RESEARCH ARTICLE Adeno-associated viral glutamate decarboxylase expression in the lateral nucleus of the rat hypothalamus reduces feeding behavior AJ Noordmans 1 , DK Song 1 , CJ Noordmans 1 , M Garrity-Moses 2 , MJ During 3 , HL Fitzsimons 3 , MJ Imperiale 4 and NM Boulis 2 1 Department of Neurology, University of Michigan, USA; 2 Department of Neurosurgery, Cleveland Clinic Foundation, USA; 3 Functional Genomics and Translational Neuroscience Laboratory, Department of Molecular Medicine and Pathology, University of Auckland, New Zealand; and 4 Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA, USA In vivo gene transfer of glutamate decarboxylase (GAD) has been explored as a means of inducing or increasing the production of the inhibitory amino-acid neurotransmitter, GABA. This strategy has been applied to neuroprotection, seizure prevention, and neuromodulation. In the present experiment, AAV2 was used to transfer the genes for green fluorescence protein (GFP) and GAD65 into the lateral nucleus of the rat hypothalamus. Microinjection of 500 nl of AAV2 resulted in transduction of a 0.2570.04 mm 3 with targeting errors of X ¼ 0.48 mm, Y ¼ 0.18 mm, Z ¼ 0.37 mm using standard stereotactic technique. Pre- and postinjection food and water consumption, urine and feces production, and weight were recorded. In comparison with rAAVCAGGFP- and PBS-injected animals, rats treated with rAAVCAG- GAD65 demonstrated reduced weight gain (Po0.014) and transiently reduced daily food consumption (Po0.007) during the postoperative period. No changes in water consumption or waste production were recorded. Effective GAD65 gene transfer was confirmed with in situ hybridization using a probe to the woodchuck post-transcriptional regula- tory element sequence included in the vector. These findings suggest that increased GABA production in lateral nucleus of the hypothalamus induced by GAD65 gene transfer may reduce weight gain through reduced feeding. Gene Therapy (2004) 11, 797–804. doi:10.1038/sj.gt.3302223 Published online 12 February 2004 Keywords: GABA; feeding; hypothalamus; glutamate decarboxylase; adeno-associated virus Introduction In the past decade, stereotactic lesions of the human brain have been largely replaced by targeted drug delivery and neurostimulation to achieve modulation of nervous system function. Ablative pain procedures have been replaced in many cases by intrathecal narcotic pumps, while ablative spasticity procedures have been partially replaced by intrathecal baclofen pumps. Palli- dotomy for the relief of Parkinsonian symptoms has been replaced by deep brain stimulation. Nonetheless, the application of mechanical prosthetics within the human nervous system has limitations. These devices are subject to a significant rate of mechanical failure and infections. In vivo gene delivery has the potential to provide an alternative means of focused neuromodulation. By inducing the expression of genes that play a role in synaptic activity, the functional role of these neurons may be altered. The most common examples of this strategy involve the transfer of genes for precursors to neuropep- tide transmitters, or for the enzymes that produce neurotransmitters. Wilson et al 1 have demonstrated that herpes simplex (HSV) vectors may be used to transfer the gene for preproenkephalin to spinal sensory neurons. Preproenkephalin gene transfer can inhibit the perception of neuropathic pain and somatic pain. 2 Many authors have pursued the transfer of genes for enzymes that produce dopamine as a means of replacing dopamine production in models of Parkinsonism. Combinations of the genes for tyrosine hydroxylase (TH), and aromatic acid decarboxylase (AADC) have successfully enhanced dopamine production and reduced the functional con- sequences of dopamine depletion in a variety of models. 3,4 Finally, the transfer of the gene for glutamate decarboxylase (GAD), the rate-limiting enzyme in GABA production, has been investigated as a means of increasing inhibitory synaptic activity. Adenoviral, retro- viral, and adeno-associated viral (AAV) GAD vectors have been designed with confirmed GABAergic activity in cell lines, 5 primary neuronal and glial cultures, 6,7 and organotypic hippocampal cultures. 6 These investigators speculate that GAD vectors might act therapeutically in a variety of disorders thought to result from excess synaptic excitation, including neurodegenerative disor- ders and epilepsy. More recently, GAD gene transfer has been applied to focused neuromodulation in models of Parkinson’s disease 8 Electrophysiological evidence sug- gests that subthalamic nucleus (STN) GAD65 gene transfer has the capacity to change the excitatory output Received 13 January 2003; accepted 14 November 2003; published online 12 February 2004 Correspondence: NM Boulis, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA Gene Therapy (2004) 11, 797–804 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt