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Excitant amino acid projections from rat amygdala and thalamus to nucleus accumbens
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Brain Research Bulk-tin, Vol. 20, pp. 467-471. 0 Pergamon Press plc, 1988.Printed in the U.S.A.
Excitant Amino Acid Projections From Rat Amygdala and Thalamus to Nucfeus Accumb~ns TIMOTHY
G. ROBINSON
AND PHILIP
M. BEART’
University of ~~~~~ar~e, Clilinical P~ar~a~~~agy and T~erapeati~s Unit Austin and repatriation Haspita~s, Heidelberg, 3084, Aastraiia Received
19 June
1987
‘I’. G. AND P. M. BEART. Excifanf amino acid projections from rut amygdala and thaiamus to nucleus affkity uptake of D-[3H]aspartate, [3H]choline and RES BULL 20(4) 467-471, 1988.-High [3H]GABA was examined in synaptosom~-confining preparations of rat nucleus accumbens septi 7 to 10 days after unilateral or bilateral N-methyl-D-aspartate lesions confined to the parataenial nucleus of the thalamus or the basolateral nucleus of the amygdala. Uptake of both D-[3H]aspartate and [3H]choline was significantly reduced (11% and 14%less than ROBINSON,
~~~~rn~e~~. BRAIN
control, respectively) by unilateral lesion of the thalamus, whereas [3H]GABA uptake was unaffected. Bilateral thalamic lesions significantly reduced D+H]aspartate uptake (11% less than control) into homogenates of the nucleus accumbens, whilst [*H]GABA uptake was unaltered. D-r3H]asparatate uptake was significantly reduced (26% less than control) following unilateral lesion of the amygdala, whereas both [$H]GABA and [3H]choline uptake were unaffected. Bilateral amygdaloid lesions si~i~cantly increased D-~3H]aspa~ate uptake (39% greater than control), whilst uptake of fW]GABA was not affected. The results implicate glutamate and/or aspartate as putative neurotransmitters in afferent projections from the basolateral amygdala and the parataenial thalamus to the nucleus accumbens. Thalamic afferents to the nucleus accumbens may also utilize acetylcholine as their transmitter. Nucleus accumbens Basolateral amygdala Acetylcholine Excitotoxin
Parataenial thalamus
Glutamate transmitter
terminals) in synaptosome-enriched preparations of the NAS, after the placement of discrete excitotoxic lesions in these nuclei. Male Sprague-Dawley rats (260-320 g) were employed. Animals were anaethetised with a mixture (2 n&kg) of amob~bit~ (66 mglkg; Lilly, Indianapolis, IN) and methohexitone (33 mg/kg; Lilly, Indianapolis, IN). The excitotoxin, N-methyl-D-aspartate (NMDA; 40 nmoles in artificial CSF, pH 7.4; Sigma, St. Louis, MO), was microinfused (200 nl over 6 min, then left in situ a further 2 min) into the BL or the FT to produce bilateral or unilateral lesions [3, 4, 261. Infusions were made through a 230 pm (o.d.) stainless steel needle at the following stereotaxic coordinates: BL at AP= -0.33, L= 50.46, DV=-0.84 cm and FYIat AP=-0.25, L= 50.07, DV= -0.52 cm (flat skull position, reference from bregma) [21]. Sham animals or the contralateral control hemisphere received at the abovementioned coordinates, an equal volume of either ar-methyl-DL-aspartate (40 nmoles; a non-excitant dicarboxylic amino acid without neurotoxic properties, Sigma, St. Louis, MO) [26], vehicle or were untreated. Animals were sacrificed 7-10 days after surgery, and their brains were rapidly removed into ice. This time point was chosen on the basis of previous studies from our laboratory where lesion-induced decreases in the synaptosomal uptake of D-r3H]asp were found for a number of excitatory
WITH the aid of retrograde and anterograde tracing techniques, the nucleus accumbens septi (NAS) has been shown to receive projections from the parataenial nucleus of the thalamus (PT) [ 11,13,19] and from the basolateral nucleus of the amygdala (BL) [IO, 14, 19, 221. Indeed, the functional inte~eiationship between the NAS and the amygdaloid complex has attracted considerable attention and has been described as a neurological interface between emotion and action [8,16]. There is, however, a paucity of literature relating to afferent pathways to the NAS, especially describing the type of neurotransmitter involved. Recent retrograde-transpo~ autoradio~phic studies in our laboratory reveal that L-glutamate and/or L-aspartate (GWAsp) is likely to be the transmitter of several accumbal afferent pathways [2,5]. In particular, we found that the retrograde transport of D++H]aspartate (D-[3H]asp), which is a transmitter-specific technique for localizing Glu/Asputilizing somata [27], discretely labels cells bodies in the amygdala (especially BL) and in midline thalamic nuclei (particularly PT) after its intra-accumbal microinjection suggesting that both pathways may utilize Glu/Asp as their transmitter [2,51. The aim of the present study was to confern the possible involvement of Glu/Asp in the pathways from the BL and the PT to the NAS by examining the high affinity uptake of D-i3Hlasp (a marker for Glu/Asp nerve ‘Requests for reprints should be addressed to Philip M. Beart,
467
46X
ROBINSON PT LESION UNIL.
BL
BILAT.
UNIL. l."_
AND BEAR-1
LESION
BILAT * *
FIG. 1. (A) Effects of unilateral and bilateral N-methyl-D-aspartate lesions of the PT on the high affinity uptake of Dt3H]aspartate, [:‘H]GABA and [“Hlchotine by synaptosome-containing preparations of the NAS. Values are expressed as the percentage change relative to control. Each value is the mean (?S.E.M.) of independent determinations (6-14 animals) performed in quadruplicate. *p
amino acid systems [3,4]. The NAS was dissected out as previously described on a chilled dissecting plate 131. For unilaterally lesioned rats, the two nuclei accumbens were processed separately and data from the lesioned NAS was always compared to the contralateral side of the same animal (paired t-test). In bilaterally lesioned rats, data was from experiments with pooled accumbens relative to separate control animals (independent r-test). The analysis of the high affinity uptake of D-[2,3-3H]asp (21 Ci/mmol; final concentration 10 PM D-asp), i3H]GABA (78 Ciimmol; final concentration 10 FM GABA) and ~H]choline (80 Cilmmol; final concentration 4 FM choline) was as described in detail elsewhere [3,4]. The uptake of [SH]GABA and [3H]choline served as controls to establish the transmitter-selective nature of lesion-induced changes. protein was estimated by the method of Lowry 1151. The remainder of the brain was frozen in dry ice cooled isopentane for subsequent histological examinatjon. Coronal sections of rat brain (40 pm) were stained with cresyl violet and counterstained with 1~x01 fast blue to reveal the extent of the lesions. All radioisotopes were from Amersham International, UK. Unilateral lesion of the PT area produced small, but consistently observed reductions in the high affinity uptake into homogenates of the NAS of both D-fH]asp and PHIcholine when compared to control values @=2.59, p
nates of the NAS, hence these data were pooled as appropriate for subsequent analyses. Figure 2 presents typical lesions of the PT in a group of 7 animats. When examined by light microscopy the lesion was predominantly localized to midline thalamic nuclei. In a few cases, additional damage was seen in the centrolateral thalamic nucleus. Less frequently, the dorsal aspect of the ventrolateral thalamic nuclei (LV) was partially lesioned. The anterior aspects of the paraventricular and intermediodorsal nuclei were largely spared. In some PT lesions (n==3), minor additional damage was revealed in the hippocampus (dentate gyrus and CA4). In these cases, the neurochemic~ data were not included. More laterally located thalamic nuclei and the habenula were never lesioned. The high affinity uptake of D-13H]asp into synaptosomalcontaining preparations of the NAS from animals with a unilateral lesion of the BL area was significantly reduced by 26% of control values (t =2.75, pCO.05, n= 15, paired r-test; Fig. IB). [3H]GABA and [3H]choline uptake were unaffected when compared to control values (Fig. lB, n=21 and n=6 respectively). Bilateral lesions of BL were performed to further establish the involvement of excitatory amino acids and the high affinity uptake of D-[Q]asp was increased by 39% (t=4.14, p
469
AMINO ACID PROJECTIONS TO THE NUCLEUS ACCUMBENS
bregma
FIG. 2. Serial coronal sections of the thalamic area (redrawn from [21]) displaying the extent of N-methyl-D-aspartate lesions in 7 animals used for neurochemistry, as revealed in cresyl-violet stained sections. Area shown by lightest stippling indicates complete necrosis in at least one rat; medium shaded area, at least 2 rats; black area, 7 rats. Abbreviations: AV, anteroventral thalamic nucleus; Cx, frontoparietal cortex motor area; G, gelatinosus nucleus thalamus; Hb, lateral habenular nucleus; LD, laterodorsal thalamic nucleus; LP, lateral posterior thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedia1 thalamic nucteus.
-4.3
1
-3.8
1
-3.3
1
-2.8
-2.3
1 bregma
FIG. 3. Serial coronal sections of the ~ygd~oid area (redrawn from [211)~spla~~ the extent of N-methyl-ample lesions in 8 animals used for neurochemistry, as revealed in cresyl-violet stained sections. Outer boundaries indicate complete necrosis in at least one rat; lightest stippling, at least 2 rats; medium shaded area, 4 rats; black area, 7 rats. Abbreviations: BLV, basolateral amygdaloid area ventral; BM, basomedial amygdaloid nucleus; CA3, field CA3 of Ammon’s horn; Ce, central amygdaloid nucleus; CP, caudate-putamen; La, lateral amygdaloid nucleus; Me, medial amygdaloid nucleus; PO, primary olfactory cortex.
470
the PT was supported by these results. GluiAsp, rather than ACh or GABA. appear to be a transmitter in the projection from the BL to the NAS. Apart from our own retrograde transport studies with D-[:‘H]asp [2,5], excitatory amino acids have not previously been implicated as the neurotransmitters of this pathway. The afferent projection from the BL to the NAS has previousIy been examined by various methods including behavioural assessment, electrophysiology, HRP studies and autoradiographic tracing procedures [8, 10, 14, 16, 19. 227. The increased uptake of D-[:‘H]asp after bilateral lesions was unexpected and might be attributable to synaptic and/or glial adaptations which follow degenerative changes. Hy~rtrophic reaction ofglia [ 17,23,2S] or plastic activity [l&28] of amygdaloid neurones (perhaps attributable to inter- or intra-amygdafoid connections [6]) might cause such an elevation of D-[Y-I/asp uptake. These phenomena have not been studied in detail, particularly as they apply to the amygdala or the NAS. However. these data when considered along with the studies employing unilateral lesions still implicate a role for GluiAsp in the projection from the BL to the NAS. Additionally, these findings are consistent with our neurochemical mapping data obtained after the intra-accumbal m~croinject~on of D-[:‘H Jasp where we observed retrogradely labelled ceil bodies in the amygdala, particularly in the BL 12.51. Our results also indicate that both GluiAsp and ACh may be the transmitters of the projection from the FT IO the NAS. Projections between the NAS and the FT have been delineated as a result of neuroanatomical tracing studies [ 1I, 13, 191. However, clear definition of the neurotransmitter(s) involved had to await our own discovery of topographic perikaryal labelling in midline thalamus by retrogradely transported D-[:‘H]asp [2,5]. Although the lesion studies of Fonnum and coileagues [9,29] do not supporr a role for
I. Barrington-Ward, S. J., J. C. Kilpatrick, 0. T. Phillipson and C. J. Pycock. Evidence that thalamic efferent neurones are noncholinergic: a study in the rat with special reference to the thalamostriatal pathway. Brain Rcs 299: 145-151, 1984. 2. Beart, P. M., R. J. Summers and M. J. Christie. Mapping of excitatory amino acid projections to central gray and nucleus accumbens by D[$H]aspartate retrograde transport. In: E.vc&f(>rvAmino Acid Tr~nsffzissi~}t~. edited bv T. P. Hicks. D. Lodge a&l H. McLennan. New York: A. R. I& 1986, pp. 361-364. 3. Christie, M. J., L. B. James and P. M. Beart. An excitant amino acid projection from the medial prefrontal cortex to the anterior nucleus accumbens in the rat. J Nemwhem 45: 477-482, 1985. 4. Christie, M. J., L. B. James and P. M. Beart. An excitatory amino acid projection from rat prefrontal cortex to ~riaqueductal gray. Bruit? Rc,s Bull 16: 127-129, 1986. 5. Christie, M. J., R. J. Summers, J. A. Stephenson, C. J. Cook and P. M. Beart. Excitatory amino acid projections to the nucleus accumbens septi in the rat: a retrograde transport study utilizing D[3H]aspartate and [3H]GABA. NPuro,sc,ic~,lc,a 22: 425-439. 1987. 6. de Olomos, J., G. F. Alheid and C. A. Beltramino. Amygdala. In: T/w Rut Nervous S>~stt~rn,edited by G. Paxinos. New York: Academic Press, 1985, pp. 223-334. and some projections of 7. Fibiger, H. C. The organization cholinergic neurons of the mammalian forebrain. Bruin RccsRev 4: 327-388, 1982. 8. Fonberg, E. The role of the amygdaloid nucleus in animaf behaviour. Prog Brain Res 22: 273-281, 1968,
ROBINSON
AND Hf~,4K’I‘
Glu/Asp in the thalamoaccumbai pathway, their thalamtc lesions wcrc somewhat more caudo~ter~~l than ours. and therefore would not have lesioned the PT. Our retrograde tracing studies with D-[XHlasp [2.5] support the present biochemical observations of reduced D-[%l]asp uptake in NAS after lesions in the PT area. Retrograde transport of D-[“HIasp results in topographic labelling of many midline thalmic nuclei [2,5] and the modest reduction t 11%) in the high affinity uptake of D-[%I]asp may be attributable to the sparing of more ventrally located nuclei (e.g.. central medial. reuniens and rhomboid), which also project to the NAS. While there has been some controversy as to whether ACh is a transmitter of thalamic efferents [I, 7, 12. 10, 241, consistent with other recent findings [20]. our data support :I role for this neurotransmitter. This study, employing kainate lesions of thalamostriatal neurones. has provided evidence for both GluiAsp and ACh as transmitters in the thalamostriatal pathway 1201. To summarize, our results employing excitotoxic lesions in conjunction with neurochemistry support recent observations from this laboratory of intense retrograde labelling of the PT and the BL after the intra-accumbal microinjection of I)-[“H]asp [2,S]. Excitatory amino acids seem to be the transmitter of several pathways convergent upon the NAS. including those from the medial prefrontal cortex. the BL and the PT 12. 3, 5.9, 291. The ro1ef.s) of GIuiAsp afferents in various behaviours mediated by the NAS wilt be of appreciable interest. .AC’KNOWJ_EDGEMENT
This work was supported by a Programme Grant from the National Health and Medical Research Council of Australia of which P.M.B. is a Principal Research Fellow.
9. Fonnum. F. and I. Walaas. Localization of neurotransmitters in nucleus accumbens. In: 7%(, N~i~~(~bj[~~~~~~ c?f’the NI~&M ACct~mbots, edited by R. B. Chronister and J. F. De France. Brunswick, ME: Haer Institute, 1981, pp. 259-315. 10. Kelley, A. E., V. B. Domesick and J. H. Nauta. The amygdalostriatal projection in the rat-an anatomical study by anterograde and retrograde tracing methods. N[‘nro,vc,ic,,lc.~, 7: 615-630, 1982. Il. Kelley, A. E. and L. Stinus. The distribution of the projection from the parataenial nucleus of the thalamus to the nucleus accumbens in the rat: an autoradiographic study. .c;X,,Brcrin Rtps 54: 499-512, 1984. 12. Kilpatrick, I. C. and 0. T. Philipson. On the transmitter chemistry of thalamos~iatal fibres. Newosci Lctt 67: 97-99, 1986. 13. Krettek, J. E. and J. L. Price. Projections from the amygdaloid complex to the cerebral cortex and the thalamus in the rat and cat. J Camp Neurd 172: 687-722, 1977. 14. Krettek, J. E. and J. L. Price. Amygdaioid projections to subcortical structures within the basal forebrain and the brainstem in the rat and cat. ./ Conrp Ncuvol 178: 225-254, 1978. IS. Lowry. 0. L., N. J. Rosenbrough, A. L. Farr and R. J. Randall. Protein measurement with the Folin phenol reagent. J Bird C’hem 193: 265-275, 1951. 16. Mogenson, G. J., D. L. Jones and C. Y. Yim. From motivation to action: functional interface between the limbic system and the motor system. Prog Nertrohid 14: 69-97, 1980. 17. Molin, S.-O., B. Nystrom, K. Haglid and A. Hamberger, Role of “glial plasticity” in amino acid content and metabolism of the deagerented dentate gyrus. .I Nerrrosci Res f 1: 1-l t, 1984.
