Tet3 mediates stable glucocorticoid-induced alterations in DNA methylation and Dnmt3a/Dkk1 expression in neural progenitors.

Developmental exposure to excess glucocorticoids (GCs) has harmful neurodevelopmental effects, which include persistent alterations in the differentiation potential of embryonic neural stem cells (NSCs). The mechanisms, however, are largely unknown. Here, we investigated the effects of dexamethasone (Dex, a synthetic GC analog) by MeDIP-like genome-wide analysis of differentially methylated DNA regions (DMRs) in NSCs isolated from embryonic rat cortices. We found that Dex-induced genome-wide DNA hypomethylation in the NSCs in vitro. Similarly, in utero exposure to Dex resulted in global DNA hypomethylation in the cerebral cortex of 3-day-old mouse pups. Dex-exposed NSCs displayed stable changes in the expression of the DNA methyltransferase Dnmt3a, and Dkk1, an essential factor for neuronal differentiation. These alterations were dependent on Tet3 upregulation. In conclusion, we propose that GCs elicit strong and persistent effects on DNA methylation in NSCs with Tet3 playing an essential role in the regulation of Dnmt3a and Dkk1. Noteworthy is the occurrence of similar changes in Dnmt3a and Dkk1 gene expression after exposure to excess GC in vivo.

Opposing effects of hMOF and SIRT1 on H4K16 acetylation and the sensitivity to the topoisomerase II inhibitor etoposide.

Various inhibitors of histone deacetylase (HDAC) activity can sensitize drug resistant cancer cells to chemotherapeutic agents. However, the mechanisms underlying such effects of distinct HDAC inhibitors (HDACi) remain poorly understood. Here we show that both the HDACi trichostatin A and valproic acid induced a sensitization of multidrug-resistant cancer cells to the topoisomerase II inhibitor etoposide/VP16. This effect was associated with increased acetylation of certain lysines on histones H3 and H4, including lysine 16 on histone H4 (H4K16). Overexpression of the histone acetyltransferase hMOF, known to target H4K16, was sufficient to mimic HDACi treatment on sensitization and H4K16 acetylation, and importantly, small-interfering RNA (siRNA)-mediated knockdown of hMOF abolished the HDACi-mediated sensitizing effects as well as the increase in H4K16 acetylation. Conversely, siRNA-mediated knockdown of the H4K16 deacetylase SIRT1 mimicked HDACi treatment whereas overexpression of SIRT1 abolished H4K16 acetylation and significantly reduced the sensitizing effects of HDACi. Interestingly, the effects of hMOF on H4K16 acetylation and sensitization to the topoisomerase II inhibitor could be directly counteracted by exogenous expression of increasing amounts of SIRT1 and vice versa. Our study results suggest that hMOF and SIRT1 activities are critical parameters in HDACi-mediated sensitization of multidrug-resistant cancer cells to topoisomerase II inhibitor and increased H4K16 acetylation.

Glucocorticoids induce long-lasting effects in neural stem cells resulting in senescence-related alterations.

Alterations in intrauterine programming occurring during critical periods of development have adverse consequences for whole-organ systems or individual tissue functions in later life. In this paper, we show that rat embryonic neural stem cells (NSCs) exposed to the synthetic glucocorticoid dexamethasone (Dex) undergo heritable alterations, possibly through epigenetic mechanisms. Exposure to Dex results in decreased NSC proliferation, with no effects on survival or differentiation, and changes in the expression of genes associated with cellular senescence and mitochondrial functions. Dex upregulates cell cycle-related genes p16 and p21 in a glucocorticoid receptor(GR)-dependent manner. The senescence-associated markers high mobility group (Hmg) A1 and heterochromatin protein 1 (HP1) are also upregulated in Dex-exposed NSCs, whereas Bmi1 (polycomb ring finger oncogene) and mitochondrial genes Nd3 (NADH dehydrogenase 3) and Cytb (cytochrome b) are downregulated. The concomitant decrease in global DNA methylation and DNA methyltransferases (Dnmts) suggests the occurrence of epigenetic changes. All these features are retained in daughter NSCs (never directly exposed to Dex) and are associated with a higher susceptibility to oxidative stress, as shown by the increased occurrence of apoptotic cell death on exposure to the redox-cycling reactive oxygen species (ROS) generator 2,3-dimethoxy-1-naphthoquinone (DMNQ). Our study provides novel evidence for programming effects induced by glucocorticoids (GCs) on NSCs and supports the idea that fetal exposure to endogenous or exogenous GCs is likely to result in long-term consequences that may predispose to neurodevelopmental and/or neurodegenerative disorders.

p57Kip2 is a repressor of Mash1 activity and neuronal differentiation in neural stem cells.

Mammalian central nervous system (CNS) development is a highly organized process involving the precise and coordinated timing of cell-cycle exit, differentiation, survival, and migration. These events require proper expression of pro-neuronal genes but also repression of alternative cell fates and restriction of cell-type-specific gene expression. Here, we show that the cyclin-dependent kinase (CDK) inhibitor p57Kip2 interacted with pro-neuronal basic helix-loop-helix (bHLH) factors such as Mash1, NeuroD, and Nex/Math2. Increased levels of p57Kip2 inhibited Mash1 transcriptional activity independently of CDK interactions and acted as a direct repressor in transcriptional assays. Proliferating telencephalic neural progenitors co-expressed basal levels of Mash1 and p57Kip2, and endogenous p57Kip2 accumulated transiently in the nuclei of neural stem cells (NSCs) during early stages of astrocyte differentiation mediated by ciliary neurotrophic factor (CNTF), independent of cell-cycle exit and at times when Mash1 expression was still prominent. In accordance with these observations, gain- and loss-of-function studies showed that p57Kip2 repressed neuronal differentiation after mitogen withdrawal, but exerted little or no effect on CNTF-mediated astroglial differentiation of NSCs. Our data suggest a novel role for p57Kip2 as a context-dependent repressor of neurogenic transcription factors and telencephalic neuronal differentiation.

Combinatorial action of the HDAC inhibitor trichostatin A and etoposide induces caspase-mediated AIF-dependent apoptotic cell death in non-small cell lung carcinoma cells.

Commonly used regimens in cancer therapy rely on the induction of apoptotic cell death, and drug resistance can be attributed, at least in part, to a disabled apoptotic program. Non-small cell lung carcinomas (NSCLC), exhibit an intrinsic resistance to chemotherapy. Here, we show that co-treatment with etoposide (VP16) and the pan-histone deacetylase (HDAC) inhibitor trichostatin A (TSA), but not valproic acid (VPA), induced apoptotic cell death in drug-resistant NSCLC cells. Co-treatment, but not single treatment, with VP16 and TSA induced apoptosis in a caspase-dependent manner accompanied by a crucial decrease in Bcl-xL expression allowing Bax activation and subsequent initiation of the apoptosis inducing factor (AIF)-dependent death pathway. Importantly, AIF proved to be required for the effects of TSA/VP16 as RNA knockdown of AIF resulted in a complete abolishment of TSA/VP16-induced apoptotic cell death in drug-resistant NSCLC cells. Our results thus provide evidence for the requirement of both caspase-dependent and caspase-independent apoptotic pathways in TSA/VP16-mediated death of drug-resistant NSCLC cells, and extend previous suggestions that HDAC inhibitors in combination with conventional chemotherapeutic drugs could be valuable in the treatment of NSCLC cancer and other malignancies in which Bcl-xL is overexpressed.

Regulation of somatic growth by the p160 coactivator p/CIP.

A family of p160 coactivators was initially identified based on ligand-dependent interactions with nuclear receptors and thought to function, in part, by recruiting CREB-binding protein/p300 to several classes of transcription factors. One of the p160 factors, p/CIP/AIB1, often amplified and overexpressed in breast cancer, also exhibits particularly strong interaction with CREB-binding protein/p300. In this manuscript, we report that p/CIP, which exhibits regulated transfer from cytoplasm to nucleus, is required for normal somatic growth from embryonic day 13.5 through maturity. Our data suggest that a short stature phenotype of p/CIP gene-deleted mice reflect both altered regulation of insulin-like growth factor-1 (IGF-1) gene expression in specific tissues and a cell-autonomous defect of response to IGF-1, including ineffective transcriptional activities by several classes of regulated transcription factors under specific conditions. The actions of p/CIP are therefore required for full expression of a subset of genes critical for regulating physiological patterns of somatic growth in mammals.

Combinatorial roles of the nuclear receptor corepressor in transcription and development.

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2.

