ABSTRACT
Both in vivo neuropharmacology and optogenetic stimulation can be used to decode neural circuitry, and can provide therapeutic strategies for brain disorders. However, current neuronal interfaces hinder long-term studies in awake and freely behaving animals, as they are limited in their ability to provide simultaneous and prolonged delivery of multiple drugs, are often bulky and lack multifunctionality, and employ custom control systems with insufficiently versatile selectivity for output mode, animal selection and target brain circuits. Here, we describe smartphone-controlled, minimally invasive, soft optofluidic probes with replaceable plug-like drug cartridges for chronic in vivo pharmacology and optogenetics with selective manipulation of brain circuits. We demonstrate the use of the probes for the control of the locomotor activity of mice for over four weeks via programmable wireless drug delivery and photostimulation. Owing to their ability to deliver both drugs and photopharmacology into the brain repeatedly over long time periods, the probes may contribute to uncovering the basis of neuropsychiatric diseases.
Subject(s)
Neuropharmacology/methods , Optogenetics/instrumentation , Wireless Technology/instrumentation , Animals , Brain/physiology , Brain Diseases , Deep Brain Stimulation/methods , Drug Delivery Systems/instrumentation , Drug Delivery Systems/methods , Implants, Experimental , Lab-On-A-Chip Devices , Locomotion , Male , Mice , Mice, Inbred C57BL , Models, Animal , Neuropharmacology/instrumentation , Optogenetics/methodsABSTRACT
Convection-enhanced delivery (CED) is a promising technique for the administration of therapeutic agents such as cytotoxics, neurotrophins and enzymes to the brain. In this study we describe the development of an implantable catheter system that is compatible with long-term intermittent CED. Catheters made from fused silica, PEEK or carbothane, and of various internal and external diameters were implanted in the striatum of rats and assessed for patency at 21 or 28 days. A high-rate of catheter blockage was observed with all fused silica and PEEK catheters. Carbothane catheters with an outer diameter of 0.6mm and an inner diameter of 0.35 mm had significantly lower rates of blockage (P≤0.01). Carbothane catheters were then implanted into 4 Large White/Landrace pigs and 4 NIH miniature pigs and infusions undertaken at monthly intervals to evaluate catheter patency and infusate distribution. Catheter patency was demonstrated for a maximum period of 163 days in one animal. Widespread and reproducible intraputamenal CED could be achieved with intermittent drug delivery at flow-rates as high as 5 µl/min. Problems were encountered using the pig model due to catheter distortion from rapid animal growth. In conclusion, it is possible to achieve intermittent high-flow CED with a chronic implanted carbothane catheter with a low rate of catheter blockage.
Subject(s)
Catheters, Indwelling/standards , Infusion Pumps, Implantable/standards , Neuropharmacology/instrumentation , Neurosurgical Procedures/instrumentation , Prosthesis Implantation/methods , Animals , Catheters, Indwelling/adverse effects , Infusion Pumps, Implantable/adverse effects , Male , Models, Animal , Neuropharmacology/methods , Neurosurgical Procedures/adverse effects , Neurosurgical Procedures/methods , Rats , Rats, Wistar , Sus scrofaABSTRACT
Microinjections are a major tool in modern neuroscience. Microinjection techniques in conscious animals typically involve four steps: (1) animal adapts to experimental setup; (2) injection system is filled and the microinjector is carefully inserted; (3) a drug solution is injected; (4) 1-2 min later the microinjector is carefully removed. Steps 2 and 4 are difficult to perform in rodents without disturbing the animal. This disruption can cause stress and accompanying tachycardia and hyperthermia - unwanted artifacts in physiological research. To reduce these effects, we altered the traditional approach. Our procedure of microinjection consisted of the following steps: (1) we filled the injection setup and fixed the microinjector in its guide cannula; (2) allowed an animal to adapt to the setup; (3) performed an experiment including microinjection(s); (4) removed the microinjector after the experiment was complete. The key change we incorporated was a 1m long piece of tubing with a small internal diameter; it allowed us to inject nanoliter volumes through the injector which had been placed into the guide cannula in advance. This way we avoided the usual manipulations related to microinjection, and minimized extraneous disturbances to the rat. In this report we describe the details of this technique in conscious rats and provide examples of the effects and the reproducibility of a 100 nL drug injection on cardiovascular function.
