Biomedical subjects

V M Pickel

Publications and source records attributed to V M Pickel.

At least 109 records · Page 6Linked to original sources

PubMed1995

Association of sorcin with the cardiac ryanodine receptor.

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A > 500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.

Animals↗

PubMed1995

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

Animals↗

PubMed1995

Co-localization of angiotensin II and gamma-aminobutyric acid in axon terminals in the rat subfornical organ.

Angiotensin II (Ag II) and gamma-aminobutyric acid (GABA) in the subfornical organ have been implicated in drinking and cardiovascular responses to changes in circulating hormones. We combined immunogold silver labeling of Ag II with immunoperoxidase detection of GABA to determine whether there might be common cellular sites for their physiological actions in this circumventricular region. Electron microscopy showed that numerous terminals in central portions of the rat subfornical organ contained both Ag II and GABA immunoreactivity. These terminals as well as others exclusively labeled for AgII or GABA formed mainly symmetric, inhibitory type synapses with unlabeled dendrites. Immunogold-silver aggregates recognizing AgII were often detected near non-synaptic portions of the plasma membrane with or without apparent association with large dense core vesicles. In contrast, the GABA immunoperoxidase labeling was most intensely localized to membranes of small clear vesicles which were aggregated near the presynaptic junction. Our results indicate that in rat subfornical organ, neuronal AgII may modulate the inhibitory postsynaptic responses to GABA following release from single axon terminals.

Angiotensin II↗

PubMed1995

Immunocytochemical localization of the renal neutral and basic amino acid transporter in rat adrenal gland, brainstem, and spinal cord.

A neutral and basic amino acid transporter (NBAT) cloned from rat kidney was recently localized to enteroendocrine cells and enteric neurons. We used an antibody directed against a synthetic peptide representing a putative extracellular domain of NBAT to determine whether this transporter was also present in other endocrine and neural tissues, including rat adrenal gland, brainstem, and spinal cord. Abundant, highly granular labeling for NBAT was observed in the cytoplasm of chromaffin and ganglion cells in the adrenal medulla. A small population of intensely labeled varicose processes was also seen in both the cortex and the medulla of the adrenal gland. More numerous, intensely labeled varicose processes were detected in brainstem and spinal cord nuclei, including the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, dorsal motor nucleus of the vagus, and intermediolateral cell column of the thoracic spinal cord. Significant perikaryal labeling for NBAT was only detected in brainstem and spinal cord following intraventricular colchicine treatment, which increased the number, distribution, and intensity of NBAT-immunolabeled cells. These NBAT-immunoreactive perikarya were most numerous in the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, and raphe nuclei. Ultrastructural examination of the nuclei of the solitary tract of normal rats showed that NBAT was localized predominantly to axon terminals. Within these labeled terminals, NBAT was associated with large dense core vesicles and discrete segments of plasma membrane. The observed localization of NBAT suggests that this renal specific amino acid transporter subserves a role as a vesicular or plasmalemmal transporter in monoamine-containing cells, including chromaffin cells and autonomic neurons.

Adrenal Medulla↗

PubMed1995

GABA-containing neurons in the ventral tegmental area project to the nucleus accumbens in rat brain.

The ventral tegmental area receives a gamma-aminobutyric acid (GABA) innervation from the nucleus accumbens and contains GABA immunoreactive neurons believed to be interneurons. We combined the immunocytochemical detection of retrogradely transported Fluoro-Gold (FG) from the nucleus accumbens (Acb) with the detection of GABA within the same section of tissue in the ventral tegmental area (VTA) of the rat brain to determine whether there might also be reciprocal GABAergic projections in the mesolimbic pathway. Immunoperoxidase labeling for FG and immunogold-silver labeling for GABA were most readily distinguished within perikarya and dendrites in sections examined by electron microscopy. Ultrastructural observations indicated that 36% (n = 110) of the FG-labeled perikarya and dendrites also contained GABA immunoreactivity. The present results provide the first evidence that GABA is contained in a subpopulation of neurons in the mesolimbic pathway from the VTA to the Acb. The reciprocity of this circuitry may provide an important feedback loop thus facilitating inhibition of motor activity.

Animals↗

PubMed1995

Ultrastructural characterization of neurons recorded intracellularly in vivo and injected with lucifer yellow: advantages of immunogold-silver vs. immunoperoxidase labeling.

Immunoperoxidase labeling of lucifer yellow provides a sensitive method for morphological characterization of neurons recorded intracellularly in vitro or in vivo. However, the reaction product is often so dense that it obscures ultrastructural details necessary for the analysis of synaptic contacts onto individually filled neurons. In the present study, we describe a silver intensification procedure using 1 nm gold labeling of lucifer yellow as an optimal means for immunocytochemically identifying single physiologically characterized neurons at the ultrastructural level. Single neurons in the frontal cortex of anesthetized rats were impaled in vivo and filled with lucifer yellow. The brains were then perfused with an acrolein fixative. Single vibratome sections through the recording site were reacted with a rabbit antibody directed against lucifer yellow followed by goat anti-rabbit 1 nm gold-labeled IgG and silver intensified. For comparison, additional sections were processed for immunoperoxidase detection of lucifer yellow. Labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The immunogold-silver label as well as peroxidase reaction product of lucifer yellow was readily detected in cell bodies, proximal and distal dendrites, and spines. However, in contrast to immunoperoxidase, the immunogold-silver reaction did not obscure subcellular organelles. Most importantly, the synaptic junctions formed by afferents to the filled neuron were more easily identifiable following the immunogold-silver procedure. This clear visualization of postsynaptic densities is essential for examining synaptic circuitry between afferents and physiologically characterized neurons.

Animals↗

PubMed1995

Monosynaptic input from Leu5-enkephalin-immunoreactive terminals to vagal motor neurons in the nucleus ambiguus: comparison with the dorsal motor nucleus of the vagus.

Vagal motor neurons in the rat dorsal motor nucleus of the vagus (DMN) are known to receive direct synaptic input from enkephalin-containing terminals. We examined 1) whether the vagal motor neurons within the nucleus ambiguus (NA) also received monosynaptic input from enkephalin-immunoreactive terminals and 2), if so, whether their ultrastructural relations differed from those in the DMN. In both regions, terminals containing Leu5-enkephalin-like immunoreactivity (LE-LI) were examined in relation to motor neurons identified by retrograde transport of wheat germ-agglutinated horseradish peroxidase (WGA-HRP) applied to the cut end of the cervical vagus nerve in single sections of the medulla oblongata of adult rats. By light microscopy, the most significant overlap between varicose processes with LE-LI and WGA-HRP-containing neurons was seen in the rostral compact portion of the NA and the DMN at the level of the obex. Thus, only these regions were examined by electron microscopy. The most distinguishing ultrastructural feature of WGA-HRP-labeled neurons in the NA compared to the DMN was their higher incidence of nonsynaptic appositions with other neurons. In both the NA and the DMN, terminals with LE-LI formed primarily symmetric synapses on smaller (presumably distal) dendrites; many of these dendrites, as well as most target perikarya, contained WGA-HRP. Additionally, in the compact portion of the NA compared to the DMN 1) multiple LE-labeled terminals more frequently contacted single perikarya or dendrites and 2) single terminals with LE-LI more commonly showed two contacts or active zones and contained more abundant LE-immunoreactive large (80-100 nm) dense-core vesicles (dcvs). In contrast to small (40-50 nm), clear vesicles, which were usually aggregated near active zones, the immunoreactive dcvs were usually located near glial processes distal to these zones. These results indicate that enkephalin immunoreactivity is intensely localized to dcvs within terminals that may have direct inhibitory (symmetric synapses) actions on vagal motor neurons in both the compact portion of the NA and the DMN. Moreover, because numbers of dcvs and active zones have been equated with synaptic strength, our findings suggest enhanced potencies of enkephalin-immunoreactive terminals in the compact portion of the NA. Our findings support a prominent role for enkephalin in the coordinated activity of esophageal motor neurons located in the compact portion of the NA.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Use of quantitative ultrastructural immunoperoxidase labeling for analysis of catecholamine neurotoxicity and plasticity.

