Hypovolemic shock: critical involvement of a projection from the ventrolateral periaqueductal gray to the caudal midline medulla.

Previous research has suggested that the ventrolateral column of the periaqueductal gray (vlPAG) plays a crucial role in triggering a decompensatory response (sympathoinhibition, hypotension, bradycardia) to severe blood loss. vlPAG excitation triggers also quiescence, decreased vigilance and decreased reactivity, the behavioral response which usually accompanies hypovolemic shock. The aim of this study was to identify, in unanesthetized rats, the main descending pathway(s) via which vlPAG neurons trigger sympathoinhibition and bradycardia in response to severe blood loss. Firstly, immediate early gene (c-Fos) expression was used to identify vlPAG neurons selectively activated by severe blood loss. Subsequently, the specific medullary projections of these vlPAG neurons were defined by combined c-Fos, retrograde tracing (double-label) experiments. It was found that vlPAG neurons selectively activated by severe hemorrhage project overwhelmingly to the vasodepressor portion of the caudal midline medulla (CMM). Previous studies indicate that this CMM region mediates behaviorally-coupled cardiovascular adjustments and the findings described here fit with the idea that CMM neurons are uniquely recruited by salient challenges, the adaptive responses to which require more than reflexive homeostatic cardiovascular adjustments.

Enhanced Fos expression in glutamic acid decarboxylase immunoreactive neurons of the mouse periaqueductal grey during opioid withdrawal.

Previous studies using c-Fos immunohistochemistry suggest that a sub-population of neurons in the midbrain periaqueductal gray region is activated during opioid withdrawal. The neurochemical identity of these cells is unknown but cellular physiological studies have implicated GABAergic neurons. The present study investigated whether GABAergic neurons are activated in the mouse periaqueductal gray during opioid withdrawal using dual-antibody immunohistochemistry for Fos and glutamic acid decarboxylase. Both chronic opioid treatment and naloxone-precipitated opioid withdrawal increased Fos expression in the periaqueductal gray, with the greatest increase being four-fold in the caudal ventrolateral subdivision following withdrawal. Neurons stained for both Fos and glutamic acid decarboxylase were greatly enhanced in all subdivisions of the periaqueductal gray following withdrawal, particularly in the lateral and ventrolateral divisions where the increase was up to 70-fold. These results suggest that activation of a subpopulation of GABAergic interneurons in the periaqueductal gray plays a role in opioid withdrawal.

Genetic blockade of the dopamine D3 receptor enhances hippocampal expression of PACAP and receptors and alters their cortical distribution.

Dopamine D3 receptors (D3Rs) are implicated in several aspects of cognition, but their role in aversive conditioning has only been marginally uncovered. Investigations have reported that blockade of D3Rs enhances the acquisition of fear memories, a phenomenon tightly linked to the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP). However, the impact of D3R ablation on the PACAPergic system in regions critical for the formation of new memories remains unexplored. To address this issue, levels of PACAP and its receptors were compared in the hippocampus and cerebral cortex (CX) of mice devoid of functional D3Rs (D3R(-/-)) and wild-types (WTs) using a series of comparative immunohistochemical and biochemical analyses. Morphometric and stereological data revealed increased hippocampal area and volume in D3R(-/-) mice, and augmented neuronal density in CA1 and CA2/3 subfields. PACAP levels were increased in the hippocampus of D3R(-/-) mice. Expression of PACAP receptors was also heightened in mutant mice. In the CX, PACAP immunoreactivity (IR), was restricted to cortical layer V in WTs, but was distributed throughout layers IV-VI in D3R(-/-) mice, along with increased mRNAs, protein concentration and staining scores. Consistently, PAC1, VPAC1 and VPAC2 IRs were variably redistributed in CX, with a general upregulation in cortical layers II-IV in knockout animals. Our interpretation of these findings is that disturbed dopamine neurotransmission due to genetic D3R blockade may enhance the PACAP/PAC1-VPAC axis, a key endogenous system for the processing of fear memories. This could explain, at least in part, the facilitated acquisition and consolidation of aversive memories in D3R(-/-) mice.

