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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: J Physiol. 2023 Jan 29;601(4):831–845. doi: 10.1113/JP283311

Impaired dendritic spike generation in the Fragile X prefrontal cortex is due to loss of dendritic sodium channels

Federico Brandalise 1,2,4, Brian E Kalmbach 1,2,5, Erik P Cook 3, Darrin H Brager 1,2,*
PMCID: PMC9970745  NIHMSID: NIHMS1863640  PMID: 36625320

Abstract

Patients with Fragile X syndrome, the leading monogenetic cause of autism, suffer from impairments related to the prefrontal cortex including working memory and attention. Synaptic inputs to the distal dendrites of layer 5 pyramidal neurons in the prefrontal cortex have a weak influence on the somatic membrane potential. To overcome this filtering, distal inputs are transformed into local dendritic Na+ spikes, which propagate to the soma and trigger action potential output. Layer 5 extratelencephalic (ET) PFC neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. We used current clamp and outside-out patch clamp recording to investigate dendritic spike generation in ET neurons from male wild type and Fmr1 knockout (FX) mice. The threshold for dendritic spikes was more depolarized in FX neurons compared to wild type. Analysis of voltage responses to simulated in vivo “noisy” current injections showed that a larger dendritic input stimulus was required to elicit dendritic spikes in FX ET dendrites compared to wild type. Patch clamp recordings revealed that the dendritic Na+ conductance was significantly smaller in FX ET dendrites. Taken together, our results suggest that the generation of Na+-dependent dendritic spikes is impaired in ET neurons of the PFC in FX mice. Considering our prior findings that somatic D-type K+ and dendritic HCN-channel function is reduced in ET neurons, we suggest that dendritic integration by PFC circuits is fundamentally altered in Fragile X syndrome.

Keywords: voltage-gated channel, dendrite, PFC

Graphical Abstract

graphic file with name nihms-1863640-f0001.jpg

Loss of dendritic sodium conductance in layer 5 ET neurons in the prefrontal cortex of Fragile X mice. Dendritic sodium channels are critical to the generation of sodium spikes in the dendrites of layer 5 pyramidal neurons in the mouse prefrontal cortex. In L5 PFC neurons of FX mice (red) the threshold for dendritic sodium spikes is depolarized due to a reduced dendritic sodium conductance.

INTRODUCTION

The prefrontal cortex (PFC) is involved in working memory tasks and contributes to goal-directed behavior by selecting and executing task-appropriate responses while suppressing maladaptive or task-inappropriate ones (Goldman-Rakic, 1990; Rainer et al., 1998; Miller & Cohen, 2001; Sakai et al., 2002; Lara & Wallis, 2015). The PFC sits at the top of the hierarchy associated with cognitive function, by exerting top-down control over numerous cortical and subcortical regions (Kesner & Churchwell, 2011). Output from the deep layers, is carried by the axons of both extratelencephalic (ET) and intratelencephalic (IT) pyramidal neurons (Molnár & Cheung, 2006; Shepherd, 2013; Baker et al., 2018). These neuron types differ in morphology, response to neuromodulation, distribution of ion channels and long-range targets (Dembrow & Johnston, 2014). L5 ET neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. Higher order thalamic nuclei receive feedforward inputs from L5 ET neurons of the PFC (Li et al., 2003; Sherman, 2016). Thalamic nuclei in turn send connections to the superficial layers (L1) of the PFC (Groenewegen, 1988; Deniau et al., 1994). Evidence suggests that there are strong reciprocal connections between the PFC and thalamic nuclei and that these thalamocortical loops are critical for cognitive processing (Schmitt et al., 2017; Collins et al., 2018).

In cortical neurons, the distal dendrites, where the majority of synaptic inputs impinge, and the axosomatic region, where action potentials are generated, are electrically isolated from each other due to both neuronal morphology and the presence of voltage-gated ion channels including h-channels (Berger et al., 2003; Harnett et al., 2013; Dembrow et al., 2015). In contrast to L5 neurons in sensory cortex, which rely on Ca2+ plateaus (Larkum et al., 1999; Larkum et al., 2003), synaptic inputs in the distal dendrites of L5 ET PFC neurons are transformed into local dendritic Na+ spikes, which can propagate to the soma and result in action potential output (Remy et al., 2009; Kalmbach et al., 2017). These dendritic nonlinearities are believed to play important roles for neuronal computations in cortical circuits (Poirazi et al., 2003a; 2003b; London & Häusser, 2005; Ujfalussy et al., 2015; Kaifosh & Losonczy, 2016).

