Lateral Periaqueductal Gray Research Articles

A hypothalamus-lateral periaqueductal gray GABAergic neural projection facilitates arousal following sevoflurane anesthesia in mice.

The lateral hypothalamus (LHA) is an evolutionarily conserved structure that regulates basic functions of an organism, particularly wakefulness. To clarify the function of LHAGABA neurons and their projections on regulating general anesthesia is crucial for understanding the excitatory and inhibitory effects of anesthetics on the brain. The aim of the present study is to investigate whether LHAGABA neurons play either an inhibitory or a facilitatory role in sevoflurane-induced anesthetic arousal regulation. We used fiber photometry and immunofluorescence staining to monitor changes in neuronal activity during sevoflurane anesthesia. Opto-/chemogenetic modulations were employed to study the effect of neurocircuit modulations during the anesthesia. Anterograde tracing was used to identify a GABAergic projection from the LHA to a periaqueductal gray (PAG) subregion. c-Fos staining showed that LHAGABA activity was inhibited by induction of sevoflurane anesthesia. Anterograde tracing revealed that LHAGABA neurons project to multiple arousal-associated brain areas, with the lateral periaqueductal gray (LPAG) being one of the dense projection areas. Optogenetic experiments showed that activation of LHAGABA neurons and their downstream target LPAG reduced the burst suppression ratio (BSR) during continuous sevoflurane anesthesia. Chemogenetic experiments showed that activation of LHAGABA and its projection to LPAG neurons prolonged the anesthetic induction time and promoted wakefulness. In summary, we show that an inhibitory projection from LHAGABA to LPAGGABA neurons promotes arousal from sevoflurane-induced loss of consciousness, suggesting a complex control of wakefulness through intimate interactions between long-range connections.

Brainstem nuclei responsive to cystometry in both endometriosis and cystitis rat models: C-fos immunohistochemistry study.

Although the co-occurrence of interstitial cystitis (IC) and endometriosis (ENDO) is remarkably high, the exact pathophysiology for this co-occurrence is unknown. The convergence of the inputs from the involved structures to the same neuronal centers may suggest neuronal hyperexcitability as a mechanism for this co-occurrence. The present study aimed to investigate the association between IC and ENDO, by studying the changes in brainstem responses to cystometry in a rat model of ENDO and cyclophosphamide (CYP)-induced IC using c-fos immunohistochemistry. Following cystometry the brainstem areas that had significant increase in c-fos expression in ENDO alone included: periaqueductal gray (PAG) nuclei, dorsal raphe nucleus, raphe obscurus nucleus, kolliker- Fuse areas, and area postrema. However, the brainstem areas that had increased significantly in the c-fos expression in the ENDO and CYP treated animals included: gigantocellular nucleus, lateral paragigantocellular nucleus, caudoventrolateral nucleus, rostroventrolateral/caudoventrolateral nucleus, lateral reticular nucleus, locus coeruleus, lateral PAG, raphe pallidus nucleus, raphe magnus nucleus, rostroventrolateral nucleus, dorsal motor nucleus of vagus, and solitary tract nucleus. Whereas only lateral parabrachial nucleus showed significant increase in c-fos expression in CYP treated animals alone. The results of the present study demonstrate the overlap of brainstem nuclei that are excited by urinary bladder under ENDO and IC conditions. The pattern of hyperexcitability of the brainstem nuclei may help in understating the pathophysiology of IC and ENDO conditions.

Whole-brain monosynaptic inputs to lateral periaqueductal gray glutamatergic neurons in mice.

