Motivated to time: Effects of reinforcer devaluation and opportunity cost on interval timing.

Prior research suggests that interval timing performance is sensitive to reinforcer devaluation effects and to the rate of competing sources of reinforcement. The present study sought to replicate and account for these findings in rats. A self-paced concurrent fixed-interval (FI) random-ratio (RR) schedule of reinforcement was implemented in which the FI requirement varied across training conditions (12, 24, 48 s). The RR requirement-which imposed an opportunity cost to responding on the FI component-was adjusted so that it took about twice the FI requirement, on average, to complete it. Probe reinforcer devaluation (prefeeding) sessions were conducted at the end of each condition. To assess the effect of contextual reinforcement on timing performance, the RR requirement was removed before the end of the experiment. Consistent with prior findings, performance on the FI component tracked schedule requirement and displayed scalar invariance; the removal of the RR component yielded more premature FI responses. For some rats, prefeeding reduced the number of trials initiated without affecting timing performance; for other rats, prefeeding delayed responding on the FI component but had a weaker effect on trial initiation. These results support the notion that timing and motivational processes are separable, suggesting novel explanations for ostensible motivational effects on timing performance.

Hyperactive MEK1 Signaling in Cortical GABAergic Neurons Promotes Embryonic Parvalbumin Neuron Loss and Defects in Behavioral Inhibition.

Many developmental syndromes have been linked to genetic mutations that cause abnormal ERK/MAPK activity; however, the neuropathological effects of hyperactive signaling are not fully understood. Here, we examined whether hyperactivation of MEK1 modifies the development of GABAergic cortical interneurons (CINs), a heterogeneous population of inhibitory neurons necessary for cortical function. We show that GABAergic-neuron specific MEK1 hyperactivation in vivo leads to increased cleaved caspase-3 labeling in a subpopulation of immature neurons in the embryonic subpallial mantle zone. Adult mutants displayed a significant loss of parvalbumin (PV), but not somatostatin, expressing CINs and a reduction in perisomatic inhibitory synapses on excitatory neurons. Surviving mutant PV-CINs maintained a typical fast-spiking phenotype but showed signs of decreased intrinsic excitability that coincided with an increased risk of seizure-like phenotypes. In contrast to other mouse models of PV-CIN loss, we discovered a robust increase in the accumulation of perineuronal nets, an extracellular structure thought to restrict plasticity. Indeed, we found that mutants exhibited a significant impairment in the acquisition of behavioral response inhibition capacity. Overall, our data suggest PV-CIN development is particularly sensitive to hyperactive MEK1 signaling, which may underlie certain neurological deficits frequently observed in ERK/MAPK-linked syndromes.

Longer operant lever-press duration requirements induce fewer but longer response bouts in rats.

Operant behavior is organized in bouts that are particularly visible under variable-interval (VI) schedules of reinforcement. Previous research showed that increasing the work required to produce a response decreases the rate at which bouts are emitted and increases the minimum interresponse time (IRT). In the current study, the minimum effective IRT was directly manipulated by changing the minimum duration of effective lever presses reinforced on a VI 40-s schedule. Contrary to assumptions of previous models, response durations were variable. Response durations were typically 0.5 s greater than the minimum duration threshold; durations that exceeded this threshold were approximately log-normally distributed. As the required duration threshold increased, rats emitted fewer but longer bouts. This effect may reflect an effort-induced reduction in motivation and a duration-induced facilitation of a response-outcome association.

Chronic stress has lasting effects on improved cued discrimination early in extinction.

Chronic stress typically leads to deficits in fear extinction when tested soon after chronic stress ends. Given the importance of extinction in updating fear memories, the current study examined whether fear extinction was impaired in rats that were chronically stressed and then given a break from the end of chronic stress to the start of fear conditioning and extinction. Male rats were chronically stressed by restraint (6 h/d/21 d) and tested soon (termed immediate, STR-IMM), or 3 or 6 wk after a rest period from restraint (termed rest or "R," STR-R3, STR-R6). In Experiment 1, STR-R3 and STR-R6 discriminated between the cue and nonshock context better than STR-IMM or control. Interestingly, STR-IMM showed high freezing to the nonshock context. Consequently, Experiment 2 investigated whether STR-IMM generalized across contexts, which was not supported. Experiment 3 determined whether STR-IMM were susceptible to second-order conditioning to a novel context, but showed that the level of second-order conditioning was similar for all groups. These findings reveal that rats exposed to chronic stress and then given a rest period of 3 or 6 wk, express unique fear extinction profiles compared to control and STR-IMM. Specifically, STR-R demonstrated excellent cue and context discrimination during extinction, and perhaps showed a stress inoculation effect. For STR-IMM, the heightened freezing under these extensive acclimation parameters was not attributed to generalization nor to second-order fear conditioning to "safe" contexts and, instead, may reflect hypervigilance.

