Exogenous monoamines affect the segregation of retinogeniculate fibers in developing rats

DevelopmentalBrain Research, 22 (1985) 135-140 Elsevier

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BRD 60088

Short Communications

Exogenous monoamines affect the segregation of retinogeniculate fibers in developing rats P. W. LAND and L. L. ROSE Centerfor Neuroscience and Department of Anatomy and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 (U.S.A.) (Accepted May 7th, 1985) Key words: retinogeniculate projection - - development - - monoamine - - norepinephrine - - 6-hydroxydopamine

The development of retinogeniculate projections was examined in rats which had norepinephrine levels altered by subcutaneous administration of 6-hydroxydopamine (6-OHDA) or exogenous norepinephrine (NE) during early postnatal life. NE, but not 6-OHDA, treatment resulted in an abnormal segregation of crossed and uncrossed axons at postnatal day 10, such that projections from the two eyes occupied extensively overlapping territory. This effect is at least partially reversible since in animals examined 30 days after cessation of NE treatment the retinogeniculate projections ultimately became segregated. A feature of many regions of the central nervous system (CNS) is that early in development, afferent projections arising from different neuronal populations initially are more diffuse and overlapping than in adults. Axons from different sources segregate from one another only gradually, as the animal matures 16,24,25,31-33. Removing one group of afferents before the final organization is achieved often results in the retention of widespread projections by remaining afferents, indicating that normal segregation patterns are brought about by competition between different groups of axons during development 7,16,26. This developmental process has been most extensively investigated in the visual system, where it has been shown that much of the segregation of inputs from the two eyes occurs prior to eye opening 21.25,31-33. This suggests that factors other than visual experience are important in interaxonal competition. Recently, central noradrenergic mechanisms have been shown to have a modulatory influence on the establishment of the adult patterns of connectivity within the CNS 2,4,12-1a.17. As yet, however, there

is little anatomical evidence for an actual change in axonal projections following alterations in norepinephrine (NE) levels. In the present study, we sought to determine whether manipulating levels of NE might affect the normal segregation of retinal ganglion cell axons from the two eyes in the dorsal lateral geniculate nucleus (dLGN) of pigmented rats. We chose to study the developing retinogeniculate projection for several reasons. First, retinogeniculate axons from the two eyes do not become segregated from one another until 10 days after birth 21. Second, it is possible to demonstrate anatomically the axonal projections from either retina with standard techniques. Third, the rat d L G N is known to receive a rich noradrenergic innervation from the locus coeruleus 15. Finally, it is possible to manipulate CNS levels of N E by subcutaneous injection of pharmacologic agents, since there is no b l o o d - b r a i n barrier to monoamines or their neurotoxic analogues throughout this periodlS, 2s. N E levels in newborn animals were either depleted through the administration of the neurotoxin 6-hydroxydopamine ( 6 - O H D A ) or

Correspondence: P. W. Land, Center for Neuroscience and Department of Anatomy and Cell Biology, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261, U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

137

Fig. 1. Darkfield photomicrographs of HRP reaction product in coronal sections through the dLGN of 10-day-old rats showing the patterns of optic fiber termination. Sections on the right are ipsilateral and sections on the left are contralateral to the injected eye. Sections ascend from rostral to caudal at 160-pm intervals through the middle third of the dLGN. In this portion of the nucleus the extent of the uncrossed projection is largest and the segregation of crossed and uncrossed fibers is most pronounced in normal animals. A projection to the ventral lateral geniculate nuclei can be seen in the extreme ventral and lateral portion of each section, a: normal rat. Note that uncrossed fibers occupy well-delimited foci and that there is a corresponding gap in the crossed projection on the left. b: NE-treated littermate of animal in (a). Note the increased extent and less distinct boundaries of the uncrossed projection in this animal. In addition, labeled axons spread diffusely through much of the ipsilateral dLGN. Note also that the crossed projection is more extensive than normal, as reflected by the smaller gap on the left. Thus there is a greater degree of overlap of crossed and uncrossed projections, c: 6-OHDA-treated littermate of animals in (a) and (b). The extent of the uncrossed projection and the degree of segregation of fibers from the two eyes are not clearly different from normal. The patterns of projection also are within the normal range of variability. Calibration bar, 200pm for a-c.

e l e v a t e d by the a d m i n i s t r a t i o n of e x o g e n o u s N E at a

d u r e p r e v i o u s l y has b e e n s h o w n to s e l e c t i v e l y de-

time w h e n retinal axons f r o m the two eyes are begin-

stroy cortical n o r a d r e n e r g i c axons while l e a v i n g do-

ning to s e g r e g a t e f r o m o n e a n o t h e r in the d L G N .

