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|Neuropsychopharmacology: The Fifth Generation of Progress|
Vasoactive Intestinal Peptide In The Central Nervous System
Douglas E. Brenneman, Joanna M. Hill, and Illana Gozes
How ironic in these rational times
to be labeled intestinal
When one has a part to play
that's so much more subliminal
What loftier, elevated role
than to keep the brain more temperate
And what more worthier goal
than help true lovers consummate
Excerpt from a poem by Dr. John A. Bevan
University of Vermont
As alludes the poet, vasoactive intestinal peptide (VIP) is a multi-functional neuropeptide with roles that extend far beyond actions in the small intestine, the source from which the peptide was isolated (121). Since its discovery in 1970, over 7000 research papers have been written on VIP, with 16% of these referring to the brain. VIP is a 28-amino acid peptide that is widely distributed in the central and peripheral nervous systems. Important actions for VIP beyond the scope of this summary have been reviewed for the cardiovascular (34), reproductive (106), pulmonary (87), immune (130) and gastrointestinal systems (129). General physiological effects include vasodilation, bronchodilation, immunosuppression, hormonal secretion and increases in gastric motility. However, our focus will be to describe the current understanding of VIP in the brain, with limited reference to related topics in the peripheral nervous system. Our purpose is to highlight recent progress in the areas of gene expression, receptor characterization, drug design, functional neuroanatomy, neuroendocrine regulation, neurotrophism, growth regulation and clinical pathology/therapeutics. Our hope is that this abbreviated summary of VIP's action in the brain will stimulate further research interest for this Very Important Peptide.
MOLECULAR CHARACTERISTICS OF VIP
VIP Family of Peptides
The VIP family of peptides is compared in Figure 1. The identical VIP structure has been observed in many species, including man, rat, mouse, pig, sheep, dog and goat. The guinea pig VIP differs slightly (three amino acids) from that of other mammals. Within the VIP family of peptides, a range of similarities is observed, with PACAP-27 (pituitary adenylate cyclase activating peptide) being the closest in structure to VIP (100). An operational mode has emerged in this field suggesting that any function described for VIP should be examined for the PACAP peptides as well. In many cases, the two peptides elicit similar pharmacological responses, varying only in potency.
VIP Gene Structure
Since the first cloning of the VIP gene (9) and chromosomal localization (53), many important advances in the understanding of the molecular biology of VIP have occurred, including gene regulation by innervation and hormonal control, splicing mechanisms and newly discovered regulatory sites. Our purpose will be to highlight aspects of gene structure and expression; a more detailed treatment of these topics has been reviewed recently (35).
The peptides of the VIP family are probably the result of exon duplication coupled to gene duplication. The VIP gene contains seven exons, each encoding a distinct functional domain in the final mRNA and protein (Figure 2). VIP and the related peptide PHM (peptide histidine methionine, human) or PHI (peptide histidine isoleucine, rat) are encoded by two adjacent exons in the genome. Recently, tissue-specific alternative splicing was demonstrated in turkey and chicken hypothalamus, where VIP-specific mRNA (devoid of PHI) and mRNA specific for both VIP and PHI were discovered (137,159). In birds, there is an increased level of VIP-specific transcripts that is regulated by nesting behavior (159).
The 3'-untranslated region (UTR) of the mammalian VIP mRNA contains three AUUUA motifs that confer message instability through formation of RNA-protein complexes. An AU-rich segment of c-fos mRNA can successfully compete with VIP mRNA for binding with cytoplasmic proteins, suggesting post-transcriptional regulation through the 3'-UTR (155). Alternatively polyadenylated VIP mRNAs have been shown to be differentially controlled at the level of stability (29), with a 1.0 kb message exhibiting greater stability than a 1.7 kb message. A larger form of VIP transcript has also been detected (55).
In the 5' flanking region of the gene, there is high conservation of upstream regulatory sequences in the human, rat (47) and mouse (125). Fink et al. (44) reported that a 17-nucleotide enhancer element [VIP cAMP-responsive element (CRE)] was located within 100 base pairs (bp) of the transcription start site. The VIP-CRE contains two conserved domains (CGTCA), in inverted orientation, which are also found in several other cAMP-responsive genes. Hahm and Eiden have also recently shown (65) a consensus PMA-responsive element (TGACTCA) 2.25 kb upstream of the transcription start site. Waschek et al. (152) have shown that sequences between -5.2 kb and -2.5 kb upstream from the transcription start site control VIP gene expression in neuroblastoma cells. Hahn and Eiden mapped this area and found a tissue specific element between -4.66 kb and—4.02 kb upstream from the transcription start site. They have further identified an octamer consensus sequence (ATGCAAAT) that binds POU homodomain proteins. Other consensus sequences included two conserved hexamers (CACCTG and CATGTG), each duplicated, that are also found in the human choline acetyltransferase gene and are suggested to be responsible for cell type-specific silencer activity. These studies were substantiated by quantitative analysis of the expression of a VIP transgene bearing the proximal two kilobases of the 5' flanking region of the human VIP gene, which did not confer tissue specificity (140).
Neuropoietic cytokines (e.g., ciliary neurotrophic factor, CNTF) activate the signal transducers and activators of transcription (STAT) family of transcription factors. CNTF increases VIP gene expression, perhaps through the VIP cytokine responsive element (CyRE), which contains a STAT binding site (115). In addition, the cholinergic differentiation factor/leukemia inhibitory factor is also involved in the regulation of VIP gene expression (135). The control of VIP gene expression by neuropoietic cytokines is interesting also from the perspective of the 3' untranslated region of the VIP gene. The 3' region contains reiterated AUUUA motifs (as stated above) that are also found in the 3' untranslated regions of lymphokines, cytokines and proto-oncogene transcripts (155).
Following transcription, splicing and polyadenylation, VIP mRNA is translated into prepro-protein. The prepro-VIP is then digested to form the active peptides (Figure 2). A recent report indicated that all the prepro-VIP-derived peptides, except PHI/PHV are expressed in the female anterior pituitary and are increased by estrogen (131). Peptide products that were measured include: prepro-VIP (22-79), peptide histidine isoleucine (PHI), peptide histidine valine (PHV), prepro-VIP (111-122), VIP, prepro-VIP (156-170). The absence of PHI and PHV was probably due to translational or post-translational modification, as PHI coding sequences were detected in the pituitary.
The multiple levels of control for the VIP gene allow tight regulation of its expression. Some tissues influencing VIP gene expression include: the thyroid gland, the adrenal gland, the ovaries (estrogen), the pituitary (prolactin) and the brain (serotonin). Environmental influences include osmotic shock (for recent review, see Davidson et al. [35). For examples of VIP gene regulation see: Georg et al. (46) for the effects of retinoic acid, Lam et al. (83) for glucocorticoids, Gozes et al. (52) for adrenal regulation, Kasper et al. (82) for estrogen regulation and Chew et al.(30) for osmotic regulation.
VIP Gene Expression: The Suprachiasmatic Nucleus and Diurnal Rhythms
VIP is expressed in the suprachiasmatic nucleus (SCN; Figure 3), a brain region involved in the regulation of circadian rhythmicity. Synaptic activity affects VIP gene expression in the SCN, as do lactation, adrenal hormones and light input (35). The possible involvement of VIP in the control of diurnal rhythms has been determined utilizing specific antagonists, antisense oligodeoxynucleotides and light/dark phase shifts. In the SCN, VIP mRNA exhibits diurnal oscillations, with high levels during the dark periods. These oscillations, however, are independent of the light /dark cycles, as the oscillations were observed in animals reared in the dark (48; Figure 3). Blindness or exposure of the animals to total darkness during development results in increased hypothalamic VIP mRNA (48). Pharmacological blockade of VIP function during development produces a loss of the 24-hour circadian rhythmicity (Figure 3; 58) that is prevented by co-treatment with an agonist. Specific VIP antagonists have been used to demonstrate the involvement of a cAMP-mediated mechanism in determining diurnal rhythmicity in vivo. Other studies have demonstrated that spontaneously hypertensive rats exhibited elevated levels of VIP mRNA in the SCN and altered circadian rhythms. Interestingly, treatment with p-chlorophenylalanine, an inhibitor of tryptophan hydroxylase that interferes with serotonergic transmission, induced circadian changes in VIP mRNA in animals maintained in constant darkness (102). Further studies demonstrated altered VIP gene expression in the fetal SCN following prenatal serotonergic deficiency (142).
