Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications.

In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast-like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in-growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre-clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586-1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Striatal dopamine nerve terminal markers in human, chronic methamphetamine users.

Methamphetamine is a drug that is significantly abused worldwide, Although long-lasting depletion of dopamine and other dopamine nerve terminal markers has been reported in striatum of nonhuman primates receiving very high doses of the psychostimulant, no information is available for humans. We found reduced levels of three dopamine nerve terminal markers (dopamine, tyrosine hydroxylase and the dopamine transporter) in post-mortem striatum (nucleus accumbens, caudate, putamen) of chronic methamphetamine users. However, levels of DOPA decarboxylase and the vesicular monoamine transporter, known to be reduced in Parkinson's disease, were normal. This suggests that chronic exposure to methamphetamine does not cause permanent degeneration of striatal dopamine nerve terminals at the doses used by the young subjects in our study. However, the dopamine reduction might explain some of the dysphoric effects of the drug, whereas the decreased dopamine transporter could provide the basis for dose escalation occurring in some methamphetamine users.

Development of a 3D human in vitro skin co-culture model for detecting irritants in real-time.

Tissue engineered materials for clinical purposes have led to the development of in vitro models as alternatives to animal testing. The aim of this study was to understand the paracrine interactions arising between keratinocytes and fibroblasts for detecting and discriminating between an irritant-induced inflammatory reaction and cytotoxicity. We used two irritants [sodium dodecyl sulphate (SDS) and potassium diformate (Formi] at sub-toxic concentrations and studied interleukin-1 alpha (IL-1 alpha) release from human keratinocytes and activation of NF-kappaB in human fibroblasts. NF-kappaB activation in fibroblast 2D cultures required soluble factors released by prior incubation of keratinocytes with either SDS or Formi. Neither cell type responded directly to either agent, confirming a paracrine mechanism. Fibroblasts were then cultured in 3D microfiber scaffolds and transfected with an NF-kappaB reporter construct linked to GFP. Findings for 3D cultures were similar to those in 2D in that soluble factors released by prior incubation of keratinocytes with SDS or Formi was required for NF-kappaB activation in fibroblasts. Similarly, direct incubation with either agent did not directly activate NF-kappaB. A technical advantage of using transfected cells in 3D was an ability to detect NF-kappaB activation in live fibroblasts. To confirm paracrine signaling a twofold increase in IL-1 alpha was measured in keratinocyte-conditioned medium after incubation with SDS or Formi, which correlated with fibroblast NF-kappaB activity. In summary, this work has value for developing 3D tissue engineered co-culture models for the in vitro testing of irritant chemicals at sub-toxic concentrations, as an alternative to in vivo models.

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering.

Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.

Retrograde amnesia and the "reminder effect": an alternative interpretation.

Recent findings suggest that amnesic agents block the retrieval of stored information. "Reminder" treatments, such as noncontingent punishments given after the production of amnesia for avoidance learning, improve the later retention performance of an animal. The data reported suggest that noncontingent treatments provide an additional learning experience which adds to the retention performance of partially amnesic or poorly trained animals.

Regionally-selective cell colonization of micropatterned surfaces prepared by plasma polymerization of acrylic acid and 1,7-octadiene.

Micropatterned surfaces have been used as a tool for controlling the extent and strength of cell adhesion, the direction of cell growth and the spatial distribution of cells. In this study, chemically micropatterned surfaces were prepared by successive plasma polymerization of acrylic acid (AA) and 1,7-octadiene (OD) through a mask. Rat vascular smooth muscle cells (VSMC), bovine endothelial cells (EC), porcine mesenchymal stem cells (MSC) or human skeletal muscle cells (HSKMC) were seeded on these surfaces in densities from 9,320 cells/cm(2) to 31,060 cells/cm(2). All cell types adhered and grew preferentially on the strip-like AA domains. Between day 1 and 7 after seeding, the percentage of cells on AA domains ranged from 84.5 to 63.3 % for VSMC, 85.3 to 73.5 % for EC, 98.0 to 90.0 % for MSC, and 93.6 to 55.0 % for HSKMC. The enzyme-linked immunosorbent assay (ELISA) revealed that the concentration of alpha-actin per mg of protein was significantly higher in VSMC on AA. Similarly, immunofluorescence staining of von Willebrand factor showed more apparent Weibel-Palade bodies in EC on AA domains. MSC growing on AA had better developed beta-actin cytoskeleton, although they were less stained for hyaluronan receptor (CD44). In accordance with this, MSC on AA contained a higher concentration of beta-actin, although the concentration of CD44 was lower. HSKMC growing on AA had a better developed alpha-actin cytoskeleton. These results based on four cell types suggest that plasma polymerization is a suitable method for producing spatially defined patterned surfaces for controlled cell adhesion, proliferation and maturation.

Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells.

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.

Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity.

alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

Photochemically modified diamond-like carbon surfaces for neural interfaces.

Diamond-like carbon (DLC) was modified using a UV functionalization method to introduce surface-bound amine and aldehyde groups. The functionalization process rendered the DLC more hydrophilic and significantly increased the viability of neurons seeded to the surface. The amine functionalized DLC promoted adhesion of neurons and fostered neurite outgrowth to a degree indistinguishable from positive control substrates (glass coated with poly-L-lysine). The aldehyde-functionalized surfaces performed comparably to the amine functionalized surfaces and both additionally supported the adhesion and growth of primary rat Schwann cells. DLC has many properties that are desirable in biomaterials. With the UV functionalization method demonstrated here it may be possible to harness these properties for the development of implantable devices to interface with the nervous system.

Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size.

While the transporters that accumulate classical neurotransmitters in synaptic vesicles have been identified, little is known about how their expression regulates synaptic transmission. We have used adenoviral-mediated transfection to increase expression of the brain vesicular monoamine transporter VMAT2 and presynaptic amperometric recordings to characterize the effects on quantal release. In presynaptic axonal varicosities of ventral midbrain neurons in postnatal culture, VMAT2 overexpression in small synaptic vesicles increased both quantal size and frequency, consistent with the recruitment of synaptic vesicles that do not normally release dopamine. This was confirmed using noncatecholaminergic AtT-20 cells, in which VMAT2 expression induced the quantal release of dopamine. The ability to increase quantal size in vesicles that were already competent for dopamine release was shown in PC12 cells, in which VMAT2 expression increased the quantal size but not the number of release events. These results demonstrate that vesicle transporters limit the rate of transmitter accumulation and can alter synaptic strength through two distinct mechanisms.

Alpha-melanocyte-stimulating hormone inhibits NF-kappaB activation in human melanocytes and melanoma cells.

Alpha-melanocyte-stimulating hormone is produced by several different cell types including neural cells, endothelial cells, monocytes, and keratinocytes. A biologic role in melanocyte pigmentation is widely recognized, but more recent studies describe a part in modulating inflammatory and immune responses. The aim of the this study was to investigate the mechanism by which alpha-melanocyte-stimulating hormone antagonizes proinflammatory cytokine action. We report that alpha-melanocyte-stimulating hormone (10-9 M) was effective in opposing a tumor necrosis factor-alpha stimulated increase in NF-kappaB DNA binding activity in: (i) normal ocular melanocytes; (ii) cells cultured from ocular melanoma tumors; and (iii) two cutaneous melanoma cell lines. NF-kappaB is activated by many inflammatory mediators and controls transcription of genes required for immune and inflammatory responses. The transcription factor complex was positively identified as the p50/p65 heterodimer, recognized to have transcriptional activating potential. Maximum reduction of NF-kappaB DNA binding activity with alpha-melanocyte-stimulating hormone was detected 2 h after cellular stimulation and varied from between 53% and 18% depending on cell type. Whereas the acute inhibitory effects could be mimicked by elevating cyclic adenosine monophosphate, alpha-melanocyte-stimulating hormone was not found to have any effect on the relative level of IkappaBalpha protein expression over 24 h. These data show that alpha-melanocyte-stimulating hormone has a pronounced effect on NF-kappaB activity in melanocytes and melanoma cells, identifying a specific dimeric complex, and suggest this to be a key pathway by which immunomodulation/anti-inflammation may operate. The results may also be considered in the broader context of general inflammatory pathologies concerning cells which express alpha-melanocyte-stimulating hormone receptors and utilize the NF-kappaB signaling pathway.

Elevated levels of tyrosine hydroxylase in the locus coeruleus in major depression.

