0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

TEL.14 – Manipulation of serotonin transmission on behavioural and neurochemical deficits…
analyzed in different brain areas to widely evaluate brain aminergic metabolisms
in different forms of HPA.
PHE blood levels will be analyzed in each mouse to evaluate the degree of
HPA.
METHODS
1) To investigate effects of 5-HTP treatment during critical postnatal
period to prevent mental retardation in phenylketonuric subjects.
Animals – Homozygote (-/-) PAH enu2 (ENU2) mice, obtained by heterozygous
mating, will be used for all experiments. At postnatal day 28, animals (sex
matched) will house 2-4 per standard breeding cage with food and water ad
libitum on a 12:12h dark: light cycle (light on 07.00 am -07.00 pm h).
Experimental groups – Three groups of ENU2 mice (WT-sal ENU2-sal, ENU2
5-HTP) will be collected from male pups derived from a total of thirty matings
(n= 36; n= 8 for each group) and exposed to experimental treatment between
14-21 postnatal days.
WT-sal and ENU2-sal mice will be i.p. treated with saline, twice to day.
ENU2-5-HTP mice will be i.p. treated with 5-HTP (20 mg/kg, twice to day).
Behavioural experiments will start when animals reached 8 weeks of age.
Drugs – Chloral hydrate and 5-HTP will be purchased from Sigma-Aldrich (St-
Luis, MO), dissolved in saline (0.9% NaCl) and injected i.p. in a volume of 10
ml/kg.
Behavioural Testing – All mice will be submitted to behavioural tests (Object
Recognition Test. Spatial Novelty Test and Morris Water Maze using modifications
of the basic protocol, in random order) to evaluate behavioural profile.
Experimental procedures are previously reported [Cabib et al. 2003;
Vorhees and Williams 2006]. All tests where videotaped by means of a camera
placed within the sound-attenuated cubicle and connected with a recorder
placed outside the cubicle. Later on, an experimenter unaware of the treatment
conditions quantified behaviour, using the Observer program (Version
3.0, System for Macintosh, Noldus, Wageningen 1997).
2) To evaluate effects of 5-HTP treatment in different forms of HPA (control,
mild HPA, mild PKU and classic PKU.
Animals – See previous paragraph.
Experimental groups – Cntr group will be composed by BTBR wild-type mice
(n=10; PHE < 4 mg/dl);
– m-HPA group will be composed by BTBR wild-type mice daily treated
with phenylalanine (1-2 mg/g body wt) for two weeks (n=10; PHE 4-10 mg/dl);
– m-PKU group will be composed by ENU2 mice maintained to a mildly
low-PHE diet (Mucedola, Italy) (n=10; PHE 4-10 mg/dl);
– c-PKU group will be composed by untreated ENU2 mice (n=10; PHE >
20 mg/dl).
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