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.     

Input from central nucleus of the amygdala efferents to pericoerulear dendrites,some of which contain tyrosine hydroxylase immunoreactivity

Light microscopic anterograde tracing studies indicate that neurons in the central nucleus of the amygdala (CNA) project to a region of the dorsal pontine tegmentum ventral to the superior cerebellar peduncle which contains noradrenergic dendrites of the nucleus locus coeruleus (LC). However, it has not been established whether the efferent terminals from the CNA target catecholamine-containing dendrites of the LC or dendrites of neurons from neighboring nuclei which may extend into this region. To examine this question, we combined immunoperoxidase labeling of the anterograde tracer biotinylated dextran amine (BDA) from the CNA with immunogold-silver labeling of the catecholamine-synthesizing enzyme tryrosine hydroxylase (TH) in the rostrolateral LC region of adult rats. By light microscopy, BDA-labeled processes were dense in the dorsal pons within the parabrachial nuclei as well as in the pericoerulear region immediately ventral to the superior cerebellar peduncle. Higher magnification revealed that BDA-labeled varicose fibers overlapped TH-labeled processes in this pericoerulear region. By electron microscopy, anterogradely labeled axon terminals contained small, clear as well as some large dense core vesicles and were commonly apposed to astrocytic processes along some portion of their plasmalemma. BDA-labeled terminals mainly formed symmetric type synaptic contacts characteristic of inhibitory transmitters. Of 250 BDA-labeled axon terminals examined where TH immunoreactivity was present in the neuropil, 81% contacted unlabeled and 19% contacted TH-labeled dendrites. Additionally, amygdala efferents were often apposed to unlabeled axon terminals forming asymmetric (excitatory type) synapses. These results demonstrate that amygdaloid efferents may directly alter the activity of catecholaminergic and non-catecholaminergic neurons in this pericoerulear region of the rat brain. Furthermore, our study suggests that CNA efferents may indirectly affect the activity of pericoerulear neurons through modulation of excitatory afferents. Amygdaloid projections to noradrenergic neurons may help integrate behavioral and visceral responses to threatening stimuli by influencing the widespread noradrenergic projections from the LC. © 1996 Wiley-Liss, Inc.     

Cellular interactions between axon terminals containing endogenous opioid peptides or corticotropin-releasing factor in the rat locus coeruleus and surrounding dorsal pontine tegmentum

Recent evidence suggests that certain stressors release both endogenous opioids and corticotropin-releasing factor (CRF) to modulate activity of the locus coeruleus (LC)-norepinephrine (NE) system. In ultrastructural studies, axon terminals containing methionine(5)-enkephalin (ENK) or CRF have been shown to target LC dendrites. These findings suggested the hypothesis that both neuropeptides may coexist in common axon terminals that are positioned to have an impact on the LC. This possibility was examined by using immunofluorescence and immunoelectron microscopic analysis of the rat LC and neighboring dorsal pontine tegmentum. Ultrastructural analysis indicated that CRF- and ENK-containing axon terminals were abundant in similar portions of the neuropil and that approximately 16% of the axon terminals containing ENK were also immunoreactive for CRF. Dually labeled terminals were more frequently encountered in the "core" of the LC vs. its extranuclear dendritic zone, which included the medial parabrachial nucleus (mPB). Triple labeling for ENK, CRF, and tyrosine hydroxylase (TH) showed convergence of opioid and CRF axon terminals with noradrenergic dendrites as well as evidence for inputs to TH-labeled dendrites from dually labeled opioid/CRF axon terminals. One potential source of ENK and CRF in the dorsal pons is the central nucleus of the amygdala (CNA). To determine the relative contribution of ENK and CRF terminals from the CNA, the CNA was electrolytically lesioned. Light-level densitometry revealed robust decreases in CRF immunoreactivity in the LC and mPB on the side ipsilateral to the lesion but little or no change in ENK immunoreactivity, confirming previous studies of the mPB. Degenerating terminals from the CNA in lesioned rats were found to be in direct contact with TH-labeled dendrites. Together, these data indicate that ENK and CRF may be colocalized to a subset of individual axon terminals in the LC "core." The finding that the CNA provides, to dendrites in the area examined, a robust CRF innervation, but little or no opioid innervation, suggests that ENK and CRF axon terminals impacting LC neurons originate from distinct sources and that terminals that colocalize ENK and CRF are not from the CNA.     

Amygdaloid Corticotropin-Releasing Factor Targets Locus Coeruleus Dendrites: Substrate for the Co-ordination of Emotional and Cognitive Limbs of the Stress Response

Corticotropin-releasing factor (CRF), the neurohormone that initiates the endocrine limb of the stress response via its actions on the anterior pituitary, also acts as a neurotransmitter in the noradrenergic locus coeruleus (LC) to activate this system during stress. Because the central nucleus of the amygdala contains numerous CRF-immunoreactive neurones, the present study examined whether CRF projections from the central nucleus of the amygdala target LC dendrites, thereby providing a mechanism for limbic-CRF modulation of brain noradrenergic activity. Retrograde tracers injected into the rostrolateral pericoerulear region, where CRF-immunoreactive fibres are dense, labelled numerous CRF-immunoreactive neurones in the central nucleus of the amygdala. Consistent with this, ultrastructural analysis of the rostrolateral pericoerulear region in sections that were dually labelled for an anterograde tracer (biotinylated dextran amine, BDA) injected into the central nucleus of the amygdala and CRF immunoreactivity revealed that a substantial percentage (35%) of amygdaloid axon terminals were CRF-immunoreactive. These terminals formed synaptic specializations with unlabelled dendrites that were more often of the asymmetric (excitatory) type. Additionally, ultrastructural analysis of sections that were dually labelled to visualize CRF-and tyrosine hydroxlase-immunoreactivity demonstrated synaptic specializations between CRF-immunoreactive terminals and LC dendrites in the rostrolateral peri-LC, which were also frequently asymmetric. Taken together with previous ultrastructural findings that LC dendrites in the rostrolateral pericoerulear region are targeted by anterogradely labelled terminals from the central nucleus of the amygdala, the present results implicate this nucleus as a source of CRF that can impact on LC activity via effects on dendrites in the rostrolateral pericoerulear region. This cellular substrate for amygdaloid-CRF modulation of brain noradrenergic activity may serve as a mechanism for the integration of emotional and cognitive responses to stress.     

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. These results provide the first direct ultrastructural evidence that axon terminals containing CRF-immunoreactivity 1) directly contact catecholamine-containing dendrites within the rostral pole of the LC, 2) may presynaptically modulate other afferents, and 3) are often enveloped by astrocytic processes. © 1996 Wiley-Liss, Inc.     

Glucocorticoid receptor-immunoreactivity in corticotrophin-releasing factor afferents to the locus coeruleus

The stress-related neurohormone, corticotropin-releasing factor (CRF), also acts as a neurotransmitter to activate the brain noradrenergic nucleus, locus coeruleus (LC). Previous electrophysiological findings demonstrating that tonic CRF secretion in the LC is increased in adrenalectomized rats suggest that activity of certain CRF afferents to the LC is under inhibitory regulation by endogenous corticosteroids. The present study was designed to identify putative CRF afferents to the LC that may be regulated by glucocorticoids. Retrograde tract tracing from the rat LC and pericoerulear regions was combined with immunohistochemistry to visualize CRF and glucocorticoid receptors in the same sections of rat brain. The retrograde tracer, wheat germ agglutinin conjugated to horseradish peroxidase coupled to gold (WGA–Au–HRP) was injected into either the nucleus LC or the rostrolateral pericoerulear region (peri-LC), where CRF-immunoreactive terminals have been demonstrated to synapse with LC dendrites. Sections were processed to visualize the tracer, as well as CRF- and glucocorticoid receptor-immunoreactivity. Following injections of WGA–Au–HRP into the nuclear LC, triple labeled neurons were observed primarily in Barrington's nucleus, where 74±4% of retrogradely labeled CRF-immunoreactive neurons colocalized glucocorticoid receptor immunoreactivity. In contrast, injections that incorporated the rostrolateral peri-LC retrogradely labeled numerous neurons that were immunoreactive for both CRF and glucocorticoid receptors in the central nucleus of the amygdala. Thus, 94±2% of the retrogradely labeled CRF-immunoreactive neurons in the central nucleus of the amygdala colocalized glucocorticoid receptor immunoreactivity. Additionally, triple labeled neurons were observed in the bed nucleus of the stria terminalis following WGA–Au–HRP injections that incorporated the rostrolateral peri-LC. The present results implicate Barrington's nucleus, the central nucleus of the amygdala and the bed nucleus of the stria terminalis as glucocorticoid-sensitive sources of CRF that can influence the LC-noradrenergic system. Alterations in glucocorticoid levels or glucocorticoid receptor function would be predicted to affect the impact of these specific CRF systems on LC activity.     

Amygdalar peptidergic circuits regulating noradrenergic locus coeruleus neurons: linking limbic and arousal centers

The endogenous opioid peptides, met- or leu-enkephalin, and corticotropin-releasing factor (CRF) regulate noradrenergic neurons in the locus coeruleus (LC) in a convergent manner via projections from distinct brain areas. In contrast, the opioid peptide dynorphin (DYN) has been shown to serve as a co-transmitter with CRF in afferents to the LC. To further define anatomical substrates targeting noradrenergic neurons by DYN afferents originating from limbic sources, anterograde tract-tracing of biotinylated dextran amine (BDA) from the central amygdaloid complex was combined with immunocytochemical detection of DYN and tyrosine hydroxylase (TH) in the same section of tissue. Triple labeling immunocytochemistry was combined with electron microscopy in the LC where BDA was identified using an immunoperoxidase marker, and DYN and TH were distinguished by the use of sequential immunogold labeling and silver enhancement to produce different sized gold particles. Results show direct evidence of a monosynaptic pathway linking amygdalar DYN afferents with LC neurons. To determine whether DYN-containing amygdalar LC-projecting neurons colocalize CRF, retrograde tract-tracing using fluorescent latex microspheres injected into the LC was combined with immunocytochemical detection of DYN and CRF in single sections in the central amygdala. Retrogradely labeled neurons from the LC were distributed throughout the rostro-caudal extent of the central nucleus of the amygdala (CeA) as previously described. Cell counts showed that approximately 42% of LC-projecting neurons in the CeA contained both DYN and CRF. Taken with our previous studies showing monosynaptic projections from amygdalar CRF neurons to noradrenergic LC cells, the present study extends this by showing that DYN and CRF are co-transmitters in monosynaptic projections to the LC and are poised to coordinately impact LC neuronal activity.     

Ultrastructure of endomorphin-1 immunoreactivity in the rat dorsal pontine tegmentum: evidence for preferential targeting of peptidergic neurons in Barrington's nucleus rather than catecholaminergic neurons in the peri-locus coeruleus

Endomorphins are opioid tetrapeptides that have high affinity and selectivity for mu-opioid receptors (muORs). Light microscopic studies have shown that endomorphin-1 (EM-1) -containing fibers are distributed within the brainstem dorsal pontine tegmentum. Here, immunoelectron microscopy was conducted in the rat brainstem to identify potential cellular interactions between EM-1 and tyrosine hydroxylase (TH) -labeled cellular profiles in the locus coeruleus (LC) and peri-LC, an area known to contain extensive noradrenergic dendrites of LC neurons. Furthermore, sections through the rostral dorsal pons, from colchicine-treated rats, were processed for EM-1 and corticotropin releasing factor (CRF), a neuropeptide known to be present in neurons of Barrington's nucleus. EM-1 immunoreactivity was identified in unmyelinated axons, axon terminals, and occasionally in cellular profiles resembling glial processes. Within axon terminals, peroxidase labeling for EM-1 was enriched in large dense core vesicles. In sections processed for EM-1 and TH, approximately 10% of EM-1-containing axon terminals (n=269) targeted dendrites that exhibited immunogold-silver labeling for TH. In contrast, approximately 30% of EM-1-labeled axon terminals analyzed (n = 180) targeted CRF-containing somata and dendrites in Barrington's nucleus. Taken together, these data indicate that the modulation of nociceptive and autonomic function as well as stress and arousal responses attributed to EM-1 in the central nervous system may arise, in part, from direct actions on catecholaminergic neurons in the peri-LC. However, the increased frequency with which EM-1 axon terminals form synapses with CRF-containing profiles in Barrington's nucleus suggests a novel role for EM-1 in the modulation of functions associated with Barrington's nucleus neurons such as micturition control and pelvic visceral function.     

Light and electron microscopic evidence for topographic and monosynaptic projections from neurons in the ventral medulla to noradrenergic dendrites in the rat locus coeruleus

Physiological studies have shown that afferents from the nucleus paragigantocellularis (PGi) in the rostral ventral medulla underlie the modulation of locus coeruleus (LC) activity by a variety of stimuli. However, there have been no anatomical demonstrations of a monosynaptic projection from neurons in the PGi to the LC. Thus, biotinylated dextran amine (BDA) was iontophoretically injected into the ventral medulla and single-tissue sections were processed for peroxidase localization of BDA and gold–silver labeling of tyrosine hydroxylase (TH). Discrete microinjections of BDA were placed into either the medial or lateral aspects of the ventral medulla. For medially placed injections, a medio-dorsal pathway to the LC was observed. This trajectory resulted in a predominant innervation of the ventral LC. Lateral injection placements yielded a fiber pathway that coursed more laterally within the medullo-pontine reticular formation and primarily innervated the dorsolateral LC. These light microscopic data suggested that neurons in the PGi use distinct pathways to innervate the LC and are topographically organized within this structure. Electron microscopic analyses of the LC region indicated that axon terminals originating from either subregion were equally likely to contact noradrenergic neurons in the LC. Approximately 57% and 62% of BDA-labeled terminals originating from the medial (n=150) or lateral (n=150) aspects of the ventral medulla, respectively, formed heterogeneous synaptic contacts (i.e., inhibitory- and excitatory-type) with dendrites containing TH. It is well known that the PGi is a functionally diverse region that is involved in sensory integration, autonomic regulation and pain modulation. It is also known that LC efferents are spatially organized with respect to their postsynaptic targets. Taken together, our findings that subdivisions of the ventral medulla topographically and monosynaptically innervate the LC suggest that regionally specific PGi neurons target subsets of LC neurons with efferent targets that may possess analogous functional correlates.     

Pro-opiomelanocortin colocalizes with corticotropin- releasing factor in axon terminals of the noradrenergic nucleus locus coeruleus

We previously demonstrated that the opioid peptide enkephalin and corticotropin-releasing factor (CRF) are occasionally colocalized in individual axon terminals but more frequently converge on common dendrites in the locus coeruleus (LC). To further examine potential opioid cotransmitters in CRF afferents we investigated the distribution of pro-opiomelanocortin (POMC), the precursor that yields the potent bioactive peptide beta-endorphin, with respect to CRF immunoreactivity using immunofluorescence and immunoelectron microscopic analyses of the LC. Coronal sections were collected through the dorsal pontine tegmentum of rat brain and processed for immunocytochemical detection of POMC and CRF or tyrosine hydroxylase (TH). POMC-immunoreactive processes exhibited a distinct distribution within the LC as compared to the enkephalin family of opioid peptides. Specifically, POMC fibers were enriched in the ventromedial aspect of the LC with fewer fibers present dorsolaterally. Immunofluorescence microscopy showed frequent coexistence of POMC and CRF in varicose processes that overlapped TH-containing somatodendritic processes in the LC. Ultrastructural analysis showed POMC immunoreactivity in unmyelinated axons and axon terminals. Axon terminals containing POMC were filled with numerous large dense-core vesicles. In sections processed for POMC and TH, approximately 29% of POMC-containing axon terminals (n = 405) targeted dendrites that exhibited immunogold-silver labeling for TH. In contrast, sections processed for POMC and CRF showed that 27% of POMC-labeled axon terminals (n = 657) also exhibited CRF immunoreactivity. Taken together, these data indicate that a subset of CRF afferents targeting the LC contain POMC and may be positioned to dually impact LC activity.     

Amygdala efferents form inhibitory-type synapses with a subpopulation of catecholaminergic neurons in the rat nucleus tractus solitarius

The central nucleus of the amygdala (CNA) integrates visceral responses to stress partially through efferent projections to portions of the medial nuclei of the solitary tracts (mNTS) containing catecholaminergic neurons. To determine anatomical sites for CNA modulation of these neurons, immunoperoxidase detection of anterogradely transported Phaseolus vulgaris leucoagglutinin (PHA- L) or biotinylated dextran amine (BDA) was combined with immunogold silver labeling of the catecholamine-synthesizing enzyme, tyrosine hydroxylase, in adult rat mNTS. From 350 anterogradely labeled terminals identified within the intermediate mNTS, 30% formed symmetric, inhibitory-type synapses and the remainder lacked recognized junctions as seen within a single plane of section. Of the terminals forming symmetric synapses, 16% were presynaptic to tyrosine hydroxylase immunoreactive dendrites and the remainder to unlabeled dendrites. The level of tyrosine hydroxylase immunoreactivity as assessed by density of gold-silver particles was significantly lower in dendrites receiving synaptic input from CNA efferents as compared with dendrites of the same sizes (2. 0 μm² in mean area) which received synapses from unlabeled terminals or lacked recognizable synaptic inputs. When separately examined without regard to afferent input, the medium- and larger-sized dendrites having mean cross-sectional areas of 1–3 μ²m also contained significantly less tyrosine hydroxylase immunoreactivity than small (<1 μ²m) dendrites. These results suggest that CNA efferents to the mNTS inhibit non-catecholamine-containing neurons and a subpopulation of catecholamin ergic neurons distinguished by their low levels of tyrosine hydroxylase. The findings also indicate that small, presumably more distal, dendrites in the intermediate mNTS may synthesize and/or release catecholamines. © 1995 Wiley-Liss Inc.     

Selective distribution of the NMDA-R1 glutamate receptor in astrocytes and presynaptic axon terminals in the nucleus locus coeruleus of the rat brain: An immunoelectron microscopic study

The regional and cellular distribution of the different classes of excitatory amino acid receptors with respect to the noradrenergic neurons of the nucleus locus coeruleus (LC) are unknown. We therefore combined immunoperoxidase labeling for the R1 subunit of the N-methyl-D-aspartate (NMDA) receptor with immunogold-silver localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), in single sections through the rat LC to determine the subcellular localization of this glutamate receptor subtype with respect to the noradrenergic neurons. At the light microscopic level, there was light to moderate labeling for the NMDA-R1-like (li) receptor in the caudal pole of the LC and dense labeling in the dorsolateral aspect of the LC adjacent to the superior cerebellar peduncle. In the rostral pole of the LC which is enriched with noradrenergic dendrites, significant overlap between both immunoreactivities could be observed. At the ultrastructural level, immunoperoxidase labeling for NMDA-R1 was selectively distributed in astrocytic processes and within presynaptic axon terminals but was rarely seen in catecholamine-containing somata or dendrites. Peroxidase labeling for NMDA-R1, however, was occasionally observed in dendrites in the rostral pole of the LC. Most of these dendrites lacked detectable levels of TH, although TH immunoreactivity was apparent in the neuropil. Dendrites containing NMDA-R1-li immunoreactivity often received asymmetric (excitatory-type) contacts from unlabeled terminals. NMDA-R1-li-immunoreactive axon terminals usually contained small clear, as well as large dense-core vesicles and were often apposed to unlabeled dendrites, axon terminals and/or glial processes. These results provide the first ultrastructural evidence that NMDA-R1-li immunoreactivity is selectively distributed within astrocytic processes and presynaptic axon terminals within the LC. © 1996 Wiley-Liss, Inc.     

