In search of the locus coeruleus: guidelines for identifying anatomical boundaries and electrophysiological properties of the blue spot in mice, fish, finches, and beyond.

Our understanding of human brain function can be greatly aided by studying analogous brain structures in other organisms. One brain structure with neurochemical and anatomical homology throughout vertebrate species is the locus coeruleus (LC), a small collection of norepinephrine (NE)-containing neurons in the brainstem that project throughout the central nervous system. The LC is involved in nearly every aspect of brain function, including arousal and learning, which has been extensively examined in rats and nonhuman primates using single-unit recordings. Recent work has expanded into putative LC single-unit electrophysiological recordings in a nonmodel species, the zebra finch. Given the importance of correctly identifying analogous structures as research efforts expand to other vertebrates, we suggest adoption of consensus anatomical and electrophysiological guidelines for identifying LC neurons across species when evaluating brainstem single-unit spiking or calcium imaging. Such consensus criteria will allow for confident cross-species understanding of the roles of the LC in brain function and behavior.

Brightness illusions drive a neuronal response in the primary visual cortex under top-down modulation.

Brightness illusions are a powerful tool in studying vision, yet their neural correlates are poorly understood. Based on a human paradigm, we presented illusory drifting gratings to mice. Primary visual cortex (V1) neurons responded to illusory gratings, matching their direction selectivity for real gratings, and they tracked the spatial phase offset between illusory and real gratings. Illusion responses were delayed compared to real gratings, in line with the theory that processing illusions requires feedback from higher visual areas (HVAs). We provide support for this theory by showing a reduced V1 response to illusions, but not real gratings, following HVAs optogenetic inhibition. Finally, we used the pupil response (PR) as an indirect perceptual report and showed that the mouse PR matches the human PR to perceived luminance changes. Our findings resolve debates over whether V1 neurons are involved in processing illusions and highlight the involvement of feedback from HVAs.

Brightness illusions evoke pupil constriction preceded by a primary visual cortex response in rats.

The mind affects the body via central nervous system (CNS) control of the autonomic nervous system (ANS). In humans, one striking illustration of the "mind-body" connection is that illusions, subjectively perceived as bright, drive pupil constriction. The CNS network driving this pupil response is unknown and requires an animal model for investigation. However, the pupil response to this illusion has long been thought to occur only in humans. Here, we report that the same brightness illusion that evokes pupil constriction in humans also does so in rats. We surveyed the role of most of rat cortex in this "mind-body" connection by recording cortex-wide EEG. These recordings revealed that, compared to a luminance-matched control stimulus, the illusion of brightness for a specific stimulus color and size, evoked a larger response in primary visual cortex (V1) and not in secondary visual, parietal, or frontal cortex. The response preceded pupil constriction suggesting a potential causal role of V1 on the pupil. Our results provide evidence that this "mind-body" connection is not confined to humans and that V1 may be part of a mammalian CNS network for bodily reactions to illusions.

Distinct ensembles in the noradrenergic locus coeruleus are associated with diverse cortical states.

The noradrenergic locus coeruleus (LC) is a controller of brain and behavioral states. Activating LC neurons en masse by electrical or optogenetic stimulation promotes a stereotypical "activated" cortical state of high-frequency oscillations. However, it has been recently reported that spontaneous activity of LC cell pairs has sparse yet structured time-averaged cross-correlations, which is unlike the highly synchronous neuronal activity evoked by stimulation. Therefore, LC population activity could consist of distinct multicell ensembles each with unique temporal evolution of activity. We used nonnegative matrix factorization (NMF) to analyze large populations of simultaneously recorded LC single units in the rat LC. NMF identified ensembles of spontaneously coactive LC neurons and their activation time courses. Since LC neurons selectively project to specific forebrain regions, we hypothesized that distinct ensembles activate during different cortical states. To test this hypothesis, we calculated band-limited power and spectrograms of local field potentials in cortical area 24a aligned to spontaneous activations of distinct LC ensembles. A diversity of state modulations occurred around activation of different LC ensembles, including a typical activated state with increased high-frequency power as well as other states including decreased high-frequency power. Thus­in contrast to the stereotypical activated brain state evoked by en masse LC stimulation­spontaneous activation of distinct LC ensembles is associated with a multitude of cortical states.

