Spreading depolarization causes reversible neuronal mitochondria fragmentation and swelling in healthy, normally perfused neocortex.

Mitochondrial function is tightly linked to their morphology, and fragmentation of dendritic mitochondria during noxious conditions suggests loss of function. In the normoxic cortex, spreading depolarization (SD) is a phenomenon underlying migraine aura. It is unknown whether mitochondria structure is affected by normoxic SD. In vivo two-photon imaging followed by quantitative serial section electron microscopy (ssEM) was used to monitor dendritic mitochondria in the normoxic cortex of urethane-anesthetized mature male and female mice during and after SD initiated by focal KCl microinjection. Structural dynamics of dendrites and their mitochondria were visualized by transfecting excitatory, glutamatergic neurons of the somatosensory cortex with bicistronic AAV, which induced tdTomoto labeling in neuronal cytoplasm and mitochondria labeling with roGFP. Normoxic SD triggered a rapid fragmentation of dendritic mitochondria alongside dendritic beading, both reversible; however, mitochondria took significantly longer to recover. Several rounds of SD resulted in transient mitochondrial fragmentation and dendritic beading without accumulating injury, as both recovered. SsEM corroborated normoxic SD-elicited dendritic and mitochondrial swelling and transformation of the filamentous mitochondrial network into shorter, swollen tubular and globular structures. Our results revealed normoxic SD-induced disruption of the dendritic mitochondrial structure that might impact mitochondrial bioenergetics during migraine with aura.

Chronic basal forebrain activation improves spatial memory, boosts neurotrophin receptor expression, and lowers BACE1 and Aß42 levels in the cerebral cortex in mice.

The etiology of Alzheimer's dementia has been hypothesized in terms of basal forebrain cholinergic decline, and in terms of reflecting beta-amyloid neuropathology. To study these different biological elements, we activated the basal forebrain in 5xFAD Alzheimer's model mice and littermates. Mice received 5 months of 1 h per day intermittent stimulation of the basal forebrain, which includes cholinergic projections to the cortical mantle. Then, mice were behaviorally tested followed by tissue analysis. The 5xFAD mice performed worse in water-maze testing than littermates. Stimulated groups learned the water maze better than unstimulated groups. Stimulated groups had 2-3-fold increases in frontal cortex immunoblot measures of the neurotrophin receptors for nerve growth factor and brain-derived neurotrophic factor, and a more than 50% decrease in the expression of amyloid cleavage enzyme BACE1. Stimulation also led to lower Aß42 in 5xFAD mice. These data support a causal relationship between basal forebrain activation and both neurotrophin activation and reduced Aß42 generation and accumulation. The observation that basal forebrain activation suppresses Aß42 accumulation, combined with the known high-affinity antagonism of nicotinic receptors by Aß42, documents bidirectional antagonism between acetylcholine and Aß42.

Plasticity of perisynaptic astroglia during ischemia-induced spreading depolarization.

High astroglial capacity for glutamate and potassium clearance aids in recovering spreading depolarization (SD)-evoked disturbance of ion homeostasis during stroke. Since perisynaptic astroglia cannot be imaged with diffraction-limited light microscopy, nothing is known about the impact of SD on the ultrastructure of a tripartite synapse. We used serial section electron microscopy to assess astroglial synaptic coverage in the sensorimotor cortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. At the subcellular level, astroglial mitochondria were remarkably resilient to SD compared to dendritic mitochondria that were fragmented by SD. Overall, 482 synapses in `Sham' during `SD' and `Recovery' groups were randomly selected and analyzed in 3D. Perisynaptic astroglia was present at the axon-spine interface (ASI) during SD and after recovery. Astrocytic processes were more likely found at large synapses on mushroom spines after recovery, while the length of the ASI perimeter surrounded by astroglia has also significantly increased at large synapses. These findings suggest that as larger synapses have a bigger capacity for neurotransmitter release during SD, they attract astroglial processes to their perimeter during recovery, limiting extrasynaptic glutamate escape and further enhancing the astrocytic ability to protect synapses in stroke.

Novel mechanism of hypoxic neuronal injury mediated by non-excitatory amino acids and astroglial swelling.

