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Electrical activity regulates the manner in which neurons mature and form connections to each other. However, it remains unclear whether increased single-cell activity is sufficient to alter the development of synaptic connectivity of that neuron or whether a global increase in circuit activity is necessary. To address this question, we genetically increased neuronal excitability of in vivo individual adult-born neurons in the mouse dentate gyrus via expression of a voltage-gated bacterial sodium channel. We observed that increasing the excitability of new neurons in an otherwise unperturbed circuit leads to changes in both their input and axonal synapses. Furthermore, the activity-dependent transcription factor Npas4 is necessary for the changes in the input synapses of these neurons, but it is not involved in changes to their axonal synapses. Our results reveal that an increase in cell-intrinsic activity during maturation is sufficient to alter the synaptic connectivity of a neuron with the hippocampal circuit and that Npas4 is required for activity-dependent changes in input synapses.
The addition of new granule cell (GC) neurons to the adult dentate gyrus (DG) of the hippocampus may serve as a substrate for memory throughout life (Imayoshi et al., 2008). These excitatory neurons display enhanced synaptic plasticity (Schmidt-Hieber et al., 2004) and integrate into existing circuitry (Jessberger and Kempermann, 2003). The production, survival, and wiring of GCs into the DG circuit are affected by activity (Kempermann et al., 1997; van Praag et al., 1999; Kee et al., 2007). Importantly, seizures alter the maturation and connectivity of adult-born DG GCs (Parent et al., 1997; Jessberger et al., 2007).
Understanding the factors underlying activity-dependent maturation and connectivity of adult-born neurons is important to understand the physiological basis of learning and the pathological basis of epilepsy. In a seizure or in behavioral paradigms used to stimulate DG activity, general levels of activity in the brain are increased, so it is unclear whether the observed changes in maturation and connectivity result directly from the increased activity of an individual new neuron, indirectly via elevated activity of other neurons in the circuit in which the new neurons are embedded, or a combination of both.
To investigate how the level of neuronal activity of a single developing neuron affects its maturation and integration into an unperturbed circuit, we have developed a system to genetically increase excitability in individual neurons by introducing a voltage-gated sodium channel (Kelsch et al., 2008; Lin et al., 2010). We recently used this system to investigate the effects of genetically increased excitability on the maturation and integration of the GCs of the olfactory bulb, a type of inhibitory neuron produced during adulthood. We demonstrated that genetically increased intrinsic excitability was sufficient to enhance the survival of the new granule neurons of the bulb but, surprisingly, did not affect their synaptic organization (Kelsch et al., 2009; Lin et al., 2010). The plasticity of excitatory and inhibitory neurons differs in many respects (Bi and Poo, 1998), and it is plausible that electrical hyperexcitability affects the morphology of excitatory but not inhibitory neurons.
To test this hypothesis, we genetically raised the intrinsic excitability of individual new GCs in the DG, a type of excitatory neuron also produced during adulthood. We observed that elevating neuronal excitability of individual new neurons during their maturation is sufficient to induce changes in synaptic connectivity such as aberrant localization of synapses and enlarged spines. Cell-autonomous hyperexcitability leads to both input and output connectivity alterations that increase inhibition on the hyperexcitable neuron and dampen its excitatory influence on its downstream targets. We then examined the genetic basis for these alterations by deleting the transcription factor Npas4 in individual new neurons in conditional NPas4 knock-out mice. We observed that the transcription factor Npas4 is required for the activity-induced changes in synaptic inputs to these neurons but not for changes to output synapses in their axons. These observations indicate that cell-autonomous increases in excitability during neuronal maturation can effect profound changes in neuronal connectivity and that separate genetic programs regulate activity-dependent changes in input and output synapses.
Materials and Methods
Retroviral vectors.
Cloning of the different constructs was performed using standard molecular techniques. The cDNA for NaChBac was obtained from David Clapham (Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, MA). NaChBac E191K was generated by PCR based on previously published sequences (Ren et al., 2001; Yue et al., 2002). Retroviral vectors were derived from a Moloney leukemia virus with an internal promoter derived from the Rous sarcoma virus (Molar) (Kelsch et al., 2007). Retroviral particles were produced and stored as described previously (Lois et al., 2002). The viral titers were ∼107 infectious units/μl. Viral constructs were generated as follows. For NaChBac–EGFP, the stop codon of NaChBac was eliminated by PCR and fused in-frame to the cDNA of EGFP. For NaChBac–Cre, the stop codon of the NaChBac–EGFP fusion was eliminated by PCR and linked by a foot-and-mouth disease (FMDV) virus 2A sequence to the cDNA of Cre recombinase. For PalmEGFP–NaChBac, the palmitoylation sequence from the GAP43 gene was first added to the N terminus of EGFP. The stop codon of the palmitoylated version of EGFP was eliminated by PCR and linked by an FMDV 2A picornavirus sequence to the cDNA of NaChBac. For Synaptophysin–GFP, the stop codon of Synaptophysin was eliminated by PCR and fused in-frame to the cDNA of EGFP. For Synaptophysin–GFP–NaChBac, the stop codon of the Synaptophysin–GFP fusion was removed by PCR and linked by a 2A sequence to the cDNA of NaChBac. For PSD-95–GFP, the stop codon of PSD-95 was eliminated by PCR and fused in-frame to the cDNA of EGFP. For PSD-95–GFP–NaChBac, the stop codon of the PSD-95–GFP fusion was removed by PCR and linked by a 2A sequence to the cDNA of NaChBac. For invertible palmitoylated mCherry (PalmMCherry), the double-floxed inverse open reading frame vector was obtained from Karl Deisseroth (Stanford University, Palo Alto, CA). The ChR2–EYFP cDNA was excised from the vector and replaced with the cDNA of PalmMCherry, which was made by adding the palmitoylation sequence from the GAP43 gene to the N terminus of the mCherry cDNA.
Retroviral labeling in vivo.
Nine- to 12-week-old female BL6 mice (Charles River), floxed NMDA receptor subunit 1 mice (Tsien et al., 1996), floxed Npas4 mice (Lin et al., 2008), and their respective wild-type littermates were stereotaxically injected at two sites per DG with 0.5 μl/site retroviral vectors, after anesthesia with avertin solution. The stereotaxic coordinates were 2.0 mm posterior from bregma, 1.5 mm lateral from the midline, and 1.95 mm ventral from the brain surface, and 2.7 mm posterior from bregma, 1.9 mm lateral from the midline, and 2.05 mm ventral from the brain surface.
Mice were administered an overdose of avertin before they were perfused intracardially, first with PBS and then with 3% paraformaldehyde (PFA). The brains were incubated with 3% PFA overnight and cut with a Leica vibratome into 40 μm frontal sections. For immunocytochemistry, the sections were first blocked with blocking solution containing bovine serum albumin (3 mg/ml PBS) and 0.25% Triton X-100 in PBS and incubated overnight in the relevant primary antibody diluted in blocking solution: polyclonal rabbit anti-GFP antibody (AB3080; 1:2000; Millipore Bioscience Research Reagents), rabbit anti-RFP antibody (LS-C60076; 1:200; Lifespan), mouse anti-vesicular GABA transporter (VGAT) antibody (catalog #131 002; 1:500; Synaptic Systems), mouse anti-parvalbumin (P3088; 1:500; Sigma), and rabbit anti-Npas4 (1:10,000; gift from Y.L.). Sections were washed four times in PBS, for 10 min each time, before a 2 h incubation at room temperature with Alexa Fluor 488 or 555 goat anti-rabbit or anti-mouse secondary antibody (Invitrogen) diluted 1:700 in blocking solution. The sections were washed four times in PBS, for 10 min each time, before being mounted on slides with mounting medium (Vectashield; Vector Laboratories).
Survival ratio analyses.
Two viruses were mixed at an approximate 1:1 ratio for survival analysis. One of the viruses carried the construct encoding mCherry, whereas the other carried either NaChBac or NaChBac E191K fused to EGFP (NaChBac–EGFP or NaChBacE191K–EGFP). Fluorescently labeled cells were quantified with the aid of the Neurolucida software (MicroBrightField). The survival ratio is defined as the total number of EGFP-positive cells (including double-labeled cells) divided by the number of single-labeled mCherry+ cells. The ratio of EGFP+ to mCherry+ neurons at 7 d post-infection (dpi) was used to normalize all data at subsequent time points for comparison; hence, ratios at all subsequent time points were relative to the 7 dpi ratio. Ten to 20 entire sections per DG were analyzed to collect at least 100 counted cells in each DG. The mean survival ratio from each DG was treated as a single sample.
For electrophysiology, viruses in which NaChBac is directly fused to GFP were used because they produce strong fluorescent signals in the soma, which is useful for targeting neurons for fluorescence-guided whole-cell recordings. Animals were given an overdose of ketamine/xylazine and then perfused intracardially with ice-cold slicing solution containing the following (in mm): 212 sucrose, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 7 MgCl2, and 10 glucose, pH 7.3 (308 mOsm). Brain slices were incubated in ice-cold cutting solution and cut into 350 μm frontal slices with a Leica microtome at a speed of 0.08 mm/s. Slices were incubated for 30 min at 35°C, for recovery, in carbogenated recording solution containing the following (in mm): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgCl2, 2 CaCl2, and 20 glucose, pH 7.3 (312 mOsm). Fluorescent-guided whole-cell patch-clamp recordings were performed with a MultiClamp 700B amplifier (Molecular Devices). The pipette solution contained the following (in mm): 2 NaCl, 4 KCl, 130 K-gluconate, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.3 Tris-GTP, and 14 Tris-phosphocreatine, pH 7.3. Successful patching onto the target cell was confirmed by identifying a fragment of fluorescent membrane trapped inside the pipette tip during or after the recording. Pipette resistance ranged from 5 to 8 MΩ, and the pipette access resistance was always <16 MΩ after series resistance compensation. The junction potential was not corrected throughout the study. For spontaneous EPSC (sEPSC) recording, the neurons were held at −77 mV, and synaptic events were collected at 25°C. sEPSCs contributed to the majority of spontaneous events because ∼98% of events could be blocked by 100 μm d,l-AP-5 and 20 μm NBQX (Sigma) at the end of the recording. Furthermore, we observed that the spontaneous synaptic events recorded with bicuculline and TTX in both control and NaChBac+ neurons have a reversal potential at approximately −4 mV. These values are consistent with those of sEPSCs mediated by the opening of glutamate receptors (Cull-Candy and Usowicz, 1989), as reported previously for sEPSPs in GCs of the rat DG (−5.5 ± 1.1 mV; Crunelli et al., 1984).
Inhibitory blockers, such as bicuculline, were not included during sEPSC recording because they triggered frequent EPSC bursting input in granule neurons, which precluded additional analysis. To record sIPSCs, intracellular 130 K-gluconate was replaced with 130 CsCl and included 20 μm NBQX and 50 μm AP-5 in the recording bath to increase the driving force for chloride efflux, enabling us to record spontaneous GABAergic input at −77 mV.
