4 research outputs found
Identification of the Pre-Botzinger Complex Inspiratory Center in Calibrated âSandwichâ Slices from Newborn Mice with Fluorescent Dbx1 Interneurons
Inspiratory active preâBötzinger 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 followâup 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 Dbx1âderived 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 0â4. Our findings provide the essential basis for future optically guided electrophysiological and fluorescence imagingâbased studies, as well as the application of other Creâdependent tools to record or manipulate respiratory rhythmogenic neurons. These resources will ultimately help elucidate the mechanisms that promote respiratoryârelated oscillations of preBötC Dbx1âderived neurons and thus breathing
Genetically Encoded Glutamate Indicators with Altered Color and Topology
Glutamate is one of the 20 common
amino acids and of utmost importance for chemically mediated synaptic
transmission in nervous systems. To expand the color palette of genetically
encoded indicators for glutamate, we used protein engineering to develop
a red intensity-based glutamate-sensing fluorescent reporter (R-iGluSnFR1).
Manipulating the topology of R-iGluSnFR1, and a previously reported
green fluorescent indicator, led to the development of noncircularly
permutated (ncp) variants. R- and R<sup>ncp</sup>-iGluSnFR1 display
glutamate affinities of 11 ÎŒM and 0.9 ÎŒM, respectively.
We demonstrate that these glutamate indicators are functional when
targeted to the surface of HEK-293 cells. Furthermore, we show that
G<sup>ncp</sup>-iGluSnFR enabled reliable visualization of extrasynaptic
glutamate in organotypic hippocampal slice cultures, while R-iGluSnFR
can reliably resolve action potential-evoked glutamate transients
by electrical field stimuli in cultures of dissociated hippocampal
neurons
Dependence on extracellular Ca2+/K+ antagonism of inspiratory centre rhythms in slices and en bloc preparations of newborn rat brainstem
The pre-Bötzinger Complex (preBötC) inspiratory centre remains active in isolated brainstemâspinal cords and brainstem slices. The extent to which findings in these models depend on their dimensions or superfusate [K+] and [Ca2+] (both of which determine neuronal excitability) is not clear. We report here that inspiratory-related rhythms in newborn rat slices and brainstemâspinal cords with defined boundaries were basically similar in physiological Ca2+ (1.2 mm) and K+ (3 mm). Hypoglossal nerve rhythm was 1 : 1-coupled to preBötC activity in slices and to cervical nerve bursts in en bloc preparations lacking the facial motonucleus (VII). Hypoglossal rhythm was depressed in brainstems containing (portions of) VII, while pre/postinspiratory lumbar nerve bursting was present only in preparations with > 79% VII. preBötC-related slice rhythms were inhibited in 1.5 mm Ca2+ solution, whereas their longevity and burst rate were substantially augmented in 1 mm Ca2+. Ca2+ depression of slice rhythms was antagonized by raising superfusate K+ to 8â10 mm. This strong extracellular Ca2+/K+ antagonism of inspiratory (motor) rhythms was also revealed in brainstemâspinal cords without VII, while the inhibition was progressively attenuated with increasing amount of rostral tissue. We hypothesize that depression of hypoglossal rhythm and decreased Ca2+ sensitivity of preBötC rhythm are probably not related to an increased content of rostral respiratory structures, but rather to larger brainstem dimensions resulting in interstitial gradients for neuromodulator(s) and K+, respectively. We discuss whether block of pre/postinspiratory activity in preparations with < 79% VII is due to impairment of the pathway from preinspiratory interneurons to abdominal muscle