sfn2017

Horner KA, Logue JB, Jenrette TA (2017) Patch compartment lesions reduce habitual sucrose consumption. Neuroscience 2017 Abstracts 689.16 / II23. Society for Neuroscience, Washington, DC.

Summary: The striatum mediates habit formation and reward association. The striatum can be divided into the patch and matrix compartment, which are two neurochemically and anatomically distinct regions that may sub-serve different aspects of behavior. For example, the patch compartment may mediate reward-related behaviors, while the matrix compartment may mediate adaptive motor functions. Furthermore, previous studies have shown that enhanced relative activation of the patch versus matrix compartment is associated with inflexible behaviors, such as stereotypy. Habitual behaviors are also inflexible in nature, but whether enhanced activation of the patch compartment contributes to habitual behavior is not known. The goal of the current study was to examine the role of patch compartment neurons in the development of habit formation. We used dermorphin-saporin to specifically ablate neurons of the patch compartment prior to training animals to self-administer sucrose on a random interval schedule of reinforcement, which has been shown to foster habit formation. Our data showed that destruction of the neurons of the patch compartment prevented the reinstatement of sucrose self-administration after sucrose devaluation, indicating that absence of the patch compartment interrupted the development of habitual behavior. Our data also indicate that c-Fos levels were decreased in the dorsolateral striatum (DLS) and sensorimotor cortex (SMC), but increased in dorsomedial striatum (DMS) and prefrontal cortex (PFC) in patch-lesioned animals that did not develop habitual behavior, indicating that diminished habit formation is associated with decreased activation of regions that participate in habitual behavior, and increased in regions associated with goal-directed behaviors. Together, these data indicate that the patch compartment participates in habit formation by altering the flow of information through basal ganglia circuits.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Falquetto B, Oliveira LM, Moreira TS, Takakura AC (2017) Role of orexinergic neurons in the chemosensory control of breathing in a Parkinson’s disease model. Neuroscience 2017 Abstracts 779.08 / HH1. Society for Neuroscience, Washington, DC.

Summary: Parkinson´s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). Non-motor symptoms such as neuropsychiatric, sleep and breathing disorders are also observed in PD. Previous study has already demonstrated that in 6-hydroxydopamine (6-OHDA)-model of PD there is a reduction in the number of phox2b neurons in the retrotrapezoid nucleus (RTN) and a decrease in the respiratory response to hypercapnia. Here, we tested the involvement of orexin cells from lateral hypothalamus/perifornical area (LH/PeF) on breathing in this model of PD. 6-OHDA (24 µg/µl) injections into the striatum reduced the number of catecholaminergic (40 days: 128 ± 10 and 60 days: 116 ± 13 vs. vehicle: 938 ± 15 neurons) and orexin-B-ir neurons (40 days: 310 ± 9 and 60 days: 258 ± 15 vs. vehicle: 412 ± 13 neurons). The injection of anti-Orexin-B saporin into the LH/PeF produces a further reduction in the number of orexinergic neurons in PD animals (79 ± 8 vs. control: 427 ± 14 neurons). The respiratory frequency (fR) at rest and in response to hypercapnia (7% CO2) was assessed 60 days after bilateral 6-OHDA or vehicle injections into the striatum and anti-Orexin-B saporin or IgG saporin into the LH/PeF during sleep and wakefulness in the dark and light phases of the diurnal cycle. Sixty days after 6-OHDA, we observed a reduction of fR at rest during sleep in the light phase only in PD animals (56 ± 2 vs. control: 66 ± 2 bpm). During the dark phase, there is a reduction in fR response to hypercapnia in PD animals with depletion of orexinergic neurons during wakefulness (119 ± 6 vs. control: 152 ± 3 bpm) and sleep (128 ± 7 vs. control: 147 ± 5 bpm). Our data suggest that orexinergic neurons are important to restore chemoreceptor function in a rat model of PD during sleep and wakefulness in rats.

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ATS Poster of the Year Winner

Del Rio R, Andrade DC, Toledo C, Diaz HS (2017) RVLM C1 neuron ablation normalizes cardiorespiratory control in heart failure. Neuroscience 2017 Abstracts 507.13 / NN21. Society for Neuroscience, Washington, DC.

