Low selenium diet increases the dopamine turnover in prefrontal cortex of the rat

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Pergamon

PII] S9086Ð9075"85#99012Ð3

Neurochem[ Int[ Vol[ 29\ No[ 5\ pp[ 438Ð444\ 0886 Þ 0886 Elsevier Science Ltd Printed in Great Britain[ All rights reserved 9086Ð9075:86 ,06[99¦9[99

LOW SELENIUM DIET INCREASES THE DOPAMINE TURNOVER IN PREFRONTAL CORTEX OF THE RAT  LICA CASTAN ½ O\ ANTONIO AYALA\ ANGE  ANTONIO RODRIGUEZ!GO  MEZ\ ANTONIO J[ HERRERA\ JOSE JOSEFINA CANO and ALBERTO MACHADO Departamento de Bioqu(mica\ Bromatolog(a y Toxicolog(a[ Facultad de Farmacia\ Universidad de Sevilla\ Sevilla 30901\ Spain "Received 3 July 0885^ accepted 29 September 0885# Abstract*It has been proposed that interaction of catecholamines and indoleamines with free radicals may result in the formation of endogenous neurotoxins[ In order to better understand the mechanisms involved in neurodegenerative disorders showing evidence of oxidative stress\ we have studied the basal concentrations and the turnover rates of dopamine\ noradrenaline\ serotonin and their metabolites in the prefrontal cortex of rats that were fed on control or low selenium diets[ Nutritional de_cit of selenium decreases the brain antioxidant protection in experimental conditions by the decrease in glutathione peroxidase activity[ The dopamine and serotonin turnover increased and noradrenaline and 4!hydroxy!2!indoleacetic acid turnover decreased compared to experimental control animals[ The increase of dopamine turnover in experimental rats was accompanied by an increase in tyrosine hydroxylase activity[ These results suggest that the decrease of brain protection against oxidative damage could induce brain damage by disturbing the turnover rate of some monoamines[ Þ 0886 Elsevier Science Ltd

The past two decades have witnessed a great amount of research trying both to link speci_c neurochemical de_cits with diseases that cause impairment of learn! ing and memory and to _nd drugs that can improve this impairment[ This is the case with Alzheimer|s disease\ whose pathogenesis remains unknown[ It has been proposed that free radicals contribute to the pathogenesis of neurodegenerative disorders in which there is substantial evidence of oxidative stress "Olanow\ 0882#[ The brain is particularly vulnerable to oxidative damage due to its high rate of oxygen consumption\ high levels of unsaturated fatty acids and the essentially nonregenerative nature of nervous tissue[ It has been reported that indoleamines and catecholamines act as free radical scavengers "DeLange and Glazer\ 0877#[ Interaction of neuro! transmitters and free radicals may result in the for! mation of endogenous neurotoxin"s# "Volicer and Crino\ 0889^ Fishman et al[\ 0880^ Chen et al[\ 0881#[ The prefrontal cortex "PFC# has an important role in complex emotional and cognitive responses[ PFC is innervated by noradrenergic neurons originating from the locus coeruleus "Fuxe et al[\ 0857^ Unger! Ð*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ To whom all correspondence should be addressed[

stedt\ 0860^ Hokfelt et al[\ 0863^ Swanson and Hart! man\ 0864^ Levitt and Moore\ 0867#[ Dopaminergic terminals have been assumed to arise from the ventral tegmental area "VTA^ A09# and the medial substantia nigra "A8# "Swanson\ 0871^ Kalsbeek et al[\ 0877#[ Serotoninergic projection _bers come from the dorsal raphe nucleus "Anden et al[\ 0855#[ Lesion of the prefrontal cortex in adulthood induces many behavioural disturbances\ which have also been observed after depletion of dopamine "DA# in adult prefrontal cortex "Glowinski et al[\ 0874#[ A number of pharmacological and biochemical _ndings have indicated that the mesoprefrontal DA system exhibits di}erent functional characteristics from other DA sys! tems "Roth\ 0876#[ So\ it has been reported to be devoid of some types of DA autoreceptors "Chiodo et al[\ 0873^ Galloway et al[\ 0875# and a higher rate of transmitter turnover "Bannon et al[\ 0870^ White and Wang\ 0873#[ Therefore\ PFC may be particularly vul! nerable to oxidant stress owing to its higher turnover\ though the oxidative metabolism of dopamine has the potential to generate cytotoxic free radicals "biogenic amines undergo oxidative metabolism that forms H1O1 in a reaction catalyzed by the monoamine oxi! dase!MAO!enzyme#[ The principal line of defence

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against these active oxygen species is the cellular anti! oxidant system\ which includes glutathione per! oxidase "GPx#[ Expression of GPx in most tissues is highly sensitive to dietary levels of selenium "Toyoda et al[\ 0878#\ which is bound to the active site as a selenocysteine and serves as the redox center in cataly! sis[ Nutritional de_ciency of Se is then accompanied by a decrease in the activity of GPx "Combs and Combs\ 0873#[ Although Se de_ciency rarely occurs\ it may be produced by a change in the diet during periods of malnutrition or some inborn metabolic errors like phenylketonuria "Reilly et al[\ 0889^ Wilke et al[\ 0881#[ Moreover\ therapy with Se and other antioxidants results in positive clinical responses in various neurological degenerative diseases associated with increased oxidative damage\ such as neuronal ceroid lipofuscinoses "Westermarck and Santavuori\ 0873#\ multiple sclerosis "Halliwell and Gutteridge\ 0873# and Batten|s disease "Clausen et al[\ 0877#[ For this reason\ in the present communication we described some of the neurochemical e}ects of a short period of Se de_ciency in rats[ We have studied the turnover of noradrenaline "NA#\ DA and its metabolites 2\3!dihydroxy! phenylacetic acid "DOPAC#\ 2!metoxytyramine "2! MT# and homovanillic acid "HVA# along with the turnover of serotonin "4!hydroxytryptamine\ 4!HT# and its main metabolite 4!hydroxy!2!indoleacetic acid "4!HIAA# in Se!de_cient animals[ This approach has been applied to the PFC since some observers believe that free radicals might be involved in cytotoxicity in Alzheimer|s and Parkinson|s diseases "Jesberger and Richardson\ 0880#[ We have used non!isotopic methods to estimate the turnover rate of DA\ NA and 4!HT as follows] "0# the rate of accumulation of DA\ NA\ 4!HT and 2!MT was measured after inhibition of monoamine oxidase by pargyline "Brodie et al[\ 0855#[ "1# The rate of decrease in the content of DOPAC\ HVA and 4!HIAA was determined after treatment with the MAO inhibitor pargyline "Tozer et al[\ 0855#[ In addition\ GPx activity in PFC has been determined[ Likewise\ tyrosine hydroxylase "TH# amount was studied by immunoblot analysis[ EXPERIMENTAL PROCEDURES

Two groups of female Wistar rats\ each weighing 199Ð 149 g\ were fed semisynthetic diets containing 9[1 mg:kg Se "control diet\ Panlab S[L[# or 09[0Ð01[3 mg:kg Se "low Se diet\ Panlab S[L[# for 04 days[ Animals were housed two per cage under controlled environmental conditions[ Food and tap water were allowed ad libitum[ The animals were regularly weighed and their food consumption recorded[ After 04 days of being fed the control or the Se!de_cient diets\ animals

