Chronic restraint stress-induced hyperalgesia is modulated by the periaqueductal gray neurons projecting to the rostral ventromedial medulla in mice.

Stress-induced hyperalgesia (SIH) is induced by repeated or chronic exposure to stressful or uncomfortable environments. However, the neural mechanisms involved in the modulatory effects of the periaqueductal gray (PAG) and its associated loops on SIH development hav e not been elucidated. In the present study, we used chronic restraint stress (CRS)-induced hyperalgesia as a SIH model and manipulated neuronal activity via a pharmacogenetic approach to investigate the neural mechanism underlying the effects of descending pain-modulatory pathways on SIH. We found that activation of PAG neurons alleviates CRS-induced hyperalgesia; on the other hand, PAG neurons inhibition facilitates CRS-induced hyperalgesia. Moreover, this modulatory effect is achieved by the neurons which projecting to the rostral ventromedial medulla (RVM). Our data thus reveal the functional role of the PAG-RVM circuit in SIH and provide analgesic targets in the brain for clinical SIH treatment.

[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.

Orexin neurons play contrasting roles in itch and pain neural processing via projecting to the periaqueductal gray.

Pain and itch are recognized as antagonistically regulated sensations; pain suppresses itch, whilst pain inhibition enhances itch. The neural mechanisms at the central nervous system (CNS) underlying these pain-itch interactions still need to be explored. Here, we revealed the contrasting role of orexin-producing neurons (ORX neurons) in the lateral hypothalamus (LH), which suppresses pain while enhancing itch neural processing, by applying optogenetics to the acute pruritus and pain model. We also revealed that the circuit of ORX neurons from LH to periaqueductal gray regions served in the contrasting modulation of itch and pain processing using optogenetic terminal inhibition techniques. Additionally, by using an atopic dermatitis model, we confirmed the involvement of ORX neurons in regulating chronic itch processing, which could lead to a novel therapeutic target for persistent pruritus in clinical settings. Our findings provide new insight into the mechanism of antagonistic regulation between pain and itch in the CNS.

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.

Characterization of Ultrasonic Vocalization-Modulated Neurons in Rat Motor Cortex Based on Their Activity Modulation and Axonal Projection to the Periaqueductal Gray.

Vocalization, a means of social communication, is prevalent among many species, including humans. Both rats and mice use ultrasonic vocalizations (USVs) in various social contexts and affective states. The motor cortex is hypothesized to be involved in precisely controlling USVs through connections with critical regions of the brain for vocalization, such as the periaqueductal gray matter (PAG). However, it is unclear how neurons in the motor cortex are modulated during USVs. Moreover, the relationship between USV modulation of neurons and anatomical connections from the motor cortex to PAG is also not clearly understood. In this study, we first characterized the activity patterns of neurons in the primary and secondary motor cortices during emission of USVs in rats using large-scale electrophysiological recordings. We also examined the axonal projection of the motor cortex to PAG using retrograde labeling and identified two clusters of PAG-projecting neurons in the anterior and posterior parts of the motor cortex. The neural activity patterns around the emission of USVs differed between the anterior and posterior regions, which were divided based on the distribution of PAG-projecting neurons in the motor cortex. Furthermore, using optogenetic tagging, we recorded the USV modulation of PAG-projecting neurons in the posterior part of the motor cortex and found that they showed predominantly sustained excitatory responses during USVs. These results contribute to our understanding of the involvement of the motor cortex in the generation of USV at the neuronal and circuit levels.

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.

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.

MRI radiomics based machine learning model of the periaqueductal gray matter in migraine patients.

Background and purpose:

The aim of the study was to investigate the question: Can MRI radiomics analysis of the periaqueductal gray region elucidate the pathophysiological mechanisms underlying various migraine subtypes, and can a machine learning model using these radiomics features accurately differentiate between migraine patients and healthy individuals, as well as between migraine subtypes, including atypical cases with overlapping symptoms?

