- Email: [email protected]
Forebrain catecholamine concentrations in the rat after neocortical and hippocampal lesions
Life Sciences, Vol. 29, pp. 2603-2607 Printed in the U.S.A,
Pergamon Press
FOREBRAIN CATECHOLAMINE CONCENTRATIONS IN THE RAT AFTER NEOCORTICAL AND HIPPOCAMPAL LESIONS Michael L. Woodruff I
Ronald H. Baisden 2 and Barbara S. Fish 3
Departments of Anatomy and Psychiatry I, Department of Anatomy 2 Quillen-Dishner College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614 and Department of Pharmacology and Toxicology~, University of Arizona, Tucson, Arizona 85721 (Received in final form October 26, 1981) Summary Spectrofluorometric procedures were used to measure forebrain content of dopamine and norepinephrine in rats after either dorsolatera1 neocort[cal ahlatLon, or combined neocortical-hippocampal ablations. The concentration of both of these catecholamines was significantly higher in the ablated rats than in uninjured control rats. I1owever, there was no difference between the rats receiving only neocortical lesions and the rats with combined lesions on this measure. Damage to restricted regions of the brain is known to cause morphological, biochemical and electrophyslological alterations in structures remote from the actual site of damage. For example, destruction of the fornix or dorsal hippocampus is followed by collateral axonal sprouting (7) and increases in catecholamine concentrations (6) in the septal region. This lesion-induced collateral sprouting in the septal nuclei also appears to produce physiologically viable synapses (13). Another example of lesion-induced neuronal plasticity is provided by the observatioh that experimental ischemic lesions caused by unilateral ]igation of the middle cerebral artery produce changes in the pattern of catecho]aminergic innervation in undamaged neocortical areas in the rat (I0, 12). Although biochemical changes have been observed in the forebrain after experimental Ischemic damage to rat neocortex and in the septal area after hippocampal ablat[on, there are presently no reports concerning the consequences for forebrain catecholamine concentrations of aspiration lesions of neocortex, or of hippocampus in addition to neocortex. Such information would be valuable because a large number of studies have been published in which the effects of such lesions on behavior have been described (3). Moreover, evidence has begun to accumulate which suggests that leslon-induced changes in catecholamine content in at least some forebrain areas which have not been damaged directly by the lesion may have consequences for behavior. For example, Baisden and Woodruff (I) have reported that hippocampal les[on-induced sprouting in the septal area may be responsible, at least in part, for the deficit in DRL performance which follows bilateral hippocampal damage (3), and Robinson and his co-workers (9,11) have demonstrated behavioral responses correlated to catecholaminergic plasticity induced by isehemlc infarction of rat neocortex. Therefore, the purpose of the present experiment was to determine the effect of large aspiration lesions of either the neocortex alone, or of the neocortex and hippocampus 0024-3205/81/252603-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2604
Brain Lesions and Catecholamines
Vol. 29, No. 25, 1981
together, upon the amount of norep[nephrtne and dopamine contained brain of rats.
in the fore-
Materials and Methods Thirty-four male Long-Evans hooded rats weighing between 350 and 450 gm at the time of surgery served as subjects. Ten of these rats served as unoperated controls (Group N), while 12 were given bilateral neocortical aspiration lesions (Group C) and 12 were given bilateral hippocampal aspiration les[ons. The procedure used in our laboratory for producing the aspiration lesions has been described in detail elsewhere (4,16). Ninety days after the les[ons were made in the rats in Group C and Group H, each rat in Groups C, H, and N was killed by means of cervical dislocation and the brain was removed rapidly over ice. The forebrain was separated from the hindbrain at the level of the Intraquadr[geminal ridge and the pes pedunculi and assayed for norepinephrine (NE) and dopamine (DA) content by means of a modification of the fluorometric method of Shellenberger and Gordon (14). The forebrain was weighed and homogenized in perchloric acid. Alumina was used to extract the NE and DA and the catecholamines were eluted from the alumlna with acetic acid. The resulting solution was divided into two 1.0 ml aliquots. One aliquot served as a test sample and the other as a tissue blank. Fluorescent derivatives of NE and DA were formed in the test samples by oxidation. Tissue blanks were submitted to reverse oxidation. In addit£on to the test samples and tissue blanks, five Internal standards contalning known amounts of NE and DA and a reagent blank contalning. 1.0 ml each of acetic acid and perchloric acid were carried through the entire procedure. Moreover, a blank and five external standards containing known amounts of NE and DA were prepared prior to the oxidation procedure. All tubes were read in quartz cuvettes at 30 sec intervals in a Farrand Spectrofluorometer. Activation and emission slits were set at 20 mm/40 mm and I0 mm/ 20 mm respectively. The excitation wavelength for NE was 380 mu and the emission wavelength 485 mu. When the tubes were reread for DA the excitation wavelength was 310 mu and the emission wavelength was 380 mu. Results The aspiration lesions were made in the same manner and by the same person as the lesions described in our previous reports during the past several years (1,16,17). Each brain was inspected before it was homogenized and the superficial appearance of the lesions in the brains from the rats in Croups H and C was similar to the appearance of the lesions described in our previous experiments. Extraction efficiency (internal standard slope divided by external standard slope) for NE ranged from 67% to 93% and for DA from 81% to 95%. The spectrofluorometrlc readings were converted to nanograms after reverse oxidation blank readings were subtracted from each sample reading. Conversion was made by the formula x = (y-b)/m, where b is the y intercept and m is the slope calculated from the external standards; y is the fluorometry reading corrected for recovery, and x is the amount for NE or DA for the sample.