Response-inhibition capacity in spontaneously hypertensive and Wistar rats: acquisition of fixed minimum interval performance and responsiveness to D-amphetamine.

Reduced response-inhibition capacity is a defining feature of attention-deficit hyperactivity disorder. The fixed minimum interval (FMI) schedule has been systematically validated to assess such capacity in rats. On each FMI trial, the first lever press initiates an inter-response time (IRT); a potentially consummatory response terminates the IRT; only IRTs longer than a target interval result in access to food. Despite task validity, steady-state FMI performance in the most common animal model of attention-deficit hyperactivity disorder, the spontaneously hypertensive rat (SHR), is similar to normotensive control performance, even though SHR performs at lower levels, especially during acquisition, in similar response-withholding tasks. To determine whether such limitations of the model are specific to stable-state performance, this experiment compared FMI 6-s performance in SHR and Wistar rats during acquisition and in steady state, and assessed the effect of acute D-amphetamine (AMP) administration (0.1, 0.5, and 1.0 mg/kg) on steady-state performance. Median latencies to first lever press were consistently shorter in SHR than in Wistar rats; IRTs were shorter for SHR than for Wistar rats during acquisition, but substantially less so during asymptotic performance. AMP dose-dependently reduced latencies, shortened IRTs, and, at the highest dose, increased the proportion of IRTs under schedule control. These results suggest that, relative to Wistar rats, SHR have a reduced capacity to learn to withhold a reinforced response; once the FMI is acquired, high doses of D-AMP disrupt withholding performance in both strains, but they also enhance the responsiveness of both strains to reinforcement contingencies.

Interval timing under a behavioral microscope: Dissociating motivational and timing processes in fixed-interval performance.

The distribution of latencies and interresponse times (IRTs) of rats was compared between two fixed-interval (FI) schedules of food reinforcement (FI 30 s and FI 90 s), and between two levels of food deprivation. Computational modeling revealed that latencies and IRTs were well described by mixture probability distributions embodying two-state Markov chains. Analysis of these models revealed that only a subset of latencies is sensitive to the periodicity of reinforcement, and prefeeding only reduces the size of this subset. The distribution of IRTs suggests that behavior in FI schedules is organized in bouts that lengthen and ramp up in frequency with proximity to reinforcement. Prefeeding slowed down the lengthening of bouts and increased the time between bouts. When concatenated, latency and IRT models adequately reproduced sigmoidal FI response functions. These findings suggest that behavior in FI schedules fluctuates in and out of schedule control; an account of such fluctuation suggests that timing and motivation are dissociable components of FI performance. These mixture-distribution models also provide novel insights on the motivational, associative, and timing processes expressed in FI performance. These processes may be obscured, however, when performance in timing tasks is analyzed in terms of mean response rates.

Chronic stress disrupts fear extinction and enhances amygdala and hippocampal Fos expression in an animal model of post-traumatic stress disorder.

Chronic stress may impose a vulnerability to develop maladaptive fear-related behaviors after a traumatic event. Whereas previous work found that chronic stress impairs the acquisition and recall of extinguished fear, it is unknown how chronic stress impacts nonassociative fear, such as in the absence of the conditioned stimulus (CS) or in a novel context. Male rats were subjected to chronic stress (STR; wire mesh restraint 6 h/d/21d) or undisturbed (CON), then tested on fear acquisition (3 tone-footshock pairings), and two extinction sessions (15 tones/session) within the same context. Then each group was tested (6 tones) in the same context (SAME) or a novel context (NOVEL), and brains were processed for functional activation using Fos immunohistochemistry. Compared to CON, STR showed facilitated fear acquisition, resistance to CS extinction on the first extinction day, and robust recovery of fear responses on the second extinction day. STR also showed robust freezing to the context alone during the first extinction day compared to CON. When tested in the same or a novel context, STR exhibited higher freezing to context than did CON, suggesting that STR-induced fear was independent of context. In support of this, STR showed increased Fos-like expression in the basolateral amygdala and CA1 region of the hippocampus in both the SAME and NOVEL contexts. Increased Fos-like expression was also observed in the central amygdala in STR-NOVEL vs. CON-NOVEL. These data demonstrate that chronic stress enhances fear learning and impairs extinction, and affects nonassociative processes as demonstrated by enhanced fear in a novel context.

