UNITS RECORDINGS Str: SOUND PROTOCOL
Author
Cecília Pardo Bellver and Gergő A. Nagy.
Experiment name
Units recordings Str.

Session 1

Session 2

10 Days 

7 x

20 min

20 min

6 h

Experiment description
A second set of mice have been implanted with head plates and recordings were performed during awake, head-fixed conditions from the posterior striatum (pStr) using silicon probes with the aim of understanding potential behaviours that might be affected by striatal function. 
Protocol description
Mobile HomeCage: Head-fixed recordings from awake mice have been performed in a Neurotar Mobile HomeCage system (https://www.neurotar.com/) using custom made homecages (30 cm diameter; 2 cm height; material: foam). The homecage was located on an AirLift table. The airflow was set to allow stability and smooth movement of the homecage (Figure 1).

Figure 1. Head-fixed apparatus.  Schematic of the apparatus used during the head-fixed protocol.

Head-fixed behavioural task: Mice were habituated to the experimenter and to the head-fixed conditions for 4-7 days following full recovery after the head plate implantation. During the recordings the animals freely explored the homecage while receiving the sound stimuli.

Session 2

20 min

6 h

Injections and histology
Implant: For the head-fixed recordings the animals were anesthetised with isoflurane (2-3 v/v%) and were implanted with stainless steel head plates. Animals were allowed a minimum of one week recovery time. On the day of the recordings the mice were anesthetised with isoflurane (2-3 v/v%) and the craniotomies were performed. The mice were implanted with a silicon probe(one shank, 32 recording sites) in the posterior striatum (AP: -1.5 mm; ML: -3.1 mm with 6ª angle; DV: -3.5 mm; coordinates from Bregma, depth measured from brain surface). The recordings were started approximately 2 hours after isoflurane administration was suspended.
Histology: After the end of the behavioural training the mice were deeply anaesthetised with an overdose of ketamine/ xylacine and transcardially perfused with 0.9 % NaCl (50 mL) followed by 100 mL of a fixative solution containing 4 % paraformaldehyde in 0.1 M phosphate buffer (PB; pH = ~7.4). The brains were removed from the skull and postfixed in the same fixative for 4 h. Coronal sections (100 μm thick) were prepared from the tissue blocks containing the entire striatal region using a vibratome. The sections were mounted and images taken with an epifluorescence microscope. 

Session 2

20 min

6 h

Data collection and analysis
The behaviour was recorded with a webcam camera and the videos were saved in .wmv file format. Name format: [AnimalShortCode]_[DateTime]_video.wmv. 
The silicon probe was connected to a 32-channel Intan Headstage (RHD2132, Intan Technologies) through an Omnetics connector for digitalisation and amplification of the data. Data acquisition was conducted with an Intan RHD USB Interface Board (Intan Technologies), digitalised at 30 kHz and recorded by the open source Open Ephys GUI (Open Ephys). The TTLs were generated with Spike2 (Cambridge Electronic Design) and recorded through one of the analog inputs in the board. Files can be read using scripts available in the Open Ephys GitHub repository (open-ephys/analysis-tools repository).  Name format: 114_CH[channel#].continuous, where channel# = 1 to 32 and for the TTL 114_ADC1.continuous.
Data was offline analysed using built-in and custom-made MATLAB (Mathworks) scripts. The data analysis code is available in the Laboratory of Network Neurophysiology GitHub: LNN_Matlab-code. Action potentials were manually sorted into putative neuronal clusters based on amplitude (peak-to-valley), waveform energy, first and second principal component features using the MClust software (A. D. Redish). The electrodes were analysed in groups of 4 and pair of 2 (see DataSet2_ElectrodesGroups.csv), Name format: GR[group#].mat, where group# = 1 to 15. Putative neurons were named relative to the group they belong, Name format: GR[group#]_[neuron#].mat, where group# = 1 to 15. Other relevant files for running MClust are also available (.clusters, .fd, .t).
The behaviour of the animal during the training was assessed and saved in table format ([AnimalShortCode]_[DateTime]_[Recording#]_Behaviour.csv). Behaviours assessed: Rest, the animal does not move; Move, position change.
Available data
o	Animals information: DataSet2_AnimalCodes.csv
o	Neurons isolated: DataSet2_NeuronsList.csv
o	Probe location: Information in DataSet2_AnimalCodes, images in DataSet2_UnitsRecordingsStr_ElectrodesLocation.zip
o	Example video: NG164_20191007_001_video.wmv, in DataSet2_UnitsRecordingsStr_SoundProtocolData.zip
o	Example recording: 114_CH1 to 100_CH32.continuous files, in DataSet2_UnitsRecordingsStr_SoundProtocolData.zip
o	Units data: Example of clustered neurons belonging to the example recording in DataSet2_UnitsRecordingsStr_SoundProtocolData.zip, refer to DataSet2_ElectrodesGroups.csv for a detailed explanation of the grouping used to analyse the neurons.
o	Decoded behaviour: Description of the behaviours in NG164_20191007_001_Behaviour.csv.

All videos, TTLs, recordings and tracking are available on demand. 
Please contact pardo-bellver.cecilia(a)koki.hu