Abstracts

Rationale: Semilunar Granule Cells (SGCs) have been proposed as a morpho-functionally distinct class of hippocampal dentate projection neurons contributing to feedback inhibition and memory processing. However, whether SGCs retain their unique structural characteristics and inhibitory inputs through postnatal development remains unresolved. Focusing on postnatal days 11-13, 28-42 and >120, corresponding with infancy, adolescence, and adulthood, we examined whether SGCs differ from Granule Cells (GCs) in somatodendritic morphology and inhibitory inputs. Methods: Whole cell recordings of synaptic and tonic GABA currents were obtained from rats aged 11-150 days. Cell morphologies of putative SGCs and GCs were reconstructed in 3D (Neurolucida 360) from biocytin filled neurons. SGCs and GCs were identified based on axons entering hilus, molecular layer dendrites with spines and somata in the inner molecular or granule cell layer, respectively. Somatodendritic morphological parameters were quantified using Neurolucida Explorer (MBF Biosciences). Morphometric parameters from 35 neurons were analyzed by unsupervised hierarchical clustering, and principal component analysis (PCA), using R packages. Results: The PCA analysis of 42 morphometric parameters showed that the first seven dimensions, retained over 90% of the variance in the dataset. Clustering on the first three dimensions resolved two subgroups defined by investigator assigned classification, while grouping based on animal age showed considerable overlap. Unsupervised hierarchical clustering on morphometry identified four clusters: two clusters included only cells identified as SGC by the investigator, one cluster included 11 GC and one putative SGC while the fourth including a mix of 5 GCs and 4 SGCs showed low silhouette score indicating an unstable cluster. There was over >85% (31 of 36) agreement between cell classification based on the unsupervised approach and that assigned by the investigator. The top 5 morphological variables which best represent the principal components included the number of primary dendrites, second and third order dendritic segments, second order nodes and soma width. These parameters showed significant differences between cell types and did not differ between age groups, (Soma width: Cell Type, F(1,33) = 4.457, p = 0.042; Age F(2,34)=0.295, p = 0.747, TW-ANOVA). In contrast, certain summed dendritic parameters including the total numbers of dendritic segments and total dendritic showed neither cell-type nor age related differences (Total dendritic terminals: Cell Type F(1,34) = 0.909, p = 0.347 ; Age F(2,34)= 1.940, p = 0.159 by TW-ANOVA). Physiologically, SGCs consistently showed a significantly higher frequency of sIPSCs (spontaneous inhibitory postsynaptic currents) compared to GCs from age matched controls at all postnatal time points examined. Both cell-types showed developmental changes in sIPSC frequency, which peaked during adolescence. In contrast, sIPSC amplitude showed a progressive reduction with age in both cell types. Conclusions: These findings highlight the distinct morphology and enhanced synaptic inhibition of SGCs through development and identify the key somatodendritic features that distinguish SGCs from GCs regardless of developmental stage. Funding: No funding