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Remote lateralized changes in cortical [3H]spiperone binding following focal frontal cortex lesions in the rat
Brain Research, 516 (1990) 127-131
127
Elsevier BRES 15446
Remote lateralized changes in cortical [3H]spiperone binding following focal frontal cortex lesions in the rat Helen S. Mayberg 1'2'4, Timothy H. Moran I and Robert G. Robinson 1'3 Department of 1Psychiatry and Behavioral Sciences, 2Neurology, 3Neuroscience and 4Radiology, Division of Nuclear Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.) (Accepted 10 October 1989)
Key words: Rat; Serotonin receptor $2; Frontal cortex; Lateralization; Behavior
Positron emission tomography (PET) studies in stroke patients have shown that right hemisphere lesions lead to increases in ipsilateral (compared to contralateral) cortical S2-serotonin receptors, while left hemisphere lesions do not. To assess whether similar lateralized'changes in cortical S2-receptors could be demonstrated in response to brain injury in the rat, [3H]spiperone (SP) autoradiography was performed 30 days after unilateral cortical suction lesions. Right lesions produced bilateral increases in total SP binding in frontal cortex (excluding the lesion site): 48% greater than after left lesions, and 23% greater than shams. Left lesions led to bilateral decreases in S2-receptors in the frontal and perirhinal cortex and these decreases were asymmetric. There was a greater decrease in the hemisphere contralateral to the lesion than in the side with the lesion. Frontal S2-receptor binding was positively correlated with running wheel activity in all animals with lesions, regardless of lesion side. These results suggest that there is a lateralized receptor and behavioral response to focal injury in rats, analogous to that previously observed in humans.
INTRODUCTION Using positron emission t o m o g r a p h y (PET), we have recently r e p o r t e d that right, but not left hemisphere s t r o k e lesions p r o d u c e an ipsilateral increase in cortical S2-serotonin receptors m e a s u r e d by the in vivo binding of (3-N-[llC]-methyl)spiperone 1°. This differential biochemical response could not be explained by differences in d e m o g r a p h i c characteristics, medications or lesion par a m e t e r s in these patients. F u r t h e r m o r e , the a s y m m e t r y of S2-receptors in the left t e m p o r a l c o m p a r e d to the right t e m p o r a l cortex was related to the severity of depression in patients with left-sided lesions 1°'15. T h e d e m o n s t r a t i o n of asymmetries in m o o d and biochemical response to unilateral brain injury in humans have b e e n paralleled by the study of behavioral and biochemical asymmetries in rats with experimental unilateral brain injury 12-14'16. L o c o m o t o r hyperactivity has been induced in rats by right, but not left sided ischemic cortical strokes or focal suction lesions. This behavioral change has b e e n associated with w i d e s p r e a d depletions in ipsilateral and sometimes contralateral cortical m o n o a mines following right, but not left hemisphere lesions 12'14. T h e p r e s e n t e x p e r i m e n t was p e r f o r m e d to d e t e r m i n e w h e t h e r changes in cortical S2-serotonin receptors also
occur following unilateral cortical suction lesions in rats, analogous to the p h e n o m e n a seen with P E T imaging in stroke patients. O u r interest was to d e t e r m i n e whether the lateralized p h e n o m e n a seen in humans could be duplicated using in vitro a u t o r a d i o g r a p h y and a well studied rodent m o d e l of focal injury, where lateralized behavioral and neurochemical changes have b e e n previously described. MATERIALS AND METHODS
Surgical lesion procedure A right (n = 6) or left (n = 5) sided frontoparietal craniotomy was performed in male Sprague-Dawley rats (300-350 g)12. Animals were first anesthetized with nembutol (50 mg/kg) and then placed in a stereotaxic apparatus. Using an internally bevelled 18-gauge needle attached to a vacuum device, tissue was removed from the lateral aspect of the frontoparietal cortex above the rhinal fissure and just anterior to the middle cerebral artery. The center of each lesion was approximately 8.5 mm anterior to the interaural line (Fig. 1). This method has been previously demonstrated to produce lesions of uniform size and comparable volumes12. Control animals (n = 5), received anesthesia followed by either right or left craniotomy without interruption of the dura. The scalp was surgically closed and the animals were returned to their individual cages for post-operative recovery.
Behavioral testing Animals were housed in individual cages containing a feeding tray, watering tube and a 34 cm diameter running wheel from which
Correspondence: H.S. Mayberg, Department of Radiology/Nuclear Medicine, Johns Hopkins Medical Institutions, Nelson Bl-130, 600 N. Wolfe St., Baltimore, MD 21205, U.S.A.
128 microscopy to confirm that all lesions were restricted to the cortex.
Regional analysis of autoradiographs
Fig. 1. Photomicrograph of 25/~m coronal rat brain section labeled with [3H]spiperone. 3H-Autoradiograph of a 25/tm coronal brain section taken through the center of a left frontocortical suction lesion. The lesion, located approximately 8.5 mm anterior to the interaural line, involves all layers of cortex and does not extend into the basal ganglia. [3H]Spiperone binding is most prominent in frontal, cingulate and perirhinal cortical regions and in the caudate/ putamen and nucleus accumbens.
cyclometer readings were taken at 24 h intervals. Animals were exposed to a regular 12 h light-dark cycle and were acclimatized to the cages for 3 weeks, prior to surgery. Baseline preoperative activity was recorded as the mean activity during the 7 days prior to craniotomy. Daily running wheel activity was also measured during the entire 30 day post-operative period. The average percent change from baseline was calculated for days 20-30 post-surgery.
