Forward into our next century—A letter from the editor-in-chief to the readers of the Journal of Comparative Neurology

Dynorphin facilitates conditioned place aversion and reduces locomotor activity through mechanisms potentially involving direct activation of target neurons or release of catecholamines from afferents in the nucleus accumbens. We examined the ultrastructural substrates underlying these actions by combining immunoperoxidase labeling for dynorphin 1–8 and immunogold silver labeling for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). The two markers were simultaneously visualized in single coronal sections through the rat nucleus accumbens. By light microscopy, dynorphin immunoreactivity was seen as patches of immunoreactive varicosities throughout all rostrocaudal levels of the nucleus accumbens. The dynorphin-immunoreactive terminals identified by electron microscopy ranged from 0.2 to 1.5 μm in cross-sectional diameter, contained numerous small (30–40 nm) clear vesicles, as well as one or more large (80–100 nm) dense core vesicles. From the dynorphin-immunoreactive terminals quantitatively examined in single sections, 74% (173/370) showed symmetric synaptic junctions mainly with large unlabeled dendrites. Of the dynorphin-immunoreactive terminals forming identifiable synapses, approximately 30% contacted more than one dendritic target. In addition, single dendrites frequently received convergent input from more than one dynorphin-labeled terminal. Irrespective of their dendritic associations, dynorphin-immunoreactive terminals also frequently showed close appositions with other axons and terminals; these included unlabeled (41%), TH-labeled (10%) or dynorphin-labeled axons (14%). In contrast to dynorphin-immunoreactive terminals, TH-labeled terminals formed primarily symmetric synapses with small dendrites and spines or lacked recognizable specializations in the plane of section analyzed. In some cases, single dendrites were postsynaptic to both dynorphin and TH-immunoreactive terminals. We conclude that dynorphin-immunoreactive terminals potently modulate, and most likely inhibit, target neurons in both subregions of the rat nucleus accumbens. This modulatory action could attenuate or potentiate incoming catecholamine signals on more distal dendrites of the accumbens neurons. The findings also suggest potential sites for presynaptic modulatory interactions involving dynorphin and catecholamine or other transmitters in apposed terminals.     

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.     

Dynorphin-immunoreactive neurons in the rat nucleus accumbens: Ultrastructure and synaptic input from terminals containing substance P and/or dynorphin

The endogenous opioid peptide dynorphin is enriched in neurons in the nucleus accumbens, for which coexistence and synaptic interactions with substance P have been postulated. We examined the immunogold-silver localization of dynorphin and immunoperoxidase labeling for substance P in single coronal sections through the core subregion of the nucleus accumbens of acrolein-fixed rat brain tissue. Dynorphin-immunoreactive somata were more prevalent than substance P-containing neurons throughout the region sampled for ultrastructural analysis. Dynorphin-labeled cells were spherical, contained unindented nuclei, and were closely apposed to other somata and dendrites, some of which also contained dynorphin immunoreactivity. The appositions were characterized by the absence of glial processes and contiguous contacts between the plasma membranes. Smooth endoplasmic reticulum and coated vesicles could also be identified in the cytoplasms on either side of the somatic or dendritic appositions. The dynorphin somata and dendrites received synaptic input from numerous unlabeled as well as dynorphin-and/or substance P-labeled axon terminals. Both types of terminals were morphologically similar in their content of small and large dense core vesicles and their formation of mainly symmetric synaptic specializations. In addition to dynorphin-immunoreactive targets, numerous dynorphin-and substance P-labeled terminals also formed synapses with unlabeled somata and dendrites. In some cases, terminals separately labeled for dynorphin and substance P converged on common targets with or without detectable dynorphin immunoreactivity. Terminals colocalizing both peptides were also found to synapse on unlabeled or dynorphin-labeled somata and dendrites. Additionally, presynaptic interactions were suggested by close appositions between dynorphin-and/or substance P-labeled terminals and other terminals that were unlabeled, dynorphin labeled, or substance P labeled. These results provide morphological data suggesting nonsynaptic communication between dynorphin-immunoreactive neurons and other neurons possibly mediated through receptive sites or second messengers associated with smooth endoplasmic reticulum in the nucleus accumbens. They also indicate that, in this region, 1) the activity of dynorphin neurons may be dependent on activation of autoreceptors for dynorphin as well as substance P and 2) additional neurons lacking dynorphin immunoreactivity are most likely inhibited (symmetric junctions) by terminals containing either one or both peptides. The findings may have implications for motor and analgesic responses to aversive tonic pain transmitted through dynorphin and substance P pathways within the nucleus accumbens. © 1995 Willy-Liss, Inc.     

