Delayed changes of chromogranin A immunoreactivity (CgA ir) in human striate cortex during postnatal development

Developmental Brain Research, 67 (1992) 333-341 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0165-3806/92/$05.00

333

BRESD 51469

Delayed changes of chromogranin A immunoreactivity (CgA ir) in human striate cortex during postnatal development Lee-Cyn Ang a, David H. George a, Deborah Shul a, De-Qun Wang b and David G.

Munoz b

Department of Pathology (Neuropathology), University of Saskatchewan and Royal University Hospital, Saskatoon, Sask. STN OWO (Canada) and bDepartment of Pathology (Neuropathology), University of Western Ontario and Uz:.iversit),Hospital London, Ont. N6A 5CI (Canada) (Accepted 25 February 1992)

Key words: Area 17, Cortical organization: Immunohistochemistry; Neuropeptide; Postnatal maturation: Visual cortex

The changes in chromogranin A expression in the human striate cortex from birth till 67 years were studied by immu~iohistochemical method in 18 autopsied patients. The first chromogranin A immunoreactivity (CgA ir) was identified at birth in layer IV (especially IVc) mainly as fine nerve terminals. By 6 months, the first perikaryal reactivity was noted in the large pyramidal neurons of layer V. The smaller neurons in layers IV, V and VI showed a progressive increase in CgA it from 15 months to about 17 years. At approximately 9 years, immunoreactivity began to be noted in supragranular neurons in layers II and III. The final laminar distribution of CgA ir seemed to be attained at about 25 years with relatively little change thereafter. The CgA ir in the striate cortex demonstrates a prolonged period of developmental changes, lasting from birth to about 25 years. INTRODUCTION Chromogranin A (CgA) belongs to a family of highly acidic, heat-stable glycoproteins secreted from chromaffin granules of the adrenal medulla a'42. It has been detected in other neuroendocrine cells in the gut, pancreas, adenohypophysis, thyroid, parathyroid and submandibular gland 6'7't1'28. In the nervous system, CgA has been demonstrated in the peripheral sympathetic neurons ~t,26, 37 cholinergic nerve terminals in skeletal muscles 49, and inner segments of the rods and cones 37. Immunohistochemicai studies have revealed widespread presence of CgA in different subpopulations of neurons in bovine and ovine brains ~. D N A hybridization studies have confirmed the presence of CgA in mammalian brains 22'24. More recently the presence of CgA in human brains has been demonstrated 35 by using a previously characterized monoclonal antibody, LK2H1027'52'~a. While its exact functions in the central nervous system (CNS) are still uncertain, it has a calcium binding property as'39, and is also known to be the precursor of at least two modulatory peptides: pancreastatin s'2L2a'25,4s, and chromostatin 14. The role of CgA as an autocrine inhibitory modulator is supported by data derived from studies on chromaffin cell cultures which demonstrate the secretion from chromaffin cells is controlled by chromogranin A-derived peptides 41. Interestingly, the chromogranin A

levels in rat brains are not altered by adrenalectomy or dexamethasone treatment (1 mg/kg for 6 days) 5°. Recently, CgA immunoreactive (ir) neurites have been noted to be a major component in senile plaques 36's~ and the ratio of CgA to synaptin/synaptophysin has also been found to be increased in the brains in Alzheimer and Pick disease 51. As there is widespread CgA expression observed in the human cortex in adults a's'a8 we seek to examine the differences in CgA ir in the striate cortex during development from childhood to adult life. MATERIALS AND METHODS

lmmunoblotting Striate cortex was obtained at autopsy from 5 individuals, age 58 to 80 years, ¢'ee of neurological disease. Postmortem interval ranged from 3 to 25 h. Histological examination of the contralateral cerebral hemisphere confirmed the absence of pathology. The tissue was frozen between two metal plates, and kept at -80°C until lyophilized. The tissues, crushed to fine powder, were homogenized (1:10, w/v) in ice-cold 20 mM Tris-HCl, 5 mM 2-mercaptoethanoi solution (pH 7.4) in a Polytron (Brinkmann Instruments) for 20 s (scale 5.5). After this, the samples were immediately boiled for 5 rain and centrifuged at 130,000 x g for 1 h. The resulting supernatants, containing the heat-stable CgA, were assayed for protein 29 and used for electrophoresis. CgA was purified from heatstable aqueous extracts of human adrenal gland by sequential ionexchange and gel-filtration chromatography as described elsewhere t9.40.so. Samples (protein concentration: 2.5 pg/l) were mixed with equal

Correspondence: L.C. Ang, Department of Pathology (Neuropathology), Royal University Hospital, Saskatoon, Sask. S7N OXO, Canada. Fax: (1) (306) 966-2223.

