Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

Bone marrow stromal cells attenuate injury-induced changes in galanin, NPY and NPY Y1-receptor expression after a sciatic nerve constriction.

Single ligature nerve constriction (SLNC) of the rat sciatic nerve triggers neuropathic pain-related behaviors and induces changes in neuropeptide expression in primary afferent neurons. Bone marrow stromal cells (MSCs) injected into the lumbar 4 (L4) dorsal root ganglia (DRGs) of animals subjected to a sciatic nerve SLNC selectively migrate to the other ipsilateral lumbar DRGs (L3, L5 and L6) and prevent mechanical and thermal allodynia. In this study, we have evaluated the effect of MSC administration on the expression of the neuropeptides galanin and NPY, as well as the NPY Y(1)-receptor (Y(1)R) in DRG neurons. Animals were subjected to a sciatic nerve SLNC either alone or followed by the administration of MSCs, phosphate-buffered saline (PBS) or bone marrow non-adherent mononuclear cells (BNMCs), directly into the ipsilateral L4 DRG. Seven days after injury, the ipsilateral and contralateral L4-5 DRGs were dissected out and processed for standard immunohistochemistry, using specific antibodies. As previously reported, SLNC induced an ipsilateral increase in the number of galanin and NPY immunoreactive neurons and a decrease in Y(1)R-positive DRG neurons. The intraganglionic injection of PBS or BNMCs did not modify this pattern of expression. In contrast, MSC administration partially prevented the injury-induced changes in galanin, NPY and Y(1)R expression. The large number of Y(1)R-immunoreactive neurons together with high levels of NPY expression in animals injected with MSCs could explain, at least in part, the analgesic effects exerted by these cells. Our results support MSC participation in the modulation of neuropathic pain and give insight into one of the possible mechanisms involved.

Galanin decreases proliferation of PC12 cells and induces apoptosis via its subtype 2 receptor (GalR2).

Galanin is a neuropeptide with a wide range of effects in the nervous and endocrine systems, mediated through three G protein-coupled receptor subtypes (GalR1-3). Interestingly, galanin and its receptors are also expressed in certain tumors. Here we studied the effects of galanin in rat pheochromocytoma (PC12) cells stably transfected with GFP-tagged GalR2. Galanin at 100 nM inhibited cell proliferation in both nontransfected and transfected cells. Conversly, both galanin and the GalR2(R3)-agonist AR-M1896 induced caspase-dependent apoptotic cell death only in GalR2-transfected cells. Western-blot analyses of downstream mediators of the G(q/11)-type G protein showed down-regulation of pAkt and pBad in galanin-exposed transfected cells. Also, the specific PI3 kinase inhibitor LY-294002 increased the level of pBad and decreased activation of caspases. In addition, p21(cip1) levels were up-regulated in galanin-exposed PC12 cells and down-regulated in galanin-exposed GalR2-transfected cells. In agreement, FACS analyses of galanin exposed cells showed occurrence of cell cycle arrest in PC12 cells and cell death in transfected cells. Finally, as shown with real-time PCR, galanin and its receptors were expressed at very high levels in human pheochromocytoma tissues as compared with normal adrenal medulla. These findings point to GalR2 as a possible target for therapeuthic interventions in pheochromocytoma.

Galanin gene transfer curtails generalized seizures in kindled rats without altering hippocampal synaptic plasticity.

