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.     

Galanin stimulates the release of vasoactive intestinal polypeptide from perifused hypothalamic fragments in vitro and from periventricular structures into the cerebrospinal fluid in vivo in the rat

Intracerebroventricular injection of galanin (2 micrograms/rat) raised plasma prolactin (PRL) levels in the rat, which was accompanied by an increase in immunoreactive vasoactive intestinal polypeptide (VIP) in the cerebrospinal fluid (CSF). Immunoreactive VIP release from superfused rat hypothalamic fragments in vitro was dose-relatedly stimulated by galanin (10(-7) and 10(-8) M). PRL release from superfused rat anterior pituitary cells was stimulated by TRH (10(-8) M) but not affected by galanin (10(-7) to 10(-5) M). These findings indicate that central galanin has a stimulating role in the release of hypothalamic VIP, which results in pituitary PRL secretion in the rat.     

Evidence for a role of the neuropeptide galanin in spatial learning.

The neuropeptide galanin coexists with acetylcholine (ACh) in the basal forebrain cholinergic neurons and modulates cholinergic activity in the forebrain. The cholinergic forebrain neurons appear to play a significant role in learning and memory, as suggested by a severe loss of these neurons in Alzheimer's disease. The involvement of endogenous galanin in learning is demonstrated here by the use of the recently synthesized high-affinity galanin antagonist M35 [galanin(1-13)-bradykinin(2-9) amide] (Kd = 0.1 nM). Intracerebroventricular (i.c.v.) administration of M35 (6 but not 3 nmol) produced a significant (P < 0.025) facilitation of acquisition in a spatial learning test (Morris swim maze) without any increase in swim speed. Thus, M35 (6 nmol) shortened the escape latency, reduced the number of failures to reach the platform, and shortened the path length to reach the hidden platform. M35 (3 and 6 nmol) tended to enhance retention performance seven days after the last training session. Receptor autoradiographic studies on the distribution of [125I]M35 following i.c.v. administration show that it binds preferentially in the periventricular regions including the hippocampus. These results suggest that galanin may modulate spatial learning and memory and that galanin antagonists may provide a new principle in the treatment of Alzheimer's disease.     

Galanin in the regulation of the hypothalamic-pituitary-adrenal axis (Review)

Galanin is a regulatory 30- or 29-amino acid peptide, widely distributed in the nervous system and gut, that acts via three subtypes of G protein-coupled receptors, named GAL-R1, GAL-R2 and GAL-R3. Findings have been accumulated that galanin regulates neuroendocrine hypothalamic axes, including the hypothalamic-pituitary-adrenal (HPA) one. Galanin and its receptors are expressed in the hypothalamic paraventricular and supraoptic nuclei, anterior pituitary and adrenal medulla. Adrenal cortex does not express galanin, but is provided with GAL-R1 and GAL-R2. The bulk of evidence indicates that galanin stimulates the activity of the central branch of the HPA axis (i.e. the release of corticotropin-releasing hormone and ACTH), thereby enhancing glucocorticoid secretion from the adrenal cortex. Investigations carried out in the rat show that galanin is also able to directly stimulate corticosterone (glucocorticoid) secretion from adrenocortical cells, through GAL-R1 and GAL-R2 coupled to the adenylate cyclase-protein kinase A signaling cascade, and nor-epinephrine release from adrenal medulla. There is indication that galanin may also enhance corticosterone release via an indirect paracrine mechanism involving the local release of catecholamines, which in turn activate beta-adrenoceptors located on adrenocortical cells. The physiological relevance in the rat of the glucocorticoid secretagogue action of galanin is suggested by the demonstration that the blockade of galanin system significantly lowers basal corticosterone secretion. There is also evidence that galanin plays a role in the modulation of HPA-axis response to stress, as well as in the pathogenesis of pituitary adenomas and perhaps of pheochromocytomas.     

