RESEARCH

In my lab at Smith, we have been investigating the molecular mechanisms that underlie the actions of general anesthetic agents in the mammalian brain. I previously studied mechanisms of volatile anesthetic action in the Franks lab , Biophysics Section, at Imperial College, Univ. London, UK. We used a variety of techniques to analyze the modulatory effects of anesthetic molecules on neuronal ion channels in vitro. These techniques included two-electrode-voltage clamp of Xenopus oocytes (frog eggs) injected with RNA/DNA encoding for a variety of ion channels, voltage-clamp experiments from cell lines transfected with GABA A receptor subunits (a, b and g), and recordings from acutely dissociated neurons in culture. At Smith, I am continuing to explore effects of volatile and intravenous agents on GABA A and glycine receptor combinations expressed in oocytes to highlight their importance as anesthetic targets.

I have also been working within the field of DNA microarrays, and my lab is currently bringing this technology to bear on some anesthetic related questions. Little is understood about the long-term side effects of general anesthetic agents (e.g. preconditioning, post-operative depression and hepatotoxicity of halothane) and some sedatives/anxiolytics (e.g. tolerance/dependence of the barbiturates, benzodiazepines). I have established a small microarray facility for broad screening of mammalian genomes to discover which genes are differentially expressed after long-term exposure to anesthetic or sedative agents.


Background and Significance
Questions for Future Research
Publications, Abstracts and Presentations

Alive but not Kicking: The Molecular Neurobiology of Anesthesia  (PDF file, requires Adobe Reader)

Background and Significance

Administration of an anesthetic is standard practice for rendering a patient unconscious prior to and during surgery. Although anesthetic agents have been used for over a 150 years, it is only recently that the molecular mechanisms for the action of these agents on the mammalian CNS are becoming understood.

It is now widely accepted that volatile anesthetics mediate their actions through direct interaction with membrane proteins rather than lipids (Franks and Lieb, 1998). Many neuronal ion channels are now recognized as likely candidate targets for anesthetic agents. At clinical concentrations, ligand-gated ion channels (e.g. GABA A, Glycine) are typically more sensitive to modulation by anesthetics (see Hall et al., 1994b) than the voltage gated family (see Hall et al., 1994a). Furthermore, it has been shown for ligand-gated channels that there are differential sensitivities to general anesthetics depending on the receptor subunit combinations that are expressed (Violet et al., 1997).

Another exciting feature of anesthetic/ligand-gated channel interactions is that receptors (e.g. GABA A) often exhibit differential modulation by the individual stereoisomers of optically active agents (e.g. isoflurane, '+' and '-' isomers, see Hall et al., 1994b). Interestingly the same stereoselective sensitivity has been reported for isoflurane in vivo where the '+' isomer a more effective anesthetic than the '-' for loss of righting reflex in rats (Dickinson et al., 2000). Therefore, the observations of modulation of ligand-gated responses (e.g. potentiation of GABA A currents in neurons) at the molecular level are likely explanations for general anesthesia in the whole animal.

Dickinson, R., White, I., Lieb, W.R. and Franks, N.P. 2000. Stereoselective loss of righting reflex in rats by isoflurane, Anesthesiology, v93, 837-843.

Franks, N.P. and Lieb, W.R. 1998. Which targets are most relvant to general anaesthesia? Toxicology, v100-101, 1-8.

Hall, A.C., Lieb, W.R. and Franks, N.P. (1994a) Insensitivity of P-type calcium channels to inhalational and intravenous general anesthetics. Anesthesiology, v81, 117-123.

Hall, A.C., Lieb, W.R. and Franks, N.P. (1994b) Stereoselective and non-stereoselective actions of isoflurane on the GABAA receptor. British Journal of Pharmacology, v112, 906-910.

Violet, J.M., Downie, D.L., Nakissa, R.C., Lieb, W.R. and Franks, N.P., 1997. Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics, Anesthesiology, v86, 866-874.


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Questions for Future Research

1. What are the critical structural components of potential anesthetic agents for potent positive modulation of GABA and glycine responses at their respective receptors?

   We have explored a variety of monoterpenoid analogues (e.g. menthol) for positive modulation of GABA A receptors and for their potential as anesthetic agents in vivo. For these investigations we use Xenopus oocytes that have been injected with RNA or DNA in order to express different GABA subunit combinations, and for in vivo assays, the loss of righting reflex in Xenopus tadpoles. Given some structural similarities between compounds like menthol and the intravenous anesthetic agent, propofol, these experiments have revealed some interesting structural requirements for anesthetic potency.
   

