Biomedical subjects

R G Wiley

Publications and source records attributed to R G Wiley.

At least 55 records · Page 3Linked to original sources

PubMed1996

Injection of IgG 192-saporin into the medial septum produces cholinergic hypofunction and dose-dependent working memory deficits.

192-IgG saporin is an anti-neuronal immunotoxin that combines the 192 monoclonal antibody to the p75 neurotrophin receptor found on terminals and cell bodies of neurons in the cholinergic basal forebrain with the ribosome-inactivating protein saporin. Injection of 100, 237.5 or 375 ng of 192-saporin into the medial septum produced dose-related deficits in a variable-delay radial-arm maze task. 192-saporin decreased the number of correct choices and increased the number of errors in the delayed non-match to sample task. These deficits persisted throughout training and were most evident in the 375 ng group. The behavioral deficits were associated with dose-dependent decreases in pre-synaptic cholinergic parameters (ie., high affinity choline uptake) in the terminal fields of the medial septum (hippocampus, cingulate, entorhinal cortex). Choline uptake was not affected in the frontal cortex or the striatum; structures not innervated by the septum. There were no changes in regional concentrations of dopamine, serotonin, or their metabolites. Site-specific injection of IgG 192-saporin is a useful approach to explore the functions of the cholinergic basal forebrain and to model diseases of cholinergic hypofunction such as Alzheimer's disease.

Acetylcholine↗

PubMed1996

The effects of immunolesions of nerve growth factor-receptive neurons by 192 IgG-saporin on sleep.

Low-affinity nerve growth factor (NGF) receptors are present on the cholinergic neurons of the basal forebrain. We studied the effects of 192 IgG-saporin, a specific immunotoxin for the NGF receptor-positive, cholinergic basal forebrain neurons, on sleep, the power spectrum of the electroencephalogram (EEG), and body temperature. After 3 d baseline recordings, 12 male rats were injected intracerebroventricularly with 4 micrograms 192 IgG-saporin. EEG, motor activity, and brain temperature were recorded for 23 h on the first, third, fifth, and seventh day after the treatment. 192 IgG-saporin did not affect the total daily amounts but altered the circadian distribution of sleep. On days 1 and 3 after the injection of the immunotoxin, the amount of non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) increased during the dark period, whereas during the light both NREMS and REMS decreased. On day 5, these changes were less pronounced and sleep completely returned to the baseline by day 7. The EEG was suppressed in each frequency band and each vigilance state, and, in contrast to sleep, these changes in EEG persisted for 7 days. Brain temperature was decreased from day 3. These results suggest that NGF receptor-positive, cholinergic basal forebrain neurons are not necessary for the maintenance of total sleep time but contribute to the generation of normal EEG and the maintenance of brain temperature.

Analysis of Variance↗

PubMed1996

The basal forebrain cholinergic system and object memory in the rat.

Rats with near complete destruction of basal forebrain cholinergic neurons from intracerebroventricular injections of 192 IgG-saporin were trained on object discrimination problems and then retrained two weeks later to measure retention. Despite dramatic reductions of acetylcholinesterase-positive fibers in hippocampus and neocortex, these animals did not differ from controls on an analysis of savings scores. Thus, the basal forebrain cholinergic system may serve functions that support non-spatial memory but are not specifically mnemonic in nature.

Acetylcholinesterase↗

PubMed1996

Loss of nucleus basalis magnocellularis, but not septal, cholinergic neurons correlates with passive avoidance impairment in rats treated with 192-saporin.

Intraventricular injection of 192-saporin, an immunotoxin directed at the low affinity neurotrophin receptor (p75NGFr), selectively destroys cholinergic neurons in the basal forebrain (CBF). In the present study, we sought to determine if there was a correlation between degree of CBF neuron destruction and degree of passive avoidance behavioral impairment. 192-saporin caused a decrease in the number of p75NGFr + neurons in both nucleus basalis magnocellularis (Nbm) and medial septal nucleus/diagonal band of Broca (MS/DBB). All rats had >95% loss of the p75NGFr + cholinergic neurons in the MS/DBB, but there was variation in the extent of the Nbm cell loss. A significant correlation was found between the severity of impairment of passive avoidance learning and the magnitude of the loss in the number of p75NGFr + neurons in the Nbm. Step-through latency also correlated significantly with the magnitude of loss of AChE histochemical staining intensity in dorsolateral neocortex ipsilateral to the injection of 192-saporin. These data show that >95% loss of cholinergic neurons in MS/DBB is not sufficient to impair passive avoidance learning. However, in the presence of severe loss of cholinergic neurons from the MS/DBB, the resulting deficit in passive avoidance behavior is proportional to the degree of cholinergic neuron loss from the Nbm. These results are interpreted as support for the hypothesis that the cholinergic projection from Nbm to neocortex plays a role in passive avoidance behavior.