BACKGROUND: Levels of tyrosine hydroxylase (TH) are regulated in the noradrenergic locus coeruleus (LC) in response to changes in the activity of LC neurons and in response to changes in brain levels of norepinephrine. To study the potential role of central noradrenergic neurons in the pathobiology of major depression, TH protein was measured in the LC from postmortem brains of 13 subjects with a diagnosis of major depression and 13 age-matched control subjects having no Axis I psychiatric diagnosis. Most of the major depressive subjects died as a result of suicide. METHODS: Protein from sections cut through multiple rostro-caudal levels of LC was transferred to Immobilon-P membrane, immunoblotted for TH, and quantified autoradiographically. RESULTS: The distribution of TH-immunoreactivity (TH-ir) along the rostro-caudal axis of the LC was uneven and was paralleled by a similar uneven distribution of neuromelanin-containing cells in both major depressive and psychiatrically normal control subjects. Amounts of TH-ir in the rostral, middle and caudal levels of the LC from major depressive subjects were significantly higher than that of matched control subjects. There were no significant differences in the number of noradrenergic cells at any particular level of the LC comparing major depressive subjects to control subjects. CONCLUSIONS: Elevated expression of TH in the LC in major depression implies a premortem overactivity of these neurons, or a deficiency of the cognate transmitter, norepinephrine.

Evidence for coexistence of GABA and dopamine in neurons of the rat olfactory bulb.

Immunoreactivities for gamma-aminobutyric acid (GABA) and the dopamine-synthesizing enzyme tyrosine hydroxylase (TH) were localized ultrastructurally and colocalized at the light microscopic level in neurons of the rat main olfactory bulb. By means of a simultaneous indirect immunofluorescence technique, GABA and TH immunoreactivities were found to coexist in a large number of neurons in the glomerular and external plexiform layers. Virtually all the TH-immunoreactive periglomerular neurons also contained GABA immunoreactivity (GABA-I) while there was an additional number of GABA-immunoreactive periglomerular cells (27%) which did not contain TH immunoreactivity (TH-I). In contrast, the numerous tufted-type neurons in the glomerular and superficial external plexiform layers which contained TH-I did not contain GABA-I. In the external plexiform layer (EPL), 41% of the immunoreactive neurons contained GABA-I alone, 24% contained TH-I alone, and 35% contained both. EPL neurons containing GABA-I only or both GABA-I and TH-I never exhibited tufted cell morphological characteristics and were generally of the short-axon type. Electron microscopic examination of GABA-I and TH-I elements in the glomerular layer detected morphologically similar periglomerular perikarya and intraglomerular processes immunoreactive for each substance and other neurons and processes of the same type containing neither GABA-I or TH-I. These data indicate that the classical neurotransmitters GABA and dopamine coexist in large numbers of neurons in the rat main olfactory bulb including characteristic periglomerular cells and certain other local-circuit neuronal types.

Ventral mesencephalic neurons containing both cholecystokinin- and tyrosine hydroxylase-like immunoreactivities project to forebrain regions.

The coexistence of cholecystokinin- and tyrosine hydroxylase-like immunoreactivities within neurons of the rat ventral mesencephalon was analyzed by using an indirect immunofluorescence technique for the simultaneous demonstration of two antigens in the same tissue section. A high degree of colocalization was observed in the substantia nigra pars compacta, in which 80-90% of all labeled neurons at rostral and up to 70% at intermediate levels contained both cholecystokinin and tyrosine hydroxylase. At caudal levels, the incidence of colocalization declined to approximately 30-50%. All of the immunoreactive perikarya in the substantia nigra pars lateralis were labeled with both substances. Other areas of the ventral midbrain that exhibited a moderate proportion of neurons immunoreactive for both cholecystokinin and tyrosine hydroxylase included the ventral tegmental area, interfascicular nucleus, and rostral and caudal linear nuclei. In addition, coexistence was occasionally observed within neurons of the central and ventral periaqueductal gray matter, supramammillary region, peripeduncular region, retrorubral field, and extremely rarely, within the substantia nigra pars reticulata. Cell bodies containing tyrosine hydroxylase-like immunoreactivity (indicative of dopamine) usually outnumbered those containing the peptide except in the supramammillary region and in the ventral periaqueductal gray matter, where the cholecystokinin perikarya were present in higher numbers. The double-labeling colocalization technique was combined with fluorescence retrograde tracing to determine some of the forebrain projections of these neurons. Ventral midbrain neurons containing both cholecystokinin and tyrosine hydroxylase were found to project to the caudate-putamen, nucleus-accumbens, prefrontal cortex, and amygdala. These projections originated from neurons located predominantly in the substantia nigra pars compacta and the ventral tegmental area. Thus, cholecystokinin occurs within the well-known dopaminergic nigrostriatal pathway in the rat. Overall, these results demonstrate that a significant proportion of the dopamine neurons giving rise to the ascending mesotelencephalic projections also contain the peptide cholecystokinin.

