Immunocytochemical detection of acetylcholine in the rat central nervous system

A specific antibody to acetylcholine was raised and used as a marker for cholinergic neurons in the rat central nervous system. The acetylcholine conjugate was obtained by a two-step immunogen synthesis procedure. An enzyme-linked immunosorbent assay was used to test the specificity and affinity of the antibody in vitro; the results indicated high affinity. A chemical perfusion mixture of allyl alcohol and glutaraldehyde was used to fix the acetylcholine in the nervous tissue. Peroxidase-antiperoxidase immunocytochemistry showed many acetylcholine-immunoreactive cells and fibers in sections from the medial septum region.     

Neurotransmitter plasticity at the molecular level

Contrary to long-held assumptions, recent work indicates that neurons may profoundly change transmitter status during development and maturity. For example, sympathetic neurons, classically regarded as exclusively noradrenergic or cholinergic, can also express putative peptide transmitters such as substance P. This neuronal plasticity is directly related to membrane depolarization and sodium ion influx. The same molecular mechanisms and plastic responses occur in mature as well as developing neurons. Further, contrary to traditional teaching, adult primary sensory neurons may express the catecholaminergic phenotype in vivo. Transmitter plasticity is not restricted to the peripheral nervous system: ongoing studies of the brain nucleus locus ceruleus in culture indicate that specific extracellular factors elicit marked transmitter changes. Consequently, neurotransmitter expression and metabolism are dynamic, changing processes, regulated by a variety of defined factors. Transmitter plasticity adds a newly recognized dimension of flexibility to nervous system function.     

Choline: high-affinity uptake by rat brain synaptosomes

Synaptosomes from rat brain accumulate choline by two kinetically distinct processes, a high-affinity uptake system [Michaelis constant (K(m)) = 1 x 10(-6)M], and a low-affinity system (K(m) = 9 x 10(-5)M). The high-affinity uptake system requires sodium, and is associated with considerable formation of acetylcholine. The low-affinity uptake system is less dependent on sodium, and does not appear to be associated with a marked degree of acetylcholine formation. The high-affinity choline uptake appears to represent selective choline accumulation by cholinergic neurons.     

Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea

Efferent feedback onto sensory organs provides a means to modulate input to the central nervous system. In the developing mammalian cochlea, inner hair cells are transiently innervated by efferent fibers, even before sensory function begins. Here, we show that neonatal inner hair cells are inhibited by cholinergic synaptic input before the onset of hearing. The synaptic currents, as well as the inner hair cell's response to acetylcholine, are mediated by a nicotinic (alpha9-containing) receptor and result in the activation of small-conductance calcium-dependent potassium channels.     

Cholinergic interneurons control local circuit activity and cocaine conditioning

Cholinergic neurons are widespread, and pharmacological modulation of acetylcholine receptors affects numerous brain processes, but such modulation entails side effects due to limitations in specificity for receptor type and target cell. As a result, causal roles of cholinergic neurons in circuits have been unclear. We integrated optogenetics, freely moving mammalian behavior, in vivo electrophysiology, and slice physiology to probe the cholinergic interneurons of the nucleus accumbens by direct excitation or inhibition. Despite representing less than 1% of local neurons, these cholinergic cells have dominant control roles, exerting powerful modulation of circuit activity. Furthermore, these neurons could be activated by cocaine, and silencing this drug-induced activity during cocaine exposure (despite the fact that the manipulation of the cholinergic interneurons was not aversive by itself) blocked cocaine conditioning in freely moving mammals.     

Grafting genetically modified cells to the damaged brain: restorative effects of NGF expression

Fibroblasts were genetically modified to secrete nerve growth factor (NGF) by infection with a retroviral vector and then implanted into the brains of rats that had surgical lesions of the fimbria-fornix. The grafted cells survived and produced sufficient NGF to prevent the degeneration of cholinergic neurons that would die without treatment. In addition, the protected cholinergic cells sprouted axons that projected in the direction of the cellular source of NGF. These results indicate that a combination of gene transfer and intracerebral grafting may provide an effective treatment for some disorders of the central nervous system.     

