Excitatory synchronization of rat hippocampal interneurons during network activation in vitro.

Introduction: Hippocampal interneurons (INs) are known to synchronize their electrical activity via mechanisms, which are poorly defined due to immense complexity of neural tissue but seem to depend on local cell interactions and intensity of network activity. Methods: Here, synchronization of INs was studied using paired patch-clamp recordings in a simplified culture model with intact glutamate transmission. The level of network activity was moderately elevated by field electric stimulation, which is probably an analogue of afferent processing in situ. Results: Even in baseline conditions, ∼45% of spontaneous inhibitory postsynaptic currents (sIPSCs) resulting from firing of individual presynaptic INs coincided between cells within ±1 ms due to simple divergence of inhibitory axons. Brief network activation induced an appearance of 'hypersynchronous' (∼80%) population sIPSCs occurring in response to coherent discharges of several INs with jitter ±4 ms. Notably, population sIPSCs were preceded by transient inward currents (TICs). Those were excitatory events capable to synchronize firing of INs, in this respect being reminiscent of so-called fast prepotentials observed in studies on pyramidal neurons. TICs also had network properties consisting of heterogeneous components: glutamate currents, local axonal and dendritic spikelets, and coupling electrotonic currents likely via gap junctions; putative excitatory action of synaptic gamma-aminobutyric acid (GABA) was not involved. The appearance of population excitatory-inhibitory sequences could be initiated and reproduced by firing of a single excitatory cell reciprocally connected with one IN. Discussion: Our data demonstrate that synchronization of INs is initiated and dominated by glutamatergic mechanisms, which recruit, in a whole-sale manner, into supporting action other excitatory means existing in a given neural system.

Integration of energy homeostasis and stress by parvocellular neurons in rat hypothalamic paraventricular nucleus.

KEY POINTS: Central regulation of energy homeostasis and stress are believed to be reciprocally regulated, i.e. excessive food intake suppresses, while prolonged hunger exacerbates, stress responses in vivo. This relationship may be mediated by neuroendocrine parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus that receive both stress- and feeding-related input. We find that hunger strongly and selectively potentiates, while re-feeding suppresses, a cellular analogue of a stress response induced by acute glucopenia in CRH neurons in rat hypothalamic slices. Neuronal activation in response to glucopenia was mediated synaptically, via the relative enhancement of glutamate over GABA input. These results illustrate how acute stress responses may be initiated in vivo and show that it is reciprocally integrated with energy balance via local hypothalamic mechanisms acting at the level of CRH neurons and their afferent terminals. ABSTRACT: Increased food intake is a common response to help cope with stress, implying the existence of a previously postulated but imperfectly understood, inverse relationship between the regulation of feeding and stress. We have identified components of the neural circuitry that can integrate these homeostatic responses. Prior fasting (∼24 h) potentiates, and re-feeding suppresses, excitatory responses to acute glucopenia in about half of the corticotropin releasing hormone (CRH)-expressing, putatively neurosecretory, stress-related neurons in the paraventricular nucleus of the hypothalamus studied. Glucoprivation stress ex vivo resulted from a preferential relative increase in excitatory (glutamatergic) over inhibitory (GABAergic) inputs. Putative preautonomic cells were less sensitive to fasting, and showed a predominant inhibition to acute glucopenia. We conclude that hunger may sensitize hypothalamic stress responses by acting via local mechanisms, at the level of CRH neurons and their presynaptic inputs. Those mechanisms involve neither presynaptic ATP-sensitive potassium channels nor postsynaptic ATP levels.

Astrocytes and presynaptic plasticity in the striatum: Evidence and unanswered questions.

One of the main functions of astrocytes is to ensure glutamate homeostasis by glutamate uptake and glutamine synthesis. However, during the past ten years it has become clear that astrocytes may also induce changes in synaptic glutamate release when respective pathways must cope with the consequences of brain damage or other alterations in their functional requirements. The loss of glutamatergic synapses in Parkinson's and Huntington's disease is likely to associate with a continuous redistribution of presynaptic activity within the pool of surviving synapses, and astrocytes may have a role in the maintenance of independent control at individual glutamate release sites. The rodent striatum should be a good model structure to analyse astrocyte-synapse interactions underlying disease-related plasticity, because it does not itself contain any glutamatergic neurons. Here we examine recent results that may shed light on the mechanisms underlying pathway-specific alterations in the corticostriatal or thalamostriatal synaptic transmission with a possible involvement of astrocytic release or uptake of glutamate. The conclusions emphasize the need of exploring the highly compartmentalised and presumably heterogeneous synapse astrocyte-interactions at a single synapse level.

