PubMed Health⌕ Search

Biomedical subjects

R G Wiley

Publications and source records attributed to R G Wiley.

At least 19 recordsLinked to original sources
PubMed2003

Intrathecal substance p-saporin attenuates operant escape from nociceptive thermal stimuli.

Destruction of neurons in the superficial dorsal horn that express substance P receptor (NK-1R) has been reported to block development of behavioral hypersensitivity following peripheral sensitization of nociceptors. Baseline sensitivity was not altered in these rat models that assessed innate reflex responses (i.e. hind-paw withdrawal to thermal or mechanical stimulation). In the present study, we evaluated effects of intrathecal substance P-saporin (SP-sap), a toxin selective for cells expressing NK-1R, on operant escape responses of rats to thermal stimulation. For comparison, lick/guard reflex testing was performed. Injection of a modest dose (175 ng) of SP-sap into the lumbar subarachnoid space produced a partial loss of lamina I/II NK-1R-expressing dorsal horn neurons but did not affect NK-1R-expressing neurons in deeper laminae. Lick/guard responses to 0.3, 44 or 47 degrees C were not affected after SP-sap treatment, but escape responses to these temperatures were significantly attenuated. Three hours after application of mustard oil to the dorsal surface of both hind paws, escape from 44 degrees C was enhanced for controls but not SP-sap-treated rats. Lick/guard responses were enhanced by mustard oil for both SP-sap and control animals. Administration of morphine (1.0 mg/kg, s.c.) before testing decreased escape responding at 47 degrees C for both controls and SP-sap rats. Thus, partial loss of NK-1R-expressing neurons in the superficial dorsal horn attenuated thermal nociceptive sensitivity and prevented secondary hyperalgesia when studied with an operant algesia assay, in contrast to innate reflexes which were less sensitive to modification by intrathecal SP-sap.

Analgesics, Opioid↗
PubMed2003

Destruction of midbrain dopaminergic neurons by using immunotoxin to dopamine transporter.

1. The ability to target specific neurons can be used to produce selective neural lesions and potentially to deliver therapeutically useful moieties for treatment of disease. In the present study, we sought to determine if a monoclonal antibody to the dopamine transporter (anti-DAT) could be used to target midbrain dopaminergic neurons. 2. The monoclonal antibody recognizes the second, large extracellular loop of DAT. The antibody was conjugated to the "ribosome-inactivating protein"; saporin, and stereotactically pressure microinjected into either the center of the striatum or the left lateral ventricle of adult, male Sprague-Dawley rats. 3. Local intrastriatal injections produced destruction of dopaminergic neurons in the ipsilateral substantia nigra consistent with suicide transport of the immunotoxin. Intraventricular injections (i.c.v.) produced significant loss of dopaminergic neurons in the substantia nigra and ventral tegmental area bilaterally without evident damage to any other aminergic structures such as the locus coeruleus and raphe nuclei. To confirm the anatomic findings, binding of [3-H]mazindol to DAT in the striatum and midbrain was assessed using densitometric analysis of autoradiograms. Anti-DAT-saporin injected i.c.v. at a dose of 21 microg, but not 8 microg, produced highly significant decreases in mazindol binding consistent with loss of the dopaminergic neurons. 4. These results show that anti-DAT can be used to target midbrain dopaminergic neurons and that anti-DAT-saporin may be useful for producing a lesion very similar to the naturally occurring neural degeneration seen in Parkinson's disease. Anti-DAT-saporin joins the growing list of neural lesioning agents based on targeted cytotoxins.

Animals↗
PubMed2001

Lack of effect of moderate Purkinje cell loss on working memory.

192 immunoglobulin G-saporin (192-sap) is an immunotoxin which targets the cholinergic basal forebrain after injection into either the ventricular system or the parenchyma of the rat brain. When injected by the i.c.v. route, 192-sap kills some cerebellar Purkinje cells in addition to its more extensive killing of the cholinergic basal forebrain. Behaviorally, i.c.v. injections of 192-sap result in impaired performance in a variety of experimental paradigms of learning and memory including a working memory task in the radial maze. The current study examined the contribution, if any, of immunotoxin-induced Purkinje cell loss to impaired performance in the radial maze. To meet this aim, we used i.c.v. injection of another immunotoxin, OX7-saporin (OX7-sap), at a dose that produced Purkinje cell loss of similar extent to that produced by i.c.v. 192-sap. We then compared these OX7-sap-injected rats with 192-sap-injected rats in a radial maze working memory task. We found a working memory impairment only in the 192-sap-injected rats. These data show that moderate Purkinje cell loss alone is insufficient to impair working memory. Furthermore, the data are consistent with the idea that the working memory deficit observed in 192-sap-injected animals is likely due to lesioning of the cholinergic basal forebrain.

