Chapter 1
Methods and Approaches to Understand Stress Processing Circuitry

James P. Herman and Brent Myers

Department of Psychiatry and Behavioral Neuroscience, University of Cincinnati, Cincinnati, Ohio, USA

Photomicrograph demonstrating the GABAergic phenotype of central amygdala (CeA) neurons projecting to the posterior bed nucleus of the stria terminalis (pBST). The retrograde tracer Fluorogold was injected into the pBST and visualized by immunohistochemistyry in the cell bodies of CeA neurons (brown). This was followed by in situ hybridization for GAD (glutamate decarboxylase) 65 mRNA (black grains), a marker for the GABAergic neurons. The co-localization of Fluorogold and GAD65 mRNA indicates that projections from the CeA to the pBST produce gamma-aminobutyric acid (GABA) and are inhibitory, putatively leading to disinhibition of the hypothalamic-pituitary-adrenocortical axis.

1.1 Introduction

Understanding mechanisms of neuroendocrine stress regulation requires the use of anatomical approaches to precisely localize neuronal populations that control physiological output. For example, the activation of the hypothalamo-pituitary-adrenocortical (HPA) axis is mediated in large part by 2000-4000 neurons (corticotropin releasing hormone neurons) located in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN), one of about 10 anatomically distinct subdivisions of this nucleus (Swanson and Sawchenko, 1983). Moreover, recent data suggest that upstream control of the HPA axis may be differentially regulated by subpopulations of neurons located within a defined brain region (e.g. the bed nucleus of the stria terminalis; Choi et al., 2007). Finally, regulatory changes of critical functional importance to HPA axis output can be subsumed by subpopulations of neurochemically distinct neurons within the PVN proper, which can be masked when the nucleus is considered as a unit. For example, vasopressin mRNA is normally expressed at very low levels in the medial parvocellular PVN, but is very abundant in neighbouring magnocellular neurons. When animals are adrenalectomized, glucocorticoid feedback inhibition of parvocellular neurons is lost, whereas magnocellular neurons are unaffected. Thus, despite the 8-fold increase in vasopressin mRNA expression in the parvocellular zone, which is involved in a massive drive of pituitary ACTH release, no net change is observed in whole-PVN vasopressin mRNA content due to dilutional effects of the magnocellular signal. All of these issues highlight the need to study central stress regulatory circuits in their anatomical context.

Understanding the anatomical context of stress processing is critical for both delineating normal homeostatic adaptive processes and those that culminate in stress pathologies, including such diverse diseases as post-traumatic stress disorder (PTSD), depression, cardiovascular disease and the metabolic syndrome. The current chapter is designed to provide an orientation on both tried and true and state-of-the-art approaches to stress circuit study, concentrating on methods used to characterize circuit activation, connectivity and function.

1.2 Assessment of stress activation

Tracing stress-regulatory circuits requires the use of methods to report activation of brain regions under study. In order for a particular set of neurons to regulate stress responses, one assumes that they should be either activated or deactivated during stimulation, be it discrete (acute) or prolonged. In the past, investigators have used measures of glucose metabolism (2-deoxyglucose autoradiography) or mitochondrial activity (cytochrome oxidase staining) to reveal activated pathways. In recent years, these rather crude methods have been replaced by use of molecular markers that precisely report cellular activation. This section will concentrate on molecular markers, as they afford single-cell resolution with the capacity to be combined with circuit mapping methods.

1.2.1 Markers of acute activation: Fos

The gold standard for mapping acute stress activated neurons employs methods to localize the immediate early gene, cfos. The cfos gene expression is driven by calcium signalling pathways, which are reliably activated during neuronal stimulation (Morgan and Curran, 1989). Post-stimulus induction of cfos gene expression is extremely rapid, due to the fact that transcription is initiated by removal of an arrest signal in the promoter region (Schroder et al., 2013). Moreover, cfos mRNA is unstable and rapidly degraded and, similarly, Fos protein has a relatively short half-life. This combination of ubiquitous localization, rapid transcription, rapid degradation and short protein half-life allows for assessment of rapid cellular activation against a background of virtually zero in the unstimulated state. These properties make cfos gene and Fos protein detection tools an excellent means by which to visualize activated neurons in the brain. While other immediate early genes (such as egr1 and arc) increase transcription after stressor exposure (Cullinan et al., 1995; Ons et al., 2004) (Table 1.1), to date cfos is the only gene that exhibits this 'on-off' property and makes it the method of choice for assessment of stress circuit activation in an anatomical context.

Table 1.1 Commonly-used Markers of Cellular Activation (partial list)

Assessment of cfos induction can be performed using either in situ hybridization for cfos mRNA or immunohistochemistry for Fos protein. In situ hybridization using radiolabelled probes has the advantage of affording quantitation of the extent of stress-induced cfos induction, using densitometric analysis (off X-ray film or phosphorimager screens) or grain count analysis from emulsion dipped sections. Since the cfos mRNA is rapidly transcribed and degraded, peak levels are typically observed within 30 minutes of stimulus (stressor) onset and generally return to baseline within 90 minutes to 2 hours. Due to the time lag between transcription, translation and generation of a detectable pool of protein, Fos immunoreactivity typically peaks 90-120 minutes after stimulus onset and returns to baseline within 4 hours (it should be kept in mind that both synthesis and degradation will be subject to modification by stressor duration or intensity, so the above estimates should be considered a general rule of thumb rather than a firm guideline). Quantification of an absolute amount of protein by immunohistochemistry is problematic. However, this method of detection is suitable for analysis of numbers of activated neurons. Moreover, detection of Fos can be combined with immunohistochemistry for other antigens (dual or triple immunofluorescence) or transgene/knock-in fluorescent markers to phenotype-activated neurons or with fluorescent tracers to identify projections of activated neurons (see Section 1.3 on 'Tract Tracing'). Immunohistochemistry is also quicker and cheaper than in situ hybridization (generally a 2-3 day procedure, with no radioisotope or disposal costs) and is generally the method of choice when information on the extent of induction is not required.

Use of cfos as an activational marker has been invaluable in identifying stress-activated pathways. However, its use comes with some caveats. First, one cannot assume that a cfos negative cell is not activated. There are numerous examples of cell populations that are electrophysiologically active after stress but do not show cfos induction (e.g. the CA1 region of the hippocampus). Second, cfos does not lend clear information on cellular inhibition, as neurons that are inhibited do not show a cfos signal. Finally, cfos is rapidly induced by the initiation of stress and, consequently, differences in post-excitation shut-off may not be visible as a reduction in the number of Fos (protein)-activated neurons detected by...