Neurotensin receptor mechanisms and its modulation of glutamate

transmission in the brain

Relevance for neurodegenerative diseases and their treatment

T. Antonelli a, K. Fuxe b,*, M.C. Tomasini a, E. Mazzoni a, L.F. Agnati c, S. Tanganelli a, L. Ferraro a

a Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Ferrara, 44100 Ferrara, Italyb Department of Neuroscience, Division of Cellular and Molecular Neurochemistry, Karolinska Institutet, S-171 77 Stockholm, Sweden

c Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, 41100 Modena, Italy

Received 4 December 2006; received in revised form 18 May 2007; accepted 19 June 2007

www.elsevier.com/locate/pneurobio

Progress in Neurobiology 83 (2007) 92–109

Abstract

The extracellular accumulation of glutamate and the excessive activation of glutamate receptors, in particular N-methyl-D-aspartate (NMDA)

receptors, have been postulated to contribute to the neuronal cell death associated with chronic neurodegenerative disorders such as Parkinson’s

disease. Findings are reviewed indicating that the tridecaptide neurotensin (NT) via activation of NT receptor subtype 1 (NTS1) promotes and

reinforces endogenous glutamate signalling in discrete brain regions. The increase of striatal, nigral and cortical glutamate outflow by NT and the

enhancement of NMDA receptor function by a NTS1/NMDA interaction that involves the activation of protein kinase C may favour the

depolarization of NTS1 containing neurons and the entry of calcium. These results strengthen the hypothesis that NT may be involved in the

amplification of glutamate-induced neurotoxicity in mesencephalic dopamine and cortical neurons. The mechanisms involved may include also

antagonistic NTS1/D2 interactions in the cortico-striatal glutamate terminals and in the nigral DA cell bodies and dendrites as well as in the nigro-

striatal DA terminals. The possible increase in NT levels in the basal ganglia under pathological conditions leading to the NTS1 enhancement of

glutamate signalling may contribute to the neurodegeneration of the nigro-striatal dopaminergic neurons found in Parkinson’s disease, especially in

view of the high density of NTS1 receptors in these neurons. The use of selective NTS1 antagonists together with conventional drug treatments

could provide a novel therapeutic approach for treatment of Parkinson’s disease.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Neurotensin agonists and antagonists; Neurodegeneration; Receptor–receptor interaction; Excitotoxicity; Glutamate; Dopamine; Basal ganglia;

Parkinson’s disease

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

2. Neurotensin receptor mechanisms and modulation of glutamate systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

3. Neurotensin modulation of striatal, nigral and cortical glutamate release in the living brain . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

4. Neurotensin induced rise of extracellular glutamate levels in primary cultures of rat cortical neurons: evidence for facilitatory

NTS1/NMDA receptor interactions in control of glutamate release. Relevance for modulation of glutamate transmission . . . . . . . . . 95

5. Neurotensin enhancement of glutamate excitotoxicity in primary cultures of rat cortical neurons and rat mesencephalic

dopaminergic neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

5.1. The model of glutamate (30 and 100 mM)-induced neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

5.2. Effects of neurotensin on glutamate (30 mM and 100 mM)-induced neurotoxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Abbreviations: CalC, calphostin C; CNS, central nervous system; DA, dopamine; ERK, extracellular signal-regulated kinase; IP3, inositol 1,4,5-trisphosphate;

MAPK, mitogen-activated protein kinases; NMDA, N-methyl-D-aspartate; NT, neurotensin; PD, Parkinson’s disease; PI, phosphoinositide; PKC, protein kinase C;

TH, tyrosine hydroxylase; TH IR, tyrosine hydroxylase immunoreactive; VT, volume transmission

* Corresponding author. Tel.: +46 8 52487078; fax: +46 8 315721.

E-mail address: Kjell.Fuxe@ki.se (K. Fuxe).

0301-0082/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pneurobio.2007.06.006

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 93

6. Relevance of the neurotensin enhancement of glutamate-induced excitotoxicity for neurodegenerative disorders . . . . . . . . . . . . . 99

7. Neurotensin mechanisms and Parkinson’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

7.1. Neurotensin mechanisms in the substantia nigra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

7.2. NT mechanisms in the striatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

7.3. A possible role of NTS1 antagonists in antiparkinsonian therapy including anti-dyskinetic and neuroprotective effects. . . 103

8. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

1. Introduction

Neurotensin (NT) immunoreactive cell body and terminal

systems and their receptors are found in many parts of the brain

and especially interact with the mesolimbic, mesocortical and

nigro-striatal dopamine (DA) neurones (Emson et al., 1985;

Nemeroff and Cain, 1985; Deutch and Zahm, 1992; Fuxe et al.,

1992a, b; Rostene et al., 1992; Binder et al., 2001; Dobner et al.,

2003; Petrie et al., 2005; Antonelli et al., 2007). Much of the

focus in relation to NT–DA interactions and brain disease has

been on schizophrenia in view of the hypothesis raised by

Nemeroff (1980) that NT may be an endogenous neuroleptic. A

recent review on NT, schizophrenia and antipsychotic drug

action has also been published (Kinkead and Nemeroff, 2006),

underlining evidence that NT receptor agonists may represent

novel antischizophrenic drugs.

However, the important role central NT receptor mechan-

isms play in modulation of glutamate transmission has not been

reviewed nor its relevance for Parkinson’s disease except for a

recent review by St-Gelais et al. (2006) which briefly addressed

this issue. The reason is probably that it has only recently

become clear that NT enhances glutamate excitotoxicity in DA

neurons and that NT receptors are involved in NMDA induced

excitotoxicity through the work of the Tanganelli group

(Antonelli et al., 2002, 2004). The progress in this field is dealt

within this paper.

2. Neurotensin receptor mechanisms and modulation of

glutamate systems

Three NT receptor subtypes have been identified, NTS1,

NTS2 and NTS3. The NTS1 and NTS2 are G protein-coupled

receptors with a shared 60% homology. The NTS1 has a high

affinity for NT, while NTS2 has a substantially lower affinity

for the peptide (Tanaka et al., 1990; Chalon et al., 1996; Vincent

et al., 1999). The non-peptide NT antagonist SR48692 binds

preferentially to the NTS1 and has proven very useful for

elucidating the functions controlled by this receptor (Gully

et al., 1993; Rostene et al., 1997). In fact, many of the known

central and peripheral effects of NT are blocked by the NT

receptor antagonist SR48692 giving evidence for a major

involvement of NTS1 known to operate mainly via activation of

phospholipase C with increase of intracellular Ca2+ release and

activation of protein kinase C (PKC) (see Rostene et al., 1997).

The NTS2 is selectively recognized by the anti-histamine H1

receptor antagonist levocabastine (Chalon et al., 1996; Richard

et al., 2001).

The NTS3 belongs to an entirely different family of proteins,

namely type1 amino acid receptors with only one transmem-

brane spanning domain, located in intracellular vesicles of

neurons and glia and appears involved in NT inactivation, cell

sorting and in trophism in cancer cells (Nouel et al., 1999;

Mazella, 2001; Navarro et al., 2001). It can be translocated to

the surface membrane where in addition to acting as a

scavenger protein for extracellular NT it can heteromerize with

NTS1 leading to modulation of the NTS1 signalling in terms of

mitogen-activated protein kinase activation (MAPK) and

control of the phosphoinositide (PI) turnover (Martin et al.,

2002). There may also exist a potential NTS4 belonging to this

family, identified as SorLA/LR11 (Jacobsen et al., 2001) only

expressed in neurons. It may have a role in neural development

by stimulating proliferation and neurite growth.