Quantitative analysis of regional grain densities was performed on digitized images using computerized microdensitometry (Amersham RAS 1000 Image Processing System) and techniques similar to those described elsewhere 2"3. The regional specificity of SP to S2-serotonin receptors in the cortex was assessed by digitally subtracting the cinanserin blocked sections (which compete with SP for the S2-sites, leaving the non-S2-sites) from the adjacent unblocked SP sections (total binding sites: $2- and non-S2-sites). This subtraction technique yielded a computer-generated image of the S2-receptor sites, which was then used for quantification. Comparisons were made between the slides incubated with SP alone (total binding sites) and those generated by the subtraction procedure (S2-sites only). Receptor measurements in the lesion and control groups were made using the brain slices incubated with SP alone to approximate the conditions used in the human PET studies (where total binding of the PET tracer (3-N-[11C]methyl)spiperone, a SP analogue, was used). Optical density readings were obtained from 3 sections (75 p m apart) for each brain area of interest. The average optical density value for each region was then converted to dpm and subsequently to femtomoles of ligand/mg tissue using the tritium standards and automated programs within the image analysis system. Quantification was performed in cortical and subcortical areas remote from the experimental lesion (approx. rostral to the 9.0 interaural line). Samples were taken from macroscopically identified cortical regions (Fig. 2) including frontal (F1, F2, F3), perirhinal (Ins, Pir), and cingulate (C) cortex, as well as from the nucleus accumbens (NA) using a standard stereotaxic rat atlass.
Data analysis The concentration of [3H]SP (fmol/mg protein) was measured in paired right and left hemisphere areas in cortical and subcortical brain structures. An asymmetry ratio of the lesion side to non-lesion side was also calculated for each region of interest. An ANOVA was performed on the data from each brain region separately. When overall significance was present, planned t-tests were carried out to determine which of the lesion groups were
A utoradiography On post-operative day 30, animals were sacrificed by decapitation and the brains were rapidly removed from the cranial vault and frozen in N-methyl butane (-20 to -40 °C). Frozen brain blocks were stored at -70 °C until sectioned. Consecutive 25/~m coronal sections, cut from the rat forebrain block (approx. 9.0-12.7 mm anterior to the interaural plane), were used for analysis of [3H]spiperone (SP) binding to the cortex and underlying striatum. Frozen sections were thaw-mounted onto gelatin-coated glass microscope slides, dessicated, and stored at -70 °C. Labeling of D2-dopamine and S2-serotonin receptors was accomplished using published methods 11. Slides with mounted tissue sections were incubated for 30 min at room temperature (approx. 22 °C) in 0.17 M Tris buffer (pH 7.7) containing 120 mM NaCI, 5 nM KCI, 2 mM CaC! 2, 1 mM MgCI 2, and 0.001% ascorbic acid and 0.4 nM [3H]spiroperidol (22.8 Ci/mmol, New England Nuclear, Boston, MA). Anatomically contiguous tissue slides were incubated in the presence of cinanserin (10/~M) or haloperidol (1 pM) to block S2-serotonin and Dz-dopamine receptors, respectively. After the 30 min incubation period, slides were dipped rapidly in distilled water, followed by 2 successive 5 min washes in cold Tris buffer (4 °C), and a final dip in distilled water to remove residual salts from the buffer solution. Slides were dried in a stream of cold dry air and dessicated in a vacuum jar overnight. Autoradiographs were generated (Fig. 1) by apposing the prepared slides to tritium-sensitive LKB Ultrofilm along with tritium standards (autoradiographic [3H]micro-scales, Amersham) for 7 weeks. After generation of the autoradiographic images, the tissue sections were stained with Cresyl violet. Representative sections through the maximum depth of the lesion were examined using light
Fig. 2. Region of interest template for autoradiographic analysis. Quantitative analysis of regional grain densities was performed on tissue samples incubated with [3H]spiperone alone (total binding) using computerized microdensitometry. Regions of interest were placed in cortical and subcortical areas remote from the experimental lesion (approx. rostral to the 9.0 interaural line). Multiple samples were taken from macroscopically identified cortical regions including frontal (F1, F2, F3), perirhinal (Ins, Pir), and cingulate (C) cortex, as well as from the nucleus accumbens (NA). Optical densities were averaged within a given slice and over approximately 75 pm to determine femtomoles ligand/mg protein for a given region of interest.
