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Effects of 6-hydroxydopamine lesions of the medial prefrontal cortex on local cerebral blood flow and D1 and D2 dopamine receptor binding in rats: a quantitative autoradiographic study
EUROPEAN NEUROPSYCHOPHARMACOLOGY ELSEVIER
European
Neuropsychopharmacology.
5 (1995)
95-101
Effects of Ghydroxydopamine lesions of the medial prefrontal cortex on local cerebral blood flow and D, and D, dopamine receptor binding in rats: a quantitative autoradiographic study Michio
Suzuki”.* “Department
, Yasuhiro
Kawasaki’,
Masahiko Hirofumi
Muratad, Mori’
Ryoko
Shibata”,
Masayoshi
of Neuropsychiatry. Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, hDepartment of Neuropsychiatry. School of Medicine, Kunazawa University, Kanazawa, Japan ‘Radioisotope Center. Kanarawa University, Kanazawa. Japan Received
27 December
1993; revised
3 January
1995;
accepted
4 January
Kurachi”, Japan
1995
Abstract The effects of lesions of the dopamine (DA) nerve terminals in the medial prefrontal cortex (MPFC) on local cerebral blood flow (LCBF) and DA receptor binding in rats were investigated. 4 pg of 6-hydroxydopamine (6-OHDA) was infused stereotaxically into the area of the bilateral MPFC of rats pretreated with desmethylimipramine, and control rats received a vehicle solution. Twenty-four days after the operation LCBFs of 23 brain regions were measured using the quantitative autoradiographic N-isopropyl-p-[‘251]’ 10d oamphetamine technique. D, and D, DA receptor binding in various brain regions was also quantified autoradiographically using [“HISCH 23390 and [?H]YM 09151-2 as the respective ligands. 6-OHDA lesions of MPFC produced significant increases in LCBF of the nucleus accumbens, the dorsolateral portion of the caudate-putamen and the anterior cingulatecortex. The lesionedanimalsdid not showdecreasedLCBF in MPFC per se. D, and D, DA receptor binding was not affected in any brain region examined. These results suggest that lesions of the DA nerve terminals in MPFC
induce an enhancementof functional activity in the terminal regionsof the subcortical DA systems,and that hypofunction of the mesocortical dopamine system does not elicit reduced metabolic activity in the prefrontal cortex. Keywords: 6-Hydroxydopamine; Medial prefrontal cortex; Autoradiography; Local cerebra1 blood Schizophrenia
1. Introduction
Several studies have suggested that the medial prefrontal cortex (MPFC), the projection area of mesocortical dopaminergic neurons, may modulate the function of mesolimbic and nigrostriatal dopamine (DA) systems. It has been reported that DA depletion in MPFC is associated with increases in DA and its metabolites in other dopaminergic terminal fields including the nucleus accumbens and striatum. as well
* Corresponding
Neuroscience,
author.
Hospital, S-171 76 Stockholm, + 48 8 34 65 63.
Elsevier Science B.V. SSDI
Present
address:
Department
Psychiatry and Psychology Section,
0924-977X(95)00003-8
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Tel.:
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flow; Dopamine
receptor;
as with enhanced spontaneous and amphetamine-induced locomotor activity in rats (Pycock et al., 1980a,b; Leccese and Lyness, 1987; Haroutunian et al., 1988). Similar subcortical biochemical and behavioral changes are also reported to be elicited transiently by excitotoxin lesions destroying intrinsic neurons in MPFC (Christie et al., 1986). These findings suggest that the mesocortical dopaminergic neurons may exert an inhibitory influence on the subcortical dopaminergic nerve terminals, presumably mediated via corticofugal projections to the subcortical sites. There is increasing evidence suggesting that the prefrontal cortex and subcortical DA systems may play an important role in the pathophysiology of schizophrenia. Many of the recent neuroimaging studies measuring regional cerebral blood flow and
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glucose utilization have revealed decreased activity and attenuated activation of the prefrontal cortex in schizophrenic patients (Ingvar and Franzen, 1974; Kurachi et al., 1985; Weinberger et al., 1986; Suzuki et al., 1992; Andreasen et al., 1992). In addition. data implicating dysregulation of the subcortical DA systems in schizophrenia have also been reported (Seeman et al.? 1984; Wong et al., 1986). However. the mechanisms underlying these findings remain to be elucidated. Although it has been hypothesized that mesocortical dopaminergic hypoactivity and the resultant mesolimbic dopaminergic hyperactivity may be involved in these phenomena (Weinberger et al., 1986), little substantial evidence is available. Therefore, it may be important to clarify the interaction between the prefrontal cortex and the subcortical DA systems in understanding schizophrenia. It is known that local cerebral blood flow (LCBF) correlates closely with local energy metabolism in cerebral tissues and represents local functional activity in the central nervous system (Sokoloff, 1981). NIsopropyl-p-[ “31]iodoamphetamine (““I-IMP) is highly trapped on first pass through rat brain when injected and is washed out slowly (Winchell et al.. 1980). Therefore the initial distribution of “‘I-IMP reflects regional brain perfusion (Kuhl et al., 1982). ““I-IMP has been used for measurements of LCBF in both humans and small animals employing single photon emission computed tomography and quantitative autoradiography, respectively. The quantitative autoradiographic ““I-IMP technique is a potentially powerful method for studying the effect of drugs or brain lesioning on local functional activity in the brain. Meanwhile, quantitative autoradiography has also been applied to determine the distribution of various neurotransmitter receptors in the brain (Young and Kuhar, 1979; Savasta et al., 1986; Unis et al., 1990). In the present study, we employed “‘I-IMP, a radiopharmaceutical labelled with 12’1 instead of “‘I, to examine the effect of 6-hydroxydopamine (6OHDA) lesions of the DA nerve terminals in MPFC on LCBF in various brain regions in rats. In addition, we applied quantitative receptor autoradiography to investigate the changes in D, and D, DA receptor binding using [‘H]SCH 23390 and [3H]YM 09151-2, respectively.
2. Experimental
procedures
2.1. Preparation
of animals
.5 (lW.5)
P-101
imipramine (25 mgikg i.p.) 30 min before the operation to protect noradrenergic terminals. The animals were anesthetized with pentobarbital (30 mgikg i.p.) and immobilized in a stereotaxic apparatus. The rats were given one of the following injections through a 30-gauge stainless steel cannula into the area of the bilateral MPFC: vehicle (2 ~1, 0.1% ascorbic acid in Ringer’s solution) or 6-OHDA (Sigma Chem. Co., St. Louis, USA) (4 pg dissolved in 2 ~1 vehicle). The stereotaxic coodinates used were A, bregma + 2.7 mm; L, 2 0.8 mm; V, 3.3-3.4 mm from dura (Paxinos and Watson, 1986). Both vehicle and 6-OHDA were infused over a 2 min period and the injection cannula was left in place for an additional 3 min after cessation of the infusion. The rats were allowed to recover, and the following studies were performed 24 days after the operation. 2.2.
