Use-dependent shift from inhibitory to excitatory GABAA receptor action in SP-O interneurons in the rat hippocampal CA3 area

Cortical inhibitory interneurons set the pace of synchronous neuronal oscillations implicated in synaptic plasticity and various cognitive functions. The hyperpolarizing nature of inhibitory postsynaptic potentials (IPSPs) in interneurons has been considered crucial for the generation of oscillations at beta (15-30 Hz) and gamma (30-100 Hz) frequency. Hippocampal basket cells and axo-axonic cells in stratum pyramidale-oriens (S-PO) play a central role in the synchronization of the local interneuronal network as well as in pacing of glutamatergic principal cell firing. A lack of conventional forms of plasticity in excitatory synapses onto interneurons facilitates their function as stable neuronal oscillators. We have used gramicidin-perforated and whole cell clamp recordings to study properties of GABAAR-mediated transmission in CA3 SP-O interneurons and in CA3 pyramidal cells in rat hippocampal slices during electrical 5- to 100-Hz stimulation and during spontaneous activity. We show that GABAergic synapses onto SP-O interneurons can easily switch their mode from inhibitory to excitatory during heightened activity. This is based on a depolarizing shift in the GABAA reversal potential (EGABA-A), which is much faster and more pronounced in interneurons than in pyramidal cells. We also found that the shift in interneuronal function was frequency dependent, being most prominent at 20- to 40-Hz activation of the GABAergic synapses. After 40-Hz tetanic stimulation (100 pulses), GABAA responses remained depolarizing for approximately 45 s in the interneurons, promoting bursting in the GABAergic network. Hyperpolarizing EGABA-A was restored >60 s after the stimulus train. Similar but spontaneous GABAergic bursting was induced by application of 4-aminopyridine (100 microM) to slices. A shift to depolarizing IPSPs by the GABAAR permeant weak acid anion formate provoked interneuronal population bursting, supporting the role of GABAergic excitation in burst generation. Furthermore, depolarizing GABAergic potentials and synchronous interneuronal bursting were enhanced by pentobarbital (100 microM), a positive allosteric modulator of GABAARs, and were blocked by picrotoxin (100 microM). Intriguingly, GABAergic bursts displayed short (<1 s) oscillations at 15-40 Hz, even though only depolarizing GABAA responses were seen in the SP-O interneurons. This beta-gamma rhythmicity in the interneuron network was dependent on electrotonic coupling, and was abolished by blockade of gap junctions with carbenoxolone (200 microM). Results here implicate the rapid activity-dependent degradation of hyperpolarizing IPSPs in SP-O interneurons in setting the temporal limits for a given interneuron to participate in beta-gamma oscillations synchronized by GABAergic synapses. Furthermore, they imply that mutual GABAergic excitation provided by interneurons may be an integral part in the function of neuronal networks. We suggest that the use-dependent change in EGABA-A could represent a form of short-term plasticity in interneurons promoting coherent and sustained activation of local GABAergic networks.     

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.     

Cortistatin-expressing interneurons require TrkB signaling to suppress neural hyper-excitability

Signaling of brain-derived neurotrophic factor (BDNF) via tropomyosin receptor kinase B (TrkB) plays a critical role in the maturation of cortical inhibition and controls expression of inhibitory interneuron markers, including the neuropeptide cortistatin (CST). CST is expressed exclusively in a subset of cortical and hippocampal GABAergic interneurons, where it has anticonvulsant effects and controls sleep slow-wave activity (SWA). We hypothesized that CST-expressing interneurons play a critical role in regulating excitatory/inhibitory balance, and that BDNF, signaling through TrkB receptors on CST-expressing interneurons, is required for this function. Ablation of CST-expressing cells caused generalized seizures and premature death during early postnatal development, demonstrating a critical role for these cells in providing inhibition. Mice in which TrkB was selectively deleted from CST-expressing interneurons were hyperactive, slept less and developed spontaneous seizures. Frequencies of spontaneous excitatory post-synaptic currents (sEPSCs) on CST-expressing interneurons were attenuated in these mice. These data suggest that BDNF, signaling through TrkB receptors on CST-expressing cells, promotes excitatory drive onto these cells. Loss of excitatory drive onto CST-expressing cells that lack TrkB receptors may contribute to observed hyperexcitability and epileptogenesis.

