Adult Rat Forebrain Research Articles

Reversible activation of glutamate transport in rat brain glia by protein kinase C and an okadaic acid-sensitive phosphoprotein phosphatase.

High-affinity L-glutamate (GLU) transport is an important regulator of excitatory amino acid (EAA) concentrations in brain extracellular fluid and may play a key role in excitatory synaptic transmission. In view of evidence that EAA transporters (EAAT) are heterogenous and contain consensus sites for phosphorylation, this investigation was undertaken to contrast the effects of transporter phosphorylation in fractions derived from glia and neurons (synaptosomes) of the adult rat forebrain. Treatment with phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), increased the maximal rate of GLU transport in glial plasmalemmal vesicles by greater than 50 percent (237+/-18 vs. 365+/-27 pmol/mg protein/90s, p < 0.05) but caused no change in synaptosomes. The effect by PDBu was concentration and time-dependent and was inhibited completely by the PKC inhibitor calphostin C. Inhibition of serine-threonine phosphoprotein phosphatases with okadaic acid produced similar effects which were not additive with PDBu. Together, these results demonstrate that glial EAAT can be regulated by multiple phosphorylation processes.

Widespread expression in adult rat forebrain of mRNA encoding high-affinity neurotensin receptor

The peptides neurotensin (NT) and neuromedin N exert effects on neurons by means of a high-affinity NT receptor (NTRH) belonging to the superfamily of G-protein-coupled receptors. In the present study, we used in situ hybridization histochemistry with sensitive riboprobe methodology to investigate the distribution of NTRH mRNA in the forebrain of adult rats. Labeled cells were abundant in the hypothalamus, epithalamus, ventral thalamus, septum, amygdala, and pallidum, including many regions where NTRH mRNA had not been detected previously. In the hypothalamus, novel sites of NTRH mRNA expression included the arcuate, periventricular, paraventricular, supraoptic, medial preoptic, anterior, ventromedial, and posterior nuclei, as well as the lateral hypothalamic area. In the thalamus, novel sites of expression included the anterodorsal nucleus, lateral habenula, and zona incerta, where labeling was much more extensive than previously reported. Novel telencephalic sites of expression included most bed nuclei of the stria terminalis, most divisions of the amygdala, the main olfactory bulb, the endopiriform nucleus, the claustrum, many parts of retrohippocampal allocortex, and limited parts of most isocortical areas. Novel sites of expression were also observed in the midbrain and pons. Taking into account expected differences in the subcellular locations of receptor mRNA and protein, the regional distribution of NTRH mRNA agrees well with that of NTRH determined previously. Our results identify many novel sites of NTRH mRNA expression in adult brain and provide a basis for investigating involvement of NT and related peptides in regulating the activity of these diverse cells, whose phenotypes remain largely undetermined.

Widespread expression in adult rat forebrain of mRNA encoding high‐affinity neurotensin receptor

Widespread expression in adult rat forebrain of mRNA encoding high‐affinity neurotensin receptor

Immunohistochemical localization of the mGluR1? metabotropic glutamate receptor in the adult rodent forebrain: Evidence for a differential distribution of mGluR1 splice variants

Alternative splicing has been shown to occur at the metabotropic glutamate receptor 1 (mGluR1) gene. Three main isoforms that differ in their carboxy-termini have been described so far and named mGluR1alpha, mGluR1beta and mGluR1c. These variants when expressed in recombinant systems all activate phospholipase C, although the [Ca2+] signals generated have different kinetics. Tissue distribution studies of specific mGluR1 splice variants are limited to the mGluR1alpha isoform. In the present work, we examined the localization of mGluR1beta in the adult rat and mouse forebrain by using a specific antipeptide antibody. Furthermore, the mGluR1beta immunostaining was compared with that obtained with antibodies specific for mGluR1alpha or with a pan-mGluR1 antibody which recognizes all isoforms. mGluR1beta-like immunoreactivity (LI) was found confined to the neuropil and neuronal perikarya and appeared discretely distributed in the rodent forebrain. Differential cellular distribution between mGluR1alpha and mGluR1beta was observed. In the hippocampus, mGluR1alpha-LI was restricted to non-principal neurons in all fields, whereas mGluR1beta-LI was strongest in principal cells of the CA3 field and dentate granule cells but absent in CA1. We have also shown that the vast majority of neurons in the striatum express mGluR1. The predominant form appeared to be mGluR1beta, with a distribution pattern reflecting the patch-matrix organization of the striatum. The specificity of the immunoreactivity described for mGluR1 splice variants was confirmed in mGluR1-deficient mice. The observation of a different cellular and regional distribution of mGluR1 splice variants, in particular in the hippocampus, suggests that they may mediate different roles in synaptic transmission.

Localization of nerve growth factor, trkA and p75 immunoreactivity in the hippocampal formation and basal forebrain of adult rats

In the immunohistochemical staining of nerve growth factor, it has been reported that fixation-dependent lability of nerve growth factor hampers its localization. In the present study, we used two different polyclonal antibodies to immunostain nerve growth factor in rat brain tissue. We found that in paraformaldehyde-fixed (immersion- or perfusion-fixed) brains, nerve growth factor-like immunoreactivity was located primarily in the cytoplasmic membrane and fiber tract of hippocampal neurons and was sparse in cortical neurons. When fresh frozen brain sections were fixed in paraformaldehyde solution, nerve growth factor-like immunoreactivity was distributed evenly in the cell body. However, when fresh frozen brain sections were fixed in acetone, immunoreactivity to nerve growth factor was present as discrete or confluent dense particles in the cell body, especially in the nuclear region. Also, when paraformaldehyde-perfusion-fixed brain sections were heat treated in salt solution before immunostaining, nerve growth factor-like immunoreactivity could be retrieved in the cytoplasmic and nuclear regions. The hippocampal formation, cerebral cortex and basal forebrain expressed nerve growth factor-like immunoreactivity. Double immunostaining in fresh frozen brains showed that the low-affinity nerve growth factor receptor (p75) co-expressed with nerve growth factor and trkA proto-oncogene in basal forebrain neurons.Our study shows that formaldehyde fixation can mask nerve growth factor antigen, and special treatment, such as heating, is needed to retrieve nerve growth factor antigen to permit immunohistochemical detection. For immunohistochemical study of nerve growth factor in rat brain tissue, successful immunostaining can be obtained by using fresh frozen brains to prevent the masking effect of fixatives or by using paraformaldehyde-fixed brains with heat treatment. It is likely that nerve growth factor is synthesized and accumulated mainly in the cell body but not in the fiber tracts, which is similar to the distribution of its messenger RNA. The co-existence of p75 with nerve growth factor and trkA in basal forebrain neurons suggests the role of low- and high-affinity receptors in regulating the trophic effect of nerve growth factor.

Neuroleptics and dopamine transporters.

The effects of neuroleptic treatments on dopamine transporters and on dopamine receptors was investigated in the forebrain of adult rats treated for 21 days with either haloperidol, clozapine or saline. The dopamine D1 receptors, labeled with [3H]SCH23390, increased in nucleus accumbens, latero-dorsal rostral neostriatum and substantia nigra, after clozapine but not haloperidol. The dopamine D2 receptors, studied with [3H]raclopride, increased in nucleus accumbens and in dorso-lateral, ventro-medial and dorso-medial quadrants of the rostral neostriatum after either haloperidol or clozapine treatments, and also in latero-ventral rostral neostriatum but only after haloperidol. Haloperidol also up-regulated D2 receptors in rostral and caudal neostriatum, but clozapine produced a more uneven increase, especially in caudal neostriatum. In contrast, the densities of dopamine uptake sites, or transporters, labeled with [125I]RTI-121, remained unchanged after both neuroleptic treatments. The observation that dopamine transporters are resistant to treatments that modify D1 and D2 receptors indicates that these uptake sites can probably be ruled out as the target of neuroleptic drugs, and that dopamine receptor up-regulations can indeed occur independently of the densities of nerve endings at the terminal fields of innervation.