L’51-IMP autoradiography
After insertion of femoral vein and artery catheters under ether anesthesia, the rats were loosely taped to a board and allowed to recover from anesthesia for at least 2 h. 1.85 MBq of 12”I-IMP (spec. act. 12.2-12.6 GBqimmol, Nihon Medi-Physics, Takarazuka, Japan) was injected intravenously, and arterial blood samples were drawn continuously for 3 min. Then the animals were decapitated, and the brains were removed rapidly and frozen in hexane chilled with dry ice. 20 pm thick coronal sections of the brain were obtained with a cryostat maintained at - 20°C. X-ray film (Sakura, Tokyo, Japan) was exposed to the sections for 1 month with gelatin standards of known 12’1 concentrations. The film was developed and the optical densitometry of the autoradiograms were measured using a CCD camera (TI-22A, NEC Co., Tokyo, Japan) connected to a personal computer (PC 9801, NEC Co., Tokyo, Japan) with an image memory board (ADS Co., Nara, Japan) and the program DIAL10 (ADS Co., Nara. Japan). Twenty-three brain regions were selected as regions of interest (Fig. 1, Table l), and anatomical regions were identified by comparing the autoradiograms with atlases of the rat brain (Kbnig and Klippel, 1963; Zilles, 1985). LCBF was calculated from brain and plasma radioactivities according to a reference sample method (Kuhl et al., 1982). 2.3. D, and D2 receptor
Male Wistar rats weighing 220-240 g at the beginning of the experiment were housed at 24 -+ 2°C with a 12 h light/12 h dark cycle and with free access to food and water. The rats were pretreated with desmethyl-
autoradiography
The rats were decapitated, and the brains were rapidly removed and frozen. Coronal tissue sections (20 pm in thickness) were prepared at - 20°C using a cryostat and were thaw-mounted onto gelatin-coated slides.
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Fig. 1. Schematic drawings of the regions of interest in LCBF measurement. AC. anterior cingulate cortex; ACC, nucleus accumbens; AMC. central amygdaloid nucleus; AML. lateral amygdaloid nucleus; CAI ( hippocampus CAI; CPA, caudate-putamen, anteromedial; CPD, caudate-putamen, dorsolatcral; DC, hippocampus, dentate gyrus; DM, dorsomedial thalamic nucleus; HT, hypothalamus; LH. lateral habenula; MC, medial geniculate hody; MPC, medial precentral cortex; MPFI. medial prefrontal cortex (rostra1 to lesion); MPF2, medial prefrontal cortex (lesioned area): NC, substantia nigra, pars compacta; NR, substantia nigra. pars reticulata; PA, primary auditory cortex; PM, primary motor cortex; PS. primary somatosensory cortex; PV, primary visual cortex; SP. septum; VT, ventral tegmental area.
D, binding sites were assayed using [3H]SCH 23390 (spec. act. 2.9-3.1 TBqimmol, DupontiNEN, Boston, MA, USA) as a ligand. The slices were preincubated for 20 min in 50 mM Tris buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl,, and 1 mM MgCl,. The sections were then incubated for 2 h at 25°C in the same buffer containing 0.3 nM [SH]SCH 23390. Ad’ latent sections were incubated in the presence of excess (100 nM) cold R( + )-SCH 23390 to determine the non-specific binding. The incubation was terminated by rinsing sections for 20 min in ice-cold buffer and the sections were dried rapidly under a stream of cold air. Labeled slides were apposed to tritium-sensitive Hyperfilm (Amersham, Arlington Heights,VA, USA) for 3 weeks to 2 months along with gelatin tritium standards with known concentrations of tritium. The film was developed and the optical densities of the autoradiograms were determined with a CCD camera (TI-22A. NEC Co., Tokyo, Japan) connected to a personal computer (Macintosh IIci, Apple Computer Inc.) with a graphics display board (RasterOps 24STV, RasterOps Co., CA) using the program Image 1.35 (National Institute of Health). Ten brain regions were selected as regions
of interest (Table 2). Specific binding values were obtained by the subtraction of non-specific binding from the corresponding total binding. The optical densities were converted into fmol SCH 23390/mg tissue by reference to the tritium standards. D, binding sites were assayed using [“H]YM 091512 (spec. act. 3.2 TBq/mmol, Dupont/NEN, Boston, MA, USA) as a ligand. The ligand concentration was 0.3 nM. The assay procedure was essentially the same as that used for D, binding except that 0.1% ascorbic acid was added in the incubation medium and 10 PM S( - )-sulpiride was used as a displacer. [“H]YM 09151-2 binding was determined in six brain regions with a relatively high Dz DA receptor density (Table 2).
2.4. Statistical analysis The effects of the 6-OHDA lesions on LCBF and D, and Dz DA receptor binding in comparison with the sham-operated animals were examined by Student’s t-test. Statistical significance was defined as
P < 0.05.
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3. Results
Mean values of LCBF in the cortical and subcortical regions of the rat brain are presented in Table 1. LCBFs in the cerebral cortices were generally higher in the 6-OHDA-lesioned rats than in the sham-operated animals, but no statistically significant differences were found in most of the regions. 6-OHDA lesions produced significantly increased LCBF only in the right anterior cingulate cortex (P < 0.05). 6-OHDA lesions did not elicit a decrease in LCBF in the lesioned MPFC per se. In subcortical regions, the rats with bilateral 6-OHDA lesions of MPFC showed significantly increased LCBF in the bilateral nucleus accumbens (right, P< 0.05; left, PCO.01). LCBF in the caudate-putamen was also higher in the lesioned animals than in the controls, with the difference in the right dorsolateral portion reaching statistical significance (P < 0.05).
4. Discussion This study examined the effects of 6-OHDA lesions of MPFC on LCBF and DA receptor bindings in rats using the quantitative autoradiography. To our knowledge, this is the first to investigate changes in LCBF after lesions of the DA nerve terminals in the MPFC. 6-OHDA lesions produced increased LCBF in the nucleus accumbens, the dorsolateral portion of caudate-putamen and the anterior cingulate cortex, while D, and D, receptor binding was not affected in any brain regions examined. Pycock et al. (1980a,b) reported that 6-OHDA lesions of MPFC induced increases in the content of DA and its metabolites in the striatum and nucleus accumbens, suggesting that lesions in the frontal cortical DA system may result in subcortical DA hyperactivity. Other investigators have duplicated these findings (Leccese and Lyness, 1987; Haroutunian et al., 1988). The dosage of 6-OHDA injected into MPFC in the present study was smaller than those
3.2. Changes in D, and Dz receptor binding Data are shown in Table 2. There was no significant change in [3H]SCH 23390 or [3H]YM 09151-2 binding
Brain
lesions
in the medial
prefrontal
region
cortex
on local Local Control
Cortical regions Medial prefrontal cortex (rostra1 to lesioned Medial precentral cortex Medial prefrontal cortex (lesioned area) Anterior cingulate cortex Primary motor cortex Primary somatosensory cortex Primary auditory cortex Primary visual cortex Subcortical regions Caudate-putamen, anteromedial Caudate-putamen. dorsolateral Nucleus accumbens Septum Lateral habenula Hippocampus. CA1 Hippocampus. dentate gyrus Dorsomedial thalamic nucleus Hypothalamus Central amygdaloid nucleus Lateral amygdaloid nucleus Medial geniculate body Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral teamental area
area)
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of the 6-OHDA lesioned rats compared with that of the sham-operated animals in any of the brain regions examined.
