1. Volume 90, Issue 4, Pages 853-865 (May 2016)
Astrocyte Intermediaries of Septal Cholinergic Modulation in the Hippocampus 
Milan Pabst, Oliver Braganza, Holger Dannenberg, Wen Hu, Leonie Pothmann, Jurij Rosen, Istvan Mody, Karen van Loo, Karl Deisseroth, Albert J. Becker, Susanne Schoch, Heinz Beck 
Neuron 
Volume 90, Issue 4, Pages (May 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Septal Cholinergic Projections Cause Long-Lasting Inhibition of Dentate Granule Cells In Vivo and In Vitro
(A) Whole-cell in vivo patch-clamp recordings of dentate granule cells and stimulation of septal cholinergic neurons with a light fiber within the MSDB. Lower panel: representative reconstruction of a biocytin-labeled dentate granule cell after in vivo recording.
(B) Light-evoked hyperpolarizations recorded in vivo in dentate granule cells. Upper panel: representative example, lower panel: hyperpolarization amplitudes, average 4.33 ± 0.68 mV, average membrane potential −62.8 ± 2.6 mV, n = 5).
(C and D) Light-evoked inhibition of tonic action potential firing in granule cells induced by injection of a small depolarizing current to achieve firing rates of ∼3 Hz (example in C). Quantification of firing rates before and during stimulation in (D). Asterisks indicate p < 0.001, paired t test, n = 3.
(E) Representative firing behavior and reconstruction of a dentate granule cell after in vitro recording.
(F) Light-evoked responses in dentate granule cells.
(G) Light-evoked outward currents were blocked by the GABAA-receptor antagonist gabazine.
(H) In 34 of 71 dentate granule cells, only hyperpolarizations were recorded. In some cases, depolarizations preceded the hyperpolarizations (amplitudes of 3.50 ± 0.58 mV versus 7.05 ± 0.42 mV, respectively).
(I) Light-evoked hyperpolarizations were not significantly reduced by the muscarinic ACh receptor antagonist atropine alone (5 μM) but were blocked by additional application of the nicotinic antagonist MLA (50 nM, lower-left panel). One-way ANOVA, p = , F(2,20) = 4.375, asterisks indicate p < 0.05, Dunnett’s multiple comparison test, n = 9. Application of α7 nicotinic AChR-specific concentrations of MLA alone (20 nM) also significantly reduced the granule cell inhibition (paired t test, n = 7, p = , lower-right panel).
(J) False color plot of membrane potential over time after stimulation for all granule cells (n = 71).
(K–M) Light-evoked inhibition of repetitive firing in granule cells evoked by prolonged current injections (example in K). Raster plot for all measured cells is shown. The quantification of the normalized number of action potentials without and with light-induced inhibition with and without the GABAA receptor antagonist gabazine (10 μM) is shown (M).
Asterisks indicate significant effects of light stimulation of firing frequency, p = , paired t test, n = 6). § indicates significant difference in the presence of gabazine compared to control recordings, p = , unpaired t test). Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
Septohippocampal Cholinergic Projections Activate Interneurons in the Dentate Hilus In Vivo
(A and B) Characteristic unit waveforms (left panels, mean ± SD) from hilar neurons classified as putative fast-spiking interneurons (A, blue) and other hilar neurons (B, green). Representative spike autocorrelograms (right panels) are shown from the interneurons depicted as filled circles in (C).
(C) Clustering of identified single units into putative fast spiking interneurons and other hilar neurons according to the mean time of the spike autocorrelogram and the spike width. The average values for spike width of the two clusters were 0.35 ± 0.13 ms and 0.94 ± 0.21 ms (mean ± SD).
(D) Raster plot showing increased spike frequency of a representative interneuron during baseline and optogenetic activation of cholinergic MSDB neurons with 20 ms pulses at 12 or 20 Hz.
(E) Spiking rate histogram of the same unit, spiking rate averaged over the trials shown in (D), 1-s time bins.
(F) Mean ± SEM of spike frequency of n = 29 putative fast spiking interneurons.
(G) Average spiking rate during baseline and during episodes of blue light stimulation at 12 or 20 Hz (binned over 10 s, baseline from −15 to −5 s before onset of light stimulation, n = 18 mice, n = 29 units, ∗∗∗p < 0.001, Wilcoxon signed-rank test).
(H) False color image displaying spiking rate Z scores of the putative fast-spiking interneurons and mean ± SEM over all units (upper panel).
