Biochemical and behavioral responses of pilocarpine at muscarinic receptor subtypes in the CNS. Comparison with receptor binding and low-energy conformations

Pilocarpine was tested biochemically in vitro for its ability to stimulate phosphoinositide (PI) turnover in the hippocampus (M1/M3 responses) where it displayed 35% of the maximal carbachol response with an EC50 value of 18 microM, and low-Km GTPase in the cortex (M2 response), where it had 50% of the maximal carbachol response with an EC50 value of 4.5 microM. Behaviorally, pilocarpine was able to restore deficits in a representational memory task (sensitive to M1 antagonists) produced by intrahippocampal injections of AF64A. Twenty-three low-energy conformations of protonated pilocarpine were generated using the program MacroModel. The data indicate that pilocarpine is a partial agonist at both M1 and M2 muscarinic receptors in the CNS. Behaviorally, with respect to the memory task, M1 effects of pilocarpine apparently predominate. It also is conceivable that different conformations of pilocarpine are active as agonists at different muscarinic receptor subtypes.     

Degeneration of the basalocortical pathway from the cortex induces a functional increase in galaninergic markers in the nucleus basalis magnocellularis of the rat.

The present work aimed 1) to evaluate whether an increase in galanin or galanin receptors could be induced in the nucleus basalis magnocellularis (nbm) by degeneration of the basalocortical neurons from the cortex and 2) to analyze the consequences of such an increase on cortical activity. First, a mild ischemic insult to the frontoparietal cortex was performed to induce the degeneration of the basalocortical system; galanin immunoreactivity, galanin binding sites, and cholinergic muscarinic receptors were quantified through immunocytochemistry and autoradiography. Second, galanin infusions in the nbm were undertaken to mimic a local increase of the galaninergic innervation; cortical acetylcholine release, cerebral glucose use, and cerebral blood flow were then measured as indices of cortical activity. As a result of the cortical ischemic lesion, the postsynaptic M1 and presynaptic M2 muscarinic receptors were found to be reduced in the altered cortex. In contrast, galaninergic binding capacity and fiber density were found to be increased in the ipsilateral nbm in parallel with a local decrease in the cholinergic markers such as the muscarinic M1 receptor density. Galanin infusion into the nbm inhibited the cortical acetylcholine release and cerebral blood flow increases elicited by the activation of the cholinergic basalocortical system but failed to affect acetylcholine release, cerebral blood flow, and cerebral glucose use when injected alone in the nbm. These results demonstrate that degeneration of the basalocortical system from the cortex induces an increase in galaninergic markers in the nbm, a result that might suggest that the galaninergic overexpression described in the basal forebrain of patients with Alzheimer's disease can result from a degeneration of the cholinergic basalocortical system from the cortex. Because galanin was found to reduce the activity of the basalocortical cholinergic system only when this one is activated, galanin might exert its role rather during activation deficits than under resting conditions such as the resting cortical hypometabolism, which is characteristic of Alzheimer's disease.     

Effect of neostigmine on the hippocampal noradrenaline release: role of cholinergic receptors

The effect of the cholinesterase inhibitor neostigmine on hippocampal noradrenaline (NA) release was studied using in vivo microdialysis. Local application of neostigmine significantly increased the release of NA. The effect was potentiated by coperfusion of the nicotinic antagonist mecamylamine but was completely blocked by the muscarinic antagonist atropine. The neostigmine-evoked NA release was not affected by the M2-selective muscarinic antagonist gallamine but was completely blocked by the M1-selective muscarinic antagonist pirenzepine. While muscarinic antagonists had no effect on the resting release of NA, mecamylamine increased it. Our data indicate that acetylcholine can stimulate the hippocampal NA release via M1 muscarinic receptors and that a population of nicotinic receptors mediate inhibitory tone on hippocampal NA release. The fact that neostigmine is able to enhance both cholinergic and noradrenergic neurotransmission may help to understand the beneficial effect of cholinesterase inhibitors in Alzheimer's disease.     

