Our recent work has shown the feasibility of using a refined immunomagnetic enrichment (IE) assay to detect cytokeratin 20-positive circulating tumour cells (CK20 pCTCs) in colorectal cancer (CRC) patients. We attempted to improve the sensitivity for CRC by detecting another intestinal-type differentiation marker, CDX2 pCTCs, using the same methodology.
CDX2 pCTCs were detected in patients with CRC, colorectal adenoma (CAD), benign colorectal diseases (BCD), other common cancers (OCC) and normal subjects (NS). Statistical analysis was used to correlate CDX2 pCTCs to the clinicohistopathological factors, recurrence, metastasis and survival after follow-up for 42 months in CRC patients.
CDX2 pCTCs were detected in 81% CRC patients (73 out of 90, median number=21.5 CTCs), 7.5% CAD patients (3 out of 40), 0% patients with BCD (0 out of 90), 2.5% patients with OCC (2 out of 80) and 0% NS (0 out of 40). Furthermore, statistical analysis showed that CDX2 pCTC numbers were associated with tumour- node-metastasis stage and lymph node status. Using the median CDX2 pCTC numbers as the cutoff points, stratified groups of CRC patients had significant differences in their recurrence and survival.
This study showed that the refined IE assay can detect CDX2 pCTCs with high sensitivity and that CDX2 pCTCs can generate clinically important information for CRC patients.
clinical significance; CDX2-positive circulating tumour cells; colorectal cancer
Patients with dural arteriovenous fistula (DAVF) are at higher risk of developing neurological deficits when there is retrograde leptomeningeal venous drainage. Our aim is to demonstrate the presence of dilated deep medullary veins in the brain on magnetic resonance imaging (MR) in this group of patients, and to assess their clinical significance. Nine patients with angiographically proven DAVF associated with leptomeningeal venous drainage who had MR before treatment were studied. MR was performed in at least two orthogonal planes before and after gadolinium administration. The dural fistula was located at the cavernous sinus in five patients, at the transverse-sigmoid sinus in three and at the tentorium in one. Dilated deep medullary veins were noted in six patients. Of these, four showed parenchymal abnormalities which included intracerebral haematoma, venous infarction, brain oedema and T2 hyperintensity in brainstem. Venous varix was present in one patient. No neurological complication or parenchymal change was observed in the three patients without dilated deep medullary veins. Therefore, in patients with intracranial DAVF associated with leptomeningeal venous recruitment, the MR finding of dilated deep medullary veins suggests a more severe degree of venous hypertension and congestion in the brain. This subgroup of patients has a much higher chance of neurological complications and warrants urgent intervention.
dural arteriovenous fistula, intracranial, magnetic resonance imaging
Knowledge of current epidemiology and spine trauma trends assists in public resource allocation, fine-tuning of primary prevention methods, and benchmarking purposes. Data on all patients with traumatic spine injuries admitted to the Alfred Hospital, Melbourne between May 1, 2009, and January 1, 2011, were collected from the Alfred Trauma Registry, Alfred Health medical database, and Victorian Orthopaedic Trauma Outcomes Registry. Epidemiological trends were analyzed as a general cohort, with comparison cohorts of nonsurvivors versus survivors and elderly versus nonelderly. Linear regression analysis was utilized to demonstrate trends with statistical significance. There were 965 patients with traumatic spine injuries with 2,333 spine trauma levels. The general cohort showed a trimodal age distribution, male-to-female ratio of 2:2, motor vehicle accidents as the primary spine trauma mechanism, 47.7% patients with severe polytrauma as graded using the Injury Severity Score (ISS), 17.3% with traumatic brain injury (TBI), the majority of patients with one spine injury level, 7% neurological deficit rate, 12.8% spine trauma operative rate, and 5.2% mortality rate. Variables with statistical significance trending toward mortality were the elderly, motor vehicle occupants, severe ISS, TBI, C1–2 dissociations, and American Spinal Injury Association (ASIA) A, B, and C neurological grades. Variables with statistical significance trending toward the elderly were females; low falls; one spine injury level; type 2 odontoid fractures; subaxial cervical spine distraction injuries; ASIA A, B, and C neurological grades; and patients without neurological deficits. Of the general cohort, 50.3% of spine trauma survivors were discharged home, and 48.1% were discharged to rehabilitation facilities. This study provides baseline spine trauma epidemiological data. The trimodal age distribution of patients with traumatic spine injuries calls for further studies and intervention targeted toward the 46- to 55-year age group as this group represents the main providers of financial and social security. The study's unique feature of delineating variables with statistical significance trending toward both mortality and the elderly also provides useful data to guide future research studies, benchmarking, public health policy, and efficient resource allocation for the management of spine trauma.
