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Pkhd1l1 is predicted to encode a very large type-I transmembrane protein, but its function has largely remained obscure. Recently, it was shown that Pkhdl1l1 is a component of the coat that decorates stereocilia of outer hair cells in the mouse ear. Consistent with this localization, conditional deletion of Pkhd1l1 specifically from hair cells, was associated with progressive hearing loss. In the zebrafish, there are two paralogous pkhd1l1 genes - pkhd1l1α and pkhd1l1β. Using CRISPR-Cas9 mediated gene editing, we generated loss-of-function alleles for both and show that the double mutants exhibit nonsense-mediated-decay (NMD) of the RNAs. With behavioural assays, we demonstrate that zebrafish pkhd1l1 genes also regulate hearing; however, in contrast to Pkhd1l1 mutant mice, which develop progressive hearing loss, the double mutant zebrafish exhibited statistically significant hearing loss even from the larval stage. Our data highlight a conserved function of Pkhd1l1 in hearing and based on these findings from animal models, we postulate that PKHD1L1 could be a candidate gene for sensorineural hearing loss (SNHL) in humans.

Differential reinforcement of low rate (DRL) responding is a schedule-controlled behavior sometimes used in timing research, but also received critics of not providing a pure measure of timing due to the influence of the subject’s motivation or inhibitory control. Nevertheless, we argue that the DRL task provides a unique approach to study how timing and emotion interact with each other. Here, we review evidence showing that male rats prenatally treated with choline supplementation had difficulty in acquiring longer criterion times in the DRL task. This was possibly due to the stronger memory strength of their previously learned shorter criterion times. Female rats, in contrast, performed better than male rats in the same task, but those receiving prenatal choline supplementation were the best performers in this task with longer criterion times because they required less training. Like all female rats, male rats treated with prenatal choline supplementation made very few burst responses, suggesting that the treatment improved male rats’ emotional regulation when facing ‘frustrating’ outcomes. The differential impulse control plus different memory strength of the rats trained in the DRL task revealed the potential interaction of sex hormones and prenatal choline supplementation, a rare combination in a single animal study on timing and time perception. In summary, although the DRL task is certainly not the best timing task, it may be useful in assisting us in better understanding how time perception participates in emotional regulation, especially relevant when the emotion is triggered by a failure in timing.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal. Ministry of Education (MOE) National Research Foundation (NRF) This work was funded by a joint grant from the Israel Science Foundation and National Research Foundation of Singapore (6276/17 and NRF2017‐NRF‐ISF002‐2676) to YG and SJ, and a Tier 1 Grant from the Singapore Ministry of Education (RG34/20) to SJ.

The habenula is an evolutionarily conserved structure of the vertebrate brain that is essential for behavioural flexibility and mood control. It is spontaneously active and is able to access diverse states when the animal is exposed to sensory stimuli. Here we investigate the dynamics of habenula spontaneous activity, to gain insight into how sensitivity is optimized. Two-photon calcium imaging was performed in resting zebrafish larvae at single cell resolution. An analysis of avalanches of inferred spikes suggests that the habenula is subcritical. Activity had low covariance and a small mean, arguing against dynamic criticality. A multiple regression estimator of autocorrelation time suggests that the habenula is neither fully asynchronous nor perfectly critical, but is reverberating. This pattern of dynamics may enable integration of information and high flexibility in the tuning of network properties, thus providing a potential mechanism for the optimal responses to a changing environment. Significance Statement: Spontaneous activity in neurons shapes the response to stimuli. One structure with a high level of spontaneous neuronal activity is the habenula, a regulator of broadly acting neuromodulators involved in mood and learning. How does this activity influence habenula function? We show here that the habenula of a resting animal is near criticality, in a state termed reverberation. This pattern of dynamics is consistent with high sensitivity and flexibility, and may enable the habenula to respond optimally to a wide range of stimuli. Ministry of Education (MOE) Published version This work was funded by the Singapore Ministry of Education through an Academic Research Fund Tier 1 Award (MOE2016-T1-001-152) and a Tier 2 Award (MOE2017-T2-1-058).

Background Detection of predator cues changes the brain state in prey species and helps them avoid danger. Dysfunctionality in changing the central state appropriately in stressful situations is proposed to be an underlying cause of multiple psychiatric disorders in humans. Methods Here, we investigate the dynamics of neural circuits mediating response to a threat, to characterize these states and to identify potential control networks. We use resonant scanning 2-photon microscopy for in vivo brain-wide imaging and custom designed behavioral assays for the study. Results We first show that 5–7 day old zebrafish larvae react to an alarm pheromone (Schreckstoff) with reduced mobility. They subsequently display heightened vigilance, as evidenced by increased dark avoidance. Calcium imaging indicates that exposure to Schreckstoff elicits stimulus-locked activity in olfactory sensory neurons innervating a lateral glomerulus and in telencephalic regions including the putative medial amygdala and entopeduncular nucleus. Sustained activity outlasting the stimulus delivery was detected in regions regulating neuromodulator release, including the lateral habenula, posterior tuberculum, superior raphe, and locus coeruleus. Conclusion We propose that these latter regions contribute to the network that defines the “threatened” state, while neurons with transient activity serve as the trigger. Our study highlights the utility of the zebrafish larval alarm response system to examine neural circuits during stress dependent brain state transitions and to discover potential therapeutic agents when such transitions are disrupted.

