Biomedical subjects

Virginia M Pickel

Publications and source records attributed to Virginia M Pickel.

At least 19 recordsLinked to original sources

PubMed2006

Subcellular distributions of adenosine A1 and A2A receptors in the rat dorsomedial nucleus of the solitary tract at the level of the area postrema.

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.

Adenosine↗

PubMed2006

Subcellular distribution of M2 muscarinic receptors in relation to dopaminergic neurons of the rat ventral tegmental area.

Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Among the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles but showed a more prominent, size-dependent plasmalemmal location in nondopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R- or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA.

Animals↗

PubMed2006

Prodynorphin storage and processing in axon terminals and dendrites.

The classical view postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans-Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN-containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and striatum, PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium-induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission.

Animals↗

PubMed2006

Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius.

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca(2+) currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca(2+) or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC(50)=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca(2+) currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca(2+) attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca(2+) currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca(2+) currents that underlie the actions of Ang II on central autonomic regulation.

Angiotensin II↗

PubMed2006

Dopamine D1 receptors have subcellular distributions conducive to interactions with prodynorphin in the rat nucleus accumbens shell.

Activation of dopamine (DA) D1 receptors (D1Rs) in the nucleus accumbens (Acb) markedly affects the levels of prodynorphin, the precursor of aversion-associated dynorphin peptides. The location of prodynorphin, specifically as related to the dopaminergic inputs and D1Rs in the Acb, is fundamental for establishing the physiologically relevant sites. To determine these sites, we examined the electron microscopic dual-immunolabeling of prodynorphin and D1R or tyrosine hydroxylase (TH), a marker of catecholamine terminals in the rat Acb shell. This subregion is targeted by mesolimbic dopaminergic inputs affecting reward-aversion responses and locomotor activity. Prodynorphin was prominently localized to large (100-200 nm) granular aggregates in somatodendritic and axonal profiles, some of which expressed dynorphin A/B. In somata and dendrites, prodynorphin was often found in punctate clusters in the cytoplasm. Of the total prodynorphin-labeled dendrites, approximately 63% expressed D1Rs, which were largely located on the plasma membranes. In comparison with dendrites, many more axon terminals contained prodynorphin, although only 15% of these terminals contained D1R-labeling. Prodynorphin terminals formed symmetric synapses with D1R-labeled or unlabeled dendrites, and also apposed TH-containing axon terminals. Our results provide ultrastructural evidence that in the Acb shell, the prodynorphin is available for cleavage to physiologically active peptides in both dendrites and terminals of neurons that express D1Rs. They also indicate that dynorphin peptides have distributions that would enable their participation in modulation of DA release or D1R-mediated postsynaptic responses in Acb shell neurons.

Animals↗

PubMed2005

Overlapping intracellular and differential synaptic distributions of dopamine D1 and glutamate N-methyl-D-aspartate receptors in rat nucleus accumbens.

The dopamine D1 receptor (D1R) in the nucleus accumbens (Acb) shell is highly implicated in psychostimulant-evoked locomotor activity and reward, whereas the D1R in the Acb core is more crucial for appetitive instrumental learning. These behavioral effects depend in part on interactions involving glutamatergic N-methyl-D-aspartate (NMDA) receptors, whose essential NR1 subunit has physical associations with the D1R. To determine the relevant sites for D1R activation and interactions involving NMDA receptors, we examined the electron microscopic immunolabeling of D1R and NR1 C-terminal peptides in rat Acb shell and core. In each Acb subdivision, the D1Rs were located principally on extrasynaptic plasma membranes of dendritic shafts and spines and more rarely were associated with cytoplasmic endomembranes. Many D1R-labeled somata and dendrites also contained NR1 immunoreactivity. In comparison with D1R, NR1 immunoreactivity was more often seen in the cytoplasm and near asymmetric synapses on somatodendritic profiles. In these profiles, notable overlapping distributions of D1R and NR1 occurred near endomembranes. The exclusively D1R- or D1R- and NR1-containing dendrites were most prevalent in the Acb shell, but were also present in the Acb core. In each region, NR1 was also detected in axon terminals without D1R, which formed excitatory-type synapses with D1R-labeled dendrites. These results provide ultrastructural evidence that D1Rs in the Acb have subcellular distributions supporting, 1) intracellular cotrafficking with NR1 and 2) modulation of the postsynaptic excitability in spiny neurons affected by presynaptic NMDA receptor activation. The region-specific differences in receptor distributions suggest a major, but not exclusive, involvement of Acb D1R in reward-related processing.

