Defensive behaviors are critical for animal survival. They are dynamic and adaptive, as environmental contexts, properties and intensity of threats, as well as expectations from past experiences can all modulate the form as well as the magnitude of defensive behaviors (Fanselow, 1994; Gross and Canteras, 2012; Tovote et al., 2016). Threat signals in the external environment are sensed by different sensory modalities through distinct sensory pathways to initiate appropriate defensive behaviors. Previous studies have mostly been focused on defensive behaviors initiated under stimulation of one individual sensory modality (Fanselow and LeDoux, 1999; Yilmaz and Meister, 2013). However, a danger may be associated with cues of multiple sensory modalities arriving at the same time, and the integration of information of these different modalities may profoundly influence the behavioral output. Intuitively, the presence of multisensory signals is helpful for strengthening defensive responses. However, neural circuit bases for the potential cross-modality interactions in defensive behaviors are largely unknown. In this study, we designed experiments to specifically examine whether tactile input can affect a well-established auditory-induced defensive behavior (Fanselow and LeDoux, 1999; Tovote et al., 2016). The vibrissal system is crucial to behaviors such as navigation and exploration (Carvell and Simons, 1990; Diamond et al., 2008), and rodents frequently collect information from surroundings using their whiskers (Prigg et al., 2002). We reason that it may be common for animals to use both vibrissal and auditory systems in sensing environmental dangers.

Zona incerta (ZI) is a major GABAergic subthalamic nucleus consisting of heterogeneous groups of cells. In rodents, four (rostral, ventral, dorsal, caudal) sectors of ZI can be loosely defined based on neurochemical expression patterns (Ma et al., 1997; Mitrofanis et al., 2004), and it has been suggested that different sectors might be involved in different circuits and functions (Liu et al., 2017; Plaha et al., 2008). Our recent study has shown that GABAergic neurons in the rostral sector of ZI (ZIr) play a role in reducing defensive behavior in an experience-dependent manner (Chou et al., 2018). It also raises a possibility that ZI might play a broader role in defensive behavior. ZI receives inputs from various cortical areas including the primary somatosensory cortex (SSp) (Kolmac et al., 1998; Shammah-Lagnado et al., 1985) as well as from the brainstem trigeminal nucleus that relays vibrissal information (Roger and Cadusseau, 1985; Smith, 1973). A recent study has demonstrated that deflecting whiskers directly induces neuronal activity in the ventral sector of ZI (ZIv) (Urbain and Deschênes, 2007), where parvalbumin (PV) positive neurons are enriched (Kolmac and Mitrofanis, 1999). In the present study, we investigated whether somatosensory input through whisker stimulation could modulate defensive behavior via recruiting ZIv PV+ neurons.

To test whether tactile input can affect defensive behavior, we employed a relatively simple behavioral test, sound-induced flight, following our previous studies (Xiong et al., 2015; Zingg et al., 2017). Such behavior has been observed in both freely moving and head-fixed conditions (Xiong et al., 2015; Zingg et al., 2017). In our first set of experiments, animals were head-fixed and placed on a smoothly rotatable plate (Chou et al., 2018; Liang et al., 2015). Loud noise sound (80 dB sound pressure level or SPL) elicited animal running, and the running speed was recorded in real time (Figure 1A, left). Tactile stimulation was applied by deflecting whiskers unilaterally with a cotton stick controlled by a piezo actuator (Figure 1—figure supplement 1A). In our control experiments, the whisker deflection per se did not elicit significant locomotion of animals (Figure 1—figure supplement 1B). Trials without and with tactile stimulation were interleaved. We found that tactile stimulation enhanced the running induced by noise sound (Figure 1A, right), as demonstrated by the increased peak speed (Figure 1B, Figure 1—figure supplement 1C) and total travel distance (Figure 1C, Figure 1—figure supplement 1D). The temporal profile of the behavioral response was not significantly affected, as shown by the quantifications of onset latency and time to peak (Supplementary file 1). Silencing the SSp contralateral to the whiskers being deflected by infusing a GABA receptor agonist, muscimol (Figure 1D, left), removed the difference in speed between conditions without and with whisker stimulation (Figure 1D–F), without altering the response temporal profile (Supplementary file 1). This suggests that the tactile enhancement of running is mediated mainly through SSp. To further demonstrate the tactile effect on flight behavior in freely moving animals, we used a two-chamber test following our previous study (Zingg et al., 2017). When the mouse was exposed to noise applied in one chamber, it quickly escaped to the other chamber by crossing through a narrow channel (Figure 1—figure supplement 2A). Trimming of all whiskers of the animal significantly decreased the average speed of the flight through the channel (Figure 1—figure supplement 2B), suggesting that tactile information through whiskers can indeed enhance flight behavior in a more natural condition.

