ABSTRACT Pyramidal neuron subtypes differ in intrinsic electrophysiology properties and dendritic morphology. However, do different pyramidal neuron subtypes also receive synaptic inputs

that are dissimilar in frequency and in excitation/inhibition balance? Unsupervised clustering of three intrinsic parameters that vary by cell subtype – the slow afterhyperpolarization, the

sag, and the spike frequency adaptation – split layer 5 barrel cortex pyramidal neurons into two clusters: one of adapting cells and one of non-adapting cells, corresponding to previously

described thin- and thick-tufted pyramidal neurons, respectively. Non-adapting neurons presented frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous

frequency ratio was higher in adapting than in non-adapting cells, suggesting a higher excitatory drive in adapting neurons. Therefore, our study on spontaneous synaptic inputs suggests a

different extent of synaptic information processing in adapting and non-adapting barrel cortex neurons, and that eventual deficits in inhibition may have differential effects on the

excitation/inhibition balance in adapting and non-adapting neurons. SIMILAR CONTENT BEING VIEWED BY OTHERS SPIKE RELIABILITY IS CELL TYPE SPECIFIC AND SHAPES EXCITATION AND INHIBITION IN THE

CORTEX Article Open access 02 January 2025 EFFECTS OF OPTOGENETIC INHIBITION OF A SMALL FRACTION OF PARVALBUMIN-POSITIVE INTERNEURONS ON THE REPRESENTATION OF SENSORY STIMULI IN MOUSE

BARREL CORTEX Article Open access 12 November 2022 CORTICAL RESPONSE SELECTIVITY DERIVES FROM STRENGTH IN NUMBERS OF SYNAPSES Article 16 December 2020 INTRODUCTION The characterization of

synaptic inputs is necessary to establish how pyramidal neurons of different subtypes participate in information processing as well as a starting point to better understand how these neurons

are affected by disease. A substantial body of research on intrinsic cell electrophysiology properties and dendritic morphology has shown that neocortical layer 5 (L5) pyramidal neurons

belong to two main subtypes referred to as adapting and non-adapting, or thin-tufted and thick-tufted, respectively1,2,3,4,5. Because the barrel cortex is a well-established model system

used for investigating basic cortical processing and neurological disorders, there is also considerable interest in the EPSCs and IPSCs of pyramidal neurons in this brain area. However, it

has not been shown if in the primary somatosensory cortex barrel field (S1BF) these two kinds of neurons differ in their spontaneous postsynaptic currents. The characterization of synaptic

inputs will aid in understanding how action potential generation is controlled in adapting and non-adapting neurons, which differ considerably in intrinsic excitability6 and have been

posited to play different roles in perception and brain state generation7. Postsynaptic current (PSC) measurements will indicate which of these pyramidal neuron functions relies more heavily

on synaptic inputs. Finally, establishing that there is a baseline difference in spontaneous PSCs between pyramidal neuron subtypes demonstrates the need to categorize the neurons in any

study involving comparisons of synaptic inputs between pyramidal neurons from groups containing different subtypes. Synaptic inputs in the neocortex consist overwhelmingly of glutamatergic

EPSCs and GABAergic IPSCs, and are processed by both dendritic morphology and intrinsic electrophysiology properties to determine the timing and strength of action potential output. Three of

the intrinsic properties most consistently shown to be differentially expressed in pyramidal neurons are the slow afterhyperpolarization (sAHP), the sag, and the spike frequency

adaptation2,3,4,5,6,8,9,10. The sAHP refers to the hyperpolarization occurring 500 ms after several spikes are fired, is apamin insensitive, has Ca+2-dependent and Na+-dependent

components11,12,13, and has been shown to increase coincidence detection in pyramidal neurons during strong background excitation14. Sag, referring to the depolarization occurring after the

onset of hyperpolarization during sustained injection of hyperpolarizing current, is caused by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The opening of these

channels reduces cellular excitability, excitatory postsynaptic potential amplitudes, and temporal summation15,16,17. Spike frequency adaptation, the gradual reduction in firing frequency

during a constant stimulus, such as constant depolarizing current injection, is mediated by a variety of mechanisms. They include inactivation of Na+ channels18,19, Ca+2-dependent K+

currents20,21,22, Na+-activated K+ current13, and M-type K+ current23,24. Adaptation has been proposed as a mechanism for preventing runaway excitation caused by recurrent excitatory

connectivity in the cortex25. Several studies have also focused on the role of adaptation in maximizing information transfer26,27,28,29,30. In neocortical pyramidal neurons the co-occurrence

