Distribution of α2-adrenergic receptor subtype gene expression in rat brain

α₂-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. α₂-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective α₂-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific α₂-adrenoceptor subtypes (α_2A, α_2B, and α_2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with ³⁵S-labeled RNA probes to map the distribution of α₂-adrenoceptor subtype gene expression in rat brain. α_2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. α_2B mRNA was observed only in the thalamus. α_2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the α₂-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of α_2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of α_2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the α₂-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.     

Effect of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin on in vivo L-[β-11C]DOPA turnover in the rat striatum with infusion of L-tyrosine

Summary L-[¹¹C]DOPA, combined with positron emission tomography (PET), has made possible the assessment of dopamine turnover in vivo. Before the evaluation of PET study with L-[¹¹C]DOPA in the primate, the effect of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH₄) and/or L-tyrosine infusion on L-[¹¹C]DOPA turnover was analyzed in the rat striatal tissue and in the striatal extracellular fluid using microdialysis. L-[¹¹C]DOPA was rapidly taken up into the brain after intravenous injection and converted to [¹¹C]dopamine, [¹¹C]DOPAC and [¹¹C]HVA in the striatal tissue. Small amount of 3-O-methyl-[¹¹C]DOPA, a product of DOPA by 3-O-methylation in peripheral tissues, was also detected in the striatal tissue. The striatum/cerebellum ratio of total radioactivity uptake was linear against time up to 40 min after L-[¹¹C]DOPA injection. The uptake ratio, increased by 6R-BH₄ administration, was further increased by L-tyrosine infusion. The in vivo microdialysis technique was further applied to determine L-[¹¹C]DOPA and its metabolites in striatal extracellular fluid (ECF). The peripheral administration of 6R-BH₄ (50mg/kg) induced elevation of [¹¹C]DOPA concentration in ECF in the early phase after injection, following higher radioactivity in [¹¹C]dopamine and [¹¹C]HVA fractions than those in control animals at late phase. The 6R-BH₄-induced elevation of [^11C]DOPA uptake and the radioactivity of its metabolites was further enhanced by the continuous infusion of L-tyrosine at a dose of 1.0 mol/min/kg. L-Tyrosine infusion alone did not induce the elevation of radioactivity. The results suggest that [¹¹C]DOPA might be a useful probe to evaluate the effect of 6R-BH₄ and/or L-tyrosine loading in the primate.     

Quantification of [11C]PIB PET for imaging myelin in the human brain: a test–retest reproducibility study in high-resolution research tomography

An accurate in vivo measure of myelin content is essential to deepen our insight into the mechanisms underlying demyelinating and dysmyelinating neurological disorders, and to evaluate the effects of emerging remyelinating treatments. Recently [¹¹C]PIB, a positron emission tomography (PET) tracer originally conceived as a beta-amyloid marker, has been shown to be sensitive to myelin changes in preclinical models and humans. In this work, we propose a reference-region methodology for the voxelwise quantification of brain white-matter (WM) binding for [¹¹C]PIB. This methodology consists of a supervised procedure for the automatic extraction of a reference region and the application of the Logan graphical method to generate distribution volume ratio (DVR) maps. This approach was assessed on a test–retest group of 10 healthy volunteers using a high-resolution PET tomograph. The [¹¹C]PIB PET tracer binding was shown to be up to 23% higher in WM compared with gray matter, depending on the image reconstruction. The DVR estimates were characterized by high reliability (outliers <1%) and reproducibility (intraclass correlation coefficient (ICC) >0.95). [¹¹C]PIB parametric maps were also found to be significantly correlated (R²>0.50) to mRNA expressions of the most represented proteins in the myelin sheath. On the contrary, no correlation was found between [¹¹C]PIB imaging and nonmyelin-associated proteins.     

[11C]-PK11195 PET: Quantification of neuroinflammation and a monitor of anti-inflammatory treatment in Parkinson's disease?

[¹¹C]-PK11195 PET has been used for in vivo brain imaging of microglia activation in Parkinson's disease (PD) patients. COX-2 inhibition has been shown to reduce neuroinflammation and neurodegeneration in animal models of PD. This pilot study assessed the use of [¹¹C]-PK11195 PET to quantify neuroinflammation and evaluate the ability of COX-2 inhibition to reduce neuroinflammation in PD patients.MethodsFourteen PD patients and eight healthy, age matched controls underwent a [¹¹C]-PK11195 PET and MRI scan. Five PD patients were scanned before and after one month of celecoxib treatment 200 mg/day. Arterial plasma sampling and metabolite analysis were performed to create plasma input curves. A 2-compartment model and Logan analysis were applied and parametric DV images were compared using t-test in SPM2. In addition a simplified reference region model (SRTM) was applied, with both the cerebellum and a reference region derived from cluster analysis.ResultsUsing the cluster analysis, PD patients showed higher contralateral putamen BP and midbrain BP compared to controls, although considerable overlap was seen and differences were not statistically significant. Unexpectedly, BP and DV after celecoxib were slightly higher. Cerebellum as reference region resulted in lower BP values and k₃/k₄ gave 10-fold higher BP values. Linearization of the data did not show differences between PD patients and controls.ConclusionsIn current practice, [¹¹C]-PK11195 seems an unsuitable tracer for accurate or reliable quantification of neuroinflammation. Refinement of [¹¹C]-PK11195 uptake analysis and, more importantly, further development of better tracers is necessary to enable accurate measurement of neuroinflammation and effects of anti-inflammatory treatment in patients.     

Chronic alterations in rat brain α-adrenoceptors following traumatic brain injury

Norepinephrine (NE) has been implicated in cerebral plasticity and recovery of function after brain injury. To examine the status of noradrenergic mechanisms in the brain following traumatic brain injury (TBI), male Sprague-Dawley rats underwent right sensorimotor cortex contusions and were observed for the next 30 days for recovery of motor function by measurement of the time taken to perform a modified beam walking task! At 30 days, their brains were assayed by receptor autoradiography for αr- and α2-adrenoceptor binding with 1 nM [3H]prazosin and 1 nM [3H]paraminoclonidine, respectively. One day after contusion, TBI rats took 60% longer to run the beam than sham-lesioned controls. Run times were directly proportional (r = 0.784; P = 0.012) to lesion volume determined at 30 days. The motor deficit persisted for 8 days, after which TBI and control rats had similar run times, largely due to increased run times in sham rats. At 30 days, TBI rats had a generalized, bilateral decrease in [3H]prazosin binding across all brain areas read (F[l,13] = 9.23; P = 0.009) with specific 12%-21% decreases in the cortex contralateral to the lesion and bilaterally in the dorsomedial hypothalamic and three thalamic nuclei. On the other hand, [3H]paraminoclonidine binding did not differ from sham lesion controls in any brain area of TBI rats. Thus, unilateral TBI is followed by widespread, bilateral changes in α1-adrenoceptor binding which would leave the animal vulnerable to any factors which reduced the access of NE to its postsynaptic adrenoceptors. This is compatible with the observation that α1-antagonists and α2-agonists can transiently reinstate the motor deficit after recovery has occurred.     

Distribution of histone deacetylases 1–11 in the rat brain

Although protein phosphorylation has been characterized more extensively, modulation of the acetylation state of signaling molecules is now being recognized as a key means of signal transduction. The enzymes responsible for mediating these changes include histone acetyl transferases and histone deacetylases (HDACs). Members of the HDAC family of enzymes have been identified as potential therapeutic targets for diseases ranging from cancer to ischemia and neurodegeneration. We initiated a project to conduct comprehensive gene expression mapping of the 11 HDAC isoforms (HDAC1-11) (classes I, II, and IV) throughout the rat brain using high-resolution in situ hybridization (ISH) and imaging technology. Internal and external data bases were employed to identify the appropriate rat sequence information for probe selection. In addition, immunohistochemistry was performed on these samples to separately examine HDAC expression in neurons, astrocytes, oligodendrocytes, and endothelial cells in the CNS. This double-labeling approach enabled the identification of specific cell types in which the individual HDACs were expressed. The signals obtained by ISH were compared to radiolabeled standards and thereby enabled semiquantitative analysis of individual HDAC isoforms and defined relative levels of gene expression in >50 brain regions. This project produced an extensive atlas of 11 HDAC isoforms throughout the rat brain, including cell type localization, providing a valuable resource for examining the roles of specific HDACs in the brain and the development of future modulators of HDAC activity.     

Aggregation of αSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons

Fibrillar αSynuclein is the major constituent of Lewy bodies and Lewy neurites, the protein deposits characteristic for Parkinson’s disease (PD). Multiplications of the αSynuclein gene, as well as point mutations cause familial PD. However, the exact role of αSynuclein in neurodegeneration remains uncertain. Recent research in invertebrates has suggested that oligomeric rather than fibrillizing αSynuclein mediates neurotoxicity. To investigate the impact of αSynuclein aggregation on the progression of neurodegeneration, we expressed variants with different fibrillation propensities in the rat substantia nigra (SN) by means of recombinant adeno-associated viral (AAV) vectors. The formation of proteinase K-resistant αSynuclein aggregates was correlated to the loss of nigral dopaminergic (DA) neurons and striatal fibers. Expression of two prefibrillar, structure-based design mutants of αSynuclein (i.e., A56P and A30P/A56P/A76P) resulted in less aggregate formation in nigral DA neurons as compared to human wild-type (WT) or the inherited A30P mutation. However, only the αSynuclein variants capable of forming fibrils (WT/A30P), but not the oligomeric αSynuclein species induced a sustained progressive loss of adult nigral DA neurons. These results demonstrate that divergent modes of αSynuclein neurotoxicity exist in invertebrate and mammalian DA neurons in vivo and suggest that fibrillation of αSynuclein promotes the progressive degeneration of nigral DA neurons as found in PD patients.     

Expression of protocadherin-γC4 protein in the rat brain

It has been proposed that the combinatorial expression of γ-protocadherins (Pcdh-γs) and other clustered protocadherins (Pcdhs) provides a code of molecular identity and individuality to neurons, which plays a major role in the establishment of specific synaptic connectivity and formation of neuronal circuits. Particular attention has been directed to the Pcdh-γ family, for which experimental evidence derived from Pcdh-γ-deficient mice shows that they are involved in dendrite self-avoidance, synapse development, dendritic arborization, spine maturation, and prevention of apoptosis of some neurons. Moreover, a triple-mutant mouse deficient in the three C-type members of the Pcdh-γ family (Pcdh-γC3, Pcdh-γC4, and Pcdh-γC5) shows a phenotype similar to the mouse deficient in whole Pcdh-γ family, indicating that the latter is largely due to the absence of C-type Pcdh-γs. The role of each individual C-type Pcdh-γ is not known. We have developed a specific antibody to Pcdh-γC4 to reveal the expression of this protein in the rat brain. The results show that although Pcdh-γC4 is expressed at higher levels in the embryo and earlier postnatal weeks, it is also expressed in the adult rat brain. Pcdh-γC4 is expressed in both neurons and astrocytes. In the adult brain, the regional distribution of Pcdh-γC4 immunoreactivity is similar to that of Pcdh-γC4 mRNA, being highest in the olfactory bulb, dentate gyrus, and cerebellum. Pcdh-γC4 forms puncta that are frequently apposed to glutamatergic and GABAergic synapses. They are also frequently associated with neuron-astrocyte contacts. The results provide new insights into the cell recognition function of Pcdh-γC4 in neurons and astrocytes.     

Is synaptic dopamine concentration the exclusive factor which alters the in vivo binding of [11C]raclopride?: PET studies combined with microdialysis in conscious monkeys.

The effects of dopamine release manipulated by drugs on the in vivo binding of [11C]raclopride in the striatum were evaluated in conscious monkeys combined with microdialysis. The in vivo binding of [11C]raclopride was evaluated by high resolution positron emission tomography (PET), and the dopamine concentrations in the striatal extracellular fluid (ECF) were measured by microdialysis in the same animals. The systemic administration of the direct dopamine enhancers, GBR12909 (a dopamine transporter (DAT) blocker, at 0.5, 2 and 5 mg/kg) or methamphetamine (a dopamine releaser, at 0.1, 0.3 and 1 mg/kg) dose-dependently increased the dopamine concentration in the striatal ECF, and decreased in vivo [11C]raclopride binding in the striatum. The administration of the indirect dopamine modulators benztropine (a muscarinic cholinergic antagonist, at 0.1, 0.3 and 1 mg/kg) or ketanserine (a 5-HT2 antagonist, at 0.3, 1 and 3 mg/kg) also increased dopamine level in the striatal ECF, and decreased [11C]raclopride binding in a dose-dependent manner. However, the plots of percentage change in dopamine concentration in striatal EFC against that in [11C] raclopride binding indicated different relationships between the effects of direct dopamine enhancers (GBR12909 and methamphetamine) and indirect dopamine modulators (benztropine and ketanserine). These results suggested that the alternation of [11C]raclopride binding in vivo as measured by PET was differently affected by different neuronal manipulations, and not simply by the synaptic concentration of dopamine.     

Lack of G protein-coupled sigma receptors in rat brain membranes: receptor-mediated high-affinity GTPase activity and [35S]GTPγS binding studies

Summary. Although sigma () receptors have been identified as an independent receptor family distinct from opioid and phencyclidine receptors, the physiological roles of these receptors are largely unknown. It is controversial whether there exist metabotropic receptors that are coupled with heterotrimeric G proteins. In the present study, the stimulatory effects of ligands on high-affinity GTPase activity and [³⁵S]GTPS binding were determined in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. In either G protein activation assay, none of the ligands examined had stimulatory effect in any brain regions, except for unambiguous concentration-dependent increase in [³⁵S]GTPS binding by (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine [(+)-3-PPP] in striatal membranes. However, the competition study clearly showed this response was mediated through dopamine D₂-like receptors, but not receptors. It is concluded that receptors are not coupled to heterotrimeric G proteins, at least those of G_i/o type.     

