Sex differences in NMDA GluN1 plasticity in rostral ventrolateral medulla neurons containing corticotropin-releasing factor type 1 receptor following slow-pressor angiotensin II hypertension.

There are profound, yet incompletely understood, sex differences in the neurogenic regulation of blood pressure. Both corticotropin signaling and glutamate receptor plasticity, which differ between males and females, are known to play important roles in the neural regulation of blood pressure. However, the relationship between hypertension and glutamate plasticity in corticotropin-releasing factor (CRF)-receptive neurons in brain cardiovascular regulatory areas, including the rostral ventrolateral medulla (RVLM) and paraventricular nucleus of the hypothalamus (PVN), is not understood. In the present study, we used dual-label immuno-electron microscopy to analyze sex differences in slow-pressor angiotensin II (AngII) hypertension with respect to the subcellular distribution of the obligatory NMDA glutamate receptor subunit 1 (GluN1) subunit of the N-methyl-D-aspartate receptor (NMDAR) in the RVLM and PVN. Studies were conducted in mice expressing the enhanced green fluorescence protein (EGFP) under the control of the CRF type 1 receptor (CRF1) promoter (i.e., CRF1-EGFP reporter mice). By light microscopy, GluN1-immunoreactivity (ir) was found in CRF1-EGFP neurons of the RVLM and PVN. Moreover, in both regions tyrosine hydroxylase (TH) was found in CRF1-EGFP neurons. In response to AngII, male mice showed an elevation in blood pressure that was associated with an increase in the proportion of GluN1 on presumably functional areas of the plasma membrane (PM) in CRF1-EGFP dendritic profiles in the RVLM. In female mice, AngII was neither associated with an increase in blood pressure nor an increase in PM GluN1 in the RVLM. Unlike the RVLM, AngII-mediated hypertension had no effect on GluN1 localization in CRF1-EGFP dendrites in the PVN of either male or female mice. These studies provide an anatomical mechanism for sex-differences in the convergent modulation of RVLM catecholaminergic neurons by CRF and glutamate. Moreover, these results suggest that sexual dimorphism in AngII-induced hypertension is reflected by NMDA receptor trafficking in presumptive sympathoexcitatory neurons in the RVLM.

Adolescent social isolation enhances the plasmalemmal density of NMDA NR1 subunits in dendritic spines of principal neurons in the basolateral amygdala of adult mice.

Social isolation during the vulnerable period of adolescence produces emotional dysregulation manifested by abnormalities in adult behaviors that require emotional processing. The affected brain regions may include the basolateral amygdala (BLA), where plasticity of glutamatergic synapses in principal neurons plays a role in conditioned emotional responses. This plasticity is dependent on NMDA receptor trafficking denoted by intracellular mobilization of the obligatory NR1 NMDA subunit. We tested the hypothesis that the psychosocial stress of adolescent social isolation (ASI) produces a lasting change in NMDA receptor distribution in principal neurons in the BLA of adults that express maladaptive emotional responses to sensory cues. For this, we used behavioral testing and dual electron microscopic immunolabeling of NR1 and calcium calmodulin-dependent protein kinase II (CaMKII), a protein predominantly expressed in principal neurons of the BLA in adult C57Bl/6 mice housed in isolation or in social groups from post-weaning day 22 until adulthood (∼3 months of age). The isolates showed persistent deficits in sensorimotor gating evidenced by altered prepulse inhibition (PPI) of acoustic startle and hyperlocomotor activity in a novel environment. Immunogold-silver labeling for NR1 alone or together with CaMKII was seen in many somatodendritic profiles in the BLA of all mice irrespective of rearing conditions. However, isolates compared with group-reared mice had a significantly lower cytoplasmic (4.72 ± 0.517 vs 6.31 ± 0.517) and higher plasmalemmal (0.397 ± 0.0779 vs 0.216 ± 0.026) density of NR1 immunogold particles in CaMKII-containing dendritic spines. There was no rearing-dependent difference in the size or number of these spines or those of other dendritic profiles within the neuropil, which also failed to show an impact of ASI on NR1 immunogold labeling. These results provide the first evidence that ASI enhances the surface trafficking of NMDA receptors in dendritic spines of principal neurons in the BLA of adult mice showing maladaptive behaviors that are consistent with emotional dysregulation.

