RESUMEN
Mild traumatic brain injury (mTBI) gives rise to a remarkable breadth of pathobiological consequences, principal among which are traumatic axonal injury and perturbation of the functional integrity of neuronal networks that may arise secondary to the elimination of the presynaptic contribution of axotomized neurons. Because there exists a vast diversity of neocortical neuron subtypes, it is imperative to elucidate the relative vulnerability to axotomy among different subtypes. Toward this end, we exploited SOM-IRES-Cre mice to investigate the consequences of the central fluid percussion model of mTBI on the microanatomical integrity and the functional efficacy of the somatostatin (SOM) interneuron population, one of the principal subtypes of neocortical interneuron. We found that the SOM population is resilient to axotomy, representing only 10% of the global burden of inhibitory interneuron axotomy, a result congruous with past work demonstrating that parvalbumin (PV) interneurons bear most of the burden of interneuron axotomy. However, the intact structure of SOM interneurons after injury did not translate to normal cellular function. One day after mTBI, the SOM population is more intrinsically excitable and demonstrates enhanced synaptic efficacy upon post-synaptic layer 5 pyramidal neurons as measured by optogenetics, yet the global evoked inhibitory tone within layer 5 is stable. Simultaneously, there exists a significant increase in the frequency of miniature inhibitory post-synaptic currents within layer 5 pyramidal neurons. These results are consistent with a scheme in which 1 day after mTBI, SOM interneurons are stimulated to compensate for the release from inhibition of layer 5 pyramidal neurons secondary to the disproportionate axotomy of PV interneurons. The enhancement of SOM interneuron intrinsic excitability and synaptic efficacy may represent the initial phase of a dynamic process of attempted autoregulation of neocortical network homeostasis secondary to mTBI.
Asunto(s)
Conmoción Encefálica , Animales , Axotomía , Interneuronas/fisiología , Ratones , Parvalbúminas , SomatostatinaRESUMEN
Mild traumatic brain injury (mTBI) affects brain structure and function and can lead to persistent abnormalities. Repetitive mTBI exacerbates the acute phase response to injury. Nonetheless, its long-term implications remain poorly understood, particularly in the context of traumatic axonal injury (TAI), a player in TBI morbidity via axonal disconnection, synaptic loss and retrograde neuronal perturbation. In contrast to the examination of these processes in the acute phase of injury, the chronic-phase burden of TAI and/or its implications for retrograde neuronal perturbation or death have received little consideration. To critically assess this issue, murine neocortical tissue was investigated at acute (24-h postinjury, 24hpi) and chronic time points (28-days postinjury, 28dpi) after singular or repetitive mTBI induced by central fluid percussion injury (cFPI). Neurons were immunofluorescently labeled for NeuroTrace and NeuN (all neurons), p-c-Jun (axotomized neurons) and DRAQ5 (cell nuclei), imaged in 3D and quantified in automated manner. Single mTBI produced axotomy in 10% of neurons at 24hpi and the percentage increased after repetitive injury. The fraction of p-c-Jun+ neurons decreased at 28dpi but without neuronal loss (NeuroTrace), suggesting their reorganization and/or repair following TAI. In contrast, NeuN+ neurons decreased with repetitive injury at 24hpi while the corresponding fraction of NeuroTrace+ neurons decreased over 28dpi. Attenuated NeuN expression was linked exclusively to non-axotomized neurons at 24hpi which extended to the axotomized at 28dpi, revealing a delayed response of the axotomized neurons. Collectively, we demonstrate an increased burden of TAI after repetitive mTBI, which is most striking in the acute phase response to the injury. Our finding of widespread axotomy in large fields of intact neurons contradicts the notion that repetitive mTBI elicits progressive neuronal death, rather, emphasizing the importance of axotomy-mediated change.
Asunto(s)
Conmoción Encefálica , Lesiones Encefálicas , Reacción de Fase Aguda/complicaciones , Reacción de Fase Aguda/metabolismo , Animales , Axones/metabolismo , Conmoción Encefálica/complicaciones , Conmoción Encefálica/metabolismo , Lesiones Encefálicas/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Ratones , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Mild traumatic brain injury (mTBI) can produce long lasting cognitive dysfunction. There is typically no cell death and only diffuse structural injury after mTBI. Thus, functional changes in intact neurons may contribute to symptoms. We have previously shown altered intrinsic properties of axotomized and intact neurons within 2 d after a central fluid percussion injury in mice expressing yellow fluorescent protein (YFP) that allow identification of axonal state prior to recording. Here, whole-cell patch clamp recordings were used to examine synaptic properties of YFP(+) layer V pyramidal neurons. An increased frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) was recorded from axotomized neurons at 1 d and intact neurons at 2 d after injury, likely reflecting an increased number of afferents. This also was reflected in the increased amplitude of the EPSC evoked by local extracellular stimulation for all neurons from injured cortex and increased likelihood of producing an action potential for intact cells. Field potentials recorded in superficial layers after online deep layer stimulation contained a single negative peak in controls but multiple negative peaks in injured tissue. The amplitude of this evoked negativity was significantly larger than controls over a series of stimulus intensities at both the 1 d and 2 d survival times. Interictal-like spikes never occurred in the field potential recordings from controls but were observed in 20-80% of stimulus presentations in injured cortex. Together, these results suggest an overall increase in network excitability and the production of particularly powerful (intact) neurons that have both increased intrinsic and synaptic excitability.
