Mesolimbic dopamine receptor increases two weeks following hippocampal kindling

Brain Research, 449 (1988)357-360 Elsevier

357

BRE 22906

Mesolimbic dopamine receptor increases two weeks following hippocampal kindling John G. Csernansky 1'2, Steven Kerr 1, Raj Pruthi 2 and Elisabeth S. Prosser 2 1Department of Psychiatry and Behavloral Sciences, Stanford University Sctzool of Medlcine, Stanford, CA 94305 (U.S.A.) and ZPaloAlto Veterans Administration Medical Center, Palo Alto, CA 94304 (U.S.A.) (Accepted 16 February 1988) Key words Kmdling:Nucleus accumbens; Receptor, -D_,~Dopamme receptor

Kindled seizures developed in rats followingrepeated electrical stimulation of the left CA1 regton of the hippocampus. Two weeks after the final kindled seizure, the densities of dopamine (DA) D_,receptors were assayed in the left and right amygdaio~darea, nucleus accumbens, and nucleus caudatus A significant increase (107%) in the density ofDA D2-receptorsin the ipsilateral nucleus accumbens occurred. This findingmay help to define the long-termneurochemicaiconsequencesof kindling.

Changes in the density of various neurotransmitter receptors following electrical kindling have been extensively studied to help determine the neurochemical basis of the kindling phenomenon (for reviews see refs. 13, 19, 24). Changes in subcortical dopamine (DA) receptors, particularly those in the nucleus accumbens, might be expected since there are direct projections from the amygdala and hippocampus to the nucleus accumbc, ns 9'21. However, two prior attempts to study changes in D A receptors following electrical kindling are difficult to interpret because the receptor binding methods used could not discriminate between dopaminergic and serotonergic components of radioligand receptor binding 1'12. We recently reported that electrical kindling of the amygdala acutely leads to an increased density of D A D 2 receptors in the nucleus accumbens ipsilateral to the site of kindling s . To extend this work, we now report that an increased density of ipsilateral nucleus accumbens D A D z : eceptors also occurs two weeks after electrical kindling of the CA1 region of the hippocampus. Electrode implantation. Bipolar electrodes of Teflon-coated stainless-steel wire wrapped around a 31gauge stainless-steel cannula (no. 32 gauge; Medi-

wire) were stereotactically implanted into the anteriot CA1 region of the left hippocampus of 31 Sprague-Dawley male rats (150 g; Simoasen Labs, Gilroy, CA) anesthetized with Vetalar, xylazine, and acepromazine (5:25:1). Coordinates for the hippocampal CA1 region (A 4.0 mm, L 2.8 mm, V 6.8 ram) were determined using an atlas, and checked histologically in preliminary experiments 14. During each animal dissection, the electrode location was checked by visual inspection. The animals were housed two to a cage in a 12/12 light-dark, temperature- and humidity-controlled facility. Food and water w e r e available ad lihitum. Kindling procedure. Sixteen implanted animals were successfui!y kindled; 12 were reserved as controls. Three imlalanted animals were stimulated but failed to achieve the kindled state. The animals were stimulated to produce an afterdischarge using a constant current stimulator and a 400/~A, 1-s train of 60 cps biphasic square waves, approximately 5 days each week. Behavioral seizures were rated on a scale of I - V , as described by Racine 23. The control animals were always handled in a manner similar to experimental animals. The experimental animals were defined as kindled

Correspondence: J. Csernansky, Department of Psychiatryand Behavioral Sciences, Stanford University Schoolof Med,cme. Stanford, CA 94305, U S A. 0006-8993/88/$03,50© 1988Elsevier Science Pubhshers B,V. (BiomedicalDivision)

