Neuron,

Vol. 7, 867-879, December,

1991, Copyright

0 1991 by Cell Press

Neuropeptides in Perspective: The Last Ten Years Tomas HGkfelt Department of Histology Karolinska Institute S-10401 Stockholm Sweden

and Neurobiology

Interest in the role of peptides in the nervous system was sparked 20 years ago when Guillemin, Schally, and their colleagues first identified the peptidergic nature of the hypothalamic releasing and inhibiting hormones (luteinizing hormone releasing hormone, thyrotropin releasing hormone, and somatostatin) (Guillemin, 1978; Schally et al., 1973). Their discovery was soon followed by the sequencing of substance P (Chang et al., 1971) and the delineation of substance Pas a possible pain transmitter. The subsequent isolation of two endogenous opioid peptides that are ligands for the morphine receptor (Hughes et al., 1975), methionineand leucine-enkephalin, suggested that peptides represent a new class of chemical transmitters and that study of this class might lead to new approaches in the treatment of pain. In addition to pain, peptides were also found to influence other important higher functions, including cognition. For example, vasopressin affected learning and memory (De Wied, 1971). Moreover, the peptides revealed new insights into the biochemical diversity of neurons. In particular, the finding of the coexistence in the same neuron of a peptide (somatostatin) and a classic small molecule transmitter (noradrenaline) (HGkfelt et al., 1977) raised important new questions about synaptic transmission: Can neurons utilize more than one transmitter? If so, do peptides exert functions in the nervous system other than fast signaling (HGkfelt et al., 1980a)? These and other questions held out the promise that peptides would provide new insights into brain function. Has this promise been sustained? How has the neurobiologyof peptides progressed in the1980s? I address this problem by focusing on the mammalian systems.

Peptides in the 1980s In the 1970s we first appreciated that peptides have an uneven distribution in the central nervous system (CNS). They are particularly abundant in certain areas such as the dorsal horn of the spinal cord, the lower brain stem, and the hypothalamus. In the hypothalamus, the parvocellular neurosecretory systems express the hypothalamic releasing and inhibitory hormones (thyrotropin releasing hormone, luteinizing hormone releasing hormone, somatostatin, corticotropin releasing hormone, and growth hormone releasing hormone) and several other peptides (Ceccatelli et al., 1989; Swanson et al., 1986). The magno-

Review

cellular systems produce oxytocin, vasopressin, and a host of other peptides (Brownstein and Mezey, 1986; Meister et al., 1990). The autonomic nervous system has peptides in all of its subdivisions (Schultzberg and Lindh, 1988). Virtually every gastrointestinal neuron has now been identified with regard to peptide and transmitter, and their projections have been determined by Costa, Furness, and their collaborators (Costa et al., 1986) (Figure 1). The chemical neuroanatomyof peptides has up until recently been based on immunohistochemistry, a technique originally devised by Albert Coons and collaborators in the’40s (Coons, 1958). Although the specificityof immunohistochemistry has been questioned frequently (Swaab et al., 1977), the patterns described over the years appear to be surprisingly correct and consistentwith the recent studies using in situ hybridization. In situ hybridization has also raised an important question concerning the localization of protein synthesis by demonstrating that oxytocin mRNA is present in nerve terminals in the posterior pituitary (Jirikowski et al., 1990). Is it possible that peptides are synthesized in nerve endings? Early estimations of the number of “transmitter peptides” present in the brain ranged in the hundreds (Snyder, 1980), but in the 198Os, after the elucidation of the precursorsof theopioid peptides, thetachykinins, and other established peptides, the rate of discovery of new peptides has slowed. In the1970s new peptides were found mostly by extracting large amounts of intestine, brain, or pituitary tissue and then monitoring the purification steps by a bioassay. In the 1980s moIecularcloningwas intr0duced.A new peptidediscovered in this way was calcitonin gene-related peptide (CGRP). Rosenfeld, Amara, Evans, and their colleagues (Rosenfeld et al., 1983) observed that the gene for the hormone calcitonin contained a structurally related sequence. They then deduced the amino acid sequence, synthesized a peptide fragment, and raised antibodies, which were then used for radioimmunoassay and immunohistochemistry. Bythis means, they demonstrated that there was a tissue-specific processing of the precursor. Calcitonin was expressed mainly in the thyroid gland, whereas CGRP had a widespread distribution in the nervous system (Rosenfeld, et al., 1983). CGRP was the first peptide to be demonstrated in motoneurons, and it was also found in a large population of primary sensory neurons and in other systems (Gibson et al., 1984; Rosenfeld et al., 1983). Functionally, CGRP turned out to be a more powerful vasodilator than substance P (Brain et al., 1985), with which it coexists in sensory neurons (see below). In the early 198Os, many additional peptides were discovered by a new strategy introduced by Mutt, Tatemoto, and their collaborators and based on the search for peptides with C-terminal amidation, i.e.,

NWKW 868

Figure 1. PeptIdes. Neurotransmitter-Synthesizing Enzymes, and Neurotransmitter5 in Intrinsic Neurons and Extrinsic Nerve5 ot the Gastrointestinal Tract

,I

DRG ,

’ I I/

PREVERT

Oral is on the left; anal on the right. Taken from Costa et al. (1986). Abbreviations are as follows: acetylcho line, ACh; calcitonln gene-related peptide. CGRP; chloramphenicol acetyltransferase, ChAT; cholecystokinin, CCK; circular mu+ cle. CM; dorsal root ganglion, DRG; dynorphin, DYN; enkephalin, ENK; ?, CRP; long]tudinal muscle, LM; mucosa, M; myenteric plexus, MP; neuropeptide tyrosine, NPY. noradrenaline, NA; prevertebral ganglion, PREVERT. G; somatostatin, SOM; splanchnit nerve, SPLANC. N, submucosa, SM: substance P, SP; vasoactive intestinal peptide, VIP;.

G

LM MP

CM

SM

M

identification on purely chemical criteria (Tatemoto and Mutt, 1988). Peptides found in this way include neuropeptide tyrosine and peptide tyrosine tyrosine, which belong to the family of pancreatic polypeptides; peptide histidine isoleucine, which has been shown by cloning (Itoh et al., 1983) to be part of the same precursor as vasoactive intestinal polypeptide (VIP) (Said and Mutt, 1970); and the 29 amino acid peptide galanin (Tatemoto and Mutt 1988). It has recently been demonstrated that human galanin comes in two forms, 19 and 30 amino acid peptides without C-terminal amidation (Bersani et al., 1991). Two new peptide families were discovered in peripheral tissues, the blood pressure-lowering, atrionatriuretic factor in the heart (de Bold et al., 1981) and the extremely potent vasoconstrictor endothelin in blood vessels (Yanagisawa et al., ,1988). Both of these seem to have corresponding peptides in the brain (Giaid et al., 1989; Shinmi et al., 1989; Sudoh et al., 1988).

After a latency of many decades, nerve growth factor (NGF), the first discovered growth factor (LeviMontalcini, 1987), has now been shown to be a member of a polypeptide family encompassing brain-derived neurotrophic factor and neurotrophin-3 (Barde, 1989; Thoenen, 1991). Neurotrophic effects of a number of other factors, such as ciliary neurotrophic factor and acidic and basic fibroblast growth factors, have also been demonstrated (Thoenen, 1991). It is likely that close interactions occur between growth factors and neuropeptides. In fact, it has been shown that NGF stimulates substance P in dorsal root ganglia (Kessler and Black, 1980) and that NGF counteracts the downregulation of substance P after axotomy (Fitzgerald et al., 1985; see below). At last:

Receptors

for Neuropeptides

That receptors for peptides exist in the nervous system has not been self-evident, in spite of the autora-

Review: The Expanding 869

Roles of Neuropeptides

diographic (Young and Kuhar, 1979) demonstration of distinct binding patterns in the brain for many peptides, e.g., opioid peptides (Goodman et al., 1980), and of multiple types of binding sites for individual peptides (Lee et al., 1982; Lord et al., 1977). However, their receptor proteins were elusive. Furthermore, in view of the coexistence between peptides and classic transmitters, peptide-bindingsitescouldtheoretically very well be located on the receptor molecules of classic transmitters. For example, the endogenous ligand for benzodiazepine-binding sites, located on y-aminobutyric acid (GABA) receptors, is a peptide (Costa et al., 1983). It was therefore important that the first peptide receptors, the receptors for substance P and substance K (Hershey and Krause, 1990; Masu et al., 1987; Yokota et al., 1989) and for neurotensin (Tanakaet al., 1990),werecloned and shown to belong to the family of G protein-coupled receptors with seven membrane-spanning segments. This made possible the study of their localization (Elde et al., 1990) and regulation. Additional studies are required to determine whether the binding of neuropeptides to their receptors involves the same transmembrane regions that have been implicated in the binding of biogenie amines to their seven transmembrane receptor. Similarly, will there turn out to be specific kinases that regulate the activity of peptide receptors in the way that BARK and rhodopsin kinases regulate adrenergic and rhodopsin receptor function? Coexistence Classic small molecule transmitters, including acetylcholine, catecholamines, serotonin, and the fast amino acid inhibitory transmitter GABA, have been found tocoexist in numerous placeswith oneor more peptides (Chan-Palay and Palay, 1984; Cuello, 1982; Hokfelt et al., 1986). The two types of messenger molecules have different subcellular storage sites (Fried et al., 1985; Lundberg et al., 1981; Matteoli et al., 1988) (Figure 2). The small, clear synaptic vesicles that are -500 A in diameter contain exclusively the classic transmitter. The larger vesicles (large, dense-core vesicles) contain peptide plus classic transmitter. Consequently, peptides can be released only when large, dense-core vesicles are “activated.“There is early evidence from peripheral systems and more recently from central and invertebrate systems that individual action potentials firing at low frequency will not release peptides. Release requires bursting or high frequency activity (Bean and Roth, 1991; Edwards et al., 1982; lverfeldt et al., 1989; Lundberget al., 1982; Whim and Lloyd, 1989) (Figure 2). The principle of frequency-dependent release is important. As a consequence, it can be predicted that at normal firing rates, peptides might not be released at all and that peptide antagonists should not have any effects under these circumstances. In general terms, the response to the classic transmitter is rapid and short lasting, whereas the peptide induces a response of longer duration

