Facilitation,and muscarinic and α-adrenergic inhibition of the secretion of 3H-acetylcholine and 3H-noradrenaline from guinea-pig ileum myenteric nerve terminals

The transmitter stores of cholinergic and noradrenergic neurons in guinea-pig ileum myenteric plexus were labelled by preincubation with ³H-choline or ³H-noradrenaline (³H-NA), respectively. Secretion of transmitter was evoked by electrical field stimulation. In the presence (but not in the absence) of eserine (10 μM) the secretion of ³H-acetylcholine (³H-ACh) per shock increased with the frequency of stimulation. Half maximal secretion was obtained at 0.7 Hz (apparent K^Freq_m). Variation in number of shocks per train did not influence the secretory response per shock (at 6 Hz). The secretion of ³H-NA per shock also increased with the frequency of stimulation (K^Freq_m= 1.5 Hz). Comparison of the inhibitory effects of oxotremorine and of exogenous NA, on the secretory responses to stimulation at frequencies close to K^Freq_m, showed that the secretory mechanisms of both neurons are 13–17-fold more sensitive to the inhibitory effect of their ‘own’, than to that of ‘foreign’, agonist. In both types of neuron the inhibitory effects of oxotremorine and of NA were competitively antagonized by atropine and by yohimbine, respectively. Dissociation constants for the respective antagonist were essentially the same, irrespectively of the type of neuron, indicating that the properties of ‘presynaptic’ muscarinic or α-receptors are independent of whether they occur on cholinergic or on noradrenergic terminals.     

Functional reactivation of the deafferented hippocampus by embryonic septal grafts as assessed by measurements of local glucose utilization

Summary Transection of the septo-hippocampal connections through fimbria-fornix damage in the rat results in profound hippocampal cholinergic deafferentation, and, when applied bilaterally, leads to severe and long-lasting impairments in learning and memory. Previous studies have shown that intrahippocampal septal grafts can reestablish a new cholinergic innervation in the inititally denervated hippocampal formation and at least partly compensate for the lesion-induced learning impairments in fimbria-fornix lesioned rats. The purpose of the present study was to determine the magnitude of lesion-induced alterations in cerebral function as reflected in local glucose use measured by (¹⁴C)-2-deoxyglucose (2-DG) autoradiography, and the degree to which this index of functional activity could be normalized following reinnervation from transplants of fetal cerebral tissue from the primordial septal region. Six months after unilateral fimbriafornix transection the rate of glucose utilization was reduced markedly throughout the ipsilateral hippocampus when compared to the intact contralateral side, while in the neocortex only the cingulate cortex showed long-lasting reductions in glucose use. Rats that received a transplant of fetal septal-diagonal band tissue at the time of fimbria-fornix transection, and were sacrificed 6 months later, displayed significantly greater glucose utilization in the ipsilateral hippocampus and cingulate cortex than was measured in these areas in rats with lesion alone. The recovery in glucose use was paralleled by a significant increase in acetylcholinesterase (AChE) staining in several areas of the ipsilateral hippocampal formation and cingulate cortex. This index of graft-induced cholinergic reinnervation was, moreover, significantly correlated with the rate of glucose use. Thus, in the fimbria-fornix transected animals the magnitude of glucose depression correlated with the extent of reduction in AChE staining, and in the grafted animals the degree of normalization of glucose use was correlated with the graft-induced increase in AChE-staining density. These results thus indicate that the 2-DG autoradiographic technique can provide a unique opportunity to map both altered functional activity in localized areas of the brain following specific lesions and the extent to which transplant-derived reinnervation of the host may induce a return to normal functional levels in the target site.ETP and Royal Society (London) visiting fellow     

Continuous depolarization induces choline acetyltransferase activity in septal and hippocampal co-cultured embryonic rat neurons

We tested the influence of continuous high-K+ treatment on acetylcholine (ACh) release and choline acetyltransferase (ChAT) activity on septal cell culture, and septal and hippocampal cell co-culture obtained from rat embryos. Continuous 9 mM K+ treatment did not affect ACh release and ChAT activity in septal culture, but increased ACh release in co-culture without affecting ChAT activity. A slight increase in extracellular K+ concentration, therefore, induced neuronal excitation. Continuous 55 mM K+ treatment increased ACh release in septal culture. This effect was due to direct excitation of septal neurons. In co-culture, 55 mM K+ treatment increased both ACh release and ChAT activity. These results indicate that hippocampal neurons are indispensable for the depolarization-induced increase in ChAT activity in the early stage of developing septal cholinergic neurons.     

