Parks Faculty

Amy Harkins, Ph.D.,<br/>Secondary Appointment

Amy Harkins, Ph.D.,
Secondary Appointment

Associate Professor from the Department of Pharmacological and Physiological Science, Secondary Faculty Appointment in Biomedical Engineering

Phone:
314-977-6480
Education:

Ph.D,
University of Pennsylvania

Areas of Research

  • Nerve regeneration in biomaterial scaffolds
  • cellular and molecular mechanisms of synaptic transmission as a measure of functional regeneration

Interests

My laboratory is currently conducting research in the fields of neuroscience, analytical chemistry and biomedical engineering.  Neurons communicate intercellularly via synaptic transmission.  An action potential firing in one neuron activates voltage-dependent calcium channels to open and calcium enters the presynaptic terminal.  Elevated intracellular calcium levels trigger the fusion of vesicles with the plasma membrane to cause release of transmitter, which diffuses across the synaptic cleft, and activates post-synaptic receptors.  The post-synaptic neuron responds to communicate the signal to the next neuron.

Research Questions

We are investigating cellular and molecular mechanism(s) that permit neurons to communicate, and we are investigating how neurons regenerate in 3-dimensional scaffolds to accurately and functionally reform neuronal connections to target tissues.

  1. What protein(s) acts as a calcium sensor to activate the release machinery and regulate release of large dense core vesicles and small clear vesicles?
  2. What 3-dimensional scaffold and growth conditions permit maximal neuronal regrowth and regeneration following nerve injury?
  3. What intracellular signaling mechanisms underlie the appropriate regrowth/regeneration and functional communication between neurons and their target tissues?

Experimental Approaches

The laboratory bridges the biophysical and biomedical engineering fields of research.  We use neurons and model secretory cells to measure release of synaptic vesicles and their transmitter contents.  Research approaches include RNA interference, and biophysical/analytical chemistry techniques such as carbon fiber amperometry, membrane capacitance and patch-clamp current measurements to detect vesicle fusion events, and subsequent release of neurotransmitters from single cells.  We use 3-dimensional scaffold biomaterials to grow neurons and study signaling molecules for appropriate and functional connectivity during regeneration.

The techniques permit us to target specific proteins for knockdown, measure vesicle fusion, neurotransmitter release and neuronal regrowth at the single cell level.  These approaches combined with the cell systems will allow us to contribute to a mechanistic understanding of how synaptic transmission functions, and what biomedically engineered materials and conditions will permit an injured nerve to regrow and reform functional connections.

Please visit the laboratory website for more information regarding the ongoing projects, people in the lab, their project websites, and a current listing of our publications

An example of our current research is shown from Mallory Smyth’s work (summer, 2010).  Dorsal root ganglion neurons were grown on 2D collagen surfaces, on 2D collagen gels, and within 3D collagen gels, and were stained with a neuronal specific antibody.

Below is a list of Amy Harkins’ selected publications:

  • Moore-Dotson JM, Papke JB, Harkins AB. (2010)
    Upregulation of synaptotagmin IV inhibits transmitter release in PC12 cells with targeted synaptotagmin I knockdown. BMC Neuroscience. Aug 24;11(1)
  • Pike CM, Grabner CP, Harkins AB. (2009)F
    Fabrication of amperometric electrodes. Journal of Visualized Experiments. May 4;(27) pii: 1040. doi 10.3791/1040
  • Samways DS, Harkins AB, Egan TM. (2009)
    Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry. Cell Calcium. Apr;45(4):319-25
  • Fox AP, Cahill AL, Currie KP, Grabner C, Harkins AB, Herring B, Hurley JH, Xie Z. (2008)
    N- and P/Q type Ca2+ channels in adrenal chromaffin cells. Acta Phyiologica (Oxf) Feb;192(2):247-61
  • Roden WH, Papke JB, Moore JM, Cahill AL, Macarthur H, Harkins AB. (2007)
    Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release. American Journal of Physiology - Cell Physiology. Dec;293(6):C1742-52
  • Cahill AL, Moore JM, Sabar FI, Harkins AB. (2007)
    Variability in RNA interference in neuroendocrine PC12 cell lines stably transected with an shRNA plasmid. Journal of Neuroscience Methods. Nov 30; 166(2):236-40
  • Moore JM, Papke JB, Cahill AL, Harkins AB. (2006)
    Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca2+-evoked release of catecholamine. American Journal of Physiology - Cell Physiology. Aug;291(2):C270-81
  • A.B. Harkins, A.L. Cahill, J.F. Powers, A.S. Tischler, A.P. Fox (2004)
    Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells. Proceedings of the National Academy of Science. Oct 19;101(42):15219-24
  • A.B. Harkins, A.L. Cahill, J.F. Powers, A.S. Tischler, A.P. Fox. (2003)
    Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line. Journal of Neurophysiology. 90:2325-2333
  • A.B. Harkins, A.P. Fox. (2002)
    Cell death in weaver mouse cerebellum. Review. The Cerebellum. 1(3):201-206
  • A.B. Harkins, A.P. Fox. (2000)
    Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells. Brain Research. 885:231-239
  • A.B. Harkins, S. Dlouhy, B. Ghetti, A.L. Cahill, L. Won, B. Heller, A. Heller, A.P. Fox. (2000)
    Evidence of elevated intracellular calcium levels in weaver homozygote mice. Journal of Physiology. 524:447-455
  • A.B. Harkins, A.P. Fox (1998)
    Activation of nicotinic acetylcholine receptors augments calcium channel-mediated exocytosis in rat pheochromocytoma (PC12) cells. Journal of General Physiology. 111:257-269
  • F. Rangwala, R.C. Drisdel, S. Rakhilin, E. Ko. P. Atluri, A.B. Harkins, A.P. Fox, S. Salman, W.N. Green (1997)
    Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors. Journal of Neuroscience. 17:8201-8212
  • R. Rao-MIrotznik, A.B. Harkins, G. Buchsbaum, P. Sterling (1995)
    Mammalian rod terminal: Architecture of a binary synapse. Neuron. 14:561-569
  • N. Kurebayashi, A.B. Harkins, S.M. Baylor (1993)
    Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. Biophysical Journal. 64:1934-1960
  • A.B. Harkins, N. Kurebayashi, S.M. Baylor (1993)
    Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophysical Journal. 65:865-881
  • A.B. Harkins, D.L. Armstrong (1992)
    Trimethyltin alters membrane properties of CA1 hippocampal neurons. Neurotoxicology. 13:569-582
  • S. Hollingworth, A.B. Harkins, N. Kurebayashi, M. Konishi, S.M Baylor (1992)
    Excitation-contraction coupling in intact frog skeletal fibers injected with mmolar concentration of Fura-2. Biophysical Journal. 63:224-234
  • M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor (1991)
    Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra. Journal of General Physiology. 97:271-301

Harkins Lab

Dr. Harkins’ laboratory is currently conducting research in the fields of neuroscience, analytical chemistry and biomedical engineering.

The primary goal of the lab is to investigate nerve regeneration in 3-dimensional scaffolds to accurately and functionally reform nerve connectivity to target tissues.

The laboratory bridges the biophysical and biomedical engineering fields of research. Our approaches include 3-dimensional scaffold biomaterials that occur naturally or are synthesized. The scaffolds are used to grow neurons and study signaling molecules for appropriate reconnection and functional activity during regeneration. To measure reconnectivity, we utilize biophysical and analytical chemistry techniques of amperometry and patch-clamp analysis for neuronal communication as the connectivity of neurons is reestablished.

If you’re interested in reading more about the projects and research questions, and how we approach the problems, please visit our official lab page.