Levels of catecholamines and the synthesizing enzyme, tyrosine hydroxylase (TH) are markedly decreased in the dorsal striatum, caudate-putamen nuclei, following neurotoxic lesions with 6-hydroxy-dopamine (6-OHDA). We examined whether pre-embedding immunoperoxidase labeling of TH could be standardized for quantitatively examining the density and ultrastructure of spared dopaminergic terminals in the striatum of lesioned rats. The peroxidase-antiperoxidase (PAP) method was used to localize rabbit antiserum against TH in caudate-putamen nuclei of adult rats given unilateral nigral injections of either vehicle or 6-hydroxydopamine in the early postnatal period. Experimental differences in fixation and immunocytochemical labeling were minimized by limiting comparisons of immunoreactivity to co-processed sections from the same litters of animals. Imaging software and a Phillips CM-10 electron microscope were used to quantitatively examine immunoreactive profiles in a narrow zone of tissue in contact with the embedding resin. Under these conditions variables attributed to differences in penetration were minimized. There were no significant differences in numbers or mean-cross sectional diameter of immunoreactive terminals in striatum ipsilateral versus contralateral to vehicle injections. Ipsilateral to the 6-OHDA injections, the density (numbers/area) of striatal TH-immunoreactive terminals was reduced by 50-90% in the majority of animals. In the most extensively lesioned rats, the cross-sectional areas of the remaining immunoreactive axons were significantly larger than in the contralateral striatum of the same animal or either hemisphere of vehicle injected controls. These results confirm and extend earlier findings on the plasticity of residual dopaminergic terminals in adult animals after neurotoxic damage. They also establish a quantitative method for ultrastructural analysis of the density of immunoreactivity in thick sections of tissue labeled prior to plastic embedding. The method has broad applicability to quantitative studies of neurotoxicity and plasticity in brain.

Animals↗

PubMed1995

Ultrastructural immunolabeling shows prominent presynaptic vesicular localization of delta-opioid receptor within both enkephalin- and nonenkephalin-containing axon terminals in the superficial layers of the rat cervical spinal cord.

Opioid peptides, Met5- and Leu5-enkephalin, are known endogenous ligands for the delta-opioid receptor (DOR) associated with opioid analgesia at the spinal level. To determine the cellular sites for DOR-mediated actions, we examined the ultrastructural localization of DOR and Met5-enkephalin (ME) in the spinal cord by combining immunoperoxidase and immunogold-silver labeling for antibodies against DOR and ME, respectively. Antibodies for DOR localization were raised in guinea pig against peptide 34-47 (p34), an amino acid sequence within the extracellular N-terminus of the DOR recently cloned from mouse neuroblastoma glioma (NG-108) cells. Selective immunoperoxidase labeling for DOR was detected by light microscopy in NG-108 cells and in the lamina I and II of the dorsal horn of the spinal cord (C2-C4). Electron microscopy of these spinal laminae revealed that the majority of the punctate varicosities seen by light microscopy were axon terminals. delta-opioid receptor-like immunoreactivity (DOR-LI) in axon terminals was most prominently associated with large dense core vesicles, and sometimes seen along the membranes of small clear vesicles and segments of the plasmalemma. A semiquantitative analysis of dually labeled sections revealed that of the terminals showing DOR-LI, 23/102 (23%) also contained Met5-enkephalin-like immunoreactivity (ME-LI). Conversely, 23/35 (66%) of the terminals showing ME-LI also showed DOR-LI. In addition to the presynaptic localization, selective postsynaptic densities within dendrites were also occasionally (9%) immunolabeled for the opioid receptor. These results provide the first ultrastructural evidence that DOR may serve autoreceptor functions on ME terminals as well as presynaptic modulation of other transmitters in the dorsal horn of the rat spinal cord. Additionally, the vesicular localization of DOR-LI in axon terminals suggests the involvement of these organelles in the transport of the receptors to the plasma membrane.