Microdialysis delivery of morphine to the hypoglossal nucleus of Wistar rat increases hypoglossal acetylcholine release

STUDY OBJECTIVES: The medullary hypoglossal nucleus (XII) innervates the genioglossal muscles of the tongue, and opioid-induced alterations in tongue muscle tone contribute to airway obstruction. Previous studies have shown that morphine causes a significant decrease in acetylcholine (ACh) release in some brain regions, but the effects of morphine on ACh release in XII have not been quantified. DESIGN: A within-subjects design was used to test the hypothesis that morphine alters ACh release in XII of anesthetized Wistar rat. ACh release during microdialysis with Ringer's solution (control) was compared to ACh release during dialysis delivery of opioids. SETTING: University of Michigan. PATIENTS OR PARTICIPANTS: N/A. INTERVENTIONS: Microdialysis delivery of opioids to XII. MEASUREMENTS AND RESULTS: Morphine caused a statistically significant, concentration-dependent increase in XII ACh release. The increase in XII ACh release caused by 10 microM morphine was blocked by the mu opioid antagonist naloxone and not blocked by the kappa opioid antagonist norbinaltorphimine. CONCLUSIONS: The data comprise the first direct measures of ACh release in XII and support the conclusion that morphine depresses hypoglossal nerve activity, in part, by increasing ACh release in XII. Activation of mu opioid receptors on inhibitory neurons within XII likely disinhibits cholinergic terminals, causing increased ACh release. The results are consistent with previous studies showing that blocking the enzymatic degradation of ACh in XII significantly inhibited tongue muscle activity.     

GABAA and GABAB receptors differentially regulate striatal acetylcholine release in vivo

Microdialysis was used to study the effects of selective GABAergic agents on striatal acetylcholine (ACh) release in awake, freely moving rats. Local perfusion with the GABA_A agonist muscimol dramatically reduced striatal ACh release, while the GABA_B agonist baclofen caused only minor decreases in ACh release. Co-perfusion with the GABA_A antagonist bicuculline diminished the muscimol-induced decrease in ACh release. Likewise, co-perfusion with the GABA_B antagonist 2-hydroxysaclofen attenuated the baclofen-induced reduction in ACh release. Bicuculline alone markedly increased striatal ACh release, but 2-hydroxysaclofen by itself had no effect. These results suggest that GABA tonically regulates striatal ACh release primarily through stimulation of inhibitory GABA_A receptors.     

Galanin lacks binding sites in the porcine pituitary and has no detectable effect on oxytocin and vasopressin release from rat neurosecretory endings.

High concentrations of immunoreactive galanin-like material in rat hypothalamus, median eminence and neurohypophysis have been reported in the literature suggesting a regulatory role of galanin on hormone release from the anterior and posterior lobe of the pituitary. We studied binding of iodinated galanin to crude membrane preparations from porcine anterior hypothalamus, anterior and neurointermediate lobe of the hypophysis. In contrast to the hypothalamus where specific binding of 125I-galanin was found, there was no displaceable galanin binding in membranes of the anterior or neurointermediate lobe of porcine pituitaries. Effects of galanin on oxytocin and vasopressin release were investigated using isolated neurosecretory endings from rat neurohypophyses. Galanin had no detectable effect on the release of oxytocin or vasopressin.     

Biphasic modulation by galanin of excitatory synaptic transmission in substantia gelatinosa neurons of adult rat spinal cord slices

