RESUMEN
AIMS: While vascular pathology is a common feature of a range of neurodegenerative diseases, we hypothesized that vascular changes occur in association with normal ageing. Therefore, we aimed to characterize age-associated changes in the blood-brain barrier (BBB) in human and mouse cohorts. METHODS: Immunohistochemistry and Evans blue assays were used to characterize BBB dysfunction (tight junction protein expression and serum plasma protein accumulation), vascular pathology (pericyte loss and vascular density) and glial pathology (astrocyte and microglial density) in ageing neurological control human prefrontal cortex (a total of 23 cases from 5 age groups representing the spectrum of young adult to old age: 20-30 years, 31-45 years, 46-60 years, 61-75 years and 75+) and C57BL/6 mice (3 months, 12 months, 18 months and 24 months, n = 5/6 per group). RESULTS: Quantification of the tight junction protein ZO-1 within the cortex and cerebellum of the mouse cohort showed a significant trend to both increased number (cortex P < 0.001, cerebellum P < 0.001) and length (cortex P < 0.001, cerebellum P < 0.001) of junctional breaks associated with increasing age. GFAP expression significantly correlated with ageing in the mice (P = 0.037). In the human cohort, assessment of human protein accumulation (albumin, fibrinogen and human IgG) demonstrated cells morphologically resembling clasmatodendritic astrocytes, indicative of BBB dysfunction. Semiquantitative assessment of astrogliosis in the cortex expression revealed an association with age (P = 0.003), while no age-associated changes in microglial pathology, microvascular density or pericyte coverage were detected. CONCLUSIONS: This study demonstrates BBB dysfunction in normal brain ageing, both in human and mouse cohorts.
Asunto(s)
Envejecimiento/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Uniones Estrechas/metabolismo , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , Astrocitos/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Pericitos/metabolismo , Adulto Joven , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND: Increasing age is associated with an increase in the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to these conditions is not fully understood. This study examined the effects of increasing age on the function of the murine anorectum. METHODS: The effects of increasing age on cholinergic, nitrergic, and purinergic signaling pathways in the murine anorectum were examined using classical organ bath assays to examine tissue function and electrochemical sensing to determine age-related changes in nitric oxide and acetylcholine release. KEY RESULTS: Nitrergic relaxation increased between 3 and 6 months, peaked at 12 months and declined in the 18 and 24 months groups. These changes were in part explained by an age-related decrease in nitric oxide (NO) release. Cholinergic signaling was maintained with age by an increase in acetylcholine (ACh) release and a compensatory decrease in cholinesterase activity. Age-related changes in purinergic relaxation were qualitatively similar to nitrergic relaxation although the relaxations were much smaller. Increasing age did not alter the response of the anorectum smooth muscle to exogenously applied ACh, ATP, sodium nitroprusside or KCl. Similarly, there was no change in basal tension developed by the anorectum. CONCLUSIONS AND INFERENCES: The decrease in nitrergic signaling with increasing age may contribute to the age-related fecal impaction and constipation previously described in this model by partially obstructing defecation.
Asunto(s)
Envejecimiento/metabolismo , Canal Anal/metabolismo , Músculo Liso/metabolismo , Recto/metabolismo , Transducción de Señal/fisiología , Acetilcolina/análisis , Acetilcolina/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Óxido Nítrico/análisis , Óxido Nítrico/metabolismoRESUMEN
We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.
Asunto(s)
Envejecimiento/fisiología , Restricción Calórica , Plexo Mientérico/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Calbindina 2 , Calbindinas , Muerte Celular/efectos de los fármacos , Masculino , Plexo Mientérico/citología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/farmacología , Proteína G de Unión al Calcio S100/metabolismoRESUMEN
To gain insight into the mechanisms regulating expression of transmitter phenotypes in the enteric nervous system, we have studied the development and birthdate of vasoactive intestinal peptide immunoreactive (VIP-IR) myenteric neurons in the chicken proventriculus (secretory portion of the avian stomach) by a combination of immunocytochemistry and radioautography. The appearance and numbers of VIP-IR neurons in whole mounts of the myenteric plexus from chick embryos and chickens were examined. We found that VIP-IR neurons first appeared at embryonic day (E) 5.5-6.5 in the distal part of the proventriculus. At E7.5, VIP-IR neurons were found singly, in pairs, or in small groups, which together with unlabeled cells formed primitive myenteric ganglia. VIP-IR fibers were found within the developing fiber tracts which connected the ganglia. The number of VIP-IR neurons was found to be maximum in the E15.5 embryo and to decline to 68% of maximum in the 4 week old chicken. Birthdate studies were performed by application of either single pulses or cumulative doses of [3H]-thymidine to embryos between E3 and E14. Whole mounts of the myenteric plexus from the proventriculus of these embryos were immunostained for VIP at E10 or E17. The whole mounts were subsequently sectioned and processed for radioautography. We found that VIP-IR myenteric neurons were born between E3 and E10 with a peak at E7. Most cells underwent terminal division between E5 and E9. These data will be useful in determining the time and conditions when cells make decisions about transmitter phenotypes.
