Dendritic Beading Research Articles

Artifactual dendritic beading in rat spinal cord induced by perfusion with cold saline and paraformaldehyde

Extensive dendritic beading of MAP2 (microtubule-associated protein 2) immunoreactivity has previously been observed in the contused rat spinal cord. However, we have also observed dendritic beading in occasional uninjured animals. The purpose of this study was to examine the possibility that perfusion conditions contributed to the dendritic beading. Under deep anesthesia, uninjured rats (adult female Long-Evans, 200–225 g) were transcardially perfused with 0.9% saline solution followed by 4% paraformaldehyde at cold (4 °C) or warm (20 °C) temperature, and at a low (20 ml/min) or high (50 ml/min) flow rate. Dendrites were visualized by MAP2 immunoreactivity. The results demonstrate that perfusion with cold solutions at a high flow rate induces pronounced dendritic beading, and when perfused at a low flow rate, results in moderate dendritic beading. Warm perfusates did not induce dendritic beading when administered at a low flow rate, but occasional beading was observed with a high flow rate. Western blots revealed spectrin breakdown, but not MAP2 loss, in rats perfused with cold saline solution at a high flow rate, conditions that also resulted in dendritic beading. These findings demonstrate that dendritic morphology is sensitive to both temperature and flow rate of the perfusate. Warm fixative and a low perfusion flow rate minimized the perfusion-induced dendritic beading.

Hippocampal seizures cause depolymerization of filamentous actin in neurons independent of acute morphological changes

Seizures may exert pathophysiological effects on dendritic spines, but the molecular mechanisms mediating these effects are poorly understood. Actin represents a major structural protein of dendritic spines, and actin filaments (F-actin) can be depolymerized by the regulatory molecule, cofilin, leading to structural or functional changes in spines in response to normal physiological activity. To investigate mechanisms by which pathophysiological stimuli may affect dendritic spine structure and function, we examined changes in F-actin and cofilin in hippocampus due to 4-aminopyridine (4-AP)-induced seizures/epileptiform activity in vivo and in vitro and investigated possible structural correlates of these changes in actin dynamics. Within an hour of induction, seizure activity caused both a significant decrease in F-actin labeling, indicating depolymerization of F-actin, and a corresponding decrease in phosphorylated cofilin, signifying an increase in cofilin activity. However, 4-AP seizures had no overt short-term structural effects on dendritic spine density. By comparison, high potassium caused a more dramatic decrease in cofilin and an immediate dendritic beading and loss of dendritic spines. These findings indicate that activation of cofilin and depolymerization of F-actin represent mechanisms by which seizures may exert pathophysiological modulation of dendritic spines. In addition to affecting non-structural functions of spines, the degree to which overt structural changes occur with actin depolymerization is dependent on the severity and type of the pathophysiological stimulus.

Calcium-dependent NMDA-induced dendritic injury and MAP2 loss in acute hippocampal slices

Excessive glutamate receptor stimulation can produce rapid disruption of dendritic morphology, including dendritic beading. We recently showed that transient N-methyl-d-aspartic acid (NMDA) exposure resulted in irreversible loss of synaptic function and loss of microtubule associated protein 2 (MAP2) from apical dendrites. The present study examined the initiation and progression of dendritic injury in mouse hippocampal slices following this excitotoxic stimulus. NMDA exposure (30 μM, 10 min) produced irregularly shaped dendritic swellings, evident first in distal apical dendrite branches, and later (20–90 min) involving most proximal dendrites. Over the same time course, immunoreactivity for the microtubule-associated protein MAP2 was progressively lost from apical dendrites, and increased in CA1 somata. This damage and MAP2 loss was Ca2+-dependent, and was not reversible within the time course of these experiments (90 min post-NMDA washout). Formation of regularly-spaced, spherical dendritic varicosities (dendritic beading) was rarely observed, except when NMDA was applied in Ca2+-free ACSF. Under these conditions, beading appeared predominant in interneurons, as assessed from experiments with GAD67-GFP (Δneo) mice. Ca2+-removal was associated with significantly better preservation of dendritic structure (MAP2) following NMDA exposure, and other ionic fluxes (sensitive to Gd3+ and spermine) may contribute to residual damage occurring in Ca2+-free conditions. These results suggest that irregularly shaped dendritic swelling is a Ca2+-dependent degenerative event that may be quite different from Ca2+-independent dendritic beading, and can be a predominant type of injury in CA1 pyramidal neurons in slices.

