Two types of cholinergic innervation in cortex, one co-localized with vasoactive intestinal polypeptide

The existence of cholinergic neuronal cell bodies in mammalian cerebral cortex was long the subject of much controversy (see ref. 1 for review). Recently, however, a specific cholinergic marker, the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT, E.C.2.3.1.6), was demonstrated by immunohistochemical methods to be present in bipolar neurones in rat cortex. Here we show that at least 80% of these intrinsic cholinergic neurones also contain immunoreactivity for vasoactive intestinal polypeptide (VIP), a neuroactive peptide found to be present in a subpopulation of cortical neurones. On the other hand, we find that the ChAT-positive cells in the basal forebrain, which are another major source of cholinergic innervation of the cortex, contain no detectable VIP-immunoreactivity. In addition, we have observed by both light and electron microscopy that some VIP- and some ChAT-positive structures in cortex are closely associated with blood vessels.     

Acetylcholine induces burst firing in thalamic reticular neurones by activating a potassium conductance

Recent studies have emphasized the role of acetylcholine (ACh) as an excitatory modulator of neuronal activity in mammalian cortex and hippocampus. Much less is known about the mechanism of direct cholinergic inhibition in the central nervous system or its role in regulating neuronal activities. Here we report that application of ACh to thalamic nucleus reticularis (nRt) neurones, which are known to receive a cholinergic input from the ascending reticular system of the brain stem, causes a hyperpolarization due to a relatively small (1-4 nS) increase in membrane conductance to K+. This cholinergic action appears to be mediated by the M2 subclass of muscarinic receptors and acts in conjunction with the intrinsic membrane properties of nucleus reticularis neurones to inhibit single spike activity while promoting the occurrence of burst discharges. Thus, cholinergic inhibitory mechanisms may be important in controlling the firing pattern of this important group of thalamic neurones.     

Cholinergic-rich brain transplants reverse alcohol-induced memory deficits

Alcohol-induced memory impairment in man has been attributed to deficiencies in subcortical noradrenergic and cholinergic systems, as well as to damage in midbrain structures. Korsakoff's psychosis, a disease in which alcohol poisoning causes apparently irreversible memory defects, is characterized by lesions in cholinergic and noradrenergic nuclei and by a decrease in the activity of choline acetyltransferase (ChAT) and the content of noradrenaline (NA) in forebrain areas such as cerebral cortex and hippocampus, innervated by these nuclei. Prolonged intake of ethanol in rodents similarly produces signs of noradrenergic and cholinergic deafferentation in the cortex and hippocampus, as well as persistent memory deficits. To test whether alcohol-induced memory impairments depend on cholinergic deafferentation, we transplanted cholinergic-rich fetal basal forebrain cell suspensions into the cortex and hippocampus of alcohol-treated rats. The substantial and persistent memory losses produced in our rats by ethanol intake were associated with an impairment of cholinergic function, and were reversed by cholinergic-rich transplants into cortex and hippocampus.     

Role of endogenous substance P in stress-induced activation of mesocortical dopamine neurones

The dopamine (DA) innervation to the forebrain arises from subpopulations of midbrain DA neurones broadly classified as nigrostriatal, mesolimbic and mesocortical. Significant differences in the autoregulatory mechanisms and neuronal inputs of these DA pathways may account for their differences in physiological and pharmacological responsiveness. For example, footshock stress can activate rat mesocortical DA cells but does not alter nigrostriatal DA turnover, while also decreasing substance P (SP) concentrations in the midbrain interpeduncular nucleus and in the adjacent ventral tegmental area (VTA), but not in the substantia nigra (SN). This suggested that the activation of the SP input to the VTA may mediate activation of certain DA systems by footshock stress; behavioural studies also had suggested an excitatory effect of SP on DA cells in the VTA. SP antagonists now available are neurotoxic and of questionable efficacy, we therefore used monoclonal antibody against SP. Antibody microinjected into the VTA prevented normal footshock-induced activation of mesocortical DA neurones, suggesting mediation by SP input to the VTA. The in vivo application of antibodies may prove valuable in studies of neuropeptides in the central nervous system (CNS).     