Subject(s)
Catheterization/methods , Microinjections/methods , Neuropharmacology/methods , Stress, Psychological/prevention & control , Animals , Catheterization/instrumentation , Catheters/standards , Catheters/trends , Male , Microinjections/instrumentation , Neuropharmacology/instrumentation , Neurosurgical Procedures/instrumentation , Neurosurgical Procedures/methods , Rats , Rats, Sprague-Dawley , Stress, Psychological/etiology , Stress, Psychological/physiopathology , Tachycardia/etiology , Tachycardia/physiopathology , Tachycardia/prevention & control , Wakefulness/physiologyABSTRACT
The use of functional MRI (fMRI) in combination with pharmacological challenges has increased exponentially in recent years, motivated by the idea not only to elucidate the neurochemical foundations of human emotional and cognitive faculties, but also to optimize human brain function in healthy individuals and identify novel drug targets, with the ultimate goal to design more specific pharmacological therapies for the various disorders of human emotion and cognition. In particular, emotional responding of the amygdala has become a central interest, and pharmacological fMRI has been used to specifically probe, and modulate, amygdala activation in response to facial expressions of emotion and emotionally laden scenes. This article reviews recent fMRI experiments manipulating the amygdala's physiological response to such stimuli by pharmacological means and lays a particular focus on monoaminergic, glutamatergic, GABAergic, and hormonal/peptidergic challenges.
Subject(s)
Amygdala/physiology , Brain Mapping , Emotions/physiology , Magnetic Resonance Imaging , Amygdala/drug effects , Emotions/drug effects , Humans , Neuropharmacology/instrumentation , Neuropharmacology/methodsABSTRACT
The forced swim test (FST) is a commonly used procedure of preclinical screening of drugs for the antidepressant activity. It has high predictive validity for a large group of antidepressant drugs blocking serotonin and noradrenaline reuptakes and improvement of immobility time evaluation in the FST is an important problem of preclinical psychopharmacology. Here a new automated version of the FST was developed. This version includes 4 inventions: (1) transmitted lighting instead of reflected lighting, (2) mouse silhouette tracking, (3) automated choice of immobility threshold and (4) the permutation test of drug's effect. Experiment was carried out on adult males of C57BL/6J and BALB/cJ mouse strains. The mice were treated with tricyclic antidepressant imipramine (15 and 30 mg/kg, i.p.). Mouse was placed in water tank, its movements were recorded by a rater and the silhouette alterations were automatically tracked. The sequence of silhouette alterations was scanned for immobility bouts with a threshold algorithm. Threshold was gradually altered and the value which maximized the difference between control and treated groups was chosen. The immobility values obtained with the procedure were compared with the permutation test. The data obtained with this procedure did not differ from those obtained by the rater. Imipramine dose dependently attenuated immobility time in C57BL/6 mice without any effect on BALB/c mice. The new procedure has been implemented in the EthoStudio software. It provides an objective automated evaluation of immobility time in the FST.
Subject(s)
Antidepressive Agents/pharmacology , Neuropharmacology/methods , Pattern Recognition, Automated/methods , Psychopharmacology/methods , Stress, Psychological/drug therapy , Stress, Psychological/physiopathology , Animals , Antidepressive Agents/administration & dosage , Disease Models, Animal , Drug Evaluation, Preclinical/instrumentation , Drug Evaluation, Preclinical/methods , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neuropharmacology/instrumentation , Psychopharmacology/instrumentation , SwimmingABSTRACT
Tetrodotoxin is one of the most potent and oldest known neurotoxins. It acts by blocking the voltage-gated sodium channels in nerve cell membranes, leading to a transient silencing of neural activity. TTX is among the most widely used pharmacological agents for the temporary and selective blocking of neural structures. As such, an exact knowledge of the spatial and temporal diffusion gradient of TTX is important when planning pharmacological interventions. Here we report a method for the direct assessment of spatio-temporal TTX diffusion gradients using immunohistochemistry. TTX injections were performed in vivo via chronically implanted guiding cannulae, placed bilaterally in the dorsal entopallium of pigeons. To determine the temporal spread, animals were perfused at different time points after TTX injections. For visualization of the TTX affected area an immunohistochemical protocol was developed. The extension of staining was assessed 1h after injection when TTX was diffused over 3mm in all directions. TTX immunolabeling could be detected for up to 32 h; after 48 h no staining was found. Our findings provide a better understanding of the temporal decay and spread of intracranial TTX injections, thereby allowing a more reliable estimation of size and duration of TTX-effects.
Subject(s)
Immunohistochemistry/methods , Microinjections/methods , Neuropharmacology/methods , Neurophysiology/methods , Sodium Channel Blockers/pharmacokinetics , Tetrodotoxin/pharmacokinetics , Animals , Antibody Specificity/immunology , Brain/drug effects , Brain/physiology , Columbidae , Diffusion , Microinjections/instrumentation , Neuropharmacology/instrumentation , Neurophysiology/instrumentation , Syringes , Time FactorsABSTRACT
Neuroscience research projects often use intracranial (IC) microinfusions to target drug delivery to specific brain areas during behavioral testing. These experiments require accurate and precisely-timed delivery of small volumes. We present here a stepper motor-powered micropump assembly for such delivery. This system is hands-free, does not use a potentially leaky fluid swivel or use long delivery tubes that are subject to peristaltic forces during animal movements, and has been applied in combination with other paradigms. This micropump system reliably delivers a wide range of fluid volumes (e.g. 50 nL to 1 microL in tissue or greater for intraventricular injections) bilaterally from two independent, commercially available microsyringes through standard surgically implanted guide cannulae. It is easy to build and disassemble for cleaning or changing microsyringes. This system can also be used for a variety of purposes, e.g. intracranial self-administration, place conditioning, and many more, with the advantage that it provides a way to gather important data in the seconds and minutes following IC microinfusion without disruption of the animal's behavior by handling.
Subject(s)
Drug Delivery Systems/instrumentation , Microinjections/instrumentation , Neuropharmacology/instrumentation , Neuropsychology/instrumentation , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Brain/drug effects , Brain/physiology , Catheterization/instrumentation , Catheterization/methods , Conditioning, Psychological/drug effects , Conditioning, Psychological/physiology , Craniotomy/methods , Drug Delivery Systems/methods , Microinjections/methods , Microsurgery/instrumentation , Microsurgery/methods , Neuropharmacology/methods , Neuropsychology/methods , Neurosurgical Procedures/instrumentation , Neurosurgical Procedures/methods , Postoperative Complications/etiology , Postoperative Complications/physiopathology , Postoperative Complications/prevention & control , Self Administration/instrumentation , Self Administration/methods , Syringes/trendsABSTRACT
The plus-maze discriminative avoidance paradigm has been used to study the relationship between aversive memory and anxiety. The present study aims to verify if the elevated plus-maze can provide information about appetitive memory and anxiety-like behavior, through a task motivated by food reward. Animals were allowed to explore an elevated plus-maze and received reinforcement in one of the enclosed arms. In a test session performed 24h later, in the absence of reward, rats showed preference for the previously rewarded enclosed arm over the neutral enclosed arm. The administration of diazepam and pentylenetetrazole before training induced, respectively, anxiolytic and anxiogenic effects (as evaluated by open-arm exploration). Both drugs induced amnestic effects, i.e., lack of preference for the rewarded arm in the test session. The results suggest that appetitive memory can be influenced by anxiety levels as well. The plus-maze appetitive discrimination task seems to be a useful model to investigate the relationship between memory and anxiety.
