Protective effect of the alpha2-adrenoceptor antagonist, dexefaroxan, against spatial memory deficit induced by cortical devascularization in the adult rat.

The alpha2-adrenoceptor antagonist, dexefaroxan, has been shown in the rat to have neuroprotective and plastic effects against degenerative structural changes in elements of the basalocortical cholinergic system that result from cortical devascularization [Neuroscience 115 (2002) 41]. The present study, using the same experimental protocol, examined the functional consequences of cortical devascularization and dexefaroxan treatment in the Morris water maze memory test. Rats were first trained to find the hidden platform in the test, and then subjected to the devascularization procedure. Thirty-one days later, lesioned rats exhibited a significant deficit in recalling the platform location, compared with sham control animals. A 28-day subcutaneous infusion with dexefaroxan (0.63, 2.5, and 10 mg rat(-1) day(-1)), starting from the moment of the devascularization, protected against this spatial memory deficit.

Inhibition of [3H]-(+)-MK 801 binding to rat brain sections by CPP and 7-chlorokynurenic acid: an autoradiographic analysis.

1. The regional binding of [3H]-(+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d)cyclohepten-5,10-imine maleate ([3H]-(+)-MK 801) to sections of rat brain was measured by an in vitro quantitative autoradiographic technique. A heterogeneous distribution of binding sites was observed. 2. High values of binding were detected in the hippocampal formation and cerebral cortex, while very low binding was found in cerebellum. [3H]-(+)-MK 801 binding was not detectable in white matter tracts or in the brain stem. 3. [3H]-(+)-MK 801 binding was inhibited by increasing concentrations of both 7-chlorokynurenate (1-1000 microM) and ((+)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (0.1-100 microM). High concentrations of both drugs were able to inhibit completely specific [3H]-(+)-MK 801 binding. 4. IC50 values calculated for both 7-chlorokynurenate and CPP-induced [3H]-(+)-MK 801 binding inhibition were similar in all brain regions analyzed. 5. The inhibitory action of 7-chlorokynurenate and that of CPP on [3H]-(+)-MK 801 binding were reversed by addition of glycine and glutamate respectively. 6. It is concluded that activation of glycine and N-methyl-D-aspartate (NMDA) receptors is obligatory for the binding of [3H]-(+)-MK 801 to occur in all of the brain regions examined in the present study. Furthermore, on the basis of the similar regional sensitivities of [3H]-(+)-MK 801 binding to the inhibitory action of 7-chlorokynurenate and CPP, a single pharmacological classification of the NMDA receptor complex in brain is suggested. The cerebellum was not included in the study due to the very low level of [3H]-(+)-MK 801 binding detected under the experimental conditions used.

Suppression of nigrostriatal and mesolimbic dopamine release in vivo following noradrenaline depletion by DSP-4: a microdialysis study.

Pretreatment of rats with the noradrenergic neurotoxin DSP-4 selectively reduced regional levels of noradrenaline in the brain by more than 75%, and decreased the concentration of endogenous DA in microdialysates of the caudate nucleus and nucleus accumbens by 52% and 28%, respectively. Results support the hypothesis that central noradrenergic mechanisms facilitate nigrostriatal and mesolimbic dopamine transmission in vivo.

Suppression of in vivo neostriatal acetylcholine release by vesamicol: evidence for a functional role of vesamicol receptors in brain.

Experiments examined the effects of peripheral and central administration of the vesicular acetylcholine transport blocker vesamicol (AH5183) on the content, synthesis, and release of acetylcholine in the rat brain in vivo. In time course studies, a single intraperitoneal dose of DL-vesamicol (5 mg/kg) rapidly and reversibly (within 2 h) doubled the content of acetylcholine in the striatum and hippocampus, without affecting choline levels or the rate of transmitter synthesis. In microdialysis experiments, the same peripheral dose of drug produced a reversible 55% reduction in endogenous striatal acetylcholine release. A similar inhibitory effect was produced by direct intrastriatal perfusion with vesamicol. Moreover, this effect of vesamicol was (a) concentration-dependent and saturable (EC50 = 68 nM), (b) rapidly reversible, (c) stereospecific for the L-isomer, and (d) poorly mimicked by a vesamicol analog with lower plasma membrane permeability. This profile of effects is consistent with an interaction with a specific vesamicol receptor as defined by previous in vitro binding studies. These results support a functional role for vesamicol receptors in modulating central cholinergic transmission in vivo.

