Dopamine and Histamine Interaction in Basal Ganglia

Institutionen för Neurovetenskap
Biomedicinprogrammet
Examensarbete för magisterexamen i medicinsk vetenskap
med huvudinriktning biomedicin
Vårterminen 2006
Interaction between dopamine
and histamine in the basal ganglia
Författare: Mario Clementi
Författare: Mario Clementi
Handledare: Docent Gilberto Fisone, Institutionen för Neurovetenskap
Examinator: Docent Ewa Ehrenborg, Institutionen för Medicin

Institutionen för Neurovetenskap
Biomedicinprogrammet
Examensarbete för magisterexamen i medicinsk vetenskap
med huvudinriktning biomedicin
Vårterminen 2006
Interaction between dopamine
and histamine in the basal ganglia
Sammanfattning
L-DOPA-inducerad dyskinesi (LID) representerar i dagsläget den största
utmaningen i behandlingen av Parkinsons sjukdom. Dopamin aktiverar D1 och D2
receptorer i striatala nervceller. D1 receptorer ökar, medan D2 receptorer minskar
signaleringen av cAMP/PKA. Denna motsatta reglering återspeglas i
fosforyleringen av jonkanaler, receptorer och enzymer som styr nervcellers
aktivitet. Striatala nervceller uttrycker också höga nivåer av histamin H3
receptorer, som är kopplade till Gi/o proteiner. Det har nyligen visats att
aktiveringen av H3 receptorer minskar D1 inducerad cAMP ackumulering i striatala
skivor från råtthjärna. Här visar vi att aktiveringen av D1 receptorer i striatala
skivor från mushjärna ökar PKA medierad fosforylering av AMPA glutamat
receptorns subenhet GluR1 på Ser-845 (p < 0,00005) samt av DARPP-32 på Thr-
34 (p < 0,001). Fosforyleringen av dessa proteiner resulterar i en ökad transmission
av AMPA glutamat receptorn. Samtidig aktivering av H3 receptorn resulterade i en
icke-signifikant (p = 0.11) minskning av GluR1 fosforyleringen, vilket tyder på att
dopamin och histamin utövar en motsatt reglering på cAMP/PKA signaleringen i
striatonigrala nervceller. Fosforyleringen av GluR1 och DARPP-32 detekterades
genom western blot analys, m.h.a. fosforyleringsspecifika antikroppar. H3
receptorn kan representera ett målprotein för behandlingen av LID, som uppvisar
en abnorm D1 medierad cAMP/PKA/DARPP-32 signalering.
Abstract
L-DOPA-induced dyskinesia (LID) currently represents the major challenge to the
treatment of Parkinson’s disease. Dopamine activates D1 and D2 receptors on
striatal neurons. D1 receptors increase, whereas D2 receptors decrease cAMP/PKA
signaling. This opposite regulation is reflected on the state of phosphorylation of
ion channels, neurotransmitter receptors and enzymes involved in the control of
neuronal activity. Striatal neurons also express high levels of histamine H3
receptors, which couple to Gi/o proteins. Activation of H3 receptors was recently
reported to reduce D1 induced cAMP accumulation in rat striatal slices. Here we
show that activation of D1 receptors in mouse striatal slices increases PKA
mediated phosphorylation of the glutamate AMPA receptor GluR1 subunit at Ser-
845 (p < 0,00005) and of DARPP-32 at Thr-34 (p < 0,001). Phosphorylation of

reported to reduce D1 induced cAMP accumulation in rat striatal slices. Here we
show that activation of D1 receptors in mouse striatal slices increases PKA
mediated phosphorylation of the glutamate AMPA receptor GluR1 subunit at Ser-
845 (p < 0,00005) and of DARPP-32 at Thr-34 (p < 0,001). Phosphorylation of
these proteins facilitates glutamate AMPA receptor transmission. Moreover,
simultaneous activation of H3 receptors resulted in a non-significant (p = 0.11)
reduction of phospho[Ser-845 ]GluR1, suggesting that dopamine and histamine
exert an opposite regulation on cAMP/PKA signaling in striatonigral neurons.
Phospho[Ser-845]GluR1 and phospho[Thr-34]DARPP-32 were detected by
western immunoblotting, using phosphorylation site-specific antibodies. The H3
receptor could represent a target for the treatment of LID, which displays abnormal
activation of D1 mediated cAMP/PKA/DARPP-32 signaling.

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CONTENTS
LIST OF ABBREVIATIONS 5
INTRODUCTION 6
SYNAPTIC TRANSMISSION 6
HISTAMINE 6
DOPAMINE 9
SLOW SYNAPTIC TRANSMISSION IN THE STRIATUM 9
DARPP-32, AN INTEGRATOR OF SYNAPTIC TRANSMISSION IN THE STRIATUM 11
PARKINSON’S DISEASE AND LEVODOPA-INDUCED DYSKINESIA 13
MODULATION OF GLUTAMATERGIC TRANSMISSION IN THE STRIATUM 14
AIMS OF THE STUDY 16
MATERIALS AND METHODS 18
DRUGS 18
TREATMENT AND TISSUE PREPARATION 18
DETERMINATION OF PHOSPHOPROTEINS 18
RESULTS 20
REGULATION OF GLUR1 PHOSPHORYLATION BY SKF-81297 AND IMMEPIP 20
REGULATION OF DARPP-32 PHOSPHORYLATION BY SKF-81297 AND IMMEPIP 21
REGULATION OF GLUR1 PHOSPHORYLATION BY SKF-81297 AND (R)-α-
METHYLHISTAMINE 22
DISCUSSION 23
LEVELS OF PHOSPHOPROTEINS IN THE STRIATAL SLICE PREPARATION 23
PHARMACOLOGICAL PROFILE OF THE HISTAMINE H3 RECEPTOR 23
IMIDAZOLE H3-AGONISTS 24
CONCLUDING REMARKS AND FUTURE DEVELOPMENTS 25
ACKNOWLEDGEMENTS 27
LIST OF REFERENCES 28

Mario Clementi Interaction between dopamine and histamine in the basal ganglia
5
List of Abbreviations
AC Adenylyl cyclase
ADHD Attention Deficit Hyperactivity Disorder
AIMs Abnormal involuntary movements
AMPA (±)-alfa-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
ATP Adenosine triphosphate
cAMP Cyclic 3’, 5’-adenosine monophosphate
BBB Blood–brain barrier
CdK-5 Cyclin-dependent kinase-5
cDNA Complementary deoxyribonucleic acid
CREB cAMP-response element binding protein
DARPP-32 (D32) Dopamine and cAMP-regulated phosphoprotein of 32 kDa
ERK Extracellular signal-regulated protein kinase
G-protein Trimeric guanyl nucleotide-binding protein
GABA Gamma-aminobutyric acid
Gi/o proteins Inhibitory pertussis toxin sensitive G-proteins
GluR1 AMPA glutamate receptor subunit 1
Golf protein Olfactory neuron-specific G-protein
GPe Globus pallidus pars externa
GPi Globus pallidus pars interna
GPCR G-protein coupled receptor
GTPγS Guanosine 5’-thiotriphosphate
IgG Gamma-heavy chain class immunoglobulin
KRB Krebs-Ringer’s bicarbonate buffer
L-Ca2+ L type calcium channel
L-DOPA L-3,4-Dihydroxyphenylalanine
LID L-DOPA -induced dyskinesia
MAPK Mitogen-activated protein kinase
mRNA Messenger ribonucleic acid
N/P-Ca2+ N/P type calcium channel
NHP Nonhuman primate
NKA Na+, K+ ATPase
NMDA N-methyl-D-aspartate
PI3K Phosphatitylinositol 3-kinase
PKA Cyclic adenosine 3’, 5’-monophosphate-dependent protein kinase
PP-1 Protein phosphatase-1
PP-2A Protein phosphatase-2A
PP-2B Protein phosphatase-2B/calcineurin
PTX Pertussis toxin
Ser Serine
SKF-81297 6-Chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine-7,8-diol
SNpc Substantia nigra pars compacta
SNpr Substantia nigra pars reticulata
STN Subthalamic nucleus
Thr Threonine
TM Tuberomammillary