of minimal spike frequency adaptation, small sAHP, high sag, and a thick apical dendrite tuft has been demonstrated3,4,5,6,8,9. Conversely, the co-occurrence of pronounced spike frequency

adaptation, large sAHP, low sag, and a thin dendritic tuft has also been shown3,4,5,6,8,10. Additionally, thick-tufted neurons have been associated with bursting firing patterns and

thin-tufted neurons with non-bursting, “regular spiking” patterns, mostly in sharp electrode recordings10,31,32,33. The fact that pyramidal neurons from the same layer have different

electrophysiological and anatomical profiles hints that their synaptic input profiles may also differ. This is also suggested more directly by the effects of synaptic inputs on sag16 and on

sAHP34,35, and by the relationship between synaptic inputs and the effect of spike frequency adaptation28. To establish if spontaneous synaptic inputs differ between L5 pyramidal neuron

subtypes in the barrel cortex we performed whole-cell patch clamp recordings in acute slices. We measured intrinsic properties (sAHP, sag, and spike frequency adaptation) as well as synaptic

inputs, and we filled a subset of L5 pyramidal cells for morphological analysis. Subsequent unsupervised cluster analysis classified the pyramidal neurons into two clusters of cells,

adapting and non-adapting neurons, with significantly different intrinsic properties and dendritic morphologies. We found significant differences in the amounts of synaptic inputs, both

excitatory and inhibitory, that these two main subtypes of L5 pyramidal neurons of S1BF receive. METHODS ANIMALS We used male and female Tg(Thy-1-EGFP)MJrs/J (GFP-M) transgenic mice, 1–6

months of age (94.5 ± 6.0 d, _n_ = 36), which present with sparse GFP labeling of L5 pyramidal neurons under the thy-1 promoter36. Mice were group housed by gender under a 12 h light, 12 h

dark cycle, and had access to nesting material as well as food and water _ad libitum_. Experiments were performed during the light hours of the cycle. All the procedures described in this

study were approved by the Institutional Animal Care and Use Committee of Tulane University, and were performed in accordance with the NIH Office of Laboratory Animal Welfare’s _Public

Health Service Policy on Humane Care and Use of Laboratory Animals_ and _Guide for the Care and Use of Laboratory Animals_. BRAIN SLICE PREPARATION Mice received general anesthesia by

isoflurane inhalation, after which they were decapitated and the brain was quickly removed and submerged in an iced sucrose solution containing (in mM): 234 sucrose, 2.5 KCl, 25 NaHCO3, 1.25

NaH2PO4, 7 MgCl2, 0.5 CaCl2, 7 glucose, pH 7.3–7.4, bubbled with 95% O2, 5% CO2. After 2 min, the brain was blocked in the coronal plane anterior and posterior to the somatosensory cortex.

The anterior plane was attached with cyanoacrylate-based glue to a detachable stage, after which the brain was sliced in 350 µm increments on a vibratome while submerged in iced sucrose

solution. These coronal brain slices were then incubated 45–60 min at 30 °C in aCSF (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 25 glucose, pH 7.3–7.4, bubbled with 95% O2,

5% CO2. Afterwards, slices were maintained for at least 1 hour at RT in aCSF prior to being moved to the recording chamber. Slices were allowed to equilibrate for at least 15 min in the

recording chamber prior to recording. CELL MORPHOLOGY Neurons were filled with biocytin (0.5% in the patch electrode solution) through the patch pipette during recordings lasting 15–20 min

in voltage clamp or current clamp mode. Subsequently, the patch pipette was slowly withdrawn. After overnight fixation in 4% paraformaldehyde, slices were rinsed in PBS and incubated in

blocking solution (2% BSA, 5% sucrose, 1% Triton X-100 in PBS) for 1 hour at RT. Slices were then incubated with Streptavidin-Alexa 594 (1/400) in blocking solution overnight at 4 °C. The

next day, the slices were thoroughly washed in PBS and mounted using Fluoromount Aqueous Mounting Medium. Images were acquired with an A1Rsi confocal microscope (Nikon Instruments Ltd.,

