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.     

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.     

Mu-opioid receptor is located on the plasma membrane of dendrites that receive asymmetric synapses from axon terminals containing leucine-enkephalin in the rat nucleus locus coeruleus

We have recently shown, by using immunoelectron microscopy, that the mu-opioid receptor (μOR) is prominently distributed within noradrenergic perikarya and dendrites of the nucleus locus coeruleus (LC), many of which receive excitatory-type (i.e., asymmetric) synaptic contacts from unlabeled axon terminals. To characterize further the neurotransmitter present in these afferent terminals, we examined in the present study the ultrastructural localization of an antipeptide sequence unique to the μOR in sections that were also dually labeled for the opioid peptide leucine-enkephalin (L-ENK). Immunogold-silver labeling for μOR was localized to extrasynaptic portions of the plasma membranes of perikarya and dendrites. The μOR-labeled dendrites were usually postsynaptic to axon terminals containing heterogeneous types of synaptic vesicles and forming asymmetric synaptic specializations characteristic of excitatory-type synapses. The majority of these were immunolabeled for the endogenous opioid peptide L-ENK. Some μOR-labeled dendrites received synaptic contacts from unlabeled axon terminals in fields containing L-ENK immunoreactivity. In such cases, the μOR-labeled dendrites were in proximity to L-ENK axon terminals that contained intense peroxidase labeling within large dense core vesicles along the perimeter of the axoplasm. These results indicate that L-ENK may be released by exocytosis from the dense core vesicles and diffuse within the extracellular space to reach μOR sites on the postsynaptic dendrite or dendrites of other neighboring neurons. The present study also reveals that unlabeled terminals apposed to μOR-labeled dendrites may contain other opioid peptides, such as methionine-enkephalin. These data demonstrate several sites where endogenous opioid peptides may interact with μOR receptive sites in the LC and may provide an anatomical substrate for the LC's involvement in mechanisms of opiate dependence and withdrawal. © 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.     

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.     

Patterns of colocalization of GABA, glutamate and glycine immunoreactivities in terminals that synapse on dendrites of noradrenergic neurons in rat locus coeruleus

Amino acid transmitters play a key role in regulating the activity of noradrenergic neurons in the locus coeruleus. We investigated the anatomical substrate for this regulation by quantifying immunoreactivity for GABA, glutamate and glycine in terminals that contacted the dendrites of tyrosine hydroxylase-immunoreactive principal neurons in rat locus coeruleus. Pre-embedding peroxidase immunocytochemistry was used to detect tyrosine hydroxylase-immunoreactivity in Vibratome sections of tissue perfused with 2.5% glutaraldehyde. GABA, glutamate and glycine were localized with postembedding immunogold labelling. Gold particle densities over terminals were measured in three semiserial ultrathin sections, each reacted for a different amino acid. More than 90% (range among rats, 89%-95%) of the terminals analyzed (n = 288) were immunoreactive for at least one amino acid. A high proportion (39%-49%) were positive for two or three amino acids. About two-thirds (60%-69%) of the boutons contained GABA, of which more than half (51%-55%) also contained glycine. More than one-third (36%-38%) of the terminals were positive for glycine. Terminals immunoreactive for glycine alone were rare (0%-2%). About one-third of the terminals showed glutamate-immunoreactivity (32%-37%). GABA and/or glycine occurred in one-fifth to one-third of these. These results show that amino acid-immunoreactivity is present in almost all of the terminals that synapse on tyrosine hydroxylase-positive dendrites in locus coeruleus. Glutamate provides a major excitatory input. The almost complete colocalization of glycine with GABA suggests that the inhibitory input to locus coeruleus is predominantly GABAergic with a contribution from glycine in about half of the GABAergic boutons.     