Age-correlated changes in dopaminergic nigrostriatal perikarya of the C57BL/6NNia mouse.

Alterations in neurotransmitter systems of the basal ganglia have been postulated to contribute to the disruption of motor function and balance associated with aging. This study examined nigrostriatal (A9) and mesolimbic (A10) dopamine neurons for qualitative age-correlated changes using fluorescence histochemistry for catecholamines and immunocytochemical techniques for the catecholamine-synthesizing enzyme, tyrosine hydroxylase. Results from this study suggest that age-correlated morphological changes in A9 but not all A10 neurons in the midbrain are present in mature adult (10-month) C57BL/6NNia mice and show a progressive increase in severity until at least 30 months of age. These changes are characterized by a progressive accumulation of lipofuscin in dopamine-containing perikarya, a markedly reduced dopamine content per cell as determined visually by histofluorescence, and an increase in the number of large, fluorescent axonal dilations in dopamine-containing fibers of the mesolimbic and nigrostriatal systems. These data suggest that heterogeneous morphological aging patterns exist within dopamine-containing neurons of the midbrain and that based upon their terminal projection sites, various regions of the striatum and cortex may be differentially affected in the aged brain. In addition, these findings support the belief that age-related changes in neural structure are not generalized to an entire brain nucleus or cell type but are selective for individual cells within an affected area.

Striatal biopterin and tyrosine hydroxylase protein reduction in dopa-responsive dystonia.

OBJECTIVE: To determine the mechanism leading to striatal dopamine (DA) loss in dopa-responsive dystonia (DRD). BACKGROUND: Although mutations in the gene GCH1, coding for the tetrahydrobiopterin (BH4) biosynthetic enzyme guanosine triphosphate-cyclohydrolase I, have been identified in some patients with DRD, the actual status of brain BH4 (the cofactor for tyrosine hydroxylase [TH]) is unknown. METHODS: The authors sequenced GCH1 and measured levels of total biopterin (BP) and total neopterin (NP), TH, and dopa decarboxylase (DDC) proteins, and the DA and vesicular monoamine transporters (DAT, VMAT2) in autopsied brain of two patients with typical DRD. RESULTS: Patient 1 had two GCH1 mutations but Patient 2 had no mutation in the coding region of this gene. Striatal BP levels were markedly reduced (<20% of control subjects) in both patients and were also low in two conditions characterized by degeneration of nigrostriatal DA neurons (PD and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated primate), whereas brain NP concentrations were selectively decreased (<45%) in the DRD patients. In the putamen, both DRD patients had severely reduced (<3%) TH protein levels but had normal concentrations of DDC protein, DAT, and VMAT2. CONCLUSIONS: The data suggest that 1) brain BH4 is decreased substantially in dopa-responsive dystonia, 2) dopa-responsive dystonia can be distinguished from degenerative nigrostriatal dopamine deficiency disorders by the presence of reduced brain neopterin, and 3) the striatal dopamine reduction in dopa-responsive dystonia is caused by decreased TH activity due to low cofactor concentration and to actual loss of TH protein. This reduction of TH protein, which might be explained by reduced enzyme stability/expression consequent to congenital BH4 deficiency, can be expected to limit the efficacy of acute BH4 administration on dopamine biosynthesis in dopa-responsive dystonia.

Striatal dopaminergic and serotonergic markers in human heroin users.

To establish whether chronic opiate exposure might impair brain dopaminergic or serotonergic function in humans, we assessed biochemical indices of monoaminergic neurotransmitter activity and integrity in post mortem striatum of nine chronic heroin users and 14 control subjects. Striatal levels of the vesicular monoamine transporter were normal, suggesting that the density of dopamine nerve terminals is not reduced in heroin users. In nucleus accumbens, levels of tyrosine hydroxylase protein (-25%) and those of the dopamine metabolite homovanillic acid (-33%) were reduced significantly together with a trend for decreased dopamine (-32%) concentration. These changes could reflect either a compensatory downregulation of dopamine biosynthesis in response to prolonged dopaminergic stimulation caused by heroin, or reduced axoplasmic transport of tyrosine hydroxylase. Striatal levels of serotonin were either normal or elevated whereas concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid were decreased by 27-38%. Our data suggest that chronic heroin exposure might produce a modest reduction in dopaminergic and serotonergic activity that could affect motivational state and impulse control, respectively.

Four isoforms of tyrosine hydroxylase are expressed in human brain.

