Neuroscience Applications Booklet Harvard Apparatus and affiliates are cited over 232,037 times in ‘Google Scholar’, and over 10,000 times in 2012 alone.*

The following booklet contains examples of Harvard Apparatus products used in Neuroscience Research, along with summarizations of the experiments and related published articles.

Thank you for considering Harvard Apparatus products for your Neuroscience Research needs.

*Data compiled November 2014. Dear Scientist, Application Briefs

he importance of your research work 2 - 25 Tcannot be overstated. Everyone’s life is impacted by a family member or friend that has a neurodegenerative disorder, a learning disability, a traumatic injury or a psychological Bibliography disorder. Your work is our only hope that future generations will be relieved of such 26 - 27 conditions. We at Harvard Apparatus congratulate and thank you for devoting your career to neuroscience research.

Harvard Apparatus and affiliated entities, have been supplying tools for neuroscience research for over 100 years. We provide researchers with an extensive product offering for animal, organ, tissue and cellular research. Whether you need to execute precise infusion into cells or animals, sample extraction, behavior analysis, or control and measure physiological parameters – Harvard Apparatus is ready to meet your needs. Our product quality, dedicated support and neuroscience expertise have made Harvard Apparatus the leader in this field. Our products have been extensively used for neuroscience experiments for decades and have been cited in thousands of publications. The enclosed is a sampling of published articles that cited the use of products from one or more of our affiliated companies. We hope that these abstracts and the bibliography at the end will assist you in your important work and that you consider Harvard Apparatus as your primary source for neuroscience research apparatus.

November 2014 Ventral Tegmental Area EXPERIMENT SUMMARIZATION Neuronal Activity Correlates to Animals’ Behavioral Response to Materials & Methods Male Sprague-Dawley rats (N=142_ at post natal day of Chronic Methylphenidate about 32 days were purchased (Harlan, Indianapolis, IN, USA) and were allowed 3-4 days of acclimation in the Recorded from Adolescent vivarium on a 12 hour light/dark schedule prior to the SD Male Rats electrode implantation. Food and water were given at libitum. The animals were housed individually in clear acrylic standard cages that served as both home cage Z. Jones et al (2014) and test cage for this study. Journal of Behavioral and Brain Science Electrodes were implanted during stereotaxic surgery on Vol 4(4); ID 45258, 22 anesthetized subjects that had their heads shaved prior to the procedure. Once electrodes were implanted, subjects were allowed 3-4 days recovery in their home cages and were connected to the wireless head stage Introduction transmitter (Triangle BioSystems (TBSI); Durham, NC, USA) for acclimation for at least 2 hours/day to the Methylphenidate (MPD) is considered as the first- behavioral and electrophysiological recording systems. line pharmacotherapy to treat ADHD. More At first experimental day, the animal’s age was about recently, MPD has also been used as a cognitive post natal 39-41 days. enhancement recreationally. Its therapeutic effects are not fully understood, nor are the long Five groups were used: saline, 0.6, 2.5, 5.0 and 10.0 term effects of the drug on brain development. The mg/kg MPD. Behavioral and neuronal activity ventral tegmental area (VTA) neuronal activity was recordings were resumed for an additional hour post recorded from freely behaving adolescent rats drug injection. The wireless TBSI head stage sent using a wireless recording system. neuronal activity signals from the 4 recording electrodes to a receiver that was connected to an analog to digital converter where data was collected and stored for analysis. The experiment lasted for 10 days. On days 2 through 6, subjects received either saline or a daily MPD injection, but without neuronal recording depending on the group. On days 7 through 9, the subjects underwent a washout period in which no injections were given, and then on day 10, a saline injection was delivered. Following the saline injection on day 10, the neuronal and behavioral baseline activity was recorded for an hour followed by a rechallenge administration of either saline or MPD dose similar to day 1, and recordings were resumed for an additional hour.

