Inhibitory-excitatory synaptic balance is shifted toward increased excitation in magnocellular neurosecretory cells of heart failure rats

Despite the well-established contribution of neurohumoral activation to morbidity and mortality in heart failure (HF) patients, relatively little is known about the underlying central nervous system mechanisms. In this study, we aimed to determine whether changes in GABAergic inhibitory and glutamatergic excitatory synaptic function contribute to altered hypothalamic magnocellular neurosecretory cell (MNC) activity in HF rats. Patch-clamp recordings were obtained from MNCs in brain slices from sham and HF rats. Glutamate excitatory (EPSCs) and GABAergic inhibitory postsynaptic currents (IPSCs) were simultaneously recorded, and changes in their strengths, as well as their interactions, were evaluated. We found a diminished GABAergic synaptic strength in MNCs of HF rats, reflected as faster decaying IPSCs and diminished mean IPSC charge transfer. Opposite changes were observed in glutamate EPSC synaptic strength, resulting in a shift in the GABA-glutamate balance toward a relatively stronger glutamate influence in HF rats. The prolongation of glutamate EPSCs during HF was mediated, at least in part, by an enhanced contribution of AMPA receptor desensitization to the EPSC decay time course. EPSC prolongation, and consequently increased unitary strength, resulted in a stronger AMPA receptor-mediated excitatory drive to firing discharge in MNCs of HF rats. Blockade of GABA(A) synaptic activity diminished the EPSC waveform variability observed among events in sham rats, an effect that was blunted in HF rats. Together, our results suggest that opposing changes in postsynaptic properties of GABAergic and glutamatergic synaptic function contribute to enhanced magnocellular neurosecretory activity in HF rats.     

Differences between the scaling of miniature IPSCs and EPSCs recorded in the dendrites of CA1 mouse pyramidal neurons

Anatomical studies have described inhibitory synaptic contacts on apical dendrites, and an abundant number of GABAergic synapses on the somata and proximal dendrites of CA1 pyramidal cells of the hippocampus. The number of inhibitory contacts decreases dramatically with distance from the soma, but the local electrophysiological characterization of these synapses at their site of origin in the dendrites is missing. We directly recorded dendritic GABA receptor-mediated inhibitory synaptic events in adult mouse hippocampal CA1 pyramidal neurons and compared them to excitatory synaptic currents recorded at the same sites. Miniature GABAergic events were evoked using localized application of a hyperosmotic solution to the apical dendrites in the vicinity of the dendritic whole-cell recording pipette. Glutamatergic synaptic events were blocked by kynurenic acid, leaving picrotoxin-sensitive IPSCs. We measured the amplitude and kinetic properties of mIPSCs at the soma and at three different dendritic locations. The amplitude of mIPSCs recorded at the various sites was similar along the somato-dendritic axis. The rise- and decay-times of local mIPSCs were also independent of the location of the synapses. The frequency of mIPSCs was 5 Hz at the soma, in contrast to < 0.5 Hz at dendritic sites, which could be increased to 10–20 Hz and 6–10 Hz, respectively, by our hyperosmotic stimulation protocol. Miniature glutamatergic events were evoked with the same protocol after blocking inhibitory synapses by bicucculine. The measured amplitudes increased along the somato-dendritic axis proportionally with their distance from the soma. The measured kinetic properties were independent of location. Consistent with the idea that IPSCs may have a restricted local effect in the dendrites, our data show a lack of distance-dependent scaling of miniature inhibitory synaptic events, in contrast to the scaling of excitatory events recorded at the same sites.     

