Marzia Martina

In Vivo Detection of Human TRPV6-Rich Tumors with Anti-Cancer Peptides Derived from Soricidin

Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemias and lymphomas.

From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip

We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip’s polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)—polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.

Recordings of cultured neurons and synaptic activity using patch-clamp chips

Planar patch-clamp chip technology has been developed to enhance the assessment of novel compounds for therapeutic efficacy and safety. However, this technology has been limited to recording ion channels expressed in isolated suspended cells, making the study of ion channel function in synaptic transmission impractical. Recently, we developed single- and dual-recording site planar patch-clamp chips and demonstrated their capacity to record ion channel activity from neurons established in culture. Such capacity provides the opportunity to record from synaptically connected neurons cultured on-chip. In this study we reconstructed, on-chip, a simple synaptic circuit between cultured pre-synaptic visceral dorsal 4 neurons and post-synaptic left pedal dorsal 1 neurons isolated from the mollusk Lymnaea stagnalis. Here we report the first planar patch-clamp chip recordings of synaptic phenomena from these paired neurons and pharmacologically demonstrate the cholinergic nature of this synapse. We also report simultaneous dual-site recordings from paired neurons, and demonstrate dedicated cytoplasmic perfusion of individual neurons via on-chip subterranean microfluidics. This is the first application of planar patch-clamp technology to examine synaptic communication.

Vesicular storage of glycine in glutamatergic terminals in mouse hippocampus.

Glycine acts as a neuromodulator to regions rich in glutamatergic synapses, such as the forebrain. However, recent evidences for synaptic release of glycine in hippocampal cultured neurons and synaptosomes argue for the existence of functional glycinergic synapses in the hippocampus. It is well established that GABA and glycine act in concert at inhibitory synapses, while the existence of synapses which utilize both glutamate and glycine is less common. The purpose of the present study was to investigate the distribution of glycine and its role in hippocampal neurotransmission. Using immunohistochemistry, we demonstrate that vesicular glycine is preferentially stored in glutamatergic, rather than GABAergic presynaptic terminals. Using the sniffer patch technique, we found that glycine could be released upon presynaptic activity. Furthermore, using whole-cell patch-clamp recordings, we show for the first time the presence of a postsynaptic strychnine-sensitive chloride current in response to presynaptic stimulation. The small amplitude of this current is likely due to the paucity of postsynaptic glycine receptors rather than a low level of glycine release. Taken together, our results suggest that glycine is stored in glutamatergic presynaptic terminals. It is likely that the major role of glycine that is released from presynaptic terminals is to modulate N-methyl-d-aspartate receptor function but may also play a role in decreasing neuronal excitability by opposing glutamatergic neurotransmission in pathological states such as epilepsy or ischemia.

Selective Pharmacological Modulation of Pyramidal Neurons and Interneurons in the CA1 Region of the Rat Hippocampus.

The hippocampus is a complex network tightly regulated by interactions between excitatory and inhibitory neurons. In neurodegenerative disorders where cognitive functions such as learning and memory are impaired this excitation-inhibition balance may be altered. Interestingly, the uncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist memantine, currently in clinical use for the treatment of Alzheimer’s disease, may alter the excitation-inhibition balance in the hippocampus. However, the specific mechanism by which memantine exerts this action is not clear. To better elucidate the effect of memantine on hippocampal circuitry, we studied its pharmacology on NMDAR currents in both pyramidal cells (PCs) and interneurons (Ints) in the CA1 region of the hippocampus. Applying whole-cell patch-clamp methodology to acute rat hippocampal slices, we report that memantine antagonism is more robust in PCs than in Ints. Using specific NMDAR subunit antagonists, we determined that this selective antagonism of memantine is attributable to specific differences in the molecular make-up of the NMDARs in excitatory and inhibitory neurons. These findings offer new insight into the mechanism of action and therapeutic potential of NMDA receptor pharmacology in modulating hippocampal excitability.

Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system

There is a need for improved therapy for acquired brain injury, which has proven resistant to treatment by numerous drugs in clinical trials and continues to represent one of the leading causes of disability worldwide. Research into cell-based therapies for the treatment of brain injury is growing rapidly, but the ideal cell source has yet to be determined. Subpopulations of cells found in amniotic fluid, which is readily obtained during routine amniocentesis, can be easily expanded in culture, have multipotent differentiation capacity, are non-tumourigenic, and avoid the ethical complications associated with embryonic stem cells, making them a promising cell source for therapeutic purposes. Beneficial effects of amniotic fluid cell transplantation have been reported in various models of nervous system injury. However, evidence that amniotic fluid cells can differentiate into mature, functional neurons in vivo and incorporate into the existing circuitry to replace lost or damaged neurons is lacking. The mechanisms by which amniotic fluid cells improve outcomes after experimental nervous system injury remain unclear. However, studies reporting the expression and release of neurotrophic, angiogenic, and immunomodulatory factors by amniotic fluid cells suggest they may provide neuroprotection and (or) stimulate endogenous repair and remodelling processes in the injured nervous system. In this paper, we address recent research related to the neuronal differentiation of amniotic fluid-derived cells, the therapeutic efficacy of these cells in animal models of nervous system injury, and the possible mechanisms mediating the positive outcomes achieved by amniotic fluid cell transplantation.

Chronically Saturating Levels of Endogenous Glycine Disrupt Glutamatergic Neurotransmission and Enhance Synaptogenesis in the CA1 Region of Mouse Hippocampus

Glycine serves a dual role in neurotransmission. It is the primary inhibitory neurotransmitter in the spinal cord and brain stem and is also an obligatory coagonist at the excitatory glutamate, N‐methyl‐D‐aspartate receptor (NMDAR). Therefore, the postsynaptic action of glycine should be strongly regulated to maintain a balance between its inhibitory and excitatory inputs. The glycine concentration at the synapse is tightly regulated by two types of glycine transporters, GlyT1 and GlyT2, located on nerve terminals or astrocytes. Genetic studies demonstrated that homozygous (GlyT1−/−) newborn mice display severe sensorimotor deficits characterized by lethargy, hypotonia, and hyporesponsivity to tactile stimuli and ultimately die in their first postnatal day. These symptoms are similar to those associated with the human disease glycine encephalopathy in which there is a high level of glycine in cerebrospinal fluid of affected individuals. The purpose of this investigation is to determine the impact of chronically high concentrations of endogenous glycine on glutamatergic neurotransmission during postnatal development using an in vivo mouse model (GlyT1+/−). The results of our study indicate the following; that compared with wild‐type mice, CA1 pyramidal neurons from mutants display significant disruptions in hippocampal glutamatergic neurotransmission, as suggested by a faster kinetic of NMDAR excitatory postsynaptic currents, a lower reduction of the amplitude of NMDAR excitatory postsynaptic currents by ifenprodil, no difference in protein expression for NR2A and NR2B but a higher protein expression for PSD‐95, an increase in their number of synapses and finally, enhanced neuronal excitability. Synapse 2011.

A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis

We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.