BackgroundDetection of predator cues changes the brain state in prey species and helps them avoid danger. Dysfunctionality in changing the central state appropriately in stressful situations is proposed to be an underlying cause of multiple psychiatric disorders in humans.MethodsHere, we investigate the dynamics of neural circuits mediating response to a threat, to characterize these states and to identify potential control networks. We use resonant scanning 2-photon microscopy for in vivo brain-wide imaging and custom designed behavioral assays for the study.ResultsWe first show that 5-7 day old zebrafish larvae react to an alarm pheromone (Schreckstoff) with reduced mobility. They subsequently display heightened vigilance, as evidenced by increased dark avoidance. Calcium imaging indicates that exposure to Schreckstoff elicits stimulus-locked activity in olfactory sensory neurons innervating a lateral glomerulus and in telencephalic regions including the putative medial amygdala and entopeduncular nucleus. Sustained activity outlasting the stimulus delivery was detected in regions regulating neuromodulator release, including the lateral habenula, posterior tuberculum, superior raphe, and locus coeruleus.ConclusionWe propose that these latter regions contribute to the network that defines the “threatened” state, while neurons with transient activity serve as the trigger. Our study highlights the utility of the zebrafish larval alarm response system to examine neural circuits during stress dependent brain state transitions and to discover potential therapeutic agents when such transitions are disrupted.

Habenula neurons are constantly active. The level of activity affects mood and behaviour, with increased activity in the lateral habenula reflecting exposure to punishment and a switch to passive coping and depression. Here, we identify GABAergic neurons that could reduce activity in the lateral habenula of larval zebrafish. GAD65/67 immunohistochemistry and imaging of gad1b:DsRed transgenic fish suggest the presence of GABAergic terminals in the neuropil and between cell bodies in the lateral habenula. Retrograde tracing with the lipophilic dye DiD suggests that the former derives from the thalamus, while the latter originates from a group of cells in the posterior hypothalamus that are located between the posterior tuberal nucleus and hypothalamic lobes. Two‐photon calcium imaging indicates that blue light causes excitation of thalamic GABAergic neurons and terminals in the neuropil, while a subpopulation of lateral habenula neurons show reduced intracellular calcium levels. Whole‐cell electrophysiological recording indicates that blue light reduces membrane potential of lateral habenula neurons. These observations suggest that GABAergic input from the thalamus may mediate inhibition in the zebrafish lateral habenula. Mechanisms governing release of GABA from the neurons in the posterior hypothalamus, which are likely to be in the tuberomammillary nucleus, remain to be defined., Two distinct populations of GABAergic neurons innervate the lateral habenula of larval zebrafish. One population, located in the anterior thalamus, terminates in neuropils. Another population, located in the posterior hypothalamus, appears to terminate on cell soma bilaterally.

Male Sprague–Dawley rats were exposed to social defeat and subordination by aggressive male Long–Evans rats. The social defeat procedure involved the continuous exposure to an aggressive resident for 10 days, while living in a protective cage within the resident’s home cage with daily brief confrontations. These stress experiences resulted in 1) reduced body weight; 2) decreased social interaction; 3) increased ultrasonic vocalizations; 4) reduced sucrose preference (anhedonia); and 5) decreased clock speed while timing 15-s and 45-s target durations in a bi-peak procedure. Treatment with ketamine (15 mg/kg, i.p.) produced a rapid reversal of anhedonia and overproduction of duration. Taken together, these data provide the first evaluation of the effects of continuous social defeat and its associated depression-like symptoms on timing and time perception using a ‘state change’ design.

Although the striatal dopamine (DA) is reportedly involved in impulsive action, little is known about the DA subtype receptors of dorsal striatum (dSTR) in the impulsive control involved in differential reinforcement of low-rate-responding (DRL) behavior. We examined the receptor-specific dopaminergic modulation of d-amphetamine (AMP)-altered DRL 10 s (DRL-10 s) performance by locally infusing SCH23390 (SCH) and raclopride (RAC), DA D1 and D2 receptor antagonists, respectively, into the rat's dSTR. Systemic injection of AMP significantly affected DRL-10 s behavior by increasing total, non-reinforced, and bust responses, as well as by decreasing reinforced responses, which correspondingly caused a leftward shift of the inter-response-time distribution curve as confirmed by a profound decrease in peak time (i.e.