Japan) using a 10X, 0.45 NA objective. ImageJ (http://rsb.info.nih.gov/ij/) was used for quantification. The tuft width was defined as the horizontal width of the full dendritic tuft,

including the tufts of all primary apical dendrites. Apical dendrite length was obtained by measuring the distance from the intersection of the base of the apical dendrite with an ellipse

inscribed in the cell body to the point where the tuft of the primary apical dendrite began. Shaft width was calculated as the mean full width at half maximum (FWHM) of the apical dendrite

at 225, 250, and 275 µm from the interception of the major and minor axes defining the aforementioned inscribed ellipse. An apical dendrite was counted as a primary apical dendrite if it

bifurcated from the main apical dendrite, and extended toward and formed a dendritic tuft near the pia. ELECTROPHYSIOLOGY The recording chamber was perfused continuously at a rate of 2 

ml/min with aCSF bubbled with 95% O2, 5% CO2, and warmed to 28–30 °C. Patch pipettes were pulled in three stages on a horizontal puller (Sutter Instruments, Novato, CA, USA) from glass

capillaries with ID of 1.2 mm and OD of 1.65 mm (KG-33, King Precision Glass, Claremont, CA, USA). When filled with patch solution (in mM: 70 K-gluconate, 70 KCl, 2 NaCl, 2 MgCl2, 10 HEPES,

1 EGTA, 2 MgATP, 0.3 Na2GTP, 290 mOsm, pH 7.3 adjusted with KOH), the pipettes had a resistance of 2.5–4.5 MΩ. In some recordings biocytin (0.5%) was included in the patch solution for

subsequent morphological analysis. Pyramidal cells were identified by their triangular shape and their apical dendrite or by the expression of GFP. Cells were patch-clamped while visualized

with a 40X immersion objective and Dodt gradient contrast in a SliceScope microscope (Scientifica, UK). GFP was visualized with a 470 nm LED passing through a filter set that consisted of an

HQ470/40X excitation filter, a dichroic mirror Q495LP, and an HQ525/50 nm emission filter. Recordings were made using a Multiclamp 700B amplifier and a Digidata 1550 digitizer controlled

with the Multiclamp Commander program and the pClamp 10 program (Molecular Devices, Sunnyvale, CA, USA). The acquisition frequency was 10 kHz. Voltage clamp traces were Bessel filtered at 2 

kHz during acquisition. The bridge was balanced automatically in Multiclamp Commander prior to attempting seal formation. Fast capacitance transients were compensated automatically in

Commander upon GΩ seal formation. Recordings were terminated if the access resistance monitored in the Clampex Membrane Test was ≥30 MΩ. The input resistance was calculated in voltage clamp

from 5 mV hyperpolarizing steps. The membrane potential was not adjusted for the liquid junction potential. Cells with resting membrane potential positive or equal to −60 mV were excluded

from the analysis. To compute the spike frequency adaptation index, we elicited spikes with a series of 2 s current pulses of amplitude increasing in 5 to 20 pA increments. The index was

taken from steps containing 12–16 action potentials (6–8 Hz). The sAHP was measured by eliciting 35 action potentials during 500 ms by injecting current via the patch pipette in 5 ms pulses.

The pulse amplitude was adjusted to elicit single action potentials. To study the sag we delivered a series of 2 s hyperpolarizing pulses from resting potential in current clamp. The pulse

amplitude was increased in 20–40 pA increments and % sag was calculated from steps in which the maximal hyperpolarization was −80 mV to −90 mV. IPSCs were recorded as inward currents at −70

mV in the presence of 20 µM 6,7-Dinitroquinoxaline-2,3-dione (DNQX). To record miniature IPSCs (mIPSCs), the voltage-gated sodium channel antagonist tetrodotoxin (TTX) (1 µM) was bath

applied until action potentials could no longer be elicited with intracellular injection in current clamp. mIPSCs were recorded subsequently in voltage clamp mode. Simultaneous application

of picrotoxin (60 µM) in addition to 20 µM DNQX eliminated all detectable spontaneous PSCs when cells were voltage-clamped at −70 mV (_n_ = 3 cells). sEPSCs were recorded at −80 mV in the

presence of 40 µM bicuculline methiodide. Application of 40 µM bicuculline and 20 µM DNQX after a control period abolished all detectable spontaneous PSCs when cells were voltage-clamped at

−80 mV (_n_ = 10). Picrotoxin, bicuculline methiodide, DNQX, and TTX were stored in frozen aliquots at 1,000X the working concentration. ELECTROPHYSIOLOGY DATA ANALYSIS To determine the

spike frequency adaptation index, we measured the interspike interval (ISI) of successive spikes, excluding the first two ISIs. Each ISI was then normalized to the third ISI and plotted as a

function of the sequential ISI number. The slope of the linear regression was multiplied by 100 to obtain the adaptation index6,10. Cells with irregular, stuttering spiking were excluded.

