Neuromodulation can be thought of as a change in the state of a n

Neuromodulation can be thought of as a change in the state of a neuron or group of neurons that alters its response to subsequent stimulation. A number of models have been proposed to explain the actions of ACh in the central nervous system (CNS). For example, ACh has been suggested to be critical for the response to uncertainty, such that an increase in cholinergic tone predicts the unreliability of predictive cues in a known context and improves the signal-to-noise

ratio in a learning environment (Yu and Dayan, 2005). Another model has suggested that ACh reinforces neuronal loops and cortical dynamics during learning by enhancing the influence of feed-forward afferent inputs to the cortex BGB324 carrying sensory information and decreasing excitatory feedback activity mediating retrieval (Hasselmo, 2006). ACh can also alter firing of neurons on a rapid time scale, as in fear-conditioning, when foot-shock results in direct cholinergic activation of interneurons in the auditory cortex that contribute to learning (Letzkus et al., 2011). All these models are consistent with a primary role of ACh as a neuromodulator that changes the state of an ensemble of neurons in response to changing environmental conditions. In this review, we will provide further support for the idea that cholinergic neurotransmission in the brain

is primarily neuromodulatory and is categorically distinct from the actions of ACh at the neuromuscular junction. We propose that the role of buy RAD001 ACh as a neuromodulator in the brain is to increase neurotransmitter release in response to other inputs, to promote burst firing, and/or suppress tonic firing, depending upon the system and the neuronal

subtypes stimulated. Further, ACh contributes to synaptic plasticity in many brain areas. The two primary sources of ACh in the brain include projection neurons that innervate distal areas and local interneurons that are interspersed among their cellular targets. Cholinergic projection neurons are found in nuclei throughout the brain, the such as the pedunculopontine and laterodorsal tegmental areas (PPTg and LDTg), the medial habenula (MHb) (Ren et al., 2011), and the basal forebrain (BF) complex (Mesulam, 1995; Zaborszky, 2002; Zaborszky et al., 2008), including the medial septum. These cholinergic neurons project widely and diffusely, innervating neurons throughout the CNS. Cholinergic interneurons are typified by the tonically active ACh neurons of the striatum and nucleus accumbens, and there is some indication from anatomical studies that cholinergic interneurons are present in the rodent and human neocortex, but not the nonhuman primate cortex (Benagiano et al., 2003; Mesulam, 1995; von Engelhardt et al., 2007).

To allow the separation of the signal evoked by each event, a bla

To allow the separation of the signal evoked by each event, a blank screen (medium gray) was presented as an interstimulus interval (ISI) for a period of 2, 3, or 4 s (picked randomly) between the CAM1, SOL, and CAM2 stages. A randomly picked intertrial interval (ITI) of 3, 4, or 5 s was also used. For determination of memory performance

in the Test session (Figure 3B), all 40 camouflage images from the stimulus set were presented to each participant. Thirty images (and their solutions) Bioactive Compound Library were therefore previously seen (in the Study session), and the remaining ten images were novel (different ones for different participants). Each camouflage image was presented for 10 s, and the participants were instructed to press a

button if they thought they identified the object in the image. Once they pressed the button, or after 10 s if they did not press it, the four-option multiple choice question appeared, and the participants were requested to identify the object in the image by pressing one of four numbers on a keypad. The question remained on the screen until a response was given, but not for longer than 10 s, and the next camouflage image was then presented. If a choice was made within the 10 s limit, participants were next asked to indicate their level of confidence: BKM120 “guess,” “fairly confident,” or “completely confident.” Regardless of the confidence level indicated, if their answer to the multiple choice question was correct, the camouflage image was Urease re-presented, with a Grid-map of numerals (1–9) superimposed on it (Grid task). Participants were asked to specify the Grid-map numeral overlaid on a specific feature of the object

(e.g., “the eye of the clown,” Figure 3B). The object features queried at the Grid questions were ones that are not likely to be distinctive by themselves, but rather those whose identification was facilitated by a holistic perception of the embedded object. The image with the Grid-map was presented until a response was provided, but not for more than 10 s. The chance level, combining the multiple choice question and the Grid task question, is only 2.75% (25% at the multiple choice question followed by 11% at the grid-map question). Therefore, in all subsequent analyses correct responses on both tests were used as indication that the observer perceived the underlying scene correctly. This classification was supported by two analyses. First, we compared the number of these images with the expected number of chance correct answers in the multiple choice task. The expected number of chance correct answers (computed individually for each participant) is the number of images that were indicated as not identified in the Study whose perceptual identification was not verified in the Grid task, divided by four. The comparison of those two sets of numbers revealed that they did not differ significantly (p = 0.15).

