Slices Of Rats Research Articles

Excitatory Effects of Astrocytic Hydrogen Sulfide on the Electrical Activity of Oxytocin Neurons in the Supraoptic Nucleus

Introduction: In the regulation of oxytocin (OT) neuronal activity, hydrogen sulfide (H₂S), a gaseous neurotransmitter, likely exerts an excitatory role. This role is associated with increased expression of astrocytic cystathionine-β-synthase (CBS), the key enzyme for H₂S synthesis. However, it remains unclear whether H₂S is mainly produced in astrocytes and contributes to the autoregulation of OT neurons. Methods: In hypothalamic slices of male rats, OT and H₂S-associated drug effects were observed on the firing activity and spontaneous excitatory postsynaptic currents (sEPSCs) of putative OT neurons in the supraoptic nucleus (SON) in whole-cell patch-clamp recording. Expression of glial fibrillary acidic protein (GFAP) in the SON was analyzed in Western blots. In addition, changes in the length of rat pups’ hypothalamic astrocytic processes were observed in primary cultures. Results: In brain slices, OT significantly increased the firing rate of OT neurons, which was simulated by CBS allosteric agonist S-adenosyl-L-methionine (SAM) and H₂S slow-releasing donor GYY4137 but blocked by CBS inhibitor aminooxyacetic acid (AOAA). L-α-aminoadipic acid (a gliotoxin) blocked SAM-evoked excitation. OT and SAM also increased the frequency and amplitude of sEPSCs; the effect of OT was blocked by AOAA. Both OT and GYY4137 reduced GFAP expression in the SON. Morphologically, OT or GYY4137 time-dependently reduced the length of astrocytic processes in primary cultures. Conclusions: These findings indicate that the auto-excitatory effect of OT on OT neurons is mediated by H₂S from astrocytes at least partially and astrocytic H₂S can elicit retraction of astrocytic processes that subsequently increase OT neuronal excitability.

Disruption of vitamin A homeostasis by the biocide tetrakis(hydroxymethyl) phosphonium sulphate in pregnant rabbits.

The biocide tetrakis(hydroxymethyl)phosphonium sulphate (THPS) and other members of the tetrakis(hydroxymethyl) phosphonium salts (THPX) family are associated with liver toxicity in several mammalian species and teratogenicity in rabbits. Malformations include skeletal changes and abnormalities in eye development and are very similar to those seen with vitamin A deficiency or excess. For this reason, it was hypothesized that teratogenicity of THPS(X) might be attributed to disturbances in retinol availability and/or metabolism as a result of maternal toxicity, for example, either due to insufficient dietary intake by the mothers or due to liver toxicity. Therefore, in the present study, liver toxicity and vitamin A homeostasis were studied in pregnant rabbits that were exposed to 13.8 or 46.0 mg/kg THPS during organogenesis and in precision-cut liver slices of rats and rabbits exposed to 0-70 μM THPS. Results show that in vivo exposure to THPS leads to a marked reduction of food intake, increased plasma concentrations of γ-glutamytransferase, degenerative changes in the liver and to changes in retinoid content in liver and plasma in the rabbits during organogenesis. In addition, THPS, both in vivo and ex vivo, caused a change in expression of proteins related to vitamin A metabolism and transport. Together, these observations could explain the birth defects observed in earlier teratogenicity studies.

Tandospirone prevents stress-induced anxiety-like behavior and visceral hypersensitivity by suppressing theta oscillation enhancement via 5-HT1A receptors in the anterior cingulate cortex in rats.

Tandospirone, a third-generation of antianxiety agent with fewer side effects, has been widely used in the treatment of anxiety. Moreover, it is interesting that tandospirone has been found to relieve gastrointestinal symptoms in patients with refractory irritable bowel syndrome who also have psychological dysfunctions. However, the underlying mechanism remains unclear. In this study, using a visceral hypersensitivity rat model induced by chronic water avoidance stress to mimic the symptoms of irritable bowel syndrome, we found that tandospirone relieved anxiety-like behavior and visceral hypersensitivity induced by stress. Meanwhile, stressed rats had increased 5-HT concentration, less 5-HT1A receptor expression, and enhanced theta oscillations in the anterior cingulate cortex (ACC). Furthermore, the power of the theta band in ACC is positively correlated with the level of visceral sensitivity. Activation of 5-HT1A receptors by its agonist, 8-OH-DPAT, to compensate for their effect in ACC reduced the enhancement of theta oscillations in ACC slices in stressed rats, whereas 5-HT1A receptor antagonist, WAY100135, facilitates theta oscillations in slices of normal rats. Tandospirone reduced the enhancement of theta band power in ACC in vitro and in vivo, thus alleviating anxiety-like behavior and visceral hypersensitivity through 5-HT1A receptors in stressed rats. These results suggest a novel mechanism by which tandospirone activates 5-HT1A receptors to relieve stress-induced anxiety and visceral hypersensitivity by suppressing theta oscillation enhancement in ACC.

Cu-Zn SOD suppresses epilepsy in pilocarpine-treated rats and alters SCN2A/Nrf2/HO-1 expression

Copper-zinc superoxide dismutase (Cu-Zn SOD) is downregulated in epilepsy, however, the role of Cu-Zn SOD in epilepsy remains unclear. Based on the pilocarpine hydrochloride-induced rat model of epilepsy, cortical-striatum brain slices of rats were examined based on field excitatory post-synaptic potentials. Pathological changes were observed by transmission electron microscope. Also using SH-SY5Y cells, flow cytometry and TUNEL staining were applied to investigate cell apoptosis, and ELISA was applied to detect SOD activity. In addition, qRT-PCR and western blot were performed to detect SCN2A/Nrf2/HO-1 gene and protein expression levels, respectively. Cu-Zn SOD over-expression suppressed epilepsy in vivo. In addition, Cu-Zn SOD knockdown notably decreased SOD activity and induced apoptosis in SH-SY5Y cells. Moreover, Cu-Zn SOD silencing decreased the levels of SCN2A, Nrf2 and HO-1. Lastly, Cu-Zn SOD was shown to modulate the NaV1.2/Nrf2/HO-1 axis in rats. In this model, Cu-Zn SOD attenuated epilepsy and was shown to alter the expression level of proteins of the NaV1.2 /Nrf2/HO-1 signalling pathway, indicating that Cu-Zn SOD might be a target for the treatment of epilepsy.

Mediation of Sinusoidal Network Oscillations in the Locus Coeruleus of Newborn Rat Slices by Pharmacologically Distinct AMPA and KA Receptors.

Brain control by locus coeruleus (LC) neurons involves afferent glutamate (Glu) inputs. In newborns, LC Glu receptors and responses may be sparse due to immaturity of the brain circuits providing such input. However, we reported, using newborn rat brain slices, that Glu and its ionotropic receptor (iGluR) agonist NMDA transform spontaneous local field potential (LFP) rhythm. Here, we studied whether α-amino-3-hydroxy-5-methyl-4-isoxazole propionic-acid (AMPA) and kainate (KA) iGluR subtypes also transform the LFP pattern. AMPA (0.25–0.5 µM) and KA (0.5–2.5 µM) merged ~0.2 s-lasting bell-shaped LFP events occurring at ~1 Hz into ~40% shorter and ~4-fold faster spindle-shaped and more regular sinusoidal oscillations. The AMPA/KA effects were associated with a 3.1/4.3-fold accelerated phase-locked single neuron spiking due to 4.0/4.2 mV depolarization while spike jitter decreased to 64/42% of the control, respectively. Raising extracellular K+ from 3 to 9 mM increased the LFP rate 1.4-fold or elicited slower multipeak events. A blockade of Cl−-mediated inhibition with gabazine (5 μM) plus strychnine (10 μM) affected neither the control rhythm nor AMPA/KA oscillations. GYKI-53655 (25 μM) blocked AMPA (but not KA) oscillations whereas UBP-302 (25 μM) blocked KA (but not AMPA) oscillations. Our findings revealed that AMPA and KA evoke a similar novel neural network discharge pattern transformation type by acting on pharmacologically distinct AMPAR and KA receptors. This shows that already the neonatal LC can generate oscillatory network behaviors that may be important, for example, for responses to opioids.

