Coexistence of catecholamine and methionine enkephalin-Arg6-Gly7-Leu8 in neurons of the rat ventrolateral medulla oblongata

Summary An overlapping distribution of catecholamine-containing cells and proenkephaline—A derived peptide-containing neurons have been identified in the rat medulla oblongata. However, it is not evident whether the coexistence of these bioactive substances occurs in the same neurons or not. Therefore, we examined the coexistence of catecholamine and methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL), a proenkephaline—A derived peptide, using a combination of histofluorescence and peroxidase-anti-peroxidase (PAP) immunohistochemical (modified formaldehyde-glutalaldehyde (Faglu)) methods on the same tissue sections. We found one third of A1/C1 catecholamine fluorescent cells show MEAGL-like immunoreactivity.     

Projection of ventrolateral medullary (A1) catecholamine neurons toward nucleus tractus solitarii

Summary The distribution and interconnections of brainstem catecholamine cell groups thought to be important in cardiovascular control were studied using histochemical and ultrastructural techniques in the rabbit. Lesions and microinjections of horseradish peroxidase (HRP) were made in the nucleus tractus solitarii in the dorsomedial medulla, and in the ventrolateral medulla. After lesions of the dorsomedial medulla the fluorescence intensity of the Al-group of catecholamine neurons was increased, and swollen axons could be seen coursing from the ventrolateral medulla toward the lesions on the same side, but not the opposite side. Most of these axons ran in a band about 2 mm in width, centered at the level of the obex. Electron microscopically, specific cells, identified as A1-catecholamine neurons, showed evidence of chromatolysis after the dorsomedial lesions. Following injection of HRP into the nucleus tractus solitarii, A1-catecholamine cells in the ventrolateral medulla on the same side contained the reaction product. Lesions of the ventrolateral medulla did not produce evidence of a reciprocal projection of A2-catecholamine neurons toward the ventrolateral medulla.Thus axons of the A1-group of catecholamine neurons in the ventrolateral medulla project toward the ipsilateral nucleus tractus solitarii in a relatively compact band at the level of the obex. On the other hand, the A2-group of catecholamine neurons in the dorsomedial medulla does not appear to send projections toward the A1-group.These studies were supported by grants from the National Heart Foundation of Australia and The Life Insurance Medical Research Fund of Australia and New Zealand, and Merck Sharp and Dohme (Australia) Pty Limited     

Evidence that enteric neurons may derive from the sympathoadrenal lineage.

The first neurons that differentiate in the embryonic foregut of mammals transiently express catecholamine biosynthetic enzymes and accumulate catecholamine. Since this transmitter is found predominantly in cells of the sympatho-adrenal (SA) lineage, it has been suggested that enteric and sympathetic neurons may derive from the same progenitor. Enteric neurons would then lose the catecholamine phenotype during further development, as the two lineages diverge. We have further investigated this possibility using the SA1 monoclonal antibody that binds selectively to SA progenitor cells in the embryonic rat. We find that SA1 binds to the tyrosine hydroxylase+, neurofilament+, and SCG10+ cells of the Embryonic Day 14.5 (E14.5) rat foregut. We also find that a marker for later neuronal differentiation in the SA lineage, B2, also appears in the myenteric plexus concomitant with the loss of SA1 staining. Thus, at least some enteric neuronal precursors may exhibit the SA1----B2 antigenic switch previously observed in developing sympathetic neurons at E14.5. SA1 staining in the foregut partially overlaps with staining for neuropeptide Y, vasoactive intestinal polypeptide, and serotonin. These results support the hypothesis that enteric and sympathetic neurons derive from a common progenitor and that as the markers for the SA lineage are down-regulated, the many types of enteric neurons begin to differentiate.     

