It is known that pain perception can be altered by mood, attention and cognition, or by direct stimulation of the cerebral cortex, but we know little of the neural mechanisms underlying the cortical modulation of pain. One of the few cortical areas consistently activated by painful stimuli is the rostral agranular insular cortex (RAIC) where, as in other parts of the cortex, the neurotransmitter gamma-aminobutyric acid (GABA) robustly inhibits neuronal activity. Here we show that changes in GABA neurotransmission in the RAIC can raise or lower the pain threshold--producing analgesia or hyperalgesia, respectively--in freely moving rats. Locally increasing GABA, by using an enzyme inhibitor or gene transfer mediated by a viral vector, produces lasting analgesia by enhancing the descending inhibition of spinal nociceptive neurons. Selectively activating GABA(B)-receptor-bearing RAIC neurons produces hyperalgesia through projections to the amygdala, an area involved in pain and fear. Whereas most studies focus on the role of the cerebral cortex as the end point of nociceptive processing, we suggest that cerebral cortex activity can change the set-point of pain threshold in a top-down manner.

At a "classical" neuronal synapse, neurotransmitter is released from presynaptic vesicles by exocytosis, crosses the synaptic cleft, and binds to receptors located postsynaptically. Implicit in this characterization is that neurotransmitter release sites at these synapses are closely apposed to the targeted receptor; neurotransmitter and receptor are separated only by the synaptic cleft and density between pre-and postsynaptic elements (1-4). Examples include glycinergic (3,4) and some glutamatergic synapses (1, 2) in the central nervous system (CNS) and cholinergic synapses in sympathetic ganglia (5) and at the adult neuromuscular junction (6).On the other hand, some -y-aminobutyric acid (GABA) receptors in the cerebellum are located at sites away from GABA-containing synapses, raising the possibility that GABA can act upon targets distant from its site of release, in a "nonsynaptic" fashion. The fact that postsynaptic densities are absent at some CNS monoaminergic synapses (7) suggests that norepinephrine and serotonin have a similar action. Peptide neurotransmitters, which often colocalize with more classical neurotransmitters (8), may also act in a diffuse, nonsynaptic manner. Thus, for example: (i) there are significant mismatches between the distribution of peptides and their respective binding sites (9-11); (ii) peptide neurotransmitters can diffuse away from their site of release (12, 13) and can even be recovered in spinal cord cerebrospinal fluid (14); (iii) binding sites for ju (15) and 8 (16) opioid peptides and for neurotensin (17) rarely overlap synaptic densities; (iv) dense core vesicles that contain neuropeptides are usually located away from the synaptic density, which is the presumed site of release of classical neurotransmitters (18,19); and (v) the locus of exocytosis of dense core vesicles can, in fact, be distant from the synaptic junction (density) (20,21). In the present report we use an antiserum directed against the substance P (SP) receptor (SPR), which corresponds to the NK-1 subtype of tachykinin receptors (22), and demonstrate that there is indeed significant mismatch at the synaptic level between peptide and peptide receptor. Furthermore, we demonstrate that the SPR decorates a large proportion of the somatic and dendritic surface of subpopulations of CNS neurons, indicating that much of the neuronal surface is a potential target of peptide neurotransmitter. METHODSThe studies were performed on male Sprague-Dawley rats (240-260 g) that were deeply anesthetized with sodium pentobarbital (60 mg/kg) and perfused through the ascending aorta with 100 ml of 0.1 M sodium phosphate-buffered saline (PBS; pH 7.4) followed by a 0.1 M sodium phosphatebuffered fixative solution containing either 4.0%o paraformaldehyde (for light microscopy) or 2.0%o glutaraldehyde, 0.5% formaldehyde, and 0.2% picric acid (for both light and electron microscopy), according to the protocol of . After the perfusion the brain and spinal cord were removed and postfixed in the same solution for 2-4 hr.The au...

The rostral agranular insular cortex (RAIC) has recently been identified as a site where local changes in GABA and dopamine levels, or application of opioids, can alter nociceptive thresholds in awake animals. The connections of the cortex dorsal to the rhinal fissure that includes the RAIC have been examined previously, with emphasis on visceral and gustatory functions but not nociception. Here we examined the afferent and efferent connections of the RAIC with sites implicated in nociceptive processing. Sensory information from the thalamus reaches the RAIC via the submedius and central lateral nuclei and the parvicellular part of the ventral posterior nucleus. The RAIC has extensive reciprocal cortico-cortical connections with the orbital, infralimbic, and anterior cingulate cortices and with the contralateral RAIC. The amygdala, particularly the basal complex, and the nucleus accumbens are important targets of RAIC efferent fibers. Other connections include projections to lateral hypothalamus, dorsal raphe, periaqueductal gray matter, pericerulear region, rostroventral medulla, and parabrachial nuclei. The connectivity of the RAIC suggests it is involved in multiple aspects of pain behavior. Projections to the RAIC from medial thalamic nuclei are associated with motivational/affective components of pain. RAIC projections to mesolimbic/mesocortical ventral forebrain circuits are likely to participate in the sensorimotor integration of nociceptive processing, while its brainstem projections are most likely to contribute to descending pain inhibitory control.

