Behavioural and neurochemical effects of chronic intraventricular injections of nerve growth factor in adult rats with fimbria lesions

Rats received bilateral lesions of the fimbria. These lesions impaired their ability to learn a radial maze. Rats given repeated intraventricular injections of nerve growth factor (NGF, 10 micrograms twice weekly during 4 weeks after the lesion) learned the maze problem more rapidly than rats with the same injury but treated with a control protein (cytochrome c). When retested after a period of 6 weeks without NGF treatment, the performance of NGF-treated and cytochrome c-treated rats with fimbria lesions did not differ. Whereas our previous study showed an increase in choline acetyltransferase (ChAT) activity in the septum and the hippocampus 4 days after the last NGF injection, the present study found that after the retest period (i.e. 10 weeks after the last NGF injection) ChAT activity was increased in the septum but not in the hippocampus. The relationship between the NGF-induced changes in ChAT activity and behaviour is discussed.     

Acetylcholine-rich transplants in the hippocampus: influence of intrinsic growth factors and application of nerve growth factor on choline acetyltransferase activity

Three groups of rats received either unilateral fimbria-fornix lesions by aspiration through the overlying cingulate cortex (group I), a fimbria-fornix lesion followed by an intrahippocampal transplant of acetylcholine (ACh)-rich embryonic septal tissue (group II), or a similar septal transplant placed into the intact hippocampus, in the absence of the denervating lesion (group III). The 3 groups were subdivided into equal subgroups receiving 6 intrahippocampal injections of nerve growth factor (NGF) at 4-day intervals, control injections of cytochrome c, or no injections. On the 28th day all animals were sacrificed and the majority taken for biochemical analysis of hippocampal choline acetyltransferase (ChAT). The animals with intact hippocampi (group III) were given a denervating fimbria-fornix lesion 3 days prior to sacrifice in order to reveal graft-derived ChAT activity from intrinsic ChAT activity. The fimbria-fornix lesions (group I) depleted hippocampal ChAT activity to 15-20% of normal, which was not influenced by NGF injections. The ACh-rich grafts placed in the denervated hippocampus (group II) restored hippocampal ChAT activity to approximately 60% of the normal level, and this was promoted to approximately 84% of NGF, but not cytochrome c, injections into the hippocampus. Grafts placed into the intact hippocampus (group III) did not raise ChAT activity above the lesion-alone level, and this was not influenced by NGF injections. Acetylcholinesterase (AChE) histochemistry showed no difference in outgrowth from the grafts in the denervated hippocampus with or without NGF injections. The results are interpreted, in agreement with observations in tissue culture, as indicating that NGF enhances ChAT activity in grafted neurons, rather than promoting survival and growth per se.     

Effect of thyroid hormone analogs on the activity of choline acetyltransferase in cultures of dissociated septal cells

Thyroid hormones influence the expression of transmitter-specific enzymes by central cholinergic neurons. Based on the fact that these cholinergic neurons degenerate selectively in human Alzheimer's disease, it was hypothesized that thyroid hormones might be beneficial in its treatment. However, since thyroid hormones influence the function of most peripheral organs, derivatives selective for central cholinergic neurons are necessary. The structural requirements of the receptor mediating the effects of the thyroid hormones on central cholinergic neurons were therefore compared with those of the receptors mediating actions on peripheral organs. Cultures were prepared of dissociated neurons from the septal area of fetal rat brains, and the differentiation of cholinergic neurons was assessed by measuring the activity of choline acetyltransferase (ChAT). Triiodothyronine (T3) was found to stimulate ChAT activity in a dose-dependent manner. The effect of T3 was additive to that of nerve growth factor. The potency of derivatives of T3 in elevating ChAT activity in the cultures was compared with their known anti-goiter activity determined in vivo and their binding affinity to the hepatic nuclear receptor measured in vitro. The findings indicate that the structural requirements of central and peripheral receptors are similar and that it therefore appears unlikely that analogs of thyroid hormones can be developed which selectively affect cholinergic neurons.     

Cross-species septal transplants: recovery of choline acetyltransferase activity

Following interruption of the fornix-fimbria pathway, hippocampal choline acetyltransferase (ChAT) activity was restored gradually by cross-species cell suspension transplants of embryonic septum. The hippocampal segment closest to the implant reached 35% of normal 17 weeks after transplantation. The overall restoration of ChAT by xenogenic cell suspension had many similarities to that reported for homogenic solid and cell suspension septal grafts. The time course of the recovery of ChAT activity was different from the time course of the ingrowth of acetylcholinesterase stained fibers reported previously.     

