Periaqueductal Grey EP3 Receptors Facilitate Spinal Nociception in Arthritic Secondary Hypersensitivity.

UNLABELLED: Descending controls on spinal nociceptive processing play a pivotal role in shaping the pain experience after tissue injury. Secondary hypersensitivity develops within undamaged tissue adjacent and distant to damaged sites. Spinal neuronal pools innervating regions of secondary hypersensitivity are dominated by descending facilitation that amplifies spinal inputs from unsensitized peripheral nociceptors. Cyclooxygenase-prostaglandin (PG) E2 signaling within the ventrolateral periaqueductal gray (vlPAG) is pronociceptive in naive and acutely inflamed animals, but its contributions in more prolonged inflammation and, importantly, secondary hypersensitivity remain unknown. In naive rats, PG EP3 receptor (EP3R) antagonism in vlPAG modulated noxious withdrawal reflex (EMG) thresholds to preferential C-nociceptor, but not A-nociceptor, activation and raised thermal withdrawal thresholds in awake animals. In rats with inflammatory arthritis, secondary mechanical and thermal hypersensitivity of the hindpaw developed and was associated with spinal sensitization to A-nociceptor inputs alone. In arthritic rats, blockade of vlPAG EP3R raised EMG thresholds to C-nociceptor activation in the area of secondary hypersensitivity to a degree equivalent to that evoked by the same manipulation in naive rats. Importantly, vlPAG EP3R blockade also affected responses to A-nociceptor activation, but only in arthritic animals. We conclude that vlPAG EP3R activity exerts an equivalent facilitation on the spinal processing of C-nociceptor inputs in naive and arthritic animals, but gains in effects on spinal A-nociceptor processing from a region of secondary hypersensitivity. Therefore, the spinal sensitization to A-nociceptor inputs associated with secondary hypersensitivity is likely to be at least partly dependent on descending prostanergic facilitation from the vlPAG. SIGNIFICANCE STATEMENT: After tissue damage, sensitivity to painful stimulation develops in undamaged areas (secondary hypersensitivity). This is found in many painful conditions, particularly arthritis. The periaqueductal gray (PAG) is an important center that controls spinal nociceptive processing, on which secondary hypersensitivity depends. Prostaglandins (PGs) are mediators of inflammation with pronociceptive actions within the PAG under normal conditions. We find that secondary hindpaw hypersensitivity in arthritic rats results from spinal sensitization to peripheral A-nociceptor inputs. In the PAG of arthritic, but not naive, rats, there is enhanced control of spinal A-nociceptor processing through PG EP3 receptors. The descending facilitatory actions of intra-PAG PGs play a direct and central role in the maintenance of inflammatory secondary hypersensitivity, particularly relating to the processing of A-fiber nociceptive information.

Objective validation of central sensitization in the rat UVB and heat rekindling model.

BACKGROUND: The UVB and heat rekindling (UVB/HR) model shows potential as a translatable inflammatory pain model. However, the occurrence of central sensitization in this model, a fundamental mechanism underlying chronic pain, has been debated. Face, construct and predictive validity are key requisites of animal models; electromyogram (EMG) recordings were utilized to objectively demonstrate validity of the rat UVB/HR model. METHODS: The UVB/HR model was induced on the heel of the hind paw under anaesthesia. Mechanical withdrawal thresholds (MWTs) were obtained from biceps femoris EMG responses to a gradually increasing pinch at the mid hind paw region under alfaxalone anaesthesia, 96 h after UVB irradiation. MWT was compared between UVB/HR and SHAM-treated rats (anaesthetic only). Underlying central mechanisms in the model were pharmacologically validated by MWT measurement following intrathecal N-methyl-d-aspartate (NMDA) receptor antagonist, MK-801, or saline. RESULTS: Secondary hyperalgesia was confirmed by a significantly lower pre-drug MWT {mean [±standard error of the mean (SEM)]} in UVB/HR [56.3 (±2.1) g/mm(2) , n = 15] compared with SHAM-treated rats [69.3 (±2.9) g/mm(2) , n = 8], confirming face validity of the model. Predictive validity was demonstrated by the attenuation of secondary hyperalgesia by MK-801, where mean (±SEM) MWT was significantly higher [77.2 (±5.9) g/mm(2) n = 7] in comparison with pre-drug [57.8 (±3.5) g/mm(2) n = 7] and saline [57.0 (±3.2) g/mm(2) n = 8] at peak drug effect. The occurrence of central sensitization confirmed construct validity of the UVB/HR model. CONCLUSIONS: This study used objective outcome measures of secondary hyperalgesia to validate the rat UVB/HR model as a translational model of inflammatory pain.

