Proclivity to self-injurious behavior in MRL-lpr mice: implications for autoimmunity-induced damage in the dopaminergic system.

Systemic lupus erythematosus is frequently accompanied by psychiatric manifestations of unknown origin. Although damage of central neurons had been documented, little is known about neurotransmitter systems affected by the autoimmune/inflammatory process. Recent studies on lupus-prone MRL-lpr mice point to imbalanced dopamine function and neurodegeneration in dopamine-rich brain regions. We follow up on anecdotal observations of singly housed mice developing chest wounds. Compulsive grooming and/or skin biting accounted for open lesions, lending itself to the operational term 'self-injurious behavior' (SIB). Low incidence of spontaneous SIB increased significantly after repeated injections of dopamine-2/3 receptor (D2/D3R) agonist quinpirole (QNP). To further probe the dopaminergic circuitry and examine whether SIB is associated with development of lupus-like disease, we compared behavioral responses among cohorts that differed in the immune status. Two-week treatment with QNP (intraperitoneal, 0.5 mg kg(-1) body weight per day) induced SIB in 60% of diseased MRL-lpr mice, and exacerbated their splenomegaly. Although increased grooming and stereotypy were observed in less symptomatic MRL+/+ controls, only one mouse (10%) developed SIB. Similarly, SIB was not seen in young, asymptomatic groups despite dissimilar ambulatory responses to QNP. In situ hybridization revealed treatment-independent upregulation of D2R mRNA in substantia nigra of diseased MRL-lpr mice. The above results suggest that development of systemic autoimmunity alters sensitivity of the dopaminergic system and renders MRL-lpr mice prone to SIB. Although pathogenic factors were not examined, we hypothesize that immune and endocrine mechanisms jointly contribute to early neuronal damage, which underlies behavioral deficiency in the adulthood.

Neurobehavioral alterations in autoimmune mice.

Inbred MRL, NZB and BXSB strains of mice spontaneously develop a systemic, lupus-like autoimmune disease. The progress of autoimmunity is accompanied with a cascade of behavioral changes, most consistently observed in tasks reflective of emotional reactivity and the two-way avoidance learning task. Given the possibility that behavioral alterations may reflect a detrimental consequence of autoimmune-inflammatory processes and/or an adaptive response to chronic malaise, they are tentatively labeled as autoimmunity-associated behavioral syndrome (AABS). It is hypothesized that neuroactive immune factors (pro-inflammatory cytokines, brain-reactive antibodies) together with endocrine mediators (corticotropin-releasing factor, glucocorticoids) participate in the etiology of AABS. Since AABS develops natively, and has a considerable face and predictive validity, and since the principal pathway to autoimmunity is known, AABS may be a useful model for the study of CNS involvement in human autoimmune diseases and by extension, for testing autoimmune hypotheses of several mental disorders (major depression, schizophrenia, Alzheimer's disease, autism and AIDS-related dementia).

Immunosuppression prevents neuronal atrophy in lupus-prone mice: evidence for brain damage induced by autoimmune disease?

An early onset of systemic, lupus-like disease in MRL-lpr mice is accompanied by deterioration in their behavioral performance and atrophy of pyramidal neurons in the parietal cortex and the hippocampal CA1 area. Using the immunosuppressive drug cyclophosphamide (CY) to attenuate the disease, we have tested the hypothesis that the autoimmune/inflammatory process is responsible for changes in brain morphology. A modified Golgi impregnation method revealed that, in comparison to saline-treated controls, immunosuppressive treatment with CY (100 mg/kg/week i.p. over 8 weeks) increased dendritic branching and spine numerical density in the CA1 region of MRL-lpr mice and MRL +/+ mice, which develop less severe manifestations of the disease. More interestingly, CY selectively prevented the atrophy and aberrant morphology of pyramidal neurons in the parietal cortex of MRL-lpr mice. The neuropathological measures (in particular reduced dendritic spine density) significantly correlated with increased serum levels of antinuclear antibodies and splenomegaly. The present results support the hypothesis that chronic autoimmune disease induces functionally important changes in neuronal morphology, and provide an empirical basis for understanding the behavioral dysfunction in systemic lupus erythematosus and autoimmune phenomena reported in some forms of mental illness.

Progressive atrophy of pyramidal neuron dendrites in autoimmune MRL-lpr mice.

The autoimmune-prone MRL-lpr substrain of mice develop an autoimmunity-associated behavioral syndrome (AABS) which resembles in many respects the behavior of animals exposed to chronic stress. The present study examined whether these mice show changes in the morphology of neuronal dendrites, as found in animals exposed to chronic stress. A modified Golgi-Cox procedure was used to visualize the dendrites of pyramidal neurons in the parietal cortex and in the CA1 hippocampal field of 5-week and 14-week old MRL-lpr mice and MRL + / + controls. Reduced dendritic branching and length, and an up to 20% loss of dendritic spines were observed in parietal and hippocampal pyramidal neurons of MRL-lpr mice at both ages. In the parietal cortex, there was an age-dependent potentiation in the reduction of basilar, but not apical, dendrite branching and length, as well as in the loss of spines on basilar segments. Loss of spines in the hippocampus followed an age-related course for apical but not basilar dendrites. Moreover, compared to age-matched controls, brain weight was smaller in MRL-lpr mice at 14 but not 5 weeks of age. Considering that dendritic atrophy becomes more extensive when autoimmune disease is florid in MRL-lpr mice, it is proposed that immune/inflammatory factor(s) produce dendritic loss. Reduced dendritic complexity may represent, at least in part, a structural basis for the altered behavioral profile of MRL-lpr mice.

Reduced corticotropin-releasing factor and enhanced vasopressin gene expression in brains of mice with autoimmunity-induced behavioral dysfunction.

The spontaneous development of autoimmune disease in MRL-lpr mice induces behavioral and endocrine changes that resemble effects of chronic stressors. To further examine the correspondence between autoimmune disease and chronic stress, we asked whether the brains of autoimmune mice show a shift in the corticotropin-releasing factor (CRF) to vasopressin (AVP) ratio. Using in situ hybridization histochemistry with 35S-labelled mouse riboprobes, the levels of mRNA transcripts encoding CRF and AVP were compared between autoimmune MRL-lpr and control MRL +/+ brains. CRF transcript levels were lower in the hypothalamic paraventricular nucleus and in the central nucleus of the amygdala in MRL-lpr mice. AVP transcript levels were higher in the paraventricular and the supraoptic nuclei in MRL-lpr mice compared to controls. CRF mRNA levels were inversely related to performance in stress-sensitive tasks and to measures of autoimmunity. As found previously for behavioral performance, immunosuppressive treatment with cyclophosphamide abolished the group difference in neuropeptide gene expression. These results indicate that an autoimmune disease process is necessary for the shift in the brain CRF:AVP ratio. Furthermore, they support the parallel between chronic stress and chronic autoimmunity/inflammation, and suggest common central mechanisms relevant to endocrine function and behavior.

Increased TUNEL staining in brains of autoimmune Fas-deficient mice.

Profound changes in brain morphology and behavior coincide with the spontaneous development of systemic autoimmune/inflammatory disease in Fas-deficient MRL-lpr mice. The dendrites atrophy, the density of hippocampal and cortical neurons decreases, and an anxious/depressive-like behavior emerges while lymphoid cells infiltrate into the choroid plexus of MRL-lpr mice. We hypothesized that the inherited lack of the Fas-dependent anti-inflammatory mechanism would lead to unsuppressed immune activity, characterized by reduced apoptosis in the MRL-lpr brain. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) method as an indicator of apoptosis, a surprisingly high incidence of TUNEL-positive cells was observed in the hippocampus, choroid plexus and periventricular regions of MRL-lpr mice, 5-10-fold higher than that found in the MRL +/+ control brain. Immunostaining with anti-CD3, CD4 and CD8 monoclonal antibodies showed limited overlap between CD-positive and TUNEL-positive cells, suggesting that the dying cells are for the most part (approximately 70%) not T-lymphocytes. Although further characterization of the phenotype of the dying cells and the mechanism of cell death are required, the present results suggest the involvement of a Fas-independent apoptotic process in neurodegeneration induced by systemic autoimmune disease.

Animal models for nervous system disease in systemic lupus erythematosus.

