Task-specific effects of biological sex and sex hormones on object recognition memories in a 6-hydroxydopamine-lesion model of Parkinson's disease in adult male and female rats.

Many patients with Parkinson's disease (PD) experience cognitive or memory impairments with few therapeutic options available to mitigate them. This has fueled interest in determining how factors including sex and sex hormones modulate higher order function in this disease. The objective of this study was to use the Novel Object Recognition (NOR) and Object-in-Place (OiP) paradigms to compare the effects of a bilateral neostriatal 6-hydroxydopamine (6-OHDA) lesion model of PD in gonadally intact male and female rats, in orchidectomized male rats and in orchidectomized males supplemented with 17ß-estradiol or testosterone propionate on measures of recognition memory similar to those at risk in PD. These studies showed that 6-ODHA lesions impaired discrimination in both tasks in males but not females. Further, 6-OHDA lesions disrupted NOR performance similarly in all males regardless of whether they were gonadally intact, orchidectomized or hormone-supplemented. In contrast, OiP performance was disrupted in males that were orchidectomized or 6-OHDA-lesioned but was spared in orchidectomized and orchidectomized, 6-OHDA lesioned males supplemented with 17ß-estradiol. The distinct effects that sex and/or sex hormones have on 6-OHDA lesion-induced NOR vs. OiP deficits identified here also differ from corresponding impacts recently described for 6-OHDA lesion-induced deficits in spatial working memory and episodic memory. Together, the collective data provide strong evidence for effects of sex and sex hormones on cognition and memory in PD as being behavioral task and behavioral domain specific. This specificity could explain why a cohesive clinical picture of endocrine impacts on higher order function in PD has remained elusive.

Domain-specific contributions of biological sex and sex hormones to what, where and when components of episodic-like memory in adult rats.

Episodic memory involves the integration and recall of discrete events that include information about what happened, where it happened and when it occurred. Episodic memory function is critical to daily life, and its dysfunction is both a first identifiable indicator and an enduring core feature of cognitive decline in ageing and in neuropsychiatric disorders including Alzheimer's disease and schizophrenia. Available evidence from human studies suggests that biological sex and sex hormones modulate episodic memory function in health and disease. However, knowledge of how this occurs is constrained by the limited availability and underutilization of validated animal models in investigating hormone impacts on episodic-like memory function. Here, adult female, adult male and gonadally manipulated adult male rats were tested on the what-where-when episodic-like memory task to determine whether rats model human sex differences in episodic memory and how the hormonal milieu impacts episodic-like memory processes in this species. These studies revealed salient ways in which rats model human sex differences in episodic memory, including a male advantage in spatial episodic memory performance. They also identified domain-specific roles for oestrogens and androgens in modulating what, where and when discriminations in male rats that were unlike those engaged in corresponding novel object recognition and novel object location tasks. These studies thus identify rats and the what-where-when task as suitable for investigating the neuroendocrine bases of episodic-like memory, and provide new information about the unique contributions that sex and sex hormones make to this complex mnemonic process.

Gonadectomy but not biological sex affects burst-firing in dopamine neurons of the ventral tegmental area and in prefrontal cortical neurons projecting to the ventral tegmentum in adult rats.