Asunto(s)
Potenciales de Acción/fisiología , Lesiones Encefálicas , Potenciales Postsinápticos Excitadores/fisiología , Neocórtex , Red Nerviosa , Células Piramidales , Animales , Axones/patología , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Masculino , Ratones , Ratones Endogámicos C57BL , Neocórtex/citología , Neocórtex/patología , Neocórtex/fisiopatología , Red Nerviosa/citología , Red Nerviosa/patología , Red Nerviosa/fisiopatología , Técnicas de Placa-Clamp , Células Piramidales/citología , Células Piramidales/patología , Células Piramidales/fisiologíaRESUMEN
Injured axons with distinct morphologies have been found following mild traumatic brain injury (mTBI), although it is currently unclear whether they reflect varied responses to the injury or represent different stages of progressing pathology. This complicates evaluation of therapeutic interventions targeting axonal injury. To address this issue, we assessed axonal injury over time within a well-defined axonal population, while also evaluating mitochondrial permeability transition as a therapeutic target. We utilized mice expressing yellow fluorescent protein (YFP) in cortical neurons which were crossed with mice which lacked Cyclophilin D (CypD), a positive regulator of mitochondrial permeability transition pore opening. Their offspring were subjected to mTBI and the ensuing axonal injury was assessed using YFP expression and amyloid precursor protein (APP) immunohistochemistry, visualized by confocal and electron microscopy. YFP(+) axons initially developed a single, APP(+), focal swelling (proximal bulb) which progressed to axotomy. Disconnected axonal segments developed either a single bulb (distal bulb) or multiple bulbs (varicosities), which were APP(-) and whose ultrastructure was consistent with ongoing Wallerian degeneration. CypD knock-out failed to reduce proximal bulb formation but decreased the number of distal bulbs and varicosities, as well as a population of small, APP(+), callosal bulbs not associated with YFP(+) axons. The observation that YFP(+) axons contain several pathological morphologies points to the complexity of traumatic axonal injury. The fact that CypD knock-out reduced some, but not all, subtypes highlights the need to appropriately characterize injured axons when evaluating potential neuroprotective strategies.
Asunto(s)
Axones/patología , Lesiones Encefálicas/patología , Encéfalo/patología , Ciclofilinas/deficiencia , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Axones/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Encéfalo/metabolismo , Lesiones Encefálicas/metabolismo , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Degeneración Walleriana/metabolismo , Degeneración Walleriana/patologíaRESUMEN
Traumatic axonal injury (TAI) is a consistent component of traumatic brain injury (TBI), and is associated with much of its morbidity. Increasingly, it has also been recognized as a major pathology of mild TBI (mTBI). In terms of its pathogenesis, numerous studies have investigated the susceptibility of the nodes of Ranvier, the paranode and internodal regions to TAI. The nodes of Ranvier, with their unique composition and concentration of ion channels, have been suggested as the primary site of injury, initiating a cascade of abnormalities in the related paranodal and internodal domains that lead to local axonal swellings and detachment. No investigation, however, has determined the effect of TAI upon the axon initial segment (AIS), a segment critical to regulating polarity and excitability. The current study sought to identify the susceptibility of these different axon domains to TAI within the neocortex, where each axonal domain could be simultaneously assessed. Utilizing a mouse model of mTBI, a temporal and spatial heterogeneity of axonal injury was found within the neocortical gray matter. Although axonal swellings were found in all domains along myelinated neocortical axons, the majority of TAI occurred within the AIS, which progressed without overt structural disruption of the AIS itself. The finding of primary AIS involvement has important implications regarding neuronal polarity and the fate of axotomized processes, while also raising therapeutic implications, as the mechanisms underlying such axonal injury in the AIS may be distinct from those described for nodal/paranodal injury.
Asunto(s)
Axones/patología , Axotomía/efectos adversos , Lesiones Encefálicas/etiología , Lesiones Encefálicas/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ancirinas/metabolismo , Axones/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Edema Encefálico/etiología , Edema Encefálico/patología , Lesiones Encefálicas/complicaciones , Moléculas de Adhesión Celular Neuronal/metabolismo , Citoesqueleto/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nódulos de Ranvier/patología , Factores de TiempoRESUMEN
Mild traumatic brain injury (mTBI) often produces lasting detrimental effects on cognitive processes. The mechanisms underlying neurological abnormalities have not been fully identified, in part due to the diffuse pathology underlying mTBI. Here we employ a mouse model of mTBI that allows for identification of both axotomized and intact neurons in the living cortical slice via neuronal expression of yellow fluorescent protein. Both axotomized and intact neurons recorded within injured cortex are healthy with a normal resting membrane potential, time constant (τ), and input resistance (R(in)). In control cortex, 25% of cells show an intrinsically bursting action potential (AP) firing pattern, and the rest respond to injected depolarizing current with a regular-spiking pattern. At 2 d postinjury, intrinsic bursting activity is lost within the intact population. The AP amplitude is increased and afterhyperpolarization duration decreased in axotomized neurons at 1 and 2 d postinjury. In contrast, intact neurons also show these changes at 1 d, but recover by 2 d postinjury. Two measures suggest an initial decrease in excitability in axotomized neurons followed by an increase in excitability within intact neurons. The rheobase is significantly increased in axotomized neurons at 1 d postinjury. The slope of the plot of AP frequency versus injected current is larger for intact neurons at 2 d postinjury. Together, these results demonstrate that intact and axotomized neurons are both affected by mTBI, resulting in different changes in neuronal excitability that may contribute to network dysfunction following TBI.