Molecular Biology of VIP Receptors
Two cloned VIP receptors have been characterized. The gene of one receptor, HVR1 (human VIP receptor 1), spans ~22 kb and is composed of 13 exons. The G+C-rich flanking region of the gene contains potential binding sites for multiple nuclear factors including: Sp1, AP2, ATF, interferon regulatory factor 1, NF-IL6, acute phase response factor and NFkB. Figure 4 (adapted from Sreedharan et al. ) depicts the organization of the HVR1 gene exons (solid bars) with respect to the seven transmembrane spanning domains of the receptor protein (I-VII). The locations of the introns relative to the cDNA structure are indicated by arrows. The final size of the mRNA is 3 kb. The HVR1 gene is located at chromosomal site 3p22, a position associated with small cell-lung cancer. Recent studies (108) have identified a negative glucocorticoid response element in the rat type 1 VIP receptor gene (located within the 126-bp 5'-flanking region of the VIP receptor gene). Dexamethasone suppresses endogenous VIP receptor mRNA expression in cultured lung cells. DNase I footprinting demonstrated that purified glucocorticoid receptor bound to the VIP1 receptor promoter between -21 bp and -36 bp relative to the transcription start site. These studies, coupled with the complex regulation of the VIP gene by glucocorticoids (described above), suggested a pivotal role for glucocorticoids and the adrenal gland in the regulation of VIP activities.
The VIP2 receptor gene is encoded by 13 exons and spans at least 40 kb. In humans, the gene is located on chromosome 7q36.3, close to the locus responsible for the craniofacial defect holoprosencephaly type 3 (HPE3). Detailed mapping studies carried out on cell lines derived from patients with HPE suggest that deletion of the VIP2 receptor gene is not the sole factor responsible for the HPE3 phenotype. However, it is possible that monosomy at the VIP2 receptor gene locus may contribute to the phenotypic symptoms (e.g., agenesis of the pituitary and the olfactory bulbs) seen in many cases of HPE3.
The major site of expression of the VR1 is the lung. The VIP receptor 2 (VR2) is predominantly distributed in other organs. The VR2 and all the other receptors in the family were recently reviewed by Harmar and Lutz (67). The protein structure, with the seven transmembrane domains of the VR2, is depicted in Figure 4. The two cloned VIP receptors are linked to adenylate cyclase. Other receptors recognizing VIP include the PACAP receptors, with six splice variants [reviewed by Journot et al. (79); Figure 4]. Some of the splice variants of the PACAP receptor molecule are linked to phosphatidyl inositol turnover and calcium mobilization.
VIP BINDING SITES
With in vitro autoradiography, VIP binding sites in several body tissues can be subdivided into two groups based on their sensitivity to GTP (71), a characteristic of receptors linked to adenylate cyclase. GTP-insensitive VIP binding sites occur throughout the brain and have a higher affinity for VIP. Although the relation between the GTP-sensitive and -insensitive binding sites to the currently cloned VR1 and VR2 receptors is unclear, the cloned receptors are linked to adenylate cyclase and probably represent the GTP-sensitive binding.
VIP's action in the central nervous system involves behavioral, electrophysiological, secretory, metabolic, vascular and mitogenic effects in many brain regions. The signal transduction mechanisms that mediate these effects also show cellular and molecular diversity. The first described and most widely examined effect of VIP is its stimulation of adenylate cyclase with the generation of cAMP (Figure 5) . VIP is often used as a control molecule to investigate the effects of cAMP on physiological processes. Significant increases in cAMP accumulation are commonly observed after treatment with VIP in the 0.01–1 mM range for both neurons and glia.
VIP action has also been associated with G-proteins, signal transducing proteins stimulating the hydrolysis of GTP to GDP. The cloned VIP receptors belong to a subset of G protein-coupled receptors linked to adenylate cyclase (133). G-proteins are involved in many signal transduction pathways, such as cAMP formation and phosphoinositide breakdown. GTP and its analogs inhibit VIP receptor binding and potentiate cAMP synthesis in response to VIP (107). In the caudate nucleus, the neuromodulatory effects of VIP are thought to involve inhibitory and stimulatory G-proteins, Gi and Gs coupled to adenylate cyclase (92). In the pineal gland, VIP-mediated increases in cAMP are mediated through pertussis toxin-sensitive G proteins (66). The interaction between VIP and other neurotransmitters is also mediated through G-proteins. For example, the stimulatory effects of VIP on cAMP accumulation in the pineal can be attenuated by neuropeptide Y acting though a pertussis toxin-sensitive G protein (66). Also, the synergistic action between VIP and leucine-enkephalin is mediated by a stimulatory GTP-binding protein (105). In locus coeruleus neurons, VIP causes direct excitation by inducing a Na+-dependent inward current, an effect which becomes irreversible in the presence of GTP-gamma-S (149). Thus, the diverse array of VIP-mediated effects in the CNS is probably due in part to the complexity and specificity conferred by the G-proteins.
VIP action is associated with other second messengers in the central nervous system. The molecular nature of these receptors is unknown. It is possible that some of these actions are mediated through PACAP receptors. Treatment of pinealocytes with low concentrations of VIP produces increases in intracellular calcium (Figure 6), concomitant with elevations in cGMP and regulation of a cyclic nucleotide-gated cation channel (123). Previous studies indicated that the survival of post-mitotic neurons in CNS cultures could be increased by a sub-nanomolar concentration of VIP. The high potency of this pharmacological response suggests that survival-promoting actions of VIP might involve a receptor and signal transduction mechanism that does not involve cAMP-dependent phosphorylations. Support for a non-cAMP mechanism resides in the great mismatch in EC50s for survival promotion (30 pM) vs. that of VIP-mediated increases in cAMP (3 mM). This large difference in effective concentrations strongly suggests an alternate mechanism. In cultured astroglial cells, treatment with sub-nanomolar amounts of VIP can elicit mobilization of intracellular calcium (Figure 5) . Astroglial heterogeneity is evident, in that only 20% of astrocytes respond with calcium increases after VIP treatment. The increase in calcium is apparently from an intracellular compartment, since the response to VIP persisted in the absence of extracellular calcium. Furthermore, thapsigargin pre-treatment, which depletes intracellular calcium stores, abolishes the VIP-induced calcium response. The VIP-elicited increases were accompanied by acute (1 min) and transient elevations of inositol triphosphate. Treatment with sub-nanomolar VIP also results in the translocation of protein kinase C (PKC) from the cytoplasm to the nucleus (104). Specific PKC isotypes were increased by VIP, with nuclear PKC alpha and, to a lesser extent, PKC delta and zeta immunoreactivity being elevated. Although cAMP may not be involved in the secretion of survival-promoting substances from astroglia, this relationship may be restricted to specific cell types.
Nitric oxide may also be involved in the mechanism of VIP in brain and other organs. Nitric oxide synthetase is co-localized with VIP in both the pineal gland and in some cells in the SCN (116). Furthermore, nitric oxide may be involved in both circadian regulation and circulatory effects with VIP.
CNS DEVELOPMENT OF VIP
VIP is a late developing peptide. In the rat occipital cortex, tritiated thymidine methods have determined that most VIP neurons are generated on embryonic day (E)19, two days before birth (25). Immunochemical (40,90) and molecular methods (71) show that VIP is low to undetectable in the prenatal rat brain, but increases rapidly postnatally. In situ hybridization histochemistry (ISHH) methods, tracing the mRNA for VIP in rat brain from E14 onward, reveal no mRNA until birth , when it becomes abundant in the superior colliculus (Figure 7) . Although the density of VIP mRNA appears relatively stable after birth in the superior colliculus, the mRNA for VIP increases in most brain regions until the levels and distribution pattern of the adult brain have been established. This is particularly evident in the thalamus and dorsal raphe (Figure 7). In the cortex, the rapid increase of mRNA for VIP up to postnatal day 14 (61) is followed by a gradual decrease until adult levels are reached. This apparent decrease in mRNA may be due to an increased distance between VIP neurons as the total volume of the cortex expands; however, a transient presence of VIP neurons in the visual cortex of the cat has been described (148). Additionally, Northern blot analyses have revealed a decrease in cortical mRNA for VIP from postnatal day 21 to adult (60). The reduction of VIP in the cortex with development suggests that some VIP neurons play a role in cortical differentiation. While generally considered a postnatal peptide in the CNS, VIP has been detected at E14 in the peripheral nervous system of the rat (141), in the hamster SCN three days before birth (12), and in the prenatal human spinal cord (27) and Macaque monkey cortex (69). The use of a sensitive ISHH technique has revealed the mRNA for VIP in the hindbrain of the E14 mouse (151). This suggests that either low levels of VIP message are present in the prenatal brain and that these levels have been too low to be detected by previously used methods or that the mouse and rat differ in the expression of VIP in the CNS.
Although the highly sensitive reverse transcription-PCR (rt-PCR) method was not able to detect VIP mRNA in any E11 rat embryo tissues (74), by E14 VIP is detected in the rat embryonic body by ISHH (71) and Northern blot techniques (61). By E16, the sphenopalatine ganglion expresses exceptionally high levels of VIP mRNA, which decrease with age (71). At E16, low levels of VIP mRNA are seen in the aorta and intestines (71).