Extracellular Vesicles From Hyperammonemic Rats Induce Neuroinflammation in Cerebellum of Normal Rats: Role of Increased TNFα Content.

Hyperammonemia plays a main role in the neurological impairment in cirrhotic patients with hepatic encephalopathy. Rats with chronic hyperammonemia reproduce the motor incoordination of patients with minimal hepatic encephalopathy, which is due to enhanced GABAergic neurotransmission in cerebellum as a consequence of neuroinflammation. Extracellular vesicles (EVs) could play a key role in the transmission of peripheral alterations to the brain to induce neuroinflammation and neurological impairment in hyperammonemia and hepatic encephalopathy. EVs from plasma of hyperammonemic rats (HA-EVs) injected to normal rats induce neuroinflammation and motor incoordination, but the underlying mechanisms remain unclear. The aim of this work was to advance in the understanding of these mechanisms. To do this we used an ex vivo system. Cerebellar slices from normal rats were treated ex vivo with HA-EVs. The aims were: 1) assess if HA-EVs induce microglia and astrocytes activation and neuroinflammation in cerebellar slices of normal rats, 2) assess if this is associated with activation of the TNFR1-NF-kB-glutaminase-GAT3 pathway, 3) assess if the TNFR1-CCL2-BDNF-TrkB pathway is activated by HA-EVs and 4) assess if the increased TNFα levels in HA-EVs are responsible for the above effects and if they are prevented by blocking the action of TNFα. Our results show that ex vivo treatment of cerebellar slices from control rats with extracellular vesicles from hyperammonemic rats induce glial activation, neuroinflammation and enhance GABAergic neurotransmission, reproducing the effects induced by hyperammonemia in vivo. Moreover, we identify in detail key underlying mechanisms. HA-EVs induce the activation of both the TNFR1-CCL2-BDNF-TrkB-KCC2 pathway and the TNFR1-NF-kB-glutaminase-GAT3 pathway. Activation of these pathways enhances GABAergic neurotransmission in cerebellum, which is responsible for the induction of motor incoordination by HA-EVs. The data also show that the increased levels of TNFα in HA-EVs are responsible for the above effects and that the activation of both pathways is prevented by blocking the action of TNFα. This opens new therapeutic options to improve motor incoordination in hyperammonemia and also in cirrhotic patients with hepatic encephalopathy and likely in other pathologies in which altered cargo of extracellular vesicles contribute to the propagation of the pathology.

Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death

Background & AimsIn cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonaemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging.MethodsTo primary cocultures of astrocytes and neurons, low concentrations (1 and 5 μM) of NH4Cl were applied. In rats with bile duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonaemia and minimal HE, acute brain slices were studied. One group of BDL rats was treated twice daily with the ammonia scavenger ornithine phenylacetate (OP; 0.3 g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (Δψm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy.ResultsNeuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction, and reduced cell viability (27.8 ± 2.3% and 41.5 ± 3.7%, respectively) compared with untreated cultures (15.7 ± 1.0%, both p <0.0001). BDL led to increased cerebral LP (p = 0.0003) and cytosolic ROS generation (p <0.0001), which was restored by OP (both p <0.0001). Mitochondrial function was severely compromised in BDL, resulting in hyperpolarisation of Δψm with consequent overconsumption of adenosine triphosphate and augmentation of mitochondrial ROS production. Administration of OP restored Δψm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP.ConclusionsOur results elucidate that low-grade hyperammonaemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonaemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development.Lay summaryThe impact of hyperammonaemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules, and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets.