Subject(s)
Anxiety/psychology , Appetitive Behavior/physiology , Discrimination Learning/physiology , Maze Learning/physiology , Neuropsychology/methods , Reward , Amnesia/chemically induced , Animals , Anti-Anxiety Agents/pharmacology , Anxiety/physiopathology , Appetitive Behavior/drug effects , Diazepam/pharmacology , Discrimination Learning/drug effects , Female , GABA Antagonists/pharmacology , Housing, Animal , Maze Learning/drug effects , Models, Animal , Neuropharmacology/instrumentation , Neuropharmacology/methods , Neuropsychology/instrumentation , Pentylenetetrazole/pharmacology , Rats , Rats, WistarABSTRACT
The identification of animal disease-like models for cognitive symptoms in schizophrenia is of central importance to the successful development of pharmacological therapies for psychosis resulting in a functional outcome in patients. Executive function is one of the most severely affected cognitive domains in schizophrenia that remains inadequately treated by existing therapies. The rat attentional set-shifting (or intra-dimensional-extra-dimensional (ID/ED)) task has been developed to test executive function in rodents and successful translation of pre-clinical data into the clinical setting now depends on the identification of a predictive animal disease-like model. The present study investigates whether a continuous 14-day mini-pump infusion of the non-competitive NMDA receptor antagonist phencyclidine (PCP) leads to a deficit in the ID/ED task, and subsequently evaluates the effect of modafinil in this model. Lister hooded rats were implanted subcutaneously with osmotic mini-pumps containing saline or PCP (15 mg/kg/day) for 14 days followed by a 7-day drug-free recovery phase. Rats were then tested in the ID/ED task following an acute injection of either vehicle or modafinil. PCP-treated animals displayed a selective deficit at the ED shift stage resembling that observed in schizophrenic patients. This deficit was reversed by an acute injection of modafinil. The PCP-induced impairment and its reinstatement by modafinil are quantitatively and qualitatively similar to that described earlier by our group following sub-chronic intraperitoneal PCP administration, indicative that sub-chronic PCP infusion via osmotic mini-pumps may represent an attractive alternative to the systemic administration protocols generally employed to date.
Subject(s)
Cognition Disorders/chemically induced , Infusion Pumps, Implantable/trends , Neuropharmacology/instrumentation , Phencyclidine/administration & dosage , Schizophrenia/chemically induced , Animals , Antipsychotic Agents/pharmacology , Attention/drug effects , Attention/physiology , Behavior, Animal/drug effects , Behavior, Animal/physiology , Behavioral Sciences/instrumentation , Behavioral Sciences/methods , Benzhydryl Compounds/pharmacology , Brain/drug effects , Brain/physiopathology , Central Nervous System Stimulants/pharmacology , Cognition Disorders/physiopathology , Disease Models, Animal , Drug Administration Schedule , Hallucinogens/administration & dosage , Hallucinogens/toxicity , Male , Modafinil , Neuropharmacology/methods , Neuropsychological Tests , Neurosciences/instrumentation , Neurosciences/methods , Phencyclidine/toxicity , Rats , Schizophrenia/physiopathology , Schizophrenic PsychologyABSTRACT
The elevated plus-maze is a device widely used to assess rodent anxiety under the effect of several treatments, including pharmacological agents. The animal is placed at the center of the apparatus, which consists of two open arms and two arms enclosed by walls, and the number of entries and duration of stay in each arm are measured for a 5-min exposure period. The effect of an anxiolytic drug is to increase the percentage of time spent and number of entries into the open arms. In this work, we propose a new measure of anxiety levels in the rat submitted to the elevated plus-maze. We represented the spatial structure of the elevated plus-maze in terms of a directed graph and studied the statistics of the rat's transitions between the nodes of the graph. By counting the number of times each transition is made and ordering them in descending frequency we represented the rat's behavior in a rank-frequency plot. Our results suggest that the curves obtained under different pharmacological conditions can be well fitted by a power law with an exponent sensitive to both the drug type and the dose used.
