The authors thank Joseph Pitock and Katie Przybysz, PhD for technical assistance during LIQ HDR development. We also thank Nicholas Petersen and Marie Doyle, PhD for many helpful conversations during the development of LIQ HDR. This work was supported by the National Institute on Alcohol Abuse and Alcoholism at the National Institutes of Health (P50 AA022538 and R01 AA029130 to EJG).

Intermittent-Access Two-Bottle Choice Drinking with Lick Detection in Rats

All authors declare no conflicts of interest.

The current protocol provides step-by-step instructions for capturing ethanol drinking data using an intermittent-access two-bottle choice home cage procedure. This method can be implemented with relative ease and with little-to-no cost or need for specialized research equipment. Using the procedures provided here for constructing and implementing LIQ HDR - a lick detection system for use in rats - researchers can capture high-resolution drinking microstructure data in addition to standard measures of intake and preference.
The intermittent-access schedule described in the current protocol is a widely used procedure in large part because it is well-known to engender higher levels of ethanol intake than many alternative schedules of ethanol access. Indeed, previous research has shown that rats with access on an intermittent schedule have higher average daily ethanol intake than rats with continuous 24 h access6,7. Nevertheless, different schedules of ethanol access may be desirable depending on the research question or other experimental design parameters. Researchers can very easily modify the current protocol to accommodate different schedules (e.g., continuous access, drinking in the dark, etc.) by simply changing the bottle on/off time and days.
Although two-bottle choice drinking is a relatively simple to implement paradigm, issues can arise that have the potential to compromise data collection. It is, therefore, crucial that experimenters inspect data on a daily basis in order to quickly identify potential problems. In particular, researchers should pay close attention to very large or very small changes in bottle weights that may be indicative of a leaky or clogged sipper, respectively. Catching signs of these issues early can prevent the exclusion of all data from a given subject from analysis. Indeed, if researchers notice drastic changes (or absence of change) in bottle weight over a 24 h period, a simple switch to a new bottle can help to confirm whether a given bottle should be removed from circulation. For these reasons, it is wise to have extra bottles and sippers on hand. However, it should be noted that some rats naturally exhibit very low levels of ethanol drinking, e.g.,35. This is particularly true for rats of certain strains, see8 for review. Thus, researchers should not be alarmed if a small population of animals in a given experiment has low or no ethanol consumption. Likewise, some animals are prone to play with the sipper/bottle in ways that can promote spillage. Although rare, repeated instances of high changes in bottle weight that are not associated with consumption may require that a particular animal be removed from the experiment.
Our detailed step-by-step protocol demonstrates the ease of implementation and use of the LIQ HDR system for rats. Researchers with no prior knowledge or skills in microcontroller electronics and programming can readily adapt this system to their experimental needs with little or no modification. Our representative data further demonstrate its successful application for rat home cage drinking studies and its powerful capacity to capture numerous drinking microstructure measures at high temporal resolution. LIQ HDR includes several design modifications relative to its mouse predecessor30. This was done not only as a necessity for successful adaptation for use in rats but also to improve functionality and user experience. In-cage components, including the 3D-printed bottle holder, sipper blocker, and electronic components, are relocated to the cage top to protect against chewing, which can result in frequent and significant data loss and hardware turnover. The addition of a laser-cut acrylic panel further helps prevent the destruction of these vulnerable components. Aside from the acrylic panel, which is the same material as a standard shoebox rat cage, all other LIQ HDR components are inaccessible to the animal from inside the cage or only accessible for a brief moment during bottle change. This design limits the potential for destruction of device components and risk to the animal via ingestion of non-food items. In contrast to the relatively labor-intensive custom-made bottles used in the LIQ HD design, LIQ HDR&#39;s 3D-printed bottle holder is designed to be compatible with commercial off-the-shelf rodent drinking bottles. Bottles are installed on the cage top and accessible to rats in much of the same way as drinking bottles are applied during standard care and husbandry. The addition of sipper blockers is a simple but effective solution that improves system reliability and prevents valuable data loss that, in the absence of the blocker, can result from drinking that occurs prior to initiating data capture on the Arduino interface or erroneous lick measures that occur when rats interact with the sippers during daily calibration. While the considerably larger rat cages necessitate scaling up the original hardware design in size, the use of significantly longer cables can negatively impact the sensitivity and accuracy of capacitive sensing due to interference, as well as the integrity of I2C communication between the daisy-chained sensor boards and Arduino interface. We circumvent this issue with the adoption of the differential I2C communication system to achieve ultra-long-range, near-lossless signal transmission through Ethernet cables. The materials used for the in-cage components allow for easy cleaning and disinfection in between uses. The lickometer can be spayed and wiped down with 70% EtOH solution, while care must be taken to ensure the electrical components, such as the conducting copper tape, remain intact. Non-electrical equipment that is in direct contact with the animal, such as the sipper blocker and acrylic panel, can be thoroughly washed with warm soap water.
