Sensory Input and Behavior
Implications of Immediate-Early Gene Induction in the Brain Following Sexual Stimulation of Female and Male Rodents

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Abstract

Induction of immediate-early genes (IEGs), such as c-fos, has been widely used to mark the activation of brain regions following different types of sexual stimulation and behavior. A relatively common set of hormone-concentrating basal forebrain and midbrain structures in female and male rodents is activated by copulatory stimulation, in particular, stimulation of sensory nerves that innervate the penis or vagina/cervix, olfactory or pheremonal stimuli, and conditioned sexual incentives. These regions include the preoptic area, lateral septum, bed nucleus of the stria terminalis, paraventricular hypothalamus, ventromedial hypothalamus, medial amygdala, ventral premammillary nuclei, ventral tegmentum, central tegmental field, mesencephalic central gray, and peripeduncular nuclei. Regions that do not contain classic intracellular steroid receptors, such as the ventral and dorsal striatum or cortex, are also activated. IEGs have also been colocalized with cytoplasmic proteins like GnRH and oxytocin, and have been used in conjunction with retrograde tracers to reveal functional pathways associated with different sexual behaviors. Steroid hormones can also alter the ability of sexual stimulation to induce IEGs. Despite the many similarities, some differences in IEG induction between sexes have also been found. We review these findings and raise the question of what IEG induction in the brain actually means for sexual behavior, that is, whether it indicates the perception of sexual stimulation, commands for motor output, or the stimulation of a future behavioral or neuroendocrine event related to the consequences of sexual stimulation. To understand the role of a particular activated region, the behavioral or neuroendocrine effects of lesions, electrical stimulation, drug or hormone infusions, must also be known.

Introduction

Much of what is known about the neuroanatomical and neurochemical mechanisms that underlie appetitive and consummatory aspects of sexual behavior comes from studies that have examined the effects of systemic or intracranial administration of hormones or drugs on animal sexual behaviors, extracellular recording in brain regions during the presentation of sexually relevant stimuli, or the effects of electrical stimulation or lesions of discrete brain areas on different sexual behaviors 72, 82. The limited application of in vivo techniques, such as microdialysis and voltammetry, has made it possible to quantify changes in extracellular concentrations of certain neurotransmitters in discrete brain regions during ongoing sexual activity (e.g., dopamine; 24, 67, 83), and to correlate such changes with specific sexual behaviors. Recent advances in immunocytochemistry and molecular histology (e.g. in situ hybridization) have also made it possible to visualize and quantify neurochemical, protein synthetic, and stimulus transduction events that occur within single neurons during sexual activity. Ex vivo detection of the mRNA or protein products of immediate-early genes (IEGs), such as those of the fos, jun, and egr-1 families, have proven exceptionally useful in marking regions of the brain that are activated during sexual behavior or as a result of certain types of sexual stimulation. In contrast to metabolic markers like 2-deoxyglucose, the protein products of these genes are expressed in the nucleus and thus provide cellular resolution. It is possible to identify neurochemical subgroups of cells that are activated by sexual stimulation by examining the colocalization of the protein products of these genes with known cytoplasmic proteins. Retrograde or anterograde tracers can also be colocalized with the protein products of these genes to provide a map of “functional” neural circuitry.

Although studies that have utilized IEGs as markers of neuronal activation have helped to localize brain regions that may be involved the control of sexual behavior, they have also created considerable uncertainty as to what IEG expression actually means. Simply observing an induction of Fos protein in a brain region following copulation does not tell us very much, except that something about the experience of copulation induced Fos in that brain region. The interpretation is made all the more complex when differences in hormone treatment, differences in the amount or intensity of sexual stimulation, or differences in the patterns of sexual behavior are considered. What the induction might mean in a particular brain region also depends on prior knowledge of the role of that region in the behavior, something that requires more information based on other techniques (e.g., effects of lesions, electrical stimulation, drug or hormone infusions, etc.). The flow of information can also work the other way. A particular sensory stimulation or behavioral act, for example, ejaculation, may induce Fos selectively within a particular brain region. If the role of that region in behavior is unknown, it can be subsequently examined using discrete lesions, or electrical or chemical stimulation. Thus, the use of a combination of techniques brings converging operations to bear on questions of function, and provides a far more powerful means to assess the role of brain regions in behavior than any of the techniques could alone. Because c-fos has been the predominant IEG examined, much of our discussion will concentrate on the induction of Fos mRNA and protein.

IEGs are currently viewed as part of the signal transduction cascade by which neurons convert extracellular chemical or electrical signals into early genomic activation [75]. Because early studies found that Fos mRNA and protein were inducible in brain by a variety of physiological and pharmacological stimuli, Fos was proposed as a marker of functional activation of neurons (e.g., [101]). However, what the “function” might be requires more information than Fos alone can provide, and the term “activation” can mean many things. Fos is transcribed by the action of several proteins, including CREB proteins (Cyclic AMP Response Element Binding proteins) that are phosphorylated in response to Ca2+ influx or cAMP turnover, Serum Response Element proteins, and also by a relatively direct action of growth factors [75]. Obviously, it is possible that neuronal depolarization can induce Fos through Ca2+ influx, for example, following glutamate release [3], although depolarization and Fos induction have never been shown together in the same neurons. Some neurotransmitters work through G-protein–coupled adenylate cyclase, which can induce Fos as a function of increased cAMP turnover (which can phosphorylate CREB proteins). Such receptor activation could result from cellular depolarization, as in the case of muscarinic receptor agonists [52], but it could also result from cellular hyperpolarization, as in the case of dopaminergic agonists that bind to receptors of the D1 family 6, 10. Thus, from an electrophysiological standpoint, Fos induction could indicate either the excitation or inhibition of neuronal firing during the signal transduction event. However, before either one of these can be said with any certainty, the colocalization of Fos with these receptor types must be shown.