Perception of species-specific vocalizations in rats : role of the cholinergic septo-hippocampal pathway and aging

The effect of a chemical lesion of the cholinergic septo-hippocampal pathway induced by ethylcholine aziridinium (AF64A) on brain potentials evoked by species-specific vocalization containing informations of high biological relevance was studied in young adult (10 months) and aged (24 months) rats by means of neocortical electroencephalographic recordings. In control rats, the perception of a rats vocalization in a life endangering situation (lasting 0.8 s) initiated an evoked potential followed by a late positive slow wave (LPSW)-complex and a direct current shift with a duration of up to 16 s. Four months after treatment with AF64A (2 nmol into each of the lateral ventricles), the main negative component of the initial acoustic evoked potential (peak latency of about 60 ms after stimulus onset) was reduced (P = 0.04) both in young adult and aged rats. Further changes included a decrease of the late positive wave amplitude in young adult rats (P = 0.001) and a shorter duration of the LPSW-complex in aged rats (P = 0.03). AF64A induced also changes in specific components revealed by Principle Component Analysis, but only in the group of young rats. A decrease in the slow wave component (factor 1, 3000–4000 ms after stimulus onset ; P = 0.02) was observed. Age per se affected the late positive potential shifts as indicated by a shorter latency of the late positive wave (P = 0.03).

A detailed analysis of the major neurotransmitter markers proved an almost exclusive reduction of the activity of choline acetyltransferase in the ventral and dorsal hippocampus (up to 60%), which was equal in young adult and aged rats. The irreversible loss in choline acetyltransferase activity, which was restricted to the hippocampus, was associated by a slight reduction in serotonergic function.

The present data suggest that the complex cognitive and emotional processes initiated by species-specific vocalization appear to be affected by aging. Furthermore, as a consequence of a cholinergic deficit in the hippocampus, the integration processes essential for the recognition of the biological meaning of a species-specific vocalization are considerably disturbed. These findings provide an experimental basis for studying disturbances in the perceptual response to stimuli of high emotional value in patients with hypocholinergic function as in Alzheimers disease.     

Highly selective effects of nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3 on intact and injured basal forebrain magnocellular neurons

Cholinergic neurons of the basal nucleus complex (BNC) respond to nerve growth factor (NCF), the first member of a polypeptide gene family that also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), NGF, BDNF, and NT-3 are enriched in hippocampus. In addition, NGF and, more recently, BDNF have been shown to stimulate the cholinergic differentiation and enhance the survival of BNC cells in vitro. The present investigation was designed to test, in a comparative fashion, the in vivo effects of human recombinant NGF, BDNF, and NT-3 with confirmed activities in vitro on cholinergic and γ-aminobutyric acid (GABA)-ergic BNC neurons. The specific questions asked were whether and, to what extent, biologically active recombinant neurotrophins stimulate the transmitter phenotypes of intact cholinergic and GABAergic neurons of the BNC, and whether, and to what extent, recombinant neurotrophins protect the transmitter phenotypes of axotomized cholinergic and GABAergic neurons of the BNC following complete transections of the fimbria-fornix (measured by ChAT mRNA hybridization). Our results confirm the profound stimulatory and p^75NGFR expression in both intact and axotomized cholinergic neurons and to exert minor effects on some cholinergic markers (e.g., ChAT immunoreactivity). NT-3 had no influence on GABAergic neurons. Taken together, these results indicate that, despite their significant sequence homologies and their shared abundance in target fields of BNC neurons, NGF, BDNF, and NT-3 show striking differences in their efficacies as cholinergic trophic factors. GABAergic neurons of the BNC are resistant to neurotrophins. The result of the present investigation establish that NGF excels among neurotrophins as a trophic factor for intact and injured basal forebrain cholinergic neurons. © 1994 Wiley-Liss, Inc.     

Time course of the elevation of nerve growth factor (NGF) content in the hippocampus and septum following lesions of the septohippocampal pathway in rats

Axotomy of cholinergic neurons in the medial septum-diagonal band and degeneration of their terminals in the hippocampus resulting from fornix-fimbria lesions induce elevation of NGF content in these two brain regions. Postlesion levels of cholinergic neuron-specific ChAT activity in the septum suggest that endogenously produced NGF may, at least partly, promote survival of axotomized cholinergic neurons or induce ChAT activity in undamaged cells or both. These findings thus support the proposed trophic role for NGF in central cholinergic neurons.     

