Analysis of Metabolic Pathways by 13C-Labeled Molecular Probes and HRMAS Nuclear Magnetic Resonance Spectroscopy: Isotopologue Identification and Quantification Methods for Medical Applications.

The use of 13C-labeled molecular probes is essential to explore altered metabolic pathways in human pathologies. The analysis of the different 13C isotopologues resulting from these changes in metabolic pathways is essential to understand the different biological processes involved. We propose an NMR methodology consisting of eight different NMR experiments performed under HRMAS conditions to explore metabolic pathways in unprocessed pathological cells and tissues. This methodology has the potential to study human pathologies in the medical field and to enable the analysis of the mode of action of therapeutic treatments.

Matrix Metalloproteinase-11 Promotes Early Mouse Mammary Gland Tumor Growth through Metabolic Reprogramming and Increased IGF1/AKT/FoxO1 Signaling Pathway, Enhanced ER Stress and Alteration in Mitochondrial UPR.

Matrix metalloproteinase 11 (MMP11) is an extracellular proteolytic enzyme belonging to the matrix metalloproteinase (MMP11) family. These proteases are involved in extracellular matrix (ECM) remodeling and activation of latent factors. MMP11 is a negative regulator of adipose tissue development and controls energy metabolism in vivo. In cancer, MMP11 expression is associated with poorer survival, and preclinical studies in mice showed that MMP11 accelerates tumor growth. How the metabolic role of MMP11 contributes to cancer development is poorly understood. To address this issue, we developed a series of preclinical mouse mammary gland tumor models by genetic engineering. Tumor growth was studied in mice either deficient (Loss of Function-LOF) or overexpressing MMP11 (Gain of Function-GOF) crossed with a transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV) (MMTV-PyMT). Both GOF and LOF models support roles for MMP11, favoring early tumor growth by increasing proliferation and reducing apoptosis. Of interest, MMP11 promotes Insulin-like Growth Factor-1 (IGF1)/protein kinase B (AKT)/Forkhead box protein O1 (FoxO1) signaling and is associated with a metabolic switch in the tumor, activation of the endoplasmic reticulum stress response, and an alteration in the mitochondrial unfolded protein response with decreased proteasome activity. In addition, high resonance magic angle spinning (HRMAS) metabolomics analysis of tumors from both models established a metabolic signature that favors tumorigenesis when MMP11 is overexpressed. These data support the idea that MMP11 contributes to an adaptive metabolic response, named metabolic flexibility, promoting cancer growth.

Comparative analysis of resuscitation using human serum albumin and crystalloids or 130/0.4 hydroxyethyl starch and crystalloids on skeletal muscle metabolic profile during experimental haemorrhagic shock in swine: A randomised experimental study.

BACKGROUND: Protection against acute skeletal muscle metabolic dysfunction and oxidative stress could be a therapeutic target in volume expansion for severely bleeding patients. OBJECTIVES: This experimental pilot study in swine aims at comparing 130/0.4 hydroxyethyl starch (HES) with 4% albumin along with crystalloid perfusion for first-line volume expansion in haemorrhagic shock with a particular emphasis on oxidative stress and muscular mitochondrial function. DESIGN: Randomised experimental study. SETTING: Digestive Cancer Research Institute Preclinical Laboratory, Strasbourg University Hospital, France, from February 2012 to June 2013. ANIMALS: Twenty large white pigs. INTERVENTION: Pressure-controlled haemorrhagic shock and volume resuscitation using either 4% human serum albumin or 130/0.4 HES along with crystalloid perfusion were performed in 20 large white pigs. MAIN OUTCOME MEASURES: Muscular biopsy of gastrocnemius muscle was performed for metabolomics screening, mitochondrial respiratory chain assessment and electron spin resonance reactive oxygen species production along with arterial and venous reactive oxygen species production at baseline, at the completion of shock, at 90 min and at 180 min after volume expansion. RESULTS: There was no difference between the two groups in measurements of skeletal muscle superoxide production. In a pooled analysis, there was a statistically significant decrease in gastrocnemius muscle creatine content from baseline to 90 min (P < 0.05) and 180 min (P < 0.05). Muscular lactate content and mitochondrial respiratory chain oxidative capacity remained constant at the respective time points. CONCLUSION: In this pilot experimental study in swine, during pressure-controlled haemorrhagic shock treated with either albumin or 130/0.4 HES in conjunction with crystalloid perfusion, skeletal muscle metabolic profile was unaltered. ETHICAL APPROVAL NUMBER: 38.2012.01.031.

