Early life exposure to high fructose diet induces metabolic dysregulation associated with sex-specific cognitive impairment in adolescent rats.

The incidence of adolescent mental health disorders is on the rise. Epidemiological studies suggest that poor nutrition is a significant contributor to this public health crisis, specifically through exposure to high level of dietary sugar, including fructose, during critical periods of development. Previous studies have shown that elevated fructose exposure during adolescence disrupts mental health. Despite these data, it is currently unknown how fructose exposure, specifically during infancy, may impact adolescent mental health. We developed a rat experimental protocol to investigate the effects of fructose exposure during infancy on behavioral, cognitive and metabolic endpoints in adolescence. We found that exposing rats to high fructose from birth to weaning resulted in higher circulating glucose, insulin and leptin levels in adolescence. High fructose during infancy also increased bodyweight, disrupted metabolic homeostasis in the basolateral amygdala (BLA) as indicated by decreased activity of the cellular energy sensor AMPK, and impaired attention and impulsivity in a male-specific manner. This impaired attention observed in adolescent male rats following neonatal fructose exposure was partially rescued by viral-mediated, in vivo expression of a constitutively active form of AMPK in principal neurons of the BLA. Our results suggest that exposure to high level of fructose during infancy may impact adolescent mental health in a male-specific manner and that manipulation of AMPK activity may mitigate this impact.

Chronic stress induces cell type-selective transcriptomic and electrophysiological changes in the bed nucleus of the stria terminalis.

Distinct regions and cell types in the anterolateral group of the bed nucleus of the stria terminalis (BNSTALG) act to modulate anxiety in opposing ways. A history of chronic stress increases anxiety-like behavior with lasting electrophysiological effects on the BNSTALG. However, the opposing circuits within the BNSTALG suggest that stress may have differential effects on the individual cell types that comprise these circuits to shift the balance to favor anxiogenesis. Yet, the effects of stress are generally examined by treating all neurons within a particular region of the BNST as a homogenoeus population. We used patch-clamp electrophysiology and single-cell quantitative reverse transcriptase PCR (scRT-PCR) to determine how chronic shock stress (CSS) affects electrophysiological and neurochemical properties of Type I, Type II, and Type III neurons in the BNSTALG. We report that CSS resulted in changes in the input resistance, time constant, action potential waveform, and firing rate of Type III but not Type I or II neurons. Additionally, only the Type III neurons exhibited an increase in Crf mRNA and a decrease in striatal-enriched protein tyrosine phosphatase (Ptpn5) mRNA after CSS. In contrast, only non-Type III cells showed a reduction in calcium-permeable AMPA receptor (CP-AMPAR) current and changes in mRNA expression of genes encoding AMPA receptor subunits after CSS. Importantly, none of the effects of CSS observed were seen in all cell types. Our results suggest that Type III neurons play a unique role in the BNSTALG circuit and represent a population of CRF neurons particularly sensitive to chronic stress.

Low frequency pulse stimulation of Schaffer collaterals in Trpm4-/- knockout rats differently affects baseline BOLD signals in target regions of the right hippocampus but not BOLD responses at the site of stimulation.

Electrical stimulation of right Schaffer collateral in Trpm4-/- knockout and wild type rats were used to study the role of Trpm4 channels for signal processing in the hippocampal formation. Stimulation induced neuronal activity was simultaneously monitored in the CA1 region by in vivo extracellular field recordings and in the entire brain by BOLD fMRI measurements. In wild type and Trpm4-/- knockout rats, consecutive 5 Hz pulse trains elicited similar neuronal responses in the CA1 region and similar BOLD responses in the stimulated right hippocampus. Stimulus-related positive BOLD responses were also found in the left dorsal hippocampus. In contrast to the right dorsal hippocampus, baseline BOLD signals in the left hippocampus significantly decreased during consecutive stimulation trains. Similarly, slowly developing significant declines in baseline BOLD signals, in absence of any positive BOLD responses, were also observed in the right entorhinal, right piriform cortex, right basolateral amygdala and right dorsal striatum whereas baseline BOLD signals remained almost stable in the corresponding left regions. Furthermore, significant declines in baseline BOLD signals were found in the prefrontal cortex and prelimbic/infralimbic cortex. Because significant baseline BOLD declines were only observed in target regions of the right dorsal hippocampus, it might reflect functional connectivity between these regions. In all observed regions the decline in baseline BOLD signals was significantly delayed and less pronounced in Trpm4-/- knockout rats when compared to wild type rats. Thus, either Trpm4 channels are involved in mediating these baseline BOLD shifts or functional connectivity of the hippocampus is impaired in Trpm4-/- knockout rats.

