Ferenc Erdélyi

Local regulation of [3H]‐noradrenaline release from the isolated guinea‐pig right atrium by P2X‐receptors located on axon terminals

In this study the regulation of cardiac sympathetic outflow by presynaptic P2X receptor‐gated ion channels was examined. ATP (30u2003μM–1u2003mM) and other P2‐receptor agonists elicited [3H]‐noradrenaline ([3H]‐NA) outflow from the isolated guinea‐pig right atrium with the potency order of ATP>2‐methyl‐thioATP>α,β‐methylene‐ATP=ADP, whereas β,γ‐methylene‐L‐ATP was inactive. Ca2+‐free conditions abolished both electrical field stimulation (EFS)‐ and ATP‐evoked release of tritium. Unlike from EFS‐induced outflow, ATP‐induced [3H]‐NA outflow was not reduced by ω‐Conotoxin‐GVIA (100u2003nM), Cd2+ (100u2003μM) and tetrodotoxin (1u2003μM). The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [3H]‐NA release was not prevented by 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 250u2003nM), 3,7‐dimethyl‐1‐propargylxanthine (DMPX, 250u2003nM), or by reactive blue 2 (RB2, 10u2003μM), antagonists of A1‐, A2‐ and inhibitory P2 receptors. Zn2+ (50u2003μM), the P2X‐receptor modulator potentiated, and P2X receptor antagonists, i.e. suramin (300u2003μM), pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 30u2003μM) and 2′‐o‐(trinitrophenyl)‐adenosine 5′‐triphosphate (TNP‐ATP, 30u2003μM) antagonized the ATP (1u2003mM)‐evoked response. RT–PCR study revealed the expression of P2X2 and P2X3 receptor mRNAs in guinea‐pig superior cervical ganglion. PPADS (30u2003μM) significantly reduced the EFS‐induced [3H]‐NA outflow in the presence DPCPX (250u2003nM) and RB2 (10u2003μM). In summary a P2X‐type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea‐pig. The pharmacological profile of the receptor resemble to homo‐oligomeric P2X3 or hetero‐oligomeric P2X2/P2X3 complexes, and provide a new target to intervene on sympathetic neuroeffector transmission at the presynaptic site.

Cholinergic modulation amplifies the intrinsic oscillatory properties of CA1 hippocampal cholecystokinin‐positive interneurons

In the mammalian hippocampus, the neurotransmitter acetylcholine (ACh) promotes learning and memory storage. During sensory processing and learning, large ACh‐dependent electrical oscillatory events are observed, which involve the synchronization of both inhibitory and excitatory neural circuits. While the actions of ACh are known on excitatory hippocampal circuits, its actions on specific inhibitory circuits are poorly understood. We show that two types of cholecystokinin‐positive local circuit inhibitory interneuron, the so‐called ‘basket cells’ and ‘Schaffer collateral‐associated’ cells, which innervate separately the cell body and dendritic regions of principal cells, are modulated similarly by cholinergic receptor activation. In both cell types activation of their muscarinic receptors triggers a general increase of excitability and intrinsic oscillatory activity, and a more efficient engagement to slow network oscillations. Knowledge of how cholinergic neuromodulation acts on neurochemically identical but morphologically distinct inhibitory interneurons will allow us to understand the role played by this important neuromodulator during hippocampal‐dependent tasks in vivo.

Visualization of stress-responsive inhibitory circuits in the GAD65-eGFP transgenic mice