Muscarinic receptor subclassification and G-proteins: significance for lithium action in affective disorders and for the treatment of the extrapyramidal side effects of neuroleptics

The classification of muscarinic receptors into M1 and M2 subtypes and the involvement of guanine nucleotide binding proteins (G-proteins) as major mediators of receptor information transduction in the cholinergic and other neurotransmitter systems have prompted us to undertake studies both at receptor and postreceptor levels that may shed light on the importance of these new findings to the pharmacotherapy of manic-depressive illness and of extrapyramidal syndromes. We searched for patterns of muscarinic selectivity among the commonly used anticholinergics (biperiden, procyclidine, trihexyphenidyl, benztropine, and methixen) through radioligand receptor studies in various rat tissues. The drugs showed a range of selectivity, from the totally nonselective methixen to the highly M1-selective biperiden. Sinus arrhythmia measurements were undertaken in psychiatric patients treated with different antiparkinsonian anticholinergics. The extent of sinus arrhythmia suppression was inversely correlated with the degree of M1 selectivity of the drugs used, advocating the use of M1-selective antiparkinsonian anticholinergics like biperiden in the treatment of extrapyramidal side effects. The implications of muscarinic receptor subclassification were further extended to include postreceptor phenomena. We have directly studied G-protein function by measuring cholinergic agonist-induced increases in guanosine triphosphate (GTP) binding to these proteins. This cholinergic agonistic effect was shown to be exerted by G-proteins other than Gs (the adenylate cyclase stimulatory G-protein), i.e., Gi (the adenylate cyclase inhibitory G-protein) or Gp [the G-protein activating phosphatidylinositol (PI) turnover], as ribosylation by pertussis toxin abolished this cholinergic effect, whereas it was unaffected by cholera toxin. Pertussis toxin-blockable, carbamylcholine-induced increases in GTP binding capacity were found to be mediated through M1 muscarinic receptors, as M1-selective antagonists were 100-fold more effective than M2 selective antagonists in blocking carbamylcholine effects. Moreover, carbamylcholine effect was exclusively detected in tissues predominantly populated by M1 receptors. Our results thus suggest that carbamylcholine-induced increases in GTP binding are exerted through M1 receptors interacting with Gp. At therapeutically efficacious concentrations, lithium completely blocked carbamylcholine-induced increases in GTP binding capacity in both in vitro and in vivo experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase C activity alters the effect of μ-opioid receptors on inhibitory postsynaptic current in the striosomes

We have previously reported that earlier blockade of protein kinase C (PKC) augments the suppressive effect of μ-opioid receptors (MORs) on the GABAergic inhibitory postsynaptic current (IPSC) in the MOR-rich striosomes of the striatum. Interestingly, striatal medium-spiny neurons have muscarinic acetylcholine receptor subtypes M1 and M4, among which M1 activates the phosphoinositide signaling pathway yielding PKC. In this study, we examined whether acetylcholine regulates the effects of MOR on presynaptic IPSC by binding to the M1 receptor, and found that IPSC suppression by the MOR agonist, [D-Ala-N-Me-Phe, Gly-ol]-enkephalin, was significantly augmented and prolonged by the PKC inhibitor chelerythrine and attenuated by the PKC activator, phorbol 12, 13-dibutyrate. This modulatory action by chelerythrine was mimicked by the muscarinic antagonist atropine and the M1-specific antagonist pirenzepine, whereas M2-M4 antagonists had no discernible effect. These results suggest that PKC activity modulates the effect of MOR by muscarinic receptors in the striosomes.     

Binding sites for [3H]AF-DX 116 and effect of AF-DX 116 on endogenous acetylcholine release from rat brain slices

The present study shows that the putative M2 ligand, [3H]AF-DX 116, binds to two classes of muscarinic sites in homogenates of rat hippocampus, striatum and cerebral cortex: one with a high affinity (Kd less than 5 nM)/low capacity (Bmax = 30-63 fmol/mg protein), and a second of lower affinity (Kd greater than 65 nM) and higher capacity (Bmax greater than 190 fmol/mg protein). In experiments which tested the effects of the muscarinic antagonists on acetylcholine (ACh) release from brain slices, the non-selective antagonist (-)-quinuclidinyl benzylate and atropine significantly enhanced the potassium (25 mM)-evoked release of ACh. This effect was mimicked by the M2 ligand AF-DX 116, but neither the M1-selective antagonist pirenzepine, nor the putative M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), altered ACh release. Also, the muscarinic agonist, oxotremorine, significantly depressed evoked ACh release from brain slices, an effect that was completely antagonized by atropine or by AF-DX 116, but not by pirenzepine or 4-DAMP. Thus, it appears that presynaptic muscarinic autoreceptors in the rat hippocampus, striatum and cerebral cortex belong to the M2 subtype of muscarinic receptors.     

