June Chan

Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding

The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5-20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization of rabbit anti-TH simultaneously with immunoperoxidase labeling of a mouse monoclonal antibody against the opiate peptide, leucine-enkephalin (LE). Vibratome sections were collected from acrolein fixed brains of adult rats. These sections were immunolabeled without use of freeze-thawing or other methods that enhance penetration, but damage ultrastructure. By light microscopy, incubations in the silver intensifier (Intense M, Janssen) for less than 10 min at room temperature resulted in a brownish-red reaction product for TH. This product was virtually indistinguishable from that seen using diaminobenzidine reaction for detection of peroxidase immunoreactivity. Longer incubations produced intense black silver deposits that were more clearly distinguishable from the brown immunoperoxidase labeling. However, by light microscopy, the gold particles seen by electron microscopy were most readily distinguished from peroxidase reaction product with shorter silver intensification periods. The smaller size of gold particles with shorter periods of silver intensification also facilitated evaluation of labeling with respect to subcellular organelles. Detection of the silver product did not appear to be appreciably changed by duration of post-fixation in osmium tetroxide. In dual-labeled sections, perikarya and terminals exhibiting immunogold-silver labeling for TH were distinct from those containing immunoperoxidase labeling for LE. These results (1) define the conditions needed for optimal immunogold-silver labeling of antigens while maintaining the ultrastructural morphology in brain, and (2) establish the necessity for controlled silver intensification for light or electron microscopic differentiation of immunogold-silver and peroxidase reaction products and for optimal subcellular resolution.

Compartment-specific localization of cannabinoid 1 (CB1) and μ-opioid receptors in rat nucleus accumbens

Interactions between cannabinoid and opioid systems have been implicated in reward and drug seeking behaviors involving neuronal circuitry in the nucleus accumbens (Acb) shell and core. To determine the relevant sites, we examined the electron microscopic localization of cannabinoid type-1 (CB1) receptors and mu-opioid receptors in each Acb compartment in rat brain. CB1 receptor immunogold labeling was seen on the plasma membrane and within the cytoplasm of neuronal and glial profiles throughout the Acb. These neuronal profiles included somata and dendrites as well as axon terminals, many of which formed excitatory-type, asymmetric synapses with notable perforations that are often associated with synaptic plasticity. The number of CB1-labeled terminals within the neuropil of the Acb shell was significantly greater than in the core. Mu-opioid receptors were also detected in axonal and dendritic profiles. These dendrites were most prevalent in the Acb shell, where mu-receptors also were located in 21% of the dendritic profiles and 3% of the axon terminals containing CB1 receptors. More of the CB1-labeled terminals contacted dendrites expressing mu-opioid receptors in the shell (19%) compared with the core (13%). Conversely, of the synaptic mu-labeled terminals, 20% in the shell and 10% in the core contacted dendrites containing CB1 receptors. These findings provide ultrastructural evidence that cannabinoid-opioid interactions are mediated by activation of CB1 and mu-opioid receptors within the same or synaptically linked neurons in the Acb shell and core. They also suggest a particularly important role for presynaptic CB1 receptors in the reward circuit of the Acb shell.

VESICULAR MONOAMINE TRANSPORTER-2 : IMMUNOGOLD LOCALIZATION IN STRIATAL AXONS AND TERMINALS

The vesicular monoamine transporter‐2 (VMAT2) mediates the reserpinesensitive neuronal uptake of monoamines into vesicles and other intracellular organelles. Accordingly, this transporter is expressed at high levels in regions that contain a dense monoamine innervation, such as the rat dorsolateral striatum. We used ultrastructural immunocytochemistry in this region to show that immunogold labeling for VMAT2 is present in varicose axonal processes, many of which also contain the catecholamine‐synthesizing enzyme tyrosine‐hydroxylase. Within these mainly dopaminergic processes, VMAT2 was associated with small synaptic vesicles (SSVs) and more rarely with large dense‐core vesicles or tubulovesicles. These findings suggest that SSVs are the major organelles involved in the storage and release of dopamine in the dorsolateral striatum. Synapse 26:194–198, 1997.

Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens.

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.

Cellular substrates for interactions between dynorphin terminals and dopamine dendrites in rat ventral tegmental area and substantia nigra

Dynorphin and other kappa opioid agonists are thought to elicit aversive actions and changes in motor activity through direct or indirect modulation of dopamine neurons in ventral tegmental area (VTA) and substantia nigra (SN), respectively. We comparatively examined the immunoperoxidase localization of anti-dynorphin A antiserum in sections through the VTA and SN of adult rat brain to assess whether there were common or differential distributions of this opioid peptide relative to the dopamine neurons. We also more directly examined the relationship between dynorphin terminals and dopamine neurons in VTA and SN by combining immunoperoxidase labeling of rabbit dynorphin antiserum and immunogold-silver detection of mouse antibodies against tyrosine hydroxylase (TH) in single sections through the VTA and SN. Light microscopy showed dynorphin-like immunoreactivity (DY-LI) in varicose processes. These were relatively sparse in VTA and were unevenly distributed in the SN, with little labeling in the pars compacta (pcSN) and the highest density of DY-LI in the medial and lateral pars reticulata (prSN). Electron microscopy established that the regional differences were attributed to differences in density (number/unit area) of immunoreactive profiles. The profiles containing DY-LI were designated as axon terminals based on having diameters greater than 0.1 micron, few microtubules and many synaptic vesicles. In both the VTA and SN, the dynorphin-labeled terminals contained primarily small (35-40 nm) clear vesicles. These vesicles were rimmed with peroxidase immunoreactivity and were often seen clustered above axodendritic synapses. These synaptic specializations were usually symmetric; however a few asymmetric densities also were formed by immunoreactive terminals in both VTA and SN. Additionally, most of the dynorphin-labeled terminals contained 1-2, but occasionally 7 or more intensely peroxidase positive dense core vesicles (DCVs). Approximately 60% of the DCVs were located near axolemmal surfaces. The axolemmal surfaces contacted by immunoreactive DCVs were more often apposed to dendrites in the VTA; while in the SN other axon terminals were the most commonly apposed neuronal profiles. In both regions, a substantial proportion of the plasmalemmal surface in contact with the labeled DCVs was apposed to astrocytic processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Phenylethanolamine N-methyltransferase-containing neurons in the rostral ventrolateral medulla. II: Synaptic relationships with GABAergic terminals

The ultrastructural morphology of terminals synthesizing gamma-aminobutyric acid (GABA), as indicated by peroxidase immunoreactivity for its synthetic enzyme L-glutamate decarboxylase (GAD), was examined in the rostral ventrolateral medulla (RVL) of the adult rat brain. The objective of the study was to determine the types of synaptic associations between the GABAergic terminals and other neurons in the RVL, particularly the C1-adrenergic neurons containing phenylethanolamine N-methyltransferase (PNMT). The brains were fixed by perfusion with 3.75% acrolein and 2.0% paraformaldehyde in phosphate buffer. Coronal Vibratome sections through the RVL were singly labeled with a sheep antiserum to GAD using the peroxidase-antiperoxidase (PAP) method. Additional sections were dually labeled using the PAP technique for the GAD antiserum and immunogold labeling for a rabbit antiserum against PNMT. Ultrastructural analysis revealed that peroxidase labeling for GAD was localized primarily to axons and axon terminals in both single and dual labeled material. The axons were small and unmyelinated. The GAD-labeled terminals were 0.5-2.0 microns in diameter and contained a large population of small clear vesicles usually associated with a few mitochondria. These terminals formed synapses with many dendrites, a few nerve cell bodies and axon terminals. The junctions were all symmetric and the postsynaptic structures failed to exhibit immunoreactivity when processed only for GAD labeling. In sections incubated with both GAD and PNMT antisera, the peroxidase-labeled GABAergic terminals formed symmetric synapses with nerve cell bodies and dendrites showing immunogold labeling for PNMT. In addition, the GAD-labeled terminals were presynaptic to other dendrites which appeared to have equal access to the antisera and gold markers, but failed to exhibit detectable immunoreactivity for PNMT. Both the PNMT-labeled and unlabeled somata and dendrites also received symmetric and asymmetric contacts from terminals containing neither GAD nor PNMT-immunoreactivity. We conclude that GABA is at least one of the inhibitory transmitters regulating adrenergic as well as non-adrenergic outflow from the RVL.

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

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

Chronic Intermittent Hypoxia Induces NMDA Receptor-Dependent Plasticity and Suppresses Nitric Oxide Signaling in the Mouse Hypothalamic Paraventricular Nucleus

Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly developing chemosensory-dependent blood pressure elevation ascribed in part to NMDA receptor-dependent plasticity and reduced nitric oxide (NO) signaling in the carotid body. The hypothalamic paraventricular nucleus (PVN) is responsive to hypoxic stress and also contains neurons that express NMDA receptors and neuronal nitric oxide synthase (nNOS). We tested the hypothesis that extended (35 d) CIH results in a decrease in the surface/synaptic availability of the essential NMDA NR1 subunit in nNOS-containing neurons and NMDA-induced NO production in the PVN of mice. As compared with controls, the 35 d CIH-exposed mice showed a significant increase in blood pressure and an increased density of NR1 immunogold particles located in the cytoplasm of nNOS-containing dendrites. Neither of these between-group differences was seen after 14 d, even though there was already a reduction in the NR1 plasmalemmal density at this time point. Patch-clamp recording of PVN neurons in slices showed a significant reduction in NMDA currents after either 14 or 35 d exposure to CIH compared with sham controls. In contrast, NO production, as measured by the NO-sensitive fluorescent dye 4-amino-5-methylamino-2′,7′-difluorofluorescein, was suppressed only in the 35 d CIH group. We conclude that CIH produces a reduction in the surface/synaptic targeting of NR1 in nNOS neurons and decreases NMDA receptor-mediated currents in the PVN before the emergence of hypertension, the development of which may be enabled by suppression of NO signaling in this brain region.

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

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

Region-Specific Changes in the Subcellular Distribution of AMPA Receptor GluR1 Subunit in the Rat Ventral Tegmental Area after Acute or Chronic Morphine Administration

Opiate addiction is characterized by progressive increases in drug intake over time suggesting maladaptive changes in motivational and reward systems. These behaviors are mediated by dopaminergic neurons originating from the ventral tegmental area (VTA), and long-term changes of these dopaminergic neurons are attributed to increased postsynaptic glutamatergic activation. Indeed, chronic morphine administration is known to increase AMPA receptor glutamate receptor 1 (GluR1) subunit in the VTA. However, there is no ultrastructural evidence that morphine affects the expression or surface availability of GluR1 subunits in VTA neurons of defined distribution or transmitter phenotype. Therefore, we examined electron microscopic immunolabeling of GluR1 and tyrosine hydroxylase (TH) in two VTA regions of rats perfused 1 h after a single injection of morphine, or chronic morphine in intermittent-escalating doses for 14 d, and appropriate saline controls. Acute morphine administration produced a significant increase in GluR1 immunogold particles at the plasma membrane and postsynaptic densities in both TH- and non-TH-containing dendrites in the parabrachial VTA, a region that contains mainly prefrontal-cortical-projecting dopaminergic neurons involved in motivation and drug-seeking behavior. Chronic morphine administration maintained the increased synaptic GluR1 labeling in the parabrachial VTA, but also increased the number of GluR1-labeled synapses and TH immunoreactivity in dendrites of the paranigral VTA where substantially more dopaminergic neurons project to limbic structures implicated in locomotor activation and reward. These results demonstrate a region- and dose-dependent redistribution of GluR1-containing AMPA receptors, which is consistent with acute morphine activation of cortical-projecting VTA neurons and chronic morphine activation of limbic-projecting VTA neurons.