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Network state-dependent inhibition of identified hippocampal CA3 axo-axonic cells in vivo

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Abstract

Hippocampal sharp waves are population discharges initiated by an unknown mechanism in pyramidal cell networks of CA3. Axo-axonic cells (AACs) regulate action potential generation through GABAergic synapses on the axon initial segment. We found that CA3 AACs in anesthetized rats and AACs in freely moving rats stopped firing during sharp waves, when pyramidal cells fire most. AACs fired strongly and rhythmically around the peak of theta oscillations, when pyramidal cells fire at low probability. Distinguishing AACs from other parvalbumin-expressing interneurons by their lack of detectable SATB1 transcription factor immunoreactivity, we discovered a somatic GABAergic input originating from the medial septum that preferentially targets AACs. We recorded septo-hippocampal GABAergic cells that were activated during hippocampal sharp waves and projected to CA3. We hypothesize that inhibition of AACs, and the resulting subcellular redistribution of inhibition from the axon initial segment to other pyramidal cell domains, is a necessary condition for the emergence of sharp waves promoting memory consolidation.

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Figure 1: Identified CA3 AACs fire less during SWR events in vivo.
Figure 2: GABAergic input to CA3 pyramidal cell AISs is withdrawn during sharp waves in vivo.
Figure 3: AACs in CA3 fire rhythmically around the peak of theta oscillations and are coupled to gamma oscillations in vivo.
Figure 4: Innervation of AAC dendrites by GABAergic and mossy fiber terminals.
Figure 5: AACs are SATB1-immunonegative, PV+ interneurons.
Figure 6: A preferential medial septal GABAergic input to the somata of AACs identified by SATB1PV+ labeling in CA3.
Figure 7: A subset of GABAergic medial septal cells increase their firing during sharp waves in vivo and project to CA3.

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  • 29 October 2013

    In the version of this article initially published online, the received date was given as 24 May 2012. The correct date is 24 May 2013. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

This paper celebrates the contributions of János Szentágothai (1912–1994) to neuroscience. We thank the excellent assistance of K. Detzner, D. Kotzadimitriou, B. Micklem and S. Biro. We thank J. Somogyi for advice on confocal microscopy and T. Forro, K. Hartwich and O. Valenti for the use of identified interneurons that they labeled in vivo. We are grateful to R. Shigemoto (National Institute for Physiological Sciences, Okazaki, Japan) and M. Watanabe (Department of Anatomy, Hokkaido University Graduate School of Medicine) for the gift of antibodies. We thank T. Ellender and P. Magill for comments on an earlier version of the manuscript. B.L. was supported by the Blaschko European Visiting Research Fellowship at Oxford; we acknowledge grant 242689 of the European Research Council, grant SCIC03 of the Vienna Science and Technology Fund and the Medical Research Council.

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Contributions

T.J.V., B.L., L.K., M.G.C., J.J.T., T.K. and P.S. collected and analyzed data and wrote the paper. To expand on the equal-contributions footnote, each of the first four authors made important—though different—contributions, and hence they should be considered equal first authors.

Corresponding authors

Correspondence to Tim J Viney, Balint Lasztoczi or Peter Somogyi.

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Integrated supplementary information

Supplementary Figure 1 A CA1 AAC recorded in a freely moving rat is inhibited during sharp waves.

(a) Left, a bouton (B) of cell TV34n filled with electron opaque HRP reaction end-product making a type II synapse (filled arrow) with a pyramidal cell AIS (identified by the dense membrane undercoating, asterisk); open arrow, a type II synapse with an unlabeled bouton. Right, another bouton of the same cell. Scale bars, 0.2 μm. (b and c) Firing patterns of the identified AAC TV34n during (b) SWS and (c) during quite wakefulness and head movement. Movement is detected by an accelerometer; spindles (s) are present in the EEG during SWS.

Supplementary Figure 2 Synaptic junctions between the boutons of identified CA3 AACs and axon initial segments of pyramidal cells.

Ten electron micrographs taken from serial sections of neurobiotin-labeled (HRP end-product) boutons (B) of AAC B45a making type II synapses (arrows) with AISs, identified by membrane undercoating (asterisks) and/or microtubule fascicles. Unlabeled boutons also make similar synapses (open arrows). Spines (s) from AISs, described previously19, also receive one or more AAC synapses. Sections were not contrasted by lead. Scale for all images: 0.5 μm.

Supplementary Figure 3 State-dependent firing rates of identified AACs in freely moving rats.

(a and b) Firing rates of identified CA2 AAC LK24g (black) and CA1 AAC TV34n (gray) during (a) network oscillations and (b) behavioral states. (c and d) Mean ± s.e.m. firing rates of 5 published identified parvalbumin-expressing basket cells20 for comparison with the AACs. Note the different rates during sharp waves when compared to theta oscillations.

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Supplementary Figure 4 Innervation of AAC dendrites by mossy fiber terminals.

(a,i) Electron micrograph of a neurobiotin-labeled AAC dendrite in sLuc (dend, cell B45a) receiving synaptic input (filled white arrows) from a large mossy fiber bouton (MF) recognized by dense vesicular filling. Unfilled white arrows, synapses made by boutons other than the large mossy terminal; s, invaginated spine from a thorny excrescence of a CA3 pyramidal cell dendrite. (a,ii) Serial section of the spine (s) with synaptic junction (asterisk). (a,iii) Enlargement of synaptic junctions in i (white arrows). Image captured at a different angle of tilt. Black arrowheads, synaptic vesicles; open arrowhead, dense core vesicle. (b) Same as in a,i; another large mossy terminal (MF) making a synapse (white arrow, see inset b,ii) onto the same dendrite. Asterisks, synapses by the mossy terminal on a pyramidal cell spine (s). Sections were not contrasted by lead. Scale bars: a,i, a,ii, b,i, 0.5 μm; a,iii, b,ii, 0.1 μm.

Supplementary Figure 5 Preferential targets of septo-hippocampal neurons and firing patterns of medial septal cells.

(a) Digital trace (cyan) of a PHA-L-labeled septal axon preferentially targeting SATB1+/PV+ interneurons (unconnected PHA-L+ axons are black). (b) Left, septal-innervated PV+ somata from boxes 1 and 2 in a. Right, SATB1+ nuclei of same cells. (c) Single optical sections of parvalbumin-immunoreactivity from b shown with and without PHA-L-immunoreactivity. PV+ septal boutons, asterisks; main axon, arrows. (d) PHA-L-labeled septal axon (cyan, arrow) targeting a SATB1–/PV+ soma (magenta, in box 2) amongst SATB2+ CA1 pyramidal cells (cyan, nuclei). Another PV+ cell (in box 1), is SATB1+ and NPY+ (both white, nucleus and Golgi apparatus, respectively) and not innervated by the septal axon. Note NPY+ axons (white, e.g. inside boxed regions). (e) Single optical sections of both PV+ cells (from boxes 1 and 2 in d). Immunoreactivity for parvalbumin and PHA-L/SATB2 (left), and SATB1 and NPY (right). The septal innervated cell (from box 1) is SATB1– and NPY–. (f and g) Single optical sections of in vivo recorded neurobiotin-labeled medial septal cells M65b (f) and M40f (g). Parvalbumin, magenta; HCN4, cyan; asterisks, somata; arrow, plasma membrane. (h) Firing patterns of cell M82f (see Figure 7) during theta oscillations recorded in CA1. (i) Firing rate versus CA1 theta phase for medial septal cells. Same color code as in Fig. 7e and those identified with axons projecting to CA3 are shown with thick lines. Confocal z-stacks (number of optical sections / thickness in μm / intensity projection mode): a, 73/29/average (montage); b box 1, 52/15/average; box 2, 83/24/average; d, 84/32/average. Scale bars (μm): a, 20; b, c, e, 5; d, f, g, 10.

Source data

Supplementary Figure 6 Synapses between medial septal terminals and an AAC identified by SATB1-PV+ labeling.

(a) Median-filtered confocal z-projection of parvalbumin-immunoreactivity (magenta) in two CA3 neurons. One neuron (asterisk) shows nuclear SATB1 immunoreactivity (white), the other has no detectable SATB1 immunoreactivity (arrow). (b) Confocal z-projection of PHA-L-labeled boutons (cyan) apposed to the PV+ (magenta) SATB1–immunonegative neuron shown in a (arrow). The PV+/SATB1+ neuron in a is not innervated by PHA-L boutons (asterisk). Both cells contain endogenous biotin (cyan, somatic labeling) that is visualized by the streptavidin-conjugated Alexa Fluor secondary antibody bound to the biotinylated anti-PHA-L primary antibody. (c) Light microscopic image z-stack of the same area after converting PHAL-immunoreactivity to diaminobenzidine-based HRP reaction product (20X objective), showing PHA-L-innervated SATB1–/PV+ cell from a/b (arrow) revealing axon (partially myelinated, m) bypassing the SATB1+/PV+ cell (asterisk). Note endogenous biotin in the soma. (d) Light microscopic z-stack image of PHA-L-innervated cell (100X objective). (e) Two-dimensional reconstruction of the medial septal-innervated cell. Four boutons in d and e (marked gj) correspond to the panels below. (f) Electron micrograph of PHA-L-labeled boutons apposed to the target cell. Boxed region is shown rotated in g. (g–j) Synaptic junctions (arrows) between PHA-L-labeled septal boutons (B) and the target AAC. Confocal z-stacks (number of optical sections / thickness in μm / intensity projection mode): a, 2 separate single optical z-sections in same x-y location superimposed / 1.1 / maximum; b, 9 (PHA-L) and 27 (parvalbumin) optical sections superimposed / 15.1 / maximum; c, 24 / 13.7 / minimum; d, 93 / 24.2 / minimum. Scale bars: a–b, d–e, 10 μm; c, 20 μm; f, 1 μm; g–j, 0.2 μm.

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Viney, T., Lasztoczi, B., Katona, L. et al. Network state-dependent inhibition of identified hippocampal CA3 axo-axonic cells in vivo. Nat Neurosci 16, 1802–1811 (2013). https://doi.org/10.1038/nn.3550

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