Fos-like immunoreactivity in the periaqueductal gray of rats exposed to a natural predator

In the present study we examined, in rats exposed to a predator (cat), the distribution of neurons expressing Fos along the continuum formed by the central gray surrounding the caudal pole of the third ventricle and the periaqueductal gray (PAG). After the predatory encounter, a distinct cluster of Fos-immunoreactive cells was observed in the precommissural nucleus. In the rostral two-thirds of the PAG, Fos expression was mostly seen in the dorsomedial and dorsolateral regions. In contrast, at caudal levels of the PAG, most of the Fos-labelled neurons were distributed in the lateral and ventrolateral PAG. These results are discussed and compared with the pattern of the PAG activation after fear conditioned to a context or elicited by aversive foot shock.     

Fos-like immunoreactive neurons following electrical stimulation of the dorsal periaqueductal gray at freezing and escape thresholds

Electrical stimulation of the dorsal regions of the periaqueductal gray (PAG) leads to defensive reactions characterized as freezing and escape responses. Until recently it was thought that this freezing behavior could be due to the recruitment of neural circuits in the ventrolateral periaqueductal gray (vlPAG), while escape would be mediated by other pathways. Nowadays, this view has been changing mainly because of evidence that freezing and escape behaviors thus elicited are not altered after lesions of the vlPAG. It has been suggested that there are at least two pathways for periaqueductal gray-mediated defensive responses, one involving the hypothalamus and the cuneiform nucleus (CnF) which mediates responses to immediate danger and another one involving the amygdala and vlPAG which mediates cue-elicited responses, either learned or innate. To examine this issue further we measured Fos protein expression in brain areas activated by electrical stimulation of the dorsolateral PAG (dlPAG) at the freezing and escape thresholds. The data obtained showed that freezing-provoking stimulation caused increases in Fos expression in the dorsomedial PAG (dmPAG), while escape-provoking stimulation led to increases at both dmPAG and dlPAG. Surprisingly, neither escape- nor freezing-provoking stimulations altered Fos expression in the central nucleus of amygdala (CeA). Escape-provoking stimulation caused increased Fos expression in the ventromedial hypothalamus (VMH), dorsal premammilary nucleus (PMd) and in the cuneiform nucleus. Significant increases in Fos labeling were found in the dmPAG and PMd following freezing-provoking stimulation. Therefore, the present data support the notion of a neural segregation for defensive behaviors in the dorsal columns of PAG, with increased Fos expression in the dmPAG following freezing, while dlPAG is affected by both freezing and escape responses. dlPAG, CnF, VMH and PMd are part of a brain aversion network activated by fear unconditioned stimuli. The present data also suggests that the defensive responses generated at the dlPAG level do not recruit the neural circuits of the vlPAG and CeA usually activated by conditioned fear stimuli.     

Differential effects of NK1 receptors in the midbrain periaqueductal gray upon defensive rage and predatory attack in the cat

This study utilized anatomical and behavioral-pharmacological methods to determine the role of NK(1)-Substance P receptors in the midbrain periaqueductal gray (PAG) in defensive rage behavior in cats. For behavioral pharmacological experiments, monopolar stimulating electrodes were implanted in the medial hypothalamus for elicitation of defensive rage behavior and cannula-electrodes were implanted in the PAG for microinjections of receptor compounds. Microinjections of the NMDA antagonist, AP-7 (2 nmol), into the dorsal PAG blocked defensive rage elicited by medial hypothalamic stimulation, thus establishing the PAG as a synaptic region that receives hypothalamic inputs linked to defensive rage behavior. Microinjections of the NK(1) agonist, GR73632, into the same injection sites facilitated defensive rage in a dose-dependent manner, and also induced spontaneous hissing in five cats. The effects of GR73632 were reduced by pretreatment of the PAG with the NK(1) antagonist, GR82334 (16 nmol), microinjected into the same sites. Microinjections of GR73632 (8 nmol) into the PAG also suppressed predatory attack elicited by stimulation of the lateral hypothalamus. Immunohistochemical methods utilized to detect Substance P and Fos immunoreactivity revealed that neurons in the PAG activated after defensive rage-inducing medial hypothalamic stimulation lie in the same region as Substance-P-immunoreactive processes. Fos immunoreactivity was highest in the dorsomedial aspect of the rostral PAG after medial hypothalamic stimulation. Cats that were unstimulated or that exhibited predatory attack after lateral hypothalamic stimulation had low c-fos expression levels in the PAG. Substance P immunoreactivity was high throughout the dorsal PAG. The results indicate that NK(1) receptors in the PAG potentiate defensive rage and suppress predatory aggression in the cat.     

Hypothalamic sites responding to predator threats--the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.     

Sensing danger through the olfactory system: the role of the hypothalamic dorsal premammillary nucleus

The dorsal premammillary nucleus (PMd) has a critical role on the expression of defensive responses to predator odor. Anatomical evidence suggests that the PMd should also modulate memory processing through a projecting branch to the anterior thalamus. By using a pharmacological blockade of the PMd with the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), we were able to confirm its role in the expression of unconditioned defensive responses, and further revealed that the nucleus is also involved in influencing associative mechanisms linking predatory threats to the related context. We have also tested whether olfactory fear conditioning, using coffee odor as CS, would be useful to model predator odor. Similar to cat odor, shock-paired coffee odor produced robust defensive behavior during exposure to the odor and to the associated context. Shock-paired coffee odor also up-regulated Fos expression in the PMd, and, as with cat odor, we showed that this nucleus is involved in the conditioned defensive responses to the shock-paired coffee odor and the contextual responses to the associated environment.     

Severe Reduction of Rat Defensive Behavior to a Predator by Discrete Hypothalamic Chemical Lesions

Nonspecific lesion and stimulation methods have suggested that the hypothalamus is critical for the expression of defensive behavior, although the organization of neural circuits mediating such behavior is unclear. In the rat hypothalamus, we found that increased Fos levels were restricted to specific cell groups following presentation of a stimulus (predator) known to elicit partly innate defensive responses. The dorsal premammillary nucleus showed the most striking increase in Fos levels, and cell body-specific chemical lesions therein virtually eliminated two major components of defensive behavior but increased exploratory behavior, suggesting that this caudal hypothalamic nucleus plays a critical role in the expression of behavioral responses sometimes critical for survival of the individual. We have previously shown that the Fos-responsive cell groups in the medial hypothalamus are interconnected in a neural system distinct from those mediating reproductive and ingestive behaviors.     

Fos expression induced by changes in arterial pressure is localized in distinct,longitudinally organized columns of neurons in the rat midbrain periaqueductal gray

The distribution of neurons expressing Fos within the periaqueductal gray (FAG) following pharmacologically induced high or low blood pressure was examined to determine (1) if PAG neurons are responsive to changes in arterial pressure (AP) and (2) the relationship of these cells to the functionally defined hypertensive and hypotensive columns in PAG. Changes in AP differentially induced robust Fos expression in neurons confined to discrete, longitudinally organized columns within PAG. Increased AP produced extensive Fos-like immunoreactivity within the lateral PAG, beginning at the level of the oculomotor nucleus. At the level of the dorsal raphe, Fos expression induced by increased AP shifted dorsally, into the dorsolateral division of PAG; this pattern of Fos labeling was maintained throughout the caudal one-third of PAG. Double-labeling for Fos and nicotinamide adenine dinucleotide phosphate diaphorase confirmed that Fos-positive cells induced by increased AP were located in the dorsolateral division of PAG at these caudal levels. Fos positive cells were codistributed, but not colocalized, with nicotinamide adenine dinucleotide phosphate diaphorase-positive cells. Decreased AP evoked a completely different pattern of Fos expression. Fos-positive cells were predominantly located within the ventrolateral PAG region, extending from the level of the trochlear nucleus through the level of the caudal dorsal raphe. Double-labeling studies for Fos and serotonin indicated that only 1–2 double-labeled cells per section were present. Saline infusion resulted in very few Foslike immunoreactive cells, indicating that volume receptor activation does not account for Fos expression in PAG evoked by changes in AP. These results indicate that (1) substantial numbers of PAG neurons are excited by pharmacologically induced changes in AP and (2) excitatory barosensitive PAG neurons are anatomically segregated based on their responsiveness to a specific directional change in AP. © 1995 Wiley-Liss, Inc.     