Analysis of electrophysiological data.
Data were acquired and analyzed with pClamp9 software (Molecular Devices), and 2 min traces of sIPSCs and sEPSCs were analyzed with Mini Analysis Program (Synaptosoft). Overall current was calculated by multiplying the average charge area per spike of each individual neuron by frequency of spikes of the same neuron.
Morphological analyses.
For confocal microscopy, confocal image stacks of 40-μm-thick DG sections were acquired by using an Olympus Fluoview confocal microscope (60× oil-immersion lens; numerical aperture 1.4; pixel size, 0.23 × 0.23 μm) and with z-step 0.25 μm. Ten to 0–30 neurons were analyzed in each DG for dendritic length, density, spine size, and perisomatic inhibitory analysis, and data from four to seven DGs were collected for each experimental condition. A typical image stack consisted of ∼80–150 image planes each of 1024 × 1024 pixels.
For image processing and quantification, after acquisition, maximal intensity projections were prepared for each image stack by using the MetaMorph analysis software (Universal Imaging).
Measurements of PSD-95:GFP clusters were performed as described in our previous publication (Kelsch et al., 2007). To attribute the GFP puncta to a particular neuron, we took advantage of the presence of low levels of diffuse PSD-95:GFP protein in the cytoplasm not detectable by its endogenous fluorescence. This diffuse PSD-95:GFP protein could be visualized by amplifying its signal with antibodies raised against GFP (coupled to a red fluorophore to distinguish it from the intrinsic green fluorescence of PSD+ puncta) and allowed us to attribute PSD+ puncta to the neurites belonging to a particular neuron. For the projection images, the threshold was set so that any possible diffuse GFP fluorescence at the dendritic shaft was below this threshold. The number of PSD-95:GFP+ clusters in a region of interest was counted by using the integrated morphometry analysis function of the MetaMorph software. The length of the respective segment of the dendritic arbor was then measured, and the density of PSD-95:GFP+ clusters was determined. All datasets were manually supervised to prevent the inclusion of nonspecific green specks.
Spine size in palmitoylated EGFP (PalmG) neurons were measured as follow. For each stack, laser intensity and detector sensitivity were set so that the fluorescence signal from the spines occupied the full dynamic range of the detector. This meant that some pixels in the dendritic branch were saturated, but no pixels were saturated within the spines. Maximum density projections of the confocal stacks were prepared. Only the areas of the spine heads flanking the dendrites were measured. Spines above or below the respective dendrite were not included in the analysis. The cross-sectional area of spine heads in a region of interest was obtained from the integrated morphometry analysis function in MetaMorph and expressed in square micrometers. To calculate spine density, we divided the number of spines by the length of the respective segment of the dendritic arbor.
Presynaptic sites in PalmG neurons were measured as follows. First, we traced (with Neurolucida) the axons present in each section and marked their presynaptic sites. On each axon, we identified these presynaptic sites as boutons or large mossy fiber terminals (LMTs) based on their characteristic morphology. Second, we divided the number of boutons or LMTs by the length of the axons traced and expressed their density as "number of presynaptic sites per micrometer" or "number of LMTs per micrometer."
Statistical analyses.
The Mann–Whitney test from OriginPro 8 (OriginLab) was used for comparing the frequency of spontaneous firing in NaChBac+ and control neurons at resting membrane potential to determine statistical significance. All other data were analyzed with the two-sample two-tailed Student's t test in Prism 5 (GraphPad Software). Data were reported as mean ± SEM.
Expression of NaChBac in adult-born DG GCs elevates neuronal excitability
Changes in brain activity, such as those triggered by increased behavioral demands or seizures, affect the maturation and connectivity of new neurons born in the adult DG (Overstreet-Wadiche et al., 2006; Kron et al., 2010). These changes in connectivity could be attributable to the increased activity of the new neurons, of the circuit in which the neurons are embedded, or a combination of both. To isolate the contribution of elevated activity in new neurons, we increased the activity of individual new neurons cell autonomously with the ion channel NaChBac. NaChBac is a bacterial voltage-gated sodium channel that has both a more negative activation threshold than native sodium channels in GCs (∼15 mV more negative) and a longer inactivation time (hundreds of milliseconds compared with <1 ms in mammalian sodium channels) (Ren et al., 2001; Bean, 2007). Because of its unique electrical properties, NaChBac was used previously to induce hyperexcitability in Drosophila pacemaker neurons (Nitabach et al., 2006). More recently, we took advantage of NaChBac-induced depolarization to investigate the maturation of the GCs of the olfactory bulb, a type of inhibitory neuron generated throughout life. Genetically increasing intrinsic excitability by NaChBac expression is sufficient to enhance the survival of the new granule neurons of the bulb but, surprisingly, does not affect their synaptic organization (Kelsch et al., 2009; Lin et al., 2010). The plasticity responses of excitatory and inhibitory neurons differ in many respects (Bi and Poo, 1998), and it is plausible that electrical hyperexcitability by NaChBac affects the morphology of excitatory but not inhibitory neurons.
To investigate whether cell-autonomous increases in excitability are sufficient to alter neuronal connectivity of excitatory neurons, we used oncoretroviruses to introduce NaChBac into individual adult-born DG GCs, a type of excitatory neuron. Because this class of retroviruses cannot transport their genetic material across the intact nuclear envelopes of nondividing cells (Lewis and Emerman, 1994), they can selectively infect dividing cells in the hilus region of the DG, labeling and effectively birthdating new GCs. We used a titer of oncoretrovirus that sparsely labeled GCs [∼100 hyperexcitable cells among ∼400,00 wild-type cells of the DG in the adult B6/57mouse (Abusaad et al., 1999)] in the DG, thus keeping the vast majority of the circuit unaltered. We performed patch-clamp electrophysiological recordings of labeled GCs and found that control, wild-type cells (mCherry+) did not exhibit any type of spontaneous activity at any times tested, between 9 and 36 d post-injection (dpi). In contrast, NaChBac+ cells exhibited spontaneous, long-lasting depolarizations starting as early as 7 dpi (Fig. 1A,B). NaChBac expression did not affect the resting membrane potential or input resistance of the neurons at any times, but it increased their capacitance (Fig. 1B).
Current injection failed to elicit action potentials in control cells at 9 dpi but triggered trains of action potentials between 24 and 36 dpi (Fig. 1C,D). In contrast, current injection triggered long-lasting depolarizations in NaChBac+ cells at all times examined (9, 28, and 36 dpi) (Fig. 1C,D). Finally, whereas the threshold to fire action potentials in wild-type, mCherry+ cells between 26 and 36 dpi is 40 pA, 20 pA is sufficient to trigger a full depolarization in NaChBac+ neurons (Fig. 1D).
In summary, NaChBac expression renders new DG hyperexcitable, confirming our previous observations in the olfactory bulb (Kelsch et al., 2009; Lin et al., 2010).
To investigate whether, as in the olfactory bulb, NaChBac expression enhances the survival of new neurons, we measured the ratio of mCherry+ (wild-type) to NaChBac+ (hyperexcitable) cells at different time points after viral injection. However, we did not find any deviations from the 1:1 ratio at any time point (7–36 dpi; data not shown). Similarly, we did not find any deviations in the ratio of mCherry+ to E191K+ [a nonconducting variant of NaChBac (Yue et al., 2002)] cells at any time points after injection. These observations suggest that NaChBac expression does not significantly affect the proliferation of neuronal progenitors or the survival advantage of young neurons in the DG, in contrast with our observations in the olfactory bulb (Lin et al., 2010).
We did not observe any seizures or any other gross behavioral changes in the animals injected with NaChBac viruses, which is not surprising given the very low percentage of cells expressing NaChBac (approximately one NaChBac+ cell for every 4000 wild-type cells in the DG). These results confirm that expression of NaChBac in small numbers of new DG GCs increases their excitability in a cell-autonomous manner, without grossly perturbing the function of the DG.
NaChBac-induced excitability results in additional GABAergic input to the cell body
In many circuits, surrounding neurons react to individual neuron activity to keep circuit activity within a range that prevents disruption of function (Turrigiano and Nelson, 2004). DG GCs only start receiving glutamatergic input late in development, ∼21 d after birth, but receive GABAergic input much earlier. These cells first receive extrasynaptic input by ambient (extrasynaptic) GABA starting 3 d after they are generated, followed by GABA-mediated synaptic inputs as early as 7 d after their birth (Ge et al., 2006). For this reason, we hypothesized that, when hyperexcitable adult-born GCs are introduced into the DG circuit, one of the earliest responses of the surrounding circuit would be to alter the GABAergic input targeted to NaChBac+ neurons. To test this hypothesis, we performed immunostaining against the VGAT, which is present in the vast majority of the presynaptic terminals of inhibitory interneurons (Chaudhry et al., 1998). We quantified VGAT+ puncta on cell bodies, because this measurement was more reliable than counting the number of contacts on dendrites.
At 9 dpi, there was no significant difference in the density of VGAT+ contacts with the soma of either control or NaChBac+ neurons (Fig. 2A). However, by 13 dpi, there were significantly more VGAT+ contacts on the soma of NaChBac+ neurons compared with neurons expressing NaChBac E191K, a nonconducting variant of NaChBac (Yue et al., 2002), and this effect persisted until at least 28 dpi (Fig. 2A). To further investigate this increased innervation, we then used antibodies against parvalbumin and GAD65. Parvalbumin+ cells are a subset of inhibitory interneurons that preferentially synapse onto the cell bodies of DG granule neurons (Freund and Buzsáki, 1996), whereas GAD65 is an isoform of glutamic acid decarboxylase (GAD), an enzyme present in a large proportion of inhibitory interneurons (Erlander and Tobin, 1991). We confirmed the trend of increased GABAergic contact in NaChBac+ neurons using parvalbumin (Fig. 2B,C) and GAD65 immunolabeling (Fig. 2C). To verify whether our observations regarding perisomatic GABAergic contact corresponded to a functional increase in inhibitory input, we performed electrophysiological recordings to measure sIPSCs of individual neurons. We coinjected a mixture of retroviruses, one carrying the construct for NaChBac fused to GFP and the other carrying the construct for mCherry, into the DG and recorded from control neurons (mCherry-only) and NaChBac+ neurons in the same DG at 17 dpi. mCherry was used to label control neurons because these cells would appear red and could be easily distinguished from the GFP-expressing NaChBac+ neurons. Indeed, there was an increase in both the frequency and amplitude of sIPSCs received by NaChBac+ GCs relative to control GCs (Fig. 3A). These results indicate that individual adult-born DG GCs with elevated neuronal excitability receive more GABAergic inputs than age-matched wild-type GCs.
