Postnatal Development of Functional Projections from Parasubiculum and Presubiculum to Medial Entorhinal Cortex in the Rat

Abstract

Neurons in parasubiculum (PaS), presubiculum (PrS), and medial entorhinal cortex (MEC) code for place (grid cells) and head direction. Directional input has been shown to be important for stable grid cell properties in MEC, and PaS and PrS have been postulated to provide this information to MEC. In line with this, head direction cells in those brain areas are present at postnatal day 11 (P11), having directional tuning that stabilizes shortly after eye opening, which is before premature grid cells emerge in MEC at P16. Whether functional connectivity between these structures exists at those early postnatal stages is unclear. Using anatomical tracing, voltage-sensitive dye imaging and single-cell patch recordings in female and male rat brain slices between P2 and P61, we determined when the pathways from PaS and PrS to MEC emerge, become functional, and how they develop. Anatomical connections from PaS and PrS to superficial MEC emerge between P4 and P6. Monosynaptic connectivity from PaS and PrS to superficial MEC was measurable from P9 to P10 onward, whereas connectivity with deep MEC was measurable from P11 to P12. From P14/P15 on, reactivity of MEC neurons to parasubicular and presubicular inputs becomes adult-like and continues to develop until P28-P30. The maturation of the efficacy of both inputs between P9 and P21 is paralleled by maturation of morphological properties, changes in intrinsic properties of MEC principal neurons, and changes in the GABAergic network of MEC. In conclusion, synaptic projections from PaS and PrS to MEC become functional and adult-like before the emergence of grid cells in MEC.SIGNIFICANCE STATEMENT Head direction information, crucial for grid cells in medial entorhinal cortex (MEC), is thought to enter MEC via parasubiculum (PaS) and presubiculum (PrS). Unraveling the development of functional connections between PaS, PrS, and MEC is key to understanding how spatial navigation, an important cognitive function, may evolve. To gain insight into the development, we used anatomical tracing techniques, voltage-sensitive dye imaging, and single-cell recordings. The combined data led us to conclude that synaptic projections from PaS and PrS to MEC become functional and adult-like before eye opening, allowing crucial head direction information to influence place encoding before the emergence of grid cells in rat MEC.

Keywords: learning; memory; ontogeny; parahippocampal region; spatial navigation.

Figure 1.

Postnatal development of projections from PaS to MEC. The presynaptic side. A–C, Representative grayscale inverted images of an area in MEC showing labeling of parasubicular axons in layers I-III in MEC at (A) P5, (B) P12, and (C) P20. A′, B′, C′, Higher-magnification images of areas indicated with white boxes in A–C. Indicated are examples of varicosities, likely reflecting presynaptic elements (white arrowheads) and axonal branching points (blue arrowheads). Insets, The anterograde tracer injection sites in PaS. Dashed lines indicate brain areas and layers. Scale bars: A–C, 500 μm; A′–C′, 100 μm. Sub, subiculum.

Figure 2.

Postnatal development of projections from PrS to MEC. The presynaptic side. A–C, Representative area in MEC showing labeling of presubicular axons in layers I-III in MEC at (A) P5, (B) P12, and (C) P20. A′, B′, C′, Higher-magnification images of areas indicated with white boxes in A–C. Indicated are an example of a growth cone (red arrowhead), varicosities, likely reflecting presynaptic elements (white arrowheads), and axonal branching points (blue arrowheads). Insets, The anterograde tracer injection sites in PrS. Dashed lines indicate brain areas and layers. Scale bars: A–C, 500 μm; A′–C′, 100 μm. DG, dentate gyrus; CA1, field CA1 of the hippocampus; Sub, subiculum.

Figure 3.

