Rescue of impaired sociability and anxiety-like behavior in adult cacna1c-deficient mice by pharmacologically targeting eIF2α.

CACNA1C, encoding the Cav1.2 subunit of L-type Ca2+ channels, has emerged as one of the most prominent and highly replicable susceptibility genes for several neuropsychiatric disorders. Cav1.2 channels play a crucial role in calcium-mediated processes involved in brain development and neuronal function. Within the CACNA1C gene, disease-associated single-nucleotide polymorphisms have been associated with impaired social and cognitive processing and altered prefrontal cortical (PFC) structure and activity. These findings suggest that aberrant Cav1.2 signaling may contribute to neuropsychiatric-related disease symptoms via impaired PFC function. Here, we show that mice harboring loss of cacna1c in excitatory glutamatergic neurons of the forebrain (fbKO) that we have previously reported to exhibit anxiety-like behavior, displayed a social behavioral deficit and impaired learning and memory. Furthermore, focal knockdown of cacna1c in the adult PFC recapitulated the social deficit and elevated anxiety-like behavior, but not the deficits in learning and memory. Electrophysiological and molecular studies in the PFC of cacna1c fbKO mice revealed higher E/I ratio in layer 5 pyramidal neurons and lower general protein synthesis. This was concurrent with reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α, an inhibitor of mRNA translation. Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC. Thus this study identifies a novel Cav1.2 mechanism in neuropsychiatric-related endophenotypes and a potential future therapeutic target to explore.

Effects of whole-brain radiation therapy on the blood-brain barrier in immunocompetent and immunocompromised mouse models.

BACKGROUND: Approximately 20% of all cancer patients will develop brain metastases in their lifespan. The standard of care for patients with multiple brain metastases is whole-brain radiation therapy, which disrupts the blood-brain barrier. Previous studies have shown inflammatory mediators play a role in the radiation-mediated increase in permeability. Our goal was to determine if differential permeability post-radiation occurs between immunocompetent and immunocompromised mice. METHODS: We utilized a commissioned preclinical irradiator to irradiate brains of C57Bl/6J wild-type and athymic nude mice. Acute (3-24 h) effects on blood-brain barrier integrity were evaluated with our in-situ brain perfusion technique and quantitative fluorescent and phosphorescent microscopy. The presence of inflammatory mediators in the brain and serum was determined with a proinflammatory cytokine panel. RESULTS: Blood-brain barrier integrity and efflux transporter activity were altered in the immunocompetent mice 12 h following irradiation without similar observations in the immunocompromised mice. We observed increased TNF-α concentrations in the serum of wild-type mice immediately post-radiation and nude mice 12 h post-radiation. The brain concentration of CXCL1 was also increased in both mouse strains at the 12-h time point. CONCLUSIONS: The immune response plays a role in the magnitude of blood-brain barrier disruption following irradiation in a time- and size-dependent manner.

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.

Angiotensin II AT-1A receptor immunolabeling in rat medial nucleus tractus solitarius neurons: subcellular targeting and relationships with catecholamines.

The angiotensin II AT-1A receptor (AT-1A) is the major mediator of the hypertensive actions of angiotensin II (ANG II) in the medial nucleus of the solitary tract (mNTS). The localization of the AT-1A receptor at surface or intracellular sites is an important determinant of its signaling properties, including intercellular or intracrine communication. However, the spatial localization of this protein, particularly within small distal or intermediate size dendrites of mNTS neurons, is unknown. Within the mNTS, ANG II and catecholamines interact in the regulation of autonomic function; however, it is unknown if AT-1A receptors are present at functional sites in catecholamine containing dendrites, or are contacted by catecholamine containing axon terminals. We compared surface and intracellular distributions of the AT-1A receptor in dendritic processes from the mNTS using immunogold electron microscopy in conjunction with immunoperoxidase labeling for tyrosine hydroxylase (TH) and morphometric analysis. Collapsed across all AT-1A-labeled dendritic profiles, immunogold labeling was more frequent in intracellular sites as compared with the plasma membrane. Small (<0.6 microm) dendritic profiles contained a higher ratio of particles associated with the surface membrane when compared with larger profiles. Approximately 27% of all AT-1A receptor-labeled dendritic profiles also contained labeling for TH. Approximately 12% of dendritic profiles single labeled for the AT-1A receptor were contacted by TH containing axons or axon terminals. The present results provide the first quantitative demonstration of select plasmalemmal and intracellular localizations of AT-1A receptors in dendritic processes of mNTS neurons, including those containing TH, or contacted by catecholaminergic axon terminals. These results suggest that AT-1A receptors are positioned for modulation of catecholamine signaling in the mNTS.

