Three Types of Cortical Layer 5 Neurons That Differ in Brain-wide Connectivity and Function

Abstract

Cortical layer 5 (L5) pyramidal neurons integrate inputs from many sources and distribute outputs to cortical and subcortical structures. Previous studies demonstrate two L5 pyramid types: cortico-cortical (CC) and cortico-subcortical (CS). We characterize connectivity and function of these cell types in mouse primary visual cortex and reveal a new subtype. Unlike previously described L5 CC and CS neurons, this new subtype does not project to striatum [cortico-cortical, non-striatal (CC-NS)] and has distinct morphology, physiology, and visual responses. Monosynaptic rabies tracing reveals that CC neurons preferentially receive input from higher visual areas, while CS neurons receive more input from structures implicated in top-down modulation of brain states. CS neurons are also more direction-selective and prefer faster stimuli than CC neurons. These differences suggest distinct roles as specialized output channels, with CS neurons integrating information and generating responses more relevant to movement control and CC neurons being more important in visual perception.

Figure 1. Three Distinct Classes of Layer 5 Pyramidal Neurons Defined by BAC Cre Transgenic Lines Exhibit Distinct Long Distance Axonal Projection Patterns

(A) Coronal sections showing eGFP labeled neurons after AAV-FLEX-eGFP (or tdTomato) injection into the primary visual cortex of three BAC transgenic Cre mice, Tlx3-Cre PL56, Glt25d2-Cre NF107, Efr3a-Cre NO108 or C57BL/6 mouse injected with Cav2-Cre virus into the superior colliculus (SC). Left and right panels for each condition correspond to photographs of the same fields of view but with the panels to the left imaged at lower brightness to illustrate dendritic morphology and cell body locations and right panels at higher brightness to better reveal axonal projections. (B) Axonal projections of eGFP⁺ labeled V1 Cre⁺ neurons to V2L, contralateral V1, ipsilateral striatum (i-str), thalamic nuclei LP and dLGN, superior colliculus (SC), and pons. Inset in i-str panel of Tlx3-Cre: contralateral striatum. Insets in i-str panels of Glt25d2-Cre and Cav2-Cre to SC: magnified images. '+' or '−' indicates presence or absence of GFP⁺ axons in each panel. (C) Partial reconstructions of L5 Efr3a-Cre⁺ neurons using Neurolucida. Dendrites: black; axons: red. (D) Schematic representation illustrating three different Cre⁺ subpopulations of layer 5 neurons projecting their axons to different structures. Abbreviations: CC, cortico-cortical; CC-NS, cortico-cortical non-striatal; CS, cortico-subcortical; dLGN, dorsal lateral geniculate nucleus; i-str, ipsilateral striatum; L5, layer 5; LP, lateral posterior thalamic nucleus; SC, superior colliculus; V1, primary visual cortex; V2L, secondary visual cortex, lateral area. Scale bars = 200 μm (A), 100 μm (B, C).

Figure 2. Morphological and Electrophysiological Properties of Three Types of Layer 5 Pyramidal Neurons

(A) Confocal images displaying soma and proximal dendrites of eGFP⁺ Tlx3-Cre⁺, Glt25d2-Cre⁺ and L5 Efr3a-Cre⁺ neurons. (B) Dendritic and cell body morphologies of Tlx3-Cre⁺, Glt25d2-Cre⁺ and L5 Efr3a-Cre⁺ neurons differ in cell soma size (μm²), apical dendrite diameter (μm) at the base, and height over width (H/W) ratio. (C) Examples of action potential trains evoked by 200 pA current injections into Tlx3-Cre⁺, Glt25d2-Cre⁺, and L5 Efr3a-Cre⁺ neurons. (D) Intrinsic electrical property differences in (left) percent sag (%), (middle) input resistance (MΩ) and (right) interspike interval at initial over interspike interval at steady state phase of Tlx3-Cre⁺, Glt25d2-Cre⁺ and L5 Efr3a-Cre⁺ neurons. Values are reported as means ± SEM for each class of neurons. Statistics were calculated from one-way ANOVAs followed by Tukey’s post-hoc tests to compare means of pairs of each L5 class. Significant differences between pairs are indicated by the P value. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001. Scale bar = 10 μm (A).

Figure 3. Monosynaptic Rabies Virus Tracing of Inputs to Layer 5 Pyramidal Neurons

(A) Schematic illustrating virus injection schemes for monosynaptic rabies virus tracing. (B) Schematic illustrating labeling of various neuronal populations following rabies virus-mediated monosynaptic input labeling. (C) Coronal sections of visual cortices of Tlx3-Cre, Glt25d2-Cre, Efr3a-Cre mice showing GFP-TVA⁺, dsRed⁺ starter neuron locations. Scale bar = 100 μm (C).

Figure 4. Brain-Wide Monosynaptic Input to Layer 5 Pyramidal Neurons Revealed by Monosynaptic Rabies Virus Tracing

(A-L) Coronal sections of a rabies virus labeling in Tlx3-Cre⁺ mice showing GFP-TVA⁺, dsRed⁺ starter neurons and local inputs in V1 (A) and long-distance inputs from V2L and AuD (A), V2MM, V2ML, RSA, and RSG (B), Au1 (C), M2 (D), TeA (E), Cl (F), VDB (G), LD (H), LP (I), dLGN (J), APTD (K) and PrS (L). (M) Coronal section diagrams along the anterior-posterior axis illustrating locations of anatomical regions shown in A-L. Note that the diagrams were chosen for illustration purposes and do not necessarily correspond exactly to figures. (N) Summary of long-range monosynaptic inputs onto Tlx3-Cre⁺, Glt25d2-Cre⁺, Efr3a-Cre⁺ neurons in the primary visual cortex. Statistics were calculated from two-way ANOVAs with Tukey’s post-hoc tests. Significant differences between pairs are indicated by the P value. **p < 0.01 and ****p<0.0001. Abbreviations are: APTD, anterior pretectal nucleus, dorsal part; Au1, primary auditory cortex; AuD, secondary auditory cortex, dorsal area; BF, basal forebrain; Cl, claustrum; dLGN, dorsal lateral geniculate nucleus; LD, laterodorsal thalamic nucleus; LP, lateral posterior thalamic nucleus; M2, secondary motor cortex; PrS, presubiculum; RSA, retrosplenial agranular cortex; RSG, retrosplenial granular cortex; V1, primary visual cortex; VDB, nucleus of the vertical limb of the diagonal band; V2L, secondary visual cortex, lateral; V2ML, secondary visual cortex, mediolateral area; V2MM, secondary visual cortex, mediomedial area; TeA, temporal association cortex. Scale bars = 500 μm (A), 200 μm (B-L).

Figure 5. Laminar Distributions Long-Range Cortical Inputs onto Layer 5 Pyramidal Neurons

(A) An example coronal section showing laminar distribution of dsRed⁺ rabies traced input neurons in V2ML onto Tlx3-Cre⁺ V1 neurons. Arrowheads indicate dsRed⁺ presynaptic neurons in L4. (A, right panel) Schematic illustrating relative proportions of input neurons from each layer as thickness of arrows for Tlx3-Cre⁺, Glt25d2-Cre⁺ and Efr3a-Cre⁺ mice. (B-E) Laminar distributions of long-range cortical inputs onto Tlx3-Cre⁺, Glt25d2-Cre⁺, Efr3a-Cre⁺ V1 neurons as proportions of total cortical inputs (%) for all cortical areas (B), visual cortices except V1 (C), other sensory cortices (D) and retrosplenial and cingulate cortices (E). Abbreviations are V1, primary visual cortex; V2ML, secondary visual cortex, mediolateral area. Scale bar = 100 μm (A).

Figure 6. Visual Responses of Layer 5 Pyramidal Neurons Assayed with Two-Photon Calcium Imaging

(A) Schematic illustration of two-photon in vivo calcium imaging set up for awake and head-fixed stationary mouse. (B) Two-photon microscope z-stack projection of Glt25d2-Cre⁺ mouse V1 after AAV-FLEX-GCaMP6 and AAV-FLEX-tdTomato injection. (C) Representative images of single cells expressing GCaMP6 in V1 of Tlx3-Cre⁺, Glt25d2-Cre⁺, Efr3a-Cre⁺ mice. Arrowheads indicate cells plotted in (D) and (E). Scale bar = 100 μm. (D) Spatial Frequency (SF) Experiments. Top: Medians for preferred SF, OSI and DSI (with interquartile ranges for OSI and DSI, at the preferred SF) for Tlx3-Cre⁺, Glt25d2-Cre⁺, L5 Efr3a-Cre⁺ neurons, as well as the percentage of cells with OSI or DSI > 0.5. Bottom left: Distributions of preferred SF, OSI and DSI (at the preferred SF) for Tlx3-Cre⁺, Glt25d2-Cre⁺, and L5 Efr3a-Cre⁺ neurons. Bottom right: SF and orientation tuning curve examples for each cell type. Values plotted as means ± SEM. Gray lines indicate average responses during blank stimulus; shading is ± SEM. (E) Temporal Frequency (TF) Experiments. Top: Medians with interquartile ranges for preferred TF, OSI and DSI (at the preferred TF) for Tlx3-Cre⁺ and Glt25d2-Cre⁺ neurons, as well as the percentage of cells with OSI or DSI > 0.5. Bottom left: Distributions of preferred TF, OSI and DSI (at the preferred TF) for Tlx3-Cre⁺ and Glt25d2-Cre⁺ neurons. Bottom right: Temporal frequency (TF) and orientation tuning curve examples for each cell type. Values plotted as means ± SEM. Gray lines indicate average responses during blank stimulus; shading is ± SEM. For median plots, statistical significances are labeled as p values after Wilcoxon rank-sum test (TF experiments) or Kruskal-Wallis test with Dunn's multiple comparisons test as post-hoc (SF experiments). *p < 0.05 and **p < 0.01. Abbreviations: c/d, cycle per degree; Hz, Hertz.

Figure 7. Connectivity and Function of Three Types of L5 Pyramidal Neurons

Three genetically-identified populations of layer 5 pyramidal neurons in V1 of Tlx3-Cre⁺, Glt25d2-Cre⁺, and Efr3a-Cre⁺ mice were characterized based on monosynaptic rabies tracing of brain-wide inputs (left) and their morphologies, axonal projections, intrinsic electrophysiology and in vivo responses to drifting gratings (right). (Left) Tlx3-Cre⁺ and Efr3a-Cre⁺ V1 neurons receive preferential long-range inputs from the extrastriate visual areas (orange/green), whereas Glt25d2-Cre⁺ V1 neurons receive preferential inputs from the retrosplenial cortex, the basal forebrain, and dLGN (purple). (Right) CC Tlx3-Cre⁺, CS Glt25d2-Cre⁺, CC-NS Efr3a-Cre⁺ V1 L5 pyramidal neurons exhibit distinct axonal projections, cell morphology, electrical properties and visual responses. Abbreviations: BF, basal forebrain; CC, cortico-cortical; CC-NS, cortico-cortical non-striatal; CS, cortico-subcortical; dLGN, dorsal lateral geniculate nucleus; RSC/CC, retrosplenial and cingulate cortex; VC, visual cortex.