JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton.

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer's disease.

Distinct populations of cortical pyramidal neurons mediate drug reward and aversion.

Processing within the anterior cingulate cortex (ACC) is crucial for the patterning of appropriate behavior, and ACC dysfunction following chronic drug use is thought to play a major role in drug addiction. However, cortical pyramidal projection neurons can be subdivided into two major types (intratelencephalic (IT) and pyramidal tract (PT)), with distinct inputs and projection targets, molecular and receptor profiles, morphologies and electrophysiological properties. Yet, how each of these cell populations modulate behavior related to addiction is unknown. We demonstrate that PT neurons regulate the positive features of a drug experience whereas IT neurons regulate the negative features. These findings support a revised theory of cortical function in addiction, with distinct cells and circuits mediating reward and aversion.

Intermittent but not continuous access to cocaine produces individual variability in addiction susceptibility in rats.

Drug addiction is a chronic disease defined by a complex set of characteristics, including loss of control over drug intake and persistent drug craving, which primarily affects a small percentage of people who try drugs. Although many models have been developed to study individual aspects of drug use, there is great translational value in having an animal model that encompasses multiple aspects of the human disease, including the variation in severity observed in humans. Here, we describe an intermittent access model of cocaine self-administration that produces a subset of rats that display many of the core features of addiction, including escalation of drug intake, a binge-like pattern of drug use, robust locomotor sensitization, and high levels of drug-seeking during cue-induced reinstatement. This group is compared with rats that have the same drug history but do not develop this pattern of drug-taking and drug-seeking, as well as rats that undergo a traditional continuous access paradigm. Finally, we observe that high levels of cocaine consumption produce long-term changes in intracellular calcium signaling in the dorsomedial striatum.

The ins and outs of the striatum: role in drug addiction.

Addiction is a chronic relapsing disorder characterized by the loss of control over drug intake, high motivation to obtain the drug, and a persistent craving for the drug. Accumulating evidence implicates cellular and molecular alterations within cortico-basal ganglia-thalamic circuitry in the development and persistence of this disease. The striatum is a heterogeneous structure that sits at the interface of this circuit, receiving input from a variety of brain regions (e.g., prefrontal cortex, ventral tegmental area) to guide behavioral output, including motor planning, decision-making, motivation and reward. However, the vast interconnectivity of this circuit has made it difficult to isolate how individual projections and cellular subtypes within this circuit modulate each of the facets of addiction. Here, we review the use of new technologies, including optogenetics and DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), in unraveling the role of the striatum in addiction. In particular, we focus on the role of striatal cell populations (i.e., direct and indirect pathway medium spiny neurons) and striatal dopaminergic and glutamatergic afferents in addiction-related plasticity and behaviors.

Motor neuron-specific overexpression of the presynaptic choline transporter: impact on motor endurance and evoked muscle activity.

The presynaptic, hemicholinium-3 sensitive, high-affinity choline transporter (CHT) supplies choline for acetylcholine (ACh) synthesis. In mice, a homozygous deletion of CHT (CHT-/-) leads to premature cessation of spontaneous or evoked neuromuscular signaling and is associated with perinatal cyanosis and lethality within 1 h. Heterozygous (CHT+/-) mice exhibit diminished brain ACh levels and demonstrate an inability to sustain vigorous motor activity. We sought to explore the contribution of CHT gene dosage to motor function in greater detail using transgenic mice where CHT is expressed under control of the motor neuron promoter Hb9 (Hb9:CHT). On a CHT-/- background, the Hb9:CHT transgene conferred mice with the ability to move and breath for a postnatal period of ∼24 h, thus increasing survival. Conversely, Hb9:CHT expression on a wild-type background (CHT+/+;Hb9:CHT) leads to an increased capacity for treadmill running compared to wild-type littermates. Analysis of the stimulated compound muscle action potential (CMAP) in these animals under basal conditions established that CHT+/+;Hb9:CHT mice display an unexpected, bidirectional change, producing either elevated or reduced CMAP amplitude, relative to CHT+/+ animals. To examine whether these two groups arise from underlying changes in synaptic properties, we used high-frequency stimulation of motor axons to assess CMAP recovery kinetics. Although CHT+/+; Hb9:CHT mice in the two groups display an equivalent, time-dependent reduction in CMAP amplitude, animals with a higher basal CMAP amplitude demonstrate a significantly enhanced rate of recovery. To explain our findings, we propose a model whereby CHT support for neuromuscular signaling involves contributions to ACh synthesis as well as cholinergic synaptic vesicle availability.

Dysarthria and dysphagia due to the opercular syndrome in multiple sclerosis.

We report a patient with relapsing-remitting multiple sclerosis (RRMS), who developed bilateral severe tongue weakness due to the anterior opercular syndrome. This was caused by a recent inflammatory demyelinating lesion in the right perisylvian juxtacortical region, superimposed on a pre-existing left perisylvian lesion, which had previously caused temporary isolated right tongue weakness.