Endogenous analgesic action of the pontospinal noradrenergic system spatially restricts and temporally delays the progression of neuropathic pain following tibial nerve injury.

Pontospinal noradrenergic neurons form part of an endogenous analgesic system that suppresses acute pain, but there is conflicting evidence about its role in neuropathic pain. We investigated the chronology of descending noradrenergic control during the development of a neuropathic pain phenotype in rats following tibial nerve transection (TNT). A lumbar intrathecal cannula was implanted at the time of nerve injury allowing administration of selective α-adrenoceptor (α-AR) antagonists to sequentially assay their effects upon the expression of allodynia and hyperalgesia. Following TNT animals progressively developed mechanical and cold allodynia (by day 10) and subsequently heat hypersensitivity (day 17). Blockade of α2-AR with intrathecal yohimbine (30 µg) revealed earlier ipsilateral sensitization of all modalities while prazosin (30 µg, α1-AR) was without effect. Established allodynia (by day 21) was partly reversed by the re-uptake inhibitor reboxetine (5 µg, i.t.) but yohimbine no longer had any sensitising effect. This loss of effect coincided with a reduction in the descending noradrenergic innervation of the ipsilateral lumbar dorsal horn. Yohimbine reversibly unmasked contralateral hindlimb allodynia and hyperalgesia of all modalities and increased dorsal horn c-fos expression to an innocuous brush stimulus. Contralateral thermal hyperalgesia was also reversibly uncovered by yohimbine administration in a contact heat ramp paradigm in anaesthetised TNT rats. Following TNT there is an engagement of inhibitory α2-AR-mediated noradrenergic tone which completely masks contralateral and transiently suppresses the development of ipsilateral sensitization. This endogenous analgesic system plays a key role in shaping the spatial and temporal expression of the neuropathic pain phenotype after nerve injury.

Transient receptor potential channel A1 and noxious cold responses in rat cutaneous nociceptors.

The role of transient receptor potential channel A1 (TRPA1) in noxious cold sensation remains unclear. Some data support the hypothesis that TRPA1 is a transducer of noxious cold whilst other data contest it. In this study we investigated the role of TRPA1 in cold detection in cutaneous nociceptors in vivo using complementary experimental approaches. We used noxious withdrawal reflex electromyography, and single fibre recordings in vivo, to test the hypothesis that TRPA1-expressing primary afferents mediate noxious cold responses in anaesthetised rats. TRPV1 and TRPM8 agonists sensitise their cognate receptors to heat and cold stimuli respectively. Herein we show that the TRPA1 agonist cinnamaldehyde applied to the skin in anaesthetised rats did not sensitise noxious cold evoked hind limb withdrawal. In contrast, cinnamaldehyde did sensitise the C fibre-mediated noxious heat withdrawal, indicated by a significant drop in the withdrawal temperature. TRPA1 agonist thus sensitised the noxious reflex withdrawal to heat, but not cold. Thermal stimuli also sensitise transient receptor potential (TRP) channels to agonist. Activity evoked by capsaicin in teased primary afferent fibres showed a significant positive correlation with receptive field temperature, in both normal and Freund's complete adjuvant-induced cutaneous inflammation. Altering the temperature of the receptive field did not modulate TRPA1 agonist evoked-activity in cutaneous primary afferents, in either normal or inflamed skin. In addition, block of the TRPA1 channel with Ruthenium Red did not inhibit cold evoked activity in either cinnamaldehyde sensitive or insensitive cold responsive nociceptors. In cinnamaldehyde-sensitive-cold-sensitive afferents, although TRPA1 agonist-evoked activity was totally abolished by Ruthenium Red, cold evoked activity was unaffected by channel blockade. We conclude that these results do not support the hypothesis that TRPA1-expressing cutaneous afferents play an important role in noxious cold responses.

