Nitric oxide modulates the cardiovascular effects elicited by acetylcholine in the NTS of awake rats.

Microinjection of acetylcholine chloride (ACh) in the nucleus of the solitary tract (NTS) of awake rats caused a transient and dose-dependent hypotension and bradycardia. Because it is known that cardiovascular reflexes are affected by nitric oxide (NO) produced in the NTS, we investigated whether these ACh-induced responses depend on NO in the NTS. Responses to ACh (500 pmol in 100 nl) were strongly reduced by ipsilateral microinjection of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 nmol in 100 nl) in the NTS: mean arterial pressure (MAP) fell by 50 +/- 5 mmHg before L-NAME to 9 +/- 4 mmHg, 10 min after L-NAME, and HR fell by 100 +/- 26 bpm before L-NAME to 20 +/- 10 bpm, 10 min after L-NAME (both P < 0.05). Microinjection of the selective inhibitor of neuronal nitric oxide synthase (nNOS), 1-(2-trifluoromethylphenyl) imidazole (TRIM; 13.3 nmol in 100 nl), in the NTS also reduced responses to ACh: MAP fell from 42 +/- 3 mmHg before TRIM to 27 +/- 6 mmHg, 10 min after TRIM (P < 0.05). TRIM also tended to reduce ACh-induced bradycardia, but this effect was not statistically significant. ACh-induced hypotension and bradycardia returned to control levels 30-45 min after NOS inhibition. Control injections with D-NAME and saline did not affect resting values or the response to ACh. In conclusion, injection of ACh into the NTS of conscious rats induces hypotension and bradycardia, and these effects may be mediated at least partly by NO produced in NTS neurons.

Identification of neurons with acetylcholinesterase and NADPH-diaphorase activities in the centrifugal visual system of the chick.

The isthmo-optic nuclei (ION) and ectopic neurons, which constitute the centrifugal visual system (CVS), are thought to be cholinoceptive and nitrergic. However, it is not clear which neurons express these markers, namely the ones that project to the retina rather than in neurons that only participate in a local circuit. Therefore, to characterize the neurochemical patterns of the centrifugal visual system in the post-hatched chick, retinopetal cells of the isthmo-optic nuclei and the ectopic region were identified via immunolabeling for cholera toxin, a neuronal tracer, which has been injected in the ocular globe. Then, double labeled with acetylcholinesterase histochemistry to reveal cholinergic synapses, or NADPH-diaphorase histochemistry as a nitrergic marker. Briefly, acetylcholinesterase activity was present mainly in cholera toxin labeled cell bodies of the isthmo-optic nucleus and the ectopic region indicating that retinal projecting neurons of centrifugal visual system comprise a cholinoceptive pathway. On the other hand, NADPH-diaphorase histochemistry was present in the neuropile and sparse cell bodies inside of the isthmo-optic nucleus and in ectopic neurons which were not cholera toxin positive suggesting their role in an intrinsic circuit of the centrifugal visual system. These data support the idea that these two neurochemical systems are present in distinct neuronal populations in the centrifugal visual system.

Cholinergic regulation of Na+-K+-ATPase activity in rat parotid gland: changes after castration.

In this study, we investigated the different signalling pathways involved in muscarinic acetylcholine M(3) receptor-dependent modulation of Na(+)-K(+)-ATPase in parotid glands from normal and castrated rats. Carbachol inhibited the enzyme activity in parotid glands from control rats while it stimulated the enzyme activity in castrated rats. The inhibition of Ca(2+) calmodulin by trifluoperazine abolished the inhibitory effect of carbachol in control rats, while the inhibition of protein kinase C by staurosporine stimulated Na(+)-K(+)-ATPase. In castrated rats, trifluoperazine inhibited the carbachol-stimulant effect while staurosporine had no effect. Results indicate that in control glands the activation of a phospholipid-Ca(2+) calmodulin-dependent protein kinase C is responsible for the inhibitory effect of carbachol on Na(+)-K(+)-ATPase activity. In castrated rats, the activation of the enzyme by carbachol is regulated by its Ca(2+) calmodulin-stimulating action, and not by activation of protein kinase C. The activation of the Na(+)-K(+)-ATPase observed in castrated rats resulted in a decrease in carbachol-induced net K(+) efflux and thereby could decrease salivary fluid production.

Effects of estrogens on choline-acetyltransferase immunoreactivity and GAP-43 mRNA in the forebrain of young and aging male rats.

