Constitutive Nitric Synthase Activity Prefrontal Cortex ...downloads.hindawi.com/journals/np/2000/637096.pdf · separategroupswereused(group 1: PFC+Hippo-campi;groupII: Amygdala+Cerebellum,seeTable

NEURAL PLASTICITY VOL. 7, NO. 3, 2000

Constitutive Nitric Oxide Synthase Activity in the PrefrontalCortex of Rats as an Index of Emotional State before Death

Roman Hauser,* Tomasz Gos, Jadwiga Hartwich, Maciej Krzyanowskiand Aldona Dembifiska-Kie6

Institute ofLegal Medicine, Medical University ofGdaisk, ul.Curie-Sktodowskiej 3a,80-210 Gdarisk, Poland; Department ofClinical Biochemistry, Jagiellonian University,

Medical Faculty, uLKopernika 15 A, 30-501 Krak6w, Poland

INTRODUCTION

The prefrontal cortex (PFC), as a part of the’limbic circuit’, plays a fundamental role inemotional and cognitive processes (Lane et al.,1997; Maguire et al., 1998). This has been shownboth in experimental animal models and in humans,using modem imaging techniques (Lane et al.,1997; Maguire et al., 1998; Middleton & Strick,1997). Numerous excitatory amino acids (EAA)neurons are present in PFC, and glutamate plays akey role in excitatory neurotransmission in thecentral nervous system (McDonald, 1996). Nitricoxide (NO) plays a significant, indirect role in thestimulation of glutamate receptors, particularly theionotropic (iGluR) and metabotropic (mGluR)glutamate receptor types. The stimulation of bothtypes of glutamate receptors increases after thegeneration of NO (Gage et al, 1997; Kendrick etal., 1997), indicating that NO may act as a

modulator of glutamatergic neurotransmission.Both glutamate and the activity of the enzyme

NO-synthase (NOS) respond to noxious, stressfulenvironmental influences. An increased level of

’Corresponding author:tel: +48 58 302 3443fax: +48 58 341 0485e-mail: [email protected]

glutamate was demonstrated post mortem in thebrain tissue of rats that had been subjected tostrong sensory stimulation (Hauser et al., 1999).An increased level of glutamate was also detectedin the intracellular space of PFC and in thehippocampus of rats that were irritated by painstimuli (Bagley & Moghaddam, 1997). Immuno-histochemical investigation of the rat hippocampusdemonstrated the increased expression of NOS inresponse to heat stress (LeGreves et al., 1997).NO-synthase was increased in the periaqueductalgray of rats that were exposed for 15 min to apredator (eat) (Chiavegatto et al., 1998).

In the present work, post-mortem constitutiveNO synthase (eNOS) activity, as well as glutamateconcentration, was assessed in the PFC of rats thathad experienced simultaneous different short-termaversive sensory (acoustic, visual, and mechanical)stimulation. The extreme character and simultaneousapplication of stimuli, applied for a time muchshorter than that reported previously (Baltrons &Garcia, 1997; Le Greves et al., 1997), were chosenbecause they correspond to the reality of forensiccases. In such cases, the victims very often are

exposed to complex stimuli of extreme intensity justbefore death.

Investigations dealing with the problem ofpost-mortem assessment of a very short, intensefear reaction caused by extreme stimulationimmediately before death are lacking (Gos &

(C)Freund & Pettman, U.K. 2000 205

206 ROMAN HAUSER ET AL.

Hauser, 1996). Biochemical ’frozen frames’ ofneurotransmission could help in the reconstructionof events just before deathnamely, was a victimof a crime aware of a life-threatening situationshortly before dying. By using the present experi-mental paradigm, a possible application of thebiochemical assessment of human brain tissuecould be developed for future forensic investigations.

EXPERIMENTAL

Animals

Tests were carried out on 84 male Wistar rats(weighing 270 g to 380 g each), whose brain massranged from 1.6 to 2.1 g. During the experiments,the animals were given free access to water and astandard laboratory diet.

