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M W Hill

Publications and source records attributed to M W Hill.

At least 19 recordsLinked to original sources

Rapid cooling aborts seizure-like activity in rodent hippocampal-entorhinal slices.

PURPOSE: As a preliminary step in the development of an implantable Peltier device to abort focal neocortical seizures in vivo, we have examined the effect of rapid cooling on seizures in rodent hippocampal-entorhinal slices. METHODS: Seizure-like discharges were induced by exposing the slices to extracellular saline containing 4-aminopyridine (50 micromol/L). RESULTS: When we manually activated a Peltier device that was in direct contact with the slice, seizures terminated within seconds of the onset of cooling, sometimes preceding a detectable decrease in temperature measured near the top of the slice. However, activation of the Peltier device did not stop seizures when slices were no longer in direct physical contact with the device, indicating that this was not a field effect. When cooling was shut off and temperature returned to 33 degrees C, bursting sometimes returned, but a longer-term suppressive effect on seizure activity could be observed. In two of our experiments, a custom computer program automatically detected seizure discharges and triggered a transistor-transistor logic pulse to activate the Peltier device. In these experiments, the Peltier device automatically terminated the slice bursting in less than 4 seconds. When the Peltier device was placed in contact with the normal, exposed cortex of a newborn pig, we found that the cortical temperature decreased rapidly from 36 degrees C to as low as 26 degrees C at a depth of 1.7 mm below the cooling unit. CONCLUSIONS: These experiments show that local cooling may rapidly terminate focal paroxysmal discharges and might be adapted for clinical practice.

Animals↗

Effects of anticonvulsant lactams on in vitro seizures in the hippocampal slice preparation.

Some 3,3-disubstituted 2-pyrrolidinones and 2-piperidinones (five- and six-membered ring lactams, respectively) possess potent in vivo anticonvulsant activity. In vitro these lactams potentiate GABA(A) receptor-mediated chloride currents, which is thought to be the mechanism by which they exert their therapeutic effects. However, the apparent affinity for these GABA(A) interactions is low: EC50s range from hundreds of micromolar to low millimolar values. In order to more completely characterize the activities of these compounds, it was necessary to know the concentrations required to curtail epileptiform activity in an intact neural network, and the mechanism by which this occurs. To address these questions, we used two methods of inducing ictal activity in hippocampal-entorhinal cortical slices: 4-aminopyridine (4-AP) and low Mg2+. We found that 3,3-diethyl-2-pyrrolidinone (diethyl-lactam) prevents seizure-like discharges with IC50s of 1.1 and 2.1 mM in the two models, respectively. These values are nearly identical to the EC50 value obtained in whole-cell studies of diethyl-lactam's GABA(A) receptor modulation. The addition of the GABA(A) antagonist picrotoxin to the low Mg2+ ACSF produced seizures which persisted during diethyl-lactam application. Neither 3-benzyl-3-ethyl-2-piperidinone (3-BEP) nor alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL), two compounds which are similar to diethyl-lactam, but demonstrate picrotoxin-insensitive inhibition of voltage-dependent currents, diminished low Mg2+/picrotoxin seizure activity. Our results support the hypothesis that diethyllactam and related compounds exert their anticonvulsant activity primarily, if not exclusively, by modulating the GABA(A) receptor.

4-Butyrolactone↗

Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.

Amino Acid Sequence↗

Contribution of subsaturating GABA concentrations to IPSCs in cultured hippocampal neurons.

The time course of EPSCs and IPSCs is at least partly determined by the concentration profile of neurotransmitter acting on postsynaptic receptors. Several recent reports have suggested that the peak synaptic cleft concentration of the inhibitory neurotransmitter GABA likely reaches at least 500 microM, a level that saturates the GABAA receptor. In the course of investigating the experimental anticonvulsant 3,3-diethyl-2-pyrrolidinone (diethyl-lactam), we have observed an important contribution to IPSC decay by subsaturating concentrations of GABA. Diethyl-lactam augments currents elicited by the exogenous application of subsaturating concentrations of GABA in voltage-clamped, cultured hippocampal neurons and significantly prolongs the decay of autaptic IPSCs and miniature IPSCs in our cultures. In addition, diethyl-lactam potentiates currents in excised outside-out membrane patches elicited by the prolonged application of low concentrations of GABA. However, when patches are exposed to 1-2 msec pulses of 1 mM GABA, diethyl-lactam does not alter current decay. Tiagabine, which blocks GABA reuptake, does not prolong IPSCs, so it is unlikely that uptake inhibition accounts for the enhancement of IPSCs. EPSCs and miniature IPSC frequency are unaffected by diethyl-lactam, again consistent with a postsynaptic site of action. We propose that during an IPSC, a substantial number of postsynaptic receptors must be exposed to subsaturating concentrations of GABA. A simplified model of GABAA receptor kinetics can account for the effects of diethyl-lactam on exogenous GABA and IPSCs if diethyl-lactam has its main effect on the monoliganded states of the GABAA receptor.

