Effects of Anti-NMDA Antibodies on Functional Recovery and Synaptic Rearrangement Following

NeuroMolecular Medicine

Effects of anti-NMDA antibodies on functional recovery and synaptic rearrangement

following hemicerebellectomy

Daniela Laricchiutaa,b,*, Virve Cavalluccib, Debora Cutulia,b, Paola De Bartolob,c, Paola

Caporalia, Francesca Fotia,b, Carsten Finked, Marcello D’Ameliob,e, Mario Mantof and

Laura Petrosinia,b a Department of Psychology, Faculty of Medicine and Psychology, University "Sapienza" of

Rome, via dei Marsi 78, 00185, Rome, Italy b IRCCS Fondazione Santa Lucia, Via del Fosso di Fiorano 64, 00143, Rome, Italy c Department of Sociological and Psychopedagogical studies, University “Guglielmo Marconi” of Rome, Via Plinio 44, 00193, Rome, Italy d Department of Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin,

Germany. e Laboratory of Molecular Neurosciences, Medical School University Campus-Biomedico, Via

Alvaro del Portillo 21, 00128, Rome, Italy f Unité d'Etude du Mouvement, FNRS Neurologie, ULB Erasme, 808 Route de Lennik 1070,

Brussels, Belgium

* Corresponding author:

Daniela Laricchiuta,

E-mail address: [email protected]

Neurological assessment of cerebellar symptomatology Twenty-four hours after the right HCb, 24 animals exhibited severe postural and motor deficits. Namely, HCb induced strong extensor hypotonia ipsilateral to the lesion side causing tilted posture to the right. Locomotion was markedly ataxic and stunted, with oscillatory movements and staggering. Dynamic tremor was also present. While stepping, the most evident symptoms were right hindlimb hyperflexion during the swing phase and joint angle fluctuations. To change direction, the right hindlimb was used as a pivot, remaining flexed under their body weight. Wide-based locomotion and collapsing on the bellies were present.

Locomotion tended also to veer to the lesion side provoking asymmetrical gait and falls. To avoid falling, animals tended to lean against support surfaces. Eye nystagmus beating to the right side was also present.

These severe postural and locomotor deficits tended to progressively diminish over time. After two post-operative months, the animals still exhibited some slight cerebellar symptoms as right extensor hypotonia, right hindlimb flexion, and wide-based locomotor posture.

Nevertheless, they succeeded in walking quite efficiently without support.

One-way ANOVA with repeated measures on the scores of all cerebellar symptoms taken into account obtained HCbed animals revealed a significant time effect (F(13,299) = 239.1; p < 0.0001), given symptom severity significantly diminished as days went by ( Fig. S1).

Twenty rats (5 animals/group) were assigned to the 4 groups devoted to biochemical analyses performed the end of the behavioral testing at 24 h after the frontal injections.

Before the injections (pre-injection day) neurological deficits provoked by the right HCb were still present in both HCb (H-S and H-A) groups, while no deficit was present in the sham (S-S and S-A) animals. A two-way ANOVA (group x day) on the scores of the behavioral rating scalerevealed significant group (F(3,16) = 131.17; p < 0.00001) and day (F(1,16) = 9.03; p < 0.01) effects. The interaction was not significant (F(3,16) = 3.71; p < 0.05). Post-hoc comparisons on interaction revealed that H-S and H-A groups were significantly different (at least p = 0.0002) from both S-S and S-A groups in the two evaluations (pre-injection and 1 stpost-injection day).

Interestingly, in both groups treated with anti-NMDA Ab (H-A, S-A) there were significant within-group differences between the two evaluations. Namely, significant (at least p = 0.03) postural and locomotor impairment was observed by comparing pre-injection and 1st post- injection valuations.

Escape Latencies. A two-way ANOVA (group x trial) on escape latency of Place 1 phase revealed a significant group effect (F(3,16) = 6.22; p < 0.01) and no significant trial effect (F(4,64) =

1.40; p = 0.24). The interaction was not significant (F(12,64) = 0.61; p = 0.83). Post-hoc comparisons on group effect revealed that S-S animals showed the lowest latencies in comparison to the remaining groups (at least p = 0.02) and that H-S and S-A animals exhibited latencies lower than H-A animals (p = 0.05).