K-shell photodetachment of Li(-): experiment and theory.

An experimental K-shell photodetachment study of Li(-) giving rise to doubly photoionized Li(+) ions has been carried out at the Advanced Light Source, using a collinear photon-ion beam apparatus. The experiment reveals dramatic structure, differing substantially both qualitatively and quantitatively from the corresponding processes above the 1s ionization threshold in Li and Li(+), as predicted by our enhanced R-matrix calculation. The experimental/theoretical comparison shows good agreement over some of the photon energy range, and also reveals some puzzling discrepancies.

Molecular cloning and characterization of a murine hemicholinium-3-sensitive choline transporter.

In cholinergic neurons, a specific requirement for precursor choline in the biosynthesis of acetylcholine (ACh) is thought to be sustained by a presynaptic, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). This transporter exhibits micromolar affinity for choline and transport activity is Na(+)- and Cl(-)-dependent. Based on the sequence information available with the recent cloning of rat and human CHTs [Okuda, Haga, Kanai, Endou, Ishihara and Katsura (2000) Nat. Neurosci. 3, 120-125; Apparsundaram, Ferguson, George Jr and Blakely (2000) Biochem. Biophys. Res. Commun. 276, 862-867; Okuda and Haga (2000) FEBS Lett. 484, 92-97], we have identified a murine CHT orthologue (mCHT) by reverse transcriptase-PCR of spinal cord mRNA and confirmed this sequence using assembled mouse genomic DNA. Inferred splice junctions for mCHT exons are conserved with those of hCHT. The mCHT cDNA encodes a protein of 580 amino acids with 93% and 98% amino acid identity to human CHT and rat CHT1, respectively. Hydropathy analysis of the predicted amino acid sequence of mCHT indicates a protein containing 13 transmembrane domains (TMDs), with the N-terminus oriented extracellularly and the C-terminus oriented intracellularly. Northern blot analysis of mouse tissues reveals the expression of mCHT as a single transcript of approximately 5 kb with highest expression in regions that are rich in cholinergic cell bodies, e.g. the spinal cord, brainstem, mid-brain and striatum, whereas hybridization signals are absent in regions lacking cholinergic soma, e.g. the cerebellum and kidney. Expression of mCHT in COS-7 cells results in high-affinity [(3)H]HC-3-binding sites (K(d)=5 nM), and Na(+)- and Cl(-)-dependent HC-3-sensitive choline uptake (K(m)=2 microM), assessed in resealed membrane vesicles. The availability of cloned, functional mCHT and its cognate genomic DNA should prove useful for studies of mCHT regulation and should open possibilities for evaluation of CHT dysfunction in murine models.

Antidepressant-like effects of the subtype-selective nicotinic acetylcholine receptor agonist, SIB-1508Y, in the learned helplessness rat model of depression.

RATIONALE: Epidemiological studies of smokers suggest that there is a link between nicotine and depression. Nonetheless, few studies have examined the potential use of nicotinic ligands in the treatment of depression. OBJECTIVES: The goal of this study was to evaluate the effects of SIB-1508Y, a novel subtype-selective ligand for high affinity nicotinic acetylcholine receptors (nAChRs), in the learned helplessness model of depression in rats. METHODS: In this model, exposure to inescapable foot-shock produces a lasting deficit in escape responses emitted in a subsequent conditioned avoidance procedure (learned helplessness). The effect of SIB-1508Y on learned helplessness was compared to the clinically used antidepressants, imipramine and fluoxetine, and the non-selective nAChR ligand, nicotine. RESULTS: Similarly to imipramine and fluoxetine, subchronic treatment (5 days) with SIB-1508Y reversed the escape deficit in the learned helplessness model in a dose dependent manner. The effect of SIB-1508Y on learned helplessness was still apparent 1 week following drug administration and was also maintained after 4 weeks of daily administration. In contrast, while nicotine was able to attenuate the learned helplessness deficit, this trend only reached statistical significance after chronic administration. The non-competitive ion channel blocker mecamylamine increased escape failures when administered alone and blocked the effects of SIB-1508Y but not imipramine. SIB- 1508Y also produced an increase in avoidance responding, which suggests an enhancement of learning. CONCLUSION: These results not only suggest a role for nAChRs in the development of a depressive-like syndrome, but also that subtype-selective nAChR agonists, such as SIB-1508Y, may offer a novel therapeutic approach to the treatment of depression.

Molecular cloning of a human, hemicholinium-3-sensitive choline transporter.

