The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-κB plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-κB activity can be used.
In this study, methods are described for the analysis of NF-κB-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.
25 Related JoVE Articles!
Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation
Institutions: University of California, Irvine, University of California, Irvine.
Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4
. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6
. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9
. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10
, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.
Neuroscience, Issue 56, glutamate uncaging, whole cell recording, GFP, transgenic, interneurons
Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury
Institutions: University of Texas Medical Branch.
Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2
Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al
has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4
We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 °C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.
Neuroscience, Issue 74, Neurobiology, Medicine, Biomedical Engineering, Anatomy, Physiology, Cellular Biology, Molecular Biology, Genetics, Surgery, Anesthesiology, Micromanipulation, Microdissection, Laser Capture Microdissection, LCM, Investigative Techniques, traumatic brain injury, TBI, hippocampus, Fluoro-Jade, gene expression analysis, gene expression, neurons, animal model
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm
Institutions: Trier University, Trier University.
Selection tasks in which simple stimuli (e.g.
letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.
Behavior, Issue 87, stimulus-response binding, distractor-response binding, response retrieval, distractor inhibition, event file, action control, selection task
A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat
Institutions: Canadian Centre for Behavioural Neuroscience, University of Lethbridge.
Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O’Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and Séguinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."
Although there is considerable evidence that the hippocampus is involved in piloting (O’Keefe and Nadel, 1978; O’Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O’Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria–fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.
Neuroscience, Issue 26, Dead reckoning, fimbria-fornix, hippocampus, odor tracking, path integration, spatial learning, spatial navigation, piloting, rat, Canadian Centre for Behavioural Neuroscience
Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice
Institutions: Fujita Health University, Core Research for Evolutionary Science and Technology (CREST), National Institutes of Natural Sciences.
The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.
Behavior, Issue 85, Fear, Learning, Memory, ImageFZ program, Mouse, contextual fear, cued fear
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory
Institutions: Boston College, Wofford College, University of Notre Dame.
Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these two factors may interact to influence consolidation. Here we present a protocol to examine the interactive influence of cortisol and sleep on memory consolidation, by combining three methods: eye tracking, salivary cortisol analysis, and behavioral memory testing across sleep and wake delays. To assess resting cortisol levels, participants gave a saliva sample before viewing negative and neutral objects within scenes. To measure overt attention, participants’ eye gaze was tracked during encoding. To manipulate whether sleep occurred during the consolidation window, participants either encoded scenes in the evening, slept overnight, and took a recognition test the next morning, or encoded scenes in the morning and remained awake during a comparably long retention interval. Additional control groups were tested after a 20 min delay in the morning or evening, to control for time-of-day effects. Together, results showed that there is a direct relation between resting cortisol at encoding and subsequent memory, only following a period of sleep. Through eye tracking, it was further determined that for negative stimuli, this beneficial effect of cortisol on subsequent memory may be due to cortisol strengthening the relation between where participants look during encoding and what they are later able to remember. Overall, results obtained by a combination of these methods uncovered an interactive effect of sleep and cortisol on memory consolidation.
Behavior, Issue 88, attention, consolidation, cortisol, emotion, encoding, glucocorticoids, memory, sleep, stress
Trace Fear Conditioning in Mice
Institutions: Baylor University, Baylor University.
In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.
Behavior, Issue 85, fear conditioning, learning, trace conditioning, memory, conditioned and unconditioned stimulus, neutral stimulus, amygdala-dependent learning
Using Affordable LED Arrays for Photo-Stimulation of Neurons
Institutions: Institut Pasteur and Centre National de la Recherche Scientifique (CNRS).
Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2
. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4
. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.
Neuroscience, Issue 57, Adult neurogenesis, Channelrhodopsin, Neural stem cells, Plasticity, Synapses, Electrophysiology
Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
Institutions: Columbia University , Columbia University , Columbia University , Harvard Medical School.
The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2
. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4,
which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6
. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7
, interaction with other purified cerebellar cells8,9
or cell death7
Neuroscience, Issue 23, cerebellum, cerebellar granule neuron progenitors, cerebellar granule neurons, external granule layer, culture, cell purification
Extinction Training During the Reconsolidation Window Prevents Recovery of Fear
Institutions: Mt. Sinai School of Medicine, New York University , New York University .
Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2
. Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3
. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4
. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9
. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16
, although this may vary depending on the strength and age of the memory15
. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11
. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14
. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e.
, extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11
. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16
, or declarative18
memories leads to interference with the original memory trace14
. We outline below a novel protocol used to block fear recovery in humans.
Neuroscience, Issue 66, Medicine, Psychology, Physiology, Fear conditioning, extinction, reconsolidation, emotional memory, spontaneous recovery, skin conductance response
GABA-activated Single-channel and Tonic Currents in Rat Brain Slices
Institutions: Uppsala University, Sweden.
channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, respectively 4,5,6,7
channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ). Two alphas, two betas and one 3rd
subunit form the functional channel 8
. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA
channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10
to study the functional properties of the GABAA
channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10
Neuroscience, Issue 53, brain, patch-clamp, ion channels, tonic current, slices, whole-cell current, single-channel current, GABAA, GABA
Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury
Institutions: Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Perelman School of Medicine at the University of Pennsylvania.
Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1
. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3
. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6
Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo
physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo
recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1
). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits.
The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.
Neuroscience, Issue 69, Medicine, Anatomy, Physiology, hippocampus, traumatic brain injury, electrophysiology, patch clamp, voltage sensitive dye, extracellular recording, high-performance liquid chromatography, gas chromatography-mass spectrometry
Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons
Institutions: Charité Universitätmedizin.
GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.
Neuroscience, Issue 91, electrophysiology, acute slice, whole-cell patch-clamp recording, neuronal morphology, immunocytochemistry, parvalbumin, hippocampus, inhibition, GABAergic interneurons, synaptic transmission, IPSC, GABA-B receptor
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration
Institutions: University of Bonn, Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE).
One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g.
the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.
Neuroscience, Issue 77, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Biomedical Engineering, Biophysics, Biochemistry, biology (general), animal biology, Nervous System, Life Sciences (General), Neurosciences, brain slices, dendrites, inhibition, excitation, glutamate, GABA, micro-iontophoresis, iontophoresis, neurons, patch clamp, whole cell recordings
Paired Whole Cell Recordings in Organotypic Hippocampal Slices
Institutions: University of Auckland, Stanford University.
Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.
Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Institutions: California Institute of Technology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, University Of Washington Medical Center, Johns Hopkins University School of Medicine.
The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for C
omputer tomography-guided f
radiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.
Neuroscience, Issue 81, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
Gene-gun Transfection of Hippocampal Neurons
Institutions: Brown University.
Neuroscience, Issue 1, brain, hippocampus, neuron, transfection, gene-gun
Studying the Integration of Adult-born Neurons
Institutions: State University of New York at Stony Brook.
Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1
). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2
). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo
. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.
Neuroscience, Issue 49, dentate gyrus, neurogenesis, newborn dentate granule cells, functional integration
Dissection of Hippocampal Dentate Gyrus from Adult Mouse
Institutions: Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST), Fujita Health University, Graduate School of Medicine, Kyoto University, Graduate School of Medicine, Kyoto University, National Institutes of Natural Sciences.
The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.
Neuroscience, Issue 33, Dentate gyrus, Hippocampus, dissection, neurogenesis, alpha-CaMKII, mouse