Impaired reach-to-grasp responses in mice depleted of striatal cholinergic interneurons

by Nilupaer Abudukeyoumu, Marianela Garcia-Munoz, Yoko Nakano, Gordon W. Arbuthnott.
Brain Mechanism for Behavior Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

Cholinergic interneurons (ChIs) are sparsely distributed within the striatum, a nucleus that plays important roles in voluntary motor control, associated learning, procedural memory, execution of movement, action selection, and planning.[1]

ChIs comprise 1-3% of all striatal neurons, are the main source of striatal acetylcholine, and have long been associated with deficits in Parkinson’s disease. A striatal imbalance between dopamine and acetylcholine has been suggested as one of the causes of parkinsonism.[2]

To selectively deplete ChIs in the dorsolateral striatum of 21-day-old male mice (C57BL/6J), we used the saporin immunotoxin that targets choline acetyltransferase (Anti-ChAT-SAP, Cat. #IT-42). Experimental animals received a stereotaxic unilateral infusion of the targeted toxin (0.3μl/3min) and sham controls received the same volume of sterile saline (sham control). Our histological analysis encompassed Weeks 2-6 postsurgery performed at 2-week intervals (Fig. 1). The loss of cells reached a stable ~70% level by 4 to 6 weeks with the additional surprising finding that axon terminals stained with a vesicular acetylcholine transporter antibody were more numerous two weeks after the injection, returning to control levels by six weeks.

Figure 1.

From a functional point of view, it will be important to find out if despite the cell loss, axon terminals sprout to invade the Anti-ChAT-SAP injected area from ~30% surviving ChIs or from ChIs in the surrounding tissue. To begin the study of dorsolateral striatal function following Anti-ChAT-SAP-induced ChI loss, we followed the same procedures as before[3] and observed the animal’s perfomance in a reach-to-grasp task (Fig. 2). Mice were divided in two control groups (intact and sham) and one experimental Anti-ChAT-SAP-injected group. Training started one week postsurgery during the animal’s active circadian cycle and following 12 hours of food deprivation.

Figure 2. Damage to ChIs impaired the use of t

Once the animals passed the initial acquisition phase, the successful performance in the reach-to-grasp task — expressed as mean ± SD percentage is shown in Fig. 3.
Controls: 51.11 ± 3.83; n = 25 [intact], 48.79 ± 4.6; n = 9 [sham]
Treated: 26.28 ± 3.74; n = 13

The significantly-impaired performance of the experimental group compared to controls was present even when the animals were pretrained. The loss of ChIs impairs the performance of striatally-mediated motor tasks, which suggests that cholinergic synaptic function is more important than non-synaptic communication in this situation. A non-synaptic cholinergic tone may be important for setting functional striatal states in other circumstances,[4] however, these specific lesions of ChI cells suggest that performance of a learned forelimb task requires that the cholinergic synaptic circuits of the striatum are intact.

References

  1. Abudukeyoumu N, Hernández-Flores T, Garcia M, Arbuthnott, G. Cholinergic modulation of striatal microcircuits. (2018). Eur J Neurosci. 10.1111/ejn.13949.
  2. Aosaki T, Miura M, Suzuki T, Nishimura K, & Masuda M. Acetylcholine-dopamine balance hypothesis in the striatum: an update. (2010). Geriatr Gerontol Int., 10 Suppl 1 S148-157. 2010/07/16.
  3. Lopez-Huerta VG, Nakano Y, Bausenwein J, Jaidar O, Lazarus M, Cherassse Y, Garcia-Munoz M, & Arbuthnott G. The neostriatum: two entities, one structure? (2016). Brain Struct Funct, 221 (3):1737-1749. 2015/02/06. PMC4819794.
  4. Pittman-Polletta BR, Quach A, Mohammed AI, Romano M, Kondabolu K, Kopell NJ, Han X, & McCarthy MM. Striatal cholinergic receptor activation causes a rapid, selective and state-dependent rise in cortico-striatal beta activity. (2018). Eur J Neurosci. 48 (8):2857-2868.