were sacri_ced by decapitation between 09[99 and 00[99 a[m[ and the brains quickly removed[ After the olfactory bulbs were cut o} at the rostral tip of the forceps minor\ the PFC was dissected as previously described "Venero et al[\ 0889# and frozen in liquid nitrogen until analysis[ The total time for isolation of the tissues was less than 2 min[ 4!HT and its metabolite 4!HIAA\ NA\ DA and its metab! olites DOPAC\ 2!MT\ HVA\ pargyline HCL\ glutathione reductase "GR#\ reduced glutathione "GSH# and NADPH were purchased from Sigma "St Louis\ MO\ U[S[A[#[ Par! gyline HCl "64 mg:Kg# was dissolved in 9[8) NaCl and injected intraperitoneally[ Injection volumes were 4 ml:kg[ All other chemicals were of the highest purity available from commercial sources[ GPx activity was measured by a modi_cation of the coupled assay procedure of Paglia and Valentine "0856#[ The brain samples were homogenized "09) w:v# in 9[14 M sucrose by ultrasonic disintegration for two pulses of 29 s at 39 W at 3>C[ Samples were centrifuged at 099\999 g at 3>C and the supernatant was used to determine enzyme activity[ The reaction mixture consisted of 49 mM potassium phos! phate bu}er "pH 6#\ 0 mM EDTA\ 0 mM NaN2\ 9[1 mM NADPH\ 0 E[U[:ml GR\ 0 mM GSH\ 9[14 mM H1O1 in a total volume of 0 ml[ Enzyme source "9[0 ml# was added to 9[7 ml of the above mixture and allowed to incubate for 4 min at room temperature before initiation of the reaction by the addition of 9[0 ml peroxide solution[ Absorbance at 239 nm was recorded for 4 min and the activity was calculated from the slope of these lines as mmol NADPH oxidized:min[ Blank reactions with enzyme source replaced by distilled water were subtracted from each assay[ TH activity was measured as described previously "Rein! hard et al[\ 0875# with some modi_cations[ Brie~y\ samples were homogenized "09) w:v# in 49 mM Tris bu}er con! taining 9[1 mM dithiothreitol "DTT# and 7) sucrose[ Homo! genate was centrifuged at 01\999 g in microfuge for 04 min at 3>C and an aliquot of supernatant was further diluted 0]4 with 29 mM Tris!acetic acid containing 9[0) Triton X!099 and incubated with 9[4 nmol of tyrosineÐHCl containing 9[3 mCi:nmol of L!ðring!2\4!2HŁ!tyrosine "35[9 Ci:mmol speci_c activity^ NEN\ U[S[A[#\ 49 nmol of cofactor 5"R#!L\!erythro! 4\5\6\7!tetrahydrobiopterin\ 4\999 units of catalase and 4 mM DTT in 099 mM potassium phosphate\ pH 5[9[ The released ð2HŁOH was separated by an aqueous slurry of acti! vated charcoal\ and the radioactivity was determined by liquid scintillation counting[ Protein was measured by the method of Lowry et al[ "0840#[ Biogenic amine analysis was performed by HPLC with electrochemical detection "Bioanalytical System model LC! 3B with a glassy carbon electrode# with an HS!2 C07 column "Merck cartridge 014×3 mm column _lled with LiChrospher RP!07\4 mm particle size material#[ The potential was set at 9[7 V against Ag:AgCl reference electrode\ and the mobile phase was prepared according to Dibussolo et al[ "0872#[ Standards were prepared in 9[0 M perchloric acid:0 mM sodium bisul_te and stored at 3>C for up to 1 months[ The detection limit of the assay was 49Ð099 pg:sample[ The brain tissue was homogenized in 9[0 M perchloric acid containing 0 mM sodium bisul_te by ultrasonic disintegration over ice using a Labsonic 0409[ Samples were centrifuged at 29\999 g for 04 min at 3>C and the supernatant was then _ltered through a 9[1 mm _lter[ Analysis were performed in the iso! cratic mode\ at a ~ow rate of 0[9 ml:min[ Concentrations of DOPAC\ HVA\ and 4!HIAA were determined after MAO inhibition by intraperitoneal injec!

Low selenium diet increases the dopamine turnover in prefrontal cortex of the rat

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tion of pargyline "64 mg:kg# "Tozer et al[\ 0855#[ Fractional turnover rates "k# were calculated by linear regression of logarithms of DOPAC\ HVA and 4!HIAA concentrations at 9\ 09 and 19 min after pargyline injection[ The slopes obtained were used to estimate total DOPAC\ HVA and 4! HIAA turnover rate "T#\ as the product of k by the estimated concentration of each metabolite at time 9 "basal con! centration#[ DA\ NA\ 2!MT and 4!HT levels were deter! mined at various times "9\ 09 and 19 min[# after intraperitoneal injection of pargyline "64 mg:kg# "Brodie et al[\ 0855#[ The turnover rate was obtained by calculating the slope of the curve by linear regression[ SDSÐPAGE was performed as described by Laemmli "0869#[ Equal amounts of protein from each individual experiment were loaded into di}erent lanes[ After elec! trophoresis\ the gels were Western blotted[ Blots were incu! bated with anti!TH antibody "monoclonal antibody from Boehringer\ Germany# followed by antimouse IgG alkaline phosphatase conjugate "Goat antimouse IgG Biotin con! jugate from Caltag\ U[S[A[#[ After developing\ blots were photographed immediately using 5×8 cm black and white _lm\ which was then analysed by densitometry using a laser computing densitometer "Molecular Dynamics\ Sunnyvalley\ CA\ U[S[A[#[ Optical density was measured using arbitrary units[ Results are mean2SD of six animals assessed in dupli! cate[ Di}erence between control and Se!de_cient rats for all the parameters studied was assessed by using the Student|s t! test[ The di}erence was considered signi_cant when P³9[94 "# or P³9[90 "#[ Least!squares linear regression was applied for the calculation of all slopes[

