Edited by: Igor Jakovcevski, Helmholtz-Gemeinschaft Deutscher Forschungszentren (HZ), Germany
Reviewed by: Maja Vulovic, University of Kragujevac, Serbia; Pavle R. Andjus, University of Belgrade, Serbia
This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology
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Maternal diabetes has been related to low verbal task scores, impaired fine and gross motor skills, and poor performance in graphic and visuospatial tasks during childhood. The primary motor cortex is important for controlling motor functions, and embryos exposed to high glucose show changes in cell proliferation, migration, and differentiation during corticogenesis. However, the existing studies do not discriminate between embryos with or without neural tube defects, making it difficult to conclude whether the reported changes are related to neural tube defects or other anomalies. Furthermore, postnatal effects on central nervous system cytoarchitecture and function have been scarcely addressed. Through molecular, biochemical, morphological, and electrophysiological approaches, we provide evidence of impaired primary motor cerebral cortex lamination and neuronal function in pups from diabetic rats, showing an altered distribution of SATB2, FOXP2, and TBR1, impaired cell migration and polarity, and decreased excitability of deep-layer cortical neurons, suggesting abnormalities in cortico-cortical and extra-cortical innervation. Furthermore, phase-plot analysis of action potentials suggests changes in the activity of potassium channels. These results indicate that high-glucose insult during development promotes complex changes in migration, neurogenesis, cell polarity establishment, and dendritic arborization, which in turn lead to reduced excitability of deep-layer cortical neurons.
Impairment in language, learning, memory, motor coordination, perception and problem-solving, as well as autism and schizophrenia, have been related to pregnancy diabetes (
Neurodevelopmental impairment affecting cognition in the offspring of diabetic mothers was first reported by
Prospective studies in early infancy reported lower mental development index (MDI) in the Bayley Scale of Infant Development in 1-year-old Israelite (
In older children,
Therefore, it appears that diabetes during pregnancy affects the neurodevelopment of offspring in the short and long term. However, this issue is difficult to study in humans due to the presence of confounding variables and the neurological complexity of the altered functions that involve several areas of the central nervios system (CNS) such as the cerebral cortex (motor, sensory, visual, and/or auditory), hippocampus, basal ganglia, amygdala, thalamus, and cerebellum. Animal models allow studying the effect of hyperglycemia at different stages of prenatal and postnatal development in a more controlled way, and obtaining brain tissue for evaluation at the cellular, functional, and molecular levels.
Few studies have analyzed neocortical development in embryos without neural tube defects or the offspring of diabetic dams.
In general, projection neurons originated during embryo corticogenesis are glutamatergic, and possess unique morphological, functional, and expression profiles (
Our research group is interested in studying the effect of maternal diabetes on motor performance, given its importance for language execution and fine and gross movements. We, therefore, explored changes in the primary motor cortex (M1), the main source of cortical synaptic information to the basal ganglia, a group of neuronal nuclei critically involved in the planning, execution, and control of voluntary movement (
We report here changes in the expression and distribution of SATB2, FOXP2, and TBR1 in M1 of neonates (Postnatal day 0; P0) and infants (P21) of diabetic rats, as well as aberrant distribution, impaired migration, and polarity establishment accompanied by electrophysiological changes, in deep cortical layer neurons in the P21 offspring of diabetic rats. These findings contribute to the knowledge of the postnatal consequences of
All experiments and animal manipulations were in accordance with the Animal Welfare Assurance (A5281-01), the ‘Guide for the Care and Use of Laboratory Animals’ (N.I.H. Publication No. 80-23, revised 1978) and ‘Norma Official Mexicana para la Producción, Cuidado y Uso de Animales de Laboratorio’ (NOM-062-ZOO-1999). The protocol was approved by the Research and Animal Care (CICUAL), Biosecurity, and Ethics committees of Instituto Nacional de Perinatología Isidro Espinosa de los Reyes (protocol 3230-21202-01-2015).