Asunto(s)
Potenciales de Acción/fisiología , Lesiones Encefálicas/patología , Fenómenos Electrofisiológicos/fisiología , Células Piramidales/lesiones , Células Piramidales/patología , Animales , Axotomía/métodos , Lesiones Encefálicas/fisiopatología , Masculino , Ratones , Neuronas/patología , Neuronas/fisiologíaRESUMEN
Traumatic axonal injury (TAI) is a consistent component of traumatic brain injury (TBI) and is associated with much of its morbidity. Little is known regarding the long-term retrograde neuronal consequences of TAI and/or the potential that TAI could lead to anterograde axonal reorganization and repair. To investigate the repertoire of anterograde and retrograde responses triggered by TIA, Thy1-YFP-H mice were subjected to mild central fluid percussion injury and killed at various times between 15 min and 28 d post-injury. Based upon confocal assessment of the endogenous neuronal fluorescence, such injury was found to result in diffuse TAI throughout layer V of the neocortex within yellow fluorescent protein (YFP)-positive axons. When these fluorescent approaches were coupled with various quantitative and immunohistochemical approaches, we found that this TAI did not result in neuronal death over the 28 d period assessed. Rather, it elicited neuronal atrophy. Within these same axotomized neuronal populations, TAI was also found to induce an early and sustained activation of the transcription factors c-Jun and ATF-3 (activating transcription factor 3), known regulators of axon regeneration. Parallel ultrastructural studies confirmed that these reactive changes are consistent with atrophy in the absence of neuronal death. Concurrent with those events ongoing in the neuronal cell bodies, their downstream axonal segments revealed, as early as 1 d post-injury, morphological changes consistent with reactive sprouting that was accompanied by significant axonal elongation over time. Collectively, these TAI-linked events are consistent with sustained neuronal recovery, an activation of a regenerative genetic program, and subsequent axonal reorganization suggestive of some form of regenerative response.
Asunto(s)
Axones/enzimología , Axones/patología , Lesión Axonal Difusa/enzimología , Lesión Axonal Difusa/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neurogénesis/genética , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 3/fisiología , Animales , Atrofia , Axones/metabolismo , Axotomía/métodos , Lesión Axonal Difusa/metabolismo , Activación Enzimática/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regeneración Nerviosa/genética , Neuronas/enzimología , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
A high membrane-to-cytoplasm ratio makes axons particularly vulnerable to traumatic injury. Posttraumatic shifts in ionic homeostasis promote spectrin cleavage, disrupt ankyrin linkages and destabilize axolemmal proteins. This study contrasted ankyrin-G and αII-spectrin degradation in cortex and corpus callosum following diffuse axonal injury produced by fluid percussion insult. Ankyrin-G lysis occurred preferentially in white matter, with acute elevation of all fragments and long-term reduction of a low kD form. Calpain-generated αII-spectrin fragments increased in both regions. Caspase-3 lysis of αII-spectrin showed a small, acute rise in cortex but was absent in callosum. White matter displayed nodal damage, with horseradish peroxidase permeability into the submyelin space. Ankyrin-G-binding protein neurofascin and spectrin-binding protein ankyrin-B showed acute alterations in expression. These results support ankyrin-G vulnerability in white matter following trauma and suggest that ankyrin-G and αII-spectrin proteolysis disrupts Node of Ranvier integrity. The time course of such changes were comparable to previously observed functional deficits in callosal fibers.
Asunto(s)
Ancirinas/metabolismo , Lesiones Encefálicas/patología , Regulación de la Expresión Génica/fisiología , Proteínas de Microfilamentos/metabolismo , Fibras Nerviosas Mielínicas/patología , Nódulos de Ranvier/patología , Proteínas de Transporte Vesicular/metabolismo , Análisis de Varianza , Animales , Barrera Hematoencefálica , Lesiones Encefálicas/metabolismo , Corteza Cerebral/metabolismo , Cuerpo Calloso/metabolismo , Modelos Animales de Enfermedad , Peroxidasa de Rábano Silvestre/metabolismo , Masculino , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Nódulos de Ranvier/metabolismo , Nódulos de Ranvier/ultraestructura , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 --> R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2(W56R) can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3'-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP2(3)('-UTR). Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.