山羊小肠内AchE和NOS阳性神经元数量分布的比较

应用乙酰胆碱酯酶(AchE)和NADPH-黄递酶组织化学方法,研究了15日龄、4月龄和12月龄山羊小肠中胆碱能神经元和NO能神经元的数量分布变化。结果显示:1)肌间神经丛和黏膜下神经丛含有丰富的AchE阳性神经元,神经元聚集成神经节,并由节间支连成网状。2)NOS(一氧化氮合成酶)阳性神经元主要分布于肌间神经丛,神经元构成的神经节也连成网状;黏膜下神经丛内NOS阳性神经元稀疏,散在分布。3)小肠肌间神经丛中,12月龄的AchE和NOS阳性神经元总数比15日龄的增加163%和137%,但阳性神经元密度降低39%(AchE)和40%(NOS)。4)比较各肠段,AchE和NOS阳性神经元密度在回肠中最高,但神经元总数以空肠最多。结果表明山羊小肠中分布有丰富的胆碱能和NO能神经元,但黏膜下神经丛中胆碱能神经元多于NO能神经元;随着山羊小肠的发育,胆碱能和NO能神经元的数量逐渐增多而密度却下降。     

Somatostatin augments the M-current in hippocampal neurons

Immunocytochemical and electrophysiological evidence suggests that somatostatin may be a transmitter in the hippocampus. To characterize the ionic mechanisms underlying somatostatin effects, voltage-clamp and current-clamp studies on single CA1 pyramidal neurons in the hippocampal slice preparation were performed. Both somatostatin-28 and somatostatin-14 elicited a steady outward current and selectively augmented the noninactivating, voltage-dependent outward potassium current known as the M-current. Since the muscarinic cholinergic agonists carbachol and muscarine antagonized this current, these results suggest a reciprocal regulation of the M-current by somatostatin and acetylcholine.     

Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit

Neural circuits regulate cytokine production to prevent potentially damaging inflammation. A prototypical vagus nerve circuit, the inflammatory reflex, inhibits tumor necrosis factor-α production in spleen by a mechanism requiring acetylcholine signaling through the α7 nicotinic acetylcholine receptor expressed on cytokine-producing macrophages. Nerve fibers in spleen lack the enzymatic machinery necessary for acetylcholine production; therefore, how does this neural circuit terminate in cholinergic signaling? We identified an acetylcholine-producing, memory phenotype T cell population in mice that is integral to the inflammatory reflex. These acetylcholine-producing T cells are required for inhibition of cytokine production by vagus nerve stimulation. Thus, action potentials originating in the vagus nerve regulate T cells, which in turn produce the neurotransmitter, acetylcholine, required to control innate immune responses.     

Plasticity of hippocampal circuitry in Alzheimer's disease

Two markers of neuronal plasticity were used to compare the response of the human central nervous system to neuronal loss resulting from Alzheimer's disease with the response of rats to a similar neuronal loss induced by lesions. In rats that had received lesions of the entorhinal cortex, axon sprouting of commissural and associational fibers into the denervated molecular layer of the dentate gyrus was paralleled by a spread in the distribution of tritiated kainic acid-binding sites. A similar expansion of kainic acid receptor distribution was observed in hippocampal samples obtained postmortem from patients with Alzheimer's disease. An enhancement of acetylcholinesterase activity in the dentate gyrus molecular layer, indicative of septal afferent sprouting, was also observed in those patients with a minimal loss of cholinergic neurons. These results are evidence that the central nervous system is capable of a plastic response in Alzheimer's disease. Adaptive growth responses occur along with the degenerative events.     

Molecular biology of synaptic receptors

A special proteolipid (a hydrophobic protein) has been extracted and purified from nerve-ending membranes and total particulate matter of gray areas of the central nervous system. Such a proteolipid shows a high affinity for binding d-tubocurarine, serotonin, and atropine and has been called receptor proteolipid. The interaction of this proteolipid with atropine sulfate was studied with light scattering and polarization of fluorescence. The changes observed, which follow a cooperative type of curve, were attributed to the aggregation of the proteolipid macromolecules. Such a phenomenon was then observed under the electron microscope. A receptor proteolipid having a high affinity for binding acetylcholine, hexamethonium, and other cholinergic drugs was isolated and purified from electric tissue of fishes and from electroplax membranes. Such a proteolipid was also extracted from membranes from which acetylcholinesterase had been removed, and it was concluded that this enzyme and the receptor proteolipid are two different macromolecules. A high affinity binding site with a dissociation constant of K1 equal to 10(-7) and about ten sites with K2 equal to 10(-5) were recognized in the receptor proteolipid. Under the electron microscope the receptor proteolipid of brain appears as a rod-shaped macromolecule which may assume paracrystalline arrays with 10(-8) molar atropine sulfate. Similarly the receptor proteolipid from electric tissue and from skeletal muscle may form paracrystalline arrays under the action of acetylcholine and hexamethonium. A model of the cholinergic receptor based on the properties of the proteolipid is presented. Preliminary work indicates the possibility of obtaining a biophysical response to acetylcholine when the receptor proteolipid is embedded in artificial bilayered lipid membrance.     

Supraoptic neurosecretory cells: adrenergic and cholinergic sensitivity

Adrenergic and cholinergic agonists and antagonists were applied microelectrophoretically to over 700 neurons in the cat supraoptic nucleus, 20 percent of which were antidromically identified as neurosecretory cells. Norepinephrine uniformly depressed all sensitive cells. Acetylcholine caused both muscarinic depression and nicotinic excitation which were antagonized by atropine and dihydro-beta-erythroidine, respectively. These results support the hypothesis that norepinephrine and acetylcholine are directly involved in controlling the release of antidiuretic hormone.     

