Neonatal enucleations reduce number, size, and acetylcholinesterase histochemical staining of neurons in the dorsal lateral geniculate nucleus of developing rats

Developmental Brain Research, 47 (1989) 209-225

209

Elsevier BRD 50915

Neonatal enucleations reduce number, size, and acetylcholinesterase histochemical staining of neurons in the dorsal lateral geniculate nucleus of developing rats Richard T. Robertson 1, Henry K.

Poon 1, M. Rebecca Duran 1 and Jen Yu 2

Departments of 1Anatomy and Neurobiology and 2physical Medicine and Rehabilitation, College of Medicine, University of California, lrvine, CA 92717 (U.S.A.) (Accepted 17 January 1989)

Key words: Acetylcholinesterase; Development; Enucleation; Lateral geniculate nucleus; Transneuronal degeneration; Visual deprivation

Previous studies have demonstrated that transient patterns of acetylcholinesterase (ACHE) activity are characteristic of geniculo-recipient regions of rat cortical area 17 during the second and third postnatal weeks of life. Neonatal enucleation results in a marked reduction of this transiently expressed cortical ACHE. Parallel studies have demonstrated that the dorsal lateral geniculate nucleus (dLGN) also expresses AChE transiently during development. The present study examines neuronal number and size as well as AChE histochemical staining in the dLGN of normal and neonatally enucleated rat pups to determine whether changes in dLGN neurons could account for the decreased visual cortical AChE staining that results from neonatal enucleation. Changes in 4 parameters in dLGN were noted after neonatal enucleation. First, a 26-37% shrinkage in the volume of dLGN occurred contralateral to enucleation. Second, enucleation resulted in a loss of 16-30% of AChE-stained neuronal somata. Third, remaining AChE-positive neuronal somata appeared shrunken by approximately 40%. Fourth, intensity of AChE histochemical staining of individual dLGN neurons was reduced by approximately 24% following neonatal enucleation. These data suggest that loss of transient AChE activity in cortical area 17 consequent to neonatal enucleation is secondary to enucleation-induced alterations in the dLGN; these alterations include loss of neurons, shrinkage of neurons, and an apparent decrease in the ability of neurons to synthesize ACHE. These data support the hypothesis that geniculocortical projection neurons express AChE transiently during development of geniculocortical connectivity and indicate that normal afferent connections and/or activity are important for the transient expression of AChE by these neurons. INTRODUCTION P r i m a r y visual cortex of the rat displays transient p a t t e r n s of acetylcholinesterase (ACHE) activity during the second and third postnatal weeks of life 55'56'58-60"62"63. The transiently expressed A C h E a p p e a r s within occipital cortex as a dense fiber-like plexus in geniculo-recipient layers of cortical area 17. T h e close c o r r e s p o n d e n c e b e t w e e n the p a t t e r n of A C h E and the distribution of geniculocortical axon terminals 48'54'74 suggests that the transient A C h E m a y be located within geniculocortical axon terminals 55"58'59. This suggestion receives support from recent e x p e r i m e n t s d e m o n s t r a t i n g that neurons of

the dorsal lateral geniculate nucleus ( d L G N ) of the rat thalamus also display strong A C h E staining, particularly during the second postnatal w e e k of life 25'55'59'63. F u r t h e r , p l a c e m e n t of lesions in the dorsal thalamus, including the d L G N , results in elimination of the transient p a t t e r n of A C h E in cortical area 1759. O t h e r recent e x p e r i m e n t s have d e m o n s t r a t e d that the transient p a t t e r n of A C h E is m a n i p u l a b l e by neonatal enucleations. N e o n a t a l unilateral eye removal results in a m a r k e d reduction of the transiently expressed A C h E activity in the m o n o c u l a r portion of area 17 contralateral to the e n u c l e a t e d orbit, while bilateral eye r e m o v a l results in a virtual

Correspondence: R.T. Robertson, Department of Anatomy and Neurobiology, College of Medicine, University of California, Irvine, CA 92717, U.S.A. 0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

210 absence of the transient AChE throughout cortical area 175s'6°'('3. Because the transiently expressed A C h E in cortical area 17 appears to be contained within terminals of geniculocortical axons, the reduction of cortical AChE that follows neonatal enucleation could be a direct result of changes at the level of the dLGN. That is, the decrease in transiently expressed cortical AChE could result from any of a variety of atrophic changes in the dLGN, including, but not limited to, (1) death of dLGN thalamocortical neurons, (2) atrophy of these dLGN neurons, and (3) decreased level of synthesis of AChE by dLGN neurons. We have investigated these possibilities by studying the effects of neonatal enucleation on neuron number, size, and AChE histochemicai staining in dLGN. Portions of these data have been presented previously in abstract form -~7. MATERIALS AND METHODS

Animals and surgical preparations Experiments were performed on 64 male and female Sprague-Dawley rat pups from 10 litters. Dams with timed pregnancies were purchased from licensed breeders and pups were born in the laboratory. Litters were adjusted to 10 pups for each dam on the day of birth, postnatal day 0 (PND 0). Within 12 h of birth, pups were anesthetized by hypothermia and had one or both eyes removed surgically or were subjected to sham lesions. Sham lesioned animals were anesthetized and had the developing palpebrai fissure opened surgically. In each litter, 2-4 pups were included in each group as unilateral enucleates, bilateral enucleates or sham-lesioned controls. On PND 6-10, rat pups were deeply anesthetized with chloral hydrate or sodium pentobarbital and perfused through the heart with 50 ml 10% formalin in 0.1 M sodium phosphate buffer (pH 7.3). Most animals received systemic injections of the irreversible AChE inhibitor diisopropylfluorophosphate (DFP; 1.5 mg/kg; i.p.) 1-8 h prior to sacrifice 7.

H&tochemistry Frozen sections of 30 #m were collected in pairs. One section from each pair was processed for a standard Nissl stain. The other section was processed

for AChE histochemistry 7'2''. The substrate was 1.0 X 1 0 - 4 M acetylthiocholine iodide and non-specific cholinesterase activity was inhibited by 1.14 × 10 4 M iso-OMPA 7. Sections were incubated for 20-36 h at room temperature. The histochemicai reaction product was developed by placing the sections in an aqueous solution of 1% ammonium sulfide for 30 s. Sections were dehydrated, coverslipped and examined under the light microscope. Density of AChE histochemical reaction product was taken as an index of the amount or relative activity of AChE 7"~.

Data analysis Material for comparisons was taken from littermates that were unilaterally or bilaterally enucleated, or were sham-lesioned controls. Quantitative assessments were made of effects of enucleations on morphological and histochemical measures in dLGN. Volume of the dLGN. The volume of dLGN was calculated from areal measurements taken from every AChE-stained section (i.e. every second section) through the dLGN. Measurements of crosssectional area of dLGN were made in two ways. In some animals drawings were made, at a final magnification of 240x, of the dLGN from each AChE-stained section. The area of the dLGN in each of these drawings was determined with the aid of a Zeiss Zidas system. Areal measurements of sections from other animals were made with a Zeiss Axiomat microscope with attached video monitor. A joystick was used to outline the perimeter of the dLGN and an attached computer determined the area of the outlined dLGN. In both methods, volume of dLGN in each section was calculated by multiplying the area by the section thickness (30 #m). No attempt was made to correct for tissue shrinkage. The values for volume of dLGN of each section were combined across all sections and multiplied by 2 (every second section was measured) to obtain an estimate of the total volume of the dLGN of each hemisphere under each experimental condition. Enucleation related changes in calculated total volume of the nucleus result from a reduction in cross-sectional area of dLGN in each section and a reduction in the number of sections in which the dLGN is present. Numbers of cells. Counts of numbers of ACHE-

211 positive neuronal somata were made in every ACHEstained section (every second section) through the dLGN of control and enucleated animals. Drawings of each section were made using a 63x objective and included every identified neuron. Cells that were counted had AChE-positive cytoplasm and a pale nucleus. In comparing adjacent sections stained for AChE and Nissl material it was evident that glial cells did not stain for ACHE. Drawings and counts were checked by two investigators and cell counts were corrected by the method of Abercrombie I.

