Anat Embryol (1991) 183:397-413

Anatomy and Embryology 9 Springer-Verlag1991

Distribution of GABA and neuropeptides in the human cerebral cortex A light and electron microscopic study* W.Y. Ong 1 and L.J. Garey 2 1 Laboratory of Neurobiology, National Institute for Medical Research, London NW7 1AA, UK 2 Department of Anatomy, Chafing Cross and Westminster Medical School, London W6 8RF, UK Accepted January 10, 1991

Summary. Antibodies were used to identify neurons in

human frontal and temporal cortex that were immunopositive to 7-aminobutyric acid (GABA) and the neuropeptides vasoactive intestinal polypeptide (VIP), substance P (SP) and somatostatin (SOM). Specimens were taken at surgical biopsy and fixed immediately after removal. The results described for both light and electron microscopy were obtained when relatively high concentrations of glutaraldehyde (2.5-3%) were present in the fixative. Specimens were examined from three adults and an infant aged 5 months. GABAergic neurons were present in all cortical layers, with fewest in layers I, deep III and V, and were mainly small, and round or oval. No labelled pyramidal neurons were detected. GABAergic puncta were common in the neuropil, probably representing axonal profiles. VIP-neurons were also found in all layers, including layer I, and were approximately twice as numerous as GABA-cells. SP-positive cells were found throughout the layers, but were sparse in layers I and VI. They were about three times commoner than GABAergic neurons. SOM-reactivity was demonstrated in about the same number of cells as that for SP. Again, this involved all layers, but layer I least. Peptidergic neurons were larger, on the average, than GABAergic cells, and were frequently pyramidal in character. In the infant, the distribution, size and frequency of immunoreactive neurons were similar to those in the adult. However, GABAergic puncta were commoner. Key words: GABA - Peptides - Immunocytochemistry - Cerebral cortex - Child - Human

Introduction

The deonstration of 7-aminobutyric acid (GABA) in mammalian neurons has been simplified by the tech* This paper represents part of a study for the degree of Ph.D. in the National University of Singapore by WYO while at the Department of Anatomy, National University of Singapore. Offprint requests to: L.J. Garey

nique of immunocytochemistry. The cerebral cortex of several species, including primates (Hendrickson et al. 1981; Houser et al. 1983; Hendry et al. 1983, 1984a, 1987; DeFelipe et al. 1985, 1986; Fitzpatrick et al. 1987; Huntley et al. 1988; Ong and Garey 1990b) has been studied with the technique, using antibodies to GABA or its synthesising enzyme, glutamic acid decarboxylase. About 25% of the cortical neurons are GABAergic and are non-pyramidal morphologically. Other studies conclude that certain GABAergic neurons also contain neuroactive peptides, such as vasoactive intestinal polypeptide (VIP), substance P (SP) and somatostatin (SOM), which may act as transmitters or neuromodulators (Hendry et al. 1984a, b; Jones and Hendry 1986). These peptides have been localised by immunocytochemistry in the cerebral cortex of various primates (Beach and McGeer 1983; Hendry etal. 1984a; Campbell etal. 1987; Huntley et al. 1988). GABA, VIP, SP and SOM have also been demonstrated in the human cerebral cortex in a number of biochemical (Emson et al. 1979, 1981; Cooper et al. 1981; Davies and Terry 1981; Geola et al. 1981) and immunocytochemical studies (Sorensen 1982; Vincent et al. 1982a; Chronwall et al. 1984; Emson and Hunt 1984; Braak et al. 1985; Roberts et al. 1985; Chan-Palay 1987; Gasper et al. 1987; Schlander et al. 1987; Manolidis and Baloyannis 1987; Schiffmann et al. 1988). However, some authors indicate that post-mortem changes can influence the quality of neuronal labelling. SOMpositive axons are poorly visualised in autopsy specimens compared with those obtained at biopsy (ChanPalay 1987), and GABAergic somata may not stain well in autopsy specimens (Schiffmann et al. 1988). To date, relatively little is known about the comparative numbers and distribution of GABAergic and VIP-, SP- and SOMimmunoreactive neurons in cortex obtained at biopsy. As alterations in the GABAergic and peptidergic systems may be associated with various pathological conditions, such as epilepsy (Van Gelder and Courtois 1972, Van Gelder et al. 1972; Emson and Joseph 1975; Lloyd eta1. 1981; Higuchi etal. 1983; Krnjevic 1983), Alzheimer's type dementia (Davies and Terry 1981; Crystal

398

9 9

".~

9 . 9 o"

~

9

9

,,

9

9

9 ..

9

~

9 1 4 9 o,

~

" ~

~

.9

9

9

9149

9

2--7. 9

9149

'

I

9

.%

9 9

i

..

: .:. 9 ...

9

III

~149

91 J

.

II

o

9176 9

9 ~

9149

9

9 ~149 ,

9

9

9

.':

;"

*."

.

.

-.

;

III

,

, -

.:. 9

I

I

..

IV

9

,~

.: 9

~m

~

9

.

9149

9

9 9 9

9

":

9

9

":

9

%' 9149

"::':"...,2 .~

.

9 ;

9 .

..

IV

9

B I 0 I

Q

I

9

Q.

i

I

9 9

Vl

I I

~ 9

9

~ ~

".

9 9 9

A

9

9

o

9

VI

9

C 9

9

."

"9

9"

9

9

9

"

"" ".'-..

"'

.

9

"

.,*

l

9

~ 9

" 9 9 "".~.."

~

9

:

""

'

"

"

"'"

*

.

.

9 9

,,

9. ,

. "9

9 ,

,

9

.

..

"'.....

,,"

9

*

9 ~ 9

9

9 9

"

9

9

. 9

" ~,

9

9 ,

9

9

9

9

.,

.

.

,*

,

9

,

9176

9

9

9. . : ' . : - . . . . . . . . . ? 9

$ D

o,

III

*,

9 ,

.

9 9 ,

9

,:

.:

* ",

-

9.

9

"

,

,%

~

9

,

9149

,',

9

,

9

,

9

9

Ill

9

~ ?

~149

9

. .'-..

.

9 9

~

~

,~

II

":

"4',.

;...

..,

,, ..

9

u

~

" g

o

9

~

9

9

9176 .".,

9

9

~

%9 9

IV

.

.

;

9

,"

.

9

9

. ,,

9 9

|

~

i

9

Q

DI

*,

9

9"

, 9

9

9

;

- 9149

".

IV

9 9149 o, 9 9~

9

~

99

~149

o

~

~

'. ;

V 9

~

9 9

~ ~176 ~176 .

~ ..