Dopamine D2 and acetylcholine α7 nicotinic receptors have subcellular distributions favoring mediation of convergent signaling in the mouse ventral tegmental area.

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) mediate nicotine-induced burst-firing of dopamine neurons in the ventral tegmental area (VTA), a limbic brain region critically involved in reward and in dopamine D2 receptor (D2R)-related cortical dysfunctions associated with psychosis. The known presence of α7nAChRs and Gi-coupled D2Rs in dopamine neurons of the VTA suggests that these receptors are targeted to at least some of the same neurons in this brain region. To test this hypothesis, we used electron microscopic immunolabeling of antisera against peptide sequences of α7nACh and D2 receptors in the mouse VTA. Dual D2R and α7nAChR labeling was seen in many of the same somata (co-localization over 97%) and dendrites (co-localization over 49%), where immunoreactivity for each of the receptors was localized to endomembranes as well as to non-synaptic or synaptic plasma membranes often near excitatory-type synapses. In comparison with somata and dendrites, many more small axons and axon terminals were separately labeled for each of the receptors. Thus, single-labeled axon terminals were predominant for both α7nAChR (57.9%) and D2R (89.0%). The majority of the immunolabeled axonal profiles contained D2R-immunoreactivity (81.6%) and formed either symmetric or asymmetric synapses consistent with involvement in the release of both inhibitory and excitatory transmitters. Of 160 D2R-labeled terminals, 81.2% were presynaptic to dendrites that expressed α7nAChR alone or together with the D2R. Numerous glial processes inclusive of those enveloping either excitatory- or inhibitory-type synapses also contained single labeling for D2R (n=152) and α7nAChR (n=561). These results suggest that classic antipsychotic drugs, all of which block the D2R, may facilitate α7nAChR-mediated burst-firing by elimination of D2R-dependent inhibition in neurons expressing both receptors as well as by indirect pre-synaptic and glial mechanisms.

The impact of adolescent social isolation on dopamine D2 and cannabinoid CB1 receptors in the adult rat prefrontal cortex.

Adolescent experiences of social deprivation result in profound and enduring perturbations in adult behavior, including impaired sensorimotor gating. The behavioral deficits induced by adolescent social isolation in rats can be ameliorated by antipsychotic drugs blocking dopamine D2 receptors in the prefrontal cortex (PFC) or by chronic administration of a cannabinoid CB1 receptor antagonist. The patterning and abundance of D2 receptors in the PFC evolves concurrently with CB1 receptors through the period of adolescence. This evidence suggests that mature expression and/or surface distribution of D2 and CB1 receptors may be influenced by the adolescent social environment. We tested this hypothesis using electron microscopic immunolabeling to compare the distribution of CB1 and D2 receptors in the PFC of adult male Sprague-Dawley rats that were isolated or socially reared throughout the adolescent transition period. Prepulse inhibition (PPI) of acoustic startle was assessed as a measure of sensorimotor gating. Social isolation reduced PPI and selectively decreased dendritic D2 immunogold labeling in the PFC. However, the decrease was only evident in dendrites that were not contacted by axon terminals containing CB1. There was no apparent change in the expression of CB1 or D2 receptors in presynaptic terminals. The D2 deficit therefore may be tempered by local CB1-mediated retrograde signaling. This suggests a biological mechanism whereby the adolescent social environment can persistently influence cortical dopaminergic activity and resultant behavior.

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.

Distribution of angiotensin type 1a receptor-containing cells in the brains of bacterial artificial chromosome transgenic mice.

