Identification of the Pre-Botzinger 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

Transcriptome of Neonatal PreBotzinger Complex Neurones in Dbx1 Reporter Mice

We sequenced the transcriptome of brainstem interneurons in the specialized respiratory rhythmogenic site dubbed preBotzinger Complex (preBotC) from newborn mice. To distinguish molecular characteristics of the core oscillator we compared preBotC 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 preBotC neurons express kappa-opioid receptors in addition to mu-opioid receptors that heretofore have been associated with opiate respiratory depression, which may have clinical applications. Second, Dbx1 preBotC neurons express the hypoxia-inducible transcription factor Hif1a at levels three-times higher than non-Dbx1 neurons, which links core rhythmogenic microcircuits to O-2-related chemosensation for the first time. Third, we detected a suite of transcription factors including Hoxa4 whose expression pattern may define the rostral preBotC border, Pbx3 that may influence ipsilateral connectivity, and Pax8 that may pertain to a ventrally-derived subset of Dbx1 preBotC neurons. These data establish the transcriptomic signature of the core respiratory oscillator at a perinatal stage of development

Welcome and Opening Remarks

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

Light activation of Arch-expressing Dbx1 preBötC neurons hyperpolarizes Dbx1 neurons and precludes respiratory rhythm.

<p><b>A</b>, Rostral slice surface of a P2 <i>Dbx1</i>^{<i>CreERT2</i>};Ai35D mouse showing Arch-GFP expression. Dotted box marks the preBötC. <b>B</b>, Dodt image of the slice in A showing location of the preBötC relative to known anatomical markers, the principal loop of the inferior olive (IOP_loop) and semicompact division of the nucleus ambiguus (scNA). The scale bar represents 150 μm and applies to both A and B. <b>C</b>, Inspiratory Dbx1 neuron visually identified by membrane-delimited EGFP expression (top), Dodt contrast microscopy (middle), and by dialysis of Alexa Fluor 568 introduced via the patch pipette solution after the onset of whole-cell recording (bottom). Scale bar represents 10 μm. <b>D</b>, Membrane potential trajectory of the neuron in C with synchronous XII output. TTX was applied at 1 μM. Voltage and time calibrations are shown <b>E</b>, A non-Dbx1 neuron lacking EGFP expression (top), identified in Dodt contrast microscopy and via Alexa Fluor 568 dialysis (bottom). Scale bar represents 10 μm. <b>F</b>, Membrane potential trajectory of the non-Dbx1 preBötC neuron in E with synchronous XII output. <b>G</b>, Membrane potential trajectory of a non-Dbx1 neuron with inspiratory modulation. Voltage calibration in D applies to F and G; separate time calibrations are shown. 589-nm light application is indicated by yellow bars in D, F, and G.</p

Arch-EGFP expresion and histology of fiber-optic implants in adult <i>Dbx1</i>^{<i>CreERT2</i>};Ai35D mice.

<p><b>A</b>, EGFP expression in 35 week-old <i>Dbx1</i>^{<i>CreERT2</i>};Ai35D mouse. Scale bar represents 500 μM. <b>A´</b>, Inset of boxed region in A showing an expanded view of the ventral region of the slice, which includes the preBötC. Scale bar represents 250 μM. <b>B</b>, Bright field image of a thionin-stained section adjacent to A. Scale bar represents 500 μM and applies to B, C, and D. <b>B´</b>, Inset of boxed region in B showing an expanded view of the ventral region of the slice, which shows visible markers that co-locate with the preBötC including the semicompact division of the nucleus ambiguus (scNA) and the principal loop of the inferior olive (IO_loop). Scale bar represents 250 μM. <b>C</b>, Bright field images of adjacent thionin-stained sections from an experimental mouse whose fiber-optics and ferrules targeted the preBötC. <b>D</b>, Bright field image of thionin-stained section from an experimental mouse whose fiber-optics and ferrules targeted medullary circuitry dorsal and rostral to the preBötC.</p

Activation of Arch in Dbx1 preBötC neurons in freely behaving awake mice.

<p><b>A</b>, Airflow, V_T, MV, and <i>f</i>_R plotted continuously during two consecutive applications of 2-s light pulses. Inspiratory airflow is plotted downward, which reflects whole-body plethysmography. <b>A</b>_<b>1</b>, <b>A</b>_<b>2</b>, Expanded airflow traces from A. Yellow bars indicate 589-nm light application. <b>B</b>_<b>1</b>, <b>B</b>_<b>2</b>, Cycle-triggered averages of airflow from each bout prior to (not highlighted) and during illumination (highlighted). Note that inspiratory airflow is attenuated, whereas expiratory airflow is not. Time calibrations are shown for each panel.</p

Light application to the dorsal medulla rostral to preBötC in sedated mice.

<p><b>A</b>, Airflow, V_T, MV, and <i>f</i>_R plotted continuously during two consecutive applications of 2-s light pulses. Inspiratory airflow is plotted upward, which reflects nose-cone measurements. <b>A</b>_<b>1</b>, <b>A</b>₂, Expanded airflow traces from A. Yellow bars indicate 589-nm light application. <b>B</b>_<b>1</b>, <b>B</b>_<b>2</b>, Cycle-triggered averages of airflow from each bout prior to (not highlighted) and during illumination (highlighted). Time calibrations are shown for each panel.</p

The respiratory effects of Arch-mediated photoinhibition.

<p>The left column shows effects in sedated animals. The right column shows effects in freely-behaving awake animals. Cyan symbols pertain to illumination of the preBötC whereas magenta symbols pertain to illumination of the dorsal medulla rostral to preBötC. Respiratory measurements include T_i (first row), MV (second row), V_T (third row), and <i>f</i>_R (fourth row). Control, light application, and recovery data are shown for all experimental subjects. Double asterisks refer to the probability of a type I statistical error with alpha < 0.01. Triple asterisks refer to the probability of a type I statistical error with alpha < 0.001. “n.s.” (i.e., not significant) refers to the probability of a type I statistical error with alpha > 0.05.</p