Light deprivation damages monoamine neurons and produces a depressive behavioral phenotype in rats.

Light is an important environmental factor for regulation of mood. There is a high frequency of seasonal affective disorder in high latitudes where light exposure is limited, and bright light therapy is a successful antidepressant treatment. We recently showed that rats kept for 6 weeks in constant darkness (DD) have anatomical and behavioral features similar to depressed patients, including dysregulation of circadian sleep-waking rhythms and impairment of the noradrenergic (NA)-locus coeruleus (LC) system. Here, we analyzed the cell viability of neural systems related to the pathophysiology of depression after DD, including NA-LC, serotoninergic-raphe nuclei and dopaminergic-ventral tegmental area neurons, and evaluated the depressive behavioral profile of light-deprived rats. We found increased apoptosis in the three aminergic systems analyzed when compared with animals maintained for 6 weeks in 12:12 light-dark conditions. The most apoptosis was observed in NA-LC neurons, associated with a significant decrease in the number of cortical NA boutons. Behaviorally, DD induced a depression-like condition as measured by increased immobility in a forced swim test (FST). DD did not appear to be stressful (no effect on adrenal or body weights) but may have sensitized responses to subsequent stressors (increased fecal number during the FST). We also found that the antidepressant desipramine decreases these neural and behavioral effects of light deprivation. These findings indicate that DD induces neural damage in monoamine brain systems and this damage is associated with a depressive behavioral phenotype. Our results suggest a mechanism whereby prolonged limited light intensity could negatively impact mood.

A neural circuit for circadian regulation of arousal.

An unknown aspect of behavioral state regulation is how the circadian oscillator of the suprachiasmatic nucleus (SCN) regulates sleep and waking. In this report, we describe the necessary elements for a circuit that provides circadian regulation of arousal. Trans-synaptic retrograde tracing revealed a prominent indirect projection from the SCN to the noradrenergic nucleus locus coeruleus (LC), a brain arousal system. Double-labeling experiments revealed several possible links between the SCN and the LC, including the dorsomedial (DMH) and paraventricular hypothalamic nuclei (PVN), as well as medial and ventrolateral pre-optic areas. Lesion studies confirmed that the DMH is a substantial relay in this circuit. Next, neurophysiology experiments revealed circadian variations in LC impulse activity. Lesions of the DMH eliminated these circadian changes in LC activity, confirming the functionality of the SCN-DMH-LC circuit. These results reveal mechanisms for regulation of circadian and sleep-waking functions.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

The bed nucleus of the stria terminalis. A target site for noradrenergic actions in opiate withdrawal.

Hyperactivity of brain norepinephrine (NE) systems has long been implicated in mechanisms of opiate withdrawal (OW). However, little is known about where elevated NE may act to promote OW. Here we report that the bed nucleus of the stria terminalis (BNST), the densest NE target in the brain, is critical for NE actions in OW. (1) Many BNST neurons become Fos+ after OW. Pretreatment with the beta antagonist, propranolol, markedly reduces OW symptoms and the number of Fos+ cells in the BNST. (2) Numerous neurons in the nucleus tractus solitarius (A2 neurons) and the A1 cell group are triple labeled for tyrosine hydroxylase, a retrograde tracer from the BNST, and Fos after OW, revealing numerous NE neurons that project to the BNST from the medulla that are stimulated by OW. Fewer such triple-labeled neurons were found in the locus caeruleus. (3) Behavioral studies reveal that local microinjections of selective beta-adrenergic antagonists into the BNST attenuate OW symptoms. In particular, withdrawal-induced place aversion is abolished by bilateral microinjection of a cocktail of selective beta 1 (betaxolol) plus the beta 2 (ICI 181,555) antagonists (1.0 nmol each/0.5 microL per side) into the BNST. Similar results were obtained with neurochemically selective lesions of the ventral ascending NE bundle, the pathway for A1 and A2 projections to the BNST. Similar lesions of the dorsal NE bundle of projections from the locus caeruleus had no effect on either aversive or somatic withdrawal symptoms. Together, these results indicate that beta-receptor activation in the BNST is critical for aversive withdrawal symptoms, and that A1 and A2 neurons in the medulla are the source of this critical NE.

Afferent projections to the rat locus coeruleus demonstrated by retrograde and anterograde tracing with cholera-toxin B subunit and Phaseolus vulgaris leucoagglutinin.

The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin. To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties. After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described. We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kölliker-Fuse nucleus, (4) mesencephalic reticular formation. Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, dorsal raphe nucleus, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus. No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract or the spinal cord, structures which were previously reported as inputs to the locus coeruleus. Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including (1) Barrington's nucleus, (2) the mesencephalic trigeminal nucleus, (3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and (4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus. These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus. These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents. These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin. Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kölliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance.

