Elisabeth J. Van Bockstaele

Convergent regulation of locus coeruleus activity as an adaptive response to stress

Although hypothalamic-pituitary-adrenal axis activation is generally considered to be the hallmark of the stress response, many of the same stimuli that initiate this response also activate the locus coeruleus-norepinephrine system. Given its functional attributes, the parallel engagement of the locus coeruleus-norepinephrine system with the hypothalamic-pituitary-adrenal axis serves to coordinate endocrine and cognitive limbs of the stress response. The elucidation of stress-related afferents to the locus coeruleus and the electrophysiological characterization of these inputs are revealing how the activity of this system is fine-tuned by stressors to facilitate adaptive cognitive responses. Emerging from these studies, is a picture of complex interactions between the stress-related neuropeptide, corticotropin-releasing factor (CRF), endogenous opioids and the excitatory amino acid neurotransmitter, glutamate. The net effect of these interactions is to adjust the activity and reactivity of the locus coeruleus-norepinephrine system such that state of arousal and processing of sensory stimuli are modified to facilitate adaptive behavioral responses to stressors. This review begins with an introduction to the basic anatomical and physiological characteristics of locus coeruleus neurons. The concept that locus coeruleus neurons operate through two activity modes, i.e., tonic vs. phasic, that determine distinct behavioral strategies is emphasized in light of its relevance to stress. Anatomical and physiological evidence are then presented suggesting that interactions between stress-related neurotransmitters that converge on locus coeruleus neurons regulate shifts between these modes of discharge in response to the challenge of a stressor. This review focuses specifically on the locus coeruleus because it is the major source of norepinephrine to the forebrain and has been implicated in behavioral and cognitive aspects of stress responses.

Functional Coupling between Neurons and Glia

Neuronal–glial interactions play an important role in information processing in the CNS. Previous studies have indicated that electrotonic coupling between locus ceruleus (LC) neurons is involved in synchronizing the spontaneous activity. The results of the present study extend the functional electrotonic coupling to interactions between neurons and glia. Spontaneous oscillations in the membrane potential were observed in a subset of glia. These oscillations were synchronous with the firing of neurons, insensitive to transmitter receptor antagonists and disrupted by carbenoxolone, a gap junction blocker. Hyperpolarization of neurons with [Met]5enkephalin blocked the oscillations in glia. Selective depolarization of glia with a glutamate transporter substrate (l-α-aminoadipic acid) increased the neuronal firing rate, suggesting that changes in the membrane potential of glia can modulate neuronal excitability through heterocellular coupling. Dye-coupling experiments further confirmed that small molecules could be transferred through gap junctions between these distinct cell types. No dye transfer was observed between neurons and oligodendrocytes or between astrocytes and oligodendrocytes, suggesting that the junctional communication was specific for astrocytes and neurons. Finally, immunoelectron microscopy studies established that connexins, the proteins that form gap junctions, were present on portions of the plasmalemma, bridging the cytoplasm of neurons and glia in LC. This heterocellular coupling extends the mechanisms by which glia participate in the network properties of the LC in which the degree of coupling is thought to influence cognitive performance.

Topography of serotonin neurons in the dorsal raphe nucleus that send axon collaterals to the rat prefrontal cortex and nucleus accumbens

Diverse physiological actions have been reported for 5-hydroxytryptamine (5-HT, serotonin) in the medial prefrontal cortex (MPFC) and the nucleus accumbens (Acb) suggesting that the 5-HT innervation of these forebrain areas may be derived from different populations of neurons. We examined this possibility by mapping the distribution of 5-HT-immunoreactive (ir) and non-5-HT-ir neurons containing retrograde labeling following injections of different tracers into both these target regions. The analysis was focused in the dorsal raphe nucleus (DRN) of the midbrain, since 5-HT pathways to the MPFC and Acb primarily originate from this area. Volume microinjections of the fluorescent retrograde tracer, Fluoro-Gold (FG), were placed into the MPFC and microinjections of cholera toxin B subunit coupled to 15 nm gold particles (CT-Au) were placed into the Acb of the same animal. Sections through the DRN containing retrogradely labeled neurons were further processed for immunofluorescent localization of 5-HT using a rhodamine marker. Neurons retrogradely labeled from the Acb were greater in number overall than those projecting to the MPFC. In addition, Acb-projecting neurons extended into the lateral wings of the DRN, whereas MPFC-projecting neurons were more restricted to the midline. Both groups of retrogradely labeled neurons, however, were more numerous in the caudal aspect of the dorsal raphe nucleus and were scattered amongst 5-HT immunoreactive perikarya. Of 783 +/- 69 CT-Au labeled cells, 15% also contained the FG label and 11% contained FG and 5-HT immunoreactivity. Of 613 +/- 48 FG labeled cells, 24% also contained the CT-Au label and 21% were also immunoreactive to 5-HT. The results suggest a more prominent input to the Acb from both 5-HT-ir and non-5-HT-ir neurons in the caudal aspect of the DRN and further indicate that while most 5-HT-ir and non-5-HT-ir neurons project differentially to both forebrain regions, a few cells also show collateralization to the MPFC and Acb. Such collateralization of single serotonergic neurons to divergent targets may integrate cognitive and motor activities in response to pharmacological manipulations of ascending serotonergic pathways.

