Immunocytochemical localization of somatostatin and cholecystokinin in the cat visual cortex

Bram Research, 332 (1985) 361-364

361

Elsevmr BRE 20735

Immunocytochemical localization of somatostatin and cholecystokinin in the cat visual cortex HILDE DEMEULEMEESTER 1:, FRANS VANDESANDE l and GUY A ORBAN-" I Laboratortum voor Neuroendocrmologte en Immunologtsche Blotechnologte, Kathoheke Umversttett te Leuven and 2Laboratortum voor Neuro- en Psychofvstologte, Kathoheke Untversttelt te Leuven, Campus Gasthulsberg, B-3000 Leuven ( Belgmm)

(Accepted November 20th, 1984) Key words tmmunocytochemlstry - - somatostatm (SRIF) - - cholecystokmln (CCK) - - areas 17.18 and 19 - - lamination

Immunocytochemlstr) was used to examine the morphology and distribution of cholecystoklnln-hke and somatostatln-hke neurons in areas 17, 18 and 19 of cat visual cortex as a function of lamination Immunoreactlve cells of both peptldes were observed in all layers of cat visual cortex While somatostatln-hke cells occurred mainly in layers II+III and VI, cholecystoklnln-hke cells were observed chiefly in the superficial layers (I+II+III) Somatostatm-hke cells displayed morphological teatures of multlpolar and bipolar vaneties, and cholecystoklnln-hke cells displayed morphological features of multlpolar and bltufted varletms Simdar results were obtained for all 3 areas

Information processing in the visual cortex has

lection, these animals and the untreated ones were

been extensively studied both physiologically and an-

killed with an overdose of pentotai and perfused

atomlcally8-m A l t h o u g h recently a small n u m b e r of

through the heart with physiological solution and

short axon cells have been identified by intracellular

then with fixative (4% p a r a f o r m a l d e h y d e in 0 1 M

injection techniques 2,5 relatively little ts known about

phosphate buffer). The brain was postflxed for 24 h

the functional role of the many short axon cells in the

by immersion in the same fixative Using a freezing

visual cortex A n important step towards this goal is

mlcrotome or v i b r a t o m e (Oxford Instruments), cor-

the identification of their transmitter. T h e role of

onal sections were cut at 3 0 - 6 0 / ~ m throughout the

G A B A m visual processing is well d o c u m e n t e d 11 14

frontocaudal extent of the visual cortex, collected in

but only 1 0 - 1 5 %

of the neurons are G A B A e r -

0.1 M Trls-sallne buffer and processed for lmmuno-

glc I In search of the cortical neurotransmltters, we

cytochemlstry 17A8. The incubation period for both

have investigated the distribution in the visual corti-

antlsera was 24 h at r o o m t e m p e r a t u r e Th e optimal

cal areas (17, 18 and 19) of two neuropeptides, soma-

dilution was 1/16000-1/32000 for C C K antiserum

tostatin (SRIF) which is believed to be inhibitory in

and 1/2000-1/8000 for the S R I F antiserum

action 4 and cholecystoklnin ( C C K ) which has been

In the untreated animals both S RI F- and CCK-lm-

shown to exert an excitatory influence on central ner-

munoreactlve

vous c e l l s : l~ Six adult cats were used Two cats were untreated

found

Two cats received an lntraventrlcular injection of

were identified according to their dendritic pattern,

colchlcine (750 and 1000 ~g, respectively), while in

as described by Peters and Reg l d o r m for Golgl prepa-

the last two cats colchlclne (24/~g) was injected directly into the lateral gyrus according to the proce-

rations of cat visual cortex. Somatostatin-hke neurons, were almost all non-

dure of Somogyl et al 16 During the colchlclne mlec-

pyramidal cells, (a few lmmunoreactxve pyramidal-

tlons the animals were anesthetized with alfateslne

like neurons were observed), comprising approxi-

(0 3 ml/kg) Forty-eight hours after the colchlclne in-

mately 0 . 2 - 0 3% of the neuronal population of cat

Correspondence

nerve

fibers

and

perlkarya

A f t er colchlclne t r e a t m e n t ,

stained more intensely

were

the perlkarya

S R I F and C C K perlkarya

G A Orban, Kathoheke Unlversltelt te Leuven, Campus Gasthulsberg, Herestraat, B-3000 Leuven. Belgium

0006-8993/85,'$03 30 © 1985 Elsevmr Science Pubhshers B V (Biomedical Division)

362

I

II \

-- . . . . . . . . . . .

