Pregnenolone sulfate increases hippocampal acetylcholine release and spatial recognition

The pregnenolone sulfate is a neurosteroid with promnesic properties. Recently, a correlation between endogenous levels of pregnenolone sulfate in the hippocampus and performance in a spatial memory task has been reported in aged rats. Cholinergic transmission is known to modulate memory processes and to be altered with age. In the present experiment we investigated the effect of increasing doses of pregnenolone sulfate on hippocampal acetylcholine release. Our results show that intracerebroventricular administrations of this neurosteroid induced a dose-dependent increase in acetylcholine release. Administration of 12 and 48 nmol of pregnenolone sulfate induced a short lasting (20 min) enhancement of acetylcholine output with a maximum around 120% over baseline and the administration of 96 and 192 nmol doses induced a long-lasting (80 min) increase that peaked around 300% over baseline. In a second experiment we have observed that the 12 nmol dose enhanced spatial memory performance, whereas the 192 nmol dose was inefficient. These results are consistent with previous work suggesting that, a modest increase in acetylcholine release facilitates memory processes, while elevation beyond an optimal level is ineffective. Nevertheless, neurosteroids may be of value for reinforcing depressed cholinergic transmission in certain age-related memory disorders.     

The neurosteroid pregnenolone sulfate infused into the medial septum nucleus increases hippocampal acetylcholine and spatial memory in rats

The effects of an infusion of the neurosteroid pregnenolone sulfate into the medial septum on acetylcholine release in the hippocampus and on spatial memory were evaluated in two experiments. Results show that pregnenolone sulfate enhanced acetylcholine release by more than 50% of baseline and improved recognition memory of a familiar environment. Therefore, our results suggest that the septo-hippocampal pathway could be involved in the promnesic properties of this neurosteroid.     

GET73 increases rat extracellular hippocampal CA1 GABA levels through a possible involvement of local mGlu5 receptor

N‐[(4‐trifluoromethyl) benzyl] 4‐methoxybutyramide (GET73) is a newly synthesized compound displaying anti‐alcohol and anxiolytic properties. In light of the importance of the hippocampal CA1 subregion in alcohol addiction and anxiety‐like behaviors—this in vivo microdialysis study characterized the effect of GET73 on extracellular GABA levels in the hippocampal CA1 region of the freely moving rat—including a possible role for mGlu5 receptor in mediating this effect. Both intraperitoneal administration (2–10 mg/kg) and local intra‐hippocampal CA1 perfusion with GET73 (50–1000 nM) were associated with a transient, step‐wise increase in dialysate hippocampal CA1 GABA levels. The GET73 (10 mg/kg)‐induced increase in GABA levels was not affected by intra‐CA1 perfusion with either the GABA reuptake inhibitor SKF89976A (0.5 mM) or by local GABA_A (bicuculline; 1μM) and GABA_B (CGP35348; 500 μM) receptor antagonists. On the contrary, the GET73‐induced increase in GABA levels was partially counteracted by the intra‐CA1 perfusion with the mGlu5 receptor negative allosteric modulator MPEP (300 µM). Interestingly, GET73 at the lowest (2 mg/kg) dose tested, by itself ineffective, fully counteracted the increase in GABA levels induced by the mGlu5 receptor agonist CHPG (1000 µM). Taken together, these findings suggest that the GET73‐induced increase in hippocampal CA1 GABA levels operates independently of local GABA reuptake and/or GABA_A or GABA_B receptors. Furthermore, the present data lead to hypothesize a possible interaction between GET73 and mGluR5‐mediated regulation of hippocampal CA1 GABA transmission, an effect which may be relevant to the ability of GET73 to reduce alcohol intake in an alcohol‐preferring rat strain. Synapse 67:678–691, 2013 . © 2013 Wiley Periodicals, Inc.     