Nucleus retroambiguus projections to the periaqueductal gray in the cat

The nucleus retroambiguus (NRA) of the caudal medulla is a relay nucleus by which neurons of the mesencephalic periaqueductal gray (PAG) reach motoneurons of pharynx, larynx, soft palate, intercostal and abdominal muscles, and several muscles of the hindlimbs. These PAG-NRA-motoneuronal projections are thought to play a role in survival behaviors, such as vocalization and mating behavior. In the present combined antero- and retrograde tracing study in the cat, we sought to determine whether the NRA, apart from the neurons projecting to motoneurons, also contains cells projecting back to the PAG. After injections of WGA-HRP in the caudal and intermediate PAG, labeled neurons were observed in the NRA, with a slight contralateral preponderance. In contrast, after injections in the rostral PAG or adjacent deep tectal layers, no or very few labeled neurons were present in the NRA. After injection of [(3)H]leucine in the NRA, anterograde labeling was present in the most caudal ventrolateral and dorsolateral PAG, and slightly more rostrally in the lateral PAG, mainly contralaterally. When the [(3)H]leucine injection site extended medially into the medullary lateral tegmental field, labeling was found in most parts of the PAG as well as in the adjoining deep tectal layers. No labeled fibers were found in the dorsolateral PAG, and only a few were found in the rostral PAG. Because the termination pattern of the NRA fibers in the PAG overlaps with that of the sacral cord projections to the PAG, it is suggested that the NRA-PAG projections play a role in the control of motor functions related to mating behavior.     

Neural activation during cat odor-induced conditioned fear and 'trial 2' fear in rats

Exposure to cat odor, an innate threat stimulus for rats, engages a conditioning process whereby the environment in which the odor was experienced comes to elicit fear. Additionally, response to cat odor appears to change with repeated exposure, with benzodiazepines having an anxiolytic effect upon first, but not second, cat odor exposure. We explored the neural correlates of these two phenomena using Fos immunohistochemistry. Rats were exposed to cat odor (a worn cat collar) and were allowed to hide from this stimulus. A ‘trial 1’ group was perfused after a single exposure, and a ‘trial 2’ group after two exposures. A ‘context’ group was exposed to cat odor once, then perfused after re-exposure to the odor-paired context. Trial 1, trial 2 and context groups showed similar defensive responses including avoidance and hiding. The trial 1 group showed Fos expression in limbic, hypothalamic and brainstem regions associated with defensive behavior. The trial 2 group showed a similar pattern although with less activation in the lateral septum, anterior and ventromedial hypothalamus, and dorsolateral periaqueductal gray. The context-exposed group showed Fos expression in a subset of the regions activated by cat odor itself: the dorsal premammillary nucleus, ventrolateral periaqueductal grey, cuneiform nucleus and locus ceruleus. Little activation was seen in the amygdala or hippocampus. These results show that stimuli associated with predatory threat come to activate similar brain regions to the threat stimulus itself.     

A role for the periaqueductal grey in opioidergic inhibition of maternal behaviour

Opiates are known to be involved in the regulation of various events surrounding parturition and lactation, such as maternal behaviour in rats. The onset of this behaviour has been closely linked to opiate action in the medial pre-optic area, where administration of morphine disrupts maternal behaviour during lactation. By combining the use of Fos protein immunohistochemical detection and pharmacological manipulations, in the present paper we show that the periaqueductal grey (PAG) is another region critically involved in the opioidergic blockade of maternal behaviour. According to our observations, a critical level of morphine-induced activation of the rostral lateral PAG appears to be required to inhibit maternal behaviour in lactating rats. This hypothesis was further confirmed in experiments showing that morphine's inhibitory effect on maternal responsiveness was blocked by unilateral naloxone injection into the rostral PAG, but not into nearby regions of the mesencephalic reticular nucleus. Therefore, only a partial inhibition of the opiate's effect on the rostral PAG was needed to block the inhibitory effect of morphine on maternal behaviour. Further studies are needed to ascertain whether the rostral lateral PAG plays a role in the natural onset of maternal behaviour, playing a complementary role to the medial pre-optic area, or merely inhibits maternal behaviour in response to this specific pharmacological challenge. Conversely, the present findings may well reflect a more general role of the PAG, seemingly providing an important piece of information for proposing a hitherto unexplored concept of the PAG as an important centre for the selection of adaptive behavioural responses.     