Gene expression mapping of histone deacetylases and co-factors, and correlation with survival time and 1H-HRMAS metabolomic profile in human gliomas.

Primary brain tumors are presently classified based on imaging and histopathological techniques, which remains unsatisfaying. We profiled here by quantitative real time PCR (qRT-PCR) the transcripts of eighteen histone deacetylases (HDACs) and a subset of transcriptional co-factors in non-tumoral brain samples from 15 patients operated for epilepsia and in brain tumor samples from 50 patients diagnosed with grade II oligodendrogliomas (ODII, n = 9), grade III oligodendrogliomas (ODIII, n = 22) and glioblastomas (GL, n = 19). Co-factor transcripts were significantly different in tumors as compared to non-tumoral samples and distinguished different molecular subgroups of brain tumors, regardless of tumor grade. Among all patients studied, the expression of HDAC1 and HDAC3 was inversely correlated with survival, whereas the expression of HDAC4, HDAC5, HDAC6, HDAC11 and SIRT1 was significantly and positively correlated with survival time of patients with gliomas. (1)H-HRMAS technology revealed metabolomically distinct groups according to the expression of HDAC1, HDAC4 and SIRT1, suggesting that these genes may play an important role in regulating brain tumorigenesis and cancer progression. Our study hence identified different molecular fingerprints for subgroups of histopathologically similar brain tumors that may enable the prediction of outcome based on the expression level of co-factor genes and could allow customization of treatment.

NMR HRMAS spectroscopy of lung biopsy samples: Comparison study between human, pig, rat, and mouse metabolomics.

PURPOSE: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy, we assessed the lung metabolome of various animal species in order to identify the animal model that could be substituted to human lung in studies on fresh lung biopsies. METHODS: The experiments were conducted on intact lung biopsy samples of pig, rat, mouse, and human using a Bruker Advance III 500 spectrometer. Thirty-five to 39 metabolites were identified and 23 metabolites were quantified. Principal component analysis, partial least-squares discriminant analysis, and analysis of variance tests were performed in order to compare the metabolic profiles of each animal lung biopsies to those of the human lung. RESULTS: The metabolic composition between human and pig lung was similar. However, human lung was distinguishable from mouse and rat regarding: Trimethylamine N-oxide and betaïne which were present in rodents but not in human lung, carnitine, and glycerophosphocholine which were present in mouse but not in human lung. Conversely, succinic acid was undetected in rat lung. Furthermore, fatty acids concentration was significantly higher in rodent lungs compared to human lung. CONCLUSION: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy on lung biopsy, samples allowed to highlight that pig lung seems to be close to human lung as regarding its metabolite composition with more similarities than dissimilarities.

Cold ischemia with selective anterograde in situ pulmonary perfusion preserves gas exchange and mitochondrial homeostasis and curbs inflammation in an experimental model of donation after cardiac death.

The aim of this study was to assess the functional preservation of the lung graft with anterograde lung perfusion in a model of donation after cardiac death. Thirty minutes after cardiac arrest, in situ anterograde selective pulmonary cold perfusion was started in six swine. The alveolo-capillary membrane was challenged at 3, 6, and 8 h with measurements of the mean pulmonary arterial pressure (mPAP), the pulmonary vascular resistance (PVR), the PaO2 /FiO2 ratio, the transpulmonary oxygen output (tpVO2 ), and the transpulmonary CO2 clearance (tpCO2 ). Mitochondrial homeostasis was investigated by measuring maximal oxidative capacity (Vmax ) and the coupling of phosphorylation to oxidation (ACR, acceptor control ratio) in lung biopsies. Inflammation and induction of primary immune response were assessed by measurement of tumor necrosis factor alpha (TNFα), interleukine-6 (IL-6) and receptor for advanced glycation endproducts (RAGE) in bronchoalveolar lavage fluid. Data were compared using repeated measures Anova. Pulmonary hemodynamics (mPAP: P = 0.69; PVR: P = 0.46), oxygenation (PaO2 /FiO2 : P = 0.56; tpVO2 : P = 0.46), CO2 diffusion (tpCO2 : P = 0.24), mitochondrial homeostasis (Vmax : P = 0.42; ACR: P = 0.8), and RAGE concentrations (P = 0.24) did not significantly change up to 8 h after cardiac arrest. TNFα and IL-6 were undetectable. Unaffected pulmonary hemodynamics, sustained oxygen and carbon dioxide diffusion, preserved mitochondrial homeostasis, and lack of inflammation suggest a long-lasting functional preservation of the graft with selective anterograde in situ pulmonary perfusion.

