FP7-PEOPLE-2012-IEF
Motor_Dev
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FP7-PEOPLE-2012-IEF Motor_Dev
T ABLE OF CONTENTS
A Summary of Activities (Months 13-24)........................................................................................... 3

Scientific summary ................................................................................................................................... 3
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Manuscripts published, submitted or in preparation. .................. Ошибка! Закладка не определена.
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Presentation of results in national and international conferencesОшибка! Закладка не определена.
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Supervision activity and collaborations ......................................... Ошибка! Закладка не определена.
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Courses .......................................................................................... Ошибка! Закладка не определена.
B Results Part I ................................................................................................................................ 6
C Figures and Figure Legends Part I .................................................................................................. 8
C Results Part II ............................................................................................................................. 15
D Figures and Figure legends Part II ................................................................................................ 17
F Experimental Protocols ............................................................................................................... 22
G Reference List ............................................................................................................................. 25
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A S UMMARY OF A CTIVITIES (M ONTHS 13-24)

Scientific summary
Part I. In the first 12 months period of activities, we developed a method enabling recording simultaneously translaminar spinal cord neuronal population activity and motor behavior in vivo, in neonatal rats. Our in vivo data suggest that the predominant function of developing spinal cord networks consists in generating spontaneous bursts in intermediate to ventral (motor) zones, which drive movements and cause, through sensory feedback, activation of dorsal (sensory) zones. This is supported by the loss of activation of sensory zones in association with movements after transection of dorsal rootlets.
While twitches represent the most prominent form of motor activity in rat pups, neonatal behavior is also characterized by more complex movement patterns mainly occurring in awake periods. Thus, we extended our analysis from twitches (essentially defined as behavioral events lasting less than 600 ms and occurring in a background of atonia) to more complex movement
(behavioral events lasting more than 600 ms).
Spontaneous activity also has been described in the neonatal isolated spinal cord preparation or spinal cord slices, in which correlated sensorimotor activity may be supported by intrinsic connections and emphatic interactions (Bos et al., 2011; Demir et al., 2002; Fellippa-Marques et al.,
2000; Kremer and Lev-Tov, 1998). Therefore, we further investigated interactions between sensory and motor zones in the isolated spinal cord in vitro, through silicone probe recordings. We found that activity in motor and sensory zones of the spinal cord in vitro are largely dissociated. Taken together, our results provide direct evidence to the hypothesis that sensory feedback from spontaneous movements is instrumental for the temporal binding of motor and sensory neurons in the developing spinal cord.
Part II. We run a complete series of experiments providing: (i) A complete developmental profile of early activity patterns in primary motor cortex (M1); (ii) Histological assessment of M1 and primary somatosensory cortex (S1); (iii) Understanding of the relationship between spontaneous movements and epileptiform activity in M1 through different developmental stages. Our data suggests that, at early stages of life, activity in primary motor cortex (M1) is mostly driven by spontaneous movements, and during this period, epileptiform discharges remain remaining largely infraclinal.
Technical advances:
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Development of electrophysiological recordings of translaminar spinal cord neuronal activity in vivo (neonatal rats); this method enables system-level investigations that might provide a more complete view of the development of core modules of sensorimotor functions;
Establishment of electrophysiological recordings of translaminar spinal cord neurons activity in vitro;
Modeling electroclinical uncoupling in neonatal rats;
Establishment of novel analytical routines using the MATLAB environment.

Manuscripts published, submitted or in preparation
During the first 12 months period, we published the following related articles:
Sitdikova G, Zakharov A, Janackova S, Gerasimova E, Lebedeva J, Inacio AR, Zaynutdinova
D, Minlebaev M, Holmes GL, Khazipov R (2014) Isoflurane suppresses early cortical activity. Ann Clin Transl Neurol 1: 15-26;
Gerasimova EV, Zakharov AV, Lebedeva YA, Inacio AR, Minlebaev MG, Sitdikova GF,
Khazipov RN (2014) Gamma oscillations in the somatosensory cortex of newborn rats. Bull
Exp Biol Med 156:295-8.
We are currently submitting a manuscript entitled “Spontaneous movements synchronize developing sensory and motor spinal cord networks” and finalizing a manuscript entitled “Delayed development of the corticospinal tract underlies electroclinical dissociation in neonatal rats”.

Presentation of results in national and international conferences
The results obtained have been or will be presented and discussed at:
- The host institute data club (INMED, Marseille, France), June 23, 2014;
- The 9 th Forum of Neuroscience, Federation of European Neuscience Society (Milan, Italy),
July 5-9, 2014;
- International Scientific Conference, Science of The Future, St. Petersburg, Russian
Federation, September 17-20, 2014;
- The host institute annual meeting (INMED, Marseille, France), September 16-18, 2015;
- The Society for Neuroscience Annual Meeting, Chicago, IL, USA, October 17-23, 2015.

Supervision activity and collaborations
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The grant holder has supervised a Master 1 student, Dounia Bougotaya, who performed electrophysiological recordings in vivo using silicone-based electrode arrays, as well as histological assessment of the electrode array placement (Part II).
Throughout the period of activities, the grant-holder has worked in tight collaboration with A.
Nasretdinov, PhD student, Kazan University, Russian Federation, on data analysis. During the one and half months stay at Kazan University, a parallel collaboration has been established with Prof. A.
Rizvanov, on models of ischemic stroke.

Courses
The grant holder has attended:
The “Connaissance de l’animal de laboratoire - méthodologie expérimentale” course, provided by INSERM;
The “Le Français pour Chercheurs et personnels étranger” course, provided by INSERM;
Neurophysiology course, levels I and II, led by Dr. C. HAMMOND.
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B R ESULTS P ART I
Motor_Dev
Sensory feedback synchronizes sensory and motor zones during complex movement sequences
Analysis of complex motor sequences (such as crawling), classified based on duration, revealed that similarly to twitches, complex movements drive firing of spinal dorsal neurons, and no fundamental difference was found neither in the patterns evoked by different types of motor activity at the single segment level nor in correlation of activity in the motor and sensory zones (Part
I, Figure 1A-C). Moreover, as for twitching, global metrics of more complex motor behavior were not affected by the deafferentiation procedure (Part I, Figure 1D).
Dissociation of sensory-motor networks function in the isolated spinal cord
Spontaneous activity in isolated spinal cords was characterized by regular bursts within the dorsal horn, occurring at 15 ± 4.9 min -1 (mean ± SD, n = 5 animals, ≈ 6860 bursts), (Part I, Figure 4A).
These were associated with a dorsal sink and MUA bursts, lasting approximately 119 ± 27.7 ms
(mean ± SD, n = 5 animals, ≈ 6860 bursts; Part I, Figure 4A-C). A main feature of activity in motor zones was its organization in "megabursts" - 3.0 ± 0.27 s long periods of oscillatory activity at 6 ± 0.1 s Hz, occurring at 1.7 ± 0.3 min -1 (mean ± SD, n = 5 animals, ≈ 760 bursts; Part I, Figure 4D). These bursts were similar to those evoked by rhythmic dorsal roots stimulation (Part I, Figure 2). Isolated, short-lasting bursts (lasting about 62 ± 14 ms) reminiscent of twitch-related events in vivo also occurred in motor zones, at a frequency of approximately 2 min -1 (n = 5 animals, ≈ 760 bursts; Part I,
Figure 4E).