Patients with Fragile X syndrome (FXS), the most common form of inherited cognitive impairment and leading monogenic cause of autism, suffer deficits related to PFC function including: attention and working memory deficits, impulsivity and behavioral inflexibility (Munir et al., 2000a; 2000b; Wilding et al., 2002; Bray et al., 2011). In rodents, sensorimotor coordination has been investigated using a number of behavioral tasks (Siegel et al., 2012; Guo et al., 2014; Murakami et al., 2014; Goard et al., 2016). In many of these behavioral tasks, when the cue is separated in time from the stimulus, the task becomes PFC-dependent and assumes the structure of a working memory task. The prevailing hypothesis is that a persistent signal to downstream brain regions is necessary to bridge the temporal gap between cue and stimulus (Weiss & Disterhoft, 2011; Siegel et al., 2012; Guo et al., 2017; Economo et al., 2018). L5 ET neurons, which project directly to thalamus and other downstream brain regions, are a prime candidate for providing this persistent signal and modulate many of the PFC-dependent behavioral phenotypes associated with FXS (Nakayama et al., 2018).

We recently showed that dendritic spike generation is impaired in CA1 pyramidal neurons of FX mice (Ordemann et al., 2021). In the present study, we investigated dendritic spike generation in ET neurons of FX mice. We found that the threshold for dendritic spikes was more depolarized, and the maximum rate of rise was slower for dendritic spikes in FX ET dendrites compared wild type. Analysis of outside-out patch clamp recordings revealed the density of dendritic voltage-gated Na+ channels was significantly lower in FX neurons compared wild type. The net consequences of these changes impact the ability of ET neurons of the PFC to generate dendritic spikes and reliably trigger action potential output with potential negative impacts on PFC circuit function in Fragile X syndrome.

Materials and Methods

Ethical Approval

All procedures involving mice were performed with the approval of the University of Texas Institutional Animal Care and Use Committee (AUP 2019-00313). All wild type and Fmr1 knockout mice (C57Bl/6 male, 2-4 months old) were housed in the satellite animal facility under the management of the University of Texas Animal Resource Center and in reversed light/dark cycles.

Acute slice preparation

Mice were anesthetized with a ketamine/xylazine (100 mg/kg, 10 mg/kg) cocktail and were perfused through the heart with ice-cold saline consisting of (in mM): 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 7 dextrose, 205 sucrose, 1.3 ascorbate and 3 sodium pyruvate (bubbled with 95% O2/5% CO2 to maintain pH at ~7.4). A vibrating tissue slicer (Vibratome 3000, Vibratome Inc.) was used to make 300 μm thick coronal sections containing the prefrontal cortex. Slices were held for 30 minutes at 35°C in a chamber filled with artificial cerebral spinal fluid (aCSF) consisting of (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 2 MgCl2, 10 dextrose and 3 sodium pyruvate (bubbled with 95% O2/5% CO2) and then at room temperature until the time of recording. Note that wild type and FX mice were interleaved daily, when possible, although the experimenter was not blind to the genotype.

Electrophysiology

Slices were placed in a submerged, heated (32–34°C) recording chamber that was continually perfused (1–2 ml/minute) with bubbled aCSF containing (in mM): 125 NaCl, 3.0 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 10 dextrose, 3 sodium pyruvate. Slices were viewed either with a Zeiss Axioskop microscope and differential interference optics. Patch pipettes (4–8 MΩ) were pulled from borosilicate glass and wrapped with Parafilm to reduce capacitance. All recordings were made from L5 ET pyramidal neurons in the anterior cingulate or prelimbic regions of mPFC.

Whole cell recordings

The pipette solution contained (in mM): 120 K-gluconate, 16 KCl, 10 HEPES, 8 NaCl, 7 K2 phosphocreatine, 0.3 Na–GTP, 4 Mg–ATP (pH 7.3 with KOH). Neurobiotin (Vector Laboratories; 0.1-0.2%) was also included for histological processing and post-hoc cell location determination. Alexa 594 (16 μM; Thermo Fisher #A10428) was also included in the internal recording solution to determine the dendritic recording location relative to the soma. Data were acquired using a Dagan BVC–700 amplifier (Dagan Inc.) and custom data acquisition software written using Igor Pro (Wavemetrics). Data were acquired at 10–50 kHz, filtered at 2–10 kHz, and digitized by an ITC-18 (InstruTech) interface. Pipette capacitance was compensated for, and the bridge was balanced during each recording. Series resistance was monitored and compensated throughout each experiment and was 10–25 MΩ for somatic recordings and 15-40 MΩ for dendritic recordings. Recordings were discarded if series resistance increased by more than 30% during the recording. Voltages are not corrected for the liquid-junction potential (estimated as ~12 mV).

White noise injections and Leaky-integrate-and-fire model

Dendritic recordings were performed with a large, zero-mean, Gaussian-distributed white-noise stimulus current injected through the recording pipette for 1–2 min. The variance of the noise current was adjusted to two levels: large (that produced somatic action potentials) and small (subthreshold with no somatic action potentials). Dendritic action potentials (dSpikes) were estimated during the noise current injection using a deconvolution method described in Kalmbach et al., 2017. This method reveals the current the dendrites generated, with large fast inward current spikes labeled as dSpikes (example dSpikes are shown in Figure 4C). Previous work found that these dSpikes were dependent on Na+ channel activation (Kalmbach et al., 2017). Hundreds of dSpikes were typically observed for each cell during the large 1–2 min noise current injection.

Figure 4. White-noise analysis reveals a higher dendritic spike threshold for FX dendrites.

Figure 4.

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A) One to two minutes of zero-mean Gaussian-distributed white-noise current was injected into the dendrites while simultaneously recording dendritic (Vdend) and somatic (Vsoma) membrane potentials. B) Location of the dendritic electrode and standard deviation (SD) of the white-noise stimulus for each recording. C) Example Vdend and Vsoma and the deconvolved current dSpikes (note that positive corresponds to an inward current). Only deconvolved current dSpikes with peak amplitudes greater than 0.5 nA were included in the analysis. As illustrated in this example, somatic APs most always followed large deconvolved dSpikes, but large dSpikes also occurred without subsequent APs. D) The average stimulus, Vdend and Vsoma aligned to the peak of the deconvolved dSpike for WT and FX recordings (dSTAs). To reduce variability, the dSTAs were computed from all recording sessions combined. The left column corresponds to dSTAs computed from deconvolved current dSpikes that preceded somatic APs, while the right column shows dSTAs using the remaining deconvolved current dSpikes with peak amplitudes greater than 0.5 nA. E) A leaky integrate-and-fire (IAF) model was used to generate dSpikes using the same stimulus for the WT and FX recordings (see Methods for model parameters). The WT and FX models both used the same leaky integrator, with either the same threshold (TFX = TWT, left) or different thresholds (TFX = 1.64 X TWT, right). STAs were then computed from each model’s spike output.

The dSpike-triggered average (dSTA) of the stimulus was the average noise current aligned to the peak of the deconvolved dSpikes. The spike-triggered average theorem states that the dSpike-triggered average of the white-noise current represents the linear integration of the dendrites.

To demonstrate that an increase in FX dSpike threshold could account for our observed changes in the dSpike-triggered average of the white-noise current, we implemented a leaky-integrate-and-fire (leaky IAF) model of the dendrites (see Figure 4E). The model simulated the effects of a change in dendritic spike threshold on dendritic integration. The leaky integrator of the model was convolved with the same white-noise current injections used in the actual recordings. Thus, the WT and FX models received the same statistical white-noise inputs as the WT and FX experiments, respectively.

The leaky integrator was modeled as a sum of two exponentials with decay constants = 0.5 and 1.8 ms, weighted by 1.1 and 1.0, respectively (shown in Fig. 4E), and was the same for both wild type and FX models. Spike thresholds were set to produce enough spikes required for the spike triggered averages (STAs; about 1000 spikes). When the convolution of the leaky integrator and the white-noise stimulus (labeled as x in Fig. 4E) crossed the threshold (T), the model produced a spike, and the leaky integrator was reset to zero. The model’s spike times were randomly jittered using a Gaussian distribution with a standard deviation of 100 us to mimic recording noise. Thresholds were initially set to be the same for both the WT and FX models, and then the FX model’s threshold was increased by 64%. We chose this increase in the FX model’s spike threshold because it mimicked the experimentally observed increase in the dSTA observed in the FX recordings.

Outside-out recordings

Outside-out recordings were made using an Axopatch 200B amplifier (Molecular Devices), sampled at 10 kHz, analog filtered at 2 kHz and digitized by an ITC-18 interface connected to a computer running Axograph X. The pipette solution contained (in mM): 90 K-gluconate, 50 CsCl, 10 HEPES, 5 EGTA, 4 NaCl, 7 K2 phosphocreatine, 0.3 Na–GTP, 4 Mg–ATP (pH 7.3 with KOH). TEA-Cl (20) and 3,4 diaminopyridine (0.1 mM) were added to the extracellular saline. INa was measured using depolarizing voltage commands from −80 to 40 mV from a holding potential of −90 mV. Patch area was estimated by fitting the decay of the capacitive transient in response to a small voltage step (assuming 1 μF cm−2; Routh et al., 2017). Activation data were fit to a single Boltzmann function using a least-squares program. Linear leakage and capacitive currents were digitally subtracted by scaling traces at smaller command voltages in which no voltage-dependent current was activated.

Statistical Analysis

Repeated measures analysis of variance (RM–ANOVA), between-subjects factors ANOVA, mixed factors ANOVA and post–hoc t–tests were used to test for statistical differences between experimental conditions. Sidak’s correction was used to correct for multiple comparisons. Pearson’s product moment correlation was used to test for statistically significant correlations between variables. Data in the text and in tables are presented as mean and standard deviation. Error bars in all figures represent standard error of the mean (SEM). Statistical analyses were performed using Prism (Graphpad) and considered significant if p<0.05. Power analyses were performed using G*power and reported as Type II error probability (β).

RESULTS

Dendritic spike generation is impaired in FX ET neurons

In many pyramidal neurons, distal dendritic voltage signals are strongly attenuated by the time they reach the soma. L5 ET neurons generate dendritic Na+ spikes to overcome the electrotonic attenuation of distal synaptic inputs (Dembrow et al., 2015; Kalmbach et al., 2017). We first compared dendritic spikes between wild type and FX ET neurons using dendritic recording and square pulse depolarizing injections (Fig. 1A). ET neurons were identified by a resonant frequency >2.2 Hz in response to a 15Hz chirp stimulus (Dembrow et al., 2010; Kalmbach et al., 2015). In agreement with our previous results (Kalmbach et al., 2015; Brandalise et al., 2020), FX ET dendrites had a higher input resistance and longer membrane time constant consistent (Table 1). The voltage threshold for dendritic spikes was calculated as 20% of the second peak of the second derivative as previously described (Gasparini et al., 2004; Ordemann et al., 2021). The threshold for dendritic spikes did not vary with distance from the soma in either wild type or FX ET neurons (Fig. 1B). Dendritic spike threshold was, however, significantly more depolarized in FX compared with wild type dendrites (wild type: −30.2 ± 3.9 mV, n=32; FX: −24.8 ± 6.19 mV, n=21; unpaired t-test: t=3.869, p = 0.003;). We grouped dendritic spikes into proximal (<250 μm) and distal (>250 μm, near the nexus) based on the distance of the recording electrode from the soma. Threshold was more depolarized for both proximal and distal dendritic spikes (Fig. 1C; wild type: proximal n = 10, distal n = 22; FX: proximal n = 11, distal n = 10; two-way ANOVA, main effect of genotype: F(1, 49)=13.26, p = 0.0007). The maximum rate of rise and peak membrane potential for dendritic spikes decreased with distance from the soma for both wild type and FX ET dendrites (Fig. 1D, F). The maximum rate of rise was significantly slower (Fig. 1E; wild type: proximal n = 10, distal n = 22; FX: proximal n = 7, distal n = 10; two-way ANOVA, main effect of genotype: F(1, 44)=14.31, p = 0.0005) and the duration significantly longer (wild type: 2.5 ± 0.35 ms, n = 12; FX: 3.4 ± 1.2 ms, n = 9; two-way ANOVA, significant interaction: F(1, 55)=4.699, p = 0.0345) of dendritic spikes in FX dendrites compared to wild type dendrites. By contrast, there was no significant difference in peak membrane potential between FX and wild type dendritic spikes (Fig. 1G; wild type: proximal n = 12, distal n = 26; FX: proximal n = 13, distal n = 10; two-way ANOVA, main effect of genotype: F(1, 57)=0.3475, p = 0.5579).

Figure 1. Dendritic spikes are altered in FX ET neurons.

Figure 1.

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A, Representative dendritic spikes recorded from wild type (black) and FX (red) ET dendrites. Threshold is indicated by the black arrow heads. B, Dendritic spike threshold as a function of dendritic location for wild type and FX dendrites. C, Dendritic spike threshold is significantly more depolarized in FX ET neurons compared to wild type ET neurons. D, Dendritic spike rate of rise (dV/dt) as a function of dendritic location for wild type and FX dendrites. E, Dendritic spike maximum dV/dt is significantly smaller in FX ET neurons compared to wild type ET neurons. F, Dendritic spike peak voltage as a function of dendritic location for wild type and FX dendrites. G, There is no significant difference in dendritic spike peak voltage between wild type and FX ET neurons.

Table 1. Comparison of the subthreshold electrophysiological properties between wild type and FX ET dendrites.

The subthreshold electrophysiological measurements are different between wild type and FX ET dendrites and consistent with a loss of dendritic Ih (Kalmbach et al., 2015; Brandalise et al., 2020).

wild type
(n=26)		 Fmr1 KO
(n=21)		 unpaired T-test
p-value
VM (mV) −59.6 ± 10.7 −61.4 ± 6.4 0.087
RN (MΩ) 48.2 ± 22.4 66.7 ± 23.3 0.0023
Membrane time constant (ms) 7.5 ± 6.1 16.3 ± 10.1 0.009
Membrane sag (%) 29.8 ± 13.2 20.3 ± 10.1 0.0086
Rebound slope (mV/mV) −0.47 ± 0.36 −0.28 ± 0.09 0.0134
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Back propagating action potentials are smaller in FX ET dendrites

To determine if the impairment of active events was limited to dendritic spikes, we measured the backpropagation of action potentials. We made dendritic current clamp recordings and elicited action potentials in wild type and FX ET neurons using extracellular stimulation in layer 5 in the presence of synaptic blockers to prevent fast glutamatergic and GABAergic transmission (Fig. 2A). We found that the peak membrane potential of backpropagating action potentials (bAP) decreased with increasing distance for both wild type (r2 = 0.17, p = 0.0047) and FX (r2 = 0.41, p < 0.0001) neurons (Fig. 2B). When recording location was binned by distance (proximal: <250 μm; distal: >250 μm), we found that the peak membrane potential of distal, but not proximal, bAPs was significantly smaller in FX neurons compared to wild type (Fig. 2C; two-way ANOVA, interaction: F(1, 76)=4.363, p = 0.0401; distal wild type: 5.3 ± 8.78 mV, n=29; distal FX: −4.4 ± 11.4 mV, n=16). The half width of bAPs increased with distance for both wild type and FX neurons and was significantly longer in FX neurons at distal, but not proximal, recording locations (Fig. 2D-E; two-way ANOVA, interaction: F(1, 76)=5.756, p = 0.0189; distal wild type: 1.7 ± 0.652 ms, n=29; distal FX: 2.4 ± 0.876 ms, n=16).

Figure 2. Backpropagating action potentials in the distal dendrites of FX ET neurons are shorter and wider.

Figure 2.

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A, Representative backpropagating action potential recorded from wild type (black) and FX (red) ET dendrites. B, Backpropagating action potential peak as a function of dendritic location for wild type and FX dendrites. C, Backpropagating action potential peak is significantly smaller at more distal locations in FX ET neurons compared to wild type ET neurons. D, Backpropagating action potential halfwidth as a function of dendritic location for wild type and FX dendrites. E, Backpropagating action potential halfwidth is significantly larger at more distal locations in FX ET neurons compared to wild type ET neurons.

Dendro-somatic coupling is reduced in FX ET neurons

We used simultaneous somatic and dendritic current clamp recordings to investigate the coupling between the soma and dendrites in wild type and FX ET neurons. We used somatic current injection to elicit single action potentials and measure their backpropagation into the dendrites (Fig. 3A). The peak voltage of action potentials in the soma was not significantly different between wild type and FX neurons (Fig. 3B; wild type: 58 ± 8.73 mV, n=7; FX: 57 ± 9.79 mV, n = 7; unpaired t-test: t=0.2951, p =0.7734). In agreement with our results using extracellular stimulation, bAPs attenuated with distance in both wild type (r2 = 0.49, p = 0.035) and FX (r2 = 0.7, p = 0.005) ET dendrites (Fig. 3C). Attenuation of bAPs was significantly greater, however, in FX ET neurons compared to wild type (F-test: F(1,14)=8.62, p = 0.01; Fig. 3C). To investigate the ability of dendritic spikes to influence somatic membrane potential, we simulated synaptic input by using double exponential dendritic current injections (Fig. 3D). In agreement with our results using square pulses (Fig. 1), the threshold for dendritic spikes was significantly more depolarized in FX ET neurons compared to wild type (Fig. 3E; wild type: −29 ± 3.57 mV, n=5; FX: −12 ± 5.03 mV, n=7; unpaired t-test: t=7.023, p =0.001). Furthermore, while dendritic spikes in wild type ET neurons reliably triggered somatic action potentials (median probability = 1.0), this coupling was significantly reduced in FX ET neurons (median probability = 0.8; Fig. 3F; Mann-Whitney test: U=4, p =0.008).

Figure 3. Dendritic-somatic coupling is reduced in FX ET neurons.

Figure 3.

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A, Representative dual somatic-dendritic recording showing dendritic and somatic membrane potential during somatic current injection to trigger action potentials in wild type and FX ET neurons. B, Somatic action potential amplitude was not significantly different between wild type (n=6) and FX (n=7) ET neurons. C, Attenuation of backpropagating action potentials during dual recording is greater in FX compared to wild type ET neurons. D, Representative dual somatic-dendritic recording showing dendritic and somatic membrane potential during dendritic current injection to trigger dendritic spikes in wild type and FX ET neurons. Traces shown on expanded time scale at the right. E, Dendritic spike threshold during dual recordings was significantly more depolarized in FX ET neurons compared to wild type (n=5 wild type and 7 FX). F, The probability of dendritic spikes triggering somatic action potentials was smaller in FX ET neurons (n=5 wild type and 7 FX).

Larger stimuli are required to trigger dendritic spikes under in vivo like conditions

In vivo intracellular recordings in cortex have shown that the dendritic membrane potential randomly fluctuates in response to complex patterns of excitatory and inhibitory synaptic inputs (Smith et al., 2013). To examine if the changes in FX dendritic spikes observed above also occur under the range of frequencies likely experienced by the dendrites in vivo, we delivered a white-noise stimulus through the dendritic electrode during simultaneous somatic and dendritic recording (Fig. 4A; see Kalmbach et al., 2017). During the white-noise current injection, it was not possible to reliably identify dendritic spikes present in the dendritic voltage waveform. Thus, we used a previously published systems-based approach that deconvolved out the nonlinear current produced by the dendrites in response to the white noise injection (Kalmbach et al., 2017). The deconvolution process revealed inward “current spikes” generated by the dendrites. It was previously demonstrated that these dendritic current spikes (referred to as dSpikes) were dependent on dendritic Na+ channels and varied in amplitude (Kalmbach et al., 2017).

A zero-mean, Gaussian-distributed white-noise current was delivered through the dendritic electrode for one to two minutes. Neither the location of the dendritic electrode or the magnitude of the noise injection was significantly different between WT and FX recordings (p = 0.85 and 0.09, respectively, two sample t-test; Fig 4B). As shown by the example recording in Fig. 4C, the noise stimulus produced both hyperpolarized and depolarized dendritic membrane potentials around rest, and both small and large dendritic current spikes were revealed by the deconvolution. As previously reported, the white-noise stimulus produced somatic action potentials that were usually preceded by large dendritic current spikes (Kalmbach et al., 2017).

To reveal the dendritic input that produced somatic action potentials, we averaged the stimulus noise aligned to each deconvolved dendritic current spike that preceded each somatic action potential (referred to as the dSpike-triggered-average or dSTA). To reduce variability, we computed the dSTA of the noise stimulus by combining thousands of dSpikes recorded from all cells. The resulting population dSTA of the noise stimulus shows that a somatic AP required a larger dendritic stimulus in FX dendrites compared to wild type (p < 1e-8, two-sample t-test; Fig. 4D, top left column). We further observed that the average stimulus that preceded a dSpike with no subsequent somatic AP was larger for FX versus WT dendrites (p < 1e-6, two-sample t-test; Fig. 4D, right column). Notably, the magnitude of the average deconvolved dSpike, dendritic and somatic potentials were nearly identical between WT and FX dendrites (Fig. 4D).

Based on the spike-triggered-average theorem, the shape of the dSTA derived using a white-noise stimulus captures the shape of the dendritic integrator. Thus, from Fig. 5 we can conclude that the timescale of the dendritic integrator was similar, and relatively fast, for both WT and FX dendrites. Only the magnitude of the stimulus dSTA was larger for FX compared to WT. As reported above, the dSpike threshold was found to be greater for FX dendrites, and we wondered is this could account for the differences in the stimulus dSTA magnitudes.

Figure 5. Neuronal morphology is not different between wild type and FX ET neurons.

Figure 5.

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A, Representative Neurolucida reconstructions of ET neurons from wild type (black) and FX (red) mice. B, Sholl analysis of dendritic length is not different between wild type and FX ET neurons. C, There is no significant difference in total dendritic length between wild type and FX ET neurons. D, Sholl analysis of dendritic branching is not different between wild type and FX ET neurons. E, There is no significant difference in total number of intersections between wild type and FX ET neurons.

To illustrate the link between a change in dSpike threshold and the magnitude of the stimulus dSTA, we simulated a simple integrate-and-fire (IAF) model using the same white-noise stimuli used in our recordings (see Methods). We set the parameters of the leaky integrator (shown in Fig. 4E) and threshold (TWT and TFX) to mimic the dSTAs observed in the data. When the stimuli used during the WT and FX recordings were presented to the two IAF models (ie., identical integrators and thresholds), the dSTAs of the noise stimulus were very similar (Fig. 4E, left). When the spike threshold for the FX model was increased by 64%, however, the FX stimulus dSTA increased compared to the WT dSTA in a similar manner to that observed in the data (Fig. 4E, right). Thus, a change in the dSTA of the noise stimulus for the FX model stimulus is consistent with an increase in dendritic spike threshold observed in FX dendrites (Figs 1 and 3). Together, the white-noise experimental and modelling results bolster the hypothesis that an increase in threshold for FX dendritic spikes alters how FX neurons process dendritic inputs.

Neuronal morphology is not different between wild type and FX neurons

Neuron morphology and dendritic branching has a strong influence the electrotonic decay of membrane potential (Rall, 1962). During recordings, neurons were filled neurobiotin for post-hoc morphological reconstructions (Fig. 5A). There was no significant difference in total dendritic length between wild type and FX ET neurons (Fig. 5B; wild type: 6.3 ± 2.08 mm, n=6; FX: 6.2 ± 0.89 mm, n = 7; unpaired t-test: t=0.1395, p =0.8916). Sholl analyses found no significant difference in dendritic length as a function of distance from the soma (Fig. 5C; two-way ANOVA, main effect of genotype: F(1, 312)=1.18e−14, p>0.99). There was no significant difference in total number of intersections between wild type and FX ET neurons (Fig. 5D; wild type: 188 ± 48.9 intersections, n=6; FX: 179 ± 15.87 intersections, n = 7; unpaired t-test: t=0.4626, p =0.6527). Sholl analyses found no significant difference in the number of intersections as a function of distance from the soma (Fig. 5E; two-way ANOVA, main effect of genotype: F(1, 312)=0.00, p>0.99). These results suggest that similar to our observations for Layer 2/3 pyramidal neurons in the PFC and CA1 pyramidal neurons in the hippocampus (Routh et al., 2017; Ordemann et al., 2021), gross dendritic morphology of L5 ET neurons in the PFC are not different in FX mice.

Dendritic Na+ conductance is lower FX ET neurons

Dendritic Na+ and K+ channels contribute to the generation and amplitude of dendritic spikes (Golding & Spruston, 1998; Golding et al., 1999; Kalmbach et al., 2017; Ordemann et al., 2021). We previously demonstrated that there were no significant differences in dendritic K+ channels between wild type and FX ET neurons (Kalmbach et al., 2015). We therefore hypothesized that the more depolarized dendritic spike threshold in FX ET neurons was due to a difference in dendritic Na+ channel function. We made outside-out patch clamp recordings of dendritic Na+ current from wild type and FX ET neurons using step voltage commands (−80 to +40 mV, Δ10 mV, 50 ms). We found that the peak sodium current in FX dendritic patches was significantly smaller beginning at −30 mV compared to wild type neurons (Fig. 6A-B; two-way repeated measures ANOVA, interaction: F(24, 180) = 11.57, p < 0.0001). The maximum sodium conductance density increased with distance from the soma in both wild type and FX patches. However, the maximum conductance density was lower in FX ET neurons compared wild type (Fig. 6C). Indeed, Na+ conductance density was lower in FX ET neurons across the range of membrane potentials tested (Fig. 6D; two-way repeated measures ANOVA, interaction: F(24, 180) = 9.180, p < 0.0001).