Three Water Restriction Schedules Used in Rodent Behavioral Tasks Transiently Impair Growth and Differentially Evoke a Stress Hormone Response without Causing Dehydration.

Water restriction is commonly used to motivate rodents to perform behavioral tasks; however, its effects on hydration and stress hormone levels are unknown. Here, we report daily body weight and bi-weekly packed red blood cell volume and corticosterone (CORT) in adult male rats across 80 days for three commonly used water restriction schedules. We also assessed renal adaptation to water restriction using postmortem histologic evaluation of renal medulla. A control group received ad libitum water. After one week of water restriction, rats on all restriction schedules resumed similar levels of growth relative to the control group. Normal hydration was observed, and water restriction did not drive renal adaptation. An intermittent restriction schedule was associated with an increase in CORT relative to the control group. However, intermittent restriction evokes a stress response which could affect behavioral and neurobiological results. Our results also suggest that stable motivation in behavioral tasks may only be achieved after one week of restriction.

Synchronous spiking associated with prefrontal high γ oscillations evokes a 5-Hz rhythmic modulation of spiking in locus coeruleus.

The brainstem noradrenergic locus coeruleus (LC) is reciprocally connected with the prefrontal cortex (PFC). Coupling between LC spiking and the depolarizing phase of slow (1-2 Hz) waves in PFC field potentials during sleep and anesthesia suggests that LC drives cortical state transition. Reciprocal LC-PFC connectivity should also allow interactions in the opposing (top-down) direction, but prior work has only studied prefrontal control over LC activity using electrical or optogenetic stimulation. Here, we describe the physiological characteristics of spontaneously occurring top-down LC-PFC interactions. We recorded LC multiunit activity (MUA) simultaneously with PFC single-unit and local field potential (LFP) activity in urethane-anesthetized rats. We observed cross-regional coupling between the phase of 5-Hz oscillations in LC-MUA and the power of PFC LFP 60-200 Hz high γ (hγ). Transient increases in PFC hγ power preceded peaks in the 5-Hz LC-MUA oscillation. Analysis of cross-regional transfer entropy demonstrated that the PFC hγ transients were predictive of a transient increase in LC-MUA. An ∼29 ms delay between these signals was consistent with the conduction velocity from the PFC to the LC. Finally, we showed that PFC hγ transients are associated with synchronized spiking of a subset (27%) of PFC single units. Our data suggest that PFC hγ transients may indicate the timing of the top-down excitatory input to LC, at least under conditions when LC neuronal population activity fluctuates rhythmically at 5 Hz. Synchronized PFC neuronal spiking that occurs during hγ transients may provide a previously unknown mode of top-down control over the LC.NEW & NOTEWORTHY The prefrontal cortex (PFC) is thought to control activity in the noradrenergic locus coeruleus (LC). Prior anatomical and prefrontal stimulation studies demonstrated the potential for PFC-LC interactions; however, it is unknown what types of PFC activity affect the LC. Here, we show that transient increases in PFC high γ power and associated changes in PFC unit-pair synchrony are a potential sign of top-down control over the LC.

Redefining Noradrenergic Neuromodulation of Behavior: Impacts of a Modular Locus Coeruleus Architecture.

The locus coeruleus (LC) is a seemingly singular and compact neuromodulatory nucleus that is a prominent component of disparate theories of brain function due to its broad noradrenergic projections throughout the CNS. As a diffuse neuromodulatory system, noradrenaline affects learning and decision making, control of sleep and wakefulness, sensory salience including pain, and the physiology of correlated forebrain activity (ensembles and networks) and brain hemodynamic responses. However, our understanding of the LC is undergoing a dramatic shift due to the application of state-of-the-art methods that reveal a nucleus of many modules that provide targeted neuromodulation. Here, we review the evidence supporting a modular LC based on multiple levels of observation (developmental, genetic, molecular, anatomical, and neurophysiological). We suggest that the concept of the LC as a singular nucleus and, alongside it, the role of the LC in diverse theories of brain function must be reconsidered.