Stimuli

Paired rats were stimulated simultaneouslywith three types of stimuli in a single stimulationsession lasting 15 s. The character of the stimuliapplied might approach that reported in forensiccases, in which the victim was very oftensubmitted to strong, aversive stimuli of differentmodalities (mainly mechanical, visual, andacoustic) acting concomitantly before sudden andviolent death (for example, traffic accidents,explosions, murders). By using the presentexperimental paradigm, the authors aimed to copyreal-life situations within the limits accepted by anEthics Committee.

The acoustic stimulus was produced by usingtwo acoustic whistlers and by scraping the metalgrid with a metal rod. The characteristics of theacoustic stimulus were measured using a Swan 910acoustic analyzer.

The first whistler generated the main harmonic

A at 2624 Hz at the acoustic level 100 dB. Thesecond harmonic B revealed the value of theacoustic level 80 dB, and the third harmonic C theacoustic level of 64 dB. The sharp break in thespectrum D was located near 11 kHz. The maximumlevel of the high frequency band E was about 60dB. This value of the acoustic level was valid forfrequencies between 14 and 16 kHz.

The second whistler generated the mainharmonic at 3840 Hz at the acoustic level 107 dB.This whistler also generated ultrasound frequenciesat the high acoustic level. For example, a frequencyof22700 Hz means that the acoustic level was 50 dB.

The scraping device generated a stable spectrumof a noise-type sound having a main frequency of200 Hz at the acoustic level 54 dB and acontinuous spectrum, which was nearly flat closeto the upper frequency ofthe acoustic analyzer.

The mechanical stimulus was produced byexerting pressure upon the animals with a plasticrod having one flat surface and the other rectangular.This rod was a model of a typical blunt weapon.The mean areas were 30 cm2 for the fiat surfaceand 0.5 cm for the rectangular surface. The forcewas measured using a dynamometric method, withcalibration of the force-measuring device usingstandard masses. The mean value of the force was3 N. The force was applied to the backs and sidesof the animal’s bodies. The stimuli were repeatedusing a frequency of Hz.

The visual stimulus was produced using astroboscope lamp S 100 W with a regulatedfrequency. The characteristics of this stimulus weremeasured using a standard photoelectric photometer(Carl Zeiss Jena), equipped with a detector used forvisual photometry. The visual stimulus was generatedusing 6 Hz as the frequency of flashes. The meanvalue of light intensity at the point located 30 cmfrom the lamp was 10000 Ix. This value was stable,with a deviation of less than 5%.

The stimulation of rats took place in atransparent Plexiglas cage with a movable lid.

CONSTITUTIVE NOS ACTIVITY IN THE RAT PREFRONTAL CORTEX 207

After the stimulation period, the cage was closedand pressurized carbon dioxide gas was infusedinto the cage through plastic tubing. Deathoccurred within min.

For each stimulation session, a pool of stressedanimals was accompanied by a control pool ofunstressed animals. Paired control rats remained for12 h overnight in the same cage as that described forthe stimulated rats. On the morning of the next day,the lid of the cage was moved down withoutdisturbing the animals, and the same terminationprocedure was performed. During the entiretermination procedure, the behavior of the animalswas observed from the neighboring room. Animalsthat slept during the termination were selected asunstressed controls.

Sample Preparation

In an effort to obtain the brain structures forthe eNOS activity assay as soon as possible, twoseparate groups were used (group 1: PFC + Hippo-campi; group II: Amygdala + Cerebellum, see Table1). Each group consisted of stressed and controlanimals. In each of the two stressed animal pools,the stress procedure was identical. The brains ofthe animals from the stressed pool of group I(n=16) were dissected for PFC and hippocampi(one sample of PFC and one of hippocampi werelost during the biochemical assay procedure). Thesame dissection was performed on animals fromthe control pool of group I (n=l 6). From this pool,only the eNOS activity values obtained from theeight animals that slept during the terminationprocedure were included in the results for this pool(Table 1).

