Orexin Neurons Respond Differentially to Auditory Cues Associated with Appetitive versus Aversive Outcomes.

Orexin (Orx) neurons are known to be involved in the promotion and maintenance of waking because they discharge in association with cortical activation and muscle tone during waking and because, in their absence, waking with muscle tone cannot be maintained and narcolepsy with cataplexy ensues. Whether Orx neurons discharge during waking in association with particular conditions, notably with appetitive versus aversive stimuli or positive versus negative emotions, is debated and considered important in understanding their role in supporting particular waking behaviors. Here, we used the technique of juxtacellular recording and labeling in head-fixed rats to characterize the discharge of Orx neurons during the performance of an associative discrimination task with auditory cues for appetitive versus aversive outcomes. Of 57 active, recorded, and neurobiotin-labeled neurons in the lateral hypothalamus, 11 were immunohistochemically identified as Orx-positive (Orx(+)), whereas none were identified as melanin-concentrating hormone-positive. Orx(+) neurons discharged at significantly higher rates during the tone associated with sucrose than during the tone associated with quinine delivered upon licking. They also discharged at high rates after the tone associated with sucrose. Across periods and outcomes, their discharge was positively correlated with EEG gamma activity and EMG activity, which is indicative of cortical activation and behavioral arousal. These results suggest that Orx neurons discharge in a manner characteristic of reward neurons yet also characteristic of arousal neurons. Accordingly, the Orx neurons may respond to and participate in reward processes while modulating cortical activity and muscle tone to promote and maintain arousal along with learned adaptive behavioral responses. SIGNIFICANCE STATEMENT: Orexin neurons play a critical role in promoting and maintaining a waking state because, in their absence, narcolepsy with cataplexy ensues. Known to discharge during waking and not during sleep, they have also been proposed to be selectively active during appetitive behaviors. Here, we recorded and labeled neurons in rats to determine the discharge of immunohistochemically identified orexin neurons during performance of an associative discrimination task. Orexin neurons responded differentially to auditory cues associated with appetitive sucrose versus aversive quinine, indicating that they behave like reward neurons. However, correlated discharge with cortical and muscle activity indicates that they also behave like arousal neurons and can thus promote cortical activation with behavioral arousal and muscle tone during adaptive waking behaviors.

Melanin-concentrating hormone neurons discharge in a reciprocal manner to orexin neurons across the sleep-wake cycle.

Neurons containing melanin-concentrating hormone (MCH) are codistributed with neurons containing orexin (Orx or hypocretin) in the lateral hypothalamus, a peptide and region known to be critical for maintaining wakefulness. Evidence from knockout and c-Fos studies suggests, however, that the MCH neurons might play a different role than Orx neurons in regulating activity and sleep-wake states. To examine this possibility, neurons were recorded across natural sleep-wake states in head-fixed rats and labeled by using the juxtacellular technique for subsequent immunohistochemical identification. Neurons identified as MCH+ did not fire during wake (W); they fired selectively during sleep, occasionally during slow wave sleep (SWS) and maximally during paradoxical sleep (PS). As W-Off/Sleep-On, the MCH neurons discharged in a reciprocal manner to the W-On/Sleep-Off Orx neurons and could accordingly play a complementary role to Orx neurons in sleep-wake state regulation and contribute to the pathophysiology of certain sleep disorders, such as narcolepsy with cataplexy.

Discharge profiles of identified GABAergic in comparison to cholinergic and putative glutamatergic basal forebrain neurons across the sleep-wake cycle.

Whereas basal forebrain (BF) cholinergic neurons are known to participate in processes of cortical activation during wake (W) and paradoxical sleep (PS or P, also called REM sleep), codistributed GABAergic neurons have been thought to participate in processes of cortical deactivation and slow-wave sleep (SWS or S). To learn the roles the GABAergic neurons might play, in relation to cholinergic and glutamatergic neurons, we juxtacellularly recorded and labeled neurons during natural sleep-wake states in head-fixed rats. Neurobiotin (Nb)-labeled cells were identified immunohistochemically as choline acetyltransferase (ChAT)+, glutamic acid decarboxylase (GAD)+, or ChAT-/GAD-. Of the latter, some were identified as glutamatergic by immunostaining of their terminals with the vesicular glutamate transporter (VGluT2). In contrast to ChAT+ neurons, which all discharged maximally during W and PS, GAD+ neurons comprised multiple sleep-wake subgroups. Some GABAergic neurons discharged maximally during W and PS, as WP-max active cells (36%), and in positive correlation with gamma electroencephalographic (EEG) activity. Some discharged maximally during SWS, as S-max active cells (28%), and in positive correlation with delta EEG activity. Others increased their discharge progressively during sleep to discharge maximally during PS, as P-max active cells (36%), and in negative association with electromyographic (EMG) activity. ChAT-/GAD- cells comprised WP-max (46%), S-max (17%), P-max (17%), and W-max active cells (14%), whose discharge was positively correlated with EMG activity. GABAergic neurons would thus play similar or reciprocal roles to other cholinergic and glutamatergic BF neurons in regulating cortical activity and muscle tone along with behavior across sleep-wake states.

GABAergic neurons intermingled with orexin and MCH neurons in the lateral hypothalamus discharge maximally during sleep.

The lateral hypothalamus (LH), where wake-active orexin (Orx)-containing neurons are located, has been considered a waking center. Yet, melanin-concentrating hormone (MCH)-containing neurons are codistributed therein with Orx neurons and, in contrast to them, are active during sleep, not waking. In the present study employing juxtacellular recording and labeling of neurons with Neurobiotin (Nb) in naturally sleeping-waking head-fixed rats, we identified another population of intermingled sleep-active cells, which do not contain MCH (or Orx), but utilize gamma-aminobutyric acid (GABA) as a neurotransmitter. The 'sleep-max' active neurons represented 53% of Nb-labeled MCH-(and Orx) immunonegative (-) cells recorded in the LH. For identification of their neurotransmitter, Nb-labeled varicosities of the Nb-labeled/MCH- neurons were sought within sections adjacent to the Nb-labeled soma and immunostained for the vesicular transporter for GABA (VGAT) or for glutamate. A small proportion of sleep-max Nb+/MCH- neurons (19%) discharged maximally during slow-wave sleep (called 'S-max') in positive correlation with delta electroencephalogram activity, and from VGAT staining of Nb-labeled varicosities appeared to be GABAergic. The vast proportion of sleep-max Nb+/MCH- neurons (81%) discharged maximally during paradoxical sleep (PS, called 'P-max') in negative correlation with electromyogram amplitude, and from Nb-labeled varicosities also appeared to be predominantly GABAergic. Given their discharge profiles across the sleep-wake cycle, P-max together with S-max GABAergic neurons could thus serve to inhibit other neurons of the arousal systems, including local Orx neurons in the LH. They could accordingly dampen arousal with muscle tone and promote sleep, including PS with muscle atonia.

The noradrenergic system is necessary for survival of vulnerable midbrain dopaminergic neurons: implications for development and Parkinson's disease.

The cause of midbrain dopaminergic (mDA) neuron loss in sporadic Parkinson's disease (PD) is multifactorial, involving cell autonomous factors, cell-cell interactions, and the effects of environmental toxins. Early loss of neurons in the locus coeruleus (LC), the main source of ascending noradrenergic (NA) projections, is an important feature of PD and other neurodegenerative disorders. We hypothesized that NA afferents provide trophic support for vulnerable mDA neurons. We demonstrate that depriving mDA neurons of NA input increases postnatal apoptosis and decreases cell survival in young adult rodents, with relative sparing of calbindin-positive subpopulations known to be resistant to degeneration in PD. As a mechanism, we propose that the neurotrophin brain-derived neurotrophic factor (BDNF) modulates anterograde survival effects of LC inputs to mDA neurons. We demonstrate that the LC is rich in BDNF mRNA in postnatal and young adult brains. Early postnatal NA denervation reduces both BDNF protein and activation of TrkB receptors in the ventral midbrain. Furthermore, overexpression of BDNF in NA afferents in transgenic mice increases mDA neuronal survival. Finally, increasing NA activity in primary cultures of mDA neurons improves survival, an effect that is additive or synergistic in the presence of different concentrations of BDNF. Taken together, our results point to a novel mechanism whereby LC afferents couple BDNF effects and NA activity to provide anterograde trophic support for vulnerable mDA neurons. Early loss of NA activity and anterograde neurotrophin support may contribute to degeneration of vulnerable neurons in PD and other neurodegenerative disorders.