Animals↗

PubMed2005

Increased AMPA GluR1 receptor subunit labeling on the plasma membrane of dendrites in the basolateral amygdala of rats self-administering morphine.

Glutamate-dependent synaptic plasticity is emerging as an important neural substrate of addiction. These drug-dependent neural adaptations may occur within brain systems that mediate reward, emotion, and cognitive function such as the amygdala complex. Modification of glutamate receptor targeting may be a key mechanism mediating neural plasticity; however, evidence for alteration of amygdala AMPA receptor localization in response to drug self-administration is lacking. High-resolution immunogold electron microscopic immunocytochemistry was used to compare surface and intracellular labeling of the calcium sensitive AMPA GluR1 receptor subunit in the basolateral (BLA) and central (CeA) nuclei of the amygdala in rats self-administering escalating doses of morphine or saline. Morphine self-administration was associated with regionally diverse effects on dendritic GluR1 targeting in the BLA and CeA. In the BLA of morphine self-administering animals, there was a significant increase in the proportion of immunogold particles for GluR1 on the plasma membrane of dendrites, particularly in association with extrasynaptic sites, which was most prominent in large (2-4 microm) profiles. In contrast, there were no significant differences in surface or intracellular immunogold labeling in the CeA between morphine self-administering and control animals. In both amygdala regions, GluR1 and the micro-opioid receptor, the major cellular target of morphine, were only infrequently colocalized. These results indicate that GluR1 targeting is a dynamic process that can be differentially affected in distinct amygdala regions in response to chronic self-administration of morphine. Homeostatic adaptations in the subcellular localization of calcium sensitive AMPA receptors within the BLA may be an important neural substrate for alterations in reward, autonomic function, and behavioral processes associated with opiate addiction.

Amygdala↗

PubMed2004

Synaptic and vascular associations of neurons containing cyclooxygenase-2 and nitric oxide synthase in rat somatosensory cortex.

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid synthesis that is present in cortical pyramidal neurons and highly implicated in control of cerebral blood flow during neural activity. We examined the electron microscopic localization of COX-2 and neuronal nitric oxide synthase (nNOS), a functionally related enzyme, in the somatosensory cortex of rat brain to determine the relevant functional sites. COX-2 immunoreactivity was detected in significantly more somatodendritic than axonal profiles, while nNOS was more often seen in axon terminals. The dendritic COX-2 was localized to endomembranes near synaptic inputs from axon terminals, some of which contained nNOS. Conversely, COX-2 terminals formed asymmetric, excitatory-type synapses with dendrites containing nNOS. The dendritic and axonal profiles containing COX-2 as well as those containing nNOS were minimally separated from penetrating arterioles and capillaries by filamentous glial processes. The perivascular COX-2 labeled terminals were among those that also formed axo-dendritic synapses, suggesting that the release of prostanoids and/or excitatory transmitters from a single terminal may simultaneously affect neuronal activity and cerebral blood flow. Thus, COX-2 has a compartmental distribution in somatosensory cortical neurons consistent with the local neuronal synthesis of prostanoids that are involved in neurovascular coupling and whose actions are modulated by nitric oxide.

Animals↗

PubMed2004

5-HT3A receptor subunits in the rat medial nucleus of the solitary tract: subcellular distribution and relation to the serotonin transporter.

