Comparison of the maturation of the adrenergic and serotonergic neurotransmitter systems in the brain: implications for differential drug effects on juveniles and adults.

Our understanding of the development of neurotransmitter systems in the central nervous system has increased greatly over the past three decades and it has become apparent that drug effects on the developing nervous system may differ considerably from effects on the mature nervous system. Recently it has become clear there are significant differences in the effectiveness of antidepressant drug classes in children and adolescents compared to adults. Whereas the selective serotonin reuptake inhibitors are effective in treating all ages from children to adults, the tricyclic antidepressants, many of which inhibit norepinephrine reuptake, have been shown to be ineffective in treating children and adolescents even though they are effective in adults. We review here the development of the noradrenergic and serotonergic nervous systems, both in terms of neurotransmitter system markers and function. Both of these neurotransmitter systems are primary targets of antidepressant medications as well as of central nervous system stimulants. It is clear from a comparison of their development that the serotonin system reaches maturity much earlier than the norepinephrine system. We suggest this may help explain the differences in response to antidepressants in children and adolescents compared to adults. In addition, these differences suggest that drugs acting preferentially on either neurotransmitter system may impact the normal course of CNS development at different time points. Consideration of such differences in the development of neurotransmitter systems may be of significance in optimizing treatments for a variety of centrally mediated disorders.

Analysis of brain adrenergic receptors in dopamine-beta-hydroxylase knockout mice.

The biosynthesis of norepinephrine occurs through a multi-enzymatic pathway that includes the enzyme dopamine-beta-hydroxylase (DBH). Mice with a homozygous deletion of DBH (Dbh-/-) have a selective and complete absence of norepinephrine. The purpose of this study was to assess the expression of alpha-1, alpha-2 and beta adrenergic receptors (alpha1-AR, alpha2-AR and beta-AR) in the postnatal absence of norepinephrine by comparing noradrenergic receptors in Dbh-/- mice with those in Dbh heterozygotes (Dbh+/-), which have normal levels of norepinephrine throughout life. The densities of alpha1-AR, alpha2-AR and beta-AR were assayed with [3H]prazosin, [3H]RX21002 and [125I]-iodo-pindolol autoradiography, respectively. The alpha2-AR agonist high affinity state was examined with [125I]-para-iodoclonidine autoradiography and alpha2-AR functionality by alpha2-AR agonist-stimulated [35S]GTPgammaS autoradiography. The density of alpha1-AR in Dbh-/- mice was similar to Dbh+/- mice in most brain regions, with an up-regulation in the hippocampus. Modest decreases in alpha2-AR were found in septum, hippocampus and amygdala, but these were not reflected in alpha2-AR functionality. The density of beta-AR was up-regulated to varying degrees in many brain regions of Dbh-/- mice compared to the heterozygotes. These findings indicate that regulation of noradrenergic receptors by endogenous norepinephrine depends on receptor type and neuroanatomical region.

Delivery of human acetylcholinesterase by adeno-associated virus to the acetylcholinesterase knockout mouse.

The purpose of this work was to develop a gene delivery system that expressed acetylcholinesterase (AChE) for prolonged periods. An adeno-associated virus (AAV) expressing human AChE was constructed by co-transfecting three plasmids into HEK 293T cells. The purified vector expressed 0.17 microg AChE per 1 million viral particles in culture medium in 23 h, or 0.8 U/ml. The AAV/hAChE was injected into muscle of adult AChE knockout mice and into the brains of 3-6 week old AChE knockout mice. Intramuscular injection yielded plasma AChE levels approaching 50% of the AChE activity of wild-type mouse plasma. The highest AChE activity was found on day 3 post-injection. AChE activity declined thereafter to a constant 7% of normal. The decreased level was accompanied by the appearance of anti-human AChE antibodies, suggesting partial clearance of AChE from plasma by antibodies. Intrastriatal injection resulted in AChE expression in the striatum. No antibodies were detected in animals treated intrastriatally. Motor coordination was improved and the lifespan of intrastriatally-treated AChE knockout mice was prolonged. Human AChE was expressed in mouse brain for up to 7 months after intrastriatal injection of an AAV/hAChE construct. Gene-therapy to supply AChE to the striatum improved motor coordination and prolonged the life of mice genetically deficient in AChE, probably by reducing their susceptibility to spontaneous seizures. This supports the hypothesis that their seizures are induced by excess acetylcholine.

Diminished conditioned responding to the nicotine stimulus by antidepressant drugs with differing specificity for the serotonin and norepinephrine transporter.