GABAergic innervation to adult-born GCs is initially depolarizing as a result of high levels of expression of the NA+/K+/Cl− cotransporter NKCC1 relative to the K+/Cl− cotransporter KCC2 (Plotkin et al., 1997; Clayton et al., 1998). The subsequent upregulation of KCC2 as cells mature lowers the intracellular concentration of Cl− and eventually makes the GABA reversal potential more negative than the resting membrane potential, rendering GABAergic innervation hyperpolarizing (Rivera et al., 1999; Wang et al., 2002). This switch from depolarizing to hyperpolarizing GABAergic inputs occurs after 14 dpi in adult-born GCs (Ge et al., 2006). As mentioned above, we observed an increase in inhibitory input to NaChBac+ GCs that occurs by 13 dpi (Fig. 2A). At 13 dpi, we also observed an increase in the number of KCC2-positive NaChBac+ GCs compared with controls (Fig. 3B). The increase in the percentage of KCC2+ cells induced by NaChBac expression suggests a premature reduction in Cl− concentration, which would in turn result in an earlier switch to inhibition by GABA. This accelerated maturation could enable the increase in GABAergic input to prematurely become inhibitory and thus dampen the hyperexcitable neurons earlier in development. In agreement with the notion of hyperexcitability accelerating the maturation of new GCs, we observed that polysialylated neural cell adhesion molecule (PSA-NCAM), a marker for immature neurons (Seki and Arai, 1993), is also downregulated earlier in NaChBac+ GCs compared with control neurons (Fig. 3C).
Our results indicate that NaChBac activity impacts the maturation and early synapse formation of DG GCs. From an early developmental stage, hyperexcitable GCs start receiving more GABAergic input from surrounding interneurons. In addition, cell-autonomous hyperexcitability speeds up development of newly born GCs.
Increased excitability leads to changes in excitatory glutamatergic input
Having discovered that NaChBac-induced hyperexcitability induces marked changes in neuronal maturation and an increase in inhibitory inputs early on, we proceeded to study how NaChBac affects the next phase of development of these GCs when they start to receive excitatory input synapses. DG GCs normally begin receiving excitatory inputs from 21 d after birth via spines along their apical dendrites that acquire mature morphology by ∼28 d. To examine the changes in excitatory input received by a neuron rendered hyperexcitable by NaChBac, we infected neural progenitors in the DG with a bicistronic retroviral vector that expresses both PalmG and NaChBac. PalmG is localized into the membranes of infected neurons, allowing us to visualize the detailed morphology of the neurons, including dendritic spines.
Using the bicistronic PalmG:NaChBac construct, we observed that NaChBac+ GCs exhibited some connectivity changes that were similar to those observed in immature DG GCs after seizures. DG GCs migrate a small distance, ∼5–10 μm, from the hilar border in the DG, in which neural progenitors reside, to the granule layer of the GC, in which they settle and integrate into the DG circuit. Seizures induce the ectopic migration of GCs to either the outer third of the GC layer or the hilar region (Parent et al., 1997; Jessberger et al., 2009). The majority of NaChBac+ neurons had cell bodies correctly localized within the granule layer of the DG. We observed no NaChBac+ neurons in the outer third of the GC layer but found occasional neurons in the hilar region (Fig. 4A), whereas no wild-type neurons were ever found there. The morphology of these ectopic neurons was similar to their counterparts in the GC layer. They were polarized and had dendrites extending in the opposite direction of their axon. The neurons were entirely in the hilus, and their dendrites were also located solely in the hilus instead of the molecular layer. Because of the location of their dendrites, the connectivity of these neurons is likely to be perturbed. As mentioned above, NaChBac activity induces premature downregulation of PSA-NCAM (Fig. 3C), which has been implicated in neuronal migration either through its role in decreasing cell–cell adhesion (Johnson et al., 2005) or in sensing growth factor gradients (Muller et al., 2000). Thus, it is possible that the downregulation of PSA-NCAM may be responsible for the ectopic location of some of these NaChBac+ neurons. In addition to cells found in the hilus, we also observed that cell bodies of NaChBac+ GCs reside closer to the hilar border than wild-type controls at 28 dpi (Fig. 4B). These observations suggest that hyperexcitability in young neurons affects the migration and settling of the GCs within the GC layer.
Mature GCs in the DG display apical dendrites that branch into the molecular layer of the DG. During their development, newly generated GCs in the adult DG transiently display basal dendrites extending into the hilus, which disappear by 4 or 5 d after the birth of the neuron (Shapiro and Ribak, 2006). Approximately 20% of NaChBac+ neurons displayed these aberrant basal dendrites, whereas control neurons expressing the E191K nonconducting channel never did (Fig. 4C). Interestingly, the presence of spiny basal dendrites emanating from the cell bodies of fully mature GCs and extending into the hilus is one of the hallmarks of seizure-related changes in the DG (Shapiro and Ribak, 2006; Jessberger et al., 2007). To investigate whether the basal dendrites of NaChBac+ neurons contained postsynaptic sites, we infected new DG GCs with viral vectors encoding both NaChBac and a fusion between GFP and PSD-95, a scaffolding protein selectively localized to the postsynaptic density of glutamatergic input synapses (Kelsch et al., 2008). We performed immunocytochemistry against the diffuse, unclustered GFP that filled the cytoplasm with a red secondary antibody to visualize the dendritic morphology, whereas PSD-95+ clusters were identified by the direct green fluorescence from GFP (Kelsch et al., 2008). We observed that the basal dendrites of NaChBac+ cells had PSD-95:GFP+ clusters (Fig. 4C). The persistence of basal dendrites suggests that hyperexcitable neurons receive additional synaptic inputs to their cell bodies, and this input is likely to be excitatory (Ribak et al., 2000; Thind et al., 2008).
Increase in neuronal activity during seizures also affects the formation of apical dendrites and their synapses. When we examined the morphology of NaChBac+ GCs in the granule layers, we observed that they had shorter apical dendrites on average (Fig. 5A). In addition, the density of protrusions on apical dendrites of NaChBac+ neurons was half of the spine density of control neurons expressing the pore-dead NaChBac E191K channel at 28 dpi (Fig. 5B). There was an increase in spine density from 28 to 42 dpi for control neurons (Fig. 5B, far right panel) but no additional change for NaChBac+ neurons. The average spine size at 28 dpi of NaChBac+ neurons was twice of that of NaChBac E191+ neurons (Fig. 5C, far right panel). Interestingly, the increase in spine size of NaChBac+ neurons resembles the increased proportion of mushroom spines observed in GCs after seizure (Jessberger et al., 2007).
To investigate whether the large protrusions on NaChBac+ neuron apical dendrites were indeed synaptic spines, we infected new DG GCs with GFP constructs fused to PSD-95 (Kelsch et al., 2008). Control neurons were infected with a virus expressing only the PSD-95–GFP fusion, whereas hyperexcitable neurons were infected with PSD-95–GFP:NaChBac, a bicistronic construct encoding both GFP-tagged PSD-95 and NaChBac. All protrusions present on the dendrites of labeled neurons expressed GFP:PSD-95 (Fig. 5C, left panels), confirming that the larger protrusions in NaChBac+ cells are postsynaptic sites. Furthermore, larger protrusions exhibited larger GFP:PSD-95+ clusters, suggesting that any observed change in spine size could possibly indicate larger postsynaptic densities and, in effect, larger synapses.
The morphological alterations we report here suggest that NaChBac+ neurons experience an overall decrease in the number of excitatory inputs. However, although fewer in number, each individual spine in NaChBac+ neurons was larger on average than those of control neurons. To examine how these morphological changes translated into functional differences, we measured the sEPSCs of individual NaChBac+ neurons by electrophysiological recording and found that overall frequency of sEPSCs is significantly reduced in NaChBac+ neurons (Fig. 6A, left graph), whereas the average amplitude of sEPSCs was increased (Fig. 6A, middle graph). These results are consistent with NaChBac+ neurons having fewer but larger synapses. We observed that these spontaneous synaptic events, in both control and NaChBac+ neurons, could be blocked by glutamate blockers such as d,l-AP-5 and NBQX and had a reversal potential at approximately −4 mV (Fig. 6B). These values are consistent with those of sEPSCs mediated by the opening of glutamate receptors (Cull-Candy and Usowicz, 1989), as reported previously for sEPSPs in GCs of the rat DG (−5.5 ± 1.1 mV; (Crunelli et al., 1984).
Because the frequency and amplitude of sEPSCs in NaChBac+ neurons changed in opposing directions, to find out what the resultant current was, we calculated the overall excitatory current that the neurons received by multiplying the average charge area per spike of each individual neuron by the frequency of spikes of the same neuron. The overall excitatory current received by NaChBac+ neurons was not significantly different from that received by controls (Fig. 6A, right graph), hinting at the existence of a mechanism that maintains a set level of excitatory drive into these neurons.
Elevated excitability leads to changes in excitatory outputs at CA3
To quantify the changes in outputs of NaChBac-expressing DG GCs, we examined the morphology of presynaptic terminals on their axons in the CA3 region, in which their main output is.
The axons of DG GCs synapse on multiple targets on CA3, on both excitatory pyramidal cells and inhibitory interneurons. The axon collaterals of dentate GCs form specialized presynaptic sites called LMTs. LMTs measure between 3 and 8 μm in their greatest dimension and form complex interdigitating connections with CA3 pyramidal cells. DG axons also have two other types of smaller output synapses that contact inhibitory neurons at CA3 called en passant boutons and filopodial terminals (Acsády et al., 1998). En passant boutons are varicosities 0.5–2 μm in diameter distributed along the axons of GCs, and filopodial terminals are thin protrusions emanating from the LMT. We focused on the effects of hyperexcitability on LMTs because, as a result of their characteristic morphology, these presynaptic sites can be unambiguously identified by membrane-bound GFP labeling. Expression of NaChBac decreased the overall density of presynaptic terminals present on the axons of adult-born dentate GCs at CA3 (Fig. 7A,B), which suggests that the hyperexcitable neurons downregulated their overall output to CA3. We observed that the overall density of LMTs in the axons of NaChBac+ neurons was significantly decreased compared with control neurons (Fig. 7A,C). In addition, NaChBac expression also appears to reduce the size of the LMTs (Fig. 7A). Finally, he proportion of total presynaptic sites that are LMTs is also significantly reduced in NaChBac+ neurons (Fig. 7D). These observations indicate that the output from hyperexcitable NaChBac+ DG GCs onto CA3 is significantly decreased.