spine trauma; epidemiology; demographics; spinal injury characteristics; neurological status; registry; prevention
A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.
cell contraction; calcium; cortical tension; myeloid precursor cell; optical stretcher; cell rheology
Background. Exercise has been suggested to be a viable treatment for depression. This study investigates the effect of supervised aerobic exercise training on depressive symptoms and physical performance among Chinese patients with mild to moderate depression in early in-patient phase. Methods. A randomized repeated measure and assessor-blinded study design was used. Subjects in aerobic exercise group received 30 minutes of aerobic training, five days a week for 3 weeks. Depressive symptoms (MADRS and C-BDI) and domains in physical performance were assessed at baseline and program end. Results. Subjects in aerobic exercise group showed a more significant reduction in depressive scores (MADRS) as compared to control (between-group mean difference = 10.08 ± 9.41; P = 0.026) after 3 weeks training. The exercise group also demonstrated a significant improvement in flexibility (between-group mean difference = 4.4 ± 6.13; P = 0.02). Limitations. There was lack of longitudinal followup to examine the long-term effect of aerobic exercise on patients with depression. Conclusions. Aerobic exercise in addition to pharmacological intervention can have a synergistic effect in reducing depressive symptoms and increasing flexibility among Chinese population with mild to moderate depression. Early introduction of exercise training in in-patient phase can help to bridge the gap of therapeutic latency of antidepressants during its nonresponse period.
Materials with negative permittivity and permeability can overcome the diffraction limit, thereby making the sub-wavelength imaging possible. In this study, we analyze the effects of gradient index on a half-infinite perfect lens. We assume that the sharp interface between the vacuum and the negative-index material is replaced by a smooth transition profile such that the index gradually changing from positive to negative. Interestingly, we find that if the graded index profile is modeled by a tanh function, we can have closed-form analytical solutions for this problem, which is a distinct advantage as numerical solutions are not accurate for evanescent waves with large transverse wave vectors. By analyzing the analytical formulas we confirm that a nonzero total absorption can occur even for a near-zero absorption coefficient in the steady-state limit and the image plane contains multiple sub-wavelength images of an object.
The conventional dose of anti-vascular endothelial growth factor treatment may slowly reduce the subretinal fluid and height of a vascularized pigment epithelial detachment (vPED), but rarely leads to its complete resolution. We report a dramatic outcome involving a high dose (2 mg) of ranibizumab for treating vPED.
This report describes three eyes with vPED that received 2 mg in 0.05 ml of ranibizumab injections on a monthly basis and were followed prospectively. Each patient received a complete ocular examination, including best-corrected standardized ETDRS testing, fundus photography (FP), fluorescein angiography (FA), optical coherent tomography (OCT), and indocyanine-green angiography at baseline. ETDRS and OCT testing were repeated monthly, while FP and FA were performed every 3 months.
Following a single intravitreal injection of 2 mg ranibizumab, there was rapid resolution of the subretinal fluid, haemorrhage, exudates, and flattening of the vPED within 10 days for Case 1, and within 1 month for Case 2 and Case 3.
Rapid and dramatic decrease in the exudative changes and collapse of the vPED may develop after a single injection of high-dose (2 mg) ranibizumab in certain eyes with a vPED. The improvement was maintained with additional monthly injections to 12 months.
age-related macular degeneration; anti-vascular endothelial growth factor therapy; high-dose ranibizumab; 2 mg ranibizumab; retinal pigment epithelial detachment; vascularized pigment epithelial detachment
Background The establishment of a spine trauma registry collecting both spine column and spinal cord data should improve the evidential basis for clinical decisions. This is a report on the pilot of a spine trauma registry including development of a minimum dataset.
Methods A minimum dataset consisting of 56 data items was created using the modified Delphi technique. A pilot study was performed on 104 consecutive spine trauma patients recruited by the Victorian Orthopaedic Trauma Outcomes Registry (VOTOR). Data analysis and collection methodology were reviewed to determine its feasibility.
Results Minimum dataset collection aided by a dataset dictionary was uncomplicated (average of 5 minutes per patient). Data analysis revealed three significant findings: (1) a peak in the 40 to 60 years age group; (2) premorbid functional independence in the majority of patients; and (3) significant proportion being on antiplatelet or anticoagulation medications. Of the 141 traumatic spine fractures, the thoracolumbar segment was the most frequent site of injury. Most were neurologically intact (89%). Our study group had satisfactory 6-month patient-reported outcomes.
