Gross morphology, histology, and ultrastructure of the olfactory rosette of a critically endangered indicator species, the Delta Smelt, Hypomesus transpacificus.

The Delta Smelt (Hypomesus transpacificus) is a small, semi-anadromous fish native to the San Francisco Bay-Delta Estuary and has been declared as critically endangered. Their olfactory biology, in particular, is poorly understood and a basic description of their sensory anatomy is needed to advance our understanding of the sensory ecology of species to inform conservation efforts to manage and protect them. We provide a description of the gross morphology, histological, immunohistochemical, and ultrastructural features of the olfactory rosette in this fish and discuss some of the functional implications in relation to olfactory ability. We show that Delta Smelt have a multilamellar olfactory rosette with allometric growth. Calretinin immunohistochemistry revealed a diffuse distribution of olfactory receptor neurons within the epithelium. Ciliated, microvillous and crypt neurons were clearly identified using morphological and immunohistochemical features. The olfactory neurons were supported by robust ciliated and secretory sustentacular cells. Although the sense of smell has been overlooked in Delta Smelt, we conclude that the olfactory epithelium has many characteristics of macrosmatic fish. With this study, we provide a foundation for future research into the sensory ecology of this imperiled fish.

Secretagogin expression in the mouse olfactory bulb under sensory impairments.

The interneurons of the olfactory bulb (OB) are characterized by the expression of different calcium-binding proteins, whose specific functions are not fully understood. This is the case of one of the most recently discovered, the secretagogin (SCGN), which is expressed in interneurons of the glomerular and the granule cell layers, but whose function in the olfactory pathway is still unknown. To address this question, we examined the distribution, generation and activity of SCGN-positive interneurons in the OB of two complementary models of olfactory impairments: Purkinje Cell Degeneration (PCD) and olfactory-deprived mice. Our results showed a significant increase in the density of SCGN-positive cells in the inframitral layers of olfactory-deprived mice as compared to control animals. Moreover, BrdU analyses revealed that these additional SCGN-positive cells are not newly formed. Finally, the neuronal activity, estimated by c-Fos expression, increased in preexisting SCGN-positive interneurons of both deprived and PCD mice -being higher in the later- in comparison with control animals. Altogether, our results suggest that the OB possesses different compensatory mechanisms depending on the type of alteration. Particularly, the SCGN expression is dependent of olfactory stimuli and its function may be related to a compensation against a reduction in sensory inputs.

Sex differences in main olfactory system pathways involved in psychosexual function.

We summarize literature from animal and human studies assessing sex differences in the ability of the main olfactory system to detect and process sex-specific olfactory signals ("pheromones") that control the expression of psychosexual functions in males and females. A case is made in non primate mammals for an obligatory role of pheromonal signaling via the main olfactory system (in addition to the vomeronasal-accessory olfactory system) in mate recognition and sexual arousal, with male-specific as well as female-specific pheromones subserving these functions in the opposite sex. Although the case for an obligatory role of pheromones in mate recognition and mating among old world primates, including humans, is weaker, we review the current literature assessing the role of putative human pheromones (eg, AND, EST, "copulin"), detected by the main olfactory system, in promoting mate choice and mating in men and women. Based on animal studies, we hypothesize that sexually dimorphic effects of putative human pheromones are mediated via main olfactory inputs to the medial amygdala which, in turn, transmits olfactory information to sites in the hypothalamus that regulate reproduction.

The Impact of Acoustic fMRI-Noise on Olfactory Sensitivity and Perception.

Sensory perception is neither static nor simple. The senses influence each other during multisensory stimulation and can be both suppressive and super-additive. As most knowledge of human olfactory perception is derived from functional neuroimaging studies, in particular fMRI, our current understanding of olfactory perception has systematically been investigated in an environment with concurrent loud sounds. To date, the confounding effects of acoustic fMRI-noise during scanning on olfactory perception have not yet been investigated. In this study we investigate how acoustic noise derived from the rapid switching of MR gradient coils, affects olfactory perception. For this, 50 subjects were tested in both a silent setting and an fMRI-noise setting, in a randomised order. We found that fMRI-related acoustic noise had a significant negative effect on the olfactory detection threshold score. No significant effects were identified on olfactory discrimination, identification, identification certainty, hedonic rating, or intensity rating.

A specific olfactory cortico-thalamic pathway contributing to sampling performance during odor reversal learning.