Identification of the glial cell types containing carnosine-related peptides in the rat brain

The cellular localization of carnosine-like immunoreactivity was investigated in the adult rat forebrain and in glial cell cultures obtained from newborn rat brain. Using double staining methods, we showed that in vivo carnosine-like immunoreactivity was occurring in a large number of both glial fibrillary acidic protein (GFAP)-positive astrocytes and 2′3′-cyclic nucleotide 3′-phosphodiesterase (CNP)-positive oligodendrocytes. In vitro, the carnosine-immunoreactive staining was restricted to a subpopulation of completely differentiated oligodendrocytes, whereas no reaction was detected in immature oligodendrocytes and in astrocytes. These observations could have profound physiopathological implications considering the role suggested for carnosine and related peptides as endogenous antioxidants, free radical scavengers and anti-glycating agents of the central nervous system (CNS).

Thyroid Hormone Regulates NGF Content and p75LNGFRExpression in the Basal Forebrain of Adult Rats

Several lines of data from human and animal studies have suggested a role of thyroid hormone in the regulation of cholinergic neurons in the adult brain. In this study we have investigated the content of nerve growth factor (NGF) and the expression of NGF low affinity receptor (p75LNGFR) in the basal forebrain of adult hypothyroid rats. We describe an increase of both NGF and p75LNGFRexpression in the basal forebrain of adult hypothyroid rats. The administration of colchicine up-regulates p75LNGFRexpression in both hypo- and control rats, whereas it fails to down-regulate choline acetyl transferase mRNA expression during hypothyroidism. These data offer a possible neurobiological explanation to cognitive defects observed during adult hypothyroidism in humans.

Prenatal exposure to ethanol causes differential effects in nerve growth factor and its receptor in the basal forebrain of preweaning and adult rats.

In this study we investigated nerve growth factor (NGF) levels in the cortex and hippocampus of the offspring of pregnant female Sprague-Dawley rats receiving a single intragastric administration of acute ethanol on the 15th day of gestation and compared them with a control group of rats that received an injection of sucrose. We also examined the distribution of the low-affinity NGF receptor, p75NGFR, on NGF-responsive neurons that are localized in the septum and the nucleus of Meynert, which receive the respective trophic support from the hippocampus and the cortex. In the ethanol-treated group, the results show that at post-natal age 15 days, the NGF septohippocampal pathways were markedly affected. At day 15, the NGF level was significantly higher in the offspring of ethanol-treated rats. By day 40, NGF values in both groups decreased to similar levels. At day 60, however, the NGF level in the ethanol-treated animals decreased to a significantly lower value than that of the control group, which remained essentially unchanged. In parallel, at day 60 the numbers of septal cholinergic neurons expressing p75NGFR were also significantly lower in ethanol-treated rats than in control animals. Because ethanol is known to induce neurological disorders, as well as deficits in cell proliferation and differentiation, the results suggest that one cause of the deleterious effects induced by ethanol is the low availability of NGF during certain stages of postnatal brain development.

Differential Effects of Protein Synthesis Inhibition on the Activity-Dependent Expression of BDNF Transcripts: Evidence for Immediate-Early Gene Responses from Specific Promoters

In the adult rat forebrain, brain-derived neurotrophic factor (BDNF) expression is very rapidly induced by neuronal activity, suggesting that this might occur without intervening protein synthesis. The rat BDNF gene has four differentially regulated promoter regions; each gives rise to an mRNA containing a unique 5' exon (I-IV) and a common 3' exon (V) that codes for mature BDNF protein. The present study used exon-specific in situ hybridization and both in vivo and in vitro preparations to determine whether activity induces BDNF as an "immediate-early gene" (IEG) from specific promoter regions and to compare the regulation of BDNF and nerve growth factor (NGF). In cultured hippocampal slices, kainic acid markedly increased pan-BDNF (exon V) and NGF mRNA content; cycloheximide attenuated the effect of kainic acid on both. In vivo stimulation of a paroxysmal afterdischarge increased both pan-BDNF and NGF mRNA levels in the dentate gyrus granule cells; pretreatment with anisomycin modestly attenuated the paroxysmal afterdischarge-induced increase of both transcripts. To determine whether partial drug effects on BDNF expression reflect the differential regulation of transcript species, levels of mRNAs containing exons I-IV were evaluated. A single afterdischarge increased exon I-IV-containing mRNA levels; anisomycin significantly attenuated the increase in exon I- and II-containing mRNAs but had no effect on the increase in exon III- and IV-containing mRNAs. These data show that for mature forebrain neurons, activity induces the expression of BDNF exon III- and IV-containing transcripts as IEG responses.

Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain

Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain

Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently described member of the EGF family that binds to the stimulates phosphorylation of the EGF receptor (EGFR). In this study, we examined the cellular localization of HB-EGF gene transcripts and protein in adult rat forebrain. In situ hybridization studies showed that neurons in various regions, including cortex, hippocampus, and deep structures, express HB-EGF mRNA. Positively labeled cells were also present in white matter, which suggests that both neurons and glia express HB-EGF mRNA. Immunohistochemical studies with an antibody specific to proHB-EGF, a transmembrane form of HB-EGF, demonstrated ubiquitous immunoreactivity in neurons and glial cells in white matter. In view of the wide expression of its cognitive receptor, EGFR, in central nervous system neurons, our results suggest that HB-EGF is an endogenous ligand for EGFR in the central nervous system and may play an important role in physiological conditions.

Distribution and steroid hormone regulation of aromatase mRNA expression in the forebrain of adult male and female rats: a cellular-level analysis using in situ hybridization.

Many of the effects of gonadal steroid hormones in the male brain are due to the actions of the testosterone metabolite estradiol, which is synthesized by the actions of the P450 enzyme aromatase. Aromatase activity is present in regions of the preoptic area, hypothalamus, and limbic system. Levels of aromatase activity in the brain are highly dependent on gonadal steroid hormones in many brain regions, but not all. We examined the distribution of aromatase mRNA in adult male and female rat brains as well as the regulation of the levels of aromatase mRNA in the brains of males by gonadal steroid hormones using in situ hybridization. This method was performed using a 35S-labelled cRNA probe, transcribed in vitro from the rat ovarian aromatase cDNA. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventromedial nucleus (VMN), the medial amygdala (mAMY), and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in females, but females tended to have a lower number of aromatase mRNA expressing cells in each region compared to males. Aromatase mRNA levels in the BNST, MPN, VMN, and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. The degree of reduction in mean levels of aromatase mRNA following castration does not simply account for the large changes measured in activity following castration. Examination of the entire population of individual cells expressing aromatase mRNA in castrated males suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of neurons within regions where activity is known to decrease following castration.

Distribution and steroid hormone regulation of aromatase mRNA expression in the forebrain of adult male and female rats: A cellular‐level analysis using in situ hybridization

Many of the effects of gonadal steroid hormones in the male brain are due to the actions of the testosterone metabolite estradiol, which is synthesized by the actions of the P450 enzyme aromatase. Aromatase activity is present in regions of the preoptic area, hypothalamus, and limbic system. Levels of aromatase activity in the brain are highly dependent on gonadal steroid hormones in many brain regions, but not all. We examined the distribution of aromatase mRNA in adult male and female rat brains as well as the regulation of the levels of aromatase mRNA in the brains of males by gonadal steroid hormones using in situ hybridization. This method was performed using a 35S-labelled cRNA probe, transcribed in vitro from the rat ovarian aromatase cDNA. In the adult male, many heavily labelled cells were found in the encapsulated bed nucleus of the stria terminalis (BNST), the medial preoptic nucleus (MPN), the ventromedial nucleus (VMN), the medial amygdala (mAMY), and the cortical amygdala (CoAMY). The regional distribution of aromatase mRNA was similar in females, but females tended to have a lower number of aromatase mRNA expressing cells in each region compared to males. Aromatase mRNA levels in the BNST, MPN, VMN, and mAMY tended to be lower in castrated males than in intact males, whereas aromatase mRNA levels were unaltered by castration in the CoAMY. The degree of reduction in mean levels of aromatase mRNA following castration does not simply account for the large changes measured in activity following castration. Examination of the entire population of individual cells expressing aromatase mRNA in castrated males suggests that aromatase mRNA may be regulated by steroid hormones differentially in specific populations of neurons within regions where activity is known to decrease following castration. © 1996 Wiley-Liss, Inc.

β‐Dystroglycan: Subcellular Localisation in Rat Brain and Detection of a Novel Immunologically Related, Postsynaptic Density‐Enriched Protein

The distribution of a glycoprotein component of the muscle dystrophin complex, beta-dystroglycan, has been determined in subcellular fractions of adult rat forebrain. The results show that beta-dystroglycan is enriched in several membrane fractions, including synaptic membranes, but in marked contrast to dystrophin is not detectable in the postsynaptic density fraction. The antiserum also recognises a second molecular species of apparent molecular mass of 164 kDa which is highly enriched in the postsynaptic density fraction. Preabsorption of the antiserum with the antigen (a 22-mer peptide corresponding to the C-terminal sequence of rabbit skeletal muscle beta-dystroglycan) abolished reactivity against both beta-dystroglycan and the 164-kDa postsynaptic density-enriched protein, confirming that the two species are immunologically related. Enzymatic removal of N-linked oligosaccharide lowered the apparent molecular mass of beta-dystroglycan by 3 kDa but did not alter the mass of the 164-kDa species.

Glycine and GABAA receptor-mediated chloride fluxes in synaptoneurosomes from different parts of the rat brain

Strychnine-sensitive, inhibitory glycine receptors have not until lately been considered to play a significant role in neurotransmission in mammalian forebrain regions. In order to investigate the role of glycine as a neurotransmitter in brain we have measured glycine induced chloride fluxes in different adult rat forebrain areas using synaptoneurosomes and a chloride-sensitive fluorescent indicator. The results have been compared to those obtained with GABA. The synaptoneurosomes from every brain area investigated responded to both glycine and GABA with chloride fluxes in a picrotoxin sensitive manner. The effect of glycine was inhibited by strychnine, which had no effect on the GABA-induced Cl − flux. Bicuculline inhibited the effect of GABA, but had no effect on the glycine-induced Cl − flux. Addition of GABA did not affect the response to glycine and vice versa. The endogenous content of glycine and GABA in the synaptoneurosome preparations was about the same and synaptoneurosomes from every brain area investigated released both glycine and GABA upon depolarisation with KCI. The depolarisation induced release of both GABA and glycine was partly Ca 2+-dependent and partly Ca 2+-independent. These results indicate that glycine can induce inhibitory Cl − fluxes distinct from GABA induced fluxes in every investigated brain area and that glycine can be released upon depolarisation.

BDNF increases monoaminergic activity in rat brain following intracerebroventricular or intraparenchymal administration

We have previously demonstrated alterations in serotonin metabolism within descending pathways following infusion of brain-derived neurotrophic factor (BDNF) into the midbrain, near the periaqueductal gray and dorsal and median raphe nuclei. The aim of the present study was to extend these studies to include a comprehensive regional examination of monoamine (serotonin, dopamine and norepinephrine) and metabolite levels in discrete areas of the intact, adult rat forebrain following direct intraparenchymal midbrain BDNF infusion. We have compared neurochemical changes following midbrain infusion of BDNF to those obtained following intracerebroventricular (i.c.v.) infusion. Significant increases in levels of 5-HIAA and/or the 5-HIAA/5-HT ratio were found in all areas examined including the hippocampus, cortex, striatum, n. accumbens, substantia nigra and hypothalamus following both midbrain and i.c.v. infusion. Changes in dopaminergic activity were also observed, but displayed more regional specificity, i.e. changes were found primarily within the striatum and cortex. The two infusion sites produced similar patterns of neurochemical effects although the magnitude of the changes did vary in some areas. These results suggest that BDNF increased synthesis and/or turnover of serotonin, and to a lesser extent dopamine, in the mature rat forebrain. Furthermore, these data point to possible functional roles for BDNF in neuropsychiatric and neurodegenerative conditions which involve a dysregulation of these monoamine systems.