Other References Using Anti-ChAT-SAP
Liu A, Aoki S, Wickens J. (2017) A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions. BioProtoc. 7(22). DOI: 10.21769/2610.
Xiao H, Li M, Cai J, Li N, Zhou M, Wen P, Xie Z, Wang Q, Chang J, Zhang W. (2017) Selective Cholinergic Depletion of Pedunculopontine Tegmental Nucleus Aggravates Freezing of Gait in Parkinsonian Rats. Neurosci Lett 659:92-98. PMID: 28803956
Abudukeyoumu N, Garcia-Munoz M, Jaidar OP, Arbuthnott G (2016) Striatal cholinergic interneurons: their depletion and its progression. Soc Neurosci Meeting Abstract 245.09
Aoki S, Liu AW, Zucca A, Zucca S, Wickens JR. (2015) Role of striatal cholinergic interneurons in set-shifting in the rat. J Neurosci 35(25):9424-9431.
Aoki S, Wickens JR. (2015) Anti-ChAT-SAP elucidates a causal role in behavioral flexibility. Targeting Trends 16(4).
Kucinski A. (2015) Impairments in gait, posture and complex movement control in rats modeling the multi-system, cholinergic-dopaminergic losses in Parkinson’s Disease. Targeting Trends 16(1).
Xu M, Kobets A, Du JC, Lennington J, Li L, Banasr M, Duman RS, Vaccarino FM, DiLeone RJ, Pittenger C. (2015) Targeted ablation of cholinergic interneurons in the dorsolateral striatum produces behavioral manifestations of Tourette syndrome. Proc Natl Acad Sci USA 112(3):893-898.
LaPlante F. (2013) Role of cholinergic neurons in the nucleus accumbens and their involvement in schizophrenic pathology. Targeting Trends 14(1).
LaPlante F, Dufresne MM, Ouboudinar J, Ochoa-Sanchez R, Sullivan RM. (2013) Reduction in cholinergic interneuron density in the nucleus accumbens attenuates local extracellular dopamine release in response to stress or amphetamine. Synapse 67(1):21-29.
LaPlante F, Zhang ZW, Huppe-Gourgues F, Dufresne MM, Vaucher E, Sullivan RM. (2012) Cholinergic depletion in nucleus accumbens impairs mesocortical dopamine activation and cognitive function in rats. Neuropharmacology 63(6):1075-1084.
LaPlante F, Lappi DA, Sullivan RM (2011) Cholinergic depletion in the nucleus accumbens: Effects on amphetamine response and sensorimotor gating. Prog Neuropsychopharmacol Biol Psychiatry 35(2):501-509.

Cover Article: The locus coeruleus: a potential link between cerebrovascular and neuronal pathology in Alzheimer’s disease.

Contributed by S C Kelly, P T Nelson, S E Counts

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that most commonly affects individuals over the age of 65. While of unknown etiology, AD is characterized by a steady decline in cognitive functions including memory, attention, executive functions, and language. There is currently no disease-modifying therapy for the disease, and treatments to date have only yielded modest, short-term symptomatic relief. Recently, the “vascular hypothesis” of AD was proposed, due to the high comorbidity of individuals with cerebrovascular disease (CVD) and AD. These two diseases share similar risk factors and can both lead to dementia, neuronal injury, and neurological dysfunction, suggesting that AD pathogenic mechanisms may involve a dysregulaton of the cerebrovasculature and compromise of neurovascular functioning. However, the potential common mechanisms linking CVD and AD remain unknown.

In this regard, the locus coeruleus (LC) projection system, which provides the sole source of norepinephrine (NE) to the forebrain, mediates attention, memory, and executive function as well as cerebrovascular function and undergoes severe cell loss in AD.1 Furthermore, LC-mediated NE signaling is thought to play a role in blood brain barrier maintenance and neurovascular coupling, suggesting that LC degeneration may impact the high comorbidity of CVD and AD. However, the extent to which LC projection system degeneration impacts neurovascular function in AD is unclear.

Figure 1:  Anti-DBH-SAP lesioned animals are nearly completely depleted of noradrenergic innervation to the PFC. A) Intact innervation of DBH fibers in PFC of control IgG lesioned animal. B) Little to no immunoreactivity for DBH observable in PFC of Anti-DBH-SAP lesioned animal.

To model these relationships in vivo, we stereotactically lesioned LC projection neurons (Fig. 1; Anti-DBH-SAP Cat. #IT-03) innervating the prefrontal cortex (PFC), a major LC projection zone, in the TgF344-19 rat model of AD (aged 6 months old) using the noradrenergic immunotoxin, dopamine-β-hydroxylase (DBH)-saporin or a control Mouse IgG-Saporin (Mouse IgG-SAP Cat. #IT-18; n = 7-9/group). Prior to sacrifice, at 1-month post-op, the rats were tested behaviorally on the Barnes maze task—a special learning and memory paradigm. Rats that received the Anti-DBH-SAP lesion were significantly slower to find the escape cage in the maze (Fig. 2). Additionally, the lesioned rats continued to investigate incorrect holes multiple times showing a deficit in working memory. This behavioral deficit in particular is indicative of LC dysfunction in the PFC. These rats showed no locomotor differences as determined by the open field test (not shown). To continue this study, postmortem PFC will be analyzed for LC fiber innervation, NE levels, and cerebrovascular (cerebral amyloid angiopathy, micro-hemorrhage, cerebral artery endothelial remodeling) and AD-like pathology (amyloid load, tau epitopes, inflammation). We will determine the extent to which CVD and AD pathologic variables correlate with noradrenergic innervation and behavioral outcomes. We hope these studies will elucidate noradrenergic pathways contributing to neurovascular pathology and cognitive decline during the onset of AD and provide therapeutic rationale for targeting LC neuroprotection to modify disease progression.