Cell type-specific postsynaptic effects of neuropeptide Y in substantia gelatinosa neurons of the rat spinal cord.

Cellular mechanisms of antinociceptive action of neuropeptide Y were investigated in substantia gelatinosa (SG) neurons in rat spinal cord slices. Somatic and synaptic effects of NPY were compared in two subpopulations of cells with different firing patterns, tonic (TFNs), and delayed firing (DFNs) neurons. For the study, TFNs were selected on morphological basis: they had appearance of central and radial but not islet cells, and are likely excitatory interneurons in dorsal horn networks. In their turn, DFNs were classified as radial and vertical cells. 0.3 µM NPY via Y1 receptors activated hyperpolarizing postsynaptic current of GIRK type in majority of TFNs (∼77%) but not DFNs (∼8%). Miniature synaptic currents in all neurons were seen as a mixture of excitatory (mEPSCs) and inhibitory (mIPSCs), the frequency of the former being ∼5 times greater. The mEPSCs were mediated by glutamate receptors of AMPA subtype, while the dominant part of mIPSCs--by glycine receptors. In all cell types, NPY moderately depressed the frequency of both mEPSCs and mIPSCs; the effects occurred via Y2 and Y1 receptors, respectively. The data suggest that behavioral NPY-evoked antinociception is achieved via postsynaptic hyperpolarization of majority of TFNs (assumingly, excitatory interneurons) via Y1 receptors and depression of the mEPSCs via Y2 receptors.

Glucosensing in parvocellular neurons of the rat hypothalamic paraventricular nucleus.

Specialized hypothalamic neurons responding to rising extracellular glucose via increases or decreases in their electrical activity [glucose-excited (GE) and glucose-inhibited (GI) cells, respectively] have been reported in the hypothalamic arcuate, ventromedial and lateral nuclei. The hypothalamic paraventricular nucleus (PVN) is an important neurosecretory and preautonomic output nucleus. We tested whether parvocellular PVN neurons also possess glucosensing properties, using patch-clamp recording and immunocytochemistry. Putative neurosecretory (p-NS) and preautonomic (p-PA) cells were identified electrophysiologically. Although parvocellular neurons were insensitive to transitions from 10 to 2.5 mm glucose, approximately 68% of p-PA cells responded directly to glucopenia (mimicked by a step to 0.2 mm glucose) with an increased membrane conductance. Of these, approximately 24% hyperpolarized (accompanied by an outward current) and thus were GE, approximately 26% depolarized (with an inward current, thus GI) and approximately 18% did not change membrane potential. The concentration dependence of the glucose response was similar for both GE and GI cells (EC(50) of 0.67-0.7 mm), but was steep, with Hill slopes of 3-4. The K(ATP) channel blockers glibenclamide and tolbutamide did not prevent, while the K(ATP) channel opener diazoxide did not mimic, the effects of low glucose on GE neurons. Moreover, the K(ATP) sulfonylurea receptor SUR1 was not detected in glucosensitive neurons. We conclude that the PVN contains previously unknown GE and GI cells that could participate in regulation of autonomic functions. GE neurons in the PVN sense ambient glucose via a unique mechanism, probably independent of K(ATP) channels, in contrast to neurons in other hypothalamic nuclei.

A-type K+ current dominates somatic excitability of delayed firing neurons in rat substantia gelatinosa.

Substantia gelatinosa neurons display three main types of intrinsic firing behavior: tonic, adapting, and delayed onset. Here, voltage-gated currents expressed by delayed firing neurons were studied in nucleated patches obtained in spinal cord slices of 3-5 weeks-old rats. Inward Na+ current was negligible under these conditions and was usually occluded by superposition of much larger outward currents. Two kinds of outward currents were found, an A-type (K(A) ) and delayed rectifier (K(DR) ) potassium currents. K(A) activated rapidly (<1.5 ms at >-20 mV) and operated at subthreshold membrane potentials; voltages of steady-state half-maximal activation and inactivation were -38.7 and -87.2 mV, respectively. Inactivation was biexponential with a dominant fast component (~90%, time constant ∼8 ms). K(DR) activated more slowly (<8 ms at >-20 mV), half-maximal activation was -23.6 mV, and decayed mono-exponentially with a time constant 70-110 ms. Maximal amplitudes of K(A) were almost 10-times larger than those of K(DR) , their respective densities were 8.5 and 0.97 µS µm⁻². Tetraethylammonium, 5 mM, blocked K(DR) but not K(A) , whereas both currents were depressed by 5 mM 4-aminopyridine. In current-clamp recordings, 4-action potential but not tetraethylammonium abolished firing delay suggesting the causative role of K(A) . Thus, the predominance of fast K(A) over other somatic currents is a distinctive feature of delayed firing neurons among all other types of substantia gelatinosa neurons and likely explains the appearance of their typical firing delay.

