Identification of direct connections between the dura and the brain.

The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance1,2. How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems.

nNOS regulates ciliated cell polarity, ciliary beat frequency, and directional flow in mouse trachea.

Clearance of the airway is dependent on directional mucus flow across the mucociliary epithelium, and deficient flow is implicated in a range of human disorders. Efficient flow relies on proper polarization of the multiciliated cells and sufficient ciliary beat frequency. We show that NO, produced by nNOS in the multiciliated cells of the mouse trachea, controls both the planar polarity and the ciliary beat frequency and is thereby necessary for the generation of the robust flow. The effect of nNOS on the polarity of ciliated cells relies on its interactions with the apical networks of actin and microtubules and involves RhoA activation. The action of nNOS on the beat frequency is mediated by guanylate cyclase; both NO donors and cGMP can augment fluid flow in the trachea and rescue the deficient flow in nNOS mutants. Our results link insufficient availability of NO in ciliated cells to defects in flow and ciliary activity and may thereby explain the low levels of exhaled NO in ciliopathies.

Astrocyte deletion of α2-Na/K ATPase triggers episodic motor paralysis in mice via a metabolic pathway.

Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.

Zika Virus Infection in the Developing Mouse Produces Dramatically Different Neuropathology Dependent on Viral Strain.

Zika virus (ZIKV) infection during pregnancy has been causally linked to a constellation of neurodevelopmental deformities in the fetus resulting in a disease termed congenital Zika syndrome (CZS). Here we detail how ZIKV infection produces extensive neuropathology in the developing mouse brain and spinal cord of both sexes. Surprisingly, neuropathology differs depending on viral strain with a French Polynesian isolate producing primarily excitotoxicity and a Brazilian isolate being almost exclusively apoptotic but occurring over a prolonged period that is more likely to produce severe hypoplasia. We also show exposure can produce a characteristic pattern of infection that mirrors neuropathology and ultimately results in gross morphological deformities strikingly similar to CZS. This research provides a valuable mouse model mirroring the clinical course of disease that can be used to test potential therapies to improve treatment and gain a better understanding of the disabilities associated with CZS.SIGNIFICANCE STATEMENT Zika virus (ZIKV) infection during pregnancy has been causally linked to a constellation of neurodevelopmental deformities in the fetus resulting in a disease termed congenital Zika syndrome. Despite its devastating effects, very little is known about how ZIKV infection produces fetal neuropathology. Here we detail the temporal progression of ZIKV infection in the mouse brain and spinal cord resulting in massive neurodegeneration of infected regions. We also report a ZIKV strain from a region of Brazil with high levels of microcephaly (abnormally small head circumference) produces particularly devastating neuropathology.

Using animal models to evaluate the functional consequences of anesthesia during early neurodevelopment.

Fifteen years ago Olney and colleagues began using animal models to evaluate the effects of anesthetic and sedative agents (ASAs) on neurodevelopment. The results from ongoing studies indicate that, under certain conditions, exposure to these drugs during development induces an acute elevated apoptotic neurodegenerative response in the brain and long-term functional impairments. These animal models have played a significant role in bringing attention to the possible adverse effects of exposing the developing brain to ASAs when few concerns had been raised previously in the medical community. The apoptotic degenerative response resulting from neonatal exposure to ASAs has been replicated in many studies in both rodents and non-human primates, suggesting that a similar effect may occur in humans. In both rodents and non-human primates, significantly increased levels of apoptotic degeneration are often associated with functional impairments later in life. However, behavioral deficits following developmental ASA exposure have not been consistently reported even when significantly elevated levels of apoptotic degeneration have been documented in animal models. In the present work, we review this literature and propose a rodent model for assessing potential functional deficits following neonatal ASA exposure with special reference to experimental design and procedural issues. Our intent is to improve test sensitivity and replicability for detecting subtle behavioral effects, and thus enhance the translational significance of ASA models.

A gyral coordinate system predictive of fibre orientations.

When axonal fibres approach or leave the cortex, their trajectories tend to closely follow the cortical convolutions. To quantify this tendency, we propose a three-dimensional coordinate system based on the gyral geometry. For every voxel in the brain, we define a "radial" axis orthogonal to nearby surfaces, a "sulcal" axis along the sulcal depth gradient that preferentially points from deep white matter to the gyral crown, and a "gyral" axis aligned with the long axis of the gyrus. When compared with high-resolution, in-vivo diffusion MRI data from the Human Connectome Project, we find that in superficial white matter the apparent diffusion coefficient (at b = 1000) along the sulcal axis is on average 16% larger than along the gyral axis and twice as large as along the radial axis. This is reflected in the vast majority of observed fibre orientations lying close to the tangential plane (median angular offset < 7°), with the dominant fibre orientation typically aligning with the sulcal axis. In cortical grey matter, fibre orientations transition to a predominantly radial orientation. We quantify the width and location of this transition and find strong reproducibility in test-retest data, but also a clear dependence on the resolution of the diffusion data. The ratio of radial to tangential diffusion is fairly constant throughout most of the cortex, except for a decrease of the diffusivitiy ratio in the sulcal fundi and the primary somatosensory cortex (Brodmann area 3) and an increase in the primary motor cortex (Brodmann area 4). Although only constrained by cortical folds, the proposed gyral coordinate system provides a simple and intuitive representation of white and grey matter fibre orientations near the cortex, and may be useful for future studies of white matter development and organisation.

Improved tractography using asymmetric fibre orientation distributions.

Diffusion MRI allows us to make inferences on the structural organisation of the brain by mapping water diffusion to white matter microstructure. However, such a mapping is generally ill-defined; for instance, diffusion measurements are antipodally symmetric (diffusion along x and -x are equal), whereas the distribution of fibre orientations within a voxel is generally not symmetric. Therefore, different sub-voxel patterns such as crossing, fanning, or sharp bending, cannot be distinguished by fitting a voxel-wise model to the signal. However, asymmetric fibre patterns can potentially be distinguished once spatial information from neighbouring voxels is taken into account. We propose a neighbourhood-constrained spherical deconvolution approach that is capable of inferring asymmetric fibre orientation distributions (A-fods). Importantly, we further design and implement a tractography algorithm that utilises the estimated A-fods, since the commonly used streamline tractography paradigm cannot directly take advantage of the new information. We assess performance using ultra-high resolution histology data where we can compare true orientation distributions against sub-voxel fibre patterns estimated from down-sampled data. Finally, we explore the benefits of A-fods-based tractography using in vivo data by evaluating agreement of tractography predictions with connectivity estimates made using different in-vivo modalities. The proposed approach can reliably estimate complex fibre patterns such as sharp bending and fanning, which voxel-wise approaches cannot estimate. Moreover, histology-based and in-vivo results show that the new framework allows more accurate tractography and reconstruction of maps quantifying (symmetric and asymmetric) fibre complexity.

Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures.

Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)- or FA (palmitoylcarnitine)-derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups. Transmission electron microscopy images also showed no ultrastructural remodeling for failing vs. donor mitochondria; however, the fraction of lipid droplets (LDs) in direct contact with a mitochondrion was reduced, and the average distance between an LD and its nearest neighboring mitochondrion was increased. Analysis of FA processing gene expression between donor and failing LVs revealed 0.64-fold reduced transcript levels for the mitochondrial-LD tether, perilipin 5, in the failing myocardium (P = 0.003). Thus, reduced FA use in heart failure may result from improper delivery, potentially via decreased perilipin 5 expression and mitochondrial-LD tethering, and not from intrinsic mitochondrial dysfunction.-Holzem, K. M., Vinnakota, K. C., Ravikumar, V. K., Madden, E. J., Ewald, G. A., Dikranian, K., Beard, D. A., Efimov, I. R. Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

Experimental subarachnoid haemorrhage results in multifocal axonal injury.

The great majority of acute brain injury results from trauma or from disorders of the cerebrovasculature, i.e. ischaemic stroke or haemorrhage. These injuries are characterized by an initial insult that triggers a cascade of injurious cellular processes. The nature of these processes in spontaneous intracranial haemorrhage is poorly understood. Subarachnoid haemorrhage, a particularly deadly form of intracranial haemorrhage, shares key pathophysiological features with traumatic brain injury including exposure to a sudden pressure pulse. Here we provide evidence that axonal injury, a signature characteristic of traumatic brain injury, is also a prominent feature of experimental subarachnoid haemorrhage. Using histological markers of membrane disruption and cytoskeletal injury validated in analyses of traumatic brain injury, we show that axonal injury also occurs following subarachnoid haemorrhage in an animal model. Consistent with the higher prevalence of global as opposed to focal deficits after subarachnoid haemorrhage and traumatic brain injury in humans, axonal injury in this model is observed in a multifocal pattern not limited to the immediate vicinity of the ruptured artery. Ultrastructural analysis further reveals characteristic axonal membrane and cytoskeletal changes similar to those associated with traumatic axonal injury. Diffusion tensor imaging, a translational imaging technique previously validated in traumatic axonal injury, from these same specimens demonstrates decrements in anisotropy that correlate with histological axonal injury and functional outcomes. These radiological indicators identify a fibre orientation-dependent gradient of axonal injury consistent with a barotraumatic mechanism. Although traumatic and haemorrhagic acute brain injury are generally considered separately, these data suggest that a signature pathology of traumatic brain injury-axonal injury-is also a functionally significant feature of subarachnoid haemorrhage, raising the prospect of common diagnostic, prognostic, and therapeutic approaches to these conditions.

Isoflurane-induced apoptosis of neurons and oligodendrocytes in the fetal rhesus macaque brain.

BACKGROUND: The authors have previously shown that exposure of the neonatal nonhuman primate (NHP) brain to isoflurane for 5 h causes widespread acute apoptotic degeneration of neurons and oligodendrocyte. The current study explored the potential apoptogenic action of isoflurane in the fetal NHP brain. METHODS: Fetal rhesus macaques at gestational age of 120 days (G120) were exposed in utero for 5 h to isoflurane anesthesia (n = 5) or to no anesthesia (control condition; n = 4), and all regions of the brain were systematically evaluated 3 h later for evidence of apoptotic degeneration of neurons or glia. RESULTS: Exposure of the G120 fetal NHP brain to isoflurane caused a significant increase in apoptosis of neurons and of oligodendrocytes at a stage when oligodendrocytes were just beginning to myelinate axons. The neuroapoptosis response was most prominent in the cerebellum, caudate, putamen, amygdala, and several cerebrocortical regions. Oligodendrocyte apoptosis was diffusely distributed over many white matter regions. The total number of apoptotic profiles (neurons + oligodendrocytes) in the isoflurane-exposed brains was increased 4.1-fold, compared with the brains from drug-naive controls. The total number of oligodendrocytes deleted by isoflurane was higher than the number of neurons deleted. CONCLUSIONS: Isoflurane anesthesia for 5 h causes death of neurons and oligodendrocytes in the G120 fetal NHP brain. In the fetal brain, as the authors previously found in the neonatal NHP brain, oligodendrocytes become vulnerable when they are just achieving myelination competence. The neurotoxic potential of isoflurane increases between the third trimester (G120) and the neonatal period in the NHP brain.

Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration.

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.

Alcohol-induced apoptosis of oligodendrocytes in the fetal macaque brain.

BACKGROUND: In utero exposure of the fetal non-human primate (NHP) brain to alcohol on a single occasion during early or late third-trimester gestation triggers widespread acute apoptotic death of cells in both gray and white matter (WM) regions of the fetal brain. In a prior publication, we documented that the dying gray matter cells are neurons, and described the regional distribution and magnitude of this cell death response. Here, we present new findings regarding the magnitude, identity and maturational status of the dying WM cells in these alcohol-exposed fetal NHP brains. RESULTS: Our findings document that the dying WM cells belong to the oligodendrocyte (OL) lineage. OLs become vulnerable when they are just beginning to generate myelin basic protein in preparation for myelinating axons, and they remain vulnerable throughout later stages of myelination. We found no evidence linking astrocytes, microglia or OL progenitors to this WM cell death response. The mean density (profiles per mm3) of dying WM cells in alcohol-exposed brains was 12.7 times higher than the mean density of WM cells dying by natural apoptosis in drug-naive control brains. CONCLUSIONS: In utero exposure of the fetal NHP brain to alcohol on a single occasion triggers widespread acute apoptotic death of neurons (previous study) and of OLs (present study) throughout WM regions of the developing brain. The rate of OL apoptosis in alcohol-exposed brains was 12.7 times higher than the natural OL apoptosis rate. OLs become sensitive to the apoptogenic action of alcohol when they are just beginning to generate constituents of myelin in their cytoplasm, and they remain vulnerable throughout later stages of myelination. There is growing evidence for a similar apoptotic response of both neurons and OLs following exposure of the developing brain to anesthetic and anticonvulsant drugs. Collectively, this body of evidence raises important questions regarding the role that neuro and oligo apoptosis may play in the human condition known as fetal alcohol spectrum disorder (FASD), and also poses a question whether other apoptogenic drugs, although long considered safe for pediatric/obstetric use, may have the potential to cause iatrogenic FASD-like developmental disability syndromes.

Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain.

OBJECTIVE: Previously we reported that exposure of 6-day-old (P6) rhesus macaques to isoflurane for 5 hours triggers a robust neuroapoptosis response in developing brain. We have also observed (unpublished data) that isoflurane causes apoptosis of cellular profiles in the white matter that resemble glia. We analyzed the cellular identity of the apoptotic white matter profiles and determined the magnitude of this cell death response to isoflurane. METHODS: Neonatal (P6) rhesus macaques were exposed for 5 hours to isoflurane anesthesia according to current clinical standards in pediatric anesthesia. Brains were collected 3 hours later and examined immunohistochemically to analyze apoptotic neuronal and glial death. RESULTS: Brains exposed to isoflurane displayed significant apoptosis in both the white and gray matter throughout the central nervous system. Approximately 52% of the dying cells were glia, and 48% were neurons. Oligodendrocytes (OLs) engaged in myelinogenesis were selectively vulnerable, in contrast to OL progenitors, astrocytes, microglia, and interstitial neurons. When adjusted for control rates of OL apoptosis, the percentage of OLs that degenerated in the forebrain white matter of the isoflurane-treated group was 6.3% of the total population of myelinating OLs. INTERPRETATION: Exposure of the infant rhesus macaque brain to isoflurane for 5 hours is sufficient to cause widespread apoptosis of neurons and OLs throughout the developing brain. Deletion of OLs at a stage when they are just beginning to myelinate axons could potentially have adverse long-term neurobehavioral consequences that might be additive to the potential consequences of isoflurane-induced neuroapoptosis.

Ultrastructural studies in APP/PS1 mice expressing human ApoE isoforms: implications for Alzheimer's disease.

Alzheimer's disease is characterized in part by extracellular aggregation of the amyloid-ß peptide in the form of diffuse and fibrillar plaques in the brain. Electron microscopy (EM) has made an important contribution in understanding of the structure of amyloid plaques in humans. Classical EM studies have revealed the architecture of the fibrillar core, characterized the progression of neuritic changes, and have identified the neurofibrillary tangles formed by paired helical filaments (PHF) in degenerating neurons. Clinical data has strongly correlated cognitive impairment in AD with the substantial synapse loss observed in these early ultrastructural studies. Animal models of AD-type brain amyloidosis have provided excellent opportunities to study amyloid and neuritic pathology in detail and establish the role of neurons and glia in plaque formation. Transgenic mice overexpressing mutant amyloid precursor protein (APP) alone with or without mutant presenilin 1 (PS1), have shown that brain amyloid plaque development and structure grossly recapitulate classical findings in humans. Transgenic APP/PS1 mice expressing human apolioprotein E isoforms also develop amyloid plaque deposition. However no ultrastructural data has been reported for these animals. Here we show results from detailed EM analysis of amyloid plaques in APP/PS1 mice expressing human isoforms of ApoE and compare these findings with EM data in other transgenic models and in human AD. Our results show that similar to other transgenic animals, APP/PS1 mice expressing human ApoE isoforms share all major cellular and subcellular degenerative features and highlight the identity of the cellular elements involved in Aß deposition and neuronal degeneration.

Repetitive closed-skull traumatic brain injury in mice causes persistent multifocal axonal injury and microglial reactivity.

Repetitive mild or "concussive" traumatic brain injury (TBI) can cause substantial neurologic impairment, but the pathological features of this type of injury are not fully understood. We report an experimental model of TBI in which the closed skulls of anesthetized male C57BL/6J mice are struck with an electromagnetically controlled rubber impactor twice with an interval of 24 hours between impacts. The mice had deficits in Morris water maze performance in the first week after injury that only partially resolved 7 weeks later. By routine histology, there was no apparent bleeding, neuronal cell loss, or tissue disruption, and amyloid precursor protein immunohistochemistry demonstrated very few immunoreactive axonal varicosities. In contrast, silver staining revealed extensive abnormalities in the corpus callosum and bilateral external capsule, the ipsilateral cortex and thalamus, and the contralateral hippocampal CA1 stratum radiatum and stratum oriens. Electron microscopy of white matter regions demonstrated axonal cytoskeletal disruption, intra-axonal organelle compaction, and irregularities in axon caliber. Reactive microglia were observed in the same areas as the injured axons by both electron microscopy and Iba1 immunohistochemistry. Quantitative analyses of silver staining and Iba1 immunohistochemistry at multiple time points demonstrated transient cortical and thalamic abnormalities but persistent white matter pathology as late as 7 weeks after injury.Thus, prominent and long-lasting abnormalities in this TBI model were underestimated using conventional approaches. The model may be useful for mechanistic investigations and preclinical assessment of candidate therapeutics.

Diffusion characteristics associated with neuronal injury and glial activation following hypoxia-ischemia in the immature brain.

To identify quantitative MRI indices of injury in the brain following neonatal hypoxic-ischemic brain injury, we subjected mouse pups to hypoxia-ischemia on postnatal day 7 and obtained conventional and diffusion-weighted in vivo images of the brain 24 h later followed by histological assessment. T(2)-weighted images showed increased signal intensity in the CA1 and CA2 regions of the hippocampus ipsilateral to the injury and adjacent white matter. In contrast, diffusion imaging showed reduced apparent diffusion coefficient (ADC) values in CA1 and CA2, but increased values in the adjacent white matter. Histological analysis showed widespread gliosis with degenerating oligodendrocytes in the ipsilateral hippocampus. In addition, white matter areas that were abnormal by MRI showed an increase in the number of activated microglia (CD45 positive cells). Activated caspase-3 immunostaining showed a marked increase in neurons in the hippocampal regions corresponding to those with reduced ADC, and a quantitative measure of staining showed a statistically significant correlation with the ADC. In contrast, ADC was higher in adjacent white matter, where histology showed activation of microglia and reactive oligodendrocytes but not caspase-3 activation. These results suggest that the ADC response differs between areas of neuronal injury as compared with those showing glial changes without marked cell death.

Axons pull on the brain, but tension does not drive cortical folding.

During human brain development, the cerebral cortex undergoes substantial folding, leading to its characteristic highly convoluted form. Folding is necessary to accommodate the expansion of the cerebral cortex; abnormal cortical folding is linked to various neurological disorders, including schizophrenia, epilepsy, autism, and mental retardation. Although this process requires mechanical forces, the specific force-generating mechanisms that drive folding remain unclear. The two most widely accepted hypotheses are as follows: (1) Folding is caused by differential growth of the cortex and (2) folding is caused by mechanical tension generated in axons. Direct evidence supporting either theory, however, is lacking. Here we show that axons are indeed under considerable tension in the developing ferret brain, but the patterns of tissue stress are not consistent with a causal role for axonal tension. In particular, microdissection assays reveal that significant tension exists along axons aligned circumferentially in subcortical white matter tracts, as well as those aligned radially inside developing gyri (outward folds). Contrary to previous speculation, however, axonal tension is not directed across developing gyri, suggesting that axon tension does not drive folding. On the other hand, using computational (finite element) models, we show that differential cortical growth accompanied by remodeling of the subplate leads to outward folds and stress fields that are consistent with our microdissection experiments, supporting a mechanism involving differential growth. Local perturbations, such as temporal differences in the initiation of cortical growth, can ensure consistent folding patterns. This study shows that a combination of experimental and computational mechanics can be used to evaluate competing hypotheses of morphogenesis, and illuminate the biomechanics of cortical folding.

A highly toxic cellular prion protein induces a novel, nonapoptotic form of neuronal death.

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.