Solanum nigrum L. in COVID-19 and post-COVID complications: a propitious candidate.

COVID-19 is caused by severe acute respiratory syndrome coronavirus-2, SARS-CoV-2. COVID-19 has changed the world scenario and caused mortality around the globe. Patients who recovered from COVID-19 have shown neurological, psychological, renal, cardiovascular, pulmonary, and hematological complications. In some patients, complications lasted more than 6 months. However, significantly less attention has been given to post-COVID complications. Currently available drugs are used to tackle the complications, but new interventions must address the problem. Phytochemicals from natural sources have been evaluated in recent times to cure or alleviate COVID-19 symptoms. An edible plant, Solanum nigrum, could be therapeutic in treating COVID-19 as the AYUSH ministry of India prescribes it during the pandemic. S. nigrum demonstrates anti-inflammatory, immunomodulatory, and antiviral action to treat the SARS-CoV-2 infection and its post-complications. Different parts of the plant represent a reduction in proinflammatory cytokines and prevent multi-organ failure by protecting various organs (liver, kidney, heart, neuro, and lung). The review proposes the possible role of the plant S. nigrum in managing the symptoms of COVID-19 and its post-COVID complications based on in silico docking and pharmacological studies. Further systematic and experimental studies are required to validate our hypothesis.

Complex Mutation Pattern of Omicron BA.2: Evading Antibodies without Losing Receptor Interactions.

BA.2, a sublineage of Omicron BA.1, is now prominent in many parts of the world. Early reports have indicated that BA.2 is more infectious than BA.1. To gain insight into BA.2 mutation profile and the resulting impact of mutations on interactions with receptor and/or monoclonal antibodies, we analyzed available sequences, structures of Spike/receptor and Spike/antibody complexes, and conducted molecular dynamics simulations. The results showed that BA.2 had 50 high-prevalent mutations, compared to 48 in BA.1. Additionally, 17 BA.1 mutations were not present in BA.2. Instead, BA.2 had 19 unique mutations and a signature Delta variant mutation (G142D). The BA.2 had 28 signature mutations in Spike, compared to 30 in BA.1. This was due to two revertant mutations, S446G and S496G, in the receptor-binding domain (RBD), making BA.2 somewhat similar to Wuhan-Hu-1 (WT), which had G446 and G496. The molecular dynamics simulations showed that the RBD consisting of G446/G496 was more stable than S446/S496 containing RBD. Thus, our analyses suggested that BA.2 evolved with novel mutations (i) to maintain receptor binding similar to WT, (ii) evade the antibody binding greater than BA.1, and (iii) acquire mutation of the Delta variant that may be associated with the high infectivity.

Role of ACE2-Ang (1-7)-Mas axis in post-COVID-19 complications and its dietary modulation.

Severe acute respiratory syndrome-coronavirus-2 (COVID-19) virus uses Angiotensin-Converting Enzyme 2 (ACE2) as a gateway for their entry into the human body. The ACE2 with cleaved products have emerged as major contributing factors to multiple physiological functions and pathogenic complications leading to the clinical consequences of the COVID-19 infection Decreased ACE2 expression restricts the viral entry into the human cells and reduces the viral load. COVID-19 infection reduces the ACE2 expression and induces post-COVID-19 complications like pneumonia and lung injury. The modulation of the ACE2-Ang (1-7)-Mas (AAM) axis is also being explored as a modality to treat post-COVID-19 complications. Evidence indicates that specific food components may modulate the AAM axis. The variations in the susceptibility to COVID-19 infection and the post-COVID its complications are being correlated with varied dietary habits. Some of the food substances have emerged to have supportive roles in treating post-COVID-19 complications and are being considered as adjuvants to the COVID-19 therapy. It is possible that some of their active ingredients may emerge as the direct treatment for the COVID-19.

Current targets and drug candidates for prevention and treatment of SARS-CoV-2 (COVID-19) infection.

Angiotensin-converting enzyme 2 (ACE2), the host cell-binding site for SAR-CoV-2, poses two-fold drug development problems. First, the role of ACE2 itself is still a matter of investigation, and no specific drugs are available targeting ACE2. Second, as a consequence of SARS-CoV-2 interaction with ACE2, there is an impairment of the renin-angiotensin system (RAS) involved in the functioning of vital organs like the heart, kidney, brain, and lungs. In developing antiviral drugs for COVID-19, ACE2, RNA-dependent RNA polymerase (RdRp), and the specific enzymes involved in the viral and cellular gene expression have been the primary targets. SARS-CoV-2 being a new virus with unusually high mortality, there has been a need to get medicines in an emergency, and the drug repurposing has been a primary strategy. Considering extensive mortality and morbidity throughout the world, we have made a maiden attempt to discover the drugs interacting with RAS and identify the lead compounds from herbal plants using molecular docking. Both host ACE2 and viral RNA-dependent RNA polymerase (RdRp) and ORF8 appear to be the primary targets for the treatment of COVID-19. While the drug repurposing of currently approved drugs seems to be one strategy for the treatment of COVID-19, purposing phytochemicals may be another essential strategy for discovering lead compounds. Using in silico molecular docking, we have identified a few phytochemicals that may provide insights into designing herbal and synthetic therapeutics to treat COVID-19.

Role of biomarkers in predicting diabetes complications with special reference to diabetic foot ulcers.

Diabetic foot ulcer (DFU) is one of the major complications of diabetes and about 1% of people with diabetes have to go for lower limb amputation. With better understanding of the pathological basis of DFU, number of biomarkers like atrial natriuretic peptides, galectin-3, and cardiac troponins for diabetic cardiomyopathy, cystatin C for diabetics nephropathy and C-reactive protein for infection and procalcitonin could aid in early and noninvasive diagnosis especially when clinical signs are misleading. Predictive role of novel biomarkers in primary prevention however, requires additional studies considering sex, age and multiple complications in DFU. The current review provides an insight about the novel and emerging biomarkers of diabetes and its complications with special reference to DFUs.

Thymic stromal lymphopoietin receptor blockade reduces allergic inflammation in a cynomolgus monkey model of asthma.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells. OBJECTIVE: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model. METHODS: An mAb against human TSLPR was generated and confirmed to be cross-reactive to cynomolgus monkey. Animals were dosed weekly with either vehicle or anti-TSLPR mAb for 6 weeks, and their responses to allergen challenge at baseline, week 2, and week 6 were assessed. RESULTS: After 6 weeks of treatment, anti-TSLPR mAb-treated animals showed reduced bronchoalveolar lavage (BAL) fluid eosinophil counts, reduced airway resistance in response to allergen challenge, and reduced IL-13 cytokine levels in BAL fluid compared with values seen in vehicle-treated animals. Expression profiling of BAL fluid cells collected before and after challenge showed a group of genes upregulated by allergen challenge that strongly overlapped with 11 genes upregulated in dendritic cells (DCs) when in vitro stimulated by TSLP (TSLP-DC gene signature). The number of genes differentially expressed in response to challenge was reduced in antibody-treated animals after 6 weeks relative to vehicle-treated animals. Expression of the TSLP-DC gene signature was also significantly reduced in antibody-treated animals. CONCLUSION: These results demonstrate promising efficacy for TSLPR blockade in an allergic lung inflammation model in which TSLP activation of mDCs might play a key role.

Unbound brain concentration determines receptor occupancy: a correlation of drug concentration and brain serotonin and dopamine reuptake transporter occupancy for eighteen compounds in rats.

It is a commonly accepted hypothesis that central nervous system (CNS) activity is determined by the unbound brain drug concentration. However, limited experimental data are available in the literature to support this hypothesis. The objective of this study was to test this hypothesis by examining the relationship between in vitro binding affinity (K(I)) and in vivo activity quantified as the drug concentration occupying 50% of the transporters (OC(50)) for 18 serotonin (SERT) and dopamine transporter (DAT) inhibitors. In vivo rat OC(50) was determined by autoradiography using [(3)H]N,N-dimethyl-2,2-amino-4-cyanophenylthiobenzylamine and [(3)H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) as the ligands to assess SERT and DAT occupancy, respectively. The unbound brain concentrations were calculated from total brain concentrations and the unbound brain fraction, which was determined by the brain homogenate method. The in vivo total brain SERT and DAT OC(50) values (mean +/- S.D.) were 408 +/- 368- and 410 +/- 395-fold greater than the K(I) values, respectively. In contrast, the in vivo unbound brain SERT and DAT OC(50) values were only 3.3 +/- 2.1- and 4.1 +/- 4.0-fold different from the K(I) values. Therefore, prediction of the biophase drug concentration by using the unbound brain concentration rather than the total brain concentration results in an approximately 100-fold improvement for the accuracy. In the present study, a 10-fold improvement was also observed by using the unbound plasma concentration rather than the total plasma concentration to predict the biophase concentration in the brain. This study supports the hypothesis that CNS activity is more accurately determined by the unbound brain drug concentration and not by the total brain drug concentration.

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-beta-erythroidine, or methyllycaconitine. Nicotine (0.8 mg/kg, 15 and 30 min after injection) increased the maximal velocity (V(max)) of [3H]DA uptake in PFC with no change in K(m), 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-beta-erythroidine, or methyllycaconitine (1.5, 8.0, and 10.0 mg/kg s.c., respectively) completely blocked the nicotine-induced increase in V(max) 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.

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, [(3)H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [(3)H]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 [(3)H]DASB and [(3)H]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 (IC(50)) 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-beta-carboxy-3-beta-(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.

Kinetic and thermodynamic assessment of binding of serotonin transporter inhibitors.

Several serotonin reuptake inhibitors are in clinical use for treatment of depression and anxiety disorders. However, to date, reported pharmacological differentiation of these ligands has focused mainly on their equilibrium binding affinities for the serotonin transporter. This study takes a new look at antidepressant binding modes using radioligand binding assays with [(3)H]S-citalopram to determine equilibrium and kinetic rate constants across multiple temperatures. The observed dissociation rate constants at 26 degrees C fall into a narrow range for all molecules. Conversely, association rate constants generally decreased with increasing equilibrium binding affinities. Consistent with this, the measured activation energy for S-citalopram association was relatively large (19.5 kcal . mol(-1)), suggesting conformational change upon ligand binding. For most of the drugs, including citalopram, the enthalpy (DeltaH(O)) and entropy (-TDeltaS(O)) contributions to reaction energetics were determined by van't Hoff analyses to be roughly equivalent (25-75% DeltaG(O)) and to correlate (positively for enthalpy) with the polar surface area of the drug. However, the binding of the drug fluvoxamine was predominantly entropically driven. When these data are considered in the context of the physicochemical properties of these ligands, two distinct binding modes can be proposed. The citalopram-type binding mode probably uses a polar binding pocket that allows charged or polar interactions between ligand and receptor with comparatively small loss in enthalpy due to dehydration. The fluvoxamine-type binding mode is fueled by energy released upon burying hydrophobic ligand moieties into a binding pocket that is flexible enough to suffer minimal loss in entropy from conformational constraint.

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.

Nicotine increases dopamine transporter function in rat striatum through a trafficking-independent mechanism.

In previous in vivo voltammetry studies, acute nicotine administration increased striatal dopamine clearance. The current study aimed to determine whether nicotine also increases [(3)H]dopamine uptake across the time course of the previous voltammetry studies and whether dopamine transporter trafficking to the cell surface mediates the nicotine-induced augmentation of dopamine clearance in striatum. Rats were administered nicotine (0.32 mg/kg, s.c.); striatal synaptosomes were obtained 5, 10, 40 or 60 min later. Nicotine increased (25%) the V(max) of [(3)H]dopamine uptake at 10 and 40 min. To determine whether the increase in V(max) was due to an increase in dopamine transporter density, [(3)H]GBR 12935 (1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride) binding was performed using rat striatal membranes; no differences were found between nicotine and saline-control groups at 5, 10 or 40 min post-injection, indicating that nicotine did not increase striatal dopamine transporter density; however, [(3)H]GBR 12935 binding assays determine both cell surface and intracellular dopamine transporter. Changes in cellular dopamine transporter localization in striatum were determined using biotinylation and subfractionation approaches; no differences between nicotine and saline-control groups were observed at 10 and 40 min post-injection. These results suggest that the nicotine-induced increase in dopamine uptake and clearance in striatum may occur via a trafficking-independent mechanism.

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 (V(max)) 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 V(max) 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 V(max) 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.

Increased capacity and density of choline transporters situated in synaptic membranes of the right medial prefrontal cortex of attentional task-performing rats.

Cholinergic neurons innervating the cortex have been conceptualized as a major component of the attention system of the brain. Because of recent evidence indicating plastic mechanisms regulating choline transporter (CHT)-mediated high-affinity choline uptake, which is the rate-limiting step of acetylcholine synthesis, the present experiment determined the capacity of cholinergic terminals to transport choline, and the proportion of choline transporters localized in the membrane of synaptic terminals, in several brain regions of rats performing a cognitive vigilance task (CVT) and a simple reaction time task (SRTT) and nonperforming (NP) rats. Compared with evidence from NP rats, increased choline transporter capacity [as indicated by maximum transporter velocity (Vmax)] and an increased density of CHTs situated in synaptic plasma membrane, relative to intracellular locations, were observed in the medial prefrontal cortex of the right but not left hemisphere of CVT-performing animals. Furthermore, right medial prefrontal Vmax values of CVT-performing rats correlated positively and left medial Vmax values correlated negatively with the animals' performance in signal trials. Measures of CHT function in the brains of SRTT-performing animals did not differ significantly from those in NP rats. The present data support the hypothesis that an increased capacity of choline transporters in the right medial prefrontal cortex, primarily attributable to increased trafficking of transporters from intracellular compartments to the terminal membrane, represents a cellular mechanism contributing to the mediation of attentional performance.

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 Parkinson's disease.

Effect of clonidine on cardiac norepinephrine spillover in isolated rat heart.

The purpose of this study is to determine the effect of clonidine on cardiac norepinephrine spillover utilizing an isolated rat heart preparation with attached cardiac sympathetic nerves. Following a 20-minute stabilization period, the sympathetic ganglion for each heart preparation was electrically stimulated with 10V and 2 Hz for 30 seconds (S1: 60 pulses). Heart rate, left ventricular developed pressure, and coronary perfusion pressure was allowed to return to baseline and the perfusate was randomly switched to Krebs buffer containing one of two treatments: placebo or clonidine (1 microM). After 10 minutes of treatment, the sympathetic ganglion was again electrically stimulated with 10V and 2 Hz for 30 seconds (S2: 60 pulses). The perfusate exiting the heart before, during, and after each electrical stimulation was collected for the determination of cardiac norepinephrine spillover. Clonidine administration significantly reduced cardiac norepinephrine spillover by approximately 50% (P < 0.05) and was associated with a 36% reduction in heart rate (P < 0.05). These findings provide evidence that clonidine can directly suppress NE spillover from cardiac sympathetic nerve terminals. Thus, suppression of cardiac NE by clonidine may be due to stimulation of presynaptic alpha2-adrenergic receptors or imidazoline subtype I receptors located on cardiac sympathetic nerve terminals. Results from our study demonstrate a reduction in cardiac NE spillover by clonidine and provide additional evidence that it can directly suppress peripheral sympathetic activity in that our results were obtained utilizing an isolated perfused heart preparation with attached cardiac sympathetic nerves devoid of any CNS input.

Regulation of choline transporter surface expression and phosphorylation by protein kinase C and protein phosphatase 1/2A.

The Na(+)/Cl(-)-dependent, hemicholinium-3-sensitive choline transporter (CHT) provides choline for acetylcholine biosynthesis. Recent studies show that CHT contains canonical protein kinase C (PKC) serine and threonine residues. We examined the ability of PKC and serine/threonine protein phosphatase 1/2A (PP1/PP2A) to regulate CHT function, surface expression, and phosphorylation. In mouse crude striatal and hippocampal synaptosomes, PKC activators beta-phorbol 12-myristate 13-acetate (beta-PMA) and beta-phorbol 12,13-dibutyrate produced time- and concentration-dependent reductions in CHT function. PP1/PP2A inhibitors okadaic acid (OKA) and calyculin A (CL-A) produced a time- and concentration-dependent decrease in CHT function. However, tautomycin (PP1 inhibitor) and cyclosporin A (PP2B inhibitor) failed to alter CHT-mediated choline uptake. Choline transport kinetic studies following beta-PMA, OKA, and CL-A treatment revealed a reduction in the maximal choline transport velocity (V(max)) with no change in K(m) for choline. These modulators also produced no change in the total levels of CHT protein in the crude hippocampal and striatal synaptosomes; however, surface biotinylation studies using the membrane-impermeant N-hydroxysuccinimide-biotin in crude synaptosomes following treatment with beta-PMA, OKA, and CL-A indicate significant reductions of CHT levels in biotinylated fractions. Pretreatment with OKA alone, but not beta-PMA, significantly augmented the phosphorylation level of CHT proteins. Our findings suggest that neuronal PKC and PP1/PP2A activity may establish the level of function and surface expression of CHT. These studies also provide the first evidence that CHT is a phosphoprotein and that the basal PP1/PP2A activity may have a dominant role in controlling the levels of CHT phosphorylation.

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.

Vesicular localization and activity-dependent trafficking of presynaptic choline transporters.

Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.

Nicotinic acetylcholine receptors interact with dopamine in induction of striatal long-term depression.

The dorsal striatum participates in motor function and stimulus-response or "habit" learning. Acetylcholine (ACh) is a prominent neurotransmitter in the striatum and exerts part of its actions through nicotinic cholinergic receptors. Activation of these receptors has been associated with the enhancement of learning and certainly is instrumental in habitual use of nicotine. Nicotinic receptors have also been suggested to be a possible therapeutic target for disorders of the basal ganglia. In this report we show that the activation of nicotinic acetylcholine receptors in the dorsal striatum contributes to dopamine (DA)- and activity-dependent changes in synaptic efficacy. High-frequency activation of glutamatergic synapses onto striatal neurons results in a long-term depression (LTD) of synaptic efficacy that is dependent on the activation of dopamine receptors. This stimulation also produces robust increases in extracellular dopamine concentration as well as strong activation of cholinergic striatal interneurons. Antagonists of nicotinic acetylcholine receptors inhibit striatal LTD. However, on coapplication of dopamine reuptake inhibitors with nicotinic receptor antagonists, activity-induced striatal LTD is restored. Dopamine release is modulated by activation of nicotinic receptors in the dorsal striatum, and activation of nicotinic receptors during high-frequency synaptic activation appears to be capable of interacting with dopaminergic actions that lead to striatal LTD. Our results suggest that stimulation of mechanisms involved in striatal synaptic plasticity is an important role for striatal nicotinic acetylcholine receptors and that these mechanisms may contribute to the enhancement of learning and habit formation produced by nicotine intake.