VI

9

~

9

Vl

9 ~ ~

B

D

Fig. 1 A - H . D i s t r i b u t i o n o f G A B A e r g i c a n d peptidergic n e u r o n s in adult a n d i n f a n t frontal cortex, f r o m c o m p u t e r p l o t s T h e cortical layers are indicated o n the right o f each section; each d o t repre-

sents one i m m u n o p o s i t i v e n e u r o n . A G A B A , infant. B G A B A , adult. C VIP, infant. D VIP, adult. E SP, infant, F SP, adult. G S O M , i n f a n t H S O M , adult 9 Scale b a r 200 ~ m

and Davies 1982; Chan-Palay etal. 1985; Morrison et al. 1985 ; Roberts et al. 1985; Chan-Palay and Yasargil 1986; Nakamura and Vincent 1986; Chan-Palay 1987), schizophrenia (Ferrier et al. 1983) and Huntington's disease (Marshall and Landis 1985), the present study was carried out, using surgical biopsy specimens of human cortex, to further elucidate the morphology and distribution of its GABAergic and peptidergic neurons, and to

compare them with the pattern in non-human primates (Ong and Garey 1990b). Materials and methods

Specimens. T h r e e

s p e c i m e n s o f a d u l t cerebral cortex a n d one f r o m a n infant were o b t a i n e d d u r i n g n e u r o s u r g i c a l operations. Some blocks f r o m each were prepared for s t a n d a r d electron m i c r o s c o p y

399 /

f

J

~ 9

,.

~

9149149

,9

'~149 9

9

") ".7

." " . '

..-,

9 .': ..."

9

9,

:

9 "" t

9149 9

9 {

9

..

."

"9

9

;

". . ' :

"

..1

9

9

:

'..'.

9

9

9

.

:-..

* .

,

"9

,,

,,,

.

9

"(.,

.9

"-.

.::

"..

" 9

9

99

.

9

9 ,

.

:,'"

," ,,,,, 9

%,

:'.:

."

.

9

:

". 9

..

":'~:

:"

,',,,

;::; i;i

". ....

9.

,...:

. . ~ ..

.,,

Vl

,,

.

III

.

.. :

9.

. %

,,

s

.

"o

"..

IV

.

:.''..

;9 9

IV

V

9

*~"

" .

.9

'' 9

:;

.

9

.:

9

."

"-~ :.. 9.

* 9

.

..:

.~176

2"

9

.."

: 9

.

9,

"', ..

.

.

.

9

"":"'':"

;. 9

9 :

9.

: . . . .

"',:"

9

~

...':..-..

9 1 7 6

9 ":"

9

III

. , .% . . 9. . o

9

,,

9

"."''

..'-..-'.

,-,

9 " n *,"

9

.

9149

9

9 ,,

9 ;

,.

":

,,,,;

,,

.-,

-.:..

"..

9

.

9

.,

II

9

,,,

9

7"

...

".,

:.:).

.~

9 ."

**~"

...-:." 9

,%

....

9

:,

.:-

9 ;.

..

9

.*.o,.

::,,,',.,

: ...:

""

: .,.... ":.-

:'.::~:".... 9

""

I

9 ~

9 ).. "-?: :'-.:. 9 "

9

~

Vl

.':

9".

.

-

.:'2..

9

.9

...:.~

,,~,

9

,.

~

,

-.

.

9 ~ ',"

G

E

~ ;

.' 9

"~

9

.'..

S

~ 1 4 9 1 4. 9

~

.....

s

..:; . . . . . : . . . . . , ~

..,

.

.

9 1 4 99 9

;

" 9

*

9 .

9

:., ~

,9

.

9

:, :,

";

:

9

;"

:

9 .9

:

, ,,

9 , 9

.

,9

;

"9

9 9 9 1 4 9 1 4. 9

:, , , , ~

:,,.

. 9149 .

,:"

IH

..

..

.

," ",

i.

..-.

-"

"

. .

9 9

9

-"

":i:

9, 9

9 ~

.

.

.

"

9

".

o

.' 9 .

"-"i"

..... !'-

;

9.'." . . . . . .. .....:. 9

9

..~. "..." :.

9

9

"" "" ...:'...'...

.

.

.9 .

.,

. ":

,

".

.

9

9

"

9

,

"':C 9

"

.

.

,

,"

,.

.o

9,

F

"" 9

9

"9

9149

9

~

.

Vl

":'

9

:,'',:',

IV

" .'.

.9

"

,

9

"

,:

' ' "

...

"

:9

-

9 9

.

.

.

.9 .

.

9

.

., 9 ~

. 9

."

" 9

9

V

,.

:

9 .

9

9

."

.

9

. 9 ,9

9

2" .'"::

9 9 9

~

9

.

,, , 2 , , ;

'"

9

.. 9

9

. : 9 9

Fig.

" ...'.

Ill

;'.. ".

"~'.: 7.-:...'.'...

9

,"

":.. .

.-; o

. 9 "s,"

II

9 o

9

9

0

9

v

:

:."

.. "." ......

"...":::...

"'""'"

...-

""

9 .9 9

,,"'.

9" .

:,, -

.

~ .

.

;::-

."

9 .

.

..'.

,:,,

9 ....

.

". :.

.9 "

"

i i::;

-: ..:'..-.;...

9

:

:~

j'.,

.

,,

. 9

,:

9

,,' 9

o,

". 9

I

,:

.

"':'"""':7".""

.

9

:

.

;,

v

-....:.

s

9

~

""

9

" : " ~ ,9o ~ 1 4"9 ' "9

9 :

9 9

9

~

9 :-.:. :.

. "~

9

9

5.:... 9149149149 9

,

; .

~

9

9

9

"" " 9

. ;.

;

9

II

9

9149

. 9 9 ""* o 9

; .

9

. ' . r

:

9 9"

:"

....: 9

!.'. 9 .-.

9

9

9 .

9149

:

9

~ o

9 :

""

"

".

VI

9

H

1 E-H

(Ong and Garey 1991), and others were used for the present study. One specimen from the middle temporal gyrus (area 21 of Brodmann 1909) was removed to provide surgical access to a deep left temporal lobe tumour in a 58-year-old man. At operation, the temporal cortex appeared normal and without oedema. A second specimen was excised from the left frontal cortex ahead of the precentral gyrus (probably area 8 or 9) during surgery for a deep frontal lobe metastatic tumour in a 61-year-old man. This cortex also appeared normal and well vascularised. The third specimen was from a 60-year-old woman with a left frontal lobe men-

ingioma, and consisted of a block of normal cortex on the margin of the tumour. The precise cortical area could not be determined. The fourth specimen was from a case of left fronto-parietal porencephalic cyst in a male infant aged 5 months. A block that included the cyst wall and a strip of adjacent normal cortex, that was probably derived from areas 9 and 44, was examined.

