Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Example electrophysiological data recorded from a ventrolateral periaqueductal grey (vlPAG) tetrode (30 kHz sampling rate, 600–3 kHz bandpass filter, Ch1-4, tetrode channels 1–4). Raw data captured during early extinction training around the time of CS+ onset (left, tone on) and CS+ offset (right, tone off). Light blue bar shows time the tone is on. ( B ) Spikes were sorted using all channels in MClust to identify individual units; in this case, four units (colour coded) were isolated. Individual units were classified as described in Materials and methods. ( C ) Violin plots showing distribution of baseline firing rates for all available single units recorded in vlPAG during three phases of fear conditioning: habituation (n = 30 units), acquisition (n = 50 control units), and extinction (n = 55 control units). Horizontal black lines show median, and blue lines IQR firing rate for each phase of conditioning. ( D ) Same as ( C ), but for single units recorded during muscimol experiments. Habituation (n = 10 units), acquisition (n = 17 units), and extinction (n = 25 units). Horizontal black lines show median, and orange lines IQR firing rate for each phase of conditioning. Figure 1—figure supplement 1—source data 1. Numerical data to support graphs in .

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Sampling, Isolation, Control

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Photomicrographs of two example tetrode tracks (arrows) in periaqueductal grey (PAG). Outlines show left half of two coronal sections (anteroposterior [AP] levels –6.5 and –7.5) of the PAG. Dotted lines indicate approximate location of the ventrolateral sector (vlPAG). Note that a lack of correspondence between the physiological and histological boundaries of the lateral border of PAG has been reported previously . ( B ) Same as ( A ) but tracks in sagittal sections (±1 mm of midline) medial cerebellar nucleus (MCN) and ( C ) example track of a cerebellar cannula. ( D ) Pairs of figurines to the left show two coronal sections (AP levels –6.5 and –7.5) of the PAG. Pairs of figurines to the right show sagittal sections of left and right cerebellum (±1 mm of midline). Tetrodes-only controls (n = 6 rats, tetrodes implanted on one side of PAG and also in the contralateral MCN), tracks colour coded for each animal, and length of each track indicates extent of unit recording in that track; reconstruction of tetrode tracks in the vlPAG (left) and contralateral MCN (right). ( E ) Same as ( D ) but for saline controls (tetrodes implanted on one side in PAG and cannulae implanted bilaterally in MCN for saline infusion; n = 4 rats). In the cerebellar sections, the whole length of each cannula track has been reconstructed, and the blue-coloured tips indicate the extension of the internal cannula for infusions. ( F ) Same as ( E ) but cannulae used for infusion of muscimol (n = 4 rats). In the cerebellar tracks, orange-coloured tips indicate the extension of the internal cannulae for infusions. ( G ) Same as ( F ) but only cannulae implanted in left and right cerebellum for infusion of muscimol (n = 4 rats).

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Saline

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Example type 1 offset unit in vlPAG recorded during extinction following acquisition where a 1 s trace was introduced between conditioned stimulus (CS) offset and the unconditioned stimulus (US) during acquisition. Raster plot and peri event time histogram (PETH) showing activity relative to CS+ offset (arrow in raster and time 0 in PETH). Time of the US during acquisition is shown by red dotted rectangle; 40 ms bins. ( B ) Same as ( A ) but mean PETH ± SEM for all type 1 offset units (n = 6). Horizontal dashed red line represents significance level (p<0.05). ( C ) Same as ( A ) but example type 4 offset unit. ( D ) Same as ( B ) but mean PETH ± SEM for all type 4 offset units (n = 6). ( E ) Bar chart plotting mean time to peak or trough in activity for all units after CS+ offset (delay, n = 19 units recorded after delay conditioning; trace, n = 12 units recorded after trace conditioning; individual units for trace experiments: light grey type 1 offset units and dark grey type 4 offset units). No significant difference in timing between trace and delay (p>0.05). Figure 1—figure supplement 3—source data 1. Numerical data to support graphs in .

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Activity Assay

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Group average ERPs recorded at CS+ onset in the MCN and vlPAG in control animals (n = 6 rats), arrows indicate time of tone onset; each waveform shows mean ± SEM; dark blue, average ERP during early extinction (EE), light blue, average ERP during late extinction (LE). ( B ) Same as ( A ) but ERPs recorded simultaneously at CS+ offset (n = 6 rats). ( C ) Plots showing mean peak-to-peak amplitude of ERPs recorded at CS+ onset in EE versus LE; left panel, MCN; right panel, vlPAG (n = 6 rats, means ± SEM). Pairs of data points connected with dashed lines show the change in mean amplitude over extinction training for individual animals. ( D ) Same as ( C ) but for CS+ offset (n = 6 rats, paired t -test, **p<0.01). ( E ) Repeated-measures correlation (r rm ) for ERPs at CS+ onset (n = 6 rats) comparing the amplitude of ERPs recorded simultaneously in MCN and vlPAG. Each colour represents data and line of best fit for an individual animal. ( F ) Same as ( E ) but for CS+ offset (n = 6 rats). Figure 3—source data 1. Numerical data to support graphs in .

