The central release of acetylcholine during stimulation of the visual pathway

1. In rabbits anaesthetized with Dial ACh has been collected from the surface of the cerebral cortex during stimulation of the visual pathways.2. The spontaneous release of ACh from the visual and non-visual areas of the cortex was found to be similar.3. Stimulation of the retinae by diffuse light produced a large increase in ACh release from the primary visual receiving areas (4.3 times the spontaneous release) and a smaller increase (1.9 times the spontaneous release) from other parts of the cortex.4. Direct unilateral electrical stimulation of the lateral geniculate body evoked a large increase in ACh release (3.4 times the spontaneous release) from the ipsilateral visual cortex and a smaller increase (1.7 times the spontaneous release) from the contralateral visual area and other regions of the cerebral cortex. The evoked increase from the contralateral cortex was not mediated by transcallosal pathways.5. The increase in ACh release evoked from the visual cortex by stimulation of the ipsilateral lateral geniculate body was dependent on the frequency of stimulation. The evoked release was smallest at low stimulus frequencies and increased to a maximum at 20 stimuli/sec.The evoked ACh release from other areas of the cortex was independent of the frequency at which the lateral geniculate body was stimulated.6. The possible central nervous pathways associated with the spontaneous release of ACh and the release evoked by stimulation of the eyes by light and by direct stimulation of the lateral geniculate body are discussed.7. It is concluded that two ascending cholinergic systems may be involved; the non-specific reticulo-cortical pathways responsible for the e.e.g arousal response, and the more specific thalamo-cortical pathways associated with augmenting and repetitive after-discharge responses. The first system is thought to be concerned with the small but widespread increase in ACh release from the cortex following stimulation of the visual pathway while the second system could give rise to the larger increases evoked from the primary receiving areas of cortex. The spontaneous release of ACh from the surface of the brain may be the result of contributions from both systems.     

The central release of acetylcholine during consciousness and after brain lesions

1. Acetylcholine (ACh) has been collected from the visual cortex of anaesthetized rabbits during stimulation of the lateral geniculate body and after cutting central nervous pathways. ACh has also been collected from the visual cortex of conscious, free-moving rabbits.2. After a unilateral ;vertical' lesion separating the geniculate body from more centrally situated nuclei, ACh release evoked from the contralateral cortex by geniculate body stimulation was abolished but evoked release from the ipsilateral cortex was only reduced.3. After a bilateral, ;horizontal' lesion separating the thalamic nuclei from the reticular formation, unilateral geniculate stimulation gave an increased ACh release from the ipsilateral but not from the contralateral visual cortex.4. The ;vertical' and ;horizontal' lesions had no permanent effect on the spontaneous release of ACh from the visual cortex.5. Unilateral destruction of the geniculate body reduced the spontaneous release of ACh from the ipsilateral cortex but did not affect the contralateral release.6. The spontaneous and directly evoked ACh release from chronically undercut areas of cortex was found to be considerably lower than from intact areas of cortex.7. A high output of ACh was obtained from the visual cortex of conscious, free-moving rabbits. The rate of ACh release was closely related to the activity and state of arousal of the animals.8. These results support an earlier suggestion that two major ascending cholinergic systems exist in the rabbit brain. One pathway is the non-specific reticulo-cortical tract responsible for cortical arousal and the other is the more specific thalamo-cortical pathway associated with augmenting and repetitive after-discharge responses. The functional significance of these two cholinergic pathways and their role in the conscious animal are discussed.     

Cholinergic mechanisms in the caudate nucleus

1. Acetylcholine (ACh), and a number of cholinomimetic and cholinolytic agents, have been applied to single neurones of the caudate nucleus, and the effects compared with those produced by stimulation of nucleus ventralis anterior thalami (VA).2. Neurones responding by excitation following ACh and VA stimulation, and others depressed by both ACh and VA stimulation have been observed. The two types of cell are located in different regions of the nucleus.3. Both types of ACh effect, and the responses to VA stimulation, are prevented by atropine.4. Taken with the earlier observation that the release of ACh from the caudate can be enhanced by VA stimulation, it has been suggested that the present results indicate a final cholinergic link in the pathway from VA to caudate.     