Postnatal development of morphological properties of neurons in MEC. The postsynaptic side. Golgi-stained material showing the neuronal morphology at different postnatal days. A, Sagittal section of a P20 animal stained with NeuN as a marker for neuronal somata. Square represents the approximate position of the Golgi images for B–F. Scale bar, 1.5 mm. High magnification of a piece of MEC stained with Golgi (for details, see Materials and Methods) of a P4 (B), P6 (C), P10 (D), P15 (E), and P21 (F) animal. The neurons appear immature from P4 to P10 and adult-like starting at P15. Apical dendrites can be seen radiating from the deep layers to the pial surface in the 2 older animals. Neurons in superficial layers of the youngest animals (B) appear substantially larger than in older animals; this is due to an incubation artifact resulting in too dense silver deposits. Both neuronal stains in younger tissue (Nissl) as well in the intracellular fill data corroborate that the neurons at these young postnatal ages are not larger. Scale bar: (in C), B–F, 200 μm.

Figure 4.

Postnatal development of physiological properties of principal neurons in MEC. The postsynaptic side. A–D, Response properties of MEC LII principal neurons. Voltage responses of one typical LII principal neuron per age group: (A) P9–P11, (B) P12–P14, (C) P15–P17, and (D) P28–P30. E–H, Response properties of MEC LIII principal neurons. Voltage responses of one typical LIII principal neuron per age group: (E) P9–P11, (F) P12–P14, (G) P15–P17, and (H) P28–P30. N indicates number of neurons measured per age group. A–H, First column, Voltage responses of typical principal neurons to a weak hyperpolarizing and depolarizing 1 s current step just reaching firing threshold. Insets, Zoom of 20 ms, displaying the AP afterpotentials. Second column, The voltage responses to a ±200 pA step of 1 s. Third column, A voltage response of the same neuron shown in the first two columns in response to a ZAP stimulus. Fourth column, Membrane fluctuations recorded just below firing threshold. In all subfigures, the average membrane potential is indicated right below the individual voltage traces.

Figure 5.

Before P9, monosynaptic membrane changes are not observed in MEC in response to PaS and PrS stimulation. A–C, Left, Semihorizontal brain section of a P6–P7 old animal with a stimulation electrode in (A) MEC, (B) PaS, and (C) PrS. Right, Optical signal traces after 20 Hz obtained from MEC LII, LIII, and LIII lateral or LV, respectively, of the boxed areas indicated to the left. B′, C′, Left, Drawings of a standard horizontal rat brain slice used to study contacts between (B′) PaS or (C′) PrS and MEC. The gray pipette stimulation electrode represents the stimulation position. A drawing of a representative principal neuron recorded from intracellularly (black pipette) is presented with dendrites and soma in black. No membrane changes are observed in MEC in response to (B′) PaS or (C′) PrS stimulation before P6/P7. Pink dotted lines indicate the time points of stimulation.

Figure 6.

Postnatal development of network and neuronal reactions in MEC in response to PaS stimulation. A, Left, Representative Nissl-stained slice illustrating the position of the bipolar stimulation electrode in PaS and the position of four pixels in LII-LVI (light to dark gray, respectively) from which the optical responses are collected. The pixel size is 0.03 mm². Right, Averaged optical signal traces in MEC after 20 Hz PaS stimulation. Dashed vertical line indicates the onset of stimulation. B, Drawings of a standard horizontal rat brain slice used to study contacts between PaS and MEC. The gray pipette stimulation electrode indicates the stimulation position in superficial PaS. For each age group, a drawing of a representative principal neuron recorded from intracellularly (black pipette) is presented with dendrites and soma in black and axons in red. C, The average waveform of an eEPSP in MEC LII, in response to 1 Hz PaS stimulation at different age groups: left, P9–P12; second left, P12–P14; third left, P15–P17; right, P28–P30. Inset (right of traces), Zoom-in of the first 20 ms shown to the left. D, The average amplitudes ± SEM of eEPSPs in response to four repetitive stimulations. *Significance and the corresponding p value. E, Single eEPSPs in response to 1 Hz (top) or 20 Hz (bottom) PaS stimulation, as recorded in LII (light gray), LIII (gray), or LV (dark gray). The membrane potential of the neuron is written. Pink dotted lines indicate the time points of stimulation. N indicates number of neurons measured per age group.

