Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry

Abstract

Introduction: The pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH.

Methods: We utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC.

Results: We found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC.

Conclusion: We provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

Keywords: desmosome-like contact; intermittent hypoxia; mitochondria; neuroplasticity; pre-Bötzinger complex; synapse; ultrastructure.

FIGURE 1

Electron micrographs showing SST and NK1R immunoreactivities in the pre-BötC. NK1R immunogold particles were localized mainly along the inner surface of the plasma membrane [arrows in panels (A,B)]. The particles were also distributed in the rough endoplasmic reticulum (C) and multivesicular body [open arrowheads in panel (D), open thin arrowheads in panel (E)]. Some multivesicular bodies had clathrin coat [double open arrowheads in panels (D,E)]. Multivesicular bodies with no NK1R immunoreactivity were also visible [open thick arrowhead in panel (E)]. SST immunoperoxidase reaction product was observed in somata [arrowheads in panels (A,B)], primary dendrites [arrowheads in panel (B)], and large dense-core vesicles in terminals [solid arrowheads in panel (D)]. SST was specifically expressed in the Golgi apparatus (C). Perforated symmetric synapses (SS, solid thick arrows in panel (D,F)] and horseshoe-shaped SS [solid thin arrow in panel (D) were visualized. Desmosome-like contacts (DLC) were seen alongside of SS (solid arrowhead in panel (E)] or between two SS [solid arrowhead in panel (F)]. Mitochondria seemed in more proximity to DLC [open arrows in panel (E)]. Nu: nucleus, Som: soma, ER: endoplasmic reticulum, G: Golgi apparatus, T: terminal, Den: dendrite. a: artificial. Scale bars in panels (A,B): 1 μm, in panels (C–F): 0.5 μm.

FIGURE 2

Electron micrographs showing CO-reactive mitochondria in the pre-BötC. Mitochondria with dark (solid thin arrows), moderate (open arrows with solid arrowheads), and light (open arrows) CO reactivity were identified in NK1R⁺ dendrites (A–C). Open arrows in panel (D) indicated mitochondria with no CO histochemistry. Thin arrowhead in panel (A) pointed to the presumable fused membrane of two mitochondria. Som: soma, Den: dendrite, T: terminal. Ly: lysosome. Scale bars: 0.5 μm.

FIGURE 3

Electron micrographs showing perforated synapses, DLC and mitochondria in the pre-BötC. Perforated synapses had discontinuous postsynaptic densities [PSDs, solid arrows in panels (A,B,D,E)]. DLC were found alongside of synapses [solid thick arrowheads in panels (A–D,F)] or between two segments of perforated synapses [solid arrowhead in panel (E)]. PSDs were seen in continuity to the dense membranes of DLC (B–D). Open arrowheads pointed to synaptic vesicles accumulated in distinct pools opposed each discrete PSD segments and DLC. Open arrows showed mitochondria in proximity to DLC. Filamentous or flocculent elements could be found between the mitochondria and the DLC [solid thin arrowheads in panels (A–C)]. Curve in panel (F) represented the distance between the DLC and the synapse, and straight lines in panel (F) showed the closest distances of mitochondria to the center of DLC and PSDs. Den: dendrite, T: terminal. Scale bars: 0.2 μm.

FIGURE 4

Electron micrographs showing perforated synapses in the pre-BötC. An SST⁺ terminal made perforated SS with a NK1R⁺ dendrite [arrows in panel (A)]. Perforated synapses were found to be localized in large dendritic shafts, and also extended into restricted spaces [thin arrows in panels (B,C)]. Synaptic vesicles could be seen accumulated in distinct pools opposed each discrete segment of PSDs [open arrowheads in panels (B,C,d1,d3)]. Solid arrowheads in panels (d1–d3) pointed to DLC. A mitochondrion was found in more proximity to a DLC than SS [open arrow in panel (d2)]. Areas labeled by panels (d1–d3 in D) were magnified in insets. T: terminal, Den: dendrite. Scale bars: 0.5 μm.

FIGURE 5

Percentages and the size of synapses formed by SST^– (A–C) or SST⁺ (D–F) terminals onto NK1R⁺ dendrites in the pre-BötC in the normoxic (NOR), daily acute intermittent hypoxia (dAIH) and chronic intermittent hypoxia (CIH) groups. Pie charts illustrated proportions of macular and perforated AS and SS (A,D). The mean length of PSDs in SST^– AS and SS showed significant increases in the dAIH group, as compared to the CIH group (Kruskal–Wallis test, p = 0.0047 in AS, p = 0.0009 in SS), but had no significant differences while compared to the NOR group (B). The mean area was also significantly increased in the dAIH group, as compared to the CIH group [(C) Kruskal–Wallis test, p < 0.0001 in AS, p = 0.0055 in SS]. Significant increases in the mean area were also identified in AS in the NOR group, as compared to the CIH group (Kruskal–Wallis test, p = 0.0159) and in SS in comparison with the dAIH group [(C) Kruskal–Wallis test, p = 0.0312]. Significant increase in the PSD size was identified only in SST⁺ SS in the dAIH group, as compared to the NOR [(E,F) Kruskal–Wallis test, p = 0.0249 in length; p = 0.0327 in area]. The mean area was also significantly increased in the dAIH group, as compared to the CIH group [(F) Kruskal–Wallis test, p = 0.021]. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

DLC relationships with synapses and mitochondria in the pre-BötC in the NOR, dAIH and CIH groups. Pie charts presented proportions of DLC in association with macular and perforated AS and SS (A). DLC were in more proximity to synapses in the dAIH than the CIH group [(B) Kruskal–Wallis test, p = 0.035]. Mitochondria were found in more proximity to DLC than synapses in terminals [(C) Mann Whitney U test, p = 0.001 in NOR, p = 0.0059 in dAIH, p = 0.0004 in CIH] and in dendrites [(C) Mann Whitney U test, p = 0.023 in NOR, p = 0.019 in dAIH, p = 0.089 in CIH]. The distance of mitochondria to isolated DLC showed no significant differences of three groups (D). Percentages of darkly, moderately and lightly CO-reactive mitochondria in proximity to DLC in terminals and dendrites were displayed in three groups (E). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Expressions of SST and NK1R in the pre-BötC in the NOR, dAIH and CIH groups. SST immunoreactivity is visualized with Alexa 488 (green) and NK1R immunoreactivity with Texas Red (red). Thin arrows presented colocalization of two fluorophores in small fusiform neurons (A–C). SST⁺ boutons were found in close association with NK1R⁺ somata (thick arrowheads) and processes (thin arrowheads). Thick arrows illustrated NK1R⁺ large neurons (B,C). Western blots showing bands detected with antibodies against SST, NK1R and actin in tissues of the ventrolateral medulla oblongata, including the pre-BötC region (D). Darker bands were found in lanes with protein extracts from the dAIH group, while lighter ones were from the CIH group. Scale bars: 20 μm, *p < 0.05, **p < 0.01.

FIGURE 8

Electron micrographs showing AS, SS and DLC formed by SST⁺ terminals in the pre-BötC. SST⁺ terminals made SS [open arrows in panel (A,B)] and AS [solid arrows in panels (C,D)] onto NK1R⁺ dendrites. Arrowheads in panels (B,D) pointed to postsynaptic dense bodies. DLC were found alongside of AS [arrowheads in panels (C,E)]. Mitochondria could be seen in proximity to DLC [open arrows in panels (C,E)]. Synaptic vesicles appeared in accumulation in distinct pools opposed to PSDs and DLC [open arrowheads in panel (E)].

FIGURE 9

Electron micrographs and histograms showing spines and associated mitochondria in the pre-BötC in three groups. Spine-shaft microdomains presented synapses in spines [synapse with T1 in panel (A), open arrowheads in panel (B)] and in parent shafts adjacent to the base of the spine neck [open arrows in panels (A,B)], which were coupled with mitochondria positioning in spines [solid arrowhead in panels (A,B)], at the spine neck [solid arrow in panel (A)] and in parent shafts near the base of the spine neck [solid arrows in panel (B)]. Multiple-synapse spines with both AS and SS were detectable [open arrowheads in panels (B,F)]. A presumed SS at the spine neck [open arrow in panel (D)] extended into a spine that also received an AS on the head [open arrowhead in panel (D)]. Mitochondria seemed successively in spines [solid arrowheads in panels (C,F)] with a follower at the spine neck [arrows in panels (C,F)]. SST⁺ terminals made synapses onto spines [synapse with T1 in panel (A), open arrowhead in panel (E)], in the spine-shaft microdomain [open arrow with T2 in panel (A)], and on the shaft [open arrow with T3 in panel (A)]. T: terminal, Den: dendrite, Spi: spine. Scale bars: 0.5 μm. Histograms showed mitochondrial positioning (G), size (H) and co reaction (I) in spine-shaft microdomains.

FIGURE 10

Electron micrographs showing mitochondrial fusion and fission in the pre-BötC. Fusion was identified between two larger mitochondria [arrows in panels (A–D)]. The fused outer membrane was clearly visible with a slight curvature toward the inner membrane [arrowheads in panels (A–C)]. Fission was found between two smaller mitochondria [arrows in panel (F)] interconnected by a mitochondrial tubule [arrowheads in panel (F)]. A cluster of mitochondria overlapped in close proximity to a perforated synapse [open arrows in panel (G)], of which the larger two (thick arrowheads) seemed in fusion, and the smaller two (thin arrowheads) in fission. The endoplasmic reticulum was commonly visible among mitochondria [solid arrow in panel (G)]. Filamentous or flocculent elements could be found between mitochondria [arrowhead in panel (E)]. Den: dendrite, T: terminal. Scale bars: 0.5 μm.