Figure 1.
Postnatal NMDA receptor blockade decreases first-spike latency in neocortical layer IV FSIs.
(A, B) Prototypical discharge patterns of FSIs from a vehicle- or MK-801-treated mouse. Traces show voltage response to 600 ms injections of current at saturating (black trace), or threshold (red trace) amplitude. Red arrows below trace indicate onset of current injection and spike threshold (defined as dVm/dt>10). Solid red bar below trace illustrates delay to first spike (C) Quantification of delay to first spike at rheobase response. Values are means ± S.E.M. *P<0.05, vs. control. (D, E). A log-log plot of first spike delay versus amplitude of injected current in layer IV FSIs from vehicle- (D) or MK-801–treated mice (E). Dashed lines on the y-axes illustrate spike latency of log 0.3 ms (or 2.0 ms log10).
Table 1.
Electrical properties of layer IV FS neurons in the Somatosensory Cortex.
Figure 2.
Postnatal NMDA receptor blockade destabilizes spike threshold in neocortical layer IV FSIs.
(A, B) Representative voltage responses of first spikes elicited from FSIs at rheobase (Ith); Ith+100 pA; Ith+200; and Ith+300 pA current steps. For clarity, only the initial spike in each trace is displayed, and the intervening, +50 pA, current steps are omitted. (C, D) Phase plots of the traces shown in A, B. Phase plots are shown for the first spike discharged during injection at Ith and Ith +: 50, 100, 150, 200, 250 and 300 pA. (E, F) Regions of interest indicated by the dashed line are displayed at higher magnification. Red dashed line indicates spike threshold (dV/dt = 10). Insets are plots of injected current amplitude versus first spike threshold of displayed cell. (G, H) Plots of injected current amplitude versus first spike threshold. (I) Mean slope of injected current amplitude versus first spike threshold in (G, H). Values are means ± S.E.M. **P<0.005, vs. control by ANOVA.
Figure 3.
Postnatal NMDA receptor blockade reduces somatic expression of Kv1.1.
(A–H) Representative confocal micrographs (maximal intensity, flattened Z-stack, z separation of 1.03 um) of somatosensory cortex from adolescent mice (PND42) neonatally injected with vehicle (A–D), or MK-801 (E–H). (D, H) Staining for the Kv1.1 potassium channel subunit is dramatically reduced in FSIs from MK-801-injected mice. Insets in (B) and (F) show higher magnification of layer IV regions of interest. Green = Parvalbumin; Red = Kv1.1. Scale bars = 100 um (A–C; E–G) or 10 um (D, H and insets in B, F).
Figure 4.
Postnatal NMDA receptor blockade increases glutamatergic input to neocortical FSIs.
(A) Representative whole-cell patch clamp recordings from FSIs in acute brain slices from the somatosensory cortex of adolescent mice (PND21-P25) neonatally-treated with vehicle or MK-801. Spontaneous activity was recorded in 2 mM (left traces) or 0 mM (right traces) of added Mg2+. Recordings were obtained in voltage clamp mode. sIPSCs (top traces) were recorded at the sEPSC reversal potential (∼+10 mV) and sEPSCs (bottom traces) were recorded at the Cl- reversal potential (∼−40 mV). (B) Bar graphs of mean sEPSCs and sIPSCs frequency obtained during 5 min recordings. Values are means ± S.E.M. *P<0.05, vs. control by ANOVA.
Figure 5.
Postnatal NMDA receptor blockade increases expression of functional NR2B receptors in neocortical FSIs.
Prototypical excitatory post-synaptic currents were evoked onto layer IV FSIs in a thalamocortical slice preparation. (A–C) AMPA-mediated responses were similar in vehicle-treated mice (black traces) and MK-801-treated mice (red traces). (VHold-AMPA = −60 mV). Traces shown are the average of 5–10 responses obtained at the same stimulus intensity from vehicle-treated mouse (black lines); or MK-801-treated mouse (red lines). Dashed boxes designate regions of interest in activation and decay kinetics. (D–F) Bar graphs of mean current amplitude and mean weighted activation tau (τwAct.) and deactivation tau (τwDeact.) of evoked AMPA current evoked from vehicle-treated mice (black bars) and MK-801-treated mice (red bars). (G, H) The kinetics of NMDA-mediated responses are slower in MK-801-treated mice (red traces). (VHold-NMDA = +60 mV). Traces shown are the average of 5–10 responses obtained at the same stimulus intensity. Dashed boxes designate regions of interest in activation and decay kinetics. (I–K) Bar graphs of mean current amplitude and mean weighted activation tau (τw) and deactivation tau (τw) of evoked NMDA current from vehicle and MK-801-treated mice. Values are means ± S.E.M. *P<0.05, vs. control by ANOVA. (L–S) Representative confocal micrographs of layer IV somatosensory cortex from mice neonatally treated with vehicle (L–O), or MK-801 (P–S). (L, P) Dashed boxes designate regions of interest in layer IV selected for higher magnification as shown in (M–O), and (Q–S). Green = Parvalbumin; Red = GluN2B. Scale bars = 100 um (L, P) or 10 um (M–O; Q–S). Images are shown as a maximum intensity projection of single images at a Z-spacing of 1.0 uM. (N, R) Arrowheads denote GluN2B staining as puncta or broad staining in brain slices from a vehicle-treated, or MK-801-treated mouse, respectively. (T–W) Representative traces of ifenprodil blockade on monosynaptic NMDA current evoked onto a FSI from an adolescent mouse neonatally treated with vehicle (T) or MK-801 (V) Traces shown are averages of 5–10 responses to the same stimulus intensity before (black lines) and after (grey lines) 10 minute wash in of 3 uM Ifenprodil. (U, W) Same traces as shown in (T) and (V) scaled on y-axis to facilitate visual comparison of the actions of ifenprodil on activation and decay kinetics. Average traces were fit, as previously described. (X, Y, Z) Quantitative analysis of impact of ifenprodil on mean peak amplitude (X); mean reduction in total charge (Y); and mean change in τw decay (Z). (N = 4 cells from 4 animals (vehicle-treated); and 3 cells from 3 animals (MK-801-treated).
Figure 6.
Schematic depiction of how neonatal MK-801 treatment impacts the spike timing of an FSI from an adolescent mouse.
(A) A significant proportion of the current injected during whole-cell patch-clamp (yellow lightning bolts) dissipates through the high density of Kv1.1-containing K+ channels expressed in FSIs from vehicle-treated mice. (B) The time required to depolarize the cell membrane to threshold is increased and first-spike latency is temporally expanded. (C) By contrast, a lower proportion of injected current dissipates FSIs from MK-801-treated mice due to reduced expression of Kv1.1-containing K+ channels. (D) The time required to depolarize to threshold is reduced and first-spike latency is therefore minimized.