The mesocortical and mesolimbic dopamine systems regulate cognitive and motivational processes and are strongly implicated in neuropsychiatric disorders in which these processes are disturbed. Sex differences and sex hormone modulation are also known for these dopamine-sensitive behaviours in health and disease. One relevant mechanism of hormone impact appears to be regulation of cortical and subcortical dopamine levels. This study asked whether this regulation of dopamine tone is a consequence of sex or sex hormone impact on the firing modes of ventral midbrain dopamine neurons. To address this, single unit extracellular recordings made in the ventral tegmental area and substantia nigra were compared among urethane-anaesthetized adult male, female, gonadectomized male rats. These comparisons showed that gonadectomy had no effect on nigral cells and no effects on pacemaker, bursty, single-spiking or random modes of dopamine activity in the ventral tegmental area. However, it did significantly and selectively increase burst firing in these cells in a testosterone-sensitive, estradiol-insensitive manner. Given the roles of prefrontal cortex (PFC) in modulating midbrain dopamine cell firing, we next asked whether gonadectomy's effects on dopamine cell bursting had correlated effects on the activity of ventral tegmentally projecting prefrontal cortical neurons. We found that gonadectomy indeed significantly and selectively increased burst firing in ventral tegmentally projecting but not neighbouring prefrontal cells. These effects were also androgen-sensitive. Together, these findings suggest a working model wherein androgen influence over the activity of PFC neurons regulates its top-down modulation of mesocortical and mesolimbic dopamine systems and related dopamine-sensitive behaviours.

Androgen effects on mesoprefrontal dopamine systems in the adult male brain.

Epidemiological data show that males are more often and/or more severely affected by symptoms of prefrontal cortical dysfunction in schizophrenia, Parkinson's disease and other disorders in which dopamine circuits associated with the prefrontal cortex are dysregulated. This review focuses on research showing that these dopamine circuits are powerfully regulated by androgens. It begins with a brief overview of the sex differences that distinguish prefrontal function in health and prefrontal dysfunction or decline in aging and/or neuropsychiatric disease. This review article then spotlights data from human subjects and animal models that specifically identify androgens as potent modulators of prefrontal cortical operations and of closely related, functionally critical measures of prefrontal dopamine level or tone. Candidate mechanisms by which androgens dynamically control mesoprefrontal dopamine systems and impact prefrontal states of hypo- and hyper-dopaminergia in aging and disease are then considered. This is followed by discussion of a working model that identifies a key locus for androgen modulation of mesoprefrontal dopamine systems as residing within the prefrontal cortex itself. The last sections of this review critically consider the ways in which the organization and regulation of mesoprefrontal dopamine circuits differ in the adult male and female brain, and highlights gaps where more research is needed.

Spontaneous Object Exploration in a Recessive Gene Knockout Model of Parkinson's Disease: Development and Progression of Object Recognition Memory Deficits in Male Pink1-/- Rats.

Cognitive impairments appear at or before motor signs in about one third of patients with Parkinson's disease (PD) and have a cumulative prevalence of roughly 80% overall. These deficits exact an unrelenting toll on patients' quality and activities of daily life due in part to a lack of available treatments to ameliorate them. This study used three well-validated novel object recognition-based paradigms to explore the suitability of rats with knockout of the PTEN-induced putative kinase1 gene (Pink1) for investigating factors that induce cognitive decline in PD and for testing new ways to mitigate them. Longitudinal testing of rats from 3-9 months of age revealed significant impairments in male Pink1-/- rats compared to wild type controls in Novel Object Recognition, Novel Object Location and Object-in-Place tasks. Task-specific differences in the progression of object discrimination/memory deficits across age were also seen. Finally, testing using an elevated plus maze, a tapered balance beam and a grip strength gauge showed that in all cases recognition memory deficits preceded potentially confounding impacts of gene knockout on affect or motor function. Taken together, these findings suggest that knockout of the Pink1 gene negatively impacts the brain circuits and/or neurochemical systems that support performance in object recognition tasks. Further investigations using Pink1-/- rats and object recognition memory tasks should provide new insights into the neural underpinnings of the visual recognition memory and visuospatial information processing deficits that are often seen in PD patients and accelerate the pace of discovery of better ways to treat them.

Biological Sex and Sex Hormone Impacts on Deficits in Episodic-Like Memory in a Rat Model of Early, Pre-motor Stages of Parkinson's Disease.

