Dr. Cristina Giaroni
PhD Program in Experimental and Applied Medicine
University of Insubria
Curriculum: Neuroscience
The project is addressed to candidates with bursary
Project title: Adaptive changes of the gastrointestinal neuromuscular function in mutant mouse models
of schizophrenia and correlation with changes in the commensal microbiota: role of ionotropic
glutamatergic receptors
State of the art
Enteric neuronal circuitries display a considerable ability to adapt to a changing microenvironment, which
comprises several cellular "players", including neurons, enteric glial cells, smooth muscle cells, interstitial
cells of Cajal, immune cells and commensal bacteria (1). In particular, the gut microbiota seems to directly
modulate the development and function of the enteric nervous system (ENS), supporting the concept that
changes in gut microbiota composition, induced by infections or antibiotics, can perturb ENS integrity and
activity, thus favouring the onset of GI disorders. Recently, the existence of a possible correlation between
the gut microbiota and the central nervous system (CNS), modulated by the ENS, which may be able to
modify superior cognitive and behavioural functions, has been suggested. The gut microbiota may
participate to the development and plasticity of the CNS as suggested by the reduction in the expression
levels of protein involved in the synaptogenesis and synaptic-vescicle maturation, such synaptophysin and
PSD-95 in the CNS of germ-free mice (2). Furthermore, in the striatum of germ-free mice, the increase in the
turnover rates of several neurotransmitters such ad noradrenaline, dopamine and serotonin has been
associated with anxiety-like behaviour and changes in motor-activity (2).
On the base of these observations, we may hypothesize that alterations of the gut microbiota may play a
key role in the development of psychiatric disorders observed, for example, in patients with gastrointestinal
problems (3).
The molecular mechanisms and role of specific receptors/mediators responsible of gut microbiota
alterations, which, in turn, may induce changes in ENS neurotransmitters pathways, are still largely
unknown. Our research group has focused on the role of the glutamatergic transmission in adaptive changes
occurring in enteric neuronal circuitries. Morphologic studies demonstrated that the entire glutamatergic
neurotransmitter pathway is present in enteric neurons (4). From a functional point of view, the aminoacid
may play a pivotal role in the modulation of both neurotransmitters release and motor parameters,
principally mediated via ionotropic AMPA and NMDA receptor activation (5,6). The functional relevance of
the enteric glutamatergic pathways remains, however, largely to be unraveled. As observed in the CNS,
alterations of the glutamatergic function may represent a critical step in the pathogenesis and/or in the
progression of symptoms related to several intestinal disorders. NMDA receptor function increases after an
ischemic or inflammatory injury and may contribute to motility dysfunctions associated with both
pathological states (7,8).
In the CNS, glutamatergic pathways may have an important role in the pathogenesis of several
disorders characterized by severe alterations of the cognitive function, such as schizophrenia. Both preclinical and clinical studies indicate an association between schizophrenia and NMDA receptor hypofunction.
Administration of NMDA antagonists, such as ketamine, in normal subjects determines psychotic
behaviours, negative symptoms and cognitive alterations such as those observed in schizophrenic patients
(9). Knock-out mice for the NMDA receptor subunit NR1, represent an experimental model reproducing the
pathological cognitive changes associated with the disorder (10). In the hippocampus and cortex of germfree mice, with stereotyped behaviours and high level of anxiety and response to stress, a decrease in the
expression of the NR2 subunit of NMDA receptor has been observed. At a peripheral level, schizophrenia is
related to gastrointestinal motor function alterations that mainly consist of delayed colonic motility
associated with megacolon and constipation. However, the possible involvement of the enteric
glutamatergic transmission as well as the mechanisms underlying reduced colonic motility in these
conditions, remain to be unraveled.
