Nature Manuscript #F02592
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SUPPLEMENTARY MATERIAL
Reversible lesions in HVC. Injections of lidocaine (2% in phosphate buffered saline)
were made from pulled glass pipettes (30-50m tip size) using a pressure injection
system (Pico Spritzer, Inc.). Injected volumes were 10–100nL. HVC localization was
determined prior to injection by recording sleep bursts in HVC interneurons.
Antidromic Identification of HVC neurons.
Bipolar stimulating electrodes were
placed in RA and/or X for antidromic identification20,31 of RA-projecting- and Xprojecting- HVC neurons. Electrical stimulation (single monopolar pulses of 0.2ms
duration) was produced using an isolated stimulation unit (AMPI, Inc), with intensities
varying from 50-500A. HVC neurons were found and isolated using ongoing 1 Hz
stimulation in RA or X to elicit spike responses. The absence of spontaneous firing in
HVC[RA] neurons, and the low spontaneous firing rates of HVC[X] neurons in the awake
non-singing animal made them nearly impossible to isolate without ongoing electrical
stimulation in RA or X. Once a cell was isolated, stimulation threshold current was
measured and stimulus intensity was set from 10% to 50% above threshold to produce a
reliable response. The response latency is defined as the interval between stimulus onset
and the first evoked spike. Latency variability is defined as the standard deviation of the
latencies measured for at least 20 stimulus trials. A criterion for antidromic activation of
RA-projecting and X-projecting neurons was a latency variability of less than 100μs.
Collision tests were done by stimulating in RA or X with some delay after a
spontaneous spike. For projection neurons, stimulating at small delays (1–3ms) resulted
in 100 percent failure rate (collisions) when the triggering event was a single spike. For
interneurons, stimulating at neither small nor intermediate delays resulted in a
significant failure rate. No other criteria were used to restrict the population of HVC
neurons in the dataset. RA neurons were isolated and identified on the basis of their
highly periodic spontaneous spike trains.27
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Nature Manuscript #F02592
31. Fuller, J. H. & Schlag, J. D. Determination of antidromic activation by the collision
test: problems of interpretation. Brain Res 112, 283-298 (1976).
Assessment of sleep state. The sleep state of birds was assessed by
measurement of the electroencephalogram (EEG). EEGs were measured differentially
between two electrodes, one placed between the bone layers near the sagittal sinus, the
other placed below the dura approximately 2mm lateral and 2mm anterior of the
bifurcation of the sagittal sinus (lambda). High-frequency bursts of spikes in RA only
occurred during sleep-like EEGs; the presence of spontaneous bursts in RA was
therefore used as an alternate criterion for the sleep state.
Calculation of conditional correlation (additional details). Why do we use a
conditional correlation measure to quantify correlations between spike trains, instead of
the more traditional cross-correlation? During sleep, RA neurons generate roughly 13
times as many bursts as HVC[RA] neurons. Thus, even if every HVC[RA] spike were
precisely aligned with an RA spike, most RA spikes would not be aligned with HVC[RA]
spikes, and the two spike trains would have a very small peak cross-correlation. For
example, in Figure (a), we see that for nearly every HVC[RA] burst, there follows an RA
burst 10ms later. However, for every RA burst we see, there are ~10 other bursts that are
not correlated with the HVC[RA] burst, and that do not appear in the raster plot. Thus,
while the cross correlation between these spike trains (Figure b) shows a peak at +10ms,
the height of the peak is only 0.16. (In figure b, the cross correlation was normalized by
the interpolated autocorrelograms. Other normalization techniques give similar or
smaller values of peak cross-correlation.) In contrast, cross-correlations of HVC
interneuron pairs give peak cross-correlation values close to unity. Thus, the small value
of cross-correlations between RA and HVC[RA] spike trains is a generic property of the
cross-correlation function for spike trains with largely different spike rates.
Nature Manuscript #F02592
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We have adopted an approach to this problem, first worked out by Brillinger, et
al, in which one chooses a reference spike train (train ‘A’ in Figure d), and determines
the conditional spike probability, i.e., the fraction of spikes in train A for which there is
one or more spikes in train B within a small time window (e.g. 10ms, shown by the gray
box in figure d). Note that, unlike the cross-correlogram, the conditional spike
probability depends on which spike train is chosen as the reference. (We always choose
the reference neuron as the type that fires fewer spikes, e.g. the HVC[RA] neuron.) Of
course, even uncorrelated spike trains may have a non-zero conditional spike
probability. Thus, we normalize the calculated conditional spike probability relative to
its expected value, to yield a number between –1 and 1, by analogy with a cross
correlation, and to yield a value of zero if the conditional spike probability is the same
as expected for suitably randomized spike trains. The resulting ‘conditional correlation’
function for the spike data of Figure (a) is shown in Figure (c). Note that this function is
qualitatively similar to the cross-correlogram, but has a peak value of 0.75, close to the
value one would intuitively expect for the data shown in the raster plot (given that 6 of
the 31 HVC[RA] burst have no associated RA burst).
In computing conditional correlations, we have excluded single spikes in the
spike trains of RA neurons and HVC interneurons. Single spikes are those not preceded
or followed by an interspike interval of less than 10ms. RA neurons and HVC
interneurons generate intrinsic periodic (~20 Hz) firing that is uncorrelated between
neurons (unpublished observations). Single spikes thus lead to an underestimate of the
conditional correlation for burst-related spikes. Only records containing more than 20
RA bursts and 8 HVC[RA] bursts were analyzed. Of these, all bursts were analyzed; there
was no additional selection criterion applied.