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Dendritic bead etiology. Portion of a CA3 O-LM cell dendrite before ( A) and 10 min after ( B) KA administration (2 pulses at 10 A, 10 msec duration at 1 Hz; 100 M KA). Beads appeared at sites that had preexisting varicosities (large arrows), as well as sites that bore preexisting spines/filopodia (arrowheads). A bead was also found to originate from the node of an apparent nascent dendritic branch (small arrows). Note that beads did not form at many preexisting spines/filopodia, and that these structures were no longer visualized after stimulus. Scale bar, 10 m.
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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...
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Context 1
... the apparent correlations between sites of bead origi- nation and pre-existing dendritic structures in this retrospective analysis, we were unable to rule out that these were purely coincidental. Given the much larger sizes of beads with respect to the often smaller spacings between such preexisting structures, it appeared inevitable that beads would have to encompass one or more of these structures in many instances. Furthermore, as can be seen in Figure 5, many dendritic sites that contained preexist- ing spines/filopodia did not bead after KA administration. Con- sidering these findings, we could not unequivocally determine whether or not beads actually arose from these preexisting struc- tures. In short, determining a priori where a bead would originate was not ...
Context 2
... analysis was undertaken to identify anatomical structures from which distal dendritic beads might arise, by identifying dendritic sites that would eventually become beaded. Beads were often found to originate at sites that contained discrete dendritic elements, including: varicosities (Figs. 4 A, 5); spines/filopodia (Figs. 2 B, 5); branch points (Fig. 5); and short dendritic undula- tions or sharp turns. For 105 beads examined (n 10 cells), 95% were found to occur at dendritic sites that contained one or more of these structures. However, five beads were found to originate at sites containing no apparent unique structures, i.e., at apparently smooth dendritic ...
Citations
... These studies indicate that some level of dendrite beading is rapidly (minutes to several hours) reversible once normal activity or blood flow is restored (Murphy et al., 2008;Zeng et al., 2007). However, more global damage (Oliva et al., 2002) or repeated spreading depolarization after initial ischemia (Risher et al., 2010) can lead to irreversible beading and dendrite loss. Thus, it is likely that dendrites are acutely and irreversibly damaged in conditions that have a large impact on human health. ...
... Of the four inhibitory subclasses, the somatostatin (SOM) group is the most diverse, as revealed by studies using SOM-specific transgenic mouse lines (Oliva et al., 2002;Halabisky et al., 2006;Ma et al., 2006;McGarry et al., 2010;Riedemann et al., 2016). In recent years SOM diversity was reinforced by the transcriptomic taxonomies. ...
Inhibitory interneurons play a crucial role in proper development and function of the mammalian cerebral cortex. Of the different inhibitory subclasses, dendritic-targeting, somatostatin-containing (SOM) interneurons may be the most diverse. Earlier studies used transgenic mouse lines to identify and characterize subtypes of SOM interneurons by morphological, electrophysiological and neurochemical properties. More recently, large-scale studies classified SOM interneurons into 13 morpho-electro-transcriptomic (MET) types. It remains unclear, however, how these various classification schemes relate to each other, and experimental access to MET types has been limited by the scarcity of type-specific mouse driver lines. To begin to address these issues we crossed Flp and Cre driver mouse lines and a dual-color combinatorial reporter, allowing experimental access to genetically defined SOM subsets. Brains from adult mice of both sexes were retrogradely dye-labeled from the pial surface to identify layer 1-projecting neurons, and immunostained against several marker proteins, allowing correlation of genetic label, axonal target and marker protein expression in the same neurons. Using whole-cell recordings ex-vivo, we compared electrophysiological properties between intersectional and transgenic SOM subsets. We identified two layer 1-targeting intersectional subsets with non-overlapping marker protein expression and electrophysiological properties which, together with a previously characterized layer 4-targeting subtype, account for about half of all layer 5 SOM cells and >40% of all SOM cells, and appear to map onto 5 of the 13 MET types. Genetic access to these subtypes will allow researchers to determine their synaptic inputs and outputs and uncover their roles in cortical computations and animal behavior.
