DOI: 10.5061/dryad.1g1jwsv4t
title: Data for: Multimodal convergence in the pedunculopontine tegmental nucleus: motor, sensory, and theta-frequency inputs influence the activity of single neurons

description: The pedunculopontine tegmental nucleus of the brainstem (PPTg) has
 extensive interconnections and neuronal-behavioural correlates. It is
 implicated in movement control and sensorimotor integration. We
 investigated whether single neuron activity in freely moving rats is
 correlated with components of skilled forelimb movement and whether
 individual neurons respond to both motor and sensory events. We found that
 individual PPTg neurons showed changes in firing rate at different times
 during the reach. This type of temporally specific modulation is like
 activity seen elsewhere in voluntary movement control circuits, such as
 the motor cortex, and suggests that PPTg neural activity is related to
 different specific events occurring during the reach. In particular, many
 neuronal modulations were time-locked to the end of the extension phase of
 the reach, when fine distal movements related to food grasping occur,
 indicating strong engagement of PPTg in this phase of skilled individual
 forelimb movements. In addition, some neurons showed brief periods of
 apparent oscillatory firing in the theta range at specific phases of the
 reach-to-grasp movement. When movement-related neurons were tested with
 tone stimuli, many also responded to this auditory input, allowing for
 sensorimotor integration at the cellular level. Together, these data
 extend the concept of the PPTg as an integrative structure in the
 generation of complex movements, by showing that this function extends to
 the highly coordinated control of the forelimb during skilled reach to
 grasp movement and that sensory and motor-related information converges on
 a single neuron, allowing for direct integration at the cellular level.
description: All procedures were approved by the University of Otago Committee
 on Ethics in Care and Use of Laboratory Animals and were in accord with
 the “Principles of Laboratory Animal Care” (National Institutes of Health
 publication number 80-23, revised 1996). The
 behavioural and single-neuron recording methods have been previously
 described in detail (Hyland & Jordan, 1997; Parr-Brownlie
 & Hyland, 2005). Male Wistar rats (~ 400 g at the time of surgery)
 were trained to reach into a 25-mm-wide, 14-mm high rectangular opening 55
 mm above the floor to retrieve chocolate flavoured breakfast cereal (Coco
 Pops™) from a tray. Once a clear paw preference for reaching had been
 established, chronic extracellular recording electrodes were implanted
 into the contralateral PPTg (anteroposterior -7.8 mm and lateral ± 2.0 mm,
 relative to bregma (Paxinos & Watson, 1997)) using sterile
 stereotaxic technique under full anaesthesia.  During
 recording sessions, signals from electrodes were amplified and filtered
 (2000 -8000 x, 0.5 - 10 kHz bandpass). Extracellular action potentials
 from single neurons were recorded, along with marker channels for
 automatically or manually entered behavioural events using SciWorks
 software (Datawave, CO). When a suitably isolated neuron was identified,
 it was first recorded while animals made ~ 50 reaches. Following this, for
 a subset of neurons recordings were made while the rats were exposed to 50
 trials of 50 ms duration 4.5 kHz tone stimuli (Med Associates Sonalert),
 with pseudorandom intertrial intervals.   Unit activity
 was analysed off-line using Spike2 software (CED, Cambridge, UK). Spikes
 belonging to single neurons were discriminated based on waveform shape
 (Fig. 1 B). For analysis of reach- and tone responses, activity was
 averaged over the reaching trials or tone presentations in peri-event time
 histograms (PETH). For reach data, PETH was centred on the interruption of
 an infrared beam at the position of the food. We chose this moment in the
 reach sequence because we were interested in determining if PPTg activity
 may correlate to distal skilled components of the reach-to-grasp movement,
 and this marks the termination of the extension phase and onset of the
 complex movements of the paw that are generated for grasping (Whishaw
 & Pellis, 1990; Hyland & Jordan, 1997; Sacrey<em> et
 al.</em>, 2009). For the reach-PETH, cell activity was averaged
 across trials, extending for 2 seconds before and after the light-beam
 interrupts generated by the preferred paw (contra-lateral to the recorded
 hemisphere) and normalised by converting to instantaneous frequency
 (spikes/s) (Fig. 1 C). The first 500 ms of each PETH was used to define
 the baseline firing rate. PETH for slow-firing cells used 50 ms bin width
 and for fast-firing cells, 25 ms was used. Peaks in the PETH were defined
 as a group of consecutive bins that were beyond + 2 standard deviations
 (SD) from the mean baseline firing rate. For fast-firing cells, three
 consecutive 25ms bins were required, for slow-firing cells, two 50 ms
 bins. For inhibitions, the same criteria were applied using a threshold at
 – 2 SD, except for cases in low-firing cells where this threshold value
 was &lt; 0 spikes/s. In these, we used the criterion defined by
 Galvan<em> et al.</em> (2016), where troughs were defined as
 periods in the PETH with zero bin counts that lasted ≥ 2 bins longer than
 any silent period in the baseline. The time of the modulation onset was
 defined as the leading edge of the first bin to cross the threshold, and
 the duration measured to the trailing edge of the last bin remained beyond
 the threshold. Onset times are expressed relative to the time of the
 light-beam interrupt which marked both the end of the ballistic extension
 phase of the reach and the onset of grasping. The amplitude of peaks was
