Pharmacological characterization of desensitization in scratching behavior induced by intrathecal administration of hemokinin-1 in the rat.

Desensitization is induced by the repeated administration of high doses of substance P (SP) or hemokinin-1 (HK-1). However, little information is available about the mechanisms involved in the induction of desensitization by these peptides. Thus, to characterize this desensitization, we examined the dose-dependent effect of these peptides, the effect of pretreatment with neurokinin 1(NK1) receptor antagonists, and the effect of pretreatment with inhibitors of protein kinases such as protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin kinase II (CaMKII) and mitogen-activated protein kinase kinase (MEK). The number of scratchings induced by 10(-3)M SP or HK-1 decreased following pretreatment with 10(-11)-10(-3)M SP or HK-1 with a marked reduction at 10(-3) and 10(-6)M SP or HK-1. The effect of NK1 receptor antagonists on desensitization induced by pretreatment with 10(-6)M SP was marked, whereas there was little effect of pretreatment with these antagonists on 10(-6)M HK-1-induced desensitization. Additionally, 10(-6)M SP- and HK-1-induced desensitization was attenuated by pretreatment with PKA, PKC and MEK inhibitors, except a CaMKII inhibitor that inhibited SP-induced desensitization. These results indicate that the receptor and kinases involved in HK-1-induced desensitization are partially different from those of SP.

Immunohistochemical expression of p63, Ki67 and ß-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder.

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or ß-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and ß-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.

Development of early lesions of microangiopathy in chronically diabetic monkeys.

A chronic diabetic state was produced in Macaca fuscatus , and these diabetic monkeys were kept without insulin treatment for up to 25 mo. The metabolic derangements were characterized by hyperglycemia, insulinopenia, hyperglucagonemia, ketonemia, and hyperlipidemia. Significant thickening of the capillary basement membrane of the gastrocnemius muscle was observed in the chronically diabetic monkeys, and became obvious in the course of diabetic state; 732 +/- 35 A in controls, 750 +/- 58 A in diabetic monkeys with duration of 4 mo, and 1165 +/- 112 A in those with duration of more than 11 mo. In addition to duration of the diabetic state, severity of hyperglycemia is also thought to play an important role in the capillary basement membrane thickening judging from the fact that diabetic monkeys with constant hyperglycemia showed a greater membrane thickening. Ultrastructural alterations, such as significant thickening of glomerular basement membrane and increase of mesangial matrix, were observed in kidney as well. These results indicate that diabetic microangiopathy has been produced by metabolic derangements characterized by chronic hyperglycemia, insulinopenia, and hyperlipidemia.

An elevation of serum immunoglobulin E provides a new aspect of hyperthyroid Graves' disease.

In hyperthyroid Graves' disease, short-term methimazole is sufficient to induce lasting remission in some patients, but even long-term treatment fails to do so in others. We have evaluated the role of autoimmune abnormalities in the helper T cell type 2 (TH2)-interleukin-13 (IL-13)-TSH receptor system in maintaining hyperthyroidism by comparing IgE levels in patients with various thyroid diseases. One hundred and ninety-three patients with hyperthyroid Graves' disease were treated with methimazole, and blood samples were obtained to measure serum levels of T4, T3, TSH, thyroglobulin, antimicrosomal antibody, TSH binding inhibitory Ig (TBII), thyroid-stimulating antibody, thyroid stimulation-blocking antibody, IgE, interferon-gamma, IL-4, and IL-13. Elevation of serum IgE (> or = 170 U/mL) was found in 35.5% of patients with hyperthyroid Graves' disease, and serum levels of T4, T:1, antimicrosomal antibody, and TBII were significantly greater in patients with IgE elevation than in those with normal serum IgE. During methimazole treatment, there was a parallel decrease in the serum T4 concentration in the presence or absence of an IgE elevation. However, there was a significantly smaller decrease in TBII in patients with elevated IgE than in those with normal IgE. As a result, the remission rate was significantly greater in patients with normal IgE than in those with IgE elevation. Serum levels of IL-13 were elevated in 64.7% of patients with IgE elevation in the absence of detectable TH1 marker, interferon-gamma. These findings suggest that in one third of patients with hyperthyroid Graves' disease, TH2 cells are stimulated and secrete excess amounts of IL-13, which subsequently stimulates B cells to synthesize more TSH receptor antibody and IgE, so that during methimazole treatment TBII decreases less in patients with IgE elevation, producing a lower remission rate.