Sprouting and synaptic reorganization in the subiculum and CA1 region of the hippocampus in acute and chronic models of partial-onset epilepsy

Repeated seizures induce permanent alterations in the hippocampal circuitry in experimental models and patients with intractable temporal lobe epilepsy (TLE). Most studies have concentrated their attention on seizure-induced reorganization of the mossy fiber pathway. The present study examined the projection pathway of the CA1 pyramidal neurons to the subiculum, which is the output of the hippocampal formation in five models of TLE. We examined the laminar pattern of Timm's histochemistry in the stratum lacunosum-moleculare of CA1 in three acute and two chronic models of TLE: intraventricular kainic acid (KA), systemic KA, systemic pilocarpine, chronic electric kindling and chronic i.p. pentylenetetrazol. The laminar pattern of Timm histochemistry in the stratum moleculare of CA1 was permanently remodeled in epileptic models suggesting sprouting of Timm containing terminals from the adjacent stratum lacunosum. Ultrastructural examination confirmed that Timm granules were localized in synaptic terminals. As the source of Timm-labeled terminals in this region was not known, sodium selenite, a selective retrograde tracer for zinc-containing terminals, was iontophoretically injected in vivo in rats exposed to systemic pilocarpine, systemic KA or chronic pentylenetetrazol. The normal projection of CA1 pyramidal neurons to the subiculum is topographically organized in a lamellar fashion. In normal rats, the extent of the injection site (terminals) and the retrogradely labeled pyramidal neurons (cell soma) corresponded to the same number of lamellas. In epileptic rats, the retrograde labeling extended 42-67% farther than the normal dorso-ventral extent including lamellas above and below the expected. This is direct evidence for sprouting of CA1 pyramidal axons into the subiculum and stratum lacunosum-moleculare of the CA1 region confirming the alterations of the laminar pattern of Timm's histochemistry. Sprouting of the CA1 projection to subiculum across hippocampal lamellas might lead to translamellar hyperexcitability, and to amplification and synchronization of epileptic discharges in the output gate of the hippocampal formation.     

Distribution of spontaneous currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons

Excitatory and inhibitory pathways have specific patterns of innervation along the somato-dendritic axis of neurons. We have investigated whether this morphological diversity was associated with variations in the frequencies of spontaneous and miniature GABAergic and glutamatergic synaptic currents along the somato-dendritic axis of rat hippocampal CA1 pyramidal neurons. Using in vitro whole cell recordings from somata, apical dendrites and basal dendrites (for which we provide the first recordings) of CA1 pyramidal neurons, we report that over 90% of the spontaneous currents were GABAergic, <10% being glutamatergic. The frequency of spontaneous GABAergic currents was comparable in the soma and in the dendrites. In both somata and dendrites, the Na(+) channel blocker tetrodotoxin abolished more than 80% of the spontaneous glutamatergic currents. In contrast, tetrodotoxin abolished most dendritic (>90%) but not somatic (<40%) spontaneous GABAergic currents. Computer simulations suggest that in our experimental conditions, events below 40pA are electrotonically filtered to such a degree that they are lost in the recording noise. We conclude that, in vitro, inhibition is massively predominant over excitation and quantitatively evenly distributed throughout the cell. However, inhibition appears to be mainly activity-dependent in the dendrites whereas it can occur in the absence of interneuron firing in the soma. These results can be used as a benchmark to compare values obtained in pathological tissue, such as epilepsies, where changes in the balance between excitation and inhibition would dramatically alter cell behaviour.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

Transition to seizures in the isolated immature mouse hippocampus: a switch from dominant phasic inhibition to dominant phasic excitation

The neural dynamics and mechanisms responsible for the transition from the interictal to the ictal state (seizures) are unresolved questions in epilepsy. It has been suggested that a shift from inhibitory to excitatory GABAergic drive can promote seizure generation. In this study, we utilized an experimental model of temporal lobe epilepsy which produces recurrent seizure-like events in the isolated immature mouse hippocampus (P8-16), perfused with low magnesium ACSF, to investigate the cellular dynamics of seizure transition. Whole-cell and perforated patch recordings from CA1 pyramidal cells and from fast- and non-fast-spiking interneurons in the CA1 stratum oriens hippocampal region showed a change in intracellular signal integration during the transition period, starting with dominant phasic inhibitory synaptic input, followed by dominant phasic excitation prior to a seizure. Efflux of bicarbonate ions through the GABA A receptor did not fully account for this excitation and GABAergic excitation via reversed IPSPs was also excluded as the prime mechanism generating the dominant excitation, since somatic and dendritic GABA A responses to externally applied muscimol remained hyperpolarizing throughout the transition period. In addition, abolishing EPSPs in a single neuron by intracellularly injected QX222, revealed that inhibitory synaptic drive was maintained throughout the entire transition period. We suggest that rather than a major shift from inhibitory to excitatory GABAergic drive prior to seizure onset, there is a change in the interaction between afferent synaptic inhibition, and afferent and intrinsic excitatory processes in pyramidal neurons and interneurons, with maintained inhibition and increasing, entrained 'overpowering' excitation during the transition to seizure.     