Effects of locus coeruleus lesions on parkinsonian signs, striatal dopamine and substantia nigra cell loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in monkeys: a possible role for the locus coeruleus in the progression of Parkinson's disease.

Six pairs of female squirrel monkeys were given a daily intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 9-14 days, beginning the same day on which they received either a bilateral 6-hydroxydopamine lesion or a sham lesion of the locus coeruleus. Sham animals developed typical parkinsonian signs (i.e. tremor, bradykinesia, hypokinesia and reduced blink rate) which largely recovered by six to nine weeks after the start of MPTP treatment. At nine weeks, post mortem levels of striatal dopamine in these same animals were partially reduced (by 45%), and this only in the putamen, compared to values obtained from three non-operated, normal control animals. Additionally, histological examination revealed a moderate loss of neuronal cell bodies in the substantia nigra, pars compacta. In marked contrast, the locus coeruleus-lesioned monkeys exhibited little or no recovery from the parkinsonian signs induced by MPTP. Post mortem examination of these animals revealed profound decreases in caudate (by 84%) and putamen (by 91%) dopamine content, and severe neuronal cell loss in the substantia nigra pars compacta of all animals. These neurological, biochemical and histological assessments indicate that lesioning of the locus coeruleus impairs the recovery which usually occurs from the parkinsonian manifestations induced by MPTP in squirrel monkeys. The results support the hypothesis that deficient locus coeruleus noradrenergic mechanisms underlie the progression of Parkinson's disease.

Effects of locus coeruleus lesions on the release of endogenous dopamine in the rat nucleus accumbens and caudate nucleus as determined by intracerebral microdialysis.

Bilateral 6-hydroxydopamine lesions of the rat locus coeruleus (a) depleted forebrain norepinephrine levels by 67%, (b) reduced the basal release of dopamine in the nucleus accumbens and caudate nucleus by 26% and 19%, respectively, and (c) reduced (+)-amphetamine-induced release in the nucleus accumbens and caudate nucleus. The locus coeruleus appears to exert a tonic excitatory influence on striatal and limbic dopamine release in vivo.

Drug effects on the release of endogenous acetylcholine in vivo: measurement by intracerebral dialysis and gas chromatography-mass spectrometry.

Intracerebral microdialysis was combined with a sensitive and specific gas chromatographic-mass spectrometric assay to measure the release of endogenous acetylcholine in the rat striatum in vivo. In rats anesthetized with urethane (1.2 g/kg i.p.), the levels of striatal acetylcholine dialyzed into a Ringer's perfusate were: (a) reliably measurable only in the presence of physostigmine; (b) stable at between 3 and 8 h of perfusion (30-75 pmol/20 min in the presence of 75 microM physostigmine); (c) reduced by calcium-free Ringer's solution, tetrodotoxin (0.1 microM), and vesamicol (1.0 microM); and (d) increased by elevated potassium (100 mM), atropine (3-300 microM), and haloperidol (0.75 mg/kg i.p.). In conscious unrestrained rats, the spontaneous release of striatal acetylcholine was not altered significantly following the administration of urethane. The changes in acetylcholine release observed in this study are consistent with the known actions of some drugs or ionic conditions on striatal cholinergic neurotransmission and are evident under the condition of urethane anesthesia. The present results demonstrate the sensitivity and suitability of this method for monitoring endogenous striatal acetylcholine release in vivo.

Modulation of mesolimbic dopaminergic projections by beta-endorphin in the rat.

Intracerebroventricular injection of beta-endorphin stimulated the metabolism of dopamine in a dose-dependent, opiate antagonist-reversible manner. Local injections into the nucleus accumbens also caused similar increases, indicating that the actions of this peptide on mesolimbic dopaminergic projections were occurring at opioid receptor sites within the nucleus accumbens. Tolerance experiments suggested that epsilon opioid receptors may be involved in mediating these effects in the n. accumbens, unlike in the striatum.

Preservation of dopamine release in the denervated striatum.