Animals↗

The diazoxide derivative 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-S,S-dioxide augments AMPA- and GABA-mediated synaptic responses in cultured hippocampal neurons.

The diazoxide derivative 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine-S,S-dioxide (IDRA21) enhances memory and learning in rodents, most likely by potentiating AMPAergic synaptic activity. We examined IDRA21's effect upon AMPAergic synaptic currents and whole-cell glutamate currents in cultured rat hippocampal neurons to determine whether IDRA21 was a partial modulator of AMPA receptor desensitization and deactivation. Comparable to cyclothiazide, IDRA21 prolonged AMPAergic autaptic currents (5.6 times control, EC50 150 microM) and slowed the rate of AMPA deactivation (3 times control) following 1-ms applications of 1 mM glutamate to excised, outside-out membrane patches. IDRA21 also augmented autaptic GABA currents by 27 +/- 8.1%, although it had two opposing effects, reducing the peak amplitude versus prolonging autaptic GABA currents. IDRA21 (200 microM) inhibited whole-cell GABA currents elicited by exogenously applied 1 mM GABA by 41 +/- 11%. At sufficient concentrations, IDRA21 reduced AMPA receptor desensitization and slowed the rate of deactivation, most consistent with full agonist activity with lower potency compared to cyclothiazide. IDRA21 slightly augments GABAergic synaptic currents.

Animals↗

Inhibition of voltage-dependent sodium channels by the anticonvulsant gamma-aminobutyric acid type A receptor modulator, 3-benzyl-3-ethyl-2-piperidinone.

3-Benzyl-3-ethyl-2-piperidinone (3-BEP) belongs to a family of compounds that includes alpha- substituted gamma-butyrolactones, gamma-thiobutyrolactones, 2-pyrrolidinones and hexahydro-2H-azepin-2-ones. Many of these drugs exhibit potent in vivo anticonvulsant activity in mice. Previous electrophysiological studies demonstrated that they potentiate gamma-aminobutyric acid- (GABA) mediated chloride currents. This GABAA receptor modulation was thought to be the main mechanism of anticonvulsant activity. We report that 3-BEP also modulates sodium channels. It decreased sodium currents in cultured rat hippocampal neurons in a voltage- and concentration-dependent manner. The drug's apparent affinity increased as neurons were depolarized. At a holding potential of -60 mV, the apparent IC50 was 487 microM. This concentration is comparable to its EC50 for GABAA modulation (575 microM). Current blockade occurred over all activation voltages tested. The steady state inactivation curve was shifted by 600 microM 3-BEP from V50 = -65.3 mV to -72.0 mV, and recovery from inactivation was slowed from tau = 4.9 to 12.8 msec. Sodium current inhibition was not observed for three related compounds, suggesting a degree of chemical specificity for this activity. We conclude that in addition to its known effects on GABAA receptors, 3-BEP modulates sodium channels. Therefore this compound may prevent seizures by both enhancing inhibition and diminishing neuronal excitability.

Animals↗

Synthesis and anticonvulsant activities of 3,3-dialkyl- and 3-alkyl-3-benzyl-2-piperidinones (delta-valerolactams) and hexahydro-2H-azepin-2-ones (epsilon-caprolactams).