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Previous studies have suggested that SSp projects to ZIv (Kolmac et al., 1998; Shammah-Lagnado et al., 1985), and that ZIv neurons respond to whisker deflections (Urbain and Deschênes, 2007). To confirm this projection, we injected AAV1-CamKII-hChR2-eYFP into SSp of PV-ires-Cre crossed with Ai14 (Cre-dependent tdTomato) reporter mice (Figure 2A). We found profuse fluorescence-labeled axons in ZIv, but few in other ZI sectors (Figure 2B). We next directly examined the effect of stimulating the SSp projection to ZIv, by placing optic fibers on top of ZIv to deliver LED light pulses (20 Hz train for 5 s) bilaterally (Figure 2C). The optogenetic activation of the SSp axons in ZIv enhanced noise-induced running (Figure 2C–E, Figure 2—figure supplement 1A–B) without affecting the response temporal profile (Supplementary file 1), but by itself had no effect on the baseline locomotion speed (Figure 2—figure supplement 1C). Infusing muscimol into ZIv bilaterally abolished the enhancement of flight response by whisker stimulation (Figure 2F–H) without affecting the response temporal profile (Supplementary file 1). Together, these results suggest that activation of the SSp-ZIv projection is sufficient and necessary for the tactile enhancement of auditory-induced flight response.

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Immuno-histological studies have suggested that PV+ neurons are a major cell type in the ventral sector of ZI (Kolmac and Mitrofanis, 1999). To test whether SSp axons innervate PV+ neurons, we performed slice whole-cell recording from ZIv PV+ neurons (labeled by tdTomato expression in PV-Cre::Ai14 animals) while optically activating ChR2-expressing SSp axons in ZI (Figure 2I). We observed that blue light pulses evoked monosynaptic excitatory postsynaptic currents (EPSCs) in most ZIv PV+ neurons recorded with tetrodotoxin (TTX) and 4-aminopyridine (4-AP) present in the bath solution. The EPSC could be blocked by an AMPA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Figure 2J–K). These results indicate that ZIv PV+ neurons receive direct excitatory input from SSp.

To investigate whether the PV+ neurons play a role in the tactile modulation of flight response, we injected AAV encoding Cre-dependent ChR2 or ArchT into ZI of PV-Cre::Ai14 mice (Figure 3A,D). The viral expression of opsins co-localized well with Cre-dependent tdTomato expression (Figure 3—figure supplement 1A), indicating PV-specific expression of opsins. Optogenetic manipulation of ZI PV+ neuron activity with blue (for the ChR2 group to activate) or green (for the ArchT group to suppress) LED light was interleaved with control trials in which only sound was delivered. The efficacies of ChR2 and ArchT were confirmed by slice whole-cell recordings showing that blue LED light evoked robust spiking in ChR2-expressing neurons and green LED light induced a strong hyperpolarization of the membrane potential in ArchT-expressing cells (Figure 3—figure supplement 1B–C). We found that activation of ZI PV+ neurons enhanced flight response induced by noise stimulation (Figure 3A–C, Figure 3—figure supplement 1D–E), whereas suppression of these neurons reduced the flight response (Figure 3D–F, Figure 3—figure supplement 1F–G). None of the manipulations affected the temporal profile of the behavioral response (Supplementary file 1). As a control, neither activation nor suppression of ZIv PV+ neurons alone significantly affected the baseline locomotion (Figure 3—figure supplement 2). We next expressed Cre-dependent inhibitory designer receptors exclusively-activated by designer drugs (DREADDi) (Zhu and Roth, 2014), hM4D(Gi), in ZI of PV-Cre mice, and intraperitoneally injected the DREADDi agonist, clozapine-N-oxide (CNO) (Figure 3G). The efficacy of DREADDi inhibition was confirmed by slice recording showing that CNO increased the threshold for spiking and decreased the number of spikes evoked by current injections (Figure 3—figure supplement 3). The chemogenetic silencing of ZIv PV+ neurons prevented the enhancement of noise-induced flight response by whisker stimulation (Figure 3G–I) without affecting the response temporal profile (Supplementary file 1).

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We next performed awake single-unit optrode recordings in ZI, following our previous study (Zhang et al., 2018). ZIv PV+ neurons were optogenetically identified by their time-locked spike responses to blue laser pulses (Figure 3J). We found that these neurons responded to both noise sound and whisker deflections and that concurrent whisker deflections increased the response level to noise (Figure 3K–L). This result indicates that ZIv PV+ neurons can integrate tactile and auditory inputs and that tactile input plays a faciliatory role, consistent with the behavioral results. Altogether, our results strongly suggest that the tactile enhancement of flight behavior is mediated primarily by ZIv PV+ neurons.

To identify which downstream target nucleus of ZIv PV+ neurons is involved in the behavioral modulation, we traced the projections from ZIv PV+ neurons by injecting AAV encoding Cre-dependent GFP in PV-Cre mice (Figure 4A). Consistent with previous results (Barthó et al., 2002; Trageser and Keller, 2004), we found that two targets, the medial posterior complex of thalamus (POm) and superior colliculus (SC), received the strongest projections from ZIv PV+ neurons (Figure 4B, Figure 4—figure supplement 1). We then specifically activated the ZIv PV+ projection to POm or SC by placing optic fibers on top of POm or SC, respectively, in PV-Cre mice injected with AAV encoding Cre-dependent ChR2 in ZI (Figure 4C,F). While activation of the ZIv-SC projection did not significantly change the flight speed (Figure 4C–E), that of the ZIv-POm projection significantly increased the flight speed (Figure 4F–H, Figure 4—figure supplement 2A–B), similar to the activation of ZIv PV+ neuron cell bodies. As a control, activation of the ZIv-POm projection alone did not change the baseline locomotion speed (Figure 4—figure supplement 2C). To confirm that the ZIv-POm projection is necessary for the tactile modulation, we expressed Cre-dependent hM4D(Gi) in ZI of PV-Cre mice and locally infused CNO into POm through implanted cannulas (Figure 4I). The chemogenetic silencing of the ZIv-POm projection prevented the enhancement of flight speed by whisker stimulation (Figure 4I–K). None of the manipulations affected the temporal profile of flight response (Supplementary file 1). Taken together, our results demonstrate that the projection of ZIv PV+ neurons to POm primarily mediates the enhancement of sound-induced flight behavior by tactile stimulation.

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In this study, we demonstrate that additional tactile stimulation enhances flight behavior triggered by threats such as loud noise. Both SSp and ZIv PV+neurons, which receive SSp input, are necessary for this modulation, and activation of the SSp-ZIv projection is sufficient for driving the enhancement of the behavior. We also demonstrate that activation of ZIv PV+ neurons alone can enhance the flight behavior and that inactivation of the PV+ neurons or their projections to POm blocks the tactile enhancement of the flight behavior. Together, our data suggest that tactile input through whisker deflections can modulate defensive flight via the SSp-ZIv-POm pathway.

Rodents frequently use their whiskers to locate and identify objects (O'Connor et al., 2013; Pammer et al., 2013). In complex sensory environments, whiskers are essential for them to gather information from surroundings as to guide their behaviors during exploration and navigation (Ahl, 1986; Diamond et al., 2008; Sofroniew et al., 2014; Yu et al., 2016). When escape behavior is concerned, specific somatosensory input plus loud sound may indicate the immediate proximity of a predator, and enhancement of flight at such moments will greatly increase survival chances of prey animals. In addition, information conveyed by the somatosensory system about the environment could be extremely useful for the prey animal to quickly choose an effective escape route (Diamond et al., 2008; Douglass et al., 2008). Therefore, the ability to integrate somatosensory input and modulate defensive flight behavior accordingly is beneficial for animals to avoid dangers. Here, we show that somatosensory input from whiskers can enhance auditory-induced flight response. Indeed, in freely moving mice, trimming of whiskers reduces the efficiency of their escape from a source of loud noise by crossing through a channel, indicating a faciliatory role of the tactile input.

The tactile-auditory cross-modality modulation relies on conveying somatosensory information primarily from SSp to ZI. ZI has been implicated in maintaining normal posture and locomotor functions (Edwards and Isaacs, 1991), as it sends dense projections to motor-related thalamic and brainstem nuclei (Kolmac et al., 1998; Shaw and Mitrofanis, 2002). The somatosensory input to ZI thus has a potential to influence motor functions (Périer et al., 2002; Supko et al., 1991). In this study, we show that SSp projections to ZI are mainly concentrated in ZIv, where PV+ neurons are a major cell type (Mitrofanis, 2005; Zhou et al., 2018). Consistent with this projection, PV+ neurons in ZIv receive direct excitatory input from SSp and respond to whisker deflections. Concurrent whisker deflections also increase their responses to sound, indicating that tactile-auditory integration takes place in ZIv PV+ neurons. Activating SSp-ZIv axon terminals or ZIv PV+ neurons directly enhances auditory-induced flight, while silencing the PV+ neurons abolishes the enhancement of flight by tactile stimulation. Therefore, our data demonstrate that via the SSp-ZIv pathway mediated mainly by ZIv PV+ neurons, somatosensory input can modulate motor functions in defensive behavior. Whether ZIv PV+ neurons are involved specifically in tactile-auditory integration or multisensory integration in general remains to be further investigated.

