Post-mortem high affinity glutamate uptake in human brain

POST-MORTEM HIGH AFFINITY GLUTAMATE UPTAKE IN HUMAN BRAIN R. SC~WRKCY.* and W. 0. *M~~r~land

Psychiatric

University tDivisions Tennessee

Research

of Maryland

School

of Ne~lropatholo~y.

School

of Medicine

Department

and Memphis

Abstract---High-affinity braln

(P2) fractions examined

prepared

of the putative

at autopsy.

under

varying

by electron-microscopy.

equivalent uptake

uptake

tissue collected

measures

from fresh rat brain

sites for ~ILitarn~~te in the human

sonicating

the

P_, suspension

integrity

of brain

changes

in e~p~rin~ental

elements.

prior

Frozen data.

tissue samples

Portions

Accumlilation indicating

prepared

the presence of the amino

tissue

which

of specimens

within

7 h after

kinetic

death.

Regional

distribution

of

uptake

sites

and

with

of sodium-dependent acid

is abolished process

without

has been

IS 2Xh ~(~,\r-i~7(~rf~1~1. display only W’,, of the uptake capacity

were also

were compared

of the

frozen

in

synaptosomal

of the P, fractions features

up to three months

from

was examined

in crude

the depeiiden~~

can be stored

samples

glutamate

and morphologtcal

specimens.

of Hospital.

were determined

tissue. The data demonstrate

to in~ubatioI1.

However.

Administration

neurotransmitter

of tissue storage.

neurochemical

and

Ullivers~ty

U.S.A.

characteristics

conditions

Both

MD

of Pathology.

excitatory

Uptake

of Psychiatry,

Baltimore.

Veterans’

TN.

JK~

De~drtrnen~

of Medicine,

Memphis.

human

WHKTSKL.

Center,

by the

appreciable dissected

collected

analysis

on

and

and frozen

in caudate

tissue

with data obtained in rat brain. The selectivity of the uptake process (non-gtutamatergic drugs do not interfere at I mM) and the absolute and relative potencies of a spectrum of specific and metabohc uptake inhibitors are virtually identical in human caudate and rat striatum. Ultrastructural examination of P, preparations from frozen human caudate tissue demonstrates the presence of synaptosomes (approximately I :3 in density) identical to those observed in fresh rat btriatum. The findings support the view that glutamate uptake measurements in quick-frozen and thawed human brain samples (i) are technically feasible and (2) may provide a tool for the examination of glu~n~ater~i~ rne~h~~nisnls in normal and neur~)p~~thologi~ai states.

(K:,, =

1.1

htM: V,,,, =

114 pmoles/min/mg

protein)

The acidic amino acid glutamate fulfilr many if not all criteria in order to he considered a neurotransmitter in the central nervous system’. Unfortunately, because of its ubiquitous presence and multiple metabolic functions, experiments devised to unravel specific neurotransmitter properties of glutamate have proved to be conceptually difficult and have often yielded equivocal results. The presence, in brain. of a selective uptake system for glutamate’.‘” currently constitutes one of the most important synaptic mechanisms related to its neurt~transmitter function. Therefore, the inactivation process has been scrutinized not only in terms of its involvement in the regulation of glutamatergic neurotransmission” but has also been used as an innovative tool for screening glutamatergic connections in the brain.2’ Very little is known about the neurotransmitter role of glutamate in the human brain---largely due to experimental problems associated with its assessment. Thus, whereas the measurement of biosynthetic enzymes or levels of some neurotransmitters in human body fluids or post-mortem brain tissue may provide valuable information concerning their funcAhhrfriclrioli:

Pz,

crude

are in agreement

tion or dysfunction.‘.~’ such an approach has as yet failed to reveal equally important it~formation on the glutamate system. Such knowledge, on the other hand, appears to be particularly relevant in view of the recently hypothesized involvement of glutamate in the etiology of the human neurodegenerative disease, Huntington’s disease.“,‘” Based on recent work on the remarkable persistence of glutamate uptake sites in rat brain after various storage and freezing and thawing conditions,” the present study was designed to evaluate human post-mortem brain tissue regarding its capabilities to actively and specifically ~~ccurnul~~te g~ut~~m~~te. Both biochemical and llltr~~structural methodoiogies were used and correlations with wellestablished equivalent parameters of fresh rat brain were made throughout the course of the project.

synaptosomal~n~itocl~ondri~~i

sediment. 1771

EXPERIMENTAL

PROCEDURES

Brains were obtained from male patients (age range 51 84~) who had died without history of neurological or psychiatric dlscase. All brain specimens were collected routinely at autopsy 2.5-7 h post-mortem unless noted otherwise (cf. Table 2). Cerebrum. brainstem and cerebellum were removed as a single unit. and the hralnstem and cere-

