References

Shramm PA, Ancheta L, Higgins D, Lappi DA (2017) A rapid, pH-sensitive screening method to detect internalization of cell surface markers for development of antibody-based pharmaceuticals to treat brain tumors. Neuroscience 2017 Abstracts 566.24 / H7. Society for Neuroscience, Washington, DC.

Summary: Some of the most potent treatments for cancers have been antibodies to cell surface proteins that cause tumor cell proliferation. Examples are cetuximab (antigen: EGFR) approved for colorectal cancer and Trastuzumab (ERBB2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized by so-called antibody mediated internalization. As such, the mitogenic cell surface protein no longer plays a role in cancer cell division. Despite the blood brain barrier challenging systemic treatment for brain tumors, intracerebroventricular injection can produce similar results. For example, Gholamin et al., (Sci Transl Med 9:381, 2017) and Kang et al. (Sci Rep 6:34922, 2016) reported down-regulation of brain tumor mitogenic agents through antibody-mediated endocytosis. The quick and efficient screening of antibodies that internalize effectively is vital for determining suitability of an antibody as a therapeutic targeting agent. Here we describe a method for the efficient determination of internalization of cell surface molecules by antibodies using a pH-dependent fluorescent reporter cross-linked to a secondary antibody in a plate-based assay with visualization of internalization in hours. This conjugate is comprised of an affinity-purified monovalent secondary antibody against both the heavy and light chain of human or mouse IgG and is conjugated to a pH -dependent fluorescent reporter. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell (thereby providing evidence of internalization). A successful assay protocol has been developed to provide an EC50 by way of a fluorescence-detecting plate reader, which could be used to explore antibody candidates as therapeutics in a quick and reproducible manner.

Related Products: Fab-pHast human (Cat. #PH-01)

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See Also:

• Gholamin S et al. Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors. Sci Transl Med 9(381):eaaf2968, 2017.
• Kang BR et al. Cell surface GRP78 as a biomarker and target for suppressing glioma cells. Sci Rep 6:34922, 2016.
• Ancheta LR et al. Basigin-2 (EMMPRIN), a prognostic marker, is a dynamic portal of entry into cancer cells. Cancer Res 71(8):5218, 2011. Proceedings of the American Association for Cancer Research Annual Meeting, Orlando, FL
• Ancheta L et al. Method for screening neuronal tumor cell surface markers for high specificity and rapid internalization as potential oncologic treatments. Neuroscience 2017 Abstracts 612.11 / SS46, 2017. Society for Neuroscience, Washington, DC

Ancheta L, Shramm PA, Lappi DA (2017) Method for screening neuronal tumor cell surface markers for high specificity and rapid internalization as potential oncologic treatments. Neuroscience 2017 Abstracts 612.11 / SS46. Society for Neuroscience, Washington, DC.

Summary: Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules involved in the growth, progression, and spread of the tumor. These therapies are often cytostatic; they block tumor cell proliferation as opposed to chemotherapy that kills the cells. A primary approach to identify potential targets is the ability to compromise a ligand/receptor relationship that causes tumor cell proliferation. There are now many examples of the use of antibodies in tumor therapy to cause a breakdown in that relationship. In clinical use against brain tumors are antibodies to cell-surface EGFR, VEGFR, PDGFR, and c-kit. These work by down-regulation of the receptor by antibody-mediated internalization. It is crucial for development of a targeted therapy to have a method to determine the suitability of an antibody to cause internalization rapidly and completely. Here we describe a method for the efficient determination of internalization of cell surface molecules by antibodies: a cytotoxicity assay utilizing an antibody labeling method to streamline the process of multiple candidate screening. Cells are chosen that have significant levels of expression of the desired marker and the assay readout is definitive: cell death is demonstrated in 72 hours. This method is designed for the rapid screening of multiple antibodies for specificity and internalization in neuronal tumor cells to explore antibody candidates as therapeutics in a quick and reproducible manner.

