Subbu Apparsundaram

Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter.

Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585–602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine‐ and dopamine‐containing neurons. Intracerebroventricular anti‐dopamine β‐hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP‐25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states. J. Comp. Neurol. 420:211–232, 2000.

Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex.

Rats raised in an enriched environmental condition (EC) exhibit a decreased (35%) maximal velocity (Vmax) of [3H]dopamine (DA) uptake in medial prefrontal cortex (mPFC) compared with rats raised in an impoverished condition (IC); however, no differences between EC and IC groups in Vmax for [3H]DA uptake were found in nucleus accumbens and striatum. Using biotinylation and immunoblotting techniques, the present study examined whether the brain region‐specific decrease in DA transporter (DAT) function is the result of a reduction in DAT cell surface expression. In mPFC, nucleus accumbens and striatum, total DAT immunoreactivity was not different between EC and IC groups. Whereas no differences in cell surface expression of DAT were found in nucleus accumbens and striatum, DAT immunoreactivity in the biotinylated cell surface fraction of mPFC was decreased (39%) in EC compared with IC rats, consistent with the magnitude of the previously observed decrease in Vmax for [3H]DA uptake in mPFC in EC rats. These results suggest that the decrease in DAT cell surface expression in the mPFC may be responsible for decreased DAT function in the mPFC of EC compared with IC rats, and that there is plasticity in the regulatory mechanisms mediating DAT trafficking and function.

Decreased plasma membrane expression of striatal dopamine transporter in aging

Aging in rodents, monkeys, and man is correlated with a reduction in dopamine transporter (DAT) ligand binding and DAT function. Using Western blot techniques, we investigated whether the source of these age-related changes in DAT was correlated with decreases in DAT protein levels in the striatum, substantia nigra (SN), nucleus accumbens (NAc), and ventral tegmental area (VTA) of 6, 18, and 24-month-old male Fischer 344 rats. The relative levels of tyrosine hydroxylase (TH) were also determined in each region. In the striatum, we also assessed [3H]-DA uptake and DAT plasma membrane expression using a membrane-impermeant biotin analog in crude synaptosomes prepared from these age groups. There was no significant age-related difference in DAT immunoreactivity per total protein or per total TH in striatum, NAc, SN, or VTA. Significant age-related changes in TH were only seen in the VTA of the 24-month-old rats (approximately 60% decrease). However, [3H]-DA uptake and DAT protein recovered in the biotinylated fraction in 24-month-old rats were significantly decreased (approximately 30%) compared to 6-month-old animals in the striatal synaptosomes. These data suggest that age-related decreases in striatal DAT function and ligand binding are related to a decrease in plasma membrane expression of DAT and not a decrease in the steady-state levels of DAT protein or loss of dopaminergic neuropil.

Neurotrophic and neuroprotective effects of the neuregulin glial growth factor‐2 on dopaminergic neurons in rat primary midbrain cultures

Glial growth factor‐2 (GGF2) and other neuregulin (NRG) isoforms have been shown to play important roles in survival, migration, and differentiation of certain neural and non‐neural cells. Because midbrain dopamine (DA) cells express the NRG receptor, ErbB4, the present study examined the potential neurotrophic and/or neuroprotective effects of GGF2 on cultured primary dopaminergic neurons. Embryonic day 14 rat mesencephalic cell cultures were maintained in serum‐free medium and treated with GGF2 or vehicle. The number of tyrosine hydroxylase‐positive (TH+) neurons and high‐affinity [3H]DA uptake were assessed at day in vitro (DIV) 9. Separate midbrain cultures were treated with 100 ng/mL GGF2 on DIV 0 and exposed to the catecholamine‐specific neurotoxin 6‐hydroxydopamine (6‐OHDA) on DIV 4. GGF2 treatment significantly increased DA uptake, the number of TH+ neurons, and neurite outgrowth when compared to the controls in both the serum‐free and the 6‐OHDA‐challenged cultures. Furthermore, three NRG receptors were detected in the midbrain cultures by western blot analysis. Immunostaining for glial fibrillary acidic protein revealed that GGF2 also weakly promoted mesencephalic glial proliferation in the midbrain cultures. These results indicate that GGF2 is neurotrophic and neuroprotective for developing dopaminergic neurons and suggest a role for NRGs in repair of the damaged nigrostriatal system that occurs in Parkinsons disease.

Antidepressants Targeting the Serotonin Reuptake Transporter Act via a Competitive Mechanism

Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099–3108, 1973). Two different SERT-selective radioligands, [3H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [3H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [3H]DASB and [3H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC50) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-β-carboxy-3-β-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.

Reduced plasma membrane surface expression of GLAST mediates decreased glutamate regulation in the aged striatum.