A growing body of evidence shows that olfactory information is processed within a thalamic nucleus in both rodents and humans. The mediodorsal thalamic nucleus (MDT) receives projections from olfactory cortical areas including the piriform cortex (PCX) and is interconnected with the orbitofrontal cortex (OFC). Using electrophysiology in freely moving rats, we recently demonstrated the representation of olfactory information in the MDT and the dynamics of functional connectivity between the PCX, MDT and OFC. Notably, PCX-MDT coupling is specifically increased during odor sampling of an odor discrimination task. However, whether this increase of coupling is functionally relevant is unknown. To decipher the importance of PCX-MDT coupling during the sampling period, we used optogenetics to specifically inactivate the PCX inputs to MDT during an odor discrimination task and its reversal in rats. We demonstrate that inactivating the PCX inputs to MDT does not affect the performance accuracy of an odor discrimination task and its reversal, however, it does impact the rats' sampling duration. Indeed, rats in which PCX inputs to MDT were inactivated during the sampling period display longer sampling duration during the odor reversal learning compared to controls-an effect not observed when inactivating OFC inputs to MDT. We demonstrate a causal link between the PCX inputs to MDT and the odor sampling performance, highlighting the importance of this specific cortico-thalamic pathway in olfaction.

Neuronal Response Latencies Encode First Odor Identity Information across Subjects.

Odorants are coded in the primary olfactory processing centers by spatially and temporally distributed patterns of glomerular activity. Whereas the spatial distribution of odorant-induced responses is known to be conserved across individuals, the universality of its temporal structure is still debated. Via fast two-photon calcium imaging, we analyzed the early phase of neuronal responses in the form of the activity onset latencies in the antennal lobe projection neurons of honeybee foragers. We show that each odorant evokes a stimulus-specific response latency pattern across the glomerular coding space. Moreover, we investigate these early response features for the first time across animals, revealing that the order of glomerular firing onsets is conserved across individuals and allows them to reliably predict odorant identity, but not concentration. These results suggest that the neuronal response latencies provide the first available code for fast odor identification.SIGNIFICANCE STATEMENT Here, we studied early temporal coding in the primary olfactory processing centers of the honeybee brain by fast imaging of glomerular responses to different odorants across glomeruli and across individuals. Regarding the elusive role of rapid response dynamics in olfactory coding, we were able to clarify the following aspects: (1) the rank of glomerular activation is conserved across individuals, (2) its stimulus prediction accuracy is equal to that of the response amplitude code, and (3) it contains complementary information. Our findings suggest a substantial role of response latencies in odor identification, anticipating the static response amplitude code.

Pepper with and without a sting: Brain processing of intranasal trigeminal and olfactory stimuli from the same source.

Humans have distinct and overlapping brain regions for the processing of intranasal olfactory or trigeminal stimuli. It may be assumed that trigeminal stimulants and "trigeminal-free" odorous stimuli from the same source are processed differently in the human brain. Using functional magnetic resonance imaging (fMRI), this study investigated the question whether the black pepper (Piper nigrum) derived trigeminal active compound piperine and the trigeminal-free pepper essential oil (pepperEO) are processed in relation to their degree of trigeminal stimulation. Twenty-one young healthy adults underwent a fMRI scan where piperine and pepperEO were delivered orthonasally using an olfactometer. Ratings for intensity, irritability, and pleasantness of each stimulus were obtained at the end of the scanning session. Results showed brain activation of the trigeminal network in response to piperine, and olfactory-related areas in response to pepperEO. PepperEO induced larger activations in the trigeminal network as compared to piperine. This was possibly due to the associative learning for the pungent sensations of pepperEO. In addition, conjunction analyses showed that the secondary somatosensory area, insula, and thalamus were involved in the processing of both piperine and pepperEO. In conclusion, the results strongly suggest that the cerebral representations of trigeminal irritants can be evoked by learned associations.

The Olfactory Mosaic: Bringing an Olfactory Network Together for Odor Perception.

Olfactory perception and its underlying neural mechanisms are not fixed, but rather vary over time, dependent on various parameters such as state, task, or learning experience. In olfaction, one of the primary sensory areas beyond the olfactory bulb is the piriform cortex. Due to an increasing number of functions attributed to the piriform cortex, it has been argued to be an associative cortex rather than a simple primary sensory cortex. In fact, the piriform cortex plays a key role in creating olfactory percepts, helping to form configural odor objects from the molecular features extracted in the nose. Moreover, its dynamic interactions with other olfactory and nonolfactory areas are also critical in shaping the olfactory percept and resulting behavioral responses. In this brief review, we will describe the key role of the piriform cortex in the larger olfactory perceptual network, some of the many actors of this network, and the importance of the dynamic interactions among the piriform-trans-thalamic and limbic pathways.