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.

In vitro functional reactivities of cutaneous mast cells from patients with mastocytosis.

Cutaneous mast cells from 3 patients with mastocytosis were evaluated for their morphologic characteristics and in vitro functional reactivities to different secretory agonists. By electron microscopy, mastocytosis mast cells appeared larger than normal skin mast cells, frequently had atypical, highly indented or bilobed nuclei, and each contained numerous, elongated cytoplasmic projections. Suspensions of mastocytosis mast cells were obtained from lesional skin biopsy specimens, and their response to both immunologic and nonimmunologic secretagogues was compared with mast cells from normal skin. Lesional skin mast cells had a net histamine release of 12.3% (+/- 1.3 SEM) and 31.1% (+/- 6.0 SEM) following stimulation with the purified human anaphylotoxin C3a and mouse monoclonal antihuman IgE antibodies, respectively. This specific release was similar to the responses observed in normal skin mast cells (11.5% +/- 4.5 SEM and 16.7% +/- 2.1 SEM, respectively). Mast cells from cutaneous lesions of mastocytosis also responded to the nonimmunologic secretagogues, morphine sulfate and calcium ionophore A23187 with a specific histamine release of 15.1% (+/- 1.2 SEM) and 39.8% (+/- 8.7 SEM), respectively. The results of this study demonstrate that mast cells from lesions of mastocytosis are morphologically atypical, but have a histamine content similar to normal skin mast cells and retain their functional reactivities to clinically relevant secretory stimuli.

UVB radiation and DNFB skin painting induce suppressor cells universally in mice.

Ultraviolet radiation (UVR)3 from within the spectrum B (UVB) has the capacity to distort the induction of contact hypersensitivity (CH) in murine skin. A damaging effect of UVB on epidermal Langerhans cells (LC) appears to be universal in all genetically defined strains of mice tested. However, while UVB impairs the induction of CH in some strains of mice, it has no apparent effect on CH in others. Thus, a disparity exists between the effects of UVB on LC and on CH. This is a paradox because LC are generally regarded to serve as the antigen-presenting cells of the skin, placing them at the earliest stages of induction of CH. One possible explanation for this paradox has been that UVB-susceptible strains of mice may generate hapten-specific suppressor T cells, whereas their UVB-resistant counterparts may not, when their skin is treated with UVR and painted with haptens such as dinitrofluorobenzene (DNFB). This possibility was excluded by examining the capacity of UVR and hapten to generate suppressor T cells in several different inbred strains of mice. The results indicate that the induction of hapten-specific afferent T suppressor cells is a universal sequela to treatment of mice with UVB and hapten, irrespective of whether the mice display the phenotype of vigorous CH or not. Thus, the genetic basis of UVB-resistance does not reside in the ability of UVR to induce suppressor T cells. Rather, attention should now be focused on its ability to interrupt induction of effector mechanisms.

Subcellular localization of alpha-2A-adrenergic receptors in the rat medial nucleus tractus solitarius: regional targeting and relationship with catecholamine neurons.

alpha-2A-adrenergic receptor (alpha2A-AR) agonists modulate diverse autonomic functions. These actions are believed to involve functionally specialized, second-order neurons in catecholamine-containing portions of the medial nucleus tractus solitarius (mNTS) at both intermediate (NTSi) and caudal (NTSc) levels. However, the cellular mechanisms subserving alpha2A-AR-mediated actions within the mNTS have yet to be established. Immunocytochemistry was employed to examine the subcellular distribution of alpha2A-AR in both the intermediate and caudal mNTS and its association with cells containing the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Quantitative regional comparison using immunogold showed that this receptor was distributed differentially to dendrites (NTSi, 46%; NTSc, 31%) and glia (NTSi, 29%; NTSc, 48%) at different levels of the NTS. Somata, axons, and terminals less frequently contained alpha2A-AR. The subcellular distribution of alpha2A-AR relative to catecholaminergic neurons also was similar within both subregions. Approximately 50% of alpha2A-AR-labeled somata also contained TH. In somatic profiles, alpha2A-AR labeling was often found in the cytosol and in association with endoplasmic reticulum and Golgi complexes, sites of receptor synthesis and trafficking. Approximately 20% of alpha2A-AR-immunoreactive dendrites also contained TH, where the receptor was often found on extrasynaptic portions of the plasma membrane near unlabeled terminals, some of which made symmetric contacts. However, TH-labeled terminals and dendrites usually were detected in the neuropil at a short distance (<10 microm) from alpha2A-AR-labeled neurons. alpha2A-AR-labeled glia frequently apposed unlabeled dendrites and terminals and were often located near TH-immunoreactive dendrites. These results indicate that, within the mNTS, alpha2A-AR is involved in a variety of autonomic processes, including postsynaptic modulation of mostly noncatecholaminergic dendrites, as well as influencing glia functions.

