Anti-glutamatergic Effects of Three Lignan Compounds: Arctigenin, Matairesinol and Trachelogenin - An ex vivo Study on Rat Brain Slices.

Arctigenin is a bioactive dibenzylbutyrolactone-type lignan exhibiting various pharmacological activities. The neuroprotective effects of arctigenin were demonstrated to be mediated via inhibition of AMPA and KA type glutamate receptors in the somatosensory cortex of the rat brain. The aim of this study was to compare the effects of arctigenin with matairesinol and trachelogenin on synaptic activity in ex vivo rat brain slices. Arctigenin, matairesinol and trachelogenin were isolated from Arctium lappa, Centaurea scabiosa and Cirsium arvense, respectively, and applied on brain slices via perfusion medium at the concentration range of 0.5 - 40 µM. The effects of the lignans were examined in the CA1 hippocampus and the somatosensory cortex by recording electrically evoked field potentials. Arctigenin and trachelogenin caused a significant dose-dependent decrease in the amplitude of hippocampal population spikes (POPS) and the slope of excitatory postsynaptic potentials (EPSPs), whereas matairesinol (1 µM and 10 µM) decreased EPSP slope but had no effect on POPS amplitude. Trachelogenin effect (0.5 µM, 10 µM, 20 µM) was comparable to arctigenin (1 µM, 20 µM, 40 µM) (p > 0.05). In the neocortex, arctigenin (10 µM, 20 µM) and trachelogenin (10 µM) significantly decreased the amplitude of evoked potential early component, while matairesinol (1 µM and 10 µM) had no significant effect (p > 0.05). The results suggest that trachelogenin and arctigenin act via inhibition of AMPA and KA receptors in the brain and trachelogenin has a higher potency than arctigenin. Thus, trachelogenin and arctigenin could serve as lead compounds in the development of neuroprotective drugs.

Synaptic proteome changes in the hypothalamus of mother rats.

To establish synaptic proteome changes associated with motherhood, we isolated synaptosome fractions from the hypothalamus of mother rats and non-maternal control females at the 11th postpartum day. Proteomic analysis by two-dimensional differential gel electrophoresis combined with mass spectrometric protein identification established 26 significant proteins, 7 increasing and 19 decreasing protein levels in the dams. The altered proteins are mainly involved in energy homeostasis, protein folding, and metabolic processes suggesting the involvement of these cellular processes in maternal adaptations. The decrease in a significantly altered protein, complement component 1q subcomponent-binding protein (C1qbp) was validated with Western blotting. Furthermore, immunohistochemistry showed its presence in hypothalamic fibers and terminals in agreement with its presence in synaptosomes. We also found the expression of C1qbp in different hypothalamic nuclei including the preoptic area and the paraventricular hypothalamic nucleus at the protein and at the mRNA level using immunohistochemistry and in situ hybridization histochemistry, respectively. Bioinformatical network analysis revealed that cytokines, growth factors, and protein kinases are common regulators, which indicates a complex regulation of the proteome change in mothers. The results suggest that maternal responsiveness is associated with synaptic proteins level changes in the hypothalamus, and that growth factors and cytokines may govern these alterations. BIOLOGICAL SIGNIFICANCE: The period of motherhood is accompanied with several behavioral, neuroendocrine, emotional and metabolic adaptations in the brain. Although it is established that various hypothalamic networks participate in the maternal adaptations of the rodent brain, our knowledge on the molecular background of these alterations remains seriously limited. In the present study, we first determined that the functional alterations of the maternal brain can be detected at the level of the synaptic proteome in the hypothalamus. Independent confirmation of synaptic localization, and also the established decrease in the level of C1qbp protein suggest the validity of the data. Common regulators of altered proteins belonging to the growth factor and cytokine family suggest that the synaptic adaptation is governed by these extracellular signals and future studies should focus on their specific roles. Our study was also the first to describe the expression pattern of C1qbp in the hypothalamus, a protein potentially involved in mitochondrial and neuroimmunological regulations of synaptic plasticity. Its presence in the preoptic area responsible for maternal behaviors and also in the paraventricular hypothalamic and arcuate nuclei regulating hormonal levels suggests that the same proteins may be involved in different aspects of maternal adaptations. The conclusions of the present work contribute to establishing the molecular alterations that determine different maternal adaptations in the brain. Since maternal changes are models of neuronal plasticity in all social interactions, the reported results can affect a wide field of molecular and behavioral neuroscience.

Maternally involved galanin neurons in the preoptic area of the rat.