Chemical bladder irritation provokes c-fos expression in the midbrain periaqueductal gray matter of the rat

We investigated the topographical localization of c-fos expression in the midbrain periaqueductal gray matter (PAG) to detect nociception-induced neural activity in the PAG. In conscious female Wistar rats, c-fos expression was induced by continuous intravesical infusion of saline or 0.1% acetic acid. Number of c-fos protein (Fos)-positive cells was counted at each coronal section of the PAG as well as Barrington's nucleus. Fos-positive cells were also counted at L1 and L6 of the spinal cord, where most of the hypogastric and pelvic nerve afferent terminals project, respectively. Compared with saline infusion, acetic acid infusion provoked irritative bladder responses characterized by a marked increase in the frequency of bladder contractions, and induced a significant increase in the number of Fos-positive cells in both L1 and L6 of the spinal cord. Following acetic acid infusion, there was a significant increase in the number of Fos-positive cells in all coronal sections of the PAG compared with saline infusion, especially in the caudal part of the PAG. The increase in the number of Fos-positive cells was mainly observed in the ventrolateral and lateral parts of the caudal PAG, and in the dorsal part of the rostral PAG. However, there was no difference in the number of Fos-positive cells in Barrington's nucleus between saline and acetic acid infusion. In conclusion, nociception induced by chemical bladder irritation influences neural activity in the PAG. Implication of topographical difference in Fos expression in the PAG and its relevance to changes in bladder function remain to be elucidated.     

Viscerotopic control of regional vascular beds by discrete groups of neurons within the midbrain periaqueductal gray

It is well established that a group of bulbospinal neurons within the rostral ventrolateral medulla plays a crucial role in the tonic and phasic control of arterial pressure. In the cat, these neurons are confined to a discrete region which has been termed the subretrofacial (SRF) nucleus. Recent evidence suggests that this nucleus is viscerotopically organized with respect to its control over different vascular beds. These observations raise the question as to whether functionally different subgroups of SRF pressor neurons receive inputs from supramedullary cell groups that also exert a specific control over particular vascular beds. To answer this question retrogradely transported tracers (i.e. rhodamine or fluorescein-labelled microspheres, wheat germ agglutinin-horseradish peroxidase) were injected into physiologically identified sites within the rostral or caudal parts of the SRF nucleus of the cat. Separate groups of neurons in the midbrain periaqueductal gray region (PAG) were found to project specifically to subgroups of cells within the rostral and caudal parts of the SRF nucleus. These findings, together with the results of recent functional studies of the PAG suggest that these distinct projections from the PAG to the SRF nucleus are involved in the expression of different patterns of emotionally coupled cardiovascular responses.     

Phosphorylated cyclic AMP response element binding protein expression induced in the periaqueductal gray by predator stress: its relationship to the stress experience, behavior and limbic neural plasticity

Electrophysiological studies in cats and recently in rats implicate neuroplasticity in the periaqueductal gray (PAG) and its afferents in stressor-induced increases in fearful behavior and anxiety-like behavior (ALB). Such increases may model aspects of affective changes following traumatic stress in humans. The present study explored the role of neuroplasticity in PAG and its connection with the central nucleus of the amygdala (ACE) in male rodent anxiety-like response to predator stress. In the first of two studies, the effects of predator stress on the induction of phosphorylated cyclic AMP response element binding protein (pCREB) were investigated. pCREB expression in the PAG and ventromedial hypothalamus (VMH) was examined immunohistochemically. Predator stress increased the degree of pCREB expression in PAG cells (measured densitometrically) but did not increase the number of cells expressing pCREB (measured stereologically). Moreover, predator stress-specific increase in pCREB-like immunoreactivity (lir) was restricted to the right lateral column of the PAG. In addition, pCREB lir in the right lateral column likely reflects aspects of the stress experience because the stressor (cat behavior) and the response to the stressor (rat defensive behavior) are highly predictive of degree of pCREB expression. There was no effect of predator stress on pCREB lir in the VMH.

Because pCREB expression has been associated with long-lasting potentiation (LLP) of neural transmission, we examined the effects of predator stress on transmission in the ACE-PAG pathway in a second study. Predator stress elevated evoked potential measures of ACE-PAG transmission in the right hemisphere but not in the left hemisphere 11–12 days after predator stress. This finding is consistent with the longer-lived effects of pharmacological stress on amygdalo-PAG transmission in the right hemisphere but not in the left hemisphere in cats. Of interest is the fact that the same aspects of the stressor experience and reaction to it, which are predictive of the degree of pCREB expression, are also highly predictive of the degree of potentiation of measures of ACE-PAG transmission. Behavioral analyses revealed that the most consistent effects of predator stress are on behavior in the plus maze (open arm exploration and risk assessment) and on startle. In addition, covariance analysis suggests that ACE-PAG potentiation mediates some but not all of the changes in ALB produced by predator stress. Because pCREB expression may be a precursor to neuroplastic changes in certain forms of memory and LLP, the present findings complement studies in the cat, showing that neuroplastic changes in the PAG underlie changes in affect following stress. Furthermore, these findings suggest that neuroplastic changes in PAG may be important mediators of predator stress-induced changes in affective behavior in rodents. Finally, consistent with cat and human studies, the right hemisphere appears particularly important in long-term response to stress.     