3.1. Changes in local cerebral blood flow
Table 1 Effects of 6.OHDA
5 (199-5)
cerebral
cerebral
blood blood
Row in the rat flow
(N = 8)
Lesion
(N = 10)
left
right
left
137.4 + 5.3 143.0 25.X 151.2 2 5.3 150.5 + 4.Y 111.4k3.3 124.4 2 4.2 1X4.5 k 5.7 133.1 -+ 5.4
133.x 136.0 151.7 149.0 114.0 127.0 1X8.9 134.6
119.2 5 4.5 118.2 + 5.2 122.0 + 6.3
123.5 k 4.3 113.2 -t 4.7 120.8 t 4.x
137.7 2 6.8 12X.9 k 6.1 144.7 + 5.0”
184.5 2 6.4 86.3” 3.5 111.3k4.7 131.3 -c 2.9 105.7 k 3.2 92.0 -+ 3.1 117.4 * 3.4 165.8 i 5.5 98.8 + 3.1 X4.6 i- 3.5 102.1 2 5.5
1X4.2 2 4.4 84.6 k 6.2 112.4 t 5.0 136.8 -c 7.6 108.9 + 7.1 92.5 r 7.9 112.3 t X.9 166.9 k 8.5 107.9 2 6.0 93.656.2 106.6 2 6.4
+ 2 k + f k 2 t
7.0 5.5 5.1 5.2 3.9 2.9 6.2 4.6
150.12 146.2 159.9 170.2 127.4 13x.4 193.7 142.2
right 9.1 27.1 2 10.1 2 7.8 -t 7.2 ? 5.0 k X.3 2 5.5
+ 6.2 f 4.3 k 4.2 -t 3.6 _f 3.8 -t 4.7 t 5.0 I 4.~5 -c 2.7 + 1.7 -t 6.3
Values represent the mean i SEM of local cerebral blood flow (ml/ 100 g tissueimin) *P < 0.05. **P < 0.01 compared with controls (Student’s t-test).
2 ‘+ + -’ t + +
9.6 6.2 9.2 7.3* 6.3 6.5 6.9 6.1
137.3 k 6.6 131.1” 5.9” 148.2 2 5.6** 122.2 2 6.2
108.2 t 3.Y 179.9 86.3 110.4 132.6 104.8 100.3 117.9 163.4 103.6 91.7 104.6
150.5 148.4 162.3 171.1 128.3 130.3 203.1 150.4
182.8 85.5 113.1 137.4 112.0 90.9 111.7” 175.0 102.7 89.9 108.2
? + + k 2 t f + -f k
6.0 6.7 6.0 8.5 7.4 6.2 7.8 8.4 5.6 5.6 5.7
M. Table 2 Effects of 6-OHDA Brain
lesions
Suzuki
in the medial
et al.
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prefrontal
region
cortex
Neuropsychopharmacology
on D, and D1 dopamine
[3H]SCH
cortex (lesioned cortex anteromedial dorsolateral
area)
f k r + 2 19.8 ? 25.3 t 1.9 +
nucleus nucleus pars compacta pars reticulata area
Values represent the means the numbers of rats.
f SEM
of specific
[‘H]SCH
binding
2.8 3.1 2.4 0.4 0.4 1.3 1.1 0.2
23390
(11) (11) (9) (9) (9) (10) (10) (9) and [‘H]YM
used in the previous reports (Pycock et al., 1980a,b; Martin-Iverson et al., 1986; Haroutunian et al., 1988), and much larger amounts of 6-OHDA have been used to make lesions in other dopaminergic terminal fields including the striatum and the nucleus accumbens (Cadet et al., 1991; Churchill and Kalivas, 1992). The dosage we used was chosen to keep the influence of 6-OHDA on other catecholamine terminals in MPFC to a minimum. Therefore dopaminergic terminals in MPFC might be only partially lesioned in our experiment. However we have also confirmed that a marked depletion of DA (6% of the control value) in MPFC and significant increases in DA. 3,4-dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens and the striatum are observed after 6OHDA lesioning of MPFC in rats treated in the same way as in the present study (Kurachi et al., 1995). Our present data indicate that lesions of the frontal DA nerve terminals may produce enhancement of not only biochemical DA turnover but also local metabolic activities in the terminal regions of the ascending mesolimbic and nigrostriatal pathways. In our results, LCBF in MPFC was not reduced in the lesioned rats, consistent with the finding of Kozlowski and Marshall (1980) that 6-OHDA injection into the ventral tegmental area in rats did not induce a significant change in local cerebral glucose utilization in the medial frontal cortex. Our results showed that the lesioned animals had rather a trend toward increased LCBF in MPFC, and a significant increase in LCBF was observed in the neighboring anterior cingulate cortex. These findings are in contrast to the results of a biochemical study which showed significant reductions in the concentrations of DA and its metabolites in MPFC (Kurachi et al., 1995). It has been indicated that areas rich in neuropil have larger energy demands and greater rates of glucose utilization than areas rich in cell bodies (Kennedy et al., 1976), and that axon terminals, not cell bodies, are the sites of enhanced metabolic activity during increased afferent activity
4.4 4.1 39.8 40.7 36.1 4.0 6.0
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in the rat [‘H]YM
Lesion
4.3 i 0.3 (10) 4.2 k 0.3 (10) 40.0 41.2 35.1 4.2 6.4
receptor
23390 binding
Control Medial prefrontal Anterior cingulate Caudate-putamen. Caudate-putamen, Nucleus accumbens Central amygdaloid Lateral amygdaloid Substantia nigra, Substantia nigra. Ventral tegmental
5 (1995)
09151-2
Control
Lesion
t 0.3 (9) t 0.2 (9) t 2.4 (12) t 2.9 (12) t2.2(11) t 0.4 (7) t 0.3 (7)
19.4 2 1.4 (9) 27.9 2 2.0 (9) 12.9 2 0.7 (8) -
20.5 t 1.9 (12) 24.5 t 2.0 (12) 2.4 t 0.2 (9)
4.6 + 0.7 (6) 1.8 + 0.3 (6) 3.8 k 0.9 (6)
09151.2
binding
-
(fmolimg
tissue).