Selective Optogenetic Dopamine Terminal Stimulation Reveals Role of GABAergic Signaling in Individual Differences in Dopamine Release

An estimated 70.5% of Americans aged 12 or older report having used an illicit substance at least once, while about 10‐20% of those individuals ultimately develop a substance use disorder (SUD). These data highlight substantial individual variability in the risk of developing SUD following initial drug use. Identifying markers of increased risk provides a significant opportunity to identify at‐risk populations and to aid in the prevention of the development of SUD. In preclinical rodent models, locomotor response to a novel environment predicts drug use vulnerability. Rodents that demonstrate higher locomotor response to an inescapable novel environment (high responders; HR) acquire self‐administration (SA) of drugs more rapidly and at lower doses compared to low responders (LR). Striatal dopamine (DA) signaling is critical for the acquisition of drug SA and tonic/phasic firing patterns of DA neurons encode information about salient environmental stimuli and rewards. HR rats show increased phasic DA signaling to reward‐predictive cues compared to LR rats. Dopamine release dynamics are tightly controlled by local modulation within the nucleus accumbens (NAc), including by nicotinic acetylcholine receptors (nAChRs), but the mechanisms by which this local modulation may contribute to differential DA release is not fully understood. Our lab has previously shown that locomotor response to a novel environment predicts nAChR modulation of phasic DA signals in the NAc. Specifically, desensitization or blockade of α6β2‐containing (α6β2*) nAChRs within the NAc was found to augment phasic DA signals in brain slices of HR rats, and reduce phasic DA signals in LR rats. However, these studies utilized non‐specific electrical stimulation to record DA release. In the present study, we used ex vivo fast‐scan cyclic voltammetry with an optogenetic approach to selectively stimulate DA neuron terminals or cholinergic interneurons (CINs) within the NAc to examine the specific contributions of acetylcholine (ACh) to the overall DA response in HR and LR rats. We find that, contrary to previous results with electrical stimulation, selective DA phasic stimulation results in lower DA release in HRs compared to LRs, while blockade of α6β2* nAChRs using light stimulation results in higher DA release in HRs. This suggests that additional circuitry is likely involved in differential DA signaling within the NAc. Given the tonic activity of CINs and presence of nAChRs on GABA interneurons, we next used pharmacological manipulations to examine the possibility of GABA signaling contributing to individual differences in DA release. Here, we find that antagonism of GABA receptors blocks the previously seen differential effects of α6β2* nAChR antagonism on DA release, suggesting that GABA interneurons do play a critical role in differential DA signaling in HRs versus LRs. In sum, these data help form a clearer understanding of the mechanisms underlying individual differences in DA signaling, which may be a primary driver of differences in sensitivity to the reinforcing effects of drugs of abuse. Investigating these mechanisms allows us to better identify behavioral and neurochemical markers of SUD vulnerability in humans and to ultimately stimulate development of individualized and effective treatments.

The prefrontal cortex shows widespread decrease in H3 histamine receptor binding densities in rats with genetic generalized epilepsies

Distributions of brain H3 histamine receptors in regions of the prefrontal cortex were studied by assessing regional binding densities for [3 H](R)α-methylhistamine in coronal brain slices of normal rats and rats with genetically determined absence and/or audiogenic epilepsies.The three groups of epileptic rats displayed widespread significant decreases in H3 histamine receptor binding densities. A 20–25% decline was seen in the rostral aspects of the lateral prefrontal cortex, namely the granular, dysgranular, and dorsal agranular insular regions. The reduction was not specific for the epilepsy types. The same was observed in the rostral part of the primary cingulate cortex and the secondary midcingulate cortex. On borders of this core effect, several seizure-type specific declines were seen. Namely, the infralimbic, prelimbic and posterior agranular insular cortices demonstrated absence-epilepsy related reductions in the H3 histamine receptor binding densities. A decrease related to audiogenic seizures was noted in the rostral part of the piriform cortex.The pattern of widespread and seizure-type unspecific decline in H3 histamine receptor binding densities points to a common part of brain loops underlying generalized convulsive and non-convulsive types of epilepsy. It also might hint at putative seizure-related changes in the release of histamine from specific fibers innervating the prefrontal area.