2. What molecular mechanisms are responsible for the long-term side effects of some anesthetic agents (e.g. preconditioning, post-operative depression, hepatotoxicity)? If these effects are the result of changes in gene expression, which genes are differentially expressed after exposure to anesthetic agents?
   
   We are currently using DNA microarray technology to address these questions. This technique relies on spotting samples (typically single DNA clones or oligonucleotides) onto a standard glass slide at very high density (ca. 100 mm spot diameter, up to 30,000 spots/slide), and each gene can be identified according to its position in the array.

   

   Two RNA samples, from control and treated tissues, are then labeled with different fluorochromes during a reverse transcriptase reaction to yield two populations of cDNA. The labeled cDNAs (targets) are then hybridised to the probe (spotted) DNAs on the slide. For individual spots, relative levels of the two fluorochromes (proportional to levels of the RNA) can be detected with a scanner, and the image data then analysed for relative levels of gene expression. It is possible to screen thousands of genes for up- and down regulation of expression in a single microarray hybridisation experiment.
   The technique is not limited to the use of DNA/RNA and can be used to measure protein/antibody interactions (typically for immunodiagnostics).

   We are interested in screening mammalian neurobiology microarray slides for differential gene expression in control and anesthetic/sedative treated neuronal tissues. These experiments will be some of the first to address the chronic effects of anesthesia in the brain. For example, there may be changes in gene expression that underlie anesthetic preconditioning, post-operative depression and the long-term tolerance/dependence of anxiolytic/sedative agents.

   Biology is undergoing a revolution where students will be required to understand, analyse, and process huge data sets and derive useful interpretations from mass screening experiments such as microarray. To tackle this profusion of information, my lab will use bioinformatics tools for post-analyses of anesthetic-induced gene regulation.

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Publications (undergraduate co-authors are underlined)

Hall, A.C., Tibbs, G.R., Dolly, J.O., Lieb, W.R. and Franks, N.P. (1993). A simple method for recording single-channel activity from synaptic plasma membranes. Journal of Neuroscience Methods, 49, 81-91.

Hall, A.C., Stow, J., Sorensen, R., Dolly, J.O. and Owen, D.G. (1994). Differential block of voltage-dependent K + currents in cultured rat sensory neurones by a- and d-dendrotoxin. British Journal of Pharmacology, 113, 959-967.

Hall, A.C., Lieb, W.R. and Franks, N.P. (1994) Insensitivity of P-type calcium channels to inhalational and intravenous general anesthetics. Anesthesiology, 81, 117-123.

Hall, A.C., Lieb, W.R. and Franks, N.P. (1994) Stereoselective and non-stereoselective actions of isoflurane on the GABA A receptor. British Journal of Pharmacology, 112, 906-910.

Dolly, J.O., Muniz, Z.M., Parcej, D.N., Hall, A.C., Scott, V.E.S., Awan, K.A. and Owen, D. (1994). Subtypes of fast-activating, voltage-gated K + channels in the nervous system. In: Neurotoxins and Neurobiology (eds. Tipton, K.F. & Dajas, F.), Ellis Horwood series in Neuroscience, pp. 103-122.

Lee, K., Khan, R.N., Rowe, I.C.M., Ozanne, S.E., Hall, A.C., Papadakis, E., Hales, C.N. and Ashford, M.L.J. (1996). Ciclazindol inhibits ATP-sensitive K + channels and stimulates insulin secretion in CRI-G1 insulin-secreting cells. Molecular Pharmacology, 49, 715-721.

Downie, D.L., Hall, A.C., Lieb, W.R. and Franks, N.P. (1996). Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes. British Journal of Pharmacology,118, 493-502.

Owen, D.G., Hall, A.C., Stephens, G., Stow, J. and Robertson, B. (1996). The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K + channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells. British Journal of Pharmacology, 120,1029-1034.

Hall, A.C., Hoffmaster, R.M., Stern, E.L., Harrington, M.E. and Bickar, D. (1997). Suprachiasmatic nucleus neurons are glucose-sensitive. Journal of Biological Rhythms, 12, 388-400.

Meyer, J.L., Hall, A.C. and Harrington, M.E. (1998). Histamine phase shifts the hamster circadian pacemaker via a NMDA-dependent mechanism. Journal of Biological Rhythms, 13, 288-295.

Hall, C.D., Lowther, N., Tweedy, B.R., Hall, A.C. and Shaw, G. (1998). The kinetics and mechanism of the phosphorus-catalysed dimerisation of acrylonitrile. Journal of Chemical Society, Perkin 2 2047-2054.