Effect of gastrin-releasing peptide on rat hippocampal extracellular GABA levels and seizures in the audiogenic seizure-prone DBA/2 mouse

Gastrin-releasing peptide (GRP), a selective agonist for the BB(2) subtype of bombesin receptor, is reported to depolarise GABAergic interneurons in the stratum oriens layer of the hippocampus. Such an action might lead to increased extracellular levels of GABA in the hippocampus, and result in an anti-convulsant effect with this peptide. We have tested this hypothesis by determining the effect of GRP on extracellular levels of GABA in the ventral hippocampus of the freely moving rat using in vivo microdialysis, and by intracerebroventricular (i.c.v.) administration of GRP to audiogenic seizure-prone DBA/2 mice prior to exposure to the noise of an electric bell. Following local perfusion in the ventral hippocampus by reverse dialysis GRP (10 microM) significantly raised levels of GABA in the recovered dialysates by approximately 40%. In the seizure studies, GRP (30-300 ng) increased the latency to tonic seizure, the number of mice convulsing and reduced the incidence of lethality. In both dialysis and seizure studies, the effects of GRP were blocked by the selective BB(2) receptor antagonist, [D-Phe(6), Leu-NHEt(13)]bombesin (6-13). These experiments provide further functional evidence that activation of the BB(2) receptor may modulate neurotransmission in the hippocampus, and that this action may confer anti-convulsant properties on agonists acting at the BB(2) receptor in the brain.     

Immobilization stress-induced increase of hippocampal acetylcholine and of plasma epinephrine, norepinephrine and glucose in rats

We investigated the role of the hippocampal cholinergic neurons during immobilization stress in rats using a microdialysis technique. Blood levels of glucose, epinephrine and norepinephrine during immobilization stress were also determined. Acetylcholine release was initially increased by immobilization stress, then gradually decreased. Plasma level of epinephrine increased gradually and reached significance at 30 min after the start of immobilization and remained at the elevated level during immobilization. Plasma level of norepinephrine initially increased and reached significance at 30 min after the start of immobilization and remained at the elevated level during immobilization. Plasma level of glucose increased gradually and reached maximum and significance 45 min after the start of immobilization, then decreased. Fifteen min after immobilization, acetylcholine release increased again, while concentrations of epinephrine and norepinephrine were still elevated. Thus the response of acetylcholine and the other responses to immobilization stress were not parallel.     

Effects of estrogen on acetylcholine release in frontal cortex of female rats: involvement of serotonergic neuronal systems

The effects of estrogen on cortically projecting cholinergic neurons were investigated using in vivo microdialysis to measure cortical basal acetylcholine (ACh) levels and serotonin (5-HT)-stimulated ACh release in frontal cortex of freely moving Wistar female rats. Bilateral ovariectomy (OVX) or sham operations were performed under anesthesia. Immediately after surgery, each OVX animal was subcutaneously implanted with pellet containing 0.1/0.5 mg of 17beta-estradiol (E(2)) or a vehicle. Nineteen days later, a transverse microdialysis probe was stereotaxically implanted in the frontal cortex (AP: +2.7 mm, DV: -2.5 mm relative to bregma). Two days later (21 days after beginning of estrogen treatment), in vivo microdialysis experimentation was conducted. Serum E(2) levels of animals with 0.1 and 0.5 mg-pellets were equivalent to those levels during diestrous and proestrous, respectively. Although the replacement of different amounts of E(2) produced significant changes in body weight, it failed to affect basal ACh levels in the frontal cortex. Systemically administered serotonin releasing agent, fenfluramine, significantly increased cortical ACh release in all animal groups. The fenfluramine's ability to increase ACh release was potentiated by E(2) replacement with a 0.5 mg-pellet. E(2)-induced enhancement was also observed when the selective 5-HT(1A) agonist, 8-hydroxy-2-(di-n-propylamino) tetralin, but not the 5-HT(2A/2C) agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, was administered. Therefore, the effect of estrogen on 5-HT-stimulated ACh release might be exerted partly via 5-HT(1A) receptors, and not via 5-HT(2) receptors. These results suggest that the positive effects of estrogen on cognitive functions might be mediated through the ACh-5-HT interactions.     

Infusions of physostigmine into the hippocampus or the entorhinal cortex attenuate avoidance retention deficits produced by intra-septal infusions of the GABA agonist muscimol

Septal γ-aminobutyric acid (GABA) receptor activation is known to disrupt memory formation, although the mechanisms underlying this impairment remain unclear. The present study explored the possibility that high levels of septal GABA receptor activity might impair memory by down-regulating acetylcholine (ACh) function in archicortex and entorhinal cortex. To test this possibility, rats were trained on an avoidance task 15 min after receiving intra-septal infusions of vehicle or muscimol (5 nmol/0.5 μl) combined with unilateral intra-hippocampal (10 μl/1 μl) or intra-entorhinal cortex (1.875 μg/0.25 μl) infusions of vehicle or the acetylcholinesterase inhibitor physostigmine. We demonstrate that these infusions do not alter acquisition performance on a continuous multiple trial inhibitory avoidance task. However, intra-septal infusions of muscimol dramatically impair retention performance 48 h later. More importantly, infusions of physostigmine into the hippocampus or the entorhinal cortex, at doses that do not influence acquisition or retention performance when infused alone, attenuate the impairing effects of the muscimol infusions on retention. We suggest that high levels of septal GABA receptor activity might impair memory by down-regulating ACh levels in the hippocampal region, and that such memory impairments can be ameliorated by increasing ACh levels in the hippocampus or entorhinal cortex.     