Hypotensive hypovolemia and hypoglycemia activate different hindbrain catecholamine neurons with projections to the hypothalamus

To better understand the involvement of hindbrain catecholamine neurons in hypovolemia-induced secretion of AVP, we injected antidopamine beta-hydroxylase saporin (DSAP) or unconjugated saporin (SAP) control solution into the hypothalamic paraventricular nucleus (PVH) of anesthetized rats to retrogradely lesion catecholamine neurons innervating magnocellular areas of the hypothalamus. Subsequently, hypotensive hypovolemia was induced by remote blood withdrawal (4.5 ml, 1 ml/min) using an intra-atrial catheter. Blood was sampled at 2, 5, 20, and 50 min after onset of blood withdrawal. The AVP response was severely impaired by DSAP. Peak responses at 50 min were 51 pg/ml in SAP control and 17 pg/ml in DSAP-lesioned rats, indicating the importance of catecholamine neurons for this response. We also measured AVP responses to osmotic challenge induced by administration of hypertonic saline (1 M, 15 ml/kg, sc) and to insulin-induced hypoglycemia. Osmotic challenge increased AVP levels, but the response was not impaired by DSAP, indicating that AVP neurons were not damaged by the DSAP injection. Insulin-induced hypoglycemia did not increase AVP levels in either DSAP- or SAP-treated rats. However, the same dose of insulin increased food intake and corticosterone secretion in SAP controls, and these responses were profoundly impaired by DSAP. Thus catecholamine neurons are required for both the AVP response to hypotensive hypovolemia and for feeding and corticosterone responses to hypoglycemia. Lack of an AVP response to insulin-induced hypoglycemia in intact rats therefore indicates that responses to hypovolemia and hypoglycemia are mediated by different catecholamine neurons under distinct sensory controls.     

Nitric oxide synthase in guinea pig sympathetic ganglia: Correlation with tyrosine hydroxylase and neuropeptides

Nitric oxide synthase (NOS) has previously been reported in a small population of postganglionic sympathetic neurons in the guinea pig. The present study of paravertebral ganglia and the inferior mesenteric ganglion aimed to classify these neurons according to their content of neuropeptides (calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide) and the rate-limiting enzyme of catecholamine synthesis, tyrosine hydroxylase, by means of immunohistochemical and histochemical double-labelling techniques. NOS-containing neurons belonged to the non-catecholaminergic population of postganglionic neurons, and partial coexistence was found with neuropeptide Y and vasoactive intestinal peptide immunoreactivities but not with calcitonin gene-related peptide. However, most of the NOS-containing neurons contained none of the neuropeptides, thus representing a hitherto unrecognized population of postganglionic neurons. The findings show that NOS is localized to small but neurochemically highly specific populations of postganglionic neurons, which most likely reflects an association with target- and function-specific pathways.     

Selective Destruction of Cultured Dopaminergic Neurons from Fetal Rat Mesencephalon by 1-Methyl-4-Phenylpyridinium: Cytochemical and Morphological Evidence

Dopaminergic neurons in cultures of dissociated cells from fetal rat mesencephalon were exposed to the principal metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenyl-pyridinium ion (MPP+), and several of its structural analogues. At concentrations between 0.01 and 0.1 microM, MPP+ inhibited catecholamine accumulation as visualized by cytofluorescence. Between 0.1 and 10.0 microM, MPP+ resulted in disappearance of tyrosine hydroxylase immunoreactivity without affecting other cells in the cultures. At concentrations higher than 10 microM, MPP+ was toxic to all cells present in the cultures. The effect of low concentrations of MPP+ on catecholamine cytofluorescence of the dopaminergic neurons was partially reversible. The intermediate concentrations produced irreversible structural changes of tyrosine hydroxylase-positive cells, resulting in complete disappearance of these neurons. The morphological changes were specific to the dopaminergic neurons and were not evident in other cells viewed with phase contrast microscopy. Of the structural analogues tested, the 1-ethyl analogue of MPP+ was effective in selectively destroying dopaminergic neurons in our culture system. The antioxidants L-acetyl-carnitine, beta-carotene, and alpha-tocopherol failed to protect against MPP+ neurotoxicity when co-incubated with the toxin.     

Granule matrix property and rapid "kiss-and-run" exocytosis contribute to the different kinetics of catecholamine release from carotid glomus and adrenal chromaffin cells at matched quantal size

Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ~100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ~200-250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca(2+) ([Ca(2+)](i)) to ~0.5 μmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca(2+)](i) triggers a form of rapid "kiss-and-run" exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Comparative anatomy of brain monoaminergic neurons in New World and Old World monkeys

A comparison of the distribution of brain monoamine neurons in several New World and Old World monkeys was undertaken using the Falck-Hillarp formaldehyde histofluorescence technique. The overall organization of the monoamine neurons was very similar in all species, although subtle variations were found. Catecholamine (noradrenaline and dopamine) and indoleamine (serotonin) cell bodies corresponding to groups A1–A7, A8–A10, and B1–B9, respectively were found throughout the brainstem. A few catecholamine (dopamine) cells equivalent to groups All and A12 in the diencephalon were also observed. Noradrenaline neurons, rather than those of the dopamine and serotonin systems, tended to be less numerous in the New World monkeys. Ascending catecholamine and indoleamine fiber bundles were observed in most monkeys. It is interesting that fibers corresponding to the “ventral noradrenaline bundle” appeared to be much finer in the common marmoset and tamarin than in other species. In addition, a substantial catecholamine (noradrenaline) innervation of the diencephalon was noted in all the Old World monkeys, while a much lower overall terminal density was apparent in the New World forms.     

