Healthy and diseased corticospinal motor neurons are selectively transduced upon direct AAV2-2 injection into the motor cortex.

Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1(G93A) transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken ß-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1(G93A) transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases.

Mechanical stability of the femoral fixation for single- and double-bundle ACL reconstruction in an in vitro experimental model.

Anterior cruciate ligament ACL reconstruction using the double-bundle (DB) technique is gaining popularity. A possible weak link in the DB technique could be that two tendon grafts of smaller diameters are used. The purpose of this study was to test different femoral fixation methods and graft diameters representing single-bundle (SB) and DB ACL reconstructions and compare their biomechanical properties. We hypothesized that SB 6-mm graft constructs had inferior biomechanical properties than SB 9-mm grafts or DB 2 × 6-mm grafts. Furthermore, we hypothesized that interference (IF) screw fixation would demonstrate less elongation and a higher stiffness than Endobutton (Smith & Nephew®, Inc., Andover, Massachusetts, USA) fixation (EBF). We performed an in vitro study using porcine knees and extensor tendons. The mechanical test consisted of a cyclic test followed by a load-to-failure test. We found that 6-mm graft constructs had an ultimate failure load that was up to 40% less than both the 9-mm and 2 × 6-mm graft constructs, despite the fixation method (P-values ≥ 0.004). Comparing fixation methods, EBF was superior to IF concerning maximum load to failure (P < 0.001); IF resulted in a higher stiffness of the femur/graft complex than the EBF (P < 0.001) but no significant difference in elongation between fixation methods. Since the two graft strands are subjected to different loads in different knee flexion angles, the reduced strength of the individual graft strands in DB ACL reconstruction could be a concern.

Pediatric reference intervals for endocrine markers in healthy children and adolescents on the Liaison XL (DiaSorin) immunoassay system.

OBJECTIVES: Prominent physiological changes occurring throughout childhood and adolescence necessitate the consideration of age and sex in biomarker interpretation. Critical gaps exist in pediatric reference intervals (RIs) for specialized endocrine markers, despite expected influence of growth and development. The current study aimed to establish and/or verify RIs for six specialized endocrine markers on a specialized immunoassay system. METHODS: Samples were collected from healthy children and adolescents (5 to <19 years) and apparently healthy outpatients (0 to <5 years) as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER). Serum samples were analysed for aldosterone, renin (plasma), thyroglobulin, anti-thyroglobulin, growth hormone, and insulin-like growth factor-1 (IGF-1) on the Liaison XL (DiaSorin) immunoassay platform. RIs (2.5th and 97.5th percentiles) were established for aldosterone, renin, thyroglobulin, anti-thyroglobulin, and growth hormone. Manufacturer-recommended pediatric RIs for IGF-1 were verified. RESULTS: Age-specific RIs were established for aldosterone, renin, and thyroglobulin, while no age-specific differences were observed for anti-thyroglobulin or growth hormone. IGF-1 was the only endocrine marker studied that demonstrated significant sex-specific differences. Manufacturer-recommended IGF-1 RIs were verified for children aged 6 to <19 years, while those for children aged 0 to <6 years did not verify. CONCLUSIONS: This study marks the first time that pediatric RIs for aldosterone and renin were established in the CALIPER cohort and highlights the dynamic changes that occur in water and sodium homeostasis during the first years of life. Overall, these data will assist pediatric clinical laboratories in test result interpretation and improve clinical decision-making for patients tested using Liaison immunoassays.

Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity.

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

Angiogenesis imaging study using interim [18F] RGD-K5 PET/CT in patients with lymphoma undergoing chemotherapy: preliminary evidence.

BACKGROUND: Our aim was to measure the impact of two cycles of standard chemotherapy on tumoural neoangiogenesis by [18F] fluorine arginine-glycine-aspartic (RGD-K5) positron emission tomography-computed tomography (PET) on patients presenting with lymphoma. Nineteen patients at Rouen's Henri Becquerel Cancer Centre were prospectively included. Fluorodeoxyglucose (FDG) and RGD-K5 PET were performed before (C0) and after (C2) two cycles of chemotherapy. End-of-treatment FDG PET was performed for final evaluation. Maximum standardised uptake value (SUVmax), SUVmean, Metabolic Tumour Volume (MTV) and Angiogenic Tumour Volume (ATV) were measured for all lesions. RGD SUVmax and SUVmean were also analysed in 13 normal organs at C0 and C2. The patient's treatment response was considered using the Deauville score (DS) at the end of FDG PET treatment (DS 1-3 were considered responders, and 4 and 5 non-responders). RESULTS: Eighteen patients had both C0 FDG and RGD PET. Twelve patients had both C2 FDG and RGD, completed the treatment protocol and were included in end-of-treatment analysis. No statistical difference was found in RGD uptake of normal organs before and after chemotherapy for SUVmax and SUVmean. On C0 RGD, apart from classical Hodgkin lymphoma (cHL; n = 5) and grey zone lymphoma (GZL; n = 1), other lymphoma sub-types (n = 12) had low RGD uptake (p < 0.001). Regarding FDG, there was no significant difference for SUVmax, SUVmean and MTV at C0 and C2 between patients with cHL and non-Hodgkin lymphoma (NHL). At C2 RGD, non-responders had higher SUVmax and SUVmean compared to responders (p < 0.001). There was no significant difference in RGD ATV between responders and non-responders. CONCLUSIONS: Our study showed significant higher initial RGD uptake in patients presenting with cHL and GZL compared to NHL. Non-responder also had higher post-chemotherapy RGD uptake compared to responders. Issues raised by RGD uptake, particularly in cHL, are yet to be explored and need to be confirmed in a larger population.

Lack of humoral immune response to the tetracycline (Tet) activator in rats injected intracranially with Tet-off rAAV vectors.

The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression.

Maternal glucocorticoid hormones influence neurotransmitter phenotypic expression in embryos.

Treatment of pregnant rats with reserpine prevented the normal disappearance of catecholamine fluorescence in presumptive neuroblasts of the embryonic gut. These cells normally express the noradrenergic phenotype transiently during embryonic development. The effect of reserpine was reproduced by treating mothers with hydrocortisone acetate. Moreover, the reserpine effect was blocked by treatment with dexamethasone, which inhibits the stress-induced increase in plasma glucocorticoids, and by mitotone, which causes adrenocortical cytolysis. It is concluded that reserpine, through the mediation of maternal glucocorticoid hormones, alters the phenotypic expression of these embryonic neuroblasts.

Adrenal medulla grafts enhance recovery of striatal dopaminergic fibers.

The drug, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), depletes striatal dopamine levels in primates and certain rodents, including mice, and produces parkinsonian-like symptoms in humans and nonhuman primates. To investigate the consequences of grafting adrenal medullary tissue into the brain of a rodent model of Parkinson's disease, a piece of adult mouse adrenal medulla was grafted unilaterally into mouse striatum 1 week after MPTP treatment. This MPTP treatment resulted in the virtual disappearance of tyrosine hydroxylase-immunoreactive fibers and severely depleted striatal dopamine levels. At 2, 4, and 6 weeks after grafting, dense tyrosine hydroxylase-immunoreactive fibers were observed in the grafted striatum, while only sparse fibers were seen in the contralateral striatum. In all cases, tyrosine hydroxylase-immunoreactive fibers appeared to be from the host rather than from the grafts, which survived poorly. These observations suggest that, in mice, adrenal medullary grafts exert a neurotrophic action in the host brain to enhance recovery of dopaminergic neurons. This effect may be relevant to the symptomatic recovery in Parkinson's disease patients who have received adrenal medullary grafts.

Substance P in principal sympathetic neurons: regulation by impulse activity.

Regulation of the putative peptide neurotransmitter substance P was examined in the superior cervical sympathetic ganglion of the neonatal rat. Surgical decentralization (denervation) of the superior cervical ganglion increased ganglion substance P content. In cultured ganglia, the amount of substance P increased more than 50-fold after 48 hours, and this rise was dependent on protein and RNA synthesis. Veratridine prevented the increase in substance P in vitro, and tetrodotoxin blocked the veratridine effect; this suggests that sodium influx and membrane depolarization prevent substance P elevation. Immunohistochemical analysis of cultured ganglia indicated that substance P was present in the perikarya of principal sympathetic neurons and in ganglionic nerve processes. Transsynaptic impulses, through the mediation of postsynaptic sodium influx, may decrease substance P in sympathetic neurons.

Dopaminergic neurons protected from degeneration by GDNF gene therapy.

Glial cell line-derived neurotrophic factor (GDNF) supports growth and survival of dopaminergic (DA) neurons. A replication-defective adenoviral (Ad) vector encoding human GDNF injected near the rat substantia nigra was found to protect DA neurons from the progressive degeneration induced by the neurotoxin 6-hydroxydopamine (6-OHDA) injected into the striatum. Ad GDNF gene therapy reduced loss of DA neurons approximately threefold 6 weeks after 6-OHDA lesion, as compared with no treatment or injection of Ad lacZ or Ad mGDNF (encoding a biologically inactive deletion mutant GDNF). These results suggest that Ad vector-mediated GDNF gene therapy may slow the DA neuronal cell loss in humans with Parkinson's disease.

Selection for quantitative trait loci associated with resistance to Stewart's wilt in sweet corn.

The objectives of this research were to identify quantitative trait loci (QTL) for Stewart's wilt resistance from a mapping population derived from a sweet corn hybrid that is highly resistant to Pantoea stewartii and to determine if marker-based selection for those QTL could substantially improve Stewart's wilt resistance in a population derived from a cross of resistant lines and a highly susceptible sweet corn inbred. Three significant QTL for Stewart's wilt resistance on chromosomes 2 (bin 2.03), 5 (bin 5.03), and 6 (bin 6.06/6.07) explained 31% of the genetic variance in a population of 110 F(3:4) families derived from the sweet corn hybrid Bonus. The three QTL appeared to be additive in their effects on Stewart's wilt ratings. Based on means of families that were either homozygous or heterozygous for marker alleles associated with the resistance QTL, the QTL on chromosomes 2 and 6 appeared to have dominant or partially dominant gene action, while the QTL on chromosome 5 appeared to be recessive. A population of 422 BC(2)S(2) families was derived from crosses of a sweet corn inbred highly susceptible to Stewart's wilt, Green Giant Code 88 (GG88), and plants from two F(3:4) families (12465 and 12467) from the Bonus mapping population that were homozygous for marker alleles associated with Stewart's wilt resistance at the three QTL. Mean Stewart's wilt ratings for BC(2)S(2) families were significantly (P < 0.05) lower for families that were homozygous for the bnlg1902 marker allele (bin 5.03) from resistant lines 12465 or 12467 than for families that were heterozygous at this marker locus or homozygous for the bnlg1902 marker allele from GG88. Resistance associated with this QTL was expressed only if F(3:5) or BC(2)S(2) families were homozygous for marker alleles associated with the resistant inbred parent (P(1)). Marker alleles identified in the F(3:5) mapping population that were in proximity to the resistance QTL on chromosomes 2 and 6 were not polymorphic in crosses of GG88 with 12465 and 12467. Selection for other polymorphic marker loci adjacent to these two regions did not improve Stewart's wilt resistance of BC(2)S(2) families.

Response of Maize Hybrids With and Without Rootworm- and Drought-Tolerance to Rootworm Infestation Under Well-Watered and Drought Conditions.

Anecdotal data in the past have suggested that the effect of the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), on maize yield is greater under drought and the effect of drought is greater under rootworm infestations, but no field experiments have controlled both moisture and rootworm levels. Field studies were conducted in 2012, 2013, and 2014 with treatments in a factorial arrangement of western corn rootworm infestation levels, and maize hybrids (with and without tolerance to drought and rootworm feeding). The experiment was repeated under well-watered and drought conditions in adjacent plots. Leaf water potential and stomatal conductance data suggested significant plant stress was achieved in the drought plots toward the end of the season each year and maize hybrids only played a minor role. In particular, in 2012 and 2013 yield was dramatically lower for the drought experiment than for the well-watered experiment. However, the impacts of rootworm infestation level and maize hybrids on water potential, stomatal conductance, and yield were variable across years and between experiments. In fact, the only year that the main effect of rootworm infestation levels significantly impacted yield was in 2014, when an extremely high infestation level was added and this was only for the well-watered portion of the experiment. Overall, rootworm infestation level played a relatively minor role in maize productivity and it did not appear that soil moisture level influenced that to a large degree.

Buspirone treatment of anxious alcoholics. A placebo-controlled trial.

BACKGROUND: Symptoms of anxiety are common in alcoholics and may contribute to relapse following initiation of abstinence. Buspirone hydrochloride, a serotonin1A partial agonist, has a pharmacologic profile that may be particularly suited to the treatment of anxious alcoholics. METHODS: We conducted a randomized, 12-week, placebo-controlled trial of buspirone in 61 anxious alcoholics, all of whom also received weekly relapse prevention psychotherapy. Outcomes were measured at the end of treatment and at a 6-month follow-up evaluation. RESULTS: Buspirone therapy was associated with greater retention in the 12-week treatment trial, reduced anxiety, a slower return to heavy alcohol consumption, and fewer drinking days during the follow-up period. CONCLUSIONS: Buspirone appears to have a useful role in the treatment of anxious alcoholics. Further research is needed to clarify which patient characteristics and concomitant treatments result in optimal response to buspirone therapy.