In the central nervous system, angiotensin II (AngII) binds to angiotensin type 1 receptors (AT(1)Rs) to affect autonomic and endocrine functions as well as learning and memory. However, understanding the function of cells containing AT(1)Rs has been restricted by limited availability of specific antisera, difficulties discriminating AT(1)R-immunoreactive cells in many brain regions and, the identification of AT(1)R-containing neurons for physiological and molecular studies. Here, we demonstrate that an Agtr1a bacterial artificial chromosome (BAC) transgenic mouse line that expresses type A AT(1)Rs (AT1aRs) identified by enhanced green fluorescent protein (EGFP) overcomes these shortcomings. Throughout the brain, AT1aR-EGFP was detected in the nuclei and cytoplasm of cells, most of which were neurons. EGFP often extended into dendritic processes and could be identified either natively or with immunolabeling of GFP. The distribution of AT1aR-EGFP cells in brain closely corresponded to that reported for AngII binding and AT1aR protein and mRNA. In particular, AT1aR-EGFP cells were in autonomic regions (e.g., hypothalamic paraventricular nucleus, central nucleus of the amygdala, parabrachial nucleus, nuclei of the solitary tract and rostral ventrolateral medulla) and in regions involved in electrolyte and fluid balance (i.e., subfornical organ) and learning and memory (i.e., cerebral cortex and hippocampus). Additionally, dual label electron microscopic studies in select brain areas demonstrate that cells containing AT1aR-EGFP colocalize with AT(1)R-immunoreactivity. Assessment of AngII-induced free radical production in isolated EGFP cells demonstrated feasibility of studies investigating AT1aR signaling ex vivo. These findings support the utility of Agtr1a BAC transgenic reporter mice for future studies understanding the role of AT(1)R-containing cells in brain function.

Differential glutamate AMPA-receptor plasticity in subpopulations of VTA neurons in the presence or absence of residual cocaine: implications for the development of addiction.

Cocaine-induced plasticity of mesocorticolimbic dopamine (DA) neurons, originating in the ventral tegmental area (VTA), persists in the absence of cocaine and may contribute to both drug-craving and relapse. Glutamate AMPA receptors (AMPARs) in these neurons are implicated in this plasticity. However, there is no ultrastructural evidence that the absence of cocaine following repeated administrations affects the critical surface/synaptic availability of AMPAR GluR1 subunits in either DA or non-DA, putative GABAergic neurons within the VTA. To assess this, we used electron microscopic immunolabeling in the VTA of adult male mice sacrificed at 30 min or 72 h after receiving the final of six (15 mg/kg) cocaine injections, a dosing paradigm that resulted in development of locomotor sensitization. At each time point, both cocaine- and saline-injected mice showed AMPAR GluR1 immunogold labeling in somatodendritic profiles, many of which contained immunoperoxidase labeling for the DA-synthesizing enzyme, tyrosine hydroxylase (TH). At 30 min after the last injection, when cocaine was systemically present, only the non-TH labeled dendrites showed a significant increase in the synaptic/plasmalemmal density of GluR1 immunogold particles. At 72 h, when systemic cocaine was depleted, synaptic GluR1 labeling was greatly enhanced in TH-containing dendrites throughout the VTA and in non-TH dendrites of the limbic-associated paranigral VTA. Our results demonstrate that systemic cocaine produces GluR1 trafficking specifically in non-DA neurons of the VTA, which may subsequently contribute to the abstinent-induced enhancement of AMPA receptor synaptic transmission in mesocorticolimbic DA neurons leading to heightened drug seeking behavior.

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.

Ultrastructural relationship between N-methyl-D-aspartate-NR1 receptor subunit and mu-opioid receptor in the mouse central nucleus of the amygdala.

The central nucleus of the amygdala (CeA) is an important neuroanatomical substrate of emotional processes that are critically involved in addictive behaviors. Glutamate and opioid systems in the CeA play significant roles in neural plasticity and addictive processes, however the cellular sites of interaction between agonists of N-methyl-d-aspartate (NMDA) and mu-opioid receptors (muOR) in the CeA are unknown. Dual labeling immunocytochemistry was used to determine the ultrastructural relationship between the essential NMDA-NR1 receptor subunit and muOR in the CeA. It was found that over 80% of NR1-labeled profiles were dendrites while less than 10% were axons. In the case of muOR-labeled profiles, approximately 60% were dendritic, and over 35% were axons. Despite their somewhat distinctive patterns of cellular location, numerous dual-labeled profiles were observed. Approximately 80% of these were dendritic, and less than 10% were axonal. Moreover, many dual-labeled dendritic profiles were contacted by axon terminals receiving asymmetric-type synapses indicative of excitatory signaling. These results indicate that NMDA and muORs are strategically localized in dendrites, including those receiving excitatory synapses, of central amygdala neurons. Thus, postsynaptic co-modulation of central amygdala neurons may be a key cellular substrate mediating glutamate and opioid interaction on neural signaling and plasticity associated with normal and pathological emotional processes associated with addictive behaviors.

Angiotensin II type 2 receptors have a major somatodendritic distribution in vasopressin-containing neurons in the mouse hypothalamic paraventricular nucleus.