Animals↗

PubMed1996

Altered NGF protein levels in different brain areas after immunolesion.

Nerve growth factor (NGF) provides critical trophic support to the cholinergic basal forebrain neurons that express high levels of the low-affinity NGF receptor (p75NGFR) in the adult rat brain. Intraventricular injection of 192 IgG-saporin, made by coupling the monoclonal antibody to p75NGFR 192 IgG to the cytotoxin saporin, selectively destroys the p75NGFR-bearing neurons in the basal forebrain and was used here to examine the effects of selective cholinergic lesions on brain NGF protein levels. We showed that 192 IgG-saporin produced significant long-lasting elevation of NGF protein levels in the hippocampus, cortex, and olfactory bulb, with profound reductions of ChAT activities representing complete cholinergic deafferentations of these areas. NGF level was maintained in the basal forebrain, even though there was almost complete loss of p75NGFR-immunoreactive cells and significant decrease of ChAT activity. In addition, a mild glial response was observed in the basal forebrain, and most of the activated astroglia expressed NGF-like immunoreactivity there. The increases in NGF protein levels in the target areas of the basal forebrain were most likely due to loss of cholinergic basal forebrain neurons and retrograde transport of NGF from these areas. Glial-derived NGF is partially responsible for the maintained level of NGF in the basal forebrain after the loss of cholinergic neurons. The accumulation of NGF protein in the target areas may have some effects on synaptic rearrangement in denervated tissues.

Animals↗

PubMed1996

Targeting toxins to neural antigens and receptors.

Immunotoxins have been used to study the targeting of biologically active substances at neurons in vivo and to make experimental neural lesions. OX7-saporin, directed against Thy 1, destroys any neuron. 192 IgG-saporin, directed against the 'low affinity' neurotrophin receptor (p 75NTR), selectively destroys neurons expressing this receptor (sympathetic, sensory, cholinergic basal forebrain, cerebellar Purkinje). Anti-D beta H-saporin, directed against dopamine beta-hydroxylase, the enzyme that converts dopamine to norepinephrine, selectively destroys noradrenergic neurons (sympathetic, CNS). These agents show that several types of neural antigens may prove useful in treating pain, and anti-D beta H-saporin may be active against pheochromocytoma or neuroblastoma.

Animals↗

PubMed1996

Immunolesioning of basal forebrain cholinergic neurons facilitates hippocampal kindling and perturbs neurotrophin messenger RNA regulation.

The immunotoxin 192 IgG-saporin induces an efficient and specific lesion of low-affinity nerve growth factor receptor-bearing cholinergic neurons in the basal forebrain. Intraventricular injection of 192 IgG-saporin, which caused a complete loss of cholinergic afferents to the hippocampus and neocortex and a partial denervation of amygdala and piriform cortex, was found to markedly facilitate the initial stages of seizure development in hippocampal kindling. In contrast, the progression of kindling process from focal to generalized seizures was not affected. In situ hybridization demonstrated that basal levels of brain-derived neutrotrophic factor messenger RNA in the hippocampal formation and piriform cortex were significantly decreased by the lesion, which also attenuated the seizure-induced increase of brain-derived neurotrophic factor messenger RNA expression in the hippocampus and frontal cortex. In the dentate gyrus, the 192 IgG-saporin lesion selectively reduced the upregulation of messenger RNAs for brain-derived neurotrophic factor exons I and III after a generalized seizure, whereas the increase of exon II messenger RNA was unchanged. The lesion abolished the seizure-evoked increase of nerve growth factor and TrkC messenger RNA levels and decrease of neutrophin-3 messenger RNA expression in dentate granule cells, while TrkB messenger RNA levels were not affected. We conclude that the basal forebrain cholinergic system (1) suppresses kindling epileptogenesis in the hippocampus, and (2) enhances both basal and seizure-evoked brain-derived neurotrophic factor synthesis in the hippocampal formation and some cortical areas through a specific pattern of activation of promoters within the brain-derived neurotrophic factor gene.