129 different from the sham controls. To assess the presence of binding asymmetry in brain regions produced by the lesions, a 2-way mixed model ANOVA was performed on the calculated asymmetry ratio across factors of lesion status (right, left and sham) and brain region (frontal, perirhinal, cingulate, and accumbens). Appropriate planned t-comparisons were again performed. Lastly, running wheel activity during post-operative days 20-30 (expressed as a percent of preoperative baseline activity) was correlated with the average regional binding in lesion animals (both the right and left lesion groups) using Pearson product coefficients. RESULTS Histological inspection of tissue sections confirmed that lesions in both the right and left lesion groups were restricted to the cortex. The cortex was intact in all sham animals. Reliable measurements of actual lesion size were not possible due to shrinkage and distortion of the lesion margins associated with the freezing and subsequent thin sectioning required for autoradiographic processing. No gross morphological changes were evident in the receptor measurement sites which were all remote from the actual lesion. The percentage of total [3H]SP binding that was specific to S2-receptors was 67% in frontal cortex, 68% in cingulate cortex, 57% in perirhinal cortex and 19% in the N A . All subsequent results relate to these 4 brain regions (all anatomically rostral to the site of the suction lesion) in slices incubated with SP alone. S2-Serotonin receptors were symmetrically distributed in all cortical and subcortical regions in the non-lesion sham group (Table I). Lesions affected [3H]SP binding depending on the side of the lesion and the brain region measured. Analysis of data from the frontal cortex demonstrated a significant effect of the type of lesion (left, right or sham) (F2,13 = 4.67, P = 0.029), overall differences between binding in the right and left hemispheres (F1,13 = 18.12, P = 0.001) and a significant interaction between the type of lesion and the receptor changes in each hemisphere (F2,13 = 11.34, P = 0.001). Planned t-comparisons demonstrated that right lesions produced significant increases in S 2-
receptors in both the right (t = 6.49, P < 0.001) and left (t = 9.4, P < 0.001) frontal cortex. In contrast, left lesions produced significant decreases in S2-receptors in both the right (t = -9.08, P < 0.001) and left (t = -2.4, P < 0.05) frontal cortices, compared to shams. Furthermore, the magnitude of the decrease in the right hemisphere was significantly greater than the decrease in the left hemisphere in response to left lesions (t = 5.95, P < 0.001). Analysis of data from the perirhinal cortex demonstrated no significant effect of lesion side on S2-receptor binding (F2A 3 = 1.07, P = 0.37). The right and left hemisphere measurements were significantly different (F1,13 = 14.4, P = 0.002), but there was no interaction between the type of lesion and the side of the measurement in this a r e a ( F 2 , 1 3 = 0.46, P = 0.64). Planned t-comparisons demonstrated that binding in the right hemisphere was decreased compared to the left hemisphere in response to both left lesions (t = 2.79, P < 0.02), and right lesions (t = 2.34, P < 0.05). There were no significant changes in the cingulate or nucleus accumhens in any of the lesion groups. A n examination of the asymmetry ratios demonstrated differences among the lesion groups (left, right, sham) (F2,13 = 10.3, P = 0.002) and a significant interaction between lesion group and the region measured (frontal, perirhinal, cingulate, accumbens) (F6,39 = 8 . 5 5 , P = 0.0001). There was, however, no effect of the region measurement alone (F3,13 ~-- 1.7, P = 0.18). Planned t-comparisons reconfirmed that greater asymmetries were present in the frontal cortex and pe'rirhinal cortex following left lesions compared to both sham lesions (frontal t = 3.8, P < 0.001; perirhinal t = 5.55, P < 0.001) and right lesions (frontal t = 4.3, P < 0.001; perirhinal t = 8.5, P < 0.001). Differences in motor activity were seen among the three groups. The activity of right lesion animals was 168% + 35 of the baseline activity (mean + S.E.M.), compared to 119% + 25 for left lesion animals and 115% + 31 for shams. The relationship between SP binding in
TABLE I Total f :3H]spiperone binding (fmol/mg protein + S. E. M. ) Region
Frontal Cingulate Perirhinal Accumbens
Left lesion group (n = 5)
Right lesion group (n = 6)
Sham lesion (n = 5)
Left
Right
L/R
Left
Right
R/L
Left
80 _+10 102 + 20 78 _+9 136 _+19
64 _+10"** 96 + 18 57 _+11 143 -+ 25
1.27 _+0.05* 1.05 _+0.02 1.47 _+0.16" 0.99 _+0.06
110 + 8* 123 + 11 87 + 5 202 + 23
105 + 9* 123 + 11 72 + 5 207 + 31
0.95 + 0.03 1.00 + 0.03 0.84 + 0.08 1.01 + 0.05
86 + 116 + 75 + 195 +
*P < 0.001 compared to corresponding sham value. **P < 0.001 compared to left hemisphere value.
5 10 4 8
Right
L/R
88 + 5 117 + 12 75 + 5 192 + 6
0.98 + 0.02 1.00 + 0.02 1.02 + 0.05 1.02 + 0.05
130 each region and motor activity was assessed in the two lesion groups (right and left). There was a positive correlation between mean daily running wheel activity during postoperative days 20-30 (expressed as a percent of pre-operative baseline activity) and the average [3H]SP binding in frontal cortex in animals with right and left sided lesions (r = 0.65, df = 11, P = 0.03). There were no significant correlations between motor behavior and SP binding in perirhinal cortex, cingulate, or nucleus accumbens. DISCUSSION In this study, right sided frontolateral cortical suction lesions produced a significant bilateral increase in [3H]SP binding in the non-lesion areas of the frontal cortex, while identical lesion of the left hemisphere produced a significant bilateral decrease. Furthermore, left sided lesions resulted in an asymmetric change in S2-serotonin receptors in frontal and perirhinal cortex due to a greater decrease in binding in the contralateral (i.e. right) hemisphere. Frontal cortical spiperone binding was significantly correlated with the degree of locomotor activity in both right and left lesion groups. Synaptic neurochemical changes in response to, but remote from the site of injury is not a novel finding. Both pre- and post-synaptic changes have been demonstrated in response to both ischemic and focal brain injury in brain regions without obvious neuropathological damage. Finklestein has demonstrated remote changes in norepinephrine, dopamine and serotonin and their metabolites in the uninjured areas of the hemisphere ipsilateral to large ventrolateral cortical suction lesions in rats 5"6. Starkstein has recently demonstrated that a right unilateral frontocortical lesion induces bilateral changes in dopamine metabolism in the NA 16. The magnitude of these chemical changes was related to the degree of motor hyperactivity seen in the animals and occurred in an area that is recognized for its connections to the basal ganglia and for its role in motor behaviors 1. Brown, using two distinct gerbil ischemia models, has described decreases in frontal cortex S2-serotonin receptors 72 h after injury 4. These postsynaptic receptor changes were seen in areas remote from the hypoxic injury in the hippocampus and did not appear to be solely related to changes in forebrain serotonin levels. Remote changes in S2-serotonin receptors in response to a frontal bisection lesion have also been reported by Leysen9. Various theories have been proposed to explain these remote neurochemical changes following relatively focal lesions. Precise mechanisms, however, have not been demonstrated. Asymmetrical behavioral and presynaptic neurochemical effects of unilateral cortical lesions have also been
