CCK-nitric oxide interaction in rat cortex,striatum and pallidum

We have chosen to study the effects of both nitric oxide (NO) and cholecystokinin neuromodulatory systems in some motor structures that are frequently involved in excitotoxic phenomena. In particular, 7-nitroindazole, a selective inhibitor of neuronal NO synthase, was administered in control and sulfated cholecystokinin octapeptide-treated rats. Cortical surface, striatal and pallidal depth bioelectric activities were examined through Fast Fourier Transform analysis. Cortical and pallidal recordings revealed an increase of rapid standard rhythms after the inhibition of neuronal NO synthase; in contrast, striatal depth recordings showed a marked increase of slow standard rhythms. All these effects were completely abolished by chronic pre-treatment with sulfated cholecystokinin octapeptide. The results suggest a functional co-operation between cholecystokinin and NO systems in the modulation of the bioelectric activity of all the motor structures examined, and the possibility of preventing excitotoxic damages induced by an anomalous balance between excitatory and inhibitory neurotransmitters in these areas.     

Proteomics of rat hypothalamus,hippocampus and pre-frontal/frontal cortex after central administration of the neuropeptide PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts pleiotropic functions, acting as a hypophysiotropic factor, a neurotrophic and a neuroprotective agent. The molecular pathways activated by PACAP to exert its physiological roles in brain are incompletely understood. In this study, adrenocorticotropic hormone (ACTH), prolactin, luteinising hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), brain-derived neurotrophic factor and corticosterone blood levels were determined before and 20, 40, 60, and 120 min after PACAP intracerebroventricular administration. PACAP treatment increased ACTH, corticosterone, LH and FSH blood concentrations, while it decreased TSH levels. A proteomics investigation was carried out in hypothalamus, hippocampus and pre-frontal/frontal cortex (P/FC) using 2-dimensional gel electrophoresis at 120 min, the end-point suggested by studies on PACAP hypophysiotropic activities. Spots showing statistically significant alterations after PACAP treatment were identified by Matrix-assisted laser desorption/ionization-Time of flight mass spectrometry. Identified proteins were consistent with PACAP involvement in different molecular processes in brain. Altered expression levels were observed for proteins involved in cytoskeleton modulation and synaptic plasticity: actin in the hypothalamus; stathmin, dynamin, profilin and cofilin in hippocampus; synapsin in P/FC. Proteins involved in cellular differentiation were also modulated: glutathione-S-transferase α and peroxiredoxin in hippocampus; nucleoside diphosphate kinase in P/FC. Alterations were detected in proteins involved in neuroprotection, neurodegeneration and apoptosis: ubiquitin carboxyl-terminal hydrolase isozyme L1 and heat shock protein 90-β in hypothalamus; α-synuclein in hippocampus; glyceraldehyde-3-phosphate dehydrogenase and prohibitin in P/FC. This proteomics study identified new proteins involved in molecular mechanisms mediating PACAP functions in the central nervous system.     

Age-dependent loss of NMDA receptors in hippocampus,striatum, and frontal cortex of the rat: Prevention by acetyl-l-carnitine

Acute i.p. administration of Acetyl-L-Carnitine (ALCAR), a component of several biological systems, has been found to modify spontaneous and evoked electrocortical activity in young rats, and, in the old rats, to improve learning ability and to increase the number of NMDA receptors in the whole brain. The present study was aimed at ascertaining the effect of chronic treatment with ALCAR added to drinking water on age-related changes in the different brain areas of rats. In twenty-four-month-old rats, ALCAR treatment for six months significantly impeded the decline in the number of NMDA receptors within the hippocampus, the frontal cortex and the striatum compared to the adult animal. This finding thus confirms the previously reported positive effect of ALCAR on the brain NMDA receptor system.     

N-Acetyl-aspartyl-glutamate in the rat dorsal striatum: topographical distribution and effect of sensorimotor cortex lesions

The dipeptide N-acetyl-aspartyl-glutamate (NAAG) has been proposed as putative neurotransmitter of some corticostriatal projections. To further explore this possibility, endogenous NAAG levels were measured in various microdissected striatal regions in normal animals and in those with bilateral lesion of sensorimotor cortex. In intact rats there was a rostro-caudal gradient for NAAG, with highest concentrations in the more caudal portions of the striatum without significant differences between the medial and lateral regions. Decortication induced no significant changes in peptide concentration in any of the striatal regions or in the respective crude synaptosomal (P2) fractions. However, decorticated animals showed a large degree of deafferentation as evidenced by a marked and significant decrease in [³H]glutamate uptake as well as in glutamate levels measured in striatal homogenates or in crude synaptosomal fractions. No changes in striatal dopamine levels were observed in lesioned animals. Thus, these findings are not in favor of the existence of corticostriatal projections arising from the sensorimotor cortex using NAAG as neurotransmitter.     

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.     

Differential effect of morphine on dopaminergic neurons in frontal cortex and striatum of the rat

Inhibition of dopamine synthesis by a single injection of α-methyl-para-tyrosine (200 mg/kg, i.p.) was complete from 30 to at least 300 min after administration. When morphine (20 mg/kg) was given intraperitonealy 30 min after α-MpT treatment an enhanced decline of dopamine was observed in frontal parts of the cortex but not in the striatum. These results indicate that morphine affects dopaminergic neurons in frontal parts of the cortex in a way differently from those in the striatum of the rat. This may be caused either by a difference in the properties of dopaminergic nerve endings in both structures or by an effect of morphine on the input to the cortical system which is lacking in the striatum.     

Acquisition of and withdrawal from cocaine self-administration regulates 5-HT1B mRNA expression in rat striatum

This study investigated how different stages of cocaine self-administration in rats affect the expression of two serotonin receptors in dorsal and ventral striatum, the 5-HT_1B and 5-HT₆ subtypes, which have both been implicated in mediating some aspects of cocaine-related behaviors. In the first experiment, rats were trained to work for saccharin (oral) or cocaine (i.v.) reinforcers. We found that continuous access to cocaine for 23 days did not change the level of 5-HT_1B mRNA expression compared to control animals receiving saccharin. However, a single cocaine session, given either by self-administration or non-contingently, increased 5-HT_1B mRNA in dorsal striatum, whereas forced abstinence for two weeks after cocaine reduced 5-HT_1B mRNA expression in the same subregion. 5-HT₆ mRNA was not changed by any of these treatments. A follow-up experiment investigated the effects of limited versus extended access to cocaine as well as forced abstinence, and we found that 14 days of forced abstinence significantly reduced 5-HT_1B mRNA throughout the dorsal and ventral striatum compared to no withdrawal. These results suggest that the influence of 5-HT_1B receptors in striatal projection neurons may be increased during cocaine acquisition and reduced after forced abstinence and may therefore be targets for pharmacological intervention in addiction.     

The effect of frontal cortex lesion on the activity of the dopaminergic system in rat striatum

The experiment was carried out on male Wistar rats weighing 180-220 g with lesion in the cortex of the frontal lobe. The activity of the dopaminergic system was studied by means of behavioural tests such as determination of spontaneous motor activity, apomorphine-induced stereotypy, haloperidol-induced catalepsy. Increased intensity of stereotypy was observed reaching a maximum 14 days after frontal lobe damage. Moreover, a slight tendency was observed for inhibition of haloperidol-induced catalepsy without changes in the spontaneous motor activity of the animals. Biochemical investigations demonstrated reduced dopamine content in the striatum on the side of the lesion.     

Estimating causal interaction between prefrontal cortex and striatum by transfer entropy

Transfer entropy (TE) is an information-theoretic measure for the investigation of causal interaction between two systems without a requirement of pre-specific interaction model (such as: linear or nonlinear). We introduced an efficient algorithm to calculate TE values between two systems based on observed time signals. By this method, we demonstrated that the TE correctly estimated the coupling strength and the direction of information transmission of two nonlinearly coupled systems. We also calculated TE values of real local field potentials (LFPs) recorded simultaneously in the lateral prefrontal cortex (LPFC) and the striatum of the behavioral monkey, and observed that the TE value from the LPFC to the striatum was stronger than that from the striatum to the LPFC, consistent with anatomical structure between the two areas. Moreover, the TE value dynamically varied dependent on behavior stages of the monkey. These results from simulated and real LFPs data suggested that the TE was able to effectively estimate functional connectivity between different brain regions and characterized their dynamical properties.     

PLG A91 and PLG M6: two new plasminogen allotypes in Japan

Summary In the Japanese two new inherited plasminogen allotypes were observed, PLG A91 and PLG M6, which migrated closely to PLG A and showed normal antigenic levels and functional activities.     

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.     

A spermatozoa-associated factor regulates proenkephalin gene expression in the rat epididymis

The gene encoding the opioid peptide precursor preproenkephalin is expressed at high levels in the initial segment of the adult rat epididymis. Expression is localized to principal cells, the secretory epithelial cells lining the epididymal duct. During development, epididymal proenkephalin mRNA levels show a pronounced increase at about 44 days of age, coincident with the initial entry of spermatozoa into the epididymal lumen. Hypophysectomy leads to a 60-fold decrease in epididymal proenkephalin mRNA levels. Testosterone replacement can prevent this decline in a manner consistent with an effect upon spermatogenesis. Castration studies demonstrate that a gonadal factor other than testosterone directly regulates epididymal proenkephalin expression, and the results of efferent duct ligation suggest that this factor must be supplied through an intact connection of the testis and epididymis. Proenkephalin mRNA levels in the epididymis correlate with the decline and reappearance of spermatozoa induced by the alkylating agent busulphan. Thus, the developmental profile of proenkephalin expression, coupled with the results of both surgical and pharmacological manipulations of the reproductive tract, indicate that spermatozoa, or a spermatozoa-associated factor, regulate proenkephalin gene expression in the epididymis.     