binding
-
Figures
18.9% 25.9 2 15.6 k 5.12 2.4 k 4.9 t in parentheses
1.8 (11) 2.0 (11) 1.5 (10)
0.2 (5) 0.3 (5) 0.7 (5) indicate
(Kadekaro et al., 1985). Using [ 14C]deoxyglucose autoradiography in rats, increased glucose utilization has been reported in the globus pallidus both after electrical stimulation of the striatum (Aiko et al., 1988) and after neurotoxic lesioning of the nigrostriatal neurons (Wooten and Collins, 1981; Porrino et al., 1987). These findings suggest increased activity in the axon terminals of the striatopallidal neurons induced by direct stimulation of the neurons in the former, and by disinhibition of the striatal efferents caused by disruption of inhibitory dopaminergic inputs to the striatum in the latter. However, it has been demonstrated that electrolytic lesions of the striatum also induce increased glucose consumption in the globus pallidus in rats (Hosokawa et al., 1984), suggesting transsynaptic disinhibition of pallidal neuronal activity following the denervation of inhibitory striatopallidal neurons. Considering the studies cited, some explanations for our findings could be suggested. In our results, diminished inhibitory dopaminergic inputs to MPFC might elicit a slight metabolic increase in cell bodies of the medial prefrontal efferent neurons and a significant increase in axon terminals of these neurons in the nucleus accumbens and striatum. However, further investigations will be needed to clarify the direct influence of corticofugal projection neurons on LCBF in the subcortical nuclei. An alternative explanation for increased LCBF in the subcortical sites is that they might reflect an augmentation of the subcortical DA turnover. Pycock et al. (1980a,b) reported an increase in [“Hlspiperone binding in the striatum and the nucleus accumbens following 6-OHDA lesions of MPFC. Haroutunian et al. (1988) also demonstrated increased [ “Hldomperidone binding in nucleus accumbens. However, in the present autoradiographic study using more specific ligands, neither D, nor D, dopamine receptor density at a single concentration of each ligand was altered. We have also performed a saturation analysis using brain homogenates and confirmed
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that 6-OHDA lesions of MPFC do not induce a significant change in B,,, and K, of [‘H]SCH23390 or [“Hlspiperone binding in the striatum and nucleus accumbens (unpublished data). Concerning D, sites, a few studies have suggested that control by the mesocortical DA system of nonDA cortico-subcortical neurons is essential to the modulation of receptor sensitivity in the nucleus accumbens and striatum (Reibaud et al., 1984; Herve et al., 1989). In addition, the lack of change in [“HISCH 23390 b’m d’mg in MPFC following prefrontal DA denervation in our results might be caused by the possible concomitant destruction of the noradrenergic innervation, which may regulate D, receptor denervation supersensitivity in the rat prefrontal cortex (Tassin et al.. 1986). Another possibility is that our method may be less sensitive to detecting slight changes in D, receptor binding in MPFC. because prefrontal D, receptor density is much lower than that in the striatum and nucleus accumbens. Previous studies have indicated that the dopaminergic denervation induces an increase in the density of D, receptors in the striatum (Creese et al.. 1977; Savasta et al.. 1987). It was possible to assume that the D, receptor density might increase also in the denervated MPFC in this study. However the D2 receptor density could not be successfully measured because of its lower abundance and relatively high non-specific binding of [“H]YM 09151-2 in MPFC under the present experimental conditions. It is known that both rapid and gradual compensatory changes occur biochemically and electrophysiologically in the nigrostriatal DA system after the destruction of DA terminals in the striatum (Zigmond and Stricker, 1989). Also in case of 6-OHDA lesions of MPFC. compensatory mechanisms may repair some effects of the lesion in the course of time. Therefore, it could be informative to further examine LCBF and DA receptor binding at various time points before and after 24 days following lesioning. The present data demonstrated that DA denervation in MPFC did not induce decreased prefrontal blood flow in the rats, suggesting that the reduced frontal blood flow and metabolism observed in schizophrenic patients may not be caused by hypofunction of the mesocortical DA system. Recent postmortem neuropathological studies have revealed decreased neuronal density without significant glial proliferation and an abnormal pattern of neuronal migration in the prefrontal cortex of schizophrenics, suggestive of disturbances in cortical development (Benes et al., 1986; Akbarian et al., 1993). In addition, both morphological and functional abnormalities have been demonstrated in the limbic regions including the amygdala, hippocampus, and parahippocampal gyrus of schizophrenic patients (Reynolds, 1983; Jakob and
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Beckmann, 1986; Suddath et al., 1990; Friston et al., 1992). Therefore, further studies exploring the role of prefrontal efferent neurons and their interaction with the subcortical DA systems and limbic areas will contribute toward the understanding of the pathophysiology of schizophrenia.
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Reivich. M., Jehle, J.W. and Sokoloff, L. (1976) Metabolic mapping of the primary visual system of the monkey by means of the autoradiographic [“Cldeoxyglucose technique. Proc. Natl. Acad. Sci. USA 73, 4230-4234. Kozlowski, M.R. and Marshall. J.F. (1980) Plasticity of [lJC]2deoxy-D-glucose incorporation into neostriatum and related structures in response to dopamine neuron damage and apomorphine replacement. Brain Res. 197, 167-183. Konig, J.F.R. and Klippel. R.A. (1963) The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. R.E. Krieger, Huntington, NY. Kuhl. D.E.. Barrio, J.R.. Huang, S.-U.. Selin, C., Ackermann, R.F., Lear. J.L., Wu. J.L., Lin. T.H. and Phelps, M.E. (1982) Quantifying local cerebral blood flow by N-isopropyl-p[‘*‘I]iodoamphetamine (IMP) tomography. J. Nucl. Med. 23, 196-203. Kurachi. M., Kobayashi, K.. Matsubara, R., Hiramatsu, H., Yamaguchi, N., Matsuda, H., Maeda, T. and Hisada, K. (1985) Regional cerebral blood fow in schizophrenic disorders. Eur. Neural. 24. 1766181. Kurachi, M., Yasui, S., Kurachi, T., Shibata. R., Murata. M., Hagino. H., Tanii, Y.. Kurata, K.. Suzuki, M. and Sakurai, Y. (1995) Hypofrontality does not occur with 6-hydroxydopamine lesions of the medial prefrontal cortex in rat brain. Eur. Neuropsychopharmacol. 5. 63-68. Leccese, A.P. and Lyness, W.H. (1987) Lesions of dopamine neurons in the medial prefrontal cortex: effects on self-administration of amphetamine and dopamine synthesis in the brain of the rat. Neuropharmacology 26. 