Postnatal development of the cerebellar Purkinje cell shape in guinea pig ontogenesis

In sagittal cerebellum sections, morphometrical study of cerebellum of mature-born animals - guinea pigs - was performed using Nissl's procedure. A change of shape and volume of Purkinje cells and their nuclei in the course of the guinea pig postnatal ontogenesis was studied. It has been shown that both the growth process itself and the rate of formation of the definite form of Purkinje cells and of their nuclei in the course of ontogenesis proceeds non-uniformly. The most intensive growth of vertical and horizontal diameters of Purkinje cells and of their nuclei is observed during the 1st and 4th weeks of postnatal life. Especially rapid is an increase of horizontal diameters of Purkinje cells and of their nuclei, which impairs the ovoid-bear-like shape to the cerebellar Purkinje cells of adult guinea pigs.     

Functional Role of Cerebellar Purkinje Cells in Ontogenesis of Postural-Motor Reactions in Mature- and Immature-Born Mammals

In experiments on guinea pigs, cats, and rats of different ages, from newborns to adults, the postural and stato-kinetic reflexes were studied with subsequent recording of electrical activity of identified cerebellar Purkinje cells (PC) in the same animals. Simultaneously in same age group of the animals, a morphometrical study of the shape and size of Purkinje cells and their nuclei was carried out. The first signs of manifestation of reflexes of standing and of the support reaction were observed in mature-born guinea pigs as early as at the first day after birth. At this time the PC fire has an irregular and low-frequency character with long inhibitory pauses and a great dispersion of the frequency-time characteristics. In the course of postnatal ontogenesis the parameter of the ratio of frequencies of simple and complex spikes increased in all the studied animals, guinea pigs, rat puppies, and kittens, but to different degrees. The complete maturation of the PC activity in the mature-born guinea pigs took 3–4 weeks after birth, whereas in the immature-born rat puppies and kittens this process took, on average, 5–6 and 8–9 weeks, respectively. By this time, all the studied postural-motor reactions were formed. The comparison of the course of formation of the frequency-time characteristics of the Purkinje cell activity in mature- (guinea pigs) and immature-born (rats and cats) animals allowed establishing that the highest rate of maturation of the Purkinje cell activity in the mature-born animals is observed during the first half, whereas in immature-born animals, during the second half of postnatal ontogenesis. A similar rate of changes also took place at maturation of postural-motor reflexes. The complete functional maturity of the Purkinje cell occurred, as a rule, at the time period when the shape and size of the Purkinje cell body became close to the definitive ones, and their vertical and horizontal diameters reached the values characteristic of adult animals.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in precocial- and altricial-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV-VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC represented by simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, from 1 to rarely 11 active PC are recorded, on average, in the single microelectrode track. At the one-week age, the highest number active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of the cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals exceeds again, although slightly of the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in mature- and immature-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV–VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC in the form of simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, on average, from 1 to rarely 11 active PC are recorded in the single microelectrode track. At the one-week age, the highest number of active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals it again exceeds, although slightly, the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

Chronic prenatal ethanol exposure-induced decrease of guinea pig hippocampal CA1 pyramidal cell and cerebellar Purkinje cell density

The brain is a key target of ethanol teratogenicity, in which ethanol can produce neurodegeneration in selected areas, including the hippocampus and cerebellum. The research objective was to test the hypothesis that chronic prenatal ethanol exposure, via maternal ethanol administration, produces differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells. Timed pregnant guinea pigs received chronic oral administration of ethanol, isocaloric-sucrose/pair-feeding, or water throughout gestation (term, about gestational day (GD) 68), and the offspring were studied at GD 62 (near-term fetus), postnatal day (PD) 1 (neonate), PD 5, and PD 12 (early postnatal life). Ethanol treatment, compared with isocaloric-sucrose/pair-feeding and water treatments, decreased brain, hippocampal, and cerebellar weights at GD 62, PD 1, PD 5, and PD 12. Hippocampal CA1 pyramidal cell linear density and cerebellar Purkinje cell linear density were unaffected at GD 62. Ethanol treatment produced 25, 30, and 30% decreases in linear density of hippocampal CA1 pyramidal cells at PD 1, PD 5, and PD 12, respectively, and a 30% decrease in linear density of cerebellar Purkinje cells at PD 12 only. At PD 5, Purkinje cell profile linear density remained unaffected; however, ethanol treatment appeared to increase linear density of apoptotic Purkinje cell nuclei, as determined by a modified TUNEL method. The data demonstrate that chronic prenatal ethanol exposure produces apparent differential time course of decreased linear density of hippocampal CA1 pyramidal cells and cerebellar Purkinje cells in the developing guinea pig.     