Asunto(s)
Embrión de Pollo/fisiología , Pollos/crecimiento & desarrollo , Neuronas/fisiología , Proventrículo/inervación , Péptido Intestinal Vasoactivo/análisis , Envejecimiento , Animales , Embrión de Pollo/citología , Desarrollo Embrionario y Fetal , Neuronas/citología , Proventrículo/embriología , Proventrículo/crecimiento & desarrolloRESUMEN
The ontogeny and distribution of nerve cell bodies and fibres which contain vasoactive intestinal polypeptide-, substance P-, enkephalin- and neurotensin-like immunoreactivity have been studied in the chicken gastrointestinal tract, using immunocytochemistry. All four peptides were found in nerve fibres, with characteristic distribution patterns, which, in the cases of vasoactive intestinal polypeptide, substance P and methionine enkephalin were similar to those described for the mammalian gut. In addition, many of these fibres were shown to arise from intrinsic neurons, since immunoreactive nerve cell bodies for each of the peptides studied were observed. Neurotensin-immunoreactive nerves were confined to the upper part of the tract and neurotensin immunoreactive cell bodies were only observed in embryonic and newly hatched chicken gut. All four peptides were first observed at 11 days of incubation, or Hamburger-Hamilton stage 37, 20 in the upper part of the tract, particularly in the gizzard. Substance P and methionine enkephalin were subsequently seen in more caudal regions, while vasoactive intestinal polypeptide developed from each end of the tract. Adult patterns of immunoreactivity in nerve fibres were achieved during the first week after hatching. A striking observation was that immunoreactive neuronal cell bodies were much more abundant in the gut of young chickens and chicken embryos than in that of adult birds.
Asunto(s)
Pollos/crecimiento & desarrollo , Sistema Digestivo/inervación , Endorfinas/metabolismo , Encefalinas/metabolismo , Hormonas Gastrointestinales/metabolismo , Neurotensina/metabolismo , Sustancia P/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Embrión de Pollo , Sistema Digestivo/embriología , Sistema Digestivo/crecimiento & desarrollo , Encefalina Metionina , Nervios Periféricos/crecimiento & desarrollo , Nervios Periféricos/metabolismoRESUMEN
We have studied the possible purinoceptor-mediated modulation of astroglial cell proliferation in neuron-glia primary cultures obtained from rat corpus striatum. Cultures were grown for three days in the presence of either 2-chloro-adenosine or alpha beta-methylene-ATP (which behave as agonists of adenosine/P1 and ATP/P2 purinoceptors, respectively), and then immunostained with an antibody to glial fibrillary acidic protein. 2-Chloro-adenosine decreased and alpha beta-methylene-ATP increased the number of astroglial cells in culture. For both derivatives, the effect was dose-dependent. The effect of alpha beta-methylene-ATP was antagonized by the trypanoside suramin, suggesting the involvement of a suramin-sensitive P2 purinoceptor, whereas the effect of 2-chloro-adenosine was not reversed by the P1 purinoceptor antagonist p-sulphonyl-phenyl-theophylline, implying the activation of a xanthine-insensitive adenosine purinoceptor subtype. In order to evaluate the extent of astrocyte proliferation in the presence of these two analogues, some cultures were incubated with bromodeoxyuridine for 24 h before fixing, and then double-immunostained for glial fibrillary acidic protein and bromodeoxyuridine. The percentage of bromodeoxyuridine positive astrocytes was significantly increased after exposure to both agents. It is therefore concluded that purines can modulate astroglial cells in opposite ways, inducing decreases or increases of cell number by activation of P1 and P2 purinoceptors, respectively. For the P2 purinoceptor-mediated effect, there was a quantitative correlation between the percentage of bromodeoxyuridine positive astrocytes and the cell number. For the P1 purinoceptor-mediated effect, no apparent correlation between these two parameters was found. This suggests the activation of independent effects, which involve other mechanisms besides the stimulation of DNA synthesis, and which eventually result in a reduction of cell number. The possible relevance of these findings to in vivo regulation of astrocyte cell function as well as in trauma- and ischaemia-associated hypergliosis is discussed.