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.     

Deoxyglucose uptake and choline acetyltransferase activity in cerebral cortex following lesions of the nucleus basalis magnocellularis

The uptake of [3H]2-deoxyglucose (2-DG) into various brain regions of rats with unilateral or bilateral lesions of the nucleus basalis magnocellularis (nBM) was measured. The activity of choline acetyltransferase (ChAT) in these brain regions was also determined. Lesions of the nBM caused a significant decrease in cortical ChAT activity but had no effect on 2-DG accumulation. Pentobarbital treatment reduced 2-DG accumulation in all brain areas examined and these reductions were not influenced by the nBM lesions. The results indicate that a decrease in the cholinergic innervation of the cortex does not influence cortical glucose utilization. It appears unlikely, therefore, that the reported decrease in cortical glucose utilization in Alzheimer's disease is related to degeneration of the nBM-cortical cholinergic projection.     

No nerve growth factor response to treatment with memantine in adult rats

Summary. Nerve growth factor (NGF) is the most widely examined neurotrophin in the experimental models of Alzheimers disease (AD) and has been shown to prevent the retrograde degeneration of cholinergic neurons. In this study we examined NGF and cholineacetyltransferase (ChAT) changes in several rat brain regions after excitotoxic lesion of the entorhinal cortex with quinolinic acid and tested the effect of memantine on spatial learning in the radial maze after lesion. We observed a significant increase (+26%, p=0.02) of NGF concentrations in the hippocampus of the lesioned rats when compared to sham-lesioned rats. Chronic treatment with memantine showed no significant effect on the NGF increase in the hippocampus (p=0.72). The ChAT activity was significantly increased in the lesioned rats when compared to controls (+16%, p<0.05) and did not depend on treatment with memantine. In spite of this, memantine improved performance of the radial maze. This indicates that memory improving effects of memantine observed in experimental animals and in clinical studies are probably not related to changes in brain NGF content, whereas the observed NGF increase in the denervated hippocampus is probably trauma-related reflecting impaired retrograde transport of hippocampal NGF.Present address: Solvay Pharmaceuticals BV, Weesp, NiederlandePresent address: Institute of Pharmacology PAN, Cracow, Poland     

Regional heterogeneity of choline acetyltransferase activity in primate neocortex

Choline acetyltransferase activity in precentral and temporal regions of primate neocortex is 2.5-fold higher than in occipital cortex. These results suggest large differences in the density of innervation in different regions of primate neocortex by the nucleus basalis of Meynert.     

Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase

The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.     

Nerve growth factor levels and choline acetyltransferase activity in the brain of aged rats with spatial memory impairments

Nerve growth factor (NGF) and choline acetyltransferase (ChAT) activity levels were measured in 7 different brain regions in young (3-month-old) and aged (2-years-old) female Sprague-Dawley rats. Prior to analysis the spatial learning ability of the aged rats was assessed in the Morris' water maze test. In the aged rats a significant, 15–30%, increase in NGF levels was observed in 4 regions (septum, cortex, olfactory bulb and cerebellum), whereas the levels in hippocampus, striatum and the brainstem were similar to those of the young rats. The NGF changes did not correlate with the behavioral performance within the aged group. Minor 15–30%, changes in ChAT activity were observed in striatum, brainstem and cerebellum, but these changes did not correlate with the changes in NGF levels in any region. The results indicate that brain NGF levels are maintained at a normal or supranormal levels in rats with severe learning and memory impairments. Ther results, therefore, do not support the view that the marked atrophy and cell loss in the forebrain cholinergic system that is known to occur in the behaviorally impaired aged rats is caused by a reduced availability of NGF in the cholinergic target areas. The results also indicate that the slightly increased levels of NGF are not sufficient to prevent the age-dependent atrophy of cholinergic neurons, although they might be important for the stimulation of compensatory functional changes in a situation where the system is undergoing progressive degeneration.     

Effect of enucleation on choline acetyltransferase activity in layers of goldfish optic tectum

Choline acetyltransferase (ChAT) activity was determined in layers of optic tectum in control goldfish and in goldfish 4-20 days following unilateral enucleation. Significant changes in activity were found in the periventricular (PV) and superficial gray and white (SGW) layers. Within 4 days, ChAT activity in the PV layer on the lesioned side was about 75% of that on the control side. By 20 days, ChAT specific activity in the SGW layer on the lesioned side was about 150-160% of that on the control side. This increase in specific activity in the SGW layer was accounted for by the decrease in volume and in density of the layer after enucleation, so that the total amount of activity in the layer did not change significantly, indicating that the optic terminals contain little to no ChAT activity. ChAT activity in the optic tract was very low and did not decrease after enucleation. These data strongly indicate that the retinotectal pathway in goldfish is not cholinergic and, therefore, that the ChAT activity in the SGW layer is related to sources other than retinal ganglion cells. It is suggested that one such source might be neurons with somata in the PV layer.     

Enhancement of choline acetyltransferase activity in coculture of rat septal and hippocampal neurons

We report that choline acetyltransferase (ChAT) activity and neuronal survival were enhanced in rat septal neurons cocultured with hippocampal neurons. The enhancement of ChAT activity also occurred as a result of the addition of hippocampal conditioned medium (HpCM). When septal neurons from embryonic day 17 (E17) rats were cocultured with hippocampal neurons, ChAT activity was increased 2-fold compared with homogeneous culture of septal neurons. By contrast, no increase in ChAT activity was observed in coculture of septal and neocortical neurons. Treatment with HpCM obtained from cultured E19 rat hippocampal neurons enhanced the ChAT activity of E17 rat septal neurons. The enhancement of ChAT activity caused by coculture with hippocampal neurons and that caused by the addition of HpCM were not blocked by the addition of anti-nerve growth factor (NGF) antibody, suggesting that NGF, which is known to increase the ChAT activity of septal neurons both in vivo and in vitro, did not participate in the increase of ChAT activity. These findings indicate that possible target-derived neurotrophic factor(s), other than NGF, from hippocampal neurons enhance(s) the ChAT activity of septal neurons.     

Interaction between nerve growth factor and GM1 monosialoganglioside in preventing cortical choline acetyltransferase and high affinity choline uptake decrease after lesion of the nucleus basalis

Monosialoganglioside GM₁ and nerve growth factor (NGF) were administered alone or concomitantly to adult male rats with a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM). High-affinity choline uptake (HACU) rate and choline acetyltransferase (ChAT) activity were measured, 4 and 21 days after surgery, respectively, in the frontal and parietal cortices of both hemispheres. A 33–34% decrease in HACU rate and a 43-39% decrease in ChAT activity was found in the ipsilateral cortices 4 and 21 days, respectively, after the lesion. If the lesioned rats received NGF (10 μg i.c.v.) twice a week or daily administrations of GM₁ (30 mg/kg, i.p.), beginning immediately after surgery the decrease in HACU rate and ChAT activity was smaller. If NGF and GM, were given concomitantly no decrease in HACU rate and ChAT activity was detected in the lesioned hemisphere and a slight increase occurred in the contralateral hemisphere. However, after the concurrent administration of NGF (10 μg i.c.v.) and the inactive dose of GM₁ 10 mg/kg i.p. no decrease in HACU and ChAT activity was also found in the lesioned rats. The latter finding indicates a potentiation by GM₁ of NGF effects on the cholinergic neurons of the IBM. The two drugs may either antagonize the neurotoxic effects of ibotenic acid or stimulate a compensatory activity in the remaining neurons.     

Estrogen treatment suppresses forebrain p75 neurotrophin receptor expression in aged,noncycling female rats

There is increasing evidence that estrogen has beneficial effects on cognition, both in humans and in rodents, and may delay Alzheimer's disease onset in postmenopausal women. Several rodent studies have utilised the ovariectomy model to show estrogen regulation of the p75 neurotrophin receptor, TrkA, and markers of acetylcholine synthesis in the cholinergic basal forebrain. We studied estrogenic effects in aged (16-17-month-old), noncycling rats. Estrogen treatment for 10 days drastically reduced p75(NTR) immunoreactivity in the rostral parts of the basal forebrain. The number of p75(NTR)-immunoreactive neurons was decreased, and those neurons remaining positive for p75(NTR) showed reduced p75(NTR) staining intensity. In vehicle-treated rats, almost all choline acetyltransferase-immunoreactive neurons were p75(NTR) positive (and vice versa), but, in estrogen treated rats, large numbers of choline acetyltransferase-immunoreactive cells were negative for p75(NTR). Similar levels of p75(NTR) down-regulation in the rostral basal forebrain were found when estrogen treatment was extended to 6 weeks. There was no reduction in the number of p75(NTR)-immunoreactive neurons in the caudal basal forebrain after 10 days of treatment. After 6 weeks of treatment, however, there was evidence of p75(NTR) down-regulation in the caudal basal forebrain. There was no evidence of hypertrophy or atrophy of cholinergic neurons even after 6 weeks of estrogen treatment. Considering the evidence for the role of p75(NTR) in regulating survival, growth and nerve growth factor responsiveness of cholinergic basal forebrain neurons, the results indicate an important aspect of estrogen's effects on the nervous system.     