People diagnosed with depression also tend to have a co-morbid nicotine addiction. Thus, there is interest in whether medications used to treat depression alter the effects of nicotine. This study assessed whether the antidepressant drugs citalopram, imipramine, and reboxetine, with differing specificity for the serotonin and norepinephrine transporter, altered responding controlled by the conditional stimulus (CS) effects of nicotine. Rats received intermixed 20-min nicotine (0.4 mg base/kg, SC) and saline sessions. On nicotine sessions, rats had intermittent access to sucrose; no sucrose was available on saline sessions. After discrimination performance stabilized and a nicotine generalization curve (0.025-0.4 mg/kg) was established, the antidepressant drugs were assessed. In these tests, rats were pretreated with citalopram (1-17 mg/kg), imipramine (1-17 mg/kg), or reboxetine (1-30 mg/kg) before the training dose of nicotine and placement in a chamber for a 4-min extinction test. At the higher doses, all three antidepressant drugs blocked responding evoked by the nicotine CS and decreased nicotine-induced hyperactivity. When these higher doses of citalopram, imipramine, and reboxetine were tested alone (no nicotine), they decreased chamber activity and/or dipper entries. Nevertheless, all three drugs produced partial or complete blockade of the CS effects of nicotine at doses that produced no effect on dipper entries or chamber entries. This finding suggests that both neurotransmitters play a role in the CS effects of nicotine and that modifications in these systems by antidepressants may be clinically relevant.

Impairment of brain endothelial glucose transporter by methamphetamine causes blood-brain barrier dysfunction.

BACKGROUND: Methamphetamine (METH), an addictive psycho-stimulant drug with euphoric effect is known to cause neurotoxicity due to oxidative stress, dopamine accumulation and glial cell activation. Here we hypothesized that METH-induced interference of glucose uptake and transport at the endothelium can disrupt the energy requirement of the blood-brain barrier (BBB) function and integrity. We undertake this study because there is no report of METH effects on glucose uptake and transport across the blood-brain barrier (BBB) to date. RESULTS: In this study, we demonstrate that METH-induced disruption of glucose uptake by endothelium lead to BBB dysfunction. Our data indicate that a low concentration of METH (20 µM) increased the expression of glucose transporter protein-1 (GLUT1) in primary human brain endothelial cell (hBEC, main component of BBB) without affecting the glucose uptake. A high concentration of 200 µM of METH decreased both the glucose uptake and GLUT1 protein levels in hBEC culture. Transcription process appeared to regulate the changes in METH-induced GLUT1 expression. METH-induced decrease in GLUT1 protein level was associated with reduction in BBB tight junction protein occludin and zonula occludens-1. Functional assessment of the trans-endothelial electrical resistance of the cell monolayers and permeability of dye tracers in animal model validated the pharmacokinetics and molecular findings that inhibition of glucose uptake by GLUT1 inhibitor cytochalasin B (CB) aggravated the METH-induced disruption of the BBB integrity. Application of acetyl-L-carnitine suppressed the effects of METH on glucose uptake and BBB function. CONCLUSION: Our findings suggest that impairment of GLUT1 at the brain endothelium by METH may contribute to energy-associated disruption of tight junction assembly and loss of BBB integrity.

Immunoregulation of a CB2 receptor agonist in a murine model of neuroAIDS.

Chronic HIV-1 infection commonly affects behavioral, cognitive, and motor functions in the infected human host and is commonly referred to as HIV-1-associated neurocognitive disorders (HAND). This occurs, in measure, as a consequence of ingress of leukocytes into brain perivascular regions. Such cells facilitate viral infection and disease by eliciting blood-brain barrier and neuronal network dysfunctions. Previous works demonstrated that the endocannabinoid system modulates neuroimmunity and as such neuronal and glial functions. Herein, we investigated CB2R receptor expression in murine HIV-1 encephalitis (HIVE) and the abilities of a highly selective CB2R agonist, Gp1a, to modulate disease. HIV-1-infected human monocyte-derived macrophages were injected into the caudate and putamen of immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL/HIVE). Brains of hu-PBL/HIVE mice showed microglial activation and increased expression of CB2R, but not CB1R or GPR55. Gp1a substantively reduced infiltration of human cells into the mouse brain and reduced HLA DQ activation. Gp1a down modulated CCR5 expression on human cells in the spleen with an increase in Fas ligand expression. Our results support the notion that CB2 receptor agonists may be a viable therapeutic candidate for HAND.

Mitochondrial fragmentation is involved in methamphetamine-induced cell death in rat hippocampal neural progenitor cells.

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca2+) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH.

Regulation of mu opioid receptor internalization by the scaffold protein RanBPM.