Asunto(s)
Adenosina Trifosfato/farmacología , Adenosina/análogos & derivados , Astrocitos/citología , Cuerpo Estriado/efectos de los fármacos , Animales , Astrocitos/metabolismo , Bromodesoxiuridina/metabolismo , Recuento de Células/efectos de los fármacos , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Cuerpo Estriado/citología , Purinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
We reported previously that myenteric plexus explants promoted striatal neurite elongation in co-culture and that this effect was abolished by tetrodotoxin (TTX). Here we demonstrate that the nitric oxide synthase blocker N-nitro-L-arginine methyl ester significantly reduced the neuritogenic effect of the myenteric plexus whereas the nitric oxide donor, sodium nitroprusside (SNP), partially reversed the blocking effect of TTX. 2-Chloroadenosine (2-CA), a stable analogue of adenosine, which is produced following release of ATP from enteric neurones, further enhanced the effect of SNP. Basic fibroblast growth factor or neurotrophin-3 in combination with 2-CA and SNP were only marginally neuritogenic in striatal cultures alone. These results suggest that NO is involved in the trophic effects of myenteric plexus explants on striatal neurones.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Cuerpo Estriado/citología , Plexo Mientérico/citología , Neuritas/efectos de los fármacos , Óxido Nítrico/metabolismo , 2-Cloroadenosina/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Células Cultivadas , Cuerpo Estriado/metabolismo , Relación Dosis-Respuesta a Droga , Plexo Mientérico/metabolismo , NG-Nitroarginina Metil Éster , Neuritas/fisiología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa , Ratas , Ratas Sprague-Dawley , Tetrodotoxina/farmacologíaRESUMEN
The effects of the stable adenosine analogue, 2-chloroadenosine (2-CA) and basic fibroblast growth factor (bFGF) on myenteric neurones in dissociated cell culture were examined. 2-CA had no effect on neuronal numbers, but increased neurite length, in a dose-dependent manner. bFGF increased both the number of myenteric neurones and neurite length. When 2-CA was applied together with bFGF, an enhanced increase in neurite outgrowth, but no additional increase in neuronal numbers was observed. 2-CA-induced effects were blocked by the adenosine antagonist 8-(p-sulphophenyl)theophylline. These results show, for the first time, that both purines and bFGF may have trophic actions on myenteric neurones and also indicate that purines enhance some effects of bFGF, in a synergistic manner.
Asunto(s)
2-Cloroadenosina/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Plexo Mientérico/citología , Neuronas/efectos de los fármacos , 2-Cloroadenosina/antagonistas & inhibidores , Animales , Células Cultivadas , Sinergismo Farmacológico , Técnica del Anticuerpo Fluorescente , Ganglios/citología , Ganglios/metabolismo , Inmunohistoquímica , Plexo Mientérico/efectos de los fármacos , Neuritas/fisiología , Neuritas/ultraestructura , Neuronas/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
Pharmacological evidence suggests that nitric oxide (NO) is involved in non-adrenergic, non-cholinergic, nerve mediated responses seen in guinea-pig trachealis muscle. The synthetic enzyme for NO (NO synthase) has recently been shown to be responsible for neuronal NADPH-diaphorase activity. Therefore, to determine whether intrinsic paratracheal neurones could be a source of NO in the trachea, expression of NADPH-diaphorase activity was examined histochemically using whole mount preparations of the tracheal plexus. Many paratracheal neurones were found to express moderate to high levels of NADPH-diaphorase activity and are thus likely to be a source of NO in this tissue. This observation provides further evidence that NO is involved in the regulation of relaxation in airway smooth muscle.
Asunto(s)
NADPH Deshidrogenasa/biosíntesis , Neuronas/enzimología , Tráquea/inervación , Animales , Animales Recién Nacidos , Ganglios Parasimpáticos/anatomía & histología , Ganglios Parasimpáticos/enzimología , Cobayas , Histocitoquímica , Técnicas In Vitro , Terminaciones Nerviosas/enzimologíaRESUMEN
In previous studies, we have demonstrated that enteric ganglia can survive when transplanted into the striatum. However, if such grafts are to be effective in clinically significant situations, it is necessary for them to survive in the brain for long periods. In this study, we have examined the corpus striatum of host rats one year after transplantation of pieces of myenteric plexus taken from young Fischer rats. NADPH-diaphorase-containing enteric neurones had survived within the CNS environment one year after grafting, and had extended axons into the surrounding striatum.
