Bilateral ablation of frontal cortex reduces concentration of cholecystokinin-like immunoreactivity in rat dorsolateral striatum

Brain Research, 452 (1988) 113-117 Elsevier

113

BRE 13632

Bilateral ablation of frontal cortex reduces concentration of cholecystokinin-like immunoreactivity in rat dorsolateral striatum Dieter K. Meyer, Karl Schultheiss and Michael Hardung Institute of Pharmacology, University of Freiburg, Freiburg (F.R.G.) (Accepted 15 December 1987)

Key words: Dorsolateral striatum; Cholecystokinin; Frontal cortical area

Rat striatum (caudatoputamen, CP) contains high concentrations of cholecystokinin-like immunoreactivity (CCK-LI) which is not synthesized in the CP itself, but is brought to it by afferent projections. Some of these have been reported to originate in the sensorimotor cortex. The source of the major part of the CCK-LI in the CP is not known. In the present study, it was investigated whether neurons in the frontal cortex send CCK-LI-containing fibers to the CP. Ablation of the frontal pole of one hemisphere did not decrease but significantly enhanced the CCK-LI in the dorsal CP. Unilateral ablation of the frontal pole combined with the ipsilateral severance of corpus callosum fibers reduced ipsilaterally the concentration of CCK-LI in the dorsolateral CP by approximately 60%. Also ablation of the frontal poles of both sides bilaterally reduced the concentration of CCK-LI in the dorsolateral CP by approximately 40%. It is concluded that the neuronal elements in the dorsolateral CP of one side, which contain CCK-LI, are in some way connected with neurons in the frontal poles of both hemispheres. This connection may be just functional or may be due to CCK-containing fibers, which originate in the frontal pole.

INTRODUCTION The c a u d o p u t a m e n (CP) of rat brain contains high concentrations of cholecystokinin-like i m m u n o r e a c tivity ( C C K - L I ) , which is mainly due to the p e p t i d e cholecystokinin-8 sulfate 2"5. W i t h the use of i m m u n o cytochemical techniques, C C K - L I has been found in axons and terminals 7'a1'21'22. While s o m e authors did not find any C C K - i m m u n o r e a c t i v i t y in striatal neuronal cell bodies 7"1l'21"22, others found only very few positive p e r i k a r y a 2°. Thus, the p e p t i d e is unlikely to be synthesized in the CP, but seems to be contained in afterent projections. This assumption was corrobo r a t e d by recent findings that m R N A specific for preprocholecystokinin could not be m a d e visible with in situ hybridization techniques in the rat CP 19. A l s o when filter hybridization techniques were used, only very small and variable amounts of this m R N A species were found in rat CP 3. Several projections to the CP containing C C K - L I have been described. Thus, a projection from the substantia nigra/ventral tegmen-

tal area to the accumbens nucleus and striatum has been r e p o r t e d 1°. It is doubtful, however, that it brings significant a m o u n t s of C C K - L I to the dorsal CP 7'15, which is the part of the CP dorsal to a horizontal plane through the anterior commissure. Bilateral projections from s e n s o r i m o t o r and prelimbic cortical areas have been described which m a y bring C C K - L I to the dorsal CP ts. Lesions severing these cortical projections r e d u c e d the content of C C K - L I of the dorsal CP by 3 0 - 5 0 % , but did not d e p l e t e it. Thus, brain areas which are main contributors of CCK-fibers to the CP are still unknown. It was the aim of the present study to investigate w h e t h e r projections from the frontal pole of rat brain bring C C K LI to the dorsal CP. MATERIALS AND METHODS Male Wistar rats (150-200 g) were k e p t u n d e r diurnal lighting conditions (lights on at 6.00 h; off at 18.00 h). Brain lesions were m a d e u n d e r e t h e r anes-

Correspondence: D.K. Meyer, Institute of Pharmacology, University of Freiburg, Hermann Herderstr. 5, D-7800 Freiburg, F.R.G.

114 thesia after removal of a bone flap covering the respective area. The following parameters were used: (A) Unilateral injection of ibotenic acid into the head of the CP. Plane angle: 2.5 mm down; 8 mm anterior to the interauricular line; 2.5 mm lateral to the midline; 5.5 mm down from the dorsal brain surface. One microliter of phosphate-buffered saline containing 15 ~g of pH-adjusted ibotenic acid was injected with a Hamilton syringe over a period of 5 rain. (B) Ablation of the right frontal pole. The lesion was made by a freehanded knife cut which completely separated the frontal pole from the rest of the brain. The frontal plane of the cut bordered the most rostral part of the CP (in the area of the forceps minor of the corpus callosum, according to Paxinos and Watson16). (C) Ablation of the right frontal pole plus an ipsilateral parasagittal lesion of the corpus callosum. The ablation of the right frontal pole was performed as described in B. The parasagittal lesion was made by a freehanded knife cut which was 1 mm parallel to the midline and extended for approximately 5 mm from the frontal pole lesion to the corpus of the CP. (D) Bilateral ablation of the frontal pole. These lesions were performed as described in B. For determination of CCK-LI, the rats were killed by cervical dislocation 6-7 days after knife cuts and 5 days after ibotenic acid injections. The brains were quickly removed, and coronal slices (1 mm) cut with a plastic template. The head of the CP was dissected as described previously 15. Briefly, after removal of any surrounding tissue, the dorsal and ventral striatum were separated by a cut along the plane of the anterior commissure. The medial part of the dorsal striatum (dorsomedial CP) was separated from the lateral part (dorsolateral CP) by a dorsoventral cut which approximately halved the slice. Then, the tissue samples were boiled for 7 min in 0.5 ml of distilled water and sonicated. After centrifugation (4000 g for 8 min), an aliquot was added to the radioimmunoassay after its appropriate dilution with assay buffer. The radioimmunoassay was performed as previously described 2. CCK-LI is expressed as the equivalent of the sulfated cholecystokinin-octapeptide. Protein contents were determined with the method of Lowry et al. 13. Concentrations of CCK-LI are given in pg/~g protein. For determination of CCK-mRNA, the rats were