Gene therapy-based overexpression of endogenous seizure-suppressing molecules represents a promising treatment strategy for epilepsy. Viral vector-based overexpression of the neuropeptide galanin has been shown to effectively suppress generalized seizures in various animal models of epilepsy. However, it has not been explored whether such treatment can also prevent the epileptogenesis. Using a recombinant adeno-associated viral (rAAV) vector, we induced hippocampal galanin overexpression under the neuron specific enolase promoter in rats. Here we report that in animals with galanin overexpression, the duration of electrographic afterdischarges was shortened and initiation of convulsions was delayed at generalized seizure stages. However, the hippocampal kindling development was unchanged. Short-term plasticity of mossy fiber-cornu ammonis (CA) 3 synapses was unaltered, as assessed by paired-pulse and frequency facilitation of field excitatory postsynaptic potentials (fEPSPs) in hippocampal slices, suggesting that despite high transgene galanin expression, overall release probability of glutamate in these synapses was unaffected. These data indicate that hippocampal rAAV-based galanin overexpression is capable of mediating anticonvulsant effects by lowering the seizure susceptibility once generalized seizures are induced, but does not seem to affect kindling development or presynaptic short-term plasticity in mossy fibers.

Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.

Completion of the Human Genome Project and recent developments in proteomics make it possible to systematically generate affinity reagents to a large portion of the proteome. Recently an antibody-based human protein atlas covering many organs including four areas of the brain has been released (www.proteinatlas.org). Due to the heterogeneity, size, and availability of tissue a more thorough analysis of the human brain is associated with considerable difficulties. Here we applied 120 antibodies raised against 112 human gene products to the smaller rat brain, a rodent animal model, where a single section represents a 'superarray' including many brain areas, and consequently allowing analysis of a huge number of cell types and their neurochemicals. Immunoreactive structures were seen in the investigated brain tissue after incubation with 56 antibodies (46.6%), of which 25 (20.8%) showed a clearly discrete staining pattern that was limited to certain areas, or subsets of brain cells. Bioinformatics, pre-adsorption tests and Western blot analysis were applied to identify non-specific antibodies. Eleven antibodies, including such raised against four 'ambiguous' proteins, passed all validation criteria, and the expression pattern and subcellular distribution of these proteins were studied in detail. To further explore the potential of the systematically generated antibodies, all 11 antibodies that passed validation were used to analyze the spinal cord and lumbar dorsal root ganglia after unilateral transection of the sciatic nerve. Discrete staining patterns were observed for four of the proteins, and injury-induced regulation was found for one of them. In conclusion, the study presented here suggests that a significant portion (10%) of the antibodies generated to a human protein can be used to analyze orthologues present in the rodent brain and to produce a protein-based atlas of the rodent brain. It is hoped that this type of antibody-based, high throughput screening of brain tissue from various rodent disease models will provide new information on the brain chemical neuroanatomy and insights in processes underlying neurological pathologies.

Generation and phenotypic characterization of a galanin overexpressing mouse.

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.

Suppressed kindling epileptogenesis in mice with ectopic overexpression of galanin.

The neuropeptide galanin has been shown to suppress epileptic seizures. In cortical and hippocampal areas, galanin is normally mainly expressed in noradrenergic afferents. We have generated a mouse overexpressing galanin in neurons under the platelet-derived growth factor B promoter. RIA and HPLC analysis revealed up to 8-fold higher levels of galanin in transgenic as compared with wild-type mice. Ectopic galanin overexpression was detected especially in dentate granule cells and hippocampal and cortical pyramidal neurons. Galanin-overexpressing mice showed retardation of seizure generalization during hippocampal kindling, a model for human complex partial epilepsy. The high levels of galanin in mossy fibers found in the transgenic mice were further increased after seizures. Frequency facilitation of field excitatory postsynaptic potentials, a form of short-term synaptic plasticity assessed in hippocampal slices, was reduced in mossy fiber-CA3 cell synapses of galanin-overexpressing mice, indicating suppressed glutamate release. This effect was reversed by application of the putative galanin receptor antagonist M35. These data provide evidence that ectopically overexpressed galanin can be released and dampen the development of epilepsy by means of receptor-mediated action, at least partly by reducing glutamate release from mossy fibers.

Expression of neurotrophin receptors in rat testis. Upregulation of TrkA mRNA with hCG treatment.

We report the expression of TrkA, TrkB and TrkC mRNAs in adult rat testis. With in situ hybridisation a low signal for TrkB and TrkC could be seen in postmeiotic cells of the seminiferous epithelium, whereas no signal for TrkA could be observed in untreated animals. Animals treated with hCG showed an induction of TrkA mRNA in premeiotic cells 12 h after the treatment, whereas an injection with EDS had no effect on the expression of Trk mRNAs. With the RNAse protection assay a low signal for TrkA was seen in whole testis of hCG treated animals. In staged tubules low expression was seen at stages VII-XI of untreated animals. Animals injected with hCG revealed that TrkA induction was highest during stages VIIcd and VIII of the cycle. The distinct expression pattern of these high-affinity neurotrophin receptors suggests different roles for neurotrophins during spermatogenesis. Induction of TrkA mRNA by hCG suggests that high-affinity binding of NGF during stages VIIcd-VIII in premeiotic cells is under control of the hypothalamic-pituitary-testicular axis.

Dopamine D(2) receptors regulate tyrosine hydroxylase activity and phosphorylation at Ser40 in rat striatum.

In the striatum, dopamine release is inhibited by activation of dopamine D(2) autoreceptors. Changes in dopamine release have been attributed to changes in the synthesis of dopamine, which is regulated via phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines. Here, we have studied the involvement of dopamine D(2) receptors in the regulation of TH phosphorylation at distinct seryl residues, using phosphorylation site-specific antibodies and a preparation of rat striatal slices. The D(2) receptor agonist, quinpirole, reduced basal TH phosphorylation at Ser40 but not at Ser19 or Ser31. Quinpirole was also able to reduce the increase in Ser40 phosphorylation caused by forskolin, an activator of adenylyl cyclase, without affecting the increase in Ser19 phosphorylation produced by the glutamate receptor agonist, N-methyl-D-aspartate (NMDA). In addition, the dopamine D(2) receptor agonist reduced both basal and forskolin-stimulated activity of TH, measured as 3,4-dihydroxyphenylalanine (DOPA) accumulation. Quinpirole decreased phosphorylation of Ser40 induced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A and Ro-20-1724, a phosphodiesterase inhibitor. In contrast, quinpirole did not affect the increase in Ser40 phosphorylation caused by the cAMP analogue, 8-Br-cAMP. These data indicate that, in the striatum, activation of dopamine D(2) receptors results in selective inhibition of TH phosphorylation at Ser40 via reduction of the activity of adenylyl cyclase. They also provide a molecular mechanism accounting for the ability of dopamine D(2) autoreceptors to inhibit dopamine synthesis and release from nigrostriatal nerve terminals.

Potent and nontoxic antisense oligonucleotides containing locked nucleic acids.

Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum.

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.

Leukemia inhibitory factor null mice: unhampered in vitro outgrowth of sensory axons but reduced stimulatory potential by nerve segments.

Leukemia inhibitory factor (LIF) is locally up-regulated after peripheral nerve injury and may be involved in the subsequent regeneration. Here, adult mice with or without LIF gene deletions were used to study the role of LIF in regeneration. The results show that axonal regeneration in vitro from dorsal root ganglia (DRGs) was unaffected by LIF deletion. However, segments from wild type mice promoted DRG axonal outgrowth better than segments from LIF deleted animals when in vivo-injured sciatic nerve segments were co-cultured with DRGs from normal adult mice. Addition of LIF could not restore the deficit. This suggests that LIF is engaged in the local regulation of regeneration but not in the regenerative events occuring at the cell body level.

Regulation of galanin and neuropeptide Y in dorsal root ganglia and dorsal horn in rat mononeuropathic models: possible relation to tactile hypersensitivity.

The expression of galanin and neuropeptide Y in rat lumbar 5 (L5) dorsal root ganglia and dorsal horn (L4-5) was studied after four types of peripheral nerve injury using immunohistochemistry and in situ hybridization. The possible correlation between these two peptides and tactile allodynia-like behaviour was analysed as well. The models employed were the Gazelius (photochemical lesion) and Seltzer and Bennett (constriction lesions) models, as well as complete sciatic nerve transection (axotomy). Two weeks after surgery, the Gazelius model rats more frequently displayed a greater tactile allodynia than the rats from the Seltzer and Bennett models. Tactile allodynia was not observed in any of the axotomized rats. A marked increase in the number of galanin-immunoreactive and galanin messenger RNA-positive neuron profiles was observed in ipsilateral dorsal root ganglia in all types of models. The increase in allodynic rats (Gazelius, Seltzer and Bennett models) was less pronounced than that after axotomy. In addition, in the Bennett model the number of galanin-immunoreactive neurons was significantly lower in allodynic rats as compared to non-allodynic rats, and the same tendency, but less obvious was found in the Seltzer model. Furthermore, an increase in galanin-immunoreactive fibres was found in the superficial laminae of the ipsilateral dorsal horn in all lesion models, especially in lamina II. A dramatic increase in the number of neuropeptide Y and neuropeptide Y messenger RNA-positive neuron profiles was also found in the ipsilateral dorsal root ganglia in all models, but no significant difference was found in peptide levels between allodynic and non-allodynic rats in any of the models. The present results suggest that the levels of endogenous galanin may play a role in whether or not allodynia develops in the Bennett model.

Leukemia inhibitory factor regulates galanin/galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology.

After transection of the sciatic nerve there is a dramatic increase in both galanin/galanin message-associated peptide-like immunoreactivities and preprogalanin messenger RNA levels in rat and mouse lumbar 4 and 5 dorsal root ganglion neurons. There is strong evidence that after nerve injury leukemia inhibitor factor is a key molecule in the control of peptide expression both in sympathetic neurons and in dorsal root ganglion neurons, although the cells of origin of endogenous leukemia inhibitory factor remain to be established. We have therefore studied the effect of leukemia inhibitory factor on galanin expression in 72 h cultured dorsal root ganglion neurons from normal mice, leukemia inhibitory factor-deficient and heterozygous mice with immunohistochemistry and in situ hybridization. In cultures of leukemia inhibitory factor-deficient (-/-) mice only 13% of the dorsal root ganglion neurons expressed galanin message-associated peptide and in cultures from heterozygous (+/-) and wild-type (+/+) mice the corresponding figures were, respectively, 24 and 40%. After addition of leukemia inhibitory factor (10 or 50 ng/ml) to the culture medium, the number of neurons expressing galanin message-associated peptide was increased (up to 41%) in cultures from (-/-) animals after the high concentration and reached similar values in cultures from heterozygous animals incubated with the low concentration. These findings were supported by parallel analysis of prepro-galanin messenger RNA levels, where similar transcript levels and effects in the various cultures were observed in the non-radioactive in situ hybridization experiments. These results support the hypothesis that leukemia inhibitory factor is an important regulator of galanin/galanin message-associated peptide expression following axotomy, and may therefore be involved in the defence mechanisms against neuropathic pain at the level of dorsal root ganglion neurons.

Intrathecal galanin alleviates allodynia-like behaviour in rats after partial peripheral nerve injury.

We have previously suggested that the neuropeptides galanin and galanin message-associated peptide (GMAP) may have an inhibitory role in spinal nociception. The present study examined the effects of intrathecal (i.t.) administration of these two peptides on allodynia-like behaviours in response to mechanical and cold stimulation in rats after photochemically induced ischaemic peripheral nerve injury. I.t. galanin significantly alleviated the mechanical- and cold-allodynia-like behaviours in nerve injured rats, and was not associated with motor impairment or sedation. I.t. GMAP relieved mechanical allodynia much less than galanin. I.t. M-35, a high-affinity galanin receptor antagonist, did not significantly alter the response of the rats to mechanical or cold stimulation. At 1 or 2 weeks postinjury, around 15% of dorsal root ganglion (DRG) neuron profiles showed galanin-like immunoreactivity. These profiles were mostly small sized. Although the number of galanin positive cells was thus increased in the DRG in the present model, the increase was substantially less than after complete sciatic nerve section, as previously shown. The present results showed that spinal administration of galanin inhibited some abnormal pain-like behaviours in rats after partial peripheral nerve injury. These results further support an inhibitory function for galanin in nociception. However, endogenous galanin may not play a significant role in suppressing nociceptive input after partial ischaemic peripheral nerve injury, as the upregulation of galanin is moderate.

