Biochemical evidence for glutamate as neurotransmitter in corticostriatal and corticothalamic fibres in rat brain

03~-~522~8l~a~863.

Neurc.~cimcs Vol. 6, No. 5. pp. 863 to 873. 1981 Printed in Great Britain

I iS02.00~0 Ltd 1981 IBRO

Persamon Press 0

BIOCHEMICAL EVIDENCE FOR GLUTAMATE AS NEUROTRANSMI~ER IN CORTIC~STRIATAL AND CORTICOTHALAMIC FIBRES IN RAT BRAIN F. FONNUM,J. STORM-MATHISEN’ and I. DIVA? Norwegian Defence Research Establishment, Division for Toxicology, N-2007, Kjeller, Norway effects of ablation of frontal, occipital or entire hemicortex on several neurotransmitter parameters in the rostra1 and caudal neostriatum, thalamus and the contralateral anterior medial cortex were investigated. In particular the effects on the high affinity uptake of D-aspartate and on the endogenous level of amino acids, especially glutamate and aspartate, were studied in order to identify Abstract-The

glutamate- or aspartate-containing nerve terminals in these regions. The results show a specific decrease in high affinity uptake of Daspartate in both rostra1 and caudal neostriatum ipsilateral to the lesion after frontal or entire hemid~orti~tion. There was also a small but significant decrease in D-aspartate uptake on the contralateral side. Only the level of endogenous glutamate decreased in the neostriatum after hemidecortication. There was a specific decrease in D-aspartate uptake in the thalamus only ipsilateral to the cortical lesions. In thalamus there was a significant decrease both in the level of glutamate and to a smaller extent in that of aspartate after hemidecortication. Anterior medial cortex showed a very active high affinity uptake of D-aspartate, which was slightly reduced after removal of the contralateral hemicortex. The high affinity uptake of D-aspartate was in all cases mainly due to uptake in synaptosomes. The results show that the neostriatum receives glutamate-containing fibres from the neocortex, particularly the frontal part. This projection is mainly ipsilateral with a small element derived from the contralateral side. The thalamus, both the rostra1 and caudal parts, receives glutamate-containing fibres from the whole extent of the ipsilateral neocortex. Some of the corticothalamic fibres may also contain aspartate. The anterior medial cortex probably contains a high proportion of glutamate- and/or aspartate-containing nerve terminals but only a low proportion of these are derived from the contralateral cortex.

?k~ NEOCORTM: sends heavy excitatory projections to 1974) have prompted us to investigate in more detail the neostriatum and the thalamus (ANDERSEN, ECCLES whether L-Glu could be the transmitter of several & SEARS, 1964; PURPURA, 1972; KITAI,Kocss, FWstypes of corticofugal fibres such as those to rostra1 TON& SUGIMORI, 1976; FEGER,DENIAU,de C&AM- and caudal neostriatum, thalamus and the contraPLAIN& FELZ, 1979). The identification of the translateral cortex. mitter of these corticofugal fibres would be of great We have therefore studied several transmitterinterest. A first step in this direction was taken when related parameters including the high affinity uptake we, in a short communication, showed that a lesion in of D-aspartate (~-Asp) and the levels of endogenous the frontal cortex was a~omp~ied by a ‘large reducamino acids in these regions after ablation of the frontion in the high affinity uptake of L-glutamate (L-GIu) tal or occipital cortices or after an entire hemidecortiin the anterior part of neostriatum (DIVAC, FONNUM cation. Kinetic studies (DAVIES& JOHNSTON,1976) & STORM-MATHISEN, 1977). This was confirmed by and deafferentation of specific fibres (YOUNG,OSTERMCGEER, MCGEER, SCHERER& SINGH (1977). Later GRANITE, HEMDEN& SNYDER,1974; LUND KARLSEN KIM, HASSLJZR, HAUG & PAIK (1977) showed that a & FONNUM, 1978; STORM-MATHISEN & WOXEN frontal cortical ablation was followed by a larger OPSAHL, 1978) have shown that the high affinity reduction in the level of L-Glu than in that of any uptake of ~-Asp as well as of L-Glu occurs mainly in other amino acid examined. These biochemical findglutamate nerve terminals and that the membrane ings, together with the well known excitatory effect transport systems of glutamate and aspartate nerve obtained by iontophoretically applied L-Glu (e.g., terminals probably do not distinguish between these Spencer, 1977; CURTIS& JOHNSTON,1974; KRNJEVIC, amino acids (Review: FONNUM,LUND-KARLSEN, MALTHE&REN~SEN,SKREDE& WALLAS,1979). The metaPresent address: ‘Anatomical Institute, University of bolically inert ~-Asp was therefore used as marker of Oslo. ‘Institute of Neurophysiology, University of Copenhigh affinity uptake into glutamate nerve terminals. hagen Since high affinity uptake of ~-Asp does not differenAbbreoiations: L-Glu, L-glutamate; ~-Asp, D-aspartate: tiate between glutamate- and aspartate-containing GABA, y-aminobutyrate; GAD, glutamate becarboxylase; nerve terminals, we have also carried out amino acid ChAT, choline acetyltransferase; AAD. amino acid decarboxylase. analysis in the striatum and the thalamus after corti863

I-‘. FONWM.

864

J. STORM-MATHIS~S and 1. DI\A(

cal lesions. The high affinity uptake of ;I-aminobutyrate (GABA) was also analysed to exclude the possibility that the changes observed in high affinity uptake of o-Asp could be due to a general effect of the lesions on uptake processes. Glutamate decarboxylase (GAD), choline acetyltransferase (ChAT) and aromatic amino acid decarboxylase (AAD) were determined to show that the effects of the lesions were restricted to glutamate neurons. EXPERIMENTAL

PROCEDURES

Surgical operarions Albino Wistar rats, weighing about 250 g, were obtained from Dr MGllergaard-Hansens, Avlslaboratorium, Vejby, Denmark. The animals were anesthetized by intraperitoneal injections of equithesin (3.3 ml/kg) (see under Materials for composition). The skull was opened and the cortex was removed down to the white matter by suction through a fine hypodermic needle under microscopic guidance. For frontal ablation the cortex was removed between the fronto-parietal suture and the frontal pole (Fig. la). In occipital ablation the cortex was removed from the occipital pole to 2 mm caudal to the bregma. The occipital and frontal ablation together do therefore not correspond to the total hemidecortication (Fig. I b). In hemidecorticated rats the whole cortex except the areas ventral to the rhinal solcus was removed (Fig. Ic). The lesions were inspected macroscopically. and the brains were photographed before the biochemical studies. During later sectioning of the brain the depth of the lesion could also be inspected. In no case did we observe that the lesion penetrated into the striatum or into the thalamus. In sham operated animals only the skull was opened. Dissection and preparations of tissue Six to eight days after surgery the animals were stunned and decapitated, and transverse brain slices (about 1.25 mm thickness) were prepared on a Sorvall tissue slicer in the cold room. Samples of neostriatum and thalamus were dissected out with broken splints of razorblades under the microscope. The anterior medial cortex was dissected out from slice I or 2 as shown. The rostra1 striatum was dissected from slices 3 and 4 (see Fig. 2) rostra1 to the crossing of the cOmmissura anterior. The caudal striatum was dissected out from the slices at a very caudal level (slice 6. 7 and 8). The ‘rostral’ thalamus was dissected from slice 6 and the ‘caudal’ thalamus from slice 7. The border between the 2 parts of thalamus is at about the beginning of the fasciculus retroflexus. The tissue samples were placed on a wet filterpaper and weighed on a Mettler Electronic Microbalance. The wet weight of the samples was usually from 20-50 mg. A 2% (wt/vol) homogenate of the tissue was prepared in 0.32 M sucrose buffered to pH 7.4 with 10 mM sodium phosphate with a glass-teflon homogenizer rotating at 8bOrpm. This homogenate was used directly in the biochemical assay for high affinity o-Asp uptake, high affinity GABA uptake, glutamate decarboxylase, choline acetyltransfcrase or aromatic amino acid decarboxylase. In separate experiments larger samples of neostriatum or thalamus were homogenized as above and treated for subcellular fractionation as dexribed by LUND KARLSEN& FONKUM(1978). The homogenate was first centrifuged at 1000 g for 5 min to remove nuclei. cell debris and large