Figure 6. Sodium conductance is lower in FX ET dendrites.

Figure 6.

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A, Representative sodium currents recorded from outside-out patches in response to voltage steps to 0 mV from wild type (black) and FX (red) ET dendrites. B, Current-voltage plot showing smaller peak sodium current at more depolarized potentials in FX ET neurons. The presence of FMRP1-298 peptide did not rescue peak sodium current. C, The maximum sodium conductance density increases with increasing distance from the soma for both wild type and FX ET neurons but was larger in wild type compared to FX. D, Summary plot showing lower sodium conductance density as a function of membrane potential in FX outside-out dendritic patches. E, Summary plot showing that there was no significant difference in rate of sodium channel inactivation between wild type and FX recordings. F, Summary plot showing that there was no significant difference in sodium channel activation between wild type and FX recordings.

Consistent with previous results, the rate of Na+ channel inactivation increased with membrane depolarization for both wild type and FX dendrites however, there was no significant difference in the rate of Na+ channel inactivation between wild type and FX patches (Fig. 6E; two-way repeated measures ANOVA, F(2, 16) = 0.4732, p = 0.6314). The voltage dependence of Na+ channel activation was not different between wild type and FX ET dendrites (Fig. 6F). There was no significant difference in voltage of half activation (Table 2; one-way ANOVA, F(2, 16) = 1.629, p = 0.2271) or slope factor (Table 2; one-way ANOVA, F(2, 16) = 1.250, p = 0.3130). These results suggest that diminished expression of dendritic Na+ channels underlie the depolarized dendritic spike threshold in FX ET neurons.

Table 2. Voltage-dependence of sodium channel activation is not different between wild type and FX ET dendrites.

The voltage of half activation (V1/2: unpaired t-test, t = 0.9458, p = 0.3615) and slope factor (k: unpaired t-test, t = 1.310, p = 0.2129) of activation curves between wild type and FX outside out patch clamp recordings

wild type FX p-value
V1/2 (mV) −22.4 ± 3.9 −24.3 ± 3.7 mV 0.3615
Slope factor (k) 12.4 ± 1.7 13.5 ± 1.6 0.2129
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DISCUSSION

Dendritic spikes are critical to information processing in the prefrontal cortex. Distal synaptic inputs onto Layer 5 ET neurons are too weak to overcome the electronic isolation and trigger somatic action potentials. To overcome this, distal synaptic inputs are integrated and transformed into local dendritic Na+ spikes. We found that the threshold for dendritic spikes was significantly more depolarized in FX ET neurons compared to wildtype. There was also a smaller proportion of strong dendritic spikes in FX dendrites compared to wild type. Simultaneous recording from the dendrite and soma revealed that dendro-somatic coupling is reduced and that a larger dendritic stimulus is necessary to elicit a dendritic spike in FX dendrites compared to wild type. Lastly, we show that dendritic Na+ conductance is lower in FX ET neurons. We suggest that the lower expression of dendritic Na+ channels contribute the impaired generation of dendritic spikes in L5 ET neurons of the FX PFC.

FMRP regulation of Na+ channels

Our data suggest that a decrease in Na+ conductance (Fig. 6) contributes to the depolarized dendritic spike threshold in L5 ET neurons in the prefrontal cortex of the Fmr1 KO mouse. FMRP modulates ion channel expression and function through translational mechanisms and through protein-protein interactions (Brager & Johnston, 2014). FMRP can both repress and promote translation of target mRNAs (Darnell et al., 2011; Banerjee et al., 2018). Na+ channels in the brain are composed of a pore-forming α subunit (NaV1.1, NaV1.2, NaV1.3, or NaV1.6) and auxiliary β subunits (NaVβ1–4) (Catterall, 2012). Two Na+ channel α subunits, NaV1.2 (SCN2A) and NaV1.6 (SCN8A), are known mRNA targets of FMRP (Darnell et al., 2011). In neurons of the medial prefrontal cortex, reduction of NaV1.2 alters dendritic excitability and produces behavioral inflexibility and social impairments consistent with autistic phenotypes (Spratt et al., 2019). If FMRP promotes translation of SCN2A or SCN8A, then its loss would decrease the expression of NaV1.2 and NaV1.6 in L5 neurons respectively. The biophysical properties and surface expression of Na+ channel α subunits is regulated in part by β subunits (Patino & Isom, 2010). Accordingly, a change in the expression or association of the Na+ channel pore-forming subunit with β subunits could alter the kinetics and/or surface expression of Na+ channels. One potential mechanism underlying the lower conductance density in FX neurons would be a decrease in the activation to inactivation balance. A slower membrane time constant due to loss of dendritic Ih in FX dendrites (Kalmbach et al., 2015; Brandalise et al., 2020) could affect the rate of sodium channel activation. We found that the rate of Na+ channel inactivation was not different between wild type and FX neurons. While it is possible that the activation rate for FX Na+ channels is slower compared to wild type neurons we could not directly test this hypothesis due to the very rapid activation rate of Na+ channels.