killed 3 days after surgery. The frontal pole contralateral to the lesion was dissected into an inner part (which is assumed to contain projection neurons) and an outer part (which is assumed to contain mainly local circuit neurons). The inner part was extracted for total RNA with guanidinium hydrochloride using a modification of a procedure described by Cheley and Anderson 4. Total R N A was run on denaturing agarose-formaldehyde gels 12 and subsequently blotted onto nylon membranes. R N A was quantified by measurement of the ethidiumbromide fluorescence of the 18S rRNA in the gel as determined by densitometry. Northern filters were probed with a 32p-labelled CCK-RNA complementary to the message sense which was derived by in vitro transcription of a CCKcDNA containing SP6-plasmid 14. To abolish unspecific signals, filters were treated with ribonuclease A 9. C C K - m R N A was quantified by densitometric scanning of the autoradiograms. CCK-mRNA was expressed by comparing the respective densitometric signals (CCK-mRNA/rRNAlss), i.e. in artibrary units. Two experiments were performed. The absolute values of these two experiments differed. To combine their data, the means of the respective control groups were calculated. Then, within one experiment the values of the control group as well as of the lesion group were expressed as percent of the respective mean. The percent values thus obtained were combined and used for statistical analysis. In all experiments, Student's t-test was used for statistical evaluation. Statistical comparison of one area of lesioned rats was made with the respective area of sham-operated controls. RESULTS AND DISCUSSION The first experiment was perfored to exclude the possibility that CCK-LI is synthesized in the dorsal CP by neurons which have not been detected with immunocytochemical or molecular biological techniques. For ttais purpose, ibotenic acid (15 ~g), a neurotoxic agent which kills neuronal cell bodies but leaves afferent axons and terminals intact 17, was injected into the CP. This treatment did not decrease the concentration of CCK-LI in the dorsomedial and dorsolateral CP, but even tended to enhance the concentrations of CCK-LI in the CPs of both sides

115 (Table I, panel A ) . This finding is in a g r e e m e n t with a previous r e p o r t that kainic acid, a n o t h e r neurotoxic agent, does not reduce the concentrations C C K - L I when injected into the CP 7. It clearly indicated that neurons in the CP do not synthesize significant amounts of C C K - L I . The ablation of the right frontal pole did not decrease the concentration of C C K - L I , but even significantly increased it in the dorsolateral CP of both sides as well as in the contralateral d o r s o m e d i a l CP. D u e to a large variation, a similar effect in the ipsilateral d o r s o m e d i a l CP was not significant (Table I, panel B). These surprising enhancements indicated that neurons in the frontal pole and the neuronal e l e m e n t s in the CP which contain C C K - L I are in some way connected. To learn m o r e about this connection, it was investigated w h e t h e r the unilateral ablation of the frontal pole affected the C C K - c o n t a n i n g neurons

TABLE I Effects of lesions on CCK-L1 in dorsal CP C, sham-operated controls; L, lesioned rats; pg/pg protein + S.E.M. (n). Percent values: L/C x 100. DL, dorsolateral CP; DM, dorsomedial CP. Part of CP

CCK-LI (pg/pg protein) Contralateral

lpsUateral

(.4) Injection of ibotenic acid into the right CP DL C 1.6 + 0.2 (5) C 2.3 + 0.4 (5) L 1.9 + 0.2 (4) 1 1 9 % L2.6 -+ 0.3 (4) DM

C 1.9 _+0.3 (5) L2.5 + 0.2 (4)

C 1.7 + 0.2 (5) 1 3 2 % L2.7 + 0.5 (4)

(B) Ablation of right frontal pole DL C 1.6 + 0.3 (5) L3.1+0.3(5)* 194% DM

C 1.3 + 0.2 (5) L3.2+0.4(5)*

246%

113% 158%

C 1.6 + 0.3 (5) L3.3-+0.7(5)*

206%

C 1.9 -+ 0.1 (5) L3.3+0.8(5)

173%

(C) Ablation of right frontal pole plus lesion of the ipsilateral corpus callosum DL C 1.2 + 0.1 (6) C 1.1 + 0.2(6) L0.9 + 0.4 (5) 75% L0.4 + 0.1 (5)* 36% DM

C 1.1 + 0.1 (6) 1 3 0 % L0.5 _+0.2 (5) *

45%

(D) Bilateral ablation of the frontal pole DL C2.1 _+0.3(6) C 1.6 -+ 0.3 (6) L 1.3 -+ 0.1(4)* 62% L0.9 _+0.1 (4)*

56%

DM *P < 0.05.

