Cortical Regulation Research Articles

Chronic Cold Stress Alters Prefrontal Cortical Modulation of Amygdala Neuronal Activity in Rats

Recent studies suggest that long-term exposure to stress can sensitize animals to subsequent novel or acute stressors. Stressors affect amygdala activity, and the prefrontal cortex has been implicated in the regulation of responses to stress. Little is known, however, about how the physiology of amygdala neurons is altered by chronic stressors or the role of the prefrontal cortex in these changes. We used in vivo extracellular recordings from neurons in the rat central and basolateral amygdala nuclei to examine the effects of chronic stress on the basal firing and responses of amygdala neurons to a novel stressor. Additionally, prefrontal cortical afferents were severed to examine its role in the modulation of the response to stressors. Chronic exposure to cold enhanced the sensitivity of central amygdala neurons to footshock. A portion of this may be due to enhanced basolateral amygdala output. Furthermore, prefrontal cortical regulation of this response is weakened by chronic stress. The physiology of the amygdala is altered by chronic stress. Furthermore, the prefrontal cortical regulation of these responses may be weakened after chronic stress. This is a potential biological substrate for abnormal affect upon chronic stress and its effect on affective disorders.

Activity and Connectivity of Brain Mood Regulating Circuit in Depression: A Functional Magnetic Resonance Study

Functional imaging studies indicate that imbalances in cortico-limbic activity and connectivity may underlie the pathophysiology of MDD. In this study, using functional Magnetic Resonance Imaging (fMRI), we investigated differences in cortico-limbic activity and connectivity between depressed patients and healthy controls. Fifteen unmedicated unipolar depressed patients and 15 matched healthy subjects underwent fMRI during which they first completed a conventional block-design activation experiment in which they were exposed to negative and neutral pictures. Next, low frequency blood oxygenation dependent (BOLD) related fluctuations (LFBF) data were acquired at rest and during steady-state exposure to neutral, positive and negative pictures. LFBF correlations were calculated between anterior cingulate cortex (ACC) and limbic regions--amygdala (AMYG), pallidostriatum (PST) and medial thalamus (MTHAL) and used as a measure of cortico-limbic connectivity. Depressed patients had increased activation of cortical and limbic regions. At rest and during exposure to neutral, positive, and negative pictures cortico-limbic LFBF correlations were decreased in depressed patients compared to healthy subjects. The finding of increased activation of limbic regions and decreased LFBF correlations between ACC and limbic regions is consistent with the hypothesis that decreased cortical regulation of limbic activation in response to negative stimuli may be present in depression.

Stress Interferes with Working Memory

The prefrontal cortex, located just above the eyes, is necessary for the higher order executive functions of the brain, including planning and working memory. Birnbaum et al. studied rats and monkeys performing tasks that require intact working memory. High activity of an intracellular signaling enzyme, protein kinase C (PKC), disrupted the animals' ability to remember. A cellular correlate of working memory, measured by recording neurons' electrical activity, showed this same sensitivity to high PKC. Because stress causes release of norepinephrine, a known activator of PKC, stress may exert its well-established inhibitory effect on prefrontal cognitive function through PKC. S. G. Birnbaum, P. X. Yuan, M. Wang, S. Vijayraghavan, A. K. Bloom, D. J. Davis, K. T. Gobeske, J. D. Sweatt, H. K. Manji, A. F. T. Arnsten, Protein kinase C overactivity impairs prefrontal cortical regulation of working memory. Science 306 , 882-884 (2004). [Abstract] [Full Text]

Protein Kinase C Overactivity Impairs Prefrontal Cortical Regulation of Working Memory

The prefrontal cortex is a higher brain region that regulates thought, behavior, and emotion using representational knowledge, operations often referred to as working memory. We tested the influence of protein kinase C (PKC) intracellular signaling on prefrontal cortical cognitive function and showed that high levels of PKC activity in prefrontal cortex, as seen for example during stress exposure, markedly impair behavioral and electrophysiological measures of working memory. These data suggest that excessive PKC activation can disrupt prefrontal cortical regulation of behavior and thought, possibly contributing to signs of prefrontal cortical dysfunction such as distractibility, impaired judgment, impulsivity, and thought disorder.