In ischemic stroke and post-traumatic brain injury (TBI), blood-brain barrier disruption leads to leaking plasma amino acids (AA) into cerebral parenchyma. Bleeding in hemorrhagic stroke and TBI also release plasma AA. Although excitotoxic AA were extensively studied, little is known about non-excitatory AA during hypoxic injury. Hypoxia-induced synaptic depression in hippocampal slices becomes irreversible with non-excitatory AA, alongside their intracellular accumulation and increased tissue electrical resistance. Four non-excitatory AA (l-alanine, glycine, l-glutamine, l-serine: AGQS) at plasmatic concentrations were applied to slices from mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Two-photon imaging, light transmittance (LT) changes, and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. radiatum were used to monitor synaptic function concurrently with cellular swelling and injury. During normoxia, AGQS-induced increase in LT was due to astroglial but not neuronal swelling. LT raise during hypoxia and AGQS manifested astroglial and neuronal swelling accompanied by a permanent loss of synaptic transmission and irreversible dendritic beading, signifying acute damage. Neuronal injury was not triggered by spreading depolarization which did not occur in our experiments. Hypoxia without AGQS did not cause cell swelling, leaving dendrites intact. Inhibition of NMDA receptors prevented neuronal damage and irreversible loss of synaptic function. Deleterious effects of AGQS during hypoxia were prevented by alanine-serine-cysteine transporters (ASCT2) and volume-regulated anion channels (VRAC) blockers. Our findings suggest that astroglial swelling induced by accumulation of non-excitatory AA and release of excitotoxins through antiporters and VRAC may exacerbate the hypoxia-induced neuronal injury.

Rapid Neuronal Ultrastructure Disruption and Recovery during Spreading Depolarization-Induced Cytotoxic Edema.

Two major pathogenic events that cause acute brain damage during neurologic emergencies of stroke, head trauma, and cardiac arrest are spreading depolarizing waves and the associated brain edema that course across the cortex injuring brain cells. Virtually nothing is known about how spreading depolarization (SD)-induced cytotoxic edema evolves at the ultrastructural level immediately after insult and during recovery. In vivo 2-photon imaging followed by quantitative serial section electron microscopy was used to assess synaptic circuit integrity in the neocortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. SD triggered a rapid fragmentation of dendritic mitochondria. A large increase in the density of synapses on swollen dendritic shafts implies that some dendritic spines were overwhelmed by swelling or merely retracted. The overall synaptic density was unchanged. The postsynaptic dendritic membranes remained attached to axonal boutons, providing a structural basis for the recovery of synaptic circuits. Upon immediate reperfusion, cytotoxic edema mainly subsides as affirmed by a recovery of dendritic ultrastructure. Dendritic recuperation from swelling and reversibility of mitochondrial fragmentation suggests that neurointensive care to improve tissue perfusion should be paralleled by treatments targeting mitochondrial recovery and minimizing the occurrence of SDs.

Reversible Disruption of Neuronal Mitochondria by Ischemic and Traumatic Injury Revealed by Quantitative Two-Photon Imaging in the Neocortex of Anesthetized Mice.

Mitochondria play a variety of functional roles in cortical neurons, from metabolic support and neuroprotection to the release of cytokines that trigger apoptosis. In dendrites, mitochondrial structure is closely linked to their function, and fragmentation (fission) of the normally elongated mitochondria indicates loss of their function under pathological conditions, such as stroke and brain trauma. Using in vivo two-photon microscopy in mouse brain, we quantified mitochondrial fragmentation in a full spectrum of cortical injuries, ranging from severe to mild. Severe global ischemic injury was induced by bilateral common carotid artery occlusion, whereas severe focal stroke injury was induced by Rose Bengal photosensitization. The moderate and mild traumatic injury was inflicted by focal laser lesion and by mild photo-damage, respectively. Dendritic and mitochondrial structural changes were tracked longitudinally using transgenic mice expressing fluorescent proteins localized either in cytosol or in mitochondrial matrix. In response to severe injury, mitochondrial fragmentation developed in parallel with dendritic damage signified by dendritic beading. Reconstruction from serial section electron microscopy confirmed mitochondrial fragmentation. Unlike dendritic beading, fragmentation spread beyond the injury core in focal stroke and focal laser lesion models. In moderate and mild injury, mitochondrial fragmentation was reversible with full recovery of structural integrity after 1-2 weeks. The transient fragmentation observed in the mild photo-damage model was associated with changes in dendritic spine density without any signs of dendritic damage. Our findings indicate that alterations in neuronal mitochondria structure are very sensitive to the tissue damage and can be reversible in ischemic and traumatic injuries. SIGNIFICANCE STATEMENT: During ischemic stroke or brain trauma, mitochondria can either protect neurons by supplying ATP and adsorbing excessive Ca2+, or kill neurons by releasing proapoptotic factors. Mitochondrial function is tightly linked to their morphology: healthy mitochondria are thin and long; dysfunctional mitochondria are thick (swollen) and short (fragmented). To date, fragmentation of mitochondria was studied either in dissociated cultured neurons or in brain slices, but not in the intact living brain. Using real-time in vivo two-photon microscopy, we quantified mitochondrial fragmentation during acute pathological conditions that mimic severe, moderate, and mild brain injury. We demonstrated that alterations in neuronal mitochondria structural integrity can be reversible in traumatic and ischemic injuries, highlighting mitochondria as a potential target for therapeutic interventions.

