Recording EMG Signals on a Computer Sound Card.

For a few dollars and in a few minutes, a simple circuit can be built to permit a cheap external sound card and a laptop computer to be used as a portable data acquisition system for recording EMG. The circuit uses a common audio amplifier integrated circuit to increase the gain of the EMG signals recorded from EMG surface electrodes and to match the impedance of the electrode-skin interface with the input impedance of the sound card. Data can be recorded using open source sound editing software and analyzed offline using simple Python code. It is hoped that such activities provide opportunities to undergraduates to gain confidence as experimentalists and as innovators.

Breadboard Amplifier: Building and Using Simple Electrophysiology Equipment.

Electrophysiology is a valuable skill for the neuroscientist, but the learning curve for students can be steep. Here we describe a very simple electromyography (EMG) amplifier that can be built from scratch by students with no electronics experience in about 30 minutes, making it ideal for incorporating into a laboratory activity. With few parts and no adjustments except the gain, students can begin physiology experiments quickly while having the satisfaction of having built the equipment themselves. Because the output of the circuit goes to a computer sound card, students can listen to electrophysiological activity as they see it on the computer screen, a feature many of our students greatly appreciated. Various applications are discussed, including dual channel recording, using streaming media platforms with remote lab partners and acquiring data in the field on a smart phone. Our students reported that they enjoyed being able to build a working device and using it to record from their own muscles.

Pencil-and-Paper Neural Networks: An Undergraduate Laboratory Exercise in Computational Neuroscience.

Although it has been more than 70 years since McCulloch and Pitts published their seminal work on artificial neural networks, such models remain primarily in the domain of computer science departments in undergraduate education. This is unfortunate, as simple network models offer undergraduate students a much-needed bridge between cellular neurobiology and processes governing thought and behavior. Here, we present a very simple laboratory exercise in which students constructed, trained and tested artificial neural networks by hand on paper. They explored a variety of concepts, including pattern recognition, pattern completion, noise elimination and stimulus ambiguity. Learning gains were evident in changes in the use of language when writing about information processing in the brain.

Compensatory plasticity restores locomotion after chronic removal of descending projections.

Homeostatic plasticity is an important attribute of neurons and their networks, enabling functional recovery after perturbation. Furthermore, the directed nature of this plasticity may hold a key to the restoration of locomotion after spinal cord injury. Here we studied the recovery of crawling in the leech Hirudo verbana after descending cephalic fibers were surgically separated from crawl central pattern generators shown previously to be regulated by dopamine. We observed that immediately after nerve cord transection leeches were unable to crawl, but remarkably, after a day to weeks, animals began to show elements of crawling and intersegmental coordination. Over a similar time course, excessive swimming due to the loss of descending inhibition returned to control levels. Additionally, removal of the brain did not prevent crawl recovery, indicating that connectivity of severed descending neurons was not essential. After crawl recovery, a subset of animals received a second transection immediately below the anterior-most ganglion remaining. Similar to their initial transection, a loss of crawling with subsequent recovery was observed. These data, in recovered individuals, support the idea that compensatory plasticity directly below the site of injury is essential for the initiation and coordination of crawling. We maintain that the leech provides a valuable model to understand the neural mechanisms underlying locomotor recovery after injury because of its experimental accessibility, segmental organization, and dependence on higher-order control involved in the initiation, modulation, and coordination of locomotor behavior.

Mechanisms contributing to the dopamine induction of crawl-like bursting in leech motoneurons.

Dopamine (DA) activates fictive crawling behavior in the medicinal leech. To identify the cellular mechanisms underlying this activation at the level of crawl-specific motoneuronal bursting, we targeted potential cAMP-dependent events that are often activated through DA(1)-like receptor signaling pathways. We found that isolated ganglia produced crawl-like motoneuron bursting after bath application of phosphodiesterase inhibitors (PDIs) that upregulated cAMP. This bursting persisted in salines in which calcium ions were replaced with equimolar cobalt or nickel, but was blocked by riluzole, an inhibitor of a persistent sodium current. PDI-induced bursting contained a number of patterned elements that were statistically similar to those observed during DA-induced fictive crawling, except that one motoneuron (CV) exhibited bursting during the contraction rather than the elongation phase of crawling. Although DA and the PDIs produced similar bursting profiles, intracellular recordings from motoneurons revealed differences in altered membrane properties. For example, DA lowered motoneuron excitability whereas the PDIs increased resting discharge rates. We suggest that PDIs (and DA) activate a sodium-influx-dependent timing mechanism capable of setting the crawl rhythm and that multiple DA receptor subtypes are involved in shaping and modulating the phase relationships and membrane properties of cell-specific members of the crawl network to generate crawling.