The 5-hydroxytryptamine 3 (5HT3) receptor is a serotonin-gated ion channel implicated in reflex regulation of autonomic functions within the nucleus of the solitary tract (NTS). To determine the relevant sites for 5-HT3 receptor mediated transmission in this region, we used electron microscopic immunocytochemistry to examine the subcellular distribution of the 5HT3 receptor subunit A (5HT3A) in relation to the serotonin transporter (SERT) in the intermediate medial NTS (mNTS) of rat brain. The 5HT3A immunolabeling was detected in many axonal as well as somatodendritic and glial profiles. The axonal profiles included small axons and axon terminals in which the 5HT3A immunoreactivity was localized to membranes of synaptic vesicles and extrasynaptic plasma membranes. In dendrites and glia, the 5HT3A immunoreactivity was located on the plasma membranes or in association with membranous cytoplasmic organelles. The dendritic plasmalemmal 5HT3A labeling was prominent within and near excitatory-type synapses from terminals including those that resemble vagal afferents. The 5HT3A-labeled glial processes apposed 5HT3A-immunoreactive axonal and dendritic profiles, some of which also contained SERT. Terminals containing 5-HT3A and/or SERT were among those providing synaptic input to 5HT3A-labeled dendrites. Thus, 5HT3A has a subcellular distribution consistent with the involvement of 5-HT3 receptors in modulation of both presynaptic release and postsynaptic responses of mNTS neurons, some of which are serotonergic. The results further suggest that the neuronal as well as glial 5HT3 receptors can be activated by release of serotonin from presynaptic terminals or by diffusion facilitated by SERT distribution at a distant from the synapse.

Animals↗

PubMed2004

Decreased plasma membrane targeting of NMDA-NR1 receptor subunit in dendrites of medial nucleus tractus solitarius neurons in rats self-administering morphine.

Opioid abuse is associated with repeated administration and escalation of dose that can result in profound adaptations in homeostatic processes. Potential cellular mechanisms and neural sites mediating opiate-dependent adaptations may involve NMDA-dependent synaptic plasticity within brain areas participating in behaviors related to consumption of natural reinforcers, as well as affective-autonomic integration, notably the medial nucleus tractus solitarius (mNTS). NMDA-dependent synaptic plasticity may be mediated by changes in the intracellular and surface targeting of NMDA receptors, particularly in postsynaptic sites including spines or small distal dendrites. High-resolution immunogold electron microscopic immunocytochemistry combined with morphometry were used to measure changes in targeting of the NMDA-NR1 (NR1) receptor subunit between intracellular and plasmalemmal sites in dendrites of neurons of the intermediate mNTS of rats self-administering escalating doses of morphine (EMSA). In control and EMSA rats, the density of plasmalemmal and cytosolic gold particles was inversely related to profile size. Collapsed across all NR1-labeled dendrites, rats self-administering morphine had a lower number of plasmalemmal gold particles per unit surface area (7.1 +/- 0.8 vs. 14.4 +/- 1 per 100 microm), but had a higher number of intracellular gold particles per unit cross-sectional area (169 +/- 6.1 vs. 148 +/- 5.1 per 100 microm2) compared to saline self-administering rats. Morphometric analysis showed that the decrease in plasma membrane labeling of NR1 was most robust in small dendritic profiles (<1 microm), where there was a reciprocal increase in the density of intracellular particles. These results indicate that the plasmalemmal distribution of the essential NR1 subunits in distal sites may prominently contribute to NMDA receptor-dependent modulation of neural circuitry regulating homeostatic processes, and targeting of these proteins can be prominently affected by morphine self-administration.

Animals↗

PubMed2004

Activity-regulated cytoskeleton-associated protein Arc is targeted to dendrites and coexpressed with mu-opioid receptors in postnatal rat caudate-putamen nucleus.

Dendritic expression of the activity-regulated cytoskeleton-associated protein (Arc) is dramatically enhanced by increased synaptic activity in adult brain. We used immunocytochemical electron microscopy to determine whether the subcellular localization of Arc in developing dendrites corresponds to the peak period of synaptogenesis in the postnatal rat caudate-putamen nucleus (CPN). The distribution was compared with that of mu-opioid receptors (MORs), whose localization in dendritic spines closely parallels excitatory synapse formation during postnatal development (Wang et al. [2003] Neuroscience 118:695-708). Sections were processed for immunocytochemical detection of antisera against Arc or MORs at the beginning (postnatal day 15; P15) and the end (P30) of the peak period of synaptogenesis in rat CPN. At P15, immunolabeling for Arc showed a punctate distribution in the cytoplasm of dendritic shafts, some of which was associated with polyribosomes. In some spiny dendrites, Arc immunoreactivity was more intensely localized in putative spines than in their parental dendrites, whereas, in other spiny dendrites, Arc labeling was restricted in the shafts. Many dendritic shafts and spines also showed immunoreactivity for MORs, although dually labeled spines were less numerous than the shafts. At P30, the proportion of singly and dually labeled spines significantly increased from 2.0% to 7.5% and from 9.5% to 21%, respectively. Arc labeling in spines was more detectable beneath the postsynaptic density or at extrasynaptic sites on the plasma membrane. Our results suggest a correlation between Arc expression in dendritic spines during postnatal development and the onset of synaptogenesis in opioid-responsive neurons in the rat CPN.

Animals↗

PubMed2004

NADPH oxidase contributes to angiotensin II signaling in the nucleus tractus solitarius.

Angiotensin II (AngII), acting through angiotensin type 1 (AT1) receptors, exerts powerful effects on central autonomic networks regulating cardiovascular homeostasis and fluid balance; however, the mechanisms of AngII signaling in functionally defined central autonomic neurons have not been fully elucidated. In vascular cells, reactive oxygen species (ROS) generated by the enzyme NADPH oxidase play a major role in AngII signaling. Thus, we sought to determine whether NADPH oxidase is present in central autonomic neurons and, if so, whether NADPH oxidase-derived ROS are involved in the effects of AngII on these neurons. The present studies focused on the intermediate dorsomedial nucleus of the solitary tract (dmNTS) because this region receives autonomic afferents via the vagus nerve and is an important site of AngII actions. Using double-label immunoelectron microscopy, we found that the essential NADPH oxidase subunit gp91phox is present in somatodendric and axonal profiles containing AT1 receptors. The gp91phox-labeled dendrites received inputs from large axon terminals resembling vagal afferents. In parallel experiments using patch clamp of dissociated NTS neurons anterogradely labeled via the vagus, we found that AngII potentiates the L-type Ca2+ currents, an effect mediated by AT1 receptors and abolished by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride. The NADPH oxidase assembly inhibitor apocynin and the peptide inhibitor gp91phox docking sequence, but not its scrambled version, also blocked the potentiation. The results provide evidence that NADPH oxidase-derived ROS are involved in the effects of AngII on Ca2+ influx in NTS neurons receiving vagal afferents and support the notion that ROS are important signaling molecules in central autonomic networks.

Angiotensin II↗

PubMed2004

Electron microscopic dual labeling of high-affinity neurotensin and dopamine D2 receptors in the rat nucleus accumbens shell.

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.

Animals↗

PubMed2004

Regional and subcellular compartmentation of the dopamine transporter and tyrosine hydroxylase in the rat ventral pallidum.