Mice deficient for the extracellular matrix glycoprotein tenascin-r show physiological and structural hallmarks of increased hippocampal excitability, but no increased susceptibility to seizures in the pilocarpine model of epilepsy

Recognition molecules provide important cues for neuronal survival, axonal fasciculation, axonal pathfinding, synaptogenesis, synaptic plasticity, and regeneration. Our previous studies revealed a link between perisomatic inhibition and the extracellular matrix glycoprotein tenascin-R (TN-R). Therefore, we here studied neuronal excitability and epileptic susceptibility in mice constitutively deficient in TN-R. In vitro analysis of populational spikes in hippocampal slices of TN-R-deficient mice revealed a significant increase in multiple spikes in the CA1 region, as compared with wild-type mice. This difference between genotypes was only partially reduced after blockade of GABA(A) receptors with picrotoxin, indicating a deficit in GABAergic inhibition and an increase in intrinsic excitability of CA1 pyramidal cells in TN-R-deficient mice. Using a battery of immunohistochemical markers and histological stainings, we were able to identify two abnormalities in the hippocampus of TN-R-deficient mice possibly related to increased excitability: the high number of glial fibrillary acidic protein-positive astrocytes and low number of calretinin-positive interneurons in the CA1 and CA3 regions. In order to test whether the revealed abnormalities give rise to increased susceptibility to seizures in TN-R-deficient mice, we used the pilocarpine model of epilepsy. No genotype-specific differences were found with regard to the time-course of pilocarpine-induced and spontaneous seizures, neuronal cell loss, aberrant sprouting and distribution of synaptic and inhibitory interneuron markers. However, pilocarpine-induced astrogliosis and reduction in calretinin-positive interneurons were less pronounced in TN-R mutants, thereby resulting in an occlusion of effects induced by TN-R deficiency and pilocarpine. Thus, TN-R-deficient mutants show several electrophysiological and morphological hallmarks of increased neuronal excitability, which, however, do not give rise to more accelerated or severe epileptogenesis in the pilocarpine model of epilepsy.     

Time-matched pre- and postsynaptic changes of GABAergic synaptic transmission in the developing mouse superior colliculus

Developmental changes in the kinetics of GABAergic postsynaptic currents have been reported for various brain structures. However, it has remained unclear whether these modifications are matched by presynaptic changes. We addressed this question by analysing evoked IPSCs (eIPSCs) in mouse superior colliculus slices between postnatal day (P) 1 and 22. eIPSCs were elicited by electrical stimulation and measured in the whole-cell patch-clamp configuration. IPSCs were analysed using the binomial model of synaptic transmission. The readily releasable pool (RRP, N ) was estimated from the cumulative eIPSC amplitude histograms during 50-Hz stimulation. Median delayed IPSC (dIPSC) amplitude was used as a quantal amplitude ( q ) estimate. The mean release probability ( p ) was determined as the mean eIPSC amplitude divided by the product of RRP and q . The experiments revealed that GABAergic synapses pass through two distinct periods of functional adjustment: (i) P1–3 (coincidental with the onset of glutamatergic spontaneous activity and a switch from depolarizing to hyperpolarizing GABA action) displayed a significant decrease of p , associated with an increase in the paired-pulse ratio (eIPSC₂/eIPSC₁); and (ii) P6–15 (the period before and shortly after eye opening) is characterized by a drastic reduction of IPSC duration. On the presynaptic side, it was accompanied by a down-regulation of asynchronous release in favour of stimulus-locked synchronous release. We conclude that postsynaptic modifications of GABAergic synaptic transmission in the superior colliculus (SC) are indeed accompanied by presynaptic changes, and this may guarantee the necessary efficacy of inhibition during the developmental reconstruction of the synaptic network in the SC.     

Astrocytic expression of cannabinoid type 1 receptor in rat and human sclerotic hippocampi

Cannabinoid type 1 receptor (CB1R), which is traditionally located on axon terminals, plays an important role in the pathology of epilepsy and neurodegenerative diseases by modulating synaptic transmission. Using the pilocarpine model of chronic spontaneous recurrent seizures, which mimics the main features of mesial temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) in humans, we examined the expression of CB1R in hippocampal astrocytes of epileptic rats. Furthermore, we also examined the expression of astrocytic CB1R in the resected hippocampi from patients with medically refractory mesial TLE. Using immunofluorescent double labeling, we found increased expression of astrocytic CB1R in hippocampi of epileptic rats, whereas expression of astrocytic CB1R was not detectable in hippocampi of saline treated animals. Furthermore, CB1R was also found in some astrocytes in sclerotic hippocampi in a subset of patients with intractable mesial TLE. Detection with immune electron microscopy showed that the expression of CB1R was increased in astrocytes of epileptic rats and modest levels of CB1R were also found on the astrocytic membrane of sclerotic hippocampi. These results suggest that increased expression of astrocytic CB1R in sclerotic hippocampi might be involved in the cellular basis of the effects of cannabinoids on epilepsy.     

Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures

The septal region of the basal forebrain plays a critical role modulating hippocampal excitability and functional states. Septal circuits may also play a role in controlling abnormal hippocampal hyperexcitability in epilepsy. Both lateral and medial septal neurons are targets of hippocampal axons. Since the hippocampus is an important epileptogenic area in temporal lobe epilepsy, we hypothesize that excessive excitatory output will promote sustained neurodegeneration of septal region neurons. Pilocarpine-induced status epilepticus (SE) was chosen as a model to generate chronic epileptic animals. To determine whether septal neuronal populations are affected by hippocampal seizures, immunohistochemical assays were performed in brain sections obtained from age-matched control, latent period (7 days post-SE) and chronically epileptic (more than one month post-SE survival) rats. An anti-NeuN (neuronal nuclei) antibody was used to study total neuronal numbers. Anti-ChAT (choline acetyltransferase), anti-GAD (glutamic acid decarboxylase) isoenzymes (65 and 67), and anti-glutamate antibodies were used to reveal cholinergic, GABAergic and glutamatergic neurons, respectively. Our results revealed a significant atrophy of medial and lateral septal areas in all chronically epileptic rats. Overall neuronal density in the septum (medial and lateral septum), assessed by NeuN immunoreactivity, was significantly reduced by approximately 40% in chronically epileptic rats. The lessening of neuronal numbers in both regions was mainly due to the loss of GABAergic neurons (80-97% reduction in medial and lateral septum). In contrast, populations of cholinergic and glutamatergic neurons were spared. Overall, these data indicate that septal GABAergic neurons are selectively vulnerable to hippocampal hyperexcitability, and suggest that the processing of information in septohippocampal networks may be altered in chronic epilepsy.     

Synaptic reorganization of calbindin-positive neurons in the human hippocampal CA1 region in temporal lobe epilepsy

The distribution, morphology, synaptic coverage and postsynaptic targets of calbindin-containing interneurons and afferent pathways have been analyzed in the control and epileptic CA1 region of the human hippocampus. Numerous calbindin-positive interneurons are preserved even in the strongly sclerotic CA1 region. The morphology of individual cells is altered: the cell body and dendrites become spiny, the radially oriented dendrites disappear, and are replaced by a large number of curved, distorted dendrites. Even in the non-sclerotic epileptic samples, where pyramidal cells are present and calbindin-immunoreactive interneurons seem to be unchanged, some modifications could be observed at the electron microscopic level: they received more inhibitory synaptic input, and the calbindin-positive excitatory afferents - presumably derived from the CA1, the CA2 and/or the dentate gyrus - are sprouted. In the strongly sclerotic tissue, with the death of pyramidal cells, calbindin-positive terminals (belonging to interneurons and the remaining excitatory afferents) change their targets. Our data suggest that an intense synaptic reorganization takes place in the epileptic CA1 region, even in the non-sclerotic tissue, before the death of considerable numbers of pyramidal cells. Calbindin-positive interneurons participate in this reorganization: they show plastic changes in response to epilepsy. The enhanced inhibition of inhibitory interneurons may result in the disinhibition of pyramidal cells or in an abnormal synchrony in the output region of the hippocampus.     

Activation of kainate receptors controls the number of functional glutamatergic synapses in the area CA1 of rat hippocampus

The expression and functions of kainate-type glutamate receptors (KARs) in the hippocampus are developmentally regulated. In particular, presynaptic KARs depressing glutamate release are tonically activated during early postnatal development, and this activity is down-regulated in parallel with maturation of the synaptic circuitry. In order to understand the physiological relevance of the tonic KAR-mediated signalling, we have here studied the effect of long-term pharmacological activation of KARs on glutamatergic synaptic connectivity in hippocampal slice cultures where presynaptic KARs are expressed but not endogenously activated. Prolonged (16–20 h) activation of the GluR5 subunit-containing KARs using the agonist ATPA (1 μ m ) caused a specific and enduring increase in the number of glutamatergic synapses in area CA1, evidenced as an increase in the frequency of action potential-independent spontaneous EPSCs (mEPSCs) and in immunostaining against synaptic marker proteins. The long-term ATPA treatment had no detectable effect on GABAergic transmission or on glutamate release probability. Further, the effect of ATPA on synaptic density was independent of action potential firing and dependent on protein kinase C. A critical role of endogenous KAR activity in synaptic development was revealed by chronic treatment of the cultures with the selective GluR5 antagonist LY382884, which caused a significant impairment of glutamatergic transmission to CA1 pyramidal neurons. Together, these data suggest a role for the GluR5 subunit-containing KARs in the formation and/or stabilization of functional glutamatergic synapses in area CA1.     

Upregulated SHP‐2 expression in the epileptogenic zone of temporal lobe epilepsy and various effects of SHP099 treatment on a pilocarpine model

Temporal lobe epilepsy (TLE) is defined as the sporadic occurrence of spontaneous recurrent seizures, and its pathogenesis is complex. SHP‐2 (Src homology 2‐containing protein tyrosine phosphatase 2) is a widely expressed cytosolic tyrosine phosphatase protein that participates in the regulation of inflammation, angiogenesis, gliosis, neurogenesis and apoptosis, suggesting a potential role of SHP‐2 in TLE. Therefore, we investigated the expression patterns of SHP‐2 in the epileptogenic brain tissue of intractable TLE patients and the various effects of treatment with the SHP‐2‐specific inhibitor SHP099 on a pilocarpine model. Western blotting and immunohistochemistry results confirmed that SHP‐2 expression was upregulated in the temporal neocortex of patients with TLE. Double‐labeling experiments revealed that SHP‐2 was highly expressed in neurons, astrocytes, microglia and vascular endothelial cells in the epileptic foci of TLE patients. In the pilocarpine‐induced C57BL/6 mouse model, SHP‐2 upregulation in the hippocampus began one day after status epilepticus, reached a peak at 21 days and then maintained a significantly high level until day 60. Similarly, we found a remarkable increase in SHP‐2 expression at 1, 7, 21 and 60 days post‐SE in the temporal neocortex. In addition, we also showed that SHP099 increased reactive gliosis, the release of IL‐1β, neuronal apoptosis and neuronal loss, while reduced neurogenesis and albumin leakage. Taken together, the increased expression of SHP‐2 in the epileptic zone may be involved in the process of TLE.     

