Onset of Pup Locomotion Coincides with Loss of NR2C/D-Mediated Cortico-Striatal EPSCs and Dampening of Striatal Network Immature Activity

Adult motor coordination requires strong coincident cortical excitatory input to hyperpolarized medium spiny neurons (MSNs), the dominant neuronal population of the striatum. However, cortical and subcortical neurons generate during development large ongoing patterns required for activity-dependent construction of networks. This raises the question of whether immature MSNs have adult features from early stages or whether they generate immature patterns that are timely silenced to enable locomotion. Using a wide range of techniques including dynamic two-photon imaging, whole cell or single-channel patch clamp recording in slices from Nkx2.1-GFP mice, we now report a silencing of MSNs that timely coincides with locomotion. At embryonic stage (as early as E16) and during early postnatal days, genetically identified MSNs have a depolarized resting membrane potential, a high input resistance and lack both inward rectifying (IKIR) and early slowly inactivating (ID) potassium currents. They generate intrinsic voltage-gated clustered calcium activity without synaptic components. From postnatal days 5–7, the striatal network transiently generates synapse-driven giant depolarizing potentials when activation of cortical inputs evokes long lasting EPSCs in MSNs. Both are mediated by NR2C/D-receptors. These immature features are abruptly replaced by adult ones before P10: MSNs express IKIR and ID and generate short lasting, time-locked cortico-striatal AMPA/NMDA EPSCs with no NR2C/D component. This shift parallels the onset of quadruped motion by the pup. Therefore, MSNs generate immature patterns that are timely shut off to enable the coordination of motor programs.


INTRODUCTION
Adult medium spiny neurons (MSNs), the GABAergic principal neurons of the striatum, have unique features required for the appropriate selection of motor programs (Grillner et al., 2005). They are highly hyperpolarized at rest and require strong coincident excitatory glutamatergic inputs for the execution of appropriate movements (Wilson and Kawaguchi, 1996). These unique features are due to strong inward rectifying (I K IR ) and slow inactivating (I D ) potassium currents and feedforward and feedback afferent inhibition that confer a low input resistance, hyperpolarized resting potential, and a long delay to initial spiking (Tepper et al., 2004). In a large range of animal species and brain structures, immature neurons are highly excitable and developing networks generate network-driven patterns that are instrumental in neuronal growth, synapse formation, and the formation of functional circuits (for review Ben-Ari, 2002;Spitzer, 2006;Huberman et al., 2008;Blankenship and Feller, 2010). These observations raise the question whether MSNs differ from other neuronal types and are silent from early developmental stages and if not when and how are the early patterns switched off to enable correct motor coordination and quadruped motion.
Medium spiny neurons originate in the lateral ganglionic eminence (Deacon et al., 1994;Olsson et al., 1998) and migrate radially into the developing striatum compartments (Van Der Kooy and Fishell, 1987). To understand MSN functional maturation, we targeted embryonic and early postnatal MSNs with the use of Nk2 homeobox 1 (Nkx2.1)-GFP Mice. Nkx2.1, also known as thyroid transcription factor-1 (TTF-1), is a protein that regulates transcription of genes and is specifically expressed by interneurons but not by MSNs in the striatum (Nobrega-Pereira et al., 2008). We determined MSNs' cellular properties using whole cell and single-channel recordings. In parallel we performed two-photon dynamic imaging that enables to determine the activity of large neuronal samples in slice preparation (Crepel et al., 2007). We Frontiers in Cellular Neuroscience www.frontiersin.org then used behavioral analysis to describe posture and onset of locomotion in newborn pups. We report an abrupt switch of cellular and synaptic properties in MSNs in parallel with the initiation of locomotion.
Since embryonic and early postnatal MSNs lack their characteristic adult K + currents, they could not be identified from their electrophysiological properties. We therefore identified them as GFP-negative neurons (Sousa et al., 2009) in striatal slices from Nkx2.1-GFP mice. To quantify the proportion of Nkx2.1positive striatal interneurons that faintly expressed GFP and were therefore considered as MSNs, we performed immunocytochemistry of Nkx2.1 (see below; Figure 1A). From a total of 2767 Nkx2.1-expressing striatal neurons labeled with TTF-1 (red), 75% were also GFP positive (yellow, n = 2077), showing that in perinatal slices from Nkx2.1-GFP mice, 25% of the Nkx2.1positive neurons, though GFP-negative, were not MSNs. In our two-photon recordings, GFP positive/fura 2-loaded neurons represented 10-20% of all fura 2-loaded cells. Therefore instead of 10-20% interneurons, we had around 13-27% interneurons in the field. We thus overestimated the proportion of MSNs by 3-7%.
