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Readers of this book cannot help but be aware of the pre-eminence of the laboratory rat in behavioral pharmacology. Rats are relatively inexpensive, docile, easy to maintain creatures, and there is a good file of behavioral and pharmacological literature pertaining to the rat. There is, also, a strong tradition in many behavioral testing programs to acquaint students with the laboratory rat at an early stage in their training. Having come from such a tradition, I worked with rats for some time until I found myself in a position of low funding with mice available ad libitum through an experimental breeding program in our biology department. To my surprise many of the standard procedures which have traditionally employed rats can be duplicated with mice. There are occasional problems associated with the small size of the mouse, but often the procedures are actually easier with mice than rats. In many cases the availability of strains of mice differing in the distribution of dopamine, serotonin, or opiate receptor sites makes studies possible that could not be accomplished with oth possible that could not be accomplished with other species.

This chapter outlines some of the situations where mice are useful and appropriate subjects and warns of some potential problems. The emphasis will be on the positive aspect of the mouse as an experimental animal in the study of drug effects on reward.

There are three basic attributes of mice which make them a valuable research tool. First, mice can be inexpensive, especially if you breed your own. Second, mice come in several strains some of which have interesting properties; for instance, CXBK mice are not analgesic at standard doses of morphine (Oliverio, Castellano, & Eleftheriou, 1975; Reith, Sershen, Vadasz, & Lajtha, 1981). And third, mice do not show stress responses when they are lightly restrained by immobilizing their tails (Moran & Straus, 1980). This makes operant work with electrical brain stimulation or microinjections much easier because there are no swivels or commutators needed. During restraint, intravenous injections can be made via the tail veins without the difficulty of surgically implanting a carotid catheter.

Mice can be prepared with stereotaxically implanted electrodes or guide cannulae using a slight modification of the methods used for rats. A standard Kopf small animal stereota for rats. A standard Kopf small animal stereotaxic instrument or probably any small stereotaxic device can be used. On the Kopf instrument the nose clamp will have to be removed (It unscrews.) as it extends over the skull of an animal as small as a mouse. The standard ear and incisor bars work, but specially designed mouse ear and incisor bars are available. To keep the mouse’s nose from rotating upward in the Kopf instrument, tape the nose down with masking tape. Stereotaxic atlases are available from either Sidman, Angevine, & Pierce (1971) or Lehmann (1974). The only differences between mouse and rat implant surgery relate to the size and metabolic rate of mice. They usually require more anesthetic (70 to 80 mg/kg of sodium pentobarbital), and screw placement is critical. The mouse skull is very thin and in order to get a good grip, the screws (0-80 by 1/8 inch) must be placed into the frontal skull over the olfactory bulbs. They can be placed immediately posterior to the lateral skull vein which is a major landmark on the mouse skull. Bilateral screws over the olfactory bulbs and one posterior screw placed laterally near the skull ridge will anchor an electrode platform securely. We rarely experience dislodged electrode platforms using this placement during the first 4 weeks of testing. Prolonged retention has not been as good. Few animals retain their electrodes longer than one year; plans to maintain animals for demonstration or screenin animals for demonstration or screening work using this technique are not feasible. For intracranial self-stimulation (ICSS) or multiple unit recording, 36 gauge Teflon-coated, Nichrome electrodes work well. We have been able to show a fair degree of anatomical specificity in ICSS work using 36 gauge electrodes (Criswell, 1982a). Guide cannula can be implanted for microinjections using standard stereotaxic techniques or, alternatively, microinjections can be made in etherized mice by making a scalp incision, by visualizing landmarks (The junction between the inferior and superior colliculi are easily visualized through the skull.), and by inserting a 26 gauge hypodermic needle through the skull followed by a 30 gauge injection cannula with a block to regulate depth (Criswell, 1976).