Animals↗

PubMed1996

Effects of neonatal lesions of the basal forebrain cholinergic system by 192 immunoglobulin G-saporin: biochemical, behavioural and morphological characterization.

Selective removal of the basal forebrain cholinergic neurons by the immunotoxin 192 immunoglobulin G-saporin has offered a new powerful tool for the study of the relationships between cholinergic dysfunction and cognitive impairments. In the present study the morphological and functional consequences of selective lesions of the basal forebrain cholinergic system during early postnatal development have been investigated following bilateral intraventricular injections of 192 immunoglobulin G-saporin to immature (four-day-old) rats. Administration of increasing doses (0.2-0.8 microgram) of the immunotoxin produced dose-dependent loss of cholinergic neurons in the septal/diagonal band area (up to 72-86%) and in the nucleus basalis magnocellularis (up to 91-93%), paralleled by marked reductions in choline acetyltransferase activity in the hippocampus and several cortical regions (73-84%). The parvalbumin-positive neurons in the septal/diagonal band area and the calbindin-positive Purkinje cells in the cerebellum were unaffected at all dose levels. Brain dopamine or noradrenaline levels were unaffected or increased by the immunotoxin treatment. At the optimal dose, 0.4 microgram, the toxin conjugate produced maximal cholinergic depletion without significant mortality. Higher doses (0.8, 1.2 and 1.6 micrograms) of toxin, on the other hand, proved to be lethal for most or all of the injected animals. When tested at three and eight months after the optimal dose, in spite of persisting cholinergic depletion, the noenatally lesioned animals showed no impairment in the water maze task or in locomotor activity and exploration as compared to normal controls, probably reflecting partial sparing of the cholinergic neurons by the neonatal immunotoxic lesion (above all in the vertical and horizontal limbs of the diagonal band area), and/or a greater degree of plasticity in the developing as compared to the mature cholinergic system. The place navigational performance of the neonatally lesioned animals in the water maze task was abolished by central muscarinic cholinergic receptor blockade (by atropine) or by a second immunotoxic lesion, which eliminated virtually all residual cholinergic neurons in the septal/diagonal band area and the nucleus basalis. Administration of 192 immunoglobulin G-saporin to similarly trained, but previously normal adult rats, produced similar cholinergic depletions but much less severe place navigation deficits, suggesting that preoperative training on the task may reduce the functional consequences of a subsequent cholinergic lesion. The results thus support the view that the basal forebrain cholinergic system may be implicated in the acquisition rather than retention of spatial memory in the water maze task.

Animals↗

PubMed1996

The regulation of hippocampal dynorphin by neural/neuroendocrine pathways: models for effects of aging on an opioid peptide system.

Previous research has demonstrated increased messenger RNA expression and peptide content in an opioid system localized to hippocampal dentate granule cells in aged rats. This altered regulation of dynorphin was correlated with the emergence of an age-related impairment in spatial learning. Considerable evidence exists for additional effects of aging on systems that provide input to the dynorphin-containing dentate granule cells. Such changes have been well documented for loss of perforant path innervation from entorhinal cortex, deterioration in septohippocampal cholinergic neurons, and high amounts of glucocorticoids that have, among their targets, receptors located in the dentate gyrus. Similar to the effects of aging on hippocampal dynorphin, age-related changes in each of these systems correlate with the severity of spatial learning impairment in aged rats. This raises the possibility that dysregulation of dynorphin in the aged brain is a reactive response to antecedant change(s) in this circuitry, a hypothesis that was examined by separately manipulating in young rats the three neural/neuroendocrine systems identified above. Of the three models examined only removal of the perforant path reproduced the effect of aging on dynorphin in the hippocampal formation. An immunotoxin was used in Experiment 1 to selectively remove septo-hippocampal cholinergic neurons in young rats. No alteration in hippocampal opioid peptides was produced by this treatment. Experiment 2 examined effects of exposure to excess corticosterone. Adrenalectomized rats exhibited a significant decrease in hippocampal dynorphin-A (1-8) content, which was reversed by corticosterone replacement at a concentration approximating normal basal levels. Dynorphin-A (1-8) content, however, was not reliably increased by exposure to excess corticosterone. In contrast, perforant path removal was found to reproduce the effect of aging on dynorphin content; either aspiration of the entorhinal cortex or knife-cut transections of the perforant path reliably increased hippocampal dynorphin content. These results support the conclusion that age-related deterioration in the septohippocampal cholinergic system and evaluated exposure to corticosterone are not sufficient to induce an elevation in hippocampal dynorphin content. Only removal of the perforant path innervation was found to reproduce the elevation in hippocampal dynorphin content observed in aged rats with hippocampal-dependent learning impairment.