Dynorphin and stress-related peptides in rat locus coeruleus: contribution of amygdalar efferents

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress-related peptide corticotropin-releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN- and CRF-containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA-positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA-containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light-level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN- and CRF-labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel.     

Subcellular targeting of kappa‐opioid receptors in the rat nucleus locus coeruleus

The dynorphin (DYN)‐kappa opioid receptor (κOR) system has been implicated in stress modulation, depression, and relapse to drug‐seeking behaviors. Previous anatomical and physiological data have indicated that the noradrenergic nucleus locus coeruleus (LC) is one site at which DYN may contribute to these effects. Using light microscopy, immunofluorescence, and electron microscopy, the present study investigated the cellular substrates for pre‐ and postsynaptic interactions of κOR in the LC. Dual immunocytochemical labeling for κOR and tyrosine hydroxylase (TH) or κOR and preprodynorphin (ppDYN) was examined in the same section of tissue. Light microscopic analysis revealed prominent κOR immunoreactivity in the nuclear core of the LC and in the peri‐coerulear region where noradrenergic dendrites extend. Fluorescence and electron microscopy revealed κOR immunoreactivity within TH‐immunoreactive somata and dendrites in the LC as well as localized to ppDYN‐immunoreactive processes. In sections processed for κOR and TH, ≈29% (200/688) of the κOR‐containing axon terminals identified targeted TH‐containing profiles. Approximately 49% (98/200) of the κOR‐labeled axon terminals formed asymmetric synapses with TH‐labeled dendrites. Sections processed for κOR and ppDYN showed that, of the axon terminals exhibiting κOR, 47% (223/477) also exhibited ppDYN. These findings indicate that κORs are poised to modulate LC activity by their localization to somata and dendrites. Furthermore, κORs are strategically localized to presynaptically modulate DYN afferent input to catecholamine‐containing neurons in the LC. These data add to the growing literature showing that κORs can modulate diverse afferent signaling to the LC. J. Comp. Neurol. 512:419–431, 2009. © 2008 Wiley‐Liss, Inc.     

Ultrastructural evidence for prominent postsynaptic localization of α2C-adrenergic receptors in catecholaminergic dendrites in the rat nucleus locus coeruleus

Alpha-2-adrenergic receptor (α₂-AR) agonists potently inhibit the activity of noradrenergic neurons of the locus coeruleus (LC), an effect that may be mediated by the A- and/ or C-subtypes of α₂-AR (α_2A- and α_2C-AR). To gain insight into the functional significance of these α₂-AR subtypes in the LC, we have examined their ultrastructural localization by using subtype-specific antibodies. We recently demonstrated that α_2A-ARs are localized prominently in axon terminals and catecholaminergic dendrites in the LC. In the present study, we sought to identify the subcellular substrates underlying α_2C-AR actions in the LC by analyzing the ultrastructural distribution of α_2C-AR immunoreactivity (α_2C-AR-IR) in sections that were dually labeled for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Alpha-2C-AR-IR was predominantly localized in dendrites, most of which also contained immunolabeling for TH. Within such dendrites, α_2C-AR-IR was associated with the plasma membrane and occasionally Golgi cisternae and tubulovesicles. The vast majority of dendrites containing α_2C-AR-IR received asymmetric (excitatory) contacts from unlabeled axon terminals that often contained dense core vesicles. Alpha-2C-AR-IR was observed in some unmyelinated axons and astrocytic processes that were apposed to TH-immunoreactive dendrites but was rarely associated with axon terminals. These results provide the first ultrastructural evidence that α_2C-ARs (1) are localized postsynaptically in catecholaminergic neurons of the LC and (2) may be strategically situated to modulate the activation of LC neurons by excitatory inputs. J. Comp. Neurol. 394:218–229, 1998. © 1998 Wiley-Liss, Inc.     