Characterizing operant hyperactivity in the Spontaneously Hypertensive Rat.

BACKGROUND: Operant hyperactivity, the emission of reinforced responses at an inordinately high rate, has been reported in children with ADHD and in the Spontaneously Hypertensive Rat (SHR), the most widely studied animal model of ADHD. The SHR emits behavior at hyperactive levels, relative to a normoactive strain, only when such behavior is seldom reinforced. Because of its dependence on rate of reinforcement, operant hyperactivity appears to be driven primarily by incentive motivation, not motoric capacity. This claim was evaluated in the present study using a novel strategy, based on the organization of behavior in bouts of reinforced responses separated by pauses. METHOD: Male SHR, Wistar-Kyoto (WKY) and Wistar rats (WIS) were exposed each to a multiple variable-interval schedule of sucrose reinforcement (12, 24, 48, 96, and 192 s) between post-natal days (PND) 48 and 93. Responding in each schedule was examined in two epochs, PND 58-62 and 89-93. Parameters of response-reinforcement functions (Herrnstein's hyperbola) and bout-organized behavior were estimated in each epoch. RESULTS: SHR emitted higher response rates than WKY and WIS, but only when rate of reinforcement was low (fewer than 2 reinforcers per minute), and particularly in the second epoch. Estimates of Herrnstein's hyperbola parameters suggested the primacy of motivational over motoric factors driving the response-rate differential. Across epochs and schedules, a more detailed analysis of response bouts by SHR revealed that these were shorter than those by WKY, but more frequent than those by WKY and WIS. Differences in bout length subsided between epochs, but differences in bout-initiation rate were exacerbated. These results were interpreted in light of robust evidence linking changes in bout-organization parameters and experimental manipulations of motivation and response-reinforcement contingency. CONCLUSIONS: Operant hyperactivity in SHR was confirmed. Although incentive motivation appears to play an important role in operant hyperactivity and motoric capacity cannot be ruled out as a factor, response-bout patterns suggest that operant hyperactivity is primarily driven by steeper delay-of-reinforcement gradients. Convergence of this conclusion with theoretical accounts of ADHD and with free-operant performance in children with ADHD supports the use of SHR as an animal model of ADHD.

Extinction learning deficit in a rodent model of attention-deficit hyperactivity disorder.

BACKGROUND: Deficient operant extinction has been hypothesized to be constitutive of ADHD dysfunction. In order to elucidate the behavioral mechanisms underlying this deficit, the performance of an animal model of ADHD, the spontaneously hypertensive rat (SHR), was compared against the performance of a control strain, the Wistar-Kyoto rat (WKY) during extinction. METHOD: Following extensive training of lever pressing under variable interval schedules of food reinforcement (reported previously), SHR and WKY rats were exposed to two sessions of extinction training. Extinction data was analyzed using the Dynamic Bi-Exponential Refractory Model (DBERM) of operant performance. DBERM assumes that operant responses are organized in bouts separated by pauses; during extinction, bouts may decline across multiple dimensions, including frequency and length. DBERM parameters were estimated using hierarchical Bayesian modeling. RESULTS: SHR responded more than WKY during the first extinction session. DBERM parameter estimates revealed that, at the onset of extinction, SHR produced more response bouts than WKY. Over the course of extinction, response bouts progressively shortened for WKY but not for SHR. CONCLUSIONS: Based on prior findings on the sensitivity of DBERM parameters to motivational and schedule manipulations, present data suggests that (1) more frequent response bouts in SHR are likely related to greater incentive motivation, and (2) the persistent length of bouts in SHR are likely related to a slower updating of the response-outcome association. Overall, these findings suggest specific motivational and learning deficits that may explain ADHD-related impairments in operant performance.