In remarkable contrast to the delayed appearance of the peptide VIP in the brain, VIP binding sites appear very early in development and are primarily, if not exclusively, localized to the CNS during the early prenatal period (71). In the developing rat CNS, both GTP-sensitive and GTP-insensitive binding sites have been reported as early as E13 (74). During development, binding sites are abundant and patterns of binding distribution in the nervous system change due to the transient appearance of both types of binding sites in regions undergoing specific ontogenic events (71). In the E13 rat, binding is not apparent in the embryo body but is abundant in the spinal cord, where GTP-insensitive binding is particularly dense in the regions of the floor and roof plates. During early embryonic development, the floor plate is composed of macroglial cells and governs tissue organization through diffusible and contact-mediated signals. The dense VIP binding sites along its extent supports the hypothesis that VIP regulates morphogenic events by releasing diffusible signals from glial cells in this region. In vitro autoradiography reveals binding sites in the E9 mouse (62) and transcripts for the VIP2 receptor mRNA have been identified as early as E14 (150) in the mouse brain. In addition, following culture of E9 mouse embryos in vitro, autoradiography reveals that VIP treatment down-regulated VIP binding sites and further indicate that growth-regulation occurs largely through GTP-insensitive binding sites (62).
While binding sites are almost uniformly dense in the brain stem and spinal cord from E13 to E16 in the rat, the brain begins to exhibit regionally specific patterns of distribution (71), and binding sites appear in several tissues throughout the body (Figure 8). VIP binding sites are abundant in neuroepithelial cell regions of the brain and in the intermediate medial thalamus, where cells are undergoing rapid division. Other areas of intense VIP binding are the floor and roof plates and in the posterior commissure (Figure 8d, Figure 8e, and Figure 8f). Late in gestation, beginning at E19, VIP binding sites become almost homogenous throughout the brain, obscuring most regional differences. This pattern remains until postnatal day 14, after which the adult pattern begins to emerge. As an example, the changes in VIP binding sites occurring during the development of the rat cortex are illustrated in Figure 9. In several regions of the adult brain, such as the ventral thalamus, cortex and suprachiasmatic nucleus, VIP mRNA and VIP binding overlap; however, other regions show little temporal or spatial relationship between VIP gene expression and VIP binding.
Perhaps the most dramatic demonstration of VIP-mediated increases in growth during development is the work of Gressens in cultured whole embryos (Figure 10) . In this study, VIP produced a five-fold increase in the number of cells in S phase in the primitive neuroepithelium and a doubling of the total DNA in the embryo. As in the case of neuronal survival in the CNS, this effect of VIP is thought to be indirect, in that the binding sites for VIP at this stage of development are confined to the CNS, yet the entire embryo undergoes growth. A classical approach to the study of the functional role of a neurochemical is to block its action with a specific antagonist or a neutralizing antibody. The importance of VIP to brain cell proliferation was investigated with such a strategy in the E9 mouse. Administration of a VIP antagonist during a critical period of neurogenesis resulted in microcephaly, with a disproportionately greater inhibition of brain growth compared to that of the body (63). Specificity was demonstrated in that co-treatment with VIP prevented the antagonist-induced reduction in brain size, DNA and protein content. Together, these studies by Gressens strongly support the view that VIP or a VIP-like molecule has a critical role in regulating mitosis in the brain of the mid-gestational mouse, a period during which a large surge in VIP concentration occurs in the maternal plasma in the rat (74).
The presence of abundant VIP binding sites in the early embryonic CNS, long before the expression of VIP neurons in the brain, coupled with the evident role of VIP in the regulation of early postimplantation growth, indicated that the brain was not the source of VIP acting on these sites. Furthermore, sensitive rt-PCR methods did not detect VIP mRNA in any tissues of the rat embryo at E11 (74), the developmental time comparable to E9 in the mouse, suggesting that the embryo body was not a source of VIP. However, in the E11 rat embryo, an age when VIP mRNA is undetectable in rat tissues, VIP was more than four times higher than at E17, an age when VIP mRNA is abundant in many body tissues (74). These data strongly suggested that VIP from extra-embryonic sources acts on CNS receptors during early neurogenesis. An examination of the VIP content of maternal serum of rats throughout pregnancy revealed a peak in VIP at days E10 to E12 that was six to 10 times the concentration found during the final third of pregnancy (74). Since this peak in serum VIP levels of the mother coincided with the critical mid-gestational period during which VIP has been shown to regulate embryonic growth, it may reflect an upregulation of VIP in extra-embryonic tissues that acts as a source of VIP during this gestational stage. As further support for this hypothesis, VIP levels in the spinal cord of the pregnant rat have been found to increase during pregnancy, with peak values occurring between days 14 and 21. These data suggest that maternal VIP, similar to other maternal hormones and cytokines, may have a role in the coordination of prenatal development.
FUNCTIONAL NEUROANATOMY OF VIP IN THE BRAIN
In the adult brain, both VIP and VIP binding sites are widely distributed (4,7,71,73). VIP neurons have been identified by immunocytochemical methods and VIP mRNA by ISHH and Northern blot analysis. In vitro autoradiography has revealed both GTP-sensitive and GTP-insensitive binding sites throughout the brain. In addition, ISHH has identified cells containing the mRNA for two cloned receptors, VP1 (133) and VP2 (67). Since the cloned receptors are linked to adenylate cyclase activation, they probably represent the GTP-sensitive sites discussed below.
The following includes a discussion of several of the major anatomical sites in which VIP plays a part in the regulation of brain chemistry. Although brain tissues from mammalian species such as cat, mouse, hamster, macaque and human have been used to study the brain chemistry of VIP, the animal receiving most careful study is the rat. Consequently, most schematics, photomicrographs, and conclusions are based on the rat, with the caveat that the rat may not be representative of mammals in general.
The cerebral cortex has among the highest reported concentrations of VIP (86). VIP peptide (88,117) and its mRNA (4,61,71) have been localized to intrinsic neurons throughout all neocortical regions, as well as in the subiculum and olfactory cortices. Within the neocortex, the fusiform, bipolar VIP neurons are rather evenly distributed in layers II–V, with their long axes oriented perpendicular to the pial surface (Figure 11). VIP neurons comprise up to 1% of the neurons in some brain regions and exhibit minimal branching except in layers I and IV–V, where arborizations extend for 60–100 mm (88). Although the dendritic arborization diverges only minimally from the main axis of the cell, areas of arborization partially overlap, resulting in what Magistretti has described as adjacent cortical columns in which very localized input-output functions of VIP can occur that can "cover" the entire cerebral cortex (Figure 12). In addition, VIP immunoreactive fibers in the corpus callosum suggest transcallosal cortical projections. VIP exhibits over 85% co-localization with acetylcholine and over 30% co-localization with g-aminobutyric acid (GABA) in rat visual cortical neurons, suggesting complex interactions among these three neurotransmitters (109). Bipolar VIP neurons receive direct thalamic input and are also influenced by cortical pyramidal neurons (Figure 11). The cortical pathway of norepinephrine (NE) neurons originating in the locus coeruleus is also illustrated in Figure 12. This pathway enters the cortex rostrally and proceeds caudally, parallel to the pial surface and, as such, can modulate activity globally. Magistretti and coworkers have described the synergistic actions of VIP and NE, wherein potentiation of cAMP levels in the cortex (88) results in the activation of a cascade of phosphorylations leading to the promotion of glycogenolysis. The morphology and columnar distribution pattern of VIP neurons in the cortex make them ideally suited to regulate local energy needs within the cortex.
In vitro autoradiography revealed a pattern of binding which supports these hypotheses. VIP binding sites are found to be abundant throughout the cortex, with the highest binding occurring in layers I, II, IV and VI (Figure 9, Figure 11, Figure 12, Figure 13) , the layers exhibiting the greatest dendritic arborization of VIP neurons. Based on GTP-sensitivity, about 50% of the VIP binding sites in the cortex are linked to adenylate cyclase, and ISHH has revealed the mRNA for the VR1 receptor (77). The VR2 receptor was found in the olfactory cortices but not in the neocortex (67).
VIP neurons in the hippocampal formation are exclusively interneurons, sparsely scattered throughout both Ammon's horn and the dentate gyrus, sending their axons to nearby pyramidal or granule cells (Figure 14). These neurons are of diverse morphology and receive input from the GABAergic septohippocampal pathway and diagonal band (64). One-third to one-half of the VIP-containing neurons also contain GABA and/or cholecystokinin (132).