Asunto(s)
Cuerpo Estriado/enzimología , Plexo Mientérico/enzimología , NADPH Deshidrogenasa/análisis , Neuronas/enzimología , Animales , Supervivencia Celular/fisiología , Cuerpo Estriado/citología , Histocitoquímica , Masculino , Plexo Mientérico/citología , Ratas , Ratas Endogámicas F344RESUMEN
Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.
Asunto(s)
Aminoácido Oxidorreductasas/análisis , Colon/inervación , Plexo Mientérico/enzimología , NADPH Deshidrogenasa/análisis , Neuronas/enzimología , Animales , Animales Recién Nacidos , Cobayas , Inmunohistoquímica , Plexo Mientérico/citología , Neuronas/citología , Óxido Nítrico Sintasa , Técnicas de Cultivo de ÓrganosRESUMEN
Dissociated striatal neurons exhibited increased neurite outgrowth when co-cultured with myenteric plexus explants. Enriched enteric neurons or enriched enteric glia produced a less marked response; non-ganglionic cells had no effect. Increases in striatal neuron and glial cell numbers were seen in all co-cultures. Tetrodotoxin abolished the neuritogenic response of myenteric plexus explants but did not affect increases in cell numbers. These observations suggest that spontaneous neuronal activity within the myenteric plexus is involved in the release of a neuritogenic factor(s), possibly from glial cells, and that this is distinct from the factor(s) affecting striatal cell numbers.
Asunto(s)
Plexo Mientérico/fisiología , Neostriado/fisiología , Neuritas/fisiología , Neuronas/fisiología , Animales , Células Cultivadas , Fibroblastos , Cobayas , Plexo Mientérico/ultraestructura , Neostriado/citología , Neostriado/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuroglía/fisiología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tetrodotoxina/farmacologíaRESUMEN
This paper describes methods for removing the ganglionated myenteric and submucous plexuses from the mammalian gut and maintaining them as explants in tissue culture. A detailed account is given of cell types, their interactions and the development of these cultures during 5 weeks in vitro. Three major cell types were identified in the cultures: neurons, glial cells and fibroblasts. The development of the plexuses in culture was studied in detail for the myenteric plexus from the guinea pig taenia coli. It followed a characteristic pattern, in which the merging of individual ganglia into a continuous monolayer of flattened neurons was accompanied and followed by the formation of an extensive outgrowth zone of flat glial cells covered by a dense mesh of outgrowing neurites. In older cultures, neuronal migration resulted in the reformation of discrete and compact aggregates, which consisted of neurons and glial cells, and were interconnected by thick neurite bundles. This arrangement resembles in many ways the original organization of enteric nervous tissue in vivo. This is the first time the enteric ganglia have been freed from the gut wall and grown in culture as explants of nervous tissue. These preparations open many new directions for investigations of the largest and most complex division of the peripheral nervous system, including studies of the molecular nature of neuronal and glial cell surfaces, analysis of cell-cell interactions, trophic factors and developmental signals.
Asunto(s)
Plexo Mientérico/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Plexo Submucoso/fisiología , Animales , Animales Recién Nacidos , Femenino , Fibroblastos/fisiología , Cobayas , Masculino , Plexo Mientérico/citología , Neuronas/citología , Técnicas de Cultivo de Órganos , Conejos , Ratas , Ratas Endogámicas , Especificidad de la Especie , Plexo Submucoso/citologíaRESUMEN
5-Hydroxytryptamine (5-HT)-like immunoreactive neurones were studied during the development of myenteric plexus explant cultures from the guinea-pig taenia coli over a period of 3 weeks in vitro, using immunofluorescence histochemistry. Brightly fluorescent 5-HT-like immunoreactive neuronal cell bodies and fibres were found in all ages of cultures examined. In mature cultures, where the histotypic organization resembles that of the plexus in vivo, the pattern of immunoreactivity was strikingly similar to that previously described for in situ preparations. These culture preparations may therefore be useful models for the study of the development of putative serotonergic neurones. High-performance liquid chromatography was used to measure 5-HT levels in fetal calf serum, a supplement of the culture medium used in this study. 5-HT levels of 0.48 X 10(-5) M to 1.74 X 10(-4) M were found in 4 batches of this serum, indicating that some of the immunoreactive neurones observed in the cultures may have selectively taken up 5-HT during development in vitro.
Asunto(s)
Ciego/inervación , Diferenciación Celular , Plexo Mientérico/citología , Serotonina/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Técnicas de Cultivo , Técnica del Anticuerpo Fluorescente , Cobayas , Neuronas/citologíaRESUMEN
Serotonin (5-HT) and substance P (SP) were visualized by indirect immunofluorescence using two fluorochromes applied sequentially on the same preparation of myenteric plexus from the guinea pig gut. Three groups of labelled neurones were found: (i) neurones with 5-HT-like immunoreactivity only; (ii) neurones with SP-like immunoreactivity only; (iii) neurones with both 5-HT- and SP-like immunoreactivity, which represented approximately one-third of the 5-HT-like immunoreactive neurones.
Asunto(s)
Colon/inervación , Plexo Mientérico/metabolismo , Serotonina/metabolismo , Sustancia P/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Cobayas , Neuronas/metabolismoRESUMEN
A combined autoradiographic and immunocytochemical procedure has been used to identify neurochemically the subpopulation of cultured myenteric neurones which expresses muscarinic receptors. Antibodies to substance P (SP), [Met]enkephalin (ENK), vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) were used to immunostain cultures that had previously been labelled with the irreversible muscarinic antagonist, [3H]propylbenzilylcholine mustard. Most neurites which displayed SP-like, ENK-like or SOM-like immunoreactivity did not possess muscarinic receptors. In contrast, many VIP-like immunopositive fibres also possessed muscarinic receptors. The identity of the majority of neurones which express muscarinic receptors, that do not contain VIP, remains to be determined.
Asunto(s)
Plexo Mientérico/metabolismo , Neuronas/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Animales Recién Nacidos , Encefalina Metionina/análisis , Técnica del Anticuerpo Fluorescente , Cobayas , Técnicas de Cultivo de Órganos , Somatostatina/análisis , Sustancia P/análisis , Péptido Intestinal Vasoactivo/análisisRESUMEN
Tissue culture preparations of the myenteric plexus from the guinea pig taenia coli have been studied by electron microscopy. Three main cell types can be identified: neurons, enteric glial cells and fibroblasts. The ultrastructure of these cells resembles that of the same cells in situ. Neuronal processes form close associations with other neurons and glial cells, but not with fibroblasts. After extended periods in culture, neurons and glial cells form aggregates of cells which resemble ganglia of the myenteric plexus in situ, having a compact neuropil and synapses between neuronal elements. Aggregates are connected to each other by thick bundles of neurites. Vesicle-containing nerve profiles are common; the majority contain a predominance of small agranular vesicles, but some contain many large granular or large opaque vesicles; profiles may also contain variable mixtures of these kinds of vesicles.
Asunto(s)
Plexo Mientérico/ultraestructura , Animales , Ganglios/citología , Ganglios/ultraestructura , Cobayas , Microscopía Electrónica , Plexo Mientérico/citología , Neuroglía/citología , Neuroglía/ultraestructura , Técnicas de Cultivo de ÓrganosRESUMEN
The maintenance of differentiated properties and survival rates of enteric neurons, grown in explant cultures for periods of up to 3 weeks, was studied. Using catecholamine fluorescence, immunohistochemistry and autoradiography, it was found that adrenergic neurons, VIP-containing neurons and putative GABAergic neurons, which constitute small subpopulations of guinea pig myenteric neurons in vivo, were all represented in plexus explants after maintenance in culture for 2-3 weeks. The pattern of expression of the transmitter-related enzymes, acetylcholinesterase and monoamine oxidase, paralleled that found in in situ studies. Investigation of neuronal structure by intracellular injection of horseradish peroxidase revealed that the cultured neurons continue to express the wide diversity in gross morphology which characterizes these cells in vivo. Employing autoradiography following uptake of [3H]GABA to label putative GABAergic neurons, their survival rate from days 1 to 15 of culturing was determined. No neuronal death was detected between days 1 and 5, while the number of neurons decreased between days 5 and 15. These observations suggest that enteric neurons maintained in explant cultures survive well and maintain to a high degree their histochemical and morphological properties.