Dynamics of hematologic parameters and of the erythrocyte deformability index at the juvenal period of rats and guinea pigs

In experiments on Wistar rats and guinea pigs, dynamics of blood hematological parameters and of erythrocyte deformability index (DI) was studied from birth to the 3-month age. In rats, significant changes were revealed in the picture of blood for the first month of postnatal ontogenesis. The number of erythrocytes and leukocytes sharply increased, the number of reticulocytes decreased, their ratio in the degree of maturity was changed. The mean erythrocyte volume and their acidic resistance also decreased. Prior to the 4-week age, DI increased, which was accompanied by an increase in the hemoglobin hydration and flattening of the erythrocyte shape. Then DI decreased and erythrocytes acquired the more spherical shape. After the critical period of 3–4 weeks the studied parameters reached plateau. In guinea pigs, at this period of ontogenesis the hematologic parameters had sufficiently stable values, except for a DI decrease from birth to 1.5 months, then it rose without reaching the initial values. Like in the case with rats, DI had a close correlation with the erythrocyte parameter of shape (O_min).     

Dynamics of hematologic parameters and of the erythrocyte deformability index at the juvenal period of rats and guinea pigs

In experiments on Wistar rats and guinea pigs, dynamics of blood hematological parameters and of erythrocyte deformability index (DI) was studied from birth to the 3-month age. In rats, significant changes were revealed in the picture of blood for the first month of postnatal ontogenesis. The number of erythrocytes and leukocytes sharply increased, the number of reticulocytes decreased, their ratio in the degree of maturity was changed. The mean erythrocyte volume and their acidic resistance also decreased. Prior to the 4-week age, DI increased, which was accompanied by an increase in the hemoglobin hydration and flattening of the erythrocyte shape. Then ID decreased and erythrocytes acquired the more spherical shape. After the critical period of 3-4 weeks the studied parameters reached plateau. In guinea pigs, at this period of ontogenesis the hematologic parameters had sufficiently stable values, except for a DI decrease from birth to 1.5 months, then it rose without reaching the initial values. Like in the case with rats, DI had a close correlation with the erythrocyte parameter of shape (O min).     

Ectopic parvalbumin-positive cells in the cerebellum of the adult mutant mouse 'nervous'

The basket and stellate cells of the 'nervous' mouse cerebellum lose 50% of their presynaptic parallel fibres and more than 90% of Purkinje cells, their major postsynaptic targets, after postnatal day 23. To study the fate of these molecular layer cells we examined the cerebellum of the mutant mouse 'nervous' with antisera against the 'marker protein' parvalbumin, which exclusively tags Purkinje, basket, and stellate cells. Ten homozygous 'nervous' mice, 12-14 weeks old, and 30 control animals of several inbred strains were examined. The number of basket and stellate cells decreased in affected areas of the molecular layer. In addition, parvalbumin-positive cells were detected in the granular layer, in the white matter of the cerebellum and in the area of the cerebellar peduncles of all 'nervous' mice, but not in those of the control animals. These cells mainly occurred in areas which still displayed degenerating Purkinje cell axons. Ectopic parvalbumin-positive cells could either represent a class of interneurons, which have changed their antigenic properties, and therefore happen to stain with antisera against parvalbumin, or stratum moleculare cells which have failed to recognise their proper position during ontogenesis. Alternatively these cells could represent postdevelopmental basket cells, which have acquired the ability to migrate.     

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.     

A developmental profile of the levels of calcium pumps in chick cerebellum

The functional expression and distribution of intracellular ATPase (sarco(endo)plasmic reticulum Ca(2+)-ATPase: SERCA) and plasma membrane Ca(2+)-ATPase (PMCA) was analyzed in the developing chick cerebellum. The activity and Ca(2+) uptake increase with development for both ATPases. However, the protein content increases with the stage of development only for SERCA, remaining constant for PMCA. Immunohistochemical assays showed that the ontogenesis of these ATPases goes along with definite stages of cerebellum histogenesis, and is complete at hatching. The SERCA is mainly distributed in Purkinje neurons, whereas the PMCA seems to be expressed initially in climbing fibers, shifting to soma and spiny branchlets of Purkinje cells at late embryonic stages. Granule cells express both ATPases according to their degree of maturity, whereas only PMCA is present in cerebellar glomeruli. These pumps are present in deep nuclei and the choroid plexus, although in this latter tissue their expression declines with development. The spatio-temporal distribution of SERCA and PMCA must be closely related to their association with the development of specific cells and processes of the chick cerebellum.     

Neuronal expression of macrophage colony stimulating factor in Purkinje cells and olfactory mitral cells of wild-type and cerebellar-mutant mice

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immuno-histochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.     

Angiostrongylus cantonensis: role of eosinophils in the neurotoxic syndrome (Gordon-like phenomenon)

The role of eosinophils in the pathophysiology of Angiostrongylus cantonensis infections was investigated in nonpermissive (guinea pig) and permissive (rat) hosts. Neurological symptoms similar to the Gordon phenomenon (ataxia, tremor, paralysis) together with a loss of Purkinje cells in the cerebellum were observed after intracraneal injection of human eosinophil extracts or after infection with A. cantonensis, only in guinea pigs and not in rats. Blood eosinophilia as well as eosinophil numbers present in the cerebellum and in the cerebrospinal fluid were higher in guinea pigs than in rats, at all times after infection with A. cantonensis. Increased levels of cytotoxicity toward L3 larvae in vitro were obtained in the presence of guinea pig eosinophils and IgE antibodies, rather than with the corresponding rat effector system. The detection of one eosinophil granule component, the eosinophil peroxidase, in the cerebrospinal fluid from infected guinea pigs but not from rats suggested that in nonpermissive hosts, neurological disorders, similar to the previously described Gordon phenomenon, might be due to eosinophil neurotoxins released after interaction of eosinophils with the parasites.     

Axonal Membrane-Skeletal Protein A60: Association with a Brain Spectrin-Binding Activity and Entry into Cerebellar Axons at a Stage After the Initiation of Axonal Growth

Abstract: A60 is a 60-kDa component of the axonal cortical cytoskeleton in CNS neurones. It appears to be neurone specific and is tightly bound to brain membranes. In this study the cytoskeletal activities and developmental expression of A60 in rat cerebellum have been examined using the monoclonal antibody DR1. A60 in a partially purified soluble extract of brain membranes interacts selectively with brain but not erythrocyte spectrin. Because erythrocyte spectrin is more closely related to the dendritic form of spectrin than the axonal form, this raises the possibility that AGO localises in axons by interaction with the axonal form of spectrin only. A60 is not found in rat cerebellum before the day of birth. However, during postnatal development of the cerebellum (days 1–13) DR1 reactivity appears progressively. On postnatal day 1, a small population of cells in the mantle layer (presumptive Purkinje cells) is DR1 positive. There is no DR1 reactivity found in Purkinje cell axons during their initial phase of growth. By postnatal day 7, Purkinje cell bodies, initial dendritic segments, and the cerebellar white matter are all positive. This pattern of labelling is strengthened up until postnatal day 13. By contrast, in adult rat cerebellum, the location of A60 has changed so that it is most concentrated in axons, and dendritic staining is lost. These data indicate that A60 is a spectrin-binding component of the adult axonal membrane skeleton, the presence of which is only required in axons after the initial phase of growth.     

