I. Introduction

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Neurotensin (NT²) was first isolated in 1973 from bovine hypothalamus by Carraway and Leeman. In 1988, the rat NT gene was isolated and sequenced (Kislauskis et al., 1988) and found to consist of a 10.2-kilobase segment containing 4 exons and 3 introns. The gene encodes a 170-amino acid precursor protein containing both the tridecapeptide NT and a closely related hexapeptide, neuromedin N (NN). The four amino acids at the carboxy terminal of NT and NN are identical, and amino acids 8-13 of NT are essential for biologic activity (Lambert et al., 1995). The NT/neuromedin N (NT/NN) gene is highly conserved between species (Dobner et al., 1987; Bean et al., 1992; Evers et al., 1995).

Elements involved in the regulation of NT/NN mRNA expression are located in the upstream 200-bp flanking region of the rat gene. In this region, several cis-regulatory elements function cooperatively to integrate multiple environmental stimuli into a concerted transcriptional response (Kislauskis and Dobner, 1990). In the rat NT/NN gene, these sites include one consensus AP-1 site, two near consensus cyclic AMP response elements, one near consensus glucocorticoid response element, and a sequence identical to the human c-jun gene autoregulatory element. Notably, the glucocorticoid response element is absent in the regulatory sequence of the human NT/NN gene (Vita et al., 1993).

In neurons, NT is stored in dense core vesicles and released in a Ca²⁺-dependent manner (Bissette and Nemeroff, 1995). NT transmission is terminated primarily by cleavage of NT by several peptidases, including neutral endopeptidase 24.11 (Almenoff et al., 1981), angiotensin-converting enzyme (Skidgel et al., 1984), metalloendopeptidase 24.15 (Orlowski et al., 1983), and metalloendopeptidase 24.16 (Checler et al., 1986b). In brain tissue, the reported half-life of NT is approximately 15 min (Checler et al., 1986a).

There are currently three characterized receptors for NT in the CNS: a receptor with low affinity for NT (NTRL or NT₂) that also binds the histamine H₁ receptor antagonist levocabastine (Chalon et al., 1996; Mazella et al., 1996; Vita et al., 1998), a levocabastine-insensitive receptor with high affinity for NT (NTRH or NT₁) (Tanaka et al., 1990; Vita et al., 1993), and a third NT receptor (NTR; NT₃) that is located intracellularly and has been identified as the previously characterized gp95/sortilin (Mazella et al., 1998; Zsürger et al., 1994). Although there is strong homology and identity between NT₁ and NT₂ across species, there are also significant interspecies differences (see Table 1). Species-selective modified peptide agonists have been identified with over 100-fold higher affinity for the rat over the human NT₁ (Cusack et al., 1995). Additionally, it is unclear whether NT is an agonist or an antagonist at NT₂. When the rat or human NT₂ is expressed in Chinese hamster ovary (CHO) cells, NT acts as an antagonist whereas both levocabastine and SR4869 (a small molecule NT₁ antagonist) act as receptor agonists (Yamada et al., 1998). When human NT₂ are expressed in this system, NT, NN, and levocabastine act as antagonists, and the NTR antagonists SR142948A and SR48692 act as agonists (Botto et al., 1998; Vita et al., 1998). In vivo, however, SR142948A has been shown to block the analgesic effects of NT in rodents, an NT effect that has been associated with activation of NT₂ (Gully et al., 1997), indicating that the NT effects seen in CHO cells may be expression system-specific.

View this table: [in this window] [in a new window]   TABLE 1 Summary of the characteristics of the NT receptor subtypes The amino acid sequence of NT and neuromedin N are provided in the top portion of the table. The lower section of the table summarizes the general characteristics, receptor agonists and antagonists for each of the three cloned NTRs (Chabry et al., 1993; Chalon et al., 1996; Gully et al., 1997; Hermans and Maloteaux, 1998; Le et al., 1997a; 1997b; Morris et al., 1998; Munck Petersen et al., 1999; Nouel et al., 1999; Petersen et al., 1997; Vincent, 1995; Vincent et al., 1999; Watson et al., 1993; Yamada et al., 1998).

Both NT₁ and NT₂ are G-protein coupled receptors with the typical 7-transmembrane configuration characteristic of these receptors. Although for the most part it is unclear which second messenger systems the NTRs are associated with in vivo, the NT system has alternately been shown to regulate cyclic AMP (Bozou et al., 1986; Yamada et al., 1993; Slusher et al., 1994), cyclic GMP (Gilbert and Richelson, 1984), phosphatidyl inositol (PI) turnover (Snider et al., 1986; Watson et al., 1992; Erwin and Radcliffe, 1993; Hermans et al., 1994), intracellular Ca²⁺ influx (Memo et al., 1986; Woll and Rozengurt, 1989; Slusher et al., 1994; Trudeau, 2000), phospholipase C (Hermans et al., 1992; Watson et al., 1992; Chabry et al., 1994), and Na⁺,K⁺-ATPase activity (Lopez Ordieres and Rodriguez de Lores Arnaiz, 2000) in vitro. NTR activation not only leads to an activation of second messenger pathways, but also changes the affinity of DA receptors via allosteric receptor/receptor interactions and modulates gene expression via the internalized NT-NTR complex. NT₃ is a type I amino acid receptor with a single transmembrane-spanning region (Mazella et al., 1998). NT₃ is located in glia, neurons, and adipocytes (Chabry et al., 1993; Morris et al., 1998) and is believed to be involved in the sorting of luminal proteins from the trans-Golgi to late endosomes (Petersen et al., 1997). NT₃ may also be involved in modulation of NT signal termination via mediation of NT uptake and degradation (Mazella et al., 1998; Mazella, 2001; Navarro et al., 2001).