The lateral periaqueductal gray (LPAG), which mainly contains glutamatergic neurons, plays an important role in social responses, pain, and offensive and defensive behaviors. Currently, the whole-brain monosynaptic inputs to LPAG glutamatergic neurons are unknown. This study aims to explore the structural framework of the underlying neural mechanisms of LPAG glutamatergic neurons. This study used retrograde tracing systems based on the rabies virus, Cre-LoxP technology, and immunofluorescence analysis. We found that 59 nuclei projected monosynaptic inputs to the LPAG glutamatergic neurons. In addition, seven hypothalamic nuclei, namely the lateral hypothalamic area (LH), lateral preoptic area (LPO), substantia innominata (SI), medial preoptic area, ventral pallidum, posterior hypothalamic area, and lateral globus pallidus, projected most densely to the LPAG glutamatergic neurons. Notably, we discovered through further immunofluorescence analysis that the inputs to the LPAG glutamatergic neurons were colocalized with several markers related to important neurological functions associated with physiological behaviors. The LPAG glutamatergic neurons received dense projections from the hypothalamus, especially nuclei such as LH, LPO, and SI. The input neurons were colocalized with several markers of physiological behaviors, which show the pivotal role of glutamatergic neurons in the physiological behaviors regulation by LPAG.

Periaqueductal gray matter and medial prefrontal cortex reflect negative prediction errors during differential conditioning.

Computational models of associative learning posit that negative prediction errors (PEs) arising from the omission of aversive outcomes weaken aversive Pavlovian associations during differential conditioning and extinction. It is possible that negative PEs may underlie exaggerated conditioned responses to the conditioned stimulus not paired with an aversitve outcome (CS-) during differential conditioning and to the conditioned stimulus originally paired with a aversive outcome (CS+) during extinction in patients with clinical anxiety disorders. Although previous research has demonstrated that manipulations of the periaqueductal gray matter (PAG) interfere with extinction learning in animals, the role of the PAG in processing negative PEs within the human brain is presently unclear. We set out to investigate how PAG responses and connectivity are impacted by negative PEs using ultra-high-field (7 T) functional magnetic resonance imaging and hierarchical Bayesian analysis. During differential conditioning, negative PEs were associated with larger responses within the lateral and dorsolateral PAG and increased connectivity between the dorsolateral PAG and medial areas of Brodmann area 9. Collectively, these results shed light on the association between activity within the PAG and medial prefrontal cortex and the omission of aversive outcomes during Pavlovian learning.

Disruption of Periaqueductal Gray-default Mode Network Functional Connectivity in Patients with Crohn's Disease with Abdominal Pain

Abdominal pain in Crohn's disease (CD) has been known to be associated with changes in the central nervous system. The periaqueductal gray (PAG) plays a well-established role in pain processing. However, the role of PAG-related network and the effect of pain on the network in CD remain unclear.Resting-state functional magnetic imaging (fMRI) data were collected from 24 CD patients in remission with abdominal pain, 24 CD patients without abdominal pain and 28 healthy controls (HCs). Using the subregions of PAG (dorsomedial (dmPAG), dorsolateral (dlPAG), lateral (lPAG) and ventrolateral (vlPAG)) as seeds, the seed-based FC maps were calculated and one-way analysis of variance (ANOVA) was performed to investigate the differences among the three groups.Results showed that the group differences were mainly involved in the FC of the vlPAG with the precuneus, medial prefrontal cortex (mPFC) as well as orbitofrontal cortex (OFC), and the FC of the right lateral PAG (lPAG) with the precuneus, inferior parietal lobule (IPL), angular gyrus and premotor cortex. The FC values of all these regions decreased successively in the order of HCs, CD without abdominal pain and CD with abdominal pain. The pain score was negatively correlated with the FC of the l/vlPAG with the precuneus, angular gyrus and mPFC in CD patients with abdominal pain.This study implicated the disrupt communication between the PAG and the default mode network (DMN). These findings complemented neuroimaging evidence for the pathophysiology of visceral pain in CD patients.

Study on the central neural pathway and the relationship between the heart and small intestine via a dual neural tracer.