Animal models have much to teach us about nervous system dysfunction in SLE. It should be stressed that the murine strains described in this review have variable expression in the onset and severity of clinical and serological features, perhaps making them more like a heterogeneous human population with SLE. With this in mind, studies involving animal models like those involving human subjects should use a sample size that ensures adequate power. It is not surprising that studies that use sample sizes as low as four to five animals per group would find discrepant results, especially in outcomes that are measured prior to the terminal phases of the disease. Similar to human SLE patients, murine models have systemic autoimmune as well as neurological manifestations. Studies with murine models must continue to consider some type of SLE disease activity measures in order to control for the effects of systemic disease on nervous system dysfunction. Because of the short time window between the earliest evidence of neurologic dysfunction and severe autoimmune disease manifestations, especially in MRL/lpr mice, the disease acceleration model may allow a more careful dissection of how immunological events are related to nervous system dysfunction. Alternatively, the study of MRL/lpr mice ultraearly (e.g., 3 weeks of age) could also provide invaluable information about the first events leading to nervous system dysfunction in SLE. Both approaches promise to identify predictors of specific nervous system manifestations that may suggest novel and more specific therapeutic interventions.

Spatial learning during the course of autoimmune disease in MRL mice.

The present study examines whether autoimmune MRL-lpr mice develop impairments in learning and memory that correlate with changing severity of lupus-like disease. MRL-lpr mice (n = 20) were tested in the Morris water-maze at 12, 14, 16 and 18 weeks of age. Age-matched controls were congenic MRL +/+ mice (n = 20) that develop the disease much later. Immune status was assessed by the presence of anti-nuclear antibodies (ANA), brain-reactive antibodies, proteinuria, and haematocrit. Learning rates and memory retention did not differ between the substrains, and did not correlate or deteriorate with advancing age and autoimmunity. However, the baseline performance level in autoimmune MRL-lpr mice was shifted, as evidenced by a consistently longer task-solving latencies. Thigmotaxic swimming (along the pool wall) was pronounced in the MRL-lpr group, and was associated with the observed difference in performance. The present study does not support the notion that learning/memory abilities of autoimmune MRL-lpr mice are impaired per se, but may support the hypothesis that the rapid progress of humoral autoimmunity affects the emotionality of lupus-prone mice.

Neurotoxic properties of cerebrospinal fluid from behaviorally impaired autoimmune mice.

The chronic, lupus-like autoimmune disease in MRL-lpr mice is associated with leucocyte infiltration into the choroid plexus, brain cell death, and deficits in motivated behavior. The presence of lymphoid cells in the ventricular lumen and the increased number of TUNEL-positive cells in periventricular areas led to the hypothesis that immune cells enter into the cerebrospinal fluid (CSF) and induce primary neuronal damage in regions bordering the cerebral ventricles. Using an in vitro approach, we presently examine the possibility that CSF from autoimmune mice is neurotoxic and/or gliotoxic. The CSF and serum from diseased MRL-lpr mice, less symptomatic MRL +/+ controls, and healthy Swiss/Webster mice (non-autoimmune controls) were frozen until their effects on the viability of pyramidal neurons and astrocytes were assessed in a two-color fluorescence assay. Significant reduction in neuronal viability (in some cases as low as 67%) was observed in the co-cultures of hippocampal neurons and astrocytes incubated for 24 h with CSF from autoimmune MRL-lpr mice. The viability of astrocytes did not differ among the groups, and the CSF from autoimmune mice appeared more toxic than the serum. The behavior of MRL-lpr mice differed significantly from the control groups, as indicated by impaired exploration, reduced intake of palatable food, and excessive immobility in the forced swim test. The present results suggest that CSF from the behaviorally impaired lupus-prone mice is neurotoxic and are consistent with the hypothesis that neuroactive metabolites are produced intrathecally in neuropsychiatric lupus erythematosus.

Blunted sensitivity to sucrose in autoimmune MRL-lpr mice: a curve-shift study.