Timing, neural timescales, and temporal cognition.

This special issue provides a representative snapshot of cutting-edge behavioral neuroscience research on sense of time, cognitive and behavioral functioning, and neural processes. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Assessing complex odor discrimination in mice using a novel instrumental patterning task.

Negative patterning tasks are a key tool to unveil the mechanisms by which stimulus representations are acquired-a central concern in Robert Rescorla's research. In these tasks, target stimuli are reinforced when presented individually (A+/B+) but not when presented in compound (AB-). The discrimination of single stimuli from their compound presentation is a challenge for theories of associative learning, because it cannot be explained by the simple accrual of associative strength. The present study examined the conditions under which mice learn this part-whole discrimination in olfactory stimuli using a novel instrumental methodology. In two experiments, reinforcement was contingent on distinct responses depending on whether a set of odor mixtures were presented in isolation or as a compound. Using C57BL/6 mice, Experiment 1 showed a mutual interference between learning a response to individual odors and learning a different response to those odors presented in compound. Using wild-type APP/PS1 mice (a control strain for a murine model of Alzheimer's disease), Experiment 2 replicated this interference and showed that it is stimulus-specific. These experiments show that the instrumental patterning task may not only complement Pavlovian negative patterning tasks but may also motivate its own questions on the representation of complex stimuli. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

High fat diet intake during pre and periadolescence impairs learning of a conditioned place preference in adulthood.

BACKGROUND: Brain regions that mediate learning of a conditioned place preference (CPP) undergo significant development in pre and periadolescence. Consuming a high fat (HF) diet during this developmental period and into adulthood can lead to learning impairments in rodents. The present study tested whether HF diet intake, consumed only in pre and periadolescence, would be sufficient to cause impairments using a CPP procedure. METHODS: Rats were randomly assigned to consume a HF or a low fat (LF) diet during postnatal days (PD) 21-40 and were then placed back on a standard lab chow diet. A 20-day CPP procedure, using HF Cheetos® as the unconditioned stimulus (US), began either the next day (PD 41) or 40 days later (PD 81). A separate group of adult rats were given the HF diet for 20 days beginning on PD 61, and then immediately underwent the 20-day CPP procedure beginning on PD 81. RESULTS: Pre and periadolescent exposure to a LF diet or adult exposure to a HF diet did not interfere with the development of a HF food-induced CPP, as these groups exhibited robust preferences for the HF Cheetos® food-paired compartment. However, pre and periadolescent exposure to the HF diet impaired the development of a HF food-induced CPP regardless of whether it was assessed immediately or 40 days after the exposure to the HF diet, and despite showing increased consumption of the HF Cheetos® in conditioning. CONCLUSIONS: Intake of a HF diet, consumed only in pre and periadolescence, has long-lasting effects on learning that persist into adulthood.

On the Appropriate Measure to Estimate Hyperbolic Discounting Rate (K) using the Method of Least Squares.

The estimation of the rate at which value declines with delay requires identifying the correct discounting model, applying the appropriate parameter estimation method, and choosing the dependent measure from which parameters are estimated. The simplest adequate discounting model is the hyperbolic model; the simplest method to estimate its sole free parameter, K, is the method of least squares. Estimates of K based on relative subjective values (RSV), although typical, are not necessarily more reliable than those obtained through other measures. We consider an alternative measure termed immediacy premium (IP): the ratio of value lost over value preserved due to outcome delay. According to hyperbolic discounting, IP is a linear function of delay. As a result, estimates of K obtained from IP circumvent the divergence between individual and aggregate estimates obtained with the RSV method. Moreover, published data suggests that estimates of K based on RSV and IP differ systematically in humans and in pigeons. Regardless of the dependent measure adopted, estimates of K obtained from nonhuman animals, but not from humans, yield residuals that conform with error-distribution assumptions of the method of least squares. Although residuals obtained using the IP method in human data diverged more from normality than those obtained using the RSV method, the sequential dependence over delays of the former was weaker than that of the latter. We therefore recommend adopting both RSV and IP when estimating hyperbolic K using the method of least squares, resorting to more elaborate estimation methods when inferences drawn from these estimates are inconsistent.

Internal-Clock Models and Misguided Views of Mechanistic Explanations: A Reply to Eckard & Lattal (2020).