Repetitive bursting was only encountered in 3% of cells recorded, and those cells were excluded. To determine the sAHP, five consecutive sweeps were averaged and the sAHP was measured at 500

ms after the last action potential, relative to the resting membrane potential just before stimulation6. % sag = 100*(peak change - steady-state change)/(peak change)6. Peak change and

steady-state change were measured from the membrane potential just prior to current injection. Principal component analysis (PCA) and hierarchical clustering were computed using MATLAB

(MathWorks, Natick, MA, USA). Values for each variable (spike frequency adaptation index, sAHP, and % sag) from each recording (sample) were scaled (zero mean and unit variance) and centered

(subtracting off the mean) before both PCA and pairwise distance between pairs of values were calculated. Singular value decomposition (SVD) algorithm was used to perform PCA. Unsupervised

hierarchical clustering was computed using _correlation_ as the distance metric and _average_ method for linkage of samples. The results from the cluster analysis were further validated

using the _clValid_ package37 on R Statistical Software (The R Foundation for Statistical Computing, Vienna, Austria; Version 3.4.1). sIPSCs and mIPSCs were detected and measured with

MiniAnalysis (Synaptosoft, Fort Lee, NJ, USA), using a detection threshold of 5 pA. The reported IPSC decay time is the time from peak amplitude to 37% of peak amplitude. Rise time was

measured from rise onset time to peak time. STATISTICS Data are provided in text, figures, and tables as mean ± SE. Statistical differences between groups were determined using the Student’s

_t_-test. Paired _t_-test was used for within-cell comparisons. Unpaired two-sample _t_-tests assuming equal variances or assuming unequal variances were used accordingly after applying the

_F_-test two-sample for variances in each case. One-way ANOVA followed by Bonferroni _post hoc_ test was used to compare adapting, thy1-GFP-positive (GFP+), and non-adapting

thy1-GFP-negative (GFP−) neurons. Statistical significance was set at _p_ < 0.05. The datasets generated during and/or analyzed during the current study are available from the

corresponding author upon reasonable request. RESULTS INTRINSIC ELECTROPHYSIOLOGY AND CLUSTERING OF L5 PYRAMIDAL NEURONS To distinguish between L5 pyramidal neuron subtypes in S1BF we

quantified three intrinsic electrophysiology parameters whose differential expression in neocortical pyramidal neurons has been extensively documented: sAHP, sag, and spike frequency

adaptation index2,3,4,5,6,8,9,10. These active membrane properties play a role in determining action potential output in response to synaptic inputs. We applied PCA to the values obtained

for the measured parameters from each individual recording (_n_ = 102 cells). The first two principal components (PC1 and PC2) explained 61.2% and 23.5% of the total variance, respectively

(Fig. 1a). The component loadings indicated that adaptation index (−0.61) and % sag (0.61) were negatively correlated as previously reported3,4,6,8, and were the variables that, making

similar contributions, influenced PC1 the most. Unsupervised cluster analysis and further validation revealed two clusters (cluster 1, C1 and cluster 2, C2) of neurons (Fig. 1a). Neurons

were then classified into the two groups based on the cluster analysis after PCA (Fig. 1b), and the average values for the properties measured were: adaptation indexC1 = 17.7 ± 1.9; sAHPC1 =

 2.7 ± 0.1 mV; % sagC1 = 10.2 ± 0.8%; and adaptation indexC2 = 2.0 ± 0.3, _p_ < 0.001; sAHPC2 = 1.2 ± 0.1 mV, _p_ < 0.001; % sagC2 = 19.6 ± 0.9%, _p_ < 0.001 (Fig. 1c–h). The

co-occurrence of small sAHP, large sag, and small adaptation index in C2 and of large sAHP, small sag, and large adaptation index in C1 confirms previous reports2,3,4,5,6,8,9,10. As already

suggested by the PC loadings, spike frequency adaptation index was the parameter with the largest fold change between clusters (~8.7 times larger in C1; Fig. 1f) and, therefore, we refer to

cells in the cluster C1 as adapting while cells in the cluster C2 are referred to as non-adapting. MORPHOLOGY OF ADAPTING AND NON-ADAPTING L5 PYRAMIDAL NEURONS To evaluate if the cells

included in these two clusters exhibited morphological differences between them, a subset of neurons were filled with biocytin during whole-cell recordings. All the neurons filled showed the

typical morphological features of L5 pyramidal neurons, i.e., pyramidal/triangular shape of the cell body and a prominent apical dendrite oriented towards and perpendicular to layer 1 (Fig.