Alterations in deprivation, glucoprivic and sucrose intake following general, mu and kappa opioid antagonists in the hypothalamic paraventricular nucleus of rats.

While opioid agonists administered into the hypothalamic paraventricular nucleus increase food intake in rats, naloxone reduces deprivation-induced intake. Ventricular administration of either mu (beta-funaltrexamine) or kappa (nor-binaltorphamine) opioid antagonists reduces spontaneous, deprivation, glucoprivic and palatable intake. The present study assessed whether microinjections of either general, mu or kappa opioid antagonists into the paraventricular nucleus altered either deprivation (24 h) intake, 2-deoxy-D-glucose hyperphagia or sucrose intake in rats. Deprivation intake was significantly reduced by nor-binaltorphamine (5 micrograms, 68 nmol, 30-33%), beta-funaltrexamine (5 micrograms, 100 nmol, 26-29%) or naltrexone (10 micrograms, 260 nmol, 26%) in the paraventricular nucleus. 2-Deoxy-D-glucose hyperphagia was significantly reduced only after 2 h by naltrexone (10 micrograms, 260 nmol, 69%), norbinaltorphamine (20 micrograms, 272 nmol, 69%) or beta-funaltrexamine (20 micrograms, 400 nmol, 83%) in the paraventricular nucleus. Sucrose intake was significantly reduced by nor-binaltorphamine (5 micrograms, 68 nmol, 27-36%), naltrexone (5-10 micrograms, 130-260 nmol, 18-31%) and beta-funaltrexamine (5 micrograms, 100 nmol, 20%) in the paraventricular nucleus. These data indicate that general, mu and kappa opioid antagonists administered into the hypothalamic paraventricular nucleus produce similar patterns of effects upon different forms of food intake as did ventricular administration, implicating this nucleus as part of the circuitry underlying opioid mediation of ingestion.

A cis-acting element within the hepatitis A virus 5'-non-coding region required for in vitro translation.

Every picornavirus studied thus far has a sequence within the 5'-non-coding region that is required for internal ribosome binding and translation of the polyprotein. In an attempt to identify this region in hepatitis A virus we constructed a truncated hepatitis A virus (HAV) cDNA clone that contains the entire 736 bp 5' non-coding region (5'-NCR) and 754 base pairs of the viral capsid coding region (P1) under control of the SP6 promoter. In vitro transcription and translation of this transcript in a rabbit reticulocyte lysate yielded a protein product of about 29 kDa as analyzed by autoradiography following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A series of mutations of this construct have defined a minimal sequence between bases 347 and 734 in the 5'-NCR that is required for efficient in vitro translation. The deleted constructs (D 523-734 and D 632-734) showed a reduced ability to translate in the rabbit reticulocyte lysate system in comparison with the full-length 5'-NCR construct, pH1489. The translation of these deleted constructs was artificially restored by the addition of a 5'-terminal methylated cap structure, m7GpppG, to the RNA. This increase in translational efficiency could be competed away with cap analog (m7GDP) thus indicating that this region is required for cap-independent internal ribosome binding for HAV translation.

The effect of naloxone on food-motivated behavior in the obese Zucker rat.

We assessed differences in food reinforced behavior between obese and lean Zucker rats with a progressive ratio schedule 3 (PR3) in which a subject emitted three additional lever-presses each time a reinforcer was delivered. The number of responses required for a reinforcer eventually exceeded its value, termed the "break point", a sensitive measure of food motivated behavior. Break points were higher in obese rats than lean controls for grain pellets (27.5 versus 9.5, P = 0.01) but not for sweet pellets (51.6 versus 38.5, P = 0.31). We determined if naloxone (0.01-3.0 mg/kg, SC), which reduces free food intake in obese Zucker rats, affects food motivated behavior in obese Zuckers and lean controls. Naloxone reduced break points in both obese and lean rats to a similar extent when working for either grain pellets or sweet pellets. Under free-access feeding conditions, naloxone again decreased pellet intake similarly in the obese and lean Zucker rats. Naloxone appeared to decrease free-access pellet consumption to a greater extent than break point in both groups. These results show that (1) obese rats exhibit higher levels of performance for food than lean rats only when working for the less valued grain pellet, (2) naloxone reduces both break points and free-access pellet consumption independent of genotype, and (3) naloxone appears to decrease food more effectively in rats given free access to food than in rats working for food.

