Mapping the circuits that form the basis of specific behaviors is essential to understand the brain and is an essential task for the neuroscience of our time. The circuitries that underlie fear are important in the clinic and consequently have become a hotspot of neuroscientific research in the last few years (Tovote et al., 2015; Mobbs et al., 2020; Silva and McNaughton, 2019). Here, we have used chemogenetics and optogenetics to show that the activation of a specific, well delimited neuronal group in the hypothalamus leads to innate defensive behavior, including tachypnea, immobility, and bradycardia.

During hypothalamic development, two neuronal migratory streams expressing the transcription factor Foxb1 form the nuclei of the mamillary region as well as one nucleus of the lateral hypothalamus (LHA). One of these streams forms most of the mamillary body while the other forms the dorsal premammillary nucleus (PMd) and the Foxb1-expressing neurons of the parvafox nucleus (parvafox^Foxb1) in the LHA (Alvarez-Bolado et al., 2000). The ventrolateral part of the PMd (vlPMd) projects to the dorsolateral periaqueductal gray (dlPAG; Canteras and Swanson, 1992) while the parvafox^Foxb1 axon terminals occupy a region straddling the dlPAG and the lPAG (Bilella et al., 2016). Solid evidence indicates that the dlPAG is associated with innate defensive responses like ‘freezing’ and escape behavior (Canteras and Goto, 1999; Vianna et al., 2001; Bittencourt et al., 2004; Deng et al., 2016; Kunwar et al., 2015; Souza and Carobrez, 2016; Evans et al., 2018).

In addition to inputs from Foxb1⁺ cells of the hypothalamus, the dlPAG also receives afferences from neurons of the ventromedial hypothalamus (VMH) expressing steroidogenic factor 1 (SF1⁺). The optogenetic activation of their cell bodies in the VMH (Kunwar et al., 2015) and of their terminal endings in the dlPAG (Wang et al., 2015a) leads to immobility. As the stimulation intensity increases, immobility is followed by bursts of activity (running and jumping; Kunwar et al., 2015). This behavioral change is ascribed to the co-activation of another axon collateral of the SF1⁺ neurons, innervating the anterior hypothalamic nucleus (AHN; Wang et al., 2015a). As animals remain immobile, increasing the intensity of the stimulation of SF1⁺ cell bodies in the VMH leads to a change of the cardiovascular responses from tachycardia to bradycardia (Wang et al., 2015b).

The third afference to the dlPAG (Schenberg et al., 2005) stems from medially located glutamatergic neurons of the superior colliculus (mSC), which is responsive to innately aversive looming stimuli (Evans et al., 2018). mSC neurons integrate threat evidence and pass the information through a synaptic threshold to the glutamatergic dlPAG neurons that initiate escape (Evans et al., 2018).

We set out to establish the function of two distinct neuronal groups identifiable through Foxb1 expression, the parvafox^Foxb1 of the LHA and the PMd^Foxb1 of the hypothalamic mamillary region, both projecting to specific regions of the PAG (Canteras and Swanson, 1992; Bilella et al., 2016). To that end, we used the specific expression of Cre recombinase in Foxb1^tm1(cre)Gabo mice as a tool.

In our study (Figure 1), projection-unspecific chemogenetic activation of the Foxb1⁺ cell bodies of the PMd and of the parvafox^Foxb1 led to tachypnea, while selective optogenetic activation of theire terminal endings in the rostral part of the dlPAG provoked immobility, accompanied by bradycardia.

Figure 1

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Chemogenetic activation of Foxb1⁺ cell bodies in the PMd and parvafox nuclei of the hypothalamus, as well as optogenetic excitation of their axonal endings in the dlPAG both lead to a phenotypic presentation congruent with the reaction observed during innate defensive behavior: tachypnea, immobility, and bradycardia.

As both CNO and clozapine have been reported to activate DREADDs (with different temporal dynamics), we have designed the experimental outline of this study in a way that would allow us to also investigate the effect of both DREADD ligands against each other and against the effect of saline injections. We have shown that injection of both DREADD ligands reproducibly led to the same phenotype in DREADD-expressing animals but not in control animals. The activation of hM3Dq (activating DREADD receptors) expressed in the parvafox^Foxb1 led to changes in several respiratory parameters, particularly to an increase in respiratory frequency (measured in breaths per minute), consistent with an increased respiratory effort. We infer that this effect is at least partly mediated by the projections of the parvafox^Foxb1 neurons to the dlPAG. In accordance with this assumption, injections of excitatory aminoacids D,L-hopmocysteic acid (DLH) in the dlPAG raise respiratory activity by an increase in respiratory rate (Iigaya et al., 2010; Subramanian and Holstege, 2014; Dampney et al., 2013).

Immobility is the absence of movements associated with an increase in respiration and muscle tone (Fanselow, 1994; Kalin and Shelton, 1989) and is consistently correlated with heart rate deceleration (Vianna and Carrive, 2005; Walker and Carrive, 2003). The body immobility observed in the open field test perturbed the execution and therefore may have influenced the results of the hot-plate test.

In our optogenetic experiments, we cannot differentiate between the respective contributions of the Foxb1-terminals belonging to the parvafox^Foxb1 versus those belonging to the PMd to the behavioral (immobility) and autonomic (bradycardia) effects observed. Are these effects generated by a single common population of neurons, for example, all Foxb1⁺ neurons together (i.e. ‘command neurons’) or instead by the distinct sub-populations of neurons in the PMd or in the parvafox^Foxb1?

A subpopulation of neurons in both nuclei express Foxb1 and a distinct subpopulation consists of parvalbumin positive cells in the parvafox (Bilella et al., 2014) and Cck positive cells in the PMd (Wang et al., 2021). Ten percent of parvalbumin neurons in the parvafox express also Foxb1, whereas the situation for Cck in the PMd is yet unknown.

The PMd and the parvafox^Foxb1 originate from the same Foxb1⁺ cell lineage have differential connectivity pattern and as a result they may assume distinct functional tasks (Bilella et al., 2014; Wang et al., 2021). The PMd receives afferences from the infralimbic and prelimbic areas of the medial frontal cortex (Comoli et al., 2000), whereas the parvafox^Foxb1 neurons receives their inputs from the lateral and ventrolateral orbitofrontal cortex (Babalian et al., 2018). The neurons of the PMd have a bifurcated output, sending their axons rostrally to the anterior hypothalamic nucleus (AHN) and caudally to the dorsolateral sector of the PAG (Canteras and Swanson, 1992). Instead, the efferences of the parvafox^Foxb1 neurons have only minor rostral projections to the septal region and no rostral projections to other hypothalamic nuclei such the SF1⁺ neurons of the VHN (Kunwar et al., 2015). Instead, the parvafox^Foxb1 rather projects caudally to a wedge-shaped field straddling the lateral (lPAG) and dorsolateral (dlPAG) column (Bilella et al., 2016). A further columnar field of terminals is located in the Su3-region of the ventromedial PAG (Bilella et al., 2016).

The Foxb1⁺ neurons of the parvafox nucleus also use glutamate as a neurotransmitter (Bilella et al., 2016) and express the gene coding for the neuropeptide adenylate cyclase-activating polypeptide1 (Adcyap1; Girard et al., 2011), found to be involved among others in stress disorders (Ressler et al., 2011) and cardiorespiratory control (Shi et al., 2021; Barrett et al., 2019).

The PMd is predestined to play a pivotal functional role in the coordination of defensive behaviors, to both innate and conditioned threats (Wang et al., 2021). It consists of a predator activated ventrolateral part (vlPMd) which project to the dlPAG and a dorsomedial part (dmPMd) activated by encounter with aggressive conspecific which avoid projecting to the dlPAG. Ibotenic-acid lesions of the PMd eliminates escape and freezing responses (Canteras et al., 1997).

