Diane A. Lane

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.

Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension

Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.

Progranulin deficiency promotes post-ischemic blood-brain barrier disruption.

Loss-of-function mutations of progranulin (PGRN) have been linked to frontotemporal dementia, but little is known about the effects of PGRN deficiency on the brain in health and disease. PGRN has been implicated in neurovascular development, inflammation, and Wnt signaling, a pathway involved in the formation of the blood–brain barrier (BBB). Because BBB alterations and inflammation contribute to ischemic brain injury, we examined the role of PGRN in the brain damage produced by ischemia-reperfusion. PGRN+/− and PGRN−/− mice underwent middle cerebral artery occlusion (MCAO) with monitoring of cerebral blood flow. Infarct volume and motor deficits were assessed 72 h later. Post-ischemic inflammation was examined by expression of inflammatory genes and flow cytometry. BBB structure and permeability were examined by electron microscopy (EM) and Evans blue (EB) extravasation, respectively. MCAO resulted in ∼60% larger infarcts in PGRN+/− and PGRN−/− mice, an effect independent of hemodynamic factors or post-ischemic inflammation. Rather, massive hemorrhages and post-ischemic BBB disruption were observed, unrelated to degradation of tight junction (TJ) proteins or matrix metalloproteinases (MMPs). By EM, TJ were 30–52% shorter, fewer, and less interlocking, suggesting a weaker seal between endothelial cells. Intracerebral injection of platelet-derived growth factor-CC (PDGF-CC), which increases BBB permeability, resulted in a more severe BBB breakdown in PGRN+/− and PGRN−/− than wild-type mice. We describe a previously unrecognized involvement of PGRN in the expression of key ultrastructural features of the BBB. Such a novel vasoprotective role of PGRN may contribute to brain dysfunction and damage in conditions associated with reduced PGRN function.

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.

The influences of reproductive status and acute stress on the levels of phosphorylated delta opioid receptor immunoreactivity in rat hippocampus

In the hippocampus, ovarian hormones and sex can alter the trafficking of delta opioid receptors (DORs) and the proportion of DORs that colocalize with the stress hormone, corticotropin releasing factor. Here, we assessed the effects of acute immobilization stress (AIS) and sex on the phosphorylation of DORs in the rat hippocampus. We first localized an antibody to phosphorylated DOR (pDOR) at the SER363 carboxy-terminal residue, and demonstrated its response to an opioid agonist. By light microscopy, pDOR-immunoreactivity (ir) was located predominantly in CA2/CA3a pyramidal cell apical dendrites and in interneurons in CA1-3 stratum oriens and the dentate hilus. By electron microscopy, pDOR-ir primarily was located in somata and dendrites, associated with endomembranes, or in dendritic spines. pDOR-ir was less frequently found in mossy fibers terminals. Quantitative light microscopy revealed a significant increase in pDOR-ir in the CA2/CA3a region of male rats 1h following an injection of the opioid agonist morphine (20mg/kg, I.P). To look at the effects of stress on pDOR, we compared pDOR-ir in males and cycling females after AIS. The level of pDOR-ir in stratum radiatum of CA2/CA3a was increased in control estrus (elevated estrogen and progesterone) females compared to proestrus and diestrus females and males. However, immediately following 30min of AIS, no significant differences in pDOR levels were seen across estrous cycle phase or sex. These findings suggest that hippocampal levels of phosphorylated DORs vary with estrous cycle phase and that acute stress may dampen the differential effects of hormones on DOR activation in females.

Subcellular plasticity of the corticotropin-releasing factor receptor in dendrites of the mouse bed nucleus of the stria terminalis following chronic opiate exposure