To quantify the proportion of fura 2-loaded cells that were astrocytes and not neurons in slices from P10 to P12 mice, and could be erroneously considered as silent neurons, we performed double loading of slices with sulforhodamine 101 (SR101, 1 μM), a specific marker of astroglia (Nimmerjahn et al., 2004) during 20 min at 35-37˚C and then with fura 2AM (Figure 1B). At P12, astrocytes represented around 25% of all the imaged cells (n = 201/815 from five slices). Among these astrocytes, around 40% were also positive for fura 2 (n = 86/201). Therefore around 10% of fura 2-loaded cells were astrocytes (n = 86/815). All these fura 2-loaded astrocytes were silent and could be erroneously counted as silent neurons.

IMMUNOCYTOCHEMISTRY AND DiI EXPERIMENTS
To reveal the neurobiotin injected during whole cell recordings, the sections were left 12 h in paraformaldehyde (3%) at 4˚C, rinsed in PBS, left 12 h in PB-sucrose 20%, and then at −80˚C for at least 2 h. They were thawed at RT, rinsed in PB and incubated Frontiers in Cellular Neuroscience www.frontiersin.org 30 min in 1% H 2 O 2 in PB. Slices were washed with PB and KPBS and incubated for 12 h in ABC complex at a dilution of 1:100 in KPBS + 0.3% triton (Abcys). They were rinsed in KPBS and incubated for approximately 10 min in 3,3 diaminobenzidine (DAB 0.7 mg/ml) with peroxide (0.2 mg/ml; Sigma Fast), rinsed, mounted in Crystal/Mount (Electron Microscopy Sciences), coverslipped, and examined. Dendritic and axonal fields were reconstructed using the Neurolucida system (MicroBrightField Inc., Colchester, VT, USA).
To visualize cortico-striatal axons, we injected small amounts of DiI crystals diluted in ethanol in the neocortex of 400 μm thick slices from E16 to P2 brains postfixed by immersion for 2-4 weeks in 4% paraformaldehyde. Slices were then incubated in the fixative solution at 32˚C for 2-3 weeks, coverslipped, and examined with a confocal microscope (Zeiss LSM 510).

ANALYSIS OF IMAGING DATA
We performed analysis of the calcium activity with custom-made software written in Matlab (MathWorks; Bonifazi et al., 2009). The contour of each loaded cell was semi-automatically detected and its fluorescence measured as a function of time. Active cells are neurons exhibiting any Ca 2+ event of at least 5% DF/F deflection within the period of recording. Ca 2+ spikes or Ca 2+ plateaus cells were neurons exhibiting at least one Ca 2+ spike or one Ca 2+ plateau within the period of recording. Ca 2+ plateaus were intrinsic correlated Ca 2+ events of long duration (see Results). Giant depolarizing potentials (GDP) cells were cells generating synchronized synapse-driven Ca 2+ spikes (GDPs). To compute the activity correlation of two cells, the onset of each event was represented by a Gaussian (s = 1 frame, to allow some jitter). The inner product of the resulting values was then calculated. The significance of each correlation value was estimated by direct comparison with a distribution computed from surrogate data sets, in which the events were randomly reshuffled in time.
To compare both network-wide and single-neuron activity between putative intrinsically driven activity at P2 (P2-P3 recordings) and synaptically driven activity at P6 (P5-P7 recordings), we used standard k-means clustering to determine how well event durations and frequencies distinguished P2 and P6 time-points. The k-means algorithm used minimization of city-block distance, with 30 replications from random starting positions, from which we retained the replicate with the minimum mean distance. We also sought evidence for differences in spontaneous neural ensembles in network-wide activity. Each recording's matrix of pair-wise correlations, computed by Gaussian convolution as above was partitioned into groups using a modified community detection algorithm, detailed in Humphries (2011), which finds the number and size of groups within the matrix that maximize benefit function Q data = (similarity within groups) − (expected similarity within Frontiers in Cellular Neuroscience www.frontiersin.org groups). The resulting partition thus corresponded to groups of neurons that were more similar in activity patterns than was expected given the total similarity of each neuron's activity to the whole data-set. We then ran a further stringent control for potentially spurious groupings, by first shuffling the inter-event intervals of each neuron in a recording, correlating the shuffled event onsets, and then running the algorithm on the shuffled data-set. This was repeated 100 times to get a distribution of Q for control data. The 95th percentile of these values was taken as the 95% confidence interval Q ctrl . Any network with Q data > Q ctrl thus contained significant ensemble structure, compared to that expected from just the firing statistics of the network.

