Neuropeptides. Their Distribution and Function in the Brain

Neuropeptides. Their Distribution and Function in the Brain D.F:. SWAAB

INTRODUCTION The recent notion that peptides might act as neurotransmitters in the brain is based on the fusion of three quite independent lines of investigation, which all started in the 1930s. ( 1 ) Although the Scharrers demonstrated neurosecretory phenomena throughout the animal kingdom, according to critics the concept of neurons secreting hormones into the bloodstream was based upon “nothing more than signs of postmortem changes or fixation artefacts” (cf. Scharrer and Scharrer, 1940). The concept of neurosecretion became well-established following Bargmann’s ( 1 949) application of Gomori staining to the brain and pituitary, which enabled Bargmann and Scharrer ( 195 I ) to conclude that the neurosecretory material was transported from the hypothalamus to the neurohypophysis. In 1954 Barry proposed the existence of Gomori-positive terminations in extrahypothalamic areas of the central nervous system originating from the paraventricular nucleus, and which he called “des synapses neurosh-Ctoires” . These remarkable observations were unfortunately published in French. They were, however, most probably also forgotten because at the time they could not be related to the concept of neurosecretion, which was just being accepted. Later all attention was drawn by the discovery of another group of hypothalamic hormones, the releasing and inhibiting factors - TRH, LH-RH, somatostatin - which had great conceptual, fundamental and clinical consequences (for review see Guillemin, I978a). (2) A reverse relation between the brain and hormones had already been proposed in the 1930s on the basis of effects of pituitary extracts on the brain and the presence of the biological activity of pituitary hormones in the brain. Apart from Cushing’s work (1932) showing strong central, mainly Parasympathetic effects of neurohypophysial extracts after direct injection into the human cerebral ventricles (Fig. l ) , attention was focused on the anterior and intermediate pituitary lobe products. Zondek (1 935 ; Zondek and Krohn, 1932) found chromatophoric activity not only in its highest concentration i n the pars intermedia of the pituitary (hence the name “intermedin”), but also in the human fetal, neonatal and adult brain (TableI). Since he could not find any hormone in the cerebrospinal fluid, Zondek concluded that “internledin” was transported from the pituitary via the pituitary stalk to the periventricular brain. Popa (1 938) proposed that the transport of “xanthomClanophorine” from the pituitary to the brain takes place both via the portal vessels and the cerebrospinal fluid (Fig. 2). At present, however, there is a wealth of data showing that opiomelanocortin peptides are not only produced by the pituitary but also by neurons in the brain (for reviews see Smyth and Zakarian, 1982; Swaab et al., 1981).

98

Fig. I . “Showing the vaso-dilator and sudorific effects (sparing the bone-flap) of 2.5 mg of pilocarpine into the cerebral ventricles : an intraventricular injection of a cubic centimeter of pituitrin in susceptible persons gives an equally marked response” (from Cushing, 1932). This was the first central effect described of neurohypophysial extract5 in the human.

Fig. 2 . Showing the release of hypophysial products (derived from “neurotropine”) to the hypothalamus. 9, route from the pars distalis of the pituitary to the hypothalamus via the long portal vessels (“hCmoneurocrinie” of Collin); 10, release of products from the pars distalis of the pituitary via the intermediate lobe and neurohypophysis to the hypothalamus (“neurocrinie” of Collin) ; 1 1 ,the route from the pars distalis to the hypothalamus via the portal vessels. (From Popa, 1938.)

99 TABLE 1 BRAIN ANALYSIS OF A 36-YEAR-OLD WOMAN WHO DIED OF AN ACUTE EMBOLISM Extraction of the hormones was with 0.25 % boiling acetic acid. Note the presence of “intermedin” (chromatophore stimulating activity) in the various brain parts (from Zondek, 1935). Part of brain

Cortex Pituitary, anterior lobe Pituitary, posterior lobe Pituitary, stalk Tuber cinereum Hypothalamus directly adjacent to tuber Periventricular gray Thalamus, deep layer Optic tract Optic nerve Mammillary body Pedunculus Choroid plexus Corpus callosum Pineal gland Floor of the 4th ventricle

2.8 0.44 0.07 0.02 0.25 1.53 2.82 2.00 0.32 0.25 0.11 1.45 0.12 1.3 0.13 0.23

XANTHOMEL AMPHOR~VE.

0 4 000 1500

10 15 50 40 0 0 0 0 0 0 0 0 0

TABLE I1

L

0 0

PEPTIDES FIRST KNOWN AS HYPOTHALAMIC HORMONES For abbreviations SCK list before references Peptide (number amino ucids)

Cell 1ml.y

"Limbic" innervation

Other projections

AVP (9)

SCN

hd, hpv, OVLT

AVP (9)

SONiPVNdp

A, DBB, Ih, OB, sl, Ce, LC, NA, NC. NTS, VHi NX, P, PBD. PVG, RD, RLM. SC, SN, spinal cord

OXT (9)

SONiPVNnlip

A , OB, sm. TS, TT

LH-RH (10)

AH, BN, DBB, MBH, A, m. nih. MPA, ms, DBB, IP, PVG, VT ar, mh, MPA, OT, P, OB, OT, OVLT, SCN, PC, S , sm, TS (gliacells SPO MPA?)

