus. Therefore, the function of the VIP neurons in Auerbach's plexus seems to differ from that in Meissner's plexus. In the oligoganglionic segment, there were a few VIP-like immunoreactive processes, but no VIP-like immunoreactive synaptic formations. VIP-like immunoreactive processes were rarely encountered in the aganglionic segment. In both the oligo-and aganglionic segments, bowel relaxation is considered to be disturbed due to the lack of synaptic contacts of VIP-like immunoreactive neurons with other neuronal components.This work was supported in part by a grant (no. 57370002) from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

Comparative immunohistochemical study of Carassius RFamide localization in teleost guts in different salinity habitats

Carassius RFamide (C-RFa) is a peptide, isolated originally from the brain of Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptides. From the physiological aspect, it is known that C-RFa has contraction-promoting action on fish intestines, but its localization in peripheral tissues is unknown. We observed the localization of C-RFa in teleost guts using an immunohistochemical technique. C-RFa-like immunoreactive (irC-RFa) sites were observed in not only the smooth muscle cells in the longitudinal muscle layer, but also in both Auerbach's and Meissner's nerve plexus in the stomach, pyloric ceca and intestine. In epithelial mucous cells, irC-RFa sites were observed in the surface mucous cells in the stomach in freshwater fish (FW), and in the goblet cells of the apical sites in the villi of the pyloric ceca and intestine in all fish. In the stomach, irC-RFa sites were found in the fundic glands of the body regions in seawater (SW) and brackish water (BW) fish, but not in FW fish. This study confirmed that one of the functions of C-RFa is the smooth muscle contraction of the longitudinal muscle layer in digestive organs. We suggest that C-RFa may have functional roles in both central and peripheral neurotransmission. In addition, it appears that the difference in C-RFa localization of SW, BW, and FW fish reflects the adaptation of the stomach function to different salinity habitats.     

Distribution and ontogeny of VIP-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris

Summary The presence, distribution and development of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris (L.) was investigated by immunohistochemical methods utilizing mammalian VIP antisera. In the gut VIP-like immunoreactivity was observed in both nerves and endocrine cells. Endocrine cells with VIP-like material were only detected in the intestinal epithelium while nerve fibres containing VIP-like material were noted along the whole gastro-entero-pancreatic system, being more numerous in the pyloric sphincter and in the intestinal portion. Immunoreactive nerve cell bodies were encountered in the stomach and intestinal portions localized in the submucosa and in the myenteric plexus. Intestinal immunoreactive endocrine cells were already present in the first developmental stage considered (embryos aged 4 months). They grow in number and before birth reach a frequency higher than in adults. Nerves and cell bodies showing VIP-like immunoreactivity, appear later, before birth, as a few elements in the smooth muscular layer, but only after birth their distribution and frequency are similar to those found in adults. The faint immunofluorescence shown by the immunoreactive endocrine cells and their developmental pattern, which is always different from that observed in nervous elements, suggest the presence of at least two VIP-like substances in the gastro-entero-pancreatic system of S. stellaris.     

大鼠肠道内NOS与AChE、VIP阳性神经元的分布关系研究

应用一氧化氮合酶 (NOS)、乙酰胆碱酯酶 (ACh E)组织化学及血管活性肠肽 (VIP)免疫组织化学方法 ,光镜下比较观察大鼠肠道内 NOS、ACh E、VIP阳性神经元的形态学特征。结果显示 ,肠肌间丛 NOS阳性神经元胞体大小不等 ,形态不一 ,NOS、ACh E和 VIP阳性神经元的分布密度为 ACh E>NOS>VIP,在不同的肠段和层次分布密度有差异 ,NOS与 ACh E存在共染。在肌间丛和粘膜下丛 ,少数 VIP与 NOS共染。在粘膜下丛 ,三种阳性神经元的分布密度为 ACh E>VIP>NOS。在肌间丛和粘膜下丛 ,可见 VIP阳性末梢环抱 NOS阳性神经元胞体 ,两者呈终扣样接触。上述结果提示 NOS阳性神经元与 ACh E、 VIP阳性神经元有密切的形态学联系。在消化道功能调节上 ,它们可能起协调作用。     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.     

A novel neuropeptide,pituitary adenylate cyclase-activating polypeptide (PACAP), in human intestine: evidence for reduced content in Hirschsprung's disease

Summary A novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exhibits sequence homology with vasoactive intestinal polypeptide (VIP) and occurs in the mammalian brain, lung and gut. The distribution of PACAP in ganglionic and aganglionic portions of the large intestine of patients with Hirschsprung's disease was examined by immunohistochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were distributed in all layers of the ganglionic and aganglionic segments of the intestine, although they were less numerous in the latter, and PACAP-immunoreactive nerve cell bodies were seen in the ganglionic portion of the intestine. The concentration of immunoreactive PACAP was lower in the aganglionic than in the ganglionic segment of the intestinal wall. PACAP and VIP were found to coexist in both ganglionic and aganglionic segments of the intestine. Apparently, PACAP participates in the regulation of gut motility. The scarcer PACAP innervation of the aganglionic segment may contribute to the defect in intestinal relaxation seen in patients with Hirschsprung's disease.     

Medullary and mesencephalic neuronal groups reacting to antibodies against VIP, somatostatin and bombesin in adult Gallus gallus domesticus

The caudo-cranially intermediate one-third of medullary dorsal region, the periaqueductal grey and the rostro-ventral portion of the midbrain tegmentum of adult chickens were studied in detail by means of the PAP-DAB procedure, to define further the main morphological features of the neuronal populations that in previous studies had shown VIP (Vasoactive Intestinal Polypeptide),-Somatostatin (SRIF)-, and Bombesin-like immunoreactivities. In the medulla, VIP-like immunoreactivity was detected within neuronal bodies and processes and extended down to the cervical spinal cord. SRIF-like immunoreactivity was seen only within nerve cell processes, at least a part of which could be sensitive fibre terminals. Bombesin-like immunoreactivity was observed only within neuronal processes. In the periaqueductal grey, all 3 immunoreactivities were detected within perikarya and neuronal processes, with a higher density cranially. In the rostro-ventral portion of the midbrain tegmentum, VIP-like and Bombesin-like immunoreactivities were detected (the latter being located somewhat more cranially) both in neuronal bodies and in processes. SRIF-like immunoreactivity was found in this region only in long neuronal processes.     

An immuno-electron-microscopic study of the localization of VIP-like immunoreactivity in the adrenal gland of the rat

Summary VIP-like immunoreactivity was revealed in a few chromaffin cells, medullary ganglion cells and a plexus of varicose nerve fibers in the superficial cortex and single varicose fibers in the juxtamedullary cortex and the medulla of the rat adrenal gland. VIP-like immunoreactive chromaffin cells were polygonal in shape without any distinct cytoplasmic processes and they appeared solitarily. Their cytoplasm contained abundant granular vesicles having a round core and the immunoreactive material was localized to the granular core. VIP-immunoreactive ganglion cells were multipolar and had large intracytoplasmic vacuoles. The immunoreactive material was localized not only in a few granular vesicles but also diffusely throughout the axoplasm. VIP-immunoreactive varicose nerve fibers in the superficial cortex were characterized by abundant small clear vesicles and some large granular vesicles, while those in the juxtamedullary cortex and medulla and the ganglionic processes were characterized by abundant large clear vesicles, as well as the same vesicular elements as contained in the nerves in the superficial cortex. The immunoreactive material was localized on the granular cores and diffusely in the axoplasm in both nerves. Based on the similarity and difference in the composition of the vesicles contained in individual nerves, it is likely that the VIP-immunoreactive nerve fibers in the medulla and the juxtamedullary cortex are derived from the medullary VIP-ganglion cells, while those in the superficial cortex are of extrinsic origin. The immunoreactive nerve fibers in both the cortex and the medulla were often in direct contact with cortical cells and chromaffin cells, where no membrane specializations were formed. The immunoreactive nerve fibers were sometimes associated with the smooth muscle cells and pericytes of small blood vessels in the superficial cortex. In addition they were often seen in close apposition to the fenestrated endothelial cells in the cortex and the medulla, only a common basal lamina intervening. Several possible mechanisms by which VIP may exert its effect in the adrenal gland are discussed.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.     

Segmental distribution of colonic neuropeptides in Hirschsprung's disease.