Here, we have revealed that a subset of GABAergic neurons in the mouse brain became activated during systemic stress response. Stress-induced expression of immediate early gene product c-Fos, as a marker of neuronal activation was visualized in a transgenic mouse line expressing enhanced green fluorescent protein (eGFP) under the control of the regulatory region of mouse glutamic acid decarboxylase (GAD) 65 gene. In most GABAergic regions egfp transgene expression corresponded to acknowledged distribution of GABA neurons. Ether inhalation, as a strong systemic stressor induced c-Fos expression throughout the stress-related circuit, and did not affect the distribution and expression of the eGFP-transgene. Stress provoked strong neuronal activation in the piriform cortex, midline thalamic nuclei, lateral septum (LS), bed nucleus of the stria terminalis (BNST), and in parvocellular part of the hypothalamic paraventricular nucleus (PVN) as revealed by c-Fos immunfluorescence. Cells in the LS, BNST, and AHA including the subparaventricular zone (SPVZ) displayed significant eGFP/c-Fos co-localization, revealing stress-responsive GABAergic neurons. None of the stress-activated neurons within the medial parvocellular subdivision of the PVN were GABAergic. Our present results suggest that stress-recruited GABAergic neuron populations are preferentially located in distinct limbic and hypothalamic regions and these neurons might be involved in an inhibitory mechanism that counteract the endocrine, autonomic and behavioral aspects of the stress response. Furthermore, the present GAD65-eGFP transgenic model seems to be a relevant tool to analyze inhibitory control of the central stress circuit at single cell level.

Novel interneuronal network in the mouse posterior piriform cortex.

The neural circuits of the piriform cortex mediate field potential oscillations and complex functions related to integrating odor cues with behavior, affective states, and multisensory processing. Previous anatomical studies have established major neural pathways linking the piriform cortex to other cortical and subcortical regions and major glutamatergic and GABAergic neuronal subtypes within the piriform circuits. However, the quantitative properties of diverse piriform interneurons are unknown. Using quantitative neural anatomical analysis and electrophysiological recording applied to a GAD65‐EGFP transgenic mouse expressing GFP (green fluorescent protein) under the control of the GAD65 promoter, here we report a novel inhibitory network that is composed of neurons positive for GAD65‐EGFP in the posterior piriform cortex (PPC). These interneurons had stereotyped dendritic and axonal properties that were distinct from basket cells or interneurons expressing various calcium‐binding proteins (parvalbumin, calbindin, and calretinin) within the PPC. The GAD65‐GFP neurons are GABAergic and outnumbered any other interneurons (expressing parvalbumin, calbindin, and calretinin) we studied. The firing pattern of these interneurons was highly homogenous and is similar to the regular‐spiking nonpyramidal (RSNP) interneurons reported in primary sensory and other neocortical regions. Robust dye coupling among these interneurons and expression of connexin 36 suggested that they form electrically coupled networks. The predominant targets of descending axons of these interneurons were the dendrites of Layer III principal cells. Additionally, synapses were found on dendrites and somata of deep Layer II principal neurons and Layer III basket cells. A similar interneuronal subtype was also found in GAD65‐EGFP‐negative mouse. The extensive dendritic bifurcation at superficial lamina IA among horizontal afferent fibers and unique axonal targeting pattern suggests that these interneurons may play a role in direct feedforward inhibitory and disinhibitory olfactory processing. We conclude that the GAD65‐GFP neurons may play distinct roles in regulating information flow and olfactory‐related oscillation within the PPC in vivo. J. Comp. Neurol. 499:1000–1015, 2006.

Homo‐ and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system

Here, we investigated how nucleotides and nucleosides affect the release of tritiated purines and endogenous adenosine 5′‐triphosphate (ATP) from superfused rat hippocampal slices. ATP elicited concentration‐dependent [3H]purine efflux from slices preloaded with [3H]adenosine. High‐performance liquid chromatography analysis of the effluent showed that the tritium label represented the whole set of adenine nucleotides and nucleosides, and ATP significantly increased the outflow of [3H]ATP. Adenosine 5′‐diphosphate, adenosine, uridine, uridine 5′‐triphosphate, α,β‐methylene‐ATP and 3′‐O‐(4‐benzoylbenzoyl)‐ATP were also active in eliciting [3H]purine release. Adenosine (300 μM) also evoked endogenous ATP efflux from the hippocampal slices. Reverse transcription‐coupled‐polymerase chain reaction analysis revealed that mRNAs encoding a variety of P2X and P2Y receptor proteins are expressed in the rat hippocampus. Nevertheless, neither P2 receptor (i.e. pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, 30 μM, suramin, 300 μM and reactive blue 2, 10 μM), nor adenosine receptor (8‐cyclopentyl‐1,3‐dipropylxanthine, 250 nM and dimethyl‐1‐propargylxanthine, 250 nM) antagonists modified the effect of ATP (300 μM) to evoke [3H]purine release. The nucleoside transport inhibitors, dipyridamole (10 μM), nitrobenzylthioinosine (10 μM) and adenosine deaminase (2–10 U ml−1), but not the ecto‐adenylate kinase inhibitor diadenosine pentaphosphate (200 μM) significantly reduced ATP‐evoked [3H]purine efflux. In summary, we found that ATP and other nucleotides and nucleosides promote the release of one another and themselves by the nucleoside transport system. This action could have relevance during physiological and pathological elevation of extracellular purine levels high enough to reverse the nucleoside transporter.