Cortical regulation during the early stage of initiation of voluntary swallowing

The aim of the present study was to reveal the spatiotemporal relations among cortical regions involved in the initiation of voluntary swallowing in humans, using magnetoencephalography (MEG). The swallowing-related activity was distributed widely for 2000 ms before the electromyogram (EMG) onset of the right suprahyoid muscle. The cingulate cortex, the insula and the inferior frontal gyrus were the main loci active prior to swallowing. These cortical loci coincide with those suggested by previous human brain mapping studies that investigated the brain mechanism, which controls swallowing. Activation in the cingulate cortex was registered in the early stage of swallowing and could be related to the cognitive process regarding the food being safe to swallow. The activation in the insula lasted for a long period of time before the initiation of swallowing. This suggests that the long-lasting activation in the insula prior to swallowing is essential for the initiation of swallowing.

Neuromagnetic analysis of the late phase of readiness field for hand precision movement using magnetoencephalography

The aim of this study was to elucidate the cortical regulation of precise finger movements using magnetoencephalography (MEG). Magnetic brain signals of the following two tasks were recorded by magnetoencephalography. The first task was to bend the right thumb once as quickly as possible (a simple movement). The second task was to alternately oppose the thumb with the index finger and the middle finger of the right hand (a precise movement). In this study, we confirmed the differences between the two tasks observed in the late phase of the readiness field (RF), especially in the magnetic field 600 ms before the onset of movement. There were obvious differences in parietal aspects and temporal aspects of the left hemisphere. In the dipole estimation, activities of the parietal aspects were in the prefrontal area and in the supplementary motor area (SMA). Activities of the temporal aspect were in the premotor area. In the precise finger movement, it is interesting to note that the prefrontal area and the SMA are active right before the onset of movement. These results indicate that communication of the motor area, the prefrontal area and the SMA before the onset of movement is necessary for precise hand movements.

Cortical regulation during the early stage of initiation of voluntary swallowing in humans.

The aim of the present study was to reveal the spatiotemporal relations among cortical regions involved in the initiation of voluntary swallowing in humans using magnetoencephalography (MEG). As a control task, finger extension movement, which is purely voluntary, was also investigated using the same techniques. The swallowing-related activity was distributed widely for 2000 ms before the electromyogram onset of the right suprahyoid muscle; however, the finger-related activity occurred in the late stage of the recording. The cingulate cortex, the insula, and the inferior frontal gyrus were the main loci active prior to swallowing. These cortical loci coincide with those suggested by previous human brain mapping studies that investigated the brain mechanism which controls swallowing. Activation in the cingulate cortex was registered in the early stage of swallowing and could be related to the cognitive process regarding the food being safe to swallow. The activation in the insula lasted for a long time before the initiation of swallowing. This suggests that the long-lasting activation in the insula prior to swallowing is essential for the initiation of swallowing.

Neuromagnetic analysis of the late phase of the readiness field for precise hand movements using magnetoencephalography.

The aim of this study was to elucidate the cortical regulation of precise finger movements by using magnetoencephalography, with particular emphasis on the late phase of the readiness field. Magnetic brain signals were recorded non-invasively by 306 channel magnetoencephalography during the following two tasks. The first task, a simple task, was to bend the right thumb once as quickly as possible. The second task, a precise one, was to alternately oppose the thumb with the index finger and the middle finger of right hand. In this study, we confirmed that the differences between the two tasks were observed in the late phase of the readiness field, especially in the magnetic field 600 ms before the onset of movement. The activity of the magnetic field of the precise movement task was higher than the activity of the simple movement task. There were obvious differences in the spatial and temporal aspects of the left hemisphere. In the simple movement, the premotor area or motor area was activated in the late phase of the time window. The average latency from the EMG onset was -98.6+/-34.0 ms (n = 5). In the precise movement, the prefrontal area and the SMA were activated in the early and/or middle phases of the time window. The average latency from the EMG onset was -292.0+/-14.9 ms (n = 3) for the prefrontal cortex and -167.8+/-38.3 ms (n = 4) for the SMA. The premotor area or motor area was activated in the late stage of the RF. The average latency from the EMG onset was -111.6+/-61.4 ms (n = 5). Many studies have been performed on the movement-related readiness field. However, the activity of the prefrontal area and the SMA had not previously been studied in the late phase of the readiness field. Our study indicated that the prefrontal area and the SMA played important roles immediately before the onset of precise finger movement. The integration of the prefrontal area, the SMA, and the premotor area is important for the onset of precise finger movement.