Characterization of volatiles of necrotic Stenocereus thurberi and Opuntia littoralis and toxicity and olfactory preference of Drosophila melanogster, D. mojavensis wrigleyi, and D. mojavensis sonorensis to necrotic cactus volatiles.

Drosophila mojavensis wrigleyi and D. mojavensis sonorensis are geographically separated races of cactophilic fruit flies. D. mojavensis sonorensis inhabits the Sonoran Desert and utilizes necrotic rots of Stenocereus thurberi Engelm. as a food source and to oviposit while D. mojavensis wrigleyi inhabits Santa Catalina Island, California and utilizes the necrotic rots of Opuntia littoralis (Engelm.) Cockerell. The objectives of this study were to determine the volatile compositions of the necrotic cacti and to determine if the volatile components show either selective toxicity or attraction toward the fruit flies. The volatile chemical compositions of field-rot specimens of both necrotic cacti were obtained by dynamic headspace (purge-and-trap) and hydrodistillation techniques and analyzed by gas chromatography - mass spectrometry. The volatile fraction of necrotic S. thurberi early rot was dominated by carboxylic acids (84.8%) and the late rot by p-cresol (32.6% in the dynamic headspace sample and 55.9% in the hydrodistilled sample). O. littoralis volatiles were dominated by carboxylic acids (86% in the dynamic headspace sample and 89.1% in the hydrodistilled sample). Fifteen compounds that were identified in the necrotic rot volatiles were used to test insecticidal activity and olfactory preference on the cactophilic Drosophila species, as well as D. melanogaster. Differences in toxicity and olfactory preference were observed between the different taxa. Both races of D. mojavensis exhibited toxicity to benzaldehyde and 2-nonanone, while butanoic acid and palmitic acid were tolerated at high concentrations. D. m. wrigleyi demonstrated a greater olfactory preference for anisole, butanoic acid, 2-heptanone, and palmitic acid than did D. m. sonorensis, while D. m. sonorensis demonstrated a greater preference for hexadecane, octanoic acid, and oleic acid than did D. m. wrigleyi.

Intermittency coding in the primary olfactory system: a neural substrate for olfactory scene analysis.

The spatial and temporal characteristics of the visual and acoustic sensory input are indispensable attributes for animals to perform scene analysis. In contrast, research in olfaction has focused almost exclusively on how the nervous system analyzes the quality and quantity of the sensory signal and largely ignored the spatiotemporal dimension especially in longer time scales. Yet, detailed analyses of the turbulent, intermittent structure of water- and air-borne odor plumes strongly suggest that spatio-temporal information in longer time scales can provide major cues for olfactory scene analysis for animals. We show that a bursting subset of primary olfactory receptor neurons (bORNs) in lobster has the unexpected capacity to encode the temporal properties of intermittent odor signals. Each bORN is tuned to a specific range of stimulus intervals, and collectively bORNs can instantaneously encode a wide spectrum of intermittencies. Our theory argues for the existence of a novel peripheral mechanism for encoding the temporal pattern of odor that potentially serves as a neural substrate for olfactory scene analysis.

Parental olfactory experience influences behavior and neural structure in subsequent generations.

Using olfactory molecular specificity, we examined the inheritance of parental traumatic exposure, a phenomenon that has been frequently observed, but not understood. We subjected F0 mice to odor fear conditioning before conception and found that subsequently conceived F1 and F2 generations had an increased behavioral sensitivity to the F0-conditioned odor, but not to other odors. When an odor (acetophenone) that activates a known odorant receptor (Olfr151) was used to condition F0 mice, the behavioral sensitivity of the F1 and F2 generations to acetophenone was complemented by an enhanced neuroanatomical representation of the Olfr151 pathway. Bisulfite sequencing of sperm DNA from conditioned F0 males and F1 naive offspring revealed CpG hypomethylation in the Olfr151 gene. In addition, in vitro fertilization, F2 inheritance and cross-fostering revealed that these transgenerational effects are inherited via parental gametes. Our findings provide a framework for addressing how environmental information may be inherited transgenerationally at behavioral, neuroanatomical and epigenetic levels.

