B.) Petr Stepanovich Kupalov. “
“The neuropeptides oxytocin (OT) and vasopressin (VP) are known to play important roles in the brain. This review examines the acute neuromodulatory effects of OT and VP, considering their activity in the context of a restricted number of behavioral systems. Following a short overview of their molecular properties, production and release, and characteristics of receptor binding and intracellular pathways,

this review will focus on their neuromodulatory modes of action. While the neuromodulatory actions of OT and VP are only beginning to be understood, they appear to have a widespread distribution of effects that seems consistent with a diffuse www.selleckchem.com/products/cx-5461.html mode of action. Thus, these neuropeptides have been thought to operate by nontargeted release from hypothalamic centers reaching receptors by long-range diffusion. Recently, however, it has become clear that controlled rapid and local release of OT is possible in different brain areas, and similar local delivery can be expected for

VP. Thus, it seems possible that their release can be targeted to selected sets of brain regions, possibly occurring in concert or in competition. In this review, I aim to provide a framework that may serve future Tofacitinib clinical trial studies to address the endogenous and targeted modes of actions of these neuropeptides. It considers their neuromodulatory effects across brain

regions in the context of distinct behavioral systems: olfaction and social interactions, fear and homeostasis, learning and memory, and sensory and motor systems. OT and VP are two closely related neuropeptides, both consisting of nine amino acids that only differ at the 3rd and 8th position (Figure 1). The difference at the 8th position is their TCL most distinguishing feature, where vasopressin possesses in most mammals an arginine and OT a leucine. They have appeared early in evolution with ancestors that can be traced back as far as the snails and annelids. A VP-like peptide, called [Lys8]conopressin, can be found in cones, leeches, and snails (van Kesteren et al., 1995). Segmented worms express the homolog peptide “annetocin” and a number of insects express “inotocin.” Invertebrates mostly have only one OT/VP homolog, whereas most vertebrates have two (Caldwell and Young, 2006). It is thought that separate genes for VP and OT have arisen by duplication of a common ancestral gene in jawless fish (cyclostomes) as long as 500 million years ago. In vertebrates, this duplication gives rise to two nona-peptide homologs that share five or more aminoacids with OT/VP (Figure 1). Thus we can find “isotocin & vasotocin” in bony fish; “mesotocin & vasotocin” in lungfish, amphibians, reptiles, and birds; and “OT & phenypressin” in marsupials (Darlison and Richter, 1999).

Thus, of the four loci reported to influence CSF tau/ptau, three show robust evidence for also altering risk for AD. In each instance the direction of effect on CSF tau and ptau, and on risk for

AD, cognitive decline, and tangle pathology was consistent; alleles KU-55933 concentration associated with lower tau and ptau were associated with reduced risk for AD, slower cognitive decline, and reduced neurofibrillary tangle pathology. While this is an exciting piece of work that supports the idea of using endophenotype to understand the basis of disease, there is still much to do. There exist exciting opportunities and challenges in taking the current findings further. GWA studies identify loci, not genes, and as such a critical next step is to identify the pathobiologically relevant gene (or genes) at the Compound Library in vivo 3q28 locus and TREM gene cluster and determine the effect of these risk alleles on that gene. For the most part, the risk alleles associated by GWA are not linked to protein-coding variability, and in all likelihood they confer their effects by altering transcript

expression. While Cruchaga et al. (2013) went some way toward testing this—by examining the effect of the identified alleles on expression of proximal transcripts—this small exploratory study requires further work. Given the recent identification of TREM2 as a risk gene for AD, the identification of multiple independent alleles at this locus that alter CSF tau/ptau is particularly exciting, and further fine-scale investigation of this locus is certainly warranted in AD. Lastly, by Cruchaga et al. (2013)’s own calculations, the four loci linked with CSF tau/ptau levels only explain a minor proportion of the variability in

CSF tau/ptau, indicating that at least some of the remaining variability can be attributed to as-yet-unknown genetic variants. Thus, there are clearly more risk alleles to be found. It is likely that the best route to identify these unknown factors will employ a combination of genetic methods, including GWA and second-generation sequencing. There is certainly great promise in these methods; however, analyzing cohorts of sufficient size will be critical; approximately 1,300 CSF samples are an impressive amount, but making substantive future gains in this area will probably require Adenosine larger cohorts and unified biological measures. In summary, Cruchaga et al. (2013) have taken an exciting step in showing that the endophenotype is a valuable and informative measure for understanding disease. Their work has provided valuable new insight into the basis of AD and promises to be the impetus for addressing a new series of research questions in this field. Given their findings, this effort certainly appears to be a valuable addition to the massive consortium-based case control analyses. “
“Neural activity recorded simultaneously across multiple neurons allows neuroscientists to study the importance of synchronous or correlated activity for the neural code.

Pooled sera per group were 500-fold diluted and used in IPMA to immunostain BSR monolayers infected with each of the nine reference AHSV strains. As expected, guinea pig sera raised against single VP2 proteins immunostained monolayers infected with the homologous AHSV serotype (Table 2). Similar to cross-neutralization of genetically related AHSV serotypes, some monolayers infected TGF-beta inhibitor with genetically related AHSV serotypes were also immunostained. In contrast to the cross-neutralization results (Table 1), AHSV-6 was not recognized by α-AHSV-9 VP2 serum (Table 2). In addition to immunostaining of genetically related

AHSV serotypes, some unrelated AHSV reference strains were also recognized in IPMA; e.g. AHSV-8 was recognized not only by α-VP2 sera of AHSV-5 and -8 but also by AHSV-4. AHSV-5 was also recognized by α-VP2 of AHSV-3. In general this immunostaining was weaker than for the respective homologous AHSV serotype (Table 2). VP2 protein of orbiviruses is the major selleck products determinant of

eliciting nAbs and has been used as recombinant protein-based vaccine in previous studies [17], [21], [22], [23] and [31]. Particularly, VP2 of AHSV serotype 4 has been studied extensively by European research groups, as the last European AHS outbreak was caused by this serotype [32]. In this report we studied the immunogenicity of VP2 proteins of all nine AHSV serotypes as a first step in the development of AHS subunit vaccines. This is the first report to show that VP2 of all nine AHSV serotypes

induce serotype specific nAbs with slight cross-neutralizing antibodies. The baculovirus expression system was used to produce recombinant VP2 protein of all nine serotypes for induction of nAbs. Further, some VP2 genes were optimized to increase protein expression. Still, quantities of soluble VP2 significantly varied between the different serotypes. Since it is generally known that recombinant VP2 protein of orbivirus is highly Rutecarpine insoluble, it is likely that quantities of soluble VP2 proteins vary by differences in expression or solubility [33]. VP2 proteins of each AHSV serotype were produced in insect cells and each induced detectable nAb titers in guinea pigs as an alternative animal model. Previously, purified AHSV VP2 seemed to be less immunogenic in rabbits [21], but as little as 5 μg of VP2 protein in insect cell lysate could protect horses from AHS by induction of nAbs [14]. In this study, guinea pigs were immunized with insect cell lysate containing 50 μg of VP2 to elicit detectable antibodies. Each VP2 elicited serotype specific Abs, but nAb titers varied considerably among different AHSV serotypes, from 37 for AHSV-2 to 1365 for AHSV-6. Further, cross-neutralization antibodies between genetically related serotypes were detected, but most of those cross-neutralizing Abs titers were considerably lower than for the respective serotype. Moreover, some expected cross-reactive nAbs were not detected.

The central importance of FoxG1 as an essential transcriptional regulator is underscored by the observation that even subtle alterations in FoxG1 expression levels can have profound effects on brain development. Mice with heterozygous mutations in the FoxG1 gene have impaired pallial development, suggesting that the cortex is highly sensitive to FoxG1 gene dosage ( Eagleson

et al., 2007, Shen et al., 2006a and Siegenthaler et al., 2008). Similarly, in humans, cases of Rett syndrome have been attributed to haploinsufficiency of FoxG1 ( Ariani et al., 2008 and Le Guen et al., 2011). Moreover, duplication of the FoxG1 locus has been found in patients with epilepsy, mental retardation, and speech impairment ( Brunetti-Pierri et al., 2011). Alisertib These observations strongly suggest that the precise regulation of FoxG1 expression is critical for proper brain development. www.selleckchem.com/products/Bortezomib.html We hypothesized that the dynamic expression of FoxG1 in pyramidal neuron precursors is critical for proper cortical development, and to test this, we utilized both genetic gain- and loss-of-function approaches. Remarkably, we find that the observed dynamic variation in FoxG1 expression during pyramidal cell migration is crucial for the development of the cerebral cortex. Specifically, we find that a failure to downregulate FoxG1 at the