 1i). The morphological analysis of these neurons indicated that non-adapting neurons (C2) possessed significantly larger apical tufts and thicker apical dendrite shafts (Fig. 1i–k) and a

larger number of primary apical dendrites terminating in a dendritic tuft near the pia (Fig. 1i,m) than adapting neurons. There was no difference in apical dendrite length between adapting

and non-adapting neurons (Fig. 1l). Therefore, in our hands too, adapting cells belonged to the thin-tufted type and non-adapting neurons corresponded to the thick-tufted type (Fig. 1i–m).

Our results confirm previous reports regarding the relationship between intrinsic electrophysiology and dendritic morphology, which indicated that large sAHP, small sag, and large adaptation

index are preferentially found in thin-tufted cells, and small sAHP, large sag, and small adaptation index are preferentially found in thick-tufted cells2,4,5,8. POSTSYNAPTIC CURRENTS IN

ADAPTING AND NON-ADAPTING L5 PYRAMIDAL CELLS There are no detailed characterizations of spontaneous and miniature postsynaptic inputs in these two types of barrel cortex neurons available in

the literature. However, this knowledge is critical for understanding information processing by the interaction of synaptic inputs and intrinsic properties. Moreover, such characterization

can demonstrate that between-cell comparisons of synaptic inputs should be made according to cell subtype. For those reasons, once we identified adapting and non-adapting pyramidal neurons,

we quantified their synaptic inputs. Given that changes in synaptic inhibition are hypothesized to occur in several neurological disorders38,39,40,41 and during

aging42,43,44,45,46,47,48,49,50,51, we focused primarily on characterization of inhibitory postsynaptic currents. Comparing adapting neurons (C1) and non-adapting neurons (C2) revealed that

the frequency of sIPSCs was approximately three-fold higher in non-adapting neurons (Fig. 2a,c and Table 1). We did not find a difference in sIPSC amplitude or decay time between the two

groups (Fig. 2a,d,e and Table 1). Are action potentials in the GABAergic neurons presynaptic to non-adapting neurons responsible for the higher IPSC frequencies in non-adapting neurons? To

test this, after measuring sAHP, sag, and spike frequency adaptation index, we blocked action potentials with bath-applied TTX (1 μM), and subsequently recorded mIPSCs. Similar to sIPSCs,

the frequency of mIPSCs was higher in non-adapting cells compared to adapting cells (Fig. 2b,f and Table 1). The mIPSC amplitude and decay time were not different between the two groups

(Fig. 2b,g,h and Table 1). Likewise, the mIPSC rise times were not different (adapting neurons: 1.47 ± 0.05 ms; non-adapting neurons: 1.52 ± 0.02 ms, Fig. 2b,i). The within-cell comparison

of IPSC frequency also showed no effect of action potential blockade, whether adapting and non-adapting cells were considered separately or pooled (_p_ > 0.05, all paired _t_-tests,

adapting _n_ = 8; non-adapting _n_ = 15). Both the lack of a TTX effect on IPSCs and the fact that mIPSCs, like sIPSCs, occurred at higher frequencies in non-adapting cells indicate that the

higher frequency of IPSCs in non-adapting cells is not caused by higher levels of activity in the presynaptic GABAergic neurons. Taken together, our data on mIPSCs suggest that the higher

frequency of IPSCs in non-adapting neurons is most likely caused by a larger number of GABAergic synapses on these neurons. On the other hand, the lack of difference in amplitude, rise time,

and decay time in mIPSCs suggest that GABAergic innervation is similarly distributed along the somatodendritic axis in adapting and non-adapting neurons, at least when assessed from

synaptic currents recorded at the soma. Although our study focused on IPSCs, we also surveyed sEPSCs to gain a first understanding of the EPSC/IPSC balance in adapting and non-adapting

barrel cortex L5 pyramidal neurons. We found that non-adapting neurons had a higher frequency of sEPSCs than adapting neurons (Fig. 2j–m and Table 1). On the other hand, sEPSC amplitude and

decay time were not different between the two types of neurons. Furthermore, while the mean sEPSC/sIPSC frequency ratio for adapting cells was 0.67, the non-adapting cells’ sEPSC/sIPSC ratio

was 0.45, indicating that non-adapting L5 pyramidal neurons receive 33% fewer excitatory currents per inhibitory current. Our results show that while non-adapting cells receive higher

frequencies of both IPSCs and EPSCs than adapting cells, the excitation/inhibition balance in adapting cells is shifted towards excitation due to a proportional higher frequency of EPSCs per

IPSC. INTRINSIC PROPERTIES AND SYNAPTIC INPUTS OF THY1-GFP L5 PYRAMIDAL NEURONS Transgenic mice carrying thy-1 gene regulatory elements are used extensively in imaging studies due to the

sparse expression pattern of fluorophores achieved in the neocortex36,52,53,54. Several neurological disorder55,56,57 and aging58 studies use these transgenic mice to monitor aberrant

structural and functional changes involving dendritic spine morphology and dynamics. Inhibitory neurotransmission has been hypothesized to modulate these dendritic spine properties59,60,61.