Episodic memory deficits are among the earliest appearing and most commonly occurring examples of cognitive impairment in Parkinson's disease (PD). These enduring features can also predict a clinical course of rapid motor decline, significant cognitive deterioration, and the development of PD-related dementia. The lack of effective means to treat these deficits underscores the need to better understand their neurobiological bases. The prominent sex differences that characterize episodic memory in health, aging and in schizophrenia and Alzheimer's disease suggest that neuroendocrine factors may also influence episodic memory dysfunction in PD. However, while sex differences have been well-documented for many facets of PD, sex differences in, and sex hormone influences on associated episodic memory impairments have been less extensively studied and have never been examined in preclinical PD models. Accordingly, we paired bilateral neostriatal 6-hydroxydopamine (6-OHDA) lesions with behavioral testing using the What-Where-When Episodic-Like Memory (ELM) Task in adult rats to first determine whether episodic-like memory is impaired in this model. We further compared outcomes in gonadally intact female and male subjects, and in male rats that had undergone gonadectomy-with and without hormone replacement, to determine whether biological sex and/or sex hormones influenced the expression of dopamine lesioned-induced memory deficits. These studies showed that 6-OHDA lesions profoundly impaired recall for all memory domains in male and female rats. They also showed that in males, circulating gonadal hormones powerfully modulated the negative impacts of 6-OHDA lesions on What, Where, and When discriminations in domain-specific ways. Specifically, the absence of androgens was shown to fully attenuate 6-OHDA lesion-induced deficits in ELM for "Where" and to partially protect against lesion-induced deficits in ELM for "What." In sum, these findings show that 6-OHDA lesions in rats recapitulate the vulnerability of episodic memory seen in early PD. Together with similar evidence recently obtained for spatial working memory, the present findings also showed that diminished androgen levels provide powerful, highly selective protections against the harmful effects that 6-OHDA lesions have on memory functions in male rats.

Region and sex differences in constituent dopamine neurons and immunoreactivity for intracellular estrogen and androgen receptors in mesocortical projections in rats.

Many cortical and prefrontal functions show sex differences in their development, adult capacity, and dysfunction in disorders like schizophrenia. Correlations between circulating gonadal hormones and certain prefrontal functions have also been identified in humans and experimental animal models. Although multiple mechanisms may be involved, such hormone sensitivities/sex differences could be related to gonadal steroid actions on another regulator of cortical/prefrontal cortical function, the mesocortical dopamine system. Thus, although it is well known that perturbations in prefrontal dopamine signaling induce behavioral deficits, it is also known that several endpoints of these afferents are sensitive to gonadal steroids and/or are sexually dimorphic. This study explored possible substrates for this in two ways: by comparing the distributions of immunoreactivity for intracellular estrogen (alpha and beta) and androgen receptors among retrogradely labeled dopaminergic and nondopaminergic mesocortical neurons projecting to prefrontal, premotor, and primary motor cortices, areas in which male rat dopamine axons are differentially hormone-sensitive; and by comparing anatomical data in males and females. These analyses revealed region-, cell-, and sex-specific specializations in receptor localization that paralleled established patterns of mesocortical hormone sensitivity, including the androgen sensitivity of dopamine axons and dopamine-dependent functions in prefrontal cortex. It was also found that the proportions of dopamine neurons making up mesocortical projections were approximately 30% in males, whereas in females, significantly more constituent cells were dopaminergic. Together, these features may be part of the neurobiology giving mesocortical afferents their hormone sensitivities and/or sex differences in physiology, function, and dysfunction in disease.

Computed tomography of amyloid plaques in a mouse model of Alzheimer's disease using diffraction enhanced imaging.

Our understanding of early development in Alzheimer's disease (AD) is clouded by the scale at which the disease progresses; amyloid beta (Abeta) plaques, a hallmark feature of AD, are small (approximately 50 microm) and low contrast in diagnostic clinical imaging techniques. Diffraction enhanced imaging (DEI), a phase contrast x-ray imaging technique, has greater soft tissue contrast than conventional radiography and generates higher resolution images than magnetic resonance microimaging. Thus, in this proof of principle study, DEI in micro-CT mode was performed on the brains of AD-model mice to determine if DEI can visualize Abeta plaques. Results revealed small nodules in the cortex and hippocampus of the brain. Histology confirmed that the features seen in the DEI images of the brain were Abeta plaques. Several anatomical structures, including hippocampal subregions and white matter tracks, were also observed. Thus, DEI has strong promise in early diagnosis of AD, as well as general studies of the mouse brain.

On again, off again effects of gonadectomy on the acoustic startle reflex in adult male rats.

Numerous studies have shown sex and/or estrous cycle differences in the acoustic startle reflex (ASR) and its prepulse inhibition (PPI) in humans and animals. However, few have examined the effects of hormone manipulations on these behaviors. This study paired gonadectomy (GDX) in adult male rats with testing for ASR and PPI at 2, 4, 9, 16, 23, 30 and 37 days after surgery. Initial studies of control, GDX and GDX rats given testosterone propionate revealed no group differences in PPI, but did reveal phasic facilitation of the ASR in GDX rats that was greatest on the first and final testing sessions and that was attenuated by testosterone. A second study addressing roles for estrogen and androgen signaling tested new control and GDX rats along with GDX rats given estradiol or the non-aromatizable androgen, 5-alpha-dihydrotestosterone and revealed no group differences in PPI, and increases in ASR in GDX rats that were largest during the first and final testing sessions and that were attenuated by both hormone replacements. However, while responses in GDX rats given testosterone were similar to those of controls, ASR in estradiol- and to a lesser extent in dihydrotestosterone-treated GDX rats were typically lower than in controls. This may suggest that hormone modulation of the ASR requires synergistic estrogen and androgen actions. In the male brain where this can be achieved by local steroid metabolism, the enzymes responsible, e.g., aromatase, could help identify loci in the startle circuitry that may be especially relevant for the hormone modulation observed.

The impact of biological sex and sex hormones on cognition in a rat model of early, pre-motor Parkinson's disease.

Parkinson's disease (PD) is well known for motor deficits such as bradykinesia. However, patients often experience additional deficits in working memory, behavioral selection, decision-making and other executive functions. Like other features of PD, the incidence and severity of these cognitive symptoms differ in males and females. However, preclinical models have not been used to systematically investigate the roles that sex or sex hormones may play in these complex signs. To address this, we used a Barnes maze spatial memory paradigm to compare the effects of a bilateral nigrostriatal dopamine lesion model of early PD on cognitive behaviors in adult male and female rats and in adult male rats that were gonadectomized or gonadectomized and supplemented with testosterone or estradiol. We found that dopamine lesions produced deficits in working memory and other executive operations, albeit only in male rats where circulating androgen levels were physiological. In males where androgen levels were depleted, lesions produced no additional Barnes maze deficits and attenuated those previously linked to androgen deprivation. We also found that while most measures of Barnes maze performance were unaffected by dopamine lesions in the females, lesions did induce dramatic shifts from their preferred use of thigmotactic navigation to the use of spatially guided place strategies similar to those normally preferred by males. These and other sex- and sex hormone-specific differences in the effects of nigrostriatal dopamine lesions on executive function highlight the potential of gonadal steroids as protective and/or therapeutic for the cognitive symptoms of PD. However, their complexity also indicates the need for a more thorough understanding of androgen and estrogen effects in guiding the development of hormone therapies that might effectively address these non-motor signs.

Rest mutant zebrafish swim erratically and display atypical spatial preferences.

The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest.

Estrogen receptor-beta immunoreactivity in the midbrain of adult rats: regional, subregional, and cellular localization in the A10, A9, and A8 dopamine cell groups.

Estrogen modulates dopamine synthesis, release, and metabolism in corticolimbic and striatal targets of midbrain dopamine neurons. The relevant sites of receptor-mediated action, however, had been elusive, because all available evidence suggested a paucity of intracellular estrogen receptors in the A8, A9, and A10 dopamine regions and their afferent targets. More recent evidence of a relative abundance of the beta isoform of the estrogen receptor (ER) in the substantia nigra and ventral tegmental area (VTA), however, suggests that this newly described receptor may be important in estrogen's stimulation of midbrain DA systems. It is unknown, however, precisely how ERbeta is distributed with respect to the functionally and neurochemically diverse cell populations of the ventral midbrain. To address these issues, this study used single- and double-label immunocytochemistry to detail the regional, subregional, and cellular distributions of ERbeta immunoreactivity in and around midbrain dopamine-containing cell groups in hormonally intact adult male and female rats. These analyses revealed that ERbeta-immunoreactive nuclei were found only in neurons, more specifically, within subsets of both dopaminergic and nondopaminergic neurons in the dorsal VTA, the parabrachial pigmented nucleus, the substantia nigra pars lateralis, the retrorubral fields, and to a lesser extent the linear midline nuclei. These regional and cellular receptor distributions thus place the ERbeta isoform in anatomical register with midbrain dopamine systems known to participate in a spectrum of motor, cognitive, and affective functions.

Mesostriatal and mesolimbic projections of midbrain neurons immunoreactive for estrogen receptor beta or androgen receptors in rats.

The dopamine (DA) inputs to the caudate putamen, the nucleus accumbens, and the amygdala in rats are sensitive to circulating estrogens and androgens. One mechanism for the hormone modulation of these systems may be via actions at cognate intracellular estrogen and androgen receptors. However, although it is known that specific subsets of midbrain DA neurons are immunopositive for estrogen receptor beta (ERbeta) or androgen receptors (ARs), it is not known where these receptor-bearing cells project. To address this issue, we combined double-label immunocytochemistry with retrograde tract tracing to identify the forebrain projections of ERbeta- or AR-immunoreactive (IR) midbrain neurons. Specifically, Fluoro-Gold and/or cholera toxin were injected into discrete subregions of the caudate-putamen, the nucleus accumbens, or the amygdala. Evaluations of the resultant midbrain labeling revealed that ERbeta-IR neurons sent collateral projections mainly to both the ventral caudate-putamen and the amygdala, but not to the dorsal caudate or nucleus accumbens. In contrast, AR-IR neurons projected either to the amygdala or the nucleus accumbens but not to the caudate-putamen. The organization of these forebrain projections concurs with some of the known hormone sensitivities of mesostriatal and mesolimbic DA systems in rats and provides an anatomical model that predicts separate influences for androgens and estrogens over mesostriatal and mesolimbic DA systems.

Regional, laminar and cellular distribution of immunoreactivity for ERbeta in the cerebral cortex of hormonally intact, postnatally developing male and female rats.

Estrogen influences cerebral cortical development. Among the receptors involved are classical (ERalpha) and beta (ERbeta) intracellular estrogen receptors. In the first 2 weeks of postnatal life, cortical ERalpha is transiently expressed at much higher levels than in adulthood. In this study, development of ERbeta was examined by mapping ERbeta immunoreactivity in relation to major cortical regions, layers and cell types in postnatal male and female rats that were 1-28 postnatal days (PND) old. These studies revealed that ERbeta-immunoreactive nuclei were present in the allocortices on PND 1 but were not detected in isocortex until PND 7. Allocortical labeling was also higher on PND 1 than at all later ages, while in isocortical areas low numbers of ERbeta nuclei were seen on PND 7 that rose to higher, near adult densities by PND 21. Finally, double labeling showed that ERalpha was expressed mainly in neurons immunopositive for calretinin, while ERbeta was localized predominantly in parvalbumin-immunoreactive cells. Thus, the postnatal cortical developments of ERbeta and ERalpha occur according to different timetables, different patterns and in association with different cortical cells. It thus seems it likely that the two also make distinct contributions to postnatal cortical development and/or sexual differentiation.