Both ICSS and self-administration studies often use operant technology where a response at some sort of manipulandum—usually a lever press—results in delivery of a reinforcer such as ICSS, intravenous or intracerebral-ventricular administration of a drug. A peculiarity of the mouse makes it an ideal subject for operant studies of drug effects on reinforcement. A mouse can be lightly restrained by taping its tail to a surface. During restraint there is little evidence of stress (Moran & Straus, 1980). By placing a mouse inside an operant chamber with its tail protruding operant chamber with its tail protruding through a hole in one wall and by taping the tail down, you have a very useful preparation. The mouse is able to emit operant responses (We use a nosepoke, but a lever press should work also.), but it is unable to circle and its tail is always available. This means that you can do electrical stimulation or recording without a commutator. Commutators are not much of a problem for stimulation, but they are often a source of noise in recording studies. Intracranial microinjections can be performed without a swivel connector (Swivel connectors are problematic with mice.); and, best of all, you can do both at the same time or even microinject through two cannulae simultaneously while stimulating or recording through a pair of electrodes. The tail is always available, and intravenous injections through a lateral tail vein are not too difficult in a mouse. One hint here is to place the mouse under a heat lamp for a short period of time to raise its body temperature. Mice regulate body temperature in part by dilation of the tail veins, and it makes insertion of an intravenous needle much easier.

Figure 1 shows the all purpose operant chamber that we have been using for a couple of years. The dimensions of the chamber (8 cm front to back by 9 cm side to side by 10 cm high) are such that when the mouse’s tail is placed through the hole in the back wall and taped down, it can reach either of the holes (ma, it can reach either of the holes (manipulanda) in the front of the cage. A light and photocell are placed across the hole so that a nosepoke response breaks the light beam and darkens the photocell.

A simple and inexpensive way to interface the operant chamber to control and data acquisition facilities is via a microcomputer. Any microcomputer with a game paddle input such as the Vic-20, Commodore-64, Apple II+, or Radio Shack color computer has a built-in photocell interface. By connecting the photocell across the paddle input, you have replaced the variable resistor in the paddle with the variable resistance of the photocell. Figure 2 shows an interface for a Vic-20 microcomputer. The Vic-20 is available for under $100 in many parts of the world. This system will allow the use of two manipulanda. If you want to use a lever, just replace the photocell with the normally closed contacts of the switch. The resistors in series with the photocell are needed because the Vic-20 cannot handle a rapid decrease in resistance to near zero. It won’t hurt the microcomputer to leave them out, but a fast mouse can confuse the computers analog/digital (A-D) converter resulting in an occasional missed response. The parallel input/output (I-O) port is capable of driving a transistor with each data bit, and the circuit shown allows the Vic-20 to control the current of a constant voltage source by placing different resistors in series with the voltagnt resistors in series with the voltage. As shown the output will change by 1 part in 16 as the number poked into the output port is incremented or decremented. The current level is controlled by the four relays. If you want more resolution, simply add one more relay with a resistance of one half that of the lowest shown and change the stimulation program to count to 32 instead of 16. The program in the Appendix will run an autotitration schedule using this apparatus. In an autotitration schedule responses at one manipulandum result in reinforcement, but the magnitude of the reinforcer is reduced with continued responding. A response at a second manipulandum resets the magnitude of reinforcement to its original level. This allows determination of the subject’s preferred magnitude of reinforcement and is similar to a threshold determination where you find the lowest reinforcement magnitude for which the animal is willing to work. If you remove line 562, it will record response rate at both manipulanda but reinforce responses at only one, and the current will not be decremented every fifth response (a simple right-left discrimination task). If you want to use this apparatus for drug self-administration, simply connect one of the relays to an infusion pump, and you have a right-left discrimination procedure where responses at one manipulandum deliver the drug and responses at the other manipulandum serve as a control for changes in dum serve as a control for changes in activity level. Figure 3 illustrates a mouse working at an autotitration task with this. Figure 4 shows a dose-response curve for morphine effects on response rate at a low current (about 10% above threshold) and high current (where response rate started to decline with increased current). Vigorous responding was obtained at all doses using the high current stimulation while it dropped considerably—but not to zero—at the lower stimulation level. Using 36 gauge electrodes in the perifornical lateral hypothalamic area, thresholds for ICSS ranged between 2 and 30 mA of 60 Hz sinewave stimulation for 1/3 second. These levels are lower than those obtained with rats using larger electrodes. The effectiveness may be related to current density, and the smaller electrode tips can be an important factor in regulation of current density. We usually start testing at a current of 6 mA rather than the 50 mA commonly used in rats.
 