Effects of thyroid status on presynapticα2-adrenoceptor function andβ-adrenoceptor binding in the rat brain

Summary The effect of thyroid status on noradrenergic synaptic function in the mature rat brain was examined by measuring presynaptic2- and post-synaptic-adrenoceptors.Repeated triiodothyronine (T₃) administration to rats (100g/kg×14 days: hyperthyroid) caused an 18% increase in striatal-adrenoceptors as shown by [³H]-dihydroalprenolol binding with no change in membranes from cerebral cortex or hypothalamus. In contrast, hypothyroidism (propyl-thiouracil, PTU×14 days) produced significant 12% and 30% reductions in striatal and hypothalamic-adrenoceptors respectively with no change in the cerebral cortex. Presynaptic2-adrenoceptor function was measured in the two dysthyroid states using the clonidine-induced hypoactivity model. Experimental hyperthyroidism increased the degree of clonidine-induced hypoactivity, and suggests increased presynaptic2-adrenoceptor function compared with control rats, whereas hypothyroidism suppressed presynaptic2-adrenoceptor function. These results show firstly that changes in thyroid status in the mature rat may produce homeostatic alterations at central noradrenergic synapses as reflected by changes in pre- and postsynaptic adrenoceptor function. Secondly, there appear to be T₃-induced changes in-adrenoceptors in the striatum where changes in dopaminergic neuronal activity have previously been demonstrated.     

Influence of O-methylated metabolite penetrating the blood–brain barrier to estimation of dopamine synthesis capacity in human L-[β-11C]DOPA PET

O-methyl metabolite (L-[β-¹¹C]OMD) of ¹¹C-labeled L-3,4-dihydroxyphenylalanine (L-[β-¹¹C]DOPA) can penetrate into brain tissue through the blood–brain barrier, and can complicate the estimation of dopamine synthesis capacity by positron emission tomography (PET) study with L-[β-¹¹C]DOPA. We evaluated the impact of L-[β-¹¹C]OMD on the estimation of the dopamine synthesis capacity in a human L-[β-¹¹C]DOPA PET study. The metabolite correction with mathematical modeling of L-[β-¹¹C]OMD kinetics in a reference region without decarboxylation and further metabolism, proposed by a previous [¹⁸F]FDOPA PET study, were implemented to estimate radioactivity of tissue L-[β-¹¹C]OMD in 10 normal volunteers. The component of L-[β-¹¹C]OMD in tissue time-activity curves (TACs) in 10 regions were subtracted by the estimated radioactivity of L-[β-¹¹C]OMD. To evaluate the influence of omitting blood sampling and metabolite correction, relative dopamine synthesis rate (k_ref) was estimated by Gjedde–Patlak analysis with reference tissue input function, as well as the net dopamine synthesis rate (K_i) by Gjedde–Patlak analysis with the arterial input function and TAC without and with metabolite correction. Overestimation of K_i was observed without metabolite correction. However, the k_ref and K_i with metabolite correction were significantly correlated. These data suggest that the influence of L-[β-¹¹C]OMD is minimal for the estimation of k_ref as dopamine synthesis capacity.     

Regulation of cytokine expression by Schwann cells in response to α2-macroglobulin binding to LRP1

Binding of activated α(2)-macroglobulin (α(2)M) to LDL receptor-related protein-1 (LRP1) in Schwann cells activates ERK/MAP kinase and Akt and thereby promotes cell survival and migration. The goal of this study was to determine whether α(2)M binding to LRP1 regulates expression of cytokines and chemokines. To assess the LRP1 response selectively, we studied primary cultures of rat Schwann cells. In a screening assay that detects 84 gene products, monocyte chemoattractant protein-1 (MCP-1/CCL2) mRNA expression was increased more than 13-fold in Schwann cells treated with activated α(2)M. The effects of α(2)M on MCP-1 expression were selective, because expression of the general proinflammatory cytokine tumor necrosis factor-α (TNF-α) was not induced. We confirmed that α(2)M selectively induces expression of MCP-1 and not TNF-α in single-target qPCR assays. MCP-1 protein accumulated at increased levels in conditioned medium of α(2)M-treated cells. LRP1 was necessary for induction of MCP-1 expression, as determined in experiments with the LRP1 antagonist receptor-associated protein, a mutated form of full-length α(2)M that does not bind LRP1, and in studies with Schwann cells in which LRP1 was silenced. Inhibiting ERK/MAP kinase activation blocked expression of MCP-1. These studies support a model in which LRP1 regulates multiple aspects of Schwann cell physiology in the response to PNS injury.     

