Supplementary Material
Brain Structure & Function
Cannabinoid receptor interacting protein Crip1a modulates CB1
receptor signaling in mouse hippocampus
Stephan Guggenhuber1, Alan Alpar2,3,4, Rongqing Chen5, Nina Schmitz1,
Melanie Wickert1, Tobias Mattheus1, Anne E. Harasta1,6, Martin Purrio1,
Nadine Kaiser1, Maurice R. Elphick7, Krisztina Monory1, Werner Kilb5, Heiko
J. Luhmann5, Tibor Harkany2,8, Beat Lutz1,*, Matthias Klugmann1,6,*
1Institute
of Physiological Chemistry, University Medical Center of the
Johannes Gutenberg University, D-55128 Mainz, Germany.
2Division
of
Molecular Neurobiology, Department of Medical Biochemistry and Biophysics,
Scheeles väg 1:A1, Karolinska Institutet, SE-17177 Stockholm, Sweden.
3Research
Group of Experimental Neuroanatomy and Developmental Biology,
Hungarian Academy of Sciences, Budapest, Hungary.
4Department
of
Anatomy, Histology and Embryology, Semmelweis University, Budapest,
Hungary. 5Institute of Physiology, University Medical Center of the Johannes
Gutenberg University, D-55128 Mainz, Germany. 6Department of Physiology
& Translational Neuroscience Facility, School of Medical Sciences, UNSW,
High Street, Randwick, NSW 2052, Sydney, Australia. 7School of Biological &
Chemical Sciences, Queen Mary University of London, London E1 4NS, UK.
8Department
of Molecular Neuroscience, Center for Brain Research, Medical
University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.
Correspondence should be addressed to Matthias Klugmann or Beat Lutz. Email: m.klugmann@unsw.edu.au or blutz@uni-mainz.de.
Materials and Methods
Behavioral testing
Behavioral testing commenced at four weeks after vector injections, when
AAV-mediated transgene expression has reached maximum and stable
levels. AAV-CRIP1a and AAV-empty animals (n=21-25) were subjected to the
tests in the order listed below. Animals were left undisturbed for at least 5
days between the different test sessions. The experimenter was blind to the
treatment of the animals. Locomotor activity and anxiety-related behavior was
tested using an open field box and the light/dark emergence test, respectively,
as described (Schneider et al. 2007). Inherent anxiety was investigated using
the elevated plus maze, consisting of two open arms (35 cm length, 6 cm
width) and two enclosed black arms (35 cm length, 6 cm width, 20 cm height)
at 1 m above the floor. The light on the floor of the open arms and the center
had about 30 lux. At the beginning of the test, the mouse was placed in a
close arm, and the behavior was video-monitored for 5 min. Recordings were
used to determine the time spent in the different arms, the entire entries to the
arms as well as head dips and risk assessment.
References
Schneider M, Spanagel R, Zhang SJ, Bading H, Klugmann M (2007) Adenoassociated virus (AAV)-mediated suppression of Ca2+/calmodulin
kinase IV activity in the nucleus accumbens modulates emotional
behaviour in mice. BMC neuroscience 8:105. doi:10.1186/1471-22028-105
Legends to Supplementary figures
Figure 1. CRIP1a does not affect excitatory synaptic transmission. A, B,
Amplitude and frequency of miniature EPSCs were not significantly different in
CA1 pyramidal cells from AAV-empty and AAV-CRIP1a transfected animals.
C, Typical current traces illustrating paired-pulse facilitation of the 2nd eEPSC.
Note comparable paired-pulse facilitation between AAV-empty and AAVCRIP1a mice. D, Bar graph illustrating paired-pulse ratio of evoked EPSCs in
AAV-empty and AAV-CRIP1a mice. n.s., non significant.
Figure 2. CRIP1a overexpression did not affect locomotion and anxiety
behavior. A, B, Animals were subjected to an open field test. The time spent
in the center (p = 0.83) and the distance travelled (p = 0.42) did not differ
between AAV-empty and AAV-CRIP1a mice. C, D, In the elevated plus maze,
there was no significant difference in the time spent in the open arm (p = 0.59)
or in the entries into the open arm (p = 0.36) between the two groups. E, F,
The light/dark test revealed that AAV-empty and AAV-CRIP1a animals did not
differ in the time spent in the lit compartment (p = 0.22) and in the entries into
the lit compartment (p = 0.73). N = 21 to 25 mice per group. Data are
expressed as means ± SEM.