Adrenalectomy↗

PubMed1996

Selective immunolesioning of the basal forebrain cholinergic system disrupts short-term memory in rats.

Selective depletion of nerve growth factor receptor-bearing neurons in the basal forebrain cholinergic system nuclei by the immunotoxin 192 IgG-saporin offers a new and highly useful tool for the study of the role of the forebrain cholinergic system in cognitive functions. In the present study, we have tested the effects of 192 IpG-saporin in an operant delayed matching-to-position task which has previously been used to discriminate between delay-dependent learning impairments and delay-independent disturbances of non-mnemonic processes. Rats were first trained to criterion performance and then received intraventricular injections of 5 microg of 192 IgG-saporin 4 weeks prior to a second testing session. Rats with 192 IgG-saporin lesions displayed a significant delay-dependent decline in performance compared to normal controls, indicating a deficit in short-term memory. Administration of the muscarinic blocker scopolamine (0.5 mg/kg, i.p.) produced more pronounced impairment in the performance of the normal control rats across all delays, and induced further impairment also in animals with 192 IgG-saporin lesions. These effects were not observed following control injections of methyl scopolamine, suggesting that the impairment induced by scopolamine was due to the blockade of central muscarinic receptors. No improvement in performance was observed in either group following systemic treatment with the muscarinic cholinergic agonist arecoline (1.00 mg/kg). Biochemical and morphological analyses confirmed the selective and severe (>90-95%) depletion of cholinergic neurons throughout the septal-diagonal band area and the nucleus basalis region by the intraventricular 192 IgG-saporin treatment. Although the immunotoxin was observed to produce additional damage to the cerebellar Purkinje cells, no gross motor abnormalities were observed that could contribute to the effects on accuracy in the task used here. In conclusion, the results show that selective combined lesions of the basal forebrain cholinergic neurons in the septal-diagonal band area and nucleus basalis produce long-lasting impairments in short-term memory, thus providing further support for a role of this system in cognitive functions.

Animals↗

PubMed1995

Behavioral and neurobiological alterations induced by the immunotoxin 192-IgG-saporin: cholinergic and non-cholinergic effects following i.c.v. injection.

192-IgG-Saporin is an anti-neuronal immunotoxin that combines the 192 monoclonal antibody to the p75 neurotrophin receptor found on terminals and cell bodies of neurons in the cholinergic basal forebrain with the ribosome-inactivating protein saporin. Bilateral intraventricular injection of the 192-saporin produced a variety of dose-related behavioral, neurochemical, and histological alterations in adult male rats. While both the 2 micrograms and 4 micrograms dose produced comparable cholinergic hypofunction only the high dose produced behavioral changes. Behavioral deficits induced by the 4 micrograms dose of 192-saporin induced alterations in rotorod performance and reactivity on the hot-plate which recovered over 8 weeks. In addition, the 4 micrograms dose produced a persistent impairment in the acquisition and performance of standard Morris water maze task as well as a cued version of the task. The neurobiological alterations induced by 192-saporin involved both cholinergic and non-cholinergic systems. Both doses of 192-saporin produced a 60-80% decrease in high affinity choline transport in the hippocampus and cortex without altering this parameter in the striatum. In addition, there was a significant dose-related decrease of norepinephrine in the hippocampus in the high dose group. 192-saporin did not alter the content of dopamine, serotonin, or their metabolites in any region examined. 192-saporin also produced a loss of Purkinje cells in the cerebellum. This cell type also expresses the p75 receptor and appears to be a target for intraventricular 192-saporin. This complex interplay of factors makes the i.c.v. model of 192-saporin very problematic for studying the functional properties of the cholinergic basal forebrain. However, recent data suggest that injection of 192-saporin directly into components of the cholinergic basal forebrain can be used to further elaborate the function of this brain system and to model disorders of cholinergic hypofunction such as Alzheimer's disease.