Female Wistar rats (230–300 g) were maintained at Instituto Nacional de Perinatología Isidro Espinoza de los Reyes animal facilities under standard conditions (12:12 h light/dark cycle, 21 ± 2°C and, 40% relative humidity) with access to water and food
At day 5 of pregnancy, rats were divided into two groups, control and diabetic, which received a single intraperitoneal injection of a citrate-buffered solution (vehicle; pH 6.4) or streptozotocin (STZ; 50 mg/kg body weight in 250 μl vehicle; Sigma Aldrich, St. Louis, MO, United States), respectively. After 24 h, plasma glucose levels were determined in a drop of blood obtained by puncture of the tail vein by using an electronic glucometer (ACCU-Check Performa, Roche Diagnostics, Basel, Switzerland). Rats injected with STZ and glucose levels higher than 200 mg/dl were assigned to the diabetic group, whereas animals with less of 200 mg/dl of glucose were discarded. Animals that received citrate-buffered solution with glucose levels 96–120 mg/dl were included in the control group.
For experiments involving embryos, pregnant rats were killed by decapitation at E14. The embryos were extracted, washed in cold phosphate-buffered saline (PBS, pH 7.4), and fixed by immersion in Boüin’s solution (15:15:1 saturated picric acid aqueous solution: 40% formalin:glacial acetic acid) followed by immersion in 15 and 30% sucrose gradients for 24 h each. After fixation, embryos were frozen in isopentane (Sigma-Aldrich) embedded in TissueTek (Sakura Finetek Europe, Flemingweg, Netherlands) and coronal slices (10 μm thick) were obtained with a cryostat (Leica CM1850 UV, Wetzlar, Germany).
For postnatal analyses, male neonates (P0) were weighted, glucose levels were measured (
Pups (P0 and P21) were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.; Laboratorios PiSA, Mexico City, Mexico), and transcardiac perfusion, through the ascending aorta, was performed with PBS (7.5 ml) followed by Boüin’s solution as fixator (7.5 ml), at room temperature and a constant flow rate of 0.5 ml/min (Masterflex L/S Digital Miniflex Pump, Vernon Hills, IL, United States). After decapitation, brains were removed and placed in Boüin’s solution for 72 h and then placed in 30% sucrose solution for 72 h for cryopreservation, embedded in TissueTek, frozen and maintained at −80°C until used.
The frontal cerebral cortex from one pup per litter (
The dynamic range was established for each amplification product to determine the fluorescence threshold and reaction efficacy. The presence of a single amplified product was ensured by a single peak in the melting curves. Preamplifier cDNA (15 cycles using the Gotaq D.N.A. polymerase; Promega) was used to evaluate changes in the relative expression of FoxP2, Tbr1, and Satb2 using the 2–ΔΔCT method (
qPCR was performed in a Rotor-Gene 6000 thermocycler (Qiagen, Germantown, MD, United States). The commercial KAPATM SYBER FAST® qPCR mix (KAPA Biosystems, Wilmington, MA, United States), the corresponding cDNA, 20 pmol of the forward (F) and reverse (R) primers, in a total volume of 10 μl was used for qPCR reactions. The amplification conditions were: 95°C for 10 min followed by 40 cycles at 95°C for 30 s, 63°C (Foxp2), 60°C (Tbr1), 57°C (Satb2) and 58°C (Gapdh) for 15 s and 72°C for 15 s.
Primer sequences were as follows:
Foxp2, F: 5′-GAAAGCGCGAGACACATCG-3′, R: 5′-GAA GCCCCCGAACAACACA-3′;
Tbr1, F: 5′-GGAAGTGAATGAGGACGGCA-3′, R: 5′-TGG CGTAGTTGCTCACGAAT-3′;
Satb2, F: 5′-CCGCACACAGGGATTATTGTC-3′, R: 5′-TCC ACTTCAGGCAGGTTGAG-3′;
Gapdh F: 5′-GGACCTCATGGCCTACATGG-3′, R: 5′-CCC CTCCTGTTGTTATGGGG-3′.