RESPONSE/ RELEASE

T+P

/ FIRING RATE

Figure 2. Frequency Dependence Transmitter and Peptide

of Response to and Release of

(Top) At low firing rates, only transmitter 0 is released, whereas at higher firing rates or bursting activity, peptide (P) is also secreted. (Bottom)A nerveending. The small synapticvesiclescontain only classic transmitter (dots), and large, dense-corevesicles contain both classic transmitter and peptide (triangles). Small vesicles release their content into the synaptic cleft, whereas large vesicles may often release their content extrajunctionally. Modified from Lundberg and Hokfelt (1983).

(Figure 2). There is some evidence that peptides are not released at active zones into the synaptic cleft, but are released extrajunctionally (Gelding and Bayraktaroglu, 1984; Thureson-Klein and Klein, 1990), and may diffuse over longer or shorter distances. In fact, it has been postulated that peptides can diffuse over very long distances to other brain regions, acting in the manner of a local hormone (Duggan et al., 1990; Fuxe and Agnati, 1991). Recent evidence suggests that in addition to containing biogenic amineand peptide, neurons may also contain a third transmitter, a fast excitatory amino acid transmitter such as glutamate. Thus, glutamate-like immunoreactivity (Nicholas et al., 1990) and the glutamate-synthesizing enzyme phosphate-activated glutaminase(Kanekoetal., 1990)are present in medullary catecholaminergic and serotonergic neurons. If this is true, these neurons may be able to release three classes of transmitters conveying fast, moderate, and slow signaling (Iversen and Goodman, 1986). In peripheral sympathetic neurons in the vas deferens, a similar situation may exist whereby ATP represents the fast transmitter, which is coreleased with nor-

NelJKNl 870

Figure 3. Film Autoradiographs of Adjacent tia Nigra of Schizophrenic Brain

Sections

of Substan-

Note the overlapping distribution of ceils positive for CCK (A) and tyrosine hydroxylase (B) mRNA. The bar indicates 1 mm. Taken from Schalling et al. (1990).

adrenaline

and

neuropeptide

tyrosine

(Kasakov

et al.,

and Westfall, 1984). Does coexistence violate Dale’s principle (Eccles, 1986), sometimes referred to as the”one neuron-one transmitter” hypothesis? Eccles based this principle on Dale’s proposal of 1932 that “anyclass of nerve cells operates at all of its synapses bythe same transmission mechanism.” Clearly, this principle was formulated before the discovery of coexistence and thus does not take the number of transmitters released into account. As a result, the principle can be formulated to indicate that a neuron releases the same combination of transmitters at all of its terminals. However, even this reformulation may not be generally applicable, as elegantly shown by Scheller and his colleagues (Sossin et al., 1990). In Aplysia two peptides produced in the same neuron may be routed into different terminal domains (Sossin et al., 1990), thus apparently violating Dale’s principle. The prohormone of egg-laying 1988;

Sneddon

hormone IS cleaved into two sets of peptides, the N-terminally derived bag cell peptides, with a local, autocrine function, and the C-terminally originating egg-laying hormone, with a more distant, hormonal action.Thesetwomessengersaresorted intodifferent vesicles which in turn are transported into separate processes, segregating autocrine and hormonal release sites (Sossin et al., 1990). Coexistence may be clinically important. Thus, cholecystokinin (CCK)-like immunoreactivity was observed early in rat mesencephalic dopaminergic neurons (H6kfelt et al., 1980b) and in other species, but not in normal human brain (Palacios et al., 1989; Schalling et al., 1990). However, in neuroleptic-treated schizophrenic brains, a limited CCK signal could be observed in dopamine neurons (Schalling et al., 1990) (Figure 3). Whether this apparent up-regulation of CCK message is related to neuroleptic treatment or the disease itself remains to be shown. A second caseof clinical interest is galanin in cholinergic forebrain neurons of several species (ChanPalay, 1988a, 1988b; Kordower and Mufson, 1990; Melander and Staines, 1986; Melander et al., 1985; Walker et al., 1989). Since these cholinergic forebrain neurons are known to be degenerated in Alzheimer’s disease (Whitehouse et al., 1982), a possible relation to this disease may be explored. In fact, galanin mRNA levels in rat have been shown to be markedly up-regulated in cholinergic forebrain and other neurons after various types of lesions (Cartes et al., 1990). Since galanin inhibits glutamate release induced by anoxia in the hippocampus (Ben-Ari, 1990), one of the projection areas of the galanin-containing cholinergic ventral forebrain neurons (Melander et al., 1985), galanin could serve to counteract the neurotoxic effects of excessive glutamate release. However, galanin also inhibits release of acetylcholine in the ventral hippocampus (Fisone etal., 1987), and this additional effect mayattenuate acquisition and learning (Mastropaolo et al., 1988; Sundstrdm et al., 1988). Since Alzheimer’s disease may involve a progressive degeneration of galaninlcholinergic forebrain neurons and a compensatory increase in neuronal activity in the nonlesioned neurons, there may be an overall increase in galanin synthesis and release in hippocampus, which could worsen the learning and memory deficits (H6kfelt et al., 1987a). Interestingly, Chan-Palay (1988a, 1988b) has observed a galanin hyperinnervation of cholinergic forebrain neurons in Alzheimer brains and proposed that an inhibitory effect of galanin may be responsible for symptoms in thisdisease. Clearly,agalanin antagonist could be beneficial, if either one or both of the above mentioned hypotheses is correct. Cooperativity

of Coexisting

Messengers

A key to the function of peptides in signaling (Iversen and Goodman, 1986) is to understand the importance of their coexistence with classic small molecule transmitters. Although in most systems the classic transmit-

Review: The Expanding 871

Roles of Neuropeptides

Figure4. Afferent stance P, sal Horn

The Synaptic Cleft of a Primary Nerve Ending Costoring SubCGRP, and Glutamate in the Dorof the Spinal Cord

A cascadeof events may enhance transmission at these synapses. These include stimulation of postsynaptic receptors (RI-R3) for the individual messenger molecules as well as presynaptic events leading to enhanced release (2-4), inhibition of substance P endopeptidase by CGRP (I), and postsynaptic sensitization of glutamate receptors by substance P (5). See text for details.

ter seems to be the important messenger, in some neurons, e.g., in the hypothalamic neurosecretory releasing hormone systems, peptides are the primary messengers. However, some corticotropin releasing hormone neurons also contain GABA (Meister et al., 1988), butcorticotropin releasing hormone iscertainly the main releaser of adenocorticotropic hormone. In fact, in some cases a peptide can directly activate a ligand-gated ion channel, as has recently been shown in Helix aspersa (Cottrell et al., 1990). In the lamprey fish, somatostatin and GABA are colocalized in nerve endings that impinge on spinal stretch receptor neurons. Both compounds hyperpolarize these neurons, somatostatin acting on a potassium current and GABA on a chloride channel (Christenson et al., 1991). There are many examples of how coexisting transmitters and peptides may interact, demonstrating both synergistic and antagonistic effects, but with the peptide as an auxiliary messenger. This has been shown especially by Lundberg and coworkers (1982), who analyzed the interactions of acetylcholine and VIP, which are released in a frequency-dependent fashion in the cat salivary gland, whereby acetylcholine induces secretion. VIP is responsible for the atropine-resistant vasodilation and thereby facilitates secretion, but VIP may also act on the secretory elements by enhancing acetylcholine binding (Lundberg et al., 1982). In some noradrenergic peripheral neurons, the coexisting peptide neuropeptide tyrosine acts as a functional endogenous antagonist by inhibiting noradrenaline release (Allen et al., 1982; Lundberg et al., 1982). One of the most impressive behavioral examples of cooperativity of coexisting messengers is the potentiation by CGRP of scratching and biting induced by substance P (Wiesenfeld-Hallin et al., 1984). Whereas the scratching effect induced by substance P alone lasts for a couple of minutes, when given together with CGRP, scratchingwill go on for more than 30 min

