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.     

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.     

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.     

Ultrastructural relationship between the AMPA-GluR2 receptor subunit and the mu-opioid receptor in the mouse central nucleus of the amygdala

Activation of GluR2-expressing non-calcium-permeable AMPA-type glutamate receptors in the central nucleus of the amygdala (CeA) may play an important role in integrating emotion and memory with goal-directed behaviors involved in opioid addiction. The location of non-calcium-permeable AMPA receptors within distinct neuronal compartments (i.e., soma, dendrite, or axon) is an important functional feature of these proteins; however, their ultrastructural location and subcellular relationship with mu-opioid receptors (μOR) in the CeA are unknown. Immunocytochemical electron microscopy was used to characterize the ultrastructural distribution of GluR2 and its association with μOR in the mouse CeA. A single-labeling analysis of GluR2 distribution employing immunoperoxidase or immunogold markers revealed that this protein was frequently affiliated with intracellular vesicular organelles, as well as the plasma membrane of CeA neuronal profiles. Among all GluR2-labeled neuronal structures, over 85% were dendrites or somata. Unlabeled axon terminals frequently formed asymmetric excitatory-type synaptic junctions with GluR2-labeled dendritic profiles. Dual-labeling immunocytochemical analysis showed that GluR2 and μOR were co-localized in neuronal compartments. Among all dual-labeled structures, approximately 80% were dendritic. Synaptic inputs to these dual-labeled dendrites were frequently from unlabeled axon terminals forming asymmetric excitatory-type synapses. The presence of GluR2 in dendritic profiles receiving asymmetric synapses suggests that activation of the non-calcium-permeable AMPA receptor plays a role in the postsynaptic modulation of excitatory signaling involving CeA neuronal circuits that coordinate sensory, affective, and behavioral processes involved in drug addiction. Given the critical role of non-calcium-permeable AMPA receptor function in neural and behavioral adaptability, their dendritic association with μOR in CeA dendrites provides a neuronal substrate for opioid-mediated plasticity.     

Dual ultrastructural localization of enkephalin and tyrosine hydroxylase immunoreactivity in the rat ventral tegmental area: multiple substrates for opiate-dopamine interactions.

Endogenous opiates modulate activity in the mesocorticolimbic dopaminergic system, and this interaction is thought to underlie major aspects of motoric, reward-seeking, and stress-coping behaviors. We sought to determine the ultrastructural substrate for this modulatory action at the level of dopaminergic perikarya in the rat ventral tegmental area (VTA). Using a dual-labeling, immunoperoxidase and immunogold-silver method, we localized antisera directed against leu5-enkephalin (ENK) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in acrolein-fixed sections through the VTA. ENK-like immunoreactivity (ENK-LI) was visualized within unmyelinated axons and in axon terminals. In terminals, ENK-LI was densely localized to one or more dense-cored vesicles and either densely or lightly detected surrounding small clear vesicles. Immunoreactive dense-cored vesicles were occasionally associated with the presynaptic specialization but were more frequently detected at distant sites along the plasmalemmal surface, often in apposition to astrocytic processes. ENK-immunoreactive terminals formed both symmetric and asymmetric synapses, most frequently on large proximal dendrites. Direct appositions without glial separation were also detected between terminals containing ENK-LI and other ENK-labeled or unlabeled terminals. In contrast to ENK-LI, immunolabeling for TH was primarily detected within perikarya and dendrites in the VTA. Of the ENK-immunoreactive terminals that formed synaptic contacts in single sections, approximately 50-60% were in association with TH-labeled dendrites. The remainder formed synapses on dendrites lacking detectable immunoreactivity for TH. Multiple ENK-immunoreactive terminals occasionally formed convergent synaptic contacts on single TH-labeled or unlabeled dendrites. Furthermore, individual ENK-labeled terminals sometimes formed divergent contacts on two TH-labeled or unlabeled dendrites. When a single ENK-immunoreactive terminal made dual synaptic contacts on TH-labeled dendrites, the latter were usually in close apposition to one another. These findings represent the first ultrastructural demonstration that opioid peptide-containing terminals provide a direct synaptic input to dopaminergic, as well as nondopaminergic, neurons in the VTA. In addition, the morphological evidence suggests that endogenous opioid peptides (1) may be released from nonsynaptic sites, (2) may modulate the release of transmitters from other terminals, and/or (3) may synchronize the activity of multiple neuronal targets in the VTA. These results provide a number of morphological substrates through which opiates may directly or indirectly regulate activity in mesocorticolimbic dopaminergic pathways.     

