Axonal transport of [3H]proteins in a noradrenergic system of the rat brain

Brain Research, 150 (1978) 55-68 © Elsevier/North-HollandBiomedicalPress

55

AXONAL TRANSPORT OF [aH]PROTEINS IN A NORADRENERGIC SYSTEM OF THE RAT BRAIN

BARRY E. LEVIN* FA Hospital, East Orange, and the Department of Neurosciences, College of Medicine and Dentistry of New Jersey, Newark, N. J. (U.S.A.)

(Accepted November 3rd, 1977)

SUMMARY Proteins synthesized from [3H]leucine injected into the rat nucleus locus coeruleus (LC) were transported through the hypothalamus in 5 successive waves at rates of 72-192, 24-48, 13-20, 3-4 and 1.4-2.9 mm/day (waves 1 through V, respectively). Waves I through IV began axoplasmic transport in the LC within the first few hours after [3H]protein synthesis began in the LC. Wave V was delayed in onset for 1.7-3.7 days and was also probably transported through the contralateral hypothalamus. Wave IV was not transported within the LC-hypothalamic axons ascending through the dorsal noradrenergic bundle since its transport was not blocked by 6OHDA lesions in this bundle, as was transport of the other 4 waves. Unilateral dorsal bundle lesions caused a well defined caudal backup of dopamine-fl-hydroxylase immunofluorescence and a fall in dopamine-fl-hydroxylase activity in the ipsilateral frontal cortex and hypothalamus of 55 70 and 9 ~, respectively. Bilateral lesions caused only a significantly further reduction in hypothalamic levels indicating crossed innervation of the hypothalamus by the LC of 27 70- Waves I and II have been classified as rapid transport and contained 33 ~ of the transported [all]protein. Wave V was slowly transported and contained 51 ~ of the transported [all]protein, while wave III was intermediate in rate and contained 16 700of transported [all]proteins.

INTRODUCTION The axoplasmic transport (AT) of proteins in the central nervous system has been studied in various experimental models and neuronal systems. Transport has generally been classified into slow and fast components but the number of waves * Requests for reprints should be sent to the author at the NeurologyService, VA Hospital, East Orange, NJ 07019

56 found to be transported has varied from two .9,t~,zl,:3.~-~" to three 7,`~2 to four :~. Ratc~ of fast transport vary from 96 to 500 mm/day 9,11,~l~22,3,~.~2.:1~. An 'intermediate" rate of 6 to 80 mm/dayT,11,t3,"2, ~,~,4~ is also reported in some systems. A majority of these studies have been performed in the optic system of various species where the eye is injected with radiolabeled amino acid precursors 7,21,~'=',:3"~,42,a~;. The present study utilizes the primarily uncrossed noradrenergic pathway whose cell bodies lie in tile nucleus locus coeruleus (LC) and whose axons travel rostrally through the dorsal noradrenergic bundle (DB) to the median forebrain bundle in the hypothalamus, and terminate primarily in diffuse cortical projections ~°,~:~,:~4.'~8,4:3,~v. This model has the advantage of allowing determinations of AT rates independent of precursor uptake and synthesis rates since transport rates are calculated on the basis of events occurring after the onset of transport 28,29. It can be lesioned specifically with 6-hydroxydopamine (6-OHDA) 4,5,47,4s to exclude extra neuronal transport and the pathways can be traced by immunofluorescent techniques utilizing specific antibodies to dopamine-fi-hydroxylase ( D B H : EC 1.14.2.2), a marker enzyme for noradrenergic neurons and cell processes in the brain~6, ~v. METHODS

Animals Male Sprague-Dawley rats were housed 4 to 6 per cage from the age of 30 days until use at 60 to 70 days of age (200-300 g) in a room at 20 °C with t2 h light-dark cycles.

Injection procedure Animals were placed in a K o p f stereotaxic frame, by the method of Koenig and Klippet z6, under light ether anesthesia, and injected with I #1 L-leucine [3,4,5-

MFB

I A7

I A5

DB

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Fig. I. Dissection planes and lesion sites. This represents a sagittal section of the rat brain showing the course of the noradrenergic pathway from the locus coeruleus (LC) through the posterior (PH) and anterior (AH) hypothalamus. Dotted lines define the planes of dissection of AH and PH sections and vertical bars, the sites of lesioning in the median forebrain bundle (MFB) and dorsal noradrenergic bundle (DB).

57 3H(N)] (New England Nuclear, 79.8 Ci/mmoles, 1/~Ci/#l) into the left LC as previously described2s-3°. The [3H]leucine was buffered, 1 in 20 (v/v), with 0.4 M carbonate buffer, pH 8.6, to a final pH of 7.0. Fig. 1 shows the locations of lesions in the LChypothalamic pathway. Rats were injected with 1 #1 6-OHDA (10 #g/#l in distilled H20 containing 0.2 #g//A ascorbic acid) in the left DB (6.2 mm below skull surface, 1.0 mm lateral to sagittal sinus, 1.8 mm anterior to the intra-aural line: H 6.2, L 1.0, A 1.8) and others were lesioned in the left median forebrain bundle (H 8.3, L 1.5, A 4.0), at various times. Other animals were sham lesioned in the DB with carrier solution (ascorbic acid in distilled H20).

Preparation of tissue Groups of 4-27 animals were killed by cervical dislocation at varying time intervals (1 h-21 days) after [3H]leucine injection. The brains were dissected into left and right LC, anterior (AH) and posterior hypothalamic (PH) sections according to the method of Levin and Stolk29, giving weight agreement of 10 ~ for LC and 1 to 3 for AH and PH sections (Fig. 1). Tissue samples were weighed, homogenized in 5 ml ice cold 10 ~ (w/v) trichloracetic acid (TCA), incubated for 30 min at 4 °C and centrifuged at 1000 × g for 10 min. The precipitate was resuspended in 5 ~o TCA, incubated for 30 min at 4 °C and centrifuged at 1000 × g. The precipitate was dissolved in 0.5 ml tissue solubilizer (Protosol, New England Nuclear) and counted in 10 ml of scintillation mixture (ACS, Amersham/Searle). A 0.3 ml portion of the original acid soluble supernatant was also counted by liquid scintillation counting after addition of 0.05 ml acetic acid. Results are expressed as disint./min of wet weight tissue and AT determined by subtracting the values of the hypothalamic sections contralateral (right) to the LC injection from the ipsilateral (left) sections to control for non-specific accumulation of activity7,28,~9,35. DBH assay and immunofluorescent histochemistry Animals were lesioned unilaterally in the left DB or bilaterally, with 1 #1 6OHDA (10/zg//~l), 14 d prior to sacrifice. Brains were dissected into left and right hypothalamic and frontal cortex sections according to Glowinski and Iversen 15. They were assayed for DBH activity by modification39 of the method of Molinoff et al. 36 using tyramine as a substrate. Final copper concentrations in the reaction mixture were 113 #M for cortex and 96 # M for hypothalamus. Units of activity are given as nmoles of octopamine formed/g of wet weight tissue/h. Sections of midbrain at the level of the DB lesions were cut in coronal section after quick freezing and processed for indirect immunofluorescence for DBH by the method of Hartman et a1.16,17. Rabbit antiserum to purified bovine adrenal DBH, prepared by the method of Foldes et al. 12. gave a single precipitin line on immunoelectrophoresis when run against crude adrenal lysate. Determination of transport rate and time of onset of transport Hypothalamic sections were cut 2 mm in thickness (Fig. 1), giving a theoretical range of 0.5--4 mm that [SH]proteins might travel between the PH and AH. Transport

58 rates were determined by dividing this range of distances by the time required for thc peaks of [3H]protein activity to pass sequentially from the PH to AH. Where peak~ overlapped in time in the PH and AH, the difference between the earliest arrival of [3H]protein activity in the PH and AH was calculated by extrapolation to baseline of slopes derived by linear regression analysis of the increasing [ZH]protein activity in each hypothalamic section. A range of possible times of onset of transport were then calculated by the formula: distance from LC to AH Departure time ~-: arrival time AH . . . . transport rate The range of possible transport rates and onset times was then narrowed by making left DB lesions with 6-OHDA at various times after the injection of [3H]leucine into the left LC. Thus certain theoretical values for onset and transport rate could be discarded when these values fell outside of the range of values possible when the particular waves were blocked or remained unaffected by DB lesions. The timing of these lesions was calculated by the formula: Time of arrival at DB = theoretical departure time +

distance from LC to DB transport rate

RESULTS

Incorporation of [aH]leucine into protein [3H]leucine incorporation begins rapidly after LC injection. Acid soluble counts have already fallen below TCA precipitable counts by 1 h suggesting that incorporation begins well before this time. Fig. 2 illustrates the incorporation and turnover of [3H]leucine and the [aH]protein formed in the left and right LC from 1 h to 21 d. Dissection technique necessarily includes tissues other than the LC so these curves represent a composite of cell types and other tissues. The right LC curve represents [ZH]protein synthesized from [3H]leucine taken up from the blood stream and cerebrospinal fluid after leakage during the injection procedure 19,2°,~9,z°. It differs from the left side only in magnitude, demonstrating that the trauma attendant to injection on the left does not significantly change incorporation or turnover. TCA soluble material disappears rapidly over the first 18 h, (half life of 6.6 h), plateaus from 18 h to 7 days and then falls to zero by day 10. Acid precipitable [3H]protein reaches peak synthesis at 2-4 h and then has a triphasic turnover. An early fall with a half life of 2.3 h occurs from 4-8 h and is followed by a plateau from 8 h to 10 days, after which it again falls from 10 to 21 days with a half life of 2.8 days. Five distinct waves of acid precipitable [3H]protein passed sequentially through the hypothalamus following injection of [3H]leucine into the left LC (Figs. 3 and 4). These are referred to, respectively, as waves I through V as adapted from the nomenclature of Karlsson and Sjostrand 2z. There was no significant transport of acid soluble activity during this 21 d period. The peaks of waves I, II and IV passed sequentially from P H to A H while waves III and V were broader and their peaks overlapped.

59

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Left Locus-TCAsol

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Fig. 2. [3H]leucine turnover and incorporation in the locus coeruleus. Groups of 4--27 animals were

injected with [3H]leucine in the left LC and sacrificed at intervals of 1 h-21 d. The left and right LC areas were dissected and the trichloroacetic acid soluble (TCA-sol) and precipitable (TCA-ppt) activity expressed as DPM/g of wet weight tissue on a semilog scale. Transport of waves I, II, III and V were blocked by 7 0 - 1 0 0 ~ by 6 - O H D A lesions in the ipsilateral DB while sham lesions had no effect (Table I). Dorsal bundle lesions caused no significant change in [aH]leucine incorporation and turnover in the LC, so that the observed changes in transport were not due to decreased [3H]protein synthesis. Wave IV was not blocked by DB lesions but was partially blocked by lesions in the ipsilateral median forebrain bundle, indicating transport through an alternate pathway from the one under study. Dorsal bundle 6 - O H D A lesions placed after [3H]leucine injections allowed a more accurate determination of the ranges for times of onset and rates of transport (Table II). Calculation of rates using an average distance of 2 m m between hypothalamic sections2S, a0 gave respective values of 96, 48, 26, 3-4 and 3 m m / d a y for the

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Fig. 3. Rapid axoplasmic transport of [3H]proteins. Groups of 4-21 animals were injected with [~H]leucine into the left locus coeruleus and sacrificed at intervals of 1-24 h. Right and left hemisections of anterior and posterior hypothalamus were dissected and the trichloracetic acid precipitable Jail]protein determined. Results are given as left minus right sided activity in DPM/g of wet wt. tissue -t- S.E.M. ( ~ ); * P < 0.05, ** P < 0.025, *** P < 0.001 (left vs. right side by paired t test).

5 waves. Dorsal bundle lesions at the times given in Table II gave a third point in time and space for determination of transport rates and onset. These results in Table II were generally in g o o d agreement with the figures calculated f r o m the average intrahypothalamic value o f 2 mm, despite the fact that they allowed a possible range of 0.5--4.0 m m for this distance. Wave IV was calculated using only the 2 m m values since it was n o t blocked by DB lesions. Wave I I I was not blocked by a lesion made 6.5 h after [aH]leucine injection and can be used as an example of how rate and onset were calculated. The onset of wave III was 10.1 h in the P H and 11.9 h in the A H or 1.8 h to transverse a possible distance of 0.5-4.0 mm. This gives a range of possible rates o f 7-53 m m / d a y , onsets o f 0.3-8.3 h and calculated times at the DB lesion (3.3 m m f r o m the L C injection site) of 6.2-9.4 h. The failure o f the D B lesions made at 6.5 h to block transport excludes all rates above 20 and below 13 mm/day. It also demonstrates that functional disconnection of the distal axon f r o m the cell b o d y had no effect on transport o f [3H]proteins in the transected segment. N o n e of these lesions significantly changed [3H]protein turnover in the LC. The 5 waves can, there-

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DAY S Fig. 4. Slow axoplasmic transport of Jail]proteins. Groups of 6-27 animals were injected with [3H]leucine into the left locus coeruleus and sacrificed at intervals of 1-21 days. Hemisections of right and left anterior and posterior hypothalamus were dissected and the trichloraeetic precipitable [SH]protein expressed as left minus right sided activity in DPM/g wet wt. tissue 4- S.E.M. ( ~ ) , * p < 0.05, ** P < 0.01, *** P < 0.001 (left vs. right side by paired t test).

fore, be clearly separated into five separate ranges. Waves I through IV began transport within the first few hours after [3H]leucine injection, during the period of rapid turnover of [3H]proteins in the LC (Fig. 2). Wave V, however, was delayed in onset from 2-4 days, a time when [3H]protein turnover in the LC had plateaued. The more rapidly transported proteins in waves I and II comprised one third of the total [aH]proteins transported within the LC-hypothalamic system (Table III). Slowly transported proteins (wave V) accounted for over half of the total, while the 'intermediate' transport in wave III contained 15 ~. Wave IV was not included since it was not transported in this pathway. Dorsal bundle lesions and brain DBH activity Six hydroxydopamine lesions in the left DB, identical to those which blocked transport, caused a fall in DBH enzyme activity in the ipsilateral frontal cortex of

62 TABLE i Effect o f 6-OHDA lesions on axoplasmic tran.v)ort Control animals were injected with 1/tl [3H]leucine into the left LC and peak values for AT determined for waves I through V (left minus right sides of the AH expressed as disint./min/g wet weight tissue ± S.E.M.). Other animals were injected with 1/fl 6-OHDA (10/~g//fl) or ascorbic acid carricr solution into the left DB or median forebrain bundle (MFB) 1 day prior to [3H]leucine injections. Negative values for left minus right results are given as zero. n : number of animals; P values calculated by Student's t-test for lesioned compared to control groups. Wave

n

Transport

1 Control 6-OHDA (DB) II Control 6-OHDA (DB) Ascorbic acid (DB) III Control 6-OHDA(DB) IV Control 6-OHDA (DB) 6-OHDA (MFB) V Control 6-OHDA (DB)

8 5 10 5 6 21 7 12 11 9 27 6

3033 ± 1052 0 ± 162 2791 ~ 1049 0 ± 617 3219 -~ 1215 3201 5:780 355_~ 355 3645 ± 907 5099 ± 1473 1610 ± 244 10479 3 1641 2744 -I 1200

Pet" cent control

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0.05 0.001 NS

11.1

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140 43.6

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TABLE II Determination o f transport rate and time o f onset Lesions were made in the left DB with 6-OHDA at varying times (hours to days) after the injection of [3H]leucine into the left LC. Animals were sacrificed at times of peak activity of [3H]protein for each wave (left minus right AH activity). Results are expressed as percent of control values (unlesioned) as given in Table I. Times of onset and rates were calculated as described in Methods. Wave IV is estimated only on the basis of transport from PH to AH. Wave

Time o f lesion

n

Percent o f control

onset

Rate (ram~day)

I II III IV V

2.5 h 6-OHDA 4.5 h 6-OHDA 6.5 h 6-OHDA Control 3.75 days 5.0 days

6 6 6 12 10 6

0'** 113.4 89.2 -20.6** 112

1.5-2.9 h 0.5-2.8 h 0.3-2.3 h 5.0-17.0 h 1.7-3.7 d --

72-192 24-48 13-20 3-4 1.4-2.9 --

* P < 0.5, ** P < 0.01, *** P < 0.001 compared to unlesioned controls.

63 TABLE III Per cent o f [3Hjprotein transported in each wave within catecholamine axons

Mean values (disint./min/g wet weight tissue) were obtained for waves I, I1, III, and V in the anterior hypothalamus (left minus right sides) at the peak of each wave. The sum of transport is represented by the total of these values and percent of the total by the fraction each peak value contributes to the total. Wave

Disint./min/g

Per cent o f total

I II III V

3603 3052 3148 10479

17.8 15.0 15.5 51.7

TABLE IV Effect o f dorsal bundle lesions on D B H activity

Animals were lesioned in the dorsal bundle with 1 #1 6-OHDA (10/~g/#l) 14 days prior to assay for D B H (nmole octopamine/g wet weight tissue/h -4- S.E.M.) in the frontal cortex and hypothalamus. n = number of tissue sections assayed. Lesion group

n

Frontal cortex

Per cent control

Unlesioned Unilateral (a) Ipsilaterai (b) Contralateral Bilateral

12

87.5 i 14.0

--

18 18 5

39.5 -4- 6.93*** 88.8 4- 5.94 34.6 -4- 3.42

45.1" 101.5 39.5*

Hypothalamus

195 ± 16.1 177 4- 8.69** 220 -4- 8.51 124.3 -4- 8.81

Per cent control

-90.6 113 63.7*

* P < 0.05 compared to unlesioned controls (Student's t-test for two means); ** P < 0.005, *** P < 0.001 compared to contralateral side (paired t-test).

55 7oo and ipsilateral hypothalamus of 9 ~o at 14 days after lesioning (Table IV). Bilateral DB lesions caused no further change in cortical levels but hypothalamic levels fell by an additional 27 ~o, suggesting that at least some of the LC-hypothalamic fibers cross the midline at a site caudal to the DB lesions. Such lesions caused backup of a discrete area of immunofluorescence to DBH caudal to the lesion (Fig. 5), without evidence of backup in any other ascending catecholamine pathways. There was no evidence of fluorescent backup rostral to the DB lesions. DISCUSSION

This study illustrates several important aspects of protein metabolism and transport within a well defined catecholamine system in the brain. Incorporation of amino acid precursors into brain protein generally has turnover patterns which occur in 2-3 phases, despite variability in route of administrationa,9-11,14, 49, type of anesthetic agent used, subject age and metabolic state 14. Protein turnover and [aH]leucine in-

64

Fig. 5. Backup of dopamine-/3-hydroxylase immunofluorescence after 6-hydroxydopamine lesions. This is a coronal section of the rat midbrain taken just caudal to a 6-hydroxydopamine lesion in the dorsal noradrenergic bundle. The tissue was processed for indirect immunofluorescence to DBH as described in the text and illustrates the pile up of D B H which occurs caudal to the lesion suggesting blockade of orthograde axonal transport. No backup was seen on the rostral side of the lesion. Dilated a n d torturous axonal stumps are shown containing D B H immunofluorescent material.

65 corporation in the LC are similar to those for whole brain. The more rapid, early phase generally has a half-life of a few hours ~,1° to a few days 18 and the rapid fall in [aH] proteins levels can be attributed, in part, to the transport of newly synthesized proteins out of the injection site as occurs here with waves I through IV. This is consistent with the finding that rapidly transported proteins are synthesizing during the first 3 h after precursor injection 35 (Table II). The plateau of [3H]protein activity in the LC from 8 h-10 days does not appear to be changed appreciably by the delayed onset of wave V, and probably reflects local turnover of [all]proteins within several elements of brain tissue within the injection area z,ls since [3H]leucine (TCA-soluble) activity is still detectable during this period. This study presents evidence for at least five well defined waves of [aH]protein transport, four of which migrate within ascending LC-hypothalamic axons lying in the dorsal noradrenergic bundle 47. Transport within noradrenergic axons has been defined here by the use of 6-OHDA, a specific toxin of catecholamine fibers4, 5,47,48. This was the basis for concluding that wave IV was not transported within this pathway. This leaves open the possibility that it could migrate within a parallel, noncatecholamine pathway such as suggested by Jones et al. 2° or a more ventral bundle of noradrenergic fibers arising from the subcoeruleus 37. Transport through the latter pathway would be blocked by a median forebrain but not a dorsal bundle lesion. Dorsal bundle lesions with 6-OHDA also allowed a more accurate assessment of the rates and onsets of transport. This was necessary because small differences in the distance travelled between the two hypothalamic sections could result in considerable differences in rate calculations. The resulting estimates show 5 distinct and separate ranges, with the faster waves generally leaving the LC at about the same time. This suggests that the proteins within each wave are transported by differing mechanisms. It has previously been shown that various waves of transport generally contain differing subcellular constituents. Wave I corresponds to the rates generally accepted for rapidly transported proteins contained in vesicles s,9,25, presynaptic membrane components 7,9,2~,42 and synaptosomal and microsomal particles 2~,45,46. Wave II can also be considered as rapidly transported, despite its slower rate, since it, and wave I, correspond to the rates of two waves of [3H]fucosyl-glycoproteins transported within this same system zs and since glycoproteins are generally considered to migrate only by rapid transport13,23, 5~. The relatively short half-life of waves I and II in the hypothalamus also agrees with the finding that rapidly migrating proteins are transported primarily to the nerve terminals2, 7-9 and only a minority of the LC neurons innervate the hypothalamus as shown by the lesion studies here. Interestingly, wave II also corresponds to the rate at which norepinephrine is transported in this system 29 and supports the possibility that this wave may contain DBH ~a, a vesicle bound1,6, ~6 glycoprotein 5°,51 which appears to be transported together with norepinephrine in peripheral nerves 27. The proximal backup of DBH immunofluorescence and distal fall of DBH activity following 6-OHDA lesions of the DB suggest that DBH does undergo orthograde transport within this system, although no conclusion as to transport rate can be made. Wave V conforms to the generally accepted characteristics of slowly transported

66 proteins. It comprises more than half of the proteins transported within this pathway and its long half-life in the hypothalamus is consistent with deposition of the soluble and particulate axoplasmic constituents carried by slow transportV,"-',='4,'~r','~5,'~%long the nonterminal portion of the axon. The delayed onset of wave V tbr 2-4 days conflicts with previous autoradiographic studies showing onset within the first postinjection day 1°, but the DB lesion studies make it unlikely that onset occurs before 2 days. Wave III does not appear to belong to either group of transport rates. It has no counterpart in the study of rapidly transported glycoproteins in this system 2~ and has a significantly longer half-life in the hypothalamus than waves l and II. It's rate is clearly different from the slowly transported wave V and the small amount of [3H]protein it contains is unlike most slowly transported protein waves. For this reason, wave III is probably classified as intermediate in rate and function 7,1 J, 13,23,:Jr,.4s. The fall in hypothalamic DBH levels which follows DB lesioning supports prior studies showing noradrenergic innervation of the hypothalamus by the LC ~9,41. although studies to the contrary exist 31,32. The present findings give little evidence tbr crossed innervation of the frontal cortex 41 but support a limited degree of crossed innervation of the hypothalamus 19,20. Further support for such crossed hypothalamic innervation comes from the fact that [all] protein levels in the right (contralateral) hypothalamus show a three fold rise above baseline levels during the passage of wave V through the left hypothalamus. This would also suggest that the calculated amount of [all]protein transported in wave V (Table III) is actually an under-estimate since it was calculated on a left minus right basis. The relatively smaller amount of cortical innervation by the LC found here (55-60 ~ ) compared to other studies (70-85 ~ ) is probably attributable to differences in the type and site of pathway lesion 2°,21,4°,41 or the volume of 6-OHDA injected31,aL The completeness of the present lesions is supported by the effective blockade of transport they caused. In conclusion, this study confirms the presence of multiple waves of protein transport in a specific neuronal pathway in the central nervous system 7,~,4~,'~ and supports previous experiments showing two rates of 'rapid' axonal transport as demonstrated by the transport of fucosyl glycoproteins 21,28. ACKNOWLEDGEMENTS This project was supported by VA Grant No. MRIS 5220 (01). REFERENCES l Belmar, J., DePotter, W. P. and De Schaepdryver, A. F., Subcellular distribution of noradrenaline and dopamine-fl-hydroxylase in the hypothalamus of the rat. Evidence for the presence of two populations of noradrenaline storage particles, J. Neurochem., 23 (1974) 607-609. 2 Bennett, G., Di Giamberardino, L., Koenig, H. L. and Droz, B., Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]fucoseand [SH]glucosamine, Brain Research, 60 (1973) 129-146.

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