Animals↗
PubMed2001

Colocalization of mu-opioid receptors and activated G-proteins in rat cingulate cortex.

Anterior cingulate cortex (ACC) has a role in pain processing, however, little is known about opioid system organization and actions. This rodent study defines opioid architecture in the perigenual and midcingulate divisions of ACC, relates mu-opioid receptor binding and G-protein activation, and localizes such binding to afferent axons with knife-cut lesions and specifically to noradrenergic terminals with immunotoxin lesions (anti-dopamine beta-hydroxylase-saporin; anti-DBH-saporin). [(3)H]Tyr-D-AlaGly-MePhe-Gly-ol (DAMGO) binding was highest in perigenual areas 32 and 24 with a peak in layer I. Midcingulate area 24' and posterior cingulate area 29 had overall lower binding in each layer. In contrast, DAMGO-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding in area 24' was similar to that in area 24, whereas area 29 had low and homogeneous binding. Undercut lesions reduced [(3)H]DAMGO binding in all layers with the greatest loss in layer I (-65%), whereas DAMGO-stimulated [(35)S]GTPgammaS binding losses occurred in only layers I-III. Anti-DBH-saporin reduced [(3)H]DAMGO binding in layer I of area 24; DAMGO-stimulated [(35)S]GTPgammaS binding was unchanged in areas 24' and 29. Correlation analysis of receptor and G-protein activation before and after undercut lesions suggested there were a greater number of DAMGO receptor sites for each G-protein on axons, than on somata and proximal dendrites. Finally, perigenual and midcingulate cortices have different opioid architectures due to a higher proportion of mu-opioid receptors expressed by afferent axons in areas 24 and 32.

Analgesics, Opioid↗
PubMed2000

Neuronal lesioning with axonally transported toxins.

Axonally transported toxins can be used to make selective lesions of the nervous system. Collectively, these techniques are termed 'molecular neurosurgery' because they exploit the surface molecular identity of neurons to selectively destroy specific types of neurons. Suicide transport, is anatomically selective but not type-selective. The most widely used suicide transport agents are the toxic lectins (ricin, volkensin) and the immunotoxin, OX7-saporin. The toxic lectins and saporin are ribosome inactivating proteins that irreversibly inhibit protein synthesis. The toxic lectins have binding subunits but saporin requires a targeting vector to gain entrance into cells. Immunolesioning uses monoclonal anti-neuronal antibodies to deliver saporin selectively into neurons that express a particular target surface antigen. Neuropeptide-saporin conjugates selectively destroy neurons expressing the appropriate peptide receptors. Notable experimental uses of these agents include analysis of the function of the cholinergic basal forebrain (192-saporin) and pain research (anti-DBH-saporin, substance P-saporin). It is likely that more immunolesioning and neuropeptide-toxin conjugates will be developed in the near future.

Animals↗
PubMed2000

Immunolocalization of the cocaine- and antidepressant-sensitive l-norepinephrine transporter.