Subject(s)
Anti-Anxiety Agents/pharmacology , Behavior, Animal/physiology , Ethology/methods , Maze Learning/physiology , Neuropharmacology/methods , Neuropsychology/methods , Animals , Anxiety/drug therapy , Anxiety/psychology , Behavior, Animal/drug effects , Data Interpretation, Statistical , Ethology/instrumentation , Exploratory Behavior/drug effects , Exploratory Behavior/physiology , Housing, Animal , Mathematics , Maze Learning/drug effects , Models, Animal , Models, Theoretical , Neuropharmacology/instrumentation , Neuropsychology/instrumentation , RatsABSTRACT
Analysis of the interplay between cell proliferation and death has been greatly advantaged by the development of CNS slice preparations. In slices, interactions between neurons and neurons and the glial cells are fundamentally preserved in a fashion close to the in vivo situation. In parallel, these preparations offer the possibility of an easy experimental manipulation. Two main types of slices are currently in use: the acute slices, which are short living preparations where the major functions of the intact brain (including neurogenesis) are maintained, and the organotypic cultures, where the maturation and plasticity of neuronal circuitries in relation to naturally occurring neuronal death and/or experimental insults can be followed over several weeks in vitro. We will discuss here the main advantages/disadvantages linked to the use of CNS slices for histological analysis of neuronal proliferation and death, as well as the main findings obtained in the most popular types of preparations, i.e. the cortical, hippocampal, cerebellar and retinal slices.
Subject(s)
Cell Proliferation , Central Nervous System/cytology , Central Nervous System/physiology , Neurosciences/methods , Animals , Cell Death/physiology , Microscopy/instrumentation , Microscopy/methods , Neurons/cytology , Neurons/physiology , Neuropharmacology/instrumentation , Neuropharmacology/methods , Neurosciences/instrumentation , Organ Culture Techniques/methods , Staining and Labeling/instrumentation , Staining and Labeling/methodsABSTRACT
We developed an inexpensive computer vision-based method utilizing an algorithm which differentiates drug-induced behavioral alterations. The mice were observed in an open-field arena and their activity was recorded for 100 min. For each animal the first 50 min of observation were regarded as the drug-free period. Each animal was exposed to only one drug and they were injected (i.p.) with either amphetamine or cocaine as the stimulant drugs or morphine or diazepam as the inhibitory agents. The software divided the arena into virtual grids and calculated the number of visits (sojourn counts) to the grids and instantaneous speeds within these grids by analyzing video data. These spatial distributions of sojourn counts and instantaneous speeds were used to construct feature vectors which were fed to the classifier algorithms for the final step of matching the animals and the drugs. The software decided which of the animals were drug-treated at a rate of 96%. The algorithm achieved 92% accuracy in sorting the data according to the increased or decreased activity and then determined which drug was delivered. The method differentiated the type of psychostimulant or inhibitory drugs with a success ratio of 70% and 80%, respectively. This method provides a new way to automatically evaluate and classify drug-induced behaviors in mice.
Subject(s)
Algorithms , Behavior, Animal/drug effects , Neuropharmacology/methods , Pattern Recognition, Automated/methods , Psychotropic Drugs/pharmacology , Video Recording/methods , Amphetamine/pharmacology , Animals , Behavior, Animal/physiology , Central Nervous System Stimulants/pharmacology , Cocaine/pharmacology , Diazepam/pharmacology , Exploratory Behavior/drug effects , Exploratory Behavior/physiology , Hypnotics and Sedatives/pharmacology , Male , Mice , Motor Activity/drug effects , Motor Activity/physiology , Neuropharmacology/instrumentation , SoftwareABSTRACT
INTRODUCTION: Electroencephalography (EEG) recording and drug administration is commonly used for neurological experiments in rats, but is typically cumbersome due the use of multiple lines. We have developed a unique system, which allows long-term simultaneous video-electroencephalography recording, drug infusion and blood sampling in rats. METHODS: The vEEG/drug infusion system was designed and tested on two contrasting animal models of epilepsy. Animals were implanted with EEG-electrodes and a jugular vein cannula fixed in a head cap, avoiding an additional cable for tethering. In an acute infusion study (n=16), repeated blood samples were taken after i.v. bolus injection of valproate. In a subset of these rats (n=10), paired blood samples were removed from the jugular vein and the heart after valproate administration. In a chronic infusion study (n=38), heparinised (4IU/h) saline or valproate (42mg/kg/h) was infused continuously for up to 17 days. RESULTS: In the acute study, repeated blood samples showed a decrease in plasma valproate levels over time following bolus injection. In the chronic study, high quality continuous EEG was achieved and 79% of animals were successfully infused throughout the planned infusion period (13-17 days), with 66% of projected blood samples able to be taken during the infusion. There was a high correlation between the jugular vein and cardiac plasma levels of valproate (Spearman test, r=0.69; p<0.05). CONCLUSION: This system is ideal for pharmacokinetic/dynamic studies and long-term drug infusion where simultaneous EEG and/or frequent blood sampling are desired.