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Introduction
Synaptic transmission
There are two categories of chemical transmission between nerve cells—fast and
slow synaptic transmission. It is estimated that about 50% of the fast synapses in
the brain are excitatory and the majority of them use glutamate as their
neurotransmitter. In contrast, the remaining 50% of the fast synapses are inhibitory
and utilize GABA (gamma-aminobutyric acid) as their neurotransmitter.
Fast synaptic transmission occurs in less than one millisecond when fast acting
neurotransmitters bind to ligand-gated ion channels (also known as ionotropic
receptors) located in the plasma membrane of postsynaptic nerve cells. Ligand-
gated ion channels are heteromeric assemblies of 4-5 subunits arranged around an
aqueous pore. Binding of neurotransmitters causes a conformational change in the
ion channel, which transiently increases its permeability to particular ions.
Activation of glutamate receptors results in a net inward current carried mainly by
positively-charged sodium ions, which depolarises the target cell thereby
increasing the probability to generate an action potential. In contrast, activation of
GABA receptors results in increased permeablity towards negatively-charged
chloride ions, generating a hyperpolarizing signal in the postsynaptic nerve cell,
which reduces the probability to generate an action potential.
Slow synaptic transmission occurs over periods of time ranging between
hundreds of milliseconds to minutes. It has been estimated that there are
approximately 150 known neurotransmitters in the brain and most of them are
slow-acting and produce their effects by binding to receptors which couple to
trimeric guanyl nucleotide-binding proteins (GPCRs; also known as metabotropic
receptors or seven-transmembrane-helix [7TM] receptors) thereby initating a signal
transduction cascade. Secondary messengers carry the signal inside the cell and
activate distinct classes of protein kinases, which phosphorylate and thereby
change the properties of ion channels, ion pumps, neurotransmitter receptors and
transcription factors.
These relatively slow enzymatic reactions, such as protein phosphorylation and
dephosphorylation, are events critically involved in the control of neuronal
activity. The reason for this, is that they modulate, rather than mediate, fast
synaptic transmission and they do it in two ways. One way is by regulating the
state of phosphorylation of synapsins and other key proteins situated in the
presynaptic terminal, thereby modulating the efficacy of neurotransmitter release.
A second way is by regulating the state of phosphorylation of neurotransmitter
receptors located in the postsynaptic terminal, thereby modulating the
responsiveness of these receptors to the released neurotransmitter.
Histamine
One slow-acting neurotransmitter which has important actions in the central
nervous system (CNS) is the biogenic amine histamine (2-[4-
imidazolyl]ethylamine). This has been known since the discovery that classical
antihistamines produced sedative actions. The physiological role of histamine as
the cause of allergic reactions was discovered in 1910 by Henry Dale (1875-1968)
and Patrick Playfair Laidlaw (1881-1940) in London. They found that toxic doses of
histamine produced something similar to anaphylactic shock in animals.

7
Researchers then sought to find substances that could counteract the effects of
histamines. Around 1944 Paul Charpentier and his collaborators at the French firm
Rhône Poulenc carried out investigations on a number of non-oxidised
phenothiazines which possessed potent antihistaminic properties. The most
important product of this work was promethazine, produced in 1946, which could
coltrol motion sickness. In 1946, the swiss-born Italian pharmacologist Daniel Bovet
(1907-; winner of the 1957 Nobel Prize for medicine) and his colleagues found
another phenothiazine analogue—diethazine—that was effective in Parkinson’s
disease. In 1949 Henri Laborit, a French navy surgeon, become particularly
impressed by promethazine’s ability to relieve his patient’s anxiety. This led Rhône
Poulenc’s scientists in 1950 to develop the promethazine derivative
chlorpromazine. This compund became the first successful drug in the treatment of
schizophrenia (Swazey, 1975; Estes, 1995).
Despite the findings that antihistamic compounds had important actions in the
central nervous system, the histaminergic system was initially neglected when
research on the aminergic systems started to flourish in the middle of the 1960s.
The reason for this was that the fluorimetric assay that revealed the anatomical
identity of the catecholaminergic and serotonergic neurons and their projections
failed to determine the location of histamine in the brain, because the reagents that
were used to detect this diamine crossreacted with spermidine—a uniformly
distributed polyamine that occurs at high concentrations (Green, 1970). The
histaminergic system gained general acceptance only in 1984, after the
immunohistochemical demonstration that the tuberomammillary (TM) nucleus,
which is part of the posterior hypothalamus, was the sole seat of histaminergic
neurons and the origin of the widely distributed histaminergic projections (Fig. 1;
Panula et al., 1984; Watanabe et al., 1984). Four histamine receptors have now been
cloned (H1–H4). The H1–H3 receptors are widely expressed in distinct patterns in
the mammalian brain, whereas the H4 receptor expression is almost exclusively
restricted to hematopoietic cells and is suggested to mediate functions of the
immune system.
Figure 1. The histamine pathways in the brain. (Adapted from Purves et al., 2001)

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The human H3 receptor (Fig. 2) was recently cloned by Lovenberg et al., 1999, and
its complementary DNA (cDNA) contains an open reading frame of 445 amino
acids with only limited homology (21–22%) with the H1 and H2 receptor genes and
similarly low sequence homology (20–27%) to other known biogenic amine GPCRs
(Lovenberg et al., 1999). In fact, the human H3 receptor was initially considered a
new muscarinic receptor based on homology to the rat M1 receptor. Eventually the
H3 receptor was shown to be a member of the large super-family of GPCRs and to
couple to members of the pertussis toxin (PTX) sensitive Gi/o family of
heterotrimeric G-proteins to mediate the inhibition of adenylyl cyclase (the o in Gi/o
stands for other because it was the second PTX sensitive G-protein that was
discovered [Jiang et a l ., 2001]). The histamine H3 receptor was initially
characterized as an inhibitory autoreceptor controlling histamine synthesis and
release in the brain (Arrang et al., 1983, 1987). However histamine H3 receptor
mRNA is also expressed in the vast majority of striatal medium spiny neurones
(Pollard et al., 1993; Pillot et al., 2002).
Recently the H3 receptor has gained particular interest of many pharmaceutical
companies (reviewed by Leurs et al., 2005) due to the fact that this receptor is
involved in the regulation of arousal state, brain energy metabolism, locomotor
activity, autonomic and vestibular functions, feeding, drinking, sexual behavior,
and analgesia (Hough, 1988; Schwartz et al., 1991; Wada et al., 1991). It is therefore
believed that selective H3 receptor ligands could have therapeutic potential for the
treatment of various important neurological and psychiatric diseases. However,
histamine H3 receptor pharmacology, functions and biochemistry are far from
being fully understood.
Figure 2. The histamine H3 receptor. Alternative splicing sites are indicated with different
colours (modified from Leurs et al., 2005).
One important hurdle in the identification of the molecular mechanisms used by
brain histamine is the large variety of H3 receptor isoforms that might have
different pharmacological profiles (reviewed by Hancock et al., 2003). The number
of possible H3 receptor isoforms is high owing to the simultaneous occurrence of
multiple splicing events in the same H3 receptor mRNA molecule. So far, at least 20
different isoforms have been described on the basis of detection of varying H3
receptor mRNAs or using H3 receptor isoform-specific antibodies (Chazot et al.,
2001; Shenton et al., 2004). However, the exact expression patterns of the various H3
receptor isoforms remains elusive at this time. The H3(445) receptor isoform
described by Lovenberg et al., 1999, is currently the best characterized H3 receptor