Noradrenergic ensemble-based modulation of cognition over multiple timescales.

Cognition fluctuates over relatively faster and slower timescales. This is enabled by dynamic interactions among cortical neurons over similarly diverse temporal and spatial scales. Fast and slow cognitive processes, such as reorienting to surprising stimuli or using experience to develop a behavioral strategy, are also sensitive to neuromodulation by the diffusely-projecting brainstem noradrenergic nucleus, Locus Coeruleus. However, while a dynamic, multi-scale cortical ensemble code influences cognition over multiple timescales, it is unknown to what extent LC neuronal activity operates in this regime. An ensemble code within the LC may permit an interface with cortical ensembles allowing noradrenergic modulation of fast and slow cognitive processes. Alternatively, given that LC neurons are thought to spike synchronously, there may be a mismatch between LC and cortical neuronal codes that constrains how the noradrenergic system can influence cognition. We review new evidence that clearly demonstrates cell type-specific ensemble activity within LC occurring over a range of behaviorally-relevant timescales. We also review recent studies demonstrating that sub-sets of LC neurons modulate specific forebrain targets to control behavior. A critical target for future research is to study the temporal dynamics of projection-specific LC ensembles, their interactions with cortical networks, and the relevance of multi-scale coerular-cortical dynamics to behaviors over various timescales.

The Locus Coeruleus Is a Complex and Differentiated Neuromodulatory System.

Diffuse projections of locus coeruleus (LC) neurons and evidence of synchronous spiking have long been perceived as features of global neuromodulation. Recent studies demonstrated the possibility of targeted modulation by subsets of LC neurons. Non-global neuromodulation depends on target specificity and the differentiated spatiotemporal dynamics within LC. Here, we characterized interactions between 3,164 LC cell pairs in the rat LC under urethane anesthesia. Spike count correlations were near zero and only a small proportion of unit pairs had synchronized spontaneous (15%) or evoked (16%) discharge. We identified infra-slow (0.01-1 Hz) fluctuations of LC unit spike rate, which were also asynchronous across the population. Despite overall sparse population synchrony, we report the existence of LC ensembles and relate them to forebrain projection targets. We also show that spike waveform width was related to ensemble membership, propensity for synchronization, and interactions with cortex. Our findings suggest a partly differentiated and target-specific noradrenergic signal.

Atomoxetine accelerates attentional set shifting without affecting learning rate in the rat.

RATIONALE: Shifting to a new rule is a form of behavioral flexibility that is impaired in numerous psychiatric and neurological illnesses. Animal studies have revealed that this form of flexibility depends upon norepinephrine (NE) neurotransmission. Atomoxetine, a NE reuptake inhibitor, improves performance of humans in set shifting tasks. OBJECTIVE: Our objective was to validate its effects in a rodent set shifting task. METHODS: We tested the drug effect using an operant task that required a shift from a visual cue-guided behavior to a novel location-guided rule. RESULTS: A 1.0-mg/kg dose significantly accelerated rule shifting without affecting learning strategies, such as win-stay or lose-shift. Fitting behavioral performance with a learning function provided a measure of learning rate. CONCLUSION: This novel analysis revealed that atomoxetine accelerated shifting to the new rule without affecting learning rate.

Unilateral electrical stimulation of rat locus coeruleus elicits bilateral response of norepinephrine neurons and sustained activation of medial prefrontal cortex.