Both VIP1 and VIP2 receptors are located in the dense pyramidal and granule cells of the hippocampal formation. In vitro autoradiography has revealed VIP binding sites throughout the hippocampal formation, with very high binding in the molecular layer of the dentate gyrus. Within Ammon's horn, VIP binding is also most dense in the molecular layer (Figure 13, Figure 14). This pattern of distribution is consistent with the receptors being present on the dendritic arborizations of the pyramidal and granule cell neurons containing mRNA for VIP receptors. Within the hippocampal formation, binding is primarily of the higher affinity, GTP-insensitive type (Figure 13). Since the molecular layers are composed mostly of the dendritic arborizations of pyramidal and granule cells, the dense VIP binding sites in these regions, coupled with the close interaction of VIP-containing neurons with GABAergic systems, indicate a role for VIP in the regulation of electrical activity in the hippocampal formation and its effects on cognitive function.
In the thalamus, numerous neurons with abundant VIP mRNA are found throughout the ventromedial, ventrolateral, ventroposterior lateral, reticular, geniculate and gelatinosus thalamic nuclei (4,71) [Figure 7 and Figure 15]. A rapid turnover of VIP in these cells could account for the low levels of VIP reported with immunological methods (86). The cells containing VIP appear to be large projecting neurons (80) [Figure 15]. Many VIP neurons in the ventral thalamus co-localize with cholecystokinin (22), and those in the reticular nucleus co-localize with GABA. The thalamic regions expressing these cells integrate both sensory and motor signals relayed to the cortex, and these in turn are regulated by cortical and basal ganglion input (Figure 15). The density of VIP binding sites ranges from moderate to very high and shows much regional variation throughout the thalamus (Figure 13 and Figure 15), with the highest levels in the paraventricular, geniculate, ventrolateral and ventroposterior lateral, mediodorsal, lateral dorsal and reuniens nuclei. In most thalamic nuclei, VIP binding sites are primarily of the GTP-insensitive type (Figure 13) , suggesting that VIP acts at many non-cyclase linked receptors. However, the VR2 receptor, and to a much lesser extent the VIP1 receptor, have a significant presence in the thalamus, indicating an enrichment of cyclase-linked VIP binding sites.
The distribution of VIP and VIP binding sites within the thalamus indicates a role for VIP in the relay of sensory information to the cortex (ventrobasal complex and the geniculate bodies) and motor information from the basal ganglia and cerebellum (ventrolateral nuclei). In addition, the presence of dense VIP binding sites in the paraventricular thalamic and mediodorsal nuclei indicate the involvement of VIP in the relay of information from the hypothalamus, hippocampus and other regions to the cingulate and prefrontal cortices, regions involved in the regulation of complex behaviors.
Hypothalamus: Suprachiasmatic Nucleus
Within the hypothalamus, the SCN has the highest density of VIP neurons and the highest density of VIP binding sites (Figure 16). Within these densely clustered cells, VIP mRNA is abundant and appears to be expressed in almost all of the neurons in the ventral region of the nucleus. GABA is co-localized with VIP in these neurons, some of which also contain galanin, AVP and/or gastrin releasing peptide (161). This nucleus is the major pacemaker of the mammalian brain and, through its diverse afferent and efferent connections (Figure 16), coordinates both hormonal and circadian rhythms. The synchronization of the SCN to the dark-light cycle is regulated by two incoming visual pathways. The VIP neurons of the SCN appear to receive direct, primary, photic input from the retina and also relay information from the lateral geniculate nucleus of the thalamus (Figure 16). The VIP neurons are also influenced by serotonergic input from the raphe and GABAergic interneurons. Dense neurotensin receptors are also found on these VIP neurons (Figure 16). Cellular levels of VIP immunoreactivity and VIP mRNA vary over the day/night cycle, with the highest levels achieved during the night (48).
Many VIP-immunoreactive axons terminate on adjacent neurons (including VIP neurons) within the SCN and in the region immediately surrounding the SCN, the preoptic area and heavily to a "subparaventricular zone" extending posteriorly from the SCN to the paraventricular nucleus (Figure 16). This region contains more than three-quarters of all SCN efferents, projects to the same regions as the SCN, and is thought to constitute an amplification mechanism for the circadian signal. Other projection sites include the anterior hypothalamus, the parvocellular regions of the paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus, the hypothalamic LHRH system and the paraventricular thalamic nucleus (Figure 16).
The pattern of VIP binding sites is consistent with the description of VIP action in the SCN. VIP binding occurs with moderate to high densities in the nuclei receiving VIP projections, with a particularly high density of binding within the SCN itself and in the paraventricular nucleus of the thalamus (Figure 13 and Figure 16) . About 50% of the binding in the SCN is GTP-insensitive (Figure 13) , and this nucleus is also enriched in the VIP2 receptor, indicating that VIP in the SCN stimulates both cyclase and non-cyclase second messenger systems.
The exact role of VIP in the generation the pacemaker signal in the SCN is unclear. It has been hypothesized that SCN circadian rhythms are orchestrated by a cocktail of peptides, including VIP, PHI and gastrin releasing peptide (2). VIP may relay photic information to the pacemaker. An endogenous VIP rhythm in the SCN has been shown (58,127). This endogenous VIP rhythm may be important for the generation of various circadian rhythms. For example, chronic injections of a VIP antagonist to newborn rat pups result in a loss of circadian motor behavior (58). Recent work suggests that arginine vasopressin and VIP are both oscillating in the SCN, but are under the control of different circadian oscillators (128). The phase relationship between the two peptide rhythms is also different after treatment with antimitotic drugs or N-methyl-D-aspartate. These results suggest two distinct oscillators in the rat SCN.
The role of VIP in the human SCN has received exceptional attention in recent work by Zhou and colleagues (161). These workers have shown sex differences in the number of VIP neurons as a function of age. Females exhibited a very stable number of VIP neurons throughout life, while VIP neurons in the SCN of males varied significantly. Young males (10–40 years of age) had twice as many VIP neurons in the SCN than did females. However, this was followed in middle aged males by a dramatic decrease, such that females had significantly more VIP neurons than did males. After 65 years of age, the number of VIPergic neurons did not differ between sexes.
Hypothalamus: Hypothalamic-pituitary Axis
Some of the first physiological data published on VIP as a neuropeptide concerned a potential regulation of anterior pituitary release of prolactin. A dose-related increase in plasma prolactin levels occurred following intravenous or intracerebroventricular (ICV) administration of VIP. In addition, VIP was found in hypothalamic nerve endings, reported to be in high concentrations in the median eminence, and was ten times more concentrated in the pituitary portal circulation than in systemic circulation. Immunocytochemical studies in the rat have subsequently shown that parvocellular and magnocellular neurons in the paraventricular nucleus contain VIP neurons, as do cells of the supraoptic nucleus (26) [Figure 17]. However, in the rat and mouse, VIP usually can be visualized in these structures only after colchicine treatment or following adrenalectomy, hypophysectomy, or during lactation. In other species, such as mink and sheep, large numbers of magnocellular VIP-containing neurons have been reported in the paraventricular nucleus (PVN). Furthermore, in contrast to the scant VIP observed in the rat median eminence, a high concentration of VIP is found in the human median eminence (122).
VIP-containing fibers have been visualized in the external layer of the median eminence. Furthermore, numerous studies of stress conditions support a role for hypothalamic VIP in the regulation of anterior pituitary function, where it is thought to influence the release of prolactin, ACTH, growth hormone, and LH and to influence anterior pituitary GABA concentration (37). Thus, similar to other hypothalamic releasing factors, VIP may be released in the median eminence and, by transfer into the portal capillary circulation, exert effects on the release of anterior pituitary hormones. VIP immunoreactivity is also found in the internal zone of the median eminence, as well as in the posterior pituitary (91) (Figure 17) where it may influence the release of oxytocin and vasopressin and/or be released into the systemic circulation. An enrichment of VIP-containing axons innervating the portal vessels of the hypothalamic pituitary stalk indicates that VIP may also influence pituitary function through the regulation of vasodilation of these vessels; however, a peripheral origin of these neurons is assumed.
VIP binding sites are particularly dense in the supraoptic nucleus and are almost exclusively GTP-sensitive, which is consistent with the identification of VIP2 receptors in this nucleus (Figure 13 and Figure 17). The PVN has a lower density of VIP binding sites that are also primarily of the GTP-sensitive type, and the mRNA for VIP2 receptors has also been identified here. Further indications that hypothalamic VIP may influence anterior pituitary function are evident in the presence of binding sites for VIP in the median eminence and anterior pituitary (Figure 13 and Figure 17). In the anterior pituitary, however, high concentrations of VIP displaced only 25% of radiolabeled VIP, suggesting another receptor type.
Bed Nucleus of the Stria Terminalis and Amygdala
The bed nucleus of the stria terminalis (BNST) and the amygdala (Figure 18) contain among the highest concentrations of VIP in the brain (86). The BNST contains few VIP neurons and a moderately high density of GTP-sensitive binding sites; however, it has dense VIP-immunoreactive fibers. Through the stria terminalis and other pathways, this nucleus communicates with widespread brain regions, including the hypothalamus, amygdala and dorsal raphe.