9
isoform. Most isoforms differ from the H3(445) isoform by large deletions of one or
more stretches of amino acids. The isoforms with deletions in the third intracellular
loop (Fig. 2) have gained particular interest due to the involvement of this domain
in G-protein coupling (Drutel et al., 2001). As a result, these isoforms possess
different pharmacological profiles (reviewed by Hancock et al., 2003), such as
agonist potencies (Wellendorph et al., 2002), signaling properties (Drutel et al., 2001)
and constitutive activity (Morisset et al., 2000).
Dopamine
One of the most extensively studied slow-acting neurotransmitters is the biogenic
amine dopamine. Four major neurological and psychiatric diseases are associated
with perturbations in the dopaminergic neurotransmission: Parkinsonism,
schizophrenia, Attention Deficit Hyperactivity Disorder (ADHD), and drug abuse
(Carlsson, 2000). Parkinsonism is associated with the death of dopamine-producing
nerve cells projecting to the striatum (Marsden, 1986). Administration of L-3,4-
dihydroxyphenylalanine (levodopa; L-DOPA), the metabolic precursor of
dopamine which penetrates the blood–brain barrier, is still the most effective
therapeutic option (Lang et al., 1998). Most drugs currently used for the treatment
of schizophrenia are antagonists of a subclass of dopamine receptors. ADHD is
treated with Ritalin, which mainly works by stimulating the release of dopamine.
Most of the drugs of abuse give rise to abnormal dopaminergic signaling.
Dopaminergic neurons originating from the substantia nigra pars compacta and
the ventral tegmental area innervate the striatum. This is the major component of
the basal ganglia, a collection of subcortical structures involved in the control of
voluntary movements as well as in motivational, emotional and cognitive aspects
of motor behavior. The striatum expresses high levels of dopamine receptors,
which belong to the rhodopsin-like family of GPCRs. Dopamine receptors are
grouped into two classes: D1–class (D1– and D5–subclass) and D2–class (D2–, D3–
and D4–subclass; Gerfen, 1992).
Slow synaptic transmission in the striatum
GABA–ergic medium-sized spiny neurons comprise about 95% of striatal nerve
cells and integrate excitatory glutamatergic inputs from cortex, thalamus and
limbic areas with dopaminergic inputs originating in the substantia nigra pars
compacta and in the ventral tegmental area. In addition, the striatum receives
modulatory input from the histaminergic system arising in the tuberomamillary
nucleus of the hypothalamus. Medium-sized spiny neurons are the only efferent
pathway for conveying information out of the striatum (Fig. 3).
In the dorsal striatum, medium spiny neurons give rise to two major outputs
responsible for fine motor control: the direct pathway, which contains GABA and
dynorphine and projects to the substantia nigra pars reticulata/internal globus
pallidus (Gpi), and the indirect pathway, which contains GABA and enkephalins
and projects to the external globus pallidus (Gpe; Fig. 3).
These two pathways exert opposite effects on motor activity via modulation of
thalamocortical neurons. Activation of the direct striatonigral pathway will
disinhibit thalamocortical neurons and thereby facilitate motor activity, whereas
activation of the indirect striatopallidal pathway will increase the inhibition of
thalamocortical neurons and reduce motor activity (Gerfen, 1992; Fig. 3).

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Considerable functional and morphological evidence indicates that the
GABA/dynorphine neurons of the direct pathway express dopamine receptors of
the D1–subclass, whereas the GABA/enkephalin neurons of the indirect pathway
express most of dopamine receptors of the D2–subclass (Gerfen, 1992). In addition
to this, there is some evidece that histamine H3 and dopamine D1 receptors are co-
localized on the striatonigral projection neurones of the direct pathway (Ryu et al.,
1994).
Dopamine produces its effects on medium spiny neurons by binding to and
activating two subclasses of dopamine receptors, designed D1 and D2. D1 and D2
receptors have opposite regulation on the dopamine-sensitive type V adenylyl
cyclase (AC; Kebabian and Greengard, 1971), the enzyme that converts adenosine
triphosphate (ATP) to cyclic 3’, 5’-adenosine monophosphate (cAMP; Fig. 4).
Dopamine, acting on D1 receptors increases the catalytic activity of AC. This effect
is mediated by the α-subunit of the olfactory neuron-specific G-protein (Gαolf;
Herve et al. 1993; Zhuang et al., 2000). In contrast, activation of dopamine D2
receptors coupled to PTX sensitive Gi proteins results in inhibition of AC (Kebabian
and Calne, 1979). The formation of cAMP activates the cyclic adenosine 3’, 5’-
monophosphate-dependent protein kinase (PKA). This enzyme is present in the
brain at very high concentrations if compared to, for instance, the liver. In addition
to this, PKA is concentrated in the synaptic region of nerve cells (Miyamoto et al.,
1969).
Figure 3. Diagram illustrating the functional organization of the basal ganglia. The striatum
receives an excitatory glutamatergic input (green) from cerebral cortex and thalamus (not
shown), and a modulatory dopaminergic input (black) from the substantia nigra pars compacta
(SNpc). The striatum is largely composed of two distinct subpopulations of GABAergic (red)
medium-sized spiny neurons expressing high levels of either dopamine D1 or D2 receptors.
These neurons innervate either directly (D1), or indirectly (D1)—via the external globus pallidus
(Gpe) and subthalamic nucleus (STN)—the substantia nigra pars reticulata (SNpr) and the Gpi.
Dopamine activates, via D1 receptors, the direct striatonigral pathway and inhibits, via D2
receptors, the indirect striatopallidal pathway. This opposite regulation disinhibits thalamo-
cortical glutamatergic neurons and promotes motor activity (Adapted from Fisone et al., 2004).

