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.     

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla of the rat. I. Normal ultrastructure

The electron microscopic localization of the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) was examined in the rostral ventrolateral medulla (RVL) of adult rats. The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were immunocytochemically labeled using a rabbit polyclonal antiserum to PNMT and the peroxidase-antiperoxidase method. A semi-quantitative ultrastructure analysis revealed that the perikarya constituted 9% of the total immunoreactive profiles observed in the RVL. The labeled somata were large (18-24 microns) and were characterized by an indented nucleus and abundant cytoplasm with numerous mitochondria. An average of 136.8 +/- 11.6 mitochondria were present per 100 microns2 cytoplasm, which is 38% greater than the numbers found for PNMT-immunoreactive neurons in the nucleus of the solitary tract. Moreover, the labeled somata were often found in direct apposition to the basal lamina of small capillaries and neighboring astrocytic processes. The remaining labeled profiles were neuronal processes of which 72% were dendrites. Both the PNMT-labeled somata and dendrites received primarily symmetric contacts from unlabeled axon terminals. Only a few axons and terminals containing immunoreactivity for PNMT were observed. The axons were both unmyelinated and myelinated. The PNMT-immunoreactive terminals were characterized by a mixed population of vesicles and by the formation of synaptic junctions with both unlabeled dendrites and PNMT-labeled perikarya and dendrites. The ultrastructural morphology and proximity to blood vessels and glia suggest a high metabolic activity and possibly a chemosensory function of PNMT neurons in the RVL. The existence of myelinated and unmyelinated axons could imply that PNMT-containing neurons have different conduction velocities in efferent pathways to the spinal cord or other brain regions. Furthermore, the multiple types of synaptic interactions between labeled and unlabeled axons and dendrites support the concept that adrenergic neurons modulate and are modulated by neurons containing the same or other putative transmitters in the RVL.     

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.     

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

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

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)     

Mu-opioid receptors in the ventral tegmental area are targeted to presynaptically and directly modulate mesocortical projection neurons.

Mesocorticolimbic projections originating from dopaminergic and GABAergic neurons in the ventral tegmental area (VTA) play a critical role in opiate addiction. Activation of mu-opioid receptors (MOR), which are located mainly within inhibitory neurons in the VTA, results in enhanced dopaminergic transmission in target regions, including the medial prefrontal cortex (mPFC). We combined retrograde tract-tracing and electron microscopic immunocytochemistry to determine if neurons in the VTA that project to the mPFC contain MOR or receive input from MOR-containing terminals. Rats received unilateral injections of the retrograde tracer Fluoro-Gold (FG) into the mPFC. Tissue sections throughout the VTA were then processed for electron microscopic examination of FG and MOR. Immunoperoxidase labeling for FG was present in VTA cell bodies that contained immunogold-silver particles for MOR that often were contacted by profiles exclusively immunoreactive for MOR, including somata and axon terminals. The majority of dually labeled profiles were dendrites that received convergent input from unlabeled axon terminals forming either symmetric or asymmetric type synapses. Within retrogradely labeled cell bodies and proximal dendrites, MOR immunoreactivity was mainly sequestered within the cytoplasm. In contrast, distal retrogradely labeled dendrites contained MOR gold particles located along the plasma membranes. These data suggest that opiates active at MOR in the VTA modulate cortical activity through 1) presynaptic actions on MOR in terminals contacting mesocortical cell bodies, and 2) direct activation of MOR in distal dendrites of projection neurons.     

Ultrastructural immunocytochemical localization of neurotensin and the dopamine D2 receptor in the rat nucleus accumbens