Animals↗

PubMed1995

Effects of suicide transport lesions of the striatopallidal or striatonigral pathways on striatal ultrastructure.

In the basal ganglia, centrally active suicide transport agents produce selective lesions of the striatopallidal and striatonigral pathways based on receptor binding and neuropeptide mRNA studies. Anatomical analyses indicate a selective, albeit modest, loss of projection neurons. In the present study, we sought to determine the ultrastructural sequelae in the striatum of suicide transport injections of the globus pallidus (GP) or substantia nigra (SN). Neostriata of adult rats were examined 10 days after lesions of the striatopallidal or striatonigral pathways with OX7-saporin or volkensin. Controls consisted of normal unoperated rats and animals injected into either target with ricin, a toxic lectin that is not transported in the central nervous system. Injections with OX7-saporin or volkensin into the GP or SN produced a decrease in striatal synaptic density of approximately 20%, relative to the contralateral side. Dark degenerating profiles, though very rare in the contralateral striata, were present throughout the neuropil in the ipsilateral striata. In animals with striatopallidal lesions, axospinous synapses of both the asymmetric and symmetric type were decreased in density, while the number of synapses formed with dendritic shafts was unaffected. In addition, the number of striatal mitochondrial profiles was decreased ipsilateral to the lesions. In animals with striatonigral lesions, the number of axospinous and axodendritic synapses of the asymmetric type was decreased ipsilateral to the lesions. Synaptic density and ultrastructural integrity remained unaffected in the striata of animals receiving ricin injections and in the contralateral striata of animals receiving OX7-saporin or volkensin injections. Our results, taken together with previous studies showing marked loss of receptors, uptake sites and mRNA, suggest that while most synapses are present and intact, the efficacy of synaptic transmission may be altered.

Animals↗

PubMed1995

192IgG-saporin immunotoxin-induced loss of cholinergic cells differentially activates microglia in rat basal forebrain nuclei.

To characterize the specificity of a novel cholinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the low-affinity nerve growth factor receptor with the cytotoxic protein saporin), coronal sections through the basal forebrain of adult rats, that received a single intracerebro-ventricular injection of 4 micrograms of 192IgG-saporin conjugate, were subjected to histochemical and immunocytochemical procedures to evaluate cholinergic (choline acetyltransferase (ChAT)-immunoreactive, acetylcholinesterase-positive, NADPH-diaphorase-positive) and GABAergic structures (parvalbumin-immunoreactive, labeling of perineuronal nets with Wisteria floribunda agglutinin) as well as microglia (visualized with Griffonia simplicifolia agglutinin) and astrocytes (immunostaining for glial fibrillary acidic protein). Seven days following injection of the immunotoxin, ChAT-immunoreactive cells nearly completely disappeared throughout the magnocellular basal forebrain complex, including globus pallidus, as compared to vehicle-injected controls. However, there was no significant difference in the number of ChAT-positive cells in the adjacent ventral pallidum and in the caudate-putamen of immunolesioned and control animals. NADPH-diaphorase-containing cells, including a significant subpopulation of cholinergic cells, also strikingly decreased in number by more than 90% in the magnocellular basal forebrain complex following immunolesion, and only a few noncholinergic diaphorase-positive cells survived in the medial septum, vertical and horizontal diagonal band, and nucleus basalis of Meynert. In contrast, the number of parvalbumin-containing GABAergic projection neurons in the septum-diagonal band of Broca complex and nucleus basalis of Meynert from immunolesioned rats was not different from that of vehicle-injected control animals. Immunolesioning also did not result in any change in either number or shape of cells surrounded by perineuronal nets, which are frequently associated with parvalbumin-containing GABAergic neurons. Seven days following injection of the immunotoxin, a very strong activation of microglia with an identical distribution pattern was observed in all experimental animals. Large numbers of activated microglia were found in all magnocellular basal forebrain nuclei, corresponding to the distribution of degenerating cholinergic cells. Additionally, immunolesioning also resulted in a dramatic activation of microglia in the lateral septal nuclei, which are known to be almost free of cholinergic cells, but not of penetrating cholinergic dendrites in adjacent zones, and in the ventral pallidum, where there was no observed loss of cholinergic cells. There was no significant increase in microglia activation in striatum and cortical areas, and no astrocytic response in any of the basal forebrain nuclei at this particular time point of survival. These results suggest that 192IgG-saporin specifically destroys basal forebrain cholinergic neurons and does not suppress their neuronal activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: behavioural, biochemical and stereological studies in the rat.