One or two E14, P0 or P21 animals were used per litter from 3–5 different litters (
Brain slices were washed with PBS, blocked and permeabilized (10% normal goat serum, and 0.3% Triton-X100 in PBS) during 1 h. Autofluorescence was blocked with TrueBlack lipofuscin autofluorescence quencher (Biotium, Hayward, CA, United States), and slices were then incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-SATB2 (1:250,
Micrographs of the frontal cortex (E14) and M1 (P0 and P21) were obtained using an epifluorescence microscope (Olympus IX81, Shinjuku, Tokyo, Japan) with a charge-coupled device camera (ORCA-FLASH 2.8 CCD model C11440; Hamamatsu, Honshu, Japan) and the images were processed with Adobe Photoshop CS6 (San Jose, CA, United States).
For fluorescence quantification, the exposition and gain values used for the first micrograph from a control sample for each marker were maintained for all subsequent acquisitions, and the fluorescence was measured using Fiji software (
Total protein was obtained from the fresh frontal cerebral cortex of one pup from five different litters (
Electrophoresis of protein samples (20 μg for SATB2 and TBR2, or 80 μg for FOXP2) and 2.5 μl of a commercial molecular weight standard (1610376, Precision Plus ProteinTM WesternCTM Blotting Standards, Bio-Rad or GTX50875, Trident Prestained Protein Ladder, GeneTex) was performed in denaturalizing 10% polyacrylamide gels with the Mini-Protean II system (Bio-Rad, Hercules, CA, United States). Proteins were transferred to nitrocellulose membranes (AmershamTM Hybond TM-ELC, Buckinghamshire, United Kingdom) using the
To evaluate dendritic orientation and to perform Sholl and dendritic complexity index (DCI) analyses, coronal brain slices from P21 pups from control and diabetic dams were analyzed with the FD Rapid GolgiStainTM Kit (FD Neurotechnologies, Columbia, MD, United States) following the manufacturer instructions. Animals were decapitated, the brain was rapidly removed, and coronal sections (60 μm thick) were obtained with a cryostat. The slices were placed in the developer solution for 1 min and then washed in distilled water for 5 min, dehydrated in alcohol (50, 75, 96, and 100%; 5 min each), transferred to xylol and mounted with Entellan (Merck, Darmstadt, Germany).
Reconstructions of several micrographs (20×) in different planes following the pyramidal neuron arborization were made, scale printed, and the drawings obtained from the complete neurons were scanned for analysis. At least six neurons from the M1 per pair of layers (I–II, III–IV, and V–VI) of three pups from three different litters per group were analyzed.
To evaluate dendritic orientation, the angle of insertion of the apical dendrite to the cell body was quantified using Fiji software (
To evaluate the morphological characteristics of the dendritic arbor of the pyramidal neurons, the Sholl intersection profile was performed, by counting the intersections of the dendritic arbor with an imaginary circle of a given radius (10 μm increases), as a function of the distance from the soma (
Based on the Sholl analysis, the dendritic complexity index (DCI), was calculated with the equation (
The ordinal value of a dendrite refers to the order in which a branch appears in relation to the first branch or primary dendrite (
Three P21 pups from three different dams (
The hemispheres were separated along the middle sagittal line, and the forebrain was cut and fixed to a vibratome (VT1000S; Leica, Nussloch, Germany) to obtain coronal slices (385 μm thick) containing the M1. The slices were incubated at 34°C for 30 min in a solution containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM Na2HPO4, 26 mM NaHCO3, 4 mM MgCl2, 10 mM D-(+)-glucose and 1 mM CaCl2, saturated with 95% O2 and 5% CO2 for 30 min at 34°C, followed by at least 60 min at room temperature.