The reliability and effectiveness of any lick detection system requires proper construction and installation as well as extensive testing, diligent error monitoring, and effective troubleshooting. All DIY components need to be made and assembled with skill and care. This is especially important for steps involving soldering, which may require practice for beginners. Regardless, extensive testing of all lickometers is required before use in actual experiments. If the system must be modified in order to adapt to individual experimental needs, the sensor settings (e.g., touch and release thresholds) may need to be further fine-tuned for accurate lick detection. For first-time users, we suggest testing the system in a brief, small-sample pilot study using a highly palatable solution that will engender high levels of drinking. Lick detection can be verified in real-time by visually inspecting the sensor board as the LED flashes whenever a lick is registered. Strong, significant, and consistent correlations between lick number and change in bottle weight should be used as a benchmark for proper hardware and software setup. Even after the setup is finalized, we recommend initiating recording for a given experiment on a day when rats receive two bottles containing water in order to troubleshoot equipment issues that may be evident at the beginning of an experiment. It may also be advantageous to test lickometer function on the water day associated with cage change since this requires that lickometers be disconnected and reconnected to the sensor. Random failure of individual lickometer or sensor, which would result in very obvious outlier data (e.g., no or few licks detected despite a substantial change in bottle weight) consistently for the same lickometer, is indicative of faulty component(s). We recommend preparing extra components for each element of the lickometer system in advance to enable easy replacement as necessary. Finally, the importance of making sure that sensor cables do not overlap or bend excessively cannot be overemphasized. Doing so can result in random instances of poor lick detection, evident by an unreasonably low lick number for a given change in bottle weight or extremely large lick durations (close to 60,000 ms for a 1-min bin) spanning minutes or hours. Nevertheless, small losses of data are inevitable in studies that collect data over 24 h for many animals across many days using either the standard or lickometer-equipped approach. Researchers can exclude erroneous data for the rare drinking sessions when bottle weights and/or licks are clearly not reflective of intake (e.g., sipper clog, calibration error, etc.). On occasions such as these, the average value for a given measure (e.g., intake (g/kg), licks, etc.) across the study period for that particular subject can be substituted for the missing value during data analysis. Mixed-effects ANOVA can also be used as an alternative to accommodate missing data points.
One of the advantages of the two-bottle choice drinking paradigm is the voluntary aspect of ethanol intake. This affords excellent face validity, particularly in light of findings showing that experimenter-administered alcohol can produce significantly different effects on brain function and behavior compared to voluntarily self-administered alcohol42. Moreover, the voluntary nature of the paradigm allows researchers to capture individual differences in intake similar to the variability in alcohol preference and drinking patterns observed in humans. These individual differences can provide a powerful opportunity for researchers to correlate consumption (intake and/or patterns) with the magnitude of pathological effects observed.
Nevertheless, some limitations of the two-bottle home cage drinking paradigm should be considered. For example, although individual variability in ethanol intake can be advantageous for some experiments, it may be a disadvantage in other experiments where a consistent level of intoxication or ethanol exposure may be desirable. This is particularly true for experiments examining the consequences of dependence and withdrawal from chronic ethanol exposure. Importantly, two-bottle choice drinking is not characterized as a model of dependence - even in rats with access to ethanol for prolonged periods of time. Thus, in the absence of accompanying symptoms of withdrawal, researchers should use caution in interpreting the consequences of prolonged ethanol consumption as effects of dependence and/or withdrawal. Indeed, it is possible, if not plausible, that rats drinking relatively large quantities of ethanol during the two-bottle choice procedure exhibit symptoms of withdrawal but that rats drinking lower total volumes in a given 24 h period do not show similar signs of dependence. Drinking patterns can also play an important role in this consideration, as two animals drinking the same amount over a 24 h period can experience vastly different levels of intoxication (i.e., BEC) depending on the pattern of their ethanol intake22. This highlights the utility of LIQ HDR in the two-bottle choice procedure as it can readily identify rats that consume a given dose of ethanol at a fast rate (i.e., gulp) versus those who consume the same dose at a much slower rate (i.e., sip). Although potentially undesirable for studies investigating the effects of a controlled dose of ethanol, detection of changes in drinking patterns (e.g., reduced binge episodes) following a particular experimental intervention has significant translational value given the harm reduction approach that is increasingly pursued in the clinic. Finally, effort is not required in order for animals to gain access to ethanol in home cage drinking paradigms, including the one described here. Therefore, the utility of this paradigm is limited to analysis of drinking alone, whereas operant methods are favored for instances when researchers are interested in examining alcohol seeking - an important feature of AUD.
In summary, the protocol described here provides researchers with all the information and resources necessary to measure ethanol intake in rats in the home cage setting. We present options for relatively low- and high-resolution approaches providing researchers with the flexibility to choose the method that is most suitable to their research needs.

