Distribution of putative neurotransmitters in the neocortex

COMMENTARY DISTRIBUTION

OF PUTATIVE NEUROTRANSMITTERS IN THE NEOCORTEX P. C. EMSON

M.R C. Neurochemical Pharmacology University

Unit, Department of Pharmacology, of Cambridge. Cambridge, England

Medical

School,

Hills Rd..

and 0. LINI>VALL Department of Histology. University of Lund Biskopsgatan 5. S-22362 Lund, Sweden Acetylcholine Amino acids Glutamate and aspartate ;,-Aminobutyric acid, glycine and taurine Amine\ (‘atecholamines Z-Hydroxytryptamine Other aminec Peptides Substance P Vasoacti\e intestinal polypeptide (‘holecystokinin Neurotensin Somatostatin Other peptidcs ConcluGon

THL OBJECT of this commentary is to consider the data available on the identity of neurotransmitters in the neocortex. The distribution and localization of putative neurotransmitters and, where appropriate, their associated enzymes will be considered along with data which indicate the origin of transmittercontaining pathways to and from the cortex. We shall mainly discuss recent neurochemical and histochemical studies on the localization of putative transmitters and receptors in the cortex. Where appropriate, some physiological experiments concerned with establishing the identity of transmitter candidates will be discussed, in particular when these extend the earlier reviews of PHILLIS (1970), HEBB (1970), DE FEUIXS (197.5) and KRNJEVI~. (1974). This commentary has been prompted by recent discoveries indicating the presence of dopaminergic and a variety of peptidergic fibres and neurones in the neocortex. which considerably expand the list of possible cortical transmitters. It should be emphasized that the evidence in favour of a transmitter role for some of the candidates (e.g. acetylcholine and dopamine) is strong, in other cases (e.g. vasoactive intestinal polypeptide or the cholecystokinin octapeptide) it is very preliminary. N\, 4 I \

ACETYLCHOLINE HEB~ (1970) discussed the evidence presented by KRNJEVIC’(1967) that the cholinergic nerve fibres of the cortex originate in the ascending reticular system. In aggreement with this idea is a large amount of work correlating cerebral activity, arousal and acetylcholine. In particular a number of authors (KANAI & SZF.RB, 1965; CELESIA & JASPER, 1966; PHILLIS, 1968) have demonstrated that acetylcholine is released from the cortex by stimulation of the reticular formation. Additional support for the idea of a cholinergic pathway from the reticular formation to the cortex was provided by the histochemical mapping of acetylcholinesterase-containing pathways in the CNS, in particular by SHUTE & LEWIS (1967). Shute and Lewis showed clearly that a number of the cell groups in the phylogenetically older regions of the brain (substantia nigra, ventral tegmental area, mesencephalic reticular formation) were acetylcholinesterase-positive. Further, hemisections through the forebrain showed a substantial accumulation of acetylcholinesterase caudal to the cut indicating the presence of acetylcholinesterase in ascending axons in the medial

1

l’orehr,un

hnndlc

(WC

1 IS. I AI. Hov,c\cr.

L\orh ha\ \hoM n that m,lii! of the ccl1 the ‘asccndiriy

rctlcular

\\slein

acti\;~tiiig

Illoll~~aminu-~o~~tainlng.

zubsqucnt

gr-otiph forming

art’ in fact

e.g. the cells 111the suh\tantla 1iucI011\ lociis

tinuous .jecting

cont:urr

lion on .Amine~.

Tliia

acet~lcholincst~7-~~sc

thnsc

;issoci;ilion (WC

III

tlic

of monoamincs

BL lC.111K 6i

xitl

BILI /IKJIA\.

19751 15 so fill- uneXplilincd: II does. hob\evcr. indicate the halard of using ;I non-spccitic marker \uch ;I> acet! lcholinr\tcraac to map choliiicrgic pathfia! s. As mentioned. hcmiscction\ made irostral to the huhstantia nigra Irc~~~lt111marked accumulation of xet! Icholine\tcrac-rich material drztal to the cut. HowcLer. iiiicrodis~c.ctlc,n~ c01iih11ied lvith the au;i> 01‘the cliolint ;~cet>Itransfcrase content of the medial gorcbrain bundle and liuclci roatral to the lesion (t‘ohPul,lt. WA\I.AAS & I\ I K’lf.1. 19771 indicated no depletion of the choline ;icet!ltr~iiisf~r~isc content of arcas Fuch ;IS the diagon‘tl band. the olfactor! tuborclc. the preoptic area and the l’rontai iortcs. which arc rich 111choline The! ~IISO failed to ohser\e an! ;ICacet! Itran4cr;lsc. cumulallon ol‘choline acet~ltr~in~fcrnsc in the medial forebrain bundle ca11da1 to the cut. although there has a depletion of monoaniin~-;l~aociatcd enl> mes rostra1 to the cut. (C‘holinc acct\lrr~lnsfcrasc. the \bnthetic cn/!mc IOr acctylchollnc. represents. in the central net3 oii\ h~stciii ;it least. ;I spccitic marker for cholincrgic pathwahs.1 ‘This ev~dencc Indicates that the mescncephahc part of the asccndmg reticular system IS monoaniinergic. HoweLcr. it \hould bc emphasired that although the description b> SW II & LI WIS (1967) of the ascending mesenccphalic ‘cholinergic’ sh4tem W;I~ 111error because of the prcsencc of acctylcholinesterarc. hit not choline accth Itransferasc in monoamine neuroncs pro_iecting to the forebrain, their de\criptlon of cholinergic prolcctmna from forebrain nuclei do correspond to cholinergic projections dcmonstrablc by depletion of choline acet) Itransfer;ISC and acctylcholinestcr~lsc (SW below). Additionally their descriptions of the distribution of ucetylcholinester;lse-cont;~ining ncuroncs and axon:, in the mcsencephalon corresponds closely to Lvhat is known by Huorcscencc histochemical techniques about the distribution of tnonoaminc-contnjnjng neurones and proJections. Indeed under certain circumstances it is possible to demonstrate parallel loss of acetylcholincstcrasc-rich dopamine-containing terminals from the caudatc nucleus b! histochemistrl or biochemical assa) for either acetylcholinester~isc or dopamine.

a number

neuronc cell bodies

cocruleuh contain norndrcnalinc. and thohc in the raphe nuclc‘i contain 3- Ii!~irox~tr!ptaminl:: bee see-

nipra

dopam~nc.

of large acetylcholinesterasc-rictl 1967: .lA(.of%oM III. & PALUWIIS. 1974; DI\K. 1975: MI:SIII.A\~ L! V:%N HOFSIN. 1976) which apparentI> form one co71-

contain

system

of forebrain

cholinergic

net1roncs

pro-

the cortex (SW Tnblc 1I [In primates. the scattered acctyIcholincsterasc-positivc neuroncs which. in the rat. are found in the globu\ pallidus and entopeduncular nucleus. are located in the nucleus basalis of the substantia inominata (MESLKA~I 6i V,\S HOESFN. l976).] The evidence for these prqjections is based mainly on studies using ;I combination of histochemical techniques. including ((I) pretreatment of rats with low doses of diisoprop! IIluorophosphate to reduce the background cn~yme activit) in acetylcholinesterasc-rich areas (Brlr-~WI:K& BILUIRJIAIL, 1975) to facilitate the \isualiLation of acetylcholinestcrase-rich ncuroncs. (h) study of the depletion of acetylcholinestcrasc frotn forebrain areas after lesions, and (c) injections of horseradish pcroxidasc into the cortex to label neurones projecting to cortical areas (DIL.AC. 19751. In addition. lesion studies to demonstrate parallel changes m the content and distribution, in the different cortical arcah. of the nonspecific marker acetylcholinesterase and the specific marker choline acetyltransferasc have ~1x0 been used. Obviously the ideal tool for this t! pc of study would be an antiserum to purified choline xxtyltransferase to enable the immunohistochemical \istopographically

ualization

to

of cholinergic

neurones

and terminals.

as

has been carried

out successfull) for the monoamine synthesizing enzymes. tlrosine hydroxylase and dopamine /I-hydroxq lase (see section on Amines). IJnfortunately, although progress is being made towards production of specific antibodies to choline acetkltransferase (MCGWK M&EFK. Sruc;kr & (‘HAY 1974: ENG, UYl.11~. C‘IIAO & WOLFGKACI, 1974; C‘OT.ZAKI & HARTVIA~. I977) no detailed immunohistochemical information on the localization of cortical choline acetyltransferase is available so that the indirect approach of lesions, acetylcholinesterase staining and choline acetyltransferase determinations has still to be used.

Although these lesion and biochemical studies indicate the existence of cholinergic forebrain neurones projecting to the cortex they do not tell us anything about the organization of these projections within the various cortical layers and, also. if there are intrinsic cortical cholinergic neurones (MCGEER ~‘7 trl., 1974). In order organization

Recent studies (summarized in Table I) suggest that the various magnocellular forebrain nuclei represent the origins of cholinergic projections to the cortex and forebrain. The magnocellular nuclei concerned include. in the rat. the nucleus of the diagonal band, the medial and lateral preoptic nuclei, the nucleus basalis and the entopeduncular m&us. These nuclei

(SHLTI &k L.IWIS.

to

obtain of

information cholinergic

about afferents

the

laminar

BEIBLEL

&

EMSON (I 975) measured the choline acetyltransferase and acetylcholinesterase content of the layers of the sensorimotor cortex (Table 2). Examination of the distribution of the more specific marker enzyme choline acetyltransferase indicated that its activity was concentrated in the upper layers of the sensorimotor cortex ()I~-IV). This distribution matches reasonably with

AChE rich cell\. ChAT content 50” of striatal leveLJ ‘.’ ”

_.

labelling

labelled

in nucleus

bv HRP injections’in dorsal frontal cortex.-’

Ncurones

Neurones in nucleus labelled by HRP in jections in piriform. cntorhinal and lateral neocortex.’

Neurones on border of nucleus labelled by HRP injections in dorsal and lateral neocortex.3

Peroxidase

experiments

(‘tits between diagonal band nucleus and tract deplete dorsal medial frontal cortex and lateral septum of ChAT and produce a ChAT depletion in the ipsilateral hippocampus.‘.”

Lesions transecting pre-optic area deplete piriform cortex and amqgdala of ChAT and A(‘hE: lesions in prcoptic area deplete olfactory bulb and anterior olfactory nucleus of AChE.”

Lesions separating globus pallidus from caudate nucleus deplete lateral and dorsal neocortex of AChE and ChAT.’ Kainic acid injections deplete dorsal and lateral cortex of ChAT.”

Lesion

Rc~fiwv~ces:

area

6. ~I(.!JIM[!I?A.