The hypothalamic paraventricular nucleus (PVN) and angiotensin II (AngII) play critical roles in cardiovascular and neurohumoral regulation ascribed in part to vasopressin (VP) release. The AngII actions in the PVN are mediated largely through angiotensin II type 1 (AT1) receptors. However, there is indirect evidence that the functionally elusive central angiotensin II type 2 (AT2) receptors are also mediators of AngII signaling in the PVN. We used electron microscopic dual immunolabeling of antisera recognizing the AT2 receptor and VP to test the hypothesis that mouse PVN neurons expressing VP are among the cellular sites where this receptor has a subcellular distribution conducive to local activation. Immunoreactivity for the AT2 receptor was detected in somatodendritic profiles, of which approximately 60% of the somata and approximately 28% of the dendrites also contained VP. In comparison with somata and dendrites, axons, axon terminals, and glia less frequently contained the AT2 receptor. Somatic labeling for the AT2 receptor was often seen in the cytoplasm near the Golgi lamellae and other endomembrane structures implicated in receptor trafficking. AT2 receptor immunoreactivity in dendrites was commonly localized to cytoplasmic endomembranes, but was occasionally observed on extra- or peri-synaptic portions of the plasma membrane apposed by astrocytic processes or by unlabeled axon terminals. The labeled dendritic plasmalemmal segments containing AT2 receptors received asymmetric excitatory-type or more rarely symmetric inhibitory-type contacts from unlabeled axon terminals containing dense core vesicles, many of which are known to store neuropeptides. These results provide the first ultrastructural evidence that AT2 receptors in PVN neurons expressing VP and other neuromodulators are strategically positioned for surface activation by AngII and/or intracellular trafficking.

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.

Spatial and intracellular relationships between the alpha7 nicotinic acetylcholine receptor and the vesicular acetylcholine transporter in the prefrontal cortex of rat and mouse.

The alpha 7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) is expressed in the prefrontal cortex (PFC), a brain region where these receptors are implicated in cognitive function and in the pathophysiology of schizophrenia. Activation of this receptor is dependent on release of acetylcholine (ACh) from axon terminals that contain the vesicular acetylcholine transporter (VAChT). Since rat and mouse models are widely used for studies of specific abnormalities in schizophrenia, we sought to determine the subcellular location of the alpha7nAChR with respect to VAChT storage vesicles in axon terminals in the PFC in both species. For this, we used dual electron microscopic immunogold and immunoperoxidase labeling of antisera raised against the alpha7nAChR and VAChT. In both species, the alpha7nAChR-immunoreactivity ((-)ir) was principally identified within dendrites and dendritic spines, receptive to axon terminals forming asymmetric excitatory-type synapses, but lacking detectable alpha7nAChR or VAChT-ir. Quantitative analysis of the rat PFC revealed that of alpha7nAChR-labeled neuronal profiles, 65% (299/463) were postsynaptic structures (dendrites and dendritic spine) and only 22% (104/463) were axon terminals or small unmyelinated axons. In contrast, VAChT was principally localized to varicose vesicle-filled axonal profiles, without recognized synaptic specializations (n=240). Of the alpha7nAChR-labeled axons, 47% (37/79) also contained VAChT, suggesting that ACh release is autoregulated through the presynaptic alpha7nAChR. The VAChT-labeled terminals rarely formed synapses, but frequently apposed alpha7nAChR-containing neuronal profiles. These results suggest that in rodent PFC, the alpha7nAChR plays a major role in modulation of the postsynaptic excitation in spiny dendrites in contact with VAChT containing axons.

Mu-opioid and corticotropin-releasing-factor receptors show largely postsynaptic co-expression, and separate presynaptic distributions, in the mouse central amygdala and bed nucleus of the stria terminalis.