Cross-sectional area of dLGN neuronal somata. AChE-stained sections placed on a Zeiss Axiomat microscope were viewed with the aid of a video camera and monitor. We focused our attention on the monocular portion of dLGN, i.e. the dorsal and

lateral portion of the dLGN 52, in transverse sections taken from the middle third of the rostro-caudal extent of the nucleus. Individual neurons were identified by the presence of AChE-positive cytoplasm and a pale nucleus. The individual neuronal soma under study was defined by outlining its perimeter with a joystick. The computer system calculated and recorded the area of the soma under study. The cross-sectional areas of 50-100 neurons in animals from each experimental group were determined by the computer system. AChE densitometry. AChE-stained sections placed on a Zeiss Axiomat microscope were viewed with the aid of a video camera and monitor. The dLGN is easily identified in sections stained for ACHE, and pretreatment with DFP allows the

Fig. 1. Photomicrographs illustrating the patterns of AChE staining in dLGN of infant and adult rats. A: transverse section through

dLGN of a normal PND 10 rat pup. Also indicated are the ventral lateral geniculate nucleus (vLGN), lateral posterior nucleus (LP), and ventrobasal complex (VB). B: section of dLGN from a PND 8 rat pup sacrificed 8 h after systemic treatment with 1.5 mg/kg DFP. Calibration bar in B = 250/~m for A and B. C: transverse section through dLGN of a normal adult rat. D: section of dLGN from an adult rat 8 h after 1.5 mg/kg DFP. Calibration bar in D = 250/~m for C and D.

213 TABLE I

Volume (in mm 3) of the dorsal lateral geniculate nucleus of rats at postnatal days 6, 8, 9 and 10 Means and standard errors are shown for calculated volumes of dLGNs ipsilateral (Uni, Ips) and contralateral (Uni, Contr) to the enucleated orbit for animals unilaterally enucleated at PND 0, for sham-operated control animals (Sham), and for animals enudeated bilaterally at PND 0 (Bilat).

Age (PND) 6 8 9 10

No.

2 3 2 3

Uni, ips

0.425 0.618 0.867 1.096

Uni, contr

Sham

Bilat

S.E.M.

~

S.E.M.

.~

S.E.M.

~

S.E.M.

0.019 0.009 0.032 0.082

0.316 0.399 0.599 0.772

0:006 0.007 0.018 0.035

0.460 0.647 0.870 1.150

0.029 0.021 0.021 0.072

0.319 0.413 0.612 0.820

0.017 0.012 0.019 0.060

borders of individual neurons to be clearly seen. The area under study, either the entire dLGN or an individual neuronal soma containing a clearly visible nucleus, was defined by outlining its perimeter with a joystick. The identified area of the video-recorded image then was digitized and a deAnza densitometric system determined the relative grey value for this area. The converted grey value varied from '0' (white) to '250' (black) and indicated the density of AChE histochemical reaction product within the outlined area. Intensity of illumination, contrast of the image and sensitivity of the densitometer were adjusted for each brain so that measurements of AChE reaction product density from the internal capsule would produce readings of 38-42. In cases of unilateral enucleation, density readings of the internal capsules of the two hemispheres were found to be equivalent. Density of AChE histochemical reaction product was determined both for the entire dLGN for each section and for 50-100 individual neural somata within dLGN. As with cell measurements (above), densitometric measurements were taken from neurons in the dorsal and lateral portions of dLGN in sections taken from the middle third of the rostrocaudal extent of the nucleus. Particular attention was paid to comparing the density of AChE histochemical reaction product in

enucleated and control cases. Direct comparisons were confined to littermates that were unilaterally or bilaterally enucleated, or were sham operated controls. These littermates were sacrificed at the same age and the brains were processed together through the same histochemical procedures. RESULTS

AChE staining in the dLGN of laboratory rats develops postnatally. AChE staining above apparent background levels first appears at about PND 3, reaches peak intensity at PND 8 - t 0 , and declines to adult levels by PND 1655. We chose to focus our studies of AChE staining in dLGN on material from rats sacrificed at PND 8-10, when AChE staining is most intense. Fig. 1 presents representative material from unoperated developing rat pups and from adult rats and demonstrates that the quality of AChE staining in the adult appears different f r o m that in the developing rat. In normal animals, without DFP treatment, the dLGN Of both developing (Fig. 1A) and adult (Fig. 1C) rats stain positively for ACHE. However, density o f the ~,histoehemical reaction product appears greater ,in t h e infant. Further, staining in the adult is associated primarily with fibers while the infant displays more evidence of somal staining. Systemic pretreatment with DFP

,(.._ Fig. 2. Photomicrographs of AChE-reacted transverse sections through dorsal thalamus of PND 10 rat pups. A: low magnification photomicrograph of posterior thalamus of a unilaterally enucleated animal. Note the shrinkage in the right dLGN, eontralateral to the enucleated orbit. Calibration bar = 1 mm. B and C: higher power views of the left (B) and right (C) dLGN from A. D: the dLGN from a control animal that underwent a sham lesion at birth. E: the dLGN from an animal enucleated bilaterally at birth. Calibration bar in E = 200/~m for B - E .

214 reduces staining of neuropil and results in clear A C h E staining of neuronal somata in developing rats (Fig. 1B), but not in the adult (Fig. 1D). Fig. 2 presents examples of A C h E histochemical staining of the d L G N in sham-operated control and enucleated animals. These 3 animals were littermates and were all sacrificed on P N D 10, without administration of DFP. Without DFP pretreatment, A C h E activity at this age is so intense that staining of individual neurons is obscured by dense neuropil staining, particularly in dorsal portions of d L G N . Fig. 2 A - C illustrates the pattern of A C h E staining in an animal that received a unilateral enucleation of the left orbit on P N D 0. Examples of d L G N staining also are shown from a control animal that received a sham lesion on P N D 0 (Fig. 2D) and an animal subjected to bilateral enucleation at birth (Fig. 2E). Two points can be discerned from these photomi-

A 250 " 200 • g¢ Q Pi RI J¢ 0

Uni, Ips

Uni, Contr Sham Con~tlon

Bilat

Uni, Contr Sham Condition

Biiat

B 120 t 100 I=

80i

8 uJ ,11 O <

Uni, JpS

Fig. 3. Histograms showing results of densitometric analyses of AChE-reacted transverse sections through dLGN from PND 10 rat pups. AChE reaction product density (in converted densitometric units) is presented for dLGN ipsilateral (Uni, Ips) and contralateral (Uni, Contr)to the enucleated orbit for unilaterally enucleated animals, for sham operated control animals (Sham), and for animals enucleated bilaterally (Bilat). A: material from animals not treated with DFP. B: material from animals treated with DFP 7 h prior to sacrifice.