Hypercapnia and acetylcholine release from the cerebral cortex and medulla

1. The cerebral cortex and medulla of fifty-eight anaesthetized dogs released ACh spontaneously through push-pull cannulae after perfusion with the anticholinesterase, sarin. Hypercapnia (12% CO(2)) evoked a significant release of ACh above the basic spontaneous level, from the medullary and cortical areas. Hypercapnia + hypoxia (12% CO(2) + 8% O(2)), in combination, produced an ACh release comparable to hypercapnia; hypoxia (8% O(2)) had no effect in any region.2. Areas in the medullary reticular formation responsive to injections of CO(2)-bicarbonate solutions (;respiratory responsive areas') produced a significant increase of ACh after exposure to hypercapnia or hypercapnia + hypoxia, over that obtained from either the ;non-respiratory responsive areas' of the medulla or the cerebral cortex.3. The evidence supports the concept that ACh may participate as a neurotransmitter within the cerebral cortex and medulla. Also the results would suggest but do not prove, that a cholinergic factor may be a component in respiratory control under certain circumstances, such as exposure to hypercapnia.     

Ionotropic glutamate receptors regulating labeled acetylcholine release from rat striatal tissue in vitro: possible involvement of receptor modulation in magnesium sensitivity

This study evaluated the role of glutamate ionotropic receptors on the control of [3H]acetylcholine ([3H]ACh) release by the intrinsic striatal cholinergic cells. [3H]-choline previously taken up by chopped striatal tissue and converted to [3H]ACh, was released under stimulation by glutamate, N-methyl-d-aspartate (NMDA), kainate and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Experiments were conducted in the absence of choline uptake inhibitors or acetylcholinesterase inhibitors. A paradigm of two stimulations was employed, the first in control conditions and the second after 9 min of perfusion with the test agents MK-801, 2-amino-5-phosphonopentanoic acid (AP-5), tetrodotoxin (TTX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]quinoxaline-7-sulfonamide (NBQX), glycine and magnesium. Our results support that (1) in the absence of Mg2+, NMDA is the most effective agonist to stimulate [3H]ACh release from striatal slices (2) magnesium effectively antagonized kainate and AMPA stimulation suggesting that at least part of the kainate and AMPA effects might be attributed to glutamate release (3) besides NMDA, kainate receptors showed a more direct involvement in [3H]ACh release control based on the smaller dependence on Mg2+ and less inhibition by TTX and (4) stimulation of ionotropic glutamate receptors may induce long lasting biochemical changes in receptor/ion channel function since the effects of TTX and/or Mg2+ ions on [3H]ACh release were modified by previous exposure of the tissue to agonists.     

Cholinergic and non-cholinergic transmission in the medial geniculate nucleus of the cat

1. Studies involving the electrophoretic administration of antagonists of ACh (atropine, DHbetaE) and cholinesterase inhibitors (neostigmine, physostigmine) to MGN neurones indicate that ACh is an excitatory transmitter in the feline MGN, most probably released from fibres which originate in or traverse the mesencephalon.2. Auditory afferents to the MGN, cortico-geniculate fibres and the excitatory fibres which mediate ;spontaneous' firing of MGN neurones are unlikely to be cholinergic.3. Almost all geniculo-cortical relay cells are excited by ACh, this excitation being mediated by receptors which have both muscarinic and nicotinic properties. The excitation of relay cells by ACh is sometimes preceded or followed by a depression of firing which is resistant to atropine and DHbetaE, but the significance of this depression is unknown.4. The firing of many unidentified MGN neurones is depressed by ACh in the absence of any excitation, and this depression is blocked by both atropine and DHbetaE, and potentiated by anticholinesterases. This type of depression by ACh may be related to cholinergic inhibition, but this possibility has yet to be investigated.     

Inactivation of prefrontal cortex abolishes cortical acetylcholine release evoked by sensory or sensory pathway stimulation in the rat