Sociosexual stimuli and gonadotropin-releasing hormone/luteinizing hormone secretion in sheep and goats.

Sociosexual stimuli have a profound effect on the physiology of all species. Sheep and goats provide an ideal model to study the impact of sociosexual stimuli on the hypothalamic-pituitary-gonadal axis because we can use the robust changes in the pulsatile secretion of luteinizing hormone as a bioassay of gonadotropin-releasing hormone secretion. We can also correlate these changes with neural activity using the immediate early gene c-fos and in real time using changes in electrical activity in the mediobasal hypothalamus of female goats. In this review, we will update our current understanding of the proven and potential mechanisms and mode of action of the male effect in sheep and goats and then briefly compare our understanding of sociosexual stimuli in ungulate species with the "traditional" definition of a pheromone.

Objective assessment of olfactory function using functional magnetic resonance imaging.

OBJECTIVE: To show the results of a device that generates automated olfactory stimuli suitable for functional magnetic resonance imaging (fMRI) experiments. MATERIAL AND METHODS: Ten normal volunteers, 5 women and 5 men, were studied. The system allows the programming of several sequences, providing the capability to synchronise the onset of odour presentation with acquisition by a trigger signal of the MRI scanner. The olfactometer is a device that allows selection of the odour, the event paradigm, the time of stimuli and the odour concentration. The paradigm used during fMRI scanning consisted of 15-s blocks. The odorant event took 2s with butanol, mint and coffee. RESULTS: We observed olfactory activity in the olfactory bulb, entorhinal cortex (4%), amygdala (2.5%) and temporo-parietal cortex, especially in the areas related to emotional integration. CONCLUSIONS: The device has demonstrated its effectiveness in stimulating olfactory areas and its capacity to adapt to fMRI equipment.

Experience induces functional reorganization in brain regions involved in odor imagery in perfumers.

Areas of expertise that cultivate specific sensory domains reveal the brain's ability to adapt to environmental change. Perfumers are a small population who claim to have a unique ability to generate olfactory mental images. To evaluate the impact of this expertise on the brain regions involved in odor processing, we measured brain activity in novice and experienced (student and professional) perfumers while they smelled or imagined odors. We demonstrate that olfactory imagery activates the primary olfactory (piriform) cortex (PC) in all perfumers, demonstrating that similar neural substrates were activated in odor perception and imagination. In professional perfumers, extensive olfactory practice influences the posterior PC, the orbitofrontal cortex, and the hippocampus; during the creation of mental images of odors, the activity in these areas was negatively correlated with experience. Thus, the perfumers' expertise is associated with a functional reorganization of key olfactory and memory brain regions, explaining their extraordinary ability to imagine odors and create fragrances.

Effects of Bax gene deletion on social behaviors and neural response to olfactory cues in mice.

Bax is a pro-death protein that plays a crucial role in developmental neuronal cell death. Bax(-/-) mice exhibit increased neuron number and lack several neural sex differences. Here we examined the effects of Bax gene deletion on social behaviors (olfactory preference, social recognition, social approach and aggression) and the neural processing of olfactory cues. Bax deletion eliminated the normal sex difference in olfactory preference behavior. In the social recognition test, both genotypes discriminated a novel conspecific, but wild-type males and Bax(-/-) animals of both sexes spent much more time than wild-type females investigating stimulus animals. Similarly, Bax(-/-) mice were more sociable than wild-type mice in a social approach test. Bax deletion had no effect on aggression in a resident/intruder paradigm where males, regardless of genotype, exhibited a shorter latency to attack. Thus, the prevention of neuronal cell death by Bax gene deletion results in greater sociability as well as the elimination of sex differences in some social behaviors. To examine olfactory processing of socially relevant cues, we counted c-Fos-immunoreactive (Fos-ir) cells in several nodes of the accessory olfactory pathway after exposure to male-soiled or control bedding. In both genotypes, exposure to male-soiled bedding increased Fos-ir cells in the posterodorsal medial amygdala, principal nucleus of the bed nucleus of the stria terminalis and medial preoptic nucleus (MPN), and the response in the MPN was greater in females than in males. However, a reduction in Fos-ir cells was seen in the anteroventral periventricular nucleus of Bax(-/-) mice.

Complementary sensory and associative microcircuitry in primary olfactory cortex.

The three-layered primary olfactory (piriform) cortex is the largest component of the olfactory cortex. Sensory and intracortical inputs converge on principal cells in the anterior piriform cortex (aPC). We characterize organization principles of the sensory and intracortical microcircuitry of layer II and III principal cells in acute slices of rat aPC using laser-scanning photostimulation and fast two-photon population Ca(2+) imaging. Layer II and III principal cells are set up on a superficial-to-deep vertical axis. We found that the position on this axis correlates with input resistance and bursting behavior. These parameters scale with distinct patterns of incorporation into sensory and associative microcircuits, resulting in a converse gradient of sensory and intracortical inputs. In layer II, sensory circuits dominate superficial cells, whereas incorporation in intracortical circuits increases with depth. Layer III pyramidal cells receive more intracortical inputs than layer II pyramidal cells, but with an asymmetric dorsal offset. This microcircuit organization results in a diverse hybrid feedforward/recurrent network of neurons integrating varying ratios of intracortical and sensory input depending on a cell's position on the superficial-to-deep vertical axis. Since burstiness of spiking correlates with both the cell's location on this axis and its incorporation in intracortical microcircuitry, the neuronal output mode may encode a given cell's involvement in sensory versus associative processing.

¿Cómo se conecta la olfacción? Mecanismos celulares y moleculares que dirigen el desarrollo de las conexiones sinápticas desde la nariz a la corteza (II) / How is the sense of smell connected? Cellular and molecular mechanisms guiding the development of the synaptic connections from the nose to the cortex (II)

Como iniciamos en la primera parte de esta revisión, el desarrollo del sistema olfativo presenta una serie de peculiaridades fascinantes, lo que lo convierte en uno de los modelos más estudiados para entender los mecanismos del desarrollo del sistema nervioso. Si en la primera parte revisamos los diferentes mecanismos por contacto (lamininas, moléculas de adhesión celular, efrinas, etc.) y secretables (semaforinas, slits, factores de crecimiento, etc.) que intervienenen la formación de las conexiones sinápticas entre el epitelio olfativo, el bulbo olfativo y la corteza olfativa, en esta segunda parte revisaremos los mecanismos moleculares responsables de las conexiones intracorticales del sistema olfativo principal, así como la limitada información disponible acerca del sistema olfativo accesorio. También revisaremos los mecanismos implicados en la migración de los precursores de interneuronas desde la zona subventricular del cerebro anterior hasta el bulbo olfativo, otro de los eventos fundamentales en el desarrollo de este sistema (AU)

As discussed in the first part of this review, the development of the olfactory system offers a series of fascinating peculiarities that make it one of the models that has been most widely studied in order to reach an understanding of the mechanisms involved in the development of the nervous system. In the first part we reviewed the different mechanisms based on contact (laminins, cell adhesion molecules, ephrins, etc.) and on secretion (semaphorins, slits, growth factors, etc.) that are involved in the formation of the synaptic connections among the olfactory epithelium, the olfactory bulb andthe olfactory cortex. In this second part we will review the molecular mechanisms responsible for the intracortical connections in the main olfactory system, as well as the limited information available concerning the accessory olfactory system. We shall also review the mechanisms involved in the migration of the interneuron precursors from the subventricular area of the forebrain to the olfactory bulb, which is another crucial event in the development of this system (AU)

¿Cómo se conecta la olfacción? Mecanismos celulares y moleculares que dirigen el desarrollo de las conexiones sinápticas desde la nariz a la corteza (I) / How is the sense of smell connected? Cellular and molecular mechanisms guiding the development

Resumen. Dentro del conjunto del sistema nervioso central, el sistema olfativo resulta fascinante por sus particularidades fisiológicas durante el desarrollo, siendo uno de los modelos más estudiados para entender los mecanismos relacionados con la guía y el crecimiento axonal hacia sus objetivos apropiados. Se conoce una constelación de mecanismos, unos mediados por contacto (lamininas, moléculas de adhesión celular, efrinas, etc.) y otros secretables (semaforinas, slits, factores de crecimiento, etc.), por desempeñar diversas funciones en el establecimiento de las interacciones sinápticas entre el epitelio olfativo, el bulbo olfativo y la corteza olfativa. También se han propuesto al respecto otros mecanismos específicos de este sistema, incluida la increíble familia de cerca de 1.000 receptores olfativos distintos. En los últimos años, diferentes revisiones se han concentrado en los elementos parciales de este sistema, sobre todo en los mecanismos implicados en la formación del nervio olfativo, echándose en falta una revisión detallada de aquellos relacionados con el desarrollo de las conexiones entre las distintas estructuras olfativas (epitelio, bulbo y corteza). En esta primera parte de la revisión, abordamos este tema desde el siguiente enfoque: los diversos mecanismos celulares y moleculares que dirigen la formación del nervio olfativo y el tracto olfativo lateral (AU)

