Incorporating the image formation process into deep learning improves network performance.

We present Richardson-Lucy network (RLN), a fast and lightweight deep learning method for three-dimensional fluorescence microscopy deconvolution. RLN combines the traditional Richardson-Lucy iteration with a fully convolutional network structure, establishing a connection to the image formation process and thereby improving network performance. Containing only roughly 16,000 parameters, RLN enables four- to 50-fold faster processing than purely data-driven networks with many more parameters. By visual and quantitative analysis, we show that RLN provides better deconvolution, better generalizability and fewer artifacts than other networks, especially along the axial dimension. RLN outperforms classic Richardson-Lucy deconvolution on volumes contaminated with severe out of focus fluorescence or noise and provides four- to sixfold faster reconstructions of large, cleared-tissue datasets than classic multi-view pipelines. We demonstrate RLN's performance on cells, tissues and embryos imaged with widefield-, light-sheet-, confocal- and super-resolution microscopy.

Circuit-Specific Plasticity of Callosal Inputs Underlies Cortical Takeover.

Injury induces synaptic, circuit, and systems reorganization. After unilateral amputation or stroke, this functional loss disrupts the interhemispheric interaction between intact and deprived somatomotor cortices to recruit deprived cortex in response to intact limb stimulation. This recruitment has been implicated in enhanced intact sensory function. In other patients, maladaptive consequences such as phantom limb pain can occur. We used unilateral whisker denervation in male and female mice to detect circuitry alterations underlying interhemispheric cortical reorganization. Enhanced synaptic strength from the intact cortex via the corpus callosum (CC) onto deep neurons in deprived primary somatosensory barrel cortex (S1BC) has previously been detected. It was hypothesized that specificity in this plasticity may depend on to which area these neurons projected. Increased connectivity to somatomotor areas such as contralateral S1BC, primary motor cortex (M1) and secondary somatosensory cortex (S2) may underlie beneficial adaptations, while increased connectivity to pain areas like anterior cingulate cortex (ACC) might underlie maladaptive pain phenotypes. Neurons from the deprived S1BC that project to intact S1BC were hyperexcitable, had stronger responses and reduced inhibitory input to CC stimulation. M1-projecting neurons also showed increases in excitability and CC input strength that was offset with enhanced inhibition. S2 and ACC-projecting neurons showed no changes in excitability or CC input. These results demonstrate that subgroups of output neurons undergo dramatic and specific plasticity after peripheral injury. The changes in S1BC-projecting neurons likely underlie enhanced reciprocal connectivity of S1BC after unilateral deprivation consistent with the model that interhemispheric takeover supports intact whisker processing.SIGNIFICANCE STATEMENT Amputation, peripheral injury, and stroke patients experience widespread alterations in neural activity after sensory loss. A hallmark of this reorganization is the recruitment of deprived cortical space which likely aids processing and thus enhances performance on intact sensory systems. Conversely, this recruitment of deprived cortical space has been hypothesized to underlie phenotypes like phantom limb pain and hinder recovery. A mouse model of unilateral denervation detected remarkable specificity in alterations in the somatomotor circuit. These changes underlie increased reciprocal connectivity between intact and deprived cortical hemispheres. This increased connectivity may help explain the enhanced intact sensory processing detected in humans.

Incubation of Fear Is Regulated by TIP39 Peptide Signaling in the Medial Nucleus of the Amygdala.

Fear-related psychopathologies such as post-traumatic stress disorder are characterized by impaired extinction of fearful memories. Recent behavioral evidence suggests that the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39), via its receptor, the parathyroid hormone 2 receptor (PTH2R), modulates fear memory. Here we examined the anatomical and cellular localization of TIP39 signaling that contributes to the increase in fear memory over time following a traumatic event, called fear memory incubation. Contextual freezing, a behavioral sign of fear memory, was significantly greater in PTH2R knock-out than wild-type male mice 2 and 4 weeks after a 2 s 1.5 mA footshock. PTH2R knock-out mice had significantly reduced c-Fos activation in the medial amygdala (MeA) following both footshock and fear recall, but had normal activation in the hypothalamic paraventricular nucleus and the amygdalar central nucleus compared with wild-type. We therefore investigated the contribution of MeA TIP39 signaling to fear incubation. Similar to the effect of global TIP39 signaling loss, blockade of TIP39 signaling in the MeA by lentivirus-mediated expression of a secreted PTH2R antagonist augmented fear incubation. Ablation of MeA PTH2R-expressing neurons also strengthened the fear incubation effect. Using the designer receptor exclusively activated by designer drug pharmacogenetic approach, transient inhibition of MeA PTH2R-expressing neurons before or immediately after the footshock, but not at the time of fear recall, enhanced fear incubation. Collectively, the findings demonstrate that TIP39 signaling within the MeA at the time of an aversive event regulates the increase over time in fear associated with the event context. SIGNIFICANCE STATEMENT: Fear-related psychopathologies such as post-traumatic stress disorder (PTSD) are characterized by excessive responses to trauma-associated cues. Fear responses can increase over time without additional cue exposure or stress. This work shows that modulatory processes within the medial nucleus of the amygdala near the time of a traumatic event influence the strength of fear responses that occur much later. The modulatory processes include signaling by the neuropeptide TIP39 and neurons that express its receptor. These findings will help in the understanding of why traumatic events sometimes have severe psychological consequences. One implication is that targeting neuromodulation in the medial amygdala could potentially help prevent development of PTSD.

Neuropathic and inflammatory pain are modulated by tuberoinfundibular peptide of 39 residues.

Nociceptive information is modulated by a large number of endogenous signaling agents that change over the course of recovery from injury. This plasticity makes understanding regulatory mechanisms involved in descending inhibition of pain scientifically and clinically important. Neurons that synthesize the neuropeptide TIP39 project to many areas that modulate nociceptive information. These areas are enriched in its receptor, the parathyroid hormone 2 receptor (PTH2R). We previously found that TIP39 affects several acute nociceptive responses, leading us to now investigate its potential role in chronic pain. Following nerve injury, both PTH2R and TIP39 knockout mice developed less tactile and thermal hypersensitivity than controls and returned to baseline sensory thresholds faster. Effects of hindpaw inflammatory injury were similarly decreased in knockout mice. Blockade of α-2 adrenergic receptors increased the tactile and thermal sensitivity of apparently recovered knockout mice, returning it to levels of neuropathic controls. Mice with locus coeruleus (LC) area injection of lentivirus encoding a secreted PTH2R antagonist had a rapid, α-2 reversible, apparent recovery from neuropathic injury similar to the knockout mice. Ablation of LC area glutamatergic neurons led to local PTH2R-ir loss, and barley lectin was transferred from local glutamatergic neurons to GABA interneurons that surround the LC. These results suggest that TIP39 signaling modulates sensory thresholds via effects on glutamatergic transmission to brainstem GABAergic interneurons that innervate noradrenergic neurons. TIP39's normal role may be to inhibit release of hypoalgesic amounts of norepinephrine during chronic pain. The neuropeptide may help maintain central sensitization, which could serve to enhance guarding behavior.

Anxiety- and depression-like behavior and impaired neurogenesis evoked by peripheral neuropathy persist following resolution of prolonged tactile hypersensitivity.

Pain and depression are frequently associated with and often persist after resolution of an initial injury. Identifying the extent to which depression remains causally associated with ongoing physical discomfort during chronic pain, or becomes independent of it, is an important problem for basic neuroscience and psychiatry. Difficulty in distinguishing between effects of ongoing aversive sensory input and its long-term consequences is a significant roadblock, especially in animal models. To address this relationship between localized physical discomfort and its more global consequences, we investigated cellular and behavioral changes during and after reversing a mouse model of neuropathic pain. Tactile allodynia produced by placing a plastic cuff around the sciatic nerve resolved within several days when the cuff was removed. In contrast, the changes in elevated O-maze, forced-swim, Y-maze spontaneous alternation and novel-object recognition test performance that developed after nerve cuff placement remained for at least 3 weeks after the nerve cuffs were removed, or 10-15 d following complete normalization of mechanical sensitivity. Hippocampal neurogenesis, measured by doublecortin and proliferating cell nuclear antigen expression, was also suppressed after nerve cuff placement and remained suppressed 3 weeks after cuff removal. FosB expression was elevated in the central nucleus of the amygdala and spinal cord dorsal horn only in mice with ongoing allodynia. In contrast, FosB remained elevated in the basolateral amygdala of mice with resolved nociception and persisting behavioral effects. These observations suggest that different processes control tactile hypersensitivity and the behavioral changes and impaired neurogenesis that are associated with neuropathic allodynia.

Ethanol produces corticotropin-releasing factor receptor-dependent enhancement of spontaneous glutamatergic transmission in the mouse central amygdala.

BACKGROUND: Ethanol (EtOH) modulation of central amygdala (CeA) neurocircuitry plays a key role in the development of alcoholism via activation of the corticotropin-releasing factor (CRF) receptor (CRFR) system. Previous work has predominantly focused on EtOH × CRF interactions on the CeA GABA circuitry; however, our laboratory recently showed that CRF enhances CeA glutamatergic transmission. Therefore, this study sought to determine whether EtOH modulates CeA glutamate transmission via activation of CRF signaling. METHODS: The effects of EtOH on spontaneous excitatory postsynaptic currents (sEPSCs) and basal resting membrane potentials were examined via standard electrophysiology methods in adult male C57BL/6J mice. Local ablation of CeA CRF neurons (CRF(CeAhDTR) ) was achieved by targeting the human diphtheria toxin receptor (hDTR) to CeA CRF neurons with an adeno-associated virus. Ablation was quantified post hoc with confocal microscopy. Genetic targeting of the diphtheria toxin active subunit to CRF neurons (CRF(DTA) mice) ablated CRF neurons throughout the central nervous system, as assessed by quantitative reverse transcriptase polymerase chain reaction quantification of CRF mRNA. RESULTS: Acute bath application of EtOH significantly increased sEPSC frequency in a concentration-dependent manner in CeA neurons, and this effect was blocked by pretreatment of co-applied CRFR1 and CRFR2 antagonists. In experiments utilizing a CRF-tomato reporter mouse, EtOH did not significantly alter the basal membrane potential of CeA CRF neurons. The ability of EtOH to enhance CeA sEPSC frequency was not altered in CRF(CeAhDTR) mice despite a ~78% reduction in CeA CRF cell counts. The ability of EtOH to enhance CeA sEPSC frequency was also not altered in the CRF(DTA) mice despite a 3-fold reduction in CRF mRNA levels. CONCLUSIONS: These findings demonstrate that EtOH enhances spontaneous glutamatergic transmission in the CeA via a CRFR-dependent mechanism. Surprisingly, our data suggest that this action may not require endogenous CRF.

Synaptoporin and parathyroid hormone 2 as markers of multimodal inputs to the auditory brainstem.

The distribution of the synaptic vesicle protein synaptoporin was investigated by immunofluorescence in the central auditory system of the mouse brainstem. Synaptoporin immunostaining displayed region-specific differences. High and moderate accumulations of were seen in the superficial layer of the dorsal cochlear nucleus, dorsal and external regions of the inferior colliculus, the medial and dorsal divisions of the medial geniculate body and in periolivary regions of the superior olivary complex (SOC). Low or absent labeling was observed in the more central parts of these structures such as the principal nuclei of the SOC. It was conspicuous that dense synaptoporin immunoreactivity was detected predominantly in areas, which are known to be synaptic fields of multimodal, extra-auditory inputs. Target neurons of synaptoporin-positive synapses in the SOC were then identified by double-labelling immunofluorescence microscopy. We thereby detected synaptoporin puncta perisomatically at nitrergic, glutamatergic and serotonergic neurons but none next to neurons immunoreactive for choline-acetyltransferase and calcitonin-gene related peptide. These results leave open whether functionally distinct neuronal groups are accessed in the SOC by synaptoporin-containing neurons. The last part of our study sought to find out whether synaptoporin-positive neurons originate in the medial paralemniscal nucleus (MPL), which is characterized by expression of the peptide parathyroid hormone 2 (PTH2). Anterograde neuronal tracing upon injection into the MPL in combination with synaptoporin- and PTH2-immunodetection showed that (1) the MPL projects to the periolivary SOC using PTH2 as transmitter, (2) synaptoporin-positive neurons do not originate in the MPL, and (3) the close juxtaposition of synaptoporin-staining with either the anterograde tracer or PTH2 reflect concerted action of the different inputs to the SOC.

Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP.

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase-activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

Dissociable control of motivation and reinforcement by distinct ventral striatal dopamine receptors.

Dopamine release in striatal circuits, including the nucleus accumbens (NAc), tracks separable features of reward such as motivation and reinforcement. However, the cellular and circuit mechanisms by which dopamine receptors transform dopamine release into distinct constructs of reward remain unclear. Here, we show that dopamine D3 receptor (D3R) signaling in the NAc drives motivated behavior by regulating local NAc microcircuits. Furthermore, D3Rs co-express with dopamine D1 receptors (D1Rs), which regulate reinforcement, but not motivation. Paralleling dissociable roles in reward function, we report non-overlapping physiological actions of D3R and D1R signaling in NAc neurons. Our results establish a novel cellular framework wherein dopamine signaling within the same NAc cell type is physiologically compartmentalized via actions on distinct dopamine receptors. This structural and functional organization provides neurons in a limbic circuit with the unique ability to orchestrate dissociable aspects of reward-related behaviors that are relevant to the etiology of neuropsychiatric disorders.

Regulation of hypothalamic signaling by tuberoinfundibular peptide of 39 residues is critical for the response to cold: a novel peptidergic mechanism of thermoregulation.

Euthermia is critical for mammalian homeostasis. Circuits within the preoptic hypothalamus regulate temperature, with fine control exerted via descending GABAergic inhibition of presympathetic motor neurons that control brown adipose tissue (BAT) thermogenesis and cutaneous vascular tone. The thermoregulatory role of hypothalamic excitatory neurons is less clear. Here we report peptidergic regulation of preoptic glutamatergic neurons that contributes to temperature regulation. Tuberoinfundibular peptide of 39 residues (TIP39) is a ligand for the parathyroid hormone 2 receptor (PTH2R). Both peptide and receptor are abundant in the preoptic hypothalamus. Based on PTH2R and vesicular glutamate transporter 2 (VGlut2) immunolabeling in animals with retrograde tracer injection, PTH2R-containing glutamatergic fibers are presynaptic to neurons projecting from the median preoptic nucleus (MnPO) to the dorsomedial hypothalamus. Transneuronal retrograde pathway tracing with pseudorabies virus revealed connectivity between MnPO VGlut2 and PTH2R neurons and BAT. MnPO injection of TIP39 increased body temperature by 2°C for several hours. Mice lacking TIP39 signaling, either because of PTH2R-null mutation or brain delivery of a PTH2R antagonist had impaired heat production upon cold exposure, but no change in basal temperature and no impairment in response to a hot environment. Thus, TIP39 appears to act on PTH2Rs present on MnPO glutamatergic terminals to regulate their activation of projection neurons and subsequent sympathetic BAT activation. This excitatory mechanism of heat production appears to be activated on demand, during cold exposure, and parallels the tonic inhibitory GABAergic control of body temperature.

Behavioural actions of tuberoinfundibular peptide 39 (parathyroid hormone 2).

Tuberoinfundibular peptide of 39 residues (TIP39) acts via its endogenous class B G-protein coupled receptorthe parathyroid hormone 2 receptor (PTH2R). Hence, it is also known as parathyroid hormone 2. The peptide is expressed in the brain by a small number of neurons with a highly restricted distribution, which in turn project to a large number of brain regions that contain PTH2R. This peptide neuromodulator system has been extensively investigated over the past 20 years including its behavioural actions, such as its role in the control of nociception, fear and fear incubation, anxiety and depression-like behaviours, and maternal and social behaviours. It also influences thermoregulation and potentially auditory responses. TIP39 probably exerts direct effect on the neuronal networks controlling these behaviours based on the localization of PTH2R and local TIP39 actions. In addition, TIP39 also affects the secretion of several hypothalamic hormones providing the basis for indirect behavioural actions. Recently developed experimental tools have stimulated further behavioural investigations, and novel results obtained are discussed in this review.

A thalamo-preoptic pathway promotes social grooming in rodents.

Social touch is an essential component of communication. Little is known about the underlying pathways and mechanisms. Here, we discovered a novel neuronal pathway from the posterior intralaminar thalamic nucleus (PIL) to the medial preoptic area (MPOA) involved in the control of social grooming. We found that the neurons in the PIL and MPOA were naturally activated by physical contact between female rats and also by the chemogenetic stimulation of PIL neurons. The activity-dependent tagging of PIL neurons was performed in rats experiencing physical social contact. The chemogenetic activation of these neurons increased social grooming between familiar rats, as did the selective activation of the PIL-MPOA pathway. Neurons projecting from the PIL to the MPOA express the neuropeptide parathyroid hormone 2 (PTH2), and the central infusion of its receptor antagonist diminished social grooming. Finally, we showed a similarity in the anatomical organization of the PIL and the distribution of the PTH2 receptor in the MPOA between the rat and human brain. We propose that the discovered neuronal pathway facilitates physical contact with conspecifics.

Reduced fear memory and anxiety-like behavior in mice lacking formylpeptide receptor 1.