Dopamine metabolism and release were determined in the striata of rats sustaining varying damage to the nigrostriatal dopamine (DA) projection. DA metabolism, inferred from concentrations of dihydroxy-phenylacetic acid (DOPAC) or homovanillic acid (HVA), decreased with DA denervation of more than 20%. Dopamine release, inferred from the concentration of 3-methoxytyramine (3-MT), did not decrease unless the denervation was at least 80%. The amount of 3-MT per surviving neuron exceeded that for DOPAC over most of the denervation range. Thus, striatal DA release is preserved at normal levels with the survival of only 20% of the striatal DA innervation. Decreases in DA release, rather than decreases in DA metabolism or the density of dopamine innervation, coincide with the appearance of behavioral impairments.

[3H]vesamicol binding in brain: autoradiographic distribution, pharmacology, and effects of cholinergic lesions.

An autoradiographic analysis of high-affinity binding sites for the vesicular acetylcholine transport blocker [3H]vesamicol (2-(4-phenylpiperidino) cyclohexanol; AH 5183) was conducted in rat brain. [3H]Vesamicol binding was displaced 52-99% by DPPN [( 2,3,4,8]-decahydro-3-(4-phenyl-1-piperidinyl)-2-napthalenol) (IC50 = 14 nM) and by ketanserin (500 nM), haloperidol (43 nM), and vesamicol analogs, but not by drugs selective for adenosine, adrenergic, amino acid, calcium channel, monoaminergic, opioid, PCP, sigma, or several other receptor classes. [3H]Vesamicol binding was most concentrated in the interpeduncular nucleus and fifth and seventh cranial nerve nuclei. Moderate binding was found in the lateral caudate-putamen, medial nucleus accumbens, olfactory tubercle, vertical and horizontal diagonal bands of Broca, and basolateral amygdala. The distribution of [3H]vesamicol binding was similar to distributions of acetylcholine (r = 0.88), acetylcholine esterase (r = 0.97), choline acetyltransferase (ChAT) (r = 0.97), and [3H]hemicholinium-3 binding sites (r = 0.95-0.99). Lower correlations were obtained between [3H]vesamicol and muscarinic receptor densities (r = 0.50-0.70). Few exceptions to the match between binding and cholinergic neuronal markers were found, e.g., the molecular layer of the cerebellum and the thalamus. Lesions of cholinergic neuronal projections to the neocortex or hippocampus reduced [3H]vesamicol binding in each of these regions, but to a lesser extent than reductions in ChAT. [3H]Vesamicol binding sites appear to be anatomically associated with brain cholinergic neurons, a locus that is consistent with the control by this site of vesicular acetylcholine uptake.

Injections of deuterated tryptamine into the nucleus accumbens of the rat: effects on locomotor activity and monoamine metabolism.

Previous studies have shown that the systemic injection of tryptamine stimulates locomotion in rats and that the nucleus accumbens, a region involved in locomotion, contains the largest concentrations of binding sites for tryptamine in the brain of the rat. The present study examined the behavioral and neurochemical effects of bilateral injections into the accumbens of a deuterated analog of tryptamine, a,a-[2H]tryptamine. Injections of 25 micrograms a,a-[2H]tryptamine increased movements in rats at 25-70 min after injection and increased vertical (rearing) activity at 25-40 min. Injections of 50 micrograms of a,a-[2H]tryptamine produced a transient suppression of movement and vertical activity at 5-15 min, followed by increases in these activities at 40-65 min after injection that were comparable to the increases elicited by 10 micrograms of d-amphetamine. At 30 min after the injection of 50 micrograms a,a-[2H]tryptamine the concentration of dopamine in the nucleus accumbens was increased by 87%, and was preceded by a transient decrease in the level of the metabolite of dopamine homovanillic acid. The levels of 5-hydroxytryptamine and its major metabolite, 5-hydroxyindoleacetic acid in the nucleus accumbens were not changed. Thus, a,a-[2H]tryptamine may interact with tryptamine receptors in the nucleus accumbens to modulate locomotor behavior through mesolimbic dopamine neurons.

Intracerebral dialysis: direct evidence for the utility of 3-MT measurements as an index of dopamine release.

Intracerebral dialysis was used to monitor the in vivo efflux of striatal dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) in the pentobarbital anesthetized rat. In untreated rats, there were low levels of extra-cellular DA and 3-MT which were increased 15-fold by treatment with amphetamine. Under basal and drug-stimulated conditions, 3-MT concentrations were maintained at approximately 30% of the extracellular DA levels. These data agree with in vivo turnover estimates which indicate that 20 to 30% of DA turnover is through the 3-MT pool in the striatum. In contrast, extracellular DOPAC and HVA levels were reduced only slightly by amphetamine and with a delayed onset. Our data support the hypothesis that striatal DOPAC is an accurate index of intraneuronal DA metabolism and that 3-MT is an index of the extracellular concentration of DA.

