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.     

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)     

Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents

Neurons containing gamma-aminobutyric acid (GABA) in the medial portion of the adult rat nucleus accumbens were characterized with respect to their ultrastructure, sites of termination, and catecholaminergic input. Antisera against GABA-conjugates and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), were localized within single sections by means of peroxidase-antiperoxidase (PAP) and immunoautoradiographic labeling methods. Peroxidase reaction product indicating GABA-like immunoreactivity (GABA-LI) was seen in medium-size (15-20 microns) perikarya containing either round and unindented or invaginated nuclear membranes. The cells with invaginated nuclei were few in number and usually exhibited more intense peroxidase reaction product in sections collected at the same distance from the surface of the tissue. Reaction product for GABA was also detected in proximal (1.5-3.0 microns) dendrites, axons, and terminals. Terminals with GABA-LI formed symmetric junctions on perikarya, proximal dendrites, and dendritic spines of neurons that usually lacked detectable immunoreactivity. Many of the GABAergic terminals also were apposed directly to other unlabeled terminals and to terminals exhibiting either peroxidase labeling for GABA or immunoautoradiographic labeling for TH. Many of the unlabeled terminals associated with the GABAergic axons formed asymmetric junctions on dendritic spines. From 138 TH-labeled, principally dopaminergic terminals that were examined in the medial nucleus accumbens, 4% were associated with the somata of GABAergic neurons and another 14% formed symmetric junctions with proximal dendrite showing GABA-LI. The remaining TH-immuno-reactive terminals either lacked recognizable densities or formed symmetric synapses on unlabeled dendrites and spines. A few of the unlabeled dendrites, as well as those containing GABA-LI, received symmetric synapses from both catecholaminergic and GABAergic terminals. We conclude that in the medial portion of the rat nucleus accumbens, GABA is localized to two morphologically distinct types of neurons, one or both of which receive monosynaptic input from catecholaminergic afferents, and that GABAergic terminals form symmetric synapses on other principally non-GABAergic neurons. The results also support earlier physiological evidence showing that GABA may modulate the output of other GABAergic and non-GABAergic neurons through presynaptic associations.     

C-terminal tail of beta-adrenergic receptors: immunocytochemical localization within astrocytes and their relation to catecholaminergic neurons in N. tractus solitarii and area postrema.

beta-Adrenergic receptors (beta AR) in the medial nuclei of tractus solitarii (m-NTS) and area postrema (AP) may bind to catecholamines released from neurons, whereas only the AP has fenestrated capillaries allowing access to circulating catecholamines. Since varied autonomic responses are seen following beta AR activation of the dorsal vagal complex, including the m-NTS and AP, we hypothesized that there might be a cellular basis for varied responses to beta AR stimulation that depends on the differential access to circulating catecholamines. Therefore, we comparatively examined the ultrastructural localization of the beta AR in relation to catecholaminergic neurons in these regions. An antibody directed against the C-terminal tail (amino acids 404-418) of hamster beta-adrenergic receptor (beta AR404) was used in this study. The localization of beta AR404 was achieved by the avidin-biotin peroxidase complex (ABC) technique in combination with a pre-embed immunogold labeling method to localize tyrosine hydroxylase (TH), the catecholamine-synthesizing enzyme. Within m-NTS and at subpostremal border, labeling for beta AR404 was evident along the intracellular surface of plasma membranes of small, apparently distal, astrocytic processes. Astrocytic processes with beta AR404-immunoreactivity formed multiple, thin lamellae around TH-labeled and non-TH neuronal cell bodies and dendrites. beta AR404-immunoreactive astrocytes also extended end-feet around blood vessels and surrounded groups of axon terminals that were directly juxtaposed to each other. Some, but not all, of these axons demonstrated TH-immunoreactivity. Fewer beta AR404-immunoreactive astrocytes were detected in AP, regardless of their proximity to catecholaminergic processes or blood vessels. The present astrocytic localization of beta AR404, together with the earlier, neuronal localization of beta AR's third intracellular loop, suggest that the beta AR may be substantially different between neurons and astrocytes. The regional difference in the prevalence of beta AR404-immunoreactive astrocytes suggests that these receptive sites may either: (i) be preferentially activated by catecholamines released from terminals rather than circulating catecholamines; or (ii) be down-regulated in AP due to blood-born substances, such as catecholamines. The extensive localization of beta AR in the border between m-NTS and AP also suggests that catecholaminergic activation of these astrocytes may dictate the degree of diffusion of catecholamines which are of neuronal or vascular origin. The specific localization of beta AR404-immunoreactivity to the more distal portions of astrocytes suggests the possibility that astrocytes have restrictive distributions of beta AR and that the beta-adrenergic activation lead to morphological or chemical changes that are also localized to the distal portions of astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural localization of phenylethanolamine N-methyltransferase in sensory and motor nuclei of the vagus nerve

The ultrastructural localization of phenylethanolamine N-methyltransferase (PNMT), the enzyme used in the final step in the synthesis of adrenaline, was examined in the medial nuclei of the solitary tracts (m-NTS) and in the dorsal motor nuclei of the vagus. Adult rats were anesthetized with Nembutal (50 mg/kg intraperitoneally), and the brains were fixed by vascular perfusion with a solution containing 3.75% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer. Coronal Vibratome sections were collected through the intermediate portions of the m-NTS at the level of the area postrema. These sections were immunocytochemically labeled employing a rabbit polyclonal antiserum against PNMT and the peroxidase-antiperoxidase method. Immunoreactivity was detected in perikarya, dendrites, and axon terminals in the intermediate portion of the m-NTS. The labeled perikarya were either small (10-15 microns diameter) and oval or large 20-30 microns) with two or more proximal processes. The PNMT-containing dendrites received synaptic input from unlabeled, small (0.5-1.0 microns) and large (2-3 microns) vagal-like afferents as well as from a few terminals, which also showed PNMT immunoreactivity. Axons and axon terminals containing immunoreactive PNMT were more frequently observed than the perikarya or dendrites in the m-NTS and were the only labeled profiles in the dorsal motor nuclei. In both regions the PNMT-labeled terminals formed principally symmetric synapses with unlabeled dendrites. However, a few asymmetric axodendritic and symmetric axosomatic synapses also were detected. These findings indicate that the adrenergic neurons may have multiple, but principally inhibitory, actions on other neurons within cardiovagal portions of baroreflex pathways.     

Differences in membrane structure between excitatory and inhibitory components of the reciprocal synapse in the olfactory bulb

The reciprocal dendro-dendritic synapse between granule and mitral or tufted dendrites in the external plexiform layer of the olfactory bulb consists of an excitatory mitral-to-granule synaptic contact and an adjacent inhibitory granule-to-mitral synaptic contact. The pre- and postsynaptic membranes of both synaptic contacts were identified in replicas of freeze-fractured external plexiform layer in rabbits, mice, and chinchillas. At the excitatory synaptic contact there is a prominent specialization in the postsynaptic memberane, represented by an aggregate of homogeneous particles associated with the external half of the membrane. In contrast, the postsynaptic membrane at the inhibitory granule-to-mitral synaptic contact lacks evident internal specializations, and the distribution of particles on both fracture faces resembles that at non-synaptic regions. Less marked differences in particle distribution characterized the cytoplasmic half of the presynaptic membranes. These differences probably reflect diversity in the nature or distribution of membrane proteins at excitatory and inhibitory synapses. Protuberances on the external half of the presynaptic membrane, possibly sites of vesicle interaction with the plasma membrane, surrounded but were not coextensive with both types of synaptic contact. A few gap junctions connected proximal dendrites of mitral or tufted cells with granule cell dendrites.     

Cellular basis for interactions between catecholaminergic afferents and neurons containing Leu-enkephalin-like immunoreactivity in rat caudate-putamen nuclei.