Connections of the precommissural nucleus.

The connections of the precomissural nucleus (PRC) have been examined with anterograde and retrograde axonal tracing methods in the rat. Experiments with cholera toxin B subunit (CTb) indicate that the PRC shares a number of common afferent sources with the dorsolateral periaqueductal gray (PAG). Thus, we have shown that the nucleus receives substantial inputs from the prefrontal cortex, specific domains of the rostral part of the lateral septal nucleus, rostral zona incerta, perifornical region, anterior hypothalamic nucleus, ventromedial hypothalamic nucleus, dorsal premammillary nucleus, medial regions of the intermediate and deep layers of the superior colliculus, and cuneiform nucleus. Moreover, the PRC also receives inputs from several PAG regions and from neural sites involved in the control of attentive or motivational state, including the laterodorsal tegemental nucleus and the ventral tegmental area. The efferent projections of the PRC were analyzed by using the Phaseolus vulgaris-leucoagglutinin (PHA-L) method. Notably, the PRC presents a projection pattern that resembles in many ways the pattern described previously for the rostral dorsolateral PAG in addition to projections to a number of targets that also are innervated by neighboring pretectal nuclei, including the rostrodorsomedial part of the lateral dorsal thalamic nucleus, the ventral part of the lateral geniculate complex, the medial pretectal nucleus, the nucleus of the posterior commissure, and the ventrolateral part of the subcuneiform reticular nucleus. Overall, the results suggest that the PRC might be viewed as a rostral component of the PAG, and the possible functional significance of the nucleus is discussed in terms of its connections.     

Estrous cycle stage influences on neuronal responsiveness to repeated anxiogenic stress in female rats

Experiments were carried out to investigate (i) whether estrous cycle stage influences nociceptive responsiveness to anxiogenic stress and (ii) whether prior experience of the stressor modifies the response. Exposure to mild anxiogenic vibration stress evoked hyperalgesia, reflected by a decrease in tail flick latency, only in animals in the late diestrus phase. Stress evoked hyperalgesia in late diestrus regardless of whether the rat was experiencing the stress for the first time or had been exposed to the stress previously, when in another cycle stage. Whilst the behavioral response to stress appeared to be determined exclusively by estrous cycle stage, the stress-evoked pattern of Fos expression in the periaqueductal grey matter (PAG) depended not only on cycle stage but also on whether the rat had previous experience of the stress. First exposure to stress in late diestrus evoked a 50% decrease in Fos expression compared to non-stressed controls, particularly in the lateral and dorsolateral sectors of the rostral PAG. In contrast, in experienced rats in late diestrus the pattern of Fos expression increased up to 4-fold, particularly in the ventral half of the caudal PAG but also in the lateral and dorsolateral parts. At other cycle stages Fos expression was not changed except for an increase in rats in proestrus. The results suggest that in females, changes in gonadal hormone levels during the estrous cycle impact significantly on the processing of fear-inducing stimuli by the PAG. These hormonal influences may also influence how the PAG responds to a subsequent anxiogenic challenge.     

Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator

Innate fear stimulus induces activation of neurons containing the neuronal nitric oxide synthase enzyme (nNOS) in defensive‐related brain regions such as the dorsolateral periaqueductal gray (dlPAG). Intra‐dlPAG administration of nitric oxide synthase (NOS) inhibitors and glutamate antagonists induce anxiolytic‐like responses. We investigated the involvement of nitric oxide (NO) and glutamate neurotransmission in defensive reactions modulated by dlPAG. We tested if intra‐dlPAG injections of the selective nNOS inhibitor, N‐propyl‐L ‐arginine (NP), or the glutamate antagonist, AP7 (2‐amino‐7‐phosphonoheptanoic acid), would attenuate behavioral responses and cellular activation induced by predator exposure (cat). Fos‐like immunoreactivity (FLI) was used as a marker of neuronal functional activation, whereas nNOS immunohistochemistry was used to identify NOS neurons. Cat exposure induced fear responses and an increase of FLI in the dlPAG and dorsal premammillary nucleus (PMd). NP and AP7 attenuated the cat‐induced behavioral responses. Whereas NP tended to attenuate FLI in the dlPAG, AP7 induced a significant reduction in cellular activation of this region. The latter drug, however, increased FLI and double‐labeled cells in the PMd. Cellular activation of this region was significantly correlated with time spent near the cat (r = 0.7597 and 0.6057 for FLI and double‐labeled cells). These results suggest that glutamate/NO‐mediated neurotransmission in the dlPAG plays an important role in responses elicit by predator exposure. Blocking these neurotransmitter systems in this brain area impairs defensive responses. The longer time spent near the predator that follows AP7 effect could lead to an increased cellular activation of the PMd, a more rostral brain area that has also been related to defensive responses. © 2009 Wiley‐Liss, Inc.     

Localization of the neurons active during paradoxical (REM) sleep and projecting to the locus coeruleus noradrenergic neurons in the rat

Locus coeruleus (LC) noradrenergic neurons are active during wakefulness, slow their discharge rate during slow wave sleep, and stop firing during paradoxical sleep (PS). A large body of data indicates that their inactivation during PS is due to a tonic GABAergic inhibition. To localize the neurons responsible for such inhibition, we first examined the distribution of retrogradely and Fos double-immunostained neurons following cholera toxin b subunit (CTb) injection in the LC of control rats, rats selectively deprived of PS for 3 days, and rats allowed to recover for 3 hours from such deprivation. We found a significant number of CTb/Fos double-labeled cells only in the recovery group. The largest number of CTb/Fos double-labeled cells was found in the dorsal paragigantocellular reticular nucleus (DPGi). It indeed contained 19% of the CTb/Fos double-labeled neurons, whereas the ventrolateral periaqueductal gray (vlPAG) contained 18.3% of these neurons, the lateral paragigantocellular reticular nucleus (LPGi) 15%, the lateral hypothalamic area 9%, the lateral PAG 6.7%, and the rostral PAG 6%. In addition, CTb/Fos double-labeled cells constituted 43% of all the singly CTb-labeled cells counted in the DPGi compared with 29% for the LPGi, 18% for the rostral PAG, and 10% or less for the other structures. Although all these populations of CTb/Fos double-labeled neurons could be GABAergic and tonically inhibit LC neurons during PS, our results indicate that neurons from the DPGi constitute the best candidate for this role.     

Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat.

Previous anatomical and physiological studies have revealed a substantial projection from the periaqueductal gray (PAG) to the nucleus paragigantocellularis (PGi). In addition, physiological studies have indicated that the PAG is composed of functionally distinct subregions. However, projections from PAG subregions to PGi have not been comprehensively examined. In the present study, we sought to examine possible topographic specificity for projections from subregions of the PAG to PGi. Pressure or iontophoretic injections of wheat germ agglutinin-conjugated horseradish peroxidase, or of Fluoro-Gold, placed into the PGi of the rat retrogradely labeled a substantial number of neurons in the PAG from the level of the Edinger-Westphal nucleus to the caudal midbrain. Retrogradely labeled neurons were preferentially aggregated in distinct subregions of the PAG. Rostrally, at the level of the oculomotor nucleus, labeled neurons were i) compactly aggregated in the ventromedial portion of the PAG corresponding closely to the supraoculomotor nucleus of the central gray, ii) in the lateral and ventrolateral PAG, and iii) in medial dorsal PAG. More caudally, retrogradely labeled neurons became less numerous in the dorsomedial PAG but were more widely scattered throughout the lateral and ventrolateral parts of the PAG. Only few retrogradely labeled neurons were found in the ventromedial part of the PAG at caudal levels. Injections of retrograde tracers restricted to subregions of the PGi suggested topography for afferents from the PAG. Injections into the lateral portion of the PGi yielded the greatest number of labeled neurons within the rostral ventromedial PAG. Medially placed injections yielded numerous retrogradely labeled neurons in the lateral and ventrolateral PAG. Injections placed in the rostral pole of the PGi (medial to the facial nucleus) produced the greatest number of retrogradely labeled neurons in the dorsal PAG. To examine the pathways taken by fibers projecting from PAG neurons to the medulla, and to further specify the topography for the terminations of these afferents in the PGi, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoretically deposited into subregions of the PAG that contained retrogradely labeled neurons in the above experiments. These results revealed distinct fiber pathways to the rostral medulla that arise from the dorsal, lateral/ventrolateral, and ventromedial parts of the PAG. These injections also showed that there are differential but overlapping innervation patterns within the PGi. Consistent with the retrograde tracing results, injections into the rostral ventromedial PAG near the supraoculomotor nucleus yielded anterograde labeling immediately ventral to the nucleus ambiguus in the ventrolateral medulla, within the retrofacial portion of the PGi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chronic tooth pulp inflammation causes transient and persistent expression of Fos in dynorphin-rich regions of rat brainstem

