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. 2016 Jun 10;5:e14592.
doi: 10.7554/eLife.14592.

Anatomical Organization of Presubicular Head-Direction Circuits

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Free PMC article

Anatomical Organization of Presubicular Head-Direction Circuits

Patricia Preston-Ferrer et al. Elife. .
Free PMC article

Abstract

Neurons coding for head-direction are crucial for spatial navigation. Here we explored the cellular basis of head-direction coding in the rat dorsal presubiculum (PreS). We found that layer2 is composed of two principal cell populations (calbindin-positive and calbindin-negative neurons) which targeted the contralateral PreS and retrosplenial cortex, respectively. Layer3 pyramidal neurons projected to the medial entorhinal cortex (MEC). By juxtacellularly recording PreS neurons in awake rats during passive-rotation, we found that head-direction responses were preferentially contributed by layer3 pyramidal cells, whose long-range axons branched within layer3 of the MEC. In contrast, layer2 neurons displayed distinct spike-shapes, were not modulated by head-direction but rhythmically-entrained by theta-oscillations. Fast-spiking interneurons showed only weak directionality and theta-rhythmicity, but were significantly modulated by angular velocity. Our data thus indicate that PreS neurons differentially contribute to head-direction coding, and point to a cell-type- and layer-specific routing of directional and non-directional information to downstream cortical targets.

Keywords: head-direction; in-vivo electrophysiology; neurons and circuits; neuroscience; rat; spatial navigation.

Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1.. Anatomical organization and projection targets of superficial PreS neurons.
(A) Top, parasagittal section through the dorsal PreS stained for calbindin (Cb, green) and NeuN (red). Scale bar = 500 μm. Bottom, outline of the PreS (grey) from the section shown above. RS29 indicates the subfield of RS cortex which was targeted for retrograde tracing experiments. See Figure 1—figure supplement 1 for more details. (B) Superimposed staining for calbindin (green) and NeuN (red) showing the clustering of neuronal somata in L2 of PreS and the more homogeneous distribution of cells in L3. Right, close-up magnification of the single channels for panel 1 (red, NeuN; green, calbindin). Scale bars: 100 μm (left) and 50 μm (right). (C) Parasagittal section through PreS stained for calbindin (green) showing retrogradely-labeled neuronal somata following injection of CTB-Alexa 555 (red) in ipsilateral MEC (‘ipsi-MEC’). Left panels, single channels; right panel, overlay. Scale bars: 200 μm. (D) Bar-graph showing the % of retrogradely-labelled (CTB-positive) neurons in L2 and L3 of PreS, following tracer injection in ipsi-MEC (as shown in C; 4497 total counted neurons, n = 4 brains). Error bars indicate SEM. (E) Parasagittal section through PreS stained for calbindin (green) showing retrogradely-labeled neuronal somata following injection of CTB-Alexa 555 (red) in contralateral PreS (‘contra-PreS’). Scale bar: 50 μm. Right panel, close-up magnification of the inset shown on the left, showing three retrogradelly-labelled neurons (red) positive for the marker calbindin (green). Scale bar: 10 μm. (F) Bar-graph showing the % of calbindin-positive (Cb+) and calbindin-negative (Cb-) L2 neurons, which were retrogradely-labelled following tracer injection in contra-PreS (as shown in E; 159 total counted neurons, n = 3 brains). Error bars indicate SEM. (G) Left panels, close-up magnification PreS L2 neurons following injection of CTB-Alexa 555 (red) in contralateral RS29 and stained for calbindin (green). One calbindin-positive (asterisk) and two calbindin-negative neurons (arrowheads) are indicated. Scale bar: 10 μm. Right, bar-graph showing the % of calbindin-positive (Cb+) and calbindin-negative (Cb-) L2 neurons, which were retrogradely-labelled following tracer injection in contra-RS29 (896 total counted neurons, n = 4 brains). Error bars indicate SEM. DOI: http://dx.doi.org/10.7554/eLife.14592.002
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Immunohistochemical analysis and outline of the PreS.
(A )Parasagittal section through the dorsal PreS (~3.0 mm lateral from the midline) stained for calbindin (Cb, green) and NeuN (red). Top panels show single channels, bottom panel shows the overlay. Scale bar = 500 μm. (B) Outline of the PreS (grey) from the section shown in (A). RS29 indicates the subfield of RS cortex which was targeted for retrograde tracing experiments (see Materials and methods, Figure 1G and Figure 1—figure supplement 2). (B–D) and (E–F) same as in (A) but for more lateral sections (~3.3 and 3.7 lateral from the midline, respectively; Paxinos and Watson, 2007). Scale bars = 500 μm. PreS, presubiculum; RS29, retrosplenial cortex area 29; WM, white matter; Sub, subiculum; PaS, parasubiculum; MEC, medial entorhinal cortex. DOI: http://dx.doi.org/10.7554/eLife.14592.003
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. Neuroanatomical markers outlining the rostral and caudal PreS borders.
(A) Parasagittal section through the dorsal PreS stained for calbindin (Cb, green) and paravalbumin (PV, red). Top panels show single channels, bottom panel shows the overlay. Scale bar = 500 μm. (B) Parasagittal section through the dorsal PreS stained for calbindin (Cb, green) and Wolframin (Wfs-1, red). Top panels show single channels, bottom panel shown the overlay. Scale bar = 500 μm. (C) Parasagittal section through the dorsal PreS processed for zinc histochemistry. Scale bar = 500 μm. (D) Outline and location of the PreS (indicated in grey) from the section shown in (A). The arrowheads point to the rostral and caudal PreS borders, which can be assessed based on differential expression of the neuroanatomical markers shown in (A–C). PreS, presubiculum; RS29, retrosplenial cortex area 29; WM, white matter; Sub, subiculum; PaS, parasubiculum. DOI: http://dx.doi.org/10.7554/eLife.14592.004
Figure 1—figure supplement 3.
Figure 1—figure supplement 3.. Layer distribution of retrogradely-labeled neurons in the contralateral PreS.
(A) Left panel, parasagittal section through the dorsal PreS showing retrogradely-labelled neurons following injection of CTB in the contralateral PreS. Note the presence of retrogradely-labelled neurons within PreS L2 and the sparser labeling within RS29. Right panel, overlay with the calbindin-staining (Cb, green). Scale bar = 200 μm. (B) High-magnification picture from the section shown in (A) (left panel) showing the presence of retrogradely-labeled neurons (red) within the contralateral PreS L2. Scale bar = 50 μm. (C) Distribution of retrogradely-labeled neurons across the PreS layers following injection of CTB in the contralateral PreS (n = 3 experiments). Error bars indicate SEM. (D) Representative injection site for the experiment shown in (A–B). Red, CTB; green, calbindin (Cb). Scale bar = 200 μm. (E) same as in (A) but for CTB injection in the contralateral RS29. Note the presence of retrogradely-labelled neurons within PreS L2 (see also panel F below) and the denser labeling within RS29. Scale bar = 200 μm. (F) High-magnification picture from the section shown in (E). Scale bar = 50 μm. (G) same as in (C) but for CTB injection in the contralateral RS29 (n = 4 experiments). Error bars indicate SEM. (H) Representative injection site for the experiment shown in (E–F). Red, CTB; green, calbindin (Cb). Scale bar = 200 μm. PreS, presubiculum; RS29, retrosplenial cortex area 29; WM, white matter; Sub, subiculum; PaS, parasubiculum. DOI: http://dx.doi.org/10.7554/eLife.14592.005
