NO regulates the strength of synaptic inputs onto hippocampal CA1 neurons via NO-GC1/cGMP signalling.

GABAergic interneurons are the predominant source of inhibition in the brain that coordinate the level of excitation and synchronization in neuronal circuitries. However, the underlying cellular mechanisms are still not fully understood. Here we report nitric oxide (NO)/NO-GC1 signalling as an important regulatory mechanism of GABAergic and glutamatergic synaptic transmission in the hippocampal CA1 region. Deletion of the NO receptor NO-GC1 induced functional alterations, indicated by a strong reduction of spontaneous and evoked inhibitory postsynaptic currents (IPSCs), which could be compensated by application of the missing second messenger cGMP. Moreover, we found a general impairment in the strength of inhibitory and excitatory synaptic inputs onto CA1 pyramidal neurons deriving from NO-GC1KO mice. Finally, we disclosed one subpopulation of GABAergic interneurons, fast-spiking interneurons, that receive less excitatory synaptic input and consequently respond with less spike output after blockage of the NO/cGMP signalling pathway. On the basis of these and previous findings, we propose NO-GC1 as the major NO receptor which transduces the NO signal into cGMP at presynaptic terminals of different neuronal subtypes in the hippocampal CA1 region. Furthermore, we suggest NO-GC1-mediated cGMP signalling as a mechanism which regulates the strength of synaptic transmission, hence being important in gating information processing between hippocampal CA3 and CA1 region.

Alterations in membrane and firing properties of layer 2/3 pyramidal neurons following focal laser lesions in rat visual cortex.

Focal cortical injuries are well known to cause changes in function and excitability of the surviving cortical areas but the cellular correlates of these physiological alterations are not fully understood. In the present study we employed a well established ex vivo-in vitro model of focal laser lesions in the rat visual cortex and we studied membrane and firing properties of the surviving layer 2/3 pyramidal neurons. Patch-clamp recordings, performed in the first week post-injury, revealed an increased input resistance, a depolarized spike threshold as well as alterations in the firing pattern of neurons in the cortex ipsilateral to the lesion. Notably, the reported lesion-induced alterations emerged or became more evident when an exciting perfusing solution, known as modified artificial cerebrospinal fluid, was used to increase the ongoing synaptic activity in cortical slices. Conversely, application of glutamatergic or GABAA receptor blockers reduced the observed alterations and GABAB receptor blockers abolished the differences completely. All together the present findings suggest that changes in synaptic receptors function, following focal cortical injuries, can modulate membrane and firing properties of layer 2/3 pyramidal neurons. This previously unknown functional interplay between synaptic and membrane properties may constitute a novel cellular mechanism to explain alterations in neuronal network function and excitability following focal cortical injuries.

Metaplasticity of horizontal connections in the vicinity of focal laser lesions in rat visual cortex.

Focal cortical injuries are accompanied by a reorganization of the adjacent neuronal networks. An increased synaptic plasticity has been suggested to mediate, at least in part, this functional reorganization. Previous studies showed an increased long-term potentiation (LTP) at synapses formed by ascending fibres projecting onto layers 2/3 pyramidal cells following lesions in rat visual cortex. This could be important to establish new functional connections within a vertical cortical column. Importantly, horizontal intracortical connections constitute an optimal substrate to mediate the functional reorganization across different cortical columns. However, so far little is known about their potential implication in the functional rewiring post-lesion. Here, we investigated possible alterations of synaptic plasticity of horizontal connections in layers 2/3 in an 'ex vivo-in vitro' model of focal laser lesion in rat visual cortex. LTP at these synapses was found to be enhanced post-lesion, whereas long-term depression (LTD) was impaired, revealing a metaplastic shift toward strengthening of these synapses. Furthermore, we disclosed a prolonged decay-time constant of NMDAR-dependent currents, which can contribute to the enhanced LTP. Taken together these data revealed that a laser lesion-induced focal damage of the visual cortex is accompanied by a facilitated potentiation of horizontal synaptic connections in the vicinity of the focal injury. This specific strengthening of synaptic plasticity at horizontal connections in layers 2/3 might be one important cellular mechanism to compensate focal injury-mediated dysfunction in the cerebral cortex.

