Regulatory Peptides, 36 (1991) 153-164

153

© 1991 Elsevier Science Publishers B.V. All rights reserved 0167-0115/91/$03.50

REGPEP 01100

Review

The neurobiology of neurotensin" focus neurotensin-dopamine interactions

on

J o h n K a s c k o w I and Charles B. N e m e r o f f 2 INeurobiochemistry Group, UCLA School of Medicine, Los Angeles, CA (U.S.A.)and 2Department of Psychiatry, Emory University School of Medicine, Atlanta, GA (U.S.A.) (Received l0 July 1991; revised version received 23 July 1991; accepted 24 July 199 l)

Key words: Neurotensin; Dopamine; Schizophrenia; Transduction; Pharmacology; Behavior

Contents I.

Summary

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153

IL

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154

III.

Neurotensin receptors . . . . . . . . . . . . . . . . . . . . . . . .

154

IV.

Neurotensin signal transduction . . . . . . . . . . . . . . . . . . . .

155

V.

Neurotensin - dopamine interactions . . . . . . . . . . . . . . . . . .

156

VI.

Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . .

160

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . .

160

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Summary Neurotensin (NT) is a tridecapeptide which fulfills many of the requisite criteria for a role as a central nervous system (CN S) neurotransmitter. It is closely associated with CN S dopamine neurons and has been shown to interact with dopamine at physiological, anatomical and behavioral levels. Neurotensin is colocalized with dopaminergic neurons in the hypothalamus and midbrain. In addition, it blocks behaviors associated

Correspondence: C.B. Nemeroff, Department of Psychiatry, Emory University School of Medicine, Box AF, Atlanta, Georgia 30322, U.S.A.

154 with activation of the dopaminergic pathways. Centrally administered NT has been shown to mimic many of the actions of antipsychotic drugs. In addition, the concentration of NT in cerebrospinal fluid is decreased in patients with schizophrenia. Administration of clinically effective antipsychotic drugs increases concentrations of NT in the caudate nucleus and nucleus accumbens. NT has been shown to play a role in signal transduction by mostly mobilizing calcium stores following inosital phosphate formation. This has been linked to subsequent events in protein phosphorylation. Lipophillic NT receptor agonists may represent a novel approach to the development of a new class of antipsychotic drugs.

II. Introduction

Neurotensin (NT) is a tridecapeptide originally isolated from bovine hypothalamic extracts [1,2]. It exists in all classes of vertebrates, and other organisms as well [3,4]. In addition to its well-known distribution in the central nervous system (CNS), it is found in gut, pancreas, adrenals and plasma [5]. Systemic injection of NT leads to a wide array of actions, including hypotension, hyperglycemia and decreases in both gut motility and gastric acid secretion [6]. In the CNS, NT is distributed heterogeneously. High concentrations can be detected in the substantia nigra (SN), periaqueductal gray matter, amygdala, nucleus accumbens, as well as some hypothalamic nuclei. Moderate concentrations are present in the caudate, hippocampus and globus pallidus. Low concentrations of NT are present in the cerebral cortex and cerebellum [7]. NT has been identified immunocytochemically [8], and prepro NT mRNA has been identified by in situ hybridization methods [9]. Numerous NT-containing pathways have been identified, including one from the ventral tegmental area (VTA) to the nucleus accumbens [ 10]. Other pathways include those from the central amygdaloid nucleus to the bed nucleus of the stria terminalis [ 11 ] and those from the subiculum to the alveus, fimbria, fornix and mamillary bodies [ 12]. Much evidence has accumulated implicating NT as a neurotransmitter. NT is known to be cleaved from a larger precursor molecule synthesized in the perikaryon. Synaptosomal localization has been demonstrated [ 13], as well as release upon depolarization of NT neurons [ 14]. Degradation of NT by peptidases has been demonstrated [ 15]. Furthermore, NT has been observed in nerve terminal vesicles by electron microscopy [11].