Recording Ca²⁺-transients in PMd^Cck neurons with fiber photometry, the group of Adhikari (Wang et al., 2021), revealed that these neurons are activated during escape but not during freezing and that their chemogenetic inhibition decreases escape speed from threats (Wang et al., 2021). The optogenetic activation of the PMd^Cck cells presumably leads to the activation of the glutamatergic neurons in the dlPAG, which then provoke the fleeing. In our experiments, however, activation of the larger group of Foxb1⁺ neurons in parvafox^Foxb1 and PMd led to immobility and not to escape. How can this paradoxical effect be explained? One potential explanation would be, that the parvafox^Foxb1 neurons, at least in the rostral dlPAG, innervate a different group of neurons, namely the abundant inhibitory GABA-cells (Barbaresi, 2005). It is to be assumed that these short-axon cells, with overlapping distribution in the dlPAG, would locally inhibit the activity of the glutamatergic neurons activated by the PMd^Cck neurons. Another subgroup of nitric oxide expressing neurons in the dlPAG (Onstott et al., 1993) also display a tonic inhibitory effect, via a potentiation of GABAergic synaptic inputs (Xing et al., 2008). The role of a fourth group of acetylcholinesterase positive neurons in the dlPAG (Illing, 1996) is unknown. The immobility of the mouse would then derive from the local inhibition of the excitatory elements of the dlPAG. Future work will be required to clarify this issue. A second potential explanation is rooted in the expression pattern of Foxb1 and Cck within the PMd. Our reanalysis of the scRNA seq data of the murine ventral-posterior hypothalamus has revealed, that there is a substantial number of Foxb1 and Cck single-positive cells in the PMd. Since our study and the one from Adhikari’s group both made use of single-gene Cre knock-in mouse lines, the optogenetic stimulation in both studies led to activation of both single-positive and double-positive neurons. We therefore propose that the observed effects in both studies may be exclusively mediated by single-positive neuronal populations within the PMd. Using an intersectional approach combining Cre- and Flp-recombination dependent expression of optogenetic tools, would allow to investigate whether the same behavioral effects can also be elicited by exclusively activating double-positive (Foxb1⁺/Cck⁺) neurons within the PMd.

Which other parts of the brain are recruited by the dlPAG neurons to initiate bradycardia and immobility? The dlPAG projects to the cuneiform nucleus (Meller and Dennis, 1991), which is involved in defensive locomotion (Jordan, 1998) through its connection with the lateral gigantocellular reticular nuclei (Tovote et al., 2016), implicated in high-speed locomotion (Caggiano et al., 2018). Additional targets of the cuneiform projections are the motor nucleus of the vagus and the nucleus tractus solitarius (Korte et al., 1992) and a few immunostained boutons were found around the nucleus ambiguus in the rostroventrolateral medullary nucleus (Figure 5K in Korte et al., 1992). These projections are probably involved in the bradycardic response to the optogenetic stimulation in the dlPAG. The projections of the cuneiform nucleus to the rostral ventrolateral medulla promote sympathetic vasomotor activity (Verberne, 1995). An alternative pathway for the expression of the behavioral and autonomic effects of dlPAG activation is through a link with the dorsomedial hypothalamus (DMH; Dampney et al., 2013).

Bradycardia starts immediately after the beginning of the optogenetic activation of the Foxb1⁺ terminals in the dlPAG. The increase in the HRV during the optogenetic stimulation indicates an intervention of the parasympathetic system on the sinoatrial node of the heart (Standish et al., 1995). The parasympathetic impact on the heart is mediated by acetylcholine neurotransmission (Wu et al., 2019) and has a very short latency of response, with peak effect at about 0.5 s and return to baseline within 1 s (Pumprla et al., 2002).

During ultrasonic vocalization tests in our laboratory (data not reported in this study), we observed that the Foxb1-Cre mice did not escape the ChR2-induced state of immobility, even when another genotype-matched and novel intruder mouse was placed into the ChR2-expressing mouse’s home cage. When approached by the intruder mouse, the mice were still able to move their heads under photo-stimulation, did sniff and interacted with the intruder mouse but did not actively follow the intruder. A mouse displaying complete immobility remained completely motionless even when the intruder was actively approaching it and even when it started digging into the cage bedding underneath the resident mouse.

In an interesting recent publication of Chen’s group (Liu et al., 2022), the projection of the parvafox^Foxb1 to the lateral PAG (lPAG) indeed has been found to drive social avoidance in mice. Inhibition of the parvafox^Foxb1 neurons by the GABAergic input from the lateral septum reversed the deficit in social novelty preference induced by chronic social defeat stress. The authors conclude that activation of the parvafox^Foxb1 leads to avoidance of general social situations.

Hence, our observations of a reduced social interaction phenotype agree with the results from Chen’s group. However, since activation of the parvafox^Foxb1 terminals in the dlPAG also leads to immobility of the mouse in the absence of any social stimulus, we argue that the outcome of the social avoidance test could have been influenced by the failing motor performance of the Foxb1-Cre mice and might not be of pure social nature. In light of these observations, it is further interesting to note, that the results from our hot plate experiments indicate that the immobile/hyporeactive phenotype of these mice can indeed be escaped, when an adequate stimulus is presented (e.g. thermal stimulus intensity reaching nociceptive levels).