(I) Cumulative cell density plot of firing rate Z scores during light stimulation of cholinergic MSDB neurons at 12 and 20 Hz. Rectangle marks example cell in (A), (D), and (E).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Cholinergic Septohippocampal Fibers Activate Hilar Inhibitory Interneurons and Mossy Cells
(A and C–E) Effects of light-based stimulation of cholinergic fibers on different types of dentate interneurons assessed using patch-clamp recordings in hippocampal slices. Leftmost panels always show morphological reconstructions of representative recorded neurons (A, hilar inhibitory interneuron; C, hilar mossy cell; D, dentate molecular layer interneuron; E, semilunar granule cell). Note that only a short portion of the axon of the semilunar granule cell was recovered. Gray areas correspond to dentate granule cell layer. Middle panels show the corresponding discharge behavior elicited by current injections; insets in (C) and (E) show higher magnification of distinctive action potential firing. Rightmost panels display the effects of optogenetic stimulation of cholinergic axons (stimulation time point shown in blue, insets in A and C show the excitatory responses to optogenetic stimulation at higher magnification).
(B) Effects of 20 nM MLA on light-evoked responses in hilar interneurons. Left panel, representative example; right panel, quantification of light-evoked EPSP magnitude.
(F) Magnitude of the light-induced PSPs in the different cell types. Note that action potentials were only generated by light stimulation in a subset of hilar interneurons and mossy cells, never in other types of neurons.
Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4 Granule Cell Inhibition Involves a Glutamatergic Relay
(A and B) Blocking glutamate receptors with combined application of CNQX (10 μM) and D-AP5 (50 μM) strongly reduces optogenetically evoked EPSPs in hilar interneurons (A, asterisks indicate, p = , paired t test, n = 11), and in hilar mossy cells (B, asterisks indicate p = , paired t test, n = 4).
(C) CNQX and D-AP5 also potently reduces light-evoked IPSPs in dentate granule cells (asterisks indicate p = , paired t test, n = 13).
Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 5 Granule Cell Inhibition Involves an Astrocytic Intermediary
(A) Optogenetic stimulation of ACh release from septohippocampal fibers (indicated by dashed line) stimulates Ca2+ increases in numerous SR101+ astrocytes. Astrocytes in which responses were detected are shown as black traces; gray traces indicate examples without responses. Numbered traces correspond to the astrocytes in the photomicrographs. Scale bar, 50 μm.
(B) In a different experiment, examples of early and late Ca2+ elevations, gray and black arrows indicate slow and fast transients, respectively. Bar graph shows quantification of the number of astrocytes in which early and late Ca2+ elevations could be observed after light stimulation (in the presence of glutamatergic blockers).
(C) Effects of sequentially blocking glutamate receptors and muscarinic and nicotinic ACh receptors (asterisk indicates significant differences in ANOVA with Dunnett’s post test, p < 0.05, n = 52 astrocytes in four slices).
(D) Control experiments without virus injection or without light stimulation revealed no Ca2+ transients.
(E) SR101-labeled astrocytes expressing GCaMP6f. Scale bars, 25 and 12.5 μm.
(F) Representative example of light-evoked Ca2+ transients in GCaMP6f-expressing astrocytes. Gray, single sweep measurements from the astrocyte in (E); black line, average Ca2+ transient.
Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6 Granule Cell Inhibition Involves an Astrocytic Intermediary
(A) Disrupting astrocytic function by incubation of slices with FAc reduces dentate inhibition. Representative recordings from interleaved experiments with and without FAc incubation are shown. Bar graph shows average DG inhibition with FAc incubation compared to alternating control experiments without FAc pre-incubation (lower panel, ∗∗∗p = , unpaired t test).
(B–D) Buffering astrocytic Ca2+ increases reduces DG inhibition. Dual patch-clamp recordings were obtained from astrocytes at the CA3-hilar border with an intracellular solution containing the Ca2+ chelator BAPTA (40 mM), and from a granule cell (B). Responses of granule cells and astrocytes to optogenetic stimulation were simultaneously monitored (C). IPSPs in granule cells were significantly reduced after >30 min of astrocytic dialysis with BAPTA (D).
(E) Quantification of the experiments comparing recordings with astrocytes dialyzed with BAPTA (astrocytic BAPTA, open circles), recordings where astrocytes were recorded without BAPTA (control, astrocytic recording, empty squares), and prolonged control recordings without additional astrocyte recordings (filled circles). ∗∗Significant differences to initial control values measured with repeated-measures ANOVA and Dunnett’s post test. §Significant group differences at the 40-min time point determined with ANOVA and Bonferroni post hoc tests.
(F) Dialysis of the astrocytic syncytium with BAPTA reduces activation of hilar inhibitory interneurons. Bar graph shows the average magnitude of light-evoked EPSPs in hilar interneurons with prolonged (>40 min) astrocytic recordings with 40 mM BAPTA and without astrocytic dialysis (asterisk indicates p < 0.05, unpaired t test, n = 5 and 31, respectively).
Data are represented as mean ± SEM.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions