The corticopontine projection: Axotomy-induced loss of high affinity l -glutamate and d -aspartate uptake, but not of γ-aminobutyrate uptake, glutamate decarboxylase or choline acetyltransferase, in the pontine nuclei

0306-4522;83/030449-09$03.00/O PergamonPressLtd

b’ieuroscierwVol. 8, No. 3, pp. 449to 457, 1983 Printedin Great Britain

IBRO

THE CORTICOPONTINE PROJECTION: AXOTOMY-INDUCED LOSS OF HIGH AFFINITY L-GLUTAMATE AND D-ASPARTATE UPTAKE, BUT NOT OF y-AMINOBUTYRATE UPTAKE, GLUTAMATE DECARBOXYLASE OR CHOLINE ACETYLTRANSFERASE, IN THE PONTINE NUCLEI W, THANGNIPON*, T. TAXES, P. BRODAL and J. STORM-MATHISEN~ Anatomical Institute, University of Oslo, Karl Johansgt. 47, Oslo I, Norway

Abstract-The corticopontine fibres were severed in the crus cerebri in rats and mice by a stereotaxically operated retractable wire-knife. The pontine nuclei were microscopically dissected from fresh slices of rats and synaptosome-containing homogenates were prepared. The high affinity uptake of radiolabelled L-glutamate (L-Glu) and D-aspartate (D-ASP) was heavily reduced five days after the lesions. The uptake was further reduced after bilateral (-75% for u-Asp and -65% for L-Glu) than after unilateral lesions (-55% for ~-Asp and -45 to -50% for L-Glu on the lesioned side). The molar ratio of the uptakes of ~-Asp and L-Glu was consistently lower in pons after transection of the cortical afferents than normally (-287; after bilateral lesions). y-Aminobutyrate uptake and glutamic acid decarboxylase were not changed. Choline acetyltransferase was increased (+53%) after unilateral lesions, but not altered after bilateral lesions. Autoradiograms of slices from mice, incubated with tritium-labelled amino acids and fixed in glutaraldehyde, showed high affinity uptake sites for u-Asp to be enriched in the pontine nuclei, compared to neighbouring structures. After partial lesion of the crus cerebri the uptake was reduced in the area with degenerated corticopontine afferents. y-Aminobutyrate uptake sites were relatively less concentrated in the pontine nuclei than D-ASP uptake sites. The results indicate, along with the previous demonstration of Ca-dependent K-induced release of D-[3H]aspartate from the corticopontine terminals,41 that glutamate and/or aspartate may be transmitters in this pathway. The results also suggest that acidic amino acid uptake sites may differ in their relative transport rates for aspartate and glutamate.

The corticopontine pathway is one of the main efferent projections of the cerebral cortex. It accounts for most of the afferents to the pontine nuclei, and contains fibres from most areas of the cortex.1*3 Stimulation in the cerebral cortex causes monosynaptic excitation of neurons in the pontine nuclei.‘s3’ Biochemical studies have implied glutamate (Glu) and/or aspartate (Asp) to be the transmitter of several corticocortical and corticofugal fibre systems. 7~9~‘o~‘5~20~29~30 The same is true of several projection systems within and from the archicortex. 5.9.11,21,22,26,34,37,43,45 However, until

* W. T. was on leave of absence from the Faculty of Science, Mahidol University, Rama VI Road, Bangkok 4, Thailand. Present address: MRC Developmental Neurobiology Unit, Institute of Neurology, 33 John’s Mews, London WClN 2NS, England. t Institute of Physiology. University of Oslo, Oslo 1, Norway. $ Correspondence to J. Storm-Mathisen. Abbreviations: Glu, glutamate; Asp, aspartate; GABA, y-aminobutyric acid; GAD, glutamic acid decarboxylase; ChAT, choline acetyltransferase. 449

recently40.42.4”.47

similar data had not been presented for the long projection pathways from the cerebral cortex. High affinity uptake of radiolabelled G~u~!‘~ has been extensively used as a marker for alleged glutamatergic neurons4s ~-Asp has been introduced6338 as a metabolically inert substrate for the high affinity membrane transport systems for L-Glu and ~-Asp, which are biochemically very similar. In the present paper, we have studied the effect of transection of the corticopontine fibres in the crus cerebri upon the high affinity uptake of radiolabelled L-Glu and ~-Asp in the pontine nuclei. Some of the results have been presented in abstract form.40.42 EXPERIMENTAL