The possibility of antidromic propagation of ChR2-mediated action potentials poses a major caveat to the interpretation of results obtained from experiments in which presynaptic terminals are optogenetically activated. This mechanism could lead to coincident activation and realease of neurotransmitters from collaterals originally not intended to be targeted by the intervention. The use of inhibitory optogenetic tools, however, circumvents such a mechanism of uncontrolled backpropagating action potentials (Rost et al., 2022). In our optogenetic experiments in which we have used the inhibitory opsin ArchT3.0, we detected statistically significant differences to the baseline conditions in multiple locomotive parameters. This observation of bidirectional modulation is an indication of at least some baseline activity of the Foxb1⁺ neurons in the hypothalamus in our experimental settings. Importantly, these results and observations additionally support the notion that the locomotor phenotype is due to the specific projection of Foxb1⁺ neurons onto the PAG. If the hypolocomotor phenotype in ChR2 expressing mice was driven by backpropagation-mediated activation of collaterals, the presynaptic ArchT3.0-mediated inhibition of the Foxb1⁺ projections to the PAG would be unlikely to cause an effect opposing the ChR2-mediated phenotype. In our ArchT3.0 experiments, we specifically employed a stimulation paradigm in which we had light illumination on for 10 s and then off for another 10 s. This was done to reduce pH-mediated side effects of ArchT3.0 axonal activation observed with sustained stimulation, like spontaneous release of neurotransmitters (Mahn et al., 2016). If such side effects had occured in our experiments, we would expect to see a phenotype that resembles the one observed in the ChR2-mediated activation. However, in our experiments, we saw no indication of such a behavior. Recently, two new optogenetic tools have been developed that allow efficient and targeted presynaptic silencing through GPCRs without the alteration in intracellular pH levels (Mahn et al., 2021). For future experiments, such tools might be more suitable for presyaptic silencing experiments.

In summary, our results demonstrate evidence for a role of the hypothalamic Foxb1⁺ neuronal population located in the parvafox^Foxb1, and the PMd in the expression of bradycardia and immobility, as well as in increasing respiratory rate. This effect is in accordance with distinctive coping patterns, the freezing-like behavior accompanied by bradycardia being associated with innate defensive behavior.

In view of the recent finding that PMd neurons are activated during escape (Wang et al., 2021), the immobility that we observe could be mediated by the terminal endings of the parvafox^Foxb1, synapsing on the inhibitory GABA-interneurons of the rostral third of the dlPAG or could be the result of the activation of the Foxb1 single-positive PMd neuronal population (i.e. Foxb1⁺/Cck^-). Further investigations on the neuronal activity of these hypothalamic Foxb1⁺ populations during exposure to natural threats will allow for a better understanding of the differential roles of Foxb1⁺ and Cck⁺ PMd neurons. Such experiments could for example consist of fiber photometry calcium recordings or microendoscopic calcium imaging experiments during predator exposure in Foxb1 and/or Cck Cre-driver lines.

Raw data generated in this study was deposited on Dryad at: https://doi.org//10.5061/dryad.rv15dv4fn.Custom written code generated for this publication are available on GitHub in the following repository: https://github.com/rbcola/Parvafox-eLife-2024, (copy archived at Cola, 2024).

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   Chemo- and optogenetic activation of hypothalamic Foxb1-expressing neurons and their terminal endings in the rostral-dorsolateral PAG leads to tachypnea, bradycardia, and immobility.

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Author details

1. Reto B Cola

   Anatomy and program in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   For correspondence

   reto.cola@uzh.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1419-2480

2. Diana M Roccaro-Waldmeyer

   Anatomy and program in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland

   Contribution

   Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

3. Samara Naim

   Anatomy and program in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Alexandre Babalian

   Anatomy and program in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Petra Seebeck

   Zurich integrative Rodent Physiology (ZIRP), University of Zürich, Zürich, Switzerland

   Contribution

   Methodology

   Competing interests

   No competing interests declared

6. Gonzalo Alvarez-Bolado

   Institute of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany

   Contribution

   Resources, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3044-1603

7. Marco R Celio

   Anatomy and program in Neuroscience, Faculty of Science and Medicine, University of Fribourg, Fribourg, Switzerland

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Writing – original draft, Writing – review and editing

   For correspondence

   marco.celio@unifr.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3623-842X

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