Arousal state transitions occlude sensory-evoked neurovascular coupling in neonatal mice

Abstract In the adult sensory cortex, increases in neural activity elicited by sensory stimulation usually drive vasodilation mediated by neurovascular coupling. However, whether neurovascular coupling is the same in neonatal animals as adults is controversial, as both canonical and inverted responses have been observed. We investigated the nature of neurovascular coupling in unanesthetized neonatal mice using optical imaging, electrophysiology, and BOLD fMRI. We find in neonatal (postnatal day 15, P15) mice, sensory stimulation induces a small increase in blood volume/BOLD signal, often followed by a large decrease in blood volume. An examination of arousal state of the mice revealed that neonatal mice were asleep a substantial fraction of the time, and that stimulation caused the animal to awaken. As cortical blood volume is much higher during REM and NREM sleep than the awake state, awakening occludes any sensory-evoked neurovascular coupling. When neonatal mice are stimulated during an awake period, they showed relatively normal (but slowed) neurovascular coupling, showing that that the typically observed constriction is due to arousal state changes. These result show that sleep-related vascular changes dominate over any sensory-evoked changes, and hemodynamic measures need to be considered in the context of arousal state changes

Development of dendrite polarity in Drosophila neurons

<p>Abstract</p> <p>Background</p> <p>Drosophila neurons have dendrites that contain minus-end-out microtubules. This microtubule arrangement is different from that of cultured mammalian neurons, which have mixed polarity microtubules in dendrites.</p> <p>Results</p> <p>To determine whether Drosophila and mammalian dendrites have a common microtubule organization during development, we analyzed microtubule polarity in Drosophila dendritic arborization neuron dendrites at different stages of outgrowth from the cell body <it>in vivo</it>. As dendrites initially extended, they contained mixed polarity microtubules, like mammalian neurons developing in culture. Over a period of several days this mixed microtubule array gradually matured to a minus-end-out array. To determine whether features characteristic of dendrites were localized before uniform polarity was attained, we analyzed dendritic markers as dendrites developed. In all cases the markers took on their characteristic distribution while dendrites had mixed polarity. An axonal marker was also quite well excluded from dendrites throughout development, although this was perhaps more efficient in mature neurons. To confirm that dendrite character could be acquired in Drosophila while microtubules were mixed, we genetically disrupted uniform dendritic microtubule organization. Dendritic markers also localized correctly in this case.</p> <p>Conclusions</p> <p>We conclude that developing Drosophila dendrites initially have mixed microtubule polarity. Over time they mature to uniform microtubule polarity. Dendrite identity is established before the mature microtubule arrangement is attained, during the period of mixed microtubule polarity.</p

Normal Spastin Gene Dosage Is Specifically Required for Axon Regeneration

Axon regeneration allows neurons to repair circuits after trauma; however, most of the molecular players in this process remain to be identified. Given that microtubule rearrangements have been observed in injured neurons, we tested whether microtubule-severing proteins might play a role in axon regeneration. We found that axon regeneration is extremely sensitive to levels of the microtubule-severing protein spastin. Although microtubule behavior in uninjured neurons was not perturbed in animals heterozygous for a spastin null allele, axon regeneration was severely disrupted in this background. Two types of axon regeneration—regeneration of an axon from a dendrite after proximal axotomy and regeneration of an axon from the stump after distal axotomy—were defective in Drosophila with one mutant copy of the spastin gene. Other types of axon and dendrite outgrowth, including regrowth of dendrites after pruning, were normal in heterozygotes. We conclude that regenerative axon growth is uniquely sensitive to spastin gene dosage

Mitochondria and Caspases Tune Nmnat-Mediated Stabilization to Promote Axon Regeneration

<div><p>Axon injury can lead to several cell survival responses including increased stability and axon regeneration. Using an accessible Drosophila model system, we investigated the regulation of injury responses and their relationship. Axon injury stabilizes the rest of the cell, including the entire dendrite arbor. After axon injury we found mitochondrial fission in dendrites was upregulated, and that reducing fission increased stabilization or neuroprotection (NP). Thus axon injury seems to both turn on NP, but also dampen it by activating mitochondrial fission. We also identified caspases as negative regulators of axon injury-mediated NP, so mitochondrial fission could control NP through caspase activation. In addition to negative regulators of NP, we found that nicotinamide mononucleotide adenylyltransferase (Nmnat) is absolutely required for this type of NP. Increased microtubule dynamics, which has previously been associated with NP, required Nmnat. Indeed Nmnat overexpression was sufficient to induce NP and increase microtubule dynamics in the absence of axon injury. DLK, JNK and fos were also required for NP. Because NP occurs before axon regeneration, and NP seems to be actively downregulated, we tested whether excessive NP might inhibit regeneration. Indeed both Nmnat overexpression and caspase reduction reduced regeneration. In addition, overexpression of fos or JNK extended the timecourse of NP and dampened regeneration in a Nmnat-dependent manner. These data suggest that NP and regeneration are conflicting responses to axon injury, and that therapeutic strategies that boost NP may reduce regeneration.</p></div

Reducing mitochondria in dendrites increases axotomy-induced neuroprotection.

<p>(A-A”) A schematic of the axotomy-induced neuroprotection/NP assay is shown. (A) Without pre-axon injury, dendrites degenerate within 18h after injury. (A’) An axon injury 8h prior to dendrite injury induces NP so that dendrite degeneration is delayed; this timeline is the standard one used to assay NP throughout. (A”) When 48h elapses between the axon injury and dendrite severing, very little NP is observed and most dendrites are gone 18h after they are removed. Laser-induced injury is indicated by red lightning bolts. The ddaE neuron is drawn in green and other neurons labeled by 221-Gal4, which was used in most experiments to drive expression, are drawn in grey. (B and C) The NP assay as illustrated in Fig 1A’ was performed in wide-type (yw indicates control neurons that do not express an RNAi hairpin, and Rtnl2 indicates neurons that express a control RNAi hairpin) and Miro RNAi neurons. Neurons were labeled with EB1-GFP under the control of 221-Gal4. In control conditions half the neurons have a dendrite that remains at 18h, and (top row) and half have a fully degenerated dendrite (bottom row in B). Red arrows are the site of axon injury; purple arrows mark the site of dendrite injury. Green lines indicate stabilized dendrites. The scale bar is 20 μm. (D) Quantification of NP is shown. The number of neurons analyzed for each genotype is indicated above the bars. A Fisher’s exact test was used to determine statistical significance. Rtnl2 RNAi was used as a control as it targets a non-essential gene for which we have never observed phenotypes. * p<0.05. (E) Dendrites in ddaE neurons expressing EB1-GFP and control or Miro RNA hairpins were severed without prior axon injury. The presence of intact dendrites (no breaks in continuity) was scored at 4h, 7h and 11h after dendrites were severed. The numbers above the bars are the numbers of cells analyzed; one cell per animal.</p

Nmnat is required for stabilization of dendrites in response to axotomy.

<p>(A) Left, dendrites of Nmnat RNAi neurons were severed without axon pre-cut. Neurons were labeled with EB1-GFP. The scale bar is 20 μm. A purple arrow marks the site of dendrite injury. Right, quantification of dendrite degeneration at various time points is shown with control data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g001" target="_blank">Fig 1E</a> for comparison. * The numbers above the bars are the number of cells analyzed for each condition. (B) Left, the NP assay was performed in Nmnat RNAi neurons labeled with EB1-GFP. A red arrows shows the site of axon injury, a purple arrow, dendrite injury. The scale bar is 20 μm. Right, quantification of NP is shown, with control data from Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g001" target="_blank">1D</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g002" target="_blank">2F</a>. The numbers of neurons analyzed are indicated on the bars. ** p<0.01, determined by Fisher’s exact test. (C) The NP assay was performed in neurons expressing Dronc RNAi in conjunction with a control RNAi or Nmnat RNAi in cells labeled with EB1-GFP. Images of neurons 18hpd are shown. The green line indicates a stabilized dendrite. The scale bar is 20 μm. Right, quantification of NP is shown. The numbers of neurons analyzed are indicated above the bars. A Fisher’s exact test was used to determine statistical significance. ** p<0.01, *** p<0.001. (D) Left, kymographs of EB1-GFP in the dendrites of control and Nmnat RNAi neurons before and 8h after axon injury are shown; the cell body is off to the right in each image. The trajectory of EB1 comets appears white. The X- and Y- axes represent distance and time, respectively. Right, microtubule dynamics is quantified. The numbers of neurons analyzed are indicated above the bars. *** p<0.001, determined by unpaired t test. Error bars are SD. (E) A proposed model that summarizes the finding so far is shown. Steps that cannot be definitely resolved with the data are indicated by question marks.</p

Axotomy-induced mitochondrial fission inhibits neuroprotection.

<p>(A and A’) Representative images of dendritic mitochondria in control (A) and Drp1 RNAi (A’) neurons before and 8h post axon injury (hpa) are shown. mito-GFP and mCD8-RFP were coexpressed in ddaE neurons under the control of 221-Gal4 in order to visualize mitochondria and the cell membrane, respectively. Orange, blue and magenta arrows indicate long (>1.5 μm), medium (1–1.5 μm) and short (<1 μm) mitochondria respectively. Scale bars are 10 μm. (B-D’) The length, length distribution, and total number of mitochondria in control (B-D) and Drp1 RNAi neurons (B’-D’) before and after axon injury were measured. Statistical significance was determined using a Fisher’s exact test (C and C’), or a t test (B, B’, D and D’). * p<0.05, ** p<0.01, *** p<0.001, N.S. not significant. Error bars represent SD. (B, B’, D and D’) The numbers of neurons analyzed are indicated above the bars. (C) 227 and 229 mitochondria from 9 neurons were analyzed for uninjured and 8hpa, respectively. (C’) 173 and 169 mitochondria from 8 neurons were analyzed for uninjured and 8hpa, respectively. (E) The NP assay was performed in Drp1 RNAi neurons labeled with EB1-GFP. Red arrows indicate the site of axon injury and purple arrows, dendrite injury. Green lines mark stabilized dendrites. Scale bar, 20 μm. (F) Quantification of NP is shown with control data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g001" target="_blank">Fig 1D</a> for comparison. * p<0.05 and **p<0.01, determined by Fisher’s exact test. The numbers of neurons are indicated above the bars. For the RNAi experiment, Rtnl2 data is shown as the matched control and yw (no RNAi hairpin) control data was used for the overexpression comparison. (G) Dendrite injury was performed in Drp1 RNAi neurons without pre-axotomy. Dendrite degeneration assayed at the indicated times. Control data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g001" target="_blank">Fig 1E</a> is included for comparison. The numbers of cells analyzed for each condition are shown above the bars.</p

