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. 2023 Sep 29:17:1277375.
doi: 10.3389/fncel.2023.1277375. eCollection 2023.

Mild hypoxia-induced structural and functional changes of the hippocampal network

Alexandra Hencz  1 , Andor Magony  1   2 , Chloe Thomas  3 , Krisztina Kovacs  3 , Gabor Szilagyi  4 , Jozsef Pal  1 , Attila Sik  1   2   3
Affiliations

Mild hypoxia-induced structural and functional changes of the hippocampal network

Alexandra Hencz et al. Front Cell Neurosci. .

Abstract

Hypoxia causes structural and functional changes in several brain regions, including the oxygen-concentration-sensitive hippocampus. We investigated the consequences of mild short-term hypoxia on rat hippocampus in vivo. The hypoxic group was treated with 16% O2 for 1 h, and the control group with 21% O2. Using a combination of Gallyas silver impregnation histochemistry revealing damaged neurons and interneuron-specific immunohistochemistry, we found that somatostatin-expressing inhibitory neurons in the hilus were injured. We used 32-channel silicon probe arrays to record network oscillations and unit activity from the hippocampal layers under anaesthesia. There were no changes in the frequency power of slow, theta, beta, or gamma bands, but we found a significant increase in the frequency of slow oscillation (2.1-2.2 Hz) at 16% O2 compared to 21% O2. In the hilus region, the firing frequency of unidentified interneurons decreased. In the CA3 region, the firing frequency of some unidentified interneurons decreased while the activity of other interneurons increased. The activity of pyramidal cells increased both in the CA1 and CA3 regions. In addition, the regularity of CA1, CA3 pyramidal cells' and CA3 type II and hilar interneuron activity has significantly changed in hypoxic conditions. In summary, a low O2 environment caused profound changes in the state of hippocampal excitatory and inhibitory neurons and network activity, indicating potential changes in information processing caused by mild short-term hypoxia.

Keywords: dark neuron; electrophysiology; hippocampus; mild hypoxia; network oscillation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
(A) Changes in tissue O2 levels were measured in the hippocampal regions of urethane-anaesthetized animals (n = 20). There is a statistically significant difference between normoxic (21%) and hypoxic (16%) conditions. Error bars represent mean ± SEM. ***p < 0.001 The location of 32-channel probes in CA1-hilus axis (B) and in the CA3b region (C). Traces of DiI-coated 4 shanks are visible in the fluorescent image. Scale bar: 600 μm.
Figure 2
Figure 2
Gallyas silver-stained images of the control (21% O2) and hypoxic hippocampi after exposure to 16% O2 for 1 h. Very few ‘dark’ neurons are visible in the dentate gyrus (A) CA1 (B) or the CA3 areas (C) in the control hippocampus. (D) Numerous neurons are silver-impregnated in the subgranular layer (examples indicated by arrows) and in the hilus in the hypoxic hippocampus. (E) Examples of ‘dark’ neurons are pointed by arrows within CA1 str. radiatum exposed to 16% O2. (F) ‘Dark’ neurons are present in large numbers in the CA3 area with long dendrites descending into the str. radiatum when animals are exposed to 16% O2 for 1 h. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SL, stratum lucidum; SLM, stratum lacunosum-moleculare; SM, stratum moleculare; SG, stratum granulare; DG, dentate gyrus. Scale bars: 100 μm.
Figure 3
Figure 3
Quantification of the number of dark neurons in the hippocampus in normoxic (21% O2) and hypoxic (16% O2) conditions. (A) There is a statistically significant difference in the number of damaged ‘dark’ neurons between the 21 and 16% experimental groups. (B) Area analysis shows that there are fewer ‘dark’ neurons in regions of CA1 than in the hilus and CA3. A statistically significant difference exists between the CA1 str. radiatum, str. pyramidale, str. oriens and CA3 regions, hilus. CA1SO, CA1 stratum oriens; CA1SP, CA1 stratum pyramidale; CA1SR, CA1 stratum radiatum; CA3SO, CA3 stratum oriens; CA3SP, CA3 stratum pyramidale; SL, stratum lucidum; CA3SR, CA3 stratum radiatum. Error bars are represented as mean ± SEM. *p < 0.05 and ***p < 0.001.
Figure 4
Figure 4
(A) Some ‘dark’ neurons in the dentate hilus are SST-immunopositive. Immunofluorescent staining against SST from the 16% O2 experimental group shows several immunopositive cells that are also silver-impregnated on (B) (arrows). (C) Quantitative analysis of SST-immunopositive, ‘dark’ neurons and double-labeled cells in hippocampal regions. Only the hilus contained double-labeled neurons. Scale bars: (A) 100 μm, (B) 100 μm.
Figure 5
Figure 5
Spectral characteristics of the slow component and the theta oscillation in normoxic (21%) and hypoxic (16%) O2 conditions. Colors represent spectral power ranging from blue (low) to red (high) on a common scale (20–50 dB/Hz). The frequency shift of the slow component is demonstrated with dashed lines and black triangles.
Figure 6
Figure 6
(A) Example of single channel hippocampal raw recording (top) and corresponding spectrogram (bottom) displaying theta activity and a slow component. (1) RAW (wideband), (2) slow component filter (1–4 Hz), (3) theta filter (4–8 Hz), (4) spectrogram. Dashed lines delineate the area of interest where the slow component activity can be seen (black rectangle). (B,C) Comparison of spectral power (dB/Hz) theta oscillation (B) and slow component (C) at 21 and 16% O2 concentrations. There is a significant shift in the frequency but not in the power of the slow component between normoxic and hypoxic conditions Error bars represent mean ± SEM. *p < 0.05.
Figure 7
Figure 7
(A) The unit activity of pyramidal cells in the CA1 region in hypoxic and normoxic conditions. Inter-spike interval (ISI) and standard deviation values significantly decreased in a hypoxic environment meaning that hypoxia increases firing frequency and CA1 pyramidal cells fire more regularly. (B) Pyramidal cells in the CA3 region change activity in hypoxia compared to normoxic conditions. Inter-spike interval (ISI) and standard deviation values were analyzed. Based on the analysis, the frequency of the action potential of the hypoxic CA3 pyramidal cell increased (ISI decreased) and pyramidal cells fired more regularly (SD decreased). (C) The activity of one group of putative inhibitor interneurons in the CA3 region in hypoxic and normoxic environments. Inter-spike interval (ISI) and standard deviation values were analyzed Based on the analysis, the frequency of the action potential of hypoxic interneurons decreased (ISI increased) and fired more irregularly (SD increased). (D) The activity of another group of putative inhibitory interneurons in the CA3 region. The frequency of the action potential of hypoxic interneurons increased (ISI decreased) in a hypoxic environment and the activity showed more regularity (SD decreased). (E) Unit activity of hilar interneurons in normoxic and hyperoxic conditions has been compared based on ISI and SD values. ISI values significantly increased indicating that the activity of hypoxic interneurons decreased. (F) Example epoch of a single unit comparing its firing characteristics due to change in O2 concentration (21% on the left, 16% on the right). The firing rate (ISI 78.57 ms at 21% O2 and 37.49 ms at 16% O2) increased but not the shape of the unit. Error bars represent mean ± SEM. A, **p < 0.005; B, **p < 0.005 and ***p < 0.001; C, **p < 0.005; D, **p < 0.005 and ***p < 0.001; E, **p < 0.005.
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