A non-image-forming visual circuit mediates the innate fear of heights in male mice.

The neural basis of fear of heights remains largely unknown. In this study, we investigated the fear response to heights in male mice and observed characteristic aversive behaviors resembling human height vertigo. We identified visual input as a critical factor in mouse reactions to heights, while peripheral vestibular input was found to be nonessential for fear of heights. Unexpectedly, we found that fear of heights in naïve mice does not rely on image-forming visual processing by the primary visual cortex. Instead, a subset of neurons in the ventral lateral geniculate nucleus (vLGN), which connects to the lateral/ventrolateral periaqueductal gray (l/vlPAG), drives the expression of fear associated with heights. Additionally, we observed that a subcortical visual pathway linking the superior colliculus to the lateral posterior thalamic nucleus inhibits the defensive response to height threats. These findings highlight a rapid fear response to height threats through a subcortical visual and defensive pathway from the vLGN to the l/vlPAG.

Cell-type-specific hypothalamic pathways to brainstem drive context-dependent strategies in response to stressors.

Adaptive behavioral responses to stressors are critical for survival. However, which brain areas orchestrate switching the appropriate stress responses to distinct contexts is an open question. This study aimed to identify the cell-type-specific brain circuitry governing the selection of distinct behavioral strategies in response to stressors. Through novel mouse behavior paradigms, we observed distinct stressor-evoked behaviors in two psycho-spatially distinct contexts characterized by stressors inside or outside the safe zone. The identification of brain regions activated in both conditions revealed the involvement of the dorsomedial hypothalamus (DMH). Further investigation using optogenetics, chemogenetics, and photometry revealed that glutamatergic projections from the DMH to periaqueductal gray (PAG) mediated responses to inside stressors, while GABAergic projections, particularly from tachykinin1-expressing neurons, played a crucial role in coping with outside stressors. These findings elucidate the role of cell-type-specific circuitry from the DMH to the PAG in shaping behavioral strategies in response to stressors. These findings have the potential to advance our understanding of fundamental neurobiological processes and inform the development of novel approaches for managing context-dependent and anxiety-associated pathological conditions such as agoraphobia and claustrophobia.

The role of periaqueductal gray astrocytes in anxiety-like behavior induced by acute stress.

Astrocytes in the central nervous system play a vital role in modulating synaptic transmission and neuronal activation by releasing gliotransmitters. The 5-HTergic neurons in the ventrolateral periaqueductal gray (vlPAG) are important in anxiety processing. However, it remains uncertain whether the regulation of astrocytic activity on vlPAG 5-HTergic neurons is involved in anxiety processing. Here, through chemogenetic manipulation, we explored the impact of astrocytic activity in the PAG on the regulation of anxiety. To determine the role of astrocytes in the control of anxiety, we induced anxiety-like behaviors in mice through foot shock and investigated their effects on synaptic transmission and neuronal excitability in vlPAG 5-HTergic neurons. Foot shock caused anxiety-like behaviors, which were accompanied with the increase of the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs), the area of slow inward currents (SICs), and the spike frequency of action potentials (AP) in vlPAG 5-HTergic neurons. The chemogenetic inhibition of vlPAG astrocytes was found to attenuate stress-induced anxiety-like behaviors and decrease the heightened synaptic transmission and neuronal excitability of vlPAG 5-HTergic neurons. Conversely, chemogenetic activation of vlPAG astrocytes triggered anxiety-like behaviors, enhanced synaptic transmission, and increased the excitability of vlPAG 5-HTergic neurons in unstressed mice. In summary, this study has provided initial insights into the pathway by which astrocytes influence behavior through the rapid regulation of associated neurons. This offers a new perspective for the investigation of the biological mechanisms underlying anxiety.

[Inhibition of glutamatergic neurons in the dorsomedial periaqueductal gray alleviates excessive defensive behaviors of mice with post-traumatic stress disorder].

