targeted-toxins

Works SJ, Wellman CL (2001) Differential dendritic atrophy in frontal cortex after cholinergic lesion in young adult and aged rats. Neuroscience 2001 Abstracts 101.17. Society for Neuroscience, San Diego, CA.

Summary: Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: three months after surgery, ibotenic acid lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of aged rats relative to young adults. To determine whether the differential effect of the lesion was due specifically to loss of cholinergic input from the NBM, we assessed dendritic morphology in frontal cortex after cholinergic depletion in young adult and aged male rats. Rats received unilateral lesions of the NBM using 192 IgG-saporin, and sham lesions of the contralateral NBM. Two weeks after surgery, brains were stained using a Golgi-Cox procedure. Pyramidal neurons in lamina II-III of frontal cortex were drawn and dendritic morphology was quantified in three dimensions. In young adults, lesions did not alter overall branch number or length. However, in aged rats, lesions decreased basilar dendritic number and length, by 17% and 25% respectively. Furthermore, young adults demonstrated a lesion-induced redistribution of basilar dendrites: dendritic material proximal to the soma was decreased 15%, while distal dendritic material was increased as much as threefold relative to the sham-lesioned hemisphere. Alternatively, lesions in aged rats decreased the amount of distal dendritic material by 25%. Thus, the dendritic atrophy resulting from NBM lesions in aged rats occurs within two weeks after lesion, and results specifically from loss of cholinergic innervation.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Helm KA, Han JS, Gallagher M (2001) Selective cholinergic deafferentation affects GR mRNA expression in the rat brain. Neuroscience 2001 Abstracts 175.10. Society for Neuroscience, San Diego, CA.

Summary: Two common features of the aging process include progressive dysfunction of both the basal forebrain cholinergic (BFC) system and suprahypothalamic feedback on the hypothalamic-pituitary-adrenal (HPA) axis. Prior research has shown that an age-related reduction in glucocorticoid receptor (GR) mRNA expression occurs in cortical target sites of the BFC system, including the hippocampus, prefrontal cortex, and anterior olfactory cortex, which are highly correlated with both spatial learning impairments and a blunted negative feedback of the stress response among aged rats. Selective deafferentation of the BFC system in young rats produces a similar reduction in both GR mRNA expression in the hippocampus and the efficiency of the stress response, as measured by a protracted increase in the levels of plasma corticosterone following acute stress. The current study investigated the possibility that loss of cholinergic input from cells in the basal forebrain alters GR mRNA expression in other BFC target structures, including the medial prefrontal cortex and the anterior olfactory cortex. Lesions of the BFC system were made by microinjections of the immunotoxin IgG-192-saporin into the medial septum/vertical limb of the diagonal band, and the substantia innominata/nucleus basalis. Basal levels of plasma corticosterone measured in the morning and evening 3 weeks later did not reveal any differences between lesioned and non-lesioned rats. The abundance of GR mRNA in sections processed for quantitative in situ hybridization will include a full analysis of cortical as well as subcortical sites to reveal the extent of effects of cholinergic lesions on GR mRNA expression throughout the brain. Supported by NIA PO1-AG09973.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Janni G, Jasmin L, Ohara PT (2001) Schwann cells can enter the demyelinated spinal cord from dorsal roots via scar tissue. Neuroscience 2001 Abstracts 157.4. Society for Neuroscience, San Diego, CA.

Summary: We have studied the routes of entry of Schwann cells into the demyelinated spinal cord. Following application of the toxin CTB-Sap (B fragment of Cholera toxin conjugated to Saporin) into the intrathecal space of adult rats there occurs massive loss of oligodendrocytes with secondary demyelination of the lumbar spinal cord with sparing of axons. Concurrent with the demyelination, an arachnoiditis develops that results in dorsal roots becoming adherent to the lateral spinal cord. Light and electron microscopy showed that Schwann cells in the dorsal roots were always separated from the demyelinated axons by a mesenchymal interface part of which was formed by the dorsal root perineurium. Within 15 days of the demyelination, Schwann cell precursors (p75 immunopositive) migrated from the dorsal roots into the spinal cord via the adhesions and were found to divide within the cord. Schwann cell myelination of demyelinated central axons was consistently observed by day 30. Therefore the Schwann precursors were able to migrate into demyelinated spinal cord through non-neuronal cellular barriers without being in direct contact with demyelinated axons. These findings suggest that, under appropriate conditions, Schwann cells might be introduced therapeutically into the demyelinated spinal cord via intrathecal application and avoid direct spinal injection.