P~rlform. cntorhinnl lateral neocortex olFlctor\ ‘ _ bulb.‘~”

Dorsal and lateral neocortex. ’

Projection

,4hhrrr~ic~tior7s: AChE. acet~lcholinesterase: ChAT, choline acetyltransferase; HRP. horseradish peroxidase. I. SH~I~I & Ltwrs. 1967. 2. .IAC‘OBOWITZ& PAIXOVITS. 1974. 3. DILAC, 1975. 4. I.‘~NN~!M TV u/.. 1977. 5. P C. EMSO\ unpublished observation\. KIM, SAI-~O. HIKANO, 1~0 & NAKAHARA, 1978. 7. EMSON. 1978. 8. EMSON. PAXINOS, LE GAL LA SAI.L~. BEN-ARI & SII.VFR. 197X. 9. WFNI(. Mb.\r’t-~ & BIGL. 1977.

band

Diagonal nuclei

Nucleus itself not AChErich but is bordered by AChE rich cells.‘~’ ChAT content 2S?,, of striatal level.4,5,h

content ____~~~~

AC‘hE-rich cells. C‘hAT content 50”,, ol striatal level.’ i,h

pallidus

Enzyme

Lateral preoptic areai entopeduncular nucleus

Globus

Nucleus

w

distribution of cholinoceptive cells identified electrophysiologically in the cat and rat ncocortcx (KKSJI,VI(: & PHILLlS, 1963~: STONF. lY72). although these electrophysiological studies prohabl! tend to ovcremphasize the number of cholinocepti\c units in the lower laqers (IV V). where the larger neuroncs ma) be more readily detected. The presence of nppreciablc amounts ol’ choline acetyltransferase in the upper layers is consistent with cholinergic pro,jections to these layers. Acetylcholine is usually considered to hc an excitatory neurotransmittcr in the cortex. although ncuronal inhibition by acet) Icholinc has been reported in the upper cortical la\crs (P~nr LIS. 196X). In this situation KKNJI:W’ (1974) has suggested that acetylcholine may activate the deeper cortical ncurones by direct facilitation and by disinhibition involving a small percentage of superficial inhibitor! neurones. The distribution of nicotinic receptors in the cortex as revealed b) [‘~51]1-bungarotoxin binding and autoradiography (S. HUN t. personal communication) indicates these nicotinic sites arc conccntrnted in layers I. V and VI. This distribution would be coc sistent with the localization of the nicotinic receptors on the apical dendrites (layer\ I- II) and the ncuronal cell bodies in layers V and VI. of the deep large pyramidal neurones. The presence of muscarinic receptors in the cortex has been demonstrated using hindmg of radioactive muscarinic ligands [-‘Hlquinuclidinylhenzilate (YAMAMUKA & SNI IEK. 1974) and r3H]propylbenzilycholine mustard (HILI \. 1976). It IX clear that, on the basis of estimates of the numbers of nicotinic and muscarinic binding sites. the mqjoritl of cortical acetylcholine receptors arc muscarinic. Synaptosome (pinched oR‘ nerve endings) prcparations from the neocortex 01‘dityerent animals (rat. cat, bovine) have been shown to contam acctylcholine. which can be released in ;I calcium-dependent manner by physiological stimulation: a sodiumdependent high affinity uptake system for choline: and the acctylcholinc synthcsi7ing enrlmc. choline

acetqltransferase (set review bq K~:HAK. lY76). The amount of synaptosomal choline acetb Itransrera~c has aIso been used to provide some indication of the proportion of choline acethltransferasc which might IX in nerve terminals in the cortex. On this basks some 70”,, of choline acctyltransfcrasc in the rat (Fou~r \I & MALTHI.-SCIWWI:X. 1971) is particulate. ,~nd at most some 30”,, of choline ncetyltransferasc could he in the cell bodies and processes of intrinsic neuroncs. Cortical undercutting in the cat (Hr~c. KKh.11\ I(’ Kc SILVEK. 1963) indicated that the bulk of choline acetyltransferase (90”,,) is lost after lesioning alfcrents to the cortex. Our own studies in the rat indicated depletions 0r choline acetyltransfcrasc smaller following undercutting cortical (5&X0”,, depletion) areas (see Table I). However. these results \uggc\t that if there arc intrinsic cholinergic ncurones in the cortex they provide only ;I small pcrccntagc of the cholinergic ncrvc terminals in the cortex. Against these obsertations are those of McGI,I.K. McGt I K. SCHFIWK & SINCZI (1977) who rcportcd no depletion of choline acetyltransferasr after undercutting the l’rontal cortex. This paradoxical result cannot rcadih he explained. McGeer’s group have also reported on the cxistencc of intrinsic cholinergic neurones m the cortex on the basih of choline acctyltransfcra\e immunohistochemistrq (MCGILK of ill.. 1974). Unl’ortunately. doubt has been thrown on the spccilicit! of this antiserum to choline acetyltransferasc (Rossu K. 1975), so the possible existence of intrinsic cholinergic ncurones in the cortex will have to wait further invcstigations. using well-charactertzed choline acetyltran+ ferase antisera. The major doubt concerning the antIserum used by McGeer’s group concerns their apparent inability to observe the disappearance 01’mlmunoreactive cholinergic terminals in well-estabhshcd cholinergic pathways such as the septohippocampnl pathway following lesions removing the cholinerglc projectiotis. Such doubts about specificity do not exist for antisera raised against other transmitter-synthcsir-

FIG. 1. (A) Unilateral hemisection through the medial forebrain bundle of the rat to show accumulation of acetylcholinesterase-positive material distal to the lesion. (MFB. medial forebrain bundle.) (B) Depletion of acetylcholinesterase from the lateral and dorsal frontal cortex of the rat after a lesion separating the globus pallidus from the caudate nucleus. (C) Knife lesion separating the piriform cortex from the am!gdala in the rat. Note acetylcholinesterase depletion of piriform cortex and accumulation of acetylcholinesterase-positive material in the amygdala area. (BL. basolateral amygdaloid nucleus; (‘E. central amygdaloid nucleus: and PC, plriform cortex.) (D) Acetylcholinesterasc-rich cells in the lateral prcoptic area of the rat. Technique of BI:TTHI.R & BII.I z[hJlAv (19751.

F-IG 3. Neuronal (A) neurones

cell bodies accumulating

in layers I and

II:

[“HI?-aminobutyric

(B) detail

01” neurones

laker VI. (* labelled neurones.)

acld In rat cerebral

in layers WM.

(L)

111 and

white matter.

cortex

IV:

layers

I VI.

((‘1 neuroncs

tn

FIG. 4. Photomicrographs types of pattern (A) Noradrenergic are shown. dopaminergic

taken from coronal

and laminar terminals

distribution from

sections of rat cerebral cortex rhowing

formed

by the neocortical

the locus coeruleus

In (B) and (C) the diRerent

distribution

systems are shown in the supragenual Compare

in the rostra1 part patterns la)er

antcromedial

.it the top

innervation\.

of the supragenual

of the supragenual

and pregenual

with text. Molecular

the three different

catecholaminergic

cortex

and

anteromedial

cortex.

respectIveI!

FIG. 9. Immunofluoresccnt the prefrontal

cortex

mtcrographs

al’ter Incubation

to gastrin.

and (DI

of (A. B) the parietal aith

antiserum

to \uh\tancc

(A. B) to

P. .A x 126 B.C.

cortex.

(c‘) the dentate

vaaoactivc

intestinal

gyrus

and

polypeptide

( x 315)and CD) x 360.

(DI ((‘1.

bn the neocortcl

Neurotransmittcr\ TABLE

3.

AMINO

ACID CONTENT

OF NORMAL

ANI)

I.-.I)I+CCI

I PIRIFORM

CORTEX OF 1HF R?\I

Amino Taurine y-Aminobutyric Glutamic acid Glutamine Aspartic acid Glycine

acid

acid

llnits are pmol amino acid per gram. (From G. PAXINOS and P. C. EMSOX.107X. unpublished ttons.)

ing enzymes or putative transmitter peptides to be discussed later (for example, tyrosine hydroxylase, dopamine /,‘-hydroxylase, tryptophan hydroxylase. substance P and the enkephalins). where immunoreactivity disappears following lesions to the appropriate pathway.

AMINO ACIDS The neocortex has an amino acid content characteriTed by the presence of substantial amounts of glutamic acid (glutamate). aspartic acid (aspartate) and glutamine. and lower amounts of y-aminobutyric acid, taurine and glycine (Table 3). Of these amino acids. glutamate and aspartate have been suggested as cortical excitatory neurotransmitters, whereas y-aminobu11ric acid and taurine (but not glycine) have been suggested as cortical inhibitory transmitters.

The evidence implicating glutamate and possibly aspartalc as excitatory cortical neurotransmitters is quite strong [the electrophysiological evidence has been reviewed by PHILLIS (I 970), KRNJEVIC’(I 974) and M~LENUAN (1975): the neurochemical evidence by JOHUSTON (1972): CURTIS & JOHNSTON (1974) and DAVISON(1976)]. The most interesting recent developments have centred around the possibility that changes in the density of high-affinity uptake sites for glutamate or aspartate after lesions can be used to map possible glutamate pathways. The initial studies were carried out on the hippocampus. where two groups (NAIILER, VACA, WHITE, LYNCH & CorMAN. 1976: WHITE. NAULER, HAMBERGER.COTMAN & (‘~:MMINS,1977: STORM-MATHISEN’1977) , showed that there is a selective loss of high-affinity glutamate uptake in the molecular layer of the dentate gyrus following lesions of the entorhinal cortex (perforant path). Amino acid analysis of the dentate gyrus, which is the termination for the perforant path terminals. revealed no significant changes in amino acid content except for a small but significant depletion of glutamate. Autoradiographic analysis of hippocampal slices incubated in C3H]glutamate revealed that there was ;I significant loss of autoradiographic silver grains from the area known to receive the perforant path

ohser\:r-

terminals after lesions of this pathway (STORM-MATHISEN. 1977). As histological studies have shown that no significant neuronal cell death occurred in the hippocampus after this lesion it seemed reasonable to conclude that the loss of glutamate uptake. and the decrease in glutamate content of the dener\ated hippocampus, represent the loss of a population of glutamate-rich nerve terminals. The major difference of these studies from previous studies aimed at looking for glutamate pathways (see review b> JoHNsroU. 1972) was that all the histological informatIon made it possible to exclude non-specific neuronal death and terminal loss as the cause of the glutamate depletions. Following these findings. a number of laboratories (DIVAC,. FOTW~A 81 S~ORWMATHNV,

1977: MCGE~R

et ul., 1977: KIM. HASSLER. PAII< & SCHR~IXR. 1977)

have applied this approach to the ncocortex of the rat. All three groups are agreed that the cortex sends glutamate-containing projections to the caudate nttcieus. Thus. undercutting the cortex (MCGI.ER cr r/i.. 1977) or removing it bq suction (DI\.Ac L~Iill., 1977) result3 in a loss of glutamate uptake sites from the striatum and ;I selective reduction in the glutamate content of the striatum. This result is unlikely to be due to retrograde neuronal loss. as cortical lesions do not result in significant neuronal losx in the stri,rturn (EMSO\ & JOSEPH. 1975). MCGEER t’t (I/. 1lY77) also reported ;I significant reduction in glutamate uptake sites within the cortex following undercutting. This result could be interpreted as evidence for
projections. does not exclude the possibilit! that aspartate IS the transmitter. since this amino acid is also taken up by the same high-afinitl uptake %)\tern Apart from the uptake studies. wmc mrerestlng recent work has been carried out by BK,AIM)KI) and co-workers (DODD& BRADFORD. I Y7h) on the cortical release of glutamate in the freely moving rat following sensory stimuli and on glutamate release liwn the piriform cortex stimulated in rifro. Bradford and colleagues have developed an elegant apparatus to allow continuous perfusion of the cortex in the freclk moving rat. This technique coupled with an extremeI> hensitive ammo acid analyser. has enabled them to demonstrate glutamate release from cortical epileptic foci (P. R. DODD, Personal communication) (contirming the earlier results of KOYAMA. 1973 More

F

recently they have demonstrated glutamate relcaac from the visual cortex in response to light stimulatwn and from the motor cortex in response to stimulation of the Icg (BR~ZIXORII & DWII. 1978). In addition. using ;\I) irk cilw system, BRALPORD & RIG IIAIWS the calcium-dependent (1976) ha\e demonstrated releaw 01‘ glutamate from the piriform cortex 171 stimulation of the lateral olfactory tract. Eurther work has revealed the presence of glutamatc-responsive sttes (RIC.HARDS, 1977) near the termination OF the olfactory tract terminals and reduction in glutamate content in the piriform cortex after olfactory bulb ablation (BRADFORD & RI(.HAKDS. 1976). An interesting recent development has been the characterization of a number of rigid glutamate analogues uhich have heen used as glutamate receptor

r\

OB

OB

LGB -a a

FIG. 2. Schematic diagram to show propoaed

D SC

D

cortical glutamatergic projections in the rat brain. The diagram shows proposed glutamatergic projections for which there are supporting biochemical data (including data to show reduction in glutamate content m the projection area after lesions of the appropriate pathway and also reductions in high affinity [“Hlglutamate uptake in the projection area after pathway lesions), It would seem likely that each cortical area will send glutamatergic projections to its associated thalamic nucleus but this remains to be elucidated. (Based on data from DIVAC cr al., 1977, MCGEEH PI ~1.. 1977: FONN~~M PI trl.. 1977 and LUNII-KARLSEN & FONNUM, 1978.) Ahhreriations u.sed: AC, nucleus accumbens: CC. corpus callosum; CS, corpus striatum; FC. frontal cortex: HF. hippocampdl formation: LGB. lateral geniculate body: LS, lateral septum: MB, mammillary body: OB, olfactor) cortex; T, thalamus: and VC. visual cortex.

agonists (JOHNSTON, CURTIS,DAVIES& MCCULLWH. 1974; SIMON,CONTRERA & KUHAR, 1976). One of the most useful has been kainic acid, which has proved to be a selective neurotoxin (COYLE & SCHWARTZ. 1976; HENDRON & COYLE, 1977). Kainic acid is believed by MCGEER rt al. (1977) to cause a selective destruction of neurones which possess glutamate receptors. It is believed to cause a massive loss of glutamate from glutamate-containing nerve terminals leading to a glutamate-induced neuronal loss. presumably due to convulsive discharge and consequent hypoxia.