The elucidation of the functional role of the basal forebrain cholinergic system will require access to a highly specific and efficient cholinergic neurotoxin. Recently, selective depletion of the nerve growth factor (NGF) receptor-bearing cholinergic neurons in the rat basal forebrain and a dramatic loss of cholinergic innervation in the related cortical regions have been obtained following intraventricular injection of a newly introduced immunotoxin, 192 IgG-saporin. Here we extend these initial findings and report that administration of increasing doses (1.25, 2.5, 5.0 or 10 micrograms) of the 192 IgG-saporin conjugate into the lateral ventricles of adult rats induced dose-dependent impairments in the water maze task and passive avoidance retention, but only weak and inconsistent effects on locomotor activity. These behavioural changes were paralleled by a reduction in choline acetyltransferase activity in hippocampus and several cortical areas (up to 97%) and selective depletions of NGF receptor-positive cholinergic neurons in the septal-diagonal band area and nucleus basalis magnocellularis (up to 99%). By contrast, the non-cholinergic parvalbumin-containing neurons in the septum were completely spared, and other cholinergic projection systems (such as in the striatum, thalamus, brainstem and spinal cord) were unaffected even at the highest dose. The observed changes in the water maze and passive avoidance tasks, as well as the cholinergic cell loss, were maintained up to at least 8 months following the intraventricular injection of a single dose (5 micrograms) of the immunotoxin. The results confirm the usefulness of the 192 IgG-saporin toxin for selective and profound lesions of the basal forebrain cholinergic neurons and provide further support for a role of the basal forebrain cholinergic system in cognitive functions.

Acetylcholinesterase↗

PubMed1995

Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat.

The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forebrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre- and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuropil. Neurons expressing m2 immunoreactivity received synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre- and postsynaptic sites.

Animals↗

PubMed1995

Differential changes in cholinergic markers from selected brain regions after specific immunolesion of the rat cholinergic basal forebrain system.

The aim of this study was to characterize the effects of cortical cholinergic denervation on cholinergic parameters in the cerebral cortex and basal forebrain using a novel immunotoxin (conjugate of the monoclonal antibody 192IgG against the low-affinity nerve growth factor receptor armed with cytotoxin saporin) to efficiently and selectively lesion cholinergic neurons in rat basal forebrain. Seven days following an intracerebroventricular injection of the cholinergic immunotoxin 192IgG-saporin the binding levels of nicotinic and M1- and M2-muscarinic acetylcholine receptors (mAChR), high-affinity choline uptake sites, as well as the m1-m4 mAChR mRNA were determined in coronal brain sections by both receptor autoradiography and in situ hybridization, and quantified by image analysis. Hemicholinium-3 binding to high-affinity choline uptake sites was decreased by up to 45% in all cortical regions and in the hippocampus after a single injection of the immunotoxin compared to controls. In contrast, M1-mAChR sites were increased over the corresponding control value in the anterior parts of cingulate, frontal, and piriform cortex by about 20%, in the hindlimb/forelimb areas (18%), in the parietal cortex (35%), in the occipital cortex area 2 (17%), as well as in the temporal cortex (25%) following immunolesion. M2-mAChR levels were found to be significantly increased in the posterior part of the parietal cortex area 1 (by about 22%) and in the occipital cortex area 2 (20%) only. With respect to laminar cortical localization, M2-mAChRs and choline uptake sites were altered in all cortical layers, whereas M1-mAChRs were preferentially affected in the upper cortical layers by the immunolesion. The increase in M1-mAChR binding in the temporal and occipital cortex as a consequence of the immunolesion was complemented by an increase in the amount of m1 and m3 mAChR mRNA of about 20% in these regions. The elevated levels of M2-mAChR sites in the occipital and temporal cortex following immunolesion were accompanied by an increase in the m4 (by 25%) but not m2 mAChR mRNA. There was no effect of the immunolesion on the m1-m4 mAChR mRNA in frontal cortical regions. in the basal forebrain, however, immunolesioning caused about a 40% decrease in the level of m2 mAChR mRNA in the medial and lateral septum as well as in the vertical and horizontal limb of the diagonal band, whereas M1- and M2-mAChR binding and the levels of m1, m3, and m4 mAChR mRNA were not affected by the immunolesion in any of the basal forebrain nuclei studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Localization of Mu and delta opioid receptors to anterior cingulate afferents and projection neurons and input/output model of Mu regulation.

