Electrophysiology and morphology of human cortical supragranular pyramidal cells in a wide age range

After birth, the brain undergoes developmental changes for a prolonged time that involve a series of complex and accurately orchestrated processes (Rakic, 2009). The production and migration of neurons is largely complete at the beginning of postnatal development, and then the intrauterine developmental processes continue: gray and white matter thickening, myelination, synaptogenesis, pruning, and establishment of the basic anatomical architecture for initial neural pathway function. Subsequently, local connections within cortical circuits are fine-tuned, and increasingly complex, longer-term connections are established between circuits (Stiles and Jernigan, 2010). After that, the changes do not end, but continue throughout human life. They are mostly driven by environmental influences and experiences and lead to changes in metabolic activities (Kuzawa et al., 2014), changes in functional connectivity patterns (Kelly et al., 2009) and, with the maturation of white matter (Beck et al., 2021; Yeatman et al., 2014) changes in the speed of long-distance transmission (van Blooijs et al., 2023). The final phase is aging, where it slowly declines with advancing age, leading to a decline in cognitive signal processing functions and often resulting in neurodegenerative diseases (Peters, 2006). The cortical supragranular glutamatergic cell (or pyramidal cell) provides the excitatory synaptic inputs for local inhibitory circuitry and other pyramidal cells by which they create distinct subnetworks (Yoshimura et al., 2005). The development and formation of dendrites (Petanjek et al., 2008; Koenderink and Uylings, 1995) and synapses (Huttenlocher and Dabholkar, 1997) of pyramidal cells in the human cerebral cortex has been documented to some extent by postmortem studies, besides much less is known about their biophysical maturation and electrical properties in the early stages of development and the subsequent change or maintenance in later ages. Numerous studies demonstrated in non-primate animal models that the electrical characteristics of neurons change prominently in the early postnatal stage (Molnár et al., 2020). Changes in the intrinsic membrane properties (Picken Bahrey and Moody, 2003), the input resistance or the kinetics of the elicited action potentials were reported (Kroon et al., 2019; Elston and Fujita, 2014) in connection with maturation of macaque and rodent pyramidal cells. To date, however, no cross-age studies have been conducted on the electrophysiological parameters of human pyramidal neurons. We have studied in detail the postnatal lifetime profile of the physiological and morphological properties of supragranular (layer 2/3) neurons of human pyramidal cells from neurosurgical resections. To this end, we performed whole-cell patch-clamp recordings and 3D anatomical reconstructions of human cortical pyramidal cells from 109 patients aged 1 m to 85 y for comprehensive data analysis to obtain the morphoelectric lifetime profile of supragranular pyramidal cells.

Prior work has characterized postnatal brain development, including synaptogenesis, pruning, myelination, and network maturation across the lifespan. Postmortem human studies described dendritic and synaptic development of pyramidal neurons and suggest early establishment of dendritic architecture, with prolonged synaptogenesis and later pruning (Huttenlocher, 1979; Huttenlocher and Dabholkar, 1997; Petanjek et al., 2008, 2011). In animal models, intrinsic electrophysiological properties undergo marked changes early postnatally, including shifts in resting membrane potential, input resistance, time constant, and AP kinetics (McCormick and Prince, 1987; Huguenard et al., 1988; Kroon et al., 2019). Depth-dependent differences within human L2/3 pyramidal neurons and species differences in h-current have been reported (Kalmbach et al., 2018; Berg et al., 2021; Moradi Chameh et al., 2021). However, before this study there were no comprehensive cross-age analyses of intrinsic electrophysiological parameters of human supragranular pyramidal cells. Morphologically, dendritic trees of human L2/3 pyramidal neurons are relatively mature early, while spine density follows an overproduction and pruning trajectory, with aging-associated changes in spine types and densities linked to cognitive decline (Benavides-Piccione et al., 2013; Jacobs et al., 1997; Luebke et al., 2015; Dumitriu et al., 2010).