Although intrathecally administrated galanin modulates nociceptive transmission in a biphasic manner, this has not been fully examined previously. In the present study, the action of galanin on synaptic transmission in the substantia gelatinosa (SG) neurons of adult rat spinal cord slices was examined, using the whole cell patch-clamp technique. Galanin concentration-dependently increased the frequency of spontaneous excitatory postsynaptic current (EPSC; EC(50) = 2.0 nM) without changing the amplitude, indicating a presynaptic effect. This effect was reduced in a Ca(2+)-free, or voltage-gated Ca(2+) channel blocker La(3+)-containing Krebs solution and was produced by a galanin type-2/3 receptor (GalR2/R3) agonist, galanin 2-11, but not by a galanin type-1 receptor (GalR1) agonist, M617. Galanin also concentration-dependently produced an outward current at -70 mV (EC(50) = 44 nM), although this appeared to be contaminated by a small inward current. This outward current was mimicked by M617, but not by galanin 2-11. Moreover, galanin reduced monosynaptic Aδ-fiber- and C-fiber-evoked EPSC amplitude; the former reduction was larger than the latter. A similar action was produced by galanin 2-11, but not by M617. Spontaneous and focally evoked inhibitory (GABAergic and glycinergic) transmission was unaffected by galanin. These findings indicate that galanin at lower concentrations enhances the spontaneous release of l-glutamate from nerve terminals by Ca(2+) entry from the external solution following GalR2/R3 activation, whereas galanin at higher concentrations also produces a membrane hyperpolarization by activating GalR1. Moreover, galanin reduces l-glutamate release onto SG neurons from primary afferent fibers by activating GalR2/R3. These effects could partially contribute to the behavioral effect of galanin.     

Galanin of the homologous species inhibits insulin secretion in the rat and in the pig.

It is known that pig galanin inhibits insulin secretion in dogs, rats and mice. The present study examined whether species-specific, homologous, galanin inhibits insulin secretion. Thus, the effects of rat galanin were examined in the rat, and the effects of pig galanin were examined in the pig, both in vivo and in vitro. In conscious rats, synthetic rat galanin (2 nmol kg-1) abolished the glucose- (0.56 mmol kg-1) induced increase in plasma insulin levels. In vitro, rat galanin (10(-9) to 10(-6) mol l-1) inhibited glucose- (8.3 mmol l-1) stimulated insulin release from isolated rat islets. In anaesthetized pigs, 15 min infusion of synthetic pig galanin (207 pmol min-1) into the pancreatic artery decreased the insulin output with a subsequent recovery. In vitro, pig galanin (10(-6) mol l-1) inhibited glucose- (8.3 mmol l-1) stimulated insulin release from isolated pig islets. We conclude that homologous galanin inhibits insulin secretion in both the rat and the pig.     

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH-5183), tityustoxin and ouabain on the release of acetylcholine and its mobilization from cytoplasmic and vesicular pools of rat brain cortical slices

The effect of vesicular acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH-5183) on the subcellular storage and release of ACh was studied in rat brain cortical slices. AH-5183 reduced the release of ACh from cortical slices stimulated by tityustoxin and ouabain. Tissue stimulated in the presence of AH-5183 contained more ACh in both the nerve terminal synaptic vesicles and cytoplasmic fraction than did tissue stimulated in drug's absence. Thus, AH-5183 blocked the tityustoxin and ouabain induced release of ACh from both cytoplasmic and vesicular pools. AH-5183 also depressed the spontaneous release of ACh from incubated slices and, in this condition, the drug had no effect in the subcellular distribution of ACh. It is suggested that AH-5183 interferes with the process of ACh release independent of its blocking action on ACh transport into the synaptic vesicles.     

Acetylcholine release in the pontine reticular formation of C57BL/6J mouse is modulated by non-M1 muscarinic receptors

Pontine acetylcholine (ACh) contributes to the regulation of electroencephalographic and behavioral arousal in all mammals so far investigated. The mouse is recognized as a powerful model for pharmacogenomics but the synaptic mechanisms regulating ACh release in mouse pontine reticular formation have not been characterized. Drug delivery by microdialysis was used in isoflurane-anesthetized C57BL/6J (B6) mice (n=33) to test the hypothesis that muscarinic autoreceptors modulate ACh release in the pontine reticular nucleus, oral part (PnO). Dialysis delivery of tetrodotoxin to the PnO significantly decreased ACh by 58% below control levels, confirming that measured ACh reflected neurotransmitter release. The muscarinic antagonist scopolamine increased ACh release in the PnO by 21% (3 nM), 48% (10 nM), 56% (30 nM), and 104% (100 nM). The muscarinic agonist bethanechol dialyzed into the PnO significantly decreased ACh release by 60% compared with control. Dialysis delivery of relatively subtype selective muscarinic antagonists to the PnO revealed the following order of potency for increasing ACh release: scopolamine (3 nM)>AF-DX 116 (100 nM)=pirenzepine (100 nM). These data support the conclusion that ACh release in PnO of B6 mouse is modulated by non-M1 muscarinic receptors.     