Descending control of nociception: Specificity, recruitment and plasticity.

The dorsal horn of the spinal cord is the location of the first synapse in pain pathways, and as such, offers a very powerful target for regulation of nociceptive transmission by both local segmental and supraspinal mechanisms. Descending control of spinal nociception originates from many brain regions and plays a critical role in determining the experience of both acute and chronic pain. The earlier concept of descending control as an "analgesia system" is now being replaced with a more nuanced model in which pain input is prioritized relative to other competing behavioral needs and homeostatic demands. Descending control arises from a number of supraspinal sites, including the midline periaqueductal gray-rostral ventromedial medulla (PAG-RVM) system, and the more lateral and caudal dorsal reticular nucleus (DRt) and ventrolateral medulla (VLM). Inhibitory control from the PAG-RVM system preferentially suppresses nociceptive inputs mediated by C-fibers, preserving sensory-discriminative information conveyed by more rapidly conducting A-fibers. Analysis of the circuitry within the RVM reveals that the neural basis for bidirectional control from the midline system is two populations of neurons, ON-cells and OFF-cells, that are differentially recruited by higher structures important in fear, illness and psychological stress to enhance or inhibit pain. Dynamic shifts in the balance between pain inhibiting and facilitating outflows from the brainstem play a role in setting the gain of nociceptive processing as dictated by behavioral priorities, but are also likely to contribute to pathological pain states.

Separation of A- versus C-nociceptive inputs into spinal-brainstem circuits.

This study was designed to determine the organization of nociceptive inputs with different behavioral significance into spinal-brainstem circuits in the rat. Induction of Fos protein was used to localize spinal dorsal horn and hypothalamic neurons activated by noxious heating of the hind paw dorsum at rates known to preferentially activate C- or A-heat nociceptors. This was combined with retrograde transport of cholera toxin subunit B from the dorsolateral/lateral- (DL/L-) or the ventrolateral- (VL-) periaqueductal gray (PAG) in order to map the organization of A- and C-fiber input to spinal-brainstem circuits. The majority of dorsal horn heat-activated neurons were located in laminae I and II. A significantly larger proportion of C-fiber-activated neurons projected to the VL-PAG (P<0.05) compared with its DL/L-sector. In contrast, there was no columnar separation in the projections of A-fiber-activated neurons. However, a significantly greater proportion of A-fiber-activated neurons (P<0.05) were retrogradely labeled from the DL/L-PAG, when compared with C-fiber-activated neurons. A large proportion (25-50%) of A- and C-fiber-activated neurons in the lateral spinal nucleus projected to the PAG. A-fiber-activated neurons were found throughout the rostral hypothalamus but those projecting to the PAG were focused in the lateral area of the anterior hypothalamus (LAAH), from where approximately 20% projected to the VL-PAG, which was significantly more than to the DL/L PAG (P<0.05). We hypothesize that the organization of A- versus C-fiber inputs to the PAG enables the coordination of coping strategies appropriate to meet the demands imposed by these different noxious stimuli. Hypothalamic-PAG projections activated by A-fiber inputs did not reflect this level of organization and we suggest that this may relate to their role in thermoregulation as opposed to autonomic responses to particular nociceptive inputs.

Noxious somatic inputs to hypothalamic-midbrain projection neurones: a comparison of the columnar organisation of somatic and visceral inputs to the periaqueductal grey in the rat.

The induction of Fos protein was used to localise hypothalamic neurones activated by noxious somatic stimulation. This was combined with retrograde transport of fluorescent latex microspheres from identified 'pressor' and 'depressor' sites in the dorsolateral/lateral or ventrolateral columns of the periaqueductal grey (PAG). Fos-positive neurones were found throughout the rostral hypothalamus. Of those neurones activated by noxious somatic stimuli that projected to the PAG all but one was retrogradely labelled from sites that included the lateral column. Only one neurone was double labelled following injection of tracer at a depressor site in the ventrolateral PAG. This is in marked contrast to visceroresponsive hypothalamic neurones, a larger proportion of which project to the PAG and which, as reported previously, preferentially target depressor sites in the ventrolateral sector. These results are discussed in relation to the roles of the anterior hypothalamus and the different functional columns of the PAG in co-ordinating autonomic and sensory functions in response to nociceptive inputs originating in different peripheral domains.

C-nociceptor activation of hypothalamic neurones and the columnar organisation of their projections to the periaqueductal grey in the rat.

The induction of Fos protein was used to localise hypothalamic neurones activated by ramps of noxious skin heating delivered at a rate of 2.5 degrees C s(-1) to preferentially activate C-nociceptors. This was combined with retrograde transport of cholera toxin subunit B from identified 'pressor' and 'depressor' sites in the dorsolateral/lateral or the ventrolateral columns of the periaqueductal grey. Fos-positive neurones were found throughout the rostral hypothalamus. Despite this wide distribution, those neurones double labelled retrogradely from the periaqueductal grey were focused in the lateral area of the anterior hypothalamus. More than 20 % of Fos-positive neurones in this region projected to depressor sites in the ventrolateral periaqueductal grey, and 10 % projected to its dorsolateral/lateral sector. These results are discussed in relation to the peripheral inputs to hypothalamic-midbrain pathways and their role in the cardiovascular responses to different components of the pain signal.

Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli.

Three genes, ipgD, mxiC, and mxiA, all in the invasion region of the Shigella virulence plasmid, were sequenced from strains representing a range of Shigella serotypes and from two enteroinvasive Escherichia coli (EIEC) isolates. The plasmids can be classified into two relatively homogeneous sequence forms which are quite distinct. pINV A plasmids are found in Shigella flexneri strains F6 and F6A, S. boydii strains B1, B4, B9, B10, B14, and B15, S. dysenteriae strains D3, D4, D6, D8, D9, D10, and D13, and the two EIEC strains (M519 and M520). pINV B plasmids are present in S. flexneri strains F1A, F2A, F3A, F3C, F4A, and FY, two S. boydii strains (B11 and B12), and S. sonnei. The D1 pINV plasmid is a recombinant with ipgD gene more closely related to those of pINV A but with mxiA and mxiC genes more closely related to those of pINV B. The phylogenetic relationships of the plasmid and those of the chromosomal genes of Shigella strains are largely consistent. The cluster 1 and cluster 3 strains tested (G.M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000) have pINV A and pINV B plasmids, respectively. However, of the three cluster 2 strains (B9, B11, and B15), B9 and B15 have pINV A while B11 has a pINV B plasmid. Those Shigella (D8 and D10 and S. sonnei) and EIEC strains which do not group with the main body of Shigella strains based on chromosomal genes were found to have plasmids belonging to one or the other of the two types and must have acquired these by lateral transfer.

Inhibitory effects evoked from the rostral ventrolateral medulla are selective for the nociceptive responses of spinal dorsal horn neurons.