Lupus-prone MRL-lpr mice show an autoimmunity-associated behavioral syndrome that has many features similar to the effects of chronic stress. The present study evaluated whether autoimmune MRL-lpr mice show reduced responsiveness to sucrose, as observed in normal animals exposed to chronic mild stress. Sixteen-week old MRL-lpr mice and their age-matched congenic MRL +/+ controls were given 0%, 0.5%, 1%, 2%, 4%, 8%, or 16% sucrose solution to drink every 48 h in a one-bottle test. The MRL-lpr mice drank less than controls at all concentrations, except at 16%. The amount of sucrose consumed vs. solution concentration followed a saturation curve. Estimates were obtained for the concentration yielding the half-maximum response (X50) and the response at saturating concentration of sucrose (Rmax). The X50 was significantly higher in MRL-lpr than in MRL +/+ mice, indicating a shift to the right of the concentration-intake curve. The Rmax did not differ significantly between substrains, suggesting that the autoimmune process did not affect performance capacity. Pretreatment with the immunosuppressant cyclophosphamide diminished the substrain difference in X50, suggesting that reduced sensitivity to sucrose is related to autoimmune/inflammatory factors. These results support the similarity between autoimmunity-associated behavioral syndrome and behavioral changes produced by chronic stress, and suggest common neuroendocrine mechanisms. Because reduced sensitivity to palatable stimulus may reflect blunted hedonic responsiveness ("anhedonia"), it is hypothesized that an autoimmune/inflammatory factor(s) produces the depression found in human lupus, and some cases of affective disorder.

Reduced preference for sucrose in autoimmune mice: a possible role of interleukin-6.

In a continuous one-bottle sucrose intake test, 4-month-old autoimmune MRL-Ipr mice show a shift to the right along the X-axis of the concentration-intake function, compared to congenic MRL +/+ controls. Using a brief (60-min) and a continuous (48-h) two-bottle test, the present report examines potential factors that could account for the reduced responsiveness to a palatable stimulus. Study 1 examines whether preference for sucrose is associated with age, changes in food/water intake, or impaired renal function. Reduced preference for sucrose was observed in 5-6-week-old MRL-Ipr males, although food/water intake or blood creatinine levels did not differ from control values. Immunosuppressive treatment abolished this deficit, suggesting a role of immune factor(s). Study 2 tests the hypothesis that chronic upregulation of the neuroactive cytokine interleukin-6 (IL-6), reported to occur from 3 weeks of age in young MRL-Ipr mice, reduces preference for sucrose. Sustained administration of IL-6 was produced by infecting healthy MRL +/+, C3H.SW and Balb/C mice with adenovirus vector carrying cDNA for murine IL-6. This resulted in high serum IL-6 levels over 5 days, a rapid decline in preference for sucrose and low blood glucose levels. The results from Study 1 indicate that impaired sensitivity to sucrose in MRL-Ipr mice can be detected before autoimmune disease is florid in MRL-Ipr mice. The results from Study 2 are consistent with altered motivation/emotional states after infection, and point to sustained IL-6 production as an early mechanism in behavioral alterations during chronic autoimmune/inflammatory conditions.

Reduced aggressiveness and low testosterone levels in autoimmune MRL-lpr males.

Autoimmune, lupus-prone MRL-lpr mice float excessively in the forced swim test, explore novel objects and places less, and show blunted responsiveness to palatable stimuli, which is consistent with the hypothesis that the development of chronic autoimmune disease alters emotional reactivity and/or motivation. The present study measures isolation-induced fighting, a model of "affective" aggression, in lupus-prone MRL-lpr and control MRL +/+ males. When compared with controls, autoimmune MRL-lpr mice show reduced aggressiveness, as evidenced by fewer fighting contacts, longer attack latency, shorter fighting episodes and shorter duration of fighting. In addition, reduced testosterone levels accompany serological signs of autoimmunity in the MRL-lpr males. The present results support the hypothesis that affective responsiveness is altered in lupus-prone mice and may suggest limbic system dysfunction during chronic autoimmune/inflammatory disease. The question of whether immune activation alters behavior by a direct effect on the nervous system, or also via the endocrine system, requires further study.

Joint pathology and behavioral performance in autoimmune MRL-lpr Mice.