mitochondna. The supernatant was added IO 5 ml tube< filled each with I ml of 0.8. 1.0, I.? and I.4 M sucrose and centrifuged for ?Omin at 80.00Og. The tubes were punctured at the bottom and samples collected. For amino acid analysis the animals were killed hy decapitation and the head dropped into liquid nitrogen for IO ‘I to cool the brain to near the freezing point (TA~AI~ASHI % APRISOS.1964). The brain was rapidly dissected out. Slices were cut with a razorblade and the samples were dIssected from slices comprising a large part of the neostriatum or middle thalamus. The samples were homogenlred in 200 mM sodium bicarbonate:acetone (I : I ) and placed at _ 20 C for at least I h. The amino acid content of the protein free supernatant was determined b> the doublelabelled dansylation technique as described by FOSSL M & WALAAS(1978). In some animals. samples were homogenized in ?“,, trichloroacetic acid. x-amino adipic acid was added as in internal standard. and the samples analyzed on an automatic amino acid analyzer (Kontron). The sample was developed with increasing concentrations of lithium citrate buffers (0.2 M Li pH 2.65, 0.2 M Li pH 2.90. 0.4 M Li pH 3.35 and 0.85 M Li pH 4.10). The amino actd was detected by fluorescence after reaction with O-phthalaldehyde (Ron!. 19711. Biochemicul

anulysrc

High affinity uptakes of ~-Asp and GABA were assayed essentially as described by S~RM-MATHISES & Woxt~ OPSAHL(1978). The homogenates (5 pl) were incubated In Krebs- tris buffer, pH 7.4 for 3 min at 25°C with a mixture of 0.1 pM [‘HI-D-ASP and I PM [U’“C] y-aminobutyric acid. Under these conditions the uptake of the 2 amino acids did not interfere with each other. The uptake was terminated by filtration through Millipore filters (0.45 pm). the samples washed and the filters counted in a Packard Tri Carb Scintillation Counter fitted with an Absolute Activity Analyzer. For enzyme analysis, the homogenates or the samples from the subcellular fractionation were treated with Triton X-100 (final cont. 0.2:< w/v) to release all activity. ChAT was assayed as described by FO~;NUM(1975). GAD as described by FONIGUM. WALAAS& IVERSEN(1977) and AAD as described by BROCH& FONI\‘CM(1972). Carnitine acetyltransferase was assayed by Kalignost extraction at acid pH (see FOSNUM.1975). Materials. Equithesin contains (I) 0.972 g sodium pentobarbital in Il.5 ml 95% ethylalcohol: (2) 4.25 g chloral hydrate in 42.8 ml propylene glycol; (3) 2.126 g MgSO, in 45.7 ml HIO. The three solutions are mixed in the order I. 2 and 3. Stufisfical rreatmenf. There were no differences between the similar samples from the 2 sides in sham operated animals. and the experimental results were therefore compared with the results from both sides in sham operated controls. The samples were compared by the Wilcoxon rank-sum test (HODGES8~ LEHMANN.19641. RESULTS Neostriatum

The rostra1 and caudal parts of neostriatum in control animals differed in the levels of several transmitter parameters. The high affinity uptake of u-Asp and AAD activity were significantly higher in the rostral than caudal part of neostriatum (Table 1). In contrast the GAD activity was slightly higher in the cau-

FIG. 1.

surface view of the different cortical ablations used (a) frontal cortex ablation (TVocc:ipital cortex ablation (c) hemidecortication. 865

FIG. 2. Samples dissected from 1-1.25 mm thick frontal slices prepared by a tissue chopper. The medial frontal cortex was dissected from slice 1 and 2, rostra1 striaium from slice 3 and 4, caudal striatum from slice 6, 7 and 8, rostra1 thalamus from slice 6 and caudal thahunus from slice 7. A, undissected slices, B. dissected slices.

866

867

Corticofugal glutamate neurons THALAMUS

STRIATUM D-Asp uptake

ChAT 20 > .z 3 P ” 1.5 E Y :: 0, 1.0 .? 3 & a5

II

HOM H

03 0.6 0.8 1.2 IA

HOM

s

n

0.3 0.6 0.8 1.2 1.4

FIG. 3. Subcellular fractionation of striatal and thalamic samples taken from unoperated (0) or hemidecorticated (0) rats. The results are expressed as relative specific activities (enzyme activity per mg protein). The relative specific activity of each parameter in the homogenates from unoperated animals is taken as unity. The results are expressed as single values (0,O) or as mean values (line drawings) Horn, homogenates; Pl, nuclear fraction: 0.3, 0.6, 0.8, 1.2 and 1.4 correspond to fractions in Molar sucrose after density gradient centrifugation. Notice that the only major changes are due to a decrease in ~-Asp uptake, particularly in the synaptosome fraction.

da1 part, but this difference was not significant. Previous studies have shown that GAD activity is concentrated in the ventrocaudal part of the neostriatum (FONNUM, GCYTTESFELD & GROFOBA, 1978). ChAT did not differ in the rostro-caudal direction (Table 1) in agreement with SCALLY, ULUS, WURTMAN & F%TIBONE

(1978).