Sensory or motor bursts-triggered PSTHs of MUA, and cross-correlation analysis of bursts in both areas revealed weakly (or failed to reveal any) correlated firing between the two zones (Part I,
Figure 5A-D). Indeed, only about 3 % of bursts within sensory zones were followed (within maximum lag of 100 ms) by bursts within motor zones (average, n = 5 animals, for which in total ≈ 6860 events were detected). Moreover, in average, only 5 % of mega- and 16 % of short-lasting motor bursts were preceded by sensory bursts. Further cross-correlation analysis of units in sensory and motor zones referenced to entire recording sessions showed only a weak correlation, with sensory neurons firing 2 to 46 ms ahead of motor units (peak value of 5.4 spikes s -1 ). This is in striking contrast to the activity recorded in vivo, where motor units leaded sensory activity both during twitching and complex movement episodes, with a lag of 58 ± 22 ms (peak value of 24 ± 17 spikes ms -1 , mean ± SD, n=15 animals). It also differs from the overall activity pattern through entire recording sessions after transecting dorsal rootlets in vivo, where the correlation between the two zones was dramatically decreased or completely lost (Part I, Figure 5E).
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Pharmacological analysis of the spontaneous activity in the isolated spinal cord confirmed that: (i) combined application of ionotropic glutamate receptor antagonists CNQX and APV completely suppressed network-driven events both in sensory and motor zones (Part I, Figure 6); (ii) blockade of GABA(A) receptors with gabazine eliminated regular bursts in sensory zones and significantly increased LFP power and MUA during motor megabursts, with loss of the prominent within-burst 6Hz oscillation; during these epileptiform discharges in motor zones evoked by disinhibition (Bracci et al., 1996), activation of neurons in sensory zones was not observed (Part I,
Figure 7).
Thus, in contrast to the in vivo situation, activity in the isolated spinal cord was characterized by much weaker interactions between sensory and motor zones, and by a temporal lead of sensory neurons in the isolated sensory-motor network, inverse to the in vivo situation, where motor zones headed population activity. Taken together, data obtained in the isolated spinal cord provide strong support to the hypothesis that sensorimotor integration in spinal cord under physiological conditions is primarily driven by sensory feedback resulting from motor unit-driven movements.
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C F IGURES AND F IGURE L EGENDS P ART I
Motor_Dev
Figure 1_ Sensory feedback during complex movement sequences. A) Mean LFP traces and CSD maps triggered by the onsets of complex movement sequences recorded before and after deafferentation (n before
= 33 and n after
= 24 complex movements, single animal). B) Corresponding normalized PETHs of MUA and complex movement waveforms (each gray trace corresponds to a single movement – absolute values - trace, and the back trace corresponds to the mean). Bottom
left: Complex movement onset-triggered time histograms of sensory, intermediate and motor zones MUA in the control condition (n = 3 animals, 69 complex movements, mean ± SD). Bottom right: Complex movement onset-triggered time histograms of sensory, intermediate and motor zones MUA after deafferentation (n = 3 animals, 136 complex movements, mean ± SD). A-B) Only stereotypical movements lasting 900 - 1500 ms were included in this analysis. C) Cross-correlogram of peri-movement motor and sensory spikes times (n = 3 animals, mean ± SD). Left: Control condition. Right: After transection of dorsal rootlets. D) Frequency (min -1 ), maximal amplitude, power (normalized to duration) and duration of complex movements after versus before transection of afferent fibers (presented as % of control).