PROCEDURES

Operations

Adult male Wistar rats (about 200g) or albino mice (about 30 g) were anaesthetized with pentobarbital, In pilot experiments the cerebral peduncle was cut by hemitransection of the brain, slightly more caudal than described before,33 by means of a spatula. In the main part of the study corticopontine fibres were severed bilaterally or unilaterally by transecting the crus cerebri selectively in the rostra] mesencephalon (Fig. 1) with a retractable wire-knife

4%)

W. Thangnipon

similar to that described by SclaTanl & Grossman.“’ The knife conslsted of a series of concentric tubes. the inner one being a stainless steel cannula of external diameter 0.4 mm holdin& a 0.12 mm tungsten wire (Lamp Metals. Ltd., Gateshead. En&land). The cannula was sharply bent, then cut off? leavm& a hole at the side of the tip. through which the wire could be extruded to form a fine hook, The knife assembly was clamped to the electrode holder of a stereotaxic Instrument (Stoelting), and tilted 20 laterally from the sagittal plane and 10 backwards from the coror,al plane. the tip of the inner cannula pointing medially and rostrally. Aiming at grazing the ventrolateral surface of the peduncle. the cannula was lowered through a hole drilled through the edge of the parietal bone, in rats 1.3 mm anterior to the parieto-occipital suture. Once the cannula tip was in place. at 5 mm below the parietal bone, the tungsten wire was extruded about 2.5 mm in the dorsomedial direction and the cannula was passed downwards about 2 mm (or until the cannula tip touched the bone), and then up again, across the ventrolateral surface of the peduncle. Subsequently the wire was retracted and the cannula withdrawn. In sham-operated animals, the skull was opened, but no lesion was made. Lesions in mice (for autoradiography) were made similarly.

The animals were anaesthetized with ether and decapitated. The brain was dissected out, cooled in sucrose solution on ice and inspected macroscopically for completeness of the lesion (Fig. 1A). Only animals with apparently complete lesions were analysed biochemically. Histological examination of a few animals confirmed that the macroscopic evaluation was reliable (Fig. lB), and that the crus cerebri appeared completely degenerated in FinkpHeimer8 stained sections (Fig. 1C). The pontine nuclei were dissected (Fig. 2) from 3-4 transverse 400 /Irn thick slices of the brain stem cut in the cold on a Sorvall TC2 tissue sectioner. Care was taken to include as much as possible of the pontine nuclei in each sample and to avoid contamination from adjacent tissue. To produce suspensions containing nerve ending particles, samples from each side were homogenized in lo@ 150 ~1 of 0.32 M sucrose with 5 mM sodium phosphate buffer pH 7.4. in a glass-Teflon microhomogenizer. The volume of fluid depended on a visual evaluation of the sample size to obtain about IO mgtis-

(‘i cl/

at 25 C for 3 min the samples were diluted with J m1 01 cold 0.94, NaCl containing 5 mM sodium phosphate bulTer pH 7.4 and OS m&/ml bovine serum albumin. collected on Millipore filters (0.45 pm). and rinsed with anothc:l two portions of 4ml of the same solution. The tiltera \bcrc eluted with 3.5 ml Dilusolve (Packard) and counted in a Packard liquid scintillation spectrometer. The channel settings and relative amounts of ‘H and “‘C were such that m no case did the overlap from the other isotope exceed 5”,, of the counts recorded. Correction for overlap &as thus unnecessary. Experiments showed that the uptake activities were proportional to the amount of tissue added under the present conditions. In pontine nuclear homo&enates. the addition of a 20-fold molar excess of GABA reduced the uptake of I /tM Glu by less than 25”,,. and ric,e ~CTVI.Thea confirms that the uptake of one of these amino acids will be negligibly affected by the presence of an equimolar concentration of the other.’ Protein, glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) were measured as described before.Statistical significance of the observed differences was assessed by the Wilcoxon rank-sum or signed-rank tests.‘” Autorudiograph!