Parallel circuits mediating distinct emotional coping reactions to different types of stress.

All animals, including humans, react with distinct emotional coping strategies to different types of stress. Active coping strategies (e.g. confrontation, fight, escape) are evoked if the stressor is controllable or escapable. Passive coping strategies (e.g. quiescence, immobility, decreased responsiveness to the environment) are usually elicited if the stressor is inescapable and help to facilitate recovery and healing. Neural substrates mediating active versus passive emotional coping have been identified within distinct, longitudinal neuronal columns of the midbrain periaqueductal gray (PAG) region. Active coping is evoked by activation of either the dorsolateral or lateral columns of the PAG; whereas passive coping is triggered by activation of the ventrolateral PAG. Recent anatomical studies indicate that each PAG column receives a distinctive set of ascending (spinal and medullary) and descending (prefrontal cortical and hypothalamic) afferents. Consistent with the anatomy, functional studies using immediate early gene expression (c-fos) as a marker of neuronal activation have revealed that the preferential activation of a specific PAG column reflects (i) the type of emotional coping reaction triggered, and (ii) whether a physical or psychological stressor was used.

Spatiotemporal pattern of ontogenetic expression of calbindin-28/kD in the retinorecipient layers of rat superior colliculus.

Using an antibody against calbindin-28kD, we have studied the spatial pattern of expression of this protein in the superior colliculi (SC) of four strains of mature laboratory rats. In all four strains, calbindin-expressing cells (CECs) formed horizontally oriented tiers in the retinorecipient and intermediate gray layers but were diffusely distributed throughout the deep layers. Ontogenetically, calbindin-28kD was expressed for the first time in the retinorecipient layers at postconceptional day 20 (PCD 20), by cells located in the rostrolateral region where the first born retinal ganglion cells (RGCs) are represented. Although on the day of birth (PCD 22/23), the CECs were distributed more widely, they were still absent in the most medial part of the SC, that is, the region where the latest born RGCs are represented. The spatial distribution of CECs became adultlike only by PCD 29, that is, at the end of the period of the naturally occurring death of the RGCs. Monocular eye enucleations on PCD 23 prevented the expression of calbindin in the medial fifth of the retinorecipient layers of the contralateral SC, while the unilateral removal of the visual cortices had no discernable effect on the numbers and distribution of the CECs in either SC. Thus, the spatiotemporal pattern of ontogenetic expression of calbindin-28kD in the retinorecipient layers of SC reflects the spatiotemporal pattern of generation of the RGCs, and the retinal input appears to induce neuronal expression of calbindin-28kD in these layers.

Common patterns of increased and decreased fos expression in midbrain and pons evoked by noxious deep somatic and noxious visceral manipulations in the rat.

Immunohistochemical detection of the protein product (Fos) of the c-fos immediate early gene was used to study neuronal activation in the rostral pons and midbrain of halothane-anesthetised rats following noxious deep somatic or noxious visceral stimulation. In animals exposed only to halothane anesthesia, Fos-like immunoreactive (IR) neurons were located in the midbrain periaqueductal gray matter, tectum, and parabrachial nucleus. Following noxious stimulation of hindlimb muscle, knee joint, vagal cardiopulmonary, or peritoneal nociceptors, there was, compared to halothane-only animals, a significant increase in the numbers of Fos-like (IR) cells in the caudal ventrolateral periaqueductal gray and the intermediate gray lamina of the superior colliculus. Given the general agreement that increased Fos expression is a consequence of increased neuronal activity, the finding that a range of noxious deep somatic and noxious visceral stimuli evoked increased neuronal activity in a discrete, caudal ventrolateral periaqueductal gray region is consistent with previous suggestions that this region is an integrator of deep noxious evoked reactions. The noxious deep somatic and noxious visceral manipulations also evoked, compared to halothane-only animals, reductions in the numbers of Fos-like IR cells in the stratum opticum of the superior colliculus and the unlaminated portion of the external subnucleus of the inferior colliculus. To our knowledge this is the first report of reductions in Fos-expression in the tectum evoked by noxious stimulation. In separate experiments, the effects of noxious deep somatic and noxious visceral manipulations on arterial pressure and heart rate were measured. The noxious visceral manipulations evoked substantial and sustained falls in arterial pressure (15-45 mmHg), and heart rate (75-100 bpm), whereas the depressor and bradycardiac effects of the noxious deep somatic manipulations were weaker, not as sustained, or entirely absent. As similar distributions and numbers of both increased and decreased Fos-like IR cells were observed after each of the deep noxious manipulations, it follows that the deep noxious evoked increases and decreases in Fos expression were not secondary to the evoked depressor or bradycardiac effects.