Disentangling the role of TRPM4 in hippocampus-dependent plasticity and learning: an electrophysiological, behavioral and FMRI approach.

Hippocampal long-term potentiation (LTP) has been extensively studied as a cellular model of learning and memory. Recently, we described a central function of the Transient Receptor Potential M4 (TRPM4) channel in hippocampal LTP in mice in vitro. Here, we used Trpm4 knock-out (Trpm4-/-) rats to scrutinize TRPM4's role in the intact brain in vivo. After having confirmed the previous in vitro findings in mice, we studied hippocampal synaptic plasticity by chronic recordings in freely moving rats, hippocampus-dependent learning by a behavioral battery and hippocampal-cortical connectivity by fMRI. The electrophysiological investigation supports an involvement of TRPM4 in LTP depending on the induction protocol. Moreover, an exhaustive analysis of the LTP kinetics point to mechanistic changes in LTP by trpm4 deletion. General behavior as measured by open field test, light-dark box and elevated plus maze was inconspicuous in Trpm4-/- rats. However, they showed a distinct deficit in spatial working and reference memory associated to the Barnes maze and T-maze test, respectively. In contrast, performance of the Trpm4-/- in the Morris water maze was unaltered. Finally, fMRI investigation of the effects of a strong LTP induction manifested BOLD responses in the ipsilateral and contralateral hippocampus and the prefrontal cortex of both groups. Yet, the initial BOLD response in the stimulated hippocampal area of Trpm4-/- was significantly enhanced compared to WT rats. Our findings at the cellular, behavioral and system level point to a relevant role for TRPM4 in specific types of hippocampal synaptic plasticity and learning but not in hippocampal-prefrontal interaction.

Repeated shock stress facilitates basolateral amygdala synaptic plasticity through decreased cAMP-specific phosphodiesterase type IV (PDE4) expression.

Previous studies have shown that exposure to stressful events can enhance fear memory and anxiety-like behavior as well as increase synaptic plasticity in the rat basolateral amygdala (BLA). We have evidence that repeated unpredictable shock stress (USS) elicits a long-lasting increase in anxiety-like behavior in rats, but the cellular mechanisms mediating this response remain unclear. Evidence from recent morphological studies suggests that alterations in the dendritic arbor or spine density of BLA principal neurons may underlie stress-induced anxiety behavior. Recently, we have shown that the induction of long-term potentiation (LTP) in BLA principal neurons is dependent on activation of postsynaptic D1 dopamine receptors and the subsequent activation of the cyclic adenosine 5'-monophosphate (cAMP)-protein kinase A (PKA) signaling cascade. Here, we have used in vitro whole-cell patch-clamp recording from BLA principal neurons to investigate the long-term consequences of USS on their morphological properties and synaptic plasticity. We provided evidence that the enhanced anxiety-like behavior in response to USS was not associated with any significant change in the morphological properties of BLA principal neurons, but was associated with a changed frequency dependence of synaptic plasticity, lowered LTP induction threshold, and reduced expression of phosphodiesterase type 4 enzymes (PDE4s). Furthermore, pharmacological inhibition of PDE4 activity with rolipram mimics the effects of chronic stress on LTP induction threshold and baseline startle. Our results provide the first evidence that stress both enhances anxiety-like behavior and facilitates synaptic plasticity in the amygdala through a common mechanism of PDE4-mediated disinhibition of cAMP-PKA signaling.

TRPM4-dependent post-synaptic depolarization is essential for the induction of NMDA receptor-dependent LTP in CA1 hippocampal neurons.