Immunocytochemical colocalization of histamine, histidine decarboxylase, 5-hydroxytryptamine and tyrosine hydroxylase in the superior cervical ganglion of the rat

Summary The coexistence of histamine, histidine decarboxylase (the enzyme synthesizing histamine), 5-hydroxytryptamine and tyrosine hydroxylase (the rate-limiting enzyme in catecholamine synthesis), was studied in the rat superior cervical ganglion with the indirect immunofluorescence method. Possible colocalization was examined by staining consecutive sections with two different antibodies, or alternatively in the same section by eluting the first antibody with a mild solution containing potassium permanganate and sulphuric acid, and by staining the same section with another antibody. It was shown that tyrosine hydroxylase immunoreactivity was found both in large principal nerve cells and in small cells, which on the basis of their size and high nucleus—cytoplasm ratio corresponded to small intensely fluorescent (SIF) cells. Histamine, histidine decarboxylase and 5-hydroxytryptamine immunoreactivities were observed only in SIF cells. Those SIF cells which were immunoreactive for histamine, histidine decarboxylase or 5-hydroxytryptamine also contained tyrosine hydroxylase immunoreactivity. On the other hand, all tyrosine hydroxylase-immunoreactive SIF cells were also immunoreactive for histidine decarboxylase or 5-hydroxytryptamine. Some of the SIF cells, which were non-reactive for histamine, were immunoreactive for tyrosine hydroxylase.     

Aminergic and Indoleamine Accumulating Neurons in the Retina of the River Lamprey (Lampetra fluviatilis)

Tissues were processed for fluorescence microscopy of biogenic amines according to the method of Falck and Hillarp. Normal animals, and animals injected with α-methylnoradrenaline or 5,6-dihydroxytryptamine were used. Catecholamine containing neurons (junctional cells) occur in the innermost rows of cell bodies of the inner nuclear layer (INL) and close to the vitreous surface. Catecholamine containing fibers occur in three layers: (1) an outer layer around the innermost perikarya of the INL, which is a condition not found in retinas of gnathostome chordates; (2) a middle layer within the outer third of the inner synaptic layer (ISL), separated from the outer layer by ganglion cell axons; (3) a sparse inner layer within the innermost third of the ISL. A few catecholamine containing fibers were seen to extend from the innermost region of the INL to the outer synaptic layer. The position of the junctional cells in the lamprey corresponds to that in gnathostome chordates, but whereas all catecholamine containing fiber layers in gnathostomes are located sclerally to the optic fiber layer and within the ISL, the middle and the inner fiber layers in the lamprey occur vitreally to the optic fiber layer. Indoleamine accumulating neurons occur in the innermost row of perikarya of the INL and close to the vitreous surface. Those of the INL send fine, varicose branches to the ISL forming a network which is somewhat denser at the inner and outer borders of the ISL than in its middle. The indoleamine accumulating terminals do not ramify within the INL in contrast to the catecholamine containing terminals.     

Characterization of the vesicular monoamine transporter in cultured rat sympathetic neurons: persistence upon induction of cholinergic phenotypic traits

