Changes in murine anorectum signaling across the life course.

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.

Age-associated changes in the blood-brain barrier: comparative studies in human and mouse.

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.

Changes in the innervation of the mouse internal anal sphincter during aging.

BACKGROUND: The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS: Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS: In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES: The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.

Myenteric neuron numbers are maintained in aging mouse distal colon.

BACKGROUND Age-associated myenteric neuronal loss has been described in several species. In some studies,cholinergic neurons have been reported to be selectively vulnerable, whereas nitrergic neurons are spared. Aging of the mouse enteric nervous system(ENS) and the subtypes of mouse myenteric neurons that may be lost have been little studied. We therefore investigated changes in the numbers of total neurons and two neuronal subpopulations in the mouse distal colon during aging. METHODS Wholemount preparations from 3­4-, 12­13-, 18­19-, and 24­25-month-old C57BL/6 mice were double immunolabeled with HuC/D antibody to identify the total neuronal population and antisera to either calbindin or neuronal nitric oxide synthase (nNOS) to identify myenteric neuronal subpopulations. Samples were analyzed by confocal microscopy. New procedures were employed to ensure unbiased counting and to correct for changes in gut dimensions with age and stretch during sample preparation. The density of nerve fibers in the tertiary plexus was also studied. KEY RESULTS No significant change in numbers of total neurons or of either subpopulation with age was measured, but because of gut growth, the density of myenteric neurons decreased between 3­4 and 12­13 months. The density of nNOS-immunoreactive nerve fibers in the tertiary plexus increased significantly with age, up to 18­19 months. Numerous swollen processes of CB and nNOS-immunoreactive neurons were observed in 18­19- and 24­25-month-old animals. Conclusions &Inferences These results indicate that aging does not result in a loss of myenteric neurons in mouse distal colon at the ages studied, although neurodegenerative changes, which may impact on neuronal function, do occur.

Effects of aging on cholinergic neuromuscular transmission in isolated small intestine of ad libitum fed and calorically-restricted rats.

BACKGROUND: Age-associated losses of enteric neurons have been described. In rat ileum, myenteric neurons lost during aging have been reported to be predominantly cholinergic, and caloric restriction (CR) has been shown to protect against these losses. Cholinergic myenteric neurons include excitatory motor neurons, so the aim of this work was to determine whether neuronal loss in ad libitum (AL)-fed animals is reflected in dysfunctional cholinergic neuromuscular transmission, and if CR reduces any such dysfunction. METHODS: Effects of electrical field stimulation (EFS) and applied acetylcholine (ACh) were examined in the longitudinal muscle of isolated ileal segments from 6-month-old rats and from 13- and 24-month-old rats fed either AL or CR diets. KEY RESULTS: Contractile responses to EFS were abolished by atropine and potentiated by the acetylcholinesterase inhibitor, eserine. Frequency-response relationships were not significantly different amongst the three age-groups. Sensitivity to applied ACh, however, was three-fold lower in the oldest animals (P < 0.05). Eserine potentiated responses to ACh; there were no statistically significant differences amongst the sensitivities to ACh in its presence. No significant differences between AL- and CR-fed animals were measured, although variability was less in CR-fed than in AL-fed groups. CONCLUSIONS & INFERENCES: The cholinergic system supplying the rat ileum longitudinal muscle did not appear to be impaired in old age. Decreased sensitivity to applied ACh in old tissues may have been due to increased acetylcholinesterase activity. Caloric restriction had no significant effect on responses to EFS or applied ACh. The implications of these results are discussed.

Reactive oxygen species, dietary restriction and neurotrophic factors in age-related loss of myenteric neurons.

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.

Trophic actions of neurotrophin-3 on postnatal rat myenteric neurons in vitro.

A number of neurotrophic factors have been implicated in the prenatal development of the enteric nervous system. Although several of these factors continue to be expressed in the gut during postnatal life, their actions on postnatal enteric neurons are not understood. One such factor is the neurotrophin, NT-3. Both NT-3 and its high affinity receptor, trk C, are expressed in the postnatal gut at a time when changes in the density of intestinal innervation are occurring. We have therefore examined the effects of NT-3 on postnatal myenteric neurons, using dissociated cell cultures of ganglia isolated from 6-8 day postnatal rat small intestine. Effects of NT-3 on neurite outgrowth and neuronal and glial cell numbers were measured after 2 days in vitro. The proportion of neurons was increased in NT-3 treated cultures, as was the proportion of neurons that extended processes. NT-3 treatment, at concentrations of between 0.1 ng and 10 ng/ml, also resulted in a significant increase in mean total neurite length. These results indicate that NT-3 may play a role in the postnatal development of the enteric nervous system.

