Loretta M. Flanagan-Cato

Immunohistochemical mapping of angiotensin type 2 (AT2) receptors in rat brain.

Recently developed antisera selective for angiotensin Type 2 (AT2) receptors were used to localize AT2 receptors in rat brain by immunohistochemistry. While the results from these experiments were largely consistent with previous autoradiographic and radioligand binding analyses of AT2 receptor populations in brain, there were also some notable differences in the distribution of immunoreactivity. More specifically, in agreement with previous studies, AT2 antisera detected apparent receptor populations in the locus coeruleus and the bed nucleus of the accessory olfactory tract, whereas AT2 receptor-immunoreactivity in the cerebellum was primarily associated with the Purkinje cell layer and the deep cerebellar nuclei rather than the molecular layer as has been previously reported in autoradiographic studies. Other regions with prominent immune-staining included all subfields of the hippocampus, which had been previously reported to contain exclusively AT1 receptors. Limbic structures such as the amygdala, thalamic areas such as the rhomboid thalamic nucleus, the paraventricular thalamic nucleus, hypothalamic areas such as the paraventricular hypothalamic nucleus, and the supraoptic nucleus also exhibited prominent AT2-immunoreactivity. In the paraventricular hypothalamic nucleus, AT2 receptor staining appeared to be associated primarily with the magnocellular neurons. In all regions examined, AT2 receptor immunoreactivity was associated with the cytoplasm and cell membrane and was not localized within the nucleus. Collectively, these results confirm and extend the neuroanatomical resolution of previous autoradiographic studies as well as identify new AT2 receptor populations in rat brain.

The Synaptic Organization of VMH Neurons That Mediate the Effects of Estrogen on Sexual Behavior

Estrogen acts in the hypothalamic ventromedial nucleus (VMH) of female rats to promote sexual behavior, as typified by the lordosis response. Morphological changes in the VMH, such as increased synaptic profiles and increased dendritic spines, suggest that estrogen may modulate behavior by altering VMH synaptic organization. To understand the significance of these changes, this laboratory has been investigating the functional classes of lordosis-relevant neurons and their local connectivity. A neurotropic virus, pseudorabies virus (PRV), was used to transneuronally label the CNS network that controls the lordosis-producing muscles. When PRV was placed in the lumbar epaxial muscles, it was sequentially detected in the lumbar ventral horn, the medullary reticular formation, the periaqueductal gray, and finally the VMH. Subsequent analysis showed that the population of VMH neurons that were initially infected with PRV largely resided beyond the cluster of estrogen receptor-containing neurons. In a separate study, VMH neurons were visualized with Lucifer yellow, and their morphology was analyzed using confocal microscopy. Such analysis confirmed that estrogen treatment increased dendritic spines in the VMH. The particular VMH neurons in this study did not express nuclear estrogen receptor, which suggests that estrogen can increase spine density indirectly. These results represent initial steps toward unraveling the local circuit that mediates estrogenic action on a specific reproductive behavior.

Editorial comment: oxytocin, vasopressin and social behavior.

Few brain chemicals have experienced as vibrant a renaissance in neuroendocrinology as the neurohypophyseal peptides oxytocin and vasopressin. Oxytocin (derived from Greek, meaning quick birth) is named for its role in the progression of labor. Oxytocin was the first peptide to have its structure defined and to be synthesized, resulting in a Nobel prize for Vincent du Vigneaud in 1955. The structure and synthesis of vasopressin (derived from Latin, meaning vessel pressure) soon followed, placing these peptides on center stage for chemists and endocrinologists alike. Both peptides have had tremendous success clinically for promoting labor and lactation and for treating diabetes insipidus.

Reverse engineering the lordosis behavior circuit

Reverse engineering takes the facts we know about a device or a process and reasons backwards to infer the principles underlying the structure-function relations. The goal of this review is to apply this approach to a well-studied hormone-controlled behavior, namely the reproductive stance of female rodents, lordosis. We first provide a brief overview on the considerable amount of progress in the analysis of female reproductive behavior. Then, we propose an analysis of the mechanisms of this behavior from a reverse-engineering perspective with the goal of generating novel hypotheses about the properties of the circuitry elements. In particular, the previously proposed neuronal circuit modules, feedback signals, and genomic mechanisms are considered to make predictions in this manner. The lordosis behavior itself appears to proceed ballistically once initiated, but negative and positive hormonal feedback relations are evident in its endocrine controls. Both rapid membrane-initiated and slow genomic hormone effects contribute to the behaviors control. We propose that the value of the reverse-engineering approach is based on its ability to provide testable, mechanistic hypotheses that do not emerge from either traditional evolutionary or simple reductionistic perspectives, and several are proposed in this review. These novel hypotheses may generalize to brain functions beyond female reproductive behavior. In this way, the reverse-engineering perspective can further develop our conceptual frameworks for behavioral and systems neuroscience.