The only NTR agonists to date are modified subfragments of the NT peptide itself (Cusack et al., 1993, 2000; Tyler et al., 1999). Conversely, several nonpeptide NTR antagonists have been identified of which SR48692 and SR142948A are the best characterized (Gully et al., 1993, 1997). Both of these antagonists possess nanomolar affinity for NT₁ in different tissues and cells from various species (Gully et al., 1993, 1997). SR142948A, however, has a 90-fold higher affinity for NT₁ than SR48692, and only SR142948A binds NT₂ with nanomolar affinity. Despite the fact that SR4892 has a low binding affinity for NT₃ (Mazella et al., 1998), there is some evidence that in cancer cell lines expressing only NT₃, SR48692 blocks NT-induced cell growth (Dal Farra et al., 2001). Table 1 provides further information on agonists and antagonists of the cloned NTRs.

Dopamine (DA), like epinephrine and norepinephrine, is a cathecholamine neurotransmitter [for extensive reviews of the DA system see Cooper et al. (1991) and Wolf et al. (1987)]. The rate-limiting enzyme for DA synthesis, tyrosine hydroxylase (TH) is common for all cathecholamines and its activity is tightly regulated by multiple feedback mechanisms. TH immunoreactivity is a useful marker of DA neurons in brain areas lacking significant adrenergic (epinephrine and norepinephrine) input. The localization of TH in the cell body and along the length of the axon allows the identification of DA perikarya as well as DA-ergic projections.

DA-ergic axons are generally characterized by the presence of multiple varicosities. The number and diameter of these varicosities, as well as the extent of collateral branching varies between terminal regions. Synaptic junctions occur en passant with punctate membrane specializations. DA is stored in synaptic vesicles and released in a Ca²⁺-dependent manner and signal transduction is terminated by fast reuptake of DA into the terminal by the DA transporter. DA is then converted to dihydroxyphenylacetic acid (DOPAC) by an intraneuronal monoamine oxidase (MAO). Extraneuronally, DA is metabolized to DOPAC and homovanillic acid (HVA) by combined activity of a cathechol-O-methyl aminotransferase and an MAO. Increased levels of these metabolites reflect increased DA neurotransmission, and changes in DOPAC and HVA in specific brain regions are closely correlated with changes in impulse flow in the corresponding DA-ergic projections.

Currently, five DA receptors (designated D₁-D₅) have been structurally characterized with elucidation of their gene sequence and corresponding amino acid sequence; all are G-protein coupled receptors (Baldessarini and Tarazi, 1996; Jaber et al., 1996). Historically, two families of DA receptors have been described based on their effect on adenylate cyclase (AC) activity. Activation of D₁-type receptors (D₁ and D₅) increases AC activity via G_s-type G-proteins. In contrast, D₂-type receptors [D₂ (D₂ short and D₂ long isoforms), D₃, and D₄] decrease AC activity via G_i-type G-proteins. It is now clear that DA receptors are also associated with G-proteins other than G_s and G_i and can affect multiple second messenger systems in a brain region-specific manner. In addition to increasing cAMP, D₁ activation increases PI turnover, and D₁ receptors have been found coupled to G_o-type G-proteins in certain systems. D₂-type receptors have been reported to increase PI hydrolysis and may also regulate phospholipase A2, intracellular Ca²⁺ levels, and K⁺ currents.

DA receptors are located on DA neurons (DA autoreceptors) as well as postsynaptically on a variety of different neuronal populations including GABA-ergic, glutamatergic, serotonergic, cholinergic, and peptidergic neurons (Baldessarini and Tarazi, 1996; Jaber et al., 1996). Postsynaptic DA receptors consist of all five subtypes whereas only D₂ and D₃ receptors serve as autoreceptors. DA autoreceptors are found on the perikarya, dendrites and axon terminals of DA neurons. Autoreceptor activation tonically inhibits DA transmission by decreasing DA release, firing rate, and TH synthesis in DA neurons. Compared with postsynaptic D₂ receptors, D₂ autoreceptors have a 5- to 10-fold higher affinity for DA and certain DA receptor agonists. Relatively selective autoreceptor agonists and antagonists are available. At low doses of DA receptor agonists, activation of autoreceptors predominates leading to diminished DA function, whereas at higher doses postsynaptic DA receptors are also activated, resulting in enhanced DA transmission (Wolf et al., 1987).