Figure 1: All-purpose two-manipulandum operant chamber with a mouse prepared for simultaneous electrical stimulation and intracerebral and intravenous injectrical stimulation and intracerebral and intravenous injections.

Figure 2: Interface circuitry for a Vic-20 microcomputer. A shows the input to the computer. Two photocells are connected to the game port. The transistor drives a Sonalert Alarm (SA) which signals a correct response. B is an array of switches (SW 1-4) which controls the resistance in series with a constant voltage source (STIM). Detail of the switches is shown in C. An NPN transistor acts as an inverting buffer for the output port and drives a S.T.D.P. relay. When the relay is on, it passes current through the resistors (R). SW1 passes the least current; SW2 passes twice as much, et cetera. The value of the resistors must be in multiples of 2. We use 8 megaohms for SW1, 4 for SW2, 2 for SW3, and 1 megaohm for SW4.

Figure 3: A typical testing station. The stimulation cable is hanging from a ring stand. Since the mouse cannot rotate in the cage, electrical or chemical stimulation of discrete brain sites is a simple procedure.

Figure 4: Dose-response curves for the effect of morphine on high and low current ICSS in ICR mice using a nosepoke response.

Use of the nosepoke response results in a rapid acquisition of the response when ICSS is used as a reinforcer and may be superior to the more commonly used lever-press response. The nosepoke, wheel-turn (Kornetsky, Esposito, McLean, & Jacobson, 1979), and shuttling responses (Levitt, Baltzer, Evers, Stillwell, & Furby, 1978) are all natural, easily acquired responses for rats. We have found that mice rapidly acquire shuttling (Criswell & Starnes, 1980) and nosepoking (Criswell, 1983).

Allowing animals to control ICSS duration and time between stimuli in a shuttle-box offers a convenient way to simultaneously measure appetitive and aversive aspects of ICSS. Levitt et al., (1977)imultaneously measure appetitive and aversive aspects of ICSS. Levitt et al., (1977) have examined opiate effects on ICSS in rats using this procedure, and it has been adapted for mice by Cazala, Cazals, and Cardo (1974), Cazala and Garrigues (1980, 1983), Cazala and Guenet (1980). A workable shuttle-box for mice can be constructed by making a Plexiglas box 26 cm long, 10 cm wide, and 10 cm high with a grid floor 1 cm above the bottom of the box. A hinged top with a 1/2 cm slit running longitudinally and a similar slit joining the open side forming a "t" will allow you to place a mouse in the cage and to slide the stimulation cable into the slit in the top. If you then close the top and tape the short arm of the "t" so that the cable will not catch in it as it slides by, you will have an inexpensive and useful shuttle-box. The box can be interfaced to recording and control equipment by balancing it on a fulcrum and by resting one end on a microswitch. As the mouse shuttles, the microswitch will be on when it is on one side of the cage and off when it is on the other. The simplest interface is via a microcomputer. Our system is run by an antique SYM-1 microcomputer which is no longer in production so I cannot include a program, but it should be a simple process to program any microcomputer with a built-in clock to rack on- and off-times and to supply current at the proper times. The relay system described in Figure 2 will work for control of ICSS, and closure of a microswitch can beS, and closure of a microswitch can be sensed by replacing the photocells shown in the figure with the normally closed contacts of a microswitch.

By simply counting cage crossings without stimulation, you can use the shuttle-box to measure activity level. Figure 5 shows the effects of morphine on activity level for two strains of mouse. By making electrical stimulation contingent on the mouse being in one side of the cage, you can measure the effects of drugs on the preferred duration of stimulation (on-time) and time between stimuli (off-time). In this situation it is a good idea to have your control apparatus automatically turn the stimulus on if the animal leaves it off for more than some criterion time (I use 30 seconds.) or they may just lay down on the off side and sleep or groom. As number of shuttles as a function of time is allowed to vary, on- and off-times are independent. Figure 6 shows differences in the effects of morphine on on- and off-times of two different mouse strains. You might want to compare the effects of morphine on on- and off-times to its effect on activity level for these two strains (Figure 5). Note that the morphine selectively increases on-times in BALB/c mice while decreasing them in C57BL/6 mice. The BALB/c mice acted like rats (Baltzer, Levitt, & Furby, 1977), but the C57BL/6 mice acted quite differently. Off-times were not affected in either strain. We only obtain this pattern of responses relnly obtain this pattern of responses reliably with electrodes within 0.5 mm of the fornix (Criswell, 1983a).
 