SIGNIFICANCE STATEMENT
Inhibitory neurons are critically important for proper development and function of the cerebral cortex. Although a minority population, they are highly diverse, which poses a major challenge to investigating their contributions to cortical computations and animal and human behavior. As a step towards understanding this diversity we crossed genetically modified mouse lines to allow detailed examination of genetically-defined groups of the most diverse inhibitory subtype, somatostatin-containing interneurons. We identified and characterized three somatostatin subtypes in the deep cortical layers with distinct combinations of anatomical, neurochemical and electrophysiological properties. Future studies could now use these genetic tools to examine how these different subtypes are integrated into the cortical circuit and what roles they play during sensory, cognitive or motor behavior.
... While the in vivo relevance of our findings remains to be firmly established, a significant proportion of work related to KA-induced neurotoxicity is carried out in isolated tissues (brain slices or acutely dissociated primary cells), with concentrations of KA often exceeding those employed in our experiments (Gepdiremen et al. 1997;Oliva et al. 2002;Hou 2011;Smialowska et al. 2012). Considering the seemingly high incidence of trace metal contamination in commonly used external solutions (Paoletti et al. 1997;Shcheglovitov et al. 2012), chelation by KA could obviously contribute to the observed effects and disinhibit Ca v 2.3 as well as other targets tonically suppressed by low nanomolar concentrations of Cu 2+ or Zn 2+ (Ca v 3.2, postsynaptic NMDA-receptors) (Spedding and Paoletti 1992;Nelson et al. 2007). ...
Kainic acid (KA) is a potent agonist at non‐N‐methyl‐D‐aspartate (non‐NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Cav2.3 voltage‐gated calcium channels have been implicated in the pathogenesis of KA‐induced seizures. At physiologically relevant concentrations, endogenous transition metal ions (Cu²⁺, Zn²⁺) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage‐dependent gating. Using whole‐cell patch‐clamp recordings in human embryonic kidney (HEK‐293) cells stably transfected with human Cav2.3d and β3‐subunits, we identified a novel, glutamate receptor‐independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu²⁺ and produces a hyperpolarizing shift in channel voltage‐dependence by about 10 mV, thereby reconciling the effects of Cu²⁺ chelation with tricine.
When tricine was used as a surrogate to study the receptor‐independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b‐wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Cav2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu²⁺‐induced allosteric suppression, presumably via formation of stable kainate‐Cu²⁺ complexes, could contribute to the receptor‐mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA in vitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.
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... Network hyperactivity can induce neuronal injury, in particular in GABA neurons (36), and is typified by dendritic swellings. In keeping with the increased network activity (GDPs, sEPSCs, and sIPSCs) received by GABA neurons, we consistently found large varicosities (suggesting swelling) along the dendrites of all GFP-expressing neurons in GIN mice (Fig. 3J) and recorded biocytin-filled interneurons (Fig. 3L) in caffeine-exposed offspring at P6. ...
Consumption of certain substances during pregnancy can interfere with brain development, leading to deleterious long-term neurological and cognitive impairments in offspring. To test whether modulators of adenosine receptors affect neural development, we exposed mouse dams to a subtype-selective adenosine type 2A receptor (A2AR) antagonist or to caffeine, a naturally occurring adenosine receptor antagonist, during pregnancy and lactation. We observed delayed migration and insertion of γ-aminobutyric acid (GABA) neurons into the hippocampal circuitry during the first postnatal week in offspring of dams treated with the A2AR antagonist or caffeine. This was associated with increased neuronal network excitability and increased susceptibility to seizures in response to a seizure-inducing agent. Adult offspring of mouse dams exposed to A2AR antagonists during pregnancy and lactation displayed loss of hippocampal GABA neurons and some cognitive deficits. These results demonstrate that exposure to A2AR antagonists including caffeine during pregnancy and lactation in rodents may have adverse effects on the neural development of their offspring.
... One alternative mechanism to homeostatic plasticity would be that intense synchronized network activity injures developing dendrites and this could lead to branch elimination (Zeng et al., 2007;Guo et al., 2012). One argument against this possibility is that, when very intense neuronal activity occurs, large amounts of glutamate are released that lead to excitotoxic injury and neuronal death, and at these times dendrites swell and form beads along their length Oliva et al., 2002;Zeng et al., 2007;Hoskison et al., 2007). However, we have not observed dendritic beading in disinhibited slice cultures. ...