Neuropeptide Y-like immunoreactivity in neurons of the solitary tract nuclei: vesicular localization and synaptic input from GABAergic terminals

The ultrastructural localization of neuropeptide Y-like immunoreactivity (NPY-LI) was examined in the medial nuclei of the solitary tracts (mNTS) of adult rat brain. Peroxidase-antiperoxidase (PAP) reaction product was localized extensively to the central lumen of large (100-150 nm), dense-core vesicles. The labeled vesicles were seen in axon terminals of untreated, control animals and in perikarya and dendrites of rats receiving intraventricular injections of colchicine 24 h prior to sacrifice. The labeled terminals were of two types. The first type contained numerous small, clear vesicles that were rimmed with peroxidase product and 1-6 large, dense-core vesicles that were labeled throughout their central lumen. The second type contained a more homogeneous population of labeled large, dense-core vesicles. Axon terminals showing NPY-LI formed either asymmetric synapses with unlabeled dendrites or were without recognized junctions. Within labeled terminals, as well as within perikarya and dendrites, the majority of the dense-core vesicles were located near non-synaptic portions of the plasmalemma that were heavily ensheathed with glial processes. Only a few unlabeled terminals penetrated the glial investments to form synaptic contacts on soma or dendrites containing NPY-LI. These synaptic contacts were of both symmetric and asymmetric types. Combined immunoperoxidase labeling for glutamic acid decarboxylase and immunogold labeling for NPY further established that at least some of the terminals forming symmetric junctions on the NPY-immunoreactive dendrites were GABAergic. These results provide ultrastructural evidence that in the mNTS, NPY-LI is localized principally to large dense-vesicles within neurons whose output is partially regulated by GABA. The preferential distribution of the labeled vesicles along non-synaptic, glial-invested portions of the plasmalemma suggests that neuronal NPY may modulate the activity of nearby astrocytes. Additionally, the localization of NPY-LI in terminals containing a mixed population of synaptic vesicles and forming asymmetric axodendritic junctions suggests that NPY and/or coexisting transmitter may also exert certain known hypotensive effects by excitation of local intrinsic or projection neurons in this brain region.     

Ultrastructural characterization of substance P-like immunoreactive neurons in the rostral ventrolateral medulla in relation to neurons containing catecholamine-synthesizing enzymes

