Life Science" Vol. 15, pp . 223-230Printed in the U.S .A .

INOORPORATION OP 3H-LYSINS INTO A RAPIDLY LABHLLING NEURONAL

PROTEIN FRACTION IN VISUAL CO~FTBX IS SUPPRHSSHD

IN DARL RHARBD RATS

Steven P .R . hose and Arun L. Sinha

Braie Research Group

Biology Department

The Open University

Malton Hall

Miltoa Leynea ML7 6AA

Hngland(Received in final form 12 Jvne 1974)

SUmmaiy

In norwal rat cortex, incorporation of 3H-lysine into protein iehigher is neurone than neuropil at periods up to 2 hra, but ishither in neuropil than neurana at later 'times .

This obeerva-tioa implies the presence of a rapidly labelling neuronalproteia fraction which is aubeequently absent from the neurons,possibly due to transport into other cell compartmeata . Whenrata are reared in the desk, incorporation into neuronalproteins is suppressed in the visual cortex but not in the votorcortex ; this suppression appears to include the rapidly-labelling fraction .

Pergamon Preae

Dart rearing of rate for 7 weeks from birth results in a depressioa of

incorporation of 3H-lysine into protein in the visual cortex neurone

relative to neuropil (1,2) . When the dark-reared rata are brought out into

the light, there is a transient elevation of incorporation into protein is

three stages of the visual pathway, retina, lateral geniculate and visual

cortex (3) . This elevation is not general, but is confined to certain

specific proteia fractions, separable by polyacrylanide gel electrophoresis

(4 ,5) and in the visual cortex appears to be exclusively s aeuroaai pheao~eaon

(2) .

These observations have focussed our att ration on the nature of the

223

224

Neuronal Protein Synthesis, Visual Stimulation

Vol. 15, No . 2

neuronal proteins(s) which change in incorporation rate, and hence probably

in synthetic rate, on dark rearing and during light exposure, and the present

paper reports evidence xhich leads ua to conclude that the neuronal proteins

xhoae synthesis are suppressed during dark rearing form a rapidly labelling

fraction composed of insoluble proteins .

In one aeries of experiments, we have folloxed the incorporation of 3iI~lysine

into protein in vivo into neurons and neuropil isolated from the whole cortex

of normally reared rats as a function of pulse length (6) . We have then

performed a similar experiment using dark reared animals . We have also

compared incorporation into motor and visual cortex neurons and neuropil in

the dark reared animals .

In the first aeries, male Wistar rats, bred in the Department, were raised

from birth on a normal 12 hr light/dark cycle, and xere used xhen their body

weight xas 180-200g .

Animals were injected intraperitoneally xith 50 ~Ci

of lysine-4,5-3H (s .a . 11Cî/m mole ; Radiochemical Centre, Amersham, Sucks)

in 1 .Om1 0 .9°X, saline . At varying times after the injection, the animals

xere tilled, their cortices removed, dissggregated into an ice-cold Picoll

median and subjected to gradient centrifugation for the preparation of

enriched neuronal and neuropil fractiana exactly as described previously

(1,2,7) . Portions of the initial cell suspension and the neuronal and neuro-

pil fractions xere taten for protein deteneination and counting of acid-

precipitable radioactivity (2) . Practions xere prepared from each animal

individually and results expressed as specific radioactivity (dp~/ng protein)

in initial cell suspension, neuronal and neuropil fractions corrected for

body weight . The neuronal/neuropil incorporation ratio xas then calculated

for each animal, and for simplicity, only this index is presented here for

the normal animals .

Under these circumstances, neuronal specific radioactivity xaa higher than

vol . 15, No . 2

Neuronal Protein Spnthesis, visual Stimulation 225

aeuropil at early times (30 minutes - 2 hours) xheo the neuronsl/aeurppil

incorporation ratio xaa around 1 .5 .

Prom this time onxards, hoxever there

xas a fall in the ratio, eo that for pulses beyond 4 bra in durttian neuropil

incorporation xae greater than neuronal, and remained so for up to 8 days ;

over thin period the ratio scarcely changed . Data for 1,4 sad 24 hra are

shorn io Table 1 . These ctlculatione, the data on xhich they are based, and

related observations by other xorkere(8) have led ue to suggest that there is

s rapidly labelling protein fraction is neuronal perikarya from normal

animals, xhich is absent is aeuropil . The subaequeat fate of this fraction,

and evidence for its transport out of the perikaryon, is discussed elsewhere

(9) .

fie had earlier observed that, in dart reared animus and using a 1 hr pulse,

the neuroaal/aeuropil incorporation ratio in visual cortex xaa lar ; only

0 .63 + 0.06 compared to a figure of more than trice thin is matched, normally

reared controls (1,2) . This ratio xta clone to that observed after a 4 hr

pulse in the normal animals . fiaa this because dark rearing suppressed the

rapidly labelling fraction? If so, then prolonging the pulse length is the

dark reared animals should be xithout effect on the ratio. If, oa the other

hand there xae a general auppreeaioa of incorporation into visual cortex

neuronal protein in the dark reared animals, then iacretaing the pulse length

to 4 hra should result in a reduction of the neuronal/neuropil ratio by some

70~ to about 0 .2 .

Accordingly, 50 day old dark reared anisala (for conditiaaa of rearing, see

1-5) xere injected xith 3H-lysine as in the first experiments (except that

the dose xae iacreseed to 250/,�Cî/animal to compensate for the smaller amount

of tissue) . The animals xere returned to individual dark boxes and xere

killed, 1,4 or 24 hours after injection, and neuronal tad aeuropil fractions

prepared from the visual cortex . As before, tissue from etch animal was

fractionated separately . Table 1 shoxa the incorporation ratios obttined for

226

Neuronal Protein Synthesis, Visual Stimulation

Vol. 15, No . 2

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Vol. 15, No . 2

Neuronal Protein Synthesis, Visual Stimulation 227

1,4 and 24 hour pUlaeb in the visual cortex of dark reared animals compared

with values for whole cortex in the normale . It trill be Been that by contrast

with the normale, in the visual cortex of the dark-reared animals there wss no

change in the neuronal/neuropil iacorporatiaa ratio at either 4 or 24`hrs

compared with 1 hr . At 4 hrs, the ratio was 10996 of that at 1 hr, ~fiereaa in

the normals the ratio at 4 hrs was 2696 of that at 1 hr . Ae a consequence of

thin, whereas at 1 hr the incorporation ratio in dark reared animals was only

54~, of that in normally reared animals, by 4 hrs the figures had risen to 22796.

To test whether the depression of incorporation at 1 hr in dark reared anisala

was specific to the visual cortex, the first part of the experiment was re-

peated using rotor cortex ; Tabie 1 shows that, by contrast with the situation

in visual cortex, dart rearing did not result in say depression in the rotor

cortex neuroaal/neuropil incorporation ratio. At 1 hr, the ratio in the motor

cortex of the dark reared animals was 99~ of that in the normale .

(Aa the

data on motor and visual cortex cannot be obtained from the sane or paired

animals is these experiments sad the results are sot 'normalized', only the

ration and not the specific activities are etrictiy comparable between the

brain regions) .

In normal animals, we have unpublished data to show that the rapidly labelling

neuronal protein fraction is amongst the water-insoluble proteins of the cell .

It therefore aemed relevant to see whether in dark rested anisals,the neuronal

protein fraction whose labelling seemed to be suppressed is the visual cortex

was itself soluble or ineoiubie . To follow this, as experiment was carried

out which also had the additional advantage of being able to compare visual

and rotor cortex neuronal and neuropil protein incorporation in the acme

anisai ; that ia, to use each anisai as its own control . Litters containing

6-8 dark reared animale were injected with 3H-lysine as befase and returned

to the dark boxes ; after 1 hr the animals were killed, the visual sad motor

cortices respectively from ail aaisale pooled (to produce sanageable

228 Neumnal Protein Synthesis, Visual Stimulation Vol. 15, No . 2

quantities of protein) and cell separations performed . The cell fractions,

and portions of the starting suspension, were homogenised with large volumes

of distilled water and further centrifuged for 1 hr at 105,000 g. The super-

natants and pellets were collected and protein a:d radioactivity determinations

made on them as before .