Dopaminergic afferents to the dorsal striatum, caudate-putamen nuclei, are known to modulate the levels and synthesis of endogenous opiate peptides (Leu5 and Met5-enkephalins). We examined the dual immunocytochemical localization of antisera raised against Leu5-enkephalin and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), to determine the cellular substrates for these and/or other functional interactions. The antisera were identified by combined immunogold-silver and immunoperoxidase labeling in single coronal sections through the caudate-putamen nuclei of adult rats. These animals were given intraventricular injections of colchicine, and the brains were fixed by acrolein perfusion prior to immunocytochemical labeling. By light microscopy, perikarya and processes containing enkephalin-like immunoreactivity (ELI) were seen in close proximity to varicose processes immunoreactive for TH. Electron microscopy further demonstrated that the ELI was localized to perikarya, dendrites, and axon terminals, whereas the TH was exclusively in axons and terminals. The dendrites containing ELI were postsynaptic to terminals that were either (1) without detectable immunoreactivity, or (2) immunoreactive for TH or enkephalin. Nonsynaptic portions of the dendrites containing ELI were covered with astrocytic processes or were in direct apposition to unlabeled dendrites. Terminals containing ELI were densely immunoreactive and were in direct contact with (1) unlabeled and occasionally enkephalin-labeled proximal dendrites, and (2) TH-labeled and unlabeled terminals. In comparison with the opiate terminals, most catecholaminergic terminals were lightly immunoreactive for TH and usually contacted more distal unlabeled dendrites or spines and, more rarely, dendrites containing ELI. In a few favorable planes of section, the terminals containing ELI and those containing TH (1) converged on common unlabeled dendrites, or (2) formed dual contacts on two different labeled or unlabeled targets. Junctions formed by terminals containing ELI and TH were sometimes characterized by symmetric synaptic densities. However, numerous other dendritic and all axonal appositions were without recognized membrane densities. The findings of the study provide anatomical substrates for multilevel interactions between catecholamines, mostly dopamine, and enkephalin in rat dorsal striatum. These include (1) monosynaptic input from dopaminergic terminals to neurons containing enkephalin, (2) presynaptic modulation of transmitter release through axonal appositions, and (3) dual regulation of common targets through convergent input. In addition, the findings suggest that both enkephalin and dopamine may have similar modulatory roles in synchronizing the activity of dual targets postsynaptic to individual axon terminals. Alterations in any one of these multiple types of interactions could account for noted motor or sensory symptoms in neurological disorders characterized by depletion of dopamine or endogenous opiate peptides, or both.     

Ultrastructural localization of tyrosine hydroxylase in rat nucleus accumbens

Immunocytochemical localization of tyrosine hydroxylase (TH) was used to determine the ultrastructural morphology and synaptic associations of catecholaminergic terminals in the nucleus accumbens of the rat. The brains were fixed by vascular perfusion with 4% paraformaldehyde and 0.2% glutaraldehyde. Coronal sections cut with a vibrating microtome were incubated with rabbit antiserum to TH then immunocytochemically labeled by the peroxidase-antiperoxidase method. Immunoreactivity for the enzyme was found within unmyelinated axons and axon terminals. These terminals contained either all small clear or combined small clear and large dense core vesicles. Approximately 40% of the labeled terminals formed symmetric synapses with unlabeled proximal or distal dendritic shafts. The dendrites showed a spare distribution of spines. Axosomatic synapses and axonal associations of the TH-containing terminals also were detected. The recipient perikarya were usually 10-20 micrometers in diameter and contained an indented nucleus and abundant cytoplasm. The content of large dense vesicles and synaptic associations with somata and proximal dendrites suggest that a certain proportion of the TH-containing terminals within the nucleus accumbens are morphologically distinct from catecholaminergic terminals within the dorsal striatum. These differences are discussed in relation to neuropeptides and functions of the dopaminergic mesolimbic and nigrostriatal pathways.     