Franks, N.P., Dickinson, R., Sousa, S.L.M., Hall, A.C. and Lieb, W.R. (1998). How does the ‘inert’ gas xenon produce general anaesthesia? Nature, v 396, 324

Hall, A.C., Earle-Cruikshanks, G. and Harrington, M.E. (1999). Role of membrane conductances and protein synthesis in subjective day phase advances of the hamster circadian clock by neuropeptide Y. European Journal of Neuroscience, v 11, 3424-3432

Diaz-Munoz, M., Dent, M.A.R., Granados-Fuentes, D., Hall, A.C., Hernandez-Cruz, A., Harrington, M.E., Aguilar-Roblero, R. (1999). Circadian rhythm of expression of the ryanodine receptor type 2 in neurons from the suprachiasmatic nuclei of rodents. Neuroreport, 10, 481-486.

Harrington, M.E., Hoque, S., Hall, A.C., Golombek, D. and Biello, S.M. (1999). Pituitary adenylate ctclase activating peptide phase shifts circadian rhythms in a manner similar to light.. Journal of Neuroscience, 19, 6637-6642

Hall, C.D., Kirkovits, G.J. and Hall, A.C. (1999) . Towards a redox ion channel. Chemical Communications, 18 1897-1898.

Dickinson , R., Sousa, S.L.M., Hall, A.C., Lieb, W.R. & Franks, N.P., (2000). Is the NMDA Receptor a Molecular Target for the “Inert” Gas Xenon? . Progress in Anesthetic Mechanisms , 6 , 389-394

Hall, A.C., Suarez, C., Hom-Choudhory, A., Manu, A.N.A., Hall, C.D., Kirkovits, G.J., Ghiriviga, I. (2003). Cation transport by a redox-active synthetic ion channel. Organic and Biomolecular Chemistry, 1, 2973-2982.

Hall, A.C. and M.E. Harrington (2003) ‘Experimental Methods in Neuroscience’: an undergraduate neuroscience laboratory course for teaching data collection, statistical analyses and report writing. Journal of Undergraduate Neuroscience Education, 2, A1-7.

Hall, A.C., Rowan, K.C ., Stevens, R.J.N., Kelley, J.C., Harrison, N.L. (2004). Isoflurane relieves slow desensitization in wild-type not mutant (S270H- a1) GABA A receptors in Xenopus oocytes. Anesthesia and Analgesia, 98: 1297-1304..

Hall, A.C., Turcotte, C.M., Betts, B.A., Yeung, W-Y., Agyeman, A.S. and Burk, L.A. (2004). Modulation of Human GABAA and glycine receptor currents by menthol and related monoterpenoids. European Journal of Pharmacology, 506, 9-16.

Hall, A.C., Stevens, R.J.N., Betts, B.A., Yeung W-Y., Kelley, J.C., Harrison, N.L. (2005). Subunit-dependent block by isoflurane of wild-type and mutant a 1 S270H GABA A receptor currents in Xenopus oocytes. Neuroscience Letters, 382, 332-337.

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Abstracts and Presentations (undergraduate co-authors are underlined)


Owen, D.G., Hall, A.C., Sorensen, R.G. and Stow, J. (1990). Inhibition of outward currents in dorsal root ganglion cells by dendrotoxin homologues. Society for Neuroscience Abstracts, 16, 156.8.

Hall, A.C., Stow, J., Dolly, J.O. and Owen, D.G. (1991). Dendrotoxin homologues block voltage-dependent potassium currents in cultured rat dorsal root ganglion cells by different mechanisms. 10th World Congress on Animal, Plant and Microbial Toxins, Abstract 371.

Owen, D.G., Robertson, B., Hall, A.C. and Scott, J.A. (1992). Blockade by peptidic toxins of MK1 potassium currents in CHO cells. Society for Neuroscience Abstracts, 18, 335.1.

Ozanne, S.E., Lee, K., Hall, A.C., Rowe, I.C.M. Ashford, M.L.J. and Hales, C.N. (1994). Ciclazindol inhibits the ATP-sensitive potassium channel in the CRI-G1 insulin-secreting cell line. Diabetic Medicine, 11 (Supp. 1), P71.

Tippins, J.R., Hall, A.C., Bowen, P.R. and Dolly, J.O. (1994). In vitro cardiovascular effects of Toxin I from Dendroaspis polylepis polylepis. British Journal of Pharmacology, 111, 243P.

Lee, K., Khan, R.N., Rowe, I.C.M., Hall, A.C. and Ashford, M.L.J. (1995). Inhibition of K ATP currents by ciclazindol in the CRI-G1 insulin-secreting cell line. Proceedings of the British Pharmacological Society, P92.