Substance P (SP) and catecholamines, particularly adrenaline, have been implicated in cardiovascular responses mediated by neurons in the rostral ventrolateral medulla (RVL). Immunoperoxidase labeling of an antiserum against SP and/or immunoautoradiographic localization of catecholamine (tyrosine hydroxylase-TH)- or adrenaline (phenylethanolamine N-methyltransferase-PNMT)-synthesizing enzymes were examined histologically to determine the cellular basis for a functional interaction involving either synaptic or intracellular relations between these putative transmitters in the adult rat RVL. Peroxidase labeling for SP was localized in perikarya, dendrites, and axon terminals. Most of these perikarya were located medial and ventral to those labeled with TH or PNMT within the same section. However, as others have previously demonstrated by light microscopy, colocalization of SP-like immunoreactivity (SPLI) and PNMT was seen in a few perikarya of colchicine treated animals. Both single- and dual-labeled perikarya contained abundant dense core vesicles. The terminals with SPLI were 0.4-1.4 micron in diameter and contained a few mitochondria, a large population of small, clear vesicles, and from three to 11 large dense core vesicles. In some cases the terminals were seen in continuity with more proximal processes of neurons in the RVL. These terminals formed synapses with a few perikarya and many dendrites, some of which also contained SPLI. In the material dually labeled for TH and SP, terminals with SPLI (n = 32) formed synaptic junctions primarily with TH-labeled dendrites (69%); the remainder were with TH-labeled perikarya (6%) or with unlabeled dendrites (25%). The axosomatic junctions were exclusively symmetric, whereas the majority of axodendritic junctions were primarily asymmetric on small dendrites (0.8-1.0 micron in diameter) or dendritic spines. In sections dually labeled for PNMT and SP, the terminals containing SPLI (n = 37) formed synaptic associations with PNMT-labeled perikarya (11%), PNMT-immunoreactive dendrites (59%), or with perikarya and dendrites lacking PNMT immunoreactivity (30%). The axosomatic junctions were all symmetric and most often associated with the spinous portion of the soma. The axodendritic junctions were primarily asymmetric and were found both on the spinous portion of the PNMT-labeled dendrites. In addition, both TH- and PNMT-labeled somata and dendrites received symmetric and asymmetric contacts from terminals lacking SPLI.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Ultrastructural localization of cannabinoid CB1 and mGluR5 receptors in the prefrontal cortex and amygdala

Stimulation of the postsynaptic metabotropic glutamate receptor mGluR5 triggers retrograde signaling of endocannabinoids that activate presynaptic cannabinoid CB1 receptors on juxtaposing axon terminals. To better understand the synaptic structure that supports mGluR5 mediation of CB1 activation in the prefrontal cortex (PFC) and basolateral amygdala (BLA), we examined electron microscopic dual immunolabeling of these receptors in the prelimbic PFC (prPFC) and BLA of adult male rats. CB1 immunoreactivity was detected in axon terminals that were typically large, complex, and contained dense-core and clear synaptic vesicles. Of terminals forming discernible synaptic specializations, 95% were symmetric inhibitory-type in the prPFC and 90% were inhibitory in the BLA. CB1-immunoreactive terminals frequently contacted dendrites containing mGluR5 adjacent to unlabeled terminals forming excitatory-type synapses. Because most CB1-containing terminals form inhibitory-type synapses, the unlabeled axon terminals forming asymmetric synapses are the likely source of the mGluR5 ligand glutamate. In the prPFC, serial section analysis revealed that GABAergic CB1-containing axon terminals targeted dendrites adjacent to glutamatergic axon terminals, often near dendritic bifurcations. These observations provide ultrastructural evidence that cortical CB1 receptors are strategically positioned for integration of synaptic signaling in response to stimulation of postsynaptic mGluR5 receptors and facilitation of heterosynaptic communication between multiple neurons.     

Ultrastructural examination of enkephalin and substance P input to cholinergic neurons within the rat neostriatum.

Enkephalin and substance P-containing inputs to cholinergic perikarya were examined in the rat neostriatum using an ultrastructural immunocytochemical double-labeling protocol. Sections of rat neostriatum were double-labeled for either choline acetyltransferase (ChAT) and substance P or ChAT and enkephalin using silver intensified colloidal gold and peroxidase as labels. Regions containing both ChAT-positive neurons and peroxidase reaction product were identified in the light microscope prior to sectioning for electron microscopy. Substance P-containing terminals which contained round synaptic vesicles and made symmetrical synaptic contacts were commonly observed in the neostriatum. Substance P synapses onto ChAT-positive perikarya and dendrites were frequently observed: up to 5 synaptic contacts were observed onto a ChAT-positive dendrite. Enkephalin labeling was also seen in a population of axon terminals containing round synaptic vesicles and exhibiting symmetrical synaptic specializations. In contrast to substance P-containing terminals, relatively few synaptic contacts were observed onto ChAT-positive labeled perikarya and dendrites although enkephalin-labeled terminals were seen in frequent contact with perikarya and dendrites of unlabeled spiny neurons. Since enkephalin and substance P are contained within different populations of striatal spiny neurons, the results of the present study suggest that these two types of neurons differ in their intrinsic striatal connections.     