GENERATION OF THE RECEPTIVE FIELDS OF SUBPIAL CELLS IN TURTLE VISUAL CORTEX

The visual cortex of turtles contains cells with at least two different receptive field properties. Superficial units are located immediately below the pial surface. They fire in response to moving bars located anywhere in binocular visual space and to two spots of light presented with different spatiotemporal separations. Their location in the cortex suggests that superficial units correspond to a distinct class of inhibitory interneurons, the subpial cells, that are embedded in geniculocortical axons as they cross the visual cortex of turtles. This study used a detailed compartmental model of a subpial cell and a large-scale model of visual cortex to examine the cellular mechanisms that underlie the formation of superficial units on the assumption that they are subpial cells. Simulations with the detailed model indicated that the biophysical properties of subpial cells allow them to respond strongly to activation by geniculate inputs, but the presence of dendritic beads on the subpial cells decreases their sensitivity and allows them to integrate the inputs from many geniculate afferents. Simulations with the large-scale model indicated that the responses of subpial cells to simulated visual stimuli consist of two phases. A fast phase is mediated by direct geniculate inputs. A slow phase is mediated by recurrent excitation from pyramidal cells. It appears that subpial cells play a major role in controlling the information content of visual responses.

Roles of Glia‐Derived Cytokines on Neuronal Degeneration and Regeneration

Accumulation of activated microglia and reactive astrocytes is observed around degenerating neurons in various inflammatory or degenerative disorders in the central nervous system. These reactive glial cells may play either neurotoxic or neuroprotective roles. In this study, we examined the effects of glia-derived cytokines on neuronal degeneration and regeneration. Neuron-rich cultures were stimulated with supernatant of microglia and astrocytes stimulated with LPS, or a various concentrations of recombinant cytokines. Neurotoxicity was evaluated by an MTS assay. Neuronal damage was also evaluated by a frequency of dendritic beading, which was found to be an early feature of neuronal damage toward cell death. Effects of the cytokines on production of neurotrophic factors by astrocytes were also examined by RT-PCR for the expression of mRNA. Supernatant of LPS-stimulated microglia induced neuronal cell death. However, all the recombinant cytokines examined did not induce cell death, while IFNgamma and TNFalpha induced dendrite beading, an early feature of neuronal damage. IL-1beta and TNFalpha enhanced the production of neurotrophic factors by astrocytes. These observations suggest that glial cell-derived cytokines may synergistically function in neuronal degeneration with other toxic factors produced by activated microglia, and that some of them may also function in regeneration by inducing neurotrophic factors.

Cooling produces minimal neuropathology in neocortex and hippocampus

Cooling is a potential treatment for several neurological diseases. We have examined rodent and cat neocortex, cooled to 5 and 3 degrees C, respectively, to identify a lower limit for safely cooling brain. Rat neocortex, intermittently cooled with a thermoelectric device for 2 h, showed no signs of neuronal injury after cresyl violet or TUNEL staining. Neurons were also preserved in cat cortex cooled for up to 2 h daily for 10 months. Cooled rat and cat cortex showed glial proliferation, but this was also observed in sham-operated rat cortex. When hippocampal slices from mice expressing the Green Fluorescent Protein (GFP) in neurons were cooled to 5 degrees C, but not higher temperatures, we saw reversible dendritic beading and spine loss after 15-30 min. While there may be biochemical and functional alterations in brain cooled as low as 5 degrees C, the neuropathological consequences of brain cooling appear to be insignificant.