Intrastriatal grafts of rat colonic smooth muscle lacking myenteric ganglia stimulate axonal sprouting and regeneration.

Grafts of living or freeze-killed freshly dissected colonic smooth muscle from young inbred Fischer rats were implanted into the corpus striatum of adult Fischer rats. Sections of brain were examined electron microscopically 3 and 6 wk after implantation. At both times, living grafts were vascularised and contained healthy differentiated smooth muscle cells, fibroblasts, interstitial cells of Cajal and some macrophages. Large bundles of small nonmyelinated axons, identified as CNS axonal sprouts, could be observed in the brain at and near the interface between the living smooth muscle and the CNS tissue. Bundles of regenerating CNS axons, often associated with astrocyte processes, had grown into the grafts. Some axons within the grafts had matured, enlarged and become myelinated by oligodendrocyte processes or Schwann cells. In some cases, smooth muscle cells were observed in close and intricate association with axons. In contrast to the living grafts, grafts of freeze-killed smooth muscle, examined 3 and 6 wk after implantation, contained macrophages, fibroblasts, collagen and large amounts of cellular debris, but no living muscle cells, astrocytes or Schwann cells. The striatal neuropil around freeze-killed grafts did not contain large bundles of CNS axonal sprouts and bundles of axons were not observed within the freeze-killed graft. This study demonstrates that cells from the smooth muscle layers of the colon, in the absence of myenteric ganglia, can stimulate a vigorous regenerative response from CNS axons when implanted into the corpus striatum of adult rats.

A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract.

The myenteric plexus is not only essential for gastrointestinal functions, but it is also a very interesting model for the study of neuronal circuits and neuron-glial interrelationships and may be a valuable source of donor tissue, for grafting into different regions of the central nervous system. For both grafting and culture procedures it is a great advantage to obtain the maximum amount of tissue. To date, most studies have isolated the myenteric plexus by manual microdissection after collagenase digestion. Using this method, it has only been possible to obtain relatively small amounts of the myenteric plexus, mostly from the cecum and proximal colon of the guinea-pig or rat. We present here a new method, which enables much greater quantities of the plexus from the small intestine and colon to be obtained. The myenteric plexus of the entire small intestine can be isolated by a combination of enzymatic digestion and mechanical agitation. The method works from birth up to 3 week old pups, and with some modifications tissue from older or even adult animals can also be processed. Another advantage over the microdissection method is that the myenteric plexuses of the different parts of the intestine can be cultured and studied separately.

Neurite outgrowth of striatal neurons in vitro: involvement of purines in the growth-promoting effect of myenteric plexus explants.

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.

NADPH-diaphorase-containing enteric neurones survive for a year in the adult rat striatum.

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.

Effects of ATP analogues and basic fibroblast growth factor on astroglial cell differentiation in primary cultures of rat striatum.

We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and alpha beta-methylene-ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose-dependent. The effect of alpha beta-methylene-ATP was antagonized by the trypanoside suramin and mimicked by 2-methyl-thio-ATP, suggesting the involvement of a suramin-sensitive P2-purinoceptor. Neither an additive nor a synergistic effect between alpha beta-methylene-ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways. It is concluded that, like basic fibroblast growth factor, ATP can promote the maturation of astrocytes towards a more differentiated phenotype characterized by the presence of longer astrocytic processes. These findings might have interesting implications for astroglial cell differentiation during brain development and for ischemia- and trauma-associated hypergliosis.

Distribution of NADPH-diaphorase activity in the embryonic chicken gut.

The appearance and distribution of NADPH-diaphorase activity in neuronal cells and fibres in different regions of the embryonic chicken gut was studied histochemically using whole mount preparations and cryostat sections. NADPH-diaphorase activity was detected in neuronal cell bodies as early as embryonic day 5.5 (E5.5 - the earliest age examined), mainly in the foregut, although some positive cells were also seen in the hindgut at this stage. NADPH-diaphorase-positive fibres were first detected in the developing nerve tracts which connect the ganglia at E5.5. The complexity of the network was maximal in the proventriculus-gizzard junction. By E9.5, NADPH-diaphorase-positive fibres were found in the circular muscle layer. NADPH-diaphorase-positive submucosal neurons were first detected at E11.5. The density of innervation was maximal at E15.5 and declined later development. The expression of neuronal NADPH-diaphorase activity progressed in a craniocaudal direction and followed a developmental pattern similar to that previously described for several neuropeptides in the avian gut.

Distribution and colocalization of NADPH-diaphorase activity, nitric oxide synthase immunoreactivity, and VIP immunoreactivity in the newly hatched chicken gut.

