Orexins (also called hypocretins) are hypothalamic neuropeptides that carry out essential functions in the central nervous system; however, little is known about their release and range of action in vivo owing to the limited resolution of current detection technologies. Here we developed a genetically encoded orexin sensor (OxLight1) based on the engineering of circularly permutated green fluorescent protein into the human type-2 orexin receptor. In mice OxLight1 detects optogenetically evoked release of endogenous orexins in vivo with high sensitivity. Photometry recordings of OxLight1 in mice show rapid orexin release associated with spontaneous running behavior, acute stress and sleep-to-wake transitions in different brain areas. Moreover, two-photon imaging of OxLight1 reveals orexin release in layer 2/3 of the mouse somatosensory cortex during emergence from anesthesia. Thus, OxLight1 enables sensitive and direct optical detection of orexin neuropeptides with high spatiotemporal resolution in living animals.

Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: ‘Hom’) wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents (‘Het’, derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors.

BACKGROUND AND PURPOSEMice with functional ablation of substance P-preferring neurokinin-1 receptors (NK1R−/− mice) display behavioural abnormalities resembling those in attention deficit hyperactivity disorder (ADHD). Here, we investigated whether the ADHD treatment, guanfacine, alleviated the hyperactivity and impulsivity/inattention displayed by NK1R−/− mice in the light/dark exploration box (LDEB) and 5-choice serial reaction–time task (5-CSRTT), respectively. Following reports of co-morbid anxiety in ADHD, we also investigated effects of guanfacine on anxiety-like behaviour displayed by NK1R−/− and wild-type (WT) mice in the elevated plus maze (EPM).EXPERIMENTAL APPROACHMice were treated with guanfacine (0.1, 0.3 or 1.0 mg·kg−1, i.p.), vehicle or no injection and tested in the 5-CSRTT or the LDEB. Only the lowest dose of guanfacine was used in the EPM assays.KEY RESULTSIn the 5-CSRTT, a low dose of guanfacine (0.1 mg·kg−1) increased attention in NK1R−/− mice, but not in WT mice. This dose did not affect the total number of trials completed, latencies to respond or locomotor activity in the LDEB. Impulsivity was decreased by the high dose (1.0 mg·kg−1) of guanfacine, but this was evident in both genotypes and is likely to be secondary to a generalized blunting of behaviour. Although the NK1R−/− mice displayed marked anxiety-like behaviour, guanfacine did not affect the behaviour of either genotype in the EPM.CONCLUSIONS AND IMPLICATIONSThis evidence that guanfacine improves attention at a dose that did not affect arousal or emotionality supports our proposal that NK1R−/− mice express an attention deficit resembling that of ADHD patients.LINKED ARTICLESThis article is part of a themed section on Animal Models in Psychiatry Research. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-20

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism.

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators.Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 Cterminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABA A Rs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABA A Rs during neuron maturation in a Cdc42 dependent mechanism. Author Summary:GABAergic inhibition regulates distinct stages of brain development; however, cellular mechanisms downstream of GABA A receptors (GABA A Rs) that influence neuronal migration, maturation and synaptogenesis are less clear. ArfGEF9 encodes for RhoGEF with Cdc42 and TC10 GTPase as its substrates. Interestingly, ArhGEF9 is the only known RhoGEF essential for GABAergic synapse formation and maintenance. We report that during brain development ArfGEF9 mRNA splicing regulation generates different ratios of CB1 and CB2 splice isoforms. CB1 mRNA splicing is enhanced during early brain developmental, while CB2 levels remains constant throughout brain development. We also show that CB1 protein has shorter half-life and ubiquitin proteasome system restricts CB1 abundance within developing neuron to modulate neuron migration and distributing GABAergic synapse along the proximal-distal axis.On the other hand, CB2 isoform although expressed abundantly throughout brain development is essential for neuron dendrite maturation. Together, our data identifies specific post-transcriptional and post-translational mechanisms downstream of GABA A Rs influencing ArhGEF9 function to regulate distinct aspects of neuronal maturation process.