Responses evoked by localized NMDA application in vitro are the r

Responses evoked by localized NMDA application in vitro are the result of activation of both synaptic and extrasynaptic receptors. We next carried out experiments in acute slices to confirm the presence of synaptic NMDAR-mediated

currents ex vivo. GluN2A-containing NMDARs have been shown to traffic to autaptic synapses in GluN2B null hippocampal cultures and in cultured cortical slices following knockdown of GluN2B (Tovar et al., 2000 and Barria and Malinow, 2002). However, their ability to traffic to GluN2B null synapses in vivo had not been tested. We recorded whole-cell currents from layer II/III cortical neurons in acute brain slices in response to presynaptic stimulation in layer IV of 2B→2A animals (P12–P16) (Figure 3). Voltage clamping at depolarized potentials to remove the Mg2+ block of NMDARs and electrically stimulating afferent axons allowed us to record APV-sensitive currents in 2B→2A cortex (Figures 3A and 3B). The peak amplitude of the synaptic NMDAR-mediated current at +50mV was not significantly different in 2B→2A mice compared

AZD8055 order to controls (Figure 3B). Consistent with a pure population of GluN2A-containing receptors, these recordings revealed a lack of sensitivity to the GluN2B selective antagonist ifenprodil (Figure 3C) and to NMDAR-mediated currents that exhibited significantly faster decay times (decreased tau) (Figure 3D) (Vicini et al., 1998). Faster decay resulted in a slight, but statistically significant, decrease in integrated current measured at +50mV over the initial 200 ms

of the evoked response, but not over the initial 100 ms of the response (Figure 3D). These and data show that our genetic strategy was successful in removing GluN2B and driving precocious expression and synaptic incorporation of GluN2A-mediated NMDAR-receptors while recovering a significant amount of synaptic NMDAR-mediated current at cortical synapses in vivo. GluN2B and GluN2A interact with, and activate, distinct signaling cascades at excitatory synapses in order to control the composition and strength of synaptic contacts. The dominant way by which NMDARs regulate synaptic strength is through bidirectional trafficking of AMPARs. In light of this, we examined AMPAR-mediated currents in 2B→2A mice. For these experiments, cortical neuron cultures were prepared from both homozygous GluN2B knockout and 2B→2A embryos, as well as from WT littermate embryos. We isolated AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs) between 11 and 15 days in vitro (DIV) using 0.5 μM tetrodotoxin (TTX) + 50 μM picrotoxin.

Consistent with these results, and independent of the olfactory-a

Consistent with these results, and independent of the olfactory-auditory integration, a significantly higher percentage of neurons from lactating mothers responded to at least one sound (Figure 6A, left; Table S1). This increase was significant when considering both responses to pure tones and to natural Androgen Receptor Antagonist sounds separately (Figure 6A, middle and right). Because this amplified response of the population to sounds is not evident in experienced virgins, it could not be explained by mere exposure to pup odors. Because this increase also did not appear in mothers

following weaning, we consider it to be a transient effect. This transient effect may well be associated with the transient endocrinal changes that occur during pregnancy and after parturition (Brunton and Russell, 2008 and Miranda

and Liu, 2009). Because different sounds bear different behavioral meanings, we analyzed how the different sounds (two different natural sounds and one reference nonnatural sound) were represented in A1 of all four experimental groups. BBN and WCs were overrepresented by ∼2-fold in lactating mothers as compared to all other groups (Figure 6B, left and middle). USVs were considerably overrepresented 3-MA manufacturer (by ∼10-fold) in lactating mothers relative to their representation in naive virgins (Figure 6B, right). Interestingly, USVs were also overrepresented (by ∼4-fold, relative to their representation in naive virgins) in experienced virgins and in mothers following weaning, albeit still significantly lower than in lactating mothers (Figure 6B, right). This analysis indicated that maternal-induced plasticity may facilitate responses to specific sounds rather than act as a general gain control to the whole circuit. Therefore, we next analyzed whether the olfactory-auditory Cell press integration also induced specific effects on the detection