Hemidercortication.

In these animals,

a substan-

tial decrease of high affinity uptake of ~-Asp was found in both the rostra1 and caudal ipsilateral samples (Table 2). There was also a small but significant reduction in both regions on the contralateral side. This lesion was not accompanied by any changes in the other transmitter parameters, apart from a small decrease of GAD in the caudal sample on both sides. The small decrease of the high affinity GABA

TABLE 1. THE DISTRRIBI~:TION OF

TRANSMITY~~R

MARKERS

IN ROSIRAL

OPERATED

HA v-ASP uptake tOgd.p.m.,/h& protein Rostra1 Caudal

AND CAUDAL

HA-GABA uptake t09d.p.m.lhig protein

GAD pmole%/g protein

1.33 -t 0.10 --1.66 _t 0.19

414 fi 18 443 _+23

330.4 * 8,6+* 279.9 2 10.3

SAMPLES FROM TM

NE~S’~RMTI;M

r\ SHAM

ANIMALS

ChAT pmoie!h!g protein

-

AAD

fimaieihlg protein -l_l____-__l

310 * 7

99 f. PC

290+ I2

62 f z

The results are mean f S.E.M.of 12 samples in each group. HA, high affinity. ** P < 0.01.

uptake in the caudai sample of the ipsilateral side was not statistically significant. Ablution of the frontal cortex. This led to a similar decrease in the uptake of rasp in the rostra1 sample and a smaller decrease in the caudal sample ipsilateral ta the lesion (Table 2). Again there was a small but sign&ant decrease in the rasp uptake in both the contralateral samples. After this lesion there were no other changes in the other transmitter parameters. Ablation of the occipital cortex. This was not accompanied by signi&cant changes in the r;>-Aspuptake when the samples inch&d the whole dorso-ventral extent of the neostriatum (Table 2) Apart from a slight decrease of GAD and high aEnity uptake of GABA in the caudal sample on the ipsilateral side,

Lesion uptake: ~em~~ort~c~tion (5) Frontal cortex ablation (5) Occipital cortex ablation (7)

there were no significant changes in the other transmitter parameters. The high affinity uptake of ~-Asp was studied in different subcelht~r fractions from unoperated and operated animals; To have enough ma+al for anafysis, the neostriatal sampk incl commissural neostriatum ipsita these animals, the cortical lesions were less complete and the decrease in high affinity up-take of ~-Asp was slightly smaller than in Table 2. The high affinity uptake of ~-Asp in unoperated &mats followed closely the distribution of the osomal marker CXAT (Fig. 3), and was very d&rent hum that of the mitochondrial marker carnitine acetyhransferase. A proportion of the uptake of ~-Asp was also found

tJnoperated side Operated side Rostra1 Rostra! Caudal Caudal Per cent of sham operated animals

HA &ASP

30 f 3*** 35 _e s++ 107 2 7

25 2 2*** 48 f 3*** 99 f 6

87 f 7* 7f + 8* 112 + 8

80 & 4** 86 It: 4* 114 -1_r3

Hemidecortication (5) Frontal cortex abfatiott (5) occipital cortex ablation (71

87 _c 15 99 + II 115 & 6

70 i: 8 93 i 7 78 2 5”

124 k 4* 121 f IIf 126 i 5*

901 fl 95 rt: 7 101 f 10

GAD activity: Hcmidwortication (5) Frontal cortex ablation (5) Occipital cortex ablation (5)

118 & 13 108 f 11 93 It: 7

71 + v* 107 f 5 74 + 5+*

99+_5 115 + 8 91 _t3

to2 * 6 102 3 9 111 +4

82 I 18 97 + 9 122 f 10

110 24 109 2 8 113 & 4

92 ri: x4 117 _c7 116 ss 12

92 4 8 92 f 8 85 * 8

95 i: 17 113 * 5 118 +7

94_i9 106 4 13 137Q 20

99c 15 112 2 8 119 & 7

HA GABA

ChAT

uptake:

70 _zt:4** 86 & 3 74 + 3**

aid&y:

H~~d~orticat~on (5) Frontal cortex ablation (5) Occipital cortex ablation (7) Aromatic amino acid decarboxykne:

Hemi&corticatiaa (5) Frontal cortex ablation (5) ckc@w GXwtexab&ion (7)

The results are expressed BS percentage of sham operated animals and are mean + S&M. The number of ss&e&n parent. HA, high affinity. +++P < 0.60t. l + P < 0.01. = P < 0.05.

869

Corticofugal glutamate neurons TABLE3. AMIWOACIDS

Amino acid

IN THE NEOSTRIATUM AFTER HEMIDECORTICATION ON RIGHT SIDE

Unoperated Control (8) (pmol/g protein)

The results are expressed samples in parentheses. ** P < 0.01.

The thalamus was separated into a rostra1 and cau-

85 f 55 + 105 f 228 + 118 + 166 f

I 4** 18 39** 11 15** of

da1 sample as shown in Fig 2. The rostra1 sample of the thalamus had a much higher high affinity uptake of o-Asp than the caudal sample (Table 4), the neostriatal samples (Table 1) and the cortical sample (Table 7). The other transmitter parameters showed less variation than rasp between the rostra1 and cauda1 samples of thalamus; GAD was slightly higher in the caudal part whereas ChAT was slightly higher in the rostra1 part (Table 4). Hemidecortication. There was a large reduction in rasp uptake in both ipsilateral thalamic samples (Table 5). No significant changes were found in the other transmitter parameters in the rostra1 sample, but a decrease was observed in GAD and GABA uptake in the caudal sample on both sides. Since the major part of the glutamate projection to neostriatum came from the frontal part of cortex, we also studied the effect of ablation of frontal and occi-

TABLE 4. THE DISTRIBUTION OF TRANSMITTER MARKERS ANIMALS

Rostra1 Caudal

29 21 14 16 24 14

as mean values k S.E.M. Number

together with ChAT in the low speed pellet fraction PI which contains cell nuclei and fragments, heavy mitochondria, myelin sheaths as well as synaptosomes. In operated animals the high affinity uptake of o-Asp was reduced both in the homogenates and particularly in the synaptosomal fractions (Fig. 3). Carnitine acetyltransferase and ChAT were distributed similarly in unoperated and operated animals. Amino acid analysis by the double isotope dansylation technique showed a specific fall in the level of Glu, but not in that of the other amino acids including Asp and glutamine (Table 3). In 3 animals changes in amino acid content were also examined by an amino acid analyzer with similar results. In percent of unoperated: taurine 106, Asp 98, Glu 61, GABA 95. Thalamus

(per cent of unoperated) 124 k 103 * 94 * 121 f 116 f 121 +

22 + 2 114 &-5 11 &2 10 + 1 19 + 1 6-l k 3

Aspartate Glutamate Glycine Alanine GABA Glutamine

Operated Animal (4) Left Right

IN THALAMIC

SAMPLES IN SHAM OPERATED

HA D-ASP Uptake lO’d.p.m./h/g protein

HA GABA Uptake 109d.p.m./h/g protein

GAD pmole/h/g protein

ChAT pmole/h/g protein

780.2 k 29.2*+ 420.2 f. 81.8

2.18 k 0.25 2.95 k 0.13

449 + 34** 640f58

12.4 f 4.5’* 60.6 f 2.4

The results are mean + ** P < 0.01.

S.E.M.

of 8 samples in each group. HA, high affinity.

TABLE5. THE EFFECT

OF HEMIDECORTKATION ON TRANSMITTER MARKERS IN THALAMIC SAMPLES

Operated Unoperated Rostra1 Rostra1 Caudal Caudal (per cent of sham operated) HA ~-Asp uptake HA GABA uptake GAD ChAT

35 f 5** 87 + I 99 * 10 10 If: 5

29 + 8:’ 61 k 2* 65 f 7’ llOk6

83 f 98 k 102 k 93 *

5 10 6 4

The results are mean &-S.E.M.of 4 samples in each group. * P < 0.05. ** P < 0.01.

116 f 14 f 66 f 91 *

20 8* -I* 14

x70

F. FOVNI:M. J. STORM-MATHISENand 1. DI\A(. TABLE 6. AMINO A~IIX IN --.

THAI.AM~JS ASTER THt RIGHT SlOt _-.--

-.-.---

HI:MII)ECOHII(‘ATIO~ .

-.

Operated

llnoperated Control (8) Amino acid

I.eft

(/lmol!‘g protein)

ox

Animal (4) Right

(per cent of unoperatcd)

-

Aspartate Glutamate Glycine Alanine GABA Glutamine

The results are expressed samples in parentheses * P < 0.05. l * P < 0.01.

as mean

pital cortex on the high affinity uptake of ~-Asp in the thalamic samples in a few animals. Both types of partial cortical lesion produced a reduction in ~-Asp uptake. Frontal cortex ablation (2 animals) gave 28 and 18% reduction in the rostra1 thalamic sample and o&pita1 cortex ablation (2 animals) gave a 35 and 56% reduction in the caudal thalamic samples respectively. Subcellular fractionation of a thaiamic sample comprising both the rostra1 and caudal regions gave the same results as with the neostriatal sample (Fig. 3). Thus ~-Asp uptake was distributed in a similar way to the synaptosomal marker ChAT and differently from the mitochondrial marker camitine acetyltransferase. Following hemidecortication the fall in ~-Asp uptake was most pronounced in the synaptosomal fractions. Amino acid analysis by the double isot& dansyktion technique showed a large fall in the level of glutamate and a smaller but significant fall in the level of aspartate. There were no changes in the other amino acids tested, apart from an increase in glutamine (Table 6). Analysis with an amino acid analyzer gave similar results (two animals). In percent of contralateral side: taurine: 103, Asp: 80, Glu: 66 and GABA: 116. Frontal cortex

THE

Hemidecorticated

r f k + + f

5’ 3** II IY 4 15

values f S.E.M. Number

of

tions, although smaller, in GABA uptake, GAD and ChAT activities (Table 7). The results do not pennit any firm conclusions about the afferents from the contralateral cortex, excupt that there could well be a small crossed giutamate/aspartate pathway. We did therefore not proceed with subcellular fractionation or amino acid analysis of this region. DISCUSSION There are strong reasons to believe that the high affinity uptake of ~-Asp or L-GIu is an inherent prop erty of glutamergic/aspartergic nerve terminals. The uptakes of ~-Asp and L-Glu are apparently indistinguishable biochemically, but inhibited by only a few structural analogues (BALCAR & JOHNSTON.1972) and therefore relatively specific. Autoradiographic studies at the electron microscopic level have shown a selective uptake of L-Glu into nerve terminals in the hippocampal region (ST~JRM-MATHISEN & IVERSEN. 1979) or synaptosomes from pigeon tectum (BEART. 1976). Selective destruction of a number of fibres suspected of using ~-Asp or L-Glu as transmitters such as the perforant path and Schaffer collaterals in the hippocampus (STORM-MATHISM. 1977; FONNUM & WALAA$ 1978), hippocampo-septal fibres (FONNUM& 1978), allocortico-accumbens

(10) (7)

566.5 + 19.5 73 + 4”’

fibres (WALAAS & Fos-

NUM, 1979; WALAAS, 1981). cortico-tectal

and cortico-

geniculate fibres (LUND KARUEN 8c FONNCM.197X) and the parallel fibres in cerebellum (YOVSC.ef ~1..

EFFECT0~ HEklrDEf33RTrc~n0N0N NEUROTRANSM~ HA DASP Uptake lo9 d.p.m.,/h/g protein

Sham operated

79 62 95 loo x4 II0

WALAA~, 1978; STORM-MATHISEN & WOXEN OPSAHL.

Samples from the anterior medial cortex showed relatively high activities of ~-Asp uptake. This uptake was slightly reduced after hemidecortication on the contralateral side. There were, however, also reduc-

TARLE 7.

101 * 9 01 _c4 YO & 8 01 *x YO & x 115 k 6

26 f 1 III +3 I2k I Xkl 17 = I 58 + 2

MARKERSIN

HA GABA Uptake 10gd.p.m./h/g protein

THE CONTRALATERAL.

GAD pmole/h/g_/protein

FRONTALC0RTF.x ChAT pmole;h,‘g protein

4.08 f 0.30 260 + 10 Per cent of sham operated 77 + 8 89 f 8

The results are expressed as mean + S.E.M. The number of samples in parentheses. l ** P < 0.001.