11
Figure 4. Signaling pathways activated by dopamine in medium spiny neurons. Dopamine
produces its effects by binding to D1 and D2 receptors, which have opposite regulation on
adenylyl cyclase (AC). Dopamine, acting on D1 receptors coupled to Gαolf proteins increases the
catalytic activity of AC, resulting in formation of cAMP, which in turn activates PKA. In contrast,
activation of dopamine D2 receptors coupled to Gi proteins results in inhibition of AC, reduced
formation of cAMP and therefore decreased activation of PKA. The contrasting regulation of AC
by D1 and D2 receptors influences the state of PKA mediated phosphorylation of downstream
target proteins (Modified from Fisone et al., 2004).
The contrasting regulation of AC by the different subclasses of dopamine
receptors is reflected on the activity of PKA, which in turn influences the state of
phosphorylation of downstream target proteins involved in the control of the state
of excitability of medium spiny neurons. As a result, dopamine activates PKA in
the direct striatonigral pathway, but not in the indirect striatopallidal pathway.
This is a crucial feature of dopamine and for this reason it promotes motor activity
(Fig. 3 and 4).
DARPP-32, an integrator of synaptic transmission in the striatum
One important PKA substrate is the protein named DARPP-32, an acronym for
dopamine and cAMP-regulated phosphoprotein of 32 kDa. DARPP-32 is a crucial
mediator of the actions of dopamine and has been a key for understanding the
mechanism of action of dopamine and its interactions with other neurotransmitters,
therapeutic drugs, and drugs of abuse. Rat DARPP-32 is a protein consisting of 205
amino acids and its sequence has been highly conserved within mammals.
Moreover, DARPP-32 is highly concentrated in striatal medium spiny neurons.
Threonine 34 (Thr-34) of DARPP-32 is phosphorylated by PKA and
dephosphorylated by protein phosphatase-2B (PP-2B; calcium/calmodulin-
dependent protein phosphatase; calcineurin). Phosphorylation of DARPP-32 at
Thr-34 profoundly changes its biological properties, converting it from an inactive
molecule into a very potent inhibitor of protein phosphatase 1 (PP-1) with a Ki of
about 10-9 M (Hemmings et al., 1984). Since the concentration of DARPP-32 in
medium spiny neurons is greater than 10-5 M, this means that a small burst of
activity in dopaminergic neurons will result in significant phosphorylation of
DARPP-32 at Thr-34 and in inhibition of PP-1.

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PP-1 has a very broad substrate specificity and controls the state of
phosphorylation and activity of target proteins such as neurotransmitter receptors,
voltage-gated ion channels, ion pumps and transcription factors (Fig. 5). For
instance, PP-1 inhibits glutamate receptors, which are important mediators of
synaptic transmission and plasticity in the striatum. Glutamate receptors are
classified as NMDA (N-methyl-D-aspartate), AMPA [(±)-alfa-amino-3-hydroxy-5-
methylisoxazole-4-propionic acid], kainate and metabotropic receptors. The first
three are ionotropic receptors, named according to their specific agonists (Fig. 5).
Therefore activation or inactivation of the cAMP/PKA/DARPP-32 cascade results
in inhibition or activation, respectively, of PP-1. This in turn affects the activity of,
among others, glutamate receptors.
Work carried out in the last decade has shown that regulation of the state of
DARPP-32 phosphorylation provides a crucial mechanism in integrating the
actions of dopamine, glutamate, therapeutic drugs, and drugs of abuse (reviewed
by Svenningsson et al., 2004; Fig. 5). For instance, the physiological importance of
the DARPP-32/PP-1 cascade has been demonstrated in mice where the DARPP-32
gene has been deleted. In these knockout mice all the physiological, biochemical
and pharmacological responses to dopamine, the psychostimulant drugs of abuse
and antischizophrenic drugs, which can be observed in normal mice, are either
greatly diminished or even abolished (Fienberg et al., 1998).
Figure 5. Signaling pathways in medium spiny neurons. Activation (green arrow) by
dopamine of the D1 subclass of dopamine receptors stimulates PKA catalyzed phosphorylation
of DARPP-32 at Thr-34 (see text). Activation by dopamine of the D2 subclass of dopamine
receptors causes the dephosphorylation of DARPP-32 through two synergistic mechanisms: (1)
D2 receptor activation prevents (orange arrow) the A2A receptor-induced increase in cAMP
formation (see text), and (2) raises intracellular calcium, which activates a calcium-dependent
protein phosphatase, namely PP-2B. Activated PP-2B dephosphorylates DARPP-32 at Thr-34.
The effects of dopamine are mimicked by the selective dopamine D1 receptor agonist SKF-
81297 and by the selective dopamine D2 receptor agonist quinpirole. The selective histamine H3
receptor agonist immepip has been shown to reduce SKF-81297 induced cAMP accumulation
in vitro.
Glutamate acts as both a fast-acting and slow-acting neurotransmitter. Activation by
glutamate of AMPA receptors causes a rapid response through influx of sodium ions,

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depolarization of the membrane, and firing of an action potential. Slow synaptic transmission, in
response to glutamate, results in part from activation of the AMPA and NMDA subclasses of
glutamate receptor, which increases intracellular calcium and the activity of PP-2B, and causes
the dephosphorylation of DARPP-32 on Thr-34.
Antischizophrenic drugs affect the physiology of these neurons, and also regulate the state of
phosphorylation of DARPP-32 on Thr-34. For example, the antischizophrenic drug haloperidol,
which blocks the activation by dopamine of the D2 subclass of dopamine receptor, increases
DARPP-32 phosphorylation, but requires tonic activation of adenosine A2A receptors which are
anatomically colocalized with dopamine D2 receptors. Caffeine promotes motor activity through
blockade of A2A receptor–mediated increases in cAMP and the resultant decreases in DARPP-
32 phosphorylation at Thr-34 in striatopallidal neurons of the indirect pathway. Drugs of abuse
such as cocaine and amphetamine, through increasing extracellular dopamine levels, increase
DARPP-32 phosphorylation in striatonigral neurons of the direct pahway. Marijuana, nicotine,
alcohol, and LSD, all of which affect the physiology of the dopaminoceptive neurons, also
regulate DARPP-32 phosphorylation. Lastly, all drugs of abuse have greatly reduced biological
effects in animals with targeted deletion of the DARPP-32 gene. Abbreviations: NKA, Na
+
, K
+
ATPase; L- and N/P-Ca
2+
, L type and N/P type calcium channels (Modified from Greengard,
2001).
Parkinson’s disease and levodopa-induced dyskinesia
The degeneration of dopaminergic nigrostriatal neurons associated with
Parkinson’s disease causes a depletion of striatal dopamine, which results in
bradykinesia, rigidity and tremor. These symptoms are a direct consequence of the
lack of dopaminergic control on striatal outputs which, in turn, results in increases
and decreases of function in the indirect and direct pathways, respectively
(Bergman et al., 1990). Parkinson’s disease is treated with the dopamine precursor
L-DOPA. This compoud is very helpful in the initial phase of the disease, but its
therapeutic efficacy wanes with time. Such “wearing-off” imposes an escalation in
the dosage of the drug, which ultimately results in the appearance of abnormal
involuntary movements (AIMs) or dyskinesia (reviewed by Bezard et al., 2001).
“Peak dose” L-DOPA-induced dyskinesia (LID) currently represents one of the
major challenges to the treatment of Parkinson’s disease (Obeso et al., 2000).
Recently the plasticity of corticostriatal synapses in rats was investigated. High-
frequency stimulation of cortical afferents induced long-term potentiation (LTP) of
corticostriatal synapses in both dyskinetic and non-dyskinetic L-DOPA-treated
animals. Control and non-dyskinetic rats showed synaptic depotentiation in
response to subsequent low-frequency synaptic stimulation, but dyskinetic rats did
not. Moreover, the striata of dyskinetic rats contained abnormally high levels of
phospho[Thr-34]-DARPP-32. The inability of dyskinetic animals to undergo
synaptic depotentiation may cause a pathological storage of nonessential motor
information, that would normally be erased, leading to the development and/or
the expression of abnormal motor patterns. This loss of bidirectional synaptic
plasticity is attributable to specific changes occurring along the dopamine D1
receptor signaling pathway leading to abnormally high levels of phospho[Thr-34]-
DARPP-32 and consequent inhibition of PP-1 activity. In support to this
hypothesis, it was also shown that the depotentiation seen in both L-DOPA-treated
non-dyskinetic rats and intact controls was prevented by activation of the D1
subclass of dopamine receptors or pharmacological inhibition of PP-1 and PP-2A
(Picconi et al., 2003).
Additional supporting data have come from biochemical studies carried out on
tissues obtained from a nonhuman primate (NHP) brain bank constituted to study
the pathophysiology of LID. These brain tissues show an increased expression of

