Colin J. Saldanha

Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody.

In songbirds, aromatase (estrogen synthase) activity and mRNA are readily detectable in the brain. This neural aromatization presumably provides estrogen to steroid‐sensitive targets via autocrine, paracrine, and synaptic mechanisms. The location of immunoreactive protein, however, has been difficult to describe completely, particularly in distal dendrites, axons, and terminals of the forebrain. Here we describe the neuroanatomical distribution of aromatase in the zebra finch by using a novel antibody raised specifically against zebra finch aromatase. The distribution of aromatase‐positive somata in the zebra finch brain is in excellent agreement with previous reports. Additionally, this antibody reveals elaborate, spinous dendritic arbors, fine‐beaded axons, and punctate terminals of telencephalic neurons that may synthesize estrogen. Some of these axon‐like fibers extend into the high vocal center (HVC) and the robust nucleus of the archistriatum (RA) in males and females, suggesting a role for presynaptic aromatization in cellular processes within these loci. Adult males have more aromatase‐positive fibers in the caudomedial neostriatum (NCM) and the preoptic area (POA) compared to females, despite the lack of detectable sex differences in the number of immunoreactive somata at these loci. Thus, the compartmentalization of aromatase in dendrites and axons may serve a sexually dimorphic function in the songbird. Finally, in adult males, aromatase expression is down‐regulated by circulating estradiol in the hippocampus, but not in the NCM or POA. The distribution of aromatase suggests a role for aromatization in the regulation of pre‐ and postsynaptic function in steroid sensitive areas of the songbird forebrain. J. Comp. Neurol. 423:619–630, 2000.

Aromatase is pre-synaptic and sexually dimorphic in the adult zebra finch brain

Oestrogens organize and activate circuits within the vertebrate central nervous system. Oestrogen synthesis occurs via the expression of aromatase, a P450 enzyme detected in microsomes and more recently in pre-synaptic boutons. Synaptic aromatase has only been described in brain regions that express aromatase in many subcellular compartments, so its function remains poorly understood. To more thoroughly study the role of oestrogen synthesis at synaptic terminals, we examined the ultrastructural compartmentalization of aromatase in the zebra finch; a species in which high aromatase activity can be measured in brain areas that do not contain somal aromatase. Here, we report the presence of aromatase in pre-synaptic boutons in the hippocampus and the high vocal centre brain areas with low and undetectable somal aromatase, respectively, in addition to areas with abundant somal aromatase such as the preoptic area and caudomedial nidopallium. At these brain areas, males had more total synapses, more aromatase pre-synaptic boutons and importantly, the proportion of total synaptic profiles that expressed aromatase was significantly higher in males relative to females. Aromatase-positive pre-synaptic boutons were always observed innervating aromatase-negative post-synaptic elements. We conclude that oestrogen may be provided to discrete oestrogen-sensitive targets by synaptic aromatization. Further, some targets may be exposed to more oestrogen in males. The expression of aromatase in individual synapses of projection neurons represents a unique mechanism of neuroendocrine action. Neurons with steroidogenic capability may modulate distant targets with the specificity of axonal innervation.

Rapid upregulation of aromatase mRNA and protein following neural injury in the zebra finch (Taeniopygia guttata).

The expression of aromatase (oestrogen synthase) within the vertebrate central nervous system (CNS) is key in the provision of local oestrogens to neural circuits. Aromatase expression appears to be exclusively neuronal under normal conditions. However, some in vitro studies suggest the presence of astrocytic aromatase in songbirds and mammals. Recently, aromatase in reactive astrocytes has been demonstrated in response to neural injury in the mammalian CNS. Since the glial aromatase expression first documented in cultures of the songbird telencephalon may reflect processes similar to those in response to mammalian neural injury, we investigated whether injury alters the pattern of aromatase‐expression in the zebra finch, a species with very high levels of forebrain aromatase expression. Adult males received a penetrating neural injury to the right hemisphere and were killed either 24 or 72 h later. Controls were anaesthetized and otherwise unmanipulated. We determined the expression of aromatase mRNA and protein using in situ hybridization and immunocytochemistry, respectively. Both the transcription and translation of aromatase is dramatically upregulated around the lesion site in response to neural injury in the zebra finch forebrain. This effect is robust and rapid, occurring within 24 h of the injury itself. Cells that upregulate aromatase appear to be reactive astrocytes based upon morphology. The hemisphere contralateral to the injury and both hemispheres in control birds showed the normal, exclusively neuronal pattern of aromatase expression. The upregulation of aromatase in astrocytes may provide high levels of oestrogen available to modulate processes such as CNS repair. Injury‐induced upregulation of astrocytic aromatase may be a general characteristic of the injured vertebrate brain.