Opioids and food intake: distributed functional neural pathways?

Agonists of the mu, delta, kappa and ORL(1)opioid receptors increase food intake while opioid receptor blockade decreases food intake. The majority of the collected data related to opioids and feeding has led to the speculation that opioids are involved in meal maintenance and orosensory reward; however, some data suggest that opioids may impact feeding associated with energy needs. Based on the wide distribution of CNS opioid receptors and the presence of other neuropeptides in the vicinity of opioidergic pathways, it seems likely that opioids affect multiple feeding systems. For example, opioids in the hindbrain might be involved in both sensory and metabolic aspects of food intake, those in the amygdala in processing of 'emotional' properties of foods, and those in the hypothalamus in energy needs. In this review we present data which support functional diversity of opioids in feeding behavior.

Central opioid receptor subtype mediation of isoproterenol-induced drinking in rats.

Opioid receptor subtype antagonists differentially alter different types of water intake such that mu2 receptors modulate deprivation-induced water intake, kappa receptors modulate hypertonic saline-induced water intake, and mu2, delta1 and kappa receptors modulate water intake following Angiotensin II (ANG II). Water intake stimulated by peripheral administration of the beta-adrenergic agonist, isoproterenol is attenuated by naloxone and is thought to be mediated by release of renin and production of ANG II. The present study examined whether systemic and i.c.v. administration of general opioid antagonists and central administration of specific opioid receptor subtype antagonists would selectively alter water intake following isoproterenol in rats. Both systemic (1 mg/kg s.c.) and central (1-20 micrograms) naltrexone reduced water intake induced by isoproterenol (25 micrograms/kg s.c.) over a 2-h period. The mu receptor antagonist, beta-funaltrexamine (B-FNA: 1-20 micrograms), but not the mu1 antagonist, naloxonazine (50 micrograms), dose-dependently reduced isoproterenol drinking. Both the kappa antagonist, nor-binaltorphamine (Nor-BNI, 5-20 micrograms) and the delta1 antagonist, [D-Ala2, Leu5, Cys6]-enkephalin (DALCE, 1-40 micrograms) also dose-dependently reduced isoproterenol drinking. These data implicate mu2, kappa and delta1 sites in the opioid modulation of isoproterenol drinking.

General, mu and kappa opioid antagonists in the nucleus accumbens alter food intake under deprivation, glucoprivic and palatable conditions.

Ventricular microinjection studies found that whereas mu (beta-funaltrexamine, B-FNA), mu1 (naloxonazine) and kappa (nor-binaltorphamine, Nor-BNI) opioid receptor antagonists, but not delta antagonists, reduce deprivation-induced intake, kappa and mu, but not mu1 or delta antagonists reduce both 2-deoxy-D-glucose (2DG) hyperphagia and sucrose intake. Since opioid agonists stimulate spontaneous food intake in the accumbens, the present study examined whether administration of either naltrexone, B-FNA or Nor-BNI in the accumbens altered intake under deprivation (24 h), glucoprivic (2DG: 500 mg/kg, i.p.) or palatable sucrose (10%) conditions. Naloxonazine's effects in the accumbens were also evaluated for deprivation-induced intake. Deprivation-induced intake was significantly decreased over 4 h by naltrexone (5-20 micrograms, 44%), B-FNA (1-4 micrograms, 55%) and Nor-BNI (4 micrograms, 31%) but not naloxonazine (10 micrograms) in the accumbens. 2DG hyperphagia was significantly decreased by naltrexone (10-20 microgram, 79%), B-FNA (1-4 micrograms, 100%) and NOR-BNI (104 micrograms, 75%) in the accumbens. Sucrose intake was significantly decreased by naltrexone (50 micrograms, 27%) and B-FNA (1-4 micrograms, 37%), but not NOR-BNI in the accumbens. These data suggest that mu receptors, and particularly the mu2 binding site in the accumbens are responsible for the opioid modulation of these forms of intake in this nucleus, and that this control may be acting upon the amount of intake per se.

The development of a successful non-university-based ambulatory in vitro fertilization/embryo transfer program: phase I.