OBJECTIVE: To investigate the role of glutamatergic neurons in the dorsomedial periaqueductal grey (dmPAG) in regulating excessive defensive behaviors in mice with post-traumatic stress disorder (PTSD). METHODS: Eight-week-old male C57BL/6 mice were subjected to stereotactic injections of different recombinant adeno- associated viral vectors (rAAV2/9-CaMKII-mCherry, rAAV2/9-CaMKII-hM3Dq-mCherry and rAAV2/9-CaMKII-hM4Di-mCherry) into the bilateral dmPAG for chemogenetic activation or inhibition of the glutamatergic neurons, followed 2 weeks later by PTSD modeling by single prolonged stress. The looming test, response to whisker stimulation test and contextual fear conditioning (CFC) test were used to observe changes in defensive behaviors of the PTSD mice. The activity of glutamatergic neurons in the dmPAG were observed using immunofluorescence staining. RESULTS: Compared with the control mice, the mouse models of PTSD showed a shortened latency of flights with increased time spent in the nest, response scores of defensive behaviors and freezing time (all P<0.01). Immunofluorescence staining revealed significantly increased c-fos-positive glutamatergic neurons in the dmPAG of PTSD mice with defensive behaviors. Activation of the glutamatergic neurons in the dmPAG (in PTSD hM3Dq group) did not cause significant changes in the latency of flights or time in nest but obviously increased response scores of defensive behaviors and freezing time of the mice, whereas inhibiting the glutamatergic neurons in the dmPAG (in PTSD hM4Di group) caused the reverse changes and obviously alleviated defensive behaviors in the PTSD mice (P<0.05 or 0.01). CONCLUSION: Inhibiting the activity of glutamatergic neurons in the dmPAG can alleviate defensive behaviors in mice with PTSD.

Functional representation of trigeminal nociceptive input in the human periaqueductal gray.

The periaqueductal gray (PAG) is located in the mesencephalon in the upper brainstem and, as part of the descending pain modulation, is considered a crucial structure for pain control. Its modulatory effect on painful sensation is often seen as a systemic function affecting the whole body similarly. However, recent animal data suggest some kind of somatotopy in the PAG. This would make the PAG capable of dermatome-specific analgesic function. We electrically stimulated the three peripheral dermatomes of the trigemino-cervical complex and the greater occipital nerve in 61 humans during optimized brainstem functional magnetic resonance imaging. We provide evidence for a fine-grained and highly specific somatotopic representation of nociceptive input in the PAG in humans and a functional connectivity between the individual representations of the peripheral nerves in the PAG and the brainstem nuclei of these nerves. Our data suggest that the downstream antinociceptive properties of the PAG may be rather specific down to the level of individual dermatomes.

Control of feeding by a bottom-up midbrain-subthalamic pathway.

Investigative exploration and foraging leading to food consumption have vital importance, but are not well-understood. Since GABAergic inputs to the lateral and ventrolateral periaqueductal gray (l/vlPAG) control such behaviors, we dissected the role of vgat-expressing GABAergic l/vlPAG cells in exploration, foraging and hunting. Here, we show that in mice vgat l/vlPAG cells encode approach to food and consumption of both live prey and non-prey foods. The activity of these cells is necessary and sufficient for inducing food-seeking leading to subsequent consumption. Activation of vgat l/vlPAG cells produces exploratory foraging and compulsive eating without altering defensive behaviors. Moreover, l/vlPAG vgat cells are bidirectionally interconnected to several feeding, exploration and investigation nodes, including the zona incerta. Remarkably, the vgat l/vlPAG projection to the zona incerta bidirectionally controls approach towards food leading to consumption. These data indicate the PAG is not only a final downstream target of top-down exploration and foraging-related inputs, but that it also influences these behaviors through a bottom-up pathway.

The functional and anatomical characterization of three spinal output pathways of the anterolateral tract.

The nature of spinal output pathways that convey nociceptive information to the brain has been the subject of controversy. Here, we provide anatomical, molecular, and functional characterizations of two distinct anterolateral pathways: one, ascending in the lateral spinal cord, triggers nociceptive behaviors, and the other one, ascending in the ventral spinal cord, when inhibited, leads to sensorimotor deficits. Moreover, the lateral pathway consists of at least two subtypes. The first is a contralateral pathway that extends to the periaqueductal gray (PAG) and thalamus; the second is a bilateral pathway that projects to the bilateral parabrachial nucleus (PBN). Finally, we present evidence showing that activation of the contralateral pathway is sufficient for defensive behaviors such as running and freezing, whereas the bilateral pathway is sufficient for attending behaviors such as licking and guarding. This work offers insight into the complex organizational logic of the anterolateral system in the mouse.

Midbrain neurons important for the production of mouse ultrasonic vocalizations are not required for distress calls.

A fundamental feature of vocal communication is that animals produce vocalizations with different acoustic features in different behavioral contexts (contact calls, territorial calls, courtship calls, etc.). The midbrain periaqueductal gray (PAG) is a key region that regulates vocal production, and artificial activation of the PAG can elicit the production of multiple species-typical vocalization types.1,2,3,4,5,6,7,8,9 How PAG circuits are organized to regulate the production of different vocalization types remains unknown. On the one hand, studies have found that partial PAG lesions abolish the production of some vocalization types while leaving others intact,3,8,10,11 suggesting that different populations of PAG neurons might control the production of different vocalization types. On the other hand, electrophysiological recordings have revealed individual PAG neurons that increase their activity during the production of multiple vocalization types,12,13,14 suggesting that some PAG neurons may regulate the production of more than one vocalization type. To test whether a single population of midbrain neurons regulates the production of different vocalization types, we applied intersectional methods to selectively ablate a population of midbrain neurons important for the production of ultrasonic vocalizations (USVs) in mice. We find that, although ablation of these PAG-USV neurons blocks USV production in both males and females, these neurons are not required for the production of distress calls. Our findings suggest that distinct populations of midbrain neurons control the production of different vocalization types.

A distinct cortical code for socially learned threat.

Animals can learn about sources of danger while minimizing their own risk by observing how others respond to threats. However, the distinct neural mechanisms by which threats are learned through social observation (known as observational fear learning1-4 (OFL)) to generate behavioural responses specific to such threats remain poorly understood. The dorsomedial prefrontal cortex (dmPFC) performs several key functions that may underlie OFL, including processing of social information and disambiguation of threat cues5-11. Here we show that dmPFC is recruited and required for OFL in mice. Using cellular-resolution microendoscopic calcium imaging, we demonstrate that dmPFC neurons code for observational fear and do so in a manner that is distinct from direct experience. We find that dmPFC neuronal activity predicts upcoming switches between freezing and moving state elicited by threat. By combining neuronal circuit mapping, calcium imaging, electrophysiological recordings and optogenetics, we show that dmPFC projections to the midbrain periaqueductal grey (PAG) constrain observer freezing, and that amygdalar and hippocampal inputs to dmPFC opposingly modulate observer freezing. Together our findings reveal that dmPFC neurons compute a distinct code for observational fear and coordinate long-range neural circuits to select behavioural responses.

Midbrain node for context-specific vocalisation in fish.

Vocalizations communicate information indicative of behavioural state across divergent social contexts. Yet, how brain regions actively pattern the acoustic features of context-specific vocal signals remains largely unexplored. The midbrain periaqueductal gray (PAG) is a major site for initiating vocalization among mammals, including primates. We show that PAG neurons in a highly vocal fish species (Porichthys notatus) are activated in distinct patterns during agonistic versus courtship calling by males, with few co-activated during a non-vocal behaviour, foraging. Pharmacological manipulations within vocally active PAG, but not hindbrain, sites evoke vocal network output to sonic muscles matching the temporal features of courtship and agonistic calls, showing that a balance of inhibitory and excitatory dynamics is likely necessary for patterning different call types. Collectively, these findings support the hypothesis that vocal species of fish and mammals share functionally comparable PAG nodes that in some species can influence the acoustic structure of social context-specific vocal signals.

Distinct patterns of activity within columns of the periaqueductal gray are associated with functionally distinct birdsongs.