& WOLFF, 1978: RIHAL 1978). The tkpcs of lahelled cells seen arc illustrated in Fig. 3. There appear to be ;-aminobutqrate-accumulating or gluramic acid decarhoxylasc-containing neurones in all la!ers of the cortex. One interesting feature of both the autoradiographic ((‘HKO~‘W,ZLI. & WOLPF. 1978) and inlni~inohistochcmical techniques (RIBAR. 197X1i\ ~hc prc\cnce of significant numbers of ;‘-;lminohut!r~ttc-~on1;1inIng neurr,nes in la!er L’I and the caIlosal \vhltc mallt’r helo\\ laqcr VI. Previous biochemical :I~s;L!\ 01 !hc ;‘-~unlnohut)ratc content of the cortical la!cr\ 11ai Indicatcd a lov,er content in the deeper corrrcal ia! cr\ and white matter (HIRS(‘H & Rorct~s, IYQ). )_he\c t\ro results are not neccxsarily incompatible. ;I\ IU p~t~rai the content of an! putative neurotran~ml(ter w(~:~ld The evidence in favour of y-aminobutyric acid be expected to be highest in the ncr\c terminals. 50 rather than glycine as the major cortical inhibitor] that presumably the chemical estimation (11 ;--aminotransmitter is summarized by KRNJEVIC’(1974). In butyrlc acid distribution mostly rellec& the numbc.rs brief, the pharmacological properties of cortical inhibition and the demonstration of a release of y-aminoof nerve terminals in ;I la!cr. The tpc\ <)i’ielI\ labelled h! the antiserum to glutamic acid do_~~rho~\butyric acid during visual inhibition (I~ERSEN, MITlase or b! the accumulation of [“H I;!-amlnohut! ric (‘HILL & SRINI~ASAN. 1971) are consistent with the acid include horizontal ncuronc\ ((‘:~jal Rcrtlu\ ccils~ involvement of ;,-aminobutyric acid rather than glytine in cortical inhibition. and mall spiny \lrllates in la>cr I: ~1 Lnt‘r 11 IF ~-.~,,linohuf~,rcrre. Techniques aimed at localizing a number of fusiform cells with \ertlc:tlll arranged y-aminobutbric acid-containing cells and receptors dendrites and in la!er VI a \arict! 01 ccl15 can be are being rapidly developed as is the neuropharmacorecognized with round or fuslform sotnala I(‘~~Ho\WALI & Wor,r;r-. 197X). All the cell t!pe\ \l:unet! IILI) logy of y-aminobutyric acid (JOHNSTON, 1978). The most promising technique for localization of y-aminocorrespond to various stcllate neurones dczcrlbcJ tn butyric acid-containing neurones is the use of antiGolgi studies (RAM~)u 1 CAJAI, 19 Ii I. i u. C‘,tjal Kc-tbodies directed against the y-aminobutyric acid synzius cells. horizontal cells. Martinotll ccII\. ha&et thetic enzyme glutamic acid decarboxylase (ROBERTS. cells and double bouquet dendrite cells. Thcs~’ results have come from the visual cortex of the rat. Hone\er. 1976). Glutamic acid decarboxylase is known to be concentrated in y-aminobutyrale-containing neurones it seems likely that this pattern of dl\tribution [>I and terminals and its activity falls following lesions y-aminobutyrate-containing neurones \\ilI hc f thcsc graphic technique and the immunohistochemical technique seem to be in good agreement (CHRONWALL ;a-aminohutyric acid receptor site5 5rilhln l!?c c~~r!c\.

12

P. C’ t-MS<)\ nnci 0. LlUl)\ \I I

and it may be that as high specific activity labelled bicuculline is available an autoradiographic study of binding sites would be practical. This ;,-aminobutqric acid receptor assay has also provided a useful additional tool with which to Investigate posstblc y-aminobutyric acid agonists and antagonists. It is thus possible to test new y- aminobutyric acid agonists for their potency when applied on to cortical neurones by iontophoresis and also for their ability to displace y-aminobutyric acid bound to brain membranes (JOHNSTON,1978). GIWW. In contrast to y-aminobutyric acid. glycine. as mentioned above, is unlikely to be a major cortical inhibitory amino acid. Although it is not possible to rule out a selective distribution of glycine-containing neurones in one cortical area or layer. the evidence against glycine being a major cortical inhibitor! transmitter is strong. This evidence includes the absence of high-affinity glycine uptake sites in the cortex (i.e. no mechanism for transmitter inactivation), the low potency and reversal level of glycine (K~LLI & BEART, 1975) the elevation of cortical glqcine following cortical lesions, i.e. glycinc levels parallel glial cell proliferation (ULMAR. 1976) and the low number of strychnine binding sites in the cortex. [Strychnine is an antagonist believed to be selective for the glycine receptor (SNWER & BENNIITT. 1976).] Glycine. like glutamate. is a universal constituent of animal cells so that it may not prove possible to demonstrate an enzyme selectively concentrated in glycine-producing neurones to which antisera might be raised for immunohistochemical localization of glycincrgic neurones. Thus. autoradiographic localization of the high affinity [3H]glycine uptake sites may be the only histochemical technique for the selective visualization of glycinergic neurones. Tuurine Like y-aminobutyric acid, the sulphur amino acid taurine produces inhibitory effects on cortical neurones which are sensitive to bicuculline (HAAS & H&LI, 1973). This inhibitory effect of iontophoretitally applied taurine can be interpreted as the action of taurine on y-aminobutyric acid receptors on cortical neurones. The levels of cortical taurine are much higher than those of y-aminobutyric acid (Table 3) but only a small percentage (IO?!,) may be associated with nerve terminals (RASSIN, STURMAN & GAULL, 1977). The remainder is believed to be in glial cells, and/or blood vessels and serving a general metabolic role in neurones. Cortical taurine levels do not fall following undercutting (Table 3), indicating that there are no major ascending taurine-containing pathways. A high-affinity uptake system for taurine is found in cortex which is separate from that for y-aminobutyric acid (KACZMAREK & DAVISON, 1972) and taurine can be released from the cortex by electrical stimulation or potassium-induced depolarization (KACZMAREK & DAVISON, 1972). Autoradiographic localization of taurine has been attempted (EHINGER. 1973): the

results are consistent ulth ;I mainly g11al locali/atlotl. MANIN (197X) has prcsentcd blochcmical CL~ticn~e that there ma! IX ;I tarlrlne-c~)ntarrllrig pathua\ lrom the olfactor)

bulb

to the plrlform

cortex.

h;~wl

on

the deplction o( piriform cortex taurine Ic\cl~ aftct bulb ablation (MANIX t,. 197s) Such a po\sihle ‘t:t~rinergic’ pathwa! may allm 115to approach pharmacologicall! the site of action of taurine. and to dccidc whether taurinc mimics the action of the naturally, occurrinu > lransmitter.

AMINtS

Catecholamine-containing neurones can be demonstrated with high sensitivity and specificit! in the microscope using fluorescence histochemical methods based on condensation with formaldehyde (FAI.(.K. HILLAKP. THIEM~ & TORP. 1962: FAIXX. 1962) or glyoxylic acid (LINIWALL & BJ~RKI.~,NI). 1974tr). In the standard formaldehyde method cortical catecholamine-containing terminals arc not. however. casil! demonstrated

and later studies

have shown

that the

tluorescence picture obtained with this technique IS incomplete. The introduction of the glyoxblic acid fluorescence method (LIPII)\AI.L 81 BJOKKL~VI). 1974~) and various moditications of the formaldehyde method (H~)KFI:LI & LJII’L(iI)AHI.. 1972h; LoKI’+. BJ~~RI(LIISI). FAWK & LIW\AI.I.. 1976) have grcatl! expanded the possibilities for studies on cortical catccholaminc terminals. Due to their high scnsitlvitb. these techniques have revealed the noradrcnergic innervation in greater detail. and have In addition demonstrated previously unknown dopaminergic projections to the cerebral cortex. In addition to tluorescence histochemical methods. the dopaminergic and noradrenergic cortical mnervations have also been visualized b\i means of immunohistochemical techniques for demonstration of t! rosine hqdroxylase (H~~)wIIII. JOHANSSOX.Fr:x~ (;()I I)STEIN & PARK. 1977~1)and dopaminc [Ghqdrox~lase (SWANSON& HARTMAN. 1975). respectiveI>. A difl’crent methodological approach for the study of noradrenergic terminals in the cortex has been devclopcd by DESVARRWS& LAPWKKI (I 973). Topical appllcation of tritiated noradrenalinc allows the radioautographic visualization of cortical noradrenaline tcrminals at both the light and electron microscopic level (D~SC.ARWS & LAPIERKI.. 1973; LAPIEKKI,.B~.Aw DET, DEMIANVZUK& DIWARRIES. 1973). The neocortex receives both dopaminergic and noradrenergic afferent fibres. No adrenaline-containing neurones have been demonstrated in the cerebral cortex (HBKWLT. Fr!x~, C~OI.IISTEIN & JOHANSWU. 1974u). Thr noradrenalinc innervation has hecn known for several years (FAI.(K. MVHI:LXISH\,II.I & OWMAN, 1965: FLIXI. 1965: ARH~ITHNOTT.1966: Fr:x~:. HAMBERC~ER & H&FELT. 1968~) whereas the cortical dopamine innervation was discovered recently (HiitiW-T, LJI:N(;I)AHL. Fcixr: & JOHANSSON.1974c: Hiir<-

Neurotransmitters FELT. F~xE, JOHANSSON& LJUNGDAHL, 19746:

LIND-

in the nc‘ocortes

13

fibres are observed

in the neocortex

(SEGAI,. PI~K~L

VALL & BJ~RKL~NO, 1974h; LINIIVALL, BJ~~RKLUNI). & BL(x)M. 1973; PICKEL. SEGAL & BLOOM. 1974: Jolts MOORE &

STENEVI, 1974;

BERWR. TASSIN. BLANC.

MOYNE & THIERRY. 1974; LINDVALL, BJ~IRKLUND& DIVAC, 1978;

BER~ER. THIERRY. TASSIN &

1976) thanks

to the previous

THIERRY and S-LINUS &

co-workers

GLOWINSKI.

biochemical

MOYNE.

findings

of

(THIERRY. BI.ANC, S~BEL. 1973tr:

THIFRKY.