Functional and neurochemical cortical cholinergic impairment following neurotoxic lesions of the nucleus basalis magnocellularis in the rat

The effect of kainic and quinolinic acid on cortical cholinergic function was examined following injections of these agents into the nucleus basalis magnocellularis (nbm) or into the frontoparietal cortex. The release of cortical ³H-acetylcholine (³H-ACh), high affinity choline uptake (HACU) and acetylcholinesterase was measured 7 days following injections of saline (control), kainic acid (4.7 nmoles) and quinolinic acid (60, 150 and 300 nmoles) into the nbm. These cortical cholinergic parameters were also examined after injections of saline (control), kainic acid (9.4 nmoles) and quinolinic acid (300 nmoles) into the fronto-parietal cortex. The release of ³H-ACh, HACU and AChE was significantly reduced in animals injected with kainic or quinolinic acid into the nbm. Histological examination of stained sections showed a loss of cell bodies in the region of the nbm and the globus pallidus. The size of the lesion produced by quinolinic acid was proportional to the dose injected into the nbm. In animals injected with kainic acid or quinolinic acid into the cerebral cortex, the release of ³H-ACh, HACU and AChE was not significantly reduced when compared with control animals, although histological examination of stained cortical sections showed a marked loss of cortical neurons. Th results show that quinolinic acid, an endogenous neuroexcitant, produces a deficit of cholinergic function similar to that described in the cortical tissue of patients with senile dementia of Alzheimer's type. The toxic effects of quinolinic acid on cortical cholinergic function are due to its action on cholinergic cell bodies in the nbm. The cortical slice preparation from quinolinic acid-treated animals showing impairment of ³H-ACh release, may be useful in assessing the action of drugs designed to improve cholinergic function.     

Facilitation, and muscarinic and alpha-adrenergic inhibition of the secretion of 3H-acetylcholine and 3H-noradrenaline from guinea-pig ileum myenteric nerve terminals

The transmitter stores of cholinergic and noradrenergic neurons in guinea-pig ileum myenteric plexus were labelled by preincubation with 3H-choline or 3H-noradrenaline (3H-NA), respectively. Secretion of transmitter was evoked by electrical field stimulation. In the presence (but not in the absence) of eserine (10 microM) the secretion of 3H-acetylcholine (3H-ACh) per shock increased with the frequency of stimulation. Half maximal secretion was obtained at 0.7 Hz (apparent Km Freq). Variation in number of shocks per train did not influence the secretory response per shock (at 6 Hz). The secretion of 3H-NA per shock also increased with the frequency of stimulation (Km Freq = 1.5 Hz). Comparison of the inhibitory effects of oxotremorine and of exogenous NA, on the secretory responses to stimulation at frequencies close to Km Freq, showed that the secretory mechanisms of both neurons are 13-17-fold more sensitive to the inhibitory effect of their 'own', than to that of 'foreign', agonist. In both types of neuron the inhibitory effects of oxotremorine and of NA were competitively antagonized by atropine and by yohimbine, respectively. Dissociation constants for the respective antagonist were essentially the same, irrespectively of the type of neuron, indicating that the properties of 'presynaptic' muscarinic or alpha-receptors are independent of whether they occur on cholinergic or on noradrenergic terminals.     

Muscarinic autoreceptor regulates acetylcholine release in rat hippocampus: in vitro evidence

Release of ³H-ACh from isolated nerve endings of rat hippocampus was evoked by incubation in Krebs-Ringer's buffer containing 25 or 35 mM potassium. The release was Ca²⁺-dependent and could be inhibited by Mg²⁺ (20 mM). The muscarinic antagonist, atropine (10^-10–10^-6 M), enhanced ³H-ACh-release. The muscarinic agonist, carbachol (10^-5–10^-3 M) inhibited ³H-ACh release via interaction with muscarinic receptors: this effect could be blocked by atropine (10^-6 M). The presence of the feed-back regulation of ³H-ACh release in a cell-free preparation provides further evidence that the presynaptic regulation is exerted by muscarinic autoreceptors localized on the cholinergic nerve ending itself. The feed back inhibition of the ³H-ACh release does not require the presence of intact neurons or neuronal loops as tetrodotoxin (2.5. 10^-6 M) does not affect the above results.     