The slices were then placed in a perfusion chamber and perfused (2–3 ml/min) with artificial cerebrospinal fluid at 32–33°C [composition in mM: 125 NaCl, 2.5 KCl, 1.25 Na2HPO4, 26 NaHCO3, 2 MgCl2, 10 D-(+)-glucose and 2 CaCl2; pH 7.4 after saturation with 95% O2 and 5% CO2]. Neurons were visualized with a Nikon FN-S2N microscope coupled to an infra-red contrast camera (Nikon, Tokyo, Japan) and an immersion objective 40×. Borosilicate pipettes with a resistance of 5–8 MΩ were obtained with a horizontal puller (Flaming-Brown P-97; Sutter Instrument, Novato, CA, United States) and filled with an intracellular solution containing 135 mM
Somatic recordings of pyramidal neurons from layers V and Vl (50–150 μm depth from the surface) were recorded in the whole-cell configuration of the patch clamp technique. Voltage- and current-clamp recordings were obtained with an Axoclamp 200B amplifier (Molecular Devices, Palo Alto, CA, United States), sampled and digitized at 10 kHz and filtered at 5 kHz (Digidata 1440A; Molecular Devices). Signals were acquired and analyzed off-line with pCLAMP 11.1 software (Molecular Devices). Additional filtering (50–60 Hz) was performed with a Hum Bug Electrical Noise eliminator (Quest Scientific, North Vancouver, BC, Canada). The resting membrane potential (RMP) was measured after initial break-in from gigaseal to whole-cell configuration. Pyramidal neurons were considered for analysis only if the access resistance was 5–9 MΩ and did not change 15% from the original value. Following the stabilization period (∼5 min), the cells were switched to the current-clamp mode, and different protocols were applied to determine input resistance (Rin), membrane time constant (τ), and the current required to elicit an action potential (rheobase) at the RMP of each recorded cell.
Rin was computed from the slope value obtained by plotting the voltage response to current injections of −30, 0, and +30 pA. The τ value was determined by adjusting an exponential function to the capacitive response elicited by current injection (30 pA, averaged from 10 consecutive sweeps). The I–V curves were constructed by applying current steps from −300 pA (30 pA steps, 1 s duration) until an action potential was elicited. Finally, the sag conductance or voltage drop was determined in response to a hyperpolarizing current step of -300 pA. The rheobase value and the action potential frequency were determined by applying current pulses from 0 to 500 pA (50 pA steps 1 s duration). The passive and active electrophysiological membrane properties were analyzed under basal or stimulated (histamine 1–100 μM) conditions, and analyses were strictly performed after the stabilization period.
Histamine acts as neuromodulator and neurotransmitter that, among several other functions, participates in spontaneous locomotion. The cerebral cortex is densely innervated by histaminergic fibers and expresses high levels of H1 receptors (H1Rs) (
Phase plots were constructed by plotting the time derivative of the somatic membrane potential (dV/dt) versus the somatic membrane potential of the first and fifth action potentials, and the mean ± the associated standard error (SEM) of the maximum rate values of the depolarization (Max DP dV/dt) or repolarization (Max RP dV/dt) phases of the action potential were plotted for the control and diabetic groups under basal and stimulated conditions.
The frontal cerebral cortex of one P21 offspring from 5 different litters (5 experiments) per group was dissected, and the tissue was homogenized in a hypotonic solution (10 mM Tris-HCl, 1 mM EGTA, pH 7.4) and centrifuged (20,000 ×
H1R density was determined in membrane aliquots incubated for 60 min at 30°C in 100 μl incubation buffer containing 10 nM of the selective H1R antagonist [3H]-mepyramine (PerkinElmer, Waltham, MA, United States) in the presence or absence of 10 μM unlabeled mepyramine to determine non-specific and total binding, respectively. The incubation was terminated by filtration through Whatman GF/B glass microfiber filters (GE Healthcare Life Science, Marlborough, MA, United States) soaked in 0.3% polyethyleneimine for 2 h. The radioactivity retained in the filters was determined by scintillation counting (
Histamine content in the frontal cerebral cortex was determined by ELISA (cat. 17-HISRT-E01-RES. sensitivity 0.2 ng/ml, 100% histamine specificity), following the protocol recommended by the supplier (ALPCO® immunoassays, Salem, NH, United States).