334 TABLE I Clinicopathologial data

Case

Age

Sex

PM interval (h)

Cause of death Diaphragmatic hernia and hypoplastic left lung postoperative death Congenital heart disease - single ventricle Pneumocystis of the lungs Ventricular septal defect - postoperative death Drowning Biliary atresia Tricuspid atresia Blunt abdominal trauma - transection of inferior vena cava Hepatocellular carcinoma Drowning Acute iron intoxication Chest and abdominal trauma Bronchial asthma with bilateral pneumothorax Chest and abdominal trauma Chest trauma Coronary heart disease Acute carobon monoxide poisoning Coronary heart disease

1

1 day

F

12

2

3 mos

F

17

3 4

6 mos 15 mos

M M

11 9

5 6 7 8

2 yrs 2.5 yrs 3 yrs 7 yrs

M M F M

24 21 17 7

9 10 11 12

7 yrs 9 yrs 14 yrs 17 yrs

M M F F

19 23 24 4.5

13

19 yrs

F

6

14

25 yrs

M

11

15 16 17

35 yrs 41 yrs 56 yrs

F M M

3 5 18

18

67 yrs

M

4

volumes of 2x sample buffer (20 mM Tris-HCI. pH 8.0. 2 mM EDTA. 5% SDS, 10~ 2-mercaptoethanol. 0.02% Bromophenol blue, final pH 7.05), belled for 5 rain and centrifuged at 1,000 × g for 10 rain. Electrophotesis was carried out on an automated horizontal apparatus (Pha:aSystem, Pharmacia, Bale d'Urfe, Oue.), using preformed 8-25% gradient acrylamide gels (PhastGels). The

94._

proteins were transferred from the gel to tlybond-ECL (Amersham, Oakville, Ont.) nitrocellulose membrane. The lane containing transferred standard protein markers were cut from the rest of the membrane, and the markers were stained in a solution of 0.1% India ink, 0.3% Tween 20, 0.9% NaCI in 0.0I sodium phosphate buffer, pH 7.4. The rest of the membrane was allowed to air d r y at room temperature for 30 min, fixed in 10% acetic acid, 25% isopropanol solution for 10 min, washed in H20 for 3 × 15 min, blocked in 5% dry milk - TPBS (0.3% Tween 20 in 50 mM PBS, pH '1.4) overnight, washed in TPBS for 3 x 15 min, incubated in LK2H10 antibody diluted 1:500 in TPBS for 90 min, washed in TPBS as above, incubated in 1:200 diluted goat anti-mouse IgGHRP antibody, diluted 1:200 for 90 min, washed in TPBS as above, incubated in a 1:1 mixture of ECL solutions A and B (ECL Western blotting detection system, Amersham) for I min, and exposed in the dark to ECL Hyperfilm (Amersham) for 30 s.

lmmunohistochemistry

Fig. 1. lmmunoblots utilizing the CgA antibody LK2HI0. Left lane: purified CgA from human adrenal medulla. Right lane: heatstable proteins from human striate cortex. Molecular weight standards are indicated by the lines on the left. representing 94, 67, 43.30, 20.1 and 14.4 kDa, starting from the top.

Striate cortices were obtained from brains of 18 autopsied patients (7 females and I1 males) with postmortem intervals ranging from 3 to 24 h (mean 13.1 h). These patients, whose ages ranged from 1 day to 67 years, had died from non.neurological causes, in particular with no history of blindness (congenital or acquired), mental retardation or dementia (Table I). Ten of them were below 10 years, and 7 were 3 years and below. The gross appearance of these striate cortices and subsequent microscopical examination had to be morphologically normal before they were included in the study. The striate cortices were disected from coronal sections of tile cerebral hemispheres approximately 1.5 cm from the occipital pole and immediately fixed in picric acid-paraf°rmaldehyde-glu" taldehyde (PPG) mixture for 48 h 43. This mixture has been used successfully in CgA studies both in human and other mammalian brains 35"3~'44. These tissue blocks were then washed four times in

335

!

RESULTS

Electrophoresis and immunoblotting Heat-stable extracts of striate cortex demonstrated multiple bands on acrylamide gels, but on Western blots immunostained with LK2H10 a single major band with apparent molecular weight 72 kDa was present, homologous to the band in the preparations of purified CgA from adrenal medulla (Fig. 1). Three of five cases showed an additional very faint band with apparent molecular weight 85 kDa, presumably corresponding to the proteoglycan form of CgA sl (not shown).

Immunohistochemistry

25Y J

ib I

Fig. 2. The striate and peristriate cortex at 2, 25 and 56 years. Note the intense CgA ir in IVc in the striate cortex~which is less apparent in the peristriate cortex. The cortex shows intense CgA ir whereas the white matter is unstained in all 3 cases.

0.01 M phosphate buffer (PB) pH 7.4. Blocks were cryoprotected by overnight incubation in 30% sucrose in PB, embedded in Tissue-Tek OCT (Miles Scientific, Napierville, IL) and left at -20°C until frozen. The blocks were then cut in a cryostat at 20 and 50 g m thicknesses. Free-floating 50/~m sections were immunostained with LK2H10 (Hybritech Inc., San Diego, C,~) at 1:10,000 dilution according to the avidin-biotin-peroxidase L~ethodafter endogenous peroxide activity was blocked by incubation with ~ydrogen peroxide2°'3s. Parallel 20 gm sections were stained with Nissl preparation.

Despite the marked differences in postmortem intervals that existed among the various patients, the CgA ir in the striate cortices remained relatively stable within similar age groups. This could be due to the fact that most of these patients died in hospital and the bodies were refrigerated within a few hours of death. Even though the laminar striatification was better developed in the adult striate cortex, in all the cortic-es studied (irrespective of ages) there was a distinct demarcation between the striate cortex and the adjacent pe~istriate, highlighted by the presence in IVc in the striate cortex of intense CgA Jr, which was not apparent in the peristriate cortex (Fig. 2). Furthermore, there was selective immunoreactivity in the cortex as opposed to the white matter, which was largely unstained. At day 1, definite CgA ir in IVc was evident as fine reticulate and granular staining in the neuropil (Figs. 3a and 4). The immunoreactivity ended abruptly at the border with layer V but merged imperceptibly with the rest of the supragranular layers including layers I and II which also showed some degree of immunostaining. No perikaryon was highlighted at this stage. The striate cortex at 3 a~d 6 months showed somewhat similar neuropil staining, but in addition, in the 6 months cortex, scattered large pyramidal-shaped neurons were identified in layer V, especially Vb (Figs. 3b,c and 4). The CgA ir in these neurons was diffusely expressed in a granular pattern in the perikarya and the proximal dendrites (Fig. 5). No axonal staining could be seen. By 15 months, there was a definite staining of CgA in a number of smaller neurons in layer V and V! (Figs. 3d and 4). The neuronal staining of layer IVc at this stage was most inten,~e, At this age, the large pyramidal neurons in layer V had well-defined proximal axons which could be traced to the subjacent white matter (Fig. 5). At 2 and 3 years, the CgA ir was quite similar to that at 15 months except for the definite condensation of reactivity in the perikarya of layer IVc while the neuropil

336 a

ld b

3mo c

6mo d 15mo o

2,/

f

7y

3y g

I II Ul

III

IV

IV

V

V

VI

VI

h

9y i

14y j

17y k

I

19y I

25y m

35y n

56y I

II Ill

III

IV IV

V

!