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.     

Evaluation of cholinergic markers in Alzheimer's disease and in a model of cholinergic deficit

Cognitive deficits in neuropsychiatric disorders, such as Alzheimer's disease (AD), have been closely related to cholinergic deficits. We have compared different markers of cholinergic function to assess the best biomarker of cognitive deficits associated to cholinergic hypoactivity. In post-mortem frontal cortex from AD patients, acetylcholine (ACh) levels, cholinacetyltransferase (ChAT) and acetylcholinesterase (AChE) activity were all reduced compared to controls. Both ChAT and AChE activity showed a significant correlation with cognitive deficits. In the frontal cortex of rats with a selective cholinergic lesion, all cholinergic parameters measured (ACh levels, ChAT and AChE activities, "in vitro" and "in vivo" basal ACh release) were significantly reduced. AChE activity was associated to ChAT activity, and even more, to "in vivo" and "in vitro" basal ACh release. Quantification of AChE activity is performed by an easy and cheap method and therefore, these results suggest that determination of AChE activity may be used as an effective first step method to evaluate cholinergic deficits.     

Graft-induced behavioral recovery from subcallosal septohippocampal damage in rats depends on maturity stage of donor tissue.

Long-Evans female rats sustained electrolytic lesions of the fimbria and the dorsal fornix and, 10-14 days later, received intrahippocampal suspension grafts of septal-diagonal band tissue from either 14-day-old (Group S14, n = 8) or 16-day-old fetuses (Group S16, n = 10), or of parietal cortex from 16-day-old fetuses (Group Cx, n = 10). Sham-operated (Group S, n = 10) and lesion-only (Group Fifo, n = 21) rats served as non-grafted controls. Spontaneous alternation was assessed in a T-maze at three weeks and two months post-grafting. Home cage and open field activity as well as radial maze learning were assessed from two months post-grafting onwards. Fimbria-fornix lesions induced lasting hyperactivity in both the open field and the home cage, impaired radial maze learning and transiently reduced spontaneous alternation rates. Neither type of graft significantly affected home cage activity. Septal-diagonal band grafts improved open field habituation (within trial decline of ambulatory activity) and radial maze learning; the former was observed only in S16 rats, whereas the latter was observed only in S14 rats. Acetylcholinesterase histochemistry revealed an initial lesion-induced depletion of hippocampal acetylcholinesterase (eight days post-surgery) which was no longer observed at the end of the experiment. Acetylcholinesterase positivity was similar in S14 and S16 grafts, which also contained many choline acetyltransferase-positive neurons. Cortical grafts were found to be almost devoid of acetylcholinesterase positivity and no well-stained choline acetyltransferase-positive neurons could be identified. Septal-diagonal band grafts from 14-day-old fetuses and cortical grafts contained more parvalbumin-positive neurons than septal-diagonal band grafts provided by 16-day-old fetuses. These results suggest that grafts rich in cholinergic neurons may promote behavioral recovery from fimbria-fornix lesion-induced deficits. However, such a recovery may concern different behavioral deficits as a function of the age of the implanted tissue, suggesting that the maturity stage of the donor may critically influence the functional expression in the lesioned recipient. Also, such a recovery does not appear to be related solely to cholinergic hippocampal (re)innervation and might depend on the presence, not only of cholinergic neurons, but also of non-cholinergic neuronal populations, such as parvalbumin-positive (probably GABAergic) neurons.     