Ultrastructural relationships between terminals immunoreactive for enkephalin, GABA, or both transmitters in the rat ventral tegmental area

The ventral tegmental area (VTA) receives extensive afferent input from neurons containing the opioid peptide enkephalin (Enk) and/or GABA. We examined the ultrastructural basis for known functional interactions between these inhibitory neuromodulators using a combined immunoperoxidase and immunogold-silver technique. As visualized with either marker in single sections, Enk-immunolabeled terminals contained numerous small clear vesicles and one or more intensely immunoreactive dense-cored vesicles. Enk-labeled terminals formed either symmetric or asymmetric synapses on small or large unlabeled dendrites. The immunoreactive dense-cored vesicles were usually detected away from these sites of synaptic contact. Terminals singly immunoreactive for GABA, or dually labeled for Enk and GABA, showed similar morphological features but formed primarily symmetric axo-dendritic synapses. In many instances, GABA- and/or Enk-immunolabeled terminals were in direct apposition to each other and formed synapses on immediately adjacent parts of a common dendrite. Close appositions were also noted between GABA- and Enk-immunoreactive axons and varicosities that did not form synapses with either common or divergent dendrites in single sections. Immunoreactive dense-cored vesicles were frequently detected at the apposed plasmalemmal surfaces between these axon terminals. The findings suggest that Enk and GABA are released from the same or convergent terminals and co-regulate the activity of common target neurons within the rat VTA. The results are also consistent with potential presynaptic interactions between these transmitters.     

Ultrastructural basis for interactions between central opioids and catecholamines. II. Nuclei of the solitary tracts

Interactions between central opioids and catecholamines are thought to underlie the ability of adrenergic agonists both to lower blood pressure and alleviate certain symptoms of opiate withdrawal. We examined the cellular substrate for interactions between neurons containing enkephalin-like opioid peptides and catecholamines in cardiovascular portions of the medial nuclei of the solitary tracts (m-NTS) of adult rats. Single sections were dually labeled using a double-bridged peroxidase method for the localization of a monoclonal leucine (Leu5)-enkephalin-antibody and immunoautoradiography for the localization of polyclonal antibodies against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Light microscopy revealed a few perikarya and numerous varicosities containing Leu5-enkephalin-like immunoreactivity (LE-LI). These were distributed among TH-labeled perikarya and processes throughout the rostrocaudal NTS. Electron microscopy of the m-NTS at the level of the area postrema further established the single as well as dual localization of TH and LE-LI in individual perikarya, dendrites, and axon terminals. Silver grains indicative of TH-labeling were usually distributed throughout the cytoplasm, whereas the peroxidase reaction product for LE-LI was localized principally to large (80-150 nm), dense-core vesicles. Immunoautoradiographic labeling for TH was detected in 118 terminals within a series of sections containing 183 terminals with LE-LI. Of these, 26% of the TH-labeled terminals and 32% of the enkephalin-containing terminals formed symmetric synapses with unlabeled dendrites, while only 7% of each type formed symmetric synapses with TH-labeled dendrites. In favorable planes of sections, the unlabeled as well as TH-labeled dendrites received convergent input from both types of terminals. A few of the remaining terminals that contained either TH or LE-LI formed asymmetric junctions with unlabeled distal dendrites; the others were without recognizable synaptic specializations within the plane of section. Approximately 20% of the TH-labeled terminals and 6% of the terminals containing LE-LI were dually labeled for both antibodies. These were invested with astrocytic processes characterized by bundles of intermediate filaments. We conclude that within cardiovascular portions of the m-NTS, opioid peptides and catecholamines contained within the same or separate terminals modulate the activity of target neurons through direct symmetric, probably inhibitory, synaptic junctions and may additionally modulate the activity of neighboring astrocytes through exocytotic release from large dense-core vesicles.(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.     