Hall, A.C., Downie, D.L., Dickinson, R., Tomlin, S.L., Franks, N.P. and Lieb, W.R. (1995). Action of the volatile anesthetic isoflurane on neuronal & cloned glycine receptors. Society for Neuroscience Abstracts, 21, 634.5.

Hall, A.C. and Harrington, M.E. (1996). Neuropeptide Y activates large conductance channels in suprachiasmatic nucleus (SCN) neurons. Society for Neuroscience Abstracts, 22, 808.5.

Hall, A.C. and Harrington, M.E. (1997). Neuropeptide Y activates ATP-sensitive K + channels in hamster suprachiasmatic nucleus neurons: an essential step for non-photic phase advances in vitro. Gordon Conference for Chronobiology, New London, NH.

Hall, A.C. and Harrington, M.E. (1997). ATP-sensitive K + channels and glucose-sensitivity in hamster SCN neurons. Society for Neuroscience Abstracts, 23, 99.9.

Lei, C., Hall, A.C. and Powell, J.A. (1997). The control of triad-associated protein expression by the a1-subunit of the dihydropyridine receptor (DHPR) in skeletal muscle in culture. Biophysical Society Meeting, A378, P177.

Meyer, J.L., Hall, A.C. and Harrington, M.E. (1998). Histamine phase shifts the hamster circadian pacemaker via a NMDA-dependent mechanism. VIth Meeting Society for Research on Biological Rhythms, P187.

Hall, A.C. (1998).In vitro function of NPY receptors in the SCN. Proceedings of the FASEB Meeting, ‘Neurobiology of vertebrate circadian rhythms’, Snowmass, CO.

Hall, A.C. (2002). ‘Experimental methods in Neuroscience’: a new undergraduate behavioral neuroscience laboratory course for teaching data collection, statistical analyses and report writing. Society for Neuroscience Abstracts, 32, 22.31.

Stevens, R.J.N., Rowan, K.C., Turcotte, C.M. and Hall, A.C. (2002) Effects of the Volatile Anesthetic Isoflurane on Desensitized Human GABA A Receptors Expressed in Xenopus Oocytes. MIT and Harvard Hippocratic Society Meeting.

Stevens, R.J.N., Rowan, K.C., Turcotte, C.M. and Hall, A.C. (2003) Isoflurane relieves slow desensitization of GABA A receptors expressed in Xenopus ooctyes. Society for Neuroscience Abstracts, 33, 48.8.

Betts, B.A., Agyeman, S.A., Turcotte, C.M., Yeung, W-Y., Burk, L.A.. and Hall, A.C. (2004) Modulation of Human GABA-A and Glycine Receptors Currents by Monoterpenoids. N.E.U.R.O.N. meeting, Wheaton College.

Scordilis,S.P., Aung, L.L., Hall A.C. (2004) Global gene expression during unweighting and unloading. IBE Meeting, Austin TX.

Sandstrom , N.J., Turgeon S.M., George S.A.., Thornton, J.A., Smith D.A., Hall A.C., Harrington, M.E. (2004). A Comparison of Neuroscience Programs at Amherst, Oberlin, Smith and Williams Colleges. Society for Neuroscience Abstracts, 34, 28.17.

LaDow, E.S., Edmands, S.D., Hall, A.C. (2004). Differential gene expression in a mouse neuroblastoma cell line after exposure to the volatile anesthetic isoflurane. Society for Neuroscience Abstracts, 34, 343.9.

Edmands, S.D., Hall, A.C. (2005) DNA microarray analysis of isoflurane induced differential gene expression in rat liver, kidney, and heart. University of Massachusetts Program in Molecular and Cellular Biology, † Smith College, Dept Biological Sciences, Neuroscience Program, American Society of Anesthesiologists Meeting, Atlanta GA.

M.E. Harrington, B. Powell, A.C. Hall (2005). Teaching Research Skills to Students in Neuroscience, Neuroscience Program, Depts. Psychology and Biological Sciences, Smith College, Northampton, MA 01063, USA. Society for Neuroscience Abstracts, 35, 20.13.

Betts B.A., Yeung W-Y., Agyeman A.S., Turcotte C.M., Burk L.A., Hall, A.C.. (2005). Modulation of Human GABAA and Glycine Receptor Currents by Menthol and Related Monoterpenoids. Departments of Biological Sciences and Chemistry, Smith College, Northampton, MA 01062, USA. Society for Neuroscience Abstracts, 35, 488.17

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