Norepinephrine (NE) transporters (NETs) constitute the primary mechanism for inactivation of synaptically released NE, are targets for multiple antidepressants and psychostimulants, and have been reported to be deficient in affective and autonomic disorders. Although the regional distribution of NETs has been defined through synaptosomal transport and autoradiographic approaches, NET protein expression has yet to be characterized fully in the central nervous system (CNS). We identified a cytoplasmic NET epitope (amino acids 585-602) and corresponding antibody (43411) that permits cellular localization of endogenous NET expression in the CNS and periphery. In the adult rat brain, NET labeling was confined to noradrenergic neuronal somata, axons, and dendrites, including extensive arborizations within the hippocampus and cortex, but was absent from epinephrine- and dopamine-containing neurons. Intracerebroventricular anti-dopamine beta-hydroxylase/saporin, a treatment that destroys a majority of noradrenergic neurons and their projections, validated the specificity of the 43411 antibody. At the level of light microscopy, 43411 labeling colocalized with the axonal markers syntaxin, synaptophysin, and SNAP-25. Indirect immunofluorescence revealed a nonuniform pattern of NET expression along axons, particularly evident within sympathetic fibers of the vas deferens, reflecting a high degree of spatial organization of NE clearance. NET labeling in somata was intracellular and absent from plasma membranes. Among nonneuronal cells, glial cells lacked NET immunoreactivity, whereas CNS ependymal cells were an unexpected site of labeling. NET immunoreactivity was also evident in a subset of adrenal chromaffin cells where labeling appeared to be predominantly associated with intracellular vesicles. Initial ultrastructural evaluation via preembedding immunogold techniques also revealed substantial cytoplasmic NET immunoreactivity in axon terminals within the prelimbic prefrontal cortex, consistent with postulates of regulated trafficking controlling neurotransmitter clearance. NET visualization should be of significant benefit in evaluating neuronal injury resulting from chronic drug exposure and in disease states.

Animals↗
PubMed2000

Molecular heterogeneity of protein kinase C expression in human ventricle.

OBJECTIVE: Although activation of protein kinase C (PKC) modulates the function of normal cardiac myocytes and likely plays a role in the pathogenesis of cardiomyopathic disease states, the molecular basis of PKC expression in human ventricle has not been examined in detail. METHODS: We have performed Western analysis and immunohistochemistry on explanted human cardiac tissue from nondiseased and diseased specimens using isoform-specific antibodies directed against all known PKC isozymes. RESULTS: In homogenates from left and right ventricle, all isoforms except PKC-gamma and theta were detected by immunoblotting, with confirmation using a second antibody directed against a different epitope when possible. For PKC-betaII, delta, and epsilon, data indicated that these isoforms were variably phosphorylated in vivo, resulting in multiple bands during immunoblotting. Because of potential antibody cross-reactivity, reverse transcriptase polymerase chain reaction (RT-PCR) was performed which confirmed expression of PKC-alpha, betaI, and zeta. Immunohistochemistry demonstrated that all isoforms detected in ventricular homogenate by Western analysis could be localized to cardiac myocytes. From a methodologic standpoint, significant degradation of PKC isoforms could be demonstrated when samples were either frozen or allowed to remain at room temperature, compared to immediate subcellular fractionation. CONCLUSIONS: These findings indicate that the PKC expression in human ventricular myocytes is remarkably diverse, with multiple conventional, novel, and atypical isoforms present, and highlight the importance of sample preparation in comparative studies of PKC isoform expression.

Adolescent↗
PubMed2000

Impaired acquisition of a Morris water maze task following selective destruction of cerebellar purkinje cells with OX7-saporin.

Spatial learning in the Morris water maze task is believed to be dependent on an intact hippocampal system. However, evidence from human studies and animal experiments suggests a potential cerebellar involvement in spatial processing, place learning, and other types of 'higher-order' cognition. In order to investigate this possibility, intraventricular injections (ICV) of the anti-neuronal immunotoxin OX7-saporin were used to selectively destroy cerebellar Purkinje cells, without affecting other brain areas believed to be critically involved in spatial learning and memory. Bilateral ICV injections of 2 microg OX7-saporin (4 microg total) in adult male rats produced substantial loss of Purkinje cells (56%) throughout the cerebellum without affecting hippocampal morphology or biochemical indices of cholinergic, serotonergic, or catecholaminergic function in the hippocampus, frontal cortex, or striatum. ICV OX7-saporin significantly impaired acquisition and performance of the standard Morris water maze task (though the impairment was less severe than reported in earlier studies that used alternate lesion methods or mutant mice species), but did not alter performance on the cued version of the task, or locomotor activity. In addition, lesioned animals spent significantly less time in the target quadrant on probe trial days 4 and 7 and the average distance to target scores (ADT) were significantly greater than controls on those days. Swim speed was not affected. Based on the specificity of the behavioral and neurobiological alterations, these data support the hypothesis that the cerebellum is involved in spatial processing and place learning.

Adult↗
PubMed2000

Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin.