Amino Acid Sequence↗

PubMed1994

Ultrastructure of Met5-enkephalin terminals in the caudate-putamen nuclei of adult rats receiving neonatal intranigral 6-hydroxydopamine.

Destruction of dopamine neurons of the nigrostriatal pathway in the early postnatal rat enhances the levels of Met5-enkephalin in the adult dorsal striatum (caudate-putamen nuclei) and may contribute to the abnormal self-injurious behavior seen in humans with Lesch-Nyhan disease. We examined whether there were ultrastructural changes in Met5-enkephalin immunoreactive terminals in the rat model that might reflect cellular sites for enhanced activity of these opioid neurons. At 3 days postnatal, 10-20 nl injections of a 1% solution of the dopamine neurotoxin, 6-hydroxydopamine (6-OHDA), or vehicle were placed unilaterally in the region of the substantia nigra of 25 litters of male rat pups. In adulthood (72-80 days postnatal), the brains of these animals were fixed by vascular perfusion with an aldehyde solution. Met5-enkephalin immunolabeling was examined in coronal sections at three rostrocaudal levels through the caudate-putamen nuclei of control (ipsilateral and contralateral to vehicle and contralateral to 6-OHDA) and experimental (ipsilateral to 6-OHDA) groups. In selectively lesioned animals, there was a significant increase in the relative optical density of immunoautoradiographic labeling for enkephalin throughout the rostrocaudal striatum ipsilateral to 6-OHDA as compared to control groups. Electron microscopy revealed immunoperoxidase labeling for enkephalin in axon terminals and more rarely in soma and dendrites irrespective of drug treatment. In both experimental and control striatal tissues, the enkephalin immunoreactive terminals formed primarily symmetric synapses with unlabeled dendrites or spines. However, ipsilateral to 6-OHDA injections there was a small (5.4%), but significant increase in the proportion of enkephalin immunoreactive terminals contacting dendritic spines, the known targets of dopamine terminals. Appositions were commonly detected between enkephalin immunoreactive terminals and other morphologically heterogeneous axons in the striatum ipsilateral to 6-OHDA and in control tissues. Met5-enkephalin immunoreactive terminals in adult striatum ipsilateral to 6-OHDA injections showed a 214% increase in volume as compared to vehicle-injected controls. Concurrently, there was a small (13%), but significant increase in the numerical density (number/volume) of enkephalin-labeled terminals both contralateral and ipsilateral to 6-OHDA injections. These results suggest that a change in bouton size is the major mechanism by which striatal enkephalin neurons alter their synaptic efficacy and target associations to compensate for damage to the nigrostriatal dopamine neurons.

Animals↗

PubMed1994

Synaptic structure and connectivity of serotonin terminals in the ventral tegmental area: potential sites for modulation of mesolimbic dopamine neurons.