Corticotropin-releasing hormone (Crh), a 41-residue polypeptide, activates two G-protein-coupled receptors, Crhr1 and Crhr2, causing (among other transductional events) phosphorylation of the transcription factor Creb. The physiologic role of these receptors is only partially understood. Here we report that male, but not female, Crhr2-deficient mice exhibit enhanced anxious behaviour in several tests of anxiety in contrast to mice lacking Crhr1. The enhanced anxiety of Crhr2-deficient mice is not due to changes in hypothalamic-pituitary-adrenal (HPA) axis activity, but rather reflects impaired responses in specific brain regions involved in emotional and autonomic function, as monitored by a reduction of Creb phosphorylation in male, but not female, Crhr2-/- mice. We propose that Crhr2 predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of Crh mediated by Crhr1. Neither male nor female Crhr2-deficient mice show alterations of baseline feeding behaviour. Both respond with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of Crhr2 in vascular permeability is independent of gender.

Distribution of the transcription factor signal transducer and activator of transcription 3 in the rat central nervous system and dorsal root ganglia.

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that acts as an intracellular signalling molecule after receptor activation by several cytokines, e.g., interleukin-6, leptin and ciliary neurotrophic factor. We have investigated the localization of STAT3 in the rat central nervous system and dorsal root ganglia. Light microscopic immunohistochemistry showed that STAT3-like immunoreactivity (STAT3-LI) was present in the nucleus and cytoplasm of neurons. STAT3-LI was seen both in cell bodies and in proximal and distal dendrites. Many structures involved in motor functions, such as the ventral horn of the spinal cord, the motor cranial nerve nuclei, the red nucleus and the Purkinje cells of the cerebellum showed STAT3-LI. STAT3-LI was also present in many regions involved in autonomic regulation, such as the intermediolateral cell column of the spinal cord, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus nerve, the area postrema, the locus coeruleus, the Barrington's nucleus and the arcuate, the lateral, the dorsomedial, the ventromedial, and the paraventricular hypothalamic nuclei. Other structures showing STAT3-LI were the dorsal root ganglia, the thalamus (the anterodorsal and paraventricular nucleus), the cerebral neocortex (layer 5) and the olfactory bulb. The wide distribution of STAT3-LI in the nervous system is consistent with reports of cytokine actions in the brain, but the present findings further suggest novel roles for STAT3 in mediating influences of cytokines on specific neuronal circuits regulating motor, sensory and autonomic functions.

Progesterone receptor expression in the brainstem of the female rat.

By using in situ hybridization and immunohistochemistry, the presence of neurons expressing progesterone receptor mRNA (PR mRNA) and progesterone receptor-like immunoreactivity (PR-LI) was examined in the brainstem and spinal cord of female rats. Neurons expressing PR mRNA and PR-LI were seen in the ventrolateral medulla, the parvocellular reticular formation and the nucleus of the solitary tract. PR mRNA, but not PR-LI, was seen in the hypoglossal nucleus, the inferior olive, the locus coeruleus and the parabrachial nucleus. No consistent labeling was present in the spinal cord. These findings show that progesterone receptors are expressed in brainstem areas involved in various functions, including autonomic regulation and pain modulation.

Expression of LMO-4 in the central nervous system of the embryonic and adult mouse.

LMO (LIM-only) proteins constitute a family of coregulators thought to act as adapters in transcriptionally active complexes. In this manuscript, we describe in situ hybridization studies to elucidate the expression of the recently identified LMO-4 in embryonic and adult mouse brain. We found that LMO-4 expression was very widespread at early (E10.5-11.5) embryonic stages with more intense hybridization signal in certain regions. The expression became more regionalized over mid (E14.5-15.5) and late (E17.5-P0) embryonic stages. Throughout development the ventricular zone, consisting of less differentiated cells, showed less intense signal than the subventricular and marginal zones. Further, expression of LMO-4 in and surrounding myelinating axons, representing Schwann cells, was seen at late embryonic stages. In the adult brain, a restricted number of regions displayed significant LMO-4 expression. Interestingly, these were the same regions that showed most consistent and intense hybridization signal during embryonic development. These regions included the cerebral cortex (layer 2-6), the hippocampus (e.g., the CA3 layer), the dentate gyrus, the basal ganglia, the lateral amygdala, olfactory structures, the paraventricular, dorsomedial and ventromedial hypothalamic nuclei, zona incerta, intralaminar and reticular nuclei of the ventral thalamus, medullary motor structures and the cerebellum. In summary, the regionalized expression of LMO-4 during development suggest a role for this gene in differentiation and specification of specific brain regions involved in various functions, such as autonomic, motor and neuroendocrine regulation.

