Phyllis K. Sher

Successful Treatment of Giggle Incontinence With Methylphenidate

AbstractPurpose: Giggle incontinence, the second most common type of childhood enuresis unrelated to disease, is notoriously difficult to treat. However, the association of laughter or emotion precipitated alteration of muscle tone is suggestive of a functional relationship to cataplexy, a part of the narcoleptic syndrome complex that may respond to stimulant medication.Materials and Methods: Two boys and 5 girls (mean age 10.9 years) with giggle incontinence, a positive family history of giggle incontinence (4 patients) and no evidence of urological disease were treated with methylphenidate for 1 to 5 years.Results: All patients responded positively with complete cessation of enuresis to varying dose schedules of methylphenidate.Conclusions: These results suggest that giggle incontinence is a centrally mediated and likely hereditary disorder that may share a common pathophysiological basis with the narcolepsy/cataplexy syndrome.

EEG Changes and Seizure Exacerbation in Young Children Treated with Carbamazepine

Summary: Carbamazepine (CBZ) has been reported to exacerbate some seizure types in children. We studied the correlation between CBZ‐associated EEG changes and seizure exacerbation in 59 children aged <6 years treated with CBZ. All patients had EEGs before and after initiation of treatment; initial EEGs were not significantly different among the patients. In 33 children (56%), the subsequent EEGs were either unchanged or improved or demonstrated minor changes (Group I), and excellent to complete seizure control was achieved in 67% of patients. In 26 children (44%), the EEG became significantly more abnormal and was characterized predominantly by new appearance of generalized spikelpolyspike‐and‐wave discharges (group 11). The majority of these patients (65%) experienced seizure exacerbation (p <0.001). For group I, symptomatic partial epilepsy, idiopathic focal epilepsy, and complex febrile seizures were significantly more common; in group 11, cryptogenic seizure disorders were more common (p < 0.005). Children in group I were more likely to remain on CBZ or to be weaned from medication after successful treatment, whereas children in group II required additional medication(s) or complete discontinuation of CBZ. Our results suggest that new appearance of generalized paroxysmal discharges after treatment is highly correlated with seizure exacerbation or suboptimal control as well as with adverse outcome. Conversely, absence of significant EEG deterioration on CBZ is usually associated with good seizure control.

Properties of [3H]diazepam binding sites on cultured murine glia and neurons

[3H]Diazepam binding was assayed in situ on living cultures of fetal mouse cerebral cortex or glia in an attempt to further characterize the high and low affinity binding sites. Mixed neuronal-glial cultures were found to have a high (Kd approximately equal to 10 nM) as well as a low (Kd approximately equal to 240 nM) affinity binding site. Glial cultures also had a similarly high affinity site (Kd 13 nM). In both types of cultures, the high affinity site was Ro 5-4864 sensitive and clonazepam resistant. Since Ro 5-4864 has particular affinity for non-neuronal elements and clonazepam for neuronal elements, the data suggest that the high affinity binding site may be localized to glial elements and the low affinity site primarily neuronal.

Chronic hypoxia in neuronal cell culture metabolic consequences

Cerebral cortical cell cultures obtained from fetal mice were subjected to 5% O2 for 24 h at a developmental stage equivalent to that of the human neonate. Immediately after the hypoxic insult (HI), medium lactate was elevated and pH, partial pressure of oxygen, and bicarbonate concentration were depressed compared to controls. At this time, the cultures evidenced a modest reduction in high-affinity GABA uptake but minimal morphologic or other biochemical evidence of cellular dysfunction. Within 24 h of restitution of normoxia, there was prominent disruption of neuronal integrity as well as significant reductions in benzodiazepine (BDZ) receptor binding, clonazepam-displaceable (CLO) BDZ binding, high-affinity uptake of GABA and beta-alanine, choline acetyltransferase (ChAT) activity, and total protein. Except for the neuronal marker CLO, GABA uptake was depressed more than other parameters for the subsequent 72 h. In contrast, the non-neuronal marker, Ro5-4864-displaceable BDZ binding, was always increased. Both morphologic and biochemical changes occurred independent of correction of pH, bicarbonate, and lactate. These data suggest that chronically hypoxic nervous tissue in vitro exhibits considerable delay in the evolution of maximal abnormality but that a population of GABAergic cells may be relatively more vulnerable. Although glial cell stimulation may contribute to neuronal survival, it also is possible that, because of the temporal association with cellular dysfunction, restitution of normoxia may contribute to nervous tissue injury.