Ultrastructural localization of neuropeptide Y Y1 receptors in the rat medial nucleus tractus solitarius: relationships with neuropeptide Y or catecholamine neurons

Neuropeptide Y (NPY) Y1 receptor (Y1-R) agonists influence cardiovascular regulation. These actions may involve NPY- and catecholamine-containing neurons in the medial nucleus of the solitary tract (mNTS), at the level of the area postrema. The cellular sites through which Y1-R agonists may interact with NPY and catecholamines in the mNTS, however, are not known. To determine potential sites of action for Y1-R agonists, and their relationship to NPY or catecholamines in the mNTS, we used electron microscopic immunocytochemistry for the detection of sequence-specific antipeptide antisera against Y1-R alone or in combination with antisera against NPY or the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Analyses were conducted in the rat mNTS, at the level of the area postrema. Y1-R was found mainly in small unmyelinated axons and axon terminals but also in some somata and dendrites as well as a small number of glia. Within axon terminals, labeling for Y1-R was often present on dense core vesicles and small synaptic vesicles as well as extrasynaptic areas of the plasmalemma. Some Y1-R-labeled terminals also contained NPY or TH, suggesting that agonists of Y1-R may influence the release of NPY or catecholamines in the mNTS. In addition, Y1-R was found in dendrites that received asymmetric excitatory-type synapses from unlabeled axon terminals. Some of these dendrites contained NPY or TH, which indicates that Y1-R may be targeted for functional activation within NPY- or catecholamine-expressing neurons in the mNTS. These results demonstrate that Y1-R is a presynaptic receptor in NPY- or catecholamine-containing axon terminals within the mNTS as well as a postsynaptic receptor on NPY- or catecholamine-containing neurons that are contacted by axon terminals that likely contain excitatory amino acid transmitters. Agonists of Y1-R in the mNTS may thus affect cardiovascular regulation by modulating NPY, catecholamine, and excitatory amino acid transmission.     

Immunocytochemical localization of enkephalin in the neostriatum of rat brain: A light and electron microscopic study

The peroxidase-antiperoxidase (PAP) immunocytochemical technique was used to determine the light and electron microscopic localization of antisera directed against either methionine [Met⁵]- or leucine [Leu⁵]-enkephalin in the neostriatum of brains from untreated rats. By light microscopy, neuronal perikarya and processes showing enkephalin-like immunoreactivity (ELI) were unevently distributed throughout the neostriatum. The greatest accumulation of neuronal structures showing ELI was in the ventro- and caudo- lateral portions of the nucleus. The labeled perikarya measured 10–15 μm in diameter and constituted about 15–20% of the total neurons in the enostriatum. By electron microscopy, examination of three areas from horizontal and coronal sections revealed no regional differences in types of neurons showing ELI or in their synaptic organization. All labeled neurons showed a relatively low intensity reaction product which was diffusely distributed throughout the perikarya and dendrites. The cytoplasm contained relatively few organelles, which included mitochondria, endoplasmic reticulum and numerous “alveolate” vesicles. The dendrites had many spiny processes which formed asymmetric synapses with unlabeled axon terminals containing all small clear vesicles. In contrast to the perikarya and dendrites a dense accumulation of reaction product was present in a few myelinated and numerous unmyelinated axons and axonal varicosties. Approximately 75% of the labeled varicosities did not form a specialized synaptic junction in a single plane of section. The remaining 25% of the labeled terminals formed asymmetric junctions primarily with unlabeled dendrites and rerely with unlabeled perikarya or axons. The morphology and synaptic relations of the neurons showing ELI suggest that they may belong to the general group of medium-sized spiny cells characterized in Golgi studies by Kemp and Powell ('71a). At least some of the peptide-containing neurons may also have a myelinated efferent axon.     