Conclusion The minimum dataset had high completion rates, was practical and feasible to collect. This pilot study is the basis for the development of a spine trauma registry at the Level 1 trauma center.
registry; spine trauma; minimum dataset; Victorian Orthopaedic Trauma Outcomes Registry
The gravity of Earth is responsible for the formation of water waves and usually difficult to change. Although negative effective gravity was recently predicted theoretically in water waves, it has not yet been observed in experiments and remains a mathematical curiosity which is difficult to understand. Here we experimentally demonstrate that close to the resonant frequency of purposely-designed resonating units, negative effective gravity can occur for water waves passing through an array of resonators composing of bottom-mounted split tubes, resulting in the prohibition of water wave propagation. It is found that when negative gravity occurs, the averaged displacement of water surface in a unit cell of the array has a phase difference of π to that along the boundary of the unit cell, consistent with theoretical predictions. Our results provide a mechanism to block water waves and may find applications in wave energy conversion and coastal protection.
Heroin abuse and natural aging exert common influences on immunological cell functioning. This observation led to a recent and untested idea that aging may be accelerated in abusers of heroin. We examined this claim by testing whether heroin use is associated with premature aging at both cellular and brain system levels. A group of abstinent heroin users (n=33) and matched healthy controls (n=30) were recruited and measured on various biological indicators of aging. These measures included peripheral blood telomerase activity, which reflects cellular aging, and both structural and functional measures of brain magnetic resonance imaging. We found that heroin users were characterized by significantly low telomerase activity (0.21 vs 1.78; 88% reduction; t(61)=6.96, P<0.001; 95% confidence interval=1.12–2.02), which interacted with heroin use to affect the structural integrity of gray and white matter of the prefrontal cortex (PFC; AlphaSim corrected P<0.05), a key brain region implicated in aging. Using the PFC location identified from the structural analyses as a ‘seed' region, it was further revealed that telomerase activity interacted with heroin use to impact age-sensitive brain functional networks (AlphaSim corrected P<0.05), which correlated with behavioral performance on executive functioning, memory and attentional control (Pearson correlation, all P<0.05). To our knowledge, this study is the first to attempt a direct integration of peripheral molecular, brain system and behavioral measures in the context of substance abuse. The present finding that heroin abuse is associated with accelerated aging at both cellular and brain system levels is novel and forms a unique contribution to our knowledge in how the biological processes of drug abusers may be disrupted.
addiction; aging; heroin; MRI; prefrontal cortex; resting state; telomerase
The symptoms of Parkinson’s disease (PD) are related to changes in the frequency and pattern of activity in the reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN). In idiopathic and experimental PD the GPe and STN exhibit hypo- and hyper-activity, respectively, and abnormal synchronous rhythmic burst firing. Following lesion of midbrain dopamine neurons abnormal STN activity emerges slowly and intensifies gradually until it stabilizes after 2–3 weeks. Alterations in cellular/network properties may therefore underlie the expression of abnormal firing. Because the GPe powerfully regulates the frequency, pattern and synchronization of STN activity, electrophysiological, molecular and anatomical measures of GPe-STN transmission were compared in the STN of control and 6-hydroxydopamine-lesioned rats and mice. Following dopamine depletion: 1) the frequency (but not the amplitude) of mIPSCs increased by ~70%; 2) the amplitude of evoked IPSCs and isoguvacine-evoked current increased by ~60% and ~70%, respectively; 3) mRNA encoding α1, β2 and γ2 GABAA receptor subunits increased by 15–30%; 4) the density of postsynaptic gephyrin and γ2 subunit co-immunoreactive structures increased by ~40%, whereas the density of vesicular GABA transporter and bassoon co-immunoreactive axon terminals was unchanged; 5) the number of ultrastructurally defined synapses per GPe-STN axon terminal doubled with no alteration in terminal/synapse size or target preference. Thus, loss of dopamine leads, through an increase in the number of synaptic connections per GPe-STN axon terminal, to substantial strengthening of the GPe-STN pathway. This adaptation may oppose hyperactivity but could also contribute to abnormal firing patterns in the parkinsonian STN.