Changes in intracellular calcium transients and LTP in the surround of visual cortex lesions in rats.

Injury and loss of neurons are observed in the center of a cerebral cortical lesion. Mechanisms of early functional reorganization post-lesion involve changes in the strength of synaptic coupling as measured in long-term potentiation (LTP). Since these changes in LTP may depend on the intraneuronal calcium concentration ([Ca2+]I), the present study analyzed the strength of synaptic LTP combined with measurements of the stimulus-induced peak calcium influx in slices from rat visual cortex in vitro. Slices were analyzed 1-7 days post-lesion by use of electrophysiological and calcium fluorescence imaging techniques. A theta-burst stimulus (TBS) was electrically applied to cortical layer IV, while changes in extracellular field potentials (FPs) and in the corresponding peak calcium influx were recorded in layers II/III. Both the strength of LTP and of the FP mediated peak calcium influx were significantly enhanced 1-6 days post-lesion at a distance of 4 mm from the lesion border. Pharmacological experiments revealed that the expression of LTP was dependent on the activation of NMDA receptors. The area of increased stimulus-evoked peak calcium influx correlated with the enhanced LTP, suggesting that changes in [Ca2+]I mediate the strength of long-term synaptic plasticity following a cortical lesion. This mechanism may support synaptic reorganization in the surround of the deafferented region in rat visual cortex.

Increased synaptic plasticity in the surround of visual cortex lesions in rats.

Lesion-induced functional loss is reduced when new synaptic connections are established in the surround of a cortical lesion. For this, long-term synaptic plasticity can play a key role. We studied long-term potentiation (LTP) and long-term depression (LTD) in slices of rat visual cortex with small cortical lesions. Surprisingly, the normal balance between LTP and LTD was significantly altered in the first week following cortical injury. Theta-burst induced LTP was increased, whereas LTD evoked by low frequency stimulation was not affected. The increased potentiation of subcortical inputs after cortical lesions opens a window for facilitated early functional reorganization by repetitive visual training.

Intracellular calcium signals in the surround of rat visual cortex lesions.

Focal lesions of the visual cortex induce deafferentiation, excitotoxic cell death as well as functional reorganization in the surrounding tissue. The intracellular second messenger calcium is involved in a wide range of cellular responses including excitotoxicity and functional reorganization following cortical injuries. We investigated the intracellular calcium concentration [Ca2+]i in neurons of the visual cortex using fluorescence imaging of fura-2 signals in a slice preparation obtained from lesioned and sham-operated cortices. We observed an increase in resting and stimulus evoked [Ca2+]i in the surround of the lesion, which were mediated by NMDA and non-NMDA ionotropic glutamate receptors. This increase in [Ca2+]i might be an important factor for lesion-induced functional reorganization in the rat visual cortex.

Lesion-induced changes in NMDA receptor subunit mRNA expression in rat visual cortex.

Focal lesions in the visual cortex are well known to induce pronounced perilesional reorganization of the neuronal circuitry. Since NMDA receptors crucially control synaptic plasticity and reorganization, we studied lesion-induced changes in their subunit expression and biophysical properties. Between 8 and 10 days after focal thermolesioning, pyramidal neurones in the near surround of the lesion were studied in acute brain slices. We found a significant decrease in the ratio of NR2A and NR2B subunit mRNA as compared to neurones from sham operated animals. Interestingly, no significant differences in the properties of NMDA receptor-mediated postsynaptic currents (NMDA PSCs) were observed between lesioned and sham operated animals. Thus, the observed perilesional changes in the NR2A/NR2B mRNA ratio appear to be subthreshold to result in significant changes in the functional properties of NMDA receptors.

Interaction between intracellular tetanization and pairing-induced long-term synaptic plasticity in the rat visual cortex.