Intramuscular grafts of myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclerosis.

Effects of ex vivo GDNF gene delivery on the degeneration of motoneurons were studied in the G1H transgenic mouse model of familial ALS carrying a human superoxide dismutase (SOD1) with a Gly93Ala mutation (Gurney et al., 1994). Retroviral vectors were made to produce human GDNF or E. coli beta-galactosidase (beta-Gal) by transient transfection of the Phoenix cell line and used to infect primary mouse myoblasts. In 6-week-old G1H mice, 50,000 myoblasts per muscle were injected bilaterally into two hindlimb muscles. Untreated G1H and wild-type mice served as additional controls. At 17 weeks of age, 1 week before sacrifice, these muscles were injected with fluorogold (FG) to retrogradely label spinal motoneurons that maintained axonal projections to the muscles. There were significantly more large FG-labeled alpha motoneurons at 18 weeks in GDNF-treated G1H mice than in untreated and beta-Gal-treated G1H mice. A morphometric study of motoneuron size distribution showed that GDNF shifted the size distribution of motoneurons toward larger cells compared with control G1H mice, although the average size and number of large motoneurons in GDNF-treated mice were less than that in wild-type mice. GDNF also prolonged the onset of disease, delayed the deterioration of performance in tests of motor behavior, and slowed muscle atrophy. Quantitative, real-time RT-PCR and PCR showed persistence of transgene mRNA and DNA in muscle for up to 12 weeks postgrafting. These observations demonstrate that ex vivo GDNF gene therapy in a mouse model of FALS promotes the survival of functional motoneurons, suggesting that a similar approach might delay the progression of neurodegeneration in ALS.

Human fetal astrocytes as an ex vivo gene therapy vehicle for delivering biologically active nerve growth factor.

The therapeutic use of neurotrophic factors for neurodegenerative diseases is promising, however, optimal methods for continuous delivery of these substances to the human central nervous system (CNS) remains problematic. One approach would be to graft genetically engineered human cells that continuously secrete high levels of a biologically produced and processed neurotrophic factor. This ex vivo gene therapy approach has worked well in animal models of neurodegenerative diseases using a variety of nonneuronal cell types to deliver the transgene. In our studies, we have been investigating the potential of astrocytes, a cell type normally present in the CNS, as a vehicle for ex vivo gene therapy. Here, we demonstrate that astrocytes in the human fetal cortex can be isolated and efficiently infected with an amphotropic retrovirus harboring mouse beta-nerve growth factor (NGF). These transduced astrocytes express high levels of NGF mRNA and secrete bioactive NGF protein as demonstrated by stimulation of neurite outgrowth from adrenal chromaffin cells. NGF ELISA showed that these astrocytes secrete NGF protein at a rate of 41 ng/day per 10(5) cells after 2 weeks in vitro, whereas NGF is undetectable in medium conditioned by normal astrocytes. These data suggest that human fetal astrocytes can be used for delivering biologically produced neurotrophic factors to the human CNS.

Adenovirus-mediated transgene expression in nonhuman primate brain.

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

Placebo-controlled trial of fluoxetine as an adjunct to relapse prevention in alcoholics.

OBJECTIVE: The authors tested the hypothesis that fluoxetine, when used in combination with relapse prevention psychotherapy, directly reduces relapse frequency and severity for alcoholics. METHOD: The authors conducted a randomized, placebo-controlled trial of fluoxetine (up to a maximum of 60 mg/day) for 12 weeks in combination with weekly psychotherapy for 101 alcohol-dependent subjects who were not selected on the basis of comorbid major depression. Outcomes were measured at the end of treatment and 6 months after treatment. RESULTS: Placebo-treated subjects were more complaint with the medication regimen and remained in the study longer than fluoxetine-treated subjects. There was significantly less alcohol consumption in both groups during treatment than before treatment. These effects persisted during the posttreatment period. Although fluoxetine treatment had no significant effects on alcohol consumption, it reduced Hamilton Depression Rating Scale scores more than placebo treatment among subjects with current major depression. CONCLUSIONS: Fluoxetine at a dose of 60 mg/day is probably not of use for relapse prevention in alcoholics with mild to moderate alcohol dependence and no comorbid depression. In alcoholics with major depression, the drug may reduce depressive symptoms. Subsequent studies with fluoxetine should probably focus on more severely alcohol-dependent subjects or those with comorbid depression.