Direct Innervation of GnRH Neurons by Encephalic Photoreceptors in Birds

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal that brain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.

The Passerine Hippocampus is a Site of High Aromatase: Inter- and Intraspecies Comparisons☆

The vertebrate hippocampus (HP) plays a critical role in the organization of memory. Estrogens alter synaptic morphology and function in the mammalian HP and may potentiate memory performance. Previous studies suggest that the songbird HP itself is a site of significant aromatase expression, intimating that local estrogen synthesis may provide a source of this steroid to estrogen-sensitive neural circuits. To explore the potential role of local estrogen synthesis on HP structure and function, we have characterized aromatase message and activity in the zebra finch HP. Toward this end we have compared (a) HP aromatase mRNA with that at other neural loci, (b) HP aromatase activity between adults of both sexes, and (c) HP and hypothalamic preoptic area (HPOA) aromatase activity among songbirds and nonsongbirds. Finally we asked whether aromatase activity was intrinsic to the HP by maintaining it in culture, isolated from the rest of the telencephalon. The HP of every songbird studied expresses aromatase, with comparable levels across sexes. Notably, aromatase activity was found at higher levels in the songbird HP than in the HPOA. In both nonsongbird species investigated, however, HP aromatase activity was undetectable under identical assay conditions. Additionally, the developing songbird HP continues to express aromatase when cultured in isolation from the rest of the telencephalon. The data suggest that HP aromatase is characteristic of passeriformes and, as in the HPOA, may represent a mechanism whereby estrogen is made available to neural circuits. Passerines may prove invaluable animal models for investigations of the estrogenic modulation of HP structure and function.

Rapid Effects of Corticosterone on Cache Recovery in Mountain Chickadees (Parus gambeli)

Environmental perturbations increase adrenal activity in several vertebrates. Increases in corticosterone may serve as a proximate trigger whereby organisms can rapidly adapt their behavior to survive environmental fluctuations. In food-caching songbirds, inclement weather may present the need to alter caching and/or retrieval behaviors to ensure food supplies. We hypothesized that corticosterone may increase the rate of caching and/or retrieval behaviors in the mountain chickadee, a food-storing songbird, and tested if these potential effects were mediated by alterations in appetite, activity, or memory for cache sites. Corticosterone or vehicle was administered to subjects 5 min prior to either caching or recovery in a naturalistic laboratory paradigm during which we recorded the number of caching events, sites visited, and seeds eaten (caching) or caches recovered, total sites visited, cache-related visits, and non-cache-related visits (recovery). Data were analyzed using nested ANOVA for treatment within sequential trial. There was no effect on any caching behaviors following treatment. However, birds treated with corticosterone during retrieval recovered more seeds and tended to visit more cache-related sites than did controls. Since groups did not differ in the number of seeds eaten or the total number of sites visited, it seems unlikely that corticosterone affected appetite or activity. Rapid surges in corticosterone may increase the efficacy of an underlying memory process for cache sites which is reflected in higher cache recovery in corticosterone-treated birds than in controls. Thus, rapid alterations in plasma corticosterone following environmental change may alter memory-reliant behaviors which promote survival in the food-caching mountain chickadee.

Detection and transduction of daylength in birds

Daylength is an important environmental cue used by temperate zone avian species to time the onset of seasonal reproductive activity. Photic cues are detected by extra-retinal, extra-pineal central nervous system elements, and are rapidly transduced to an efferent signal. In this paper, we describe the brain locus of putative encephalic photoreceptors in birds, and explore the pathway of information transfer from photic input to the reproductive axis. To this end, we examine how photoreceptors might communicate with the hypothalamic-pituitary axis, and how brain peptides vary seasonally. Recent studies indicate that brain photoreceptors lie in the lateral septum and in the tuberal hypothalamus, and co-express proteins characteristic of retinal photoreceptors, as well as vasoactive-intestinal polypeptide (VIP). At the light microscopic level, photoreceptor cells appear to communicate with gonadotropin-releasing hormone (GnRH) neurons, and vice versa. Expression of VIP-like immunoreactivity is highest in photorefractory animals while GnRH-like immunoreactivity is highest in photosensitive birds. Expression of these CNS peptides is correlated with changes in plasma prolactin and luteinizing hormone (LH), suggesting a mechanism mediating seasonal cyclicity.