Several facts indicate that DA-ergic synapses (at least in some brain regions) favor paracrine or volume transmission. First, DA is released from synaptic densities and extrasynaptic sites. Second, although DA terminals form classic symmetric as well as asymmetric synapses, less than 10% of DA receptors are located in postsynaptic densities, and DA transporters are not concentrated solely around synapses (Pickel et al., 1996; Zoli et al., 1998). DA synapses, therefore, appear to be "open" synapses allowing for the diffusion of DA into the extracellular fluid and activation of DA receptors distant from the actual release site.

Pharmacologic manipulation of the DA system is possible at almost every level of DA transmission (Cooper et al., 1991): from modulation of DA synthesis to activity at postsynaptic DA receptors. Relatively selective toxins of DA neurons, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) allow specific lesions of DA-ergic projections. Vesicular stores of DA can be depleted using reserpine and tetrabenzine. The indirect DA receptor agonists such as cocaine, amphetamine, and nomifensin release DA and/or block its reuptake. These compounds are not completely selective and also bind to other monoamine transporters. Direct DA receptor agonists or antagonists have evolved from nonsubtype-specific drugs to compounds with selective activity at D₁- versus D₂-type receptors. Ligands (especially agonists) that are completely selective for specific members of the D₁ or D₂ receptor families are not yet available.

	II. Neurotensin/Dopamine Anatomy

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Within the CNS, DA-containing cells are found in the mesencephalon (cell groups A8-A10), diencephalon (A11-A14), olfactory bulb (A16), and retina (A17) (for extensive review, see Björklund and Lindvall, 1984; Fallon and Loughlin, 1995) (Fig. 1).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Origin and major projections of the mesocorticolimbic and nigrostriatal DA systems. There are three DA midbrain nuclei: the SNc and SNl (=A9), the VTA (=A10), and the RRF (=A8). These neurons give rise to the nigrostriatal and mesocorticolimbic DA projection systems. PIR, piriform cortex; OTu, olfactory tubercles.

Remarkably, 80 to 90% of all DA neurons are found in the midbrain. There are three DA midbrain nuclei; the substantia nigra pars compacta (SNc) and pars lateralis (SNl) (=A9), the ventral tegmental area (VTA) (=A10), and the retrorubral field (RRF) (=A8). These neurons give rise to the nigrostriatal and mesocorticolimbic DA projection systems. The cell bodies of the nigrostriatal DA system are located in the SNc and RRF and project with a medial to lateral topography to the dorsolateral aspects of the caudate/putamen (CPu). DA cell bodies in the SNl project primarily to the amygdala. The mesocorticolimbic DA system has cell bodies primarily in the VTA and terminal fields in the nucleus accumbens (NAcc), olfactory tubercles and ventromedial CPu, bed nucleus of the stria terminalis (BNST), septum, amygdala, and limbic cortical areas such as prefrontal, cingulate, entorhinal, and piriform cortices. In addition to these two major projection systems originating in the SN and VTA, the SNc also innervates the subthalamic nucleus, and the VTA innervates the habenula and the locus coeruleus. Historically the cell bodies of the nigrostriatal and mesocorticolimbic DA system were thought to be strictly segregated to the SNc and RRF or VTA, respectively. It now appears that this segregation is not as strict and that the terminal fields of the A8/A9 and A10 cell groups overlap to a considerable extent. The SNc provides input to limbic and cortical regions such as the amygdala and anterior cingulate cortex; the RRF projects to the amygdala and entorhinal cortex. Midbrain DA neurons differ not only in their projection fields, but also in the inputs they receive, the receptors they express and the neuropeptides they colocalize. For example, the density of DA autoreceptors varies greatly among DA neurons (Fallon and Loughlin, 1995). DA neurons located in the medial VTA that project to prefrontal cortical areas have very few, if any autoreceptors. These neurons have a higher spontaneous firing rate, display more burst firing, and are less sensitive to manipulations by DA-ergic drugs than other DA neurons.

Three DA projection systems originate in the diencephalon, the tuberohypophyseal, the incertohypothalamic, and the medullary-periventricular system. The cell bodies of the tuberohypophyseal or tuberoinfundibular systems (A12) are located in the arcuate nucleus and adjacent areas of the periventricular nucleus, and project to the median eminence and the neural and intermediate lobes of the pituitary. In the median eminence, DA released into the hypophyseal portal system inhibits prolactin release from the anterior pituitary. The incertohypothalamic DA system has cell bodies in the posterior hypothalamus (A13 + the periventricular A14 neurons) that innervate the anterior hypothalamus and lateral septal nuclei. The medullary-periventricular system cell bodies (A11) are located periventricularly in the caudal thalamus, posterior dorsal hypothalamus, and periaqueductal gray. A11 neurons project locally as well as to the spinal cord.

Outside these nuclei, DA-containing cells are found in the olfactory bulb and the retina. In the olfactory bulb, periglomerular DA cells (A16) surround the glomerulae, and specialized DA-ergic amacrine cells (A17) are located in the inner portion of the nuclear layer of the retina.