358 after they had had 5 stage V seizures. The median (range) number of stimulations reqmred for kindling was 35 (23-46). Each kindled animal was sacrificed for biochemical analyses 14 days after its last stage V seizure. Control animals were sacrificed at an analogous timepoint after electrode implantation. f f H ] S p i p e r o n e b i n d i n g . The animals were sacrificed by decapitation. Discrete brain regions were dissected on ice by hand using landmarks derived from an atlas l~. The right and left nucleus caudatus (NC), nucleus accumbens ( N A ) , and amygdaloid areas (A) (posterior olfactory tubercle) were stored separately m 2 ml of 10 mM p h o s p h a t e - s a l i n e buffer (pH 7.4) a t - 7 0 °C until assay. All 6 brain regions for a given animal were thawed on the same day. To achieve a predetermined, adequate protein concentration for assay, tissues from 2 - 4 animals were pooled, based on wet weight. Pooled tissue samples were placed in 10 ml of 10 mM phosphate buffer containing 0.9% NaCI at pH 7.4, and homogenized for 10 s (Brinkmann polytron, setting 5). The homogenates were then centrifuged for 20 min at 50,000 g and the pellets were rinsed twice with 2 ml of buffer Pellets were resuspended in 150 volumes (w/v) of the same p h o s p h a t e - s a l i n e buffer. Specific [3H]spiperone bmding was d e t e r m i n e d in the presence or absence of 5 ~M sulplride using a modification of the method of Burt et al. 5. Seven concentrations of [3H]spiperone (88 Ci/mmol; Amersham) (0.05-3.2 nM) were added to assay tubes containing 5 BM sulpinde or buffer, and 100-200 l~g/ml of protein t7 in a total volume of 0.45 ml. The use of sulpiride as counterligand excluded serotonin S, sites from specific binding, and allowed the identification ~g L;-. ~,~ o,.dtng sites as D A D, receptors 2~. The samples were incubated for 10 rain at 37 °C and rapidly illtered and rinsed through Whatman GF/B glass fiber filters using a Brandel cell harvester. Filters were placed m 4 ml of Optifluor (Packard) scmtdlation fired 12 h prior to counting. Scatchard analysis of the data was performed using an ~terative curve-fitting computer program ( E B D A L I G A N D , Elsevier-Biosoft). Equilibrium dlssoclanon constant (Kd) and the receptor density (Bin, 0 were determined for all 6 brain regions. Difference, between mean values were analyzed using the Student's t-test (two-tailed). In all cases, the Scatchard plots were linear, sug-

TABLE I Mean (+ S E M.I o f FH]~pqJerone B ...... value~ (mnol/I mg proteol)

L-NA** R-NA L-NC R-NC L-A R-A*

Kindled (n = 5)

Control (n = 6)

0.11668 + 0.0135 0.0494 + 0.0052 I1 17911___11.11149 0 1662 +_0 01177 11.11524+_0.111146 0 0621 + 11.01194

110322 + 0 0053 11.0382 + 0.01172 11.1571 + 0 0128 0.1444 +_0 0116 11.04511+_0 01115 0 0400 + 0 0045

* t = 2.23, P = 01152 ** t = 2.56, P = 0 031. gesting a single binding site. [tH]Splperone Bma ~ and K d values are summarized in Tables I and I1. In the nucleus accumbens, ipsilateral to the stimulated hippocampus, the Bin, Xvalue for the k m d l e d animals was significantly greater ( + 107%) than controls. A trend toward a significant increase was also found in the contralateral amygdaloid area. No statistically sigmficant changes in Bm,x values were observed in other brain areas. Statistically significant changes in K o values were found in both caudate nuclei. However, these changes were not of the magnitude usually associated with a significant physiological change in receptor function. No change in Ko value was found m the lpsllateral nucleus accumbens These data suggest that electrical kindling of the h~ppocampus produces an increase in the density of D A D 2 receptors m the ipsilateral nucleus accumbens. Because the increase was present two weeks after the last kindled seizure, this change is very likely related to the long-lasting kindled state, rather than to the seizures. Burnham et al. 4 have suggested the use of two-week persistence as a criterion for persistent neurochemical changes related to the kindled

TABLE II Mean (+ S E M ) o! [~H]~ptpelone K a Lalue~ OtMt

L-NA R-NA L-NC ~ R-NC *~ L-A R-A

Kindled (n = 5J

(._bmtol 01 = 61

0_055 + 0.016 01158+11014 0 055 _+0 1108 0.044+11009 (11141 + 0.007 0 1168_+0 016

0 1167 + 0 1125 (I 115 +011211 0 147 _+0 11311 (I Ill +~1018 (I 113 + 0.033 0 07711+ I).1118

* t = 2 7 5 , P=0,022, ~* t = 3 17, P=0011

359 state. N o statistically significant changes in [3H]spiperone sites were found in o t h e r structures, although some non-significant increases were seen (i.e. contralateral amygdala). The use of additional animals for assay, or the choice of some o t h e r ttme during the kindhng p r o c e d u r e might have revealed additional statistically significant changes. This finding complements o u r previous work mdicating that amygdaloid kindling also produces a change in D A Dz receptors in the tpsilateral nucleus accumbens s. A change in nucleus accumbens D A function after both hippocampal and amygdala kindhng is consistent wtth the presence of convergent input from these two limbic structures to the nucleus accumbens 9 -" O t h e r changes in nucleus accumbens D A function are possible. Increases m postsynaptic D A Dz receptors might well be preceded by changes in presynaptic D A function (i.e. transmitter turnover, presynaptic a u t o r e c e p t o r acttvity). Early studies H~ suggested the amygdaloid D A levels were decreased in amygdala-kindled rats. However others have failed to find changes in brain D A levels in kindled rats "-'3"6"16"'-5. Mintz and H e r b e r g z° have recently shown indirect evtdence of a relationship between subcortical D A function and amygdala kindling. Assymetric turning behavior after amphetamine administration predicted the rate of kindling. Interestingly, a specific mvestigation of D A levels in the tpsilateral nucleus accumbens has not yet been emphasized despite the importance of hmbic output to this structure. Such experiments are now underway to complement the present findings. The increase m nucleus accumbens D A D,_ recep-

1 Ashton, D , Leysen, J.E and Wauqtncr, A Neurotran,,miners and receptor binding in am~gdalotd kindled rats serotoncrgic and noradrenergic modulatory effects, LzJe Scl., 27 (1980) 1547-1556. 2 Blnckwood, D , The role ot noradrenahnc and dopummc m amygdalold kindling In P L Morselh, K G Lloyd. W Loscher, B Meldrom ;rod E N Reynolds (Eds), Neurotransnutter~. Setzures, and Epdepsr, Raven. New York, 1981. pp 283-299 3 Burnham, W M , King, G A and Lloyd. K G . Extra-focal catecholamme levels in kindled rat forebrams, Prog Neuropsy'_'hopharmacol. 5 ( 1981) 537-541 4 Burnham, W.M., Racine, R.J and Okazaki, M.O., Kindling mechamsms- I1 Biochemical studies In J A Wada tEd ), Kmdhng 3, Raven, New York, 1986, pp 283-299. 5 Burt, D.R , Creesc, I and Snyder, S H . Properucs ol 3H-

tors seen here m a y represent an ongoing adaptation of the mesolimbic D A pathway to the abnormal function of a kmdled hippocampus. Recent work of Yim and Mogenson 31-3-"indicates that an important role of the mesolimbic D A pathway is to modulate limbic activity. This finding m a y have relevance for o u r understanding of neuropsychiatric dtseases where abnormalities of brain D A have been implicated. Increases in mesolimbic D A D2 function have been postulated to play a role in the production of psychosis in schizophrenia 77-s'zg. Increases in D A D_, receptors have been found in schizophrenic brain using post-mortern 15--'2--'7 and more recently PET techmques ~°. However, it should be kept in mind that not all investigatmns have found such alterations ~. or have attributed them to neuroleptic treatment Is. Animal models such as kindling may be of particular value in helpmg to define potential endogenous mechanisms whereby increases in mesolimbic D A function occur.

This work was supported by a grant from the National Institute of Mental Health, MH-30854, to the M e n ' a l Health Clinical Research Center at Stanford I_J..~versity ( M H C R C ) , a grant from the Research Service of the Veterans Administration to the Schizophrenia Biologic Research Center (SBRC) at the Palo Alto Veterans Admimstration Medtcal Center, and a grant from the John D. and Catherine T. M a c A r t h u r Foundation to the Stanford Node of the M a c A r t h u r Network on the Psychobiology of Depression. The authors thank Pamela J. Elliott for manuscript preparation and editorial advice.

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