Effects of pharmacologic increases in brain GABA levels on cocaine-induced changes in extracellular dopamine

Cocaine-induced increases in extracellular dopamine (DA) concentrations were measured using in vivo microdialysis techniques in the nucleus accumbens (NACC) of freely moving rats. In control animals, cocaine increased extracellular DA concentrations approximately 482% 60 min following administration, returning to baseline values 200 min later. When administered 2 h following an acute dose of γ-vinyl-GABA (GVG, Vigabatrin), cocaine-induced increases in extracellular DA were reduced to approximately 365% of baseline values. Chronic GVG administration further dose-dependently attenuated the effects of cocaine but did not alter the rate of increase or the rate of return to baseline values. These results indicate that GVG, a drug that increases brain GABA concentrations, is effective in attenuating the effect of cocaine on NACC DA. Taken with our earlier findings, these results support the targeting of brain GABAergic systems as a potentially effective pharmacologic treatment strategy for cocaine addiction. Synapse 28:60–65, 1998. © 1998 Wiley-Liss, Inc.     

Delta(9)-tetrahydrocannabinol decreases extracellular GABA and increases extracellular glutamate and dopamine levels in the rat prefrontal cortex: an in vivo microdialysis study

Cannabinoid modulation of prefrontal cortex and hippocampus neuronal functioning has been correlated to the disruptive action of marijuana on memory tasks. This study investigates the effects of delta(9)-tetrahydrocannabinol (delta(9)-THC) on dopamine, glutamate and GABA levels in vivo by brain microdialysis in the prefrontal cortex. Delta(9)-THC (1 mg/kg, i.v.) significantly increased extracellular dopamine and glutamate levels and decreased GABA levels. These effects were prevented by the cannabinoid antagonist SR141716A (1 mg/kg, i.v.), which per se was ineffective. These results suggest that delta(9)-THC disrupt the normal interplay between neurotransmitters in this area and may bear relevance in understanding neuronal mechanisms underlying cannabinoid-induced cognitive deficits.     

Changes in extracellular glutamate and GABA levels in the hippocampal CA3 and CA1 areas and the induction of glutamic acid decarboxylase-67 in dentate granule cells of rats treated with kainic acid

For the evaluation of glutamatergic and GABAergic transmission during seizures, rat hippocampal CA₁ and CA₃ areas were separately assessed by brain microdialysis, and extracelluar glutamate and GABA were measured through the course of the seizures after a systemic administration of kainic acid (KA). The generalized convulsion started at about 1.5 h and was suppressed by diazepam at 2 h after the KA treatment. In the CA₃ area, extracellular glutamate started to increase soon after the KA injection and returned to the control level at about 1.5 h. A decrease and then slight increase of the extracellular glutamate level in CA₃ followed the diazepam injection. In the CA₁ area, in contrast, a long-lasting decrease of extracellular glutamate was observed. The extracellular GABA concentration in the CA₃ area increased immediately after the systemic administration of KA and returned to the normal level at about 3.5 h. A second increase in the extracellular GABA in this area began at about 4.5 h after the KA treatment. In the CA₁ area, an increase of extracellular GABA began at about 3.5 h after KA administration (much later than that observed in the CA₃ area) and was maintained throughout the observation. In situ hybridization showed a transient expression of glutamic acid decarboxylase (GAD)-67 mRNA in the granule cell layer of the dentate gyrus at 4 and 6 h, whereas GAD65 mRNA was unaffected. GABA immunoreactivity in the same area and mossy fibers in the CA₃ were increased most significantly at 8 h after administration of KA. The possible relation of GABA induction in mossy fibers with the delayed increase in extracellular GABA in CA₃ was discussed.     