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s e m e i T e d h i i a s o t f n n d o o i y t u r s w A o a f a p h b a h o m f V T c R L s n c s Step-By-Step EXPERIMENT SUMMARIZATION Instructions for Retina Recordings with Perforated Multi Materials & Methods Electrode Arrays The setup for pMEA recordings consists of two perfusion loops: An upper loop to supply the tissue with fresh Katja Reinhard, Alexandra Tikidji-Hamburyan, solution and a lower loop to adjust the proper negative Hartwig Seitter, Saad Idrees, Marion Mutter, Boris pressure. We provide an overview of this dual perfusion Benkner, Thomas A. Münch system and a detailed list of the components we used to build our setup. Werner Reichardt Centre for Integrative Neuroscience and Bernstein Center for Computational The procedure on how to use the system is described in Neuroscience, University of Tübingen, detail. Except for the constant vacuum pump the Tübingen, Germany amplifier baseplate that allows vacuum application, and some small components, no additional material is (2014) PLoS ONE 9:e106148 needed compared to conventional MEA recordings. The upper perfusion system supplies the retina with fresh solution during the recordings. The lower perfusion system is only used before the experiment to fill the MEA Introduction chamber with solution without introducing air bubbles into the vacuum system. The vacuum system provides negative pressure to pull the retina towards the Multi-electrode arrays are a state-of-the-art electrodes. This negative pressure needs to be constant tool in retina research. The structure of the to avoid fluctuations, and high enough to ensure good retina has allowed the use of multi-electrode tissue-electrode contact without tearing the tissue. arrays for high-throughput, parallel recordings Constant negative pressure is provided by a Constant of retinal responses to presented visual stimuli, Vacuum Pump (CVP) and is further reduced by an and has led to significant new insights into additional fine flow control between the CVP and the retinal organization and function. However, MEA baseplate. The most important step for ensuring using conventional arrays can be associated reliable negative pressure is the removal of air bubbles: with three main problems: (1) low signal-to- any air bubble in the vacuum system will degrade the noise ratio due to poor contact between stable negative pressure. Setting up for pMEA recordings electrodes and tissue, especially in the case of takes approximately 40–60 minutes. Except for the steps strongly curved retinas from small animals, e.g. involving the vacuum system and preparation of the rodents; (2) insufficient oxygen and nutrient filter paper, all steps are very similar to conventional supply to cells located on the bottom of the MEA recordings. Further, no coating of the MEA is recording chamber; and (3) displacement of the necessary for pMEA recordings. Overall, pMEA tissue during recordings. Perforated multi- recordings require about 10 minutes more preparation electrode arrays (pMEAs) have been found to time than conventional MEAs. alleviate all three issues in brain slice recordings. Over the last years, we have been Results & Conclusions using such perforated arrays to study light evoked activity in the retinas of various species pMEAs provide good signal-to-noise ratios including mouse, pig, and human. In this article, The vacuum applied through the perforation of pMEAs we provide detailed step-by-step instructions greatly enhances the contact between the tissue and the for the use of perforated MEAs to record visual electrodes. On good recording electrodes, we can detect responses from the retina, including spike recordings from retinal ganglion cells and in 4 vitro electroretinograms (ERG). phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 5 - c , e l

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o o o a h e . . i r o 6 9 3 n p e r a s H p T f T T h 2 C d T 2 p 1 r Implementation of a EXPERIMENT SUMMARIZATION Two-dimensional Behavior Matrix to Distinguish Materials & Methods Individuals with Differential Male C57BL/6 inbred mice at seven weeks of age (Eumsung, Depression States in a Chungbuk, Korea) were used in this investigation. Subjects were restrained for 2h daily for 14 days and depression Rodent Model of Depression states of individual mice were assessed using the U-field test, behavioral assessment developed to measure animal’s Jin-Young Park et al. (2014) sociability and the tail suspension test and/or forced swim test, which are the typical methods that measure Exp Neurobiol . Sep; 23(3); 215-223 psychomotor withdrawal states.