VIP neurons are found throughout the amygdala, with the possible exception of the central nucleus. VIP binding sites occur throughout the amygdaloid nuclei, with the highest densities found in the basolateral and medial amygdala, most of which are GTP-insensitive (Figure 13). Immunocytochemical techniques reveal particularly dense fibers within the central nucleus of the amygdala; however, autoradiography demonstrates few VIP binding sites within this nucleus. The enrichment of VIP fibers in the central amygdala may be related to the dense VIP fibers in the stria terminalis, the major bi-directional pathway linking the amygdala with the BNST, medial hypothalamus and pre-optic area. This suggests that VIP plays a role in the in processing of autonomic, endocrine and somatomotor information as it relates to motivation and emotional processing.
Both immunocytochemistry (86) and in situ hybridization techniques (71) have identified large VIP neurons in the periaqueductal gray and throughout the region of the dorsal raphe, a region of moderate VIP binding (Figure 18). The large size of these neurons, coupled with the presence of VIP-immunoreactive fibers in the medial forebrain bundle, suggest that these neurons contribute to this important bi-directional pathway connecting the brainstem to the BNST, ventral forebrain, amygdala, hypothalamus and perhaps the cortex. Although VIP immunoreactivity has not been reported in the cerebellum, the VIP raphe neurons may travel with the well-known aminergic raphe system and act on the dense, GTP-sensitive VIP binding sites of the cerebellum.
The superficial layer of the superior colliculus (stratum griseum superficiale) is enriched with VIP neurons (71,86) and contains dense VIP binding sites (Figure 18) which are almost exclusively of the GTP-insensitive type (Figure 13). VIP immunoreactivity disappeared from the superior colliculus following eye enucleation (101). In addition, some VIP neurons project to the lateral geniculate nucleus of the thalamus (99), a brain region having a very high density of VIP binding sites (73). These data indicate that VIP may be involved in the motor coordination of visual reflexes.
In the spinal cord, VIP has been localized to primary afferent neurons in the dorsal root ganglia projecting to the dorsal horn (co-localized with galanin ), sympathetic neurons (5), and neurons in the dorsal horn, lamina X and the intermediolateral nucleus. VIP binding sites have been found throughout spinal cord (158) in a rostro-caudal gradient, with the highest binding occurring in the lumbar and sacral regions. Within the cord, the highest density of binding is found in laminae I and II, around the central canal/lamina X. Very high densities are also found in the parasympathetic lateral horn of the sacral region. These data implicate VIP in sensory transmission and the regulation of autonomic functions.
Progress in discerning the biological function of many neurochemicals has had its historical foundation in the development of drugs which can either block or stimulate specific receptors that mediate the substance's action. These drugs have also formed the basis for therapeutic intervention for the treatment of human disease. With regard to known VIP antagonists, all have a portion of VIP or a peptide family member as a part of their structure. The following have been identified as VIP antagonists: 1) [4-Cl-D-Phe6,Leu17]-VIP, 2) [Ac-Tyr1,D-Phe2]-Growth Hormone Releasing Factor 1-29 amide, 3) VIP(10-28), 4) Neurotensin6-11-VIP7-28 (hybrid peptide antagonist), and 5) Stearyl, Norleucine17-hybrid antagonist (SNH). The first three have been widely used, and their actions have been reviewed recently (56). Transfection of the cloned VP1 and VP2 receptor cDNAs into cell lines, coupled with cAMP measurements, have shown that [4-Cl-D-Phe6,Leu17]-VIP and [Ac-Tyr1,D-Phe2]-Growth Hormone Releasing Factor 1-29 amide both are effective, whereas the VIP(10-28) and the hybrid peptide antagonist are not (144). As previously noted, not all actions of VIP in the brain are mediated through cAMP. These data suggest both tissue specificity and the existence of additional receptors for VIP.
The hybrid neurotensin/VIP peptide antagonists appear to exhibit specificity for VIP receptors in the brain, and they have been used to study the effects of VIP during development and behavior. The rationale for the formulation of the VIP hybrid antagonist was that the chemical nature of the neurotensin N-terminal portion may augment membrane permeability of the VIP portion of the hybrid molecule (56). Despite the presence of a neurotensin sequence, the VIP hybrid antagonist apparently does not interact with neurotensin receptors in the CNS. This compound also proved to be a useful adjunct to confirm the neurotrophic action of VIP in CNS cultures. Treatment of developing spinal cord cultures with the hybrid antagonist produced a 35–50% reduction in the number of surviving neurons. The antagonist-induced cell death was competitively prevented by co-treating cultures with VIP (59). Similar findings were also observed with a C-terminal fragment, VIP(10-28) (13). The specificity of the hybrid antagonist on central nervous system-associated receptor functions has been examined: 1) the hybrid antagonist did not inhibit VIP-mediated effects on lymphoid cell proliferation; 2) the antagonist did not inhibit VIP-stimulated amylase secretion from pancreatic acini, exhibiting minimal agonistic activity (59); 3) prenatal administration of the hybrid early in development (E9–E11) produced severe microcephaly that was prevented by co-treatment with VIP, while producing smaller decreases in body growth (63); and 4) co-treatment with PACAP did not block the hybrid-induced microcephaly.
Two VIP agonists have been developed that show therapeutic promise: a lipophilic VIP analogue (stearyl, norleucine17-VIP [SNV])  that has neuroprotective properties and Ro25-1553, a substance developed as a bronchodilator for the treatment of asthma (10). The Ro 25-1553 is a cyclic compound that was designed to enhance the stability of VIP to enzymatic degradation involving Ser25 and Ile26.
Ro 25-1553 combines amino acid substitutions to the VIP structure and conformational restrictions imposed by the cyclization of amino acids at a key cleavage site for VIP. Ro 25-1533 has a 385-fold greater potency than native VIP in relaxing human airway smooth muscle. The activity of this compound in the brain is unknown. Focusing on the compound that has demonstrated activity in the brain, stearyl-Nle17-VIP (SNV) exhibited a 100-fold greater potency than VIP, with maximal neuroprotective action observed at 1 pM. SNV was effective over a broader concentration range than VIP. Studies with SNV provided the most definitive evidence that VIP neurotrophism was operating through a mechanism that did not involve cAMP as a second messenger. As SNV incorporated two structural modifications in the VIP molecule, each of the two compounds containing the individual changes (stearyl-VIP and Nle-VIP) was tested separately. Each of the analogues showed a 10-fold greater potency than VIP in protecting neurons from death. When tested for cAMP formation in cortical astrocytes, both SNV and stearyl-VIP were essentially inactive, whereas Nle-VIP increased cAMP levels at 10-7 M. SNV not only produced a dramatic increase in the potency to promote neuronal survival, but also an unexpected increase in both its specificity (excluding adenylate cyclase activation) and biological effectiveness (activity over a much broader range of concentration than VIP) .
CELLULAR EFFECTS ON BRAIN CELLS
Electrophysiology of VIP
VIP is a neurotransmitter and neuromodulator in the central nervous system. The effects of VIP on electrical activity are highly divergent, both excitatory and inhibitory responses have been observed in neurons from the same brain area. Some of this diversity can be attributed to the modulatory effect of VIP on other neuroactive substances, the net effect being contingent on the co-neurotransmission. In early studies on the spinal cord, VIP depolarized dorsal root terminals and motoneurons (110). Iontophoretic application of VIP to CA1 neurons in rat hippocampal slices produced rapid depolarization and a large increase in membrane conductance (36). Surveying the preoptic, septal and midbrain central gray neurons, VIP produced a mixture of responses, some excitatory and others inhibitory (68). The frequency and duration of calcium-dependent action potentials were increased by VIP treatment of a pituitary cell line. Several voltage-sensitive conductances were involved in this response, and they may mediate secretory events in these cells (70). In the cortex, VIP responses have been investigated in situ in the anesthetized rat (126). VIP was found to elicit a wide range of effects on background firing rates, including increases, decreases and biphasic responses. Application of VIP and norepinephrine together produced predominantly (70%) inhibitory responses to firing rates. Both the inhibitory effects of GABA and the excitatory responses to acetylcholine were enhanced by VIP, supporting the peptide's role as a neuromodulator. In the locus coeruleus, VIP and other cAMP-inducing agents increased the firing rate of noradrenergic neurons by inducing an inward current (149). The inward current produced by VIP was non-additive to that produced by other cAMP-active substances. In addition, intracellular application of an inhibitor to cAMP-dependent protein kinase attenuated the response to VIP. In contrast, VIP-mediated depolarization and membrane resistance in the retinal horizontal neurons were not elicited by analogs of cAMP or forskolin (84). These studies again suggest that regional and cell-specific mechanisms mediate the electrophysiological responses to VIP, only some of which involve cAMP-linked phosphorylations.