The aim of the present study was to determine whether or not descending control of spinal dorsal horn neuronal responsiveness following neuronal activation at pressor sites in the rostral ventrolateral medulla is selective for nociceptive information. Extracellular single-unit activity was recorded from 49 dorsal horn neurons in the lower lumbar spinal cord of anaesthetized rats. The 30 Class 2 neurons selected for investigation responded to noxious (pinch and radiant heat) and non-noxious (prod, stroke and/or brush) stimulation within their cutaneous receptive fields on the ipsilateral hindpaw. The excitatory amino acid, DL-homocysteic acid, was microinjected into either the rostral or the caudal rostral ventrolateral medulla at sites that evoked increases in arterial blood pressure. Effects of neuronal activation at these sites were then tested on the responses of Class 2 neurons to noxious and non-noxious stimulation within their excitatory receptive fields. The noxious pinch and radiant heat responses of Class 2 neurons were depressed, respectively to 13+/-3.8% (n=23) and to 16+/-3.7% (n=18) of control, following stimulation at sites in the rostral rostral ventrolateral medulla. In contrast, the low-threshold (prod) responses of eight Class 2 neurons tested were not depressed following neuronal activation at the same sites. When tested, control injections of the inhibitory amino acid, GABA, at the same sites in the rostral rostral ventrolateral medulla had no significant effects on neuronal activity. Neither intravenous administration of noradrenaline (to mimic the pressor responses evoked by DL-homocysteic acid microinjections in the rostral ventrolateral medulla) nor activation at pressor sites in the caudal rostral ventrolateral medulla had any significant effect on neuronal responsiveness. With regard to sensory processing in the spinal cord, these data suggest that descending inhibitory control that originates from neurons in pressor regions of the rostral rostral ventrolateral medulla is highly selective for nociceptive inputs to Class 2 neurons. These data are discussed in relation to the role of the rostral ventrolateral medulla in executing the changes in autonomic and sensory functions that are co-ordinated by higher centres in the CNS.

Visceral inputs to neurons in the anterior hypothalamus including those that project to the periaqueductal gray: a functional anatomical and electrophysiological study.

The present study was designed to examine peripheral, in particular noxious visceral, inputs to neurons in the hypothalamus that project to the midbrain periaqueductal gray. The induction of Fos protein was used to localize hypothalamic neurons that were activated by noxious visceral stimulation. This was combined with retrograde transport of fluorescent latex microspheres from identified "pressor" and "depressor" sites in the dorsolateral/lateral or ventrolateral columns of the periaqueductal gray. A second series of electrophysiological experiments examined the receptive field characteristics, including the incidence of viscerosomatic convergence, of neurons in the ventral part of the anterior hypothalamus. Noxious visceral stimulation (intraperitoneal acetic acid) induced Fos-like immunoreactivity in significantly more neurons in the hypothalamus than control stimuli (intraperitoneal saline and intravenous phenylephrine). Particularly high numbers of Fos-positive neurons were found in the paraventricular nucleus, the supraoptic nucleus and ventral regions of the anterior hypothalamus. When combined with retrograde tracing from "depressor" sites in the ventrolateral periaqueductal gray, the highest numbers of double-labelled neurons were localized in the paraventricular nucleus and the lateral area of the anterior hypothalamus. However, the regions that contained the greatest proportions of Fos-positive neurons that projected to "depressor" sites in the ventrolateral periaqueductal gray were the lateral area of the anterior hypothalamus and its rostral extension, the lateral preoptic area. Fewer double-labelled neurons were localized in the hypothalamus after retrograde transport from sites in the dorsolateral/lateral periaqueductal gray compared to the results obtained from injections of tracer in the ventrolateral periaqueductal gray. Furthermore, the numbers of Fos-positive hypothalamic neurons that projected to the dorsolateral/lateral periaqueductal gray were very similar in experimental and control animals. The electrophysiological study confirmed that a large proportion of neurons in and around the lateral area of the anterior hypothalamus can be driven by noxious visceral stimulation and demonstrated a high incidence of viscerosomatic convergence in these cells (66% of cells driven from somatic structures were also driven by electrical stimulation of the splanchnic nerve). Somatic receptive fields of these neurons were generally large, often including all four limbs and the face. The results of the functional anatomical and electrophysiological studies have identified neurons in an area of the ventral anterior hypothalamus that are a focus of nociceptive visceral input and which project to the midbrain periaqueductal gray, in particular to its ventrolateral column. These results are discussed in relation to the roles of the anterior hypothalamus and the different longitudinal columns of the periaqueductal gray in co-ordinating autonomic and sensory functions in response to visceral pain.