Microinfusion of serotonin (5-hydroxytryptamine; 5-HT) into the ventral tegmental area enhances the release of dopamine in the nucleus accumbens, a major target of midbrain dopamine neurons. We examined the synaptic basis for 5-HT modulation of neurons in the ventral tegmental area which either (i) project to the nucleus accumbens or (ii) contain the catecholamine synthesizing enzyme tyrosine hydroxylase, a marker of dopamine neurons in this brain region. In the first study, immunoperoxidase labeling of 5-HT in the ventral tegmental area was combined with retrograde transport of gold particles following unilateral injections of the tracer into the nucleus accumbens of adult rats. The gold particles had been previously coupled to wheat germ agglutinin conjugated to inactive horseradish peroxidase. Gold particles were enlarged for visualization using a silver enhancement procedure. By brightfield microscopy, retrogradely labeled neurons contained black punctate granules within their perikarya and proximal processes. The labeled cells were scattered ipsilateral to the injection within the paranigral and parabrachial subdivisions of the ventral tegmental area. Both regions also contained 5-HT immunoreactive varicosities. By electron microscopy, irrespective of the ventral tegmental subdivision, 5-HT labeling was seen primarily in unmyelinated axons and axon terminals. The terminals contained small, clear and large dense core vesicles and ranged from 0.3 micron to 1.4 microns in cross-sectional diameter. 22% (n = 250) of the axon terminals containing 5-HT immunoreactivity formed synaptic contacts with neurons containing the retrograde label. Of these 5-HT terminals, 16% formed asymmetric type contacts and 6% formed symmetric junctions on the retrogradely labeled neurons. The remaining 5-HT terminals were either apposed to (but lacked recognized synapses on) perikarya and large dendrites containing the retrogradely transported protein-gold tracer or contacted unlabeled neurons. In the second set of experiments combining immunoperoxidase of 5-HT and immunogold silver for tyrosine hydroxylase, 32% (n = 250) of the 5-HT-labeled terminals formed synaptic junctions with perikarya or dendrites containing tyrosine hydroxylase immunoreactivity. Of these 5-HT terminals, 23% formed asymmetric type junctions. The remainder were either symmetric or lacked recognized membrane densities. The prominence of asymmetric junctions formed by 5-HT-labeled terminals on neurons projecting to the nucleus accumbens and those containing tyrosine hydroxylase in the ventral tegmental area suggests a cellular basis for serotonergic excitation of mesoaccumbens dopamine neurons. Additionally, the multiplicity of junctions formed by 5-HT terminals on targets with or without retrograde labeling or tyrosine hydroxylase immunoreactivity is consistent with known diverse physiological actions of 5-HT in the tegmental area.

Animals↗

PubMed1994

Met5-enkephalin is localized within axon terminals in the subfornical organ: vascular contacts and interactions with neurons containing gamma-aminobutyric acid.

Met5-enkephalin inhibits sodium and water excretion and antagonizes the central actions of angiotensin II in subfornical organ of rat brain. We examined the ultrastructural basis for enkephalin modulation in this circumventricular region. Additionally, we examined the possibility that there might be cellular sites for functional interactions involving Met5-enkephalin and gamma-aminobutyric acid (GABA), a known inhibitory transmitter throughout the central nervous system. Met5-enkephalin and GABA were identified in single coronal sections through the subfornical organ using immunoperoxidase and silver-enhanced immunogold labeling methods, respectively. Enkephalin-like immunoreactivity was most prominently localized within axon terminals. These were distributed primarily in the central, highly vascular, regions of the subfornical organ. Enkephalin-labeled terminals were apposed to the basement membranes of fenestrated capillaries and also formed symmetric, inhibitory type synapses with neurons. In terminals associated with either blood vessels or neurons, the enkephalin immunoreactivity was enriched in large (80-150 nm) dense core vesicles. The immunoreactive vesicles were usually located within portions of the axon in close proximity to astrocytic processes. In contrast, smaller vesicles in the same terminals were more often aggregated near the basement membrane of the capillaries and the active zone of the synapse. The targets of enkephalin-immunoreactive terminals were either unlabeled or GABA-labeled dendrites of local neurons. Enkephalin was also co-localized with GABA in perikarya and in axon terminals. Terminals containing only GABA were far more abundant than those containing enkephalin or enkephalin and GABA. GABA-immunoreactive terminals formed symmetric synapses on unlabeled dendrites some of which also received convergent input from terminals containing enkephalin. Additionally, the enkephalin-immunoreactive terminals were closely apposed to GABA-labeled and unlabeled terminals. These results suggest sites for nonsynaptic release of Met5-enkephalin from dense core vesicles in contact with astrocytes near blood vessels and synaptic complexes in the rat subfornical organ. Moreover, the observed dual localization and pre- and postsynaptic associations between neurons containing Met5-enkephalin and GABA indicate that inhibitory effects of opioids in the subfornical organ may be mediated or potentiated by GABA.

Animals↗

PubMed1994

Dynorphin-immunoreactive terminals in the rat nucleus accumbens: cellular sites for modulation of target neurons and interactions with catecholamine afferents.

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 microns 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.

Animals↗

PubMed1994

Analysis of synaptic inputs and targets of physiologically characterized neurons in rat frontal cortex: combined in vivo intracellular recording and immunolabeling.