Morphophysiologic properties of islet cells in substantia gelatinosa of the rat spinal cord.

Substantia gelatinosa (SG) neurons of the spinal cord are highly heterogeneous in their morphophysiologic properties and could be categorized on several subtypes. Here the properties of islet cells in rat SG (approximately 11%) are described with the use of confocal microscopy and patch-clamp recording. The cells had significantly longer and thicker dendritic trees among all other neurons. Only these cells expressed slow inward current activated by hyperpolarization, which could be blocked by Cs+ but not Ba2+, presumably representing H-current (Ih). Possibly due to Ih, islet cells had peculiar membrane and firing responses. Of note the membrane potential showed a sag in response to hyperpolarization while depolarization triggered action potentials (APs) in a tonic-like pattern. APs, however, occurred with larger maximal frequencies and in response to broader stimulation intensities than in other tonically firing neurons. Neuronal variability in SG and possible functional roles of islet cells are discussed.

Developmental switch in neuropeptide Y and melanocortin effects in the paraventricular nucleus of the hypothalamus.

Homeostatic regulation of energy balance in rodents changes dramatically during the first 3 postnatal weeks. Neuropeptide Y (NPY) and melanocortin neurons in the arcuate nucleus, a primary energy homeostatic center in adults, do not fully innervate the paraventricular nucleus (PVN) until the third postnatal week. We have identified two classes of PVN neurons responsive to these neuropeptides, tonically firing neurosecretory (NS) and burst-firing preautonomic (PA) cells. In neonates, NPY could inhibit GABAergic inputs to nearly all NS and PA neurons, while melanocortin regulation was minimal. However, there was a dramatic, age-dependent decrease in NPY responses specifically in the PA neurons, and a 3-fold increase in melanocortin responses in NS cells. These age-dependent changes were accompanied by changes in spontaneous GABAergic currents onto these neurons. This primarily NPYergic regulation in the neonates likely promotes the positive energy balance necessary for growth, while the developmental switch correlates with maturation of homeostatic regulation of energy balance.

RNA editing produces glycine receptor alpha3(P185L), resulting in high agonist potency.

The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

Selective postsynaptic inhibition of tonic-firing neurons in substantia gelatinosa by mu-opioid agonist.

BACKGROUND: Spinal substantia gelatinosa (SG) is a site of action of administered and endogenous opioid agonists and is an important element in the system of antinociception. However, little is known about the types of neurons serving as specific postsynaptic targets for opioid action within the SG. To study the spinal mechanisms of opioidergic analgesia, the authors compared the action of mu-opioid agonist [D-Ala, N-Me-Phe, Gly-ol]-enkephalin (DAMGO) on SG neurons with different intrinsic firing properties. METHODS: Whole cell patch clamp recordings from spinal cord slices of Wistar rats were used to study the sensitivity of SG neurons to DAMGO. RESULTS: Three groups of neurons with distinct distributions in SG were classified: tonic-, adapting-, and delayed-firing neurons. DAMGO at 1 microm concentration selectively hyperpolarized all tonic-firing neurons tested, whereas none of the adapting- or delayed-firing neurons were affected. The effect of DAMGO on tonic-firing neurons was due to activation of G protein-coupled inward-rectifier K conductance, which could be blocked by 500 microm Ba and 500 microm Cs but increased by 50 microm baclofen. As a functional consequence of DAMGO action, a majority of tonic-firing neurons changed their pattern of intrinsic firing from tonic to adapting. CONCLUSIONS: It is suggested that tonic-firing neurons, presumably functioning as excitatory interneurons, are primary postsynaptic targets for administered and endogenous opioid agonists in spinal SG. Functional transition of cells in this group from tonic to adapting firing mode may represent an important mechanism facilitating opioidergic analgesia.

Mechanism of spike frequency adaptation in substantia gelatinosa neurones of rat.