Despite very different functions, studies increasingly report that there may be a potential central nervous anatomical connection between the heart and the small intestine. In this study, the central nervous anatomical relationship between the heart and small intestine was studied via a viral tracer. Pseudorabies virus (PRV) syngeneic strains with different fluorescent reporter genes (eGFP or mRFP) were microinjected into the heart walls and small intestinal walls of male C57BL/6J using glass microelectrode. The results showed that the co-labeled nuclei in the brain were lateral periaqueductal gray (LPAG) and ventrolateral periaqueductal gray (VLPAG) in the midbrain, mesencephalic trigeminal nucleus (Me5), and motor trigeminal nucleus anterior digastric Part (5Adi) in the pons. The co-labeled sites in the spinal cord were intermediolateral column (IML) in the second thoracic vertebra, IML and lamina 7 of the spinal gray (7SP) in the third thoracic vertebra, and IML in the fourth thoracic vertebra. Our data show that there is a neuroanatomical connection between the small intestine and the heart in the central nervous system (CNS). Neuroanatomical integration of the heart and small intestine may provide a basis for revealing the physiological and pathological interactions between the circulatory and digestive systems. The interactions may be mediated more effectively through sympathetic nerves.

Regional hypothalamic, amygdala, and midbrain periaqueductal gray matter recruitment during acute pain in awake humans: A 7-Tesla functional magnetic resonance imaging study

Over the past two decades, magnetic resonance imaging (MRI) studies have explored brain activation patterns during acute noxious stimuli. Whilst these human investigations have detailed changes in primarily cortical regions, they have generally not explored discrete changes within small brain areas that are critical in driving behavioural, autonomic, and endocrine responses to pain, such as within subregions of the hypothalamus, amygdala, and midbrain periaqueductal gray matter (PAG). Ultra-high field (7-Tesla) MRI provides enough signal-to-noise at high spatial resolutions to investigate activation patterns within these small brain regions during acute noxious stimulation in awake humans. In this study we used 7T functional MRI to concentrate on hypothalamic, amygdala, and PAG signal changes during acute noxious orofacial stimuli. Noxious heat stimuli were applied in three separate fMRI scans to three adjacent sites on the face in 16 healthy control participants (7 females). Images were processed using SPM12 and custom software, and blood oxygen level dependent signal changes within the hypothalamus, amygdala, and PAG assessed. We identified altered activity within eight unique subregions of the hypothalamus, four unique subregions of the amygdala, and a single region in the lateral PAG. Specifically, within the hypothalamus and amygdala, signal intensity largely decreased during noxious stimulation, and increased in the lateral PAG. Furthermore, we found sex-related differences in discrete regions of the hypothalamus and amygdala. This study reveals that the activity of discrete nuclei during acute noxious thermal stimulation in awake humans.

Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization.

During threat exposure, survival depends on defensive reactions. Prior works linked large glutamatergic populations in the midbrain periaqueductal gray (PAG) to defensive freezing and flight, and established that the overarching functional organization axis of the PAG is along anatomically-defined columns. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains diverse cell types that vary in neurochemistry. How these cell types contribute to defense remains unknown, indicating that targeting sparse, genetically-defined populations may reveal how the PAG generates diverse behaviors. Though prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found in mice that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (CCK) cells selectively caused flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-CCK cells reduced predator avoidance without altering other defensive behaviors like freezing. Lastly, l/vlPAG-CCK activity decreased when approaching threat and increased during movement to safer locations. These results suggest CCK cells drive threat avoidance states, which are epochs during which mice increase distance from threat and perform evasive escape. Conversely, l/vlPAG pan-neuronal activation promoted freezing, and these cells were activated near threat. Thus, CCK l/vlPAG cells have opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar boundaries. In addition to the anatomical columnar architecture of the PAG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.

Impaired visceral pain-related functions of the midbrain periaqueductal gray in rats with colitis

The midbrain periaqueductal gray (PAG) is a key structure involved in the supraspinal modulation of pain. Previous studies have reported the association of gut inflammation-triggered chronic abdominal pain with structural and neuronal alterations within the PAG. However, whether PAG-executed visceral nociception processing and descending modulation are altered in gut pathology is not known. We used c-Fos immunohistochemistry and extracellular microelectrode recording in urethane-anesthetized male Wistar rats to evaluate the colitis-induced changes in visceral pain-related neuronal properties of the PAG and its descending outflow to visceral nociceptive neurons of the caudal ventrolateral medulla (CVLM). Analysis of c-Fos protein expression in inflamed animals has shown diminished activation of the lateral and ventrolateral PAG columns by noxious colorectal distension (CRD), although the nonstimulated c-Fos labeling in these PAG subdivisions was enhanced compared with that in controls. Microelectrode recording in the ventrolateral PAG revealed a colitis-elicited decrease in the proportion of CRD-excited neurons accompanied by an increase in the number of unresponsive cells and weakened reactions to the stimulation of CRD-inhibited PAG units. Colonic inflammation has also been found to cause a shift in the effects of ventrolateral PAG electrostimulation on CRD-excited CVLM neurons from being mostly inhibitory under normal conditions to excitatory in colitis. These findings identify impaired PAG functioning in ascending and descending visceral nociception control that may contribute to gut injury-associated visceral hyperalgesia. The data obtained can benefit a better understanding of the supraspinal mechanisms involved in the pathogenesis of postinflammatory chronic abdominal pain.

Periaqueductal gray neurons encode the sequential motor program in hunting behavior of mice

Sequential encoding of motor programs is essential for behavior generation. However, whether it is critical for instinctive behavior is still largely unknown. Mouse hunting behavior typically contains a sequential motor program, including the prey search, chase, attack, and consumption. Here, we reveal that the neuronal activity in the lateral periaqueductal gray (LPAG) follows a sequential pattern and is time-locked to different hunting actions. Optrode recordings and photoinhibition demonstrate that LPAGVgat neurons are required for the prey detection, chase and attack, while LPAGVglut2 neurons are selectively required for the attack. Ablation of inputs that could trigger hunting, including the central amygdala, the lateral hypothalamus, and the zona incerta, interrupts the activity sequence pattern and substantially impairs hunting actions. Therefore, our findings reveal that periaqueductal gray neuronal ensembles encode the sequential hunting motor program, which might provide a framework for decoding complex instinctive behaviors.

Evidence that increased cholecystokinin (CCK) in the periaqueductal gray (PAG) facilitates changes in Resident–Intruder social interactions triggered by peripheral nerve injury

Individual differences in the effects of a chronic neuropathic injury on social behaviours characterize both the human experience and pre-clinical animal models. The impacts of these changes to the well-being of the individual are often underappreciated. Earlier work from our laboratory using GeneChip® microarrays identified increased cholecystokinin (CCK) gene expression in the periaqueductal gray (PAG) of rats that showed persistent changes in social interactions during a Resident-Intruder encounter following sciatic nerve chronic constriction injury (CCI). In this study, we confirmed these gene regulation patterns using RT-PCR and identified the anatomical location of the CCK-mRNA as well as the translated CCK peptides in the midbrains of rats with a CCI. We found that rats with persistent CCI-induced changes in social behaviours had increased CCK-mRNA in neurons of the ventrolateral PAG and dorsal raphe nuclei, as well as increased CCK-8 peptide expression in terminal boutons located in the lateral and ventrolateral PAG. The functional significance of these changes was explored by microinjecting small volumes of CCK-8 into the PAG of uninjured rats and observing their Resident-Intruder social interactions. Disturbances to social interactions identical to those observed in CCI rats were evoked when injection sites were located in the rostral lateral and ventrolateral PAG. We suggest that CCI-induced changes in CCK expression in these PAG regions contributes to the disruptions to social behaviours experienced by a subset of individuals with neuropathic injury.

Reciprocal connectivity of the periaqueductal gray with the ponto-medullary respiratory network in rat

Synaptic activities of the periaqueductal gray (PAG) can modulate or appropriate the respiratory motor activities in the context of behavior and emotion via descending projections to nucleus retroambiguus. However, alternative anatomical pathways for the mediation of PAG-evoked respiratory modulation via core nuclei of the brainstem respiratory network remains only partially described. We injected the retrograde tracer Cholera toxin subunit B (CT-B) in the pontine Kölliker-Fuse nucleus (KFn, n = 5), medullary Bötzinger (BötC, n = 3) and pre-Bötzinger complexes (pre-BötC; n = 3), and the caudal raphé nuclei (n = 3), and quantified the descending connectivity of the PAG targeting these brainstem respiratory regions. CT-B injections in the KFn, pre-BötC, and caudal raphé, but not in the BötC, resulted in CT-B-labeled neurons that were predominantly located in the lateral and ventrolateral PAG columns. In turn, CT-B injections in the lateral and ventrolateral PAG columns (n = 4) produced the highest numbers of CT-B-labeled neurons in the KFn and far fewer numbers of labeled neurons in the pre-BötC, BötC, and caudal raphé. Analysis of the relative projection strength revealed that the KFn shares the densest reciprocal connectivity with the PAG (ventrolateral and lateral columns, in particular). Overall, our data imply that the PAG may engage a distributed respiratory rhythm and pattern generating network beyond the nucleus retroambiguus to mediate downstream modulation of breathing. However, the reciprocal connectivity of the KFn and PAG suggests specific roles for synaptic interaction between these two nuclei that are most likely related to the regulation of upper airway patency during vocalization or other volitional orofacial behaviors.

The blockade of D1/D2-like dopamine receptors in the lateral periaqueductal gray region affects morphine self-administration with and without exercise in rats

Introduction: The periaqueductal gray (PAG) region plays an essential role in the modulation of nociception. Also, lateral PAG (lPAG) is involved in reward circuitry by the dopaminergic system in addiction. The present study investigated the blockade of D1/D2-like dopamine receptors in the lateral PAG region affects morphine self-administration with and without exercise. Methods: Rats were divided into six groups. The rats were initially trained to receive small pellets of food by pressing an active lever in the self administration apparatus. Exercise groups were run on a treadmill at 20m/min, 5 days/week, for 4 weeks before the surgery. Then rats were bilaterally implanted with cannulae in lPAG. The SCH23390 and sulpiride were microinjected into the lPAG, 5min before receiving morphine. Afterward, the animals were allowed to self administer morphine in 2h sessions over 11 consecutive days. At last, the numbers of lever pressing, infusion times and withdrawal symptoms were measured. Results: The results showed the number of active lever pressing was significantly increased in the morphine group compared to other groups in self-infusion during 11 days. Exercise significantly reversed the detrimental effects of morphine self-administration after five days. However, the synergistic effect of injected sulpiride into the lPAG region with exercise training was more pronounced on the amelioration of morphine than on the combinatory effect of SCH23390 with exercise. Conclusion: The findings suggested that the D2 dopamine receptor in the lPAG region was involved in the morphine addiction via the dopaminergic system and exercise training in combination with antagonists could reduce the rewarding properties of morphine.

Input-output connections of LJA5 prodynorphin neurons.

Sensory information is transmitted from peripheral nerves, through the spinal cord, and up to the brain. Sensory information may be modulated by projections from the brain to the spinal cord, but the neural substrates for top‐down sensory control are incompletely understood. We identified a novel population of inhibitory neurons in the mouse brainstem, distinguished by their expression of prodynorphin, which we named LJA5. Here, we identify a similar group of Pdyn+ neurons in the human brainstem, and we define the efferent and afferent projection patterns of LJA5 neurons in mouse. Using specific genetic tools, we selectively traced the projections of the Pdyn‐expressing LJA5 neurons through the brain and spinal cord. Terminal fields were densest in the lateral and ventrolateral periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), caudal pressor area, and lamina I of the spinal trigeminal nucleus and all levels of the spinal cord. We then labeled cell types in the PAG, LPB, medulla, and spinal cord to better define the specific targets of LJA5 boutons. LJA5 neurons send the only known inhibitory descending projection specifically to lamina I of the spinal cord, which transmits afferent pain, temperature, and itch information up to the brain. Using retrograde tracing, we found LJA5 neurons receive inputs from sensory and stress areas such as somatosensory/insular cortex, preoptic area, paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus, PAG, and LPB. This pattern of inputs and outputs suggest LJA5 neurons are uniquely positioned to be activated by sensation and stress, and in turn, inhibit pain and itch.

Descending forebrain projections targeting respiratory control areas in the midbrain and brainstem of rats.

Breathing can be voluntarily modulated via descending inputs from the forebrain to evoke respiratory‐related behaviours, such as vocalization, sniffing, swallowing or breath‐holding. Such behaviors require controlled laryngeal adduction and thus, are conducted during the post‐inspiratory phase of respiratory cycle. However, descending pathways that connect forebrain regions with primary post‐inspiratory control areas such as the pontine Kölliker‐Fuse nucleus (KF) and the medullary Bötzinger complex (BötC) remain to be identified. Here, we investigated the topography of forebrain descending projection neurons to a variety of bulbar respiratory nuclei.We locally microinjected the conventional retrograde tracer cholera toxin subunit B (CT‐B, 100–150nL) into the BötC, KF, the pre‐Bötzinger complex (pre‐BötC), the midline raphé nuclei and the midbrain periaqueductal gray (PAG). Twelve days after unilateral CT‐B injections, brains were sectioned (40μm) and immunohistochemically stained with an anti‐CT‐B antibody. The strength of descending projections was qualitatively assessed: as strong (+++), moderate (++) or weak (+) numbers of CT‐B labeled cell bodies.Retrogradely labelled neurons after unilateral injections into the lateral PAG confirmed the predominantly ipsilateral location of strong and moderate descending projection neurons in the cingulate (+++), pre‐limbic (+++), ectorhinal (++), motor (+++) and insular (++) cortices, the lateral septum (++), amygdala (+++) and hypothalamus (+++). In comparison, retrogradely labeled neurons after unilateral KF injection were also found ipsilaterally in the motor (++), prelimbic (++) and insular cortices (+++), the amygdala (++) and hypothalamus (+++). However, amongst all analysed descending target areas, only the KF receives substantial inputs from the ectorhinal (+++) and endopiriform (++) cortices. In addition, the medullary BötC receives weaker inputs from prelimbic (+) and insular (+) cortices and receives moderate inputs from the amygdala and hypothalamus. Descending projection neurons to the pre‐BötC were in accordance with the literature: motor (+) and insular (+) cortices, amygdala (+++) and hypothalamus (++). Finally, descending inputs to the medullary raphé obscurus and raphé magnus nuclei also arose from motor, prelimbic and insular cortices, amygdala and hypothalamus. However, these projections were significantly weaker compared to KF or PAG.The results suggest that descending forebrain projections into respiratory control areas are organized in general pathways that originate from motor, prelimbic and insular cortices as well as the amygdala and hypothalamus. However, only the KF, a key area for the gating of post‐inspiratory activity and respiratory plasticity, receives projections arising from the endopiriform and ectorhinal cortex. The functional implications of these descending control pathways need to be explored in future studies.Support or Funding InformationMelbourne Research Scholarship (University of Melbourne) [181858] to PT‐B.

Single prolonged stress alters neural activation in the periacqueductal gray and midline thalamic nuclei during emotional learning and memory.