Figure 5: Dose-response curve for the effect of morphine and morphine plus naloxone on activity level of BALB/c and C57BL/6 mice.

Figure 6: Dose-response curves for the effect of morphine and morphine plus naloxone on preferred stimulus duration (ON-times) and preferred time between stimuli (OFF-times) in BALB/c and C57BL/6 mice.

There are certainly other operant techniques which can be used with mice, and the non-operant procedures such as conditioned place preference described in previous chapters should be easily adaptable to the mouse. In cases where cost of maintaining an animal colony is important or where only small quantities of an experimental drug are available, the momal colony is important or where only small quantities of an experimental drug are available, the mouse has much to recommend it. The other prime reason for using mice as experimental subjects is the availability of several specialized strains of mouse.

The common Swiss-Webster or ICR mouse is available through most lab animal suppliers. They breed easily and have large litters (12 to 16 pups) and are, therefore, inexpensive. Current prices from suppliers run about a dollar each. They can be bred in a standard laboratory animal facility without difficulty. They can be comfortably housed five to a cage in disposable plastic rodent cages. Mice make nests whenever possible and have sticky urine. For those reasons it is probably not wise to keep them in standard screen floored rat cages. Mice can be safely picked up by the tail but should not be carried for long distances that way. It is convenient to set the mouse on the back of a hand while transporting it between cages. These mice are albino and have rather poor vision. Other strains would be more appropriate for visual discrimination work. Also, the ICR or Swiss mouse is an outbred strain, and there is quite a bit of variability between mice. Where variability in either behavior or biochemistry is a primary concern, one of the inbred strains would be more appropriate.

One of the most useful features of the mouse is the availability of several inbred strains which show different pharmacological and behavioral responses to abused drugs. Many of the inbred strains are difficult to breed and may be expensive or only occasionally available. They are typically designated by sometimes obscure codes. One of the more commonly used inbred strains is the BALB/c. BALB stands for Bag albino, and the letters after the slash represent a particular substrain. In spite of the origin of BALB mice from one pair, different breeding colonies produce BALBs with different characteristics. A BALB/cJ (J for Jackson Laboratories) is a different animal from a BALB/byJ. Some strains such as the CXBK are f2 backcrosses from two inbred strains and are only produced periodically and may run several dollars each when available. In general, the inbred strains are smaller (20 to 30 grams) than the Swiss or ICR mouse (30 to 40 grams). The particularly interesting areas where strain effects have been documented involve responses to opiates, dopaminergic compounds, and ethanol.

I have compiled a non-exhaustive set of references regarding the use of mice in ICSS and self-administration studies (see Table 1). I have also listed studies relating strain differences in responses to opiates, biogenic amines, and ethanol.
 

Baltzer, J. H., Levitt, R. A., & Furby, J. E. (1977). Etorphine and shuttle-box self-stimulation in the rat. Pharmacology Biochemistry & Behavior, 7, 413-416.

Baran, A., Schuster, L., Eleftheriou, B. E., & Bailey, D. W. (1975). Opiate receptors in mice: Genetic differences. Life Sciences, 17, 633-640.

Berger, B., Herve, D., Dolphin, A., Barthelemy, C., Gay, M., & Tassin, J. P. (1979). Genetically determined differences in noradrenergic input to the brain cortex: A histochemical and biochemical study in two inbred strains of mice. Neuroscience, 4, 877-888.

Carroll, B. J., & Sharp, P. T. (1972). Monoamine mediation of the morphine-induced activation of mice. British Journal of Pharmacology, 46, 124-139.

Castellano, C. (1980). Dose-dependent effects of heroin on memory in two inbred strains of mice. Psychopharmacology, 67, 235-239.

Castellano, C. (1981). Strain dependent effects of the enkephalin analogue FK33-824 on locomotor activity in mice. Pharmacology Biochemistry & Behavior, 15, 729-734.