Presynaptic inhibition of acetylcholine release

High potassium (51 mM) has been shown to evoke release of acetylcholine ([3H]ACh and endogenous ACh) from cholinergic nerves in rat bronchial smooth muscle. The release of [3H]ACh was reduced by 85% when the Ca2+ concentration was changed from 2 to 0.1 mM. The veratridine-induced release was completely inhibited by tetrodotoxin, but tetrodotoxin did not reduce the potassium-evoked release. The muscarinic agonist, oxotremorine, reduced the potassium stimulated release of [3H]ACh, without affecting the basal release. In contrast, scopolamine substantially potentiated the potassium-evoked release. Adenosine had a dual effect in the rat bronchi. Adenosine inhibited the potassium-evoked release of [3H]ACh and this presynaptic effect of adenosine was antagonized by 8-phenyltheophylline. Adenosine also induced contraction of the bronchial smooth muscle and there was potentiation by adenosine of the ACh-induced contraction. The results indicate that cholinergic nerve terminals in the rat bronchi possess muscarinic receptors which inhibit the release of ACh. Adenosine may have analogous effects, e.g. presynaptic inhibition of transmitter release in addition to postsynaptic enhancement of bronchial smooth muscle contraction.     

The release of endogenous acetylcholine from the medial septum/diagonal band of rat brain

This study sought to establish whether cholinergic neurons in the medial septum/vertical limb of the diagonal band (ms/vdB) release endogenous acetylcholine (ACh) locally, and whether the release was modulated by presynaptic feedback mechanisms. Release of ACh from slices of the ms/vdB was assessed by gas chromatography-mass spectrometry (GC-MS). Potassium depolarization resulted in a 20- to 25-fold increase in ACh release above spontaneous levels. Omission of Ca2+ from the incubation medium decreased this release by 91%. In the presence of 4 microM atropine, potassium-induced ACh release was enhanced by 48%. These results indicate that ACh is released in the ms/vdB by a Ca2+-dependent and atropine-sensitive process.     

Influence of presynaptic receptors on neuromuscular transmission in rat

The presence and physiological significance of acetylcholine (ACh) receptors on motor nerve terminals was examined at the rat diaphragm neuromuscular junction. Intracellular recording techniques were used to monitor end-plate potentials (EPP), miniature end-plate potentials (MEPP), and resting potentials of the muscle fibers. Muscle action potentials were blocked by the cut-muscle technique. Quantal release was determined by the ratio EPP/MEPP, after correcting for nonlinear summation. Blockade of acetylcholinesterase with eserine and neostigmine was tested to determine the influence of residual ACh on transmitter release. Partial blockade of ACh receptors with curare was examined to further clarify the role of these presynaptic receptors. The experiments demonstrate that residual ACh inhibits transmitter release and that blockade of ACh receptors enhances transmitter release. It is concluded that presynaptic ACh receptors exist and that they serve an important physiological function. It is suggested that the presynaptic ACh receptors normally serve to limit transmitter release in a negative feedback pathway.     

Sodium ion transport participates in non-neuronal acetylcholine release in the renal cortex of anesthetized rabbits

This study examined the mechanism of release of endogenous acetylcholine (ACh) in rabbit renal cortex by applying a microdialysis technique. In anesthetized rabbits, a microdialysis probe was implanted into the renal cortex and perfused with Ringer’s solution containing high potassium concentration, high sodium concentration, a Na⁺/K⁺-ATPase inhibitor (ouabain), or an epithelial Na⁺ channel blocker (benzamil). Dialysate samples were collected at baseline and during exposure to each agent, and ACh concentrations in the samples were measured by high-performance liquid chromatography. High potassium had no effect on renal ACh release. High sodium increased dialysate ACh concentrations significantly. Ouabain increased dialysate ACh concentration significantly. Benzamil decreased dialysate ACh concentrations significantly both at baseline and under high sodium. The finding that high potassium-induced depolarization does not increase ACh release suggests that endogenous ACh is released in renal cortex mainly by non-neuronal mechanism. Sodium ion transport may be involved in the non-neuronal ACh release.     

