Shanker Karunanithi - Arizona Research Laboratories Division of Neurobiology

Shanker Karunanithi

Assistant Professor of Neurobiology

B.E., Electrical Engineering, University of Sydney, Australia, 1988
Ph.D., Physiology, University of Sydney, Australia, 1994

Office:

Gould-Simpson Bldg. Rm. 430

Email:

shanker@neurobio.arizona.edu

Phone:

(520) 626-5699

Fax:

(520) 621-8282

Determinants of synaptic strength under normal and stressful conditions

A synapse is a specialized connection facilitating information transfer from a neuron to a target cell by releasing neurotransmitters from synaptic vesicles. Learning and memory formation, stress adaptation, drug addiction and withdrawal, depression, and fear conditioning are examples of behavioral changes reinforced in part by altering the strength of the transmitted signal at individual synapses (synaptic strength). For example, learning and memory formation are correlated with increases in synaptic strength. Intriguingly, individual synapses differ in their signaling strengths, and the extent to which their strengths can be modified. Reconciling the diversity in strengths amongst different synapses and their capacity for functional alteration with changes in behavior remains one of the greatest of challenges. The broad research interests of the laboratory are to elucidate the determinants of synaptic strength under normal and stressful conditions. In particular, to determine: (a) the factors generating differences in strengths amongst synapse types; (b) how synapses functionally adapt to acquire heat-resistance.

Detailed Research Description

a) Synaptic Differentiation

Each synapse is functionally unique. The extent to which molecules and ultrastructure contribute towards generating functional differences in strength amongst synapses is poorly understood. To study synaptic differentiation, we utilize a simple preparation in which a target muscle cell receives inputs from two functionally and structurally different motor neurons; the terminals of one motor neuron are composed of big boutons, whereas those of its partner are composed of small boutons. Synaptic strength is strongly influenced by neurotransmitter release efficacy (the average number of synaptic vesicles releasing neurotransmitter) and quantal size (the average size of the synaptic response generated in the target cell when individual synaptic vesicles secrete neurotransmitter). We discovered that quantal size differences between the two bouton types correlates with synaptic vesicle size differences, and vesicle size is in turn correlated to its transmitter content. Our current results indicate that synaptic vesicle size differences could result in part from differences in the levels of neuronal activity between the two motor neurons. In the near future, investigations will be undertaken to determine the mechanisms by which neuronal activity regulates vesicle size.

Synapse and vesicle sizes were identified to be regulated by the tumor suppressor gene, discs-large (dlg). The DLG protein is homologous to the mammalian PSD-95 synaptic scaffolding protein found in central synapses. Mutant synapses generated larger quantal currents than controls, by releasing more glutamate from larger vesicles. Future work will determine whether the signaling pathways by which dlg, and other identified genes regulating vesicle size work independently or in concert.

b) Role of Heat Shock Proteins in Conferring Neuroprotection to Synapses

Synapses are critical sites of information transfer in the nervous system and their functionality needs to be preserved under stress. Synapses are irreversibly damaged when exposed to lethal temperatures. However, a prior heat shock (brief exposure to sublethal temperatures) preserved synaptic function at higher than normal temperatures by altering synaptic properties. In the future we wish to determine how synapses adapt functionally to acquire thermotolerance?

Prior heat shock causes downregulation in synthesis of most proteins, except for a class of proteins called heat shock proteins (Hsps), whose levels are highly induced. In Drosophila, heat shock protein 70 (Hsp70) is the most abundantly expressed protein following heat shock (its levels are below detection in unshocked animals); in our previous work, Hsp70 was shown in part to afford synaptic thermotolerance. Surprisingly, using a mutant where Hsp induction is arrested, substantial synaptic thermotolerance was detected following heat shock. The latter finding revealed a mechanism for acquiring thermotolerance in the absence of production of induced Hsps. Current work utilizing DNA microarrays has discovered previously unidentified genes activated by heat shock, which may confer synaptic thermotolerance. Using mutants to these genes, investigations are being undertaken: a) to identify the proteins which afford synaptic thermotolerance; b) to identify target synaptic proteins which receive thermoprotection. We also wish to explore whether similar mechanisms of neuroprotection operate under different forms of stress, such as, hypoxia and hypothermia.
Clinically, the knowledge gained from this work may prove to be useful in developing methods which help maintain normal synaptic function under conditions which may not otherwise sustain it, such as, aging, neurodegeneration, high fever, ischemia, and stroke.

Synapse Model: Drosophila Neuromuscular Junction (NMJ)

The Drosophila nmj is composed of individual synaptic boutons, which functionally resemble mammalian central glutamatergic synapses. An advantage of the Drosophila nmj is that individual boutons are readily accessible for physiological recordings, whereas accessing individual, naturally formed synaptic boutons in the mammalian brain is extremely difficult.

Techniques Utilized in the Laboratory

Focal macropatch recording from individual synaptic boutons, intracellular recording, voltage clamping, Monte Carlo simulations in computer modeling, calcium imaging, UV flash photolysis of caged compounds, serial reconstruction of synapses using electron microscopy, confocal microscopy, immunohistochemistry, DNA microarrays, gel electrophoresis, generation of transgenic Drosophila, RNA interference (RNAi) to silence gene function.

Publications

Dawson-Scully K Lin Y Imad M Zhang J Marin L Horne JA Meinertzhagen IA Karunanithi S Zinsmaier KE Atwood HL. Jan 2007. Morphological and functional effects of altered cysteine string protein at the Drosophila larval neuromuscular junction. Synapse, 61:1-16

Macleod GT, Chen L, Karunanithi S, Peloquin JB, Atwood HL, McRory JE, Zamponi GW, Charlton MP. Jun 2006. The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation. Eur J Neurosci, 23:3230-44

Neal SJ, Karunanithi S, Best A, So AK, Tanguay RM, Atwood HL, Westwood JT. Apr 2006. Thermoprotection of synaptic transmission in a Drosophila heat shock factor mutant is accompanied by increased expression of Hsp83 and DnaJ-1. Physiol Genomics,2006 Apr 4;

Suster ML, Karunanithi S, Atwood HL, Sokolowski MB. Oct 2004. Turning behavior in Drosophila larvae: a role for the small scribbler transcript. Genes Brain Behav, 3:273-86

Karunanithi S, Marin L, Wong K, Atwood HL. Dec 2002. Quantal size and variation determined by vesicle size in normal and mutant Drosophila glutamatergic synapses. J Neurosci, 22:10267-76

Atwood HL, Karunanithi S. Jul 2002. Diversification of synaptic strength: presynaptic elements. Nat Rev Neurosci, 3:497-516

Karunanithi S, Barclay JW, Brown IR, Robertson RM, Atwood HL. Apr 2002. Enhancement of presynaptic performance in transgenic Drosophila overexpressing heat shock protein Hsp70. Synapse, 44:8-14

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