Asunto(s)
Plexo Mientérico/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Transporte Axonal , Diferenciación Celular , Supervivencia Celular , Cobayas , Histocitoquímica , Peroxidasa de Rábano Silvestre , Monoaminooxidasa/metabolismo , Plexo Mientérico/enzimología , Neuronas/enzimología , Técnicas de Cultivo de Órganos , Péptido Intestinal Vasoactivo/análisis , Ácido gamma-Aminobutírico/análisisRESUMEN
Explant cultures of the myenteric plexus from beneath the taenia coli of newborn guinea-pig caeca were grown on glass substrates in a serum-free, hormone-supplemented culture medium. The growth of enteric neurons, glial cells and fibroblasts and their interactions in this medium were studied over 3 weeks in vitro, and compared with those of cells cultured in the presence of foetal calf serum and in serum-free medium in the absence of added hormones. Enteric neurons, glial cells and fibroblasts survived in the serum-free, hormone-supplemented medium, but this was dependent on the presence of the hormone supplements and also on a brief initial period of exposure to serum-supplemented medium. The pattern of development of the cultures grown in serum-free medium, however, differed markedly from that of cultures grown in serum-supplemented medium: the numbers of enteric glial cells and fibroblasts were substantially reduced in the absence of serum; neurites grew extensively on the glass substrate rather than being restricted to a carpet of glial cells; and reaggregation of neurons and glial cells into compact groups resembling differentiated enteric ganglia did not occur. This ability to grow enteric neurons in a defined chemical environment, without the presence of unknown serum components, thus offers an opportunity for detailed study of the effects of growth and trophic factors on neuronal survival, neurite outgrowth and neurochemical differentiation in the entericnervous system, and also for study of the factors involved in enteric ganglion formation.
RESUMEN
We have shown previously that a soluble factor(s) released by the myenteric plexus promotes neurite outgrowth from postnatal striatal neurons, and that this effect was abolished by tetrodotoxin. We have now investigated the possible involvement of purines in the mediation of this neuritogenic response, by examining their effect on neurite length of striatal neurons both in co-culture with myenteric plexus explants and cultured alone. Both ATP and 2-chloroadenosine partially reversed the inhibitory effect of tetrodotoxin in co-cultures with whole myenteric plexus, while the stable ATP analogue, alpha, beta-methylene ATP, had no effect, suggesting that ATP was being broken down to adenosine before exerting its action. Further support for this view was that the ATP (P2) purinoceptor antagonist suramin did not reverse the effects of ATP, while the adenosine (P1) purinoceptor antagonist 8-(p-sulphophenyl)theophylline did antagonize the effects of ATP in tetrodotoxin-treated co-cultures. Further, both 8-(p-sulphophenyl)theophylline and adenosine deaminase reduced the effect of the myenteric plexus on striatal neurons in the absence of tetrodotoxin, and the adenylate cyclase activator forskolin completely reversed the effect of tetrodotoxin in our co-culture system. The neurite outgrowth-promoting effect of 2-chloroadenosine in tetrodotoxin-treated co-cultures was not further enhanced by a combination of neuropeptides. Serotonin and GTP were without effect on striatal neurons in the presence or absence of myenteric plexus explants. In experiments without myenteric plexus, both 2-chloroadenosine and forskolin caused a slight increase in striatal neurite length; ATP and GTP were ineffective. Basic fibroblast growth factor, nerve growth factor, neurotrophin-3 or neurotrophin-4/5 had no effect on neurite outgrowth in postnatal striatal cultures after two days in vitro. When these growth factors were added in combination with 2-chloroadenosine, the observed increase in mean neurite length did not exceed that induced by 2-chloroadenosine alone. Both 2-chloroadenosine and the ganglioside mix AGF1 increased neurite elongation of striatal neurons after two days in vitro, but an inhibition of enhanced neurite outgrowth was observed when both substances were added together. Both laminin and fibronectin were not neuritogenic for postnatal striatal neurons under our culture conditions. These observations suggest that a factor other than the growth factors tested here is involved in the promotion of striatal neurite outgrowth in co-culture with myenteric plexus explants. In summary, adenosine (probably acting through the A2 subclass of the P1 purinoceptor) leads to increased striatal neurite outgrowth in co-culture with myenteric plexus and we propose that it does so either (1) by triggering the release of a neuritogenic factor, possibly from enteric glial cells, or (2) by acting synergistically with such a growth factor. Adenosine acts via P1 purinoceptors, which leads to changes in cyclic AMP, and the response to forskolin suggests that cyclic AMP is probably involved in the events leading to increased striatal neurite outgrowth.