The anxiolytic effects of opiates active at the mu-opioid receptor (mu-OR) may be ascribed, in part, to suppression of neurons that are responsive to the stress-associated peptide, corticotropin releasing factor (CRF), in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). The corticotropin releasing factor receptor (CRFr) and mu-OR are expressed in both the CeA and BNST, but their subcellular relationship to each other is not known in either region. To address this question, we used dual electron microscopic immunolabeling of mu-OR and CRFr in the mouse lateral CeA and anterolateral BNST. Immunolabeling for each receptor was detected in the same as well as in separate somatic, dendritic and axonal profiles of neurons in each region. CRFr had a plasmalemmal or cytoplasmic distribution in many dendrites, including those co-expressing mu-OR. The co-expression of CRFr and mu-OR also was seen near excitatory-type synapses on dendritic spines. In both the CeA and BNST, over 50% of the CRFr-labeled dendritic profiles (dendrites and spines) contained immunoreactivity for the mu-OR. However, less than 25% of the dendritic profiles containing the mu-OR were labeled for CRFr in either region, suggesting that opiate activation of the mu-OR affects many neurons in addition to those responsive to CRF. The dendritic profiles containing CRFr and/or mu-OR received asymmetric, excitatory-type synapses from unlabeled or CRFr-labeled axon terminals. In contrast, the mu-OR was identified in terminals forming symmetric, inhibitory-type synapses. Thus, in both the CeA and BNST, mu-OR and CRFr have strategic locations for mediation of CRF and opioid effects on the postsynaptic excitability of single neurons, and on the respective presynaptic release of excitatory and inhibitory neurotransmitters. The commonalities in the synaptic location of both receptors in the CeA and BNST suggest that this is a fundamental cellular association of relevance to both drug addiction and stress-induced disorders.

Preferential relocation of the N-methyl-D-aspartate receptor NR1 subunit in nucleus accumbens neurons that contain dopamine D1 receptors in rats showing an apomorphine-induced sensorimotor gating deficit.

Sensorimotor gating as measured by prepulse inhibition (PPI) to startle-evoking auditory stimulation (AS) is disrupted in schizophrenia and in rodents receiving systemic administration of apomorphine, a dopamine D1/D2 receptor agonist, or MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist. The functional analogies and our prior results showing apomorphine- and AS-induced relocation of the dopamine D1 receptor (D1R) in the nucleus accumbens (Acb) shell suggest that apomorphine and AS may affect the subcellular distribution of the NMDA receptor NR1 subunit, a protein that forms protein-protein interactions with the D1R. We quantitatively compared the electron microscopic immunogold labeling for NR1 in dendritic profiles distinguished with respect to presence of D1R immunoreactivity and location in the Acb shell or core of rats receiving a single s.c. injection of vehicle (VEH) or apomorphine (APO) alone, or combined with AS (VEH+AS, APO+AS). The rats in the APO+AS group were previously shown to have PPI deficits, whereas the rats in the VEH+AS group had normal PPI. A significantly higher percentage of plasmalemmal and a lower percentage of cytoplasmic NR1 immunogold particles were seen in D1R-labeled dendritic spines in the Acb shell of the APO+AS group compared with all other groups. D1R-containing small dendrites in the Acb shell of the APO+AS group also showed a significantly higher density of plasmalemmal and a lower density of cytoplasmic NR1 immunogold particles compared with VEH or APO groups. In the Acb core, the APO+AS group had significantly fewer dendritic spines co-expressing NR1 and D1R compared with VEH or VEH+AS groups. These results, together with our earlier findings, suggest that NMDA receptors are preferentially mobilized in D1R-containing Acb neurons of rats showing apomorphine-induced disruption of PPI in a paradigm using acoustic stimulation.

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.

Dendritic distributions of dopamine D1 receptors in the rat nucleus accumbens are synergistically affected by startle-evoking auditory stimulation and apomorphine.

Prepulse inhibition of the startle response to auditory stimulation (AS) is a measure of sensorimotor gating that is disrupted by the dopamine D1/D2 receptor agonist, apomorphine. The apomorphine effect on prepulse inhibition is ascribed in part to altered synaptic transmission in the limbic-associated shell and motor-associated core subregions of the nucleus accumbens (Acb). We used electron microscopic immunolabeling of dopamine D1 receptors (D1Rs) in the Acb shell and core to test the hypothesis that region-specific redistribution of D1Rs is a short-term consequence of AS and/or apomorphine administration. Thus, comparisons were made in the Acb of rats killed 1 h after receiving a single s.c. injection of vehicle (VEH) or apomorphine (APO) alone or in combination with startle-evoking AS (VEH+AS, APO+AS). In both regions of all animals, the D1R immunoreactivity was present in somata and large, as well as small, presumably more distal dendrites and dendritic spines. In the Acb shell, compared with the VEH+AS group, the APO+AS group had more spines containing D1R immunogold particles, and these particles were more prevalent on the plasma membranes. This suggests movement of D1Rs from distal dendrites to the plasma membrane of dendritic spines. Small- and medium-sized dendrites also showed a higher plasmalemmal density of D1R in the Acb shell of the APO+AS group compared with the APO group. In the Acb core, the APO+AS group had a higher plasmalemmal density of D1R in medium-sized dendrites compared with the APO or VEH+AS group. Also in the Acb core, D1R-labeled dendrites were significantly smaller in the VEH+AS group compared with all other groups. These results suggest that alerting stimuli and apomorphine synergistically affect distributions of D1R in Acb shell and core. Thus adaptations in D1R distribution may contribute to sensorimotor gating deficits that can be induced acutely by apomorphine or develop over time in schizophrenia.