Neuronal pannexin-1 channels are not molecular routes of water influx during spreading depolarization-induced dendritic beading.

Spreading depolarization-induced focal dendritic swelling (beading) is an early hallmark of neuronal cytotoxic edema. Pyramidal neurons lack membrane-bound aquaporins posing a question of how water enters neurons during spreading depolarization. Recently, we have identified chloride-coupled transport mechanisms that can, at least in part, participate in dendritic beading. Yet transporter-mediated ion and water fluxes could be paralleled by water entry through additional pathways such as large-pore pannexin-1 channels opened by spreading depolarization. Using real-time in vivo two-photon imaging in mice with pharmacological inhibition or conditional genetic deletion of pannexin-1, we showed that pannexin-1 channels are not required for spreading depolarization-induced focal dendritic swelling.

Chloride Cotransporters as a Molecular Mechanism underlying Spreading Depolarization-Induced Dendritic Beading.

Spreading depolarizations (SDs) are waves of sustained neuronal and glial depolarization that propagate massive disruptions of ion gradients through the brain. SD is associated with migraine aura and recently recognized as a novel mechanism of injury in stroke and brain trauma patients. SD leads to neuronal swelling as assessed in real time with two-photon laser scanning microscopy (2PLSM). Pyramidal neurons do not express aquaporins and thus display low inherent water permeability, yet SD rapidly induces focal swelling (beading) along the dendritic shaft by unidentified molecular mechanisms. To address this issue, we induced SD in murine hippocampal slices by focal KCl microinjection and visualized the ensuing beading of dendrites expressing EGFP by 2PLSM. We confirmed that dendritic beading failed to arise during large (100 mOsm) hyposmotic challenges, underscoring that neuronal swelling does not occur as a simple osmotic event. SD-induced dendritic beading was not prevented by pharmacological interference with the cytoskeleton, supporting the notion that dendritic beading may result entirely from excessive water influx. Dendritic beading was strictly dependent on the presence of Cl(-), and, accordingly, combined blockade of Cl(-)-coupled transporters led to a significant reduction in dendritic beading without interfering with SD. Furthermore, our in vivo data showed a strong inhibition of dendritic beading during pharmacological blockage of these cotransporters. We propose that SD-induced dendritic beading takes place as a consequence of the altered driving forces and thus activity for these cotransporters, which by transport of water during their translocation mechanism may generate dendritic beading independently of osmotic forces. SIGNIFICANCE STATEMENT: Spreading depolarization occurs during pathological conditions such as stroke, brain injury, and migraine and is characterized as a wave of massive ion translocation between intracellular and extracellular space in association with recurrent transient focal swelling (beading) of dendrites. Numerous ion channels have been demonstrated to be involved in generation and propagation of spreading depolarization, but the molecular machinery responsible for the dendritic beading has remained elusive. Using real-time in vitro and in vivo two-photon laser scanning microscopy, we have identified the transport mechanisms involved in the detrimental focal swelling of dendrites. These findings have clear clinical significance because they may point to a new class of pharmacological targets for prevention of neuronal swelling that consequently will serve as neuroprotective agents.