Autoradiography was performed in mice since the small size facilitates cutting and processing of the slices of whole brain stem. Slices (200 pm) were incubated with 2.8 btM of D-C3H]Asp (16.8 Ci mmol-‘) or 1.4 PM [2.3-3H]GABA (New England Nuclear, 34.5 Ci mmol- I) in Krebs’ solution for 15 min at room temperature, rinsed and fixed in 5”,, distilled glutaraldehyde in Krebs’ solution. Autoradio&rams of the surfaces of the slices were produced following the previously described procedure34,39 with slight modifications. The slices were exposed for 45 days. and processed in solutions of about equal osmolarity (about I500 mOsM) to minimize swelling of the tissue slices. After developing in D19B (Kodak) diluted with an equal volume of Hz0 the slices were washed for 1 min in a SB4 hardening stopbath

sueml-‘.

High atlinity uptake of L-Glu, II-ASP and GABA were measured in rats as discussed elsewhere.35 Briefly, 5 ~1 of homogenate was added to 500 ~1 ice cold Krebs’ phosphate solution. containing 140 mM NaCI. S mM KCI. 1.2 mM CaCI,. 1.2 mM MgSO,. 15mM sodium phosphate buffer pH 7.4. 5 mM glucose and 0.5 mgjml bovine serum albumin. The reaction was started by adding 5Oiil of distilled water containing L-[2.3-‘H]Glu (New England Nuclear, Boston. MA, 23.3 Ci mmol-‘. diluted with unlabelled monosodium glutamate to give a specific radioactivity of 2.66 Ci mmol-‘) and [U-‘4C]GABA (The Radiochemical Centre, Amersham, U.K., 224 mCi mmol- ‘) to give final concentrations of 1 PM, and placing the tubes on a shaking water bath. In some experiments, samples were incubated with 11-[2,3-~Hl Asp (New England Nuclear, 16.8 Ci mmol-’ diluted with unlabelled ~-Asp to a specific radioactivity of 5.21 Ci mmol-- ‘) and L-[U-“%I Glu (The Radiochemical Centre, 285 mCi mmol-I). After incubation

Fig. 2. Schematic drawing of a section through pons (based on KGnig & Klippel, Fig. 52a) I’ illustrating dissection of samples (hatched area). Symbols: CC, caudal continuation of crus cerebri; cgm, corpus geniculatum mediale; cs, colliculus superior; for, formatio reticularis; FLM, fasciculus longitudinalis medialis; LM. lemniscus medialis; mr, nucleus medianus raphes; np, nuclei pontis; pag, substantia grisea centralis; PCM, pedunculus cerebellaris medius; PCS, pedunculus cerebellaris superior; III, nucleus principalis nervi oculomotorii.

Fig. 1. Illustrations of transection of crus cerebri in the rostra1 mesencephalon of the rat by means of wire-knife. A. Fresh brain with lesion on right side (1). B. Transverse section through lesion site. C. Transverse section through pons showing degeneration of the caudal continuation of crus cerebri (CC), 7 days survival. The sections were stained by the Fink & Heimer method. sn, substantia nigra; other symbols, see Fig. 2. Scale bars I mm.

451

453

Glutamate and aspartate uptake in the pons

Table 1, Glutamate, y-aminobutyric acid and acetylcholine markers in the pontine nuclei 5 days after transection of the crus cerebri in rat [‘H]Glu

