Unravelling cell type-specific responses to Parkinson's Disease at single cell resolution.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder. The pathological hallmark of PD is loss of dopaminergic neurons and the presence of aggregated α-synuclein, primarily in the substantia nigra pars compacta (SNpc) of the midbrain. However, the molecular mechanisms that underlie the pathology in different cell types is not currently understood. Here, we present a single nucleus transcriptome analysis of human post-mortem SNpc obtained from 15 sporadic Parkinson's Disease (PD) cases and 14 Controls. Our dataset comprises ∼84K nuclei, representing all major cell types of the brain, allowing us to obtain a transcriptome-level characterization of these cell types. Importantly, we identify multiple subpopulations for each cell type and describe specific gene sets that provide insights into the differing roles of these subpopulations. Our findings reveal a significant decrease in neuronal cells in PD samples, accompanied by an increase in glial cells and T cells. Subpopulation analyses demonstrate a significant depletion of tyrosine hydroxylase (TH) enriched astrocyte, microglia and oligodendrocyte populations in PD samples, as well as TH enriched neurons, which are also depleted. Moreover, marker gene analysis of the depleted subpopulations identified 28 overlapping genes, including those associated with dopamine metabolism (e.g., ALDH1A1, SLC6A3 & SLC18A2). Overall, our study provides a valuable resource for understanding the molecular mechanisms involved in dopaminergic neuron degeneration and glial responses in PD, highlighting the existence of novel subpopulations and cell type-specific gene sets.

Transcriptome of neonatal preBötzinger complex neurones in Dbx1 reporter mice.

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBötzinger Complex (preBötC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBötC neurons derived from Dbx1-expressing progenitors that are respiratory rhythmogenic to neighbouring non-Dbx1-derived neurons, which support other respiratory and non-respiratory functions. Results in three categories are particularly salient. First, Dbx1 preBötC neurons express κ-opioid receptors in addition to µ-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBötC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBötC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBötC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development.

Fate mapping neurons and glia derived from Dbx1-expressing progenitors in mouse preBötzinger complex.

The brainstem preBötzinger complex (preBötC) generates the inspiratory breathing rhythm, and its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors. To study the neural bases of breathing, tamoxifen-inducible Cre-driver mice and Cre-dependent reporters are used to identify, record, and perturb Dbx1 preBötC neurons. However, the relationship between tamoxifen administration and reporter protein expression in preBötC neurons and glia has not been quantified. To address this problem, we crossed mice that express tamoxifen-inducible Cre recombinase under the control of the Dbx1 gene (Dbx1CreERT2) with Cre-dependent fluorescent reporter mice (Rosa26tdTomato), administered tamoxifen at different times during development, and analyzed tdTomato expression in the preBötC of their offspring. We also crossed Rosa26tdTomato reporters with mice that constitutively express Cre driven by Dbx1 (Dbx1Cre) and analyzed tdTomato expression in the preBötC of their offspring for comparison. We show that Dbx1-expressing progenitors give rise to preBötC neurons and glia. Peak neuronal tdTomato expression occurs when tamoxifen is administered at embryonic day 9.5 (E9.5), whereas tdTomato expression in glia shows no clear relationship with tamoxifen timing. These results can be used to bias reporter protein expression in neurons (or glia). Tamoxifen administration at E9.5 labels 91% of Dbx1-derived neurons in the preBötC, yet only 48% of Dbx1-derived glia. By fate mapping Dbx1-expressing progenitors, this study illustrates the developmental assemblage of Dbx1-derived cells in preBötC, which can be used to design intersectional Cre/lox experiments that interrogate its cellular composition, structure, and function.

Transient Suppression of Dbx1 PreBötzinger Interneurons Disrupts Breathing in Adult Mice.

Interneurons derived from Dbx1-expressing precursors located in the brainstem preBötzinger complex (preBötC) putatively form the core oscillator for inspiratory breathing movements. We tested this Dbx1 core hypothesis by expressing archaerhodopsin in Dbx1-derived interneurons and then transiently hyperpolarizing these neurons while measuring respiratory rhythm in vitro or breathing in vagus-intact adult mice. Transient illumination of the preBötC interrupted inspiratory rhythm in both slice preparations and sedated mice. In awake mice, light application reduced breathing frequency and prolonged the inspiratory duration. Support for the Dbx1 core hypothesis previously came from embryonic and perinatal mouse experiments, but these data suggest that Dbx1-derived preBötC interneurons are rhythmogenic in adult mice too. The neural origins of breathing behavior can be attributed to a localized and genetically well-defined interneuron population.

Dbx1 precursor cells are a source of inspiratory XII premotoneurons.

All behaviors require coordinated activation of motoneurons from central command and premotor networks. The genetic identities of premotoneurons providing behaviorally relevant excitation to any pool of respiratory motoneurons remain unknown. Recently, we established in vitro that Dbx1-derived pre-Bötzinger complex neurons are critical for rhythm generation and that a subpopulation serves a premotor function (Wang et al., 2014). Here, we further show that a subpopulation of Dbx1-derived intermediate reticular (IRt) neurons are rhythmically active during inspiration and project to the hypoglossal (XII) nucleus that contains motoneurons important for maintaining airway patency. Laser ablation of Dbx1 IRt neurons, 57% of which are glutamatergic, decreased ipsilateral inspiratory motor output without affecting frequency. We conclude that a subset of Dbx1 IRt neurons is a source of premotor excitatory drive, contributing to the inspiratory behavior of XII motoneurons, as well as a key component of the airway control network whose dysfunction contributes to sleep apnea.

Synaptic Depression Influences Inspiratory-Expiratory Phase Transition in Dbx1 Interneurons of the preBötzinger Complex in Neonatal Mice.

The brainstem preBötzinger complex (preBötC) generates the rhythm underlying inspiratory breathing movements and its core interneurons are derived from Dbx1-expressing precursors. Recurrent synaptic excitation is required to initiate inspiratory bursts, but whether excitatory synaptic mechanisms also contribute to inspiratory-expiratory phase transition is unknown. Here, we examined the role of short-term synaptic depression using a rhythmically active neonatal mouse brainstem slice preparation. We show that afferent axonal projections to Dbx1 preBötC neurons undergo activity-dependent depression and we identify a refractory period (∼2 s) after endogenous inspiratory bursts that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1 preBötC neurons. We demonstrate that the duration of the refractory period-but neither the cycle period nor the magnitude of endogenous inspiratory bursts-is sensitive to changes in extracellular Ca(2+). Further, we show that postsynaptic factors are unlikely to explain the refractory period or its modulation by Ca(2+). Our findings are consistent with the hypothesis that short-term synaptic depression in Dbx1 preBötC neurons influences the inspiratory-expiratory phase transition during respiratory rhythmogenesis. SIGNIFICANCE STATEMENT: Theories of breathing's neural origins have heretofore focused on intrinsically bursting "pacemaker" cells operating in conjunction with synaptic inhibition for phase transition and cycle timing. However, contemporary studies falsify an obligatory role for pacemaker-like neurons and synaptic inhibition, giving credence to burst-generating mechanisms based on recurrent excitation among glutamatergic interneurons of the respiratory kernel. Here, we investigated the role of short-term synaptic depression in inspiratory-expiratory phase transition. Until now, this role remained an untested prediction of mathematical models. The present data emphasize that synaptic properties of excitatory interneurons of the respiratory rhythmogenic kernel, derived from Dbx1-expressing precursors, may provide the core logic underlying the rhythm for breathing.

Identification of the pre-Bötzinger complex inspiratory center in calibrated "sandwich" slices from newborn mice with fluorescent Dbx1 interneurons.

Inspiratory active pre-Bötzinger complex (preBötC) networks produce the neural rhythm that initiates and controls breathing movements. We previously identified the preBötC in the newborn rat brainstem and established anatomically defined transverse slices in which the preBötC remains active when exposed at one surface. This follow-up study uses a neonatal mouse model in which the preBötC as well as a genetically defined class of respiratory interneurons can be identified and selectively targeted for physiological recordings. The population of glutamatergic interneurons whose precursors express the transcription factor Dbx1 putatively comprises the core respiratory rhythmogenic circuit. Here, we used intersectional mouse genetics to identify the brainstem distribution of Dbx1-derived neurons in the context of observable respiratory marker structures. This reference brainstem atlas enabled online histology for generating calibrated sandwich slices to identify the preBötC location, which was heretofore unspecified for perinatal mice. Sensitivity to opioids ensured that slice rhythms originated from preBötC neurons and not parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN) cells because opioids depress preBötC, but not pFRG/RTN rhythms. We found that the preBötC is centered ~0.4 mm caudal to the facial motor nucleus in this Cre/lox reporter mouse during postnatal days 0-4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imaging-based studies, as well as the application of other Cre-dependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratory-related oscillations of preBötC Dbx1-derived neurons and thus breathing.

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice.

To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality. Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors. Here, we show that selective photonic destruction of Dbx1 preBötC neurons in neonatal mouse slices impairs respiratory rhythm but surprisingly also the magnitude of motor output; respiratory hypoglossal nerve discharge decreased and its frequency steadily diminished until rhythm stopped irreversibly after 85±20 (mean ± SEM) cellular ablations, which corresponds to ∼15% of the estimated population. These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations. We also establish quantitative cellular parameters that govern network viability, which may have ramifications for respiratory pathology in disease states.

Transmission of the respiratory rhythm to trigeminal and hypoglossal motor neurons in the American Bullfrog (Lithobates catesbeiana).

Spatially distinct, interacting oscillators in the bullfrog medulla generate and coordinated buccal and lung ventilatory rhythms, but how these rhythms are transmitted onto trigeminal and hypoglossal motor neurons is unknown. Using a vertically-mounted isolated brainstem preparation, the Sheep Dip, we identified the regions of the brainstem containing motor nuclei using a solution capable of blocking synaptic release and, following washout, locally exposed these regions to 5 µM NBQX and/or 50 µM AP5. Local application of NBQX significantly reduced the amplitude of buccal and lung bursts on the trigeminal nerve, and lung bursts on the hypoglossal nerve. Local AP5 caused a significant reduction in lung burst amplitude on both nerves, but for buccal bursts, hypoglossal amplitude increased and trigeminal amplitude was unchanged. Local co-application of NBQX and AP5 eliminated fictive respiratory motor output completely in both nerves. These results are consistent with mammalian data, suggesting a critical role for glutamate in transmission of respiratory activity from oscillators to motor neurons.

Evidence for a distributed respiratory rhythm generating network in the goldfish (Carsssius auratus).

Central pattern generators located in the brainstem regulate ventilatory behaviors in vertebrates. The development of the isolated brainstem preparation has allowed these neural networks to be characterized in a number of aquatic species. The aim of this study was to explore the architecture of the respiratory rhythm-generating site in the goldfish (Carassius auratus) and to determine the utility of a newly developed isolated brainstem preparation, the Sheep Dip. Here we provide evidence for a distributed organization of respiratory rhythm generating neurons along the rostrocaudal axis of the goldfish brainstem and outline the advantages of the Sheep Dip as a tool used to survey neural networks.