130331308. Martin-Iverson, M.T.. Szostak. C. and Fibiger, H.C. (1986) 6Hydroxydopamine lesions of the medial prefrontal cortex fail to influence intravenous self-administration of cocaine. Psychopharmacology 88. 310-314. Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press. Sydney. Porrino, L.J., Burns, R.S., Crane. A.M., Palombo. E., Kopin, 1.J. and Sokoloff. L. (1987) Changes in local cerebral glucose utilization associated with Parkinson’s syndrome induced by I-methyl-4. phenyl-1.2,3,6-tetrahydropyridine (MPTP) in the primate. Life Sci. 40, 1657-1664. Pycock, C.J.. Kerwin, R.W. and Carter, C.J. (1980a) Effect of lesion of cortical dopamine terminals on subcortical dopamine receptors in rats. Nature 286, 74-77. Pycock, C.J.. Carter. C.J. and Kerwin, R.W. (1980b) Effect of h-hydroxydopamine lesions of the medial prefrontal cortex on neurotransmitter systems in subcortical sites in the rat. J. Neurothem. 34. 91-99. Reibaud, M., Blanc, G.. Studier, J.-M., Glowinski, J. and Tassin, J.-P. (1984) Non-DA prefronto-cortical efferents modulate D, receptors in the nucleus accumbens. Brain Res. 305, 43-50. Reynolds, G.P. (1983) Increased concentrations and lateral asymmetry of amygdala dopamine in schizophrenia. Nature 305. 527-529. Savasta, M., Dubois, A. and Scatton, B. (1086) Autoradiographic
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localization of D, dopamine receptors in the rat brain with [‘HISCH 23390. Brain Res. 375, 291-301. Savasta, M., Dubois, A., Feuerstein. C., Manier. M. and Scatton, B. (1987) Denervation supersensitivity of striatal D? dopamine receptors is restricted to the ventroand dorsolateral regions of the striatum. Neurosci. Lett. 74, 180-186. Seeman, P.. Ulpian. C., Bergeron. C., Riederer, P., Jellinger. K., Gabriel, E.. Reynolds, G.P. and Tourtelotte, W.W. (1984) Bimodal distribution of dopamine receptor densities in brains of schizophrenia. Science 225. 728-731. Sokoloff, L. (1981) Relationships among local functional activity, energy metabolism, and blood flow in the central nervous system. Fed. Proc. 40. 2311-2316. Suddath, R.L., Christison, G.W., Torrey, E.F., Casanova. M.F. and Weinberger, D.R. (1990) Anatomical abnormalities in the brains of monozygotic twins discordant for schizophrenia. New Engl. .I. Med. 322, 789-794. Suzuki. M.. Kurachi, M.. Kawasaki, Y.. Kiba, K. and Yamaguchi, N. (1992) Left hypofrontality correlates blunted affect in schizophrenia. Jpn. J. Psychiatry Neurol. 46, 6533657. Tassin. J.P., Studler, J.M., Herve. D.. Blanc, G. and Glowinski, J. (1986) Contribution of noradrenergic neurons to the regulation of dopaminergic (D,) receptor denervation supersensitivity in rat prefrontal cortex. J. Neurochem. 46, 243-248. Unis. A.S., Vincent, J.G. and Dillon. B. (1990) Brain receptor autoradiography with [‘H]YM 09151-2: a ligand for labeling dopamine D-2 receptors. Life Sci. 47. PL-151-155. Weinberger. D.R.. Berman, K.F. and Zec, R.F. (1986) Physiological dysfunction of dorsolateral prefrontal cortex in schizophrenia. I. Regional cerebral blood flow evidence. Arch. Gen. Psychiatry 33, 134-124. Winchell, H.S., Horst, W.D., Braun. L.. Oldendorf, W.H., Hattner. R. and Parker, H. (1980) N-Isopropyl-p-[ ‘?]iodoamphetamine: Single pass brain uptake and washout; Binding to brain synaptosomes and localization in dog and monkey. J. Nucl. Med. 21, 947-952. Wong. D.F., Wagner, H.N.. Tune, L.E.. Dannals, R.F., Pearlson, G.D., Links, J.M.. Tamminga, C.A., Broussolle, E.P., Ravert. H.T.. Wilson. A.A., Toung, J.K.T., Malat, J.. Williams. J.A., O’Tuama, L.A., Snyder. S.H., Kuhar, M.J. and Gjedde, A. (1986) Positron emission tomography reveals elevated D, dopamine receptors in drug-naive schizophrenics. Science 234, 1558-1563. Wooten, G.F. and Collins, R.C. (1981) Metabolic effects of unilateral lesion of the substantia nigra. J. Neurosci. 1, 285-291. Young III. W.S. and Kuhar. M.J. (1979) A new method for receptor autoradiography: [‘Hlopioid receptors in rat brain. Brain Res. 179. 255-270. Zigmond. M.J. and Stricker. E.M. (1989) Animal models of parkinsonism using selective neurotoxins: clinical and basic implications. Int. Rev. Neurobiol. 31, l-79. Zilles. K. (1985) The Cortex of the Rat. A Stereotaxic Atlas. Sprmger-Verlag, Berlin.
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Effects of Ghydroxydopamine lesions of the medial prefrontal cortex on local cerebral blood flow and D, and D, dopamine receptor binding in rats: a quantitative autoradiographic study Michio
Suzuki”.* “Department
, Yasuhiro
Kawasaki’,
Masahiko Hirofumi
Muratad, Mori’
Ryoko
Shibata”,
Masayoshi
of Neuropsychiatry. Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, hDepartment of Neuropsychiatry. School of Medicine, Kunazawa University, Kanazawa, Japan ‘Radioisotope Center. Kanarawa University, Kanazawa. Japan Received
27 December
1993; revised
3 January
1995;
accepted
4 January
Kurachi”, Japan
1995
Abstract The effects of lesions of the dopamine (DA) nerve terminals in the medial prefrontal cortex (MPFC) on local cerebral blood flow (LCBF) and DA receptor binding in rats were investigated. 4 pg of 6-hydroxydopamine (6-OHDA) was infused stereotaxically into the area of the bilateral MPFC of rats pretreated with desmethylimipramine, and control rats received a vehicle solution. Twenty-four days after the operation LCBFs of 23 brain regions were measured using the quantitative autoradiographic N-isopropyl-p-[‘251]’ 10d oamphetamine technique. D, and D, DA receptor binding in various brain regions was also quantified autoradiographically using [“HISCH 23390 and [?H]YM 09151-2 as the respective ligands. 6-OHDA lesions of MPFC produced significant increases in LCBF of the nucleus accumbens, the dorsolateral portion of the caudate-putamen and the anterior cingulatecortex. The lesionedanimalsdid not showdecreasedLCBF in MPFC per se. D, and D, DA receptor binding was not affected in any brain region examined. These results suggest that lesions of the DA nerve terminals in MPFC
induce an enhancementof functional activity in the terminal regionsof the subcortical DA systems,and that hypofunction of the mesocortical dopamine system does not elicit reduced metabolic activity in the prefrontal cortex. Keywords: 6-Hydroxydopamine; Medial prefrontal cortex; Autoradiography; Local cerebra1 blood Schizophrenia
1. Introduction
Several studies have suggested that the medial prefrontal cortex (MPFC), the projection area of mesocortical dopaminergic neurons, may modulate the function of mesolimbic and nigrostriatal dopamine (DA) systems. It has been reported that DA depletion in MPFC is associated with increases in DA and its metabolites in other dopaminergic terminal fields including the nucleus accumbens and striatum. as well
* Corresponding
Neuroscience,
author.
Hospital, S-171 76 Stockholm, + 48 8 34 65 63.
Elsevier Science B.V. SSDI
Present
address:
Department
Psychiatry and Psychology Section,
0924-977X(95)00003-8
Sweden.