and discrimination of the sounds we presented. To that end, we isolated the effects of pup odors on BBN-, WC-, and USV-evoked responses in lactating mothers (this analysis was limited to this group because of the relatively low sample size of neurons responding to USV stimuli in all other groups; Table S2). Pup odors did not significantly affect the average detectability of neurons to BBN and WC stimuli because some neurons increased and others decreased their detectability (Figure 6C). In contrast, pup odors had a more homogeneous effect on responses to USV stimuli. Specifically, pup odors induced consistent increase for USV detectability in most neurons (Figure 6C; Table S2). For example, 12/15 neurons from lactating mothers increased their responses to USVs (Figure 6D, neurons marked with arrows pointing upwards, and Figure 4A, top left).

Intriguingly, treatment with PI-PLC strongly reduced the growth c

Intriguingly, treatment with PI-PLC strongly reduced the growth cone collapse rate of T→N interactions to a level similar to those observed for the other three interactions (Figure 2). While PI-PLC treatment affects all GPI-anchored proteins, the most parsimonious explanation seems to be that the removal of ephrinAs from nasal axons in these cultures essentially reduces growth cone collapse to baseline levels. Our in vitro data therefore confirm F. Bonhoeffer’s early in vitro findings and offer a good molecular candidate, i.e., ephrinAs, for the growth

cone collapse-inducing activity of nasal axons. To analyze the role of ephrinAs on RGC axons in vivo, we used ephrinA5 conditional “KO-first” mice (Skarnes et al., 2011) (Figure S2A available online), which we obtained from the International Knockout Dabrafenib mouse Mouse Consortium (IKMC) and the European Conditional Mouse Mutagenesis (EUCOMM)

project. In these mice, loxP sites flank the second exon of the ephrinA5 gene, which contains most of the coding region of ephrinA5, while the first exon contains only the first 20 amino acids (aa) of the mature protein, which comprises in total 228 aa. Thus, a conditional, Cre-mediated excision of exon 2 abolishes the synthesis of a functional ephrinA5 transcript ( Figure S2C). For the widely published full KO of ephrinA5, a comparable approach was taken, that is, deletion of exon BI 2536 mouse 2 by homologous recombination ( Feldheim et al., 1998 and Frisén et al., 1998). In addition, these KO-first mice harbor a splice acceptor-IRES-lacZ cassette, flanked by frt sites, in the intron preceding exon 2 (Figure S2A) (Skarnes et al., 2011), which abolishes the normal splicing from exon 1 to exon 2. Since we were interested here in analyzing retinocollicular mapping after a conditional

next inactivation of ephrinA5 in either the retina, the SC, or both (Figure S2C), we first removed this SA-IRES-lacZ cassette by breeding them with mice expressing flp recombinase ubiquitously ( thereby restoring the normal splicing/expression of the ephrinA5 gene while retaining the loxP sites flanking exon 2 ( Figure S2B). In order to abolish expression of ephrinA5 in the retina, a mouse line was chosen in which Cre is expressed under the control of the rx promoter (rx:cre) (Swindell et al., 2006), and for inactivation of ephrinA5 in the SC, the En1cre/+ line, in which Cre is expressed from the endogenous engrailed-1 promoter (Basson et al., 2008). Both lines have been extensively characterized (Basson et al., 2008 and Swindell et al., 2006). Expression of rx starts at E8.5 in the entire prospective optic vesicle; accordingly Cre will be expressed in all RGCs. While there is additional expression in the entire forebrain (see also Ackman et al., 2012 and Pinter and Hindges, 2010), expression of ephrinA5 in the SC is unaffected.