Amphetamine-stimulated cortical acetylcholine release: role of the basal forebrain

Systemic administration of amphetamine results in increases in the release of acetylcholine in the cortex. Basal forebrain mediation of this effect was examined in three experiments using microdialysis in freely-moving rats. Experiment 1 examined whether dopamine receptor activity within the basal forebrain was necessary for amphetamine-induced increase in cortical acetylcholine by examining whether intra-basalis perfusion of dopamine antagonists attenuates this increase. Systemic administration of 2.0 mg/kg amphetamine increased dopamine efflux within the basal forebrain nearly 700% above basal levels. However, the increase in cortical acetylcholine efflux following amphetamine administration was unaffected by intra-basalis perfusions of high concentrations of D1- (100 microM SCH 23390) or D2-like (100 microM sulpiride) dopamine receptor antagonists. Experiments 2 and 3 determined whether glutamatergic or GABAergic local modulation of the excitability of the basal forebrain cholinergic neurons influences the ability of systemic amphetamine to increase cortical acetylcholine efflux. In Experiment 2, perfusion of kynurenate (1.0 mM), a non-selective glutamate receptor antagonist, into the basal forebrain attenuated the increase in cortical acetylcholine produced by amphetamine. Experiment 3 revealed that positive modulation of GABAergic transmission by bilateral intra-basalis infusion of the benzodiazepine receptor agonist chlordiazepoxide (40 microg/hemisphere) also attenuated the amphetamine-stimulated increase in cortical acetylcholine efflux. These data suggest that amphetamine increases cortical acetylcholine release via a complex neuronal network rather than simply increasing basal forebrain D1 or D2 receptor activity.     

Effects of tachykinins on phosphoinositide metaboism in the hypothalamus: is the NK1 receptor involved?

Substance P (SP) has been shown to stimulate the hydrolysis of inositol phospholipids in peripheral tissues and in the brain. In mammalian peripheral tissues, three tachykinin receptor subclasses, neurokinin 1 (NK1), neurokinin 2 (NK2) and neurokinin 3 (NK3), have been identified. The purpose of our study was to pharmacologically characterize the SP receptors in the hypothalamus using phosphoinositide breakdown as a functional response. SP, previously described as a NK1 agonist, and Neurokinin A (NKA), previously described as a NK2 agonist, stimulated phosphoinositide breakdown in the hypothalamus in a dose-dependent fashion, with SP being more potent than NKA. The NK2-selective antagonist L-659,877, at a dose of 10⁻⁶ M, abolished the effect of SP (10⁻⁸ M) without affecting basal phosphoinositide breakdown. However, this NK2-selective antagonist did not inhibit the NKA-induced stimulation on phosphoinositide metabolism. The NK-1-selective antagonist L-668,169 stimulated phosphoinositide metabolism at a concentration of 10⁻⁶ M, but not at 10⁻⁸ M. This NK1-receptor antagonist did not significantly inhibit the effect of SP on phosphoinositide metabolism. Spantide II, another NK1-selective antagonist, also stimulated phosphoinositide metabolism at a dose of 10⁻⁶ M. Like L-668,169, spantide II failed to inhibit the SP-induced stimulation of phosphoinositide metabolism, and even potentiated the response to SP. The blockade of the SP-induced phosphoinositide breakdown by the NK2-receptor antagonist (L-659,877), the absence of blockade of the NKA-induced phosphoinositide metabolism by the NK2 receptor antagonist and the absence of inhibition by the NK1 receptor antagonists (L-688,169 and spantide II) suggest that the SP-induced phosphoinositide metabolism in the hypothalamus is mediated by a receptor subclass different from those mediating the peripheral actions of SP.     

