Selective lesioning of nucleus incertus with corticotropin releasing factor-saporin conjugate

The nucleus incertus (NI), a brainstem nucleus found in the pontine periventricular grey, is the primary source of the neuropeptide relaxin-3 in the mammalian brain. The NI neurons have also been previously reported to express several receptors and neurotransmitters, including corticotropin releasing hormone receptor 1 (CRF₁) and gamma-aminobutyric acid (GABA). The NI projects widely to putative neural correlates of stress, anxiety, depression, feeding behaviour, arousal and cognition leading to speculation that it might be involved in several neuropsychiatric conditions. On the premise that relaxin-3 expressing neurons in the NI predominantly co-express CRF₁ receptors, a novel method for selective ablation of the rat brain NI neurons using corticotropin releasing factor (CRF)-saporin conjugate is described. In addition to a behavioural deficit in the fear conditioning paradigm, reverse transcriptase polymerase chain reaction (RT-PCR), western blotting (WB) and immunofluorescence labelling (IF) techniques were used to confirm the NI lesion. We observed a selective and significant loss of CRF₁ expressing cells, together with a consistent decrease in relaxin-3 and GAD65 expression. The significant ablation of relaxin-3 positive neurons of the NI achieved by this lesioning approach is a promising model to explore the neuropsychopharmacological implications of NI/relaxin-3 in behavioural neuroscience.