Using tight-seal recordings from rat spinal cord slices, intracellular labelling and computer simulation, we analysed the mechanisms of spike frequency adaptation in substantia gelatinosa (SG) neurones. Adapting-firing neurones (AFNs) generated short bursts of spikes during sustained depolarization and were mostly found in lateral SG. The firing pattern and the shape of single spikes did not change after substitution of Ca2+ with Co2+, Mg2+ or Cd2+ indicating that Ca2+-dependent conductances do not contribute to adapting firing. Transient KA current was small and completely inactivated at resting potential suggesting that adapting firing was mainly generated by voltage-gated Na+ and delayed-rectifier K+ (KDR) currents. Although these currents were similar to those previously described in tonic-firing neurones (TFNs), we found that Na+ and KDR currents were smaller in AFNs. Discharge pattern in TFNs could be reversibly converted into that typical of AFNs in the presence of tetrodotoxin but not tetraethylammonium, suggesting that lower Na+ conductance is more critical for the appearance of firing adaptation. Intracellularly labelled AFNs showed specific morphological features and preserved long extensively branching axons, indicating that smaller Na+ conductance could not result from the axon cut. Computer simulation has further revealed that down-regulation of Na+ conductance represents an effective mechanism for the induction of firing adaptation. It is suggested that the cell-specific regulation of Na+ channel expression can be an important factor underlying the diversity of firing patterns in SG neurones.

Ionic basis of tonic firing in spinal substantia gelatinosa neurons of rat.

Ionic conductances underlying excitability in tonically firing neurons (TFNs) from substantia gelatinosa (SG) were studied by the patch-clamp method in rat spinal cord slices. Ca(2+)-dependent K(+) (K(CA)) conductance sensitive to apamin was found to prolong the interspike intervals and stabilize firing evoked by a sustained membrane depolarization. Suppression of Ca(2+) and K(CA) currents, however, did not abolish the basic pattern of tonic firing, indicating that it was generated by voltage-gated Na(+) and K(+) currents. Na(+) and K(+) channels were further analyzed in somatic nucleated patches. Na(+) channels exhibited fast activation and inactivation kinetics and followed two-exponential time course of recovery from inactivation. The major K(+) current was carried through tetraethylammonium (TEA)-sensitive rapidly activating delayed-rectifier (K(DR)) channels with a slow inactivation. The TEA-insensitive transient A-type K(+) (K(A)) current was very small in patches and was strongly inactivated at resting potential. Block of K(DR) rather than K(A) conductance by 1 mM TEA lowered the frequency and stability of firing. Intracellular staining with biocytin revealed at least three morphological groups of TFNs. Finally, on the basis of present data, we created a model of TFN and showed that Na(+) and K(DR) currents are sufficient to generate a basic pattern of tonic firing. It is concluded that the balanced contribution of all ionic conductances described here is important for generation and modulation of tonic firing in SG neurons.

Chronic treatment with ionotropic glutamate receptor antagonist kynurenate affects GABAergic synaptic transmission in rat hippocampal cell cultures.

It is well documented that prolonged treatment with antagonists of ionotropic glutamate receptors activates a number of homeostatic mechanisms including alteration of glutamatergic transmission. We studied whether this treatment can also affect GABAergic transmission. Using whole-cell voltage clamp recording and local extracellular stimulation we investigated evoked inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons grown in the presence of ionotropic glutamate receptor antagonist kynurenate (1 mM) and in control conditions. Chronic kynurenate treatment did not significantly affect the amplitude of evoked IPSCs and IPSC reversal potentials. In contrast we found that the paired-pulse depression was increased by 67% in cultures treated with kynurenic acid. We conclude that additional mechanism(s), alteration of GABAergic synaptic transmission, may contribute to homeostatic plasticity induced by chronic block of ionotropic glutamate receptors.

Post-tetanic depression of GABAergic synaptic transmission in rat hippocampal cell cultures.

The effect of tetanic stimulation (30 Hz, 4 s) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. It was found that tetanic stimulation elicited post-tetanic depression (PTD) of the evoked IPSCs with a duration of more than 50 s in about 60% of the connections tested; post-tetanic potentiation was induced in 25% of the connections. We propose that the opposite effects of tetanization on IPSC amplitude are due to differences in the type of the interneuron that was tetanized. Since PTD in our experiments was usually accompanied by changes in the IPSC coefficient of variation and changes of a paired pulse depression, which are thought to reflect presynaptic mechanisms of modulation, we suggest that part of the PTD is due to a presynaptic mechanism(s).