S~INCS,

BLANC & GLOWINSKI, 1973h). Noradrenuline. The noradrenergic innervation of the neocortex has been described by FCIXE of ul. (1968~) using the FalckkHillarp formaldehyde method and by LEVI-IT & MOORE (1978) and LINI)~ALL et al. (1978) using the glyoxylic acid method. The terminals are distributed in all cortical areas but vary in density between various regions and cortical layers (see also DESCARRIES& LAPIERRF. 1973; LAPIERREcd (I/.. 1973). Some of the very fine fibres typically pass perpendicularly to the surface. divide in a T-shaped manner in the molecular layer and then have a horizontal course parallel to the outer surface of the brain. Others distribute in a more diffuse manner. As a rule most of the noradrenaline-containing terminals are found in the molecular layer (Fig. 4A). The highest density of not-adrenaline terminals exists in the cingulate cortex. particularly m its anterior part, and in the frontal cortex. Axons in the cingulum can be seen to pass medially into the cingulate cortex. give ofI’collaterals in layers 111 and IV and enter the molecular layer where they divide to form a rich plexus of fibres. In the molecular layer. axons of a preterminal appearance can be followed for considerable distances. The noradrenaline innervation in the sensorimotor. auditory and visual cortex is arranged similarly but the terminal desnity is clearly lower. These results on the distribution and arrangement of the noradrenalinecontaining cortical fibres obtained with the formaldehqdc and glyoxylic acid tnethods have been confirmed bq immunofluorescence histochemical studies. using dopamine fl-hydroxylase as a marker for noradrenergic neurones (SWANSON& HARTMAN, 1975). It is generally agreed that the neocortical noradrenaline innervation originates in the nucleus locus coeruleus in the pons. Thus, it has been demonstrated that after lesions of the locus coeruleus or the dorsal tegmental bundle (which originates exclusively in the locus coeruleus) there is a marked decrease of histochemically demonstrable noradrenaline-containing terminals in the neocortex (UNGERSTEIX, 1971; MAEIIA & SHIMIZli, 1972: LIVBRINK & JOXSSON, 1974; LINI)VAI.L @‘t(II.. 1978) and a loss of biochemically measurable noradrenaline (ANLEZARK. CROW & GREENWAY. 1973: MOORI:. 1973; KOBAYASHI.PALKOvm. KOPIN & JACOBOWITZ, 1974: LII)BRINK & JONSSON. 1974. LFVITT & MOORE. 1978) and of the major metabolite 4-hydroxy-i-methoxynoradrenaline phenylglycol (ARBUTHNOrT. CHRISTIE,CROW. ECCLESTON & WAI.-~I-:R.1973; KORF, AGHAJANIA~ & ROTH, 1973). Furthermore. after injections of radioactively labelled amino acids into the locus coeruleus. labelled

& MCXIRE. 1977), and horseradish peroxidase injected into the neocortex is transported in a retrograde direction to the noradrenergic cell bodies in the locus coeruleus (LI.A~~As. REIYOSO-%AREZ Rr MARI’INWMORENO. 1975; LINDVALL rt ul.. 1978). The coerukr-cortical axons ascend in the dorsal tegmental bundle up to the diencephalon. Several routes of cntr! to the nrocortex have been proposed (Fig. 5). The ma.jor branch continues rostrallq in the medidl forobrain bundle up to the level of the rostra1 septum. where the fibres run dorsally into the cingulum (J-I XI. H~IKFI;I.T& UNGCRSINT, 1969: UNCX.RsTEl)‘r. 197 I : LIYIN~LL & BJ~RKL.CNII. 1974h; Jout s & MOORE.. 1977). Some fibres enter the internal capsule. traverse the caudate nucleus and reach the neocortex .it the lateral part of the corpus callos~tm (JA~OROWI-I-Z.1973: SACHS. JONSSOU & F~IXF, 1973: TOHYAMA, MAYI)A & SHIMIZI:, 1974; JON~:S6i Momt. 1977). Some of the locus axons follow the ventral amygdaloid bundle and ansa peduncularis to the neocortex: some axons which follow the same route in-

FIG. 5.

Schematic representation

ways for the locus coeruleus

of the various axon pathneurones

projecting

1~) the

neocortex.

ilhhr~~iutiom:

C, cingulum;

cholamme-containing hippocampus:

bundle:

DTB, dorsal tegmental cateEC, entorhinal cortex: H.

Ic‘. internal capsule: MFB. bundle:

PFC.

piriform

medial forebrain

cortex.

14

ANTERO-

POSTERO-

MEDIAL

I

MEDIAL

CORTEX

I,

CORTEX

PG ,SG:

MEDIAL

SURFACE

LATERAL

? ?anteromedial ? ?supragenual FIG. 6. Terminology of the neocortical

? ?perirhinal

DA system

m

DA system

used for the various subdickons dopaminerglc

SURFACE

systems in illustrated

DA system

suprarhinal DA system

of the medial cortex. schematically.

PG.

In addition. pregenual:

the distribution SG. aupragcnual.

A8920

A 7190

A7890 FIG. 7. Schematic in the neocortex. anterior

representatmn

Areas of termination

to the interaural

ilhhreviurwrl,s: pallidus: termmalis;

in eight coronal

AM.

of dopaminergic

terminal\

give the distance in microns

line.

anteromedial

capsule:

A 4110

planes of the distribution

arc indicated h) hatchings. Numbers

ac. nucleus accumhcn>;

ncp, nucleus caudatus

EC, external

A 5660

FMI.

ace. central

putamen.

dopamin&gic forceps mmor:

RF. rhinal fissure: SC. supragcnual

amygdaloid

ot,

olfactory tubercle: system: CA. anterior

HI.

dopaminergic intermediary

hippocampus:

nucleus:

IC. internal

system; SR. suprarhinal olfactoq

tract.

cl. claustrum:

gp. glohus

at. interstitial nucleus of the stria commishurc: C‘C. corpus callosum; capsule:

OT.

dopaminerglc

optic

tract:

system; TOI.

nervate the piriform cortex. entorhinal cortex, the amygdala and part of the hippocampus (LINDVALL & BJ~~RKLLJND, 19746; .IONES& MOORE,1977). Locus coeruleus axons have also been traced from the lateral hypothalamus along the posterior limb of the anterior commissure to the external capsule (TOHYAMAet al.. 1974: PKWI. clt al.. 1974). Dopur~~ine. The dopaminergic terminal axons differ from the noradrenergic ones in several ways. e.g. with respect to distribution, arrangement and morphology (LITWVALLer d., 1974; 197X; FUXE, HBKFI:LT, JOHANSSON. JONSSON. LWBRINK & LJUNGDAHL. 1974: Hij~~I:LT ot crl., 1074h: BERGERet al., 1974; 1976). In contrast to the noradrenaline-containing terminals, which have a widespread distribution to all cortical areas. the dopamincrgic ones are restricted to certain welldefined regions where they represent the majority of catecholamine nerve terminals (LINWALL et (I[., 1974: IY7X) (Fig. 4B and C). It should be tnentioned in this context that by studying the electrically induced release of [3H]dopamine from slices obtained from various cortical regions in the rat, SALIIATE & ORREG~ (1977) have obtained evidence for a widespread and diffuse dopaminergic innervation of the neocortex. Their findings suggested the presence of dopamine-containing axon terminals in the frontal. parietal and occipital cortex. rhc density of fibres diminishing in the rostra-caudal direction. These data are in partial conflict with the fluorescence histochemical (see LINDVALL et u/., 1978) and immunohistochemical (H~~KFELT et LI/.. 19770) studies where no dopaminergic projections to the parietal or occipital cortices have been found. Dopaminergic terminals are found mainly in the frontal lobe and in the ventral entorhinal area. In the entorhinal cortex. the dopamine-containing fibres form a series of clusters which are located principally in the 2nd and 3rd layers but also extend into the molecular laqer (H~KFELT or al., 1974h; LEWVALL et ul.. 1974: COLLIER & ROUTTENBERG,1976). lnterestin&l!. COLLIER & ROUWENBERG (1976) have shown islands of non-fluorescent nerve cells closely associated with the dopamine fibre aggregations in the ventral entorhinal area. The dopaminergic innervation has been found to originate in the A IO cell group (COL.LIER& RCKWENBERG, 1976; 0. LINIWALL 8~ A. BJ<)RKLI’W. unpublished observations: for description of the cell groups. see DAHLSTR~M& FUXE, 1964). In the frontal lobe three different dopdminergic terminal systems have been distinguished on the basis of fibre morphology, dirtribution of the fibres and locahzation of the cell bodies of origin (LINDVALL t’t ul., 1978) (Figs 3B and C’, 6 and 7). The w~tcmmedial doparuirw s~sr~wr is mainly found in the pregenual part of the anteromedial cortex (see also BERGER rr al., 1976; H~~KFI;LTc’t al.. 1974b; 1977a; LINWALL et rrl., 1974). It extends to the frontal pole but the majority of fibres are seen around the external capsule. The dopaminergic fibres are distributed through the Znd-hth layers with the higher density in the 5th and

6th layers; some scattered fibres are also found in the molecular layer. A minor part of the system extends with a progressively decreasing density into the supragenual part of the anteromedial cortex. Here. the fibres are distributed only in the basal cortical layers. T/w ,suprarhinu/ dopaminr .s>~strrc is found dorsal to the rhinal fissure in the frontal lobe and can be regarded as a direct lateral continuation of the anteromedial system (LINIWALL ct trl.. 1974: BERGIR Y( nl.. 1976). This dopaminergic innervation is most dense in the basal cortical layers but some terminal axons extend into the molecular layer. The suprarhinal qstem can be followed from a coronal level lust rostra1 to the nucleus accumbens 10 the most rostra1 part of the caudate-putamen. It continues caudallj around the rhinal sulcus in close relation to the claustrum and is then called the perirhinal dopamine system (Fig. 6). At caudal levels, this dopaminergic innervation is continuous with that of the piriform cortex. In contrast to the other dopaminergic terminal sqsterns. the fibres of rku .suprugunuu/ dopurnirw ,sn~t’m are distributed in the superficial cortical layers. The highest densit) is found in the 3rd layer but tibres extend through the 2nd layer to invade the molecular layer (Fig. 4B). The supragenual system is found in a restricted area of the supragenual part of the anteromedial cortex (anterior cingulate cortex) from the rostral part of the genu about 2 mm caudallp. The system extends about I .Smm dorsally from the corpus callosum. It is well established that the neocortical dopamine afferents originate in the mesencephalon (LINIWALL et ul.. 1074: F~xt et nl.. 1974: BERGER 1’1 (I/.. 1974: BECKST~AI). 1976; SIMON. LEMOAL, GALE). & CARDO, 1976: (-,\RTER & FIBIGER. 1977) (Fig. 8). Using various

F : anteromedial system R: suprarhinal system C: supragenual system FIG. 8. Relative location of dopamine cell bodies in ventral mesencephalon projecting to neocortical areas. Ahhruiarioms: IP, interpeduncular nucleus: ML. medial lemniscus: SN, substantia nigra.

types of lesions and retrograde peroxidase

Ihe localization

has recently VALL

tracing

been delineated

er 01..

in greater

197X). The supragenual.

suprarhinal

dopaminc

of horseradish

of the cell bodies of origin

f&-e

detail

systems originate

ferent parts of the A 9 and A 10 cell groups. giving

rise to Ihe antcromcdial

in the A 10 cell group. dS0

LINDVALL

STEAD.