Cazala, P., Cazals, Y., & Cardo, B. (1974). Hypothalamic self-stimulation in three inbred strains of mice. Brain Research, 81, 159-167.

Cazala, P., & Garrigues, A. M. (1980). An apparent genetic relationship between appetitive and aversive effects of lateral hypothalamic stimulation in the mouse. Physiology & Behavior, 25, 357-361.

Cazala, P., & Garrigues, A. M. (1983). Effects of apomorphine, clonidine or 5-methoxy-NN-Dimethyltryptamine on approach and escape components of lateral hypothalamic and mesencephalic central gray stimulation in two inbred strains of mice. Pharmacology Biochemistry & Behavior, 18, 87-93.

Cazala, P., & Guenet, J. (1980). The recombinant inbred strains: A tool for the genetic analysis of differences observed in the self-stimulation behavior of the mouse. Physiology & Behavior, 24, 1057-1060.

Cheng, R. S., & Pomeranz, B. (1979). Correlation of genetic differences in endorphin systems with analgesic effects of D-amino acids in mice. Brain Research, Brain Research, 177, 583-587.

Collins, R. L., & Whitney, G. (1978). Genotype and test experience determine responsiveness to morphine. Psychopharmacology, 56, 57-60.

Crabbe, J. C., Janowsky, J. S., Young, E. R., Kosobud, A., Stack, J., & Rigter, H. (1982). Tolerance to ethanol hypothermia in inbred mice: Genotypic correlations with behavioral responses. Alcoholism: Clinical and Experimental Research, 6, 446-458.

Criswell, H. E. (1976). Analgesia and hyperactivity following morphine microinjection into mouse brain. Pharmacology Biochemistry & Behavior, 4, 23-26.

Criswell, H. E. (1982a). A simple methodology for opiate self-administration and electrical brain stimulation in the mouse. Life Sciences, 31, 2391-2394.

Criswell, H. E. (1982b). Effect of opiates on perifornical reward areas. Anatomical Record, 204, 395-396.

Criswell, H. E., & Ridings, R. (1983). Intravenous self-administration of morphine by naive mice. Pharmacology Biochemistry & Behavior, 18, 467-470.

Criswell, H. E., & Starnes, D. M. (1980). Effect of morphine on preferred duration of electrical brain stimulation in the mouse. Society for Neuroscience Abstracts, 6, 309.

Daszuta, A., Faudon, M., & Hery, F. (1984). In vitro H-serotonin (5-HT) synthesis and release in BALBc and C57BL mice. -HT) synthesis and release in BALBc and C57BL mice. II. Cell body areas. Brain Research Bulletin, 12, 565-570.

Daszuta, A., Hery, F., & Faudon, M. (1984). In vitro H-serotonin (5-HT) synthesis and release in BALBc and C57BL mice. I. Terminal areas. Brain Research Bulletin, 12, 559-563.

Filibeck, U., Castellano, C., & Oliverio, A. (1981). Differential effects of opiate agonists-antagonists on morphine-induced hyperexcitability and analgesia in mice. Psychopharmacology, 73, 134-136.

Frischknecht, H. R., Siegfried, B., Riggio, G., & Waser, P. G. (1983). Inhibition of morphine-induced analgesia and locomotor activity in strains of mice: A comparison of long-acting opiate antagonists. Pharmacology Biochemistry & Behavior, 19, 939-944.

Gentry, R. T., Rappaport, M. S., & Dole, V. P. (1983). Elevated concentrations of ethanol in plasma do not suppress voluntary ethanol consumption in C57BL Mice. Alcoholism: Clinical and Experimental Research, 7, 420-423.

Gilliam, D. M., & Collins, A. C. (1982). Circadian and genetic effects on ethanol elimination in LS and SS mice. Alcoholism: Clinical and Experimental Research, 6, 344-349.

Gilliam, D. M., & Collins, A. C. (1983). Concentration-dependent effects of ethanol in long-sleep and short-sleep mice. Alcoholism: Clinical and Experimental Research, 7, 337-342.

Gwynn, G. J., & Domino, E. F. (1984a). Genotype-dependent behavioral sensitivity to mu vs. kappa opiate agonists. I. Acute and chronic effects on mouse locomotor activity. Journal of Pharmacology and Experimental Therapeutics, 231, 306-311.

