The modulation of neuronal activity by means of electrical stimulation is a successful therapeutic approach for patients suffering from a variety of central nervous system disorders. Prototypic networks formed by cultured cortical neurons represent an important model system to gain general insights in the input-output relationships of neuronal tissue. These networks undergo a multitude of developmental changes during their maturation, such as the excitatory-inhibitory shift of the neurotransmitter GABA. Very few studies have addressed how the output properties to a given stimulus change with ongoing development. Here, we investigate input-output relationships of cultured cortical networks by probing cultures with and without functional GABAAergic synaptic transmission with a set of stimulation paradigms at various stages of maturation. On the cellular level, low stimulation rates (<15 Hz) led to reliable neuronal responses; higher rates were increasingly ineffective. Similarly, on the network level, lowest stimulation rates (<0.1 Hz) lead to maximal output rates at all ages, indicating a network wide refractory period after each stimulus. In cultures aged 3 weeks and older, a gradual recovery of the network excitability within tens of milliseconds was in contrast to an abrupt recovery after about 5 s in cultures with absent GABAAergic synaptic transmission. In these GABA deficient cultures evoked responses were prolonged and had multiple discharges. Furthermore, the network excitability changed periodically, with a very slow spontaneous change of the overall network activity in the minute range, which was not observed in cultures with absent GABAAergic synaptic transmission. The electrically evoked activity of cultured cortical networks, therefore, is governed by at least two potentially interacting mechanisms: A refractory period in the order of a few seconds and a very slow GABA dependent oscillation of the network excitability.

Noradrenaline (NA) modulates the spinal motor networks for locomotion and facilitates neuroplasticity, possibly assisting neuronal network activation and neuroplasticity in the recovery phase of spinal cord injuries. However, neither the effects nor the mechanisms of NA on synaptic transmission and neuronal excitability in spinal ventral horn (VH) neurons are well characterized, especially in rats aged 7 postnatal days or older. To gain insight into NA regulation of VH neuronal activity, we used a whole-cell patch-clamp approach in late neonatal rats (postnatal day 7-15). In voltage-clamp recordings at -70 mV, NA increased the frequency and amplitude of excitatory postsynaptic currents via the activation of somatic α1- and β-adrenoceptors of presynaptic neurons. Moreover, NA induced an inward current through the activation of postsynapticα1- and β-adrenoceptors. At a holding potential of 0 mV, NA also increased frequency and amplitude of both GABAergic and glycinergic inhibitory postsynaptic currents via the activation of somatic adrenoceptors in presynaptic neurons. In current-clamp recordings, NA depolarized resting membrane potentials and increased the firing frequency of action potentials in VH neurons, indicating that it enhances the excitability of these neurons. Our findings provide new insights that establish NA-based pharmacological therapy as an effective method to activate neuronal networks of the spinal VH in the recovery phase of spinal cord injuries.

GABAergic system plays a part in synaptic plasticity in the hippocampus. We had reported a long-term potentiation (LTP)-like facilitation in vivo, known as synaptic plasticity, through GABAA receptor blockade by bicuculline and the expression of proteins involved with this synaptic plasticity in mouse hippocampus. In the present study, we aimed to show improvement of impaired synaptic plasticity through GABAA receptor blockade and to clarify the molecular mechanisms involved with this improvement in the hippocampus of mice overexpressing human amyloid precursor protein with the E693Δ mutation (APPOSK-Tg) as an Alzheimer's disease model showing impaired synaptic plasticity. Electrophysiological study showed that the LTP-like facilitation expressed with application of bicuculline in vivo was significantly greater than impaired tetanic LTP in APPOSK-Tg mice, which was improved by bicuculline. Proteomic analysis showed that the expression of 11 proteins in the hippocampus was significantly changed 8 h after bicuculline application to APPOSK-Tg mice. The identified proteins could be functionally classified as chaperone, cytoskeletal protein, energy metabolism, metabolism, neuronal development, and synaptic component. Additionally, western blotting validated the changes in four proteins. We therefore propose that the improvement of impaired synaptic plasticity through GABAA receptor blockade could be mediated by the changed expression of these proteins.

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1β and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABAA receptors could be a suitable target for improving neuroinflammation in MHE.

A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established.

