The immune/inflammatory signaling molecule tumor necrosis factor α (TNFα) is an important mediato... more The immune/inflammatory signaling molecule tumor necrosis factor α (TNFα) is an important mediator of both constitutive and plastic signaling in the brain. In particular, TNFα is implicated in physiological processes, including fever, energy balance, and autonomic function, known to involve the hypothalamic paraventricular nucleus (PVN). Many critical actions of TNFα are transduced by the TNFα type 1 receptor (TNFR1), whose activation has been shown to potently modulate classical neural signaling. There is, however, little known about the cellular sites of action for TNFR1 in the PVN. In the present study, high-resolution electron microscopic immunocytochemistry was used to demonstrate the ultrastructural distribution of TNFR1 in the PVN. Labeling for TNFR1 was found in somata and dendrites, and to a lesser extent in axon terminals and glia in the PVN. In dendritic profiles, TNFR1 was mainly present in the cytoplasm, and in association with presumably functional sites on the plasma ...
Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates co... more Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates corticosterone-sensitive uptake of monoamines including norepinephrine, epinephrine, dopamine, histamine and serotonin. OCT3 is expressed widely throughout the amygdaloid complex and other brain regions where monoamines are key regulators of emotional behaviors affected by stress. However, assessing the contribution of OCT3 to the regulation of monoaminergic neurotransmission and monoamine-dependent regulation of behavior requires fundamental information about the subcellular distribution of OCT3 expression. We used immunofluorescence and immuno-electron microscopy to examine the cellular and subcellular distribution of the transporter in the basolateral amygdaloid complex of the rat and mouse brain. OCT3-immunoreactivity was observed in both glial and neuronal perikarya in both rat and mouse amygdala. Electron microscopic immunolabeling revealed plasma membrane-associated OCT3 immunoreact...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2015
Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulat... more Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neuron...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in t... more Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in the production of stable nitrotyrosine (NT) moieties that are immunochemically detectable in many regions of normal brain and enriched in those areas containing constitutive nitric oxide synthase (cNOS). These include the caudate-putamen nucleus (CPN) and the globus pallidus, which receives major inhibitory input from the CPN. To determine the functional sites for NT production in these critical motor nuclei, we examined the electron microscopic immunocytochemical localization of NT and cNOS in rat brain. In the CPN, NT was localized to the somata and dendrites of cNOS-containing interneurons and spiny neurons, some of which received input from cNOS-labeled terminals. The NT immunoreactivity was most prevalent on outer mitochondrial membranes and nearby segments of the plasma membranes in dendrites and within asymmetric synapses on dendritic spines. In the CPN and globus pallidus, there w...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999
The expression of a brain-specific high-affinity Na+-dependent (and Cl--dependent) L-proline tran... more The expression of a brain-specific high-affinity Na+-dependent (and Cl--dependent) L-proline transporter (PROT) in subpopulations of putative glutamatergic neurons in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (). To gain insights into potential sites where PROT may function, we used a C-terminal domain antipeptide antibody to determine the regional distribution and subcellular localization of PROT in rat forebrain. PROT immunoreactivity was seen in processes having a regional light microscopic distribution comparable to that of known glutamatergic projections within the cortex, caudate putamen nucleus (CPN), hippocampal formation, and other forebrain regions. In all regions examined by electron microscopy (cortex, CPN, and the stratum oriens of CA1), PROT labeling was observed primarily within subpopulations of axon terminals forming asymmetric excitatory-type synapses. Immunogold labeling for PROT was detected in close contact wi...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1998
Molecular cloning has recently identified a vertebrate brain-specific Na+-dependent inorganic pho... more Molecular cloning has recently identified a vertebrate brain-specific Na+-dependent inorganic phosphate transporter (BNPI). BNPI has strong sequence similarity to EAT-4, a Caenorhabditis elegans protein implicated in glutamatergic transmission. To characterize the physiological role of BNPI, we have generated an antibody to the protein. Immunocytochemistry of rat brain sections shows a light microscopic pattern that is suggestive of reactivity in nerve terminals. Excitatory projections are labeled prominently, and ultrastructural analysis confirms that BNPI localizes almost exclusively to terminals forming asymmetric excitatory-type synapses. Although BNPI depends on a Na+ gradient and presumably functions at the plasma membrane, both electron microscopy and biochemical fractionation show that BNPI associates preferentially with the membranes of small synaptic vesicles. The results provide anatomic evidence of a specific presynaptic role for BNPI in glutamatergic neurotransmission, ...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1997
