Using ICSS to Study Mood States in Rodents
William A. Carlezon Jr., Ph.D. and Elena H. Chartoff, Ph.D. Department of Psychiatry Harvard Medical School McLean Hospital Belmont, MA

It is becoming increasingly important to assess mood states in laboratory animals. Dramatic advances in molecular biology now offer unprecedented opportunities to manipulate gene and protein expression in discrete areas of the brain. In turn, these new techniques enable fine-grain analyses of how molecules affect behavior. Tests that reflect reward, reduced ability to experience reward (anhedonia) and/or aversion (dysphoria) are in high demand because many psychiatric conditions that are currently intractable in humans (e.g., major depression, bipolar disorder, addiction) are characterized by dysregulated motivation. For these reasons, we utilize intracranial self-stimulation (ICSS) to understand how pharmacological or molecular manipulations affect the function of brain reward systems.

ICSS is an operant paradigm in which rodents self-administer rewarding electrical stimulation (often referred to as brain stimulation reward [BSR]) through electrodes implanted into the brain. Though often used interchangeably, ICSS is the behavior and BSR is what is earned. Many brain areas will support ICSS, but we implant the stimulating electrodes into the medial forebrain bundle (MFB) for our studies. Stimulation of this pathway produces reliable ICSS at relatively low current intensities, and is associated with few (if any) of the motor side effects that can occur with other electrode placements (e.g., ventral tegmental area [VTA]). Presumably, MFB stimulation activates glutamate inputs to the mesolimbic dopamine system, thereby transynaptically activating this brain reward pathway. We have shown that manipulations in projection regions of the VTA (e.g., the nucleus accumbens [NAc]) have profound effects on ICSS behavior (Carlezon and Wise, 1996; Todtenkopf et al., 2006), confirming that this pathway plays an important role in BSR. ICSS has several other practical advantages, including the fact that BSR is relatively impervious to anxiety or satiation, unlike many natural rewards (food, sexual behavior). Well-trained rats or mice will engage in ICSS for hours (or, if allowed, even days), often to the exclusion of every other behavior.

Several ICSS methodologies can be used to quantify BSR, including the curve-shift, detection threshold, and autotitration variants. Investigators who use ICSS tend to be extremely loyal to their preferred method. In our case, we use the curve-shift variant because it enables us to generate “response rate-frequency” relationships across a range of stimulation parameters that are analogous to “dose-effect” functions in pharmacology. We vary the frequency (Hz) rather than the intensity (µA) of the stimulation because we want to keep constant the population of neurons that is activated. The device used to deliver the stimulation is an important consideration. We use Programmable ICSS Stimulators available from Med Associates (PHM-152). In conjunction with MED PC control software, these devices can be programmed to deliver the precise stimulus trains that are critical to make this test work reliably. We use these stimulators together with Med Associates operant conditioning chambers (ENV-307CT for mice, or ENV-007CT for rats) fitted with levers for rats (ENV-110M), response wheels for mice (ENV-113AM), and stimulus lights (e.g. ENV-315M).

Typically, we test our animals (rats, mice) in a series of 15 1-min trials, each at a different stimulation frequency. Each 1-min trial (Fig. A) comprises a 5-sec period where the animal receives free (priming) trains (1 per sec) of the cathodal (monopolar) stimulation that is available, followed by a 50-sec period where the number of responses (lever presses for rats, wheel revolutions for mice) is counted, followed by a 5-sec time out period during which the frequency is lowered in a 0.05-log unit step (this time out period, during which no stimulation is available, can be conceptualized as either the end of a trial or the beginning of the next one). Each 15-min series of frequencies (trials) produces a “response rate-frequency” function: animals typically press at maximal rates for high frequencies, intermediate rates for moderate frequencies, and negligible rates for low frequencies (Fig. B). There are several ways to quantify stimulation “thresholds”, defined here as the minimum frequency of stimulation required to sustain responding at some arbitrary rate. The most intuitive measure is “half maximum” (or M₅₀), which is analogous to an ED₅₀ in pharmacology. However, manipulations that affect response capabilities (e.g., cause increases or decreases in maximal response rates) can cause artificial shifts in this measure. Accordingly, we quantify ICSS thresholds using Theta-0 (T₀), which provides an estimate of the theoretical frequency at which the stimulation becomes rewarding (i.e., response rates become greater than 0). T0 is minimally sensitive to treatment-induced alterations in response capabilities (Fig. C) (Miliaressis et al., 1986).