14
DARPP-32. However—for technical reasons—it was not possible to determine the
levels of phospho[Thr-34]-DARPP-32. Moreover these tissues showed an increased
activity of Gαolf in terms of binding to radiolabelled guanosine 5’-thiotriphosphate
(GTPγS). Interestingly, the D1 agonist-induced Gαolf activity was linearly related to
dyskinesia, whereas D1 receptor expression was not (Aubert et al., 2005). Taken
together, this evidence suggest that LID results from increased dopamine D1
receptor–mediated transmission at the level of the direct pathway.
Modulation of glutamatergic transmission in the striatum
In the striatum, dopamine exerts a complex modulation of glutamatergic
transmission by regulating different subtypes of glutamate ionotropic receptors.
Previous studies have shown that, in striatonigral neurons, activation of dopamine
D1 receptors enhances responses mediated by NMDA (Cepeda et al., 1993; Colwell
and Levine, 1995; Cepeda et al., 1998; Flores-Hernández et al., 2002) and AMPA
glutamate receptors (Yan et al., 1999; Lin et al., 2003). The AMPA glutamate
receptor is an oligomeric complex of four homologous subunits designated
GluR1–4 (glutamate receptor 1–4). Phosphorylation of the GluR1 subunit plays an
essential role in the regulation of AMPA receptors. PKA catalyses phosphorylation
of Serine 845 (Ser-845) at the C-terminus of GluR1. Phosphorylation of this residue
results in a 40% potentiation of the peak current through GluR1 homomeric
channels (Roche et al., 1996). D1 receptor mediated activation of the PKA/DARPP-
32 cascade—which promotes GluR1 phosphorylation—prevents current rundown,
a phenomenon where the amplitude of AMPA currents gradually declines (Yan et
al., 1999; Snyder et al., 2000). In addition, it has been reported that PKA-dependent
phosphorylation of GluR1 is involved in dopamine D1 receptor-mediated insertion
of AMPA receptors onto the surface of striatal medium spiny neurons
(Mangiavacchi and Wolf, 2004). In conclusion, it is possible that the appearance of
levodopa-induced dyskinesia may involve potentiation of AMPA receptors in
striatonigral projection neurons of the direct pathway due to abnormal activation
of the cAMP/PKA/DARPP-32 signaling pathway.
In the indirect striatopallidal pathway dopamine exerts an opposite effect on the
reguation of AMPA receptors. The dopamine D2-like agonist quinpirole decreases,
whereas haloperidol—an antipsychotic drug with dopamine D2 receptor
antagonistic properties—increases the phosphorylation of GluR1 at the PKA site
Ser845. The latter effect is acheived by removing the inhibitory tone exerted by
dopamine D2 receptors on the PKA/DARPP-32 cascade (Håkansson et al., 2006). In
addition, it requires tonic activation provided by adenosine acting on A2A
receptors, which are anatomically colocalized with dopamine D2 receptors in
striatopallidal projection neurons (Gerfen, 1990) and positively coupled to AC via
Golf-proteins (Fig. 6). The reduction of locomotor activity—an unwanted side effect
produced by haloperidol—may therefore involve facilitation of glutamate AMPA
receptor transmission in striatopallidal neurons. Moreover, the increase in Ser-845
phosphorylation produced by haloperidol is abolished in DARPP-32 knockout
mice, or in mice in which the PKA phosphorylation site on DARPP-32 (i.e. Thr-34)
has been mutated (Thr-34 → Ala mutant mice).

15
Figure 6. Regulation of GluR1 phosphorylation by haloperidol and adenosine in
striatopallidal neurons. Blockade of Gi-coupled dopamine D2 receptors results in the
stimulation of adenylyl cyclase through the tonic activity provided by adenosine on A2A
receptors. This, in turn, increases the production of cAMP. Activated PKA catalyzes
phosphorylation of Thr-34, which converts DARPP-32 (D32) into an inhibitor of PP-1 (left
illustration). The increase in GluR1 phosphorylation at Ser-845 produced by haloperidol is
abolished in DARPP-32 knockout mice (right illustration) (Modified from Fisone et al., 2004).

16
Aims of the Study
In rat striatal slices it has recently been shown that the selective histamine H3
receptor agonist immepip (4-[1H-imidazol-4-ylmethyl] piperidine; Fig. 7; Vollinga
et al., 1994), inhibits cAMP accumulation (Sánchez-Lemus and Arias-Montaño,
2004) and stimulation of GABA release (Arias-Montaño et al., 2001) elicited by the
selective dopamine D1 receptor agonist SKF-81297 (6-Chloro-2,3,4,5-tetrahydro-1-
phenyl-1H-3-benzazepine-7,8-diol; Fig. 7; Reavill et al., 1993). Taken together this
evidence indicates the existence of an important functional interaction between
dopamine and histamine at the level of striatonigral projection neurons. We
therefore expected that this could be reflected on the regulation of PKA activity
and, in turn, on the state of phosphorylation of downstream target proteins such as
the GluR1 subunit of the glutamate AMPA receptor and DARPP-32 (Fig. 8). These
two proteins are phosphorylated by PKA—events that facilitate glutamate AMPA
receptor transmission in striatonigral neurons of the direct pathway. Histamine H3
receptor agonists may therefore be interesting candidate drugs for the attenuation
of dyskinesia.
Figure 7. Test substances used in this study. As for most H3 receptor ligands developed so
far, immepip and (R)-α-methylhistamine closely resemble histamine and contain an imidazole
ring.
The aim of this project was to examine the effects produced by histamine on the
state of phosphorylation of GluR1 and DARPP-32 at their PKA sites. We started by
examining the effect of the dopamine D1 receptor selective agonist SKF-81297 on
the state of phosphorylation of GluR1 at Ser-845 and of DARPP-32 at Thr-34.
Because of the positive regulation exerted by D1 receptors on AC in striatonigral
projection neurons, we expected SKF-81297 to increase GluR1 and DARPP-32
phosphorylation at Ser-845 and at Thr-34 respectively. We then proceeded by
testing the ability of two selective histamine H3 receptor agonists immepip and (R)-
α-methylhistamine (Fig. 7; Arrang et al., 1987) to reduce the increase in
phosphorylation of GluR1 and DARPP-32 produced SKF-81297. Due to the
negative regulation exerted by H3 receptors on the cAMP pathway, we expected
immepip and (R)-α-methylhistamine to reduce the increase in GluR1 and DARPP-
32 phosphorylation produced by SKF-81297.