The dorsal aorta initiates a molecular cascade that instructs sympatho-adrenal specification

The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel-specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell-derived factor-1 (SDF -1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.     

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.     

Distribution of ChAT in the Diencephalon of the One Day Chicken

The distribution of choline-acetyltransferase(ChAT)-like immunoreactive structure in the one day chicken diencephalons was examined by the method of streptavidin-Perosidase(SP)immunocytochemistry.It was found that ChAT immunoreactive neurons and fibers presented in many regions of one day chicken.Neural structure containing cholinergic neurons with varying density and immunoreactivity were the N.habexularis lateralis and N.habexularis medialis of the epithalamus,the N.rotundus,N.Ovoidalis,N.subrotundus and N.geniculatus lateralis of the thalamus,the hypothalamus,the N.decussationis supraopticae dorsalis of stratathalamus.There were also many varicose and nonvaricose fibers and terminals in the N.geniculatus lateralis and the hypothalamus.The results suggested that the diencephalons of one day chicken is richly innervated by the cholinergic neuron system.     

Ectodermal Wnt function as a neural crest inducer

Neural crest cells, which generate peripheral nervous system and facial skeleton, arise at the neural plate/ectodermal border via an inductive interaction between these tissues. Wnts and bone morphogenetic proteins (BMPs) play roles in neural crest induction in amphibians and zebrafish. Here, we show that, in avians, Wnt6 is localized in ectoderm and in vivo inhibition of Wnt signaling perturbs neural crest formation. Furthermore, Wnts induce neural crest from naive neural plates in vitro in a defined medium without added factors, whereas BMPs require additives. Our data suggest that Wnt molecules are necessary and sufficient to induce neural crest cells in avian embryos.     

Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo

Tritiated acetylcholine was used to measure binding sites with characteristics of nicotinic cholinergic receptors in rat brain. Regulation of the binding sites in vivo was examined by administering two drugs that stimulate nicotinic receptors directly or indirectly. After 10 days of exposure to the cholinesterase inhibitor diisopropyl fluorophosphate, binding of tritiated acetylcholine in the cerebral cortex was decreased. However, after repeated administration of nicotine for 10 days, binding of tritiated acetylcholine in the cortex was increased. Saturation analysis of tritiated acetylcholine binding in the cortices of rats treated with diisopropyl fluorophosphate or nicotine indicated that the number of binding sites decreased and increased, respectively, while the affinity of the sites was unaltered.     

Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels

Although it is generally agreed that general anesthetics ultimately act on neuronal ion channels, there is considerable controversy over whether this occurs by direct binding to protein or secondarily by nonspecific perturbation of lipids. Very pure optical isomers of the inhalational general anesthetic isoflurane exhibited clear stereoselectivity in their effects on particularly sensitive ion channels in identified molluscan central nervous system neurons. At the human median effect dose (ED50) for general anesthesia, the (+)-isomer was about twofold more effective than the (-)-isomer both in eliciting the anesthetic-activated potassium current IK(An) and in inhibiting a current mediated by neuronal nicotinic acetylcholine receptors. For inhibiting the much less sensitive transient potassium current IA, the (-)-isomer was marginally more potent than the (+)-isomer. Both isomers were equally effective at disrupting lipid bilayers.     

Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats

The origin, termination, and length of axonal growth after focal central nervous system injury was examined in adult rats by means of a new experimental model. When peripheral nerve segments were used as "bridges" between the medulla and spinal cord, axons from neurons at both these levels grew approximately 30 millimeters. The regenerative potential of these central neurons seems to be expressed when the central nervous system glial environment is changed to that of the peripheral nervous system.     

In situ hybridization to study the origin and fate of identified neurons

Egg-laying behavior in Aplysia is mediated by a set of peptides, including egg-laying hormone (ELH), which are released by a cluster of identified neurons, the bag cells. A family of neuropeptide genes which includes the gene encoding ELH along with two additional genes encoding the A and B peptides thought to initiate the egg-laying process has been isolated and their nucleotide sequence has been determined. In situ hybridization and immunofluorescence was used to explore the origin and distribution of the neurons that express this family of genes. The ELH genes are expressed, not only in the bag cells, but in an extensive system of neurons distributed in four of the five ganglia of the central nervous system. The genes for ELH are expressed in these cells early in the animal's life cycle. As a result, it was possible to use in situ hybridization to trace the cells expressing ELH to their site of origin. The cells originate outside the central nervous system in the ectoderm of the body wall and appear to migrate to their final locations within the central nervous system by crawling along strands of connective tissue.