Life Scieaces Vol . 22, pp . 1011-1014 Printed in the U .S .A .
Pergamon Press
NEUROCHEMICAL CHANGFS FOLTAYfING KAINIC ACID LESIONS OF THE NiTCLEUS ACCUNISENS : IMPLICATIONS FOR A GABAERGIC ACCUMBAIr-VEN7RAL ~M~e L PATIiWAY John L. Waddington and A] .an J. Cross Division of Psychiatry, MRC Clinical Research Centre, Harrow, Middlesex HA1 3IIJ, II .K . (Received in final form February 6, 1978) Summary
Unilateral injection of kainic acid into the nucleus accumbens of the rat produced moderate depletions of the GABA synthesising enzyme glutamic acid decarboxylase in the accumbens and ventral tegmental area, but failed to alter these parameters in the striatum or substantia nigra. Similar injections into the striatum produced opposite effects to those seen following accumbens injections . These results are consistent with a GABA-ergic accumbal-ventral tegmental pathway analogous to the well defined striatonigral pathway . However, alternative interpretations, possibly in terms of a non-GABAergic accumbal-ventral tegmental pathway modulating GABA interneurons intrinsic to the tegmentum, must be considered . There is considerable anatomical, electrophysiological and pharmacological evidence for inhibition of activity in nigrostriatal dopamine (DA) neurones mediated by ~( -aminobutyric acid (GABA) neurons ; this inhibitory influence is exerted by both a striatonigral GABA pathway and by GABA neurones intrinsic to the striatum (1-7) . hfhilst there is pharmacological evidence for DA-GA.BA interactions in the so called 'mesolimbic' DA system projecting from the ventral tegmental area (VTA) to the nucleus accumbens (8,9), the mechanism mediating these effects is less well defined, particularly with regard to the existence of a O:ABAergic accumbal-VTA pathway. The finding that electrical stimulation of the accumbens produces an inhibition in the firing rate of VTA neurones which is attenuated by the GABA antagonist bicuculline (10) is consistent with such a postulated GABAergic pathway . Like the substantia nigra (SN) the VTA contains the GABA-synthesising enzyme glutamic acid decarboxylase (GAD) (x,11) . However, transaction of the rat brain between the accumbens and VTA fails to reduce GAD levels in the VTA, whilst they are substantially reduced in the SN (7) . Following intracerebral injections, kainic acid has been shown to be toxic for neuronal cell bodies, but not nerve fibres or terminals (12,13) . Lesions of the striatum with kainic acid reduce GAD levels both in the striatum and SN, due to destruction of GABA neurones originating in the striatum (12,13) . As evidence of the existence of an accumbal-VTA pathway is of some importance, with particular regard to a possible role in schizophrenia (8,14,15), we have made kainic acid lesions of the nucleus accumbens and measured GAD levels in both the accumbens and VTA. Lesion procedure
Materiale and Methods
0300-9653/78-0320-0947$02 .00/0 Copyright p Pergamon Press
1012
Neurochemical Changes, Accumbene Lesion
Vol . 22, No . 11, 1978
Male Sprague-Dawley rate, 150-200g, were anaesthetised with pentobarbital (50mg/kg i .p .) and received unilateral etereotaxic injections of kainic acid into the accumbens, co-ordinates A~ .S ; V-0 .8 ; L 1 .2, or striatum, coordinates Ap .9 ; V0 .4 ; L 2 .6 . (Konig and Klippel) . Kainic acid (Sigma) was dissolved in physiological saline and injected in a dose of 1y.g/0 .5y.L for the accumbens and 2;ag/1uL for the striatum . Injections were made by slow motion infusion at a rate oß 1uL/min . After the te r+~~nA tion of the injection, the injection unit was retained in position for 1 min to allow diffusion of drug . Tissue preparation Rate were decapitated ~ days after lesion, their brains rapidly extracted and frozen . Frozen brains were fixed on a cryostat and from 500-90041 frozen sections of each brain, the following regions were dissected from each side under magnification : accumbens, striatum, substantia nigra and ventral tegmental area . The region dissected ae VTA was that defined by McGeer et al (11) . Tissues were homogenised in 200W1 ice-cold 58 mM potassium phosphate buffer pH 6 .5 . Assay procedure GAD activity was assayed by direct estimation of 14C-GABA produced from 14 C-glutamic acid . To 25W1 of tissue homogenate, 25p1 of reaction mixture containing dithiotheitol (20 n moles), pyridoxal phosphate (20 n moles) and L- II-14C glutamic acid (340 n moles, 0 .025;iCi) was added . After incubation at 37 °C for 1 hour the reaction was stopped by the addition of 500;11 of icecold distilled water . The reaction mixture was then passed over a column of Dowex=1 acetate (100-200 mesh, 3 cma x 0 .5 cms) which retains the labelled substrate and wa~had with 1 ml of distilled water . The filtrate and wash containing the 14C-GABA, were collected in a scintillation vial, to which was added 12 .0 mls of a triton-toluene based scintillant for quantifying by liquid scintillation counting. Mean GAD activity (p moles/ug protein/hr, mean t SD) is the various areas on the control side of the rat brains was : S .N . ; 3e4 t 59 ; accumbens 159 t 56 ; VTA 118 t 26 and striatum 112 t 13 (n = 9 in all cases) . These absolute values were not directly comparable to those of other studies as the substrate concentration ie subsaturating ; however the relative regional distribution of GAD activity was similar to that found in other studies (7,11, 16) . Results kaiaic acid lesions sin GAD levels follow As reported by Schwarz & Coyle 12,13 , kainic acid lesions of the striates significantly reduced GAD levels in both the striatum and SN . We report that such lesions do not significantly alter GAD levels in accumbens or VTA . Kainic acid lesions of the accumbene, however, significantly reduced GAD levels in both the accumbens and VTA without significantly altering those in the striatum or SN (Table 1) . C
Levels of GAD in the regions indicated were compared between lesioned and non-leeioned sides and the significaaces of changes determined by the students ~t~ test (2-tailed) . Analyses of variance revealed significant interactions between site of lesion X site of GAD depletion for both SN v/s VTA (F (1,~) _ 12 .59, pC0 " 01) sad striatum v/e accumbens (F (1,~)=74 .47, p
Pergamon Press
NEUROCHEMICAL CHANGFS FOLTAYfING KAINIC ACID LESIONS OF THE NiTCLEUS ACCUNISENS : IMPLICATIONS FOR A GABAERGIC ACCUMBAIr-VEN7RAL ~M~e L PATIiWAY John L. Waddington and A] .an J. Cross Division of Psychiatry, MRC Clinical Research Centre, Harrow, Middlesex HA1 3IIJ, II .K . (Received in final form February 6, 1978) Summary
Unilateral injection of kainic acid into the nucleus accumbens of the rat produced moderate depletions of the GABA synthesising enzyme glutamic acid decarboxylase in the accumbens and ventral tegmental area, but failed to alter these parameters in the striatum or substantia nigra. Similar injections into the striatum produced opposite effects to those seen following accumbens injections . These results are consistent with a GABA-ergic accumbal-ventral tegmental pathway analogous to the well defined striatonigral pathway . However, alternative interpretations, possibly in terms of a non-GABAergic accumbal-ventral tegmental pathway modulating GABA interneurons intrinsic to the tegmentum, must be considered . There is considerable anatomical, electrophysiological and pharmacological evidence for inhibition of activity in nigrostriatal dopamine (DA) neurones mediated by ~( -aminobutyric acid (GABA) neurons ; this inhibitory influence is exerted by both a striatonigral GABA pathway and by GABA neurones intrinsic to the striatum (1-7) . hfhilst there is pharmacological evidence for DA-GA.BA interactions in the so called 'mesolimbic' DA system projecting from the ventral tegmental area (VTA) to the nucleus accumbens (8,9), the mechanism mediating these effects is less well defined, particularly with regard to the existence of a O:ABAergic accumbal-VTA pathway. The finding that electrical stimulation of the accumbens produces an inhibition in the firing rate of VTA neurones which is attenuated by the GABA antagonist bicuculline (10) is consistent with such a postulated GABAergic pathway . Like the substantia nigra (SN) the VTA contains the GABA-synthesising enzyme glutamic acid decarboxylase (GAD) (x,11) . However, transaction of the rat brain between the accumbens and VTA fails to reduce GAD levels in the VTA, whilst they are substantially reduced in the SN (7) . Following intracerebral injections, kainic acid has been shown to be toxic for neuronal cell bodies, but not nerve fibres or terminals (12,13) . Lesions of the striatum with kainic acid reduce GAD levels both in the striatum and SN, due to destruction of GABA neurones originating in the striatum (12,13) . As evidence of the existence of an accumbal-VTA pathway is of some importance, with particular regard to a possible role in schizophrenia (8,14,15), we have made kainic acid lesions of the nucleus accumbens and measured GAD levels in both the accumbens and VTA. Lesion procedure
Materiale and Methods
0300-9653/78-0320-0947$02 .00/0 Copyright p Pergamon Press
1012
Neurochemical Changes, Accumbene Lesion
Vol . 22, No . 11, 1978
Male Sprague-Dawley rate, 150-200g, were anaesthetised with pentobarbital (50mg/kg i .p .) and received unilateral etereotaxic injections of kainic acid into the accumbens, co-ordinates A~ .S ; V-0 .8 ; L 1 .2, or striatum, coordinates Ap .9 ; V0 .4 ; L 2 .6 . (Konig and Klippel) . Kainic acid (Sigma) was dissolved in physiological saline and injected in a dose of 1y.g/0 .5y.L for the accumbens and 2;ag/1uL for the striatum . Injections were made by slow motion infusion at a rate oß 1uL/min . After the te r+~~nA tion of the injection, the injection unit was retained in position for 1 min to allow diffusion of drug . Tissue preparation Rate were decapitated ~ days after lesion, their brains rapidly extracted and frozen . Frozen brains were fixed on a cryostat and from 500-90041 frozen sections of each brain, the following regions were dissected from each side under magnification : accumbens, striatum, substantia nigra and ventral tegmental area . The region dissected ae VTA was that defined by McGeer et al (11) . Tissues were homogenised in 200W1 ice-cold 58 mM potassium phosphate buffer pH 6 .5 . Assay procedure GAD activity was assayed by direct estimation of 14C-GABA produced from 14 C-glutamic acid . To 25W1 of tissue homogenate, 25p1 of reaction mixture containing dithiotheitol (20 n moles), pyridoxal phosphate (20 n moles) and L- II-14C glutamic acid (340 n moles, 0 .025;iCi) was added . After incubation at 37 °C for 1 hour the reaction was stopped by the addition of 500;11 of icecold distilled water . The reaction mixture was then passed over a column of Dowex=1 acetate (100-200 mesh, 3 cma x 0 .5 cms) which retains the labelled substrate and wa~had with 1 ml of distilled water . The filtrate and wash containing the 14C-GABA, were collected in a scintillation vial, to which was added 12 .0 mls of a triton-toluene based scintillant for quantifying by liquid scintillation counting. Mean GAD activity (p moles/ug protein/hr, mean t SD) is the various areas on the control side of the rat brains was : S .N . ; 3e4 t 59 ; accumbens 159 t 56 ; VTA 118 t 26 and striatum 112 t 13 (n = 9 in all cases) . These absolute values were not directly comparable to those of other studies as the substrate concentration ie subsaturating ; however the relative regional distribution of GAD activity was similar to that found in other studies (7,11, 16) . Results kaiaic acid lesions sin GAD levels follow As reported by Schwarz & Coyle 12,13 , kainic acid lesions of the striates significantly reduced GAD levels in both the striatum and SN . We report that such lesions do not significantly alter GAD levels in accumbens or VTA . Kainic acid lesions of the accumbene, however, significantly reduced GAD levels in both the accumbens and VTA without significantly altering those in the striatum or SN (Table 1) . C
Levels of GAD in the regions indicated were compared between lesioned and non-leeioned sides and the significaaces of changes determined by the students ~t~ test (2-tailed) . Analyses of variance revealed significant interactions between site of lesion X site of GAD depletion for both SN v/s VTA (F (1,~) _ 12 .59, pC0 " 01) sad striatum v/e accumbens (F (1,~)=74 .47, p