Remission and recurrence of hyperthyroid Graves' disease during and after methimazole treatment when assessed by IgE and interleukin 13.

We analyzed the relationship between serum IgE concentrations and the remission or recurrence of Graves' disease. One hundred seven patients with Graves' disease were treated with methimazole (MMI). Serum IgE concentration greater than 170 IU/ml was found in 41 of 107 untreated patients (38.3%). However, the presence of TSH-binding inhibiting immunoglobulin or thyroid-stimulating antibody did not correlate with the IgE concentrations. Remission was found in 20 of 41 patients with elevated IgE concentrations (48.8%) after 18 months of MMI treatment, as opposed to 53 of 66 patients with normal concentrations (80.3%) (P = 0.0014). MMI treatment was discontinued in 73 patients who were followed for 26-48 months. The recurrence of Graves' disease was found in 13 patients, whereas the remaining 60 were still in remission. The rate of long-standing remission was lower in patients with elevated than normal IgE concentration (34.1% vs. 69.7%, P = 0.0007). We also analyzed serum levels of interleukin (IL)-13. Although IL-13 was not detected in all patients, the detection rate was higher in patients without remission and in those with recurrence than in those with long-standing remission (47.1%, 38.5%, and 13.3%, respectively; P = 0.0012). More patients with elevated IgE were positive for allergic diseases and for family history of allergic diseases in their first-degree relatives. We conclude that the elevation of IgE and the higher detection rate of IL-13 are associated with both remission and recurrence of Graves' disease.

Opioid peptide gene expression in rat trigeminal nucleus caudalis neurons: normal distribution and effects of trigeminal deafferentation.

Preproenkephalin (preproenkephalin A) and preprodynorphin (preproenkephalin B) are the opioid peptide genes expressed in neurons of the nucleus caudalis of the trigeminal nuclear complex. We have used recently developed techniques for quantitative in situ hybridization to identify the neurons in laminae I and II of the nucleus caudalis that display the mRNA products of each of these genes. The specificity of these hybridization patterns is supported by several biochemical features, and by qualitative and quantitative parallels with previous immunohistochemical results. In animals killed 4 days after unilateral lesions of the trigeminal ganglion, neuronal expression of both preproenkephalin and preprodynorphin is altered in the nucleus caudalis. Decreases in preproenkephalin mRNA are due to a decline in the number of neurons that appear to express this gene. Conversely, preprodynorphin mRNA increases by adding a significant population of expressing neurons. These deafferentation-induced changes in gene expression may provide clues to the role of primary afferent information in modulating the functions of nucleus caudalis neurons containing opioid peptides.

Cholecystokinin and neurotensin mRNAs are differentially expressed in subnuclei of the ventral tegmental area.

Immunohistochemical studies of ventral tegmental area (VTA) neurons indicate that individual cells can contain dopamine as well as the neuropeptide neurotransmitters cholecystokinin (CCK) and neurotensin (NT). We have defined the distribution of the cells expressing the mRNAs encoding these two dopamine cotransmitter peptides in each of the subnuclei of the ventral tegmental area, and quantitated the extent of expression of each gene by using in situ hybridization methods. These studies reveal significant differences in the patterns of expression of each of these two genes within various subdivisions of the VTA. The rostral linear nucleus contained numerous CCK positive cells, some of which appeared to express preproCCK-mRNA at a very high level, but this nucleus contained relatively few NT-expressing cells. The parabrachialis pigmentosus contained numerous NT and CCK positive cells. The paranigralis and interfascicularis nuclei displayed positive CCK cells but with expression at only modest levels. NT cells were very few in these nuclei. The caudal linear nuclei contained the highest number of NT-expressing neurons and these cells expressed very high levels of NT mRNA. The selective distribution of these peptide genes within the VTA subnuclei may have specific consequences. Studies of the connectivity of neurons in the VTA show that the different subnuclei of this region project to several functionally and architectonically different regions of the cerebral cortex and subcortically to nuclei related to the limbic system. Results from our study show very prominent expression of CCK mRNAs in those subnuclei that project heavily to the prefrontal, other cortical areas, and the amygdaloid complex. The NT gene is expressed prominently in those subnuclei of VTA that project heavily to the entorhinal cortex and amygdaloid complex. These results provide support for a differential role for the NT-expressing neurons than that of CCK-expressing neurons of VTA in "reward" mechanisms and in drug-seeking and motivational behavior. These observations could be applied to create working hypotheses and experimental paradigms to test the differential functional activity of the subdivisions of VTA and their potential roles in the pathogenesis and treatment of drug-seeking behavior and other neuropsychiatric disorders.