VI

Fig, 3. The distribution of CgA ir in the striate cortex at various ages. a: day I; b: 3 months; c: 6 months; d: 15 months; e: 2 years; f: 3 years', g: 7 years: h: 9 years: i: 14 years: j: 17 years: k: 19 years: h 15 years; m: 35 years and n: 56 years. The period of significant changes in pattern of CgA ir lasted from day 1 to 25 years.

staining in this layer became less intense (Figs. 3e,f and 4). By 7 and 9 years, CgA ir was noted in an increasing number of the smaller neurons of layers V and VI, and more intense neuropil staining was noted in the supragranular layers (Figs. 3g,h, 4 and 6). Occasional immunoreactk ~ neurons were also noted in the supragranular layers at 9 years (Fig. 6). The CgA ir at 14 years identified more perikarya in layer VI (Fig. 3i). From 14 years to 25 years, there was a significant increase in perikaryal staining of neurons in layers II and III (Figs. 3i-l and 6). A vague laminar pattern of neuropil staining gradually emerged at 19 years: staining was most intense in layers I, II and III, moder-

ately intense in IV and VI and least intense in V (Fig. 3k). From 25 years onwards, the laminar pattern of CgA ir was more or less maintained throughout adulthood up to 67 years (Fig. 31-n). In the adult cortex, perikaryal CgA ir was noted in all layers except layer I, though the neuropil staining was seen in all cortical layers. There was, however, some suggestion of an increase of CgA ir in the neuropii with increased age even in adulthood (Figs. 2, 3 and 6), and this appeared to be unrelated to background staining as the white matter was still largely unstained. The large neurons in IVb, known to project to the ipsilateral superior temporal sulcus, showed no Cg ir 46. In contrast to the major changes in CgA ir, there was

337

ld

6mo

15mo

2y

7y

9y

• :'?:

IV

IV

Li," VI

l aoo

.:j!l VI

I" .

.'.

• " ":

" " :'

"~," i

"" 1

Fig. 4. Higher magnification com ~anng the CgA ir in layers IV, V and VI in striate cortices from day 1 to 9 years. Note the granular and reticulate neuropil staining at day 1 to 15 months compared to more definitive perikaryal staining from 2 years onwards in the IVc. More cell bodies are also visible in layers IV and VI at 9 years. The large pyramidal cells in layer V become apparent only at 6 months.

very little change of laminar pattern in the striate cortex from birth to adulthood in the Nissl preparations (Fig.

7). DISCUSSION We confirm the presence of CgA in Western blots of heat-stable extracts from human striate cortex using a monoclonal antibody. Weiler et al. have demonstrated

similar findings in the Western blot using a polyclonal antiserum 51 against CgA. The higher molecular mass form of CgA previously identified as the proteoglycan form of CgA 5°'5~ is present as a minor fraction only in the normal adult cerebral cortex. This latter result may not apply to other regions of the cortex as major differences exist in the rat between cortical regions in respect to the ratio of the proteoglycan to other forms of CgA 5°. Even though a previous study on ovine and bovine

Fig. 5. The CgA ir in a large pyramidal neuron in V with immunostaining of perikaryon at 6 months, and additional immunostaining of the proximal dendrites and a segment of axon (arrow) at 15 months.

338

7y

9y

17y

25y

II III

III

IV

IV

Fig. 6. Highe: magnificationcomparing the CgA ir in layers I, II, III and IV in the striate cortices at 7, 9, 17, 25 and 56 years. Note the increasing perikaryal staining in layers II. III, IVa and IVb from 9 to 25 years.

brains demonstrated definite perikaryal CgA in the neecortex, it described only sparse CgA ir nerve fibers in the neuropil~. In adult human cortex, there is intensive and extensive CgA ir not only in the perikarya but also in the neuropii 35. This intense neuropil staining in human cortex has been attributed to staining of distal dendritic arbors and minute puncta 3s. The difference in CgA ir between ungulate and human cortex could be duo to intrinsic variations between species or to the different antibodies used in these experiments. In this study, CgA ir exhibits variability in regional distribution corresponding to different cytoarchitectonic areas such as the striate cortex and the peristriate cortex. However, the most dramatic finding is the markedly delayed acquisition of the adult pattern of CgA ir in the striate cortex during development. While the marked variability of observed immunostaining between different ages might be attributed to tissue quality, fixation artefacts, staining artefacts, etc., the progressive nature of the changes and similarities in the pattern among patients in the same age groups suggest the CgA ir is related to age differences. The earliest CgA expression noted in the neuropil of layer IV, namely IVc, has been interpreted as nerve terminals 35'44. The first perikaryal immunostaining, however, was noted in the large pyramidal neurons at layer V. These are projection neurons, whose axons extend into the subcortical white matter to terminate mainly in the subcortical areas, such as the brainstem (superior colliculi) involved in extraocular movement ~5.46.Some of the smaller CgA ir pyramidal neurons in layer VI, first seen at 15 months, probably have projections to the thai-