beginning of the multipolar phase transiently stalls pyramidal neuron precursors within the lower intermediate zone as a result of failure to express Unc5D. Cells perturbed in this fashion were ultimately displaced to more superficial layers, Oxygenase and their laminar identity was respecified accordingly. Whereas the downregulation of FoxG1 was essential for pyramidal cell migration, the reinitiation of FoxG1 expression following Unc5D expression was also critical for cells to leave the multipolar cell phase and to enter into the cortical plate. Taken together, our findings demonstrate that the dynamic expression of FoxG1 during the postmitotic multipolar cell phase critically regulates

the assembly and integration of pyramidal neuron precursors into the cortical network. In the developing cerebral cortex, we found that FoxG1 expression is transiently downregulated in nascent pyramidal neuron precursors located at the lower portion of the intermediate zone (E14.5 Figures 1A and 1B; see other embryonic stages for Figures S1A, S1B, and S1C, available online). By comparing the expression of FoxG1 to other transcription factors expressed within the ventricular (VZ) and intermediate (IZ) zones (Hevner et al., 2006), such as Neurogenin2 (Neurog2) ( Hand et al., 2005, Miyata et al., 2004 and Nguyen et al., 2006), Tbr2 ( Arnold et al., 2008 and Sessa et al., 2008) ( Figure 1C), and NeuroD1 ( Mattar et al., 2008) ( Figure 1D), we found that NeuroD1 expression, which is restricted to postmitotic cells ( Mattar et al., 2008), is complementary to FoxG1 ( Figures 1B and 1B′).

Smad7 alone could

slightly decrease BMPR1a and β-catenin protein levels. When cotransfected with Smurf1, Smad7 substantially downregulated BMPR1a and β-catenin steady-state protein levels (Figure 7D). Similarly, the level of p-Smad is also reduced (Figure 7D), indicating that a decrease of BMP-Smad signaling parallels with downregulation of the BMPR1a level, possibly underlying a reduced sensitivity to BMPs (Figure 7D). Consistently, expression of Smad7 together with Smurf1 was found to reverse the inhibition of expression of myelin genes Mbp, Mag, and Plp in rat OPC culture exposed to BMP4 ( Figure 7E). In addition, Smad7/Smurf1 expression antagonized the inhibitory effects mediated by BMPRCA-Smad1/p300 expression on the Mbp promoter activity while repressing the Hes1 promoter activity ( Figure 7F). These data agree fully with other biochemical studies in the TGF-β field that inhibitory Smads negatively regulate receptor-activated Dinaciclib in vivo Smad signaling in BMP-stimulated cells ( Massagué et al., 2005). Collectively,

our observations suggest that Smad7 is a critical downstream target of Sip1 and promotes oligodendrocyte differentiation indirectly by inhibiting BMP-Smad signaling and perhaps β-catenin-mediated negative regulatory pathways. To further determine whether Smad7 is required for oligodendrocyte development, we generated and analyzed conditional Smad7 knockout mice, with the Smad7 allele deleted in the VX-770 price oligodendrocyte lineage by Olig1-Cre ( Chen et al., 2009b) ( Figure 8A). Conventional Smad7 null embryos die in utero due to multiple defects in cardiovascular development ( Chen et al., science 2009b). Although Smad7cKO (Smad7flox/flox;Olig1Cre+/−) mice are viable, they developed tremors at

postnatal week 2. To determine the role of Smad7 in oligodendrocyte development, we examined expression of the markers for mature oligodendrocytes and their precursors in the CNS of Smad7cKO animals at P7. In the brains and spinal cord of Smad7cKO mice, the expression of the myelin genes Mbp and Plp1 was diminished in the white matter in contrast to robust expression in control mice ( Figure 8B). In contrast, the OPC marker PDGFRα was detected throughout the spinal cord and the number of positive cells was comparable to that of control littermates ( Figure 8B). We did not detect any significant alteration of astrocytic GFAP expression in the spinal cord of Smad7 mutant mice (data not shown). The severe downregulation of myelin gene expression in Smad7cKO mice suggests that Smad7 is critically required for oligodendrocyte differentiation. BMP, Wnt, and Notch signaling activation is a major obstacle for remyelination by oligodendrocytes in acute and subacute demyelinating lesions, as these pathways inhibit oligodendrocyte precursor differentiation (Fancy et al., 2010, Franklin and Ffrench-Constant, 2008 and Kotter et al., 2011).