Caudate nucleus and social cognition: neuropsychological and SPECT evidence from a patient with focal caudate lesion.

Most studies in social cognition have focused on developmental diseases or analyzed the consequences of acquired frontal lesions on the integrity of Theory of Mind (ToM), but, to our knowledge, none to date has addressed the eventual consequences of damage to the basal ganglia on ToM. To investigate the possible consequences of such lesions on social cognition, we tested a selected patient, MVG, a 44-year-old man with a focal caudate nucleus (CN) lesion following stroke. In the aftermath of this stroke, MVG shows loss of empathy and difficulties recognizing emotions in others. The dual aims of this study were first, to evaluate the implications of CN on ToM and recognition of emotion, and second, to discuss these results as a consequence of a disconnection of the sub-cortical orbito-frontal (OF) loop due to caudate damage. We performed a complete neuropsychological assessment of MVG, as well as different tasks evaluating social cognition, such as the Faux-Pas Test and the Reading the Eyes in the Mind Test. No deficits were found in the neuropsychological tests. However, on tasks assessing social cognition, MVG showed impairments in the "warm" or "affective" part of ToM as well as in the ability to recognize negative emotions (i.e., sadness and fear). These results indicate that damage to the head of the left CN can lead to impairment of ToM and emotion recognition. Furthermore, the data shows that, in MVG, such impairment appears to be due to a disconnection of the sub-cortical OF circuit resulting from damage to the CN. Neuro-imaging data tends to confirm this hypothesis by bringing out a hypo-perfusion in both, the territory of his left CN and prefrontal (i.e., ventromedial) brain areas.

The assessment of the quality of the graft in an animal model for lung transplantation using the metabolomics 1H high-resolution magic angle spinning NMR spectroscopy.

Standards are needed to control the quality of the lungs from nonheart-beating donors as potential grafts. This was here assessed using the metabolomics 1H high-resolution magic angle spinning NMR spectroscopy. Selective perfusion of the porcine bilung block was set up 30 min after cardiac arrest with cold Perfadex®. Lung alterations were analyzed at 3, 6, and 8 h of cold ischemia as compared to baseline and to nonperfused lung. Metabolomics analysis of lung biopsies allowed identification of 35 metabolites. Levels of the majority of the metabolites increased over time at 4°C without perfusion, indicating cellular degradation, whereas levels of glutathione decreased. When lung was perfused at 4°C, levels of the majority of the metabolites remained stable, including levels of glutathione. Levels of uracil by contrast showed a reverse profile, as its signal increased over time in the absence of perfusion while being totally absent in perfused samples. Our results showed glutathione and uracil as potential biomarkers for the quality of the lung. The metabolomics 1H high-resolution magic angle spinning NMR spectroscopy can be efficiently applied for the assessment of the quality of the lung as an original technique characterized by a rapid assessment of intact biopsy samples without extraction and can be implemented in hospital environment.

SPECT assessment of brain activation induced by caffeine: no effect on areas involved in dependence.

Caffeine is not considered addictive, and in animals it does not trigger metabolic increases or dopamine release in brain areas involved in reinforcement and reward. Our objective was to measure caffeine effects on cerebral perfusion in humans using single photon emission computed tomography with a specific focus on areas of reinforcement and reward. Two groups of nonsmoking subjects were studied, one with a low (8 subjects) and one with a high (6 subjects) daily coffee consumption. The subjects ingested 3 mg/kg caffeine or placebo in a raspberry-tasting drink, and scans were performed 45 min after ingestion. A control group of 12 healthy volunteers receiving no drink was also studied. Caffeine consumption led to a generalized, statistically nonsignificant perfusion decrease of 6% to 8%, comparable in low and high consumers. Compared with controls, low consumers displayed neuronal activation bilaterally in inferior frontal gyrus-anterior insular cortex and uncus, left internal parietal cortex, right lingual gyrus, and cerebellum. In high consumers, brain activation occurred bilaterally only in hypothalamus. Thus, on a background of widespread low-amplitude perfusion decrease, caffeine activates a few regions mainly involved in the control of vigilance, anxiety, and cardiovascular regulation, but does not affect areas involved in reinforcing and reward.

Pathogenesis and pharmacology of epilepsy in the lithium-pilocarpine model.