Table 2 shows the ratio of visual/motor cortex specific radioactivitiea in

the whole cell fractions and the supernatant and pellet fractions . In the

whole suspension, and both cell types, some 27-37~ of the protein was present

in the supernatant after homogenisation and centrifugation and there were no

differences between visual and motor cortex fn distribution or recovery of

protein . Comparing the specific radioactivities in the whole suspension, it

will be seen that the incorporation into.the pellet fraction wns depressed in

visual compared to motor cortex . ~rnia is compatible with previous observations

that the elevation which occurs in dark reared light exposed animals is riboso-

molly bound (10) . There were no differences in the specific radioactivity of

neuropil or its subfractions between the brain regions, though the pellet was

very wriable . Both neuronal and neuronal pellet specific radioactivity were

much reduced in visual compared with motor cortex . The consequence of these

changes xas that the whole neuronal/neuropil incorporation ratio, 1 .75 + 0 .24

in motor cortex, was only 0 .81 + 0 .12 in the visual cortex (p ~ 0 .01) .

Por

the supernatant fractions, there was no significant difference between visual

and motor incorporation ratios, but in the pellet fractions, the neuronal/

neuropil incorporation ratio fell from 4 .57 + 1 .56 in the motor cortex to

1 .26 + 0 .19 in the visual cortex (p -C 0 .05) .

Vol. 15, No . 2

Neuronal Protein Synthesis, Vioual Stimulation

249

TAHLS 2

Supernatant and pellet specific radioactivitiee in visual

Dark reared rata were injected xith 3H-lysine and after 1 hr neuronal andneuropil fractions separated, fractionated into soluble and insolublecoaponeote and counted ae described in the text . Ratio of visual to motorcortex specific activities is _+ s .e.u . where n ~ 6;

6-8 aaimls from asingle litter were pooled for each experiment . n .e . = not significant .

These results are consistent xith the hypothesis that incorporation into the

rapidly labelling insoluble protein fraction present in cortical neurone in

normally reared animals is specifically suppressed in the visual but not

the rotor cortex of dark reared rate . It is of interest to the interpretation

of this data to note that recently elevations in incorporation of orotic acid

into RNA have been found is the visual cortex of dark reared sad light exposed

rats in as analogous experimental situation to ours (11,12) .

This may

indicate that for the rapidly labelling neuronal protein fraction to be

synthesized, new messenger is required .

and rotor

Practioa

cortex neuronal and neuropil proteins

Specific activity (dpn4/mg psot)visual castex/notor cortex

in dark reared rata

P

Suspension 92 .7+ 8.5 na

Supernatant 96 .3+16.7 na

Pellet 80 .2= 7.7 ,0 .02

Neuropü 95 .0+10.0 ne

Supernatant 85 .2+19 .6 ns

Pellet 146 .8+34 .4 na

Neuronal 60 .0+17.9 < 0 .05

Supernatant 109 .2+30 .1 na

Pellet 43 .0+ 8.1 ~ 0 .001

230

Neuronal Protein Synthesis, Visual Stimulation

Vol. 15, No . 2

Acknarsdgementa

We thank Mra . Layla Sinha for skilled technical help and meabers of the

Brain Research Group for helpful discussions . This research xas supported by

a grant from the Medical Research Council .

References

1 .

SINHA, A.K. and ROSB, S .P .R . Life Sci . 11 (2) 663-668 (1972) .

2 .

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3 .

RICHARDSON, K . and ROSB, S.P .R. Brain Res . 44,

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RICHARDSON, K . and ROSB, S .P .R . J . Neurochem. 21, 521-530 . (1973) .

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ROSS, S .P .R . Biochem . J. 102, 33-43 (1967)

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DROZ, H . and KOBNIG, H.L . in Protein Metabolism of the Nervous System .ed . Lajtha, A . Plenum Press . Nex York . 93-108 .

(1970) .

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ROSH, S .P .R . and SINHA, A.K . .I . Neurochem. In Press .

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DBWAR, A.J . and RBADII~, H.W . Bxp . Neurol .

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