N-formylpeptide receptor 1 (FPR1) is a G protein-coupled receptor that mediates pro-inflammatory chemotactic responses by phagocytic leukocytes to N-formylpeptides produced by bacteria or mitochondria. Mice lacking Fpr1 (Fpr1 (-/-) mice) have increased susceptibility to challenge with certain bacteria. FPR1 is also a receptor for annexin-1, which mediates the anti-inflammatory effects of glucocorticoids as well as negative feedback by glucocorticoids of the hypothalamic-pituitary-adrenocortical axis. However, homeostatic functions of FPR1 in the neuroendocrine system have not previously been defined. Here we show that in systematic behavioral testing Fpr1 (-/-) mice exhibited increased exploratory activity, reduced anxiety-like behavior, and impaired fear memory, but normal spatial memory and learning capacity. Consistent with this, the homeostatic serum level of corticosterone in Fpr1 (-/-) mice was significantly lower compared with wild-type mice. The data implicate Fpr1 in modulation of anxiety-like behavior and fear memory by regulating glucocorticoid production.

Parathyroid hormone 2 receptor is a functional marker of nociceptive myelinated fibers responsible for neuropathic pain.

We have previously demonstrated that parathyroid hormone 2 (PTH2) receptors are expressed in dorsal root ganglion (DRG) neurons and that its endogenous agonist tuberoinfundibular peptide of 39 residues (TIP39) causes nociceptive paw flexor responses after intraplantar administration. Here we found that the PTH2 receptor is selectively localized on myelinated A-, but not unmyelinated C-fibers using immunohistochemical labeling, based on PTH2 receptor expression on antibody N52-positive medium/large-sized DRG neurons, but not on TRPV1, substance P, P2X(3) receptor or isolectin B4-binding protein-positive small-sized DRG neurons. Pharmacological studies showed that TIP39-induced nociceptive responses were mediated by activation of G(s) and cAMP-dependent protein kinase. We also found that nociceptive responses induced by TIP39- or the cAMP analog 8-bromo-cAMP were significantly greater following partial sciatic nerve injury induced neuropathic pain, without changes in PTH2 receptor expression. Together these data suggest that activation of PTH2 receptors stimulates nociceptive A-fiber through G(s)-cAMP-dependent protein kinase signaling, and this pathway has elevated sensitization following nerve injury.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

Tuberoinfundibular Peptide of 39 residues is required for germ cell development.

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a PTH 2 receptor ligand. We report that mice with deletion of Tifp39, the gene encoding TIP39, are sterile. Testes contained Leydig and Sertoli cells and spermatogonia but no spermatids. Labeling chromosome spreads with antibodies to proteins involved in recombination showed that spermatogonia do not complete prophase of meiosis I. Chromosomes were observed at different stages of recombination in single nuclei, a defect not previously described with mutations in genes known to be specifically involved in DNA replication and recombination. TIP39 was previously shown to be expressed in neurons projecting to the hypothalamus and within the testes. LH and FSH were slightly elevated in Tifp39(-/-) mice, suggesting intact hypothalamic function. We found using in situ hybridization that the genes encoding TIP39 and the PTH 2 receptor are expressed in a stage-specific manner within seminiferous tubules. Using immunohistochemistry and quantitative RT-PCR, TIP39 expression is greatest in mature testes, and appears most abundant in postmeiotic spermatids, but TIP39 protein and mRNA can be detected before any cells have completed meiosis. We used mice that express Cre recombinase under control of a spermatid-specific promoter to express selectively a cDNA encoding TIP39 in the testes of Tifp39(-/-) mice. Spermatid production and fertility were rescued, demonstrating that the defect in Tifp39(-/-) mice was due to the loss of TIP39. These results show that TIP39 is essential for germ cell development and suggest that it may act as an autocrine or paracrine agent within the gonads.

Tuberoinfundibular peptide of 39 residues in the embryonic and early postnatal rat brain.

