Abstract List for 2015 meeting

#339: Keeping axon alive from inside and outside the neuron

Beirowski, Bogdan K ;

Hunter James Kelly Research Institute, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

In my presentation I will talk about maintenance mechanisms of axons which are the longest cellular projections of neurons relaying electrical and biochemical signals in nerves and white-matter tracts of the nervous system. Axons do not exist in isolation, but are inextricably and intimately associated with their enwrapping glia (Schwann cells and oligodendrocytes) to form an unique axon-glia unit. Because of their incredible length and energetic demand, axons are very vulnerable and at continuous risk of damage. Many debilitating neurodegenerative disorders share the common feature of early damage and demise of axons. The most relevant neurological symptoms in a number of these conditions are due to compromised axon integrity. Thus, neuroprotective therapies promoting axon stability have great potential for more efficient treatment. My laboratory is investigating the cell-autonomous and non-cell-autonomous mechanisms of the degeneration of axons. In other words, we are attempting to elucidate what causes axon breakdown from within neurons, and which external (glial) events trigger axon loss. In my presentation I will exemplify these models on the basis of our most recent discovery that metabolic dysregulation exclusively in Schwann cells is sufficient to trigger axon breakdown.

#322: Age and Sex Influence Ultrasonic Vocalization Detection by CBA/CaJ Mice

Kobrina, Anastasiya ; Dent, Micheal ;

Department of Psychology, SUNY University at Buffalo;

Age-related hearing loss (ARHL), or prysbycusis, is a universal feature of mammalian aging. This condition is characterized by the progressive decrease in hearing sensitivity, as well as a decrease in the perception of complex auditory signals, such as speech. Mice are frequently used as an animal model for human hearing research, yet their hearing capabilities have not been fully explored. Previous studies have established auditory threshold sensitivities for pure tone stimuli using behavioral methodologies. Further, pure tone thresholds have been measured in the CBA/CaJ strain using ABRs across the lifespan. This strain shows sex differences in hearing loss similar to humans, with males having high frequency hearing loss earlier than females. Nonetheless, little is known about how this strain perceives their own ultrasonic vocalizations (USVs) or how aging and sex influence that perception. Our aim was to establish auditory threshold sensitivity for several USV types, as well as to track these thresholds across the mouse’s lifespan. We expected thresholds to vary across USVs, and to see sex differences in USV thresholds, with males having higher thresholds than females. In order to determine how well mice detect these complex communication stimuli, several CBA/CaJ mice were trained and tested at various ages in a detection task using operant conditioning procedures. The detection of seven USV call types, as well as a 42 kHz pure tone stimulus, were measured. Results showed that mice are sensitive to their complex vocalizations even into old age. Thresholds differed for the different USV types. Mice were able to detect some USVs at a lower intensity than pure tones of the same frequency range. Elderly male mice showed higher thresholds for some USVs compared to females. In conclusion, the results highlight the importance of USVs for survival and communication, and lay the groundwork for future studies on complex signal perception and aging in this species.

#335: Modeling Progressive Supranuclear Palsy

MacLaren, Duncan A A; Ljungberg, Trisha ; Santini, Joseph A ;

Department of Pharmacology and Toxicology, SUNY University at Buffalo;

Progressive Supranuclear Palsy (PSP; the most common atypical Parkinsonism) is characterized by motor and cognitive deficits typical of Parkinson’s Disease (PD). However, standard therapies for PD (eg dopaminergic replacement) are ineffective in PSP. Moreover, there is no animal model of PSP. In both diseases there is degeneration of neurons within the pedunculopontine tegmentum (PPTg). In PD, this degeneration affects all neuronal subtypes (glutamatergic, cholinergic and GABAergic) while in PSP it is selective for the cholinergic population. There are other clear pathological differences: PSP is a tauopathy with progressive accumulation of malformed tau protein, which is not seen in PD. Behaviorally, one differentiator between PSP and PD is the response to startling acoustic stimuli: PSP patients have reduced startle amplitude while PD patients have a normal startle amplitude but a reduced ability to control the startle (impaired pre-pulse inhibition – PPI). We have previously shown that rats with damage to all neuronal types in PPTg have a normal startle amplitude but impaired PPI (mirroring PD) while rats with selective loss of cholinergic PPTg neurons have a near-absent startle response (mirroring PSP). In order to model the progressive accumulation of tau in PSP, using a viral seeding technique we overexpressed human wildtype tau selectively in rat cholinergic PPTg neurons. This may recapitulate PSP in a number of ways: 1) deposit tau in the same brain regions via trans-synaptic spread and so produce progressive neuronal loss, 2) result in the selective death of PPTg cholinergic neurons, 3) produce a model with a progressive onset of symptoms. Histologically, we have shown that 3 months after seeding, the tau is pathological and is deposited in PPTg projection sites. Currently, we are assessing the longitudinal effects of tau-seeding on PSP related behaviors. These are the first steps towards a model of PSP which could facilitate the discovery of therapeutics.