Although moderate alcohol use has historically been associated with moderate health benefits, recent large-scale comprehensive studies have revealed that no amount of alcohol consumption is safe1,2. In fact, alcohol use is the 7th leading risk factor for death and disability globally1 with individuals who drink even small amounts of alcohol having increased risk for cancers, infectious diseases, and injury1,3. In the United States, deaths from alcohol use increased by almost 30% between 2016 and 20214. Importantly, the risk of death and disability increases monotonically with increased consumption1.
Although studies in humans have provided valuable insight into the neurobiology of alcohol use and misuse, the experimental control afforded by animal models is crucial for an in-depth understanding of the mechanisms underlying drinking behavior and risk for heavy drinking. These models are also valuable tools for the development of treatments aimed at reducing uncontrolled consumption. Two-bottle choice home cage drinking is one of the most widely used preclinical paradigms to study alcohol consumption in rodents. This is due, in large part, to its ease of implementation as it allows for the measurement of total voluntary alcohol drinking and preference without the need for specialized research equipment or complex analyses. Using this approach, researchers can collect measurements at their desired intervals (e.g., hours, days, weeks) by calculating the change in bottle weight from the beginning to the end of a drinking session. Variations on this basic method have been used by many in the field to facilitate low to moderate to binge levels of alcohol intake over both short and long periods of time. For example, continuous and intermittent two-bottle choice procedures have been used to facilitate low and moderate levels of alcohol intake, respectively, in both mice5 and rats6,7,8. The same paradigms can promote high levels of intake in alcohol-preferring strains7,9. Alternatively, an adaptation of this method that limits alcohol access to 2-4 h daily beginning approximately 3 h after the start of the dark cycle has been shown to engender binge drinking in mice (i.e., Drinking in the Dark)10,11. Additional variations on these methods have been used in conjunction with methods that facilitate alcohol dependence (i.e., chronic intermittent ethanol vapor exposure) to examine the escalation of voluntary alcohol drinking during withdrawal12,13,14,15,16,17,18,19.
Although widely adopted by the field, the standard approach of measuring alcohol intake by change in bottle weight is limited in resolution to total consumption per drinking session. Consequently, this approach is unable to capture drinking patterns, which are well known to impact both the risk for and severity of alcohol use disorder (AUD)20,21,22. Indeed, an individual&#39;s average intake over time can be a consequence of several heavy drinking episodes or many drinking episodes each associated with relatively low consumption and these nuances go undetected when consumption is measured as total intake per drinking session. Importantly, significantly more drinking episodes associated with large drinking bouts (i.e., gulping) are observed in individuals with AUD relative to healthy controls23 and data in nonhuman primates suggests that individuals who establish a pattern of alcohol consumption that includes large drinking bouts early in their drinking history are at significant risk for AUD22. Moreover, drinking episodes that include large bouts of consumption significantly increase risk for morbidity and mortality independent of AUD diagnosis21. Altogether, these data have led to increased interest in examining drinking patterns in addition to levels of overall intake in preclinical models and has prompted the development of a number of systems of varying complexity that are able to capture drinking data at the level of individual licks.
Among the first was the development of a two-bottle system equipped with photobeam lick detection designed for use in mice24. This system was subsequently adapted by a separate group for use in rats25. In the years since, variations on this theme have been used to develop similar systems contained within custom rodent housing26, that allow for lick detection of individual socially housed animals27,28, and that capture both feeding and drinking behavior29. Among these, LIQ HD (Lick Instance Quantifier Home cage Device)30 has garnered significant attention for offering a system for use in mice that parallels the ease of implementation and affordability of the system developed by Godnyuck et al.24. This system utilizes capacitive sensing to directly detect contact between the tongue and the conductive metal sipper tube, as opposed to indirect measures such as sipper approach or interaction that are afforded using the photobeam detection system. The authors provide data demonstrating that their capacitive sensing system affords significantly greater precision and sensitivity in lick detection, producing stronger correlations between lick number and change in bottle weight than photobeam detection. The authors further demonstrate the ability to use this system in conjunction with a continuous access two-bottle choice home cage drinking paradigm to capture measures of alcohol drinking microstructure in addition to measures of overall daily intake30.
Here, we describe procedures for two-bottle choice home cage alcohol drinking using a standard approach as well as methods that enable lick detection (Figure 1). We also introduce methods for building and implementing LIQ HDR - an adaptation of LIQ HD30 for use in rats that includes hardware modifications to accommodate larger cage sizes and a redesigned 3D-printed bottle holder compatible with standard off-the-shelf bottles. These methods provide researchers with the flexibility to use either a standard or lickometer-equipped approach depending on their experimental resources and data collection needs.
Below is a description of the materials needed and step-by-step instructions for capturing home cage drinking using the intermittent-access two-bottle choice drinking procedure adapted from Simms et al.7. In this procedure, rats are provided 24 h access to two drinking bottles 7 days a week. On Mondays, Wednesdays, and Fridays (MWF), rats receive one bottle containing water (H2O) and another bottle containing ethanol (EtOH). These bottles are removed on Tuesdays, Thursdays, and Saturdays (TRS) and replaced with two bottles of water. Rats drink from the same two bottles of water on Saturdays and Sundays. Note that these methods can easily be adjusted for alternate schedules of ethanol access (e.g., continuous, binge, etc.) if desired. Separate instructions are provided for standard and lickometer-equipped approaches.

<table><tbody><tr><td>&lt;strong&gt;Commercially Available Components&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Quantity&lt;/strong&gt;</td></tr><tr><td>12-Key capacitive touch sensor breakout (MPR121)</td><td>Adafruit</td><td>1982</td><td>3</td></tr><tr><td>2-Pin cable matching pair (JST XH 2.5mm pitch)</td><td>Adafruit</td><td>4872</td><td>18</td></tr><tr><td>4-Pin breadboard jumper (Qwiic)</td><td>SparkFun</td><td>PRT-17912</td><td>1</td></tr><tr><td>4-Pin cable (100mm Qwiic)</td><td>SparkFun</td><td>PRT-17259</td><td>1</td></tr><tr><td>4-Pin cable (50mm Qwiic)</td><td>SparkFun</td><td>PRT-17260</td><td>3</td></tr><tr><td>5V-to-3V level shifter breakout</td><td>Adafruit</td><td>5637</td><td>1</td></tr><tr><td>Capacitive touch shield (2.8inch)</td><td>Adafruit</td><td>1947</td><td>1</td></tr><tr><td>Copper conductive tape (1/4inch)</td><td>DigiKey</td><td>4393-CFT-1/4-ND</td><td>1</td></tr><tr><td>Data logging shield</td><td>Adafruit</td><td>1141</td><td>1</td></tr><tr><td>Differential I2C communication breakout kit (QwiicBus)</td><td>SparkFun</td><td>KIT-17250</td><td>1</td></tr><tr><td>Electrical tape</td><td>DigiKey</td><td>3M156004-ND</td><td>1</td></tr><tr><td>Heat shrink tubing (3/16inch various colors)</td><td>Cable Ties and More</td><td>HS357-S10</td><td>6</td></tr><tr><td>Heat shrink tubing kit (4inch black)</td><td>Jameco</td><td>2095963</td><td>1</td></tr><tr><td>Lithium coin battery (3V 12.5mm)</td><td>DigiKey</td><td>1908-CR1220JAUCHSB-ND</td><td>1</td></tr><tr><td>M5 flat washer</td><td>Grainger</td><td>54FN66</td><td>1</td></tr><tr><td>M5 hex nut</td><td>Grainger</td><td>6CA72</td><td>1</td></tr><tr><td>M5 machine screw (12mm)</td><td>Grainger</td><td>6GU88</td><td>1</td></tr><tr><td>M5 machine Screw (25mm)</td><td>Grainger</td><td>&amp;nbsp;6GU91</td><td>2</td></tr><tr><td>Male/male jumper wire</td><td>Adafruit</td><td>1957</td><td>1</td></tr><tr><td>Microcontroller board (Arduino Mega 2560 Rev3)</td><td>DigiKey</td><td>1050-1018-ND</td><td>1</td></tr><tr><td>PETG filament (2.85mm silver)</td><td>MatterHackers</td><td>M-DDH-UZR2</td><td>2</td></tr><tr><td>PLA filament (2.85mm black)</td><td>MatterHackers</td><td>M-SEW-RUAW</td><td>2</td></tr><tr><td>PLA filament (2.85mm silver)</td><td>MatterHackers</td><td>M-66P-6CLE</td><td>1</td></tr><tr><td>Power supply (DC 9V 1A)</td><td>Newegg</td><td>9SIBPPKJYR5991</td><td>1</td></tr><tr><td>SD/microSD memory card (8GB)</td><td>Adafruit</td><td>1294</td><td>1</td></tr><tr><td>Shield stacking headers</td><td>Adafruit</td><td>85</td><td>1</td></tr><tr><td>Solid-core wire (22AWG red)</td><td>Adafruit</td><td>288</td><td>1</td></tr><tr><td>Stranded-core wire (22AWG