Paul M. Forlano

Brain aromatase: New lessons from non-mammalian model systems

This review highlights recent studies of the anatomical and functional implications of brain aromatase (estrogen synthase) expression in two vertebrate lineages, teleost fishes and songbirds, that show remarkably high levels of adult brain aromatase activity, protein and gene expression compared to other vertebrate groups. Teleosts and birds have proven to be important neuroethological models for investigating how local estrogen synthesis leads to changes in neural phenotypes that translate into behavior. Region-specific patterns of aromatase expression, and thus estrogen synthesis, include the vocal and auditory circuits that figure prominently into the life history adaptations of vocalizing teleosts and songbirds. Thus, by targeting, for example, vocal motor circuits without inappropriate steroid exposure to other steroid-dependent circuits, such as those involved in either copulatory or spawning behaviors, the neuroendocrine system can achieve temporal and spatial specificity in its modulation of neural circuits that lead to the performance of any one behavior.

Distribution of estrogen receptor alpha mRNA in the brain and inner ear of a vocal fish with comparisons to sites of aromatase expression

Among vertebrates, teleost fish have the greatest capacity for estrogen production in the brain. Previously, we characterized the distribution of the estrogen‐synthesizing enzyme aromatase in the brain of the midshipman fish. Here, we investigated the distribution of estrogen receptor alpha (ERα). A partial cDNA of ERα was cloned and used to generate midshipman‐specific primers for RT and real‐time PCR which identified transcripts in liver and ovary, the CNS, and the sensory epithelium of the main auditory endorgan (sacculus). In situ hybridization revealed abundant expression throughout the preoptic area, a vocal‐acoustic site in the hypothalamus, amygdala homologs of the dorsal pallium, the pineal organ, the inner ear, the pituitary, and the ovary. Weaker expression was found in the midbrains nucleus of the medial longitudinal fasciculus and in the dimorphic vocal motor nucleus. ERα expression in the pineal, gonad, and pituitary axis may function to time seasonal abiotic cues to reproductive state, while expression in the vocal motor and auditory systems support neurophysiological evidence for estrogen as a modulator of vocal motor and auditory encoding mechanisms in midshipman fish. While ERα is restricted to specific nuclei, aromatase expression is abundant in glial cells throughout the entire forebrain, and high in midbrain and hindbrain – spinal vocal regions. The only site of aromatase‐containing neurons is in the peripheral auditory system, where it is localized to ganglion cells in the auditory nerve. Estrogen production proximal to ERα‐positive neurons may provide for focal sites of estrogen effects on reproductive‐, vocal‐, and auditory‐related neurons. J. Comp. Neurol. 483:91–113, 2005.

Distribution of androgen receptor mRNA expression in vocal, auditory, and neuroendocrine circuits in a teleost fish

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive‐related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context‐dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain‐spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase‐containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. J. Comp. Neurol. 518:493–512, 2010.

Multiple ERβ antisera label in ERβ knockout and null mouse tissues

In the process of characterizing a custom-made affinity-purified antiserum for estrogen receptor beta (ERbeta), ck5912, we used a number of common tests for specificity of ck5912 along with that of 8 commercially available ERbeta antisera: Affinity Bioreagents PA1-310B, Invitrogen D7N, Upstate 06-629, Santa Cruz H150, Y19, L20, 1531, and Abcam 9.88. We tested their recognition of recombinant ERbeta (rERbeta) versus rERalpha, ERbeta versus ERalpha transfected into cell lines, as well as labeling in wildtype (WT) versus estrogen receptor beta knockout (betaERKO) and null (ERbeta(ST)(L-/L-)) mouse ovary, hypothalamus, and hippocampus. To our surprise, we found that while most of these antisera passed some tests, giving the initial impression of specificity, western blot analysis showed that all of them recognized apparently identical protein bands in WT, betaERKO and ERbeta(ST)(L-/L-) tissues. We share these results with the goal of helping other researchers avoid pitfalls in interpretation that could come from use of these ERbeta antisera.

Quantitative analysis of pre- and postsynaptic sex differences in the nucleus accumbens.

The nucleus accumbens (NAc) plays a central role in motivation and reward. While there is ample evidence for sex differences in addiction‐related behaviors, little is known about the neuroanatomical substrates that underlie these sexual dimorphisms. We investigated sex differences in synaptic connectivity of the NAc by evaluating pre‐ and postsynaptic measures in gonadally intact male and proestrous female rats. We used DiI labeling and confocal microscopy to measure dendritic spine density, spine head size, dendritic length, and branching of medium spiny neurons (MSNs) in the NAc, and quantitative immunofluorescence to measure glutamatergic innervation using pre‐ (vesicular glutamate transporter 1 and 2) and postsynaptic (postsynaptic density 95) markers, as well as dopaminergic innervation of the NAc. We also utilized electron microscopy to complement the above measures. Clear but subtle sex differences were identified, namely, in distal dendritic spine density and the proportion of large spines on MSNs, both of which are greater in females. Sex differences in spine density and spine head size are evident in both the core and shell subregions, but are stronger in the core. This study is the first demonstration of neuroanatomical sex differences in the NAc and provides evidence that structural differences in synaptic connectivity and glutamatergic input may contribute to behavioral sex differences in reward and addiction. J. Comp. Neurol. 518:1330–1348, 2010.

Conserved neurochemical pathways involved in hypothalamic control of energy homeostasis

The melanocortin system, which includes α‐melanocyte‐stimulating hormone (α‐MSH) and its endogenous antagonist, agouti‐related protein (AgRP), is fundamental for the central control of energy homeostasis in mammals. Recent studies have demonstrated that many neuropeptides involved in the control of ingestive behavior and energy expenditure, including melanocortins, are also expressed and functional in teleost fishes. To test the hypothesis that the underlying neural pathways involved in energy homeostasis are conserved throughout vertebrate evolution, the neuroanatomical distribution of α‐MSH in relation to AgRP was mapped in a teleost (zebrafish, Danio rerio) by double‐label immunocytochemistry. Zebrafish α‐MSH‐ and AgRP‐immunoreactive (ir) cells are found in discrete populations in the ventral periventricular hypothalamus, the proposed arcuate homologue in teleosts. Major ascending projections are similar for both peptides, and dense ir‐fibers innervate preoptic and ventral telencephalic nuclei homologous to paraventricular, lateral septal, and amygdala nuclei in mammals. Furthermore, α‐MSH and AgRP‐ir somata and fibers are pronounced at 5 days post fertilization when yolk reserves are depleted and larvae begin to feed actively, which supports the functional significance of these peptides for feeding behavior. The conservation of melanocortin peptide function and projection pathways further support zebrafish as an excellent genetic model system to investigate basic mechanisms involved in the central regulation of energy homeostasis. J. Comp. Neurol. 505:235–248, 2007.

Neural and hormonal mechanisms of reproductive-related arousal in fishes

The major classes of chemicals and brain pathways involved in sexual arousal in mammals are well studied and are thought to be of an ancient, evolutionarily conserved origin. Here we discuss what is known of these neurochemicals and brain circuits in fishes, the oldest and most species-rich group of vertebrates from which tetrapods arose over 350 million years ago. Highlighted are case studies in vocal species where well-delineated sensory and motor pathways underlying reproductive-related behaviors illustrate the diversity and evolution of brain mechanisms driving sexual motivation between (and within) sexes. Also discussed are evolutionary insights from the neurobiology and reproductive behavior of elasmobranch fishes, the most ancient lineage of jawed vertebrates, which are remarkably similar in their reproductive biology to terrestrial mammals.

Alternative Reproductive Tactics: Neuroendocrine mechanisms of alternative reproductive tactics: the chemical language of reproductive and social plasticity

The wide range of variation in reproductive tactics displayed among teleost fishes has provided a rich source of natural experiments for investigating the neural mechanisms of alternative reproductive tactics (ARTs). These studies have mainly focused on identifying the location and extent of neuropeptide-containing cells in the forebrain’s preoptic area (POA), in part, because of the wellestablished influence of these neurons on reproductive mechanisms. We first review the ARTs of teleost species that have served as model systems for investigating the neural mechanisms of reproductive plasticity and then the general organization of the POA of vertebrates. Comparative surveys then show how life-history trajectories and reproductive tactics vary with interand intrasexual dimorphisms in the size and number of POA neurons that synthesize either arginine vasotocin (AVT) or gonadotropin-releasing hormone (GnRH). The emerging evidence for the potential role of neurosteroids in mechanisms of reproductive plasticity inclusive of ARTs is then considered before concluding with a listing of a suite of neuroendocrinological traits that may provide proximate mechanisms essential to the widespread evolution of ARTs among teleost fish.

Neuroendocrine control of seasonal plasticity in the auditory and vocal systems of fish

Seasonal changes in reproductive-related vocal behavior are widespread among fishes. This review highlights recent studies of the vocal plainfin midshipman fish, Porichthys notatus, a neuroethological model system used for the past two decades to explore neural and endocrine mechanisms of vocal-acoustic social behaviors shared with tetrapods. Integrative approaches combining behavior, neurophysiology, neuropharmacology, neuroanatomy, and gene expression methodologies have taken advantage of simple, stereotyped and easily quantifiable behaviors controlled by discrete neural networks in this model system to enable discoveries such as the first demonstration of adaptive seasonal plasticity in the auditory periphery of a vertebrate as well as rapid steroid and neuropeptide effects on vocal physiology and behavior. This simple model system has now revealed cellular and molecular mechanisms underlying seasonal and steroid-driven auditory and vocal plasticity in the vertebrate brain.

Catecholaminergic connectivity to the inner ear, central auditory, and vocal motor circuitry in the plainfin midshipman fish porichthys notatus

Although the neuroanatomical distribution of catecholaminergic (CA) neurons has been well documented across all vertebrate classes, few studies have examined CA connectivity to physiologically and anatomically identified neural circuitry that controls behavior. The goal of this study was to characterize CA distribution in the brain and inner ear of the plainfin midshipman fish (Porichthys notatus) with particular emphasis on their relationship with anatomically labeled circuitry that both produces and encodes social acoustic signals in this species. Neurobiotin labeling of the main auditory end organ, the saccule, combined with tyrosine hydroxylase immunofluorescence (TH‐ir) revealed a strong CA innervation of both the peripheral and central auditory system. Diencephalic TH‐ir neurons in the periventricular posterior tuberculum, known to be dopaminergic, send ascending projections to the ventral telencephalon and prominent descending projections to vocal–acoustic integration sites, notably the hindbrain octavolateralis efferent nucleus, as well as onto the base of hair cells in the saccule via nerve VIII. Neurobiotin backfills of the vocal nerve in combination with TH‐ir revealed CA terminals on all components of the vocal pattern generator, which appears to largely originate from local TH‐ir neurons but may include input from diencephalic projections as well. This study provides strong neuroanatomical evidence that catecholamines are important modulators of both auditory and vocal circuitry and acoustic‐driven social behavior in midshipman fish. This demonstration of TH‐ir terminals in the main end organ of hearing in a nonmammalian vertebrate suggests a conserved and important anatomical and functional role for dopamine in normal audition. J. Comp. Neurol. 522:2887‐2927, 2014.