crographs. Note firstly that the cross-sectional area of d L G N contralateral to an enucleated orbit is smaller than that of d L G N contralateral to an intact eye. This difference can be seen in the d L G N contralateral to a unilateral enucleation, as compared with the d L G N ipsilateral to the enucleation (Fig. 2 A - C ) or in the d L G N of bilateral enucleates, as compared to d L G N from a sham control animal (Fig. 2D,E). Note secondly that shrunken d L G N s contralateral to enucleated orbits appear to stain somewhat more intensely for A C h E than do d L G N s contralateral to intact eyes, especially in the dorsal portion of dLGN. We calculated the volumes of d L G N in this series of animals and in other cases. The results are presented in Table I. These data indicate that the volume of d L G N s contralateral to unilaterally enucleated orbits is 64-74% of the volume of d L G N s ipsilateral to unilaterally enucleated orbits. Put another way, d L G N s contralateral to enucleation appear to shrink by 26-36%. A paired sample Student's t-test revealed this difference to be statistically significant (t = 7.98; df = 9; P < 0.001). The volume of d L G N s ipsilateral to enucleated orbits did not differ significantly from the volume of d L G N s from sham-operated control animals. Further, bilateral enucleation results in d L G N s with volumes approximately 63-70% of the volumes of d L G N s from sham operated control animals (Table I). Shrinkage, then, is 3 0 - 3 7 % and again this difference is statistically significant (t = 10.9; df = 9; P < 0.001). The volume of d L G N s of bilateral enucleates did not differ significantly from the volume of d L G N s in the hemisphere contralateral to unilateral enucleations. Densitometric analyses of this material were undertaken to quantify changes in A C h E staining. As illustrated in Fig. 3A, neonatal enucleation appears to result in a slight increase in the intensity of A C h E staining per unit area of the d L G N . The dLGNs from bilaterally enucleated animals and the d L G N contralateral to a unilateral enucleation both show slightly higher A C h E density measurements than do control animals. However, M a n n - W h i t n e y U-tests on material from 5 sets of animals (a set consists of unilaterally enucleated, bilaterally enucleated, and sham-operated littermates) show that these slight differences are not statistically significant.

215 Figs. 4 and 5 present examples of material from a set of PND 9 animals sacrificed 7 h after DFP treatment. Again, this set of animals includes littermates that were bilaterally enucleated, unilaterally enucleated, or sham operated at PND 0. Fig. 4A,B demonstrates again that unilateral enucleation results in a marked shrinkage of the contralateral dLGN, relative to the dLGN ipsilateral to the enucleated orbit. Similarly, bilateral enucleation results in shrinkage of dLGN (Fig. 4D) compared to the control animal (Fig. 4C). Results from analyses of the volume of tissue occupied by the dLGN in each experimental condition of this series are included in Table I. We detected no evidence indicating that pretreatment with DFP affected the volumes of d L G N in any of the 3 experimental conditions. Results of densitometric analysis of AChE histo-

d

chemical staining of sections from these cases are illustrated in Fig. 3B. A C h E density is p r e s e n t e d for each of the 4 conditions. In comparing these results with those presented in Fig. 3A, it can be seen that the overall density of A C h E staining is much reduced with DFP pretreatment. More interestingly, AChE staining density appears greater in dLGNs contralateral to intact eyes than in dLGNs contralateral to enucleated orbits. Mann-Whitney U-tests of these data and similar data from 3 other sets of animals reveal that average AChE density readings from dLGNs ipsilateral to enucleated orbits are significantly greater than are A C h E densities from dLGNs contralateral to enucleated orbits (U = 1; P < 0.05). Similarly, A C h E densities from dLGNs of sham-operated animals are greater than AChE densities from dLGNs of animals enucleated bilat-

D

L Fig. 4. Photomicrographs of AChE-reacted transverse sections through dorsal thalamus of PND 10 rat pups. These animals received

systemic DFP (1.5 mg/kg; i.p.) 8 h prior to sacrifice. A and B: the dLGN ipsilateral (A) and contralateral (B) to the enucleated orbit from an animal unilaterally enucleated at PND 0. C: the dLGN from a sham-lesioned control animal. D: a dLGN from a bilaterally enucleated animal. Calibration bar in D = 100/zm for A-D.

216

L~

)

Fig. 5. Photomicrographs of AChE-reacted transverse sections through dLGN of PND 1(1 rat pups. These animals received systemic DFP (1.5 mg/kg; i.p.) 8 h prior to sacrifice. A and B: the dLGN ipsilateral (A) and contralateral (B) to the enucleated orbit in an animal unilaterally enucleated at PND (L C: the dLGN from a sham-lesioned control animal. D: a dLGN from a bilaterally enucleated animal. Calibration bar in D = 50 ~m for A-D.

erally at birth ( U = 6; P < 0.011. Analyses done on material from animals sacrificed 1 h after D F P

TABLE II Number of acetylcholinesterase histoehemically stained neurons in the dorsal lateral geniculate nucleus of rats at postnatal days 8 and 10

Means and standard errors are shown for calculated total numbers of dLGN neurons ipsilateral (Uni, Ips) and contralateral (Uni, Contr) to the enucleated orbit for animals unilaterally enucleated at PND 0, for sham-operated control animals (Sham), and for animals enucleated bilaterally at PND 0 (Bilat). Age ( PND)

8 10

Mean (S.E.M.) Mean (S.E.M.)

Uni, ips

Uni, contr

Sham

Bilat

23,738 (390) 22,354 (338)

18,142 (305) 18,906 (315)

25,238 (713) 22,902 (575)

17,665 (555) 18,354 (4211

treatment revealed very little A C h E staining in the d L G N , or in the rest of the thalamus. Densitometric analyses of the relatively light A C h E staining present detected no differences in staining between sham-operated and enucleated animals at this short time after D F P treatment. These results suggest that d L G N s of enucleated animals are not pharmacologically more sensitive to DFP than are d L G N s of control animals. A n advantage of this D F P pretreated material is that the D F P reduces A C h E staining in the neuropil, allowing observations to be made on A C h E staining of individual n e u r o n s 7. Results of counts of ACHEstained d L G N neurons from 2 series of animals sacrificed at PND 8 and 2 series sacrificed at PND 10 are presented in Table II. These counts reveal that dLGNs contralaterai to unilateral enucleations contain 16-24% fewer AChE-positive n e u r o n s than do the dLGNs ipsilateral to enucleations. Further,

217 dLGNs from bilaterally enucleated animals contain 20-30% fewer AChE-positive neurons than do dLGNs of sham operated control animals. A Student's t-test, combining counts of all sham operates and comparing with combined counts from all bilateral enucleates, demonstrates the difference in cell number to be significant (t = 9.71; df = 6; P < 0.001). Similarly, a t-test comparing dLGNs contralateral to unilateral enucleations with those ipsilateral to enucleations demonstrates that the differences between these groups are statistically significant (t = 33.11; df = 3; P < 0.001).