Under many physiological circumstances, Na(+)- and Cl(-)-dependent, hemicholinium-3 (HC-3)-sensitive, high-affinity choline uptake (HACU) in cholinergic neurons is thought to be rate-limiting in the biosynthesis of acetylcholine (ACh). Based on sequence information provided by the Human Genome Project and the recently reported rat CHT1 (rCHT1), we cloned a human CHT cDNA from spinal cord. The hCHT cDNA encodes a protein of 580 amino acids having 93% identity to rCHT1 and 51% identity to the Caenorhabditis elegans homolog CHO-1, and is distantly related to members of the Na(+)-coupled glucose transporter (SGLT) gene family of Na(+)-coupled glucose (SGLT), nucleoside and iodide transporters. Northern blot analysis reveals the expression of a approximately 5 kb transcript in human brain regions rich in cholinergic neurons including the putamen, spinal cord, and medulla. Expression of hCHT cDNA in COS-7 cells results in saturable, Na(+)/Cl(-)-dependent choline uptake (K(m) = 1.2 microM) in membrane vesicles and [(3)H] HC-3 binding (K(d) = 4 nM) in membrane fractions, consistent with characteristics reported in mammalian cholinergic neurons. Using radiation hybrid mapping techniques, we localized the hCHT gene to human chromosome 2q12. These studies elucidate the primary structure and chromosomal localization of hCHT and provide a basis for mechanistic analysis of HACU regulation and an investigation of the role of hCHT in disease states.

Knowledge, attitudes, and behaviors of Alabama's primary care physicians regarding cancer genetics.

PURPOSE: To determine Alabama's primary care physicians' knowledge, attitudes, and behaviors regarding cancer genetics. METHOD: A questionnaire was mailed to a random sample of 1,148 physicians: family and general practitioners, internists, and obstetrician-gynecologists. RESULTS: Of the surveyed physicians, 22.1% responded. Of the respondents, 63% to 85% obtained family histories of cancer from 76% to 100% of their patients. Obstetrician-gynecologists referred more patients for cancer genetic testing (p = .008) and were more confident in their abilities to tailor preventive recommendations based on the results (p = .05) than were the other physicians. Primary care physicians were more likely than were obstetrician-gynecologists to identify lack of time during the patient visit as hindering efforts to do genetic counseling (p = .01). Physicians in practice for ten years or less were more confident in explaining genetic test results than were those in practice for more than 20 years (p = .01). CONCLUSION: These data validate gaps in primary care practices in obtaining family history of cancer, as well as lack of confidence in explaining genetic test results and in tailoring recommendations based on the tests.

Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts.

The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated.

Mast cell cholangiopathy: another cause of sclerosing cholangitis.

A 75-year-old woman with known systemic mastocytosis presented with abdominal pain, ascites, and bile duct thickening on computed tomography and ultrasonography. A liver biopsy specimen showed infiltration with mast cells. Endoscopic retrograde cholangiography showed ductal changes compatible with those found in primary sclerosing cholangitis. Brush cytology of the intrahepatic bile ducts confirmed mast cell infiltration. Systemic mastocytosis can infiltrate the biliary system, producing a cholangiopathy radiographically similar to primary sclerosing cholangitis.

Fiber-optic interferometric sensor for the detection of acoustic emission within composite materials.

An optical-fiber Michelson interferometric acoustic emission sensor is described. The sensor uses ordinary singlemode fiber and is embedded in the composite material under test. Signals are demodulated through the active homodyne. This system provides a novel approach for material nondestructive evaluation.

Use of hexadeuterated valproic acid and gas chromatography-mass spectrometry to determine the pharmacokinetics of valproic acid.

Di-[( 3,3,3-2H3]propyl)acetic acid, a hexadeuterated analogue of valproic acid, was synthesized and its pharmacokinetic properties compared with valproic acid. Concentrations of valproic acid and [2H]valproic acid in serum and saliva were determined by GC-MS using selected-ion monitoring. Saliva drug levels were measured with good precision down to 0.1 microgram/mL. Kinetic equivalence of valproic acid and [2H]valproic acid was demonstrated in a single-dose study in a human volunteer. An isotope effect was observed for omega-oxidation, but the difference in metabolism was not sufficient to make [2H]valproic acid biologically nonequivalent. The application of [2H]valproic acid to determine the kinetics of valproic acid under steady-state concentrations was evaluated in the same volunteer. The kinetic data obtained with [2H]valproic acid was consistent with previously reported values for valproic acid including kinetic differences observed between single-dose and steady-state experiments. Saliva levels of valproic acid were found to give a good correlation (r = 0.953) with total serum valproic acid under multiple-dose conditions. A concentration dependence was found for the ratio of saliva valproic acid to free valproic acid in serum, low ratios being observed at high serum concentrations of valproic acid.

Outcomes and indications of corpus callosum section for intractable seizure control.

Callosotomy (CCS) was performed in 9 cases of rigorously classified drug-refractory epilepsy not eligible for focal cortical resection. Complete CCS was carried out in two stages in 8 patients. 1 case had an anterior CCS only. None of the 9 patients has become seizure free. In 7, seizure frequency has diminished by a factor of 3-60 for at least one seizure type. Drop attacks in 2 cases have ceased. Clinical seizure patterns appeared more helpful in predicting outcome than preoperative EEG and stereoelectroencephalographic studies. Complete CCS may result in prolonged behavioral disturbances in the areas of language, hemisphere competition and attention-memory sequencing with impact on the sphere of daily living.