-

Putative centrcrl functions

References

water balance, food in- Buijs, 1978, 1980; Buijs et a]., 1978; Hoortake neman and Buijs, 1982; Raichle and Grubb, 1978 ; (see all references on AVP in next paragraph) blood pressure, avoidance behavior, memory, brain devclopment. pain sensitivity, thermoregulation, water balance

Armstrong ct al.. 1980; Berntson and Berson. 1980: Boerctal.. 1980a,b; Britoet al., 1981 ; Buijs, 1978, 1980; Buijs ct al.. 1978, 1980; Buijs and Swaab, 1979; Buijs and Pevet, 1980; Cooper el al.. 1979; DK W e d , 1965, 197 I ; Jolles and Verhoeven, 1982; Kasting et al.. 1980; Kovacs et al., 1979: Lcgros ct al., 1978; Lipton and Glyn, 1980; Merkcr et al., 1980: Morris et al.. 1980; Oliveros et al., 1980; Raichle and Grubb, 1978; Swanson and Sawchenko, 1980; Van Praag and Verhoeven, I980 ; Versteeg et al., I979 ; Weingartner et al., 1981.

Ce. NA, NTS. NX, P. maternal behavior, PBD, PVG. RLM, SC. prolactin release, meSN, spinal cord mory, avoidance behavior, blood pressure

Armstrong et al., 1980; Buijs, 1978. 1980; Buijs et al., 1978; Buijs and Swaab, 1979; Buijs and Pevet. 1980; Ferrier et al., 1980; Kovacs et al., 1979; Lipton and Glyn, 1980; Pedersen and Prange, 1979; Salisbury et al., 1980; Swanson and Sawchenko, 1980; Van Praag and Verhoeven, 1980; Versteeg et al., 1979.

ovulation and SKX beha- Barry, 1976, 1978, 1979; Barry et al., 1973; vior influence on cho- Kastin et al., 1979; Krisch, 1980a; Krisch and roid plexus Leonhardt, 1980; Merchenthaler et al., 1980; Pevet et al., 1980; Phillips et al., 1980; Sakuma and Pfaff, 1980: Silverman and Krey. 1978.

a. A. AH, ar, Dr, Hi, A. CAI. CA2, HT. NC, SG. T. NTS. NA. reduction REM sleep, retina influence choroid hpv. hv. LiC. MPA, SFO. OVLT. OT. a NC, npv, OT. OTr, P, plexus function PVNp, S. SCN, Str. retina

Alpertet al.. 1976; Dierickx and Vandesande. I979 ; Eskay et al., I980 : Finley et al., I978 ; Guillemin et al., 1978b; HOkfelt et al.. 1978; Johansson and HBkfelt, I980 ; Krisch, I 980b ; Krisch and Leonhardt, 1980; Petrusz et al.. 1977; Pevet et al., 1980; Rezek et al., 1976.

hd, P. PF, SCN, PN. a. hd, hv, PF, P V N d p . zi, motoneurons, retina thermoregulation, shi- Boschi and Rips, 1981 ; Brown et al., 1977; hpv, hv. MO(raphe), PV, SCN. sl. BN vering, running devel- Cooper and Boyer, 1978; Eskay et al., 1980: opment, antidepressive Hiikfelt et al., 1975, 1978; Jackson, 1980; retina Johansson and HBkfelt, 1980; Pevet et al., 1980: Spindel and Wurtman. 1980: Stratton et al., 1976; Youngblood et al., 1979.

TABLE In PEPTIDES FIRST KNOWN AS PITUITARY HORMONES For abbreviations see list before references Peptideiprotein (number amino acids)

Cell body

“Limbic” innervation

Other projections

Putative central functions

References

ACTWa-8-y-MSH, P-LPW a-8-yendorphin, dynorphin (265 + 13 + smaller fragments?)

ar, choroid plexus, Dr, A, AH, ar, BN, hd, Ce, LC, NTS, PVG, hv, MBH, MO, moto- hpv, HT, m, MPA, PF, SN, spinal cord, T , ventricular surface neurons, P , PV, Zi PN, PVN, sl, st (I-HI), Zi

pain control, temperature regulation, thermal reception and its development, avoidance behavior, brain development, attention, antipsychotic, prolactin and LH release

Alde and Celis, 1980; Benjannet et al., 1980; Bugnon et al., 1979; De Wied, 1965; Dupont et al.. 1980; Goldstein et al.. 1979; Jacohowitz and O’Donohue, 1978; Joseph, 1980; Kastin et al., 1979; Lipton and Glyn, 1980; O’Donohue and Jacohowitz, 1980; Pacold et al., 1978; Pevet et al., 1980; Sandman et al., 1979; Swaah, 1980; Swaah and Fisser, 1978; Swaah et al., 1981; Tilders et al., 1977; Van Praag and Verhoeven, 1980; Van Vugt et al.. 1981 ; Walker et al., 1980: Watson and M i l , 1980; Watsonet al., 1977, 1979; Wardet al., 1979.

Enkephalin (MedLeu)

A, ar, BN, cp, hd, hv, A, C A I , CA2. HT, Ih, cp, gp, MO, spinal LiC, m, MO, NTS, PF, mh, OT, sl cord, T, Zi, retina PN, PNT, prem, pf, PVNm, sl, spinal cord, VNLG, hpv. PVG, retina

neuroendocrine, motor, sensory, antiamnestic, disinhihition of inhibitory interneurons

Akiletal., 1979: Dupontetal., 1980;Eskayet al., 1980; Hokfelt et al., 1978, 1979; Johansson and Hokfelt, 1980; Larsson et al., 1979; Micevych and Elde, 1980; Nicoll et al., 1980: Rigter, 1978; Van Leeuwen et al., 1982; Wamsley et al., 1980.