An investigation into asymmetrical cortical regulation of salivary S-IgA in conscious man using transcranial magnetic stimulation

Transcranial magnetic stimulation (TMS) can activate discrete areas of the cerebral cortex through the intact skull of healthy conscious volunteers. A magnetic coil generates a brief and focused magnetic field that penetrates the skull to activate the specific area of cerebral cortex beneath. This non-invasive procedure is painless, well tolerated by participants and now widely used to explore brain function. We used TMS to investigate asymmetrical cortical regulatory influences on one aspect of immune function: secretion of the antibody immunoglobulin A (S-IgA) into saliva. The right and left temporo-parietal-occipital cortex of 16 healthy, conscious subjects was stimulated on two different occasions, at least 1 week apart. There was an immediate increase in S-IgA concentration following both right and left stimulation. Saliva volume was reduced immediately post-right but not left stimulation. When secretion (μg/min) of S-IgA was calculated (controlling for changing saliva volume) an increase was apparent following left but not right hemisphere stimulation. Furthermore, a significant difference between the relationship between S-IgA concentration and volume of saliva post-left and right stimulation was observed. We conclude that TMS can be used as a tool to investigate cortical regulation of autonomic and immune function in healthy, conscious human subjects and that secretion of saliva and S-IgA is differentially affected by stimulation of the left and right cerebral cortex.

Functional circuitry involved in the regulation of whisker movements.

Neuroanatomical tract-tracing methods were used to identify the oligosynaptic circuitry by which the whisker representation of the motor cortex (wMCx) influences the facial motoneurons that control whisking activity (wFMNs). Injections of the retrograde tracer cholera toxin subunit B into physiologically identified wFMNs in the lateral facial nucleus resulted in dense, bilateral labeling throughout the brainstem reticular formation and in the ambiguus nucleus as well as predominantly ipsilateral labeling in the paralemniscal, pedunculopontine tegmental, Kölliker-Fuse, and parabrachial nuclei. In addition, neurons in the following midbrain regions projected to the wFMNs: superior colliculus, red nucleus, periaqueductal gray, mesencephalon, pons, and several nuclei involved in oculomotor behaviors. Injections of the anterograde tracer biotinylated dextran amine into the wMCx revealed direct projections to the brainstem reticular formation as well as multiple brainstem and midbrain structures shown to project to the wFMNs. Regions in which retrograde labeling and anterograde labeling overlap most extensively include the brainstem parvocellular, gigantocellular, intermediate, and medullary (dorsal and ventral) reticular formations; ambiguus nucleus; and midbrain superior colliculus and deep mesencephalic nucleus. Other regions that contain less dense regions of combined anterograde and retrograde labeling include the following nuclei: the interstitial nucleus of medial longitudinal fasciculus, the pontine reticular formation, and the lateral periaqueductal gray. Premotoneurons that receive dense inputs from the wMCx are likely to be important mediators of cortical regulation of whisker movements and may be a key component in a central pattern generator involved in the generation of rhythmic whisking activity.

Prefrontal cortical regulation of hypothalamic-pituitary-adrenal function in the rat and implications for psychopathology: side matters.

In recent years, dysfunction of hypothalamic-pituitary-adrenal (HPA) axis function has been implicated in a wide variety of psychiatric conditions. The importance of this system in responding to and coping with stress is well documented, and the integrity of such systems is of obvious significance to good mental health. The prefrontal cortex (PFC) is also heavily implicated in numerous psychopathological conditions. There is thus a growing interest in the potential role the PFC might play in regulating HPA function, and whether abnormalities of these systems are linked. The present paper reviews a number of recent animal studies which have attempted to elucidate the role of the PFC in regulation of HPA axis function, and how these systems may interact. It is concluded that the PFC is involved both in activating HPA responses to stress and in the negative feedback regulation of this system. Cerebral laterality is an important feature of this regulation, with the right PFC being most directly linked to stress-regulatory systems. On this basis, a number of parallels are drawn to the human literature, where asymmetrical disturbances in PFC activity may help explain associated patterns of HPA dysfunction.

Nuclear and cortical regulation in doublets of Paramecium: II. When and how do two cortical domains reorganize to one?