The next section provides a detailed overview of anatomical NT/DA interactions in DA cell body regions and DA terminal areas with an emphasis on midbrain and striatal areas. Most of the electrophysiologic, neurochemical, and behavioral differences in NT/DA interactions between the nigrostriatal and the mesocorticolimbic DA systems can be explained based on prominent anatomical differences between these two projection systems.

NT cell bodies in the mesencephalon are unequally distributed between the A9 and A10 DA systems (Table 2). In contrast to the significant number of NT-positive cells in the VTA, very few NT-positive cells are detected in the SNc, SNl, and RRF (detectable NT neurons in the SNc are located mostly in its medial aspects) (Uhl et al., 1977; Jennes et al., 1982; Uhl, 1982). Interestingly, the few NT neurons found in the SNc do not colocalize TH (Seroogy et al., 1988). The vast majority of NT-positive cells in the VTA colocalize TH and the neuropeptide cholecystokinin (CCK), however, NT/DA/CCK neurons represent only a small fraction of DA-ergic cells³ (Hökfelt et al., 1984; Seroogy et al., 1988; Seroogy et al., 1987). These mixed NT/DA neurons have been shown to project to the prefrontal cortex (PFC), entorhinal cortex (ERC), NAcc, basolateral nucleus of the amygdala, and lateral septum (LS) (Fallon, 1988; Febvret et al., 1991). NT/DA projections overlap mesocorticolimbic DA projections with the exception of the central nucleus of the amygdala and the NAcc core where there are no mixed projections (Fallon, 1988; Asan, 1998).

View this table: [in this window] [in a new window]   TABLE 2 Anatomical association between the DA system and the NT system Table 2 summarizes the anatomical association of NT with the mesocorticolimbic, nigrostriatal, and diencephalic DA systems in their origin and projection areas (given in column one). The second column summarizes the presence of NT cell bodies, transmitters colocalized with NT, projection areas of NT-ergic neurons and types of DA receptors expressed by these neurons whenever known. NT cell bodies: (), not detectable; (+), few; (++), moderate; (+++), high; (ND), not determined. The third column indicates the presence of NT or mixed NT/DA fibers and their origin. NT fibers: NT/DA, NT colocalized with DA; (), not detectable; (+), sparse; (++), moderate; (+++), dense; (ND), not determined. The last column summarizes the density of NTR binding and the expression of NT1 or NT2 mRNA in each of these regions. NT receptors: (), not detectable; (+), low; (++), moderate; (+++), dense; (ND), not determined. The cellular location of the receptors is given whenever possible.

A dense network of NT fibers innervates both the VTA and SNc (Hökfelt et al., 1984; Woulfe and Beaudet, 1989). These fibers do not colocalize TH, suggesting that they originate from outside the midbrain (Bayer et al., 1991; Woulfe and Beaudet, 1992). A recent retrograde labeling study indicates that neurons projecting from the rostral lateral septum, the preoptic area, and the lateral hypothalamus may be the source of NT innervation to the VTA (Zahm et al., 2001).

The vast majority of DA neurons in the VTA and SNc express NTRs, predominantly the NT₁-subtype, and 80 to 95% of midbrain NTRs are expressed on DA-ergic neurons (Palacios and Kuhar, 1981; Uhl, 1982; Quirion, 1983; Quirion et al., 1985; Hervé et al., 1986; Moyse et al., 1987; Szigethy and Beaudet, 1989; Brouard et al., 1992; Nicot et al., 1995; Fassio et al., 2000). The remaining NTRs are found on non-DA-ergic axon terminals (most likely GABA-ergic striatonigral projections) and glial cells (Boudin et al., 1998). NT₁ immunoreactive neurons are also located in the RRF, the SN pars reticulata and to a lesser extent in the SNl (Fassio et al., 2000). In addition to high levels of NT₁ mRNA expression, NT₂ mRNA is also abundant in the midbrain, although the cellular location of NT₂ in the midbrain has not yet been clarified (Walker et al., 1998; Lépée-Lorgeoux et al., 1999). There is also pharmacologic and behavioral evidence for additional NTR subtypes in the midbrain (discussion later in this review).

Of the NT terminals contacting DA-ergic, non-DA-ergic, and mixed DA/NT cells in the midbrain, only a small fraction actually exhibit synaptic specialization (Woulfe and Beaudet, 1992). Less than 10% of these rare synaptic contacts are with TH-positive neurons (Woulfe and Beaudet, 1992). Nonetheless, 60% of NT terminals are within 5 µm of DA cells, which would allow for NT to act on DA cells via paracrine transmission (Woulfe and Beaudet, 1992). Electron microscopic localization of NTRs demonstrates that these receptors are not clustered opposite nerve terminals but are more or less evenly distributed over the perikarya and dendrites of DA cells (Dana et al., 1989; Fassio et al., 2000) supporting paracrine signaling and/or dendritic release as the major mode of NT neurotransmission in the mesencephalon.