Differential regulation of neuropeptide Y mRNA expression in the arcuate nucleus and locus coeruleus by stress and antidepressants

In rats, circulating corticosterone and insulin are involved in regulation of the hypothalamic neuropeptide Y (NPY) system, which in turn, is involved in regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Since the HPA axis and stress responsivity is altered in diseases such as depression, we investigated interactions between the effects of stress and antidepressant drug treatment on arcuate nucleus and locus coeruleus NPY mRNA expressions using in-situ hybridization histochemistry. After acute (2 h) and repeated immobilization (2 h daily, for 14 days), plasma concentrations of corticosterone increased, and those of insulin decreased. The expression of NPY mRNA was significantly increased in the arcuate nucleus, but was unchanged in the locus coeruleus following acute and repeated immobilization. Adrenalectomized rats with systemic corticosterone replacement (ADX+CORT), whose corticosterone concentration was maintained at approximately 50-100 ng/ml during repeated stress, showed a decrease in plasma insulin and an increase in arcuate nucleus NPY mRNA similar to that observed in sham rats, suggesting that changes in NPY mRNA levels are more closely tied to circulating insulin than to circulating corticosterone. In contrast, locus coeruleus NPY mRNA expressions in ADX+CORT rats were significantly higher than those in sham rats after repeated stress. Desmethylimipramine (DMI) treatment for 24 days did not affect basal plasma concentrations of corticosterone or insulin, or arcuate nucleus NPY mRNA expressions, but significantly decreased basal levels of locus coeruleus NPY mRNA compared to saline-treated rats. After repeated immobilization (2 h daily, for 4 days), DMI significantly reduced the stress-induced rise in locus coeruleus NPY mRNA levels, but potentiated the stress-induced rise in arcuate nucleus NPY mRNA expression. These results demonstrate that: (1) the increase in arcuate nucleus NPY mRNA expressions in stressed rats closely follows the decrease in plasma concentrations of insulin; (2) increases in NPY mRNA expressions occur in the absence of changes in plasma corticosterone; and (3) desipramine treatment potentiated the effect of stress on arcuate nucleus NPY mRNA expressions, but blocked the repeated stress-induced increase in locus coeruleus NPY mRNA expressions. Thus, NPY mRNA expression in the arcuate nucleus and the locus coeruleus is sensitive to the effects of stress and to the antidepressant drug desipramine, but the arcuate nucleus NPY system is regulated by different mechanisms than the locus coeruleus NPY system. The results provide further evidence for the importance of circulating insulin in the regulation of the arcuate nucleus NPY system.     

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.     

Synaptic contacts of substance P-immunoreactive axon terminals in the nucleus tractus solitarius onto neurons projecting to the caudal ventrolateral medulla oblongata in the rat

The possibility that substance P (SP)-immunoreactive axon terminals in the nucleus tractus solitarius (NTS) make synaptic contacts onto NTS neurons projecting to the catecholaminergic cell region in the caudal ventrolateral medulla oblongata (CVLM) was examined in the rat using a retrograde tract-tracing method combined with immunohistochemistry. After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex (WGA-HRP-gold), into the CVLM region where tyrosine hydroxylase-immunoreactive neurons were situated, many retrogradely labeled neurons were detected in the dorsal parts of the NTS, especially at levels between 1.0 mm caudal and 0.5 mm rostral to the obex. Immunoelectron microscopy revealed synaptic contacts between SP-immunoreactive axon terminals and WGA-HRP-gold-labeled neurons in the NTS. These findings indicated that SP regulates NTS neurons which project to the catecholaminergic cell region of the CVLM. ©1997 Elsevier Science B.V. All rights reserved.     

Substance P in the rat nucleus accumbens: ultrastructural localization in axon terminals and their relation to dopaminergic afferents

Dual labeling electron microscopic immunocytochemistry was used to investigate the cellular substrate for functional interactions between substance P (SP) and dopamine in the rat nucleus accumbens. Coronal vibratome sections from acrolein-fixed brains were sequentially processed for the localization of: (1) a rat monoclonal antiserum against SP identified by the peroxidase--anti-peroxidase immunocytochemical method, and (2) a rabbit polyclonal antiserum against tyrosine hydroxylase (TH) identified by immunoautoradiography. The monoclonal rat antiserum recognized principally SP, but also exhibited cross-reactivity with certain other tachykinins such as substance K. Terminals showing SP-like immunoreactivity (SPLI) were 0.2-1.5 microns in diameter and contained numerous small (30-40 nm), round vesicles; one or more large (80-150 nm), dense-core vesicles; and an occasional membrane-bound multivesicular body. From a total of 114 SP-labeled terminals that were quantitatively analyzed, 30.1% formed symmetric synapses with dendrites; whereas only 8% formed asymmetric junctions with dendritic spines. Terminals showing SPLI also occasionally formed junctions with dendrites receiving synaptic input from other terminals that were similarly labeled for the peptide or from terminals immunoautoradiographically labeled for TH. In contrast to the low frequency of postsynaptic relationships, 39.8% of the terminals containing SPLI showed close associations with other unlabeled or TH-labeled terminal or preterminal axons. The axonic contacts were characterized by equally spaced membranes that were not separated by glial processes. Within the terminals containing SPLI, vesicles were located near the axonic contacts; whereas vesicles in unlabeled terminals were located more distally with respect to these appositions. We conclude that in the rat nucleus accumbens SP or a closely related tachykinin subserves principally inhibitory functions at postsynaptic sites as indicated by the prominence of symmetric junctions. The abundance of axonic associations and sparsity of convergent input from TH- and SP-labeled terminals at closely spaced sites on dendrites supports the concepts that a SP-like tachykinin also may modulate the release of dopamine through direct or indirect presynaptic mechanisms. The possibility that there may be more extensive postsynaptic associations through convergence at widely spaced sites on common neurons is discussed.     

Evidence against specificity of electrical stimulation of the nucleus locus coeruleus in activating the sympathetic nervous system in the rat

Electrical stimulation of the central nucleus locus coeruleus (LC) was previously shown to increase activity of the peripheral sympathetic nervous system (SNS) as measured by increases in plasma levels of the norepinephrine (NE) metabolite 3-methoxy-4-hydroxyphenthyleneglycol (MHPG) in the rat⁵. Four experimental approaches were designed to test the specificity of the LC cell group in activating the SNS in the stimulation paradigm. Varying the stimulation current amplitude, varying the site of stimulating electrode placement, and electrolytic lesions of the LC yielded results consistent with the hypothesis that the site of SNS activation was within the anatomical region of the LC cell group. Neurochemical lesioning with intraventricular 6-hydroxydopamine, however, did not effectively block the plasma MHPG increase observed after stimulation of the LC region. The possibility that non-noradrenergic cells, fibers of passage, or terminals in the LC region of the midbrain may be responsible for SNS activation when the LC is electrically stimulated is discussed. These studies are pertinent to all studies of LC function which employ electrical stimulation of the LC nucleus, including investigations of the role of the LC in social behavior, intracranial self-stimulation, and blood pressure regulation.     

Agonist-induced internalization of corticotropin-releasing factor receptors in noradrenergic neurons of the rat locus coeruleus

Corticotropin-releasing factor (CRF) acts within the locus coeruleus (LC), to modulate activity of the LC-norepinephrine (NE) system. Combining molecular and cellular approaches, we demonstrate CRF receptor (CRFr) mRNA expression in Sprague-Dawley rat LC and provide the first in vivo evidence for agonist-induced internalization of CRFr. CRFr mRNA was detected in LC micropunches by RT-PCR. In dual labelling immunofluorescence studies, tyrosine hydroxylase (TH) containing neurons exhibited CRFr labelling. At the ultrastructural level, immunogold-silver labelling for CRFr was localized to the plasma membrane of TH-immunoperoxidase labelled dendrites. CRF (100 ng) injection into the LC produced a robust neuronal activation that peaked 10-15 min after injection and was maintained for the duration of the recording. This was associated with CRFr internalization in LC neurons that was apparent at 5 and 30 min after injection. By 5 min after injection the ratio of cytoplasmic to total dendritic CRFr-labelling was 0.81 +/- 0.01 in rats injected with CRF and 0.59 +/- 0.02 in rats injected with artificial cerebrospinal fluid (ACSF; P < 0.0001). Enhanced internalization of CRFr was maintained at 30 min after CRF injection, with the ratio being 0.86 +/- 0.02 for CRF-injected cases and 0.57 +/- 0.03 for ACSF-injected cases (P < 0.0001). Internalized CRFr was associated with early endosomes, indicative of degradation or recycling. Agonist-induced CRFr internalization in LC neurons may underlie acute desensitization to CRF or stress. This process may be a pivotal target by which stressors or pharmacological agents regulate the sensitivity of the LC-NE system to CRF and subsequent stressors.     

Ovarian hormone-induced reorganization of oxytocin-labeled dendrites and synapses lateral to the hypothalamic ventromedial nucleus in female rats

Central oxytocin (OT) modulates many social behaviors, including female rat sexual receptivity, quantified as the copulatory stance known as lordosis. The expression of the lordosis response is modulated by OT action in the ventromedial nucleus of the hypothalamus (VMH), as demonstrated by behavioral pharmacology experiments. However, the subcellular localization of OT in this brain region has been unclear. We tested the hypothesis that ovarian hormones reorganize OT-labeled pre- or postsynaptic elements in the fiber complex lateral to the VMH by using immunoelectron microscopy. OT immunolabeling occurred in axonal boutons identified by the presence of small, clear synaptic vesicles and double labeling with the presynaptic markers synaptophysin and vesicular glutamate transporter 2. OT immunoreactivity also was observed in dendritic profiles, verified with double labeling for the dendrite-specific marker microtubule-associated protein 2. Ovarian hormones did not alter the density of axonal boutons; however, estradiol treatment reduced the density of dendritic profiles by 34%. This effect was reversed when progesterone was given subsequent to estradiol. The effect of estradiol treatment was specific to dendrites that lacked OT immunostaining; the density of OT-labeled dendritic profiles remained constant during estradiol treatment. With the estradiol-induced exit of non-OT-labeled dendritic profiles, the remaining OT-labeled dendritic profiles experienced an increase in their number of synaptic contacts. Thus, hormone treatments that mimic the 4-day rat estrous cycle provoke a chemically coded reorganization of dendrite innervation in the fiber plexus lateral to the VMH that may underlie the hormone-specific effect of OT on reproductive behavior.     

Accurate three-dimensional reconstruction of neuronal distributions in brain: Reconstruction of the rat nucleus locus coeruleus

A technique is described which permits construction of accurate, quantified 3-dimensional maps of the distribution of neuronal cell groups in brain. The cartesian coordinates of landmarks and individual neurons are obtained from serial histological sections utilizing a computer-linked digitizing microscope. The digitized images of these sections are displayed on a computer graphics picture system where they are aligned so that spatial relationships within the nucleus are essentially identical to those of the intact brain. This is accomplished using information about landmarks obtained from photomicrotomy. As a consequence of the alignment procedure, each neuron is assigned a 3-dimensional coordinate representing its position in the reconstituted nucleus, and the reconstruction is oriented in a stereotaxic coordinate system. Nuclei from different brains can then be registered to one another, assigned coordinates relative to this standard coordinate space, and be compared statistically. Differences between nuclei in the spatial distribution of neurons in toto, or in the distribution of anatomically or physiologically defined subpopulations of neurons, can then be visualized with greater accuracy and in more detail than that permitted by traditional techniques. In addition, such comparisons can easily be quantified and statistically evaluated using, for example, analysis-of-variance techniques. For illustrative purposes, the technique is applied to the rat nucleus locus coeruleus as reconstructed from serial Nissl-stained sections.     

Ultrastructure of serotonin-immunoreactive terminals in the core and shell of the rat nucleus accumbens: Cellular substrates for interactions with catecholamine afferents

The nucleus accumbens is composed of a core region involved in motor functions and a shell region implicated in emotional and motivational processes. Both of these regions receive serotonin- and dopamine-containing afferents. We examined whether the serotonin innervation or relation to catecholamine (mainly dopamine) axons in the nucleus accumbens shows common features or specializations corresponding to the noted functional differences in core and shell subregions. To address this question, we examined the ultrastructure of serotonin-containing axons and their relation to catecholamine-containing afferents in either the core or shell of the nucleus accumbens. Single coronal sections through the rat forebrain were processed for immunoperoxidase labeling of serotonin and immunogold silver labeling of tyrosine hydroxylase, the catecholamine-synthesizing enzyme. Varicose processes showing peroxidase product for serotonin by light microscopy were confirmed to be axons and terminals by electron microscopy. In a quantitative analysis of serotonin-immunoreactive terminals forming one or more contacts in single sections, some common features were observed. For the core (n = 120) and the shell (n = 82), 41% formed synaptic junctions with unlabeled dendrites, 75% were in apposition with unlabeled terminals, which often formed asymmetric junctions, and 20% were in apposition with axons or terminals containing tyrosine hydroxylase. Thus, in both the core and shell of the nucleus accumbens, serotonin terminals synapse on postsynaptic neurons and are likely to modulate or be modulated by presynaptic interactions with excitatory axons forming asymmetric junctions and by catecholaminergic afferents. Marked differences in the morphology of serotonin axons were also seen in the core versus shell of the nucleus accumbens. By light microscopy, serotonin-immunoreactive axons were thicker and more varicose than those found in the core. Ultrastructural analysis confirmed that, in contrast to the core, serotonin-immunoreactive axons and terminals in the shell were larger in cross-sectional diameter size (0.7 μm vs. 0.3 μm). Additionally, serotonin axon terminals in the shell contained more numerous immunoreactive large dense core vesicles and more frequently formed symmetric as opposed to asymmetric contacts with dendrites. The larger size and more numerous dense core vesicles in serotonin-immunoreactive terminals in the shell support the concept that serotonin or co-existing neurotransmitter may be more tonically released in the shell versus core of the nucleus accumbens. © 1993 Wiley-Liss, Inc.     

Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals

In the nucleus raphe dorsalis of the cat, an electron microscopic immunocytochemistry method was used to identify the fine structure of serotoninergic dendritic profiles and axon terminals analyzed in serial sections. Two classes of serotoninergic dendrites were distinguished in the nucleus. The first class was constituted by conventional serotonin (5-HT) dendrites that were contacted by unlabeled axon terminals containing differing populations of synaptic vesicles. The second class consisted of serotoninergic dendrites that contained vesicles in their dendritic shafts. Such 5-HT dendrites were further subdivided into two groups according to their synaptic contacts. In some 5-HT vesicle-containing dendrites, the vesicles were densely packed in small clusters and were associated with a well-defined synaptic specialization. These dendrites were classified as serotoninergic presynaptic dendrites and established synaptic contacts with unlabeled and labeled dendrites and were contacted by unlabeled axon terminals. In other 5-HT vesicle-containing dendrites, extensive serial section examination showed that the vesicles could be observed near the membrane but were never found to be associated with any synaptic membrane specialization. Serotoninergic axon terminals that were presumed to be recurrent collaterals of 5-HT neurons were present in the nucleus. Some of them were observed in synaptic contact with dendrites or dendritic protrusions whereas others did not exhibit synaptic specializations. The existence of serotoninergic dendrodendritic synaptic contacts and axon terminals suggests direct local interactions between serotoninergic neurons within the nucleus raphe dorsalis.     

