Ronald G. Wiley

Immunolesioning: selective destruction of neurons using immunotoxin to rat NGF receptor

192 IgG, a monoclonal antibody to the rat nerve growth factor (NGF) receptor, was disulfide-coupled to saporin, a ribosome-inactivating protein. Systemic injection of 192 IgG-saporin destroyed sympathetic postganglionic neurons and some sensory neurons. Injection of 192 IgG-saporin into the lateral ventricle destroyed cholinergic neurons of the basal forebrain. These results show that antineuronal immunotoxins are a powerful approach that may prove useful in a variety of neurobiological applications.

Selective immunotoxic lesions of basal forebrain cholinergic cells: Effects on learning and memory in rats.

Male Long-Evans rats were given injections of either 192 IgG-saporin, an apparently selective toxin for basal forebrain cholinergic neurons (LES), or vehicle (CON) into either the medial septum and vertical limb of the diagonal band (MS/VDB) or bilaterally into the nucleus basalis magnocellularis and substantia innominata (nBM/SI). Place discrimination in the Morris water maze assessed spatial learning, and a trial-unique matching-to-place task in the water maze assessed memory for place information over varying delays. MS/VDB-LES and nBM/SI-LES rats were not impaired relative to CON rats in acquisition of the place discrimination, but were mildly impaired relative to CON rats in performance of the memory task even at the shortest delay, suggesting a nonmnemonic deficit. These results contrast with effects of less selective lesions, which have been taken to support a role for basal forebrain cholinergic neurons in learning and memory.

Differential effects on spatial navigation of immunotoxin-induced cholinergic lesions of the medial septal area and nucleus basalis magnocellularis

The effects on anatomy and behavior of a ribosomal inactivating protein (saporin) coupled to a monoclonal antibody against the low-affinity NGF receptor (NGFr) were examined. In adult rats, NGFr is expressed predominantly in cholinergic neurons of the medial septal area (MSA), diagonal band nuclei, and nucleus basalis magnocellularis (nBM), but also in noncholinergic cerebellar Purkinje cells. Rats with immunotoxin injections to the MSA, nBM, and lateral ventricle were compared to controls on a spatial and cued reference memory task in the Morris maze. Toxin injections to the MSA slightly impaired the initial, but not asymptotic, phase of spatial navigation. Injections to the nBM impaired all phases of spatial navigation. Cued navigation, however, was not affected in either the MSA or nBM group. The ventricular injections severely affected spatial and cued navigation. Acetylcholinesterase (AChE) histochemistry and NGFr and choline acetyltransferase immunohistochemistry revealed a loss of (1) almost all NGFr-positive cholinergic neurons in the MSA and AChE fibers in hippocampus (MSA group); (2) almost all NGFr neurons in the nBM, some in the MSA, most AChE fibers in neocortex and some in the hippocampus (nBM group), and (3) almost all NGFr neurons in the MSA and nBM and their corresponding hippocampal and cortical AChE fibers (ventricular group). Cholinergic nBM projections to the amygdala were largely preserved in all groups. The amount of cholinergic fiber loss in the cortex correlated modestly, but significantly, with the severity of impairment of the asymptotic phase of performance of the spatial task. An unambiguous interpretation of the anatomical locus of behavioral deficits was not possible because of damage to cholinergic striatal interneurons (nBM group) and to noncholinergic cerebellar Purkinje cells (ventricular group). These data suggest that the cholinergic cortical system is critical to the performance of this spatial memory task. Cholinergic denervation of the hippocampus alone, however, is not sufficient to impair markedly performance of this task.

Spatial learning impairments in rats with selective immunolesion of the forebrain cholinergic system

A monoclonal antibody to the low-affinity NGF receptor, 192 IgG, coupled to a cytotoxin, saporin, was recently introduced as an efficient selective neurotoxin for the NGFr-bearing cholinergic neurones in the rat basal forebrain. In the present study we report that an intracerebroventricular injection of this 192 IgG-saporin conjugate induces a severe, long-lasting spatial learning impairment, as assessed in the Morris water-maze task. This behavioural impairment was associated with 65-90% depletion of choline acetyltransferase activity (ChAT) in the hippocampus and cortex. ChAT activity associated with other cholinergic neurone systems in the brain (striatum, mesencephalon, spinal cord), was left virtually unaffected. This new immunotoxin holds great promise as a tool for selective and efficient lesions of the forebrain cholinergic system in functional and behavioural studies.

192 immunoglobulin G-saporin produces graded behavioral and biochemical changes accompanying the loss of cholinergic neurons of the basal forebrain and cerebellar Purkinje cells

Immunolesions of the cholinergic basal forebrain were produced in rats using various intraventricular doses of the immunotoxin 192 immunoglobulin G-saporin: 0.34, 1.34, 2.0, 2.7 and 4.0 micrograms/rat. A battery of behavioral tests, chosen on the basis of reported sensitivity to conventional medial septal or nucleus basalis lesions, was administered. Dose-dependent impairments were found in acquisition, spatial acuity and working memory in the water maze. Dose-dependent hyperactivity in the open field and in swimming speed was observed. The highest dose group (4.0 micrograms) exhibited motoric disturbances which were particularly apparent in swimming and in clinging to an inclined screen. Response and habituation to acoustic startle were diminished in the three higher dose groups. Histological results from acetylcholinesterase and low-affinity nerve growth factor receptor staining showed that the lesion was selective for cholinergic neurons bearing p75 nerve growth factor receptors in the basal forebrain nuclei. However, some Purkinje cells in the superficial layers of the cerebellum were also destroyed at the higher doses of immunotoxin. The activity of choline acetyltransferase, used as a marker of cholinergic deafferentation in regions innervated by the basal forebrain nuclei, was decreased with increasing doses to a plateau level of about 90% (average depletion) for the two highest dose groups. These two groups were the only ones to exhibit consistent and severe behavioral impairments on all behavioral tests performed. Thus, for a relatively selective cholinergic basal forebrain lesion, almost a 90% reduction in choline acetyltransferase activity is needed to produce substantial behavioral deficits. It appears that either a considerable safety factor exists or robust compensatory mechanisms can ameliorate behavioral deficits from a major, but incomplete loss of cholinergic basal forebrain innervation.