Ultrastructural immunocytochemical localization of μ opioid receptors and Leu5-enkephalin in the patch compartment of the rat caudate-putamen nucleus

To delineate the cellular sites for the motor effects of opiates acting at the μ opioid receptor (MOR) in the rat caudate-putamen nucleus, we examined the ultrastructural immunogold and immunoperoxidase labeling of an antipeptide antiserum specific for the MOR. We also combined these labeling methods to examine the subcellular relationship between the MOR and the endogenous opioid peptide, Leu⁵-enkephalin (LE). By light microscopy, MOR-labeling was seen in a heterogeneous patchy distribution. Electron microscopic analysis of these patches showed that more than 80% of the total neuronal profiles (n = 1,586) containing MOR-like immunoreactivity (MOR-IR) were dendrites and dendritic spines. The remaining labeled profiles included a few perikarya and many axon terminals. MOR-IR was predominantly localized to extrasynaptic plasma membranes of dendrites, and to both synaptic vesicles and plasma membranes in terminals. Ten percent of the total MOR-labeled terminals (n = 272) formed asymmetric synapses with unlabeled or MOR-labeled dendritic spines. Terminals containing LE-IR formed synapses, in almost equal proportions, on MOR-labeled dendrites and dendritic spines, while over 80% of the unlabeled terminals formed synapses on MOR-labeled dendritic spines. Moreover, colocalization of MOR- and LE-IR was often seen in both dendrites and terminals. These results indicate that in patch compartments of the caudate-putamen nucleus, the MOR is mainly involved in extrasynaptic modulation of spiny neurons, including those that contain LE. In addition, the findings provide a cellular basis for presynaptic opioid modulation of neurotransmitter release through MOR located on axon terminals. © 1996 Wiley-Liss, Inc.     

Pre- and postsynaptic sites for serotonin modulation of GABA-containing neurons in the shell region of the rat nucleus accumbens

The shell of the nucleus accumbens receives a dense serotonergic innervation and contains abundant gamma-aminobutyric acid (GABA)-immunoreactive neurons. Moreover, serotonin (5-hydroxytryptamine: 5-HT) and GABA have been implicated in a variety of common motivational and motor-related functions partially ascribed to this brain area. We used immunoelectron microscopy of antisera directed against 5-HT and GABA in the same section of tissue to examine whether there were cellular substrates that might indicate more specific sites for functional interactions involving these transmitters in the shell region of the rat nucleus accumbens. Immunogold-silver labeling for GABA was localized to perikarya, dendrites, axons and axon terminals, whereas immunoperoxidase labeling for 5-HT was restricted to axons and axon terminals. Approximately half (187/366) of the 5-HT-immunoreactive axon terminals apposed or formed synaptic junctions with postsynaptic neurons. These junctions were mainly of the symmetric-type (83/187) characteristic of inhibitory transmitters, and were equally prevalent on dendrites with and without detectable gold-silver labeling for GABA. Of the 187 5-HT-labeled axon terminals with recognized synaptic contacts, 36% also showed convergence on a common dendrite with a GABA-labeled axon terminal. In addition, 5-HT- and GABA-immunoreactive axon terminals were commonly (83/366) identified in direct apposition to one another. Within a single plane of section, 41% of the apposed GABA-immunoreactive axon terminals formed symmetric-type junctions with dendrites or somata, whereas, the apposed 5-HT-labeled axon terminals rarely showed postsynaptic contacts. These results indicate that 5-HT-containing axon terminals may postsynaptically inhibit GABAergic neurons and their targets within the shell of the rat nucleus accumbens. Additionally, our results strongly suggest that, in this brain region, appositions between 5-HT and GABA axons and axon terminals may facilitate presynaptic interactions between these transmitter systems. © 1996 Wiley-Liss, Inc.     

Ultrastructural localization of phenylethanolamine N-methyltransferase-like immunoreactivity in the rat locus coeruleus

Adrenergic afferents from the rostral ventrolateral medulla are known to modulate the activity of noradrenergic neurons of the locus coeruleus (LC). The light and electron microscopic localization of a polyclonal antiserum directed against the adrenaline synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) was used to determine the identity and targets of the adrenergic afferents to the LC of the rat brain. By light microscopy, varicose processes showing intense PNMT-like immunoreactivity (LI) were seen throughout the neuropil surrounding neuronal perikarya which in adjacent sections were shown to contain immunoreactivity for the noradrenaline synthesizing enzyme, dopamine-beta-hydroxylase. Electron microscopy confirmed that these labeled varicose processes were primarily axon terminals. Terminals containing PNMT-LI constituted 30% (141 out of 464) of all identifiable terminals within the LC. These terminals were 0.5-1.8 micron in diameter and contained many small, clear and from 2 to 10 larger dense-core vesicles. The targets of the terminals with PNMT-LI were principally unlabeled (i.e. non-PNMT-containing) perikarya and dendrites. The synaptic junctions on perikarya were rare and exclusively symmetric; whereas, those on proximal (large) dendrites were somewhat more numerous and included symmetric as well as asymmetric membrane specializations. However, the vast majority (85% from a total of 141) of the terminals with PNMT-LI formed asymmetric synaptic junctions on unlabeled distal (small) dendrites and dendritic spines. In rare instances, the PNMT-immunoreactive terminals also formed synaptic junctions with other similarly labeled terminals. These findings provide the first ultrastructural evidence that adrenergic terminals in the LC (1) are one of the more prevalent synaptic inputs to the principally noradrenergic neurons; (2) have both symmetric and asymmetric synaptic specializations conventionally associated with inhibition and excitation, respectively; and (3) may modulate other adrenergic terminals through presynaptic mechanisms. In addition to the varicose processes, light microscopy revealed diffuse PNMT-LI throughout the LC. The ultrastructural correlate of this labeling was seen as patches of peroxidase product within the cytoplasm of a few perikarya and dendrites and throughout the cytoplasm of astrocytes identified by their discrete bundles of microfilaments. The detection of PNMT-LI in cells that are not known to synthesize adrenaline is surprising and suggests either a functional diversity for PNMT or amino acid sequence homologies with related enzymes which are enriched in the LC.     

Post-embedding immunogold labeling of gamma-aminobutyric acid in lamina II of the spinal trigeminal subnucleus pars caudalis: I. A qualitative study

This study examines the normal synaptic organization of the feline spinal trigeminal nucleus pars caudalis (PC). A primary goal of this study is to identify and characterize the synaptic complexes within PC based on their specific neurotransmitter content. Post-embedding immunogold techniques are utilized with electron microscopy to determine the ultrastructural localization of gamma-aminobutyric acid (GABA) immunoreactivity within lamina II of PC. The colloidal gold particles (10 nm) are randomly distributed over immunoreactive (IR) profiles without preference toward membranous or cytoplasmic regions. GABA immunoreactivity occurs on small unmyelinated axons, on terminals which form synaptic contacts, and on some vesicle-containing dendrites. The GABA-IR terminals form symmetric (type II) contacts onto unlabeled somata and dendrites of various sizes, and onto other unlabeled axon terminals. The GABA-IR terminal in axo-axonic complexes is presynaptic to a round vesicle-containing terminal, which itself may form a type I asymmetric contact onto an unlabeled dendrite or soma. A proportion of vesicle-containing dendrites show GABA-immunoreactivity and are postsynaptic to unlabeled terminals with round vesicles. Other, but far fewer, vesicle-containing dendrites are GABA negative and postsynaptic to GABA-IR terminals. In summary, the findings are consistent with the localization of GABA in intrinsic neurons, and may be associated with presynaptic and postsynaptic inhibition within nociceptive related pathways. © 1996 Wiley-Liss, Inc.     