(5)

A

Prolactin (1 98)

increase REM-sleep, Drucker-Colin et al , , I975 ; Hoddes , 1979 : memory effects Mendelson et al., 1980; Pacold et al., 1978; Stem et al., 1975.

ar, hd, hpv, hv, PVNm, A, BN, HT (hd, ar), Is, LC, NTS, NX, PVG, T maternal behavior SON prem, SCN

Fuxe et al., 1977a; Hokfelt et al., 1978; Pacoldetal., 1978;Terkelet al., 1979:Toubeau et al., 1979; Yogev et al., 1980;Zarrow et al., 1971.

I03 (3) The third line of peptide research started when, during a search for acetylcholine, substance P was found by Von Euler and Gaddum (193 1) since brain extracts lowered the rabbit’s blood pressure, while this effect was resistant to atropine. The discovery that at least 10 times more substance P was present in the dorsal than in the ventral root opened, in the 1950s, the possibility that substance P was a transmitter of the primary sensory neuron (Lembeck, 1978). How similar the morphological basis of these three neuronal peptidergic systems really was, only became apparent after the isolation and characterization of the peptides and their subsequent immunocytochemical light and electron microscopical localization in the brain during the last few years. Ironically, however, it also became increasingly obvious, with insights in specificity problems which are inherent to immunocytochemistry, that we are not sure at all about the chemical nature of the stained compounds. Since an antibody recognizes an antigenic site, and not a compound as such, a positive immunocytochemical staining in fact does not prove at all that the expected compound is present (not even that a related compound is present !). On the other hand, in the absence of immunocytochemical staining the compound may yet be present, e.g. because fixation diminished antigenicity. Although I will not explicitly deal with this problem in the present paper for the various peptidergic pathways in the brain, there is certainly reason for doubt on the exact chemical nature of many, if not most, of the localized compounds mentioned in Tables 11-IV (Swaab et al., 1977 ; Pool et al., 1982). Examples of immunocytochemical staining of certain peptides in the brain which are most probably due to cross-reaction of the antibodies are: ( 1) the claimed presence of somatostatin or ACTH in the magnocellular supraoptic and paraventncular neurons, which appeared to be due to staining of neurophysins (for references see Swaab, 1982) ; (2) the assertion of Burlet et al. ( 1 980) that vasotocin is present in the suprachiasmatic nucleus of the rat, which should be based upon staining of vasopressin in this area, as vasotocin is not present at all in the mammalian brain (Dogterom et al., 1980); and (3) the observation of Martin and Voigt (1981) that enkephalins coexist with vasopressin and oxytocin in the neurohypophysial nerve terminals of the rat, which might be due to cross-reaction of their antibody to the neurohypophysial hormones and their neurophysins (see Van Leeuwen, 1982). In general, the specificity problem has received too little attention in the studies which propose (e.g. Hokfelt et al., 1980) the coexistence of different peptides or the presence of peptides in combination with other putative neurotransmitters within one neuron, in one synapse, or even in one granule, for which at present the evidence is as poor as the specificity procedures are. Adding to this the problem of heterogeneity of peptides by biotransformation in the brain (Trent and Weir, I98 1 ; Burbach et al., 1980), it will be clear that one cannot be sure of anything at this moment if the evidence is only based on an immunocytochemical staining. PEPTIDE TRANSPORT IN THE BRAIN The gradually changing notion that peptides apart from being neurohormones, might also act as neurotransmitters can best be illustrated with the changing ideas on their route of transportation of vasopressin into the brain. When De Wied showed in 1965 - the period when the neuroendocrine concept of the Scharrers found general recognition - that posterior lobectomy in the rat resulted in a more rapid extinction of conditioned shuttlebox avoidance response, and subsequently that the behavioral deficit could be substituted by vasopressin (Lande et al., 1971), it was only logical to explain the central effects of vasopressin by their release from the neural lobe and transport

TABLE IV PEPTIDES NOT PRIMARILY KNOWN AS HORMONES For abbreviations see list before the references Peptide (number amino ucids)

Cell body a, A, DBB, Dr,HT(hd, hv, PV, prem, SCN), ip, mh, MO, pons, PVG, S, st, TO, retina

"Limbic" innervation

Other projections

Pututive centrnl functions

A, BN, HT (a, AH, ar, cp, ip, MO. NC. pons, sensoryipain transmis. hd, hpv, hv, PN, SCN, SN, spinal cord sion, somatostatin reSON), LiC, OB, sl, st, lease, learning TO, VHi

References

Ben-An et al., 1977; Eckstein et al., 1980; Eskay et al., 1980; Hokfclt ct al., 1978: Huston and Staubli, 1978. 1979; Ljungdahl et al., 1978; Paulin et al., 1980; Saito and Saito, 1980; Stiubli and Huston, 1979. 1980; Szrcniawski ct al., 1980.