Calcium-binding proteins: differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy

Calbindin, parvalbumin, and calretinin, members of EF-hand calcium-binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium-binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin-immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin-immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium-binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal.     

Tyrosine-ester sulfotransferase from rat liver: bacterial expression and identification.

A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.     

Functional connectivity between prefrontal cortex and striatum estimated by phase locking value

The interplay between the prefrontal cortex (PFC) and striatum has an important role in cognitive processes. To investigate interactive functions between the two areas in reward processing, we recorded local field potentials (LFPs) simultaneously from the two areas of two monkeys performing a reward prediction task (large reward vs small reward). The power of the LFPs was calculated in three frequency bands: the beta band (15–29 Hz), the low gamma band (30–49 Hz), and the high gamma band (50–100 Hz). We found that both the PFC and striatum encoded the reward information in the beta band. The reward information was also found in the high gamma band in the PFC, not in the striatum. We further calculated the phase-locking value (PLV) between two LFP signals to measure the phase synchrony between the PFC and striatum. It was found that significant differences occurred between PLVs in different task periods and in different frequency bands. The PLVs in small reward condition were significant higher than that in large reward condition in the beta band. In contrast, the PLVs in the high gamma band were stronger in large reward trials than in small trials. These results suggested that the functional connectivity between the PFC and striatum depended on the task periods and reward conditions. The beta synchrony between the PFC and striatum may regulate behavioral outputs of the monkeys in the small reward condition.     

Antipsychotic drug treatment induces differential gene expression in the rat cortex

Antipsychotic drug treatment is known to modulate gene expression in experimental animals. In this study, candidate target genes for antipsychotic drug action were searched using microarrays after acute clozapine treatment (1, 6 and 24 h) in the rat prefrontal cortex. Microarray data clustering with a self-organizing map algorithm revealed differential expression of genes involved in presynaptic function following acute clozapine treatment. The differential expression of 35 genes most profoundly regulated in expression arrays was further examined using in situ hybridization following acute clozapine, and chronic clozapine and haloperidol treatments. Acute administration of clozapine regulated the expression of chromogranin A, synaptotagmin V and calcineurin A mRNAs in the cortex. Chronic clozapine treatment induced differential cortical expression of chromogranin A, son of sevenless (SoS) and Sec-1. Chronic treatment with haloperidol regulated the mRNA expression of inhibitor of DNA-binding 2 (ID-2) and Rab-12. Furthermore, the expression of visinin-like proteins-1, -2 and -3 was regulated by chronic drug treatments in various brain regions. Our data suggest that acute and chronic treatments with haloperidol and clozapine modulate the expression of genes involved in synaptic function and in regulation of intracellular Ca2+ in cortex.     

Quinolinate-like neurotoxicity produced by aminooxyacetic acid in rat striatum

Summary The endogenous tryptophan metabolite quinolinic acid elicits in rodent brain a pattern of neuronal degeneration which resembles that caused by L-glutamate. Its qualities as a neurotoxic agent raised the hypothesis that quinolinic acid might be involved in the pathogenesis of human neurodegenerative disorders. Kynurenic acid, another endogenous tryptophan metabolite and preferential N-methyl-D-aspartate (NMDA) antagonist, has been shown to block quinolinic acid neurotoxicity. Here we report that microinjections of aminooxyacetic acid (AOAA), an inhibitor of kynurenine transaminase and of other pyridoxal phosphate-dependent enzymes, into the rat striatum produce neuronal damage resembling that caused by quinolinic acid. AOAA-induced striatal lesions can be prevented by kynurenic acid and the selective NMDA antagonist 2-amino-7-phosphonoheptanoic acid. These results suggest that AOAA produces excitotoxic lesions by depleting brain concentrations of kynurenic acid (inhibition of synthetic enzyme) or due to impairment of intracellular energy metabolism (depletion of cell energy resources). The concept of deficient neuroprotection due to metabolic defects might help to clarify the pathogenesis of human neurodegenerative disorders and to develop strategies that may be useful in their treatment.This work was supported by research grant from the Polish Academy of Sciences.These data have been communicated to the International Congress on Amino Acid Research held in Vienna in August 7–12, 1989.