Birth-related expression of c-fos, c-jun and substance P mRNAs in the rat brainstem and pia mater: possible relationship to changes in central chemosensitivity.

In situ hybridization was used to characterize respiration-related areas of the brainstem activated around the time of birth as well as their postnatal sensitivity to CO2. Levels of mRNA corresponding to the immediate early genes (IEG), c-fos and c-jun, and of substance P precursor, ppt-A, were determined in rat fetuses (E21) and neonatal pups (1 h, 1 day and 6 days after normal birth) and after exposure to hypercapnia (12% CO2 for 1 h). Transient increases in c-fos mRNA were observed in the central chemoreceptor area of the ventral medullary surface (VMS), in the lateral reticular nucleus (LRN), in the nucleus of the solitary tract (NTS), and in the nucleus raphé pallidus (RPA) 1 h after birth. Increased expression of c-fos mRNA in the VMS could also be evoked by hypercapnia and this response was particularly pronounced 1 day after birth. On the other hand, c-jun mRNA could be detected already at E21 in the hypoglossal nucleus (XII) and LRN and these levels were not significantly altered at 1 h after birth. There was, however, an increase in the expression of c-jun mRNA in the pia mater surrounding the brainstem after birth. At 1 day after birth, c-jun mRNA levels had decreased in the LRN and pia mater, and later on (6 days after birth) in XII. Furthermore, the ppt-A mRNA level in NTS increased immediately after birth and remained high 1 and 6 days later. These results suggest that (a) the central chemoreceptor area of the VMS, as well as the NTS, LRN, RPA and pia mater are activated following birth; (b) the VMS, but not the other structures examined, can be activated immediately after birth by hypercapnia; and (c) increased expression of ppt-A mRNA may be related to the transition of respiratory control at birth.

Cloning of a second nm23-M1 cDNA: expression in the central nervous system of adult mouse and comparison with nm23-M2 mRNA distribution.

Nm23 has been identified as a gene family encoding different isoforms of the nucleoside diphosphate kinase. This protein is a key enzyme in the control of cellular concentrations of nucleoside triphosphates. Moreover, it has been shown to play important roles in various cellular functions such as differentiation and metastasis. In the present study, a second cDNA for nucleoside diphosphate kinase A (Nm23-M1) was isolated from a cDNA library of mouse embryonic stem cells. This clone encodes the same putative 152 aminoacids long protein as an already published cDNA but is longer in both its 5' and 3' untranslated regions. Tissue and cellular distribution of nm23-M1 mRNA was investigated by using Northern blot analysis and in situ hybridization. Nm23-M1 transcripts were found to be widely distributed throughout the mouse central nervous system with prominent expression in several restricted areas. No differences were noticed between the distribution of long and short transcripts. Furthermore, a similar pattern of expression was described in the central nervous system for nm23-M2 mRNA, encoding a second isoform of the nucleoside diphosphate kinase. However, the transcript of this isoform displayed a wider distribution and was expressed in all organs analysed by northern blotting. The possible involvement of nm23-M1 in differentiation of mouse nervous system is further discussed.

Neurocircuitries of the basal ganglia studied in organotypic cultures: focus on tyrosine hydroxylase, nitric oxide synthase and neuropeptide immunocytochemistry.