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Control

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Example ERP recorded in the ventrolateral periaqueductal grey (vlPAG) during early extinction training at CS+ offset. Average of 14 trials. ( B ) Example single-unit data recorded simultaneously from the same vlPAG recording site. Vertical dashed line and arrowhead indicate CS+ offset at time 0. Raster plot 14 trials, peri event time histogram (PETH), 10 ms bin width.

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques:

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Schematic showing a sagittal section of the cerebellum injection into the MCN of an anterograde tracer (AAV-CAG-tdTomato viral vector). To the left, high-power photomicrograph showing transfected neurons in MCN. To the right, coronal section shows representative pattern of terminal labelling in the contralateral PAG in one animal (dlPAG, lPAG, vlPAG; dorsolateral, lateral, and ventrolateral PAG). ( B ) Coronal sections of the PAG at different anteroposterior (AP) levels showing the pattern of terminal labelling in all available animals (n = 7 rats). The darker the red shading, the larger the number of animals with terminal labelling in that area.

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Injection, Plasmid Preparation, Transfection

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Histological reconstruction of cannulae placement in periaqueductal grey (PAG) in control (n = 9 rats, tracks colour coded blue to black for different animals) and DREADD animals (n = 10 rats, tracks colour coded orange to red for different animals). For each track, the tips indicate the extension of the internal cannula for infusions. ( B ) Percentage of the total time in an elevated plus maze spent in the open arm (left) and in the closed arm (right) for control (n = 9 rats) versus DREADD (n = 9 rats) experiments. Individual data points show percentage of time per animal. Bars show mean ± SEM (open arm, t(16) = 1.036, p=0.316; closed arm, t(16) = 1.036, p=0.316; unpaired t -test). ( C ) Time spent in the centre versus the periphery of the open field arena for control versus DREADD animals. Individual data points show total time per animal. Bars show mean ± SEM (centre, t(12) = 0.145, p=0.887; periphery, t(12) = 0.463, p=0.651, unpaired t -test). ( D ) The total distance moved in the open field arena in control (n = 8 rats) versus DREADD (n = 6 rats) experiments. Individual data points show total distance per animal. Bars show mean ± SEM (t(12) = 0.267, p=0.794, unpaired t -test). ( E ) Beam walking traversal times in control (n = 9 rats) and DREADD (n = 9 rats) experiments before (baseline) and after infusion of clozapine N-oxide (CNO) into ventrolateral PAG (vlPAG) for the two groups of animals. Pairs of data points connected by dotted lines show change in mean crossing time per animal between baseline and CNO. Bars show mean ± SEM (time to traverse, t(16) = 0.508, p=0.618, unpaired t -test). ( F ) Same as ( E ) but beam traversal times in control (n = 9 rats) and DREADD (n = 9 rats) experiments before (baseline) and intraperitoneal injection of CNO. Unpaired t -test **p<0.001. Figure 6—figure supplement 2—source data 1. Numerical data to support graphs in .

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Control, Injection

Journal: eLife

Article Title: Cerebellar modulation of memory encoding in the periaqueductal grey and fear behaviour

doi: 10.7554/eLife.76278

Figure Lengend Snippet: ( A ) Schematic timeline representing the fear conditioning protocol for the DREADDs experiment. ( B ) The effect of clozapine N-oxide (CNO) delivery into the ventrolateral PAG (vlPAG) during acquisition on freezing behaviour measured during presentation of the paired conditioned stimulus-unconditioned stimulus (CS-US) (upper plot), and the inter-tone interval (ITI) (lower plot). Blue plot, control animals (n = 9 rats); red plot, DREADD (hM4D(Gi)) animals, n = 10 rats; data points mean ± SEM. ( C ) The number of ultrasonic vocalisations (USVs) recorded in control versus DREADD animals during CS-US presentation (upper panel) and during the ITI (lower panel); bars show median ± IQR. Mann–Whitney, one-tailed test, *p<0.05. Figure 6—source data 1. Numerical data to support graphs in .

Article Snippet: To investigate the relationship within animals between unit response size and freezing activity and the relationship between MCN and vlPAG ERPs, repeated-measures correlation (using RStudio; see ) was calculated between response area at tone onset and offset and percentage of time spent freezing during blocks of seven tones (i.e. block 1 = tones 1–7; block 2 = tones 8–14, etc.).

Techniques: Control, MANN-WHITNEY, One-tailed Test