AMINO ACID PROJECTIONS
TO THE NUCLEUS
ACCUMBENS
18. Nadler, J. V., C. W. Cotman and G. S. Lynch. Biochemical plasticity of short intemeurons: increased glutamate decarboxylase activity in the denervated area of rat dentate gyrus following entorhinal lesions. Exp Neural 45: 403-413, 1974. 19. Newman, R. and S. S. Winans. An experimental study of the ventral striatum of the golden hamster. 1. Neuronat connections of the nucleus accumbens. J Camp Ne~rof 191: 167-192,198O. 20. Nieoullon, A., E. Scarfone, L. Kerkerian, M. Errami and N. Dusticier. Changes in choline acetyltransferase, glutamic acid decarboxylase, high-affinity glutamate uptake and dopaminergic activity induced by kainic acid lesion of the thalamostriatal neurons. Neurosci Lett 58: 299-304, 1985. 21. Paxinos, G. and C. Watson. The Rat Brain in Stereotaxit Co~~r~fnat~s. Sydney: Academic Press, 1982. 22. Phillipson, 0. T. and A. C. Griffiths. The topographic order of inputs to nucleus accumbens in the rat. Neuroscience 16: 275296, 1985. 23. Rose, G., G. Lynch and C. W. Cotman. Hypertrophy and redistribution of astrocytes in the deafferented dentate gyrus. Brain Res Bull 1: 87-92, 1976.
24. Saelens, J. K., S, Edwards-Neale
25.
26.
27.
28.
29.
471
and J. P. Simke. Further evidence for choline&c thalamo-striatal neurons. J Neuruchem 32: 1093-1094, 1979. Sandberg, M., H. K. Ward and H. F. Bradford. Effect of cortico-striate pathway lesion on the activities of enzymes involved in synthesis and metabolism of amino acid transmitters in the striatum. .I Neuro~hem 44: 42-47, 1985. Schwarz, R., D. Scholz and J. T. Coyle. Structure-activity relations for the neurotoxicity of kainic acid derivatives and glutamate analogues. Neuropharmacology 17: 141-151, 1978. Streit, P. Selective retrograde labelling indicating the transmitter of neuronal pathways. J Comp Neural 191: 429-463, 1980. Vincent, S. R., J. I. Nagy and H. C. Fibiger. Increased striatal glutamate decarboxylase after lesions of the nigrost~at~ pathway. Brain Res 143: 168-173, 1978. Walaas, I. and F. Fonnum. The effects of surgicaf and chemical lesions on neurotransmitter candidates in the nucleus accumbens of the rat. Neuroscience 4: 209-216, 1979.
Brain Research Bulk-tin, Vol. 20, pp. 467-471. 0 Pergamon Press plc, 1988.Printed in the U.S.A.
Excitant Amino Acid Projections From Rat Amygdala and Thalamus to Nucfeus Accumb~ns TIMOTHY
G. ROBINSON
AND PHILIP
M. BEART’
University of ~~~~~ar~e, Clilinical P~ar~a~~~agy and T~erapeati~s Unit Austin and repatriation Haspita~s, Heidelberg, 3084, Aastraiia Received
19 June
1987
‘I’. G. AND P. M. BEART. Excifanf amino acid projections from rut amygdala and thaiamus to nucleus affkity uptake of D-[3H]aspartate, [3H]choline and RES BULL 20(4) 467-471, 1988.-High [3H]GABA was examined in synaptosom~-confining preparations of rat nucleus accumbens septi 7 to 10 days after unilateral or bilateral N-methyl-D-aspartate lesions confined to the parataenial nucleus of the thalamus or the basolateral nucleus of the amygdala. Uptake of both D-[3H]aspartate and [3H]choline was significantly reduced (11% and 14%less than ROBINSON,
~~~~rn~e~~. BRAIN