The binding of [3H]dihydrotetrabenazine ([3H]TBZOH), a specific ligand of the reserpine-sensitive monoamine transporter in brain and adrenal medulla storage vesicles, has been measured in cultured sympathetic neurons from newborn rat in relation to their neurotransmitter phenotype. As shown previously, neurons cultured in the absence of muscle-conditioned medium displayed high activities in catecholamine synthesizing enzymes and low levels of choline acetyltransferase, and neurons cultured in conditioned medium displayed the reverse pattern (J. P. Swerts, A. Le Van Thai, A. Vigny, and M. J. Weber, Dev. Biol. 100, 1-11, 1983). The density of [3H]TBZOH binding sites as well as their subcellular distribution were identical in both types of cultures. Two other structures rich in choline acetyltransferase, the electric organ of Torpedo and the ciliary ganglion of the chick embryo did not contain measurable amounts of [3H]TBZOH binding sites, suggesting that the monoamine transporter is not an ubiquitous component of cholinergic synaptic vesicles. These data suggest that the synthesis of the monoamine transporter in sympathetic neurons is not coregulated with the syntheses of the three norpinephrine synthesizing enzymes. It is proposed that the same population of synaptic vesicles can accumulate acetylcholine or catecholamine, depending only upon which neurotransmitter synthesizing enzymes are expressed by sympathetic neurons.     

Transmitters and modulators in the superior cervical ganglion of the rat

Several transmitters and modulators have been found to exist in the superior cervical ganglion of the rat. It has been shown that noradrenaline is present in the principal neurons and dopamine is the main catecholamine in the small intensely fluorescent cells. 5-hydroxytryptamine and histamine have been investigated immunohistochemically and found to be present only in the small intensely fluorescent cells of an adult rat, in the same cells which are also immunoreactive to tyrosine hydroxylase. On the other hand, enkephalins which were studied using highly specific antibodies against methionine-enkephalin-arginine-phenylalanine and methionine-enkephalin-arginine-glycine-leucine were found in the principal neurons and nerve fibres. Ligation studies showed that enkephalins in the superior cervical ganglion of the rat are both of intrinsic and extrinsic origin. It is evident that the transmission in the sympathetic ganglion is complex. The possible function of the transmitter and modulator candidates is discussed.     

Demonstration of enkephalin-, substance p- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

Summary The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of norphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Demonstration of enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of morphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Effect of chronic and acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on the function and structure of cultured chromaffin cells

Cultured bovine adrenal chromaffin cells were treated chronically with various concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the culture medium for 2–8 days or acutely for 10–15 min. Culture of cells with MPTP for periods of 2–8 days resulted in a marked loss of total cellular catecholamines and a parallel reduction in secretory response, but not the ratio of stimulated to unstimulated secretion. By the eighth day in culture, at the highest MPTP concentration (1000 μM), cell catecholamine content and secretion were only about 10% that of untreated cells. The proportion of total cellular catecholamines secreted was not altered by MPTP, suggesting that the secretory process was unaffected by the drug. The loss of secretory output was not prevented by inhibitors of monoamine oxidase or catecholamine uptake, drugs known to prevent MPTP-induced damage to central dopaminergic neurons. The subcellular organelles of MPTP-treated cells appeared relatively normal except for extensive depletion of the vesicle contents, in agreement with the biochemical data. The severity of the depletion appeared to be lessened in cells treated with monoamine oxidase inhibitors.Short term exposure to MPTP at concentrations less than 100 μM had little effect on secretion induced by carbachol. Higher concentrations of MPTP increased unstimulated release and reduced stimulated release. Pretreatment of the cells with MPTP resulted in a lasting reduction in their subsequent secretory responsiveness. MPTP alone, at concentrations greater than 100 μM induced catecholamine release that was unaffected by pretreatment of the cells with monoamine oxidase inhibitors or the catecholamine uptake inhibitor desipramine. MPTP-induced secretion by intact cells was calcium-dependent, while the small increase by permeabilized cells was not.     

Characterization of neurotransmitter phenotype during neuronal differentiation of embryonal carcinoma cells

Embryonal carcinoma cells are useful in the study of embryogenesis and development, and their differentiation into neurons serves as a model of neuronal development. Retinoic acid was used to differentiate P19S18O1A1 embryonal carcinoma cells into neuronal, glial, and fibroblast-like cells and the phenotype of the neuronal population was examined. Neuron-specific enolase was present in the neuronal cells, suggesting that these neurons had reached some degree of maturity. A population (approximately 70%) of the neurons showed positive immunocytochemistry for tyrosine hydroxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, three enzymes in the pathway of catecholamine synthesis. Therefore a population of the neurons appeared to be adrenergic. These neurons also showed a low level of histofluorescence for endogenous catecholamines and exhibited an exogenous catecholamine reuptake system. In order to determine the phenotype of other neuron-like cells found to be negative for the adrenergic properties examined, immunocytochemistry for neuropeptides and neurotransmitters known to coexist within central neurons was performed. Serotonin, vasoactive intestinal peptide, glutamic acid decarboxylase, and choline acetyltransferase were all absent from retinoic acid-treated P19S18O1A1 neuronal cultures. These studies, along with those that compare the effects of retinoic acid and other growth modulators on neuronal differentiation of embryonal carcinoma cells, should aid in the understanding of neuronal induction and development in vivo.     