To confirm that we were quantifying actual presynaptic sites in our measurements, we injected adult mice with retroviral vectors expressing Synaptophysin–GFP, a protein selectively localized to presynaptic neurotransmitter vesicles (Wiedenmann and Franke, 1985). Neurons were infected with a bicistronic virus encoding both Synaptophysin–GFP and NaChBac to visualize the presynaptic sites on axons of hyperexcitable cells. Cells in a separate DG infected with a virus encoding Synaptophysin–GFP were used as controls. Using a red fluorescent secondary antibody against the diffuse, unclustered GFP that filled the cytoplasm, we were able to visualize the full morphology of the axons at CA3, as well as determine the location of Synaptophysin-positive presynaptic sites, which showed up as green GFP-positive clusters. All structures resembling presynaptic terminals as labeled by PalmG were positive for GFP:synaptophysin in both wild-type neurons and those expressing NaChBac (Fig. 7E). This confirms that the structures we quantified corresponded to presynaptic terminals on the axons of these adult-born GCs.
Our observations of output connectivity at CA3 suggest that an increase in intrinsic excitability in adult-born GCs leads to a decrease in excitatory output at CA3. However, the output does not depend exclusively on the number of synaptic contacts but also on the properties of each of these contacts. For instance, the properties of voltage-gated potassium channels in the terminal are critical in determining the amount of release (Geiger and Jonas, 2000). Additional experiments involving electrophysiological recordings from the postsynaptic neurons in CA3 may allow to investigate whether the output is indeed reduced.
Activity-induced changes in input connectivity are dependent on cell-autonomous Npas4 signaling
Two of our observations in NaChBac+ neurons led us to hypothesize that the early increase of GABAergic synapses triggered by hyperexcitability could be related to the later changes in synaptic connectivity observed in dendrites and axons. First, one of the earliest changes observed in the development of NaChBac+ neurons was the increase in perisomatic GABAergic inputs at ∼13 dpi (Fig. 2). At 17 dpi, the overall current of sIPSCs received by NaChBac+ neurons was 10 times that of controls (Fig. 3A, bottom panel, right). An alteration of this magnitude early in neuronal development could have a significant impact on subsequent integration. Second, the premature upregulation of KCC2 (Fig. 3B) suggests that the action of GABA could be hyperpolarizing earlier in the maturation of NaChBac+ neurons. Rendering GABA hyperpolarizing on immature neurons by altering chloride concentration is known to affect the dendritic development of adult-born GCs in the DG (Ge et al., 2006).
The transcription factor Npas4 was a likely candidate underlying the increase of inhibition in NaChBac+ neurons, because it is regulated by activity and is involved in the activity-dependent regulation of inhibitory synapses in hippocampal neurons (Lin et al., 2008). We hypothesized that the increased inhibition observed in hyperexcitable new DG GCs could be attributable to the expression of Npas4.
To study the effects of expressing NaChBac in the absence of Npas4, we expressed Cre recombinase, NaChBac, and a fluorescent protein in individual neurons in the DG of Npas4 conditional knock-out mice. The expression level of a tricistronic vector containing the three abovementioned genes was too low for visualization of the labeled neurons. To achieve stronger expression of fluorescent proteins, we injected a mixture of viruses into the DG of Npas4 conditional knock-out mice. The first virus carried a bicistronic construct expressing GFP and Cre recombinase, and the second virus carried an invertible cassette with a bicistronic construct encoding both PalmMCherry and NaChBac. The invertible cassette is in the reverse 3′ to 5′ orientation with respect to the retroviral promoter except in the presence of Cre recombinase when it flips to the correct 5′ to 3′ orientation and expresses both mCherry and NaChBac (Fig. 8). In this manner, the presence of Cre leads to the expression of mCherry and NaChBac and, simultaneously, to the deletion of the Npas4 locus in individual GCs in the Npas4 conditional knock-out mice (Fig. 8). The PalmMCherry protein localizes to the membranes of such neurons, enabling the identification of fine structural features, such as synaptic spines. In this experiment, we used the same dual virus strategy in both wild-type mice and conditional knock-out mice and compared results between them.
As described above, expression of NaChBac in wild-type adult mice results in an increase in VGAT+ perisomatic inhibitory terminals on new DG GCs (Fig. 2A). In contrast, the deletion of Npas4 in individual NaChBac+ new GCs blocked the increase in VGAT terminals triggered by NaChBac at both 17 and 28 dpi (Fig. 9A). Knocking out Npas4 alone in DG GCs, using a virus carrying only a GFP–Cre recombinase construct, has no effect on the number of VGAT+ puncta at 17 dpi and leads to a very small increase at 28 dpi (Fig. 9A). Furthermore, Npas4 signaling within individual adult-born neurons in the DG is necessary to trigger the changes in dendritic morphology induced by NaChBac+ neurons (Fig. 9B,D,E). Deletion of Npas4 in new GCs blocked the changes caused by NaChBac expression in dendritic length, spine density, and size. Dendritic length and spine density in NaChBac+Npas4− neurons are indistinguishable from those of control neurons expressing the pore-dead version of NaChBac (Fig. 9B,D).
Interestingly, Npas4 was not required for activity-dependent changes in the output synapses, because NaChBac+Npas4− neurons still exhibited a dramatic reduction in density of LMTs at CA3 (Fig. 9C). Knocking out Npas4 alone has no effect on either input or output synapses (Fig. 9B–E). These results demonstrate that Npas4 signaling specifically regulates activity-dependent formation of synaptic inputs to DG GCs.
Increase in electrical activity of a single new neuron in the DG is sufficient to induce changes in maturation and connectivity
Global manipulations of brain activity via behavioral paradigms or seizures have demonstrated the influence of neuronal activity on the maturation, integration, and connectivity of adult-born neurons in the DG (Kee et al., 2007; Kron et al., 2010). However, it is unclear whether these changes resulted directly from cell-autonomous increased firing of new neurons, indirectly through the elevated activity in the surrounding circuit, or from a combination of both. Here, we genetically modulate the electrical activity in individual adult-born DG GCs and show that an increase in cell-intrinsic activity of new neurons is sufficient to cause dramatic changes in their maturation and connectivity. NaChBac activity-induced connectivity changes in the adult DG appear to be homeostatic, because NaChBac induces an increase in inhibitory inputs and decrease in excitatory outputs. Furthermore, at 13 dpi, when NaChBac+ neurons display a significantly higher number of perisomatic VGAT+ puncta, more NaChBac+ neurons express KCC2 than control neurons (Fig. 3B). This observation is consistent with previous reports indicating that the timing of KCC2 expression is activity dependent (Ganguly et al., 2001). Because the upregulation of KCC2 relative to NKCC1 is correlated with the switch between GABAergic inputs being depolarizing to hyperpolarizing (Rivera et al., 1999; Wang et al., 2002), this suggests that the GABAergic input to NaChBac+ neurons becomes inhibitory earlier in their development than in control cells, which could serve to dampen the heightened excitability of these neurons.
The results of a previous study in vitro seemed to suggest that global activity alterations are necessary to affect changes in GABAergic terminals because suppression of single-cell activity in dissociated hippocampal cultures using the potassium channel Kir2.1 did not alter GABAergic inputs (Hartman et al., 2006). Our results show that, in new DG neurons in vivo, elevating single-cell activity is sufficient to induce changes in GABAergic input (Fig. 2). It is not possible to directly compare the results from these two experiments because they were produced in different conditions and systems, namely adult-generated DG neurons rendered hyperexcitable in vivo versus embryonic hippocampal neurons silenced in vitro. However, in agreement with this previous study, when we silenced adult-generated DG neurons in vivo via retroviral expression of the Kir2.1 channel, we did not observe any changes in either dendritic or axonal morphology (data not shown). These observations suggest that the regulation of synaptic input to a single adult-born DG GC may be modulated by increases, but not decreases, in intrinsic activity.
In our study, we observed that sEPSCs received by NaChBac+ neurons were of a lower frequency but of increased amplitude than those received by control neurons, and this was consistent with the decreased spine density but increased spine size on their dendrites (Fig. 5B,C). Interestingly, the overall excitatory current received was not significantly different compared with controls (Fig. 6A, right graph). In contrast, the overall GABAergic current received by NaChBac+ neurons was ∼10 times that of control neurons (Fig. 3A, right graph). These findings suggest that there may exist mechanisms to ensure that new DG neurons receive a set value of excitatory input and that modulating inhibition may be the primary method by which the activity of an adult-born neuron in the DG is regulated.
An activity-dependent genetic program involving immediate early gene Npas4 governs neuronal connectivity of adult-born neurons to the mature DG circuit
Our results reveal an intermediate step between neuronal activity and changes in synaptic connectivity, which involves a transcription factor whose expression is activity dependent. The connectivity changes triggered by an increase in cell-intrinsic excitability in neurons are dependent on the immediate early gene Npas4. The role of Npas4 in these connectivity changes is activity dependent; deletion of Npas4 does not affect the formation of synapses in control cells that have baseline excitability, but it blocks synaptic alterations triggered by NaChBac-induced hyperexcitability. Although Npas4 has been shown to regulate the formation of inhibitory inputs to CA1 pyramidal cells in vitro (Lin et al., 2008), our results in DG GCs suggests that knocking out Npas4 alone in individual neurons does not decrease the number of inhibitory VGAT+ contacts on its soma (Fig. 9A). Npas4 is expressed at extremely low levels at baseline in DG GCs, but it is upregulated by strong stimuli, such as during kainic acid-induced seizures (Ramamoorthi et al., 2011). These observations suggest that, in young DG GCs, the role of Npas4 in inducing an increase in inhibitory contacts may require exceeding a threshold of activity that is only achieved by hyperexcitable neurons.
Finally, Npas4 is involved in activity-dependent changes to input connectivity to adult-born DG GCs but not to their output connectivity in CA3 (Fig. 9C). This observation suggests that there are independent programs governing input and output synapses, and this finding could have important implications for the structural alterations triggered by epilepsy.
This work was supported by a NIDCD RO1 Grant to C.L. and funded in part with a grant from the MIT Simons Initiative on Autism and the Brain (Y.L. and C.L.). We thank Alberto Stolfi and Drew Friedman for help with the engineering of the viral constructs, David Clapham (Harvard Medical School, Boston, MA) for providing us with the NaChBac cDNA, and Karl Deisseroth (Stanford University, Palo Alto, CA) for providing the double-floxed inverse ORF vector.
The authors declare no competing financial interests.
- Correspondence should be addressed to Carlos Lois, Neurobiology Department, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605. carlos.lois{at}umassmed.edu


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Prophet Joel on The End-Time Outpouring of the Spirit
How to Respond to Global Crisis (Joel 2:12-17)
God's Government Through Corporate Intercessory Worship God has given the human race great dignity. We have a free will, which means we have been given the ability to make real choices that make a real difference. These choices for righteousness or for sin will bring either blessing or evil to us and those around us. [...]
Military Invasions in God's End-Time Plan
The Fivefold Action Plan God Wants from Us
Lament like a virgin girded with sackcloth for the husband of her youth … 13 Gird yourselves and lament, you priests; wail, you who minister before the altar; come, lie all night in sackcloth, you who minister to my God; for the grain offering and the drink offering are withheld from the house of your [...]
6 pages

subset: commoncrawl


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Fish & Richardson has received top "Band 1" national rankings for its Intellectual Property (IP) practice and its International Trade Commission (ITC) patent litigation practice in the 2019 edition of Chambers USA.
Fish also received top band rankings for its regional practices in California, Delaware, District of Columbia, Georgia, Massachusetts, Minnesota, New York, and Texas. Thirteen Fish attorneys – Juanita Brooks, Ruffin Cordell, John Dragseth, Michael Florey, Christopher Green, Danielle (DJ) Healey, David Hoffman, Martina Hufnal, Thad Kodish, Douglas McCann, Michael McKeon, Frank Scherkenbach, and Dorothy Whelan – were also selected for inclusion in the directory as leaders in their practice areas.
Chambers notes that Fish stands out for its "cutting-edge litigation practice with an excellent reputation" and "vast experience before the district, federal and ITC courts, in addition to administrative proceedings." The firm is "admired for notable trial successes… and continues to impress with its handling of ITC investigations for a number of the nation's leading brands." An "additional strength is advising on strategic management of IP portfolios including patent prosecutions and licensing agreements."
Fish is known for its "phenomenal trial lawyers" and is "a firm we go to for more of our high-level, more complex matters." Clients praised the firm's ITC practice which "is among the best in ITC litigation; it has a team of exceptional attorneys with the ability to understand all relevant legal concepts and technical issues, an in-depth industry knowledge, and can assess how this fits into the client's business objectives."
The Chambers Guides have been ranking the best law firms and lawyers since 1990, and cover 190 countries across the world. Inclusion in Chambers is based solely on the findings of its research teams, who conduct extensive, confidential interviews with thousands of lawyers and clients to identify the leading law firms and lawyers worldwide. Individual lawyers are ranked based on their legal knowledge and experience, ability, effectiveness, and client service. Law firms are ranked on the same qualities, as well as overall effectiveness and capability. Rankings are grouped in bands from 1-6, with 1 being the best.
Related Attorneys
Martina Tyreus Hufnal
Ruffin B. Cordell
DJ Healey
Senior Principal
Juanita Brooks
Michael J. McKeon
Dorothy P. Whelan
Senior Principal
David M. Hoffman
Thad C. Kodish
Christopher O. Green
Frank E. Scherkenbach
John A. Dragseth
Senior Principal
Michael E. Florey
Senior Principal
Douglas E. McCann
Related Services
- Litigation
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- Product Liability and Mass Torts
- Trademark, Copyright, and Media Litigation
- Trade Secret Litigation


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The Australian Graphic Design Association (AGDA) quite often produces posters to advertise their various events or conferences they are supporting, which are then sent out to each of their members. They're usually of an extremely high quality. The one that I received today is no exception and thought that it needed to be shared.
This particular poster is to advertise ICOGRADA Design Week being held in Brisbane (Queensland, Australia) on October 11-17 2010. Through the theme of 'Optimism', Icograda Design Week Brisbane 2010 aims to promote dialogue on issues shaping the nature and relevance of communication design practice today.
The poster was designed by the talented folk at Studio Bomba.
Bomba is a studio of brand boffins that spend every last second of their work-a-day life thinking about how people can best use design to close the gap between '?' and '!'.
You can click on the image below to view a larger PDF version. The PDF was created by taking numerous scans of the A1 sized poster. The original poster was printed on 120gsm ecoStar 100% PCW:FSC CoC: paper made Carbon Neutral from Raleigh Paper.
I'd love to here what you think of this graphic or if you've seen any others of late that are particularly clever, leave a comment and let me know.


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Our office is proud to be a provider of the Invisalign system. Invisalign offers orthodontic treatment to correct a wide range of problems, including crooked teeth, gaps between your teeth, overcrowding, and bite problems.
Invisalign offers you a way to straighten your teeth without the unsightly and uncomfortable brackets and wires involved in traditional braces. The Invisalign aligners are comfortable, clear plastic trays custom made to fit your teeth. The clear plastic trays are practically invisible when they're in place, allowing you to correct your teeth without anyone knowing you're undergoing orthodontic treatment.
Every few weeks, you switch to the next set of aligners in the series, gradually adjusting your teeth into the right position through small changes in the fit of each set of aligners. The gentle, gradual adjustments will help your teeth and gums grow accustomed to the new positions, helping to keep overall treatment times down.
Other benefits of Invisalign include:
- Gentle tooth adjustments that lead to less pain during your orthodontic treatment
- Removable aligners, which don't limit what you can eat while using Invisalign
- No need to perform adjustments, meaning fewer visits and quicker dental appointments during treatment
If you are interested in finding out if you are a good candidate for the Invisalign system, call our office today and schedule a consultation exam.


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66th Birthday Cupcake Toppers – Rainbow LGBTQ
66th birthday toppers featuring rainbow colors and fun big dots with glowing lights at the top and retro style text for a big ole gay birthday party.
(gay, lesbian, trans, 66 year old birthday ideas, diy party, favor tags and party circles, instant download, picks)
This is an Instant Download digital file. Just click the download link on your receipt to start printing!
You Get: 1 PDF file with twelve 2.1" circles on the page to punch out with a 2" circle punch or cut with scissors. Just print, cut, then tape a toothpick to the back...or if making favor tags, punch a hole and attach with ribbon.


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Landholder duty
You pay landholder duty when you make a relevant acquisition in a landholder.
Landholder duty was introduced on 1 July 2011 to replace land rich duty.
A landholder is a corporation or listed unit trust that has land-holdings in Queensland with an unencumbered value of $2 million or more.
There are 2 types of landholders:
- private landholder - an unlisted corporation (i.e. a company not listed on a recognised stock exchange)
- public landholder - a listed unit trust or listed corporation.
Land-holdings commonly include:
- an interest in land, other than
- a security interest
- an interest in a trust
- anything fixed to the land (e.g. pipelines, plant and equipment), whether or not the landholder has an interest in it
- an interest in land, including anything fixed to the land, that is the subject of a purchase or sale agreement
- the land-holdings of a subsidiary of the landholder
- land-holdings held on trust by the landholder, where the landholder (or a subsidiary of the landholder) is a beneficiary of the trust
- land-holdings held by the landholder for a partnership, regardless of the interest that the landholder has in the partnership.
This guide explains what a relevant acquisition is, how landholder duty is calculated and when an exemption may apply.
- Last reviewed: 22 Feb 2022
- Last updated: 26 Jun 2019
I want to...
Queensland Revenue Office
- Call 1300 300 734 (Australia) or
+61 7 3179 2500 (overseas)
- Send an email using our online enquiry form.


