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Exp Neurobiol 2022; 31(6): 376-389
Published online December 31, 2022
© The Korean Society for Brain and Neural Sciences
Myungmo An1,2†, Hyun-Kyung Kim1,2†, Hoyong Park3†, Kyunghoe Kim1,2, Gyuryang Heo1, Han-Eol Park1, ChiHye Chung3* and Sung-Yon Kim1,2*
1Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, 2Department of Chemistry, Seoul National University, Seoul 08826, 3Department of Biological Sciences, Konkuk University, Seoul 05029, Korea
Correspondence to: *To whom correspondence should be addressed.
Sung-Yon Kim, TEL: 82-2-880-4994
ChiHye Chung, TEL: 82-2-450-0432
†These authors contributed equally to this article.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The lateral septum (LS) is a forebrain structure that has been implicated in a wide range of behavioral and physiological responses to stress. However, the specific populations of neurons in the LS that mediate stress responses remain incompletely understood. Here, we show that neurons in the dorsal lateral septum (LSd) that express the somatostatin gene (hereafter, LSd
Keywords: Stress, Norepinephrine, Somatostatin, Lateral septum, Locus coeruleus
Stress—commonly defined as a disruption of homeostasis—impacts a variety of behavioral and physiological parameters, ranging from cardiac and respiratory patterns to arousal and emotional states [1-8]. While adaptive stress responses are essential for the survival and well-being of all animals, excessive and maladaptive stress responses contribute to the etiology of numerous disorders, including anorexia, depression, and anxiety disorder [7, 9-13]. As such, considerable effort has been extended in gaining a mechanistic understanding of stress responses, which is still an active research area [14-23].
A body of research has established that multiple forebrain structures coordinately orchestrate stress responses [24-27]. Among these brain regions, many distinct subregions and cell types of the LS have been implicated in diverse stress-related functions [28-36]. Recent studies have revealed a role of the LSd in stress-related behaviors [29, 30, 34, 36], where neurons expressing somatostatin (a neuropeptide also linked to stress in different parts of the brain [37-42]) are concentrated [28-30, 36, 43-46]. Indeed, LSd
Norepinephrine (NE) is a key neuromodulator in stress responses [47-54]. The LS is densely innervated by noradrenergic fibers, and some LS neurons express adrenergic receptors [55-59]. Besides, studies over the past several decades have identified the functional relevance of NE signaling to LS neurons in stress [60, 61]. However, the specific identity of LS neurons that receive NE signals has not been identified. The locus coeruleus (LC) is a pontine brain area that serves as a major source of NE to many forebrain areas, including the LS [53-56]. Since noradrenergic neurons in the LC are well known to be activated upon diverse stressors (such as restraint stress, innate fear, and footshock) [49, 51-54], their connections to the LS may represent an important component of stress reactions.
Here, we explored the role of LSd
All procedures were approved by the Seoul National University Institutional Animal Care and Use Committee. Adult wild-type or heterozygote mice from C57BL/6J background (C57BL/6J mice, JAX #000664; Ssttm2.1(cre)Zjh/J, JAX #013044) were groups housed (except for the restraint experiment) with
The recombinant adeno-associated virus (AAV) vector expressing GCaMP6m (AAV1-hSyn-FLEX-GCaMP6m, 1.2×1013 copies/ml) was purchased from the Penn Vector Core, and the AAVs expressing channelrhodopsin (AAV5-EF1α-DIO-hChR2(H134R)-eYFP, 6.2×1012 copies/ml), eYFP (AAV5-EF1a-DIO-eYFP, 3.5×1012 copies/ml), optimized rabies G protein (AAV8-CA-Flex-RG, 1.8×1012 copies/ml) and TVA receptor (AAV8-EF1a-FLEX-TVAcherry, 5.4×1012 copies/ml) were obtained from the UNC vector core. The AAV expressing mRuby-fused synaptophysin (AAV-DJ-hSyn-FLEX-GFP-2A-Synaptophysin-mRuby, 4.0×1013 copies/ml) was purchased from the Stanford Vector Core. The recombinant EnvA-pseudotyped G-deficient rabies virus vector expressing GFP (RV-EnvA-ΔG-GFP) was purchased from the Salk vector core or generously provided by B. K. Lim (UCSD).
