The other subjects who did not fit into the model had recurrent ankle sprains, but did not present with mechanical or functional instability. Among the 108 ankles used to fit the updated model, the percentage of the classifications see more was 42.6% (46) for perceived instability, 30.5% (33) for perceived instability plus recurrent sprain, 11.1% (12) for perceived instability plus mechanical instability and recurrent sprain,

9.3% (10) for mechanical plus perceived instability, 2.8% (3) for recurrent sprain, 2.8% (3) for mechanical instability, and 0.9% (1) for mechanical instability plus recurrent sprain.3 In addition to the expanded sub-groups, functional instability is referred to as perceived instability in the newer model “because functional instability is now used with widely different meanings”.3 Several limitations were acknowledged by the authors. The model was tested retrospectively using data from previous studies. Only one method was used to test mechanical instability, perceived instability and recurrent sprain in the original data sets. Mechanical instability was examined using an anteroposterior manual testing method. The model was tested with data

from limited age and activity groups. Finally, the sample size for some sub-groups was rather small. Although research interest in CAI has increased steadily in recent years, the results are rather inconsistent.2 This may be largely related to the different

criteria used to define functional instability, which Selleckchem Capmatinib may have led to subject groups with different instability characteristics. In a recent extensive literature review of 118 studies on the inclusion criteria of CAI studies, Delahunt et al.2 showed that the most common descriptors for ankle instability and functional instability are frequent ankle sprains and ankle joint giving way. However, most of the studies using the concept most of giving way did not actually define or describe the concept. It is also unclear if giving way is the same as a feeling of ankle instability. Therefore, in order to avoid confusion, these authors provided operational definitions for mechanical instability, functional instability, CAI, recurrent ankle sprain, “giving way” of the ankle, the feeling of ankle instability, and acute lateral ankle sprain.2 These clearly defined terms may help minimize discrepancies in the targeted populations, and select more homogenous subject cohorts in future CAI studies. In addition to having clearly defined operational terms, the usage of ankle instability surveys such as the Foot and Ankle Ability Measure,7 Ankle Joint Functional Assessment Tool,8 and Cumberland Ankle Instability Tool9 can quantify functional instability and further differentiate CAI patients from healthy controls. For mechanical instability, its presence should be assessed through instrumented measures or manual testing.

Neurons were transfected with GFP, a marker for cytoplasmic volume, and stained for endogenous dynactin and dynein. In the distal neurite, we observed a striking enrichment of dynactin but not of dynein, as compared to soluble GFP (Figure 2A). We saw a similar distal enrichment of dynactin in primary cortical, motor, and dopaminergic neurons, suggesting that this is a generally conserved mechanism (Figure S2). Line-scan analysis of the DRG neurons showed that dynactin accumulates in the

distal neurite significantly more than dynein (Figure 2B). These data suggest that dynactin is specifically recruited and/or retained in the distal neurite. Next, we asked whether the CAP-Gly domain is necessary for this distal enrichment of dynactin. We overexpressed wild-type or ΔCAP-Gly p150Glued in primary DRG neurons using a bicistronic vector that also expresses GFP. Wild-type p150Glued VX-770 cost selleckchem was clearly enriched at the neurite tip, while neurons expressing ΔCAP-Gly p150Glued did not show a similar accumulation (Figure 2C). We quantified this difference using line-scan analysis and showed that wild-type p150Glued is significantly enriched over the

distal 10 μm of the neurite tip as compared to ΔCAP-Gly p150Glued (Figure 2D). These data demonstrate that the CAP-Gly domain functions to properly localize dynactin in the distal neurite. Motors from the kinesin superfamily, including kinesin-1 and kinesin-2, drive the fast axonal transport of vesicular cargos. The anterograde movement of cytosolic proteins via slow axonal transport is also dependent on kinesin-1 (Scott et al.,

