Layer 6 corticothalamic neurons activate a cortical output layer, layer 5a

Abstract

Layer 6 corticothalamic neurons are thought to modulate incoming sensory information via their intracortical axons targeting the major thalamorecipient layer of the neocortex, layer 4, and via their long-range feedback projections to primary sensory thalamic nuclei. However, anatomical reconstructions of individual layer 6 corticothalamic (L6 CT) neurons include examples with axonal processes ramifying within layer 5, and the relative input of the overall population of L6 CT neurons to layers 4 and 5 is not well understood. We compared the synaptic impact of L6 CT cells on neurons in layers 4 and 5. We found that the axons of L6 CT neurons densely ramified within layer 5a in both visual and somatosensory cortices of the mouse. Optogenetic activation of corticothalamic neurons generated large EPSPs in pyramidal neurons in layer 5a. In contrast, excitatory neurons in layer 4 exhibited weak excitation or disynaptic inhibition. Fast-spiking parvalbumin-positive cells in both layer 5a and layer 4 were also strongly activated by L6 CT neurons. The overall effect of L6 CT activation was to suppress layer 4 while eliciting action potentials in layer 5a pyramidal neurons. Together, our data indicate that L6 CT neurons strongly activate an output layer of the cortex.

Keywords: corticothalamic; inhibitory interneurons; layer 4; layer 5; somatosensory cortex; visual cortex.

Figure 1.

Cre expressing neurons in the cortex of Ntsr1-Cre mice are L6 CT neurons. A, F, Experimental configuration. Neuronal tracers were injected into the LGN (A, B) or VPM (F, G) to retrogradely label L6 CT neurons in visual cortex (C, D) or somatosensory cortex (H, I) of Ntsr1-Cre mice crossed with a tdTomato reporter line. Images of the injection sites (B, G; cortex, Ctx). Low (C, H) and high-magnification (D, I) views of Ntsr1+ neurons (red) and retrogradely labeled L6 CT neurons (green) in visual cortex (C, D) and somatosensory cortex (H, I). E, J, Summary data showing the overlap between Ntsr1+ neurons and neurons retrogradely labeled from the LGN (E; n = 3 mice) or VPM (J; n = 3 mice). In visual cortex, 82.6 ± 3.8% of Ntsr1+ cells were retrogradely labeled, and 95.0 ± 2.1% of retrogradely labeled L6 CT neurons were Ntsr1+. In the somatosensory cortex, 91.0 ± 2.0% of Ntsr1+ cells were retrogradely labeled, and 92.7 ± 0.9% of retrogradely labeled L6 CT neurons were Ntsr1+. Scale bars: (in B, G) 500 μm; (all other images) 100 μm.

Figure 2.

The axonal processes of L6 CT neurons primarily ramify in L5a. Low (A, E) and high-magnification (B, F) views of thalamocortical axons (green) and L6 CT neurons (red) in visual cortex (A, B) and somatosensory cortex (E, F). A normalized intensity plot centered over L4 and L5a for the red and green channels averaged along the horizontal axis is shown (B, F, right). C, L6 CT neurons (red) and retrogradely labeled L5b corticotectal neurons (green) in visual cortex. G, IR-DIC (left) and epifluorescent (right) views of the same slice of barrel cortex in an Ntsr1-Cre mouse crossed with a ChR2-YFP reporter line. The axons of L6 CT neurons in visual cortex (D; n = 4 mice) and barrel cortex (H; n = 9 mice) revealed using a synaptophysin-tdTomato reporter line. Low- (I) and high-magnification (J) images of cytochrome C oxidase-stained barrel cortex and the axonal processes of L6 CT neurons identified by crossing Ntsr1-Cre mice with a synaptophysin-tdTomato reporter line (n = 3 mice). Note that the intracortical axonal band of L6 CT neurons is located below the barrels. Scale bars: (in I) 300 μm; (in all other images) 100 μm.

Figure 3.

The synaptic impact of L6 CT neurons is markedly greater in L5a than in L4. A, Experimental configuration. B, Two examples of paired recordings composed of one L5a pyramidal neuron and one L4 excitatory cell. Inset, Onset of the first responses. C, Summary data of the amplitudes of the first response for pairs of L4 and L5a excitatory cells in visual cortex and barrel cortex. D, Single examples of responses before and during bath application of TTX (left), and summary data (right; L5a: n = 4 cells; disynaptic inhibition in L4: n = 4 cells). E, Single examples of responses before, during, and after bath application of glutamate receptor antagonists (left) and summary data (right; L5a: n = 7 cells, disynaptic inhibition in L4: n = 6 cells). F, Single example (left) and summary data (right) comparing the latency of the response in recorded pairs of L5a and L4 excitatory neurons showing monosynaptic responses. G, Single example (left) and summary data (right) comparing the latency of the response in recorded pairs of L5a and L4 excitatory neurons in which the L4 neuron exhibited a disynaptic inhibitory response.

Figure 4.

Activation of L6 CT neurons evokes action potentials in L5a pyramidal neurons. A, Experimental configuration. B, Example response from a paired recording of a L5a pyramid and a L4 excitatory cell. C, Average number of action potentials per photostimulation elicited in each pair of L5a and L4 neurons (L5a: 0.81 ± 0.16, L4: no action potential elicited, n = 13 pairs, p = 0.0002). D, The average probability of firing an action potential is shown for L4 and L5a neurons for each photostimulation in the train (n = 13 pairs).

Figure 5.

Activation of L6 CT neurons selectively stimulates fast-spiking inhibitory interneurons. Experimental configuration (A, E) and sample recordings (B, F) from an excitatory neuron and an inhibitory FS interneuron in L5a (A–D) and L4 (E–H). Amplitudes of the first response for L5a pairs (C; n = 30 pairs, p < 0.0001) and L4 pairs (G; n = 23 pairs, p < 0.0001). Experimental configuration (I), sample recording (J) and summary data (K; n = 8 pairs, p = 0.0112) from an excitatory pyramidal neuron and an inhibitory somatostatin-expressing GIN interneuron in L5a. Summary data of the short-term synaptic plasticity measured from L5a and L4 excitatory and inhibitory neurons following L6 CT photostimulation (D, H, L). The data for L5a pyramidal neurons in L is replotted from D.

Figure 6.

Activation of L6 CT neurons evokes action potentials in L5a pyramidal neurons and L5a FS inhibitory interneurons. A, Experimental configuration. B, Example responses from a paired recording of a L5a pyramid and a L5a FS cell. C, Average number of action potentials per photostimulation elicited in each pair of L5a pyramid and L5a FS cell (L5a pyramids: 0.57 ± 0.19; L5a FS cells: 0.63 ± 0.31, n = 8 pairs, p = 0.8105).

Figure 7.

Schematic illustrating the proposed circuit organization. L6 CT neurons provide strong input to L5a pyramidal neurons, as well as FS cells in L4 and L5. Somatostatin-expressing inhibitory interneurons and L4 excitatory neurons receive weak L6 CT input. The net effect of this synaptic organization is that L6 CT activation evokes action potentials in L5a pyramidal neurons while suppressing L4 excitatory cells.