The neuroleptic-like effects of neurotensin (NT) are thought to be due to interactions with dopamine (DA) acting primarily at D₂ receptors within the nucleus accumbens septi (Acb). Using electron microscopic dual labeling immunocytochemistry, we sought to demonstrate cellular substrates for functional interactions involving NT and DA D₂ receptors in the adult rat Acb. Peroxidase reaction product representing D₂ receptor-like immunoreactivity (D₂-LI) was seen along membranes of Golgi lamellae and multivesicular bodies of perikarya containing immunogold labeling representing NT-LI. Dually labeled somata usually contained highly indented nuclei, a characteristic of aspiny neurons. Dendrites also occasionally colocalized the two immunomarkers. Other somata, dendrites, and all axon terminals were singly labeled with either NT-LI or D_2-LI. In distinct sets of terminals, NT-LI was commonly associated with large, dense-cored vesicles, whereas D₂-LI was found along the plasmalemma and over nearby small clear vesicles. Each type of terminal comprised ∼ 20% of synaptic input to NT-immunoreactive dendrites. Similar proportions of terminals containing NT-LI or D₂-LI contacted unlabeled (∼ 55%) or NT-labeled (∼ 35%) dendrites and, occasionally, were observed converging onto common dendrites. Terminals containing NT-LI or D₂-LI also were often closely apposed. These findings provide the first ultrastructural evidence that: (1) NT and D₂ receptors are colocalized in aspiny neurons and dendrites, (2) NT may produce a direct postsynaptic effect on neurons receiving input from terminals which are presynaptically modulated by DA via D₂ receptors, and (3) NT and DA acting at D₂ receptors may interact through presynaptic modulation of common axon terminals. © 1996 Wiley-Liss, Inc.     

Presumptive GABAergic centrifugal input to the lamprey retina: a double-labeling study with axonal tracing and GABA immunocytochemistry

The distribution of GABA-like immunoreactivity (GABA-LI) was performed in the lamprey retinopetal system which was previously identified by either anterograde or retrograde axonal tracing methods. This study was carried out at the ultrastructural level for the retina and under both the light and electron microscope for the mesencephalic retinopetal centers (M5 and RMA). The GABA-LI was distributed in about 40% of anterogradely HRP-labeled axon terminals in the inner retina. These made synaptic contacts upon either HRP-labeled ganglion cell dendrites or mostly on GABA-LI or on immunonegative amacrine cell dendrites and somata. The other immunonegative HRP-labeled axon terminals also established synaptic contacts on amacrine cell dendrites and somata. The mesencephalic retinopetal neurons, retrogradely labeled with HRP or [³H]proline, were GABA-LI in 65% of M5 somata and only in 15% of RMA neurons. M5 and RMA retinopetal neurons and dendrites, either GABA-LI or immunonegative, were contacted: (1) asymmetrically by HRP-labeled or unlabeled axon terminals containing rounded synaptic vesicles, always immunonegative and (2) symmetrically by HRP-unlabeled axon terminals containing pleiomorphic synaptic vesicles, which were either GABA-LI or immunonegative. The role of GABA as a putative neurotransmitter in the centrifugal visual system is discussed.     

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

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

Galanin-containing axons synapse on tyrosine hydroxylase-immunoreactive neurons in the hypothalamic arcuate nucleus of the rat

Prolactin (PRL) secretion by the anterior pituitary gland is dependent upon the tonic inhibitory influence of the tuberoin-fundibular dopaminergic (TIDA) neuronal system. TIDA neurons, in turn, are regulated by various afferent neuronal systems. To support the concept that the recently-discovered neuropeptide, galanin (GAL), is one of the neurotransmitter/neuromodulator substances which might synaptically regulate the function of the TIDA system, immunocytochemical double-labeling studies were carried out in the hypothalamic arcuate nucleus (AN) of the male rat. The analysis of light microscopic preparations revealed the overlapping of GALergic and dopaminergic (detected by tyrosine hydroxylase immunoreactivity) neuronal elements in both the dorsomedial and ventrolateral parts of the AN. TH-containing perikarya and dendrites were contacted by varicose GAL-IR axons in these regions. The electron microscopic studies of ultrathin sections demonstrated axosomatic and axodendritic synapses between GALergic axons and TH-IR neurons. These findings support the view that GAL may modulate PRL release, acting as a neurotransmitter/neuromodulator in synaptic afferents to the TIDA system.     