The brain stem nucleus locus coeruleus (LC) is thought to modulate cortical excitability by norepinephrine (NE) release in LC forebrain targets. The effects of LC burst discharge, typically evoked by a strong excitatory input, on cortical ongoing activity are poorly understood. To address this question, we combined direct electrical stimulation of LC (LC-DES) with extracellular recording in LC and medial prefrontal cortex (mPFC), an important cortical target of LC. LC-DES consisting of single pulses (0.1-0.5 ms, 0.01-0.05 mA) or pulse trains (20-50 Hz, 50-200 ms) evoked short-latency excitatory and inhibitory LC responses bilaterally as well as a delayed rebound excitation occurring ∼100 ms after stimulation offset. The pulse trains, but not single pulses, reliably elicited mPFC activity change, which was proportional to the stimulation strength. The firing rate of ∼50% of mPFC units was significantly modulated by the strongest LC-DES. Responses of mPFC putative pyramidal neurons included fast (∼100 ms), transient (∼100-200 ms) inhibition (10% of units) or excitation (13%) and delayed (∼500 ms), sustained (∼1 s) excitation (26%). The sustained spiking resembled NE-dependent mPFC activity during the delay period of working memory tasks. Concurrently, the low-frequency (0.1-8 Hz) power of the local field potential (LFP) decreased and high-frequency (>20 Hz) power increased. Overall, the DES-induced LC firing pattern resembled the naturalistic biphasic response of LC-NE neurons to alerting stimuli and was associated with a shift in cortical state that may optimize processing of behaviorally relevant events.

Adolescent behavior and dopamine availability are uniquely sensitive to dietary omega-3 fatty acid deficiency.

BACKGROUND: Understanding the nature of environmental factors that contribute to behavioral health is critical for successful prevention strategies in individuals at risk for psychiatric disorders. These factors are typically experiential in nature, such as stress and urbanicity, but nutrition--in particular dietary deficiency of omega-3 polyunsaturated fatty acids (n-3 PUFAs)-has increasingly been implicated in the symptomatic onset of schizophrenia and mood disorders, which typically occurs during adolescence to early adulthood. Thus, adolescence might be the critical age range for the negative impact of diet as an environmental insult. METHODS: A rat model involving consecutive generations of n-3 PUFA deficiency was developed on the basis of the assumption that dietary trends toward decreased consumption of these fats began 4-5 decades ago when the parents of current adolescents were born. Behavioral performance in a wide range of tasks as well as markers of dopamine-related neurotransmission was compared in adolescents and adults fed n-3 PUFA adequate and deficient diets. RESULTS: In adolescents, dietary n-3 PUFA deficiency across consecutive generations produced a modality-selective and task-dependent impairment in cognitive and motivated behavior distinct from the deficits observed in adults. Although this dietary deficiency affected expression of dopamine-related proteins in both age groups in adolescents but not adults, there was an increase in tyrosine hydroxylase expression that was selective to the dorsal striatum. CONCLUSIONS: These data support a nutritional contribution to optimal cognitive and affective functioning in adolescents. Furthermore, they suggest that n-3 PUFA deficiency disrupts adolescent behaviors through enhanced dorsal striatal dopamine availability.

Distinct prestimulus and poststimulus activation of VTA neurons correlates with stimulus detection.

Dopamine neurons of the ventral tegmental area (VTA) signal the occurrence of a reward-predicting conditioned stimulus (CS) with a subsecond duration increase in post-CS firing rate. Important theories about reward-prediction error and reward expectancy have been informed by the substantial number of studies that have examined post-CS phasic VTA neuron activity. On the other hand, the role of VTA neurons in anticipation of a reward-predicting CS and analysis of prestimulus spike rate rarely has been studied. We recorded from the VTA in rats during the 3-choice reaction time task, which has a fixed-duration prestimulus period and a difficult-to-detect stimulus. Use of a stimulus that was difficult to detect led to behavioral errors, which allowed us to compare VTA activity between trials with correct and incorrect stimulus-guided choices. We found a sustained increase in firing rate of both putative dopamine and GABA neurons during the pre-CS period of correct and incorrect trials. The poststimulus phasic response, however, was absent on incorrect trials, suggesting that the stimulus-evoked phasic response of dopamine neurons may relate to stimulus detection. The prestimulus activation of VTA neurons may modulate cortical systems that represent internal states of stimulus expectation and provide a mechanism for dopamine neurotransmission to influence preparatory attention to an expected stimulus.

Preparatory attention relies on dynamic interactions between prelimbic cortex and anterior cingulate cortex.

