Molecular mechanisms of nociception in the rat brain : anatomical connections and trans-synaptic regulation of gene expression of neurons in the pontine parabrachial nucleus

• The pontine parabrachial nucleus (PB) is a major recipient of fibers fromnociceptive (pain-responsive) spinal and trigeminal dorsal horn neurons. With the use of combinations of molecular and morphological techniques, the anatomical connections of PB neurons and their expression of neuropeptide genes and transcription factors were studied, both in naive rats and in rats that had been subjected to nociceptive stimulation.A large number of neurons in PB expressed preproenkephalin mRNA(ppENK), which implies that these neurons synthesize the opioid enkephalin. One region of PB, the internal lateral subnucleus, contained many ppENKexpressing neurons that projected to the thalamic intralaminar nuclei, suggesting a role for these neurons in attentional mechanisms. Another region, the Kölliker-Fuse subnucleus (K-F), contained many ppENK-expressing neurons with descending projections to regions in the ventral medulla and spinal cord involved in pain control and autonomic modulation. The ppENKexpression in K-F increased after nociceptive stimulation of the neck or tail, suggesting that supraspinal opioidergic neurons with descending projections are activated by pain stimuli.Stimulation of the K-F region has been shown to elicit analgesia inanimals and humans. This analgesia has previously been ascribed to theactivation of noradrenergic neurons located in and close to K-F (the A7 cell group). Double-labeling procedures demonstrated that the ppENK-expressing neurons constituted a separate, much larger population, interspersed with the noradrenergic neurons. Thus, analgesia elicited by stimulation of K-F must involve the activation of opioidergic neurons.In search for the intracellular proteins that mediate the noxious-inducedup-regulation of ppENK, the expression of FOS in PB was studied after various types of nociceptive stimulation. FOS expression is low in PB of the naive animal, but FOS is rapidly produced after trans-synaptic activation, and it can bind to the DNA site that regulates ppENK transcription. However, noxiousevoked FOS was predominantly expressed in ppENK-negative neurons in PB. Another protein, CREB, can also regulate ppENK transcription. Therefore, the expression of active, phosphorylated, CREB was investigated in PB after nociceptive stimulation and was found to be present in the majority of theppENK-expressing neurons in K-F and the internal lateral subnucleus. These findings suggest that phosphorylation of CREB can mediate noxious-induced up-regulation of ppENK transcription in PB.Following nociceptive stimulation of the neck, tail, and hindlimb, most ofthe FOS was found in the dorsal and the superior lateral subnuclei of PB. The results suggest that neurons in these subnuclei are activated trans-synaptically by a direct spinal nociceptive input. The expression of FOS in PB after nociceptive stimulation of the face was found to be different from that seen after nociceptive stimulation of other parts of the body. Most of the FOSexpressing neurons were now detected in K-F, the external lateral, and the external medial subnuclei. This result is coherent with the termination pattern of fibers from the trigeminal dorsal horn, and suggests that neurons that are activated by nociceptive stimulation of the face have other functions than those activated by stimulation of other regions.Since FOS can bind to the ppCCK gene and regulate cholecystokininproduction, the parabrachial expression of preprocholecystokinin mRNA(ppCCK), which encodes the neuropeptide cholecystokinin, was investigated. Many ppCCK-expressing neurons were present in the superior lateral subnucleus, and these neurons displayed FOS after noxious stimulation. In addition, many ppCCK-expressing neurons in PBsl displayed the second messenger Ca2+/calmodulin-dependent kinase Il (CaMKII) and, after noxious stimulation, phosphorylated CREB. These findings point to a putative intracellular route for noxious-induced calcium-mediated regulation of ppCCK transcription through FOS induction via CaMKII and CREB. The ppCCKexpressing neurons in the superior lateral subnucleus were shown to project to the ventromedial hypothalamic nucleus, suggesting that they influence blood glucose homeostasis as a response to tissue damage.In search for a neuropeptide that is expressed by noxious-activated(FOS-1abeled) neurons in the dorsal lateral subnucleus, the preprodynorphin mRNA (ppDYN) expression was investigated. ppDYN encodes the opioid dynorphin, and the transcription of ppDYN can similar to other neuropeptide genes be regulated by the FOS protein. The dorsal lateral subnucleus was rich in ppDYN-expressing neurons, and many of the ppDYN-expressing neurons contained FOS, which implies that they are activated by nociceptive stimulation. The ppDYN-expressing neurons were found to project to the hypothalamic median preoptic nucleus, suggesting an involvel)lent of these neurons in cardiovascular regulation.In summary, the present study demonstrates that both opioidergic andnon-opioidergic neurons in a brainstem region, PB, are trans-synapticallyactivated by nociceptive stimulation in the awake rat. It is shown thatnociresponsive neurons in PB are organized in distinct cell groups that display different anatomical connections, intracellular signaling mechanisms, and gene expressions, suggesting different functions for these cell groups. It is suggested that nociresponsive parabrachial neurons influence the tuning and excitability of autonomic reflex circuits as part of an integrated response to tissue damage.

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