The presence at the cellular level of NTS1 and D2 receptors

in the same axon terminal and dendrites (Delle Donne et al.,

2004) together with the demonstrated antagonistic intramem-

brane NTS1/D2 receptor–receptor interactions using biochem-

ical radioligand binding analysis in striatal membranes (Agnati

et al., 1983; von Euler and Fuxe, 1987; Tanganelli et al., 1989,

1993; Jansson et al., 2002; Fuxe et al., 1992b; Li et al., 1995;

Diaz-Cabiale et al., 2002; Antonelli et al., 2007) gives indirect

evidence for the existence of NTS1/D2 heteromerization. A

possible direct interaction between D2 and NTS1 receptors

with the formation of heteromers has also been considered by

Jomphe et al. (2006) as one of the possible but not exclusive

mechanisms underlying the functional control of striatal

dopamine D2 mediated transmission by NT. Such a possible

mechanism may be hypothesized since there is now strong

evidence that D2 receptors form homo- and hetero-oligomers

with different receptors like adenosine A2A (Fuxe et al., 1998),

somatostatin SSTR5 (Rocheville et al., 2000) and cannabinoid

CB1 (Kearn et al., 2005) thus influencing their ligand binding

pharmacology, intracellular signal transduction and trafficking

(Agnati et al., 2003; Fuxe et al., 2007). However, the NTS1/D2

receptor interaction in the DA neurons also involves a protein

kinase C and calcium dependent mechanism and prior

activation of D2 inhibits NTS1 signalling suggesting a

bidirectional regulation of D2 and NTS1 receptors (Jomphe

et al., 2006) in line with previous work on DA/NT interactions

at the recognition level of NT receptors in striatal membranes

(Agnati et al., 1985).

Most of the in vitro and in vivo studies on the role played by

NT in the central nervous system (CNS) have clearly indicated

that NTS1 regulates dopaminergic function both in nucleus

accumbens and in dorsal striatum by reducing D2 autoreceptor

Fig. 1. Effects of local perfusion with neurotensin (NT) alone or in combina-

tion with intraperitoneal administration of SR48692 on striatal (Panel A) or

substantia nigra pars reticulata (Panel B) extracellular glutamate levels in the

awake rat. NT was perfused into the striatum or the substantia nigra for 60 min

(solid bar) while SR48692 was i.p. administered at the arrow. Control rats were

perfused with normal Ringer perfusion medium throughout the experiment.

The results are expressed as percentage of the mean of the three basal values

before treatment. Each point represents the mean � S.E.M. of 6–8 animals.

The significance for the peak effects (maximal responses) is represented.

(Panel A) *p < 0.05; **p < 0.01 significantly different from control and

NT + SR48692 0.2 mg/kg; op < 0.05 significantly different from

NT + SR48692 0.1 mg/kg (Ferraro et al., 1995); (Panel B) **p < 0.01 sig-

nificantly different from control as well as SR48692 groups; op < 0.01

significantly different from NT + SR48692 group according to ANOVA

followed by the Newman–Keuls test for multiple comparisons (Ferraro

et al., 2001).

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–10994

function as well as the activity of postjunctional D2 receptors

located on glutamate terminals (Fuxe et al., 1992a,b; Wagstaff

et al., 1996; Goulet et al., 1999; Binder et al., 2001; Dobner

et al., 2003). The NT induced interference of inhibitory D2

autoreceptor signalling is postulated to take place mainly via

the existence of a NTS1/D2 autoreceptor heteromeric complex

in the plasma membrane of terminals and cell bodies/dendrites

of nigro-striatal dopamine neurons leading to increased DA

release. In a similar way it may be hypothesized that

postjunctional NTS1 via the formation of a possible NTS1/

D2 heteromeric receptor complex likely located mainly on the

plasma membrane of striatal glutamate terminals, antagonizes

the inhibitory D2 receptor mediated signalling on the glutamate

terminals, leading to an increase of glutamate release.

Facilitatory NTS1/N-methyl-D-aspartate (NMDA) receptor–

receptor interactions may also exist in cortico-striatal glutamate

terminals further enhancing glutamate release (Antonelli et al.,

2004). This mechanism may exist both at the membrane and

cytoplasmic level (involvement of PKC) and may play a

substantial role for enhancement of central glutamate

transmission in discrete brain regions (see below).

3. Neurotensin modulation of striatal, nigral and

cortical glutamate release in the living brain

Recently, by using the microdialysis technique, it has been

possible to show that NT plays a crucial role also in the

regulation of the aminoacidergic transmission in the basal

ganglia and cerebral cortex (Chapman and See, 1996; Ferraro

et al., 1997, 1998; Petrie et al., 2005; Chen et al., 2006). Thus,

the parent peptide NT(1–13) and its biologically active

carboxyl-terminal fragment NT(8–13) (Carraway and Leeman,

1975; Granier et al., 1982), enhance striatal glutamatergic

transmission possibly via the activation of local NTS1 receptors

on striatal glutamate terminals belonging to the cortico-striatal

glutamate system, since the effects of NT(1–13) are fully

counteracted by the selective NTS1 antagonist SR48692

(Ferraro et al., 1995; Fig. 1A). Similar results on glutamate

release were obtained after intranigral perfusion of NT in the

absence or presence of SR48692 in the pars reticulata of the

substantia nigra (Fig. 1B). This probably reflects the existence

of NTS1 on the glutamate terminals of the subthalamic-pars

reticulata system. Furthermore, NT enhances glutamate

signalling in the cerebral cortex. Thus, in rat cortical slices

(Ferraro et al., 2000) NT(1–13) slightly increases spontaneous

glutamate release but potassium (35 mM)-evoked glutamate

release was clearly increased by NT(1–13) with a bell-shaped

concentration response curve (Fig. 2A). The biologically active

NT fragment (8–13) mimicked the actions of NT(1–13), while

the biologically inactive fragment lacked effects (Fig. 2B). All

the effects were blocked by the NTS1 antagonist SR48692

(Fig. 2B). Thus, NT via NTS1 also facilitates cortical glutamate

transmission. Taken together, striatal, nigral and cortical

glutamate transmission may be strengthened by NT in circuits

where NTS1 exists, again underlining a role for NTS1 in

modulation of glutamate transmission, which in the case of

striatal glutamate transmission may involve, as previously

Fig. 2. Effects of neurotensin NT(1–13) (Panel A), its related peptide fragments

NT(8–13) and NT(1–7), SR48692 (100 nM) alone and in the presence of NT(1–

13) or NT(8–13) (Panel B) on K+ (35 mM)-evoked endogenous glutamate

release from rat cerebral cortex slices. The peptides were added to the super-

fusion medium 5 min before St2 and maintained until the end of the collection

period. The selective NT receptor antagonist SR48692 was added alone or

20 min before the peptides and maintained until the end of the collection period.

The data are presented as histograms of the St2/St1 ratio. K+-evoked glutamate

release were expressed as percent increase over the spontaneous (i.e. basal)

glutamate release, as calculated by the mean of the two fractions collected prior

to the depolarising stimulus. Each data point is the mean � S.E.M. of 5–7

experiments. (Panel A) **p < 0.01 significantly different from control and

NT(1–13) (1 and 1000 nM); op < 0.05 from NT(1–13) (100 nM). (Panel B)**p < 0.01 significantly different from all the other groups according to

ANOVA followed by the Newman–Keuls test for multiple comparisons (Ferraro

et al., 2000).

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 95

hypothesized, antagonistic NTS1/D2 like receptor–receptor

interactions in the cortico-striatal glutamate terminals.