A two-way ANOVA (group x trial) on escape latency of Cue phase revealed a significant group effect (F(3,16) = 3.50; p < 0.05) and not significant trial effect (F(2,32) = 1.57; p = 0.22). The interaction was not significant (F(6,32) = 0.94; p = 0.48). Post-hoc comparisons on group effect revealed that the S-S animals showed the lowest latencies in comparison to the remaining groups (p < 0.05).

Total distance. A two-way ANOVA (group x trial) on total distance of Place 1 phase revealed significant group (F(3,16) = 3.88; p < 0.05) and trial (F(4,64) = 5.36; p = 0.001) effects. The interaction was not significant (F(12,64) = 0.79; p = 0.65). Post-hoc comparisons on group effect revealed that S-S animals showed the lowest total distances in comparison to the remaining groups (at least p = 0.04) and that H-S animals showed total distances lower than H-A animals (p < 0.05).

A two-way ANOVA (group x trial) on total distance of Cue phase revealed a significant group effect (F(3,16) = 3.22; p = 0.05) and no significant trial effect (F(2,32) = 1.22; p = 0.31). The interaction was not significant (F(6,32) = 0.92; p = 0.49). Post-hoc comparisons on group effect revealed that S-S animals showed total distances lower than S-A (p < 0.05) and H-A (p <

0.05) animals.

Peripheral distance. A two-way ANOVA (group x trial) on peripheral distance of Place 1 phase revealed significant group (F(3,16) = 4.82; p = 0.01) and trial (F(4,64) = 3.01; p < 0.05) effects. The interaction was not significant (F(12,64) = 1.34; p = 0.22). Post-hoc comparisons on group effect revealed that S-S animals showed the lowest percentage of peripheral distance in comparison to the remaining group (at least p = 0.03). Two-way ANOVA on peripheral distance of Cue phase revealed no significant group (F(3,16) = 2.20; p = 0.12) and trial (F(2,32) =

0.03; p = 0.96) effects. Also the interaction was not significant (F(6,32) = 1.1; p = 0.38).

Swimming velocity. A one-way ANOVA on mean swimming velocity did not reveal

significant differences (F(3,16) = 1.36; p = 0.22) among S-S ( x = 23.6 cm/s), S-A ( x = 23.2), H-

S ( x = 21.2) and H-A ( x = 21.8) animals.

Total distance during Probe Phase. A one-way ANOVA on percentage of total distance travelled in the previously rewarded quadrant during the Probe phase revealed significant difference among groups (F(3,16) = 5.89; p < 0.01). Post-hoc comparisons revealed that S-S animals ( x = 32.33%) showed percentage of total distance higher than S-A ( x = 25.59%), H-

S ( x = 21.34%) and H-A ( x = 20.61%) (at least p = 0.05) animals. Navigational strategies. In the Place 1 phase, while S-S animals put into action even efficient navigational strategies, S-A, H-S and H-A animals exhibited high percentages of the inefficient Circling. Thus, the anti-NMDA Ab injections had not only a decompensating effect on HCbed animals but also an impairing effect on intact animals.

A two-way ANOVA on navigational strategies of Place 1 phase revealed a not significant group effect (F(3,16) = 1.25; p = 0.32) and a significant strategy effect (F(2,32) = 25.49; p <

0.00001). The interaction was significant (F(6,32) = 4.77; p < 0.001). Post-hoc comparisons on interaction revealed that S-A, H-S and H-A groups, but not S-S animals, put into action mainly the Circling strategy in comparison to other strategies (at least p = 0.006).

A two-way ANOVA on navigational strategies of Cue phase revealed not significant group

(F(3,16) = 0.71; p = 0.55) and strategy (F(2,32) = 2.57; p = 0.09) effects. The interaction was not significant (F(6,32) = 1.40; p = 0.24).