Synaptic Plasticity and Excitation-Inhibition Balance in the Dentate Gyrus: Insights from In Vivo Recordings in Neuroligin-1, Neuroligin-2, and Collybistin Knockouts

The hippocampal dentate gyrus plays a role in spatial learning and memory and is thought to encode differences between similar environments. The integrity of excitatory and inhibitory transmission and a fine balance between them is essential for efficient processing of information. Therefore, identification and functional characterization of crucial molecular players at excitatory and inhibitory inputs is critical for understanding the dentate gyrus function. In this minireview, we discuss recent studies unraveling molecular mechanisms of excitatory/inhibitory synaptic transmission, long-term synaptic plasticity, and dentate granule cell excitability in the hippocampus of live animals. We focus on the role of three major postsynaptic proteins localized at excitatory (neuroligin-1) and inhibitory synapses (neuroligin-2 and collybistin). In vivo recordings of field potentials have the advantage of characterizing the effects of the loss of these proteins on the input-output function of granule cells embedded in a network with intact connectivity. The lack of neuroligin-1 leads to deficient synaptic plasticity and reduced excitation but normal granule cell output, suggesting unaltered excitation-inhibition ratio. In contrast, the lack of neuroligin-2 and collybistin reduces inhibition resulting in a shift towards excitation of the dentate circuitry

Differential Postnatal Expression of Neuronal Maturation Markers in the Dentate Gyrus of Mice and Rats

The dentate gyrus (DG) is a unique structure of the hippocampus that is distinguished by ongoing neurogenesis throughout the lifetime of an organism. The development of the DG, which begins during late gestation and continues during the postnatal period, comprises the structural formation of the DG as well as the establishment of the adult neurogenic niche in the subgranular zone (SGZ). We investigated the time course of postnatal maturation of the DG in male C57BL/6J mice and male Sprague-Dawley rats based on the distribution patterns of the immature neuronal marker doublecortin (DCX) and a marker for mature neurons, calbindin (CB). Our findings demonstrate that the postnatal DG is marked by a substantial maturation with a high number of DCX-positive granule cells (GCs) during the first two postnatal weeks followed by a progression toward more mature patterns and increasing numbers of CB-positive GCs within the subsequent 2 weeks. The most substantial shift in maturation of the GC population took place between P7 and P14 in both mice and rats, when young, immature DCX-positive GCs became confined to the innermost part of the GC layer (GCL), indicative of the formation of the SGZ. These results suggest that the first month of postnatal development represents an important transition phase during which DG neurogenesis and the maturation course of the GC population becomes analogous to the process of adult neurogenesis. Therefore, the postnatal DG could serve as an attractive model for studying a growing and functionally maturing neural network. Direct comparisons between mice and rats revealed that the transition from immature DCX-positive to mature CB-positive GCs occurs more rapidly in the rat by approximately 4–6 days. The remarkable species difference in the speed of maturation on the GC population level may have important implications for developmental and neurogenesis research in different rodent species and strains

Nuclear size measurement as a valuable tool to discriminate between early and late stages in structural maturation and age of newborn DGCs.

<p>(A, D) The correlation of nuclear size with structural stage and cell age is illustrated as a dot plot (each dot represents a single cell, pooled from all animals per group) to highlight the variability. (B, E) Newborn DGCs were pooled in an early (DCX stage 1–3, cell age 7–14 dpi) and a late phase (DCX stage 4–6, cell age 21–77 dpi). The mean nuclear sizes of each group were determined and used to calculate the equidistance between early and late phases, which was then used as a threshold to discriminate and assign newborn DGCs to the early or the late phase of development (shown as red dashed line). (C, F). Based on that threshold, cells were categorized into true positive, false positive and false negative predictive values. True positive classifications were found with a reliability of about 70% across all stages. Number of animals: (A, D) DCX stage 1: n = 5, stage 2: n = 7, stage 3: n = 4, stage 4: n = 6, stage 5: n = 9, stage 6: n = 11; cell age: n = 3 per group. (B, E) DCX stage 1–3: n = 8, stage 4–6: n = 12; cell age 7–14 dpi: n = 6, age 21–77 dpi: n = 18. Error bars represent SEM.</p

