Persistently increased post-stress activity of paraventricular thalamic neurons is essential for the emergence of stress-induced alterations in behaviour

Short-term effects of predator odour stress exposure (POSE)

In this study, we used a well-established, behaviourally relevant model of stress, predatory odour stress exposure (POSE) [35–38]. We analysed the changes of spontaneous behaviour in the home cage of the animals and compared the pre- and post-stress periods in order to avoid interference of different stressors by exposing the animals to another stressful situation (e.g., elevated plus maze). We utilised optogenetic perturbation of PVT/CR+ neuronal activity timed after the stress event. Our manipulations and recordings were centred on middle portion of PVT (AP, BR: −0.8 mm to 1.8 mm) and did not include the rostral 500 μm sector of the nucleus or its most caudal end (see Methods section for).

After recording the pre-stress, behaviour in the home cage (PRE 1–5 days) we exposed mice to 2-methyl-2-thiazoline (2MT) for 10 min [39,40] in a novel cage and compared home cage activity between pre- and post-stress days. In order to test the role of post-stress PVT/CR+ neuronal activity in stress-induced alteration of behaviour, we perturbed PVT/CR+ firing after POSE (1 h) when the animals were returned to their home cage. We used intermittent activation (2 s ON, 13 s OFF) of the inhibitory opsin SwiChR in CR-Cre mice that was previously proved to be effective [13] (Figs 1A and S1). Since we applied optogenetic inhibition after, not during, POSE we avoided interference with perceiving the stress exposure. Tetrode recordings were used to demonstrate the effect of SwiChR activation. PVT/CR+ cells decreased their firing rate during the laser ON periods (S1 Fig), as expected. As a control for the SwiChR group, we used AAV-DIO-EYFP injected stressed animals (EYFP or non-inhibited group, Fig 1A and 1B). As a control for the stress situation, we used a no odour exposure (NOE) group that was exposed to the novel cage without 2MT and a home cage groups which remained in the home cage (see Methods) as well.

Fig 1. Short term effects of inhibiting PVT/CR+ neurons after the predatory odour stress exposure.

(A) Scheme of the experiment. AAVs were injected into PVT and behaviour was assessed 4 weeks later. Following a 5 day baseline period to assess home cage behaviours (PRE5), mice were submitted to POSE. Immediately after the POSE both SwiChR and EYPF groups received a 1 h long, photoinhibition of PVT/CR+ neurons in the home cage. Created with BioRender.com. (B, C) Representative confocal images showing EYFP and SwiChR expression in CR-positive (CR+) PVT neurons. Magenta represents CR immunostaining, while green represents GFP immunostaining, respectively. MD, mediodorsal thalamic nucleus; Hab, habenula; D3V, third ventricle. Scale bars, 250 μm. (D, E) Quantification of time spent with defensive behaviour during predator odour (2MT) stress exposure (POSE) in EYFP (D, t[13] = 21.05) and SwiChR (E, t[6] = 9.35) mice compared to a 2 min baseline (BL) period. (F) Same for the NOE (without 2MT presentation) group (t[4] = 1.822). (G) Bar graph showing the duration of hyperventilation in the Home Cage control group (n = 7), EYFP (n = 7), SwiChR (n = 6), and NOE control (n = 5) in their home cage after POSE (K-W = 21.58, p n = 7), EYFP (n = 7), SwiChR (n = 6), and NOE (n = 5) in their home cage after POSE (F(3,29) = 2.961, p p n = 7); EYFP (n = 7); SwiChR (n = 6); NOE control (F(3,20) = 50.00, p p S1 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. *p p p p

https://doi.org/10.1371/journal.pbio.3002962.g001

Both the EYFP and the SwiChR groups displayed robust defensive behaviours [41] (escape jumps and freezing) during the 10-min exposure to 2MT (Figs 1B–1E and S2A and S1 Movie) since the SwiChR group was not inhibited during the POSE. Freezing accounted for 95% of the total time spent in defensive behaviours. An increase in defensive behaviours was not observed in the NOE group (Fig 1F and S2 Movie) showing that the novel environment per se did not contribute to the defensive behaviours. Following the return of the animals to their home cage, on the day of the stress (POST0 day), EYFP mice displayed significantly more high-frequency respiration (hyperventilation, Fig 1G and S3 Movie), and a trend to elevated sleep onset latency (Fig 1H) compared to the SwiChR group (Fig 1H and S4 Movie) which underwent 1 h long photoinhibition after POSE. This indicates that inhibition of PVT/CR+ neurons after the stress event can ameliorate the immediate impact of stress on behaviour. During the optogenetic inhibition, the SwiChR animals did not display overt behavioural alterations, they showed normal locomotor activity (S4 Movie). However, agreeing with a previous report [13], they moved less compared to the identical period of their last pre-stress day (PRE5) (Fig 1I).

Next, we tested the impact of POSE and post-stress photoinhibition on c-Fos activation and hormonal changes. One hour after POSE PVT/CR+ neurons, the EYFP animals displayed significantly increased c-Fos expression (Fig 1J and 1K) relative to home cage and NOE controls. In the EYFP mice, c-Fos expression also increased in the paraventricular hypothalamic nucleus (PVH) (Fig 1L) together with significantly elevated blood corticosterone (CORT) levels (Fig 1M) as in earlier studies [42,43]. Photoinhibition of PVT/CR+ neurons for 1 h after stress abolished the increase in c-Fos expression of PVT/CR+ cells (Fig 1K). A decrease in c-Fos expression was most prominent under the fibre optic targeting PVT (S2B and S2C Fig). Activation of SwiChR in PVT/CR+ cells, however, did not result in decreased c-Fos activity in PVH (Fig 1L) or decreased blood corticosterone levels (Fig 1M). This in line with previous reports indicating that PVT is involved in the CORT response to acute stress [22], but the acute activation of the hypothalamic–pituitary–adrenal axis (HPA) is controlled by neuronal mechanisms other than PVT/CR+ neuronal activity [21,44–46].

These short-term changes demonstrated that POSE could induce behavioural, hormonal, and gene expression alterations consistent with a strong stress exposure. They also show that post-stress photoinhibition of PVT/CR+ neurons is able to alleviate the immediate behavioural changes induced by POSE without influencing the hormonal response to stress.

Short-term effects of POSE on PVT/CR+ neuronal activity

Next, we aimed to identify how the activity of PVT/CR+ neurons is altered immediately after the post-stress period relative to the pre-stress period. Earlier data indicated decreased GABA-A receptor-mediated inhibition in PVT neurons following restraint stress [47]. Thus, we tested functional GABA-A receptor-mediated synaptic currents after POSE in PVT/CR+ cells using in vitro slice preparations 2 h after POSE (Fig 2A) and compared these to a NOE group. We found that the frequency of spontaneous inhibitory synaptic currents (sIPSCs) significantly decreased in PVT/CR+ neurons (Fig 2B and 2C), while their amplitude remained unaltered (Fig 2D). These data show that the PVT/CR+ cells receive decreased inhibitory synaptic activity immediately after the stress.

Fig 2. Short-term changes in PVT/CR+ unit activity after predator odour stress exposure.

(A) Scheme of the experiments representing the in vitro and in vivo recordings. Created with BioRender.com. (B) Example traces of sIPSC recordings from NOE and POSE mice. (C) Bar graphs of average sIPSC frequency (U = 11) and (D) amplitude (U = 30) in PVT/CR+ neurons recorded ex vivo from NOE (n = 8 neurons from n = 2 mice) and POSE mice 2 h after POSE (n = 9 neurons from n = 2 mice). (E) Waveforms of 3 optotagged PVT/CR+ neurons recorded by the same tetrode (TT1). The neurons were recorded for 2 consecutive days (top vs. bottom row). Channel numbers (Ch) and similarity scores (see Methods) between the 2 days are shown. (F–H) Alteration in the firing rate of PVT/CR+ neurons recorded for 1 h both on the PRE5 (black dots) and the POST 0 days after the POSE (red dots) in the (F) wake, (G) nest, and (H) sleep states (F, t[19] = 1.499; G, t[19] = 2.871; H, t[19] = 3.378; n = 20 units from n = 4 mice). The firing rate is normalised to the mean wake population average of the PRE 5 day. (I–K) Same as E–G, for HFA (I, t[19] = 2.463; J, t[19] = 1.847; K, t[19] = 2.411). Underlying data can be found in S2 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. *p p

https://doi.org/10.1371/journal.pbio.3002962.g002

To test whether this decreased inhibition can have an immediate consequence on the post-POSE firing activity of PVT/CR+ cells, we used tetrode recordings and optogenetically tagged individual PVT/CR+ neurons. We recorded the same PVT/CR+ activity before the POSE (pre-stress fifth day) and on POST0 day immediately after POSE (n = 20 neurons from 4 animals; Figs 2A, 2E, and S3). For these experiments, CR-Cre animals were injected with AAV-DIO-ChR2-EYFP (S3A Fig) which allowed optotagging and clustering (Figs 2E and S4) of PVT/CR+ neurons. We separately analysed firing activity during the wake state outside the nest (wake), wake state inside the nest (nest), and non-rapid eye movement (NREM) sleep inside the nest (sleep).

We found that compared to the PRE5 day activity, on average, PVT/CR+ neurons displayed a significant increase in their firing rate during the nest and sleep states but not during the wake state (Fig 2F–2H) on POST 0 day after the stress exposure. We also analysed the alterations in the firing pattern of PVT/CR+ neurons. Since high-frequency activity (HFA, above 100 Hz) exert disproportionally large effects on their postsynaptic targets and their synaptic plasticity [48,49], we quantified the occurrence of HFA (spike doublets or triplets) in the same PVT/CR+ cell population at PRE5 and POST 0 days. We found that compared to PRE5 day individual PVT/CR+ neurons displayed significantly more HFA clusters in the wake and sleep states (Fig 2I–2K) immediately after the stress.

These data together show that in parallel with the behavioural and hormonal changes PVT/CR+ cell activity is elevated on P0 after the stress event. These data also demonstrate that elevation of c-Fos in PVT/CR+ cells on P0 (Fig 1J and 1K) is paralleled by increased neuronal activity.

Long-term effects of POSE on spontaneous behaviour

In order to test if POSE is able to persistently alter spontaneous behaviour, we measured and compared the spontaneous wake and sleep behaviours of the EYFP mice during the 5 pre- (PRE1-5), and the 4 post-stress days (POST1-4) using intragroup comparison. All measurements were performed in the home cages of the mice (Fig 3A). We found significant alterations in all measures of spontaneous behaviour in the post-stress period (Fig 3). Compared to PRE1–5 days, the horizontal locomotor activity of the EYFP animals remained significantly elevated for the entire duration of the POST period (POST1–4 days) (Fig 3B–3E). After the stress, the animals frequently moved along the edge of their cages (Fig 3C) which was rare in the pre-stress period (PRE period) (Fig 3B). After POSE, the animals needed significantly more time to fall asleep (time between entering the nest and falling asleep, referred to as nest time, Fig 3F) and displayed significantly less nest-building behaviour (Fig 3G and S5 Movie). They increased the time spent with freezing (defined as at least 2 s motionless period) in their nest (Fig 3H and S6 Movie). Both the frequency of the freezing bouts and their total duration increased during the POST period (Fig 3H and 3I). Day-by-day comparison of behavioural data between the PRE and POST periods (RM-ANOVA) also yielded significant differences with the exception of nest building (S5 Fig).

Fig 3. Long-term behavioural alterations after predatory odour stress exposure.

(A) Scheme of the experiment. Created with BioRender.com. (B, C) Top, Representative horizontal locomotor activity during a 3 h session in 1 EYFP mouse within the home cage during one of the PRE days. Nest onset (NO) and sleep onset (SO) are indicated with black arrows. Bottom, Tracked movements of the same animal in the first hour. The dots show the spatial position of the head of the mouse in every frame. Colour of the dots indicate time elapsed (s), from red to blue; small black rectangle marks the nest area; big black rectangle marks cage area. NO and SO, black arrows. (C) Same as B during a POST day. Head positions outside the cage indicate events when the animal was moving along the edge of its cage. (D) Temporal dynamics of averaged, normalised horizontal locomotor activity of EYFP the animals (n = 14) during the PRE1–5 days (black) vs. the POST1–4 days (red) periods (30 min, t[13] = 2.438; 45 min, t[13] = 2.392). (E) The averaged, normalised horizontal locomotor activity of EYFP the animals before nest onset (W = 85). Dots represent the averaged daily values of individual animals. (F–J) (F) Nest time (t[13] = 3.579), (G) nest building time (W = −89), (H) freezing time (t[13] = 5.692), (I) number of freezing bouts (t[9] = 3.569) and (J) steepness of the NREM EEG spectral slope (t[13] = 1.086) in EYFP animals (n = 14) during the PRE (black dots) and POST (red dots) period. Dots represent the averaged daily values of individual animals. Underlying data can be found in S3 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. *p p p p

https://doi.org/10.1371/journal.pbio.3002962.g003

We also measured the quality of sleep by the frequency characteristics (spectral slope) of the EEG (see Methods) [50]. On the stress day (POST0), the spectral slope of the EEG was significantly increased during NREM sleep (S2D Fig) consistent with the earlier data [51,52]. This value, however, did not remain significant during the rest of the post-stress days (POST1–4) indicating normal sleep depth during this period (Fig 3J).

As a control experiment, we compared the behaviour of the NOE group in the PRE and POST period. Our analysis revealed no significant differences in any of the measured behavioural variables during the POST period compared to the PRE period (S6 Fig).

These data collectively demonstrate long-term, stress-induced changes in the spontaneous behaviour of mice within their home cage that occur when the mice are exposed to the predatory odour in a novel environment (see Methods).

Long-term effects of POSE on PVT/CR+ neuronal activity

We found that the firing activity of PVT/CR+ cells was elevated immediately after POSE (Fig 2E–2K). To investigate whether the neuronal activity of PVT/CR+ cells return to pre-stress levels, as expected, based on the process of homeostatic regulation, or, if it persists for extended periods, we performed long-term recordings of PVT/CR+ neurons. We optotagged n = 69 PVT/CR+ neurons during the PRE1–5 days and n = 57 PVT/CR+ neurons during the POST 1–4 days across 4 animals (Fig 4A) and measured their activity. These cells included those which were used to identify short-term changes in PVT/CR+ firing activity after stress (Fig 2). Some of the neurons (n = 13 tagged cells) could be recorded for up to 3 days (PRE5, POST0, and POST1 days; Figs 4D, 4F, 4H, S3B and S3C).

Fig 4. Long-term changes of PVT/CR+ unit activity after predator odour stress exposure.

(A) Scheme of the experiment. Created with BioRender.com. (B) Change in the firing rate of an example PVT/CR+ neuron recorded on PRE 5 day and POST 1 day. (C) Mean firing rate values of PVT/CR+ neurons (n = 126 neurons in 4 animals) during wake, nest, and sleep during the PRE 1–5 and the POST 1–4 days periods. Dots represent individual cells. The data are normalised to the mean wake firing rate of the PRE period (wake, U = 1357, p = 0.0026; nest, U = 1,041, p p p = 0.0009; wake/sleep SMI, U = 1,304, p = 0.0011; nest/sleep SMI, U = 1,814, P = 0.4576). (F) Change in HFA of an example PVT/CR+ neuron recorded on PRE 5 and POST 1 days. Red lines (spikes) show HFAs, black lines mark the spikes with longer interspike intervals than 10 ms. (G) Frequency of HFAs in the entire PVT/CR+ population during the entire PRE and POST periods (wake, U = 1,309, p = 0.0011; nest, U = 992, p p n = 448 pairs) during the PRE and POST period. (U = 13,625, p p p S4 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. **p p p

https://doi.org/10.1371/journal.pbio.3002962.g004

We found that during the entire post-stress period (POST1–4 days), the mean firing rate of PVT/CR+ cell population did not return to the pre-stress levels. PVT/CR+ neuronal activity remained significantly elevated in all 3 states (wake, nest, and during sleep) (Fig 4B and 4C) during the entire post-stress period. As indicated above (Fig 2F–2H) during the pre-stress period PVT/CR+ neurons displayed state-dependent activity. Firing rate was highest during wake, lower in the nest and lowest during non-REM sleep (Fig 4C). In order to test whether increased firing rate observed after the stress affects all the 3 states to a similar degree, we calculated state modulation indices (SMI, see Method) for each pair of states (wake-to-nest, nest-to-sleep, wake-to-sleep) for every recorded PVT/CR+ cells for the PRE and POST periods. Compared to the pre-stress period after the stress PVT/CR+ neurons displayed significantly less state modulation in the wake-to-nest and wake-to-sleep relations as shown by their lower SMI values (Fig 4D and 4E). Less state modulation was largely due to a larger post-stress increase in firing rates recorded during the resting states (nest and sleep) relative to the wake state (Fig 4C).

Next, we analysed whether alteration in firing patterns can also be demonstrated during the post-stress period. We found that relative to the PRE period the occurrence of HFA remained elevated in all 3 behavioural states during the POST1–4 days (Figs 4F, 4G, S7A and S7B). Next, we analysed the correlated activity (CA, spikes of 2 simultaneously recorded neurons within 5 ms) among simultaneously recorded PVT/CR+ cells. Similar to HFA, we found that the occurrence of CA also significantly increased in all 3 states (Fig 4H and 4I). After appropriate normalizations of HFA and CA with the background firing activity (see Methods), these changes remained significant only in the nest CA values (S7C and S7D Fig). This indicates that a general increase of the mean firing rate in PVT/CR+ neurons is largely responsible for the elevated occurrence of HFA and CA.

These data together show that the activity PVT/CR+ cells display state-dependent changes persisting for days, after a single stress exposure paralleling the observed behavioural changes. Neurons postsynaptic to PVT/CR+ cells receive more HFA and CA from the PVT/CR+ cells for several consecutive days.

Effect of post-stress photoinhibition on long-term changes in behaviour

Prolonged alteration of behaviour in the post-stress period (Fig 3) correlated with persistent, stress induced changes of PVT/CR+ neuronal activity (Fig 4). Thus, next, we asked whether reducing the activity of PVT/CR+ cells for 1 h after the stress event can prevent long-term changes in the behaviour during the POST1–4 days as well (Fig 5A). We found that post-stress inhibition on the day of the stress was able to prevent the development behavioural alterations during the entire post-stress period. Compared to the PRE 1–5 days SwiChR mice exhibited no significant, stress-induced behavioural changes during the POST1–4 days. Locomotion decreased, not increased after the stress (Fig 5B–5E). There was no alteration in the nest time (Fig 5F) or in the nest building (Fig 5G). Frequency of freezing bouts and their duration did not change during the POST period relative to the PRE period (Fig 5H and 5I). Day-by-day comparison of behavioural data between the PRE and POST periods (RM-ANOVA) yielded no significant differences (S8 Fig). Post-stress inhibition, however, significantly, increased the NREM EEG spectral slope during the POST period (Fig 5J) indicating a prolonged effect on sleep.

Fig 5. Post-stress photoinhibition of PVT/CR+ neurons prevents stress induced, long-term behavioural changes.

(A) Scheme of the experiment. Created with BioRender.com. (B) Top, Changes in horizontal locomotor activity during 3 h in one SwiChR mouse within the home cage on a PRE day. Nest onset (NO) and sleep onset (SO) are indicated with black arrows. Bottom, Tracked movements of the animal in the first hour. The dots show the spatial position of the head of the mouse in every frame and the colour of the dots indicate lasting time (s), from red to blue; small black rectangle marks the nest area; big black rectangle marks cage area. Nest onset (NO) and sleep onset (SO) are indicated with black arrows. (C) Same as (B) on a POST day. (D) The dynamics of averaged, normalised horizontal locomotor activity (E) of SwiChR mice (n = 7) during the PRE (black) vs. the POST (blue) periods (t[6] = 2.835). (E) Averaged, normalised horizontal locomotor activity of SwiChR mice during the PRE (black) vs. the POST (blue) periods (t[6] = 2.944). (F) Nest time (t[6] = 0.4886), (G) nest building time (t[6] = 0.1084), (H) freezing time (t[6] = 1.142), (I) number of freezing bouts (t[5] = 1.347), and (J) steepness of NREM EEG spectral slope (t[6] = 2.969) in SwiChR mice during the PRE and POST periods. Dots represent the average daily values of individual animals. Underlying data can be found in S5 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. *p

https://doi.org/10.1371/journal.pbio.3002962.g005

As a control, we performed post-stress photoinhibition in the NOE group and compared their behaviour in the PRE and POST period. This manipulation resulted in decreased nest time, reduced freezing behaviour and increased nest-building activity (S9 Fig). Among these behaviours, the changes in freezing and nest-building activity were statistically significant.

These data indicate that post-stress photoinhibition immediately after the stress event has prolonged effects on stress-induced behaviour. Interference with PVT/CR+ neuronal activity, even in the absence of predatory odour, can significantly reduce stress-related behaviours for several days.

Effect of post-stress photoinhibition on long-term changes in PVT/CR+ neuronal activity

Next, we investigated whether post-stress inhibition of PVT/CR+ cells for 1 h can also prevent the long-term alterations in the firing activity of PVT/CR+ cells (Fig 6A) for the entire POST period similar to the behavioural changes. For these experiments, we used SwiChR injected animals implanted with tetrodes. We found that the mean average firing rate of PVT/CR+ neurons in the SwiChR animals calculated for the entire POST period (POST1–4 days) did not significantly change (n = 39 neuron in 2 mice) in any state relative to the PRE period (PRE1–5 days, n = 38 in 2 mice) (Fig 6B). The marked post-POSE alteration in the state-dependent firing, observed in EYFP animals (Fig 4E) was also abolished, thus state-modulation of neuronal activity did not decrease during the post-POSE period (Fig 6C). In fact, in the case of wake/sleep SMIs PVT/CR+ neurons increased not decreased their state modulation indices after stress in the SwiChR group (Fig 6C). Post-POSE HFA activity (Figs 6D and S10A) and CA of PVT/CR+ cells also remained unaltered during the nest and sleep state (Fig 6E). We found only one measure, CA during the wake state, which increased significantly compared to the PRE period similar to the EYFP group (Figs 6E and S10B).

Fig 6. Effect of post-stress photoinhibition on long-term changes in PVT/CR+ neuronal activity.

(A) Scheme of the experiment. (B) Post-POSE photoinhibition of PVT/CR+ neurons on POST0 day prevents alterations in the firing rate, (C) SMI, (D) occurrence of HFA, and (E) CA during the PRE (black, n = 38 neurons in 2 mice) vs. the POST (blue, n = 39 in 2 mice) periods (all p > 0.05 unless indicated otherwise). Underlying data can be found in S6 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. *p p

https://doi.org/10.1371/journal.pbio.3002962.g006

These data show that inhibiting the activity of PVT/CR+ cells immediately after stress was able to prevent not only the emergence of stress-induced behavioural consequences but also the alteration of the firing activity in PVT/CR+ neurons for the entire POST period as well.

Role of PVT/CR+ neurons in stress-induced changes of their forebrain targets

Next, we aimed to identify the impact of altered post-stress firing of PVT/CR+ neurons on their forebrain targets by measuring changes c-Fos expression in the EYFP (n = 7 animals) and SwiChR (n = 6 animals) groups (Figs 7A and S11) 1 h after the POSE. We also included home cage (n = 7) and no odour (n = 5) controls in these experiments. We selected 3 postsynaptic regions in which significant proportions of the subcortical glutamatergic inputs arise from PVT/CR+ cells (prelimbic cortex (PrL), basolateral amygdala (BLA), and shell of the nucleus accumbens (NAcS)) (Fig 7B) [13] and another two, the central amygdala (CeA) and bed nucleus of stria terminalis (BNST) to which, substantial glutamatergic inputs arrive from subcortical centres other than PVT [53]. Since PVT/CR+ neurons were labelled in these animals, we specifically quantified c-Fos positive neuron numbers in those downstream areas within these structures that contained labelled PVT/CR+ axons, hence were under direct excitatory PVT influence (Fig 7B). We found that compared to home cage and NOE controls POSE markedly increased c-Fos expression in all investigated downstream brain regions except the BNST (Fig 7C–7G). Post-POSE photoinhibition of PVT/CR+ neurons significantly reduced c-Fos expression in the PrL (Fig 7C), BLA (Fig 7D), and NAcS (Fig 7E), but not in the CeA (Fig 7F) and BNST (Fig 7G). This is consistent with the fact that most subcortical excitatory inputs to PrL, BLA, and NAcS arise in PVT. SwiChR inhibition of PVT/CR+ cells in these regions alleviates stress-related elevation of excitatory activity resulting in lower c-Fos expression (Fig 7C–7G). In contrast, CeA and BNST receive other major subcortical glutamatergic inputs [53], which can transmit stress signals independently of PVT/CR+ input, allowing c-Fos expression to persist despite reduced PVT/CR+ activity (Fig 7C–7G).

Fig 7. Role of PVT/CR+ neurons in stress-induced molecular changes.

(A) Scheme of the experiments representing the timeline for GABA-A receptor and c-Fos immunostainings. Created with BioRender.com. (B) Top, Representative, low power (upper panel) and high power (bottom panel) confocal images showing c-Fos expression (magenta) in the nucleus accumbens shell (NacS) from Home Cage control, EYFP, and SwiChR mice. Cyan (left) or green (middle and right) indicates CR- or GFP immunostaining, respectively, labelling PVT axons in NacS. (C–G) Quantification of c-Fos positive neurons in (C) PrL, (D) BLA, (E) NacS, and (F) CeA, (G) BNST in Home Cage (n = 7), EYFP (n = 7), SwiChR (n = 6), and NOE (n = 5) mice on POST0 day (PrL, F(3,21) = 17.10, p p = 0.0013, NacS, F(3,21) = 14.49, p p = 0.0073; oval nucleus of the BNST F(3,20) = 0.848, p = 0.4837). (H) Representative low and high power fluorescent images from the PVT of AAV5-DIO-SwiChRCA-EYFP injected mice. Blue, Hoechst counterstaining. (I, J) Changes in the fluorescence intensity of (H) GABA-A α1 and (I) GABA-A γ2 immunostainings on POST1 day (GABA-A α1, F(2,7) = 23.08, p = 0.0008; GABA-A γ2, F(2,7) = 6.692, p = 0.0237; Home Cage n = 3 mice; EYFP n = 3 mice; SwiChR n = 4 mice). (K) Representative confocal images showing GABA-A α1 and GABA-A γ2 immunostainings in the PVT from Home Cage control, EYFP, and SwiChR mice on POST1 day. Underlying data can be found in S7 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. *p p p p

https://doi.org/10.1371/journal.pbio.3002962.g007

These data illustrate that altered PVT/CR+ activity immediately after the stress can lead to increased c-Fos activity in their postsynaptic targets. Reducing the activity of PVT/CR+ neurons is able to alleviate the stress-induced molecular activation in key forebrain regions which control adaptive behaviours.

Stress-induced changes in homeostatic GABA-A receptor expression

We observed a prolonged increase in the firing activity of PVT/CR+ neurons (Fig 4). Increased activity is linked to a compensatory, homeostatic adjustments in synaptic inhibition [54]. Thus, using high-resolution immunocytochemistry we tested how the expression levels of α1 and γ2 subunits of synaptic GABA-A receptor proteins are altered in PVT/CR+ neurons as a result of the elevated post-POSE activity on POST1 (Figs 7A and S11) in EYFP (n = 3), SwiChR (n = 4), and home cage control (n = 3) groups (Fig 7H). It is known that γ2 subunits exhibit primarily synaptic localization, whereas α1 subunits, which co-assemble with γ2 to form functional receptors are found both synaptically and extrasynaptically [55,56]. We found that the expression of both GABA-A receptor subunits significantly increased in the EYFP animals after POSE compared to home cage controls (Fig 7I–7K), suggesting a homeostatic up-regulation of GABA-A receptors potentially evoked by the increased firing of PVT/CR+ neurons 1 day after the stress event. Similar to other post-stress changes post-POSE SwiChR activation was able to prevent these alterations in α1 and γ2 expression (Fig 7I–7K). These data suggest a homeostatic up-regulation of GABA-A receptor expression in PVT/CR+ cells 24 h after the stress which is absent in the inhibited group.

Effects of late inhibition of PVT/CR+ cells on the stress-induced behavioural consequences

The data above demonstrate that the altered activity of PVT/CR+ neurons immediately after the stress have a critical role in establishing stress-induced alteration of behaviour for several days. It is still unclear, however, whether PVT/CR+ neurons are actively involved in the maintenance of stress-induced phenotype at later time points, or, alternatively later during the post-stress period other brain regions take over this role. To gain insight into this question, we applied a 1-h bout of photoinhibition not immediately after POSE but 5 days later (LATE group; Figs 8A and S12), when mice still exhibited robust stress-induced behavioural phenotype and altered PVT/CR+ firing activity. We examined spontaneous behaviour for 5 more days after the late inhibition.

Similar to P0 photoinhibition, late inhibition had an immediate effect on locomotor activity (Fig 8B) but late inhibition was also able to induce prolonged effects for the entire late period (POST 5–9 days). Compared to the POST1–4 days following the late inhibition (POST 5–9 days), the horizontal activity (Fig 8C and 8D) and freezing behaviour (Fig 8E) were significantly reduced. Day-by-day comparison of the data between the POST1–4 and POST 5–9 periods (RM-ANOVA) also yielded significant differences (S13 Fig). In contrast, nest time (Fig 8F) and nest building time (Fig 8G) were not significantly affected.

Fig 8. Effect of late inhibition of PVT/CR+ neurons on stress-induced behavioural changes.

(A) Scheme of the experiment. Created with BioRender.com. (B) Averaged, normalised horizontal activity during the photoinhibition in LATE0 day compared to the preceding day (POST4) (t[6] = 2.474, (n = 7)). (C) Dynamics of averaged, normalised horizontal locomotor activity of SwiChR mice during the POST (POST 1–4 days, red) vs. the LID (LID 1–5 days, blue) periods (15 min, t[6] = 2.738, p = 0.0338; 30 min, t[6] = 2.443, p = 0.0503). (D) Averaged, normalised horizontal locomotor activity of SwiChR mice during the POST (POST 1–4 days, red) vs. the LID (LID 1–5 days, blue) periods (W = −26). (E) Freezing behaviour (t[6] = 6.909, p = 0.0005), (F) nest time (t[6] = 2.031), and (G) nest building time (W = −5) in SwiChR mice during the POST (POST 1–4 days, red dots) and LID (LID 1–5 days, blue dots) periods. Dots represent the average daily values of individual animals. Underlying data can be found in S8 Data. See S10 Data for the full results of the statistical tests. Data are shown as mean ± SEM. Ns means no significant difference between groups, #p p p

https://doi.org/10.1371/journal.pbio.3002962.g008

These observations suggest that 5 days after the stress the activity of PVT/CR+ cells still contribute to the maintenance of the stress-induced behavioural profile. They also demonstrate that similar to the P0 intervention, the effect of a single bout of photoinhibition in PVT/CR+ cells can last for several days. In contrast to the P0 group, however, not all stress-induced changes were ameliorated in the LATE cohort.

Subjects

Adult male Calb2-IRES-Cre (CR-Cre, C57Bl/6J, n = 73) mice (postnatal 3 to 5 months) were used in the experiments from the Jackson Laboratory (RRID: IMSR_JAX:10774). All animals were group-housed (3 to 4/cage) in Plexiglas chambers at constant temperature (22 ± 1°C) and humidity (40% to 60%) under a circadian light-dark cycle (lights-on at 9 AM, lights-off at 9 PM). All mice were single housed after electrode and optical fibre implantation. For the animals, the food (Sniff) and water were provided ad libitum.

Viral vectors

For control mice in the behavioural, sleep, corticosterone, c-Fos, and GABA-receptor localization experiments, we used AAV5.EF1a.DIO.eYFP.WPRE.hGH (Addgene27056) vector (EYFP mice), whereas for optogenetic inhibition in the same experiments, AAV5.EF1a.DIO.SwiChRCA.TS.EYFP.WPRE (UNC Vector Core) vector was injected (SwiChR mice). In the optrode experiments for the not inhibited, control animals (n = 4 ChR2 animals), we utilised AAV5.EF1a.DIO.hChR2(H134R)-eYFP.WPRE.hGH (Addgene20298P) vector for optical tagging, whereas for the inhibited animals with unit recordings, the same SwiChR construct was utilised to phototagging as above (n = 2 SwiChR animals).

Surgery

Mice were deeply anesthetised with ketamine-xylazin (intraperitoneal injection, ketamine, 83 mg/kg; xylazine, 3.3 mg/kg). Depth of sleep was monitored throughout the surgery, if needed supplemental dose (ketamine, 28 mg/kg; xylazine, 1.1 mg/kg) was injected intramuscularly. Externally applied lidocaine was used on the head and ears. The mice were placed in a stereotaxic frame (Kopf Instruments, Tujunga, California, United States of America) on a heating pad to prevent hypothermia. We used eye protection gel against drying. The scalp in the midline was removed (from Bregma to Lamda) and the skull thoroughly cleaned and treated with 3% hydrogen peroxide for implantation.

Viral injection

A craniotomy was drilled above the PVT and the virus was slowly injected (1 nl/min) into the PVT (AP: −0.9, ML: 0, AP and ML taken from the bregma). We injected 50 to 50 nl in 2 dorsoventral positions (DV: 3.1 and 2.9, DV taken from the brain surface) in the behavioural, corticosterone, c-Fos, and GABA-A receptor localization experiments and 150–150 nl in the same positions in case of the tetrode experiments. Virus was allowed to express for a minimum of 3 weeks before behavioural and tetrode experiments (S2A, S3A, S6, S9, S11 and S12 Figs).

Implantation

In the behavioural, corticosterone, c-Fos, and EEG experiments, 2 optic fibres were implanted above the PVT (AP: −0.9 and −1.4, ML: 0, DV: 2.8, 10° angle), together with 3 EEG screw electrodes (2 above the frontal lobe, at AP: +0.2, ML: +/− 1.8 and 1 above the parietal lobe at AP: −1.1, ML: +/− 1.8) and 1 EMG wire electrode in the neck muscle (directly behind the skull). The EEG and EMG electrodes were soldered to Omnetics connectors (Omnetics). In the tetrode experiments, tetrodes were also implanted above the PVT (AP: −0.9, ML: 0, DV: 2.7, 9.5°angle) and gradually screwed down to PVT until the start of the experiment. Optical fibres, EEG and EMG electrodes were fixed with dental cement (Metabond), tetrode wires were fixed with flexible glue first and then also with cement (Figs 2A, 3A, 4A, 5A, 6A, 8A, S6 and S9). After surgery, the animals were given saline and painkillers for 1 to 5 days according to their condition. Recovery from surgery lasted 2 weeks.

Recording apparatus

All mice were recorded in single-animal boxes with sound and electrical noise isolation. Cameras (FlyCapture) and LEDs were built in the boxes, providing an ambient 200 lux (50 cm from the cage) light. Electrophysiological recordings were provided via Intan Interface Board, Intan cables and chips (Intan Technologies). To avoid entanglement of wires and fibres optics as well as to reduce the weight of the cables and connectors, a dual (wire and fibre optic) commutator system was used (Imetronic), connecting to the Intan-based recording system and the head implants of the animals. We recorded the electrophysiological signal with a custom-made script of the Bonsai visual-programming software.

Predatory odour stress exposure (POSE)

We assessed defensive behaviour to an ecologically pertinent aversive stimulus namely exposure to predator odour stress (POSE), using a synthetic analogue of a fox anogenital product (2-methyl-2-thiazoline; 2MT). The assessments were conducted in a transparent plexiglass arena measuring 32 × 12.5 × 15 cm. The testing environment featured a fume hood with background sound set at 62 dB and high-intensity light ranging from 280 to 320 lux. To standardise odour exposure across subjects, testing occurred in covered cages. At start, mice were placed in the testing cage and allowed to freely explore for a 2-min baseline period. This initial period served as a reference point. Subsequently, each subject was exposed to fox odour (2-MT) for a duration of 10 min [39,40]. For this presentation, 2 μl of pure, undiluted 2-MT (sc-251779, Santa Cruz Biotechnology) was pipetted onto a 2 × 2 cm filter paper, which was then placed into a 1.5 ml Eppendorf tube. After a 2-min baseline period, the Eppendorf was placed into the left corner of the test cage. Mouse behaviour was recorded (Bonsai) and quantified (Solomon) using a video-based measurement system. We used 2 behavioural variables to characterise the innate behavioural responses: freezing and escape jumps. The mice were considered to freeze if movement was not detected for 2 s. Identical procedure was used for the NOE experiments, but in this case 2-MT was omitted. In case of home cage controls, the animals were left in their home cages until the beginning of the perfusion.

Behavioural protocol

In the behaviour experiments, we distinguished 3 major states: the pre-stress period (5 days PRE 1–5 days), the day of POSE (POST 0 day), and the post-stress period (4 days POST 1–4 days) (Figs 1A, 2A, 3A, 4A, 5A, 6A, 8A, S6 and S9). In case of the LATE group, we made the optogenetic inhibition 5 days after the POSE and then we continued recording in the next 5 consecutive days, the same way as detailed above (Fig 8A).

Animals were habituated to the recording apparatus for 3 days (3 h/day) prior to the experiments. The home cages of the animals were placed to sound insulated boxes, their lids were removed, and the cables were connected to the head implants of the animals. Animals were allowed to move freely and to climb to the edge of their home cages. During the pre-stress periods, the baseline activity of the mice (EEG/EMG and behaviour in n = 28 animals, PVT/CR+ unit activity and behaviour in n = 6 animals) were recorded for 3 h every day starting at the beginning of their light phase (ZG time 0) for 5 days. On the day of the POSE animals were transferred to a new room and were placed into a new cage for POSE (see above). After POSE, we put the animals back to their come cages and continued recording for the next 3 h. In the first hour, the laser protocol was performed, which meant inhibition in case of the SwiChR group. On the POST period, we recorded the activity of the mice in the same way as on the PRE period. POST period lasted for 4 days.

Tetrode recording

In the tetrode experiments, movable drives were built and implanted, and 28 channels of unit activity, 1 channel of EEG, and 1 channel of EMG were recorded (Figs 2A, 4A and 6A).

The tetrode drives consisted of 7 tetrodes (28 channels), an optical fibre (105 μm core diameter, 0.22 NA, Thorlabs, New Jersey, USA), an EMG wire electrode, EEG screw electrode, a ground, and a reference cable attached to the electrode interface board. The tetrodes were positioned 300 micrometres below the tip of the optical fibre. The end of the optical fibre was milled into a cone shape. The tetrodes were gold-plated the day before implantation to a resistance below 80 k ohms

In tetrode recording, animals were subjected to the same behavioural test as described above (see in Behavioural protocol section). Phototagging was used to identify PVT/CR+ cells (see in Optogenetic inhibition and phototagging section). Data were recorded with OpenEphys board and software. Video was recorded in the same way as in the behavioural experiments (see in Behavioural protocol section).

Optogenetic inhibition and phototagging

In all experiments, immediately after the POSE the animals were returned to their home cage and 2 external optical fibres (105 μm core diameter, 0.22 NA, Thorlabs, New Jersey, USA) were coupled to the implanted optical fibres with a ceramic sleeve. A rotary joint (Doric Lenses, Québec, Canada) was built in the commutator system to avoid the coiling of optical fibres and Intan cables. Optical manipulations were provided with a 473 nm DPSS laser (LaserGlow Technologies). Before every use laser intensity was checked by a manual photometer (Thorlabs, New Jersey, USA). Laser intensity was also monitored online during the stimuli via built in photosensors (Thorlabs, New Jersey, USA). Lasers were controlled via Matlab with a National Instrument Board (Matlab) and Arduino (Arduino Uno). Photoinhibition was maintained by laser pulses and the same laser protocol was performed in case of the SwiChR group (stressed and inhibited) and also in EYFP group (stressed) for 60 min. Both for EYFP (stressed, n = 43 mice) in SwiChR (stressed and inhibited n = 30 mice) mice trains of 2 s ON and 13 s OFF light pulses were used (10 to 15 mW).

For phototagging in ChR2-expressing mice (n = 4), we utilised 4 Hz, 1 ms long pulses (0.5 to 5 mW; 120 to 150 stimulation) at the beginning and at the end of the recordings (S4D Fig). For phototagging in SwiChR animals (n = 2), we used 4, 2 s long (2 s ON and 13 s OFF) pulses (0.5 to 5 mW) during wakefulness as well as during sleep (4–4 trains) (S1 Fig).

Histological verification of viral infection and optic fibre/tetrode placement

Mice were anesthetised with 2% isoflurane followed by an intraperitoneal injection of an anesthetic mixture (ketamine, 83 mg/kg; xylazine, 3.3 mg/kg) to achieve deep anesthesia. Next, mice were perfused transcardially with 0.9% saline for 2 min followed by 100 ml of 4% PFA solution. After perfusion, brains were removed from the skull, and thalamic section was immersion-fixed in 2% PFA for 30 min. Brains were cut into 50 μm sections using a vibrating microtome (Vibratome 3000). To verify the viral infection and cannula/fibre optic placement every third sections were stained from each animal. Sections were washed 4 times in 0.1 M phosphate buffer (PB) and incubated in a blocking solution containing 2% TritonX-100 and 10% normal donkey serum (Jackson ImmunoResearch, USA) in PB for 2 h at room temperature. Sections were then incubated (24 h) with anti-calretinin guinea pig (1:1,000, 214104, Synaptic Systems, RRID: AB_10635160), anti-GPF chicken (1:1,000, A10262, Thermo Fisher Scientific, RRID: AB_2534023), and anti-IBA1 rabbit (1:500, 27030, Wako, RRID: AB_2314667) primary antibodies dissolved in 2% normal donkey serum solution. Afterward, sections were washed in PBS 4 times (10 min each time), then incubated with secondary antibodies. The primary antibodies were visualised using the following secondary antibodies: Alexa Fluor 488-conjugated donkey anti-chicken (1:1,000, 703-545-155, Jackson ImmunoResearch), Cy3-conjugated donkey anti-rabbit (1:1,000, 711-165-152, Jackson ImmunoResearch), and Alexa Fluor 647- conjugated donkey anti-guinea pig (1:1,000, 706-605-148, Jackson ImmunoResearch) antibodies. After being washed with PBS (phosphate buffer saline), the sections were mounted on glass slides and cover slipped using Mowiol 4–88 (81381, Sigma-Aldrich) fluorescent mounting medium. Images were acquired on Panoramic Digital Slide Scanner (Zeiss, Plan-Apochromat 10X/NA 0.45, xy: 0.65 μm/pixel, Panoramic MIDI II; 3DHISTECH, Budapest, Hungary).

c-Fos mapping with corticosterone measurement

Following POSE, neuronal activation and stress hormone reactivity were investigated using c-Fos immunohistochemistry and serum corticosterone measurement, respectively (Figs 1A and S2). The following experimental groups were included:

(1) Home cage control group (Home Cage, n = 7). These animals were left in their home cage for the whole duration of the experiment. They were not moved to a new cage on the day of the experiment, received no POSE or laser stimulation.

(2) No odour exposure (NOE) group (n = 5). These animals were moved to the new cage, were not exposed to 2 MT there but received laser stimulation when they returned to their home cage using the same protocol as the other 2 groups.

(3) EYFP group (n = 7). These animals were moved to the new cage, were exposed to 2 MT, and received laser stimulation when they returned to their home cage. Since these animals were injected with EYFP containing virus PVT/CR+ cell did not experience optogenetic inhibition.

(4) SwiChR group (n = 6). These animals were moved to the new cage, were exposed to 2 MT, and received laser stimulation when they returned to their home cage. The activity of PVT/CR+ cells were reduced by optogenetic inhibition (Fig 7A).

All groups were submitted the same habituation protocol to familiarise the mice with the recording environment (for details, see Behavioural protocol section). Mice were decapitated 60 min after POSE. Trunk blood was collected for CORT radioimmunoassay (RIA) and brains were fixed in 4% PFA for 3 days. c-Fos, CR, GFP triple staining was performed on 50-μm thick coronal sections selected by a 1:6 fractionator sampling covering the whole rostral to caudal extension of PVT and the forebrain regions targeted by PVT/CR+ neurons including prelimbic cortex (PrL, +2.22 mm to +1.54 mm from Bregma), nucleus accumbens shell (NAcS, +1.94 mm to+ 0.86 mm from Bregma), paraventricular hypothalamic nucleus (PVH, –0.58 mm to –1.22 mm from Bregma), central amygdala (CeA, –1.22 mm to –1.94 mm from Bregma), basolateral amygdala (BLA, –0.58 mm to –1.94 mm from Bregma), and bed nucleus of the stria terminalis (BNST, 0.26 mm to 0.02 mm from Bregma).

For immunocytochemistry, free-floating brain sections were washed in 0.1 M PB and blocked in 10% normal donkey serum (NDS) in PB containing and 2% Triton-X 100 for 2 h at room temperature. The primary antibodies against c-Fos (rabbit, 1:1,000, 226003, Synaptic Systems, RRID: AB_2231974), calretinin (guinea pig, 1:1,000, 214104, Synaptic Systems, RRID: AB_10635160), and GFP (chicken, 1:1,000, A10262, Thermo Fisher Scientific, RRID: AB_2534023) were diluted in PB containing 2% NDS. Twenty-four hours later, sections were washed multiple times and were incubated with Alexa Fluor 488-conjugated donkey anti-chicken (1:1,000, 703-545-155, Jackson ImmunoResearch), Cy3-conjugated donkey anti-rabbit (1:1,000, 711-165-152, Jackson ImmunoResearch), Alexa Fluor 647-conjugated donkey anti-guinea pig (1:1,000, 706-605-148, Jackson ImmunoResearch) antibodies, and with Hoechst 33258 (1:2,000, Sigma-Aldrich) for 2 h at room temperature. After further PB washes, sections were mounted on glass slides and coverslipped using Mowiol 4–88 (81381, Sigma-Aldrich) fluorescent mounting medium. Images were taken using a Panoramic Digital Slide Scanner (Zeiss, Plan-Apochromat 10X/NA 0.45, xy: 0.65 μm/pixel, Panoramic MIDI II; 3DHISTECH, Budapest, Hungary). Every third section was stained and analysed per animal. To assess c-Fos activation in the PVT/CR+ neuron projections targets, we delineated and identified regions of interest (PrL, NAcS, BLA, and CeA) based on axonal labelling and on neuroanatomical landmarks using Hoechst counterstaining on fluorescent pictures applying CaseViewer 2.3 software (3DHISTECH). c-Fos signal was counted bilaterally in covering the whole anteroposterior extension of the actual region/subregion.

To assess the POSE-induced activation of PVT/CR+ neurons, c-Fos immunoreactivity of CR expressing cells were analysed manually in 20× confocal images below the optic fibre using a Nikon A1R confocal microscope (CFI Plan Apo VC20X/N.A. 0.75, xy: 0.31 μm/pixel, Nikon Europe, Amsterdam, the Netherlands).

To quantify c-Fos density throughout the anteroposterior extension of the PVT, anti-c-Fos (rabbit; 1:20,000, PC38, Calbiochem, RRID: AB_2106755) was developed with DABNi as a chromogen. All sections used for quantification were developed together for the same duration. The section was dehydrated and then mounted with DePex (Serva, Heidelberg, Germany). The number of c-Fos-labelled cells was analysed using a custom-written ImageJ (RRID:SCR_003070) script.

Corticosterone measurement

Serum corticosterone was measured by direct RIA as described [74]. Briefly, the corticosterone antibody was raised in rabbits against corticosterone-carboxymethyloxime bovine serum albumin conjugate. ¹²⁵I-labelled corticosterone-carboxymethyloxime-tyrosine methyl ester was used as tracer. The interference from plasma transcortin was eliminated by inactivating transcortin at a low pH. Assay sensitivity was 1 pmol, the intraassay coefficient of variation was 7.5%.

GABA-A receptor subunit expression analysis

To investigate the effect of stress on GABAA receptor subunit expression levels in the PVT, we used SwiChR (n = 4) and EYFP (n = 3) injected animals which underwent POSE as described previously (Figs 7A and S11); 24 h after POSE, injected and not injected home cage control (Home Cage, n = 3) mice were anesthetised with 2% isoflurane followed by an intraperitoneal injection of an anesthetic mixture (ketamine, 83 mg/kg; xylazine, 3.3 mg/kg) to achieve deep anesthesia. Next, mice were perfused transcardially with 0.9% saline for 2 min followed by 100 ml of 1% PFA solution. After perfusion, brains were removed from the skull, and thalamic section was immersion-fixed in 2% PFA for 30 min. Brains were cut into 50 μm sections using a vibrating microtome (Vibratome 3000). Perfusion-fixed sections were washed 4 times in 0.1 M PB for 1 h. For detection of GABA_A-receptor γ2 and α1 subunits, sections were pretreated with 0.2 M HCl (hydrogen-cloride) solution containing 0.2 mg/ml pepsin (Dako) at 37°C for 2 min, followed by a 2 h long blocking period in 10% normal donkey serum in PB as described previously [75]. This was followed by an overnight incubation at room temperature in a solution containing 2% NDS, 0.05% sodium azide, and a mixture of primary antibodies: rabbit anti-γ2 (1:2,000, 224003, Synaptic System, RRID:AB_2263066) [75], guinea-pig anti-α1 (1:500, MSFR101550, GABAARa1-GP, Frontier Institute) [76], and chicken anti-GFP (1:1,000, A10262, Thermo Fisher Scientific, RRID:AB_2534023) to enhance the endogenously expressed EYFP signal. The primary antibodies were visualised using the following secondary antibodies: Alexa Fluor 488-conjugated donkey anti-chicken (1:1,000, 703-545-155, Jackson ImmunoResearch), Cy3-conjugated donkey anti-rabbit (1:1,000, 711-165-152, Jackson ImmunoResearch), and Alexa Fluor 647-conjugated donkey anti-guinea pig (1:1,000, 706-605-148, Jackson ImmunoResearch) antibodies. After being washed with PBS, the sections were mounted on glass slides and cover slipped using Mowiol 4–88 (81381, Sigma-Aldrich) fluorescent mounting medium. Images from PVT were obtained using a Nikon A1R confocal microscope (Plan Apo VC 60× oil objective; numerical aperture, 1.40; xy, 0.10 μm/pixel; z-step size, 0.10 μm). Images were taken through a z-plane (1.5 μm) at the centre of the tissue containing 20 stacks within each region. During image acquisition gain, offset, laser intensity, zoom, and pinhole were kept constant. In each animal, 12 to 30 regions of interest (ROIs, rectangular frame) restricted to the PVT were analysed from 3 brain sections. To determine the expression levels of GABA_A-receptor subunits, the images were thresholded and gray values of pixels were registered in ROIs defined in appropriate optical sections of image stacks in ImageJ software (RRID:SCR_003070).

Ex vivo slice preparations

Ex vivo electrophysiology was performed on adult CR/IRES-Cre mice (n = 4) infused in the PVT with the AAV5.EF1a.DIO.SwiChRCA.TS.EYFP.WPRE virus (Fig 2A). Coronal brain slices (300 μm) including the PVT were prepared minimum 4 weeks following surgery. Following anesthesia with isoflurane, the brain was quickly removed in an ice-cold bicarbonate-buffered solution (BBS) containing the following (mM): 116 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 30 glucose, 1.6 CaCl₂, 1.5MgCl₂, and 5 × 10−5 minocycline (pH 7.4, bubbled with 95% O₂, 5% CO₂). Slices were then cut using a vibrating blade microtome (Campden model 7000smz2) in an ice-cold solution containing (mM): 150 sucrose, 10 choline-Cl, 2.5 KCl, 3.1 Na-pyruvate, 11.6 ascorbic acid, 1.25 NaH₂PO₄, 26 NaHCO₃, 30 glucose, 0.5 CaCl₂, 7 MgCl₂, supplemented with D-APV (25 μm). They were then transiently immersed (10/15 min) in a bath containing the same solution at 32° to 34°C (pH 7.4, bubbled with 95% O₂, 5% CO₂). Finally, slices were transferred to warm BBS (32° to 34°C) for the rest of the experimental day.

In our hands, SwiChR was tonically activated by ambient light. Throughout the experimental day, we thus paid particular attention to protect slices from all light sources.

For electrophysiology, slices were moved to a recording chamber mounted on an upright Olympus BX51WI microscope (Olympus France) and continuously perfused with bubbled BBS (~1/1.5 ml/min; 32 to 35°C). Cells were visualised with a combination of Dodt contrast and an on-line video contrast enhancement. Whole-cell (WC) recordings of PVT/CR+ cells were performed with an EPC-10 double amplifier (Heka Elektronik, Lambrecht/Pfalz, Germany) run by the PatchMaster software (Heka). PVT cells could be easily identified in the transmitted deep red light (∼750 nm) with which slices were visualised using a CoolSnap HQ2 CCD camera (Photometrics, Trenton, New Jersey, USA) run by MetaMorph (Universal Imaging, Downington, Pennsylvania, USA).

Whole-cell (WC) recordings were performed with pipettes (resistance: 2/3 MΩ) filled with an intracellular solution containing (mM): 75 NMDG, 4.6 MgCl₂, 10 HEPES, 0.5 EGTA, 4 Na₂-ATP, 0.4 Na₂-GTP, 1 QX-314, ~300 mOsm, and pH 7.3 with HCl. This solution maximises the driving force for chloride ions by containing a close to symmetrical concentration with respect to the external BBS.

Spontaneous inhibitory postsynaptic currents (IPSCs) were recorded at a holding potential of −70 mV in the continuous presence of the AMPA receptor blocker NBQX (5/10 μm) in the bath. Only SwiChR-expressing neurons were recorded, identified by an inward current developing in response to 470 nm diode pulses (Thorlabs Maisons-Laffitte, France). Light pulses were relayed to the recording chamber via the epifluorescence pathway of the microscope. During recordings, the series resistance was partially compensated (max 65%), whereas liquid junction potentials were not corrected. The analysis was performed using custom-built routines in Igor (Wavemetrics, Lake Oswego, USA).

Data analysis

Sleep electroencephalogram (EEG) spectral slope analysis

Periods of NREM sleep EEG spectral slope was defined according to a previously published approach [50]: the best linear fit to the equidistantly oversampled (piecewise cubic Hermite interpolation) double logarithmic plot of the average power spectral density (Welch periodogram based on 4 s Han windowing and Fast Fourier Transformation). Fitting was based on the 2 to 6 Hz and 18 to 48 Hz ranges, in order to exclude frequencies characterised by frequent upward deflections caused by NREM sleep-specific spectral peaks. The slope of the linear is known to reflect the depth of sleep (sleep intensity) [50,77].

Horizontal activity

The horizontal movement of the animals was calculated from the video. Using a neural network-based program package called DeepLabCut, we calculated the X and Y coordinates of the animal’s head for each video frame (Figs 3B, lower panel; 3C, lower panel; 5B, lower panel; and 5C, lower panel). DeepLabCut’s neural network was pre-trained on our own data. We then filtered the coordinates based on their likelihood (at least 0.8) and calculated the magnitude of displacement for each frame. For Figs 3D, 5D and 8D, we averaged 15, 1-min long displacement data. For Figs 3E, 5E and 8E, the displacement data set was normalised by the averaged nest onset time of each animal calculated for the PRE period.

Analysis of behaviour in the nest

We defined the following behavioural variables:

Nest onset: Nest onset was defined as the moment when an animal enters its nest and did not leave it for at least 20 s.

Sleep onset: We defined sleep onset as the time point when the first 2 min of uninterrupted sleep was initiated. Sleep onset was defined using the combination of EEG and EMG (3 times larger amplitude of the EEG activity compared to baseline and EMG OFF state).

Nest time: Nest time was defined as the time between nest onset and sleep onset.

For the detailed analysis of the behaviour, we used the Solomon Coder software from the beginning of the recording until the sleep onset of the animals. During the analysis, we differentiated and quantified the following behaviours:

Nest building: A stereotyped mouse behaviour, during which the mouse usually reaches out, pulls in or gathers nest material to build its nest (S5 Movie).

Freezing in the nest: During this freezing-like behaviour in the nest, the animals were awake; the body of the animal did not perform horizontal movements for at least 2 s (range 2 to 60 s). During this type of behaviour, the animals sometimes perform sudden jerky movements, head-bobbing or turning but no nest building, tramping, nest tromping, comfort movements or rearing (S6 Movie) [78,79].

Hyperventilation: On POST 0 day, immediately after the stress animals displayed hyperventilation visible in the video [80]. This was detected as a rhythmic movement of the thorax interspersed with short whole body movements. This type of activity could be detected only on POST 0 day (S3 Movie).

Unit clustering

The clustering was done in a semi-automatic way. The 3 h of measurement were recorded in 2 files of 1.5 h each. These files were automatically analysed with SpyKING Circus. SpyKING Circus automatically generated clusters. These clusters were then manually curated using the Phy program [81]. All clusters with an average amplitude below 50 μV were discarded (S4A and S4E Fig). Noise was then filtered out from the clusters by displaying the different PCA component features of the channels and cutting the resulting clusters (S4E Fig). In doing so, it was checked that a given unit really belonged to a single cell and that the same cell was not scattered in several clusters (using Amplitude View and Feature View). Then, the autocorrelograms (S4C Fig) and inter spike interval distributions of the cells were checked (no spike within 2 ms and ISI distribution with Gaussian curve shape were accepted).

Unit activity analysis

Statistics

Data are represented as mean ± SEM. All the statistical analysis was done using GraphPad Prism version 9. First, normality of the data distribution was checked, using Shapiro–Wilk normality test. When comparing 2 groups, if both groups showed a normal distribution, a t test was used. Paired t tests were used when the same animal or cell was tested over several days (indicated by a connecting line between the 2 points in the figures) and unpaired t tests in all other cases. If the requirements of t test were not fulfilled, then Mann–Whitney test or Wilcoxon matched-pairs signed rank tests were used. To compare multiple groups, one-way ANOVA was used followed by Tukey’s post hoc test. The significance level was set at p 0.05. Individual data points are shown in the figures. All the statistics used in figures and supplementary figures are shown in tables of statistics (S10 Data).