#327: Functional human Oligodendrocyte differentiation gene identification using transcriptional networks.

Pol, Suyog U ¹; Shayya, Hani ²; Welliver, Ross ²; Bratton, David ²; Milliron, Aberlee ²; O'Bara, Melanie ²; Sim, Fraser ²;

¹Department of Biomedical Engineering, ²Department of Pharmacology and Toxicology, SUNY University at Buffalo;

In demyelinating diseases such as multiple sclerosis, identification of molecular signals that regulate human oligodendrocyte (Ols) progenitor (hOPC) fate may provide novel means to induce remyelination. In this study, we isolated CD140a+ OPCs from human fetal brain and extracted RNA each day during differentiation in vitro for microarray. As expected, we observed large up-regulation of Myelin Basic Protein (MBP) mRNA (315 fold, p-value=0.005, n=4). Weighted Gene Co-expression Network Analysis was applied to define cross-species conserved network modules. We tested the functional importance of genes within the pro-differentiation modules by lentiviral over-expression in hOPCs. Over-expression of GNB4, a G-protein ß subunit, significantly increased the O4+ mature OLs from 16 ± 2% in control mCherry overexpressing cells to 24 ± 3% (mean ± SEM, t-test p<0.003).To confirm a functional role in vivo, the above hOPCs were transplanted into neonatal shiverer/rag2 mice. Strikingly, GNB4-infected hOPC transplanted animals exhibited a greater than 3 fold increase in the area of MBP staining within the corpus callosum (unpaired t-test p < 0.05, n=4). Thus GNB4 can provide a novel insight to link GPCR Muscarinic Receptor type 3 OPC fate regulation with intra cellular signaling. Furthermore, to determine whether progenitor module member SULF2 was regulated during remyelination, we analyzed mice spinal cord after lysolecithin-induced demyelination. SULF2 mRNA and protein was found in demyelinated lesions. A significant increase in Proteolipid protein 1  expressing mature OLs at 14 days post-lesion in OPC-specific SULF1/2 conditional knockout mice suggest that sulfatase expression regulates OPC fate following demyelination (unpaired t-test p < 0.05, n≥3). Also sulfatases aid BMP and WNT signaling in OPCs suggesting a regulatory mechanism. As such, sulfatase inhibitors may be ideally positioned to interfere with differentiation inhibitory factors to promote remyelination.

#338: A role for salivary proteins in taste and feeding

Torregrossa, Ann-Marie M ;

Department of Psychology, SUNY University at Buffalo;

Under normal feeding and drinking conditions, taste compounds invariably mix with saliva before reaching their receptor targets. This sets the stage for salivary constituents to modulate the taste signal at the most fundamental level.  However, very little work has been conducted examining how salivary proteins may modulate taste perception. My work has begun to explore how salivary proteins are modified by experience and how these proteins then modify diet acceptance and choice. My lab has been using classical behavioral techniques, including psychophysics, as well as proteomics and whole-nerve electrophysiology to explore the role of salivary proteins in bitter taste specifically. While some bitters signify toxins, a large number of nutritionally significant food sources contain bitter phytochemicals, making variation in bitter taste perception key in influencing healthy diet selection.