1976;

genual

system

along

CARTER

suprarhinal

The proterminal Ihe

fibres

and in the lateral

to originate

the

rostromedial

axons

forebrain

ventral

further

‘;js-

(LI~~)\A~.I

and

rostrally

laterally

:und

into

tex medial to the cingulum

some

runs

la)erh

or sweep

of car-

feature

cortical

dopaminergic

recently

discovered

of the organization innervation

convergence

tex of mesencephalic

of the

is given

b!

in the prefrontal

dopamine

neuroncs

the cor-

and neur-

n~~cleus (DIVAC.

ones from

the mediodorsal

LINUVALL,

BJCIR~~LUYVI)& PASSINCII~AM, 197.5: DI\ AC.

BJ~~RKLI:&I). LINIIVALI. &

as the

from

the

neocortical

194X: DIVAC.

of

the

frontal

lobe:

the

in the

in three different and

findings

in

to

rcspectikely.

ferentially

also

it seems that the

a

of central

been known.

Whether

deGpraniin<.

of the uptake

(HORU.

(‘olt.~

&

noradrcnalinc cortlcat

&

of

found

neocortex prefrontal

is of interest

dopamine

neurones

dopamme

the

ficial a

laminar

distribution

above:

;IS 21 FMSO~

cortex precursor &

Kootj.

rn-

cortcr,

i\

cortex.

dopamincrgic corlical the

in

s>+ la>cr\.

:mtcromedlal

the

deep ulth

ia!er5

(hi\.

the

\\cre high both in the \upcrbasal

cortical

layer\

(E’llsoil

hydroxq tase actI\ iL> h;td

Gmilar

to that were

of dopamme. highest

111 the

in the \upragenual

cor-

lower in the deeper layers. Legmental arc;1 (A IO) caused

decrease of dopamine

pregenual

present

I’! ir/.. 197X). 45

of

15 found

tex) but were only slightly

the

of dopamincrgic

\upcrticial

concentrations

Lesion5 of the ventral

FVSOV

laycrh ~15UC ths hlghcxt

lakers (particularI!

a prominent

I IIC

various

h!

antcromedlal

extcn\lon

and in the most

the

part of the dopamine

& KCX)H, 197X). The tyrosine

superficial

also exists

the

Ihe

111 ~11

In the ~upragcnual

concentrations

area for

caudal

HI ;\%c

the histochcm-

CJIirl., 197X). In agreement

Noradrenaline

and

in system

pre-

systems than has prcviouslq such a specificity

the

dopaminergic

p,rt~‘nt

I LO. HoR-,.

tn

distribution

concentrations. present

I\

;I

lobe.

Mctl with

pregcnual

In (hat

ncuroncs

as dctcrmincd

in the basal cortical

dopamine

anterv-

to dopununc.

dopnniinc

cortex

the major

tn the

I,

(‘(‘I

(sec. e.g. l.i\i)\‘,~I.t.

above.

C~\

cortcu.

Mere \imiIar

the frontal

(197X) correlates

described

1971:

concentraironh

concentration

Ktx)tc

ncuronc;

\\ hich

1974). In contrast

Iayerb of the frontal

ot

~II 111~

1973. T%sslh. Twin t
arcas within

The

l’ou~l

dopamlne-uptdkc

,I

Kr Sut~)trc.

GL0WIusk;l.

rht

cc)iict‘iilr,tti~)II\

intO noradrenergic

MA(YCA~ 6i 111Kst.\.

to ihc

~NII

\uprarhinal

was

lo

manil\

and supragenual

the

was also

bq

much more precise and ‘specific’ organiza-

tion of the dopaminergic

in

blocker

I 11~’

tintlingb

studies (see e.g. L.IUI)\AL I I’m

these areas there

(LiPjl)\ALt,

1977: BJOR~CLI’YI). DIYAC, &

1978). The convergence

the classification points to

in

and

resistant

project

1tie dopaniincrgic

of

cortices

In

in the mcsencephalon.

in the monkey

terminate

(BROWS & GOLIIMAN. LINLIVALL.

:trea innervated

determinations

afferents

medial

whereas

resemble

nucleus also receives dopa-

biochemical

areas

111tiop.~-

changes.

h~drouq lase arc

shown by histochemical

nervatmn

the mediodorsal From

terminal

tyrosinc

Tile>

and non-..

(1978) II;~\c

K(H)II

lobe. the highc\t

terminals

nvtsworthq

presumed

originating

and

MO

CLIP\

hiaLochcniical

neurons\ 6i

obser\scd laminnr

cortex

dopaminergic

the frontal

with

tern

and primates.

thalamic

E:moh

l&c.

;ixon

each species. the frontal afferents

that rhc dopamincrgic frontal

Gmilar

The

and tree shrew are particularly

minergic

agreement

iii

tir Hi<. 1.1111-

the cortex

Since the ratio

\houed

tally

that

Ihc scnxwniotol-,

doparn~~~

on the other hand. the suprcpcnual

these

of mammals

data arc

c~tlpu-

from the rostra1 (0 the

in the opossum,

because

are

to noradrenaline

ilic

anti

1rans\crsc’

or both

caudal end of the ncocortcx. mine

lwiid

tronral

in

decreased gradualI!

II-<

i ;II’IOU\

ill

\\ere lower.

bh three

WOOLSI:\.

anteromedial

tree shrew.

animals

parts

projections

to topographicall)

opossum ancestors

is recog-

(ROSI, &

in the rat. dorsolareral

and frontopolar

BJ~RK-

and tree shrew) the medlodorsal

nucleus prqjects

suprarhinal

receix ing

nucleus

1972). It has been shown

species (rat. opossum thalamic

area

mediodorsal

cortex

the \alws cortices

found that concentrations

PASSI~~G~IAM, 197X: Btrc,I;-

DIVAC. 1977. The prefrontal

nized

areas

&

1976: BERGER t’/ trl.. 1976: L[NI)VAI.I,

STEAD, LUNII

thalamic

;IIIIIIIC\

~1 the

it/., 197X). i.e. in the pregenuai

c~~~dally

BJ~~RKL~;~I).

Rr

\;IILIC\

whereas

different

dopammc

1974h). An interesting

four

region

either

III~W

ytrrs~ & (‘.~KISSOS (1976) subdl\~ded

in

the

of

ill

oni-

M ith rr’cs:tlt hlo-

i & ~100~1 / l9,Xi

Li:l,ri

highest noraclrenalinc

within

the supragenual

(L,IUI)\ALI.

tictt’ri7iinatiori~

late cortices

di~trihliliori

;Lrc in good agreemcni

region\.

Other

the deep

cortex

callosum

chemical cortical

the

,111

nnradrcn;iliri~-c~l~~~~i~i~l~~~ tiwi

dnd

the neocortcx

cortex.

accumbcns Into

&

lenticularis

ansa

entorhinal

anteromedial

the corpus

the

dopaniine-

k,ir,,c,iro/tr,lri!r~,~1.11~lliii~!

in/ itala

histochemical

adrenaline

to the cortex

bundle

to the nucleus

dorsorostrally the pregenual

in

A IO cell group.

dopaminergic

cscencc

visual and auditor!

A IO. The

with the axons of the mcsohmbic

continue

above

supra-

distributed

1974h). In the lateral hypothalamuh

innervate

(see

of the pars compacta

fibres leave the system and pin to

part

1977). The

has been found

medial

BJBRKLCNI),

The cells

1976: Bi:( E;-

ccl1 bodies

in the dorsolatcral

ascend together in

in extent

nigra

system

cells localized

c’f rr/..

F‘IRIGLR.

the mediolateral

of the substantia

tern

&

originates

m dif-

m its medial

SlMOh

1974:

Yf rd..

and

s>stcm arc localired

mainlq

Bioc /~~Jilii~.~f/tr,ftrll.\i\

(Lihij-

anreromcdial

leaving

concentration

only

the

in noradrenergic 197X). The effect

neuroncs III

111

dopamine the

icf.

xupra-

genual cortex was much less marked. On the other hand. lesions of the substantia nigra caused significant depletions of dopamine from the superficial layers of the supragenual cortex but did not appreciably effect the dopamine content of the deeper layers. This is in agreement with the neuroanatomical data on the origin and distribution of the various dopamincrpic terminal systems. As described above the prefrontal cortex of the rat receives both noradrenergic and dopaminergic afferent fibres. The highest densities of the t\vo innervations arc found in the superficial and deep cortical layers, respectitcly. Recently. BUNNEI. & II~;~~.~A~~~~ (1976) suggesrcd a method for differentiation between dopamine and noradrenaline innervated cells in this area based on microiontophoretic techniques. They found a clear correspondence between the inhibitory response of a cell to dopamine and noradrenaline and the la>er in which tht: cell was located. Thus cells in laqcrs II and III were much more sensitive to noradrenaline th;ln dopamine. whereas the opposite was true for lalers V and VI. Thus. in the experiments of BI ~sr\r & !~C;tlAJA~l,4V (1976) the responsiveness of the cells within the prefrontal cortex to dopamine and noradrenaline seems rather well correlated with the densit! of the dopaminergic and noradrenergic inputs to the cortical layers where the cells are located (cf. abme). C‘t~t~~c~k~larl~rnr ~~eptors. Both a- and /I-adrenoceptars have been characterized in the neocortex of the & SN~DEK, 1976: U’PRICHARI) & rat (BILXYI) SN~.IXR. lY77: IJ’PRICHARI). GREENBERG.SHE:E~AN& S~~IXR. 1077). Single neurones can respond both with excitation probably mediated via x-adrenoceptors and depression probably via /I-adrenoceptors to noradrenaline (K RxJt:vI(’ & PHILLIS, 196%: JOHNSON. ROBFKTS. SOHII-.SLI:K& STRAIIGHAN. lY6Y: BEVAN. BHAIISHAW. ROWIUS

&

SZABAIN, 1974:

SZARAIX,

BEGAN 8r BRAIXHAW. 1977). Most neurones seem to ha\e both t) pes of receptor (SZABAIX et ol.. 1977) and thus. the response of a neurone to noradrenaline may rellect the relationship between the two functionally opposite receptor populations. Cortical neurones are sensitive to dopamine. which can give both excitatory and depressant responses (BEVAN. BRAIXHAW & SZAtsAl)I, 1975). In the somatosensory cortex noradrenaline stems to bc more potent than dopamine (BRAI+ SHAW, Bt:vAU & SZABAIII. 1977). The excitatory rehponsc to dopamme is probably mediated tia a receptor difJerent from the ,r-adrenoceptor mediating the noradrenaline response (BRAIXHAW et rr/.. 1977). It should also bc mentioned that a dopamine-sensitive aden)late qclase system. i.e. a specific dopamine receptor enz!mc system, has been found in the cerebral cortex of both rat (VON HUNGEN & ROBERTS. 1973) and monkey MAKMAN. 1975:

(MISHKA. DEMIRJIAN, KATZMAN & WEINRYH &

MICHEL. 1976). Using

a Huorescent /j-adrenergic receptor antagonist it has recently become possible directly to localize /I-adrenergic receptors in the rat cerebral cortex

(MELAMED. LAHAV & ATLAS, 1977). In the neocortex,

the fluorescence was observed in layers III~VI and in the inner part of layer 11. whereas in the outer part of la!er II and in the molecular layer, fluorescence was almost undetectable. The fluorescent dots were mainly localized on cell bodies and possibly also on dendritic ramifications of pyramidal neurones. between the Thus. there is a poor correlation P-adrenoceptor densit! and the fibre density and noradrenaline concentrations in the various cortical laqers (cf. above). However. a poor correlation between regional variations in receptor binding sites and endogenous Ievelc of neurotransmitters has been obscr\cd prekiouslb. e.g. for noradrenaline (U’PRICHAKI) c’t rrl.. 1977). 5-hydroxytryptamine (BEX:UFTJ acid (ENNA & & Si~\rI>I:R. 1976) and y-aminobutyric SN~IIFR. 1975). The cingulate cortex showed a unique labelling pattern (MFI AMEl) P’I d., 1977) with 21 high densit! of tluorescent dots in a distinct strip in layer II.