previously demonstrated. Right, but not left frontolateral cortical suction lesions in the rat have been shown to produce significant depletions of norepinephrine and dopamine in the locus coeruleus, substantia nigra and cerebral cortex 13'14A6. We do not yet know the mechanism by which right hemisphere lesions produce a different presynaptic response than left hemisphere lesions. Our present findings that both the direction and magnitude of cortical S2-receptor change depends on which hemisphere is injured further support the hypothesis that the right and left hemisphere are biochemically different in their response to focal injury. The regional pattern of change in SP binding following focal brain injury was slightly different in rats compared to that seen in humans. In our previous PET study of cortical S2-serotonin receptors, we found that right hemisphere but not left hemisphere lesions led to an increase in [11C-n-methyl]spiperone binding in umnjured areas of the ipsilateral temporal and parietal cortex, but not the frontal cortex 1°. Because of the obvious differences between the rat and human lesions used in these studies, the implications of the present results for understanding the changes produced by ischemic strokes is not straightforward. In this study we were directly addressing whether focal lesions in rats produce comparable receptor changes to those seen in human ischemic stroke. We found that right hemisphere lesions produced an increase in spiperone binding bilaterally and most prominently in the frontal cortex. This is the first demonstration of an asymmetric postsynaptic neurochemical change following focal brain injury in rats, and these results are similar to what was seen using PET. The suction lesion method, although ideal for creating lesions of consistent size and location 12, is not identical to ischemic lesions in stroke patients. This type of lesion however, has been shown to produce many of the same behavioral and biochemical changes found following middle cerebral artery ligations in rats a3'14. The density of S2-serotonin receptors were estimated in this experiment using total [3H]SP binding in cortex. Therefore, we cannot be sure that the receptor changes were exclusively or even primarily in S2-receptors. We were however, specifically interested in cortical brain regions where S2-serotonin receptors are known to be highly concentrated. It has been previously demonstrated that SP binds with high affinity to D2-dopamine and Sz-serotonin receptors, as well as to the pharmacologically uncharacterized spirodecanone site 2"11. In vivo binding 7, classical quantitative autoradiography n, and autoradiography using computerized digital subtraction methods z'3 have demonstrated that binding of [3H]SP in cortex is predominantly to S2-serotonin receptor sites, with the highest concentration of receptors in frontal and
131 perirhinal regions. In addition, it has been shown that spirodecanone binding is most prominent in layer I of the cortex, whereas S2-serotonin receptors are found in cortical layer IV 11. We have confirmed that total [3H]SP binding is a reasonable estimate of the S2-receptors in these regions. We have not, however, evaluated the specific contribution of the spirodecanone site to our observed changes in total spiperone binding. Despite this limitation of our experimental method, there is no physiologic or pharmacologic precedent for either a regional or lateralized change in spirodecanone binding following focal brain injury. One could also argue that a selective S2-antagonist might have been a more appropriate choice for these studies. The primary goal of this particular study, however, was to determine whether the changes in S2-serotonin receptors that we have previously reported in stroke patients using P E T and (3-N[ltC]methyl)spiperone could be produced in a rat model of stroke.
In summary, the findings from these studies in the rat are consistent with the lateralized changes in cortical S2-serotonin receptors demonstrated with P E T in stroke patients. These results suggest that focal brain injury produces significant changes in both pre- and postsynaptic aspects of the central monoaminergic system. The magnitude and direction of these changes vary, depending on the side of the injury. A t this point, we do not know what the relationship is between these chemical changes and behavioral outcome following either rat or human brain injury. However, all of these factors may contribute to the development of, or protection against the lateralized behavioral syndromes seen following specific cortical lesions.
REFERENCES
Williams and Wilkins, Baltimore, 1983. 9 Leysen, J.E., Geerts, R., Gommeren, W., Verwimp, M. and VanGompel, P., Regional distribution of serotonin-2 receptor binding sites in the brain and effects of neuronal lesions, Arch. Int. Pharmacodyn. Ther., 256 (1982) 301-305. 10 Mayberg, H.S., Robinson, R.G., Wong, D.W., Parikh, R., Bolduc, P., Starkstein, S.E., Price, T., Dannals, R.E, Links, J.M., Wilson, A.A., Ravert, H.T. and Wagner, H.N., PET imaging of cortical $2 serotonin receptors after stroke: lateralized changes and relationship to depression, Am. J. Psychiatry, 145 (1988) 937-943. 11 Palacios, J.M., Niehoff, D.L. and Kuhar, M.J., [3H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors, Brain Research, 213 (1981) 277-289. 12 Pearlson, G.D., Kubos, K.L. and Robinson, R.G., Effect of anterior-posterior lesion location on the asymmetrical behavioral and biochemical response to cortical suction ablations in the rat, Brain Research, 293 (1984) 241-250. 13 Robinson, R.G., Differential behavioral effects of right versus left hemispheric cerebral infaraction: evidence for cerebral lateralization in the rat, Science, 205 (1979) 707-710. 14 Robinson, R.G. and Coyle, J.T., The differential effect of right versus left hemispheric cerebral infaraction on catecholamines and behavior in the rat, Brain Research, 188 (1980) 63-78. 15 Robinson, R.G., Kubos, K.L., Starr, L.B., Rao, K. and Price, T.R., Mood disorders in stroke patients: importance of location of lesion, Brain, 107 (1984) 81-93. 16 Starkstein, S.E., Moran, T.H., Bowersox J.A. and Robinson, R.G., Behavioral abnormalities induced by frontal cortical and nucleus accumbens lesions, Brain Research, 473 (1988) 74-80.