When injured, fish release an alarm substance (Schreckstoff) that elicits fear in members of their shoal. Although Schreckstoff has been proposed to be produced by club cells in the skin, several observations indicate that these giant cells function primarily in immunity. Previous data indicate that the alarm substance can be isolated from mucus. Here we show that mucus, as well as bacteria, are transported from the external surface into club cells, by cytoplasmic transfer or invasion of cells, including neutrophils. The presence of bacteria inside club cells raises the possibility that the alarm substance may contain a bacterial component. Indeed, lysate from a zebrafish Staphylococcus isolate is sufficient to elicit alarm behaviour, acting in concert with a substance from fish. These results suggest that Schreckstoff, which allows one individual to unwittingly change the emotional state of the surrounding population, derives from two kingdoms and is associated with processes that protect the host from bacteria., When injured, fish release an alarm substance produced by club cells in the skin that elicits fear in members of their shoal. Here, the authors show that mucus and bacteria are transported from the external surface into club cells, and bacterial components elicit alarm behaviour, acting in concert with a substance from fish.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism., Small open reading frame-encoded peptides (SEPs), shorter than 100 amino acids, are involved in many cell biological processes. Here the authors identify 16 nuclear-encoded mitochondrial SEPs, including BRAWNIN, an essential regulator of respiratory chain complex III assembly and ATP production.

Confocal microscopy files and tracking data to accompany the paper.

The habenula is an evolutionarily conserved structure of the vertebrate brain that is essential for behavioural flexibility and mood control. It is spontaneously active and is able to access diverse states when the animal is exposed to sensory stimuli or reward. Here we analyze two-photon calcium imaging time-series of the habenula of larval zebrafish and find that percolation occurs, indicating the presence of long-range spatial correlations within each side of the habenula, with percolation occurring independently in each side. On the other hand, the analysis of neuronal avalanches suggests that the system is subcritical, implying that the flexibility in its dynamics may result from other dynamical processes.

Background Optical silencing of activity provides a way to test the necessity of neurons in behaviour. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown to potently inhibit activity in cultured mammalian neurons and in Drosophila. Here, we test the usefulness of these channels in larval zebrafish, using spontaneous coiling behaviour as the assay. Results When the GtACRs were expressed in spinal neurons of embryonic zebrafish and actuated with blue or green light, spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 μW/mm2 of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required slightly stronger illumination. No inhibition was seen in non-expressing siblings. After light offset, the movement of GtACR-expressing fish increased, which suggested that termination of light-induced neural inhibition may lead to activation. Consistent with this, two-photon imaging of spinal neurons showed that blue light inhibited spontaneous activity in spinal neurons of GtACR1-expressing fish, and that the level of intracellular calcium increased following light offset. Conclusions These results show that GtACR1 and GtACR2 can be used to optically inhibit neurons in larval zebrafish with high efficiency. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property can provide insight into the effects of transiently stimulating a circuit. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0430-2) contains supplementary material, which is available to authorized users.

Previous studies have shown that d-amphetamine, a dopamine (DA) indirect agonist, alters operant responding with respect to the behavior maintained on a differential reinforcement of low-rate (DRL) schedule of reinforcement. These behavioral changes have been presumed to result from drug-induced hyperdopaminergia that leads to activation of DA receptors. This study investigated the acute dose effects of DA receptor subtype-selective agonists on the performance of DRL 10-sec behavior by rats. SKF38393 (a D1 receptor agonist) and quinpirole (a D2/D3 receptor agonist) were able to dose-dependently disrupt DRL 10-sec behavior by decreasing the total responses, the non-reinforced responses, and the peak rate of response. Bromocriptine (a D2/D3 receptor agonist) produced a significantly different pattern of behavioral changes when examined during two distinct time phases (15 min and 3 hr after the drug injection). DRL responding was only altered at higher doses of bromocriptine in the second phase as indicated by decreasing reinforced responses and peak rate, together with an increase of burst responses. In contrast to the D1 and D2/D3 receptor agonists, PD168077 (a D4 receptor agonist) did not affect DRL 10-sec behavior. None of these tested drugs affected DRL 10-sec behavior in a manner similar to that of d-amphetamine. These findings show that there are differential effects on the performance of DRL 10-sec behavior when drugs are able to preferentially activate D1, D2/D3 and D4 receptors, supporting the assertion that there is functional heterogeneity of the DA receptor subtypes.

In behavioral analysis, optical electrical silencing provides a way to test neuronal necessity. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown—in neuronal culture and in Drosophila—to inhibit neurons potently. Here, we test the usefulness of these channels in zebrafish. When the GtACRs were expressed in motor neurons and actuated with blue or green light, fish spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 µW/mm2 of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required stronger illumination. After light offset, GtACR-expressing fish movement increased; this suggested that termination of light-induced neural inhibition may lead to depolarization. Consistent with this, two-photon imaging of spinal neurons showed that intracellular calcium also increased following light offset. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property may help provide insight into the effects of stimulating a circuit transiently. These results show that GtACR1 and GtACR2 can be used to optically inhibit neurons in zebrafish, and thus to test neural circuit necessity.