Of the neurons in the striatum, 95% are medium spiny GABA-ergic projection neurons that are highly collateralized. The striatum can be divided into distinct subregions based on several criteria, including DA-ergic innervation, glutamatergic input, projection areas and neurochemical markers. The ventral and dorsal striatum are the two major subdivisions and these two brain regions have distinct functional roles (Fig. 2). The dorsal striatum is part of the basal ganglia and mostly involved in motor coordination. The ventral striatum is part of the limbic system and although it plays a role in motor coordination, its major function is the processing of emotion and cognition (Paxinos and Watson, 1986).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Striatal subdivisions based on the origin of their DA-ergic and glutamatergic input, projection areas, and function.

The distribution of NT cell bodies, NT fibers, and NTRs differs between the dorsal and the ventral striatum. NT/NN mRNA expression, NT-positive neurons and NT axon terminals are primarily restricted to the ventral striatum (olfactory tubercles, the Nacc, and the ventromedial and ventrolateral CPu) (Zahm, 1987, 1992). The number of NT-positive neurons and fibers is extremely low in the dorsal striatum but is dramatically enhanced after specific pharmacologic manipulations (Zahm, 1992). Microdialysis studies also report higher extracellular NT concentrations in the NAcc compared with the dorsal CPu (Huang and Hanson, 1997; Radke et al., 1998). In contrast to the density of NT terminals, NTR binding is more dense in the CPu than in the NAcc (Uhl, 1982; Quirion et al., 1985; Schotte and Leysen, 1989). One possible explanation for this apparent mismatch between the levels of NT peptide and NT binding is increased basal neuropeptide release resulting in ligand-induced receptor internalization (Dournaud et al., 1998). This hypothesis is supported by a report from Boudin et al. (1998) that compared the distribution of NT₁ receptor immunoreactivity and NTR binding as measured by NTR autoradiography in the SNc. This study demonstrated that in dendrites of DA neurons in the SNc, an anti-NT₁ receptor antibody detected internalized receptors in the absence of radioligand binding. This would suggest that internalized NTRs are less accessible to the radioligand than cell surface NTRs. Thus, neuropeptide receptor autoradiography may underestimate the number of receptors in areas of high peptide release.

There are discordant results for the cellular location of NTRs in the striatum. A number of in situ and immunohistochemical studies are in agreement that within the ventral striatum, NT₁ receptors are located primarily on medium spiny output neurons as well as aspiny GABA-ergic or cholinergic interneurons and are only rarely detected on nerve terminals (Nicot et al., 1994; Boudin et al., 1996; Delle Donne et al., 1996; Alexander and Leeman, 1998). There is mounting evidence, however, that up to 60% of NT₁ are located on DA-ergic, glutamatergic, and GABA-ergic axons in the ventral striatum (Pickel et al., 2001). In the dorsal striatum, NT₁ receptors are restricted to DA terminals (Boudin et al., 1996). There is a moderate density of NT₂ receptor mRNA in the NAcc and somewhat less in the CPu (Walker et al., 1998). Throughout the striatum, NT₂ receptors appear to be located primarily on glial cells (Schotte et al., 1988). In contrast to in situ hybridization and immunohistochemical studies, 6-OHDA midbrain DA lesion studies report decreases in NTR binding in the NAcc ranging from 0 to 80%, whereas DA terminal numbers (as determined by DA transporter density) were drastically reduced in all studies. Thus some of these reports indicate that NTRs may be located on DA terminals in the NAcc to some extent (Hervé et al., 1986; Schotte et al., 1988; Schotte and Leysen, 1989; Cadet et al., 1991; Pickel et al., 2001). There are several possible explanations for these conflicting data. First, there may be regional differences in NTR location within subregions of the NAcc. Dilts and Kalivas (1989) reported that 6-OHDA lesioning did not affect NTR binding in the NAcc shell but decreased NTR binding by over 50% in the NAcc core. Second, these discrepancies could be explained by varying extents of DA cell lesions between various studies. This appears unlikely, however, because comparative low levels of DA transporter after 6-OHDA lesions have been associated with both unchanged (Schotte et al., 1988) and reduced (Cadet et al., 1991) NTR binding in the NAcc. Third, unchanged NT binding kinetics in the NAcc after 6-OHDA lesions could be a combination of the loss of NTRs on DA terminals and increases in postsynaptic receptors in the NAcc due to a prolonged reduction in DA-ergic tone. NTRs have been shown to be up-regulated in the NAcc after subchronic D₂ receptor antagonist (haloperidol) administration (Hervé et al., 1986). It is therefore important to consider the time point after 6-OHDA lesions when comparing these studies. Fourth, increased NTR binding could be due to increased glial associated NT₂ binding (Nouel et al., 1999). Under basal conditions, the majority of NT₂ binding in the NAcc is associated with neurons. In response to neuronal injury, there is an increase in the number of astrocytes expressing NT₂ and in the amount of NT₂ mRNA expressed per astrocyte.