The dopamine transporter (DAT) regulates extracellular dopamine concentrations, transports neurot... more The dopamine transporter (DAT) regulates extracellular dopamine concentrations, transports neurotoxins, and acts as a substrate for cocaine reinforcement. These functions are known to differ in the limbic-associated shell and motor-associated core compartments of the nucleus accumbens (NAc). Previous studies have shown differential expression of DAT in the NAc shell and core but were limited in resolution to the regional level. Thus, it is not known whether there are differences in the amount, subcellular localization, or plasmalemmal targeting of DAT within individual dopaminergic axons in the two regions. We used high-resolution electron microscopic immunocytochemistry to investigate these possibilities. We show that in both the shell and core, DAT immunogold labeling is present in tyrosine hydroxylase-immunoreactive varicose axons that form symmetric synapses. Within these labeled axons, most DAT gold particles are located on extrasynaptic plasma membranes, but some are associate...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine a... more The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine and, thus, in the termination of normal dopaminergic neurotransmission. DAT is also a major binding site for cocaine and other stimulants, the psychoactive effects of which are associated primarily with the inhibition of dopamine reuptake within mesocorticolimbic dopaminergic neurons. We used electron microscopy with an anti-peptide antiserum directed against the N-terminal domain of DAT to determine the subcellular localization of this transporter in the rat ventral tegmental area (VTA), the region that contains the cell bodies and dendrites of these dopaminergic neurons. We show that in the VTA, almost 95% of the DAT immunogold-labeled profiles are neuronal perikarya and dendrites, and the remainder are unmyelinated axons. Within perikarya and large proximal dendrites, almost all of the DAT immunogold particles are associated with intracellular membranes, including saccules of Golgi and...
Midbrain dopaminergic neurons are known to release dopamine from somata and/or dendrites located ... more Midbrain dopaminergic neurons are known to release dopamine from somata and/or dendrites located in the substantia nigra (SN) and the ventral tegmental area (VTA). There is considerable controversy, however, about the subcellular sites for somatodendritic dopamine storage in these regions. In the present study, we used dual-labeling electron microscopic immunocytochemistry to localize the vesicular monoamine transporter-2 (VMAT2), a novel marker for sites of intracellular monoamine storage, within identified dopaminergic (tyrosine hydroxylase-containing) neurons in the rat SN and VTA. In dopaminergic perikarya, immunogold labeling for VMAT2 was localized to the Golgi apparatus, tubulovesicles that resembled smooth endoplasmic reticulum (SER), and the limiting membranes of multivesicular bodies. In dopaminergic dendrites, VMAT2 was extensively localized to tubulovesicles that resembled saccules of SER, and less frequently localized to isolated small synaptic vesicles (SSVs) or large dense-core vesicles (DCVs). In rare cases, VMAT2-immunoreactive SSVs were clustered within the cytoplasm of an SN or a VTA dendrite. Dopaminergic dendrites in the VTA contained a significantly higher number of immunogold particles for VMAT2 per unit area than those in the SN. Together, these observations support the proposal that dopamine is stored in and may be released from dendritic SSVs and DCVs, but suggest that the SER is the major site of dopamine storage within midbrain dopaminergic neurons. In addition, they provide new evidence that dopaminergic dendrites in the VTA may have greater potential for reserpinesensitive storage and release of dopamine than those in the SN.
Interactions between cannabinoid and opioid systems have been implicated in reward and drug seeki... more 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-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.-Opioid receptors were also detected in axonal and dendritic profiles. These dendrites were most prevalent in the Acb shell, where-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-opioid receptors in the shell (19%) compared with the core (13%). Conversely, of the synaptic-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-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.