ANALYSIS OF ELECTROPHYSIOLOGICAL DATA
We determined series resistance (R s ), membrane capacitance (C m ), and input resistance (R input ) by on-line fitting analysis of the transient currents in response to a 5-mV pulse at −70 mV. Criteria for considering a recording included R input > 100 MΩ, R s < 25 MΩ, with R s < 30% change. We analyzed spontaneous postsynaptic currents (sPSCs) in 180 s recordings at a given membrane potential with the Mini-Analysis program (version 5.1.4; Synaptosoft, Decatur, GA, USA). Events were characterized by the following parameters: rise time (10-90%), amplitude, and decay time (τ). We discriminated mixed AMPA/KA events in the absence of specific receptor antagonists from the SD given by the fit of each event to determine whether one or two exponentials best fitted the decays (Epsztein et al., 2005). E16-P5 MSNs generating sEPSCs (61 out of 131) or sIPSCs (33 out of 79) with a frequency lower than 0.05 Hz were not included. We filtered the single-channel currents at 1 kHz (GABA A channels) or 3 kHz (NMDA channels) and digitized them at 10 kHz. We discarded multilevel and short (2 ms) openings during the analysis. To obtain unitary current-voltage (I-V) relationships, we measured the amplitude of unitary GABA and NMDA currents evoked by steps from −120 to +40 mV. Histograms of cursormeasured amplitudes allowed determination of the mean unitary current amplitude at each voltage tested.
Measurements of V rest and E GABA(A) : to determine the action of GABA in a given neuron (depolarizing or hyperpolarizing), one must measure the reversal potential of the GABA A -mediated current [E GABA(A) ] and the resting membrane potential (V rest ). However, conventional whole cell recordings introduce a number of errors in these measures in particular in developing neurons. We therefore estimated the value of V rest from cell-attached recordings of the single-channel NMDA current (iNMDA), which is known to reverse at a membrane potential (V m ) close to 0 mV. We plotted the relationship between iNMDA and the extracellular potential applied to the patch of membrane (V p ) from experimental data (see Figure 9B). This curve , we plotted the relationship between the single-channel GABA A current (iGABA A ) and V p . This curve [iGABA A = f(V p )] gives the value of V p when iGABA A = 0 pA (see Figure 9A), because by definition when iGABA A is null, the driving force (DF) of chloride ions through the GABA A channel (Tyzio et al., 2003). Therefore, when iGABA A = 0 pA, DF GABA = −V p . Knowing V rest and DF GABA , it is easy to calculate E GABA(A) = DF GABA + V rest .
To obtain whole cell current-voltage (I -V ) relationships, we measured voltages at the end of each hyperpolarizing current step (950 ms). The inward rectification was detected when the I -V relationship was not linear between −90 and −120 mV. To compare immature and adult MSNs delay of firing in response to depolarizing steps, we measured the first interspike interval (ISI) of the response. We then pooled the slope values of the linear regression lines and compared their distribution as a function of time (Belleau and Warren, 2000). We estimated the threshold potential for Na + spikes in whole cell current clamp recordings at V rest (−70 mV for E16-P7 slices and −80 mV for the adult slices) by applying successive intracellular depolarizing steps (duration 950 ms) or in response to cortical stimulation.

MOTOR BEHAVIOR
Motion development was assessed in Swiss newborn mice (n = 16) from two different litters (Janvier SAS, Le Genest Saint Isle, France) between postnatal day 2 (P2, day of birth: P0) and P12. C57BL/6 pups could not be tested because of their low weight. We used an open field test to assess overall activity. We tested each pup twice a day, with a 20-min delay between the two tests. We placed the pup on a translucid acrylic plate (24 cm × 16 cm) covered with a silicone gel. Two cameras were placed below the plate. One acquired the pup's contact points with the floor which appeared as highly contrasted areas, based on the frustrated total internal reflection (FTIR) principle (Han, 2005). We identified the mouse abdominal contact points using custom-made software. The second camera acquired the trajectory of the pup. We calculated from this trajectory the total distance traveled, and the total distance traveled along straight line segments.
The membrane potential trajectory attributable to the activation of the inwardly rectifying K + current (I K IR ) was not detected in any MSNs at E14, was present in 17% of MSNs at P7 (n = 3/44) and in all MSNs at P10 and P30 (n = 8/8 and 6/6, respectively). It greatly increased from P10 to P30 (Figures 4A,B). Accordingly, bath application of cesium (3 mM), a blocker of I K IR , increased the proportion of active cells by 240% at P6-P10 (from 4.3 ± 1.1 to 10.4 ± 1.7%, 1349/6; p = 0.04 paired Student's t -test; data not shown). The mean input resistance (R m ) of MSNs was high until P6 and significantly decreased by approximately 75% between P6 and P10 (from 982 ± 129 MΩ at P6, n = 21 to 263 ± 19 MΩ at P10, n = 11; p = 0.004, one way ANOVA; Figure 4C). Spiking threshold (V threshold ) did not change from P2 to P30 (P2: −32.2 ± 1.7 mV, n = 14 and P30: −30.8 ± 0.8 mV, n = 10; p = 0.51 one way ANOVA) but the instantaneous firing frequency did. The first ISI of a firing train as a function of injected current was stable until P6 and was then significantly reduced between P6 and P10 (1.44 ± 0.14 Hz/pA, n = 11 at P6 vs. 0.91 ± 0.08 Hz/pA, n = 7 at P10; p = 0.03, one way ANOVA) showing an increased delay to spiking at P10 (Figures 4D,E). The depolarization needed to generate spikes calculated as (V threshold − V rest ) from whole cell recordings (Figures 4F,G) was significantly lower until P6

EARLY POSTNATAL MSNs GENERATE CORRELATED Ca 2+ SPIKES WHEN THEY EXPRESS THE NR2C/D NMDA SYNAPTIC CURRENT AND MEMBRANE PROPERTIES ARE STILL IMMATURE
The first synapse-driven pattern observed in postnatal MSNs was correlated Ca 2+ spikes (GDPs), that appeared at P5-P7 (10% of the recordings, 969/5; mean frequency: 0.10 ± 0.05 Hz and mean duration: 0.70 ± 0.10 s, 102/5), and subsequently disappeared. The vast majority of active striatal neurons (84.0 ± 9.7%) were engaged in this activity with a large number of correlated cell pairs (24%), attesting to the large neuronal ensemble that fired together during these events (Figure 5A left). Electrophysiological activity underlying each GDP consisted of bursts of 2-3 Na + spikes ( Figure 5B). Ionotropic glutamate and GABA receptor antagonists abolished GDPs, but left other Ca 2+ events (uncorrelated Ca 2+ spikes or Ca 2+ plateaus) intact (p = 0.02 and 0.52 respectively, paired Student's t -test; Figures 5A-D). Since GDPs were also suppressed by APV (40 μM) alone, suggesting that NMDA receptors were heavily involved, we tested their sensitivity to PPDA (Feng et al., 2004), the preferential antagonist of NR2C/D NMDA receptors. At 100 nM PPDA removed most of the correlations between neurons (from 45 to 11%). Most of the GDP cells stopped their activity and the few who did not, evoked Ca 2+ spikes at a much lower frequency (from 0.17 ± 0.01 to 0.07 ± 0.01 Hz; p = 0.0003, Mann-Whitney test; Figures 5C,D).
Frontiers in Cellular Neuroscience www.frontiersin.org To understand the time course of development of the spontaneous synaptic activities afferent to MSNs and that could play a role in the transiently expressed GDPs, we performed separate recordings of glutamatergic and GABAergic synaptic events. Glutamatergic events: the fraction of MSNs exhibiting AMPA-or KA-mediated sEPSCs steadily increased from E16 to E18 (38 and 50%) to a maximum at P5-P7 (100%). The mean frequency of AMPA or KA receptor-mediated EPSCs gradually increased from E16 to E18 (0.11 ± 0.04 and 0.3 ± 0.1 Hz; n = 5 and 5 MSNs) to P30 (1.3 ± 0.7 and 0.9 ± 0.2 Hz; n = 5 and 6 MSNs; p = 0.03 and p = 0.004 respectively, one way ANOVA) but their amplitude (p = 0.3 for AMPA, p = 0.07 for KA, one way ANOVA), rise times and decay times remained constant (Figures 6A-E left and  middle). In contrast, the fraction of MSNs showing spontaneous NMDA receptor-mediated EPSCs shifted from 40% at P0 (5/12 cells) to more than 90% at P5-7 (16/18), and decreased thereafter to 30% at P30 (4/12; Figures 6A,B right). Their frequency stayed constant and low (around 0.1 Hz, p = 0.9; one way ANOVA) and also their amplitude (p = 0.9) and rise times but their decay time significantly decreased in the same period (Figures 6C-E right).

GABAergic events
The fraction of MSNs exhibiting spontaneous GABA A R-mediated currents steadily increased from E16 to a maximum at P5-P7 and the mean frequency of these currents (GABA A PSCs) progressively increased from E16 up to P30 (519 MSNs; Figures 8A-C).
The DF of GABA (DF GABA ), determined from single-channel recordings of GABA A Rs (see Materials and Methods; Tyzio et al., 2003), was similarly depolarizing by 15 mV at P2 (+15.3 ± 3.4 mV; n = 8 MSNs, Figures 9A,B) and P30 (Dehorter et al., 2009; +16.1 ± 1.9 mV, n = 11; p = 0.8, Student's t -test). To estimate (E GABA A ), we plotted the relationship between the single-channel GABA A current (iGABA A ) and the pipette potential (V p ). This curve [iGABA A = f(V p )] gives the value of V p when iGABA A = 0 pA (Figures 9A,B right), because when iGABA A is null, V m = E GABA A . Therefore, when iGABA . By definition, E GABA A − V rest = DF GABA A , the DF of chloride ions through the GABA A channel (Tyzio et al., 2003). Therefore, when I GABA A = 0 pA, DF GABA A = −V p . Knowing V rest and DF GABA A , we can calculate the reversal potential for GABA A , E GABA A = DF GABA A + V rest . The reversal potential of GABA A currents was Frontiers in Cellular Neuroscience www.frontiersin.org 10 mV more depolarized at P2 than at P30 (Figure 9C). These values are based on the assumption that the NMDA current reverses at 0 mV in MSNs. Although an error of 5 mV may exist (Tyzio et al., 2003), the comparison of V rest and E GABA obtained with the same methods at P2 and P30 confirms the validity of our conclusions. Therefore, GABAergic synapses depolarize MSNs from V rest to a value closer to the action potential threshold than in the adult (Misgeld et al., 1982;Koos and Tepper, 1999). However, GABA does not excite immature MSNs. Focal applications of the GABA A receptor agonist isoguvacine failed to generate action potentials in cell-attached recordings from P2 (n = 6, Figure 9D) or P5 (n = 7, data not shown) MSNs. Similarly, stimulation of the striatal neuropil failed to evoke action potentials in cell-attached recordings of MSNs in the continuous presence of ionotropic glutamate receptors antagonists (data not shown).
The above data show that striatal neurons generate GDPs during a transient period when all MSNs are connected to glutamatergic and GABAergic neurons and still exhibit immature membrane properties (see Figure 4). There is a more efficient GABAergic depolarizing drive than at P30 that may participate in the GDPs of P5-P7 MSNs (together with glutamatergic EPSPs; Bracci and Panzeri, 2006) but these events are mainly driven by cortico-striatal synapses at a time where cortical neurons also generate GDPs (Allene et al., 2008).

INTRINSIC AND SYNAPSE-DRIVEN IMMATURE ACTIVITY DERIVE FROM DISTINCT NEURAL ENSEMBLES
Because approximately 90% of the P5-P7 striatal networks did not generate GDPs at the time of recording, we wanted to understand whether synapse formation in some way altered the dynamics of the network. We compared network-wide activity at P2-P3 (when synapse density is low) to that at P5-P7 (when all MSNs are innervated by glutamatergic and GABAergic inputs). We found that both frequency and duration of events changed from P2-P3 to P5-P7 (Figures 10A,B). The distributions of mean event frequency significantly differed between P2-P3 and P5-P7 (Mann-Whitney U -test; n P2 = 18, n P6 = 33; U = 320; p = 0.02). Similarly, the distributions of median event duration significantly differed between P2-P3 and P5-P7 (U = 569.5, p = 0.0046). To gage the size of these differences between P2-P3 and P5-P7, we used kmeans clustering to assign each recording to either P2-P3 or P5-P7 on the basis of its event statistics. We found that both duration (70% correct assignments) and frequency (64% correct) reliably indicated a recording's developmental stage. Thus, the switch from intrinsically to synaptically driven activity reliably decreased duration and increased the frequency of calcium events in the immature striatal network.
Both P2-P3 and P5-P7 networks were capable of showing spontaneous formation of neural ensembles (Figures 10C,D). We found markedly more P5-P7 (43%, 9/21) than P2-P3 (29%, 4/18) recordings with significant ensemble structure. Conversely, these P2-P3 recordings contained more ensembles (range 5-9) than the P5-P7 recordings (range 2-6). We also found that the P5-P7 recordings with GDP-driven, network-wide synchronization could be sub-divided into ensembles that indicated the delay in a neuron's participation of the network-wide synchronization ( Figure 10E). These results suggest that the transition from intrinsic to synaptically driven activity promoted the appearance of putative cell assemblies. Figures 11A-E  at P10, entering an adult-like phase. AMPA or KA receptormediated signals are probably not involved in the extinction of early spontaneous activity in MSNs because their frequency continuously increases with age. In contrast, the switch in other properties such as the abrupt loss of NR2C/D, expression of I K IR, decrease in R m , hyperpolarization of V rest , and hyperpolarization of E GABA A all coincided with the silencing of MSNs. In order to identify when the transition of MSNs from immature to adult-like state occurs in relation to a motor behavioral output, we quantified the maturation of pup body contacts and motion. At P2, prior to the onset of quadruped ambulation, the duration of abdominal contact with the cage surface was high (35.3 ± 4.9% of total time), then progressively decreased at P3-P6 (17.8 ± 1.6%) and P7-P8 (2.9 ± 0.7%), disappearing by P9-P10 (n = 16; p = 1 × 10 −8 between P3-P6 and P7-P8; one way ANOVA; Figures 11F,G, top). Body motion was practically absent at P2, with pivoting and crawling predominating in P3-P7 pups. The total distance pups traveled along straight line segments underwent a marked increase from P3 to P6 (0.2 ± 0.1 cm, n = 56) to P9-P10 (7.9 ± 1.6 cm, n = 28; p = 0.0002, one way ANOVA test). By P12 pups traversed relatively large distances (28.9 ± 3.5 cm, n = 14; p = 1 × 10 −12 compared to P9-P10; one way ANOVA test; Figures 11F,G, bottom). Thus, spontaneous displacement with the ventral surface of the body held above the floor was first observed in P9-P10 mouse pups, in parallel with the chronology of MSN silencing.