What are the minimal criteria to establish a claim for axon regen

What are the minimal criteria to establish a claim for axon regeneration? ISRIB cost First, it is critical to provide compelling evidence that the axons that extend past a lesion are not spared. Criteria for this have been described (Steward

et al., 2003) and are reasonably well accepted by the field. Next, how does one prove that growth involves “regeneration”; that is, that an axon growing into or beyond a lesion site originated from a transected axon? Regeneration can be proven when all of the axons of a projecting system are lesioned (i.e., no axons are spared), and growth of labeled axons from an identified source is observed into or around the lesion site. Usually, this involves tract tracing to identify the origin, course, and termination of axons (Figures 2A–2D). Studies in which pathways are labeled by genetically driven fluorescent markers provide an alternative approach providing that the identity of the labeled axons can be definitively established, and it can be confirmed that the lesions completely interrupt the genetically labeled pathway (more on this below). Somewhat less satisfying, but still reasonably compelling evidence of regeneration can be obtained through a combination of double click here retrograde tracing. For example, in the case of studies of regeneration of descending pathways after SCI, a retrograde tracer is injected before the lesion (Figure 2E) to identify the cells of

origin of a pathway that will subsequently be lesioned.

After the lesion is performed and sufficient time has passed to allow potential axonal regeneration, a second (different) retrograde tracer is injected at the site of the original tracer injection. Hypothetically, an axon that has regenerated below a complete lesion of the system will exhibit labeling of the neuronal somata with both tracers (Figure 2E). A shortcoming of this approach is that it is not possible to determine the point of origin of the axons that grow or the course of the axons past the lesion. For all assessments, Ketanserin it is critical to confirm that the experimental lesion completely transects the pathway being studied. Important evidence in this regard can be obtained by an analysis of axon distribution at different times postinjury. Long-distance axon regeneration will take some time, including the time required for (1) recovery from the axonal injury, (2) molecular changes required for a shift to a growth mode, and (3) elongation of the axon. Ramon y Cajal provided estimates of the timing of growth of regenerating peripheral nerves that sound quite plausible today: (1) preparation of the dividing phase and growth of sprouts within the central stump (proximal to the injury; 2–5 days); (2) growth through the scar (velocity of 0.25 mm per day); elongation within the supportive environment of the peripheral stump (2.64 mm/day) (Ramon y Cajal, 1928). Even under “regeneration enabled” circumstances, the rate of elongation may be slower in the CNS.

, 2003) We generated hemagglutinin (HA)-DAXX constructs expressi

, 2003). We generated hemagglutinin (HA)-DAXX constructs expressing nonphosphorylatable (S669A) and phosphomimetic (S669E) DAXX mutants. Whereas S669E DAXX migrated like hyperphosphorylated DAXX, migration of the S669A mutant corresponded to hypophosphorylated DAXX (Figure 5H). Overexpression of an active form of calcineurin led to reduced migration of wild-type (WT) DAXX but did not affect the two mutants (Figure 5H). Similarly, coexpression NVP-BGJ398 in vitro of HIPK1 promoted hyperphosphorylation of WT DAXX only (Figure 5H). These results indicate that DAXX S669 phosphorylation is modulated by calcineurin. We next explored whether the phosphorylation status of DAXX regulates its interaction with H3.3 and ATRX. As shown in Figure 3A, we found

an enrichment of endogenous hypophosphorylated DAXX in YFP-H3.3 immunoprecipitates in neurons. Similar findings were obtained with exogenously expressed WT DAXX in 293T cells (Figure 5I) as well as in neurons (Figure S5B). HIPK1 overexpression led to DAXX hyperphosphorylation, but only a small proportion of hyperphosphorylated DAXX was found to be check details associated with H3.3 (Figure 5I). This enrichment did not appear due to reduced H3.3 affinity for hyperphosphorylated DAXX, because similar levels of S669E and S669A mutants were found to be associated with H3.3 (Figure 5I). Finally, we failed

to detect any effect of DAXX phosphorylation status on its ability to interact with ATRX (Figure S5C). Because DAXX/H3.3 complexes are enriched in hypophosphorylated DAXX, we reasoned that DAXX phosphorylation status could play a role in the regulation of H3.3 deposition. To test this hypothesis, we performed rescue experiments in DAXXFlox/Flox neurons. CRE promoted efficient deletion of endogenous DAXX in cells coinfected either with a green fluorescent protein (GFP) vector or DAXX constructs ( Figures S6A–S6C). Similar expression levels of WT, S669A, and S669E DAXX were achieved in transduced neurons ( Figure 6A). Upon membrane depolarization, migration