The alpha-7 nicotinic acetylcholine receptor (α7nAChR) and the dopamine D 2 receptor (D 2 R) are ... more The alpha-7 nicotinic acetylcholine receptor (α7nAChR) and the dopamine D 2 receptor (D 2 R) are both implicated in attentional processes and cognition, mediated in part through the prefrontal cortex (PFC). We examined the dual electron microscopic immunolabeling of α7nAChR and either D 2 R or the vesicular acetylcholine transporter (VAChT) in rodent PFC to assess convergent functional activation sites. Immunoreactivity (ir) for α7nAChR and/or D 2 R was seen in the same as well as separate neuronal and glial profiles. At least half of the dually labeled profiles were somata and dendrites, while most labeled axon terminals expressed only D 2 R-ir. The D 2 R-labeled terminals were without synaptic specializations or formed inhibitory or excitatory-type synapses with somatodendritic profiles, some of which expressed the α7nAChR and/or D 2 R. Astrocytic glial processes comprised the majority of nonsomatodendritic α7nAChR or α7nAChR and D 2 Rlabeled profiles. Glial processes containing α7nAChR-ir were frequently located near VAChTlabeled terminals and also showed perisynaptic and perivascular associations. We conclude that in rodent PFC α7nACh and D 2 R activation can dually modulate (1) postsynaptic dendritic responses within the same or separate but synaptically linked neurons in which the D 2 R has the predominately presynaptic distribution, and (2) astrocytic signaling that may be crucial for synaptic transmission and functional hyperemia.
Opiate addiction is characterized by progressive increases in drug intake over time suggesting ma... more 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 longterm 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.
Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly develop... more 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-2Ј,7Ј-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 (NPY) and dynorphin elicit regionally selective presynaptic modulation in the hipp... more 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)
Ultrastructural immunocytochemical identification of transmitters in afferent terminals and targe... more 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.
The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophreni... more The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophrenia and in psychostimulant-induced drug-seeking behavior, both of which are affected by activation of the functionally opposed high-affinity neurotensin receptor (NTS1). To determine the functionally relevant sites, we examined the dual electron microscopic immunocytochemical localization of D2R and NTS1 in the NAc shell of rat brain. Immunolabeling for each receptor was seen in association with cytoplasmic organelles, or more rarely, on the plasma membrane of both axonal and somatodendritic profiles. Some of the axonal and many of the dendritic processes colocalized the two receptors. The dually labeled axon terminals often formed symmetric synapses or appositional contacts with unlabeled dendritic profiles. The morphology of these terminals suggests that they contain either inhibitory amino acids or dopamine. Other axonal profiles expressing exclusively NTS1 or D2R were without synaptic specializations or formed asymmetric, excitatory-type synapses mainly on unlabeled dendritic spines. In addition, however, several D2R-immunoreactive terminals were observed presynaptic to dendrites containing NTS1. The somatodendritic profiles immunolabeled for NTS1 and/or D2R had morphological features typical of inhibitory spiny projection neurons in the NAc. These results suggest that activation of NTS1 and D2R can dually modulate transmitter release from the same or separate phenotypically distinct axon terminals in the NAc shell. These presynaptic receptors as well as the postsynaptic NTS1 distribution in neurons that also contain or receive input from terminals containing D2R may mediate the opposing actions of neurotensin and dopamine in the NAc.
Adenosine A1 and A2A receptors mediate distinct cardiovascular components of defense reactions th... more Adenosine A1 and A2A receptors mediate distinct cardiovascular components of defense reactions that are ascribed, in part, to opposing actions within the nucleus tractus solitarius. To assess the cellular sites of relevance to these actions, we examined the light and electron microscopic immunolabeling of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema (dmNTS-AP), a region crucial for cardiovascular regulation involving vagal baroreceptor afferents. Immunoreactivity for each receptor was independently localized to distinct segments of plasma membranes and endomembranes in somatodendritic, axonal, and glial profiles. The dendritic labeling for each receptor also was detected within and near asymmetric, excitatory-type synapses. Of all peroxidase labeled profiles exclusive of somata, approximately 58% were A1- and 39% were A2A-labeled dendrites. Dendrites and astrocytic glia were the profiles that most often expressed both subtypes of adenosine receptors. The axonal labeling for A2A receptors was seen mainly in unmyelinated axons, whereas the A1 receptors were prominently localized within axon terminals. These terminals often formed single or multisynaptic excitatory-type junctions or single symmetric synapses on dendrites, a few of which expressed A1 and A2A receptors. These results provide the first ultrastructural evidence that A1 and A2A receptors have distributions conductive to their dual involvement in modulating the output of single neurons and glial function in the dmNTS-AP, where the predominate presynaptic effects of adenosine are mediated through A1 receptors.
Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles b... more Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles by the vesicular monoamine transporter-2 (VMAT2). Somatodendritically released dopamine in this region binds to the D2 receptor (D2R) to modulate ongoing neurotransmission. Although autoregulation of mesocortical dopaminergic neurons in the parabrachial VTA (PB-VTA) is known to be less efficacious than that of mesolimbic dopaminergic neurons in the paranigral (PN-VTA), the cellular basis for this regional heterogeneity is not known. For this reason, we used electron microscopic immunocytochemistry to determine the subcellular localization of the dopamine storage vesicles (identified by the presence of VMAT2) in relation to the D2R in these VTA subdivisions. In both regions, D2R immunoreactivity was principally located on extrasynaptic dendritic plasma membranes near excitatory-type synapses. Equivalent percentages (72 and 74%) of the D2R-labeled dendrites in each region contained VMAT2-immunoreactive tubulovesicles. Of the total VMAT2-labeled dendrites, however, a significantly lower percentage in the PB-VTA (26%) than in the PN-VTA (38%) contained D2R labeling. In contrast, a significantly higher number of VMAT2 immunogold-silver deposits was seen within individual dendrites in the PB-VTA than in PN-VTA. In both regions, D2R immunoreactivity was also detected in VMAT2-negative axon terminals that formed synapses on dendrites containing VMAT2. Our results are the first to demonstrate that within VTA neurons and their afferents the D2R is strategically positioned for activation by dopamine released from dendritic storage vesicles. These findings also suggest that the potential for D2R activation may affect the expression levels of VMAT2 in VTA dendrites.
The immune/inflammatory signaling molecule tumor necrosis factor α (TNFα) is an important mediato... more The immune/inflammatory signaling molecule tumor necrosis factor α (TNFα) is an important mediator of both constitutive and plastic signaling in the brain. In particular, TNFα is implicated in physiological processes, including fever, energy balance, and autonomic function, known to involve the hypothalamic paraventricular nucleus (PVN). Many critical actions of TNFα are transduced by the TNFα type 1 receptor (TNFR1), whose activation has been shown to potently modulate classical neural signaling. There is, however, little known about the cellular sites of action for TNFR1 in the PVN. In the present study, high-resolution electron microscopic immunocytochemistry was used to demonstrate the ultrastructural distribution of TNFR1 in the PVN. Labeling for TNFR1 was found in somata and dendrites, and to a lesser extent in axon terminals and glia in the PVN. In dendritic profiles, TNFR1 was mainly present in the cytoplasm, and in association with presumably functional sites on the plasma ...
Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates co... more Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates corticosterone-sensitive uptake of monoamines including norepinephrine, epinephrine, dopamine, histamine and serotonin. OCT3 is expressed widely throughout the amygdaloid complex and other brain regions where monoamines are key regulators of emotional behaviors affected by stress. However, assessing the contribution of OCT3 to the regulation of monoaminergic neurotransmission and monoamine-dependent regulation of behavior requires fundamental information about the subcellular distribution of OCT3 expression. We used immunofluorescence and immuno-electron microscopy to examine the cellular and subcellular distribution of the transporter in the basolateral amygdaloid complex of the rat and mouse brain. OCT3-immunoreactivity was observed in both glial and neuronal perikarya in both rat and mouse amygdala. Electron microscopic immunolabeling revealed plasma membrane-associated OCT3 immunoreact...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2015
Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulat... more Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neuron...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2000
Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in t... more Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in the production of stable nitrotyrosine (NT) moieties that are immunochemically detectable in many regions of normal brain and enriched in those areas containing constitutive nitric oxide synthase (cNOS). These include the caudate-putamen nucleus (CPN) and the globus pallidus, which receives major inhibitory input from the CPN. To determine the functional sites for NT production in these critical motor nuclei, we examined the electron microscopic immunocytochemical localization of NT and cNOS in rat brain. In the CPN, NT was localized to the somata and dendrites of cNOS-containing interneurons and spiny neurons, some of which received input from cNOS-labeled terminals. The NT immunoreactivity was most prevalent on outer mitochondrial membranes and nearby segments of the plasma membranes in dendrites and within asymmetric synapses on dendritic spines. In the CPN and globus pallidus, there w...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999
The expression of a brain-specific high-affinity Na+-dependent (and Cl--dependent) L-proline tran... more The expression of a brain-specific high-affinity Na+-dependent (and Cl--dependent) L-proline transporter (PROT) in subpopulations of putative glutamatergic neurons in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (). To gain insights into potential sites where PROT may function, we used a C-terminal domain antipeptide antibody to determine the regional distribution and subcellular localization of PROT in rat forebrain. PROT immunoreactivity was seen in processes having a regional light microscopic distribution comparable to that of known glutamatergic projections within the cortex, caudate putamen nucleus (CPN), hippocampal formation, and other forebrain regions. In all regions examined by electron microscopy (cortex, CPN, and the stratum oriens of CA1), PROT labeling was observed primarily within subpopulations of axon terminals forming asymmetric excitatory-type synapses. Immunogold labeling for PROT was detected in close contact wi...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1998
Molecular cloning has recently identified a vertebrate brain-specific Na+-dependent inorganic pho... more Molecular cloning has recently identified a vertebrate brain-specific Na+-dependent inorganic phosphate transporter (BNPI). BNPI has strong sequence similarity to EAT-4, a Caenorhabditis elegans protein implicated in glutamatergic transmission. To characterize the physiological role of BNPI, we have generated an antibody to the protein. Immunocytochemistry of rat brain sections shows a light microscopic pattern that is suggestive of reactivity in nerve terminals. Excitatory projections are labeled prominently, and ultrastructural analysis confirms that BNPI localizes almost exclusively to terminals forming asymmetric excitatory-type synapses. Although BNPI depends on a Na+ gradient and presumably functions at the plasma membrane, both electron microscopy and biochemical fractionation show that BNPI associates preferentially with the membranes of small synaptic vesicles. The results provide anatomic evidence of a specific presynaptic role for BNPI in glutamatergic neurotransmission, ...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1997
The dopamine transporter (DAT) regulates extracellular dopamine concentrations, transports neurot... more The dopamine transporter (DAT) regulates extracellular dopamine concentrations, transports neurotoxins, and acts as a substrate for cocaine reinforcement. These functions are known to differ in the limbic-associated shell and motor-associated core compartments of the nucleus accumbens (NAc). Previous studies have shown differential expression of DAT in the NAc shell and core but were limited in resolution to the regional level. Thus, it is not known whether there are differences in the amount, subcellular localization, or plasmalemmal targeting of DAT within individual dopaminergic axons in the two regions. We used high-resolution electron microscopic immunocytochemistry to investigate these possibilities. We show that in both the shell and core, DAT immunogold labeling is present in tyrosine hydroxylase-immunoreactive varicose axons that form symmetric synapses. Within these labeled axons, most DAT gold particles are located on extrasynaptic plasma membranes, but some are associate...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1997
The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine a... more The dopamine transporter (DAT) plays an important role in the plasmalemmal reuptake of dopamine and, thus, in the termination of normal dopaminergic neurotransmission. DAT is also a major binding site for cocaine and other stimulants, the psychoactive effects of which are associated primarily with the inhibition of dopamine reuptake within mesocorticolimbic dopaminergic neurons. We used electron microscopy with an anti-peptide antiserum directed against the N-terminal domain of DAT to determine the subcellular localization of this transporter in the rat ventral tegmental area (VTA), the region that contains the cell bodies and dendrites of these dopaminergic neurons. We show that in the VTA, almost 95% of the DAT immunogold-labeled profiles are neuronal perikarya and dendrites, and the remainder are unmyelinated axons. Within perikarya and large proximal dendrites, almost all of the DAT immunogold particles are associated with intracellular membranes, including saccules of Golgi and...