Mu opioid receptors (MOP) are transducers of the pharmacological effects of many opioid drugs, including analgesia and tolerance/dependence. Previously, we observed increased MOP signaling during postnatal development that was not associated with increased MOP or G protein expression. A yeast two-hybrid screen of a human brain cDNA library using the MOP C-terminus as bait identified RanBPM as a potential MOP-interacting protein. RanBPM has been recognized as a multi-functional scaffold protein that interacts with a variety of signaling receptors/proteins. Co-immunoprecipitation studies in HEK293 cells indicated that RanBPM constitutively associates with MOP. Functionally, RanBPM had no effect on MOP-mediated inhibition of adenylyl cyclase, yet reduced agonist-induced endocytosis of MOP. Mechanistically, RanBPM interfered with beta arrestin2-GFP translocation stimulated by MOP but not alpha(1B)-adrenergic receptor activation, indicating selectivity of action. Our findings suggest that RanBPM is a novel MOP-interacting protein that negatively regulates receptor internalization without altering MOP signaling through adenylyl cyclase.

RanBPM, a scaffolding protein in the immune and nervous systems.

We review the literature for Ran Binding Protein in the Microtubule-Organizing Center (RanBPM; RanBP9), a 90-kDa protein that possesses many characteristics of a scaffolding protein, including protein-interaction motifs, a cytoskeletal-binding domain, and multiple canonical docking sites for signaling intermediates. We focus on studies that have examined functional interactions between RanBPM and other proteins. These studies suggest that RanBPM provides a platform for the interaction of a variety of signaling proteins, including cell surface receptors, nuclear receptors, nuclear transcription factors, and cytosolic kinases. These studies indicate that RanBPM acts as a scaffolding protein and is important in regulating cellular function in both the immune system and the nervous system.

Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase.

Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.

A transient expression of functional alpha2-adrenergic receptors in white matter of the developing brain.

Norepinephrine is a neurotransmitter with peripheral and central actions mediated by alpha-1, alpha-2, and beta-adrenergic receptors. In this paper, we report an expression of alpha-2 adrenergic receptors in developing white matter tracts as revealed by [(3)H]RX821002 autoradiography. In rats, these receptors are present in the corpus callosum and anterior commissure at gestational day 20. Quantification of their postnatal expression reveals peak expression in the corpus callosum at postnatal day 1, which decreases with maturation and disappears by postnatal day 21. Expression in the anterior commissure is persistently elevated throughout the first ten days of postnatal development and then decreases to near background levels by postnatal day 21. Further characterization of the receptors by agonist-stimulated [(35)S]GTPgammaS binding verifies alpha-2 adrenergic receptors are functionally coupled to G proteins early in development and therefore are mature receptors. In situ hybridization did not detect mRNA for any of the alpha-2 adrenergic receptor subtypes (A, B, and C) in white matter tracts of postnatal day 5 brain. [(3)H]RX821002 emulsion autoradiography demonstrated autoradiographic grains that were of comparable density between cells and over cell bodies. Collectively, these data suggest that alpha-2 adrenergic receptors in neonatal commissures are synthesized at sites distant from their white matter expression and may be guiding the maturation of these brain commissures.

Mu opioid receptor coupling to Gi/o proteins increases during postnatal development in rat brain.

Mu opioid receptors are densely expressed within rat striatum and are concentrated in anatomically discrete patches called striosomes. The density of striosomal mu receptors remains relatively constant during postnatal development, but little is known about their functional maturation. We examined the extent of G protein coupling by mu opioid receptors in rat brain during development, focusing on striosomes within the striatum because of receptor density. The mu receptors were quantified using [(3)H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) autoradiography. Adjacent sections were analyzed for DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding to assess mu receptor activation of G(i/o) proteins. Striosomal mu receptor expression increased only slightly between postnatal day 5 and adult. In contrast, mu receptor-stimulated [(35)S]GTPgammaS binding increased from 0.13 to 2.6 fmol/mg tissue over the same period, a 20-fold difference. The ratio of specific DAMGO-stimulated [(35)S]GTPgammaS binding to [(3)H]DAMGO binding, representing the relative number of G proteins activated per receptor, increased 19-fold between postnatal day 5 and adult. Similar patterns were observed throughout the striatum and other brain regions such as the nucleus accumbens, although the extent of change varied from region to region. These data indicate that mu opioid receptors exhibit enhanced function in the adult rat brain compared with the neonate. These data also suggest that this increase in G protein coupling is developmentally regulated and that in the developing rat brain the density of mu opioid receptor expression may not necessarily correlate with receptor activation of G proteins.