Related Products: Fab-pHast human (Cat. #PH-01)

Hulsey D, Shedd M, Mong J, Rennaker RL, Hays SA, Kilgard MP (2017) Vagus nerve stimulation dependent enhancement of motor cortex plasticity requires noradrenergic innervation. Neuroscience 2017 Abstracts 317.06 / HH5. Society for Neuroscience, Washington, DC.

Summary: Pairing forelimb movements with vagus nerve stimulation (VNS) drives robust plasticity within primary motor cortex (M1). VNS activates cholinergic circuits, which are required for VNS-depended enhancement of plasticity. However, there may be multiple neuromodulatory mechanisms required for VNS-dependent enhancement of plasticity. Norepinephrine regulates plasticity, and the noradrenergic locus coeruleus is driven vigorously by VNS. However, the role of norepinephrine in VNS-dependent enhancement of plasticity is unknown. We hypothesize that noradrenergic innervation of M1 and/or basal forebrain is necessary for M1 plasticity associated with VNS pairing. To test this, we trained rats on a skilled lever press task emphasizing use of the proximal forelimb. After demonstrating proficiency on the task, rats received M1 injections of vehicle or DBH-Saporin to selectively deplete norepinephrine in motor cortex, and underwent implantation of a stimulating cuff electrode on the vagus nerve. Sham and NE- lesioned rats resumed training one week after surgery. After returning to pre-surgical performance, both groups received 10 sessions of training with VNS paired on successful trials. Intracortical microstimulation was performed to derive M1 maps within 24 hours of the final training session. Initial data suggests that sham lesioned animals who receive VNS pairing with successful trials show a robust expansion of proximal forelimb movements represented in M1. Noradrenergic lesion of M1 blocks this VNS- dependent expansion of proximal forelimb representation, indicating that cortical norepinephrine innervation is necessary for VNS driven plasticity. Ongoing experiments will determine whether noradrenergic input to the central cholinergic systems is required for VNS-dependent enhancement of plasticity.

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

Krajewski-Hall SJ, Blackmore EM, McMinn JR, McMullen NT, Rance NE (2017) Increased core temperature following ablation of neurokinin 3 receptor-expressing neurons in the mouse median preoptic nucleus and adjacent preoptic area (MnPO/POA). Neuroscience 2017 Abstracts 414.02 / PP19. Society for Neuroscience, Washington, DC.

Summary: We have previously proposed that KNDy neurons play a role in the generation of hot flushes via neurokinin 3 receptor (NK3R) signaling in the preoptic hypothalamus. This hypothesis is strongly supported by recent clinical studies showing that the number and severity of hot flushes is reduced by treatment with NK3R antagonists. To determine if preoptic NK3R neurons modulate thermoregulation in the mouse, we selectively ablated them using injections of saporin conjugated to a NK3R agonist (NK3-SAP). NK3-SAP was stereotaxically injected into Tacr3-EGFP mice to target the MnPO/POA. Controls received injections of BLANK-SAP. The mice were ovariectomized (OVX) and a telemetry probe was implanted i.p. to measure core temperature (TCORE) and activity. Skin temperature (TSKIN) was monitored using a temperature data-logger attached to the surface of the tail. In experiment 1, circadian temperature rhythms were monitored over a 3 day period in mice housed in their home cages (12 light:12 dark). In experiment 2, mice were exposed to three temperatures, 18, 28 and 35oC, in an environmental chamber. Mice were then implanted s.c. with estradiol (E2) capsules and the experiments repeated. We verified by immunohistochemistry and quantitative microscopy that approximately 80% of the EGFP-NK3R neurons in the MnPO were ablated using NK3-SAP. Ablation of NK3R neurons significantly elevated TCORE during the light phase in both OVX and OVX + E2 mice (OVX: BLANK-SAP, 36.7 + 0.1 vs NK3-SAP 37.4 + 0.1; OVX+E2: Blank-SAP 36.1 + 0.1 vs NK3-SAP 36.8 + 0.1). NK3-SAP injections had no significant effect on TCORE during the dark phase. Ablation of NK3R neurons also increased TCORE during the light phase in mice exposed to 18oC and 28oC. All mice exhibited hyperthermia at 35oC. In contrast, ablation of NK3R neurons in the MnPO/POA had no effect on TSKIN or activity regardless of experimental treatment. These data suggest that NK3R neurons in the MnPO/POA participate in the thermoregulatory axis by promoting heat loss during the day and provide further insight into the CNS thermoregulatory pathways that may be activated during the generation of hot flushes.

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Wan L, Jin H, Liu X, Jeffry J, Barry D, Shen K, Peng J, Liu X, Jin J, Sun Y, Kim R, Meng Q, Mo P, Yin J, Tao A, Bardoni R, Chen Z (2017) Distinct roles of NMB and GRP in itch transmission. Sci Rep 7:15466.. doi: 10.1038/s41598-017-15756-0

Summary: To examine the role of NMBR+ dorsal horn neurons in itch and pain transmission, the authors treated mice with NMB-SAP (Cat. #IT-70; 2–3 μg, i.t.) at which no side effects were observed. NMB-SAP treated mice barely showed scratching behaviors, whereas control mice exhibited NMB-induced scratching with a rapid onset of scratching responses. These data demonstrate an important role of NMBR+ neurons in itch transmission. In contrast to notable deficits in itch transmission, NMB-SAP treated mice exhibited normal behavioral responses to acute mechanical stimuli, thermal stimuli and tail immersion. The authors also examined inflammatory pain behaviors of NMB-SAP treated mice and found normal nocifensive behaviors.

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Tapp D, McMurray M (2017) Serotoninergic projections to the OFC and BLA modulate reversal learning. Neuroscience 2017 Abstracts 423.01 / TT12. Society for Neuroscience, Washington, DC.

Summary: Behavioral flexibility, the ability to adapt to changing reward contingencies, is a critical aspect of choice behavior. Such ability is disrupted in numerous psychiatric disorders, such as substance abuse disorders, attention deficit disorder, and obsessive- compulsive disorder. The orbitofrontal cortex (OFC) and the basolateral amygdala (BLA) have been implicated as key regulators for this behavior. Additionally, the neurotransmitter serotonin is known to influence behavioral flexibility, and is disrupted in numerous psychiatric disorders. While serotonin and these brain regions have been examined separately, they have yet to be directly linked in this behavioral context. Using a rat model, this study examined such a relationship by selectively leisoning serotoninergic projections to the OFC, BLA, or both regions with a SERT-conjugated Saporin, and assessing behavioral flexibility in a probabilistic spatial reversal-learning task. Preliminary results indicated that the loss of serotonergic projections to either the OFC, BLA, or both impaired behavioral flexibility. Based on these results, we determined that serotonin regulates reversal learning through its action in the OFC and BLA. Therefore, the serotonergic system may serve as a future therapeutic target for diseases in which behavioral flexibility is impaired, and may explain the effectiveness of serotonin modulators in the treatment of these diseases.

Related Products: Anti-SERT-SAP (Cat. #IT-23)

Beselia G, Dashniani M, Burjanadze M, Solomonia R, Kruashvili L, Chkhikvishvili N (2017) Modulation of GluN2B subunit-containing NMDA receptors expression and spatial long-term memory in medial septal immunolesioned rats. Neuroscience 2017 Abstracts 428.01 / UU39. Society for Neuroscience, Washington, DC.