Despite continued research, the pathophysiologic mechanism responsible for functional obstruction in the aganglionic segment of bowel in Hirschsprung's disease remains controversial. Narrowing of the affected segment is thought by many investigators to be the result of loss of intrinsic inhibitory innervation. For this hypothesis to be consistent, inhibitory neuropeptides should be present in the dilating, transitional segment of bowel. In order to quantitate reported changes in peptidergic nerve staining in Hirschsprung's disease, we measured concentrations of five neuropeptides (vasoactive intestinal peptide, peptide histidine-methionine, met5-enkephalin, substance P and bombesin-like immunoreactivity) by radioimmunoassay in the affected segments of bowel from six patients with Hirschsprung's disease. Tissue extracts were prepared using gut obtained at surgery from the: (1) constricted, aganglionic segment, (2) dilating, aganglionic transitional segment and (3) dilated, proximal ganglionic segment. Concentrations of vasoactive intestinal peptide, peptide histidine-methionine, substance P and met5-enkephalin were significantly reduced in both the muscularis externa and the mucosal-submucosal layers from the constricted aganglionic segment. By contrast, concentrations of the candidate inhibitory neuropeptides, vasoactive intestinal peptide and peptide histidine-methionine, were minimally reduced in the dilating, aganglionic transitional segment. These results are consistent with the hypothesis that constriction of the aganglionic segment is due to loss of intrinsic inhibitory innervation. Concentrations of bombesin-like immunoreactivity were similar in the three segments of human gut, suggesting the presence of this immunoreactive neuropeptide in extrinsic nerve fibers.     

Synaptic relationships between proopiomelanocortin- and ghrelin-containing neurons in the rat arcuate nucleus

Both proopiomelanocortin (POMC) and ghrelin peptides are implicated in the feeding regulation. The synaptic relationships between POMC- and ghrelin-containing neurons in the hypothalamic arcuate nucleus were studied using double-immunostaining methods at the light and electron microscope levels. Many POMC-like immunoreactive axon terminals were found to be apposed to ghrelin-like immunoreactive neurons and also to make synapses with ghrelin-like immunoreactive neuronal perikarya and dendritic processes. Most of the synapses were symmetrical in shape. A small number of synapses made by ghrelin-like immunoreactive axon terminals on POMC-like immunoreactive neurons were also identified. Both the POMC- and ghrelin-like immunoreactive neurons were found to contain large dense granular vesicles. These data suggest that the POMC-producing neurons are modulated via synaptic communication with ghrelin-containing neurons. Moreover, ghrelin-containing neurons may also have a feedback effect on POMC-containing neurons through direct synaptic contacts.     

GABA, Glutamic Acid Decarboxylase, and GABA Transaminase Levels in the Myenteric Plexus in the Intestine of Humans and Other Mammals

Abstract: Regional distribution of endogenous γ- aminobutyric acid (GABA), its synthesizing enzyme, glutamic acid decarboxylase (GAD), and metabolic enzyme, GABA transaminase (GABA-T), were determined in the intestinal tract of guinea pigs and cats and the findings compared with the number of ganglion cells in Auerbach's plexus. There were positive correlations among the GABA contents and the numbers of neural cells of the plexus. The precise localization of GABA and GAD in individual layers (mucosa, circular and longitudinal muscles, and Auerbach's plexus) in the human and cat colon was also determined. The endogenous GABA contents and GAD activity were the highest in Auerbach's plexus in tissues of both species. These results indicate that GABA is synthesized and localized in Auerbach's plexus and probably plays a significant role in the enteric nervous system.     

Chagasic megacolon: enteric neurons and related structures

Megacolon, the irreversible dilation of a colonic segment, is a structural sign associated with various gastrointestinal disorders. In its hereditary, secondary form (e.g. in Hirschsprung’s disease), dilation occurs in an originally healthy colonic segment due to an anally located, aganglionic zone. In contrast, in chronic Chagas’ disease, the dilated segment itself displays pathohistological changes, and the earliest and most prominent being found was massive loss of myenteric neurons. This neuron loss was partial and selective, i.e. some neurons containing neuronal nitric oxide synthase and/or vasoactive intestinal peptide (VIP) were spared from neuron death. This disproportionate survival of inhibitory neurons, however, did not completely correlate with the calibre change along the surgically removed, megacolonic segments. A better correlation was observed as to potentially contractile muscle tissue elements and the interstitial cells of Cajal. Therefore, the decreased densities of α-smooth muscle actin- and c-kit-immunoreactive profiles were estimated along resected megacolonic segments. Their lowest values were observed in the megacolonic zones itself, whereas less pronounced decreases were found in the non-dilated, transitional zones (oral and anal to dilation). In contrast to the myenteric plexus, the submucosal plexus displayed only a moderate neuron loss. Neurons co-immunoreactive for VIP and calretinin survived disproportionately. As a consequence, these neurons may have contributed to maintain the epithelial barrier and allowed the chagasic patients to survive for decades, despite their severe disturbance of colonic motility. Due to its neuroprotective and neuroeffectory functions, VIP may play a key role in the development and duration of chagasic megacolon.     