Previous theoretical and empirical studies have indeed shown that

functional interactions between brain regions are particularly crucial for cognitive processes and can occur in the absence of changes in local activity parameters, such as discharge phosphatase inhibitor library rate and oscillation amplitude (Hipp et al., 2011; Lima et al., 2011). Recent advances in EEG and MEG approaches have now allowed the noninvasive mapping of changes in the large-scale networks during perceptual and higher cognitive processes (Figure 2). Support for the distinction between local oscillatory versus long-range synchronization processes comes from studies that have examined the frequencies at which neuronal ensembles oscillate. Local processes tend to be associated with increased oscillations at gamma-band frequencies (25–200 Hz) while long-range interactions tend to involve a larger spectrum of frequency bands comprising theta (4–7 Hz), alpha (8–12 Hz), and beta (13–25 Hz) frequencies (von Stein and Sarnthein, 2000). One reason could be that larger networks cannot support selleck products synchronization with very high temporal precision as a result of long conduction times. This is because lower frequencies put fewer constraints on the precision of timing since the phases of increased and reduced excitability are longer (Kopell et al., 2000). Recent theoretical (Vicente et al., 2008) and empirical work (Buschman and Miller, 2007), however,

indicates that long-range synchronization can also occur at substantially higher frequencies (>30 Hz) and that even zero phase-lag synchronization is compatible with conduction Tolmetin delays. It is therefore conceivable that the nesting of local high-frequency oscillations in more global, lower-frequency oscillations serves

the binding of local processes into more integrated global assemblies. This possibility is supported by the growing evidence on the existence of cross-frequency coupling, the amplitude, frequency or phase of high-frequency oscillations being modulated by slower oscillatory processes (Canolty et al., 2006; Canolty and Knight, 2010; Jensen and Colgin, 2007; Palva et al., 2005). Neuron clusters can participate in several networks oscillating at different frequencies by engaging in partial coherence with both of them. This concatenation of rhythms has been observed in the hippocampus for gamma- and theta-band oscillations (Wang and Buzsáki, 1996), between different cortical laminae (Roopun et al., 2008) and for both low- and high-frequency activity (Canolty et al., 2006; Jensen and Colgin, 2007; Palva et al., 2005). Much work has been devoted to the analysis of synaptic mechanisms and circuits that support the generation of oscillatory activity and its synchronization over short and long distances, respectively, which makes it possible to relate abnormalities of these dynamic phenomena to specific neuronal processes (Sohal et al., 2009; Traub et al., 2004; Vicente et al., 2008; Wang and Buzsáki, 1996).

To estimate the causal strength of prefrontal-hippocampal interactions and its dynamics during development, Granger causality analysis, a powerful method for studying directed interactions between brain areas (Ding et al., 2000 and Anderson et al., 2010), was carried out for pairs of signals in theta-frequency band from the PFC and Hipp. Whereas the peak Granger causality values from the neonatal Cg to the CA1, denoted as Cg → Selleck MK 2206 Hipp (n = 5 pups), were not significantly different from those in the opposite direction, denoted as Hipp → Cg (Figure 5), a different causal relationship was found for the interaction between the neonatal PL and Hipp. The hippocampal theta bursts drove stronger

the prelimbic SB and NG than vice versa, since the peak Granger causality values were significantly higher for Hipp → PL than for the reciprocal connection PL → Hipp in all 10 investigated pups (Figure 5). The results are in line with the stable coupling between the PL and Hipp as revealed by coherence and cross-correlation analysis and support the driving role

of hippocampal theta bursts for the prelimbic oscillations. Toward the end of the second postnatal week the peak values of Granger causality for pairs of signals from the Cg or PL and Hipp were significant Selleck Decitabine in all investigated pups (n = 14), but similar for both directions (Figure 5). Thus, we suggest that with progressive maturation, prefrontal and hippocampal networks mutually influence each other. To identify the mechanisms underlying the directed communication between the developing PFC and Hipp, we assessed the spike-timing