Figure 7.

Postnatal development of network and neuronal reactions in MEC in response to PrS stimulation. A, Left, Representative Nissl-stained slice illustrating the position of the bipolar stimulation electrode in PrS and the position of four pixels in LII-LVI (light to dark blue, respectively) from which the optical responses are collected. The pixel size is 0.03 mm². Right, Averaged optical signal traces in MEC after 20 Hz PrS stimulation. Dashed vertical line indicates the onset of stimulation. B, Drawings of a standard horizontal rat brain slice used to study contacts between PrS and MEC. The gray pipette stimulation electrode indicates the stimulation position in superficial PrS. For each age group, a drawing of a representative principal neuron recorded from intracellularly (black pipette) is presented with dendrites and soma in black and axons in red. C, The average waveform of an eEPSP in MEC LIII, in response to 1 Hz PrS stimulation at different age groups: left, P9–P12; second left, P12–P14; third left, P15–P17; right, P28–P30. Inset (right of traces), Zoom-in of the first 20 ms shown to the left. D, The average amplitudes ± SEM of eEPSPs in response to four repetitive stimulations. *Significance and the corresponding p value. E, Single eEPSPs in response to 1 Hz (top) or 20 Hz (bottom) PrS stimulation, as recorded in LII (light blue), LIII (blue), or LV (dark blue). The membrane potential of the neuron is indicated. Pink dotted lines indicate the time points of stimulation. N indicates number of neurons measured per age group.

Figure 8.

Network reactions in adult MEC. Averaged optical signal traces in MEC after 20 Hz PaS stimulation (left) and PrS stimulation (right) from age group P59–P61. Dashed vertical line indicates the onset of stimulation.

Figure 9.

Overview summarizing the development of functionality and functional projections from PaS and PrS to MEC in the rat. Top row, Head direction cells are present in PaS and PrS from P11 onward. P11 was the first day of recordings performed by Bjerknes et al. (2015). Polar plots showing distribution of firing rate for one representative head direction cell (peak firing rate indicated). Directional tuning stabilizes shortly after eye opening. Adapted with permission from Bjerknes et al. (2015). Second row, Grid cells are also present from the first day of recordings (P16), but grid regularity increases throughout the first 4–5 weeks of postnatal development. Rate maps of grids (top row) and spatial autocorrelations (bottom row) from P16 to P18 old rats show grid-like characteristics, but the regularity and specificity of the grid increase with age. Grid scores: blue represents r = −1; red represents r = 1. Adapted with permission from Langston et al. (2010) and Ainge and Langston (2012). Third row, Schematic overview of the development of the presynaptic (PaS and PrS) and postsynaptic (MEC) anatomy. Orange represents the somata of MEC neurons together with their neurites. The dendritic morphology of P4–P6 old MEC neurons is poorly developed. The majority of somata with recognizable neurites are located in layers II and III. For all neurons, the number of neurites is low and the neurites are poorly developed, remaining mostly within the layer of origin. Dendrites of mainly superficial layers show sparse branching that does not extend over long distances. Axons from PaS (gray tones) and PrS (blue tones) emerge from P4 to P6 but continue to develop over time. Indicated are varicosities (filled circles) and synapses (axonal branching points with synapse shaped terminals contacting the postsynaptic MEC neuron). From P9/P10 onward, the morphology of neurons in MEC layers II and III looks more mature, but the neuritic tree morphology together with the presynaptic morphology indicates that the connections are still not adult-like. From P12–P15 onward, the gross neuronal morphology looks more adult-like. Fourth row, The averaged optical signal traces in MEC after 20 Hz PaS stimulation (gray) and PrS stimulation (blue) from the different age groups. Bottom row, Averaged voltage trace of an eEPSP in MEC LII (gray; top) and LIII (blue; bottom), in response to 1 Hz PaS (gray; top) and PrS (blue; bottom) stimulation, respectively, at the different age groups: left, P9–P12; second left, P12–P14; third left, P15–P17; right, P28–P30.