Translaminar inhibitory cells recruited by layer 6 corticothalamic neurons suppress visual cortex

Abstract

In layer 6 (L6), a principal output layer of the mammalian cerebral cortex, a population of excitatory neurons defined by the NTSR1-Cre mouse line inhibit cortical responses to visual stimuli. Here we show that of the two major types of excitatory neurons existing in L6, the NTSR1-Cre line selectively targets those whose axons innervate both cortex and thalamus and not those whose axons remain within the cortex. These corticothalamic neurons mediate widespread inhibition across all cortical layers by recruiting fast-spiking inhibitory neurons whose cell body resides in deep cortical layers yet whose axons arborize throughout all layers. This study reveals a circuit by which L6 modulates cortical activity and identifies an inhibitory neuron able to regulate the strength of cortical responses throughout cortical depth.

Figure 1. Photostimulation of NTSR1-Cre neurons suppresses thalamus independent cortical activity in vivo

(A) Illustration of in vivo recording configuration from V1 in an adult NTSR1-Cre mouse conditionally expressing ChR2. The ChR2-expressing layer 6 pyramidal cell (L6PC; red triangle) was photoactivated while recording from a L2/3 neuron (grey triangle). (B) Left traces: Response of a L2/3 neuron recorded in vivo in the whole-cell current clamp configuration (scale bar 200pA 20mV/250ms) to current injection (150pA; top) and to current injection with photoactivation of L6PCs (blue bar 0.5s). Right: The average firing rate is plotted against time (black: control; blue: with photostimulation; asterisks indicate significant difference; p=0.0002, 0.0002, 0.0074; n=10 cells, 6 mice; blue bar: duration of photostimulation). (C) IPSC recorded in vivo in a L2/3 neuron voltage clamped at +7mV (scale bar 200pA/250ms) in response to photoactivation of L6PCs (blue bar 1.5s). 5 superimposed sweeps (blue). Average trace in black. See also Figure S1. (D) Inhibition could be mediated by the activation of either cortico-thalamic or intracortical L6PCs.

Figure 2. The NTSR1-Cre line selectively targets layer 6 cortico-thalamic pyramidal cells

(A) Top left: Schematic of thalamic injection of fluorescent microspheres into adult NTSR1-Cre X tdTomato reporter mouse in vivo. Green fluorescent microspheres are retrogradely transported to the soma of cortico-thalamic neurons. Bottom left: Confocal image illustrating the thalamic injection site on a coronal section of the brain (Red: tdTomato; Green: fluorescent microspheres; Yellow: superimposition of Red and Green; scale bar 500μm). (B) Left: confocal image illustrating a coronal section through V1. Note the accumulation of microspheres in L6 (Yellow fluorescence; scale bar 100μm). Right: Magnification of area delineated by white square to left. Top right: Red channel: confocal image of L6PCs expressing tdTomato (NTSR1+; scale bar 50μm; the white and black arrows indicate cell bodies that express or do not express tdTomato, respectively). Right middle: microspheres. Right bottom: overlay of red tdTomato expression, green microspheres and blue nuclear counter stain (DAPI). Note that while all tdTomato expressing cell bodies contained microspheres, most cell bodies lacking tdTomato expression do not contain beads. (C) Summary histogram: Left: 154 of 154 tdTomato expressing cells (red column; 4 mice) contained microspheres while only 9 of 197 non-expressing cells contained microspheres (4 mice; p<0.0001). Right: 154 of 163 cells that contained microspheres expressed tdTomato (4 mice). (D) What inhibitory interneurons are being recruited by L6CTs to suppress the visual cortex?

Figure 3. Selective recruitment of deep layer fast spiking cells by layer 6 cortico-thalamic pyramidal cells in vivo

(A) Schematic illustrates in vivo extracellular recording from V1 in NTSR1-ChR2 anesthetized mouse during visual stimulation and photo-activation of L6 cortico-thalamic pyramidal cells (L6CTs). Histogram shows separation of fast spiking (FS) from regular spiking (RS) units based on trough to peak latency (102 units, 9 mice). Dotted line indicates the chosen divider for defining a unit as FS or RS. Bottom: 30 FS units (grey) and 30 RS units (pink) shown on bottom with representative example shown in bold (scale bar 0.5ms). (B) Peristimulus time histogram of the response of two example FS units to visual stimulation (black bar, 1.5s) with (blue) and without (black) photo-activation of L6CTs (blue bar, 0.5s). Note that while the top FS unit (i) is suppressed the lower one (ii) is facilitated by photo-activation of L6CTs. (C) Fold change in firing rate (LED on/LED off, log scale; black circles denotes FS units, red triangles RS units) in response to photoactivation of L6CTs during visual stimulation shown for all units in A. Note that the only units whose firing rate increases during photo-activation of L6CTs are FS units. Recording depth of individual FS units are shown in bottom panel against the log scale of their fold change. Example units from B are labeled next to their corresponding depths (red circles). Cross indicates outlier moved from 296 to 100 fold change. (D) In vivo unit recordings from awake mouse (n=146 RS units 92 FS units; 4 mice) presented as in C.