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.     

Identification of γ-aminobutyric acid-immunoreactive axon endings associated with mesencephalic periodontal afferent terminals and morphometry of the two types of terminals in the cat supratrigeminal nucleus

A previous study has shown that mesencephalic periodontal afferent terminals receive contacts more frequently from axonal endings containing pleomorphic, synaptic vesicles (P-endings) in the supratrigeminal nucleus (Vsup) than in the trigeminal motor nucleus, suggesting that interneurons in Vsup play an important role in modulating the jaw-closing reflex. The present study was attempted to identify neurotransmitters in P-endings associated with mesencephalic periodontal afferents in cat Vsup through the use of intracellular staining of horseradish peroxidase combined with the postembedding immunogold methods. A morphometric analysis was carried out to compare the ultrastructural features of these two types of terminals. Serial sections of 31 labeled boutons and of their associated 38 P-endings were examined. They were processed for postembedding immunogold labeling with antibodies to the neurotransmitter γ–aminobutyric acid (GABA). The 38 P-endings presynaptic to periodontal afferents showed GABA-like immunoreactivity, but the afferent terminals were free from the labeling. The morphometric analysis indicated that bouton volume, apposed surface area, total active zone size, and mitochondrial volume were smaller in GABA-immunoreactive P-endings than in periodontal afferents, but the pooled data of the two types of terminals showed that each synaptic parameter was highly correlated in a positive, linear manner with bouton volume. These observations provide evidence that P-endings presynaptic to mesencephalic periodontal afferents contain the neurotransmitter GABA and that their axoaxonic synapses are organized in accordance with the ultrastructural “size principle” proposed by Pierce and Mendell (Pierce and Mendell [1993] J. Neurosci. 13:4748–4763) on Ia-motoneuron synapses. J. Comp. Neurol. 389:127–138, 1997. © 1997 Wiley-Liss, Inc.     

Ultrastructural evidence of direct synaptic contact of catecholamine terminals with oxytocin-containing neurons in the parvocellular portion of the rat hypothalamic paraventricular nucleus

Ultrastructural evidence of the direct interaction between catecholamine (CA) terminals and oxytocin (OX)-containing neurons in the parvocellular portion of the rat hypothalamic paraventricular nucleus was demonstrated using immunohistochemistry combined with false transmitter (5-hydroxydopamine, 5-OHDA) histochemistry. At the parvocellular portion, 5-OHDA labelled CA terminals make synaptic contact with proximal dendrites or somas of OX-positive cells, suggesting that the ascending CA system monosynaptically regulates the extrahypothalamic OX system.     

Ultrastructure of glutamate and GABA immunoreactive axon terminals of the rat nucleus tractus solitarius, with a note on infralimbic cortex afferents

The principal fast neurotransmitters in the CNS are glutamate and GABA. Our aim was to provide a baseline account on the ultrastructure of the axon terminals immunoreactive to glutamate or GABA present in the nucleus tractus solitarius (NTS) of the rat. In addition, we wanted to complete our study of cortico-solitary afferents at the electron microscopic level, by analyzing the inputs from the infralimbic cortex. Using post-embedding immunogold, we found that nearly 61% of the axon terminals were glutamatergic, and 36% were GABAergic in the rat visceral NTS. In general, axons making asymmetric synaptic contacts were enriched in glutamate, compared to axons involved in symmetric synapses. In contrast, the vast majority of the GABAergic axon terminals made symmetric synaptic contacts. We could discern five types of glutamatergic and two types of GABAergic axon terminals that differed in their fine structure. Afferents from the infralimbic cortex were small, with clear synaptic vesicles and no dense core vesicles; they made asymmetric contacts with fine dendrites, and were glutamatergic. We conclude that most axon terminals in the NTS use glutamate or GABA as fast transmitters, in addition to being a heterogeneous population of morphological types.