Mice were placed in a stereotaxic frame (Kopf Instruments) while resting on a heat pad under 1.5~3.0% isoflurane anesthesia. Following hair removal and alcohol disinfection, craniotomy was performed using a hand drill (Saeshin, 208B), and 250~300 nl of viral vectors were injected to the LS using a pressure injection system (Nanoliter 2000) with a pulled glass capillary at 50 nl/min. After injection, the capillary was retracted slowly (0.01 mm/s) to prevent the virus from flowing backward. The coordinates were +1.00 mm antero-posterior (AP), 0 mm medio-lateral (ML), -2.70 mm dorso-ventral (DV) for LSd stimulation, anterograde projection mapping, and rabies tracing experiments, except for the fiber photometry group.
For fiber photometry recordings from the LSd, recombinant AAVs expressing GCaMP6m were unilaterally injected into the LSd of
For optogenetic stimulation experiments, recombinant AAVs expressing channelrhodopsin (ChR2) were injected into the LSd of
For anterograde projection mapping, AAVs expressing GFP and mRuby-fused synaptophysin were injected, and after 3~4 weeks, mice were euthanized and processed for histology. For rabies tracing experiments, AAVs expressing Cre-dependent TVA and G were injected into the LSd of
The incision was sutured, and antibiotics and analgesics were given to the mice. Mice were kept in their home cages for four weeks to allow for recovery and adequate viral expression.
Fiber photometry recordings were performed as previously described [62, 63]. Briefly, excitation lights from 470-nm and 405-nm LEDs (Thorlabs, M470F3/M405F1) that were sinusoidally modulated by the RZ5P processor (Tucker Davis Technologies) at 211 Hz and 531 Hz, respectively, were delivered to the target region of mice via a low-autofluorescence fiberoptic patch cord and cannula (Doric Lenses, 400 μm-core, 0.48 NA). The light intensity was maintained at a maximum of 20 μW during recordings. The emitted fluorescence was detected by a femtowatt photoreceiver (Newport, 2151). The resulting signal was demodulated, amplified, and collected at ~1 kHz by the RZ5P processor. To correlate the photometry signals with behavior, behavioral experiments were recorded using a video camera, and the location and activity of the mice were automatically tracked by video tracking software (Noldus Ethovision). A TTL pulse generated by a pulse generator (Sanworks, Pulse Pal) was split and fed into the RZ5P processor and a TTL-triggered blue LED was placed in the field of view where mice could not see. For the footshock, event timestamps marking shock deliveries were used.
For optogenetic stimulations, 5 or 10 mW blue light (159 mW/mm2 at the tip of the patch cords) was generated by a 473-nm laser (MBL-III-473; OEM Laser Systems) and delivered to mice through fiberoptic patch cords (0.22 NA, 200 μm diameter; Newdoon) connected by a rotary joint (Doric Lenses). Light pulses (5 or 10 ms pulse trains at 15 Hz) were generated by controlling the blue laser with a pulse generator (Pulse Pal, Sanworks). Light pulse trains were delivered throughout the experimental session unless otherwise stated.
To reduce stress caused by experimenters, all mice were handled for at least 5 days prior to behavior experiments. To reduce the stress caused by the patch cord connection, mice were first connected to a patch cord and placed in a new cage for 5 min before being introduced to the behavior arena. To avoid interactions between experiments, different behavioral assays were performed at least two weeks apart. Behavioral tests that cause severe stress in mice (e.g., footshock, restraint stress, and tail suspension) were carried out at the end (specifically, for the footshock experiment) or in a separate cohort (for the restraint stress and tail suspension experiments). For all behavior assays where video analysis was appropriate, video tracking software (Noldus, EthoVision XT) was used to track the location and activity of mice.
For the elevated plus maze test, mice were placed in a plus-shaped plastic maze, consisting of two open and closed arms (30×5 cm) extending from a central platform elevated from the ground by 50 cm. Each mouse was initially placed in closed arms, and the behavior was recorded for 10 min for all experiments.
For the open field test, mice were placed in an open field chamber (50×50×50 cm), where the center zone was defined as a square at the center (20×20 cm). Each mouse was placed at the corner at the beginning of the session. Mouse behavior was recorded for 10 min for fiber photometry experiments. For stimulation experiments, mice were recorded for 20 min, in which laser stimulation was applied at the second and fourth 5-min epochs; the two laser-off and laser-on epochs were pooled for analysis.
To conduct the fiber photometry test during the restraint test, mice were introduced into a clear Plexiglas tube (3 cm inner diameter) and two black plastic gates with holes for the nose and tail were inserted to hold mice in place tightly. For the baseline recording, single-housed mice were connected to the patch cord, returned to the home cage, and the fiber photometry recording began after 5 min. After baseline recording in the home cage for 10 min, mice were briefly anesthetized with isoflurane and placed into the restraint apparatus. The restraint apparatus was then placed in a behavioral chamber (25×25×25 cm) for 45 min. Mice were then returned to the home cage and the recording was continued to monitor the post-restraint responses.
For the footshock test, the mice were placed in a behavioral chamber (18×20×36 cm) with a metal grid floor connected to an electric shock generator (Precision animal shocker, Coulbourn). After 10 min of baseline recording, a footshock (0.2 mA, 2 s) was delivered at a pseudo-random interval (90 s on average) 5 times.
For the tail suspension test, the tail of mice was attached to a bar that was 40 cm elevated from the ground. A 2-cm tube was inserted through the tail to prevent the mice from climbing up during the test. The session was divided into three 3-min epochs, where the laser was turned off, on, and off, respectively.
For the real-time place preference test, mice were placed in a white plastic arena (50×25×25 cm) consisting of two identical chambers with a slit to freely move across the chambers for 15 min. One chamber was paired with laser stimulation, and the choice of the stimulation-paired chamber was counterbalanced across mice.
To measure heart and respiratory rates, pulse oximetry (MouseOx Plus, Starr Life Sciences) was used. First, mice were shaved around the neck, then habituated to head fixation and the collar sensor for 30 min a day, at least for 3 days. For each recording session, mice were acclimated to the head fixation and collar sensor for 5 min, then the recording began. Heart and respiratory rates were measured by a collar sensor attached to the animal’s neck for 9 min, in which 3-min laser stimulation was applied from 3 min after the recording began.
Mice were anesthetized with isoflurane and the brains were extracted. Acute 300 μm-thick coronal slices were obtained using a vibratome (Leica VT1200S) in an ice-cold dissection solution (in mM; 212 sucrose, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 7 MgCl2, and 10 glucose, gassed with 95% O2, and 5% CO2). Slice was recovered in artificial cerebrospinal fluid (aCSF) (in mM; 118 NaCl, 2.5 KCl, 11 glucose, 1 NaH2PO4, and 26.2 NaHCO3, gassed with 95% O2 and 5% CO2) at 35℃ for an hour, and then maintained at room temperature. All electrophysiological recording was made under the constant perfusion of aCSF heated to 30℃. Neurons were visualized with an upright microscope (Nikon, Eclipse FN1) equipped with both DIC optics and a filter set for visualizing eYFP (ChR2) and tdTomato, using a 40× water-immersion objective and an sCMOS camera (Andor, Zyla 4.2).
Whole-cell recordings were made from fluorophore-labeled or non-labeled LSd neurons, using patch pipettes (2~6 MΩ) filled with either potassium-based internal solution (for current-clamp analyses; in mM; 130 K-gluconate, 10 HEPES, 0.6 EGTA, 5 KCl and 2.5 MgCl2, pH 7.3) or cesium-based internal solution (for voltage-clamp experiments; in mM; 115 Cs-methanesulphonate, 20 CsCl, 10 HEPES, 2.5 MgCl2, 0.6 EGTA, 5 QX314, 4 Na2-ATP, 0.4 Na2-GTP, and 10 Na-phosphocreatin, pH 7.3). Series resistance was typically 10-15 MΩ. Neuronal activity was filtered at 2 kHz, sampled at 20 kHz, and recorded to disk using Multiclamp 700B and Clampex 10.3 (Molecular Devices).
To record optogenetically evoked action potential, 3.5~5.5 mW blue light generated by a 473-nm blue laser was delivered to slices through fiberoptic patch cords, while recording from ChR2-expressing neurons at a holding current of 0 pA. Pulsed input signals (5-ms pulse trains at 15 or 30 Hz) were generated by pClamp (Molecular Devices). To record miniature excitatory postsynaptic current (mEPSCs), LSd neurons were recorded at a holding potential of -70 mV, with aCSF containing 50 μM picrotoxin and 1 μM tetrodotoxin. Recorded data were analyzed using Minianalysis 6.0.7 (Synaptosoft).
All data were analyzed with custom-written Matlab (Mathworks) code. The photometry signal was analyzed as previously described [62, 63]. Briefly, data were low-pass filtered at 2 Hz, downsampled to 100 Hz, and a linear function scaled the 405-nm signal to the 470-nm signal to obtain the fitted 405-nm signal. The ΔF/F was calculated as (raw 470 nm signal – fitted 405 nm signal) / (fitted 405 nm signal). Peri-event time plots were created using either the TTL timestamps generated by shocker-triggering pulse generators or timestamps marked by manual video analysis.
In anxiety tests, ΔF/F for specific zones was normalized using average and standard deviation of ΔF/F values from the whole-session data. For peri-event plots, ΔF/F was normalized using mean and standard deviation from -5 s to -2.5 s relative to zone entrance events. Only zone entrance events with intervals longer than 5 s were included in the analysis, to ensure that another entrance event does not influence the defined baseline. To examine the correlation between the calcium activity and the movement velocity of mice, normalized ΔF/F and velocity values from the open field test were binned into 1-s intervals.
In the restraint stress experiment, ΔF/F values were normalized by subtracting the average ΔF/F of the baseline recording (from the first homecage recording) and dividing the difference by the baseline standard deviation. For the analysis of the restraint epoch, recordings from the last 30 min were used to exclude the periods that could have been potentially affected by brief anesthesia preceding restraint. The peak time and amplitude of individual calcium transients were identified based on the maximum value of the thresholded signal within each transient, using the code generously provided by Rothschild .
In the footshock test, ΔF/F during shock was normalized using the mean and standard deviation of ΔF/F values from the 10-min baseline recordings. For peri-event time plots, the baseline was defined as 10 s preceding shock delivery in footshock tests.
Mice were anesthetized and transcardially perfused using ice-cold 1× PBS and 4% paraformaldehyde (PFA) solution in PBS. Brains were extracted and fixed overnight in PFA solution, and equilibrated in 30% sucrose solution, before cutting into 50 µm-thick sections using a freezing microtome (Leica, SM2010R). Slices were stored in a cryoprotectant solution (a 5:6:9 mixture of glycerol, ethylene glycol and PBS) at 4℃. Sections were then washed in PBS, incubated for >25 min in 1: 50,000 DAPI solution, washed again in PBS and mounted on microscope slides with PVA-DABCO. Confocal images were obtained on a Zeiss LSM 880 laser scanning microscope using 10×/0.45 NA objective lens. Only mice with restricted expression of the desired transgene (GCaMP and ChR2) in the LSd were included in the study. Note that while we did not demonstrated the specificity of Cre and Cre-dependent transgene expressions in
Statistical analyses and linear regressions were performed using Matlab (Mathworks) or Prism (GraphPad). We used a two-tailed Wilcoxon rank-sum test, one-way repeated measures ANOVA, two-way repeated measures ANOVA with subsequent Bonferroni post-tests, or Pearson correlation depending on the experimental paradigm. *p<0.05, **p<0.01, ***p<0.001. Data were presented as mean±s.e.m. unless otherwise noted. No statistics to determine sample size, blinding or randomization methods were used. Viral expression and implant placement were verified by histology before mice were included in the analysis.
To directly assess if LSd
We first examined if the activity of LSd
We then asked if LSd
To gain insight into how LSd
Next, to probe the monosynaptic inputs of LSd
We next sought to determine the causal function of LSd
To explore the behavioral effect of activating LSd
In this study, we investigated the role of LSd
Several recent studies have also investigated the function and anatomy of LSd
In addition, Besnard and colleagues  found that optogenetic stimulation of LSd
Simultaneously, the study by Besnard and colleagues  revealed significant functional heterogeneity within the LSd
Numerous studies have revealed that NE is released upon stressors and modulates a variety of behavioral and physiological functions [49, 72-76]. We observed that NE, but not corticosterone, increased excitatory synaptic transmission of LSd
However, we observed that optogenetic stimulation of all LSd
Collectively, our results have shown the activity pattern of LSd
M. A., H. -K. K., and H. P. contributed equally. M. A., H. -K. K., H. P., C. C. and S. -Y. K. designed the project, interpreted the data, and wrote the paper with input from all authors. M. A., H. -K. K., and K. K. performed behavior, pharmacology, fiber photometry, and histology experiments with contributions from G. H. and H. -E. P., H. P. and C. C. performed ex vivo electrophysiology experiments. H. -E. P. established the fiber photometry setup. S. -Y. K. supervised all aspects of the work.