Imaging Reactive Oxygen Radicals in Excised Mouse Lung Trapped by Reaction with Hydroxylamine Probes Using 1 GHz Rapid Scan Electron Paramagnetic Resonance.

PURPOSE: Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease. PROCEDURES: Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology. RESULTS: The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×1014 CP• and (9.9 ± 1.0) ×1014 DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells. CONCLUSION: The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.

Cyclic Disulfide-Bridged Dinitroxide Biradical for Measuring Thiol Redox Status by Electron Paramagnetic Resonance.

Among low-molecular-weight thiols, glutathione (GSH) is the main antioxidant in the cell, and its concentration is an indicator of the redox status. A cyclic disulfide-linked dinitroxide was designed for monitoring GSH by electron-paramagnetic resonance (EPR) spectroscopy. Reaction of the disulfide with GSH and three other thiols was measured at 9.6 GHz (X-band) and shown to be of first order in thiols. It is proposed that the reaction of the disulfide with 1 equiv of thiolate produced a short-lived intermediate that reacts with 1 equiv of thiolate to produce the cleavage product. The equilibrium ratio of the cleaved and intact disulfide is a measure of the redox state. Since the long-term goal is to use the disulfide to probe physiology in vivo, the feasibility of EPR spectroscopy and imaging of the disulfide and its cleavage product was demonstrated at 1 GHz (L-band).

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in the adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity," which has been examined during or after the classic "critical period." Because peripheral perturbations can alter the auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters the ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo widefield imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activities in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs, shifting the excitation-inhibition balance toward excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

PURPOSE: Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. PROCEDURES: In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10 mg/kg). At 24 h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. RESULTS: As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. CONCLUSIONS: We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity", which has been examined during or after the classic 'critical period'. Because peripheral perturbations can alter auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the classic critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activity in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs shifting the excitation-inhibition balance towards excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Cortical inhibitory but not excitatory synaptic transmission and circuit refinement are altered after the deletion of NMDA receptors during early development.

Neurons in the cerebral cortex form excitatory and inhibitory circuits with specific laminar locations. The mechanisms underlying the development of these spatially specific circuits is not fully understood. To test if postsynaptic N-methyl-D-aspartate (NMDA) receptors on excitatory neurons are required for the development of specific circuits to these neurons, we genetically ablated NMDA receptors from a subset of excitatory neurons in the temporal association cortex (TeA) through in utero electroporation and assessed the intracortical circuits connecting to L5 neurons through in vitro whole-cell patch clamp recordings coupled with laser-scanning photostimulation (LSPS). In NMDAR knockout neurons, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated connections were largely intact. In contrast both LSPS and mini-IPSC recordings revealed that γ-aminobutyric acid type A (GABAA) receptor-mediated connections were impaired in NMDAR knockout neurons. These results suggest that postsynaptic NMDA receptors are important for the development of GABAergic circuits.

Age-related changes in excitatory and inhibitory intra-cortical circuits in auditory cortex of C57Bl/6 mice.

A common impairment in aging is age-related hearing loss (presbycusis), which manifests as impaired spectrotemporal processing. Aging is accompanied by alteration in normal inhibitory (GABA) neurotransmission, and changes in excitatory (NMDA and AMPA) synapses in the auditory cortex (ACtx). However, the circuits affected by these synaptic changes remain unknown. Mice of the C57Bl/6J strain show premature age-related hearing loss and changes in functional responses in ACtx. We thus investigated how auditory cortical microcircuits change with age by comparing young (∼ 6 weeks) and aged (>1 year old) C57Bl/6J mice. We performed laser scanning photostimulation (LSPS) combined with whole-cell patch clamp recordings from Layer (L) 2/3 cells in primary auditory cortex (A1) of young adult and aged C57Bl/6J mice. We found that L2/3 cells in aged C57Bl/6J mice display functional hypoconnectivity of both excitatory and inhibitory circuits. Compared to cells from young C57Bl/6 mice, cells from aged C57Bl/6J mice have fewer excitatory connections with weaker connection strength. Whereas young adult and aged C57Bl/6J mice have similar amounts of inhibitory connections, the strength of local inhibition is weaker in the aged group. We confirmed these results by recording miniature excitatory (mEPSCs) and inhibitory synaptic currents (mIPSCs). Our results suggest a specific reduction in excitatory and inhibitory intralaminar cortical circuits in aged C57Bl/6J mice compared with young adult animals. We speculate that these unbalanced changes in cortical circuits contribute to the functional manifestations of age-related hearing loss.