Acute stress-mediated increases in extracellular glutamate levels in the rat amygdala: differential effects of antidepressant treatment

Depressive illness is associated with changes in amygdalar volume, and stressful life events are known to precipitate depressive episodes in this patient population. Stress affects amygdalar synaptic plasticity and several neurotransmitter systems have been implicated in stress-mediated changes in the brain, including the glutamatergic system. However, the role of the glutamatergic system in stress-mediated plasticity in the amygdala remains to be determined. Accordingly the current study examined the stress modulation of extracellular glutamate levels in the basolateral nucleus (BLA) and the central nucleus (CeA) of the amygdala by in vivo microdialysis. Acute stress increased extracellular glutamate levels in the BLA and CeA, although the dynamics of these stress-mediated changes were dramatically different in these amygdalar nuclei. Tetrodotoxin administration reduced basal, and completely eliminated stress-mediated increases in glutamate efflux in the amygdala, demonstrating that stress effects are dependent on local axonal depolarization. Moreover, stress-mediated increases in glutamate efflux in the BLA were inhibited by the antidepressant tianeptine but not by the selective serotonin-reuptake inhibitor fluoxetine. Collectively, these data demonstrate that stress-induced modulation of glutamate neurochemistry reflects a fundamental pathological change that may contribute to the aetiology and progression of depressive illness, and suggest that some antidepressants such as tianeptine may elicit their clinical effects by modulation of glutamatergic neurotransmission.     

Extracellular GABA concentrations in area 17 of cat visual cortex during topographic map reorganization following binocular central retinal lesioning

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of mammals, plays an important role in cortical reorganization following sensory deprivation, by regulating the level of cortical inhibition and gating changes in receptive field size and synaptic efficacy. In cats it has been shown that 2 weeks after the induction of binocular retinal lesions, GABAergic inhibition, as determined by immunocytochemistry, is decreased in the deafferented region of area 17, whereas 3 months post-lesion, normal GABAergic control is restored within the cortical scotoma. In this study we used in vivo microdialysis to investigate the extracellular GABA concentrations 1–2 months post-lesion, in the sensory-deprived and remote, non-deprived region of area 17. Data were collected at those sample times and sites for which the extracellular glutamate concentrations had been determined in a previous investigation to elucidate the role of this excitatory neurotransmitter in cortical reorganization. As for glutamate, we observed significantly increased extracellular GABA concentrations in non-deprived area 17, whereas in deafferented area 17, extracellular GABA concentrations were comparable to those observed in normal, control subjects. These data suggest that 1–2 months post-lesion the deafferented cortex behaves like normal visual cortex, in contrast to remote, non-deprived cortex. Notwithstanding the increase in extracellular GABA concentration of 134%, the parallel increase in glutamate concentration of 269% could give rise to a net increase in excitability in remote area 17. We therefore suggest that LTP-like mechanisms, and thereby cortical reorganization, might still be facilitated, while possible excessive hyperexcitability is balanced by the moderately increased GABAergic control.     

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: An intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

An investigation of the origin of extracellular GABA in rat nucleus accumbens measuredin vivo by microdialysis

Summary GABA transmission in the nucleus accumbens is believed to play a central role in motivational processes and the expression of psychostimulant drug action. Here we report measurements of extracellular GABA in nucleus accumbens of the rat and investigate its origin. Extracellular GABA was detected using microdialysis in combination with a novel HPLC-based assay. In the awake rat, GABA in the microdialysates (1) increased 10-fold following perfusion with 0.5 mM nipecotic acid, a GABA releasing agent and uptake blocker, (2) increased 7-fold following local perfusion with 50 mM KCl, (3) decreased 50% following perfusion with tetrodotoxin, (4) decreased 50% following perfusion with a Ca²⁺-free medium and (5) decreased 40% following perfusion with high (12.5mM) MgCl. Finally, in the anaesthetized rat, GABA in the microdialysates decreased 50% following i.p. injection of 100mg/kg 3-mercap-toproprionic acid, a GABA synthesis inhibitor. We conclude that GABA in microdialysates from nucleus accumbens of the rat (awake) responds appropriately to selected pharmacological agents and derives at least in part (50%) from neurones.     