Autocrine Neuromodulation and Network Activity Patterns in the Locus Coeruleus of Newborn Rat Slices.

Already in newborns, the locus coeruleus (LC) controls multiple brain functions and may have a complex organization as in adults. Our findings in newborn rat brain slices indicate that LC neurons (i) generate at ~1 Hz a ~0.3 s-lasting local field potential (LFP) comprising summated phase-locked single spike discharge, (ii) express intrinsic ‘pacemaker’ or ‘burster’ properties and (iii) receive solely excitatory or initially excitatory–secondary inhibitory inputs. μ-opioid or ɑ2 noradrenaline receptor agonists block LFP rhythm at 100–250 nM whereas slightly lower doses transform its bell-shaped pattern into slower crescendo-shaped multipeak bursts. GABAA and glycine receptors hyperpolarize LC neurons to abolish rhythm which remains though unaffected by blocking them. Rhythm persists also during ionotropic glutamate receptor (iGluR) inhibition whereas <10 mV depolarization during iGluR agonists accelerates spiking to cause subtype-specific fast (spindle-shaped) LFP oscillations. Similar modest neuronal depolarization causing a cytosolic Ca2+ rise occurs (without effect on neighboring astrocytes) during LFP acceleration by CNQX activating a TARP-AMPA-type iGluR complex. In contrast, noradrenaline lowers neuronal Ca2+ baseline via ɑ2 receptors, but evokes an ɑ1 receptor-mediated ‘concentric’ astrocytic Ca2+ wave. In summary, the neonatal LC has a complex (possibly modular) organization to enable discharge pattern transformations that might facilitate discrete actions on target circuits.

Sevoflurane Effects on Neuronal Energy Metabolism Correlate with Activity States While Mitochondrial Function Remains Intact.

During general anesthesia, alterations in neuronal metabolism may induce neurotoxicity and/or neuroprotection depending on the dose and type of the applied anesthetic. In this study, we investigate the effects of clinically relevant concentrations of sevoflurane (2% and 4%, i.e., 1 and 2 MAC) on different activity states in hippocampal slices of young Wistar rats. We combine electrophysiological recordings, partial tissue oxygen (ptiO2) measurements, and flavin adenine dinucleotide (FAD) imaging with computational modeling. Sevoflurane minimally decreased the cerebral metabolic rate of oxygen (CMRO2) while decreasing synaptic transmission in naive slices. During pharmacologically induced gamma oscillations, sevoflurane impaired network activity, thereby decreasing CMRO2. During stimulus-induced neuronal activation, sevoflurane decreased CMRO2 and excitability while basal metabolism remained constant. In this line, stimulus-induced FAD transients decreased without changes in basal mitochondrial redox state. Integration of experimental data and computer modeling revealed no evidence for a direct effect of sevoflurane on key enzymes of the citric acid cycle or oxidative phosphorylation. Clinically relevant concentrations of sevoflurane generated a decent decrease in energy metabolism, which was proportional to the present neuronal activity. Mitochondrial function remained intact under sevoflurane, suggesting a better metabolic profile than isoflurane or propofol.

Activation of lactate receptor HCAR1 down-modulates neuronal activity in rodent and human brain tissue

Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca2+ spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy.

Afterhyperpolarization Promotes the Firing of Mitral Cells through a Voltage-Dependent Modification of Action Potential Threshold.

