Rapid developmental emergence of stable depolarization during wakefulness by inhibitory balancing of cortical network excitability

Abstract

The ability to generate behaviorally appropriate cortical network states is central to sensory perception and plasticity, but little is known about the timing and mechanisms of their development. I paired intracellular and extracellular recordings in the visual cortex of awake infant rats to determine the synaptic and circuit mechanisms regulating the development of a key network state, the persistent and stable subthreshold membrane potential (Vm) depolarization associated with wakefulness/alertness in cortical networks, called the "desynchronized" or "activated" state. Current-clamp recordings reveal that the desynchronized state is absent during the first 2 postnatal weeks, despite behavioral wakefulness. During this period, Vm remains at the resting membrane potential >80% of the time, regardless of behavioral state. Vm dynamics during spontaneous or light-evoked activity were highly variable, contained long-duration supratheshold plateau potentials, and high spike probability, suggesting an unstable and hyperexcitable early cortical network. Voltage-clamp recordings reveal that effective feedforward inhibition is absent at these early ages despite the presence of feedback inhibition. Stable membrane depolarization during wakefulness finally emerges 1-2 d before eye opening and is statistically indistinguishable from that in adults within days. Reduced cortical excitability, fast feedforward inhibition, and the slow cortical oscillation appear simultaneously with stable depolarization, suggesting that an absence of inhibitory balance during early development prevents the expression of the active state and hence a normal wakeful state in early cortex. These observations identify feedforward inhibition as a potential key regulator of cortical network activity development.

Figure 1.

Rapid and simultaneous development of stable membrane depolarization during wakefulness and sleep. A, Intracellular recording of V_m in a layer 2/3 neuron from a P10 rat V1 in the following three states of vigilance: anesthetized (1% isoflurane), awake, and sleeping (quiet/slow-wave sleep). Action potentials are clipped for better resolution. V_rest is determined during anesthesia, which is subsequently removed. Simultaneous monitoring of the local field potential in layer 4 (L4), and of movement by piezo-electric and EMG signals is used to determine behavioral state and to identify cortical activity patterns. Note the absence of depolarization during wakefulness and sleep, and the presence of large-amplitude, unstable, discrete events. B, Similar recording at P14. Note stable and persistent depolarization during wakefulness, and up–down state transitions during sleep. C, Histograms of V_m during three vigilance periods: 1% isoflurane anesthesia (blue); 60 s before movement during recovery from isoflurane anesthesia (green); and wakefulness (red). Development of unimodal V_m distribution during wakefulness develops between P11 and P13. Clear separation between active and down states during unconsciousness is visible by P13 but stabilizes over the next week.

Figure 2.

V_m dynamics show rapid development of adult-like depolarization during wakefulness. A, B, Expanded V_m traces from Figure 1 show quantification metrics used during wakefulness (color) and examples of the terms for cortical network states used in the present study (italics). C, Waking depolarization (blue arrows) was quantified as the peak of the V_m distribution (for examples, see Fig. 1C) during wakefulness vs V_rest. D, Time depolarized (red bars) is the percentage of total time the cell was >5 mV above V_rest. E, Silent periods (blue line) are the percentage of periods that the cell spent at V_rest that lasted >1 s. F, The incidence of plateau potentials (green box) is the percentage of the time depolarized the cell spent above the action potential (AP) threshold. G, The depolarization instability of depolarization periods (purple arrow) is the σ of a Gaussian distribution fit to the V_m distribution during the time depolarized. H, Spike density is the number of APs per second of depolarization. In line with previous literature, the active state (B, right column) is a stable, mostly subthreshold depolarization that occurs during the synchronized state in alternation with down-states (B, right column) to form the slow-wave (Haider and McCormick, 2009). The desynchronized state (or activated state) is a persistent depolarization resembling a continuous active state during wakefulness. By these definitions, young animals lack a desynchronized state as well as an active state. Instead, wakefulness (and sleep) is characterized by a persistent down-state, interrupted by large-amplitude unstable activities that spend significant time above the AP threshold. We have previously called these activities SATs (Colonnese and Khazipov, 2010).

Figure 3.

Development of cortical active states attenuates visual responses. A, V_m during evoked activity during wakefulness and sleep before development of cortical active states (P11). Colored lines are as in Figure 1. B, V_m responses after cortical active state development (P14). During wakefulness (top), a brief evoked membrane depolarization rides on a stable membrane depolarization. During unconsciousness (0.5% isoflurane anesthesia), stimulation during down-states can trigger the occurrence of an active state, but does not cause the large, unstable responses of younger animals. C, Quantification of evoked V_m during wakefulness. Top, Proportion of time V_m > AP threshold (plateau potential, green) or >5 mV above V_rest (time depolarized, red) following stimulation. Bottom, Number of action potentials during 1 s following stimulation. **p < 0.01. Bars indicate the SEM.

Figure 4.

Development of resting membrane potential and action potential threshold. A–C, V_rest (A), action potential threshold (B), and distance between the two (V_rest − threshold, C) for each neuron by age.

Figure 5.

Spontaneous but not light-evoked inhibitory currents in early cortex. A, Light-evoked (top traces) and spontaneous (bottom) synaptic currents and their associated LFP during wakefulness in V1 of P11 rat. Presumptive GABAergic currents (red) and glutamatergic (Glu; blue) currents are recorded for each neuron, and synaptic events with similarly sized LFPs are shown for comparison. Despite large GABAergic currents during spontaneous activity, light (green bars) evokes little synaptic response. B, Same analysis for a neuron from a P13 rat. C, Distribution of spontaneous (gray bars) and evoked (colored bars) currents for GABAergic (top four graphs) and glutamatergic (Glu; bottom four graphs) currents. Example animals from early (P11) and later (P13) ages are shown. D, Median evoked current (relative to peak spontaneous current) for each neuron. GABA currents are shown above in red; glutamate current are shown below in blue. E, Peak spontaneous current used to calculate relative amplitude in D. F, I/E ratio during spontaneous activity for each neuron.

Figure 6.

Rapid development of fast feedforward inhibition. A, Mean visually evoked currents for GABA (top red) and glutamate (Glu, top blue), and the mean visually evoked LFP from layer 4 (bottom) for the same neuron for the matched trials from a P11 (left) and P13 (right) rat. C, D, Relationship between excitatory and inhibitory currents is quantified as “Delay,” and “I @ Emax,” and “Imax,” as shown. B, Similar layout for P13. C, Delay between excitatory and inhibitory currents by age. D, Ratio of mean inhibitory current amplitude to mean excitatory current amplitude measured at the peak of the excitatory current (I@Emax, orange) or measured at the peak of each (Imax, red).