Vol. 29, No. 25, 1981
Brain Lesions and Catecholamines
2605
I100 I000 900800" 700 600 ng/g 500 400 300 200 I00
Norm. Cort.
Hipp.
Norepinephrine
Norm, Cort.
Hipp.
Dopamine
FIG. I Concentration of NE and DA (ng/g) in the forebrain of normal rats (NORM.), rats with dorsolateral neocortical lesions (CORT.), or combined neocortical-hippocampal lesions (HIPP.). The mean and standard error NE and DA per gm of tissue weight is presented for each group in Figure I. One-way analyses of varLance followed by simple contrast tests were conducted for the data for each transmitter. A significant overall effect was found for NE (F (2,31) = 19.36, p .001). Applicat[on of the simple contrasts tests revealed that Group N differed from Croup C (F = 32.56, p .0001) and from Group H (F = 26.97, p .0001), but Group C did not differ stat[stLcally from Group H (F = 0.29, p .05). The same pattern of effect was found for DA forebrain concentrations. A significant overall effect was found (F (2,31) = 16.98, p .001). Group N differed from Group C (F = 29.80, p .001) and from Group H (F = 21.97, p .001), but Groups H and C did not dLffer from each other (F = 0.66, p .05). Discuss ion The results of this study indicate that both dorsolateral neocortical lesions and combined dorsolateral neocortical-hippocampal lesions significantly increase the amount of DA and NE found Ln the forebrain of the rat. Because of the relatively long survival time employed in the present experiment, the change in transmitter content is probably permanent, but the methodology
2606
Brain Lesions and Catecholamines
Vol. 29, No. 25, 1981
employed does not permit preclse localization of the change withln the forebrain, nor does it allow the elucidation of the cause of the change. However, these data are heuristically useful in that they suggest that substantial and unpredicted blochemical changes occur after these lesions. Two explanations for these changes are offered below. The cell bodles of origin of the NE- and DA-conta[ning axons and terminals destroyed by the ablations made in the present experiment are found in the pons and midbrain (2,5) and each of these neurons has a relatlvely large terminal field. Destructlon of the terminal field of some of these neurons could lead either to enhanced levels of transmitter in the remaining axons and terminals, or to actual increase in the number of terminals. Catecholaminergic neurons have been reported to undergo reconstruction after damage to terminal areas (6,15) and the work of Robinson and hls colleagues (9,10,11,12) indicates that the number of axonal varicosities increases significantly as a result of ischemic neocortical damage. For these reasons, [t seems more likely that the increases in NE and DA observed in the present experiment are due to actual morphological expansion of terminal zones of catecholalminergic axons remaining after the ablations, than to slmple enhancement of catecholamine content in existing, morphologically static, neurons. H1stofluorescent mlcroscopy should be applied to test thls hypothesis. There were no statistically significant dlfferences in forebrain concentrations of catecholamines between rats given only neocortical ablations and rats with combined neocortical-hippocampal aspirations. This finding is somewhat surprising because of the substantial dlfferences in effect upon behavior that these lesions have (3), and because of the difference in amount of tissue removed in the two preparations. Restricted neocortical damage appears to produce effects limited primarily to undamaged neocortex (9,10,11,12), whereas it seems likely that hippocampal removal causes increases in catecholamine levels in other brain regions, possibly restricted to subcortical sites such as the septum, which compensate for loss of hippocampal catecholamines due to dlrect effect of the lesion. These data may be complementary to the observations of Reinstein et al. (8) who found that rats with either dorsolateral neocortical damage, or combined neocortical and hippocampal damage, exhibited lowered uptake of 2-deoxyglucose in the occipital neocortex and greater uptake of this substance In the parletal cortex than did unablated control rats. However, the brain-damaged groups did not differ from each other in uptake of 2-deoxyglucose. Acknowledsements This research was supported by Biomedical Research Development Grant 1-508RR09171-03. References i. 2. 3. 4. 5. 6. 7. 8.