Androgen Metabolism in the Juvenile Oscine Forebrain: A Cross-Species Analysis at Neural Sites Implicated in Memory Function

Juvenile songbirds are useful models for studying the neural bases of memory. Memory-reliant behaviors demonstrated at this stage include song learning (most songbirds) and food caching (food-storing songbirds). Sex steroids are implicated in the modulation of memory processes in several vertebrates. The songbird forebrain expresses aromatase, 5alpha-reductase and 5beta-reductase, enzymes which convert testosterone to estradiol, 5alpha-, and 5beta-dihydrotestosterone, respectively. To explore the role of local androgen metabolism on memory processes, we documented the activities of these enzymes in the anterior neostriatum (NAN), caudomedial neostriatum (NCM), and hippocampus (HP) of four species of juvenile songbird, two of which are food storers. Areas were dissected, homogenized, and provided with radiolabeled substrate; and formed estrogens, and 5alpha- and 5beta-reduced androgens were measured. In the NAN, 5beta-reductase was the predominant enzyme, suggesting that local inactivation of testosterone may preserve the sensitive period of song acquisition. In the NCM, estrogens were formed in abundance despite high 5beta-reductase, suggesting that locally high estrogen synthesis may play a role in processes of song perception. In the HP, both estrogens and 5alpha reduced androgens were formed, suggesting that HP function may be modulated by both estrogens and androgens. Finally, a derived measure of steroid-differential reveals that food-storing songbirds differ from nonstorers in the steroidal milleiu within the HP, but not in the NAN or NCM. Thus, distinct loci within the juvenile songbird forebrain are exposed to different patterns of androgen metabolites. This local conversion may play a role in the neuroendocrine modulation of memory in these birds.

Inhibition of Hippocampal Aromatization Impairs Spatial Memory Performance in a Male Songbird

Recent studies have revealed the presence and regulation of aromatase at the vertebrate synapse, and identified a critical role played by presynaptic estradiol synthesis in the electrophysiological response to auditory and other social cues. However, if and how synaptic aromatization affects behavior remains to be directly tested. We have exploited 3 characteristics of the zebra finch hippocampus (HP) to test the role of synaptocrine estradiol provision on spatial memory function. Although the zebra finch HP contains abundant aromatase transcripts and enzyme activity, immunocytochemical studies reveal widespread pre- and postsynaptic, but sparse to undetectable somal, localization of this enzyme. Further, the superficial location of the avian HP makes possible the more exclusive manipulation of its neurochemical characteristics without perturbation of the neuropil and the resultant induction of astroglial aromatase. Last, as in other vertebrates, the HP is critical for spatial memory performance in this species. Here we report that local inhibition of hippocampal aromatization impairs spatial memory performance in an ecologically valid food-finding task. Local aromatase inhibition also resulted in lower levels of estradiol in the HP, but not in adjacent brain areas, and was achieved without the induction of astroglial aromatase. The observed decrement in acquisition and subsequent memory performance as a consequence of lowered aromatization was similar to that achieved by lesioning this locus. Thus, hippocampal aromatization, much of which is achieved at the synapse in this species, is critical for spatial memory performance.

Telencephalic Aromatase but Not a Song Circuit in a Sub-Oscine Passerine, the Golden Collared Manakin (Manacus vitellinus)

In oscine passerines, the telencephalon expresses high levels of the estrogen synthetic enzyme aromatase. In contrast, forebrain aromatase is limited to low levels at discrete limbic loci in non-passerines. The function of forebrain aromatase in oscines is unknown, however, estrogen-sensitive elements of the telencephalic song circuit (an oscine characteristic) may be influenced by local aromatization. Very few studies have investigated the neuroendocrine characteristics of sub-oscine passerines. Species of this passerine sub-order are taxonomically similar to oscines, but do not appear to learn how to sing as oscines, and show no evidence of a song circuit. The neural expression of aromatase in these birds is unknown. We asked whether the golden-collared manakin, a sub-oscine, (a) showed evidence of a song circuit, and (b) expressed aromatase in the telencephalon at high levels like the zebra finch (oscine passerine) or at low levels like the quail (non-passerine). Nissl stains and immunocytochemistry for microtubule associated proteins showed no evidence of a song circuit in manakins of either sex, whereas both techniques delineate all song nuclei in the zebra finch. However, biochemical and immunocytochemical measures reveal that in the manakin, several telencephalic loci, including the hippocampus, caudomedial neostriatum, nucleus taeniae, and the lateral neostriatum express aromatase. Assays run in parallel show low to undetectable levels of aromatase in the telencephalon of the quail (nonpasserine) and abundant levels in the zebra finch (oscine passerine), suggesting a dissociation between the presence of a song circuit and forebrain aromatase expression in this sub-oscine. These data suggest that forebrain aromatase may have evolved in sub-oscine songbirds before the evolution of a song circuit and singing behavior in oscines. Alternatively, forebrain aromatase may serve functions distinct from singing behavior.