Midbrain dopaminergic neurons are known to release dopamine from somata and/or dendrites located ... more Midbrain dopaminergic neurons are known to release dopamine from somata and/or dendrites located in the substantia nigra (SN) and the ventral tegmental area (VTA). There is considerable controversy, however, about the subcellular sites for somatodendritic dopamine storage in these regions. In the present study, we used dual-labeling electron microscopic immunocytochemistry to localize the vesicular monoamine transporter-2 (VMAT2), a novel marker for sites of intracellular monoamine storage, within identified dopaminergic (tyrosine hydroxylase-containing) neurons in the rat SN and VTA. In dopaminergic perikarya, immunogold labeling for VMAT2 was localized to the Golgi apparatus, tubulovesicles that resembled smooth endoplasmic reticulum (SER), and the limiting membranes of multivesicular bodies. In dopaminergic dendrites, VMAT2 was extensively localized to tubulovesicles that resembled saccules of SER, and less frequently localized to isolated small synaptic vesicles (SSVs) or large dense-core vesicles (DCVs). In rare cases, VMAT2-immunoreactive SSVs were clustered within the cytoplasm of an SN or a VTA dendrite. Dopaminergic dendrites in the VTA contained a significantly higher number of immunogold particles for VMAT2 per unit area than those in the SN. Together, these observations support the proposal that dopamine is stored in and may be released from dendritic SSVs and DCVs, but suggest that the SER is the major site of dopamine storage within midbrain dopaminergic neurons. In addition, they provide new evidence that dopaminergic dendrites in the VTA may have greater potential for reserpinesensitive storage and release of dopamine than those in the SN.
Interactions between cannabinoid and opioid systems have been implicated in reward and drug seeki... more 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-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.-Opioid receptors were also detected in axonal and dendritic profiles. These dendrites were most prevalent in the Acb shell, where-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-opioid receptors in the shell (19%) compared with the core (13%). Conversely, of the synaptic-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-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.
The alpha-7 nicotinic acetylcholine receptor (α7nAChR) and the dopamine D 2 receptor (D 2 R) are ... more The alpha-7 nicotinic acetylcholine receptor (α7nAChR) and the dopamine D 2 receptor (D 2 R) are both implicated in attentional processes and cognition, mediated in part through the prefrontal cortex (PFC). We examined the dual electron microscopic immunolabeling of α7nAChR and either D 2 R or the vesicular acetylcholine transporter (VAChT) in rodent PFC to assess convergent functional activation sites. Immunoreactivity (ir) for α7nAChR and/or D 2 R was seen in the same as well as separate neuronal and glial profiles. At least half of the dually labeled profiles were somata and dendrites, while most labeled axon terminals expressed only D 2 R-ir. The D 2 R-labeled terminals were without synaptic specializations or formed inhibitory or excitatory-type synapses with somatodendritic profiles, some of which expressed the α7nAChR and/or D 2 R. Astrocytic glial processes comprised the majority of nonsomatodendritic α7nAChR or α7nAChR and D 2 Rlabeled profiles. Glial processes containing α7nAChR-ir were frequently located near VAChTlabeled terminals and also showed perisynaptic and perivascular associations. We conclude that in rodent PFC α7nACh and D 2 R activation can dually modulate (1) postsynaptic dendritic responses within the same or separate but synaptically linked neurons in which the D 2 R has the predominately presynaptic distribution, and (2) astrocytic signaling that may be crucial for synaptic transmission and functional hyperemia.
Opiate addiction is characterized by progressive increases in drug intake over time suggesting ma... more 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 longterm 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.
Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly develop... more 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-2Ј,7Ј-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 (NPY) and dynorphin elicit regionally selective presynaptic modulation in the hipp... more 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)
Ultrastructural immunocytochemical identification of transmitters in afferent terminals and targe... more 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.
The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophreni... more The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophrenia and in psychostimulant-induced drug-seeking behavior, both of which are affected by activation of the functionally opposed high-affinity neurotensin receptor (NTS1). To determine the functionally relevant sites, we examined the dual electron microscopic immunocytochemical localization of D2R and NTS1 in the NAc shell of rat brain. Immunolabeling for each receptor was seen in association with cytoplasmic organelles, or more rarely, on the plasma membrane of both axonal and somatodendritic profiles. Some of the axonal and many of the dendritic processes colocalized the two receptors. The dually labeled axon terminals often formed symmetric synapses or appositional contacts with unlabeled dendritic profiles. The morphology of these terminals suggests that they contain either inhibitory amino acids or dopamine. Other axonal profiles expressing exclusively NTS1 or D2R were without synaptic specializations or formed asymmetric, excitatory-type synapses mainly on unlabeled dendritic spines. In addition, however, several D2R-immunoreactive terminals were observed presynaptic to dendrites containing NTS1. The somatodendritic profiles immunolabeled for NTS1 and/or D2R had morphological features typical of inhibitory spiny projection neurons in the NAc. These results suggest that activation of NTS1 and D2R can dually modulate transmitter release from the same or separate phenotypically distinct axon terminals in the NAc shell. These presynaptic receptors as well as the postsynaptic NTS1 distribution in neurons that also contain or receive input from terminals containing D2R may mediate the opposing actions of neurotensin and dopamine in the NAc.
Adenosine A1 and A2A receptors mediate distinct cardiovascular components of defense reactions th... more Adenosine A1 and A2A receptors mediate distinct cardiovascular components of defense reactions that are ascribed, in part, to opposing actions within the nucleus tractus solitarius. To assess the cellular sites of relevance to these actions, we examined the light and electron microscopic immunolabeling of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema (dmNTS-AP), a region crucial for cardiovascular regulation involving vagal baroreceptor afferents. Immunoreactivity for each receptor was independently localized to distinct segments of plasma membranes and endomembranes in somatodendritic, axonal, and glial profiles. The dendritic labeling for each receptor also was detected within and near asymmetric, excitatory-type synapses. Of all peroxidase labeled profiles exclusive of somata, approximately 58% were A1- and 39% were A2A-labeled dendrites. Dendrites and astrocytic glia were the profiles that most often expressed both subtypes of adenosine receptors. The axonal labeling for A2A receptors was seen mainly in unmyelinated axons, whereas the A1 receptors were prominently localized within axon terminals. These terminals often formed single or multisynaptic excitatory-type junctions or single symmetric synapses on dendrites, a few of which expressed A1 and A2A receptors. These results provide the first ultrastructural evidence that A1 and A2A receptors have distributions conductive to their dual involvement in modulating the output of single neurons and glial function in the dmNTS-AP, where the predominate presynaptic effects of adenosine are mediated through A1 receptors.
Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles b... more Throughout the ventral tegmental area (VTA), dopamine is packaged within subcellular organelles by the vesicular monoamine transporter-2 (VMAT2). Somatodendritically released dopamine in this region binds to the D2 receptor (D2R) to modulate ongoing neurotransmission. Although autoregulation of mesocortical dopaminergic neurons in the parabrachial VTA (PB-VTA) is known to be less efficacious than that of mesolimbic dopaminergic neurons in the paranigral (PN-VTA), the cellular basis for this regional heterogeneity is not known. For this reason, we used electron microscopic immunocytochemistry to determine the subcellular localization of the dopamine storage vesicles (identified by the presence of VMAT2) in relation to the D2R in these VTA subdivisions. In both regions, D2R immunoreactivity was principally located on extrasynaptic dendritic plasma membranes near excitatory-type synapses. Equivalent percentages (72 and 74%) of the D2R-labeled dendrites in each region contained VMAT2-immunoreactive tubulovesicles. Of the total VMAT2-labeled dendrites, however, a significantly lower percentage in the PB-VTA (26%) than in the PN-VTA (38%) contained D2R labeling. In contrast, a significantly higher number of VMAT2 immunogold-silver deposits was seen within individual dendrites in the PB-VTA than in PN-VTA. In both regions, D2R immunoreactivity was also detected in VMAT2-negative axon terminals that formed synapses on dendrites containing VMAT2. Our results are the first to demonstrate that within VTA neurons and their afferents the D2R is strategically positioned for activation by dopamine released from dendritic storage vesicles. These findings also suggest that the potential for D2R activation may affect the expression levels of VMAT2 in VTA dendrites.
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Papers by June Chan