The nigrostriatal and mesolimbic systems of the rat were reconstructed using an organotypic culture model, whereby neonatal brain tissue was grown in vitro for approximately one month. The nigrostriatal system comprised of tissue from the substantia nigra, the dorsal striatum and the frontoparietal cortex, while the mesolimbic system included the ventral tegmental area, ventral striatum (including the fundus striati, accumbens nucleus, olfactory tubercle, lateral septum, ventral pallidum and piriform cortex) and cingulate cortex. These regions were also cultured alone or in pairs. The cultures were monitored in vitro, and after one month fixed in a formalin-picric acid solution, and processed for immunohistochemistry using antibodies raised against tyrosine hydroxylase, nitric oxide synthase, preprocholecystokinin, glutamate decarboxylase, neuropeptide Y, dopamine- and cyclic AMP-regulated phosphoprotein-32 and glial fibrillary acidic protein. The tissue survived in single, double or triple cultures, although differences were found depending upon the source and combination of cultured region. Neurons had localization and shape as in vivo. Local networks were especially prominent in the mesencephalon, where both tyrosine hydroxylase-positive axons spread from the "substantia nigra" to the rest of the tissue, and where nitric oxide synthase-positive networks also surrounded tyrosine hydroxylase-positive neurons. Glutamate decarboxylase-positive nerve terminals formed dense networks around tyrosine hydroxylase-positive neurons. In the striatum, nitric oxide synthase and dopamine- and cyclic AMP-regulated phosphoprotein-32 neurons were surrounded by tyrosine hydroxylase-positive nerve terminals. The nigral and ventral tegmental area dopamine neurons projected to striatal and cortical structures, but the projection from the ventral tegmental area to the cingulate cortex was more prominent. With regard to co-existence, preprochole-cystokinin-like immunoreactivities was found in many tyrosine hydroxylase-positive neurons and neuropeptide Y- and nitric oxide synthase-like immunoreactivity co-existed in striatal and cortical tissues. In general terms, the chemical neuroanatomy in the cultures was similar to that described earlier in vivo. Nitric oxide synthase staining was particularly intense. Taken together, the organotypic model captures many of the morphological and neurochemical features seen in vivo, providing a valuable model for studying neurocircuitries of the brain in detail, where 'normal' and 'pathological' conditions can be simulated.

Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo.

Peptide nucleic acids (PNAs) form stable and tight complexes with complementary DNA and/or RNA and would be promising antisense reagents if their cellular delivery could be improved. We show that a 21-mer PNA, complementary to the human galanin receptor type 1 mRNA, coupled to the cellular transporter peptides, transportan or pAntennapedia(43-58), is efficiently taken up into Bowes cells where they block the expression of galanin receptors. In rat, the intrathecal administration of the peptide-PNA construct results in a decrease in galanin binding in the dorsal horn. The decrease in binding results in the inability of galanin to inhibit the C fibers stimulation-induced facilitation of the rat flexor reflex, demonstrating that peptide-PNA constructs act in vivo to suppress expression of functional galanin receptors.

Distribution of a glucocorticoid-induced orphan receptor (JP05) mRNA in the central nervous system of the mouse.

JP05 (originally referred to as glucocorticoid-induced receptor gene or cDNA clone 4.2) designates a gene originally isolated from murine thymoma WEHI-7TG cells after being treated with glucocorticoids and forskolin. This gene is also induced by dexamethasone (a potent glucocorticoid receptor agonist) in isolated normal murine thymocytes. The predicted amino acid sequence was found to share significant similarity to the family of G-protein-coupled receptors, in particular to the tachykinin receptors NK-1, NK-2 and NK-3, with which it has an overall identity of 32%, 31% and 33%, respectively. The results of the present in situ hybridization analysis reveal that JP05 mRNA containing cells are extensively distributed throughout the rostrocaudal extension of the brain and spinal cord. However, the vast majority of the areas with high to moderate levels of JP05 mRNA were localized in the forebrain, primarily within limbic system structures, the dorsal and ventral striatum and in some hypothalamic nuclei. These results are discussed in relation to the central nervous system distribution of glucocorticoid receptor-containing cells and to the tachykinin system.