Illustration of the patient data on the samples utilized

(A) Illustration of the defined age groups.

(B) Number of patients involved in age groups, (n = 6, 7, 7, 12, 22, 22, 33 from infant to late adulthood, respectively). Dots show the number of human layer 2/3 pyramidal cells in our dataset regarding the defined age groups (n = 74, 33, 51, 63, 92, 66, 120 from infant to late adulthood, respectively).

(C) Distributions of patient ages within age groups.

(D) Brain model indicates the number of surgically removed tissues from the cortical lobes. Colors indicate age groups.

(E) The distribution of recovered cell bodies distance from the L1/2 border.

Subthreshold membrane properties vary across life stage

(A-D), Boxplots show resting membrane potential (A), input resistance (B), tau (C), and sag ratio (D) distributions in various age groups. (B) Inset shows representative voltage traces from each group. Scale bar: 5 mV; 20 ms. Asterisks indicate significance (Kruskal–Wallis test with post-hoc Dunn test, * P < 0.05, ** P < 0.01, *** P < 0.001).

Subthreshold membrane properties

Table showing the mean and standard deviation of the passive parameters across the age groups.

Age related differences in the action potential kinetics.

(A-D) Boxplots show differences in rheobase (A), action potential half-width (B), action potential up-stroke (C), and action potential amplitude (D) between the age groups. Asterisks indicate statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

(E) Representative action potentials aligned to threshold potential onset (scale: x axis: 1ms, y axis: 20 mV) (top) and phase plots of the representative APs (scale: x axis: 10 mV, y axis: 100 mV/ms) (bottom).

(F) Uniform Manifold Approximation and Projection (UMAP) of 32 (Table 4) (top) and 8 selected electrophysiological properties (resting Vm, input resistance, tau, sag ratio, rheobase, AP half-width, AP up-stroke and AP amplitude) (bottom) with data points for 331 cortical L2/3 pyramidal cells, colored with the corresponding age groups.

Action potential and firing pattern parameters

Table showing the mean and standard deviation of the suprathreshold properties across the age groups.

Morphological properties

Table showing the mean and standard deviation of the morphological characteristics across the age groups.

Examined electrophysiological properties

Age-dependency of the AP firing pattern parameters

(A) Representative membrane potential responses to an 800 ms long rheobase (middle) (left to right: infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood, late adulthood), and increased current steps (bottom) Colored respectively to the age groups. Scale bar top: 1ms, 100 pA, bottom: 1ms, 20mV.

(B-D) Boxplots show changes across the age groups in f-I slope (B), first AP latency (C), and adaptation of APs (D). Asterisks indicate statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

Morphological features of layer 2/3 pyramidal cells in different stages of life

(A) Representative reconstructions of L2/3 pyramidal cells (from left to right) from infant (n = 7), early childhood (n = 8), late childhood (n = 11), adolescence (n = 11), young adulthood (n = 9), middle adulthood (n = 9), and late adulthood (n = 8) patients.

(B-I) Boxplots show summarized data from all the reconstructed cells (Suppl. Fig. 7) of total dendritic length (B), apical dendritic length (C), total basal dendritic length (D), the total number of nodes on the apical and basal dendrites (E), the maximal horizontal (F), and the maximal vertical (G) extension of dendrites, the average length of the apical (H) and basal (I) terminal dendritic segments. Asterisks indicate statistical significance (* P < 0.05, ** P < 0.01, Kruskal-Wallis test with post-hoc Dunn test).

Comparison of dendritic spine densities in pyramidal cells from infant and late adulthood samples.

(A) Anatomical 3D reconstruction of human L2/3 pyramidal cells from the infant (left), and the late adulthood (right) age groups.The histogram (in the middle) demonstrates the distribution of dendritic spines on the two representative cells according to their distance from the soma (μm) on the apical and basal dendrites.

(B) Boxplots of the average spine densities on the apical (top), and basal (bottom) dendritic branches from the n = 3 infant (blue) and n = 3 late adulthood (red) L2/3 pyramidal cells. The symbols are color-coded by the 6 individual cells.

(C-G) The plots show the distribution of mushroom (C), thin (D), filopodium (E), branched (F), and stubby (G) dendritic spine types on the apical dendrites of the reconstructed infant (n = 3, blue) and late adult (n = 3, red) pyramidal cells. Top, schematic illustration and representative images of the examined dendritic spine types. Center, age dependent distribution of spine types. Asterisks indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

(H-L) Same as C-G but on basal dendrites.

Patient metadata

Table showing the patients age, gender, brain region (F - frontal, T - temporal, P - parietal, O - occipital, PO - parieto-occipital, FT - fronto-temporal, TO - temporo-occipital, FP - fronto-parietal), hemisphere and the surgical procedure.

Number of dendritic spines on the examined cells.

Table showing the overall number of dendritic spines, the total number of spines on the apical and basal dendritic arbour and the number of dendritic spines from different phenotypic subtypes of the individual pyramidal cells (n = 3 infant and n = 3 late adulthood).

Patient metadata

Distribution of patients by gender (female: infant n = 2, early childhood n = 4, late childhood n = 2, adolescence n = 7, young adulthood n = 13, middle adulthood n = 13, late adulthood n = 23, male: infant n= 4, early childhood n = 3, late childhood n = 5, adolescence n = 5, young adulthood n = 9, middle adulthood n = 9, late adulthood n = 10) (A), surgical procedure (tumor removal: infant n = 2, early childhood n = 2, late childhood n = 5, adolescence n = 7, young adulthood n = 13, middle adulthood n = 12, late adulthood n = 19, VP (ventriculoperitoneal) shunt: infant n= 4, early childhood n = 5, late childhood n = 1, adolescence n = 4, young adulthood n = 6, middle adulthood n = 9, late adulthood n = 12, other: infant n= 0, early childhood n = 0, late childhood n = 1, adolescence n = 1, young adulthood n = 3, middle adulthood n = 1, late adulthood n = 2) (B), hemisphere (left: infant n = 3, early childhood n = 3, late childhood n = 3, adolescence n = 2, young adulthood n = 8, middle adulthood n = 6, late adulthood n = 8, right: infant n= 3, early childhood n = 4, late childhood n = 4, adolescence n = 10, young adulthood n = 14, middle adulthood n = 16, late adulthood n = 25) (C) and brain area (frontal: infant n = 2, early childhood n = 2, late childhood n = 4, adolescence n = 5, young adulthood n = 11, middle adulthood n = 7, late adulthood n = 11, temporal: infant n = 2, early childhood n = 2, late childhood n = 2, adolescence n = 3, young adulthood n = 8, middle adulthood n = 7, late adulthood n = 11, parietal: infant n = 1, early childhood n = 3, late childhood n = 1, adolescence n = 2, young adulthood n = 2, middle adulthood n = 4, late adulthood n = 6, occipital: infant n = 1, early childhood n = 0, late childhood n = 0, adolescence n = 2, young adulthood n = 1, middle adulthood n = 4, late adulthood n = 5)(D). Stacked columns are colored regarding the age groups shown on the colorbar.

Distribution of subthreshold electrophysiological features with age.

(A-D) Scatter plots showing the passive electrophysiological characteristics: resting membrane potential (A), input resistance (B), tau (C), and sag ratio (D) throughout the lifespan. Dots are colored according to the age groups, age is represented in years on a logarithmic scale.

Electrophysiological differences during the first year of life

A-D, Boxplots showing differences in passive properties, resting membrane potential (A), input resistance (B), tau (C) and sag ratio (D) within the infant age group (* P < 0.05, ** P < 0.01, *** P < 0.001, two sample t-test (A, B, C) or Mann-Whitney test (D)).

E-H, Differences in the action potential kinetics, Rheobase (E), AP half-width (F), up-stroke (G), and AP amplitude (H) between the cells from the first and the second half of the first year of life. Asterisks indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney test).

I-K, Deviations in AP firing pattern parameters, F-I slope (H), first AP latency (I), and adaptation

(J) during infancy.

Distribution of suprathreshold electrophysiological features with age.

(A-D) Diagrams show changes through age in rheobase (A), action potential half-width (B), action potential up-stroke (C), and action potential amplitude (D). Dots are colored according to the age groups, age is represented in years on a logarithmic scale.

Relationship between patient metadata and electrophysiology.

Uniform Manifold Approximation and Projection (UMAP) of 8 electrophysiological properties (resting Vm, input resistance, tau, sag ratio, rheobase, AP half-width, AP up-stroke, and AP amplitude) with data points for 331 cortical L2/3 pyramidal cells, colored with the corresponding age groups (A), surgical procedures (B), brain area (C) and gender (D).

Distribution of firing pattern characteristics with age.

(A-C) Plots show F-I slope (A), first AP latency (B), and adaptation of APs (C) depending on age. Dots are colored according to the age groups, age is represented in years on a logarithmic scale.

Comparison of the subthreshold properties of the cells as a function of their distance from the layer border.

A-D: Box plots show resting membrane potential (A), input resistance (B), tau (C), and sag ratio

(D) of the pyramidal cells whose soma is located at a distance greater than and less than 200 μm from the L1 border. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, Mann-Whitney test).

Comparison of the action potential properties of the cells as a function of their distance from the layer border.

A-C : Boxplots show rheobase (A), action potential half-width (B), action potential upstroke (C) and action potential amplitude (D) of cells whose soma is located at a distance greater than and less than 200 μm from the L1 border. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood. Asterisks indicate significance (* P < 0.05, ** P < 0.01, Mann-Whitney test).

Comparison of the firing pattern characteristics of the cells as a function of their distance from the layer border.

A-C : Boxplots show: F-I slope (A), latency of the first AP (B) and adaptation of APs (C) of cells whose soma is located at a distance greater than and less than 200 μm from the L1 border.