Development of different types of muscle fiber in the postnatal ontogeny of guinea pigs

As demonstrates estimation of myosin ATPase and SDG activity, the guinea pig is already born with differentiated muscle fibers (MF), and the first histochemical differences between them take place in the uterine 10 days before birth. Tonic oxidative fibers of the first type, arranging hexagonally, develop especially quickly at early stages of postnatal ontogenesis. Their relative contents up to the end of the observations (185 days) do not change, and area of their transversal section increases but slightly in comparison to the phasic fibers. The main age changes of the muscle tissue are connected with formation and rearrangement of the phasic fibers. The most intensive reconstructions of the phasic fibers coincide with the period of game activity and sex maturation. In mixed muscles the part of the glycolytic fibers increase during the postnatal ontogenesis. In the process of ontogenesis the soleus muscle fully consists of oxidative fibers. The definitive level of the MF development is established after the guinea pigs have reached their sex maturation. Comparing the results of the given investigation with the previous data on development of MF in rats, it is possible to conclude that term and premature animals have various rates in development of the muscle system, however, main stages of myogenesis coincide, though they are connected with various phases of ontogenesis.     

Mathematical analysis of myelin structure of nerves of the brachial plexus in antenatal and early postnatal ontogenesis of man]

A gradual increase of the information entropy indices and a decrease of the excess percentage is observed in the course of antenatal and early postnatal ontogenesis of man. The indices of the information entropy reach their maximum and the excess percentage reaches its minimum in children younger than 3 years. At that time the myelinated fibres have the most various diameters. In the antenatal development of man the prevalence of fine (1--4 mkm) myelinated fibres makes the structure of the humeral plexus nerves more definite while in the postnatal ontogenesis their structure is less definite since the myelinated fibres of different diameter are met with almost the same probability.     

The role of hyaluronate in morphogenesis of the neurons

The data about organization of the extracellular matrix (ECM) components and their interplay in the mammalian brain are rather limited. Hyaluronate (HA) is one of the main ECM glycosaminoglycans. Its location and function in the brain are believed to be mediated through its interaction with HA-binding proteins and proteoglycans. In this report, we describe distribution of the total HA-binding activity in the cells in the course of postnatal development of the rat brain and the effect of HA on cultured neurons. A high level of the HA-binding activity was found in the newborn cerebellum, but it quickly decreased after postnatal day 1. On postnatal day 5, strong HA-binding activity was demonstrated only in apical parts of growth cones of Purkinje cells. The data showed rapid down-regulation of HA-binding activity at the first stage of cerebellum maturation (migration of granule cells and beginning of differentiation of neurons). To obtain more information concerning a key role of HA in morphogenesis of neurons, low density cell cultures of the hippocampal neurons were used. The presence of HA in the substrate led to an increase in the cell adherence. However, a part of the cells got differentiated later. These data allow us to suggest that interactions between extracellular HA and cell-surface receptors can regulate motility and differentiation of the neurons.     

Neurotransmitters, neuropeptides and calcium binding proteins in developing human cerebellum: a review

Many endogenous neurochemicals that are known to have important functions in the mature central nervous system have also been found in the developing human cerebellum. Cholinergic neurons, as revealed by immunoreactivities towards choline acetyltransferase or acetylcholinesterase, appear early at 23 weeks of gestation in the cerebellar cortex and deep nuclei. Immunoreactivities gradually increase until the first postnatal month. Enkephalin is localized in the developing cerebellum, initially in the fibers of the cortex and deep nuclei at 16–20 weeks and then also in the Purkinje cells, granule cells, basket cells and Golgi cells at 23 weeks onward. Another neuropeptide, substance P, is localized mainly in the fibers of the dentate nucleus from 9 to 24 weeks but substance P immunoreactivity declines thereafter. GABA, an inhibitory neurotransmitter of the central nervous system, starts to appear at 16 weeks in the Purkinje cells, stellate cells, basket cells, mossy fibers and neurons of deep nuclei. GABA expression is gradually upregulated toward term forming networks of GABA-positive fibers and neurons. Catecholaminergic fibers and neurons are also detected in the cortex and deep nuclei at as early as 16 weeks. Calcium binding proteins, calbindin D28K and parvalbumin, make their first appearance in the cortex and deep nuclei at 14 weeks and then their expression decreases toward term, while calretinin appears later at 21 weeks but its expression increases with fetal age. The above findings suggest that many neurotransmitters, neuropeptides and calcium binding proteins (1) appear early during development of the cerebellum; (2) have specific temporal and spatial expression patterns; (3) may have functions other than those found in the mature neural systems; and (4) may be able to interact with each other during early development.     