Side

Operation Bilateral Sham Unilateral Sham

Both Lesioned side Contralateral

[“C]GABA uptake

n

uptake

8 4

35 + 3%* 183 + 7

95 * 9% 125 * 17

5 5 5

49 + 6%t 86 + 8% 162 + 10

98 It 15% 83 + 7% 115 & 8

GAD

ChAT

Protein

81 f 15% 361 * 90

10.5& 15% 513 t 93

88 k 3% 1.00 * 0.07

III &9% 101 * 11% 568 + 103

1.53+ 24%:: 112 + 16% 557 + 85

86 k 12%II 79 + l2%11 1.29 f 0.10

For iesioned animals, values are given as per cent of simultaneously processed sham operated controls (calculated for each experiment). For sham operated animals, values represent nmol/min/g protein or pg protein/PI homogenate. All values are expressed as mean & s.e.m. of N samples from 4 (bilateral) or 5 (unilateral) lesioned and control animals. Statistical significance was assessed by the Wilcoxon signed-rank test when comparing right and left side in the same animals and with the rank-sum test when comparing lesioned and control animals. For differences not indicated, P > 0.1. * Lower than sham P = 0.002; lower than unilateral, lesioned side P = 0.015. t Lower than sham P = 0.004; lower than contralateral P = 0.031. $ Higher than sham P = 0.028; higher than contralateral P = 0.031. j/Lower than sham P < 0.05.

with Na,S04 added and fixed for 5 min in Kodafix diluted with 9 parts of H,O. Then the slices were washed in (NH&SO, solutions of decreasing concent~tion from 1500 to 500 mOsM and mounted in ‘glycerine jelly’. RESULTS Biochemical

&ects of axotomy

In initial experiments the corticopontine pathway was severed by a hemitransection of the brain at the level of the mammillary body by means of a spatula. The uptake of I.,-Glu in the pontine nuclei samples on the operated side was reduced to 40-70x of unoperated controls (average 56x, 5 animals) at survival times ranging between 5 and 13 days. There was no significant reduction in GABA uptake (average 107%). In order to cut the descending fibres more selectively, the crus cerebri was transected by means of the stereotaxically operated retractable wire-knife at the level of the rostra1 end of the substantia nigra (Fig. 1). At 5 days survival time a highly significant reduction in Glu uptake was obtained on the operated side (Tables 1 and 2). The uptake of L-Glu showed slightly reduced vaiues also in the pontine nuclei contralateral to the lesion (P = 0.06 with the Wilcoxon signed-rank test). The per cent reduction in Glu uptake observed at 7 and 11 days survival time was slightly less than at 5 days. There was no significant change in GABA uptake or GAD, but there was a signi~~ant increase

in the activity of ChAT. The concentrations of protein in the homogenates were slightly lower in the operated animals, possibly because of some loss of tissue substance and oedema. A series of animals with bilateral lesions were prepared to investigate whether part of the residual Glu uptake could be due to the small contingent of crossed corticopontine fibresz3 Indeed, the uptake of L-Glu was signi~cantly more reduced in these animals than in the animals with unilateral lesions (Tables 1 and 2). For GABA uptake, GAD and protein, the values were similar to those in animals with unilateral lesions, but there was no increase in ChAT activity. The uptake mechanisms of L-Glu and ~-Asp behave very similarly biochemically4 and have been found to be reduced to the same extent after transection of fibres that have Glu uptake capacity.37 However, when the uptake activities for ~-[~HlAsp and L-[‘~C]G~U were compared in double labelling experiments in the pontine nuclei, all experiments showed a greater relative reduction in the uptake of ~-Asp than in that of L-Glu (Table 2) after transection of the corticopontine fibres (P = 0%X% for pooled results of unilateral and bilateral lesions). In some of the animals the uptake of L-[~H]GIu was measured separately in the same homogenates. This showed a percentage reduction very similar to that in ~-[r~C]Glu and smaller than that in ~-[~HlAsp. -

Fig. 3. Autoradiograms of slices of mouse pons after incubation with ‘H-labelled amino acids and fixation in glutaraldehyde. A. Reduction of D-[~HJAsP uptake in the ventromedial parts (*) of nuclei pontis (np) on the right side 5 days after a lesion restricted to the medial parts of right crus cerebri in the rostra1 mesencephalon. Macroscopically, the lesion was estimated to comprise about 50% of the crus cerebri. B. Distribution of uptake sites for n-E3H]Asp in the ports of a normal mouse. Note the darkness of the np. C. Uptake of C3H]GABA in pons of a normal mouse. Note that the np are less heavily labelled than the more dorsally-located grey areas. (The transverse pontine fibres are partly torn off in this preparation, which is from a level slightly rostra1 to A & B.) Abbreviations: see Fig. 2. Scale bars

45-l

W. Thangnipon

Table 2, Comparison

of the uptake

of aspartate

and glutamate

~‘r (~1.