An emerging view of prefrontal cortex (PFC) function is that multiple PFC areas process information in parallel, rather than as distinct modules. Two key functions assigned to the PFC are the regulation of top-down attention and stimulus-guided action. Electrophysiology and lesion studies indicate the involvement of both the anterior cingulate cortex (ACC) and prelimbic cortex (PL) in these functions. Little is known, however, about how these cortical regions interact. We recorded single unit spiking and local field potentials (LFPs) simultaneously in rodents during a sustained attention task and assessed interactions between the ACC and PL by measuring spike-LFP phase synchrony and LFP-LFP phase synchrony between these areas. We demonstrate that the magnitude of synchrony between the ACC and PL, before stimulus onset, predicts the subjects' behavioral choice after the stimulus. Furthermore, neurons switched from a state of beta synchrony during attention to a state of delta synchrony before the instrumental action. Our results indicate that multiple PFC areas interact during attention and that the same neurons may participate in segregated assemblies that support both attention and action.

The influence of NMDA and GABA(A) receptors and glutamic acid decarboxylase (GAD) activity on attention.

RATIONALE: Attention dysfunction is the hallmark of cognitive deficits associated with major psychiatric illnesses including schizophrenia. Cognitive deficits of schizophrenia have been attributed to reduced function of the N-methyl-D-aspartate (NMDA) receptor or reduced expression of the gamma-aminobutyric acid (GABA)-synthesizing enzyme glutamic acid decarboxylase-67, which presumably leads to attenuated neurotransmission at GABA(A) receptors. OBJECTIVE: The present study used a rodent model to compare the inhibition of NMDA and GABA(A) receptors, and GAD activity on attention. We tested the impact of inhibiting these proteins brain wide or in the anterior cingulate cortex (ACC), a prefrontal cortex region critical for attentional processing. METHODS: Rats were trained on the three choice serial reaction time task (3-CSRT), an attention test. The impact of systemic or intra-ACC injection of drugs on performance was measured in well-trained rats. RESULTS: Reducing GABA(A) receptor function within the ACC with the direct antagonist SR95531 (1 or 3 ng/side) or brain wide using systemic injection of the benzodiazepine inverse agonist FG7142 (5 mg/kg) impaired accuracy and increased omissions. Systemic or intra-ACC inhibition of NMDA receptors using MK-801 (at 3 mg/kg or 3 µg, respectively) also impaired performance. Inhibition of GAD with 3-mercaptopropionic acid, even at high doses, had no effect on 3-CSRT accuracy or omissions when administered systemically or within the ACC. CONCLUSIONS: These data demonstrate that, while tonic stimulation of NMDA and GABA(A) receptors within the ACC are critical for attentional performance, reduction in GAD activity may have little functional significance and is not indicative of reduced GABA neurotransmission.

Anterior cingulate neurons represent errors and preparatory attention within the same behavioral sequence.

The anterior cingulate cortex (ACC) has been implicated in both preparatory attention (i.e., selecting behaviorally relevant stimuli) and in detecting errors. We recorded from the rat ACC and medial prefrontal cortex (mPFC), which is functionally homologous to the primate dorsolateral PFC, during an attention task. The three-choice serial reaction time task requires a rat to orient toward and divide attention between three brief (300 ms duration) light stimuli presented in random order across nose poke holes in an operant chamber. In both the ACC and mPFC, we found that neural activity was related to the level of preparatory (precue) attention and subsequent correct or incorrect choice, in that the magnitude of the single units' response to the cue was lower on incorrect trials and was not different than baseline on unattended trials. This preparatory neural activity consisted of both excitatory and inhibitory phasic responses. The number of units responding to the cue was similarly graded, in that fewer units exhibited phasic responses to the cue on incorrect and unattended trials, compared with correct trials. Although preparatory activity was found in both the ACC and mPFC, activity after incorrect nose pokes, which may be related to error detection, were only observed in the ACC. Thus, during the same behavioral sequence, the ACC encodes both error-related events and preparatory attention, whereas the mPFC only participates in preparatory attention. The finding of substantial inhibitory activity during the preparatory period suggests a critical role for inhibition of pyramidal cells in PFC-mediated cognitive functions.