Basic intrinsic cellular properties of dentate granule cells with hilar basal dendrites.

<p>(A) Overview of a mature entorhino-hippocampal slice culture (blue, TOPRO nuclear stain; EC, entorhinal cortex; DG, dentate gyrus; CA1, hippocampal subfield CA1). Scale bar: 500 µm. (B) Dentate gyrus of a mature entorhino-hippocampal slice culture shown at higher magnification. Schematic representations of dentate granule cells. Granule cell somata are located in the granule cell layer (GCL) while granule cell dendrites extend into the molecular layer (ML). A subset of mature dentate granule cells may exhibit additional dendrites, which emerge from the basal portion of the soma and extend into the hilar region (hilar basal dendrites; hBDs; blue, TOPRO nuclear stain). Scale bar: 100 µm. (C) Hilar basal dendrites (hBDs) were defined using the following objective criteria. The granule cell soma was divided into quarters by connecting the origin of the axon with the apical pole of the soma and by an orthogonal centerline trough this line. The two quarters next to the origin of the axon were called basal quarters (red). Dendrites emerging from the basal quarters, reaching into the hilar region and not crossing the centerline were considered as hBDs. (D, E) 2D-projected confocal image stacks of dentate granule cells filled with Alexa568 (red). Asterisk indicates patch electrode. Arrow points to a hBD. Scale bar: 50 µm. (F) Sample traces showing input-output curves of a hBD-GC. Voltage traces (top) in response to a 1 sec current pulse. Protocol is shown underneath the traces. (G–J) Granule cells with and without hBDs were indistinguishable in their (G) actionpotential (AP)-threshold, (H) AP frequency, (I) time to first spike or (J) inter spike interval (n = 9 GCs without hBDs and n = 12 GCs with hBDs; in 7 cultures). (K–N) Evaluation of action potential properties did not show a significant difference between the two GC populations in (L) AP-amplitude, (M) afterhyperpolarization (AHP) amplitude or AP width (N) measured at three different positions (indicated with I, II and III in panel K).</p

The morphology of the apical dendritic tree of granule cells with hilar basal dendrites.

<p>(A, B) Alexa-filled dentate granule cells were reconstructed in confocal image stacks using Neuronstudio software. 2D-projected image stacks (left) and the corresponding reconstructed skeletons (right) are shown for a granule cell without (A) and with a hBD (B). Scale bar: 50 µm. (C–E) No significant difference in total dendritic branch length (TDBL), number of segments per branch order or mean length per branch order was observed between granule cells with and without basal dendrites (n = 21 GCs without hBDs and n = 21 GCs with hBDs; in 12 cultures). (F, G) The Sholl-analysis of the two GC populations showed a comparable complexity of the dendritic trees (distance of circles/spheres in F, 25 µm).</p

Spine density in the outer molecular layer is not altered in granule cells with hilar basal dendrites.

<p>(A) 2D-projected confocal image stacks of individual dendritic segments of dentate granule cells imaged in the outer molecular layer. Scale bar: 2 µm. (B) Spine density analysis of granule cells with and without hBDs (n = 13 segments from no-hBD and 10 segments from hBD-GCs; in 10 cultures). Spine densities were not significantly different.</p

Morphological properties of hilar basal dendrites.

<p>Hilar basal dendrites (hBDs) contribute to ∼8–10% of the total dendritic branch length of hBD-GCs. Most hBD-GCs revealed a single hBD emerging from the basal pole of the soma. In one case two and in another three first order hBD segments were observed. Second order branches were found in 50% of cases. In two cases third order segments and in one case fourth order segments were observed (n = 14 hBD-GCs were analyzed; in 9 cultures).</p

Excitatory and inhibitory synaptic strength of dentate granule cells with hilar basal dendrites.

<p>(A–C) Sample traces of miniature excitatory postsynaptic currents (mEPSC). Cumulative distribution of the mEPSC amplitudes and mean values of mEPSC amplitude and frequency (n = 7 GCs without hBDs and n = 9 GCs with hBDs; in 6 cultures). (D–F) Sample traces of miniature inhibitory postsynaptic currents (mIPSC). Cumulative distribution of the mIPSC amplitudes and mean values of mIPSC amplitude and frequency (n = 8 GCs without hBDs and n = 7 GCs with hBDs; in 4 cultures). No significant difference between granule cells with and without hBDs was observed in these experiments.</p

Granule cells with hilar basal dendrites are not heavily innervated by mossy fibers.

<p>(A) GC axons, i.e., mossy fibers, contact mossy cells in the hilus and CA3 pyramidal neurons (not shown) without connecting to other GCs. However, under pathological conditions hilar basal dendrites (hBDs) may be innervated by mossy fibers. Computational studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048500#pone.0048500-Morgan1" target="_blank">[15]</a> suggests that a strong innervation of hBDs by mossy fibers could promote hyperexcitability in the dentate gyrus network. (B) Paired recordings between neighboring granule cells with and without hBDs revealed no evidence for a heavy GC-to-GC connectivity in the dentate gyrus of mature entorhino-hippocampal slice cultures. The proximity of axons and hBDs was documented in confocal image stacks prior to recordings. In 48 probed connections only one functional connection was found. However, this connection was indeed found on a hBD-GC, thus confirming that functional GC-to-GC connectivity is possible on hBD-GCs (asterisk, patch pipette). Scale bar: 50 µm. (C) Flash photolysis of caged-Glutamate and caged-GABA was performed over hBDs (boxed area 1 in the inset) and in the neuropil in the neighborhood of hBDs (boxed area 2 in the inset) while recording evoked responses from the soma of the same hBD-GC (asterisk, patch pipette). Scale bar: 50 µm. (D, E) Sample traces for GABA and Glutamate uncaging experiments. Evoked responses were blocked by the AMPA-receptor antagonist CNQX or the GABA_A-receptor blocker SR95531 respectively (in 3 independent experiments each).</p