Ultrastructural immunocytochemical identification of transmitters in afferent terminals and targets of individual physiologically characterized neurons is essential for understanding the complex circuitry within the mammalian neocortex. For this type of analysis, we examined the utility of combining in vivo intracellular recording and biocytin injections with silver intensified 1 nm immunogold labeling of GABA and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). These transmitters are found to local neurons and afferents known to prominently modulate the activity of pyramidal neurons in the neocortex. Individual neurons were physiologically characterized and filled with biocytin in the frontal cortex of anesthetized rats. The brains were then preserved by vascular perfusion with aldehydes. Single vibratome sections through the recording site were reacted (1) for immunoperoxidase detection of biocytin and (2) for immunogold labeling of GABA or TH. Dually labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The dense peroxidase product for biocytin was detected in pyramidal neurons. These were located in superficial as well as deep cortical laminae, and were readily distinguished from immunogold silver labeling. GABA labeled terminals formed symmetric synapses with larger biocytin filled dendrites, whereas the TH labeled terminals contacted distal dendrites and spines. Peroxidase labeling for biocytin also was seen in a few axon terminals forming synapses with unlabeled and with GABA immunoreactive dendrites. These results suggest that single pyramidal neurons of the rat frontal cortex receive dual input from both GABA and catecholamine terminals. Additionally, this study demonstrates the usefulness of silver enhancement of 1 nm colloidal gold prior to plastic embedding for electron microscopic detection of neurotransmitters within afferents and targets of neurons physiologically characterized in vivo.

Animals↗

PubMed1994

Immunolabeling of retrogradely transported Fluoro-Gold: sensitivity and application to ultrastructural analysis of transmitter-specific mesolimbic circuitry.

Fluorescence microscopy shows extensive filling of perikarya and distal dendrites following injections of Fluoro-Gold (FG) into their terminal fields. However, elucidation of synaptic contacts onto identified projection neurons has been limited by the lack of compatibility between electron-dense markers required for ultrastructural analysis and morphology preservation. The recent advent of antisera to FG has revealed numerous potential applications for analyzing chemically defined synaptic circuitry. To take advantage of the high sensitivity of this retrograde tracer in ultrastructural studies, we extended and detailed the original description of single immunocytochemical labeling of FG by comparing the advantages of immunodetection of an antiserum against FG using 2 distinct electron-dense markers: (1) avidin-biotin peroxidase (ABC) reacted with 3,3'-diaminobenzidine and darkened with osmium tetroxide, or (2) silver-intensified 1 nm colloidal gold particles. We subsequently examined the utility of combining these markers in single sections for detection of transmitters (e.g., gamma-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT)) in axon terminals presynaptic to retrogradely labeled neurons. Both analyses were carried out on the well-characterized mesolimbic pathway originating from perikarya in the ventral tegmental area (VTA) that project to the nucleus accumbens. Injections of FG were stereotaxically placed in the nucleus accumbens of anesthetized adult rats. From these animals, vibratome sections of aldehyde-fixed brains were examined for light-microscopic detection of FG using: (1) epi-fluorescence without immunocytochemistry, (2) immunoperoxidase, or (3) immunogold-silver. All 3 methods revealed circumscribed injections in the nucleus accumbens. Additionally, both immunocytochemical methods appeared to be as sensitive as epi-fluorescence in light-microscopic detection of retrogradely labeled perikarya and fine-caliber dendrites extending for 2-3 branch points beyond the soma. Electron microscopy showed that the FG was detectable not only in lysosomes but also throughout the cytoplasmic matrix of perikarya and dendrites using either immunoperoxidase or immunogold-silver labeling methods. In the second part of this analysis, single sections of tissue were processed for dual labeling using either immunoperoxidase or immunogold-silver for detection of FG in conjunction with the converse label for GABA or 5-HT, respectively. Regardless of the labeling combinations, the peroxidase and gold-silver reactions were readily distinguished within sections examined by light or electron microscopy. Synaptic junctions from unlabeled or from GABA or 5-HT labeled terminals were most readily identified when the targets were lightly immunoreactive for peroxidase or labeled using silver-intensified colloidal gold.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