Fixation. Specimens were fixed by immersion within seconds of surgical removal. The first was fixed in 1% paraformaldehyde (PF) and 2.5% glutaraldehyde (GA) in 0.1 M phosphate buffer. The

400 second specimen provided four blocks that were each treated differently, in order for the effect of fixation to be evaluated. Fixation was in, respectively, 4% PF, 4% PF plus 0.5% GA, 4% PF plus 1.25% GA, and 2% PF plus 3% GA. The third and fourth specimens were divided into three blocks each and were fixed in 4% PF, 4% PF plus 0.5% GA, and 2% PF plus 3% GA. Blocks were divided into smaller blocks a few minutes later. Except for the second specimen, which was sectioned 7 days after the biopsy, all were cut one day later at 100 gm by Vibratome perpendicular to the pial surface. Sections from the first specimen were incubated with antibody against GABA only, while sections from the other blocks were incubated with antibodies against GABA, VIP, SP and SOM.

Immunocytochemistry. Sections were washed for 3 h in three changes of phosphate buffered saline (PBS) to remove free aldehydes. They were then incubated for a day at 4~ with rabbit primary antibodies against VIP (ICN ImmunoBiologicals), SP, SOM and GABA (Sera-lab), diluted 1 : 500 with PBS. Sections were washed with three changes of PBS and incubated for 1 h at room temperature in a 1:100 dilution of biotinylated goat anti-rabbit serum, followed by three changes of PBS to remove unreacted secondary antibody. They were then reacted for I h at room temperature with an avidin-biotinylated horseradish peroxidase complex (Hsu et al. 1981). The reaction was visualised by treatment for 5 rain in 0.05% 3,3 diaminobenzidine tetrahydrochloride solution in tris buffer containing 0.05% hydrogen peroxide. In order to control for immunospecificity, the following experiments were performed : 1. Some sections were reacted as above, except that the primary antibody solution was substituted with PBS; they showed a complete absence of immunostaining. 2. Sections from all but the first case were also incubated with a 1:500 dilution of antiserum to leucine-enkephalin (Sera-Lab). These sections showed very few immunopositive cell bodies, but some stained fibres.

Light microscopy. The sections were washed in PBS, dehydrated and mounted with Permount. Sections fixed with 2.5% or 3% GA showed more immunostaining, especially for peptides, and were further analysed. Examples of different neuronal types were photographed and drawn using a drawing tube. Selected sections in which neurons appeared clearly in a single focal plane near the surface of the block, where the immune reaction was strong, were used for quantitative analysis by entering the coordinates of immunopositive neurons into a computer via a digital graphics tablet (Fig. 1). GABAergic and peptidergic neurons were counted in a 0.5 by 2 mm rectangle through the thickness of the cortex. The size of labelled neurons was measured in the third adult specimen and in the infant.

Table 1. Number of neurons immunoreactive to different antibodies (Ab) in a 0.5 by 2 mm rectangle through the thickness of the cortex, assessed in a fixed focal plane Ab

Adult

Infant

GABA VIP SP SOM

113 255 332 343

128 153 313 373

II, the superficial p o r t i o n o f I I I , a n d IV (Fig. 1 B, 2 A ) . M o d e r a t e n u m b e r s o f labelled cells were p r e s e n t in layerVI, while layer I, the d e e p aspect o f layer I I I a n d l a y e r V c o n t a i n e d o n l y small n u m b e r s .

Vasoactive intestinal polypeptide (VIP). M a n y V I P - p o s i tive n e u r o n s were f o u n d in all layers o f the cortex, inc l u d i n g layer I (Fig. 1 D), b u t were densest in layer II, the m i d d l e o f layer I I I a n d in layers IV, V a n d VI. T h e r e was a t e n d e n c y for the n e u r o n s to be a r r a n g e d in vertical clusters o r c o l u m n s . T h e r e were a b o u t twice as m a n y V I P - n e u r o n s as G A B A e r g i c n e u r o n s (Table 1). Substance P (SP). Even larger n u m b e r s o f n e u r o n s were labelled for SP in all layers o f the cortex, a l t h o u g h there were few in layer I (Fig. 1 F). M o s t positive s o m a t a were f o u n d in two h o r i z o n t a l b a n d s . T h e first was in layer II a n d the superficial t h i r d o f I I I ; the second, b r o a d e r b a n d e n c o m p a s s e d the d e e p p o r t i o n o f layer III, layer IV a n d the superficial h a l f o f V. R e l a t i v e l y few labelled n e u r o n s were f o u n d in the m i d d l e o f l a y e r I I I a n d layer VI. T h e d i s t r i b u t i o n was t h e r e f o r e u n l i k e t h a t o f V I P - p o s i t i v e cells, a n d they were n o t o r g a n i s e d in vertical c o l u m n s . S P - n e u r o n s were a b o u t three times m o r e n u m e r o u s t h a n G A B e r g i c n e u r o n s (Table 1). Somatostatin (SOM). N e u r o n s labelled for S O M were p r e s e n t in a b o u t the s a m e n u m b e r s as S P - n e u r o n s (Table 1). T h e y were d i s t r i b u t e d t h r o u g h o u t the d e p t h o f the cortex, t h o u g h t h e y were i n f r e q u e n t in layer I (Fig. 1 H). S O M n e u r o n s d i d n o t f o r m o b v i o u s h o r i z o n tal o r vertical a g g r e g a t e s (Figs. 1 H, 3 A).

Electron microscopy. Some sections from the second cortical specimen, fixed with 2% PF and 3% GA and incubated with antibodies against peptides, were further processed for electron microscopy. They were trimmed into smaller rectangular blocks, with the long axes perpendicular to the pia. These blocks were osmicated, stained with uranyl acetate, dehydrated in an ascending series of ethanol and embedded in Araldite. Semithin sections were cut for general survey before obtaining thin sections for electron microscopy. The thin sections were counterstained with lead citrate.

Results

Distribution of labelled neurons in the adult cortex GABA. T h e p a t t e r n o f d i s t r i b u t i o n o f G A B A e r g i c neur o n s in the t e m p o r a l a n d f r o n t a l cortices was similar. T h e y were f o u n d in all layers, t h o u g h m o s t were in layer

Size of neurons in the adult cortex (Table 2) In the f o l l o w i n g d e s c r i p t i o n , small n e u r o n s refer to those t h a t were less t h a n 12 g m in l o n g e s t d i a m e t e r , m e d i u m n e u r o n s to t h o s e b e t w e e n 12 a n d 17 ~tm, a n d large neurons to those t h a t were g r e a t e r t h a n 17 ~tm.