Long-term changes in synaptic transmission in slices of rat visual cortex were induced either by pairing the excitatory postsynaptic potentials with postsynaptic depolarization or by intracellular tetanization without synaptic stimulation. Changes in the excitatory postsynaptic potential amplitude induced by any of the protocols applied in isolation persisted for longer than 1 h. Pairing-induced long-term potentiation was input specific. We studied the interaction between intracellular tetanization and pairing-induced plasticity by applying the two protocols one after the other at 10-min intervals. The pairing procedure applied after intracellular tetanization did not lead to any further potentiation, but to a depotentiation of the potentiated inputs. A second pairing protocol applied 10 min later led to further depotentiation, while previously unaffected inputs became weakly depressed. If intracellular tetanization was applied after the pairing procedure, the synaptic responses did not change immediately, but a slow return of the excitatory postsynaptic potential amplitude to the control level could be observed. Therefore, intracellular tetanization is not capable of inducing further potentiation after pairing, and pairing cannot further potentiate the inputs which have already been potentiated by intracellular tetanization. The maintenance of long-term potentiation induced by any of the protocols was impaired by successive application of another procedure. These results suggest a similarity of the mechanisms of synaptic changes induced by the two protocols and demonstrate that the direction of synaptic gain change depends on the history of the synapse.

Reorganization in the visual cortex after retinal and cortical damage.

Retinal and cortical lesions are completely different events that trigger visual cortical plasticity. We therefore compared the cortical effects of homonymous lesions of the central retina with effects of cortical lesions. All in vivo experiments were performed in anaesthetized, adult cats. Retinal lesions were made with a Xenon-light photocoagulator, and cortical lesions were induced by focal application of heat or ibotenic acid injection. Both, in cortical regions representing the retinal scotoma and at the border of small focal cortical lesions single neuron activity was initially suppressed and accompanied by a narrow area of increased activity adjacent to the region of functional loss during the first 1-2 weeks. At the same time an increased glutamatergic NMDA response and a reduction of GABA(A) and GABA(B) responses was observed around the cortical lesions in vitro. At an early stage long-term potentiation (LTP) is facilitated in those regions that were characterized by local upregulation of excitation and downregulation of inhibition after cortical lesions. Similarly, at the border of cortical scotomas in area 17 an increased glutamate level was found while inside the scotoma GAD levels were reduced. Shifts in topography of retinal representation as well as increases of receptive field size were detected as signs of lesion-induced neuronal reorganization after retinal and cortical lesions with longer survival times. A common cascade of events is triggered in the visual cortex by retinal as well as cortical lesions: reduced GABAergic inhibition and increased glutamatergic excitation, leading to increased spontaneous activity and visual excitability that is accompanied by facilitated LTP, and appears to initiate local cortical reorganization after functional disturbances in the visual system.

Long-term cellular dysfunction after focal cerebral ischemia: in vitro analyses.

The long-term (< or = six months) functional consequences of permanent middle cerebral artery occlusion were studied with in vitro extra- and intracellular recording techniques in adult mouse neocortical slices. After survival times of one to three days, 28 days and six months, intracellular recordings from layers II/III pyramidal cells in the vicinity of the infarct did not reveal any statistically significant changes in the intrinsic membrane properties when compared to age-matched control animals. However, a pronounced hyperexcitability could be observed upon orthodromic synaptic stimulation in neocortical slices obtained from mice 28 days after induction of ischemia. Low-intensity electrical stimulation of the afferents elicited particularly in this group epileptiform extracellular field potential responses and intracellular excitatory postsynaptic potentials, that were longer in duration as compared to the controls. When the N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potential was pharmacologically isolated in a bathing solution containing 0.1 mM Mg2+ and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione, the synaptic responses were longer and larger in the ischemic cortex as compared to the controls. Higher stimulus intensities evoked in normal medium a biphasic inhibitory postsynaptic potential, that contained in the 28 days post-ischemia group a prominent amino-phosphonovaleric acid-sensitive component, indicating a strong concurrent activation of a N-methyl-D-aspartate receptor-mediated excitatory postsynaptic potential. This pronounced co-activation could only be observed in the 28 days ischemic group, and neither after one to three days or six months post-ischemia nor in the controls. The quantitative analysis of the efficiency of stimulus- evoked inhibitory postsynaptic potentials recorded in amino-phosphono-valeric acid revealed a reduction of GABA-mediated inhibition in ischemic cortex. Although this reduction in intracortical inhibition may already contribute to an augmentation of N-methyl-D-aspartate receptor-mediated excitation, our results do also indicate that the function of N-methyl-D-aspartate receptors is transiently enhanced in the ischemic cortex. This transient hyperexcitability does not only cause cellular dysfunction in the vicinity of the infarct, but may also contribute to neuronal damage due to excitotoxicity.

Long-term changes of ionotropic glutamate and GABA receptors after unilateral permanent focal cerebral ischemia in the mouse brain.

Long-term hyperexcitability was found after unilateral, permanent middle cerebral artery occlusion in exofocal neocortical areas of the adult mouse [Mittmann et al. (1998) Neuroscience 85, 15-27]. The aim of the present study was to test the hypothesis in an identical paradigm of ischemia. whether alterations in the densities of both excitatory and inhibitory amino acid receptors may underlie these pathophysiological changes. Alterations in densities of [3H]dizocilpine, [3H]D,L-amino-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, [3H]kainate and [3H]muscimol binding sites were demonstrated with quantitative in vitro receptor autoradiography. All binding sites were severely reduced in the core of the ischemic lesion. A completely different reaction was found in the exofocal, histologically inconspicuous parts of the somatosensory cortex and the more remote neocortical areas of both hemispheres. The [3H]muscimol binding sites were significantly reduced four weeks after ischemia in the motor cortex, hindlimb representation area and exofocal parts of the primary and secondary somatosensory cortices of both hemispheres. The focus of the reduction in [3H]muscimol binding sites was found in lower layer V and upper layer VI. Contrastingly, the densities of [3H]dizocilpine binding sites were found to be increased in these areas, whereas those of [3H]D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and [3H]kainate binding sites did not show significant changes. The [3H]dizocilpine binding site density increased predominantly in layers III and IV. All binding sites were also reduced in the retrogradely reacting, gliotic part of the ipsilateral ventroposterior thalamic nucleus, whereas the [3H]D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites were increased in the surround of the ipsilateral nucleus and no changes in binding sites were seen in the whole contralateral nucleus. We conclude that permanent local ischemia leads to a long-term and widespread impairment of the normal balance between binding sites of excitatory and inhibitory neurotransmitter receptors in neocortical areas far away from the focus of the post-ischemic tissue damage. The imbalance comprises an up-regulation of the [3H]dizocilpine binding sites in the ion channels of N-methyl-D-aspartate receptors and a down-regulation of [3H]muscimol binding sites of the GABA(A) receptors in the ipsi- and contralateral neocortex. These changes at the receptor level explain the previously observed hyperexcitability with the appearance of epileptiform field potentials and the long duration of excitatory postsynaptic potentials four weeks after ischemia.

Muscarinic inhibition of persistent Na+ current in rat neocortical pyramidal neurons.

Muscarinic modulation of persistent Na+ current (INaP) was studied using whole cell recordings from acutely isolated pyramidal cells of rat neocortex. After suppression of Ca2+ and K+ currents, INaP was evoked by slow depolarizing voltage ramps or by long depolarizing voltage steps. The cholinergic agonist, carbachol, produced an atropine-sensitive decrease of INaP at all potentials. When applied at a saturating concentration (20 microM), carbachol reduced peak INaP by 38% on average. Carbachol did not alter the voltage dependence of INaP activation nor did it interfere with the slow inactivation of INaP. Our data indicate that INaP can be targeted by the rich cholinergic innervation of the neocortex. Because INaP is activated in the subthreshold voltage range, cholinergic inhibition of this current would be particularly suited to modulate the electrical behavior of neocortical pyramidal cells below and near firing threshold.

Evidence for persistent Na+ current in apical dendrites of rat neocortical neurons from imaging of Na+-sensitive dye.

Evidence for a persistent Na+ current (I(NaP)) in the apical dendrite of neocortical neurons was sought with the use of fluorescence imaging to measure changes in intradendritic Na+ concentration. Neurons in neocortical brain slices were filled iontophoretically through an intracellular recording microelectrode with the Na+-sensitive dye benzofuran isophthalate (SBFI), and fluorescence images were recorded with a cooled charge-coupled device camera system using 380-nm illumination. In the presence of Ca2+ and K+ channel blockers, a short depolarizing current pulse evoked a single action potential followed by a plateau depolarization (PD) lasting >1 s. This tetrodotoxin (TTX)-sensitive PD is known to be maintained by I(NaP). A single action potential caused no detectable SBFI fluorescence change, whereas the PD was associated with an SBFI fluorescence change in the soma and apical dendrite indicating increased intracellular Na+ concentration. Determination of the full spatial extent of the dendritic fluorescence change was prevented by our inability to detect the dim fluorescence signal in the distal regions of the apical dendrite. In each experiment the fluorescence change extended into the apical dendrite as far as dye could be visualized (50-300 microm). A slow, depolarizing voltage-clamp ramp that activated I(NaP) caused similar fluorescence changes that were eliminated by TTX, indicating that the SBFI fluorescence changes are caused by Na+ influx due to I(NaP) activation. We conclude that I(NaP) can be generated by the apical dendritic membrane to at least 300 microm from the soma.

Impairment of intracortical GABAergic inhibition in a rat model of absence epilepsy.

The WAG/Rij rat strain is characterized in its EEG by the manifestation of spike-wave discharges which resemble in their spontaneous appearance and pharmacological sensitivity the absence epilepsy observed in humans. In order to test the hypothesis whether cellular intrinsic membrane and/or synaptic network properties in the neocortex are modified in this form of epilepsy, we analyzed with extra- and intracellular recording techniques the functional status of neocortical slices obtained from adult epileptic WAG/Rij rats and compared them with the data acquired from non-epileptic control Wistar rats. Intrinsic membrane properties, like resting membrane potential, neuronal input resistance and basic cellular firing characteristics, did not differ between these two strains. However, the analysis of extra- and intracellularly recorded synaptic responses revealed an intracortical hyperexcitability which was accompanied by a significant reduction in the efficiency of GABAergic inhibition. Our data indicate that the imbalance between intracortical excitatory and inhibitory mechanisms may at least contribute to the expression and augmentation of spike-wave discharges in epileptic WAG/Rij rats.

Ischaemia-induced long-term hyperexcitability in rat neocortex.

The long-term structural and functional consequences of transient forebrain ischaemia were studied with morphological, immunohistochemical and in vitro electrophysiological techniques in the primary somatosensory cortex of Wistar rats. After survival times of 10-17 months postischaemia, neocortical slices obtained from ischaemic animals were characterized by a pronounced neuronal hyperexcitability in comparison with untreated age-matched controls. Extra- and intracellular recordings in supragranular layers revealed all-or-none long-latency recurrent responses to orthodromic synaptic stimulation of the afferent pathway. These responses were characterized by durations up to 1.7 s, by multiple components and by repetitive synaptic burst discharges. The reversible blockade of this late activity by DL-amino-phosphonovaleric acid (APV) suggested that this activity was mediated by N-methyl-D-aspartate (NMDA) receptors. The peak conductance of inhibitory postsynaptic potentials was significantly smaller in neurons recorded in neocortical slices obtained from ischaemic animals than those from the controls. However, the average number of parvalbumin (PV)-labelled neurons per mm3, indicative of a subpopulation of GABAergic interneurons, and the average number and length of dendritic processes arising from PV-containing cells was not significantly different between ischaemic and control cortex. The prominent dysfunction of the inhibitory system in ischaemic animals occurred without obvious structural alterations in PV-labelled cells, indicating that this subpopulation of GABAergic interneurons is not principally affected by ischaemia. Our data suggest a long-term down-regulation of inhibitory function and a concurrent NMDA receptor-mediated hyperexcitability in ischaemic neocortex. These alterations may result from structural and/or functional properties of inhibitory non-PV-positive neurons or permanent functional modifications on the subcellular molecular level, i.e. alterations in the phosphorylation status of GABA and/or NMDA receptors. The net result of these long-term changes is an imbalance between the excitatory and inhibitory systems in the ischaemic cortex with the subsequent expression and manifestation of intracortical hyperexcitability.

Lesion-induced transient suppression of inhibitory function in rat neocortex in vitro.

The structural and functional consequences of a local thermolesion were examined in rat neocortex with electrophysiological in vitro techniques and immunocytochemistry. Age-matched untreated and sham-operated animals served as controls and were analysed in the same way. The lesions consisted of a core of coagulated tissue 2-3 mm in diameter and reached ventrally into the deep cortical layers. After two days reactive astrocytes and after nine days a dense gliosis were observed in the immediate vicinity. Modifications in the intrinsic membrane characteristics and the synaptic network properties were investigated with intra- and extracellular recording techniques after survival times of one to eight days. Neurons recorded in the surrounding of lesions in neocortical slices revealed a significantly more depolarized resting membrane potential and a higher neuronal input resistance. In comparison to cells in control slices, maximal discharge rates to injection of depolarizing current pulses of neurons close to a focal lesion were not significantly altered and intrinsic burst firing was never observed. However, between postlesion days 1 and 5, neurons in the surroundings of lesions showed a transient increase in synaptic excitability. This hyperactivity was most clearly pronounced at a distance of 2-3 mm from the centre of the lesion (i.e. about 1-1.5 mm away from the lesion border) and characterized by long-duration field potential responses and multiphasic long-lasting excitatory postsynaptic potentials to orthodromic stimulation of the afferent input. This lesion-induced hyperexcitability was associated with a significant reduction in the peak conductance of the Cl(-)-dependent fast inhibitory postsynaptic potential and the K(+)-dependent long-latency inhibitory postsynaptic potential, suggesting that the intracortical GABAergic system was functionally impaired. The decrease in synaptic inhibition was associated with prolonged N-methyl-D-aspartate receptor-mediated activity, which could be reversibly blocked by D-amino-phosphonovaleric acid. In addition, neurons recorded in the vicinity of the lesion responded to an orthodromic synaptic stimulus with a long-lasting burst. The lesion-induced disturbance in the balance between the excitatory and inhibitory system may not only have profound influences on the mechanisms of intracortical information processing, but may also lead to the expression of epileptiform activity and long-term functional deficits.

Role of NMDA receptors and voltage-activated calcium channels in an in vitro model of cerebral ischemia.

In an in vitro model of cerebral ischemia we investigated the functional consequences of repeated hypoxias and the potential protective effect of the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) and the calcium channel blocker verapamil in preventing the expression of pathophysiological activity. Rat neocortical slices were exposed to nitrogen for 2-13 min and the hypoxia-induced functional modifications were monitored in layer II/III by recording the extracellular DC potential, the extracellular calcium concentration ([Ca2+]o) and the stimulus-evoked synaptic responses. Hypoxia caused a reversible 2.4-24.6 mV negative shift in the extracellular DC potential associated with a [Ca2+]o decrease from 1.2 to 0.2 mM and a complete loss of synaptic responsiveness. Repeating hypoxias induced an increase in the amplitude of this anoxic depolarization (AD) and a significant decrease in the AD onset latency. Synaptic responses partially recovered at 20 and 60 min intervals between subsequent hypoxic periods, indicating that the initial AD did not induce any short-term irreparable functional deficits. Verapamil (50 microM) caused an increase in the AD onset latency. However, in comparison to untreated controls, verapamil induced a reduction of excitatory and inhibitory responses during hypoxia probably by blocking voltage-activated calcium conductances. In addition, verapamil did not have any significant effect on the hypoxia-induced reduction of [Ca2+]o. Bath application of D-APV (30 microM) prevented the significant reduction in the AD onset latency to the second hypoxia, but had no significant effect on the AD amplitude and duration. The hypoxia-induced decrease in [Ca2+]o was not altered after addition of D-APV to the bathing medium. These data indicate that the influx of calcium through voltage-activated calcium channels and the NMDA receptor-gated ionophore does not significantly contribute to the massive depolarization observed under hypoxic conditions.