Neurokinin2-R in medial septum regulate hippocampal and amygdalar ACh release induced by intraseptal application of neurokinins A and B

The neurokinin receptors (NK-R), NK(2)- and NK(3)-R, have been implicated in behavioral processes, but apparently in opposite ways: while NK(2)-R agonism disrupts memory and has anxiogenic-like action, NK(3) -R agonists facilitate memory and display anxiolytic-like effects. Systemic application of NK(2)-R antagonists block the release of acetylcholine (ACh) in the hippocampus, which is induced by intraseptal administration of the NK(2)-R ligand, neurokinin A (NKA). We investigated the effects of medial septal injection of NKA and a preferred ligand of NK(3)-R, neurokinin B (NKB), on the activity of cholinergic neurons of the basal forebrain and assessed the role of the medial septal NK(2)-R in the control of extracellular ACh levels in cholinergic projection areas. ACh was dialysed in the frontal cortex, amygdala and hippocampus of anesthetized animals and was analysed by HPLC-EC. ACh levels in hippocampus and amygdala, but not in frontal cortex were increased after intraseptal injection of either NKA or NKB (0.1, 1, 10 μM). Application of the nonpeptidic NK(2)-R antagonist, saredutant SR48968 (1, 10, 100 pM), followed by NKA (1 μM) or NKB (10 μM) injection into the medial septum, blocked the ACh increase in hippocampus and amygdala. These results indicate that medial septal NK(2)-R have an important role in mediating ACh release, for one, via the septal-hippocampal cholinergic projection and, secondly, via direct or indirect route to the amygdala, but not frontal cortex. They also support the hypothesis that hippocampal cholinergic neurotransmission controls amygdala function suggesting that this interaction is regulated via NK(2)-R in the medial septum.     

Progress toward a high-affinity allosteric enhancer at muscarinic M1 receptors

Loss of forebrain acetylcholine is an early neurochemical lesion in Alzheimer's disease (AD). As muscarinic acetylcholine receptors are involved in memory and cognition, a muscarinic agonist could therefore provide a "replacement therapy" in this disease. However, muscarinic receptors occur throughout the CNS and the periphery. A selective locus of action of a muscarinic agonist is therefore crucial in order to avoid intolerable side effects. The five subtypes of muscarinic receptors, M1-M5, have distinct regional distributions with M2 and M3 receptors mediating most of the peripheral effects. M1 receptors are the major receptor subtype in the cortex and hippocampus-the two brain regions most associated with memory and cognition. This localization has led to a, so far unsuccessful, search for a truly M1-selective muscarinic agonist. However, acetylcholinesterase inhibitors, such as donepezil (Aricept), which potentiate cholinergic neurotransmission, do have a therapeutic role in the management of AD and so the M1 receptor remains a viable therapeutic target. Our approach is to develop muscarinic allosteric enhancers-compounds that bind to the receptor at an "allosteric" site, which is distinct from the "primary" site to which ACh binds, and which enhance ACh affinity (or efficacy). Having discovered that a commercially available compound, WIN 62577, is an allosteric enhancer with micromolar potency at M3 receptors, we report here some results of a chemical synthesis project to develop this hit. Modification of WIN 62577 has led to compounds with over 1000-fold increased affinity but, so far, none of these extremely potent compounds are allosteric enhancers.     

A critical role for D1 receptors in the 5-HT1A-mediated facilitation of in vivo acetylcholine release in rat frontal cortex

Subcutaneous administration of 8-OH-DPAT dose-dependently increased acetylcholine (ACh) output in frontal cortex of awake rats. The maximal effect of 8-OH-DPAT (0.5 mg/kg, s.c.) was prevented by the 5-HT_1A antagonist WAY 100635 (1 mg/kg, s.c.) and by the D₁ antagonists SCH 23390 or SCH 39166 (both 0.3 mg/kg, s.c.) but not seven days after chemical lesion of the raphe serotoninergic neurons. It is postulated that the 8-OH-DPAT activation of postsynaptic 5-HT_1A receptors enhances the release of dopamine which, by acting at D₁ receptors, stimulates the release of ACh in the frontal cortex.     