Angintensin II (8)

A, epcndym (AP, NV. hd, MBH. A, OVLT P, PF. pituicytcs, PVNpim, tanycytes (3rd ventr.)

teg- drinking behavior, milk intake, temperature regulation, cardiovascular, AVPiACTH secretion

Changaris et al., 1978; Fuxe et al., 1980a; HBkfclt et al.. 1978; Landas ct al.. 1980: Lin, 1980: McDonald ct al., 1980: Misantone et al., 19x0; Ramsay et al.. 1979: Sladek and Joynt, 1979 (rcnin in Ce, m , MO, oligodcndrocytes, PVN, SON - Fuxe et al., 1980b; Inagami et al., 1980).

Neurotcnsin (13)

A,AH,ar,hd.hpv,HT, A, hpv, m , MBH, PN. MO, NC, NX, PVG, somatostatin release, SG, T , m MPA. pf, PVNp, st, sl hemodynamics, temtegmentum perature, locomotion, pain, vascular permeability

Fahrenkrug ct al., 1980; Kahn et al., 1980; Mason et al., 1980; Ncmeroff ct al., 1977; Saito and Saito, 1980; Sims et al., 1980; Uddman et al., 1980; Uhl et al., 1977.

Bombesin

-

HT. LiC

Ce.

MO, SG, mentum, cp, LC

T

temperature, satiety

(14)

Martin and Gibbs, 1980; Polak and Bloom, 1979; Walsb et al., 1979.

Vasoactive intestinal A , ar, BN, DR, Hi, hv, a, A , AH, BN, Hi. LiC, cerebrovascular nerves, cortical functions, vaAP, NC, neostriatum, sodilatation, prolactin LiC, NC, OB, OT, MBH, P , PN, SCN peptide (VIP) PVG, SCN, sl, T , sup. NTS, spinal cord, T , P release (28) colliculus

Bataille et al., 1981 ; Fuxc ct al., 1977b: Larsson et al., 1976a.b; Roberts et al.. 1980: Rotsztejn et al.. 1980: Samson et al.. 1980: Sims et al., 1980.

Sleep promoting pep- AVT, supposed to induce REM sleep (Pavel, 1979) is not present in the mammalian brain Delta sleep-inducing peptide (DSIP) : no immunocytochemistry available. tides Growth hormone and somatostatin: see Tdbk 11.

Dogterom et al.. 1980: Negro-Vilar et al., 1979.

GastridCCK (

17133-8-4)

A, A8. A9. AIO. a. A . Hi. hpv. HT (hd. cp. ip. mesencephalon, satiety, analgesia. hy- Beinfeld et al.. 1980: Della-Fera and Baile, C A I 4 . Hi. hpv. LiC. hv). LiC. olfactory MO. NC. NTS, NX. perglyccmia. cortical I979 : Innis ct A , . I979 ; Loren el al.. I979 : MO. NC, PVC. PVNm, siructures. O T , P N . S , RD, spinal cord. st, T facilitation. regulation Morlcy and Levine. 1981 : Ooinura ct al.. ncurohypophysial hor- 1978: Rehfeld et al., 1979, 1980: Straus and SCN. SON. subilicum SCN. nt

mone release Glucagon (29)

A , A H , ependyma, T, AH, HT (hd, hpv. tanycytea PVNndp), MPA, SCN, SON

Carnosine (2)

olfactory nerve

OB

Bradykinin (9)

HT, T

H (hd). sl. LiC

Yalow, 1979; Vanderhaeghen et al., 1980; Zetler, 1980.

glucoregulation. tem- Conlon et al.. 1979; Dorn et al., 1980; Lipton perature regulation and Glyn, 1980; Loren et al., 1979. olfaction

T. cp, NC. PVG

E m w n , 1979; MacLeod, 1978; Margolis, 1978.