Compared to the catecholamine neurones very little is currently known about the distribution and organization of S-hqdroxytryptamine-containing neurones in the rat neocortex. The sensitivity of the fJuorescence histochemical methods (the formaldehyde and glyoxylic acid methods) is markedly lower for S-hydroxytqptamine than for the catecholamines. and furthermore the 5-hydroxytryptamine fluorophore shows a much higher rate of photodecomposition. Therefore. with available fluorescence methods, the distribution of S-h\droxytryptamine neurones in various brain areas is only partially revealed. Jmmunohistochemistry using tryptophan hydroxylase as a marker for 5-hydroxytryptamine-containing neurones has hitherto not given any additional data on the distribution of these neurones in the cortex. 5-hydroxytryptamine-containing terScattered minals have been demonstrated in the superificial laqers using the formaldehyde method (FUXL l965), most of them being found in the outer part of the molecular layer (FIIXE, H~KFELT & UUGERSTEIYI. 196Xh). .4 similar distribution of 5-hydroxytryptamine-containing terminals. localized mainly in the molecular layer. has been shown after pharmacological treatments which enhance the fluorescence intensity of S-hydroxqtryptamine (KUHAR, AGHAJANIAN& ROTH. 1972). The preterminal axons to the cortex have been traced after intraventricular and intracerebral in.jections of neurotonic dihydroxytryptamines (BJBRKLIND. Noms & STENEVI, 1973; FCXE & JONSSON, 1973). In these cases there is an accumulation of amine in the lesioned 5-hydroxytryptamine-containing axons. which now become visible after treatment with formaldehyde vapour. The fibres ascend in the medial forebrain bundle and at the level of the rostra1 septum they turn dorsally through the diagonal band and the septohypothalamic tract. Some

1X

1’. (

I

v’;o\

clnll 0. Llhl)\

of the fibres run along the cingulum giving 00 branches to the overlying cortex. others run rostrally into the lower part of the molecular layer of the frontal cortex (BJ~~R~~LuNI)(‘1 al.. 19731. In addition to fluorescence histochcmical dcmonstration of 5-hydroxytryptamine. indolaminr neurones can also be localized by autoradiograph>. This can be performed after intraventricular administration (AGHAJANIAN,BLOOM,LOV~LL. SHEARI) & FRIXI)MAN. 1966; FUXE & UNC;ERST~I)~-.19686: BI.(H)M. HOFFER. SIC;GINS, BARKER & NI~OLL, 1972: CHANPALAY. 197.5: 1978). local instillation (Mo~!RL:NMATHIE~:. LLGER & DESCARRIES. 1976) and topical application (DESCARRIFS. BEAI:I)E~ & WAXINS. 1975: BEAUIET & DESCARRIES.1976) of [“HIS-hydroxytryptamine. Using autoradiography. presumed 5-hydroxytryptamine-containing terminal varicosities have been found within all cortical layers of the frontoparietal neocortex of the rat except in la!er VI (BEAL’IX~ & DESCARRI~.S,1976). The intralaminar densit! of the 5-hydroxytryptamine innervation increases progressively from layer V to layer 1 in a distribution pattern suggestive of unspecific afferents. The highest densit) of indolamine plexuses after intraventricuiar administration of [3H]S-hydroxytrqptamine followed bq autoradiography has been found in the cingulate cortex whereas other parts contain only low densities (CHAN-PALAY. 1978). Lesion experiments indicated that the cortical 5-hydroxytryptamine innervation originates in cell bodies situated in the raphe nuclei of the mesencephaIon (ANII~:. DAHLSTR~M.Fuxr. LARSSON,OLSON & UNGERSTH)T.1966: UNGERSTMX.1971 : KLIHAKVI trl.. 1972; FUXE.& JONSSON. 1974). In the cat. BOBII.LII:R & CO-WORKERS (BOHILLIER.StCiUIN. PETI.~JI:AN,SAI.VERT, Tou~tr & JOCIVET. 1976) have injected [‘“Clleucine stereotaxically in several raphe nuclei and studied the efferent connections of these nuclei using the autoradiographic tracing method (DOKZ & LEBLONII.1963: LASEK..JOSEPH& WHITLO(X. 1968: COWAN.GOTTLIEB.HI’NURIC‘KSON, PKI~~: & Woo~sr y. 1972). After injections in the nucleus raphe centralis superior there was a labeliing of high densit) in the frontal neocortex, of moderate density in the gyrus cinguli, and of low density in the rest of the ncocortex. Injections in the nucleus raphe dorsalis gave labelling of high density in the frontal neocortex and of IOR density in the gyrus cinguli and the rest of the neocortex. In contrast to this, injections in either the nucleus raphe magnus or raphe pontis gave no or very low labelling in the neocortical areas. It should be pointed out that the labelling most probably also takes place in neurones that are not S-hqdroaytryptamine producing since in addition to the %hydrox)tryptamine-containing cells other, non-indolaminergic ncurones are present within the raphe nuclei. Therefore, this autoradiographic technique cannot give a pure demonstration of 5-hydroxytryptamine pathways.

,A, i

Dopamine. noradrenalmc ~1lId 5-hydrox) tr! pr.. amine arc now fait11 well established ah tranhmittcrx in the neocortex ot the rat. Honcvcr. other ;tminv\ that could possibly have a transmitter role ha\c ;11so been identified in the mammalian brain, such ;I!. hi\t+ mine (for references see GRr.r3. 1970: %I t1f.R 6i Pz> f0R. 1972: SCWWARTZ. 1975. S~,HWAKIZ. BARfjh. GARBARC;.POLLARI~, ROSE & VERIXRF, l97h), tr!ptamine (BOLLTON & MAJF.K, 1971: MAKTI~. SLOAU. CHRISTIAN6t C’LI;ME!+rs,1972: SAAL.EI)RA& Axt LROI). 1972: SAAL.EI)RA & AXELROI). lY73a: SVOIXIRASS & HORN, 1973; MARSLEU & CI:KZON. 1974: PIIII 111s. DC’RIXN & B~ULTON. 19746). S-methoxqtr\;ptaminc (GREEN. K~SLOW & C~S~A, 1973: Kosr.ow. 1971). melatonin (GREX c’r trl.. lY71). I,‘-phenqleth~lamlnc (NAKAJIMA, KAKIMOTO & SANO. 1964; Df:Rf)t ‘\i. PHILIPS &

BOI’LION.

1973: ~)WAKI)S

MOSNAIM. INWANC; &

SABELLI,

&

hAI_

1974:

197.3:

SAA\I:DKA.

1974~: WILLNEK. L~F~VRF & COSI A. 1974). /&phenql-

ethanolamine WlLLNER

(SAAVEDRA

&

AXI:LKOI).

19)73h:

(‘I u/., l974),

octopaminc (MoI_luo~t & AXELROI), 1972: SAAV~IXA. 1974h; Buck. ML.K~IIL & MOLINOFF. 1977) and the ~rlefu- and prrru-isomer> of tyramine (AXELSW~, BJ~~RKLLNI) & SEILER. 1973: PHILIPS, DURIXN

&

BOULTON. 1974~: ht.ff%.

DA\ IS.

DIXIXN & BO~;LTON.1975). The possible function of these amines as neurotransmitters in the neocortex IS as yet largely unknown. One major problem is the difficulty of demonstrating the intraneuronal locahzation of these compounds. Melatonin and its ptccur\ol N-acetyl-5-hydroxytryplamine have been demonstrated in the brain of the rat b> an immunohrstochemica1 method BuRENlfc. BROWSE& GROTA (1976) hut no neurones containing these amines were found with this technique in the neocortcx. InrlolruMw~. Fluorescence histochemical studies (B~ij~ti~f &I). FAIXK & STt:Ni:\ 1, 1970: 19710.11) have provided evidence for a population of indolaminc neurones in the rat CNS with microspectrofluorometric and pharmacological properties ditTerent from those of the %hydroxqtrjptamine-containing ncurones. These so-called B-type neurones hate their ccl1 bodies of origin in the raphe region and send asccnding axons through the mcsencephalon and hypothalamus (BJijRtif_f ~11 CI a/.. 1970). No neocortical projections have so far been demonstrated. The H-type Ruorophore has spectral and histochcmical properties of. among other indolamines. 5-methox!tr~ptammo (BJBRKLI-XI) or al.. 197lh) and it is tempting to spcculate that the B-type neurones reallq store S-methoxbtryptamine (cf. KOSLOW, 1974). To what t‘xtcnt thi\ is the case is as yet unknown. However. evidence has recently been presented (BJBRKLIINIX AxI.f.ssoh & FAL.CK. 1976) for a significant production of 5-mcthoxytryptamine and;or tryptamine in intracerehral structures outside the 5-hydroxytryptaminc and catecholamine neurone systems. The results indicated that this biosynthesis of indolamines IS ar Icast partly neuronal.

Histarninr. During the last few years, there has been growing evidence that histamine is a neurotransmitter in the mammalian brain, including the cortex (for references see. e.g. SCHWARTZ ef nl., 1976). Since the available fluorescence histochemical method for histamine is not sensitive enough for the demonstration of neuronal stores of this amine, the presumed histaminergic neurones have so far not been localized in the microscope. Instead the neurochemical effects of lesions have been studied and it has been shown that interruption of the medial forebrain bundle results in a decrease in histamine content, histidine decarboxylase activity and C3H]histamine synthesis in the ipsilateral cortex of the rat (GARBARG, BARBIN, FEGER & SCHWARTZ 1974; GARBARG, BARRIN, BIS~HOFF. POLLARD & SCHWARTI: 1976). However, the decrease in histidine decarboxylase activity is much more pronounced than the decrease in cortical histamine levels (GARBARG et al., 1976). This discrepancy could be explained by the presence of the amine in other neuronal systems and/or in non-neuronal cells, not affected by the lesion. This additional compartment is characterized by a high histamine content and a low L-histidine decarboxylase activity and may be localized in mast cells (GARBARG et ul.. 1976). Histamine-containing non-neuronal cells, presumably mast cells, have been demonstrated in the rat brain using histological (CAMMERMEYER,1972: IBRAHIM. 1974: KRUGER, 1974: EDVINSS~N. CERVOS-NAVARRO.LARSSON, OWMAN 8~ RBNNBERG, 1977) and biochemical (MARTRES. BALII)RY & S(.HWARTZ. 1975; VERIXERE. ROSE & SCHWARTZ, 1975) techniques. At present, it thus seems likely that histamine in the cortex is localized partly in a histaminergic neuron system ascending in the medial forebrain bundle and partly in nonneuronal cells, presumably mast cells. The respective sizes of these tw’o compartments appear in the rat cortex to be approximately the same (GARBARC; et a[.. 1976). In the cortex. two classes of specific histamine receptors, the H, and Hz receptors, have been found. the stimulation of which induces accumulation of cyclic 2’,3’-adenosinemonophosphate (BAUDRY, MARTRES & SCHWARTZ. 1975). Histamine seems to have a depressant action on the vast majority of cortical neurones, this effect being mediated mainly by H, receptors (HAAS & WOLF, 1977).

somatostatin (growth hormone release inhibiting factor). substance P, neurotensin, vasoactive intestinal polypeptide and cholecystokinin (for references see later). This list is undoubtedly incomplete and as more detailed dissections and immunohistochemistry is carried out it seems clear that the cortex will be shown t<>contain further peptides.