Figure 2: Anti-DBH-SAP lesioned animals are significantly impaired on the Barnes Maze task compared to performance of control saporin (IgG) injected animals A) IgG Animals were significantly faster to find target hole than Anti-DBH-SAP lesioned animals on the probe trial (P= 0.476) B) Anti-DBH-SAP lesioned animals revisited more holes that they had already investigated indicating a deficit in working memory (P=0.0110)

Reference:

  1. Bondareff W, Mountjoy CQ, and Roth M (1981) Selective loss of neurones of origin of adrenergic projection to cerebral cortex (nucleus locus coeruleus) in senile dementia. Lancet 1(8223):783-4.

Accelerating Your Research

As we start a new year, we at Advanced Targeting Systems are more dedicated than ever to providing our customers with the innovative, cutting-edge tools that will accelerate and optimize research. Scientists around the world have published exciting new data to advance our knowledge of how specific cell types affect behavior and disease. With each new discovery, possibilities move closer to realities for unraveling the molecular basis for behavioral abnormalities and cures for devastating diseases.

Which targeting tool will advance your research?

  • Targeted Toxin: a powerful tool to specifically eliminate cells. Most frequently used in vivo to discover function of a particular cell type.Typically, cell function diminishes by the 3rd day following treatment, reaches minimum activity by 7 days and is maintained indefinitely. Anatomical changes are usually complete after 14 days. If you want to know what a cell does, get rid of it, and study what happens when you do.
  • Internalization Assay Kit:  a screening tool to identify whether a targeting agent is internalized in a cell of interest. These “Z-kits” are most frequently used in vitro to discover optimal cell-surface targeting agent (antibody, peptide, ligand, etc.) for a particular cell type. The power behind this kit is a ZAP conjugate — a non-targeted saporin conjugate that piggybacks on to your primary targeting agent (antibody or biotinylated material).
  • Customized Targeting:  a made-to-order tool to meet specific research demands.  Custom conjugate – Biotinylation – Fluorescent labeling – Contract assay services.

Tell us what you want and we will do everything we can to make it so.

These are just a few examples of the ATS tools available to accelerate your research. You can browse all products on our website. Use the Search form to find an antibody or other research tool compatible with your interests.

It is an honor to have served the scientific community for almost 23 years. Thank you for the opportunity. Let me know if there is anything more we can do to help keep your research on target.

My best to you in 2017,
Denise Higgins
President

P.S.   Don’t forget to check out the current promotions the ATS Product Managers have designed to make the most of your research budget.

Strategic Partners

This issue of Targeting Trends brings an exciting report of new partners that will bring more products, more expertise, and more service to our customers around the world. The primary purpose for making this change is to better facilitate the various functions that serve you, our customer. This restructure will also challenge and reward our long-time employees with the responsibility of managing their own strategic unit.

Brian Russell, celebrating his 16th anniversary at ATS on October 2nd, will be managing BioSyntheSys. His team will provide custom conjugation services: ADCs, saporin conjugates, biotinylations, fluorescent labeling, etc. Log on to BioSyntheSys.com to enter your contact information to discuss your next conjugation service.

Leonardo Ancheta, celebrated his 13th anniversary at ATS on September 2nd, and will be managing CytoLogistics’ contract services division. Leonardo’s team will provide flow cytometry services, cell culture, laboratory assays, antibody production, and GLP contract services. Log on to CytoLogistics.com to reserve your next service.

Doug Lappi (Founder and President Emeritus of ATS) will be heading up the Research Division of CytoLogistics. His team will work on product development and offer stellar laboratory expertise and consulting in Biochemistry, Molecular Biology, assay development, Cell biology and standard laboratory skills.

Tom Cobb and Chelsea Friedman will be co-managing TLC Shipping & Storage. Tom has extensive experience with shipping logistics and will ensure that all orders are packaged, shipped and tracked to their destinations worldwide. Chelsea is a scientist with an excellent background in storage and handling of research reagents (biologics, antibodies, etc.) and will manage the inventory tracking of multiple temperature storage units. Log on to TLCshipstorage.com to see the services they have to offer.

As for me, I will be continuing to manage Advanced Targeting Systems with an emphasis on administrative support for all of our partners. By consolidating all the administrative functions (customer orders, sales & marketing, purchasing, payroll, licensing, legal, etc.) for multiple entities, there is greater efficiency and organization in the processes. We are all excited about the prospects for each of our partners and for the greater service we can provide to our customers.

Strategic Partners

Four partners with unique strengths combine to complete the Targeting Puzzle:
Advanced Targeting Systems will continue to provide excellent Customer Service
BioSyntheSys will provide quality conjugation services
CytoLogistics will provide consistent, reliable antibody production, flow cytometry, and other laboratory services
TLC Shipping & Storage will ensure all products are stored, packaged and shipped properly

Cover Article: Targeted lesioning reveals role of nucleus incertus in the anxiogenic effect of buspirone

By Jigna Rajesh Kumar, [a, b, c, d] Ramamoorthy Rajkumar, [a, b, c] Liying Corinne Lee, [a, b, c] Gavin S. Dawe [a, b, c, d]
[a] Dept Pharmacology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, 117600, Singapore [b] Neurobiology and Ageing Programme, Life Sciences Institute, National University of Singapore, 117456, Singapore [c] Singapore Institute for Neurotechnology (SINAPSE), 117456, Singapore [d] NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 117456, Singapore

The nucleus incertus (NI), located strategically at the prepontine brainstem, has widespread connections across the forebrain to various structures involved in arousal, behavioral regulation, anxiety, appetite, and cognition.[5, 6, 9, 11-12, 18-20] The NI expresses one of the highest density of corticotropin releasing factor receptor 1 (CRF1) in the brain. which raised interest in this structure and suggested its possible role in the extra-pituitary behavioral stress response.[16] The NI is the chief source of the neuropeptide relaxin-3, and the NI/relaxin-3 system is highly conserved phylogenetically, pointing to a critical functional role that is presently not well understood.[15, 22]

CRF-saporin (CRF-SAP; Cat. #IT-13) was stereotaxically injected into the NI to enable selective lesioning of the CRF1-expressing NI cells (Fig. 2).[13] This procedure established at our laboratory was found to show significant reduction in the expression of CRF1, relaxin-3, GAD 65 as well as relaxin-3 in a representative target structure, the medial septum.[13] Based on the anxiogenic effect of CRF-SAP lesioning of the NI, as depicted by the significantly reduced time spent, and entries into the open arms of the elevated plus maze, it can be inferred that the NI may act to reduce anxiety physiologically (Fig. 1).

Fig. 1. Rats stereotaxically injected with CRF-SAP (Lesion) or Saporin (Cat. #PR-01; Sham) in the NI are tested in the elevated plus maze. Lesioning of the NI has an anxiogenic effect based on the reduced time spent and number of entries into the open arms. Systemic buspirone (3mg/kg; BUS) treatment reduces the anxiety levels of lesioned rats, increasing the time spent in, and number of entries into, the open arms.

We further utilized this technique to determine if the NI was involved in mediating the anxiety-modulating effects of buspirone, a clinically prescribed novel anxiolytic whose mechanism of action is not well understood.[3-4] Buspirone is a anxioselective drug that acts specifically on symptoms of anxiety without affecting cognition, motor ability, and reward pathways thus indicating that it likely acts on structures that regulate physiological anxiety. Buspirone is a 5-HT1A partial agonist and a D2 receptor antagonist;[14] both receptors are expressed in the NI.[13, 17] Buspirone tends to show anxiolytic effects at a narrow low dose range and anxiogenic effects at a wide high dose range, the latter effect being robust and reproducible.[1, 4, 7, 10, 21] The anxiolytic effects are widely thought to be mediated by the agonism of the 5-HT1A autoreceptors at the raphe nuclei, particularly the median raphe.[2, 8, 23] A 3 mg/kg intraperitoneal dose of buspirone was found to induce a strong anxiogenic effect on various anxiety paradigms, namely the elevated plus maze, the open field, and the light-dark box.[12] This dose was also found to robustly induce c-Fos expression and therefore activate the NI. The anxiogenic effect of systemic buspirone was attenuated when the NI was lesioned by CRF-SAP, thus indicating that the NI plays a role in the effects of buspirone (Fig. 1). Infusing buspirone (5 mcg) into the NI produced increased anxiety as well, suggesting that buspirone may be acting directly on the NI.[12] Pharmacological interaction studies conducted with a 5-HT1A antagonist, NAD 299, and D2/D3 agonist, quinpirole, indicated that these effects are mediated through the 5-HT1A receptors. Intra-NI infusion of NAD 299 attenuated the anxiogenic effects of systemic buspirone while intra-NI quinpirole did not have any effect.[12] Therefore, the NI is likely to be part of the physiological anxiety circuit and the 5-HT1A receptors may be particularly important in mediating this function.