Aging of cholinergic synapses in the avian iris. Part I--Biochemical studies

The present experiments were designed to test acetylcholine (ACh) release from cholinergic nerve endings of the chicken iris during aging. Either a one-step K+ stimulated release scheme or a more strenuous two-step depletion-reloading-release scheme were used. First, we found that independent of the scheme of release, as demonstrated by a lower peak release, slower time to peak and a lower rate of increase, the 3-year-old iris released less 3H-ACh than the 4-month-old iris. In order to analyze this effect of aging, we determined five basic parameters of ACh metabolism, including total acetylcholine (ACh) and choline (Ch) levels, 3H-ACh, 3H-Ch and 3H-phosphorylcholine (PhCh) formed from exogenous 3H-Ch, for each experimental type and for the two ages. There appear to be major differences in the handling of both the total Ch and newly labelled (3H-Ch) pools between the two ages. In addition, the homeostatic control system for Ch levels in the older tissue appears to be less efficient and incapable of handling strenuous release tests. This defect of the older tissue to preserve its Ch pool may, in part, be responsible for the drastic decrease in ACh levels seen in the older iris. Together with the demonstrated reduction in neurotransmitter level and in uptake of Ch, the reduction in release demonstrated here forms a characteristic triade of age-related defects which may have important implications for our understanding of aging processes in cholinergic terminals.     

Selective cholinergic denervation, independent from oxidative stress, in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is characterized by increases in amyloid-beta (Abeta) peptides, neurofibrillary tangles, oxidative stress and cholinergic deficits. However, the selectivity of these deficits and their relation with the Abeta pathology or oxidative stress remain unclear. We therefore investigated amyloidosis-related changes in acetylcholine (ACh) and serotonin (5-HT) innervations of hippocampus and parietal cortex by quantitative choline acetyltransferase (ChAT) and 5-HT immunocytochemistry, in 6, 12/14 and 18 month-old transgenic mice carrying familial AD-linked mutations (hAPP(Sw,Ind)). Further, using manganese superoxide dismutase (MnSOD) and nitrotyrosine immunoreactivity as markers, we evaluated the relationship between oxidative stress and the ACh deficit in 18 month-old mice. Thioflavin-positive Abeta plaques were seen in both regions at all ages; they were more numerous in hippocampus and increased in number (>15-fold) and size as a function of age. A majority of plaques exhibited or were surrounded by increased MnSOD immunoreactivity, and dystrophic ACh or 5-HT axons were seen in their immediate vicinity. Counts of immunoreactive axon varicosities revealed significant decreases in ACh innervation, with a sparing of the 5-HT, even in aged mice. First apparent in hippocampus, the loss of ACh terminals was in the order of 20% at 12/14 months, and not significantly greater (26%) at 18 months. In parietal cortex, the ACh denervation was significant at 18 months only, averaging 24% across the different layers. Despite increased perivascular MnSOD immunoreactivity, there was no evidence of dystrophic ACh varicosities or their accentuated loss in the perivascular area. Moreover, there was virtually no sign of tyrosine nitration in ChAT nerve terminals or neuronal cell bodies. These data suggest that aggregated Abeta exerts an early, non-selective and focal neurotoxic effect on both ACh and 5-HT axons, but that a selective, plaque- and oxidative stress-independent diffuse cholinotoxicity, most likely caused by soluble Abeta assemblies, is responsible for the hippocampal and cortical ACh denervation.     

A comparison of behavioural effects and morphological features of grafts rich in cholinergic neurons placed in two sites of the denervated rat hippocampus