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DISCUSSION
Our results show that MSNs, the dominant neuronal population of the striatum, generate immature patterns of activity at embryonic and early postnatal stages that are reminiscent of the patterns observed in developing cortical structures (Garaschuk et al., 2000;Corlew et al., 2004;Allene et al., 2008). This confirms the similarity between developmental activities of networks independently of their neuronal structure and final function (Ben-Ari, 2001). In the middle of the second postnatal week, MSNs shift to an adultlike pattern characterized by little activity in vitro (Carrillo-Reid et al., 2008), just before pups lift their body and begin to walk. Underlying this transition is a change in the fundamental characteristics of MSNs (Figure 11). This suggests that the development of MSNs and striatal network activity parallels the development of locomotor structures and pathways (Grillner et al., 2005). Two features of immature MSNs emerge as central players in this progression. (i) Intrinsic voltage-gated Ca 2+ currents: we propose that the reduced K + currents and the consequent depolarized resting membrane potential allow spontaneous opening of N and L-type voltage-gated Ca 2+ channels, then quieted near P10 by resting membrane potential hyperpolarization and/or developmental changes of Ca 2+ channel properties; (ii) The transient NR2C/Dmediated cationic current: the expression of long lasting NMDA EPSCs mediated by the NR2C/D receptor subunit is a general feature of different developing brain structures (Monyer et al., 1994;Nansen et al., 2000;Logan et al., 2007;Dravid et al., 2008). NMDA receptor-mediated EPSCs are conspicuous in cortico-striatal neurons as early as P2 (see Hurst et al., 2001) for contradictory results). Using a dose of PPDA (100 nM) that preferentially antagonizes NR2C/D subunits (K D = 0.096 and 0.130 μM, respectively; Traynelis et al., 2010), we demonstrate that the window of operation of NR2C/D-mediated events is highly restricted to P5-P8 (Dunah et al., 1996). Therefore, as in other brain structures, immature neurons first generate long lasting synapse-driven patterns of activity that include large NMDA receptor driven currents. These currents, together with voltage-gated Ca 2+ currents, trigger the large calcium fluxes needed for a wide range of essential developmental functions including neuronal growth, synapse formation, and the formation of neuronal ensembles (Spitzer, 2006). Indeed, we demonstrated that during this P5-P8 window, network-wide changes in calcium event statistics correlated with the reliable formation of neural ensembles.
Our observations also provide interesting insights concerning the generation of GDPs that have been observed in a wide range of brain networks but investigated primarily in cortical structures . GDPs are generated both by depolarizing GABAergic and glutamatergic notably NMDA receptors-mediated currents. The striatum is an interesting site to investigate the debated role of glutamate in GDPs generation because it has in contrast with other investigated structures no internal glutamatergic neurons. Clearly, the maturation of the glutamatergic cortico-striatal inputs is instrumental in the emergence of GDPs and particularly the long lasting NR2C/D component. Adesnik et al. (2008) suggested that modest activity through NMDA receptors prevents the constitutive trafficking of AMPA receptors to the postsynaptic density via an LTD type mechanism. This ensures that synapses become functional only after strong or correlated activity, when enough calcium entry through these NMDA receptors overrides the inhibitory pathway and drives AMPA receptor insertion. This surge is provided by bursts of action potentials during GDPs and NMDA-mediated corticostriatal EPSPs as shown here. From this perspective the elimination of the long lasting NR2C/D component in cortico-striatal EPSPs would constitute a gating device to induce the expression of AMPAergic currents in MSNs.
Therefore, our results suggest an intrinsic program that switches MSN activity from an immature low threshold activation state to a high threshold state in the adult with a low activity profile during resting conditions, coincident with the emergence of locomotion. In a more conceptual frame, in addition to ubiquitous Frontiers in Cellular Neuroscience www.frontiersin.org developmental patterns of activity, there would be a superimposed sequence, unique to each brain structure, which takes over at an appropriate time to enable the generation of patterns required for specific functions. The adult-like state described here is accompanied by several additional factors including the development of the dendritic arbor and spines of MSNs and the increased density of asymmetric glutamatergic synapses (Tepper et al., 1998;Belleau and Warren, 2000), a second wave of nigro-striatal dopaminergic innervation (Moon and Herkenham, 1984), the development of thalamo-cortical loops, and sensori-motor cortex (Gianino et al., 1999;Vinay et al., 2002;Allene et al., 2008;Evrard and Ropert, 2009). It is also accompanied functionally by a dopamine-and D2 receptor-dependent decrease in the efficacy of glutamatergic transmission that takes place in vivo during weeks 2-3 of postnatal development and is a consequence of a number of physiological changes in the maturing striatum (Choi and Lovinger, 1997;Tang et al., 2001). Also, comparison of the present data with that obtained in rodents lacking dopaminergic substantia nigra neurons (pitx3 −/− mice for example, Smidt et al., 2004), will allow understanding of the early role of endogenous dopamine on the development of the striatal network (Ohtani et al., 2003;Goffin et al., 2010). Finally, present observations may be of clinical relevance because adult MSNs from the R6/2 rodent model of Huntington's disease resemble immature MSNs described here in several respects, including an increased input resistance, depolarized resting membrane potential, low level of inwardly and outwardly rectifying K + currents (Ariano et al., 2005), increased sensitivity to NMDA receptor activation  and decreased sensitivity of NMDA receptors to Mg 2+ block (Starling et al., 2005).