Mineralocorticoids and Glucocorticoids Cooperatively Increase Salt Intake and Angiotensin II Receptor Binding in Rat Brain

Mineralocorticoids, such as deoxycorticosterone acetate (DOCA), and angiotensin II (AngII) act synergistically in the brain to elicit salt appetite. Glucocorticoids, such as dexamethasone (DEX), also may enhance the behavioral effects of DOCA and AngII. However, the brain regions involved in these behavioral interactions have not been elucidated. This study tested the hypothesis that DEX potentiates the effects of DOCA on AngII binding, especially at the AT1 receptor. We confirmed that DEX potentiated the effects of DOCA on salt appetite. Concomitantly, steroid-specific and region-specific changes in AT1 binding were noted. Specifically, in the hypothalamic paraventricular nucleus, treatment with DEX or DOCA + DEX increased AT1 binding. In the subfornical organ (SFO) and area postrema, there was an increase in AT1 binding when both steroids were combined, but not when given individually. However, there was no change in AT2 binding in any brain region studied and no change in AT1 or AT2 binding to either receptor subtype in the pituitary. The results indicate that DOCA and DEX may increase the sensitivity of the brain to the behavioral and physiological actions of AngII by upregulating AT1 receptors in the SFO and area postrema.

Sex differences in the neural circuit that mediates female sexual receptivity

Female sexual behavior in rodents, typified by the lordosis posture, is hormone-dependent and sex-specific. Ovarian hormones control this behavior via receptors in the hypothalamic ventromedial nucleus (VMH). This review considers the sex differences in the morphology, neurochemistry and neural circuitry of the VMH to gain insights into the mechanisms that control lordosis. The VMH is larger in males compared with females, due to more synaptic connections. Another sex difference is the responsiveness to estradiol, with males exhibiting muted, and in some cases reverse, effects compared with females. The lack of lordosis in males may be explained by differences in synaptic organization or estrogen responsiveness, or both, in the VMH. However, given that damage to other brain regions unmasks lordosis behavior in males, a male-typical VMH is unlikely the main factor that prevents lordosis. In females, key questions remain regarding the mechanisms whereby ovarian hormones modulate VMH function to promote lordosis.

Estrogen-induced dendritic spine elimination on female rat ventromedial hypothalamic neurons that project to the periaqueductal gray.

Neurons of the ventromedial hypothalamic nucleus (VMH) that project to the periaqueductal gray (PAG) form a crucial segment of the motor pathway that produces the lordosis posture, the hallmark of female rat sexual behavior. One suggested mechanism through which estrogen facilitates lordosis is by remodeling synaptic connectivity within the VMH. For instance, estrogen alters VMH dendritic spine density. Little is known, however, about the local VMH microcircuitry governing lordosis nor how estrogen alters synaptic connectivity within this local circuit to facilitate sexual behavior. The goal of this study was to define better the neuron types within the VMH microcircuitry and to examine whether estrogen alters synaptic connectivity, as measured by dendritic spine density, on VMH projection neurons. A retrograde tracer was injected into the PAG of ovariectomized rats treated with vehicle or estradiol. Retrogradely labeled VMH neurons were filled with Lucifer yellow, then immunostained for estrogen receptor‐α (ERα). VMH neurons that project to the PAG had more dendrites than functionally unidentified neurons. Additionally, VMH projection neurons could be subdivided into those located within the cluster of ERα‐containing neurons and those medial to the cluster. Estrogen decreased spine density by 57% on the long primary dendrites of VMH projection neurons located within the ERα cluster but not on projection neurons medial to the cluster. Only 4% of the VMH projection neurons expressed ERα. These results suggest that estrogen may facilitate sexual behavior by decreasing spines selectively, via an indirect mechanism, on a subset of VMH neurons that project to the PAG. J. Comp. Neurol. 447:234–248, 2002.