Figure 2.
Figure 2.. HD tuning of PreS neurons.
(A) Histogram showing the distribution of HD Indices for all PreS neurons which met the HD criteria (n = 186; see Materials and methods). The median HD index is indicated and shown by the red line. Three recordings from putative FS INs contributed weakly-directional responses (blue; see also Figure 2—figure supplement 1). (B) Polar plots showing firing rate as a function of HD for the neuron with the highest HD index (top) and a representative FS IN (bottom; see also Figure 2—figure supplement 1). For the cell shown on the top panel, all spikes (n = 22) were fired within a narrow HD angle (~10 degrees). HD indices and peak firing rates are indicated. (C) Color-coded distribution of preferred direction for all HD cells (n = 186). Each row represents the firing rate of a single neuron (normalized relative to its peak firing rate; red), ordered by the location of their peak firing rates relative to the rat's HD. (D) Spike-trajectory plot for a HD cell, sequentially recorded during passive rotation (‘head-fixed’, HF) and free-behavior (‘freely-moving’, FM). The circular trajectory of the rat’s head during passive rotation is indicated in black, while the rat’s trajectory during free behavior in gray. Spikes fired during head-fixation and free-behavior are indicated as blue and red dots, respectively. (E) Superimposed spike waveforms (top), polar plots showing firing rate as a function of HD (middle) and linear velocities (bottom) computed from the passive rotation (left) and freely-moving session (right) for the recording shown in (E). Note the stability of the spike-shape and the similar HD tuning between the head-fixed and freely-moving session (the Pearson’s correlation coefficient, p value and peak firing rates are indicated). DOI: http://dx.doi.org/10.7554/eLife.14592.007
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Activity of identified and putative fast-spiking interneurons during passive rotation.
(A) Left, scatter-plot showing the distribution of spike-widths (assessed by ‘peak-to-trough’ times) as a function of average firing rates during passive rotation for all active neurons (n = 301). Red dots indicated identified principal neurons (PCs; n = 44), blue dots indicate identified interneurons (INs; n = 6) and grey dots indicate non-identified recordings (n = 251). The dotted lines indicate the thresholds used for classification of FS INs (n = 20). Right, representative average spike waveforms of a PC and a FS IN. Double-arrowheads indicate the peak-to-trough times. Note the narrower waveform of the FS IN compared to the PC. (B) Polar plots showing firing rate as a function of HD for the neurons indicated in (A). Neurons 1–3 were identified and classified as ‘regular-spiking’ INs based on their broad spike waveforms (see A). Neurons 4–6 were classified as putative FS INs (see A) and met the HD classification criteria. Peak firing rates and p values for HD tuning are indicated. (C) Average firing rates of the identified and putative FS INs (n = 20) during rest and passive rotation. P value is indicated (Mann-Whitney U test). (D) Theta-indices for the identified and putative FS INs (n = 20). Red line indicates the median. Note the large majority of neurons displayed weak or no theta-rhythmicity (theta index <5; as in Boccara et al., 2010; Tukker et al., 2015). (E) A morphologically and cytochemically identified theta-rhythmic FS IN (‘theta cell’). Left panel, high-magnification fluorescence micrograph of the labeled neuron (Nb, Neurobiotin), positive for PV expression (arrowhead). Right, representative spike-trace (top) and spike autocorrelogram (bottom) for the neuron shown on the left. (F) A representative recording from a non-theta-rhythmic FS IN. The narrow spike waveform (left), representative spike-trace and spike autocorrelogram (right panels) are shown. DOI: http://dx.doi.org/10.7554/eLife.14592.008
Figure 3.
Figure 3.. Identified HD cells in PreS layer 3.
(A) Morphological reconstruction of a representative layer 3 pyramidal HD cell (dendrites, red; axon, blue). Scale bar: 100 µm. (B) Angular HD (top) and angular speed (bottom) as a function of time. Spikes (red dots) are indicated. Note the sharp tuning to HD. (C) Polar plots showing firing rate as a function of HD for the neuron in (A). Peak firing rate is indicated. (D) Polar plots showing firing rate as a function of HD computed or the two halves of the recording session for the neuron in (A). The Pearson’s correlation coefficient between the two HD tuning curves and peak firing rates are indicated. (E–H) same as A–D but for another neuron. Scale bar: 100 µm. DOI: http://dx.doi.org/10.7554/eLife.14592.009
Figure 4.
Figure 4.. Long-range axonal projections of identified PreS HD cells to MEC.
(A) Polar plots showing firing rate as a function of HD for the two neurons shown in (B). (B) Morphological reconstruction of two representative layer 3 pyramidal HD cell (dendrites, black; axons, red and blue) which send long-range axonal projections to MEC. Grey lines indicate the outline of the sections relative to the PreS (~3 mm lateral from midline) while axons are aligned to the target area (~4 mm lateral from midline). WM, white matter. Asterisk indicates the rostrally-travelling axonal branch. Scale bar: 200 µm. (C) High-magnification micrograph of a DAB stained axon form an identified PreS HD cell, showing branching upon entry in MEC L3. Note the axonal varicosities present in MEC L3 (bottom) and L1 (arrowheads, top). Scale bars, 20 μm (bottom) and 5 μm (top). (D) Morphological reconstruction of long-range axonal projections from identified PreS HD cells (n = 8 axons from 8 neurons; blue) which were traced until the superficial layers of MEC. Scale bar: 200 µm. DOI: http://dx.doi.org/10.7554/eLife.14592.010