Subject(s)
Blood Specimen Collection/methods , Drug Delivery Systems/methods , Electroencephalography/methods , Neuropharmacology/instrumentation , Neuropharmacology/methods , Video Recording/methods , Animals , Anticonvulsants/blood , Anticonvulsants/pharmacokinetics , Catheterization , Disease Models, Animal , Drug Delivery Systems/instrumentation , Epilepsy/drug therapy , Epilepsy/physiopathology , Female , Infusions, Intravenous/methods , Jugular Veins/anatomy & histology , Jugular Veins/physiology , Jugular Veins/surgery , Male , Rats , Rats, Wistar , Valproic Acid/blood , Valproic Acid/pharmacokineticsABSTRACT
We sought an optimal method for targeted delivery into dorsal root ganglia (DRGs) for experimental studies, in terms of precision of delivery and avoidance of behavioral disturbances. We examined three approaches for injection into rat DRGs: percutaneous injection without surgical exposure, injection after deep exposure, and injection following deep exposure and partial laminectomy. Coomassie blue and Fast Blue were injected into DRGs for validation. At necropsy, the spread of Coomassie blue and Fast Blue was investigated under stereomicroscope and fluorescent microscope, respectively. We found that percutaneous approach did not provide any successful DRG injections. Deep exposure prior to intraganglionic injection provided variable results, but intraganglionic injection after deep exposure plus partial laminectomy was successful in 100% of attempts. Our subsequent skeletal analysis showed that the anatomical location of DRG is not compatible with successful DRG injection without surgical exposure. Neither of the methods using surgical exposure caused behavioral disturbances. Based on these results we conclude that partial laminectomy offers the most precise method of injecting DRG and does not produce behavioral evidence of nerve damage. Intraganglionic injection after deep exposure alone is less predictable, while percutaneous approaches only allow injection in the peripheral nerve.
Subject(s)
Drug Delivery Systems/methods , Ganglia, Spinal/drug effects , Laminectomy/methods , Microdissection/methods , Microinjections/methods , Neurosurgical Procedures/methods , Amidines/pharmacokinetics , Animals , Coloring Agents/pharmacokinetics , Drug Delivery Systems/instrumentation , Fluorescent Dyes/pharmacokinetics , Ganglia, Spinal/cytology , Ganglia, Spinal/surgery , Microinjections/instrumentation , Microscopy, Fluorescence/methods , Neuropharmacology/instrumentation , Neuropharmacology/methods , Rats , Rats, Sprague-Dawley , Rosaniline Dyes/pharmacokinetics , Sensory Receptor Cells/cytology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/physiology , Spine/anatomy & histology , Spine/surgery , Staining and Labeling/methodsABSTRACT
The administration of 6-hydroxydopamine (6-OHDA) into the nigrostriatal pathway is a rat model of Parkinson's disease (PD). The footfault test is a behavioural task in which rodents have their motor functions assessed. Here, we observed that unilaterally 6-OHDA-lesioned animals show a context-induced ipsilateral rotational behaviour when placed on the footfault apparatus for 3 min and this may be used as index to detect lesioned animals. Our results showed a sensitivity and specificity of 100% for lesions higher than 94% and 64%, respectively (ROC curve: AUC=0.988). A binary logistic regression model showed an expB=1.116 (95% CI, 1.007-1.236) and C=-9.081+/-4.554 (p=0.046) using the nigral tyrosine hidroxylase immunocontent as standard (each unit represents a 10%-lesion extension). Additionally, the footfault test was more sensitive than apomorphine challenging at 1mg/kg when these tests were carried out days apart and it was less sensitive than methylphenidate at 40 mg/kg (sign test, p<0.05). Therefore, the footfault test may be very useful in the PD animal model for screening animals since it is fast and simple and it does not require a drug to induce rotational activity.