Caspases inhibit axotomy-induced neuroprotection.

<p>(A) Left, images of control and Dronc RNAi neurons in which dendrites were severed without axon pre-cut are shown. Neurons were labeled with EB1-GFP. Purple arrows indicate the site of dendrite injury. The scale bar is 20 μm. Right, presence of intact dendrites was scored at different time points. Control data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g001" target="_blank">Fig 1E</a> is included for comparison. The numbers of neurons analyzed for each condition are shown above the bars. (B) The NP assay was performed in Dronc RNAi neurons labeled with EB1-GFP. Red arrows indicate site of axon injury; purple arrows, dendrite injury; green lines, stabilized dendrites. The scale bar is 20 μm. (C) Quantification of NP is shown. The numbers on the bars indicate the numbers of neurons analyzed. Control data (Rtnl2 for RNAi and yw for other genotypes) from Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g001" target="_blank">1D</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g002" target="_blank">2F</a> is shown for comparison. A Fisher’s exact test was used to determine statistical significance. * p<0.05. N.S. not significant. (D) Left, images of mitochondria in the dendrites of Dronc RNAi neurons before and 8h post axon injury are shown. Orange, blue and magenta arrows indicate long, medium and short mitochondria respectively. The scale bar is 10 μm. Right, quantification of mito-GFP length is shown. The numbers of neurons analyzed are indicated above the bars. ** p<0.01, determined with a t test. Error bars are SD.</p

Effects of JNK (bsk) overexpression on NP and axon regeneration, and a summary model.

<p>(A) A 48h NP assay was performed in bsk-overexpressing neurons. The green line indicates a stabilized dendrite. Statistical significance was determined by a Fisher’s exact test and the numbers above the bars indicate numbers of neurons analyzed. *** p<0.001. Control data is from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006503#pgen.1006503.g007" target="_blank">Fig 7D</a>. (B) Axon regeneration assays were performed in neurons overexpressing bsk paired with either mCD8-RFP or Nmnat RNAi. UAS-mCD8-RFP was expressed as a control for Nmnat RNAi to keep the number of UAS-controlled transgenes constant and rule out Gal4 dilution effects. Red arrows mark sites of axon injury. The green star indicates the tip of the converted dendrite. Statistical significance was determined by a Mann-Whitney test. Error bars represent SD. *** p<0.001. (C) A summary model of the results is shown. Axon injury activates the DLK/bsk/fos response pathway. The AP-1 transcription factor fos turns on early injury responses that include Nmnat-mediated NP (indicated by darker cell outline in middle image), microtubule dynamics (short green lines in middle image) and mitochondrial fission. NP is mediated by Nmnat, which, if unchecked dampens subsequent regeneration. Caspases and mitochondrial fission counteract NP. (D) Reduction of caspases, increased bsk or fos, or increased Nmnat result in excess or longer than normal NP. Unbalanced NP dampens regeneration.</p

Nmnat is sufficient to delay dendrite degeneration and increase microtubule dynamics.

<p>(A) Dendrites were severed in neurons expressing GFP-Nmnat-B-delta N without prior axotomy. An example of a cell immediately after dendrite injury and then 18h later is shown with the injury site indicated by a purple arrow and persistent dendrite with a green line. The scale bar is 20 μm. Quantitation of dendrite degeneration is shown at the right. In control (yw) and Nmnat overexpressing neurons, the number of intact dendrites was scored 18h after dendrites were severed. A Fisher’s exact test was used to calculate significance with *** indicating p<0.001. The number of cells analyzed for each genotype is shown above the bars. (B) Movies of EB1-GFP were acquired in the trunk of the ddaE comb dendrite. Neurons expressed a control protein, Kaede, GFP-Nmnat-B-deltaN or Wlds. Kymographs from a portion of the dendrite are shown with the cell body to the right. Quantitation of comet number in the different genetic backgrounds is shown at the right. The central line shows the mean and the error bars are the SD. Numbers of cells analyzed are shown above the plots. Significance was calculated with an unpaired t test and * indicates p<0.05.</p