Clinical and preclinical studies that have examined the neurobiology of persistent fear memory in posttraumatic stress disorder (PTSD) have focused on the medial prefrontal cortex, hippocampus, and amygdala. Sensory systems, the periaqueductal gray (PAG), and midline thalamic nuclei have been implicated in fear and extinction memory, but whether neural activity in these substrates is sensitive to traumatic stress (at baseline or during emotional learning and memory) remains unexplored. To address this, we used the single prolonged stress (SPS) model of traumatic stress. SPS and control rats were either subjected to fear conditioning (CS-fear) or presented with CSs alone (CS-only) during fear conditioning. All rats were then subjected to extinction training and testing. A subset of rats were euthanized after each behavioral stage and c-Fos and c-Jun used to measure neural activation in all substrates. SPS lowered c-Jun levels in the dorsomedial and lateral PAG at baseline, but the elevated c-Jun expression in the PAG during emotional learning and memory. SPS also altered c-Fos expression during fear and extinction learning/memory in midline thalamic nuclei. These findings suggest changes in neural function in the PAG and midline thalamic nuclei could contribute to persistent fear memory induced by traumatic stress. Interestingly, SPS effects were also observed in animals that never learned fear or extinction (i.e., CS-only). This raises the possibility that traumatic stress could have broader effects on the psychological function that are dependent on the PAG and midline thalamic nuclei.

Functional Involvement of Human Periaqueductal Gray and Other Midbrain Nuclei in Cognitive Control.

Recent theoretical advances have motivated the hypothesis that the periaqueductal gray (PAG) participates in behaviors that involve changes in the autonomic control of visceromotor activity, including during cognitively demanding tasks. We used ultra-high-field (7 tesla) fMRI to measure human brain activity at 1.1 mm resolution while participants completed a working memory task. Consistent with prior work, participants were less accurate and responded more slowly with increasing memory load-signs of increasing task difficulty. Whole-brain fMRI analysis revealed increased activity in multiple cortical areas with increasing working memory load, including frontal and parietal cortex, dorsal cingulate, supplementary motor area, and anterior insula. Several dopamine-rich midbrain nuclei, such as the substantia nigra and ventral tegmental area, also exhibited load-dependent increases in activation. To investigate PAG involvement during cognitive engagement, we developed an automated method for segmenting and spatially normalizing the PAG. Analyses using cross-validated linear support vector machines showed that the PAG discriminated high versus low working memory load conditions with 95% accuracy in individual subjects based on activity increases in lateral and ventrolateral PAG. Effect sizes in the PAG were comparable in magnitude to those in many of the cortical areas. These findings suggest that cognitive control is not only associated with cortical activity in the frontal and parietal lobes, but also with increased activity in the subcortical PAG and other midbrain regions involved in the regulation of autonomic nervous system function.SIGNIFICANCE STATEMENT Functional neuroimaging in humans has shown that cognitive control engages multiple corticostriatal networks and brainstem nuclei, but theoretical advances suggest that the periaqueductal gray (PAG) should also be engaged during cognitively demanding tasks. Recent advances in ultra-high-field fMRI provided an opportunity to obtain the first evidence that increased activation of intermediate and rostral portions of lateral and ventrolateral PAG columns in humans is modulated by cognitive load. These findings suggest that cognitive control is not solely mediated by activity in the cortex, but that midbrain structures important for autonomic regulation also play a crucial role in higher-order cognition.

Larger Numbers of Glial and Neuronal Cells in the Periaqueductal Gray Matter of μ-Opioid Receptor Knockout Mice

Background: μ-opioid receptor knockout (MOP-KO) mice display baseline hyperalgesia. We have recently identified changes in tissue volume in the periaqueductal gray matter (PAG) using magnetic resonance imaging voxel-based morphometry. Changes in the structure and connectivity of this region might account for some behavior phenotypes in MOP-KO mice, including hyperalgesia.Methods: Adult male MOP-KO and wild-type (WT) mice were studied. Immunohistochemistry was performed to detect microglia, astrocytes, and neurons in the PAG using specific markers: ionized calcium-binding adaptor molecule 1 (Iba-1) for microglia, glial fibrillary acidic protein (GFAP) for astrocytes, and the neuronal nuclei antigen (NeuN; product of the Rbfox3 gene) for neurons, respectively. Cell counting was performed in the four parallel longitudinal columns of the PAG (dorsomedial, dorsolateral, lateral, and ventrolateral) at three different locations from bregma (−3.5, −4.0, and −4.5 mm).Results: The quantitative analysis showed larger numbers of well-distributed Iba1-IR cells (microglia), NeuN-IR cells (neurons), and GFAP-IR areas (astrocytes) at all the anatomically distinct regions examined, namely, the dorsomedial (DM) PAG, dorsolateral (DL) PAG, lateral (L) PAG, and ventrolateral (VL) PAG, in MOP-KO mice than in control mice.Conclusions: The cellular changes in the PAG identified in this paper may underlie aspects of the behavioral alterations produced by MOP receptor deletion, and suggest that alterations in the cellular structure of the PAG may contribute to hyperalgesic states.

Anatomical projections of the dorsomedial hypothalamus to the periaqueductal grey and their role in thermoregulation: a cautionary note.

The DMH is known to regulate brown adipose tissue (BAT) thermogenesis via projections to sympathetic premotor neurons in the raphe pallidus, but there is evidence that the periaqueductal gray (PAG) is also an important relay in the descending pathways regulating thermogenesis. The anatomical projections from the DMH to the PAG subdivisions and their function are largely elusive, and may differ per anterior–posterior level from bregma. We here aimed to investigate the anatomical projections from the DMH to the PAG along the entire anterior–posterior axis of the PAG, and to study the role of these projections in thermogenesis in Wistar rats. Anterograde channel rhodopsin viral tracing showed that the DMH projects especially to the dorsal and lateral PAG. Retrograde rabies viral tracing confirmed this, but also indicated that the PAG receives a diffuse input from the DMH and adjacent hypothalamic subregions. We aimed to study the role of the identified DMH to PAG projections in thermogenesis in conscious rats by specifically activating them using a combination of canine adenovirus‐2 (CAV2Cre) and Cre‐dependent designer receptor exclusively activated by designer drugs (DREADD) technology. Chemogenetic activation of DMH to PAG projections increased BAT temperature and core body temperature, but we cannot exclude the possibility that at least some thermogenic effects were mediated by adjacent hypothalamic subregions due to difficulties in specifically targeting the DMH and distinct subdivisions of the PAG because of diffuse virus expression. To conclude, our study shows the complexity of the anatomical and functional connection between the hypothalamus and the PAG, and some technical challenges in studying their connection.

Brief optogenetic inhibition of rat lateral or ventrolateral periaqueductal gray augments the acquisition of Pavlovian fear conditioning.

The midbrain periaqueductal gray (PAG) coordinates the expression and topography of defensive behaviors to threat and also plays an important role in Pavlovian fear learning itself. Whereas the role of PAG in the expression of defensive behavior is well understood, the relationship between the activity of PAG neurons and fear learning, the exact timing of PAG contributions to learning during the conditioning trial, and the contributions of different PAG columns to fear learning are poorly understood. We assessed the effects of optogenetic inhibition of lateral (LPAG) and ventrolateral PAG (VLPAG) neurons on fear learning. Using adenoassociated viral vectors expressing halorhodopsin, we show that brief optogenetic inhibition of LPAG or VLPAG during delivery of the shock unconditioned stimulus (US) augments acquisition of contextual or cued fear conditioning, and we also show that this inhibition augments postencounter defensive responses to a nonnoxious threat. Taken together, these results show that LPAG and VLPAG serve a key role in the regulation of Pavlovian fear learning at the time of US delivery. These findings provide strong support for existing models that state that LPAG and VLPAG contribute to a fear prediction error signal determining variations in the effectiveness of the aversive US in supporting learning. (PsycINFO Database Record