Light microscopic and ultrastructural localization of GABA-like immunoreactive input to retrogradely labeled sympathetic preganglionic neurons

The organization of gamma-aminobutyric acid-like immunoreactive (GABA-LIR) processes was studied within the sympathetic preganglionic neuropil of male Sprague-Dawley rats and pigeons (Columba livia). Sympathetic preganglionic neurons were retrogradely labeled following horseradish peroxidase (HRP) injections into either the adrenal medulla or superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. GABA-LIR staining was visualized using peroxidase-antiperoxidase (PAP), avidin-biotin complex (ABC), or post-embedding immunogold methods. The pigeon preganglionic neuropil contained a dense network of GABA-LIR processes with punctate swellings that encircled sympathetic preganglionic perikarya within the principal preganglionic cell column (column of Terni) and the nucleus intercalatus spinalis. GABA-LIR spinal neurons were intermingled among HRP-labeled sympathetic preganglionic neurons within the column of Terni and throughout the zona intermedia. In the rat the density of the GABA-LIR processes within the four thoracic sympathetic preganglionic nuclei was less than that observed in the pigeon. Nevertheless, GABA-LIR profiles distinctively dotted preganglionic perikarya within the nuclei intermediolateralis pars principalis and pars funicularis, nucleus intercalatus spinalis and the central autonomic nucleus. GABA-LIR neurons were rarely observed within the nucleus intermediolateralis pars principalis, but were numerous in the zona intermedia and area X. No GABA-LIR spinal neurons in either vertebrate were retrogradely labeled with HRP. The ultrastructural arrangements of GABA-LIR processes within the sympathetic preganglionic neuropils of pigeons and rats were similar. GABA-LIR boutons formed symmetrical synaptic contacts and contained small round electron-lucent vesicles (50 nm) and one to several larger dense-core vesicles (80 nm). GABA-LIR terminals contacted HRP-labeled sympathetic preganglionic perikarya in all spinal nuclear regions in both vertebrates. More frequently, GABA-LIR boutons synapsed on dendrites. Occasionally, axo-axonic configurations were observed; each time only one of the axonal elements was GABA-LIR. Numerous unmyelinated and some thinly myelinated GABA-LIR axons coursed through the sympathetic preganglionic neuropils of both vertebrates. Synapses between GABA-LIR processes were present within the sympathetic preganglionic neuropil of both vertebrates. GABA-LIR dendrites were contacted by unlabeled terminals (predominantly small spherical vesicles with asymmetric synaptic specializations) and GABA-LIR terminals on GABA-LIR dendrites were similar in appearance to those synapsing on sympathetic preganglionic cell bodies and dendrites.     

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)     

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.     

Fine structural studies of growth-hormone-releasing-factor (GRF)-immunoreactive neurons and their synaptic connections in the guinea pig arcuate nucleus

The fine structure of neurons containing human growth-hormone-releasing factor (hGRF) immunoreactivity located in the arcuate nucleus of the guinea pig was studied by means of the preembedding immunohistochemical technique. The perikaryon of labeled neurons was fusiform or ovoid; the nucleus was regular in shape and contained a prominent nucleolus. The main ultrastructural features of the hGRF-immunoreactive neurons were the presence of numerous labeled secretory granules (100-120 nm in diameter) and the abundance and the enlargement of the organelles involved in the synthesis of the peptides: a well-developed rough endoplasmic reticulum and a conspicuous Golgi apparatus. Synaptic inputs were observed on immunoreactive perikarya but, above all, on the labeled dendrites. The unstained presynaptic nerve endings most often contained only small clear vesicles and formed symmetrical contacts. In rare cases, the presynaptic terminals exhibited both small clear and large dense vesicles and constituted asymmetrical contacts. Immunoreactive nerve endings were also observed in this area: the synaptic boutons contained large, stained vesicles and small, unlabeled, clear vesicles. These axon terminals made synaptic contacts with unstained dendritic processes; the contacts were symmetrical. The results indicate that hGRF-immunoreactive neurons of the guinea pig arcuate nucleus present morphological features of neuroendocrine cells. Moreover, the presence of hGRF-labeled nerve endings in the arcuate nucleus itself suggests that a substance related to hGRF might be a neuromodulator, at least in this area.     

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

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

Synaptic connections between corticogeniculate axons and interneurons in the dorsal lateral geniculate nucleus of the cat

Anatomical evidence is provided for direct synaptic connections by axons from visual cortex with interneurons in lamina A of the cat's dorsal lateral geniculate nucleus. Corticogeniculate axon terminals were labeled selectively with 3H-proline and identified by means of electron microscopic autoradiography. Interneurons in the lateral geniculate nucleus were stained with antibodies that had been raised against gamma aminobutyric acid (GABA). We found that corticogeniculate terminals synapsed with dendrites stained positively for GABA about three times as often as with unstained dendrites. Of the corticogeniculate terminals that contacted GABA-positive dendrites, 97% made synaptic connections with dendritic shafts. Only 3% synapsed with F profiles, the vesicle-filled dendritic appendages characteristic of lateral geniculate interneurons. These results suggest that the corticogeniculate pathway in the cat is directed primarily at interneurons and is organized synaptically to influence the integrated output of these cells, rather than the local interactions in which their dendritic specializations participate.     

Association of dopaminergic terminals and neurons releasing nitric oxide in the rat striatum: An electron microscopic study using NADPH-diaphorase histochemistry and tyrosine hydroxylase immunohistochemistry

To examine synaptic input and association of terminals containing dopamine and other transmitters to rat striatal nitric oxide synthase-expressing neurons, an electron microscopic study using tyrosine hydroxylase (TH) immunohistochemistry combined with histochemistry for NADPH-diaphorase (NADPHd) was performed. NADPHd-positive neurons had medium-sized cell bodies containing a highly invaginated nucleus and received relatively sparse synaptic input; 3.6% of boutons apposed to the NADPHd-positive neurons were TH-immunoreactive. Of these TH-immunoreactive boutons, two synaptic contacts showing symmetrical synaptic specializations were found on a cell body and a proximal dendrite of a NADPHd-positive neuron. Other nonsynaptic TH-immunoreactive boutons were occasionally associated with unlabeled terminals adjacent to the NADPHd-positive dendrites and also forming asymmetric synaptic contacts with unlabeled spinous or dendritic profiles. These results suggest that activity of the striatal neurons that release nitric oxide may be regulated by direct synaptic input from dopaminergic neurons and also suggest that the TH-immunoreactive terminals associated with the dendrites of nitric oxide synthase-expressing neurons provide the sites where nitric oxide influences dopamine release from neighboring terminals.     

Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: evidence for a cholinergic innervation of intracerebral blood vessels

The ultrastructural morphology and vascular associations of cholinergic neurons in the horizontal limb of the nucleus of the diagonal band of Broca (nDBBhl) and amygdala of rat were determined by the immunocytochemical localization of choline acetyltransferase (ChAT), the acetylcholine biosynthetic enzyme. Within the nDBBhl peroxidase reaction product was distributed throughout the cytoplasm of selectively labeled neuronal perikarya and dendrites. Labeled perikarya were characterized by an oval cell body (7-10 microns X 17-26 microns in diameter) in which was located a large nucleus and often a prominent nucleolus. Dendrites were by far the most numerous immuno-labeled profiles in the nDBBhl. The labeled dendrites had a cross-sectional diameter of 0.4-4.6 microns and contained numerous mitochondria and microtubules. Approximately 10% of all immunolabeled dendrites received synaptic contacts from unlabeled presynaptic boutons. In contrast to the relatively large number of ChAT-labeled dendrites within the nDBBhl, ChAT-positive axons were less frequently observed and immunolabeled axon terminals were never detected. The labeled axons had an outside diameter of 0.4-1.4 micron and were myelinated. The absence or relative paucity of immunolabeled terminals in the nDBBhl indicates that most if not all of the cholinergic perikarya within this nucleus are efferent projection neurons. The nDBB is known to have widespread projections to many areas of the neocortex, hippocampus, and amygdala. In the present study we examined the amygdala and observed many ChAT-labeled axon boutons. The immunolabeled varicosities contained numerous agranular vesicles and although ChAT-positive terminals were in direct contact with unlabeled neuronal elements within the amygdala, few if any synaptic densities were detected in a single plane of section. With respect to the vasculature, immunolabeled perikarya and dendrites within the nDBBhl and axon terminals in the amygdala were often in direct apposition to blood vessels. In many instances the labeled profile was observed lying directly on the basal lamina of a capillary endothelial cell. In no instance, however, were membrane densities observed. The presence of cholinergic neuronal elements contacting the vessel wall provides morphologic evidence suggesting that the neurogenic control of cerebral vasculature is in part mediated via a cholinergic mechanism.     

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

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