In cells that are not terminally differentiated, IEGs like Fos play a causal role in bringing about different phases of the cell cycle (e.g., [78]). This has led to the general idea that IEGs can induce long-term changes in cell function and morphology. It is unknown what long-term effects IEG induction could have on neuronal function, although it is possible that structure, receptor affinity or number, enzymes, and neurotransmitter synthesis could all be affected. However, such long-term changes would be specific to the proteins transduced, rather than the induction of IEGs per se. In addition, for Fos induction to produce any genomic changes in a neuron, proteins of the jun family (e.g., Jun, Jun-B, Jun-D) must be present in that neuron. Fos and Jun proteins form stable heterodimers around pallindromic AP1 sites (TGACTCA) found in the promoter regions of different genes. Thus, genes that contain AP1 sites within neurons that express Fos and Jun proteins following a particular stimulus could be subsequently transduced and translated into protein.

The relevance of IEG induction to behavior or sensory stimulation is equally complex. Induction of IEGs in neurons localized to a particular brain region indicate that a signal transduction event has taken place in that region. Regarding sexual behavior, such induction could be related to neural activity induced by different types of sensory stimulation during sexual behavior. It could be related to neural activity required for motor outputs during sexual behavior. It could also be an early indication of the activation of brain regions that contribute to various future behaviors or neuroendocrine events associated with the consequences of sexual stimulation (e.g., sexual reward, sexual exhaustion, estrus termination, pregnancy, or pseudopregnancy). In all cases, such induction could be enhanced or blunted by treatment with steroid hormones or drugs that alter the ability of neurons to process sensory inputs, even if the hormones or drugs themselves do not induce the IEG.

Finally, IEGs are sometimes not induced in brain regions that have previously been reported to contain neurons that increase their firing rates during sensory stimulation or behavior (e.g., [50]). In many cases, such regions (e.g., the nucleus paragigantocellularis in the brainstem) consist of neurons with spontaneous and high baseline firing rates prior to stimulation [79]. Such neurons can express IEGs following high-level electrical stimulation (unpublished observations), but sensory stimulation does not move these neurons above a critical threshold for the induction. Thus, in rare cases where Fos or other IEGs are not observed within a particular region, it does not necessarily mean that the region is not activated, especially if prior electrophysiological studies have shown significant increases in cell firing rate or lesion studies have indicated the involvement of the region. Similarly, there is sometimes a functional mismatch between IEG activation in a particular region and the results of lesion studies. For example, lesions of the retrorubral field (RRF) of male gerbils eliminate mounting [33], but Fos is induced in that region by handling alone, with no further increases observed following copulation [45]. Thus, both electrophysiological and functional mismatches can occur. Therefore IEG induction alone cannot unequivocally determine the function of an area.

Section snippets

Functional Analysis of Brain Regions Activated by Sexual Stimulation

Numerous studies since 1991 have shown that a relatively common set of brain regions express Fos or Jun in male and female rats and hamsters, and male gerbils, following specific types of sexual stimulation (Fig. 1) This is perhaps the most important and unexpected finding of this literature, because the behaviors that stimulate the inductions are quite different. This suggests that (1) different behavioral patterns contribute to a common set of afferent sensory inputs, (2) different sensory

IEG Induction and the Consequences of Sexual Stimulation

At first glance, the observation that IEGs are induced in discrete brain regions as a function of different types of sexual stimulation comes as no surprise. Surely, the activation of regions known from lesion and electrical stimulation studies to be involved in male or female sexual behavior, such as the MPOA or VMH, would be expected. But what of regions known to inhibit sexual behavior, such as the lateral septum? What if the Fos is actually induced by a signal transduction event that

Conclusions

Induction of Fos and other IEGs have helped researchers identify discrete regions in the brain activated by particular types of sexual stimulation. Such information is of critical importance because it allows the temporal relationship between sensory stimulation and neural activation to be specified. Clearly, more work is required to identify sex- and species-related differences in IEG induction, and to identify the exact sensory inputs to, and outputs from, activated brain regions. However, to

Acknowledgements

The experiments from our respective laboratories reported in this article were supported by grants from the Medical Research Council of Canada (MT-13125) and Fonds pour la Formation de Chercheurs et l’Aide à la Recherche du Québec (96-CE-103) to J.G.P. at Concordia University, NIMH Training Grant (MH-14599) to the Department of Psychobiology at the University of California, Irvine, and NIMH MERIT Award (MH-26481) and NIMH RSDA Award (MH-00478) to MMH’s advisor, Dr. Pauline Yahr. We would like

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