1 Alexander, G.E., DeLong, M.R. and Strick, EL., Parallel organization of functionally segregated circuits linking basal ganglia and cortex, Annu. Rev. Neurosci., 9 (1986) 357-381. 2 Altar, C.A., Kim, H. and Marshall, J.F., Computer imaging and analysis of dopamine (D2), serotonin ($2) binding sites in rat basal ganglia or neocortex labeled by [3H]spiroperidol, J. Pharmacol. Exp. Ther., 233 (1985) 527-538. 3 Altar, C.A., O'Neil, S.O. and Marshall, J.E, Brain dopamine and serotonin receptor sites revealed by digital subtraction autoradiography, Science, 228 (1985) 597-600. 4 Brown, C.M., Kilpatrick, A.T., Martin, A. and Spedding, M., Cerebral ischaemia reduces the density of 5ht2 binding sites in the frontal cortex of the gerbil, Neuropharmacology, 27 (1988) 831-836. 5 Finklestein, S., Campbell, A., Stoll, A.L., Baldessarini, R.J., Stinus, L., Paskevitch, P.A. and Domesick, V.B., Changes in cortical and subcortical levels and monoamines and their metabolites following unilateral ventrolateral cortical lesions in the rat, Brain Research, 271 (1983) 279-288. 6 Finklestein, S., Campbell, A., Baldessarini, R.J., Moya, K.L. and Haber, S.N., Late changes in cerebral monoamine metabolism following focal ventrolateral cerebrocortical lesions in rats, Brain Research, 344 (1985) 205-210. 7 Frost, J.J., Smith, A.C., Kuhar, M.J., Dannals, R.E and Wagner, H.N., In vivo binding of 3H-n-methylspiperone to dopamine and serotonin receptors, Life Sci., 40 (1987) 987-995. 8 K6nig, J.ER. and Klippel, R.A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem,
Acknowledgements. The authors thank Barbara and Sandra Loats of LOATS Associates, Westminster, MD for technical assistance with computer digitization of the autoradiographs. This work is supported by NIH Grants NS15178 and MH00163, and a generous gift from Mrs. Samuel Hecht.
127
Elsevier BRES 15446
Remote lateralized changes in cortical [3H]spiperone binding following focal frontal cortex lesions in the rat Helen S. Mayberg 1'2'4, Timothy H. Moran I and Robert G. Robinson 1'3 Department of 1Psychiatry and Behavioral Sciences, 2Neurology, 3Neuroscience and 4Radiology, Division of Nuclear Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.) (Accepted 10 October 1989)
Key words: Rat; Serotonin receptor $2; Frontal cortex; Lateralization; Behavior
Positron emission tomography (PET) studies in stroke patients have shown that right hemisphere lesions lead to increases in ipsilateral (compared to contralateral) cortical S2-serotonin receptors, while left hemisphere lesions do not. To assess whether similar lateralized'changes in cortical S2-receptors could be demonstrated in response to brain injury in the rat, [3H]spiperone (SP) autoradiography was performed 30 days after unilateral cortical suction lesions. Right lesions produced bilateral increases in total SP binding in frontal cortex (excluding the lesion site): 48% greater than after left lesions, and 23% greater than shams. Left lesions led to bilateral decreases in S2-receptors in the frontal and perirhinal cortex and these decreases were asymmetric. There was a greater decrease in the hemisphere contralateral to the lesion than in the side with the lesion. Frontal S2-receptor binding was positively correlated with running wheel activity in all animals with lesions, regardless of lesion side. These results suggest that there is a lateralized receptor and behavioral response to focal injury in rats, analogous to that previously observed in humans.
INTRODUCTION Using positron emission t o m o g r a p h y (PET), we have recently r e p o r t e d that right, but not left hemisphere s t r o k e lesions p r o d u c e an ipsilateral increase in cortical S2-serotonin receptors m e a s u r e d by the in vivo binding of (3-N-[llC]-methyl)spiperone 1°. This differential biochemical response could not be explained by differences in d e m o g r a p h i c characteristics, medications or lesion par a m e t e r s in these patients. F u r t h e r m o r e , the a s y m m e t r y of S2-receptors in the left t e m p o r a l c o m p a r e d to the right t e m p o r a l cortex was related to the severity of depression in patients with left-sided lesions 1°'15. T h e d e m o n s t r a t i o n of asymmetries in m o o d and biochemical response to unilateral brain injury in humans have b e e n paralleled by the study of behavioral and biochemical asymmetries in rats with experimental unilateral brain injury 12-14'16. L o c o m o t o r hyperactivity has been induced in rats by right, but not left sided ischemic cortical strokes or focal suction lesions. This behavioral change has b e e n associated with w i d e s p r e a d depletions in ipsilateral and sometimes contralateral cortical m o n o a mines following right, but not left hemisphere lesions 12'14. T h e p r e s e n t e x p e r i m e n t was p e r f o r m e d to d e t e r m i n e w h e t h e r changes in cortical S2-serotonin receptors also
occur following unilateral cortical suction lesions in rats, analogous to the p h e n o m e n a seen with P E T imaging in stroke patients. O u r interest was to d e t e r m i n e whether the lateralized p h e n o m e n a seen in humans could be duplicated using in vitro a u t o r a d i o g r a p h y and a well studied rodent m o d e l of focal injury, where lateralized behavioral and neurochemical changes have b e e n previously described. MATERIALS AND METHODS
Surgical lesion procedure A right (n = 6) or left (n = 5) sided frontoparietal craniotomy was performed in male Sprague-Dawley rats (300-350 g)12. Animals were first anesthetized with nembutol (50 mg/kg) and then placed in a stereotaxic apparatus. Using an internally bevelled 18-gauge needle attached to a vacuum device, tissue was removed from the lateral aspect of the frontoparietal cortex above the rhinal fissure and just anterior to the middle cerebral artery. The center of each lesion was approximately 8.5 mm anterior to the interaural line (Fig. 1). This method has been previously demonstrated to produce lesions of uniform size and comparable volumes12. Control animals (n = 5), received anesthesia followed by either right or left craniotomy without interruption of the dura. The scalp was surgically closed and the animals were returned to their individual cages for post-operative recovery.