The U-field test was monitored using a computerized video tracking system (SMART, Panlab, Spain). Introduction A webcam was used to record behavioral performances in the TST and FST. Behavioral tests were performed Animal models of depression are used to study during the light cycle (9AM-3PM). The behavior testing pathophysiology of depression and to advance room was lit with indirect illumination by 20 lux for the therapeutic strategies. Stress-induced depression U-field test and 250 lux for TST and FST, and sound was models in rodents are widely used. However, masked with 65 dB of white noise. Subjects were given amenable behavior criteria and experimental 20 minutes to adapt to the behavior testing room prior procedures that are suitable for animal models to the start of each behavioral test. have not been established. Given that depression is clinically diagnosed by multiple symptomatic Results & Conclusions criteria and stress effects are imposed to the brain non-specifically in stress-induced depression The majority of these mice showed severe depressive models, analyses of depression states in rodents behaviors in both tests, a significant proportion of them, using multiple symptomatic criteria may provide which were all inbred mice and received the same more power than any methods relying on a single amount of restraints, expressed differential depression symptomatic criterion. states in the sociability test and psychomotor withdrawal tests. To easily readout differential depression states of individuals in two different tests, a standard method and basic parameters required to construct two-way behavior matrix were introduced.

The two-way behavioral matrix can be used not only to measure behavioral performance of a group of animals, but also to differentiate different depression states of individuals. For example, the two-way matrix differentiates behavioral states with normal sociability and psychomotor withdrawal, which was referred to as (i) RST-RSL individuals, (ii)behavioral states with normal sociability and high psychomotor withdrawal, (iii)behavioral states with low sociability and normal psychomotor withdrawal and (iv)behavioral states with low sociability and high psychomotor withdrawal, referred to “RST-DD” (Fig 3A and B)The RST-DD and

88 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 9 9 s t

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m m o i a S S e e s f d y y u n s a w m o s w I d T s i p b s R Protective effects of EXPERIMENT SUMMARIZATION remote ischemic conditioning against ischemia/reperfusion-in - Materials & Methods A total of 81 adult male Sprague-Dawley rats were duced retinal injury in rats randomly assigned to ischemia/reperfusion with or without remote LRIC arms. Subjects were anesthetized, Xuxiang Zhang et al. (2014) orally intubated and mechanically ventilated (Rodent Ventilator Model 683, Harvard Apparatus). The retinal Visual Neurocience 31, 03, 246-262 ischemic model was generated through right middle cerebral artery occlusion (MCAO) and pterygopalatine artery occlusion for 60 minutes followed by 1, 3, and 7 days of subsequent reperfusion.

Introduction Paraffin sections were stained to quantify the number Limb remote ischemic conditioning (LRIC) provides of cells in the retinal ganglion cells (RGCs) layer a physiologic strategy for harnessing the body’s throughout the duration of the study. Cellular endogenous protective capabilities against injury expression of glial fibrillary acidic protein (GFAP) was induced by ischemia-preperfusion in the central detected and examined through immunohistochemistry . nervous system. This study seeks to determine if Protein expression was analyzed via Western blot. LRIC played a role in protecting the retina from ischemia-reperfusion injury. The role of astrocytes and cellular pathways was analyzed, which influence the severity of ischemia/reperfusion injury (Eng et al., 2000). Retinal ischemia promotes increased proliferation and differentiation of astrocytes. The increase in astrocyte activity following ischemia causes an increase in glial fibrillary acidic protein (GFAP) transcription and translation.

10 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 1 1 3 8 6

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r s d s d w C - u o r r h c d e u e I e e h a e u l u e t r o b n e l j d e e l y v v O u u c e e h n a R l u o h u t a h o n n s e e a t i p i c n L v c n H T c i m l s s l s f l R A Intrahippocampal EXPERIMENT SUMMARIZATION glucocorticoids generated by 11 -HSD1 affect memory β in aged mice Materials & Methods Male mice homozygous for a targeted disruption of J. Yau et al (2014) hsd11b1, congenic on the C57BL/6J genetic background and wild-type (WT) C57BL/6J mice were bred in-house Neurobiology of Aging under standard conditions (12 hours light-dark cycle) available online July 15, 2014 with food and water ad libitum until experimentation at either 6-8 months old (young) or 24-26 months old (aged) (Carter et al., 2009 and Kotelevtsev et al., 1997).