Anterior cingulate cortex (ACC) has one of the highest densities of opioid receptors in the CNS and it has been implicated in acute and chronic pain responses. Little is known, however, about which neurons express opioid receptors in their dendrites and axon terminals. The present studies employed experimental techniques to remove afferent axons or classes of projection neurons from rat ACC area 24 followed by coverslip autoradiography to localize changes in binding of [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO) to mu receptors and 2-[3H]D-penicillamine-5-D-penicillamine-enkephalin (DPDPE) to delta receptors. Removal of all afferents to area 24 with undercut lesions did not alter DPDPE binding, but significantly reduced binding of DAMGO in layers I, III, and V. In contrast, removal of all cortical neurons with the excitotoxin ibotenic acid almost abolished DPDPE binding in all layers. The same lesions reduced DAMGO binding in most layers; however, there was a postlesion bimodal distribution in binding with high levels of binding in layer I and moderate levels in layer VI. These data suggest that delta receptors are expressed by cortical neurons, while mu receptors are expressed by both cortical neurons and afferent axons. To explore the distribution of postsynaptic receptors, immunotoxin lesions were made in area 24 by injection of OX7-saporin into the caudate and/or thalamic nuclei. Almost complete removal of projection neurons to these targets in layers Vb and VIa did not alter DPDPE binding, while the lesions reduced DAMGO binding in all but layer II. Removal of layer Vb corticostriatal projection neurons with caudate OX7-saporin injections reduced binding only in this layer. It is proposed that opioidergic circuits in area 24 are organized according to an input/output model for mu opioid regulation. In this model mu receptors regulate axon terminal activity from the thalamus in layer Ia and the locus coeruleus in layers Ic and II, whereas cortical outputs to the thalamus are modulated via postsynaptic receptors expressed in all layers by thalamocortical projection neurons with somata in layer VI. These opioidergic circuits in ACC are of particular importance because they may regulate responses to chronic nociceptive activity and associated pain perceptions.

Afferent Pathways↗

PubMed1995

Selective immunolesion of the basal forebrain cholinergic neurons: effects on hippocampal activity during sleep and wakefulness in the rat.

Intracerebroventricular injection of the toxin 192 IgG-saporin (4 micrograms) kills the cholinergic neurons of the basal forebrain bearing the low affinity NGF receptor (NGFr). The effect of this cholinergic denervation on the hippocampal and cortical electrical activity (EEG) was studied during sleep and wakefulness. EEG was recorded under freely-moving conditions in lesioned (n = 10) and control (n = 6) rats (8-16 days post-injection). In lesioned rats, active (AW) and quiet (QW) wakefulness episode durations were similar to those of controls whereas the REM sleep duration was reduced, 8 days post-lesion (P < 0.01). Bouts of REM sleep were more numerous but shorter. The hippocampal theta activity was still present in lesioned-rats during AW (type 1 theta), QW (type 2 theta) and REM sleep. The frequency was unchanged but the amplitude of the three types of theta was significantly reduced (P < 0.01). Type 2 theta occurred with shorter and less regular bouts (P < 0.05). Abnormal slow waves (2-4 Hz) were observed during wakefulness. Histology showed a dramatic loss of NGFr-positive neurons in the basal forebrain and a decline in hippocampal and cortical acetylcholinesterase activity. These results suggest that the cholinergic septohippocampal input is not the primary pacemaker for the hippocampal theta rhythm.

Acetylcholine↗