Estrogen-induced alterations of spinal cord enkephalin gene expression.

Enkephalin-synthesizing neurons in the superficial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P<0.05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P<0.05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.

Distribution of preprovasopressin mRNA in the rat central nervous system.

Vasopressin released in the central nervous system has been shown to be involved both in homeostatic mechanisms (e.g., water balance, thermoregulation, cardiovascular regulation, metabolism, and antinociception) and in higher brain functions (e.g., social recognition and communication, and learning and memory). Many nuclear groups have been proposed to synthesize vasopressin, but available data are conflicting. We have used a sensitive in situ hybridization technique to identify the distribution of the neurons that may be the origin of the vasopressin in the central nervous system of the male Sprague-Dawley rat. Vasopressin mRNA-expressing neurons were most abundant in the hypothalamus (e.g., the paraventricular, supraoptic, and suprachiasmatic nuclei) but were also seen in the medial amygdaloid nucleus, the bed nucleus of stria terminalis, and the nucleus of the horizontal diagonal band. Previously unreported vasopressinergic neurons were seen in the entorhinal and piriform cortices, the ventral lateral portion of the parabrachial nucleus, the pedunculopontine nucleus, and the rostral part of the ventral periaqueductal gray matter and the adjacent portion of the mesencephalic reticular nucleus. Vasopressin mRNA expression suggestive of neuronal labeling was seen in the pyramidal layer of the CA1-3 fields and the dentate gyrus of the hippocampus. In addition, vasopressin mRNA expression, probably representing axonal mRNA, was detected over the hypothalamopituitary tract. No or insignificant preprovasopressin mRNA expression was present in the cerebellum, locus coeruleus, subcoeruleus, or the spinal cord. These findings provide novel information on the distribution of vasopressin neurons that are important for our understanding of how vasopressin acts in the brain.

Distribution of CREB-binding protein immunoreactivity in the adult rat brain.

We demonstrate the expression of the co-activator CREB-binding protein (CBP) in the nuclei of a large number of neurons and glial structures in the rat brain and spinal cord. Immunoblotting of nuclear extracts revealed a single band at 265 kDa, the size of CBP. We found that CBP immunoreactivity was localized to cholecystokinin mRNA-expressing neurons in the hippocampus and the thalamus, suggesting that CBP may be involved in long-term memory and modulation of cortical activity. However, CBP-labeling was not ubiquitous, and many brain regions, including several mesencephalic and diencephalic nuclei, showed sparse labeling. Further, the number of neurons displaying intense CBP-labeling varied across animals in some regions, e.g., the hippocampus and the amygdala. Since competition for limited amounts of CBP and CBP-related molecules has been shown to be important for the integration of intracellular signaling pathways with transcriptional regulation, the present results suggest that varying endogenous levels of CBP in post-mitotic neurons is an important parameter in neuronal transcriptional regulation.

Preprocholecystokinin mRNA-expressing neurons in the rat parabrachial nucleus: subnuclear localization, efferent projection, and expression of nociceptive-related intracellular signaling substances.

The pontine parabrachial nucleus (PB) is a major target for ascending fibers from nociresponsive dorsal horn neurons. Several different neuropeptides have been identified in the PB. By using double-labeling methods that combine in situ hybridization histochemistry with retrograde tract tracing and immunohistochemistry, we have examined the subnuclear localization of preprocholecystokinin mRNA (ppCCK)-containing neurons, investigated their efferent projection, and analyzed their expression of intracellular signaling substances that may be of importance for nociceptive processing. The results show that neurons containing ppCCK are preferentially localized to the superior lateral subnucleus (PBsl), whereas other subnuclei, such as the dorsal lateral, external lateral, central lateral, and ventral lateral subnuclei, and the Kölliker-Fuse nucleus, contain only moderate to small numbers of such neurons. Injections of the retrograde tracer cholera toxin subunit b into the ventromedial hypothalamus demonstrated that ppCCK-containing neurons in PBsl were projection neurons. Following nociceptive stimulation, the ppCCK-containing neurons expressed FOS protein as well as phosphorylated cyclic AMP-responsive element-binding protein (CREB). In addition, Ca2+/calmodulin-dependent kinase II (CaMKII) was heavily and rather selectively expressed in PBsl and was co-localized to ppCCK-containing neurons. These observations show that nociceptive stimuli activate a cholecystokinin pathway from the parabrachial nucleus to the ventromedial hypothalamus that may be important for homeostatic responses to tissue damage, and point to a putative intracellular route for Ca2+-mediated FOS transcription via CaMKII and CREB for the regulation of ppCCK transcription.