Many manipulations can affect the efficacy of BSR. This is reflected by shifts in the functions that relate response rates to stimulation frequency (Fig. B). For simplicity, these shifts are often expressed (or described) as “percent baseline threshold”. Generally, each day the animal is first tested to establish baseline (pretreatment) thresholds, and then again immediately after the treatment. Alternatively, chronic treatment effects can be compared to a pre-manipulation baseline, since baseline thresholds tend to be remarkably stable over weeks and months in well-trained animals. Treatment-induced leftward shifts in ICSS thresholds (Fig. B, green curve) imply that the stimulation is more rewarding as a result of a treatment (reflecting hyperfunction of brain reward systems), whereas rightward shifts (Fig. B, red curve) imply that it is less rewarding (reflecting hypofunction of reward systems). Drugs of abuse decrease the amount of stimulation required to sustain responding, indicated by leftward shifts in rate-frequency functions and decre ased ICSS thresholds. Conversely, agents that block drug reward (e.g., dopamine antagonists) increase the amount of stimulation required to sustain responding, indicated by rightward shifts in rate-frequency functions and increased thresholds. These agents also block the ability of rewarding drugs to cause leftward shifts. Drug withdrawal—which produces the symptoms of major depression in humans—also cause rightward shifts and elevations in ICSS thresholds. Thus ICSS is sensitive to manipulations that cause increased reward, decreased reward (anhedonia), or increased aversion (dysphoria).

It is important to remember BSR is simply one type of reward. When interpreting ICSS data, the conclusions must be inferential, and carefully considered within the context of this paradigm. That is, the fact that drugs of abuse such as cocaine reduce ICSS thresholds implies that the rewarding effects of cocaine add to the rewarding effects of the stimulation, making lower amounts of the stimulation more effective. Thus cocaine has reward-related (or reward-facilitating) effects—rather than rewarding effects, per se—in this assay.

Outstanding reviews on the effects of drugs of abuse or drug withdrawal on ICSS are available (e.g., Wise, 1996; Barr et al., 2003). Recently, we have been using ICSS to examine how molecular manipulations affect BSR and, by extension, the function of brain reward systems. As one example, we have used viral vectors to induce selective alterations in the expression of glutamate receptor subunits in the NAc, which produce profound effects on ICSS (Todtenkopf et al., 2006). We have also successfully utilized ICSS in mutant mice, to establish causal relationships between genetics and behavior. Mice with a defect in the function of their circadian gene CLOCK show mania-like signs: not only are they more sensitive to the reward-related effects of cocaine, they are also substantially more sensitive to BSR itself, requiring far less stimulation to sustain responding (Roybal et al., 2007). The combined use of ICSS and molecular approaches may yield improved models of psychiatric illness and addiction, ultimately leading to an improved understanding of brain function.

References

Barr AM, Markou A, Phillips AG (2003) A 'crash' course on psychostimulant withdrawal as a model of depression. Trends Pharmacol Sci 23:475-482

Carlezon WA Jr, Wise RA (1996) Microinjections of phencyclidine (PCP) and related drugs into nucleus accumbens shell potentiate brain stimulation reward. Psychopharmacol 128: 413-420

Miliaressis E, Rompre, P-P, Laviolette P, Philippe L, Coulombe D (1986) The curve-shift paradigm in self-stimulation. Physiol Behav 37: 85-91

Roybal K, Theobold D, Birnbaum S, DiNieri JA; Graham A, Russo S, Vaishnav K, Kumar A, Peevey J, Oehrlein N, Vitaterna M, Orsulak P, Takahashi JS, Nestler EJ, Carlezon WA Jr, McClung CA (2007) Mania-like behavior induced by disruption of CLOCK function. Proc Natl Acad Sci USA 104: 6406-6411

Todtenkopf MS, Parsegian A, Neve RL, Carlezon WA Jr (2006) Brain reward regulated by glutamate receptor subunits in the nucleus accumbens shell. J Neurosci 26: 11665-11669

Wise RA (1996) Addictive drugs and brain stimulation reward. Ann Rev Neurosci 19: 319-340