Quantitative autoradiographic distribution of [3H]AF-DX 116 muscarinic-M2 receptor binding sites in rat brain

The distribution of muscarinic-M2 receptors in rat brain was investigated by in vitro autoradiography using [3H]AF-DX 116, a putative probe for the muscarinic-M2 receptor subtype. Incubation of rat brain coronal sections with 10 nM [3H]AF-DX 116 showed highest binding site densities in discrete areas such as the superior colliculus and certain thalamic and brainstem nuclei, similar to the distribution reported for [2H]acetylcholine/M2 sites. [3H]AF-DX 116 site densities were markedly lower in forebrain areas such as cortex, striatum, and hippocampus, in contrast to the distribution seen for [3H]pirenzepine-M1 binding sites, which were concentrated in these forebrain areas; however, differential patterns of labeling were observed for the two muscarinic-M2 probes, [3H]AF-DX 116 and [3H]acetylcholine, in the hippocampal formation. Although [3H]AF-DX 116 binding was broadly distributed in multiple subfields of the hippocampus, [3H]acetylcholine binding was discretely distributed in a manner resembling that of acetylcholinesterase staining. This suggests the existence of muscarinic-M2 subtypes in the CNS, especially in the hippocampal formation.     

A model of prefrontal cortex dopaminergic modulation during the delayed alternation task

Working memory performance is modulated by the level of dopamine (DA) D1 receptors stimulation in the prefrontal cortex (PFC). This modulation is exerted at different time scales. Injection of D1 agonists/antagonists exerts a long-lasting influence (several minutes or hours) on PFC pyramidal neurons. In contrast, during performance of a cognitive task, the duration of the postsynaptic effect of phasic DA release is short lasting. The functional relationships of these two time scales of DA modulation remain poorly understood. Here we propose a model that combines these two time scales of DA modulation on a prefrontal neural network. The model links the cellular and behavioral levels during performance of the delayed alternation task. The network, which represents the activity of deep-layer pyramidal neurons with intrinsic neuronal properties, exhibits two stable states of activity that can be switched on and off by excitatory inputs from long-distance cortical areas arriving in superficial layers. These stable states allow PFC neurons to maintain representations during the delay period. The role of an increase of DA receptors stimulation is to restrict inputs arriving on the prefrontal network. The model explains how the level of working memory performance follows an inverted U-shape with an increased stimulation of DA D1 receptors. The model predicts that (1) D1 receptor agonists increase perseverations, (2) D1 antagonists increase distractability, and (3) the duration of the postsynaptic effect of phasic DA release in the PFC is adjusted to the delay period of the task. These results show how the precise duration of the postsynaptic effect of phasic DA release influences behavioral performance during a simple cognitive task.     

Induction of activity-dependent LTD requires muscarinic receptor activation in medial prefrontal cortex

The medial prefrontal cortex (mPFC) forms part of a neural circuit involved in the formation of lasting associations between objects and places. Cholinergic inputs from the basal forebrain innervate the mPFC and may modulate synaptic processes required for the formation of object-in-place memories. To investigate whether acetylcholine regulates synaptic function in the rat mPFC, whole-cell voltage-clamp recordings were made from pyramidal neurons in layer V. Bath application of the cholinergic agonist carbachol caused a potent and long-term depression (LTD) of synaptic responses that was blocked by the muscarinic receptor antagonist scopolamine and was mimicked, in part, by the M(1) receptor agonists McN-A-343 or AF102B. Furthermore, inhibition of PKC blocked carbachol-mediated LTD. We next determined the requirements for activity-dependent LTD in the prefrontal cortex. Synaptic stimulation that was subthreshold for producing LTD did, however, result in LTD when acetylcholine levels were enhanced by inhibition of acetylcholinesterase or when delivered in the presence of the M(1)-selective positive allosteric modulator BQCA. Increasing the levels of synaptic stimulation resulted in M(1) receptor-dependent LTD without the need for pharmacological manipulation of acetylcholine levels. These results show that synaptic stimulation of muscarinic receptors alone can be critical for plastic changes in excitatory synaptic transmission in the mPFC. In turn, these muscarinic mediated events may be important in the formation of object-in-place memories. A loss of basal forebrain cholinergic neurons is a classic hallmark of Alzheimer's dementia and our results provide a potential explanation for the loss of memory associated with the disease.     