The mass of brain tissue needed for applyingbiochemical methods excluded the simultaneousassay of eNOS activity and glutamate concentrationin the same PFC. Therefore, the glutamateconcentration assay was performed in PFC that

were obtained from a third group of animals(group III, see Table 2), consisting of both stressedand control animal pools. The stress-evokingparadigm in the third group was the same as that inthe two groups of animals that were disposed forthe eNOS activity assay. The brains of animalsfrom stressed (n=14) and control (n=14) poolsfrom group III were dissected for PFC. From thispool, only the glutamate concentrations obtainedfrom the six animals that slept during thetermination procedure were included in the results(Table 2).

Brains were removed from all animals anddissected on ice. For PFC collection, the frontalparts of the hemispheres were dissected at thelevel bregma 3.2, according to the coordinates ofPaxinos and Watson (1998), and supplied for thebiochemical assay after the removal of their basalparts at the level of rhinal fissures. The brainstructures were weighed and placed in liquidnitrogen, using a container of the Dewar CP65type (Tayor-Wharton). The time span from themoment of the death of the animals until freezingthe brain structures did not exceed 5 min.

NOS Activity Assay

The NOS activity in the homogenates of braintissue was assayed by monitoring the conversionof [3H]L-arginine to [3H]L-citrulline, with modi-fication of published techniques (Hudetz et al.,1998; Northington et al., 1997). The frozen tissuesamples were homogenized in ice-cold Tris/HCl

buffer (0.5 M, pH 7.5) and then centrifuged for 3min at 10000 g at 4C. The supematant obtainedwas used for the NOS activity assay. Briefly, 50

tL of the supematant was preincubated for 5 minwith or without L-methyl-arginine (1 mM) beforethe addition of (ca. 260000 cpm).[3H]L-arginineIncubations were performed for 30 min at 37C inTris/HCl buffer (0.5 M, pH 5.5) containing 0.03

208 ROMAN HAUSER ET AL.

mM unlabeled L-arginine, I mM NADPH, 0.03mM tetrahydrobiopterin, 100 units calmodulin,and 2 mM CaCI. The reaction was terminatedwith Tris/HCl buffer (0.5 M, pH 5.5) at 4C. The[3H]L-citrulline was separated from [3H]L-arginineusing a Dowex 50 chromatography column. Theradioactivity in the column eluate was counted byliquid scintillation spectrometry (LKB TACKBETA) using Ultima Gold XR scintillation fluid(Canberra Electronic E.U.). The NOS activity wasdefined as the difference between the controls andthe L-methyl-arginine blanks, indicating the NOS-dependent formation of [3H]L-citrulline. Theresults were expressed as the amount of radiolabeledL-citrulline formed during 30 min of incubation permilligram protein (cpm/30/min/mg protein).

The glutamate concentration in the PFC wasevaluated using glutamate dehydrogenase andNAD, according to the Bernt and Bermeyer (1974)method used hitherto (Hauser et al., 1999).

The Student’s t test was used to compare themean values from the tests after the evaluation of

the normal distribution and the equality ofvariances (with p=0.05) matched in pooled results.

All efforts were made to minimize animalsuffering and to reduce the number of animalsused. All procedures were approved by theMedical University of Gdansk Animal Use andCare Committee in accordance with EuropeanCommunity standards and guidelines.

RESULTS

The respective eNOS activities and glutamateconcentrations are presented in Table 1 and Table2. After 15 s of simultaneous mechanical+acoustic+visual stimulation, both the PFC and theamygdala of stimulated rats showed statisticallysignificant increases of eNOS activity versuscontrol animals. Such an increase was not obtainedfor the hippocampi and cerebella. The concentrationof glutamate in PFC of stimulated versus controlanimals was also significantly increased.

TABLE 1

Activity ofeNOS (measured as the amount of [3H]L-citrulline) in tissues from animalssubjected to sensory stimuli (S) for 15 s and in tissues from unstimulated control animals (N)

GROUP I

Prefrontal cortex

15

N

Hippocampi

S N

15 8

GROUP II

Amygdala

N

12

Cerebellum

12

N

[3H]L-citrulline (cpm/30 min/mg protein)

Mean

SD

124980

23620

89650

23940

<0.01

148910

51880

142375

30789

>0.05

227300

43500

178700

29000

<0.05

324920

83800

300710

83710

>0.05

n number of individuals; SD standard deviation; p significance level

CONSTITUTIVE NOS ACTIVITY 1N THE RAT PREFRONTAL CORTEX 209

TABLE 2

Glutamate concentrations in the prefrontal cortex from animals subjected tosensory stimuli (S) for 15 s and from unstimulated control animals (N)

14

GROUP III

N

Glutamate concentration (nmol/mg protein)

Mean

SD

185.5

17.1

<0.02

164

10.8

n number of individuals; SD standard deviation; p significance level

DISCUSSION

The results of the experiments carried out heredemonstrated a very rapid increase in cNOSactivity in the PFC and the amygdala of rats afterexposure to stressful sensory stimulation comparedwith control animals. The absence of this stress-induced cNOS upregulation in the hippocampi andcerebellum argues against a generalized metaboliceffect. As the responsive brain regions, the PFCand the amygdala, process cognitive and emotionalaspects of environmental influences, the observedchanges may be linked to the characteristics of thepresented stimuli.

Because the method applied here does notpermit an explanation of the mechanism involved,whether the increase in activity is related toendothelial nitric oxide synthase (eNOS) or toneuronal nitric oxide synthase (nNOS) is not clear.Neuronal NOS is located exclusively in the solublefraction of cell homogenates, but only a very small

part of eNOS is located there (Giulivi et al., 1998;Northington et al., 1997). Although the presentstudy did not use high-speed or differentialcentrifugation to separate the supematant andparticulate fractions, the percentage of endo-thelium versus brain tissue in the sample wouldargue in favor of nNOS activity. Presumably, thesample used here also includes the nNOS activityof astrocytes (Baltrons & Garcia, 1997; Yamada etal., 1997).

The sensory stimuli had an aversive characterand caused fear (McNish et al., 1997). Fear isaccompanied by an increase in the blood flow ofthe PFC (Lane et al., 1997). Up-regulated nNOSactivity in neurons and perivascular nerves mayplay a key role in the regulation of blood flowdistribution (Hudetz et al., 1998; Jun-Ge et al.,1997).

In a preliminary study, (Hauser et al., 1999),increased post-mortem levels of glutamate wereseen in brain hemispheres after and 10 min

210 ROMAN HAUSER ET AL.

duration of the same aversive simulation as thatpresented here. In the present study, such increaseswere detected in the PFC of rats after 15 s ofstimulation (Table 2). An increase in extracellularglutamate occurs in PFC during pain stimulation(Bagley & Moghaddam, 1997). Coupling of theNMDA receptor-mediated calcium influx andnNOS activation is postulated to be due to aphysical coupling of the receptor and the enzymeby an intermediary adaptor (PSD 95), in additionto the well-known Ca+/calmodulin mechanism.Presumably, NMDA receptor-mediated NO releaseinto the synaptic space must be preceded by thetranslocation of nNOS to synaptic structures bybinding to PSD 95 (Jaffrey et al., 1998).

The catalytic activity of NOS measured in thehomogenates, in the presence of excess calciumand NADPH, represents near-maximum activityand may not correlate with that in vivo.Nevertheless, in the experimental model appliedhere, the measured NOS activityas well as theglutamate concentrationin the brain tissue couldbe taken as indices of the emotional state beforedeath. The increase of cNOS activity in theamygdalaa key structure of the central fearresponse (LeDoux, 1998)--argues in favor of sucha hypothesis (Table 1).

In this study, the very short emotional arousalexisting in experimental conditions immediatelybefore death has been assessed for the first time.Whether similar increases are found in manremains unknown and requires further post-mortem biochemical ifvestigation of human brainstructures. Insight into the emotional state that iscreated in the victim’s brain immediately beforedeath could be very helpful for analyzing thecircumstances leading to death. Additionally,further experiments should be carried out to clarifythe dependence between cNOS activity, themorphological origin of cNOS, and the level ofglutamate in the PFC during the response todifferent types of aversive stimuli.

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