Direct Measurement and Imaging of Redox Status with Electron Paramagnetic Resonance.

Significance: Fundamental to the application of tissue redox status to human health is the quantification and localization of tissue redox abnormalities and oxidative stress and their correlation with the severity and local extent of disease to inform therapy. The centrality of the low-molecular-weight thiol, glutathione, in physiological redox balance has long been appreciated, but direct measurement of tissue thiol status in vivo has not been possible hitherto. Recent advances in instrumentation and molecular probes suggest the feasibility of real-time redox assessment in humans. Recent Advances: Recent studies have demonstrated the feasibility of using low-frequency electron paramagnetic resonance (EPR) techniques for quantitative imaging of redox status in mammalian tissues in vivo. Rapid-scan (RS) EPR spectroscopy and imaging, new disulfide-dinitroxide spin probes, and novel analytic techniques have led to significant advances in direct, quantitative imaging of thiol redox status. Critical Issues: While novel RS EPR imaging coupled with first-generation molecular probes has demonstrated the feasibility of imaging thiol redox status in vivo, further technical advancements are desirable and ongoing. These include developing spin probes that are tailored for specific tissues with response kinetics tuned to the physiological environment. Equally critical are RS instrumentation with higher signal-to-noise ratio and minimal signal distortion, as well as optimized imaging protocols for image acquisition with sparsity adapted to image information content. Future Directions: Quantitative images of tissue glutathione promise to enable acquisition of a general image of mammalian and potentially human tissue health.

Novel nitroxide-bile acid conjugates inform substrate requirements for human bile acid transporters.

Transport of bile acids within the enterohepatic circulation from the liver to the intestines via the gallbladder and back to the liver via the portal vein plays a critical role in bile acid regulation and homeostasis. Deficiency of fibroblast growth factor 19 (FGF19), a hormone whose role is to suppress de novo hepatic bile acid synthesis to maintain homeostatic levels, results in bile acid diarrhea (BAD). FGF19 also modulates gallbladder motility so that bile acids are concentrated in the gallbladder until postprandial contraction. To assess bile acid transport and diagnose ailments like BAD that are associated with altered bile acid synthesis and transport, we created bile acid conjugates with nitroxide radicals. Because nitroxides are paramagnetic and can promote proton relaxation, we reasoned that these paramagnetic conjugates should act as contrast agents in in vivo magnetic resonance imaging (MRI). We tested substrate capability by assessing the inhibitory potential of these novel agents against taurocholate uptake by the apical sodium dependent bile acid transporter (ASBT) and the Na+/taurocholate cotransporting polypeptide (NTCP). Surprisingly, neither the paramagnetic compounds CA-Px-1 and CA-Px-2, nor their reduced forms, CA-Px-1H and CA-Px-2H, inhibited hASBT- or hNTCP-mediated taurocholate uptake. Therefore, the new conjugates cannot serve as contrast agents for MRI in vivo. However, our findings identify important structural constraints of transportable bile acid conjugates and suggest potential modifications to overcome these limitations.

Perinatal Opioid Exposure Results in Persistent Hypoconnectivity of Excitatory Circuits and Reduced Activity Correlations in Mouse Primary Auditory Cortex.