Tel.:
of Clinical
Karolinska
+ 46 8 729 20 00: Fax:
flow; Dopamine
receptor;
as with enhanced spontaneous and amphetamine-induced locomotor activity in rats (Pycock et al., 1980a,b; Leccese and Lyness, 1987; Haroutunian et al., 1988). Similar subcortical biochemical and behavioral changes are also reported to be elicited transiently by excitotoxin lesions destroying intrinsic neurons in MPFC (Christie et al., 1986). These findings suggest that the mesocortical dopaminergic neurons may exert an inhibitory influence on the subcortical dopaminergic nerve terminals, presumably mediated via corticofugal projections to the subcortical sites. There is increasing evidence suggesting that the prefrontal cortex and subcortical DA systems may play an important role in the pathophysiology of schizophrenia. Many of the recent neuroimaging studies measuring regional cerebral blood flow and
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glucose utilization have revealed decreased activity and attenuated activation of the prefrontal cortex in schizophrenic patients (Ingvar and Franzen, 1974; Kurachi et al., 1985; Weinberger et al., 1986; Suzuki et al., 1992; Andreasen et al., 1992). In addition. data implicating dysregulation of the subcortical DA systems in schizophrenia have also been reported (Seeman et al.? 1984; Wong et al., 1986). However. the mechanisms underlying these findings remain to be elucidated. Although it has been hypothesized that mesocortical dopaminergic hypoactivity and the resultant mesolimbic dopaminergic hyperactivity may be involved in these phenomena (Weinberger et al., 1986), little substantial evidence is available. Therefore, it may be important to clarify the interaction between the prefrontal cortex and the subcortical DA systems in understanding schizophrenia. It is known that local cerebral blood flow (LCBF) correlates closely with local energy metabolism in cerebral tissues and represents local functional activity in the central nervous system (Sokoloff, 1981). NIsopropyl-p-[ “31]iodoamphetamine (““I-IMP) is highly trapped on first pass through rat brain when injected and is washed out slowly (Winchell et al.. 1980). Therefore the initial distribution of “‘I-IMP reflects regional brain perfusion (Kuhl et al., 1982). ““I-IMP has been used for measurements of LCBF in both humans and small animals employing single photon emission computed tomography and quantitative autoradiography, respectively. The quantitative autoradiographic ““I-IMP technique is a potentially powerful method for studying the effect of drugs or brain lesioning on local functional activity in the brain. Meanwhile, quantitative autoradiography has also been applied to determine the distribution of various neurotransmitter receptors in the brain (Young and Kuhar, 1979; Savasta et al., 1986; Unis et al., 1990). In the present study, we employed “‘I-IMP, a radiopharmaceutical labelled with 12’1 instead of “‘I, to examine the effect of 6-hydroxydopamine (6OHDA) lesions of the DA nerve terminals in MPFC on LCBF in various brain regions in rats. In addition, we applied quantitative receptor autoradiography to investigate the changes in D, and D, DA receptor binding using [‘H]SCH 23390 and [3H]YM 09151-2, respectively.
2. Experimental
procedures
2.1. Preparation
of animals
.5 (lW.5)
P-101
imipramine (25 mgikg i.p.) 30 min before the operation to protect noradrenergic terminals. The animals were anesthetized with pentobarbital (30 mgikg i.p.) and immobilized in a stereotaxic apparatus. The rats were given one of the following injections through a 30-gauge stainless steel cannula into the area of the bilateral MPFC: vehicle (2 ~1, 0.1% ascorbic acid in Ringer’s solution) or 6-OHDA (Sigma Chem. Co., St. Louis, USA) (4 pg dissolved in 2 ~1 vehicle). The stereotaxic coodinates used were A, bregma + 2.7 mm; L, 2 0.8 mm; V, 3.3-3.4 mm from dura (Paxinos and Watson, 1986). Both vehicle and 6-OHDA were infused over a 2 min period and the injection cannula was left in place for an additional 3 min after cessation of the infusion. The rats were allowed to recover, and the following studies were performed 24 days after the operation. 2.2.
L’51-IMP autoradiography
After insertion of femoral vein and artery catheters under ether anesthesia, the rats were loosely taped to a board and allowed to recover from anesthesia for at least 2 h. 1.85 MBq of 12”I-IMP (spec. act. 12.2-12.6 GBqimmol, Nihon Medi-Physics, Takarazuka, Japan) was injected intravenously, and arterial blood samples were drawn continuously for 3 min. Then the animals were decapitated, and the brains were removed rapidly and frozen in hexane chilled with dry ice. 20 pm thick coronal sections of the brain were obtained with a cryostat maintained at - 20°C. X-ray film (Sakura, Tokyo, Japan) was exposed to the sections for 1 month with gelatin standards of known 12’1 concentrations. The film was developed and the optical densitometry of the autoradiograms were measured using a CCD camera (TI-22A, NEC Co., Tokyo, Japan) connected to a personal computer (PC 9801, NEC Co., Tokyo, Japan) with an image memory board (ADS Co., Nara, Japan) and the program DIAL10 (ADS Co., Nara. Japan). Twenty-three brain regions were selected as regions of interest (Fig. 1, Table l), and anatomical regions were identified by comparing the autoradiograms with atlases of the rat brain (Kbnig and Klippel, 1963; Zilles, 1985). LCBF was calculated from brain and plasma radioactivities according to a reference sample method (Kuhl et al., 1982). 2.3. D, and D2 receptor
Male Wistar rats weighing 220-240 g at the beginning of the experiment were housed at 24 -+ 2°C with a 12 h light/12 h dark cycle and with free access to food and water. The rats were pretreated with desmethyl-
autoradiography
The rats were decapitated, and the brains were rapidly removed and frozen. Coronal tissue sections (20 pm in thickness) were prepared at - 20°C using a cryostat and were thaw-mounted onto gelatin-coated slides.
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Fig. 1. Schematic drawings of the regions of interest in LCBF measurement. AC. anterior cingulate cortex; ACC, nucleus accumbens; AMC. central amygdaloid nucleus; AML. lateral amygdaloid nucleus; CAI ( hippocampus CAI; CPA, caudate-putamen, anteromedial; CPD, caudate-putamen, dorsolatcral; DC, hippocampus, dentate gyrus; DM, dorsomedial thalamic nucleus; HT, hypothalamus; LH. lateral habenula; MC, medial geniculate hody; MPC, medial precentral cortex; MPFI. medial prefrontal cortex (rostra1 to lesion); MPF2, medial prefrontal cortex (lesioned area): NC, substantia nigra, pars compacta; NR, substantia nigra. pars reticulata; PA, primary auditory cortex; PM, primary motor cortex; PS. primary somatosensory cortex; PV, primary visual cortex; SP. septum; VT, ventral tegmental area.
D, binding sites were assayed using [3H]SCH 23390 (spec. act. 2.9-3.1 TBqimmol, DupontiNEN, Boston, MA, USA) as a ligand. The slices were preincubated for 20 min in 50 mM Tris buffer (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl,, and 1 mM MgCl,. The sections were then incubated for 2 h at 25°C in the same buffer containing 0.3 nM [SH]SCH 23390. Ad’ latent sections were incubated in the presence of excess (100 nM) cold R( + )-SCH 23390 to determine the non-specific binding. The incubation was terminated by rinsing sections for 20 min in ice-cold buffer and the sections were dried rapidly under a stream of cold air. Labeled slides were apposed to tritium-sensitive Hyperfilm (Amersham, Arlington Heights,VA, USA) for 3 weeks to 2 months along with gelatin tritium standards with known concentrations of tritium. The film was developed and the optical densities of the autoradiograms were determined with a CCD camera (TI-22A. NEC Co., Tokyo, Japan) connected to a personal computer (Macintosh IIci, Apple Computer Inc.) with a graphics display board (RasterOps 24STV, RasterOps Co., CA) using the program Image 1.35 (National Institute of Health). Ten brain regions were selected as regions
of interest (Table 2). Specific binding values were obtained by the subtraction of non-specific binding from the corresponding total binding. The optical densities were converted into fmol SCH 23390/mg tissue by reference to the tritium standards. D, binding sites were assayed using [“H]YM 091512 (spec. act. 3.2 TBq/mmol, Dupont/NEN, Boston, MA, USA) as a ligand. The ligand concentration was 0.3 nM. The assay procedure was essentially the same as that used for D, binding except that 0.1% ascorbic acid was added in the incubation medium and 10 PM S( - )-sulpiride was used as a displacer. [“H]YM 09151-2 binding was determined in six brain regions with a relatively high Dz DA receptor density (Table 2).