Behavioral testing Animals were housed in individual cages containing a feeding tray, watering tube and a 34 cm diameter running wheel from which
Correspondence: H.S. Mayberg, Department of Radiology/Nuclear Medicine, Johns Hopkins Medical Institutions, Nelson Bl-130, 600 N. Wolfe St., Baltimore, MD 21205, U.S.A.
128 microscopy to confirm that all lesions were restricted to the cortex.
Regional analysis of autoradiographs
Fig. 1. Photomicrograph of 25/~m coronal rat brain section labeled with [3H]spiperone. 3H-Autoradiograph of a 25/tm coronal brain section taken through the center of a left frontocortical suction lesion. The lesion, located approximately 8.5 mm anterior to the interaural line, involves all layers of cortex and does not extend into the basal ganglia. [3H]Spiperone binding is most prominent in frontal, cingulate and perirhinal cortical regions and in the caudate/ putamen and nucleus accumbens.
cyclometer readings were taken at 24 h intervals. Animals were exposed to a regular 12 h light-dark cycle and were acclimatized to the cages for 3 weeks, prior to surgery. Baseline preoperative activity was recorded as the mean activity during the 7 days prior to craniotomy. Daily running wheel activity was also measured during the entire 30 day post-operative period. The average percent change from baseline was calculated for days 20-30 post-surgery.
Quantitative analysis of regional grain densities was performed on digitized images using computerized microdensitometry (Amersham RAS 1000 Image Processing System) and techniques similar to those described elsewhere 2"3. The regional specificity of SP to S2-serotonin receptors in the cortex was assessed by digitally subtracting the cinanserin blocked sections (which compete with SP for the S2-sites, leaving the non-S2-sites) from the adjacent unblocked SP sections (total binding sites: $2- and non-S2-sites). This subtraction technique yielded a computer-generated image of the S2-receptor sites, which was then used for quantification. Comparisons were made between the slides incubated with SP alone (total binding sites) and those generated by the subtraction procedure (S2-sites only). Receptor measurements in the lesion and control groups were made using the brain slices incubated with SP alone to approximate the conditions used in the human PET studies (where total binding of the PET tracer (3-N-[11C]methyl)spiperone, a SP analogue, was used). Optical density readings were obtained from 3 sections (75 p m apart) for each brain area of interest. The average optical density value for each region was then converted to dpm and subsequently to femtomoles of ligand/mg tissue using the tritium standards and automated programs within the image analysis system. Quantification was performed in cortical and subcortical areas remote from the experimental lesion (approx. rostral to the 9.0 interaural line). Samples were taken from macroscopically identified cortical regions (Fig. 2) including frontal (F1, F2, F3), perirhinal (Ins, Pir), and cingulate (C) cortex, as well as from the nucleus accumbens (NA) using a standard stereotaxic rat atlass.
Data analysis The concentration of [3H]SP (fmol/mg protein) was measured in paired right and left hemisphere areas in cortical and subcortical brain structures. An asymmetry ratio of the lesion side to non-lesion side was also calculated for each region of interest. An ANOVA was performed on the data from each brain region separately. When overall significance was present, planned t-tests were carried out to determine which of the lesion groups were
A utoradiography On post-operative day 30, animals were sacrificed by decapitation and the brains were rapidly removed from the cranial vault and frozen in N-methyl butane (-20 to -40 °C). Frozen brain blocks were stored at -70 °C until sectioned. Consecutive 25/~m coronal sections, cut from the rat forebrain block (approx. 9.0-12.7 mm anterior to the interaural plane), were used for analysis of [3H]spiperone (SP) binding to the cortex and underlying striatum. Frozen sections were thaw-mounted onto gelatin-coated glass microscope slides, dessicated, and stored at -70 °C. Labeling of D2-dopamine and S2-serotonin receptors was accomplished using published methods 11. Slides with mounted tissue sections were incubated for 30 min at room temperature (approx. 22 °C) in 0.17 M Tris buffer (pH 7.7) containing 120 mM NaCI, 5 nM KCI, 2 mM CaC! 2, 1 mM MgCI 2, and 0.001% ascorbic acid and 0.4 nM [3H]spiroperidol (22.8 Ci/mmol, New England Nuclear, Boston, MA). Anatomically contiguous tissue slides were incubated in the presence of cinanserin (10/~M) or haloperidol (1 pM) to block S2-serotonin and Dz-dopamine receptors, respectively. After the 30 min incubation period, slides were dipped rapidly in distilled water, followed by 2 successive 5 min washes in cold Tris buffer (4 °C), and a final dip in distilled water to remove residual salts from the buffer solution. Slides were dried in a stream of cold dry air and dessicated in a vacuum jar overnight. Autoradiographs were generated (Fig. 1) by apposing the prepared slides to tritium-sensitive LKB Ultrofilm along with tritium standards (autoradiographic [3H]micro-scales, Amersham) for 7 weeks. After generation of the autoradiographic images, the tissue sections were stained with Cresyl violet. Representative sections through the maximum depth of the lesion were examined using light
Fig. 2. Region of interest template for autoradiographic analysis. Quantitative analysis of regional grain densities was performed on tissue samples incubated with [3H]spiperone alone (total binding) using computerized microdensitometry. Regions of interest were placed in cortical and subcortical areas remote from the experimental lesion (approx. rostral to the 9.0 interaural line). Multiple samples were taken from macroscopically identified cortical regions including frontal (F1, F2, F3), perirhinal (Ins, Pir), and cingulate (C) cortex, as well as from the nucleus accumbens (NA). Optical densities were averaged within a given slice and over approximately 75 pm to determine femtomoles ligand/mg protein for a given region of interest.