Male songbirds produce female-directed songs in spring that convey a state of sexual motivation. Many songbirds also sing in fall flocks in affiliative/gregarious contexts in which song is linked to an intrinsic positive affective state. The periaqueductal gray (PAG) in mammals, which is organized into functional columns, integrates information from multiple brain regions and relays this information to vocal motor areas so that an animal emits a vocal signal reflective of its affective state. Here, we test the hypothesis that distinct columns in the songbird PAG play roles in the distinct affective states communicated by sexually motivated and gregarious song. We quantified the numbers of immediate early gene ZENK-positive cells in 16 PAG subregions in male European starlings (Sturnus vulgaris) after singing gregarious or sexually motivated song. Results suggest that distinct PAG columns in songbirds context-specifically regulate song, agonistic, and courtship behaviors. A second exploratory, functional tract-tracing study also demonstrated that inputs to the PAG from specific subregions of the medial preoptic nucleus may contribute to gregarious song and behaviors indicative of social dominance. Together, findings suggest that conserved PAG columns and inputs from the preoptic nucleus may play a role in context-specific vocal and other social behaviors.

Role of the rostral dorsomedial column of the periaqueductal gray during social defeat in rats.

Previous studies showed that the dorsal premammillary nucleus of the hypothalamus (PMD) is involved in social passive defensive behaviors likely to be meditated by descending projections to the periaqueductal gray (PAG). We focused on the rostral dorsomedial PAG (rPAGdm) to reveal its putative neural mechanisms involved in mediating social defensive responses. By combining retrograde tracing and FOS expression analysis, we showed that in addition to the PMD, the rPAGdm is influenced by several brain sites active during social defeat. Next, we found that cytotoxic lesions of the rPAGdm drastically reduced passive defense and did not affect active defensive responses. We then examined the rPAGdm's projection pattern and found that the PAGdm projections are mostly restricted to midbrain sites, including the precommissural nucleus, different columns of the PAG, and the cuneiform nucleus (CUN). Also, we found decreased FOS expression in the caudal PAGdm, CUN, and PMD after the rPAGdm was lesioned. The results support that the rPAGdm mediates passive social defensive responses through ascending paths to prosencephalic circuits likely mediated by the CUN. This study provides further support for the role of the PAG in the modulation of behavioral responses by working as a unique hub for influencing prosencephalic sites during the mediation of aversive responses.

A cholinergic circuit that relieves pain despite opioid tolerance.

Chronic pain is a tremendous burden for afflicted individuals and society. Although opioids effectively relieve pain, significant adverse outcomes limit their utility and efficacy. To investigate alternate pain control mechanisms, we explored cholinergic signaling in the ventrolateral periaqueductal gray (vlPAG), a critical nexus for descending pain modulation. Biosensor assays revealed that pain states decreased acetylcholine release in vlPAG. Activation of cholinergic projections from the pedunculopontine tegmentum to vlPAG relieved pain, even in opioid-tolerant conditions, through ⍺7 nicotinic acetylcholine receptors (nAChRs). Activating ⍺7 nAChRs with agonists or stimulating endogenous acetylcholine inhibited vlPAG neuronal activity through Ca2+ and peroxisome proliferator-activated receptor α (PPAR⍺)-dependent signaling. In vivo 2-photon imaging revealed that chronic pain induces aberrant excitability of vlPAG neuronal ensembles and that ⍺7 nAChR-mediated inhibition of these cells relieves pain, even after opioid tolerance. Finally, pain relief through these cholinergic mechanisms was not associated with tolerance, reward, or withdrawal symptoms, highlighting its potential clinical relevance.

Voltage-Seq: all-optical postsynaptic connectome-guided single-cell transcriptomics.

Understanding the routing of neuronal information requires the functional characterization of connections. Neuronal projections recruit large postsynaptic ensembles with distinct postsynaptic response types (PRTs). PRT is typically probed by low-throughput whole-cell electrophysiology and is not a selection criterion for single-cell RNA-sequencing (scRNA-seq). To overcome these limitations and target neurons based on specific PRTs for soma harvesting and subsequent scRNA-seq, we created Voltage-Seq. We established all-optical voltage imaging and recorded the PRT of 8,347 neurons in the mouse periaqueductal gray (PAG) evoked by the optogenetic activation of ventromedial hypothalamic (VMH) terminals. PRTs were classified and spatially resolved in the entire VMH-PAG connectome. We built an onsite analysis tool named VoltView to navigate soma harvesting towards target PRTs guided by a classifier that used the VMH-PAG connectome database as a reference. We demonstrated Voltage-seq by locating VMH-driven γ-aminobutyric acid-ergic neurons in the PAG, guided solely by the onsite classification in VoltView.