This study compared the morphological characteristics and the behavioural effects of intrahippocampal septal cell suspension grafts injected either just above the pyramidal cell layer of the hippocampal region CA1 or within the dorsal leaf of the dentate gyrus (DG) in rats subjected to electrolytic fimbria-fornix lesions. The behavioural tests determined home-cage and open-field activity, as well as radial-maze performance. Cresyl-violet staining, acetylcholinesterase (AChE) histochemistry, and parvalbumin, glial fibrillary acidic protein and glutamic acid decarboxylase immunocytochemistry were used for morphological assessments. The cross-sectional area of the grafts was measured between 0.8 mm and 5.3 mm posterior to Bregma and used as an index of their development. Whether injected into CA1 or DG, the grafts provided the partially denervated hippocampus with a dense AChE-positive reinnervation. Both types of grafts were devoid of reactive astrocytes (although reactive astrocytes were found close to the graft-host interface), contained almost no parvalbumin-positive neurons and showed a high density of GAD-positive terminals. One of the main differences between the two groups of grafted rats was that the suspension injected into the DG yielded grafts that, in the vicinity of the injection sites (between 2.3 mm and 4.3 mm posterior to Bregma), had a cross ectional area exceeding that of the grafts placed into CA1 by about 63–110% (average 79%), the latter being more dispersed than the former in the coronal plane. In addition, rats with grafts in the DG exhibited granule cell degeneration in the vicinity of the injection sites, whereas rats with grafts in region CA1 showed no damage near the injection sites. Concerning the behavioural data, we found that fimbria-fornix lesions induced hyperactivity in both the home cage and the open field and impaired radial-maze performance. Compared with the lesion-only rats, the grafted rats in both groups had further increased open-field and home-cage activity. While the grafts placed into region CA1 slightly, but significantly, accentuated the lesion-induced deficit in radial-maze performance, those placed into the DG had no effect. These results suggest that intrahippocampal grafts may, in some (still unspecified) conditions, produce adverse behavioural effects or no behavioural effects, despite an acceptable graft-induced cholinergic reinnervation of the hippocampus. They do not allow a clear answer to the question of whether intra-DG and intra-CA1 septal suspension grafts exhibiting almost comparable morphological features (except in their size and their dispersion in the vicinity of the injection sites) induce behavioural effects that would depend on intrahippocampal location of the grafts. They suggest, however, that the granule cell degeneration caused by the implantation procedure, in conjunction with the intragyral development of the graft, probably does not account for some of the reported adverse behavioural effects of intrahippocampal basal forebrain grafts. Finally, the finding that septal cell suspensions placed into the DG yielded larger grafts than when an equivalent number of cells was injected into CA1 might be explained by a larger lesion-induced neurotrophic activity in DG than in region CA1, although both regions had undergone a similar degree of cholinergic denervation.     

Asynaptic feature and heterogeneous distribution of the cholinergic innervation of the globus pallidus in primates

The internal (GPi) and external (GPe) segments of the primate globus pallidus receive a significant cholinergic (ACh) innervation from the brainstem pedunculopontine tegmental nucleus. The present immunohistochemical study describes this innervation in the squirrel monkey (Saimiri sciureus), as visualized with an antibody raised against choline acetyltransferase (ChAT). At the light microscopic level, unbiased stereological quantification of ChAT positive (+) axon varicosities reveals a significantly lower density of innervation in GPi (0.26 ± 0.03 × 10⁶) than in GPe (0.47 ± 0.07 × 10⁶ varicosities/mm³ of tissue), with the anterior half of both segments more densely innervated than the posterior half. Neuronal density of GPi (3.00 ± 0.13 × 10³ neurons/mm³) and GPe (3.62 ± 0.22 × 10³ neurons/mm³) yields a mean ratio of ChAT+ axon varicosities per pallidal neuron of 74 ± 10 in the GPi and 128 ± 28 in the GPe. At the electron microscopic level, the pallidal ChAT+ axon varicosities are significantly smaller than their unlabeled counterparts, but are comparable in size and shape in the two pallidal segments. Only a minority of ChAT+ varicosities displays a synaptic specialization (12 % in the GPi and 17 % in the GPe); these scarce synaptic contacts are mostly of the symmetrical type and occur exclusively on pallidal dendrites. No ChAT+ axo-axonic synaptic contacts are observed, suggesting that ACh exerts its modulatory action on pallidal afferents through diffuse transmission, whereas pallidal neurons may be influenced by both volumic and synaptic delivery of ACh.     

Pre- and postsynaptic actions of nifedipine at an identified cholinergic central synapse of Aplysia

The effects of the dihydropyridine (DHP) Ca²⁺ channel antagonist, nifedipine, were studied on the cholinergic synapse between the presynaptic neurones B4/B5 and the postsynaptic neurones B3/B6 located in the buccal ganglion of Aplysia californica. Nifedipine (10 M) decreased the presynaptic Ca²⁺ current by 30%–40%. Blockade of DHP-sensitive Ca²⁺ channels, however, did not affect quantal transmitter release from the presynaptic neurones. Thus, at this synapse, DHP-sensitive Ca²⁺ channels appear not to be involved in acetylcholine (ACh) release. The postsynaptic response to an ionophoretic application of ACh was decreased by nifedipine, pointing to a blocking action of the drug on the postsynaptic receptor/channel complex. Nifedipine was also found to activate protein kinase C, which in turn induces an increase in the nifedipine-resistant presynaptic Ca²⁺ influx and in the number of released ACh quanta. These effects of nifedipine could be prevented by a previous application of 1, 5-(isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a protein kinase blocker.     