C 1.0 + 0.1 (6) L 1.3 _+0.7 (5)

C 2.2 + 0.2 (6) L 1.0 + 0.3 (4)*

45%

C 1.5 + 0.3 (6) L 1.3 + 0.2 (4)

86%

in the contralateral frontal pole cortex. F o r this purpose, m R N A specific for p r e p r o c h o l e c y s t o k i n i n ( C C K - m R N A ) was m e a s u r e d in the inner part of the contralateral frontal cortex 3 days after unilateral frontal pole ablation. This part of the cortex (app r o x i m a t e l y layers IV to VI) was chosen b e c a u s e p r o j e c t i o n neurons are generally assumed to be located here. The lesion did not change the concentration of C C K - m R N A as c o m p a r e d to the controls (controls vs lesioned rats: 100 _ 7% (n = 8) vs 97 _+ 12% (n = 6), respectively; for calculation of d a t a see Materials and M e t h o d s ) . This result indicated that the CCK-synthesis of the respective C C K - n e u r o n s was unchanged. Thus, the bilateral e n h a n c e m e n t of the C C K - L I concentration in the dorsal CP after unilateral frontal pole ablation is unlikely to be due to the increased activity of a C C K - c o n t a i n i n g p r o j e c tion from the remaining frontal pole. Instead, C C K neurons in o t h e r parts of the brain seem to be involved. In the next e x p e r i m e n t , the hypothesis was tested that projections from the contralateral side a n d / o r midline areas were involved in the ipsilateral increase in C C K - L I induced by the unilateral frontal pole ablation. T h e r e f o r e , a lesion was m a d e which c o m b i n e d the ablation of one frontal pole with the ipsilateral parasagittal severance of the rostral half of the corpus callosum. This c o m b i n e d lesion i n d u c e d a dramatic change in the concentrations of C C K - L I in the CP. It was r e d u c e d by 64 and 55% in the ipsilateral dorsolateral and d o r s o m e d i a l CP, respectively (Table I, panel C). T h e reduction in the ipsilateral d o r s o m e d i a l CP confirms the previous finding that the separation of this area from the ipsilateral p r e l i m bic cortex and/or the anterior cingulate gyrus significantly reduces its concentration of C C K - L I aS. The p r o n o u n c e d reduction in the dorsolateral CP indicated that fibers from the contralateral side and/or midline areas affect its C C K - L I content. It should be p o i n t e d out that a similar lesion of the corpus callosum alone does not change the concentration of C C K - L I in the ipsilateral dorsolateral CP 15. To test the hypothesis that neuronal afferents from both frontal poles affect the C C K - L I content of the dorsolateral CP, a bilateral frontal pole ablation was made in the next e x p e r i m e n t . This c o m b i n e d lesion significantly r e d u c e d the concentration of C C K - L I bilaterally in the dorsolateral CP and in the contralat-

116 eral dorsomedial CP (Table I, panel D). The extent of the reduction in the d o r s o l a t e r a l CP was similar to that observed after unilateral frontal pole ablation plus severance of the corpus callosum ( e x p e r i m e n t

C). In summary, unilateral ablation of the frontal pole did not reduce the concentration of C C K - L I in the CP - - only the additional lesion of the corpus callosum or of the contralateral frontal pole had this effect. It is n o t e w o r t h y that also a lesion of the sensorim o t o r cortex only p r o v e d to be effective in the dorsolateral CP, if the corpus callosum was additionally severed 15. Thus, the d o r s o l a t e r a l CP seems to receive C C K - L I containing fibers from both hemispheres. If only one side is lesioned, the remaining projections m a y c o m p e n s a t e the lesion-induced loss. These findings also indicate that neurons in the frontal poles of both sides affect the content of C C K - L I in the dorsolateral CP. H o w e v e r , the nature of this connection is unclear. T h e r e are 3 possible explanations: (1) frontal pole neurons m o d u l a t e the activity of striatal neurons which synthesize C C K - L I . Since ibotenic acid should have killed all n e u r o n a l cell bodies 17 in the CP, but did not decrease the concentration of C C K - L I (see e x p e r i m e n t A ) , this assumption is rather unlikely. (2) F r o n t a l pole neurons affect CCK-synthesizing neurons in o t h e r parts of the brain which project to the CP. This possibility is difficult to evaluate, since until now only s e n s o r i m o t o r areas have been shown to contribute relevant amounts (approximately 30%) of C C K - L I to the dorsolateral CP 15. Lesions of the projections from o t h e r cortical and sub-

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