Electron microscopy of immunoreactivity patterns for glutamate and γ-aminobutyric acid in synaptic glomeruli of the feline spinal trigeminal nucleus (subnucleus caudalis)

We studied the ultrastructure of the synaptic organization in the feline spinal trigeminal nucleus, emphasizing specific neurotransmitter patterns within lamina II of the pars caudalis/medullary dorsal horn. Normal adults were perfused, and Vibratome sections from pars caudalis were processed for electron microscopy. Ultrathin sections were reacted with antibodies for the excitatory neurotransmitter glutamate (Glu) and for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) by using postembedding immunogold techniques. Both single- and double-labeled preparations were examined. Results with single labeling show that Glu-immunoreactive terminals have round synaptic vesicles and form asymmetric synaptic contacts onto dendrites. GABA-immunoreactive axon terminals and vesicle-containing dendrites have pleomorphic vesicles, and the axon terminals form symmetric contacts onto dendrites and other axons. Double labeling on a single section shows glomeruli with central Glu-immunoreactive terminals that are presynaptic to dendrites, including GABA⁺ vesicle-containing dendrites. These Glu⁺ terminals are also postsynaptic to GABA⁺ axon terminals, and these GABA-immunoreactive terminals may also be presynaptic to the GABA⁺ vesicle-containing dendrites. Quantitative analyses confirm the specificity of the Glu and GABA immunoreactivities seen in the various glomerular profiles. The results suggest that a subpopulation of Glu-immunoreactive primary afferents (excitatory) may be under the direct synaptic influence of a GABA-immunoreactive intrinsic pathway (inhibitory) by both presynaptic and postsynaptic mechanisms. © 1996 Wiley-Liss, Inc.     

Neuropeptide Y in the rat nucleus accumbens: ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals

The ultrastructure, afferent input, and sites of termination of neurons containing neuropeptide Y-like immunoreactivity (NPY-LI) were examined in the adult rat nucleus accumbens by using the peroxidase-antiperoxidase (PAP) method. The NPY-LI was seen in sparsely distributed, spindle-shaped perikarya having cross-sectional diameters of 15-20 microns. These perikarya exhibited highly invaginated nuclear membranes and thin rims of cytoplasm containing Golgi lamellae, dense-core vesicles, and other organelles. A few large, principally aspiny, dendrites also showed NPY-LI. The dendrites received synaptic input from unlabeled terminals forming both symmetric and asymmetric junctions. Immunolabeling for NPY was evident in other processes that were not clearly differentiated as dendrites or axons. These were seen primarily near glial processes and the basal laminae of blood vessels. A few myelinated and many unmyelinated axons and axon terminals also were labeled for NPY. These terminals contained numerous, small (40-60 nm), clear and one or more large (80-100 nm) dense core vesicles. Forty-seven percent (27 out of 57) of the terminals containing NPY-LI formed symmetric junctions with unlabeled dendrites or dendritic spines. The remainder lacked recognizable densities within single planes of section. The neurons exhibiting NPY-LI in the nucleus accumbens were characterized further with respect to their afferent input from terminals labeled for the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). Immunogold labeling of a rabbit antiserum against NPY and PAP labeling for a sheep antiserum to GAD were sequentially applied to the same sections. The GAD-labeled terminals formed symmetric junctions primarily with the more numerous unlabeled dendrites. However, a few synaptic junctions also were detected between the GAD-labeled terminals and dendrites showing immunogold labeling for NPY. We conclude (1) that in the rat nucleus accumbens, NPY-LI is found principally in neurons of the aspiny type and (2) that the output from these presumably intrinsic neurons to other neighboring neurons or blood vessels is at least partially modulated by GABA.     