Time course of adaptations in dopamine biosynthesis, metabolism, and release following nigrostriatal lesions: implications for behavioral recovery from brain injury.

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.

Quantitative autoradiography of brain binding sites for the vesicular acetylcholine transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH5183).

2-(4-Phenylpiperidino)cyclohexanol (AH5183) is a noncompetitive and potent inhibitor of high-affinity acetylcholine transport into cholinergic vesicles. It is reported here that [3H]AH5183 binds specifically and saturably to slide-mounted sections of the rat forebrain (Kd = 1.1 to 2.2 X 10(-8) M; Bmax = 286 to 399 fmol/mg of protein). The association and dissociation rate constants for [3H]AH5183 binding are 8.6 X 10(6) M-1 X min-1 and 0.18 min-1, respectively. Bound [3H]AH5183 can be displaced by nonradioactive AH5183 and by the structural analog (2 alpha,3 beta,4A beta,8A alpha)-decahydro-3-(4-phenyl-1-piperidinyl)-2- naphthalenol but not by 10 microM concentrations of the cholinergic drugs acetylcholine, choline, atropine, hexamethonium, eserine, or hemicholinium-3 or by the structurally related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, (+/-)-N-allylnormetazocine (SKF 10,047), levoxadrol, or dexoxadrol. Quantitative autoradiography reveals that [3H]AH5183 binding sites are distributed heterogenously throughout the rat forebrain and are highly localized to cholinergic nerve terminal regions. At the level of the caudate nucleus-putamen, the highest concentrations of saturable [3H]AH5183 binding (713-751 fmol/mg of protein) are found in the vertical limb of the diagonal band and the olfactory tubercle, with lesser amounts (334-516 fmol/mg of protein) in the caudate-putamen, nucleus accumbens, superficial layers of the cerebral cortex, and the primary olfactory cortex. At day 7 after transsection of the left fimbria, [3H]AH5183 binding and choline acetyltransferase activity in the left hippocampus were reduced by 33 +/- 6% and 61 +/- 7%, respectively. These findings indicate that [3H]AH5183 binds to a unique recognition site in rat brain that is topographically associated with cholinergic nerve terminals.

Picomolar affinity of 125I-SCH 23982 for D1 receptors in brain demonstrated with digital subtraction autoradiography.

Iodinated SCH 23390, 125I-SCH 23982 (DuPont-NEN), was examined using quantitative autoradiography for its potency, selectivity, and anatomical and neuronal localization of binding to the dopamine D1 receptor in rat brain sections. 125I-SCH 23982 bound to D1 sites in the basal ganglia with very high affinity (Kd values of 55-125 pM), specificity (70-85% of binding was displaced by 5 microM cis-flupenthixol), and in a saturable manner (Bmax values of 65-176 fmol/mg protein). Specific 125I-SCH 23982 binding was displaced by the selective D1 antagonists SCH 23390 (IC50 = 90 pM) and cis-flupenthixol (IC50 = 200 pM) and the D1 agonist SKF 38393 (IC50 = 110 nM) but not by D2-selective ligands (I-sulpiride, LY 171555) or the S2 antagonist cinanserin. Compared with 3H-SCH 23390, the 5- to 10-fold greater affinity for the D1 site and 50-fold greater specific radioactivity of 125I-SCH 23982 makes it an excellent radioligand for labeling the D1 receptor. The concentrations of D1 sites were greatest in the medial substantia nigra and exceeded by over 50% the concentration of D1 sites in the lateral substantia nigra, caudoputamen, nucleus accumbens, olfactory tubercle, and entopeduncular nucleus. Lower concentrations of D1 sites were present in the internal capsule, dorsomedial frontal cortex, claustrum, and layer 6 of the neocortex. D1 sites were absent in the ventral tegmental area. Intrastriatal injections of the axon-sparing neurotoxin, quinolinic acid, depleted by 87% and by 46-58% the concentrations of displaceable D1 sites in the ipsilateral caudoputamen and medial and central pars reticulata of the substantia nigra, respectively. No D1 sites were lost in the lateral substantia nigra. Destruction of up to 94% of the mesostriatal dopaminergic projection with 6-hydroxydopamine did not reduce D1 binding nor, with one exception, increase striatal or nigral D1 receptor concentrations. 125I-SCH 23982 selectively labels D1 binding sites on striatonigral neurons with picomolar affinity, and these neurons contain the majority of D1 sites in rat brain.