The cerebral cortex from one offspring from different litters (
Data are presented as means ± the associated standard error (SEM). Unpaired Student’s
We first analyzed by immunofluorescence the laminar distribution of markers for superficial (SATB2) and deep (FOXP2 and TBR1) cortical layers in the M1 of neonates (P0) and P21 rats from control and diabetic dams.
As expected, at P0 for both the control and diabetic groups the layer distribution of these transcription factors in M1 was different from that reported for the adult cortex as well as from other cortical areas of P0 rats (
SATB2, FOXP2, and TBR1 expression and distribution in the neonatal primary motor cortex from control and diabetic dams.
The analysis by qRT-PCR of the expression of the transcription factors showed a significant increase in Foxp2 and Tbr1 mRNAs in the diabetic group compared with the control group (2.45 ± 0.20- and 4.17 ± 0.75-fold of control values, respectively), with no significant change for Satb2 expression (0.76 ± 0.09 of control values;
To explore the distribution of the transcription factors at P21, we performed immunofluorescence assays in pups from diabetic and control dams. As expected for the motor cortex, deep layers were thicker than in other cortical areas, as evaluated by the immunoreactivity to the deep cortical layer markers FOXP2 and TBR1 in the control group (
SATB2, FOXP2, and TBR1 distribution in the primary motor cortex of 21-days-old offspring from control and diabetic rats.
Changes observed in the distribution of the cortical markers suggested impaired cell differentiation and migration. Hence, apical dendrites were visualized at P0 by MAP2 immunofluorescence. The immunoreactivity showed increased MAP2 expression and angled dendrites (
Migration and cell polarity markers in pups and embryos from control and diabetic rats.
To evaluate impaired migration and dendritic growth, Reelin immunofluorescence was performed in neonates, along Nestin staining to detect RG scaffolding for cortical neuron migration. In neonates from diabetic rats, the Reelin signal significantly decreased to 53 ± 12% of that observed in the control group (
To corroborate impaired neuron migration, the angle of insertion of the apical dendrite into the cell body of pyramidal neurons was evaluated in Golgi-Cox stained neurons from M1 of P21 pups. A large fraction of these neurons had an inadequate angle of insertion in the diabetic group (55%, 2.80-fold of control value;
Next, we determined the dendritic arborization and complexity by reconstructing Golgi-Cox staining neurons (
Dendritic arbor analysis in 21-days-old offspring from control and diabetic rats.
To determine whether DCI alterations resulted in changes in the electrophysiological membrane properties of deep-layer neurons, we examined the passive and active properties of layers V and VI pyramidal neurons in the absence and presence of histamine, which modulates neuronal excitability (
Current/voltage relationship in deep cortical primary motor cortex neurons of 21-days-old offspring from control and diabetic rats.
Electrophysiological properties of deep cortical layer neurons in P21 offspring of control and diabetic rats.