p a m i n e r g i c fibers intacttL L i t t e r m a t e s of t h e s e ani-

O u r results s h o w that e x o g e n o u s N E alters the segregation of r e t i n o g e n i c u l a t e fibers by a l l o w i n g axons

mals w e r e i n j e c t e d e i t h e r with N E (n = 9) o r v e h i c l e (n = 3; 0 . 5 % a s c o r b a t e in 0 . 5 % saline) or w e r e unin-

f r o m b o t h r e t i n a e to o c c u p y m o r e t h a n the usual vol-

j e c t e d (n = 7). N E - t r e a t e d animals r e c e i v e d s u b c u t a -

u m e of the d L G N . L o n g - E v a n s b l a c k - h o o d e d rats w e r e d e p l e t e d of

n e o u s i n j e c t i o n s of e i t h e r 12.5 or 25 m g / k g N E [ ( - ) - a r t e r e n o l , Sigma; free base in 0 . 5 % a s c o r b a t e ]

N E (n = 6) by 4 s u b c u t a n e o u s i n j e c t i o n s of 6 - O H D A

at 12-h intervals on days 0 - 4 . In o r d e r to slow the de-

(Regis or Sigma; 100 m g / k g free base in 0 . 5 % ascorbic acid) on p o s t n a t a l days 0, 1, 2 and 3. This p r o c e -

g r a d a t i o n of e x o g e n o u s N E , t h e s e animals r e c e i v e d s u b c u t a n e o u s i n j e c t i o n s of the m o n o a m i n e oxidase

138 (MAO) inhibitor iproniazid phosphate (Sigma; 100 mg/kg) 30 rain prior to N E injectionW. A n additional 4 animals received iproniazid phosphate (100 mg/kg) alone on postnatal days 0 - 4 , so that we could assess the effects of briefly raising endogenous N E levels. Subcutaneous injections avoided unintended, direct damage to CNS structures which might be produced by intracranial injections. The effects of these various treatments on the segregation of retinal axons were examined, in most cases, on postnatal day 10, by which time the pattern of segregation normally resembles that of adults. Several animals also were examined at 35 days of age. The right eye of animals in all treatment groups was injected with 1 - 2 ~1 of 30% horseradish peroxidase (HRP) in 2% dimethylsulfoxide (DMSO) on postnatal day 9. The animals were sacrificed the following day (day 10) by perfusion with 0.5% glutaraldehyde in phosphate buffer followed by 2% glutaraldehyde in phosphate buffer. Frozen sections through the d L G N were cut at 40 ~ m and reacted with tetramethylbenzidine22. The 35-day-old animals were similarly prepared after monocular injection of 5 ~tl HRP. Camera iucida drawings of the d L G N and the region occupied by the ipsilateral retinal projection were measured using an Apple II microcomputer, graphics tablet and modified graphics software package. Contralateral projections were not measured because of the difficulty in establishing the boundary of the gap in this projection, especially in the NE-treated animals. The pattern of crossed and uncrossed retinal fiber termination in the d L G N of animals in our control groups was identical to that described in previous reports from this laboratory 21. On day 10, axons from the ipsilateral eye (Fig. la, right) occupy a compact region of the nucleus that has been vacated by the fibers of the contralateral eye. The region containing uncrossed fibers represents an average of 13.8% of the volume of the d L G N (Fig. 2). The absence of a contralateral projection in the regions of ipsilateral fiber termination is reflected by a corresponding 'gap' in the contralateral projection (Fig. la, left). No clear differences in the retinogeniculate projection patterns of the 6 - O H D A - t r e a t e d animals could be discerned (Fig. lc). While the uncrossed projection occasionally appears less dense than in

55

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x mmm "~, ¢L ...J

20-

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15

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5

6-OHDA

CONTROL

NE

Fig. 2. Graph showing the mean volume of dLGN occupied by ipsilaterally projecting fibers in the 3 treatment groups. Bars represent S.E.M. *: the average volume of the dLGN occupied by uncrossed fibers in 6-OHDA-treated animals was not significantly different from the control value. Student's t-test, ts = 0.77, P > 0.05. S.D. for the 6-OHDA data is 1.26. **: S.D. for control animals is 1.80. ***: the average volume of the clLGN occupied by the ipsilateral retinal projection in NE-treated animals is significantly different from the volume occupied in control animals. Student's t-test, t H = 3.59, P < 0.01, S.D. for the NE data is 3.67.

controls, it occupies an average 12.9% of the d L G N , which is not significantly different from the control value (t 8 = 0.77, P > 0.05; Fig. 2). By contrast, extensive alterations in retinal projection patterns were observed in the animals that received the 25 mg/kg dose of NE (Fig. lb). At day 10, the ipsilateral projection in these animals occupies a significantly larger region of the geniculate than it does in control animals, averaging 20,2% of the d L G N (tll = 3.59, P < 0.01; Fig. 2). In addition, contralaterat fibers also are present in the region of ipsilateral fiber termination, resulting in a degree of overlap that is not seen in control animals at this age. For example, in Fig. lb (left), note that there is only a small region devoid of label in the d L G N contralateral to eye in-