Pedunculopontine Chx10+ neurons control global motor arrest in mice.

Arrest of ongoing movements is an integral part of executing motor programs. Behavioral arrest may happen upon termination of a variety of goal-directed movements or as a global motor arrest either in the context of fear or in response to salient environmental cues. The neuronal circuits that bridge with the executive motor circuits to implement a global motor arrest are poorly understood. We report the discovery that the activation of glutamatergic Chx10-derived neurons in the pedunculopontine nucleus (PPN) in mice arrests all ongoing movements while simultaneously causing apnea and bradycardia. This global motor arrest has a pause-and-play pattern with an instantaneous interruption of movement followed by a short-latency continuation from where it was paused. Mice naturally perform arrest bouts with the same combination of motor and autonomic features. The Chx10-PPN-evoked arrest is different to ventrolateral periaqueductal gray-induced freezing. Our study defines a motor command that induces a global motor arrest, which may be recruited in response to salient environmental cues to allow for a preparatory or arousal state, and identifies a locomotor-opposing role for rostrally biased glutamatergic neurons in the PPN.

Selective activation of AKAP150/TRPV1 in ventrolateral periaqueductal gray GABAergic neurons facilitates conditioned place aversion in male mice.

Aversion refers to feelings of strong dislike or avoidance toward particular stimuli or situations. Aversion can be caused by pain stimuli and has a long-term negative impact on physical and mental health. Aversion can also be caused by drug abuse withdrawal, resulting in people with substance use disorder to relapse. However, the mechanisms underlying aversion remain unclear. The ventrolateral periaqueductal gray (vlPAG) is considered to play a key role in aversive behavior. Our study showed that inhibition of vlPAG GABAergic neurons significantly attenuated the conditioned place aversion (CPA) induced by hindpaw pain pinch or naloxone-precipitated morphine withdrawal. However, activating or inhibiting glutamatergic neurons, or activating GABAergic neurons cannot affect or alter CPA response. AKAP150 protein expression and phosphorylated TRPV1 (p-TRPV1) were significantly upregulated in these two CPA models. In AKAP150flox/flox mice and C57/B6J wild-type mice, cell-type-selective inhibition of AKAP150 in GABAergic neurons in the vlPAG attenuated aversion. However, downregulating AKAP150 in glutamatergic neurons did not attenuate aversion. Knockdown of AKAP150 in GABAergic neurons effectively reversed the p-TRPV1 upregulation in these two CPA models utilized in our study. Collectively, inhibition of the AKAP150/p-TRPV1 pathway in GABAergic neurons in the vlPAG may be considered a potential therapeutic target for the CPA response.

Play and tickling responses map to the lateral columns of the rat periaqueductal gray.

The persistence of play after decortication points to a subcortical mechanism of play control. We found that global blockade of the rat periaqueductal gray with either muscimol or lidocaine interfered with ticklishness and play. We recorded vocalizations and neural activity from the periaqueductal gray of young, playful rats during interspecific touch, play, and tickling. Rats vocalized weakly to touch and more strongly to play and tickling. Periaqueductal gray units showed diverse but strong modulation to tickling and play. Hierarchical clustering based on neuronal responses to play and tickling revealed functional clusters mapping to different periaqueductal gray columns. Specifically, we observed play-neutral/tickling-inhibited and tickling/play-neutral units in dorsolateral and dorsomedial periaqueductal gray columns. In contrast, strongly play/tickling-excited units mapped to the lateral columns and were suppressed by anxiogenic conditions. Optogenetic inactivation of lateral periaqueductal columns disrupted ticklishness and play. We conclude that the lateral periaqueductal gray columns are decisive for play and laughter.

[Research progress on the mechanism of pain related neural pathways above the spinal cord].

Pain is a multi-dimensional emotional experience, and pain sensation and pain emotion are the two main components. As for pain, previous studies only focused on a certain link of the pain transmission pathway or a certain key brain region, and there is a lack of evidence that connectivity of brain regions is involved in pain or pain regulation in the overall state. The establishment of new experimental tools and techniques has brought light to the study of neural pathways of pain sensation and pain emotion. In this paper, the structure and functional basis of the neural pathways involved in the formation of pain sensation and the regulation of pain emotion in the nervous system above the spinal cord level, including thalamus, amygdala, midbrain periaqueductal gray (PAG), parabrachial nucleus (PB) and medial prefrontal cortex (mPFC), are reviewed in recent years, providing clues for the in-depth study of pain.

