Electron microscopic localization of corticotropin-releasing factor (CRF) and CRF receptor in rat and mouse central nucleus of the amygdala.

Corticotrophin-releasing factor (CRF) is expressed in the central nucleus of the amygdala (CeA), where the CRF receptor (CRFr) plays an important role in anxiety- and stress-related behaviors. To determine the subcellular sites of CRFr activation in this region, we examined the electron microscopic immunolabeling of antisera recognizing CRF or CRFr. The ultrastructural analysis was principally conducted in the lateral subdivision of the rat CeA, with comparisons being made in mice so as to optimally utilize mutant mice in control experiments. The CRFr labeling was seen in many small dendrites and dendritic spines as well as in a few somata, large dendrites, axons, and axon terminals or more rarely in glial processes. Approximately 35% of the CRFr-labeled dendrites contained CRF immunoreactivity, which was distributed diffusely throughout the cytoplasm, or specifically affiliated with either endomembranes or large dense-core vesicles. The CRF-immunoreactive vesicles also were present in somata and axon terminals with or without CRFr labeling. The CRF immunoreactivity was usually absent from both terminals and dendrites joined by asymmetric, excitatory-type synapses, where a postsynaptic location of the CRFr was commonly observed. Numerous terminals containing both CRF and CRFr were seen, however, within the neuropil and sometimes apposing the excitatory synapses. These results provide ultrastructural evidence for a primary involvement of CRF receptors in modulation of the postsynaptic excitability of CeA neurons, an effect that may be limited by the availability of CRF. The findings have important implications for understanding CRF mediation of rapid responses to stress.

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.

Chronic administration of morphine is associated with a decrease in surface AMPA GluR1 receptor subunit in dopamine D1 receptor expressing neurons in the shell and non-D1 receptor expressing neurons in the core of the rat nucleus accumbens.

The nucleus accumbens (Acb) is an extensively studied neuroanatomical substrate of opiate reward and the neural plasticity associated with chronic opioid use. The cellular mechanisms mediating opioid-dependent plasticity are uncertain, however AMPA-type glutamate receptor trafficking in dopamine D1 dopamine receptor (D1R) expressing neurons may be a potential cellular pathway for these adaptations, although there is no evidence for this possibility. Immunogold electron microscopy was used to quantify the surface expression of the AMPA GluR1 subunit in dendritic profiles of neurons in the Acb in response to intermittent 14-day non-contingent injections of escalating doses of morphine, a model that parallels opioid self-administration. To determine if changes in GluR1 trafficking occurred in neurons potentially sensitive to dopamine-induced D1R activation, immunoperoxidase labeling of D1R was combined with immunogold labeling of GluR1. Immunogold quantification was performed in two distinct Acb subregions, the shell, an area involved in processing incentive salience related to rewarding stimuli, and the core, an area involved in reward-seeking behaviors. We provide the first report that chronic morphine administration is associated with a receptor-phenotypic decrease in surface trafficking of GluR1 in Acb subregions. When compared to saline injected animals, morphine produced a decrease in plasma membrane GluR1 labeling in medium- and large-sized D1R expressing dendritic profiles in the Acb shell. In contrast, in the Acb core, surface GluR1 was decreased in small-sized dendrites that did not express the dopamine receptor. These results indicate that chronic intermittent injection of escalating doses of morphine is accompanied by ultrastructural plasticity of GluR1 in neurons that are responsive to glutamate and dopamine-induced D1R activation in the Acb shell, and neurons capable of responding to glutamate but not D1R receptor stimulation in the Acb core. Thus, AMPA receptor trafficking associated with chronic opiate exposure in functionally distinct areas of the Acb may be distinguished by D1R receptor activation, suggesting the potential for differing neural substrates of reward and motor aspects of addictive processes involving glutamate and dopamine signaling.

Contingent and non-contingent effects of heroin on mu-opioid receptor-containing ventral tegmental area GABA neurons.

Opiate activation of mu-opioid receptors (muORs) in the ventral tegmental area (VTA) modulates gamma-aminobutyric acid (GABA) neurotransmission within the mesocorticolimbic dopamine (DA) reward system. We combined in vivo extracellular electrophysiological recordings in anesthetized and freely behaving rats with intracellular Neurobiotin filling and immunocytochemistry to characterize the effects of opiates on VTA GABA neurons, evaluate their discharge activity during opiate self-administration, and identify the cellular sites for opiate activation. We identified a subpopulation of VTA GABA neurons that was characterized by location, spike discharge profile, activation by microelectrophoretic DA, and response to internal capsule (IC) stimulation. Systemic administration of heroin or microelectrophoretic application of the selective muOR agonist [d-Ala2, N-Me-Phe4, Gly-ol]-Enkephalin (DAMGO) reduced VTA GABA neuron firing rate (heroin IC(50) = 0.35 mg/kg) and was blocked by the muOR antagonist naloxone. Heroin also reduced IC-evoked post-stimulus spike discharges, a manifestation of gap-junction-mediated electrical coupling between VTA GABA neurons. The baseline firing rate of VTA GABA neurons significantly increased (239%) following the acquisition of heroin self-administration behavior and transiently increased during each response for heroin (105%), but decreased (49%) following heroin, similar to non-contingent heroin. Electrophysiologically characterized VTA GABA neurons were filled with Neurobiotin and labeled dendrites contained plasmalemmal muOR immunoreactivity. Dually labeled muOR dendrites contained dendrodendritic appositions characteristic of gap junctions. These findings indicate that inhibition of this population of GABAergic neurons by opiates acting on dendritic muORs has implications for modulation of electrical coupling between VTA GABA neurons and dopamine (DA) neurotransmission in the VTA and terminal field regions.

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.

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.

Alpha-2A-adrenergic receptors are present on neurons in the central nucleus of the amygdala that project to the dorsal vagal complex in the rat.

The descending pathway between the central nucleus of the amygdala (CeA) and the dorsal vagal complex (DVC) is an important substrate for autonomic functions associated with emotion. Activity in this circuit is crucially modulated by catecholamines and agonists of the alpha-2A-adrenergic receptor (alpha(2A)-AR), which relieve cardiovascular and gastrointestinal symptoms associated with experience of aversive stimuli. The subcellular distribution of alpha(2A)-AR within the CeA, however, has not been characterized. It is also not known if any alpha(2A)-AR-expressing neurons in the CeA project to the dorsal vagal complex. In order to address these questions, we examined the immunocytochemical labeling of alpha(2A)-AR in the CeA of rats receiving microinjection of the retrograde tracer fluorogold (FG) into the dorsal vagal complex at the level of the area postrema, an area involved in cardiorespiratory and gastrointestinal functions. Of all alpha(2A)-AR-labeled profiles in the CeA, the majority were either dendrites (42%) or somata (24%). alpha(2A)-AR labeling was often present on the plasmalemma in dendrites and was mainly found in endosome-like organelles in somata. Of all alpha(2A)-AR immunoreactive somata, 62% also contained immunolabeling for FG and 23% of all dendrites also showed labeling for the retrograde tracer. The intracellular distribution of alpha(2A)-AR did not differ in somata or dendrites with or without detectable FG. The remaining singly labeled alpha(2A)-AR profiles consisted of axons (11%), axon terminals (12%), and glial processes (13%). In numerous instances, alpha(2A)-AR-labeled glia or axon terminals were apposed to DVC projecting neurons. Together, this evidence suggests that the principal site for alpha(2A)-AR activation is at extrasynaptic sites on dendrites of CeA neurons, many of which project to the DVC and also show endosomal receptor labeling. In addition, these results indicate that activation of alpha(2A)-AR in the CeA may influence the activity of DVC projecting neurons through indirect mechanisms, including changes in presynaptic transmitter release or glial function. These results suggest that alpha(2A)-AR agonists in the CeA may modulate numerous processes including stress-evoked autonomic reactions and feeding behavior.

Kappa-Opioid and NMDA glutamate receptors are differentially targeted within rat medial prefrontal cortex.

Activation of kappa-opioid receptors (KOR) in the medial prefrontal cortex (mPFC) modulates excitatory transmission, which may involve interactions with N-methyl-D-aspartate (NMDA) glutamate receptors. We investigated possible anatomical correlates of this modulation by using dual labeling electron microscopy to examine the cellular distributions of antibodies raised against KOR and the R1 subunit of the NMDA receptor (NR1). KOR immunoreactivity primarily was localized to plasma and vesicular membranes of axons and axon terminals that were morphologically heterogeneous. A small proportion of KOR immunoreactivity was associated with cytosolic compartments of dendrites and membranes of glial processes. NR1 labeling was mainly postsynaptic, associated most often with membranes of cytoplasmic organelles in cell bodies and large dendrites and plasmalemmal surfaces of distal dendrites. The remaining NR1-labeled profiles were axonal profiles and glial processes. Of all cellular associations between labeled profiles, the majority were KOR-labeled axons that contacted NR1-immunoreactive dendrites or cell bodies. Occasionally the two antigens were colocalized in axon terminals that formed either asymmetric synapses or displayed varicose morphology. KOR and NR1 also were colocalized within dendrites, and rarely were observed in the same cell bodies. Occasionally glial processes coursing adjacent to axo-spinous appositions expressed both KOR and NR1 immunoreactivity. These results indicate that ligand activation of KOR or NMDA receptors differentially modulates excitatory transmission in the mPFC through pre- and postsynaptic mechanisms, respectively. The data also suggest more minor roles for colocalized KOR and NMDA receptors in shared regulation of presynaptic transmitter release, postsynaptic responsivity, and glial function.