#329: Validation of parametric imaging obtained from DSA images using Doppler ultrasound on neurovascular phantoms

Balasubramoniam, Anusha ¹; Bednarek, Daniel R ²; Rudin, Stephen ²;

¹Biomedical Engineeering, ²Department of Physiology and Biophysics, SUNY University at Buffalo;

To validate Digital Subtraction Angiography (DSA) based parametric images using blood obtained from Doppler ultrasound in neurovascular phantoms. A silicone neurovascular phantom was embedded in ballistic gel which has properties equivalent to human tissue. The phantom contained the internal carotid artery, middle cerebral artery and anterior communicating artery. A peristaltic pump, simulating physiological flow conditions and pumping blood mimicking fluid through the phantom was connected to obtain Color Doppler images, the gel prevented movement of the phantom. For parametric images, at various flow rates (100, 120 and 160 ml/min) water was pumped through the phantom. 10 ml contrast boluses were manually injected. DSA images were obtained at 10 frames/sec from the Toshiba C-arm. The image sequences obtained were input into a LabVIEW software to get parametric maps from time-density curves. At the internal carotid artery, the parametric maps were compared with velocities determined from Doppler ultrasound. Velocities obtained from Doppler ultrasound were 38, 48 and 65 cm/s at flow rates of 100, 120 and 160 ml/min, respectively. For a 20% increase in flow rate, blood velocity percentage change measured by Doppler ultrasound was 26.3%. There was a 20% decrease of Bolus Arrival Time (BAT) and 14.3% decrease of Mean Transit Time (MTT). This showed strong inverse correlation with velocity obtained from Doppler ultrasound. The parameters obtained from parametric imaging are quite sensitive to velocity changes and are well correlated to the velocities obtained from Doppler ultrasound.

#340: Characterization of an Alternate PLCβ Signaling Pathway in Taste Receptor Cells

Benfey, Eric D ; Medler, Kathryn F ;

Biology Department, SUNY University at Buffalo, College of Arts and Sciences;

Taste receptor cells use multiple signal recognition mechanisms to identify taste stimuli. Salty and sour taste qualities are ionic, and interact directly with ion channels to elicit a response, while chemically complex taste qualities (bitter, sweet, and umami) activate G-protein coupled receptors and initiate a PLCβ  signaling cascade. PLCβ  generates IP3, which activates IP3 receptors located on intracellular calcium stores. Once activated, the IP3 receptor releases calcium into the cytoplasm. This increase in intracellular calcium leads to neurotransmitter release from the taste receptor cells, which relays taste information to the gustatory nerves. The isoforms PLCβ2 and IP3R3 are co-expressed in taste receptor cells that recognize chemically complex tastants. However, studies have shown that in the absence of either PLCβ2 or IP3R3, mice are capable of detecting taste qualities, though at a diminished capacity. This suggests that an alternative pathway for taste detection exists within taste receptor cells. Previous work from our lab has indicated that PLCβ3 is co-expressed in taste receptor cells with IP3R1 (Hacker et. Al. 2008). We characterized the calcium responses in isolated taste receptor cells from wild type mice and mice containing an IP3R3 knock-out. We found that responses to bitter compounds occur less frequently in cells from mice lacking IP3R3, while sweet responses are nearly all abolished. Our results suggest the PLCβ2 pathway is the main pathway that detects sweet stimuli, while the alternate PLCβ pathway makes major contributions to the bitter detection pathway.

#348: Enhanced ER stress in BM angiogenic progenitor cells in long-term type 2 diabetes mouse model

Bhatta, Maulasri M ; Ma, Jacey Hongjie ; Wang, Joshua J ; Sakowski, Jonna ; Zhang, Sarah X ;

Ross Eye Institute, Department of Ophthalmology, SUNY University at Buffalo;

Bone marrow-derived circulating angiogenic cells (CACs) play an important role in vascular repair. In diabetes, compromised functioning of the CACs contributes to the development of diabetic retinopathy; however, the underlying mechanisms are poorly understood. We examined whether endoplasmic reticulum (ER) stress, which has recently been linked to endothelial injury, is involved in diabetic angiogenic dysfunction.The numbers of bone-marrow progenitors and CACs were significantly reduced in db/db mice. Vascular density was markedly decreased in the retinas of db/db mice, and this was accompanied by vascular beading. The production of ER stress markers (glucose-regulated protein-78 [GRP-78], phosphorylated inositol-requiring enzyme-1α [p-IRE-1α], phosphorylated eukaryotic translation initiation factor-2α [p-eIF2α], activating transcription factor-4 [ATF4], C/EBP homologous protein [CHOP] and spliced Xbox binding protein-1 [XBP1s]) was significantly increased in bone marrow-derived EOCs from db/db mice. In addition, mouse EOCs cultured in high-glucose conditions demonstrated higher levels of ER stress, reduced colony formation, impaired migration and increased apoptosis, all of which were largely prevented by the chemical chaperone 4-phenylbutyrate. Taken together, our results indicate that diabetes increases ER stress in bone marrow angiogenic progenitor cells. Thus, targeting ER stress may offer a new approach to improving angiogenic progenitor cell function and promoting vascular repair in diabetes.