in the pontine

nuclei after transection

of the crus cerrbrl

in

rat ~--~_~

Operation

II-[“HI Asp

uptake

L-[‘~C]GIU

uptake

Sham

Denervated

1.1 I + 0.07 (4) 1.05 f 0.07 (5)

Bilateral Unilateral

‘?, Reduction

0.79 k 0.04 (8)* 0.85 f 0.05 (5)1

28 19

“<, Remaining uptake D-[‘H] Asp I-[‘4C]Glu 25 + I (8)* 45 f 3(5)q

35 + I (8)*: 55 F 5(5It

_ _____

activities L-[‘H]

Glu

37 + I (8)*: 57 * 3(J),

The uptake ratios were calculated from the molar uptake rates in double labelling experiments. The uptake activities remaining after lesions are based on protein and expressed as per cent of activities in sham operated controls. Values are given as mean k s.e.m. of(n) samples from 4 (bilateral) or 5 (unilateral) lesioned and control animals (not the same as for Table 1). Survival time was 5 days, except for 2 animals with unilateral lesions which survived for 7 days. Statistical significance was assessed with the Wilcoxon rank-sum test. * Lower than sham P < 0.004. t Lower than sham P < 0.05. $ Less reduced than II-[“H] Asp, P = 0.004 (P Q 0.001 for pooled results of bilateral and unilateral lesions). 11 Less reduced than after bilateral lesions P < 0.006.

Autoradiography Since

the uptake

of ~-Asp

was highly

localized

in

~-Asp is less quickly metabolized than L-Glu, ~-[~HlAsp was selected for autoradiographic studies. In normal animals the pontine nuclei stood out as very heavily labelled compared to most other structures in the brain stem (Figs 3A, B). In animals with lesion of the crus cerebri there was a conspicuous reduction in labelling intensity in the pontine nuclei ipsilateral to the lesion (Fig. 3A). Autoradiograms of slices from normal animals incubated with ~-Asp contained clusters of autoradiographic grains suggestive of nerve endings. Slices from normal animals incubated with C3H]GABA showed a different autoradiographic pattern. Thus, the pontine nuclei were relatively less strongly labelled and the raphe nuclei more strongly labelled (Fig. 3C). the

corticopontine

fibres,

and

since

DISCUSSION

Uptake of glutamate and aspartate The biochemical results demonstrate that fibres descending in the crus cerebri are responsible for most of the high affinity uptake of L-Glu and ~-Asp in homogenates of the pontine nuclear complex. The loss of uptake of ~-Asp after transection of crus cerebri was also visualized autoradiographically in slices. The localization of autoradiographic grains suggested the presence of the uptake sites in boutons. Previous studies in other regions lo have shown that particles responsible for high affinity uptake of ~-Asp in homogenates sediment in the nerve ending fraction, in normal material as well as in material with loss of uptake after axotomy. Furthermore, analysis of electron-microscopic autoradiograms of slices incubated with L-[~H]G~u in conditions similar to those in the present study have suggested that nerve endings and unmyelinated axons contain some 80% of the L-Glu

taken up in the slices. 3h We therefore conclude that the uptake of L-Glu and ~-Asp observed in the present experiments represents uptake into nerve endings and (fragments of) preterminal axons. In addition, in separate experiments” we have found that ~-[~HlAsp taken up into slices of the pontine nuclei can be released in a Ca’+-dependent manner on K’-induced depolarization, and that the structures responsible for such release are mainly those which degenerate after transection of the crus cerebri. Anatomical data1’3.25 imply that the pontine afferents descending via the crus cerebri are essentially the fibres of the corticopontine pathway, and that these afferents constitute the greater part of the afferents of the pontine nuclei. It therefore seems likely that the loss of uptake observed after transection of the crus cerebri represents loss of uptake in the degenerating corticopontine pathway. Although this pathway is mainly uncrossed, there is anatomical evidence for a small crossed contingent in the rat.‘” The slight reduction in L-Glu and ~-Asp uptake observed in the present study in the pontine nuclei contralateral to a crus cerebri transection and the greater reduction in the uptake activities in bilateral than in unilateral lesions are consistent with the existence of a minor contingent of crossed corticopontine fibres. Furthermore, sprouting of the remaining fibres might occur, as has been observed after neonatal cerebellar lesions.” The residual activity of acidic amino acid uptake in the pontine nuclei after complete bilateral transection of the corticopontine fibres could be due. in part, to other afferents, e.g. from the central cerebellar nuclei.” In addition, intrinsic neurons, or local collaterals of pontocerebellar fibres, might contain acidic amino acid uptake activity. Axons of any of these neurons could proliferate in the denervated pontine nuclei, Reactive proliferation may start already after a few days (see e.g. Cotman & Nadler4) and might also in part explain the tendency for less reduction in uptake activity at survival times longer than 5 days. Increased uptake activity in intact nerve terminals