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as the endogenous ligand of parathyroid hormone 2 receptor. We have recently demonstrated that TIP39 expression in adult rat brain is confined to the subparafascicular area of the thalamus with a few cells extending laterally into the posterior intralaminar thalamic nucleus (PIL), and the medial paralemniscal nucleus (MPL) in the lateral pontomesencephalic tegmentum. During postnatal development, TIP39 expression increases until postnatal day 33 (PND-33), then decreases, and almost completely disappears by PND-125. Here, we report the expression of TIP39 during early brain development. TIP39-immunoreactive (TIP39-ir) neurons in the subparafascicular area first appeared at PND-1. In contrast, TIP39-ir neurons were detectable in the MPL at embryonic day 14.5 (ED-14.5), and the intensity of their labeling increased thereafter. We also identified TIP39-ir neurons between ED-16.5 and PND-5 in two additional brain areas, the PIL and the amygdalo-hippocampal transitional zone (AHi). We confirmed the specificity of TIP39 immunolabeling by demonstrating TIP39 mRNA using in situ hybridization histochemistry. In the PIL, TIP39 neurons are located medial to the CGRP group as demonstrated by double immunolabeling. All TIP39-ir neurons in the AHi and most TIP39-ir neurons in the PIL disappear during early postnatal development. The adult pattern of TIP39-ir fibers emerge during postnatal development. However, fibers emanating from PIL can be followed in the supraoptic decussations towards the hypothalamus at ED-18.5. These TIP39-ir fibers disappear by PND-1. The complex pattern of TIP39 expression during early brain development suggests the involvement of TIP39 in transient functions during ontogeny.

Evidence for the expression of parathyroid hormone 2 receptor in the human brainstem.

BACKGROUND AND PURPOSE: The parathyroid hormone 2 receptor (PTH2R) is a G protein coupled receptor. Pharmacological and anatomical evidence suggests that the recently identified tuberoinfundibular peptide of 39 residues is, and parathyroid hormone and parathyroid hormone-related peptide are not, its endogenous ligand. Initial functional studies suggest that the PTH2R is involved in the regulation of viscerosensory information processing. As a first step towards clinical applications, herein we describe the presence of the PTH2R in the human brainstem. MATERIAL AND METHODS: Total RNA was isolated from postmortem human cortical and brainstem samples for RT-PCR. Good quality RNA, as assessed on formaldehyde gel, was reverse transcribed. The combined cDNA products were used as template in PCR reactions with primer pairs specific for the human PTH2R. In addition, PTH2R immunolabelling was performed on free floating sections of the human medulla oblongata using fluorescent amplification immunochemistry. RESULTS: Specific bands in the RT-PCR experiments and sequencing of PCR products demonstrated the expression of PTH2R mRNA in the human brainstem. A high density of PTH2R-immunoreactive fibers was found in brain regions of the medulla oblongata including the nucleus of the solitary tract, the spinal trigeminal nucleus, and the dorsal reticular nucleus of the medulla. CONCLUSION: Independent demonstration of the presence of PTH2R mRNA and immunoreactivity supports the specific expression of the PTH2R in the human brainstem. The distribution of PTH2R-immunoreactive fibers in viscerosensory brain regions is similar to that reported in mouse and rat suggesting a similar role of the PTH2R in human as in rodents. This finding will have important implications when experimental data obtained on the function of the TIP39-PTH2R neuromodulator system in rodents are to be utilized in human.

Distribution of tuberoinfundibular peptide of 39 residues and its receptor, parathyroid hormone 2 receptor, in the mouse brain.

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a potent parathyroid hormone 2 receptor (PTH2R) agonist. Existing anatomical data also support the suggestion that TIP39 is the PTH2R's endogenous ligand, but a comprehensive comparison of TIP39 and PTH2R distributions has not been performed. In the present study, we compared the distributions of TIP39 and PTH2R on adjacent mouse brain sections. In addition, we determined the locations of PTH2R-expressing cell bodies by in situ hybridization histochemistry and by labeling beta-galactosidase driven by the PTH2R promoter in knockin mice. An excellent correlation was found between the distributions of TIP39-containing fibers and PTH2R-containing cell bodies and fibers throughout the brain. TIP39 and the PTH2R are abundant in medial prefrontal, insular, and ectorhinal cortices, the lateral septal nucleus, the bed nucleus of the stria terminalis, the fundus striati, the amygdala, the ventral subiculum, the hypothalamus, midline and intralaminar thalamic nuclei, the medial geniculate body, the periaqueductal gray, the ventral tegmental area, the superior and inferior colliculi, the parabrachial nuclei, the locus coeruleus, subcoeruleus and periolivary areas, and the nucleus of the solitary tract. Furthermore, even the subregional distribution of TIP39- and PTH2R-immunoreactive fibers in these regions showed remarkable similarities, providing anatomical evidence that TIP39 may act on the PTH2R. Based on these observations and on previous pharmacological data, we propose that TIP39 is an endogenous ligand of the PTH2R and that they form a neuromodulator system, which is optimally positioned to regulate limbic, endocrine, and auditory brain functions. Published 2007 Wiley-Liss, Inc.

Development of a rat parathyroid hormone 2 receptor antagonist.

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine(4), tyrosine(5), tryptophan(6), histidine(7)-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.