In the olfactory bulb, mitral cells (MCs) display a spontaneous firing that is characterized by bursts of action potentials (APs) intermixed with silent periods. Intraburst firing frequency and duration are heterogeneous among MCs and increase with membrane depolarization. By using patch-clamp recording on rat slices, we dissected out the intrinsic properties responsible for this bursting activity. We showed that the threshold of AP generation dynamically changes as a function of the preceding trajectory of the membrane potential. In fact, the AP threshold became more negative when the membrane was hyperpolarized and had a recovery rate inversely proportional to the membrane repolarization rate. Such variations appeared to be produced by changes in the inactivation state of voltage-dependent Na+ channels. Thus, AP initiation was favored by hyperpolarizing events, such as negative membrane oscillations or inhibitory synaptic input. After the first AP, the following fast afterhyperpolarization (AHP) brought the threshold to more negative values and then promoted the emission of the following AP. This phenomenon was repeated for each AP of the burst making the fast AHP a regenerative mechanism that sustained the firing, AHP with larger amplitudes and faster repolarizations being associated with larger and higher-frequency bursts. Burst termination was found to be because of the development of a slow repolarization component of the AHP (slow AHP). Overall, the AHP characteristics appeared as a major determinant of the bursting properties.

HP16: Estimating synaptic transmission changes in the epileptic brain with and without stimulation

Computational modeling is a powerful tool for exploring brain mechanisms underlying neurophysiological observations. Nowadays, it is increasingly used to design and evaluate the effect of therapeutic interventions. In this contribution, the computational approach is used to investigate the effects of single pulse and repetitive electrical stimulation on excitation, slow dendritic and fast somatic inhibition in a rodent model of epilepsy. Method: Building on the modeling work of Wendling et al. (J Clin Neurophysiol 2005;22(5):343–356), we replicated local field potential recordings of hippocampal rat slices with and without stimulation. Through an automated comparison of simulated and real data segments, we classified different types of epileptic activity and estimated levels of excitatory and inhibitory synaptic gains across time. Result: Spontaneous transitions from interictal to ictal activity were associated with consistent patterns of excitatory and inhibitory dynamics. Interestingly, model results suggest that an increase of excitation close to seizure onset is preceded by a slow build-up of peri-dendritic inhibition. Single pulse stimulation with varying intensity led to seizure facilitation, accompanied by a significant increase in excitation pre-to-post stimulation. Repetitive stimulation at 1 Hz with varying intensity and duration led to seizure prevention, locking synaptic communication to a characteristic state post stimulation. During stimulation, we observed an increase of excitation, followed by a drop in somatic and an increase in dendritic inhibition, prolonging the duration of the inter-seizure interval. Conclusion: A slow build-up of dendritic inhibition might be linked to ictogenesis. Opposing effects of brain stimulation are associated with characteristic changes in synaptic transmission.

High, in Contrast to Low Levels of Acute Stress Induce Depressive-like Behavior by Involving Astrocytic, in Addition to Microglial P2X7 Receptors in the Rodent Hippocampus.

Extracellular adenosine 5′-triphosphate (ATP) in the brain is suggested to be an etiological factor of major depressive disorder (MDD). It has been assumed that stress-released ATP stimulates P2X7 receptors (Rs) at the microglia, thereby causing neuroinflammation; however, other central nervous system (CNS) cell types such as astrocytes also possess P2X7Rs. In order to elucidate the possible involvement of the MDD-relevant hippocampal astrocytes in the development of a depressive-like state, we used various behavioral tests (tail suspension test [TST], forced swim test [FST], restraint stress, inescapable foot shock, unpredictable chronic mild stress [UCMS]), as well as fluorescence immunohistochemistry, and patch-clamp electrophysiology in wild-type (WT) and genetically manipulated rodents. The TST and FST resulted in learned helplessness manifested as a prolongation of the immobility time, while inescapable foot shock caused lower sucrose consumption as a sign of anhedonia. We confirmed the participation of P2X7Rs in the development of the depressive-like behaviors in all forms of acute (TST, FST, foot shock) and chronic stress (UCMS) in the rodent models used. Further, pharmacological agonists and antagonists acted in a different manner in rats and mice due to their diverse potencies at the respective receptor orthologs. In hippocampal slices of mice and rats, only foot shock increased the current responses to locally applied dibenzoyl-ATP (Bz-ATP) in CA1 astrocytes; in contrast, TST and restraint depressed these responses. Following stressful stimuli, immunohistochemistry demonstrated an increased co-localization of P2X7Rs with a microglial marker, but no change in co-localization with an astroglial marker. Pharmacological damage to the microglia and astroglia has proven the significance of the microglia for mediating all types of depression-like behavioral reactions, while the astroglia participated only in reactions induced by strong stressors, such as foot shock. Because, in addition to acute stressors, their chronic counterparts induce a depressive-like state in rodents via P2X7R activation, we suggest that our data may have relevance for the etiology of MDD in humans.

Biophysical and synaptic properties of regular spiking interneurons in hippocampal area CA3 of aged rats

Neuronal processing from the dentate gyrus to the hippocampus is critical for storage and recovery of new memory traces. In area CA3, GABAergic interneurons form a strong barrage of inhibition that modulates pyramidal cells. A well-established feature of aging is decreased GABAergic inhibition, a phenomenon that contributes to the exacerbated excitability of aged pyramidal cells. In hippocampal slices of aged rats (22–28 months old) we examined the properties of regular spiking CA3 interneurons with patch-clamp whole-cell recordings. We found enhanced firing discharge without altering the maximal firing rate of aged regular spiking interneurons. In the mossy fibers (MF) to interneurons synapse, a switch in the AMPA receptor subunit composition was found in aged interneurons. Young regular spiking interneurons predominantly express CP AMPA receptors and MF LTD. By contrast, aged regular spiking interneurons contain a higher proportion of CI AMPA receptors and respond with MF LTP. We show for the first time that the specialized MF terminals contacting interneurons, retain synaptic capabilities and provide a novel insight of the interneuron's function during aging.

Serotonin Discharge Regulation by Additional Neurotransmitters of Rat Hippocampus Associated With the Continence Central Circuit.

PurposeThe lower urinary tract is believed to be centrally regulated with the involvement of a range of neurotransmitters. The parasympathetic excitatory input to the urinary bladder is suppressed when the serotonergic system is activated, and thereby voiding is blocked. In healthy people, continence is usually underpinned by hippocampal formation (circuit 3). In order to advance knowledge of how serotoninergic neurons and additional nerve fibers were correlated, the purpose of the present work was to research how the discharge of serotonin from hippocampal slices was affected by different neurotransmitters in rat models.MethodsThe adopted procedure involved administration of the central neurotransmitters acetylcholine, norepinephrine, dopamine, N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA), glycine, and neuropeptide Y as well as monitoring of the alterations in the discharge of [3H]5-hydroxytryptamine (5-HT). Furthermore, to determine whether the effect of the neurotransmitters was influenced by interneuron, tetrodotoxin was also employed.ResultsAcetylcholine (10-5M) did not alter [3H]5-HT discharge, whereas more 5-HT was discharged from the hippocampal slices of rats under stimulation by norepinephrine (10-5M) as well as dopamine (10-5M) and tetrodotoxin (10-6M) did not inhibit the discharge. By contrast, tetrodotoxin inhibited the discharge of [3H]5-HT that was exacerbated by NMDA (10-4M). Meanwhile, compared to control, GABA (10-5M), glycine (10-5M), or neuropeptide Y (10-6M) did not alter the [³H]5-HT discharge.ConclusionsFrom the research findings, it can be concluded that 5-HT discharge from rat hippocampus is enhanced by norepinephrine and dopamine through direct effect on the 5-HT neuronal terminal. By contrast, 5-HT discharge is intensified by NMDA by activating interneurons.

Mechanical actions of dendritic-spine enlargement on presynaptic exocytosis.