Analysis of the Excitatory and Inhibitory Components of Postsynaptic Currents Recorded in Pyramidal Neurons and Interneurons in the Rat Hippocampus

Postsynaptic currents recorded from interneurons and pyramidal cells in hippocampal slices by local voltage clamping were found to be the sum of excitatory (EPSC) and inhibitory (IPSC) components. An approach allowing quantitative assessment of the amplitude and time course of EPSC and IPSC without pharmacological blockade of the major postsynaptic receptors involved in generating these currents was developed. The approach is based on the existence of a significant difference between reversion potentials of cationic and anionic currents and the presence of a linear zone in the voltage-current characteristics of responses to excitatory and inhibitory transmitters. Comparison of the results of this calculation-based method with those of classical pharmacological analysis of the excitatory and inhibitory components of postsynaptic currents showed them to be virtually identical, which allows synaptic currents in defined neurons to be studied without altering the state of synaptic connections throughout the brain slice. IPSC was found to make a smaller contribution to the total postsynaptic current recorded in interneurons as compared with pyramidal neurons in rat hippocampal field CA1.__________Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 90, No. 8, pp. 945–956, August, 2004.     

Network interactions mediated by new excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy

Axon sprouting and synaptic reorganization in the hippocampus are associated with the development of seizures in temporal lobe epilepsy. Synaptic interactions among CA1 pyramidal cells were examined in fragments of hippocampal slices containing only the CA1 area from saline- and kainate-treated rats. Glutamate microapplication to the pyramidal cell layer increased excitatory postsynaptic current (EPSC) frequency, but only in rats with kainate-induced epilepsy. In bicuculline, action potentials evoked in single pyramidal cells increased the frequency of network bursts only in slices from rats with kainate-induced epilepsy. These data further support the hypothesis that excitatory connections between CA1 pyramidal cells increase after kainate-induced status epilepticus.     

Characterization of inhibitory circuits in the malformed hippocampus of Lis1 mutant mice

Heterozygous mutation or deletion of a lissencephaly gene (Lis1) in humans is associated with a severe disruption of cortical and hippocampal lamination, cognitive deficit, and severe seizures. Mice with one null allele of Lis1 (Lis1(+/-) mice) exhibit significant brain malformations and slowed migration of interneuron precursors. Although hyperexcitability was demonstrated in dysplastic hippocampal slices from Lis1(+/-) mice, little is known about synaptic function in these animals. Here we analyzed GABA-mediated synaptic inhibition. We recorded isolated whole cell inhibitory postsynaptic currents (IPSCs) on visually identified pyramidal neurons in disorganized CA1 regions of hippocampal slices prepared from Lis1(+/-) mice. We observed a 32% increase in spontaneous IPSC frequency in Lis1(+/-) mice compared with normotopic CA1 pyramidal neurons in age-matched controls. This increase was not associated with a change in spontaneous IPSC decay or miniature IPSC frequency. Mean IPSC amplitude was increased, and event histograms indicated a greater number of large (>125 pA) events. Tonic inhibition, response to paired-pulse stimulation and evoked IPSC decay kinetics were not altered. Consistent with increased synaptic inhibition, Lis1(+/-) interneurons also exhibited more spontaneous firing in cell-attached recordings and increased excitation as measured by voltage-clamp recording of spontaneous excitatory postsynaptic currents (EPSCs) onto interneurons. Our results reveal a significant alteration in the function of inhibitory circuits within the malformed Lis1(+/-) hippocampus. Given that precisely coordinated GABAergic activity is vital to generation of oscillatory activity and place field precision in hippocampus, these alterations in synaptic inhibition may contribute to seizures and altered cognitive function in type I Lissencephaly.     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Spontaneous synaptic activity is required for the formation of functional GABAergic synapses in the developing rat hippocampus

