Long-term potentiation in the neonatal rat barrel cortex in vivo

Abstract

Long-term potentiation (LTP) is important for the activity-dependent formation of early cortical circuits. In the neonatal rodent barrel cortex, LTP has been studied only in vitro. We combined voltage-sensitive dye imaging with extracellular multielectrode recordings to study whisker stimulation-induced LTP in the whisker-to-barrel cortex pathway of the neonatal rat barrel cortex in vivo. Single whisker stimulation at 2 Hz for 10 min induced an age-dependent expression of LTP in postnatal day (P) 0 to P14 rats, with the strongest expression of LTP at P3-P5. The magnitude of LTP was largest in the activated barrel-related column, smaller in the surrounding septal region, and no LTP could be observed in the neighboring barrel. Current source density analyses revealed an LTP-associated increase of synaptic current sinks in layer IV/lower layer II/III at P3-P5 and in the cortical plate/upper layer V at P0-P1. Our study demonstrates for the first time an age-dependent and spatially confined LTP in the barrel cortex of the newborn rat in vivo.

Figure 1.

Mechanical deflection of a single whisker for 10 min at 2 Hz elicits LTP in barrel cortex of newborn rats in vivo. A, Schematic diagram of the experimental setup illustrating selective mechanical stimulation of the C2 whisker (A₁) and simultaneous VSDI in the barrel cortex (A₂). The exposed barrel cortex was stained with the voltage-sensitive dye RH1691. A single-whisker deflection of the C2 whisker elicits a local VSDI response in a P3 rat. The green dot indicates the center of the C2 barrel-related cortical column. The black dot is the electrode insertion position. Orange represents the region of the C2 whisker stimulation-evoked VSDI response. A₃ shows the same area after termination of the electrophysiological recording and retraction of the recording electrode. The red dot and arrowhead show the electrode insertion point. A₄, Stimulation protocol for induction of LTP. During baseline recording, the whisker was deflected twice per 5 min at 1 min intervals for 30 min. For LTP induction, the whisker was deflected at a frequency of 2 Hz for 10 min. Afterward, the same two stimuli per 5 min were used again for 60 min during the poststimulation recording period. B, Time course of FP responses before and after induction of LTP. B₁, Representative FP recording during baseline and the 5–30 and 35–60 min phases after 2 Hz stimulation (red) or without 2 Hz stimulation (black) in a P4 rat. B₂, Relative FP slopes recorded in different age groups and in P3–P5 control group. Data are expressed as mean ± SEM. B₃, Box plots of FP slopes in P3–P5 control and LTP groups of different ages with baseline and 35–60 min poststimulation.

Figure 2.

Spatial properties of LTP induced by repetitive single-whisker stimulation. A, Photograph of the 4 × 4-channel Michigan electrode array (A₁). A local VSDI response was elicited by C2 single-whisker deflection in a P4 rat (A₂). The green dot indicates the center of the C2 barrel-related cortical column. The black dots are the electrode insertion positions. The green circle represents the region of the C2 whisker stimulation-evoked VSDI response. A₃ shows the same area as in A₂ after termination of the electrophysiological recordings and retraction of the recording electrode. The red dots and arrowheads show the electrode insertion points. B₁, Photomicrograph of tangential section through layer IV of the barrel field of a P5 rat after processing the tissue for 5-HTT staining. Colored arrowheads indicate different locations of the four shanks of the 4 × 4-channel Michigan electrode. B₂, FP responses recorded in the activated barrel-related column, in the neighboring septum, and in an adjacent barrel during baseline and post-LTP induction periods from the same P5 rat as shown in B₁. C₁, Mean ± SEM of relative FP responses recorded in the stimulated barrel (square), in the surrounding septal region (circle), and in the neighboring barrel (diamond) of P3–P5 rats. C₂, Box plots of FP slopes recorded in barrel, septa, and neighboring barrel of P3–P5 rats averaged for baseline and 35–60 min post-LTP induction. D₁, Mean ± SEM of relative FP slopes recorded in various distances from the center of the activated barrel in P0–P1 rats. D₂, Box plots of FP slopes in various distances from the center of the stimulated barrel in P0–P1 rats averaged for baseline and 35–60 min post-LTP induction.

Figure 3.

Representative FP response depth profiles and corresponding CSD analyses before and after induction of LTP in a P4 (B) and P0 (C) rat. A, photograph of the 1 × 16-channel Michigan electrode array (left). Digital photomontage reconstructing the location of the DiI-covered electrode in coronal Nissl-stained section (right). B, C, Depth profiles of FP responses to single-whisker stimulation and corresponding CSD analyses in a P4 and P0 rat. The interelectrode distance was 100 μm in the P4 rat and 50 μm in the P0 rat. B₁, C₁, Control data obtained during baseline recording. B₂, C₂, Data after LTP induction. In CSD analyses, current sinks (blue) are downward-going and current sources (red) are upward-going. D, Relative FP slopes recorded in layer II/III and IV at P3–P5. E, Relative FP slopes recorded in the cortical plate and deep layer at P0–P1. Data are expressed as mean ± SEM. Box plots represent FP slopes during baseline and 35–60 min post-LTP induction (right). MZ, marginal zone; WM, white matter; SP, subplate; CP, cortical plate.