2011). We therefore tested whether the distal enrichment of dynactin is dependent on kinesin-1 activity by expressing either the dominant-negative kinesin-1 inhibitor, KHC-tail, or the KHC-stalk, mafosfamide which does not inhibit the motor and was used as a control (Konishi and Setou, 2009). We found that expression of KHC-tail disrupts the distal localization of dynactin, while expression of KHC-stalk had no effect on dynactin localization (Figure 3A). Line-scan analysis confirmed a significant difference in the distal accumulation of dynactin after expression of the KHC-tail, as compared to localization in neurons expressing either the vector and or the KHC-stalk (Figure 3B). Kinesin-1 has not been shown to directly interact with dynactin, nor did we observe co-immunoprecipitation of the motor with p150Glued expressed in COS7 cells (Figure S3). Thus the mechanism leading to kinesin-1-dependent distal localization of dynactin is likely to be indirect. In contrast, previous work has identified a direct interaction between kinesin-2 and p150Glued (Deacon et al., 2003). Therefore, we tested whether kinesin-2 may also contribute to the anterograde transport of dynactin. Expression of Kif3A-HL, a dominant-negative inhibitor of kinesin-2 lacking the motor domain (Nishimura et al.

, 1998, Klein and Aplin, 2009 and Riento et al., 2005b). Moreover, RhoA had been previously implicated in the control of cortical neuron migration (Hand et al., 2005 and Kholmanskikh et al., 2003), although how RhoA activity is regulated in migrating neurons had remained unclear. By using an in vivo rescue assay, we provide evidence that Rnd3 antagonizes RhoA pathway in neurons through an interaction with the Rho GTPase-activating protein p190RhoGAP or with another unknown inhibitor that also requires T55 to interact with Rnd3. Other potential interactors with Rnd proteins in migrating neurons

Ruxolitinib datasheet include the semaphorin receptors, Plexins, which have been implicated in cortical DAPT neuron migration (Hirschberg et al., 2010) and have been shown to be bound and regulated by Rnd proteins in neurons and other cell types (Oinuma et al., 2003, Tong et al., 2007 and Uesugi et al., 2009). The finding that Rnd2, like Rnd3, promotes migration of cortical neurons by inhibiting RhoA was more unexpected because Rnd2 does not interfere with RhoA activity in fibroblasts (Chardin, 2006 and Nobes et al., 1998). The mechanism by which Rnd2 inhibits RhoA in neurons, which does not involve interaction with p190RhoGAP and is therefore

different from that of Rnd3, remains to be characterized. Although both Rnd2 and Rnd3 inhibit RhoA in migrating neurons, several lines of evidence indicate that they exert different functions: (1) the two genes cannot replace each other in shRNA rescue experiments; (2) knockdown of Rnd2 and Rnd3 results in very different morphological defects that appear during distinct phases of migration, and (3) the migration defect of Rnd3-silenced mafosfamide neurons, but not that of Rnd2-silenced neurons, can be corrected by F-actin depolymerization. We explain this apparent paradox by the fact that Rnd2 and Rnd3 have different subcellular localizations and only Rnd3 inhibits RhoA at the plasma membrane. In support of this hypothesis, we show that Rnd2 can replace

Rnd3 in migrating neurons if it is targeted to the plasma membrane by replacement of its carboxyl-terminal domain with that of Rnd3. Localization of active RhoA is dynamically regulated in migrating fibroblasts ( Pertz et al., 2006). The finding that Rnd3 and Rnd2 control different phases of radial migration by inhibiting RhoA in different cell compartments suggests that in cortical neurons as well, RhoA acts dynamically in different cellular domains to control different aspects of the migratory process. Analysis of the morphological defects of knockdown neurons provides clues to the function of Rnd3 in cortical neuron migration. Rnd3-silenced neurons present an increased average distance between the centrosome and the nucleus, suggesting that nucleokinesis is disrupted in these cells.

The test tubes were then turned upside down to remove the excess conidial suspension/formulation through absorption by the cotton plug. The eggs were held at 27 ± 1 °C and RH ≥80%. The biological parameters evaluated were: incubation period; hatching period; and hatching percentage. The methodology used in the bioassay with larvae was similar to that used in the egg bioassay. Larval treatment was performed on the tenth day after total larval hatching. The tubes with hatching

percentage below 95% were discarded. Mortality IWR-1 mw was evaluated every five days up to day 20 after treatment. Dead engorged females, eggs, and larvae from all treatment groups were incubated at 25 ± 1 °C and RH ≥80% to allow fungal growth and further evaluations of their characteristics (Samson and Evans, 1982). The periods of egg incubation and hatching were assessed using analysis of variance (ANOVA) followed by the Student–Newman–Keuls test (SNK) with a significance level of 5% (p ≤ 0.05). The hatching percentage, NI, EPI, and mortality percentage of larvae were assessed by the Kruskal–Wallis test selleck inhibitor followed by the Student’s t-test with a significance level of 5% (p ≤ 0.05) ( Sampaio, 2002). Aqueous conidial suspensions of M. anisopliae s.l. and B. bassiana were 100% viable within 24 h at 25 ± 1 °C, and RH ≥80% while oil-based conidial formulations were 100% viable after 48 h of incubation under the same conditions. R. microplus engorged