Glial Function and VIP
VIP can regulate and stimulate glial function. Since VIP is confined to neurons and has not been detected in glia, neuroglial communication via VIP is unidirectional. However, VIP-stimulated glia communicate with neurons and other cells through secreted substances (Figure 19). Astrocytes have at least two classes of binding sites for VIP (32,59). The lower affinity VIP receptor is linked to adenylate cyclase (113), while the high affinity site has been correlated with calcium mobilization and phosphatidyl inositol turnover (42).
Glial functions influenced by VIP can be divided into four categories: electrophysiology, metabolism, gene regulation and secretion. High concentrations of VIP produced hyperpolarization in astroglia (76) and Schwann cells (41). This effect on Schwann cells was blocked by a VIP antagonist. The hyperpolarization elicited by VIP is thought to be mediated by cAMP and is insensitive to changes in the concentration of extracellular calcium. However, the responses are potentiated by lithium ions, suggesting the involvement of polyphosphoinositols in the membrane (41).
Secretion is an important function of astroglia that is only beginning to be explored. Although the mechanism by which VIP and other secretagogues mediate secretory processes in these cells is still not understood, it is clear that the released substances include cytokines, growth factors, protease inhibitors, and extracellular matrix proteins (Figure 19). Several groups have shown that interleukin-6 (IL-6) is released by VIP (51,124). IL-6 is a pleiotropic cytokine that may act as a trophic factor or influence immune responses in the brain. This effect of VIP is thought to be mediated through cAMP (124). Interleukin-1 alpha and interleukin-1 beta are also released from astroglia, but at picomolar VIP concentrations far lower than those needed to stimulate adenylate cyclase (16). These contrasting findings suggest that multiple mechanisms exist for cytokine secretion from astroglia. IL-1 alpha increased the survival of neurons in electrically blocked spinal cord cultures. In addition, interferons alpha and beta are released by VIP. This effect has implications for the brain's defense mechanism against viruses (28).
VIP also increased the release of protease nexin-1 (PN-1) from astroglia (43). This is particularly significant in that PN-1, a serine protease inhibitor, has marked effects on neurite extension and neuronal survival. The presumed mechanism of action of PN-1 involves inhibition of thrombin-like protease activity in the CNS. Indeed, thrombin has been shown to decrease neuronal survival in developing spinal cord cultures (43). The role of thrombin in neuronal plasticity and neuronal survival is an active area of research in both developmental neurobiology and studies of neuronal injury. In Schwann cells, VIP can upregulate the expression of PN-1 through a mechanism that may involve regulation of angiotensin II receptors (8).
Growth factors comprise a third class of molecules released or regulated by VIP in astroglia. High concentrations of VIP can downregulate the expression of ciliary neurotrophic factor in cultured rat brain astrocytes (95). This effect is mimicked by cAMP-linked receptor agonists. In contrast, sub-nanomolar concentrations of VIP release a novel growth factor, recently isolated from the conditioned medium of VIP-stimulated astrocyte cultures: activity-dependent neurotrophic factor (ADNF) . Whereas all other molecules shown thus far to be released by VIP were previously identified substances, ADNF is novel, not only in structure but also as a representative of a new concept in neuroprotection: extracellular stress proteins. ADNF is a 14 kD protein that has a sequence similarity to heat shock protein 60, an intracellular molecule recognized as important in protein folding and cellular protection against thermal stress and toxic substances. The pharmacology of ADNF is highly unusual in its extraordinary potency, exhibiting an EC50 at or below femtomolar (10-15 M) concentrations.
One of the first processes found to be regulated by VIP is energy metabolism in astrocytes (89). Both norepinephrine and VIP help control local energy homeostasis through stimulation of glycogenolysis, which occurs within minutes of treatment. This effect of VIP has an EC50 in the nanomolar range. Following glycogenolysis, VIP stimulates the resynthesis of glycogen, which can reach 5–10-fold higher levels than before treatment with the peptide (89). Glycogen resynthesis is a transcription-dependent process regulated by cAMP. Recent studies indicated that this effect of VIP is mediated through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. Both C/EBP beta and C/EBG delta behave as cAMP-inducible immediate-early genes in astrocytes (23). VIP treatment of astrocytes also produced changes in their morphology from flat to predominantly process-bearing cells. Recent reports also indicated that VIP is a regulator of extracellular adenosine levels in cortical cultures. This effect may help determine the inhibitory tone of the cerebral cortex (31). In summary, VIP is a key neuroglia messenger that is of fundamental importance to homeostasis, development and repair.
VIP BRAIN FUNCTIONS
Perhaps no other area has received greater attention for VIP than its effect on neuroendocrine function, particularly in regard to its secretagogue action on prolactin and growth hormone from the anterior pituitary. VIP released from the hypothalamus into the portal blood reaches the pituitary and regulates hormone secretion. The progression of research in this important area of VIP action is summarized in several reviews (119,120,154).
In the mammalian pituitary, simultaneous inhibition of pituitary VIP mRNA expression and VIP release may be a necessary mechanism for the dopaminergic inhibition of prolactin mRNA expression and prolactin release (6). Another inhibitor of prolactin release in the mammal is dexamethasone, acting through lipocortin (a protein shown to mediate the anti-inflammatory and anti-proliferative actions of glucocorticoids) to reduce the VIP-induced prolactin increases (139). In 1992, Lam et al. (83) demonstrated divergent effects of glucocorticoids on VIP gene expression in cerebral cortex, hypothalamus and pituitary. Four weeks following adrenalectomy in five-week-old rats, VIP mRNA content increased in the anterior pituitary but did not change in the cerebral cortex or hypothalamus. Dexamethasone treatment for ten days abolished the adrenalectomy effect in the pituitary and caused a decrease in VIP mRNA in pituitaries of control rats. In the cerebral cortex, dexamethasone induced an increase in VIP mRNA in adrenalectomized and also in control rats; in contrast, hypothalamic mRNA remained unchanged. Parallel changes have been observed in VIP. Serum prolactin paralleled pituitary VIP. Other studies have shown that adrenalectomy of adult rats (three months old) resulted in about a two-fold decrease in VIP mRNA in the SCN. Corticosterone or a glucocorticoid agonist (RU 28362) did not affect the decrease in VIP mRNA (52). Thus, the glucocorticoid effects are age-dependent and time-dependent, suggesting complex regulatory mechanisms (see also section on VIP receptor).
Recent studies have shown that pretreatment with a VIP antagonist significantly reduces the food-induced increases in ACTH and corticosterone. These results suggest that one potential role of hypothalamic VIP involves activation of hypothalamic releasing factors to regulate ACTH and corticosterone levels during or after a meal (3). Furthermore, during cold stress, blood levels of ACTH, aldosterone and corticosterone increased in the rat, and administration of a VIP antagonist [Ac,Tyr1,D-Phe2-GRF(1-29) amide] caused a marked depression in the response of the HPA axis to the cold stress. Thus, VIP may play a pivotal role in the mechanism associated with response to cold stresses (97). Both VIP and PHM act as ACTH releasing factors, although PHM is less potent (153). However, pretreatment with the VIP antagonist [Lys1, Pro2,5, Arg3,4, Tyr6]-VIP totally suppressed VIP-induced ACTH secretion but had no effect on PH-induced release, suggesting two different receptors (3). Furthermore, the dose-response curve to PHI-mediated release for ACTH and corticosterone secretion was bell-shaped, similar to the response observed with VIP in neuronal survival assays (see Figure 20). As for VIP, the maximal reduction in corticosterone secretion was observed with the lowest dose of the VIP antagonist used, increasing doses of the antagonist were less and less effective, also suggesting bell-shaped dose dependency. The bell- shaped response may involve a mechanism of receptor desensitization (e.g., 156).
Most hypnogenic agents are either growth factors or influence growth factor production (118). The growth factors are humoral agents that interact with neural circuits to regulate sleep. This association between growth factors and sleep is believed to be involved with synaptic plasticity, a functional reorganization of neuronal connections that is considered an important function of sleep. Since VIP has both neurotrophic and hypnogenic properties, the peptide fits this generalized association between sleep regulators and growth factors. After central administration, VIP enhances rapid eye movement (REM) sleep, also called paradoxical sleep (PS). Recent evidence suggests that one of the targets for this action is the nucleus dorsal raphe (nDR), a structure recognized to have several permissive components that allow sleep to occur and which has extensive VIP innervation (38). Administration of VIP to the nDR increases the duration of PS and slow-wave sleep, although these two types of sleep are increased at differing concentrations of VIP. Insomnia produced by treatment of rats with a serotonergic synthesis inhibitor can be prevented by ICV administration of VIP (39). Other studies have suggested that VIP accumulates in the cerebrospinal fluid during waking or sleep deprivation and provides a signal for the production of PS (78). As with many other functions of VIP, the effects on sleep are indirect, in this case involving prolactin. VIP is a known secretagogue for pituitary prolactin, acting at the level of both the hypothalamus and the pituitary gland. VIP-mediated increases in PS sleep can be prevented by ICV co-administration of antiserum to prolactin (98). Together, these studies suggest that VIP plays a role in the regulation of paradoxical sleep that is mediated, in part, through the action of prolactin, although the mechanism has not yet been established.