The naturally occurring steroid solasodine induces neurogenesis in vitro and in vivo.

In this study, we explored the capacity of the naturally occurring compound solasodine to promote neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to solasodine for 2 days followed by a 5-day washout differentiated into cholinergic neurons that expressed specific neuronal markers and displayed important axonal formation that continued growing even 30 days after treatment. In vivo, a 2-week infusion of solasodine into the left ventricle of the rat brain followed by a 3-week washout resulted in a significant increase in bromodeoxyuridine uptake by cells of the ependymal layer, subventricular zone, and cortex that co-localized with doublecortin immunostaining, demonstrating the proliferative and differentiating properties of solasodine on neuronal progenitors. In addition, these data demonstrate that under our experimental conditions adult ependymal cells retrieved their proliferative and differentiating abilities. The GAP-43/HuD pathway was activated both in vitro and in vivo, suggesting a role in the differentiating process triggered by solasodine. Solasodine treatment in rats resulted in a dramatic increase in expression of the cholesterol- and drug-binding translocator protein in ependymal cells, suggesting a possible role played by neurosteroid production in solasodine-induced neurogenesis. In GAD65-GFP mice that express the green fluorescent protein under the control of the glutamic acid decarboxylase 65-kDa promoter, solasodine treatment increased the number of GABAergic progenitors and neuroblasts generated in the subventricular zone and present in the olfactory migratory tract. Taken together, these results suggest that solasodine offers an interesting approach to stimulate in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy.

Hippocampal “cholinergic interneurons” visualized with the choline acetyltransferase promoter: anatomical distribution, intrinsic membrane properties, neurochemical characteristics, and capacity for cholinergic modulation

Release of acetylcholine (ACh) in the hippocampus (HC) occurs during exploration, arousal, and learning. Although the medial septum-diagonal band of Broca (MS-DBB) is the major extrinsic source of cholinergic input to the HC, cholinergic neurons intrinsic to the HC also exist but remain poorly understood. Here, ChAT-tauGFP and ChAT-CRE/Rosa26YFP (ChAT-Rosa) mice were examined in HC. The HC of ChAT-tauGFP mice was densely innervated with GFP-positive axons, often accompanied by large GFP-positive structures, some of which were Neurotrace/DAPI-negative and likely represent large axon terminals. In the HC of ChAT-Rosa mice, ChAT-YFP cells were Neurotrace-positive and more abundant in CA3 and dentate gyrus than CA1 with partial overlap with calretinin/VIP. Moreover, an anti-ChAT antibody consistently showed ChAT immunoreactivity in ChAT-YFP cells from MS-DBB but rarely from HC. Furthermore, ChAT-YFP cells from CA1 stratum radiatum/stratum lacunosum moleculare (SR/SLM) exhibited a stuttering firing phenotype but a delayed firing phenotype in stratum pyramidale (SP) of CA3. Input resistance and capacitance were also different between CA1 SR/LM and CA3 SP ChAT-YFP cells. Bath application of ACh increased firing frequency in all ChAT-YFP cells; however, cholinergic modulation was larger in CA1 SR/SLM than CA3 SP ChAT-YFP cells. Finally, CA3 SP ChAT-YFP cells exhibited a wider AP half-width and weaker cholinergic modulation than YFP-negative CA3 pyramidal cells. Consistent with CRE expression in a subpopulation of principal cells, optogenetic stimulation evoked glutamatergic postsynaptic currents in CA1 SR/SLM interneurons. In conclusion, the presence of fluorescently labeled hippocampal cells common to both ChAT-tauGFP and ChAT-Rosa mice are in good agreement with previous reports on the existence of cholinergic interneurons, but both transgenic mouse lines exhibited unexpected anatomical features that departed considerably from earlier observations.