The effect of the parapineal lesion on habenula response to blue light. a Visualization of the parapineal (yellow arrow), which is located adjacent to the left habenula and innervates the dorsal neuropil. b, c Two-photon lesioning of the parapineal. b Before lesioning. c After lesioning, which led to formation of a bubble (arrow). d, e Habenula cells segmented from five fish, overlaid on top of each other, showing responses before and after the lesion. Cells responding to light ON are shown in blue and to OFF in pink. f, g Heatmaps of the habenula cells, in the five fish, responding to light ON and OFF before (f) and after (g) lesioning the parapineal. Horizontal black lines divide data from different fish. h Percentage of cells showing ON and OFF responses before and after parapineal lesioning. i, j Histogram showing distribution of mean intensity in habenula neurons during light ON (e) and OFF (f) before and after lesion. Insets show cumulative distribution from all fish. P values and test statistic (D) were obtained using the Kolmogorov–Smirnov test. pp parapineal, lHb left habenula, rHb right habenula, cv circumventricular organ, a anterior, p posterior. Scale bar = 25 μm. (PDF 417 kb)

Examples of signals that were excluded from visualization of K-means clusters. a–e Pixels showing stimulus-independent activity in the thalamus, at five different focal planes. Pixels are colored according to the traces in panel f. For clarity, these signals were excluded from the visualization of clusters representing light-evoked activity shown in Fig. 4a–e. g Stimulus-independent activity in the habenula. Pixels are colored according to the traces in panel h. For clarity, these signals were excluded from the visualization of clusters representing light-evoked activity shown in Fig. 1e–f. f, h Cluster centers that did not represent light-evoked activity in the thalamus and habenula, obtained by running K-means on the time series of pixels in panel a–e and g. Th Thalamus, lHb left habenula, rHb right habenula, a anterior, p posterior. (PDF 404 kb)

The ability to control when to start an action and when to stop is crucial in human and animal behavior. A failure to suppress premature behavior or to carry out an action in a timely manner is commonly seen in several neuropsychological disorders. Despite the phenomenon, the exact neural mechanisms underlying this timing impulsivity remain to be elucidated. Systemic injection of d-amphetamine (AMP) has been shown to disrupt rat's performance in the differential reinforcement of low-rate (DRL) task that requires both optimal timing and proper impulsive control as measured by peak time and non-reinforced responses, respectively. By directly infusing selective D1 or D2 receptor antagonists (SCH23390 and raclopride, respectively) into three brain areas, we aimed to uncover which brain regions and which dopamine receptor subtypes are involved in counteracting the rat's deficit of DRL performance induced by the systemic injection of AMP. We found that D1, but not D2 receptors in the dorsal hippocampus (dHIP) and nucleus accumbens (NAC) played an important role in impulsive control as well as in timing. In the medial prefrontal cortex (mPFC), both D1 and D2 receptors played an equal role in impulsive control, but only mPFC D1 was critical in the control of timing. Together, our data revealed a regional-dependent and dopamine receptor subtype specific effect across each region tested in the mesocorticolimbic circuits on the deleterious effect of AMP in the DRL task. The current findings further advance our understanding of the neurobehavioral mechanisms involved in timing impulsivity.

Interval timing within the seconds-to-minutes range involves the interaction of the prefrontal cortex and basal ganglia via dopaminergic–glutamatergic pathways. Because the secreted protein brain-derived neurotrophic factor (BDNF) is able to modulate dopamine release as well as glutamatergic activity, we hypothesized that BDNF may be important for these timing mechanisms. Recently, the calcium-responsive transcription factor (CaRF) was identified as an important modulator of BDNF expression in the cerebral cortex. In this study, a strain of Carf knockout mice was evaluated for their ability to acquire the ‘Start’ and ‘Stop’ response thresholds under sequential and simultaneous training conditions, using multiple (15-second and 45-second) or single (30-second) target durations in the peak-interval procedure. Both Carf+/− and Carf−/− mice were impaired in their ability to acquire timed response thresholds relative to Carf+/+ mice. Additionally, control mice given microinjections of BDNF antisense oligodeoxynucleotide to inhibit protein expression in the prefrontal cortex showed timing impairments during acquisition similar to Carf mice. Together, these results suggest that the inhibitory processes required to update response thresholds and exert temporal control of behavior during acquisition may be dependent on CaRF regulation of genes including Bdnf in cortico-striatal circuits.