Predominant surface distribution of neurokinin-3 receptors in non-dopaminergic dendrites in the rat substantia nigra and ventral tegmental area.

Neurokinin-3 (NK(3)) receptors are prevalent within the substantia nigra (SN) and ventral tegmental area (VTA), where their activation can affect motor and motivational behaviors as well as cardiovascular function and stress responses. These actions are mediated, in part, by dopaminergic neurons in each region. To determine the relevant sites for activation of these receptors, we examined the electron microscopic localization of NK(3) receptors and tyrosine hydroxylase (TH), the catecholamine synthesizing enzyme in dopaminergic neurons in the SN and VTA of rat brain. In each region, immunogold-silver labeling for NK(3) receptors was detected in many somatodendritic profiles, some of which contained TH-immunoreactivity. NK(3)-immunogold particles were largely associated with endomembranes resembling smooth endoplasmic reticulum, and only occasionally located on the plasma membrane in TH-labeled dendrites. In comparison with these dendrites, non-TH immunoreactive dendrites contained significantly more total (VTA) and more plasmalemmal (VTA and SN) NK(3)-immunogold particles. In each region, NK(3) gold particles also were seen in axonal as well as glial profiles, some of which contacted TH-immunoreactive dendrites. The NK(3)-labeled axon terminals formed either symmetric or asymmetric, excitatory-type synapses, the latter of which were significantly more prevalent in the VTA, compared with SN. These results provide the first ultrastructural evidence indicating that NK(3) receptors are available in cytoplasmic reserve in dopaminergic neurons, but more immediately accessible at the plasmalemmal surface of non-dopaminergic dendrites in both the SN and VTA. The activation of these receptors, together with the NK(3) receptors in either the presynaptic axon terminals or glia may contribute to the diverse physiological effects of tachykinins in each region, and most prominently involving excitatory inputs to the VTA.

Subcellular localization of nicotinamide adenine dinucleotide phosphate oxidase subunits in neurons and astroglia of the rat medial nucleus tractus solitarius: relationship with tyrosine hydroxylase immunoreactive neurons.

Superoxide produced by the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase mediates crucial intracellular signaling cascades in the medial nucleus of the solitary tract (mNTS), a brain region populated by catecholaminergic neurons, as well as astroglia that play an important role in autonomic function. The mechanisms mediating NADPH oxidase (phagocyte oxidase) activity in the neural regulation of cardiovascular processes are incompletely understood, however the subcellular localization of superoxide produced by the enzyme is likely to be an important regulatory factor. We used immunogold electron microscopy to determine the phenotypic and subcellular localization of the NADPH oxidase subunits p47(phox), gp91(phox,) and p22(phox) in the mNTS in rats. The mNTS contains a large population of neurons that synthesize catecholamines. Significantly, catecholaminergic signaling can be modulated by redox reactions. Therefore, the relationship of NADPH oxidase subunit labeled neurons or glia with respect to catecholaminergic neurons was also determined by dual labeling for the superoxide producing enzyme and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In the mNTS, NADPH oxidase subunits were present primarily in somatodendritic processes and astrocytes, some of which also contained TH, or were contacted by TH-labeled axons, respectively. Immunogold quantification of NADPH oxidase subunit localization showed that p47(phox) and gp91(phox) were present on the surface membrane, as well as vesicular organelles characteristic of calcium storing smooth endoplasmic reticula in dendritic and astroglial processes. These results indicate that NADPH oxidase assembly and consequent superoxide formation are likely to occur near the plasmalemma, as well as on vesicular organelles associated with intracellular calcium storage within mNTS neurons and glia. Thus, NADPH oxidase-derived superoxide may participate in intracellular signaling pathways linked to calcium regulation in diverse mNTS cell types. Moreover, NADPH oxidase-derived superoxide in neurons and glia may directly or indirectly modulate catecholaminergic neuron activity in the mNTS.