With the exception of sleep regulation and circadian rhythm-controlled behaviors, the influence of VIP on other types of behavior has received little experimental attention. Earlier studies suggest that VIP may inhibit stress-motivated behaviors. ICV administration of nanogram amounts of VIP decreased rearing behavior, inhibited a step-through passive avoidance task, and increased extinction of pole-jumping active avoidance behavior (33). An intriguing and controversial effect of VIP is its potential role in learning and memory. Central administration of VIP can produce an impairment in previously learned behaviors. VIP injected directly into the rostral portion of the hippocampus produced an "amnesia" in rats trained on a foot shock avoidance task T-maze (45). The timing of treatment relative to training is important, in that VIP given 24 hours after training failed to impair behavioral retention measured a week later. Animals injected with a VIP receptor antagonist (4-Cl-D-Phe6,Leu17VIP) into the rostral hippocampus showed enhanced retention in the T-maze paradigm. Decreased performance in the Morris water maze (a test of spatial memory) was observed after chronic infusion of VIP into the cerebral ventricle (136). In the same study, VIP apparently inhibited the acquisition of new learning in the swim maze. More recently, several studies indicated that VIP was important to successful memory acquisition. Chronic administration of the hybrid VIP antagonist to mature rats produced a significant delay in the learning response in the Morris swim maze (49). Transgenic mice designed to overexpress VIP by harboring a chimeric VIP gene driven by a polyoma promoter showed a marked impairment of performance in the swim maze (57). Paradoxically, the VIP content of the brain was reduced by 20% in these transgenic mice. These studies strongly suggest important compensatory mechanisms that were activated by the overproduction of VIP in the brains of transgenic animals. In addition, transgenic mice carrying a diphtheria toxin-encoding sequence driven by the rat VIP promoter (which decreases VIP expression) also exhibited increased latencies in performance in the swim maze test. These studies support the idea that VIP is important to the successful acquisition of memory, and that the regulation of VIP content in the brain is still enigmatic. In other studies, spatial cognitive deficits produced by scopolamine in a radial arm maze paradigm were prevented by ICV administration of VIP. Importantly, the inhibition produced by VIP was associated with a bell-shaped dose response curve, strongly indicating the importance and necessity of assessing VIP-related behaviors with extensive concentration-effect curves (157). All of the studies indicate that VIP can influence memory and learning, although the behavioral consequences apparently depend upon the amount, location and duration of the VIP/VIP antagonist treatment.
A unifying concept in neurobiology is that many molecules that are important in the regulation of neuronal development are also upregulated in neural injury and are critical for nerve repair. This relationship is most often acknowledged with the neurotrophin family of growth factors, where it is clear that these substances have fundamental roles in organizing the developing nervous system. Neurotrophins are increased after neural injury and have neuroprotective properties. However, this concept is certainly not confined to the neurotrophins. Rather, it can be extended to many cytokines and neuropeptides. VIP also exhibits this important neurotrophic dualism between development and repair. A wide array of events fundamental to brain development (including cell proliferation, differentiation, neurite outgrowth, and neuronal survival) can be regulated by VIP (55). Furthermore, VIP is upregulated after neural injury and has potent neuroprotective properties. Although many of these developmental effects were first demonstrated in cell cultures, more recent studies have extended the in vitro observations to investigations of embryonic growth, behavioral development and maintenance of neuronal integrity. Many of the neurotrophic and neuroprotective properties of VIP have been attributed to the secretagogue action of the peptide. In addition, VIP appears to be an integrative regulator that can orchestrate the growth of an entire embryo during a restricted period of development, again through indirect mechanisms.
The first description of a neurotrophic action of VIP was on neuronal survival in cultures derived from the embryonic spinal cord (13). As shown in Figure 20, sub-nanomolar amounts of VIP increased the survival of spinal cord neurons treated with tetrodotoxin. Both in this case and in others (20,21), the neurotrophic properties were apparent only when the neurons were exposed to a neurotoxic substance or if the action of endogenous VIP was interfered with by a neutralizing antibody or a receptor antagonist (13,59). The neuroprotective action of VIP was contingent on the presence of astroglia (18). Stimulation of high-affinity VIP receptors on astroglia (59) resulted in the release of many neurotrophic substances, including cytokines (16), protease nexin I (43) and activity-dependent neurotrophic factor (15). Neurotrophic effects of VIP have been observed in spinal cord, hippocampal and cerebral cortical cultures. VIP exhibits potent survival-promoting activity with an EC50 of 30 pM, a concentration unusual, in that most other actions of VIP have been observed at much higher amounts (nanomolar to micromolar). The neurotrophic action of VIP appears to be correlated with the mobilization of calcium and the translocation of specific isozymes of protein kinase C. These effects contrast with those observed in the developing sympathetic nervous system, where cAMP increases were associated with trophic properties (see below). VIP promoted the survival of some neurons from the ciliary and dorsal root ganglia, an action that is again contingent on the presence of non-neuronal cells (143).
More recently, VIP has been shown to prevent apoptosis (programmed cell death) in cerebellar granule cell cultures grown under conditions (serum-free medium or low potassium medium) that did not support their survival (24). However, the amounts required to produce this effect were very high, compared with those required in cultures derived from spinal cord or hippocampus. Indeed, another VIP-like peptide, PACAP-38 (pituitary adenylate cyclase activating peptide) increased neuronal survival in cerebellar cultures at much greater potency, suggesting that this peptide may be the preferred ligand for this effect. Similarly, studies of sympathetic neuroblasts also have shown that VIP increased the proliferation and survival at high concentrations, whereas PACAP-38 was much more potent (111). In contrast to the survival effects of VIP that are contingent on glia, the effects of VIP on neuroblasts are apparently mediated by cAMP, with direct actions of the peptide eliciting the survival effects (112). In related studies, neuronal cell death produced by deprivation of nerve growth factor was prevented by high (micromolar) concentrations of VIP (138).
VIP is a mitogen and a mitogenic inhibitor. Such a dichotomy of responses immediately indicates cell-specific responses that predict dramatically different physiological functions and pathological consequences. VIP in many cases acts as a differentiation factor that inhibits cellular proliferation (94,150), while in other systems VIP can stimulate mitosis at critical times during neuronal development. As is the case with many cytokines and neuropeptides, both autocrine and paracrine effects are involved in the proliferative actions of VIP. Many of the early studies indicated that VIP either inhibited mitosis in mesenteric lymphocytes and splenocytes or inhibited the proliferative actions of other mitogens (19). With regard to brain cells, VIP was an astroglial mitogen at sub-nanomolar concentrations (19). For neurons, the proliferation of sympathetic neuroblasts was increased by VIP, an effect that may be mediated by cAMP (112).
VIP can stimulate neurite outgrowth. As in the case of neuronal survival, the mechanism through which VIP mediates these events may differ among neuronal targets. In sympathetic neuroblasts, high concentrations of VIP increased neurite sprouting (111). VIP stimulated neurite outgrowth in numerous model systems, including several human neuroblastoma cell line and PC-12 (pheochromocytoma) cells (103). In the PC-12 model, 50 nM VIP induced neurite outgrowth within one hour. This effect was associated with phosphorylation of MAP kinases. In contrast, many neuroendocrine culture systems are induced to differentiate by high concentrations of VIP (150). Many of these effects are believed to be associated with cAMP-mediated phosphorylations. However, that is not always the case. VIP releases a substance that can stimulate neurite outgrowth (protease nexin I) from glial cells (43). Similarly, VIP releases several cytokines, including interleukin-1 alpha, interleukin-1 beta (16) and interleukin-6 (51). These cytokines may mediate the differentiation effects of VIP. With regard to potential physiological significance, very low concentrations (1–100 pM) of VIP can produce the release of cytokines (16). At these concentrations, no VIP-mediated increases in cAMP can be detected.