Recent selective stimulation and ablation of galanin neurons in the preoptic area of the hypothalamus established their critical role in control of maternal behaviors. Here, we identified a group of galanin neurons in the anterior commissural nucleus (ACN), and a distinct group in the medial preoptic area (MPA). Galanin neurons in ACN but not the MPA co-expressed oxytocin. We used immunodetection of phosphorylated STAT5 (pSTAT5), involved in prolactin receptor signal transduction, to evaluate the effects of suckling-induced prolactin release and found that 76 % of galanin cells in ACN, but only 12 % in MPA were prolactin responsive. Nerve terminals containing tuberoinfundibular peptide 39 (TIP39), a neuropeptide that mediates effects of suckling on maternal motivation, were abundant around galanin neurons in both preoptic regions. In the ACN and MPA, 89 and 82 % of galanin neurons received close somatic appositions, with an average of 2.9 and 2.6 per cell, respectively. We observed perisomatic innervation of galanin neurons using correlated light and electron microscopy. The connection was excitatory based on the glutamate content of TIP39 terminals demonstrated by post-embedding immunogold electron microscopy. Injection of the anterograde tracer biotinylated dextran amine into the TIP39-expressing posterior intralaminar complex of the thalamus (PIL) demonstrated that preoptic TIP39 fibers originate in the PIL, which is activated by suckling. Thus, galanin neurons in the preoptic area of mother rats are innervated by an excitatory neuronal pathway that conveys suckling-related information. In turn, they can be topographically and neurochemically divided into two distinct cell groups, of which only one is affected by prolactin.

A Thalamo-Hypothalamic Pathway That Activates Oxytocin Neurons in Social Contexts in Female Rats.

Oxytocin is released from neurons in the paraventricular hypothalamic nucleus (PVN) in mothers upon suckling and during adult social interactions. However, neuronal pathways that activate oxytocin neurons in social contexts are not yet established. Neurons in the posterior intralaminar complex of the thalamus (PIL), which contain tuberoinfundibular peptide 39 (TIP39) and are activated by pup exposure in lactating mothers, provide a candidate projection. Innervation of oxytocin neurons by TIP39 neurons was examined by double labeling in combination with electron microscopy and retrograde tract-tracing. Potential classic neurotransmitters in TIP39 neurons were investigated by in situ hybridization histochemistry. Neurons activated after encounter with a familiar conspecific female in a familiar environment were mapped with the c-Fos technique. PVN and the supraoptic nucleus oxytocin neurons were closely apposed by an average of 2.0 and 0.4 TIP39 terminals, respectively. Asymmetric (presumed excitatory) synapses were found between TIP39 terminals and cell bodies of oxytocin neurons. In lactating rats, PIL TIP39 neurons were retrogradely labeled from the PVN. TIP39 neurons expressed vesicular glutamate transporter 2 but not glutamic acid decarboxylase 67. PIL contained a markedly increased number of c-Fos-positive neurons in response to social encounter with a familiar conspecific female. Furthermore, the PIL received ascending input from the spinal cord and the inferior colliculus. Thus, TIP39 neurons in the PIL may receive sensory input in response to social interactions and project to the PVN to innervate and excite oxytocin neurons, suggesting that the PIL-PVN projection contributes to the activation of oxytocin neurons in social contexts.

Thalamic neuropeptide mediating the effects of nursing on lactation and maternal motivation.

Nursing has important physiological and psychological consequences on mothers during the postpartum period. Tuberoinfundibular peptide of 39 residues (TIP39) may contribute to its effects on prolactin release and maternal motivation. Since TIP39-containing fibers and the receptor for TIP39, the parathyroid hormone 2 receptor (PTH2 receptor) are abundant in the arcuate nucleus and the medial preoptic area, we antagonized TIP39 action locally to reveal its actions. Mediobasal hypothalamic injection of a virus encoding an antagonist of the PTH2 receptor markedly decreased basal serum prolactin levels and the suckling-induced prolactin release. In contrast, injecting this virus into the preoptic area had no effect on prolactin levels, but did dampen maternal motivation, judged by reduced time in a pup-associated cage during a place preference test. In support of an effect of TIP39 on maternal motivation, we observed that TIP39 containing fibers and terminals had the same distribution within the preoptic area as neurons expressing Fos in response to suckling. Furthermore, TIP39 terminals closely apposed the plasma membrane of 82% of Fos-ir neurons. Retrograde tracer injected into the arcuate nucleus and the medial preoptic area labeled TIP39 neurons in the posterior intralaminar complex of the thalamus (PIL), indicating that these cells but not other groups of TIP39 neurons project to these hypothalamic regions. We also found that TIP39 mRNA levels in the PIL markedly increased around parturition and remained elevated throughout the lactation period, demonstrating the availability of the peptide in postpartum mothers. Furthermore, suckling, but not pup exposure without physical contact, increased Fos expression by PIL TIP39 neurons. These results indicate that suckling activates TIP39 neurons in the PIL that affect prolactin release and maternal motivation via projections to the arcuate nucleus and the preoptic area, respectively.