Here we examine the role of the spontaneous synaptic activity generated by the developing rat hippocampus in the formation of functional γ-aminobutyric acid (GABA) synapses. Intact hippocampal formations (IHFs) were dissected at birth and incubated for 1 day in control or tetrodotoxin (TTX)-supplemented medium at 25°C. After the incubation, miniature GABA_A-mediated postsynaptic currents (mGABA_A-PSCs) were recorded in whole-cell voltage-clamped CA3 pyramidal neurones from IHF-derived slices. After 1 day in vitro in control medium, the frequency of mGABA_A-PSCs was similar to that recorded in acute slices obtained 1 day after birth, but significantly higher than the frequency recorded from acute slices just after birth. These results suggest that the factors required in vivo for the formation of functional GABAergic synapses are preserved in the IHFs in vitro . The frequency increase was prevented when IHFs were incubated for 1 day with TTX. TTX treatment affected neither the morphology of CA3 pyramidal neurones nor cell viability. The TTX effects were reproduced when IHFs were incubated in the presence of glutamatergic or GABAergic ionotropic receptor antagonists or in high divalent cationic medium. The present results indicate that the spontaneous synaptic activity generated by the developing hippocampus is a key player in the formation of functional GABAergic synapses, possibly via network events requiring both glutamatergic and GABAergic receptors.     

Consequences of Epileptic Activity in vitro

Hippocampal sclerosis: a cause or consequence of epileptic activity? This question has concerned neurologists and pathologists for over 150 years. This paper reviews data from an in vitro model system regarding the consequences of epileptic activity of known origin. Exposure of organotypic hippocampal slice cultures to convulsants, such as bicuculline or picrotoxin for three days leads to pronounced neuronal degeneration and a reversible loss of dendritic spines. A similar pathology has been described in hippocampal tissue removed from patients suffering from severe, drug refractory epilepsy. The consequences of such pathological changes are not self-sustaining epileptic activity, as might be expected if such sclerosis caused epilepsy, but rather a selective decrease in synaptic excitation. Inhibitory synaptic transmission and GABAergic interneurons, in contrast, are preserved. At (east two mechanisms contribute to the depression of synaptic excitation: morphological changes in dendritic spines and a decrease in the expression of genes for some glutamatergic receptors. It is hoped that this model will allow the characterization of the mechanisms underlying the pathological consequences of epileptic activity, and lead to useful therapeutic strategies.     

Adenosine down-regulates giant depolarizing potentials in the developing rat hippocampus by exerting a negative control on glutamatergic inputs

Adenosine is a widespread neuromodulator that can be directly released in the extracellular space during sustained network activity or can be generated as the breakdown product of adenosine triphosphate (ATP). Whole cell patch-clamp recordings were performed from CA3 principal cells and interneurons in hippocampal slices obtained from P2-P7 neonatal rats to study the modulatory effects of adenosine on giant depolarizing potentials (GDPs) that constitute the hallmark of developmental networks. We found that GDPs were extremely sensitive to the inhibitory action of adenosine (IC(50) = 0.52 microM). Adenosine also contributed to the depressant effect of ATP as indicated by DPCPX-sensitive changes of ATP-induced reduction of GDP frequency. Similarly, adenosine exerted a strong inhibitory action on spontaneous glutamatergic synaptic events recorded from GABAergic interneurons and on interictal bursts that developed in CA3 principal cells after blockade of gamma-aminobutyric acid type A (GABA(A)) receptors with bicuculline. All these effects were prevented by DPCPX, indicating the involvement of inhibitory A1 receptors. In contrast, GABAergic synaptic events were not changed by adenosine. Consistent with the endogenous role of adenosine on network activity, DPCPX per se increased the frequency of GDPs, interictal bursts, and spontaneous glutamatergic synaptic events recorded from GABAergic interneurons. Moreover, the adenosine transport inhibitor NBTI and the adenosine deaminase blocker EHNA decreased the frequency of GDPs, thus providing further evidence that endogenous adenosine exerts a powerful control on GDP generation. We conclude that, in the neonatal rat hippocampus, the inhibitory action of adenosine on GDPs arises from the negative control of glutamatergic, but not GABAergic, inputs.