Therefore, we wanted to characterize the intrinsic electrophysiology and inhibitory synaptic inputs of the barrel cortex L5 thy1-GFP neurons. We recorded from both GFP+ and GFP− cells, and

found that all 18 GFP+ cells belonged to the non-adapting cluster C2 (Fig. 3a,b). GFP+ cells and GFP− cells from the non-adapting group did not differ from each other in sAHP, sag, and

adaptation index, but both of these cell groups had different sAHP, sag, and adaptation index compared to adapting L5 pyramidal neurons (Fig. 3b–e and Table 2). GFP+ and GFP− cells from the

non-adapting group did not differ in sIPSC frequency, but both had higher sIPSC frequencies compared to adapting L5 pyramidal neurons in C1 (Fig. 3f and Table 3). sIPSC amplitude and decay

time were not different in the three groups of cells (Table 3). We also extended our experiment to recordings of excitatory inputs, and found that GFP+ neurons and GFP− non-adapting

pyramidal neurons did not differ in sEPSC frequency, but both of these cell groups had a higher sEPSC frequency than adapting neurons (Fig. 3g and Table 3). As was the case for sIPSCs, we

found no difference in sEPSC amplitude or decay time between the three groups of cells (Table 3). Our data show that thy1-GFP neurons in L5 S1BF are non-adapting, in accord with previous

reports on thy1-YFP neurons9,62. Like GFP− non-adapting neurons, L5 thy1-GFP neurons have higher sIPSC and sEPSC frequencies than adapting L5 pyramidal neurons. SYNAPTIC INPUTS TO S1BF L5

PYRAMIDAL NEURONS OF FEMALE AND MALE MICE Because we used both female and male mice we asked if there were gender-specific differences in the frequency of sIPSCs or sEPSCs in either adapting

or non-adapting L5 pyramidal neurons. However, this was not the case. There was no difference in adapting cells’ sIPSCs (6.8 ± 1.1 vs. 4.8 ± 0.6 Hz; _n_ = 11, 10 cells; _p_ > 0.05),

non-adapting cells’ sIPSCs (17.2 ± 2.7 vs. 17.2 ± 4.6 Hz; _n_ = 18, 14 cells; _p_ > 0.05), adapting cells’ sEPSCs (4.1 ± 1.2 vs. 3.8 ± 0.7 Hz; _n_ = 5, 8 cells; _p_ > 0.05), or

non-adapting cells’ sEPSCs (8.8 ± 1.3 vs. 6.2 ± 1.1 Hz; _n_ = 14, 10 cells; _p_ > 0.05) between female and male mice, respectively. DISCUSSION Two main subtypes of L5 pyramidal neurons

have been established based on their projections: intratelencephalic (IT) neurons, whose axons stay in the ipsi- or contralateral cortex and/or the striatum, and pyramidal tract-type (PT)

neurons, whose axonal branches project to the ipsilateral cortex and striatum and beyond the telencephalon2,4,5,6,9,10,62. While the intrinsic properties and morphological features of these

two types of neurons have been extensively described and used for further subdivision into additional subtypes of L5 neurons4,7,63, little is known about the frequency, amplitude, and

balance of spontaneous excitatory and inhibitory synaptic inputs these neurons receive. In this study we used unsupervised clustering analysis after PCA of three intrinsic properties (sAHP,

sag, and spike frequency adaptation index) of S1BF L5 pyramidal neurons and identified two clusters of neurons that morphologically and electrophysiologically conform to the aforementioned

IT and PT neuron types. We characterized the synaptic inputs to neurons from these two clusters, which we refer to as adapting and non-adapting neurons, and that correspond to the IT and PT

subtypes, respectively. We found that non-adapting L5 pyramidal neurons receive higher frequency of both, sIPSCs and sEPSCs, than adapting neurons. Furthermore, the sEPSC/sIPSC frequency

ratio was ~33% lower in non-adapting neurons, indicating that the E/I balance is shifted to favor inhibition over excitation in non-adapting L5 pyramidal neurons compared with adapting

neurons. Similarly to previously published studies, spike frequency adaptation proved to be an appropriate parameter to catalogue cells residing in L5. In fact, adaptation index and sag,

while negatively correlated, were the parameters that contributed the most to the principal component and therefore had the largest influence on the final clustering of the cells (Fig. 