Uridine modulates neuronal activity and inhibits spike-wave discharges of absence epileptic Long Evans and Wistar Albino Glaxo/Rijswijk rats.

Pharmacological and functional data suggest the existence of uridine (Urd) receptors in the central nervous system (CNS). In the present study, simultaneous extracellular single unit recording and microiontophoretic injection of the pyrimidine nucleoside Urd was used to provide evidence for the presence of Urd-sensitive neurons in the thalamus and the cerebral cortex of Long Evans rats. Twenty-two neurons in the thalamus (24% of recorded neurons) and 17 neurons in the cortex (55%) responded to the direct iontophoresis of Urd. The majority of Urd-sensitive neurons in the thalamus and cortex (82% and 59%, respectively) increased their firing rate in response to Urd. In contrary, adenosine (Ado) and uridine 5'-triphosphate (UTP) decreased the firing rate of all responding neurons in the thalamus, and the majority of responding neurons in the cortex (83% and 87%, respectively). Functional relevance of Urd-sensitive neurons was investigated in spontaneously epileptic freely moving Long Evans and Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Intraperitoneal (i.p.) injection of 500mg/kg Urd decreased epileptic activity (210-270min after injection) in both rat strains. Intraperitoneal administration of 1000mg/kg Urd decreased the number of spike-wave discharges (SWDs) between 150-270min and 90-270min in Long Evans and WAG/Rij rats, respectively. The effect of Urd was long-lasting in both rat strains as the higher dose significantly decreased the number of SWDs even 24h after Urd injection. The present results suggest that Urd-sensitive neurons in the thalamus and the cerebral cortex may play a role in the antiepileptic action of Urd possibly via modulation of thalamocortical neuronal circuits.

Tuberoinfundibular peptide of 39 residues is activated during lactation and participates in the suckling-induced prolactin release in rat.

Tuberoinfundibular peptide of 39 residues (TIP39) and the PTH-2 receptor (PTH2R) constitute a peptide-receptor neuromodulator system. Based on the abundance of TIP39 fibers and axonal terminals as well as PTH2R-containing neurons and their processes in the hypothalamic para- and periventricular and arcuate nuclei TIP39 has been suggested to play a role in neuroendocrine regulation. We showed previously that TIP39 expression decreased dramatically by adulthood. In the present study, using in situ hybridization histochemistry, real-time RT-PCR, and immunohistochemistry, we found that TIP39 mRNA and peptide expression levels are markedly elevated in the posterior intralaminar complex of the thalamus (PIL) of lactating dams, one of the three locations of TIP39-containing cell bodies in the brain. In addition, in mother rats, these TIP39 neurons showed Fos expression in response to pup exposure. Transection of TIP39 fibers originating in the PIL resulted in an ipsilateral disappearance of TIP39 immunoreactivity throughout the mediobasal hypothalamus of mother rats, suggesting that TIP39 fibers there arise from the PIL. To elucidate the function of TIP39 activation in dams, mothers separated from their pups for 4 h on postpartum d 9 received injection of a PTH2R antagonist into the lateral ventricle 5 min before returning the pups. Blood samples were taken seven times during the experimental period through jugular cannulae. The PTH2R antagonist administered in two different concentrations markedly inhibited suckling-induced elevation of plasma prolactin levels in a dose-dependent manner. These results suggest that TIP39 neurons in the PIL may regulate suckling-induced prolactin release in rat dams.

Acoustic stress activates tuberoinfundibular peptide of 39 residues neurons in the rat brain.