Glutamate and aspartate uptake in the pons

would be indistinguishable from sprouting in the present experiments. It is also possible that some of the residual activity could be due to uptake into glial elements. Glial proliferation probably occurs in the denervated pontine nuclei, and there is ample evidence that astroglia can accumulate acidic amino acids.‘2,‘4*36 In the conditions used, glial uptake is very low compared to uptake in nerve endings and axons in regions with high overall uptake activity, such as the cerebral cortex and hippocampus, 36,44 but it might be quantitatively more important in the pontine nuclei, where the overall uptake activity is relatively low. Several transmitter markers, such as GAD and ChAT, are also relatively low in the pontine nuclei, at least partly due to the dilution of the neuronal elements by myelin. (The high content of myelin is evident when the pontine neuropil is compared with the neuropil in other regions, in sections visualizing myelin.16) The larger percentage reduction in ~-Asp than in L-Glu uptake after degeneration of the corticopontine fibres is the first evidence that brain tissue elements with acidic amino acid uptake activity may differ in their relative uptake rates for Asp and Glu. Previously it has been found that the uptake of ~-Asp” and ~-Asp~ is more influenced by Li+ ions than is the uptake of L-Glu. It is worth noting that in pons a comparatively high concentration of Asp has been found in synaptosome and synaptic vesicle fractions.” Recently, rods in goldfish retina have been found to accumulate ~-Asp.~~”

L-Glu in preference

to

L-

or

455

there is little evidence for the existence of shortaxoned neurons3*28 or recurrent inhibition’ in the pontine nuclei, terminals with elongated vesicles and symmetrical synaptic thickenings have been observed.24 Choline acetyltransferase

An interesting incidental observation was that ChAT activity increased after unilateral lesions. This may be a sign of denervation-induced sprouting of cholinergic nerve endings, as observed in other regions.4 Surprisingly, the increase was not seen after bilateral lesions, possibly indicating that the sprouting is directed towards compensating for asymmetric activation of the pontine nuclei. Conclusions

The present study shows that the uptake activity for acidic amino acids is highly concentrated in the afferents from the cerebral cortex to the pontine nuclei. Together with the finding that the accumulated amino acid can be released on stimulation in a Ca’+dependent manner,41 this is partial evidence that the corticopontine neurons may use Glu and/or Asp as their transmitter(s). As indicated in the introduction, this may be a general property of corticofugal fibres. After the present results were obtained, a loss of 20% in high affinity uptake of Glu has been reported46s47 in punches of the ventral pons after pericruciate cortical lesions in the cat. Concomitantly, 20-60x losses of Glu uptake occurred in several other targets of corticofugal fibres. Acknowledgements-We

Uptake of y-aminobutyrate

From the present results it is safe to conclude that most of the GABA uptake and GAD activity in the pontine nuclear complex resides in structures other than the afferents descending in the crus cerebri. These activities could be due to GABA neurons projecting to the pontine nuclei from elsewhere, or to the presence of local inhibitory GABA neurons. Although

are grateful to T. Eriksen for making the wire-knife designed for our stereotaxic instrument. We would also like to thank S. P. Grossman for supplying an unpublished description of his knife and G. Paxinos for a sample of a handoperated wire-knife. During this study W. Thangnipon was a research fellow of the Norwegian Agency for International Development, who in addition gave travelling support. Supported also by the Norwegian Council for Science and the Humanities.

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