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CREB-binding protein (CBP) is a multifunctional cofactor implicated in many intracellular signal transduction pathways. We aimed to investigate the involvement of CBP in the cAMP response element-binding protein (CREB)-mediated pathway. The point mutation Tyr658Ala in the CREB-binding domain (CBD) was shown to abolish the binding activity of CBP to phospho-CREB, the activated form of CREB. By using a mutant Cre/loxP recombination system, this point mutation was aimed to be generated in the mouse genome in a tissue- and time-specific manner. A targeting construct in which CBD exon 5 and inverted exon 5* containing the point mutation flanked by two mutant loxP sites (lox66 and lox71) oriented in a head-to-head position was generated. When Cre recombinase is present, the DNA flanked by the two mutant loxP sites is inverted, forming one loxP and one double mutated loxP site. As the double mutated loxP site shows low affinity for Cre recombinase, the favorable reaction leads to a product where the mutated exon 5* is placed into the position to be correctly transcribed and spliced. Inversion was observed to be complete in both bacteria and mouse embryonic stem cells. Our results indicate that this Cre- mediated inversion method is a valuable tool to introduce point mutations in the mouse genome in a regulatable manner.

Kallmann syndrome is a human genetic disorder characterized by a defect in olfactory system development due to an abnormality in the migration of olfactory axons and of gonadotropin releasing hormone (Gn-RH) producing neurons. The predicted protein product of the recently isolated gene for X-linked Kallmann syndrome shares motifs with molecules involved in neural development. We have isolated the chicken Kallmann syndrome gene which shows an overall 77% amino acid identity with the human homologue. The degree of identity increases within the putative functional domains, strongly supporting their functional relevance. Expression studies have been performed by Northern analysis, RT-PCR, RNase protection and in situ hybridization in chicken embryos at different developmental stages. In the developing and adult chicken, high levels of expression of this gene were found in the olfactory bulb and in the cerebellar cortex, both areas affected in patients with Kallmann syndrome. In the olfactory bulb, the gene is expressed by the mitral cells, the target of olfactory axons; in the cerebellum, expression was found in the Purkinje cells. The site and the timing of expression of the Kallmann syndrome gene within the olfactory system suggest that its protein product is a signal molecule required for proper neuronal targeting throughout life.

Kallmann syndrome is a human genetic disorder characterized by the association of hypogonadism with the inability to smell, and is due to defects in the olfactory system development (i.e. incomplete migration of olfactory axons and of gonadotropin-releasing hormone producing neurons from the olfactory epithelium to the forebrain; aplasia or hypoplasia of olfactory bulbs and tracts). The human X-linked Kallmann syndrome gene and its chicken homologue have been cloned. Their protein products contain fibronectin type III repeats and a ‘four-disulfide-core’ domain also found in molecules that are involved in neural development. Consistent with the human phenotype, the chicken Kallmann gene is expressed in the developing olfactory bulb. At present the molecular and cellular mechanism of action of the Kallmann syndrome gene product is unknown. Based on expression studies and the characteristic domains of the predicted protein, it is hypothesized that the protein may be involved in targeting olfactory axons to the bulb. Alternatively, the Kalunann protein could be an extracellular matrix component required for the proper formation of the multilayered structure of the olfactory bulb.

The amygdala is a key area of the brain where the emotional memories are stored throughout the lifespan. It is well established that synapses in the lateral nucleus of amygdala (LA) can undergo long-term potentiation, a putative cellular correlate of learning and memory. However, a type of short-term synaptic plasticity, known as depolarization-induced suppression of excitation (DSE), has not been studied previously in the amygdala in general and in the LA in particular. In this study we aimed to prove either the absence or the presence of this phenomenon in the LA. Our data demonstrate for the first time that DSE is present in the LA and that it modulates the cortical excitatory synaptic input into this region. The existence of this type of retrograde neurotransmission in glutamatergic pyramidal neurons of the LA suggests that the axonal terminals of cortical inputs do possess functional type 1 cannabinoid receptors, and provides a novel insight regarding inputs into the LA. Further experiments indeed revealed endocannabinoids as the messenger for this retrograde signaling in the LA. In conclusion, the DSE may play a functional role in synaptic plasticity and related emotional memory processing in the LA.