Fusion-related release of glutamate from astrocytes

Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle- or fusion-related mechanism.

Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain.

Physiological and immunohistochemical studies have suggested that corticotropin‐releasing factor (CRF), the hypophysiotropic peptide that initiates endocrine responses to stress, may serve as a neurotransmitter to activate noradrenergic neurons in the nucleus locus coeruleus (LC). We combined immunoperoxidase labeling for CRF and immunogold‐silver localization of the catecholamine‐synthesizing enzyme tyrosine hydroxylase (TH) in single sections through the rat LC to determine potential substrates for interactions between these two transmitters. Light microscopic analysis indicated that CRF processes are dense and highly varicose in the rostral LC region in the vicinity of noradrenergic dendrites. Electron microscopy of this rostral region revealed that immunoperoxidase labeling for CRF was mainly restricted to axons and axon terminals and was rarely seen in somata or dendrites. Axon terminals containing CRF immunoreactivity varied in size, content of synaptic vesicles, and formation of synaptic specializations. The postsynaptic targets of the CRF‐labeled axon terminals consisted of both TH‐labeled dendrites and dendrites lacking detectable TH‐immunoreactivity. Of 113 CRF‐immunoreactive axon terminals, approximately 70% were in direct contact with TH‐labeled and unlabeled dendrites. Of the CRF‐labeled axon terminals forming synapses with TH‐labeled and unlabeled dendrites, they were either of the asymmetric (excitatory type; 19%) or symmetric (inhibitory type; 11%) variety or did not form identifiable contacts in the plane of section analyzed. Unlabeled axon terminals and glial processes were also commonly located adjacent to the plasma membranes of CRF‐labeled axon terminals.

Efferent projections of the nucleus of the solitary tract to peri-locus coeruleus dendrites in rat brain: evidence for a monosynaptic pathway.

Locus coeruleus (LC) neurons respond to autonomic influences, are activated by physiological stressors, and discharge in parallel with peripheral sympathetic nerves. The circuitry underlying modulation of LC activity by physiological manipulations (i.e., hemodynamic stress, hypovolumia) remains unclear. Specifically, monosynaptic projections from primary baroreceptor centers to the LC have been suggested by electrophysiological studies but have not been unequivocally established. Light microscopic anterograde tract‐tracing studies have previously shown that neurons originating in the nucleus of the solitary tract (NTS) project to a region of the rostrodorsal pontine tegmentum, which contains noradrenergic dendrites of the LC; however, it is not known whether these NTS efferents specifically target LC dendrites. Therefore, we combined peroxidase labeling of biotinylated dextran amine (BDA) or Phaseolus vulgaris–leucoagglutinin (PHA‐L) from the NTS with gold–silver labeling for tyrosine hydroxylase (TH) in the rostrolateral peri‐LC region. Injections placed into neighboring nuclei (nucleus gracilis, hypoglossal nucleus) served as controls. Only injections centered in the NTS produced anterograde labeling in peri‐LC regions containing TH processes. By electron microscopy, BDA‐ or PHA‐L‐labeled axon terminals originating from the NTS contained small, clear, and some large dense‐core vesicles and formed heterogeneous synaptic contacts characteristic of both excitatory‐ and inhibitory‐type transmitters. Approximately 19% of the BDA and PHA‐L axon terminals examined originating from the commissural portion of the NTS formed synaptic specializations with dendrites exhibiting TH immunoreactivity in the peri‐LC. These results demonstrate that neurons projecting from the cardiovascular‐related portion of the NTS target noradrenergic dendrites, indicating that barosensitive NTS neurons may directly modulate the activity of LC neurons and may serve to integrate autonomic responses in brain by influencing the widespread noradrenergic projections of the LC. In addition, these findings demonstrate that extranuclear dendrites are an important termination site for afferents to the LC. J. Comp. Neurol. 412:410–428, 1999.

Anatomic basis for differential regulation of the rostrolateral peri–locus coeruleus region by limbic afferents