Estrogen inhibition of noradrenaline release in the rabbit oviduct1

In the present study, we investigated the influence of estrogen on ³H-noradrenaline (³H-NA) release induced in the oviductal isthmus by electrical stimulation, potassium and calcium. The fractional release of ³H-NA was measured in oviducts isolated from ovariectomized rabbits and from ovariectomized rabbits treated with estradiol cypionate, 70 μg/kg im 72 h before an experiment. Electrical field stimulation of the intramural nerves induced muscle contraction and augmented the release of labelled NA from the muscle. The ³H-NA release was reduced after estrogen treatment when reuptake of NA into the nerve terminals was blocked by desipramine, 10^-6 M. Estrogen also reduced the ³H-NA release evoked by exposure of the oviducts to 121 mM KCI in the presence of calcium (2.5 mM) and in a high potassium, calcium-free medium upon the addition of 2.5 mM calcium. In the presence of desipramine a small fraction of ³H-NA was released in high potassium, calcium-free medium. This release was unaffected by estrogen. These results suggest that estrogen reduces the release of NA from the adrenergic nerves within the oviduct and that this action is exerted primarily on the calcium-dependent release. It therefore might be due to a reduction in the entry of calcium into the nerve terminal.     

Spatial learning and memory following fimbria-fornix transection and grafting of fetal septal neurons to the hippocampus

Summary The ability of intrahippocampal grafts of fetal septal-diagonal band tissue, rich in developing cholinergic neurons, to ameliorate cognitive impairments induced by bilateral fimbria-fornix transections in rats was examined in three experiments using the Morris water-maze to test different aspects of spatial memory. Experiment 1. Rats with fimbria-fornix lesions received either septal cell suspension grafts or solid septal grafts; normal rats and rats with lesions alone were used as controls. Sixteen weeks after surgery, the rats' spatial learning and memory were tested in the water-maze using a place test, designed to investigate place navigation performance, in which rats learned to escape from the water by swimming to a platform hidden beneath the water's surface. After 5 days of training, the rats were given a spatial probe test in which the platform was removed from the tank to test spatial reference memory. Experiment 2. The same rats used in Exp. 1 were tested in a delayed-match-to-sample, working memory version of the water-maze task. The platform was located in one of two possible locations during each trial, which was composed of 2 swims. If the rat remembered the location of the platform on the 2nd swim of a trial, it should find the platform more quickly on that swim, and thereby demonstrate working memory. Experiment 3. Prior to receiving fimbria-fornix lesions, normal rats were trained in a modification of the water-maze task using alternating cue navigation and place navigation trials (i.e., with visible or non-visible escape platforms). The retention and reacquisition of the place task and the spatial probe test were examined in repeated tests up to 6 months after the lesion and intrahippocampal grafting of septal cell suspensions. The effects of central muscarinic cholinergic receptor blockade with atropine were also tested. Normal rats performed well in both the place and spatial probe tests. In contrast, rats with fimbria-fornix lesions only were unable to acquire or retain spatial information in any test. Instead, these rats adopted a random, nonspatial search strategy, whereby their latencies to find the platform decreased in the place navigation tasks. Sixty to 80% of the rats with septal suspension or solid grafts had recovered place navigation, i.e., the ability to locate the platform site in the tank, in Exp. 1 and 3, and they showed a significantly improved performance in the working memory test in Exp. 2. Atropine abolished the recovered place navigation in the grafted rats, whereas normal rats were impaired to a lesser extent. In contrast, atropine had no effect on the non-spatial strategy adopted by rats with fimbria-fornix lesions only. The results show that: (1) fimbria-fornix lesions disrupt spatial learning and memory in both naive and pretrained rats; (2) with extended training the fimbria-fornix lesioned rats develop an efficient non-spatial strategy, which enables them to reduce their escape latency to levels close to those of intact controls; (3) intrahippocampal septal grafts can restore the ability of the lesioned rats to use spatial cues in the localization of the platform site; and (4) the behavioural recovery produced by grafts is dependent upon an atropine sensitive mechanism.     