Eckard and Lattal's Perspectives on Behavior Science, 43(1), 5-19 (2020) critique of internal clock (IC) mechanisms is based on narrow concepts of clocks, of their internality, of their mechanistic nature, and of scientific explanations in general. This reply broadens these concepts to characterize all timekeeping objects-physical and otherwise-as clocks, all intrinsic properties of such objects as internal to them, and all simulatable explanations of such properties as mechanisms. Eckard and Lattal's critique reflects a restrictive billiard-ball view of causation, in which environmental manipulations and behavioral effects are connected by a single chain of contiguous events. In contrast, this reply offers a more inclusive stochastic view of causation, in which environmental manipulations are probabilistically connected to behavioral effects. From either view of causation, computational ICs are hypothetical and unobservable, but their heuristic value and parsimony can only be appreciated from a stochastic view of causation. Billiard-ball and stochastic views have contrasting implications for potential explanations of interval timing. As illustrated by accounts of the variability in start times in fixed-interval schedules of reinforcement, of the two views of causality examined, only the stochastic account supports falsifiable predictions beyond simple replications. It is thus not surprising that the experimental analysis of behavior has progressively adopted a stochastic view of causation, and that it has reaped its benefits. This reply invites experimental behavior analysts to continue on that trajectory.

Failure to find a distance effect in pigeon choice: Manipulating amount and delay of reinforcement.

The choice behavior of primates, including humans, displays a distance effect: Latency to choose between alternatives appears to increase with smaller differences in value. There is, so far, no demonstration of this effect in birds. Tests of distance effects in birds have been conducted in binary choice situations with a dominant alternative, where one alternative is superior to the other in all aspects that meaningfully contribute to value (e.g., provides access to the same reinforcer, but with a shorter delay). The present study considers the possibility that including dominant alternatives in choice tests precludes distance effects. Four pigeons were presented with binary choices between alternatives that varied in amount and delay. Some choices had a dominant alternative (smaller-sooner or larger-later vs. smaller-later) and some did not (smaller-sooner vs. larger-later). Across phases, only the delay to the smaller-sooner reinforcer varied. Distance effects were expected to be expressed as longer latencies as choice between smaller-sooner and larger-later reinforcers approached indifference. Despite the sensitivity of choice to differences in amount and delay, no distance effect was observed. Alternative explanations for the failure to find a distance effect in pigeon choice, including the Sequential Choice Model (SCM), are discussed.

Past outcomes and time flexibly exert joint control over midsession reversal performance in the rat.

In a midsession reversal task, subjects choose between two stimuli on every trial; only responses to one stimulus are reinforced. Halfway throughout the session, contingencies are reversed: previously reinforced responses are now extinguished and vice versa. Both, the outcome of the previous trial and the time elapsed since the beginning of the session, may predict the availability of reinforcement and determine choice. Thus, this task has typically been used to study cognitive flexibility and the temporal organization of behavior. This study assessed how past outcomes and time interact for behavioral control when each cue predicts the availability of reinforcement to a different extent. Eight rats were trained in four variations of the midsession reversal task differing in the reliability of outcomes and time as predictors of the reinforced response. We manipulated the reliability of the outcomes by providing either continuous or partial reinforcement, and the reliability of time by fixing the moment of reversal (middle of the session) or making the reversal unpredictable (semi-random trial). Results suggest that behavioral control alternates between outcomes and time according to the relative reliability of each cue. Model simulations show that outcomes and time may jointly determine behavior, and that momentary reinforcement rate may determine their relative influence.

Dimensions of childhood trauma and their direct and indirect links to PTSD, impaired control over drinking, and alcohol-related-problems.