BackgroundNeural activity in the vertebrate habenula is affected by changes in ambient illumination. The nucleus that links photoreceptors with the habenula is not well characterized. Here, we describe the location, inputs and potential function of this nucleus in larval zebrafish.ResultsHigh-speed calcium imaging shows that onset and offset of light evokes a rapid response in the dorsal left neuropil of the habenula, indicating preferential targeting of this neuropil by afferents mediating response to change in irradiance. Injection of a lipophilic dye into this neuropil led to bilateral labeling of a nucleus in the anterior thalamus that responds to onset and offset of light, and that receives innervation from the retina and pineal organ. Lesioning the neuropil of this thalamic nucleus reduced the habenula response to light. Optogenetic stimulation of the thalamus with channelrhodopsin-2 caused depolarization in the habenula, while manipulation with anion channelrhodopsins inhibited habenula response to light and disrupted climbing and diving that is evoked by irradiance change.ConclusionsA nucleus in the anterior thalamus of larval zebrafish innervates the dorsal left habenula. This nucleus receives input from the retina and pineal, responds to increase and decrease in irradiance, enables habenula responses to change in irradiance, and may function in light-evoked vertical migration.

Existing evidence suggests that interval timing, processing of temporal information in the hundredth of milliseconds-to-minutes range, recruits broad brain regions such as cortico-striatal circuits via dopaminergic-glutamatergic pathways. In this review, we summarize recent findings on the neurobiological basis of interval timing with a special focus on dopaminergic modulations of temporal information. Two properties of interval timing - accuracy and precision - are used to examine recent results from manipulations of dopaminergic signaling and the resulting distortions in temporal processing. Fil: Agostino, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina Fil: Cheng, Ruey-Kuang. Institute Of Molecular And Cellular Biology A*star; Singapur

Serotonergic neurons have been implicated in a broad range of processes, but the principles underlying their effects remain a puzzle. Here, we ask how these neurons influence the tendency of larval zebrafish to swim in the light and avoid regions of darkness. Pharmacological inhibition of serotonin synthesis reduces dark avoidance, indicating an involvement of this neuromodulator. Calcium imaging of tph2-expressing cells demonstrates that a rostral subset of dorsal raphe serotonergic neurons fire continuously while the animal is in darkness, but are inhibited in the light. Optogenetic manipulation of tph2 neurons by channelrhodopsin or halorhodopsin expression modifies preference, confirming a role for these neurons. In particular, these results suggest that fish prefer swimming in conditions that elicits lower activity in tph2 serotonergic neurons in the rostral raphe.

Although fear-producing treatments (e.g., electric shock) and pleasure-inducing treatments (e.g., methamphetamine) have different emotional valences, they both produce physiological arousal and lead to effects on timing and time perception that have been interpreted as reflecting an increase in speed of an internal clock. In this commentary, we review the results reported by Fayolle et al. (2015):Behav. Process., 120, 135–140) and Meck (1983: J. Exp. Psychol. Anim. Behav. Process., 9, 171–201) using electric shock and by Maricq et al. (1981: J. Exp. Psychol. Anim. Behav. Process., 7, 18–30) using methamphetamine in a duration-bisection procedure across multiple duration ranges. The psychometric functions obtained from this procedure relate the proportion ‘long’ responses to signal durations spaced between a pair of ‘short’ and ‘long’ anchor durations. Horizontal shifts in these functions can be described in terms of attention or arousal processes depending upon whether they are a fixed number of seconds independent of the timed durations (additive) or proportional to the durations being timed (multiplicative). Multiplicative effects are thought to result from a change in clock speed that is regulated by dopamine activity in the medial prefrontal cortex. These dopaminergic effects are discussed within the context of the striatal beat frequency model of interval timing (Matell & Meck, 2004:Cogn. Brain Res.,21, 139–170) and clinical implications for the effects of emotional reactivity on temporal cognition (Parker et al., 2013:Front. Integr. Neurosci., 7, 75).

The dopamine transporter (DAT) is the major regulator of the spatial and temporal resolution of dopaminergic neurotransmission in the brain. Hyperdopaminergic mice with DAT gene deletions were evaluated for their ability to perform duration discriminations in the seconds-to-minutes range. DAT -/- mice were unable to demonstrate temporal control of behavior in either fixed-interval or peak-interval timing procedures, whereas DAT +/- mice were similar to DAT +/+ mice under normal conditions. Low to moderate-dose methamphetamine (MAP) challenges indicated that DAT +/- mice were less sensitive to the clock-speed enhancing effects of MAP compared with DAT +/+ mice. In contrast, DAT +/- mice were more vulnerable than DAT +/+ mice to the disruptive effects of MAP at high doses as revealed by the elevation of response rate in the right hand tail of the Gaussian-shaped timing functions. Moreover, this treatment made DAT +/- mice functionally equivalent to DAT -/- mice in terms of the loss of temporal control. Taken together, these results demonstrate the importance of dopaminergic control of interval timing in cortico-striatal circuits and the potential link of timing dysfunctions to schizophrenia and drug abuse.

Aged male rats at 10, 20, and 30 mo of age were trained on a 2.0 vs. 8.0-s duration bisection procedure using both auditory and visual signals and were then tested with visual signal durations in which the spacing of the intermediate signal durations was held constant as the short (S) and long (L) anchor durations were moved progressively closer to each other across blocks of sessions. Auditory clicks also preceded some trials in order to determine the potential effects of arousal and/or distraction on the timing of visual signals. The consequences of aging, reducing the S:L ratio, and auditory clicks were to increase the likelihood of observing reversals in response classifications around the geometric mean of the anchor durations. Taken together, these results suggest that the bisection "reversal effect" is dependent upon the calculation of the subjective mid-point between the two anchor durations and the differential setting of response thresholds around this category boundary as a function of temporal context.

We all have a sense of time. Yet, there are no sensory receptors specifically dedicated for perceiving time. It is an almost uniquely intangible sensation: we cannot see time in the way that we see color, shape, or even location. So how is time represented in the brain? We explore the neural substrates of metrical representations of time such as duration estimation (explicit timing) or temporal expectation (implicit timing). Basal ganglia (BG), supplementary motor area, cerebellum, and prefrontal cortex have all been linked to the explicit estimation of duration. However, each region may have a functionally discrete role and will be differentially implicated depending upon task context. Among these, the dorsal striatum of the BG and, more specifically, its ascending nigrostriatal dopaminergic pathway seems to be the most crucial of these regions, as shown by converging functional neuroimaging, neuropsychological, and psychopharmacological investigations in humans, as well as lesion and pharmacological studies in animals. Moreover, neuronal firing rates in both striatal and interconnected frontal areas vary as a function of duration, suggesting a neurophysiological mechanism for the representation of time in the brain, with the excitatory-inhibitory balance of interactions among distinct subtypes of striatal neuron serving to fine-tune temporal accuracy and precision.

Understanding the role of sleep in memory consolidation is an important topic in cognitive neuroscience because it could help us understand the functions of sleep as well as how memory is being consolidated off-line during sleep. Here we review recent studies that focus on the neurophysiological changes that occur during sleep and how these sleep-dependent changes could benefit memory consolidation. In particular, rapid eye movement (REM) sleep provides a preferential time window for memory consolidation across molecular, cellular and synaptic levels while slow-wave sleep (SWS) provides a second time window for memory consolidation at the neural-network level. It is proposed that high-frequency oscillations in the hippocampus (e.g., ripple events) serve as an endogenous trigger for long-term potentiation that plays a critical role in connecting a variety of sleep-dependent neurophysiological processes. We further suggest that repeated alternation of SWS and REM are needed in order to get a “good night’s sleep”. New findings from studies examining how prenatal choline supplementation produces organizational changes in hippocampal function, may provide a useful model for validating the importance of hippocampal ripple events in facilitating memory consolidation during both REM sleep and SWS. These data support our general conclusion that the types of neural plasticity supported by prenatal choline supplementation and sleep are vital for cognitive health, because they produce a host of neurophysiological changes, some of which are also important for memory consolidation.

Choline supplementation of the maternal diet has long-term facilitative effects on spatial and temporal memory processes in the offspring. To further delineate the impact of early nutritional status on brain and behavior, we examined effects of prenatal-choline availability on hippocampal oscillatory frequency bands in 12 month-old male and female rats. Adult offspring of time-pregnant dams that were given a deficient level of choline (DEF=0.0 g/kg), sufficient choline (CON=1.1 g/kg) or supplemental choline (SUP=3.5 g/kg) in their chow during embryonic days (ED) 12-17 were implanted with an electroencephalograph (EEG) electrode in the hippocampal dentate gyrus in combination with an electromyograph (EMG) electrode patch implanted in the nuchal muscle. Five consecutive 8-h recording sessions revealed differential patterns of EEG activity as a function of awake, slow-wave sleep (SWS) and rapid-eye movement (REM) sleep states and prenatal choline status. The main finding was that SUP rats displayed increased power levels of gamma (30-100 Hz) band oscillations during all phases of the sleep/wake cycle. These findings are discussed within the context of a general review of neuronal oscillations (e.g., delta, theta, and gamma bands) and synchronization across multiple brain regions in relation to sleep-dependent memory consolidation in the hippocampus.

The indirect dopamine (DA) agonist methamphetamine (MAP) is evaluated in terms of its impact on the speed of temporal processing across multiple time scales involving both interval and circadian timing. Behavioral and neuropharmacological aspects of drug abuse, habit formation, neurotoxicity, and the potential links between interval and circadian timing are reviewed. The view that emerges is one in which the full spectrum of MAP-induced effects on timing and time perception is both complex and dynamic in as much as it involves DA-glutamate interactions and gene expression within cortico-striatal circuitry spanning oscillation periods ranging from milliseconds to multiple hours. The conclusion is that the psychostimulant properties of MAP are very much embedded within the context of temporal prediction and the anticipation of reward.