Exposure to inescapable but not escapable shock increases extracellular levels of 5-HT in the dorsal raphe nucleus of the rat

The effects of escapable and yoked inescapable electric tailshocks on extracellular levels of serotonin (5-HT) in the dorsal raphe nucleus were measured by in vivo microdialysis. In comparison to either control rats or to their own preshock baseline, rats exposed to inescapable shock showed an increase in extracellular 5-HT within 25 min of shock initiation, and 5-HT levels continued to rise during the remainder of the shock session. Rats that were exposed to comparable shock treatment, but that were given the opportunity to escape, did not show an increase in 5-HT. Rats that were restrained but not shocked also did not show an increase in 5-HT. These results add further support to suggestions that serotonergic changes occur in the dorsal raphe nucleus during inescapable shock and that such changes may contribute to the behavioral effects of inescapable shock.     

Neuropharmacodynamic evaluation of the centrally active thyrotropin-releasing hormone analogue [Leu]TRH and its chemical brain-targeting system

The centrally active thyrotropin-releasing hormone (TRH) analogue pGlu-Leu-Pro-NH(2) ([Leu(2)]TRH) showed a significant increase in the extracellular acetylcholine concentration during its perfusion to the hippocampus in rats, and this effect was manifested upon the delivery of the analogue in much smaller quantities compared to TRH when measured by in vivo intracranial microdialysis. The neuropharmacodynamic efficacy of [Leu(2)]TRH upon intravenous administration was augmented by the use of a brain-targeting derivative in which the progenitor sequence of the mature peptide was embedded in a molecular architecture that promoted enhanced brain delivery, retention and in situ generation of the pharmacologically active molecule. Compared to the unmodified peptide, the targeting system significantly improved the cumulative effect of the treatment on extracellular acetylcholine levels in rats.     

Ca-sensitive inhibition by Pb of α7-containing nicotinic acetylcholine receptors in hippocampal neurons

In the present study the patch-clamp technique was applied to cultured hippocampal neurons to determine the kinetics as well as the agonist concentration- and Ca²⁺-dependence of Pb²⁺-induced inhibition of α7 nicotinic receptors (nAChRs). Evidence is provided that more than two-thirds of the inhibition by Pb²⁺ (3–30 μM) of α7 nAChR-mediated whole-cell currents (referred to as type IA currents) develops rapidly and is fully reversible upon washing. The estimated values for τ_onset and τ_recovery were 165 and 240 ms, respectively. The magnitude of the effect of Pb²⁺ was the same regardless of whether acetylcholine or choline was the agonist. Pre-exposure of the neurons for 800 ms to Pb²⁺ (30 μM) decreased the amplitude and accelerated the decay phase of currents evoked by moderate to high agonist concentrations. In contrast, only the amplitude of currents evoked by low agonist concentrations was reduced when the neurons were exposed simultaneously to Pb²⁺ and the agonists. Taken together with the findings that Pb²⁺ reduces the frequency of opening and the mean open time of α7 nAChR channels, these data suggest that Pb²⁺ accelerates the rate of receptor desensitization. An additional reduction of type IA current amplitudes occurred after 2-min exposure of the neurons to Pb²⁺. This effect was not reversible upon washing of the neurons and was most likely due to an intracellular action of Pb²⁺. Pb²⁺-induced inhibition of α7 nAChRs, which was hindered by the enhancement of extracellular Ca²⁺ concentrations, may contribute to the neurotoxicity of the heavy metal.     

Downregulation of muscarinic- and 5-HT1B-mediated modulation of [H]acetylcholine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts of septal origin

Adult Long-Evans female rats sustained electrolytic fimbria-fornix lesions and, two weeks later, received intrahippocampal suspension grafts of fetal septal tissue. Sham-operated and lesion-only rats served as controls. Between 6.5 and 8 months after grafting, both the [³H]choline accumulation and the electrically evoked [³H]acetylcholine ([³H]ACh) release were assessed in hippocampal slices. The release of [³H]ACh was measured in presence of atropine (muscarinic antagonist, 1 μM), physostigmine (acetylcholinesterase inhibitor, 0.1 μM), oxotremorine (muscarinic agonist, 0.01 μM–10 μM), mecamylamine (nicotinic antagonist, 10 μM), methiothepin (mixed 5-HT₁/5-HT₂ antagonist, 10 μM), 8-OH-DPAT (5-HT_1A agonist, 1 μM), 2-methyl-serotonin (5-HT₃ agonist, 1 μM) and CP 93129 (5-HT_1B agonist, 0.1 μM–100 μM), or without any drug application as a control. In lesion-only rats, the specific accumulation of [³H]choline was reduced to 46% of normal and the release of [³H]ACh to 32% (nCi) and 43% (% of tissue tritium content). In the grafted rats, these parameters were significantly increased to 63%, 98% and 116% of control, respectively. Physostigmine reduced the evoked [³H]ACh release and was significantly more effective in grafted (−70%) than in sham-operated (−56%) or lesion-only (−54%) rats. When physostigmine was superfused throughout, mecamylamine had no effect. Conversely, atropine induced a significant increase of [³H]ACh release in all groups, but this increase was significantly larger in sham-operated rats (+209%) than in the other groups (lesioned: +80%; grafted: +117%). Oxotremorine dose-dependently decreased the ([³H]ACh) release, but in lesion-only rats, this effect was significantly lower than in sham-operated rats. Whatever group was considered, 8-OH-DPAT, methiothepin and 2-methyl-serotonin failed to induce any significant effect on [³H]ACh release. In contrast, CP 93129 dose-dependently decreased [³H]ACh release. This effect was significantly weaker in grafted rats than in the rats of the two other groups. Our data confirm that cholinergic terminals in the intact hippocampus possess inhibitory muscarinic autoreceptors and serotonin heteroreceptors of the 5-HT_1B subtype. They also show that both types of receptors are still operative in the cholinergic terminals which survived the lesions and in the grafted cholinergic neurons. However, the muscarinic receptors in both lesioned and grafted rats, as well as the 5-HT_1B receptors in grafted rats show a sensitivity which seems to be downregulated in comparison to that found in sham-operated rats. In the grafted rats, both types of downregulations might contribute to (or reflect) an increased cholinergic function that results from a reduction of the inhibitory tonus which ACh and serotonin exert at the level of the cholinergic terminal.     

6-fluoroDOPA metabolism in rat striatum: time course of extracellular metabolites

6-[18F]Fluoro-L-DOPA (FDOPA) is an imaging agent used in the study of dopamine terminals in the living brain using positron emission tomography (PET). To better understand the role of tracer metabolism in dynamic FDOPA PET studies, the pharmacokinetics of individual FDOPA metabolites in extracellular space in the striata of anesthetized rats was investigated using in vivo microdialysis. Brain tissues were also analysed to obtain FDOPA metabolite distribution in the combined intracellular and extracellular spaces. Total extracellular [18F] radioactivity in rat striata was observed to rise and peak at 30 min post-injection (p.i.) and declined with clearance half-life of 2 h. In the extracellular space, the dominant FDOPA metabolite at early times was FDOPAC, followed by FHVA at 50 min, then F-sulfoconjugates at 70 min and finally 3-O-methyl-6-Fluoro-L-DOPA (3OMFD) at later times. These results are consistent with the sequential metabolism and brain clearance of L-DOPA and its metabolites. Analysis of whole striatal tissue confirmed the intraneuronal localization of fluorodopamine most likely stored in vesicles. A new but not unexpected finding was the enrichment of 3OMFD in intraneuronal striatal space which is perhaps a factor in its slow cerebral clearance. Since FDOPA PET data reflects the overall pharmacokinetics of several [18F]-metabolites, the observed different rates of formation and clearance and also different neuronal localization of each metabolite contribute to the measures obtained in dynamic FDOPA PET studies. These metabolic steps and their role in tracer kinetics are, thus, important factors to consider in ascribing physiologic significance to PET-derived measures.     

Nitric oxide increases stimulation-evoked acetylcholine release from rat hippocampal slices by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an endothelium-derived relaxing factor and its main mechanism of action is activation of soluble guanylyl cyclase. NO and NO-related compounds have been reported to affect several neuronal functions in the central nervous system. In this study, we investigated the effects of NO donors (sodium nitroprusside (SNP) and (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409)) on acetylcholine (ACh) release from rat hippocampal slices. SNP (10⁻⁵ M) and FK409 (10⁻⁴ M) increased electrical stimulation-evoked ACh release without affecting basal release. As dibutyryl cyclic GMP inhibited stimulation-evoked ACh release, the effects of these NO donors were not due to soluble guanylyl cyclase activation. Atropine increased stimulation-evoked ACh release by blocking presynaptic muscarinic autoreceptors, and SNP increased stimulation-evoked ACh release in the presence of atropine, suggesting that SNP and atropine increase stimulation-evoked ACh release by different mechanisms. The present results indicate that NO enhances some part of the excitation-secretion coupling pathway without inducing ACh release directly and these effects are mediated by cyclic GMP-independent mechanism.