control, respectively) by unilateral lesion of the thalamus, whereas [3H]GABA uptake was unaffected. Bilateral thalamic lesions significantly reduced D+H]aspartate uptake (11% less than control) into homogenates of the nucleus accumbens, whilst [*H]GABA uptake was unaltered. D-r3H]asparatate uptake was significantly reduced (26% less than control) following unilateral lesion of the amygdala, whereas both [$H]GABA and [3H]choline uptake were unaffected. Bilateral amygdaloid lesions si~i~cantly increased D-~3H]aspa~ate uptake (39% greater than control), whilst uptake of fW]GABA was not affected. The results implicate glutamate and/or aspartate as putative neurotransmitters in afferent projections from the basolateral amygdala and the parataenial thalamus to the nucleus accumbens. Thalamic afferents to the nucleus accumbens may also utilize acetylcholine as their transmitter. Nucleus accumbens Basolateral amygdala Acetylcholine Excitotoxin
Parataenial thalamus
Glutamate transmitter
terminals) in synaptosome-enriched preparations of the NAS, after the placement of discrete excitotoxic lesions in these nuclei. Male Sprague-Dawley rats (260-320 g) were employed. Animals were anaethetised with a mixture (2 n&kg) of amob~bit~ (66 mglkg; Lilly, Indianapolis, IN) and methohexitone (33 mg/kg; Lilly, Indianapolis, IN). The excitotoxin, N-methyl-D-aspartate (NMDA; 40 nmoles in artificial CSF, pH 7.4; Sigma, St. Louis, MO), was microinfused (200 nl over 6 min, then left in situ a further 2 min) into the BL or the FT to produce bilateral or unilateral lesions [3, 4, 261. Infusions were made through a 230 pm (o.d.) stainless steel needle at the following stereotaxic coordinates: BL at AP= -0.33, L= 50.46, DV=-0.84 cm and FYIat AP=-0.25, L= 50.07, DV= -0.52 cm (flat skull position, reference from bregma) [21]. Sham animals or the contralateral control hemisphere received at the abovementioned coordinates, an equal volume of either ar-methyl-DL-aspartate (40 nmoles; a non-excitant dicarboxylic amino acid without neurotoxic properties, Sigma, St. Louis, MO) [26], vehicle or were untreated. Animals were sacrificed 7-10 days after surgery, and their brains were rapidly removed into ice. This time point was chosen on the basis of previous studies from our laboratory where lesion-induced decreases in the synaptosomal uptake of D-r3H]asp were found for a number of excitatory
WITH the aid of retrograde and anterograde tracing techniques, the nucleus accumbens septi (NAS) has been shown to receive projections from the parataenial nucleus of the thalamus (PT) [ 11,13,19] and from the basolateral nucleus of the amygdala (BL) [IO, 14, 19, 221. Indeed, the functional inte~eiationship between the NAS and the amygdaloid complex has attracted considerable attention and has been described as a neurological interface between emotion and action [8,16]. There is, however, a paucity of literature relating to afferent pathways to the NAS, especially describing the type of neurotransmitter involved. Recent retrograde-transpo~ autoradio~phic studies in our laboratory reveal that L-glutamate and/or L-aspartate (GWAsp) is likely to be the transmitter of several accumbal afferent pathways [2,5]. In particular, we found that the retrograde transport of D++H]aspartate (D-[3H]asp), which is a transmitter-specific technique for localizing Glu/Asputilizing somata [27], discretely labels cells bodies in the amygdala (especially BL) and in midline thalamic nuclei (particularly PT) after its intra-accumbal microinjection suggesting that both pathways may utilize Glu/Asp as their transmitter [2,51. The aim of the present study was to confern the possible involvement of Glu/Asp in the pathways from the BL and the PT to the NAS by examining the high affinity uptake of D-i3Hlasp (a marker for Glu/Asp nerve ‘Requests for reprints should be addressed to Philip M. Beart,
467
46X
ROBINSON PT LESION UNIL.
BL
BILAT.
UNIL. l."_
AND BEAR-1
LESION
BILAT * *
FIG. 1. (A) Effects of unilateral and bilateral N-methyl-D-aspartate lesions of the PT on the high affinity uptake of Dt3H]aspartate, [:‘H]GABA and [“Hlchotine by synaptosome-containing preparations of the NAS. Values are expressed as the percentage change relative to control. Each value is the mean (?S.E.M.) of independent determinations (6-14 animals) performed in quadruplicate. *p
amino acid systems [3,4]. The NAS was dissected out as previously described on a chilled dissecting plate 131. For unilaterally lesioned rats, the two nuclei accumbens were processed separately and data from the lesioned NAS was always compared to the contralateral side of the same animal (paired t-test). In bilaterally lesioned rats, data was from experiments with pooled accumbens relative to separate control animals (independent r-test). The analysis of the high affinity uptake of D-[2,3-3H]asp (21 Ci/mmol; final concentration 10 PM D-asp), i3H]GABA (78 Ciimmol; final concentration 10 FM GABA) and ~H]choline (80 Cilmmol; final concentration 4 FM choline) was as described in detail elsewhere [3,4]. The uptake of [SH]GABA and [3H]choline served as controls to establish the transmitter-selective nature of lesion-induced changes. protein was estimated by the method of Lowry 1151. The remainder of the brain was frozen in dry ice cooled isopentane for subsequent histological examinatjon. Coronal sections of rat brain (40 pm) were stained with cresyl violet and counterstained with 1~x01 fast blue to reveal the extent of the lesions. All radioisotopes were from Amersham International, UK. Unilateral lesion of the PT area produced small, but consistently observed reductions in the high affinity uptake into homogenates of the NAS of both D-fH]asp and PHIcholine when compared to control values @=2.59, p
nates of the NAS, hence these data were pooled as appropriate for subsequent analyses. Figure 2 presents typical lesions of the PT in a group of 7 animats. When examined by light microscopy the lesion was predominantly localized to midline thalamic nuclei. In a few cases, additional damage was seen in the centrolateral thalamic nucleus. Less frequently, the dorsal aspect of the ventrolateral thalamic nuclei (LV) was partially lesioned. The anterior aspects of the paraventricular and intermediodorsal nuclei were largely spared. In some PT lesions (n==3), minor additional damage was revealed in the hippocampus (dentate gyrus and CA4). In these cases, the neurochemic~ data were not included. More laterally located thalamic nuclei and the habenula were never lesioned. The high affinity uptake of D-13H]asp into synaptosomalcontaining preparations of the NAS from animals with a unilateral lesion of the BL area was significantly reduced by 26% of control values (t =2.75, pCO.05, n= 15, paired r-test; Fig. IB). [3H]GABA and [3H]choline uptake were unaffected when compared to control values (Fig. lB, n=21 and n=6 respectively). Bilateral lesions of BL were performed to further establish the involvement of excitatory amino acids and the high affinity uptake of D-[Q]asp was increased by 39% (t=4.14, p
469
AMINO ACID PROJECTIONS TO THE NUCLEUS ACCUMBENS
bregma
FIG. 2. Serial coronal sections of the thalamic area (redrawn from [21]) displaying the extent of N-methyl-D-aspartate lesions in 7 animals used for neurochemistry, as revealed in cresyl-violet stained sections. Area shown by lightest stippling indicates complete necrosis in at least one rat; medium shaded area, at least 2 rats; black area, 7 rats. Abbreviations: AV, anteroventral thalamic nucleus; Cx, frontoparietal cortex motor area; G, gelatinosus nucleus thalamus; Hb, lateral habenular nucleus; LD, laterodorsal thalamic nucleus; LP, lateral posterior thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedia1 thalamic nucteus.
-4.3
1
-3.8
1
-3.3
1
-2.8
-2.3
1 bregma
FIG. 3. Serial coronal sections of the ~ygd~oid area (redrawn from [211)~spla~~ the extent of N-methyl-ample lesions in 8 animals used for neurochemistry, as revealed in cresyl-violet stained sections. Outer boundaries indicate complete necrosis in at least one rat; lightest stippling, at least 2 rats; medium shaded area, 4 rats; black area, 7 rats. Abbreviations: BLV, basolateral amygdaloid area ventral; BM, basomedial amygdaloid nucleus; CA3, field CA3 of Ammon’s horn; Ce, central amygdaloid nucleus; CP, caudate-putamen; La, lateral amygdaloid nucleus; Me, medial amygdaloid nucleus; PO, primary olfactory cortex.
470
the PT was supported by these results. GluiAsp, rather than ACh or GABA. appear to be a transmitter in the projection from the BL to the NAS. Apart from our own retrograde transport studies with D-[:‘H]asp [2,5], excitatory amino acids have not previously been implicated as the neurotransmitters of this pathway. The afferent projection from the BL to the NAS has previousIy been examined by various methods including behavioural assessment, electrophysiology, HRP studies and autoradiographic tracing procedures [8, 10, 14, 16, 19. 227. The increased uptake of D-[:‘H]asp after bilateral lesions was unexpected and might be attributable to synaptic and/or glial adaptations which follow degenerative changes. Hy~rtrophic reaction ofglia [ 17,23,2S] or plastic activity [l&28] of amygdaloid neurones (perhaps attributable to inter- or intra-amygdafoid connections [6]) might cause such an elevation of D-[Y-I/asp uptake. These phenomena have not been studied in detail, particularly as they apply to the amygdala or the NAS. However. these data when considered along with the studies employing unilateral lesions still implicate a role for GluiAsp in the projection from the BL to the NAS. Additionally, these findings are consistent with our neurochemical mapping data obtained after the intra-accumbal m~croinject~on of D-[:‘H Jasp where we observed retrogradely labelled ceil bodies in the amygdala, particularly in the BL 12.51. Our results also indicate that both GluiAsp and ACh may be the transmitters of the projection from the FT IO the NAS. Projections between the NAS and the FT have been delineated as a result of neuroanatomical tracing studies [ 1I, 13, 191. However, clear definition of the neurotransmitter(s) involved had to await our own discovery of topographic perikaryal labelling in midline thalamus by retrogradely transported D-[:‘H]asp [2,5]. Although the lesion studies of Fonnum and coileagues [9,29] do not supporr a role for
I. Barrington-Ward, S. J., J. C. Kilpatrick, 0. T. Phillipson and C. J. Pycock. Evidence that thalamic efferent neurones are noncholinergic: a study in the rat with special reference to the thalamostriatal pathway. Brain Rcs 299: 145-151, 1984. 2. Beart, P. M., R. J. Summers and M. J. Christie. Mapping of excitatory amino acid projections to central gray and nucleus accumbens by D[$H]aspartate retrograde transport. In: E.vc&f(>rvAmino Acid Tr~nsffzissi~}t~. edited bv T. P. Hicks. D. Lodge a&l H. McLennan. New York: A. R. I& 1986, pp. 361-364. 3. Christie, M. J., L. B. James and P. M. Beart. An excitant amino acid projection from the medial prefrontal cortex to the anterior nucleus accumbens in the rat. J Nemwhem 45: 477-482, 1985. 4. Christie, M. J., L. B. James and P. M. Beart. An excitatory amino acid projection from rat prefrontal cortex to ~riaqueductal gray. Bruit? Rc,s Bull 16: 127-129, 1986. 5. Christie, M. J., R. J. Summers, J. A. Stephenson, C. J. Cook and P. M. Beart. Excitatory amino acid projections to the nucleus accumbens septi in the rat: a retrograde transport study utilizing D[3H]aspartate and [3H]GABA. NPuro,sc,ic~,lc,a 22: 425-439. 1987. 6. de Olomos, J., G. F. Alheid and C. A. Beltramino. Amygdala. In: T/w Rut Nervous S>~stt~rn,edited by G. Paxinos. New York: Academic Press, 1985, pp. 223-334. and some projections of 7. Fibiger, H. C. The organization cholinergic neurons of the mammalian forebrain. Bruin RccsRev 4: 327-388, 1982. 8. Fonberg, E. The role of the amygdaloid nucleus in animaf behaviour. Prog Brain Res 22: 273-281, 1968,
ROBINSON
AND Hf~,4K’I‘
Glu/Asp in the thalamoaccumbai pathway, their thalamtc lesions wcrc somewhat more caudo~ter~~l than ours. and therefore would not have lesioned the PT. Our retrograde tracing studies with D-[XHlasp [2.5] support the present biochemical observations of reduced D-[%l]asp uptake in NAS after lesions in the PT area. Retrograde transport of D-[“HIasp results in topographic labelling of many midline thalmic nuclei [2,5] and the modest reduction t 11%) in the high affinity uptake of D-[%I]asp may be attributable to the sparing of more ventrally located nuclei (e.g.. central medial. reuniens and rhomboid), which also project to the NAS. While there has been some controversy as to whether ACh is a transmitter of thalamic efferents [I, 7, 12. 10, 241, consistent with other recent findings [20]. our data support :I role for this neurotransmitter. This study, employing kainate lesions of thalamostriatal neurones. has provided evidence for both GluiAsp and ACh as transmitters in the thalamostriatal pathway 1201. To summarize, our results employing excitotoxic lesions in conjunction with neurochemistry support recent observations from this laboratory of intense retrograde labelling of the PT and the BL after the intra-accumbal microinjection of I)-[“H]asp [2,S]. Excitatory amino acids seem to be the transmitter of several pathways convergent upon the NAS. including those from the medial prefrontal cortex. the BL and the PT 12. 3, 5.9, 291. The ro1ef.s) of GIuiAsp afferents in various behaviours mediated by the NAS wilt be of appreciable interest. .AC’KNOWJ_EDGEMENT
This work was supported by a Programme Grant from the National Health and Medical Research Council of Australia of which P.M.B. is a Principal Research Fellow.
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