To try to identify the critical structures during epileptogenesis, we used the lithium-pilocarpine model that reproduces most clinical and neuropathological features of temporal lobe epilepsy (TLE). We used imaging techniques as well as a disease modifying approach and pharmacological strategy. With [14C]-2-deoxyglucose autoradiography, we assessed changes in cerebral glucose utilization. T2-weighted magnetic resonance imaging (MRI, 4.7 T) allowed follow-up of structures involved in epileptogenesis. A potential disease-modifying effect was studied using preconditioning with brief seizures (amygdala kindling, maximal electroshocks) and pharmacological strategies including vigabatrin (250 mg/kg), caffeine (0.3 g/L in drinking water), topiramate (10-60 mg/kg), pregabalin (50 mg/kg followed by 10 mg/kg), or RWJ-333369 (10-120 mg/kg). In adult and PN21 rats that became epileptic, entorhinal, and piriform cortices were the initial structures exhibiting significant signal changes on MRI scans, from 6 h after status epilepticus (SE) onset, reflecting neuronal death. In PN21 rats that did not become epileptic, no signal occurred in parahippocampal cortices. In hippocampus, MRI signal change appeared 36-48 h after SE, and progressively worsened to sclerosis. During the latent and chronic phases, the metabolic level in the hilus of adult and PN21 epileptic rats was normal although neuronal loss reached 60-75%. Protection limited to CA1 and/or CA3 (caffeine, topiramate, vigabatrin, amygdala kindling) did not affect the latency to spontaneous seizures. Protection limited to the entorhinal and piriform cortices (pregabalin) delayed epileptogenesis. The combined protection of Ammon's horn and parahippocampal cortices (RWJ-333369) prolonged the latency before the onset of seizures in a dose-dependent manner or, in some cases, prevented the epilepsy. The entorhinal and piriform cortices are critically involved in the early phase of the epileptogenesis while the hilus may initiate and/or maintain epileptic seizures. Pharmacological protection of the basal cortices is necessary for a beneficial disease-modifying effect but this must be combined with protection of the hippocampus to prevent epileptogenesis in this model of TLE.

Neural responses associated with positive and negative emotion processing in patients with left versus right temporal lobe epilepsy.

Studies on emotion processing in patients with temporal lobe epilepsy have dealt mainly with the processing of negative emotions. To further understand the neural basis of emotional disorders in temporal lobe epilepsy, we studied patterns of brain activation induced by implicit processing of negative and positive emotions perceived through facial expressions and emotionally salient stimuli in candidates for surgical treatment of intractable epilepsy. Using functional MRI, we compared, in patients with mesial temporal lobe epilepsy and healthy subjects, the patterns of brain activation elicited by the implicit processing of fearful, sad, and happy faces and pleasant and unpleasant scenes. The results revealed different patterns of activation in patients with left and right mesial temporal lobe epilepsy, compared with healthy subjects, suggesting that the left and right mesial temporal regions are involved differently in emotion processing, which could be related to different contributions in emotional arousal.

Ictal and interictal perfusion variations measured by SISCOM analysis in typical childhood absence seizures.

Single photon emission computed tomography (SPECT) is currently used in the presurgical evaluation of medically intractable partial epilepsies, but not very often, in generalized epilepsy. In the present study, we used the SISCOM procedure, which represents the fusion of MRI and ictal-interictal difference SPECT images using (99m)Tc-ECD, to study cerebral blood flow changes during the ictal and postictal phases of typical childhood absence seizures. The study was performed on four children with typical, difficult to treat absence seizures, aged 10-13 years at the time of scan. The delay between the onset of absence seizures and the injection of (99m)Tc-ECD was carefully noted. One scan was performed during the ictal phase and showed diffuse blood flow decreases, while the three other scans performed during the postictal phase, showed generalized blood flow increase. These data are consistent with most previous data reporting generalized changes in functional activity, not limited to the thalamo-cortical circuit in which absence seizures originate, and a decrease in cerebral blood flow during the ictal phase. Our data are concordant with the hypothesis that neuronal activity underlying the occurrence of spike-and-wave discharges does not seem to require an increase in metabolic demand and blood flow rates. [Published with videosequences].

Hypothalamic response to experimental allergic encephalomyelitis: role of substance P.