Long-Term Adeno-Associated Viral Vector-Mediated Expression of Truncated TrkB in the Adult Rat Facial Nucleus Results in Motor Neuron Degeneration

Adult facial motor neurons continue to express full-length TrkB tyrosine kinase receptor (TrkB.FL), the high-affinity receptor for the neurotrophins BDNF and neurotrophic factor-4/5 (NT-4/5), suggesting that they remain dependent on target-derived and locally produced neurotrophins in adulthood. Studies on the role of TrkB signaling in the adult CNS have been hampered by the early lethality of bdnf, nt-4/5, and trkB knock-out mice. We disrupted TrkB.FL signaling in adult facial motor neurons using adeno-associated viral vector-mediated overexpression of a naturally occurring dominant-negative TrkB receptor, TrkB.T1. Expression of TrkB.T1 resulted in neuronal atrophy and downregulation of NeuN (neuronal-specific nuclear protein) and ChAT expression in facial motor neurons. A subset of transduced neurons displayed signs of motor neuron degeneration that included dendritic beading and rounding of the soma at 2 months of TrkB.T1 expression. Cell counts revealed a significant reduction in motor neuron number in the facial nucleus at 4 months after onset of expression of TrkB.T1, suggesting that a proportion of TrkB.T1-expressing motor neurons became undetectable as a result of severe atrophy or was lost because of cell death. In contrast, overexpression of TrkB.FL did not result in a decrease in facial motor neuron number. Our results indicate that a subset of facial motor neurons remains dependent on TrkB ligands for the maintenance of structural and molecular characteristics in adulthood.

Synaptic activity becomes excitotoxic in neurons exposed to elevated levels of platelet-activating factor

Neurologic impairment in HIV-1-associated dementia (HAD) and other neuroinflammatory diseases correlates with injury to dendrites and synapses, but how such injury occurs is not known. We hypothesized that neuroinflammation makes dendrites susceptible to excitotoxic injury following synaptic activity. We report that platelet-activating factor, an inflammatory phospholipid that mediates synaptic plasticity and neurotoxicity and is dramatically elevated in the brain during HAD, promotes dendrite injury following elevated synaptic activity and can replicate HIV-1-associated dendritic pathology. In hippocampal slices exposed to a stable platelet-activating factor analogue, tetanic stimulation that normally induces long-term synaptic potentiation instead promoted development of calcium- and caspase-dependent dendritic beading. Chemical preconditioning with diazoxide, a mitochondrial ATP-sensitive potassium channel agonist, prevented dendritic beading and restored long-term potentiation. In contrast to models invoking excessive glutamate release, these results suggest that physiologic synaptic activity may trigger excitotoxic dendritic injury during chronic neuroinflammation. Furthermore, preconditioning may represent a novel therapeutic strategy for preventing excitotoxic injury while preserving physiologic plasticity.

Dendritic and Synaptic Pathology in Experimental Autoimmune Encephalomyelitis

Evidence has shown that excitotoxicity may contribute to the loss of central nervous system axons and oligodendrocytes in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Because dendrites and synapses are vulnerable to excitotoxicity, we examined these structures in acute and chronic models of EAE. Immunostaining for microtubule-associated protein-2 showed that extensive dendritic beading occurred in the white matter of the lumbosacral spinal cord (LSSC) during acute EAE episodes and EAE relapses. Retrograde labeling confirmed that most motoneuron dendrites were beaded in the white matter of the LSSC in acute EAE. In contrast, only mild swelling was observed in the gray matter of the LSSC. Dendritic beading showed marked recovery during EAE remission and after EAE recovery. In addition, synaptophysin, synapsin I, and PSD-95 immunoreactivities were significantly reduced in both the gray and white matter of the LSSC during acute EAE episodes and EAE relapses, but showed partial recovery during EAE remission and after EAE recovery. Pathologically, both dendritic beading and the reduction in synaptic protein immunoreactivity were well correlated with inflammatory cell infiltration in the LSSC at different EAE stages. We propose that dendritic and synaptic damage in the spinal cord may contribute to the neurological deficits in EAE.