Human neocortical tissue was obtained from neurosurgical resections adjacent to pathology (tumors, hydrocephalus, apoplexy, cysts, arteriovenous malformations) with ethics approval and informed consent. Samples were primarily from frontal and temporal lobes, with additional parietal and occipital tissue. Patients (n=109; age range 1 month to 85 years; 64 female, 45 male) contributed slices. Slices (320 µm) were prepared in ice-cold sucrose-based solution, incubated, and maintained in carbogenated ACSF. Whole-cell current-clamp recordings at ~36 °C were obtained from visually identified L2/3 pyramidal cells using HEKA EPC 9/10 amplifiers; pipettes (3–5 MΩ) contained K-gluconate-based internal with biocytin (8 mM) for post hoc anatomy. Step currents (800 ms, 20 pA increments from −100 pA) probed subthreshold and suprathreshold properties. Series resistance (Rs) averaged 24.93±11.18 MΩ (max 63.77 MΩ) for general analyses; fast AP-kinetic analyses used Rs≤30 MΩ cells (n=331; Rs 19.42±6.2 MΩ). Electrophysiological features (resting Vm, input resistance, tau, sag ratio, rheobase, AP half-width, AP up-stroke, AP amplitude, F–I slope, first AP latency, adaptation, and additional parameters; Table 4) were extracted with Fitmaster and custom MATLAB scripts. Somatic depth was quantified relative to the L1/L2 border for recovered somata (36% recovered; mean distance 129.69±130.77 µm). Age groups: infant <1 y; early childhood 1–6 y; late childhood 7–12 y; adolescence 13–19 y; young adulthood 20–39 y; middle adulthood 40–59 y; late adulthood ≥60 y. For morphology, only biocytin-filled cells without truncation and with complete apical dendrites were included. 3D reconstructions (Neurolucida/NeuroExplorer) were performed on 63 cells (ages 0–73 y: infant n=7, early childhood n=8, late childhood n=11, adolescence n=11, young adulthood n=9, middle adulthood n=9, late adulthood n=8). Morphometrics included total/apical/basal dendritic length, total number of nodes, maximal horizontal/vertical extents, apical/basal terminal segment lengths. Spine analyses were performed on 6 fully reconstructed cells (infant group: one 83-day-old patient, n=3 cells; late adulthood: 3 patients aged 64.3±2.08 y, n=3 cells). Spine density (spines/µm) and spine type distributions (mushroom, thin, filopodia, branched, stubby) were quantified on apical and basal branches; only fully visible spines were classified. Statistics: normality (Lilliefors), ANOVA with Bonferroni post hoc, Kruskal–Wallis with Dunn post hoc, two-sample t-test, Mann–Whitney tests (significance p<0.05). Additional analyses assessed potential influences of soma depth, pathology (tumor vs hydrocephalus), and sex on electrophysiological and morphological measures. Data visualization included UMAP projections of electrophysiological features. Histology: standard DAB processing with ABC, OsO4 postfixation, uranyl acetate staining, resin embedding, and 3D reconstruction; spine density computed between bifurcations; terminal segment length measured from last branch point to tip.

• Dataset and sampling: 498 recorded L2/3 pyramidal cells from 109 patients (1 month–85 years). Electrophysiology analyzed in 457 cells from 99 patients; AP kinetics on 331 higher-quality recordings. Seven age groups defined.
• Subthreshold properties (Figure 2; Table 1): Infant group significantly differed from other ages (Kruskal–Wallis): resting Vm p=3.53×10⁻⁸; input resistance p=1.29×10⁻¹⁶; tau p=1.31×10⁻¹⁵; sag ratio p=5.2×10⁻⁴. Means (Infant vs older): • Resting Vm: −60.64±9.86 mV (infant) vs −65 to −68.69 mV in older groups (more negative with age up to young adulthood). • Input resistance: 257.25±188.06 MΩ (infant) dropping sharply thereafter to ~65–90 MΩ (e.g., adolescence 64.79±41.82 MΩ; middle adulthood 90.14±54.37 MΩ). • Tau: 23.88±14.7 ms (infant) decreasing to ~8.5–10.4 ms in older groups. • Sag ratio: increases with age; late adulthood 0.12±0.10 higher than early life.
• Suprathreshold/AP properties (Figure 3; Table 2): Significant age effects (Kruskal–Wallis): rheobase p=8.71×10⁻¹²; AP half-width p=9.57×10⁻²⁵; AP up-stroke p=1.63×10⁻¹²; AP amplitude p=2.24×10⁻¹¹. Trends: • Rheobase: lower in early life, rises to adolescence maximum 306.22±147.21 pA (infant 104.51±103.18 pA; infant vs adolescence p=4.15×10⁻¹³), then declines (late adulthood 207.71±107.83 pA; adolescence vs late adulthood p=5.23×10⁻⁴). • AP half-width: large in infants (1.68±0.71 ms), decreases across development (young adulthood 0.76±0.27 ms; middle adulthood 0.62±0.17 ms). • AP up-stroke: slower in infants (247.43±127.27 mV/ms) vs other ages (~379–435 mV/ms). • AP amplitude: lower in infants (74.62±13.4 mV), higher in childhood/young adulthood (~88–90 mV), declines in late adulthood (82.02±8.49 mV).
• Firing pattern measures (Figure 4; Table 2): • F–I slope: no significant differences across ages (p=0.055). • First AP latency: higher in infants than all other groups (overall p=7.67×10⁻⁴; most pronounced vs late adulthood p=8.41×10⁻⁶). • Adaptation: differs between younger (<13 y) and older groups, lowest in early childhood (p=0.032).
• Depth control: Within-group comparisons by soma distance from L1 border showed no overall depth-related confounds, with a few exceptions (e.g., middle adulthood input resistance p=0.02 and AP up-stroke p=0.04; adolescence AP amplitude p=0.02 and adaptation p=0.009).
• Morphology (Figure 5; Table 3): Across ages (0–73 y), no significant changes in total dendritic length (p=0.37), apical length (p=0.6), basal length (p=0.28), total number of nodes (p=0.18), maximal horizontal (p=0.64) or vertical (p=0.51) extent. Significant differences only in average apical terminal segment length (p=0.033); basal terminal segment length not different (p=0.85).
• Spine density and types (Figure 6; n=6 cells): Infant (83 d; n=3 cells) vs late adulthood (~64 y; n=3 cells): higher total spine density in infants (p=7.57×10⁻⁴⁰) on both apical (p=2.02×10⁻³¹) and basal (p=3.8×10⁻¹²) dendrites. Spine-type distributions differed: mushrooms higher in late adulthood (apical p=4.4×10⁻⁹; basal p=9.04×10⁻⁸); thin spines and filopodia higher in infants (apical thin p=7.34×10⁻¹⁴; apical filopodia p=1.11×10⁻³⁹; basal thin p=2.46×10⁻⁸; basal filopodia p=2.14×10⁻¹²); branched spines higher in infants (apical p=1.64×10⁻¹¹; basal p=8.9×10⁻⁵); stubby spines higher in elderly (apical p=7.19×10⁻⁵; basal p=6.97×10⁻⁹).
• Pathology and sex comparisons: Most features showed no systematic differences between tumor and hydrocephalus groups; sporadic differences in select parameters (e.g., tau, AP half-width, rheobase, F–I slope, first AP latency, adaptation). Sex-related differences were also sporadic in certain age groups (e.g., resting Vm in adolescent females higher; input resistance lower in late childhood females; some AP metrics varied by sex; firing-pattern differences in specified groups).