17
Figure 8. Hypothesis of dopamine and histamine interaction. The selective histamine H3
receptor agonist immepip has been reported to inhibit D1-induced cAMP accumulation in rat
striatal slices. We therefore expected that H3 receptor agonists could reduce the state of
phosphorylation of PKA substrates such as the GluR1 subunit of the glutamate AMPA receptor
and DARPP-32. Phosphorylation of these two proteins at their respective PKA sites are events
that facilitate glutamate AMPA receptor transmission in striatonigral neurons of the direct
pathway. (Modified from Fisone et al., 2004).

18
Materials and methods
Drugs
SKF-81297 hydrobromide, immepip dihydrobromide and (R)-α-methylhistamine
dihydrobromide were purchased from Tocris Cookson Ldt., Northpoint, UK. For
the treatment of striatal slices, the drugs were dissolved in deionized water.
Treatment and tissue preparation
For the biochemical experiments we used the striatal slice preparation. Male mice
C57/BL/6 (25-30 g, B&K, Stockholm) were decapitated with a guillotine. The
brains were rapidly removed and immediately immersed in ice-cold, freshly
gassed Krebs-Ringer’s bicarbonate buffer (KRB; 118 mM NaCl, 4.7 mM KCl, 1.3
mM CaCl2, 1.5 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3 and 11.7 mM
glucose, equilibrated with 95% O2/5% CO2, pH 7). Coronal slices (250 µm) were
prepared using a vibrating blade microtome (vibratome) Leica VT1000 S (Leica
Microsystems AB, Sollentuna). Previous to the slice preparation, the brain was cut
with a razor blade at the level of the rostral corpus callosum and caudally at the
level of medial thalamus. The brain was then rapidly glued to the floor plate of the
vibratome with the caudal side facing down. The floor plate was rapidly placed
inside a bath filled with ice-cold, freshly gassed KRB. Coronal slices (250 µm) were
obtaied with a vibrating razor blade moving slowly in the dorsal-ventral axis. The
slices were transferred by means of a brush into a Petri dish filled with ice-cold,
freshly gassed KRB. Dorsal striata were then dissected out from each slice under a
microscope using fine tweezers. Two dorsal striatal slices, equivalent to one
sample, were placed by means of a brush in individual 5-ml polypropylene tubes
containing 2 ml of ice-cold, freshly gassed KRB. The test tubes were immersed in a
bath at 30°C for 30 min and the samples were constanty perfused with 95% O2/5%
CO2 by means of polyethylene tubes with an inner diameter of 1.67 mm (Becton
Dickinson, Stockholm) connected to each test tube and immersed in the medium.
Thereafter the medium was replaced with 2 ml freshly gassed KRB at 30°C and the
samples were kept for 30 more min under constant perfusion with 95% O2/5%
CO2. The medium was replaced one more time before incubation with the test
substances. This procedure prevents adenosine and purines to accumulate in the
medium. Adenosine activates the cAMP/PKA signaling in striatopallidal
projection neurons (Fig. 6) and its concentration can be as much as 100 µM 15 min
following cutting (Fredholm et al., 1984). Test substances were added for various
intervals as described below. After incubation, the solutions were rapidly removed
(30 s) with a Pasteur pipette, and the samples were immediately frozen on dry ice.
The total time from decapitation to the sample freezing was approximately 150
min. Frozen tissue samples were sonicated in 75 µl of 1% sodium dodecyl sulfate
and boiled for 10 min.
Determination of phosphoproteins
Aliquots (3 µl) of the homogenate were used for protein content determination
using the bicinchoninic acid (BCA) assay kit (Pierce Europe, Oud Beijerland, the
Netherlands). Equal amounts (50 µg) of protein from each sample were loaded onto
10% polyacrylamide gels, and the proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidine
difluoride membranes (Amersham Pharmacia Biotech, Uppsala, Sweden).

19
The membranes were immunoblotted using either affinity-purified monoclonal
antibodies that selectively detect phospho[Thr-34]-DARPP-32 (diluted 1:1,000;
preparation described by Snyder et al., 1992). These antibodies are a gift from Prof
Paul Greengard (Laboratory of Molecular and Cellular Neuroscience, The
Rockefeller University, New York, NY, USA). For the detection of phospho[Ser-
845]-GluR1 we used affinity-purified polyclonal (diluted 1:750; LuBioScience
GmbH, Lucerne, Switzerland).
Antibody binding to phospho[Thr-34]-DARPP-32 was revealed by incubation
with goat anti-mouse horseradish peroxidase-linked gamma-heavy chain class
immunoglobulin (IgG) antibodies (diluted 1:10,000; Pierce Europe, Oud Beijerland,
the Netherlands) and the enhanced chemiluminescence ECL Plus immunoblotting
detection method. Chemiluminescence was detected by autoradiography.
Antibody binding to phospho[Ser-845]-GluR1 was revealed by incubation with
Alexa Fluor 680 goat anti-rabbit IgG coniugated antibodies (diluted 1:10,000;
Invitrogen Ldt, Paisley, UK) and the Odyssey direct infrared fluorescence
detection system (Westburg BV, Leusden, the Netherlands). Quantification of the
immunoreactivity corresponding to the phospho[Thr34]-DARPP-32 and
phospho[Ser845]-GluR1 bands respectively, was done by densitometry, using NIH
Image (version 1.61) software.

20
Results
Regulation of GluR1 phosphorylation by SKF-81297 and immepip
As shown in Figure 9, incubation of striatal slices (n = 17) for 5 min with the
dopamine D1 receptor agonist SKF-81297 (1 µM) significantly increased (p <
0,00005, Student’s t test) the state of phosphorylation of GluR1 at Ser-845 (282 ±
54%) compared to control slices incubated 5 min with Krebs-Ringer’s bicarbonate
buffer (n = 13). Simultaneous incubation of striatal slices (n = 11) with SKF-81297
and the histamine H3 receptor agonist immepip (1 µM) for 5 min, did not produce
any significant decrease (p = 0,43, Student’s t test) in the state of phosphorylation of
GluR1 at Ser-845 (276 ± 59%), compared to slices incubated with SKF-81297 alone.
Moreover, simultaneous treatment (5 min) of striatal slices ( n = 3) with
immepip—at a concentration of 5 µM—and SKF-81297 (1 µM) did still not produce
a significant decrease in the state of phosphorylation of GluR1 (data not shown).
Figure 9. Effect of D1- and H3 receptor activation on the state of phosphorylation of GluR1
in striatal slices. Slices were incubated for 5 min with Krebs-Ringer’s bicarbonate buffer in the
presence or absence of drugs under test. The upper panel shows representative
autoradiograms of Western blots obtained using polyclonal antibodies against phospho[Ser-
845]-GluR1. The lower panel shows summaries of data expressed as means ± SEM. The
number of samples (= two striatal slices) per group varied from 11 to 17. The amount of
phosphorylated GluR1 is expressed as a percentage of that determined after vehicle incubation.
Values are combined from five experiments with three replicates for each condition. *p <
0,00005 vs. respective vehicle-treated group (Student’s t test).