Gwynn, G. J., & Domino, E. F. (1984b). Genotype-dependent behavioral sensitivity to mu vs. kappa opiate agonists. II. Antinociceptive tolerance and physical dependence. Journal of Pharmacology and Experimental Therapeutics, 231, 312-316.

Huidobro-Toro, J. P., & Way, E. L. (1981). Comparative study on the effect of morphine and the opioid-like peptides in the vas deferens of rodents: Species and strain differences, evidence for multiple opiate receptors. Life Sciences, 28, 1331-1336.

Kiianmaa, K., Hoffman, P. L., & Tabakoff, B. (1983). Antagonism of the behavioral effects of ethanol by naltrexone in BALB/c, C57BL/6, and DBA/2 mice. Psychopharmacology, 79, 291-294.

Kokkinidis, L., & Zacharko, R. M. (1980). Intracranial self-stimulation in mice using a modified hole-board task: Effects of d-amphetamine. Psychopharmacology, 68, 169-171.

Kornetsky, C., Esposito, R. U., McLean, S., & Jacobson, J. O. (1979). Intracranial self-stimulation thresholds: A model for the hedonic effects of drugs of abuse. Archives of General Psychiatry, 26, 289-292. General Psychiatry, 26, 289-292.

Lehmann, A. (1974). Atlas stereotaxique du cerveau de la souris. Paris: Centre National De La Recherche Scientifique.

Levitt, R. A., Baltzer, J. H., Evers, T. M., Stillwell, D. J., & Furby, J. E. (1977). Morphine and shuttle-box self-stimulation in the rat: A model for euphoria. Psychopharmacology, 54, 307-311.

Maarten, E. A., Reith, H. S., Vadasz, C., & Lajtha, A. (1981). Differences in opiate receptors in mouse brain. European Journal of Pharmacology, 74, 377-380.

McSwigan, J. D., Crabbe, J. C., & Young, E. R. (1984). Specific ethanol withdrawal seizures in genetically selected mice. Life Sciences, 35, 2119-2126.

Millard, W. J., & Dole, V. P. (1983). Intake of water and ethanol by C57BL mice: Effect of an altered light-dark schedule. Pharmacology Biochemistry & Behavior, 18, 281-284.

Moran, R. E., & Straus, J. J. (1980). A method for establishing prolonged intravenous infusions in mice. Laboratory Animal Science, 30, 865-867.

Oliverio, A., Castellano, C., & Eleftheriou, B. E. (1975). Morphine sensitivity and tolerance: A genetic investigation in the mouse. Psychopharmacologia, 42, 219.

Reggiani, A., Battaini, F., Kobayashi, H., Spano, P., & Trabucchi, M. (1980). Genotype-dependent sensitivity to morphine: Role of different opiate receptor populatorphine: Role of different opiate receptor populations. Brain Research, 189, 389-294.

Reith, E. A., Sershen, H., Vadasz, C., & Lajtha, A. (1981). Strain differences in opiate receptors in mouse brain. European Journal of Pharmacology, 74, 377-380.

Sansone, M., Ammassari-Teule, M., Renzi, P., & Oliverio, A. (1981). Different effects of apomorphine on locomotor activity in C57BL/6 and DBA/2 mice. Pharmacology Biochemistry & Behavior, 14, 741-743.

Seale, T. W., McLanahan, K., Johnson, P., Carney, J. M., & Rennert, O. M. (1984). Systematic comparisons of apomorphine-induced behavioral changes in two mouse strains with inherited differences in brain dopamine receptors. Pharmacology Biochemistry & Behavior, 21, 237-244.

Severson, J. A., Randall, P. K., & Finch, C. E. (1981). Genotypic influences on striatal dopaminergic regulation in mice. Brain Research, 210, 201-215.

Sidman, R., Angevine, J. B., & Pierce, E. T. (1971). Atlas of the mouse brain and spinal cord. Cambridge, MA: Harvard University Press.

Siegfried, B., Frischknecht, H., & Waser, P. G. (1984). Defeat, learned submissiveness, and analgesia in mice: Effect of genotype. Behavioral and Neural Biology, 42, 91-97.

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