regulation blood pres- Innis and Snyder. 1980 sure, temperature, analgesia

106 via the bloodstream back into the brain. For the neurohypophysial hormones this route became seriously doubtful since: (1) Van Wimersma Greidanus et al. (1979) could not find a correlation between peripheral vasopressin levels and passive avoidance behavior; (2) the blood levels of neurohypophysial hormones were extremely low (e.g. Dogterom et al., 1977) ; while (3) these peptides do not readily cross the “blood-brain barrier”, which is as effective to neuropeptides as, e.g., to the catecholamines (Pardridge et al., 1981). Moreover, (4) retrograde flow via the portal blood vessels from the neurohypophysis to the median eminence is not of importance as a route of transportation (Dornhorst et al., 1981). Such arguments against the bloodstream as the route of transportation exists also for other peptides, like those of the proopiomelanocortin family. Since ( 5 ) in addition, the concentration in the brain of “pituitary peptides” such as a-MSH and ACTH is unchanged after hypophysectomy (for references see Swaab et al., 1981) the brain came to be accepted more and more as the primary source of centrally active peptides, although some contribution of pituitary peptides to brain concentrations may not be fully disregarded (De Rotte et al., 1980). The second possible route, via the cerebrospinal fluid (CSF), was also not supported experimentally, as illustrated again with vasopressin as an example, since : (1) the inhibition of passive avoidance behavior by intracerebroventricular administration of anti-vasopressin, which was first explained by inactivation of vasopressin by binding to the antibodies in the CSF (Van Wimersma Greidanus et al., 1975), could also be accounted for by the rapid (300 pmlh) transportation of antibodies from the ventricles into the brain tissue (Swaab and Boer, 1980); (2) the high vasopressin levels in the CSF of hypophysectomized rats (Dogterom et al., 1977) apparently did not fit in with the disturbed avoidance behavior of hypophysectomized or posterior lobectomized animals (De Wied, 1965) ; and (3) neurohypophysial hormone-containing fibers on the ventricular surface seem to be present in early development (Boer et al., 1980a, b ; Buijs et al., 1980) whereas fiber terminations on the CSF in the region of the median eminence were, in spite.of much effort, not found in the adult rat (Buijs, 1980), although such structures were considered to be the morphological substrate for the CSF route in adulthood (Goldsmith and Zimmerman, 1975 ;Rodriguez, 1976). (4) It has always been hard to imagine how peptides could be transported effectively from the proposed site of release into the CSF upstream to the septum and other proposed sites of action. Recent data (Wang et al., 1981) confirm indeed that vasopressin released into the CSF has a different origin (probably the extrahypothalamic release sites) than that released into blood. ( 5 ) The blood-CSF barrier is an even more improbable route of transportation than the blood-brain barrier since its surface is 5 000-fold smaller (Pardridge et al., 1981). Only 0.01 5% of the intravenously injected a-MSH seems to reach the CSF (De Rotte et al., 1980). In the meantime, immunocytochemical observations revealed, however, an alternative site of production for vasopressin -the suprachiasmatic nucleus (Swaab et al., 1975 ; Vandesande et al., 1975) - and rediscovered the existence of extensive exohypothalamic pathways of neurohypophysial hormone-containing fibers terminating from the olfactory bulb down to the spinal cord (Kozlowski et a]., 1978 ;Buijs et al., 1978 ;Buijs, 1978, 1980; Sterbaet al., 1980). The same appeared to hold good for the other neuropeptides (cf. Tables 11, III and IV). Immunocytochemistry at light and electron microscopical level revealed that these peptide-containing fibers terminate in brain areas on other neurons by means of synapses mainly on the dendritic tree, but also on the cell bodies. The peptidergic synapses are morphologically indistinguishable from the conventional amine- or amino acid-containing specimens, except for the fact that the synaptic vesicles contain vasopressin or oxytocin (Buijs and Swaab, 1979; Buijs, 1982), substance P (Chan-Palay and Palay, 1977) or enkephalin (Pickel et al., 1979). Although final proof is lacking, these peptidergic pathways are currently generally considered

I07 to be the most probable route for the central effects of endogenous neuropeptide since: ( 1 ) other routes of transportation can be excluded or are highly inefficient (see before) ; (2) for a behavioral effect of peptides 200-1 000 times less peptide is needed when intracerebroventricular administration is used than after peripheral administration (De Wied, 1977), while another reduction of a factor of 20 can be obtained when the peptide is administered directly into the specific brain area (Jolles and Verhoeven, 198 I ) ;(3) the localization of the brain areas in which central effects are found correlate very well to the sites of termination of the various peptides (for examples : Buijs, 1980, and Tables 11, IT1 and IV) ; and (4) peptides can indeed be released from the central sites of termination of the peptidergic pathways (Buijs, 1982). Little is known about the exact course of the peptidergic pathways and the exact connection between their sites of production and termination, a topic which should be studied in the coming years by lesions and by combined imniunocytochemical and anatomical tracing techniques. Substantial evidence can certainly not be obtained by simply following fibers in thick sections. The vasopressinergic innervation of the lateral septum (Table II), which was thus thought to be derived from the suprachiasmatic nucleus (Buijs, 1978; Sofroniew and Weindl, 1978), did not diminish at all after electrolytic lesion of that area (Hoorneman and Buijs, 1982) and will thus probably come from either the supraoptic or the paraventricular nucleus or from an as yet unidentified source. Neuropeptides are compounds produced by nerve cells and acting within the nervous system, probably mainly via direct peptidergic pathways. Only for historical reasons they can still be subdivided into the three following groups. ( I ) Peptides which were first known as hypothalumic hormones, i.e. vasopressin, oxytocin, LH-RH, somatostatin and TRH (TableII). In contrast to reports in which vasotocin was, and still is, considered to be present as a fetal neurohypophysial hormone in the mammalian brain and remains present as a pineal hormone in development and adulthood (Perks and Vizsolyi. I973 : Skowsky and Fisher, 1973 ;Legros et al., 1976) to which REM sleep-inducing potentialities are ascribed (Pavel, 1979 ; Pavel et al., 1980),this peptide appears not to be present in the mammalian brain in any appreciable amount (Negro-Vilar et al., 1979 ; Dogterom et al., 1980; Pevet et al., 1980). Bowie and Herbert’s (1976) immunocytochemical localization of vasotocin in the rat pineal is thus another example of a “false positive identification” of a compound by this procedure, although the presence of a vasotocin-like peptide in the pineal is, of course, certainly not excluded. (2) Peptides which were first known as pituitary hormones, but are also produced by the nervous system. To this groups belongs the ever enlarging family of peptides derived from the opiomelanocortin prohormone(s), e.g. GI-, b-, y-MSH; ACTH; b-, y-LPH; a-, b-, y-endorphin, dynorphin and MedLeu-enkephalin (Table 111; for review see Smyth and Zakarian, 1982), while the “neuroproteins” , growth hormone and prolactin, might also be considered to belong to this group. When the line is drawn from the more conventional group of transmitters -the amino acids -to the more recently proposed group of putative neurotransmitters -the neuropeptides - , proteins indeed seen1 to be a logical new group of neurotransmitter candidates to be considered. (3) Peptides (Table IV) which were not prirnar-ily known as hormones, like substance P , angiotensin 11, neurotensin, bombesin, vasoactive intestinal peptide (VIP), delta sleep-inducing peptide (DSIP), peptides of the gastridCCK family, glucagon, carnosine and bradykinin. The way this group is defined is only determined historically and certainly does not exclude that such peptides have endocrine effects ; substance P may alter prolactin and growth hormone release from the pituitary (Eckstein et al., 1980). Angiotensin II stimulates vasopressin release (Sladek and Joynt, 1979). Neurotensin is thought to modulate the pituitary release