R.H. BAISDEN and M.L. WOODRUFF, Physiol. ps~chol. ~ 33-39 (1980). A. DAHLSTROM and K. FUXE, Acta Physiol. Scand. Suppl. 232 1-55 (1964). R.L. ISAACSON, The Limbic System, P ienum Press, New Yo~'~-(1974). R.L. ISAACSON and M.L. WOODRUFF, Experimental Psychobiolo$y, B. HART (ED.) 102-109 Freeman, San Francisco (1976). O. LINDVALL and A. BJORKLUND, Acta Physiol, Scan~. S u r f . 4i2 1-48 (1974). R.Y. MOORE, A. BJORKLUND, and U. STENEVl, Brain Res. 33 I~Z'35 (1971). G. RAISMAN, Brain Res. 14 25-48 (1969). D.K. REINSTEIN, R.L. ISAACSON and A.J. DUNN, Brain Res. 175 392-397 (1979).
Vol. 29, No. 25, 1981
Brain Lesions and Catecholamines
2607
969-976
9.
R.G. ROBINSON and F~E. BLOOM, ~. comp. physiol. Psychol. 92 (1978).
I0.
R.G. ROBINSON, F.E. BLOOM AND E.L.F. BATTENBERG, Brain Res. 132 259-272 (1977). R.G. ROBINSON and J.T. COYLE, Life Sci. 24 943-950 (1979). R.G. ROBINSON, W.J. SHOEMAKER and M. SCHLUMPF, Brain Res. 181 202-208 (1980). M. SEGAL, J. Physiol. 261 617-631 (1976). M.K. SHELL~NBERGER and J.H. GORDON, Anal~t. B[ochem. 39 356-372 (1971). M.L. WOODRUFF and R.H. BAISDEN, Anat. Rec. 196 255A (1980). M.L. WOODRUFF and R.L. ISAACSON, Behav. B[ol. 7 489-501 (1972). M.L. WOODRUFF, B.S. FISH and A. ALDERMAN, Physiol. Behav. 19 401-410 (1977).
Ii. 12. 13. 14. 15. 16. 17.
Pergamon Press
FOREBRAIN CATECHOLAMINE CONCENTRATIONS IN THE RAT AFTER NEOCORTICAL AND HIPPOCAMPAL LESIONS Michael L. Woodruff I
Ronald H. Baisden 2 and Barbara S. Fish 3
Departments of Anatomy and Psychiatry I, Department of Anatomy 2 Quillen-Dishner College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614 and Department of Pharmacology and Toxicology~, University of Arizona, Tucson, Arizona 85721 (Received in final form October 26, 1981) Summary Spectrofluorometric procedures were used to measure forebrain content of dopamine and norepinephrine in rats after either dorsolatera1 neocort[cal ahlatLon, or combined neocortical-hippocampal ablations. The concentration of both of these catecholamines was significantly higher in the ablated rats than in uninjured control rats. I1owever, there was no difference between the rats receiving only neocortical lesions and the rats with combined lesions on this measure. Damage to restricted regions of the brain is known to cause morphological, biochemical and electrophyslological alterations in structures remote from the actual site of damage. For example, destruction of the fornix or dorsal hippocampus is followed by collateral axonal sprouting (7) and increases in catecholamine concentrations (6) in the septal region. This lesion-induced collateral sprouting in the septal nuclei also appears to produce physiologically viable synapses (13). Another example of lesion-induced neuronal plasticity is provided by the observatioh that experimental ischemic lesions caused by unilateral ]igation of the middle cerebral artery produce changes in the pattern of catecho]aminergic innervation in undamaged neocortical areas in the rat (I0, 12). Although biochemical changes have been observed in the forebrain after experimental Ischemic damage to rat neocortex and in the septal area after hippocampal ablat[on, there are presently no reports concerning the consequences for forebrain catecholamine concentrations of aspiration lesions of neocortex, or of hippocampus in addition to neocortex. Such information would be valuable because a large number of studies have been published in which the effects of such lesions on behavior have been described (3). Moreover, evidence has begun to accumulate which suggests that leslon-induced changes in catecholamine content in at least some forebrain areas which have not been damaged directly by the lesion may have consequences for behavior. For example, Baisden and Woodruff (I) have reported that hippocampal les[on-induced sprouting in the septal area may be responsible, at least in part, for the deficit in DRL performance which follows bilateral hippocampal damage (3), and Robinson and his co-workers (9,11) have demonstrated behavioral responses correlated to catecholaminergic plasticity induced by isehemlc infarction of rat neocortex. Therefore, the purpose of the present experiment was to determine the effect of large aspiration lesions of either the neocortex alone, or of the neocortex and hippocampus 0024-3205/81/252603-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2604
Brain Lesions and Catecholamines
Vol. 29, No. 25, 1981
together, upon the amount of norep[nephrtne and dopamine contained brain of rats.