Adjuvant-induced arthritis (AA) is thought to be a model for experimental chronic stress that has as main features decreased adrenocorticotropin hormone (ACTH) plasma levels and a rise in median eminence content of arginine vasopressin (AVP) due to the activity of substance P. In experimental allergic encephalomyelitis (EAE), another chronic stress model, the role of substance P action is not clear. In this paper we tried to clarify the role of substance P in Lewis rats, which are susceptible to this disease. EAE was induced using myelin basic protein plus complete Freund's adjuvant injected into the hind limbs. One day later injections of an antagonist to substance P (RP 67580), saline, and substance P were administered daily for 12-14 days through a stainless steel cannula into the lateral ventricle of the brain, and then the rats were killed. The rats were divided into groups of controls, sham, diseased controls (no intracerebroventricular injections) and EAE (injected intracerebroventricularly). Plasma was used for the quantification of ACTH and corticosterone but not AVP which was assayed in hypothalamic median eminence extracts. In noninjected diseased rats the plasma levels of ACTH and corticosterone were significantly higher than in noninjected control rats, whereas the AVP concentrations in the median eminence were unchanged. The substance P antagonist did not affect the levels of these hormones in plasma or the median eminence. Substance P decreased the plasma levels of ACTH and corticosterone but did not increase the median eminence content of vasopressin. Administration of the antagonist 30 min before an equivalent dose of substance P increased the plasma levels of the two hormones, but did not change the content of AVP. Based on the lack of response to the antagonist RP 67580 we suggest that the substance P has different roles in EAE and AA at least in the later stages of EAE (after 11 days of immunization).

In the lithium-pilocarpine model of epilepsy, brain lesions are not linked to changes in blood-brain barrier permeability: an autoradiographic study in adult and developing rats.

Lithium-pilocarpine-induced status epilepticus (SE) leads to the genesis of massive neuronal loss in adult rats and to a lesser extent in P21 rats. Neuronal damage occurs mainly via a process of necrosis in limbic forebrain, cerebral cortex, thalamus, and substantia nigra. It is not known, however, whether damage is the result of local excitotoxic hyperactivity or if leakage at the blood-brain barrier (BBB) could participate in the damaging process. Therefore, we investigated the permeability of the BBB in adult and P21 rats using [alpha-(14)C]aminoisobutyric acid, which does not cross an intact BBB, at 90 min after the onset of SE. At both ages, BBB opening occurred both in structures that will undergo damage (thalamus, septum, amygdala) and structures that will not be injured (globus pallidus, hypothalamus). In addition, neuronal damage occurs in the absence of increased BBB permeability in hippocampus, entorhinal cortex, and substantia nigra. Moreover, the increase in the intensity and distribution of BBB permeability changes is age-related, suggesting a differential activation of seizure circuits in adult and P21 rats. In summary, there is no clear correlation between the anatomical distribution of BBB opening and the occurrence of neuronal damage which, in this model, appears to rather depend on excitotoxic mechanisms due to major neuronal hyperexcitability.

Predictive value of cortical injury for the development of temporal lobe epilepsy in 21-day-old rats: an MRI approach using the lithium-pilocarpine model.

PURPOSE: Patients with temporal lobe epilepsy (TLE) usually had an initial precipitating injury in early childhood. However, epilepsy does not develop in all children who have undergone an early insult. As in patients, the consequences of the lithium-pilocarpine-induced status epilepticus (SE) are age dependent, and only a subset of 21-day-old rats will develop epilepsy. Thus with magnetic resonance imaging (MRI), we explored the differences in the evolution of lesions in these two populations of rats. METHODS: SE was induced in 21-day-old rats by the injection of lithium and pilocarpine. T2-weighted images and T2 relaxation-time measurements were used for detection of lesions from 6 h to 4 months after SE. RESULTS: Three populations of rats could be distinguished. The first one had neither MRI anomalies nor modification of the T2 relaxation time, and these rats did not develop epilepsy. In the second one, a hypersignal appeared at the level of the piriform and entorhinal cortices 24 h after SE (increase of 49% of the T2 relaxation time in the piriform cortex) that began to disappear 48-72 h after SE; epilepsy developed in all these animals. The third population of rats showed a more moderate increase of the T2 relaxation time in cortices (14% in the piriform cortex) that could not be seen on T2-weighted images. Epilepsy developed in all these rats. Only in a subpopulation of the 21-day-old rats with epilepsy did hippocampal sclerosis develop. CONCLUSIONS: These results suggest that the injury of the piriform and entorhinal cortices during SE play a critical role for the installation of the epileptic networks and the development of epilepsy.

Correlation between PET and SISCOM in temporal lobe epilepsy.