The Role of Sogo-Zaibatsu in the Economic Development of Modern Japan

<div><p>Adult neurogenesis is frequently studied in the mouse hippocampus. We examined the morphological development of adult-born, immature granule cells in the suprapyramidal blade of the septal dentate gyrus over the period of 7–77 days after mitosis with BrdU-labeling in 6-weeks-old male Thy1-GFP mice. As Thy1-GFP expression was restricted to maturated granule cells, it was combined with doublecortin-immunolabeling of immature granule cells. We developed a novel classification system that is easily applicable and enables objective and direct categorization of newborn granule cells based on the degree of dendritic development in relation to the layer specificity of the dentate gyrus. The structural development of adult-generated granule cells was correlated with age, albeit with notable differences in the time course of development between individual cells. In addition, the size of the nucleus, immunolabeled with the granule cell specific marker Prospero-related homeobox 1 gene, was a stable indicator of the degree of a cell's structural maturation and could be used as a straightforward parameter of granule cell development. Therefore, further studies could employ our doublecortin-staging system and nuclear size measurement to perform investigations of morphological development in combination with functional studies of adult-born granule cells. Furthermore, the Thy1-GFP transgenic mouse model can be used as an additional investigation tool because the reporter gene labels granule cells that are 4 weeks or older, while very young cells could be visualized through the immature marker doublecortin. This will enable comparison studies regarding the structure and function between young immature and older matured granule cells.</p></div

Doublecortin-labeling does not co-localize with Thy1-GFP expression.

<p>(A) Frontal section of the dorsal hippocampal formation from a Thy-1-GFP mouse. Thy1-GFP expression was observed in a subpopulation of dentate granule cells (DGCs) and was expressed throughout dendritic processes of DGCs which extend into the inner molecular layer (IML) and outer molecular layer (OML) toward the hippocampal fissure (hif). Prospero homeobox protein 1 (Prox1, magenta), a specific nuclear marker of granule cells, was confined to granule cell nuclei of the granule cell layer (GCL). Doublecortin (DCX, cyan) labeled young maturing cells that are positioned in the subgranular zone (SGZ). (B) There was no co-localization of DCX and Thy1-GFP which suggests that Thy1-GFP is generally expressed in more mature (DCX-) DGCs. Both DCX+ and Thy1-GFP+ granule cells co-localized with Prox1 even during early stages of DCX expression (see small DCX+ cells in the SGZ). Scale bars: (A) 100 μm; (B) 20 μm. CA1, Cornu Ammonis area 1; H, hilus.</p

PERIOD1 coordinates hippocampal rhythms and memory processing with daytime

In species ranging from flies to mammals, parameters of memory processing, like acquisition, consolidation, and retrieval are clearly molded by time of day. However, mechanisms that regulate and adapt these temporal differences are elusive, with an involvement of clock genes and their protein products suggestive. Therefore, we analyzed initially in mouse hippocampus the daytime-dependent dynamics of parameters, known to be important for proper memory formation, like phosphorylation of the "memory molecule" cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) and chromatin remodeling. Next, in an effort to characterize the mechanistic role of clock genes within hippocampal molecular dynamics, we compared the results obtained from wildtype (WT) -mice and mice deficient for the archetypical clock gene Period1 (Per1-mice). We detected that the circadian rhythm of CREB phosphorylation in the hippocampus of WT mice disappeared completely in mice lacking Per1. Furthermore, we found that the here for the first time described profound endogenous day/night rhythms in histone modifications in the hippocampus of WT-mice are markedly perturbed in Per1-mice. Concomitantly, both, in vivo recorded LTP, a cellular correlate for long-term memory, and hippocampal gene expression were significantly altered in the absence of Per1. Notably, these molecular perturbations in Per1-mice were accompanied by the loss of daytime-dependent differences in spatial working memory performance. Our data provide a molecular blueprint for a novel role of PER1 in temporally shaping the daytime-dependency of memory performance, likely, by gating CREB signaling, and by coupling to downstream chromatin remodeling

Nuclear size and soma position are positively correlated with structural maturation and age of newborn DGCs.