Different ZI sectors are dominated by distinct cell types (Mitrofanis, 2005; Ricardo, 1981). It has been suggested that different ZI cell types or sectors may exhibit different connectivity patterns (Mitrofanis, 2005), contributing to ZI’s multiplex roles in various physiological functions. For example, it has been shown that activation of GABAergic neurons in the rostral sector of ZI (ZIr), which are likely PV-negative, can induce binge-like eating via its projections to the periventricular nucleus of thalamus (Zhang and van den Pol, 2017), while Lhx6-expressing neurons in ZIv, which are also PV-negative, can regulate sleep through their projections to hypothalamic areas (Liu et al., 2017). Different sectors or cell types may also play different roles in defensive behavior. Indeed, we have previously shown that activation of ZIr GABAergic neurons reduces noise-induced flight via their projections to the periaqueductal gray (PAG) (Chou et al., 2018). This effect is opposite to that of activating ZIv PV+ neurons, which have few projections to PAG (Figure 4—figure supplement 1). More recently, using conditioned freezing response as a model, a study of ZIv PV+ neurons has shown that both silencing the PV+ neuron output and silencing the amygdala inhibitory input to the PV+ neurons disrupt fear memory acquisition as well as recall of remote fear memory (Zhou et al., 2018). In the current study, the behavior we examined is an innate defensive behavior. Therefore, ZIv PV+ neurons can play a role in regulating both innate and learned defensive behaviors, which are generated under different contexts and likely engage different upstream pathways. It would be interesting to investigate in the future how ZI, through interactions among its different cell-types/subdivisions, regulates behaviors in complex sensory and behavioral environments.

We have identified POm as a target of ZIv PV+ neurons that is mainly responsible for the tactile enhancement of flight behavior. Silencing of the projection from ZIv PV+ neurons to POm prevents the faciliatory effect of tactile stimulation. PV+ neurons in ZI are GABAergic (Barthó et al., 2002) and provide inhibition to their target neurons. To achieve the effect of enhancing the behavioral output, disinhibitory circuits may be involved. POm is known to project to the striatum to modulate locomotion (Ohno et al., 2012; Smith et al., 2012). The inhibitory nature of striatal neurons makes them a good candidate for engaging disinhibition of distant output responses (Grillner et al., 2005; Kreitzer and Malenka, 2008). Furthermore, we have shown previously that the noise-induced flight behavior depends on a pathway from the auditory cortex (AC) to the cortex of inferior colliculus (ICx) and then to PAG (Xiong et al., 2015). It is possible that the ZIv-POm pathway directly or indirectly connect to midbrain areas downstream of the AC-ICx-PAG pathway (Marchand and Hagino, 1983; Roseberry et al., 2016). As such, somatosensory information carried by the ZIv-POm pathway can modulate the auditory-induced behavior mediated by the AC-ICx-PAG pathway. It would be interesting to investigate in the future whether and how the POm-striatal circuit is involved in this modulation.

Overall, ZI has complex input and output connectivity patterns (Chou et al., 2018; Nicolelis et al., 1992; Roger and Cadusseau, 1985; Shammah-Lagnado et al., 1985; Zhou et al., 2018). Through convergent and divergent connectivity with various brain areas, ZI may be able to carry out multiple physiological and behavioral functions synergistically.

All experimental procedures used in this study were approved by the Animal Care and Use Committee at the University of Southern California. Male and female wild-type (C57BL/6) and transgenic (PV-ires-Cre; Ai14-tdTomato) mice aged 8–16 weeks were obtained from the Jackson Laboratory. Mice were housed on 12 hr light/dark cycle, with food and water provided ad libitum.

All data generated or analysed during this study are included in the manuscript and supporting files. The data for each figure have been provided as source data files and the code used for data analysis can be found at https://github.com/xiaolinchou/flight-speed-calculation (copy archived at https://github.com/elifesciences-publications/flight-speed-calculation).

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Author details

1. Xiyue Wang

   1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States
   2. Graduate Program in Neuroscience, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation

   Contributed equally with

   Xiaolin Chou

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5805-0778

2. Xiaolin Chou

   1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States
   2. Graduate Program in Neuroscience, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation

   Contributed equally with

   Xiyue Wang

   Competing interests

   No competing interests declared

3. Bo Peng

   1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States
   2. Graduate Program in Neuroscience, University of Southern California, Los Angeles, United States

   Contribution

   Data curation

   Competing interests

   No competing interests declared

4. Li Shen

   Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Data curation

   Competing interests

   No competing interests declared

5. Junxiang J Huang

   1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States
   2. Graduate Program in Biomedical and Biological Sciences, University of Southern California, Los Angeles, United States

   Contribution

   Data curation

   Competing interests

   No competing interests declared

6. Li I Zhang

   1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States
   2. Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Funding acquisition, Writing—review and editing

   For correspondence

   liizhang@usc.edu

   Competing interests

   No competing interests declared

7. Huizhong W Tao

   1. Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, United States
   2. Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Funding acquisition, Writing—review and editing

   For correspondence

   htao@usc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3660-0513

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