The ventral pallidum (VP) is a major intermediary in the prefrontal cortical circuitry regulating sensorimotor gating and locomotor behavior, both of which are potently modulated by catecholamines. The VP catecholaminergic innervation is derived from midbrain dopaminergic neurons that differ in expression levels of the dopamine transporter (DAT) and from brainstem noradrenergic neurons without DAT. The preferentially low level of DAT in dopaminergic terminals in the prefrontal cortex and in striatal regions projecting more extensively to the VP medial (VPm) compared with VP lateral (VPl) compartment suggests possible region-specific differences in VP axonal distribution of DAT. To test this hypothesis, we examined the electron microscopic localization of DAT and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the VPm and VPl of rat brain. In both regions, DAT and TH were localized primarily in small unmyelinated axons and morphologically heterogeneous axon terminals. DAT-immunogold particles were few in number, but mostly located on the plasma membrane. In contrast, TH immunoreactivity was distributed in the cytoplasm of individual profiles, many of which were without detectable DAT. In comparison with TH, the mean area density of DAT-labeled axons was low throughout the VP. The mean area density of DAT-immunogold axon terminals, however, was significantly higher in VPl than in VPm, whereas that of TH-labeled axons was higher in VPm than in VPl. This dissociation suggests that, compared to the VPl, the VPm receives the greatest input from catecholaminergic afferents that are either nondopaminergic or characterized by having low levels or less terminal distributions of DAT.

Animals↗

PubMed2004

Ultrastructural localization of Leu5-enkephalin immunoreactivity in mesocortical neurons and their input terminals in rat ventral tegmental area.

Enkephalin (ENK) immunoreactivity is widely distributed in the ventral tegmental area (VTA), where endogenous ENK and dynorphin opioid peptides are known to have opposing actions in reward, stress, cognition, and fear-related behaviors. Many neurons in the VTA give rise to mesocortical projections terminating in the medial prefrontal cortex (mPFC), and these projections have been implicated to varying extents in all these functions. To determine whether there is a synaptic basis for ENK and/or dynorphin modulation of cortically projecting neurons within the VTA, we combined retrograde tract-tracing from the mPFC with dual immunocytochemical-labeling electron microscopy in the rat VTA. The retrograde tracer Fluorogold (FG) was microinjected into mPFC. At optimal survival periods, sections through the VTA were processed for immunolabeling of anti-FG and a Leu(5)-ENK antibody recognizing both ENK and dynorphin peptides. Over 26% of the retrogradely labeled neuronal somatodendritic profiles (n = 177) were contacted by ENK-immunoreactive axonal profiles including small axons and axon terminals. The axon terminals varied in their subcellular distribution of ENK immunoreactivity and also differed in forming either inhibitory-type (symmetric) or excitatory-type (asymmetric) synapses. Many of the axonal profiles also were apposed to FG-labeled somata or dendrites without forming recognizable synapses. Approximately one-third of the mesocortical neuronal perikarya also showed sparsely distributed somatodendritic ENK-immunoreactivity. Our results provide ultrastructural evidence that ENK and possibly dynorphin in the rat VTA have distributions consistent with involvement in diverse physiological actions affecting the output of mesocortical neurons, some of which also contain one or both peptides.

Animals↗

PubMed2003

Ultrastructural localization of delta-opioid receptors in the rat caudate-putamen nucleus during postnatal development: relation to synaptogenesis.

During development, delta-opioid receptors (DORs) in the rat caudate-putamen nucleus (CPN) appear later than mu-opioid receptors (MORs), whose developmental pattern specifically relates to synaptogenesis. We used electron microscopic immunocytochemistry to determine whether there are also age-related changes in subcellular localization of DORs in the rat CPN. Sections from postnatal day (P) 0-P30 and adult dorsomedial CPN were immunogold-silver labeled to examine the plasmalemmal and cytoplasmic distribution of these receptors. In addition, immunoperoxidase labeling was used to determine the numerical density of synapses relative to DOR-labeled profiles. Immunolabeling for DOR was undetectable at P0, light at P5, and dense from P10 onward. The labeling during P5-P10 was mainly localized in somatodendritic profiles but also was readily seen in axon terminals, most of which formed asymmetric synapses with dendrites. From P15, a few immunogold particles were seen in contact with postsynaptic densities in spines, and the proportion of these particles significantly increased in P30 and adult CPN. Other particles were localized in the cytoplasm of dendrites and terminals without significant age-related changes. Stereological analysis showed that compared with labeled dendritic shafts and spines, labeled axon terminals have a closer correlation with synapse formation. These results are in marked contrast with MORs, which show an age-related increase in association with dendritic plasma membrane and a good correlation in the developmental pattern of MOR-labeled spines with synapse formation (Wang et al. [2003] Neuroscience 118:695-708). Together, our results suggest receptor-type specific roles for endogenous opioids acting at both pre- and postsynaptic sides in the developing CPN.