Opioid use by pregnant women results in neonatal opioid withdrawal syndrome (NOWS) and lifelong neurobehavioral deficits including language impairments. Animal models of NOWS show impaired performance in a two-tone auditory discrimination task, suggesting abnormalities in sensory processing in the auditory cortex. To investigate the consequences of perinatal opioid exposure on auditory cortex circuits, we administered fentanyl to mouse dams in their drinking water throughout gestation and until litters were weaned at postnatal day (P)21. We then used in vivo two-photon Ca2+ imaging in adult animals of both sexes to investigate how primary auditory cortex (A1) function was altered. Perinatally exposed animals showed fewer sound-responsive neurons in A1, and the remaining sound-responsive cells exhibited lower response amplitudes but normal frequency selectivity and stimulus-specific adaptation (SSA). Populations of nearby layer 2/3 (L2/3) cells in exposed animals showed reduced correlated activity, suggesting a reduction of shared inputs. We then investigated A1 microcircuits to L2/3 cells by performing laser-scanning photostimulation (LSPS) combined with whole-cell patch-clamp recordings from A1 L2/3 cells. L2/3 cells in exposed animals showed functional hypoconnectivity of excitatory circuits of ascending inputs from L4 and L5/6 to L2/3, while inhibitory connections were unchanged, leading to an altered excitatory/inhibitory balance. These results suggest a specific reduction in excitatory ascending interlaminar cortical circuits resulting in decreased activity correlations after fentanyl exposure. We speculate that these changes in cortical circuits contribute to the impaired auditory discrimination ability after perinatal opioid exposure.SIGNIFICANCE STATEMENT This is the first study to investigate the functional effects of perinatal fentanyl exposure on the auditory cortex. Experiments show that perinatal fentanyl exposure results in decreased excitatory functional circuits and altered population activity in primary sensory areas in adult mice. These circuit changes might underlie the observed language and cognitive deficits in infants exposed to opioids.

Transient Coupling between Infragranular and Subplate Layers to Layer 1 Neurons Before Ear Opening and throughout the Critical Period Depends on Peripheral Activity.

Cortical layer 1 (L1) contains a diverse population of interneurons that can modulate processing in superficial cortical layers, but the intracortical sources of synaptic input to these neurons and how these inputs change over development and with sensory experience is unknown. We here investigated the changing intracortical connectivity to L1 in the primary auditory cortex (A1) of mice of both sexes in in vitro slices across development using laser-scanning photostimulation. Before postnatal day (P)10, L1 cells receive excitatory input from within L1, L2/3, L4, and L5/6 as well as from subplate. Excitatory inputs from all layers increase, especially from L4, and peak during P10-P16, around the peak of the critical period for tonotopy. Inhibitory inputs followed a similar pattern. Functional circuit diversity in L1 emerges after P16. In adults, L1 neurons receive ascending inputs from L2/3 and L5/6, but only few inputs from L4. The transient hyperconnectivity from deep layers but not L2/3 is absent in deaf mice. Our results demonstrate that deep excitatory and superficial inhibitory circuits are tightly linked in early development and might provide a functional scaffold for the layers in between. These results suggest that early thalamically driven spontaneous and sensory activity in subplate can be relayed to L1 from the earliest ages on and shape L1 connectivity from deep layers. Our results also reveal a period of high transient columnar hyperconnectivity after ear opening coinciding with the critical period, suggesting that circuits originating in deep layers might play a key role in this process.SIGNIFICANCE STATEMENT L1 contains a diverse population of interneurons that can modulate processing in superficial cortical layers but the sources of synaptic input to these neurons and how these inputs change over development is unknown. We found that during the critical period a large fraction of excitatory inputs to L1 originated in L5/6 and the cortical subplate. This hyperconnectivity is absent in deaf mice. Our results directly demonstrate that deep excitatory and superficial inhibitory circuits are tightly linked in early development and might provide a functional scaffold for the layers in between.

Impaired Hearing and Altered Subplate Circuits During the First and Second Postnatal Weeks of Otoferlin-Deficient Mice.

Sensory deprivation from the periphery impacts cortical development. Otoferlin deficiency leads to impaired cochlear synaptic transmission and is associated with progressive hearing loss in adults. However, it remains elusive how sensory deprivation due to otoferlin deficiency impacts the early development of the auditory cortex (ACX) especially before the onset of low threshold hearing. To test that, we performed in vivo imaging of the ACX in awake mice lacking otoferlin (Otof-/-) during the first and second postnatal weeks and found that spontaneous and sound-driven cortical activity were progressively impaired. We then characterized the effects on developing auditory cortical circuits by performing in vitro recordings from subplate neurons (SPN), the first primary targets of thalamocortical inputs. We found that in Otof-/- pups, SPNs received exuberant connections from excitatory and inhibitory neurons. Moreover, as a population, SPNs showed higher similarity with respect to their circuit topology in the absence of otoferlin. Together, our results show that otoferlin deficiency results in impaired hearing and has a powerful influence on cortical connections and spontaneous activity in early development even before complete deafness. Therefore, peripheral activity has the potential to sculpt cortical structures from the earliest ages, even before hearing impairment is diagnosed.