(Wiesenfeld-Hallin et al., 1984). It has also been shown that CGRP and substance P synergistically modulate the nociceptive flexor reflex (Woolf and WiesenfeldHallin, 1986). These effects may be achieved by a cascade of interactions (Figure 4) related to a population of primary sensory neurons in which the two excitatory peptides substance P (Henry et al., 1975; Pernow, 1983; Takahashi et al., 1974) and CGRP (Miletic and Tan, 1988; Ryu et al., 1988; Woolf and WiesenfeldHallin, 1986) and presumably the excitatory amino acid glutamate (Salt and Hill, 1983; Schneider and Perl, 1985) may coexist (De Biasi and Rustioni, 1988). Thus, CGRP inhibits an endopeptidase that cleaves substance P (Le Greves et al., 1985). Furthermore, CGRP potentiates release of substance P in the dorsal horn in vitro (Oku et al., 1987) and enhances release of glutamate and aspartate from the dorsal horn in vitro (Kangrga and Randic, 1990). This may be related to findings that CGRP can increase calcium levels in dorsal horn nerveendings, resulting in enhanced releaseof transmitterslpeptides (Oku et al., 1988). Substance P in turn increases release of glutamate and aspartate in the dorsal horn, as seen with in vivo dialysis (Smullin et al., 1990), and may enhance the strength of glutamatemediated excitatory transmission in the dorsal horn (Randic et al., 1990). Interestingly, both substance P and excitatory amino acids are involved in generating a slow nociceptive ventral root potential, and both types of receptors have to be activated to generate a full response (Woodley and Kendig, 1991). Neuropeptide Compounds

Antagonists:

New

Nonpeptide

Much of the knowledge of the functional role of classic transmitters is based on pharmacological experiments, especially with specific high affinity antagonists. Such drugs have not been available for peptides, an exception being opioid peptidelopiate antagonists

such as naloxone. In fact, one reason for knowing so little about the physiological significance of peptides has been the lack of drugs that can influence peptideinduced events at synapses. Attempts to develop drugs with agonistic and antagonisticactionsfor peptides fall mainly intotwocategories, nonpeptides and peptides. The latter have some drawbacks, such as high biodegradability (cannot be given orally), high molecular weight (expensive synthesis), and failure to pass the blood brain barrier. One example is the work by Folkers, Rosell, and collaborators, who produced substance P antagonists by substituting with o-amino acids at various positions (Folkers et al., 1982). These antagonists have been widely used experimentally, and antagonists of a peptide nature are now available for several neuropeptides, including recently developed galanin antagonists, which are chimeric peptides (Bartfai et al., 1991). Perhaps the most exciting development has, however, been a number of nonpeptide antagonists. Particularly successful efforts are related to CCK (Wang and Schoenfeld, 1987). Three new drugs have ignited the field. One, a new naturally occurring benzodiazepine, asperlicine, was found by random screening to have CCK antagonistic activity. A new competitive nonpeptide antagonist, L-364,718 (Devazepide, MK-329) that has a high potency and selectivity for the peripheral type of CCK (CCK-A) receptor has been developed (Evans et al., 1986). A sister compound selective for the CCK-B receptor has been produced (Bock et al., 1989). The B type receptor is present mainly in the CNS but is indistinguishable from the gastrin receptor in the periphery, and the CCK-A receptor is mainly peripheral, but occurs also in discrete areas in the brain (Woodruff et al., 1991). Second, using a novel strategy starting with the CCK octapeptide molecule and reducing it to a minimal size and then modifying its structure, Hughes and coworkers (1990) have succeeded in developing a potent and selective antagonist (Cl-988; PD 134308) at the CCK-B receptor with dissociation constants in the nanomolar range. Finally, a nonpeptide compound that is a selective antagonist for the NKI receptor has recently been found (Snider et al., 1991). All of these antagonists are stable and pass the blood brain barrier. Blockade of CCK and of tachykinin receptors with these antagonists leads to behavioral effects, suggesting a role for endogenously released peptides. Thus, the CCK-B receptor antagonist L-365,260 postpones satiety (Dourish et al., 1989). Cl-988 is a potent anxiolytic drug in various animal tests (Hughes et al., 1990), which fits with the observations that CCK-4, which is a small molecule and therefore may partially pass the blood brain barrier, elicits panic attacks both in patients with panic disorders and in a majority of healthy volunteers (Bradwejn et al., 1990; de Montigny, 1989). Moreover, no withdrawal effects were observed (Hughes et al., 1990), in contrast to benzodiazepines, the drugs most often used for treatment of anxiety. In fact, Cl-988 can counteract withdrawal

effects induced by a variety of drugs (Hughes et al., 1990). Both L-365,260and Cl-988enhance the analgesic effect of morphine and prevent morphine tolerance (Dourish et al., 1990; Wiesenfeld-Hallin et al., 1990; Xu et al., 1991). The NKI antagonist CP-96,345 produces thermal analgesia (Lecci et al. 1991). Antagonists acting on neuropeptide receptors may therefore emerge as important drugs, since they will act preferentially on (pathologically) up-regulated systems (see below). Peptides and Neuronal Plasticityon Primary Sensory Neurons

Focus

Neurons can vary in the peptides they express. An identified neuron in the earthworm expresses enkephalin-like immunoreactivity during the spring and gastrin-like immunoreactivity during the fall (Gesser and Larsson, 1985). In some central neurons, peptides are regulated by steroid hormones and thus may vary with the estrous cycle (Micevych et al., 1988; Simerly and Swanson, 1987). Thus, some neurons in the amygdaloid complex express two peptides, CCK and substance P, only one of which is influenced by prenatal steroid hormone levels, offering interesting possibilities for variations in the message (substance P versus CCK) that these neurons release. To illustrate this point, I focus on primary sensory neurons that exhibit pathway-specific patterns of peptide coexistence (Gibbins et al., 1987) and in which it has been shown that the type of peptide expressed is dependent on factors in the peripheral tissues/nerve sheaths (McMahon and Gibson, 1987). These sensory neurons show dramatic changes in expression of their peptides after injury. In untreated rats, substance P, somatostatin, and CCRP can be easily visualized, whereas VIP/peptide histidine isoleucine and galanin can be seen only in a small number of neurons (Dalsgaard, 1988). After peripheral axotomy, that is, a lesion that occurs when a smaller or bigger injury is afflicted to the body, there is a marked down-regulation of substance P and CGRP (Jesse11 et al., 1979; Noguchi et al., 1990). In contrast, the VIP levels are dramatically up-regulated in primary sensory neurons (Shehab and Atkinson, 1986). Since VIP induces glycogenolysis (Magistretti et al., 1981), increases CAMP (Magistretti and Schorderet, 1984), causes vasodilation (Said and Mutt, 1970), and enhances survival of neurons in culture (Brenneman and Eiden, 1986), it seems possible that it serves to promote survival and regeneration (Shehab and Atkinson, 1986). Galanin and its mRNAarealsostronglyup-regulated after axotomy (Hokfelt et al., 1987b; Villar et al., 1989a). However, there is so far no evidence that galanin exerts atrophic action or promotes survival. But studies on the nociceptive flexor reflex (Wall and Woolf, 1984) suggest that galanin suppresses pain by acting as an endogenous antagonist to substance P and CGRP (Xu et al., 1989,1990), both of which have been implicated as pain messengers in the dorsal horn of the spinal cord (Henry et al., 1975; Woolf and Wiesenfeld-Hallin,

Review: The Expanding Roles of Neuropeptides 873

1986). Furthermore, galanin enhances the effect of morphine at the spinal cord level (Wiesenfeld-Hallin et al., 1990). A galanin antagonist blocks the effect of not only exogenous galanin (Bartfai et al., IVVI), but also endogenously released galanin (WiesenfeldHallin et al., submitted). More importantly, this effect is dramatically increased after axotomy (WiesenfeldHallin et al., submitted). Thus, endogenous galanin seems to exert a tonic inhibition on spinal cord excitability, especially after injury. This effect may be a response of the body to the hyperexcitability of injured afferents (Wall and Gutnick, 1974) that may underlie neuropathic pain. Consequently, insufficient galanin-induced inhibition in the dorsal horn may contribute to chronic pain states, and compounds with galanin agonistic activity may be potential analgesic drugs. Under normal circumstances, galanin may be only of minor functional significance, and rapid suppression of pain may be mediated via opioid peptides released from dorsal horn neurons. Only after some time, thedramatic up-regulation of galanin after peripheral axotomy (or nerve crush) may result in increased peptide levels in the dorsal horn and an analgesic effect, preventing pain messages from reaching second order neurons in the spinal cord.

Neuropeptides

Have Trophic

Effects

Increasing evidence supports a role for peptides in trophic processes. For example, there is evidence for involvement of opioid peptides during CNS development and neuronal growth (Vertes et al., 1982; Zagon and McLaughlin, 1983). Moreover, in the periphery tachykinins stimulate the growth of neurites (Narumi and Maki, 1978) and fibroblasts and smooth muscle cells (Nilsson et al., 1985) and the synthesis of interleukins and tumor necrosis factor-a in monocytes (Lotz et al., 1988). VIP influences bone mineralization (Hohmann et al., 1986), stimulates the growth of human keratocytes (Haegerstrand et al., 1989), and promotes neuronal survival in cell culture (Brenneman and Eiden, 1986; Pincus et al., 1990). These effects may occur in vivo after release of these peptides from the nerve endings of sensory and/or autonomic neurons (Hill et al., 1991). Interestingly, central neurotoxic effects induced by 6-hydroxydopamine or b amyloid protein can be counteracted by substance P (Jonsson and Hallman, 1982; Kowall et al., 1991). Another example is CGRP, which is present in motoneurons (Gibson et al., 1984; Rosenfeld et al., 1983) and has binding sites on striated muscle cells (Jennings and Mudge, 1989; Popper and Micevych, 1989; Roa and Changeux, 1991). In addition to direct influences on neuromuscular transmission, there are effects of a more trophic nature. Thus, CGRP increases the synthesis of acetylcholine receptors in cultured chick muscle cells (Fontaine et al., 1986; New and Mudge, 1986). This presumably occurs via stimulation of adenylate cyclase and CAMP production (Laufer

and Changeux, 1987; Takami et al., 1986). In the chick this may be an important function during embryogenesis, since CGRP expression reaches a peak between embryonic days 11 and IV (Villar et al., 198Vb), a period when motor endplates become established. In fact, it has been proposed that CGRP may represent an “anterograde” neural factor released from nerve endings to regulate acetylcholine receptor gene expression via the subjunctional areas of the motor endplate (Fontaine et al., 1987). Finally, CGRP prevents diseaseinduced sprouting of motor nerve terminals (Tsujimoto and Kuno, 1988). A marked plasticity of CGRP has been found in adult motoneurons. Peripheral axotomy induces increased levels of CGRP peptide as well as mRNA (Arvidsson et al., 1990; Haas et al., 1990; Marlier et al., 1990; Moore, 1989; Noguchi et al., 1990; Piehl et al., 1991; Streit et al., 1989). Since it is assumed that proteins necessary for restitution of the axon and for survival are increased after neuronal damage (Barron, 1983; Lieberman, 1971), this could be taken as an indication of a trophic role of CGRP. It is also in contrast to the effect of axotomy on CGRP in sensory neurons, where, as discussed above, a marked down-regulation occurs. The exact putative trophic effect of CGRP is at present unclear, but its powerful vasodilatory effect could improve regeneration. Perhaps it is related to the recentlydiscovered stimulatoryeffectof CGRPoncAMP formation in glial cells associated with a transformation of the astrocytes into multipolar cells with many long processes (Lazar et al., 1991). Denis-Donini (1989) has observed that CGRP in nanomolar concentrations can mimic olfactory epithelial neurons in inducing the dopaminergic phenotype in olfactory bulb neurons. Thus, CGRP may beadifferentiation factor for some dopaminergic neurons. As a final example of a trophic/metabolic role of a neuropeptide, I will consider neurotensin and the nigrostriatal system. Neurotensin injected into the rat striatum is retrogradely transported in an unchanged form to the dopaminergic neurons in the substantia nigra (Caste1 et al., IVVO), where the peptide increases tyrosine hydroxylase gene expression (Burgevin et al., submitted). Since neurotensin is present in the striaturn (Kobayashi et al., 1977) and dopaminergic neurons have neurotensin receptors (Palacios and Kuhar, 1981), it is possible that this peptide represents a retrograde signal produced in the striatum, internalized by the striatal dopaminergic nerve endings, and after retrograde transport to the cell bodies, affecting the dopamine-synthesizing enzyme tyrosine hydroxylase. Such retrograde, long-distance signaling via receptor-mediated internalization would be a new function for neuropeptides (Burgevin et al., submitted), similar to mechanisms operating for NCF (Hendry et al., 1974). Perspective Our

knowledge

of the distribution

of peptides,

their

Neuron 074

NEURONAL

ACTIVITY

NORMAL/BURSTING

POSSIBLE I ; t

SITUATIONS

NEUROSECRETION (LHRH etc.) Growth, development Trophic influence PHYSICAL EXERCISE Learning STRESS

ANXIETY PAIN

HIGH/BURSTING

t

Narcotrc addiction

effects

EXCESSIVE

NEURONAL DAMAGE Degeneration Regeneration Figure 5. Situations under Which Peptides May Be Synthesized in Increased Amounts and Released, Ranging from Normal Neuronal Activity to Pathologic States with Neuronal Damage States indicated with capital letters on the right list situations supported by experimental evidence, in some cases including specific antagonists.

coexistence, and their actions has increased steadily during the last decade. Development of novel peptide antagonists has allowed us to analyze the physiological roles of neuropeptides. In addition to their putative role as auxiliary messengers in synaptic and nonsynaptic signaling, increasing evidence suggests that neuropeptides exert trophic actions. The concept of cotransmission has gained further definition by the finding that chemical transmission is fine-tuned with a frequency-dependent release of a fast transmitter and a peptide. Thus, peptides are often not released under basal conditions, but become released only upon activation, at high frequency or bursting firing. In some cases they may play a role when injury or other strong stresses are imposed on the neuron (see Figure 5). In fact, they may be synthesized only under such conditions, exhibiting a high degree of plasticity. Even for peptides with an established physiological role, such as the hypothalamic releasing hormones involved in control of anterior pituitary hormone secretion, it seems likely that their release occurs under situations with bursting activity, since their secretion is known to be episodic. If the’50s were the decade of acetylcholine, the’60s of catecholamines and serotonin, and the ’70s of the inhibitory amino acid transmitters GABA and glycine, some of us anticipated that the’80s would become the decade of the peptides. However, although a wealth of data were collected, a breakthrough in understanding was not achieved. Instead, the excitatory amino acids entered the scene and grasped the attention with their implications for learning and memory and for involvement in brain pathology. The cloning of several neuropeptide receptors and the (hopefully) accelerating development of new peptide antagonists should strongly stimulate the neuropeptide field. Will the ’90s be the decade of the neuropeptides?

Acknowledgments I would like to acknowledge the advice and help In preparing this review by collaborators, whose contributions in part are included in this article. Special thanks go to Dr. Robert Elde and Marcel0 Villar. For expert secretarial help, I thank Ms. Ida Engqvist and Jacqueline Nicholas. This work was supported by theSwedishMedicalResearchCouncil(04X-28871andtheUSPHS (NIMH-43230).

References Allen, J., Tatemoto, K., Polak, J., Hughes, J., and Bloom, S. (1982). Two novel related peptides, neuropeptide Y (NPY) and peptide YY (PYY) inhibit the contraction of the electrically stimulated mouse vas deferens. Neuropeptides 3. 71-77. Arvidsson, U., Johnson, H., Piehl, F., Cullheim, S., Hiikfelt, T Risling, M., Terenius, L., and Ulfhake, B. (1990). Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons Exp. Brain Res. 79, 212-216. Barde, Y.-A. (1989). Trophic ron 2, 1525-1534.

factors

and neuronal

survival.

Neu-

Barron, K. D. (1983). Comparative observations on the cytologic reactions of central and peripheral nerve cells to axotomy. In Spinal Cord Reconstruction, C. C. Kao, R. P. Bunge, and P. I Reier, eds. (New York: Raven Press), pp. 7-40. Bartfai, T., Bedecs, K., Land, T., Langel, U., Bertorelli, R., Girottl. P., Consolo, S., Xu, X., Wiesenfeld-Hallin, Z., Nilsson, S., Pierlbone, V., and Hbkfelt, T. (1991). M-15: high-affinity chimeric peptidethat blocks the neuronal actionsof galanin in the hippocampus, locus coeruleus, and spinal cord. Proc. Natl. Acad. Sci. USA, in press. Bean, A. J., and Roth, R. H. (1991). Extracellular dopamine and neurotensin in rat prefrontal cortex in vivo: effects of medial forebrain bundle stimulation frequency, stimulation pattern, and dopamine autoreceptors. J. Neurosci. 77, 2694-2704. Ben-Ari, Y. (1990). Galanin and glibenclamide modulate the anoxic release of glutamate in rat CA3 hippocampal neurons. Eur. J. Neuroscl. 2, 62-68. Bersani, M., Johnsen, A. H., HBjrup, P., Dunning, B. E., Andreasen, J. J., and Holst, J. J. (1991). Human galanin: primary structure and identification of two molecular forms. FEBS Lett. 283, 189-194. Bock, M. C., DlPardo, R. M., Evans, B. E.. Rittle, K. E., Whitter. W. L., Veber, D. F., Anderson, P. S., and Freidinger, R. M. (1989). Benzodiazepine gastrin and brain cholecystokinin receptor ligands: L-365,260. J. Med. Chem. 32, 13-16. Bradwejn, J., Koszycki, D., and Meterissian, G. (1990). Cholecy\tokinin-tetrapeptide induces panic attacks in patients with panic disorder. Can. J. Psychiatry 35, 83-85. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R., and MacIntyre, I. (1985). Calcitonin gene-related peptide is a potent vasodilator. Nature 373, 54-56. Brenneman, D. E., and Eiden, L. E. (1986). Vasoactlve lntestlnai peptide and electrical activity influence neuronal survival. Proc. Natl. Acad. Sci. USA 83, 1159-1162. Brownstein, M. J.. and Mezey, E. (1986). Multiple chemical messengers in hypothalamic magnocellular neurons. Prog. Brain Res. 68, 161-168. Castel, M. N., Malgouris, C., Blanchard, J. C., and Laduron. P.M. (1990). Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat. Neuroscience 36,425430. Ceccateili, S., Eriksson, M., and Htikfelt, 1. (1989). Distribution and coexistence of corticotropin-releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus. Neuroendocrinology 49. 309~-323.

Review: The Expanding 875

Roles of Neuropeptides

Chart-Palay, V. (1988a). Neurons with galanin innervate cholinergic ceils in the human basal forebrain and galanin and acetylcholine coexist. Brain Res. Bull. 27, 465-472. Chan-Palay, V. (1988b). Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer’s and Parkinson’s disease: a hypothesis for the role of galanin in accentuating cholinergic dysfunction in dementia. J. Comp. Neurol. 273, 543-557. Chart-Palay, V., and Palay, S., eds. (1984). Coexistenceof tive Substances in Neurons (New York: Wiley). Chang, M. M., Leeman, sequence of substance

Neuroac-

S. E., and Niall, H. D. (1971). Amino-acid P. Nature New Biol. 232, 86-87.

Christenson, J., Alford, S., Grillner, S., and Hokfelt, T. (1991). Co-localized GABA and somatostatin use different ionic mechanisms to hyperpolarize target neurons in a vertebrate spinal cord. Neurosci. Lett., in press. Coons, A. (1958). Fluorescent antibody methods. In General Cytochemical Methods, J. Danielli, ed. (New York: Academic Press, Inc.), pp. 399-422. Cortes, R., Villar, M. J., Verhofstad, A., and Hokfelt, T. (1990). Effects of central nervous system lesions on the expression of galanin: a comparative in situ hybridization and immunohistochemical study. Proc. Natl. Acad. Sci. USA 87, 7742-7746. Costa, E., Corda, M., and Guidotti, A. (1983). On a brain polypeptide functioning as a putative effector for the recognition sites of benzodiazepines and beta-carboline derivatives. Neuropharmacology 22, 1481-1492. Costa, M., Furness, J., and Cibbins, I. (1986). Chemical enteric neurons. Prog. Brain Res. 68, 217-239.

coding

of

Cottrell, G. A., Green, K. A., and Davies, N. W. (1990). The neuropeptide PheMet-Arg-Phe-NH, (FMRFamide) can activate a ligandgated ion channel in Helix neurones. Pflugers Arch. 476, 612614. Cuello,

A., ed. (1982). Co-Transmission

(London:

Macmillan).

Edwards, A. V., Jarhult, J., Andersson, P.-O., and Bloom, S. R. (1982). The importance of the pattern of stimulation in relation to the response of autonomic effecters. In Systemic Role of Regulatory Peptides, S. R. Bloom, J. M. Polak, and E. Lindenlaub, eds. (Stuttgart: Schattauer), pp. 145-148. Elde, R., Schalling, M., Ceccatelli, S., Nakanishi, S., and Hokfelt, T. (1990). Localization of neuropeptide receptor mRNA in rat brain: initial observations using probes for neurotensin and substance P receptors. Neurosci. Lett. 720, 134-138. Evans, B. E., Bock, M. C., Rittle, K. E., DiPardo, R. M., Whitter, W. L., Veber, D. F., Anderson, P. S., and Freidinger, R. M. (1986). Design of potent, orally effective, nonpeptidal antagonists of the peptide hormonecholecystokinin. Proc. Natl. Acad. Sci. USA83, 4918-4922. Fisone, G., Wu, C. F., Consolo, S., Nordstrom, O., Brynne, N., Bartfai, T., Melander, T., and Hokfelt, T. (1987). Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies. Proc. Natl. Acad. Sci. USA 84, 7339-7343. Fitzgerald, M., Wall, P., Goedert, M., and Emson, P. (1985). Nerve growth factor counteracts the neurophysiological and neurochemical effects of chronic sciatic nerve section. Brain Res. 332, 131-141. Folkers, K., Hbrig, J., Rampold, C., Lane, P., Rosell, S., and Bjorkroth, U. (1982). Design and synthesis of effective antagonists of substance P. Acta Chem. Stand. 36, 389-395. Fontaine, B., Klarsfeld, A., Hokfelt, T., and Changeux, J. P. (1986). Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes. Neurosci. Lett. 71, 59-65. Fontaine, B., Klarsfeld, A., and Changeux, J.-P. (1987). Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor alpha-subunit mRNA levels by distinct intracellular pathways. J. Cell Biol. 105, 1337-1342.

Dalsgaard, C.-J. (1988). The sensory system. In Handbook of Chemical Neuroanatomy, Vol. 6. The Peripheral Nervous System, A. Bjorklund, T. Hokfelt, and C. Owman, eds. (Amsterdam: Elsevier), pp. 599-636.

Fried, G., Terenius, L., Hokfelt, T., and Goldstein, M. (1985). Evidence for differential localization of noradrenaline and neuropeptideY in neuronal storagevesicles isolated from ratvas deferens. J. Neurosci. 5, 450-458.

De Biasi, S., and Rustioni, A. (1988). Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord. Proc. Natl. Acad. Sci. USA 85, 7820-7824.

Fuxe, K., and Agnati, L. (1991). Two principal modes of electrochemical communication in the brain: volume versus wiring transmission. In Volume Transmission in the Brain: Novel Mechanisms for Neural Transmission, K. Fuxe and L. Agnati, eds. (New York: Raven Press), pp. l-9.

de Bold, A., Borenstein, H. B., Veress, A. T., and Sonnenberg, H. (1981). A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci. 28, 89-94. de Montigny, C. (1989). Cholecystokinin panic-like attacks in healthy volunteers. Arch. Cen. Psychiatry 46, 511-517.

tetrapeptide Preliminary

induces findings,

De Wied, D. (1971). Long term effect of vasopressin on the maintenance of aconditioned avoidance response in rats. Nature232, 58-60. Denis-Donini, S. (1989). Expression of dopaminergic phenotypes in the mouse olfactory bulb induced by the calcitonin gene related peptide. Nature 339, 701-703. Dourish, C. T., Rycroft, W., and Iversen, S. D. (1989). Postpone ment of satiety by blockade of brain cholecystokinin (CCK-B) receptors. Science 245, 1509-1511. Dourish, C. T., O’Neill, M. F., Coughlan, J., Kitchener, S. j., Hawley, D., and Iversen, S. D. (1990). The selective CCK-B receptor antagonist L-365,260 enhances morphine analgesia and prevents morphine tolerance in the rat. Eur. J. Pharmacol. 776, 35-44. Duggan, A. W., Hope, P. J., Jarrott, B., Schaible, H. G., and Fleetwood, W. S. (1990). Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation. Studies with antibody microprobes. Neuroscience 35,195-202. Eccles, J. (1986). Chemical transmission Progress in Brain Research, T. Hokfelt, eds. (Amsterdam: Elsevier), pp. 3-13.

and Dale’s principle. In K. Fuxe, and B. Pernow,

Gesser, B. P., and Larsson, L. I. (1985). Changes from enkephalinlike to gastrin/cholecystokinin-like immunoreactivity in snail neurons. J. Neurosci. 5, 1412-1417. Giaid, A., Gibson, S. J., Ibrahim, B. N., Legon, S., Bloom, S. R., Yanagisawa, M., Masaki,T., Varndell, I. M., and Polak, J. M. (1989). Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia. Proc. Natl. Acad. Sci. USA 86, 7634-7638. Gibbins, I. L., Furness, J. B., and Costa, M. (1987). Pathwayspecific patterns of the co-existence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig. Cell Tissue Res. 248, 417-437. Gibson, S. J., Polak, J. M., Bloom, S. R., Sabate, I. M., Mulderry, P. M., Ghatei, M. A., McGregor, C. P., Morrison, J. F., Kelly, J. S., Evans, R. M., and Rosenfeld, M. (1984). Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J. Neurosci. 4, 3101-3111. Golding, D., and Bayraktaroglu, E. (1984). Exocytosis of secretory granules. A probable mechanism for the release of neuromodulators in invertebrates neuropil. Experientia 40, 1277-1280. Goodman, R. R., Snyder, S. H., Kuhar, M. J., and Young, W. S., III (1980). Differentiation of delta and mu opiate receptor localizations by light microscopic autoradiography. Proc. Natl. Acad. Sci. USA 77, 6239-6243.

Neuron 876

Guillemin, R. (1978). Biochemical and physiological correlates ot hypothalamic peptides. The new endocrinology of the neuron. In The Hypothalamus, S. Reichlin, R. 1. Baldessarini, and I. B. Martin, eds. (New York: Raven Press), pp. 155-194. Haas, C. A., Streit, W. J., and Kreutzberg, G. W. (1990). Rat facial motoneurons express increased levels ofcalcitonin gene-related peptide mRNA in response to axotomy. I. Neurosci. Res. 27,270275. Haegerstrand, A., Jonzon, B., Dalsgaard, C. J., and Nilsson, J. (1989).Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes. Proc. Natl. Acad. Sci. USA 86, 5993-5996.

Signalling Press).

in the Nervous

System (New York: Oxtord

University

Jennings, C. (,., and Mudge, A. W. (1989). Chick myotubes 111 culture express high-affinity receptors for calcitonin gene related peptlde. Brain Res. 504, 199-205. Jessell, T., Tsunoo. A., Kanazawa, I., and Otsuka, M. (1979). Sub stance P: depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neuron5 Brain Res. 768, 247-259. Jirikowskl, (*. F.. Sanna, P. P., and Bloom, F. L. 11990). mRNA coding for oxytocin is present in axons of the hypothalamcineurohypophysial tract. Proc. Natl.Acad. SCI. USA87,7400-7404.

Hendry, I. A., Stdckel, K., Thoenen, H., and Iversen, L. L. (1974). The retrograde axonal transport of nerve growth factor. Brain Res. 68, 103-121.

Jonsson. G., and Hallman, H. (1982). Substance neurotoxin damage on norepinephrine neurons during ontogeny. Science 275, 75-76.

Henry, j. L., Krnjevic, and spinal neurones.

Kaneko, T. Akiyama, H., Nagatsu, I., dnd Mizuno, N. (1990). 1111 munohistochemical demonstration of glutaminase in catechoi aminergic and serotonergic neurons of rat brain. Brain Res. 507 141-154

K., and Morris, Can. I. Physiol.

M. E. (1975). Substance P Pharmacol. 53, 423-432.

Hershey, A. D., and Krause, J. E. (1990). Molecular characterization of a functional cDNA encoding the rat substance P receptor. Science 247,958-962. Hill, E. L.,Turner, R., and Elde, R. (1991). Effects of sympathectomy and capsaicin treatment on bone remodeling in rats. Neuroscience 44, 747-756. Hohmann, E. L., Elde, R. P., Rysavy, J. A., Einzlg, S., and Cebhard, R. L. (1986). Innervation of periosteum and bone by sympathetic vasoactive intestinal peptide-containing nerve fibers. Science 232, 868-871. HGkfelt, T., Elfvin, L. G., Elde, R., Schultzberg, M., Goldstein, M., and Luft, R. (1977j.Occurrenceof somatostatin-like immunoreactivity in some peripheral sympathetic noradrenergic neurons. Proc. Natl. Acad. Sci. USA 74, 3587-3591.

P counteract< in the rat braln

Kangrga, I dnd Randic, M. (1990). lachyklnln\ dnd calcitonltl gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice. J. Neurosc I 10, 2026-2038 Kasakov, L., tll15, J., KirkpatrIck, K., Mllner. P., and Burnstock, c.. (1988). Direct evidenceforconcomitant releaseof noradrenaline. adenosineS-triphosphateand neuropeptidey from sympathetic nervesupplyingtheguinea-pigvasdeferens.J.Auton. Nerv.Sysl. 22, 75-82. Kessler, J., and Black, I. B. (1980). Nerve growth tactor stimulates development of substance P in sensory ganglia. Proc. Natl. Acad Sci. USA 77. 649-652.

HGkfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J. M., and Schultzberg, M. (1980a). Peptidergic neurones. Nature 284, 515521.

Kobayashi. R. M., Brown, M., and Vale, W. (1977). Regional dlstrrbution of nrurotensin and somatostatin in rat brain. Brain Res. 126, 584-548.

HBkfelt, T., Skirboll, L., Rehfeld, J. F., Goldstein, M., Markey, K., and Dann, 0. (1980b). A subpopulation of mesencephalic dopamine neurons projecting to limbic areas contains a cholecystokinin-like peptide: evidence from immunohistochemistry combined with retrograde tracing. Neuroscience 5, 2093-2124.

Kordower. J. H.. dnd Mu&on, t. J. (1990). Galanln-like Immunoreactivity withln the primate basal forebrain: differential staining patterns between humans and monkeys. 1. Comp. Neurol. 294, 281-292.

HGkfelt, T., Fuxe, K., and Pernow, B., eds. (1986). Coexistence of Neuronal Messengers: A New Principle in Chemical Transmission (Amsterdam: Elsevier).

Kowall, N., Beal, M., Busciglio,J., Dutty, L., and Yankner, B. (1991,. An in vivo model for the neurodegenerative effects of B amyloid and protection by substance P. Proc. Natl. Acad. Sci. USA 88, 7247-7251.

HGkfelt, T., Millhorn, D., Seroogy, K., Tsuruo, Y., Ceccatelli, S., Lindh, B., Meister, B., Melander, T., Schalling, M., Bartfai, T., and Terenius, L. (1987a). Coexistence of peptides with classical neurotransmitters. Experientia 43, 768-780.

Laufer, R., and Changeux, J.-P. (1987). Calcitonln gene-related peptide elevates cyclic AMP levels in chick skeletal muscle: possible neurotrophic role for a coexisting neuronal messenger. EMBO J. 6, 901-906.

Hakfelt, T., Wiesenfeld-Hallin, Z., Villar, M., and Melander, T. (1987b). Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci. Lett. 83, 217-220.

Lazar, P., Reddington, M., Streit, W., Ralvich, C., and Kreutzberg, C. W. (1991). The action of calcitonin gene-related peptide on astrocyte morphology and cyclic AMP accumulation in astrocyte cultures from neonatal rat brain. Neurosci. Lett. 730, 99-102.

Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A., and Morris, H. R. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258, 577-580.

Le Greves, P., Nyberg, F., Terenius. L., and HBkfelt, T. (1985). Calcitonin gene-related peptide is a potent inhibitor of substance P degradation. Eur. J. Pharmacol. 775, 309-311.

Hughes,J., Boden, P., Costall, B., Domeney,A., Kelly, E., Horwell, D. C., Hunter, j. C., Pinnock, R. D., and Woodruff, G. N. (1990). Development of a class of selective cholecystokinin type B recep torantagonists having potent anxiolyticactivity. Proc. Natl. Acad. Sci. USA 87, 6728-6732. Itoh, N., Obata, K., Yanaihara, N., and Okamoto, H. (1983). Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature 304, 547-549. Iverfeldt, K., Serfozo, P., Diaz, A. L., and Bartfai, T. (1989). Differential releaseof coexisting neurotransmitters: frequency dependence of the efflux of substance P, thyrotropin releasing hormone and ‘H-serotonin from tissue slices of rat ventral spinal cord. Acta Physiol. Stand. 737, 63-71. Iversen,

L., and Goodman,

E., eds. (1986). Fast and Slow Chemical

Lecci, A., Ciuliani, S., Patacchini, R., Viti, G., and Maggi, C. A, (1991). Role of NK, tachykinin receptors in thermonociception: effect of (* )-CP 96,345, a non-peptide substance P antagonist, on the hot plate test in mice. Neurosci. Lett. 129, 299-302. Lee, C., Iversen, L., Handley, M., and Sandberg, B. (1982). The possible existence of multiple receptors for substance P. Naunyn-Schmiedeberg’s Arch. Pharmacol. 378, 281-287. Levi-Montalcini, R. (1987). The nerve growth Science 237, 1154-1162.

factor 35 years later.

Lieberman, A. R. (1971). The axon reaction: a review of the princi pal features of perikaryal responses to axon injury. Int. Rev. Neurobiol. 74, 49-124. Lord, J., Waterfield, A., Hughes, I., and Kosterlitz, H. (1977). Endogenous opioid peptides: multiple agonists and receptors. Nature 267, 495-499.

Review: The Expanding 877

Roles of Neuropeptides

Lotz, M., Vaughan, J. H., and Carson, D.A. (1988). Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science 241, 1218-1221.

tonin gene-related tation of neurons 169-172.

Lundberg, J. M., and Hokfelt, T. (1983). Coexistence of peptides and classical transmitters. Trends Neurosci. 6, 325-333.

Moore, R. Y. (1989). Cranial motor neurons galanin- or calcitonin gene-related peptide-like ity. J. Comp. Neurol. 282, 512-522.

