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Acute off-target effects of neural circuit manipulations

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Rapid and reversible manipulations of neural activity in behaving animals are transforming our understanding of brain function. An important assumption underlying much of this work is that evoked behavioural changes reflect the function of the manipulated circuits. We show that this assumption is problematic because it disregards indirect effects on the independent functions of downstream circuits. Transient inactivations of motor cortex in rats and nucleus interface (Nif) in songbirds severely degraded task-specific movement patterns and courtship songs, respectively, which are learned skills that recover spontaneously after permanent lesions of the same areas. We resolve this discrepancy in songbirds, showing that Nif silencing acutely affects the function of HVC, a downstream song control nucleus. Paralleling song recovery, the off-target effects resolved within days of Nif lesions, a recovery consistent with homeostatic regulation of neural activity in HVC. These results have implications for interpreting transient circuit manipulations and for understanding recovery after brain lesions.

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Figure 1: Motor skills that survive motor cortical lesions are acutely affected by transient manipulations of motor cortex.
Figure 2: Transient inactivations of Nif severely degrade adult zebra finch song, while permanent lesions have no noticeable effect when singing resumes two days later.
Figure 3: Initial disruption and subsequent recovery in vocal performance and HVC dynamics following Nif lesions.
Figure 4: Homeostatic regulation of spiking activity in HVC neurons can account for the functional recovery after Nif lesions.

Change history

  • 10 December 2015

    The PDF was replaced on 10 December 2015 to amend corrupted figures.


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We thank E. Soucy and J. Greenwood for technical assistance. We are grateful to M. Meister, J. Sanes, N. Uchida, K. Blum, A. Dhawale, M. Josch, A. Kampff, and E. Feinberg for their feedback on our manuscript. This work was supported by a McKnight Scholars Award to B.P.Ö., HSFP and EMBO fellowships to S.B.E.W., an NRSA fellowship to R.K., and a Rubicon fellowship from the Netherlands Organization for Scientific Research to S.M.H.G.

Author information




B.P.Ö. and T.M.O. designed the study with input from all authors. T.M.O. collected and analysed the data from songbirds with help from A.K. S.M.H.G. did initial pilot experiments in songbirds that inspired the study. C.P. implemented the HVC network model. S.B.E.W. performed the optogenetics experiments in rats and analysed the data. J.Y.R. and R.K. performed the pharmacological inactivation experiments in rats and analysed the data. B.P.Ö. supervised and coordinated the project. B.P.Ö., T.M.O., and S.B.E.W. wrote the paper with input from the other authors.

Corresponding author

Correspondence to Bence P. Ölveczky.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Light stimulation of motor cortical neurons expressing the optogenetic activator Chrimson.

a, Representative example of AAV-injections into motor cortex, showing Chrimson-tdTomato expression at different magnifications in a coronal brain section (~1.5 mm anterior to bregma). The scheme of the brain (right) is adapted from Paxinos’ rat atlas. The estimated spread of the injections was 8.3 ± 1.3 mm3 (mean ± s.d., n = 2 rats), with an average of 31 ± 2% infected cells (Methods). b, Heatmap showing the instantaneous firing rates of 28 single units recorded in an anaesthetized rat in response to a 1 s light pulse, averaged over 30 stimulations (Methods). c, A custom-built battery-operated wireless optogenetic stimulation device, consisting of a printed circuit board with integrated IR sensor and LED (λ = 615 nm). The IR sensor gates the circuit and allows the LED to be triggered by an IR light-source placed on top of the rat’s cage. During surgery, the LED is affixed atop a small craniotomy above motor cortex.

Extended Data Figure 2 Both brief and sustained optogenetic stimulation of motor cortex cause significant performance deficits in our task.

a, Optogenetic stimulation was triggered on the first lever press in a trial, and lasted for either 50 ms or 1 s. b, Both sustained (1 s, left, compare Fig. 1h, n = 5 rats, P = 3 × 10−5, paired t-test) and brief (50 ms, right, n = 3 rats, P = 0.01, paired t-test) optogenetic activation of motor cortex interfered with normal task performance. c, Comparing the effects of the two stimulation protocols on task performance (ratio light on/light off) shows that sustained stimulation has a significantly larger effect (1 s: n = 5; 50 ms: n = 3, P = 0.004, unpaired t-test). Error bars represent s.e.m.