Most 6-OHDA lesion studies indicate that the majority of NTRs in the CPu are located on presynaptic DA terminals (Hervé et al., 1986; Dilts and Kalivas, 1989; Schotte and Leysen, 1989; Masuo et al., 1990b; Cadet et al., 1991). Studies lesioning either intrinsic striatal neurons or cortico-striatal projections, however, are not in agreement with an exclusive location of NTRs on DA terminals (Goedert et al., 1984; Masuo et al., 1990b). These studies report that 30% of NTRs may be located on DA terminals, 50% on intrinsic neurons, and 20% on cortico-striatal projections.

The dorsal CPu and the NAcc can be further divided into distinct subregions in which the anatomical interactions of NT and DA vary greatly. The next sections discuss the anatomical compartmentalization of NT/DA interactions in these regions. This regional heterogeneity is of paramount importance for the interpretation of functional and behavioral aspects of NT/DA interactions, discussed in subsequent sections.

1. The Nucleus Accumbens. The NAcc can be divided into three distinct subregions (the cone, the shell, and the core) based on criteria similar to that used for subdivisions of the striatum. Our review will focus primarily on the shell and core subdivisions of the NAcc. Whereas the NAcc shell is closely related to the limbic system, the NAcc core resembles the CPu and is usually considered part of the basal ganglia (Heimer et al., 1997). The type of anatomical NT/DA interaction varies greatly between, as well as within, these subregions.

2. Caudate/Putamen. NT/NN mRNA expression and NT-positive cell bodies are found only in the ventral CPu, an area that receives mixed innervation from the SNc and VTA (Zahm, 1987; Alexander et al., 1989; Merchant et al., 1992a). These neurons appear to project to the globus pallidus, where a thin strip of NT-positive fibers can be detected in the medial globus pallidus (Eggerman and Zahm, 1988). NTR binding and NT₁ mRNA expression have also been reported in the globus pallidus (Moyse et al., 1987; Alexander and Leeman, 1998). Although very few NT neurons and fibers are detected in the CPu, microdialysis studies report the presence of basal NT release (Huang and Hanson, 1997; Radke et al., 1998). In the absence of prominent NT input (Zahm, 1987), extracellular NT is most likely released from local striatal NT neurons. Numerous NT-positive neurons become detectable in the CPu after pharmacologic manipulation of DA transmission (Zahm, 1992; Castel et al., 1993b). Several regionally distinct NT neuron subpopulations respond differentially after DA-ergic stimuli.

Cortical areas receive two major types of DA innervation. Mesocortical projections from the VTA innervate deep cortical layers (V and VI) of the PFC, the piriform cortex and the entorhinal cortex. DA-ergic projections from the SNc on the other hand, innervate superficial layers (II and III) of the anterior cingulate cortex and to a lesser extent the premotor, visual, and retrosplenial cortices (Björklund and Lindvall, 1984). Pyramidal as well as nonpyramidal neurons expressing D₁, D₂, and D₄ receptors receive direct DA-ergic input (Gaspar et al., 1995; Vincent et al., 1995; Defagot et al., 1997). Hippocampal areas (including the subiculum) receive a sparse DA-ergic innervation (Björklund and Lindvall, 1984).

In general, NT-positive cell bodies are scarce in cortical and hippocampal areas and are reported in the subiculum, cingulate, and piriform cortices only after colchicine administration (Febvret et al., 1991). High levels of NT/NN mRNA expression are detected in the dorsal subiculum and CA1, and moderate amounts are found in the piriform and the cingulate cortex (Alexander et al., 1989). Despite high levels of NT/NN mRNA expression, no NT-immunoreactive neurons can be detected in CA1, and NT-positive neurons are relatively scarce in the subiculum. NT synthesis and mRNA processing are intact in these neurons and it is therefore likely that the CA1 and subiculum primarily contribute NT innervation to projection areas such as the NAcc A, entorhinal cortex and VTA.

There are dense NT fibers overlapping DA terminal fields in limbic cortical regions (Studler et al., 1988; Febvret et al., 1991). NT is colocalized with DA in all of the mesocortical projections to deep cortical layers, i.e., in DA terminals in the PFC, entorhinal cortex, and piriform cortex (Febvret et al., 1991; von Euler et al., 1991). In contrast, NT is not colocalized with DA in projections to superficial layers of the anterior cingulate cortex where NT fibers in general are sparse and restricted to layer VI (Febvret et al., 1991). Although all NT-positive fibers in the PFC colocalize DA, NT-only terminals have been reported in the entorhinal cortex, subiculum, and retrosplenial cortex (Febvret et al., 1991). The extent of NT/DA colocalization in afferents to the PFC is somewhat controversial varying from 30 to 100% of all DA terminals (Studler et al., 1988; Febvret et al., 1991).

NTR binding overlaps the distribution of NT terminals in cortical areas. NT₁ mRNA expression is found in layer VI of all neocortical areas, but its expression is highest in the limbic cortex in layers II, III, V, and VI and in the subiculum (Nicot et al., 1994; Alexander and Leeman, 1998). In cortical areas, NTRs are located on terminals (most likely DA-ergic) and on cortical cell bodies and dendrites. In the PFC and entorhinal cortex, NT₁-like immunoreactivity has been described on pyramidal cells in layers II, III, V, and VI. In layer VI of the anterior cingulate cortex on the other hand, NT₁ receptors were detected only on terminals, despite the presence of NT₁ mRNA expression in this brain region (Boudin et al., 1996).

One common role of the amygdala, BNST, and LS is the transduction of higher cognitive and emotional processes into peripheral, autonomic responses. All three brain regions receive DA-ergic input from the VTA. DA innervation is the densest in the central nucleus of the amygdala, the dorsal aspect of the lateral segment of the BNST, and the intermediate and ventral parts of the lateral septum (Uhl and Snyder, 1979; Roberts et al., 1982; Björklund and Lindvall, 1984; Kohler and Eriksson, 1984; Asan, 1998). Interestingly, in all these regions, areas with dense DA innervation correspond to the areas with the highest number of NT-positive neurons, and TH-positive fibers contact NT-positive neurons (Uhl and Snyder, 1979; Asan, 1998). Mixed NT/DA neurons project from the VTA to the amygdala, where they terminate in the basolateral nucleus and are only rarely seen in the central nucleus of the amygdala, and the lateral septum (Tay et al., 1989; Bayer et al., 1991; Jakab and Leranth, 1995; Asan, 1998). NT terminals in the amygdala (predominantly local axon collaterals and not extrinsic afferents) make axo-axonic, axo-dendritic, and axo-somatic contacts with mostly NT-positive cells and axons, suggesting NT-ergic autoregulation (Tay et al., 1989; Bayer et al., 1991). In the BNST and lateral septum, the exact relationship between DA and NT has not yet been investigated.

The central nucleus of the amygdala, BNST, and lateral septum all display relatively dense NTR binding (Moyse et al., 1987). NT₁ mRNA is present in all these regions whereas NT₂ mRNA expression is not detectable (Alexander and Leeman, 1998; Walker et al., 1998). Besides providing local axon collaterals, NT neurons in the amygdala, BNST, and lateral septum project to autonomic and endocrine centers in the brainstem and hypothalamus such as the dorsal vagal complex, the parabrachial nucleus, the central gray, and the lateral hypothalamus (Uhl et al., 1979; Moga and Gray, 1985; Gray and Magnuson, 1987, 1992; Moga et al., 1989; Shimada et al., 1989; Jakab and Leranth, 1995). NT could therefore be an important transmitter in the conversion of DA-ergic cognitive and emotional information to peripheral visceral and autonomic responses.

We refer the reader to an excellent extensive review by Rosténe and Alexander (1997) for information on NT in the diencephalon. Briefly, of the three diencephalic DA systems, only the tuberoinfundibular DA system originating in the dorsomedial arcuate nucleus and the adjacent periventricular nucleus, appears to be associated with the NT system. The majority of NT neurons in the diencephalon are found in the arcuate nucleus, with some neurons located in the periventricular nucleus and the parvo-cellular paraventricular nucleus (Kahn et al., 1980). NT neurons in the dorsomedial section of the arcuate nucleus colocalize TH and are believed to be part of the tuberoinfundibular DA system. The reported extent of NT and DA colocalization in these neurons varies from extensive to rare (Ibata et al., 1983; Hökfelt et al., 1984).

NT neurons in the arcuate nucleus project to the external zone of the median eminence whereas NT neurons in the periventricular nucleus project to the intermediate lobe of the pituitary. NT neurons within both these nuclei also have dense local axon collaterals (Kahn et al., 1980). NT can therefore be released locally in the hypothalamus, into the portal circulation, or directly into the intermediate lobe of the pituitary.

NTR binding is moderately dense throughout the hypothalamus, including the dorsomedial arcuate nucleus, periventricular nucleus and the intermediate lobe of the pituitary (Goedert et al., 1985). NT₁ receptors are located on cell bodies, dendrites, and terminals in the arcuate nucleus and only on terminals in the median eminence (Boudin et al., 1996). Previous studies suggested that in contrast to mesencephalic DA neurons, TH-positive cells in the hypothalamus do not express NT₁ mRNA (Nicot et al., 1995) and that NT₁ mRNA expression in general is very low in these nuclei (Nicot et al., 1994). Nonetheless, a more recent in situ hybridization study detected abundant NT₁ mRNA throughout the hypothalamus including the arcuate nucleus (Alexander and Leeman, 1998). These same authors reported that NT₁ mRNA is colocalized with TH in tuberoinfundibular DA neurons (Alexander, 1997). The arcuate nucleus also contains one of the highest levels of NT₂ mRNA expression in the rat brain suggesting an important role of NT₂ in NT regulation of tuberoinfundibular DA neurons (Walker et al., 1998).

The anatomical comparison of NT/DA interactions between the two midbrain DA systems suggests that NT might play a more important role in the physiologic regulation of the mesocorticolimbic than the nigrostriatal DA system. In the latter, NT neurotransmission may only be of importance in pharmacologic or pathologic situations. Figure 3 summarizes the main anatomical differences between the NT and DA systems in mesolimbic, mesocortical, and nigrostriatal DA projections.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Representation of the primary anatomical differences in NT and DA interactions in mesocortical (A), mesolimbic (B), and nigrostriatal (C) DA projections. Within the mesocorticolimbic DA system, NT is colocalized within VTA DA neurons projecting to both the nucleus accumbens and the prefrontal cortex. In contrast, NT is not colocalized in DA cell bodies located in the substantia nigra. In addition, the location of the NTRs in the terminal regions of these two DA systems is very different. In the prefrontal cortex, NTR binding is equally distributed pre- and postsynaptically. In the NAcc, studies indicate the NTRs are located both pre- and postsynaptically. In the CPu, the NTRs are located primarily presynaptically on DA terminals.

It is also important to note that the mesolimbic and nigrostriatal DA systems are not isolated systems but part of larger, heavily interconnected neural circuits (Heimer et al., 1995). Although there is a striking overlap between the anatomical location of NT and DA systems, NT is also associated with other neurotransmitter systems within these interconnected circuits. NT can therefore indirectly effect DA-ergic transmission via other neurotransmitters (see Fig. 4). For example, NT-positive fibers, neurons, and NTRs are present in the dorsal raphe and NT has been shown to have an excitatory effect on these serotonergic neurons (Jolas and Aghajanian, 1996). Serotonergic projections originating in the dorsal raphe in turn innervate DA neurons in the midbrain as well as striatal, pallidal, and cortical areas.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Examples of direct and indirect associations between NT projections and the mesocorticolimbic DA system. The cross-hatched lines represent projections colocalizing either NT and DA (black and red) or NT and GABA (yellow and red).

	III. Functional Interactions between the Neurotensin and Dopamine Systems

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The functional interactions between the NT and DA systems are as complex as the close and interconnected anatomical association between these two systems indicates are possible. The NT and DA systems reciprocally modulate each other in a heterogeneous manner in all brain regions in which these two systems coexist. In this section, we will first discuss the effects of NT on the DA system, followed by the details of how DA transmission affects the NT system.

1. Mechanism of Action of Neurotensin. Once NT binds to the NT₁ receptor, NT has been shown to act via several distinct mechanisms (Fig. 5); 1) internalization of the NT-NTR complex leading to regulation of gene expression, 2) allosteric receptor/receptor interactions between the activated NT receptor and DA D₂-type receptors leading to decreased D₂ receptor agonist binding affinity, and 3) alteration of cell firing via activation of second messenger cascades and ion channels. Although NT₁ receptors are located not only on DA neurons, but also pre- and postsynaptically to them, our discussion will be restricted to the mechanisms by which NT has been shown to act directly on DA neurons.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Mechanisms by which NT modulates DA transmission. , once NT binds to the NT1 receptor, the NT-NT1 complex is rapidly internalized. The neurochemical consequences of NT-NT1 complex internalization have been examined in nigrostriatal neurons. The NT-NT1 complex dissociates after being internalized, and NT and NT1 segregate to separate intracellular trafficking pathways. Following internalization in a DA axon terminal, NT is retrogradely transported to the cell body. All internalized NT eventually moves to surround the nucleus of the cell, and in DA neurons, this process is followed by up-regulation of TH gene expression. , the NT-NT1 complex decreases the agonist binding affinity of the DA D2 receptor via allosteric receptor/receptor interactions. , binding of NT to NT1 located on DA cell bodies in the mesencephalon has been shown to decrease the conductance of an inward rectifying K+ channel (Ih). Decreasing the Ih current leads to a slow depolarization of the neuron, and antagonism of D2 receptor agonist-induced autoinhibition of DA cell firing. , at slightly higher doses of NT than is needed to decrease Ih, NT binding to the NTR increases the conductance of a nonselective cation channel, transduced by activation of Gq and/or G-11 G-protein subtypes and IP3. An increase in the conductance of the nonselective cation channel leads to cell depolarization, increased firing rate, and eventually depolarization block. , NT interacts with the extracellular portion of the D2 receptor via hydrophobic mode matches. This interaction leads to a change in the Kd of D2 receptor antagonist binding in a manner similar to that of noncompetitive antagonists.

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View larger version (39K): [in this window] [in a new window]   Fig. 6.   Summary of the electrophysiologic and neurochemical effects of NT administered into either the ventral tegmental area (A10) or substantia nigra (A9). Effects are shown at the sight of the injection and in the prefrontal cortex (A), nucleus accumbens (B), and caudate/putamen (C). Low dose NT depolarizes midbrain DA neurons without increasing cell firing (). Intermediate doses of NT increase the number and rate of spontaneously firing DA neurons (). There is a corresponding increase in DA turnover in the terminal regions of the midbrain DA neurons. At high doses, NT decreases the firing rate of DA neurons in the VTA projecting to the nucleus accumbens (). This decrease in firing rate is most likely due to depolarization block. There is an associated decrease in DA release in the nucleus accumbens.