Dopamine D1 receptors co-distribute with N-methyl-D-aspartic acid type-1 subunits and modulate synaptically-evoked N-methyl-D-aspartic acid currents in rat basolateral amygdala.

Activation of dopamine D1 or glutamate, N-methyl-d-aspartic acid (NMDA) receptors in the basolateral amygdala (BLA) can potently influence affective behaviors and associative learning. Physical protein-protein interactions also can occur between C-terminal peptides of D1 receptors and the NMDA-receptor subunit-1 (NR1), suggesting intracellular associations of direct relevance to dopaminergic modulation of NMDA currents. We examined this possibility by combining electron microscopic immunolabeling of the D1 and NR1 C-terminal peptides with in vitro patch-clamp recording in the rat BLA. In the in vivo preparations, D1 and NR1 were localized to the surface or endomembranes of many of the same somata and dendrites as well as a few axon terminals, including those forming asymmetric, excitatory-type synapses. In vitro analysis of physiologically characterized projection neurons revealed an excitatory response to bath application of either dopamine or the preferential D1 receptor agonist, dihydrexidine. In these neurons, dopamine also selectively reduced stimulation-evoked isolated NMDA receptor-mediated currents, but not isolated non-NMDA receptor-mediated currents or the response to exogenous NMDA application. The selective reduction of the NMDA receptor-mediated currents suggests that this effect occurs at a postsynaptic locus. Moreover, both D1 and NR1 were localized to postsynaptic surfaces of biocytin-filled and physiologically characterized projection neurons. Our results provide ultrastructural evidence for D1/NR1 endomembrane associations that may dynamically contribute to the attenuation of NMDA receptor-mediated currents following prior activation of D1 receptors in BLA projection neurons. The potential for postsynaptic cross-talk between D1 and NMDA receptors in BLA projection neurons as well as a similar interaction in presynaptic terminals could have important implications for the formation and extinction of affective memories.

Subcellular distribution and plasticity of neurokinin-1 receptors in the rat substantia nigra and ventral tegmental area.

Neurokinin-1 receptors show activity-dependent changes in their surface distributions that are critical in spinal pain mechanisms, and also may play an important role in the motor and affective behaviors influenced by dopaminergic projections from the substantia nigra and ventral tegmental area. To determine the relevant sites for neurokinin-1 receptor activation in these midbrain regions, we examined the electron microscopic immunolabeling of neurokinin-1 receptors and the dopamine-synthesizing enzyme, tyrosine hydroxylase in normal rats. We also examined whether neurokinin-1 receptor distributions in one or both regions are affected by (1) startle-evoking intense auditory stimulation or (2) acute administration of apomorphine, a dopamine D1/D2 agonist that enhances startle while paradoxically reducing the prepulse inhibition produced by low intensity conditioning stimuli in rat models of schizophrenia. In each region, neurokinin-1 immunogold was located on the plasma membrane and endomembranes of somatodendritic profiles with or without tyrosine hydroxylase. As compared with controls, animals receiving intense auditory stimulation either alone or together with smaller low intensity prepulses showed a significant increase in neurokinin-1-plasmalemmal labeling in non-dopaminergic dendrites of both regions, and a reduction in this labeling in dopaminergic dendrites of the ventral tegmental area. Both effects were diminished following apomorphine administration. In absence of the intense auditory stimulation, however, apomorphine increased neurokinin-1-immunogold particles on the plasma membrane of the non-dopaminergic dendrites exclusively in the substantia nigra. Our results are the first to show that neurokinin-1 receptors have plasmalemmal distributions in dopaminergic and non-dopaminergic neurons that can be differentially modified by startle-evoking auditory stimulation. They suggest that while apomorphine can independently affect neurokinin-1 receptor trafficking in substantia nigra motor circuits, its effects on neurokinin-1 receptor distributions in the ventral tegmental area are exclusively dependent on sensory activation.