In addition to enucleation affecting the number of AChE-stained neurons, further analyses demonstrate that the remaining neurons in dLGNs from enucleated cases appear to be both smaller and less intensely stained for A C h E than are neurons of control cases. Results of computer analysis of crosssectional areas of AChE-stained dLGN neurons from 1 set of unilaterally and bilaterally enucleated and sham control animals are presented in Fig. 6. While the population of AChE-positive neurons in each experimental condition exhibits considerable variation in cross-sectional area, it is apparent that

B

A 14 12 10 e-

¢= ,.i o-

8 6

u,,.

k/.

4 2 0 4

5

6

7

8

9

10

Cell Area (sq I~

11

12

13

14

$

lS

X 1 0)

6

7

$

9

10 11

12

13

14

C e l l A r e a ( s q i=rn X 1 O)

C

D ~k~

14

I ~.in~ I t~-M

14

='el LGN 12

12

10

10

~"

8

O" ~..

6

,"

8 6

IJ. 4

4

2

2

0

4

S

S

7

8

9

10

11

12

C e l l A r e a ( s q i m l X 1 O)

13

14

15

0

4

S

6

7

$

9

10

tl

12

13

14. 1S

Cell Area (sq pm X 10)

Fig. 6. Histograms showing cross-sectional areas of d L G N neurons from each experimental condition. Animals were sacrificed on PND 10, 7 h after D F P treatment. Number of neurons is presented on the ordinate for each cell-area bin presented on the abscissa. A: d L G N ipsilateral to a unilateral enucleation on PND 0. B: d L G N contralateral to unilateral enucleation; C: d L G N from a sham operated control animal; D: d L G N from an animal bilaterally enucleated on PND 0.

218 the p o p u l a t i o n of A C h E - p o s i t i v e neurons from d L G N s contralateral to enucleated orbits is smaller in comparison with the population of stained neurons from d L G N s contralateral to intact orbits. Using a within-animal comparison in unilaterally e n u c l e a t e d animals, cross-sectional areas of ACHEpositive neurons in d L G N contralateral to the e n u c l e a t e d orbit (Fig. 6B) were found to be smaller (mean = 62.1 /.tm 2) than areas of A C h E - p o s i t i v e neurons ipsilateral (Fig. 6A) to the enucleation (mean = 102.7 ktm2). This reduction of approximately 40% is statistically significant (t = 17.02; df = 198; P < 0.001). Similarly, a between-animals

A

Unilat Enucl LGN, Ipsi

comparison revealed that cross-sectional areas of A C h E - p o s i t i v e d L G N neurons from bilaterally enucleated animals (Fig. 6D) (mean = 61.8/~m 2) are smaller than those from A C h E - p o s i t i v e d L G N neurons of s h a m - o p e r a t e d control animals (Fig. 6C) (mean = 101.6 urn2). This difference is also significant (t = 15.47; df = 198; P < 0.001). No statistically significant differences in cross-sectional areas were detected when we c o m p a r e d d L G N s of shamo p e r a t e d control animals with the d L G N s contralateral to intact orbits in unilaterally enucleated animals. Further, no differences in cross-sectional areas of A C h E - p o s i t i v e neurons were detected between

I~

100 -

=-, ¢,/)

100

~,_ TB% ® ~ . . = _= ,,,_~0~ ~ ®=" ~==a"~,'t, '"%"= ="

90

°

80

s-,

70 nl .¢: ¢..) ,¢{

.>, "== 8o

mB m ma

a

60

D D

Unllat Entml LGN, Contra

,0

~ []

a

70

.c O ~

60

50

=11~., = .

50-

4O

i

4O

•

60

=

-

i

80

•

100

i

120

•

I

140

•

I

40

•

160

20

I

40

Cell Area (sq g m )

C 100 -

m

U

rn

i

-

~

•

~

•

i

120

140

120

140

BIIM Enu¢l LGN

90 80

8O °

t-

•

100

B ra

90

J

60 80 100 C e l l Area (eq lam)

D

Sham Lesion LGN m

=>,

•

O

70

7O ~1 m mm

iii tj ,,¢

60

D

m

m

J¢ la

="

60 50

5O

m

40 4O

60

80

100

120

Cell Area (sq gm)

140

160

20

40

60 80 100 Cell Area (eq lain)

Fig. 7. Scatter plots showing the relationship between neuronal soma size and AChE staining density in dLGN neurons from control animals and animals enucleated at PND 0. AChE density in converted densitometric numbers is on the ordinate; cross-sectional cell area is on the abscissa. A: neurons from the dLGN ipsilateral to a unilateral enucleation; B: neurons from the dLGN contralateral to a unilateral enucleation; C: neurons from dLGN of a sham-lesioned animal; D: neurons from dLGN of a bilaterally enucleated animal.

219 dLGNs of bilaterally enucleated animals and the dLGNs contralateral to unilaterally enucleated orbits. In sum, it appears that dLGNs contralateral to enucleated orbits contain AChE-positive neurons that are approximately' 40% smaller in average cross-sectional area than are the neurons of dLGNs contralateral to intact eyes. Densitometric analyses of individual ACHEstained neurons in dLGN reveal differences in staining intensity. Although a considerable variation in staining intensity was seen between neurons from the same animal, it is clear that, as a population, neurons from dLGN contralateral to enucleated orbits showed lower scores for A C h E density than did neurons from dLGN contralateral to intact orbits. AChE staining density in dLGN neurons of bilaterally enucleated animals (mean = 61.8 in arbitrary units) was significantly less intense than was A C h E staining in dLGN neurons of shamoperated animals (mean = 78.2) (t = 12.6; df = 198; P < 0.001). The results of these between-animals comparisons must be interpreted cautiously because of problems inherent in equalizing reference points and overall quality of fixation and staining between animals. However, these problems are considerably less formidable when analyzing AChE staining between the two dLGNs of unilaterally enucleated animals. In unilaterally enucleated animals, average optical density scores for AChE histochemical staining in d L G N neurons contralateral to the enucleation (mean = 61.5 units) was significantly less than average density of staining of neurons ipsilateral to enucleation (mean = 80.9 units) (t = 14.6; DF = 198; P < 0.001). AChE staining intensities of individual neurons are even more interesting when optical density of staining is related to size of neurons. The data presented in Fig. 7 demonstrate that dLGNs contralateral to an intact orbit display a positive correlation between cross-sectional area of neurons and density of AChE staining (Fig. 7B,D). That is, larger neurons stain more intensely for AChE than do smaller neurons. A different relationship exists in dLGN contralateral to enucleated orbits (Fig. 7A,C). Not only are the neurons smaller and stain less intensely in these cases, but the relationship between cell area and AChE staining is a negative correlation. That is, larger neurons tend to be less intensely stained.