Synaptic transmission involves cell-to-cell communication at the synaptic junction between two neurons, and chemical and electrical forms of this process have been extensively studied. In the brain, excitatory glutamatergic synapses are often made on dendritic spines that enlarge during learning1-5. As dendritic spines and the presynaptic terminals are tightly connected with the synaptic cleft6, the enlargement may have mechanical effects on presynaptic functions7. Here we show that fine and transient pushing of the presynaptic boutons with a glass pipette markedly promotes both the evoked release of glutamate and the assembly of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins8-12-as measured by Förster resonance transfer (FRET) and fluorescence lifetime imaging-in rat slice culture preparations13. Both of these effects persisted for more than 20 minutes. The increased presynaptic FRET was independent of cytosolic calcium (Ca2+), but dependent on the assembly of SNARE proteins and actin polymerization in the boutons. Notably, a low hypertonic solution of sucrose (20 mM) had facilitatory effects on both the FRET and the evoked release without inducing spontaneous release, in striking contrast with a high hypertonic sucrose solution (300 mM), which induced exocytosis by itself14. Finally, spine enlargement induced by two-photon glutamate uncaging enhanced the evoked release and the FRET only when the spines pushed the boutons by their elongation. Thus, we have identified a mechanosensory and transduction mechanism15 in the presynaptic boutons, in which the evoked release of glutamate is enhanced for more than 20 min.

Group I metabotropic glutamate receptors contribute to the antiepileptic effect of electrical stimulation in hippocampal CA1 pyramidal neurons

Low-frequency deep brain stimulation (LFS) inhibits neuronal hyperexcitability during epilepsy. Accordingly, the use of LFS as a treatment method for patients with drug-resistant epilepsy has been proposed. However, the LFS antiepileptic mechanisms are not fully understood. Here, the role of metabotropic glutamate receptors group I (mGluR I) in LFS inhibitory action on epileptiform activity (EA) was investigated. EA was induced by increasing the K+ concentration in artificial cerebrospinal fluid (ACSF) up to 12 mM in hippocampal slices of male Wistar rats. LFS (1 Hz, 900 pulses) was delivered to the bundles of Schaffer collaterals at the beginning of EA. The excitability of CA1 pyramidal neurons was assayed by intracellular whole-cell recording. Applying LFS reduced the firing frequency during EA and substantially moved the membrane potential toward repolarization after a high-K+ ACSF washout. In addition, LFS attenuated the EA-generated neuronal hyperexcitability. A blockade of both mGluR 1 and mGluR 5 prevented the inhibitory action of LFS on EA-generated neuronal hyperexcitability. Activation of mGluR I mimicked the LFS effects and had similar inhibitory action on excitability of CA1 pyramidal neurons following EA. However, mGluR I agonist's antiepileptic action was not as strong as LFS. The observed LFS effects were significantly attenuated in the presence of a PKC inhibitor. Altogether, the LFS' inhibitory action on neuronal hyperexcitability following EA relies, in part, on the activity of mGluR I and a PKC-related signaling pathway.

Opposing Changes in Synaptic and Extrasynaptic N-Methyl-D-Aspartate Receptor Function in Response to Acute and Chronic Restraint Stress.

A pertinent mechanism by which stress impacts learning and memory is through stress-induced plastic changes in glutamatergic transmission in the hippocampus. For instance, acute stress has been shown to alter the expression, binding, and function of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR). However, the consequences of chronic stress, which could lead to various stress-related brain disorders, on NMDAR function remain unclear. While most studies on NMDARs focused on these receptors in synapses (synaptic NMDARs or sNMDARs), emerging findings have revealed functional roles of NMDARs outside synapses (extrasynaptic NMDARs or exNMDARs) that are distinct from those of sNMDARs. Using a restraint stress paradigm in adult rats, the objective of the current study is to examine whether sNMDARs and exNMDARs in the hippocampus are differentially regulated by acute and chronic stress. We examined sNMDAR and exNMDAR function in dorsal CA1 hippocampal neurons from brain slices of adult rats that were acutely (1 episode) or chronically (21 daily episodes) stressed by restraint (30 min). We found that acute stress increases sNMDAR but suppresses exNMDAR function. Surprisingly, we only observed a reduction in exNMDAR function after chronic stress. Taken together, our findings suggest that sNMDARs and exNMDARs may be differentially regulated by acute and chronic stress. Most importantly, the observed suppression in exNMDAR function by both acute and chronic stress implies crucial but overlooked roles of hippocampal exNMDARs in stress-related disorders.