Figure 4. Selective recruitment of layer 6 fast spiking cells by layer 6 cortico thalamic pyramidal cells in vitro

(A) Left: Schematic illustration of loose patch recordings from GFP expressing neurons in V1 slice from a NTSR1-ChR2 x GAD67-GFP mouse. A single loose-patch recording was made on a GFP-positive inhibitory neuron in either L2/3, L4, L5 or L6 while photoactivating L6CTs. Center: Example recordings from GFP expressing neurons in each layer of one example slice during photo-activation of L6CTs (blue bar, 1.5s). Note that only neurons in deeper layers fire in response to photo-activation (Scale bars 50pA/500ms). Right: Summary histogram showing percentage of GFP expressing neurons recruited by photo-activation of L6CTs (L1 n=42, L2/3 n=45, L4 n=41, L5 n=54, L6 n=72; 7 mice). See also Figures S2 and S3. (B) Left: Waveforms of action potentials (average of first 5 spikes; recorded in loose patch) of all responding GFP expressing neurons (GAD67(+); gray) and of directly photo-activated L6CTs (NTSR1(+); red, for comparison; scale bar 0.5ms). Bold lines are averages. Middle: Peak-to-trough height ratio is plotted against trough-to-peak latency for GFP+ cells (green circles) and NTSR1+ cells (red triangles). (C) Do L6 FS cells extend their axons throughout layers to inhibit also superficial neurons?

Figure 5. Translaminar axonal projections from fast spiking cells recruited by layer 6 cortico-thalamic pyramidal cells

(A) Morphological reconstructions of 11 FS cells with translaminar axonal arborization that were recruited above threshold for spike generation upon photostimulation of L6CTs in vitro. Nine of the eleven translaminar FS cells were recorded in the GAD67-GFP line and expressed GFP. The remaining two FS cells (top row, 1^st cell; Bottom row, 2^nd cell) were recorded in the G42 line and also expressed GFP. Dendrites and somas are shown in black with axons in grey (scale bars 50μm, medial is to the right). Thin grey tics to right of each cell indicate layer boundaries. (B) Average heat map of axons (left) and dendrites (middle) of the eleven reconstructed translaminar FS cells after normalizing for differences in layer depths. Right panel shows overlay of axons (red) and dendrites (colored green; yellow where overlapping with axons). Left: shows the relative density of neurite length for each layer for dendrites (black) and axons (grey) of all 11 cells. The relative density is the fraction of total neurite length divided by the fractional layer thickness; the fractional layer thickness is computed as the thickness of a layer divided by the cortical thickness, measured along the radial axis from the pia to the layer 6 white matter border.

Figure 6. Electrophysiological properties of layer 6 translaminar FS cells

(A) Responses to steps of current injection are shown for L6CTs, locally-projecting L6 FS cells and translaminar FS cells. Translaminar FS cells (n=11) did not significantly differ from locally-projecting FS cells (n=16) with respect to firing rate adaptation, firing rate or afterhyperpolarization following an action potential (bottom traces), although they did significantly differ from regular spiking pyramidal cells (n=10) in all these characteristics (p=0.0124, p<0.0001 and p=0.0002, respectively; see methods for analysis parameters). (B) Left: dV/dt of action potentials recorded in current clamp in translaminar FS cells (black traces; n = 11), L6CTs (red races; n = 13) and locally projecting FS cells (green traces; n = 16). Traces from translaminar FS cells are superimposed with those of L6CTs (top) and from locally projecting FS cells (bottom) for comparison. Center: The peak to trough ratio (p/t ratio) is plotted against the trough to peak latency of the dV/dt waveform (inset illustrates parameters measured). Right: averages and statistical comparison to right. No statistically significant difference was noted between locally-projecting and translaminar FS cells. Translaminar FS cells did significantly differ from L6CTs in the peak-to-trough ratio (p<0.0001), and in the trough to peak latency (p=0.0002). (C) Firing rate of FS cells in response to L6CT photo-activation: Translaminar FS cells fired at significantly higher rates than locally projecting FS cells (p=0.0037).

Figure 7. Model

L6CTs suppress responses in the visual cortex by recruiting FS cells located in L6 some of which extend large translaminar axon throughout all cortical layers.