Plasmalemmal mu-opioid receptor distribution mainly in nondopaminergic neurons in the rat ventral tegmental area

Opiate-evoked reward and motivated behaviors reflect, in part, the enhanced release of dopamine produced by activation of the mu-opioid receptor (muOR) in the ventral tegmental area (VTA). We examined the functional sites for muOR activation and potential interactions with dopaminergic neurons within the rat VTA by using electron microscopy for the immunocytochemical localization of antipeptide antisera raised against muOR and tyrosine hydroxylase (TH), the synthesizing enzyme for catecholamines. The cellular and subcellular distribution of muOR was remarkably similar in the two major VTA subdivisions, the paranigral (VTApn) and parabrachial (VTApb) nuclei. In each region, somatodendritic profiles comprised over 50% of the labeled structures. MuOR immunolabeling was often seen at extrasynaptic/perisynaptic sites on dendritic plasma membranes, and 10% of these dendrites contained TH. MuOR-immunoreactivity was also localized to plasma membranes of axon terminals and small unmyelinated axons, none of which contained TH. The muOR-immunoreactive axon terminals formed either symmetric or asymmetric synapses that are typically associated with inhibitory and excitatory amino acid transmitters. Their targets included unlabeled (30%), muOR-labeled (25%), and TH-labeled (45%) dendrites. Our results suggest that muOR agonists in the VTA affect dopaminergic transmission mainly indirectly through changes in the postsynaptic responsivity and/or presynaptic release from neurons containing other neurotransmitters. They also indicate, however, that muOR agonists directly affect a small population of dopaminergic neurons expressing muOR on their dendrites in VTA and/or terminals in target regions.     

Ultrastructural localization of tyrosine hydroxylase immunoreactivity in the rat diagonal band of Broca.

The present study sought to establish the cellular basis for the catecholaminergic (i.e., noradrenaline and dopamine) modulation of neurons in the horizontal limb of the diagonal band of Broca (HDB) in the rat brain. The light and electron microscopic localization of antigenic sites for a polyclonal antibody directed against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), were examined in the HDB using a double-bridged, peroxidase-antiperoxidase method. By light microscopy, numerous punctate, varicose processes with intense TH-immunoreactivity (TH-I) were detected in the HDB. Additionally, a few small, bipolar, or multipolar TH-immunoreactive neurons were observed. Ultrastructural analysis of single sections revealed that the TH-labeled processes were axons and axon terminals. Axons (n = 134) with TH-I were primarily unmyelinated. Terminals with TH-I (n = 169) were 0.3-1.4 microns in diameter and contained many small, clear vesicles and 0-5 larger dense-core vesicles. The types of associations (i.e., asymmetric synapses, symmetric synapses, and appositions which lacked a membrane specialization in the plane of section analyzed) formed by the TH-labeled terminals were quantitatively evaluated. The TH-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (134 out of 169), (2) were closely apposed without glial intervention to unlabeled and TH-labeled terminals (11 out of 169), or (3) had no neuronal associations in the plane of section analyzed (24 out of 169). The relatively rare (n = 4) associations with unlabeled perikarya were mostly characterized by symmetric synaptic specializations. The majority of the TH-labeled terminals were associated with the shafts of small dendrites (66% of 134). Moreover, most of the associations on dendrites and dendritic spines were further characterized by asymmetric synaptic specializations; however, many were also appositions without any apparent glial intervention in the plane of section analyzed. Additionally, the TH-labeled terminals were often associated with only one dendrite, which, in the same plane of section, was sparsely innervated by other terminals. Astrocytic processes usually surrounded the portions of the terminals and dendrites not involved in the region of association. The TH-immunoreactive perikarya were small (7-12 microns), ovoid, and had an indented nucleus with some heterochromatin. Their scant cytoplasm contained mitochondria, Golgi complexes, and endoplasmic reticulum. A few immunoreactive dendrites, presumably derived from the local neurons, were also detected. Both TH-immunoreactive perikarya and dendrites were associated primarily with unlabeled terminals, although a few terminals with TH-I also contacted them.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [³H]5-HT. The [³H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [³H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [³H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 μm in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [³H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.     

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.     

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

We sought to determine the ultrastructural localization and the extrinsic sources of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the lateral parabrachial region (PBR) of adult male rats. In the first portion of the study, a rabbit antiserum to TH was immunocytochemically localized in coronal sections through the lateral PBR from acrolein-fixed brains using the peroxidase-antiperoxidase method. Electron-microscopic analysis revealed that perikarya and dendrites with peroxidase immunoreactivity for TH constituted only 17% of the total labeled profiles. Afferents to the TH-labeled perikarya and dendrites usually failed to exhibit immunoreactivity and were thus considered noncatecholaminergic. Somatic synapses were most commonly detected on small immunoreactive perikarya in the central lateral nucleus of the PBR. Other labeled perikarya located in the dorsal lateral or ventral lateral nuclei received few somatic synapses and were morphologically distinct in terms of their larger size, infolded nuclear membrane, and abundance of cytoplasmic organelles. Axons and axon terminals with peroxidase immunoreactivity constituted the remaining labeled profiles in the lateral PBR. These terminals primarily formed symmetric synapses with unlabeled and a few labeled dendrites. The labeled axon terminals were categorized into 2 types: Type I was small (0.3-0.6 micron), contained many small clear vesicles, and exhibited few well-defined synaptic densities. The second type was large (0.8-1.4 micron), contained both small clear and large dense core vesicles, and exhibited well-defined synaptic densities. The 2 types of terminals were morphologically similar to dopaminergic terminals. The location of catecholaminergic neurons contributing to the TH-labeled terminals was determined by combining peroxidase-antiperoxidase immunocytochemistry for TH with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The tracer was unilaterally injected into the PBR of anesthetized adult rats. Immunocytochemical labeling for TH was seen as a brown reaction product within neurons in known catecholaminergic cell groups. A black granular reaction product formed by a cobalt-intensified and diaminobenzidine-stabilized tetramethyl benzidine reaction for WGA-HRP was evident within many TH-labeled and unlabeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

The fine structural organization of tyrosine hydroxylase immunoreactive neurons in rat arcuate nucleus

The fine structure of the tyrosine hydroxylase (TH) immunoreactive neurons of the hypothalamic arcuate nucleus was examined by means of immunocytochemistry [peroxidase-antiperoxidase (PAP) method], utilizing an antibody against TH. Immunolabeled axon terminals were observed infrequently and were located predominantly in the lateral region, whereas numerous labeled perikarya and dendrites were found throughout the nucleus. The labeled terminals, containing primarily clear and occasionally dense core vesicles, were never observed in synaptic contact. On the other hand, unlabeled axon terminals were frequently seen synapsing on labeled dendrites. In addition, the labeled dendrites were often seen in direct apposition to other neuronal elements such as both labeled and unlabeled perikarya. In contrast, unlabeled dendrites were never seen apposed to labeled perikarya. Labeled dendrites also occurred in direct contact with one another and with unlabeled dendrites. Moreover, numerous labeled dendrites were encountered along tanycytic processes. Dendrites engaged in tanycytic appositions were occasionally partially encompassed by thin sheaths emanating from the tanycytic process. The extensive contact made by the labeled dendritic profiles on both labeled perikarya and dendrites suggests that tubero-infundibular dopaminergic (TIDA) cells may communicate with each other by means of dendritic release of dopamine. The presence of appositions between labeled dendrites and both unlabeled perikarya and dendrites suggests that the TIDA system also influences other neuronal populations through its dendrites. Finally, the dendrotanycytic relationship suggests that the TIDA system may play some role in the regulation of tanycytic function.     