black)</td><td>Adafruit</td><td>2976</td><td>1</td></tr><tr><td>Stranded-core wire (22AWG red)</td><td>Adafruit</td><td>3068</td><td>1</td></tr><tr><td>Twist cap sipper tube with ball</td><td>Alternative Design</td><td>TCCN8.5-ST2.5SB</td><td>38</td></tr><tr><td>Water bottle (8oz)</td><td>Alternative Design</td><td>WB8FS</td><td>38</td></tr><tr><td>&lt;strong&gt;Custom 3D-Printed Components&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Quantity&lt;/strong&gt;</td></tr><tr><td>3D-printed Arduino case bottom</td><td>N/A</td><td>ARDUINO_Bottom.stl</td><td>1</td></tr><tr><td>3D-printed Arduino case top</td><td>N/A</td><td>ARDUINO_Top.stl</td><td>1</td></tr><tr><td>3D-printed cable clip L</td><td>N/A</td><td>CLIP_L.stl</td><td>18</td></tr><tr><td>3D-printed cable clip nut</td><td>N/A</td><td>CLIP_Nut.stl</td><td>18</td></tr><tr><td>3D-printed cable clip R</td><td>N/A</td><td>CLIP_R.stl</td><td>18</td></tr><tr><td>3D-printed lickometer bottle holder</td><td>N/A</td><td>LICKOMETER_Bottle Holder.stl</td><td>19</td></tr><tr><td>3D-printed lickometer cable protector L</td><td>N/A</td><td>LICKOMETER_Cable Protector_L.stl</td><td>6</td></tr><tr><td>3D-printed lickometer cable protector R</td><td>N/A</td><td>LICKOMETER_Cable Protector_R.stl</td><td>12</td></tr><tr><td>3D-printed lickometer letters</td><td>N/A</td><td>LICKOMETER_Letters.stl</td><td>19</td></tr><tr><td>3D-printed mount screw</td><td>N/A</td><td>Mount Screw.stl</td><td>4</td></tr><tr><td>3D-printed sensor case A bottom</td><td>N/A</td><td>SENSOR_A_Bottom.stl</td><td>1</td></tr><tr><td>3D-printed sensor case A top</td><td>N/A</td><td>SENSOR_A_Top.stl</td><td>1</td></tr><tr><td>3D-printed sensor case B bottom</td><td>N/A</td><td>SENSOR_B_Bottom.stl</td><td>1</td></tr><tr><td>3D-printed sensor case B top</td><td>N/A</td><td>SENSOR_B_Top.stl</td><td>1</td></tr><tr><td>3D-printed sensor case C bottom</td><td>N/A</td><td>SENSOR_C_Bottom.stl</td><td>1</td></tr><tr><td>3D-printed sensor case C top</td><td>N/A</td><td>SENSOR_B_Top.stl</td><td>1</td></tr><tr><td>3D-printed sensor case letters</td><td>N/A</td><td>SENSOR_Letters.stl</td><td>1</td></tr><tr><td>3D-printed sipper blocker</td><td>N/A</td><td>SIPPER_Blocker.stl</td><td>36</td></tr><tr><td>3D-printed sipper blocker base</td><td>N/A</td><td>SIPPER_Base.stl</td><td>18</td></tr><tr><td>3D-printed sipper blocker gear</td><td>N/A</td><td>SIPPER_Gear.stl</td><td>18</td></tr><tr><td>3D-printed sipper blocker nut</td><td>N/A</td><td>SIPPER_Nut.stl</td><td>18</td></tr><tr><td>3D-printed sipper blocker worm</td><td>N/A</td><td>SIPPER_Worm.stl</td><td>18</td></tr><tr><td>Laser-cut lickometer acrylic panel</td><td>N/A</td><td>LICKOMETER_Acrylic Panel_Sketch.dxf</td><td>19</td></tr></tbody></table>

