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Serotonergic Hyperinnervation of the FrontalCortex in an Animal Model of Depression,the Bulbectomized RatDan Zhou,1 Gisela Grecksch,2 Axel Becker,2 Christian Frank, 1 Jurgen Pilz,1and Gerald Huether1*1Psychiatric Clinic of the University of Go¨ttingen, Gottingen, Germany2Pharmacological Institute of the University of Magdeburg, Magdeburg, Germany

We studied the influence of olfactory bulbectomy inrats on three different parameters of serotonin (5-HT)presynapses, 5-HT transporter density, tryptophanhydroxylase apoenzyme concentration, and the levelsof 5-HT and 5-hydroxyindole acetic acid (5-HIAA) invarious brain regions. Compared with sham-operatedcontrols, the Bmax values of [3H]paroxetine binding,the apoenzyme concentration of tryptophan hydroxy-lase and the level of 5-HIAA, and, therefore, the5-HIAA/5-HT ratio were significantly and selectivelyincreased in the frontal cortex of bulbectomized rats,measured 12 weeks after surgery. The most likelyexplanation of the concomitant increase in levels of allthree markers of 5-HT presynapses in the frontalcortex is an increased density of 5-HT innervation inthis remote projection field of the raphe nuclei. It issuggested that the bulbectomy-associated axotomy of5-HT fibers projecting to the bulb stimulates collateralsprouting and synaptogenesis, especially in the frontalcortex. The resulting 5-HT hyperinnervation must beexpected to alter global neuronal activity in this regionand to impair the balance of information flow betweenthis and other brain regions, resulting in a multitudeof secondary behavioral and neurochemical changes.The frontocortical abnormalities observed by brainimaging studies in the brains of depressed patientsmay also be explained by a selective 5-HT hyperinner-vation of this brain region. J. Neurosci. Res. 54:109–116, 1998. r 1998 Wiley-Liss, Inc.

Key words: serotonergic innervation; presynapticmechanisms; bulbectomy; depression; animal model

INTRODUCTIONOlfactory bulbectomy in rats causes a syndrome of

behavioral, endocrine, and neurochemical abnormalities.These slowly developing, long-lasting changes are not a

simple consequence of anosmia but rather the result oflesion-induced alterations of neuronal activity and thesubsequent reorganization of neuronal connectivity inother brain regions. At the behavioral level, this syn-drome is characterized by increased irritability, exagger-ated reactivity to novel stimuli, increased muricidalbehavior, altered sexual and aggressive behaviors, motorhyperactivity, abnormal exploratory behavior, deficits inpassive avoidance and food-reinforced learning, andnumerous other learning deficits and behavioral abnormali-ties (for reviews see Archer et al., 1984; Cain, 1974;Jesberger and Richardson, 1988). In addition to thesebehavioral changes, a number of remarkable physiologi-cal and endocrine alterations were noticed in bulbecto-mized rats, e.g., decreased heart rate and blood pressure(Kawasaki et al., 1980), hyperphagia with lack of accom-panying weight gain (Robinzon et al., 1977), reducedREM sleep (Sakurada et al., 1976), altered thermoregula-tion (Forster et al., 1985), altered neuroendocrine activityof the hypothalamic-pituitary-gonadal system (Hortonand Shepherd, 1979; Lumia et al., 1992) and the hypotha-lamic-pituitary-adrenal system (Cattarelli and Demael,1986; Song et al., 1994), as well as impaired immunefunctions (for review see Song and Leonard, 1995). Thesewidespread and far-reaching physiological and behav-ioral consequences are associated with changes in centraltransmitter systems, with lesion-induced degenerationand regeneration of neuronal connections, and the reorga-nization of neuronal networks in various brain regions,only some of which receive direct projections from the

Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contractgrant number: Hu 351/9–2; Contract grant sponsor: Eli-Lilly Int. Fdn.

*Correspondence to: Prof. Dr. G. Huether, Psychiatric Clinic, Univer-sity Gottingen, von-Siebold-Str. 5, 37075 Go¨ttingen, Germany.

Received 1 June 1998; Revised 13 July 1998; Accepted 15 July 1998

Journal of Neuroscience Research 54:109–116 (1998)

r 1998 Wiley-Liss, Inc.

bulb (for reviews see Hirsch, 1980; Jesberger and Richard-son, 1988).

Many of the behavioral, endocrine, and centraltransmitter abnormalities have striking similarities todisturbances observed in humans with affective illness(Jesberger and Richardson, 1988) and are reversed withan amazing degree of specificity by long-term administra-tion of antidepressant drugs (Leonard et al., 1989; vanRiezen and Leonard, 1990). This suggests that bothdepressed patients and olfactory bulbectomized rats arecharacterized by abnormal patterns of neural connectiv-ity, involving altered activities of several neurotransmit-ter systems, which are attenuated or corrected by thesame drugs with the same time course. Because thereorganization of neuronal connectivity after bulbectomyis the most likely cause of the observed long-lasting andpersisting neurochemical, endocrine, and behavioral alter-ations, the bulbectomized rat offers a unique possibilityfor studying central reorganization processes and theirrelationship to depressive symptoms in an animal model(Jesberger and Richardson, 1988).

Several observations point to a particular role of theserotonergic system in these reorganization processes.First, many of the biochemical and behavioral effectsfound after surgical ablation of the bulbs are likewiseproduced by the selective destruction of its serotonergicinnervation by intrabulb injections of 5,6-dihydroxytryp-tamine (Cairncross et al., 1978). Second, several behav-ioral changes found in bulbectomized rats have beenattributed to an imbalance between the central serotoner-gic and noradrenergic systems (for review see Cairncrosset al., 1979). Third, long-term administration of selectiveserotonin reuptake inhibitors reportedly attenuates orreverses the impairments in behavior, neurotransmitters,and immune functions in bulbectomized rats (Song andLeonard, 1994). Finally, the time course of normalizationof altered behavioral responsiveness in bulbectomizedrats caused by long-term desipramine administrationcoincides with the down-regulation of serotonin (5-HT2A)receptors but not ofb-adrenergic receptors in the frontalcortex (Mudunkotuwa and Horton, 1996).

In the ascending serotonergic projections originat-ing in the pontine and midbrain raphe nuclei, extensivecollateral sprouting and regeneration phenomena havebeen observed after lesions along the course of the medialforebrain bundle. The reinnervated projection fields werefound to contain normal to supernormal amounts of 5-HTseveral months after the lesion (for review see Bjorklundand Stenevi, 1979). As shown recently, the combinedmeasurement of 5-HT content, of the density of presynap-tic 5-HT uptake sites, and of tryptophan hydroxylaseapoenzyme levels is a useful strategy for the assessmentof alterations in the density of 5-HT presynapses ex-pressed in distant projection fields of the raphe nuclei

(Huether et al., 1997). In the present study, we have usedthis strategy to investigate the influence of olfactorybulbectomy on the 5-HT innervation density in differentregions of the rat brain.

MATERIALS AND METHODSAnimals and Treatments

Male Wistar rats (250–300 g, Moellegard BreedingCenter Deutschland G.m.b.H.) were housed five pergroup under standardized conditions of temperature andlighting with free access to food and water. Bilateralolfactory bulbectomy was performed as described byO’Connor and Leonard (1986). Briefly, rats were anesthe-tized with pentobarbital (40 mg/kg intraperitoneally), anda midline skin incision was made to expose the skulloverlying the bulbs. Two 2-mm-diameter holes weredrilled above the bulbs (6.5 mm anterior to the bregmaand 2 mm on both sides of the midline). The olfactorybulbs were cut and removed by aspiration. The resultingspaces were filled with hemostatic sponges, and the skinwas closed with tissue adhesion. Sham-operated rats weretreated in the same manner, including piercing of thedura, but the bulbs were left intact. Eight weeks aftersurgery, rats were killed by decapitation, and their brainswere removed, frozen in liquid nitrogen, and stored at280oC until use. All procedures were conducted inaccordance with German laws for the care and use oflaboratory animals as approved by the Tierschutzkommis-sion des Landes Sachsen-Anhalt.

Sample PreparationIndividual brain regions were dissected from frozen

brains (50–300 mg, depending on the size of the region)and homogenized by sonification in a Branson Sonifier(model 240, set 5.0; Branson, Inc.) for 10 seconds in 5volumes of ice-cold phosphate-buffered saline (PBS;potassium phosphate 10 mM plus 0.9% NaCl [pH 7.4]containing 0.1 mM phenylmethylsulfonyl fluoride [PMSF]and 0.02% thimerosal). A 100-µl aliquot was removedand immediately precipitated with perchloric acid forhigh-performance liquid chromatography (HPLC) analy-sis (see below). The remaining homogenate was centri-fuged at 40,0003 g for 20 min at 4oC, and thesupernatant was used for enzyme-linked immunosorbentassay (ELISA) measurements of tryptophan hydroxylaseapoenzyme concentrations.

The pellet was resuspended in 30 vol of ice-coldbuffer (50 mM Tris/HCl [pH 7.4] containing 120 mMNaCl and 5 mM KCl). After a low-speed centrifugation(1,0003 g, 20 min, 4oC), the supernatant was collectedand centrifuged again at 40,0003 g for 10 min at 4oC.The pellet was washed twice in the same buffer, and the

110 Zhou et al.

final pellet was resuspended in buffer to a final concentra-tion of 60–70 mg wet wt/ml for further binding assays.Both binding and ELISA samples were stored at280oCuntil use.

For quantitative estimation of the tryptophan hy-droxylase apoenzyme contents, midbrain samples ofseveral Wistar rats were pooled, and the apoenzyme oftryptophan hydroxylase was purified as described byCash et al. (1985) and used as standards. Protein concen-trations in ELISA and binding samples were measured byusing the method of Lowry et al. (1951).

[3H]Paroxetine Binding AssaysAliquots of the stored membrane suspensions were

further diluted to 15–20 mg wet wt/ml in the bindingbuffer (0.2–0.3 mg protein/ml) and incubated for 2 hr atroom temperature with [3H]paroxetine (specific activity,20.2 Ci/mmol; New England Nuclear, Boston, MA) in theabsence or presence of 200 µM 5-HT (5-hydroxytrypta-mine hydrochloride; Sigma Chemical Co., St. Louis,MO) for the estimation of specific binding. The reactionmixture consisted of 100 µl of membrane suspension(20–30 µg protein), 100 µl of [3H]paroxetine containingbuffer (six concentrations covering a range of final ligandconcentrations in the assay system from 0.05 to 1.00 nM),and 100 µl of buffer or 5-HT solution in buffer (600 µM).After incubation, the reaction mixtures were readilyfiltered through Whatman GF/B filters using a 12-channelCombi Cell Harvester (model 11025; Skatron Instru-ments A.S., Lier, Norway), and the filters were washedwith 20 vol of ice-cold buffer. The radioactivity trappedby the filters was determined by liquid scintillationspectroscopy.

Tryptophan Hydroxylase ELISANunc Maxisorp-immuno F96 plates were used for

this sandwich ELISA and processed as follows.

● Coating: 80 µl PBS/well containing 10 µg/mlaffinity-purified donkey anti-mouse immuno-globulin G (H1L) serum (Paesel and LoreiG.m.b.H. & Co., Hanau, Germany) and 0.02%thimerosal (Sigma), pH 7.4; incubation overnightat 4oC.

● Blocking: 200 µl PBS/well containing 1.0%gelatin and 0.02% thimerosal; incubation for 30min at room temperature.

● Monoclonal antibody adsorption: 100 µl/wellPBS containing 5.0 µg/ml mouse anti–rat braintryptophan hydroxylase monoclonal antibody(Oncogene Science, Inc., Cambridge, MA) and0.02% thimerosal; incubation for 2 hr at roomtemperature.

● Antigen: 100 µl/well standard or supernatantsamples were added; plates were incubated atroom temperature for 2 hr.

● Polyclonal antibody: 100 µl/well sheep anti–ratbrain tryptophan hydroxylase serum (BiogenesisLtd., Poole, England) diluted 1:1,500 in PBScontaining 0.02% thimerosal; incubation at roomtemperature for 2 hr.

● Peroxidase-conjugated antibody: 100 µl/well an-tibody diluted (according to manufacturer8s in-structions) in PBS containing 0.02% thimerosaland 0.2% Tween-20; incubation for 2 hr at roomtemperature.

After application of monoclonal antibody, antigen-containing samples, polyclonal antibody, and peroxidase-conjugated antibody, the wells were rinsed three timeseach with PBS containing 0.2% Tween-20. The plateswere incubated in a shaker (Rotomix, model 92406;Bioblock Scientific, IA) at 700 rpm. Color-developmentwas performed by the addition of OPD (o-phenylenedi-amine dihydrochloride; Sigma) in 0.05 M phosphatecitrate buffer (pH 5.0, containing 0.03% sodium perbo-rate; Sigma) according to the manufacturer’s instructions.Spectrophotometer (MRX; Dynatech Laboratories Inc.,Chantilly, VA) readings were recorded every 10 min up to30 min. Blanks were obtained by preliminary coating ofthe wells without antigen; their values were subtractedfrom the sample values. Blanks were performed induplicate, and all other measurements in triplicate. Thestandards were obtained by serial dilution of the initialapoenzyme solution (1.0 µg–0.5 ng apoenzyme/ml inPBS containing 0.1 mM PMSF and 0.02% thimerosal).

HPLC Measurements of 5-HT and 5-HydroxyindoleAcetic Acid

Aliquots (100 µl) of the original homogenates werereadily mixed with 100 µl of cold 1.0 M perchloric acidcontaining 0.2% sodium metabisulfite and 0.02% ehtylene-diaminetetraacetic acid (EDTA). Precipitates were spundown at 20,0003 g for 20 min at 4oC, and thesupernatants were loaded to the autosampler of the HPLCapparatus (Gynkothek, Germering, Germany). Twentymicroliters was injected onto a C18 reversed-phase col-umn (Nucleosil; 3 µm, 1203 3 mm; Biometra, Go¨t-tingen, Germany) and eluted with a citrate-based mobilephase (72 mM citric acid, 36 mM sodium dihydrogenphos-phate, 0.25 mM EDTA, and 0.9 mM octane sulfonic acidin 19% methanol [pH 2.4]) at a flow rate of 0.4 ml/minand a temperature of 25oC. The separated indoles weremeasured with a dual on-line detection system consistingof an initial fluorescence detector (model F1000; Merck-Hitachi, Darmstadt, Germany) atlex 5 285 nm andlem5345 nm, followed by an electrochemical detector (model

5-HT Hyperinnervation in Bulbectomized Rats 111

M20; Gynkothek, Germany) at 750 mV and 0.04 nA fullscale. Recoveries were calculated from measurements ofexternal and internal standards.

Data Analysis and StatisticsThe affinity and capacity parameters (dissociation

constant [KD] and Bmax values) of [3H]paroxetine bindingwere derived from Scatchard plots of saturation isothermsof specific binding data measured over a concentrationrange of 0.05–1.00 nM by least-squares regression analy-sis (NCSS 5.9 software, J. L. Hintze, Kaysville, UT), andthe results of tryptophan hydroxylase ELISA were calcu-lated by use of Biolinx 2.0 software (Dynatech). Data areexpressed as means6 SD. The statistical significantdifferences were tested by one-way ANOVA followed bypost hoct-test.

RESULTSSpecific binding of [3H]paroxetine to membrane

preparations was saturable and of high affinity. At equilib-rium, specific binding represented approximately 80% oftotal binding at a [3H]paroxetine concentration of 1.0 nM.The saturation curves were better fitted by a one-siterather than a two-site model, with Hill coefficients (nH)very close to 1. Scatchard transformation of the bindingdata gave a single straight unbroken line, indicating a

single apparent class of binding sites with no evidence ofcooperativity.

The double-sandwich ELISA procedure used formeasurements of tryptophan hydroxylase apoenzymeconcentrations in supernatants of brain homogenates useda specific monoclonal antibody, was very sensitive (detec-tion limit 0.1 ng/well), and gave highly reproducibleresults (intra-assay variance,5%; interassay variance,10%).

HPLC measurements of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels, because of the combinationof electrochemical and fluorescence detection, were verysensitive and reliable (detection limits: 5-HT$15 pg/mgwet wt, 5-HIAA $30 pg/mg wet wt; variance of repeatedmeasurements,5% ).

The KD values of [3H]paroxetine binding measuredin different brain regions were rather similar, and nosignificant differences between sham-operated and bulbec-tomized rats were found in any region (Table I).

The Bmax values of [3H]paroxetine binding differedsubstantially between individual regions. Lowest valueswere measured in the cerebellum, and highest densitieswere found in hypothalamus, brain stem and frontalcortex (Table I). Bulbectomy had no effect on thisparameter in most brain regions studied, excepted thefrontal cortex where the Bmax values of [3H]paroxetinebinding were found to be significantly higher than thevalues measured in sham-operated control rats (Table I).

TABLE I. [ 3H]Paroxetine Binding and Tryptophan Hydroxylase Apoenzyme Content in Brainsof Sham-Operated Control and Bulbectomized Rats†

Brain region Treatment

[3H]Paroxetine binding Tryptophanhydroxylase

(ng/mg protein)Bmax

(fmol/mg protein)KD

(nM)

Frontal cortex Control 1112.46 168.0 0.086 0.03 2.796 0.48Bulbectomy 1443.76 257.8* 0.096 0.04 4.106 0.29*

Occipital cortex Control 841.66 109.8 0.096 0.03 2.026 0.31Bulbectomy 701.06 41.3 0.106 0.02 2.136 0.28

Hippocampus Control 644.66 29.9 0.076 0.02 1.716 0.28Bulbectomy 673.16 92.1 0.066 0.03 1.816 0.32

Hypothalamus Control 1358.76 186.5 0.086 0.03 3.696 0.40Bulbectomy 1413.06 146.0 0.096 0.02 3.726 0.33

Midbrain Control 921.76 105.4 0.066 0.03 2.916 0.39Bulbectomy 956.16 26.7 0.076 0.03 2.856 0.43

Brainstem Control 1074.36 132.5 0.086 0.02 2.976 0.29Bulbectomy 1183.36 95.9 0.106 0.04 3.196 0.37

Cerebellum Control 145.76 26.6 0.056 0.02 0.046 0.02Bulbectomy 128.36 20.8 0.066 0.03 0.036 0.02

†Values are means6 SD of 6 rats per group. The densities of [3H]paroxetine binding sites and the tryptophanhydroxylase apoenzyme contents were measured by binding and ELISA assays performed in triplicate asdescribed in Materials and Methods. The significance of the differences was investigated by one-way ANOVAfollowed by post hoct-test. Significantly different values of the density of [3H]paroxetine binding sites and thetryptophan hydroxylase apoenzyme contents were found only in the frontal cortex between sham-operated controland bulbectomized rats (*P , 0.05). No significant differences of these two parameters were observed in otherbrain regions between sham-operated control and bulbectomized rats.

112 Zhou et al.

The lowest concentrations of tryptophan hydroxy-lase apoenzyme were also found in the cerebellum, andthe highest levels were measured in hypothalamus, mid-brain, brainstem, and frontal cortex (Table I). Similar tothe alterations of 5-HT transporter densities observed inbulbectomized rats, the concentration of this second,independent parameter of 5-HT presynapses was selec-tively and significantly elevated in the frontal cortex. Theincrease in tryptophan hydroxylase apoenzyme concentra-tion in the frontal cortex of bulbectomized rats was evensomewhat more pronounced (43%) than the elevation ofthe Bmax values of [3H]paroxetine binding (29%).

The levels of serotonin and of its major degradationproduct, 5-HIAA, measured in the various brain regionswere lowest in the cerebellum and highest in the hypothala-mus and midbrain (Table II). Bulbectomy had no signifi-cant effect on 5-HT concentration in any region. The levelof 5-HIAA, however, was significantly elevated in thefrontal cortex of bulbectomized rats, as was the ratiobetween 5-HIAA and 5-HT (Table II).

DISCUSSIONWe have recently shown that the combined measure-

ment of 5-HT transporter density, tryptophan hydroxylaseapoenzyme concentration, and 5-HT and 5-HIAA levelsis a useful approach for the assessment of experimentallyinduced alterations of the 5-HT innervation density in agiven 5-HT projection field (Zhou et al., 1996). Amongthese parameters, the 5-HT content, because it fluctuatesconsiderably and is influenced by many other factors, wasfound to be the least reliable measure of changes in thedensity of 5-HT terminals (Huether et al., 1997).

The simultaneous increase of 5-HT transporterdensity, tryptophan hydroxylase apoenzyme levels, and5-HIAA levels in the frontal cortex of bulbectomized ratsis a very strong indication of a higher 5-HT innervationdensity of this cortical region. Presumably, the bulbec-tomy-associated axotomy of 5-HT fibers projecting intothe olfactory bulb is followed by collateral sprouting andthe formation of new 5-HT synapses, especially in thefrontal cortex. Collateral sprouting, regeneration, andsynaptogenesis have been observed after lesions at differ-ent sites along the ascending projections of the 5-HTneurons of the raphe nuclei (Azmitia and Whitaker-Azmitia, 1991; Bjorklund et al., 1981; Jacobs and Azmi-tia, 1992). Such lesion-induced regeneration and sprout-ing of 5-HT axons has been shown to result in theformation of new synapses and the functional recovery of5-HT afferents in the denervated projection fields (Nygrenet al., 1974; Wuttke et al., 1977). In addition to thesprouting observed at the proximal stump of damagedaxons, collateral sprouting and compensatory changes in5-HT innervation density have also been observed atrather remote sites of selective 5-HT lesions in the brain(Bjorklund and Stenevi, 1979). In particular the nonjunc-tional, free 5-HT nerve endings in these areas seem toelongate and establish functional connections with targetsites (Wiklund and Mollgard, 1979). The number of suchnonjunctional 5-HT varicosities, and perhaps thereforethe degree of compensatory changes in the 5-HT innerva-tion density, differs between brain regions and is particu-larly high in the frontal cortex (Seguela et al., 1989). Thelesion-induced rearrangement of 5-HT innervation den-sity, especially the hyperinnervation of the frontal cortex

TABLE II. Regional Differences of 5-HT and 5-HIAA Contents and 5-HIAA/5-HT Ratios in Brainsof Sham-Operated Control and Bulbectomized Rats†

Brain region Treatment5-HT

pg/mg wet wt5-HIAA

pg/mg wet wt 5-HIAA/5-HT

Frontal cortex Control 267.86 29.7 347.36 47.9 1.316 0.24Bulbectomy 252.36 24.9 383.86 39.6* 1.526 0.14*

Occipital cortex Control 165.76 48.6 359.56 55.2 2.266 0.40Bulbectomy 166.36 54.0 337.06 22.8 2.356 0.80

Hippocampus Control 282.26 12.2 397.06 66.0 1.366 0.24Bulbectomy 303.56 47.6 382.06 42.6 1.286 0.21

Hypothalamus Control 498.36 52.9 599.36 53.3 1.206 0.11Bulbectomy 446.86 71.6 632.36 38.8 1.456 0.20

Midbrain Control 358.36 39.7 581.76 51.3 1.616 0.14Bulbectomy 370.06 24.8 666.86 68.1 1.806 0.09

Brain stem Control 289.56 10.5 400.06 25.3 1.386 0.12Bulbectomy 247.76 35.6 363.36 66.2 1.646 0.20

Cerebellum Control 31.06 16.4 64.06 18.5 2.136 0.65Bulbectomy 35.56 13.2 89.86 13.9 2.756 0.97

†Values are means6 SD of the HPLC measurements as described in Materials and Methods (n5 6). Thesignificance of the differences was investigated by one-way ANOVA followed by post hoct-test. Significantdifferences were only found for 5-HIAA content and 5-HIAA/5-HT ratio in frontal cortex between bulbectomizedand sham-operated control rats (*P , 0.05).

5-HT Hyperinnervation in Bulbectomized Rats 113

observed in the present study, may play an important rolein the neurochemical and functional consequences ofolfactory bulb lesions. This assumption is underlined bythe fact that many of the long-term behavioral physiologi-cal neuroendocrine and neurochemical consequences ofbulbectomy are also found after the selective destructionof 5-HT nerve endings and axons by local injections ofthe 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT)into the bulb (Cairncross et al., 1978). On the basis of ourfindings in the present study and numerous reports ofcollateral sprouting and reorganization phenomena after5,7-DHT–induced lesions in other brain regions (Bjork-lund et al., 1973), it is very likely that after the selectivechemical destruction of the 5-HT afferents in the olfac-tory bulb a similar compensatory 5-HT hyperinnervationof the frontal cortex will occur.

The consequences of this 5-HT hyperinnervation oncortical information processing are difficult to assess. Thedense, predominantly nonjunctional 5-HT innervation ofthe cortex is capable of modulating the activity of vastcellular assemblies throughout the cortex (for review seeSeguela et al., 1989). An imbalance in the innervationdensity between these projection fields may have wide-spread global influences on brain function. According toSpoont’s concept of the modulatory role of 5-HT (Spoont,1992), cortical 5-HT activity acts to constraint neuronalinformation processing. It stabilizes signal propagationthrough its inhibitory actions on neuronal activity andprevents impingement of the system by exogenous signalsources. Its activity ensures that only signals of sufficientintensity are able to interfere with current informationflow. Very low levels of 5-HT activity may thereforeimpair the ability of neuronal networks to maintain theintegrity of the signal flow pattern and increase thelikelihood of switching to unstable information process-ing. Regional 5-HT hyperactivity, as seen here in thefrontal cortex of bulbectomized rats, may facilitate theformation of regionally restricted limit cycles in informa-tion flow. Under such conditions, neuronal informationprocessing, e.g., in the hyperinnervated frontal cortex, ispresumably characterized by redundant signal propaga-tion and maintenance of subthreshold response patterns(for a more detailed description of the modulatory role of5-HT in neuronal information processing, see Spoont,1992).

On the basis of these considerations about the roleof the 5-HT system in cortical information processing, the5-HT hyperinnervation of the frontal cortex in bulbecto-mized rats will presumably lead to a decrease of generalneuronal activity and therefore glucose metabolization inthis cortical region. Even though the bulbectomized rathas been used widely as an animal model of depression,no published data on altered regional blood flow andmetabolic rate are currently available. It has, however,

been shown that the lowered cerebral glucose metaboliza-tion in cortical areas after cortical lesions is rapidlyincreased by the administration of an inhibitor of 5-HTsynthesis (Pappius, 1991). Interestingly, PET and SPECTstudies in depressed patients have revealed a localreduction of cerebral blood flow in anterior prefrontalcortical areas (Bench et al., 1993; Drevets et al., 1994;Mayberg et al., 1994). In these patients, very little ispresently known about alterations of the 5-HT innerva-tion density of this cortical region. Although PET studiesof presynaptic monoamine metabolism revealed an in-creased (not decreased) accumulation of [11C] 5-HTP inthe medial prefrontal cortex of depressed patients (Agrenand Reibring, 1994), a globally reduced activity of thecentral 5-HT system is still tacitly assumed by mostresearchers in patients with major depression. This as-sumption is based mainly on 5-HIAA measurements incerebrospinal fluid (for reviews see Meltzer and Lowy,1987; van Praag, 1984), on postmortem studies of 5-HTtransporters (Leake et al., 1991) or postsynaptic 5-HTreceptors (Cheetham et al., 1988; Cheetham et al., 1990;Yates et al., 1990), on the 5-HT–facilitating mode ofaction of long-term antidepressant treatment (for reviewsee Blier and de Montigny, 1994), and on the reappear-ance of depressive symptoms after tryptophan depletionin patients who have a remission (Delgado et al., 1990).However, none of these observations excludes the possi-bility that depression is associated with an imbalanced5-HT innervation density between different brain regions.As indicated from studies on another animal model ofdepression, ‘‘learned helplessness’’ in rats, such imbal-ances may also occur between hypothalamic and hippo-campal densities of presynaptic markers of 5-HT projec-tions (Edwards et al., 1992). Wherever such disruptionsof the balanced 5-HT input between different brainregions occur, they would lead to a multitude of adaptiveresponses, reaching from up- or down-regulation ofpostsynaptic 5-HT receptor expression to alterations insignal transduction by other transmitter systems, includ-ing a multitude of functional and neurochemical changesin many different brain regions. A better understandingand more correct interpretation of this amazingly com-plex pattern of changes requires a better knowledge of theinherited and acquired variability in regional innervationdensities by monoaminergic projections. Furthermore,the primary (presynaptic) effects of experimental manipu-lations ought be separated from their secondary (postsyn-aptic) consequences.

ACKNOWLEDGMENTSSupported by a grant from the Deutsche Forsch-

ungsgemeinschaft (Hu 351/9–2) and from Eli-Lilly Int.Fdn.

114 Zhou et al.

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