VIP like immunoreactive nerves in human respiratory tract. Light and electron microscopic study

The present study provides light and electron microscopical evidence of Vasoactive Intestinal Peptide - (VIP) like immunoreactive nerves in human lower respiratory tract. Peroxidase antiperoxidase (PAP) technique was used to localize VIP-like immunoreactivity light microscopically and ultrastructurally. Under light microscopy, VIP-like immunoreactive nerves were observed in the smooth muscle layer of secondary bronchi to small bronchioli, and in bronchial glands. In addition, positive immunoreactive nervous network to VIP was found around nerve cell bodies in small microganglia. The bronchial epithelium of airway tract did not receive any VIP positive nerve fibers. Ultrastructurally VIP-like positive immunoreaction was localized in large granular vesicles ranging from 90 to 210 nm. Usually VIP-like positive immunoreactive nerve profiles contained several immunoreactive large vesicles (100-210). However, nerve profiles containing only a few positive large vesicles (80-150) were also observed. Under electron microscopy VIP-positive nerve profiles corresponded ultrastructurally to nerve profiles containing large granular vesicles observed in conventional electronmicroscopy. The present study provides new information about the innervation of human lower airway tract and widens the concept of their functional regulation on the anatomical basis reported here.     

Cholecystokinin activates specific enteric neurons in the rat small intestine

Cholecystokinin (CCK) is a peptide hormone released from the I-cells of the upper small intestine. CCK evokes a variety of physiological responses, such as stimulation of pancreatic secretion, reduction of food intake and inhibition of gastric emptying. Previously, we reported that CCK activates enteric neurons in the rat. However the specific subpopulations of enteric neurons activated by CCK have not been identified. In the work reported here, we utilized immunohistochemical detection of nuclear Fos, a marker for neuronal activation, and selected phenotypic markers to identify some of the neuronal subpopulations activated by CCK. The phenotypic markers that we examined were: nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calbindin (Cal), Calretinin (Calr), and neurofilament-M (NF-M). We found that in the myenteric plexus of the rat duodenum and jejunum, CCK activated NOS immunoreactive neurons. In the submucosal plexus of duodenum and jejunum, CCK activated Cal, Calr and NF-M immunoreactive neurons. CCK failed to activate NK-1R immunoreactive neurons in either plexus. Our results indicate that CCK activates distinct enteric neurons in the rat upper small intestine. Furthermore the fact that NOS immunoreactive neurons were activated suggests that CCK modulates the activity of inhibitory motor neurons in the myenteric plexus. Expression of Fos immunoreactivity in Calr and Cal immunoreactive neurons is consistent with a role for CCK in modulation of intrinsic sensory and/or secretomotor neuronal activity in the submucosal plexus.     

Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.     

Septal afferents to the area dentata terminate on vasoactive intestinal polypeptide (VIP)-like immunoreactive,non-pyramidal neurons

Summary Thermic lesions in the medial septum of adult rats result in dark degeneration of terminal boutons in the stratum moleculare and hilus of the area dentata. While most of the degenerating terminals are in synaptic contact with non-reactive cells, part of them end on dendrites of VIP-like immunoreactive neurons.     

Morphology and synaptic connectivity of nitric oxide synthase-immunoreactive neurons in the guinea pig retina

Immunocytochemical methods with an antiserum against neuronal nitric oxide synthase (NOS) were applied to identify the morphology and synaptic connectivity of NOS-like immunoreactive neurons in the guinea pig retina. In the present study, two types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long, sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). The somata of type 2 cells (smaller diameters) were located in the INL. Some displaced amacrine cells in the ganglion cell layer were labeled. The soma size of the displaced amacrine cells was similar to that of the type 2 amacrine cells. However, processes originating from type 2 amacrine cells and displaced amacrine cells stratified mainly in strata 1 and 5, respectively. Some cone bipolar cells were weakly NOS-immunoreactive. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Cone bipolar cells were postsynaptic to NOS-labeled amacrine cells in all strata of the IPL. Labeled amacrine cells synapsing onto ganglion cells were found only in sublamina b. A few synaptic contacts were observed between labeled cell processes. In the outer plexiform layer, dendrites of labeled bipolar cells made basal contact with cone pedicles or formed a synaptic triad opposed to a synaptic ribbon of cone pedicles.