In contrast to nonprimate species, the RNA for human tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, can undergo alternative splicing to produce four different types of mRNA. Although types 1 and 2 of these human tyrosine hydroxylase mRNAs have been identified in human brain, whether types 3 and 4 human tyrosine hydroxylase mRNAs are present in the central nervous system remains controversial. Furthermore, little is known about the expression of the protein products of these mRNAs in human brain. In this study we used antibodies raised against different octapeptide sequences from each of the predicted human tyrosine hydroxylase protein forms to determine the presence and distribution of each human tyrosine hydroxylase isoforms in several regions of human brain. Control immunocytochemical and blot immunolabeling experiments demonstrated that each antibody selectively recognized the human tyrosine hydroxylase isoform against which it was directed. In immunocytochemical studies, all four human tyrosine hydroxylase isoforms were clearly detectable in neurons of both the substantia nigra and locus coeruleus. The presence of all four isoforms in these nuclei was confirmed with blot immunolabeling studies. Single-label immunocytochemical studies of adjacent sections as well as dual-label comparisons of immunoreactivity for human tyrosine hydroxylase type 1 with type 2, type 3, or type 4 suggested that at least some neurons in these brain regions contain all four human tyrosine hydroxylase isoforms. In contrast, some neurons of the mesencephalon appeared to be selectively immunoreactive with the antibodies against type 1. In the caudate nucleus and putamen, the terminal zones of the dopaminergic projection from the substantia nigra, all four isoforms were detected, although in immunocytochemical studies type 1 appeared to be the predominant isoform present in axons and terminals. These findings demonstrate that human brain contains four distinct isoforms of human tyrosine hydroxylase and that the presence or relative amount of each isoform may differ among catecholaminergic cell populations and between catecholaminergic neurons and terminal fields. These patterns of expression may have important implications for understanding the regulation of catecholamine biosynthesis in human brain both in normal and pathological states.

Immunohistochemical studies on phosphorylation of tyrosine hydroxylase in central catecholamine neurons using site- and phosphorylation state-specific antibodies.

Antibodies raised to phosphorylated forms of tyrosine hydroxylase, the first and rate-limiting enzyme in the catecholamine biosynthesis, were applied in immunohistochemical studies on rat brain slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and on forskolin on formalin-perfused rat brains. Four antisera/antibodies were used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase. In the in vitro studies THS40p-like immunoreactivity was not observed unless slices were treated with IBMX-forskolin after which a dense fibre network was found in the striatum, and immunoreactive cell bodies were found in the ventral mesencephalon, especially in the ventral tegmental area. Although these cells were pan-tyrosine hydroxylase-positive, several of them were not stained with the tyrosine hydroxylase-monoclonal antibody. Moreover, there was a marked reduction of tyrosine hydroxylase-monoclonal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody does not recognize the serine 40-phosphorylated form of tyrosine hydroxylase. Treated slices did not show any THS40p-immunoreactive cell bodies in the dopaminergic A11 cell group and only a few, weakly fluorescent neurons were observed in locus coeruleus. However, a sparse fibre plexus was observed in locus coeruleus, possibly reflecting epinephrine fibres. In the perfused brains THS40p-like immunoreactivity could be visualized in some dopamine neurons in the ventral mesencephalon, especially the A10 area, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was found in all catecholamine groups studied, similar to the results obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody. In forebrain areas known to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres were observed, whereas THS19p-immunoreactive fibres were found in subregions of the striatum, olfactory tubercle and nucleus accumbens, essentially overlapping with dopamine fibres previously shown to contain cholecystokinin-like immunoreactivity. The present results suggests that antibodies directed against phosphorylated forms of tyrosine hydroxylase can be used to evaluate the state of tyrosine hydroxylase phosphorylation in individual neuronal cell bodies and processes both in vitro and in vivo.

On the functional coupling of neurotransmitter uptake and release in brain.

1 Isolated synaptosomal fractions from mouse forebrains were incubated [14C]-gamma-aminobutyric acid ([14C]-GABA). Release of the accumulated label in high potassium solution was measured. 2 The fractional release dependent upon calcium was decreased by raising the concentration of [14C]-GABA during labeling but was not affected by altering the time allowed for labelling or the time between labelling and stimulation. 3 These data suggest that extracellular GABA gains rapid access to available intraterminal pools. The relative distribution of the accumulated GABA in differerent pools can be influenced by the concentration of GABA in the incubation medium but, once (stored", there is no net redistribution of accumulated GABA in the absence of stimulation.