BACKGROUND: The distribution and colocalization of nitric oxide synthase and NADPH-diaphorase have been investigated quite extensively in the mammalian gut; however, no such study has been undertaken in the avian gut. In the present report, we have therefore studied the distribution and coexpression of nitric oxide synthase (NOS), NADPH-diaphorase, and vasoactive intestinal polypeptide (VIP) in enteric neurons of the newly hatched chicken gut. METHODS: Immunohistochemical methods were used to detect NOS immunoreactivity (NOS-IR) and VIP immunoreactivity (VIP-IR). NADPH-diaphorase activity was detected using a histochemical technique. RESULTS: Neurons expressing NADPH-diaphorase activity, NOS-IR, and VIP-IR were detected in both the myenteric and submucous plexus of all regions of the gastrointestinal tract examined. All NADPH-diaphorase positive neurons were also NOS-IR and all NOS-IR neurons were NADPH-diaphorase positive, in both plexuses, indicating that NADPH-diaphorase can be used as a marker for NOS containing neurons in the chicken gut. The majority of VIP-IR neurons also expressed NADPH-diaphorase activity. Only few neurons that expressed NADPH-diaphorase activity did not express VIP-IR. The proportion of VIP immunopositive neurons that were NADPH-diaphorase negative increased anally and these neurons were more prominent in the submucous than the myenteric plexus ganglia. NADPH-diaphorase positive, NOS-IR, and VIP-IR nerve fibres were detected in the circular muscle, but very few, if any, were present in the longitudinal muscle. VIP-IR, but not NOS-IR or NADPH-diaphorase activity, was detected in mucosal fibres, in contrast to the situation in the mammalian gut. CONCLUSIONS: These results indicate that in birds, as in mammals, nitric oxide may play a role in the neural control of the gut musculature, but that it is unlikely to be involved in the nervous control of mucosal activity.

The neuritogenic effect of myenteric plexus on striatal neurones in co-culture involves nitric oxide.

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.

Trophic actions of 2-chloroadenosine and bFGF on cultured myenteric neurones.

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.

NADPH diaphorase and nitric oxide synthase are expressed by the majority of intramural neurons in the neonatal guinea pig urinary bladder.

The distribution of neurons expressing NADPH diaphorase activity was examined histochemically in whole mount preparations of the neonatal guinea pig urinary bladder. NADPH diaphorase positive neurons were abundant in the intramural ganglia in both the detrusor and trigone regions of the bladder. Labelled nerve fibres were found in the ganglion interconnectives and in smooth muscle bundles. Mucosal epithelial cells and endothelial cells lining the blood vessels supplying the bladder were also found to express NADPH diaphorase activity. In order to verify that NADPH diaphorase activity represented the presence of nitric oxide synthase in bladder neurons, a well characterised tissue culture preparation was employed. This also provided an opportunity to estimate the proportion of the total population of bladder neurons which expressed NADPH diaphorase activity. Using a combination of histochemical and immunohistochemical techniques, NADPH diaphorase positive neurons were found to constitute approximately 90% of the total neuronal population, which was identified by labelling with an antiserum to the general neuronal marker protein gene product 9.5. Almost all neurons (99%) which expressed NADPH diaphorase activity in culture were also found to be immunoreactive for nitric oxide synthase. These findings indicate that nitric oxide may play a role in the neural control of bladder function, and this possibility is discussed.

Transplantation of the postnatal rat myenteric plexus into the adult rat corpus striatum: an electron microscopic study.

Since the enteric nervous system (ENS) has been demonstrated to display many similarities with the central nervous system (CNS), we have undertaken investigations examining the possibility of using the ENS as a source of neurons for transplantation into the brain in neurodegenerative diseases. Grafts of freshly dissected myenteric plexus taken from young inbred Fischer rats were implanted in the corpus striatum of adult Fischer rats, some of which had received a quinolinic acid lesion 1 to 2 weeks prior to implantation. Brain sections from the grafted animals were examined electron microscopically 3 and 6 weeks after implantation. Grafts contained many enteric neurons and glial cells in ganglia morphologically similar to those seen within the intestine. Although a glia limitans could be observed in parts of the interface between graft and brain, ganglia at the surface of the graft were observed to be closely apposed to the surrounding striatum, with no intervening basal lamina. CNS axonal sprouts were identified in the corpus striatum near the interface with the grafted enteric ganglia and bundles of similar axons, some containing fibers that had enlarged and become myelinated, could be seen within the grafts. A smaller number of CNS axonal sprouts were also observed around the grafts in the quinolinic-acid-lesioned striata. This study confirms that even in the absence of surrounding layers of smooth muscle, enteric ganglia grafted into the corpus striatum survive and stimulate the production of axonal sprouts from striatal and other neurons, which subsequently grow into the grafts.

Myenteric plexus explants promote neurite elongation and survival of striatal neurons in vitro.

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.