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Figure 2_ Nature of activity patterns induced by dorsal root stimulation in the in vitro spinal cord preparation. A)
Overlaid dark-field and epifluorescence (DiI, red) images representing the typical positioning of the electrode array in the in
vitro spinal cord preparation (in this case, L3 segment), equivalent to that of the in vivo experiments. B) Dorsal root (L3) stimulus-triggered mean LFP and CSD map (n ≈ 50 stimuli). Note the short-latency LFP deflections and current sinks evoked in dorsal horn (sensory zone). C) Top: Associated normalized PSTH of MUA. Bottom: Average MUA response times, with graphed onsets and offsets (mean ± SD, n = 5 animals). The sensory potential was associated with a MUA burst, at a latency of 3.6 ± 0.65 ms and lasting 42 ± 14 ms post-stimulus (mean ± SD, n = 5 animals), essentially limited to the dorsal horn
(depth range of 100 - 400 µm). D) Original LFP traces denoting spinal responses to rhythmic dorsal root stimulation. Motor zones were weakly activated by single stimulus. However, rhythmic stimulation of dorsal roots (at least 5 stimuli at 1-2 Hz) evoked long-lasting rhythmic oscillatory bursts in motor zones, with coherence between motor units and oscillation. E)
Normalized PSTHs of MUA (Z-scores) demonstrating the effect of stimulus intensity (as indicated in the figure) in firing rates in sensory and motor zones (Z-scores, n = 3 animals). F) Ventral root (L3) stimulus-triggered mean LFP traces (n ≈ 50 stimuli), revealing clear anterograde population spikes in ventral horn (motor zone). Generally, electrical stimulation of the respective ventral root evoked anterograde population spikes in the ventral horn (depth range: 900 - 1100 mkm, n = 5 animals), showing peak amplitude at 2.8 ± 0.45 ms after stimulus (mean ± SD, n = 5 animals).
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Figure 3. Nature of activity patterns induced by dorsal root stimulation in the in vitro spinal cord preparation:
specificity. For each preparation, dorsal roots anterior and posterior, as well as contralateral to the root innervating the hemi-segment in which the electrode array was inserted also were electrically stimulated. Figure 3A-C refers to Figure 2. A)
Anterior dorsal root (L2) stimulation-triggered mean LFP and PSTH of MUA (n ≈ 50 stimuli, same animal as in Figure 2). B)
Contralateral dorsal root (L3) stimulation-triggered mean LFP and PSTH of MUA (n ≈ 50 stimuli, same animal as in Figure 2).
Note that equivalent stimulations of anterior, posterior or contralateral roots evoked no or a weaker spinal response. C)
Dorsal root (L3) stimulation triggered mean LFP traces (n ≈ 50 stimuli) before (black traces) and after combined bath application of the ionotropic glutamate receptor antagonists APV and CNQX (purple traces). Response suppression in the presence of AP-V and CNQX confirmed its dependence on synaptic transmission.
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Figure 4_ Spontaneous sensory and motor events in the spinal cord in vitro. A) Original LFP traces representing stereotypical sensory zone bursts and heterogeneous motor zone events (S - short lasting motor event; L - long lasting motor even). B) Autocorrelograms of spike times in selected sensory and motor zones recording sites (as highlighted in
Figure 10 A). C) Histogram of inter-sensory event intervals (with a peak at 3 s). D) Top: Original LFP traces and MUA (red); the long-lasting motor event indicated in Figure 10 A is shown here in more detail. Bottom left: Filtered LFP trace and coherent MUA at a recording depth of 900  m. Bottom right: Mean power spectrum of bursts referent to a selected motor zone recording site (mean ± SD, n = 5 animals, ≈ 760 bursts). E) Original LFP traces and MUA (red); examples of isolated, short-lasting motor events.
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Figure 5_ Spontaneous in vitro spinal cord events: dissociation of dorsal and ventral activities. A) Sensory burststriggered: mean LFP and CSD map (single animal), as well as normalized PETH of MUA across all spinal depths (single animal); normalized PETHs of MUA of sensory, intermediate and motor zones (Z-scores, one trace corresponds to a single animal, n = 5), as well as the percentage of detected surrounding motor events (mean ± SD, n = 5 animals). B and C)
Equivalent graphics instead triggered by long- and short-lasting motor events, respectively. D) Left: Representative histograms of inter-sensory event intervals during bursting and non-bursting periods in motor zone (black-filled and orange-lined bars, respectively; single animal). Right: Mean inter-event interval values during bursting and non-bursting periods in motor zone per animal (n = 4). E) Cross-correlograms of all motor and sensory units recorded per animal in vitro
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(mean ± SEM, n = 5), in vivo under control conditions (mean ± SEM, n = 15) and in vivo after deafferentation ((mean ± SEM, n = 3). * p < 0.05.