Evolution of neuronal and astroglial disruption in the peri-contusional cortex of mice revealed by in vivo two-photon imaging.

In traumatic brain injury mechanical forces applied to the cranium and brain cause irreversible primary neuronal and astroglial damage associated with terminal dendritic beading and spine loss representing acute damage to synaptic circuitry. Oedema develops quickly after trauma, raising intracranial pressure that results in a decrease of blood flow and consequently in cerebral ischaemia, which can cause secondary injury in the peri-contusional cortex. Spreading depolarizations have also been shown to occur after traumatic brain injury in humans and in animal models and are thought to accelerate and exacerbate secondary tissue injury in at-risk cortical territory. Yet, the mechanisms of acute secondary injury to fine synaptic circuitry within the peri-contusional cortex after mild traumatic brain injury remain unknown. A mild focal cortical contusion model in adult mouse sensory-motor cortex was implemented by the controlled cortical impact injury device. In vivo two-photon microscopy in the peri-contusional cortex was used to monitor via optical window yellow fluorescent protein expressing neurons, enhanced green fluorescent protein expressing astrocytes and capillary blood flow. Dendritic beading in the peri-contusional cortex developed slowly and the loss of capillary blood flow preceded terminal dendritic injury. Astrocytes were swollen indicating oedema and remained swollen during the next 24 h throughout the imaging session. There were no recurrent spontaneous spreading depolarizations in this mild traumatic brain injury model; however, when spreading depolarizations were repeatedly induced outside the peri-contusional cortex by pressure-injecting KCl, dendrites undergo rapid beading and recovery coinciding with passage of spreading depolarizations, as was confirmed with electrophysiological recordings in the vicinity of imaged dendrites. Yet, accumulating metabolic stress resulting from as few as four rounds of spreading depolarization significantly added to the fraction of beaded dendrites that were incapable to recover during repolarization, thus facilitating terminal injury. In contrast, similarly induced four rounds of spreading depolarization in another set of control healthy mice caused no accumulating dendritic injury as dendrites fully recovered from beading during repolarization. Taken together, our data suggest that in the mild traumatic brain injury the acute dendritic injury in the peri-contusional cortex is gated by the decline in the local blood flow, most probably as a result of developing oedema. Furthermore, spreading depolarization is a specific mechanism that could accelerate injury to synaptic circuitry in the metabolically compromised peri-contusional cortex, worsening secondary damage following traumatic brain injury.

New method of measuring water permeability of adhesive resin films.

PURPOSE: To adapt a simple gravimetric method to measuring the permeability of adhesive resin films to liquid water, and to compare this to the water vapor permeability of the same resins. METHODS: Using commercially-available permeability cups designed for industrial permeability testing, the loss of mass of water vapor or liquid water from a stainless steel cup sealed with a resin film was measured over 1-2 days. The permeabilities of Parafilm (control), Clearfil SE Bond adhesive, Xeno IV and One-Up Bond F were compared. RESULTS: The lowest resin film permeability was obtained with Clearfil SE Bond adhesive films. The permeabilities ofXeno IV and One-Up Bond F to liquid water were 2.76 and 3.27-fold higher (P < 0.001) than that of Clearfil SE Bond adhesive. Liquid water permeability was always 2.8-3.8-fold higher (P < 0.05) than water vapor transmission rate. It was concluded that quantitative comparisons of the permeability properties of resin films can be made gravimetrically. The large permeability cups that are available commercially may be reduced in size in the future for measuring dentin adhesive films with smaller surface areas that are less liable to contain imperfections.

Potent inhibition of anoxic depolarization by the sodium channel blocker dibucaine.

Recurring waves of peri-infarct depolarizations (PIDs) propagate across gray matter in the hours and days following stroke, expanding the primary site of injury. Ischemic depolarization (termed anoxic depolarization or AD in live brain slices) is PID-like but immediately arises in the more metabolically compromised ischemic core. This causes dramatic neuronal and astrocyte swelling and dendritic beading with spine loss within minutes, resulting in acute cell death. AD is evoked in rodent neocortical slices by suppressing the Na(+)/K(+)-ATPase pump with either oxygen/glucose deprivation (OGD) or exposure to ouabain. The process driving AD and PIDs remains poorly understood. Here we show that dibucaine is a potent drug inhibiting AD because of its high binding affinity to the Na(+) channel. Field recording reveals that, when superfused with ouabain (5 min), neocortical slices pretreated with 1 µM dibucaine for 45 min display either no AD or delayed AD onset compared with untreated controls. If ouabain exposure is extended to 10 min, 1 µM dibucaine is still able to delay AD onset by ∼ 60%. Likewise, it delays OGD-evoked AD onset by ∼ 54% but does not depress action potentials (APs) or evoked orthodromic field potentials. Increasing dibucaine to 10 µM inhibits AP firing, gradually putting the slice into a stasis that inhibits AD onset but also renders the slice functionally quiescent. Two-photon microscopy reveals that 10 µM dibucaine pretreatment prevents or helps reverse ouabain-induced structural neuronal damage. Although the therapeutic range of dibucaine is quite narrow, dibucaine-like drugs could prove therapeutically useful in inhibiting PIDs and their resultant neuronal damage.

Push-out strength of modified Portland cements and resins.

PURPOSE: Modified calcium-silicate cements derived from white Portland cement (PC) were formulated to test their push-out strength from radicular dentin after immersion for 1 month. METHODS: Slabs obtained from 42 single-rooted extracted teeth were prepared with 0.6 mm diameter holes, then enlarged with rotary instruments. After immersion in EDTA and NaOC1, the holes were filled with modified PCs or ProRoot MTA, Vitrebond and Clearfil SE. Different concentrations of phyllosilicate (montmorillonite-MMT) were added to experimental cements. ProRoot MTA was also included as reference material. Vitrebond and Clearfil SE were included as controls. Each group was tested after 1 month of immersion in water or PBS. A thin-slice push-out test on a universal testing machine served to test the push-out strength of materials. Results were statistically analyzed using the least squares means (LSM) method. RESULTS: The modified PCs had push-out strengths of 3-9.5 MPa after 1 month of immersion in water, while ProRoot MTA had 4.8 MPa. The push-out strength of PC fell after incubation in PBS for 1 month, while the push-out strength of ProRoot MTA increased. There were no significant changes in Clearfil SE Bond or Vitrebond after water or PBS storage.

Use of electrochemical impedance spectroscopy to evaluate resin-dentin bonds.

Electrochemical impedance spectroscopy (EIS) offers a potentially nondestructive quantitative method for measuring the stability of resin films and or resin-bonded dentin over time. The purpose of this study was to measure the electrical impedance of five experimental dental adhesives of increasing hydrophicities as 30-microm films and as resin-bonded coatings on acid-etched dentin. Resin films or resin-coated dentin disks were placed in U-shaped chambers containing pairs of Ag-AgCl electrodes in 0.1M KCl. Electrical impedance spectra were run at day 0, 1, 7, 14, and 21 days. All resin films and resin-bonded dentin showed increases in capacitance during the first day of storage in electrolyte. This was usually associated with an increase in the pore resistance of the resins. Generally, resin-bonded dentin gave lower impedance values than their respective resins (resins 1-4) but solvated resin 5 bonded to water-saturated dentin gave higher capacitance and impedance values than resin 5 films. However, solvated resin 5 films gave higher impedance values than resin 5-bonded dentin. This behavior was confirmed by TEM examinations of silver uptake into films of neat resin 5 vs. ethanol-solvated resin 5, where water tree-like structures seen in the former were not seen in the latter. EIS is useful in examining changes in the capacitance and electrical impedance of very hydrophilic, ionic methacrylate resins. Its utility in detecting degradation in resin-bonded dentin interfaces remains to be determined in longer term studies.

Water sorption and solubility of methacrylate resin-based root canal sealers.

The water sorption and solubility characteristics of three contemporary methacrylate resin-based endodontic sealers, EndoREZ, Epiphany, and InnoEndo, were compared with those obtained from Kerr EWT, Ketac-Endo (positive control), GuttaFlow, and AH Plus (both negative controls). Ten disks of each material were dehydrated in Drierite for 24 h and weighed to constant dry mass. They were placed in water and weighed periodically until maximum water sorption was obtained. The disks were dehydrated again to determine their mass loss (solubility) at equilibrium. Epiphany exhibited the highest apparent water sorption (8%) followed by Ketac-Endo (6.2%), InnoEndo (3.4%), EndoREZ (3.0%), AH Plus (1.1%), GuttaFlow (0.4%), and Kerr EWT (0.3%). Significantly higher solubility (3.5-4%) were observed for all three methacrylate resin-based sealers and Kerr EWT (3.95%), compared with Ketac-Endo (1.6%), AH Plus (0.16%), and GuttaFlow (0.13%). American Dental Association specifications require<3% solubility for endodontic sealers. Only Ketac-Endo, AH Plus, and GuttaFlow met that criterion.

Dentine sealing provided by smear layer/smear plugs vs. adhesive resins/resin tags.

The aim of this study was to evaluate the ability of five experimental resins, which ranged from hydrophobic to hydrophilic blends, to seal acid-etched dentine saturated with water or ethanol. The experimental resins (R1, R2, R3, R4, and R5) were evaluated as neat bonding agents (100% resin) or as solutions solvated with absolute ethanol (70% resin/30% ethanol). Fluid conductance was measured at 20 cm H(2)O hydrostatic pressure after sound dentine surfaces were: (i) covered with a smear layer; (ii) acid-etched; or (iii) bonded with neat or solvated resins, which were applied to acid-etched dentine saturated with water or ethanol. In general, the fluid conductance of resin-bonded dentine was significantly higher than that of smear layer-covered dentine. However, when the most hydrophobic neat resins (R1 and R2) were applied to acid-etched dentine saturated with ethanol, the fluid conductance was as low as that produced by smear layers. The fluid conductance of resin-bonded dentine saturated with ethanol was significantly lower than for resin bonded to water-saturated dentine, except for resin R4. Application of more hydrophobic resins may provide better sealing of acid-etched dentine if the substrate is saturated with ethanol instead of with water.

Comparison of seal after obturation techniques using a polydimethylsiloxane-based root canal sealer.

A polydimethylsiloxane-based sealer (GuttaFlow) was used for filling single-rooted teeth with four obturation techniques. Prefitted gutta-percha master-cones were employed in groups 1 to 3 with backfilling of the sealer. Apical third of the roots were first filled with GuttaFlow, and sealer-coated master cones were seated with up-and-down motion (group 1) or rotated to place (group 2). Master-cones were used as carriers for introducing GuttaFlow into the canals (group 3). Canals were filled with GuttaFlow without master-cones (group 4). Canals were filled with AH Plus/gutta-percha using warm vertical compaction (control group 5). All obturated canals were tested for leakage after 1, 6, and 12 weeks using the fluid filtration technique. All groups exhibited equivalent seal regardless of time except for more extensive leakage in group 3. The use of GuttaFlow with a single gutta-percha master cone creates an apical seal that is equivalent to that produced with gutta-percha/AH Plus sealer using warm vertical compaction.

Evaluation of the effect of water-uptake on the impedance of dental resins.

UNLABELLED: Electrical impedance spectroscopy (EIS) offers a quantitative method of measuring the stability of resin films in aqueous solution over time. PURPOSE: The purpose of this study was to measure the EIS of five experimental dental adhesive films (ca. 17 microm thick) of increasing hydrophilicity (ranked by their Hoy's solubility parameters), and how much these values change over 3 weeks in aqueous buffer. METHODS: The resin films were placed in a U-shaped chamber and a pair of Ag-AgCl electrodes was used for EIS. The EIS results were confirmed by immersing the films in 50% AgNO3 for 24 h to trace the distribution of any water absorption into the resins by TEM observations. RESULTS: The resistance (Rr) of the resins 1-4 films increased most during the first day, and varied from 1x10(11) ohm for resin 1, to 40Omega for resin 5 at day 1. The day 1 Rr values of resins 1-4 were inversely proportional to their Hoy's solubility parameter for hydrogen bonding forces. Electrical impedance values of resins 1-3 and 5 varied widely but were relatively constant over time, while those of resin 4 decreased more than 99% from day 1 to 21 (p<0.05). Capacitance (Cr) of films of resins 1-4 all increased over the first day and then were relatively unchanged over the 20 days (except for resin 4 that continued to increase) and were between 0.01 and 1 nF. Silver uptake by TEM revealed the development of water-filled branching structures that formed in resins 4 and 5 over time.