GABA. M o s t G A B A e r g i c n e u r o n s in the a d u l t g r a n u l a r a n d s u p r a g r a n u l a r layers were small o r m e d i u m (Table 2). L a y e r s V a n d VI c o n t a i n e d g r e a t e r n u m b e r s o f large n e u r o n s . Neuropeptides. T h e size d i s t r i b u t i o n o f n e u r o n s labelled for V I P differed f r o m t h a t o f G A B A (Table 2). M o r e large n e u r o n s were f o u n d in layers I I I a n d V, while the

401 Table 2. Size distribution of immunopositive neurons for different antibodies (Ab) in the various layers of the adult frontal cortex. Figures at the top of the columns indicate the range of longest somatic diameter (e.g.: 12/17=12 to 17 btm). Figures in the columns indicate the percentage of labelled neurons in each layer that fall within a given range of diameters. %>17=percentage of neurons 17 gm or more in longest diameter. N=number of neurons measured for each antibody Ab

Layer

Longest diameter (gin) 8/11 12/17 18/22 23/27 28/32 %>17

GABA N=291

VIP N= 269

SP N=496

SOM N= 312

I II III IV V VI I II III IV V VI I II III IV V VI I II III IV V VI

63 41 37 56 21 16 50 23 9 37 15 25 82 51 15

57 16 15 78 76 17 58 27 12

37 58 60 44 69 44 50 74 49 56 51 40 18 45 41 36 45 31 22 24 41 42 45 45

1 3 10 37

3

10 40

3 37 2 31 33

5 5 3 2

3 42 7 34 35

4 39 7 34 39

5 15

4 44 7 39 54

31

10

11 42

19 41

7 2

28 43

5

proportion of small neurons in each cortical layer was less, except in layer VI. VIP-positive small and medium neurons were found in approxmately equal numbers in layer I. SP- and SOM-positive neurons followed a similar size distribution to VIP, with large neurons in layers III, V and VI, and mainly small in layers I, II and IV.

Description of labelled neurons in the adult cortex GABA. GABAergic neurons were identified by a dense accumulation of label in the nuclei and cytoplasm (Fig. 2A). There were, however, some neurons that, although obviously immunopositive, were more lightly labelled. Most labelled neurons had small, oval-shaped somata. M a n y exhibited a single heavily labelled superficial or deep stem dendrite (Fig. 4A, B), which sometimes branched after a short distance, but others were frankly bipolar (Fig. 4C, D). Other GABAergic neurons were multipolar with three or more stem dendrites from various aspects of the soma. Bipolar and multipolar neurons constituted the majority of the large neurons in layers III, V and VI. Small punctate profiles, probably GABAergic terminals (Fig. 4A, B), were sometimes found around the cell bodies of neurons, some possibly in contact with the perikarya.

General features of peptidergic neurons. Neurons labelled with antibodies against the various peptides were found in all layers of the cortex. Material fixed with high concentrations of G A (3%) and a delay of less than 24 h between fixation and commencement of immunocytochemistry yielded large numbers of neurons with heavy labelling in both nucleus (except the nucleolus) and cytoplasm. Where there was a delay of 7 days, and in specimens fixed with low concentrations of G A (0.5%), labelling was often confined to the cytoplasm. Vasoactive intestinal polypeptide (VIP). VIP-immunopositive neurons were of different shapes and sizes. Some had no labelled processes, while most had only one. This was c o m m o n l y directed towards the pia (Fig. 5A, B), but dendrites in other directions were also observed. Some primary dendrites were given off smoothly from the soma, tapering to continue as small diameter dendrites. Other stem dendrites were given off more abruptly, with distinct " s h o u l d e r s " . They were usually of small diameter and most arose from rounded somata. Some neurons, particularly in layer VI, had a fusiform appearance due to the presence of superficial and deep dendritic trunks. Multipolar neurons were also encountered, with three or more dendrites of small diameter given off from various aspects of the soma. Some neurons in layer II and superficial layer I I I had morphological features of pyramidal neurons. They had oval or triangular somata, and an ascending dendrite arising smoothly from their superficial aspects (Fig. 5 A). These dendrites could not usually be traced beyond one or two somatic diameters, but in some neurons the density of the reaction product increased again after a short distance. Short segments of labelled dendrites were also c o m m o n l y found in the neuropil, probably the continuations of these dendrites. The majority of large neurons in layers III, V and VI were pyramidal, fusiform or multipolar. Substance P (SP). Neurons labelled for SP were observed in all cortical layers. M a n y resembled those described for VIP in having a superficially directed dendrite (Fig. 5 C) and some had morphological features of pyramidal neurons (Fig. 5 D). Others had a second stem dendrite emerging from another aspect of the soma (Fig. 5 E). M a n y neurons in layer VI had prominent descending dendrites. Others were fusiform with superficial and deep dendritic trunks given off smoothly from the soma. In all layers of the cortex, labelled multipolar neurons were found with four or five dendrites radiating from the soma (Fig. 5 F). As for VIP-neurons, the dendrites of SP-neurons could not be traced for more than one or two somatic diameters from the cell body, although m a n y vertical and horizontal labelled processes were found within the neuropil which could represent the continuations of these dendrites. As for VIP, most large neurons in layers III, V and VI were pyramidal, fusiform or multipolar.

Somatostatin (SOM). Neurons with a variety of morphologial features were labelled with antibody against SOM. Only one labelled dendrite could be distinguished

402

Fig. 2A, B. Photomontages showing the distribution of GABAergic neurons in the adult (A) and infant (B) cerebral cortex. Scale bar 200 gm

403

Fig. 3A, B. Photomontages showing the distribution of SOM neurons in the adult (A) and infant (B) cerebral cortex. Scale bar 200 gm

in the majority (Fig. 6), although the faint outline of another stem dendrite could sometimes be seen. Labelled dendrites showed the same variations in the manner in which they arose from the soma as described for the other peptidergic systems : some were given off smoothly while others were more abrupt. Some were directed vertically (Fig. 6B, E), while others were horizontal or oblique (Fig. 6A, C, D). Multipolar neuron in layers II

to VI had three or more stem dendrites from various aspects of the soma. Other neurons in layer VI had fusiform outlines (Fig. 6 E). The long axes of these cells were usually oriented radially, but sometimes obliquely. Immunopositive pyramidal neurons were also observed (Fig. 6B, F), as were neurons that resembled inverted pyramidal cells, with a single prominent deep dendritic trunk in the infragranular layers.