1a,b). The co-occurrence of small adaptation index, large sag, and small sAHP in non-adapting and of large adaptation index, small sag, and large sAHP in adapting L5 pyramidal neurons is in

agreement with previous reports2,4,6,8. In addition, the analysis of morphological features showed that adapting neurons present thinner primary apical dendrites and less elaborated

dendritic arbors than non-adapting neurons. Our results corroborate previous studies indicating that large adaptation index, small sag, and large sAHP are preferentially found in adapting

thin-tufted cells, and small adaptation index, large sag, and small sAHP, are preferentially found in non-adapting thick-tufted cells2,4,5,8. The characterization of synaptic inputs showed

that the amplitude and decay of sIPSCs and sEPSCs were comparable between these two types of L5 pyramidal neurons. However non-adapting neurons presented higher frequencies of both sIPSCs

and sEPSCs. The difference in IPSC frequency that we observed is maintained during action potential blockade, matching results from genetically labeled neurons in L5 of the primary motor

cortex of mice and from neurons classified by the presence or absence of burst firing in L5 of the primary auditory cortex of rats64,65. A possible cause of the elevated mIPSC frequency in

non-adapting neurons is a higher number of GABAergic synapses on these cells, which have been shown to have wider dendritic tufts with a dendritic area significantly larger than that of

thin-tufted neurons1,10,66,67, consistent with our results (Fig. 1i,j). The inhibitory microcircuit organization has been proposed as a key factor in the differential activity regulation of

the main two subtypes of L5 pyramidal neurons by particular interneuron types68 and it may explain the different mIPSC frequencies in adapting and non-adapting pyramidal neurons found in our

study. While intrinsic-bursting corticofugal L5 pyramidal neurons receive mostly thalamic-driven inhibitory inputs from fast-spiking parvalbumin interneurons, regular-spiking corticocortico

L5 neurons receive mostly intracortical-driven inhibition likely from interneurons other than fast-spiking neurons68. Another potential source of difference in the IPSC frequency reported

here could be different release probabilities from GABAergic neurons presynaptic to adapting and non-adapting neurons. This hypothesis is intrinsically linked to and difficult to separate

from the notion that different types of interneurons, such as fast-spiking and somatostatin-expressing neurons with high and relatively low probability of release, respectively,

preferentially innervate different types of pyramidal cells66,69,70,71,72. The higher frequency of IPSCs in non-adapting neurons may provide a more extensive substrate for information

processing in these cells, and a higher number of GABAergic synapses would provide more opportunities for plasticity. Moreover, the higher level of inhibition also enables a larger range of

inhibition for situations requiring robust shifts in activity. Similarly, the higher frequency of sEPSCs detected in non-adapting neurons could be explained by the fact that these neurons

have more complex dendritic arbors (Fig. 1i,j) and by synaptic inputs coming from neurons with different firing rates, i.e., thalamic inputs predominantly projecting onto non-adapting

neurons _versus_ intracortical inputs onto adapting neurons, as previously suggested68,73. Interestingly, the differences in sIPSC and sEPSC frequencies between adapting and non-adapting

neurons were not equally proportional, resulting in different E/I ratios. While adapting L5 pyramidal neurons presented a mean sEPSC/sIPSC frequency ratio of 0.68, non-adapting neurons

presented a mean E/I ratio of 0.45, an approximately 33% lower E/I ratio than adapting neurons. There is no theoretical reason that we are aware of that would suggest that the elevated IPSC

frequency will lead to a higher adaptation index. However, as it has been suggested, the higher frequency of EPSCs in non-adapting neurons may result in a homeostatic upregulation of the

hyperpolarization–activated current (Ih) that diminishes excitability in response to higher frequencies of EPSCs16, and therefore keeps spike frequency more constant. This possible role of

Ih in non-adapting neurons is supported by reports describing: 1) the expression of larger numbers of HCN channels in the more extensive dendritic arbors of corticospinal thick-tufted

neurons17; 2) no spike frequency adaptation and a large Ih mediating the large sag potentials observed in corticocollicular L5 pyramidal neurons in auditory cortex74; and 3) increased