Figure 6_ Spontaneous sensory and motor events in the spinal cord in vitro: suppression of network driven
events by APV and CNQX. A) Original LFP traces before and following combined application of AP-V and CNQX. B) Time course of drug effect on bursting and spiking activity (min -1 ) within sensory and motor zones (blue and black traces, respectively).
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Figure 7_ Spontaneous sensory and motor events in the spinal cord in vitro: suppression of sensory events and
emergence of epileptiform motor events in the presence of gabazine. A) Top: Original LFP traces before and following bath application of gabazine (single animal). Bottom: Exemplary motor events prior to and after gabazine application. B)
Ventral root stimulation triggered mean LFP traces (n ≈ 50 stimuli), identifying motor neuron pool depths. C) Mean power spectrum of detected bursts referent to a selected motor zone recording site before and after gabazine application (mean
± SD, single animal), showing a loss of event structural stereotypy in the presence of the GABAA antagonist. D) Time course of drug effect on bursting and spiking activity (min -1 ) within sensory and motor zones (blue and black traces, respectively).
D) Motor event duration for each of the analyzed conditions (mean ± SD, single animal). E) Gabazine: motor burst-triggered mean LFP and CSD map, as well as normalized PETH of MUA across all spinal depths (single animal).
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C R ESULTS P ART II
Motor_Dev
Data referent to part II are still under analysis, and thus only a preliminary description of the results will be provided.
Spontaneous movements trigger M1 spindle-bursts in neonatal rats
To study the relationship between neuronal activity in M1 and spontaneous movements of neonatal rats, we performed simultaneous recordings of LFP and MUA from the hindlimb representation in M1, using silicone-based electrode arrays (Part II, Figure 1), and the corresponding hindlimb movements in P5-P6 rats. Coordinates for recordings were based on a priori functional mapping using cortical microstimulations (data not shown). At P5-6, electrical activity in M1 was characterized by discontinuous temporal pattern reminiscent of “tracé discontinu” described in premature human neonates (Lamblin et al., 1999), with bursts of activity alternating with periods of silence. Each burst of activity was typically organized in short-lived oscillation reminiscent of
“spindle-bursts” previously described for the primary somatosensory cortex (S1, (Khazipov et al.,
2004). M1 spindle-bursts were associated with a sharp elevation in MUA locked to the troughs of oscillation (Part II, Figure 2) and their phase reversal was observed typically at a depth of 1000 µm
(Part II, Figure 2).
M1 spindle-bursts were highly correlated with spontaneous movements of the corresponding extremity (Part II, Figure 2). Cross-correlation analysis revealed a clear correlation between limb movements and M1 spindle-bursts (Part II, Figure 5), with movements preceding the vast majority of M1 spindle-bursts. Thus, M1 behaves very similarly to the somatosensory cortex at this developmental stage (Khazipov et al., 2004;Mohns and Blumberg, 2010;Yang et al., 2009), with most of the activity organized in spindle-bursts which are typically preceded by spontaneous movements. This phenomenon also has been recently described by An et al., 2014.
Developmental profile of population activity in M1
We further addressed the developmental changes in M1 activity and its relation to spontaneous movements by recording from rats between P3 to P21.
During the first week, the characteristic pattern of activity in M1 was spindle-bursts, triggered by spontaneous movements. Developmental changes during this period included: (i) an increase in the ongoing activity that was also manifested in a decrease of discontinuity, and (ii) a decrease in the correlation between spindle-bursts and movements manifested in an increase of spontaneous – i.e. not preceded by movements – negative LFP troughs of M1.
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At around P8, both motor and M1 activity rapidly changed. First, the occurrence of spontaneous movements decreased (Figure 4). Second, electrical activity in M1 became more continuous (Figure 4A). The less frequent twitches were still associated with an increase of M1 activity. However, due to an increase in the ongoing M1 activity and to a decrease in the frequency of spontaneous movements, the correlation between M1 events and spontaneous movements decreased, and the number spontaneous, i.e. not preceded by movements, cortical events increased dramatically.
Spontaneous movements trigger bicuculline-induced M1 epileptiform events in neonatal rats
We further addressed the relationship between spontaneous movements and epileptiform activity in M1. Epileptiform activity was induced in M1 by local, topical application or intracortical injection of the GABA(A) receptor antagonist bicuculline (1 mM, ≈1 µL). In keeping with previous observations obtained in vitro (Wells et al., 2000) and in vivo (Minlebaev et al., 2007;Minlebaev et al., 2009), blockade of GABA(A) receptors caused recurrent epileptiform events (Part II, Figure 3).
The electrographic phenotype of the epileptiform events was age-dependent. At P3-4, epileptiform events largely maintained a spindle-bursts oscillatory structure yet became longer-lasting. From P5-
6, large amplitude population spikes often followed by lower amplitude oscillatory events started to emerge (Part II, Figure 2). Cross-correlation analysis between movement of the left hindlimb and epileptiform events in the corresponding area of M1 revealed that: (i) at P4-P5, epileptiform events were either spontaneous or triggered by hindlimb movements, (ii) after P10, virtually all (98±2%, n=5 rats) M1-right forelimb population spikes were followed by a myoclonic twitch of the right forelimb,
(iii) during transitory period at P6-10, some bicuculline-evoked population spikes preceded movements, while others followed. While the frequency of myoclonic twitches remained unchanged in P3-7 rats, starting from P8 local M1 bicuculline application increased jerks frequency (Figure 5).
These results indicate that during the first postnatal week, local epileptiform events are triggered by spontaneous physiological jerks and remain largely infraclinical, whereas from the second postnatal week, epileptiform events in M1 cortex start to drive epileptic jerks.
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D F IGURES AND F IGURE LEGENDS P ART II
Motor_Dev
Figure 1_ Recordings of neuronal population activity in M1 (P3-P21). A) A linear silicone-based electrode array (16 recording sites; center-to-center separation of 100 μm) was inserted vertically in M1, in order to record LFP and MUA.
Overlaid epi-fluorescence images (Hoechst, inverted gray-scale image; DiI, red) and scheme of the different recording sites in relation to all cortical layers in a P6 animal. Note the dense Hoechst signal corresponding to layer IV in primary somatosensory cortex (S1), and lack of this dense layer in M1, where the silicone-based array was inserted.
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Figure 2_ Spontaneous movement trigger activity in M1 over the first postnatal week. A) LFP traces and CSD maps denoting cortical events in relation to spontaneous movements in a P6 animal. Left: Control condition. Right:
Following topical application of bicuculline. Note that under both conditions, cortical events were reliable preceded by movement (see also Figure 5), although some events occurred spontaneously (i.e., not associated with movements). B)
Corresponding LFP power before and after bicuculline application. Note the dramatic increase in power. C) Typical intercortical event interval (900 μm depth) in the presence of bicuculline. D) Histograms of movement durations in the control condition and following application of bicuculline, showing similar distributions (Wilcoxon rank-sum test = 0.638).
Movement frequency also did not differ prior to or after bicuculline delivery (13.8 and 14.5 events per min, respectively, mean, single animal); A-D) Single animal (P6).
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Figure 3_ Global effect of local bicuculline delivery. A) LFP and corresponding hindlimb traces denoting, in a P14 animal, the penetration of topically applied bicuculline, in M1, and the striking increase in movements amplitude ad frequency associated with a striking increase in LFP power in deeper (≈ 1000 μm) cortical layers.
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Figure 4_ Epilepform discharges in M1 reliably trigger movements during second postnatal week. A) LFP traces and CSD maps denoting cortical events in relation to spontaneous movements in a P14 animal. Top Left: Control condition.
Top right: Following topical application of bicuculline. Bottom: Filtered LFP trace and coherent MUA at a recording depth corresponding to Layer V (1000 μm), and respective movement trace. B) Corresponding LFP power before and after bicuculline application. Note the dramatic increase in power. C) Typical inter-cortical event interval (1000 μm depth) in the presence of bicuculline. D) Histograms of movement durations in the control condition and following application of bicuculline, showing similar distributions (Wilcoxon rank-sum test = 0.599). However, movement frequency increased after bicuculline delivery (3.15 events per min before and 20 events per min after delivery of convulsive agent, mean, single animal); note the consistency between inter-cortical event interval and movement frequency after delivery of bicuculline.
A-D) Single animal (P14).
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Figure 5_ Developmental shift in the temporal relationship between cortical events and spontaneous
movements. A) Cross-correlograms of spontaneous movement onset and cortical event onset (normalized to movements) in a P6 and In P14 animal reveling peaks of correlation after and before movements, respectively.
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F E XPERIMENTAL P ROTOCOLS
Motor_Dev
ETHICAL CONSIDERATIONS
All experiments were performed in accordance with protocols approved by the Ethical
Committee for Animal Research of Marseille (reference 00708.01). Subjects were male and female
Wistar rats, 3 to 21 days old (i.e., P3 to P21, with the day of birth being considered as P0), from multiple litters.
SPINAL CORD
Extracellular recordings, in vitro
Animals (P5 to P7, n = 6) were anesthetized using isoflurane, decapitated and eviscerated. The spinal cord, along with dorsal and ventral roots, was obtained through dorsal laminectomy and transferred to a holding chamber where it was maintained for at least 1 hour. Thereafter, the spinal cord was placed, dorsal side up, in an interface recording chamber (Cunningham et al., 2008). During dissection and all subsequent steps, the spinal cord was continuously perfused with carbogenated
(95% O
2
, 5% CO
2
) artificial cereberal spinal flduid (ACSF) containing, in mM: 130 NaCl, 4 KCl, 2 CaCl
2
,
1.3 MgSO
4
, 0.58 NaHPO
4
, 25 NaHCO
3
and 10 glucose. We recorded extracellular, local field potential
(LFP) and multiunit activity (MUA) using a linear silicone-based electrode array (16 recording sites with a center-to-center separation of 100 μm, A1x16-5mm-100-703-A16, Neuronexus, Ann Arbor,
MI, USA). Labeling of the electrode array with the fluorescent dye 1,1′-Dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiI, Sigma-Aldrich, Europe) prior to insertion facilitated the later anatomical localization of each recording site. The electrode array and implantation procedure were as described for our previous in vivo recordings (see Mid-Term Report and Final
Report).
Data acquisition. Recordings were initiated 15-30 min after electrode implantation and performed at approximately 23°C; signals were amplified using a xCellAmp64 amplifier and digitized at 10 kHz (DIPSI). Dorsal and ventral roots were stimulated using a bipolar 50 µm nichrome-wire electrode; electrical pulses (50 µs) were generated by a Grass Products stimulator and stimulus isolator and delivered at 0.2-2 Hz. In a subset of experiments, recordings were performed under control conditions and in the presence of (2R)-amino-5-phosphonopentanoate (APV, 50 µm) and 6cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µm), or 4-[6-imino-3-(4-methoxyphenyl)pyridazin-1yl] butanoic acid hydrobromide (gabazine, 5 µm), all from Sigma-Aldrich.
Histology
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Transverse spinal cord slices (200 μm-thick) were imaged (for an initial DiI detection), after which complementary ChAT and NeuN immunostochemistry was performed. The anatomical location of each recording site of the electrode array was estimated based on insertion coordinates and corresponding or age-matched histological assessment (as demonstrated in Part I, Figure 2).
MOTOR CORTEX
Electrophysiological recordings, in vivo
Animals (P3 - P21, n = 32) were anesthetized using isoflurane and given the analgesic buprenorphine. A lidocaine solution was additionally used to provide local anesthesia. The parietal and frontal bones were exposed, and the periosteum gently removed. Two metal or plastic adaptors were fixed onto the skull, using a thin layer of cyanoacrylate and dental adhesive. Then, the adaptors were attached to a customized stereotactic frame, enabling head stabilization. A small craniotomy was performed over M1 (hindlimb representation), as determined a priori through intra-cortical microstimulation experiments. A linear silicone-based electrode array (16 recording sites with a center-to-center separation of 100 μm, Neuronexus) was inserted vertically in M1. Labeling of the electrode array with DiI prior to insertion facilitated the later anatomical localization of the array track. Throughout the experiments, body temperature was kept at 37°C using a heating pad, and buprenorphine was re-administered every 6 hours (when applicable). Animals were, at all times, surrounded by a cotton nest, mimicking the presence of the mother and littermates.
Data acquisition. Recordings were initiated after a minimum waiting period of 30 min following implantation of the electrode array and performed essentially as described previously
(Khazipov et al., 2004). Briefly, wideband neurophysiological signals were amplified through a xCellAmp64 amplifier and digitized at 10 kHz (DIPSI). Tactile stimuli were provided through a 0.6 mm-diameter metal bar driven by a piezoelectric bending actuator, which was controlled by a square pulse (5 ms, delivered generally at 5 s -1 , depending on the age of the animal). Spontaneous activity was recorded 30 to 60 min after discontinuing anesthesia, first under control conditions and secondly upon local intracortical delivery of the GABAA receptor antagonist bicuculline. Bicuculline
(1 mM, ≈ 1 μL, Tocris) was delivery either topically or using a glass pipette pulled from a glass capillary that was lowered to a depth consistent with the location of layer V and close to the recording electrode array.
Motor behavior
Ispilateral hindlimb movements were recorded through a piezoelectric transducer, which was capable of detecting the smallest visible movements, including respiration-related ones.
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Histology
Brain slices (150 μm-thick) were incubated with the nuclear florescent marker Hoechst, and imaged (for DiI and DAPI) under standardized conditions.
DATA ANALYSIS
Tactile stimulus or twitch/complex movement onsets (in vivo), as well as electrical stimulation of dorsal and ventral roots or spontaneous sensory and motor bursts onsets (in vitro) were used as triggers to compute mean LFP and CSD maps, as well as individual trigger spectrograms of field. CSD was computed for each recording site and smoothed with a triangular kernel of length 3. Spectral analysis was carried out using the Chronux toolbox. Spectral power and coherence were given by direct multi-taper estimators (5 Hz bandwidth, 3 tapers, 200 ms spectral window); to remove the slow frequency envelope, the LFP was filtered (5-100 Hz). Behavioral events were detected semiautomatically: isolated events lasting a minimum of 600 ms were considered complex movement sequences; behavioral events that last less 600 ms and that were precede and followed by at least 1 s of behavioral quiescence were considered as twitches (See Final Report Part I, Figure S3). Electrical stimulation of dorsal roots (in vitro) allowed identification of spinal cord “sensory zone” (the 2 recording sites/depths showing short-latency, high frequency MUA responses). Analogously, stimulation of ventral roots (in vitro) allowed, by evoking antidromic population spikes, identification of recording sites/depths referent to motoneuron pools. These zones were used for subsequent analysis (including MUA bursts quantification and cross-correlation of spike times and). In the case of
M1, analysis was performed for all recording sites/cortical layers, with particular emphasis on layers
IV and V. Multiunit bursts were generally detected by combined analysis of spike trains and significant field defections. Group data summary statistics, specific number of animals, and statistical tests used are indicated in the figure legends. Results were considered significant when p<0.05.
An extended version of the experimental procedures is provided in the Final Report.
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