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 μg) 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 μg) 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.

Central noradrenergic lesioning using anti-DBH-saporin: anatomical findings

The ability to create lesions of discrete neuronal populations is an important strategy for clarifying the function of these populations. The power of this approach is critically dependent upon the selectivity of the experimental lesioning technique. Anti-neuronal immunotoxins offer an efficient way to produce highly specific neural lesions. Two previous immunotoxins have been shown to be effective in both the CNS and PNS. They are OX7-saporin, which is targeted at Thy1, and 192-saporin, which is targeted at the low affinity neurotrophin receptor, p75NTR. In the present study, we sought to determine if an immunotoxin targeted at the neurotransmitter synthesizing enzyme, dopamine beta-hydroxylase (DBH), could selectively destroy central noradrenergic neurons after intraventricular administration. This immunotoxin, which consists of a monoclonal antibody to DBH coupled by a disulfide bond to saporin (a ribosome inactivating protein), has been shown to be selectively toxic to peripheral noradrenergic sympathetic neurons in rats after systemic injection. In the present study, immunohistochemical and Cresyl violet staining showed that the noradrenergic neurons of the locus coeruleus are destroyed bilaterally after intraventricular (i.c.v.) injection of 5, 10, and 20 micrograms of anti-DBH-saporin (alpha-DBH-sap) into rats. Complete bilateral lesioning of the A5 and A7 cell groups occurred at the two higher doses. Lesions of the A1/C1 and A2/C2/C3 cell groups were incomplete at all three doses. Dopaminergic neurons of the substantia nigra and ventral tegmental area and serotonergic neurons of the raphé, all monoaminergic neurons that do not express DBH, survived all alpha-DBH-sap doses. The cholinergic neurons of the basal forebrain, which are selectively killed by i.c.v. injection of 192-saporin, and cerebellar Purkinje cells which are killed by OX7-saporin, were not killed by alpha-DBH-sap. These results show that alpha-DBH-sap efficiently and selectively destroys CNS noradrenergic neurons after i.c.v. injection. The preferential destruction of locus coeruleus, A5, and A7 over A1/C1 and A2/C2/C3 may be due to more efficient access of the immunotoxin to these neurons and their terminals after i.c.v. injection.

Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat ☆

A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 IgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 IgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (ChAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 IgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKT1-saporin-injected controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 IgG-saporin. ChAT immunohistochemistry was confirmative of major cell loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the selectivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.

Selective localization of striatal D1 receptors to striatonigral neurons.

A new technique for producing anatomically selective lesions within the brain was used to investigate the cellular localization of the D1 and D2 receptor. The cytotoxic lectin, volkensin, is taken up by nerve terminals and retrogradely transported, killing those neurons projecting to the site of injection. Comparison of D1 and D2 binding following a unilateral volkensin injection into the substantia nigra has demonstrated that striatal D1 binding sites are selectively localized to striatonigral projection neurons.

Studies on the cellular localization of spinal cord substance P receptors

Substance P-immunoreactivity and specific substance P binding sites are present in the spinal cord. Receptor autoradiography showed the discrete localization of substance P binding sites in both sensory and motor regions of the spinal cord and functional studies suggested an important role for substance P receptor activation in autonomic outflow, nociception, respiration and somatic motor function. In the current studies, we investigated the cellular localization of substance P binding sites in rat spinal cord using light microscopic autoradiography combined with several lesioning techniques. Unilateral injections of the suicide transport agent, ricin, into the superior cervical ganglion reduced substance P binding and cholinesterase-stained preganglionic sympathetic neurons in the intermediolateral cell column. However, unilateral electrolytic lesions of ventral medullary substance P neurons which project to the intermediolateral cell column did not alter the density of substance P binding in the intermediolateral cell column. Likewise, 6-hydroxydopamine and 5,7-dihydroxytryptamine, which destroy noradrenergic and serotonergic nerve terminals, did not reduce the substance P binding in the intermediolateral cell column. It appears, therefore, that the substance P binding sites are located postsynaptically on preganglionic sympathetic neurons rather than presynaptically on substance P-immunoreactive processes (i.e. as autoreceptors) or on monoamine nerve terminals. Unilateral injections of ricin into the phrenic nerve resulted in the unilateral destruction of phrenic motor neurons in the cervical spinal cord and caused a marked reduction in the substance P binding in the nucleus. Likewise, sciatic nerve injections of ricin caused a loss of associated motor neurons in the lateral portion of the ventral horn of the lumbar spinal cord and a reduction in the substance P binding. Sciatic nerve injections of ricin also destroyed afferent nerves of the associated dorsal root ganglia and increased the density of substance P binding in the dorsal horn. Capsaicin, which destroys small diameter primary sensory neurons, similarly increased the substance P binding in the dorsal horn. These studies show that the cellular localization of substance P binding sites can be determined by analysis of changes in substance P binding to discrete regions of spinal cord after selective lesions of specific groups of neurons. The data show the presence of substance P binding sites on preganglionic sympathetic neurons in the intermediolateral cell column and on somatic motor neurons in the ventral horn, including the phrenic motor nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)