DISCUSSION

Technical considerations AChE has been studied extensively during the past few decades 7'66 and a variety of methods are available for its localization in tissue 7"29'63'66. We have chosen to use a modification of the basic Koelle and Friedenwald 29 method primarily because of its reliability and because this technique produces a histochemical reaction product that appears of graded intensity, depending on such factors as animal survival time after DFP administration, length of time of tissue incubation in the histochemical medium, and age of the animal at time of sacrifice. The characteristic of producing a reaction product of graded intensity is of great importance when investigating perhaps subtle differences in AChE staining between neurons. However, these histochemical techniques are not able to distinguish changes in quantity of the enzyme from changes in activity of existing levels of the enzyme. Thus, parametric measures of differences between dLGN neurons of enucleated and control cases must be interpreted cautiously. Counts of cells in experimental and control cases were done from material histochemically stained for ACHE. AChE-stained material was chosen for two principle reasons. First, our primary motivation for this study was our observation that neonatal enucleations result in absence of transient A C h E activity in visual cortex, and thus it seems appropriate to measure the AChE content in the dLGN. Second, cells of the dLGN are still in a rapid phase of development at PND 6--103,4'39,46,47,64 and it may be difficult to distinguish immature neurons from glial cells. The morphology of AChE-stained cells in dLGN (i.e. relative abundance of AChE-rich cytoplasm, relatively large nucleus, and presence of dendritic processes) indicates that they are neurons. Our experience 55"59'62 has indicated that glial cells in the dorsal thalamus of the rat do not stain for AChE (or other cholinesterases) and thus presence of AChE appears to be a reliable criterion by which neurons and glia can be distinguished. Further, current work in this laboratory 61 has identified dLGN neurons by [3H]thymidine labeling on embryonic days 13-15. We detect a loss of approximately 25-35% of [3H]thymidine-labeled d L G N neurons

220 following neonatal enucleation. On the other hand, however, our choice of measuring AChE-positive neurons means that we missed any neurons that may be present but not stained by these procedures. Although we believe all geniculocortical relay neurons of dLGN express AChE transiently during development, we have no data pertinent to local circuit neurons ~8'45. It is possible that local circuit neurons in dLGN do not express AChE during development and these neurons may have been excluded from our analysis. We chose to use the DFP pretreatment method of Butcher 7 in order to study the AChE content of individual cells. In this method, DFP is administered systemically to irreversibly inhibit activity of existing ACHE. Animals sacrificed less than 1 h after DFP treatment show only marginal staining for ACHE, and no differences in intensity of staining were detected between sham-operated and enucleated animals. Thus, differences in AChE staining between experimental groups appears not to result from differential effects of the DFP. In the period of several hours between DFP administration and sacrifice, cell bodies that have the cellular machinery to synthesize AChE presumably will do so and this recently synthesized AChE will be detected in somata by histochemical techniques. In this regard, we believe our studies reveal the ability of neurons to synthesize ACHE. It is clear from the present data that enucleation reduces the histochemical staining of, and hence the presumed ability to synthesize, AChE in these dLGN neurons. A related problem concerns the interpretation of reduced numbers of AChE-positive neurons in dLGNs contralateral to enucleated orbits. Does enucleation result in death of these neurons or in a decrease of synthesis of AChE such that the cells are no longer detectable by AChE histochemistry? We currently are unable to choose between these two possibilities. We have seen both a shrinkage of AChE-positive neurons, which may suggest atrophy associated with cell death, and we have seen more lightly AChE-stained neurons, which may suggest that other cells have decreased their staining beyond the point of detection. Thus, the present data do not answer conclusively the question of cell death, although they do demonstrate a reduction in ACHEpositive neurons.

The validity of between-animal comparisons always must be questioned, but we believe the present comparisons are justified for two reasons. First, the range accepted for the standard reference point (readings of 38-42 in the internal capsule) was considerably less than the difference in mean scores of bilaterally enucleated (61.8 units) and shamoperated control animals (78.2 units). Second, differences between bilateral enucleated animals and sham-operated controls were similar to differences between the two hemispheres of unilateral enucleated animals. In unilateral enucleates, we can assume that variation in quality of fixation and histochemicai staining between the two hemispheres are minimal and that the same reference point is effective for both hemispheres.

Normal development of dLGN The data presented in this paper come from tissue collected at phases of rapid growth of the dLGN. Results from experiments using thymidine autoradiography indicate that most neurons destined to comprise the dLGN undergo their final mitoses on embryonic days 13-152,6,39,44. Migration times of these neurons to the dLGN are not well documented, but it is likely that the vast majority of dLGN neurons would be in residence within the dLGN nucleus by the time the enucleations were done at PND 0. The number of identifiable dLGN neurons increases during the early postnatal periods to peak at approximately PND 10 64. In concert with the marked increase in the number of identifiable neurons in the early postnatal period, these neurons also are growing and undergoing other maturational changes. Studies of Nissl-stained 46'64 and Golgiimpregnated 47 material reveal rapid growth of the diameters and surface areas of dLGN neurons during the second postnatal week. Dendritic development, including growth and increased complexity of the dendritic arbors, approaches the mature state by the end of the third postnatal week 47. In addition to the growth of neurons within dLGN, afferents to the nucleus also are in a dynamic phase of development. The dLGN receives retinal afferents beginning on approximately embryonic day 176 and this ingrowth probably continues through the early postnatal period. Further complicating the developmental process, loss of more than 50% of the

221 optic nerve fibers occurs during early postnatal development 13. Thus, enucleation-induced degeneration of optic tract fibers is superimposed upon normally present degeneration. It is clear that the dLGN during the second postnatal week is a site of dynamic growth and change and sham-operated control animals do not offer a baseline of a stable population of neural elements. Thus, interpretation of lesion-induced changes in dLGN must be made cautiously in this very dynamic developing system.

Enucleation and L G N morphology The present results demonstrate that neonatal enucleations result in dLGNs with smaller than normal volume and with fewer and smaller ACHEpositive neurons than dLGNs from sham-operated control animals. Morphological changes in the dLGN following eye removal or optic nerve section represent a classic example of anterograde transneuronal degeneration 12A4-16'19'35'42'43'67'7°-73.The present experiments contribute to the rather extensive literature in this field by presenting quantitative data on the effects of early enucleation and by demonstrating that profound effects can be detected in infant rats as early as 6-10 days following eye removal. Our studies suggest that the reduced volume of dLGN following enucleation results from both a reduced number of neurons and a shrinkage of remaining neurons. A number of studies have demonstrated that enucleations, tetrodotoxin treatment, and even less traumatic eyelid sutures, result in a decreased number of neurons and shrinkage in remaining neurons in d L G N 9'12'16'25'35'42"71'72. Although there is general agreement among these studies that enucleation or deprivation results in atrophy of neurons in dLGN, it is clear that differences between species and age of animals result in considerable variability in severity and time of onset of neuron atrophy and lOSS10"12"35"42. In general, greater dLGN cell loss results from enucleation in younger animals than in older animals 12'23"35. Greatest cell atrophy may occur when retinal afferents are eliminated prior to their arrival in dLGN, however the presence of identifiable dLGN in anopthalmic animals 28 demonstrates that some development occurs in absence of retinal afferents. Further, longer postenucleation survival times result

in relatively greater cell loss and atrophy than do shorter survival times, indicating that at least some aspects of the degenerative process can be slow. Perhaps the most confusing issue relates to species differences in degree of dLGN atrophy following enucleation. Enucleation in primates and rodents leads to severe atrophy, with loss of approximately half the dLGN neurons 12'23'42'49"50. In contrast, enucleations in tree shrews, ferrets and minks result in dLGNs with reduced lamination but with surprisingly normal appearing neurons 5'2°. Thus, while these latter studies did not report cell counts, the results certainly do not indicate cell loss similar to that reported for rodents and primates. The discrepancies in these cases cannot be attributed to differences in timing of removal of retinal projections because, at least in some cases, enucleations were made on fetal animals before ganglion cell axons had reached the dLGN 2°. The possibility remains that other afferents (perhaps from cerebral cortex or brainstem regions) to the dLGN may develop earlier in tree shrews, minks and ferrets, and these other afferents may be able to sustain the development of dLGN neurons. However, until we have a better understanding of the development of these other afferents, these discrepancies may have to remain in the vague realm of 'species differences'. Surprisingly few data are available regarding quantitative aspects of cell loss in rat dLGN following enucleation. In the mouse, Heumann and Rabinowicz 23 report a loss of approximately 15% of dLGN neurons 10 days after neonatal enucleation. Interestingly, no cell loss was detected at PND 5, and apparent cell loss increased to approximately 27% by 180 days after the lesion. These figures are remarkably similar to the loss of 16-30% of cells reported in the present study. In related work, Jeffery27 and Fukuda and Hsiao 16 report that dLGNs contralateral to enucleated orbits contain fewer retrogradely labeled neurons following HRP injections in visual cortex than do dLGNs contralateral to intact eyes. Further, Fukuda and Hsiao 16 report that cortically projecting dLGN cells contralateral to enucleation had cross-sectional areas 17% smaller than control dLGN cells.