necrobiosis lipoidica; granulomatous inflammation
A number of ecological studies have found a pattern of increasing suicide rates after suicides of several Asian entertainment celebrities. However, the finding may be subject to positive outcome bias where cases with no perceived impact may be routinely excluded. In this study, we deploy interrupted time-series analysis using ARIMA transfer function models to investigate systematically the impact of thirteen celebrity suicides on subsequent suicide rates in South Korea. We find that three out of eleven cases were found to be followed by a significant increase in suicide rate, while controlling for seasonality, secular trends, and unemployment rates. Such significant increases could last for nine weeks. Non-significance cases may be attributable to the small amount of media coverage, the “displacement” effect of preceding case, and the negative connotation of celebrity deaths. We therefore conclude that whether or not the impacts were detected may be largely conditioned by various contextual factors. Current evidence based on ecological studies is insufficient to draw a firm conclusion. Further studies using multiple approaches should be developed.
Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists, providing a powerful means of dissecting complex neural circuits in the brain. Recently, it was reported that one popular line of these mice – mice possessing a BAC transgene with a D2 dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter – had abnormal striatal gene expression, physiology and motor behavior. Unlike most of the work using BAC mice, this interesting study relied upon mice backcrossed on the outbred Swiss Webster strain that were homozygous for the Drd2-eGFP BAC transgene.The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported, SW mice were very sensitive to transgene expression. However, in more commonly used inbred strains of mice (C57BL/6, FVB/N) that were hemizygous for the transgene, the Drd2-eGFP BAC transgene did not alter striatal gene expression, physiology or motor behavior. Thus, the use of inbred strains of mice which are hemizygous for the Drd2 BAC transgene provide a reliable tool for studying basal ganglia function.
Optical coherence tomography (OCT) is an imaging technology with the potential to provide high-resolution images of the skin non-invasively. With this device, it is possible to identify a host of skin structures including tumors. In this case report, we demonstrate the use of an OCT device in delineating a lateral tumor margin of an ill-defined basal cell carcinoma prior to Mohs micrographic surgery. Following surgery, the OCT images are compared to histologic sections to confirm their accuracy. OCT technology has the potential to be a vital tool for dermatologists and particularly Mohs surgeons in identifying tumor margins and potentially reducing the number of invasive procedures needed.
Optical coherence tomography; Mohs surgery; High-resolution imaging technology; Basal cell carcinoma
There is growing interest in the use of technology to enhance the tracking and quality of clinical information available for patients in disaster settings. This paper describes the design and evaluation of the Wireless Internet Information System for Medical Response in Disasters (WIISARD).
Materials and methods
WIISARD combined advanced networking technology with electronic triage tags that reported victims' position and recorded medical information, with wireless pulse-oximeters that monitored patient vital signs, and a wireless electronic medical record (EMR) for disaster care. The EMR system included WiFi handheld devices with barcode scanners (used by front-line responders) and computer tablets with role-tailored software (used by managers of the triage, treatment, transport and medical communications teams). An additional software system provided situational awareness for the incident commander. The WIISARD system was evaluated in a large-scale simulation exercise designed for training first responders. A randomized trial was overlaid on this exercise with 100 simulated victims, 50 in a control pathway (paper-based), and 50 in completely electronic WIISARD pathway. All patients in the electronic pathway were cared for within the WIISARD system without paper-based workarounds.
WIISARD reduced the rate of the missing and/or duplicated patient identifiers (0% vs 47%, p<0.001). The total time of the field was nearly identical (38:20 vs 38:23, IQR 26:53–1:05:32 vs 18:55–57:22).
Overall, the results of WIISARD show that wireless EMR systems for care of the victims of disasters would be complex to develop but potentially feasible to build and deploy, and likely to improve the quality of information available for the delivery of care during disasters.
Electronic Health records; disasters; wireless; mobile computing
assessment; novel radiosensitisers; early; preclinical; clinical
Globus pallidus (GP) neurons fire rhythmically in the absence of synaptic input, suggesting that they may encode their inputs as changes in the phase of their rhythmic firing. Action potential afterhyperpolarization (AHP) enhances precision of firing by ensuring that the ion channels recover from inactivation by the same amount on each cycle. Voltage-clamp experiments in slices showed that the longest component of the GP neuron’s AHP is blocked by apamin, a selective antagonist of calcium-activated SK channels. Application of 100 nm apamin also disrupted the precision of firing in perforated-patch and cell-attached recordings. SK channel blockade caused a small depolarization in spike threshold and made it more variable, but there was no reduction in the maximal rate of rise during an action potential. Thus, the firing irregularity was not caused solely by a reduction in voltage-gated Na+ channel availability. Subthreshold voltage ramps triggered a large outward current that was sensitive to the initial holding potential and had properties similar to the A-type K+ current in GP neurons. In numerical simulations, the availability of both Na+ and A-type K+ channels during autonomous firing were reduced when SK channels were removed, and a nearly equal reduction in Na+ and K+ subthreshold-activated ion channel availability produced a large decrease in the neuron’s slope conductance near threshold. This change made the neuron more sensitive to intrinsically generated noise. In vivo, this change would also enhance the sensitivity of GP neurons to small synaptic inputs.
