Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely behaving mouse models of neuropathology.
Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely behaving mouse models of neurodegenerative diseases.
The development of technology to manipulate genes has produced transgenic and knockout mouse models of almost every neurodegenerative and neurological diseases. This has necessitated translation of electrophysiological research techniques previously used in larger rodent species to the mouse animal model. One such neurophysiological investigation technique is the use long-term potentiation (LTP) to test the efficacy of synaptic connections within neuronal networks involved in various neuropathological disorders. This protocol describes techniques for reliable electrophysiological investigation of LTP in freely behaving mice. The advantage of this protocol over others is that it is simple and easy to implement; it is also rather less costly as it does not require neither the use of expensive computer-controlled microdrive systems nor field effect transistor headstages; and, to our knowledge, is the first video protocol of chronic electrophysiological recordings to study LTP in freely behaving mice. To this end, we describe in this article simple methodologies for studying long-term potentiation in freely behaving mice. These methodologies can readily be translated to transgenic and knockout mouse models of neuropathological disorders.
This protocol is appropriate for mice of 3 and 18 months of age and approximate body weight of 30-50 g). Mice can be obtained from The Jackson Laboratory (Bar Harbor, ME). All surgical and experimental protocols were approved by the Trinity College Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
1. Animal Preparation and Surgical Procedures
2. LTP Induction
Table 1 shows the coordinates for DG and mPP as used in this protocol. Figure 1A shows the markings for the target structures on the skull; also shown are the location of the ground and reference electrodes. Figure 1B illustrates representative evoked response traces both pre- and posttetanization in the same animal. Note that the posttetanization evoked response is larger than the pretetanization response which is indicative of LTP induction2. Figure 1C illustrates the method used to quantify the response amplitude. Indeed, Figure 2 shows percent change in response amplitude over a time course spanning both pre- and posttetanization time periods. It is to be noted that peak LTP values exceeded 100%. These results of enhanced response amplitude following tetanization indicate that this protocol is successful and therefore reliable for studying LTP in the freely behaving mouse model.
Table 1. Coordinates for target structures. Anterior-posterior (AP) coordinates for dentate gyrus (DG) and REF are given relative to bregma while those for medial perforant path (mPP) and GND are relative to Lambda. All DV coordinates are relative to the cortical surface below dura.
Figure 1. Electrode location and representative traces of the evoked response. A) Diagrammatic illustration of the relative location of electrodes on the mouse skull. B) Typical traces of the evoked response both pre- and posttetanization. C) Algorithm used to quantify the amplitude of the evoked response.
Figure 2. LTP in the medial perforant path-dentate gyrus synapse. Representative result of LTP induction in mPP-DG synapse of a freely behaving mouse. Note posttetanic stimulation enhancement of the evoked response amplitude indicative of LTP induction.
In this protocol, we have demonstrated a reliable and simple method for studying LTP in DG in freely behaving mice. While many studies of LTP in awake rats have been performed3,4, very few have been conducted in awake mice primarily due to the technical complexity posed by the limited cranial real estate in mice and the weight of electrode headstages relative to the average weight of mice5. The few studies that have demonstrated LTP in DG in freely behaving mice utilized either microdrive electrode systems or junction field effect transistor (jFET) preamplifiers integrated in the headstage which necessarily adds to the electrode payload burden to the animal6-10. The significance of the present protocol is that it represents an improvement over existing methods for inducing LTP in the DG in freely behaving mice as it avoids the use of microdrive electrode systems or a headstage JFET’s.
It is important to highlight the critical steps of this protocol. These include: 1) mounting of the mouse’s head in the stereotaxic frame such that it is bilaterally rigid (this step may be the most difficult as it requires practice and familiarity with the technique); 2) optimal positioning of electrodes to maximize response magnitude can be enhanced by insuring that bregma and lambda are in the same plane which can usually be achieved by adjusting the stereotaxic frame tooth bar such that the difference in dorsoventral positioning of bregma and lambda is no greater than 0.1 mm; 3) during the recording phase of the experiment, it is recommended that animals be allowed time to habituate to the recording environment because the novelty of the recording environment may insinuate fluctuations in the evoked response data collected; 4) suitable selection of electrodes will improve signal quality and fidelity: bipolar electrodes (stainless steel hypodermic tubing with 0.2 mm stainless steel wire insert with a tip separation of 0.5 mm) are preferred for stimulating while monopolar electrodes (epoxylite-insulated single strand tungsten wire) are used for recording in nervous tissue; and 5) mice can be anesthetized using an intraperitoneal injection of a mixture of ketamine (25 mg/ml), xylazine (2.5 mg/ml) and acepromazine (0.5 mg/ml). This anesthetic mixture should then be administered at a dosage of 1ml/kg which is usually effective in about 20 min supplemented by 0.2 ml/kg every 45 min to maintain a stable depth of anesthesia.
There are a few limitations of this protocol which bear mentioning. This protocol does not give any insight on ion channel mechanisms or receptor protein synthesis that may subserve LTP. Therefore scant information is available concerning the actual numbers of neurons in the population being recorded. Another limitation is that since evoked responses are being collected in awake animals it is difficult to ascertain and dissect out the effect of factors such as stress, handling, or contamination of the recorded signal by spurious sensorimotor activity. These limitations can be overcome by ensuring that evoked responses are recorded only when the animal is the inactive but alert state, otherwise known as the quiet waking vigilance state.
Nonetheless, the techniques described in this protocol provide the most physiologically relevant platform for investigating brain electrical activity underlying behavior. Following the steps outlined in this protocol, any brain structure can be targeted by using the proper coordinates as given by an atlas of the mouse brain1. It is important to note that the adult rat stereotaxic frame can be used in mice provided that suitable infant rat ear cuffs are used instead of the regular ear bars. The ear cuffs will immobilize the head without damaging the mouse’s ears. Finally, the methodologies presented here can be readily translated to electrophysiological investigations in transgenic and knockout mouse models of a host of neurological disorders.
The authors have nothing to disclose.
The authors wish to acknowledge the following: Dr. Joseph Bronzino, Dr. Khamis Abu-Hassaballah, Mr. R.J. Austin-LaFrance, and Ms. Jessica Koranda.
Ketamine (100 mg/ml) | Henry Schein | 10177 | |
Xylazine (20 mg/ml) | Henry Schein | 33197 | |
Acepromazine (10 mg/ml) | Henry Schein | 2177 | |
Dental acrylic powder | Lang Dental Manufacturing Co. | 1330CLR | |
Dental acrylic liquid | Lang Dental Manufacturing Co. | 1306CLR | |
Tungsten wire (0.127 mm) | World Precision Instruments | TGW0515 | |
Stainless Steel Hypodermic Tubing (0.286 mm) | World Precision Instruments | 832400 | |
Flunixin (50 mg/ml) | Henry Schein | 14165 | |
Epoxilyte | Superior Essex | EP 6001-M | |
Stainless steel wire insert (0.2 mm) | World Precision Instruments | 792900 | |
Stereotaxic frame apparatus | Kopf Instruments | Model 902 | |
Ear cuffs (ear cups) | Kopf Instruments | Model 921 | |
Electrophysiological stimulator | Astro-Med, Inc. | S88 | |
Digital oscilloscope | B & K Precision Corp. | 2542 | |
Current isolation unit | Astro-Med, Inc. | PSIU-6 | |
Differential amplifier | World Precision Instruments, Inc. | DAM-50 | |
Commutator | Plastics One | SLC6 | |
Dental drill | Stoelting | 58650 |