Chronic opiate administration alters the expression levels of the stress-responsive peptide, corticotropin-releasing factor (CRF), in the bed nucleus of the stria terminalis (BNST). This brain region contains CRF receptors that drive drug-seeking behavior exacerbated by stress. We used electron microscopy to quantitatively compare immunolabeling of the corticotropin-releasing factor receptor (CRFr) and CRF in the anterolateral bed nucleus of the stria terminalis (BSTal) of mice injected with saline or morphine in escalating doses for 14 days. We also compared the results with those in non-injected control mice. The tissue was processed for CRFr immunogold and CRF immunoperoxidase labeling. The non-injected controls had a significantly lower plasmalemmal density of CRFr immunogold particles in dendrites compared with mice receiving saline, but not those receiving morphine, injections. Compared with saline, however, mice receiving chronic morphine showed a significantly lower plasmalemmal, and greater cytoplasmic, density of CRFr immunogold in dendrites. Within the cytoplasmic compartment of somata and dendrites of the BSTal, the proportion of CRFr gold particles associated with mitochondria was three times as great in mice receiving morphine compared with saline. This subcellular distribution is consistent with morphine,- and CRFr-associated modulation of intracellular calcium release or oxidative stress. The between-group changes occurred without effect on the total number of dendritic CRFr immunogold particles, suggesting that chronic morphine enhances internalization or decreases delivery of the CRFr to the plasma membrane, a trafficking effect that is also affected by the stress of daily injections. In contrast, saline and morphine treatment groups showed no significant differences in the total number of CRF-immunoreactive axon terminals, or the frequency with which these terminals contacted CRFr-containing dendrites. This suggests that morphine does not influence axonal availability of CRF in the BSTal. The results have important implications for drug-associated adaptations in brain stress systems that may contribute to the motivation to continue drug use during dependence.

Acute and chronic cocaine differentially alter the subcellular distribution of AMPA GluR1 subunits in region-specific neurons within the mouse ventral tegmental area.

Cocaine administration increases AMPA GluR1 expression and receptor-mediated activation of the ventral tegmental area (VTA). Functionality is determined, however, by surface availability of these receptors in transmitter- and VTA-region-specific neurons, which may also be affected by cocaine. To test this hypothesis, we used electron microscopic immunolabeling of AMPA GluR1 subunits and tyrosine hydroxylase (TH), the enzyme needed for dopamine synthesis, in the cortical-associated parabrachial (PB) and in the limbic-associated paranigral (PN) VTA of adult male C57BL/6 mice receiving either a single injection (acute) or repeated escalating-doses for 14 days (chronic) of cocaine. Acute cocaine resulted in opposing VTA-region-specific changes in TH-containing dopaminergic dendrites. TH-labeled dendrites within the PB VTA showed increased cytoplasmic GluR1 immunogold particle density consistent with decreased AMPA receptor-mediated glutamatergic transmission. Conversely, TH-labeled dendrites within the PN VTA showed greater surface expression of GluR1 with increases in both synaptic and plasmalemmal GluR1 immunogold density after a single injection of cocaine. These changes diminished in both VTA subregions after chronic cocaine administration. In contrast, non-TH-containing, presumably GABAergic dendrites showed VTA-region-specific changes only after repeated cocaine administration such that synaptic GluR1 decreased in the PB, but increased in the PN VTA. Taken together, these findings provide ultrastructural evidence suggesting that chronic cocaine not only reverses the respective depression and facilitation of mesocortical (PB) and mesolimbic (PN) dopaminergic neurons elicited by acute cocaine, but also differentially affects synaptic availability of these receptors in non-dopaminergic neurons of each region. These adaptations may contribute to increased cocaine seeking/relapse and decreased reward that is reported with chronic cocaine use.

Cannabinoid-1 receptor gene deletion has a compartment-specific affect on the dendritic and axonal availability of μ-opioid receptors and on dopamine axons in the mouse nucleus accumbens.

Cannabinoid‐type 1 (CB1) receptors are implicated in μ‐opioid receptor (μ‐OR)‐dependent reward ascribed partially to mesolimbic dopamine release in the nucleus accumbens (Acb) shell. Thus, CB1 receptor gene deletion may preferentially alter the availability of μ‐ORs and/or dopamine innervation in this brain region, which is functionally distinct from the motor‐associated Acb core. To test this hypothesis, we examined the electron microscopic immunolabeling of the μ‐OR and the dopamine‐synthesizing enzyme, tyrosine hydroxylase (TH) in Acb shell, and core of adult C57BL/6J wild‐type (WT) and CB1‐knock‐out (KO) mice. The μ‐OR‐immunogold particles were observed in the cytoplasm and on the plasmalemma in dendrites, dendritic spines, and axon terminals throughout the Acb. Compared to WT, the Acb shell of CB1‐KO mice showed a lower cytoplasmic density of μ‐ORs in dendrites and fewer μ‐OR labeled, but not unlabeled, dendritic spines. In this region, the CB1‐KOs had a significantly enhanced plasmalemmal density of μ‐OR‐immunogold in axon terminals, 70% of which formed excitatory‐type synapses. However, the number of both μ‐OR‐labeled terminals and TH‐labeled small varicosities was significantly reduced in the Acb shell of CB1‐KOs. These adaptations were not seen in the Acb core, where CB1‐KOs had a preferentially lower dendritic plasmalemmal and total spine density of μ‐OR immunogold. Our results indicate that constitutive deletion of the CB1 receptor gene has a major impact on the pre and postsynaptic availability of μ‐ORs at axospinous synapses and on the dopamine innervation of the Acb shell as well as the dendritic surface expression of μ‐ORs in Acb core of mature rodents. Synapse 64:886–897, 2010.