Estrogen-induced remodeling of hypothalamic neural circuitry.

For decades, sexual behavior has been a valuable model system for behavioral neuroscientists studying the neural basis of motivated behaviors. One striking example of a change in motivation is the binary switch in sexual receptivity that occurs during the estrous cycle in female rats. Investigations of the neural basis of this change in behavior have fundamentally advanced our understanding of both behaviorally relevant neural pathways and basic mechanisms of steroid action in the brain. These advances have made this behavioral model system a staple of neuroendocrinology. A challenge that remains before us, given our current understanding of the circuitry and chemistry, is to develop a coherent model of how neural plasticity in the hypothalamus contributes to the dependence of this behavior on motivational state. This review will focus on the ventromedial nucleus of the hypothalamus, especially its ventrolateral subdivision. First, the anatomical, neurochemical, and functional aspects of the macro- and microcircuitry of this brain region will be discussed, followed by a discussion of the likely mechanisms of estrogen action within the ventrolateral VMH. Then, the evidence for estrogen-induced neural plasticity will be considered, including a comparison with the effects of estrogen on synaptic organization in other brain regions. Finally, a working model of neural plasticity within the ventrolateral VMH microcircuitry will be presented as a starting point for future experiments to verify or, more likely, revise and expand.

Functionally-defined compartments of the lordosis neural circuit in the ventromedial hypothalamus in female rats.

Sexual behavior in female rats, typified by the lordosis reflex, is dependent upon estrogen action in the ventromedial nucleus of the hypothalamus (VMH) and its surrounding neuropil. However, the synaptic organization of this brain region remains unclear. Pseudorabies virus (PRV) was used to transneuronally label the neural network that innervates the lumbar epaxial muscles that execute the lordosis response. PRV-labeled neurons were identified within and subjacent to the VMH four days after injection of PRV into the back muscles. The pattern of labeling was defined in relation to three landmarks: the VMH core, as defined by Crystal Violet staining; the shell, as defined by the oxytocin fiber tract; and the cluster of estrogen receptor-containing cell nuclei. The pattern of PRV labeling in the VMH displayed a striking rostral-caudal gradient. In general, many of the PRV-labeled neurons were found in the oxytocin fiber tract, with far fewer in the core of the VMH. Furthermore, PRV-labeled neurons were rarely found in the cluster of estrogen receptor-containing neurons, and less than 3% of the PRV-labeled neurons were double labeled for estrogen receptor. The results suggest that oxytocin may directly influence these lordosis-relevant VMH projection neurons, whereas estrogen may have transsynaptic effects.

Ovarian hormone‐induced reorganization of oxytocin‐labeled dendrites and synapses lateral to the hypothalamic ventromedial nucleus in female rats

Central oxytocin (OT) modulates many social behaviors, including female rat sexual receptivity, quantified as the copulatory stance known as lordosis. The expression of the lordosis response is modulated by OT action in the ventromedial nucleus of the hypothalamus (VMH), as demonstrated by behavioral pharmacology experiments. However, the subcellular localization of OT in this brain region has been unclear. We tested the hypothesis that ovarian hormones reorganize OT‐labeled pre‐ or postsynaptic elements in the fiber complex lateral to the VMH by using immunoelectron microscopy. OT immunolabeling occurred in axonal boutons identified by the presence of small, clear synaptic vesicles and double labeling with the presynaptic markers synaptophysin and vesicular glutamate transporter 2. OT immunoreactivity also was observed in dendritic profiles, verified with double labeling for the dendrite‐specific marker microtubule‐associated protein 2. Ovarian hormones did not alter the density of axonal boutons; however, estradiol treatment reduced the density of dendritic profiles by 34%. This effect was reversed when progesterone was given subsequent to estradiol. The effect of estradiol treatment was specific to dendrites that lacked OT immunostaining; the density of OT‐labeled dendritic profiles remained constant during estradiol treatment. With the estradiol‐induced exit of non‐OT‐labeled dendritic profiles, the remaining OT‐labeled dendritic profiles experienced an increase in their number of synaptic contacts. Thus, hormone treatments that mimic the 4‐day rat estrous cycle provoke a chemically coded reorganization of dendrite innervation in the fiber plexus lateral to the VMH that may underlie the hormone‐specific effect of OT on reproductive behavior. J. Comp. Neurol. 518:4531–4545, 2010.