Development of cells containing catecholamines and somatostatin-like immunoreactivity in neural crest cultures: relationship of DNA synthesis to phenotypic expression

The goal of our work is to understand the mechanisms which regulate the differentiation of embryonic neural crest cells into a number of adult cell types, including several classes of neurons. As one aspect of this analysis, the relationship between DNA synthesis and the ontogeny of cells with catecholamines and somatostatin-like immunoreactivity (SLI) in neural crest cell cultures has been investigated. Most of the precursors of the catecholamine- and SLI-positive cells carry out DNA synthesis. As these cells differentiate, their ability to carry out DNA synthesis declines. However, a small percentage of cells continue to synthesize DNA after they become catecholamine or SLI positive. There is no apparent difference between the temporal pattern of DNA synthesis in the precursors of catecholamine-positive cells with SLI and those without SLI. Thus, the time of withdrawal from the cell cycle does not distinguish the lineage of cells that are catecholamine and SLI positive from those that are catecholamine positive and SLI negative.     

Catecholamines and catecholamine-synthesizing enzymes in guinea-pig sensory ganglia

Summary Cranial and spinal sensory ganglia of the guinea-pig were investigated by means of histochemistry and biochemistry for the presence of catecholamines and catecholamine-synthesizing enzymes. Sensory neurons exhibiting immunoreactivity to the rate-limiting enzyme of catecholamine synthesis, tyrosine nydroxylase (TH), were detected by immunohistochemistry in lumbo-sacral dorsal root ganglia, the nodose ganglion and the petrosal/jugular ganglion complex. The carotid body was identified as a target of TH-like-immunoreactive (TH-LI) neurons by the use of combined retrograde tracing and immunohistochemistry. Double-labelling immunofluorescence revealed that most TH-LI neurons also contained somatostatin-LI, but TH-LI did not coexist with either calcitonin gene-related peptide- or substance P-LI. TH-LI neurons did not react with antibodies to other enzymes involved in catecholamine synthesis, i.e., aromatic amino acid decarboxylase (AADC), dopamine--hydroxylase (DH), and phenylethanolamine-N-methyltransferase (PNMT). Petrosal neurons as well as their endings in the carotid body lacked dopamine- and L-DOPA-LI. Sensory neurons did not display glyoxylic acid-induced catecholamine fluorescence. Ganglia containing TH-LI neurons were kept in short-term organ culture after crushing their roots and the exiting nerve in order to enrich intra-axonal transmitter content at the ganglionic side of the crush. However, even under these conditions, catecholamine fluorescence was not detected in axons projecting peripherally or centrally from the ganglia. Sympathetic noradrenergic nerves entered the ganglia and terminated within them. Accordingly, biochemical analyses of guinea-pig sensory ganglia revealed noradrenaline but no dopamine. In conclusion, catecholamines within guinea-pig sensory ganglia are confined to sympathetic nerves, which fulfill presently unknown functions. The TH-LI neurons themselves, however, lack any additional sign of catecholamine synthesis, and the presence of enzymatically active TH within these neurons is questionable.     

多种神经活性物质在幼年猫和鸡视网膜神经...

An improved post-embedding immunocytochemical technique was used to examine the coexistence of multi-neuroactive substances in neurons of young cat and newly hatched chicken retinas. We found that two classical neurotransmitters-glycine (Gly) and r-aminobutyric acid (GABA) and one neuropeptide, neurotensin (NT) were located in the amacrine cells of cat and chicken retinas. Among them, some cells contain Gly and GABA (Gly/GABA AC), some cells contain Gly and NT (Gly/NT AC) or GABA and NT (GABA/NT AC), and the other cells, a few of them contain Gly, GABA and NT (Gly/GABA/NT AC). In addition, two classical neurotransmitters Gly and GABA were also located or coexisted in the horizontal cells of cat retina. These results indicated that the coexistence of two neurotransmitters or one neurotransmitter and one neuropeptide or two neurotransmitters and one neuropeptide was presented in neurons of developing can and chicken retinas.