108

of growth hormone, prolactin, LH, TSH and FSH (Kahn et al., 1980; Snyder, 1978), while VIP is found in hypophysial portal blood (Shimatsu et al., 1981) and may be of physiological importance in prolactin release (Rotzstejn et al., 1980; Samson et al., 1980; Bataille et al., 1981), and CCK can alter plasma, LH, prolactin, growth hormone and TSH (Vijayan et al., 1979). The sites of termination of the various neuropeptides as given in Tables 11-IV show that the peptidergic systems do certainly not confine themselves either to a predominant innervation of the limbic system or to a presence in phylogenetically old systems as has often been claimed in the literature. Fields of termination are found practically everywhere in the nervous system, from the olfactory bulb down to the spinal cord, and are in no systematically spatial way related to the fields of termination of the classical transmitters. These observations do not, consequently, provide any support to the idea that peptides would act via particular classical transmitters. The list of neuropeptides as given in Tables 11-IV, however extensive they may be, is probably still far from being complete. In the first place, the data mentioned are mainly limited to the literature on rat and man. Secondly, other peptides like calcitonin (Galan Galan et a]., 198 1) and motulin (Chan-Palay and Palay, personal communication) are claimed to be present in the brain. A third addition to the list comes from the comparative literature. Many peptides related to those in the mammalian pituitary and brain were also found in non-mammalian neurons (e.g. Boer et al., 1979; Schot et al., 198 I ) . Recently it proved to be also a fruitful idea to test whether the mammalian brain contains “invertebrate peptides” . Examples are at present not only the amphibian skin peptide bombesin, which is found in the rat brain (see Table IV), but also the hydra head-activator-like peptide which is present in the fetal rat brain (Schaller et al., 1977) and in the human hypothalamus (H. Bodenmuller, personal communication). In addition, avian pancreatic polypeptide, isolated originally from the pancreas, was subsequently identified in neurons of invertebrates (e.g. Schot et al., I98 1) and recently also in the rat brain and spinal cord (Hokfelt et al., 198 I ) , while the molluscan cardioexcitatory tetrapeptide FRMF-amide was not only found in the snail, insects and a fish, but also in nerve fibers of the mouse nucleus pardbrachialis and the nucleus of the solitary tract (H.H. Boer et al., 1980). It goes without saying that the specificity of the immunocytochemical stainings is an even greater problem in such cross-species studies than it is in the mammalian literature.

THE DOUBLE FUNCTION OF PEPTIDES - HORMONES AND NEUROTRANSMITTERS The extensive and varied regional peptidergic innervation of the brain suggests already that a single and sharply defined central function of a particular neuropeptide is unthinkable, in the same way as it is for the amines and amino acids. Yet, the administration of a peptide may elicit or modulate, under particular circumstances, a certain behavioral pattern which may even bear a logical relationship to the peripheral endocrine effect of the same peptide. Especially data from the first group of neuropeptides suggest that such a double function might be of importance for coupling central and peripheral adaptive mechanisms. In situations of stress vasopressin produced by the paraventricular nucleus and released in the external zone of the median eminence might induce ACTH release (Vandesande et al., 1974; Gillies and Lowry, 1979; Dornhorst et al., 1981), which stimulates the adrenal and signals stress back to the brain, while centrally released vasopressin might enable adaptation of autonomic functions