The effects of acidosis on chronically hypoxic neurons in culture

Neuropathologic changes associated with perinatal hypoxic-ischemic events in the human infant most often result from chronic hypoxia rather than from acute asphyxia. To characterize the effects of acidosis associated with chronic hypoxia in developing neurons, cerebral cortical cultures obtained from fetal mice were exposed to 5% O2 continuously for either 24 or 48 h at 10 days after plating. At the conclusion of the hypoxic insult (HI), neuronal morphology was relatively intact for both conditions even though culture medium reflected significant reductions in pH and bicarbonate with elevation of lactate; cultures exposed to the longer HI manifested statistically greater aberrations from control values. Total benzodiazepine (BDZ) binding and clonazepam (CLO)-displaceable BDZ binding, reflecting the neuronal component of the receptor, were only modestly reduced immediately after HI, but were thereafter significantly and progressively lower over the 72 h normoxic recovery period. Although neuronal integrity was progressively diminished with both insults, morphology was always more normal and CLO higher in cultures subjected to 48-h HI compared to the 24-h HI (34.0 +/- 9.8 vs 1.8 +/- 1.1% of control values at 72 h, respectively; P less than 0.001). In contrast, values obtained for the glial marker Ro5-4864-displaceable BDZ binding were higher than control values for both conditions. Outcome was not influenced by removal of acidotic medium, nor by normalization of lactate. These data suggest that, with time, neurons in vitro adapt to severe hypoxia and that alterations in pH, lactate, and bicarbonate by themselves are probably not neurotoxic since the most acidotic cultures exhibited improved survival.

Benzodiazepine receptor development in murine glial cultures.

Benzodiazepine (BDZ) receptor binding characteristics were determined from glial cultures prepared from the cerebral hemispheres of newborn mice. Receptor binding and saturation analyses were performed at various ages in culture on intact cells. Utilizing 5 nM 3H-diazepam at 0.4 degrees C, specific binding reached a plateau at 16-21 days after plating. A single high-affinity binding site was identified with Kd 25.3 +/- 2.6 nM and Bmax 7,575 +/- 410 fmol/mg protein. Inhibition studies utilizing clonazepam indicated that this ligand, thought to have affinity exclusively for neurons, displaces more than 20% of the specific BDZ binding at concentrations as low as 300 nM, although the IC50 was 1.5-2.0 microM. In contrast, the IC50 for Ro5-4864 was 10-20 nM.

Autoradiographic Localization of Benzodiazepine Receptor Binding in Dissociated Cultures of Fetal Mouse Cerebral Cortex

Abstract: Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 μM) and Ro 5‐4864 (0.1 μM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58‐68% of specific grains over background cells and Ro 5‐4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines.

Characteristics of benzodiazepine receptor binding in living cultures of mouse cerebral cortex at physiologic temperature

Benzodiazepine (BDZ) receptor binding was assayed in situ in dispersed cultures of fetal mouse cerebral cortex at 37 degrees C and at 0 degrees C and compared. Contrary to data obtained from disrupted tissues, receptor binding at physiologic temperature was reduced only to 40% of that observed in the cold when intact tissues were used for assay. Neuronal (clonazepam-displaceable) receptor binding was essentially unaffected by changes in temperature; however, non-neuronal (Ro5-4864-displaceable) binding was reduced to 25% of that of 0 degrees C. A relatively high-affinity (Kd approximately 24 nM) as well as a low-affinity (Kd approximately 200 nM) binding site were identified; the numbers of binding sites were only modestly reduced at physiologic temperature and probably reflect a predominant reduction in non-neuronal sites.

Long-term exposure of cortical cell cultures to clonazepam reduces benzodiazepine receptor binding

To determine if reduced drug efficacy after long-term exposure to clonazepam may be a consequence of benzodiazepine receptor alterations, cerebral cortical cell cultures were exposed to the drug (200 nM) for 14 days. Receptor binding was assayed on living cells in situ. After drug exposure, binding in experimental cultures differed markedly from controls with respect to total, specific, and clonazepam-displaceable (neuronal) benzodiazepine binding (60%, 53%, and 6% of control values, respectively) but recovered within 96 h of drug removal. RO5-4864-displaceable (nonneuronal) binding was modestly reduced at 0 time (72% of control), but returned to control values in 24 h. The differences in binding could be attributable to a relatively reduced affinity of the high-affinity binding site (Kd approximately 18 nM for controls and approximately 30 nM for drug-exposed cultures) but not to changes in the low-affinity binding site or to reduced numbers of receptors.

GABA accumulating neurons are relatively resistant to chronic hypoxia in vitro: An autoradiographic study

Whether there is preferential loss of certain types of nerve cells or specific cellular functions after hypoxic or ischemic insults remains unclear. To evaluate this phenomenon in vitro, the vulnerability of GABAergic neurons to hypoxia was investigated both quantitatively and with autoradiography. Immature neuronal cortical cultures obtained from fetal mice were subjected to chronic hypoxia (5% O2) for 24 h or 48 h and then returned to the normoxic condition for 48 h. The shorter hypoxic exposure resulted in significantly reduced numbers of neurons in comparison to the longer exposure and also to controls (29% and 26%, respectively; p less than 0.001). LDH efflux, a reliable indicator of cell damage, also was higher after the shorter exposure insult. Nevertheless, in these same 24 h hypoxic cultures there was prominent sparing of those neurons which accumulate GABA: by 48 h of recovery GABAergic neurons constituted 29.3 +/- 2.0% of the remaining neuronal population in comparison to 11.6 +/- 0.6 and 14.4 +/- 0.8% for controls and 48 h hypoxia, respectively; (p less than 0.001). Although total GABA uptake per neuron was significantly decreased after both types of insult, there was a concomitant increase in glial GABA uptake (i.e., that which could be displaced by beta-alanine). These observations suggest that certain GABAergic cortical neurons are relatively more resistant to chronic hypoxia than the general neuronal population and that depression of overall neuronal GABA uptake may be associated with enhanced glial GABA uptake.