Spinal afferents to functionally distinct periaqueductal gray columns in the rat: an anterograde and retrograde tracing study.

The segmental and laminar organization of spinal projections to the functionally distinct ventrolateral (vlPAG) and lateral periaqueductal gray (lPAG) columns was examined by using retrograde and anterograde tracing techniques. It was found 1) that spinal input to both vlPAG and lPAG columns arose predominantly from neurons in the upper cervical (C1-4) and sacral spinal cord; 2) that there was a topographical separation of vl-PAG projecting and lPAG-projecting neurons within the upper cervical spinal cord; but 3) that below spinal segment C4, vlPAG-projecting and lPAG-projecting spinal neurons were similarly distributed, predominantly within contralateral lamina I, the nucleus of the dorsolateral fasciculus (the lateral spinal nucleus) and the lateral (reticular) part of lamina V. Consistent with the retrograde results, the greatest density of anterograde label, within both the vlPAG and lPAG, was found after tracer injections made either in the superficial or deep dorsal horn of the upper cervical spinal cord. Tracer injections made within the thoraco-lumbar spinal cord revealed that the vlPAG column received a convergent input from both the superficial and deep dorsal horn. However, thoraco-lumbar input to the lPAG was found to arise uniquely from the superficial dorsal horn; whereas the deep dorsal horn was found to innervate the "juxta-aqueductal" PAG region rather than projecting to the lPAG. These findings suggest that similar to spino-parabrachial projections, spinal projections to the lPAG (and juxta-aqueductal PAG) are topographically organised, with distinct subgroups of spinal neurons projecting to specific lPAG or juxta-aqueductal PAG subregions. In contrast, the vlPAG receives a convergent spinal input which arises from the superficial and deep dorsal horn of cervical, thoracic, lumbar, and sacral spinal segments.

Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the periaqueductal gray in the rat.

We utilised retrograde and anterograde tracing procedures to study the origin and termination of prefrontal cortical (PFC) projections to the periaqueductal gray (PAG) in the rat. A previous study, in the primate, had demonstrated that distinct subgroups of PFC areas project to specific PAG columns. Retrograde tracing experiments revealed that projections to dorsolateral (dlPAG) and ventrolateral (vlPAG) periaqueductal gray columns arose from medial PFC, specifically prelimbic, infralimbic, and anterior cingulate cortices. Injections made in the vlPAG also labeled cells in medial, ventral, and dorsolateral orbital cortex and dorsal and posterior agranular insular cortex. Other orbital and insular regions, including lateral and ventrolateral orbital, ventral agranular insular, and dysgranular and granular insular cortex did not give rise to appreciable projections to the PAG. Anterograde tracing experiments revealed that the projections to different PAG columns arose from specific PFC areas. Projections from the caudodorsal medial PFC (caudal prelimbic and anterior cingulate cortices) terminated predominantly in dlPAG, whereas projections from the rostroventral medial PFC (rostral prelimbic cortex) innervated predominantly the vlPAG. As well, consistent with the retrograde data, projections arising from select orbital and agranular insular cortical areas terminated selectively in the vlPAG. The results indicate: (1) that rat orbital and medial PFC possesses an organisation broadly similar to that of the primate; and (2) that subdivisions within the rat orbital and medial PFC can be recognised on the basis of projections to distinct PAG columns.

Orbitomedial prefrontal cortical projections to hypothalamus in the rat.