109 (Bohus, 1980; Swanson and Sawchenko, I980), causes antinocicepsis (Berntson and Berson, 1980) and facilitates the recollection of stressful situations (Weingartner et al., 1981 ; De Wied, 1980). Oxytociri released from the neurohypophysis is involved in milk ejection (Lincoln and Wakerley, 1974) while centrally released oxytocin facilitates this reflex (Freund-Mercier and Richard, 198 I ) and induces maternal behavior (Pedersen and Prange, 1979). Suckling seems consequently to induce maternal behavior by oxytocin release and by prolactin release (Zarrow et al., 1971), the latter also being stimulated by oxytocin (Salisbury et al., 1980). In this way suckling seems to couple milk ejection, milk production and maternal behavior, by means of a number of positive feedback loops that are only interrupted by weaning. Another example of a peptide with coupled central and peripheral effects is LH-RH, which induces ovulation by its action on the release of pituitary gonadotropic hormones and may facilitate lordosis behavior via the LH-RH-containing exohypothalamic pathways to the periaqueductal gray (Sakuma and Pfaff, 1980). Since TRH does not only stimulate thyroid function, but also elevates body temperature (Brown et al., 1977) and induces shivering behavior (Cooper and Boyer, 1978): probably via the exohypothalamic fibers terminating on niotoneurons (Hokfelt et al., I975), TI2H might be the crucial factor for an integrated adaptive reaction to a cold environment. Similarly coupled central and peripheral actions are proposed for neuropeptidea from the other groups ;peptides of the ACTHiMSH family which are known to be released during novel or conflicting environmental stimuli induce also enhanced grooming behavior (Jolles et al., 1981), the gastridCCK peptides would be involved in tractus digestivus physiology and the central regulation of appetite (Straus and Yalow. 1979), while alterations of glucoregulation at the periphery are supposed to be reflected by changes in the same peptides in the brain (glucagon, somatostatin) which are also involved in pancreatic function. INVOLVEMENT OF PEPTIDES IN DEVELOPMENT Increasing evidence shows that, like the classical neurotransmitters (the amines and amino acids), sex hormones and thyroid hormones, also neuropeptides obey the rule (cf. Swaab, 1980) that factors which are of importance for adult brain function are also involved in brain development. In view of this and the data presented in Tables 11-IV, it may not be surprising that the effects of neuropeptides on the developing brain, which probably concern the formation of cells, neurites and synapses, will often result in more general, rather than in well-defined, functional changes of the brain. Permanent effects have been described after neonatal administration of vasopressin, TRH, a-MSH, ACTH and analogues, which are - if the authors paid any attention to this point - often reported to be sex-dependent (for reviews see Swaab and Martin, 1981 ; Swaab and Ter Borg, 1982). Recent observations suggest a morphological basis for such sex-dependent effects. In addition to the sex difference in cell density in the suprachiasmatic nucleus (Gorski et al., 1980), a permanent sex difference was found in the vasopressinergic innervation of the lateral septum and lateral habenula from postnatal day 10 onwards (De Vries et al., 1981). This difference - the male rats having a much denser innervation than the female rats - can be manipulated by sex steroids in the neonatal period (Best and De Vries, 1982). The various trophic effects of neuropeptides on the developing brain (cf. Swaab and Martin, 198 I ) , the accelerated regeneration when ACTH 1-39 was given following crush denervation of peripheral nerves (Strand and Kung, 1980) and the positive effect of naloxone following

110

spinal injury (Faden et al., 1981), bear promise for neuropeptides as possible tools in the restoration of developmental disturbances of the nervous system. On the other hand, detrimental effects of neuropeptides on the developing brain are also known. ACTH, when given to children with petit ma1 epilepsy, caused reversible enlargement of the cerebral ventricles of the subarachnoidal space, apathy, drowsiness and pseudodementia (Lagenstein et al., 1979), while deleterious actions of opiates, methadone and naloxone on fetal brain development (Slotkinetal., 1979; Groveet al., 1979; Dingesetal., 1980;Kaltenbachetal., 1979; Strauss et al., 1979; Chasnoff et al., 1980; Hetta and Terenius, 1980) might also be explained by an action via this peptide family. Since, moreover, neonatal administration of P-endorphin or naloxone to the rat causes a permanent insensibility to temperature stimuli (Sandman et al., 1979), the proposed clinical use ofp-endorphin as an analgesic at the time of delivery (Oyama et al., 1980) or naloxone to improve fetal heart rate (Goodlin, 1981) should be discouraged forcefully. For the reasons mentioned above one should, in general, be very reserved with the use of peptides during pregnancy and in the developing child. It will take some time to get gynecologists used to the idea that even oxytocin can considered to be a psychopharmacon (cf. Buijs and Swaab, 1979), and that even this compound can at present not be excluded to be detrimental for the developing brain (Cerutti et al., 1979 ;Friedman et a]. , 1979), possibly via a direct action of oxytocin on the child’s brain. Apart from warning for the unnecessary use of medicines, research in the field of clinical behavioral teratology should be stimulated in order to study the possible effects on the child of the enormous amounts of medicines used during pregnancy and labor. PEPTIDES, AGING AND DEMENTIA In the last years, much effort has been made by an increasing number of groups into the description of changes in peptidergic systems in aging and dementia. Again in common with the classical neurotransmitters, the peptidergic systems show changes that suggest degenerative alterations in both conditions. In the rat cellular changes and alterations in the amount of peptides were reported for the supraoptic nucleus (Davies and Fotheringham, 1980), vasopressin (Turkington and Everitt, 1976), oxytocin (Watkins and Choy, 1980), LH-RH (Barnea et al., 1980; Merchenthaler et al., 1980; Hoffman and Sladek, 1980), somatostatin (Hoffman and Sladek, 1980), P-endorphin, ACTH (Gambert et al., 1980) and a-MSH (Barnea et al., 1979). In man cerebrospinal fluid oxytocin was found to be diminished in dementia (Unger et al., 1971) and vasopressin to be enhanced (Tsuji et al., 1981), neurophysin blood vessels decreased after the 50s (Legros, 1979), a diminution of somatostatin was found in the cerebral cortex of Alzheimer patients (Davies et al., 1980), and the substance P concentration is decreased in the substantia nigra of Huntington patients (Kanazawa et al., 1977). Recently we observed impressive changes in the vasopressinergic cells of the human hypothalamus during aging (E. Fliers et al., unpublished observations). All these changes do, however, as such, not enlarge our insight into the muse of degenerative changes during aging or dementia, nor do they provide at present a theoretical framework to justify as such the administration of peptides to aging or demented patients. Nevertheless, some positive effects have already been reported of peptide treatments. Lysine-vasopressin (LVP) administered by nasal spray improved attention, concentration and motoric abilities in normal controls of 50-65 years old (Legros et al., 1978), while scores in some memory tests would correlate with the neurophysin levels in blood (Legros, 1979). However, Legros could not obtain positive results in a second experiment in an older age group (personal communication). Delwaide et al. reported an effect in