amus, especially the lateral geniculate nuclei ~5'4~. By 3 years, the perikarya of neurons in layer IV were better defined by CgA-li; these are likely to be stellate neurons. The last group of neurons defined by CgA ir at 9 years and later were those in layers II and III which have projections to other association visual cortices such as areas 18 and 19ts'46. The final laminar pattern of the striate cortex was attained at about 25 years, and this pattern was maintained essentially unchangec' up to 67 years. The early postnatal changes in CgA ir could be related to the 'critical period' of cortical visual development and evolution of mental functions during early childhood. Studies on the neocortex have suggested that such early changes could be related to dendritic maturation 2'°, which in turn is dependent on the arrival of cortical affere~lts t7'54. Changes in other neuropeptide distribution such as vasoactive intestinal polypeptide (VIP), somatostatin, cholecystokinin (CCK), avian pancreatic polypeptide, and substance P in mammalian striate cortex have been shown to occur during early postnatal neural development9't6'3°'3t'32'33. Postnatal increase of noradrenergic, serotonergic, glutamatergic, GABAergic and cholinergic innervation of the visual cortex in monkeys have also been demonstrated ~2'~3. Similarly, there are vision-dependent changes affecting the glutamic acid receptor (NMDA) in cat visual cortex4. Calcium-binding proteins such parv~lbumin and caibindin-D28k have been observed to change their distribution during early revel. opment in cat visual cortex 45. An immunohistochemical study with SMI-32 has revealed the progressive changes in the expression of neurofilament protein in the first 15

339

a

ld

b

3mo c

6too d

15mo e

2y

3y a

7y

Ul

III IV

IV

V

V Vl

VI

h

9y

i

14yi

17y k

19y I

25y m

35y n

56y I

II III

IV

III

IV

V

V

VI

Vl

Fig. 7. Nissl stain on striate cortex at ! day, 3 months, 6 months, 15 months, 2 years, 3 years, 7 years, 9 years, 14 years, 17 years, 19 years, 25 years, 35 years and 56 years with very little change in overall staining pattern.

months of life in the human striate cortex ~. Therefore, the changes of CgA ir during early development are comparable to those of other neuropeptides and neuroactive substances, and like many of these substances the development of CgA expression is a postmigratory phenomenon occurring after birth. Following the early cortical changes, there was a period of continuous development in CgA ir up to age 25 years. However, major changes in CgA expression wi~re especially evident from about 9 to 25 years, when the

final laminar pattern and immunostaining of layers II and III neurons were established. Unlike the earlier postnatal period of cortical development, there is no definite correlate for these late developmental changes in CgA it. In fact, there are very few neuroanatomical substrates for late CNS development in humans. One of these is the increase in dendritic branchings noted with Golgi studies that could be observed until late adulthood s. This finding correlates well witF~other experimental ~.udies indicating that the capacity for synaptogene-

340 sis in the invertebrate and m a m m a l i a n CNS continues beyond early development 16. It is also known that the commissural fibers in the corpus callosum are not fully myelinated until the teens, and the myelination of association fibers may continue until early adulthood "ss. More recently Mesulam and Geula 34 have provided yet another morphological correlate for late cortical development using acetylcholinesterase (Ach E) histochemistry in the study of the hippocampus and auditory association cortex. In their study, Ach E-reactive neurons were virtually absent in the cortex during early postnatal life: these neurons became established by adolescence, and increased in density through early adulthood. They considered Ach E-reactivity in the cortex to represent a

morphological correlate for the late mental development occurring in the teens and early adult life associated with acquisition of cognitive and behavioral skills characteristic of adults. The CgA is perhaps the first neuropeptide that exhibits demonstrable alterations during late cortical development and therefore could provide another morphological correlate for studies in neural development during the period from late childhood to early adulthood.

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Acknowledgements. This work was supported by grants from the Saskatchewan Health Research Board and College of Medicine. University of Saskatchewan. The authors thank Robert van den Beuken for photographic assistance and Mavis Hopeweli and Deanna Turetski for preparing the manuscript.

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