A series of 3-substituted 2-piperidinone (delta-valerolactam) and hexahydro-2H-azepin-2-one (epsilon-caprolactam) derivatives were prepared and evaluated as anticonvulsants in mice. In the 2-piperidinone series, 3,3-diethyl compound 7b is the most effective anticonvulsant against pentylenetetrazole-induced seizures (ED50, 37 mg/kg; PI (TD50/ED50), 4.46), and 3-benzyl compound 4c (ED50, 41 mg/kg; PI, 7.05) is the most effective anticonvulsant against seizures induced by maximal electroshock. By contrast, none of the epsilon-caprolactams tested had anticonvulsant effects below doses causing rotorod toxicity. log P values were correlated with neurotoxicity and [35S]TBPS displacement, but not with anticonvulsant activity. Electrophysiological evaluations of selected compounds from each series indicated that both the delta-valero-lactams and epsilon-caprolactams potentiated GABA-mediated chloride currents in rat hippocampal neurons.

Animals↗

3,3-Dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones: a new class of anticonvulsant agents.

A series of 3,3-dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones (lactams) have been prepared and evaluated for their anticonvulsant activities. In the pentylenetetrazole mouse seizure model, 3,3-diethyl lactam 7c and 3-benzyl-3-ethyl lactam 7j are the most effective anticonvulsants (ED50 = 46 and 42 mg/kg, respectively) and have protective index (PI = TD50/ED50) values of 5.65 and 3.00, respectively. These protective index values compare favorably to those of the clinically used antiepileptic drugs ethosuximide (ED50 = 161 mg/kg), phenobarbital (ED50 = 22 mg/kg), and valproic acid (ED50 = 133 mg/kg), which have PI values of 2.35, 4.00, and 2.12, respectively. The benzyl compounds [3-substituents are Bn, H (7h); Bn, Me (7i); and Bn, Et (7j)] are also very effective anticonvulsants against seizures induced by maximal electroshock (ED50 = 41, 55, and 74 mg/kg, respectively) and have PI values of 3.51, 3.04, and 1.70, respectively. The corresponding PI values for phenobarbital and valproic acid are 1.37 and 5.18, respectively. As a class of anticonvulsants, the 3,3-disubstituted 2-pyrrolidinones have a broad spectrum of action and may be useful for the treatment of human epilepsies.

Animals↗

The effects of vinblastine in assessment of the influence of age on proliferative activity of murine palate and footpad epithelium.

Data concerning changes in the rate of cell proliferation of stratified epithelia with increasing age are conflicting. In the present study young (3-month-old) and old (22-month-old) C57Bl/6NNia male mice were injected intraperitoneally with 2, 3, 4 or 8 mg vinblastine sulfate/kg body weight and killed after 1.5, 3, 4.5 or 6 h. The number of arrested metaphase figures per 1000 basal cells was counted in histological sections. Data were analysed using a multivariate analysis of variance. There was a significant difference between the accumulation of mitotic figures in footpad epidermis and palate epithelium and both tissues contained an increased number of mitotic figures with increasing periods of accumulation at all dose levels. In the footpad epidermis neither the age of the animal nor the dose of vinblastine had a significant effect on the number of mitotic figures. In contrast, for palate epithelium the accumulation of mitotic figures was significantly less in the old mice compared with the young mice and at a dose of vinblastine of 2 mg/kg compared with the higher doses. There was a statistically significant interaction between the dose of vinblastine and its period of action. It was concluded that the different tissues manifest a differential sensitivity to vinblastine and that only palate epithelium showed a significant reduction in proliferative activity with age.

Aging↗

The influence of subepithelial connective tissues on epithelial proliferation in the adult mouse.

Subepithelial connective tissue is capable of modulating the pattern of histodifferentiation of stratified epithelia from adult animals, but it is not known whether the supporting connective tissue also influences epithelial proliferative activity. Epithelial and connective tissues of murine skin and oral mucosa, differing in their morphology and proliferative activity, were separated and heterotypically recombined prior to grafting to histocompatible hosts. After 3 or 8 weeks in situ, mitotic activity was determined following the administration of vinblastine sulfate. Although the mitotic activity in each of the epithelia could be modulated by some connective tissues, there was no distinct pattern of behavior. In combination with connective tissues from tongue or palate, the ear epidermis acquired a significantly increased mitotic activity. In contrast, when oral epithelia with high mitotic activity were recombined with dermal connective tissue, there was usually a significant reduction in proliferative activity. As there was no apparent association between mitotic activity and the induced changes in either organization or histodifferentiation, it is suggested that subepithelial connective tissue is capable of directly influencing the mitotic activity in the overlying epithelium.