Dopaminergic neurons modulate GABA neuron migration in the embryonic midbrain

Neuronal migration, a key event during brain development, remains largely unexplored in the mesencephalon, where dopaminergic (DA) and GABA neurons constitute two major neuronal populations. Here we study the migrational trajectories of DA and GABA neurons and show that they occupy ventral mesencephalic territory in a temporally and spatially specific manner. Our results from the Pitx3-deficient aphakia mouse suggest that pre-existing DA neurons modulate GABA neuronal migration to their final destination, providing novel insights and fresh perspectives concerning neuronal migration and connectivity in the mesencephalon in normal as well as diseased brains.

Identification of the adrenoceptor subtypes expressed on GABAergic neurons in the anterior hypothalamic area and rostral zona incerta of GAD65-eGFP transgenic mice

GABA is a major neurotransmitter in the hypothalamus. In particular, neurons in the paraventricular nucleus (PVN) of the hypothalamus receive dense GABAergic inputs from peri-PVN regions. The noradrenergic system has been reported as a modulator of GABAergic transmission to the PVN. Previous electrophysiological and morphological studies support the presence of adrenoceptors on GABAergic neurons innervating the PVN. In this study, we identified three adrenoceptors on GABAergic neurons in the peri-PVN region, focusing on the anterior hypothalamic area (AHA) and rostral zona incerta (ZIr). GABAergic neurons were identified using enhanced green fluorescent protein (eGFP), followed by single cell RT-PCR analysis of the GABA synthetic enzymes, glutamic acid decarboxylase (GAD)65 and/or GAD67. Single cell RT-PCR data revealed the expression of alpha(1A)-, alpha(1B)- and alpha(2A)-adrenoceptor mRNA on GABAergic neurons in AHA and ZIr. Additionally, immunohistochemical studies showed that the immunoreactivities of alpha(1A)-, alpha(1B)- and alpha(2A)-adrenoceptor were colocalized with eGFP-expressing neurons in AHA and ZIr. The present findings suggest the contribution of adrenoceptors to the modulation of GABAergic neurons in AHA and ZIr.

Direct Corticosteroid Modulation of GABAergic Neurons in the Anterior Hypothalamic Area of GAD65-eGFP Mice

Corticosterone is known to modulate GABAergic synaptic transmission in the hypothalamic paraventricular nucleus. However, the underlying receptor mechanisms are largely unknown. In the anterior hypothalamic area (AHA), the sympathoinhibitory center that project GABAergic neurons onto the PVN, we examined the expression of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) of GABAergic neurons using intact GAD65-eGFP transgenic mice, and the effects of corticosterone on the burst firing using adrenalectomized transgenic mice. GR or MR immunoreactivity was detected from the subpopulations of GABAergic neurons in the AHA. The AHA GABAergic neurons expressed mRNA of GR (42%), MR (38%) or both (8%). In addition, in brain slices incubated with corticosterone together with RU486 (MR-dominant group), the proportion of neurons showing a burst firing pattern was significantly higher than those in the slices incubated with vehicle, corticosterone, or corticosterone with spironolactone (GR-dominant group; 64 vs. 11~14%, p< 0.01 by χ(2)-test). Taken together, the results show that the corticosteroid receptors are expressed on the GABAergic neurons in the AHA, and can mediate the corticosteroid-induced plasticity in the firing pattern of these neurons. This study newly provides the experimental evidence for the direct glucocorticoid modulation of GABAergic neurons in the AHA in the vicinity of the PVN.