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.     

Modification of adenosine modulation of acetylcholine release in the hippocampus of aged rats

Adenosine is a neuromodulator acting through inhibitory A₁ receptors (A₁Rs) and facilitatory A_2ARs. Since A_2AR antagonists attenuate memory deficits in aged animals and memory deficits might involve a decreased cholinergic function, we investigated how aging affects the density and function of adenosine receptors in rat hippocampal cholinergic terminals. In young adult (2 months) rats, 64 and 36% of cholinergic terminals (immunopositive for vesicular ACh transporters) possessed A₁Rs and A_2ARs, respectively. In aged (24 months) rats, the percentage of cholinergic terminals with A₁Rs was preserved, whereas that with A_2ARs was larger (49%). In young adults adenosine only tonically inhibited ACh release through A₁Rs, whereas in aged rats there was a greater A₁R-mediated inhibition and a simultaneous A_2AR-mediated facilitation of ACh release. Thus, the enhanced A_2AR density and facilitation compensates for the greater tonic A₁R modulation, preserving the global adenosine modulation of ACh release in aged rats. Furthermore, since A_2AR antagonists inhibit ACh release, the beneficial effects of A_2AR antagonists on memory in aged rats might not result from ACh release modulation.     

Both presynaptic nicotinic-like and muscarinic-like autoreceptors regulate acetylcholine release at an identified neuro-neuronal synapse ofAplysia

The possible involvement of cholinergic presynaptic receptors regulating evoked quantal acetylcholine (ACh) release was investigated at an identified cholinergic neuroneuronal synapse in the buccal ganglion ofAplysia, using cholinergic agonists (carbachol, pilocarpine, oxotremorine) and/or antagonists (curare, atropine, hexamethonium). Bath applied carbachol or pilocarpine (10^–8 M to 10^–4 M) induced a decrease in the evoked quantal release of ACh. As the effects of carbachol were prevented by atropine (5 · 10^–6 M) and not by curare (10^–5 M), it was concluded that carbachol activated presynaptic muscarinic-like receptors implicated in a negative feed-back on ACh release. On the contrary, oxotremorine (up to 10^–4 M) induced a potentiation of ACh release which was suppressed by curare (4 · 10^–6 M) or hexamethonium (10^–5 M) but not by atropine (5 · 10^–6 M) pointing to the activation of presynaptic nicotinic-like receptors implicated in a positive feed-back on ACh release. Moreover, in the presence of curare, oxotremorine decreased ACh release: this suggested that oxotremorine also activated the presynaptic muscarinic-like receptors. These results revealed the conjoint presence, on the same terminal, of both muscarinic-like and nicotinic-like autoreceptors.     

The turnover of acetylcholine in ligated sciatic nerves of the rat

The accumulation of acetylcholine (ACh) and choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities proximal to a crush on rat sciatic nerves was investigated after superfusion of the nerves in situ with or without inhibitors of ACh synthesis and/or AChE. 9 h after crushing of nerves, the ACh-content of the 5 mm segment of nerve immediately proximal to the crush was increased from 37±5 to 80±4 pmol (mean ± SE), while ChAT-activity was increased to 112±10% and AChE-activity to 198±19% over that in non-ligated nerves. Superfusion of the nerves for 8 h with Krebs' bicarbonate medium had no effect on enzyme accumulations, but reduced the ACh content to 59±4 pmol. The presence of hemicholinium 3 (HC-3) (2×10^-5 M) in the superfusion medium reduced the ACh content markedly (to 17±2 pmol), but had no effect on enzyme accumulations at the crush. Adding eserine (10^-5 M) or soman (10^-6 M) to the superfusion medium increased ACh content to 133±8 pmol and 101±8 pmol, respectively, and markedly reduced AChE-activity; ChAT activity was not effected. Superfusion with a combination of HC-3 and eserine caused a marked reduction in ACh content compared with eserine alone; the effect was less with soman. The results are consistent with the view that the ACh which accumulates proximal to crush exists in a protective organelle, but that there is a continuous turnover of ACh due to leakage of ACh from the organelle, both during axonal transport and after accumulation.