#313: Toxoplasma gondii Infections Alter GABAergic Synapses and Signaling in the Central Nervous System

Brooks, Justin M ;

Department of Microbiology and Immunology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Activity in the brain is controlled by a combination of excitatory and inhibitory neurotransmission and when this balance becomes uncoordinated, electrical activity increases and seizures develop.  Humans suffering from cerebral toxoplasmosis develop seizures, although mechanisms of how Toxoplasma dysregulates synaptic transmission are unknown.  To address this question, we first tested whether Toxoplasma-infected mice develop seizures.  We found that mice develop spontaneous and long-lasting seizures 30 days post infection.  We next used immunohistochemistry to compare synaptic structures between brains of mock and parasite-infected animals.  Excitatory glutamatergic synaptic terminals in the CNS appeared normal in Toxoplasma-infected mice.  In contrast, infection led to a global deficit of the GABA biosynthetic enzymes GAD67 and GAD65, which are normally clustered at presynaptic release sites of inhibitory GABAergic synapses.  These changes appear to be due to altered localization rather than protein expression since GAD67 protein levels were unaltered by infection.   We found that mislocalization of GAD67 was not observed in brains of mice infected with the Toxoplasma type III strain, CTG indicate that polymorphic parasite protein(s) were involved.  The finding that Toxoplasma specifically affected GABAergic synapses suggested that they would be more susceptible to drugs that inhibited GABAergic signaling. Thus, the magnitude of seizures that develop in response to low-dose pentylenetetrazol (PTZ) were compared between mock and parasite-infected mice.  We found that parasite-infected mice developed more severe seizures after PTZ treatment.  Taken together, these data support a model in which seizures and other neurological complications seen in Toxoplasma-infected individuals are due to changes in GABAergic signaling.

#350: A combinatorial viral vector technique for targeting striatal direct medium spiny neurons in WT rats

Bruno, Michael J ; Bass, Caroline E ;

Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Medium spiny neurons (MSNs) account for over 90% of the neurons in the striatum and are classified as either direct (dMSN) or indirect (iMSN) projecting. dMSNs can be identified based on the expression of dynorphin and substance P, while iMSNs are enkephalin positive. Recent evidence suggests that dMSN and iMSN may mediate different aspects of drug associated behaviors. However, it has been difficult to study their respective roles because of a general lack of tools to manipulate each MSN type individually, particularly in rats, which are used extensively in behavioral studies of drug abuse. Recently our laboratory has created a combinatorial adeno-associated virus (AAV) approach to target dMSN or iMSNs in rats. One virus delivers a designer receptor exclusively activated by designer drug (DREADD) contained in a CRE recombinase dependent (DIO) cassette. In this construct DREADD expression is driven by a strong generalized promoter, but only when CRE is present. The second virus delivers CRE-recombinase under the control of either the dynorphin (dyn) or enkephalin (enk) promoter, which will drive expression only in dMSN or iMSNs, respectively. This dual technique compensates for the relatively small size of the AAV and the relatively weak activity of the dynorphin and enkephalin promoters. Immunohistochemistry (IHC) for substance P (a dMSN marker), enkephalin (an iMSN marker), and mCherry was performed on rats co-injected with the DIO-DREADD + Dyn-CRE viruses. Our results demonstrate that DREADD expression was restricted primarily to dMSNs. Specifically, approximately 83% of DREADD positive neurons (as indicated by the presence of mCherry) were dyn positive, while 17% appeared dyn negative. These preliminary results indicate that our dual targeting approach can be an effective method for targeting gene delivery to dMSNs.