2.4. Statistical analysis The effects of the 6-OHDA lesions on LCBF and D, and Dz DA receptor binding in comparison with the sham-operated animals were examined by Student’s t-test. Statistical significance was defined as
P < 0.05.
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3. Results
Mean values of LCBF in the cortical and subcortical regions of the rat brain are presented in Table 1. LCBFs in the cerebral cortices were generally higher in the 6-OHDA-lesioned rats than in the sham-operated animals, but no statistically significant differences were found in most of the regions. 6-OHDA lesions produced significantly increased LCBF only in the right anterior cingulate cortex (P < 0.05). 6-OHDA lesions did not elicit a decrease in LCBF in the lesioned MPFC per se. In subcortical regions, the rats with bilateral 6-OHDA lesions of MPFC showed significantly increased LCBF in the bilateral nucleus accumbens (right, P< 0.05; left, PCO.01). LCBF in the caudate-putamen was also higher in the lesioned animals than in the controls, with the difference in the right dorsolateral portion reaching statistical significance (P < 0.05).
4. Discussion This study examined the effects of 6-OHDA lesions of MPFC on LCBF and DA receptor bindings in rats using the quantitative autoradiography. To our knowledge, this is the first to investigate changes in LCBF after lesions of the DA nerve terminals in the MPFC. 6-OHDA lesions produced increased LCBF in the nucleus accumbens, the dorsolateral portion of caudate-putamen and the anterior cingulate cortex, while D, and D, receptor binding was not affected in any brain regions examined. Pycock et al. (1980a,b) reported that 6-OHDA lesions of MPFC induced increases in the content of DA and its metabolites in the striatum and nucleus accumbens, suggesting that lesions in the frontal cortical DA system may result in subcortical DA hyperactivity. Other investigators have duplicated these findings (Leccese and Lyness, 1987; Haroutunian et al., 1988). The dosage of 6-OHDA injected into MPFC in the present study was smaller than those
3.2. Changes in D, and Dz receptor binding Data are shown in Table 2. There was no significant change in [3H]SCH 23390 or [3H]YM 09151-2 binding
Brain
lesions
in the medial
prefrontal
region
cortex
on local Local Control
Cortical regions Medial prefrontal cortex (rostra1 to lesioned Medial precentral cortex Medial prefrontal cortex (lesioned area) Anterior cingulate cortex Primary motor cortex Primary somatosensory cortex Primary auditory cortex Primary visual cortex Subcortical regions Caudate-putamen, anteromedial Caudate-putamen. dorsolateral Nucleus accumbens Septum Lateral habenula Hippocampus. CA1 Hippocampus. dentate gyrus Dorsomedial thalamic nucleus Hypothalamus Central amygdaloid nucleus Lateral amygdaloid nucleus Medial geniculate body Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral teamental area
area)
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of the 6-OHDA lesioned rats compared with that of the sham-operated animals in any of the brain regions examined.
3.1. Changes in local cerebral blood flow
Table 1 Effects of 6.OHDA
5 (199-5)
cerebral
cerebral
blood blood
Row in the rat flow
(N = 8)
Lesion
(N = 10)
left
right
left
137.4 + 5.3 143.0 25.X 151.2 2 5.3 150.5 + 4.Y 111.4k3.3 124.4 2 4.2 1X4.5 k 5.7 133.1 -+ 5.4
133.x 136.0 151.7 149.0 114.0 127.0 1X8.9 134.6
119.2 5 4.5 118.2 + 5.2 122.0 + 6.3
123.5 k 4.3 113.2 -t 4.7 120.8 t 4.x
137.7 2 6.8 12X.9 k 6.1 144.7 + 5.0”
184.5 2 6.4 86.3” 3.5 111.3k4.7 131.3 -c 2.9 105.7 k 3.2 92.0 -+ 3.1 117.4 * 3.4 165.8 i 5.5 98.8 + 3.1 X4.6 i- 3.5 102.1 2 5.5
1X4.2 2 4.4 84.6 k 6.2 112.4 t 5.0 136.8 -c 7.6 108.9 + 7.1 92.5 r 7.9 112.3 t X.9 166.9 k 8.5 107.9 2 6.0 93.656.2 106.6 2 6.4
+ 2 k + f k 2 t
7.0 5.5 5.1 5.2 3.9 2.9 6.2 4.6
150.12 146.2 159.9 170.2 127.4 13x.4 193.7 142.2
right 9.1 27.1 2 10.1 2 7.8 -t 7.2 ? 5.0 k X.3 2 5.5
+ 6.2 f 4.3 k 4.2 -t 3.6 _f 3.8 -t 4.7 t 5.0 I 4.~5 -c 2.7 + 1.7 -t 6.3
Values represent the mean i SEM of local cerebral blood flow (ml/ 100 g tissueimin) *P < 0.05. **P < 0.01 compared with controls (Student’s t-test).
2 ‘+ + -’ t + +
9.6 6.2 9.2 7.3* 6.3 6.5 6.9 6.1
137.3 k 6.6 131.1” 5.9” 148.2 2 5.6** 122.2 2 6.2
108.2 t 3.Y 179.9 86.3 110.4 132.6 104.8 100.3 117.9 163.4 103.6 91.7 104.6
150.5 148.4 162.3 171.1 128.3 130.3 203.1 150.4
182.8 85.5 113.1 137.4 112.0 90.9 111.7” 175.0 102.7 89.9 108.2
? + + k 2 t f + -f k
6.0 6.7 6.0 8.5 7.4 6.2 7.8 8.4 5.6 5.6 5.7
M. Table 2 Effects of 6-OHDA Brain
lesions
Suzuki
in the medial
et al.