Ultrastructural localization of tyrosine hydroxylase in the rat ventral tegmental area: relationship between immunolabeling density and neuronal associations

Dopaminergic neurons of the A 10 cell group in the rat ventral tegmental area (VTA) exhibit electrical and dye coupling. Also, the activity of these neurons at least partially reflects their content of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. We examined the ultrastructural localization of TH to determine the morphological features of dopaminergic neurons in the VTA and the relationships between their TH immunoreactivity content and afferent input. Antiserum against the trypsin-treated form of TH was localized using peroxidase-antiperoxidase (PAP) and immunoautoradiographic methods. Immunoreactivity was detected in perikarya, dendrites, and terminals. The perikarya contained the usual organelles, as well as cilia, lamellar bodies, and subsurface cisterns. Qualitative evaluation of peroxidase reaction product and quantitative analysis of the number of silver grains/unit area revealed varying amounts of TH immunoreactivity in nuclei and cytoplasm. Lightly or intensely labeled nuclei were not necessarily associated with corresponding cytoplasmic labeling density. However, cytoplasmic labeling directly corresponded to the relative frequencies of neuronal appositions and synaptic input. Those neurons with less dense cytoplasmic PAP product received fewer synaptic contacts and were less frequently in apposition to other TH-labeled soma and dendrites than neurons displaying relatively more dense cytoplasmic PAP product. Analysis of single sections revealed that 67% (n = 71) of all TH-labeled somata and 15% (n = 2431) of all TH-labeled dendrites were in apposition to other TH-labeled soma or dendrites. TH-labeled terminals were rarely detected and contained relatively low levels of immunoreactivity. The majority of labeled terminals (n = 29/46) formed synapses with labeled soma and dendrites. Unlabeled terminals (n = 2424) in contact with TH-labeled dendrites appeared to form predominantly symmetric synapses. Ten percent (n = 248) of the unlabeled terminals dually synapsed onto adjacent immunoreactive dendrites, perikarya, or dendrite and perikaryon. We conclude that in the rat VTA, (1) detected TH immunoreactivity in cytoplasm, but not nucleus, corresponds to the level of feedback principally from nondopaminergic afferents; (2) dendrodendritic as well as axodendritic synapses between TH-immunoreactive neurons may mediate dopaminergic autoinhibition; and (3) gap junction-like appositions between neurons and convergent inputs from unlabeled terminals onto TH-immunoreactive profiles provide an anatomical substrate whereby cellular activities might be coordinated under certain conditions.     

Ultrastructural immunocytochemical localization of the dopamine D2 receptor and tyrosine hydroxylase in the rat ventral pallidum.

The mesopallidal dopamine system plays a role in locomotor activity and reward. To understand the potential contribution of the dopamine D2 receptor (D2R) to the action of dopamine in the ventral pallidum (VP), we used electron microscopic immunocytochemistry to examine the cellular and subcellular localization of an antipeptide antiserum against the D2R in both ventromedial and dorsolateral VP compartments. In each region the majority of the total D2R-labeled profiles (n = 1,132) were axon terminals (55%) and small unmyelinated axons (27%). These terminals were often apposed to other axon terminals or dendrites and formed almost exclusively symmetric, inhibitory-type axodendritic synapses. Immunogold D2R labeling in axon terminals was seen on the plasmalemma and membranes of nearby synaptic vesicles. In ventral pallidal sections processed for dual detection of D2R peptide and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH), D2R labeling was detected in a few axons and axon terminals containing TH immunoreactivity as well as in axons contacted by TH-labeled terminals. In most cases, however, the D2R-labeled profiles were located at a distance from small axons and terminals containing TH. Our results provide the first ultrastructural evidence that D2Rs in the two VP subterritories are strategically located for primary involvement in modulation of the presynaptic release of nondopaminergic inhibitory transmitters. They also suggest that in this region the presynaptic D2 receptors are 1) minimally involved in autoregulation of dopaminergic transmission, and 2) differentially activated by dopamine, depending in part on levels and distance from release sites.     