HA, high affinity.

256 f

16

83 +_ 6

Corticofugal glutamate neurons 1974) are all accompanied by a selective decrease in the high affinity uptake of ~-Asp or L-Glu. At the same time there was a significant reduction of the endogenous level of L-Glu, but not of other amino acids including ~-Asp. There is, however, also an uptake of these amino acids into glial cells which could interfere. In terms of uptake the most active glial cell preparations are the astrocytes cultured from dissociated mouse brain hemisphere (SCHOUSBOE & HERTZ, 1981). However, these glial cells seem to have a relatively low affinity for the amino acid (K,,, for glutamate approx 27-90 PM compared to 2-4 PM for synaptosomes (STORM-MATHISEN& WOXEN OPSAHL, 1978)). Also, experiments in rat retina have indicated that the glial uptake to a large extent is lost on homogenization (LUND KARLSEN, 1978). In addition WEILER,NYSTR~M& HAMBERGER (1979) have reported that ~-Asp and L-Glu uptakes are considerably lower in glial fractions than in synaptosomes. Degeneration of nerve fibres is usually accompanied by gliosis, rather than a loss of glial cells. Thus we do not think that under our conditions the uptake in glial cells will contribute significantly to the observed decrease of high affinity uptake of rasp. The present results show that destruction of corticofugal fibres by ablation of different parts of cortex is accompanied by a large loss in high affinity uptake of ~-Asp in the ipsilateral neostriatum and thalamus (Table 2 and 5) and a small but significant decrease also on the contralateral side of neostriatum. These effects were found in both the rostra1 and caudal parts of both subcortical formations. In both striatum and thalamus the synaptosome fraction was responsible for the major part of the uptake and also for the decrease after lesion. It was significant that the lesions did not produce any other reduction in the transmitter parameters, with the exception of small decreases in GAD and GABA uptake in the caudal parts of both thalamus and neostriatum. In the striaturn the decrease of GABAergic parameters was obtained only, when the lesion comprised occipital cortex, and in thalamus the decrease of GAD occurred both ipsi- and contralateral to the lesion. In both cases the changes in these parameters therefore differed from the changes in the high affinity uptake of D-Asp. The corticostriatal projection The anatomical data support the view that the corticofugal fibres to neostriatum contains a high capacity for o-Asp uptake. The neocortex projects to the neostriatum in a somatotopical order so that the frontal cortical areas project mainly to the head of the nucleus caudatus and the posterior region mainly to the body and tail (WEBSTER,1961). The frontal corti&l projection has, however, a wider distribution than expected (GOLDMAN& NAUNTA,1977; KEMP & PowELL, 1970; CARMAN,COWAN, POWELL & WEBSTER, 1965). In agreement we found a large loss of high affinity o-Asp uptake in both the rostra1 and caudal

871

part of neostriatum after hemidecortication which included the whole mediolateral extent of the neocortex (see HEDREEN, 1977). Removal of the frontal neocortex was accompanied by a similar decrease in ~-Asp uptake in the rostra1 neostriatum and a smaller decrease of rasp uptake in the caudal neostriatum underlining the predominant nature of the frontal cortex input. We did not find a significant fall in o-Asp uptake when the lesion was limited to the occipital cortex. This part of cortex projects to the tail of the neostriatum in rat (WEBSTER,1961) although the quantitative importance of such an input has not been evaluated. Radioautographic studies after labelled leucine injection indicate that the visual cortex only projects to a thin layer in the dorsal part of neostriaturn (HOLL~DER, TIEZE& DISEL, 1979). An altemative explanation is that the cells in the occipital cortex projecting to the neostriatum do not use L-Glu as a transmitter. At present this seems unlikely in view of the general importance of the glutamate as a transmitter of several corticofugal systems (see e.g. thalamus section). Both frontal cortex ablation and hemidecortication led to a significant decrease of o-Asp uptake in neostriatum contralateral to the lesion. This is in agreement with anatomical studies showing that the neostriatum receives some input from the contralateral cortex (CARMAN,COWAN,POWELL& WEBSTER. 1965; K~~NZLE,1975; WISE & JONE$ 1977). Since the decrease in high affinity uptake of ~-Asp cannot discriminate between glutamergic and aspartergic nerve terminals, amino acid analysis of neostriatal tissue was carried out after cortical lesion. Both the use of a double isotope dansylation technique and an amino acid analyser showed a large and significant decrease only in the levels of glutamate. We therefore believe that the corticostriatal fibres are glutamergic. The present investigation therefore supports and expands previous investigations on the role of glutamate as a neurotransmitter in the corticostriatal pathway (DIVAC et al., 1977; KIM et a[., 1977; MCGEERet al., 1977). A further discussion of the dorsoventral distribution of these fibres will be found in the subsequent paper (WALAAS,1980). Our conclusion is also in agreement with electro-physiological studies in that intra- and extra-cellular recordings from units in striatum after stimulation in the cerebral cortex give predominantly excitatory postsynaptic potentials (BLAKE, ZARZECKI & SOUTEN,1976; KITAI et al., 1976). Further, SPENCER (1976) showed that the excitatory response to cortical stimulation could be suppressed by iontophoretic application of glutamate diethylester. The corticothalamic projection