4. Neurotensin induced rise of extracellular glutamate

levels in primary cultures of rat cortical neurons:

evidence for facilitatory NTS1/NMDA receptorinteractions in control of glutamate release. Relevance

for modulation of glutamate transmission

The following findings strengthen the evidence for a

functional role of NT receptors in modulating the neuronal

activity of glutamatergic neuronal cells in the cerebral cortex

(Antonelli et al., 2004). NT increases basal endogenous

glutamate release from rat cortical cell cultures. In fact, the

parent peptide NT(1–13) (0.1–100 nM) induced an increase in

extracellular glutamate levels, displaying a bell-shaped con-

centration–response curve (Table 1). These data are consistent

with previous findings (Faggin and Cubeddu, 1990) demon-

strating in the same range of concentrations that the NT-induced

increase of K+-evoked glutamate release and of electrically

evoked [3H]DA release display bell-shaped concentration

response curves. The reduced responsiveness of glutamatergic

Table 1

Treatment Glutamate levels (% of

basal values)

(A)

Control 101 � 3

NT(1–13) 0.1 nM 116 � 6

NT(1–13) 1 nM 148 � 7**,o

NT(1–13) 10 nM 145 � 5**,o

NT(1–13) 100 nM 112 � 7

NT(1–7) 10 nM 98 � 6

NT(1–7) 100 nM 101 � 7

NT(8–13) 10 nM 147 � 8**,o

(B)

Control 99 � 7

NT(1–13) 10 nM 144 � 6**

Ca2+ 0.2 mM 102 � 5

Ca2+ 0.2 mM + NT(1–13) 10 nM 104 � 6

(Panel A) Effects of NT(1–13) and its related peptides NT(1–7) and NT(8–13)

on extracellular glutamate levels in primary cultures of rat cerebral cortex

neurons. Glutamate levels measured in the third (30 min) fraction are reported

and expressed as percentage of the basal values (0.107 � 0.012 mM) calculated

from the mean of the first two fractions. NT(1–13) and its fragments were

applied at the onset of the third fraction and maintained for 30 min. Each point

represents the mean � S.E.M. of 14 experiments for each group. **p < 0.01

significantly different from control; op < 0.05 significantly different from 0.1

and 100 nM NT(1–13), according to one-way ANOVA followed by the New-

man–Keuls test for multiple comparisons. (Panel B) Effects of a low-calcium

medium (Ca2+ 0.2 mM) on NT(1–13)-induced increase of extracellular gluta-

mate levels in primary cultures of rat cerebral cortex neurons. Glutamate levels

measured in the third (30 min) fraction are reported and expressed as percentage

of the basal values (0.094 � 0.012 mM), calculated from the mean of the first

two fractions. NT(1–13) was applied at the onset of the third fraction and

maintained for 30 min. The culture medium was replaced with Krebs–Ringer

solution containing 0.2 mM Ca2+ from the onset of the experiment and it was

maintained until the end of the experiment. Each column represents the

mean � S.E.M. of six experiments for each group. **p < 0.01 significantly

different from the other groups, according to one-way ANOVA followed by the

Newman–Keuls test for multiple comparisons (Antonelli et al., 2004).

Fig. 3. Effects of treatments with 0.1 nM NT(1–13), 0.01 and 1 mM NMDA and

100 nM SR48692, alone and in combination, on extracellular glutamate levels

in primary cultures of rat cerebral cortex neurons. Glutamate levels measured in

the third (30 min) fraction are reported and expressed as percentage of the basal

values calculated from the mean of the first two fractions. NT(1–13) was applied

at the onset of the third fraction and maintained for 30 min, while SR48692 was

added 20 min before the agonist. NMDA was added to the Krebs–Ringer

solution 10 min before the end of the third fraction. Each column represents

the mean � S.E.M. of 13 or 14 experiments for each group. **p < 0.01

significantly different from all the other groups according to one-way ANOVA

followed by the Newman–Keuls test for multiple comparisons (Antonelli et al.,

2004).

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–10996

neurones to the highest concentrations of NT(1–13) may be due

to a rapid desensitization of the NT receptors which may

involve an internalization of the NT/NTS1 complex (Beaudet

et al., 1994). This phenomenon seems to occur also when the

effects of the peptide were assessed on other neuronal systems

in various brain regions (Faggin and Cubeddu, 1990; Deutch

and Zahm, 1992; Heaulme et al., 1997, 1998).

An additional explanation for the disappearance of NT-

induced glutamate release by high concentrations of the peptide

may be related to the activation of low affinity NTS2 receptors.

In fact, Perron et al. (2007) have recently provided evidence

that formation of NTS1/NTS2 heterodimerization, without

affecting NT-induced internalization of NTS1, modulates the

NTS1 receptor trafficking by making it more similar to that of

NTS2 and decreases the cell surface density of NTS1.

The involvement of NT receptors appears to be further

supported by the effects of NT fragments since NT(8–13)

(10 nM) similarly to the parent peptide increased glutamate

release whereas NT(1–7) (10 and 100 nM) was without

effect (Table 1). The NT(1–13) induced increase in extra-

cellular glutamate levels was prevented by the replacement

of the normal Krebs–Ringer bicarbonate buffer with a low-

Ca2+ (0.2 mM) medium suggesting the involvement of a

Ca2+-dependent mechanism (Table 1). The activation of NTS1,

which is functionally coupled to the inositol 1,4,5-trispho-

sphate (IP3) cascade, induces an increase of intracellular

Ca2+ ([Ca2+]i) in a biphasic way. There is an initial rise of

Ca2+ intracellularly by release from the endoplasmatic

reticulum followed by a second long-lasting Ca2+ plateau

due to a Ca2+ store-dependent entry from the extracellular

milieu (Pigozzi et al., 2004).

The potential modulation by NT of the glutamatergic

receptor signalling, in particular the responsiveness of the

NMDA receptors, was also evaluated. The possible existence of

a reciprocal interaction between NTS1 and NMDA receptor

mediated signals could play a relevant physio-pathological role

in cortical neuronal function, the NMDA receptors being

especially important for the toxic actions of glutamate. The

application of NMDA (0.01–10 mM) to cortical cell cultures

induced a concentration-dependent increase in endogenous

extracellular glutamate levels. This effect was enhanced even in

the presence of a subthreshold concentration (0.1 nM) of NT

(1–13) (Fig. 3), an action blocked by the NTS1 antagonist

SR48692 (Fig. 3). These results suggest that NT enhances the

NMDA-receptor signalling via activation of the NTS1 subtype

as shown by the SR48692-induced counteraction of this action

and indicate the existence of facilitatory NTS1/NMDA

interactions at the membrane and/or the cytoplasmic level

through interactions between the signalling pathways of the two

receptors involving an NTS1 mediated activation of PKC

leading to phosphorylation of the NMDA receptor. NTS1 has

also an extrasynaptic localization (Dana et al., 1989; Beaudet

and Woulfe, 1992; Boudin et al., 1998; Caceda et al., 2006) and

therefore NTS1 may possibly form complexes with extra-

synaptic NMDA receptors rich in NR2B subunits activated by

glutamate spillover and by glutamate released from astrocytes

(Stocca and Vicini, 1998; Del Arco et al., 2003). Such

extrasynaptic NMDA receptors trigger silencing of CREB and

activation of cell death pathways (Hardingham et al., 2002). A

direct facilitatory NTS1/NMDA interaction may therefore

produce plastic changes in glutamate transmission and, if

excessive, produce increases in glutamate-induced excitotoxi-

city. However, at the moment it remains to be shown if NTalone

can still increase glutamate release in presence of an NMDA

receptor antagonist. The lack of a NT-mediated enhancement of

glutamate outflow in the presence of NMDA blockade will

further support our hypothesis that NT would be a true

modulator of NMDA signalling.

It can be postulated that under physiological conditions a

NTS1/NMDA heteromeric complex and/or a receptor interac-

tion through signalling pathways (involvement of PKC), may

modulate metaplasticity which is another main mode of

homeostatic plasticity (see Perez-Otano and Ehlers, 2005),

which serves to establish that receptor plasticity may exist in a

proper working range, avoiding, e.g. a dramatic NMDA

receptor internalization and downregulation. Since phospho-

lipase C-PKC-IP3 pathway is the major signal transduction

pathway known to be activated by NTS1 and the existence of an

mGluR1-mediated potentiation of NMDA receptors was

demonstrated involving the activation of PKC (Skeberdis

et al., 2001), the role of the PKC activity in the interactions

between NT and NMDA receptors was also investigated

(Matsuyama et al., 2002) and found to be involved in the

interaction. Thus, the inhibitor of PKC, calphostin C (CalC;

0.1 mM), prevented the NTS1/NMDA synergism in increasing

extracellular glutamate levels suggesting that phosphorylation

of the NMDA receptors or associated proteins was part of the

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 97

molecular mechanism. Thus, NTS1 induced PKC activation

can play a substantial role and PKC activation is known to cause

a sixfold increase in NMDA receptor surface expression

involving phosphorylation of the receptor itself or a receptor

associated protein (Lan et al., 2001). In view of the fact that the

NT induced rise of glutamate release is dependent on

extracellular Ca2+ it can also be suggested that the small

activation of phospholipase C by 0.1 nM NT leads to a rise of

intracellular Ca2+ too small to release glutamate unless a certain

influx of Ca2+ passes through activated NMDA channels

(Matsuyama et al., 2002).

In the present primary cortical cultures the NT receptors may

be located both at the somadendritic and terminal level of

cortical neurons together with NMDA and/or D2 receptors.

Based on the paper by Legault et al. (2002) our findings on NT

enhancement of glutamate release with a calcium dependency,

may be related to the production of an inward current and

increase of the firing rate of the cortical neurons associated with

an increase in the frequency of spontaneous glutamate receptor

mediated EPSCs. Based on the above paper reporting that NT

can reduce the ability of terminal D2 receptors to inhibit the

action potential evoked EPSCs we can also suggest that a NT

induced reduction of the inhibitory D2 postjunctional function

at the glutamate terminal level can contribute to the

enhancement of glutamate release observed in the present

primary cortical cultures. Furthermore, in view of the failure of

NT in the above paper to change the frequency of miniature

EPSCs it seems likely that such a NTS1/D2 receptor–receptor

interaction is a major action of NT in our model of primary

cortical neurons.

It is also known that increased PKC activity can lead to

activation of the Raf-MEK-ERK cascade via activation of the

small G protein Ras, which have important consequences for

neuronal plasticity (Sweatt, 2001; Colucci-D’Amato et al.,

2003). It is therefore of substantial interest that subthreshold

concentrations of NT and NMDA when given together could

synergistically activate extracellular signal-regulated kinase

(ERK) in primary cortical cultures (unpublished data). This will

lead to phosphorylation of a number of membrane associated

and cytosolic proteins as well as a number of transcription

factors once it becomes translocated into the nucleus, leading

also to regulation of gene transcription. It should be noted that

NT also by increasing intracellular Ca2+ levels can activate

ERK, and ERK signalling is in turn essential for Ca2+ activated

transcription, another mechanism used to produce plastic

responses in nerve cells.

5. Neurotensin enhancement of glutamate excitotoxicity

in primary cultures of rat cortical neurons and rat

mesencephalic dopaminergic neurons

The above in vitro and in vivo findings on NT/glutamate

interactions have led to the hypothesis that a NTS1-mediated

amplification of glutamate signalling could underlie a

pathophysiological role of NT in cortical, striatal and nigral

regions (Ferraro et al., 1995, 1998). In fact, the substantial

elevation in extracellular glutamate and, consequently, the

excessive stimulation of glutamate receptors, especially

NMDA receptors, are implicated in the neuronal cell death

during degenerative processes. Experiments will be summar-

ized showing the NT enhancement of the vulnerability of

cultured mesencephalic dopaminergic neurons and cortical

neurons to excitotoxic injury (a 10 min exposure to glutamate)

(Antonelli et al., 2002, 2004). In view of the potential

significance of a delayed form of glutamate neurotoxicity, the

cell damage was evaluated through different morphological,

biochemical and immunocytochemical parameters 24 h follow-

ing toxic exposure.

5.1. The model of glutamate (30 and 100 mM)-induced

neurotoxicity

Glutamate has been the major focus of research into the

excitotoxic basis of neurodegenerative disease (Choi, 1988a;

Meldrum and Garthwaite, 1990). In this context, several studies

suggested a link between glutamate excitotoxicity and the

degeneration of nigro-striatal dopamine cells during Parkin-

son’s disease (Blandini et al., 1996; Sonsalla et al., 1998; Doble,

1999). This view is supported by the fact that nigro-striatal

dopaminergic cells have been found to contain different types

of glutamate receptors (both ionotropic and metabotropic)

(Christoffersen and Meltzer, 1995; Meltzer et al., 1997; Yung,

1998; Mateu et al., 2000) with the NMDA receptors being

especially important for the toxic action of glutamate. Together

with these findings, the observation that NT enhances glutamate

signalling in the basal ganglia (Ferraro et al., 1995, 1998) led to

the hypothesis that an amplification of glutamate signals could

underlie a pathophysiological role of NT in this brain region.

Thus, mesencephalic cell cultures, which contain dopaminergic

neurons and express functional NT receptors (Brouard et al.,

1992, 1994; Nouel et al., 1997; Nalivaiko et al., 1998) and

glutamate receptor subtypes (Christoffersen and Meltzer, 1995;

Meltzer et al., 1997; Yung, 1998; Mateu et al., 2000), provide a

suitable model for testing the influence of NT on the degree of

DA cell damage induced by exogenous glutamate through the

evaluation of [3H]DA uptake and tyrosine hydroxylase (TH)

immunocytochemistry (Antonelli et al., 2002). The [3H]DA

uptake analysis in mesencephalic cell cultures represents a safe

test to evaluate DA cell damage in view of the fact that among

dissociated mesencephalic cells mainly DA cells take up and

accumulate [3H]DA (Antonelli et al., 2002). The DA

transporter system in mesencephalic cultures may therefore

give an index of the structural and metabolic state of the DA

cells in these cultures and be used to study neurotoxic actions

on the DA cells. TH positive cell counts make it possible to

measure DA cell survival or loss of phenotype (Bowenkamp

et al., 1996). A brief exposure of cultured DA mesencephalic

cells to increasing concentrations of glutamate led to a

concentration dependent reduction of [3H]DA uptake. In

contrast, only 100 mM glutamate reduced significantly the

number of TH IR cells. This discrepancy with the two methods

used may be due to the fact that with the low concentrations

used glutamate causes only an initial excitotoxic phase where

the cells are viable but with an impaired DA uptake system. A

Fig. 4. (Panel A) Effects of neurotensin (NT) on glutamate (Glu) 30 mM-

induced reduction of [3H]DA uptake in cultured mesencephalic cells. (Panel B)

Effect of NT alone and in the presence of SR48692 on the 100 mM glutamate

(Glu)-induced reduction in the number of tyrosine hydroxylase (TH)-immu-

noreactive (IR) cells. The cultures were exposed to Glu for 10 min, and NT was

added 50 min prior to Glu and maintained in contact with the cells during the

Glu exposure (total treatment time 60 min). When required, the non-peptide NT

receptor antagonist SR48692 (by itself ineffective, data not shown) was added

to the medium 20 min prior to the peptide and maintained in contact with the

cells during NT and Glu exposure (total treatment time 80 min). [3H]DA uptake

or TH immunocytochemistry was assessed 24 h later. Mean � S.E.M. values of

16–20 determinations are shown. (Panel A) **p < 0.01 significantly different

from Glu, NT as well as from NT (1 nM) + Glu-treated group according to one-

way ANOVA, followed by the Newman–Keuls test for multiple comparisons.

(Panel B) **p < 0.01 significantly different from control as well as NT;oop < 0.01 significantly different from Glu alone as well as from

SR48692 + NT + Glu treated groups according to one-way ANOVA, followed

by the Newman–Keuls test for multiple comparisons (Antonelli et al., 2002).