RESULTS

There were no signi_cant di}erences in the food intake between control and experimental rats[ The results were] control\ 08[021[6 g:animal:day^ exper! imental\ 08[729[2 g:animal:day[ There were no di}erences in the corporal weight gain[ The results were] controls\ 16[622[4 g:animal:04 days^ Se! de_cient animals\ 15[621[5 g:animal:04 days[ Comparing to control animals\ PFC of Se!de_cient rats showed decreased GPx activity from 26[4920[32 to 29[9820[11 mU:mg of protein "−08[7)\ P³9[90#[ The DA accumulation was linear in both control and Se!de_cient rats after pargyline administration "Fig[ 0a#[ The basal concentration remained without signi_cant changes between control and treated rats[ However\ the turnover rate was higher in Se!de_cient rats "28[0)\ P³9[94# than in controls "Table 0#[ Basal concentration and turnover rate of 2!MT were lower in Se!de_cient than in control rats "−14[2)\ P³9[94 and−52[6)\ P³9[94 respectively^ Table 0#[ After the pargyline administration\ the decline of DOPAC lev! els was exponential in both control and treated rats "Fig[ 0b#[ The basal concentrations and the turnover rates remained without signi_cant changes between both groups[ However\ the fractional turnover rate

Fig[ 0[ E}ects of pargyline administration "64 mg:kg# on DA "A#\ DOPAC "B# and HVA concentrations "C# in the prefrontal cortex of control "# and Se!de_cient rats "ž#[

was higher in Se!de_cient rats than in controls "48[4)\ P³9[90^ Table 0#[ The HVA declination was exponential in both control and treated rats after par! gyline administration "Fig[ 0c#[ Se!treated rats showed lower basal concentrations than control rats "−36[0)\ P³9[90#[ The fractional turnover rate and the turnover rate did not change with diet "Table 0#[ The NA basal concentrations remained without sig! ni_cant changes between control and treated rats[ However\ the NA turnover rate was lower in Se! de_cient than in control animals "−24[8)\ P³9[90^ Table 0#[ The 4!HT basal concentrations remained without signi_cant changes between control and treated rats but the turnover rate was higher in Se!de_cient rats "22[1)\ P³9[94# than in control rats "Table 0#[ The

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Table 0[ Turnover of DA\ 2!MT\ DOPAC\ HVA\ NA\ 4!HT and 4!HIAA in the prefrontal cortex of control and Se!de_cient rats after pargyline administration Basal concentration "pmol:g# Control Se de_cient DA 2!MT DOPAC HVA NA 4!HT 4!HIAA

133[0234[0 88[7205[3 003[7219[3 064[4214[1 558[1248[4 0877[72284[3 0104[82017[4

137[5260[8 63[4207[7 85[4200[8 81[826[2 559[62091[6 0696[42614[9 660[52024[8

Fractional turnover rate "h!0# Control Se de_cient * * 2[9529[28 9[6129[26 * * 1[6829[18

* * 3[7729[45 9[6829[08 * * 0[8729[32

Turnover rate "pmol:g:h# Control Se de_cient 371[82033[5 62[2223[1 240[22096[1 015[3272[0 0696[62086[5 3226[52594[8 2281[32580[5

560[52031[4 15[5222[3 369[82001[0 62[0212[3 0983[62156[7 4667[12844[4 0416[72599[8

The basal concentration values "pmol:g# are mean2SD of six animals injected with saline and killed immediately "t9 min#[ The rats injected with pargyline were killed after 09 or 19 min[ The turnover has been calculated using three data points over a period of 19 min "t9\ 09\ 19 min# for all the substances studied[ The fractional turnover rates "h−0# of DOPAC\ HVA and 4!HIAA are slope2SD of linear regression of logarithms of metabolites concentrations using six animals per data point[ The turnover rates "pmol:g:h# of DOPAC\ HVA and 4! HIAA are calculated multiplying the basal concentration levels by the fractional turnover rates\ and are mean2SD[ The turnover rates "pmol:g:h# of DA\ 2!MT\ NA and 4!HT are slope2SD of the linear regression obtained using six animals per data point[ Statistical signi_cance "Student|s t!test#] P³9[94^ P³9[90 comparing control and Se!de_cient rats[

4!HIAA in Se!de_cient rats showed signi_cant decreases in the basal concentrations "−25[4)\ P ³ 9[90#\ the fractional turnover rate "−18[9)\ P ³ 9[90# and the turnover rate "−44[9)\ P ³ 9[90# compared with the control rats "Table 0#[ The increase in the turnover rate of DA in Se! de_cient rats is in accordance with the increase in TH activity[ The Se!de_cient rats show an increase of 74[3) "P³9[94# in TH activity when compared with control animals "9[97129[914 nmol DA:g prot:h for control group^ 9[04129[933 nmol DA:mg prot:h for experimental group#[ Western blot analysis was used in order to know whether this increase in TH activity was due to the increase in the amount of enzyme[ For Western analysis\ equal amounts of protein from each individual experiment were loaded into di}erent lanes

as described in Experimental Procedures[ Results obtained from a typical experiment are shown in Fig[ 1[ There was no signi_cant change in the amount of TH protein between groups "041[925[5 and 025[3205[5 arbitrary units for control and Se! de_cient animals\ respectively#[ DISCUSSION

The e}ect of Se dietary restriction on antioxidant status was evaluated by quantitative activity of the antioxidant enzyme\ GPx[ The GPx activity decreased signi_cantly in PFC[ The decrease of brain GPx activity after 5Ð7 weeks of Se dietary restriction has been previously reported "Sutphin and Buckman\ 0880#[ This decrease in activity produces an impair!

Fig[ 1[ Western analysis of TH amount in PFC[ TH was detected by the monoclonal antibody[ Proteins were separated by SDSÐPAGE and transferred onto nitrocellulose membrane as described in Experimental Procedures[ Speci_city of the TH!band "marked by an arrow# was ensured by omitting the primary antibody "Lane A#[ Low!selenium diet did not induce any change in TH enzyme amount in PFC "Lane C# when compared with control animals "Lane B#[ Loading conditions] 39 mg[

Low selenium diet increases the dopamine turnover in prefrontal cortex of the rat

ment to the antioxidative enzymatic system\ though GPx is the major protective enzyme against hydrogen peroxide toxicity "Damier et al[\ 0882#[ Our results show changes in some biogenic amines basal concentration and turnover in PFC following a short period of Se!de_cient diet[ The neurotransmitter DA shows a signi_cant increase in turnover in Se! de_cient rats "28[0)#[ This increase of DA turnover was accompanied by a signi_cant increase in the frac! tional turnover rate of DOPAC "48[4)# and a decrease of HVA "−36[0)# turnover[ It is interesting to point out that in the PFC there is a preponderance of HVA over DOPAC\ which seems to be unique to this structure "Sharp et al[\ 0875^ Abercrombie et al[\ 0878^ Venero et al[\ 0889#[ The reason for the low DOPAC levels found in PFC is unknown\ although it has been explained by a di}use DA innervation in this structure "Maisonneuve et al[\ 0889#[ These results could be a sign of degeneration\ since changes in the same direction "increase in DA turnover and changes in the ratio DOPAC:HVA# have been described dur! ing the ageing process "Venero et al[\ 0882#[ Increased DA turnover and metabolism produces H1O1 through MAO activity that under the Se!de_cient condition could generate neuronal membrane lipid peroxi! dation\ resulting in neuronal damage[ The increase in DA turnover in rats fed with low Se diet was accompanied by an increase in TH enzyme activity "74[3) compared to controls#[ However\ this change in activity was not accompanied by an increase in the amount of the TH protein[ These results are partially in agreement with those described in hippocampus where the decrease in antioxidative capacity "by decrease in Se intake# produced the increase in DA turnover along with the increase in TH activity and amount "Castan½o et al[\ 0884#[ In the PFC the amount of TH did not change in similar circumstances\ sug! gesting that the long!term regulation of TH in this structure could be di}erent[ So\ biochemical and elec! trophysiological studies suggest that the meso! prefrontal dopamine system may be devoid of some types of autoreceptors and only contains release!mod! ulating autoreceptors "Chiodo et al[\ 0873^ Galloway et al[\ 0875#[ However\ the increase in TH activity produces the same results\ the increase in DA turn! over[ It could be also important to indicate that an increase in the turnover of DA has also been reported in degenerative stages\ where there was an increase in the biosynthetic activity of DA in order to compensate for the loss of some dopaminergic neurons "Zigmond and Striker\ 0878#[ At the same time\ the relationship between degenerative process\ oxidative deamination of DA\ and glutathione level have been described "Eli!