Ill

senile dementia, but other investigators could not find an effect in Alzheimer patients (for references see Jolles and Verhoeven, 198 1). LVP would improve memory in amnesic patients (Oliveros et al., 1978; Le Bceuf et al., 1979; Timsit-Berthier et al., 1980),but this treatment might be effective only in the lighter cases (Jolles and Verhoeven, 198 1 ) . ACTH4-joand a 4 9 analogue improve attention in healthy testees and patients with congenital disturbances. In aging these peptides diminished fear and depression (for references see Jolles and Verhoeven, 198 1). Evaluation of the often preliminary and sometimes conflicting results on the effects of peptides in aging and dementia makes clear that, on the one hand the behavioral tests used can be improved considerably (cf. Jolles and Verhoeven, 1981); on the other hand one could, however, question whether substitution of a transmitter or even many transmitters could ever fully restore higher brain functions in degenerative illnesses. Since, if it would be possible to replace a neuron totally by its product, the neurotransmitter, for what purpose d o we then have neurons which are thought to integrate information from the rest of the brain, the body and the outside world and to react accordingly to, for example, adaptive principles ? ABBREVIATIONS USED IN TABLES 11-1V a A ACTH AH AP ar AVP AVT BN CA IICA2 CCK Ce CP DBB Dr

gP hd Hi hPv HT hv iP LC Ih LH-RH LiC LPH

m MB MBH mh MO MPA MSH NA NC

nucleus accumbens septi aniygdala adrenocorticotrophic hormone anterior hypothalamic area area postrema nucleus arcuatus arginine vasopressin (= ADH) arginine vasotocin bed nucleus of the stria terminalis carnu amonis (hippocampus) cholecystokinin cerebellum nucleus caudatus putamen diagonal band of Broca dorsal root spinal ganglion globus pallidus dorsomedial nucleus of the hypothalamus hippocampus nucleus periventricularis (hypothalami) hypothalamus nucleus ventromedialis (hypothalami) interpeduncular nucleus locus coemleus lateral habenular nucleus luteinizing hormone-releasing hormone limbic cortex (cingulate gyrus, (pre)pyriform, pen-amygdaloid, entorhinal) lipotrophic hormone nucleus mammillaris mammillary body mediobasal hypothalamus nucleus medialis habenulae medulla oblongata medial preoptic area melanophore stimulating hormone nucleus ambiguus neocortex

NTS NX OB OT OTr OVLT OXT P PBD PC Pf PF PN PNT prem PVG PVNm PVNp RD RLM S SC SCN SFO

SG sl sm

SN SOM SON SPO st Str T

nucleus of the solitary tract dorsal motor nucleus of the vagus olfactory bulb olfactory tubercle olfactory tract organum vasculosum of the lamina terminalis oxytocin pineal dorsal parabrachial nucleus peri-commissural area parafascicular nucleus perifornical area preoptic nucleus posterior nucleus of the hypothalamus premammillary body periventricularicentral grey nucleus paraventricularis, pars magnocellularis nucleus paraventricularis, pars parvocellularis dorsal raphe nucleus lateral magnocellular reticular nucleus septum superior colliculus suprachiasmatic nucleus subfornical organ substantia gelatinosa (spinal cord) nucleus septi lateralis nucleus septi medialis substantia nigra somatostatin supraoptic nucleus sulcus paraolfactorius nucleus interstitialis striae terminalis striatum thalamus

112 TRH

TS TT VHi

thyroid stirnulaling hormone-releasing hormane triangular nucleus of the scplum taenia tectae ventral hippocampus

VIP VNLG VT Zi

vasoactive intestinal peptide ventral nucleus of the lateral geniculate body ventral tegmentum zona incerta

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.

DISCUSSION J.J. DREIFUSS : In homozygous Brattleboro rat. are there any major differences in extrahypothalamic projections of oxytocincrgic neurons and/or of the abnormal vasopressinergic cell ?

D.F. SWAAB: The oxytocinergic innervation of the homorygous Brattleboro is generally the same as in the normal Wistar rat brain, except that an occasional oxytocin-containing fiber is seen in the area postrema of homozygous Bruttleboros (Buijs. 1978). Concerning the fiber tracts of the abnormal cells we do not have any information at prescnt. We are collecting homozygoua Brattleboro pituitarics in order to produce antibodies to the compound(s) that are made by these cells but are different from biologically or imniunologically assayable vasopresain. Anybody who has Brattleboro material may send i t in to Amsterdam !

122 A . CUELLO: You showed a picture of a neuron stained for vasopressin where the pan close to the external membrane appears devoid of peptide and you said that there is an accumulation of lipofuscin. Since lipofuscin content in neurons increases with age, did you observe a decrease of peptide content in relationship to age?

D.F. SWAAB: Although oxytocin and vasopressin cells are intermingled, e.g. i n the paraventricular nucleus, only the vasopressinergic cells show a clearcut loss of immunocytochemically stainable vasopressin in the cell body and neurites in very old individuals. The oxytocinergic cells also accumulate lipofuscin but the inununoreactivity for oxytocin remains rather unchanged. So it is not simply a matter of non-specific cellular changes one meets in eachcell. REFERENCE Buijs, R.M. (1978) Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Pathways to the limbic system, medulla oblongata and spinal cord. Cell Tiss. Res., 192: 423-435.