relationships between prefrontal neurons and hippocampal theta bursts as well as between pairs of neurons from the two areas. Due to the very low firing rate of cingulate neurons and the results of Granger causality analysis, we focused the investigation on the prelimbic neurons. For this, we performed acute multitetrode recordings from the PL and Hipp of P7–8 (n = 7 pups) and P13–14 (n = 5 pups) rats. In a first instance, we tested whether prelimbic neurons are phase-locked to the hippocampal theta bursts. The analysis revealed that ∼9% of prelimbic neurons were Calpain significantly phase-locked to the hippocampal theta burst at both neonatal and prejuvenile age. In a second instance, we tested the impact of hippocampal firing on prelimbic cells and calculated the standardized cross-covariance (Qi,j) between all pairs (i, j) of simultaneously recorded prelimbic and hippocampal neurons (52 prelimbic neurons and 59 hippocampal neurons in P7–8 rats, 201 prelimbic neurons and 63 hippocampal neurons in P13–14 rats). In neonatal rats, only few neurons (287 PL-Hipp pairs from 3 pups) had a firing rate exceeding the set threshold of 0.05 Hz and were used for further analysis. The cross-covariance computed for all prelimbic-hippocampal pairs revealed no consistent spike timing of prelimbic neurons relative to the hippocampal cells, but yielded to a rather broad peak centered at ∼0 ms lag.

g., Womelsdorf and Fries, 2006 and Jones et al., 2007). In this issue of Neuron, Doucette and colleagues (2011) demonstrate a phenomenon that is more striking and exciting: as awake mice learn that one of two proffered odors predicts the presence of reward at a lick spout, the number of synchronous spikes

(SS) fired by pairs of olfactory bulbar (OB) neurons CX 5461 comes to reflect whether the odor is associated with reward; SS dips below spontaneous activity for unrewarded odors and hops above spontaneous for rewarded odors. This dissociation is unavailable in the firing rates of the individual OB neurons in the same trials. The beauty of this work lies in the two basic ways in which it challenges dogma. First, the results represent unusually powerful evidence for population temporal coding. Information here is uniquely available in pairs of neurons which, while typically located in the same region of the bulb, may be separated by multiple glomeruli (the functional processing units of OB spatial coding, see e.g., Wang et al., 1998). This is an easily understood and implemented population temporal code, the decoding of which simply requires downstream coincidence

detectors, connected to decision-making networks, that take input from both members of the neuron pair. Such coincidence-detecting neurons would by their very nature be preferentially sensitive and responsive to the incoming reward-related spikes. Second, these responses Tofacitinib solubility dmso reflect not odor identity per se, but rather learned reward relationships. Thus, these are important, novel data added to a growing corpus suggesting that “sensory” coding is as much about the stimulus in context as what the stimulus physically is (Kay and

TCL Laurent, 1999 and Haddad et al., 2010). The fact that the authors are recording from putative OB mitral cells, the direct recipients of olfactory information from receptor neurons in the nose, serves to drive home the point that the dividing line between sensation and perception may be found outside the brain. That is, while receptor neurons may respond to purely physical aspects of sensory stimuli, even the earliest stages of neural processing intrinsically pertain to what that stimulus means to the organism under current contingencies. Clearly, neural responses to a stimulus do not need to undergo extensive hierarchical processing to reach a point at which their relationship to reward can be identified. Note, however, that the expression of this code by OB neuron pairs does not mean that OB works alone in figuring out learned reward relationships.

, 2011 and von Philipsborn et al., 2011). Male courtship behavior is influenced by a range of sensory inputs (Krstic et al., 2009 and Koganezawa et al., 2010), especially the olfactory system (Billeter et al., 2009). Photoactivatable GFP

has been used to trace connectivity from the olfactory receptors that detect female flies through the antennal lobe to second order projections (Datta et al., 2008 and Ruta et al., 2010). Anatomical analysis suggests a compartment-level convergence of FruM neurons (Yu et al., 2010) and expression of dendritic Ruxolitinib order and synaptic reporters in candidate partners suggests connectivity (von Philipsborn et al., 2011). New understandings of the neural basis for courtship behavior have been reviewed (Manoli et al., 2006, Dickson, 2008 and Benton,