Summary: The hippocampus is important in the formation of spatial memory in both humans and animals. The N-methyl-D-aspartate (NMDA) type of glutamate receptors in the hippocampus has been reported to be essential for spatial learning and memory as well as for the induction of synaptic plasticity. Evidence accumulated from recent studies suggest that GluN2A and GluN2B subunit-containing NMDA-Rs preferentially contribute to the induction of hippocampal LTP and LTD. Using a Morris water maze (MWM) task, the LTP- blocking GluN2A antagonist had no significant effect on any aspect of performance, whereas the LTD-blocking GluN2B antagonist impaired spatial memory consolidation.1The present study was designed to investigate the effect of selective immunolesions of cholinergic and GABA-ergic1septohippocampal projection neurons [using 192 IgG-saporin (SAP) or GAT1-1 saporin (GAT), respectively] on spatial memory assessed in MWM and NMDA receptor GluN2B subunit expression in the rat hippocampus. We used MWM training protocol with eight training trials. One day after training, probe test with the platform removed was performed to examine long-term spatial memory retrieval. We found that immunolesions of medial septal cholinergic or GABAergic neurons did not affect spatial learning as exhibited by a decreased latency to find the hidden platform across the eight training trials. Trained control and SAP treated rats spent significantly longer than chance (15 s) performances such as swimming time in test sector (where the hidden platform was1located). Moreover, they spent significantly longer in test sector than the opposite sector, confirming the establishment of long-1term memory. In contrast, the preference for test sector was abolished in medial septal GAT treated rats. Because GAT treated rats learned the location of the hidden platform during training, the result suggest that GAT level of NR2B subunit of NMDA receptor in the hippocampus was decreased significantly in the GAT treated group compared with the control and SAP treated groups.1In conclusion, our findings suggest that immunolesion of medial septal GABAergic neurons can interrupt hippocampus dependent1spatial memory, possible through modulation of NMDA receptor subunit expression in the hippocampus. Moreover, our finding that selective lesions of medial septal GABAergic neurons affect probe-test performance but not spatial learning, suggests that septohippocampal GABAergic projections are involved specifically in the consolidation or retrieval, but not in the acquisition of long- term memory.

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Barnett WH, Molkov YI, Lemes E, Falqueto B, Colombari E, Takakura AT, Moreira TS, Zoccal DB (2017) Local glutamatergic transmission in the RTN/pFRG is critical for active expiration and sympathetic overactivity during hypercapnia. Neuroscience 2017 Abstracts 233.1 / FF22. Society for Neuroscience, Washington, DC.

Summary: The retrotrapezoid nucleus (RTN) contains chemosensitive cells that distribute CO -dependent excitatory drive to the brainstem respiratory network. This drive facilitates the function of the respiratory central pattern generator (CPG), modulates sympathetic activity and determines the emergence of active expiration during hypercapnia via activation of the late expiratory (late-E) oscillator in the parafacial respiratory group (pFRG). However, the microcircuitry responsible for distribution of the chemoreflex signal to the pFRG and the respiratory CPG is not well understood. Previously, we developed a computational model of the brainstem respiratory network, which was subsequently extended to include the central and peripheral chemoreflexes as well as presympathetic circuits. We present here experiments performed on the decerebrated, arterially-perfused in situ rat, aimed to test a key assumption of this model that chemosensitive and late-E neurons in the RTN/pFRG are two distinct populations, and the latter receives local glutamatergic input from the former. The model predicts: (1) suppression of RTN chemosensitive neurons will diminish the changes to the respiratory pattern and the emergence of active expiration associated with hypercapnia; (2) the disruption of local glutamatergic neurotransmission in the RTN will specifically suppress active expiration and the appearance of late-E discharges in the sympathetic motor output. To test prediction (1) we lesioned NK1 -positive chemosensitive neurons of the RTN with microinjections of substance P-saporin (SSP-SAP) conjugate. This suppressed the emergence of late-E activity in abdominal (AbN) and sympathetic nerves, and attenuated the increase in phrenic burst amplitude during hypercapnia. However, SSP-SAP and control animals exhibited late-E AbN activity in response to peripheral chemoreflex activation. Prediction (2) was tested with bilateral microinjections of kynurenic acid (Kyn, 100 mM) in the RTN/pFRG, which suppressed the emergence of late-E AbN activity but not the change in phrenic nerve amplitude during hypercapnia. Our results support the notion that RTN chemosensitive neurons are critical for inspiratory and expiratory reflex responses to hypercapnia. Our findings indicate that activation of late-E neurons in the pFRG during hypercapnia requires glutamatergic inputs from a separate neuronal population in the RTN that intrinsically detects changes in CO . During peripheral chemoreflex stimulation, pFRG late-E neurons are activated via excitatory pathways bypassing the RTN central chemoreceptors. We recapitulate these results in our computational model.