Evidence for coexistence of enkephalin and glutamate in axon terminals and cellular sites for functional interactions of their receptors in the rat locus coeruleus

The authors previously showed that a subset of axon terminals in the locus coeruleus (LC) contains methionine5-enkephalin (ENK) and gamma-aminobutyric acid (GABA) immunoreactivities. However, numerous ENK-labeled terminals lacked GABA and exhibited synaptic specializations that were characteristic of excitatory-type transmitters. To determine whether ENK coexists with glutamate in the LC, preembedding immunoperoxidase detection of ENK or immunogold-silver was combined with postembedding identification of glutamate using a gold marker. Indeed, 28% of the ENK-labeled axon terminals examined (n = 250 axon terminals) also contained glutamate. To define further sites for functional interactions between opiate ligands and excitatory amino acid receptors, the ultrastructural localization of the mu-opioid receptor (MOR) was examined with respect to either the kainate receptor (KAR) or the R1 subunit of the N-methyl-D-aspartate (NR1)-type glutamate receptor in the LC. Gold-silver labeling for MOR and peroxidase labeling for either KAR or NR1 indicated that the MOR often was localized to the plasma membrane of dendrites that also exhibited immunolabeling for either glutamate receptor subtype. In contrast to the KAR, which was identified primarily in somata and dendrites, NR1 immunoreactivity also was found frequently in axon terminals as well as in glial processes. Glial processes containing NR1 occasionally exhibited immunolabeling for MOR and sometimes were directly apposed to MOR-containing dendrites in the LC. Furthermore, NR1-labeled receptors in axon terminals sometimes were presynaptic to MOR-labeled dendrites. The authors concluded that ENK and glutamate may be cotransmitters in LC afferents. Moreover, ligands at the KAR may modulate directly MOR-containing neurons in the LC, whereas actions at NR1 receptors may affect opioid-sensitive neurons through multiple cellular mechanisms, i.e., through presynaptic, postsynaptic, or glial actions.     

GABAergic neurons in rat nuclei of solitary tracts receive inhibitory-type synapses from amygdaloid efferents lacking detectable GABA-immunoreactivity

Gamma-aminobutyric acid (GABA) is a prominent inhibitory transmitter in both the central nucleus of the amygdala (Ce) and the medial nuclei of the solitary tracts (mNTS). These regions are reciprocally connected by anatomical pathways mediating the coordinated visceral responses to emotional stress. To further determine whether GABA is present in the amygdaloid efferents or their targets in the mNTS, we combined peroxidase labeling of Phaseolus vulgaris leucoagglutinin (PHA-L) or biotinylated dextran amine (BDA) anterogradely transported from the Ce with immunogold-silver detection of antibodies against GABA in the rat mNTS. By light microscopy, peroxidase labeling for either PHA-L or BDA was seen in varicose processes, whereas immunogold-silver labeling for GABA was detected in perikarya and processes throughout the rostrocaudal mNTS. The intermediate mNTS at the level of the area postrema, a region receiving mainly cardiorespiratory and gastric visceral afferents, were examined by electron microscopy. In this region, anterograde labeling was observed exclusively in unmyelinated axons and axon terminals. These terminals lacked detectable GABA-immunoreactivity, but formed symmetric synapses that are associated with inhibition. The targets of the anterogradely labeled terminals were medium-sized dendrites both with and without GABA-labeling. These dendrites often also received convergent input from terminals that were intensely GABA-immunoreactive. We conclude that visceral activation accompanying emotional response to stress is likely to involve inhibition of GABAergic neurons in the mNTS by non-GABA-containing amygdaloid efferents. Furthermore, our results indicate that the inhibition of these GABAergic neurons may be further augmented by release of GABA from other converging terminals in the mNTS. © 1996 Wiley-Liss, Inc.     

Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic

Interactions between stress and the mesocorticolimbic dopamine (DA) system have been suggested from behavioral and electrophysiological studies. Because corticotropin-releasing factor (CRF) plays a role in stress responses, we investigated possible interactions between neurons containing CRF and those producing DA in the ventral tegmental area (VTA). We first investigated the cellular distribution of CRF in the VTA by immunolabeling VTA sections with anti-CRF antibodies and analyzing these sections by electron microscopy. We found CRF immunoreactivity present mostly in axon terminals establishing either symmetric or asymmetric synapses with VTA dendrites. We established that nearly all CRF asymmetric synapses are glutamatergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed the vesicular glutamate transporter 2, and that the majority of CRF symmetric synapses are GABAergic, insofar as the CRF-immunolabeled axon terminals in these synapses coexpressed glutamic acid decarboxylase, findings that are of functional importance. We then looked for synaptic interactions between CRF- and DA-containing neurons, by using antibodies against CRF and tyrosine hydroxylase (TH; a marker for DA neurons). We found that most synapses between CRF-immunoreactive axon terminals and TH neurons are asymmetric (in the majority likely to be glutamatergic) and suggest that glutamatergic neurons containing CRF may be part of the neuronal circuitry that mediates stress responses involving the mesocorticolimbic DA system. The presence of CRF synapses in the VTA offers a mechanism for interactions between the stress-associated neuropeptide CRF and the mesocorticolimbic DA system.     

Ultrastructural evidence for a role of gamma-aminobutyric acid in mediating the effects of corticotropin-releasing factor on the rat dorsal raphe serotonin system

The dorsal raphe nucleus (DRN) serotonin (5-HT) system has been implicated in acute responses to stress and in stress-related psychiatric disorders such as anxiety and depression. Substantial findings suggest that the neuropeptide corticotropin-releasing factor (CRF) is instrumental in modulating the activity of this system during stress. Because the DRN is neurochemically heterogeneous, dual immunoelectron microscopy was used to examine cellular substrates for interactions between CRF and either 5-HT or gamma-aminobutyric acid (GABA) in the dorsolateral and ventromedial DRN. CRF immunoreactivity was identified primarily within axon terminals, where immunolabeling was particularly enriched in dense-core vesicles. Although CRF terminals targeted 5-HT-containing dendrites in the dorsolateral DRN (16%; n = 251 terminals), synaptic contacts with dendrites that lacked detectable 5-HT immunolabeling were more numerous (48%). In contrast, dual labeling for CRF and GABA (n = 240 terminals) in the dorsolateral DRN revealed that substantially more CRF terminals contacted GABA dendrites (42%) as opposed to unlabeled dendrites (29%). In the ventromedial DRN, contacts between CRF axon terminals and either 5-HT-labeled dendrites or GABA-containing dendrites were fewer than in the dorsolateral DRN. As in the dorsolateral DRN, CRF terminals more frequently contacted GABA dendrites than 5-HT dendrites (30% vs. 8%, respectively). The findings support physiological studies suggesting that CRF has both direct and indirect effects on DRN-5-HT neurons and further implicate GABA as a primary mediator by which CRF and stressors alter the activity of the DRN-5-HT system.