Aims
The first objective of this project is to demonstrate the relevance of the enteric glutamatergic system in
gastrointestinal dysfunctions occurring in animal models of schizophrenia. To this end we will evaluate by
means of biomolecular and immunohystochemical approaches the distribution and expression of ionotropic
glutamatergic receptors of the NMDA, AMPA and kainate type in colonic segments of heterozygote mice for
dopamine transporter (Het-DAT), catechol-o-methyltransferase (Het-COMT) and for both genes (Het-DATCOMT), which represent schizophrenia experimental models (11). The effect of ionotropic glutamate
receptors agonists and antagonist on both motor functions and on electrically-stimulated and basal
endogenous glutamate release will be investigated. A second objective will be to evaluate possible changes
involving enteric ionotropic glutamate receptor induced by short and long term alterations of the gut
microbiota following antibiotic treatments. This part of the study aims to further verify whether changes of
the gut microbiota may contribute to the development of local abnormalities and also to sustain CNS
derangements underlying the pathogenesis and/or the progression of diseases with clinical and social
impact, e.g. schizophrenia.
Experimental design
Objective 1
Animal models
1- mice heterozygote for dopamine transporter (Het-DAT) and wild type mice;
2mice
heterozygote
for catechol-o-methyltransferase
(HET-COMT)
and wild type;
3mice
heterozygote
for
both
genes
(Het-DAT-COMT)
and
wild
type;
Heterozygote mice carrying mutations for catecholamine transporters and the related systems of
enzymatic degradation, with a reduced, but not totally deleted function, may represent experimental
models more relevant to human clinical situations, where the complete loss of biological functions caused
by genetic mutations may rarely be observed. Genetic polymorphisms are associated with a reduced
catecholamine transporter function rather than with a total loss of their activity.
Objective 2
Animal models
-C57Bl/6 male mice (21 days old) will be orally treated for two weeks with an antibiotic course consisting of
ampicillin, neomycin, metronidazole, vancomycin, to induce massive microbiota depletion.
The effect on specific enteric pathways will be evaluated after 1 day (acute effect of microbiota depletion)
and after 4-8 weeks (long-term effect of microbiota depletion on neuroplasticity) of treatment
discontinuation.
Parameters evaluated in the different experimental groups
1- Characterization and quantification of neuron immunopositive for the different subunits of NMDA,
AMPA and kainate receptors using immunohystochemical analysis performed with fluorescent microscopy
and confocal microscopy in colonic longitudinal muscular myenteric plexus (LMMP) preparations and by
immunohystochemistry on colonic sections. The distribution of immunoreactivity for the following
neuronal markers HuC/D (pan-neuronal marker), calbindin (marker of sensory enteric neurons), calretinin
(marker of enteric motor neurons and interneurons) will be evaluated;
2- Protein levels of the different subunits of NMDA, AMPA and kainate receptors and of their mRNAs by
means of Western Blotting and real time RT-PCR in LMMP colonic preparations, cortex, hippocampus and
striatum;
3- Efficiency of the colonic peristalsis in vitro and of the efficiency of the gastrointestinal transit in vivo in
the presence and absence of NMDA, AMPA and kainate receptor agonists and antagonists;
4- Measurement of the colon longitudinal and circular muscle electrically induced-contractions in presence
and absence of NMDA, AMPA and kainate receptor agonists and antagonists;
5- Study of the basal and electrically-stimulated endogenous glutamate overflow from colonic isolated
segments in presence and absence of NMDA, AMPA and kainate receptor agonists and antagonists, by
means High-Performance-Liquid-Chromatography associated with fluorimetric detection (HPLC-FD);
References
1- Giaroni et al, Gastroenterology 1999;117:1438-58.
2- Diaz Heijtz et al, Proc Natl Acad Sci U S A 2011; 108: 3047-3052
3- Douglas-Escobar et al, JAMA Pedriatr. 2013; 167: 374-379
4- Kirchgessner, Curr. Opin. Pharmacol 2001;1:591-6
5- Giaroni et al, Life Sci. 2000 25;67:1747-57
6-Giaroni et al, Eur J Pharmacol 2003;476:63-69
7- Giuliani et al, Neurochem Int. 2006;48:191-200
8- Kaszaki et al, J Neural Transm 2012;119:211-23
9- Coyle et al, Handb Exp Pharmacol 2012; 213:267-295
10- Rampon et al, Nat Neurosci 2000;3:238-244
11- Papaleo et al, Neuropharmacology 2012;62:1204-20