Three 50 nl injections (75 nl/min) were made as follows: −1.3 to −1.4 mm from bregma ☐.9 mm from midline and −5.30, −5.15, and −5.00 mm from skull surface. A hole was drilled in the skull, and a borosilicate glass pipette (20–40 μm tip diameter) lowered into LH. The 4- to 6-week-old male and female mice were anesthetized with isofluorane and placed in a stereotaxic frame (David Kopf Instruments).
1 A) ( Atasoy et al., 2008) were infused into lateral hypothalamus (LH) of orexin-cre mice ( Matsuki et al., 2009 Sasaki et al., 2011), bred in het-WT breeding pairs with C57BL/6J mice. Adenoviruses with “FLEX-switch” channelrhodopsin-2 (ChR2) constructs ( Fig. By combining whole-cell patch-clamp recordings from HA neurons with optogenetic stimulation of hcrt/orx cells in acute mouse brain slices, we asked whether hcrt/orx axons release fast transmitters, and whether changes in electrical activity of hcrt/orx cells can influence the firing rate of HA neurons.Īnimal procedures followed United Kingdom Home Office regulations.
However, it is unknown how the physiological output (firing rate) of HA cells is affected by endogenous firing of hcrt/orx cells at physiological frequencies. HA neurons are innervated by hcrt/orx cell axons and express hcrt/orx type-2 receptors, while HA signaling is important for arousal induced by exogenously applied hcrt/orx ( Eriksson et al., 2001 Huang et al., 2001 Yamanaka et al., 2002). We explored the effects of fast and selective optogenetic stimulation of hcrt/orx cell axons on one of their key projection targets: the histamine (HA) neurons in the tuberomammillary hypothalamus.
To study the postsynaptic consequences of electrical activity in hcrt/orx cell axons, here we used recently developed optogenetic tools ( Yizhar et al., 2011 Bernstein et al., 2012) for selective excitation of hcrt/orx cells within their endogenous neural network. Functional studies of this issue are important because neurochemical data suggest that hcrt/orx neurons may contain both excitatory and inhibitory transmitters, such as hcrt/orx, dynorphin ( Chou et al., 2001), glutamate ( Torrealba et al., 2003 Henny et al., 2010), and GABA ( Harthoorn et al., 2005). How the endogenous firing of hcrt/orx cells regulates their projection targets is unclear, due to technical difficulty of selectively stimulating hrct/orx cell axons in situ. Loss of hcrt/orx cells leads to narcolepsy and obesity ( Nishino et al., 2000 Thannickal et al., 2000 Hara et al., 2001) their selective optogenetic or pharmacogenetic silencing induces slow-wave sleep, while their optogenetic or pharmacogenetic stimulation causes awakening ( Adamantidis et al., 2007 Sasaki et al., 2011 Tsunematsu et al., 2011). Hypothalamic hypocretin/orexin (hcrt/orx) neurons project widely throughout the brain and orchestrate sleep–wake cycles, reward seeking, and body energy balance ( de Lecea et al., 2006 Sakurai, 2007). These results provide the first functional evidence that hcrt/orx neurons are capable of fast glutamatergic control of their projection targets, and suggest that variations in electrical activity of hcrt/orx axons can induce rapid changes in long-range signals generated by HA neurons. Furthermore, stimulation of hcrt/orx axons at physiological frequencies rapidly and reversibly increased action potential firing in HA cells, an effect that was abolished by blockade of AMPA receptors. These inputs depended on tetrodotoxin-sensitive action potentials, had kinetics typical of glutamatergic responses mediated by AMPA receptors, were blocked by the AMPA receptor blocker CNQX, and displayed multiple forms of short-term plasticity (depression in ≈70% trials, facilitation in ≈30% trials, both often in the same cell). Stimulation of ChR2-containing fibers with millisecond flashes of blue light produced fast postsynaptic currents in HA neurons, with a high connection probability (≈60% of HA cells were connected to ≈40% of hcrt/orx cells expressing ChR2). To study the impact of the electrical activity in hcrt/orx cells on HA neurons, we genetically targeted the light-activated excitatory ion channel channelrhodopsin-2 (ChR2) to the plasma membrane of hcrt/orx cells, and performed patch-clamp recordings from HA cells in acute mouse brain slices. A major pathway by which hcrt/orx neurons are thought to promote arousal is through projections to tuberomammillary histamine (HA) neurons. Neurochemical data suggest that hcrt/orx cells contain several transmitters, but what hcrt/orx cells release onto their projection targets is unknown. Hypothalamic hypocretin/orexin (hcrt/orx) neurons coordinate sleep–wake cycles, reward seeking, and body energy balance.
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