Homopolar doublets with twofold rotational symmetry were generated in Paramecium tetraurelia and in P. undecaurelia by electrofusion or by arrested conjugation. These doublets underwent a complex cortical reorganization over time, which led to their reversion to singlets. This reorganization involved a reduction in number of ciliary rows, a progressive inactivation and loss of one oral meridian, and a reduction and eventual disappearance of one cortical surface (semicell) situated between the two oral meridians. The intermediate steps of this reorganization included some processes that resemble those previously described in regulating doublets of other ciliates, and others that are peculiar to members of the "P. aurelia" species-group and some of its close relatives. The former included a disappearance of one cortical landmark (a contractile vacuole meridian) and transient appearance of another (a third cytoproct) within the narrower semicell. The latter included a reorganization of the paratene zone and the associated invariant (non-duplicating) region to occupy the entire narrower semicell and a redistribution of zones of most active basal-body proliferation within the opposite, wider semicell. The final steps of reorganization involved anterior displacement, invagination, and resorption of one of the two oral apparatuses and eventual disappearance of the associated oral meridian. An oral meridian deprived of its oral apparatus, either by spontaneous resorption or microsurgical removal, could persist for some time in "incomplete doublets" before regulating to the singlet condition. The phylogenetically widespread events encountered in the regulation of doublets to singlets suggest that Paramecium shares some of the global regulatory properties that are likely to be ancestral in ciliates. The more specific events are probably associated with the complex cytoskeletal architecture of this organism and with the frequent occurrence of autogamy that was described in the preceding study (Prajer et al. 1999).

Motor cortex excitability correlates with an anxiety-related personality trait

Background: In an earlier study comparing obsessive-compulsive disorder (OCD) patients to psychiatrically screened normals, we found lowered motor evoked potential (MEP) threshold to transcranial magnetic stimulation (TMS) and decreased intracortical inhibition in OCD. We sought to determine whether this pattern was specific to OCD. Methods: We measured the threshold and amplitude of MEPs to single and paired (subthreshold-suprathreshold; 3, 4, 10, 15 msec intervals) TMS in 46 healthy volunteers (23 women, 23 men) who were given the NEO-PI-R personality inventory. Nineteen of the men also received cognitive and motor tests. Results: The paired-pulse conditioned/unconditioned MEP amplitude ratios correlated with Neuroticism (N), a stable measure of trait-level anxiety and other negative emotions, in the whole sample ( r = 0.48; p = 0.0006), and in the men ( r = 0.63; p = 0.0009). There were no other significant correlations. Conclusions: This relationship reflects a factor that contributes to both personality and cortical regulation. It was not statistically significant in women, probably because of confounding hormonal influences on excitability. Decreased intracortical inhibition may be related more to trait anxiety and depression, which are high in OCD, than to OCD itself. However, the MEP threshold (significantly lowered in OCD) was unrelated to N.

Current issues on epileptic women.

Issues linked to epileptic women are being reviewed. Ovarian steroid hormones have a number of effects on the brain that predispose to epileptic activity. In particular, estradiol produces changes in the hippocampus synapses predisposing hyperexcitability associated with seizures. Also, menses and menopause periods, in which there are changing levels of steroid ovarian hormones, are associated with a particular appearing of seizures (catamenial epilepsy) and with phenotypic changes of previous ones. Epilepsy can affect the reproductive system, inducing endocrinal abnormalities (through disruption of cortical regulation of hypothalamus hormone release, and changes in the central nervous system concentration of steroid hormones induced by antiepileptics), infertility (linked to abnormalities in menstrual cycle or to the occurrence of polycytic ovaries, particularly in association with valproate treatment), and sexual disfunction (namely related to physiologic defects). Oral hormonal contraceptives should be performed using a pill with > 50mg of estrogen in order to prevent its potential loss of efficacy induced by enzyme-inducing antiepileptics. Concerning pregnancy, some topics should be discussed with, and advised to epileptic women, including: the possibility of withdrawal antiepileptics and the need of folic acid supplementation when planning a pregnancy; the risk of increased seizure frequency during pregnancy, and of the occurrence of obstetric complications; the increased risk of teratogenesis associated with antiepileptic therapy (mainly if in polytherapy); the need of vitamin K supplementation during the last month of pregnancy in order to avoid newborn haemorrhages; and the general absence of risk of breastfeeding even under sustained antiepileptic therapy.

Striatal nitric oxide signaling regulates the neuronal activity of midbrain dopamine neurons in vivo.

A major component of the cortical regulation of the nigrostriatal dopamine (DA) system is known to occur via activation of striatal efferent systems projecting to the substantia nigra. The potential intermediary role of striatal nitric oxide synthase (NOS)-containing interneurons in modulating the efferent regulation of DA neuron activity was examined using single-unit recordings of DA neurons performed concurrently with striatal microdialysis in anesthetized rats. The response of DA neurons recorded in the substantia nigra to intrastriatal artificial cerebrospinal fluid (ACSF) or drug infusion was examined in terms of mean firing rate, percent of spikes fired in bursts, cells/track, and response to electrical stimulation of the orbital prefrontal cortex (oPFC) and striatum. Intrastriatal infusion of NOS substrate concurrently with intermittent periods of striatal and cortical stimulation increased the mean DA cell population firing rate as compared with ACSF controls. This effect was reproduced via intrastriatal infusion of a NO generator. Infusion of either a NOS inhibitor or NO chelator via reverse microdialysis did not affect basal firing rate but increased the percentage of DA neurons responding to striatal stimulation with an initial inhibition followed by a rebound excitation (IE response) from 40 to 74%. NO scavenger infusion also markedly decreased the stimulation intensity required to elicit an IE response to electrical stimulation of the striatum. In single neurons in which the effects of electrical stimulation were observed before and after drug delivery, NO antagonist infusion was observed to decrease the onset latency and extend the duration of the initial inhibitory phase induced by either oPFC or striatal stimulation. This is the first report showing that striatal NO tone regulates the basal activity and responsiveness of DA neurons to cortical and striatal inputs. These studies also indicate that striatal NO signaling may play an important role in the integration of information transmitted to basal ganglia output centers via corticostriatal and striatal efferent pathways.

Gating of information flow within the limbic system and the pathophysiology of schizophrenia

Although first thought of as a dopaminergic disorder, there is little direct evidence to support a primary pathology in the dopamine system as the etiological factor in schizophrenia. In contrast, evidence is amassing in support of a cortical disturbance in this disorder; one consequence of which is a disruption in the cortical regulation of subcortical dopamine systems. Our studies show that the hippocampus plays a major role in this interaction, in that, along with the dopamine system, it provides a gating influence over information flow from the prefrontal cortex at the level of the nucleus accumbens. Moreover, chemically-induced disruption of the development of the hippocampus and entorhinal cortex were found to lead to pathophysiological changes in these interactions in the limbic system of adult rats. Therefore, schizophrenia is proposed to be a developmentally-related disorder, in which disruption of the hippocampal influence over the limbic system during ontogeny results in a pathological alteration of corticoaccumbens interactions in the adult organism.

Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders.

There is abundant evidence for abnormalities of the norepinephrine (NE) and serotonin (5HT) neurotransmitter systems in depression and anxiety disorders. The majority of evidence supports underactivation of serotonergic function and complex dysregulation of noradrenergic function, most consistent with overactivation of this system. Treatment for these disorders requires perturbation of these systems. Reproducible increases in serotonergic function and decreases in noradrenergic function accompany treatment with antidepressants, and these alterations may be necessary for antidepressant efficacy. Dysregulation of these systems clearly mediates many symptoms of depression and anxiety. The underlying causes of these disorders, however, are less likely to be found within the NE and 5HT systems, per se. Rather their dysfunction is likely due to their role in modulating, and being modulated by, other neurobiologic systems that together mediate the symptoms of affective illness. Clarification of noradrenergic and serotonergic modulation of various brain regions may yield a greater understanding of specific symptomatology, as well as the underlying circuitry involved in euthymic and abnormal mood and anxiety states. Disrupted cortical regulation may mediate impaired concentration and memory, together with uncontrollable worry. Hypothalamic abnormalities likely contribute to altered appetite, libido, and autonomic symptoms. Thalamic and brainstem dysregulation contributes to altered sleep and arousal states. Finally, abnormal modulation of cortical-hippocampal-amygdala pathways may contribute to chronically hypersensitive stress and fear responses, possibly mediating features of anxiety, anhedonia, aggression, and affective dyscontrol. The continued appreciation of the neural circuitry mediating affective states and their modulation by neurotransmitter systems should further the understanding of the pathophysiology of affective and anxiety disorders.

Consequences of altered cerebellar input for the cortical regulation of motor coordination, as reflected in EEG potentials.

The cerebellum is certainly involved in fine coordination of movements, but has no efferences of its own to the muscles. Thus, it can exert its influence only via other cerebral areas that have those efferences. This study investigated in patients with cerebellar atrophy how cortical motor areas are affected by dysfunction of the cerebellum. The main question was whether the patients' slow cortical electroencephalogram (EEG) potentials during key-press preparation and execution would be generally altered or would be specifically altered when fine coordination was needed. In the coordination task, right- and left-hand keys had to be pressed simultaneously with different forces, under visual feedback. Control tasks were to press with both hands equally or with one hand only. The patients indeed had a performance deficit in the coordination task. Their cortical EEG potentials were already drastically reduced in the simple tasks, but were enhanced by the same amount as in healthy subjects when more coordination was needed. These results suggest that the cerebellum is not exclusively active in fine coordination, but is generally involved in any kind of preparatory and executive activity, whereas the motor cortex becomes more active with fine coordination. The role of the cerebellum might be to provide the motor cortex with information needed for coordinating movements. In cerebellar atrophy, this altered input may be sufficient for the motor cortex in controlling simple tasks, but not for complex ones.

Blockade of N-methyl- d-aspartate receptors in the insular cortex disrupts taste aversion and spatial memory formation

The present experiments examined the effects of direct intracortical microinjections of the N-methyl- d-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid directly into the insular cortex of rats, before or immediately after training of conditioned taste aversion and the water maze spatial learning task. In the first series of experiments animals received bilateral injections of 2-amino-5-phosphonovaleric acid prior to taste aversion conditioning or spatial training. A strong disruptive effect was found in the acquisition of training tasks. To determine the possible involvement of N-methyl- d-aspartate receptors in the early post-training processes taking place in the cortex during both learning paradigms, in a second series of experiments, animals received bilateral 2-amino-5-phosphonovaleric acid microinjections 30, 60 or 120 min after the acquisition trial, and 15 min before the retention test. For spatial learning successive treatments were independently done either starting at the onset of the asymptotic phase of the learning curve, 0, 30 or 120 min after finishing the training session, as well as 15 min before the retention test trial. The conditioned taste aversion task remained sensitive to N-methyl- d-aspartate blockade during a period of at least 2 h after the first presentation of the gustatory stimulus, while in the case of the spatial learning task, a gradually decreasing effect was observed from the onset of the asymptotic phase onwards. Taken together, these results provide direct evidence for N-methyl- d-aspartate receptor involvement in cortical regulation of memory formation. Furthermore, our results suggest that in the same cortical region, a different time-course for the activation of N-methyl- d-aspartate-dependent mechanisms occurs during the early formation of cortically mediated memories, depending on the particular behavioural task.

Nuclear and cortical regulation in doublets of Paramecium: I. Interactions between macronucleus and contractile vacuoles and their implication on the frequency of autogamy

Summary The macronucleus of ciliates is a huge “bag” of DNA whose mode of segregation during cell division is still not understood (no chromosome condensation, no spindle, no centrosomes or functional equivalents). We unexpectedly obtained some new hints on this process through the analysis of doublets of Paramecium . We had previously noted [32] that autogamy occurred at a seemingly higher frequency in regulating lines. In the present work, we studied this process in P. tetraurelia and in P. undecaurelia which has a long, more convenient, interautogamous interval. We confirmed a highly significant decrease in duration of the interautogamous interval in both species and tried to determine its causes. The process of regulation which concerns the return of doublet state to the singlet one was previously described in part [24. 61, 35]. We observed its course through successive vegetative and sexual cycles. We first described precisely the contractile vacuole (CV) systems, and their number, extension and relations with the nuclear division events, in normal cells and in tam mutant cells of P. tetraurelia which are known to show abnormal nuclear division [6, 58]. We deduced that cortical and nuclear events are correlated during division by means of the contractile vacuole system (immunocytology and TEM observations). In regulating doublets, the enlarged cell volume leads to an increase in the number of CVs on two meridians M1, M2 and to the changes of their positioning and extension. CVM2 disappears and the number of CVs decreases when the angle between both oral apparatuses decreases from 180° towards 90°. Our results strongly suggest that abnormal number, size and position of macronuclei in doublets are related to number, spreading and positioning of the CVs. During division of doublets, the macronuclei appear to be “quartered” between both dorsal surfaces bearing the CVMs, and this leads to gross asymmetries in the distribution of the macronuclear DNA to the two daughter doublets [8]. Abnormal numbers of micronuclei and macronuclear anlagen are observed along with the evolution of the regulation process and, in turn, appear to lead to numerous anomalies at autogamy, a process which also requires correct intracellular positioning of meiotic and postmeiotic nuclei. From such observations, we deduced that contractile vacuoles, through their microtubular rootlets, exert a major function in ensuring proper macronuclear segregation during amitosis, such as the role played by “atractophores” in other protists during extranuclear pleuromitosis [57].