females treated with M. anisopliae s.l. oil-based formulations including 15 and 20% mineral oil started showing fungal growth on the cuticle three days after treatment while fungal growth on the cuticle of females treated with the oil-based Fossariinae formulations at 10% commenced four days after treatment. Conspicuous fungal growth was noted initially on the cuticle of engorged females

treated with M. anisopliae s.l. aqueous suspensions at six days post-treatment. Finally, engorged females treated with the aqueous suspension and oil-based formulations of B. bassiana showed fungal growth on their cuticle until 14 days after treatment. M. anisopliae s.l. oil-based formulations reduced 14 and 12 times the percentage of larval hatching as compared to the control groups and the group treated with the aqueous fungal suspension, respectively ( Table 1). The NI and EPI of females treated with M. anisopliae s.l. oil-based formulations declined significantly (p < 0.01; degree of freedom [df] = 7) in comparison with the control groups. A significant reduction (p < 0.05; df = 7) of these biological parameters was also observed when the formulations with 15 and 20% oil were compared with the M. anisopliae s.l. aqueous suspension. However, no significant difference (p < 0.05; df = 7) was observed between the group treated with the M. anisopliae s.l. 10% oil formulation and the same aqueous fungal suspension ( Table 1). The NI was the only biological parameter statistically affected (p < 0.05; df = 7) by both the B.

5 Hz; Figure S5J). Together, these data demonstrate that a circuit intrinsic to the OT can generate and maintain persistent gamma oscillations. If the generator of these oscillations is indeed located in the OT, then the pharmacological manipulations that

altered the structure of the oscillations in the intact slice (Figure 3) should alter them in the same way when applied specifically to the OT. First, we tested the effects of the NMDA-R blocker APV on induced gamma oscillations in the isolated OT. In transected slices, bath application of APV substantially reduced the duration (11.1% of control, p < 0.001, n = 8; Figures 7, S6A, S6B, and S6C) and power Veliparib (49.3% of control, p < 0.001, Friedman test, n = 8) of activity in the i/dOT. Moreover,

increasing the strength of afferent stimulation by 3–4× in the presence of APV did not increase the duration of the oscillations (Figure S6D), suggesting that the effect of APV was not merely to reduce the general excitability of the OT circuitry. In sum, these data suggested that NMDA-R mediated glutamatergic transmission in the i/dOT was essential for the persistence of the oscillations. Next, we tested the effects of focal application of the GABA-R blocker PTX to the OT in intact slices. Recall that PTX, when bath-applied to intact slices, eliminated gamma periodicity (Figure 3A). We puffed PTX focally onto either the OT or the lpc with a micropipette while recording activity in the sOT in intact midbrain slices. Both the OT and the Ipc are innervated by GABAergic circuits, as indicated by the presence of parvalbumin immunoreactivity in both structures Cytoskeletal Signaling inhibitor Isotretinoin (Figure 8A). When applied to the OT, puffs of PTX transiently changed gamma oscillations into episodes of high-frequency spiking, mimicking the results of bath application

(Figures 8B, 8C, and S7A; duration: 33% of control, p > 0.5; power: 31% of control, p < 0.001, Friedman test, n = 7). In contrast, puffs of PTX applied to the Ipc in the same slice did not alter the periodic structure of gamma oscillations in the sOT (Figures 8D, 8E, and S7B; duration: 88% of control, p > 0.3; power: 74% of control, p > 0.5, n = 7). These results demonstrate conclusively that inhibition in the OT regulates the gamma periodicity of the midbrain oscillator. This study demonstrates that gamma oscillations can be induced in an in vitro slice preparation of the avian midbrain network and that these oscillations strongly resemble those induced by salient sensory stimuli in vivo. The synaptic mechanisms that regulate the frequency, power, and duration of the oscillations are similar to those that regulate gamma oscillations in mammalian forebrain structures. The source of the midbrain oscillations is the i/dOT. Rhythmic output from the i/dOT entrains periodic burst firing in the cholinergic nucleus Ipc, and the Ipc broadcasts the oscillations to the sOT.

