1. On the role of NMDA glutamate receptors
in drug-induced motor disorders
Francesca Rossi
Karolinska Institutet, Stockholm, Sweden
Supervisor: Prof. Luca Nicola Ferraro
Assistant supervisor: Prof. Gilberto Fisone

2. The Basal Ganglia (BG)
• The basal ganglia has the ability to
collect, integrate and feedback
information coming from the cerebral
cortex
• The BG is critically involved in motor
control
• In the BG the main receiving station is
represented by the GABAergic
medium spiny neurons (MSNs) of the
striatum. These cells are innervated by
excitatory glutamatergic fibers from
cortex and thalamus, and modulatory
DA fibers from the midbrain (mostly
from substantia nigra pars compacta)
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https://syntaptogaming.files.wordpress.com/2012/01/basalganglia_image1.jpg

3. GABA
D1
NMDA
Direct pathway
(striatonigral)
promotes
motor activity
Indirect pathway
(striatopallidal)
inhibits
motor activity
D2
Direct and indirect pathways
NMDA
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4. Chronic treatment with L-DOPA
and Haloperidol causes severe side-effects
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Parkinson’s Disease Schizophrenia
L-DOPA Haloperidol
D1 – direct pathway
Disinhibition of movement
Dyskinesia
D2 – indirect pathway
Inhibition of movement
Catalepsy

5. • NMDA receptors: glutamate ion
channels
- key elements in the regulation of
synaptic function in the central
nervous system.
- highly permeable to Ca2+
(essential for
synaptic plasticity)
• Prolonged exposure to glutamate
causes excessive influx of Ca2+
through
NMDA receptor > cell death
• NR1 subunit: unique role in
determining the activation/activity of
NMDA receptors
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NMDA receptors

6. [1] Material and Methods
•C57BL/6J background
•Mice lacking the NR1 subunit specifically in D1 and D2
striatal cells of MSNs were generated in the laboratory
•Genetic mouse models: essential to unravel the
interactions between PD-related neurodegenerative
changes and pathways of maladaptive plasticity induced
by PD treatment
•NR1 D1 KO
•NR1 D2 KO
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Mice
Mouse carring a Cre recombinase
gene controlled by a tissue-specific
promoter X
Mouse carrying conditional (floxed)
alleles of gene Y

7. [2] Material and Methods
Drugs and dyskinesia
•Unilateral injection with the
toxin
6-OHDA in the striatum
•9 days lasting L-DOPA
treatment
Behavioural test
•L-DOPA-induced dyskinesia (LID)
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Behavioural test
•Bar test
Drugs and catalepsy
•Haloperidol (D2R)

8. [3] Material and Methods
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1. Sample preparation
2. Protein quantification
3. Gel preparation
4. Running of the gel
5. Transfer to membrane
6. Protein Staining
7. Developing images
8. Protein Quantification
PCR Genotyping and protein Western Blotting

9. Control tests
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Weight
1 Box-and- whiskers plot of weight, in grams, for the five different mouse lines used in these
experiments showing the distribution of our data. The middle line shows the median of the sample.
The top and the bottom of the box show the 75th and 25th percentiles, respectively.
In this graph the top and bottom of the whiskers show the maximum and minimum values.
The marks represent the different animals’ weights at 3 months of age.
Total NR1
Striatal levels of total NR1 protein in NR1 D1 and NR1 D2 KO mice.
The graph shows the levels of NR1 protein detected in the striatum
of NR1 D1 KO (A) and NR1 D2 KO (B) mice as measured by Western blotting.

10. Results – Behaviour
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L-DOPA-induced dyskinesia
Bar graphs show the ability of a chronic L-DOPA treatment (9 days) to induce LID. The X axis represents the different days when LID was scored:
on day 3, 6 and 9 the animals were observed for 3 hours after the injection and their LID was scored as locomotor (A) and integrated
(axial plus limb plus orofacial: ALO) (B). The red bars represent the NR1 D2 KO animals while the white bars represent the control group D2-Cre.
Statistical analysis indicate that ALO is significantly reduced in NR1D2 KO mice compared to their controls.
| L-DOPA |Lesion
2 weeks 3 days
3
6
9

11. Results – Biochemistry
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L-DOPA-induced dyskinesia
Striatal levels of phospho Thr202/Tyr204 ERK (44/42) induced by chronic L-DOPA treatment in NR1 D2 KO mice.
The graph shows the levels of phosphor Thr202/Tyr204 ERK (pERK 42/44) protein detected in the striatum
Of NR1 D2 KO mice, and measured by Western blotting
Striatal levels of phospho S235/236 S6 induced by chronic L-DOPA treatment in NR1 D2 KO mice.
The graph shows the levels of phospho Ser235/236 S6 (pS6 235/236) protein detected in the striatum
Of NR1 D2 KO mice, and measured by Western blotting

12. Results – Behaviour
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Line graph showing the time course of drug effect in NR1 D1 (Panel A) and NR1 D2 (Panel B) KO animals. Control groups are presented by dark lines while
our KO animals are represented in green and red, NR1 D1 KO and NR1 D2 KO respectively. The time to descent from the bar is represented in seconds with
a cut off of 3 min. Non-cataleptic animals descend from the bar immediately after placement while cataleptic animals stay immobile with their front paws
placed over the bar.
Catalepsy

13. Results – Biochemistry
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Catalepsy
bar graphs showing the increase of phosohoGluAr1 at Ser 845 in the striatum
in NR1 D1 and NR1 D2 KO mice after treatment with haloperidol.
Treatment effect was observed in both experiments.
Striatal levels of phoshoSer235/236 S6 induced by haloperidol in NR1 D1
and NR1 D2 KO mice. The graph shows the levels of phosphoSer235/236 (pS6 235/236)
protein detected in the striatum of NR1 D1 KO and NR1 D2 KO mice,
and measured by Western blotting.

14. Conclusions
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Inactivation of NMDA receptors in the medium spiny neurons of the indirect pathway prevents the
development of L-DOPA-induced dyskinesia.
Inactivation of NMDA receptors in the medium spiny neurons of the indirect pathway prevents the
immobility (catalepsy) induced by haloperidol, which is a surrogate marker of extrapyramidal side-
effects.
In contrast, the inactivation of NMDA receptors in the medium spiny neurons of the direct pathway
does not prevent immobility (catalepsy) induced by haloperidol.
These results suggest that the NMDA receptor could be an important target in the treatment of
motor side effects linked to antiparkinsonian and antipsychotic drugs.

15. Thank you
Prof. Gilberto Fisone
Débora Masini
Dr. Giada Spigolon