The importance of glial cells in the generation and maintenance of neuropathic pain is becoming widely accepted. We examined the role of glial-specific gap junctions in nociception in the rat trigeminal ganglion in nerve-injured and -uninjured states. The connexin 43 (Cx43) gap-junction subunit was found to be confined to the satellite glial cells (SGCs) that tightly envelop primary sensory neurons in the trigeminal ganglion and we therefore used Cx43 RNA interference (RNAi) to alter gap-junction function in SGCs. Using behavioral evaluation, together with immunocytochemical and Western blot monitoring, we show that Cx43 increased in the trigeminal ganglion in rats with a chronic constriction injury (CCI) of the infraorbital nerve. Reducing Cx43 expression using RNAi in CCI rats reduced painlike behavior, whereas in non-CCI rats, reducing Cx43 expression increased painlike behavior. The degree of painlike behavior in CCI rats and intact, Cx43-silenced rats was similar. Our results support previous suggestions that increases in glial gap junctions after nerve injury increases nociceptive behavior but paradoxically the reduction of gap junctions in normal ganglia also increases nociceptive behavior, possibly a reflection of the multiple functions performed by glia.

Neurons in sensory ganglia are surrounded by satellite glial cells (SGCs) that perform similar functions to the glia found in the CNS. When primary sensory neurons are injured, the surrounding SGCs undergo characteristic changes. There is good evidence that the SGCs are not just bystanders to the injury but play an active role in the initiation and maintenance of neuronal changes that underlie neuropathic pain. In this article the authors review the literature on the relationship between SGCs and nociception and present evidence that changes in SGC potassium ion buffering capacity and glutamate recycling can lead to neuropathic pain-like behavior in animal models. The role that SGCs play in the immune responses to injury is also considered. We propose the term gliopathic pain to describe those conditions in which central or peripheral glia are thought to be the principal generators of principal pain generators.

Transneuronal tracing of a nociceptive pathway, the spino-(trigemino)-parabrachio-amygdaloid pathway, was performed using an ␣-herpes virus, the Bartha strain of pseudorabies virus (PRV). Microinjection of PRV into the central nucleus of the amygdala (Ce) resulted in progressive retrograde and transneuronal infection of a multisynaptic circuit involving neurons in the brainstem and spinal cord as detected immunocytochemically. At short survival (26 hr), retrogradely labeled neurons were concentrated in the external lateral nucleus of the parabrachial complex (elPB) but were absent from both the trigeminal nucleus caudalis (TNC) and the spinal cord. At longer survivals (52 hr), labeled cells were present in lamina I of both the TNC and spinal dorsal horn. Retrograde labeling from the Ce with Fluorogold demonstrated that elPB neurons have long dendrites extending laterally into the terminal field of spinal and trigeminal afferents, where transneuronal passage of PRV to these afferents could occur. Even longer survivals (76 hr) resulted in a columnar pattern of cell labeling in the TNC and spinal dorsal horn that extended from lamina I into lamina II. At this longest survival, primary sensory neurons became infected. Bilateral excitotoxic lesions of the elPB blocked almost all viral passage from the Ce to superficial laminae of the TNC and spinal dorsal horn. These results demonstrate that nociceptive input to the amygdala is relayed from neurons in lamina I through the elPB. We propose that this modular arrangement of lamina I and II neurons may provide the basis for spinal processing of peripheral input to the amygdala. Key words: pain pathways; amygdala; superficial dorsal horn; parabrachial nucleus; spinal processing; viral tracer; PRV; pseudorabies virusThe central nucleus of the amygdala (Ce) is the terminal area of a major ascending nociceptive pathway, the spino-(trigemino)-parabrachio-amygdaloid tract . Most Ce neurons respond to noxious, but not innocuous, stimuli (Bernard and Besson, 1990;. The main source of afferents to the Ce is the external lateral parabrachial nucleus (elPB) (Saper, 1995). Previous anatomical studies have, however, failed to demonstrate a significant projection from the spinal dorsal horns and trigeminal nucleus caudalis (TNC) to the elPB (Blomqvist et al., 1989). Rather, afferents from nociceptive regions of the dorsal horn, laminae I and V, terminate in nuclei located in the lateral parabrachial area surrounding the elPB, none of which project to the amygdala (Bernard et al., 1995;Feil and Herbert, 1995;Saper, 1995).To explain this discrepancy, it has been proposed that elPB neurons might have long dendrites that extend into the external nuclear layer where spinal and trigeminal afferents terminate (Blomqvist et al., 1989;Saper, 1995). There is ultrastructural evidence that spinal and trigeminal afferents contact amygdala projection neurons in the lateral parabrachial area (Ma and Peschanski, 1988), but in this study, neither the laminae of origin of the spinal and trigeminal neur...

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K(+) channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca(2+)-activated K(+) channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K(+)) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain.

Growing evidence suggests that changes in the ion buffering capacity of glial cells can give rise to neuropathic pain. In the CNS, potassium ion (K ϩ ) buffering is dependent on the glia-specific inward rectifying K ϩ channel Kir4.1. We recently reported that the satellite glial cells that surround primary sensory neurons located in sensory ganglia of the peripheral nervous system also express Kir4.1, whereas the neurons do not. In the present study, we show that, in the rat trigeminal ganglion, the location of the primary sensory neurons for face sensation, specific silencing of Kir4.1 using RNA interference leads to spontaneous and evoked facial pain-like behavior in freely moving rats. We also show that Kir4.1 in the trigeminal ganglion is reduced after chronic constriction injury of the infraorbital nerve. These findings suggests that neuropathic pain can result from a change in expression of a single K ϩ channel in peripheral glial cells, raising the possibility of targeting Kir4.1 to treat pain in general and particularly neuropathic pain that occurs in the absence of nerve injury.