L2/3-driven spiking after inhibition of Ih in L5 corticospinal neurons but not in corticostriatal neurons in motor cortex17. The thy1-GFP-M mouse line as well as many other lines of

transgenic mice derived from the same thy-1 construct36 are widely used for _in vivo_ imaging of neuronal structure and function and optogenetic studies due to the intense, yet sparse

labeling of specific subsets of neurons52,58,75,76. These mouse lines present a robust expression of fluorophores like GFP and YFP, calcium sensors like GCaMP, or light-gated ion channels

like channelrhodopsin throughout L5 pyramidal neurons of the cortex. Our data indicate, however, that the expression of GFP seems to be restricted to non-adapting L5 neurons. It is important

then to characterize the intrinsic properties of this subtype of thy1-GFP+ pyramidal neurons and the inputs to them to better understand and interpret the results from imaging and

electrophysiology studies targeting these neurons. Our results from thy1-GFP+ neurons: 1) confirm previously reported data from thy1 neurons9,62, indicating their PT-like, non-adapting,

nature; 2) provide detailed characterization of the intrinsic properties of this subtype of pyramidal neuron; and 3) describe the significantly higher frequency of IPSCs and EPSCs in these

neurons compared with adapting pyramidal neurons from the same cortical layer. In summary, our data reveal striking differences in the frequency of synaptic inputs, both excitatory and

inhibitory, between adapting and non-adapting L5 pyramidal neurons in S1BF, suggesting the need to identify pyramidal neurons’ subtype in any between-cell comparisons of IPSC or EPSC

frequency. These differences in synaptic input frequency may also have possible implications for the excitation/inhibition balance, an important determinant of neuronal activity. A loss of

comparable absolute levels of inhibition in adapting and non-adapting cells would leave a larger unbalanced excitatory current in adapting cells. Likewise, a comparable increase in

inhibition in the two cell types will leave a larger unbalanced inhibitory current in adapting cells. Thus, L5 pyramidal neurons of different subtype and function may have their

excitation/inhibition balance of synaptic inputs differentially disturbed by drugs which boost GABAergic inputs, such as benzodiazepines, in conditions where brain oscillations are altered,

or in conditions impairing inhibitory synaptic transmission. CHANGE HISTORY * _ 15 JANUARY 2018 A correction to this article has been published and is linked from the HTML version of this

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circuit mechanisms for complex forelimb movement and motor map topography. _Neuron_ 74, 397–409 (2012). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We wish to

thank Dr. Jeffrey G. Tasker, Dr. Laura Schrader, Dr. Shi Di, Dr. Andrei Derbenev, Dr. Rebecca Green, and Alexis Ducote for useful discussions and kind loans of equipment and drugs. This work

was supported by a Louisiana Board of Regents Graduate Research Fellowship LEQSF (2013–18)-GF-17 to R.V., by a Professor’s International Training Mobility Program from the Universidad

Francisco de Vitoria to R.P., and by grants from the National Institute on Aging (R01AG047296), Louisiana Board of Regents RCS (LEQSF(2016-19)-RD-A-24), COBRE on Aging and Regenerative

Medicine (5P20GM103629), and by The Oliver Fund Scholars Award of Tulane University to R.M. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pharmacology, Tulane University School

of Medicine, New Orleans, 70112, USA Ion R. Popescu, Rocío Palenzuela, Rebecca Voglewede & Ricardo Mostany * Neuroscience Program, Brain Institute, Tulane University, New Orleans,

70118, USA Kathy Q. Le & Rebecca Voglewede * School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, 28223, Madrid, Spain Rocío Palenzuela * Brain

Institute, Tulane University, New Orleans, 70118, USA Ricardo Mostany Authors * Ion R. Popescu View author publications You can also search for this author inPubMed Google Scholar * Kathy Q.

Le View author publications You can also search for this author inPubMed Google Scholar * Rocío Palenzuela View author publications You can also search for this author inPubMed Google

Scholar * Rebecca Voglewede View author publications You can also search for this author inPubMed Google Scholar * Ricardo Mostany View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS I.R.P. conceived the study, performed experiments, analyzed data, and wrote the paper. K.Q.L. conceived the study, performed experiments,

analyzed data, and wrote the paper. R.P. conceived the study, performed experiments, analyzed data, and wrote the paper. R.V. performed experiments, analyzed data, and wrote the paper. R.M.

conceived the study, analyzed data, wrote the paper, and oversaw the study. CORRESPONDING AUTHOR Correspondence to Ricardo Mostany. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare that they have no competing interests. ADDITIONAL INFORMATION CHANGE HISTORY: A correction to this article has been published and is linked from the HTML version of this paper. The

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synaptic inhibition distinguishes non-adapting from adapting layer 5 pyramidal neurons in the barrel cortex. _Sci Rep_ 7, 14959 (2017). https://doi.org/10.1038/s41598-017-14971-z Download

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