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.

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.

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.

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

The effect of noxious stimulation on the expression of FOS-like immunoreactivity (FOS-LI) in neurons of the parabrachial nucleus (PB) was studied in awake, freely moving rats. In one series of experiments, the rats were subjected to noxious mechanical stimulation (pinch) of either the nape of the neck or the base of the tail for 20 seconds every 5 minutes for 90 minutes, and then they were killed by transcardial perfusion after 45-210 minutes. Control animals received innocuous mechanical stimulation (brush) of the tail. Noxious stimuli resulted in FOS-LI in neurons in the dorsal part of the lateral PB, with heavy labeling in the superior lateral (PBsl) and the dorsal lateral (PBdl) subnuclei. FOS-LI was also elicited in the central lateral subnucleus (PBcl) and, although much more sparsely, in the external lateral subnucleus and the Kölliker-Fuse nucleus. Tail and neck stimulation resulted in similar labeling patterns, but more neurons, particularly in PBsl, expressed FOS-LI after pinch of the tail than of the neck. In another series of experiments, rats received injection of 5% formalin into one hindpaw. After 75-90 minutes, FOS-LI was seen in the same parts of PB as after noxious mechanical stimulation. The heaviest labeling was seen on the side contralateral to the injection side, with statistically significant (P < 0.05) side differences present in PBsl and PBdl. In a third series of experiments, rats were hemisected at low cervical-upper thoracic segments, allowed 2 weeks to recover, and then given formalin injections in both hindpaws. Significantly more neurons were FOS-labeled in PBdl, PBsl, and PBcl on the side contralateral to the hemisection than on the ipsilateral side. These observations are discussed in relation to the organization of the spinal afferent input and the efferent connections of PB. It is concluded that the FOS-LI expression in PBdl and PBsl and probably also in PBcl, to a large extent, is evoked by the ascending spinal nociceptive input to PB. Because these subnuclei project to several hypothalamic regions, it is suggested that neurons in PB that express FOS after noxious mechanical and chemical stimulation primarily are involved in autonomic and homeostatic responses to behavioral situations that involve tissue-damaging stimuli.

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.

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.

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.

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.

Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.

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.

Preproenkephalin mRNA-expressing neurones in the rat thalamus.

We investigated the expression of preproenkephalin mRNA in the rat thalamus by in situ hybridization histochemistry using a radiolabelled full-length cRNA probe. Enkephalinergic neurones were present in the ventral thalamus, epithalamus, and intralaminar and midline nuclei of the dorsal thalamus. The largest number of labelled cell bodies was found in the zona incerta, the fields of Forel, and the ventral lateral geniculate, thalamic reticular, central medial and central lateral nuclei. Sparse labelling was present in the parafascicular, paracentral, paraventricular, and lateral habenular nuclei. The principal nuclei of the dorsal thalamus were unlabelled. The distribution of the enkephalinergic neurones suggests an involvement in arousal and attentional mechanisms, being in line with the sedative effects of exogenous opioids.

Ca2+/calmodulin-dependent kinase II immunoreactivity in the rat hypothalamus.

The distribution of Ca2+/calmodulin-dependent kinase II (CaMKII) in the rat hypothalamus was studied using immunohistochemistry. Differences in labelling density were seen both between different nuclei and within nuclei. Dense labelling of cell bodies and dendrites was present in several nuclei, including the medial preoptic, suprachiasmatic, ventromedial and arcuate nuclei. In the paraventricular nucleus many labelled neurones were seen in some parvocellular regions, whereas its posterior magnocellular part was unlabelled. Dense terminal-like labelling was present in, for example, the ventromedial nucleus and the supramammillary nucleus. We suggest that differences in CaMKII distribution reflect differences in the mechanisms of calcium signalling in these neurones, which may be related to their different neurochemical phenotypes and/or connective relationships.

Activation of a bulbospinal opioidergic projection by pain stimuli in the awake rat.

We examined the expression of preproenkephalin mRNA in pain-modulating regions of the rat's brainstem using in situ hybridization histochemistry. We found that neurones in the Kölliker-Fuse nucleus, which receives projections from spinal nociceptive-specific cells, express enkephalin mRNA, that this expression is increased by noxious pinch applied to the skin of awake animals, and that these enkephalinergic neurones are part of a descending system that terminates in the rostroventral medulla and the spinal cord. These findings show that natural noxious stimuli activate opioidergic bulbospinal neurones that could directly modulate spinal nociceptive transmission.

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.

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.

Enkephalinergic and catecholaminergic neurons constitute separate populations in the rat Kölliker-Fuse/A7 region.

Using a double-labeling immunohistochemical and in situ hybridization technique for the simultaneous detection of tyrosine hydroxylase and preproenkephalin mRNA, we demonstrate that catecholaminergic and enkephalinergic neurons in the Kölliker-Fuse nucleus (K-F)/A7 region in the dorsolateral pons constitute separate populations. The enkephalinergic cell group is much larger than the catecholaminergic cell group. Most of the enkephalinergic neurons are located caudal to the catecholaminergic neurons, but enkephalinergic neurons are also interspersed among the catecholaminergic neurons. Taken together with previous demonstrations that the enkephalinergic neurons in K-F give rise to descending projections to the ventrolateral medulla and spinal cord, the current observations suggest that the antinociceptive effects that result from electrical stimulation of K-F may be a consequence of the activation of enkephalinergic neurons, either alone or in conjunction with catecholaminergic neurons.

Estrogen receptor-like immunoreactivity in the medullary and spinal dorsal horn of the female rat.

Using an immunohistochemical technique, we demonstrate that large numbers of neurons in the laminar spinal trigeminal nucleus and spinal gray matter of the female rat express estrogen receptors (ER). Densely packed ER-immunoreactive neurons were present in lamina II, but labeled neurons were also present in lamina I, the neck of the dorsal horn, and in lamina X. Labeling was present throughout the length of the spinal cord, with the exception of segments caudal to S1, which were unlabeled. The distribution of ER-containing neurons to areas that are involved in processing of primary afferent nociceptive information suggests that the pain modulatory effects of estrogen may be exerted at the spinal level.

Preprovasopressin mRNA is not present in dorsal root ganglia of the rat.

Immunohistochemical studies on colchicine-treated rats have suggested that more than half of the neurons in dorsal root ganglia (DRG) contain vasopressin. Thus, vasopressin would be the most commonly found peptide in DRG neurons. In the present study we have reexamined the presence of vasopressin in DRG neurons, using a sensitive in situ hybridization method employing long riboprobes that will detect very small amounts of mRNA. The C3, C6, T2, T12, L2 and L5 DRG were studied. None of these ganglia contained any preprovasopressin mRNA. Yet, dense labeling for preprovasopressin mRNA was seen on simultaneously processed hypothalamic sections and a heavy preprotachykinin mRNA expression was seen in adjacent DRG sections. These findings demonstrate that vasopressin is not produced in DRG in normal rats.

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.