Choline supplementation of the maternal diet has a long-term facilitative effect on timing and temporal memory of the offspring. To further delineate the impact of early nutritional status on interval timing, we examined effects of prenatal choline supplementation on the temporal sensitivity of adult (6 months) male rats. Rats that were given sufficient choline in their chow (CON: 1.1 g/kg) or supplemental choline added to their drinking water (SUP: 3.5 g/kg) during embryonic days (ED) 12-17 were trained with a peak-interval procedure that was shifted among 75%, 50%, and 25% probabilities of reinforcement with transitions from 18 s-->36 s-->72 s temporal criteria. Prenatal choline supplementation systematically sharpened interval timing functions by reducing the associative/non-temporal response enhancing effects of reinforcement probability on the Start response threshold, thereby reducing non-scalar sources of variance in the left-hand portion of the Gaussian-shaped response functions. No effect was observed for the Stop response threshold as a function of any of these manipulations. In addition, independence of peak time and peak rate was demonstrated as a function of reinforcement probability for both prenatal choline-supplemented and control rats. Overall, these results suggest that prenatal choline supplementation facilitates timing by reducing impulsive responding early in the interval, thereby improving the superimposition of peak functions for different temporal criteria.

Methamphetamine intoxication has long-term consequences on dopaminergic function and corticostriatal-mediated behaviors in humans and other animals. In order to determine the potential impact on timing and temporal memory, we examined methamphetamine dose regimens that have been linked to neurotoxicity in adult (8 months) male rats. Rats that were given repetitive, high-dose methamphetamine (3.0 mg/kg ip × 4 injections/2 h) or saline injections were trained on a 2-s vs 8-s bisection procedure using auditory and visual signal durations. Following the high-dose regimen, baseline timing performance was reestablished prior to the rats' receiving reversal training in which the spatial/temporal mapping of the anchor durations (2 s and 8 s) to response options (left or right lever) was reversed. Low-dose methamphetamine (0.5 mg/kg ip) or saline injections were subsequently used to evaluate the effectiveness of the neurotoxic doses in terms of modifying the horizontal leftward shifts associated with increases in clock speed. Overall, the results indicate that MAP intoxication leads to reduced auditory/visual differences in clock speed, deficits in reversal learning, distortions in temporal memory, and lowered dopaminergic regulation of clock speed consistent with damage to prefrontal cortex and corticostriatal circuitry.

The present study examined the clock-speed modulating effects of acute cocaine administration in groups of male rats that received different amounts of baseline training on a 36-s peak-interval procedure prior to initial drug injection. After injection of cocaine (10, 15, or 20mg/kg, ip), rats that had received a minimal amount of training (e.g.,or=30 sessions) prior to drug administration displayed a horizontal leftward shift in their timing functions indicating that the speed of the internal clock was increased. In contrast, rats that had received an extended amount of training (e.g.,or=180 sessions) prior to cocaine (15 mg/kg, ip) administration did not produce this "classic" curve-shift effect, but instead displayed a general disruption of temporal control following drug administration. Importantly, when co-administered with a behaviorally ineffective dose of ketamine (10mg/kg, ip) the ability of cocaine to modulate clock speed in rats receiving extended training was restored. A glutamate "lock/unlock" hypothesis is used to explain the observed dopamine-glutamate interactions as a function of timing behaviors becoming learned habits.

Drugs that modulate the effective level of dopamine (DA) in cortico-striatal circuits have been shown to alter the perception of time in the seconds-to-minutes range. How this relationship changes as a function of repeated experience with the reinforcement contingencies and the gradual adaptation of the underlying neural circuits remains unclear.The present study examined the clock-speed enhancing effects of methamphetamine (MAP 0.5 or 1.0 mg/kg, ip) in groups of rats that received different levels of baseline training (20, 60, or 120 sessions) on a 50-s peak-interval (PI) procedure before initial drug administration.A curvilinear relationship was observed such that rats that received either minimal or intermediate levels of training (or=60 sessions) displayed dose- x training-related horizontal leftward shifts in their timing functions, suggesting that the speed of the internal clock was increased. In contrast, rats that had received an extended level of training (or=120 sessions) did not show this "classic" DA agonist curve-shift effect, but instead displayed a dose-dependent disruption of temporal control after MAP administration. A transition from DA-sensitive to DA-insensitive mechanisms is proposed to account for the loss of control of clock speed, as timing behaviors associated with the PI procedure gradually become learned habits through the strengthening of DA-glutamate connections.