Perception of external stimuli is often mediated through the activity of a G protein-coupled receptor in response to its ligand. Receptor-mediated internalization allows the insertion of toxins that cause the elimination of receptor-expressing neurons. Using this technique new information on systems biology can be discovered and with this, new therapeutics developed.

Animals↗
PubMed2000

Enhancement of cortical plasticity by behavioral training in acetylcholine-depleted adult rats.

Trimming all whiskers except two on one side of an adult rat's face results in cortical plasticity in which the spared whiskers, D2 and one D-row surround whisker (either D1 or D3), evoked responses containing more spikes than the response evoked by the cut whisker (called whisker pairing plasticity). Previously we have reported that acetylcholine (ACh) depletion in cortex prevents surround D-row whisker plasticity from developing within the barrel cortex. In this study we examined whether the animal's active use of its two intact whiskers can restore some aspects of plasticity in the ACh-depleted cortex. To achieve this goal, ACh was depleted from barrel field cortex, and 14 days after the depletion surgery, whiskers were trimmed and animals were trained on a whisker-dependent gap crossing task. After 7 days of training, animals were anesthetized with urethan and prepared for single-unit recording. Training the ACh-depleted, whisker-paired animals resulted in a significant enhancement of responses to paired surround whiskers: the D-paired whisker-evoked response contained more spikes than the D-cut evoked response. We conclude that training whisker paired rats has a positive impact on response properties of neurons in S1 cortex, even in ACh-depleted animals.

Acetylcholine↗
PubMed2000

Preferential destruction of cerebellar Purkinje cells by OX7-saporin.

Saporin, a plant toxin derived from Saponaria officinalis, disrupts protein synthesis by inactivating the 60S portion of the ribosomal complex. OX7 is a mouse monoclonal antibody directed against the Thy-1.1 receptor that is differentially expressed on subpopulations of central nervous system neurons. Disulfide conjugation of OX7 to saporin permits delivery of saporin to target neurons. OX7-saporin was used to study the behavioral and morphological consequences of selective destruction of cerebellar Purkinje cells which abundantly express the Thy-1.1 antigen. Male Sprague-Dawley rats received bilateral intraventricular injections of 1- or 2 microg OX7-saporin or 8 microl artificial CSF. Rats were tested for behavioral changes 1 week before and 1, 2, and 8 weeks post-treatment. OX7-saporin treatment resulted in dose- and time-dependent changes in motor performance, activity, and negative geotaxis, but did not affect foot splay. Following behavioral testing, cerebellar sections were prepared for microscopic examination and the pattern of Purkinje cell loss was determined in anatomically matched sections. OX7-saporin induced dose-dependent death of Purkinje cells, particularly in the anterior and superior portions of cerebellar folia 1-6 and folium 9. Other brain regions appeared largely unaffected. Data suggest that intraventricular injection of rats with OX7-saporin is an effective model with which to examine the consequences of Purkinje cell destruction.

Animals↗
PubMed1999

Targeting neurokinin-1 receptor-expressing neurons with [Sar9,Met(O2)11 substance P-saporin.

Neurons expressing neurokinin-1 receptors (NK-1R) are selectively destroyed by substance P (SP) coupled to the ribosome inactivating protein, saporin. SP-saporin produces incomplete lesions of striatal NK-1R-expressing neurons even at doses that produce non-specific damage. In the present study, we sought to determine if the more stable, NK-1R-specific SP analog conjugated to saporin, [Sar9,Met(O2)11]-SP (SSP-saporin), would selectively destroy cells expressing NK-1R, in vitro and in vivo. The results show that SSP-saporin is highly effective and selective, producing extensive ablation of striatal NK-1R expressing interneurons at doses that do not cause loss of other striatal neurons suggesting advantages over SP-saporin as a selective lesioning agent. SSP-saporin will be useful in larger species and for intraparenchymal injections.

Animals↗
PubMed1999

Threshold relationship between lesion extent of the cholinergic basal forebrain in the rat and working memory impairment in the radial maze.

The cholinergic basal forebrain (CBF) degenerates in Alzheimer's Disease (AD), and the degree of this degeneration correlates with the degree of dementia. In the present study we have modeled this degeneration in the rat by injecting various doses of the highly selective immunotoxin 192 IgG-saporin (192-sap) into the ventricular system. The ability of 192-sap-treated rats to perform in a previously learned radial maze working memory task was then tested. We report here that 192-sap created lesions of the CBF and, to a lesser extent, cerebellar Purkinje cells in a dose-dependent fashion. Furthermore, we found that rats harboring lesions of the entire CBF greater than 75% had impaired spatial working memory in the radial maze. Correlational analysis of working memory impairment and lesion extent of the component parts of the CBF revealed that high-grade lesions of the hippocampal-projecting neurons of the CBF were not sufficient to impair working memory. Only rats with high-grade lesions of the hippocampal and cortical projecting neurons of the CBF had impaired working memory. These data are consistent with other 192-sap reports that found behavioral deficits only with high-grade CBF lesions and indicate that the relationship between CBF lesion extent and working memory impairment is a threshold relationship in which a high degree of neuronal loss can be tolerated without detectable consequences. Additionally, the data suggest that the CBF modulates spatial working memory via its connections to both the hippocampus and cortex.

Animals↗
PubMed1999

Septal cholinergic deafferentation of the dentate gyrus results in a loss of a subset of neuropeptide Y somata and an increase in synaptic area on remaining neuropeptide Y dendrites.

Removal of cholinergic septal inputs using the immunotoxin 192 IgG-saporin reduces the number of interneurons containing neuropeptide Y (NPY)-immunoreactivity in the rat dentate gyrus by approximately 30% [Milner et al., J. Comp. Neurol. 386 (1997) 48-59]. The goal of the present study was to determine if NPY-containing neurons that survive deafferentation have any distinguishing morphological and/or microenvironmental features. For this, 2 or 24 weeks after intracerebroventricular injections of 192 IgG-saporin, NPY-immunolabeled neurons in the hilus of the dentate gyrus were examined by electron microscopy. Neither the size nor morphological traits of NPY-labeled perikaryal or dendritic profiles from lesioned compared to control rats at either time-point differed significantly. However, at both time-points, NPY-containing somatal profiles from immunolesioned rats compared to controls had a reduced percentage of their plasmalemmal surface apposed to unmyelinated axon profiles and an increased percentage of their surface occupied by astrocytic profiles. At the 24 week time-point, these differences were statistically significant. The primary contributing factor for these changes was the absence of a subgroup of NPY-labeled somatal profiles in lesioned rats compared to controls which was: (a) distinguished by frequent appositions of unmyelinated axons (from 15 to 35%) to the plasmalemmal surface; and (b) located primarily in the central hilar region. Unlike NPY-containing somata, changes associated with NPY-labeled dendritic profiles were exclusively related to associated presynaptic profiles at the 24 week time-point. In lesioned rats compared to controls at this time-point, NPY-containing dendritic profiles had a concurrent increase in the percentage of the plasmalemmal surface occupied by active zones and the size of terminals contacting them. The present results combined with those of our earlier study suggest that septal cholinergic deafferentation results in: (a) the loss of a distinct subpopulation of hippocampal NPY-containing neurons; and (b) an increase in total active zone area suggesting a strengthening of synaptic connections to the surviving population of NPY-containing neurons in the long term.

Acetylcholine↗
PubMed1999

Lesions of the C1 catecholaminergic neurons of the ventrolateral medulla in rats using anti-DbetaH-saporin.

Phenylethanolamine-N-methyltransferase (PNMT)-containing neurons in the rostral ventrolateral medulla (RVLM) are believed to play a role in cardiovascular regulation. To determine whether injection of anti-dopamine beta-hydroxylase (DbetaH)-saporin directly into the RVLM in rats could selectively destroy these cells and thereby provide an approach for evaluating their role in cardiovascular regulation, we studied rats 2 wk after unilateral injection of 21 ng anti-DbetaH-saporin into the RVLM. There was an approximately 90% reduction in the number of PNMT-positive neurons in the RVLM, although the number of non-C1, spinally projecting barosensitive neurons of this area was not altered. The A5 cell group was the only other population of DbetaH-containing cells that was significantly depleted. The depressor response evoked by injection of tyramine into the RVLM was abolished by prior injection of toxin. The pressor response evoked by injection of glutamate into the RVLM was attenuated ipsilateral to the toxin injection but was potentiated contralateral to the toxin injection. Thus anti-DbetaH-saporin can be used to make selective lesions of PNMT-containing cells, allowing for the evaluation of their role in cardiovascular regulation.

Animals↗