The study analyzed initial MRI images of individuals taken after their first migraine diagnosis, and additional MRI scans were acquired from healthy subjects. Radiomics modeling was applied to analyze all the MRI images in the periaqueductal gray region. The dataset was randomized, and oversampling was used if there was class imbalance between groups. The optimal algorithm-based feature selection method was employed to select the most important 5-10 features to differentiate between the two groups. The classification performance of AI algorithms was evaluated using receiver operating characteristic analysis to calculate the area under the curve, classification accuracy, sensitivity, and specificity values. Participants were required to have a confirmed diagnosis of either episodic migraine, probable migraine, or chronic migraine. Patients with aura, those who used migraine-preventive medication within the past six months, or had chronic illnesses, psychiatric disorders, cerebrovascular conditions, neoplastic diseases, or other headache types were excluded from the study. Additionally, 102 healthy subjects who met the inclusion and exclusion criteria were included. 

The algorithm-based information gain method for feature reduction had the best performance among all methods, with the first-order, gray-level size zone matrix, and gray-level co-occurrence matrix classes being the dominant feature classes. The machine learning model correctly classified 82.4% of migraine patients from healthy subjects. Within the migraine group, 74.1% of the episodic migraine-probable migraine patients and 90.5% of the chronic migraine patients were accurately classified. No significant difference was found between probable migraine and episodic migraine patients in terms of the periaqueductal gray region radiomics features. The kNN algorithm showed the best performance for classifying episodic migraine-probable migraine subtypes, while the Random Forest algorithm demonstrated the best performance for classifying the migraine group and chronic migraine subtype.

A radiomics-based machine learning model, utilizing standard MR images obtained during the diagnosis and follow-up of migraine patients, shows promise not only in aiding migraine diagnosis and classification for clinical approach, but also in understanding the neurological mechanisms underlying migraines. 

Premotor projections from the locus coeruleus and periaqueductal grey are altered in two rat models with inborn differences in emotional behavior.

Emotionally motivated behaviors rely on the coordinated activity of descending neural circuits involved in motor and autonomic functions. Using a pseudorabies (PRV) tract-tracing approach in typically behaving rats, our group previously identified descending premotor, presympathetic, and dual-labeled premotor-presympathetic populations throughout the central rostral-caudal axis. The premotor-presympathetic populations are thought to integrate somatomotor and sympathetic activity. To determine whether these circuits are dysregulated in subjects with altered emotional regulation, subsequent neuroanatomical analyses were performed in male subjects of two distinct genetic models relevant to clinical depression and anxiety: the Wistar Kyoto (WKY) rat and selectively bred Low Novelty Responder (bLR) rat. The present study explored alterations in premotor efferents from locus coeruleus (LC) and subdivisions of the periaqueductal grey (PAG), two areas involved in emotionally motivated behaviors. Compared to Sprague Dawley rats, WKY rats had significantly fewer premotor projections to hindlimb skeletal muscle from the LC and from the dorsomedial (DMPAG), lateral (LPAG), and ventrolateral (VLPAG) subdivisions of PAG. Relative to selectively bred High Novelty Responder (bHR) rats, bLR rats had significantly fewer premotor efferents from LC and dorsolateral PAG (DLPAG). Cumulatively, these results demonstrate that somatomotor circuitry in several brain areas involved in responses to stress and emotional stimuli are altered in rat models with depression-relevant phenotypes. These somatomotor circuit differences could be implicated in motor-related impairments in clinically depressed patients.

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.

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.

The deconstruction of chronic orofacial pain and a hiding inhibition pathway of orofacial pain: the trigeminal proprioceptive mesencephalic periaqueductal gray pathway.

Chronic orofacial pain has a significant negative impact that influences individuals' quality of life and our society. The prevalence is around 11.2% to 33.2% and remains high in females. Currently, there are two main diagnostic classification systems that are used internationally for chronic pain: the International Classification of Diseases, 11th Revision (ICD-11), which was published by the World Health Organization (WHO) in 2018, and the International Classification of Orofacial Pain, which was published by the International Association for the Study of Pain (IASP) in 2020. Deficits in ascending and descending pain modulation pathways may be involved in the chronic pain pathophysiology. A newly described "trigeminal proprioceptive mesencephalic periaqueductal gray pathway" is considered to be the mechanism of action of occlusal appliance in managing orofacial pain. The genetic basis of chronic orofacial pain is not yet fully understood, but a genetic susceptibility involving multiple genes among the peripheral nerves, brainstem and higher brain regions to regulate and suppress the transmission of pain signals, thereby modulating the perception of pain, is likely.

Low-Frequency Stimulation of Trpv1-Lineage Peripheral Afferents Potentiates the Excitability of Spino-Periaqueductal Gray Projection Neurons.

High-threshold dorsal root ganglion (HT DRG) neurons fire at low frequencies during inflammatory injury, and low-frequency stimulation (LFS) of HT DRG neurons selectively potentiates excitatory synapses onto spinal neurons projecting to the periaqueductal gray (spino-PAG). Here, in male and female mice, we have identified an underlying peripheral sensory population driving this plasticity and its effects on the output of spino-PAG neurons. We provide the first evidence that Trpv1-lineage sensory neurons predominantly induce burst firing, a unique mode of neuronal activity, in lamina I spino-PAG projection neurons. We modeled inflammatory injury by optogenetically stimulating Trpv1+ primary afferents at 2 Hz for 2 min (LFS), as peripheral inflammation induces 1-2 Hz firing in high-threshold C fibers. LFS of Trpv1+ afferents enhanced the synaptically evoked and intrinsic excitability of spino-PAG projection neurons, eliciting a stable increase in the number of action potentials (APs) within a Trpv1+ fiber-induced burst, while decreasing the intrinsic AP threshold and increasing the membrane resistance. Further experiments revealed that this plasticity required Trpv1+ afferent input, postsynaptic G protein-coupled signaling, and NMDA receptor activation. Intriguingly, an inflammatory injury and heat exposure in vivo also increased APs per burst, in vitro These results suggest that inflammatory injury-mediated plasticity is driven though Trpv1+ DRG neurons and amplifies the spino-PAG pathway. Spinal inputs to the PAG could play an integral role in its modulation of heat sensation during peripheral inflammation, warranting further exploration of the organization and function of these neural pathways.

Alamandine injection in the periaqueductal gray and rostral ventromedial medulla attenuates allodynia induced by sciatic nerve ligation in rats.

Alamandine, a peptide known to interact with Mas-related G protein-coupled receptor subtype D (MrgD), has been implicated in moderating inflammatory signals. MrgD receptors are abundantly found in pain transmission pathways, but the role of alamandine/MrgD in pain modulation has not been thoroughly explored. This study aimed to investigate the effects of alamandine (10, 40, and 100 pmol) in a rat model of allodynia induced by sciatic nerve ligation, with a specific focus on examining the involvement of MrgD receptors, NMDAR1, and serotonin transporter (SERT) in the ventrolateral periaqueductal gray (vlPAG) and rostral ventromedial medulla (RVM). Microinjection of alamandine into the vlPAG at a dose of 100 pmol and into the RVM at doses of 40 and 100 pmol resulted in a significant increase in paw withdrawal threshold (PWT). Additionally, co-administration of D-Pro7-Ang-(1-7) at 50 pmol, an MrgD receptor antagonist, effectively blocked the analgesic effects of alamandine. Immunofluorescence analysis confirmed the presence of MrgD receptors in both the vlPAG and RVM regions. Importantly, an upregulation of MrgD receptor expression was observed following allodynia induction, suggesting a potential compensatory mechanism in response to pain. Our findings support the co-localization of MrgD receptors with NMDAR1 in vlPAG neurons, suggesting their ability to initiate analgesic pathways similar to those activated by NMDA receptors in the vlPAG. Furthermore, our results underscore a significant co-localization of MrgD receptors with the SERT in the RVM, underscoring their potential impact on serotonergic neurons involved in promoting analgesic effects.

The anterior cingulate cortex and its interface with the dorsal periaqueductal grey regulating nitric oxide-mediated panic-like behaviour and defensive antinociception.

The anterior cingulate cortex (ACC) Cg1 (24b) area modulates glutamate-mediated unconditioned fear and antinociception organised by hypothalamus. However, it remains unknown whether 24b area also modulates these latter defensive responses through connections with the dorsal periaqueductal grey matter (dPAG), a midbrain structure implicated in the genesis of innate fear-induced defence. The aim of this work is to examine the correlation between the behavioural effects of intra-ACC microinjections of vehicle, NMDA (1 nmol) or lidocaine (2%) with Fos protein expression and nitrergic activity in the dPAG of male C57BL/6 mice that were threatened by snakes. In addition, the 24b area-dPAG pathways were also characterised by neural tract tracing procedures. Finally, the effect of dPAG pretreatment with the neuronal nitric oxide synthase inhibitor N(omega)-propyl-l-arginine (NPLA; 0.2, 0.4 or 0.8 nmol) 10 min before 24b area treatment with NMDA on behavioural and nociceptive responses of threatened mice was studied. The activation of 24b area N-methyl-d-aspartic acid receptors facilitated escape and freezing rather than risk assessment, and enhanced Fos expression and nitrite levels in dPAG, while lidocaine decreased escape and risk assessment as well as Fos and nitrergic activity in dPAG. In addition, dPAG pretreatment with NPLA suppressed intra-24b NMDA-facilitated panicogenic effects while increased nociception. Infusions of an antegrade neurotracer into 24b area showed axonal fibres surrounding both dorsomedial and dorsolateral PAG perikarya. Neurons were identified in 24b area after deposits of a retrograde neurotracer into dPAG. Our findings suggest that the ACC/24b area modulates innate defensive responses through the recruitment of dPAG nitrergic neurons.

