Person:

Fuller, Patrick

Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to the rat locus coeruleus (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

Wakefulness, along with fast cortical rhythms and associated cognition, depend on the basal forebrain (BF). BF cholinergic cell loss in dementia and the sedative effect of anti-cholinergic drugs have long implicated these neurons as important for cognition and wakefulness. The BF also contains intermingled inhibitory GABAergic and excitatory glutamatergic cell groups whose exact neurobiological roles are unclear. Here we show that genetically targeted chemogenetic activation of BF cholinergic or glutamatergic neurons in behaving mice produced significant effects on state consolidation and/or the electroencephalogram but had no effect on total wake. Similar activation of BF GABAergic neurons produced sustained wakefulness and high-frequency cortical rhythms, whereas chemogenetic inhibition increased sleep. Our findings reveal a major contribution of BF GABAergic neurons to wakefulness and the fast cortical rhythms associated with cognition. These findings may be clinically applicable to manipulations aimed at increasing forebrain activation in dementia and the minimally conscious state.

Armodafinil is the pharmacologically active R-enantiomer of modafinil, a widely prescribed wake-promoting agent used to treat several sleep-related disorders including excessive daytime sleepiness associated with narcolepsy, shift work sleep disorder, and obstructive sleep apnea/hypopnea syndrome. Remarkably, however, the neuronal circuitry through which modafinil exerts its wake-promoting effects remains unresolved. In the present study, we sought to determine if the wake-promoting effects of armodafinil are mediated, at least in part, by inhibiting the sleep-promoting neurons of the ventrolateral preoptic (VLPO) nucleus. To do so, we measured changes in waking following intraperitoneal administration of armodafinil (200 mg/kg) or the psychostimulant methamphetamine (1 mg/kg) in rats with cell-body specific lesion of the VLPO. Rats with histologically confirmed lesions of the VLPO demonstrated a sustained increase in wakefulness at baseline, but the increase in wakefulness following administration of both armodafinil and methamphetamine was similar to that of intact animals. These data suggest that armodafinil increases wakefulness by mechanisms that extend beyond inhibition of VLPO neurons.

Work in animals and humans suggest the existence of a slow–wave sleep (SWS) promoting/EEG synchronizing center in the mammalian lower brainstem. While sleep–active GABAergic neurons in the medullary parafacial zone (PZ) are needed for normal SWS, it remains unclear if these neurons can initiate and maintain SWS or EEG slow wave activity (SWA) in behaving mice. We used genetically targeted activation and optogenetic–based mapping to uncover the downstream circuitry engaged by SWS–promoting PZ neurons, and we show that this circuit uniquely and potently initiates SWS and EEG SWA, regardless of the time of day. PZ neurons monosynaptically innervate and release synaptic GABA onto parabrachial neurons that in turn project to and release synaptic glutamate onto cortically–projecting neurons of the magnocellular basal forebrain; hence a circuit substrate is in place through which GABAergic PZ neurons can potently trigger SWS and modulate the cortical EEG.

The advent of genetically engineered systems, including transgenic animals and recombinant viral vectors, has facilitated a more detailed understanding of the molecular and cellular substrates regulating brain function. In this review we highlight some of the most recent molecular biology and genetic technologies in the experimental “systems neurosciences,” many of which are rapidly becoming a methodological standard, and focus in particular on those tools and techniques that permit the reversible and cell-type specific manipulation of neurons in behaving animals. These newer techniques encompass a wide range of approaches including conditional deletion of genes based on Cre/loxP technology, gene silencing using RNA interference, cell-type specific mapping or ablation and reversible manipulation (silencing and activation) of neurons in vivo. Combining these approaches with viral vector delivery systems, in particular adeno-associated viruses (AAV), has extended, in some instances greatly, the utility of these tools. For example, the spatially- and/or temporally-restricted transduction of specific neuronal cell populations is now routinely achieved using the combination of Cre-driver mice and stereotaxic-based delivery of AAV expressing Cre-dependent cassettes. We predict that the experimental application of these tools, including creative combinatorial approaches and the development of even newer reagents, will prove necessary for a complete understanding of the neuronal circuits subserving most neurobiological functions, including the regulation of sleep and wake.

In this study we uncovered, through targeted ablation, a potential role for corticospinal, cerebello-rubro-spinal, and hypothalamic A11 dopaminergic systems in the development of restless legs syndrome (RLS)-like movements during sleep. Targeted lesions in select basal ganglia (BG) structures also revealed a major role for nigrostriatal dopamine, the striatum, and the external globus pallidus (GPe) in regulating RLS-like movements, in particular pallidocortical projections from the GPe to the motor cortex. We further showed that pramipexiole, a dopamine agonist used to treat human RLS, reduced RLS-like movements. Taken together, our data show that BG-cortico-spinal, cerebello-rubro-spinal and A11 descending projections all contribute to the suppression of motor activity during sleep and sleep-wake transitions, and that disruption of these circuit nodes produces RLS-like movements. Taken together with findings from recent genomic studies in humans, our findings provide additional support for the concept that the anatomic and genetic etiological bases of RLS are diverse.

Corticotropin-releasing hormone (CRH) signaling in the hippocampus has been established to be important for mediating the effects of stress on learning and memory. Given our laboratory’s recent characterization of a subset of hippocampal CRH neurons as a novel class of GABAergic interneurons, we hypothesized that these local GABAergic hippocampal CRH neurons may influence hippocampal function. Here we applied an array of molecular tools to selectively label and manipulate hippocampal CRH neurons in mice, in order to assess this interneuron population’s impact on hippocampus-dependent behaviors and hippocampal network excitability. Genetically-targeted ablation of hippocampal CRH neurons in vivo impaired object recognition memory and substantially enhanced the severity of kainic acid-induced seizures. Conversely, selective activation of CRH neurons in vitro suppressed the excitability of the mossy fiber-CA3 pathway. Additional experiments are needed to reconcile the functions of GABA and CRH signaling of hippocampal CRH neurons on hippocampal function. However, our results indicate that this interneuron population plays an important role in maintaining adaptive network excitability, and provide a specific circuit-level mechanism for this role.

Basic and clinical observations suggest that the caudal hypothalamus comprises a key node of the ascending arousal system, but the cell types underlying this are not fully understood. Here we report that glutamate-releasing neurons of the supramammillary region (SuMvglut2) produce sustained behavioral and EEG arousal when chemogenetically activated. This effect is nearly abolished following selective genetic disruption of glutamate release from SuMvglut2 neurons. Inhibition of SuMvglut2 neurons decreases and fragments wake, also suppressing theta and gamma frequency EEG activity. SuMvglut2 neurons include a subpopulation containing both glutamate and GABA (SuMvgat/vglut2) and another also expressing nitric oxide synthase (SuMNos1/Vglut2). Activation of SuMvgat/vglut2 neurons produces minimal wake and optogenetic stimulation of SuMvgat/vglut2 terminals elicits monosynaptic release of both glutamate and GABA onto dentate granule cells. Activation of SuMNos1/Vglut2 neurons potently drives wakefulness, whereas inhibition reduces REM sleep theta activity. These results identify SuMvglut2 neurons as a key node of the wake−sleep regulatory system.