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prefrontal
region
cortex
Neuropsychopharmacology
on D, and D1 dopamine
[3H]SCH
cortex (lesioned cortex anteromedial dorsolateral
area)
f k r + 2 19.8 ? 25.3 t 1.9 +
nucleus nucleus pars compacta pars reticulata area
Values represent the means the numbers of rats.
f SEM
of specific
[‘H]SCH
binding
2.8 3.1 2.4 0.4 0.4 1.3 1.1 0.2
23390
(11) (11) (9) (9) (9) (10) (10) (9) and [‘H]YM
used in the previous reports (Pycock et al., 1980a,b; Martin-Iverson et al., 1986; Haroutunian et al., 1988), and much larger amounts of 6-OHDA have been used to make lesions in other dopaminergic terminal fields including the striatum and the nucleus accumbens (Cadet et al., 1991; Churchill and Kalivas, 1992). The dosage we used was chosen to keep the influence of 6-OHDA on other catecholamine terminals in MPFC to a minimum. Therefore dopaminergic terminals in MPFC might be only partially lesioned in our experiment. However we have also confirmed that a marked depletion of DA (6% of the control value) in MPFC and significant increases in DA. 3,4-dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens and the striatum are observed after 6OHDA lesioning of MPFC in rats treated in the same way as in the present study (Kurachi et al., 1995). Our present data indicate that lesions of the frontal DA nerve terminals may produce enhancement of not only biochemical DA turnover but also local metabolic activities in the terminal regions of the ascending mesolimbic and nigrostriatal pathways. In our results, LCBF in MPFC was not reduced in the lesioned rats, consistent with the finding of Kozlowski and Marshall (1980) that 6-OHDA injection into the ventral tegmental area in rats did not induce a significant change in local cerebral glucose utilization in the medial frontal cortex. Our results showed that the lesioned animals had rather a trend toward increased LCBF in MPFC, and a significant increase in LCBF was observed in the neighboring anterior cingulate cortex. These findings are in contrast to the results of a biochemical study which showed significant reductions in the concentrations of DA and its metabolites in MPFC (Kurachi et al., 1995). It has been indicated that areas rich in neuropil have larger energy demands and greater rates of glucose utilization than areas rich in cell bodies (Kennedy et al., 1976), and that axon terminals, not cell bodies, are the sites of enhanced metabolic activity during increased afferent activity
4.4 4.1 39.8 40.7 36.1 4.0 6.0
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in the rat [‘H]YM
Lesion
4.3 i 0.3 (10) 4.2 k 0.3 (10) 40.0 41.2 35.1 4.2 6.4
receptor
23390 binding
Control Medial prefrontal Anterior cingulate Caudate-putamen. Caudate-putamen, Nucleus accumbens Central amygdaloid Lateral amygdaloid Substantia nigra, Substantia nigra. Ventral tegmental
5 (1995)
09151-2
Control
Lesion
t 0.3 (9) t 0.2 (9) t 2.4 (12) t 2.9 (12) t2.2(11) t 0.4 (7) t 0.3 (7)
19.4 2 1.4 (9) 27.9 2 2.0 (9) 12.9 2 0.7 (8) -
20.5 t 1.9 (12) 24.5 t 2.0 (12) 2.4 t 0.2 (9)
4.6 + 0.7 (6) 1.8 + 0.3 (6) 3.8 k 0.9 (6)
09151.2
binding
-
(fmolimg
tissue).
binding
-
Figures
18.9% 25.9 2 15.6 k 5.12 2.4 k 4.9 t in parentheses
1.8 (11) 2.0 (11) 1.5 (10)
0.2 (5) 0.3 (5) 0.7 (5) indicate
(Kadekaro et al., 1985). Using [ 14C]deoxyglucose autoradiography in rats, increased glucose utilization has been reported in the globus pallidus both after electrical stimulation of the striatum (Aiko et al., 1988) and after neurotoxic lesioning of the nigrostriatal neurons (Wooten and Collins, 1981; Porrino et al., 1987). These findings suggest increased activity in the axon terminals of the striatopallidal neurons induced by direct stimulation of the neurons in the former, and by disinhibition of the striatal efferents caused by disruption of inhibitory dopaminergic inputs to the striatum in the latter. However, it has been demonstrated that electrolytic lesions of the striatum also induce increased glucose consumption in the globus pallidus in rats (Hosokawa et al., 1984), suggesting transsynaptic disinhibition of pallidal neuronal activity following the denervation of inhibitory striatopallidal neurons. Considering the studies cited, some explanations for our findings could be suggested. In our results, diminished inhibitory dopaminergic inputs to MPFC might elicit a slight metabolic increase in cell bodies of the medial prefrontal efferent neurons and a significant increase in axon terminals of these neurons in the nucleus accumbens and striatum. However, further investigations will be needed to clarify the direct influence of corticofugal projection neurons on LCBF in the subcortical nuclei. An alternative explanation for increased LCBF in the subcortical sites is that they might reflect an augmentation of the subcortical DA turnover. Pycock et al. (1980a,b) reported an increase in [“Hlspiperone binding in the striatum and the nucleus accumbens following 6-OHDA lesions of MPFC. Haroutunian et al. (1988) also demonstrated increased [ “Hldomperidone binding in nucleus accumbens. However, in the present autoradiographic study using more specific ligands, neither D, nor D, dopamine receptor density at a single concentration of each ligand was altered. We have also performed a saturation analysis using brain homogenates and confirmed
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that 6-OHDA lesions of MPFC do not induce a significant change in B,,, and K, of [‘H]SCH23390 or [“Hlspiperone binding in the striatum and nucleus accumbens (unpublished data). Concerning D, sites, a few studies have suggested that control by the mesocortical DA system of nonDA cortico-subcortical neurons is essential to the modulation of receptor sensitivity in the nucleus accumbens and striatum (Reibaud et al., 1984; Herve et al., 1989). In addition, the lack of change in [“HISCH 23390 b’m d’mg in MPFC following prefrontal DA denervation in our results might be caused by the possible concomitant destruction of the noradrenergic innervation, which may regulate D, receptor denervation supersensitivity in the rat prefrontal cortex (Tassin et al.. 1986). Another possibility is that our method may be less sensitive to detecting slight changes in D, receptor binding in MPFC. because prefrontal D, receptor density is much lower than that in the striatum and nucleus accumbens. Previous studies have indicated that the dopaminergic denervation induces an increase in the density of D, receptors in the striatum (Creese et al.. 1977; Savasta et al.. 1987). It was possible to assume that the D, receptor density might increase also in the denervated MPFC in this study. However the D2 receptor density could not be successfully measured because of its lower abundance and relatively high non-specific binding of [“H]YM 09151-2 in MPFC under the present experimental conditions. It is known that both rapid and gradual compensatory changes occur biochemically and electrophysiologically in the nigrostriatal DA system after the destruction of DA terminals in the striatum (Zigmond and Stricker, 1989). Also in case of 6-OHDA lesions of MPFC. compensatory mechanisms may repair some effects of the lesion in the course of time. Therefore, it could be informative to further examine LCBF and DA receptor binding at various time points before and after 24 days following lesioning. The present data demonstrated that DA denervation in MPFC did not induce decreased prefrontal blood flow in the rats, suggesting that the reduced frontal blood flow and metabolism observed in schizophrenic patients may not be caused by hypofunction of the mesocortical DA system. Recent postmortem neuropathological studies have revealed decreased neuronal density without significant glial proliferation and an abnormal pattern of neuronal migration in the prefrontal cortex of schizophrenics, suggestive of disturbances in cortical development (Benes et al., 1986; Akbarian et al., 1993). In addition, both morphological and functional abnormalities have been demonstrated in the limbic regions including the amygdala, hippocampus, and parahippocampal gyrus of schizophrenic patients (Reynolds, 1983; Jakob and
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Beckmann, 1986; Suddath et al., 1990; Friston et al., 1992). Therefore, further studies exploring the role of prefrontal efferent neurons and their interaction with the subcortical DA systems and limbic areas will contribute toward the understanding of the pathophysiology of schizophrenia.
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