Serotonergic transmission in the periaqueductal gray matter in relation to aversive behaviour: morphological evidence for direct modulatory effects on identified output neurons.

Intracellular recordings were made from 21 cells in the dorsolateral periaqueductal gray matter in coronal midbrain slices. In the majority (n = 20) bath application of 5-hydroxytryptamine (30 or 150 mM) evoked either hyperpolarizing (n = 11) or depolarizing (n = 9) responses. Reconstructions of 11 neurons in the dorsolateral periaqueductal gray matter after filling with biocytin revealed a population of output neurons whose axons followed a dorsolateral trajectory towards the perimeter of the ipsilateral periaqueductal gray matter. In seven cells, the axon could be followed into the adjacent mesencephalic reticular formation. At the light microscopic level, immunostaining for 5-hydroxytryptamine revealed immunoreactive processes throughout the dorsolateral periaqueductal gray matter but no labelled somata or dendrites. Close associations (i.e. no discernible gap) were observed between serotonergic profiles and the somata and dendrites of biocytin-filled cells. At the ultrastructural level, serial sections through 21 appositions on to biocytin-filled dendrites in three slices revealed 19 true appositions (i.e. having closely parallel plasma membranes with no intervening glial cell profiles) with the biocytin-filled dendrite. Only four of the appositions (21%) showed evidence of synaptic specializations which included aggregations of synaptic vesicles, and some thickening of the apposing membrane. The dense reaction product in the biocytin-filled cells precluded identification of the ultrastructure of postsynaptic elements. However, examination of contacts between 5-hydroxytryptamine-immunoreactive profiles and unlabelled elements in material taken from the contralateral side of the periaqueductal gray matter (i.e. no biocytin present) or in material taken from perfusion-fixed whole brain, in which ultrastructural preservation was superior compared with slices, revealed a similar incidence (21% and 23%, respectively) of synaptic specializations. The data indicate that serotonergic transmission on to output neurons in the dorsolateral periaqueductal gray matter is largely mediated by non-junctional contacts, suggesting that the actions of 5-hydroxytryptamine on these cells are mediated predominantly by volume rather than wiring transmission.

Excitatory projections from the anterior hypothalamus to periaqueductal gray neurons that project to the medulla: a functional anatomical study.

The present study was designed to investigate the organization of excitatory projections from regions of the anterior hypothalamus that are known to co-ordinate autonomic and sensory functions to medullo-output neurons in the periaqueductal gray. The induction of Fos protein was used to identify neurons in the periaqueductal gray that were activated synaptically by chemical stimulation at sites in the anterior hypothalamus from which either increases or decreases in arterial blood pressure were evoked (pressor sites and depressor sites, respectively). This was combined with retrograde tracing using fluorescent latex microspheres from sites in the medulla. When compared to control animals, neuronal activation at pressor sites in the anterior hypothalamus evoked Fos-like immunoreactivity in significantly more neurons in all but one sub-division of the periaqueductal gray (P at least < 0.05). The majority of Fos-positive neurons following a pressor response were located in the caudal half of the periaqueductal gray where significantly more neurons contained Fos-like immunoreactivity in lateral than in any other sub-division (P < 0.01). In all but two of 14 subdivisions of the periaqueductal gray, the numbers of neurons that expressed Fos-like immunoreactivity following stimulation at depressor sites in the anterior hypothalamus were not significantly different from controls. When neuronal activation at pressor or depressor sites in the anterior hypothalamus was combined with retrograde tracing from the rostral ventrolateral medulla, nucleus raphe magnus and/or nucleus raphe obscurus the majority of double-labelled neurons were located in the caudal half of the periaqueductal gray. Comparisons between the numbers of double-labelled neurons that resulted from different combinations of hypothalamic and medullary injection sites revealed that neuronal activation at pressor sites in the anterior hypothalamus combined with retrograde tracing from the rostral ventrolateral medulla resulted in the greatest numbers of double-labelled neurons. The identification of double-labelled neurons indicates that medullo-output neurons in the periaqueductal gray receive excitatory inputs predominantly from pressor compared to depressor sites in the anterior hypothalamus. These results are discussed in relation to the roles of the different longitudinal columns of the periaqueductal gray, and the organisation of their projections to the medulla, in the co-ordination of autonomic and sensory functions.