1. Previous work demonstrated that estradiol (E2) treatment prevented the abnormal response to stress and the reduction of glucocorticoid receptors (GR) in hippocampus from aging male rats. The mechanisms originating these effects were unknown. 2. In the present work, we investigated the E2 effects on the cholinergic, growth-associated protein (GAP-43) expressing neurons of the medial septum (MS) and vertical limb of diagonal band of Broca (VDB). These areas project to the hippocampus, and may be involved in the mentioned E2 effects in aging animals. Therefore, the response to E2 of choline-acetyltransferase (ChAT) in neurons and cell processes and GAP-43 mRNA as a marker of neurite outgrowth was studied in young and old male rats. 3. Young (3-4 months) and old (18-20 months) male Sprague-Dawley rats remained untreated or were implanted s.c. with a 14 mg pellet of E2 benzoate during 6 weeks. We used immoucytochemistry to determine ChAT and isotopic in situ hybridization to analyze GAP-43 mRNA expression. 4. Aging males showed a reduction in the number and length of ChAT-immunoreactive cell processes, but not in the number of positive neurons in MS and VDB. E2 reverted both parameters in old rats to levels of young animals. Regarding basal levels of GAP-43 mRNA, they were similar in old and young animals, but E2 treatment up-regulated GAP-43 mRNA expression in MS and VDB of old animals only. 5. Our data suggest that prolonged E2 treatment may affect hippocampal function of aging male rats by regulating in part the plasticity of cholinergic, GAP-43 expressing neurones of the basal forebrain. Without discarding a direct E2 effect on the limbic tissue, effects on the cholinergic system may have a pronounced impact on the neuroendocrine and stress responses of the aging hippocampus.

Localization of choline acetyltransferase (ChAT) immunoreactivity in the brain of a caecilian amphibian, Dermophis mexicanus (Amphibia: Gymnophiona).

The organization of the cholinergic system in the brain of anuran and urodele amphibians was recently studied, and significant differences were noted between both amphibian orders. However, comparable data are not available for the third order of amphibians, the limbless gymnophionans (caecilians). To further assess general and derived features of the cholinergic system in amphibians, we have investigated the distribution of choline acetyltransferase immunoreactive (ChAT-ir) cell bodies and fibers in the brain of the gymnophionan Dermophis mexicanus. This distribution showed particular features of gymnophionans such as the existence of a particularly large cholinergic population in the striatum, the presence of ChAT-ir cells in the mesencephalic tectum, and the organization of the cranial nerve motor nuclei. These peculiarities probably reflect major adaptations of gymnophionans to a fossorial habit. Comparison of our results with those in other vertebrates, including a segmental approach to correlate cell populations across species, shows that the general pattern of organization of cholinergic systems in vertebrates can be modified in certain species in response to adaptative processes that lead to morphological and behavioral modifications of members of a given class of vertebrates, as shown for gymnophionans.

Translocation of protein kinase C by halothane in cholinergic cells.

Protein kinase C (PKC) is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for volatile anaesthetic actions. However, the effects of these agents on PKC activity are not yet fully understood. Volatile anaesthetics increase intracellular calcium concentration ([Ca(2+)](i)) in a variety of cells, thus their effects on PKC activity may be indirect due to [Ca(2+)](i) increase. Alternatively, the anaesthetics could directly stimulate PKC activity. In order to distinguish these two possibilities in intact cells, we used a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII) and confocal microscopy to evaluate the dynamic redistribution of PKC in living SN56 cells, a cholinergic cell line, in response to halothane. Halothane induced PKC translocation in SN56 cells transfected with GFP-PKCbetaII. This effect was not suppressed by dantrolene, a drug that blocks halothane-induced Ca(2+) release from intracellular stores in these cells. These findings indicate that halothane induces PKC translocation in SN56 cells independently of its ability to release calcium from internal stores.

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.

Postnatal development of cholinergic system in mouse basal forebrain: acetylcholinesterase histochemistry and choline-acetyltransferase immunoreactivity.

The distribution of acetylcholinesterase histochemistry and choline-O-acetyltransferase immunohistochemistry in the basal forebrain was studied in newborn mice (P0) and until 60 days of postnatal life (P60). A weak acetylcholinesterase activity was found at P0 and P2 in the anterior and intermediate parts of the basal forebrain, and higher in the posterior region. The intensity of labeling, neuronal size and dendritic growth seems to increase progressively in all regions of basal forebrain from P4 to P10. The AChE+ cell count shows that in the anterior portion of the magnocellular basal nucleus the number of cells does not vary significantly from birth to the second month of postnatal life. However, in the intermediate and posterior portions of the nucleus the mean number of labeled cells increases significantly from birth to the end of the second week of postnatal life (P13). The choline-acetyltransferase immunoreactivity appears only detectable at the end of the first week (P6) as a slight immunoreaction, which increases progressively in intensity at P8, and at P10 seems to attain the same intensity of labeling found at P60. These results seem to indicate that the acetylcholinesterase could have a non-classic cholinergic role in the first stages of postnatal development, acting as a growth and cellular differentiation factor.

Postnatal development of the basal forebrain cholinergic projections to the medial prefrontal cortex in mice.

The postnatal development of basal forebrain cholinergic projections to the medial prefrontal cortex in mice was analyzed by means of the double labeling track-tracing study. The tracer was injected into the medial prefrontal cortex of mice, on the day of birth (P0) to 60 days after birth. The total number of basal forebrain neurons increased from P4 to P8, and began to decrease until P13 (52.9% vs. the maximal average (P8)). After P13, the mean average remains stable up to P60. On the other hand, differential pattern of frontocortical projections of the anterior, intermediate, and posterior regions can be observed.

Ontogeny of cholinergic amacrine cells in the oppossum (Didelphis aurita) retina.

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT-immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub-population of ChAT-immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT-immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole-mounted preparations, cholinergic cell density was 263 +/- 13 cells/mm2 in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT-immunoreactive somata showed a random pattern of distribution.

[The action of epidermal growth factor on the development of cultured striatum cells]. / Acción del factor de crecimiento epidérmico sobre el desarrollo de las células estriatales en cultivo.

INTRODUCTION: Epidermic growth factor (EGF) has a neurotrophic mitogenic effect on different cell populations in the nervous system. This is modulated by the stage of development and microenvironment of the cells. OBJECTIVE AND METHODS: In this paper we describe the action of EGF on embryonic striatum cells of a culture system dissociated from neurons and glias. The cell culture is prepared from 16-17 day rat embryos. In the system used, the cell population was cultured for 20-24 hours in a medium containing serum. This medium was later replaced by a mixture of specific nutrients and treated for 6 days with 20 mg/ml of EGF. RESULTS AND CONCLUSIONS: The substitution of serum during the initial period of development led to an appreciable reduction in the living cells in the treated cultures and in the controls. The surviving cells were mainly cellular precursors, taking into account their morphological characteristics and capacity for proliferation. The effect of EGF was seen in an increase in the number of cells and was shown to be a stimulus to the proliferation of neuronal and astrocyte precursors. The specific activity of choline acetyl-transferases determined in the cultures at 16 days showed differentiation of a cholinergic neurons subpopulation, which responded to treatment with nerve growth factor with an increase in the activity of this enzyme.

Noradrenergic and acetylcholinesterase-positive nerve fibres of the uterus in sexually immature and cycling rats.

The distribution and density of the noradrenergic and acetylcholinesterase-positive nerve fibres were histochemically studied in different uterine regions of prepubertal and cycling rats in dioestrus and oestrus. Besides the rich and double innervation of blood vessels, both types of nerve fibre were found in the myometrium and cervical musculature. The non-vascular noradrenergic network looked denser at the tubal end of the horns and at the cervix, whereas the acetylcholinesterase-positive innervation was poor at the tubal end, increasing toward the cervix. Contrasting with the middle third of the uterine horn, at the tubal end, the myometrial longitudinal layer was much more innervated than the circular one, especially by the noradrenergic nerve fibres. The prepubertal rats presented an adult pattern of uterine autonomic innervation. In the cycling animals, this innervation was nearly the same during oestrus and dioestrus regarding both the density of nerve fibres and intensity of the histochemical reactions.

Neurochemical and behavioral correlates of unilateral striatal acetylcholinesterase inhibition by fasciculin in rats.

Fasciculin 2 (FAS) an anticholinesterase peptide isolated from the venom of the Green mamba (Dendroaspis angusticeps) was injected into the right striatum of albino rats (1.5 micrograms total amount). The inhibition of acetylcholinesterase (AChE) activity was 86 and 60% 24 h and 7 days after FAS injection, respectively. The treatment with apomorphine (APO) (2 mg/kg s.c.) 24 h after FAS provoked a moderate circling towards the lesioned side that was reverted by atropine (30 mg/kg i.p.). The same dose of APO 7 days after FAS, provoked an inconstant contralateral circling. Neither dopamine nor serotonin nor their metabolites were significantly affected 24 h or 7 days after FAS injection. Radioligand binding assays of dopamine, muscarinic and benzodiazepine receptors only showed a decrease of the density of the muscarinic ones 7 days after FAS. These results are interpreted as showing that the changes provoked by FAS would be compensated but the system would remain in an unsteady state only demonstrable after pharmacological challenge. The chronic down-regulation of muscarinic receptors would compensate the increased cholinergic activity and would therefore block its behavioral expression.