Postnatal changes of nicotinic acetylcholine receptor 2, α3, α4, α7 and β2 subunits genes expression in rat brain

Postnatal changes of nicotinic acetylcholine receptor (nAChR) α2, α3, α4, α7 and β2 subunits mRNAs were investigated in rat brain using ribonuclease protection assay. Multiple developmental patterns were observed: (1) transient expression during the first few postnatal weeks; α2 in the hippocampus and brain stem, α3 in the striatum, cerebellum and cortex, α4 in the hippocampus, striatum and cerebellum, α7 in the cerebellum and β2 in the striatum. (2) Constant expression across development; α2 and α3 in the thalamus, α4 in the cortex, thalamus and brain stem, α7 in the thalamus and brain stem and β2 in all brain regions except striatum. (3) Non-detection across development; α2 in the cortex, striatum and cerebellum. (4) Increase with age; α7 in the cortex and hippocampus. (5) Bell-shaped development; α7 in the striatum. Postnatal changes of nAChR isoforms in different brain regions of rat were investigated by receptor binding assays. The developmental patterns of [³H]epibatidine and (−)-[³H]nicotine binding sites were similar to each other in each brain region, but different from that of [³H]α-bungarotoxin binding sites. No obvious correlation was observed between the developmental patterns of [³H]α-bungarotoxin, [³H]epibatidine and (−)-[³H]nicotine binding sites and corresponding subunits mRNAs. These results indicate that multiple mechanisms are involved in changes of gene expression of nAChRs subunits in the brain of developing rats.     

The acute effect of sertindole on brain 5-HT2, D2 and α1 receptors (ex vivo radioreceptor binding studies)

Summary The ability of sertindole to influence the ex vivo binding of³H-ketanserin,³H-prazosin and³H-spiperone to 5-HT₂ receptors, ₁-adrenoceptors and DA D₂ receptors, respectively, in rat brain has been studied after acute treatment.Sertindole is a potent, long acting compound which readily passes the blood-brain barrier. It dose-dependently binds to all three receptors types. In line with in vivo behavioural experiments sertindole has the most pronounced effect on 5-HT₂ receptors, lower effect on ₁-adrenoceptors and the lowest effect on striatal D₂ receptors.     

Lipophilicity as a determinant of binding of procaine analogs to rat α3β4 nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structure-activity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Affinity values (log[1/IC50]) of these compounds for the α(3)β(4) nAChR were determined by their competition with [(3)H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structure-activity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity.     

Affinity and selectivity of [¹¹C]-(+)-PHNO for the D3 and D2 receptors in the rhesus monkey brain in vivo

Although [¹¹C]‐(+)‐PHNO has enabled quantification of the dopamine‐D3 receptor (D3R) in the human brain in vivo, its selectivity for the D3R is not sufficiently high to allow us to disregard its binding to the dopamine‐D2 receptor (D2R). We quantified the affinity of [¹¹C]‐(+)‐PHNO for the D2R and D3R in the living primate brain. Two rhesus monkeys were examined on four occasions each, with [¹¹C]‐(+)‐PHNO administered in a bolus + infusion paradigm. Varying doses of unlabeled (+)‐PHNO were coadministered on each occasion (total doses ranging from 0.09 to 5.61 μg kg^?1). The regional binding potential (BP_ND) and the corresponding doses of injected (+)‐PHNO were used as inputs in a model that quantified the affinity of (+)‐PHNO for the D2R and D3R, as well as the regional fractions of the [¹¹C]‐(+)‐PHNO signal attributable to D3R binding. (+)‐PHNO in vivo affinity for the D3R (K_d/f_ND ～ 0.23–0.56 nM) was 25‐ to 48‐fold higher than that for the D2R (K_d/f_ND ～ 11–14 nM). The tracer limits for (+)‐PHNO (dose associated with D3R occupancy ～ 10%) were estimated at ～0.02–0.04 μg kg^?1 injected mass for anesthetized primate and at 0.01–0.02 μg kg^?1 for awake human positron emission tomography (PET) studies. Our data enabled a rational design and interpretation of future PET studies with [¹¹C]‐(+)‐PHNO. Synapse, 2012. © 2011 Wiley Periodicals, Inc.