Sensory stimulation and electrical stimulation of sensory pathways evoke an increase in acetylcholine release from the corresponding cortical areas. The pathways by which such sensory information reaches the cholinergic neurons of the basal forebrain that are responsible for this release are unclear, but have been hypothesized to pass through the prefrontal cortex (PFC). This hypothesis was tested in urethane-anesthetized rats using microdialysis to collect acetylcholine from somatosensory, visual, or auditory cortex, before and after the PFC was inactivated by local microdialysis delivery of the GABA-A receptor agonist muscimol (0.2% for 10 min at 2 microl/min). Before PFC inactivation, peripheral sensory stimulation and ventral posterolateral thalamic stimulation evoked 60 and 105% increases, respectively, in acetylcholine release from somatosensory cortex. Stimulation of the lateral geniculate nucleus evoked a 57% increase in acetylcholine release from visual cortex and stimulation of the medial geniculate nucleus evoked a 72% increase from auditory cortex. Muscimol delivery to the PFC completely abolished each of these evoked increases (overall mean change from baseline = -7%). In addition, the spontaneous level of acetylcholine release in somatosensory, visual, and auditory cortices was reduced by 15-59% following PFC inactivation, suggesting that PFC activity has a tonic facilitatory influence on the basal forebrain cholinergic neurons. These experiments demonstrate that the PFC is necessary for sensory pathway evoked cortical ACh release and strongly support the proposed sensory cortex-to-PFC-to-basal forebrain circuit for each of these modalities.     

Responses of identified spinal neurones to acetylcholine applied by micro-electrophoresis.

1. The responses of identified cells in the cat Clarke's column and dorsal horn to micro-electrophoretically applied cholinomimetics and anti-cholinergic substances have been investigated. 2. Both antidromically identified (DSCT neurones) and synaptically activated neurones from the region of the Clarke's column of the spinal cord were excited by ACh. However, the proportion of ACh excited cells was greater in units synaptically activated by ipsilateral dorsolateral funiculus stimulation (78%) than in DSCT neurones (50%). In addition, about 55% of neurones activated either antidromically or synaptically by ipsilateral dorsal column stimulation were excited by ACh. 3. In contrast to a relatively weak excitatory potency on the DSCT neurones (maximum firing frequency did not exceed 130% of the control activated by ipsilateral dorsolateral funiculus stimulation (maximum firing frequency reached 430% of the control level). 4. ACh has a relatively quick and rapidly reversible excitatory effect on Clarke's column neurones and some types of dorsal horn interneurones, which can be obtained also with nicotine. However, the action of nicotine is frequently delayed in onset and recovery. This excitatory action of ACh can be blocked or markedly depressed by dihydro-beta-erythroidine. These results and those obtained with acetyl-beta-methylcholine and atropine seem to suggest that the receptors mediating excitation of the cholinoceptive spinal cells activated either antidromically or synaptically by ipsilateral dorsolateral funiculus stimulation besides predominantly nicotinic have also weak muscarinic properties. 5. Desensitization with repeated applications of ACh and nicotine has been observed in both DSCT neurones and units antidromically activated by ipsilateral dorsal column stimulation. 6. About 11% of units antidromically activated by ipsilateral dorsolateral funiculus stimulation were depressed by ACh. In addition, the depressant effect of ACh was more frequently encountered in the cells unresponsive either to the dorsolateral funiculus or dorsal column stimulation. ACh depression was also seen in units activated either antidromically or synaptically by ipsilateral dorsal column stimulation. In contrast, none of the units synaptically activated by the ipsilateral dorsolateral funiculus stimulation were depressed by ACh. The same was true for spinal neurones receiving convergent peripheral inputs activated either antidromically or synaptically by ipsilateral dorsolateral or dorsal column stimulation. 7. The findings that ACh depression of all tested DSCT neurones is blocked by atropine and readily evoked by acetyl-beta-methylcholine indicates that receptors mediating the effect are of muscarinic type.     

Effects of cholinergic and adrenergic nerve stimulations on amylase release from superfused small segment of rat parotid gland

The effects of the cholinergic and adrenergic nerve stimulations on amylase release from the segment isolated from the rat parotid gland were investigated, employing the combined techniques of electrical field stimulation (FS) and tyramine application with automated fluorescence method for measuring amylase. The maximum amylase release in response to FS for a short period (1 min) was attained at 16 Hz frequency, 2 ms pulse width and 8 V strength stimulation in the absence of any autonomic antagonist. Periodic short-lasting FS using these parameters at intervals of about 15 min could reproduce similar sizes of amylase release for about 2 h. Continuous long-lasting FS (3 V, 2 ms, 16 Hz) caused a transient sharp increase in amylase release followed by a sustained one. The FS-evoked amylase release was completely abolished by tetrodotoxin (TTX) (10(-7) g/ml) and markedly reduced by atropine (7 X 10(-6) M) or by propranolol (10(-5) M), while it was scarcely affected by hexamethonium (3 X 10(-4) M) and phentolamine (10(-5) M). The maximum stimulus frequency of short-lasting FS for amylase release in the presence of propranolol was similar to that (16 Hz) in the control, but it was higher (32 Hz) in the presence of atropine. Reduced response in amylase release to FS by propranolol was completely restored by the superimposed addition of dibutyryl cyclic AMP (10(-4) M). Application of tyramine (5 X 10(-4) M) evoked amylase release in the presence of atropine, which was blocked mostly by propranolol and partly by phentolamine, while tyramine was ineffective in the tissue segment from rats pretreated with 6-hydroxydopamine. From these results the following were suggested; that the intrinsic cholinergic neurotransmitter activates a muscarinic receptor of the acinar cell, while the adrenergic neurotransmitter stimulates mainly a beta-adrenergic and partly an alpha-adrenergic receptor, resulting in amylase release.     

Atropine-resistant longitudinal muscle spasms due to excitation of non-cholinergic neurones in Auerbach's plexus

1. In accordance with the dual histology of Auerbach's plexus (Dogiel, 1899; Hill, 1927) two types of neurone can be shown to be humorally active in plexus-containing preparations of longitudinal muscle from guinea-pig ileum, taken at measured distances up to 95 cm above the ileocaecal valve, when such preparations are stimulated electrically under different conditions.2. The rapid twitch, lasting 3-8 sec, which is elicited by single shocks of 0.1 or 0.2 msec pulse width, and the effect of 5-15 ng doses of acetylcholine which matched this twitch, were both extinguished equally effectively and completely by atropine sulphate (0.4-1 x 10(-8) g/ml.) or by hyoscine hydrobromide. This twitch-response is therefore caused by an excitation of cholinergic motor neurones of normal susceptibility to atropine. These are believed to be the Dogiel (1899) Type II cells of Auerbach's plexus, as suggested by Hill (1927).3. After extinction of the twitch by the invariably effective atropineblock, a second type of muscle response was revealed by tetanic stimulation with 1 sec trains of 50 pulses of the same voltage and of pulse width preferably 0.2 msec. The tetanic responses consisted of spasms of longer delay and duration (20-60 sec). These spasms could be matched by doses of acetylcholine of the order of 200 ng. However, if the atropine concentration was now raised to 10(-7), or even 10(-6) g/ml., the effect of 200-1000 ng of acetylcholine was abolished, but the tetanic spasms persisted without decrease in amplitude. In other experiments the height of the spasms remained constant as the concentration of atropine sulphate was raised from 10(-8) to 10(-6) g/ml. and was only slightly decreased by 10(-5) g/ml. Hence, these tetanic contractions are not due to a surmounting of the atropine-block by the increased release of acetylcholine following the 50 pulses.4. The tetanic spasms originate from excitation of non-cholinergic neurones, perhaps the associative Dogiel Type I cells of Auerbach's plexus (Hill, 1927), since the spasms were abolished reversibly by tetrodotoxin 2 x 10(-7) g/ml. and were absent from plexus-free, nicotine-insensitive preparations of the longitudinal muscle, both before and after atropinization.5. The tetanic spasms were not reduced by ganglion-block with paralysing doses of nicotine, with (+)-tubocurarine or with hexamethonium.6. The tetanic spasms are not mediated by a release of 5-hydroxytryptamine (5-HT) or of histamine, since they persisted in concentrations of methysergide and mepyramine adequate to block matching doses of histamine or 5-HT, or multiples thereof. Catecholamines were also excluded.7. The tetanic spasms are not mediated by a release of a prostaglandin, because they were not reduced by 0.5-2 x 10(-6) g/ml. of patulin (Ambache, 1957), which blocked the contractions evoked by matching doses of prostaglandins PGE(2) or PGF(2alpha); even after this block, PGE(2) still potentiated subsequent tetanic responses.8. The tetanic spasms were reduced or virtually abolished by strychnine in concentrations which did not depress the twitch.     

Quantal transmitter release by glioma cells: quantification of intramembrane particle changes

Glial cells in situ are able to release neurotransmitters such as glutamate or acetylcholine (ACh). Glioma C6BU-1 cells were used to determine whether the mechanisms of ACh release by a glial cell line are similar or not to quantal release from neurones. Individual C6BU-1 cells, pre-filled with ACh, were moved into contact with a Xenopus myocyte that was used as a real-time ACh detector. Upon electrical stimulation, C6BU-1 cells generated evoked ACh impulses which were Ca(2+)-dependent and quantal (quantal steps of ca. 100 pA). Changes in plasma membrane ultrastructure were investigated by using a freeze-fracture technique designed for obtaining large and flat replicas from monolayer cell cultures. A transient increase in the density of medium and large size intramembrane particles--and a corresponding decrease of small particles--occurred in the plasma membrane of C6BU-1 cells stimulated for ACh release. Changes in interaction forces between adjacent medium and large particles were investigated by computing the radial distribution function and the interaction potential. In resting cells, the radial distribution function revealed a significant increase in the probability to find two particles separated by an interval of 24 nm; the interaction potential suggested repulsive forces for intervals shorter than 24 nm and attractive forces between 24 and 26 nm. In stimulated cells, this interaction was displaced to 21 nm and made weaker, despite of the fact that the overall particle density increased. The nature of this transient change in intramembrane particles is discussed, particularly with regard to the mediatophore proteolipid which is abundant in the membranes C6-BU-1 like in those of cholinergic neurones. In conclusion, evoked ACh release from pre-filled C6-BU-1 glioma cells is quantal and Ca(2+)-dependent. It is accompanied by a transient changes in the size distribution and the organisation of intramembrane particles in the plasma membrane. Thus, for the release characteristics, glioma cells do not differ fundamentally from neurones.     

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.     

Cortical acetylcholine release and electroencephalographic arousal

1. In cats anaesthetized with N₂O-halothane acetylcholine (ACh) release from the parietal cortex was measured. In addition, low and high-frequency electroencephalographic (e.e.g.) activity was recorded quantitatively.

2. Stimulation of the mesencephalic reticular formation at 30, 60 and 100/sec produced an identical increase in cortical ACh output, while 300/sec stimulation was about ⅓ as effective and 10/sec stimulation failed to increase ACh output.

3. Reticular formation stimulation at 60 and 100/sec reduced the low-frequency and increased the high-frequency e.e.g. activity. Stimulation at 30 and 300/sec was less effective, while 10/sec stimulation had no effect on e.e.g.

4. Acute undercutting of the cortex did not affect the resting output of ACh but greatly reduced the increase due to reticular formation stimulation as compared to the contralateral intact side. Cutting the cortex around the collection area did not affect the increase in ACh output due to reticular formation stimulation.

5. Stimulation of the hypothalamus, medial thalamus and septum at 100/sec also increased cortical ACh output while stimulation of the dorsal hippocampus and caudate nucleus failed to do so.

6. Low frequency cortical e.e.g. activity was reduced by stimulating the reticular formation, the hypothalamus, the medial thalamus and slightly by septum stimulation. High-frequency e.e.g. activity was increased by stimulating the reticular formation and the hypothalamus.

7. It is concluded that the ACh measured originates from the neural tissue underlying the collection area. The increased release is concomitant to e.e.g. activation but the pathways involved in cortical e.e.g. activation and increased ACh release are distinct, since the two phenomena do not vary in a parallel fashion when the reticular formation is stimulated at different frequencies or when different subcortical areas are stimulated.

8. The effectiveness of septal stimulation in increasing ACh release indicates that at least part of the cortical cholinergic fibres traverse this area on their way to the cortex.

     

Brain stem stimulation and the acetylcholine-evoked inhibition of neurones in the feline nucleus reticularis thalami

1. In cats anaesthetized with halothane and nitrous oxide, the responses to iontophoretically applied acetylcholine (ACh) and to high-frequency stimulation of the mid-brain reticular formation (MRF) were tested on spontaneously active neurones in the nucleus reticularis thalami and underlying ventrobasal complex.2. The initial response to MRF stimulation of 90% of the ACh-inhibited neurones found in the region of the dorsolateral nucleus reticularis was an inhibition. Conversely, the initial response of 82% of the ACh-excited neurones in the ventrobasal complex was an excitation. Neurones in the rostral pole of the nucleus reticularis were inhibited by both ACh and RMF stimulation.3. The mean latency (and s.e. of mean) for the MRF-evoked inhibition was 13·7 ± 3·2 ms (n = 42) and that for the MRF-evoked excitation, 44.1 ± 4.2 ms (n = 35).4. The ACh-evoked inhibitions were blocked by iontophoretic atropine, in doses that did not block amino acid-evoked inhibition. In twenty-four ACh-inhibited neurones the effect of iontophoretic atropine was tested on MRF-evoked inhibition. In all twenty-four neurones atropine had no effect on the early phase of MRF-evoked inhibition but weakly antagonized the late phase of inhibition in nine of fourteen neurones.5. Interspike-interval histograms showed that the firing pattern of neurones in the nucleus reticularis was characterized by periods of prolonged, high-frequency bursting. Both the ACh-evoked inhibitions and the late phase of MRF-evoked inhibitions were accompanied by an increased burst activity. In contrast, iontophoretic atropine tended to suppress burst activity.6. The possibility is discussed that electrical stimulation of the MRF activates an inhibitory cholinergic projection to the nucleus reticularis. Since neurones of the nucleus reticularis have been shown to inhibit thalamic relay cells, activation of this inhibitory pathway may play a role in MRF-evoked facilitation of thalamo-cortical relay transmission and the associated electrocortical desynchronization.     

Prefrontal cortical modulation of acetylcholine release in posterior parietal cortex

Attentional processing is a crucial early stage in cognition and is subject to "top-down" regulation by prefrontal cortex (PFC). Top-down regulation involves modification of input processing in cortical and subcortical areas, including the posterior parietal cortex (PPC). Cortical cholinergic inputs, originating from the basal forebrain cholinergic system, have been demonstrated to mediate important aspects of attentional processing. The present study investigated the ability of cholinergic and glutamatergic transmission within PFC to regulate acetylcholine (ACh) release in PPC. The first set of experiments demonstrated increases in ACh efflux in PPC following AMPA administration into the PFC. These increases were antagonized by co-administration of the AMPA receptor antagonist DNQX into the PFC. The second set of experiments demonstrated that administration of carbachol, but not nicotine, into the PFC also increased ACh efflux in PPC. The effects of carbachol were attenuated by co-administration (into PFC) of a muscarinic antagonist (atropine) and partially attenuated by the nicotine antagonist mecamylamine and DNQX. Perfusion of carbachol, nicotine, or AMPA into the PPC did not affect PFC ACh efflux, suggesting that these cortical interactions are not bi-directional. These studies demonstrate the capacity of the PFC to regulate ACh release in the PPC via glutamatergic and cholinergic prefrontal mechanisms. Prefrontal regulation of ACh release elsewhere in the cortex is hypothesized to contribute to the cognitive optimization of input processing.     

Non-quantal release of acetylcholine in guinea-pig airways: role of choline transporter

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ～75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.     

Amyloid β peptide levels and its effects on hippocampal acetylcholine release in aged, cognitively-impaired and -unimpaired rats

Excessive extracellular deposition of amyloid β (Aβ) peptide in neuritic plaques and degeneration of forebrain cholinergic neurones, which innervate the hippocampus and the neocortex, are the invariant characteristic features of Alzheimer's disease (AD). Studies of the pathological changes that characterize AD, together with several other lines of evidence, indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration observed in the AD brain. However, the underlying mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurones in AD brains remain obscure. We reported earlier that nM concentrations of Aβ-related peptides, under acute conditions, can potently inhibit K⁺-evoked endogenous acetylcholine (ACh) release from the hippocampus and the cortex but not from striatum in young adult rats (J. Neurosci. 16 (1996) 1034). In the present study, to determine whether the effects of Aβ peptides alter with normal aging and/or cognitive state, we have measured Aβ_1–40 levels and the effects of exogenous Aβ_1–40 on hippocampal ACh release in young adult as well as aged cognitively-unimpaired (AU) and -impaired (AI) rats. Endogenous levels of Aβ_1–40 in the hippocampus are significantly increased in aged rats. Additionally, 10 nM Aβ_1–40 potently inhibited endogenous ACh release from the hippocampus of the three groups of rats, but the time-course of the effects clearly indicate that the cholinergic neurones of AI rats are more sensitive to Aβ peptides than either AU or young adult rats. These results, together with earlier reports, suggest that the processing of the precursor protein of Aβ peptide alters with normal aging and the response of the cholinergic neurones to the peptide possibly varies with the cognitive status of the animals.     

Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent

The medial septal diagonal band area (MS/DB), made up of GABAergic and cholinergic neurones, plays an essential role in the generation and modulation of the hippocampal theta rhythm. To understand the part that the cholinergic neurones might play in this activity, we sought to determine whether postsynaptic nicotinic receptor responses can be detected in slices of the rodent MS/DB by puffing on acetylcholine (ACh). Neurones were characterized electrophysiologically into GABAergic and cholinergic neurones according to previous criteria. Responses of the MS/SB neurones to ACh were various combinations of fast depolarizations (1.5–2.5 s), fast hyperpolarizations (3–4 s) and slow depolarizations (20–30 s), the latter two being blocked by atropine. The fast depolarizations were partially or not blocked with cadmium and low calcium, tetrodotoxin, and antagonists of other ionotropic receptors, and were antagonized with 25 μ m mecamylamine. Pharmacological investigation of the responses showed that the α7* nicotinic receptor type is associated with cholinergic neurones and 10% of the GABAergic neurones, and that nonα7* nicotinic receptor subtypes are associated with 50% of the GABAergic neurones. Pharmacological dissection of evoked and spontaneous postsynaptic responses, however, did not provide evidence for synaptic nicotinic receptor transmission in the MS/DB. It was concluded that nicotinic receptors, although prevalent on the somatic and/or dendritic membrane compartments of neurones in the MS/DB, are on extrasynaptic sites where they presumably play a neuromodulatory role. The presence of α7* nicotinic receptors on cholinergic neurones may also render these cells specifically vulnerable to degeneration in Alzheimer's disease.     

Heterogeneity of presynaptic muscarinic receptors involved in modulation of transmitter release

In order to extend the characterization of muscarinic receptors at presynaptic sites their inhibitory effect on the stimulation-evoked release of [3H]noradrenaline and [3H]acetylcholine from different axon terminals was studied and the dissociation constants and potencies of different antagonists were estimated, in guinea-pig and rat. While oxotremorine reduced the release of [3H]acetylcholine and [3H]-noradrenaline in a concentration-dependent manner from different release sites (Auerbach plexus, noradrenergic neurons in the right atrium, cerebral cortex), McN-A 343, an M1 receptor agonist, enhanced their release evoked by field stimulation. When the inhibitory effect of oxotremorine on transmitter release was studied, pancuronium, pirenzepine and atropine were competitive antagonists of presynaptic muscarinic receptors located on the noradrenergic axon terminals of the atrium. While atropine and pirenzepine inhibited the muscarinic receptors of cholinergic axon terminals in the Auerbach plexus, pancuronium and gallamine had a very low affinity. Significant differences were found in the affinity constants of antagonists for muscarinic receptors located in the cholinergic axon terminals of Auerbach plexus and cerebral cortex, and noradrenergic axon terminals of the atrium. While atropine and pirenzepine exerted similar effects on these presynaptic sites, pancuronium, gallamine and (11-(2-[diethylamino)-methyl)-1-piperidinyl)acetyl)-5, 11-dihydro-6(1-pyrido(2,3-b)(1,4)-benzodiazepin-6-on) were much more effective on muscarinic receptors controlling acetylcholine release from the cerebral cortex and noradrenaline release from the heart. There was more than 100-fold (2.0 pA2 units) difference in affinities of these antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine innervation and activation of septohippocampal GABAergic neurones: involvement of local ACh release

Recent studies indicate that the histaminergic system, which is critical for wakefulness, also influences learning and memory by interacting with cholinergic systems in the brain. Histamine-containing neurones of the tuberomammillary nucleus densely innervate the cholinergic and GABAergic nucleus of the medial septum/diagonal band of Broca (MSDB) which projects to the hippocampus and sustains hippocampal theta rhythm and associated learning and memory functions. Here we demonstrate that histamine, acting via H₁ and/or H₂ receptor subtypes, utilizes direct and indirect mechanisms to excite septohippocampal GABA-type neurones in a reversible, reproducible and concentration-dependent manner. The indirect mechanism involves local ACh release, is potentiated by acetylcholinesterase inhibitors and blocked by atropine methylbromide and 4-DAMP mustard, an M₃ muscarinic receptor selective antagonist. This indirect effect, presumably, results from a direct histamine-induced activation of septohippocampal cholinergic neurones and a subsequent indirect activation of the septohippocampal GABAergic neurones. In double-immunolabelling studies, histamine fibres were found in the vicinity of both septohippocampal cholinergic and GABAergic cell types. These findings have significance for Alzheimer's disease and other neurodegenerative disorders involving a loss of septohippocampal cholinergic neurones as such a loss would also obtund histamine effects on septohippocampal cholinergic and GABAergic functions and further compromise hippocampal arousal and associated cognitive functions.