Young autoimmune MRL-lpr mice perform more poorly than age-matched controls in tests of exploration, spatial learning, and emotional reactivity. Impaired behavioral performance coincides temporally with hyperproduction of autoantibodies, infiltration of lymphoid cells into the brain, and mild arthritic-like changes in hind paws. Although CNS mechanisms have been suggested to mediate behavioral deficits, it was not clear whether mild joint pathology significantly affected behavioral performance. Previously we observed that 11-week-old MRL-lpr mice showed a trend for disturbed performance when crossing a narrow beam. The first aim of the present study was to test the significance of this trend by increasing the sample size and, second, to examine the possibility that arthritis-like changes interfere with performance in brief locomotor tasks. For the purpose of the second goal, 18-week-old mice that differ widely in severity of joint disease were selectively taken from the population and tested in beam walking and swimming tasks. It was expected that the severity of joint inflammation would be positively correlated with the degree of locomotor impairment. The larger sample size revealed that young MRL-lpr mice perform significantly more poorly than controls on the beam-walking test, as evidenced by more foot slips and longer traversing time. However, significant correlation between joint pathology scores and measures of locomotion could not be detected. The lack of such relationship suggests that mild joint pathology does not significantly contribute to impaired performance in young, autoimmune MRL-lpr mice tested in short behavioral tasks.

Immunosuppressive treatment prevents behavioral deficit in autoimmune MRL-lpr mice.

MRL-lpr mice, which develop severe autoimmune disease, explore novel objects less well than do their congenic MRL(-)+/+ controls, in which symptoms of the disease are relatively mild. Moreover, diminished exploration in MRL-lpr mice correlates with the elevated titers of antinuclear antibodies (ANA) in their sera, suggesting that this behavioral deficit is caused by the autoimmune process. To test the hypothesis that autoimmunity affects behavior, in this study we examine whether treatment of the autoimmune process will reduce the difference in performance between the two MRL substrains in the novel-object test. Forty mice in each substrain were treated from 4 to 10 wk of age with the immunosuppressive drug, cyclophosphamide (100 mg/kg/wk, IP) or a saline vehicle. The immunosuppressive treatment reduced ANA titers to low levels and eliminated ANA production completely in 55% of MRL-lpr mice, suggesting an attenuation of the autoimmune process. In addition, treatment with cyclophosphamide, but not saline, abolished significant differences in exploration between the MRL-lpr and MRL +/+ groups, as measured by the latency to touch a novel object and the time spent exploring it. Thus, the present results suggest that a treatment which ameliorates autoimmune symptoms can concurrently remove the substrain difference in behavior. The effect of cyclophosphamide in the MRL-lpr group is believed to reflect the suppression of pathogenic immune factor(s) that alter behavior during the onset of autoimmune disease.

Brain-reactive antibodies and behavior of autoimmune MRL-lpr mice.

The hypothesis that brain-reactive autoantibodies (BRA) impair behavior was examined in MRL-lpr mice, which develop spontaneous autoimmune disease. Circulating BRA were measured as in vitro serum reactivity to Neuro-2A neuroblastoma cell line, and behavioral competence was assessed in activity monitors, open field, beam walking, and Morris water maze task. Mice with BRA in serum (BRA positive) exhibited slower spontaneous locomotion in a novel environment, shorter grooming episodes, and less exploration of the open field centre when compared to age-matched 7-11-week-old BRA-negative cagemates. Moreover, when initially exposed to the large swimming pool, BRA-positive mice showed increased swimming along the wall, but had no difficulty in learning the water maze task or in traversing a narrow beam. Brain-reactive autoantibodies titre and behavioral measures were not correlated, suggesting that the concentration of serum BRA is not reflective of the magnitude of behavioral impairment. Nevertheless, the present study suggests that the presence of circulating BRA is associated with impaired exploration and/or enhanced emotional reactivity in MRL-lpr mice. It also supports the hypothesis of a pathogenic role of BRA in various mental disorders.

Disturbed emotionality in autoimmune MRL-lpr mice.

MRL-lpr mice develop symptoms of autoimmune lupus-like disease early in the life and MRL(-)+/+ mice develop it substantially later. The present study examines our previous suggestion that autoimmune MRL-lpr mice show altered emotional reactivity. In addition, it aims to identify the set of measures which best discriminate the behavior of MRL-lpr mice from their congenic controls (MRL +/+). Behavior of males from these two substrains (n = 40/substrain; 3-4 mo of age) was compared on a battery of tests presumed to be reflective of emotional reactivity. MRL-lpr mice explored the open field less, spent more time at home-base, and defecated less in comparison to congenic MRL +/+ controls. Moreover, MRL-lpr mice hesitated to step down from an elevated platform and to make contact with a novel object. They also visited open-arms of a plus-maze less often and showed extensive floating in the Porsolt's swim test. Discriminant analysis revealed that the performance of the MRL-lpr and MRL +/+ mice differed most profoundly on measures taken in the Porsolt's swim and step-down tests. In addition, in the MRL-lpr group high titers of serum antinuclear antibodies were associated with impaired exploration of a novel object. These results are consistent with the previously proposed notion of increased "timidity" in autoimmune MRL-lpr mice and of an immune factor contribution to altered emotional reactivity. Considering that behavior of autoimmune MRL-lpr mice resembles behavior of stressed animals, it is speculated that disturbed emotional reactivity reflects the effect of autoimmunity on the hypothalamic-pituitary-adrenal axis.