Substance P is an unadecapeptide originally described by VON EULER & GADDUM (19.71) and later purified and synthesized by CHANG & LEEMAN(1970). OTS~IKA. KONISHI & TAKAHASHI(1972) and OTSUKA & TARHASHI (1977) have produced good evidence that it may be one of the primary afferent sensory transmitters in the spinal cord. In the cortex the presence of substance P-positive nerve terminals in human cortical biopsies was described by H~~KFELT. MEYERSON,NILSSON.PERNOW & SACHS. 1976). In the rat the presence of material cross reacting with antisera raised against substance P has been demonstrated tn medial prefrontal, cingulate, entorhinal and piriform cortex areas (P. C. EMSON, unpublished observations). Preliminary immunohistochemical studies have revealed the presence of substance P-positive nerve tertninals in the rat prefrontal cortex (PAxI~~s. EMSON & CWLLO, 1978). The distribution of terminals containing substance P-like material was similar to that described for the dopaminergic terminals in this area (EMSON & KOOB, 1978~. This finding raised the possibility that this substance P-positive material might actually be in the dopaminergic terminals in this cortical area. However. lesions placed caudal to the substantia nigra/ventral tegmental area depleted the substance P immunoreactivity in the medial frontal cortex, but preserved the dopamine content (EMSON. 1978). The organization of the substance P terminals (Fig. 9D) in the frontal cortex is very similar to that of the dopamine terminals (Fig. 5B). suggesting that the substance P and dopamine terminals may interact. possibly by a presynaptic effect of substance P on the dopamine terminals or rice ww. However, although the distribution of substance P in the rat prefrontal cortex corresponds well to the known distribution of the anteromedial dopamine system it also corresponds to the distribution of presumed serotoninergic fibres originating from the dorsal raphe nucleus (AZMITIA, 1978); it is. therefore. noteworthy that cell bodies occur in other raphe nuPUTATIVE PEPTIDE TRANSMITTERS clei which contain both substance P and 5-hydroxyIn recent years there has been a tremendous tryptamine (H~KFELT, LJUNGDAHL. STUNBUSCH. VERupsurge in interest in peptides as neurotransmitter HOFSTAD,NILSSON,PERNOW& GOLDSTEIN1978h). The origin of the cortical substance P appears to lie in candidates, particularly following the discovery of the the brain stem as hemisections below the brain stem natural ‘opiate-like’ peptides, the enkephalins and endorphis (see review by HUGHES & KOSTERLITZ. raphe nuclei do not change the substance P content of the frontal cortex (PAXINOSet al., 1978). Iontophor1977), and the work of OTSLJKA& TAKAHASHI(1977) etic application of substance P to cortical Betz cells and their collaborators suggesting a role for substance produces prolonged excitation (PHILLIS& LIMACKER. P as a primary afferent transmitter. The rat cerebral 1974). The long duration and slow onset of excitations cortex contains cell bodies or structures that may be nerve terminals which cross react with antisera to elicited by substance P seem to be characteristic

(WALKFR, KEMP, YAJIMA. KITAGAWA 6i WOODRUFF, 1976: O~S~IKA& TAKAHASHI. 1977: BEN-ARI, LL GAL LA SALLE & L~:vHsQ~‘~., 1978). This suggests thilt substance P may be exerting its excitatory effects by a presynaptic mechanism modifying or modulating the response of the cell to another neurotransmitter. or alternatively substance P may evoke a second messenger response.

Vasoactive intestinal polypcptide is a polgpeptide containing 2X amino acids originally isolated from the gut by MIJTT & SAID (1974) and characterized and synthesized by BODANSZK~, KLAI.SNER & SAIII (1973). It produces a range of biological effects including vasodilation. hypotension and hyperglycaemia. The availability of the pure synthetic peptide enabled workers to raise antisera to vasoactive intestinal polypeptide and to use radioimmunoassay and immunohistochemical techniques for the study of its distribution. As its name implies. vasoactive intestinal polypeptide is found in neurones and terminals in the gut, but subsequent immunohistochemical and radioimmunoassay work has shown it to be present in a wide range of brain areas and especially in the cerebral cortex which has very high levels of vasoactive intestinal polypeptide immunoreactivity (SAII) & ROSENRERG, 1976: FAHRENKRUC; & SCHAFFALIT~KY IIF MUCKADI:L.L. 1978) (Table 4). According to Fuxt. HBKFELT, SAID & Muir (1977) vasoactive intestinal polypeptide positive neurones and terminals are found in all cortical areas in the rat, with a frequency of I -5 nerve cell bodies per 100. The positive neurones arc concentrated in layers If~lV in the neocortex (Fig. 9A, B), but in the phylogenetically older areas of the cortex (prefrontal lobe, cingulate, piriform cortex) the positive neurones were in all layers. If vasoactive intestinal polypeptide is a neurotransmitter and if it is found in as high a percentage of neurones and as 5”,,, then together with the cholecystokiningastrin-like peptides we may have to consider these peptides as major cortical neurotransmitters. Undercutting the frontal cortex of the rat had no significant effect on the amount of vasoactive intestinal polypeptide immunoreactive material in the frontal cortex, indicating that the peptide is intrinsic to the cortex (P, C. EMSON,J. FAHRENKRUG& G. PAXINOS,unpublished observations). The cerebral cortex also contains an adenylate cyclase which is stimulated by vasoactive intestinal polypeptide (BLOOM. IVERSEN& QUIK, 1978). This would be consistent with the presence of vasoactive intestinal polypeptide receptors in the cortex. So far, studies of the effects of iontophoretic application of vasoactive intestinal polypeptide on to cortical neurones have not been reported. It remains to be seen if vasoactive intestinal polypeptide is a neurotransmitter, but its presence in vesicles and its calcium-dependent release from brain slices would support this suggestion (EMSON,FAHRENKRUG,SCHAFFALITZKYDE MUCKADELL. JESSEL.L& IVERSEN, 1978).

As described for vasoactive intestinal polqpepttdc. the availability of antisera to cholecystokinin. gastrin and related pcptides has revealed the presence 01 neurones and terminals. particularly in the cortex (SIRAN M~IL.I.I:R.(‘~IoI, PAKoNt I IO & YALOW.1077). showing a positice reaction to these antisera (Fig. YC). Sephadcx chromatography of porcine bratn extracts has rccealed the prcsencc of peptides cross reacting with both cholecystokinin and the carboxytcrminal octapeptide of cholecystokintn (M~!LI.ER. S-IRA[;S & YAI.OW. 1977). R~HFFLD (1978u, h) has confirmed the findings of MIILLITRcat CI(.(19771 that the major cholecystokinin fraction in the cerebral cortex corresponds to the octapeptidc but hc has also found mmor amounts of the tricontatriapeptide (5”,,) and the carboxyterminal tetrapeptide. Rehfeld’s results also tndicate that the cerebral cortex does not contain gastrm and that the cholecystokintn octapeptide is the tnojt abundant known peptide in the brain (Table 4). The absence of gastrin in the cortex indicates that the cells described by SAC~~S,H&FF~.I. ME\~~RSO~. FI 111 bi REHFI:I.II ( 1978) as gastrin-positive in fact probably contain the octapeptide fragment of cholecqstokinin. The precise role of these cholecystokinin-like peprtdcs needs further investigation before a ncurotransmtttcr role is suggested. However. It is known that rhese peptides can be demonstrated immunohistochemicall) (SACHS v/ ul.. 197X) in structures that ma> he nerve terminals.

Neurotensin is a potent vasoactive peptide which was discovered during the experiments of Leeman’s group which lead to the isolation of substance P (CHAYG & LEEMAN. 1970; CARRAWA~ & LI LMAS. 1973). It was subsequently purified and shown to be a tridecapeptide (CARRAWAY & LEHMAN.1975). Only relatively small amounts of neurotensin have been demonstrated in the rat and bovine corticcs b> radioimmunoassay ((‘ARRAWA~, & L~~MAu. 1976: UHL & SN’~.DI:R.1976). However. neurotensin-positive structures that may be terminals have been observed near the pial surface and in the deeper layer of the cortex in the rat (UHL. KUHAR & SNYIEH, 1977). In addition the bovine cortex contains binding sites which show specific high affinity neurotensin binding (dissociation constant. K,,, of 1 nM) which could represent a btologically active neurotensin receptor (t!HI. & SNYINK. 1976). Neurotensin was also localized in synaptosomal fractions made from bovine brain (UII~. & SN’~DEK. 1976). All these data suggest that ncurotensin may have a neurotransmittcr role in the cercbra1 cortex.

Somatostatin is a tetradecapeptide isolated from the hypothalamus in Guillemin’s laboratory (BCRGIJ. LING. BUTCHER & GUILLEMIN, 1973:

BRAZ~AL. VALF.

B~JRCXS. LING. BUTCHER. RIVER & GULI.F.MIN. 1973).

71

j :

t’ (

22

I

kl\OI

and 0.

It was originall! characterized b\ its abllit! to mhlbit growth hormone secretion irr ~,irro but it IS found in considerable quantities outside the hypothalamus ilIld can be considered as ;I possible ncllrotranstnittcr ;IS well as neurohormone. Irntnunclhi~tclclieii~ic~~l studies have shown the prescncc ol somatostatin-positlvc neurones and presumptibc terminals 111the cerebral cortex of rats and humans (SACIIS 01 t/l.. 197X: Hii~FEt I- of 01.. 197x0). Somatostatm 15 concentrated in nerve terminal fractions made from rat brain (EPF:LI~ACIM. BRAZI AL. TSANG. BRAWI.R & MAKIIN. 1977). In addition somatostatin can he released from brain fashion IvF.RStb. slices in ;I calcium-dependent IV~RSI:N. BI.(H)M, DO~GI.AS. BROWV & V.ALI (197X). No information is yet available about the regional distribution of somatostatin-containing neurones in the cortex. Until this information is available nothing can be said about an) physiological role for somatostatin in cortical function.

As indicated in the introduction to this section the mammalian brain contains several other peptides which have been visualized in the CNS (but not the cerebral cortex) by immunohistochemical techniques. These peptides. which may all be considered as neurotransmitter candidates. include the enkephalins (leutine and methionine) corticotropin-like peptides (peptides related to adrenocorticotrophic hormone), bombesin and carnosine. Although these peptides have not been discussed here. with more detailed regional dissection of cortical areas in different mammals or improved immunohistochemical techniques some of these peptides may well turn out to be localized in terminals or neurones in the cerebral cortex.