Nanowires precisely grown on the ends of microwire electrodes permit the recording of intracellular action potentials within deeper neural structures.

AIMS: Nanoelectrodes are an emerging biomedical technology that can be used to record intracellular membrane potentials from neurons while causing minimal damage during membrane penetration. Current nanoelectrode designs, however, have low aspect ratios or large substrates and thus are not suitable for recording from neurons deep within complex natural structures, such as brain slices. MATERIALS & METHODS: We describe a novel nanoelectrode design that uses nanowires grown on the ends of microwire recording electrodes similar to those frequently used in vivo. RESULTS & DISCUSSION: We demonstrate that these nanowires can record intracellular action potentials in a rat brain slice preparation and in isolated leech ganglia. CONCLUSION: Nanoelectrodes have the potential to revolutionize intracellular recording methods in complex neural tissues, to enable new multielectrode array technologies and, ultimately, to be used to record intracellular signals in vivo.

A structured-inquiry approach to teaching neurophysiology using computer simulation.

Computer simulation is a valuable tool for teaching the fundamentals of neurophysiology in undergraduate laboratories where time and equipment limitations restrict the amount of course content that can be delivered through hands-on interaction. However, students often find such exercises to be tedious and unstimulating. In an effort to engage students in the use of computational modeling while developing a deeper understanding of neurophysiology, an attempt was made to use an educational neurosimulation environment as the basis for a novel, inquiry-based research project. During the semester, students in the class wrote a research proposal, used the Neurodynamix II simulator to generate a large data set, analyzed their modeling results statistically, and presented their findings at the Midbrains Neuroscience Consortium undergraduate poster session. Learning was assessed in the form of a series of short term papers and two 10-min in-class writing responses to the open-ended question, "How do ion channels influence neuronal firing?", which they completed on weeks 6 and 15 of the semester. Students' answers to this question showed a deeper understanding of neuronal excitability after the project; their term papers revealed evidence of critical thinking about computational modeling and neuronal excitability. Suggestions for the adaptation of this structured-inquiry approach into shorter term lab experiences are discussed.

Assessing development of an interdisciplinary perspective in an undergraduate neuroscience course.

Neuroscience is an intrinsically interdisciplinary (ID) field yet little has been published regarding assessment of ID learning in undergraduate neuroscience students. This study attempted to empirically assess the development of an interdisciplinary perspective in 25 undergraduate neuroscience students in a neuroscience program core course. Data were collected using two simple assessment instruments: 1) written responses to the open-ended question "What is neuroscience?" and 2) a term-discipline relevance survey in which students indicated all disciplinary perspectives to which terms (such as electrode, taste, dx/dt) were relevant. Comparison of student responses early in the course (week 1 or 5) and at the end of the course (week 15) showed evidence of development of an interdisciplinary perspective, with students using significantly more integrative terms in their responses and demonstrating an increased awareness of the complexity of the field of neuroscience.

Biogenic amines modulate pulse rate in the dorsal blood vessel of Lumbriculus variegatus.

The biogenic amines are widespread regulators of physiological processes, and play an important role in regulating heart rate in diverse organisms. Here, we present the first pharmacological evidence for a role of the biogenic amines in the regulation of dorsal blood vessel pulse rate in an aquatic oligochaete, Lumbriculus variegatus (Müller, 1774). Bath application of octopamine to intact worms resulted in an acceleration of pulse rate, but not when co-applied with the adenylyl cyclase inhibitor MDL-12,330a. The phosphodiesterase inhibitor theophylline mimicked the effects of OA, but the polar adenosine receptor antagonist 8(p-sulphophenyl)theophylline was significantly less potent than theophylline. Pharmacologically blocking synaptic reuptake of the biogenic amines using the selective 5-HT reuptake blocker fluoxetine or various tricyclic antidepressants also accelerated heart rate. Depletion of the biogenic amines by treatment with the monoamine vesicular transporter blocker reserpine dramatically depressed pulse rate. Pulse rate was partially restored in amine-depleted worms after treatment with octopamine or dopamine, but fully restored following treatment with serotonin. This effect of 5-HT was weakly mimicked by 5-methoxytryptamine, but not by alpha-methylserotonin; it was completely blocked by clozapine and partially blocked by cyproheptadine. Because they are known to orchestrate a variety of adaptive behaviors in invertebrates, the biogenic amines may coordinate blood flow with behavioral state in L.variegatus.

Multiple spike initiation zones in a neuron implicated in learning in the leech: a computational model.

Sensitization of the defensive shortening reflex in the leech has been linked to a segmentally repeated tri-synaptic positive feedback loop. Serotonin from the R-cell enhances S-cell excitability, S-cell impulses cross an electrical synapse into the C-interneuron, and the C-interneuron excites the R-cell via a glutamatergic synapse. The C-interneuron has two unusual characteristics. First, impulses take longer to propagate from the S soma to the C soma than in the reverse direction. Second, impulses recorded from the electrically unexcitable C soma vary in amplitude when extracellular divalent cation concentrations are elevated, with smaller impulses failing to induce synaptic potentials in the R-cell. A compartmental, computational model was developed to test the sufficiency of multiple, independent spike initiation zones in the C-interneuron to explain these observations. The model displays asymmetric delays in impulse propagation across the S-C electrical synapse and graded impulse amplitudes in the C-interneuron in simulated high divalent cation concentrations.

Serotonergic modulation of afterhyperpolarization in a neuron that contributes to learning in the leech.

Modulation of afterhyperpolarization (AHP) represents an important mechanism by which excitability of a neuron can be regulated. In the leech brain, sensitization enhances excitability of the S-cell, an interneuron thought to play an important role in this form of nonassociative learning. This increase in excitability is serotonin (5-HT) dependent, but it is not known whether changes in AHP contribute to 5-HT-mediated enhancement of excitability. Therefore electrophysiological recordings and computational modeling were used to determine whether 5-HT enhances excitability via modulation of AHP. 5-HT reduced S-cell AHP and this decrease in the AHP corresponded with an increase in excitability. Little or no AHP is observed in the presence of Ca2+-free saline, suggesting the involvement of Ca2+-dependent K+ channels. Furthermore, AHP amplitude decreased following treatment with drugs (tubocurare and charybdotoxin) that block Ca2+-dependent K+ channel activity. The S-cell also exhibits an afterdepolarization (ADP), which is usually masked by the AHP, and was inhibited by the Na+ channel blocker saxitoxin. A model of the S-cell AHP was constructed using two Ca2+-dependent K+ currents and a Na+-driven ADP current. Reduction of the model conductances underlying the AHP to mimic the effects of 5-HT was sufficient to enhance excitability. These findings were confirmed in occlusion experiments in which pretreatment with tubocurare was able to block 5-HT-mediated decreases in mAHP levels and increases in excitability. These data show that modulation of S-cell AHP can contribute to 5-HT-mediated increases in excitability and that the S-cell afterpotential is due to the combined effects of AHP- and ADP-producing currents.

Pros and cons of a group webpage design project in a freshman anatomy and physiology course.

To generate motivation and promote the development of written communication skills, students in a freshman-level anatomy and physiology course for nonmajors created group webpages describing historically important diseases. After the groups had been formed, each individual was assigned specific components of the disease (e.g., causes or treatments), which were subsequently combined into a final product. Interviews and questionnaires were used to document students' previous educational experiences regarding, and attitudes toward, the project. Students learned more about website design than about anatomy and physiology, but students preferred the assignment over traditional term papers. Although most students could find relevant information for this project on the internet, they were uncritical in judging the accuracy of the information they found.

Serotonin delays habituation of leech swim response to touch.

Serotonin, acting through a cAMP-signaling pathway, delayed habituation to criterion of the leech's swim response to touch. This delay was reversed by crushing the connective between serotonin-exposed and serotonin-naive ganglia, and correlated with an increase in spontaneous impulse activity in this connective. We suggest that increased activity in intersegmental interneurons may play a role in maintaining swim responsiveness when concentrations of serotonin are elevated.

Beyond the central pattern generator: amine modulation of decision-making neural pathways descending from the brain of the medicinal leech.

The biological mechanisms of behavioral selection, as it relates to locomotion, are far from understood, even in relatively simple invertebrate animals. In the medicinal leech, Hirudo medicinalis, the decision to swim is distributed across populations of swim-activating and swim-inactivating neurons descending from the subesophageal ganglion of the compound cephalic ganglion, i.e. the brain. In the present study, we demonstrate that the serotonergic LL and Retzius cells in the brain are excited by swim-initiating stimuli and during spontaneous swim episodes. This activity likely influences or resets the neuromodulatory state of neural circuits involved in the activation or subsequent termination of locomotion. When serotonin (5-HT) was perfused over the brain, multi-unit recordings from descending brain neurons revealed rapid and substantial alterations. Subsequent intracellular recordings from identified command-like brain interneurons demonstrated that 5-HT, especially in combination with octopamine, inhibited swim-triggering neuron Tr1, as well as swim-inactivating neurons Tr2 and SIN1. Although 5-HT inhibited elements of the swim-inactivation pathway, rather than promoting them, the indirect and net effect of the amine was a reliable and sustained reduction in the firing of the segmental swim-gating neuron 204. This modulation caused cell 204 to relinquish its excitatory drive to the swim central pattern generator. The activation pattern of serotonergic brain neurons that we observed during swimming and the 5-HT-immunoreactive staining pattern obtained, suggest that within the head brain 5-HT secretion is massive. Over time, 5-HT secretion may provide a homeostatic feedback mechanism to limit swimming activity at the level of the head brain.

A 3-synapse positive feedback loop regulates the excitability of an interneuron critical for sensitization in the leech.

Sensitization of reflexive shortening in the leech has been linked to serotonin (5-HT)-induced changes in the excitability of a single interneuron, the S cell. This neuron is necessary for sensitization and complete dishabituation of reflexive shortening, during which it contributes to the sensory-motor reflex. The S cell does not contain 5-HT, which is released primarily from the Retzius (R) cells, whose firing enhances S-cell excitability. Here, we show that the S cell excites the R cells, mainly via a fast disynaptic pathway in which the first synapse is the electrical junction between the S cell and the coupling interneurons, and the second synapse is a glutamatergic synapse of the coupling interneurons onto the R cells. The S cell-triggered excitatory postsynaptic potential in the R cell diminishes and nearly disappears in elevated concentrations of divalent cations because the coupling interneurons become inexcitable under these conditions. Serotonin released from the R cells feeds back on the S cell and increases its excitability by activating a 5-HT7-like receptor; 5-methoxytryptamine (5-MeOT; 10 microM) mimics the effects of 5-HT on S cell excitability, and effects of both 5-HT and 5-MeOT are blocked by pimozide (10 microM) and SB-269970 [(R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol] (5 microM). This feedback loop may be critical for the full expression of sensitization of reflexive shortening.

A cephalic projection neuron involved in locomotion is dye coupled to the dopaminergic neural network in the medicinal leech.

It is widely appreciated that the selection and modulation of locomotor circuits are dependent on the actions of higher-order projection neurons. In the leech, Hirudo medicinalis, locomotion is modulated by a number of cephalic projection neurons that descend from the subesophageal ganglion in the head. Specifically, descending brain interneuron Tr2 functions as a command-like neuron that can terminate or sometimes trigger fictive swimming. In this study, we demonstrate that Tr2 is dye coupled to the dopaminergic neural network distributed in the head brain. These findings represent the first anatomical evidence in support of dopamine (DA) playing a role in the modulation of locomotion in the leech. In addition, we have determined that bath application of DA to the brain and entire nerve cord reliably and rapidly terminates swimming in all preparations exhibiting fictive swimming. By contrast, DA application to nerve cords expressing ongoing fictive crawling does not inhibit this motor rhythm. Furthermore, we show that Tr2 receives rhythmic feedback from the crawl central pattern generator. For example, Tr2 receives inhibitory post-synaptic potentials during the elongation phase of each crawl cycle. When crawling is not expressed, spontaneous inhibitory post-synaptic potentials in Tr2 correlate in time with spontaneous excitatory post-synaptic potentials in the CV motor neuron, a circular muscle excitor that bursts during the elongation phase of crawling. Our data are consistent with the idea that DA biases the nervous system to produce locomotion in the form of crawling.

Distribution and development of dopamine- and octopamine-synthesizing neurons in the medicinal leech.

Although the medicinal leech is a well-studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA-immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA-beta-hydroxylase (DbetaH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA-ir neurons. This antibody appears to cross-react with the closely related invertebrate enzyme tyramine-beta-hydroxylase, which converts tyramine to OA, suggesting that the OA-ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DbetaH antiserum selectively immunostained the OA-ir neurons, but not the DA-synthesizing cells, our results also indicate that the DA-ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47-48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH-ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.