Cellular substrates for interactions between neurons containing phenylethanolamine N-methyltransferase and GABA in the nuclei of the solitary tracts

Adrenaline and gamma-aminobutyric acid (GABA) have been implicated in autonomic functions involving the intermediate and caudal portions of the medial nuclei of the solitary tracts (m-NTS). We sought to determine whether there was a cellular basis for direct intracellular or synaptic interactions between these transmitters in neurons in the m-NTS of rat brain by using dual-labeling immunocytochemical methods. Light microscopy revealed immunoautoradiographic labeling for the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) in perikarya and processes in close proximity to cells demonstrating peroxidase reaction product for GABA. Electron microscopy of the intermediate m-NTS at the level of the area postrema further established the localization of immunoautoradiographic and peroxidase labels for PNMT and GABA in common as well as separate perikarya and dendrites. All axon terminals were labeled separately for PNMT and GABA. The PNMT-labeled terminals formed both symmetric and asymmetric synapses, whereas the GABA-labeled terminals formed exclusively symmetric synapses. Twenty-four percent (n = 42) of the PNMT- and 39% (n = 128) of the GABA-labeled terminals formed synaptic junctions on unlabeled soma and dendrites. Occasionally both types of terminals converged on a common unlabeled dendrite and on GABA-labeled dendrites. Only 3% of the PNMT- and 12% of the GABA-containing terminals formed synapses on PNMT-labeled soma and dendrites, whereas 7% of each type formed synapses with GABA-labeled profiles. The remaining labeled terminals lacked synaptic relations within the sections examined. The autoradiographic results were confirmed and extended by means of immunogold labeling for PNMT in combination with peroxidase-antiperoxidase localization of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). GAD-labeled terminals formed symmetric synapses with dendrites that were either unlabeled or contained low levels of PNMT (gold particles) or PNMT and GAD. We conclude that in caudal, more cardiovascular portions of the NTS, adrenaline and GABA may coexist, but they are more commonly detected in separate populations of neurons having receptive sites for both transmitters and innervating certain common target neurons.     

Ultrastructural localization of choline acetyltransferase in the rat rostral ventrolateral medulla: evidence for major synaptic relations with non-catecholaminergic neurons

Pharmacological and biochemical studies suggest that interactions between cholinergic and catecholaminergic neurons, particularly those of the C1 adrenergic cell group, in the rostral ventrolateral medulla (RVL) may be important in cardiovascular control. Ultrastructural localization of choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and its relation to neurons exhibiting immunoreactivity for catecholamine- (tyrosine hydroxylase; TH) or adrenaline (phenylethanolamine-N-methyltransferase; PNMT) -synthesizing enzymes were examined in the RVL using dual immunoautoradiographic and peroxidase anti-peroxidase (PAP) labeling methods. By light microscopy, the ChAT-immunoreactive neurons were located both dorsally (i.e. the nucleus ambiguus) and ventromedially to those labeled with TH or PNMT (TH/PNMT). A few ChAT-labeled processes were dispersed among TH/PNMT-containing neurons with the majority of overlap immediately ventral to the nucleus ambiguus. By electron microscopy, ChAT-immunoreactivity (ChAT-I) was detected in neuronal perikarya, dendrites, axons and axon terminals and in the vascular endothelial cells of certain blood vessels. The ChAT-labeled perikarya in the ventromedial RVL were medium-sized (15–20 μm), elongated, contained abundant cytoplasm and had slightly indented nuclei. Synaptic junctions on ChAT-immunoreactive perikarya and dendrites were primarily symmetric with 64% (45 out of 70) of the presynaptic terminals unlabeled. The remaining terminals were immunoreactive for ChAT (30%) or TH/PNMT (6%). Terminals with ChAT-I were large (0.8–2.0 μm) and contained numerous small clear vesicles and 1–2 dense core vesicles. Seventy-seven percent (112 out of 145) of the ChAT-labeled terminals formed symmetric synapses with unlabeled perikarya and dendrites, whereas only 8% were with TH/PNMT-labeled perikarya and dendrites, and 15% were with ChAT-immunoreactive perikarya and dendrites. We conclude (1) that cholinergic neurons in the RVL principally terminate on and receive input from non-catecholaminergic neurons, and (2) that the reported sympathetic activation following application of cholinergic agents to the RVL may be mediated by cholinergic inhibition of local inhibitory interneurons. The observed synapses between ChAT and TH/PNMT-containing neurons suggests that cholinergic and adrenergic neurons additionally may exert a minor reciprocal control on each other and thus may modulate their response to the more abundant input from afferents containing other transmitters.     

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.     

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)     

Distribution patterns of individual medial lemniscal axons in the ventrobasal complex of the monkey thalamus

Somatostatin-immunoreactive neurons in the rat neostriatum were studied by correlated light and electron microscopy using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactivity was localized in neuronal perikarya and processes. The perikarya were of spindle or fusiform shape (average length 16.9 microns) and were found in all parts of the neostriatum. From each neuron there arose two to four straight immunoreactive dendritelike processes, which could frequently be traced as far as about 130 microns from their perikaryon. Immunoreactive varicose axonlike processes were occasionally found, some of which were proximal axons of identified immunoreactive cells. Nine of the light microscopically identified neurons showing somatostatin-immunoreactivity were studied in the electron microscope; two of them had proximal axons with varicosities. Each neuron had an oval or elongated nucleus, which was always indented. These morphological features correspond well to those of certain "medium-size aspiny" neurons classified by Golgi studies. Although the immunoreactive endproduct was diffusely located throughout the neuron, it was characteristically located in the saccules and large granules (diameter 133 nm) of the Golgi apparatus, and large immunoreactive vesicles of similar size to those in the Golgi apparatus frequently occurred in all parts of axon. Very little synaptic input was found on the perikarya and dendrites of somatostatin-immunoreactive neurons. The perikarya and proximal dendrites received both symmetrical and asymmetrical synaptic input, while the distal dendrites usually received boutons that formed asymmetrical contacts. The somatostatin-immunoreactive boutons contained pleomorphic electron-lucent vesicles (diameter 39.3 nm) and a few large immunoreactive granular vesicles; these boutons always formed symmetrical synapses. Their postsynaptic targets were dendritic shafts, spines, and unclassified dendritic profiles. On the other hand, the varicosities of identified proximal axons of somatostatin-positive neurons did not form typical synapses, since they lacked clusters of small vesicles, but some of them were in direct apposition (via membrane specializations) to unlabelled perikarya or dendrites. It is concluded that somatostatin is a useful marker for a particular type of neuron in the neostriatum. The presence of somatostatin immunoreactivity in synaptic boutons is consistent with the view that somatostatin could be a neurotransmitter in the neostriatum.     

Ultrastructural evidence of dopaminergic input to enkephalinergic neurons in rat neostriatum

The synaptic relationship between neuronal structures reacting with antibodies to tyrosine hydroxylase (TH) and Leu- or Met-enkephalin (ENK) was studied by the 'mirror technique' in adjacent sections of rat neostriatum. TH-immunoreactive (TH-IR) axonal boutons surrounding the neural perikarya and proximal dendrites of ENK-immunoreactive (ENK-IR) neurons were very thin (0.1-0.4 micrograms). They contained many small clear vesicles and sometimes had symmetrical membrane specializations. This provides morphological evidence for catecholaminergic, presumably dopaminergic inputs to rat striatal enkephalin neurons.     

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.     

Cholinergic neurons in the rat septal complex: ultrastructural characterization and synaptic relations with catecholaminergic terminals.