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Kong C, Shin J, Lee J, Koh C-S, Yoon M-S, Na Y, Chang J, Chang W (2017) Improvements in memory after focused ultrasound are associated with changes in hippocampal cholinergic activity and neurogenesis. Neuroscience 2017 Abstracts 201.12 / C29. Society for Neuroscience, Washington, DC.

Summary: Abstract Introduction: Alzheimer’s disease is characterized pathologically by neurofibrillary tangles, amyloid plaques, gliosis, synaptic loss and cholinergic deficits. Recently, cell proliferation and neurogenesis was reported to have increased when the blood brain barrier (BBB) was disrupted by Focused ultrasound (FUS) with microbubbles. Previously, we have demonstrated that the cholinergic cell decreases in 192 IgG-saporin rat model, and that decrease in cholinergic cell is associated to decrease in cognitive behavior. The purpose of this study was to determine if the learning and memory abilities of the 192 IgG-saporin rat model are improved by FUS. Materials and Methods: Animals were divided into the four groups: Sham group (PBS injection), Lesion group (saporin injection), FUS-3 and FUS-10 groups (After 3 and 10 days after saporin injection, FUS treatment). Sprague-Dawley rats (200-250g) were injected bilaterally with 192 IgG-saporin into the ventricle. Rats were sonicated using a single-element transducer (frequency 0.5 MHz) with microbubble. The acoustic parameters used for each sonication are: pressure amplitude 0.3 MPa, pulse length 10 ms, burst repetition frequency 1 Hz, and a duration of 120 s. To confirm cell proliferation, BrdU was intraperitoneally injected 2 times per day for 4 consecutive days starting 24 hours after FUS sonication. Two weeks after IgG-saporin administration, spatial memory was tested with the Morris water maze training for 5 days and the final test was performed. Results: In the water maze test, the FUS groups had a higher number of crossing times and staying time in the platform zone than the lesion group. Also, the FUS-3 group was higher than for the FUS-10 group. We confirmed that the amounts of DCX , NeuN , and BrdU were different between the FUS group and the lesion group. Conclusion: Our results suggest that FUS sonication facilitates recovery of memory and learning abilities in cholinergic deficits rat model. Moreover, the results suggest that neurogenesis is correlated with the mechanism of cognitive behavior recovery.

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

Fogo G, Gall AJ (2017) Behavioral effects following ablation of retinal ganglion cells in diurnal grass rats. Neuroscience 2017 Abstracts 237.03 / HH34. Society for Neuroscience, Washington, DC.

Summary: Light influences behavior and physiology in mammals by entraining circadian rhythms and also through direct and acute inhibition or stimulation of activity, a process called masking. Although there has been substantial progress elucidating the mechanisms responsible for the workings of the circadian system in nocturnal species, less is known about the mechanisms that support the diurnal profile of activity of mammals, especially as they relate to the retina. We recently showed that the intergeniculate leaflet (IGL) is critical for the display of normal patterns of daily activity in diurnal grass rats (Arvicanthis niloticus). Specifically, IGL lesions reverse the activity patterns of these animals such that they became night-active; this occurred through their effects on both circadian mechanisms and masking. The IGL is a thalamic structure that receives direct inputs from the melanopsin containing intrinsically photosensitive retinal ganglion cells, known as ipRGCs. Our current approach takes advantage of a diurnal mammalian model, the Nile grass rat, to test the novel hypothesis that melanopsin is critical for the expression of diurnal behavior and physiology, and is involved in masking responses to light. We will achieve this goal by injecting the immunotoxin anti-melanopsin-saporin intraocularly in grass rats and examining behavior following this experimental manipulation. Animals will be placed in various lighting conditions, including 12:12 light-dark conditions, and will be given pulses of light to test for effects of masking. We predict that controls will exhibit more general activity during the day, consistent with a diurnal species, and will exhibit increased activity following acute pulses of light. We predict that animals with the melanopsin toxin in the retina will be out of phase with controls in behavior following acute pulses of light, similar to animals with IGL lesions. Altogether, we are building a model to understand the mechanisms underlying the normal display of diurnal behavior, and we hope to add to this knowledge by examining how melanopsin contributes to the display of diurnal behavior in grass rats.

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