Animals↗

Glucose metabolism and protein synthesis in stratified squamous epithelia from young and old mice.

Glucose metabolism in regionally distinct epidermis and oral epithelia of young adult and aged mice was studied using 5-3H glucose and differentially labelled 14C-glucose. The results obtained with 3H-glucose indicate there is active utilization of glucose by all the epithelia examined. However, an enhanced utilization of glucose was observed in old ear epidermis when compared to that in young mice. The measurements of respiratory 14CO2 in the young and old epidermis indicated that aerobic glycolysis was significantly greater in ear epidermis and buccal epithelium from old than from young mice. Pentose phosphate activity was significantly reduced in palate epithelium from old animals when compared with that of young animals. Incorporation of radioactively labelled leucine and histidine revealed some regional differences between the epithelia examined. However, no age-associated differences were observed in any of the tissues. It is concluded that the observed metabolic changes reflect regionally specific adaptations to local factors rather than representing a programmed biological event.

Aging↗

Influence of age on the morphology and transit time of murine stratified squamous epithelia.

To determine whether stratified squamous epithelia from aged animals differ from those of young animals, specimens of skin from the pinna of the ear, the back and footpad and mucosa from the palate, cheek and ventral surface of the tongue were excised from 10 young (3-4 month-old) and 10 old (23-24 month-old) C57B1/6NNia mice and prepared for light microscopy. Tracings were prepared of the nucleated cell compartment and epithelial thickness, the number of nucleated cells/mm2 surface, the basement membrane: surface ratio, cell density and the number of basal cells/mm basement membrane were determined. To evaluate the epithelial labelling index and tissue renewal, a further group of young and old mice were injected with 1 microCi/g [3H]-thymidine and killed after 1 h or 2, 4, 5, 6 or 8 days and sections were prepared for autoradiography. Whereas the epidermis from the ear and footpad showed a statistically-significant increase in thickness, the epithelium from the palate was thinner in the old animals. The other tissues examined showed no change. Cell density decreased with age in the palate; cell size increased with age in the ear and footpad. No statistically-significant differences in labelling index or minimum transit time were observed between young and old animals in any of the tissues. Thus, there is no single age-associated change in epithelial structure or renewal common to all epithelia.

Aging↗

Age-associated changes in Langerhans cells of murine oral epithelium and epidermis.

Oral mucosa and skin of older individuals are immunologically less responsive to a range of allergens, but it is not known whether this is due to changes in the number of Langerhans cells or to impaired cell function. EDTA-separated epithelial sheets from the cheek and palate mucosa, and from ear aN< footpad skin of three-month-old and 24-month-old C57BL/6NNia mice were stained for ATPase, beta-glucuronidase activity and Iab-surface antigen to demonstrate Langerhans cells. The general distribution of such cells was unchanged with age, but those in epithelia from the old mice were more varied in shape, with irregular celL bodies and more elongated dendritic processes. The numerical density of Langerhans cells in old mice was reduced by 30-59 per cent compared with that in young mice.

Aging↗

Carcinoma cells may modulate their supporting connective tissue.

The patterns of growth of two chemically-induced murine squamous cell carcinoma cell lines and their effect on a deep connective tissue graft bed were examined in syngeneic C57Bl/6 mice. The two cell lines displayed markedly different patterns of histo-differentiation but in vivo the poorly-differentiated line (FS) gave rise to tumours with much lower inocula than the well-differentiated cell line (A5). To evaluate the effect of the tumour cells on the graft bed a bioassay involving transplantation of epidermal sheets was used. Whereas the pretreatment of the deep connective tissue graft bed with the FS cell line facilitated the subsequent growth of the epidermal sheets, beds treated with the A5 cell line, lethally irradiated tumour cells or receiving no treatment failed to support continued growth of normal epidermis. We suggest that this ability of a carcinoma cell line to modify the connective tissue may facilitate the establishment of metastatic deposits.

Animals↗

Auditory evoked potentials recorded from conscious sheep.

The auditory evoked potentials produced in response to a click stimulus were measured in the conscious sheep. The early components, the brain stem evoked potentials (BSEPs) were reproducible and similar to those observed in the other animals as well as man and appeared to have frequencies that are the three subharmonics of the main frequency component of the click stimulus. The longer latency components were more variable in latency and amplitude, but the frequencies were similar to that observed in humans.

Animals↗