TRPM4 is a calcium-activated but calcium-impermeable non-selective cation (CAN) channel. Previous studies have shown that TRPM4 is an important regulator of Ca(2+)-dependent changes in membrane potential in excitable and non-excitable cell types. However, its physiological significance in neurons of the central nervous system remained unclear. Here, we report that TRPM4 proteins form a CAN channel in CA1 neurons of the hippocampus and we show that TRPM4 is an essential co-activator of N-methyl-D-aspartate (NMDA) receptors (NMDAR) during the induction of long-term potentiation (LTP). Disrupting the Trpm4 gene in mice specifically eliminates NMDAR-dependent LTP, while basal synaptic transmission, short-term plasticity, and NMDAR-dependent long-term depression are unchanged. The induction of LTP in Trpm4 (-/-) neurons was rescued by facilitating NMDA receptor activation or post-synaptic membrane depolarization. Accordingly, we obtained normal LTP in Trpm4 (-/-) neurons in a pairing protocol, where post-synaptic depolarization was applied in parallel to pre-synaptic stimulation. Taken together, our data are consistent with a novel model of LTP induction in CA1 hippocampal neurons, in which TRPM4 is an essential player in a feed-forward loop that generates the post-synaptic membrane depolarization which is necessary to fully activate NMDA receptors during the induction of LTP but which is dispensable for the induction of long-term depression (LTD). These results have important implications for the understanding of the induction process of LTP and the development of nootropic medication.

The Ca(2+)-activated cation channel TRPM4 is a negative regulator of angiotensin II-induced cardiac hypertrophy.

Cardiac muscle adapts to hemodynamic stress by altering myocyte size and function, resulting in cardiac hypertrophy. Alteration in myocyte calcium homeostasis is known to be an initial signal in cardiac hypertrophy signaling. Transient receptor potential melastatin 4 protein (TRPM4) is a calcium-activated non-selective cation channel, which plays a role in regulating calcium influx and calcium-dependent cell functions in many cell types including cardiomyocytes. Selective deletion of TRPM4 from the heart muscle in mice resulted in an increased hypertrophic growth after chronic angiotensin (AngII) treatment, compared to WT mice. The enhanced hypertrophic response was also traceable by the increased expression of hypertrophy-related genes like Rcan1, ANP, and α-Actin. Intracellular calcium measurements on isolated ventricular myocytes showed significantly increased store-operated calcium entry upon AngII treatment in myocytes lacking the TRPM4 channel. Elevated intracellular calcium is a key factor in the development of pathological cardiac hypertrophy, leading to the activation of intracellular signaling pathways. In agreement with this, we observed significantly higher Rcan1 mRNA level, calcineurin enzyme activity and protein level in lysates from TRPM4-deficient mice heart compared to WT after AngII treatment. Collectively, these observations are consistent with a model in which TRPM4 is a regulator of calcium homeostasis in cardiomyocytes after AngII stimulation. TRPM4 contributes to the regulation of driving force for store-operated calcium entry and thereby the activation of the calcineurin-NFAT pathway and the development of pathological hypertrophy.

TRPM4.

TRPM4 is a Ca(2+)-activated nonselective cation channel. The channel is activated by an increase of intracellular Ca(2+) and is regulated by several factors including temperature and Pi(4,5)P2. TRPM4 allows Na(+) entry into the cell upon activation, but is completely impermeable to Ca(2+). Unlike TRPM5, its closest relative in the transient receptor potential family, TRPM4 proteins are widely expressed in the body. Currents with properties that are reminiscent of TRPM4 have been described in a variety of tissues since the advent of the patch clamp technology, but their physiological role is only beginning to be clarified with the increasing characterization of knockout mouse models for TRPM4. Furthermore, mutations in the TRPM4 gene have been associated with cardiac conduction disorders in human patients. This review aims to overview the currently available data on the functional properties of TRPM4 and the current understanding of its physiological role in healthy and diseased tissue.

Chronic administration of anticholinergics in rats induces a shift from muscarinic to purinergic transmission in the bladder wall.

BACKGROUND: First-line pharmacotherapy for overactive bladder consists of anticholinergics. However, patient compliance is exceptionally low, which may be due to progressive loss of effectiveness. OBJECTIVE: To decipher the involved molecular mechanisms and to evaluate the effects of chronic systemic administration of anticholinergics on bladder function and on muscarinic and purinergic receptors expression in rats. DESIGN, SETTING, AND PARTICIPANTS: Female Wistar rats were implanted with an osmotic pump that chronically administered vehicle (Vehc), 0.36 mg/kg per day oxybutynin (Oxyc), or 0.19 mg/kg per day fesoterodine (Fesoc) for 28 d. INTERVENTIONS: For cystometry experiments, a small catheter was implanted in the bladder. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Urologic phenotype was evaluated by the analysis of the micturition pattern and urodynamics. Expression of muscarinic and purinergic receptors was assessed by Western blot analysis of detrusor membrane protein. Functional responses to carbachol and adenosine triphosphate (ATP) were evaluated using muscle-strip contractility experiments. RESULTS AND LIMITATIONS: The number of voided spots was transiently decreased in Oxyc rats. In Oxyc rats, the effect of an acute high dose of oxybutynin (1mg/kg intraperitoneally [IP]) on the intermicturition interval was abolished. Expression experiments revealed a decrease of muscarinic acetylcholine receptors M2 (mAChR2) and M3 (mAChR3), whereas the purinergic receptor P2X, ligand-gated ion channel, 1 (P2X1) was enhanced in Oxyc and Fesoc rats compared to Vehc rats. In concordance with the modification of the expression pattern in Oxyc rats, the force generated by carbachol and ATP in muscle-strip contractility experiments was, respectively, lower and higher. Urodynamics revealed that the effects of systemic administration of the purinergic blocker pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (50mg/kg IP) were enhanced in Oxyc rats. As rat bladder physiology is different from that of humans, it is difficult to directly extrapolate our findings to human patients. CONCLUSIONS: Chronic administration of anticholinergics in rats induces receptor loss of efficiency and a shift from muscarinic to purinergic transmission.

TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis.

In multiple sclerosis, an inflammatory disease of the central nervous system (CNS), axonal and neuronal loss are major causes for irreversible neurological disability. However, which molecules contribute to axonal and neuronal injury under inflammatory conditions remains largely unknown. Here we show that the transient receptor potential melastatin 4 (TRPM4) cation channel is crucial in this process. TRPM4 is expressed in mouse and human neuronal somata, but it is also expressed in axons in inflammatory CNS lesions in experimental autoimmune encephalomyelitis (EAE) in mice and in human multiple sclerosis tissue. Deficiency or pharmacological inhibition of TRPM4 using the antidiabetic drug glibenclamide resulted in reduced axonal and neuronal degeneration and attenuated clinical disease scores in EAE, but this occurred without altering EAE-relevant immune function. Furthermore, Trpm4(-/-) mouse neurons were protected against inflammatory effector mechanisms such as excitotoxic stress and energy deficiency in vitro. Electrophysiological recordings revealed TRPM4-dependent neuronal ion influx and oncotic cell swelling upon excitotoxic stimulation. Therefore, interference with TRPM4 could translate into a new neuroprotective treatment strategy.

TRPs in the Brain.

The Transient receptor potential (TRP) family of cation channels is a large protein family, which is mainly structurally uniform. Proteins consist typically of six transmembrane domains and mostly four subunits are necessary to form a functional channel. Apart from this, TRP channels display a wide variety of activation mechanisms (ligand binding, G-protein coupled receptor dependent, physical stimuli such as temperature, pressure, etc.) and ion selectivity profiles (from highly Ca(2+) selective to non-selective for cations). They have been described now in almost every tissue of the body, including peripheral and central neurons. Especially in the sensory nervous system the role of several TRP channels is already described on a detailed level. This review summarizes data that is currently available on their role in the central nervous system. TRP channels are involved in neurogenesis and brain development, synaptic transmission and they play a key role in the development of several neurological diseases.

Best1 is a gene regulated by nerve injury and required for Ca2+-activated Cl- current expression in axotomized sensory neurons.

We investigated the molecular determinants of Ca(2+)-activated chloride current (CaCC) expressed in adult sensory neurons after a nerve injury. Dorsal root ganglia express the transcripts of three gene families known to induce CaCCs in heterologous systems: bestrophin, tweety, and TMEM16. We found with quantitative transcriptional analysis and in situ hybridization that nerve injury induced upregulation of solely bestrophin-1 transcripts in sensory neurons. Gene screening with RNA interference in single neurons demonstrated that mouse Best1 is required for the expression of CaCC in injured sensory neurons. Transfecting injured sensory neurons with bestrophin-1 mutants inhibited endogenous CaCC. Exogenous expression of the fusion protein green fluorescent protein-Bestrophin-1 in naive neurons demonstrated a plasma membrane localization of the protein that generates a CaCC with biophysical and pharmacological properties similar to endogenous CaCC. Our data suggest that Best1 belongs to a group of genes upregulated by nerve injury and supports functional CaCC expression in injured sensory neurons.