Expression of functional tyrosine kinase B receptors by rhythmically active respiratory neurons in the pre-Bötzinger complex of neonatal mice

Abstract

Genetic loss of brain-derived neurotrophic factor (BDNF) severely disrupts brainstem control of respiratory rhythmogenesis in newborn mice; however, the sites at which BDNF acts to regulate respiratory rhythmogenesis are unknown. Using immunochemical and multiplex RT-PCR analysis in mouse brainstem slices, we report that the BDNF receptor, Tyrosine kinase B (TrkB), is strongly expressed in the pre-Bötzinger complex (PBC), the presumed site for rhythm generation, and colocalizes with neurokinin 1 (NK1), a marker of neurons critical for breathing. The period of the respiratory rhythm generated by PBC neurons in vitro was increased by 30% after BDNF treatment (100 ng/ml) and not by nerve growth factor (100 ng/ml) or BDNF (100 ng/ml) in the presence of the tyrosine kinase inhibitor K252a (200 nm). Both synaptic and voltage-dependent properties of PBC neurons were modified by BDNF. Synaptic currents underlying spontaneous rhythmic bursts and glutamate-evoked currents were enhanced by 66 and 33%, respectively. BDNF reduced the Ih current amplitude in rhythmic neurons by 46% and shifted its activation curve by -17 mV. All neurons expressing TrkB mRNA (n = 8) also expressed mRNAs for the Ih current [hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective channel (HCN1)], and three of four NK1-positive neurons coexpressed TrkB and HCN mRNA. Six of 16 PBC neurons expressed BDNF mRNA, supporting the possibility of autocrine and paracrine actions of BDNF within the respiratory pattern generator. Our data demonstrate that BDNF can modulate respiratory network activity through TrkB signaling in rhythmic PBC neurons.

Figure 3.

BDNF affects excitatory processes in PBC neurons. A1, Synaptic currents (top traces) recorded from a PBC neuron during a burst of PBC population activity (bottom traces; integrated signal) were recorded in control conditions (left), in 100 ng/ml BDNF (middle), and after 30 min washout (right). Dashed lines indicate the different amplitudes obtained under each condition. A2, Histogram depicting the mean current amplitude measured in control conditions (CTL; white bar), in the presence of BDNF (black bar), and after 30 min washout (gray bar). B1, Current evoked by pressure application of 1 mm glutamate (black squares) in control conditions (left), in the presence of 100 ng/ml BDNF (middle), and after 30 min washout (right). Unclamped action potentials were truncated. B2, Same as in A2. C1, Stimulation protocol used to evoke the I_h current and I_h raw current traces. C2, Graph of I_h versus voltage. Evoked currents were measured as described in Materials and Methods in control conditions (○) and after 30 min exposure to 100 ng/ml BDNF (•). C3, I_h current activation curves obtained in control conditions (○) and in the presence of 100 ng/ml BDNF (•). The data were fitted to a first order Boltzmann relation (lines). ^*p < 0.05; one-way ANOVA.

Figure 1.

NK1 and TrkB immunoreactivity in the newborn mouse caudal medulla. A, TrkB is prominent in the PBC region (shown at higher magnification in B) and hypoglossal nucleus (XII). C, NK1 in the PBC. D, Merged image of B and C; yellow indicates colocalization of NK1 and TrkB. Scale bars: A, 1 mm; B-D, 50 μm.

Figure 2.

BDNF decreases respiratory frequency in vitro. A, Intracellular recordings from a PBC neuron (top traces) recorded simultaneously with integrated population activity (bottom traces) in control (A1) and in the presence of 100 ng/ml BDNF (B1). A2, Bar histograms showing the mean period of rhythmic activity in the presence of either BDNF or NGF and their respective control (CTL) (BDNF control, white bar; n = 12), 100 ng/ml BDNF (black bar; n = 12), NGF control (light gray bar; n = 6), and 100 ng/ml NGF (dark gray bar, n = 6). B2, Bar histograms depicting the mean period of rhythmic activity (n = 6) in control conditions (white bar), 200 nm K252a (gray bar), and 200 nm K252a plus 100 ng/ml BDNF (black bar). BDNF effects are abolished in the presence of K252a. ^*p < 0.05.

Figure 4.

PBC neurons express TrkB, HCN, NK1, and BDNF mRNAs. A, Left, Schematic diagram of a transverse brainstem slice. The rectangle outlines the PBC, illustrated at higher magnification in the right panel. Right, Intracellular labeling with Alexa Fluor 594 of seven rhythmic PBC neurons analyzed by RT-PCR (asterisks). Labeled, unmarked neurons were not rhythmic and therefore not analyzed by RT-PCR. B, Graph depicting the numbers of TrkB-positive PBC neurons expressing different combinations of HCN, NK1, and BDNF mRNAs. C, Agarose gel analysis of the multiplex RT-PCR products of three PBC neurons. Φ indicates molecular weight markers. IO, Inferior olive; NA, nucleus ambiguus; PBC, pre-Bötzinger complex; Sp5, spinal trigeminal nucleus; X, dorsal motor nucleus of vagus; XII, hypoglossal nucleus.