Most of the current in vitro fertilization and embryo transfer (IVF-ET) programs are university-based. The establishment of a successful ambulatory IVF program in association with a busy, two-man general obstetrics-gynecologic practice is described. Seventy-one infertile couples were screened between February 1 and October 15, 1983. Forty-three couples were judged eligible for IVF-ET. Forty-three women underwent a single attempt at ET. The first 13 of these women underwent controlled ovarian hyperstimulation (COH) with 150 mg clomiphene citrate and human chorionic gonadotropin (hCG), and the remaining 30 underwent COH with human menopausal gonadotropin (hMG) and hCG. One of the 13 patients who underwent COH with clomiphene citrate conceived but subsequently miscarried early in the first trimester, for an 8% pregnancy rate. There were 12 pregnancies among the 30 patients who received hMG and hCG, for a 40% pregnancy rate. Only three of these pregnancies miscarried in the early first trimester, and three of the nine viable pregnancies are twin gestations. The possible factors responsible for the high pregnancy rate with IVF-ET, using COH with hMG and hCG, are discussed, and the feasibility of its performance in a well-controlled, non-university program is demonstrated.

Analysis of central opioid receptor subtype antagonism of hypotonic and hypertonic saline intake in water-deprived rats.

Intake of either hypotonic or hypertonic saline solutions is modulated in part by the endogenous opioid system. Morphine and selective mu and delta opioid agonists increase saline intake, while general opioid antagonists reduce saline intake in rats. The present study evaluated whether intracerebroventricular administration of general (naltrexone) and selective mu (beta-funaltrexamine, 5-20 micrograms), mu, (naloxonazine, 50 micrograms), kappa (nor-binaltorphamine, 5-20 micrograms), delta (naltrindole, 20 micrograms), or delta 1 (DALCE, 40 micrograms) opioid receptor subtype antagonists altered water intake and either hypotonic (0.6%) or hypertonic (1.7%) saline intake in water-deprived (24 h) rats over a 3-h time course in a two-bottle choice test. Whereas peripheral naltrexone (0.5-2.5 mg/kg) significantly reduced water intake and hypertonic saline intake, central naltrexone (1-50 micrograms) significantly reduced water intake and hypotonic saline intake. Water intake was significantly reduced following mu and kappa receptor antagonism, but not following mu 1, delta, or delta 1 receptor antagonism. In contrast, neither hypotonic nor hypertonic saline intake was significantly altered by any selective antagonist. These data are discussed in terms of opioid receptor subtype control over saline intake relative to the animal's hydrational state and the roles of palatability and/or salt appetite.

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.

Identification of a trans-acting activity from liver that stimulates hepatitis A virus translation in vitro.

Hepatitis A virus (HAV), a picornavirus, is the causative agent of infectious hepatitis, generally a self-limiting disease of the liver. Recently, sequences within the 5' noncoding region that affect the translation of the viral genome have been identified using in vitro systems. In this report we demonstrate that extracts prepared from mouse liver cytoplasm specifically stimulate HAV RNA translation in a rabbit reticulocyte lysate in vitro. This activity appears to act specifically on HAV sequences and is not found in other mouse tissue and several cell lines of tissue culture origin.

Ultrastructural morphometric analysis of human mast cells in normal skin and pathological cutaneous lesions.

Electron micrographs of human mast cells in normal neonatal and adult skin and in cutaneous lesions of basal cell carcinoma (BCC), hemangioma and mastocytosis were assessed by morphometric analysis. Using this quantitative histologic approach, adult skin mast cells were found to be significantly larger (47.7 microns 2 +/- 2.4 SEM vs. 38.3 microns 2 +/- 1.8 SEM, p less than or equal to 0.001) and have larger granules (0.63 micron +/- .02 SEM vs. 0.53 micron +/- .02 SEM, p less than or equal to 0.001) than infant mast cells while both mast cell populations had comparable nuclear sizes (13.7 microns 2 +/- 0.9 SEM vs. 14.3 microns 2 +/- 0.8 SEM) and numbers of cytoplasmic granules (72 +/- 4.0 SEM vs. 66 +/- 4.0 SEM). Morphometric analysis of mast cell infiltrates in the adult skin lesions of BCC and hemangioma revealed that these cells were larger than neonatal mast cells but were similar to normal adult controls. Cutaneous mast cells from 2 mastocytosis patients, however, had significantly larger mean cell surface areas (78.0 microns 2 +/- 3.4 SEM and 70.6 microns 2 +/- 3.2 SEM, p less than or equal to 0.001), nuclear areas (20.8 microns 2 +/- 1.1 SEM and 21.3 microns 2 +/- 1.2 SEM p less than or equal to 0.001) and granule diameters (0.82 micron +/- 0.4 SEM and 0.83 micron +/- .03 SEM, p less than or equal to 0.001) when compared with mast cells in normal adult skin and in the other pathologic lesions. No difference in the total number of cytoplasmic granules was observed in the different mast cell populations.(ABSTRACT TRUNCATED AT 250 WORDS)