Evidence for the coexistence of glutamate decar☐ylase and Met-enkephalin immunoreactivities in axon terminals of rat ventral pallidum

Evidence for the coexistence of glutamate decarboxylase (GAD) and Met-enkephalin (Met-Enk) in axon terminals of ventral pallidum was demonstrated by colocalization of anti-GAD and anti-Met-Enk immunoreactivities in alternate adjacent 1 micron serial sections. Conventional electron microscopy of immunostained ventral pallidum confirmed that the immunoreactive structures were boutons which made predominantly symmetrical synapses on ventral pallidal cell bodies and dendrites.     

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.     

Electron microscopic evidence for coexistence of leucine-enkephalin and γ-aminobutyric acid in a subpopulation of axon terminals in the rat locus coeruleus region

We recently described ultrastructural evidence for morphologically heterogeneous axon terminals containing the endogenous opioid peptide, methionine⁵-enkephalin (ENK), that formed synapses with neurons containing the catecholamine synthesizing enzyme, tyrosine hydroxylase, in the locus coeruleus (LC) of the rat brain. The morphological characteristics of these terminals suggested that ENK may be co-localized with either an excitatory or inhibitory amino acid. To further test this hypothesis, we combined immunogold-silver localization of γ-aminobutyric acid (GABA) and immunoperoxidase labeling for ENK in single sections through the LC, in the present study, to determine whether ENK and GABA were contained within single axon terminals. Light microscopic analysis of ENK and GABA immunoreactivities in the LC indicated that both transmitters were enriched in the dorsal pons. Although electron microscopy revealed that ENK and GABA were located primarily in axon terminals, some dendrites also contained immunolabeling for GABA. The dense core vesicles were consistently the most immunoreactive in ENK containing axon terminals and were identified toward the periphery of the axon terminal distal to the synaptic specialization. Axon terminals containing either ENK or GABA immunoreactivities contained pleomorphic vesicles as well as large dense core vesicles, varied in size and formed heterogeneous types of synaptic specializations (i.e. asymmetric vs. symmetric). Approximately 38% (n=76) of the axon terminals containing ENK immunoreactivity (n=200) also contained GABA. Some axon terminals containing peroxidase labeling for ENK (22%; n=44) converged on common targets with GABA-labeled axon terminals. Finally, a few ENK-labeled axon terminals (14%; n=28) formed asymmetric (excitatory-type) synapses with dendrites containing gold-silver labeling for GABA. The results, therefore, indicate that the opioid peptide, ENK, and the inhibitory amino acid, GABA, may influence LC neurons by concerted actions via (1) release from a common axon terminal, and (2) via separate sets of afferents converging on similar portions of the plasmalemma of target neurons. Furthermore, these studies also suggest a cellular substrate for opioid inhibition of LC neurons via activation (i.e. asymmetric synapses) of inhibitory GABAergic neurons. Future studies are required to determine whether the receptive sites for ENK and GABA are located at similar sites on the plasma membranes of LC neurons pre- or postsynaptically and whether there is differential release of either transmitter from single terminals in the LC.     

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.     

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.     

Peptide coexistence in axon terminals within the superficial dorsal horn of the rat spinal cord.

The somata of primary sensory neurons have been shown to contain up to four (and possibly more) neuroactive peptides. Although each of these peptides has been separately located in axon terminals within the superficial dorsal horn of the spinal cord, it is not clear whether multiple peptide coexistence is also a feature of terminal varicosities. The aim of this study was to determine whether the peptides substance P (SP) and calcitonin gene-related peptide (CGRP), which are colocalized in the somata of a large number of primary sensory neurons, coexist in the central terminals of these neurons in the spinal cord. The protein A-gold technique of antigen localization was used to screen single boutons in laminae I and II of the rats spinal cord for SP- and CGRP-like immunoreactivity at the ultrastructural level. The results show that SP and CGRP are colocalized within a large number of synaptic boutons in the superficial dorsal horn. Furthermore, evidence was obtained to suggest that both SP and CGRP may be found in the same synaptic vesicle within these boutons. These findings indicate that both SP and CGRP may be coreleased from single terminals in the superficial dorsal horn. This is of considerable interest in view of the reported interaction between SP and CGRP in nociceptive behavioral responses in the rat.     