Animals↗

PubMed2003

Morphine acutely regulates opioid receptor trafficking selectively in dendrites of nucleus accumbens neurons.

Morphine stimulates the internalization of mu-opioid receptors (MORs) in transfected cell models to a lesser degree than opioid peptides and other analgesic drugs, such as methadone, and previous studies have reported that morphine does not produce a detectable redistribution of MORs in neural tissue after either acute or chronic administration. Nevertheless, morphine produces profound physiological effects, raising the question of whether receptor trafficking plays any role in the in vivo actions of morphine. We investigated the effects of opiate drugs on recombinant and native opioid receptors in the nucleus accumbens, which plays an important role in mediating the behavioral effects of opiate drugs. Morphine and methadone differed in their effects on the internalization of epitope-tagged MORs in cell bodies, introduced by viral gene transfer and imaged by fluorescence microscopy. A mutation of the cytoplasmic tail that confers morphine-induced internalization in cultured cells had a similar effect on receptor trafficking in nucleus accumbens cell bodies. Surprisingly, in contrast to its failure to affect MOR distribution detectably in cell bodies, acute morphine administration produced a pronounced change in MOR distribution visualized in the processes of the same neurons. A similar effect of acute morphine administration was observed for endogenously expressed MORs by immunoelectron microscopy; the acute administration of morphine increased the density of MORs associated with internal membrane structures specifically in dendrites. These results provide the first evidence that morphine regulates the distribution of MORs in neuronal processes, suggesting that "compartment-selective" membrane trafficking represents a previously unanticipated type of opioid receptor regulation contributing to the in vivo effects of opiate drugs on a physiologically relevant population of CNS neurons.

Animals↗

PubMed2003

Ultrastructural localization of serotonin 2A and N-methyl-D-aspartate receptors in somata and dendrites of single neurons within rat dorsal motor nucleus of the vagus.

Both glutamate and serotonin are potent modulators of autonomic functions involving the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMNV) at the level of the area postrema. Moreover, many of the dendrites in this NTS region express both N-methyl-D-aspartate (NMDA) and serotonin (5HT) 2A receptors, and some of these dendrites may arise from the adjacent DMNV. Thus, single neurons in DMNV may also express both receptors. To test this hypothesis, we used electron microscopic immunocytochemistry for dual localization of the essential R1 subunit of the NMDA receptor (NR1) and the 5HT2A receptor in rat intermediate DMNV, a region serving mainly gastrointestinal functions. Gold particles representing NR1 and peroxidase reaction product for 5HT2A receptors were seen in the cytoplasm, as well as on distinct segments of the plasma membrane of many dendrites. Of the NR1-labeled dendrites, 31% (254/814) also contained 5HT2A immunoreactivity; among the 5HT2A-labeled dendrites, 52% (254/485) expressed NR1. The 5HT2A labeling was also present in numerous small unmyelinated axons, axon terminals, and glial processes. These profiles were largely without NR1 immunoreactivity, although NR1 was detected in some of the dendrites postsynaptic to 5HT2A-labeled terminals. Our results suggest that calcium entry through NMDA channels and 5HT2A receptor activation may dramatically affect postsynaptic excitability of single neurons in the DMNV. In addition, the findings also indicate that the 5HT2A receptor is strategically positioned for involvement in modulation of the presynaptic release of neurotransmitters affecting the postsynaptic activity of DMNV neurons responsive to NMDA activation.

Animals↗