A previous study in the rat revealed that distinct orbital and medial prefrontal cortical (OMPFC) areas projected to specific columns of the midbrain periaqueductal gray region (PAG). This study used anterograde tracing techniques to define projections to the hypothalamus arising from the same OMPFC regions. In addition, injections of anterograde and retrograde tracers were made into different PAG columns to examine connections between hypothalamic regions and PAG columns projected upon by the same OMPFC regions. The most extensive patterns of hypothalamic termination were seen after injection of anterograde tracer in prelimbic and infralimbic (PL/IL) and the ventral and medial orbital (VO/MO) cortices. Projections from rostral PL/IL and VO/MO targeted the rostrocaudal extent of the lateral hypothalamus, as well as lateral perifornical, and dorsal and posterior hypothalamic areas. Projections arising from caudal PL/IL terminated within the dorsal hypothalamus, including the dorsomedial nucleus and dorsal and posterior hypothalamic areas. There were also projections to medial perifornical and lateral hypothalamic areas. In contrast, it was found that anterior cingulate (AC), dorsolateral orbital (DLO), and agranular insular (AId) cortices projected to distinct and restricted hypothalamic regions. Projections arising from AC terminated within dorsal and posterior hypothalamic areas, whereas DLO and AId projected to the lateral hypothalamus. The same OMPFC regions also projected indirectly, by means of specific PAG columns, to many of the same hypothalamic fields. In the context of our previous findings, these data indicate that, in both rat and macaque, parallel but distinct circuits interconnect OMPFC areas with specific hypothalamic regions, as well as PAG columns.

Spinal sources of noxious visceral and noxious deep somatic afferent drive onto the ventrolateral periaqueductal gray of the rat.

Studies utilizing the expression of Fos protein as a marker of neuronal activation have revealed that pain of deep somatic or visceral origin selectively activates the ventrolateral periaqueductal gray (vlPAG). Previous anatomical tracing studies revealed that spinal afferents to the vlPAG arose from the superficial and deep dorsal horn and nucleus of the dorsolateral funiculus at all spinal segmental levels, with approximately 50% of vlPAG-projecting spinal neurons found within the upper cervical spinal cord. This study utilized detection of Fos protein to determine the specific populations of vlPAG-projecting spinal neurons activated by noxious deep somatic or noxious visceral stimulation. Pain of cardiac or peritoneal (i.e., visceral) origin activated neurons in the superficial and deep dorsal horn and nucleus of the dorsolateral funiculus of the thoracic cord, whereas pain of hindlimb (i.e., deep somatic) origin activated neurons in the same laminar regions but in the lumbosacral cord. Each of these deep noxious manipulations also activated neurons in the superficial and deep dorsal horn and nucleus of the dorsolateral funiculus of the upper cervical spinal cord. In a second set of experiments, the combination of retrograde tracing and Fos immunohistochemistry revealed that vlPAG-projecting spinal neurons activated by deep somatic pain were located in both the upper cervical and lumbosacral cord, whereas those activated by visceral pain were restricted to the thoracic spinal cord. Thus pain arising from visceral versus deep somatic body regions influences neural activity within the vlPAG via distinct spinal pathways. The findings also highlight the potential significance of the upper cervical cord in integrating pain arising from deep structures throughout the body.

The ventrolateral periaqueductal gray projects to caudal brainstem depressor regions: a functional-anatomical and physiological study.