C‘ONC‘LUSION The number of putative cortical neurotransmitters has increased considerably during recent years with the discovery of dopaminergic and histaminergic pathways to the cerebral cortex and especially with the recognition of a number of cortically located peptides. Peptides such as vasoactive intestinal polypeptide and substance P are located within neurones and

LINII\ \I

I

arc concentrated m varicose fihro\ which are prcsun-ably terminals synapsing on other neurones. In thl\ situation it \~ould be difficult to classif> these pcpt~dc\ as neurohotmones, and It vzen~ much more I’C;IYOIF able to regard them as neurotr~tncmittor~. It rcm;un\ to be seen whether these peptldcs hate the \;amc q,ort of role as the more classical neurotransiiiitter~ such as acetylcholinc or noradrenalme. It i\ posaiblc that peptide neurotransmittcrs ma\ provide ;I longer acting signal. prolsiding for the modification of ;I setting or gate rather than an all or none response
3ck,low[edyr,llr,lt.vWe arc indebted to Drs L. L. IVI RSIN and A. BJ~~RKI.~I~I>for their Interest in and encouragement during the writmg of this commentary and for readmg and criticising the manuscript at different stages in itr pl-oduction. We are particularly indebted to Dr Bmr C‘HK~INUALL for the photographs used in Fig. 3. and to Dr Tohl.ks HOKt-t.~~ for several photographs for Fig. 9. We are also particutarll grateful to friends and colleagues aho provided reprints and preprint!, of article> reteiant to this comnlc~v tar). These included Drs V. CHAN-PAI A). L. Drst AKK1t.s. J. FAHKLNKWI:C~.F. Fou\rru. S. Ht ~1. R. Lt;\!)-K*aI.~t Y. E. MCGI,IK. G. PAXINOS. J. RttlFI LI). J. SIoKht-MAtHtst \ and S. Shr III-K. The stimulus for thib work wab provldcd by the European Training Programme In Brain and BehaLiour Research who provided support in the form of :i leltoeship to one of us (PC‘E) and a twinning grant between our laboratories. In addition financial
REFERENCES AGHAJANIAN G. K., BLOOM F. E., LO~LLL R. A., SHhARo M. H. & FREEDMAN D. X. (1966) The uptake of 5-h>tdroxytryptamine-3H from the cerebral ventricles: autoradiographic localization. Biockem. Pharmuc. 15, 1401~1403. ANU~N N.-E., DAHLSTR~~MA., Fuxr- K.. LARSSON K.. OLSON L. & UNGERSTEUT U. (1966) Ascending monoamine neurons to the telencephalon and diencephalon. Actu phj~iol. stand. 67, 313- 326. ANLEZARK G. M.. CROW T. J. & GREENWAY A. P. (1973) Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions. Science. N.Y. 181, 682-684. ARBUTHNOTT G. W. (1966) A histochemical study of catechoiamines in the somatosensory area of rat cerebral cortex. J. Physiol., Lond. 186, 11%I 19. ARBUTHNOTT G. W., CHRISTIE J. E.. CROW T. J.. E~~LESTON D. & WALTER D. S. (1973) Lesions of the locus coeruleuq and noradrenaline metabolism in cerebral cortex. Expl Nrurol. 41, 41 I-417. AXELSSON S.. BJBRKLUNDA. & SElLER N. (1973) Identification of the para-isomer of tyramine in rat brain. L$> Sci. 13, 141 I-1419.

Neurotransmitters

in the neocortex

23

AZMI~IA E. (1978) The serotonin producing neurones of the median and dorsal raphe. In Handbook of‘ P~~chophur,f~rrc,olop (eds IVERSEN L. L., IVERSEN S. D. & SNYDER S. H.). Vol. 9, pp.233-304. Plenum Press, New York. BARKER J. L. (1977) Physiological roles of peptides in the nervous system. In Peptides in Neurohiologv (ed. GAIWR H.), pp. 295- 332. Plenum Press, New York. BAIX~RY M.. MARTRES M.-P. & SCHWARTZ J.-C. (1975) H, and H, receptors in the histamine-induced accumulation of cyclic AMP in guinea pig brain slices. Nuture. Lond. 253. 362- 363. data on serotomn nerve terminals in adult rat neocortex. Bnrirl BEA~,IXT A. & DESCARRIES L. (1976) Q uantitative Res. 111, 301-309. BEC.KSTEAI>R M. (1976) Convergent thalamic and mesencephalic projections to the anterior medial cortex m the rat. J. romp. Neural. 166, 403-416. BFESLE~ P. W. & EMSON P. C. (1975) Distribution of transmitter related enzymes in the rat sensorimotor cortex. Biochem. Sot. Trans. 3, 936939. BEN-AKI Y.. LE GAL. 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24

1’ t

!

VV’.

:Ititl

0

l.l\ll\

,;

1

~~I)WAKI)S D. J. & BLAI! K. (1973) Phenylethvlamines tonurla-like

characteristics.

F:HINC~~K B. (lY73) EMSON

Cilia1 uptake

P. C. (1978)

cortex Raven

Bioch~vt~. J. 13;.

and septum. Pres\,.

In

pollpcptide

in the rabbit

distribution

ISN

(VIP)

Dopc~min~,

Lesicular

t ‘8. t,wsoruP. ( Kr .IosI-w M.

retina.

experimentally

induced

phcn\ike-

Hrcl~,~ Rc). 60. 51 7 516.

of dopammc. .Svnpo\irrnz

J.. SCHAFFALITZhI

EMSON P. <‘.. FAHKrh’k:KI’c; mtestinal

of taurine

Complementary

in hi-am and lover of rats v.ith

95 ~100.

substance

P and

acetylcholme

R~)IIFKIS

P

WO~I)R~I~.F

(eds

1)1 Muck.4111 IL 0.

localizltlon

and

8:

13.. JI $9 I I T

pota\siun-

i,\okcd

in the G.

\‘I & I\txSlh

reled\e

from

r,tt

rat

IL’.)

prefrontal

pp. 37

J()O

I.. L. (19781 Va\~)aclr\e h)k7othalamus

Brr[l~~ I;,,,.

143. 174

mduced

F-MSOY P. Hr~rl

(‘. &

Rc,.

~blv)u

III

cp~lcps!

K(H)B G

f’. C’.. f’~rl!~oS

The

origin

I

and

projections

I IIA C’.-T..

fIuore\cent

and

distribution

S J. &

from

of enzymes

SNli)IR

Properties

in the porcine

formaldehyde.

F A., (‘h 13.. Ml-kit I)/ rahhil.

central

nerLou$

I,t.Istlvn.I

G. I. & OWMAN

1~11 p/l~~r,~~c. rou. 231.

F. &

MAI

IHF-SoREVsFN

s> stem

(1965)

K

HA\II~R(;~

Fuxr.

K.,

amine

of the cellular A. (1967)

Kc,\ (in press). and immuno-

bindlnf

in rat

hram

c!naptic

dl\trihution

of I-;lili~)i~nlnu~i~~~~~\‘~\.Ihlc

qtudie\

on \:l%):lcti\c

~nlc~linal

pal!-

(in prcqr).

Ioc:ili/atron

t‘iuorescence

c>f mi~noatninc‘;

of calechot,lmines

h! a Iluore
and

rctared

me~h
conip~~und\

cow

10, 34X 35-1.

(19721Molecular

D.

for

properties

gnthesis.

Proq.

E. (19771 Localization

the existence

terminals

K B. &

demon\tration

of adrenerglc

neriey

in cortc\-pla

of Gab,lcrgic.

and

acct\ ttransferase

of choline

their

lnipivtance

Ii 77.

Brrrirr Kc,.\. 36,

choliner-plc

,md aminergic

\Iruclurc\

in the

of monoamine

in the ner\ouI;

system.

H
neuron\

in

4c,rtr $11 \iol

Distribution

the

centrat

nervous

.~rr~d. 64, Suppl

of noradren,t)ine

s>rtem

IV

Dtstrlhurlon

247, 39 X5

ni’r!c

terminalr

in cortical

area>

(>I’

175 131.

Km. 8,

Hi)klm.i

T.. JOHANSSON 0..

!ermlnals

ner\r

ancl ;tcet!,Icho-

Brtrin

J. .Y<~t~rochr/tl. 29, 221 730.

nerve

the rat. Bruit:

complex.

receptor

Immunochemical

./. Vt,lrr&Ic!~

(‘H. (1965) Hiutochcmicai

of acetylcholme

Evidence

OI monoamme Fr’xt

cclrtc\

133-142

F.. WSI .US I. c;i I~IKSEN

mesohmblc K.

frontal

chcbline acct!ltransferase

:lcld IGABAI

B. (19%)

system.

.I. I-lisrochr!~~. (‘~Toc&!u.

for the corlip;trtmontatir)n

F‘i;xl

to the

Bruit7 Rec. 126. 309- 323.

c h t3.(19/12r Obvzr\ations on the pos>lhltltle\ 4(,/r/plrl~wd wml. 56. Suppl. 197. Ftr c h H.. 1111 I &ict’ N.-A. THIt ML
FO~C’IIXI

of c(jh;ltt

250, 243 245.

of y-aminobut!ric

F RI

tl

Lad.

to hoLIne

P.. TSANG D., BRAWFK J. & MARTIN J B. 1lY771 Suhcettular

in rat brain.

(VIP1

of the rat to the am~gdalold

Vcttwc.

t AIIRfNhRI G J & ScHAbl.AurZK;\r IN? Mrrch.4r)t.r I_ 0.

FO\\LIXI

tie\etopmcnt

U~tri,~ Rec. 100. XI 97

J.. RRuIAI

ull

the

I’. & SII \I II A. (197X) (‘holincacet~ltransferasc

the basal forebrain

in neurones.

S. H. (1975)

fractton\

v)mato\tatln

dt:ns~d

during

of c’(~p:l~,~ine-cont~lll~~~~~ afferentr

L. P. & WOLFGKAW F. (197-I) Antlboci!

(‘HAO

tocatvatron

mcmhranc

pcptide

rnori~h~~l,~f~~;~t ch:tnpc\

10.

G.. Lt GAL LA SALId (3.. Bm-ARI

containing

Fuc; I_ F.. l‘b

Epr 1 HAI’M

(1978)

Neurochemlcal

Re\. 93. 91

142. 24Y 267.

ilnehtcrass

t\h,\

H. (1975)

the rat. Brui,l

in limbic

JONSSON G.,

and

frontal

LnmRINK

cortex.

P. 6r LJI’N(xl14HI

Evidence

for

meso-cortico

‘1. (19741

The

ifopamine

neurons

origin

of

the

dop-

Hr~rlr~ Rc’\. X2.

349 355 Fr XI K.. Hi)ht-FL.1 T.. SAII, S. I. & M(JTT V. (1977) Vasoactibe tochemical cortcu Fvsr

cvltlence

1r

T. & LN(;t:RsrEC)T

(cds GRAI

co/m;\ K.

h\

HbhFbI.1

ml

K. & JoVSSoh

G.

Intravcntricular

G,\I i J. S.. BIR:I In controls

In

Mrrcrholivrr

(1974)

.‘ltlr.

K. & Uuc,f:Rsrt~lIT

after

U. (1968h)

pnlypeptldc

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V. Localization

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and peripheral

6, Part

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Further

Rio&m.

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of Amines

mapping

of monoamine\

in /he

of central

Pv~dwphurrt~.

I

10,

Histochemical Hivtoclwnic~

studies

Brrhr

neuronal

106. ii7

pathway

Chorea.

h)pothalamua.

forebrain

J. R;curoc,ho,l.

L. &

In

in the mammalian

5-h~tir~,~tr\ptaminc

It/rir~c L’X 11: Pliirrm-

central

neuron\:

norLou\

Macmrtlan.

itudicq

,hstcm London.

Lvith rhc neuroto\tc

on the drhiribuiion

,md

,~fc~ltecholamines

5-h?dr-o\tr)pt;lnllile

Rf. (147x1 Hum,m

brain

suh\tance

P

di\trlbutl,,n

30. 633 ~634

cells evidenced

C‘.

f lY76)

h> lesions

Dual

localiratlon

ill the lateral

of histamine

in .m

are,~.

hypothalamic

RVCII,I

Bruin

m rat brain

evidenced

$h IC\IOTI.,

S. H. &

COSTA

E. (1973)

quanritation

and

Identification

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In rat

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(19):li) CNS

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In

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of Nruroc~hrmisrr~

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CNS.

13. 16-28.

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iii

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GREI u A. R.. Kosr.ow

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HO1’PI.R ‘3.1. pp. t (!- 22.

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B4RAIN G.. FFGER J. & SCHWARTZ J. C. (1974) Histammergic

Ihe mrdi.ll

(;RFFN Ncu

s!s!em:

neuron\

12.

E. G.. IL~,RSI h L. L. & J~SSFLL T

E. D.. StvtiTs

;md Huntington’s

GARRARG M of

intracorticat

of indoleaik\ilam~ncs

G~RHAKG M.. BARHI\: G.. BISC’HOFF S., Pm 1.~~1) H. & SC.HWARTZ J Rc\.

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al the cellular

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1

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122.

Prehs.

htrqchnme 26Y 179

‘6

I’. (‘ Fw)u

and 0

I.l\lr\

.\i

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