Subject(s)
Behavioral Sciences/methods , Neuropharmacology/methods , Oxidopamine/toxicity , Parkinsonian Disorders/diagnosis , Parkinsonian Disorders/physiopathology , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Behavioral Sciences/instrumentation , Disease Models, Animal , Dopamine/biosynthesis , Dopamine Agonists/pharmacology , Drug Evaluation, Preclinical/instrumentation , Drug Evaluation, Preclinical/methods , Gait/drug effects , Gait/physiology , Gait Disorders, Neurologic/chemically induced , Gait Disorders, Neurologic/diagnosis , Gait Disorders, Neurologic/physiopathology , Male , Nerve Degeneration/chemically induced , Nerve Degeneration/enzymology , Nerve Degeneration/pathology , Neurons/drug effects , Neurons/enzymology , Neurons/pathology , Neuropharmacology/instrumentation , Parkinsonian Disorders/chemically induced , Rats , Rats, Wistar , Substantia Nigra/drug effects , Substantia Nigra/enzymology , Substantia Nigra/pathology , Sympatholytics/toxicity , Tyrosine 3-Monooxygenase/drug effects , Tyrosine 3-Monooxygenase/metabolismABSTRACT
OBJECTIVE: To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a (hASIC1a) current in HEK293 cells with a modified perfusion methods. METHODS: With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASIC1a currents evoked by low pH external solution. RESULTS: Using cell floating method, the amplitude of hASIC1a currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is (21+/-5) ms and (270+/-25) ms, respectively. Inactivation time constants are (496+/-23) ms and (2284+/-120) ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC1a [IC50 is (3.4+/-1.1) micromol/L and (2.4+/- 0.9) micromol/L, respectively]. Both recording methods have similar pH activation EC50 (6.6+/-0.6, 6.6+/-0.7, respectively). CONCLUSION: ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC1a current was re-confirmed and the biophysical and pharmacological properties of hASIC1a channel in HEK293 cells were precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.
Subject(s)
Cell Membrane/drug effects , Cell Membrane/metabolism , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/metabolism , Sodium Channels/drug effects , Sodium Channels/metabolism , Acid Sensing Ion Channels , Amiloride/pharmacology , Biophysics/instrumentation , Biophysics/methods , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Line , Cell Membrane/chemistry , Culture Media/chemistry , Culture Media/pharmacology , Extracellular Fluid/chemistry , Extracellular Fluid/metabolism , Humans , Hydrogen-Ion Concentration/drug effects , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nerve Tissue Proteins/chemistry , Neuropharmacology/instrumentation , Neuropharmacology/methods , Patch-Clamp Techniques/instrumentation , Patch-Clamp Techniques/methods , Perfusion/instrumentation , Perfusion/methods , Sodium Channel Blockers/pharmacology , Sodium Channels/chemistry , Time FactorsABSTRACT
We describe a device for assessing the effects of diffusible molecules on electrophysiological recordings from multiple neurons. This device allows for the infusion of reagents through a cannula located among an array of micro-electrodes. The device can easily be customized to target specific neural structures. It is designed to be chronically implanted so that isolated neural units and local field potentials are recorded over the course of several weeks or months. Multivariate statistical and spectral analysis of electrophysiological signals acquired using this system could quantitatively identify electrical "signatures" of therapeutically useful drugs.