Control ( |
Diabetic ( |
|||||||
Basal | Histamine (μM) |
Basal | Histamine (μM) |
|||||
1 | 10 | 100 | 1 | 10 | 100 | |||
RMP (mV) | −70.0 ± 1.2 | −69.1 ± 0.7 | −68.6 ± 0.7 | −64.1 ± 0.8** | −74.6 ± 1.5 | −69.0 ± 0.8* | −70.3 ± 1.9 | −69.9 ± 1.3*,a |
Rin (MΩ) | 95.5 ± 11.8 | 122.9 ± 11.0* | 126.6 ± 10.0** | 144.6 ± 15.4*** | 132.0 ± 16.0 | 146.1 ± 18.5 | 151.6 ± 18.7 | 137.7 ± 20.5 |
τ (s) | 15.5 ± 2.3 | 14.6 ± 32.7 | 17.8 ± 3.7 | 14.1 ± 3.0 | 11.5 ± 3.9 | 12.8 ± 3.4 | 13.1 ± 2.3 | 14.0 ± 2.6 |
Rheobase (pA) | 172.2 ± 18.8 | 150.0 ± 14.4 | 138.8 ± 21.6 | 155.5 ± 37.6 | 255.5 ± 38.5 | 216.6 ± 42.4 | 244.4 ± 36.7 | 266.6 ± 42.4 |
Sag (mV) | 3.83 ± 0.46 | 4.86 ± 0.38 | 5.37 ± 0.59* | 5.68 ± 0.77** | 3.78 ± 0.85 | 5.06 ± 0.78 | 4.18 ± 0.84 | 3.65 ± 0.56 |
To analyze changes in the ionic currents that underlie the depolarization (Na+) and repolarization (K+) phases of the action potential, we constructed phase plots from the first and fifth action potentials elicited by current injection (300 pA), in the absence and presence of histamine (10 μM;
Phase plot analysis of action potentials.
Histamine increases neuronal excitability in several regions of the CNS such as the thalamus (
Analysis of action potential frequency in deep cortical neurons.
To evaluate if reduced H1R density underlies the altered response to histamine in the diabetic group, H1R and histamine levels were determined in the cerebral cortex of P21 pups. Binding assays showed no difference between groups in H1R density (50 ± 4 and 52 ± 9 fmol/mg of protein for the control and diabetic groups, respectively). Interestingly, a significantly higher amount of histamine was determined in the offspring of diabetic rats compared to the control group (354 ± 19 and 261 ± 35 ng/mg of protein, respectively;
In humans, maternal diabetes has been related to postnatal neurological impairment and psychiatric disorders due to fetal programming promoted by an inadequate environment (
At the cytoarchitectonic level, we found alterations in the distribution and expression of the transcription factors SATB2, FOXP2, and TBR1 at P0 and P21 that suggest dysregulation of cortical neurogenesis during embryo development associated with increased neurogenesis and impaired migration.
Increased neurogenesis is supported by higher SATB2, FOXP2, and TBR1 levels in M1, and by results previously reported for embryos of diabetic mice and rats (
In the diabetic group, SATB2 showed a significant increase by immunofluorescence and Western blot, with no change in mRNA levels, which could be explained by post-transcriptional regulation and degradation of Satb2 mRNA through microRNAs. Although microRNAs have not been assessed in brain tissues from the offspring of diabetic mothers, increased expression of miR-15a and -15b has been reported in the skeletal muscle of adult offspring of diabetic women (
An
The aberrant distribution of the transcription factors observed in the offspring of diabetic dams may have additional consequences for the establishment of neuronal networks. SATB2 regulates the development of cortico-thalamic and cortico-striatal pathways, as well as the formation of callosal projections in the superficial layers (
During corticogenesis, FOXP2 has been related to the acquisition and execution of motor sequences related to language in humans, singing in birds and ultrasonic vocalization in rodents (
Of note, FOXP2 regulates dopamine and cortico-striatal functions related to motor learning and working memory (
Transcription factors are expected to be nuclear, however, post-transcriptional modifications, such as SUMOylation and phosphorylation, can affect nucleocytoplasmic transport. Here we observed a perinuclear/cytoplasmic localization of SATB2 and FOXP2 in control P0 offspring 2 h after birth, a phenomenon more evident in the diabetic group, suggesting that a fraction of these transcription factors may be inactive or have different gene targets in neonatal rats.
Both SATB2 and FOXP2 possess SUMOylation and phosphorylation consensus sequences (
Interestingly, SUMOylation is involved in SATB2 perinuclear localization (
Phosphoproteomic analysis of neonatal murine brain (
Ubiquitination and phosphorylation are reversible, so the time after birth for tissue collection during P0 can also contribute to the differences observed in this study. However, this possibility is difficult to discuss since not all studies indicate the time after birth at which samples were obtained or due to differences in the day reported among studies.