404

Fig. 4A-D. GABAergic neurons in the adult cortex. A, B Frontal cortex; C-D area 21 of the left temporal cortex. A neuron in layer IV with a single well-labelled, descending dendrite. The faint outline of a weakly labelled, ascending dendrite can, however, be discerned. B neurons in layer II, one with a triangular soma, with

its base towards the pia. C bipolar neuron in layer III, with two diametrically opposed stem dendrites. D neuron in layer III with two labelled processes, one directed obliquely, and the other towards the white matter. Scale bar 40 Ixm. Pial surface towards the top of the page

Electron miroscopy

Axons and dendrites. Many dendrites and myelinated ax-

Cell bodies. Optimum staining was seen only in sections within a few micrometers of the surface of immunoreacted blocks. Semithin sections showed that some neurons were labelled for peptides while others in the same sections were not. This was confirmed by electron microscopy. Both pyramidal and non-pyramidal cells were labelled with VIP, SP and SOM (Fig. 7A, B). Label was associated with free ribosomes and ribosomes on the rough endoplasmic reticulum, but was absent from mitochondria and the Golgi apparatus. We confirmed the observation made by light microscopy that some neurons contained heavily labelled nuclei, while other nuclei were less densely marked.

ons (Fig. 7C) were positive to antibodies against VIP, SP and SOM. In the axons, label was associated with filaments, while in the dendrites microtubules were labelled. The peptides were also found as a dense accumulation against the postsynaptic membranes in small dendrites or spines (Fig. 7 D). Peptide-positive axon terminals were also observed, although they were relatively less common.

The infant cortex (Table 3) The mean thickness of our specimen of infant cortex, measured at six locations, was 1.62 ram, a value compa-

Fig. 5A-F. VIP- and SP-containing neurons of the adult frontal cortex (A-B VIP; C - F SP). A labelled neurons in layer II. Some of these neurons are characterised by the presence of a superficial dendritic trunk. B labelled neurons in layer II. C neuron with an oblique dendrite. D probable pyramidal neuron in layer III. The

density of the labelled antibody decreases rapidly after half a somatic diameter. E neuron with single horizontal labelled dendrite 9 (arrow). F multipolar neurons in layer III. Scale bar: A 100 gin; B - F 50 gin. Pial surface towards the top of the page

Fig. 6A-F, SOM-containing neurons in the adult frontal cortex. A labelled neurons in layer III; note one with an oblique dendrite. B pyramidal neuron at the layer III/IV junction. C, D neurons with horizontal and oblique stem dendrites. E fusiform neuron

(arrowed) in layer Vii. F a low power micrograph to illustrate the large numbers of labelled segments of dendrites in the neuropil. Scale bar: A - E 50 lain. F 100 J-tin. Pial surface towards the top of the page

Fig. 7 A - D . Electron m i c r o g r a p h s o f pcptidergic n e u r o n s a n d processes. A p y r a m i d a l n e u r o n labeled for VIP. I,abel is associated with the free r i b o s o m e s b u t a b s e n t f r o m the Golgi a p p a r a t u s (arrow). B L a r g e n o n - p y r a m i d a l n e u r o n labelled for VIP. T h e s e neur o n s are characterised by the presence o f relativcly large n u m b e r s o f c y t o p l a s m i c organelles. C dendrite labelled for S O M . T h e anti-

b o d y is associated with the m i c r o t u b u l e s , b u t a b s e n t from the miloc h o n d r i o n (arrow). D A s y m m e t r i c a l s y n a p s e on a small d i a m e t e r dendrite or dendritic spine, labelled for S O M . T h e labelled antib o d y is present as a dense a c c u m u l a t i o n a g a i n s t the p o s t s y n a p t i c m e m b r a n e , b u t is a b s e n t frona the s y n a p t i c cleft a n d the p r e s y n a p t i c a x o n terminal. Scale bar: A 1.8 t.tm. B 3.6 lain. C i ~tm. D 0.6 lain

Fig. 8A-E. GABAergic neurons in the infant frontal cortex. A photomontage showing different types of neurons in layers I III. B--E higher power micrographs of the various elements seen in A. B immunopositive process with multiple varicosities, running vertically in layer II. C, D bipolar neurons, but with labelling mainly

confined to only one of the stem dendrites. Arrows indicate faintly labelled descending dendrites. E GABAergic puncta in apparent contact with the cell body of an unlabelled pyramidal neuron (arrow). Scale bar: A 80 lam. B 40 gm. C, D, E 20 gm. Pial surface towards the top of the page

Fig. 9A-F. Peptidergic neurons in the infant frontal cortex. A, B SP. C - F SOM. A labelled neurons in layers III-IV. The layer III/IV junction is indicated by the arrow. B higher power of a labelled pyramidal neuron in A. C labelled neurons in layer III. Some have morphological features of pyramidal cells. D higher power of labelled pyramidal neurons. In one, the labelling of the dendritic

trunk suddenly decreases after about one somatic diameter, but increases again, to produce the appearance of a labelled segment of dendrite (arrowed). E small round neurons in layer IV. These neurons are characterised by the presence of large nuclear cytoplasmic ratios. F labelled neurons in layer VI. Scale bar: A, C 100 I-tin. B, D, E, F 50 gm. Pial surface towards the top of the page

410 Table 3. Size distribution of immunopositive neurons in the infant frontal cortex, expressed as in Table 2. Ab

Effects of fixation

Layer Longest diameter (gm) 8/11 12/17 18/22 23/27 28/32 %>17

GABA N= 345

VIP N=268

SP N= 425

SOM N=405

Discussion

I II III IV V VI

49 36 27 44 10 7

51 64 54 56 60 51

19

19

30 42

30 42

I II III IV V VI

50 18 6 20 10 12

50 79 24 75 67 40

3 46 5 19 42

I II III IV V VI

81 15 9 4 11 5

19 79 12 88 39 33

6 62 8 28 57

I II III IV V VI

71 18 10 38 25 11

29 77 15 60 47 42

5 26 2 25 40

18

6

4 6

16

1

22 5

42 3 7

7

3 70 5 23 48 6 79 8 50 62 5 75 2 28 47

rable to that of adult cortex (1.61 mm). The infant cortex was also similar to the adult cortex in terms of the size distribution of the neurons, and in the morphological characteristics of GABAergic and peptidergic neurons. The laminar distribution of GABAergic neurons resembled that of adults (Figs. 1 A, B and 2A, B). A single labelled process was seen emerging from the superficial or deep aspects of most of these neurons, while some neurons had two or more well-labelled processes (Fig. 8 A, C, D). Small puncta were found in the neuropil, perhaps GABAergic terminals, often forming short chains in contact with the outlines of unlabelled pyramidal neurons (Fig. 8 E). Vertical, labelled proceses with multiple varicosities were also seen (Fig. 8 A, B). Large numbers of peptidergic neurons were found in the infant cortex (Figs. 1 A, C, E, G, 3 B, and 9). The number of SP- and SOM-immunopositive neurons was similar to that of adults, while fewer VIP-containing neurons were present in the infant (Table 1). The spatial distribution of cells in all three peptidergic systems was similar to that of the adult: there was a similar tendency for some layers to contain more of a given cell type, and for relatively large numbers of VIP-neurons to be present in layer I. SP neurons also occurred in layers II to V in the infant. The labelled neurons also displayed the same variations in dendritic morphology as in the adult (Fig. 9), and immunopositive pyramidal cells were common (Fig. 9 A D ) .