From left to right: data from all ages, infancy, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood. Asterisks indicate significance (* P < 0.05, ** P < 0.01, Mann-Whitney test).

Human cortical L2/3 pyramidal cell dendritic reconstructions.

Reconstructions (n = 63) of the examined human cortical pyramidal cells, from top to bottom: infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood age groups. Black lines are representing the pia mater, gray dashed lines represent the L1-L2 border.

Morphological comparison of the examined infant cells.

A-H: Boxplots show morphological features: total (A), apical (B), total basal (C) dendritic length, total number of nodes (D), maximal horizontal (E), and vertical (F) extension, average apical (G) and basal (H) terminal dendritic segment from infant patient during the first and second half of the first year of life.

Distribution of morphological features with age

A-F: Scatterplots show the distribution of total (A), apical (B), total basal dendritic length (C), total number of bifurcations (D), maximum horizontal extension (E), maximum vertical extension of the dendritic arborisation (F). The age is represented in years on a logarithmic scale.

G-H: Scatterplots show the average apical (G) and basal (H) terminal segment length of the cells, dots indicate the mean segment length of cells, vertical lines show the standard deviation.

(A) Anatomical 3D reconstructions of the examined n = 3 human L2/3 pyramidal cells from the infant age group.

(B) Anatomical reconstructions (n = 3) from the late adulthood group.

(C) Boxplots showing the spine density on the six individual cells, the infant pyramidal cell shown with blue, the late adulthood pyramidal cell with red (cells are numbered according to figure A, B), on the apical (top) and the basal (bottom) dendrites. Asterisks indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.001, Kruskal-Wallis test with post-hoc Dunn test).

(D-H) The plots show the distribution of mushroom (D), thin (E), filopodium (F), branched (G), and stubby (H) dendritic spine types on the apical dendrites of the reconstructed infant (n = 3, blue) and late adult (n = 3, red) pyramidal cells. Top, schematic illustration and representative images representation of the examined dendritic spine types. Bottom, spine distributions on the individual cells that were examined. Asterisks indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.001, Kruskal-Wallis test with post-hoc Dunn test).

(I-M) Same as D-H but on basal dendrites.

Comparison of subthreshold electrophysiological properties of the examined cells from patients with tumor removal or VP shunt surgical procedures.

A-D : Box plots show resting membrane potential (A), input resistance (B), tau (C), and sag ratio

(D) from patients with tumor removal (tumor) or VP shunt (shunt) surgical procedures. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, Mann-Whitney test).

Comparison of action potential properties of the examined cells from patients with tumor removal or VP shunt surgical procedures.

A-D : Boxplots show rheobase (A), action potential half-width mean (B), action potential up-stroke (C) and action potential amplitude (D) from patients with tumor removal (tumor) or VP shunt (shunt) surgical procedures. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney test).

Comparison of firing pattern characteristics of the examined cells from patients with tumor removal or VP shunt surgical procedures.

A-C : Box plots showing: F-I slope (A), first AP latency (B), and adaptation of APs (C) from patients with tumor removal (tumor) or VP shunt (shunt) surgical procedures. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, ** P < 0.01, Mann-Whitney test).

Morphological comparison of the examined cells from patients with tumor removal or VP shunt surgical procedures.

A-H: Boxplots showing morphological features: total (A), apical (B), total basal (C), dendritic length, total number of nodes (D), maximal horizontal (E), and vertical (F) extension, average apical (G) and basal (H) terminal dendritic segment from patients with tumor removal (tumor) or VP shunt (shunt) surgical procedures. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, Mann-Whitney test).

Comparison of subthreshold electrophysiological properties of the examined cells from female and male patients.

A-D : Box plots show resting membrane potential (A), input resistance (B), tau (C), and sag ratio

(D) from female and male patients. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, ** P < 0.01 Mann-Whitney test or two sample t-test).

Comparison of action potential properties of the examined cells from female and male patients.

A-D: Boxplots show rheobase (A), action potential half-width mean (B), action potential up-stroke (C) and action potential amplitude (D) from female and male patients. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood and late adulthood groups. Asterisks indicate significance (* P < 0.05, ** P < 0.01, *** P < 0.001, Mann-Whitney test or two sample t-test).

Comparison of firing pattern characteristics of the examined cells from female and male patients.

A-C: Box plots showing: F-I slope (A), first AP latency (B), and adaptation of APs (C) from female and male patients. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence, young adulthood, middle adulthood, and late adulthood groups. Asterisks indicate significance (* P < 0.05, ** P < 0.01, Mann-Whitney test or two sample t-test).

Morphological comparison of the examined cells from female and male patients.

A-H: Boxplots showing morphological features: total (A), apical (B), total basal (C), dendritic length, total number of nodes (D), maximal horizontal (E), and vertical (F) extension, average apical (G) and basal (H) terminal dendritic segment from female and male patients. From left to right: data from all age groups, infant, early childhood, late childhood, adolescence and late adulthood groups.