The reaction of shock, a precipitous, life-threatening fall in arterial pressure and heart rate, is evoked often by the combination of deep pain and blood loss following traumatic injury. A similar "shock-like" pattern of response can be evoked by excitation of the ventrolateral midbrain periaqueductal gray. Further, ventrolateral periaqueductal gray neurons are selectively activated by deep somatic or visceral pain and haemorrhage. The pathways mediating ventrolateral periaqueductal gray evoked hypotension and bradycardia are not known. In this study, the projections from the ventrolateral periaqueductal gray to "cardiovascular" regions in the caudal medulla of the rat were examined. Injections of the anterograde tracer, biotinylated dextran amine at physiologically-defined, ventrolateral periaqueductal gray depressor sites, revealed strong projections to the caudal midline medulla and to the depressor region of the caudal ventrolateral medulla. Injections of excitatory amino acids established that substantial falls in arterial pressure could be evoked from the ventrolateral periaqueductal gray-recipient parts of the caudal midline medulla. Injections of the retrograde tracer, cholera toxin subunit B at physiologically-defined, depressor sites in the caudal midline medulla and the caudal ventrolateral medulla confirmed the existence of substantial projections from the ventrolateral periaqueductal gray. Although previous studies have emphasized the importance of projections from the ventrolateral periaqueductal gray to the pressor region of the rostral ventrolateral medulla, this study has revealed the existence of strong ventrolateral periaqueductal gray projections to depressor regions within the caudal medulla (caudal midline medulla and caudal ventrolateral medulla) which likely contribute to ventrolateral periaqueductal gray-mediated hypotension and bradycardia.

Medullary catecholaminergic projections to the ventrolateral periaqueductal gray region activated by halothane anaesthesia.

Under anaesthesia, blood loss and deep pain can evoke a premature, centrally-mediated sympathoinhibition leading to decompensated shock and sometimes even death. The central circuits evoking premature vasodepressor syncope are unknown, although medullary catecholaminergic pathways have been implicated. The ventrolateral periaqueductal gray region is one of only three brain regions in which catecholamine content is increased during halothane anaesthesia. The ventrolateral periaqueductal gray also contains neurons which are selectively activated by blood loss and deep pain, and recent work from our laboratory has suggested that it is a pivotal structure in central sympathoinhibitory circuits. Using retrograde tracing techniques combined with the immunohistochemical detection of: (i) the catecholamine synthetic enzyme, tyrosine hydroxylase and (ii) the protein product of the immediate-early gene c-fos as a marker of neuronal activation; the results of this study indicate that catecholaminergic projections from the A1, C1 and C2 regions of the medulla to the ventrolateral periaqueductal gray are activated by halothane anaesthesia. These data are consistent with the hypotheses that ascending catecholaminergic projections to the ventrolateral periaqueductal gray: (i) are a component of the central neural circuitry responsible for the sympathoinhibitory effects of halothane anaesthesia, and (ii) may contribute to the premature elicitation of vasodepressor syncope following blood loss and deep pain under conditions of anaesthesia.

Caudal midline medulla mediates behaviourally-coupled but not baroreceptor-mediated vasodepression.

Within the caudal medulla there are two regions whose activation leads to vasodepression and bradycardia, the caudal ventrolateral medulla and a discrete region of the caudal midline medulla. This study investigated, in the halothane anaesthetized rat, the contribution of these two vasodepressor regions to "homeostatic" and "behaviourally-coupled" cardiovascular regulation. In an initial set of experiments the contribution of each of these two regions to the hypotension and bradycardia evoked by acute hypovolaemia (15% haemorrhage) was investigated. It was found that inactivation of the caudal midline medulla significantly attenuated (cobalt chloride) or completely blunted (lignocaine) the hypotension and bradycardia evoked by acute hypovolaemia. In contrast, inactivation of the caudal ventrolateral medulla using cobalt chloride, although attenuating the magnitude of the hypotension and completely blocking the bradycardia, did not delay the onset of the hypotension evoked by acute hypovolaemia. The caudal ventrolateral medulla is known to be critical in homeostatic cardiovascular control through the expression of the "baroreceptor reflex" and the hypotension and bradycardia evoked by activation of cardiopulmonary afferents. In a second series of experiments we found inactivation of the caudal midline medulla played no role in baroreflex-evoked bradycardia (i.v. phenylephrine) or the hypotension and bradycardia evoked by cardiopulmonary afferent activation (i.v. 5-hydroxytryptamine). These data suggest that the caudal midline medulla and caudal ventrolateral medulla play different roles in cardiovascular control. The caudal ventrolateral medulla is involved in mediating cardiovascular changes associated with a variety of stimuli including "homeostatic" and "behaviourally-coupled" cardiovascular changes, whereas the caudal midline medulla is critical for mediating "behaviourally-coupled" changes in arterial pressure and heart rate.