Strong acoustic stimulation (105 dB SPL white noise) elicited c-fos expression in neurons in several acoustic system nuclei and in stress-sensitive hypothalamic nuclei and limbic areas in rats. In the present study, using this type of loud noise for 30 min, Fos-like immunoreactivity (Fos-ir) was investigated in neurons that synthesize tuberoinfundibular peptide of 39 residues (TIP39) in the rat brain: in the subparafascicular area of the thalamus, the posterior intralaminar complex of the thalamus and the medial paralemniscal nucleus in the lateral part of the pons. By double labeling, Fos-ir was shown in nearly 80% of TIP39-positive cells in the medial paralemniscal nucleus, 43% in the posterior intralaminar complex and 18.5% in the subparafascicular area 30 min after the end of a 30-min loud noise period. In control rats, only few neurons, including 0-4% of TIP39-positive neurons showed Fos-ir. While the majority of the Fos-ir neurons were TIP39-positive in the subparafascicular area and medial paralemniscal nucleus, a fairly high number of TIP39-immunonegative, chemically uncharacterized neurons expressed c-fos in the subparafascicular area and the posterior intralaminar complex of the thalamus. These observations clearly show that some TIP39 neurons in the so-called "acoustic thalamus" and the majority of TIP39 neurons in the medial paralemniscal nucleus are sensitive to loud noise and they may participate in the central organization of responses to acoustic stress. Furthermore, the present data suggest that non-TIP39-expressing neurons may play a prevalent role in the activity of the "acoustic thalamus".

The distribution and neurochemistry of the parathyroid hormone 2 receptor in the rat hypothalamus.

This study reports the distribution of parathyroid hormone 2 receptor (PTH2R)-immunoreactive fibers in the hypothalamus using fluorescent amplification immunocytochemistry. The pattern of immunolabeling is strikingly similar to that of tuberoinfundibular peptide of 39 residues (TIP39), a peptide recently purified from bovine hypothalamus and proposed to be a ligand of the PTH2R based on pharmacological data. To investigate the anatomical basis of suggestions that TIP39 affects the secretion of several hypophysiotropic hormones we performed double-labeling studies and found that only somatostatin fibers contain PTH2R in the median eminence, which suggests that somatostatin release could be directly regulated via the PTH2R. However, several hypothalamic nuclei projecting to the median eminence contain a high density of both TIP39 and PTH2R fibers and terminals. We report here, that the PTH2R terminals also contain vesicular glutamate transporter-2, and suggest that TIP39 terminals are ideally positioned to modulate glutamatergic influences on hypophysiotropic neurons.

Expression and distribution of tuberoinfundibular peptide of 39 residues in the rat central nervous system.

Tuberoinfundibular peptide of 39 residues (TIP39) has been recently purified and identified as a selective ligand for the parathyroid hormone 2 receptor. As a next step toward understanding its functions, we report the expression and distribution of TIP39 in the rat central nervous system. In situ hybridization histochemistry and immunocytochemistry revealed TIP39-containing cell bodies in three distinct areas. The major one comprises the subparafascicular area posterior through the intralaminar nucleus of the thalamus; a second is the medial paralemniscal nucleus at the pontomesencephalic junction; and a third is in the dorsal and dorsolateral hypothalamic areas, which contained a few, scattered cell bodies. We found, in contrast to the highly restricted localization of TIP39-containing cell bodies, a much more widespread localization of TIP39-containing fibers. The highest density of fibers was observed in limbic areas such as the septum, the amygdala, and the bed nucleus of the stria terminalis; in areas involved in endocrine regulation, such as the hypothalamic dorsomedial, paraventricular, periventricular, and arcuate nuclei; in auditory areas, such as the ectorhinal and temporal cortices, inferior colliculus, medial geniculate body, and some of the nuclei of the superior olivary complex; and in the dorsolateral funiculus of the spinal cord. The localization of TIP39-containing nuclei and fibers provides an anatomical basis for previously demonstrated endocrine and nociceptive effects of TIP39 and suggests additional functions for TIP39, one apparent candidate being the regulation of auditory information processing.

Emerging functions for tuberoinfundibular peptide of 39 residues.

Tuberoinfundibular peptide of 39 residues (TIP39), a neuropeptide recently purified from the hypothalamus, appears to be an endogenous ligand for the parathyroid hormone 2 (PTH2) receptor. PTH2 receptors are present in several central nervous system and peripheral areas and are particularly concentrated in the hypothalamus, limbic areas and the outer layers of the spinal cord dorsal horn. TIP39-containing neuronal cell bodies have been identified in the subparafascicular area and the medial paralemniscal nucleus, two brainstem regions that project widely through the entire neuraxis. Treatment of hypothalamic explants with TIP39, and intraventricular injection of the peptide, suggest that it might stimulate hypothalamic-releasing factor secretion. Injection of TIP39, and sequestration of endogenous TIP39 by intrathecal injection of an antibody to TIP39, have provided evidence that it is involved in some aspects of pain sensitivity. Thus, TIP39 might be a new neuromodulator.