Inhibitory effects evoked from the anterior hypothalamus are selective for the nociceptive responses of dorsal horn neurons with high- and low-threshold inputs.

The aim of the present study was to examine the selectivity of descending control of nociceptive information in the spinal dorsal horn following neuronal activation at "pressor" sites in the anterior hypothalamus. Extracellular single-unit activity was recorded from 11 dorsal horn neurons in the lower lumbar spinal cord of anesthetized rats. Neurons selected for investigation were those that responded to noxious (pinch and radiant heat >46 degrees C) and nonnoxious (prod, stroke, and/or brush) stimulation within their cutaneous receptive fields on the ipsilateral hind paw. These are referred to as Class 2 neurons. Micropipettes were inserted stereotaxically into the anterior hypothalamus at sites where injection of the excitatory amino acid L-homocysteic acid (L-HCA) evoked increases in arterial blood pressure. The effects of microinjection of L-HCA at "pressor" sites in the anterior hypothalamus were then tested on the responses of Class 2 neurons to noxious and nonnoxious stimulation of their excitatory receptive fields. The high-threshold (pinch and/or radiant heat) responses of 7/7 Class 2 neurons tested were inhibited by an average of 66.3 +/- 8.8% (mean +/- SE) by neuronal activation at hypothalamic pressor sites. The low-threshold (prod) responses of 10/10 Class 2 neurons tested were not inhibited by neuronal activation at hypothalamic pressor sites; in 6 of these cells the response to low-intensity stimulation was increased by between 4 and 20%. Control injections of the inhibitory amino acid gamma-aminobutyric acid (GABA) at the same hypothalamic pressor sites had no significant effects on arterial blood pressure or neuronal activity. With regard to sensory processing in the spinal cord, these data suggest that descending inhibitory control that originates from neurons in pressor regions of the anterior hypothalamus is highly selective for nociceptive inputs to Class 2 neurons.

Responses of neurones in the medullary raphe nuclei to inputs from visceral nociceptors and the ventrolateral periaqueductal grey in the rat.

The ventrolateral periaqueductal grey matter (PAG) is believed to have a role in mediating cardiovascular responses to noxious visceral stimuli. The present study was carried out as a first stage in establishing whether the ventrolateral PAG may exert these influences after a relay in the caudal medullary raphe nuclei (nucleus raphe obscurus and nucleus raphe pallidus). Single unit extracellular recordings were made from neurones in the caudal raphe nuclei and, for comparison, in the more rostral nucleus raphe magnus in Saffan-anaesthetized and paralysed rats. Neurones in the mid-line medulla were tested for their responses to electrical stimulation at chemically identified depressor sites in the ventrolateral PAG and to noxious visceral stimuli (distensions of the urinary bladder and electrical stimulation of the greater splanchnic nerve). Fifty-two per cent of caudal and 74% of rostral mid-line neurones gave short latency excitatory responses to stimulation of depressor sites in the ventrolateral PAG. Of the neurones that were also tested with noxious visceral stimuli, 5% of the caudal and 47% of the rostral neurones responded to bladder distension, while 33 and 35%, respectively, of caudal and rostral neurones responded to splanchnic nerve stimulation. These results indicate that many mid-line medullary neurones receive inputs from both the ventrolateral PAG and visceral nociceptors and may, therefore, be part of the output pathway by which the ventrolateral PAG produces integrated physiological responses to noxious visceral stimuli.