UNLABELLED: The subtraction of interictal from ictal SPECT coregistered to 3-dimensional (3D) MRI (SISCOM) and (18)F-FDG PET are 2 techniques that are involved in the definition of the epileptogenic zone in refractory partial temporal lobe epilepsy (TLE). The aim of this study was compare, region by region, the functional patterns obtained by both strategies, SISCOM and PET, in patients with unilateral TLE. METHODS: Perfusion data using SISCOM and metabolic data using (18)F-FDG PET scans were acquired from 17 patients with unilateral TLE. The functional metabolism and perfusion maps were overlaid onto a 3D rendering of the patient's anatomic MRI scans. RESULTS: The functional patterns that were observed with PET and SISCOM were found mainly in the ipsilateral and contralateral temporal lobes, in the orbitofrontal and insular cortices. Despite the high rate of concordance, in some cases discrepancies could be observed between PET and SISCOM abnormalities: In the mesial regions, as in the anterior neocortex, PET abnormalities were found more frequently than SISCOM changes. SISCOM abnormalities were found more frequently in the posterior temporal neocortex. In the extratemporal cortex, SISCOM abnormalities were more widespread. CONCLUSION: The marked rate of concordance between PET and SISCOM abnormalities observed in relatively extensive regions shows that, in TLE, seizures were generated and spread in metabolically abnormal regions.

Magnetic resonance imaging in the study of the lithium-pilocarpine model of temporal lobe epilepsy in adult rats.

PURPOSE: In temporal lobe epilepsy, it remains to be clarified whether hippocampal sclerosis is the cause or the consequence of epilepsy. We studied the temporal evolution of the lesions in the lithium-pilocarpine model of epilepsy in the rat with magnetic resonance imaging (MRI) to determine the progressive morphologic changes occurring before the appearance of chronic epilepsy. METHODS: MRI was performed on an MR scanner operating at 4.7 T. We followed the evolution of lesions using T(2)- and T(1)-weighted sequences before and after the injection of gadolinium from 2 h to 9 weeks. RESULTS: At 2 h after status epilepticus (SE), a blood-brain barrier breakdown could be observed only in the thalamus; it had disappeared by 6 h. At 24 h after SE, edema was present in the amygdala and the piriform and entorhinal cortices together with extensive neuronal loss; it disappeared progressively over a 5-day period. During the chronic phase, a cortical signal reappeared in all animals; this signal corresponded to gliosis, which appeared on glial fibrillary acidic protein (GFAP) immunohistochemically stained sections as hypertrophic astrocytes with thickened processes. In the hippocampus, the correlation between histopathology and T(2)-weighted signal underscored the progressive constitution of atrophy and sclerosis, starting 2 days after SE. CONCLUSIONS: These data show the reactivity of the cortex that characterizes the initial step leading to the development of epilepsy and the late gliosis that could result from the spontaneous seizures. Moreover, it appears that hippocampal sclerosis progressively worsened and could be both the cause and the consequence of epileptic activity.

Co-occurrence of infantile epileptic seizures and childhood paroxysmal choreoathetosis in one family: clinical, EEG, and SPECT characterization of episodic events.

The co-occurrence of infantile convulsions and childhood paroxysmal choreoathetosis (ICCA syndrome) has recently been reported in several families. The pattern of familial clustering observed is consistent with a single locus mutation which has been mapped onto the pericentromeric region of chromosome 16. We studied the main clinical, electroencephalogram (EEG), and single photon emission computed tomography (SPECT) characteristics of episodic events in a new family presenting clinical features similar to that described in the ICCA syndrome. In the first year of life, a mother and her two daughters suffered from rare afebrile seizures lasting from 30 seconds to 15 minutes. Ictal EEG recording in one daughter at 7 months of age showed bilateral polyspikes with a posterior predominance. In the three patients, epileptic seizures regressed within a few weeks, and never reoccurred. At the age of 7 and 12 years, respectively, the two daughters presented daily brief (20 seconds to 1 minute) involuntary choreoathetotic episodes. In 10 of these attacks, EEG did not show any epileptiform abnormalities. In both sisters, an ictal SPECT was performed during a choreoathetotic episode. Subtracting the ictal SPECT from the interictal SPECT coregistered to magnetic resonance imaging (MRI) revealed significant modifications in the local cerebral perfusion in the sensorimotor cortex, the supplementary motor areas, and pallidum. Carbamazepine completely suppressed paroxysmal dyskinesias. These observations, together with literature data, suggest that in this syndrome, depending on brain maturation, the same genetic abnormality may result in different paroxysmal neurological symptoms.