We report here electrophysiological changes in deep cortical layer neurons in the P21 offspring of diabetic rats. Passive and active electrophysiological properties are tightly related to neuron morphology (
Although there were no significant differences in the passive properties between groups under the basal condition, both the membrane input resistance (Rin) and time constant (τ) showed tendencies that match the change in DCI. The Rin showed a marked drift to increase compared to control cells, and the time constant is faster and requires less time to reach its charging value. Together, these modifications suggest a reduction in neuronal area that might be related to higher K+ conductance at resting membrane potential.
The lack of statistical significance may also be related to the age of the animals, and it is likely that these changes consolidate in adult animals. In this regard, the adult brain (≥90 days old) might present a more significant reduction in the dendritic arbor of pyramidal neurons with pronounced changes in passive/active electrophysiological membrane properties.
In contrast, the electrophysiological active properties were altered leading to lower excitability under basal and stimulated conditions, which may be explained by an increase in K+ conductances that contribute to repolarization in the cell soma, such as BK calcium-activated potassium channels, Kv4 family channels that mediate the A-type current (
In several brain nuclei histamine increases neuronal excitability through a dual-action mediated by H1 and H2 receptors (see for instance
Furthermore, the lower firing rate of cortical neurons observed here under basal and stimulated conditions in the diabetic group may be due to changes in their morphology. This notion is supported by a study reporting reduced excitability of postnatal cortical pyramidal neurons following altered neuronal migration, probably due to reduced afferent connectivity, suggesting that synaptic integration and circuit formation are impaired (
The increase in action potential frequency promoted by histamine in deep-layer cortical neurons is in accordance with the increased excitability previously reported for neurons in other regions of the CNS, namely thalamus (
The consequences of the increased expression and changes in the laminar distribution of SATB2, TBR1, and FOXP2 are challenging to explain because most of the information previously reported relates to low or null expression in mutant animals. However, the abnormal distribution of the transcription factors and the electrophysiological alterations reported in this study suggest aberrant pathway formation and impaired cortical functions in the offspring of diabetic dams.
In summary, the expression and distribution of SATB2, TBR1, and FOXP2 suggest that high-glucose insult during development promotes complex changes in migration, neurogenesis, cell polarity establishment, and dendritic arborization, which in turn lead to reduced excitability of deep layers neurons. According to the role of the transcription factors investigated, our data suggest alterations in the cortico-cortical and extra-cortical innervation, a notion supported by the lower excitability of deep-layer neurons found. The postnatal effects of high glucose during fetal development
All datasets generated for this study are included in the article/
The animal study was reviewed and approved by the Research and Animal Care (CICUAL), Biosecurity, and Ethics committees of Instituto Nacional de Perinatología Isidro Espinosa de los Reyes (Protocol number 3230-21202-01-2015).
All authors participated in results analysis and discussion and drafting of the manuscript. RV-B performed and analyzed most of the experiments. BM-V conducted and analyzed the E14 experiments. AF-C performed P21 immunofluorescence and processed data. GG-L completed image acquisition and processing. GH-L, EG, and EJG performed electrophysiology experiments and analysis. ND, J-AA-M, and AM-H participated in all steps of the study, including conception, critical revision of the manuscript, experimental analysis, and supervision. AM-H approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We acknowledge funding by INPer and CONACyT to AM-H and CONACyT fellowship to RV-B (576845). We thank Talía Estrada Rojas (INPer) for assisting with animal care.
The Supplementary Material for this article can be found online at:
central nervous system
COUP-TF-interacting protein 2
Cut like homeobox 1
embryo day
Forkhead box protein P2
primary motor cortex
microtubule-associated protein 2
marginal zone
neural stem cells
radial glia
special AT-rich sequence-binding protein 2
T-box brain 1
ventricular zone.