INTRODUCTION: Post-Traumatic Stress Disorder (PTSD) develops after experiencing events that evoke fear, helplessness, or horror. The Hyperarousablity Hypothesis suggests that those with PTSD may drink more to dampen physiological reactivity. We examined the direct and indirect relationships between childhood trauma (e.g., physical-neglect, emotional-abuse, physical-abuse, sexual-abuse) versus an emotionally-supportive-family on PTSD, impaired control over drinking (IC), alcohol-use, and alcohol-related-problems. IC reflects consuming more alcohol than one originally intended. METHODS: We fit a multiple-group SEM to data on 835 participants. Mediational analyses were conducted by using the (K = 20,000) bootstrap technique with confidence intervals. RESULTS: Physical-neglect was directly linked to more IC among both genders. Emotional abuse was also found to be directly linked to more PTSD among both genders. Furthermore, PTSD was directly linked to more impaired control over alcohol use (IC) among both genders. Mediational analyses showed that physical-neglect was indirectly linked to more alcohol-related-problems through increased IC. Having an emotionally supportive family was directly linked to fewer PTSD symptoms among women. For both genders, emotional abuse was indirectly linked to more alcohol-related-problems through more PTSD symptoms, impaired control over alcohol use difficulties, and in turn, more alcohol-use. Sexual abuse was indirectly linked to increased alcohol-related- problems through increased PTSD symptoms and more IC, and in turn, more alcohol-use among men. CONCLUSIONS: Recalled childhood trauma (sexual and emotional abuse) may contribute to PTSD symptoms and dysregulated drinking. In conclusion, our data suggest that reducing PTSD symptoms may assist individuals in regaining control over their drinking.

Anti-craving effects of environmental enrichment.

We hypothesized that environmental enrichment in rats may reduce cocaine-seeking behaviour elicited by cocaine-priming injections and by cocaine-associated cues. Rats trained to self-administer cocaine while housed in isolated conditions were then assigned to live in isolation or an enriched environment for 21 d of forced abstinence. Subsequently, extinction and reinstatement of cocaine-seeking behaviour (operant responses without cocaine available) were assessed. Expt 1 showed that enrichment resulted in less cocaine-seeking behaviour during extinction and cue-elicited reinstatement compared to continued isolation housing, but had no effect on cocaine-primed reinstatement. A subsequent experiment, which included a pair-housed group to control for potential isolation stress, again demonstrated that enrichment attenuated cocaine seeking during extinction, but not cocaine-primed reinstatement, relative to both isolation and pair-housed conditions. The findings suggest that enrichment reduces the impact of cocaine-associated environmental stimuli, and hence it may be a useful intervention for attenuating cue-elicited craving in humans.

A computational formulation of the behavior systems account of the temporal organization of motivated behavior.

The behavior systems framework suggests that motivated behavior-e.g., seeking food and mates, avoiding predators-consists of sequences of actions organized within nested behavioral states. This framework has bridged behavioral ecology and experimental psychology, providing key insights into critical behavioral processes. In particular, the behavior systems framework entails a particular organization of behavior over time. The present paper examines whether such organization emerges from a generic Markov process, where the current behavioral state determines the probability distribution of subsequent behavioral states. This proposition is developed as a systematic examination of increasingly complex Markov models, seeking a computational formulation that balances adherence to the behavior systems approach, parsimony, and conformity to data. As a result of this exercise, a nonstationary partially hidden Markov model is selected as a computational formulation of the predatory subsystem. It is noted that the temporal distribution of discrete responses may further unveil the structure and parameters of the model but, without proper mathematical modeling, these discrete responses may be misleading. Opportunities for further elaboration of the proposed computational formulation are identified, including developments in its architecture, extensions to defensive and reproductive subsystems, and methodological refinements.

The differential role of the dorsal hippocampus in initiating and terminating timed responses: A lesion study using the switch-timing task.

This study investigated the role of the dorsal hippocampus (dHPC) in the temporal entrainment of behavior, while addressing limitations of previous evidence from peak procedure experiments. Rats were first trained on a switch-timing task in which food was obtained from one of two concurrently available levers; one lever was effective after 8 s and the other after 16  s. After performance stabilized, rats underwent either bilateral NMDA lesions of the dHPC or sham lesions. After recovery, switch-timing training resumed. In a subsequent condition, the switch-timing task was modified such that food was available after either 8 or 32 s. Although dHPC lesions had subtle and complex effects on when rats stopped seeking for food at the 8-s lever (departures), it more systematically reduced the time when rats started seeking for food at the 16-s and 32-s lever (switches). No systematic effect of dHPC lesions were observed on the coefficient of quartile variation (normalized dispersion) of latencies to switch. Within the context of the pacemaker-accumulator framework of interval timing, these findings suggest that partially or wholly independent mechanisms control the initiation and termination of timed responses, and that the dHPC is primarily involved in encoding the time to start responding.