The present study examined the effects of photoperiod on spatial and temporal memory in adult Sprague-Dawley rats that were conceived and reared in different day lengths, i.e., short day (SD-8:16 light/dark) and long day (LD-16:8 light/dark). Both male and female LD rats demonstrated increased spatial memory capacity as evidenced by a lower number of choices to criterion in a 12-arm radial maze task relative to the performance of SD rats. SD rats also demonstrated a distortion in the content of temporal memory as evidenced by a proportional rightward shift in the 20 and 60 s temporal criteria trained using the peak-interval procedure that is consistent with reduced cholinergic function. The conclusion is that both spatial and temporal memory are sensitive to photoperiod variation in laboratory rats in a manner similar to that previously observed for reproductive behaviour.

The present experiment examined the effects of cocaine (0.0 and 15 mg/kg, i.p.) and ketamine (0.0, 10.0 and 15 mg/kg, i.p.) on timing behavior using a 12-s differential reinforcement of low rates (DRL) procedure and a 2- vs. 8-s bisection procedure in rats. DRL (time production) and bisection (time perception) procedures are sensitive to effects of dopaminergic drugs and provide an assessment of the accuracy and precision of interval timing as well as the subject's level of impulsivity. When administered to rats trained on either the DRL or the bisection procedure, cocaine shifted the psychophysical functions leftward relative to control conditions. In contrast, ketamine produced no change in the temporal control of behavior on either procedure. These differential effects of cocaine and ketamine are consistent with previous reports suggesting that dopamine levels in the dorsal striatum, but not in prefrontal cortex, ventral striatum or hippocampal regions, are crucial for the regulation of the speed of an internal clock.

We previously showed that prenatal choline supplementation could increase the precision of timing and temporal memory and facilitate simultaneous temporal processing in mature and aged rats. In the present study, we investigated the ability of adult rats to selectively control the reinforcement-induced resetting of an internal clock as a function of prenatal drug treatments designed to affect the α7 nicotinic acetylcholine receptor (α7 nAChR). Male Sprague-Dawley rats were exposed to prenatal choline (CHO), nicotine (NIC), methyllycaconitine (MLA), choline + nicotine (CHO + NIC), choline + nicotine + methyllycaconitine (CHO + NIC + MLA), or a control treatment (CON). Beginning at 4-mo-of-age, rats were trained on a peak-interval timing procedure in which food was available at 10-, 30-, and 90-sec criterion durations. At steady-state performance there were no differences in timing accuracy, precision, or resetting among the CON, MLA, and CHO + NIC + MLA treatments. It was observed that the CHO and NIC treatments produced a small, but significant increase in timing precision, but no change in accuracy or resetting. In contrast, the CHO + NIC prenatal treatment produced a dramatic increase in timing precision and selective control of the resetting mechanism with no change in overall timing accuracy. The synergistic effect of combining prenatal CHO and NIC treatments suggests an organizational change in α7 nAChR function that is dependent upon a combination of selective and nonselective nAChR stimulation during early development.

Learning how to actively avoid a predictable threat involves two steps: recognizing the cue that predicts upcoming punishment and learning a behavioral response that will lead to avoidance. In zebrafish, ventral habenula (vHb) neurons have been proposed to participate in both steps by encoding the expected aversiveness of a stimulus. vHb neurons increase their firing rate as expectation of punishment grows but reduce their activity as avoidance learning occurs. This leads to changes in the activity of raphe neurons, which are downstream of the vHb, during learning. How vHb activity is regulated is not known. Here, we ask whether the neuromodulator Kisspeptin1, which is expressed in the ventral habenula together with its receptor, could be involved. Kiss1 mutants were generated with CRISPR/Cas9 using guide RNAs targeted to the signal sequence. Mutants, which have a stop codon upstream of the active Kisspeptin1 peptide, have a deficiency in learning to avoid a shock that is predicted by light. Electrophysiology indicates that Kisspeptin1 has a concentration-dependent effect on vHb neurons: depolarizing at low concentrations and hyperpolarizing at high concentrations. Two-photon calcium imaging shows that mutants have reduced raphe response to shock. These data are consistent with the hypothesis that Kisspeptin1 modulates habenula neurons as the fish learns to cope with a threat. Learning a behavioral strategy to overcome a stressor may thus be accompanied by physiological change in the habenula, mediated by intrinsic neuromodulation. ASTAR (Agency for Sci., Tech. and Research, S’pore) MOE (Min. of Education, S’pore) Published version

Time-based decision-making in peak-interval timing procedures involves the setting of response thresholds for the initiation (“Start”) and termination (“Stop”) of a response sequence that is centered on a target duration. Using intracerebral infusions of the protein synthesis inhibitor anisomycin, we report that the acquisition of the “Start” response depends on normal functioning (including protein synthesis) in the dorsal striatum (DS), but not the ventral striatum (VS). Conversely, disruption of the VS, but not the DS, impairs the acquisition of the “Stop” response. We hypothesize that the dorsal and ventral regions of the striatum function as a competitive neural network that encodes the temporal boundaries marking the beginning and end of a timed response sequence.