Copaifera langsdorffii Desf. tree oleoresin-induced antinociception recruits µ1- and κ -opioid receptors in the ventrolateral columns of the periaqueductal gray matter.

Popular medicine has been using oleoresin from several species of copaíba tree for the treatment of various diseases and its clinical administration potentially causes antinociception. Electrical stimulation of ventrolateral (vlPAG) and dorsolateral (dlPAG) columns of the periaqueductal gray matter also causes antinociception. The aim this study was to verify the antinociceptive effect of oleoresin extracted from Copaifera langsdorffii tree and to test the hypothesis that oleoresin-induced antinociception is mediated by µ1- and κ-opioid receptors in the vlPAG and dlPAG. Nociceptive thresholds were determined by the tail-flick test in Wistar rats. The copaíba tree oleoresin was administered at different doses (50, 100 and 200 mg/kg) through the gavage technique. After the specification of the most effective dose of copaíba tree oleoresin (200 mg/kg), rats were pretreated with either the µ1-opioid receptor selective antagonist naloxonazine (at 0.05, 0.5 and 5 µg/ 0.2 µl in vlPAG, and 5 µg/ 0.2 µl in dlPAG) or the κ-opioid receptor selective antagonist nor-binaltorphimine (at 1, 3 and 9 nmol/ 0.2 µl in vlPAG, and 9 nmol/ 0.2 µl in dlPAG). The blockade of µ1 and κ opioid receptors of vlPAG decreased the antinociception produced by copaíba tree oleoresin. However, the blockade of these receptors in dlPAG did not alter copaíba tree oleoresin-induced antinociception. These data suggest that vlPAG µ1 and κ opioid receptors are critically recruited in the antinociceptive effect produced by oleoresin extracted from Copaifera langsdorffii.

PAC1 receptor modulation of freezing and flight behavior in periaqueductal gray.

The midbrain periaqueductal gray (PAG) region is a critical anatomical regulator of fear-related species-specific defensive reactions (SSDRs). Pituitary adenylate-cyclase-activating polypeptide (PACAP), and its main receptor PAC1, play an important role in fear-related behavior and anxiety disorders. However, the function of the PACAP-PAC1 system within the PAG with regards to SSDRs has received little attention. To address this gap, we used transgenic PAC1flox/flox mice to examine both conditional and unconditional defensive reactions. We performed conditional PAC1 gene deletion within the ventrolateral(vl)PAG of PAC1flox/flox mice using an adeno-associated virus (AAV) coding for Cre recombinase. Following viral expression, we used a white noise fear conditioning preparation that produces both an unconditional activity burst to the onset of noise that is followed by conditional freezing. On Day 1, mice received five white noise foot-shock pairings, whereas on Day 2, they were exposed to white noise five times without shock and we scored the activity burst and freezing to the white noise. Following behavioral testing, histology for immunofluorescent analysis was conducted in order to identify PACAP positive cells and stress-induced c-fos activity respectively. We found that PAC1 deletion in vlPAG increased the unconditional activity burst response but disrupted conditional freezing. PAC1 deletion was accompanied by higher c-fos activity following the behavioral experiments. Furthermore, a significant portion of PACAP-EGFP positive cells showed overlapping expression with VGAT, indicating their association with inhibitory neurons. The findings suggested that intact PACAP-PAC1 mechanisms are essential for SSDRs in vlPAG. Therefore, midbrain PACAP contributes to the underlying molecular mechanisms regulating fear responses.

Play behavior: Tickle and play in the periaqueductal gray.

A new study has identified the periaqueductal gray as an important brain region for play and tickle behavior in rats.

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.