Physiological and pharmacological studies have suggested that catecholamines modulate cholinergic neurons in the medial septal and diagonal band nuclei (i.e., the septal complex). Thus, the ultrastructural morphology of neurons containing choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and their relation to catecholaminergic terminals exhibiting immunoreactivity for the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat septal complex. Dual immunoautoradiographic and peroxidase anti-peroxidase labeling methods were used to simultaneously localize antibodies raised in rabbits against TH and from rat-mouse hybridomas against ChAT in single sections. At least two types of perikarya with ChAT-immunoreactivity (ChAT-I) were observed. The first type were large (20-30 microns), elongated or round, and contained a small indented nucleus with an abundant cytoplasm and an occasional lamellar body. The second type was also either ovoid or round but was medium-sized (15-20 microns) and contained a larger indented nucleus and a smaller amount of cytoplasm than the first type. Both types of perikarya as well as dendrites with ChAT-I were surrounded by astrocytic processes apposed to most of their plasmalemmal surfaces. The distribution and types of terminal associations (i.e., asymmetric synapses, symmetric synapses and appositions which lacked a membrane specialization in the plane of section analyzed) with ChAT-labeled perikarya and dendrites were quantitatively evaluated. The majority (68% of 197) of the presynaptic terminals were unlabeled; the remaining terminals were immunoreactive for TH (25%) or ChAT (7%). All three types of terminals contacted primarily the shafts of small dendrites and more rarely ChAT-labeled perikarya and large dendrites. ChAT-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (31% of 176); (2) formed associations with perikarya and dendrites with ChAT-I (7%); (3) contacted the same unlabeled perikarya and dendrite as a TH-containing terminal (21%); (4) were in apposition to TH-labeled terminals (25%); or (5) were either in apposition to unlabeled or ChAT-labeled terminals or lacked associations with any processes. The majority of associations formed by the terminals with ChAT-I were on the shafts of small dendrites. Moreover, most of the associations formed were either symmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. These findings provide cellular substrates in the septal complex (1) for sparse synaptic input relative to astrocytic investment of cholinergic neurons and (2) for direct synaptic modulation of cholinergic and non-cholinergic neurons by catecholamines and/or acetylcholine. These findings have direct relevance to catecholaminergic-cholinergic interactions and to the neuropathological basis for Alzheimer's disease.     

Serotoninergic terminals: Ultrastructure and synaptic interaction with catecholamine-containing neurons in the medial nuclei of the solitary tracts

The ultrastructural morphology of serotoninergic terminals and their synaptic relation with catecholaminergic neurons were examined in the medial nuclei of the solitary tracts (m-NTS) using combined autoradiographic and immunocytochemical methods. Adult rats were pretreated with a monoamine oxidase inhibitor and subjected to a 2-hour intraventricular infusion of 50 nM tritiated 5-hydroxytryptamine (³H-5HT). At the termination of the infusion, the brains were fixed by aortic arch perfusion with a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde. Coronal Vibratome sections through the NTS and more rostral raphe nuclei were immunocytochemically labeled with specific antiserum to serotonin or tyrosine hydroxylase and then processed for autoradiography. By light microscopy, concentrations of reduced silver grains indicating uptake of ³H-5HT usually paralleled the localization of peroxidase immunoreactivity for serotonin in neuronal perikarya of the rostral raphe nuclei and in varicosities in the brainstem. The ³H-5HT-containing varicosities were found throughout the medial and commissural portions of the NTS, where they were frequently associated with processes showing immunoreactivity for the catecholamine-synthesizing enzyme tyrosine hydroxylase. Ultrastructural examination of the m-NTS revealed that the silver grains for ³H-5HT were accumulated over axon terminals. The 5HT-labeled terminals contained a heterogeneous population of vesicles and formed both symmetric and asymmetric synapses with dendrites. The recipient dendrites were either unlabeled or showed immunoreactivity for tyrosine hydroxylase. These findings support a direct serotoninergic modulation of catecholaminergic neurons within the rat m-NTS.     

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-siver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10–12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (<1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptdies. © 1993 Wiley-Liss, Inc.     