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–10998

similar difference in the sensitivity of a biochemical versus a

morphological parameter to detect a toxic insult has previously

been reported also for cultured cortical cells (Tomasini and

Antonelli, 1998). It was not possible to distinguish between

apoptotic versus necrotic processes in this analysis. However, in

cultured cortical neurons glutamate produces a pattern of

excitotoxicity that occurs across an insult-dependent con-

tinuum with prevalent necrosis after intense toxic insults and

widespread apoptosis after mild insults (20–50 mM) (Cheung

et al., 1998). We have therefore challenged cortical nerve cells

with 30 mM glutamate and were able to induce a prevalent

apoptotic cell death revealed by nuclear chromatin condensa-

tion (control: 16 � 3% apoptotic cells, n: 9; glutamate 30 mM:

49 � 5%, n: 10).

5.2. Effects of neurotensin on glutamate (30 mM and

100 mM)-induced neurotoxicity

As shown in Fig. 4A, the neurotoxic effects of glutamate on

the dopaminergic neurons are exacerbated by NT (10 nM).

With 30 mM glutamate the reduction of [3H]DA uptake was

augmented with NT (10 nM) that by itself was ineffective.

Evidence has been obtained (Antonelli et al., 2002) that the NT-

mediated potentiation of glutamate excitotoxicity may be

mediated by PKC-induced phosphorylation(s). Thus, the PKC

inhibitor CalC (0.1 mM) prevented the NT action on [3H]DA

uptake. Specifically, it may suggest that phosphorylation of

NMDA receptors may be involved in the NT-induced

potentiation of glutamate neurotoxicity in view of the CalC-

induced inhibition of the NTS1-mediated enhancement of

NMDA-induced increases of extracellular glutamate levels in

cortical cultures (see above). However, it should also be

considered that PKC activity may inhibit DA transporter

activity which may contribute to the reduction of the [3H]DA

uptake observed.

Nevertheless, the potential pathological role of NT has been

confirmed by the analysis of morphological parameters both in

mesencephalic and cortical cultures (Antonelli et al., 2002,

2004). As seen in Fig. 4B, the enhancing action of NT on

glutamate-induced toxicity in DA cells could be demonstrated

by the increased disappearance of TH positive nerve cells found

in mesencephalic cultures after combined treatment with

glutamate 100 mM and NT 10 nM that at this concentration was

ineffective. In cultured cortical neurons 10 nM NT in

combination with glutamate increased the number of apoptotic

nerve cells to 62 � 3% indicating an involvement of NT

receptors in the glutamate-induced apoptosis. Evidence has

been obtained that the selective non-peptide NTS1 antagonist

SR48692 (100 nM) counteracts the enhancing effects of NT on

glutamate-induced morphological and biochemical neurotoxic

changes in DA and cortical neurons including apoptotic

neuronal death (Antonelli et al., 2002, 2004). Thus, NT

receptors may participate in the excitotoxic process leading to

cell death and in particular in apoptotic mechanisms.

Recently, it has been reported (Pigozzi et al., 2004) that

agents mobilising Ca2+ from the endoplasmatic reticulum are

able to accelerate apoptosis in CHO cells through the

expression of GADD153 (growth arrest and DNA damage

inducible gene 153) and the activation of KCa channels. Since

NTS1 activation induces an IP3-Ca2+ release from endoplas-

matic reticulum followed by a Ca2+ store-dependent entry of

Ca2+, this could represent still another mechanism by which NT

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 99

receptor activation accelerates the apoptotic process induced by

glutamate. It should also be considered that stress of the

endoplasmatic reticulum is connected to downstream apoptotic

events like 12, 9 and 3 caspase activation (Nakagawa et al.,

2000; Morishima et al., 2002) and release of cytochrome c from

nearby mitochondria (Hajnoczky et al., 2000).

In this context, recent findings have shown that, in an in vitro

model of cortical ischemia, NTS1 receptors activation enhances

both extracellular glutamate levels and apoptotic nerve cell

death (Antonelli et al., submitted for publication).

6. Relevance of the neurotensin enhancement ofglutamate-induced excitotoxicity for neurodegenerative

disorders

Evidence has accumulated that glutamate, the major

excitatory neurotransmitter in the CNS of vertebrates, is an

important mediator of neuronal injury. The extracellular

accumulation of glutamate and the excessive activation of

glutamate receptors, particularly NMDA receptors (Choi et al.,

1988b; Choi et al., 1990; Doble, 1999; Sattler and Tymianski,

2001) has been postulated to contribute to the neuronal cell

death associated with chronic neurodegenerative disorders

including Alzheimer’s, Parkinson’s and Huntington’s diseases

(Arias et al., 1998; Sonsalla et al., 1998; Schiefer et al., 2002)

and pathologic events such as hypoxia and ischemia (Johnston

et al., 2001).

In cultured cortical neurons glutamate-induced excitotoxi-

city, via NMDA receptors, induces apoptosis or necrosis

depending on the intensity of the insult. Mild glutamate insults

lead to an apoptotic cell death while an intense glutamate insult

predominantly leads to a necrotic cell death (Cheung et al.,

1998). Therefore, endogenous compounds able to modulate

glutamatergic transmission may interfere with glutamate-

induced cell death. In view of the enhancing effects of NT

on glutamate transmission and glutamate-induced neurotoxi-

city reported above, this peptide may play a relevant role in

reinforcing the effects exerted by glutamate on a variety of CNS

functions and pathologies, in particular on glutamate-mediated

excitotoxicity. Thus, it may be postulated that NT via NTS1

may cause such effects via several mechanisms:

1. N

TS1 activation increases glutamate release in several brain

regions.

2. N

TS1 enhances NMDA receptor signalling through direct

NTS1/NMDA receptor interactions and/or PKC activation

followed by phosphorylation of NMDA receptors and

increased trafficking to the surface membrane.

3. N

Fig. 5. Neurotensin (NT) mechanisms in the substantia nigra. Compensatory

activation of NT release from nigral NT terminals in response to onset of

Parkinson’s disease. Increased dopamine (DA) cell firing by, e.g. activation of

inhibitory NTS1/D2 autoreceptor interactions and of facilitatory NTS1/NMDA

(receptor–receptor and/or cytoplasmic) interactions on glutamate (Glu) term-

inals and nigral DA cells. Increased excitotoxicity in the DA nerve cells may

develop via increased glutamate release and NMDA signalling increasing DA

cell degeneration. NTS1 antagonists are neuroprotective.

TS1 via PKC activation can activate the Raf-MEK-ERK

cascade and NMDA can activate this cascade via calcium

influx through NMDA channels with activation of calmo-

dulin kinases followed by ERK activation. In this way ERK

activation may be increased and prolonged and its

subcellular localization altered. Such events may strongly

contribute to the increased excitotoxicity observed, since

chronic activation of ERK with an aberrant localization in

the cell coupled with failure to translocate to the nucleus due

to binding to anchor proteins may trigger a program of cell

death (see Colucci-D’Amato et al., 2003). Chronic ERK

activation may therefore lead to neurodegenerative disease.

In view of above it may be suggested that NTS1 activation

may be involved in the etiology or progression of neurode-

generative pathologies and that treatment with NTS1 receptor

antagonists could perhaps provide a novel therapeutic approach

against neurodegenerative disease considering the demonstra-

tion that the NTS1 antagonist SR48692 could counteract NT

amplified glutamate excitotoxicity.

7. Neurotensin mechanisms and Parkinson’s disease

7.1. Neurotensin mechanisms in the substantia nigra

Parkinson’s disease (PD) is a neurodegenerative disorder

characterized by a progressive loss of nigro-striatal dopami-

nergic neurons (see Calne et al., 1992). Fernandez et al. (1995,

1996) found increased NT levels in the substantia nigra of PD

patients and suggested that these changes may be an integral

part of the pathology rather than a consequence of the DA

neuron degeneration. Nor does it seem to be the result of the

pharmacotherapy (Fernandez et al., 1996). However, a rise of

striatal NT has not been consistently observed in PD. Fernandez

et al. (1995) did not observe an increase of NT in striatum nor in

globus pallidus in Parkinsonian patients versus controls.

Furthermore, other groups failed to detect any NT changes

in basal ganglia including the substantia nigra (Bissette et al.,

1985; Emson et al., 1985). Thus, conflicting results exist in this

area. However, in hemiparkinsonian rats NT is instead

increased in the striatum and not in the substantia nigra,

showing the complexity arising in models of PD (Engber et al.,

1991; Taylor et al., 1992). In contrast, the NT receptors are

markedly reduced in the substantia nigra and striatum of PD

patients (Chinaglia et al., 1990; Yamada and Richelson, 1995)

Fig. 6. (Panel A) NTS1 containing dopamine (DA) and glutamate (GLU) nerve terminals in local circuits of the striato-pallidal GABA neurons showing the DA, GLU

and neurotensin (NT) signalling in the physiological state. One DA and one glutamate synapse are indicated. Low NT tone and baseline release of DA and glutamate

are indicated. The low density of NTS1 in the striato-pallidal GABA neurons is not indicated. VT, volume transmission. (Panel B) In Parkinson’s disease, loss of DA

terminals followed by reduced D2 activity leads to an increased synthesis and release of NT from the dendrites and dendritic spines via increase of adenosine A2A

signalling due to reduced D2 inhibition of A2A signalling. Increased NT signalling (VT) with increased inhibition of D2 autoreceptor function via NTS1/D2

antagonism in the DA terminal: partial rescue of DA release. Increased NT signalling (VT) with increased facilitatory NTS1/NMDA (receptor–receptor and/or

cytoplasmic) interaction in GLU terminal: increase of glutamate release to compensate for partial loss of GLU synapses. Increased antagonistic NTS1/D2 interactions

at the GLU terminal to reduce D2 signalling to further favour enhancement of GLU release. (Panel C) After treatment with D2 antagonists, the blockade of the D2

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109100

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 101

and of animals with lesions of the ascending DA pathways to

the striatum (Palacios and Kuhar, 1981; Cadet et al., 1991; Tanji

et al., 1999). This disappearance is caused by the fact that a high

density of NTS1 exists in the DA cell bodies and terminals of

the nigro-striatal DA systems. The following NT mechanism

may therefore be proposed to be in operation in the substantia

nigra contributing to the degeneration of the nigro-striatal DA

cells in PD.

As a response to a reduced striatal DA transmission in PD

the NT terminal system innervating the substantia nigra (Emson

et al., 1985) becomes activated resulting in increased nigral NT

levels and release with subsequent increased activation of the

large numbers of NTS1 located on the nigral DA cell bodies

where the majority of the nigral NTS1 receptors are located

(Fig. 5) (Boudin et al., 1996; Alexander and Leeman, 1998).

The antagonistic action of NT on the function of D2 receptor

located on nigral DA cell bodies can contribute to the ability of

intraventricular injections of NT agonists to reduce tremor and

rigidity in rat models of PD (Jolicoeur et al., 1991; Rivest et al.,

1991) by increasing DA cell firing and striatal DA release

(Fig. 5). However, NT has been found to increase nigral

glutamate release (Ferraro et al., 2001) and the site of action has

been postulated to be located on NTS1 receptors present on

nigral glutamatergic afferents. Thus, NT may in this way

enhance glutamate-induced excitotoxicity either through an

increase of glutamate release or a facilitatory NTS1/NMDA

receptor interactions. The activation of NTS1 receptor located

on nigral glutamatergic terminals may therefore contribute to

the degeneration of the nigral DA cells in PD (Fig. 5). In fact,

there exists a large literature giving evidence that nigral

glutamate afferents play an important role in enhancing

neurodegeneration of DA cells in animal models of PD

(Gardoni and Di Luca, 2006; Doble, 1999; Przedborski, 2005).

There exists inter alia strong evidence that NMDA receptor

antagonists prevent neurodegeneration of DA cells in the

substantia nigra in models of PD (see e.g. Zeevalk et al., 2000)

which probably is related to the fact that the DA cells are

metabolically compromised in these models.

An increase in energy demands by the NTS1 activation of

the DA cells may also contribute to such an increase in

degeneration due to an increased firing rate in the DA cells

caused also by reduction of the D2 autoreceptor signalling

(Tanganelli et al., 1989; Shi and Bunney, 1990; Fuxe et al.,

1992a, b) via activation of the antagonistic NTS1/D2

autoreceptor interaction (Fig. 5) (Diaz-Cabiale et al., 2002).

It has been demonstrated that NT receptor binding especially in

the nigro-striatal DA system is reduced in brains of PD patients

and in the basal ganglia of hemiparkinsonian rats and MPTP-

treated mice probably as a result of the ongoing degeneration of

autoreceptors and the postjunctional D2 receptors located on the dendritic spines a

increased activation of the adenosine A2A receptors (no longer restrained in their

signalling from GLU receptors like the NMDA receptors on the dendritic spines lead

dendritic spines. Via diffusion (VT) increased amounts of NTwill reach the NTS1 on

and DA release via NTS1 signalling and facilitatory NTS1/NMDA (receptor–recepto

the striato-pallidal D2-rich GABA pathway. Junctional and nonjunctional DA term

operates via VT. Instead, junctional DA terminal operates mainly via synaptic transm

extrasynaptic release (see Fuxe and Agnati, 1991; Agnati et al., 2000).

the nigral DA cells in which it may actively participate (see

above). This hypothesis therefore postulates that NT receptor

antagonists should have a neuroprotective potential against

nigro-striatal DA neuron degeneration if used in treatment of

PD. It should be underlined that chronic ERK activation may

contribute to neuronal cell death as in DA cell degeneration in

PD (Stanciu et al., 2000; Kulich and Chu, 2001; Zhu et al.,

2002; Bezard et al., 2005). Thus, one mechanism for NT to

contribute to DA cell death via activation of NTS1 receptors

may be the production of ERK activation, results which in fact

has been obtained in cortical cell cultures in the present

laboratory (unpublished data). Action of NT on NT receptor

subtypes located on astroglia and microglia should also be

considered in the mechanisms underlying the action of NT on

neurodegeneration (Martin et al., 2003; St-Gelais et al., 2004).

7.2. NT mechanisms in the striatum

Surmeier and colleagues (Day et al., 2006) have recently

obtained evidence for a selective elimination of glutamate

synapses on striato-pallidal GABA neurons in models of PD

with strong loss of spines. The mechanism was shown to be

disinhibition of Cav1.3 L-type calcium channels caused by loss

of D2 receptor activity located on such spines. The mechanism

may involve a rise of Ca2+ levels in the spines resulting in a

breakdown of the cytoskeleton. It is therefore of substantial

interest that DA depletion in animal models of PD (Fig. 6A and

B) and in particular blockade of D2 receptors by typical

antipsychotic drugs cause a rise of NT neurotransmission which

is associated with a NTS1-mediated induction of c-fos

expression in the dorsal striatum (Fig. 6C) (Fadel et al.,

2001; Dobner et al., 2001; Binder et al., 2004) and in the

accumbens (Govoni et al., 1980; Frey et al., 1986; Radke et al.,

1998). Such an increase in NT levels and c-fos expression has

been predominantly observed in the striato-pallidal GABA

neurons (Brog and Zahm, 1996; Merchant and Miller, 1994;

Robertson et al., 1995; Dobner et al., 2003). It should be noted

that atypical antipsychotic drugs such as clozapine and

olanzapine increase extracellular NT levels only in the nucleus

accumbens (Radke et al., 1998).

NTS1 (Boudin et al., 1996; Alexander and Leeman, 1998)

and D2 receptors likely exist on the striatal glutamate terminals

synapsing on the spines of the striato-pallidal GABA neurons. It

may be that the rise of NT synthesis and release in the dorsal

striatum upon loss of D2 receptor activity is a mechanism to

compensate for the loss of D2 activity and of glutamate

synapses in PD (Fig. 6A and B). Thus, released NT can increase

DA release via the antagonistic NTS1/D2 autoreceptor

interaction (see Diaz-Cabiale et al., 2002; Binder et al.,

nd on the GLU terminals induces a rise of DA and GLU release and also an

signalling by the D2 receptors, since they are blocked). A2A signalling and

to increased Fos expression with increases of NT synthesis and release from the

the DA and GLU terminals where the activation will aim to further increase GLU

r and/or cytoplasmic) interactions resulting in a further rise of the excitation of

inals are represented (Descarries et al., 1996) The nonjunctional DA terminal

ission and probably also via volume transmission through synaptic spillover and

Fig. 7. (Panel A) NTS1 containing dopamine (DA) and glutamate (GLU) terminals in local circuits of the striato-nigral GABA neurons and neurotensin (NT), DA and

GLU signalling in these circuits in the physiological state Low NT signalling and baseline release of DA and GLU. (Panel B) NTS1 containing DA and GLU terminals

in local circuits of the striato-nigral GABA neurons and NT, DA and GLU signalling in these circuits after treatment with D2 antagonists. After blockade of the D2

autoreceptors and the postjunctional D2 receptors located on the GLU terminals there is a rise of DA and GLU release with increased activation of the D1 receptors

and glutamate receptors like the NMDA receptors on the dendritic spines leading to increased Fos expression with increases of NT synthesis and release. Via diffusion

(volume transmission: VT) increased amounts of NTwill reach the NTS1 on the DA and GLU terminals where the activation will aim to further increase GLU and DA

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109102

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 103

2004). It may be suggested that the enhancement of NT release

and induction of c-fos expression induced by haloperidol

administration may be due to the direct activation of NTS1 in

striatal neurons and/or NTS1 located on glutamatergic cortico-

striatal projections. As discussed above, NT has been found to

increase also nigral glutamate release (Ferraro et al., 2001) and

this action has been postulated to be mediated via NTS1

receptors present on nigral glutamatergic afferents. Thus, NT

may in part via this mechanism increase DA cell firing and also

enhance glutamate-induced excitotoxicity in the nigral DA

cells. In this context, the basic elements of our hypothetical

mechanism suggesting that endogenous NT signalling by

increasing glutamatergic transmission may be involved in

neurodegeneration and excitotoxicity in the basal ganglia are in

line with previous models proposed by other authors to

illustrate the involvement of endogenous NT in neuronal

activation following antipsychotic drug and psychostimolant

administration (Fadel et al., 2001; Dobner et al., 2001, 2003;

Binder et al., 2004; Fadel et al., 2006). In particular, the

interesting paper of Dobner et al. (2003), indicating that NT is

crucial for the ability of antipsychotic drugs and psychosti-

mulants to activate discrete subpopulations of striatal neurons,

gives NT mechanisms that are similar to those proposed in the

present paper to be involved in neurodegenerative disease.

In addition, the enhancement of endogenous NT signalling

may be triggered by the reduced D2 mediated DA transmission

in PD and after treatment with antipsychotic drugs by setting

free the A2A signalling in the A2A/D2 heteromer (Fuxe et al.,

2005) present in the striato-pallidal GABA pathway and the

striatal glutamate terminals leading to increase in AC and PKA

activity followed by phosphorylation of CREB contributing to

the increase in c-fos expression observed. Thus, there exists an

AP-1 consensus sequence in the promotor region of the NT/

neuromedin N gene (Kislauskis and Dobner, 1990) which when

activated by c-fos can substantially contribute to the increase of

striatal NT synthesis and release observed with D2 antagonists

(see above).

However, it should be considered that impact of the increase

of NT synthesis and release induced by a deficit in D2 receptor

function as in PD (Fig. 6B) may be self-limited since a

postulated antagonistic NTS2/NTS1 receptor–receptor inter-

action favouring interalia the decrease in the cell surface

density of NTS1 (Perron et al., 2007) together with a possible

internalization of NTS1 may act to limit the influence of

excessive NT on glutamate and DA release in PD.

Thus, in early-moderate PD NT may be released as a volume

transmission (VT) signal in these local circuits counteracting

postjunctional inhibitory D2 signalling at the glutamate

terminal (Wagstaff et al., 1996; Goulet et al., 1999) via an

antagonistic NTS1/D2 interaction (Fuxe et al., 1992b; Diaz-

Cabiale et al., 2002) and enhancing NMDA signalling via a

postulated facilitatory NTS1/NMDA receptor interaction, both

release via NTS1 signalling and facilitatory NTS1/NMDA (receptor–receptor and/or c

nigral D1-rich GABA pathway. Junctional and nonjunctional DA terminals are rep

volume transmission. Instead junctional DA terminal operates mainly via synaptic tra

and extrasynaptic release (see Fuxe and Agnati, 1991; Agnati et al., 2000).

of them favouring glutamate release and thus enhancement of

glutamate transmission (Fig. 6A and B). The drawback is,

however, that glutamate signalling may become too dominant

over DA D2 transmission with dysregulation of the striato-

pallidal neurons. In the same local circuit the diffusing NT may

inhibit D2 autoreceptor function at the DA nerve terminal via a

NTS1/D2 autoreceptor interaction leading to increased DA

release with a certain restoration of D2 receptor signalling in

the striato-pallidal GABA neurons and reduced dysregulation

of the Cav1.3 L-type calcium channels. NTS1 receptors may

exist in low density in the striato-pallidal GABA neurons

leading to reduced antagonism of their D2 signalling by NT

(Herve et al., 1986; Boudin et al., 1996; Alexander and Leeman,

1998; Tanji et al., 1999). By probably inhibiting D2

autoreceptor function also in the DA nerve terminals

innervating the direct pathway NT will increase D1 receptor

activity by increasing the amounts of diffusing DA in the local

circuits of the striato-nigral GABA neurons (Fig. 7A) which

will favour increased activity in this pathway resulting in

increased motor initiation. D1 transmission may dominate over

D2 transmission in a state of increased NT VT, since no

antagonistic postjunctional NTS1/D1 receptor interactions

have been demonstrated (Fuxe et al., 1992a,b; Diaz-Cabiale

et al., 2002). Instead, the increase of NT signalling reduces the

signalling of the inhibitory D2 receptors on glutamate terminals

in view of the likely existence of an antagonistic NTS1/D2

receptors interactions on these terminals, favouring an

increased glutamate drive of the striato-pallidal GABA neurons

and thus motor inhibition.

7.3. A possible role of NTS1 antagonists in

antiparkinsonian therapy including anti-dyskinetic and

neuroprotective effects

In view of the neuroprotective potential of NTS1 antagonists

in treatment of PD (see above) it becomes relevant to discuss if

NTS1 antagonists may have acute symptomatic antiparkinso-

nian properties. Behavioural evidence in fact exists that the

NTS1 antagonist SR48692 given systemically or intrastriatally

enhances the levodopa induced hyperlocomotion and stereo-

typies (only systemic treatment tested) in DA-deficient mice

(Chartoff et al., 2004). These results indicate that endogenous

NT attenuates DA dependent motor behaviours in line with the

present hypothesis (see below) that NT may be involved in the

activation of the striato-pallidal GABA neurons. In agreement,

systemically administered synthetic NT analogues are able to

reduce apomorphine-induced controlateral and amphetamine

induced ipsilateral rotational behaviours in hemiparkinsonian

rats (Boules et al., 2001). Furthermore, the neuroleptic-like

actions of NT as blockade of amphetamine induced locomotor

activity (Prange and Nemeroff, 1982; Binder et al., 2001;

Kinkead and Nemeroff, 2004) are counteracted by NTS1

ytoplasmic) interactions resulting in a further rise of the excitation of the striato-

resented (Descarries et al., 1996). The nonjunctional DA terminal operates via

nsmission and probably also via volume transmission thought synaptic spillover

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109104

antagonists (Gully et al., 1993, 1997; McCormick and Stoessl,

2003). Thus, it seems likely that these effects of the antagonists

may represent antiparkinsonian actions caused by the blockade

of the antagonistic NTS1/postjunctional D2 interaction in the

surface membrane of the striatal glutamate terminals. Thus, the

D2 inhibition of glutamate release is restored and in addition

the NT releasing action of glutamate via its own signalling in

the glutamate terminal is blocked (Ferraro et al., 1995). As a

consequence the glutamate drive of the striato-pallidal GABA

neurons is reduced and the motor inhibition caused via

activation of the indirect pathway is reduced and an

antiparkinsonian action in response to the NTS1 antagonist

can develop. However there also exist conflicting findings,

since systemic administration of the synthetic NT analogues

blocks haloperidol-induced catalepsy (Cusack et al., 2000).