Related Products: CTB-SAP (Cat. #IT-14)

Burk JA, Herzog CD, Porter MC, Mahoney J, Bruno JP, Sarter M (2001) Interactions between partial cortical cholinergic deafferentation and aging on sustained attention performance in rats. Neuroscience 2001 Abstracts 202.5. Society for Neuroscience, San Diego, CA.

Summary: Previous studies have provided only limited support for the idea that aging alone impairs the functions of basal forebrain corticopetal cholinergic neurons. Conversely, aging has been hypothesized to exacerbate the functional consequences of prior insult to, or degenerative processes in, the basal forebrain cholinergic system. The present study assessed the effects of aging on the sustained attention performance of rats with moderate lesion-induced loss of cortical cholinergic inputs. Previous studies on the effects of extensive (> 70 %) cortical cholinergic deafferentation indicated that the integrity of this system is necessary for the animals’ ability to detect rare and unpredictable visual signals. In the present longitudinal experiment, moderate (< 50 %) 192 IgG-saporin-induced loss of cortical cholinergic inputs, produced in well-trained, 16 month-old rats, did not – as was intended - immediately affect their attentional performance. Animals continued to undergo attentional performance training for the remainder of their lives. At the age of 31 months, impairments in performance began to emerge in lesioned animals. Compared to their sham-lesioned counterparts, lesioned animals exhibited a steeper decrement in their ability to detect hits in the course of a test session. At this age, the performance of sham-lesioned animals did not differ from their earlier performance prior to and immediately after the lesion. These results support the general hypothesis that aging serves to exacerbate the effects of pre-existing degeneration of the basal forebrain cholinergic system.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Rinaman L, Wonders CP (2001) Targeted destruction of A2/C2 catecholamine neurons alters hypothalamic responses to vagal stimulation. Neuroscience 2001 Abstracts 131.4. Society for Neuroscience, San Diego, CA.

Summary: Central catecholamine (CA) pathways participate in viscerosensory modulation of hypothalamic neuroendocrine function. Different brainstem CA cell groups may relay different types of viscerosensory signals to different classes of hypothalamic effectors. The present study sought to determine the role of dorsal medullary A2/C2 neurons in hypothalamic responses to exogenous cholecystokinin (CCK), which activates gastrointestinal vagal sensory inputs to the caudal brainstem. Saporin toxin conjugated to dopamine-beta-hydroxylase antibody (anti-DbH-sap; 10 ng in 100 nl) or control toxin was microinjected unilaterally or bilaterally into the A2/C2 region of the dorsal vagal complex in adult male rats. After 10-14 days, rats were injected i.p. with CCK (10 ug/kg) and perfused with fixative 1 hr later. Brainstem and forebrain sections were processed for dual immunocytochemical detection of cFos (a marker of neural activation) and DbH (to define the lesion). Additional forebrain sections were processed for cFos and either oxytocin (OT), vasopressin (AVP), or corticotropin-releasing factor (CRF) to identify hypothalamic neurons activated by CCK. Anti-DbH-sap destroyed the majority of A2/C2 neurons within the microinjection site(s), with minimal non-specific damage. A2/C2 lesions markedly attenuated CCK-induced activation of OT neurons and, to a lesser extent, attentuated CRF activation. Conversely, CCK-induced cFos expression was significantly increased in AVP neurons. The latter effect was observed only after bilateral lesions. These results indicate that A2/C2 neurons participate in vagal sensory-mediated stimulation of OT neurons and CRF neurons, and inhibition of AVP neurons.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Waite JJ, Chen AD (2001) Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei. Brain Res 918:113-120. doi: 10.1016/s0006-8993(01)02968-7 PMID: 11684049

Summary: 192-Saporin (Cat. #IT-01) is used extensively to eliminate the cholinergic neurons of the basal forebrain in rats. Waite and Chen compare the degree of loss between 192-Saporin (6 or 8.2 µg in 10 µl into left lateral ventricle) and control (Saporin, 1.82 µg into left lateral ventricle; Cat. #PR-01) using three methods: Assay of post mortem choline acetyltransferase activity, in vivo microdialysis of extracellular acetylcholine (ACh), and in vivo assessment of the rate of ACh synthesis. The infusion of saporin alone had no effect. After fifteen weeks, the authors report compensation of cholinergic activity in lesioned animals occurs in the hippocampus, but not in the frontal cortex as determined by measurement of the rate of ACh synthesis.

Related Products: 192-IgG-SAP (Cat. #IT-01), Saporin Goat Polyclonal (Cat. #AB-15), Saporin Chicken Polyclonal, affinity-purified (Cat. #AB-17AP), Saporin (Cat. #PR-01)

Pang KCH, Nocera R, Secor AJ, Yoder RM (2001) GABAergic septohippocampal neurons are not necessary for spatial memory. Hippocampus 11:814-827. doi: 10.1002/hipo.1097

Summary: The medial septum and diagonal band of Broca (MSDB) are necessary for spatial memory. Both cholinergic and GABAergic neuronal populations are present in the MSDB. 192-Saporin (Cat. #IT-01) was used to eliminate cholinergic populations and kainic acid was used to reduce numbers of GABAergic neurons. Both agents were injected (independently or in combination) into the medial septum and each diagonal band of rats (192-Saporin 250 ng MS, 150 ng DB) to determine the importance of GABAergic neurons in the MSDB for spatial memory. The results showed elimination of GABAergic neurons has no impact on spatial memory, while elimination of cholinergic neurons has a mild impact.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Berger-Sweeney JE, Stearns NA, Murg SL, Floerke-Nashner LR, Lappi DA, Baxter MG (2001) Selective immunolesions of cholinergic neurons in mice: effects on neuroanatomy, neurochemistry, and behavior. J Neurosci 21(20):8164-8173. doi: 10.1523/JNEUROSCI.21-20-08164.2001

Summary: 192-Saporin (Cat. #IT-01) has long been an effective agent for elimination of cholinergic neurons in the basal forebrain of rats. Until the development of mu p75-SAP (Cat. #IT-16) there was no equivalent agent for use in mice. The authors tested mu p75-SAP in vitro and in vivo (1.8-3.6 µg in right lateral ventricle), using cytotoxic, histochemical, and behavioral assays. The data shows that mu p75-SAP is a highly selective and efficacious lesioning agent for cholinergic neurons in the mouse. The authors conclude that mu p75-SAP will be a powerful tool to use in combination with genetic modification to investigate cholinergic damage in mouse models of Alzheimer’s disease.

Related Products: mu p75-SAP (Cat. #IT-16), 192-IgG-SAP (Cat. #IT-01)

Q: How long does it take to see the cell death occurring from the use of targeted toxins using saporin? Is there a time course of hours or days?

A: Details of the time course of early events have not been extensively studied. After ricin injections into the cervical vagus nerve, the proximal nerve becomes unresponsive to electrical stimulation between 36 and 48 hours. After septal injection of 192-Saporin (192-IgG-SAP, Cat. #IT-01), hippocampal theta rhythm begins to diminish on the third postoperative day and reaches a minimum by 7 days which is maintained indefinitely. Anatomical disintegration is complete within 10-14 days after injection of most toxins.

Q: Will this time course be the same regardless of the targeted toxin used or the method of administration?

A: Presumably, injection of toxin into the vicinity of target cell bodies and dendrites should produce effects somewhat sooner than toxin injections into axonal terminal fields where retrograde axonal transport must first deliver toxin to the perikarya. In the cervical vagus, based on transport times for ricin and inhibition of toxin transport by vincristine, we concluded that fast axonal transport is involved. Colchicine coinjected intraventricularly with 192-Saporin (192-IgG-SAP, Cat. #IT-01) prevents destruction of cholinergic basal forebrain neurons suggesting that fast axonal transport also is involved with i.c.v. toxin injections. Consequently, the delay introduced by injecting toxin into axon terminal fields is usually a few hours at most.

Q: What are some assays/methods to use to be able to graphically demonstrate cell death?

A: Toxin-induced cell death can be observed and documented with a variety of techniques. Often the easiest is simple Nissl staining because all of the RIP toxins (ricin, volkensin, saporin) produce profound chromatolysis that is readily apparent in Nissl stains (i.e. cresyl violet).

Electron microscopy can demonstrate details of neuron degeneration including loss of axon terminals at a distance from the cell body which can be useful in anatomic tracing studies.

Typically, target neurons express proteins that can be visualized with immunocytochemical techniques. Thus, immunofluorescence or peroxidase immunohistochemistry can be useful in detecting loss of staining for target molecules and co-expressed molecules in the neurons being targeted. The use of multiple markers is recommended to insure that cell loss occurred rather than down regulation of marker expression.

See: Targeted Toxins

Sloviter R (2001) Featured Article: Immunolesioning hippocampal inhibitory interneurons. Targeting Trends 2(4)

Related Products: SSP-SAP (Cat. #IT-11), SP-SAP (Cat. #IT-07)

Read the featured article in Targeting Trends.

See Also:

• Martin JL et al. Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of substance P. J Comp Neurol 436:127-152, 2001.