R. Schwartz and W. 0. Whetsell Jr

1772

bellum separated from the cerebrum by a high rostra1 transection of the midbrain. A single coronal section was then made through the cerebrum from ventral to dorsal at the level of the mammillary bodies. Individual regions were dissected from the fresh specimen as follows: (f) right middle frontaf gyrus (cortex only); (2) right superior parieta1 lobule (cortex only); (3) right caudate nucleus (head removed through right lateral ventricle); (4) right putamen; (5) right globulus pallidus (lateral segment); (6) right anterior thalamus; (7) bilateral anterior hypothalamus; (8) right hippocampal formation (removed from temporal horn of lateral ventricle and excluding hippocampal cortex): (9) bilateral substantia nigra (removed from mad-to”rostral midbrain and dissected away from tegmentum and cerebral peduncles); (10) right cerebellar hemisphere (cortex); and (11) caudal medulla (complete transection). Dissected brain specimens contained at least 100 mg of tissue (wet weight). They were placed in separate f cc plastic vials, labelied and immediately frozen at - 80°C in a Revco freezer. All specimens were thus stored until they were to be used for preparation of tissue homogenates. Tissue preparation and uptake procedure

Individual brain samples were thawed and kept at 4°C. Tissues were then gently homogenized in 20~01. (w/v) 0.32M buffered sucrose solution (pH 7.4) with a Teflonglass homogenizer. The suspension was centrifuged at 1000 x g for 10min to remove nuciei and cell fragments, the supernatant carefully decanted into new tubes and centrifuged for 30min at 20,000 x 9. The resulting P2 pellet was gently rehomogenized in the original volume of buffered sucrose (see above) and 25 ~1 of the suspension, containing approx. 3Ogg protein, used for the following uptake experiment. Routinely, the tissue was incubated in 1 ml Krebs buffer (120 mM NaCI, 25 mM NaHCO,, 5 mM KCI, 2 mM CaCI,, 1 mM KHZPOL, 1mM MgS04 and 10% o-glucose), pH 7.4, for 4 min at 25°C in the presence of 3 x lo-’ M glutamate (approx. 0.3 $_?i [3H]glutamatel. For kinetic analyses, the concentrations used were 0.05,O.I, 0.3, 0.5, 1 and 3pM. Medium in which equimolar choline chloride was substituted for sodium chloride was used to measure non-specific uptake which served as blanks. For the computation of all kinetic, pharmacological and other data, calculations were made after deduction of these sodium-substituted blanks. For assessment of the pharmacological properties of the uptake process. 10~1 of drug solutions containing specific or metabolic inhibitors were added to the incubation mixture. Accumulation of glutamate was terminated by placing the incubation tubes in an ice-water bath and subsequent centrifugation at 20,000 x g for 10min. The pellets were washed twice with 3 ml cold sucrose, solubilized in 0.5 ml Protosol (New England Nuclear (NEN), Boston, Mass.) and the radioactivity determined by liquid scintillation spectrometry. In one experiment, P, fractions underwent sonication (20 KHz; 30 s) prior to uptake measurement. Glutamate uptake measurements in fresh rat striata using 0.05 &i [3H]glutamate; see Schwarczis) were performed in an analogous fashion for comparative purposes. In all experiments, 25 pl aliquots of the Pz suspension were removed and processed for protein determination according to the method of Lowry. Rosebrough, Farr & Randall.“’ Eleotrort

microscopic

undysis

1 ml of each of the resuspended P,-peilets (see above) of

two caudate preparations was transferred to new tubes and centrifuged at 4°C at 20,000 x 9 for IOmin. The aupernatant was discarded, replaced by 2 ml 2’?;, phosphate buffered glutaraldehyde and the tubes kept at 4 C. After 2 h, the tixative was removed, the ttssue rinsed with buffer. diced into small pieces and post-fixed in I”,, osmium in phosphate buffer at 4°C for 1 h. Following post-fixation. the tissue was dehydrated in graded alcohols and propylene oxide and embedded in Embed-812 resin, each piece an individual block. Finally, thick (0.5 p) and thin (5OOA) sections were prepared on a Sorvall MT-5000 microtome and observed by light- and electron-microscopy. Two specimens of different fresh rat striata were prepared in a manner identical to the procedure described for human material. Materiuls

[3H]glutamic acid (40 Ci:mmol) was purchased from NEN. Three-3-hydroxy-DL-aspar~ic acid and ui-aspartate~-hydroxamic acid were kindly supplied by Dr P. J. Roberts, Southampton. England. All other chemtcals were obtained from Sigma Chemical Co.. St. Louis. MO. RESULTS ~~d~~~-de~e~dent

~~tuke:

rqqionul

distributing

nnd

kinetic characteristics

P, preparations isolated from quick-frozen and thawed human brain could accumulate glutamate in a

fashion reminiscent of animal data. Approximately l-5nCi [3H)glutamate were accumulated per tube under our assay conditions. The process was linear up to at least five minutes and Na+-dependent since S&90% of the uptake were lost in sodium-free medium (data not shown). Sonication of the suspension prior to incubation resulted in a virtual abolition of Na+-dependent uptake (Fig. 2), indicating that structuraf integrity of the isolated nerve endings is essential for the observed process to take place. Whereas no age-related differences could be detected in the course of our study, there existed pronounced regional variation: uptake in the hippocampal formaTable 1. Regional distribution of high afftnity glutamate uptake in human brain

Brain region Hippocampus Parietal Cortex Thalamus Frontal Cortex Cerebellum Putamen Caudate Nucleus Hypothalamus Globus Pallidus Substantia Nigra Medulla

Uptake (pmoles/min/mg protein) 60.5 + 5.5 53.0 _t 7.8 44.5 4 12.0 40.2 + 4.8 40.0 1 5.2 37.8 1: 3.5 37.0 + 5.5 20.2 t 2.8 15.0 z 1.5 13.5 & 1.2 8.8 f 1.0

Values are the mean + S.E.M. of 7-.9 tissue arations. Uptake was determined in P, suspensions final glutamate concentration of 3 x l-0. ’ kf. The was incubated in Krebs-phosphate buffer. pH 7.4, for at 25°C as detailed in Experimental Procedures.

prepat a tissue 4 min

Post-mortem

glutamate

uptake

in human

1773

brain

which was dissected and frozen 15-28 h post-mortem displayed only 65% of the uptake capacity of specimens collected and processed within 7 h after death. PharmacoloyJ

vsx 10.’ M Fig. I. Double reciprocal plot of high affinity glutamate uptake into P, fractions of thawed human caudate specimens. Individual points represent specific uptake after subtraction of no sodium blanks and are the mean _t S.E.M. of individual experiments on five different brains. Velocity (v) IS expressed as pmoles!min:mg protein.

tion and three cortical areas was 5-6 times higher than in the least ‘efficient’ brain regions, namely the globus pallidus, substantia nigra and medulla. Caudate, putamen, thalamus and cerebellum displayed intermediate values (Table 1). Given these regional differences it appears noteworthy that the standard errors for each brain area were reasonably small, reflecting high intersubject reproducibility. A separate study on glutamate uptake in 20 consecutive 1 mm thick coronal slices of caudate tissue confirmed this notion: the pattern of glutamate accumulation was remarkably consistent between the four caudates examined, with anterior parts displaying slightly higher values than slices from more posterior sections (data not shown). Lineweaver-Burk plots indicated a single high-affinity site with an apparent K, of 1.1 PM and an apparent V,,,,, of 114 pmoles/mg protein/min (Fig. 1). StOt'fUJt.of

The selectivity for glutamate of the sodium-dependent uptake process was assessed by using established specific and metabolic inhibitory agents. As shown in Fig. 2 in a comparative fashion, the stereoselectivity of glutamate and the absolute and relative potencies of D- and L-glutamate, threo-3-hydroxy-DL-aspartate, DL-aspartate-j-hydroxamate L-aspartate at and 10e5 M were virtually identical in human caudates and fresh rat striatum. The same held true for a series of other structurally related glutamate analogs or metabolic inhibitors tested at 10e3 M (Table 3). Importantly, a spectrum of neuroactive substances not related to glutamatergic function could be demonstrated to be devoid of any blocking activity at 1 mM (see legend to Table 3). Ultrastructural

analysis

Electron-microscopic study of P2 pellets from two different human caudate preparations showed fields packed with membranes, some forming empty rings and some encircling aggregates of flocculent material,

tissue

Specimens of frontal cortex and putamen could be stored at -80°C for up to 3 months without any appreciable loss in glutamate uptake capacity. Storage of samples of the same brains for another 3 months. however. resulted in a 4(tSO”A decrease in uptake values in both brain regions (Table 2). As shown in Table 2. P, fractions prepared from tissue

Table 2. Effects of dissection

Frontal cortex Putamen

Fig. 2. EtTects of sonication and of inhibition by specific uptake blockers (lo-’ M) of glutamate uptake. Hatched bars: thawed human caudate. Open bars: fresh rat striaturn. S.E.M. is indicated on each bar. Aj?H: DL-aspartate-/jhydroxamate; THA. threo-3-hydroxy-oL-aspartate; GLU: glutamate: ASP: aspartate. Number of independent experiments is indicated under each bar.

and storage

intervals

on experimental

data

< 10 days

“/,

< 10 days*

p;

3 months

“4

6 months

“/;,

40.2 f 4.8

100

26.3 i 2.5

65

45.0 + 1.8

112

23.5 + 2.0

58

37.8 + 3.5

100

24.5 + 3.8

65

35.5 + 2.5

94

18.5 f

49

I.8

Data are expressed in pmoles/min/mg protein and represent the mean + S.E.M. of 7 individual brain samples. Percentages refer to the standard values in the first vertical column. Assays were performed at the post-mortem times indicated. Brains were routinely collected, dissected and frozen within 7 h after death. * Brains were collected. dissected and frozen within 15-28 h after death.

Table 3. Inhibition of glutamate uptake m P2 fractions prepared from thawed human caudate and fresh rat striatum on Inhibition Rat Human caudate striatum Competitive inhibitors Cysteine sulfinic acid u-aspartate Kainic acid DL-homocysteic acid Glutamine Metabolic inhibitors Mersalyl acid Carbonylcyanide-mchlorophenylhydrazone Ouabain

+ I i I &5 + 5 f 4

100-t I look I 82 i I

87 ) 4

97 rt 3

90 + 4 8i2

76 i 3 20 + 5

98 76 64 30 29

16 F 2 27 _+4

Data represent the mean + S.E.M. of at least three separate tissue preparations. All blocking agents were used at IO-‘M. The following compounds did not display any significant inhibitory potency at IO’ ‘M in either tissue preparation: dopamine. choline, GABA, serotonin. glycine. taurine. norepinephrine. 6-aminolevulinic acid. pyroglutamic acid.

groups of vesicles and occasional mitochondrial fragments (Fig. 3A). In this cellular debris were scattered distinct synaptosomes (Figs 3A, B, C) which could be identified by their characteristic morphology consisting of the presynaptic element with its aggregate of small rounded or ovoid clear vesicles, a presynaptic membrane, a synaptic cleft, a post-synaptic membrane and the post-synaptic thickening. Synaptosomes were not abundant in this material. An estimate of the frequency of their occurrence was obtained by analyzing 60 electron-micrographs from each specimen: 30 at !S,OOO x magnification and 30 at 35,COOx magnifcation. Similar analysis of two fresh rat brain striata indicated that there was a 2-3 fold greater frequency of synaptosomes in the fresh rat brain homogenates than in the quick-frozen and thawed human tissue preparations. There also appeared to be a greater incidence of membrane rings containing aggregates of clear round or ovoid vesicles in the rat specimens. Otherwise the appearance of the electron-microscopic fields was identical to those of the human caudate specimens (Figs 3 and 4). DISCUSSION The uptake process

The present study clearly demonstrates the persistence after freezing and thawing of sodium-dependent high-affinity glutamate uptake in human brain tissue. All features investigated indicate that the active accumulation of glutamate described here displays qualitative and quantitative properties known from fresh’v3 and frozen’s rat brain. Thus, sodiumdependency, inhibition by a spectrum of specific uptake blockers or metabolic inhibitors and kinetic analysis are all very similar or identical to data obtained in the

rat. Notably. cndogenous ncuroactlvc substances which are devoid of inhibitory activity in the rat. also do not appear to block glutamate uptake In human brain. Bectiusc of the anatomical differences between rat and human brain, the regional distribution of uptake sites cannot be compared in a rigorous fashion. Howcvcr. the gross relative rank order IS quite similar hctwccn the two spccics. with cortical areas. slriatum (or- caudate-putamen). hippocampus and cerebellum displaying higher and brain stem (or medulla), hypothalamus and substantia nigra lower glutamate uptake values.” As reflected by the rather large variation In experimental values (Table I). thalamic samples --unlike other brain areas --yielded rather widespread data, perhaps indicating differing densities of glutamatergic innervation to various thalamic nuclei.J.” From our data, an estimate can be made as to the efficiency of the high affinity transport system for glutamate in the brain of adult men. One may assume that the fraction of uptake in human tissue, which survives the freezing and thawing process is similar to the II --25’!, (of uptake into fresh tissue) detectable in frozen and thawed rat brain;18 thus, in the caudate of normal humans. glutamate uptake should be approximately I50 pmolesjmg protein/min (at 3 x IO-’ M extracellular glutamate) and the V,,, 900 pmoles!mg proteinimin. If this calculation is valid, the process may well bc suited for maintaining low levels of extracellular glutamate during synaptic resting periods2’ in spite of the high total concentration of the amino acid in human brain.” By the same token. possible defects in this high-affinity glutamate transport system may result in an cnhanccd (toxic) accumulation of glutamate in the synaptic cleft, which in turn may lead to the degeneration of postsynaptic neurons.5.‘9 Which pwtides

mtr_vuccumultrte glutamate:’

The physical integrity of the brain elements responsible for glutamate accumulation appears to play an essential role in the present study since the uptake process was virtually abolished by prior sonication of the incubation suspension. The same effect of ultrasonic treatment could be observed in rat preparations. This result in both human and rat tissue favors the interpretation that our data does indeed reflect uptake and not binding phenomena. In the past, several studies on post-mortem changes in the morphology of brain tissue have been performed. Distinct ultrastructural alterations in synaptic regions can clearly be observed within minutes after death.‘*,” However, subcellular particles, containing a spectrum of enzymes closely resembling fresh brain in both activity and distribution. can be isolated from rodent” and human10*24 post-mortem brain. Moreover, high affinity uptake processes for choline” and glutamate” remain unchanged in rat brain for hours after death. indicating the functional persistence of

Fig. 3. Homogenate of human caudate. A: Ultrastructural appearance of a portion of human caudate nucleus which was quick-frozen, stored. then thawed before homogenization and fixation. The arrow locates a synaptosome in the field. x 18.600. B. C: details of synaptosomes found scattered in this material. x 35,650. 1775

Fig. 4. Homogenate of rat striatum. A: Ultrastructural appearance of a portion of rat striatum whrch was homogenized in the fresh state before fixation. The arrows locate sy~~pt~somes in the field. x 18.400: B. C: details of synwptosomes in this material. x 35.650.

t 776

Post-mortem

the elements capable of accumulating

1777

glutamate uptake in human brain

the neurotrans-

mitter. Only

one brief mention could be found in the literature on the effect of freezing on the ultrastructural organization of human brain tissue. Freezing at - 196°C and thawing was reported to ‘highly disrupt nerve endings’.2 5 Regrettably, no accompanying micrograph was shown in that paper. Basically, our present electron-microscopic analysis conforms with the assessment of Weiner et nl,: Pt fractions are indeed severely disrupted by the freezing (at -8OC) and thawing process, but intact synaptosomes, virtually indistinguishable in appearance from those seen in fresh rat brain, can be observed in every single section. Thus, it appears plausible that these surviving organelles accumulate radiolabeiled neurotransmitters. However, contribution of glia to the uptake measured in thawed human brain specimens has to be considered: glial cells are known actively to take up glutamate from extracellular space and the pharmacological profiles of glial and neuronal transport processes are qualitatively similar.16 On the other hand, our previous work conducted on frozen and thawed rat tissue, employing quantitative pharmacological measures as well as kainate lesions,‘* suggests only negligible glial participation in the uptake process described in this study.

As shown here, glutamate uptake measurements can be performed in human post-mortem tissue, thus adding an important parameter to the battery of neurochemical measures (enzymes, neurotransmitters, neurotransmitter metabolites, receptors) already accessible to detailed scrutiny of other neurotransmitter

systems. ‘foci Brain specimens can remain in the frozen state for up to three months without appreciable deterioration of uptake capacity. However, the brains ought to be collected, dissected and frozen within approximately seven hours post-mortem since extended storage prior to freezing appears to result in a pronounced decrease in experimental values (Table 2). The procedure described here should in most cases provide a sufficient time span for the collection and, if necessary, shipment of appropriate tissue samples. Based on the present study one can therefore anticipate an increased interest in several aspects of the glutamate transport process (see Introduction) including its possible involvement in neuropsychiatric disease states. l g Finally, it appears that similar studies may be performed in the future on uptake of ~-aminobutyrate in post-mortem human brain. Preliminary data indicate2$ that s~ium-de~ndent ~-aminobutyrate transport can be demonstrated in human samples in much the same fashion as is known from animal experiments.’ In contrast, monoaminergic nerve terminals appear to be far more vulnerable to freezing and thawing as assessed in human*’ and rat (R. Schwartz, unpublished observation) brain samples. These transport processes can hardly be detected after initial exposure of the tissue to low temperature and therefore -unlike amino acid uptake sites-do not appear to lend themselves to extensive analyses under such strenuous experimental

conditions.

Acknowiedgemenrs-We gratefully acknowledge the expert technical assjstance of G. B. Saylor. This work was supported by USPHS-grant NS 16941 and a grant (to R.S.) from the Wills Foundation,

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