The study addresses whether intrinsic biophysical and morphological properties of human supragranular (L2/3) pyramidal neurons change across the lifespan. The largest electrophysiological shifts occur during the first postnatal year: resting membrane potential becomes more negative, input resistance and membrane time constant decrease sharply, APs become faster and larger, and rheobase increases—indicating maturing pyramidal cells become less excitable yet more temporally precise. With aging, some trends reverse: sag ratio increases (consistent with enhanced HCN current contribution), AP amplitude declines, and rheobase decreases from an adolescent peak, producing an overall inverted U-shaped trajectory for several active properties. These changes likely reflect age-dependent alterations in ion-channel composition and density (e.g., Na+ and K+ channels; HCN), and increased membrane area with maturation. Morphologically, gross dendritic metrics (lengths, complexity, spatial extent) remain largely stable across ages in L2/3 cells, while apical terminal segment length shows modest age dependence. Spine analyses demonstrate a developmental decrease in overall spine density from infancy to old age, alongside a shift from thin/filopodial/branched spines (infancy) toward mushroom and stubby spines (late adulthood), consistent with synaptic maturation, pruning, and aging-related synaptic remodeling. Together, these findings indicate that while the gross dendritic scaffold remains stable across life stages, synaptic-level structure and intrinsic electrophysiological properties undergo pronounced, stage-specific modulation, shaping neuronal input–output transformations and potentially contributing to developmental and aging-related changes in cognition and cortical circuit dynamics.

This work provides a comprehensive morpho-electrophysiological profile of human cortical L2/3 pyramidal neurons across the lifespan (birth to 85 years). The principal contributions are: (1) identification of marked, early-life changes in intrinsic properties that reduce excitability and increase temporal precision; (2) demonstration of age-related shifts in active properties (rheobase, AP kinetics, sag) with an inverted U-shaped trajectory; (3) evidence that gross dendritic morphology remains largely conserved across ages in L2/3 neurons; and (4) quantitative spine-density and spine-type changes from infancy to late adulthood reflecting synaptic maturation and aging. These results enhance our understanding of human cortical neuron development and aging and provide constraints for biologically realistic models of human cortical function. Future work could broaden sampling across cortical areas and layers, expand spine analyses beyond two age groups, integrate molecular profiling of ion channels and receptors across ages, and link cellular properties to in vivo network activity and cognitive performance.

• Tissue source and pathology: Samples were obtained from neurosurgical resections adjacent to lesions, with differing predominant pathologies across ages (children: hydrocephalus; adults/elderly: tumors). Although procedures were standardized and pathology-based comparisons showed mostly no systematic differences (with sporadic exceptions in selected parameters), potential pathology-related influences cannot be fully excluded.
• Depth and regional sampling: While soma depth relative to L1 was quantified and generally did not account for observed age differences (with a few exceptions), most cells originated from upper L2/3 and mainly from frontal/temporal cortex, which may limit generalizability across depths and regions.
• Morphology and spine analyses sample size: Full 3D morphologies were available for 63 cells; spine analyses were limited to 6 fully reconstructed cells (3 infant from one patient; 3 late adulthood from three patients), constraining statistical power and generalization of spine findings across all ages.
• Cross-sectional design: The study is cross-sectional across individuals rather than longitudinal within the same subjects; inter-individual variability and region-specific maturation differences may influence results.
• Sex-related effects: Some sex differences appeared in specific measures within certain age groups; the study was not primarily powered to dissect sex effects across all features and ages.