<p>(A) Examples of a 21-day-old stage 1 DCX/Prox1+ cell that is located in the subgranular zone (SGZ) and has no dendritic processes (arrowheads, upper panel) and a 21-day-old stage 6 DCX/Prox1+ cell (arrows, lower panel) with a dendrite extending into the outer molecular layer (OML; arrowheads, lower panel). Asterisk denotes the soma of an intensively labeled Thy1-GFP+ cell. (B) Nuclear size (determined with the nuclear marker Prox1, green) increased with structural maturity. There were significant differences in nuclear size between stages 1 and 6, as well as between stages 1 and 5, stages 2 and 6, and stages 3 and 6 (Kruskal-Wallis Dunn's multiple comparison test between animals, *P < 0.05; stage 1: n = 5 animals, stage 2: n = 7, stage 3: n = 4, stage 4: n = 6, stage 5: n = 9, stage 6: n = 11). (C) In BrdU/Prox1+ DGCs, nuclear size increased gradually with age until it reached a plateau at 35 dpi (n = 3 per group). (D, E) The majority of newborn DGCs was positioned in the SGZ and the inner half of the granule cell layer (GCL), regardless of structural stage and age. Error bars represent SEM. Scale bars in (A): 10μm. IML, inner molecular layer.</p

Survival rate of newborn dentate granule cells decreases over the first 4 weeks.

<p>(A, B) Newly born DGCs labeled with the mitosis marker BrdU (cyan) frequently displayed DCX-expression (magenta) at 7 days post BrdU injection (7 dpi; A), while there was no co-localization of BrdU and DCX at 77 dpi (B). All of the counted BrdU+ cells were Prox1+ (green; A, B). Arrows in (A) point to BrdU/DCX/Prox1+ cells. Arrowheads in (B) point to a BrdU/Thy1-GFP+ cell. Due to their intense somato-dendritic labeling, Thy1-GFP+ cells could be easily distinguished from the green nuclear Prox1 immunostaining. (C) Quantification of BrdU/Prox1+ cells revealed a decline in survival of newborn DGCs between 7 and 35 dpi, whereas the total number of BrdU+ cells did not change between 35 and 77 dpi. Compared to the first week post BrdU injection, 14% of BrdU/Prox1+ cells were retained at 35 dpi, after which there was no further cell loss. (D) The number of BrdU/Prox1+ DGCs that expressed DCX also decreased between 7 and 35 dpi. No DCX/BrdU/Prox1+ cells could be detected between 35 and 77 dpi. All analyses were performed in the suprapyramidal blade of the right dorsal dentate gyrus (n = 3 animals for each group, 3 sections per animal). Error bars represent SEM. Scale bars in (A, B): 10 μm. GCL, granule cell layer; SGZ, subgranular zone.</p

Structural maturation of DCX-expressing newborn DGCs is correlated with cell age.

<p>(A) DCX+ cells were categorized into six stages according to the degree of their structural maturation. Cells were considered to be in stage 1 when the soma was positioned in the subgranular zone (SGZ) and no dendritic processes were visible; stage 2 when the cell displayed short processes that were located within the SGZ; stage 3 when the principal dendritic process projected into the inner half of the granule cell layer (GCL); stage 4 when the leading dendrite reached the outer half of the GCL; stage 5 when the leading dendrite extended into the inner molecular layer (IML); and stage 6 when the leading dendrite reached the outer molecular layer (OML). (B, C) Staging of newborn DCX+ DGCs at different time points revealed a marked shift in stage distribution according to cell age. At 7 dpi, the majority of newborn DCX+ DGCs were classified as stage 1 or 2, while at 14 dpi, the majority of cells were classified as stage 5 or 6. At 28 dpi, about 92% of the DCX+ cells were classified as stage 6, but a small percentage of DCX+ cells were classified as stage 3. (D, E) The distribution of DCX+ cell ages according to each stage illustrates the prevalence of DCX stages 1–4 at 7 dpi and DCX stages 5–6 at 14 to 28 dpi. No BrdU/DCX+ DGCs were observed after 28 dpi. Notably, DCX+ cells of stages 1–6 co-existed at the same time points (7–21 dpi), suggesting a variability in the maturation time course of individual neurons. All data were obtained from 3 animals (n = 3) per group, and 3 sections per animal. Error bars represent SEM. Scale bar in (A): 20μm.</p