Physiological consequences

We provide the first direct comparison of dendritic spikes between wild type and FX KO neurons in the PFC. We found that the threshold was more depolarized, and the maximum dV/dt was slower for dendritic spikes in FX ET dendrites compared with wild type. We previously showed that dendritic Ih is lower in FX ET dendrites (Kalmbach et al., 2015; Brandalise et al., 2020). A downregulation of Ih increases neuronal excitability in part by increasing input resistance (e.g., Magee, 1998). It is possible that the higher RN would affect Na+ channel inactivation due to greater membrane depolarization. Given that our dendritic current clamp and outside patch recordings are from a fixed membrane potential this would not appear to be a significant contributing factor. We previously showed that dendritic spikes in CA1 pyramidal neurons of the hippocampus also have a depolarized threshold and reduced maximum dV/dt (Ordemann et al., 2021). In CA1 dendrites however, there is no difference in dendritic Na+ conductance. The change in dendritic spike generation in CA1 neurons is due to a shift in the activation of dendritic A-type K+ channels (Routh et al., 2013; Ordemann et al., 2021). We have previously shown, however, that dendritic A-type K+ channels are not different between wild type and FX ET neurons (Kalmbach et al., 2015). This suggest that although both L5 ET neurons of the PFC and CA1 pyramidal neurons of the hippocampus display the same dendritic spike phenotype, the biophysical mechanisms that underlie this change are different.

The distal dendrites of L5 ET neurons in the PFC receive inputs from the contralateral prefrontal cortex and both the medial dorsal and ventromedial thalamus (Dembrow et al., 2015; Collins et al., 2018). The dendritic geometry and biophysical properties of the L5 ET dendrites restrict the ability of these distal synaptic inputs to change the somatic membrane potential. Instead, synaptic inputs can be summated to produce local dendritic spikes which are able to reliably propagate to the soma and contribute to action potential firing. In wild type mouse and rat, the high density of h-channels in the distal dendrites of L5 ET neurons reduces the window for temporal integration (Kalmbach et al., 2013; 2015; Brandalise et al., 2020). Accordingly, L5 ET neurons function as coincidence detectors (Dembrow et al., 2015). We previously showed that there is a loss of dendritic h-channels and somatic D-type K+ channels in FX ET neurons (Kalmbach et al., 2015; Brandalise et al., 2020; Kalmbach & Brager, 2020). The effect of these changes would increase the window for temporal integration, changing the neuron from a coincidence detector to an integrator, and modulate the threshold for action potential generation. In this study we show that the ability of ET neurons in FX mice to reliably trigger dendritic spikes is impaired due to a loss of dendritic Na+ channels. This would effectively reduce the ability of dendritic inputs to trigger somatic action potential output. We suggest that the combined effect of Na+, K+ and h-channelopathies in Fragile X syndrome impair the processing of information by PFC circuits.

Supplementary Material

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KEY POINTS.

  • Dendritic spike threshold is depolarized in Layer 5 PFC neurons in FX mice

  • Simultaneous somatic and dendritic recording with white noise current injections revealed that larger dendritic stimuli were required to elicit dendritic spikes in FX ET neurons

  • Outside-out patch clamp recording revealed that dendritic sodium conductance density was lower in FX ET neurons

Acknowledgments

We thank James Ding for the neuronal reconstructions and Daniel Johnston for critical reading of the manuscript.

Funding

This work supported by National Institutes of Health Grants R01/R56 MH100510 (D.H.B.) and Swiss National Science Foundation Grants P2ZHP3_168621 and P400PB_180785 (F.B.).

Biography

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Dr. Federico Brandalise received his PhD in neuroscience from the University of Zurich for work in the lab of Urs Gerber and Fritjof Helmchen. For his doctoral dissertation, he studied the role of recurrent CA3 and mossy fiber synapses in the induction of dendritic NMDA spikes and their role in synaptic plasticity. Dr. Brandalise was awarded a Swiss National Foundation Fellowship and moved to the lab of Dr. Darrin Brager in the Department of Neuroscience at the University of Texas at Austin. Dr. Brandalise focused on the consequences of changes in dendritic ion channels in a mouse model of Fragile X syndrome with a focus on how channelopathies affected dendritic integration and the induction of local nonlinear membrane potential dynamics.

Footnotes

This paper is dedicated to the memory of Dr. Rick Gray.

Competing Interests

The authors declare they have no competing interests.

Data Availability Statement

The data that support the findings of this study are available upon request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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NIHMS1863640-supplement-supinfo2.xlsx (51.3KB, xlsx)
supinfo1
NIHMS1863640-supplement-supinfo1.pdf (371.3KB, pdf)

Data Availability Statement

The data that support the findings of this study are available upon request from the corresponding author.

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