Such anticataleptic effects of the NT analogues may, however,

be explained by postulating that activation of NTS1 receptors

on the nigral DA cells by the NT analogues may increase the

haloperidol-induced dendritic DA release. The diffusing DA

signal may then via VT reach the D1 receptors on the GABA

terminals of the direct pathway. Their activation will increase

GABA release inhibiting the nigro-thalamic GABA pathway to

the motor thalamus with a certain disinhibition of the excitatory

glutamate pathways to the cortical motor areas even in the

presence of widespread blockade of the D2 receptors in the

basal ganglia (see Fig. 8).

It was early on proposed that the extrapyramidal like side

effects of chronic treatment with typical antipsychotic drugs

were related to the long-lasting rise of NT levels in the dorsal

striatum (Fig. 6A and C) (Frey et al., 1986). The novel

neurochemical evidence presented here showing that NT via

Fig. 8. Role of neurotensin (NT) transmission in the substantia nigra after treatment w

bodies and dendrites and the postjunctional D2 receptors on the cell bodies and dendr

excitatory glutamate (GLU) motor drive to the cerebral cortex. Excitatory NT transmi

D2 autoreceptors leading to increases in DA release which via volume transmission a

enhancing synaptic GABA release (Radnikow and Misgeld, 1998). This leads to red

the thalamo-cortical glutamate pathway to the motor regions of the cerebral corte

NTS1 may increase the glutamate drive of the striato-pallidal

GABA neurons via facilitatory NTS1/NMDA interaction and

antagonistic NTS1/D2 receptor interactions at the striatal

glutamate terminal support this view (Fig. 6A and C). In

agreement, Stoessl (1995) has shown that vacuous chewing

movements similar to the ones seen in patients with tardive

dyskinesias can develop by injection of NT into the

ventrolateral striatum of rats. In addition, such dyskinesias

developed after chronic treatment in animals with typical

antipsychotic drugs are counteracted by NTS1 antagonists.

Based on above it is suggested that the mechanism for the

dyskinesia development involved may be the rather unique

ability of NTS1 activation to increase the glutamate drive

especially of the striato-pallidal GABA neurons including also

removal of the inhibitory D2 tone on glutamate terminals

(Fig. 6A and C) (see above), favouring motor inhibition. At the

same time NT increases activity in the direct D1-rich GABA

pathway favouring motor initiation via increasing glutamate

drive also of this system. Furthermore, increased D1 signalling

can help in this activation of the direct pathway due to the NT

induced increases in DA release (Fig. 7A and B). Models of

dyskinesias in rabbits are in fact characterized by a high D1/D2

ratio (see Ferre et al., 1994). Thus, there may be a role of NTS1

receptors in tardive dyskinesias and thus also a role for NT

receptor antagonists in its treatment (Frey et al., 1986;

McCormick and Stoessl, 2003). Dobner et al. (2001) have,

however, pointed out that the failure to modify haloperidol-

induced catalepsy in NT knockout mice argues against this

hypothesis. In our mind, however, this acute behavioural

response to D2 blockade may not be altered since the

antagonistic NTS1/D2 autoreceptor interaction and the

ith haloperidol blocking the D2 autoreceptors on the nigral dopamine (DA) cell

ites of the nigro-thalamic GABA pathway to the motor thalamus, controlling the

ssion via NTS1 receptors may become more dominant in the presence of blocked

ctivates D1 receptors on the striato-nigral GABA terminals to the zona reticulata

uced firing in the nigro-thalamic GABA pathway and increased disinhibition of

x with increases in motor drive reducing the halperidol induced catalepsy.

T. Antonelli et al. / Progress in Neurobiology 83 (2007) 92–109 105

antagonistic NTS1/D2 postjunctional interaction would not

matter since these D2 receptors are already fully blocked by

haloperidol and the glutamate drive to the striato-pallidal

GABA neurons may be sufficiently activated to produce the

catalepsy and the postulated anticataleptic actions of NT in the

substantia nigra are no longer present (see above). In line with

this view, it has been demonstrated that the NTS1 antagonist

SR48692 enhances haloperidol-induced catalepsy at subopti-

mal doses of haloperidol, suggesting that endogenous NT

normally opposes haloperidol-induced catalepsy (Casti et al.,

2004). According to the present hypothesis given above (see

Fig. 8) this action of NT is related to enhancement of nigral DA

release with a D1 mediated GABA release from the direct

pathway causing an increased inhibition of the nigro-thalamic

GABA pathway. Finally, Mesnage et al. (2004) in an

exploratory study reported that NT receptor antagonist could

not improve parkinsonian motor disability. However, in this

paper the authors reported that the lack of efficacy of NTS1

receptor antagonists could be attributed to the low dose used, as

demonstrated by the absence of adverse events observed in any

of the patients tested. In fact, it was concluded that further

studies with higher doses of NT receptor antagonists are

needed.

8. Conclusions

NT may be involved in increasing the degeneration of

dopaminergic mesencephalic neurons and cortical neurons by

enhancing glutamate signalling leading to excitotoxicity

probably via a rise of intracellular calcium and/or to an

amplification of the NMDA-mediated glutamate signalling.

Morphological and biochemical findings obtained in primary

cultures of rat cortical neurons and rat mesencephalic

dopaminergic neurons (Antonelli et al., 2002, 2004) strengthen

the evidence of an involvement of NT in neurodegenerative

processes.

The observed increase in NT levels in the basal ganglia and

cerebral cortex in pathological conditions leading to the NT

receptor enhancement of glutamate signalling may be in part

responsible for the neurodegeneration of the dopaminergic

neurons and cortical neurons occurring in PD and in pathologic

events such as hypoxia, ischemia and neurotrauma, respec-

tively. Also the increased striatal NT transmission seen in

dyskinesias after treatment with classical antipsychotic drugs

may have an important role in the dyskinetic development. The

use of selective NTS1 antagonists in combination with

conventional drug treatments could provide a novel therapeutic

approach especially for the treatment of PD in view of the high

densities of NTS1 receptors found in the nigro-striatal DA cells

and may result in both neuroprotective, symptomatic and

antidyskinetic therapy of this disease. This strategy is based in

part on the existence of antagonistic NTS1/D2 autoreceptor

interactions and postulated antagonistic NTS1/D2 like post-

junctional receptor interactions mainly at the level of the

cortico-striatal glutamate terminal and postulated facilitatory

NTS1/NMDA receptor interactions in the nigral DA cells and in

the striatum. Their blockade in PD by NTS1 antagonists may

reduce the excessive activation of the nigral DA cells associated

with excitotoxicity and the increased activation of the striato-

pallidal GABA neurons leading to motor inhibition.

Acknowledgements

This research has been supported by grants from the

Swedish Research Council (Sweden) and University of Ferrara

(Ricerca locale ex-60%). The authors thank Sanofi-Aventis

(France; also for SR48692 supply), ‘‘Fondazione Cassa di

Risparmio di Ferrara’’ (Italy) and IRET-Foundation (Italy).

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