Psychosis of Alzheimer's disease is associated with elevated muscarinic M2 binding in the cortex

OBJECTIVES: Results from recent drug trials suggest a role for the cholinergic system in the manifestation of neuropsychiatric symptoms in AD. To date, the status of muscarinic acetylcholine receptor subtypes in AD in relation to accompanying behavioral disturbances is unknown. This study aimed to measure alterations of muscarinic M(1) and M(2) receptor binding in the frontal and temporal cortex of AD and to correlate the neurochemical findings with clinical features. METHODS: The cognitive and behavioral features of 26 patients with AD were assessed prospectively using standardized tests. Together with 14 matched controls, the status of muscarinic M(1) and M(2) receptors in the postmortem frontal and temporal cortex of these patients were measured by radioligand binding assays and were correlated with clinical data. RESULTS: Compared with controls, M(2) receptor density was reduced only in the frontal cortex of AD, whereas M(1) was unaffected. Within the AD group, the neurochemical variables were not affected by demographic factors, disease severity, or cognition. Instead, M(2) receptor density was increased in the frontal and temporal cortex of patients with AD with psychotic symptoms compared with those without these symptoms. CONCLUSIONS: This study suggests a role for M(2) receptors in the psychosis of AD and may provide the rationale for treatment of behaviorally perturbed patients with AD with cholinomimetics and M(2) antagonists.     

Postnatal development of D1 dopamine receptors in the medial prefrontal cortex, striatum and nucleus accumbens of normal and neonatal 6-hydroxydopamine treated rats: a quantitative autoradiographic analysis.

The postnatal development of dopamine (DA) D1 receptors in the medial prefrontal cortex (mPFC), striatum (STR) and nucleus accumbens (NAC) of control and perinatally 6-hydroxydopamine (6-OHDA) lesioned rats was examined using quantitative autoradiography of 3H-SCH 23390 binding. D1 receptors are present at one week and increase only slightly to a stable level by 2 weeks in the STR and NAC. Their ontogeny is not altered by intracisternal injection of 6-OHDA 5 days after birth. A biphasic pattern of appearance of D1 receptors was found in the mPFC. D1 receptors are present in the mPFC at 1 week, increase 3-fold by 2-3 weeks, and then decline at 4 and 6 weeks. 6-OHDA lesions do not significantly alter this pattern. At all postnatal ages. D1 receptor binding in the mPFC exhibits a laminar distribution with increased receptor density in deep cortical layers (V, VI) compared to more superficial cortical layers (I, II). Both superficial and deep layers of D1 receptors in the mPFC show similar postnatal developmental patterns. DA turnover rates are consistently about 10-fold higher in frontal pole compared to remainder of forebrain at all postnatal ages. Early 6-OHDA lesions increase DA turnover in forebrain, but lead to a persistent reduction in DA turnover in frontal pole by 2 weeks of age.     

Immunopharmacology--antibodies for specific modulation of proteins involved in neuronal function

The application of antibodies to living neurones has the potential to modulate function of specific proteins by virtue of their high specificity. This specificity has proven effective in determining the involvement of many proteins in neuronal function where specific agonists and antagonists do not exist, e.g. ion channel subunits. We discuss studies where antibodies modulate functions of voltage gated sodium, voltage gated potassium, voltage gated calcium hyperpolarisation activated cyclic nucleotide (HCN gated) and transient receptor potential (TRP) channels. Ligand gated channels studied in this way include nicotinic acetylcholine receptors, purinoceptors and GABA receptors. Antibodies have also helped reveal the involvement of different intracellular proteins in neuronal functions including G-proteins as well as other proteins involved in trafficking, phosphoinositide signalling and neurotransmitter release. Some suggestions for control experiments are made to help validate the method. We conclude that antibodies can be extremely valuable in determining the functions of specific proteins in living neurones in neuroscience research.     

Nicotinic receptor-mediated regulation of dopamine transporter activity in rat prefrontal cortex