3-mercaptopyruvate sulfurtransferase of S669A and S669E DAXX mutants was not affected, whereas levels of hyperphosphorylated WT DAXX decreased ( Figure 6A). Furthermore, no significant differences in association with Bdnf Exon IV and c-Fos regulatory regions were detected in between the constructs both at steady state and upon KCl treatment ( Figure 6B). As expected, WT DAXX rescued H3.3 loading at Bdnf Exon IV and c-Fos regulatory regions in CRE-infected DAXXFlox/Flox neurons ( Figure 6C). Notably, S669A DAXX had a more pronounced rescuing activity at most regions analyzed ( Figure 6C). Conversely, S669E DAXX failed to rescue loading at all regions ( Figure 6C). We then tested whether DAXX phosphorylation also affected its ability to regulate transcription. WT and S669A DAXX rescued expression of Bdnf Exon IV and c-Fos. In contrast, S669E DAXX was impaired in this function ( Figure 6D). Notably, S669A DAXX was more potent in rescuing c-Fos induction compared to WT DAXX ( Figure 6D).

The cells had to meet a specific criteria to be included in the a

The cells had to meet a specific criteria to be included in the analysis, such as well separated clusters, spikes with broad widths (peak-to-trough width > 300 μs), and presence of complex bursts with 2–7 spikes within 5–15 ms. To ensure that we recorded from the same place cell across sessions, we confirmed that the properties such as autocorrelation, waveform, firing rate and firing location were similar in both sessions. The inhibitory interneurons were easily identified by their high frequency of firing with narrow waveform width and place nonspecific firing. Position data of the mice, tracked by two colored LEDs, were collected at 50 Hz and sorted into 3 × 3 cm Paclitaxel in vitro bins. Each sorted place cell

was visualized by plotting its firing rate on top of an animal’s walking path, with heat map colors ranging from blue (little or no firing) to red (high firing rate). A normalized firing rate map was obtained by dividing the spiking activity with

the animal’s position at a particular place. Firing rate maps were smoothed with a filter such that 1 cm equaled 2 pixels. Place field size was measured as in previous studies (Muller et al., 1987). Briefly, we calculated the number of pixels inside the enclosure where place cells fired normalized with the number of pixels the mice visited. Only selleck kinase inhibitor the top 80% of the firing peak with at least 8 contiguous pixels was used and defined as the place field. The pixel area covered by the mice in the box or track enclosure was converted to the respective percentage (%) of total enclosure area for cross-comparison. Two separate measures were used for calculating place field stability. First, a peak-shift measure was used where the firing field peak of session 1 was compared with the firing peak of session 2. before A shift (in cm) in peak 1 to peak 2 was calculated by the formula (x1−x2)2+(y1−y2)2,where x1, x2 are the x coordinates and y1, y2 are the y coordinates

of peaks 1 and 2. Second, a cross-correlation measure was used. Prior to applying this measure the firing maps were normalized to a standard size (Figure 4C). From the place field rate map only the in-field firing map (top 80% of the place field peak) was extracted and scaled down to a standard size of about 20 cm using the centroid as the midpoint. This was done to eliminate cross-correlation bias; correlation of larger place fields would produce better stability scores as there are more bins available. Normalized maps from session 1 were compared with session 2 using Pearson’s product moment correlations, given by the formula: r=1n−1∑i=1n[(Xi−X¯σX)(Yi−Y¯σY)]where Xi−X¯/σX,X¯ and σx are the standard score, sample mean, and sample standard deviation of data X, respectively. Spatial coherence estimates smoothness of a place field. It was calculated by correlating the firing rate in each pixel with firing rates averaged with its neighboring 8 pixels.

Future molecular studies are needed to explore how each

Future molecular studies are needed to explore how each this website mechanism contributes to neurodegeneration and pathological TDP-43 aggregation. Moreover, evaluation of larger numbers of patients with FTD and ALS associated with the expanded GGGGCC hexanucleotide repeat in C9ORF72 is warranted to further delineate the range of phenotypes and prevalence of these disorders, and to investigate the potential of the repeat for

properties such as anticipation and spontaneous mutation. Finally, we suggest that in future publications this genetic defect be referred to as “c9FTD/ALS. While our manuscript was in preparation we learned of another group who independently