Reciprocal changes in corticotropin-releasing factor (CRF)-like immunoreactivity and CRF receptors in cerebral cortex of Alzheimer's disease

Alzheimer's disease is a progressive degenerative disease of the nervous system characterized neuropathologically by the presence of senile plaques and neurofibrillary tangles in amygdala, hippocampus and neocortex. Dysfunction and death of basal forebrain cholinergic neurones projecting to forebrain targets are associated with marked decreases in cholinergic markers, including the activity of choline acetyltransferase (ChAT). Although cortical levels of somatostatin and somatostatin receptors are reduced in Alzheimer's, no consistent changes have been reported in other neuropeptide systems. We have now examined in control and Alzheimer's brain tissues pre- and postsynaptic markers of corticotropin-releasing factor (CRF), a hypothalamic peptide regulating pituitary-adrenocortical secretion which also seems to act as a neurotransmitter in the central nervous system (CNS). We have found that in Alzheimer's, the concentrations of CRF-like immunoreactivity (CRF-IR) are reduced and that there are reciprocal increases in CRF receptor binding in affected cortical areas. These changes are significantly correlated with decrements in ChAT activity. Our results strongly support a neurotransmitter role for CRF in brain and demonstrate, for the first time, a modulation of CNS CRF receptors associated with altered CRF content. These observations further suggest a possible role for CRF in the pathophysiology of the dementia. Future therapies directed at increasing CRF levels in brain may prove useful for treatment.     

Age-impaired impulse flow from nucleus basalis to cortex

Recent studies have renewed interest in the role of acetylcholine (ACh) in the cognitive changes associated with ageing and dementia. Deficits in cortical choline acetyltransferase (ChAT) in Alzheimer's disease have been consistently demonstrated, while other research has suggested a connection between deterioration of cortical ACh fibres and dementia. However, despite clear biochemical and anatomical evidence for a fall in ACh in dementia, results of therapeutic trials with cholinergic agonists, precursors and cholinesterase inhibitors have been inconsistent. Such findings suggest that cortical cholinergic disorders are not wholly a function of simple biochemical change; alterations of impulse flow along cholinergic fibres could well be as debilitating. An important extrinsic source of cortical ACh innervation derives from neurones diffusely located in rat basal forebrain, denoted the nucleus basalis (NB). We have now investigated the impulse conduction properties of cortically projecting, putatively cholinergic NB axons in adult and aged rats and have found that conduction latencies from NB to frontal cortex are significantly longer (by 51%) in aged animals. In addition, systematic analysis varying cortical stimulation depth revealed that these longer latencies are due entirely to decreased conduction velocities in the subcortical fibre projections. Indeed, intracortical velocities were virtually identical in the two groups. Our results indicate that ageing occasions a decrease in the temporal fidelity of impulse flow in the cholinergic input to the cortex from the NB, a previously overlooked but potentially important element in cognitive deficits that occur with age.     

Corticotropin regulates transsynaptically the activity of septo-hippocampal cholinergic neurones

The activity of septo-hippocampal neurones is affected by the action on cholinergic perikarya in the septum of a variety of putative neurotransmitters, including substance P and beta-endorphin. (The latter is released in the septal region from neurones which originate in the medial basal hypothalamus.) It has also been reported that two other neuropeptides, corticotropin (ACTH1-24) and alpha-melanotropin (alpha-MSH), affect acetylcholine turnover in septo-hippocampal neurones in a manner that is not blocked by transection of the afferents to the hippocampus, from which it has been inferred that the neurotransmitters act directly on the hippocampus. We now describe experiments with corticotropin which show that the effect is rather the influence on septo-hippocampal cholinergic neurones of peptidergic neurones within the septum.     

Substance P raises neuronal membrane excitability by reducing inward rectification

Much interest has recently centred on the properties of peptides that modulate the excitability of nerve cells. Such compounds include the undecapeptide substance P, which is particularly well established as an excitatory neurotransmitter, and we examine here its effects on magnocellular cholinergic neurones taken from the medial and ventral aspects of the globus pallidus of newborn rats and grown in dissociated culture. These neurones have previously been shown to respond to substance P3 and are analogous to the nucleus basalis of Meynert in man, which gives a diffuse projection to the cerebral cortex and whose degeneration is the likely cause of Alzheimer's disease. Substance P depolarizes these cultured neurones by reducing an inwardly rectifying potassium conductances; this conductance has been found in several neuronal types and has similar properties to those of certain other cells. As discussed below, modulation of inward (or anomalous) rectification by substance P implies a self-reinforcing element to the depolarization caused by the peptide.     

淫羊藿总黄酮增强双侧卵巢摘除模型小鼠学习记忆功能的作用

研究淫羊藿总黄酮(TFE)对双侧卵巢摘除模型小鼠学习记忆功能的影响,并探讨其可能的作用机制.将50只昆明种雌性小鼠随机分为模型对照组,雌激素对照组(0.3 mg·kg-1),淫羊藿总黄酮(40、80、160mg·kg-1)3个剂量组,采用背部切除双侧卵巢,另取10只小鼠作为假手术对照组,建立卵巢功能减退小鼠模型,术后各治疗组进行灌胃给药治疗3个月,假手术组和模型对照组均给予0.9%NaCl.给药结束后采用Morris水迷宫观察小鼠行为学变化;生化法和放射免疫法分别测定脑组织中AChE活性和ChAT活性;放射免疫法检测小鼠血液中雌激素的水平;HE染色光镜观察海马神经元病理形态.实验结果显示:与假手术组相比,模型对照组小鼠学习记忆能力、ChAT活性、血清雌激素水平和皮层及海马区神经细胞数量均显著下降,而脑组织中AChE活性明显升高;与模型对照组相比,TFE 40、80、160mg·kg-1剂量组各组小鼠学习记忆能力均显著增强;小鼠脑组织AChE活性下降明显,而ChAT活性却显著增加,血液中雌激素水平上升显著.TFE可能是通过升高血中雌激素水平,增强乙酰胆碱(ACh)合成酶ChAT的活性,抑制ACh水解酶AChE活性,提高大脑中神经递质乙酰胆碱的含量,延缓和改善内分泌系统和大脑胆碱能系统功能,从而促进卵巢去势小鼠学习记忆能力的提高.     

Cycloheximide-sensitive synthesis of substance P by isolated dorsal root ganglia

Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) may be used as a neurotransmitter by certain primary afferent neurones, particularly those carrying pain impulses. Substance P-like immunoreactivity has been localised to the cell bodies of one population of dorsal root ganglion neurones by immunocytochemistry. It is contained in vesicles in the central terminals of these neurones, and has also been demonstrated in the peripheral terminals. As axons and terminals have very little capacity for peptide biosynthesis, it is possible that substance P is synthesised and packaged in the perikaryon and transported to the terminals by an axoplasmic transport process. Consistent with this is the finding that substance P accumulates proximal to a ligature placed on the dorsal root. There has, however, been no direct demonstration of the biosynthesis of substance P in the nervous system. We report here that rat dorsal root ganglia incorporate 35S-methionine into substance P, characterised as authentic by immunoprecipitation followed by HPLC. There is a delay of 1-2 h between addition of label and its incorporation into substance P. Synthesis is blocked by cycloheximde suggesting that, in dorsal root ganglia, substance P is synthesised by a conventional ribosomal process. Synthesis of substance P is reduced by some 90% in ganglia from rats treated neonatally with capsaicin, a drug which is thought to destroy a population of primary afferent neurones.     

Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer disease and Alzheimer senile dementa

Both Alzheimer's disease and senile dementia of the Alzheimer type (AD/SDAT) are progressive dementias characterized neuropathologically by the presence in the cerebral cortex of numerous neurofibrillary tangles and neuritic plaques. We use the abbreviation AD/SDAT to denote all such cases, irrespective of age of onset. Studies of neurotransmitter-related parameters in autopsied brain tissues from patients with AD/SDAT have, to date, been confined to five putative transmitter systems. Acetycholine-releasing neurones seem to be most markedly and consistently affected, as judged by the extensive reductions in choline acetyltransferase (ChAT) and acetylcholinesterase activities that have been reported. Despite numerous studies, there is no consistent evidence for the involvement of neurones releasing dopamine, noradrenaline, serotonin, or gamma-aminobutyric acid in AD/SDAT, nor for loss of muscarinic cholinergic receptors. Thus, the involvement of cholinergic neurones in AD/SDAT seems to be specific. However, the possible involvement of neurones using other chemicals as transmitters has yet to be explored. The recent recognition of the existence of so-called 'peptidergic neurones' in the mammalian brain (for review see ref. 8) and the availability of radioimmunoassay (RIA) techniques for studying these peptides, have led us to begin a systematic investigation of neuropeptides in autopsied brain tissue from cases of AD/SDAT, and from neurologically normal individuals. We report here results obtained with a RIA for somatostatin, showing that somatostatin-like immunoreactivity in the cerebral cortex is reduced in tissue from AD/SDAT patients.     

人胃胆碱能神经支配的研究

选用成年健康男性尸体8例,死后迅速取下胃,并将其分成胃底、胃体、胃窦及胃幽门管4部分。光镜ChAT、AChE染色,显微图像分析仪对胃各部位肌间神经丛神经细胞内酶含量进行定量分析,同时进行透射电镜标本制备。结果表明,胃各部位胆碱能神经元的分布及细胞内酶含量不同,神经纤维与平滑肌间形成远距离突触,电镜下AChE定位于粗面内质网。最后对实验结果分别进行了讨论。     

Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus

The transmission of visual information from retina to cortex through the dorsal lateral geniculate nucleus (LGNd) is controlled by non-retinal inputs. Enhanced visually evoked responses in cat LGNd relay cells during periods of increased alertness have been attributed in large part to increased rate of acetylcholine (ACh) release by fibres ascending from the brainstem reticular formation. ACh can modulate geniculate visual responses in vivo, but comparatively little is known about the underlying ionic mechanisms of these cholinergic actions. Although direct excitation of LGNd relay neurons has been shown in vitro, the situation is complicated because cholinergic axons form numerous and complex synapses not only with relay cells, but also with inhibitory interneurons, and electrical activation of the brainstem cholinergic neurons reduces inhibitory postsynaptic potentials in the LGNd. We report here that morphologically characterized interneurons in the cat LGNd possess distinctive electrophysiological properties in comparison with those of relay cells and are inhibited by ACh through a muscarinic receptor-mediated increase in potassium conductance. Together the direct excitation of relay cells and inhibition of intrageniculate interneurons allow the ascending cholinergic system to exert a powerful facilitatory influence over the transfer of visual information to the cerebral cortex.     

Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor

In aged rodents, impairments in learning and memory have been associated with an age-dependent decline in forebrain of cholinergic function, and recent evidence indicates that the cholinergic neurons in the nucleus basalis magnocellularis, the septal-diagonal band area and the striatum undergo age-dependent atrophy. Thus, as in Alzheimer-type dementia in man, degenerative changes in the forebrain cholinergic system may contribute to age-related cognitive impairments in rodents. The cause of these degenerative changes is not known. Recent studies have shown that the central cholinergic neurons in the septal-diagonal band area, nucleus basalis and striatum are sensitive to the neurotrophic protein nerve growth factor (NGF). In particular, intraventricular injections or infusions of NGF in young adult rats have been shown to prevent retrograde neuronal cell death and promote behavioural recovery after damage to the septo-hippocampal connections. It is so far not known, however, whether the atrophic cholinergic neurons in aged animals are responsive to NGF treatment. We report here that continuous intracerebral infusion of NGF over a period of four weeks can partly reverse the cholinergic cell body atrophy and improve retention of a spatial memory task in behaviourally impaired aged rats.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo

Cutting the axons of the cholinergic neurons that project to the hippocampal formation results in death of most of these cells. Previous studies have shown that administration of nerve growth factor before or at the same time as the lesion will prevent this cell death. Here we demonstrate that basic fibroblast growth factor (FGF) administered into the brain reduces the death of cholinergic neurons in the medial septum and diagonal band of Broca after transection of their axons, in both young adult and aged rats. Moreover, FGF can partially protect against death of cholinergic neurons even when administered two days after axonal transection. These results indicate a possible function for FGF in the normal support of basal forebrain cholinergic neurons, but its range of activity could be wider, for FGF also supports noncholinergic neurons in vitro, it is localized in many of the central nervous system neurons, and it is found in relatively high concentrations in the brain.     

A subpopulation of rat dorsal root ganglion neurones is catecholaminergic

The neurotransmitters used by the sensory neurones of the dorsal root ganglia (DRG) are unknown. A proportion of these cells contain physiologically active peptides; for example, subpopulations of small-diameter neurones contain substance P or somatostatin. Although these peptides probably have some influence on synaptic transmission in the dorsal horn of the spinal cord, their status as neurotransmitters is uncertain and it is possible that they coexist with conventional neurotransmitters. In addition, the neurones containing identified peptides account for only a fraction of the DRG sensory neurones. There is evidence that the DRG contain catecholamines within fibres thought to be autonomic, but these substances have not been found within the sensory cell bodies themselves. Moreover, the apparently inappropriate, inhibitory physiological effect of catecholamines in the dorsal horn has argued against their being primary sensory neurotransmitter molecules. We have used here antisera against tyrosine hydroxylase (TH; EC 1.14.16.2) and dopamine-beta-hydroxylase (DBH; EC 1.14.17.1), two enzymes specific to catecholaminergic cells, to show that a subpopulation of rat DRG neurones is catecholaminergic and that the neurotransmitter they make is probably dopamine. We believe this to be the first report of catecholaminergic sensory neurones.     

"补肾活血"对老年鼠脑乙酰胆碱代谢的影响

为研究“补肾活血”对老年鼠脑乙酰胆碱(Ach)代谢的影响,将老年大鼠分别用补肾益精、行气活血、补肾活血方剂灌胃30d,采用高效液相色谱技术检测大鼠脑内Ach摩尔质量浓度、分光光度法检测胆碱乙酰转移酶(ChAT)和胆碱酯酶(AChE)的活性．研究发现老年大鼠脑内Ach摩尔质量浓度、ChAT活性显著低于青年鼠,AChE活性显著高于青年鼠;补肾益精、行气活血、补肾活血均可改善老年鼠脑内Ach代谢,补肾活血法明显优于补肾益精法、行气活血法．