碱性成纤维生长因子和神经生长因子联合应用培养成年大鼠海马神经干细胞向神经元样细胞的分化

背景：影响神经干细胞向神经元分化的因素很多,各种营养因子可以不同程度地刺激神经干细胞向神经元分化,如何使神经干细胞大量分化为神经元是研究的热点问题。 目的：观察联合应用碱性成纤维生长因子和神经生长因子对成年大鼠海马神经干细胞为神经元的影响。 方法：无菌条件下分离大鼠脑海马组织,传至第4代克隆球直径约为200 μm时,滴加DMEM/F12+2% B27+20 μg/L表皮生长因子+20 μg/L碱性成纤维细胞生长因子,进行单细胞克隆培养,传代的神经干细胞分成空白对照组、碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子+神经生长因子组。观察传代后的克隆球进行神经干细胞免疫细胞化学染色鉴定,计数神经元特异性烯醇化酶阳性细胞率,检测神经干细胞向神经元的分化情况。 结果与结论：①单细胞克隆培养后,克隆球细胞表达巢蛋白,诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白均呈阳性表达。②与空白对照组神经干细胞分化为神经元的比例比较,碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子组+神经生长因子组均明显提高(P < 0.05),且碱性成纤维细胞生长因子组+神经生长因子组神经元的比例最高(P < 0.05)。提示,碱性成纤维细胞生长因子可以提高神经生长因子诱和神经生长因子均可促进神经干细胞向神经元分化,且二者联合应用效果更佳。     

The effect of selective lesions on vestibular nuclear complex choline acetyltransferase activity in the rat

Biochemical, physiological and behavioral evidence suggests that acetylcholine (ACh) may play a neurotransmitter role in central vestibular function. However, the anatomic basis for a possible cholinergic influence on the vestibular nuclear complex (VNC) is unknown. To investigate vestibular cholinergic anatomy, we have made selective lesions of neurons intrinsic to the VNC, and of most known afferents to the VNC, and we have measured the activity of choline acetyltransferase (ChAT), a specific marker for cholinergic neurons, following such lesions. We found that a kainic acid lesion of the VNC, and lesions of vestibular afferents, including the VIIIth cranial nerve, cerebellum, spinal cord, vestibular commissure and the interstitial nucleus of Cajal, did not affect VNC ChAT activity. We conclude that there are no cholinergic neurons intrinsic to the VNC, and that these lesioned afferents are not cholinergic. It is likely, therefore, that a cholinergic projection to the VNC arises from a region other than those lesioned; possibilities include the nuclei of the reticular formation, the upper cervical cord and local pontomedullary nuclei.     

A comparison of brain choline acetyltransferase activity in Alzheimer's disease,multi-infarct dementia,and combined dementia

Summary Brain choline acetyltransferase (ChAT) activity was determined in 43 patients with Alzheimer's disease (AD), 14 with multi-infarct dementia (MID), and 15 with combined dementia (CD) and in 53 age-matched controls. The activity of ChAT declined in the hippocampus, temporal and frontal cortex in patients with AD and CD compared to the controls. In the AD group the reduced activity of ChAT in all brain areas was associated with a greater number of cortical neurofibrillary tangles. The degree of dementia had a negative correlation with the activity of ChAT in the frontal cortex in both AD and CD patients. The activity of ChAT in the temporal cortex of CD patients was negatively associated with the cortical tangle counts. In contrast, the activity of ChAT and MID patients was not essentially different from that of the controls. Neither did the various clinical and neuropathological variables show any significant correlation with ChAT activity in MID patients. Thus, in this study the reduction in the activity of ChAT seems to be associated with Alzheimer-type pathology but not with dementia due to vascular changes.