Endocrine Abstracts (2002) 3 P186

The pattern of green fluorescent protein (GFP) expression following intrahypothalamic injection of adeno-associated virus-GFP

HL Ward, JV Gardiner, WM Kong, KG Murphy & SR Bloom


Endocrine Unit, Imperial College Faculty Medicine, Hammersmith Hospital, London, UK.


Adeno-associated virus (AAV) has been used to express GFP in a variety of tissues. AAV has a tropism for neurones and we have been using AAV-GFP to determine the anatomical localisation of gene transfer after injection into the hypothalami of Wistar rats. The long term aim is to use this viral vector as a tool for investigating the hypothalamic control of appetite, energy expenditure and pituitary function.

Male Wistar rats were anaesthetised and injected unilaterally into either the paraventricular (PVN) or arcuate (ARC) nuclei of the hypothalamus. Each animal (n=5 per group) received 1microlitre of AAV-GFP (viral titre 106/microlitre). Coordinates: PVN 1.8mm posterior to bregma/0.2mm or 0.5mm lateral to bregma; ARC 3.3mm posterior/0.3mm lateral to bregma. Six weeks post-injection rats were perfused, brains removed and 40-micron sections taken. Alternate sections were mounted and GFP visualised by direct fluorescence.

GFP was seen on at least one section in 86.7 % of the brains. We observed a wider pattern of distribution following injection of AAV-GFP into the medial compared to lateral PVN or ARC injections. When GFP was clearly seen within neurones of the medial PVN further GFP-containing axons were observed in the median eminence (ME). Closer examination revealed GFP-containing axons exited the lateral aspect of the PVN following a lateral arc into the ME. In those brains where GFP was seen within the ME extension of GFP into the infundibular stalk was also observed. Adenovirus mediated GFP expression within the PVN following injection into the neurohypophysis has been reported. We are currently performing studies to determine if GFP is expressed within the pituitary gland.

In conclusion, we have demonstrated GFP expression in the ME following injection of AAV-GFP into the medial portion of the PVN of the hypothalamus, which provides another tool to investigate pituitary function.

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