The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of behavioural arousal. The PF-LHA contains several cell types including neurones expressing the peptides, hypocretin (HCRT; also called orexin) and melanin-concentrating hormone (MCH). Evidence suggests that most of the PF-LHA neurones, including HCRT neurones, are active during waking and quiescent during non-rapid eye movement (non-NREM) sleep. The PF-LHA contains local GABAergic interneurones and also receives GABAergic inputs from sleep-promoting regions in the preoptic area of the hypothalamus. We hypothesized that increased GABA-mediated inhibition within PF-LHA contributes to the suppression of neuronal activity during non-REM sleep. EEG and EMG activity of rats were monitored for 2 h during microdialytic delivery of artificial cerebrospinal fluid (aCSF) or bicuculline, a GABAA receptor antagonist, into the PF-LHA in spontaneously sleeping rats during the lights-on period. At the end of aCSF or bicuculline perfusion, rats were killed and c-Fos immunoreactivity (Fos-IR) in HCRT, MCH and other PF-LHA neurones was quantified. In response to bicuculline perfusion into the PF-LHA, rats exhibited a dose-dependent decrease in non-REM and REM sleep time and an increase in time awake. The number of HCRT, MCH and non-HCRT/non-MCH neurones exhibiting Fos-IR adjacent to the microdialysis probe also increased dose-dependently in response to bicuculline. However, significantly fewer MCH neurones exhibited Fos-IR in response to bicuculline as compared to HCRT and other PF-LHA neurones. These results support the hypothesis that PF-LHA neurones, including HCRT neurones, are subject to increased endogenous GABAergic inhibition during sleep. In contrast, MCH neurones appear to be subject to weaker GABAergic control during sleep.

Neurobehavioral abnormalities are commonly associated with intractable childhood epilepsy. Studies from numerous labs have demonstrated cognitive and socialization deficits in rats and mice that have experienced early-life seizures. However, the cellular and molecular mechanisms underlying these effects are unknown. Previously, experiments have shown that recurrent seizures in infancy suppress the growth of hippocampal dendrites at the same time they impair learning and memory. Experiments in slice cultures have also demonstrated dendrite growth suppression. Here, we crossed calcineurin B1 (CaNB1) floxed and Thy1GFP-M mice to produce mice that were homozygous for the both the floxed CaNB1 and the Thy1GFP-M transgene. Littermates that were homozygous for wild-type CaNB1 and Thy1GFP-M served as controls. Hippocampal slice cultures from these mice were transfected with an AAV/hSyn-mCherry-Cre virus to eliminate CaNB1 from neurons. Immunohistochemical results showed that CaNB1 was eliminated from at least 90% of the transfected CA1 pyramidal cells. Moreover, the CaN-dependent nuclear translocation of the CREB transcription coactivator, CREB-regulated transcriptional coactivator 1 (CRTC1), was blocked in transfected neurons. Cell attach patch recordings combined with live multiphoton imaging demonstrated that the loss of CaNB1 did not prevent neurons from fully participating in electrographic seizure activity. Finally, dendrite reconstruction showed that the elimination of CaNB1 prevented seizure-induced decreases in both dendrite length and branch number. Results suggest that CaN plays a key role in seizure-induced dendrite growth suppression and may contribute to the neurobehavioral comorbidities of childhood epilepsy.

The intact hippocampal formation (IHF) of neonatal or young rats can be kept alive for an extended period in a fully submerged chamber with excellent morphological preservation. Field or patch-clamp recordings, intracellular Ca2+ measurements, and 3-D reconstruction of biocytin-filled neurons can be performed routinely. The generation and propagation of network-driven activities can be studied within the IHF or between connected intact structures such as the septum and the hippocampus or two hippocampi, and the use of a dual chamber enables the application of drugs separately to each structure. This preparation will be useful to study intact neuronal networks in the developing hippocampus in vitro.

The sympathetic nervous system (SNS) regulates body functions in normal and pathological conditions and is characterized by the presence of a neuroplastic phenomenon, termed ganglionic long-term potentiation (gLTP). In hypertension, either in spontaneously hypertensive rats (SHR) or in humans, sympathetic hyperfunction, such as elevated SNS outflow and changes in synaptic plasticity have been described. Because enhanced SNS outflow is detected in the hypertensive stage and, more importantly, in the prehypertensive phase of SHR, here we explored whether synaptic plasticity, particularly gLTP, was modified in the superior cervical ganglia (SCG) of prehypertensive SHR. Furthermore, considering that GABA modulates sympathetic synaptic transmission and gLTP in Wistar rats, we studied whether GABA might modulate gLTP expression in SHR. We characterized gLTP in the SCG of young prehypertensive 6-week-old (wo) and adult hypertensive (12 wo) SHR and in the SCG of Wistar Kyoto (WKy) normotensive control rats of the same ages. We found that gLTP was expressed in 6 wo SHR, but not in 12 wo rats. By contrast, in WKy, gLTP was expressed in 12 wo, but not in 6 wo rats. We also found that gLTP depends on GABA modulation, as blockade of GABA-A subtype receptors with its antagonist bicuculline unmasked gLTP expression in adult SHR and young WKy. We propose that (1) activity-dependent changes in synaptic efficacy are altered not only during hypertension but also before its onset and (2) GABA may play a modulatory role in the changes in synaptic plasticity in SHR, because the blockade of GABA-A receptors unmasked the expression of gLTP. These early changes in neuroplasticity and GABA modulation of gLTP could be part of the sympathetic hyperfunction observed in hypertension.

Memory for the mating male's pheromones in female mice is thought to require synaptic changes in the accessory olfactory bulb (AOB). Induction of this memory depends on release of glutamate in response to pheromonal exposure coincident with release of norepinephrine (NE) in the AOB following mating. A similar memory for pheromones can also be induced artificially by local infusion of the GABA(A) receptor antagonist bicuculline into the AOB. The natural memory formed by exposure to pheromones during mating is specific to the pheromones sensed by the female during mating. In contrast, the artificial memory induced by bicuculline is non-specific and results in the female mice recognizing all pheromones as if they were from the mating male. Although protein synthesis has been shown to be essential for development of pheromone memory, the gene expression cascades critical for memory formation are not known. We investigated changes in gene expression in the AOB using oligonucleotide microarrays during mating-induced pheromone memory (MIPM) as well as bicuculline-induced pheromone memory (BIPM). We found the set of genes induced during MIPM and BIPM are largely non-overlapping and Ingenuity Pathway Analysis revealed that the signaling pathways in MIPM and BIPM also differ. The products of genes induced during MIPM are associated with synaptic function, indicating the possibility of modification at specific synapses, while those induced during BIPM appear to possess neuron-wide functions, which would be consistent with global cellular changes. Thus, these results begin to provide a mechanistic explanation for specific and non-specific memories induced by pheromones and bicuculline infusion respectively.

Vigabatrin (gamma-vinyl GABA) is an antiepileptic drug and blocks GABA transaminase activity resulting in elevations in cellular GABA levels in the brain. Nipecotic acid (NPA) promotes release of GABA from neonatal optic nerve astrocytes, resulting in a bicuculline-sensitive depolarization of the optic nerve axons. The NPA-induced depolarization of vigabatrin-treated rats (100 mg/kg, i.p.) more than doubled, suggesting an elevation in free GABA levels; the GABA transporter inhibitor, NO-711 reduced the depolarization. These results are consistent with the known ability of vigabatrin to block the GABA degradation enzyme GABA-transaminase, suggesting that vigabatrin elevates astrocytic GABA levels, thereby favoring greater release of GABA through the GABA transporter.

Several early studies suggested that spikes can be generated in the dendrites of CA1 pyramidal neurons, but their functional significance and the conditions under which they occur remain poorly understood. Here, we provide direct evidence from simultaneous dendritic and somatic patch-pipette recordings that excitatory synaptic inputs can elicit dendritic sodium spikes prior to axonal action potential initiation in hippocampal CA1 pyramidal neurons. Both the probability and amplitude of dendritic spikes depended on the previous synaptic and firing history of the cell. Moreover, some dendritic spikes occurred in the absence of somatic action potentials, indicating that their propagation to the soma and axon is unreliable. We show that dendritic spikes contribute a variable depolarization that summates with the synaptic potential and can act as a trigger for action potential initiation in the axon.

Spinal phrenic nerve activity (PNA) drives the diaphragm but cranial hypoglossal nerve activity (HNA) also expresses synchronous activity during inspiration. Here, we investigated the effects of local disinhibition (bilateral microinjections of bicuculline) of the nucleus tractus solitarius (NTS), the pre-Bötzinger complex and Bötzinger complex core circuit (pre-BötC/BötC) and the Kölliker-Fuse nuclei (KFn) on the synchronization of PNA and HNA in arterially-perfused brainstem preparations of rats. To quantitatively analyze the bicuculline effects on a putatively distributed inspiratory central pattern generator (i-CPG), we quantified the phase synchronization properties between PNA and HNA. The analysis revealed that bicuculline-evoked local disinhibition significantly reduced the strength of phase synchronization between PNA and HNA at any target site. However, the emergence of desynchronized HNA following disinhibition was more prevalent after NTS or pre-BötC/BötC microinjections compared to the KFn. We conclude that the primary i-CPG is located in a distributed medullary circuit whereas pontine contributions are restricted to synaptic gating of synchronous HNA and PNA.

Evoked striatal field potentials are seldom used to study corticostriatal communication in vivo because little is known about their origin and significance. Here we show that striatal field responses evoked by stimulating the prelimbic cortex in mice are reduced by more than 90% after infusing the AMPA receptor antagonist CNQX close to the recording electrode. Moreover, the amplitude of local field responses and dPSPs recorded in striatal medium spiny neurons increase in parallel with increasing stimulating current intensity. Finally, the evoked striatal fields show several of the basic known properties of corticostriatal transmission, including paired pulse facilitation and topographical organization. As a case study, we characterized the effect of local GABA(A) receptor blockade on striatal field and multiunitary action potential responses to prelimbic cortex stimulation. Striatal activity was recorded through a 24 channel silicon probe at about 600 µm from a microdialysis probe. Intrastriatal administration of the GABA(A) receptor antagonist bicuculline increased by 65±7% the duration of the evoked field responses. Moreover, the associated action potential responses were markedly enhanced during bicuculline infusion. Bicuculline enhancement took place at all the striatal sites that showed a response to cortical stimulation before drug infusion, but sites showing no field response before bicuculline remained unresponsive during GABA(A) receptor blockade. Thus, the data demonstrate that fast inhibitory connections exert a marked temporal regulation of input-output transformations within spatially delimited striatal networks responding to a cortical input. Overall, we propose that evoked striatal fields may be a useful tool to study corticostriatal synaptic connectivity in relation to behavior.

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the mature brain. At an early developmental period, it acts in an excitatory manner that influences many processes of proliferation, migration, and differentiation of the neurons. Previous evidence indicated that manipulation of the GABAergic system function by activation or blockade of its receptors during developmental periods leads to behavioral and cognitive abnormality in adulthood. Therefore, we examined the effects of neonatal blockade of GABA-A receptors by bicuculline on behavior, cognitive function, and hippocampal and prefrontal cortex (PFC) brain-derived neurotrophic factors level (BDNF) in adulthood. As a result, neonatal rats were treated with either bicuculline (75,150, and 300 μg/kg) or DMSO on postnatal days 7,9, and 11. These groups underwent the behavioral (open field, elevated plus maze, and hot plate) and learning and memory (passive avoidance learning and memory) tests in postnatal days (PNDs) 61-70. After the ending of the behavioral tests, the rats were sacrificed under deep anesthesia and the hippocampi and prefrontal cortex (PFC) of the brain were removed for assessing the BDNF mRNA expression. Our results indicated that neonatal administration of bicuculline at the highest dose increased passive avoidance memory and hippocampal BDNF level. Meanwhile, this drug at a low dose impaired this type of memory and increased PFC BDNF level. Besides, treatment with bicuculline during postnatal days increased anxiety and pain sensitivity in a dose-dependent manner. Taken together, these findings confirmed the notion that GABA-A receptors during the developmental period are important for programming neurobehavioral phenotypes in adult life.

The effects of neurotensin (NT) on the release of acetylcholine (ACh), aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) from the hippocampus of freely moving rats were studied by transversal microdialysis. ACh was detected by High Performance Liquid Chromatography (HPLC) with electrochemical detection while GABA, glutamate and aspartate were measured using HPLC with fluorometric detection. Neurotensin (0.2 and 0.5 microM) administered locally through the microdialysis probe to the hippocampus produced a long-lasting and concentration-dependent increase in the basal extracellular levels of GABA and ACh but not of glutamate and aspartate. The increase in the extracellular levels of GABA and ACh produced by 0.5 microM neurotensin in the hippocampus reached a maximum of about 310% for GABA and 250% for ACh. This stimulant effect of NT was antagonized by the NT receptor antagonist SR 48692 (100 microg/kg, i.p.). Local infusion of tetrodotoxin (1 microM) decreased the basal release of ACh, GABA, Asp, Glu and prevented the 0.2 microM NT-induced increase in GABA and ACh release. The effect of NT on the release of ACh was blocked by the GABA(A) receptor antagonist bicuculline (2-10 microM). Our findings indicate for the first time that neurotensin plays a neuromodulatory role in the regulation of GABAergic and cholinergic neuronal activity in the hippocampus of awake and freely moving rats. The potentiating effects of neurotensin on GABA and ACh release in the hippocampus are probably mediated by (i) NT receptors located on GABAergic cell bodies and (ii) through GABA(A) receptors located on cholinergic nerve terminals.

Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA- and AMPA-type glutamate receptors, GABA(B) receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against the GABA(A) receptor in human sera from two patients diagnosed with encephalitis who presented with cognitive impairment and multifocal brain MRI abnormalities. Both patients had antibodies directed against the extracellular epitope of the β3 subunit of the GABA(A) receptor. The β3-subunit-containing GABA(A) receptor was a major target of the patients' serum antibodies in rat hippocampal neurons because the serum reactivity to the neuronal surface was greatly decreased by 80% when the β3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABA(A) receptor antibodies, one patient also had a low level of LGI1 antibodies, and the other also had CASPR2 antibodies. Application of the patients' serum at the time of symptom presentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABA(A) receptors. Furthermore, treatment of neurons with the patients' serum selectively reduced miniature IPSC amplitude and frequency without affecting miniature EPSCs. These results strongly suggest that the patients' GABA(A) receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABA(A) receptor encephalitis and expands the pathogenic roles of GABA(A) receptor autoantibodies.

In the dorsal lateral geniculate nucleus (dLGN), GABA(C) receptors seems to be specifically expressed by local GABAergic interneurons. Although the presence of GABA(C) receptors has been demonstrated, a quantitative estimation of their contribution to inhibition in dLGN is lacking. Because the amount of inhibition mediated by these receptors might reflect their functional importance we performed whole-cell patch clamp recordings from dLGN cells acutely dissociated from brain slices. We focally applied the GABA receptor agonist muscimol and quantified effects mediated through either GABA(C) or GABA(A) receptors. Because their basic dendritic morphology was preserved, we tried to morphologically differentiate between thalamocortical cells and local interneurons. In the majority of multipolar cells, representing thalamocortical projection neurons, the specific GABA(A) receptor antagonist bicuculline completely blocked muscimol induced currents. In contrast, in most of the bipolar cells, representing interneurons, bicuculline blocked only 70-80% of the muscimol induced currents. The remaining currents were blocked by co-application of TPMPA, a specific GABA(C) receptor antagonist, or picrotoxin, an unspecific GABA(A) and GABA(C) receptor blocker. The latter neurons were also sensitive to the selective GABA(C) receptor agonist cis-aminocrotonic acid. These results indicate that in those dLGN neurons that express GABA(C) receptors, these receptors contribute considerably to GABAergic inhibitory inputs.

Alterations of glutamate-mediated synaptic transmission occur in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Here we investigated whether intracerebroventricular (Icv) administration of cladribine has effects on EAE. Icv infusion of cladribine reduced the clinical deficits of EAE mice and reversed EAE-induced enhancement of excitatory postsynaptic current (sEPSC) frequency, a neurophysiological measure of glutamatergic synaptopathy associated with central inflammation. Cladribine failed to interfere with EAE-induced microglial and astroglial activation, but blocked EAE synaptic alterations by interfering with interleukin-1β effects. Cladribine possesses neuroprotective properties in experimental MS that are independent of its peripheral immunosuppressant action.

Long-term depression (LTD) at parallel fibre synapses on a cerebellar Purkinje cell has been regarded as a cellular basis for motor learning. Although Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the LTD induction as an important Ca(2+)-sensing molecule, the underlying signalling mechanism remains unclear. Here, we attempted to explore the potential signalling pathway underlying the CaMKII involvement in LTD using a systems biology approach, combined with validation by electrophysiological and FRET imaging experiments on a rat cultured Purkinje cell. Model simulation predicted the following cascade as a candidate mechanism for the CaMKII contribution to LTD: CaMKII negatively regulates phosphodiesterase 1 (PDE1), subsequently facilitates the cGMP/protein kinase G (PKG) signalling pathway and down-regulates protein phosphatase 2A (PP-2A), thus supporting the LTD-inducing positive feedback loop consisting of mutual activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This model suggestion was corroborated by whole-cell patch clamp recording experiments. In addition, FRET measurement of intracellular cGMP concentration revealed that CaMKII activation causes sustained increase of cGMP, supporting the signalling mechanism of LTD induction by CaMKII. Furthermore, we found that activation of the cGMP/PKG pathway by nitric oxide (NO) can support LTD induction without activation of CaMKII. Thus, this study clarified interaction between NO and Ca(2+)/CaMKII, two important factors required for LTD.