Distally Directed Dendrotoxicity Induced by Kainic Acid in Hippocampal Interneurons of Green Fluorescent Protein-Expressing Transgenic Mice

Excitotoxicity, resulting from the excessive release of glutamate, is thought to contribute to a variety of neurological disorders, including epilepsy. Excitotoxic damage to dendrites, i.e., dendrotoxicity, is often characterized by the formation of large dendritic swellings, or "beads." Here, we show that hippocampal interneurons that express the neuropeptide somatostatin are highly vulnerable to the excitotoxic effects of the ionotropic glutamate receptor agonist kainate. Brief, focal iontophoretic application of kainate rapidly induced bead formation in dendrites of somatostatinergic interneurons that express green fluorescent protein (GFP) from mice of the transgenic line GIN (GFP-expressing inhibitory neurons). Surprisingly, beads often did not form at the site of kainate application or even in the dendritic segment to which kainate was applied; instead, dendritic beading occurred more distally, often encompassing all branches distal to the application site. We have termed this phenomena, "distally directed dendrotoxicity." Distally directed beading was induced regardless of the branch order of the site of application and was found to be dependent on activation of voltage-gated sodium channels. Subsequent to induction, distally directed beading would reverse in most cells; in other cells, however, beading irreversibly invaded proximal dendritic segments and gradually encompassed the entire dendritic tree. These results demonstrate that distal dendritic segments are highly vulnerable to excitotoxic injury and imply that excessive excitatory activity originating in one synaptic pathway can impact synapses at more distal dendritic segments of the same neuron. The discovery of this phenomenon will likely be important in understanding interneuronal dysfunction following excitotoxic injury.

Imaging and preventing spreading depression independent of cerebral blood flow

A moving cortical inactivation termed spreading depression (SD) is considered the physiological event responsible for migraine aura. SD is a mass depolarization of neurons and glia lasting a minute or more. It arises focally and migrates as a wave across gray matter at 2–5 mm/min. SD is generated by a sudden increase in cell membrane permeability to small ions in neurons and glia. This neurogenic origin does not preclude SD being initiated by local vascular change. However, the brain slice preparation does permit the study of SD independent of changes to blood flow or the microvasculature. When arising under near-normoxic conditions, the SD of aura dissipates within 30 min and causes no neuronal damage. In contrast, during the first 3–4 h following stroke, the combined metabolic stress of recurring SD and energy deprivation exacerbates ischemic injury to neurons in the penumbra. Purpose: To image and pharmacologically block SD under normoxic and stroke-like conditions in slices of rodent cerebral hemisphere where blood flow is not a modulating factor. Methods: Coronal brain slices are submerged in flowing artificial cerebrospinal fluid (aCSF). Normoxic SD is induced by briefly raising [K +] o. An ischemic version of SD, the anoxic depolarization (AD), is induced by removing O 2/glucose from the aCSF for 10 min. Intrinsic optical signals (IOSs) represent change in the way tissue scatters light. Light transmittance (LT), essentially unscattered light, is imaged using a charge-coupled device (CCD) to measure second-by-second regional ΔLT during periods of 1 h or more. The front of the propagating SD or AD event is imaged as an elevated LT, caused by cell swelling. A negative voltage shift, the electrophysiological signature of SD, is simultaneously recorded extracellularly. In the wake of the AD, the LT increase is overridden by a reduction in LT. This is caused by dendritic beading which efficiently scatters light, thereby revealing neuronal damage not seen following classic SD. Results: Normoxic SD evoked by elevated bath K + arises multifocally in neocortical layer II/III and migrates as a wave at 2–5 mm/min. It can be elicited repeatedly with no dendritic beading or loss of evoked electrical activity. The AD also initiates multifocally in neocortical gray (and 1–3 min later, in striatum or hippocampus); in its wake, dendritic beading develops and evoked electrical activity is permanently lost. Potentially useful therapeutics can be assessed in terms of whether they block the SD or reduce damage following AD. Glutamate receptor antagonists do not block the AD or resultant damage. However, sigma 1 receptor (σ 1R) ligands such as 10–100 μM dextromethorphan, carbetapentane, or 4-IBP block the AD and resultant damage. Normoxic SD is also blocked. σ 1R antagonists oppose AD or SD blockade by these ligands. Conclusions: Recurring SD is innocuous in normoxic slices. However, the metabolic stress of O 2/glucose deprivation (OGD) for 10 min and a single ischemic SD event (the AD) induces acute neuronal damage in slices. We propose that damage in the ischemic core is difficult to prevent because the AD arises upon stroke onset and normally resists pharmacological blockade. By dissociating the mass depolarization of AD from the ischemia, σ 1R ligands offer a possible way to reduce early stroke damage.

Two different mechanisms underlie reversible, intrinsic optical signals in rat hippocampal slices.

Intrinsic optical signals (IOSs) induced by synaptic stimulation and moderate hypotonic swelling in brain tissue slices consist of reduced light scattering and are usually attributed to cell swelling. During spreading depression (SD), however, light-scattering increases even though SD has been shown to cause strong cell swelling. To understand this phenomenon, we recorded extracellular voltage, light transmission (LT), which is inversely related to light scattering, and interstitial volume (ISV) simultaneously from the same site (stratum radiatum of CA1) in both interface and submerged hippocampal slices. As expected, moderate lowering of bath osmolarity caused concentration-dependent shrinkage of ISV and increase in LT, while increased osmolarity induced opposite changes in both variables. During severe hypotonia, however, after an initial increase of LT, the direction of the IOS reversed to a progressive decrease in spite of continuing ISV shrinkage. SD caused by hypotonia, by microinjection of high-K(+) solution, or by hypoxia, was associated with a pronounced LT decrease, during which ISV shrinkage indicated maximal cell swelling. If most of the extracellular Cl(-) was substituted by the impermeant anion methylsulfate and also in strongly hypertonic medium, the SD-related decrease in LT was suppressed and replaced by a monotonic increase. Nevertheless, the degree of ISV shrinkage was similar in low and in normal Cl(-) conditions. The optical signals and ISV changes were qualitatively identical in interface and submerged slices. We conclude that there are at least two mechanisms that underlie reversible optical responses in hippocampal slices. The first mechanism underlies light-scattering decrease (hence enhancing LT) when ISV shrinks (cell swelling) under synaptic stimulation and mild hypotonia. Similarly, as result of this mechanism, expansion of ISV (cell shrinkage) during mild hypertonia leads to an increased light scattering (and decreased LT). Thus optical signals associated with this first mechanism show expected cell-volume changes and are linked to either cell swelling or shrinkage. A different mechanism causes the light-scattering increase (leading to a LT decrease) during severe hypotonia and various forms of SD but with a severely decreased ISV. This second mechanism may be due to organelle swelling or dendritic beading but not to cell-volume increase. These two mechanisms can summate, indicating that they are independent in origin. Suppression of the SD-related light-scattering increase by lowering [Cl(-)](o) or severe hypertonia unmasks the underlying swelling-related scattering decrease. The simultaneous IOS and ISV measurements clearly distinguish these two mechanisms of optical signal generation.

Neurofibrillary Degeneration Can Be Arrested in an In Vivo Cellular Model of Human Tauopathy by Application of a Compound Which Inhibits Tau Filament Formation In Vitro

Although tau filament formation is a central event in familial tauopathies and Alzheimer's disease (AD), the cellular consequences of neurofibrillary tangle (NFT) formation are poorly understood because of the unavailability of mammalian in vivo cellular models of neurofibrillary degeneration (NFD). We have shown that human tau forms filaments and is associated with cytodegeneration when overexpressed chronically in identified neurons (ABCs) in the lamprey central nervous system (CNS). In this model, degeneration occurs according to a stereotyped sequence that closely resembles the pattern seen in tangle-bearing neurons in AD, with both tau deposition and fragmentation beginning in distal dendrites and progressing proximally over time. This sequence has been divided into four stages ranging from (1) mild beading of terminal dendrites only through (4) extensive dendritic fragmentation and loss. Here, we show that lipid-soluble, low-molecular-weight (approx 300 Da) proprietary compounds that have been demonstrated to block tau filament formation in vitro can significantly retard the progressive degeneration of ABCs that express human tau23. Bath application of one of these compounds for periods of up to 50 d after plasmid injection prevented degeneration beyond stage 2 in 90% of all treated cells, whereas over half of control cells showed severe degeneration by this time. This provides the first in vivo experimental evidence directly supporting a causal role for tau filament formation in the pathogenesis of NFD and suggests that intensive effort toward developing therapeutic agents for AD and other NFDs targeted at blocking tau filament formation is warranted.

N-methyl-D-aspartate receptor-dependent and -independent cytotoxic effects of Dictyostelium discoideum differentiation-inducing factor-1 on rat cortical neurons.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (small lipophilic hormone) that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. Recent studies have revealed that DIF-1 inhibits growth and induces the differentiation of mammalian tumor cells. The present study examines the effects of DIF-1 on rat cortical neurons in primary culture. We found that DIF-1 induced rapid neuronal cell death. The release of lactate dehydrogenase (LDH), as an indicator of cell death, increased dose-dependently with DIF-1. The release of LDH was inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists MK801 and AP5, suggesting that the NMDA receptor is involved in the induction of cell death by DIF-1. However, glutamate cytotoxicity could not explain the entire action of DIF-1 on neurons because the estimated concentration of glutamate around DIF-1-treated neurons was below 50 microM and DIF-1 caused more severe cell death than 500 microM glutamate. We discovered that another portion of DIF-1 cytotoxicity is independent of the NMDA receptor; that is, coaddition of DIF-1 and MK801 induced dendritic beading and increased expression of the immediate early genes c-fos and zif/268. These results indicate that DIF-1 induces rapid cell death via both NMDA receptor-dependent and -independent pathways in rat cortical neurons.

Intrinsic optical signals in respiratory brain stem regions of mice: neurotransmitters, neuromodulators, and metabolic stress.

In the rhythmic brain stem slice preparation, spontaneous respiratory activity is generated endogenously and can be recorded as output activity from hypoglossal XII rootlets. Here we combine these recordings with measurements of the intrinsic optical signal (IOS) of cells in the regions of the periambigual region and nucleus hypoglossus of the rhythmic slice preparation. The IOS, which reflects changes of infrared light transmittance and scattering, has been previously employed as an indirect sensor for activity-related changes in cell metabolism. The IOS is believed to be primarily caused by cell volume changes, but it has also been associated with other morphological changes such as dendritic beading during prolonged neuronal excitation or mitochondrial swelling. An increase of the extracellular K(+) concentration from 3 to 9 mM, as well as superfusion with hypotonic solution induced a marked increase of the IOS, whereas a decrease in extracellular K(+) or superfusion with hypertonic solution had the opposite effect. During tissue anoxia, elicited by superfusion of N(2)-gassed solution, the biphasic response of the respiratory activity was accompanied by a continuous rise in the IOS. On reoxygenation, the IOS returned to control levels. Cells located at the surface of the slice were observed to swell during periods of anoxia. The region of the nucleus hypoglossus exhibited faster and larger IOS changes than the periambigual region, which presumably reflects differences in sensitivities of these neurons to metabolic stress. To analyze the components of the hypoxic IOS response, we investigated the IOS after application of neurotransmitters known to be released in increasing amounts during hypoxia. Indeed, glutamate application induced an IOS increase, whereas adenosine slightly reduced the IOS. The IOS response to hypoxia was diminished after application of glutamate uptake blockers, indicating that glutamate contributes to the hypoxic IOS. Blockade of the Na(+)/K(+)-ATPase by ouabain did not provoke a hypoxia-like IOS change. The influences of K(ATP) channels were analyzed, because they contribute significantly to the modulation of neuronal excitability during hypoxia. IOS responses obtained during manipulation of K(ATP) channel activity could be explained only by implicating mitochondrial volume changes mediated by mitochondrial K(ATP) channels. In conclusion, the hypoxic IOS response can be interpreted as a result of cell and mitochondrial swelling. Cell swelling can be attributed to hypoxic release of neurotransmitters and neuromodulators and to inhibition of Na(+)/K(+)-pump activity.

Dendritic Spines Lost during Glutamate Receptor Activation Reemerge at Original Sites of Synaptic Contact

During cerebral ischemia, neurons undergo rapid alterations in dendritic structure consisting of focal swelling and spine loss. We used time-lapse microscopy to determine the fate of dendritic spines that disappeared after brief, sublethal hypoxic or excitotoxic exposures. Dendrite and spine morphology were assessed in cultured cortical neurons expressing yellow fluorescent protein or labeled with the fluorescent membrane tracer, DiI. Neurons exposed to NMDA, kainate, or oxygen-glucose deprivation underwent segmental dendritic beading and loss of approximately one-half of dendritic spines. Most spine loss was observed in regions of local dendritic swelling. Despite widespread loss, spines recovered within 2 hr after termination of agonist exposure or oxygen-glucose deprivation and remained stable over the subsequent 24 hr. Recovery was slower after NMDA than AMPA/kainate receptor activation. Time-lapse fluorescence imaging showed that the vast majority of spines reemerged in the same location from which they disappeared. In addition to spine recovery, elaboration of dendritic filopodia was observed in new locations along the dendritic shaft after dendrite recovery. Spine recovery did not depend on actin polymerization because it was not blocked by application of latrunculin-A, which eliminated filamentous actin staining in spines and blocked spine motility. Throughout spine loss and recovery, presynaptic and postsynaptic elements remained in physical proximity. These results suggest that elimination of dendritic spines is not necessarily associated with loss of synaptic contacts. Rapid reestablishment of dendritic spine synapses in surviving neurons may be a substrate for functional recovery after transient cerebral ischemia.

Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain slice.

Focal ischemia evokes a sudden loss of membrane potential in neurons and glia of the ischemic core termed the anoxic depolarization (AD). In metabolically compromised regions with partial blood flow, peri-infarct depolarizations (PIDs) further drain energy reserves, promoting acute and delayed neuronal damage. Visualizing and quantifying the AD and PIDs and their acute deleterious effects are difficult in the intact animal. In the present study, we imaged intrinsic optical signals to measure changes in light transmittance in the mouse coronal hemi-brain slice during AD generation. The AD was induced by oxygen/glucose deprivation (OGD) or by ouabain exposure. Potential neuroprotective strategies using glutamate receptor antagonists or reduced temperature were tested. Eight minutes of OGD (n = 18 slices) or 4 min of 100 microM ouabain (n = 14) induced a focal increase of increased light transmittance (LT) in neocortical layers II/III that expanded concentrically to form a wave front coursing through neocortex and independently through striatum. The front was coincident with a negative voltage shift in extracellular potential. Wherever the LT front (denoting cell swelling) propagated, a decrease in LT (denoting dendritic beading) followed in its wake. In addition the evoked field potential was permanently lost, indicating neuronal damage. Glutamate receptor antagonists did not block the onset and propagation of AD or the extent of irreversible damage post-AD. Lowering temperature to 25-30 degrees C protected the tissue from OGD damage by inhibiting AD onset. This study shows that anoxic depolarization evoked by global ischemia-like conditions is a spreading process that is focally initiated at multiple sites in cortical and subcortical gray. The combined energy demands of O(2)/glucose deprivation and the AD greatly exacerbate neuronal damage. Glutamate receptor antagonists neither block the AD in the ischemic core nor, we propose, block recurrent PID arising close to the core.

A role for sodium and chloride in kainic acid-induced beading of inhibitory interneuron dendrites

Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decreases in their synaptic activation and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity, at least in principal neurons of the hippocampus and cortex, is focal or varicose swellings of dendritic arbors. In experiments reported here, transient (1 h) exposure of hippocampal explant cultures to kainic acid produced marked focal swellings of the dendrites of parvalbumin-immunoreactive pyramidal basket cells in a highly reproducible and dose-dependent manner. At 5 mM kainic acid, more than half of the immunopositive apical dendrites in area CA 1 had a beaded appearance. However, the somal volumes of these cells were unaltered by the same treatment. The presence of focal swellings was reversible with kainate washout and was not accompanied by interneuronal cell death. In contrast, exposure to much higher concentrations (300 mM) of kainic acid resulted in the total loss of parvalbumin-positive interneurons from explants. Surprisingly, kainic acid-induced dendritic beading does not appear to be mediated by extracellular calcium. Beading was unaltered in the presence of N-methyl- d-aspartate receptor antagonists, the l-type calcium channel antagonist, nimodipine, cadmium, or by removing extracellular calcium. However, blockade of voltage-gated sodium channels by either tetrodotoxin or lidocaine abolished dendritic beading, while the activation of existing voltage-gated sodium channels by veratridine mimicked the kainic acid-induced dendritic beading. Finally, the removal of extracellular chloride prevented the kainic acid-induced dendritic beading. Thus, we suggest that the movement of Na + and Cl −, rather than Ca 2+, into cells underlies the focal swellings of interneuron dendrites in hippocampus.

Human tau filaments induce microtubule and synapse loss in an in vivo model of neurofibrillary degenerative disease.

The intracellular accumulation of tau protein and its aggregation into filamentous deposits is the intracellular hallmark of neurofibrillary degenerative diseases such as Alzheimer's Disease and familial tauopathies in which tau is now thought to play a critical pathogenic role. Until very recently, the lack of a cellular model in which human tau filaments can be experimentally generated has prevented direct investigation of the causes and consequences of tau filament formation in vivo. In this study, we show that human tau filaments formed in lamprey central neurons (ABCs) that chronically overexpress human tau resemble the 'straight filaments' seen in Alzheimer's Disease and other neurofibrillary conditions, and are distinguishable from neurofilaments by their ultrastructure, distribution and intracellular behavior. We also show that tau filament formation in ABCs is associated with a distinctive pattern of dendritic degeneration that closely resembles the cytopathology of human neurofibrillary degenerative disease. This pattern includes localized cytoskeletal disruption and aggregation of membranous organelles, distal dendritic beading, and the progressive loss of dendritic microtubules and synapses. These results suggest that tau filament formation may be responsible for many key cytopathological features of neurofibrillary degeneration, possibly via the loss of microtubule based intracellular transport.

Glutamate does not mediate acute neuronal damage after spreading depression induced by O2/glucose deprivation in the hippocampal slice.

This study argues that, in contrast to accepted excitotoxicity theory, O2/glucose deprivation damages neurons acutely by eliciting ischemic spreading depression (SD), a process not blocked by glutamate antagonists. In live rat hippocampal slices, the initiation, propagation, and resolution of SD can be imaged by monitoring wide-band changes in light transmittance (i.e., intrinsic optical signals). Oxygen/glucose deprivation for 10 minutes at 37.5 degrees C evokes a propagating wave of elevated light transmittance across the slice, representing the SD front. Within minutes, CA1 neurons in regions undergoing SD display irreversible damage in the form of field potential inactivation, swollen cell bodies, and extensively beaded dendrites, the latter revealed by single-cell injection of lucifer yellow. Importantly, glutamate receptor antagonists do not block SD induced by O2/glucose deprivation, nor do they prevent the resultant dendritic beading of CA1 neurons. However, CA1 neurons are spared if SD is suppressed by reducing the temperature to 35 degrees C during O2/glucose deprivation. This supports previous electrophysiologic evidence in vivo that SD during ischemia promotes acute neuronal damage and that glutamate antagonists are not protective of the metabolically stressed tissue. The authors propose that the inhibition of ischemic SD should be targeted as an important therapeutic strategy against stroke damage.