139 jection. This small gap occurs only at the position of the densest accumulation of uncrossed fibers (cf. Fig. lb, right). A higher dose of NE (100 mg/kg) produced similar alterations of retinal projection patterns, but often resulted in the death of up to 50% of animals prior to day 10. On the other hand, animals receiving only 12.5 mg/kg NE did not exhibit any clear changes in projection patterns. The retinal projections of the 4 animals that received only the MAO inhibitor also did not differ significantly from normal. This may reflect the relatively low endogenous transmitter content of immature catecholaminergic neurons and terminals during the perinatal period in ratsS.6. Taken together, these data suggest that some threshold increase in NE may be necessary to produce alteration in retinal axon segregation. Interestingly, in the NE-treated animals which were allowed to survive for 35 days, the distribution of crossed and uncrossed retinogeniculate axons appeared essentially normal. This suggests that it may be necessary to chronically treat animals with exogenous NE to prolong the period during which segregation is arrested. Alternatively, NE-treated animals may exhibit some transient retardation of neural development. This in itself would be interesting since we noticed that in these animals eye opening occurred on the same day as in control littermates (day 12). Our results clearly demonstrate that exogenous NE alters the segregation of retinal afferents to the geniculate. We were surprised that this process was not also affected by NE depletion. However, subsequent studies 27 have shown that the 6 - O H D A regimen used in this study, while causing a permanent depletion of cortical NE, produces only a transient disappearance of noradrenergic axons from the dLGN (see also ref. 29). It will thus be necessary to devise other methods for destroying noradrenergic fibers within the thalamus in order to more fully investigate the effects of NE depletion. The present findings differ in an important way from those obtained by directly manipulating the visual system during normal periods of segregation. Early surgical lesions |6,26 and various forms of altered visual experience during a critical period of development 9.20 all result in abnormally extensive connections. However, in these experiments only axons associated with the one intact or non-deprived eye are able to occupy more than the usual amount of termi-

nal space. This occurs at the expense of other axons that normally compete for the same territory. In contrast, our results present a situation in which axons from both eyes are able to occupy more than the usual volume of their postsynaptic target. In some respects, the present data appear to differ from those of Bear and Daniels 3 and Kasamatsu et al. 12-14that indicate that in the absence of NE, cat visual cortical neurons lose their ability to accept novel patterns of functional connections in response to monocular deprivation (MD) during a critical period of development. Connections do not change without NE. Here, normal patterns of axon segregation fail to develop in the presence of exogenous NE. In neither case is the mechanism of action of monoamines well understood. It will be especially important to better understand the anatomical substrate underlying alterations in ocular dominance before direct comparisons can be made. It is possible, nevertheless, to draw an interesting parallel between the results presented here and those reported previously for the visual cortex. Reintroduction of NE into the cortex of 6-OHDA-treated cats restores the sensitivity of cortical neurons to MD ~4. A similar effect is seen if exogenous NE is infused into the cortex of normal cats that are beyond the critical period 13. Thus, NE appears to return the cortex to a relatively immature, more plastic state. In the present study NE maintains an immature configuration of retinal axons. It will be interesting in this regard to determine whether optic fibers also remain more plastic during the period of NE treatment. This could perhaps be addressed by testing whether exogenous NE prolongs the period during which unilateral enucleation results in the formation of abnormal retinal projections 19. At present, we do not know the mechanism by which NE allows the effects we observed. Competition between developing axons can be influenced by a variety of factors, including the availability of trophic substances8 or synaptic sites 30, or by the electrical activity of developing neurons 23. All of these potentially can be influenced by monoamines 1. Furthermore, some of the effects we observed on axon segregation may in some way be due to peripheral action of the exogenously applied amine, such as alterations in blood pressure or heart rate. Biochemical experiments currently are under way to determine the

140 n o r m a l levels of N E in t h e t h a l a m u s a n d t h e d e g r e e

opment

to w h i c h t h e y a r e a l t e r e d b y t h e m a n i p u l a t i o n s u s e d

c h a n g e s in C N S c o n n e c t i v i t y .

can

produce

anatomically

demonstrable

in t h e p r e s e n t i n v e s t i g a t i o n . T h e r e s u l t s o f t h e s e s t u d ies s h o u l d h e l p l e a d to e x p e r i m e n t s u s i n g i n t r a c r a n i a l

We thank Joan Eldridge for technical assistance,

injections of appropriate NE concentrations. Never-

Barbara Bartoldi for typing the manuscript and Larry

theless, o u r f i n d i n g s i n d i c a t e t h a t f a c t o r s w h i c h i n t e r -

Middaugh and Dan Simons for helpful comments.

fere w i t h n o r m a l m o n o a m i n e

Supported by NIH Grant EY05280.

levels d u r i n g d e v e l -

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