Expression of low-molecular-weight neurofilament (NF-L) mRNA during postnatal development of the mouse brain

A regional Northern blot analysis demonstrated that the highest levels of NF-L mRNA in the adult mouse brain are present in brain stem followed by mid-brain, with lower levels found in neocortex, cerebellum, and hippocampus. The study was extended to the cellular level over the time course of postnatal development using in situ hybridization. This developmental analysis revealed that the expression of NF-L mRNA closely follows the differentiation pattern of many large neurons during postnatal neurogenesis. Neurons which differentiate early such as Purkinje, mitral, pyramidal, and large neurons of brain stem and thalamic nuclei, expressed high levels of NF-L mRNA at postnatal day 1. Early expression of NF-L mRNA may be required for the maintenance of the extensive neurofilament protein networks that are detected within the axons of larger neurons. Smaller neurons which differentiate later, such as dentate gyrus granule cells, small pyramidal and granule cells of the neocortex, and granule cells of the cerebellum, exhibit a delayed expression of NF-L mRNA.To whom to address reprint requests.     

GAP-43，Netrin-1，Collapsin-1和Neuropilin-1在出生后小鼠小脑中的动态表达

GAP-43,netrin-1,collapsin-1和neuropilin-1被认为在成网络分布的神经联系中发挥重要的作用．在年幼的啮齿类动物中,小脑包含5种不同的集中分布层：白质、内颗粒细胞层(IGL)、浦肯野氏细胞层(PCL)、分子层(ML)和外颗粒细胞层(EGL)．与浦肯野氏神经元在出生前产生这一点不同的是,EGL中的细胞在出生后产生,它们接受从前脑olivary核团发出的攀援纤维的主要神经投射,以及从内颗粒细胞发出的平行纤维的神经投射．这些神经投射主要在出生后的前3个星期内建立,同时还有浦肯野氏细胞的发育和成熟．而GAP-43,netrin-1,collapsin-1和neuropilin-1在出生后小脑发育的潜在作用仍然不清楚．为了更加清楚地探讨上述问题,检验了GAP-43,netrin-1,collapsin-1和neuropilin-1的mRNA与蛋白质在出生后5,10,20天和成年小鼠小脑中的表达情况．研究结果显示,这4种分子在小鼠出生后的小脑中有不同的时间和空间表达形式,这些结果与出生后发育和成年期间的轴突发生、延伸以及突触形成都有关联．通过免疫组织化学双标染色,发现小鼠出生后10天的小脑中,GAP-43阳性的浦肯野氏细胞也显示netrin-1或collapsin-1阳性,并且collapsin-1阳性的细胞也对 netrin-1 阳性．上述研究结果证明这4种分子可能参与了小脑的出生后发育．     

Effects of methylazoxymethanol given at different stages of postnatal life on development of the rat brain. Comparison with those of thyroid deficiency

Newborn rats were treated at different stages of their development with low doses of methylazoxymethanol acetate. The postnatal increase of the DNA content of the cerebrum did not differ from that of controls. In the cerebellum, the DNA content was transitorily reduced, but later, the external granular layer became thicker and DNA deposition increased in comparison with controls; finally, the cerebellar DNA returned to a normal value. Morphological abnormalities of the cerebellum, abnormal orientation of migrating cells, scattering of Purkinje cell bodies within the internal granule cells and specially striking abnormalities of the morphology and orientation of Purkinje cell dendrites were noted in rats treated with MAM from birth to day 3. The effects on the Purkinje cell morphogenesis persisted but were much less marked when MAM was given from 4 to 7 or from 8 to 11 days. Neonatal thyroid deficiency, as MAM-treatment between days 0 and 3, leads to an abnormal position of Purkinje cell bodies within the cerebellar cortex; it also leads to morphological abnormalities of their dendritic arborization which closely resemble those observed after MAM-treatment during the second postnatal week. It also alters the cell formation in the cerebellum. Thyroid deficiency probably exerts its effect on cell formation earlier than previous biochemical studies have shown. On another hand, the morphological abnormalities of Purkinje cell arborizations in the thyroid-deficient animals may be partly due to the perturbations of cell formation which persist later in the cerebellum.