The central projection of masticatory afferent fibers to the trigeminal sensory nuclear complex and upper cervical spinal cord.

Retrograde and anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) conjugate was used to study the organization of primary afferent neurons innervating the masticatory muscles. HRP applied to the nerves of jaw-closing muscles--the deep temporal (DT), masseter (Ma), and medial pterygoid (MP)--labeled cells in the trigeminal ganglion and the mesencephalic trigeminal nucleus (Vmes), whereas HRP applied to nerves of the jaw-opening muscles--anterior digastric (AD) and mylohyoid (My)--labeled cells only in the trigeminal ganglion. Cell bodies innervating the jaw-closing muscles were found with greater frequency in the intermediate region of the mandibular subdivision, while somata supplying the jaw-opening muscles were predominant posterolaterally. The distribution of their somatic sizes was unimodal and limited to a subpopulation of smaller cells. Projections of the muscle afferents of ganglionic origin to the trigeminal sensory nuclear complex (TSNC) were confined primarily to the caudal half of pars interpolaris (Vi), and the medullary and upper cervical dorsal horns. In the Vi, Ma, MP, AD, and My nerves terminated in the lateral-most part of the nucleus with an extensive overlap in projections, save for the DT nerve, which projected to the interstitial nucleus or paratrigeminal nucleus. In the medullary and upper cervical dorsal horns, the main terminal fields of individual branches were confined to laminae I/V, but the density of the terminals in lamina V was very sparse. The rostrocaudal extent of the terminal field in lamina I differed among the muscle afferents of origin, whereas in the mediolateral or dorsoventral axis, a remarkable overlap in projections was noted between or among muscle afferents. The terminals of DT afferents were most broadly extended from the rostral level of the pars caudalis to the C3 segment, whereas the MP nerve showed limited projection to the middle one-third of the pars caudalis. Terminal fields of the Ma, AD, and My nerves appeared in the caudal two-thirds of the pars caudalis including the first two cervical segments, the caudal half of the pars caudalis and the C1 segment, and in the caudal part of the pars caudalis including the rostral C1 segment, respectively. This rostrocaudal arrangement in the projections of muscle nerves, which corresponds to the anteroposterior length of the muscles and their positions, indicates that representation of the masticatory muscles in lamina I reflects an onion-skin organization. These results suggest that primary muscle afferent neurons of ganglionic origin primarily mediate muscle pain.(ABSTRACT TRUNCATED AT 400 WORDS)

Oral and facial representation within the medullary and upper cervical dorsal horns in the cat.

Transganglionic transport of HRP was used to study the patterns of termination of somatic afferent fibers innervating oral and facial structures within the trigeminal nucleus caudalis and upper cervical dorsal horn of the cat. In separate animals, the superior alveolar, pterygopalatine, buccal, inferior alveolar, lingual, frontal, corneal, zygomatic, infraorbital, mental, mylohyoid, and auriculotemporal branches of the trigeminal nerve were traced in this experiment. The organization of the primary afferents innervating the oral structures is not uniform across laminae and at different rostrocaudal levels of the nucleus caudalis. The superior alveolar and pterygopalatine nerves mainly terminate in laminae I, II, and V at the level of the rostral one-third of the caudalis. By contrast, the lingual, inferior alveolar, and buccal nerve terminate in laminae I-V of, respectively, the rostral third, the entire length, and caudal two-thirds of the caudalis. In addition, the lingual, buccal, and pterygopalatine nerves terminate in the dorsal and middle parts of the interstitial islands or pockets of lamina I neuropil extending to the rostral levels parallel to the nucleus interpolaris. Mediolaterally, in laminae I, II, and V of the rostral third an extensive overlap of projections was found between the branches from each trigeminal division, and some overlap was observed between projections from the mandibular and maxillary divisions. On the other hand, the projections of primary afferents innervating the facial structures are arranged in a somatotopic fashion in rostrocaudal and mediolateral axes over the laminae (I-IV) through the nucleus caudalis and upper cervical dorsal horn. Fibers from the perioral and perinasal regions terminate most rostrally in caudalis, and fibers from progressively more posterior facial regions terminate at successively lower levels. A mediolateral somatotopic arrangement was observed, with fibers from the ventral parts of face ending in the medial regions and fibers from the progressively more dorsal parts of the face ending in successively more lateral regions of the medullary and upper cervical dorsal horns. Corneal afferent terminals are concentrated in the outer parts of lamina II at the levels of the rostral parts of the caudal two-thirds of the caudalis and the interstitial islands of lamina I. The maxillary division terminates first at the most caudal level of the caudalis, followed by the ophthalmic division descending as far as the C2 segment and the mandibular division reaching the most caudal level of the C2 segment.(ABSTRACT TRUNCATED AT 400 WORDS)

Oral and facial representation in the trigeminal principal and rostral spinal nuclei of the cat.

Transganglionic transport of horseradish peroxidase (HRP) was used to study the patterns of termination of somatic afferent fibers innervating oral and facial structures within the principal nucleus (Vp), nucleus oralis (Vo), and nucleus interpolaris (Vi). The primary trigeminal afferent fibers that innervate the oral cavity supplied by the pterygopalatine, superior alveolar, lingual, buccal, and inferior alveolar branches, as well as the facial skin supplied by the frontal, corneal, zygomatic, infraorbital, auriculotemporal, mylohyoid, and mental branches, were traced in this experiment. The results show that trigeminal afferent nerves that innervate the oral cavity project mainly to the principal nucleus, the rostrodorsomedial part (Vo.r) and dorsomedial division (Vo.dm) of pars oralis, and the dorsomedial region of pars interpolaris, while an extensive overlap of projections is found in the Vo.r, Vo.dm, and rostral Vi. The central processes of fibers innervating the anterior face (i.e., mental, infraorbital, and frontal nerves) terminate in the ventral division of principalis (Vpv), caudal region pars oralis (Vo.c), and ventrolateral Vi, with the largest numbers of terminals being found in the Vpv and Vi. In contrast, the central projection patterns of the corneal, zygomatic, mylohyoid, and auriculotemporal afferents are different from those of other afferent nerves examined, and present a discrete projection to the trigeminal sensory nuclear complex (TSNC). The corneal, mylohyoid, and auriculotemporal afferents mainly project to the restricted regions of principalis and caudal Vi, while zygomatic afferent nerve fibers project to the caudal third of pars interpolaris. The typical somatotopic organization with the face of the mouth open inverted is represented in the rostrocaudal midlevels of the Vpv and caudal pars interpolaris. The Vpd receives topographical projection from primary afferent nerves that innervate the oral structure only, while this projection was organized in a complicated manner. The relationship between the functional segregation and the cytoarchitectonic differentiation of the TSNC is discussed, particularly with respect to this somatotopic organization, combined with the characteristics of projecting cells in the TSNC.

Topographic representation of lower and upper teeth within the trigeminal sensory nuclei of adult cat as demonstrated by the transganglionic transport of horseradish peroxidase.

Transganglionic transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA) entrapped in hypoallergenic polyacrylamide gel was used to study the patterns of termination of primary afferents that innervate the lower and upper tooth pulps within the trigeminal sensory nuclear complex (TSNC). HRP injections were made into the inferior and superior alveolar nerves in order to compare the central projections of the whole nerve with those from tooth pulps. In addition, the relationship between the distribution of the trigeminothalamic tract cells and the projection sites of the tooth pulp afferents was investigated by injecting HRP into the posterior ventral thalamus. HRP-labeled tooth pulp afferent fibers innervating the lower and upper teeth projected to the subnucleus dorsalis (Vpd) of pars principalis, the rostrodorsomedial part (Vo.r) and nucleus dorsomedialis (Vo.dm) of pars oralis, the medial regions of pars interpolaris, and laminae I, II, and V of pars caudalis. Terminal fields of the lower tooth pulp afferents formed a rostrocaudally running, uninterrupted column from the midlevel of Vpd to the caudal tip of caudalis. In contrast, the column of termination of upper tooth pulp afferents was discontinuous at the Vpd/Vo.r transition, and ended at the more rostral level of the caudalis than that of the lower tooth pulp afferents. The representation of the lower and upper teeth in the TSNC was organized in a somatotopic fashion which varied from one subdivision to the next, although terminal zones of the inferior and superior alveolar nerves overlapped within the Vo.r, Vo.dm, and dorsomedial part of rostral pars interpolaris. The lower and upper teeth were represented in the Vpd, Vo.r, Vo.dm, medial region of pars interpolaris, and laminae I, II, and V, in a ventrodorsal or caudorostral, dorsoventral, lateromedial, dorsoventral, and mediolateral or dorsomedial-ventrolateral sequence, respectively. The smaller, more focal terminal areas of the teeth contrasted sharply with more extensive terminal fields of the alveolar nerves. The HRP injections within the thalamus indicated that neurons in Vpd, the caudal pars interpolaris, and laminae I/V of caudalis, which are subdivisions of TSNC that receive pulpal projections, sent their axons to the ipsilateral and contralateral posterior ventral thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Preproenkephalin upregulation in nucleus caudalis: high and low intensity afferent stimulation differentially modulate early and late responses.

Nucleus caudalis expression of preproenkephalin mRNA changes following lesions depleting small-caliber primary afferent fibers and after stimulation of trigeminal afferents at different intensities. Animals treated neonatally with capsaicin display reduced preproenkephalin gene expression in nucleus caudalis neurons. Stimulation of normal animals at low intensities enhances preproenkephalin expression in a bimodal temporal pattern. High intensity stimulation is effective only at later time points in normal animals, but it causes both early and late effects on preproenkephalin expression when applied to animals neonatally lesioned with capsaicin. Transsynaptic regulation of preproenkephalin expression in pain-modulating areas of the nucleus caudalis of the trigeminal nerve thus depends on the specific type of primary afferent input. The rapid responses noted after selective large fiber stimulation appear to be suppressed by coactivation of small caliber fibers. Later responses appear less influenced by the quality of the eliciting afferent stimulus.

Thrombopoietic activity of recombinant human interleukin-11 in nonhuman primates with ACNU-induced thrombocytopenia.

The effect of recombinant human interleukin-11 (rHuIL-11) on myelosuppressive nimustine (ACNU)-induced thrombocytopenia was assessed in nonhuman primates. A single intravenous (i.v.) injection of ACNU (15 mg/kg) was administered to cynomolgus monkeys on day 0. rHuIL-11 (100 microg/kg/day) or the vehicle was given subcutaneously (s.c.) from day 1 to day 21. In monkeys receiving ACNU, the circulating platelet count decreased to a low of 42 +/- 6 x 10(9)/L by day 21 but returned to pretreatment levels (375 +/- 48 x 10(9)/L) on day 30. Administration of rHuIL-11 prevented severe thrombocytopenia; the platelet count fell only to 138 +/- 23 x 10(9)/L on day 18, and platelet recovery was faster (458 +/- 91 x 10(9)/L by day 27) compared with that of the control animals. The size of bone marrow megakaryocytes from rHuIL-11-treated animals was larger than that of the controls, indicating that rHuIL-11 stimulated megakaryopoiesis in a myelosuppressive condition. Treatment with ACNU also caused leukopenia and moderate anemia. rHuIL-11 transiently and slightly decreased the white blood cell (WBC) and red blood cell (RBC) counts. Conversely, rHuIL-11 accelerated recovery of RBC count in the late administration period. These results support the assertion that rHuIL-11 may be an important therapeutic agent for reducing the severity and duration of thrombocytopenia following cancer chemotherapy.

Simultaneous activation of N-methyl-D-aspartate and neurokinin-1 receptors modulates c-Fos and Zif/268 expression in the rat trigeminal nucleus caudalis.

We examined the acute expression of c-Fos or Zif/268 by simultaneous activation of N-methyl-D-aspartate receptor and neurokinin-1 receptor of the trigeminal nucleus caudalis in anesthetized rats. A selective N-methyl-D-aspartate receptor agonist, N-methyl-D-aspartate, and/or a selective neurokinin-1 receptor agonist, substance P, was applied topically to the dorsal surface of the spinal trigeminal tract. Immunohistochemically stained nuclei for c-Fos and Zif/268 at laminae I and II of the trigeminal nucleus caudalis were counted. Ipsilateral c-Fos and Zif/268 were increased significantly dose-dependently by N-methyl-D-aspartate (at 136 and 340 microM, and at 68, 136 and 340 microM, respectively). On the contralateral side, only Zif/268 increased significantly (at 68, 136 or 340 microM). These increases were abolished by D-2-amino-5-phosphonovaleric acid (at 25 mM), a selective N-methyl-D-aspartate receptor antagonist. Substance P (at 3.7 or 7. 4 microM) significantly increased dose-dependently ipsilateral c-Fos and Zif/268. On the contralateral side, only c-Fos was significantly increased (at 3.7 and 7.4 microM). These increases were abolished by D-2-amino-5-phosphonovaleric acid (at 25 mM) and L-703,606 (at 10 microM), a selective neurokinin-1 receptor antagonist. The combined application of N-methyl-D-aspartate 340 microM + substance P (at 0.74 or 3.7 microM) significantly increased ipsilateral c-Fos compared to either agent alone. Combined application of N-methyl-D-aspartate 340 microM + substance P at 0.74, 3.7 or 7.4 microM significantly increased ipsilateral Zif/268 expression compared to either drug alone. Other combinations did not increase c-Fos and Zif/268. Our results indicate that activation of N-methyl-D-aspartate or neurokinin-1 receptor of the trigeminal nucleus caudalis contributes to the acute induction of both c-Fos and Zif/268 on the ipsilateral superficial layer of this nucleus and simultaneous activation of both receptors by their agonists with specific concentrations produces a marked expression of these proteins. Simultaneous activation of N-methyl-D-aspartate and neurokinin-1 receptors under some specific conditions may augment synaptic transmission, contributing to long-term neuronal change.

Intracranial self-stimulation induces Fos expression in GABAergic neurons in the rat mesopontine tegmentum.

The cholinergic neurons which originate in the mesopontine tegmentum and innervate the midbrain ventral tegmental area have been proposed to play a key role in intracranial self-stimulation reward. This mesopontine area also contains GABA neurons. Detailed information is still lacking, however, about the relationship of cholinergic and GABAergic neurons in this region to self-stimulation reward. Therefore, using double immunostaining for Fos as a marker of neuronal activity and choline acetyltransferase as a marker of cholinergic neurons, or for Fos and GABA, we investigated whether self-stimulation of the medial forebrain bundle induces Fos expression within cholinergic and GABAergic neurons in two regions of the mesopontine tegmentum, i.e., pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus. Self-stimulation of the medial forebrain bundle for 1 h induced a large increase in the number of cells expressing Fos in both the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, when compared to control brains. However, the self-stimulation-induced expression of Fos was restricted mostly to GABA-, but not choline acetyltransferase-, immunostained cells. We also examined, using microdialysis, whether self-stimulation increases acetylcholine efflux in the ventral tegmental area, a terminal region of the mesopontine tegmentum cholinergic pathway. One hour of self-stimulation significantly increased acetylcholine efflux from this terminal area. These results indicate that intracranial self-stimulation of the medial forebrain bundle may increase acetylcholine release without affecting expression of Fos in cholinergic neurons, while the same stimulation may induce Fos expression in GABAergic neurons of the mesopontine tegmentum. GABAergic as well as cholinergic neurons in this area appear to be activated by self-stimulation reward in the medial forebrain bundle.

Regional differences in desensitization of c-Fos expression following repeated self-stimulation of the medial forebrain bundle in the rat.