Figure 5.
Figure 5.. Non-directional spiking patterns of identified L2 PreS neurons.
(A) Left, morphological reconstruction of a representative calbindin-positive layer 2 neuron (dendrites, red; axon, blue) recorded during passive rotation. Scale bar: 100 µm. Right, close-up magnifications of the cell’s soma (red, top panel) positive for calbindin immunoreactivity (green, middle panel) and overlay (bottom panel). Scale bar: 20 µm. (B) Angular HD (top) and angular speed (bottom) as a function of time. Spikes (red dots) are indicated. (C) Polar plots showing firing rate as a function of HD for the neuron in (A). Peak firing rate is indicated. (D–F) same as A–C but for a representative -negative L2 neuron. Scale bars in D: 100 µm (left) and 10 µm (right). DOI: http://dx.doi.org/10.7554/eLife.14592.011
Figure 6.
Figure 6.. Morphological and electrophysiological properties of L2 and L3 PreS neurons.
(A) Morphological reconstruction of a representative L2 (blue, left) and L3 (black, right) neuron, recorded during passive rotation. Scale bar = 50 μm. (B) Representative high-magnification pictures of a dendritic segment of a L2 (top) and L3 (bottom) neuron. Note the presence of spines in high density in both dendrites. Scale bars = 10 μm. (C) HD indices for all identified L2 (n = 11) and L3 neurons (n = 22). Three L3 neurons were silent, and hence not included in the analysis. Horizontal red lines represent medians and the p value is indicated (Mann-Whitney U test). (D) Representative spike-autocorrelogram for an identified L2 (top) and L3 neuron (bottom). Note the theta-rhythmicity of spiking for the L2 neuron. (E) Theta indices for all identified L2 (n = 10) and L3 neurons (n = 22) which met inclusion criteria for the theta analysis (see Materials and methods). Horizontal red lines represent medians and the p value is indicated (Mann-Whitney U test). (F) Average spike waveforms for L2 (blue, n = 11) and L3 (black, n = 22) neurons. Three L3 neurons were not included in the analysis since they were silent. Horizontal and vertical double-arrowheads indicate spike half-widths and spike negativity amplitudes, respectively. Scale bar = 1 ms. (G) Spike half-widths (left) and spike negativity amplitudes (right) for L2 (n = 11) and L3 (n = 22) neurons. Horizontal red lines represent medians and the p value is indicated (Mann-Whitney U test). DOI: http://dx.doi.org/10.7554/eLife.14592.012
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. Schematic representation of structure-function relationships within the superficial layers of PreS.
Schematic diagram showing the main principal cell types within PreS L2 and L3 (L2 calbindin-positive and calbindin-negative neurons, L3 pyramidal neurons), their corresponding long-range projection targets (MEC, contralateral Pres and contralateral RS29) and electrophysiological properties (e.g. HD versus non-HD modulated firing). Average spike waveforms and representative polar plots showing spiking activity as a function of HD for L2 and L3 neurons are also indicated. DOI: http://dx.doi.org/10.7554/eLife.14592.014
Author response image 1.
Author response image 1.. Laminar organization of projections from the PreS to MEC.
(A) Parasagittal sectionthrough MEC showing anterogradely-labeled axons following injectionof the anterogradetracer BDA -10k (red) in the ipsilateral PreS. Entorhinal layers are outlined via calbindin staining (Cb, green). Note the massive arborization of PreS afferents within L3 of the MEC. (B and C) show high-magnifications viewof the section shown in A. DOI: http://dx.doi.org/10.7554/eLife.14592.015
Author response image 2.
Author response image 2.. Dendritic morphologies of L2 neurons.
(A) Reconstructed dendritic morphologies of the L2 neurons whichdisplayed theta-rhythmic spike discharges (theta- indices ≥ 5; see Figure 6E in the revised manuscript). (B) Reconstructed dendritic morphologies of the L2 neurons, whose spiking activitywas not rhythmically entrained by theta oscillations (theta-indices ≤ 5; see Figure 6E in the revised manuscript). (C) Total dendritic lengths (left bar graph) and dendritic complexity index (calculated as in Pillai et al., 2012; right bar graph) for theta-rhythmic (‘high-theta’) and non-theta -rhythmic neurons (‘low theta’) shown in A and B, respectively. Error bars represent SD. These differences were not statistically significant. DOI: http://dx.doi.org/10.7554/eLife.14592.016
Author response image 3.
Author response image 3.. Representative electrolytic lesions and ‘recording site’, which aided identification of PreS L2 in a subset of preliminary experiments.
(A) Parasagittal section trough PreS showing the reconstructed electrode track (dotted line) and a large electrolyticlesion (dotted circle) centered on PreS L2. Green, calbindin staining. (B) High-magnifications view of the electrolytic lesion shown in A. (C) High-magnification example of another electrolytic lesion (dottedcircle and asterisk) recovered at the expecteddistance from the recording site (end of the electrode track, indicate by the arrowhead). (D) Parasagittal section trough PreS stained for Neurobiotin (red) and calbindin (green), showing a representative recording site’ within L. Here, cell identification by juxtacellular labeling failed; however, cell debris and small portions of dendrites (rig t panels; arrowheads) could be observed at the labeling site within PreS L2. DOI: http://dx.doi.org/10.7554/eLife.14592.017