AChE histochemistry of the dLGN Although the dLGN of adult rats shows consid-

222 erable AChE activity, most of this activity appears associated with afferent axons originating in the dorsal pontine nuclei 2~'4° and relatively little is associated with dLGN somata. The infant rat presents a strikingly different picture. Histochemical activity for AChE in dLGN shows a dramatic increase at about PND 4, reaches peak activity at PND 8-10, and then declines to adult levels by PND 1625'56'60'64. During this time, AChE is characteristic of dLGN neural somata, as detected with the light microscope after DFP pretreatmen¢ 6'6° and with the electron microscope 62. Transient AChE staining of dLGN somata has also been reported for the developing tree shrew 24. Available data from rat indicate that AChE is expressed transiently by all dLGN geniculocortical relay neurons during the period of time when their axons are growing into cerebral cortex and forming synapses with cortical neurons 39"55"62. Neurons of the rat dLGN appear to share this characteristic with neurons in the rat ventral basal complex 32-3455 and the ventral medial geniculate nucleus 55. Thus, transient expression of intense AChE activity appears characteristic of developing thalamocortical neurons in the 3 primary sensory systems of the rat. Similar transient expression of AChE activity is characteristic of some developing thalamocortical neurons in primates 3°'31. The data presented in Fig. 7 demonstrate a positive relationship in control animals between size of cell, as measured by cross-sectional area, and density of AChE staining. Thus, the larger geniculocortical neurons appear to express greater AChE activity, This relationship may indicate that larger cells are more mature and hence more developed in their ability to synthesize ACHE. Another interpretation relates to the possibility that transiently expressed AChE has a morphogenic function in the formation of geniculocortical connections 55. In this regard, larger neurons could have more elaborate terminal fields in cortex and hence have a greater requirement for ACHE.

Enucleation and AChE histochemistry It is clear from the morphological results presented above that enucleation results in severe atrophy in cells of the dLGN. Further, enucleation leads to decreased AChE histochemical staining in individual neurons in dLGN. In light of the shrink-

age observed in the AChE-positive dLGN neurons, it is not surprising that these cells would show a decreased level of staining for ACHE, or for many other enzymes. A neuron undergoing degenerative changes leading to cell death would be expected to show decreased activity for enzymes in general, and a neuron undergoing degenerative changes from which it recovers might be expected to shut down all but the most essential cellular machinery. Other investigators have demonstrated that other aspects of metabolic activity also are decreased by enucleation. Land 36 and Sukekawa ~'~ have reported decreased staining for the mitochondrial enzyme cytochrome oxidase following enucleation, and Cooper and Thurlow 1l have described reduced glucose metabolism in the dLGN following enucleation. The relationship between neuronal size and AChE staining intensity was clearly affected by enucleation. In dLGNs of sham-operated control rats and in dLGNs contralateral to the intact eye in unilaterally enucleated rats, larger neurons displayed more dense AChE reaction product. In contrast, dLGNs contralateral to enucleated orbits showed an inverse relationship between somal size and AChE staining density. Two factors related to degeneration may be relevant here. Larger neurons may have been undergoing some swelling, along with a decrease in AChE synthesis, as early events in their reaction to partial deafferentation. Smaller neurons may have been undergoing pyknosis, leading to a concentration of existing AChE and a resultant apparent increase in staining density.

Enucleation induced morphological and histochemical changes in light of expanded retinal projections In the normal animal, the vast majority of retinal fibers are crossed, but a significant portion of uncrossed fibers also exist 6'22'2637"38"41"51-53. Several investigators have demonstrated that unilateral eye removal in the neonate results in uncrossed retinal projections that occupy larger territories in the dLGN than in the normal animal 8,26'37"4~'51. It is generally believed that the larger than normal territories occupied by ipsilateral retinal projections in animals unilaterally enucleated at birth results from stabilization of an exuberant ipsilateral projection that normally is retracted early in postnatal development 8"26'3~'51. The expanded ipsilateral pro-

223 jection characteristic of unilaterally enucleated animals may have the ability to innervate and rescue some d L G N neurons that were denervated by removal of the contralateral eye, but relevant data are sparse. Some synapses are formed by the expanded projection 8 and detectable changes occur in electrophysiological characteristics of the d L G N 17. The present study did not address this issue directly; we chose to study regions of the d L G N of unilateral enucleates which were not likely occupied by the expanded ipsilateral projections 37"52. Further, the degree of cell loss and cell atrophy was similar in affected d L G N of unilateral enucleated and in d L G N s of bilateral enucleated animals, so if the expanded ipsilateral pathway had an effect on survival of d L G N neurons, this effect would likely be relatively minor or restricted to a small population of neurons.

results in a virtual absence of the transient A C h E throughout cortical area 17 of both hemispheres 58' 60.63 Other previous work has suggested that the transiently expressed A C h E in visual cortex is localized within geniculocortical axon terminals 59"62. The present results contribute to this body of knowledge by indicating that the reduction in cortical A C h E following eye removal appears to result from a severe reduction in A C h E content in geniculocortical neurons, including the geniculocortical axon terminals in visual cortex. The reduction in A C h E in the geniculocortical terminal field, in turn, appears to result from a loss of neurons, a reduction in size of remaining neurons, and a reduction in the ability of remaining geniculocortical neurons to synthesize ACHE. The combination of these 3 factors would result in a severe decrease in quantity of A C h E associated with the geniculocortical axon terminal field.

Implications f o r transient A C h E expression in visual cortex

ACKNOWLEDGEMENTS

Previous work from this laboratory has demonstrated that neonatal unilateral eye removal results in a marked reduction of the transient A C h E activity in the monocular portion of area 17 contralateral to an enucleated orbit, while bilateral eye removal

Supported by Grant BNS-08515 from the National Science Foundation. We thank Drs. E.G. Jones, R.A. Giolli, S.H.C. Hendry, H . D . Schwark and J.E. Swett for stimulating discussions.

REFERENCES 1 Abercrombie, M., Estimation of nuclear populations from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 Altman, J. and Bayer, S.A., Development of the diencephalon in the rat. IV. Quantitative study of the time of origin of neurons and the internuclear chronological gradients in the thalamus, J. Comp. Neurol., 188 (1979) 455-472. 3 Brauer, D., Mamori, J. and Winkelmann, E., On the ontogenetic development of the rat dorsal lateral geniculate nucleus. I. GCR-neurons at postnatal day 7: a Golgi-electron microscopic study, Anat. Embryol., 172 (1985) 39-48. 4 Briickner, G., Mares, V. and Biesold, D., Neurogenesis in the visual system of the rat. An autoradiographic study, J. Comp. Neurol., 166 (1976) 245-256. 5 Brunso-Bechtold, J.K. and Casagrande, V.A., Effect of bilateral enucleations on the development of layers in the dorsal lateral geniculate nucleus, Neuroscience, 6 (1981) 2579-2586. 6 Bunt, S.M., Lund, R.D. and Land, P.W., Prenatal development of the optic projection in albino and hooded rats, Dev. Brain Res., 6 (1983) 149-168. 7 Butcher, L.L., Acetylcholinesterase histochemistry. In: Handbook of Chemical Neuroanatomy, Vol. 1, Elsevier,

Amsterdam, 1983, pp. 1-49. 8 Campbell, G., So, K.-F. and Lieberman, A.R., Identification of synapses formed by the aberrant, uncrossed retinogeniculate projection in the hamster after neonatal monocular enucleation, Dev. Brain Res., 21 (1985) 137140. 9 Casagrande, V.A. and Condo, G.J., The effect of altered neuronal activity on the development of layers in the lateral geniculate nucleus, J. Neurosci., 8 (1988) 395-416. 10 Cook, W.H., Walker, J.H. and Barr, M.L., A cytological study of transneuronal atrophy in the cat and rabbit, J. Comp. Neurol., 94 (1951) 267-292. 11 Cooper, R.M. and Thurlow, G.A., Depression and recovery of metabolic activity in rat visual system after eye removal, Exp. Neurol., 89 (1985) 322-336. 12 Cowan, W.M., Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. In W.J.H. Nauta and S.O.E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, 1970, pp. 217-251. 13 Crespo, D., O'Leary, D.D.M. and Cowan, W.M., Changes in the numbers of optic nerve fibers during late prenatal and postnatal development in the albino rat, Dev. Brain Res., 19 (1985) 129-134. 14 Fifkova, E. and Hassler, R., Quantitative morphological changes in visual centres after unilateral deprivation, J.

224

Comp. Neurol., 135 (1969) 167-178. 15 Florence, S.L. and Casagrande, V.A., Changes in the distribution of geniculocortical projections lollowing monocular deprivation in trec shrews. Brain Res., 374 (t986) 179-184. 16 Fukuda, Y. and Hsiao, C.-E, Bilateral changes in soma size of geniculate relay cells and corticogeniculate cells after neonatal monocular enucleation in rats, Brain Re.s., 301 (1984) 13-23. 17 Fukuda, Y., Sumitomo, I. and Hsiao, C.-F., Effects of neonatal enucleation on excitatory and inhibitory organizations of the albino rat lateral geniculate nucleus, J. Neurophysiol., 50 (1983) 46-60. 18 Gabbott, P.L.A., Somogyi, J., Stewart, M.G. and Hamori, J., A quantitative investigation of the neuronal composition of the rat dorsal lateral geniculate nucleus using GABA-immunocytochemistry, Neuroscience, 19 (1986) 101-11l. 19 Guillery, R.W., The effect of lid suture upon the growth of cells in the dorsal lateral geniculate nucleus of kittens, J. Comp. Neurol., 148 (1973) 417-422. 20 Guillery, R.W., LaMantia, A.S., Robson, J.A. and Huang, K., The influence of retinal afferents upon the development of layers in the dorsal lateral geniculate nucleus of Mustelids, J. Neurosci., 5 (1985) 1370-1379. 21 Hallanger, A.E., Levey, A.I., See, H.J., Rye, D.B. and Wainer, B.H.~ The origins of cholinergic and other subcortical afferents to the thalamus in the rat, J. Comp. Neurol., 262 (1987) 105-124. 22 Hayhow, W.R., Sefton, A. and Webb, C., Primary optic centers in the rat in relation to the terminal distribution of the crossed and uncrossed optic nerve fibers, J. Comp. Neurol.. 118 (1962) 295-322. 23 Heumann, D. and Rabinowicz, T., Postnatal development of the dorsal lateral geniculate nucleus in the normal and enucleated albino mouse, Exp. Brain Res., 38 (1980) 75-85. 24 Hutchins, J.B. and Casagrande, V.A., Development of acetylcholinesterase activity in the lateral geniculate nucleus, J. Comp. Neurol., 275 (1988) 241-253. 25 Ishii, Y., The histochemical studies of cholinesterase in the central nervous system. II. Histochemical alteration of cholinesterase of the brain of rats from late fetal life to adults, Arch. Histol. Okoyama, 12 (1975) 613-637. 26 Jeffery, G., Retinal ganglion cell death and terminal field retraction in the developing rodent visual system, Dev. Brain Res., 13 (1984) 81-%. 27 Jeffery, G., Transneuronal effects of early eye removal on geniculo-cortical projection cells, Dev. Brain Res., 13 (1984) 257-263. 28 Kaiserman-Abramof, I.R., Graybiel, A.M. and Nauta, W.J.H., The thalamic projection to cortical area 17 in a congenitally anopthalamic mouse strain, Neuroscience, 5 (1980) 41-52. 29 Koelle, G.B. and Friedenwatd, J.S., A histochemical method for localizing cholinesterase activity, Proc. Soc. L~p. Biol. Med., 70 (1949)617-622. 30 Kostovic, I. and Goldman-Rakic, P.S., Transient cholinesterase staining in the mediodorsal nucleus of the thalamus and its connections in the developing human and monkey brain, J. Comp. Neurol., 219 (1983) 431-447. 31 Kostovic, I. and Rakic, P., Development of prestriate visual projections in the monkey and human fetal cerebrum revealed by transient cholinesterase staining, J. Neurosci.,

4 (1984) 25-42. 32 Kristt, D.A., Acetylcholinesterase in the ventrobasal thalamus: transience and patterning during ontogenesis, Neuroscience. 10 (1983) 923-939. 33 Kristt, D.A., AChE in immature neurons: relation to afferentation, development, regulation and cellular distribution, Neuroscience, in press. 34 Kristt. D.A. and Waldman, J.V., The origin of the acetylcholinesterase rich afferents to layer IV of infant somatosensory cortex: a histochemical analysis following lesions, Anat. Embryol., 163 (1981) 31-41. 35 Kupfer, C. and Palmer, P., Lateral geniculatc nucleus histological and cytochemical changes following afferent denervation and visual deprivation, Exp. Neurol.. 9 (1964) 400-409. 36 Land, P., Dependence of cytochrome oxidase activity in the rat lateral geniculate nucleus on retinal innervation, J. Comp. Neurol., 262 (1987) 78-89. 37 Lund, R.D., Cunningham, T.J. and Lurid, J.S., Modified optic projections after unilateral eye removal in young rats, Brain Behav. Evol., 8 (1973) 51-72. 38 Land, R.D., Lund, J.S, and Wise, R.P., The organization of the retinal projection to the dorsal lateral geniculate nucleus in pigmented and albino rats, J. Comp. Neurol., 158 (1974) 383-404. 39 Lund, R.D. and Mustari, M.J., Development of the geniculocortical pathway in rats, J. Cornp. Neurol., 173 (1977) 289-306. 40 MacKay-Sire, A., Sefton, A.J. and Martin, P.R., Subcortical projections to lateral geniculate and thalamic reticular nuclei in the hooded rat, J. Comp. Neurol., 213 (1983) 24-35. 41 Manford, M., Campbell, G. and Lieberman, A . R , Postnatal development of ipsilateral retino-geniculate projections in normal albino rats and the effects of removal of one eye at birth, Anat. Embryol., 170 (1984) 71-78. 42 Mathews, M.R., Cowan, W.M. and Powell, T.P.S., Transneuronal cell degeneration in the lateral geniculate nucleus of the macaque monkey, J. Anat. (Lond.), 94 (1960) 145-169. 43 Mathews, M.A., Narayanan, C.H., Narayanan, Y. and Sigenthaler-Mathews, D.J., Inhibition of axoplasmic transport in the developing visual system of the rat. III, Electron microscopy and Oolgi studies of retino-fugal synapses and post-synaptic neurons in the dorsal lateral geniculate nucleus, Neuroscience, 7 (1982) 405-422. 44 McAllister, J.P. II and Das, G.D., Neurogenesis in the epithalamus, dorsal thalamus and ventral thalamus of the rat: an autoradiographic and cytological study, J. Comp. Neurol., 172 (1977) 647-686. 45 Ohara, P.T., Lieberman, A.R., Hunt, S.P. and Wu, J.Y., Neural elements containing glutamic acid decarboxylase (GAD) in the dorsal lateral geniculate nucleus of the rat: immunocytochemical studies by light and electron microscopy, Neuroscience, 8 (1983) 189-211. 46 Parnavelas, J.G., Bradford, R., Mounty, E.J. and Liebermann, A.R., Postnatal growth of neuronal perikarya in the dorsal lateral geniculate nucleus of the rat, Neurosci. Lett., 5 (1977) 33-37. 47 Parnavelas, J.G., Mounty, E.J., Bradford, R. and Liebermann, A.R., The postnatal development of neurons in the dorsal lateral geniculate nucleus of the rat: a Golgi study, J. Comp. Neurol., (1977). 48 Peters. A. and Feldman, M.L., The projection of the

225

49 50 51

52

53 54 55 56

57

58

59

60

61

lateral geniculate nucleus to area 17 of the rat cerebral cortex. I. General description, J. Neurocytol., 5 (1976) 63-84. Rakic, P., Development of visual centers in the primate brain depends on binocular competition before birth, Science, 214 (1981) 928-931. Rakic, P., Specification of cerebral cortical areas, Science, 241 (1988) 170-176. Reese, B.E., The topography of expanded uncrossed retinal projections following neonatal enucleation of one eye: differing effects in dorsal lateral geniculate nucleus and superior colliculus, J. Comp. Neurol., 250 (1986) 8-32. Reese, B.E., 'Hidden lamination' in the dorsal lateral geniculate nucleus: the functional organization of this thalamic region in the rat, Brain Res. Rev., 13 (1988) 119-137. Reese, B.E. and Cowey, A., The crossed projection from the temporal retina to the dorsal lateral geniculate nucleus in the rat, Neuroscience, 20 (1987) 951-959. Ribak, C.E. and Peters, A., An autoradiographic study of the projections from the lateral geniculate body of the rat, Brain Res., 92 (1975) 341-368. Robertson, R.T., A morphogenic role for transiently expressed acetylcholinesterase in developing thalamocortical systems?, Neurosci. Lett., 75 (1987) 259-264. Robertson, R.T., Ambe, R.K. and Yu, J., Intraocular injections of tetrodotoxin reduce transiently expressed acetylcholinesterase activity in developing rat visual cortex, Dev. Brain Res., 46 (1989) 69-84. Robertson, R.T., Duran, M.R., Poon, H. and Yu, J., Effects of neonatal enucleation on transient patterns of acetylcholinesterase in the dorsal lateral geniculate nuclei of infant rats, Anat. Rec., 218 (1987) 115A. Robertson, R.T., Fogolin, R.P., Tijerina, A.A. and Yu, J., Effects of neonatal monocular and binocular enucleation on transient acetylcholinesterase activity in developing rat visual cortex, Dev. Brain Res., 33 (1987) 185-197. Robertson, R.T., Hanes, M.A. and Yu, J., Investigations of the origins of transient acetylcholinesterase activity in developing rat visual cortex, Dev. Brain Res., 41 (1988) 1-23. Robertson, R.T., HOhmann, C.M., Bruce, J.L. and Coyle, J.T., Neonatal enucleations reduce specific activity of acetylcholinesterase but not choline acetyltransferase in developing rat visual cortex, Dev. Brain Res., 39 (1988) 298-302. Robertson, R.T., Kim, G., Oh, L. and Yu, J., Transneuronal degeneration of thalamic neurons following deaffer-

62

63

64

65

66 67

68 69 70 71

72

73 74

entation: quantitative studies using 3H-thymidine autoradiography, Anat. Rec., 223 (1989) 96A-97A. Robertson, R.T., Seress, L. and Ribak, C.E., Electron microscopic studies of transiently expressed acetylcholinesterase activity in thalamus and cerebral cortex of developing rats, Soc. Neurosci. Abstr., 14 (1988) 868. Robertson, R.T., Tijerina, A.A. and Gallivan, M.E., Transient patterns of acetylcholinesterase activity in visual cortex of the rat: normal development and the effects of neonatal monocular enucleation, Dev. Brain Res., 21 (1985) 203-214. Satorre, J., Cano, J. and Reinoso-Suarez, F., Quantitative cellular changes during postnatal development of the rat dorsal lateral geniculate nucleus, Anat. Embryol., 174 (1986) 321-327. Shirokawa, T., Fukuda, Y. and Sugimoto, T., Bilateral reorganization of the rat optic tract following enucleation of one eye at birth, Exp. Brain Res., 51 (1983) 172-178. Silver, A., The Biology of Cholinesterases, North Holland, Amsterdam, 1974. Sloper, J.J., Headon, M.P. and PoweU, T.P.S., A comparison of cell size changes in central and pericentral representations within the primate lateral geniculate nucleus following early monocular deprivation, Dev. Brain Res., 40 (1988) 61-64. Storm-Mathisen, J., Quantitative histochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Neurochem., 17 (1970) 739-750. Sukekawa, K., Changes of cytochrome oxidase activity in the rat subcortical visual centers after unilateral eye enucleation, Neurosci. Lett., 75 (1987) 127-132. Tsang, Y.-U., Visual centers in blinded rats, J. Comp. Neurol., 66 (1937) 211-261. Warton, S.S., Dyson, S.E. and Harvey, A.R., Visual thalamocortical projections in normal and enucleated rats: HRP and fluorescent dye studies, Exp. Neurol., 100 (1988) 23-39. Wiesel, T.N. and Hubel, D.H., Effects of visual deprivation on morphology and physiology of cells in the cat's lateral geniculate body, J. Neurophysiol., 26 (1963) 978993. Williams, R.W. and Herrup, K., The control of neuron number, Annu. Rev. Neurosci., 11 (1988) 4232-4253. Zilles, K., Wree, A., Schleicher, A. and Divac, I., The monocular and binocular subfields of the rat's primary visual cortex: a quantitative morphological approach, J. Comp. Neurol., 226 (1984) 391-402.