Glucocorticoid receptor-immunoreactivity in C1, C2, and C3 adrenergic neurons that project to the hypothalamus or to the spinal cord in the rat.

A combined retrograde transport-double immunohistochemical staining method was used to determine the extent to which rat liver glucocorticoid receptor-immunoreactivity (GR-ir) is contained within phenylethanolamine-N-methyltransferase (PNMT)-ir neurons that project to the paraventricular nucleus of the hypothalamus (PVH) or the spinal cord. The results confirmed that cells in the C1, C2, and C3 adrenergic cell groups each contribute to the adrenergic innervation of the PVH, and indicated that the great majority of retrogradely labeled neurons in each group (80% overall) also express GR-ir. Following injections in the upper thoracic segments of the spinal cord, the bulk of adrenergic neurons that were retrogradely labeled were found in the C1 cell group, though 31% of the total number PNMT-ir cells that could be retrogradely labeled following spinal injections were localized in the C2 and C3 regions. Of these spinally projecting PNMT-ir neurons, 62% displayed GR-ir. The results suggest all three medullary adrenergic cell groups contribute projections to the spinal cord and/or the PVH, and that the capacity to express the GR phenotype is a common, though perhaps not universal, attribute of PNMT-ir neurons. No pronounced differences in the expression GR-ir were observed in adrenergic neurons as a function of their location or efferent projections. Brainstem adrenergic neurons may play a role in integrating neuronal and hormonal controls of adrenal function via ascending and descending projections.

Phenylethanolamine N-methyltransferase (PNMT)-expressing horizontal cells in the rat retina: a study employing double-label immunohistochemistry.

Phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the catecholamine biosynthetic pathway that converts norepinephrine to epinephrine, has been detected in the retinas of various vertebrate species. The expression of PNMT has generally been thought to occur in amacrine cells of the ganglion cell and inner nuclear layers. By using immunohistochemical techniques, we have found a population of PNMT- and neurofilament-positive neurons at the border of the inner nuclear layer and the outer plexiform layer in the rat retina. We have classified these cells as horizontal neurons based on their location adjacent to the outer plexiform layer, their morphology, and their expression of vimentin and neurofilaments.

Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat.

Anterograde transport, retrograde transport, and immunohistochemical techniques were used to characterize the organization of neural inputs to the paraventricular (PVH) and supraoptic (SO) nuclei from the C1, C2, and C3 adrenergic cell groups in the rostral medulla. The results are as follows: 1) Phenylethanolamine-N-methyltransferase-immunoreactive (PNMT-IR) fibers and terminals were distributed to all parts of the parvicellular division of the PVH; the dorsal and dorsal medial subdivisions received the most prominent inputs, the lateral and ventral medial parts the least. Sparse terminal fields were found consistently in the magnocellular division of the PVH and in the SO. 2) A combined retrograde transport-immunohistochemical method was used to estimate the number and proportion of cells in the regions of the C1, C2, and C3 cell groups that contribute to the PNMT-IR innervation of the PVH. On average, 232 +/- 37 retrogradely labeled cells in the C1 cell group, 73 +/- 32 in the C2 cell group, and 96 +/- 26 in the C3 group stained positively for PNMT-IR. These values comprised 70%, 84%, and 89%, respectively, of all retrogradely labeled neurons in these regions. 3) Fibers and terminals arising from the regions of each of the three adrenergic cell groups were labeled by local injections of the anterogradely transported plant lectin PHA-L. Each component projection was found to distribute in a very similar fashion and to mimic the overall distribution of PNMT-IR; differential projection patterns within the PVH or SO were not seen consistently following deposits in any of the individual adrenergic cell groups or at different rostrocaudal levels of any individual cell group. 4) A dual anterograde tracing (PHA-L)-immunohistochemical (PNMT) labeling method revealed an appreciable number of varicosities arising from the regions of C1, C2, and C3 cell groups to contain PNMT-IR. These results suggest that adrenergic inputs to the PVH and SO, while arising from distinct medullary cell groups and presumably relaying different types of sensory information, are in a position to influence similar groups of parvicellular neurosecretory and/or autonomic-related projection neurons.