Ultrastructural localization of beta-adrenergic receptor-like immunoreactivity in the cortex and neostriatum of rat brain

We sought to quantitatively examine the processes containing beta-adrenergic receptor-like immunoreactivity (beta-AR-LI) in the cerebral cortex and neostriatum using a previously characterized rabbit antiserum to frog erythrocyte beta-ARs under optimized immunolabeling conditions. Quantitative assessments of the laminar distribution of beta-AR-LI in the cortex was achieved by computer-assisted image analysis of immunoautoradiographs and by quantitative electron microscopic analysis of peroxidase-antiperoxidase (PAP) labeling in aldehyde-fixed sections and unfixed synaptosomes. In the somatosensory and anterior cingulate cortical areas, light microscopy of aldehyde-fixed sections immunolabeled by the PAP method revealed small (0.5-1.0 micron) punctate processes in all layers. In the deeper layers, rims of immunoreactivity around the plasmalemma of a population of neuronal perikarya and processes were also observed. By immunoautoradiography, labeling was seen in distinct, laminar distributions resembling the reported autoradiographic patterns using radioligands. By electron microscopy, the immunoreactive profiles in all cortical layers were primarily thick and thin postsynaptic densities (PSDs), comprising 4% of all identifiable PSDs in fixed sections and 12% in unfixed synaptosomal preparations. Also labeled were saccules of smooth endoplasmic reticulum and pinocytotic vesicles in dendrites, glial processes and lightly myelinated axons. In the neostriatum, the density of autoradiographic immunoreactivity was equivalent to the heavily labeled laminae of the cerebral cortex. Immunoreactivity detectable by light microscopy included punctate processes and rims of perikarya, as was seen in the cerebral cortex. The PAP reaction was shown by electron microscopy to be localized to the cytoplasmic surface of plasmalemma of a few proximal dendrites, but was most prominently associated with PSDs of dendritic spines. Preadsorption of the antiserum with a partially purified beta-AR preparation abolished all detectable immunoreactivity. These results provide further support for the specificity of the antiserum for beta-ARs, and are the first quantitative ultrastructural evidence for association of beta-AR-LI with PSDs in the cerebral cortex. The neostriatum, whose major catecholaminergic innervation is dopaminergic, and not noradrenergic, is also confirmed to exhibit high levels of beta-AR-LI within subcellular structures analogous to those seen in the cerebral cortex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopic immunocytochemical localization of tyrosine hydroxylase in the area postrema of rat

The ultrastructural morphology and specialized neuronal, vascular, and ventricular associations of tyrosine hydroxylase-labeled neurons are examined within the area postrema of rat brain. Specific antiserum to the purified enzyme is localized throughout the rostrocaudal and dorsoventral extent of the area postrema by means of the peroxidase-antiperoxidase technique. In all regions, peroxidase immunoreactivity for tyrosine hydroxylase is distributed throughout the cytoplasm of selectively labeled neuronal perikarya and processes. The perikarya contain a large nucleus, infolded nuclear membrane, numerous cytoplasmic organelles, and form axosomatic synapses with unlabeled terminals. The majority of the labeled processes are dendrites, which contain ribosomes, microtubules, mitochondria, and scattered vesicles. These dendrites are postsynaptic to unlabeled axon terminals and show membrane specializations with other labeled dendrites and perikarya. In contrast to dendrites, peroxidase-labeled profiles clearly distinguished as axons or axon terminals are sparse and never show membrane specializations with other neuronal or nonneuronal structures within the area postrema. Numerous large processes which could be either axons or dendrites are associated with blood vessels and the ventricular surface of the area postrema. With respect to blood vessels, processes are located either in direct apposition to the external glial membrane, or less frequently, within the perivascular space. The ventricular processes are either associated with blood vessels in the subpial space or distributed among the cilia and villi at the anterior margins of the area postrema. The neuronal and nonneuronal associations of the tyrosine hydroxylase-labeled processes are consistent with a receptor or chemosensor function for catecholamines in this circumventricular organ.