21
Regulation of DARPP-32 phosphorylation by SKF-81297 and
immepip
Very similar results were obtained on the state of phosphorylation of DARPP-32 at
Thr-34. Incubation of striatal slices (n = 3) for 5 min with SKF-81297 (1 µM)
significantly increased (p < 0,001, Student’s t test) the state of phosphorylation of
DARPP-32 at Thr-34 (171 ± 32%) compared to control slices (n = 3). However,
simultaneous incubation of slices (n = 3) with SKF-81297 and immepip (1 µM) for 5
min, did not result in a significant difference (p = 0,43, Student’s t test) in DARPP-
32 phosphorylation (189 ± 90%) compared to treatment with SKF-81297 alone (Fig.
10).
Figure 10. Effect of D1- and H3 receptor activation on the state of phosphorylation of
DARPP-32 in striatal slices. Slices were incubated for 5 min with Krebs-Ringer’s bicarbonate
buffer in the presence or absence of drugs under test. The upper panel shows representative
autoradiograms of Western blots obtained using monoclonal antibodies against phospho[Thr-
34]-DARPP-32. The lower panel shows summaries of data expressed as means ± SEM. The
number of samples (= two striatal slices) per group was 3. The amount of phosphorylated
DARPP-32 is expressed as a percentage of that determined after vehicle incubation. Values are
combined from one experiment with three replicates for each condition. *p < 0,001 vs. vehicle-
treated group (Student’s t test).

22
Regulation of GluR1 phosphorylation by SKF-81297 and (R)-α-
methylhistamine
Again incubation of striatal slices (n = 6) for 5 min with SKF-81297 (1 µM) produced
a significant increase (p < 0,0001, Student’s t test) in the state of phosphorylation of
GluR1 at Ser-845 (171 ± 31%) compared to control striatal slices (n = 6).
Simultaneous treatment of striatal slices (n = 7) with SKF-81297 and the histamine
H3 receptor agonist (R)-α-methylhistamine (1 µM) for 5 min, produced a non-
significant decrease (p = 0,11, Student’s t test) in the state of phosphorylation of
GluR1 at Ser-845 (110 ± 37%), compared to slices incubated with SKF-81297 alone
(Fig. 11).
Figure 11. Effect of D1- and H3 receptor activation on the state of phosphorylation of
GluR1 in striatal slices. Slices were incubated for 5 min with Krebs-Ringer’s bicarbonate buffer
in the presence or absence of drugs under test. The upper panel shows representative
autoradiograms of Western blots obtained using polyclonal antibodies against phospho[Ser-
845]-GluR1. The lower panel shows summaries of data expressed as means ± SEM. The
number of samples (= two striatal slices) per group varied from 6 to 7. The amount of
phosphorylated GluR1 is expressed as a percentage of that determined after vehicle incubation.
Values are combined from two experiments with 3 to 4 replicates for each condition. *p < 0,0001
vs. respective vehicle-treated group (Student’s t test).

23
Discussion
Levels of phosphoproteins in the striatal slice preparation
A critical aim for the present study was that the levels of phosphoproteins
measured by immuno-blotting would reflect the in vivo situation. Therefore, a
prerequisite in our experiments was that dephosphorylation during sampling
would be as low as possible. Degradation of ATP is one of the most rapid
dephosphorylation reactions occurring post-mortem (Lowry et a l ., 1964;
Svenningsson et al., 2000). Decapitation precedes the preparation of slices and there
are probably dramatic changes in the adenine nucleotide levels during preparation
of the slices. Therefore, immediately following slice cutting ATP levels are low,
whereas those of AMP are high, resulting in a low energy charge. The energy
charge is defined as the concentration of (ATP + 1/2 ADP)/(ATP + ADP + AMP).
90 min after cutting the energy charge is gradually normalized, in part due to the
conversion of AMP to ATP, but mainly due to breakdown to adenosine and other
purines. The viable part of the striatal slice, which accounts for about half of the
tissue, has levels of adenine nucleotides and adenosine which are similar to those
found in the intact brain (Fredholm et al., 1984). For this reason we decided to load
50 µ g of proteins onto the polyacrylamide gels in oder to detect reasonable
amounts of phosphoproteins. For striatal samples from in vivo experiments, in
contrast, 30 µ g of proteins are enough. The return of physiological function
following slice preparation is paralleled by a normalization of the energy charge,
the adenosine level and the concentration of cAMP (Fredholm et al., 1984). It is
therefore crucial to treat the tissues with drugs relatively rapidly post-mortem,
although the dissection of the striata requires great precision. In fact, during the
first set of 3-4 experiments, when the time necessary for slice preparation was
relatively long due to lack of practice, the levels of detected phosphoproteins were
low compared to the latest experiments.
Phospho[Thr-34]-DARPP-32 was detected only in one experiment, although
immunoblotting was performed in parallell with phospho[Ser-845]-GluR1. This
may be explained by the fact that we switched to a new set of primary antibodies,
although it was still obtained from the same source (a gift from Prof Paul
Greengard, Laboratory of Molecular and Cellular Neuroscience, The Rockefeller
University, New York, NY, USA; preparation described by Snyder et al., 1992). The
secondary antibody can be ruled out, because it was not changed. We even tried to
increase the concentration of the primary antibody from 1:750 to 1:300, but
unsuccessfully. Another explanation could be the fact that phospho[Ser-845]-GluR1
and phospho-[Thr-34]-DARPP-32 are dephosphorylated by different protein
phosphatases (PP-1 and PP-2B respectively). It is possible that dephosphorylation
of phospho-[Thr-34]-DARPP-32 occurs more rapidly after removal of the drugs. At
the same time it has to be pointed out that phospho-[Thr-34]-DARPP-32 is required
to preserve GluR1 phosphorylation at Ser-845, due to its inhibition of PP-1 (Figures
6 and 8). Therefore the most realistic explanation is the inability of the primary
antibody, for unknown reasons, to detect phospho-[Thr-34]-DARPP-32.
Pharmacological profile of the histamine H3 receptor
The histamine H3 receptor agonist immepip reduces dopamine D1 receptor-
mediated cAMP signaling in rat striatal slices (Sánchez-Lemus and Arias-Montaño,

24
2004). We therefore expected, that this would be reflected in the state of
phosphorylation of GluR1 and DARPP-32 at their respective PKA sites. We decided
to carry out our experiments in mouse striatal slices because this opens the
possibility to investigate effects in mice with deletions of specific genes such as, for
instance, the gene encoding for DARPP-32.
Recent pharmacological characterization of recombinant H3 receptors have
confirmed clear-cut species related differences. For instance the rat and human H3
receptors, although they show a high overall sequence homology, display distinct
ligand binding properties. Some H3 receptor antagonists/inverse agonists such as
thioperamide or ciproxifan are more potent at the rat receptor, others such as FUB
349 are more potent at the human receptor (Arrang et al. 1987; Ligneau et al. 2000;
Lovenberg et al. 2000; Ireland-Denny et al. 2001; Wulff et al. 2002; Yao et al. 2003).
Two amino acids, at positions 119 and 122 in the third transmembrane domain are
responsible for the pharmacological differences between the two species (Ligneau
et al. 2000).
We therefore beleived that the inability of immepip to reduce D1 induced
phosphorylation could be linked to species related differences between rat and
mouse. However, the seven transmembrane domains of the mouse receptor are
identical to those of the rat receptor. T his suggests that there should not be any
differences in ligand binding properties between the two receptors. Two recent
studies show that the pharmacological profile of the mouse receptor is more
similar—although not identical—to the rat receptor than to the human receptor
(Chen et al. 2003; Rouleau et al., 2004). For example, the H3 receptor antagonists
thioperamide and ciproxifan were slightly more potent (by four- to eightfold) at the
mouse receptor than at the rat receptor but much more potent (by 40- to 100-fold)
than at the human receptor (Rouleau et al., 2004).
We also thought that the existence of multiple functional isoforms could have
accounted for a different pharmacological profile between rat and mouse.
Characterization of isoforms of the mouse H3 receptor (Rouleau et al., 2004)
resulted in the finding of three isoforms: the isoform of 445 amino acids (H3(445))
previously characterized in human, guinea-pig and rat was identically expressed
also in the mouse brain. Moreover, two shorter H3(413) and H3(397) isoforms
previously found in rat were discovered also in the mouse. These shorter isoforms
were generated by deletions of 32 and 48 amino acids, respectively, located in the
third intracellular loop of the mouse H3 receptor. These deletions, although they
don’t affect the binding pocket, could result in different G-protein coupling and,
for instance, converting an agonist such as immepip, to a neutral agonist. In
addition to this, at least 20 different isoforms have been described so far (Chazot et
al., 2001; Shenton et al., 2004). It is therefore possible that mice could express
additional, different isoforms in the striatum, compared to the rat.
Moreover, southern blot analysis of the tissue distribution revealed a differential
expression of these three isoforms among mouse brain areas (Rouleau et al., 2004).
The shorter isoforms were more abundant in the striatum, than in the cerebral
cortex and the hypothalamus, suggesting that they may subserve distinct functions
in this brain area. The distribution within the striatum, however, was
homogeneous.
Imidazole H3-agonists
The histamine H3 receptor is involved in the regulation of several
neurophysiological functions, such as motor activity. In the present study we have

25
investigated the ability of two H3 receptor full agonists to reduce dopamine D1
receptor induced increase in PKA mediated phosphorylation. We choosed to
examine the two imidazole-containing agents immepip and (R)-α-methylhistamine
(Fig. 7).
So far, all H3 receptor agonists closely resemble endogenous histamine (Fig. 7)
and contain a 4(5)-substituted imidazole moiety. Small structural modifications of
the imidazole side chain of histamine can result in very potent and selective H3
receptor agonists. Methylation of the imidazole side chain resulted in (R)-α-
methylhistamine, which can be considered the archetypal agonist for
pharmacological histamine H3 receptor testing. Moreover, (R)-α-methylhistamine
displays a 15-fold higher potency than endogenous histamine at the H3 receptor
(pKi = 8.4, human H3 receptor; Arrang et al., 1987). One important limitation of (R)-
α-methylhistamine is its poor bioavailability due to the fact that it undergoes
extensive metabolism. We therefore decided to start our experiments using
immepip (pKi = 9.3, human H3 receptor; Jansen et al., 1998) for two reasons: (1) We
planned to carry out in vivo experimens and immepip displays good bioavailability
and brain penetration (Jansen et al., 1998); (2) immepip was previously reported to
reduce dopamine D1 receptor-mediated cAMP signaling in rat striatal slices
(Sánchez-Lemus and Arias-Montaño, 2004).
The inconsistent results obtained could be linked to the chemical properties of
the imidazol ring. It is well known that imidazole har a pKa near 7 (Albert and
Serjeant, 1962). Therefore the imidazole group can be uncharged or positively
charged near neutral pH depending on the experimental conditions. It is possible
that CO2 present in the gas mixture, which is converted to H2CO3 in water, may
have caused shifts in the medium’s pH below 7 due to differences in the gas flow.
Protonation of the imidazole ring may have altered the permeation properties of
the drugs across cell membranes, resulting in a reduced effect on the cAMP/PKA
signaling. This hypothesis is supported by the fact that in the two experiments
carried out with (R)-α-methylhistamine, we observed significant effects in the
reduction of phospho[Ser-845]-GluR1 in three samples, whereas in the remaining
four samples the levels of phospho[Ser-845]-GluR1 didn’t differ at all from those
found in samples treated with SKF-81297 alone. Moreover, the previous
experiment showing that immepip inhibits D1 induced cAMP accumulation in rat
striatal slices (Sánchez-Lemus and Arias-Montaño, 2004) was performed using 50
mM Tris–HCl buffer instead of the KRB. This medium has a pH of 7.4 and should
assure a reduced probability that the imidazole ring is protonated.
Concluding remarks and future developments
Our experiments with the striatal slice preparation using SKF-81297 and (R)-α-
methylhistamine should be repeated using the 50 mM Tris–HCl buffer (pH 7.4) and
increasing the concentration of (R)-α-methylhistamine. We also planned to carry
out behavioural experiments, but the difficulties encountered with the in vitro
experiments delayed our work.
The H3 receptor has been linked to other intracellular signaling pathways,
including mitogen-activated protein kinase (MAPK; Drutel et al., 2001; Giovannini
et al., 2003) and phosphatidylinositol 3-kinase (PI3K) pathways. Activation of these
pathways results in the phosphorylation of extracellular signal-regulated kinases
(ERKs) which, in turn, phosphorylate the transcription factors cAMP-response
element binding protein (CREB) and Elk-1 (Ets-domain protein Elk-1). Therefore, it
would be very useful to perform biochemical investigations in vivo in mice, in order

26
to screen the phosphorylation of different proteins such as the ERKs, CREB and
Elk-1. In this way we could obtain an overall view of H3 receptor signaling.
Another important advantage with in vivo experiments is a 10-fold amount of
tissue available compared to the slice preparation. This allows to perform more
phosphoprotein determinations from each sample, which contains the tissue from
an entire striatum.
Moreover, the laboratory of Dr. Gilberto Fisone has recently validated a mouse
model of LID (Lundblad et al., 2005). These mice display a correlation between
phosphorylation of ERKs and levodopa-induced dyskinesia (LID; manuscript in
progress). Hence, it would be interesting to investigate the effect of H3 receptor
ligands on the state of phosphorylation of these proteins in mice with LID. The H3
receptor may represent a drug target in the treatment of dyskinesia.

27
Acknowledgements
I would like to thank all the persons who contributed to my work during this
Master Thesis.
My supervisor Gilberto Fisone for teaching me the striatal slice preparation and for
being available at any time to answer my questions with his great skillfulness.
Emanuela Santini, Anders Borgkvist, Richard Andersson and Manuela Di
Benedetto for all the precious help and technical advice.
I also want to thank in particular my wife Eva, for supporting me during my entire
training programme.

28
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