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References

    1. Abbasi S, Kumar SS. Electrophysiological and morphological characterization of cells in superficial layers of rat presubiculum. Journal of Comparative Neurology. 2013;521:3116–3132. doi: 10.1002/cne.23365. - DOI - PubMed
    1. Ascoli GA, Alonso-Nanclares L, Anderson SA, Barrionuevo G, Benavides-Piccione R, Burkhalter A, Buzsáki G, Cauli B, Defelipe J, Fairén A, Feldmeyer D, Fishell G, Fregnac Y, Freund TF, Gardner D, Gardner EP, Goldberg JH, Helmstaedter M, Hestrin S, Karube F, Kisvárday ZF, Lambolez B, Lewis DA, Marin O, Markram H, Muñoz A, Packer A, Petersen CC, Rockland KS, Rossier J, Rudy B, Somogyi P, Staiger JF, Tamas G, Thomson AM, Toledo-Rodriguez M, Wang Y, West DC, Yuste R, Petilla Interneuron Nomenclature Group Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nature Reviews Neuroscience. 2008;9:557–568. doi: 10.1038/nrn2402. - DOI - PMC - PubMed
    1. Beed P, Gundlfinger A, Schneiderbauer S, Song J, Böhm C, Burgalossi A, Brecht M, Vida I, Schmitz D. Inhibitory gradient along the dorsoventral axis in the medial entorhinal cortex. Neuron. 2013;79:1197–1207. doi: 10.1016/j.neuron.2013.06.038. - DOI - PubMed
    1. Blackstad TW. Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination. Journal of Comparative Neurology. 1956;105:417–537. doi: 10.1002/cne.901050305. - DOI - PubMed
    1. Blair HT, Sharp PE. Anticipatory head direction signals in anterior thalamus: evidence for a thalamocortical circuit that integrates angular head motion to compute head direction. Journal of Neuroscience. 1995;15:6260–6270. - PMC - PubMed

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