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Eric really doesn't like it when I attempt to "celebrate" our "other" anniversary. But after spending 20 years together, this certainly won't be our first well, disagreement. So, here I am talking about it. Twenty years ago today we went on our first date. Although Eric really only likes to celebrate or wedding anniversary, I still believe there is a little something special about how long we've been together. Recently, I've been thinking about how much we've changed over 20 years.
Where We Thought We'd Be
Eric and I met at Rutgers University in May of 1997. As much as there is still a little bit of a dispute about how we met, and what exactly happened on that fateful night at The Knight Club in New Brunswick, New Jersey, one thing is certainly true. We were much different people back then.
That tends to be true for most people who've been together for almost half their lives. But for us, it is definitely true. When we met, we both knew we wanted to escape New Jersey. We grew up there, and both attended college within an hour of our home town. It was time for change.
But, we spent our time thinking about living in California, working on our careers, and raising our kids. In fact, the first summer we dated we even named our children.
Eric chose Trevor, so that our son would be named after Trevor Hoffman, a professional baseball player. Let's just say that never would have happened. I was leaning towards Kirsten, or something similarly starting with a "K" as an homage to my little sister Kayla.
We wondered about our plans for after college graduation. I started freaking out about what a duel major in History and Political Science could do for a living. I thought about law school. Within a year of dating, we were living together (don't tell my mother-in-law), moving to DC for law school, and planning our future in a way typical of people our age.
Where We Are 20 Years Later
Well, none of that really panned out, uh? We never moved to California. We certainly never had kids. So, how did we get from young college students, getting drunk at The Knight Club, to globetrotters who have lived in Bali and Bangkok.
When we first met, we had barely traveled outside of New Jersey. I traveled to England, Scotland, and France on a trip just after high school. I'd been to Mexico when I was young. But, that was about it. Eric was in the same boat. He'd traveled home to Ireland when he was a kid. Popped into Mexico once. Between the two of us we'd traveled to less than 10 countries.
Over the following decade, the travel bug spread. After our first overseas trip together to Ireland to visit family, we started to explore the world a little more. A few trips to Europe. Our first trip to Southeast Asia. Travel became a craving we had to satisfy. We took every opportunity we could to escape our jobs, even for just a weekend. Labor Day in Rome. Thanksgiving in Amsterdam. Christmas in Hong Kong.
And, the older I got, the less I wanted to have kids. Over time, our initial goal of settling down in a house in Southern California with our two kids, turned into a plan to escape the predictable life.
It was the best decision we ever made. Even if we don't remember ever specifically making a "decision."
So, here we are. Twenty years later. We've been to about 70 countries and haven't lived in the US for almost 5 years. Most important, we couldn't be happier. Both with our lives and the decisions we've made.
How Our Travel Style Changed Over 20 Years
As much as the biggest change over the last 20 years together has been the complete and total switch in our lifestyle, other things have changed as well. Namely, how we travel. When we first left the US, we were on the road, moving from place to place. We traveled on buses and trains. We stayed in hostels.
For awhile, this was fun. I assumed that all long term travelers were essentially backpackers, or minimally flashpackers, as we most likely were. But, there were noisy hostels, with uncomfortable beds and shared bathrooms.
There were chicken buses and bug attacks in Central America. Yes, we had some fun experiences. But, I've grown up. We are in our forties. And, sure, there are plenty of people who still travel as backpackers in their forties. We are just not those travelers anymore.
Now, not only do we need reliable wifi for our online jobs, but I need more comfort now than I used to. We also need fast wifi to keep in touch with friends and family online. This might mean a quick Skype call or even playing online games with friends to keep connected.
We try to walk the line between renting comfortable apartments with Airbnb (get free credit on Airbnb here), and trying the best we can to travel in an affordable luxury sort of way. Obviously, the blog helps. But even when it comes to the blog, I am a lot more choosy about who we decide to work with and who we don't. Not only do I need comfort, I also need unique experiences.
Sure, we spend some days just working on the sofa, while streaming Netflix. Not necessarily luxury. Or exotic. Then again, maybe it is. The reason why we made the decisions we did was because I didn't want my old life. I wanted more freedom. More choices. Right now, we can decide whether we want to be in Bangkok, or Bali, or Barcelona. We can decide to return to some of our favorite places, or explore some place new. We can spend a few days exploring a new city. Or, we can rent an apartment for a few months some place we love. Each day and each month seems to bring something new, and for that I am truly grateful.
The question is, what do we have to look forward to over the next 20 years??? Your guess is as good as mine.
Amber Hoffman, food and travel writer behind With Husband In Tow, is a recovering attorney and professional eater, with a passion for finding new Food and Drink Destinations. She lives with her husband, Eric, in Girona, Catalonia, Spain. Together over the last 20 years, they have traveled to over 70 countries. Amber is the author of the Food Traveler's Guide to Emilia Romagna.


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Thank you for visiting our website! Below you will be able to find the answer to Rotunda top crossword clue which was last seen on Eugene Sheffer Crossword, September 16 2016. Our site contains over 2.8 million crossword clues in which you can find whatever clue you are looking for. Since you landed on this page then you would like to know the answer to Rotunda top. Without losing anymore time here is the answer for the above mentioned crossword clue:
We found 5 possible solutions in our database matching the query Rotunda top
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On the 7th of October I celebrated the Feast of the Bacchic Martyrs, this festival is celebrating the devotees of Dionysos that have been killed for their religious beliefs. (Dionysians were persecuted well before Christians.) I cannot describe the day in detail because, frankly, I can't remember. I was so heavily entranced that my actions were not my own and guided by someone else. With limited resources (as in money) I managed to craft my first thyrsos, admittedly a lot of it is made of artificial plants, but I see a ritual meaning of plastic as it is the ultimate dead thing. The thyrsos being a symbol of life and death, seems pretty fitting. Ironic even.
I also dressed formally as Δ the festival falling roughly in the same month of confirmations of initiation as The Dionysian Artist back in 2015.
I finished the Feast with a basic but delicious vegetarian pasta dish, a plate of which was given at a crossroads by a funeral home I live next too. (Yeah, a funeral home is my neighbour, little wonder why I come in contact with the dead so often.)
Here are some photos that were *permitted* to be shared of the days activities.


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Minimization of the risk of diabetic microangiopathy in rats by Nigella sativa
Juraiporn Somboonwong1, Mariem Yusuksawad1, Somboon Keelawat2, Sirima Thongruay3, Ubon Poumsuk2
1 Department of Physiology, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand2 Department of Pathology, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand3 Department of Research and Development Center for Livestock Production Technology, Faculty of Veterinary Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
Correspondence Address:Juraiporn SomboonwongDepartment of Physiology, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330 ThailandSource of Support: None, Conflict of Interest: NoneCheck1DOI: 10.4103/0973-1296.182169
Source of Support: None, Conflict of Interest: None
Background: Microangiopathy is a chronic diabetic complication resulting from metabolic derangements, oxidative stress, and increased pro-inflammatory cytokine production. Nigella sativa Linn. is used as an herbal medicine that exerts hypoglycemic, antilipidemic, anti-inflammatory, and antioxidant effects. Objective: To examine the effects of N. sativa extract on cutaneous microvascular changes in diabetic rats. Materials and Methods: Sprague-Dawley rats were randomly assigned into the following four groups: Untreated and N. sativa-treated normal controls and untreated and N. sativa-treated rats with streptozotocin-induced diabetes. A cold-pressed N. sativa extract was then orally administered (1000 mg/kg/day). After 8 weeks of treatment, the glucose, glycosylated hemoglobin (HbA1c), tumor necrosis factor-alpha (TNF-α), insulin levels, and lipid profile were determined in cardiac blood. Dermal capillary wall thickness was measured in tail skin sections stained with periodic acid-Schiff. Endothelial apoptosis was morphologically evaluated by hematoxylin and eosin staining. Results: Diabetes significantly reduced the circulating insulin and low-density lipoprotein levels and caused elevations in the glucose, HbA1c, and triglyceride levels, accompanied by a slight increase in total cholesterol levels and no change in the high-density lipoprotein and TNF-α levels. Capillary basement membrane thickening and a decreased capillary luminal diameter despite no evidence of endothelial cell apoptosis were also observed. N. sativa treatment of diabetic rats reduced the mean HbA1cconcentration by 1.4%, enlarged the capillary lumens, and tended to attenuate dermal capillary basement membrane thickening without affecting the lipid profile or TNF-α level. Conclusion: Our results indicate that N. sativa may be used to minimize the risk of diabetic microangiopathy, potentially due in part to its glycemic control activity.SUMMARY
Keywords: Capillary basement membrane, diabetic microangiopathy, endothelial apoptosis, glycosylated hemoglobin, Nigella sativa Linn.


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Woman Arrested In Biting Attack Over Parking Space In SF Hunters Point
SAN FRANCISCO (CBS 5) - San Francisco police took a bite out of crime after an angry motorist allegedly took a bite out of another motorist in a fit of parking space rage.
San Francisco police arrested Sara Gillan, accusing her of biting another woman over a parking spot around 6 p.m. Tuesday on Jennings Street in The Bayview. Police said the fight began when a car swooped into the parking space that the suspect had apparently been waiting for.
That's when Gillan is accused of approaching a woman in the other car and biting her in the chest and the arms. The victim showed CBS 5 cameras a large bruise on her arm from the bite.
A witness told CBS 5 that teeth were not the only weapons.
"When she got loose, she got the Taser gun," said Trevell Council, who watched the brawl break out in the parking lot. "She went and got the Taser gun and everything and she was trying to get her, but they was struggling and then she dropped it."
Gillan was booked on charges of aggravated assault.
The two men and the other woman involved were released at the scene.
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Chapter One from my book, "45 Minute Business Breakthroughs," to download a copy of the entire book you can do that HERE
Let's face it. The major hot button for most small businesses these days is the ability to generate leads. All small businesses want more leads, but few of them know how to successfully attract customers to their business.
As a coach, I have in-depth knowledge and skill when it comes to generating leads. So here's the process I use to do this.
If you're like 99% of the business owners I speak with, you may often feel lost or overwhelmed as you try to navigate through all the various options available these days. Websites, social media, SEO, email marketing, Facebook, pay-per-click and so on.
Let me do you a favor right now and completely remove that overwhelm from your life forever. Are you familiar with the 80/20 rule?
For business owners, it means 20% of what you do every day is generating 80% of your total annual revenue.
In other words, you're only doing a few things daily that makes you most of your money. I can tell you specifically what makes up that 20%, and that's all you really need to focus on after today.
There are 5 areas that make up that 20% – leads, conversions, transactions, pricing and profits.
I'm going to be focusing on just 2 or 3 of these 5 areas, so you can imagine what you could actually generate revenue-wise if you implemented all 5 areas.
In fact, let me show you what's possible, and why these 5 areas are so critical.
I use a tool called a Profit Growth Calculator. Do you by chance know the exact number of leads and sales you've made over the past 12 months?
No! That's OK… let's plug in numbers for a make believe business. Let's say your business generated 1000 leads in the past year… and your average conversion rate was 25%.
Let's also say your customers bought what you sell 10 times throughout the year… and they typically paid on average around $100 per purchase.
Finally, let's say your profit margin per sale is only 25%.
Notice at the bottom that you're earning $62,500 annually. But, look what happens if we simply increase each of these 5 areas by a meager 10%.
You would see your annual revenue almost double from $62,500 to over 6 figures. By the way, that's the ballpark most business coaches play in… the 10% increase range. Nothing wrong with that either, believe me.
Most business owners would KILL to almost double their revenue, wouldn't you agree? Now, watch what happens if you could increase each of the 5 areas by 50%.
Your business would skyrocket from $62,500 to almost half a million dollars annually. Now, you may be thinking 50% gains in each of these 5 areas would be next to impossible. Let me assure you, a 50% increase is child's play, and I'm going to prove it to you right now.
When asked, most business owners tell me "word of mouth", or more often than not "referrals", are how they generate leads. Referrals are obviously an excellent lead source. In fact, it may be the best one by far, but the problem is you never know when you will get them. They're not reliable and you certainly can't generate them whenever you want.
99% of businesses today do have a website. Do you know for sure how many leads your website generates every month? Do you know for sure how many sales your website produces every month?
Can I show you why your website isn't generating leads or closing sales for you? In fact, would you like for me to give you the deeply hidden secrets that the marketing gurus DON'T want you to know?
Here's the key to successful marketing. You MUST be able to enter the conversation taking place in the head of your prospects. Or, another way to look at it is to be able to address the number one question on your prospect's mind at just the right time. So how do you do this? It's actually quite simple when you know and understand the fundamentals of marketing.
The conversation that's taking place in EVERY prospect's mind revolves around two major points. There's a problem they have, and they don't want… and there's a result they want but don't have.
Now believe it or not, there is actually a marketing formula we follow that takes these two points into account and spits out a message so compelling it practically forces your prospects to buy what you sell.
It's called the Conversion Equation, and it looks like this – Interrupt, Engage, Educate and Offer. The Interrupt is your headline – which means it's the first thing someone sees when they visit your website, read any of your marketing collateral, or hear you speak. When someone asks you what you do, it's the first words out of your mouth. That's your headline… and it MUST address the problem your prospects have that they don't want.
The Engage is your sub-headline – which is the second thing your prospects see or hear. It MUST address the result your prospect wants but doesn't have. The Educate is the information you provide… either verbally or in writing… that presents evidence to your prospects that you and your product or service are superior in every way to your competition.
Unfortunately, MOST businesses aren't different from their competitors, and that's why you MUST innovate your business to create what we refer to as a market-dominating position.
You MUST make your business unique… it MUST stand out from the crowd. It MUST make your prospects say to themselves that they would be absolute idiots to buy from anyone else but you – regardless of price. And finally, the Offer. You MUST create a compelling offer that makes it so irresistible your prospects can't turn it down.
But, here's another critical fundamental of marketing. Because of the saturation of marketing messages these days, most prospects have become numb to most marketing.
Following our Conversion Equation can dramatically overcome this, but even with this powerful tool in play, it will still take multiple "touch" points before your prospects will buy what you sell.
For most businesses today, it takes anywhere from 20 to more than 100 touch points before a prospect makes their buying decision. Following the Conversion Equation reduces the touch points to somewhere between 5 to 12 points of contact.
But here's the key… most businesses don't follow up with their prospects at all, and this provides a HUGE window of opportunity for ANY business that does follow up… to position themselves as the dominant force in their industry.
But, in order to have the opportunity to get your message in front of your prospects 5 to 12 times, you MUST find a way to collect their contact information, and that's the purpose of your Offer.
Most businesses offer something that only appeals to prospects we call NOW buyers – prospects ready to make an immediate purchase. Unfortunately, NOW buyers make up less than 1% of the total number of prospects that are in the market to buy what you sell.
These businesses typically offer prospects a free consultation, a discount, a coupon, a free assessment, a complimentary quote, or the biggest mistake of all… CALL US!
For most businesses, all of their marketing material… their website… their business card… all list their phone number as their sole offer… and that ONLY appeals to that 1% of NOW buyers. The remaining 99% of viable prospects are "investigating" and gathering information about what you sell.
They're searching for information because they want to determine who is offering the best value. You see, prospects DON'T shop price – they shop VALUE!
The only reason prospects consider price is that most businesses don't give them any other value proposition to consider except price.
Remember what I said a moment ago about making your business unique – creating a market-dominating position?
Most businesses don't do that, and since they… and all of their competitors… look exactly the same, prospects are FORCED to shop price. So, with these fundamentals in mind, let's see how your website stacks up to them.
Let me show you a website we just revised for a child psychologist so you can see what I mean… and then let's take a look at your website as a comparison. Here is the child psychologist's original website.
This is typical for this profession, and 99% of his colleagues' websites look EXACTLY like this. Notice the generic headline – Parenting Advice and Resources from Dr. John Smith.
He has to have a headline like that because he's attempting to be all things to all prospects. Basically, this doctor helps parents deal with adolescent problems. Look at the 9 areas he services – emotionally disturbed kids, behavioral problems, teen pregnancy, peer pressure and so on.
So, let's compare this site with the fundamentals we just discussed. First, you MUST create a market-dominating position. This doctor could actually create 9 of them by simply positioning his specialty in each of his 9 individual areas of treatment.
For example, let's say he decides to start with the top condition on his list… emotionally disturbed kids. These are kids that yell, scream and constantly have a highly belligerent attitude toward their parents. They scream at them and are known in some cases to threaten the parents. These kids can't be reasoned with and these poor parents have NO clue how to deal with this situation.
So, here's what this doctor needs to do. Forget the website completely – this doctor needs what we call a squeeze page. This is a single page that's online and specifically addresses ONLY this one condition. So, what should this page look like… and what should it say?
Remember the second fundamental – you MUST enter the conversation taking place in the head of your prospect. There's a problem they have they don't want… and there's a result they want but they don't have. This is where we implement the first two components of the Conversion Equation… Interrupt and Engage. The headline is the Interrupt and it must address the problem they have and don't want.
Here's the squeeze page we created for this doctor that did that.
Notice the headline – Are You Sick and Tired of the Yelling, Screaming and Belligerent Attitude of Your Child? Does that address the problem these parents have and don't want? Would you say that's a 100% bullseye?
Now, for the Engage which is the sub-headline. It MUST address the result they want but they don't have. Notice it says – Now You Can Discover the Secrets to Controlling Your Child and Instantly Restore Peace and Quiet in Your Home. Would you say that's bullseye number two?
Now, let's look at the third Conversion Equation component… Educate. In the doctor's original website, because he's trying to appeal to all prospects, his video said this – "Greeting parents. I want to welcome you to remarkable parenting. You will find tons of great information here with hundreds of pages of articles."
Think how ridiculous this sounds if I'm one of these parents with a kid that has a belligerent attitude. Do I want to read hundreds of pages of articles? Or, am I searching for a specific solution to a specific problem? Do you see why most websites these days are basically a total and complete waste of money? They don't address the things your prospects are truly looking for. Here's the new script we created for this doctor.
"As a parent, are you struggling to gain control of your child's attitude and emotions? Is your child yelling and screaming at you, while often displaying a belligerent and sometimes threatening tone that no matter what you do or try… you just can't seem to get under control?
My name is Dr. John Smith, and I help parents like you every day learn the techniques that will solve these frustrating and destructive behavioral patterns once and for all. In fact, let me prove it to you. Enter your first name and email in the box to the right, and I'll send you a series of 60-second techniques that will immediately restore peace and quiet in your home."
Think that just might get more prospects to respond to this message? And that brings us to the final component of the Conversion Equation… the Offer. Look at the doctor's original offer. It was for a free consultation. The only prospects that will accept that type of offer are those NOW buyers. Remember, they're less than 1% of the total number of prospects looking for this type of help.
When your offer is to "call me," that basically says "let me sell you" to your prospects. We are so used to getting non-stop sales pitches these days that we resist calling anyone with every fiber of our being. Most people won't answer their phone unless they recognize the caller ID. This type of offer is called an incentive offer, and incentive offers only work for common purchases, emergency situations and impulse purchases.
And remember, most prospects don't buy until they have been exposed to your messaging somewhere between 5 to 12 times. If you tell prospects to "call you," and most won't, how do you keep marketing to them? Obviously, you can't. The secret to effective marketing is to offer what most prospects truly want… INFORMATION!
Look at the last sentence in the child psychologist's video script… "enter your first name and email in the box to the right, and I'll send you a series of 60 second techniques that will immediately restore peace and quiet in your home." That offer is ZERO risk to a prospect, and it offers them something they truly want… a solution to their problem.
They can receive it by simply providing their name and email address… WITHOUT having to speak to anyone… or be subjected to any type of sales pitch. That's why the offer on this doctor's squeeze page says, "Learn the Secrets to Gaining and Maintaining Complete Control of Your Child in Less than 60 Seconds." Is that a highly compelling offer that would appeal to a majority of the prospects directed to this page?
And do you now see why we call this a squeeze page? There are NO navigation buttons on this page to distract the prospect. In fact, there is only ONE action they can take… enter their contact information. Otherwise, they have to close the page completely… and if they do, THAT is when we can redirect them to the doctor's main website to see if there is something else that might grab their attention.
That informational offer provides them with proof that this doctor can actually get them the results they're looking for, and then within that information is an offer for them to schedule a consultation with the doctor, which they are now more likely to do.
But consider these numbers for this doctor's original website. He could easily generate 300 or more leads per month using a pay-per-click campaign on Facebook. Those leads are then sent to his original website. He will then average around 10% of those leads… or 30 prospects… will see his offer for the free consultation and will call to inquire about it.
Notice I said INQUIRE about it, NOT request it. Out of that 10% that will call… only 10% of them will actually consent to the consultation… which equals 3 prospects.
Fortunately, for most professionals like this doctor, they typically convert 100% of the prospects they get in front of… so those 3 prospects will more than likely become patients. Note that out of 300 leads, the doctor winds up with 3 new clients. That is the national average today – 1% of all leads generated will typically convert into a new client. That's pathetic!
But, now let's look at the doctor's new squeeze page. First of all, let's leave his number of leads at 300 per month. That squeeze page won't impact that number whatsoever. But let me ask you this and give me your open and honest opinion.
Do you think this new page will increase the number of prospects that will request this doctor's secrets to gaining and maintaining complete control of their child? The doctor was getting 10% with his old site. What percent do you think would request this new, more compelling offer?
Most responses I get average somewhere between 50% to 70%. Well, suppose we stay really conservative and say that just 20% request the new offer.
That would mean 60 prospects would receive those secrets and actually see for themselves that this doctor's methods really work.
And once they do, what percent of those do you think might request the consultation with the doctor? Remember, that originally it was just 10%.
Again, most responses I get average between 50% to 70%. I would tend to agree with those numbers, but we know he originally converted 10%, so to be really conservative, let's just leave that conversion rate the same… 10%.
So, out of the 60 prospects requesting the doctor's secrets, 6 of them now request the consultation. And let's assume like we did originally the doctor converts all 6 of them into patients. That's an additional 3 patients per month, isn't it?
Now, let's say this doctor only charges $800 for his services, even though in reality it's typically 3 times that amount. $800 times 3 new patients is an additional $2,400 per month… which is an annual increase of $28,800. That's obviously a dramatic increase in revenue considering we're being ridiculously conservative… and all we did was make some slight changes to this doctor's site.
So, let me ask you this. Do you think we could get similar results for your business? How many leads have you generated in the last 12 months?
How many leads would you estimate you've generated this month? How many of those leads requested your offer? If we could create a similar process for your business… and offer compelling information to your prospects just like we did for the child psychologist… do you think more prospects would respond? By what percent?
Could we conservatively agree that a 10% opt-in rate is easily a no-brainer? Do you realize just that one change alone would double your current sales revenue?
And that's assuming we don't increase your number of leads or your final conversion rate… which we will. If you said your last month's revenue was $25,000… then just this one change alone adds an additional $25,000 to your bottom line.
In a recent case study I conducted, I found $58,000 in additional annual revenue just using this one simple strategy.
But consider this!
That additional revenue is NOT just a one-time increase. That's revenue the business will generate year after year after year.
And… $58,000 in additional annual revenue increases the valuation of that business somewhere in the range of $150,000 – $200,000.
This article was taken from my book, "45 Minute Business Breakthroughs," to download a FREE copy of the entire book you can do that HERE.
If you would like to explore other options of how I can help you take your business and your life to the next level, find out more about my one-on-one coaching programs by clicking HERE
Another option to consider is our online business coaching program. The E-Learning Marketing System is the most powerful and dynamic do-it-yourself client attraction program ever created. It was designed to give every small business owner unrestricted access to the tools, resources and support they need to build whatever size business they want. Check out our FREE Guided Tour to find out more about the E-Learning Marketing System by clicking HERE


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It was only January 4th, and I already found myself struggling to keep my happy, hopeful new year attitude. All it takes is one or two annoying phone calls and a fussy baby to get me off track sometimes. Thankfully I decided to keep my promise to read my Bible every night, and this is what my devotional from Pastor Bob Coy said speaking about joy:
"…Faith is joy's starting point. Joy is born and begins to grow when we place our confidence in the greatness of God, when we acknowledge all He's capable of, and when we realize all His love accomplishes on our behalf. Joy, true transcendent joy, cannot be gained apart from an abiding trust in God. It's like trying to produce light from darkness, it simply won't happen. But joy abounds limitlessly for all who make the Lord their confidence. Where there is faith in Him, there is joy…"
So here I am on January 5th trying to get my attitude back in check, trying to make sure my confidence is in God. I have a feeling this won't be the last time this year (or this week) that I have to do this.


text: Get four bone-chilling novels of psychological and supernatural suspense from New York Times bestselling author Joe Hill in one e-book, including: Heart-Shaped Box, 20th Century Ghosts, Horns, and NOS4A2. Each publication of Hill is beautiful textured, deliciously scary, and greeted with the sort of overwhelming critical acclaim that is rare for works of skin-crawling supernatural terror. Read on if you dare to see what all the well-deserved hoopla is about.


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The world is full of opportunities to live like a Victorian, if you look hard enough. For instance: The U.K. is worried about rising rates of scarlet fever.
That's according to the BBC. Public Health England warns that cases in England and Wales jumped from 4,642 in 2013, to 15,625 in 2014, to 17,586 in 2015. That's the most since 1967. And 2016 isn't shaping up any better, either: "PHE said about 600 cases are being flagged up each week in England, and further increases are expected as the infection comes into it the peak season - which typically occurs between late March and mid April," the BBC notes.
Consequently, PHE wants doctors and patients alike on the lookout. "Patients who do not show signs of improvement within a few days of starting treatment should seek urgent medical advice," warned Dr Theresa Lamagni, who noted that possible complications include pneumonia and ear infections.
This isn't a case of vaccine deniers run amuck; there's no vaccine for scarlet fever. The reasons for the spike aren't clear, either; it "may reflect the long-term natural cycles in disease incidence seen in many types of infection," according to PHE.
Good luck out there!
Contact the author at email@example.com.


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Japan's New Era
May 2019
By Yuka Hasumi, Vice President, Sales, EquiLend Asia
IT IS A TIME OF CHANGE in Japan as Emperor Akihito abdicates his position, the first to do so in over 200 years. As Japan enters the "Reiwa" Era, it continues to be a powerhouse in the Asia region from a political, technological and economic position.
In the finance markets, Japan at times tends not to attract the level of attention that its securities lending market potentially deserves. Despite being the biggest single securities lending market in Asia from a revenue perspective, it often takes a backseat in the news over hotter markets that have higher fees and greater year-over-year revenue increases.
While markets such as South Korea and Hong Kong dominate the headlines and interest from hedge funds, Japan continues to provide the securities lending market with opportunities through its depth of assets and market participants, both domestic and international. Alliances with regional exchanges in Asia provide further access and integration into Asia, reducing systematic risk. And as host of the 2020 PASLA conference, all eyes will be back on Tokyo in the coming year in the securities finance market.
In 2018, Japan represented 53% of the regional loan balance with an average on-loan value of $117.17 billion across both equities and fixed income, and the market accounted for 35% of revenue earned by lenders across the entire region in 2018, with $778.4 million.
In the broader securities markets, further changes are underway. The big news for Japanese stock markets this year is the pending operational shift from a T+3 to a T+2 (two days after a transaction) settlement cycle. It is the second chapter in a series of settlement reforms to enhance the market's international competitiveness and increase trading efficiency. The reduced settlement cycle, in line with other major global markets such as the EU and the U.S., is expected to promote increased liquidity in the market, reduce operational and systemic risks and assist counterparties in avoiding trade failures. A further development would be the possible launch of a central counterparty (CCP) model in Japan, although discussions are in the early stages.
Furthermore, firms active in Japan are embracing technology more than ever. The take-up of automated trading platforms by key participants in the Japanese markets has promoted further economic development in the market. As for the securities finance market, EquiLend's NGT trading platform, Post-Trade Suite and variety of DataLend products have seen steady interest and increasing adoption and use in Japan.
Much like its cash trading markets, Japan's securities finance market comprises both onshore (domestic) and offshore (international) participants— securities houses, trust banks, asset managers, life insurance companies, brokerage firms, custodians and online brokers.
The number and size of new entrants entering the market has grown substantially in recent years, along with holdings, and cross-border relations have improved.
The market continues to be an integral part of alternative investment strategies for domestic firms, and a leading revenue driver in the region. Furthermore, the cost of funding for local participants accepting Yen collateral provides an advantage for many on the brokerage side.
The compelling aspect of the onshore market that continues to see growth is the participation and increasing market share of retail, or online brokers, which, due to the nature of their underlying clients and growth, are able to provide access to the mid, small and micro cap equity names that so many hedge funds are searching for.
In recent years we have seen new participants enter the securities finance ring to provide access to their holdings and capture additional alpha for their clients. As a result, and given that revenue from lending Japanese equities increased an impressive 30% in 2018 versus 2017, growth and interest in the market should remain robust and perhaps increase in 2019.
With the market developments, technology adoption and growth of the securities finance market, Japan looks to be back in focus for market participants in Asia.


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If you've seen my portrait drawings on RedditGetsDrawn where I draw and paint as user ObeyMyBrain, you may have wondered, "How can I get him to draw one of my photographs?" Well wonder no longer.
Tips on choosing a reference photo:
Bright even lighting is best, unless you're going for a dark contrasty look of course.
Try to pick a photo that shows your entire head. Especially your hairline.
Portrait only at this time and just based on the reference photo. Nothing like, "Make me a pirate."
Digital Portrait
Color: $95 (one subject/person)
Black and White/Monochrome: $75
The Second subject in the same painting is $35 extra. Each additional subject beyond that is $25.
What it is: You send me a digital reference photo and I send you back a finished digital painting in the style of the examples seen here as a digital file in jpg format. It is not a paintover/tracing nor do I use photoshop filters. Entirely done using painting techniques in Adobe Photoshop and Manga Studio 5. The file will be around 4000 pixels in the long dimension. Suitable for printing greater than 11" x 14". I will also include a smaller photo for web use.
You will receive a digital file, not a print.
You can either email me the reference photos at email@example.com, or upload it/them to a gallery on imgur.com and send me the link. Once you email me your request, I will reply to you with a square invoice for the payment. I can send a paypal invoice instead if that is prefered.
I have the right to refuse a request based on subject matter or it being overly complex. In such a case I will refund completely. Ie. no large groups.
I will need at least one photo reference emailed to me along with details about what is wanted ie. color choices, etc. basically anything that will make the drawing easier.
I accept paypal but other forms of payment are possible. I can set up a listing on Etsy where Direct Credit Card Payments are accepted.
After completion if there is something not to your satisfaction I will do one minor adjustment to fix it. I will not redo the entire painting or do multiple fixes. Those will cost extra.
When complete you will be provided with a link to download your full resolution jpeg file. The file will be roughly 11x14 inches at 300 dpi. This file is provided for personal use only and is not licensed for commercial use.
If you have any questions email me at email@example.com or contact me through my facebook page.