Summary: Heart failure (CHF) is characterized by sympathoexcitation and breathing disorders. The rostral ventrolateral medulla (RVLM) is hyperactive in CHF. However, there is no direct evidence between the relationship of RVLM chronic hyperactivation, sympathoexcitation and progression of cardiac deterioration in CHF. We hypothesized that selective elimination of cathecolaminergic neurons from the RVLM delays cardiac deterioration in CHF rats. CHF was induced by volume overload in male Sprague-Dawley rats (250±20g). Ablation of C1 cells was performed by anti-dopamine-beta hydroxylase (DβH)–saporin toxin (DβH+SAP) injected into the RVLM. The degree of HF was estimated by echocardiography. Cardiac function was assessed by intraventricular PV loops. Arrhythmia index and breathing disorders were scored. Central and peripheral chemoreflex and cardiac autonomic control were also study. Partial elimination of C1 RVLM neurons (≈50%) delay the decrease in fractional shortening in CHF rats (CHF+Veh: 59±5 vs. 45±1 %, p<0.05, pre vs. post vehicle, respectively; CHF+DβH-SAP: 57±4 vs. 51±4 %, p>0.05, pre vs. post toxin, respectively). In addition, compared to CHF vehicle treated rats, CHF+DβH-SAP rats showed (CHF+Veh vs. CHF+DβH-SAP, respectively): i) a reduced cardiac sympathetic drive (-98±12 vs. -52±7 ΔHR, p<0.05), ii) an improvement in both cardiac diastolic (0.009±0.001 vs. 0.004±0.001 mmHg/µl, p<0.05) and systolic function (0.2±0.01 vs. 0.5±0.1 mmHg/µl, p<0.05), iii) a reduced number of arrhythmias (95±20 vs. 48±14 events/hour, p<0.05), and iv) a reduced incidence of breathing disorders (9±1 vs. 6±1 apneas/hour, p<0.05). Finally, the detrimental autonomic and cardiovascular effects induced by central chemoreceptors activation were abolished after C1 neurons ablation in CHF rats. Neither hypoxic nor hypercapnic ventilatory chemoreflex responses were affected by DβH- SAP treatment. Our results showed that the RVLM play a pivotal role on the progression of cardiac deterioration and in the maintenance of autonomic imbalance and breathing disorders in CHF. In addition, our results showed that the sympathoexcitation and cardiac function deterioration induced by central chemoreflex activation is related to the activation of RVLM C1 neurons.

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Suarez AN, Hsu TM, DeLartigue G, Kanoski SE (2017) Gastrointestinal vagal afferent signaling promotes hippocampal-dependent memory function in rats. Neuroscience 2017 Abstracts 510.22 / PP13. Society for Neuroscience, Washington, DC.

Summary: The vagus nerve is the primary conduit of communication between feeding-relevant gastrointestinal (GI) signals and the brain. Vagally-mediated GI satiation signals, including gastric distension and intra-gastric nutrient infusion, activate neurons in the hippocampus (HPC) through unidentified polysynaptic pathways. The functional relevance of GI-derived communication to the HPC is unknown. Here we first explored whether chronic disruption of gut-to-brain vagal tone via subdiaphragmatic vagotomy (SDV) negatively impacts HPC-dependent memory function in rats. While SDV did not impair HPC-dependent appetitive learning based on interoceptive energy status cues or social food-related cues, SDV did impair spatial working memory (Barnes maze) and contextual episodic memory (novel object in context; NOIC), two HPC-dependent tasks that involve processing of visuospatial stimuli. Next, to determine whether vagal sensory/afferent vs. motor/efferent signaling regulates HPC-dependent memory function, we employed a novel approach in which a saporin conjugated to cholecystokinin (CCK-SAP) or an unconjugated control saporin is injected into the nodose ganglia, a strategy that preserves 100% of vagal efferent signaling while eliminating ~80% of GI-derived vagal afferent signaling. Similar to SDV rats, CCK-SAP rats were impaired in both the Barne’s maze task and NOIC learning relative to controls. Consistent with the memory deficits, immunoblot protein analyses in hippocampus lysates revealed reduced neurotophic [brain- derived neurotrophic factor (BDNF)], and neurogenesis [doublecortin (DCX)] markers in both SDV and CCK-SAP rats relative to controls. These findings indicate that GI-derived vagal afferent signaling is critical in regulating HPC-dependent mnemonic function. Results have direct clinical relevance, as procedures that chronically disrupt vagus nerve signaling (e.g., vBloc) have recently been FDA-approved for obesity treatment.

Related Products: CCK-SAP (Cat. #IT-31)

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)

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.

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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.

Related Products: 192-IgG-SAP (Cat. #IT-01), GAT1-SAP (Cat. #IT-32)

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.

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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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