It was only natural that we extended our investigation also to include another major corticofugal system, namely the corticothalamic. Anatomical studies indicate that the corticothalamic projection is uncrossed and topically arranged, and that it is reciprocal to the thalamocortical projection. The con-

872

F. FONNUM. J. STORM-MATHBEN and 1. DIVA<

nections between thalamus and neocortex in rat have been well studied (LASHLEY.1941; JACOBSON & TRG JANOWSKI,1975; WISE & JONES, 1977; DIVAC, KosMAL,BJ~RKLUNLI& LINDVALL,1978). In our investigation we did not try lo separate out the different thalamic nuclei, but limited ourselves to 2 parts, 1 slice anterior and 1 slice posterior to the beginning of fasciculus retroflexus. In agreement with the anatomihemidecortication was accal investigations, companied by a large reduction in ~-Asp uptake in both the rostra1 and caudal sample ipsilateral lo the lesion. In a few animals we found significant reduction in ~-Asp uptake after ablation of either the frontal or the occipital cortex. We do not believe that the reduction in D-Asp uptake could be due lo retrograde changes in collaterals of thalamocortical fibres after the cortical lesion. Firstly, it would mean that nearly all the thalamocortical fibres degenerated within a week after cortical ablation. Secondly, the hollow of the barrels of somatosensory cortex which receives the specific thalamocortical fibres does not show any uptake of ~-Asp (SOREIDE& FONNUM,1980). Thirdly, enucleation of the neonatal animal which leads to heavy loss of thalamocortical fibres, was not accompanied by a reduction in high affinity ~-Asp or Glu uptake in visual cortex (LUND KARL~EN & FONNUM, 1978). We cannot, however, exclude that the small loss in GAD and GABA uptake in thalamus after cortical lesion could be due lo retrograde changes. The amino acid analysis after decortications showed a significant fall in both glutamate and, to a smaller extent, aspartate in the ipsilateral thalamus. This could indicate the presence of both glutamergic

and perhaps some aspartergic corticothalamic fibres. This is in agreement with the fact that some neurons in thalamus are preferentially excited by glutamate. others by aspartate, a finding indicating that both amino acids could be transmitters in the region (review: CURTIS & JOHNSTON. 1974). The findings in thalamus are comparable to previous studies in our laboratory where it was shown by the same approach that the visual cortex project glutamergic fibres to the lateral geniculate body (Luau KARLSEN & FOXSLIM. 1978). In contrast, MCGEER c’ral (1977) did not find any changes in L-Glu uptake in a thalamic sample after a frontal cortex lesion. Other

pathways

and transmitters

Although our results clearly demonstrate a very active high affinity uptake of ~-Asp in the prefrontal cortex sample, only a small proportion of this may be due lo an input from the contralateral cortex. The small changes in high affinity uptake of GABA and in GAD in the caudal neostriatum and thalamus after lesions comprising occipital cortex indicate the existence of a small contingent of corticofugal GABA fibres. The present data do not allow any firm conclusions lo be drawn on this point. Conclusions

In conclusion, the results show that corticostriatal and corticothalamic fibres have a very active high affinity uptake of ~-Asp and contain a high level of endogenous glutamate. We therefore suggest that acidic amino acids, particularly glutamate, should be regarded as strong transmitter candidates for these corticofugal systems.

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