A 2-trial Y-maze task was used to assess hippocampal- Introduction dependent spatial recognition memory (Conrad et al., 1996 and Yau et al., 2011). The size of the maze allowed Both aging and stress induce memory impairments for in vivo microdialysis in freely behaving mice. notably in hippocampus-associated cognitive functions. Stress activates the hypothalamic- Mice were anesthetized and fixed within a stereotaxic pituitary-adrenal (HPA) axis causing acute elevation frame for microdialysis guide implantation at a position of blood glucocorticoid levels, whereas in a subset just entering the dorsal hippocampus. Mice were of aged individuals, plasma glucocorticoid levels allowed to recover for 4 days before microdialysis began. are elevated, and this correlates with hippocampal- A CMA 7 microdialysis probe was introduced via the associated memory deficits (Lupien et al., 1998) pre-implanted guide cannula. Subjects were individually housed in the CMA 120 system for freely In the present study, in vivo microdialysis was used moving animals with free access to food and water. The in freely behaving mice to examine (1) the effects of probe was perfused continuously with a sterile artificial aging and/or stress on intro-hippocampal CORT cerebrospinal fluid at 0.3 µl per minute using an levels and memory during learning and retrieval; infusion pump and samples were collected via the and (2) the role of 11 -HSD1 in these processes. CMA470 Refrigerated Fraction Collector. β Histology was performed and samples were homogenized for CORT and 11 -HSD1 activity assays. β

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e v e r o h h i i y u o n 1 p g d c s m s g t l l o o a t e p u e e e h h n e a s s a a i a h h r c a b t e L o c r a c s t m e m a n d l a s T r m e 1 R T Back to basics: EXPERIMENT SUMMARIZATION Comprehensive DNA quantitation using BioDrop in the UV spectrophotometry Materials & Methods Sample preparation range, why is it worth it? Lyophilized pure genomic DNA sample (dsDNA) from calf thymus from Sigma (PN# D4764) was weighed with Boesenberg-Smith, K. A., Pessarakli, M. M. & Wolk, D. M., a precision scale (d=0.1mg) and diluted to 1000mg/ml in 2012. Assessment of DNA yield and purity: an overlooked Tris-EDTA buffer solution pH7.4. detail of PCR troubleshooting. Clinical Microbiology Newsletter , 34(1), pp. 2-5. The master sample was subsequently diluted for constituting new samples. Each sample was vortexed Haque, K. A. et al., 2003. Performance of high-throughput and centrifuged. DNA quantification methods. BMC Biotechnology , pp. 1-10. The samples were measured using 1.5 l of solution each Wilson, I. G., 1997. Inhibition and Facilitation of Nucleic Acid time. The micro-volume sample port wμas wiped using Amplification. Applied and environmental microbiology , lint free tissue after each sample measurement October, 63(10), pp. 3741-3751. Results & Conclusions

Pure dsDNA samples diluted in Tris-EDTA buffer ph7.4 Introduction were measured using BioDrop micro-volume sample port where absorbance measurements at 260, 280 and With the advent of new technologies aiming at more 230nm are systematically taken. The purity ratios clearly discerning subtle and complex information (A260/A280 and A260/A230) were directly calculated by from little amount of samples, the requirement for the BioDrop Resolution software. setting quality control processes within a particular workflow has never been greater. The first ratio 260/280, concerns protein contamination in DNA samples. Pure DNA samples have a ratio 260/280 Examples of areas where sample concentration greater than 1.8. The samples can be equally qualified as determination has seemed to expand the most are free from proteins if the values reach 2.0 as a maximum. proteomics and genomics which require lengthy When protein residues are present, the ratio is shifted as sample preparation and data analysis steps. Set as an indication of the contamination. an intermediary step, protein or nucleic acid quantitation plays a very determinant role in the The second ratio 260/230, concerns organic outcome of many downstream experiments. contaminant and solvents detection such as phenol, thiocyanates, urea, carbohydrates, B-mercaptoethanol The way the quantitation is performed is equally and guanidinium residues from chaotropic lysis buffer very important: the quality control process that can which are often responsible for poor DNA detection in potentially quantify and qualify vast amounts of further DNA sequencing processes by significantly sample types should be very quick and simple. affecting the PCR reactions (Boesenberg-Smith, et al., BioDrop µLite is a split-beam UV-Vis 2012; Wilson, 1997). spectrophotometer equipped with a built-in micro- If those ratios are checked against the sample volume sample port. The micro-volume sample concentration, within the detection range of BioDrop, a port is specifically designed for the quantitation of rapid estimation of the sample quality can be made. small sample volumes such as protein or nucleic BioDrop can accurately provide purity ratio within a acids. Set to work with as little as 0.5 µl of sample, 1000 dynamic range for DNA samples having BioDrop µLite is also conceived to deliver quick concentrations ranging from 2.0ng/ l up to 2.4 g/ l. and high quality measurements by setting only a single and fixed path length so as to reduce bias μ μ μ 14 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 and inaccuracy related to path length design. Harvard Apparatus Neuroscience Application Guide 5 1 : y n d o h