Cannabinoid-1 receptors in the mouse ventral pallidum are targeted to axonal profiles expressing functionally opposed opioid peptides and contacting N-acylphosphatidylethanolamine-hydrolyzing phospholipase D terminals.

The ventral pallidum (VP) is a major recipient of inhibitory projections from nucleus accumbens (Acb) neurons that differentially express the reward (enkephalin) and aversion (dynorphin)-associated opioid peptides. The cannabinoid-1 receptor (CB1R) is present in Acb neurons expressing each of these peptides, but its location in the VP is not known. To address this question, we used electron microscopic dual immunolabeling of the CB1R and either dynorphin 1-8 (Dyn) or Met(5)-enkephalin (ME) in the VP of C57BL/6J mice, a species in which CB1R gene deletion produces a reward deficit. We also used similar methods to determine the relationship between the CB1R and N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), an anandamide-synthesizing enzyme located presynaptically in other limbic brain regions. CB1R-immunogold was principally localized to cytoplasmic endomembranes and synaptic or extrasynaptic plasma membranes of axonal profiles, but was also affiliated with postsynaptic membrane specializations in dendrites. The axonal profiles included many single CB1R-labeled axon terminals as well as terminals containing CB1R-immunogold and either Dyn or ME immunoreactivity. Dually labeled terminals comprised 26% of all Dyn- and 17% of all ME-labeled axon terminals. Both single- and dual-labeled terminals formed mainly inhibitory-type synapses, but almost 16% of these terminals formed excitatory synapses. Approximately 60% of the CB1R-labeled axonal profiles opposed or converged with axon terminals containing NAPE-PLD immunoreactivity. We conclude that CB1Rs in the mouse VP have subcellular distributions consistent with on demand activation by endocannabinoids that can regulate the release of functionally opposed opioid peptides and also modulate inhibitory and excitatory transmission.

NEUROKININ-1 RECEPTORS IN CHOLINERGIC NEURONS OF THE RAT VENTRAL PALLIDUM HAVE A PREDOMINANTLY DENDRITIC DISTRIBUTION THAT IS AFFECTED BY APOMORPHINE WHEN COMBINED WITH STARTLE-EVOKING AUDITORY STIMULATION

Cholinergic neurons of the basal forebrain are implicated in startle reflex inhibition by a prior weak stimulus often referred to as prepulse inhibition (PPI) and used as an index of sensorimotor gating deficits in schizophrenia. Gating deficits can be produced in rodent models by acute systemic administration of apomorphine, a non-selective dopamine D1 and D2 receptor agonist that also affects trafficking of neurokinin-1 (NK(1)) receptors induced by startle evoking auditory stimulation (AS) in midbrain neurons. We used electron microscopic immunolabeling of NK(1) receptors and the vesicular acetylcholine transporter (VAchT) to test the hypothesis that the subcellular distributions of these receptors in cholinergic neurons of the rat ventral pallidum are subject to a similar regulation. In vehicle controls, NK(1) immunogold was often seen near cytoplasmic endomembranes in somata and large dendrites, but was more equally distributed in cytoplasmic and plasmalemmal compartments of medium dendrites, and principally located on the plasma membrane of small dendrites. These labeling patterns appeared to be largely independent of whether the NK(1) receptor was co-expressed with VAchT, however only the medium and small VAchT-labeled dendrites showed significant treatment-specific differences in NK(1) immunogold distributions. The NK(1) receptor immunogold particle density on the plasma membrane of medium cholinergic dendrites was significantly enhanced by combined apomorphine and AS, while neither alone affected either the plasmalemmal density or the equality of the plasmalemmal and cytoplasmic distributions of NK(1) receptors in these dendrites. Small cholinergic dendrites showed a significant AS-induced increase in both the plasmalemmal and cytoplasmic density of NK(1) gold particles, and an apomorphine-induced disruption of the preferential plasmalemmal targeting of the NK(1) receptors. These results provide ultrastructural evidence that NK(1) receptors in cholinergic neurons of the ventral pallidum have subcellular locations and plasticity conducive to active involvement in dopamine-dependent sensorimotor processing.