IlL Neurotensin receptors

NT receptors exhibit saturable, reversible and high affinity binding [ 16]. This can be demonstrated utilizing either iodinated or tritiated ligands to membrane preparations form rat and human neural tissue. The NT receptor has been isolated. Photoaffinity labeling studies of the receptor with NT revealed it to be a heterodimer consisting of a 49 kDa and 51 kDa subunit. Later it was demonstrated to be a 100 kDa polypeptide which, upon proteolysis yields the two subunits [ 17]. Mills et al. [ 18] claim that the

155 receptor is actually a 72 kDa molecule. Recently, the NT receptor structure has been elucidated and cloned [ 19]. High and low affinity NT receptor subtypes have been detected. Levocabastine inhibits the low affinity binding [20]. Schotte et al. [21] performed subcellular fractionation studies of NT receptors and showed that high affinity receptors are associated with neurons, whereas low affinity receptors are affiliated with glial cells. In almost all regions containing NT, there is a parallel distribution of NT receptors; the only exception to this is the cortex and striatum [22]. In the cortex, NT levels are low and receptor density levels are high [23]. In contrast, in the striatum NT concentrations are high and receptor levels are low [8]. The highest density of NT receptors is found in the zona compacta of the SN and the VTA, as well as in the dopamine terminal fields of the olfactory tubercles and frontal cortex [24]. NT receptors on the VTA and SN are of the high affinity type, as are the presynaptic NT receptors found on dopamine terminal fields of the caudate [25-27].

IV. Neurotensin signal transduction The biochemical events mediating signal transduction for NT receptors have begun to be characterized. NT receptors are coupled to G proteins which in turn are linked to inositol phosphate (IP 3) production. Goedert et al. [28 ] showed in whole brain slices that NT simulates IP 3 formation. There was a good correlation between the magnitude of NT stimulated phosphatidyl inositol hydrolysis and the number of specific NT binding sites in various brain regions. Kanba and Richelson [29] likewise demonstrated elevated phosphatidyl inositol hydrolysis in the neuroblastoma clone N1Ell5. They showed that this process was regulated by GTP-related events. Bozou et al. [30] revealed in HT29 colon carcinoma cells that protein kinase C mediated events do not appear to play a prominent role in NT signal transduction; however, IP 3 mediated calcium mobilization is important. Battaini et al. [31 ] also showed in rat striatal slices that NT increases the permeability of membrane calcium channels. Fewer studies have been performed aimed at characterizing the actions of NT at the phosphoprotein level. Cain et al. [32] demonstrated in rat striatal synaptosomes that NT stimulates the in vitro phosphorylation of three proteins of molecular mass 50 kDa, 72 kDa and 76 kDa [31 ]. Cain and Nemeroff [33] studied the effects of NT-sensitive protein phosphorylation in slices of rodent caudate nucleus. They found changes in three Ca z + -dependent phosphoproteins - 56 kDa, 43 kDa and 36 kDa; changes were also seen in the 43 kDa protein with cAMP treatment. The 43 kDa protein was tentatively identified as the ~ subunit of pyruvate dehydrogenase. Kasckow et al. [34] investigated the effects of NT on calcium/calmodulin-dependent protein phosphorylation in rat neostriatal slices. NT significantly altered the phosphorylation of 62 kDa and 50 kDa phosphoproteins which respectively are likely the/~ and ~ subunits of the calcium/calmodulin-dependent protein kinase. Definitive identification of all of these various NT sensitive phosphoproteins is currently underway.

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V. Neurotensin-dopamine interactions N T has been shown to interact with dopaminergic, cholinergic, serotonergic, as well as noradrenergic, neuronal systems. O f particular interest are its interactions with dopamine systems (Fig. 1) because N T may play a role in the pathophysiology of schizophrenia. [35] N T is colocalized in certain dopamine neurons in the arcuate nucleus (Al2) as well as various mesencephalic D A neurons including the VTA (Alo) and S N (m9)[36]. If one destroys the dopaminergic neurons of the VTA by application o f 6-hydroxydopamine ( 6 - O H D A ) , N T binding sites in the prefrontal cortex increase [37]. This suggests that there is a potential decrement of N T release with this treatment. Studler et al. [38 ] found decreases in prefrontal N T concentrations following 6 - O H D A treatment. In contrast, no changes in striatal and nucleus accumbens N T were evident. Bissette et al. [39] found no changes in N T concentrations in nucleus accumbens or caudate following 6 - O H D A - i n d u c e d lesions. Furthermore, in the mouse, treatment with 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine ( M P T P ) has been shown to lead to N T decrements in several dopamine terminal fields [40]. It is difficult to integrate all of these findings. Perhaps the subpopulation of neurons with both N T and dopamine are not susceptible to the neuronal degeneration induced by 6 - O H D A . Alternatively, the neurons with those colocalized transmitters may represent only a small percent of the dopamine cells. There is evidence which suggests that N T modulates dopamine binding. N T does not alter binding of the D 2 antagonist [3H]spiperone to rat brain membranes [41]. How-