Developmental Regulation of Expression of GABAA Receptor α1 and α6 Subunits in Cultured Rat Cerebellar Granule Cells

We have studied the postnatal development of GABAA receptor α1 and α6 subunits expressed by primary cultures of cerebellar granule cells originating from 2-day-old (postnatal day 2, P2) and 10-day-old (P10) rat neonates. At these ages, the granule cells are at distinct stages of cerebellar development. In both cases, GABAA receptor α1 and α6 subunit-like immunoreactivities were detected, and displayed temporal expression profiles that were correlated with the maturity of the cerebella from which the cultured granule cells were derived. Using two different specificity anti-α1 subunit-specific antibodies, immunoreactive species with Mr 53,000 Da and 54,000 Da were detected by immunoblotting. The lower 53,000-Da band co-migrated with the α1 subunit-like immunoreactivity detected in GABAA receptors purified from adult rat forebrain by benzodiazepine affinity chromatography. This 53,000-Da α1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3–5 DIV in P2 cultures. The GABAA receptor α6 subunit-like immunoreactivity (58,000 Da) was not detected until 5–7 DIV in P10 and 9–11 DIV in P2-derived cultures. The appearance of α6 subunit-like immunoreactivity was paralleled by an up-regulation of α1 subunit expression and a concomitant increase in diazepam-insensititive (DZ-IS) [3H]Ro 15-4513 binding activity, a pharmacological characteristic of α6 and α1α6-subunit-containing GABAA receptors (Pollard et al. J. Biol. Chem., 270, 21,285–21,290, (1995)). Antagonism of both non-NMDA and NMDA subtypes of ionotropic glutamate receptors did not significantly affect the developmental profile, the level of GABAA receptor α6 subunit or the total DZ-IS or DZ-S [3H]Ro 15-4513 binding activities expressed by these neurons. These results provide further evidence that the expression of specific GABAA receptor subunit genes is subject to differential regulation. Furthermore, developmental expression of the GABAA receptor α6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cue that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors. Copyright © 1996 Elsevier Science Ltd

Expression of EGFR-, p75NGFR-, and PSTAIR (cdc2)-like immunoreactivity by proliferating cells in the adult rat hippocampal formation and forebrain.

Immunocytochemical and autoradiographic techniques were used to examine proliferating cells in the adult rat brain along with the expression of specific growth factor receptors and cell cycle proteins. Two hours following an injection of [3H]thymidine ([3H]Thy), dividing cells were detected in the subgranular region of the dentate gyrus and in the subependymal region (SER) extending into the olfactory bulb. Many cells continued to divide over the next 24 h as demonstrated by the ability for thymidine-labeled cells to incorporate bromodeoxyuridine (BrdU); however, the results of BrdU, PSTAIR, and vimentin staining suggest that the majority of the progeny cells detectable by [3H]Thy autoradiography at 3 days and 1 week after injection are postmitotic and at least partially differentiated. Significant numbers of thymidine-labeled cells detected 2 h following thymidine injection in the subgranular region of the dentate gyrus and in the SER of the lateral ventricle stained positively for epidermal growth factor receptor-, vimentin-, and PSTAIR-like immunoreactivity. Significant numbers of thymidine-labeled cells in the SER also stained positively for the low-affinity neurotrophin receptor p75NGFR. No [3H]Thy/p75NGFR-labeled cells were detected in the dentate gyrus. In addition, very few [3H]Thy/PSTAIR- or [3H]Thy/ vimentin-labeled cells were detected in region CA4. These data suggest that proliferating cells located in different regions of the adult brain may not be homogeneous and may be subject to different growth factor regulation.

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.