Fig. 2. Representative images showing cannula position in the NI at 2X (A; scale=1 mm) and 10X (B; scale = 200 mm).

References

  1. Bradley BF et al. (2011). Anxiolytic and anxiogenic drug effects on male and female gerbils in the black-white box. Behav Brain Res 216: 285-292.
  2. Carli Met al. (1989). Evidence that central 5-hydroxytryptaminergic neurones are involved in the anxiolytic activity of buspirone. Br J Pharmacol 96: 829-836.
  3. Chessick CA et al. (2006). Azapirones for generalized anxiety disorder. Cochrane Database Syst Rev: CD006115.
  4. Collinson N et al. (1997). On the elevated plus-maze the anxiolytic-like effects of the 5-HT(1A) agonist, 8-OH-DPAT, but not the anxiogenic-like effects of the 5-HT(1A) partial agonist, buspirone, are blocked by the 5-HT1A antagonist, WAY 100635. Psychopharmacology (Berl) 132: 35-43.
  5. Farooq U et al. (2016). Electrical microstimulation of the nucleus incertus induces forward locomotion and rotation in rats. Physiol Behav 160: 50-58.
  6. Farooq U et al. (2013). Corticotropin-releasing factor infusion into nucleus incertus suppresses medial prefrontal cortical activity and hippocampo-medial prefrontal cortical long-term potentiation. Eur J Neurosci 38: 2516-2525.
  7. File SE et al. (1991). Low but not high doses of buspirone reduce the anxiogenic effects of diazepam withdrawal. Psychopharmacology (Berl) 105: 578-582.
  8. File SE et al. (1996). Comparative study of pre- and postsynaptic 5-HT1A receptor modulation of anxiety in two ethological animal tests. J Neurosci 16: 4810-4815.
  9. Goto M et al. (2001). Connections of the nucleus incertus. J Comp Neurol 438: 86-122.
  10. Handley SL et al. (1993). 5HT drugs in animal models of anxiety. Psychopharmacology (Berl) 112: 13-20.
  11. Kumar JR et al. (2015). Evidence of D2 receptor expression in the nucleus incertus of the rat. Physiol Behav 151: 525-534.
  12. Kumar JR et al. (2016). Nucleus incertus contributes to an anxiogenic effect of buspirone in rats: Involvement of 5-HT1A receptors. Neuropharmacology 110: 1-14.
  13. Lee LC et al. (2014). Selective lesioning of nucleus incertus with corticotropin releasing factor-saporin conjugate. Brain Res 1543: 179-190.
  14. Loane C et al. (2012). Buspirone: what is it all about? Brain Res 1461: 111-118.
  15. Ma S et al. (2007). Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat. Neuroscience 144: 165-190.
  16. Ma S et al. (2015). Ascending control of arousal and motivation: role of nucleus incertus and its peptide neuromodulators in behavioural responses to stress. Journal of neuroendocrinology 27: 457-467.
  17. Miyamoto Y et al. (2008). Developmental expression and serotonergic regulation of relaxin 3/INSL7 in the nucleus incertus of rat brain. Regul Pept 145: 54-59.
  18. Rajkumar R et al. (2013). Acute antipsychotic treatments induce distinct c-Fos expression patterns in appetite-related neuronal structures of the rat brain. Brain Res 1508: 34-43.
  19. Rajkumar R et al. (2016). Stress activates the nucleus incertus and modulates plasticity in the hippocampo-medial prefrontal cortical pathway. Brain Res Bull 120: 83-89.
  20. Ryan PJ et al. (2011). Nucleus incertus–an emerging modulatory role in arousal, stress and memory. Neurosci Biobehav Rev 35: 1326-1341.
  21. Soderpalm B et al. (1989). Effects of 5-HT1A receptor agonists and L-5-HTP in Montgomery’s conflict test. Pharmacol Biochem Behav 32: 259-265.
  22. Tanaka M et al. (2005). Neurons expressing relaxin 3/INSL 7 in the nucleus incertus respond to stress. Eur J Neurosci 21: 1659-1670.
  23. Tunnicliff G (1991). Molecular basis of buspirone’s anxiolytic action. Pharmacol Toxicol 69: 149-156.

Cover Article: Targeted depletion of hematopoietic stem cells promises safer transplantation

By Rahul Palchaudhuri, Ph.D., Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

Hematopoietic stem cell transplantation (HSCT) has been clinically used for 58 years and offers life-saving therapies for a variety of malignant and non-malignant blood disorders. Currently 50,000 transplants are performed globally per year with 90% of these for the treatment of malignancies.

Prior to receiving a transplant, a patient must be “conditioned” which serves to destroy resident hematopoietic stem cells in the marrow, in order to create niche vacancies for successful donor stem cell engraftment. Unfortunately, current conditioning strategies are non-targeted and genotoxic as they use DNA-damaging whole body irradiation and chemotherapy. As expected, these crude methods induce severe short-term and long-term conditioning-related toxicities that ultimately limit the application of hematopoietic stem cell transplantation, particularly in non-malignant conditions (e.g. sickle cell anemia, thalassemia, immunodeficiencies and autoimmune conditions).

Fig. 1: Hematoxylin and eosin staining of femur marrow sections of non-treated control, 3 mg/kg CD45-SAP or 5Gy TBI conditioned C57BL/6 mice 2 days post-conditioning. Representative images from independent experiments (n = 2 mice/group) are shown. Scale bars in top and bottom images represent 500 μm and 20 μm, respectively.1

While antibodies are potentially an appealing alternative to current conditioning methods, previous antibody-based strategies relying on naked antibodies have been met with limited success in immunocompetent animals. We therefore explored antibody-based immunotoxins created using the ribosome-inactivating protein, saporin, as a means of depleting hematopoietic stem cells in immunocompetent mice. By combining various biotinylated monoclonal antibodies with streptavidin attached to saporin (Streptavidin-ZAP, Cat. #IT-27), we created immunotoxins and screened their ability to achieve stem cell depletion in vivo. From our screen, we identified CD45-SAP as a potent stem cell-depleting agent capable of depleting >98% of hematopoietic stem cells following a single-dose administration. Using CD45-SAP we demonstrated successful donor stem cell engraftment with long-term donor chimerism levels greater than 90%.

As only hematopoietic cells express the CD45 receptor, CD45-SAP offered significant advantages with regard to toxicity compared to conventional whole body irradiation. Notably, CD45-SAP enabled quicker recovery of bone marrow cellularity, avoided damage to marrow blood vessels and other non-target marrow cells, and preserved the thymic function. Combined together, these features resulted in notably quicker recovery of B- and T-cells following CD45-SAP versus irradiation. In addition, CD45-SAP avoided neutropenia, preserving innate immunity and the ability to resist fungal infection.

To demonstrate correction of a clinically relevant disease, we employed CD45-SAP in a mouse model of sickle cell anemia and demonstrated our method achieved >90% donor cell chimerism, all mice in three groups (18/18), resulting in complete disease correction (red blood cell counts, hemoglobin levels, hematocrit levels and reticulocyte frequencies were returned to normal). Fig. 2 show hematopoietic stem cell (HSC) depletion. If these pre-clinical results can be successfully translated to the clinic, it would greatly reduce conditioning-related toxicities and expand the use of hematopoietic stem cell transplantation.

Fig. 2: HSC depletion in sickle disease mice 8 days post-administration of various doses of CD45-SAP. Data represent the mean ± SEM (n = 3 mice/dose, assayed individually).

Reference

  1. Palchaudhuri R, Saez B, Hoggatt J, Schajnovitz A, Sykes DB, Tate TA, Czechowicz A, Kfoury Y, Ruchika F, Rossi DJ, Verdine GL, Mansour MK, Scadden DT. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nat Biotechnol. 2016 Jun 6. doi: 10.1038/nbt.3584. [Epub ahead of print] PMID: 27272386.

From the President: What’s ZAP?

Wuzzup? No. What’s ZAP? Some of our products have SAP in the name, like 192-IgG-SAP (Cat. #IT-01). Some of our products have ZAP in the name, like Hum-ZAP (Cat. #IT-22).

First, what’s the same about ZAP and SAP? They both mean Saporin. The payload that Advanced Targeting Systems made famous to specifically eliminate targeted cells. For those of you new to this techology, Saporin is a ribosome-inactivating protein (Fig. 1).

Fig. 1  Saporin is obtained from the seeds of the Soapwort plant (Saponaria officinalis).  Saporin is a plant enzyme with N-glycosidase activity that depurinates a specific nucleotide in the ribosomal RNA 28S, thus irreversibly blocking protein synthesis.  It belongs to the well-characterized family of ribosome-inactivating proteins (RIPs). 

Now, what’s different about ZAP and SAP? The difference is in what the conjugate can do. A SAP conjugate has two components: 1) Saporin and 2) A targeting agent that is recognized on the cell surface and internalized. A ZAP conjugate has two components: 1) Saporin and 2) A non-specific agent that is NOT recognized on the cell surface and internalized (e.g. a secondary antibody, nonspecific peptide, or streptavidin).

If you want to make a saporin conjugate with your cell surface targeting agent, check out our ZAP products: ZAP Internalization Kits (Z-Kits) and Streptavidin products (see Page 7 for more information).

Fig. 2. The difference between SAP conjugates and ZAP conjugates. SAP conjugates target and eliminate specific cells.  ZAP conjugates need a primary targeting agent to be internalized.

From the President: A Sigh is (Not) Just a Sigh . . .

The fundamental things apply. “The Peptidergic Control Circuit for Sighing,” recently published in the prestigious journal Nature, has made us rethink our fundamental belief that sighs are only “long, deep breaths expressing sadness, relief or exhaustion.” Often prompting someone to say, “What’s wrong?” As it turns out, sighs “also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions.” Thanks to the clever researchers led by Dr. Jack Feldman at UCLA, and their collaborators at Stanford University School of Medicine, we now know a lot more about this process (see Fig. 1 below and Reference Summary on Pg. 4).

Fig. 1
On each side of the brain stem, a florescent-green marker illuminates the 200 neurons that control the sighing reflex. To determine if Neuromedin B receptor (NMBR)- and Gastric Releasing Peptide receptor (GRPR)-expressing neurons function specifically in sigh control, they were removed using Bombesin-SAP (Cat. #IT-40); Bombesin binds both receptors.
Photo Credit: Stanford/Krasnow lab

The Bötzinger Complex plays an important role in controlling breathing and was named by UCLA Professor Jack Feldman in 1978. Feldman named this area after a bottle of white wine named Botzinger present at his table (perhaps he was allowing it to breathe) during a scientific meeting in Hirschhorn, Germany, that year. Jack Feldman named the most rostral portion of the ventral respiratory group and continues to pave the way for important respiratory research. It’s a song we all need to hear, so: Play it again, . . . Jack!

Deservedly, Jack Feldman’s findings went viral. Here are just a few of the links to interviews and commentary:

NEWS ARTICLES

RADIO INTERVIEWS

TELEVISION COVERAGE

Bötzinger Complex References Using ATS Products

  1. Gray PA, Janczewski WA, Mellen N, McCrimmon DR, Feldman JL. (2001) Normal breathing requires preBotzinger complex neurokinin-1 receptor-expressing neurons. Nat Neurosci 4(9):927-930 (SP-SAP, Cat. #IT-07*).
  2. Feldman JL, Mitchell GS, Nattie EE (2003) BREATHING: Rhythmicity, Plasticity, Chemosensitivity. Annu Rev Neurosci 26:239-266 (SERT-SAP, Cat. #IT-23; SP-SAP, Cat. #IT-07*).
  3. Wenninger JM, Pan LG, Klum L, Leekley T, Bastastic J, Hodges MR, Feroah T, Davis S, Forster HV (2004) Small reduction of neurokinin-1 receptor-expressing neurons in the pre-Botzinger complex area induces abnormal breathing periods in awake goats. J Appl Physiol 97(5):1620-1628 (SP-SAP, Cat. #IT-07*).
  4. Wenninger JM, Pan LG, Klum L, Leekley T, Bastastic J, Hodges MR, Feroah TR, Davis S, Forster HV (2004) Large lesions in the pre-Botzinger complex area eliminate eupneic respiratory rhythm in awake goats. J Appl Physiol 97(5):1629-1636 (SP-SAP, Cat. #IT-07*).
  5. McKay LC, Janczewski WA, Feldman JL (2005) Sleep-disordered breathing after targeted ablation of preBotzinger complex neurons. Nat Neurosci 8(9):1142-1144 (SP-SAP, Cat. #IT-07*).
  6. McKay LC, Feldman JL (2008) Unilateral Ablation of preBotzinger Complex Disrupts Breathing During Sleep but not Wakefulness. Am J Respir Crit Care Med 178(1):89-95 (SP-SAP, Cat. #IT-07*).
  7. Montandon G, Qin W, Liu H, Ren J, Greer JJ, Horner RL. (2011) PreBotzinger complex neurokinin-1 receptor-expressing neurons mediate opioid-induced respiratory depression. J Neurosci 31(4):1292-1301 (anti-NK1r Cat. #AB-N04**).
  8. Gray PA, Hayes JA, Ling GY, Llona I, Tupal S, Picardo MCD, Ross SE, Hirata T, Corbin JG, Eugenin J, Del Negro CA (2010) Developmental origin of preBotzinger Complex respiratory neurons. J Neurosci 30(44):14883-14895 (anti-NK1r Cat. #AB-N04**).