All drugs were applied via bath perfusion. We are grateful to Pierre Apostolides and Drs. Hai Huang, Haining Zhong, and Craig Jahr for helpful discussions and to Elizabeth Brodeen-Kuo and Drs. Kevin Bender and John Williams for advice and suggestions on the manuscript. We thank Dr. Sascha du Lac for providing GIN and GlyT2-EGFP mice. This work was supported by NIH grants RO1DC004450 (L.O.T.) and F31DC010120

(S.P.K.). “
“During biogenesis most ion channels and neurotransmitter receptors undergo regulated assembly prior to insertion into the plasma membrane. In prokaryotes many PFT�� chemical structure ion channels function as homo-oligomers, which for individual subtypes range in size from dimers to hexamers, while in eukaryotes, as a consequence find more of gene duplication, appropriate members of a diverse subunit pool must be selected to form hetero-oligomeric assemblies of restricted stoichiometry and composition. The glutamate receptor ion channels (iGluRs) which mediate excitatory synaptic transmission are important examples of the biological diversity which arose from gene duplication, and these receptors play key roles in brain development,

synaptic plasticity, motor function, information processing, and memory formation. In mammals, the diverse functional roles of iGluRs are mediated by a family of 18 genes, several of which undergo alternative splicing and mRNA editing (Traynelis et al., 2010). Genetic, biochemical, and functional studies have established that individual iGluR subunits will coassemble with members of the same functional family, but not with other subtypes, to

generate the large and diverse receptor population required for normal brain activity (Ayalon et al., 2005, Ayalon and Stern-Bach, 2001, Brose et al., 1994, Burnashev et al., 1992, Leuschner and Hoch, 1999 and Partin et al., 1993). A fundamental problem in biology is to understand the mechanisms controlling this selective assembly. The major families of iGluRs STK38 were identified by classical pre-genetic techniques, using selective ligands and functional assays, leading to identification of AMPA, kainate and NMDA receptor subtypes (Watkins and Evans, 1981). For kainate and NMDA receptors, the native receptor assemblies in vivo contain subunits encoded by two or three different gene families, several of which do not generate functional ion channels when expressed as homomeric proteins. For example, GluR5, GluR6, and GluR7 (also called GluK1, GluK2, and GluK3) can form functional homomeric ion channels in heterologous expression systems (Egebjerg et al., 1991 and Schiffer et al., 1997), but in vivo they coassemble with the KA1 and KA2 subunits from a second gene family (also called GluK4 and GluK5), which also bind glutamate, but which are functionally inactive when expressed as homomeric proteins (Herb et al., 1992 and Werner et al., 1991).

Thus, RIM proteins have a so far unrecognized role in enriching voltage-gated Ca2+ channels at the presynaptic nerve terminal (see also Kaeser et al., 2011). We showed that conditional removal of RIM1/2 leads to a strong reduction of transmitter release (by ∼80%; Figure 1) and that presynaptic Ca2+ currents are strongly reduced (∼50%; Figure 2). Does the reduced transmitter release primarily reflect a reduced release probability caused by the much smaller presynaptic Ca2+ influx or are there other factors, like a reduced readily releasable pool of vesicles (Calakos et al., 2004), which

contribute to the decreased transmitter release? To investigate changes in pool size and release probability, we next used brief high-frequency trains of afferent fiber stimulation to measure the size

of the readily releasable Doxorubicin cell line pool (Figure 3; Schneggenburger et al., 1999). In control synapses, LDN-193189 mw the first EPSC was large (∼8 nA in Figure 3A), and subsequent EPSCs decreased in amplitude to a new steady-state value (Figures 3A and 3B, right). In contrast, RIM1/2 cDKO synapses had much smaller EPSCs (∼2 nA in Figure 3A), and depression usually only occurred after the third or fourth stimulus (Figures 3A and 3B, left; see also Figure 3D for the average of all cells). To quantify the onset of depression, we made line fits to relative depression curves in the range of the first to the sixth stimulus (Figure 3B, blue fit lines). This analysis gave slopes of −62% ± 26% per five stimuli (n = 8) and −27% ± 33% per five stimuli (n = 9) for control and RIM1/2 cDKO synapses, respectively (p < 0.05; see also Figure 3D, bottom, for line fits to the averaged data sets for each genotype). Thus, the onset of depression was significantly slowed in RIM1/2 cDKO synapses, which suggests a decreased release probability of any given readily releasable vesicle. We next analyzed