BACKGROUND Neurochemical and electrophysiological studies indicate that the locus coeruleus (LC)-norepinephrine system is activated by physiological and external stressors. This activation is mediated in part by corticotropin-releasing factor (CRF), the hypothalamic neurohormone that initiates the endocrine response to stress. We have previously shown that the central nucleus of the amygdala (CNA) provides CRF afferents to noradrenergic processes in the peri-LC area that may serve to integrate emotional and cognitive responses to stress. The bed nucleus of the stria terminalis (BNST) shares many anatomical and neurochemical characteristics with the CNA, including a high density of CRF-immunoreactive cells and fibers; however, recent studies have suggested that the CNA and the BNST may differentially regulate responses to conditioned and unconditioned fear, respectively, suggesting divergent neuroanatomical circuits underlying these processes. METHODS In the present study, neuroanatomical substrates subserving regulation of the LC by the BNST were examined. Anterograde tract-tracing was combined with immunoelectron microscopy to test the hypotheses that BNST efferents target noradrenergic neurons of the LC and that these efferents exhibit immunolabeling for CRF. RESULTS Ultrastructural analysis of sections that were dually labeled for the anterograde tracer biotinylated dextran amine (BDA) injected into the BNST and tyrosine hydroxylase (TH)-immunoreactivity demonstrated that BDA-labeled axon terminals formed synaptic specializations (primarily inhibitory) with TH-labeled dendrites and dendrites that lacked TH immunoreactivity. In contrast to CNA efferents that exhibited substantial immunolabeling for CRF, far fewer BDA-labeled terminals from the BNST in the rostrolateral peri-LC contained CRF. CONCLUSIONS The present results indicate that the BNST may provide distinct neurochemical regulation of the peri-LC as compared to other limbic afferents such as the CNA. These data are interesting in light of behavioral studies showing that the CNA and BNST may be differentially involved in fear versus anxiety, respectively.

Severe stress switches CRF action in the nucleus accumbens from appetitive to aversive

Stressors motivate an array of adaptive responses ranging from ‘fight or flight’ to an internal urgency signal facilitating long-term goals. However, traumatic or chronic uncontrollable stress promotes the onset of major depressive disorder, in which acute stressors lose their motivational properties and are perceived as insurmountable impediments. Consequently, stress-induced depression is a debilitating human condition characterized by an affective shift from engagement of the environment to withdrawal. An emerging neurobiological substrate of depression and associated pathology is the nucleus accumbens, a region with the capacity to mediate a diverse range of stress responses by interfacing limbic, cognitive and motor circuitry. Here we report that corticotropin-releasing factor (CRF), a neuropeptide released in response to acute stressors and other arousing environmental stimuli, acts in the nucleus accumbens of naive mice to increase dopamine release through coactivation of the receptors CRFR1 and CRFR2. Remarkably, severe-stress exposure completely abolished this effect without recovery for at least 90 days. This loss of CRF’s capacity to regulate dopamine release in the nucleus accumbens is accompanied by a switch in the reaction to CRF from appetitive to aversive, indicating a diametric change in the emotional response to acute stressors. Thus, the current findings offer a biological substrate for the switch in affect which is central to stress-induced depressive disorders.

Hypothalamic projections to locus coeruleus neurons in rat brain

Locus coeruleus (LC) neurons respond to autonomic and visceral stimuli and discharge in parallel with peripheral sympathetic nerves. The present study characterized the synaptic organization of hypothalamic afferents with catecholaminergic neurons in the LC using electron microscopy. Peroxidase labeling of axon terminals that were anterogradely labeled from the paraventricular nucleus (PVN) was combined with gold‐silver labeling of tyrosine hydroxylase in the LC. Approximately 19% of the anterogradely labeled axon terminals formed synaptic specializations with tyrosine hydroxylase‐immunoreactive dendrites in the LC. Retrograde transport from the LC combined with immunocytochemical detection of enkephalin and corticotropin‐releasing factor (CRF) suggested that most of the LC‐projecting PVN neurons (30%) were CRF immunoreactive and few (2%) were enkephalin immunoreactive. Finally, dual retrograde tracing from the LC and median eminence revealed that PVN neurons that project to the LC are a population distinct from that projecting to the median eminence. The present data suggest that a population of hypothalamic neurons is poised to directly modulate the activity of LC neurons and may integrate autonomic responses in brain by influencing LC neurons. Moreover, PVN neurons that use CRF as a neurohormone are distinct from those that use CRF as a neuromodulator to impact on the LC.

Cannabinoid receptors are localized to noradrenergic axon terminals in the rat frontal cortex.

Cannabinoid agonists exert complex actions on modulatory neurotransmitters involved in attention and cognition. Previous studies have demonstrated that acute systemic administration of the synthetic cannabinoid agonist, WIN 55,212-2, increases norepinephrine efflux in the rat frontal cortex. In an effort to elucidate whether cannabinoid (CB1) receptors are positioned to presynaptically modulate norepinephrine release in the frontal cortex, immunocytochemical detection of the CB1 receptor and the catecholamine-synthesizing enzyme dopamine-beta-hydroxylase (DbetaH) was performed using confocal immunofluorescence microscopy and immunoelectron microscopy in rat brain. Fluorescence microscopy analysis of dually labeled tissue sections from the frontal cortex indicated that individual axonal processes exhibited both CB1 receptor and DbetaH immunoreactivities. Ultrastructural analysis confirmed that one-third of axon terminals containing CB1 immunolabeling also exhibited DbetaH labeling. Cortical neurons were also found to be targeted by separately labeled CB1- and DbetaH-containing axon terminals. In conclusion, the present neuroanatomical data suggest that cortical norepinephrine release may be modulated, in part, by CB1 receptors that are presynaptically distributed on noradrenergic axon terminals.