Preprodynorphin mRNA-expressing neurones in the rat parabrachial nucleus: subnuclear localization, hypothalamic projections and colocalization with noxious-evoked fos-like immunoreactivity.

The dorsal lateral subnucleus of the rat pontine parabrachial nucleus is a major target for ascending nociceptive information from the spinal cord. With in situ hybridization histochemistry, using a radiolabelled cRNA probe, we demonstrate that neurones in and near the dorsal lateral subnucleus express preprodynorphin mRNA. The cRNA probe was constructed from a PCR product amplified from rat genomic DNA. Sequencing of the PCR product revealed that it corresponded to the sequence 466-1101 of the rat preprodynorphin gene exon 4. Tract tracing experiments, using injection of cholera toxin subunit B into the hypothalamic median preoptic nucleus, showed a retrograde labelling pattern of neurones in the parabrachial nucleus that was almost identical to that of the preprodynorphin mRNA expressing neurones. Double-labelling, combining immunohistochemical detection of tracer and in situ hybridization, revealed that the retrogradely labelled neurones expressed preprodynorphin mRNA. A similar double-labelling, combining in situ hybridization with immunohistochemical detection of noxious-evoked fos following formalin injection into one hindpaw of awake animals, showed that almost all fos-immunoreactive neurones in the dorsal lateral parabrachial subnucleus also expressed preprodynorphin mRNA. Quantitative analysis suggested that the evoked fos immunoreactivity was accompanied by an increased preprodynorphin mRNA expression. The findings provide evidence that neurones in the dorsal lateral subnucleus produce dynorphin and project to the median preoptic nucleus, and that noxious stimulation in awake animals synaptically activates the dynorphinergic neurones in this subnucleus. These observations are consistent with the idea of a functional and chemical heterogeneity among different parabrachial subnuclei that serves to produce specific homeostatic responses to stimuli that changes the physiological status of the organism, including tissue damage.

Subnuclear localization of FOS-like immunoreactivity in the parabrachial nucleus after orofacial nociceptive stimulation of the awake rat.

We used FOS-like immunohistochemistry to detect neuronal activity in the pontine parabrachial nucleus after injection of formalin into the lower lip of the awake rat and compared the labeling pattern with that seen after formalin injection into the hindpaw. One hour after a formalin injection into the lip, many FOS-immunoreactive cells were seen in the parabrachial nucleus, preferentially on the side ipsilateral to the injection site. Detailed anatomical analysis revealed that FOS-immunoreactive neurons were localized predominantly to three regions of the parabrachial nucleus: the external lateral, the external medial, and the Kölliker-Fuse subnuclei, with sparser labeling present in the dorsal and superior lateral subnuclei and in the medial parabrachial nucleus. In contrast, a formalin injection into the hindpaw resulted in dense FOS-labeling in the superior, dorsal, and central lateral subnuclei, with sparse to moderate labeling in the Kölliker-Fuse nucleus, and sparse labeling in the external lateral and external medial subnuclei, as described previously (Hermanson and Blomqvist, J. Comp. Neurol., [1996] 368:45-56). The distribution of FOS-labeled neurons after noxious orofacial stimulation corresponds to the termination pattern in the parabrachial nucleus of fibers that originate from neurons in the marginal zone of the trigeminal dorsal horn and is different from that seen after nociceptive stimulation of other body parts. Considering the differences in efferent connections of parabrachial subnuclei, the present findings imply that noxious information from the orofacial region to the parabrachial nucleus has other functional roles than noxious information from the trunk and limbs. Such roles may include the integration of somatosensory and gustatory information, which has been suggested to be of importance for feeding behavior.

Preproenkephalin messenger RNA-expressing neurons in the rat parabrachial nucleus: subnuclear organization and projections to the intralaminar thalamus.