the apparent size of the readily releasable pool by using the method of cumulative EPSC amplitudes back-extrapolated to time zero (Schneggenburger et al., 1999; Figure 3C). The back-extrapolated cumulative EPSC amplitude Mephenoxalone was 51.1 ± 16.2 nA in control mice (corresponding to ∼1700 vesicles, assuming an average mEPSC amplitude of 30 pA; n = 8 cells), but only 11.9 ± 6.9 nA in RIM1/2 cDKO mice (∼400 vesicles; n = 10 cells). Thus, there was a clear pool size reduction in RIM1/2 cDKO synapses (p < 0.001; Figure 3E). The average release probability, calculated by dividing the first EPSC amplitude by the pool size estimate (Iwasaki and Takahashi, 2001) was 0.27 ± 0.09 (n = 8) and 0.19 ± 0.05 (n = 9) in control and RIM1/2 cDKO synapses, respectively (p = 0.04; Figure 3F). These experiments with high-frequency trains show that the release deficit is primarily caused by a decreased pool of readily releasable vesicles (reduction by ∼75%; Figure 3E).

To empirically determine the functional penetrance of blue light through brain tissue in terms of neuronal activation, and to test whether we could observe an increase in

vHPC activity after illumination, we used the immediate early gene c-fos as a readout for neural activity. Although we did not observe a change in BLA somata c-fos expression induced by illumination of BLA terminals in the vHPC, c-fos expression was increased in the pyramidal layer of the vHPC extending to ∼1.5 mm below the fiber tip ( Figures S4 and S5). We complemented our c-fos readouts with estimated irradiance levels through brain tissue using an empirically based model and illumination CHIR-99021 supplier during whole-cell patch-clamp recordings (see Supplemental Experimental Procedures). While these data indicate that inhibition of BLA terminals in the vHPC can reduce anxiety-related behaviors, the illumination of ChR2-expressing processes in the vHPC could carry the potential for inducing backpropagating action potentials (Petreanu et al., 2007) or depolarization of axons of passage. To test whether the activation of BLA axon terminals synapsing locally in the vHPC was the underlying mechanism of this light-induced change in anxiety-related behavior, we combined in vivo pharmacological manipulations

with our in vivo optogenetic manipulations during anxiety assays (Figures 3A–3D). To determine whether the robust changes in anxiety-related behaviors that we observed were indeed mediated by monosynaptic, glutamatergic inputs from the BLA to selleck screening library the vHPC, rather than axons of passage or antidromic activation of BLA somata, we performed an additional heptaminol series of experiments (Figure 3). First, we expressed ChR2 in BLA projection

neurons as before and implanted a guide cannula to deliver either saline or glutamate receptor antagonists to the vHPC 30 min prior to testing and illumination on the EPM, OFT, or NSF (Figures 3A and S6). To allow for a within-subject comparison, we tested each animal twice on different days and contexts administering either saline or a glutamate receptor antagonist cocktail, counterbalanced for order. We compared saline trials to trials in which the combination of AMPA and NMDA receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and (2R)-amino-5-phosphono-pentanoate (AP5), respectively, was intracranially administered to the vHPC. In saline trials, mice replicated the light-induced anxiogenic effect on both the EPM (Figure 3B) and the OFT (Figure 3C). However, after vHPC glutamate receptor antagonism, the light-induced changes in open-arm exploration on the EPM, the time spent in the center on the OFT, and the latency to feed on the NSF test were all attenuated (Figures 3B, 3C, and 3D).

e., enhanced expression of GluA2-containing AMPARs) with far slower kinetics (>12 hr; Turrigiano et al., 1998, Wierenga et al., 2005 and Sutton et al., 2006). This notion is further supported by the observation that spatially restricted blockade of NMDAR miniature events enhances surface GluA1 expression locally ( Sutton et al., 2006). Although these observations and some theoretical considerations ( Rabinowitch and Segev, 2008) argue for a local homeostatic mechanism, there is also check details strong evidence for more global homeostatic control mechanisms in neurons that may be tuned to firing rate

( Turrigiano et al., 1998). There are unique theoretical advantages of global homeostatic mechanisms as well, particularly with regard to preserving information coding capabilities of neurons ( Turrigiano, 2008). A recent study directly assessed the impact of blocking postsynaptic firing by confining TTX treatment to the postsynaptic

cell body. Ibata et al. (2008) found such somatic AP blockade induced a transcription-dependent accumulation of GFP-tagged GluA2 at multiple sites throughout the dendritic arbor remote from the perfusion site, indicative of a cell-wide homeostatic mechanism. This transcription-dependent connection adds an interesting parallel with other evidence implicating the immediate early Fulvestrant order gene Arc in global homeostatic control ( Shepherd et al., 2006) and also distinguishes this global mechanism

with transcription-independent synaptic insertion of GluA2-lacking receptors that accompanies mini blockade ( Aoto et al., 2008). Taken together, these observations support the existence of multiple modes of homeostatic control in neurons Idoxuridine that are mediated by separate molecular pathways and implemented over distinct spatial scales. Since the discovery of polyribosomes beneath synaptic sites on dendrites, the hypothesis that dendritic protein synthesis can be engaged to adjust synaptic composition on a local level has received considerable attention. Our results indicate that in addition to allowing for fine spatial control over the postsynaptic element, local dendritic synthesis may also actively participate in controlling the function of apposed presynaptic terminals, through local synthesis of BDNF and perhaps other retrograde messengers. Thus, BDNF is both necessary and sufficient for the state-dependent presynaptic changes induced by AMPAR blockade, but acts downstream of protein synthesis. Furthermore, AMPAR blockade enhances dendritic BDNF expression in a translation-dependent manner, and a local decrease in dendritic BDNF expression accompanies spatially restricted inhibition of dendritic protein synthesis when performed coincident with AMPAR blockade.

Each bivalent vaccine candidate induced strong humoral immunity to RABV G and EBOV GP, and conferred protection from both lethal RABV and mouse-adapted EBOV challenge in mice [13]. Our primary focus is the development of an inactivated vaccine for use in humans based on the potential for superior safety and the history of the successful existing RABV vaccine that is widely used in humans, but we are also

pursuing the live attenuated vaccine candidates for use in nonhuman primate populations in Africa at risk for lethal EBOV infection [19] and [20]. Here, we expand our investigation of the immune response to the RABV vaccine candidates expressing EBOV GP. Three critical elements of an effective vaccine platform for the filoviruses were assessed: (a) the ability to induce EBOV-specific T-cell immunity, (b) coformulation of vaccine candidates to induce multivalent antibody responses,

ON-01910 solubility dmso and PF-01367338 nmr (c) induction of GP-specific immunity in the presence of pre-existing vector immunity to the RABV vaccine. The recovery and propagation of the vaccine candidates used in this study have been described previously [13] and [18]. The SADB19-derived BNSP333 virus serves as the parent rabies vaccine vector RVA (Fig. 1). RV-GP expresses the EBOV Mayinga GP ectodomain and transmembrane domain fused to the RABV G cytoplasmic domain. Inactivated RV-GP (INAC-RV-GP) was generated by treatment of sucrose purified virus because stocks with a

1:2000 dilution of beta-propiolactone (BPL) overnight at 4 °C followed by 30 min at 37 °C. RVΔG-GP expresses intact EBOV Mayinga GP and contains a deletion in the RABV G gene requiring propagation on complementary cells which express RABV G. BPL inactivated INAC-RV-HC50 expresses a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin A fused with 30 amino acids of RABV G ectodomain (ED), transmembrane domain (TM) and cytoplasmic domain (CD) [18]. A recombinant inhibitors vaccinia virus expressing EBOV Mayinga GP was constructed using published methods [21]. All mouse experiments were approved by the NIAID Division of Intramural Research Animal Care and Use Committee. Injections of 0.1 ml live or inactivated virus were administered via the intramuscular (i.m.) route, 0.05 ml in each hind leg. Live vaccines were delivered 1× at 5 × 105 FFU, and 10 μg of the killed vaccines, which are equivalent to approximately 109 FFU, were delivered on day 0 or on days 0 and 14. Groups of 10 Balb/c mice (Jackson Laboratories) were immunized with either vehicle, RVA (parent virus), RV-GP, RVΔG-GP, INAC-RV-GP or INAC-RV-GP with an additional dose at day 14. For analysis of primary T cell response, four mice per group were sacrificed at day 7 post-immunization, and splenocytes were assayed for each individual mouse by ELISPOT (Fig. 2A).