Haemorrhage-evoked compensation and decompensation are mediated by distinct caudal midline medullary regions in the urethane-anaesthetised rat.

Previous research using microinjections of excitatory amino acids suggested that the caudal midline medulla (including nucleus raphe obscurus and nucleus raphe pallidus) contained a mixed population of sympathoexcitatory and sympathoinhibitory neurones. The results of this study indicate that different anaesthetic regimes (urethane versus halothane) determine whether sympathoexcitatory (urethane only) or sympathoinhibitory (halothane only) responses are evoked by stimulation within distinct caudal midline medullary regions. In addition, anaesthetic regimes also affect the caudal midline medullary-mediated response to haemorrhage. Specifically, under conditions of urethane anaesthesia, inactivation (lignocaine) of the midline medullary region immediately caudal to the obex, prematurely triggered and dramatically potentiated the hypotension and bradycardia evoked by 15% haemorrhage; whereas under halothane anaesthesia, inactivation of the same region had no effect. In contrast, under urethane anaesthesia, inactivation of the midline medullary region immediately rostral to the obex, delayed the onset of the hypotension and bradycardia to 15% haemorrhage; inactivation of the same region under halothane anaesthesia blocked haemorrhage-evoked hypotension and bradycardia. Our findings indicate that topographically distinct parts of the caudal midline medulla contain neurones (i) that differentially regulate the timing and magnitude of the compensatory (normotensive) versus decompensatory (hypotensive) phases of the response to haemorrhage; and (ii) whose activity is altered by urethane versus halothane anaesthesia.

Swim-stress but not opioid withdrawal increases expression of c-fos immunoreactivity in rat periaqueductal gray neurons which project to the rostral ventromedial medulla.

Expression of c-fos-like immunoreactivity has been used as a marker for neuronal activation and is elevated in the periaqueductal gray following stressful and noxious stimuli, and opioid withdrawal. The present study examined the staining of c-fos-like immunoreactivity following opiate withdrawal or swim-stress (2.5-3 min at 21 degrees C) in periaqueductal gray neurons of the rat which had projections to and through the rostral ventromedial medulla identified by microinjection of the retrograde tracer, Fast Blue, into the nucleus raphe magnus prior to development of morphine dependence. Both naloxone-precipitated withdrawal and swim-stress increased numbers of neurons expressing c-fos-like immunoreactivity in periaqueductal gray. Naloxone-precipitated withdrawal did not increase the number of double-labelled neurons in periaqueductal gray suggesting that neurons excited during opioid withdrawal do not project to the ventromedial medulla. In contrast, swim-stress produced increases in double-labelled neurons in periaqueductal gray suggesting that many periaqueductal gray neurons activated by swim-stress project to the ventromedial medulla. These findings suggest that naloxone-precipitated withdrawal does not activate ventrolateral periaqueductal gray neurons which are involved in descending inhibitory pathways, consistent with behavioural observations that naloxone-precipitated withdrawal is qualitatively opposite to electrical and chemical stimulation of the ventrolateral periaqueductal gray. The results are also consistent with a role of descending projections from periaqueductal gray in stress-induced antinociception.

Convergence of deep somatic and visceral nociceptive information onto a discrete ventrolateral midbrain periaqueductal gray region.

Pain arising from deep structures (muscles, joints, viscera) is the type of pain of most clinical relevance and also the type of pain about whose central representation we have the least knowledge. In contrast to cutaneous pain which evokes defensive behaviours, hypertension and tachycardia, the physiological reactions to most deep pain (especially if persistent) usually include quiescence, hypotension, bradycardia and decreased reactivity to the environment. Excitation of neurons within a discrete ventrolateral midbrain periaqueductal gray region evokes a reaction seemingly identical to that evoked by pain arising from deep structures. We report here, using the technique of the noxious stimulus-evoked expression of the immediate-early gene, c-fos, that neurons within this same ventrolateral periaqueductal gray region are selectively activated by a range of deep somatic and visceral nociceptive manipulations. Thus we have identified a specific brain region that both receives convergent, deep somatic and visceral nociceptive input, and which mediates the behavioural and physiological reactions characteristic of most deep pain.