modeling alcohol consumption in rodents using two-bottle choice home cage drinking and microstructural analysis

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, with daily intake measured by change in bottle weight over the 24 h period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lick detection systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD (Lick Instance Quantifier Home cage Device), has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs.

Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lick detection system that enables microstructural analysis of drinking behavior.

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are ...

modeling-alcohol-consumption-rodents-using-two-bottle-choice-home

Nanyang Technological University

Research

JoVE Journal

Neuroscience

388 Views.  University of Illinois Chicago. This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lick detection system that enables microstructural analysis of drinking behavior.

Video: Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Kinematic Analysis Using 3D Motion Capture of Drinking Task in People With and Without Upper-extremity Impairments

Kinematic analysis is a powerful method for objective assessment of upper extremity movements in a three-dimensional (3D) space. Three-dimensional motion capture with an optoelectronic camera system is considered as golden standard for kinematic movement analysis and is increasingly used as outcome measure to evaluate the movement performance and quality after an injury or disease involving upper extremity movements. This article describes a standardized protocol for kinematic analysis of drinking task applied in individuals with upper extremity impairments after stroke. The drinking task incorporates reaching, grasping and lifting a cup from a table to take a drink, placing the cup back, and moving the hand back to the edge of the table. The sitting position is standardized to the individual&#39;s body size and the task is performed in a comfortable self-paced speed and compensatory movements are not constrained. The intention is to keep the task natural and close to a real-life situation to improve the ecological validity of the protocol. A 5-camera motion capture system is used to gather 3D coordinate positions from 9 retroreflective markers positioned on anatomical landmarks of the arm, trunk, and face. A simple single marker placement is used to ensure the feasibility of the protocol in clinical settings. Custom-made Matlab software provides automated and fast analyses of movement data. Temporal kinematics of movement time, velocity, peak velocity, time of peak velocity, and smoothness (number of movement units) along with spatial angular kinematics of shoulder and elbow joint as well as trunk movements are calculated. The drinking task is a valid assessment for individuals with moderate and mild upper extremity impairment. The construct, discriminative and concurrent validity along with responsiveness (sensitivity to change) of the kinematic variables obtained from the drinking task have been established.

This protocol describes an objective method to evaluate the movement performance and sensorimotor function of the upper extremity applied to individuals with stroke and healthy controls. A standardized test procedure, kinematic analysis and outcome variables for three-dimensional motion capture of drinking task are provided.

Kinematic analysis is a powerful method for objective assessment of upper extremity movements in a three-dimensional (3D) space. Three-dimensional motion ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

Medicine

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...

Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

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Chapters in this video

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

Deep Brain Stimulation with Simultaneous fMRI in Rodents

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Kinematic Analysis Using 3D Motion Capture of Drinking Task in People With and Without Upper-extremity Impairments

Intracranial Pharmacotherapy and Pain Assays in Rodents

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder