0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
EXPERIMENTAL PLAN
1. Our first specific objective is to investigate effects of 5-HTP treatment
during critical postnatal period to prevent cognitive delay in phenylketonuric
subjects.
Three groups of ENU2 mice will be used for these experiments: wild-type
treated with saline (WT-sal, n=8), ENU2 treated with saline (ENU2-sal, n=8),
and ENU2 treated with 5-HTP (20 mg/kg, twice to day; ENU2-5-HTP, n=8).
Animals to belonging to different groups will be exposed to treatment
between 14-21 postnatal days, a critical period characterized by of brain
amine deficits in PKU pups.
Effect of 5-HTP will be evaluated on behavioural test performances
(Object Recognition Test, Spatial Novelty Test, Spontaneous Alternation Test,
Morris Water Maze in standard and modified protocols) in adult ENU2 mice.
2. HPA refers any consistent excess of blood PHE levels, including classical
PKU. There are also several intermediate degrees of HPA, in which blood
PHE levels are mildly elevated.
So, our second objective is the development of animal models of different
degrees of HPA (control, mild HPA, mild phenylketonuria (PKU) and classic
PKU) on the base of plasma phenylalanine (PHE) concentrations). This aim is
based on the idea that different concentrations of blood PHE interfere differently
with cognitive functions.
– BTBR wild-type mice will be the control group (cntr, PHE < 4 mg/dl);
– BTBR wild-type mice drinking PHE-containing water (10 g/L) to
achieve and keep a status of mild hypeprhenylalaninemia (PHE 4-10 mg/dl)
for two weeks (m-HPA, n=10);
– ENU2 mice will be maintained to a mildly low-PHE diet (Mucedola,
Italy) to develop a mouse model of mild phenylketonuria (m-PKU; n=10, PHE
4-10 mg/dl);
– ENU2 mice will be the classic PKU model (c-PKU; n=10; PHE > 20
mg/dl).
Behavioural and neurochemical phenotyping will be performed on control
(cntr), mild HPA (m-HPA), mild PKU (m-PKU) and classic PKU (c-PKU)
mice.
A different group of cntr (n=10), m-HPA (n=10), m-PKU (n=10) and
c-PKU (n=10) mice will be treated with 5-HTP (20 mg/kg i.p., twice to day).
For behavioural phenotyping, all mice will be submitted to several behavioural
tests to evaluate motor (Open Field Test, Rotarod Test and Grooming
activity), cognitive (Object Recognition Test, Spatial Novelty Test, Spontaneous
Alternation Test, Morris Water Maze using modifications of the basic
protocol) and emotional (Porsolt’s Test) performances, in random order.
For neurochemical phenotyping, direct evaluation of fronto-cortical
aminergic neurotransmission will be evaluated by technique of in vivo intracerebral
microdialysis during the exposure to psychological experiences
known to affect cortical neurotransmission (restraint stress).
Moreover, tissue levels of 5-HT, DA, NE and theirs metabolites will be
790 2009