 quantified as the mean of the in-peak bins. For analysis of firing
 patterns, autocorrelation histograms were calculated from selected periods
 around or between reaching events. For tone response PETH, similar methods
 were used except that the PETH was centred on the onset of the tone and a
 2 ms bin width was used to capture the onset time of the short latency
 responses. Statistical analyses were performed using
 GraphPad Prism Version 8 (GraphPad Software LLC). To
 assist in the demarcation of the timing of overall population responses
 separately for 1<sup>st</sup> (+) and
 1<sup>st</sup> (-) responses we performed a boxcar analysis on
 the average normalized Z-score in 50 ms bins using one-sample t-tests to
 determine deviation from baseline (Z = 0), over the period – 2 to + 1.5 s
 (<em>n</em> = 71 bins), with overall alpha = 0.05. Bonferroni
 correction for multiple comparisons (p = alpha/<em>n</em>)
 yielded p = 0.0007 to reject the null hypothesis. We therefore set p =
 0.0001 as a conservative threshold to define a significant
 population-level response. To mark the onset and offset times for such
 responses we used the time at which p values crossed 0.001 before and
 after reaching the threshold. To test whether the timing of onset of the
 population (+) and (-) response components might differ, we performed a
 two-factor repeated measures ANOVA for individual neuron’s original
 (unsmoothed) PETH bin data for the factors RESPONSE TYPE (+, -) and TIME
 (71 time points from -2 to 1.5 s). To account for variation in baseline
 firing rate between neurons, data were normalised by calculating Z-score
 values for each PETH bin as the difference between the bin firing rate and
 the mean baseline rate (-2 to -1.5 s), divided by the standard deviation
 of the baseline rate. For 1<sup>st</sup> (-) neurons Z-score
 values were inverted so that the analysis focussed on each group’s
 deviation from 0, rather than the sign of the response. Sphericity was not
 assumed, with Geisser-Greenhouse correction as required.
 To objectively quantify the instantaneous frequency of regular
 repeated peaks in PETH and autocorrelation histograms we used the
 <em>Fit Data</em> function in Spike2 v7 to fit a sinusoid
 function to ranges of data that were initially visually selected as
 potentially containing oscillatory activity. The least-square fitting
 function minimised the sum of squares of the errors between the data and
 the fitted curve, and the Spike2 algorithm calculated an estimate of the
 probability that a minimum sum of squares of errors of at least this size
 would occur (Spike2 for Windows Manual v7, 2017).
description: # Data for: Multimodal convergence in the pedunculopontine tegmental
 nucleus: motor, sensory, and theta-frequency inputs influence the activity
 of single neurons
 [https://doi.org/10.5061/dryad.1g1jwsv4t](https://doi.org/10.5061/dryad.1g1jwsv4t) The files are the numerical data used for each statistical analysis reported in the paper "Multimodal convergence in the pedunculopontine tegmental nucleus: motor, sensory and theta-frequency inputs influence activity of single neurons", by Lu, Wickens, and Hyland, currently (April 2024) in production at European Journal of Neuroscience. The data has been exported to CSV format from GraphPad PRISM (the application used for the reported analyses). ## Description of the data and file structure The 2 files with the title beginning "1 Boxcar ..." are the mean PETH Z-score values of neuronal firing rate in the graphs shown in the top panel of Fig. 3C of Lu et al. for 1st (+) and 1st (-) neurons. Columns are sequential time bins across the Time axis; values in each column are the actual value for that bin (top cell) and the 9 following bins. The published analysis was a 1-sample t-test, comparing the mean of each group of 10 bins to 0, separately for each response type. The file with the title beginning "2 ANOVA ..." is the Z-score transformed values of the neuronal firing rate of the individual reach PETH of each PPTg neuron. Rows are sequential bins units are seconds; columns are neurons. Headers with "+" or "-" in name are 1st (+) and 1st (-) neurons respectively. Data were used for two-way ANOVA comparing the bin values for the two response types over time, as reported in the text. Note that values of Time &gt; 1.5 s were not included in the analysis. The file with the title beginning "3 FR-AP ..." is the baseline firing rates (spikes/s) and action potential widths (peak to trough, in seconds) for all recorded neurons, plotted in Fig. 4 B and analysed with Spearman correlation. The file with the title "4 + vs - vs 0 BFR" is the baseline firing rate (spikes/s) for each PPTg neuron tested in the reach task, separately for neurons with 1st (+) or 1st (-) responses, or no response (0), shown in Fig 4 C and analysed with Kruskal-Wallis test. The file with the title "5 + vs - vs 0 AP width." is the action potential peak-trough width (in seconds) for each PPTg neuron tested in the reach task, separately for neurons with 1st (+) or 1st (-) responses, or no response (0), shown in Fig 4 D and analysed with Kruskal-Wallis test. The file with the title beginning "6 Proportion ..." is the contingency table of numbers of neurons with reach-related (Reach R) or no reach-related activity Reach NR) that had (Tone Respond) or did not have a tone response (No Tone Respond), analysed with Fisher's exact test, as reported in the text. The file with the title "7 Theta Freq" is the oscillation frequencies (Hz) of sine wave fits to reach PETH that contained oscillations (PETH Theta) and to autocorrelation histograms for cases in which oscillation was only seen in these analyses, not in PETH (AUTO only Theta), analysed with Kolmogorov-Smirnov test, as reported in the text.