Extended Data Figure 3 Localization and lesioning of Nif.

a, Injection of fluorescently labelled cholera toxin subunit B (green) into HVC retrogradely labels Nif and anterogradely labels downstream control nucleus RA. b, Bilateral injections of the excitotoxin NMA produced focal lesions of Nif. Shown are Nissl stained sections from both hemispheres in the same example bird. Red arrows indicate the estimated boundaries of Nif; dashed green line shows the extent of the lesion.

Extended Data Figure 4 Muscimol injections into Nif.

a, Top, Nissl-stained parasagittal section of a zebra finch brain. Middle, magnified view of the region demarcated with a green square atop. Red arrows (left) indicate the estimated boundaries of Nif; violet overlay (right) shows the spread of fluorescent dye co-injected with muscimol. Orange star indicates estimated centre of injection based on brightness of the fluorescence. Bottom, estimated injection sites relative to the boundaries of Nif for all muscimol-injected birds. Colours denote different animals. b, Syllable spectrograms (left) and entropy-duration distributions (right) for a bird injected with different volumes of muscimol in Nif. Example spectrograms for 9 nl and 27 nl injections are from recordings made 3 min and 7 min after the injections, respectively. That song disruption was similarly rapid and severe for both volumes (in conjunction with the lack of effect from injections above Nif) limits the possibility that the effects on song were due to diffusion of the drug into HVC.

Extended Data Figure 5 Spectrograms of vocalizations following unilateral Nif lesion.

Data for the example bird in Fig. 3. All examples were recorded within the first hour of singing after lesion. Top, example spectrogram of a motif recorded just before lesion. Left, example spectrograms of vocalizations in which motif syllables could not be reliably identified and thus were excluded from subsequent analysis. Middle, example spectrograms of identifiable motifs that were included in the alignment-dependent analysis (Fig. 3f–j). Right, example spectrograms of songs with identifiable syllables, but truncated motifs.

Extended Data Figure 6 Different mechanisms for homeostatic regulation of neural activity produce similar effects.

a, Top, effect of Nif removal on membrane excitability during simulated songs in a model neuron (from the 40th node), smoothed with a 100-point moving average and averaged over 40 model ‘experiments’ (shaded regions denote standard deviation across ‘experiments’). A rule for homeostatic regulation of activity drives a reduction in spiking threshold after Nif removal. Middle, fraction of simulations in a 100-point window for which activity in the model HVC network propagated to the end, averaged over 40 model ‘experiments’ (Methods). Bottom, a 100-point moving average over the time to complete a full chain propagation, averaged over 40 model ‘experiments’. Orange triangle denotes time of Nif removal. Same as in Fig. 4b, d. b, c, Same as in a, but with homeostatic regulation of membrane leak conductance (b) and synaptic input strength (c) (Methods).

Supplementary information

Comparison of skill execution before and after unilateral motor cortex inactivation.

Video shows a trained rat engaging in our lever pressing task before and after injections of muscimol into left motor cortex (100 nl of 1 mM and 200 nl of 4 mM respectively). Eights trials are shown for each conditions. The videos are slowed down by a factor of two. (WMV 28810 kb)

Comparison of skill execution with and without unilateral optogenetic motor cortex stimulation.

Video shows a trained rat engaging in our lever pressing task with and without optogentic stimulation of right motor cortex. Four trials are shown. The first two are without stimulation; the last two are with a 1 second light stimulation triggered on the first lever press in the trial. (WMV 12686 kb)

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Otchy, T., Wolff, S., Rhee, J. et al. Acute off-target effects of neural circuit manipulations. Nature 528, 358–363 (2015).

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