Light-microscopic localization of somatostatin binding sites in the locus coeruleus of the rat

Somatostatin (SS14) binding sites within locus coeruleus (LC) were localized at the light microscope level by [¹²⁵I][Tyr⁰,d-Trp⁸]SS14 radioautography combined with an immunohistochemical/neurotoxic lesioning approach. In intact rats, the dense accumulation of SS14 binding sites of LC conspicuously overlapped with the cluster of tyrosine hydroxylase (TH) immunoreactive neurons; SS14 specific binding was directly proportional to the number of TH immunostained (TH+) cell bodies per mg of tissue throughout LC. Complete lesion of catecholaminergic nerve cell bodies of LC by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA) resulted in the total abolition of SS14 specific binding in the structure. In addition, specifically bound [¹²⁵I][Tyr⁰,d-Trp⁸]SS14 and TH+ cell density were quantified serially in a set of rats bearing various partial neurotoxic lesions; a highly significant correlation was found between the two parameters at each of the 16 coronal levels of LC examined. The coefficient of proportionality was identical at all levels. These results strongly suggest that somatostatin binding sites are uniformly localized on all noradrenergic neurons of LC.     

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.     

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.     

Molecular and cellular mechanisms of opiate action: Studies in the rat locus coeruleus

We have studied the molecular and cellular mechanisms underlying the acute and chronic effects of opiates on neurons of the rat locus coeruleus (LC). Acutely, opiates inhibit LC neurons by activating K⁺ channels and inhibiting a novel sodium-dependent inward current. Both of these actions are mediated via pertussis toxin-sensitive G-proteins, and regulation of the sodium current occurs through inhibition of the cyclic AMP pathway. In contrast to the acute effects of opiates, chronic treatment of rats with opiates increases levels of specific G-protein subunits, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the possibility that this upregulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these noradrenergic LC neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms by which opiates regulate the expression of these intracellular messenger proteins in the LC. These studies will lead to an improved understanding of the molecular and cellular basis of opiate addiction.     

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.     

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.     

Distribution of alpha-1 and alpha-2 binding sites in the rat locus coeruleus.

Precise anatomical distribution of alpha-1 and alpha-2 adrenergic binding sites has been investigated in the rat locus coeruleus (LC) using quantitative radioautography of brain sections incubated with 3H-prazosin or 3H-idazoxan. Distribution patterns of 3H-prazosin (alpha-1 sites) and 3H-idazoxan (alpha-2 sites) were heterogeneous and different along a postero-anterior axis in the LC. Comparison between distribution of alpha-2 binding sites and noradrenergic (NA) cellular density suggests that at least a fraction of these sites might be localized on NA perikarya or dendrites in this structure. Quantitative estimations of the binding parameters along this postero-anterior axis in the LC have revealed that the heterogeneous distributions of alpha-1 and alpha-2 binding sites are due not only to variations in the maximal densities of sites but also to variations in the affinities of these sites for their respective ligand.     

Glutamic acid decar☐ylase and enkephalin immunoreactive axon terminals in the rat neostriatum synapse with striatonigral neurons

Synaptic interactions between striatal projection neurons and axon terminals containing immunoreactive glutamic acid decar☐ylase (GAD) or Leu-enkephalin were examined in the rat neostriatum using a combined method of horseradish peroxidase retrograde transport from the substantia nigra and immunohistochemistry at the electron microscopic level. Results showed that numerous immunoreactive GAD and enkephalin boutons formed synapses with the cell bodies and dendrites of medium-sized striatonigral neurons. These findings demonstrate that within the neostriatum GABA and enkephalin directly influence caudate output pathways.     

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.     

Presence of NMDA-type glutamate receptors in cingulate corticostriatal terminals and their postsynaptic targets

The glutamatergic projection from the anterior cingulate cortex to the medial caudate-putamen nucleus (CPN) has been implicated in motor and cognitive functions, many of which are potently modulated by activation of N-methyl-D-aspartate subtype of glutamate receptors (NMDARs). To determine the functional sites for NMDAR activation within this circuitry, we combined anterograde transport of biotinylated dextran amine (BDA) from deep layers of the rat anterior cingulate cortex with immunogold labeling of NMDAR subunit, NMDAR1, in the dorsomedial CPN. BDA-containing axons were seen in patch-like clusters in a neuropil that showed more uniform immunogold-silver labeling for NMDAR1. Electron microscopy of these regions showed that BDA-labeling was present exclusively in axons and terminals, 23% (98 of 421) of which also contained NMDAR1-immunoreactivity (IR). BDA-labeled terminals often apposed NMDAR1-immunoreactive neuronal and glial profiles. These terminals also formed asymmetric excitatory-type synapses with dendritic spines. Of 155 anterogradely labeled axon terminals forming asymmetric synapses, 34% were with NMDAR1-labeled, and 66% with unlabeled dendritic spines. These results provide ultrastructural evidence for the involvement of NMDARs in presynaptic regulation of glutamate transmission, and in postsynaptic modulation of the excitability of spiny neurons in patch-like compartments of the dorsomedial CPN. These dual NMDAR-mediated actions are likely to play a major role in the acquisition of new behaviors and reward-related processes that have been associated with cortical input to the striatal patch compartments.     

Evidence for functional contact between cografted locus coeruleus and spinal cord in oculo: electrophysiological studies

The functional consequences of the locus coeruleus innervation of the spinal cord are not yet clearly understood. In a recent histological study it was shown that intraocular spinal cord grafts will become innervated by tyrosine hydroxylase-positive nerve fibers from a cografted locus coeruleus. In the present study we use this intraocular model of the descending coeruleo-spinal pathway to investigate functional contact between locus coeruleus and the spinal cord. We have pharmacologically characterized the receptor mediation of norepinephrine-induced, as well as locus coeruleus-mediated depressions of spinal cord neurons grafted in oculo. We found that electrical stimulation of the locus coeruleus part of the double grafts predominantly caused an inhibition of cografted spinal cord neurons. Norepinephrine-induced inhibition of the firing rate of single grafted spinal cord neurons was antagonized by phentolamine, an alpha-adrenergic antagonist, but was unaffected by timolol, a beta-adrenergic antagonist. Similarly, inhibition of the firing rate of grafted spinal cord neurons by stimulation of cografted locus coeruleus was antagonized by phentolamine but not by timolol. Interestingly, single spinal cord grafts were more sensitive to the depressant effects of perfused norepinephrine than was the spinal cord cografted with locus coeruleus. We conclude that spinal cord grafts can be functionally innervated by cografted locus coeruleus and that the noradrenergic inputs to spinal cord from cografted locus coeruleus are alpha-adrenergically mediated. Furthermore, the postsynaptic receptors in single spinal cord grafts appear to be supersensitive to norepinephrine application.     

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.     

Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats

The localization of the cholinergic neurons in the lower thoracic segments of the spinal cord of rats was examined by a monoclonal antibody against choline acetyltransferase (ChAT). The ChAT-immunoreactive neurons were located in the intermediate as well as anterior gray matters. In the intermediate gray the highest incidence of the immunoreactive neurons was in the nucleus intermediolateralis, followed by the nucleus intercalatus pars paraependymalis and a few immunoreactive neurons were seen in the nucleus intercalatus proprius. In the sequential immunostaining of one and the same section of the spinal cord pretreated with colchicine using the ChAT antibody and a polyclonal antibody against methionine-enkephalin-argynine-glycine-leucine (Met-Enk-Arg-Gly-Leu), substantial numbers of neurons were immunostained simultaneously by the two antibodies in the intermediate gray matter. The present finding gives strong evidence for the coexistence of acetylcholine and enkephalins in, at least, some of the preganglionic neurons projecting their axons to the periphery.     

Multiple substrates for serotonergic modulation of rat locus coeruleus neurons and relationships with kainate receptors

The ultrastructural substrates of serotonin (5-hydroxytryptamine [5-HT]) immunoreactive terminals with respect to noradrenergic neurons of the locus coeruleus (LC) have only been suggested from immunocytochemical analysis in adjacent tissue sections using antisera directed against tryptophan and tyrosine hydroxylase (TH) enzymes. Here, we conducted dual immunoelectron microscopy in the same section of tissue using antisera directed against 5-HT and TH to determine cellular substrates for proposed interactions between these two transmitter systems. Axon terminals containing peroxidase labeling for 5-HT possessed small clear as well as large dense core vesicles. Of 176 5-HT-labeled axons and terminals, 19% contacted TH-labeled dendrites. When a synaptic specialization was detectable, it was more often of the asymmetric type. Electrophysiological studies have also shown that 5-HT selectively attenuates excitatory amino acid-induced activation of neurons in the LC. Thus, to further examine the cellular relationship between 5-HT-labeled axon terminals and excitatory amino acid receptors, we conducted immunogold-silver labeling of an antibody which recognized the three identified members of the kainate receptor (KAr) subunit class (GluR 5,6,7) and peroxidase localization of 5-HT. Similar proportions of 5-HT-labeled terminals (9%) were either apposed to KAr-labeled dendrites or exhibited KAr immunoreactivity. Twenty-four percent of the 5-HT axon terminals examined were apposed by glial processes that contained KAr. These data indicate that 5-HT axon terminals are in direct contact with LC neurons and also suggest pre- and postsynaptic sites for modulation of 5-HT terminals by excitatory amino acid ligands as well as indirect sites via glial processes.