129 different from the sham controls. To assess the presence of binding asymmetry in brain regions produced by the lesions, a 2-way mixed model ANOVA was performed on the calculated asymmetry ratio across factors of lesion status (right, left and sham) and brain region (frontal, perirhinal, cingulate, and accumbens). Appropriate planned t-comparisons were again performed. Lastly, running wheel activity during post-operative days 20-30 (expressed as a percent of preoperative baseline activity) was correlated with the average regional binding in lesion animals (both the right and left lesion groups) using Pearson product coefficients. RESULTS Histological inspection of tissue sections confirmed that lesions in both the right and left lesion groups were restricted to the cortex. The cortex was intact in all sham animals. Reliable measurements of actual lesion size were not possible due to shrinkage and distortion of the lesion margins associated with the freezing and subsequent thin sectioning required for autoradiographic processing. No gross morphological changes were evident in the receptor measurement sites which were all remote from the actual lesion. The percentage of total [3H]SP binding that was specific to S2-receptors was 67% in frontal cortex, 68% in cingulate cortex, 57% in perirhinal cortex and 19% in the N A . All subsequent results relate to these 4 brain regions (all anatomically rostral to the site of the suction lesion) in slices incubated with SP alone. S2-Serotonin receptors were symmetrically distributed in all cortical and subcortical regions in the non-lesion sham group (Table I). Lesions affected [3H]SP binding depending on the side of the lesion and the brain region measured. Analysis of data from the frontal cortex demonstrated a significant effect of the type of lesion (left, right or sham) (F2,13 = 4.67, P = 0.029), overall differences between binding in the right and left hemispheres (F1,13 = 18.12, P = 0.001) and a significant interaction between the type of lesion and the receptor changes in each hemisphere (F2,13 = 11.34, P = 0.001). Planned t-comparisons demonstrated that right lesions produced significant increases in S 2-
receptors in both the right (t = 6.49, P < 0.001) and left (t = 9.4, P < 0.001) frontal cortex. In contrast, left lesions produced significant decreases in S2-receptors in both the right (t = -9.08, P < 0.001) and left (t = -2.4, P < 0.05) frontal cortices, compared to shams. Furthermore, the magnitude of the decrease in the right hemisphere was significantly greater than the decrease in the left hemisphere in response to left lesions (t = 5.95, P < 0.001). Analysis of data from the perirhinal cortex demonstrated no significant effect of lesion side on S2-receptor binding (F2A 3 = 1.07, P = 0.37). The right and left hemisphere measurements were significantly different (F1,13 = 14.4, P = 0.002), but there was no interaction between the type of lesion and the side of the measurement in this a r e a ( F 2 , 1 3 = 0.46, P = 0.64). Planned t-comparisons demonstrated that binding in the right hemisphere was decreased compared to the left hemisphere in response to both left lesions (t = 2.79, P < 0.02), and right lesions (t = 2.34, P < 0.05). There were no significant changes in the cingulate or nucleus accumhens in any of the lesion groups. A n examination of the asymmetry ratios demonstrated differences among the lesion groups (left, right, sham) (F2,13 = 10.3, P = 0.002) and a significant interaction between lesion group and the region measured (frontal, perirhinal, cingulate, accumbens) (F6,39 = 8 . 5 5 , P = 0.0001). There was, however, no effect of the region measurement alone (F3,13 ~-- 1.7, P = 0.18). Planned t-comparisons reconfirmed that greater asymmetries were present in the frontal cortex and pe'rirhinal cortex following left lesions compared to both sham lesions (frontal t = 3.8, P < 0.001; perirhinal t = 5.55, P < 0.001) and right lesions (frontal t = 4.3, P < 0.001; perirhinal t = 8.5, P < 0.001). Differences in motor activity were seen among the three groups. The activity of right lesion animals was 168% + 35 of the baseline activity (mean + S.E.M.), compared to 119% + 25 for left lesion animals and 115% + 31 for shams. The relationship between SP binding in
TABLE I Total f :3H]spiperone binding (fmol/mg protein + S. E. M. ) Region
Frontal Cingulate Perirhinal Accumbens
Left lesion group (n = 5)
Right lesion group (n = 6)
Sham lesion (n = 5)
Left
Right
L/R
Left
Right
R/L
Left
80 _+10 102 + 20 78 _+9 136 _+19
64 _+10"** 96 + 18 57 _+11 143 -+ 25
1.27 _+0.05* 1.05 _+0.02 1.47 _+0.16" 0.99 _+0.06
110 + 8* 123 + 11 87 + 5 202 + 23
105 + 9* 123 + 11 72 + 5 207 + 31
0.95 + 0.03 1.00 + 0.03 0.84 + 0.08 1.01 + 0.05
86 + 116 + 75 + 195 +
*P < 0.001 compared to corresponding sham value. **P < 0.001 compared to left hemisphere value.