/f

v

- ~ f

J I

...........

wM I

Fig 1 A-C, SRIF-lmmunoreactlve neurons, and D-F CCK-lmmunoreactlve neurons drawings made from labeled cells WM, white matter A multlpolar cell of layers II +III m area 18 (gyrus laterahs) B' multlpolar cell of upper layer VIm area 17 (sulcus splenlahs), with horizontally oriented dendrmc trees C bipolar cell oI deep layer V1 in area 17 (sulcus splemahs) with horizontally oriented dendrites D bltufted neuron of layer IIm area 17 (sulcus splenlaIis), with vertically oriented dendritic tufts. E bltufted neuron of layer I in area 19 (sulcus laterahs), wlth horizontally oriented dendrmc tufts F multipolar cell m the middle of layer II in area lq (sulcus laterahs) Cahbration bar = 50/,m visual cortex, lmmunoreact~ve somata were mainly observed in layers I I + l I I and VI SR1F-posmve neurons m layers l I + l I I have spherical cell bodies t 0 26 p m m size (Fig. 1A) While the cells occurring m layer III have their dendrites radmtmg In all directions from the soma, dendrites of cells close to the layer I border radiate laterally and upwards to the cortical surface. The SRIF-lmmunoreactive neurons of layer VI have horizontally elongated cell bodies and fall apart into two classes. SRIF-hke n e u r o n s of upper layer VI have elongated or irregularly shaped cell bodies (width + 20 ktm, length 2 0 - 3 0 pro), and most of their dendritic trunks arise from the lateral portion of the soma, r u n m n g parallel to the white matter and produce dense dendritic trees (Fig. 1B) SRIF-positive n e u r o n s of deep layer VI lay close to or in the subcortical white matter and are elongated in the horizontal direction (width + 1 2 # m , length _+ 20 pro). A primary dendritic t r u n k arises from each pole of the soma and branches a few times (Fig 1C, 2A) According the descrtptlon of Peters and Regl-

dor ~0, the SRIF-like lmmunoreacttve cells m layers I I + I I I and those m upper layer VI display the morphological features of multlpolar neurons, while cells of deep layer V1 correspond to b~polar neurons. SRIF-positlve cells m the other layers also resemble the multipolar forms, exceptionally a few horizontal bltufted neurons were observed m layer I (Table I). Approximately 2.6% of the neuron~ in layer VI were SRIF-posmve while SRIF l m m u n o r e active neurons m layers I I + I l I a m o u n t e d to 0.29~ of the cells m that layer The other layers contained TABLE I Proportton o f SRIF- and CCK-posmve neuron~ (area,~ 17. 18 and 19 pooled) Lammae

SR1F (0 3c~ ~

CCI~ (0 6c'r,)

1 II +IlI IV V Vt

0 02% 0 18°/c 0 06% 0 07% 2 6~,'~

(1 66c~ 2 2% 0 1% 0 08~'~ 0 I)7~~,

363 rectlon A few primary dendrites e m a n a t e from each

only a few SRIF-posltive cell bodies (Table I)

pole of the soma, dlvtde into daughter branches (Fig 1D, 2C) and eventually pass Into layer l Layer

Cholecystoklnin-hke n e u r o n s were also non-pyramidal cells comprising approximately 0 . 5 - 0 6% of the neuronal population of cat visual cortex. Their perikarya were locahzed in all cortical layers of the 3

I contains n e u r o n s with horizontally oriented dendritic trees. These cells he either close to the cortical

visual areas, but mamly m the superficml ones Neurons w~th vertxcally oriented p e n k a r y a were restrict-

surface or at the border with layer II Their perlkarya

ed to layers I I + I I I , most c o m m o n l y at the border

g m , length 2 0 - 3 0 ~ m ) , with dendrites arising from the left and the right side of the soma, and branching into

are elongated in the horizontal direction (width _+ 15

w~th layer I Their cell bodies (wtdth + 13j~m, length 20-30 ~m) are ovoid or elongated m the vertical d~-

dendritic tufts (Fig 1E, 2B). The remaining CCK-

P

W

I¢ )

A

ill~,!l

12

D

"