Lundberg, J. M., Fried, C., Fahrenkrug, J., Holmstedt, B., Hokfelt, T., Lagercrantz, H., Lundgren, G., and AnggPrd, A. (1981). Subcellular fractionation of cat submandibular gland: comparative studieson thedistribution of acetylcholineand vasoactive intestinal polypeptide (VIP). Neuroscience 6, 1001-1010.

peptide produces a slow and prolonged exciin the cat lumbar dorsal horn. Brain Res. 446, contain either immunoreactiv-

Narumi, S., and Maki, Y. (1978). Stimulatory effects of substance P on neurite extension and cyclic AMP levels in cultured neuroblastoma cells. J. Neurochem. 30, 1321-1326. New, H. V., and Mudge, A. W. (1986). Calcitonin gene-related peptide regulates muscle acetylcholine receptor synthesis. Natu re 323, 809-811.

Lundberg, J. M., Hedlund, B., Anggsrd, A., Fahrenkrug, J., Hokfelt, T., Tatemoto, K., and Bartfai, T. (1982). Costorage of peptides and classical transmitters in neurons. In Systemic Roleof Regulatory Peptides, S. R. Bloom, J. M. Polak, and E. Lindenlaub, eds. (Stuttgart: Schattauer), pp. 93-119.

Nicholas, A., Cuello, A., Goldstein, M., and Hokfelt, T. (1990). Glutamate-likeimmunoreactivityinmedullaoblongatacatecholamine/substance P neurons. NeuroReport 1, 235-238.

Magistretti, P. J., and Schorderet, line act synergistically to increase Nature 308, 280-282.

Nilsson, I., von Euler, A., and Dalsgaard, C. J. (1985). Stimulation of connective tissue cell growth by substance P and substance K. Nature 375, 61-63.

M. (1984). VIP and noradrenacyclic AMP in cerebral cortex.

Magistretti, P. J., Morrison, J., Shoemaker, W., Sapin, V., and Bloom, F. (1981)Vasoactive intestinal polypeptide induces glycogenolysis in the mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism. Proc. Natl. Acad. Sci. USA 78, 6535-6539. Marlier, L., Rajaofetra, N., Peretti, R. R., Kachidian, P., Poulat, P., Feuerstein, C., and Privat, A. (1990). Calcitonin gene-related peptide staining intensity is reduced in rat lumbar motoneurons after spinal cord transection: a quantitative immunocytochemical study. Exp. Brain Res. 82, 40-47. Mastropaolo, J., Nadi, N. 5, Ostrowski, N. L., and Crawley, J. N. (1988). Galanin antagonizes acetylcholine on a memory task in basal forebrain-lesioned rats. Proc. Natl. Acad. Sci USA85,98419845. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi, S. (1987). cDNAcloning of bovine substance-K recep tor through oocyte expression system. Nature 329, 836-838. Matteoli, M., Haimann, C.,Torri, T. F., Polak, J. M., Ceccarelli, B., and De Camilli, P. (1988). Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large densecore vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction. Proc. Natl. Acad. Sci. USA85,73667370. McMahon, S. B., and Gibson, S. (1987). Peptide expression is altered when afferent nerves reinnervate inappropriate tissue. Neurosci. Lett. 73, 9-15. Meister, B., Hdkfelt, T., Geffard, M., and Oertel, W. (1988). Glutamicaciddecarboxylase-andgamma-aminobutyricacid-likeimmunoreactivities in corticotropin-releasing factor-containing parvocellular neurons of the hypothalamic paraventricular nucleus. Neuroendocrinology 48, 516-526. Meister, B., Villar, M. J., Ceccatelli, S., and Hokfelt, T. (1990). Localization of chemical messengers in magnocellular neurons of the hypothalamic supraoptic and paraventricular nuclei: an immunohistochemical study using experimental manipulations. Neuroscience 37, 603-633. Melander, T., and Staines, W. A. (1986). A galanin-like peptide coexists in putative cholinergic somata of the septum-basal forebrain complexand in acetylcholinesterase-containingfibersand varicosities within the hippocampus in the owl monkey (Aotus trivirgatus). Neurosci. Lett. 68, 17-22. Melander,T., Staines, W.A., Hdkfelt,T., RBkaeus,A., Eckenstein, F., Salvaterra, P. M., and Wainer, B. H. (1985). Galanin-like immunoreactivity in cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat. Brain Res. 360, 130-138. Micevych, P., Akesson, T., and Elde, R. (1988). Distribution of cholecystokinin-immunoreactive cell bodies in the male and fe male rat: II. Bed nucleus of the stria terminalis and amygdala. J. Comp. Neurol. 269, 381-391. Miletic,

V., and Tan, H. (1988). lontophoretic

application

of calci-

Noguchi, K., Senba, E., Morita, Y., Sato, M., and Tohyama, M. (1990). Alpha-CCRPand beta-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglion. Mol. Brain Res. 7, 299-304. Oku, R., Satoh, M., Fujii, N., Otaka, A., Yajima, H., and Takagi, H. (1987). Calcitonin gene-related peptide promotes mechanical nociception by potentiating release of substance P from the spinal dorsal horn in rats. Brain Res. 403, 350-354. Oku, R., Nanayama, T., and Satoh, M. (1988). Calcitonin generelated peptide modulates calcium mobilization in synaptosomes of rat spinal dorsal horn. Brain Res. 475, 356-360. Palacios, J. M., and Kuhar, M. J. (1981). Neurotensin receptors are located on dopamine-containing neurones rn rat midbrain. Nature 294, 587-589. Palacios, J. M., Savasta, M., and Mengod, G. (1989). Does cholecystokinin colocalize with dopamine in the human substantia nigra? Brain Res. 488, 369-375. Pernow,

B. (1983). Substance

P. Pharmacol.

Rev. 35, 85-141.

Piehl, F., Arvidsson, U., Johnson, H., Cullheim, S., Villar, M., Dagerlind, A., Terenius, L., Hokfelt, T., and Ulfhake, B. (1991). Calcitonin gene-related peptide (CCRP)-like immunoreactivity and CGRP mRNA in rat spinal cord motoneurons after different types of lesions. Eur. J. Neurosci. 3, 737-757. Pincus, D. W., DiCicco, B. E., and Black, I. B. (1990). Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts. Nature 343, 564567. Popper, P., and Micevych, P. E. (1989). Localization gene-related peptide and its receptors in a striated Res. 496, 180-186.

of calcitonin muscle. Brain

Randic, M., Hecimovic, H., and Ryu, P. D. (1990). Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones. Neurosci. Lett. 777, 74-80. Roa, M., and Changeux, J.-P. (1991). Characterization and developmental evolution of a high affinity binding site for calcitonin gene related peptide (CGRP) on chick skeletal muscle membrane. Neuroscience 47, 563-570. Rosenfeld, M. C., Mermod, J. J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W., and Evans, R. M. (1983). Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304, 129-135. Ryu, P. D., Gerber, C., Murase, K., and Randic, M. (1988). Actions of calcitonin gene-related peptide on rat spinal dorsal horn neurons. Brain Res. 447, 357-361. Said, S. I., and Mutt, V. (1970). Polypeptide with broad biological activity: isolation from small intestine. Science 769, 1217-1218. Salt, T. E., and Hill, R. G. (1983). Neurotransmitter candidates of somatosensory primary afferent fibres. Neuroscience 70, 10831103. Schalling,M., Friberg, K.,Seroogy, K., Riederer, mann, S. N., Mailleux, P., Vanderhaeghen,

P., Bird, E., SchiffJ. J., Kuga, S.,

Goldstein, M., and HGkfelt, T. (1990). Analysis of expression of cholecystokinin in dopaminecells intheventral mesencephalon of several species and in humans with schizophrenia. Proc. Natl. Acad. Sci. USA 87, 8427-8431. Schally, A. V., Arimura, A., and Kastin, A. J. (1973). Hypothalamic regulatory hormones. Science 779, 341-350. Schneider, S. P., and Perl, E. R. (1985). Selective excitation of neurons in the mammalian spinal dorsal horn by aspartate and glutamate in vitro: correlation with location and excitatory input. Brain Res. 360, 339-343. Schultzberg, M., and in autonomic ganglia. Vol. 6. The Peripheral and C. Owman, eds.

Lindh, B. (1988). Transmitters and peptides In Handbookof Chemical Neuroanatomy, Nervous System, A. BjBrklund, T. HGkfelt, (Amsterdam: Elsevier), pp. 297-32?.

Shehab, S. A., and Atkinson, M. E. (1986). Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomyof the sciatic nerveoriginate from primaryafferent neurons. Brain Res. 372, 37-44. Shinmi, O., Kimura, S., Yoshizawa, T., Sawamura, T., Uchiyama, Y., Sugita,Y., Kanazawa, I., Yanagisawa, M., Goto, K.,and Masaki, T. (1989). Presence of endothelin-1 in porcine spinal cord: isolation and sequencedetermination. Biochem. Biophys. Res. Commun. 162, 340-346. Simerly, R. B., and Swanson, L. W. (1987). Castration reversibly alters levels of cholecystokinin immunoreactivity within cells of three interconnected sexually dimorphic forebrain nuclei in the rat. Proc. Natl. Acad. Sci. USA 84, 2087-2091. Smullin, D. H., Skilling, S. R., and Larson,A. A. (1990). Interactions between substance P, calcitonin gene-related peptide, taurine and excitatory amino acids in the spinal cord. Pain 42, 93-101. Sneddon, P., and Westfall, D. P. (1984). Pharmacological evidence that adenosine triphosphate and noradrenaline are cotransmitters in the guinea-pig vas deferens. J. Physiol. 347, 561580.

Tanaka, K., Masu, M., and Nakanishl, 5. (1990). Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4, 847-854. Tatemoto, K., and Mutt, V. (1988). Neuropeptide Y, peptide HI. galanin, cholecystokinin-58, neuropeptide K, and peptideYY. In Gastrointestinal Hormones, V. Mutt, ed. (San Diego: Academic Press, Inc.), pp. 421-437. Thoenen, H. (1991). The changing Trends Neurosci. 14, 165-170.

scene ot neurotrophic

factors.

Thureson-Klein, A., and Klein, R. L. (1990). Exocytosis from neuronal large dense-cored vesicles. Int. Rev. Cytol. 727, 67-126. Tsujimoto, T., and Kuno, M. (1988). Calcitonin gene-related peptide prevents disuse-induced sprouting of rat motor nerve terminals J. Neurosci. 8, 3951-3957. Vertes, Z., Melegh, C., Vertes, M., and Kovacs, S. (1982). Effect of naloxone and o-met2-pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain. Life Sci. 37,119-126. Villar, M. 1.. Cortes, R., Theodorsson, E., Wiesenfeld-Hallin, 2.. Schalling, M., Fahrenkrug, J., Emson, P. C., and HBkfelt, T. (1989a). Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience 33, 587-604. Villar, M. J., Roa, M., Huchet, M., HGkfelt, T., Changeux, J.-P.. Fahrenkrug, J., Brown, J. C., Epstein, M. C., and Hersh, L. (1989b). lmmunoreactive calcitonin gene-related peptide, vasoactive intestinal polypeptideand somatostatin in developing chicken spinal cord motoneurons. Distribution and role in regulation of CAMP in cultured muscle cells. Eur. J. Neurosci. I, 269-287. Walker, L. C., kaeus, A., and basal forebrain Comp. Neural.

Koliatsos, V. E., Kitt, C. A., Richardson, R. T., RoPrice, D. L. (1989). Peptidergic neurons in the magnocellular complex of the rhesus monkey. J. 280, 272-282.

Wall, P. D., and Cutnick, M. (1974). Ongoing activity in peripheral nerves: the physiology and pharmacology of impulses originating from a neuroma. Exp. Neurol. 43, 580-593.

Snider, R. M., Constantine, J. W., Lowe, I. J., Longo, K. P., Lebel, W. S., Woody, H. A., Drozda, S. E., Desai, M. C., Vinick, F. J., Spencer, R. W.,and Hess, H.-J. (1991).A potent nonpeptideantagonist of the substance P (NKl) receptor. Science 257, 435-437.

Wall, P. D., and Woolf, C. J. (1984). Muscle but not cutaneous C-afferent input produces prolonged increases in theexcitability of the flexion reflex in the rat. J. Physiol. 356, 443-458.

Snyder, S. H. (1980). Brain peptides 209, 976-983.

Wang, R., and Schoenfeld, R., eds. (1987). Cholecystokinin onists (New York: Alan R. Liss).

as neurotransmitters.

Science

Sossin, W. S., Sweet, C. A., and Scheller, R. H. (1990). Dale’s hypothesis revisited: different neuropeptides derived from a common prohormone are targeted to different processes. Proc. Natl. Acad. Sci. USA 87, 4845-4848. Streit, W. J., Dumoulin, F. L., Raivich, C., and Kreutzberg, C. W. (1989). Calcitonin gene-related peptide increases in rat facial motoneuronsafter peripheral nervetransection. Neurosci. Lett. 707, 143-148. Sudoh, T., Kangawa, K., Minamino, new natriuretic peptide in porcine

N., and Matsuo, H. (1988). A brain. Nature 332, 78-81.

SundstrBm, E., Archer, T., Melander, T., and HGkfelt, T. (1988). Calanin impairs acquisition but not retrieval of spatial memory in rats studied in the Morris swim maze. Neurosci. Lett. 88,331335. Swaab, D. F., Pool, C. W., and Van Leeuwen, ficity ever be proved in immunocytochemical them. Cytochem. 25, 388-391.

F. (1977). Can specistaining. J. Histo-

Swanson, L. W., Sawchenko, P. E., and Lind, R. W. (1986). Regulation of multiple peptides in CRF parvocellular neurosecretory neurons: implications for the stress response. Prog. Brain Res. 68, 169-190.

Antag-

Whim, M. D., and Lloyd, P. E. (1989). Frequency-dependent release of peptide cotransmitters from identified cholinergic motor neurons in Aplysia. Proc. Natl. Acad. Sci. USA 86,9034-9038. Whitehouse, P. l., Price, D. L., Struble, R. C., Clark, A. W., Coyle, J. T., and Delon, M. R. (1982). Alzheimer’s disease and senile dementia: loss of neurons in the basal forebrain. Science 27.5, 1237-1239. Wiesenfeld-Hallin, Z., HGkfelt, T., Lundberg, J. M., Forssmann, W. C., Reinecke, M., Tschopp, F. A., and Fischer, J. A. (1984). lmmunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat. Neurosci. Lett. 52, 199204. Wiesenfeld-Hallin, Z., Xu, X. J., Hughes, J., Horwell, D. C., and HGkfelt, T. (1990). PD134308, a selectiveantagonist of cholecystokinin type B receptor, enhances the analgesic effect of morphine and synergistically interacts with intrathecal galanin to depress spinal nociceptive reflexes. Proc. Natl. Acad. Sci. USA 87, 71057109. Woodley, S., and Kendig, J. (1991). Substance Pand NMDA recep tars mediatea slow nociceptiveventral root potential in neonatal rat spinal cord. Brain Res. 559, 17-21.

Takahashi, T., Konishi, S., Powell, D., Leeman, S. E., and Otsuka, M. (1974). ldentificationofthemotoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance P. Brain Res. 73, 59-69.

Woodruff, C. N.. Hill, D. R., Boden, P., Plnnock, R., Singh, L.. and Hughes, J. (1991). Functional role of brain CCK receptors. Neuropeptides (Suppl.) 19, 45-56.

Takami, K., Hashimoto, K., Uchida, S., Tohyama, M., and Yoshida, H. (1986). Effect of calcitonin gene-related peptide on the cyclic AMPlevelof isolated mousediaphragm. Jpn. J. Pharmacol. 42, 345-350.

Woolf, C., and Wiesenfeld-Hallin, Z. (1986). Substance P and calcitonin gene-related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat. Neurosci. Lett. 66, 226-230.

Review: The Expanding 879

Roles of Neuropeptides

Xu,X.-J., Wiesenfeld-Hallin,Z., Villar, M. J., and Hokfelt,T. (1989). lntrathecal galanin antagonizes the facilitatory effect of substance P on the nociceptive flexor reflex in the rat. Acta Physiol. Stand. 137,463-&k Xu, X.-J., Wiesenfeld-Hallin, Z., Villar, M. J., Fahrenkrug, J., and Hokfelt, T. (1998). On the role of galanin, substance P and other neuropeptides in primary sensory neurons of rat: studies with spinal reflex excitability and peripheral axotomy. Eur. J. Neurosci. 2, 733-743. Xu, X.-J., Wiesenfeld-Hallin, Z., Hughes, J., Horwell, D. C., and Hokfelt, T. (1991). PD134388, a selective antagonist of cholecystokinin typeB receptor, prevents morphine tolerance in the rat. Br. J. Pharmacol., in press. Yanagisawa, M., Kurihara, H., Kimura, S.,Tomobe,Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-415. Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R., Kakizuka, A., Ohkubo, H., and Nakanishi, S. (1989). Molecular characterization of a functional cDNA for rat substance P receptor. J. Biol. Chem. 264, 17649-17652. Young, I. W., and Kuhar, M. J. (1979). A new method for receptor autoradiography: [‘Hlopioid receptors in rat brain. Brain Res. 179, 255-270. Zagon, I. S., and McLaughlin, P. J. (1983). Increased brain sizeand cellular content in infant rats treated with an opiate antagonist. Science 227, 1179-1180. Note Added in Proof I would liketo thank my coworkers: Drs. Ulf Arvidsson, Sandra Ceccatelli, Ake Dagerlind, BjGrn Lindh, Bjijrn Meister, Tor Melander, and Martin Schalling, and collaborating groups headed by Drs. Tamas Bartfai, Jean-Pierre Changeux, Staffan Cullheim, Menek Goldstein, Jan M. Lundberg, Brun Ulfhake, and Zsuzsanna Wiesenfeld-Hallin.

Neuropeptides in perspective: the last ten years.

Neuron, Vol. 7, 867-879, December, 1991, Copyright 0 1991 by Cell Press Neuropeptides in Perspective: The Last Ten Years Tomas HGkfelt Department...
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