Electro-acupuncture modifies humoral immune response in the rat.

The immunomodulating properties of "Shangqiu" (LP-5), "Ququan" (H-8), and "Rangu" (R-2) points were studied in several experiments performed on male Wister rats treated bilaterally by acupuncture (A) or electro-acupuncture (EA). On the stimulation day 3, the sheep-red-blood cells (SRBC) antigen was injected intraperitoneally and four days later, the immunological tests (plaque forming cells-PFC, direct microhemagglutination) were performed. Also, the relative weights of lymphoid organs (spleen and thymus) and adrenal gland were derived after sacrifice. The results obtained suggest pronounced immunomodulating properties of 200 Hz cathodal current stimulation at H-8 point. The immunosuppression observed reflected in a decreased number of PFC and reduced relative weights of adrenal gland revealed in all A- or EA-treated groups.

Circulating brain-reactive autoantibodies and behavioral deficits in the MRL model of CNS lupus.

Brain-reactive autoantibodies (BRAA) are hypothesized to play a role in the neuropsychiatric manifestations that accompany systemic lupus erythematosus (SLE). The present study tests the proposed relation between circulating BRAA and behavioral deficits in lupus-prone MRL/lpr mice. Two age-matched cohorts born at different times were used to test the relationship in the context of altered disease severity. Significant correlations between autoimmunity and behavior were detected in both cohorts. These results are the first to report correlations between behavior and autoantibodies to integral membrane proteins of brain, supporting the hypothesis that BRAA contribute to the behavioral dysfunction seen in lupus.

Self-stimulation behavior: consequences upon immunity?

A variety of behavioral and emotional factors can affect the immune response by changing the brain immunoregulatory mechanisms, resulting in immunosuppression or immunopotentiation. This experiment deals with the effect of chronic self-stimulation behavior on the immune response and lymphoid tissue. Male rats were stereotaxically implanted with bipolar electrodes into the lateral hypothalamus and 7 days after surgery were screened for self-stimulation behavior. Lateral hypothalamus self-stimulating rats (LH-SS) were allowed to self-stimulate for 60 min/day for a period of 9 consecutive days: 5 days before and 4 days after immunization with 5 x 10(9) sheep red blood cells (SRBC). The animals were sacrificed and plaque-forming cell assay (PFC), microhaemmagglutination reaction with SRBC, and differential blood leucocyte counts were performed. The thymus, spleen, and inguinal lymph nodes were weighed and processed for histological examination. In the LH-SS group, an enhanced PFC response and increased anti-SRBC antibody titer were observed when compared to controls. The thymus and spleen of LH-SS rats were smaller in size in comparison with controls, but only with moderate changes in splenic cellular make-up. The relative number of lymphocytes was increased in peripheral blood of LH-SS rats when compared to intact animals. The results obtained suggest that chronic self-stimulation behavior can modulate some parameters of humoral immune response and affect the relative weight of lymphoid organs in the rat.

Behaviour of MRL mice: an animal model of disturbed behaviour in systemic autoimmune disease.

Evidence is reviewed indicating that development of lupus-like disease in the MRL-lpr substrain of mice coincides with a change in behaviour, suggestive of altered emotional reactivity and cognitive performance. The altered behaviour, termed 'autoimmunity-associated behavioural syndrome', is related to abnormal autoantibody and cytokine production. Moreover, immunosuppressive treatment with cyclophosphamide prevents the appearance of some behavioural deficits. It is argued that the MRL strain of mice constitute an animal model of behavioural dysfunction in autoimmune disease, and can be used to investigate the mechanisms by which an insidious autoimmune/inflammatory process alters mood and behaviour and may result in some forms of mental disorders.