Fig 2 A somatostatmqmmunoreacnve bipolar neuron m deep layer VI (area 17, sulcus suprasplemahs) A primary dendritic trunk emanates from each pole of the soma The cell body, as well as the dendrites are oriented parallel to the subcomcal white matter B cholecystokmm-immunoreactlve horizontally oriented Ntufted neuron m layer I (area 19, sulcus laterahs) Dendrites emanating from each pole of the soma have a horizontal course Arrow indicates the pml surface C cholecystoklnm-~mmunoreactlve vemcallv oriented bltufted neuron of layer II (area 17, sulcus splenlahs), with dendrites arising from the upper and lower half of the perlkarv,t D cholecystokmlnqmmunoreactive multipolar neuron of layer II (area 19, sulcus laterahs) w~thdendrites emanating from the cell surface m all dlrecUons Cahbrat~on bar = 25#m

364 l m m u n o r e a c t l v e n e u r o n s in l a y e r s I - V are r o u n d o r

and VI

irregularly shaped

the s a m e m

(_+ 2 0 / ~ m )

and

vary c o n s i d e r a b l y m s~ze

T h r e e o r m o r e d e n d r i t e s arise t r o m the

cell b o d y m all d~recttons (Fig

1F, 2 D )

Occasionally

n e u r o n s m u p p e r l a y e r V1 w~th horlzontall~ e l o n -

T h e d i s t r i b u t i o n p a t t e r n o f boti3 p c p l l d e s ~, a r e a s 17. 18 a n d 19 T h e C ( K a n d

the

m a j o r i t y o t the S R I F - p o s l t l ~ e cells are n,}n-pyralmdal cells, w h i c h are b e h e v e d to be local m t c r n e u r ~ m s . S R I F a n d C C K h a v e alread~ b e e n t o c a h z e d m the

g a t e d cell b o d i e s a n d d e n d r m c fields o r i e n t e d p a r a l -

vtsual c o r t e x o f o t h e r s p e c i e s T h e p a t t e r n .,,f d N n b u -

lel to the s u b c o r t t c a l w h i t e m a t t e r w e r e f o u n d to be

tton a n d t h e m o r p h o l o g y o f C C K - p o s l t w c neuron> js

CCK ~mmunoreactwe

Both the horizontally elon-

gated CCKqmmunoreactwe

n e u r o n s m layer l and

very s~mflar in cat a n d rat visual c o r t e x as ~hown by c o m p a r i s o n o f o u r m a t e r i a l with t h a t ot M c D o n a l d et

the vertically e l o n g a t e d C C K - p o s ~ t w e n e u r o n s m la~-

al 7 S R I F has also b e e n l o c a h z e d in t h e ~ isual c o r t e x

ers I I + I I I b e l o n g t o t h e N t u f t e d v a r i e t i e s d e s c r l b e o

o f o t h e r s p e c i e s In m o n k e y visual c o r t e x , t l e n d n c k -

by P e t e r s a n d R e g i d o r ~

son 3, f o u n d S R I F - i m m u n o r e a c t t v e neurcins tn l a v e r

A l t h o u g h ~t ~s n o t eas'~ to

draw the hne b e t w e e n btpolar and Ntufted neurons,

II+III

we h a d g r e a t difficulty to find c l e a r e x a m p l e s o f b t p o -

S R I F - p o s l t w e n e u r o n s m all layers. F h e s e s l m d a n -

lar n e u r o n s a m o n g s t t h e C C K q m m u n o r e a c t ~ e

neu-

ties b e t w e e n d i f f e r e n t m a m m a l s u n d e r s c o r e the func-

All t h e o t h e r C C K q m m u n o r e a c t ~ v e n e u r o n s

tional role o f t h e s e n e u r o p e p t t d e s ,n visual proces~-

rons

d~splay t h e

m o r p h o l o g i c a l f e a t u r e s o} mult~polar

a n d V I , while M c D o n a l d

el a l '