The pontine parabrachial nucleus, which is a key structure in the central processing of autonomic, nociceptive and gustatory information, is rich in a variety of neuropeptides. In this study we have analysed the distribution of parabrachial neurons that express preproenkephalin messenger RNA, which encodes for the precursor protein for enkephalin opioids. Using an in situ hybridization method, we found that preproenkephalin messenger RNA-expressing neurons were present in large numbers in four major areas of the parabrachial nucleus: the Kölliker-Fuse nucleus, the external lateral subnucleus, the ventral lateral subnucleus, and in and near the internal lateral subnucleus. Many preproenkephalin messenger RNA-expressing neurons were also seen in the central lateral subnucleus, and in the medial and external medial subnuclei. Few labeled neurons were found in the dorsal and superior lateral subnuclei. Injection of the retrograde tracer substance cholera toxin subunit B into the midline and intralaminar thalamus demonstrated that the enkephalinergic neurons in and near the internal lateral subnucleus were thalamic-projecting neurons. Taken together with the results of previous tract-tracing studies, the present findings show that many of the enkephalinergic cell groups in the parabrachial nucleus are located within the terminal zones of the ascending projections that originate from nociresponsive neurons in the medullary dorsal horn and spinal cord, as well as from viscerosensory neurons within the nucleus of the solitary tract. The enkephalinergic neurons in the parabrachial nucleus may thus transmit noci- and visceroceptive-related information to their efferent targets. On the basis of the present and previous observations, we conclude that these targets include the intralaminar and midline thalamus, the ventrolateral medulla and the spinal cord. Through these connections, nociceptive and visceroceptive stimuli may influence several functions, such as arousal, respiration and antinociception.

Thalamic-projecting preprocholecystokinin messenger RNA-expressing neurons in the dorsal column nuclei of the rat.

This study aimed at investigating the expression of preprocholecystokinin messenger RNA among thalamic-projecting neurons in the dorsal column nuclei of the rat. Thalamic-projecting neurons were identified by injection of cholera toxin subunit b into the ventroposterolateral nucleus. Following immunohistochemical detection of retrogradely transported tracer substance, the expression of preprocholecystokinin messenger RNA in the projection neurons of the dorsal column nuclei was detected by in situ hybridization, using autoradiographic visualization of a 35S-labeled RNA probe complementary to preprocholecystokinin messenger RNA. Many preprocholecystokinin-expressing neurons were seen in the dorsal column nuclei. A large proportion of these neurons were also labeled with cholera toxin. The double-labeled neurons, as well as neurons single-labeled with preprocholecystokinin messenger RNA or cholera toxin, were preferentially found within the middle region of the dorsal column nuclei, located just caudal to the obex. These findings demonstrate that neurons in the dorsal column nuclei express preprocholecystokinin messenger RNA, and show that these neurons provide a peptidergic projection from the dorsal column nuclei to the ventroposterolateral nucleus of the thalamus. These observations suggest that cholecystokinin may be involved in the transmission of somatosensory (tactile) information from the dorsal column nuclei to the thalamus.

Differential expression of the AP-1/CRE-binding proteins FOS and CREB in preproenkephalin mRNA-expressing neurons of the rat parabrachial nucleus after nociceptive stimulation.

Several subgroups in the brainstem parabrachial nucleus (PB), which is a major target for nociresponsive neurons in the medullary and spinal dorsal horn, contain large numbers of preproenkephalin (ppENK) mRNA-expressing neurons. To elucidate how noxious stimuli may regulate ppENK transcription in these neurons, we have in the present study investigated whether immunoreactivity for the transcription factors FOS and phosphorylated CREB (pCREB), respectively, is displayed in the ppENK mRNA-expressing neurons after peripheral nociceptive stimulation. Rats received injection of formalin into one hindpaw, and were killed 30-80 min later. With a combination of immunohistochemistry and in situ hybridization, we found that only a small number of ppENK mRNA-expressing neurons in PB displayed FOS-immunoreactivity after nociceptive stimulation. In contrast, large numbers of ppENK mRNA-expressing neurons displayed pCREB-like immunoreactivity after nociceptive stimulation. Most of the ppENK mRNA/pCREB-expressing neurons were found in the Kölliker-Fuse and internal lateral subnuclei, but many double-labeled cells were also seen in the ventral lateral and central lateral subnuclei. In addition, a cluster of ppENK mRNA/pCREB-expressing neurons was found in the medial part of the medial parabrachial nucleus. Our findings suggest that CREB rather than FOS regulates nociceptive-related second messenger activation of ppENK transcription in parabrachial neurons.