Extracellular L-glutamate poses a severe excitotoxic threat to neurons and glia when unregulated, therefore low synaptic levels of this neurotransmitter must be maintained via a rapid and robust transport system. A recent study from our laboratory showed a reduced glutamate uptake rate in the striatum of the aged Fischer 344 (F344) rat, yet the mechanism underlying this phenomenon is unknown. The current study utilized in vivo electrochemical recordings, immunoblotting and biotinylation in young (6 months), late-middle aged (18 months) and aged (24 months) F344 rats to elucidate the potential role that glutamate transporters (GLT-1, GLAST, and EAAC1) may play in this mechanism. Here we show that the time necessary to clear glutamate from the late-middle aged and aged striatum is significantly prolonged in comparison to the young striatum. In addition, an analysis of various sub-regions of the striatum revealed a marked dorsoventral gradient in terms of glutamate clearance times in the aged striatum, a phenomenon which was not present in the striatum of the animals of the remaining age groups. We also found that the decreased glutamate clearance time observed in the late-middle aged and aged rats is not due to a decrease in the production of total transporter protein among these three transporters. Rather, a significant reduction in the amount of GLAST expressed on the plasma membrane surface in the aged animals (approximately 55% when compared to young rats) may contribute to this phenomenon. These age-related alterations in extracellular l-glutamate regulation may be key contributors to the increased susceptibility of the aged brain to excitotoxic insults such as stroke and hypoxia.

Reduced expression and capacity of the striatal high-affinity choline transporter in hyperdopaminergic mice

Behavioral and neuronal abnormalities observed in mice exhibiting a reduced expression of the dopamine transporter model important aspects of schizophrenia, addiction, and attentional disorders. As the consequences of a chronic hyperdopaminergic tone for striatal output regulation have remained poorly understood, the present experiments were designed to determine the status of striatal interneuronal cholinergic neurotransmission in dopamine transporter knockdown animals. The high-affinity choline transporter represents the rate-limiting step of acetylcholine synthesis and release. Compared with wild type mice, striatal high-affinity choline transporter expression in dopamine transporter knockdown mice was significantly decreased. As in vivo basal striatal acetylcholine release did not differ between the strains, reduced high-affinity choline transporter expression in dopamine transporter knockdown mice was not due to reduced basal cholinergic activity. Furthermore, the proportion of high-affinity choline transporters expressed in plasma membrane-enriched versus vesicular membrane-enriched fractions did not differ from wild type animals, suggesting that changes in intracellular high-affinity choline transporter trafficking were not associated with lower overall levels of striatal high-affinity choline transporters. Synaptosomal choline uptake assays indicated a reduced capacity of striatal high-affinity choline transporters in dopamine transporter knockdown mice, and thus the functional significance of the reduced level of high-affinity choline transporter expression. Likewise, in vivo measures of the capacity of striatal high-affinity choline transporters to clear increases in extracellular choline concentrations, using choline-sensitive microelectrodes, revealed a 37-41% reduction in hemicholinium-sensitive clearance of exogenous choline in dopamine transporter knockdown mice. Furthermore, clearance of potassium-evoked choline signals was reduced in dopamine transporter knockdown mice (1.63+/-0.15 microM/s) compared with wild type animals (2.29+/-0.21 microM/s). Dysregulated striatal cholinergic neurotransmission is hypothesized to disrupt the integration of thalamic and cortical information at spiny projection neurons and thus to contribute to abnormal striatal information processing in dopamine transporter knockdown mice.

Nicotinic Receptor Activation Increases [3H]Dopamine Uptake and Cell Surface Expression of Dopamine Transporters in Rat Prefrontal Cortex

Previous research shows that nicotine increases dopamine (DA) clearance in rat prefrontal cortex (PFC) and striatum via a nicotinic receptor (nAChR)-mediated mechanism. The present study investigated whether activation of nAChRs regulates DA transporter (DAT) function through a trafficking-dependent mechanism. After nicotine administration (0, 0.3, and 0.8 mg/kg s.c., 15-1440 min after injection), DAT function and trafficking in synaptosomes of PFC and striatum were determined. nAChR mediation of the effect of nicotine on DAT function and trafficking in PFC was determined by pretreatment with mecamylamine, dihydro-β-erythroidine, or methyllycaconitine. Nicotine (0.8 mg/kg, 15 and 30 min after injection) increased the maximal velocity (Vmax) of [3H]DA uptake in PFC with no change in Km, compared with control. Biotinylation and Western blot assays showed that nicotine (0.8 mg/kg; 30 min) increased DAT cell surface expression in PFC. In contrast, a lower dose of nicotine (0.3 mg/kg; 30 min) did not alter DAT function and trafficking in PFC. Pretreatment with mecamylamine, dihydro-β-erythroidine, or methyllycaconitine (1.5, 8.0, and 10.0 mg/kg s.c., respectively) completely blocked the nicotine-induced increase in Vmax in PFC. In addition, mecamylamine completely blocked the nicotine-induced increase in DAT cell surface expression in PFC. Nicotine did not increase DAT function and cell surface expression in striatum, indicating that nicotine modulates DAT function in a brain region-specific manner. Thus, results from the present study suggest that the nicotine-induced increases in DAT function and cell surface expression in PFC may mediate some of the behavioral effects of nicotine.