5 10 4 8
Right
L/R
88 + 5 117 + 12 75 + 5 192 + 6
0.98 + 0.02 1.00 + 0.02 1.02 + 0.05 1.02 + 0.05
130 each region and motor activity was assessed in the two lesion groups (right and left). There was a positive correlation between mean daily running wheel activity during postoperative days 20-30 (expressed as a percent of pre-operative baseline activity) and the average [3H]SP binding in frontal cortex in animals with right and left sided lesions (r = 0.65, df = 11, P = 0.03). There were no significant correlations between motor behavior and SP binding in perirhinal cortex, cingulate, or nucleus accumbens. DISCUSSION In this study, right sided frontolateral cortical suction lesions produced a significant bilateral increase in [3H]SP binding in the non-lesion areas of the frontal cortex, while identical lesion of the left hemisphere produced a significant bilateral decrease. Furthermore, left sided lesions resulted in an asymmetric change in S2-serotonin receptors in frontal and perirhinal cortex due to a greater decrease in binding in the contralateral (i.e. right) hemisphere. Frontal cortical spiperone binding was significantly correlated with the degree of locomotor activity in both right and left lesion groups. Synaptic neurochemical changes in response to, but remote from the site of injury is not a novel finding. Both pre- and post-synaptic changes have been demonstrated in response to both ischemic and focal brain injury in brain regions without obvious neuropathological damage. Finklestein has demonstrated remote changes in norepinephrine, dopamine and serotonin and their metabolites in the uninjured areas of the hemisphere ipsilateral to large ventrolateral cortical suction lesions in rats 5"6. Starkstein has recently demonstrated that a right unilateral frontocortical lesion induces bilateral changes in dopamine metabolism in the NA 16. The magnitude of these chemical changes was related to the degree of motor hyperactivity seen in the animals and occurred in an area that is recognized for its connections to the basal ganglia and for its role in motor behaviors 1. Brown, using two distinct gerbil ischemia models, has described decreases in frontal cortex S2-serotonin receptors 72 h after injury 4. These postsynaptic receptor changes were seen in areas remote from the hypoxic injury in the hippocampus and did not appear to be solely related to changes in forebrain serotonin levels. Remote changes in S2-serotonin receptors in response to a frontal bisection lesion have also been reported by Leysen9. Various theories have been proposed to explain these remote neurochemical changes following relatively focal lesions. Precise mechanisms, however, have not been demonstrated. Asymmetrical behavioral and presynaptic neurochemical effects of unilateral cortical lesions have also been
previously demonstrated. Right, but not left frontolateral cortical suction lesions in the rat have been shown to produce significant depletions of norepinephrine and dopamine in the locus coeruleus, substantia nigra and cerebral cortex 13'14A6. We do not yet know the mechanism by which right hemisphere lesions produce a different presynaptic response than left hemisphere lesions. Our present findings that both the direction and magnitude of cortical S2-receptor change depends on which hemisphere is injured further support the hypothesis that the right and left hemisphere are biochemically different in their response to focal injury. The regional pattern of change in SP binding following focal brain injury was slightly different in rats compared to that seen in humans. In our previous PET study of cortical S2-serotonin receptors, we found that right hemisphere but not left hemisphere lesions led to an increase in [11C-n-methyl]spiperone binding in umnjured areas of the ipsilateral temporal and parietal cortex, but not the frontal cortex 1°. Because of the obvious differences between the rat and human lesions used in these studies, the implications of the present results for understanding the changes produced by ischemic strokes is not straightforward. In this study we were directly addressing whether focal lesions in rats produce comparable receptor changes to those seen in human ischemic stroke. We found that right hemisphere lesions produced an increase in spiperone binding bilaterally and most prominently in the frontal cortex. This is the first demonstration of an asymmetric postsynaptic neurochemical change following focal brain injury in rats, and these results are similar to what was seen using PET. The suction lesion method, although ideal for creating lesions of consistent size and location 12, is not identical to ischemic lesions in stroke patients. This type of lesion however, has been shown to produce many of the same behavioral and biochemical changes found following middle cerebral artery ligations in rats a3'14. The density of S2-serotonin receptors were estimated in this experiment using total [3H]SP binding in cortex. Therefore, we cannot be sure that the receptor changes were exclusively or even primarily in S2-receptors. We were however, specifically interested in cortical brain regions where S2-serotonin receptors are known to be highly concentrated. It has been previously demonstrated that SP binds with high affinity to D2-dopamine and Sz-serotonin receptors, as well as to the pharmacologically uncharacterized spirodecanone site 2"11. In vivo binding 7, classical quantitative autoradiography n, and autoradiography using computerized digital subtraction methods z'3 have demonstrated that binding of [3H]SP in cortex is predominantly to S2-serotonin receptor sites, with the highest concentration of receptors in frontal and
131 perirhinal regions. In addition, it has been shown that spirodecanone binding is most prominent in layer I of the cortex, whereas S2-serotonin receptors are found in cortical layer IV 11. We have confirmed that total [3H]SP binding is a reasonable estimate of the S2-receptors in these regions. We have not, however, evaluated the specific contribution of the spirodecanone site to our observed changes in total spiperone binding. Despite this limitation of our experimental method, there is no physiologic or pharmacologic precedent for either a regional or lateralized change in spirodecanone binding following focal brain injury. One could also argue that a selective S2-antagonist might have been a more appropriate choice for these studies. The primary goal of this particular study, however, was to determine whether the changes in S2-serotonin receptors that we have previously reported in stroke patients using P E T and (3-N[ltC]methyl)spiperone could be produced in a rat model of stroke.
In summary, the findings from these studies in the rat are consistent with the lateralized changes in cortical S2-serotonin receptors demonstrated with P E T in stroke patients. These results suggest that focal brain injury produces significant changes in both pre- and postsynaptic aspects of the central monoaminergic system. The magnitude and direction of these changes vary, depending on the side of the injury. A t this point, we do not know what the relationship is between these chemical changes and behavioral outcome following either rat or human brain injury. However, all of these factors may contribute to the development of, or protection against the lateralized behavioral syndromes seen following specific cortical lesions.
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Acknowledgements. The authors thank Barbara and Sandra Loats of LOATS Associates, Westminster, MD for technical assistance with computer digitization of the autoradiographs. This work is supported by NIH Grants NS15178 and MH00163, and a generous gift from Mrs. Samuel Hecht.