Neuronal networks are thought to gradually adapt to altered neuronal activity over many hours and days. For instance, when activity is increased by suppressing synaptic inhibition, excitatory synaptic transmission is reduced. The underlying compensatory cellular and molecular mechanisms are thought to contribute in important ways to maintaining normal network operations. Seizures, due to their massive and highly synchronised discharging, probably challenge the adaptive properties of neurons, especially when seizures are frequent and intense - a condition common in early childhood. In the experiments reported here, we used rat and mice hippocampal slice cultures to explore the effects that recurring seizure-like activity has on the developing hippocampus. We found that developing networks adapted rapidly to recurring synchronised activity in that the duration of seizure-like events was reduced by 42% after 4 h of activity. At the same time, the frequency of spontaneous excitatory postsynaptic currents in pyramidal cells, the expression of biochemical biomarkers for glutamatergic synapses and the branching of pyramidal cell dendrites were all dramatically reduced. Experiments also showed that the reduction in N-methyl-D-aspartate receptor subunits and postsynaptic density protein 95 expression were N-methyl-D-aspartate receptor-dependent. To explore calcium signaling mechanisms in network adaptation, we tested inhibitors of calcineurin, a protein phosphatase known to play roles in synaptic plasticity and activity-dependent dendrite remodeling. We found that FK506 was able to prevent all of the electrophysiological, biochemical, and anatomical changes produced by synchronised network activity. Our results show that hippocampal pyramidal cells and their networks adapt rapidly to intense synchronised activity and that calcineurin play an important role in the underlying processes.
... These may be compartmentalized to dendrites, axons, or perhaps specific synapses [143,144]. Focal swelling [145] is accompanied by a disruption of ion homeostasis and ATP production [138,146] causing a failure of neuritic transport [147][148][149]. Autophagosomes form at sites with collapsed cytoskeletal proteins and dendritic swelling [150], as do focal elevations in cleaved caspase-3 [151], respectively, suggesting roles for autophagy and "synaptic apoptosis" [152,153]. ...
Opiate abuse and HIV-1 have been described as interrelated epidemics, and even in the advent of combined anti-retroviral therapy, the additional abuse of opiates appears to result in greater neurologic and cognitive deficits. The central nervous system (CNS) is particularly vulnerable to interactive opiate-HIV-1 effects, in part because of the unique responses of microglia and astroglia. Although neurons are principally responsible for behavior and cognition, HIV-1 infection and replication in the brain is largely limited to microglia, while astroglia and perhaps glial progenitors can be latently infected. Thus, neuronal dysfunction and injury result from cellular and viral toxins originating from HIV-1 infected/exposed glia. Importantly, subsets of glial cells including oligodendrocytes, as well as neurons, express µ-opioid receptors and therefore can be direct targets for heroin and morphine (the major metabolite of heroin in the CNS), which preferentially activate µ-opioid receptors. This review highlights findings that neuroAIDS is a glially driven disease, and that opiate abuse may act at multiple glial-cell types to further compromise neuron function and survival. The ongoing, reactive cross-talk between opiate drug and HIV-1 co-exposed microglia and astroglia appears to exacerbate critical proinflammatory and excitotoxic events leading to neuron dysfunction, injury, and potentially death. Opiates enhance synaptodendritic damage and a loss of synaptic connectivity, which is viewed as the substrate of cognitive deficits. We especially emphasize that opioid signaling and interactions with HIV-1 are contextual, differing among cell types, and even within subsets of the same cell type. For example, astroglia even within a single brain region are heterogeneous in their expression of µ-, δ-, and κ-opioid receptors, as well as CXCR4 and CCR5, and Toll-like receptors. Thus, defining the distinct targets engaged by opiates in each cell type, and among brain regions, is critical to an understanding of how opiate abuse exacerbates neuroAIDS.
... Somewhat surprisingly, 1 hr pH 6.0 treatment had no apparent effect on dendrites ( Figure 7A). Since enlargement or "blebbing" of dendritic shafts was a typical phenomenon observed during dendrotoxicity (Oliva et al., 2002;Chen et al., 2011), we further quantified the shaft width of apical dendrites and found that pH 6 treatment had no significant effect on the width of dendritic shaft ( Figure 7A). ...