Inhibitory effects evoked from both the lateral and ventrolateral periaqueductal grey are selective for the nociceptive responses of rat dorsal horn neurones.

In rats anaesthetized with alphaxalone/alphadolone a comparative study was made of the inhibitory effects on dorsal horn neurones evoked by chemical stimulation at identified pressor and depressor sites in the lateral and ventrolateral periaqueductal grey (PAG). Stimulating micropipettes were inserted stereotaxically into the lateral or ventrolateral PAG at sites where microinjection of DL-homocysteic acid (DLH) evoked increases or decreases respectively in mean arterial blood pressure. The effects of DLH microinjection at these sites were tested against the responses of dorsal horn neurones to noxious and innocuous stimuli applied to their cutaneous receptive fields. Single unit extracellular recordings were made from 15 Class 1 (low-threshold) and 37 Class 2 (wide dynamic range) dorsal horn neurones in laminae II-VI of the lower lumbar spinal cord. The responses of Class 1 neurones to innocuous prodding of their receptive fields were unaffected by neuronal activation in either the lateral or ventrolateral PAG. The nociceptive (noxious pinch/heat) responses of most Class 2 neurones were strongly inhibited by chemical stimulation in either sector of the PAG. The low threshold (prod) responses of the same neurones were generally unaffected or only weakly inhibited by identical stimulation, regardless of stimulation site. No significant differences were found between the effects of lateral vs. ventrolateral PAG stimulation on the responses of dorsal horn neurones. These results do not support the view that dorsal horn neurones may be inhibited with different selectivities by hyper- and hypotensive regions of the PAG.

Neurones in the midbrain periaqueductal grey send collateral projections to nucleus raphe magnus and the rostral ventrolateral medulla in the rat.

Projections to the rostral ventrolateral medulla (RVLM) and nucleus raphe magnus (NRM) appear to originate from neurones with overlapping distributions in the periaqueductal grey (PAG) as demonstrated by the retrograde transport of red and green fluorescent latex microspheres. Furthermore, double-labelling studies demonstrated collateral projections from individual neurones in the PAG to the RVLM and NRM. This anatomical arrangement may allow interactions between descending control systems during specific behaviours.

Glutamatergic projections from the rostral hypothalamus to the periaqueductal grey.

Retrograde transport of fluorescent latex microspheres was combined with immunocytochemistry for glutamate to determine the organization of the projections from glutamate-containing neurones in the rostral hypothalamus to the different subdivisions of the periaqueductal grey (PAG). Double-labelled neurones, i.e. neurones immunoreactive for glutamate and projecting to the PAG, were found throughout the rostral hypothalamus. There were no apparent differences, however, in the origins of presumed glutamatergic projections from the rostral hypothalamus to the different subdivisions of the PAG.

Enkephalinergic rostral hypothalamic neurones do not project to the intermediate PAG in the rat.

Retrograde transport of fluorescent latex microspheres was combined with immunocytochemistry to determine whether enkephalinergic neurones in the rostral hypothalamus project to the intermediate periaqueductal grey. Distributions of retrogradely labelled neurones and neurones immunoreactive for enkephalins were as expected from previous studies. However, very few neurones contained both markers suggesting that enkephalinergic neurones in the rostral hypothalamus do not project to the periaqueductal grey at this level. A series of control experiments was carried out on a pathway with a known enkephalinergic component to confirm the compatibility of this combination of techniques.