in the fore-
Materials and Methods Thirty-four male Long-Evans hooded rats weighing between 350 and 450 gm at the time of surgery served as subjects. Ten of these rats served as unoperated controls (Group N), while 12 were given bilateral neocortical aspiration lesions (Group C) and 12 were given bilateral hippocampal aspiration les[ons. The procedure used in our laboratory for producing the aspiration lesions has been described in detail elsewhere (4,16). Ninety days after the les[ons were made in the rats in Group C and Group H, each rat in Groups C, H, and N was killed by means of cervical dislocation and the brain was removed rapidly over ice. The forebrain was separated from the hindbrain at the level of the Intraquadr[geminal ridge and the pes pedunculi and assayed for norepinephrine (NE) and dopamine (DA) content by means of a modification of the fluorometric method of Shellenberger and Gordon (14). The forebrain was weighed and homogenized in perchloric acid. Alumina was used to extract the NE and DA and the catecholamines were eluted from the alumlna with acetic acid. The resulting solution was divided into two 1.0 ml aliquots. One aliquot served as a test sample and the other as a tissue blank. Fluorescent derivatives of NE and DA were formed in the test samples by oxidation. Tissue blanks were submitted to reverse oxidation. In addit£on to the test samples and tissue blanks, five Internal standards contalning known amounts of NE and DA and a reagent blank contalning. 1.0 ml each of acetic acid and perchloric acid were carried through the entire procedure. Moreover, a blank and five external standards containing known amounts of NE and DA were prepared prior to the oxidation procedure. All tubes were read in quartz cuvettes at 30 sec intervals in a Farrand Spectrofluorometer. Activation and emission slits were set at 20 mm/40 mm and I0 mm/ 20 mm respectively. The excitation wavelength for NE was 380 mu and the emission wavelength 485 mu. When the tubes were reread for DA the excitation wavelength was 310 mu and the emission wavelength was 380 mu. Results The aspiration lesions were made in the same manner and by the same person as the lesions described in our previous reports during the past several years (1,16,17). Each brain was inspected before it was homogenized and the superficial appearance of the lesions in the brains from the rats in Croups H and C was similar to the appearance of the lesions described in our previous experiments. Extraction efficiency (internal standard slope divided by external standard slope) for NE ranged from 67% to 93% and for DA from 81% to 95%. The spectrofluorometrlc readings were converted to nanograms after reverse oxidation blank readings were subtracted from each sample reading. Conversion was made by the formula x = (y-b)/m, where b is the y intercept and m is the slope calculated from the external standards; y is the fluorometry reading corrected for recovery, and x is the amount for NE or DA for the sample.
Vol. 29, No. 25, 1981
Brain Lesions and Catecholamines
2605
I100 I000 900800" 700 600 ng/g 500 400 300 200 I00
Norm. Cort.
Hipp.
Norepinephrine
Norm, Cort.
Hipp.