identified repeat expansions in C9ORF72 as the cause of FTD and ALS linked to chromosome 9p ( Renton et al. 2011). Four extensive FTD and ALS patient cohorts and one control cohort were included in this study. All individuals agreed to be in the study and biological samples were obtained after informed consent from subjects and/or their proxies. Demographic and clinical information Pazopanib mw for each cohort is summarized in Table S1. The proband of chromosome 9p-linked family VSM-20 is part of a series of 26 probands ascertained at UBC, Vancouver, Canada, characterized by a pathological diagnosis of FTLD with TDP-43 pathology (FTLD-TDP) and a positive family history of FTD and/or ALS (UBC FTLD-TDP cohort). Clinical and pathological evaluations of VSM-20

were conducted at UCSF, UBC, and the Mayo Clinic ( Boxer et al., 2011). A second cohort of 93 pathologically confirmed FTLD-TDP patients independent of family history was selected from the Mayo Clinic Florida (MCF) brain bank (MCF FTLD-TDP cohort) which focuses predominantly on dementia. The clinical FTD cohort (MC Clinical FTD cohort) represents a sequential series of patients seen by the Behavioral Neurology sections at MCF (n = 197) and MCR (n = 177), the majority of whom were participants in the Mayo Alzheimer’s Disease Research Center. Members of Family 118 were participants in the Mayo Alzheimer’s Disease Patient Registry. Clinical FTD patients underwent a full these neurological evaluation, and all who were testable had a neuropsychological evaluation. Structural neuroimaging was performed in all patients and functional imaging was performed in many patients. Patients with a clinical diagnosis of behavioral variant FTD (bvFTD), semantic dementia or progressive non-fluent aphasia based on Neary criteria ( Neary et al., 1998), or patients with the combined phenotype of bvFTD and ALS were included in this study, while patients with a diagnosis of logopenic aphasia or corticobasal syndrome were excluded.

To investigate the impact of repeated cocaine on stress vulnerabi

To investigate the impact of repeated cocaine on stress vulnerability, we utilized a submaximal version of social defeat. Previous work has shown that 10 days of defeat stress induces several cardinal depressive-like behaviors, such as social avoidance and reduced sucrose preference (Berton et al., 2006 and Krishnan et al., 2007). Here, only 8 days of defeat stress were used, which in initial studies DAPT in vivo did not induce these symptoms. Next, either saline or a sensitizing regimen

of cocaine was administered prior to initiating 8 days of defeat stress (Figure 1A). Seven days of repeated cocaine (20 mg/kg/day), immediately followed by 8 days of defeat stress, revealed social avoidance (Figure 1B) and diminished sucrose preference (Figure 1D). This is

in contrast to control animals receiving saline prior to chronic stress, which showed no such deleterious behavioral responses. To further verify the potential long-lasting effects of cocaine on behavioral deficits observed after 8 days of defeat stress, RAD001 molecular weight animals were re-exposed to a low dose of cocaine (5 mg/kg) 24 hr after the social interaction test (see Figure S1A available online). Both stressed and nonstressed cocaine-treated animals displayed sensitized locomotor responses to cocaine. The social stress did not, however, potentiate cocaine-induced locomotor activity in cocaine-naive mice (Figure S1B). Animals exposed to cocaine, in the absence of later social stress, displayed more rapid social interaction (i.e., decreased

latency to interact)—an effect of cocaine that was completely reversed by exposure to 8 days of defeat stress (Figure 1C). The effects of cocaine, stress, or the combination of both stimuli had no impact on general levels of locomotor activity (Figure 1E). Cocaine, when self-administered during binges, increases thresholds for intracranial self-stimulation, indicating a withdrawal syndrome characterized by anhedonia (Markou and Koob, 1991). However, as shown in Figures 1B and Non-specific serine/threonine protein kinase 1D, we did not observe an effect of cocaine alone on social interaction or sucrose preference. We have shown recently that, following repeated (not acute) cocaine, the repressive histone modification, H3K9me2, and its associated “writer” enzymes, G9a and GLP, are reduced in NAc, leading to the activation of numerous synaptic plasticity-related transcripts, increased dendritic spine density on medium spiny neurons (MSNs), and enhanced cocaine reward (Maze et al., 2010). To validate these earlier findings, animals were treated with either saline or cocaine (20 mg/kg/day) for 7 days. At 24 hr after the final injection, NAc dissections were collected and analyzed for global alterations in G9a and H3K9me2 expression. Consistent with previous data, levels of G9a mRNA (saline versus repeated cocaine, t10 = 2.559; p < 0.