reported

mg

cells In all 3 a r e a s a b o u t 9 2 ' ~ o t t h e C C K - p o s ~ t l v e cells w e r e f o u n d m t h e s u p r a g r a n u l a r layers. While cholecystokmin occurs mainly m the suprag r a n u l a r l a y e r s , s o m a t o s t a t l n o c c u r s m layers I f + I l l 1 Fitzpatrick, D ,Lund, J S and Schmechel, D , Glutamlc acid decarboxylase lmmunoreactlve neurons and terminals m the visual cortex of monkey and eat ~oc Neuro~cl Abstr, 9 (1983) 616 2 Gilbert, C D and Wlesel, T N , Morphology and lntracortical projections of functmnally characterized neurons m the cat visual cortex, Nature (Lond), 280 (1979) 120-125 3 Hendrlckson. A , Pyramidal and stellate neurons m monkey visual cortex label for different peptldes, Soc Net, ros'tt Abstr, 9 (1983) 821 4 Hokfelt, T , Elde, R , Johansson, O , Lult, R and Arlmura, A , ImmunohlStOchemlcal evidence for the presence o! somatostatln, a powerlul inhibitory peptide, in some primary sensory neurons, Neurosct Lett, 1 (1975) 231-235 5 Martin, K A C , Somogyl, P and Whltteridge, D , Physiological and morphological properties of identified basket cells in the cat's visual cortex, Exp Brain Res, 50 (1983) 193-200 6 McDonald, J , Parnavelas, J G , Karamanlldls, A N , Brecha, N and Koemg. J I , The morphology and distribution o1 peptlde containing neurons in the adult and developing v~sual cortex of the rat I Somatostatln, J Neurocvtol. 11 (1982) 809-824 7 McDonald, J , Karamanhdis, A N , Rosenqulst, G and Brecha, N , The morphology and distribution of peptide containing neurons in the adult and developmg visual cortex of the rat I1 Cholecystoklnln, J Neurocytol. 11 (1982) 881-895 8 Meyer, G , Axonal patterns and topography of short-axon neurons In visual areas 17, 18 and 19 of the cat J cornp Neurol, 220 (1983) 405-438 9 0 r b a n , G A , Neuronal operations in the visual cortex In H B Barlow. T H Bullock, E Florey, O J Grusser and A Peters (Eds), Studies of Brain Function. ~#~l 11. Sprlnger-Verlag, Berlin. 1984, 367 pp

Antisera against both peptxdes (SRIF and C C K ) w e r e k m d l y s u p p l i e d by P r o f J J V a n d e r h a e g h e n , H D . w a s s u p p o r t e d by a L D e P o o r t e r e N e u r o s c l ence Fellowship 10 Peters, A and Regldor, J , A rcassessment of the form ol nonpyramldal neurons in area 17 of cat visual cortex, J comp Neurol, 203 (1983) 685-716 11 Sllhto, A M , The effectiveness of blcuculhne as an antagonist of GABA and visually evoked inhibition in the cat s striate cortex, J Phystol (Lond J, 250 (1975) 287-304 12 Slllito, A M , Inhibitory mechanisms underlying the directional specificity of simple, complex and hypercomplex cells in the cat's visual cortex. J Phwtol (Lond) 271 (1977) 690-720 13 Silhto, A M , The spatial extent of excitatory and mhlbitory zones in the receptive field of superficial layer hypercomplex cells, J Phystol (Lond), 273 (1977) 791-803 14 Sdhto. A M , K e m p , J A , M I l s o n , J A andBerardl, N , A re-evaluation of the mechanisms underlying simple cell orientation selectivity, Bram Research, 194 (1980) 517-5211 15 Skirboll, L R , Grace, A A , Hommer, D W , Rehfeld, J , Goldstem, M , Hokfelt, T and Bunney, B S , Peptldemonoamine coexistence studies of the action,, ol cholecystokmln-hke pepnde on the electrical activity of mIdbraln dopamlne neurons. Neuroscwnce, 6 11981) 2111-2124 16 Somogyl, P , Klsvarday, Z F , Martin, K A C and Whltteridge, D . Synaptic connectmns of morphologically ~denttfied and physiologically characterized large basket cells in the striate cortex of cat, Neurosctence, 10 (1983) 261-294 17 Vandesande, F , A critical review of lmmunocytochemical methods for light microscopy, J Neurosct Meth , 1 (19791 %23 18 VanLeeuwen, F W , P o o l , C W a n d S l m t c I , A A , E n kephahn lmmunoreactlvlty in synaptold elements on gllal cells in rat neural lobe, Neurosctence, 8 (1983) 229--241 19 Williams, J A , CholecystokinIn a hormone and a ncumtl ansmltter~, B~omed Re~ , 3 (1<)82) 107-121