Recordings from noradrenergic locus coeruleus (LC) neurons in behaving rats and monkeys revealed that these cells decrease tonic discharge during sleep and also during certain high arousal behaviors (grooming and consumption) when attention (vigilance) was low. Sensory stimuli of many modalities phasically activated LC neurons. Response magnitudes varied with vigilance, similar to results for tonic activity. The most effective and reliable stimuli for eliciting LC responses were those that disrupted behavior and evoked orienting responses. Similar results were observed in behaving monkeys except that more intense stimuli were required for LC responses. Our more recent studies have examined LC activity in monkeys performing an "oddball" visual discrimination task. Monkeys were trained to release a lever after a target cue light that occurred randomly on 10% of trials; animals had to withhold responding during non-target cues. LC neurons selectively responded to the target cues during this task. During reversal training, LC neurons lost their response to the previous target cue and began responding to the new target light in parallel with behavioral reversal. Cortical event-related potentials were elicited in this task selectively by the same stimuli that evoked LC responses. Injections of lidocaine, GABA, or a synaptic decoupling solution into the nucleus paragigantocellularis in the rostral ventrolateral medulla, the major afferent to LC, eliminated responses of LC neurons to sciatic nerve stimulation or foot- or tail-pinch. This indicates that certain sensory information is relayed to LC through the excitatory amino acid (EAA) input from the ventrolateral medulla. The effect of prefrontal cortex (PFC) activation on LC neurons was examined in anesthetized rats. Single pulse PFC stimulation had no pronounced effect on LC neurons, consistent with our findings that this area does not innervate the LC nucleus. However, trains of PFC stimulation substantially activated most LC neurons. Thus, projections from the PFC may activate LC indirectly or through distal dendrites, suggesting a circuit whereby complex stimuli may influence LC neurons. The above results, in view of previous findings for postsynaptic effects of norepinephrine, are interpreted to reveal a role for the LC system in regulating attentional state or vigilance. The roles of major inputs to LC from the ventrolateral and dorsomedial medulla in sympathetic control and behavioral orienting responses, respectively, are integrated into this view of the LC system. It is proposed that the LC provides the cognitive complement to sympathetic function.

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology.

Tract-tracing and electrophysiology studies have revealed that major inputs to the nucleus locus coeruleus (LC) are found in two structures, the nucleus paragigantocellularis (PGi) and the perifascicular area of the nucleus prepositus hypoglossi (PrH), both located in the rostral medulla. Minor afferents to LC were found in the dorsal cap of the paraventricular hypothalamus and spinal lamina X. Recent studies have also revealed limited inputs from two areas nearby the LC, the caudal midbrain periaqueductal gray (PAG) and the ventromedial pericoerulear region. The pericoeruleus may provide a local circuit interface to LC neurons. Recent electron microscopic analyses have revealed that LC dendrites extend preferentially into the rostromedial and caudal juxtaependymal pericoerulear regions. These extracoerulear LC dendrites may receive afferents in addition to those projecting to LC proper. However, single-pulse stimulation of inputs to such dendritic regions reveals little or no effect on LC neurons. Double-labeling studies have revealed that a variety of neurotransmitters impinging on LC neurons originate in its two major afferents, PGi and PrH. The LC is innervated by PGi neurons that stain for markers of adrenalin, enkephalin or corticotropin-releasing factor. Within PrH, large proportions of LC-projecting neurons stained for GABA or met-enkephalin. Finally, in contrast to previous conclusions, the dorsal raphe does not provide the robust 5-HT innervation found in the LC. We conclude that 5-HT inputs may derive from local 5-HT neurons in the pericoerulear area. Neuropharmacology experiments revealed that the PGi provides a potent excitatory amino acid (EAA) input to the LC, acting primarily at non-NMDA receptors in the LC. Other studies indicated that this pathway mediates certain sensory responses of LC neurons. NMDA-mediated sensory responses were also revealed during local infusion of magnesium-free solutions. Finally, adrenergic inhibition of LC from PGi could also be detected in nearly every LC neuron tested when the EAA-mediated excitation is first eliminated. In contrast to PGi, the PrH potently and consistently inhibited LC neurons via a GABAergic projection acting at GABAA receptors within LC. Such PrH stimulation also potently attenuated LC sensory responses. Finally, afferents to PGi areas that also contain LC-projecting neurons were identified. Major inputs were primarily autonomic in nature, and included the caudal medullary reticular formation, the parabrachial and Kölliker-Fuse nuclei, the PAG, NTS and certain hypothalamic areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Responses of primate locus coeruleus neurons to simple and complex sensory stimuli.

Single and multiple unit recordings were obtained from locus coeruleus (LC) of unanesthetized, chair-restrained monkeys during presentation of a range of sensory stimuli. Tonic activity was higher during alertness or agitation than during behavioral inattentiveness and drowsiness. Low-level, simple auditory stimuli elicited no response, while more intense stimuli evoked phasic discharges in LC activity. The most pronounced responses were elicited by aversive air puffs and by multi-modal naturalistic stimuli such as interactions with the experimenter. The results suggest that sensory stimuli effective in eliciting LC discharge have specific stimulus attributes. It is proposed that the LC is tuned to specifically respond to stimuli which are conspicuous to that species: stimuli which by their physical or behavioral properties evoke a change in the focus of attention. The LC response would thereby contribute to adaptive behavioral responses to such unexpected imperative stimuli. This hypothesis is consistent with earlier suggestions that the LC contributes to behavioral functions such as vigilance and alarm and provides a rigorous framework for future experiments.

Impulse conduction properties of noradrenergic locus coeruleus axons projecting to monkey cerebrocortex.

Antidromically driven action potentials were recorded from norepinephrine-containing locus coeruleus neurons in response to electrical stimulation of cerebrocortical and thalamic areas in anesthetized squirrel monkeys. These cells reliably conducted impulses from cortical sites of distances up to 100 mm from locus coeruleus. Monkey locus coeruleus neurons were found to exhibit several properties previously described for these cells in rat, including slow spontaneous discharge rates, characteristic impulse waveforms, antidromic activation from many target areas, a period of suppressed activity following either antidromic or orthodromic driving and responsiveness to noxious stimuli presented as subcutaneous electrical stimulation of a rear foot. However, a large population of monkey locus coeruleus neurons was found to exhibit more rapid conduction velocities than previously found for rat (e.g. approximately 34% were greater than 1 m/s), resulting in similar conduction latencies to distant target areas in the two species. This indicates that the conduction times required for locus coeruleus impulses to reach distant target areas may be conserved across different species and sizes of brains, suggesting that these latencies play an important role in the general function of the locus coeruleus system in brain and behavioral processes.