442

zan et al[\ 0889#[ Increased DA turnover in vivo is associated with increased oxidized glutathione "Cohen and Spina\ 0878#[ These e}ects are most likely mediated by the oxidation of DA\ because the pro! duction of oxidized glutathione can be suppressed by MAO inhibitors "Spina and Cohen\ 0877#[ In this context\ our results suggest that there is an increase in DA turnover by a decrease in antioxidant protection\ which takes place as result of a decrease in GPx[ In these conditions\ the decrease in antioxidant capacity could trigger the increase in TH activity\ with the consequence of an increase in DA turnover[ This cas! cade of events could be the beginning of a degenerative process\ since the increase in DA turnover and metab! olism produces H1O1 through MAO activity\ which under the Se!de_cient condition could generate neu! ronal membrane lipid peroxidation\ resulting in neu! ronal damage[ At the same time it is important to indicate that this process\ induced by a decrease of antioxidative protection\ is produced in di}erent areas of the CNS[ The increase in DA turnover has also been described for other structures of the CNS under dietary de_ciency of antioxidative compounds\ such as Se itself "Castan½o et al[\ 0882a# or vitamin E "Cas! tan½o et al[\ 0882b\c#[ On the other hand\ Se!de_cient rats also showed an increase of 4!HT and a decrease in 4!HIAA turnover in PFC[ Similar results have also been reported in some degenerative conditions such as ageing "Wor! thington and Rosemeyer\ 0863^ Zigmond and Striker\ 0878^ Venero et al[\ 0882#[ At the same time\ it was reported that the neurotoxin 4\5!dihydroxy! tryptamine "4\5!DHT# was formed endogenously in the rat brain after a single dose of methylamphetamine or p!chloroamphetamine "Commins et al[\ 0876a\b#[ Therefore\ part of the 4!HT in the synaptic cleft\ due to an alteration of the oxidative environment\ would be oxidatively converted to neurotoxic derivatives\ such as dihydroxyindoles or tryptamine!3\4!dione "partially oxidized form of 4!HT#\ with the consequent decrease of 4!HIAA[ These neurotoxins could have profound e}ects on neuronal activity "Chen et al[\ 0878#[ It is interesting to note that among all the cortical regions\ the dorsomedial prefrontal cortex "area 8# and cingulate cortex "area 13# had the greatest density of TH!labeled _bers "Morrison et al[\ 0877#[ In addition\ a large number of neurons in this cortical area can be inhibited by electrical stimulation of both DA neurons in the VTA or NA neurons in the locus coeruleus "Mantz et al[\ 0877#[ Therefore\ the changes in DA and NA turnover could be the result of a change in cortical inhibition in the Se!de_cient rats[

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Moreover\ interactions between 4!HT and DA have been observed at the level of cortical and other limbic structures rich in DA projections "Cooper and Abbot\ 0877#[ The ventral tegmental region is a major route of passage for descending and ascending _ber systems\ including the transtegmental 4!HT _bers pathway to the forebrain "Herve et al[\ 0870#[ It has been reported that DA neurons of the raphe provide a substantial input to the PFC "Yoshida et al[\ 0878#[ Experiments involving iontophoretic application of 4!HT onto cortical neurons have described both inhibitory and excitatory e}ects "Phillis\ 0873^ Lakoski and Aghajan! ian\ 0874#\ thus suggesting a more complex role for 4! HT in the cortex than was previously thought[ PFC neurotransmission is of great importance for cognitive and memory processes\ and might be sug! gested that these changes are an important factor in behavioural deviations\ particularly in the memory disturbances characteristic of some neurodegenerative diseases[ Acknowled`ement*This work was supported by a grant from the CICYT SAF 82!0085[ REFERENCES

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