... While it has been known for some time that ASICs localize to dendrites and mediate protoninduced increase in [Ca 2+ ] i there (Wemmie et al., 2002;Alvarez de la Rosa et al., 2003;Zha et al., 2006;Zha et al., 2009b), no study has addressed a potential site-specific effect of acidosis in the dendritic region. Our finding that 1 hr of pH 6.0 treatment had no obvious effect on dendritic architecture is somewhat unexpected, given the fact that dendrites are sensitive to excitotoxic stimuli (Oliva et al., 2002;Chen et al., 2011). Nevertheless, this result is consistent with one EM study showing that there was no major morphological changes in dendrites after 1-1.5 hrs of respiratory acidosis, which decreased brain pH to 6.3-6.0 (Schlote et al., 1975). ...
Acid-sensing ion channel-1a (ASIC1a) is a potential therapeutic target for multiple neurological diseases. We studied here ASIC1a glycosylation and trafficking, two poorly understood processes pivotal in determining the functional outcome of an ion channel. We found that most ASIC1a in the mouse brain was fully glycosylated. Inhibiting glycosylation with tunicamycin reduced ASIC1a surface trafficking, dendritic targeting, and acid-activated current density. N-glycosylation of the two glycosylation sites, Asn393 and Asn366, has differential effects on ASIC1a biogenesis. Maturation of Asn393 increased ASIC1a surface and dendritic trafficking, pH sensitivity, and current density. In contrast, glycosylation of Asn366 was dispensable for ASIC1a function and may be a rate-limiting step in ASIC1a biogenesis. In addition, we revealed that acidosis reduced the density and length of dendritic spines in a time- and ASIC1a-dependent manner. ASIC1a N366Q, which showed increased glycosylation and dendritic targeting, potentiated acidosis-induced spine loss. Conversely, ASIC1a N393Q, which had diminished dendritic targeting and inhibited ASIC1a current dominant-negatively, had the opposite effect. These data tie N-glycosylation of ASIC1a with its trafficking. More importantly, by revealing a site-specific effect of acidosis on dendritic spines, our findings suggest that these processes have an important role in regulating synaptic plasticity and determining long-term consequences in diseases that generate acidosis.
... Smaller varicosity size has been shown to indicate a metabolically healthier hippocampus in ischemic stroke (Davies et al., 2007 ), and indeed, our results support this idea (Figs. 4 – 8). Previous findings have shown that swelling in distal dendrites occurs first, followed by swelling in proximal dendrites (Park et al., 1996; Oliva et al., 2002). The increased number of varicosities can be explained by the fact that cypin overexpression, which results in decreased PSD-95 clustering , results in increased number of distal dendrites (Firestein et al., 1999; Chen and Firestein, 2007). ...
Focal swelling or varicosity formation in dendrites and loss of dendritic spines are the earliest indications of glutamate-induced excitotoxicity. Although it is known that microtubule dynamics play a role in varicosity formation, very little is known about the proteins that directly impact microtubules during focal swelling and dendritic spine loss. Our laboratory has recently reported that the postsynaptic protein PSD-95 and its cytosolic interactor (cypin) regulate the patterning of dendrites in hippocampal neurons. Cypin promotes microtubule assembly, and PSD-95 disrupts microtubule organization. Thus, we hypothesized that cypin and PSD-95 may play a role in altering dendrite morphology and spine number in response to sublethal NMDA-induced excitotoxicity. Using an in vitro model of glutamate-induced toxicity in rat hippocampal cultures, we found that cypin overexpression or PSD-95 knockdown increases the percentage of neurons with varicosities and the number of varicosities along dendrites, decreases the size of varicosities after sublethal NMDA exposure, and protects neurons from NMDA-induced death. In contrast, cypin knockdown or PSD-95 overexpression results in opposite effects. We further show that cypin regulates the density of spines/filopodia: cypin overexpression decreases the number of protrusions per micrometer of dendrite while cypin knockdown results in an opposite effect. Cypin overexpression and PSD-95 knockdown attenuate NMDA-promoted decreases in protrusion density. Thus, we have identified a novel pathway by which the microtubule cytoskeleton is regulated during sublethal changes to dendrites.
... It is known that dendrites can also be damaged, but it has not been clearly established whether they have a program of degeneration in the same way that axons do. Dendrite beading has been described during excitotoxicity and after ischemia (Oliva et al., 2002; Greenwood et al., 2007; Zeng et al., 2007; Li and Murphy, 2008; Murphy et al., 2008). However, this beading may not be related to that seen after axon severing as it is reversible in at least some cases (Oliva et al., 2002; Greenwood et al., 2007; Zeng et al., 2007; Li and Murphy, 2008; Murphy et al., 2008). ...
... Dendrite beading has been described during excitotoxicity and after ischemia (Oliva et al., 2002; Greenwood et al., 2007; Zeng et al., 2007; Li and Murphy, 2008; Murphy et al., 2008). However, this beading may not be related to that seen after axon severing as it is reversible in at least some cases (Oliva et al., 2002; Greenwood et al., 2007; Zeng et al., 2007; Li and Murphy, 2008; Murphy et al., 2008). Both axons and dendrites have programs of developmental disassembly known as pruning. ...
Neurons have two types of processes: axons and dendrites. Axons have an active disassembly program activated by severing. It has not been tested whether dendrites have an analogous program. We sever Drosophila dendrites in vivo and find that they are cleared within 24 h. Morphologically, this clearance resembles developmental dendrite pruning and, to some extent, axon degeneration. Like axon degeneration, both injury-induced dendrite degeneration and pruning can be delayed by expression of Wld(s) or UBP2. We therefore hypothesized that they use common machinery. Surprisingly, comparison of dendrite pruning and degeneration in the same cell demonstrated that none of the specific machinery used to prune dendrites is required for injury-induced dendrite degeneration. In addition, we show that the rapid program of dendrite degeneration does not require mitochondria. Thus, dendrites do have a rapid program of degeneration, as do axons, but this program does not require the machinery used during developmental pruning.
... Importantly, particular neuron class-specific promoters contain multiple genetic elements that support expression in different subclasses of neurons. Studies using transgenic mice have shown that the TH, GAD65, and GAD67 promoters each contain multiple genetic elements that support expression in different subclasses of catecholaminergic (Liu et al., 1997) or GABAergic (Bali et al., 2005;Chattopadhyaya et al., 2004;Di Cristo et al., 2004;Heinke et al., 2004;Kobayashi et al., 2003;Lopez-Bendito et al., 2004;Ma et al., 2006;Makinae et al., 2000;Oliva et al., 2000;Oliva et al., 2002;Tamamaki et al., 2003) neurons, respectively. ...
... Thus, further experiments, beyond the scope of this study, are now indicated to directly determine if the upstream promoter and the first intron support expression in specific, and predominately different, subclasses of glutamatergic neurons. Nonetheless, the TH, GAD65, and GAD67 promoters each contain multiple genetic elements that support expression in different subclasses of catecholaminergic (Liu et al., 1997) or GABAergic (Bali et al., 2005;Chattopadhyaya et al., 2004;Di Cristo et al., 2004;Heinke et al., 2004;Kobayashi et al., 2003;Lopez-Bendito et al., 2004;Ma et al., 2006;Makinae et al., 2000;Oliva et al., 2000;Oliva et al., 2002;Tamamaki et al., 2003) neurons, respectively. ...
Multiple applications of direct gene transfer into neurons require restricting expression to glutamatergic neurons, or specific subclasses of glutamatergic neurons. Thus, it is desirable to develop and analyze promoters that support glutamatergic-specific expression. The three vesicular glutamate transporters (VGLUTs) are found in different populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported on a plasmid (amplicon) Herpes Simplex Virus vector that contains a VGLUT1 promoter. This vector supports long-term expression in VGLUT1-containing glutamatergic neurons in rat postrhinal (POR) cortex, but does not support expression in VGLUT2-containing glutamatergic neurons in the ventral medial hypothalamus. This VGLUT1 promoter contains both the VGLUT1 upstream promoter and the VGLUT1 first intron. In this study, we begin to isolate and analyze the glutamatergic-specific regulatory elements in this VGLUT1 promoter. We show that the VGLUT1 upstream promoter and first intron each support glutamatergic-specific expression. We isolated a small, basal VGLUT1 promoter that does not support glutamatergic-specific expression. Next, we fused either the VGLUT1 upstream promoter or the first intron to this basal promoter. The VGLUT1 upstream promoter or the first intron, fused to the basal promoter, each supported glutamatergic-specific expression in POR cortex.