1-Methyl-4-phenylpyridine (MPP+): regional dopamine neuron uptake, toxicity, and novel rotational behavior following dopamine receptor proliferation.

The regional uptake and subsequent dopaminergic toxicity, receptor proliferation, and rotational behavior pharmacology following intracerebral 1-methyl-4-phenylpyridine (MPP+) administration was determined in the rat. [3H]MPP+ was transported by the high-affinity dopamine uptake system equally in the caudate-putamen (CP), nucleus accumbens (NA) and olfactory tubercle (OT), and to a lesser extent in the substantia nigra. Consistent with the equivalent uptake of [3H]MPP+ by mesostriatal and mesolimbic dopamine neurons, dopamine concentrations of the ipsilateral CP and NA were decreased equally (83-98%) following a 10, 17.5 or 25 microgram injection of MPP+ along the left medial forebrain bundle (MFB). At four weeks after a 25 microgram injection of MPP+ into the MFB, the concentration (Bmax) of D2 receptors in the left CP was increased by 42% compared with the intact hemisphere. D2 receptors did not proliferate in the denervated nucleus accumbens. The affinity (Kd) of D2 receptors was not affected in either the CP or NA. The MPP+ injection, which was restricted to the region of striatonigral efferent fibers, also produced a 60% decrease in the GABA content of the substantia nigra. Ipsiversive rotational behavior was induced in MPP+-treated rats by systemic injections of d-amphetamine. Systemic injections of neither the dopamine agonist apomorphine nor agonist prodrug formulation of 1-DOPA and carbidopa induced contraversive rotation. These behavioral and neurochemical results are identical to those observed following concomitant destruction of striatonigral GABA and mesostriatal dopamine projections, and indicate that MPP+ may be toxic to GABAergic as well as to A10 and A9 dopaminergic neurons.

125I-LSD autoradiography confirms the preferential localization of caudate-putamen S2 receptors to the caudal (peripallidal) region.

The in vitro binding of 125I-lysergic acid diethylamide (LSD) to horizontal sections of rat brain was quantified with computer-assisted autoradiography. Specific binding of 125I-LSD to D2 and S2 sites, defined with 5 microM (+)-butaclamol, was 65-94% of the total binding. Identification of S2 sites with 50 nM ketanserin showed that over 90% of the butaclamol-displaced 125I-LSD binding in the frontal, cingulate and parietal neocortex was to S2 sites (22-55 fmol/mg protein). 125I-LSD also labeled a dense population of S2 sites (16 fmol/mg protein) in the caudal caudate-putamen at the level of the globus pallidus which exceeded by 5-fold the concentration of S2 sites (3 fmol/mg protein) in more rostral portions of the caudate-putamen. The peripallidal distribution of S2 sites was identical to that observed previously with the less selective S2 label, [3H]spiperone. The dense concentration of S2 sites in the caudal caudate-putamen and their overlap with D2 binding sites indicates that the peripallidal neostriatum may play an important role in interactions between dopamine and serotonin.

Stability of dopamine in an in vitro incubation system using Medium 199.

This study investigated the stability of exogenous dopamine (DA) in vitro in a commercially available medium (Medium 199), with and without additional ascorbic acid, during incubation with pituitary halves in prolactin secretion experiments. After a 3-h incubation period, 26 and 15% of the starting concentrations of DA were degraded in the absence and presence of 1 mM ascorbic acid, respectively; in the absence of pituitary tissue, these reductions in DA concentration were 19% and 0%, respectively. In contrast, 98% of DA was degraded after 3 h of incubation in Krebs-Henseleit bicarbonate buffer which contained no tissue or no antioxidants. A number of antioxidants (including a low concentration of ascorbic acid) which are already present in Medium 199 are most likely responsible for the relative stability of DA in this medium. The loss of DA during incubation in Medium 199 is considered small for this type of study, and additional ascorbic acid only marginally improves DA stability in this medium. We have concluded that there is no need to be overly concerned about the degradation of exogenous DA in vitro for some incubation experiments using Medium 199, and the addition of extra ascorbic acid (which may be deleterious) is not necessary.