Nested circuits mediate the decision to vocalize.

Vocalizations facilitate mating and social affiliation but may also inadvertently alert predators and rivals. Consequently, the decision to vocalize depends on brain circuits that can weigh and compare these potential benefits and risks. Male mice produce ultrasonic vocalizations (USVs) during courtship to facilitate mating, and previously isolated female mice produce USVs during social encounters with novel females. Earlier we showed that a specialized set of neurons in the midbrain periaqueductal gray (PAG-USV neurons) are an obligatory gate for USV production in both male and female mice, and that both PAG-USV neurons and USVs can be switched on by their inputs from the preoptic area (POA) of the hypothalamus and switched off by their inputs from neurons on the border between the central and medial amygdala (AmgC/M-PAG neurons) (Michael et al., 2020). Here, we show that the USV-suppressing AmgC/M-PAG neurons are strongly activated by predator cues or during social contexts that suppress USV production in male and female mice. Further, we explored how vocal promoting and vocal suppressing drives are weighed in the brain to influence vocal production in male mice, where the drive and courtship function for USVs are better understood. We found that AmgC/M-PAG neurons receive monosynaptic inhibitory input from POA neurons that also project to the PAG, that these inhibitory inputs are active in USV-promoting social contexts, and that optogenetic activation of POA cell bodies that make divergent axonal projections to the amygdala and PAG is sufficient to elicit USV production in socially isolated male mice. Accordingly, AmgC/M-PAG neurons, along with POAPAG and PAG-USV neurons, form a nested hierarchical circuit in which environmental and social information converges to influence the decision to vocalize.

Brainstem Modulation of Nociception by Periaqueductal Gray Neurons Expressing the µ-Opioid Receptor in Mice.

BACKGROUND: Pharmacologic manipulations directed at the periaqueductal gray have demonstrated the importance of the µ-opioid receptor in modulating reflexive responses to nociception. The authors hypothesized that a supraspinal pathway centered on neurons in the periaqueductal gray containing the µ-opioid receptor could modulate nociceptive and itch behaviors. METHODS: The study used anatomical, optogenetic, and chemogenetic approaches in male and female mice to manipulate µ-opioid receptor neurons in the periaqueductal gray. Behavioral assays including von Frey, Hargreaves, cold plantar, chloroquine-induced itch, hotplate, formalin-induced injury, capsaicin-induced injury, and open field tests were used. In separate experiments, naloxone was administered in a postsurgical model of latent sensitization. RESULTS: Activation of µ-opioid receptor neurons in the periaqueductal gray increased jumping (least-squares mean difference of -3.30 s; 95% CI, -6.17 to -0.44; P = 0.023; n = 7 or 8 mice per group), reduced itch responses (least-squares mean difference of 70 scratching bouts; 95% CI, 35 to 105; P < 0.001; n = 8 mice), and elicited modestly antinociceptive effects (least-squares mean difference of -0.7 g on mechanical and -10.24 s on thermal testing; 95% CI, -1.3 to -0.2 and 95% CI, -13.77 to -6.70, and P = 0.005 and P < 0.001, respectively; n = 8 mice). Last, the study uncovered the role of the periaqueductal gray in suppressing hyperalgesia after a postsurgical state of latent sensitization (least-squares mean difference comparing saline and naloxone of -12 jumps; 95% CI, -17 to -7; P < 0.001 for controls; and -2 jumps; 95% CI, -7 to 4; P = 0.706 after optogenetic stimulation; n = 7 to 9 mice per group). CONCLUSIONS: µ-Opioid receptor neurons in the periaqueductal gray modulate distinct nocifensive behaviors: their activation reduced responses to mechanical and thermal testing, and attenuated scratching behaviors, but facilitated escape responses. The findings emphasize the role of the periaqueductal gray in the behavioral expression of nociception using reflexive and noxious paradigms.