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d D o o o i o a a a / c d e r s v t

n l p p v m e o o f f f d e a e a t h

r q w r o N N x i i y e g h s y a u u t b n d x x a a t o a e h o o i n n n n o m D r f e B s n e C B o t T a t s R r i f m a m l i B p v m q a B i i q s A d Temperature- EXPERIMENT SUMMARIZATION Dependent Cysteine Reactivity in the Cystic Fibrosis Transmembrane Materials & Methods Whole Cell Recordings Conductance Regulator Individual oocytes were placed in a 200 mL recording chamber (RC-1Z, Warner Instruments) and continuously Adapted with permission from Liu X, Dawson DC., perfused with Frog Ringer’s solution (4 mL/min). The Ringer’s solution contained 98 mM NaCl, 2 mM KCl, Cystic fibrosis transmembrane conductance 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES regulator: temperature-dependent cysteine (hemi-Na) (pH 7.4). CFTR channels were activated reactivity suggests different stable conformers of using 10 mM isoproterenol (a -adrenergic agonist) and the conduction pathway., 1 mM IBMX (a phosphodiesterβase inhibitor) as the Biochemistry , 50(47), 10311-7. stimulating cocktail (Isop+IBMX). An OC-725 amplifier Copyright 2011, American Chemical Society. (Warner Instruments) and the pClamp 8 data acquisition program (Axon Instruments) were used for whole cell recordings. Oocytes were maintained in the open circuit condition, and the membrane potential was periodically ramped from −120 to 60 mV over 1.8 s to Introduction construct the whole cell I−V plots. Cysteine scanning has been widely used to identify Temperature Control pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance A dual automatic temperature controller (CL-200, regulator (CFTR). Studies of this type, however, have been Warner Instruments) and an in-line solution typically conducted at room temperature rather than at heater/cooler (SC-20, Warner Instruments) were used to human body temperature. Reports of substantial effects apply acute temperature changes under a constant flow. of temperature on gating and anion conduction in CFTR The bath temperature was monitored. The temperature channels, as well as an unexpected pattern of cysteine signal was digitized and recorded using a USB Data reactivity in the sixth transmembrane segment (TM6), Acquisition Device (DI-158, DATAQ Instruments). prompted the researchers to investigate the effect of temperature on the reactivity of cysteines engineered into Fig. 1. Increased temperature dramatically increased the TM6 of CFTR. They compared reaction rates at reactivity of F337C CFTR temperatures ranging from 22 to 37 °C for cysteines toward MTSES . Oocytes expressing F33−7C CFTR were placed on either side of an apparent size-selective barrier. activated using a stimulatory cocktail (10 M Isop and 1 mM The results indicate that the reactivity of cysteines to IBMX, hatcheμd bar and cross MTSES− at several positions extracellular to the position of hairs) and then exposed to 1 mM 2-ME to reverse any the accessibility barrier are highly temperature- spurious reactions of the dependent. In addition, the apparent activation energy for substituted cysteine. (A) Exposure to 1 mM MTSES the MTSES−/cysteine reaction exceeded that expected for (dark gray bar and circles−) diffusion or thiol-disulfide exchange in solution, as well as produced no reaction, but for CFTR gating, suggesting that the change from low to 1 mM [Au(CN)2] (black bar and circles) prod−uced physiological temperatures is associated with a change in profound inhibition that was conformation of the outer vestibule of the pore. not reversed by 1 mM 2-ME but was reversed by 1 mM KCN (white bar and triangles). (B) Increasing the temperature to 32°C in the presence of 1 These data were acquired with the aid of an OC-725 oocyte mM MTSES produced a modest reaction rate. (C) Increasing clamp amplifier, an RC-1Z imaging chamber, a CL-200 dual the tempera−ture to 37°C in the presence of 1 mM MTSES produced a rapid reaction. (D) After the oocyte had bee−n channel bipolar temperature controller, and an SC-20 in- heated to 37°C and cooled to 22°C, exposure to 1 mM MTSES line solution heater/cooler from Warner Instruments. produced no evidence of a reaction. −