Summary. The physiological particularities that occur during the development of the olfactory system make it one of the most fascinating parts of the central nervous system and one of models that has been most widely studied in order to understand the mechanisms related with axonal growth and guidance towards the right targets. A variety of mechanisms are known, some mediated by contact (laminins, cell adhesion molecules, ephrins, etc.) and others that are secreted (semaphorins, slits, growth factors, etc.), to play diverse roles in establishing the synaptic interactions among the olfactory epithelium, the olfactory bulb and the olfactory cortex. In relation to this, other specific mechanisms for this system have also been proposed, including the incredible family of close to 1000 different olfactory receptors. In recent years, different reviews have focused on the partial elements of this system, especially on the mechanisms involved in the formation of the olfactory nerve. However, no detailed review of those related with the development of the connections between the different olfactory structures (epithelium, bulb and cortex) has been put forward to date. In this first part of the review, we address this topic from the following perspective: the different cellular and molecular mechanisms that guide the formation of the olfactory nerve and the lateral olfactory tract (AU)

Taste, olfactory and food texture reward processing in the brain and obesity.

Complementary neuronal recordings and functional neuroimaging in humans, show that the primary taste cortex in the anterior insula provides separate and combined representations of the taste, temperature and texture (including fat texture) of food in the mouth independently of hunger and thus of reward value and pleasantness. One synapse on, in the orbitofrontal cortex (OFC), these sensory inputs are for some neurons combined by learning with olfactory and visual inputs, and these neurons encode food reward in that they only respond to food when hungry, and in that activations correlate with subjective pleasantness. Cognitive factors, including word-level descriptions, and attention, modulate the representation of the reward value of food in the OFC. Further, there are individual differences in the representation of the reward value of food in the OFC. It is argued that overeating and obesity are related in many cases to an increased reward value of the sensory inputs produced by foods, and their modulation by cognition and attention, which overrides existing satiety signals. It is proposed that control of all rather than one or several of these factors that influence food reward and eating may be important in the prevention and treatment of overeating and obesity.

Electrophysiological analysis of the inhibitory effects of FMRFamide-like peptides on the pacemaker activity of gonadotropin-releasing hormone neurons.

Gonadotropin-releasing hormone (GnRH) neurons in the terminal nerve (TN) show endogenous pacemaker activity, which is suggested to be dependent on the physiological conditions of the animal. The TN-GnRH neurons have been suggested to function as a neuromodulatory neuron that regulates long-lasting changes in the animal behavior. It has been reported that the TN-GnRH neurons are immunoreactive to FMRFamide. Here, we find that the pacemaker activity of TN-GnRH neuron is inhibited by FMRFamide: bath application of FMRFamide decreased the frequency of pacemaker activity of TN-GnRH neurons in a dose-dependent manner. This decrease was suppressed by a blockage of G protein-coupled receptor pathway by GDP-ß-S. In addition, FMRFamide induced an increase in the membrane conductance, and the reversal potential for the FMRFamide-induced current changed according to the changes in [K(+)](out) as predicted from the Nernst equation for K(+). We performed cloning and sequence analysis of the PQRFamide (NPFF/NPAF) gene in the dwarf gourami and found evidence to suggest that FMRFamide-like peptide in TN-GnRH neurons of the dwarf gourami is NPFF. NPFF actually inhibited the pacemaker activity of TN-GnRH neurons, and this inhibition was blocked by RF9, a potent and selective antagonist for mammalian NPFF receptors. These results suggest that the activation of K(+) conductance by FMRFamide-like peptide (≈NPFF) released from TN-GnRH neurons themselves causes the hyperpolarization and then inhibition of pacemaker activity in TN-GnRH neurons. Because TN-GnRH neurons make tight cell clusters in the brain, it is possible that FMRFamide-like peptides released from TN-GnRH neurons negatively regulates the activities of their own (autocrine) and/or neighboring neurons (paracrine).