VTA

NUCLEUSACCUMBENS

SUBSTANTIA NIGRA

STRIATUM

DA PROJECTIONS . . . . . . NT PROJECTIONS

~ NT RECEPTORS JIB DA RECEPTORS

Fig. 1. Schematic drawing of selected anatomical relationships between neurotensin (NT) and dopamine (DA). Neurotensin-containing cell bodies and their projections parallel those of the mesolimbic DA system. Neurotensin receptors are present on DA cell bodies in both the m 9 and Alo region and on DA terminals in the striatum. Although it has not been definitely shown, for convenience, we have indicated here that both NT receptors and DA receptors are colocalized on the same cells in the striatum and nucleus accumbens. In addition, in the rat, but apparently not in primates, NT and DA are colocalized in certain midbrain cells in the VTA and these colocalized DA/NT cells may preferentially innervate the prefrontal cortex (modified from Nemeroff and Cain [88]).

157 ever, it does seem to cause decreased binding affinity of D I and D 2 agonists to their respective receptors. Miyoshi et al. [42] reported that NT binding to rat striatal membranes leads to conversion of some of the high affinity D~ agonist binding sites to low affinity sites. In addition, Von Euler and Fuxe [43] reported that NT decreases the affinity of D 2 receptor agonist binding. NT has been shown to stimulate dopamine neuronal firing rate in the SN and the VTA [44], as well as frontal cortex pyramidal neurons [45-47]. NT has been shown to increase tyrosine hydroxylase activity in vitro [48 ], as well as enhancing [3H ]dopamine release from brain slices in vitro [49,50]. Blaha etal. [51] demonstrated this phenomenon in vivo utilizing in vivo voltammetry and microdialysis in the nucleus accumbens. Further evidence supporting a role of NT in the regulation of dopamine neurons is provided by the studies of Bean et al. [52]. They revealed that reserpine-induced depletion of striatal dopamine is accompanied by a dose- and time-dependent increase in striatal NT concentrations. This study suggested that dopamine afferents may control the release and/or synthesis of NT within striatal NT cells. These investigators also studied the effects of reserpine on NT stores in the prefrontal cortex. They showed that this leads to decreases in NT and dopamine, suggesting that the peptide may be colocalized with dopamine in the same vesicles. Furthermore, Bean et al. [53] revealed that the mechanism of the striatal changes (vide supra) is a decrease in dopamine and NT release into the striatal extracellular fluid. When NT is administered intracerebroventricularly (icv), increased dopamine turnover occurs as evidenced by increased concentrations of the metabolites - DOPAC and HVA [54,55]. In addition, in the terminal dopaminergic fields of the mesolimbic pathways, enhanced accumulation of DOPA occurs in the presence of NT following treatment with NSD 1015, the inhibitor of L-aromatic amino acid decarboxylase [56]. In addition, microinjection of NT into the VTA leads to enhanced dopamine turnover in the nucleus accumbens and olfactory tubercles [57]. CNS NT injection leads to various behavioral changes, the outcome of which depends on whether NT is applied to dopamine terminal fields or perikarya. ICV injection leads to alterations in locomotion, feeding and drinking. In addition, it leads to decreased avoidance responding in discrete trial conditioned avoidance paradigms, antinociception, decreased body temperature and alterations in muscle tone, blood pressure and gastric acid secretion [4]. If NT is applied onto dopamine perikarya in the VTA, rats exhibit an increase in exploratory behaviors including locomotion, sniffing and rearing [58]. This is accompanied by an increase in dopamine utilization in the nucleus accumbens [59]. In contrast, ICV injection or nucleus accumbens microinjection of NT leads to a dosedependent decrease in amphetamine-induced locomotion and rearing [60]. Interestingly, Kalivas et al. [59] showed that bilateral injection of NT into the nucleus accumbens blocks the stimulatory effects of NT applied to the VTA. Although intraaccumbens NT antagonizes the increase in locomotor activity following intraperitoneal injections of D-amphetamine, intra-caudate injection of NT fails to block amphetamineinduced stereotypy. No effect of NT was observed on scopolamine- or caffeine-induced hyperactivity [61]. Thus, it appears that the actions of NT are limited to agents that

158 activate the mesolimbic dopamine system. Ervin and Nemeroff [62] showed that L-DOPA-induced locomotor activity in rats with 6-OHDA lesions is blocked by injections of N T into the nucleus accumbens. This finding provides compelling evidence that the action of N T is on neurons postsynaptic to dopamine neurons. It has become apparent that NT, behaviorally and biochemically, shares many properties with antipsychotic drugs. Both produce enhanced dopamine tumover in the nucleus accumbens, olfactory tubercle and, to a lesser extent, in the caudate. Both potentiate barbiturate and ethanol-induced sedation, and both produce hypothermia. Both also decrease locomotor activity, produce catalepsy and muscle relaxation and inhibit avoidance but not escape responding, in a conditioned avoidance paradigm [63-65]. As noted above, NT, like antipsychotic drugs, blocks the hyperactivity produced by D-amphetamine and other psychostimulants. NT differs, however, from antipsychotic drugs in several ways. NT does not directly bind to the dopamine receptor, nor does NT block amphetamine-induced stereotypy. In this manner, NT is more like the atypical antipsychotic clozapine, which also fails to block amphetamine-induced stereotypy [66]. In addition, ICV NT is a potent antinocioceptive agent, whereas neuroleptics are not. Chronic antipsychotic drug treatment in laboratory rats produces marked elevations in N T concentrations in the nucleus accumbens and caudate nucleus (Fig. 2). Single injections of haloperidol lead to N T increases within 16 h. Maximal elevations in N T levels occur after 14 days. Similar effects are observed with chlorpromazine, trifluoperazine and pimozide. No changes are seen with clinically ineffective phenothiazines such as promazine or promethazine [67]. Chronic haloperidol given for 8 months produces N T elevations in the nucleus accumbens and caudate. Thus, tolerance to this effect of antipsychotic drugs does not occur [68]. Withdrawal of haloperidol after such long-term treatment decreases N T

!T/21 NUCLEUS ACCUMBENS m ANTERIOR CAUDATE POSTERIOR CAU DATE 200

**

.d

150 Z o (D

b

100-

50

0.03 mg/kg

0.3 mg/kg

i----i

1 rng/kg

3 mg/kg

Fig. 2. The effectof chromic (21-day)treatment with various doses of haloperidol on the concentrationof neurotensin in the rat nucleus accumbens, interior caudate and posterior caudate. NT concentrations in all three brain areas were increased at doses of 1 and 3 mg/kg (*P < 0.05, **P < 0.01, ANOVA, StudentNewman-Keuls test) from Levant and Nemeroff,unpublished observations.