Cytomegalovirus (CMV) is a common opportunistic infection after lung transplant. Despite effective antiviral medications to treat CMV, invasive CMV disease contributes to lung allograft dysfunction and worse survival. Efforts to prevent CMV have led to the use of valganciclovir prophylaxis for increasingly longer periods after transplant. A pivotal concern with long-term antiviral prophylaxis is that it may prevent or delay the development of CMV specific immunity and increase the subsequent risk of late onset disease. To address this issue, we conducted a pilot study to determine if CMV specific immunity was detectable in lung transplant recipients at risk for CMV while on antiviral prophylaxis. Utilizing polychromatic flow cytometry panels, CMV specific immunity was determined by peripheral blood CD4 and CD8 T cell expression of cytokines in response to the HLA restricted CMV peptides pp65 and IE-1. We determined CMV seropositive lung transplant recipients on valganciclovir for a median of 6 months from transplant have a detectable polyfunctional CMV specific T cell response which is comparable to seropositive recipients not on antiviral medications and to healthy seropositive nontransplant controls. Thus, valganciclovir prophylaxis does not appear to impair the development of CMV specific immunity in lung transplantation.
Cytomegalovirus; valganciclovir; lung transplantation
Although age-related macular degeneration (AMD) is not a classic inflammatory disease like uveitis, inflammation has been found to have an important role in disease pathogenesis and progression. Innate immunity and autoimmune components, such as complement factors, chemokines, cytokines, macrophages, and ocular microglia, are believed to be heavily involved in AMD development. Targeting these specific inflammatory molecules has recently been explored in an attempt to better understand and treat AMD. Although antivascular endothelial growth factor therapy is the first line of defence against neovascular AMD, anti-inflammatory agents such as corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), immunosuppressive agents (eg, methotrexate and rapamycin), and biologics (eg, infliximab, daclizumab, and complement inhibitors) may provide an adjunct or alternative mechanism to suppress the inflammatory processes driving AMD progression. Further investigation is required to evaluate the long-term safety and efficacy of these drugs for both neovascular and non-neovascular AMD.
age-related macular degeneration; inflammation; corticosteroid; nonsteoridal anti-inflammatory drug; immunosuppressant; biologics
GRAIL (gene related to anergy in lymphocytes, also known as RNF128), an ubiquitin-protein ligase (E3), utilizes a unique single transmembrane protein with a split function motif, and is an important gatekeeper of T cell unresponsiveness. While it may play a role in other CD4 T cell functions including activation, survival, and differentiation, GRAIL is most well characterized as a negative regulator of TCR responsiveness and cytokine production. Here, we review the recent literature on this remarkable E3 in the regulation of human and mouse CD4 T cell unresponsiveness.
GRAIL; E3; ubiquitin-protein ligase; anergy; T cell unresponsiveness; ubiquitination; de-ubiquitinating enzymes (DUBs)
Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 – 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.
Principal medium spiny projection neurons (MSNs) of the striatum have long been thought to be homogeneous in their somatodendritic morphology and physiology. Recent work using transgenic mice in which the two major classes of MSN are labeled has challenged this assumption. To explore the basis for this difference, D1 and D2 receptor expressing MSNs in brain slices from adult transgenic mice were characterized electrophysiologically and anatomically. These studies revealed that D1 MSNs were less excitable than D2 MSNs over a broad range of developmental time points. Although M1 muscarinic receptor signaling was a factor, it was not sufficient to explain the dichotomy between D1 and D2 MSNs. Reconstructions of biocytin-filled MSNs revealed that the physiological divergence was paralleled by a divergence in total dendritic area. Experimentally grounded simulations suggested that the dichotomy in MSN dendritic area was a major contributor to the dichotomy in electrophysiological properties. Thus, rather than being an intrinsically homogenous population, striatal MSNs have dichotomous somatodendritic properties that mirror differences in their network connections and biochemistry.
medium spiny neuron; striatum; anatomical reconstruction; basal ganglia; excitability; whole-cell patch clamp recording