Dopamine
FIG. I Concentration of NE and DA (ng/g) in the forebrain of normal rats (NORM.), rats with dorsolateral neocortical lesions (CORT.), or combined neocortical-hippocampal lesions (HIPP.). The mean and standard error NE and DA per gm of tissue weight is presented for each group in Figure I. One-way analyses of varLance followed by simple contrast tests were conducted for the data for each transmitter. A significant overall effect was found for NE (F (2,31) = 19.36, p .001). Applicat[on of the simple contrasts tests revealed that Group N differed from Croup C (F = 32.56, p .0001) and from Group H (F = 26.97, p .0001), but Group C did not differ stat[stLcally from Group H (F = 0.29, p .05). The same pattern of effect was found for DA forebrain concentrations. A significant overall effect was found (F (2,31) = 16.98, p .001). Group N differed from Group C (F = 29.80, p .001) and from Group H (F = 21.97, p .001), but Groups H and C did not dLffer from each other (F = 0.66, p .05). Discuss ion The results of this study indicate that both dorsolateral neocortical lesions and combined dorsolateral neocortical-hippocampal lesions significantly increase the amount of DA and NE found Ln the forebrain of the rat. Because of the relatively long survival time employed in the present experiment, the change in transmitter content is probably permanent, but the methodology
2606
Brain Lesions and Catecholamines
Vol. 29, No. 25, 1981
employed does not permit preclse localization of the change withln the forebrain, nor does it allow the elucidation of the cause of the change. However, these data are heuristically useful in that they suggest that substantial and unpredicted blochemical changes occur after these lesions. Two explanations for these changes are offered below. The cell bodles of origin of the NE- and DA-conta[ning axons and terminals destroyed by the ablations made in the present experiment are found in the pons and midbrain (2,5) and each of these neurons has a relatlvely large terminal field. Destructlon of the terminal field of some of these neurons could lead either to enhanced levels of transmitter in the remaining axons and terminals, or to actual increase in the number of terminals. Catecholaminergic neurons have been reported to undergo reconstruction after damage to terminal areas (6,15) and the work of Robinson and hls colleagues (9,10,11,12) indicates that the number of axonal varicosities increases significantly as a result of ischemic neocortical damage. For these reasons, [t seems more likely that the increases in NE and DA observed in the present experiment are due to actual morphological expansion of terminal zones of catecholalminergic axons remaining after the ablations, than to slmple enhancement of catecholamine content in existing, morphologically static, neurons. H1stofluorescent mlcroscopy should be applied to test thls hypothesis. There were no statistically significant dlfferences in forebrain concentrations of catecholamines between rats given only neocortical ablations and rats with combined neocortical-hippocampal aspirations. This finding is somewhat surprising because of the substantial dlfferences in effect upon behavior that these lesions have (3), and because of the difference in amount of tissue removed in the two preparations. Restricted neocortical damage appears to produce effects limited primarily to undamaged neocortex (9,10,11,12), whereas it seems likely that hippocampal removal causes increases in catecholamine levels in other brain regions, possibly restricted to subcortical sites such as the septum, which compensate for loss of hippocampal catecholamines due to dlrect effect of the lesion. These data may be complementary to the observations of Reinstein et al. (8) who found that rats with either dorsolateral neocortical damage, or combined neocortical and hippocampal damage, exhibited lowered uptake of 2-deoxyglucose in the occipital neocortex and greater uptake of this substance In the parletal cortex than did unablated control rats. However, the brain-damaged groups did not differ from each other in uptake of 2-deoxyglucose. Acknowledsements This research was supported by Biomedical Research Development Grant 1-508RR09171-03. References i. 2. 3. 4. 5. 6. 7. 8.
R.H. BAISDEN and M.L. WOODRUFF, Physiol. ps~chol. ~ 33-39 (1980). A. DAHLSTROM and K. FUXE, Acta Physiol. Scand. Suppl. 232 1-55 (1964). R.L. ISAACSON, The Limbic System, P ienum Press, New Yo~'~-(1974). R.L. ISAACSON and M.L. WOODRUFF, Experimental Psychobiolo$y, B. HART (ED.) 102-109 Freeman, San Francisco (1976). O. LINDVALL and A. BJORKLUND, Acta Physiol, Scan~. S u r f . 4i2 1-48 (1974). R.Y. MOORE, A. BJORKLUND, and U. STENEVl, Brain Res. 33 I~Z'35 (1971). G. RAISMAN, Brain Res. 14 25-48 (1969). D.K. REINSTEIN, R.L. ISAACSON and A.J. DUNN, Brain Res. 175 392-397 (1979).
Vol. 29, No. 25, 1981
Brain Lesions and Catecholamines
2607
969-976
9.
R.G. ROBINSON and F~E. BLOOM, ~. comp. physiol. Psychol. 92 (1978).
I0.
R.G. ROBINSON, F.E. BLOOM AND E.L.F. BATTENBERG, Brain Res. 132 259-272 (1977). R.G. ROBINSON and J.T. COYLE, Life Sci. 24 943-950 (1979). R.G. ROBINSON, W.J. SHOEMAKER and M. SCHLUMPF, Brain Res. 181 202-208 (1980). M. SEGAL, J. Physiol. 261 617-631 (1976). M.K. SHELL~NBERGER and J.H. GORDON, Anal~t. B[ochem. 39 356-372 (1971). M.L. WOODRUFF and R.H. BAISDEN, Anat. Rec. 196 255A (1980). M.L. WOODRUFF and R.L. ISAACSON, Behav. B[ol. 7 489-501 (1972). M.L. WOODRUFF, B.S. FISH and A. ALDERMAN, Physiol. Behav. 19 401-410 (1977).
Ii. 12. 13. 14. 15. 16. 17.