2011). The courtship circuit has several advantages: a single gene (or isoform) expressed in many neural components, sexually dimorphic anatomy and behavior, some known sensory inputs, and corroborative historical data from gynandromorphs and feminization screens (Hall, 1979, Ferveur et al., 1995 and Broughton et al., 2004), but the astute use of genetic tools to manipulate neurons has led to our current understanding of the neural circuit driving male courtship behavior. Recent work has demonstrated functional imaging of FruM neurons during a facsimile of courtship behavior (Kohatsu et al., 2011), which will allow interrogation of how neurons within the circuit respond to specific sensory stimuli and how their activity correlates with behavioral output. Dasatinib molecular weight The same experimental setup could be used to deliver specific activity patterns with light-stimulated channelrhodopsin to determine how these neurons affect behavioral outcomes. Although

there is still much work to be done to connect the identified components of a courtship circuit and find the not missing links, the potential to understand how a neural circuit actually works to drive this complex behavior is unmatched. Similar smart use of the powerful tools described here should enable mapping of circuits driving a range of different behaviors. This will permit circuit comparisons, identification of neurons that participate in several circuits, and the investigation of the way decisions between behavioral programs are made. To understand how the nervous system of an animal controls a particular behavior, one needs to identify the neurons involved, determine how their activity influences the behavior, and explore how they connect to other participating neurons. The abundance of tools for spatially and temporally targeting gene expression to specific neurons, manipulating or observing their activity, and assaying behavioral consequences makes Drosophila a premier system for exploring the principles guiding the development and function of neural circuits.

In current clamp, the same uncaging stimuli produced pauses in spontaneous firing of graded duration (t = 29 ± 5 s versus 4 ± 0.3 s for 12 × 103 μm2 and 250 μm2 fields, respectively) and hyperpolarizations of graded amplitude. In individual

cells and across cells, the responses to each photolysis condition in both recording configurations were tightly correlated (Figure 3D). Furthermore, the onset kinetics of the light-evoked currents did not vary across the different uncaging stimuli (τon = 349 ± 26 ms versus 400 ± 66 ms versus 417 ± 112 ms for 12 × 103 μm2, 4.2 × 103 μm2, and 1.2 × 103 μm2 fields, respectively; one-way ANOVA p = 0.81; kinetics could not be reliably measured for responses Stem Cell Compound Library in vitro to the 250 μm2 uncaging stimulus). These results indicate that photolysis delivers LE directly to the site of action over a range of areas. The ability to tightly regulate the area over which LE is applied provides an opportunity to study the ionic conductances that underlie the mu opioid response in LC with unprecedented accuracy. Although selleck chemicals llc it has been clearly demonstrated that mu opioid receptor activation opens GIRK channels in LC neurons (Torrecilla et al.,

2002), reversal potentials determined for the evoked currents in brain slices are frequently much more negative (−140mV to −120mV) than predicted for a pure K+ conductance according to the Nernst equation (∼−105mV, typically). This observation might be accounted for by the inability to voltage-clamp currents generated in the large (Shipley et al., 1996), gap-junction-coupled dendrites (Ishimatsu and Williams, 1996 and Travagli et al., 1995) of LC neurons. Several studies suggest that inhibition of a standing, voltage-insensitive Na+ current Liothyronine Sodium may contribute 50% of the observed outward current response to enkephalin (Alreja and Aghajanian, 1993 and Alreja and Aghajanian, 1994). Thus, the complete ionic nature of the enkephalin-evoked outward currents has been a subject of debate (Alreja and Aghajanian, 1993,

Alreja and Aghajanian, 1994, Osborne and Williams, 1996, Torrecilla et al., 2002 and Travagli et al., 1995). To address this issue, we measured the reversal potential of the LE evoked outward current while restricting the uncaging area to the soma and proximal dendrites where voltage clamp is expected to be optimal (Williams and Mitchell, 2008). Importantly, the responses to the uncaging stimuli shown in Figure 3B were not significantly attenuated by the gap junction inhibitor carbenoxolone (Figure S4), suggesting that gap junctions do not contribute to the LE-mediated currents evoked by uncaging CYLE around the soma. To measure reversal potentials in the voltage range of a K+ conductance, we held cells at −55mV and applied negative voltage ramps to −140mV over 500 ms during the peak of the outward current (Figure 4A). A response to the ramp alone is presented with a response to the ramp after an uncaging stimulus corresponding to the 4.