Physiology & Behavior 89 (2006) 501–510 Activation in neural networks controlling ingestive behaviors: What does it mean, and how do we map and measure it? ⁎ Alan G. Watts , Arshad M. Khan, Graciela Sanchez-Watts, Dawna Salter, Christina M. Neuner Neuroscience Research Institute and Neuroscience Graduate Program, University of Southern California, Los Angeles, CA 90089-2520, United States Received 27 February 2006; received in revised form 5 May 2006; accepted 25 May 2006 Abstract Over the past thirty years many of different methods have been developed that use markers to track or image the activity of the neurons within the central networks that control ingestive behaviors. The ultimate goal of these experiments is to identify the location of neurons that participate in the response to an identified stimulus, and more widely to define the structure and function of the networks that control specific aspects of ingestive behavior. Some of these markers depend upon the rapid accumulation of proteins, while others reflect altered energy metabolism as neurons change their firing rates. These methods are widely used in behavioral neuroscience, but the way results are interpreted within the context of defining neural networks is constrained by how we answer the following questions. How well can the structure of the behavior be documented? What do we know about the processes that lead to the accumulation of the marker? What is the function of the marker within the neuron? How closely in time does the marker accumulation track the stimulus? How long does the marker persist after the stimulus is removed? We will review how these questions can be addressed with regard to ingestive and related behaviors. We will also discuss the importance of plotting the location of labeled cells using standardized atlases to facilitate the presentation and comparison of data between experiments and laboratories. Finally, we emphasize the importance of comprehensive and accurate mapping for using newly emerging technologies in neuroinfomatics. © 2006 Elsevier Inc. All rights reserved. Keywords: Fos; Neuroinfomatics; MAPK; Hypothalamus; Atlas; Rat; Anorexia; Brain maps 1. Introduction considering the organization of central control networks in behavior, and then consider the meaning of the term ‘activation’ A major focus for behavioral neuroscience is to understand from the viewpoint of neural physiology. We then describe the use the complete functional organization of the neural networks that and limitations of various cellular markers that are currently used initiate, maintain, and terminate specific behaviors. Clarifying to map neural activation. Finally, we will discuss strategies that neural network organization for ingestive behaviors would we can use to incorporate this type of data into standardized reveal which neurons contribute to the behavioral sequence, atlases and databases in a way that will enable investigators to when they contribute, and would provide the basis for further share and compare these complex datasets in a meaningful way, functional investigations into how collectively they can control and to construct testable models of behavioral control networks. eating and drinking. How can we clarify the organization of these networks in a way 2. Behavioral control networks and changes in neural that will help us understand their function? In this review, we will activation address this question from the perspective of mapping patterns of neural activation. We will first discuss a general model for 2.1. Behavioral control networks The sequence of motor actions that make up a behavior ⁎ Corresponding author. Neuroscience Research Institute, Hedco Neurosci- ence Building, MC 2520, University of Southern California, Los Angeles, CA ultimately derives from the changing signaling patterns with- 90089-2520, United States. Tel.: +1 213 740 1497; fax: +1 213 741 0561. in networks that control sensory transduction, central integra- E-mail address: [email protected] (A.G. Watts). tion, and motor selection and execution (Fig. 1; [1,2]). Our 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.05.025 502 A.G. Watts et al. / Physiology & Behavior 89 (2006) 501–510 constituent neurons are the building blocks of network function. Thus, if we can identify the location, chemical phenotype, and projections of the neurons that change their firing rates–or as is often more generally stated, their ‘activity’–at critical times of the behavioral sequence then it should be possible to begin defining the nature of the networks responsible for behavioral initiation, maintenance, and termination. Before we discuss tools and strategies, we will briefly consider the meaning of the term neural activation. An accurate definition becomes important for considering how the most commonly used cellular markers are related to neural function. 2.2. Neural activation The term ‘activation’ when applied to neurons (or indeed to any cell type), implies that some aspect of their function–but usually Fig. 1. A schematic representation of the neural systems and their interactions output–increases over a defined time period. Although ‘activation’ involved with controlling reflex (A) and motivated behaviors (B). Central neural is useful for general discussion, it is often used synonymously with connections are shown in black, and hormonal and feedback signals as dashed lines. The regions that constitute the central behavioral control networks in the increased firing rate, which is considered the neuron's primary brain are enclosed within the gray box in (B). Adapted from [49]. functional output. Closely examining the function of the most commonly used cellular markers together with their associated mechanisms shows that different physiological processes are understanding is clearest at the sensory and motor parts of the being tracked by each of these markers. Moreover, it is generally network because the nature of the sensory mechanisms in the accepted that the function of many commonly used markers is not brain and periphery is fairly easily probed using specific necessarily related directly to changes in firing rate. homeostatic and metabolic signals. For example, we know that Whether a neuron changes its activation state is determined angiotensin II acts on neurons in the subfornical organ (SFO) to by how it integrates its afferent inputs. Part of this integration stimulate drinking, and that leptin, insulin, and ghrelin acting on process involves changing the state of signal transduction neurons in the arcuate nucleus (ARH) have profound effects on pathways, which in turn can alter two fundamental processes feeding behavior [3,4]. Similarly, the neural bases of the simple (Fig. 2): neuronal excitability, by way of changes in membrane motor actions in the consummatory phase are relatively easily potential; and the biosynthesis of a variety of proteins and tracked in the hindbrain, and we know with some degree of peptides, which include transcription factors, proteins involved detail how several of the motor networks in the hindbrain that with transmitter release, receptor proteins, etc. A third cellular control chewing and swallowing are organized [5–9]. But process that is intimately involved with changes in neural ac- located between sensory transduction and motor output are the tivity is the neuron's energy metabolism (Fig. 2). Increases in control networks that add the adaptive value to behavior and firing rate require significant energy expenditure, particularly allow animals to survive successfully in their environment for maintaining Na+/K+-ATPase activity [10]. (Fig. 1). Despite their importance to the organization of be- havior, the constituency and function of these critical circuits 2.3. Tracking neural activation are very poorly understood, for the most part because of their great complexity and the lack of suitable tools for investigation. How do we track changes in neural activity relative to the Fig. 1 posits that the exterosensory and interosensory signals development of a behavior? And if we can do this, what will the that initiate motivated–as opposed to reflex–behaviors require results tell us about the structure and function of the networks? sophisticated processing within central control networks. In There are three main strategies that experimentalists use to track terms of network function at the absolute simplest level, a specific changes in neural activity relative to the development of a behavior is initiated when input signal processing causes some behavior. One is direct electrophysiological recording to correlate neurons within a central control network to increase their firing changes in neural firing patterns in particular brain regions with rate while others are inhibited. These firing rate changes are key the expression of a behavior. A disadvantage with regard to parts of the integration process within the network that ultimately overall network structure is that we need to know the constituents initiates a specific motor sequence. of a given neural control network in advance and with some The way that a behavior emerges from the activity of these degree of certainty; i.e. where to place the electrodes. Electro- control networks is very poorly understood. In fact for all physiology can produce complex and sophisticated sets of data ingestive behaviors our knowledge of the constituent neurons that are extremely useful for determining the signaling properties within central networks together with