Neuropeptide Y and dynorphin-immunoreactive large dense-core vesicles are strategically localized for presynaptic modulation in the hippocampal formation and substantia nigra

Neuropeptide Y (NPY) and dynorphin elicit regionally selective presynaptic modulation in the hippocampal formation and the pars reticulata of the substantia nigra, respectively. We examined potential anatomical substrates for their presynaptic modulation by determining the distribution and size of large (80–120 nm) dense-core vesicles (DCVs), organelles previously shown to be immunoreactive for each peptide. Throughout the hippocampal formation, NPY-immunoreactive DCVs were located primarily in axon terminals and were more sparingly distributed in dendrites. In comparison with other portions of the hippocampal formation, NPY-labeled DCVs were most abundant in axons and terminals of the CA1 region. The DCVs in the CA1 region of the hippocampus also more frequently had larger mean cross-sectional diameters when located along portions of the terminal in contact with unlabeled axons. In both the CA1 region of the hippocampus and the dentate gyrus, NPY-labeled DCVs in contact with portions of the axonal membrane apposed to astrocytes also were larger than those located more centrally in the axon terminal. Dynorphin-immunoreactive DCVs in axon terminals 'of the substantia nigra were significantly larger when found near portions of the axonal membrane in contact not only with other axons and astrocytic processes, but also occasionally with postsynaptic dendrites. The parallels between diameters of DCVs and known selectivity of NPY for presynaptic modulation in the CA1 region of the hippocampus suggest a direct correlation between the size and distribution of immunoreactive DCVs and their sites of exocytotic release. By analogy, we can conclude that dynorphin in the pars reticulata of the substantia nigra is also principally a presynaptic modulator, but may additionally elicit functional changes through interactions with receptive sites on astrocytes or postsynaptic neurons. © 1995 Wiley-Liss, Inc.     

Somatodendritic targeting of M5 muscarinic receptor in the rat ventral tegmental area: Implications for mesolimbic dopamine transmission

Muscarinic modulation of mesolimbic dopaminergic neurons in the ventral tegmental area (VTA) plays an important role in reward, potentially mediated through the M5 muscarinic acetylcholine receptor (M5R). However, the key sites for M5R‐mediated control of dopamine neurons within this region are still unknown. To address this question we examined the electron microscopic immunocytochemical localization of antipeptide antisera against M5R and the plasmalemmal dopamine transporter (DAT) in single sections through the rat VTA. M5R was located mainly to VTA somatodendritic profiles (71%; n = 627), at least one‐third (33.2%; n = 208) of which also contained DAT. The M5R immunoreactivity was distributed along cytoplasmic tubulovesicular endomembrane systems in somata and large dendrites, but was more often located at plasmalemmal sites in small dendrites, the majority of which did not express DAT. The M5R‐immunoreactive dendrites received a balanced input from unlabeled terminals forming either asymmetric or symmetric synapses. Compared with dendrites, M5R was less often seen in axon terminals, comprising only 10.8% (n = 102) of the total M5R‐labeled profiles. These terminals were usually presynaptic to unlabeled dendrites, suggesting that M5R activation can indirectly modulate non–DAT‐containing dendrites through presynaptic mechanisms. Our results provide the first ultrastructural evidence that in the VTA, M5R has a subcellular location conducive to major involvement in postsynaptic signaling in many dendrites, only some of which express DAT. These findings suggest that cognitive and rewarding effects ascribed to muscarinic activation in the VTA can primarily be credited to M5R activation at postsynaptic plasma membranes distinct from dopamine transport. J. Comp. Neurol. 521: 2927–2946, 2013. © 2013 Wiley Periodicals, 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.     

Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate-putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde-fixed rat brain were processed 1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine-synthesizing enzyme choline acetyltransferase (CAT) and 2) for the immunoautoradiographic localization of a rabbit polyclonal antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate-putamen versus NAS. Immunoperoxidase reaction for CAT versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2-3% of the CAT-labeled terminals (n = 136) and less than 1% of the TH-labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH-labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH-labeled terminals. Thirteen percent of the CAT-labeled and 14% of TH-labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudate-putamen and NAS cholinergic and catecholaminergic terminals 1) form symmetric, most likely inhibitory, synapses primarily with non-cholinergic neurons, 2) differentially synapse on shafts or spines of separate dendrites, and 3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic-dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.     

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

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