The objective of this study was to determine whether nicotine could selectively influence dopamine levels in the prefrontal cortex as compared with other dopaminergic areas of brain. Using a superfusion system, we found that nicotine and other agonists at nicotinic acetylcholine receptors enhanced the release of radiolabeled dopamine that was stimulated by 10 microM amphetamine from slices prepared from rat prefrontal cortex. In contrast, nicotine had no effect on amphetamine-stimulated [(3)H]dopamine release from slices of nucleus accumbens nor striatum. Under the conditions used, which included no added calcium to exclude contribution by exocytotic release, nicotine had no effect on basal release of [(3)H]dopamine. The enhancement by nicotine was concentration-dependent, reaching a maximum at 5 microM, and producing less release at higher concentrations. Enhancement by nicotine was fully reversed by 30 microM dihydro-beta-erythroidine, and by 10 microM mecamylamine, but was not affected by alpha-bungarotoxin. The potencies of nicotine, epibatidine, cytisine, and A85380 to enhance amphetamine-stimulated dopamine release, as well as the sensitivity of nicotine enhanced release to antagonists, are consistent with mediation via a high-affinity nicotinic acetylcholine receptor containing alpha 4 and beta 2 subunits, the major species of nicotinic receptor in forebrain. Since low dopaminergic activity in prefrontal cortex is correlated with cognitive deficits in schizophrenia, our findings may help explain why these deficits are improved in schizophrenics by smoking or nicotine administration.     

BDNF‐induced endocannabinoid release modulates neocortical glutamatergic neurotransmission

Endocannabinoids (eCBs) and neurotrophins, particularly brain‐derived neurotrophic factor (BDNF), are potent neuromodulators found throughout the mammalian neocortex. Both eCBs and BDNF play critical roles in many behavioral and neurophysiological processes and are targets for the development of novel therapeutics. The effects of eCBs and BDNF are primarily mediated by the type 1 cannabinoid (CB1) receptor and the trkB tyrosine kinase receptor, respectively. Our laboratory and others have previously established that BDNF potentiates excitatory transmission by enhancing presynaptic glutamate release and modulating NMDA receptors. In contrast, we have shown that BDNF attenuates inhibitory transmission by inducing postsynaptic release of eCBs that act retrogradely to suppress GABA release in layer 2/3 of somatosensory cortex. Here, we hypothesized that BDNF also induces release of eCBs at excitatory synapses, which could have a mitigating or opposing effect on the direct presynaptic effects of BDNF. We found the highest levels of expression of CB1 and trkB and receptors in layers 2/3 and 5. Surprisingly, BDNF did not increase the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) onto layer 5 pyramidal neurons in somatosensory cortex, in contrast to its effects in the hippocampus and visual cortex. However, the effect of BDNF on mEPSC frequency in somatosensory cortex was unmasked by blocking CB1 receptors or disrupting eCB release. Thus, BDNF‐trKB signaling regulates glutamate release in the somatosensory cortex via opposing effects, a direct presynaptic enhancement of release probability, and simultaneous postsynaptically‐induced eCB release that decreases release probability via presynaptic CB1 receptors.     

Cortical acetylcholine release elicited by stimulation of histamine H1 receptors in the nucleus basalis magnocellularis: a dual-probe microdialysis study in the freely moving rat

Perfusion of the nucleus basalis magnocellularis (NBM) with histamine agonists and antagonists modulates the spontaneous release of cortical acetylcholine (ACh) in freely moving rats. Perfusion of the NBM with Ringer solution containing 100 mM K+ strongly stimulated the spontaneous release of cortical ACh in freely moving rats, whereas perfusion with 1 microM tetrodotoxin reduced cortical ACh spontaneous release by more than 50%. Administration of histamine to the NBM concentration-dependently increased the spontaneous release of cortical ACh. Administration of H1 (methylhistaprodifen) but not H2 (dimaprit) or H3 (R-alpha-methylhistamine) receptor agonists to the NBM mimicked the effect of histamine. Perfusion of the NBM with either H1 (mepyramine or triprolidine) or H2 (cimetidine) receptor antagonists failed to alter ACh spontaneous release from the cortex, however, H1 but not H2 receptor antagonists antagonized the releases of cortical ACh elicited by histamine and methylhistaprodifen. Local administration of H3 receptor antagonists (clobenpropit and thioperamide) to the NBM increased the spontaneous release of ACh from the cortex; this effect was antagonized by H1 receptor antagonism. Conversely local administration of MK-801, a noncompetitive receptor antagonist of the N-methyl-D-aspartate receptor, to the NBM failed to alter ACh spontaneous release from the cortex and to antagonize ACh release elicited by histamine. This study demonstrates that activation of histamine H1 receptors in the NBM increases ACh spontaneous release from the cortex.