Hypotension following acute hypovolaemia depends on the caudal midline medulla.

Acute hypovolaemia evokes abrupt, life-threatening hypotension and bradycardia. Hypotension can be evoked also by excitation of the caudal midline medulla (CMM). This study investigated the possible contribution of the CMM depressor area to hypotension evoked by acute hypovolaemia. Inactivation of the CMM, with either lignocaine or cobalt chloride did not alter resting arterial pressure. However lignocaine injections blocked the fall in arterial pressure, and cobalt chloride injections delayed the onset and significantly attenuated the size of hypovolaemic-evoked hypotension. These findings suggest that the CMM is a key region triggering hypotension after blood loss, and that the brain areas mediating cardiovascular response to challenges such as acute hypovolaemia are not the same areas that regulate resting arterial pressure.

A calbindin immunoreactive "deep pain' recipient thalamic nucleus in the rat.

The ventrolateral periaqueductal gray (vlPAG) is the only brain region known to receive convergent deep noxious inputs and to mediate the reactions characteristic of deep pain. Injections of biotinylated dextran into the vlPAG of the rat revealed a strong projection to a discrete, calbindin terminal-immunoreactive region of the caudal ventromedial nucleus (VMc) of the thalamus. This nucleus appears homologous to the calbindin-positive, pain- and temperature-specific-posterior ventromedial thalamic region of primates. We suggest that the vlPAG to VMc projection represents an important new route via which deep noxious inputs reach thalamus. As the rat is the species of choice in most experimental studies of pain, the functional-anatomical definition of this projection should further investigation of the thalamic representation of deep pain.

C-fos expression in the spinal cord of rats exhibiting allodynia following contusive spinal cord injury.

Contusive spinal cord injury (SCI) may result in central neuropathic pain marked by allodynia-like features in the dermatomes close to the level of injury. The aim of this study was to compare the laminar distribution of activated neurons (as determined by c-fos immediate early gene expression) in the spinal cord immediately above the level of a SCI in rats with or without allodynia-like features. Non-noxious mechanical stimulation was applied to half the animals in the dermatomes corresponding to the level of injury prior to perfusion. Stimulation resulted in a significant increase in c-fos labelling in all laminae of the spinal dorsal horn in the segment immediately above the level of injury only in allodynia animals. Animals that had allodynia also demonstrated a significant increase in the level of c-fos labelling in lamina III, IV and V of the dorsal horn without stimulation. Thus, allodynia following SCI is associated with significant increases in basal and evoked c-fos expression ("neuronal activity") in response to non-noxious mechanical stimulation. The data also suggest that allodynia-like behaviour following SCI cannot be accounted for solely by changes occurring at a spinal level.

Expression of c-Fos-like immunoreactivity in the caudal medulla and upper cervical spinal cord following stimulation of the superior sagittal sinus in the cat.

Migraine is an episodic vascular headache with a well-recognized clinical picture but a poorly understood pathogenesis. Stimulation of a pain-sensitive trigeminally innervated intracranial structure, the superior sagittal sinus (SSS), was undertaken to map the higher-order neurons potentially involved in the processing of vascular head pain. The animals were prepared for stimulation by exposure of the sinus and then maintained under alpha-chloralose anaesthesia for 24 h before SSS stimulation, perfusion and immunohistochemical processing for the detection of Fos protein. Examination of the medulla and upper cervical cord revealed marked increases in Fos-like immunoreactivity in laminae I and IIo of the trigeminal nucleus caudalis and the dorsal horn of the upper cervical spinal cord. In addition, Fos-like immunoreactivity was observed in lamina X of the upper cervical spinal cord, in the commissural and medial nuclei of the solitary tract and in the nucleus retroambigualis. The use of immunohistochemical detection of Fos has allowed visualization of several populations of neurons likely to be involved in the central neural processing of vascular headache syndromes, particularly migraine.