Ultrastructural localization of δ-opioid receptor and Met-enkephalin immunoreactivity in rat insular cortex

The insular cortex has been implicated in the reinforcing properties of opiates as well as in the integration of responses to sensory-motor stimulation. Moreover, the δ-opioid receptor (DOR) and the endogenous opioid ligand, Met⁵-enkephalin (ENK) are known to be prominently distributed in insular limbic cortex. To examine the anatomical sites for opioid activation of DOR in rat insular cortex, we used immunoperoxidase for detection of an antiserum raised against a peptide sequence unique to the DOR alone, and in combination with immunogold-silver labeling for ENK. Light microscopy showed intense DOR-like immunoreactivity (DOR-LI) in pyramidal cells and interneurons in deep laminae, and in varicose processes in both superficial and deep layers of the insular cortex. Ultrastructural analysis of layers V and VI in insular cortex showed that the most prominent immunoperoxidase labeling for DOR was in dendrites. This labeling was associated with asymmetric excitatory-type junctions postsynaptic to unlabeled terminals. Dendritic DOR-LI was also distributed along selective portions of non-synaptic plasma membranes and subsurface organelles. In dually labeled sections, dendrites containing DOR-LI sometimes received synaptic input from ENK-labeled terminals or more infrequently colocalized with ENK. Other axon terminals were exclusively immunolabeled for DOR or more rarely contained both DOR and ENK immunoreactivity. Within labeled axon terminals, distinct segments of the plasma membrane and membranes of immediately adjacent synaptic vesicles showed the largest accumulation of the peroxidase reaction product for DOR. These results indicate that in rat insular cortex DOR is primarily heteroreceptive, but also serves an autoreceptive function on certain ENK-containing neurons. Our results also provide the first ultrastructural evidence that in rat insular cortex endogenous opioids interact through the DOR (1) to modulate the postsynaptic responses to other excitatory afferents and (2) to presynaptically regulate the release of other neurotransmitters. The modulatory actions on both ENK-containing and non-ENK-containing neurons may contribute significantly to the reinforcing properties of exogenous opiates acting on the DOR in limbic cortex.     

Neurotensin in the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity

We sought to determine (1) the ultrastructural localization and (2) the extrinsic sources of neurotensin-like immunoreactivity (NTLI) in the parabrachial region (PBR). The brains from untreated adult male rats and from others that received intraventricular injections of colchicine (100 micrograms/7.5 microliters saline) 24 hours prior to death were fixed by perfusion with acrolein or glutaraldehyde and paraformaldehyde. Coronal sections were immunocytochemically labeled with a polyclonal rabbit antiserum to neurotensin and the PAP method. Western dot-blots and immunocytochemical labeling with adsorbed antiserum revealed significant cross-reaction only against NT, NT8-13, and glutamine (Gln)4-NT. In the ultrastructural study, the most numerous labeled profiles were axons and axon terminals in both colchicine-treated and control animals. The terminals containing NTLI were characterized by a mixed population of small, clear and large, dense core vesicles; asymmetric junctions principally with unlabeled dendrites; and a few synaptic specializations with unlabeled axon terminals. Compared to axon terminals, relatively few perikarya or dendrites had detectable levels of NTLI in either untreated or colchicine-treated animals. The labeled perikarya measured 8-10 microns in longest cross-sectional diameter, contained NTLI throughout a narrow rim of cytoplasm, and received a few somatic synapses from unlabeled terminals. From the relative density of axon terminals and sparsity of perikarya and dendrites, we conclude that the NTLI in the PBR is principally derived from extrinsic neurons. However, the intrinsic neurons with NTLI may also contribute to the immunoreactivity in the axon terminals of the PBR. We sought to determine the precise location of the extrinsic neurons that contribute to the NTLI in axon terminals in the PBR. Following unilateral injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), dual labeling was most evident in a large population of neurons located in the dorsal, medial and commissural nuclei of the solitary tracts, ipsilateral to the side of the injection. However, a few perikarya containing both the retrogradely transported WGA-HRP and immunocytochemical labels for NT were also detected in the caudal ventrolateral reticular formation, the locus coeruleus, and the paraventricular and lateral hypothalamic nuclei. We conclude that (1) NT or a closely related peptide is present in intrinsic neurons and multiple afferent pathways to the PBR; and (2) the axon terminals with NTLI have synaptic interactions with dendrites of intrinsic neurons and with axon terminals that may have either extrinsic or intrinsic origins.