116 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 7 1 A C l 5 0 e 2 0 0 Z d 7 1 2 2 - - - - o C L C C S M R O C e r r r e e i u f b t i l a m r p s a e h m t p : C A

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p v x u e n l i i s e i e e e e

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a d 7 7 d s 2 s y l d s f e r b

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r

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s g g g m m e o a F F F e n h r i e e r e r n i 3 i i o r t l y h x x x o o o u C e e e h e h i o e i e h m t t f a c k t c d d T C R R e t C p s p c w C F C f d r o p c F e ° o t e t c t r F e r F Dynamical EXPERIMENT SUMMARIZATION modes in coupled large-scale networks Materials & Methods Mathias Mahn Master Thesis, September 30, 2011, To be able to investigate effects of electrical coupling on Technion, Haifa and LMU München separated neuronal cultures, we divided the culture Supervisors: Shimon Marom and Lars Kunz chamber of 60 electrode MEAs by inserting a plastic barrier using silicone. The MEA was placed on the Current address: Arison 303, Weizmann Institute of headstage and the aeration installed (95% air, 5 % CO2). Science, 234 Herzl Street, Rehovot 76100, Israel After placing a culture on the setup, at least 1h of spontaneous activity was recorded. To determine if the [email protected] culture shows stable spiking behaviour, event detection Adopted with permission was performed. Events were detected upon the downward crossing of a threshold set individually for each electrode to -6 x STD of the noise. Once the conditions became stable, one stimulation electrode, Introduction that efficiently evoked a network burst was chosen per compartment. The online burst detection was defined carefully. After adjusting the detection and stimulation An aim of our group is the real-time activity parameters, the closed loop program was downloaded to analysis of compartmentalized neural the DSP and the coupling experiment was started. networks in vitro and the reaction to the Cultures were used for experimentation for up to 60 h. activity by electrical stimulation with a delay in the range of ms. We chose a multi-channel Results & Conclusions electrode array (MEA) based interface on which primary cortical neurons were cultured During a closed loop experiment the voltage values from as a well-established experimental model the extracellular recordings are collected and then system. These networks show a very analyzed in a chunk by the DSP. The DSP will, in case of characteristic synchronous spiking activity the fulfillment of a given condition, trigger a which is very similar to a form of network stimulation. Here we show how fast we were able to activity found in in vivo networks during close the loop, from the time the event happened to the development of the nervous system. To be time the feedback stimulus was given. The feedback able to perform closed loop stimulation stimulus was delivered with a delay of 1-2 ms. Synaptic experiments we implemented the MEA2100 transmission delays can range from 0.3 ms, in the system, which comprises a digital signal extreme case of the calix of held, to more than 5 ms. This processor (DSP). The DSP was programmed means that the time we need to deliver a feed back is using the MathWorks Matlab and Simulink well in the physiological range of a single synaptic programming framework. We present first transmission delay. We used the separated preliminary results that are aimed at compartment MEAs to perform several experiments, illustrating the huge space of possible each lasting more than 36 hours. Out of these investigations opened by the implementation experiments we present one, which is thought to of the MEA2100 system. illustrate the space of possibilities, opened by the implementation of this system. In the example we present a network culture, which shows super network bursting (SNB) behaviour. This means that the network burst frequency is modulated by a slow oscillation, in

18 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 9 1 - r c e e e c l t

n h e r e e t

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Provided the concentration range is 50 - 800mg/ml, Add 0.01ml of sample and 0.01ml water, for the blank foodstuff extracts can be analysed with the same method. analysis, respectively and mix.

Cholesterol is determined at 500nm after After 10 minutes at 20-25 °C: enzymatic hydrolysis and oxidation, via the • Insert blank cuvette. and press green run key. formation of the indicator quinoneimine in the presence of phenol and peroxidase. • A single blank as above suffices for subsequent analyses in the same series.

• Insert sample and press green run key

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Gao, M., et. al., (2007) To test whether PFC inputs are involved in the Functional Coupling between the Prefrontal Cortex and generation of the SO in DA neurons, bilateral Dopamine Neurons in the Ventral Tegmental Area, transections were made immediately caudal to the PFC to interrupt PFC inputs to DA neurons. A slit was drilled The Journal of Neuroscience , in the skull 2.0 mm anterior to bregma. The sharp 27(20): 5414-5421 pointed tip of a 3-mm-wide scalp blade was lowered to the base of skull and passed along the slit to completely interrupt connections between the PFC and the rest of the brain. Recordings commenced 30 min after the Introduction transection. In a second group of rats, the PFC was inactivated by Stimulation of the prefrontal cortex (PFC) has been local infusion of TTX (Fisheries Science and Technology shown to have an excitatory influence on dopamine Development, Hebei, China). Silica capillary tubing (DA) neurons. We report here that, under (outer diameter, 160 µm; inner diameter, 100 µm) was nonstimulated conditions, the activity of DA filled with TTX (10 ng/µl in 0.9% saline) and inserted into neurons in the ventral tegmental area (VTA) also the medial PFC (from bregma, AP, 3.0; ML, 0.7; DV, 3.5 covaries, on a subsecond timescale, with the mm). After a stable baseline recording, TTX was infused activity of PFC cells. Thus, in 67% of VTA DA at 0.75 µl/min for 2 min The infusion was controlled by a neurons recorded in chloral hydrate-anesthetized Harvard Apparatus Pump 11 Elite syringe pump. rats, the firing of the cell displayed a slow Throughout the experiment, body temperature was oscillation (SO) that was highly coherent with the maintained at 36-38°C with a Harvard Apparatus activity of PFC neurons. The SO was suppressed by homoeothermic blanket system. transections immediately caudal to the PFC or by intra-PFC infusion of tetrodotoxin, suggesting that Effects of TTX infusion were determined by comparing it depends on inputs derived from the PFC. recordings from the same cell before and after the Unexpectedly, the SO in most VTA DA neurons was infusion. Only one cell was studied in each rat. In reversed in phase relative to PFC cell activity, separate groups of animals, saline (1.5 µl) was infused suggesting that at least part of PFC information is into the mPFC in a similar manner, or TTX (15 ng/1.5 µl) transferred to DA neurons indirectly through was infused into the lateral PFC (from bregma, AP, 3.0; inhibitory relay neurons. Further understanding of ML, 1.4; DV, 3.5). At the end of the experiment, the this coordinated activity may provide important injection site was verified using standard histology new insights into brain functions and disorders methods. thought to involve both VTA DA and PFC neurons.

22 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 3 2 h t i w

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r T e e , h h h o e h h D V D ( p p r n V t C c n t A R P ( ( Efficient Genome EXPERIMENT SUMMARIZATION Engineering of Toxoplasma gondii using CRISPR/Cas9 Materials & Methods Transfections and Plaque Assays Sidik SM, et al., (2014) Filtered T. gondii were washed and resuspended in Cytomix at 26108 parasites/ml. 56107 parasites were Plos One , volume 9, Issue 6, July 2014 combined with 100 mg of CRISPR/Cas9 plasmid supplemented with 2 mM ATP, 5 mM GSH, and 150 mM CaCl2, with or without 40 mg of double-stranded oligonucleotide, in a final volume of 400 ml. The CaCl2 concentration used here matches the original Introduction formulation of Cytomix, but was added at the time of transfection to prevent the buildup of phosphate Toxoplasma gondii is a parasite of humans and precipitates during buffer storage. Parasites were animals, and a model for other apicomplexans electroporated in 4 mm gap cuvettes (BTX Harvard including Plasmodium spp., the causative agents of Apparatus model no. 640) in an Electro Square Porator malaria. Despite many advances, manipulating the (BTX Harvard Apparatus ). 200 parasites were added to T. gondii genome remains labor intensive, and is confluent HFF monolayers in six-well dishes. In some often restricted to lab-adapted strains or lines cases, as indicated in the text, 2,000 or 20,000 parasites carrying mutations that enable selection. Here, the were used instead. If indicated, 0.2 mM Compound 2 (C2) authors have used Electroporation to introduce the or vehicle (DMSO) was added to these dishes 24 hpi. All RNA-guided Cas9 nuclease to efficiently generate plates were allowed to incubate for a total of eight days knockouts without selection, and to introduce before staining with crystal violet. point mutations and epitope tags into the T.gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes..

Figure 1. Targeted disruption of the SAG1 locus using CRISPR/Cas9. (A) Strategy for constructing the CRISPR/Cas9 plasmid pU6-SAG1:chiRNA encoding a protospacer specific to SAG1 (blue) and the Cas9 recognition motif (orange) was cloned under the upstream region of T. gondii U6. CAS9 flanked by nuclear localization signals (NLS; gray) and a FLAG tag (green) was cloned under the control of the TgTUB1 promoter. (B) Expression of Cas9 following transfection with or without pU6-SAG1. Parasites were fixed 24 hours post transfection and stained for parasite Actin (TgACT1; red), FLAG (green), and DAPI (DNA; blue). Scale bar is 10 mm. (C) Expression of SAG1 in wild-type RH parasites transfected with pU6-SAG1 Plasmid or mock-transfected parasites were allowed to grow for one lytic cycle before infecting monolayers for microscopy. Monolayers were fixed 24 hpi and stained for TgACT1 (red), SAG1 (green), and DNA. DSAG1 vacuole is indicated by an arrow. Scale bar is 10 mm. (D) Quantification of SAG1 knockout rates in RH and KU80. ∆ Two-tailed Student’s t-test; ***P>0.0005; means s.e.m., n = 3 ± experiments. (E) Quantification of transfection efficiency in the same populations 24 hours post transfection by monitoring Cas9 expression by immunofluorescence. Two-tailed Student’s t-test; means s.e.m., n = 3 experiments. £

24 phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 Harvard Apparatus Neuroscience Application Guide 5 2 n o

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i o d m d a a i n S G a S Harvard Apparatus & Affliates Contact Information

Harvard Apparatus - USA 84 October Hill Road, Holliston, MA 01746 • USA phone 508.893.8999 toll free 800.272.2775 fax 508.429.5732 email [email protected] web www.harvardapparatus.com Warner Instruments Biochrom 1125 Dixwell Avenue , Hamden , CT 06514 • USA 22 Cambridge Science Park • Milton Road phone 203.776.0664 Cambridge, CB4 0FJ • United Kingdom fax 203.776.1278 phone (44) 1223.423.723 email [email protected] fax (44) 1223.420.164 web www.warneronline.com CanadaCanada email [email protected] Harvard Apparatus Canada web www.biochrom.co.uk 6010 Vanden Abeele • Saint Laurent, Quebec • Canada phone 514.335.0792 toll free 800.361.1905 fax 514.335.3482 Coulbourn Instruments BioDrop email [email protected] 84 October Hill Road, Holliston, MA 01746 • USA web www.harvardapparatus.ca 5583 Roosevelt Street , Whitehall , PA 18052 • USA phone 610.395.3771 • toll free 800.424.3771 phone 508.893.8999 fax 610.391.1333 toll free 800.272.2775 email [email protected] fax 508.429.5732 web www.coulbourn.com email [email protected] web www.bio-drop.com Frranceance Harvard Apparatus, S.A.R.L. - France 6 Avenue des Andes • Miniparc - Bat. 8 91952 Les Ulis Cedex • France phone (33) 1-64-46-00-85 Hoefer, Inc. fax (33) 1-64-46-94-38 Hugo Sachs Elektronik 84 October Hill Road, Holliston, MA 01746 • USA U email [email protected] Gruenstrasse 1 • March-Hugstetten D-79232 • Germany phone 508.893.8999 web www.harvardapparatus.fr phone (49) 0 7665.92.00.0 toll free 800.227.4750 fax (49) 0 7665.92.00.90 fax 508.429.5732 email [email protected] email [email protected] web www.hugo-sachs.de web www.harvardapparatus.com

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