The acute self-stimulation of the medial forebrain bundle was reported to induce the expression of c-Fos, the protein product of c-fos, an immediate early gene, in the central nervous system. In the present study, we examined regional changes in c-Fos expression in several reward-related areas of rat brain in response to short- and long-term exposure to self-stimulation of the medial forebrain bundle. Short-term one-hour stimulation of the medial forebrain bundle for one day after training, which evoked steady self-stimulation behavior, significantly increased the number of c-Fos-positive neurons bilaterally in all of 15 brain structures assayed, as compared to the non-stimulation control. Among them, structures showing a larger number of the stained neurons on the stimulated side were the anterior olfactory nucleus, amygdala, medial caudate-putamen complex, lateral septum, bed nucleus of the stria terminals, ventral pallidum, substantia innominata, lateral preoptic area, medial preoptic area, lateral hypothalamus rostral to the stimulating electrodes, and substantia nigra. Long-term stimulation of the medial forebrain bundle once daily for five successive days, which maintained consistently stable self-stimulation behavior, also increased the number of c-Fos-positive neurons in the aforementioned structures, as compared to the control. However, the long-term rewarding stimulation diminished the increased number of labeled neurons, as compared to the short-term rewarding stimulation. Seven areas, medial caudate-putamen complex, ventral pallidum, substantia innominata, lateral preoptic area, medial preoptic area, rostral lateral hypothalamus and substantia nigra, showed asymmetrical, ipsilateral predominance after the short- and long-term stimulation. However, the stained neuron count in those areas after the long-term stimulation was reduced to less than 50% of that found after the short-term stimulation with the exception of lateral preoptic area and rostral lateral hypothalamus. The results suggest that the development of desensitization of c-Fos response may differ among the reward-relevant brain regions as a consequence of repeated self-stimulation. They also indicate that a larger portion of neurons in the lateral preoptic area and rostral lateral hypothalamus may be implicated in both short- and long-term self-stimulations of the medial forebrain bundle.

Nucleotide sequence of capsid protein gene of porcine parvovirus.

Approximately 90% of the genome of porcine parvovirus was cloned into bacterial cells. The nucleotide sequence of the genome from 33 map units (MU) to 95 MU was determined and shown to include the entire gene encoding the capsid proteins. The predicted amino acid sequences of the capsid proteins showed extensive homology to those of other autonomous parvoviruses.

Preproenkephalin mRNA expression in nucleus caudalis neurons is enhanced by trigeminal stimulation.

Electrical stimulation of trigeminal afferents increases expression of preproenkephalin mRNA in neurons of laminae I and II of the trigeminal nucleus caudalis in animals sacrificed 6 hours after the end of stimulation. More neurons express, and positive cells express at higher levels. These neurons thus express the mRNA corresponding to a major pain-modulating neurotransmitter gene with significant input-related plasticity.

Dopamine transporter: expression in Xenopus oocytes.

Xenopus oocytes can express biologically relevant transport activity after injection of mRNAs encoding several carrier molecules. mRNA from PC12 cells, as well as transcripts from a rat ventral midbrain library, can be expressed in these oocytes and allow them to display pharmacologically specific dopamine uptake. mRNA-injected oocytes incubated with tritiated dopamine contain tritiated dopamine and metabolites; lower amounts of radiolabeled dopamine and more radiolabeled metabolites are found in oocytes co-incubated with cocaine or in water-injected oocytes. Tritiated dopamine uptake into mRNA-injected oocytes is time, sodium, and temperature dependent. It is blocked by cocaine and mazindol, but not by haloperidol. It is not found after injection of mRNA from other brain regions. A size-selected rat midbrain library constructed in the plasma vector pCDM8 yields mRNA transcripts whose injection into oocytes causes cocaine-blockable [3H]dopamine uptake. These findings provide an assay for purification of the dopamine transporter cDNA by sib selection techniques.

Jun B, c-jun, jun D and c-fos mRNAs in nucleus caudalis neurons: rapid selective enhancement by afferent stimulation.

In situ hybridization using cDNAs complementary to specific regions of the mRNAs encoding 3 members of the jun transcription factor gene family and c-fos reveals modest levels of hybridization over superficial laminae of the nucleus caudalis of the spinal tract of the trigeminal in sections taken from unstimulated brains. Jun B expression is markedly and rapidly enhanced ipsilateral to electrical stimulation of the trigeminal ganglion. C-fos mRNA levels also show changes, especially after higher intensity stimulation. Smaller alterations in c-jun (jun A) and jun D do not reach statistical significance. In each instance of altered expression, more neurons express hybridization densities above background levels after stimulation. Parallels between these alterations and changes in the expression of preproenkephalin in these same neuronal populations are discussed.