*See alternate product: SSP-SAP (Cat. #IT-11);
**See alternate product: NK-1r affinity purified antibody (Cat. #AB-N33AP)

Cover Article: Cerebral cholinergic lesion reduces operant responses to unpleasant thermal stimuli

by Ronald G. Wiley, M.D., Ph.D., Departments of Neurology and Pharmacology, Vanderbilt University, Nashville, TN  and C. J. Vierck, Department of Neuroscience, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, USA

Degeneration of the cholinergic basal forebrain (CBF: medial septum, diagonal band of Broca, nucleus basalis of Meynert/substantia innominata) is a prominent feature of Alzheimer’s disease (AD). The CBF supplies cholinergic input to most of the cerebral cortex and hippocampus including somatosensory areas and anterior cingulate cortex that are involved in pain perception and experiencing discomfort, respectively. Clinical literature suggests that patients with AD either feel less pain or express discomfort less than comparable patients without dementia. As a result, AD patients receive less analgesics, but there is concern that AD only impairs communicating discomfort. Rats with extensive CBF lesions show impairment in a wide range of learning tasks and ability to sustain selective arousal/attention, but it is not known what role the CBF plays in central pain processing.

The present study sought to assess the impact of CBF lesions on behavioral responses to nociceptive stimuli in rats. Rats were trained on a thermal escape task where they chose whether to spend time in a dark chamber with the floor temperature at 10° C or 44.5° C (both mildly unpleasant), or move to a connected room temperature chamber with bright lighting. After establishing baseline performance on the operant task, selective CBF lesions were produced by intracerebroventricular injection of 192-saporin (192-IgG-SAP, Cat. #IT-01; Fig. 1). This immunotoxin selectively destroys neurons expressing p75NTR, the low affinity neurotrophin receptor that is uniquely expressed by CBF neurons in the forebrain. The rats were retested repeatedly over 19 weeks. On several occasions, the rats were subjected to sound stress prior to escape testing, and the rats were also tested on the thermal plate (hot/cold plate) without an escape option to measure lick guard (reflex) responses with and without preceding stress.

Fig. 1:  Representative coronal (frontal) sections from control rats (A, C) and 192-sap-treated rats (B, D) showing loss of CBF cholinergic neurons in the 192-sap rats. (A, B) Show the medial septal nucleus. (C, D) Show the nucleus basalis/substantia innominate regions. Sections were stained for demonstration of choline acetyltransferase using the immunoperoxidase technique (see text). The magnification bars in lower right corners indicate 250 lm (1).

Compared to controls, 192-IgG-SAP injected rats showed highly significant (p<0.001) loss of neurons from all subdivisions of the CBF based on post mortem brain sections stained for choline acetyltransferase. The CBF-lesioned rats escaped less than controls after 192-IgG-SAP injection (i.e. less motivated to get away from the aversively hot or cold stimuli). Reflex lick/guard responses, which are mediated at the spinal level, were not affected. The usual hyperalgesic effect of stress on the operant thermal escape task was absent in the CBF-lesioned rats. These results indicate a role for the CBF in modulating central pain processing. The loss of stress effect on thermal escape responses is consistent with loss of the arousal/attention function(s) of the CBF. These results also demonstrate the usefulness of 192-IgG-SAP for studies of the role of central (cerebral) cholinergic mechanisms in pain processing and are consistent with the idea that AD patients experience less discomfort for a given painful condition.

Reference

  1. Pain sensitivity following loss of cholinergic basal forebrain (CBF) neurons in the rat. Vierck CJ, Yezierski RP, Wiley RG. Neuroscience 319:23-34, 2016.

From the President: New Beginnings

It’s a new year. 2016. Another year passed and a brand new one to look forward to. After more than 21 years of serving the scientific community, Advanced Targeting Systems is making some changes and putting a ‘fresh face’ on things. Don’t worry, we will still provide the same high level of service and expertise to help you move your research and discovery efforts forward.

Harrison

One of the changes we have in store is a facelift for Targeting Trends, which Brian Russell (VP of Business Development) will be taking on as the new Editor in the next issue. This was going to happen with this first issue of the year, but as you can see by the beautiful picture here, he has had his hands full with the latest addition to his family. We are all so happy for him and Candi.

Besides his new role as Editor, Brian has also done a tremendous job with a redesign of the website — executed skillfully and artfully by our webmaster and database guru, Kristen Hartman. We look forward to the exciting new business development directions Brian will be unfolding.

Those are some of the new things in store for us this year. But before I close, a quick look back. Our illustrious leader, former president, founder, and scientific genius, Doug Lappi, continues to guide our science team and is enjoying a much-deserved ‘semi-retirement’ with his wife, Darlene. He still comes in every week and meets with the scientists and often gives us a challenge at the ping-pong table!

Lappi

I want to take this opportunity to state very clearly for all to read (old customers, new customers, friends, cat lovers, chronic pain drug development followers — everyone): Doug Lappi is the Sower of the Saporin seeds that bloomed into a successful company dedicated to providing quality targeting reagents for scientific research and pharmaceutical development. His contributions to science, and the research tools his work has provided, have not only made ATS a successful company, but have advanced the careers of scientists throughout the world. Thank you, Doug.