Subject(s)
Catheterization/instrumentation , Electrophysiology/instrumentation , Infusion Pumps, Implantable , Neuropharmacology/instrumentation , Signal Processing, Computer-Assisted , Action Potentials/drug effects , Action Potentials/physiology , Animals , Behavior, Animal , Brain/drug effects , Brain/physiology , Catheterization/methods , Drug Evaluation, Preclinical/methods , Electrodes, Implanted , Electrophysiology/methods , Infusion Pumps, Implantable/standards , Movement , Neurons/drug effects , Neurons/physiology , Neuropharmacology/methods , RatsABSTRACT
A beam-walking apparatus has been evaluated for its ability to detect motor impairments in mice acutely treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg, s.c., single or double administration). Mice subjected to MPTP lesioning showed deficits in motor performance on the beam-walking task, for up to 6 days post-MPTP administration, as compared to saline-treated controls. In addition, MPTP-treated mice were detected to have a marked depletion in striatal dopamine levels and a concomitant reduction in substantia nigra (SN) tyrosine hydroxylase (TH) immunoreactivity, at 7 days post-MPTP administration, indicative of dopaminergic neuronal loss. Pre-administration of the potent MAO-B inhibitor R-(-)-deprenyl at 3 or 10 mg/kg, 30 min, s.c, significantly inhibited the MPTP-induced reduction in SN TH-immunoreactivity, striatal dopamine depletions and impairments in mouse motor function. The data described in the present study provides further evidence that functional deficits following an acute MPTP dosing schedule in mice can be quantified and are related to nigro-striatal dopamine function.
Subject(s)
Brain/physiopathology , Dopamine/deficiency , Ethology/instrumentation , Gait Disorders, Neurologic/diagnosis , Neuropharmacology/instrumentation , Parkinsonian Disorders/diagnosis , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/adverse effects , Animals , Brain/metabolism , Corpus Striatum/drug effects , Corpus Striatum/pathology , Corpus Striatum/physiopathology , Dose-Response Relationship, Drug , Ethology/methods , Gait Disorders, Neurologic/physiopathology , Gait Disorders, Neurologic/prevention & control , Male , Mice , Mice, Inbred C57BL , Monoamine Oxidase Inhibitors/therapeutic use , Neural Pathways/drug effects , Neural Pathways/metabolism , Neural Pathways/physiopathology , Neuropharmacology/methods , Parkinsonian Disorders/physiopathology , Parkinsonian Disorders/prevention & control , Predictive Value of Tests , Selegiline/therapeutic use , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Substantia Nigra/physiopathology , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Microelectrode arrays (MEAs) provide a means to investigate the electrophysiological behavior of neuronal systems through the measurements from neuronal culture preparations. Changes in activity patterns of neuronal networks are usually detected by applying neural chemicals. Because of the difficulties of fabricating the arrays, and the delicate and less reliable properties of cortical neurons, MEA-based systems with cortical neuronal networks for neurophamacological applications are technically difficult, therefore restricting their utility. Here, we report a new approach to the development of such MEA-based system with sensitive and durable MEAs conveniently fabricated and the culture conditions optimized. Upon growth differentiation, cortical neurons, cultured directly on MEAs, reach a developmentally stable and reliable activity state. With this system, we monitored the global spontaneous activities of neuronal networks and demonstrated the fine discrimination for specific substances and unique property of cortical neurons, which validated both the applicability and necessity of such system in pharmacological bioassay.
Subject(s)
Action Potentials/physiology , Biological Assay/instrumentation , Biosensing Techniques/instrumentation , Cell Culture Techniques/instrumentation , Microelectrodes , Neurons/physiology , Neurotransmitter Agents/administration & dosage , Action Potentials/drug effects , Animals , Biological Assay/methods , Biosensing Techniques/methods , Cell Culture Techniques/methods , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Cerebral Cortex/physiology , Drug Evaluation, Preclinical/instrumentation , Drug Evaluation, Preclinical/methods , Electric Stimulation/instrumentation , Electric Stimulation/methods , Nerve Net/drug effects , Nerve Net/physiology , Neurons/drug effects , Neuropharmacology/instrumentation , Neuropharmacology/methods , Rats , Rats, Sprague-DawleyABSTRACT
Functional MRI (fMRI) has had a major impact in cognitive neuroscience. fMRI now has a small but growing role in clinical neuroimaging, with initial applications to neurosurgical planning. Current clinical research has emphasized novel concepts for clinicians, such as the role of plasticity in recovery and the maintenance of brain functions in a broad range of diseases. There is a wider potential for clinical fMRI in applications ranging from presymptomatic diagnosis, through drug development and individualization of therapies, to understanding functional brain disorders. Realization of this potential will require changes in the way clinical neuroimaging services are planned and delivered.