We have analyzed central Fos immunoreactivity (Fos-IR) brainstems of adult rats after three clinically relevant dental injuries: filled dentin (DF) cavities that cause mild pulp injury and heal within 1-2 weeks; open pulp exposures (PX) that cause gradual pulp loss and subsequent periodontal lesions; and filled pulp exposures (PXF). By 1 week after DF cavities, no Fos-IR remained except for sites such as lateral-ventral periolivary nucleus (LVPO) that had Fos-IR in all rats including controls. PX injury induced (1) a delayed transient expression of Fos at 1-2 weeks at three loci (ipsilateral neurons in dorsomedial nucleus oralis, paratrigeminal nucleus, and trigeminal tract), (2) persistent ipsilateral Fos for at least 4 weeks after injury in dynorphin (Dyn)-rich regions (rostral lateral solitary nucleus, periobex dorsal nucleus caudalis), and (3) late Fos-IR at 2-4 weeks (bilateral superficial cervical dorsal horn, contralateral dorsal nucleus caudalis, contralateral rostral lateral solitary nucleus). Rats with PXF injury were examined at 2 weeks, and they had greater numbers and more extensive rostro-caudal distribution of Fos neurons than the PX group. One week after PX injury, Fos-IR neurons were found in regions with strong Dyn-IR central fibers. Co-expression of Dyn and Fos was found in some unusually large neurons of the ipsilateral rostral lateral solitary nucleus, trigeminal tract, and dorsal nucleus caudalis. Immunocytochemistry for the p75 low affinity neurotrophin receptor (p75NTR) or for calcitonin gene-related peptide (CGRP) showed no consistent change in trigeminal central endings in any Fos-reactive brainstem areas, despite the extensive structural and cytochemical reorganization of the peripheral endings of the dental neurons. The Fos responses of central neurons to tooth injury have some unusual temporal and spatial patterns in adult rats compared to other trigeminal injury models.     

Periaqueductal gray matter projections to midline and intralaminar thalamic nuclei of the rat

The periaqueductal gray matter (PAG) projections to the intralaminar and midline thalamic nuclei were examined in rats. Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected in discrete regions of the PAG, and axonal labeling was examined in the thalamus. PHA-L was also placed into the dorsal raphe nuclei or nucleus of Darkschewitsch and interstitial nucleus of Cajal as controls. In a separate group of rats, the retrograde tracer cholera toxin beta-subunit (CTb) was injected into one of the intralaminar thalamic nuclei-lateral parafascicular, medial parafascicular, central lateral (CL), paracentral (PC), or central medial nucleus-or one of the midline thalamic nuclei-paraventricular (PVT), intermediodorsal (IMD), mediodorsal, paratenial, rhomboid (Rh), reuniens (Re), or caudal ventral medial (VMc) nucleus. The distribution of CTb labeled neurons in the PAG was then mapped. All PAG regions (the four columns of the caudal two-thirds of the PAG plus rostral PAG) and the precommissural nucleus projected to the rostral PVT, IMD, and CL. The ventrolateral, lateral, and rostral PAG provided additional inputs to most of the other intralaminar and midline thalamic nuclei. PAG inputs to the VMc originated from the rostral and ventrolateral PAG areas. In addition, the lateral and rostral PAG projected to the zona incerta. No evidence was found for a PAG input to the ventroposterior lateral parvicellular, ventroposterior medial parvicellular, caudal PC, oval paracentral, and reticular thalamic nuclei. PAG --> thalamic circuits may modulate autonomic-, nociceptive-, and behavior-related forebrain circuits associated with defense and emotional responses.