The importance of the delay between obtaining specimens and fixation has been emphasised (Chan-Palay and Yasargil 1986; Chan-Palay 1987; Schiffmann etal. 1988). The present study showed that the pattern of labelling of the neurons was dependent on the concentration of G A used in fixation, and the delay between fixation and immunocytochemical processing o f the specimens. Less nuclear labelling was obtained when low concentrations of G A were used in the fixative, and when there was a delay of 7 days between fixation and incubation with the primary antibody.

Specificity The following observations suggest that non-specific staining of the tissues is unlikely: 1. Although many neuronal somata were labelled for GABA, VIP, SP and SOM, only very few, faintly labelled cell bodies were found in sections incubated with rabbit anti-enkephalin. However, many enkephalin-posirive fibres were present, sometimes situated perineuronally. The paucity of enkephalin-positive cell bodies in the cerebral cortex, their weak staining, and the perineuronal distribution of enkephalin-containing fibres have been reported in the rat (Sar et al. 1978; Williams and Dockray 1983; Petrusz et al. 1985). Nonspecific binding of rabbit serum, in which the enkephalin and other peptide antibodies were raised, to the human cortical neurons is therefore unlikely. 2. Non-specific tissue binding of biotinylated goat antirabbit serum is unlikely since sections incubated with PBS in lieu of the primary antibody showed absence of staining. 3. The fact that pyramidal neurons were not labelled with anti-GABA, but were with anti-peptide, suggests that the peptide labelling of pyramidal cells is not nonspecific. Equally, the different proportions of neurons labelled with the different peptides, and their different distributions suggests specificity. 4. In both semithin sections observed by light microscopy and thin sections by electron microscopy, labelled and unlabelled neurons were frequently contiguous.

Size of neurons The size of labelled neurons is an important consideration, as there is a correlation between the size of neurons and their ultrastructural features in human cerebral cortex. Large neurons, taking a long diameter of 17 gm as a lower limit, differ from small and medium neurons in their ultrastructural characteristics, for example in having a relatively high ratio of cytoplasm to nucleus, and a greater density of cytoplasmic organelles (Ong and Garey 1991). Variations in morphological features may be due to differences in biochemical and physiologi-

411 cal characteristics of the neurons. The fact that both small and large neurons were labelled with antibody against the various neuropeptides, however, indicates that these ultrastructural variations are unlikely to be related to neuropeptide function. On the other hand, GABA-positive neurons have more constant ultrastrucrural features, at least in the supragranular layers, and most GABAergic neurons in these layers have small or medium somatic diameters. These measurements of cell sizes in the various cortical layers may serve as baseline data, to enable comparison with neurons in various pathological conditions.

Morphology and distribution of labelled somata in the adult The results of the present immunocytochemical study are similar to previous studies of GABAergic (Schlander et al. 1987; Schiffmann et al. 1988) and peptidergic (Sorensen 1982; Braak etal. 1985; Roberts etal. 1985; Chan-Palay and Yasargil 1986; Chan-Palay 1987; Gaspar et al. 1987; Manolidis and Baloyannis 1987) neurons in human cerebral cortex, in that neurons of several morphological classes were labelled. The distribution of GABAergic neurons in the adult cortex was similar to that in the temporal cortex of the macaque monkey (Ong and Garey 1990b). All labelled GABA-neurons appeared to be non-pyramidal, consistent with previous observations in the human (Schiffmann et al. 1988) and other mammals. Many neurons labelled with GABA had bipolar or multipolar morphological features. Some GABAergic neurons resembled double bouquet cells in terms of morphology of the soma and stem dendrites, and others resembled subtypes of neurons with smooth or sparsely spinous dendrites found in human temporal cortex (Ong and Garey 1990a). It appears, therefore, that many types of non-pyramidal neurons may be GABAergic, although GABA may not necessarily be distributed throughout their entire dendritic tree. The distribution of peptidergic neurons in the adult frontal and temporal cortices was similar to that of the temporal cortex in the macaque monkey (Ong and Garey 1990b). As in the monkey, greater numbers of SOMand SP-neurons were found compared with VIP-neurons. Greater numbers of neurons were also found in both the adult and infant cortex which were labelled for any one peptide than those for GABA. This is in contrast with published reports of the comparative numbers of GABAergic and peptidergic neurons, where larger numbers of the former were described (Hendry et al. 1984b; Jones and Hendry 1986; L i n e t al. 1986). The difference could be due to the fixation in our specimens, but suggests that not all peptidergic systems are colocalised with GABAergic neurons. Indeed, we find a population of pyramidal neurons labelled for neuropeptides. Peptidergic pyramidal neurons have also been reported in the cerebral cortices of a number of species, including the rat (Morrison et al. 1983; Laemle and Feldman 1985), dolphin (Garey and Revishchin 1991) and monkey (Ong and Garey 1990b), although

their existence has been questioned by some authors (Chan-Palay 1987; Jones et al. 1987). We also show labelling in certain stem dendrites, but not in others. The significance of this is unknown, although one suggestion, based on reports of colocalisation of neuropeptides in cortical neurons (Vincent etal. 1982a, b; Chronwall etal. 1984; Hendry etal. 1984b; Chan-Play 1987; K6hler et al. 1987; Papadopoulos et al. 1987), is that different neuropeptides may be present in different portions of the dendritic tree of these neurons. After fixation in relatively high concentrations of GA, reaction product was present in the nuclei of most GABA- and peptide-containing neurons, contrary to the descriptions of other authors (Meinecke and Peters 1986; Peters et al. t987), although examination of their illustrations suggests that some of these neurons also contained labelled nuclear material.