159 concentrations. This decrease is believed to be mediated by renewed stimulation of dopamine receptors rendered supersensitive by long-term haloperidol treatment which in turn leads to decreased NT synthesis. The specificity of the effect of antipsychotic drugs on NT has been explored. Thus, ( + )-butaclamol, a potent D 2 receptor antagonist, but not ( - )-butaclamol, the isomer which shares all properties with ( + )-butaclamol except D 2 receptor blockade, produces the same effect as antipsychotic drugs [69] (see Fig. 1). Bissette et al. [70] showed that haloperidol treatment leads to NT increases in both the nucleus accumbens and caudate following destruction of dopamine neurons by 6-OHDA. These latter findings imply that the postsynaptic dopamine receptors are involved in the regulation of NT levels by neuroleptics and that DA neuronal integrity is not essential for the antipsychotic drug induced increase in NT concentrations. Levant et al. [71 ] showed that the anticholinergic properties of antipsychotic drugs do not mediate the increase of NT concentrations produced by dopamine receptor blockade. Of particular interest is the finding that the atypical antipsychotic, clozapine, produces elevated NT accumulations in the nucleus accumbens without changes in the caudate. [72] This drug is known to have minimal extrapyramidal side effects with selective mesolimbic dopaminergic actions. The above study utilized a microdissection technique. However, in subsequent studies using a modification of the GlowinskiIversen dissection technique with larger tissue samples, this finding could not be confirmed. Furthermore, sulpride, a presumably selective D 2 antagonist for mesolimbic systems, has been shown to produce NT changes only in the caudate but not in the nucleus accumbens [73 ]. There is evidence suggesting that o receptor antagonist activity may account for at least part of the NT elevations because certain typical antipsychotic drugs such as haloperidol and other putative novel antipsychotics (BMY 14802) with high affinity for the a binding site, increase concentrations in the nucleus accumbens and caudate nucleus [73,74]. Both haloperidol and BMY 14802 increase prepro NT mRNA levels in these brain regions, providing evidence that the well-documented NT increase after antipsychotic drug treatment is due to increased NT biosynthesis [75,76]. Other groups have investigated the effects of other dopamine agonists, both direct and indirect, on NT concentrations. Bromocriptine produced an increase of NT levels in the SN, whereas L-DOPA, in contrast, caused decreased NT concentrations in this area [77]. Merchant et al. reported that NT concentrations and NT mRNA levels in the nucleus accumbens are elevated following high dose methamphetamine administration [78]. The reasons for these apparent discrepancies are not known but may be due to toxic effects of high dose DA agonist administration. Chronic neuroleptic treatments in rats is known to lead to elevated SN NT receptor density [79]. Pipotiazine, an antipsychotic, has been shown by Herve et al. to lead to increased density of NT receptors in the lateral prefrontal cortex, nucleus accumbens and central striatum [ 80]. However, Clapacs et al. have recently observed reduced cortical NT receptor binding density after chronic haloperidol treatment [81].

160

VI. Clinical studies Several investigators have measured NT in cerebrospinal fluid (CSF) in schizophrenic patients. Widerlov et al. [82], Lindstrom et al. [83] and Nemeroff et al. [84] measured CSF NT concentrations in drug-free schizophrenic patients. A subgroup of schizophrenic patients had lower mean CSF NT levels than age- and sex-matched controls. Widerlov et al. [82] demonstrated that the subgroup with low CSF NT levels exhibited normal levels after 1 month treatment with neuroleptics. This was recently confirmed (Breslin, Weinberger, Bissette and Nemeroff, unpublished observations). Garver et al. [85] reported that in a group of psychotic patients, low CSF NT is associated with a typical delayed, but significant treatment response to an antipsychotic drug, but not to lithium response. No changes in CSF NT levels have been noted in patients with other psychiatric disorders, including depression, Alzheimer's disease, anorexia-bulemia or premenstral syndrome. [86] In addition, Nemeroff et al. [87] demonstrated that there were increases in NT concentrations in Brodman area 32 in postmortem schizophrenic brain tissue relative to controls. These findings support the hypothesis that NT has a role in the pathophysiology of schizophrenia and/or in the mechanism of antipsychotic drug action. In conclusion, NT certainly fulfills criteria as a CNS neurotransmitter. It has been shown to be intimately associated with dopamine neurons, and can modulate dopamine action. For example, NT alters dopamine release, and the luring rate of midbrain dopamine neurons. Furthermore, NT antagonizes the behavioral action of dopamine in a manner similar to, but not identical with, antipsychotic drugs. It is even considered by some to be an 'endogenous neuroleptic'. The action of NT at the cellular level has been linked to IP 3 formation, mobilization of calcium stores and physphorylation of various protein substrates. Antipsychotic drugs reliably alter brain NT concentrations. Furthermore, NT is reduced in concentration in the CSF of a subgroup of schizophrenics, and after antipsychotic drug treatment, CSF NT concentrations often normalize. It is possible that this neuropeptide plays a role in the pathophysiology of schizophrenia. It seems equally plausible that the therapeutic effects of antipsychotic drugs are mediated in part by an action on NT systems. Further progress in the study of NT is needed. More research is needed in areas involving imaging of the NT receptor, including PET studies in humans. In addition, more exploration is needed in developing specific NT receptor agonists and antagonists. Changes in NT mRNA in postmortem brain tissue need to be measured in schizophrenia and other disease states. Greater knowledge of its actions could likely lead to a better understanding of schizophrenia and may provide us with improved insight to develop new pharmacotherapeutic agents.

Acknowledgements Supported by NIMH MH-39415. We are grateful to Nancy Winter for preparation of this manuscript.

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