The importance of VIP to postnatal brain development was suggested by studies which employed a VIP antagonist to assess the morphological and behavioral effects of blockade of VIP. Daily administration of the VIP hybrid antagonist to newborn rat pups resulted in both cortical neurodystrophy and delays in the onset of developmental milestones and behaviors. Golgi analysis of layer V cortical pyramidal neurons demonstrated a significant reduction in dendritic branch length and in the number of branches from the basilar tree after treatment with the VIP hybrid antagonist. In addition, many of the dendritic branches from the treated neurons were thickened, coarse and vacuolated. Spines frequently appeared fused and malformed (75). The morphological changes produced by treatment with the VIP hybrid antagonist strongly indicated that VIP has an important role in the formation and maintenance of dendritic arborizations during postnatal development. Additional support for this hypothesis was obtained by assessing the effect of the drug on the behavioral development of animals. In neonates treated with the hybrid VIP antagonist, delays in motor development were observed, and placing and righting behaviors were retarded (72).
Increased VIP immunoreactivity has been observed in several model systems of neuronal injury (150). It may be that the increase is a protective response of the neuron that may utilize the neurotrophic properties of VIP for repair purposes. This response has been studied extensively after peripheral nerve crush involving sensory and sympathetic neurons. In neurons of lumbar dorsal root ganglia following sciatic nerve transection, an increase in VIP synthesis occurs that can be inhibited by nerve growth factor (145). The increased expression of VIP seen in sensory neurons following peripheral nerve injury in vivo may result from deprivation of target-derived nerve growth factor, in combination with increased ciliary neurotrophic factor or leukemia inhibitory factor (LIF) released from the injured nerve (93). Within an hour of injury, LIF mRNA increased in both non-neuronal cells and in the nerve (134). The nature of the signaling process that induced the LIF is not yet known, but it appears to be a soluble factor. Recent studies have shown that LIF activates Janus kinases and members of the STAT (signal transducers and activators of transcription) family of transcription factors (114) during injury. VIP is one of the neuropeptides that is up-regulated by this pathway. The fundamental question is why the cell expresses VIP, galanin and other molecules after injury. Certainly the neurotrophic properties of VIP observed in the CNS may play some role in the repair process. In this regard, PN-1, a trophic molecule that increases neuronal survival and neurite outgrowth in some systems, was induced after lesions of the sciatic nerve. VIP is among the substances that have been shown to increase PN-1 expression in Schwann cells, the support glia of the peripheral nervous system (8). These studies strongly suggest that VIP is among the neuronal signals that mediate important cellular interactions with glia during the nerve repair process. VIP also increased the release of laminin, an important cellular matrix protein, from Schwann cells (160). Thus, as observed in the CNS, the injured PNS may utilize similar mechanisms to regulate cellular viability through VIP and related molecules.
VIP AND CLINICAL IMPLICATIONS
The clinical relevancy of any substance resides in two often related concepts: either involvement in the etiology of a pathological process or efficacy as a therapeutic agent to intervene in the progression of human disease. The action of VIP has potential in both of these avenues of medicine. With regard to brain disease, the clinical relevance of VIP resides in the following: 1) autocrine growth regulations of tumors; 2) control of cerebral blood flow; 3) involvement in learning and memory; and 4) neuroprotective/neurotrophic actions. Examples will be given for each of these as they relate to neuroblastoma, headache, Alzheimer's disease and acquired immune deficiency syndrome (AIDS).
In neuroblastoma, the most common solid malignancy in young (<5 years) children, VIP has the dual effect of either inducing differentiation or stimulating cell division, depending on the cell line and the time of application. In one tumor cell line (NMB), VIP produced dose-dependent stimulation of mitosis. Similarly, the hybrid VIP antagonist neurotensin6-11VIP6-28 inhibited neuroblastoma growth as a function of concentration (85). These studies suggest a possible therapeutic role of VIP analogues for cancer treatment.
VIP is found in cerebral arteries and has long been recognized as having powerful vasodilatory actions. Because of these actions and its anatomical localization in structures involved in pain pathways, VIP has been implicated in some kinds of headaches. In particular, studies of the etiology of cluster and migraine headaches have been investigated. During cluster headache attacks, a rare and severe disorder, monitoring of peptide levels in the external jugular revealed a three-fold increases in VIP and calcitonin gene-related peptide (CGRP) . These headaches are believed to involve the activation of the trigeminovascular system and the cranial parasympathetic nervous system. Activation of these systems leads to neurogenic inflammation within the dura mater and cerebral vasodilation, processes believed to be involved in the pathogenesis of migraine headaches. Elevated levels of peptides such as substance P, CGRP and VIP have been examined for their correlative status in these attacks. Comparisons of peptide levels in the saliva of patients suffering from migraine or cluster headache indicated that VIP levels were elevated with the cluster type but not with migraines (96). Pharmacological studies have demonstrated a relationship between effective therapeutics and VIP levels. Both sumatriptan and oxygen therapy can abort cluster headache attacks. In an isolated porcine ophthalmic artery preparation, sumatriptan inhibited VIP-induced vascular relaxation, whereas cGRP effects were not influenced (147). Similar correlations of VIP-mediated effects in this system have been observed with cyclooxygenase inhibitors (146). Although not yet established, VIP appears to have some role in the pathogenesis of cluster headaches; however, its role in migraine headaches has little support.
Unmodified VIP is a poor therapeutic agent for the treatment of brain disease because the peptide cannot cross the blood brain barrier and its half-life after systemic administration is only minutes. An important addition to VIP pharmacology, as it relates to neurotrophism, has evolved with the discovery of a lipophilic VIP agonist: stearyl-Norleucine17-VIP (SNV). Details of its design strategy are given in the pharmacology section. The mechanism by which SNV exerts its neuroprotective effects is yet to be elucidated. However, the vastly increased potency of SNV, coupled with both increased receptor specificity and its ability to enter the brain via inhalation, prompted further investigations to test for efficacy in models relevant to Alzheimer's disease (54). Beta amyloid peptide (an Alzheimer's disease neurotoxin) treatment of cultures from rat cerebral cortex produced a 70% loss in the number of neurons. This cell death was completely prevented by co-treatment with 0.1 pM SNV. These studies were extended to in vivo models of Alzheimer's disease in rats, where cholinergic deficits were induced with a cholinergic blocker (ethylcholine aziridium) . This model was chosen because a major loss in Alzheimer's disease is in cholinergic functions, a loss implicated in cognitive impairments characterizing senile patients. SNV, injected ICV or delivered intranasally, prevented impairments in spatial learning and memory associated with cholinergic blockade.
VIP also has implications for our understanding of the neurological and neuropsychiatric deficits associated with AIDS (17). Previous studies indicated that neuronal cell death, developmental delays, cortical neurodystrophy and learning impairments can be produced in rodents or CNS cultures derived from rodents treated with the envelope protein (gp120) from the human immunodeficiency virus (HIV), the recognized agent for AIDS. Importantly, VIP and VIP-like agents were shown to provide protection from the neurotoxic effects of the viral envelope protein from HIV (21). The molecular mechanism for the cytoprotective effect of VIP has not yet been resolved. However, two hypotheses, perhaps not mutually exclusive, have been considered: 1) a neurotrophic hypothesis in which a growth factor released by VIP prevents or attenuates the deleterious actions of gp120; or 2) a ligand competition hypothesis based on the sequence homology shared between VIP and the peptide T region of gp120 (17). In regard to the neurotrophic hypothesis, VIP can act as an astroglia secretagogue for substances that increase the survival of neurons (19). Among the trophic substances released by VIP is activity dependent neurotrophic factor, a novel protein that can also prevent neuronal cell killing in gp120-treated cerebral cortical cultures (15). These data suggest a pathway where gp120 toxicity is blocked by the release of ADNF that has a cytoprotective action. Alternatively, the limited sequence homology between VIP and gp120 suggests that these substances act through a common receptor. The core of this hypothesis relies on the observation that peptide T sequences were identified in all variants of gp120, and that these sequences have some homology to VIP (17). The protective action of these peptides may be attributed to their ability to block the attachment of gp120 or a neurotoxic fragment of gp120. Although these studies do not definitively address the details of how VIP protects cells from the neurotoxicity of HIV-related proteins, they do provide a novel and potentially useful strategy for the treatment of this and other diseases processes that can be prevented or attenuated by the neuroprotective properties of VIP analogs or neurotrophic substances released by VIP. As such, the neurotrophic properties related to VIP provide the clear hope for treatment of human brain disease.
VIP is a multi-functional neuropeptide that is widely distributed in the central nervous system. In addition to its role as a neurotransmitter, neuromodulator, and neuroglial messenger, VIP is an integrative growth factor capable of orchestrating embryonic growth during a critical period of brain development. VIP has pivotal roles in the regulation of sleep, circadian rhythm and neuroendocrine control of the hypothalamic-pituitary-adrenal axis. With the emergence of cloned receptors and specific agonists/antagonists for VIP, the actions of this peptide in brain physiology and pathology should gain increasing recognition. VIP is truly a Very Important Peptide.