Enhancement of acetylcholine release during paradoxical sleep in the dorsal tegmental field of the cat brain stem

Acetylcholine (ACh) in the brain stem has been implicated in the generation of paradoxical sleep (PS). In order to clarify the relationship between local ACh release in the dorsal tegmental field (FTD), a possible PS-generating locus, and sleep-wake states in 6 cats. ACh was measured by the method of in vivo microdialysis and high performance liquid chromatography-electrochemical detection. It is noteworthy that ACh release was about 2 times higher (P less than 0.001) during PS than during slow-wave sleep and wakefulness in FTD, but not in the caudate nucleus, a control region. ACh release in FTD appeared to begin to increase prior to the onset of PS. Electrical and chemical (glutamate) stimulations of the nucleus magnocellularis (MC) enhanced ACh release in FTD and shortened PS latency. These results suggest that this PS-related enhancement of ACh release in FTD is induced by some cholinergic projections from glutamate-receptive neurons in MC.     

Morphine Increases Acetylcholine Release in the Trigeminal Nuclear Complex

Study Objectives:

The trigeminal nuclear complex (V) contains cholinergic neurons and includes the principal sensory trigeminal nucleus (PSTN) which receives sensory input from the face and jaw, and the trigeminal motor nucleus (MoV) which innervates the muscles of mastication. Pain associated with pathologies of V is often managed with opioids but no studies have characterized the effect of opioids on acetylcholine (ACh) release in PSTN and MoV. Opioids can increase or decrease ACh release in brainstem nuclei. Therefore, the present experiments tested the 2-tailed hypothesis that microdialysis delivery of opioids to the PSTN and MoV significantly alters ACh release.

Design:

Using a within-subjects design and isoflurane-anesthetized Wistar rats (n = 53), ACh release in PSTN during microdialysis with Ringer''s solution (control) was compared to ACh release during dialysis delivery of the sodium channel blocker tetrodotoxin, muscarinic agonist bethanechol, opioid agonist morphine, mu opioid agonist DAMGO, antagonists for mu (naloxone) and kappa (nor-binaltorphimine; nor-BNI) opioid receptors, and GABA_A antagonist bicuculline.

Measurements and Results:

Tetrodotoxin decreased ACh, confirming action potential-dependent ACh release. Bethanechol and morphine caused a concentration-dependent increase in PSTN ACh release. The morphine-induced increase in ACh release was blocked by nor-BNI but not by naloxone. Bicuculline delivered to the PSTN also increased ACh release. ACh release in the MoV was increased by morphine, and this increase was not blocked by naloxone or nor-BNI.

Conclusions:

These data comprise the first direct measures of ACh release in PSTN and MoV and suggest synaptic disinhibition as one possible mechanism by which morphine increases ACh release in the trigeminal nuclei.

Citation:

Zhu Z; Bowman HR; Baghdoyan HA; Lydic R. Morphine increases acetylcholine release in the trigeminal nuclear complex. SLEEP 2008;31(12):1629–1637.     

Evidence for an involvement of eicosanoids in neurokinin3-receptor mediated acetylcholine release from myenteric neurons.

The release of acetylcholine (ACh) from myenteric plexus evoked by 5-hydroxytryptamine (5-HT) and senktide (a selective neurokinin3 (NK3) agonist) was depressed by mepacrine, an inhibitor for phospholipase A2 activity. Release of ACh was stimulated by arachidonic acid; this release was partially depressed by nordihydroguaiaretic acid (NDGA), which inhibits lipoxygenase activity. NDGA failed to modify the ACh secretion elicited by 5-HT. Release of ACh evoked by senktide was significantly inhibited by NDGA, suggesting an involvement of eicosanoids in the release of ACh elicited by specific neurokinin receptors in myenteric neurons.