Neonatal Hypoxia-Ischemia Causes Persistent Intracortical Circuit Changes in Layer 4 of Rat Auditory Cortex.

The connection between early brain injury and subsequent development of disorders is unknown. Neonatal hypoxia-ischemia (HI) alters circuits associated with subplate neurons (SPNs). SPNs are among the first maturing cortical neurons, project to thalamorecipient layer 4 (L4), and are required for the development of thalamocortical connections. Thus, early HI might influence L4 and such influence might persist. We investigated functional circuits to L4 neurons in neonatal rat HI models of different severities (mild and moderate) shortly after injury and at adolescence. We used laser-scanning photostimulation in slices of auditory cortex during P5-10 and P18-23. Mild injuries did not initially (P6/P7) alter the convergence of excitatory inputs from L2/3, but hyperconnectivity emerged by P8-10. Inputs from L4 showed initial hypoconnectivity which resolved by P8-10. Moderate injuries resulted in initial hypoconnectivity from both layers which resolved by P8-10 and led to persistent strengthening of connections. Inhibitory inputs to L4 cells showed similar changes. Functional changes were mirrored by reduced dendritic complexity. We also observed a persistent increase in similarity of L4 circuits, suggesting that HI interferes with developmental circuit refinement and diversification. Altogether, our results show that neonatal HI injuries lead to persistent changes in intracortical connections.

Aberrant development of excitatory circuits to inhibitory neurons in the primary visual cortex after neonatal binocular enucleation.

The development of GABAergic interneurons is important for the functional maturation of cortical circuits. After migrating into the cortex, GABAergic interneurons start to receive glutamatergic connections from cortical excitatory neurons and thus gradually become integrated into cortical circuits. These glutamatergic connections are mediated by glutamate receptors including AMPA and NMDA receptors and the ratio of AMPA to NMDA receptors decreases during development. Since previous studies have shown that retinal input can regulate the early development of connections along the visual pathway, we investigated if the maturation of glutamatergic inputs to GABAergic interneurons in the visual cortex requires retinal input. We mapped the spatial pattern of glutamatergic connections to layer 4 (L4) GABAergic interneurons in mouse visual cortex at around postnatal day (P) 16 by laser-scanning photostimulation and investigated the effect of binocular enucleations at P1/P2 on these patterns. Gad2-positive interneurons in enucleated animals showed an increased fraction of AMPAR-mediated input from L2/3 and a decreased fraction of input from L5/6. Parvalbumin-expressing (PV) interneurons showed similar changes in relative connectivity. NMDAR-only input was largely unchanged by enucleation. Our results show that retinal input sculpts the integration of interneurons into V1 circuits and suggest that the development of AMPAR- and NMDAR-only connections might be regulated differently.

Early peripheral activity alters nascent subplate circuits in the auditory cortex.

Cortical function can be shaped by sensory experience during a critical period. The onset of the critical period is thought to coincide with the onset of thalamocortical transmission to the thalamo-recipient layer 4 (L4). In early development, subplate neurons (SPNs), and not L4 neurons, are the first targets of thalamic afferents. SPNs are transiently involved in early development and are largely eliminated during development. Activation of L4 by thalamic afferents coincides with the opening of ear canal (~P11 in mice) and precedes the later critical period. Here, we show in mice that abolishing peripheral function or presenting sound stimuli even before P11 leads to bidirectionally altered functional connectivity of SPNs in auditory cortex. Thus, early sensory experience can sculpt subplate circuits before thalamocortical circuits to L4 are mature. Our results show that peripheral activity shapes cortical circuits in a sequential manner and from earlier ages than has been appreciated.

Transient Subgranular Hyperconnectivity to L2/3 and Enhanced Pairwise Correlations During the Critical Period in the Mouse Auditory Cortex.

During the critical period, neuronal connections are shaped by sensory experience. While the basis for this temporarily heightened plasticity remains unclear, shared connections introducing activity correlations likely play a key role. Thus, we investigated the changing intracortical connectivity in primary auditory cortex (A1) over development. In adult, layer 2/3 (L2/3) neurons receive ascending inputs from layer 4 (L4) and also receive few inputs from subgranular layer 5/6 (L5/6). We measured the spatial pattern of intracortical excitatory and inhibitory connections to L2/3 neurons in slices of mouse A1 across development using laser-scanning photostimulation. Before P11, L2/3 cells receive most excitatory input from within L2/3. Excitatory inputs from L2/3 and L4 increase after P5 and peak during P9-16. L5/6 inputs increase after P5 and provide most input during P12-16, the peak of the critical period. Inhibitory inputs followed a similar pattern. Functional circuit diversity in L2/3 emerges after P16. In vivo two-photon imaging shows low pairwise signal correlations in neighboring neurons before P11, which peak at P15-16 and decline after. Our results suggest that the critical period is characterized by high pairwise activity correlations and that transient hyperconnectivity of specific circuits, in particular those originating in L5/6, might play a key role.

Voltage-energized Calcium-sensitive ATP Production by Mitochondria.

Regulation of ATP production by mitochondria, critical to multicellular life, is poorly understood. Here we investigate the molecular controls of this process in heart and provide a framework for its Ca2+-dependent regulation. We find that the entry of Ca2+ into the matrix through the mitochondrial calcium uniporter (MCU) in heart has neither an apparent cytosolic Ca2+ threshold nor gating function and guides ATP production by its influence on the inner mitochondrial membrane (IMM) potential, ΔΨm. This regulation occurs by matrix Ca2+-dependent modulation of pyruvate and glutamate dehydrogenase activity and not through any effect of Ca2+ on ATP Synthase or on Electron Transport Chain Complexes II, III or IV. Examining the ΔΨm dependence of ATP production over the range of -60 mV to -170 mV in detail reveals that cardiac ATP synthase has a voltage dependence that distinguishes it fundamentally from the previous standard, the bacterial ATP synthase. Cardiac ATP synthase operates with a different ΔΨm threshold for ATP production than bacterial ATP synthase and reveals a concave-upwards shape without saturation. Skeletal muscle MCU Ca2+ flux, while also having no apparent cytosolic Ca2+ threshold, is substantially different from the cardiac MCU, yet the ATP synthase voltage dependence in skeletal muscle is identical to that in the heart. These results suggest that while the conduction of cytosolic Ca2+ signals through the MCU appears to be tissue-dependent, as shown by earlier work1, the control of ATP synthase by ΔΨm appears to be broadly consistent among tissues but is clearly different from bacteria.

Neonatal Hypoxia-Ischemia Causes Functional Circuit Changes in Subplate Neurons.

Neonatal hypoxia-ischemia (HI) in the preterm human results in damage to subcortical developing white matter and cognitive impairments. Subplate neurons (SPNs) are among the first-born cortical neurons and are necessary for normal cerebral development. While moderate or severe HI at P1 in rats leads to SPN loss, it is unclear if HI, esp. forms not associated with overt cell loss lead to altered SPN circuits. Thus, we used two HI models with different severities in P1 rats. Cauterization of the common carotid artery (CCA) causes a largely transient and thus milder ischemia (HI-Caut) while CCA ligation causes more severe ischemia (HI-Lig). While HI-Lig caused subplate damage, HI-Caut did not cause overt histological damage on the light microscopic level. We used laser-scanning photostimulation (LSPS) in acute thalamocortical slices of auditory cortex during P5-10 to study the functional connectivity of SPNs. Both HI categories resulted in hyperconnectivity of excitatory and inhibitory circuits to SPNs. Thus, alterations on the circuit level are present in the absence of cell loss. Our results show that SPN circuits are uniquely susceptible to HI. Given the key developmental role of SPNs, our results suggest that altered SPN circuits might underlie the abnormal development of cortical function after HI.

Author Correction: Voltage-energized calcium-sensitive ATP production by mitochondria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.