Labelled processes in the neuropil in the adult Many GABA-positive punctate profiles were found in the neuropil, which may represent immunopositive axons. Few putative GABAergic terminals appeared to be in direct contact with unlabelled neurons in the adult cerebral cortex and electron microscopy showed few axosomatic synapses on adult cell bodies (Ong and Garey 1991). The infant cortex Large numbers of GABAergic and peptidergic neurons were found in the cortex of the infant. The distributions of labelled neurons were broadly similar to those of the adult. However, GABAergic puncta were more commonly found against the cell bodies of unlabelled neurons in the infant, suggesting that greater numbers of inhibitory axosomatic synaptic contacts may be present in the infant. It is known that at 5 months of age the human neocortex has not attained its maximum synaptic density, but that, even then, the total synaptic number, at least in visual cortex, is considerably more than in the adult (Huttenlocher et al. 1982). GABAergic and peptidergic neurons were described in a recent study of foetal cortex in laboratory primates (Huntley et al. 1988). The authors noted considerable differences in laminar distribution, morphology, and numbers between immature and adult cortices even up to late foetal ages, and concluded that the foetal-adult transformation took place at or after birth. Our studies suggest that, except for relatively small numbers of VIPpositive neurons and fewer GABA puncta, the morphological expression of GABA and neuropeptides is already nearly complete in man at about 5 months of age. Acknowledgements. This study was supported by grants RP880314 and RP880315 from the National Universityof Singapore and 21/ 87 from the Singapore Turf Club. We should like to thank Dr. G. Baratham for his cooperation and allowing us access to the neurosurgical specimens.Ms Margaret Sim providedexcellenttechnical assistance.

412

References Beach TG, McGeer EG (1983) Neocortical substance P neurons in the baboon: an immunocytochemical finding. Neurosci Lett 41 : 265-270 Braak E, Braak H, Weindl A (1985) Somatostatin-like immunoreactivity in non-pyramidal neurons of the human isocortex. Anat Embryol 173:237-246 Brodmann K (1909) Vergleichende Lokalisationslehre der Grol3hirnrinde in ihren Prinzipien dargestellt auf Grund des Zellenbaues. Barth, Leipzig Campbell M J, Lewis DA, Benoit R, Morrison JH (1987) Regional heterogeneity in the distribution of somatostain-28- and somatostatin-281_12-immunoreactive profiles in monkey neocortex. J Neurosci 7:1133-1144 Chan-Palay V (1987) Somatostatin immunoreactive neurons in the human hippocampus and cortex shown by immunogold/silver intensification on Vibratome sections: coexistence with neuropeptide Y neurons and effects in Alzheimer-type dementia. J Comp Neuroi 260:201-223 Chan-Palay V, Yasargil G (1986) Immunocytochemistry of human brain tissue with a polyclonal antiserum against neuropeptide Y. Anat Embryol 174:27-33 Chan-Palay V, Lang W, Allen YS, Haesler U, Polak JM (1985) II. Cortical neurons immunoreactive with antisera against neuropeptide Y are altered in Alzheimer's-type dementia. J Comp Neurol 238:390-400 Chronwall BM, Chase TN, O'Donohue TL (1984) Coexistence of neuropeptide Y and somatostatin in rat and human cortical and rat hypothalamic neurons. Neurosci Lett 52:213-217 Cooper PE, Fernstrom MH, Rorstad OP, Leeman SE, Martin JB (1981) The regional distribution of somatostatin, substance P and neurotensin in human brain. Brain Res 218:219 232 Crystal HA, Davies P (1982) Cortical substance P-like immunoreactivity in cases of Alzheimer's disease and senile dementia of the Alzheimer type. J Neurochem 38:1781-1784 Davies P, Terry RD (1981) Cortical somatostatin-like immunoreactivity in cases of Alzheimer's disease and senile dementia of the Alzheimer's type. Neurobiol Aging 2:9-14 DeFelipe J, Hendry SHC, Jones EG, Schmechel D (1985) Variability in the terminations of GABAergic chandelier cell axons on initial segments of pyramidal cell axons in the monkey sensorymotor cortex. J Comp Neurol 231:364-384 DeFelipe J, Hendry SHC, Jones EG (1986) A correlative electron microscopic study of basket cells and large GABAergic neurons in the monkey sensory-motor cortex. Neuroscience 17:9911009 Emson PC, Hunt SP (1984) Peptide-containingneurons of the cerebral cortex. In : Jones EG, Peters A (eds) Cerebral Cortex, vol 2. Plenum Press, New York London, pp 145 169 Emson PC, Joseph MH (1975) Neurochemical and morphological changes during the development of cobalt-induced epilepsy in the rat. Brain Res 93:91-110 Emson PC, Fahrenkrug J, Spokes EGS (1979) Vasoactive intestinal polypeptide (VIP): distribution in normal human brain and in Huntington's disease. Brain Res 173:174-178 Emson PC, Rossor M, Lee CM (1981) The regional distribution and chromoatographic behaviour of somatostatin in human brain. Neurosci Lett, 22:319-324 Ferrier IN, Roberts GW, Crow TJ, Johnstone EC, Owens DGC, Lee YC, O'Shaughnessy D, Adrian TE, Polak JM, Bloom SR (1983) Reduced cholecystokinin-like and somtostatin-like immunoreactivity in limbic lobe is associated with negative symptoms in schizophrenia. Life Sci 33:475-482 Fitzpatrick D, Lund JS, Schmechel DE, Towles AC (1987) Distribution of GABAergic neurons and axon terminals in the macaque striate cortex. J Comp Neurol 264:73-91 Garey LJ, Revishchin AV (1991) Structure and thalamocortical relations of the cetacean sensory cortex :histological, tracer and

immunocytochemical studies. In: Thomas JA, Kastelein R (eds) Sensory Abilities of Cetaceans. Plenum Press, New York pp 1930 Gaspar P, Berger B, Lesur A, Borsotti JP, Febvret A (1987) Somatostatin 28 and neuropeptide Y innervation in the septal area and related cortical and subcortical structures of the human brain. Distribution, relationships and evidence for differential coexistence. Neuroscience 22:49-73 Geola FL, Yamada T, Warwick RJ, Tourtelotte WW, Hershman JM (1981) Regional distribution of somatostatin-like immunoreactivity in the human brain. Brain Res 229:35-42 Hendrickson AE, Hunt SP, Wu JY (1981) Immunocytochemical localization of glutamic acid decarboxylase in monkey striate cortex. Nature 292 : 605-607 Hendry SHC, Houser CR, Jones EG, Vaughn JE (1983) Synaptic organization of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex. J Neurocytol 12: 639-660 Hendry SHC, Jones EG, Emson PC (1984a) Morphology, distribution and synaptic relations of somatostatin- and neuropeptide Y-immunoreactive neurons in rat and monkey neocortex. J Neurosci 4 :2497-2517 Hendry SHC, Jones EG, DeFelipe J, Schmechel D, Brandon C, Emson PC (1984b) Neuropeptide-containing neurons of the cerebral cortex are also GABAergic. Proc Natl Acad Sci USA. 81:6526-6530 Hendry SHC, Schwark HD, Jones EG, Yan J (1987) Numbers and proportions of GABA-immunoreactiveneurons in different areas of monkey cerebral cortex. J Neurosci 7:1503 1519 Higuchi T, Sikand GS, Kato N, Wada JA, Friesen HG (1983) Profound suppression of kindled seizures by cysteamine: possible role of somatostatin to kindled seizures. Brain Res 288:359 362 Houser CR, Hendry SHC, Jones EG, Vaughn JE (1983) Morphoiogical diversity of immunocytochemically identified GABA neurons in the monkey sensory-motor cortex. J Neurocytol 12:617-638 Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 29 : 577-580 Huntley GW, Hendry SHC, Killackey HP, Chalupa LM, Jones EG (1988) Temporai sequence of neurotransmitter expression by developing neurons of fetal monkey visual cortex. Dev Brain Res 43 : 69-96 Huttenlocher PR, de Courten C, Garey LJ, Van der Loos H (1982) Synaptogenesis in human visual cortex - evidence for synapse elimination during normal development. Neurosci Lett 33:247252 Jones EG, Hendry SHC (1986) Co-localization of GABA and neuropeptides in neocortical neurons. Trends Neurosci 9:71 76 Jones EG, Hendry SHC, DeFelipe J (1987) GABA-peptide neurons of the primate cerebral cortex. A limited cell class. In: Jones EG, Peters A (eds) Cerebral Cortex, vol 6. Plenum Press, New York London, pp 237-266 K6hler C, Eriksson LG, Davies S, Chan-Palay V (1987) Co-localization of neuropeptide tyrosine and somatostatin immunoreactivity in neurons of individual subfields of the hippocampal region. Neurosci Lett 78:1-6 Krnjevic K (1983) GABA-mediated inhibitory mechanisms in relation to epileptic discharges. In: Jasper HH, Van Gelder NM (eds) Basic mechanisms of neuronal hyperexcitability. Liss, New York, pp 249-280 Laemle LK, Feldman SC (1985) Somatostatin (SRIF)-like immunoreactivity in subcortical and cortical visual centers of the rat. J Comp Neurol 233 : 45~462 Lin CS, Lu SM, Schmechel DE (1986) Glutamic acid decarboxylase and somatostatin immunoreactivities in rat visual cortex. J Comp Neurol 244:369-383 Lloyd KG, Munari C, Worms P, Bossi L, Bancaud J, Talairach

413 J, Morselli PL (1981) The role of GABA mediated neurotransmission in convulsive states. In: Costa E, DiChiara G, Gessa GL (eds) G A B A and benzodiazepine receptors. Raven Press, New York, pp 199-206 Manolidis L, Baloyannis S (1987) The neuropeptides in the acoustic cortex. I. Substance P. Acta Otolaryngol 103:481 488 Marshall PE, Landis D M D (1985) Huntington's disease is accompanied by changes in the distribution of somatostatin-containing neuronal processes. Brain Res 329:71 82 Meinecke DL, Peters A (1986) Somatostatin immunoreactive neurons in rat visual cortex : a light and electron microscopic study. J Neurocytol 15 : 121-I 36 Morrison JH, Benoit R, Magistretti P J, Bloom FE (1983) Immunohistochemical distribution of pro-somatostatin-related peptides in cerebral cortex. Brain Res 262:344~351 Morrison JH, Rogers J, Scherr S, Benoit R, Bloom FE (1985) Somatostatin in neuritic plaques of Alzheimer's patients. Nature 314 : 90-92 Nakamura S, Vincent SR (1986) Somatostatin- and neuropeptide Y-immunoreactive neurons in the neocortex in senile dementia of Alzheimer's type. Brain Res 370 : 11-20 Ong WY, Garey LJ (1990a) Neuronal architecture of the human temporal cortex. Anat Embryol 181 : 351-364 Ong WY, Garey LJ (1990 b) Pyramidal neurons are immunopositive for peptides, but not GABA, in the temporal cortex of the macaque monkey (Macacafascicularis). Brain Res 533 : 24~1 Ong WY, Garey LJ (1991) Ultrastructural characteristics of human adult and infant cerebral cortical neurons. J Anat (1991) 175:79 104 Papadopoulos GC, Parnavelas JG, Cavanagh ME (1987) Extensive co-existence of neuropeptides in the rat visual cortex. Brain Res 420 : 95-99 Peters A, Meinecke DL, Karamanlidis AN (1987) Vasoactive intestinal polypeptide immunoreactive neurons in the primary visual cortex of the cat. J Neurocytol 16:23-38 Petrusz P, Merchenthaler I, Maderdrut JL (1985) Distribution of enkephalin-containing neurons in the central nervous system.

In: Bj6rklund A, H6kfelt T (eds) Handbook of Chemical Neuroanatomy. Elsevier, Amsterdam, pp 273 334 Roberts GW, Crow TJ, Polak JM (1985) Location of neuronal tangles in somatostatin neurones in Alzheimer's disease. Nature 314:92-94 Sar M, Stumpf WE, Miller RJ, Chang KJ, Cuatrecasas P (1978) Immunohistochemical localization of enkephalin in rat brain and spinal cord. J Comp Neurol 182:17-38 Schiffmann S, Campistron G, Tugendhaft P, Brotchi J, FlamentDurand J, Geffard M, Vanderhaeghen JJ (1988) Immunocytochemical detection of GABAergic nerve cells in the human temporal cortex using a direct 7-aminobutyric acid antiserum. Brain Res 442 : 270-278 Schlander M, Thomalske G, Frotscher M (1987) Fine structure of GABAergic neurons and synapses in the human dentate gyrus. Brain Res 401:185 189 Sorensen KV (1982) Somatostatin: localization and distribution in the cortex and the subcortical white matter of human brain. Neuroscience 7:1227 1232 Van Gelder NM, Courtois A (1972) Close correlation between changing content of specific amino acids in epileptogenic cortex of cats, and severity of epilepsy. Brain Res 43 : 477-484 Van Gelder NM, Sherwin AL, Rasmussen T (1972) Amino acid content of epileptogenic human brain: focal versus surrounding regions. Brain Res 40:385-393 Vincent SR, Johansson O, H6kfelt T, Meyerson B, Sachs C, Elde RP, Terenius L, Kimmel J (1982a) Neuropeptide co-existence in human cortical neurones. Nature 298 : 65-67 Vincent SR, Sirboll L, H6kfelt T, Johansson O, Lundberg JM, Elde RP, Terenius L, Kimmel J (1982 b) Co-existence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons. Neuroscience 7:439446 Williams RG, Dockray GJ (1983) Distribution of enkephalin-related peptides in rat brain: immunohistochemical studies using antisera to Met-enkephalin and Met-enkephalin Arg6phe 7. Neuroscience 9 : 563-586