     

Selective induction of metabotropic glutamate receptor 1- and metabotropic glutamate receptor 5-dependent chemical long-term potentiation at oriens/alveus interneuron synapses of mouse hippocampus

Synaptic plasticity in inhibitory interneurons is essential to maintain a proper equilibrium between excitation and inhibition in hippocampal network. Recent studies showed that theta-burst-induced long-term potentiation (LTP) at excitatory synapses of oriens/alveus (O/A) interneurons in CA1 hippocampal region required the activation of metabotropic glutamate receptor (mGluR) 1. However these interneurons also express mGluR5 and the contribution of this receptor subtype in interneuron synaptic plasticity remains unexplored. We combined pharmacological and transgenic approaches to examine the relative contribution of mGluR1/5 in LTP at excitatory synapses on O/A interneurons. Bath-application of the selective mGluR1/5 agonist (s)-3,5-dihydroxyphenylglycine (DHPG) induced LTP of compound excitatory postsynaptic potentials. DHPG-induced LTP was not prevented by application of either mGluR1 or mGluR5 antagonists, was still present in mGluR1 knockout mice, but was blocked by co-application of both antagonists. These results indicate that LTP can be induced at O/A interneuron synapses by either mGluR1 or mGluR5 activation. As previously reported for mGluR1-dependent LTP, the mGluR5-dependent LTP was independent of N-methyl-d-aspartate receptors. Pairing DHPG application with postsynaptic depolarization induced mGluR1- and mGluR5-dependent LTP of minimally-evoked excitatory postsynaptic currents, which were composed of calcium-permeable AMPA receptor and presynaptically modulated by group II mGluRs, hence confirming that both forms of LTP occurred directly at interneuron excitatory synapses. These findings uncover a new mGluR5-dependent form of LTP at O/A interneuron synapses and indicate that activation of mGluR1 or mGluR5 is sufficient to induce LTP at these synapses. Thus, a rich repertoire of adaptive changes may take place at these interneuron synapses to regulate hippocampal feedback inhibition.     

Glycine-gated chloride channels depress synaptic transmission in rat hippocampus

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus is beginning to be appreciated. We have reported previously that CA1 pyramidal cells and GABAergic interneurons recorded in 3- to 4-wk-old rat hippocampal slices express functional GlyRs, dispelling previous misconceptions that GlyR expression ceases in early development. However, the effect of GlyR activation on cell excitability and synaptic circuits in hippocampus has not been fully explored. Using whole cell current-clamp recordings, we show that activation of strychnine-sensitive GlyRs through exogenous glycine application causes a significant decrease in input resistance and prevents somatically generated action potentials in both CA1 pyramidal cells and interneurons. Furthermore, GlyR activation depresses the synaptic network by reducing suprathreshold excitatory postsynaptic potentials (EPSPs) to subthreshold events in both cell types. Blockade of postsynaptic GlyRs with the chloride channel blocker 4, 4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS) or altering the chloride ion driving force in recorded cells attenuates the synaptic depression, strongly indicating that a postsynaptic mechanism is responsible. Increasing the local glycine concentration by blocking reuptake causes a strychnine-sensitive synaptic depression in interneuron recordings, suggesting that alterations in extracellular glycine will impact excitability in hippocampal circuits. Finally, using immunohistochemical methods, we show that glycine and the glycine transporter GlyT2 are co-localized selectively in GABAergic interneurons, indicating that interneurons contain both inhibitory neurotransmitters. Thus we report a novel mechanism whereby activation of postsynaptic GlyRs can function to depress activity in the synaptic network in hippocampus. Moreover, the co-localization of glycine and GABA in hippocampal interneurons, similar to spinal cord, brain stem, and cerebellum, suggests that this property is likely to be a general characteristic of inhibitory interneurons throughout the CNS.     

Changes in excitatory and inhibitory circuits of the rat hippocampus 12-14 months after complete forebrain ischemia.

Changes in interneuron distribution and excitatory connectivity have been investigated in animals which had survived 12-14 months after complete forebrain ischemia, induced by four-vessel occlusion. Anterograde tracing with Phaseolus vulgaris leucoagglutinin revealed massive Schaffer collateral input even to those regions of the CA1 subfield where hardly any surviving pyramidal cells were found. Boutons of these Schaffer collaterals formed conventional synaptic contacts on dendritic spines and shafts, many of which likely belong to interneurons. Mossy fibres survived the ischemic challenge, however, large mossy terminals showed altered morphology, namely, the number of filopodiae on these terminals decreased significantly. The entorhinal input to the hippocampus did not show any morphological alterations. The distribution of interneurons was investigated by neurochemical markers known to label functionally distinct GABAergic cell populations. In the hilus, spiny interneurons showed a profound decrease in number. This phenomenon was not as obvious in CA3, but the spiny metabotropic glutamate receptor 1alpha-positive non-pyramidal cells, some of which contain calretinin or substance P receptor, disappeared from stratum lucidum of this area. In the CA1 region, somatostatin immunoreactivity disappeared from stratum oriens/lacunosum-moleculare-associated cells, while in metabotropic glutamate receptor 1alpha-stained sections these cells seemed unaffected in number. Other interneurons did not show an obvious decrease in number. In stratum radiatum of the CA1 subfield, some interneuron types had altered morphology: the substance P receptor-positive dendrites lost their characteristic radial orientation, and the metabotropic glutamate receptor 1alpha-expressing cells became extremely spiny. The loss of inhibitory interneurons at the first two stages of the trisynaptic loop coupled with a well-preserved excitatory connectivity among the subfields suggests that hyperexcitability in the surviving dentate gyrus and CA3 may persist even a year after the ischemic impact. The dorsal CA1 region is lost; nevertheless hyperactivity, if it occurs, may have a route to leave the hippocampus via the longitudinally extensive axon collaterals of CA3 pyramidal cells, which may activate the subiculum and entorhinal cortex with a relay in the surviving ventral hippocampal CA1 region.     

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy

Voltage-gated sodium channels (Na(V)) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a-/- mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/- mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of Na(V)1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/- and Scn1a-/- mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of Na(V)1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/- heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.     

Action of tachykinins in the hippocampus: facilitation of inhibitory drive to GABAergic interneurons

By acting on neurokinin 1 (NK1) receptors, neuropeptides of the tachykinin family can powerfully excite rat hippocampal GABAergic interneurons located in the CA1 region and by this way indirectly inhibit CA1 pyramidal neurons. In addition to contact pyramidal neurons, however, GABAergic hippocampal interneurons can also innervate other interneurons. We thus asked whether activation of tachykinin-sensitive interneurons could indirectly inhibit other interneurons. The study was performed in hippocampal slices of young adult rats. Synaptic events were recorded using the whole-cell patch clamp technique. We found that substance P enhanced GABAergic inhibitory postsynaptic currents in a majority of the interneurons tested. Miniature, action potential-independent inhibitory postsynaptic currents were unaffected by substance P, as were evoked inhibitory synaptic currents. This suggests that the peptide acted at the somatodendritic membrane of interneurons, rather than at their axon terminals. The effect of substance P was mimicked by a selective NK1 receptor agonist, but not by neurokinin 2 (NK2) or neurokinin 3 (NK3) receptor agonists, and was suppressed by a NK1 selective receptor antagonist. In contrast to substance P, oxytocin, another peptide capable of activating hippocampal interneurons, had no effect on the inhibitory synaptic drive onto interneurons. We conclude that tachykinins, by acting on NK1 receptors, can influence the hippocampal activity by indirectly inhibiting both pyramidal neurons and GABAergic interneurons. Depending on the precise balance between these effects, tachykinins may either activate or depress hippocampal network activity.     

alpha7 nicotinic acetylcholine receptors on GABAergic interneurons evoke dendritic and somatic inhibition of hippocampal neurons.

GABAergic interneurons in the hippocampus express high levels of alpha7 nicotinic acetylcholine receptors, but because of the diverse roles played by hippocampal interneurons, the impact of activation of these receptors on hippocampal output neurons (i.e., CA1 pyramidal cells) is unclear. Activation of hippocampal interneurons could directly inhibit pyramidal neuron activity but could also produce inhibition of other GABAergic cells leading to disinhibition of pyramidal cells. To characterize the inhibitory circuits activated by these receptors, exogenous acetylcholine was applied directly to CA1 interneurons in hippocampal slices, and the resulting postsynaptic responses were recorded from pyramidal neurons or interneurons. Inhibitory currents mediated by GABA(A) receptors were observed in 27/131 interneuron/pyramidal cell pairs, but no instances of disinhibition of spontaneous inhibitory events or GABA(B) receptor-mediated responses were observed. Two populations of bicuculline-sensitive GABA(A) receptor-mediated currents could be distinguished based on their kinetics and amplitude. Anatomical reconstructions of the interneurons in a subset of connected pairs support the hypothesis that these two populations correspond to inhibitory synapses located either on the somata or dendrites of pyramidal cells. In 11 interneuron/interneuron cell pairs, one presynaptic neuron was observed that produced strong inhibitory currents in several nearby interneurons, suggesting that disinhibition of pyramidal neurons may also occur. All three types of inhibitory responses (somatic-pyramidal, dendritic-pyramidal, and interneuronal) were blocked by the alpha7 receptor-selective antagonist methyllycaconitine. These data suggest activation of these functionally distinct circuits by alpha7 receptors results in significant inhibition of both hippocampal pyramidal neurons as well as interneurons.     

Postnatal shifts of interneuron position in the neocortex of normal and reeler mice: evidence for inward radial migration

During development, interneurons migrate to precise positions in the cortex by tangential and radial migration. The objectives of this study were to characterize the net radial migrations of interneurons during the first postnatal week, and to investigate the role of reelin signaling in regulating those migrations. To observe radial migrations, we compared the laminar positions of interneurons (immunoreactive for GABA or Dlx) in mouse neocortex on postnatal days (P) 0.5 and P7.5. In addition, we used bromodeoxyuridine birthdating to reveal the migrations of different interneuron cohorts. To study the effects of reelin deficiency, experiments were performed in reeler mutant mice. In normal P0.5 cortex, interneurons were most abundant in the marginal zone and layer 5. By P7.5, interneurons were least abundant in the marginal zone, and were distributed more evenly in the cortical plate. This change was attributed mainly to inward migration of middle- to late-born interneurons (produced on embryonic days (E) 13.5 to E16.5) from the marginal zone to layers 2-5. During the same interval, late-born projection neurons (non-immunoreactive for GABA or Dlx) migrated mainly outward, from the intermediate zone to upper cortical layers. In reeler cortex, middle- and late-born interneurons migrated from the superplate on P0.5, to the deep cortical plate on P7.5. Late-born projection neurons in reeler migrated in the opposite direction, from the intermediate zone to the deep cortical plate. We conclude that many middle- and late-born interneurons migrate radially inward, from the marginal zone (or superplate) to the cortical plate, during the first postnatal week in normal and reeler mice. We propose that within the cortical plate, interneuron laminar positions may be determined in part by interactions with projection neurons born on the same day in neurogenesis.     

Inhibition and disinhibition of pyramidal neurons by activation of nicotinic receptors on hippocampal interneurons

Nicotinic acetylcholine receptors (nAChRs) are expressed in the hippocampus, and their functional roles are beginning to be delineated. The effect of nAChR activation on the activity of both interneurons and pyramidal neurons in the CA1 region was studied in rat hippocampal slices. In CA1 stratum radiatum with muscarinic receptors inhibited, local pressure application of acetylcholine (ACh) elicited a nicotinic current in 82% of the neurons. The majority of the ACh-induced currents were sensitive to methyllycaconitine, which is a specific inhibitor of alpha7-containing nAChRs. Methyllycaconitine-insensitive nicotinic currents also were present as detected by a nonspecific nAChR inhibitor. The ACh-sensitive neurons in the s. radiatum were identified as GABAergic interneurons by their electrophysiological properties. Pressure application of ACh induced firing of action potentials in approximately 70% of the interneurons. The ACh-induced excitation of interneurons could induce either inhibition or disinhibition of pyramidal neurons. The inhibition was recorded from the pyramidal neuron as a burst of GABAergic synaptic activity. That synaptic activity was sensitive to bicuculline, indicating that GABA(A) receptors mediated the ACh-induced synaptic currents. The disinhibition was recorded from the pyramidal neuron as a reduction of spontaneous GABAergic synaptic activity when ACh was delivered onto an interneuron. Both the inhibition and disinhibition were sensitive to either methyllycaconitine or mecamylamine, indicating that activation of nicotinic receptors on interneurons was necessary for the effects. These results show that nAChRs are capable of regulating hippocampal circuits by exciting interneurons and, subsequently, inhibiting or disinhibiting pyramidal neurons.     

Imaging of 4-AP-induced, GABA(A)-dependent spontaneous synchronized activity mediated by the hippocampal interneuron network.

Under conditions of increased excitability, such as application of the K(+) channel blocker 4-aminopyridine (4-AP, 100 microM), interneurons in the hippocampal slice show an additional form of synchronized activity that is distinct from the ictal and interictal epileptiform activity induced by these manipulations. In principal neurons, i.e., pyramidal and granule cells, this synchronized interneuron activity (SIA) generates large, multi-component synaptic potentials, which have been termed long-lasting depolarizations (LLDs). These LLDs are dependent on GABA(A) receptor-mediated synaptic transmission but not on excitatory amino acid (EAA) receptors. Intracellular recordings from hilar interneurons have shown that depolarizing GABA(A) receptor-mediated synaptic potentials are also largely responsible for the synchronization of interneurons. The spatiotemporal characteristics of this interneuron activity have not been investigated previously. Using a voltage-sensitive dye and optical techniques that are capable of recording spontaneous synchronized activity, we have characterized the spatiotemporal pattern of SIA (in the presence of 4-AP + EAA receptor antagonists) and compared it with interictal epileptiform activity (in 4-AP only). Like interictal activity, SIA could be observed throughout the hippocampal slice. Unlike interictal activity, which originated in area CA2/CA3 and spread from there, SIA was most prominent in area CA1 and originated either there or in the subiculum. In CA1, interictal activity was largest in and near stratum pyramidale, while SIA was mainly located in s. lacunosum moleculare. Furthermore SIA was equally likely to propagate in either direction, and multiple patterns of propagation could be observed within a single brain slice. These studies suggest that hippocampal area CA1 has the highest propensity for SIA, that multiple locations can serve as the site of origin, and that interneurons located in s. lacunosum moleculare or interneurons that specifically project to this region may be particularly important for synchronized interneuron activity.     

Excitatory GABA input directly drives seizure-like rhythmic synchronization in mature hippocampal CA1 pyramidal cells

GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABA(A) receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABA(A) receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average -38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABA(A) receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy.     

Interneuron loss reduces dendritic inhibition and GABA release in hippocampus of aged rats

Aging is associated with impairments in learning and memory and a greater incidence of limbic seizures. These changes in the aged brain have been associated with increased excitability of hippocampal pyramidal cells caused by a reduced number of gamma-aminobutyric acid-ergic (GABAergic) interneurons. To better understand these issues, we performed cell counts of GABAergic interneurons and examined GABA efflux and GABAergic inhibition in area CA1 of the hippocampus of young (3-5 months) and aged (26-30 months) rats. Aging significantly reduced high K⁺/Ca²⁺-evoked GABA, but not glutamate efflux in area CA1. Immunostaining revealed a significant loss of GABAergic interneurons, but not inhibitory boutons in stratum oriens and stratum lacunosum moleculare. Somatostatin-immunoreactive oriens-lacunosum moleculare (O-LM) cells, but not parvalbumin-containing interneurons were selectively lost. Oriens-lacunosum moleculare cells project to distal dendrites of CA1 pyramidal cells, providing dendritic inhibition. Accordingly, inhibition of dendritic input to CA1 from entorhinal cortex was selectively reduced. These findings suggest that the age-dependent loss of interneurons impairs dendritic inhibition and dysregulates entorhinal cortical input to CA1, potentially contributing to cognitive impairment and seizures.     

Nicotinic acetylcholine receptor alpha7 and alpha4beta2 subtypes differentially control GABAergic input to CA1 neurons in rat hippocampus.

The hippocampus, a limbic brain region involved in the encoding and retrieval of memory, has a well-defined structural network assembled from excitatory principal neurons and inhibitory interneurons. Because the GABAergic interneurons form synapses onto both pyramidal neurons and interneurons, the activation of nicotinic acetylcholine receptors (nAChRs) present on certain interneurons could induce either inhibition or disinhibition in the hippocampal circuitry. To understand the role of nAChRs in controlling synaptic transmission in the hippocampus, we evaluated the magnitude of nAChR-modulated GABAergic postsynaptic currents (PSCs) in pyramidal neurons and various interneurons of the CA1 region. Using whole cell patch-clamp recording and post hoc identification of neuronal types in rat hippocampal slices, we show that brief (12-s) nAChR activation by ACh (1 mM) or choline (10 mM) enhances the frequency of GABAergic PSCs in both pyramidal neurons and CA1 interneurons. The magnitude of alpha7 nAChR-mediated GABAergic inhibition, as assessed by the net charge of choline-induced PSCs, was highest in stratum lacunosum moleculare interneurons followed by pyramidal neurons and s. radiatum interneurons. In contrast, the magnitude of alpha4beta2 nAChR-mediated GABAergic inhibition, as assessed by the difference between the net charge of PSCs induced by ACh and choline, was highest in pyramidal neurons followed by s. lacunosum moleculare and s. radiatum interneurons. The present results suggest that cholinergic cues transmitted via specific subtypes of nAChRs modify the synaptic function in the hippocampus by inducing a differential degree of GABAergic inhibition in the target neurons.     

A fundamental oscillatory state of isolated rodent hippocampus

Population neuronal rhythms of various frequencies are observed in the rodent hippocampus during distinct behavioural states. However, the question of whether the hippocampus exhibits properties of spontaneous rhythms and population synchrony in isolation has not been definitively answered. To address this, we developed a novel preparation for studying neuronal rhythms in a relatively large hippocampal tissue in vitro. We isolated the whole hippocampus from mice up to 28 days postnatal age, removing the dentate gyrus while preserving the functional CA3-to-CA1 connections. Placing the hippocampal isolate in a perfusion chamber for electrophysiological assessment extracellular recordings from the CA1 revealed rhythmic field potential of 0.5 to ≤ 4 Hz that occurred spontaneously and propagated along the ventro-dorsal hippocampal axis. We provide convergent evidence, via measurements of extracellular pH and K⁺, recordings of synaptic and intracellular activities and morphological assessments, verifying that these rhythms were not the consequence of hypoxia. Data obtained via simultaneous extracellular and patch clamp recordings suggest that the spontaneous rhythms represent a summation of GABAergic IPSPs originating from pyramidal neurons, which result from synchronous discharges of GABAergic inhibitory interneurons. Similar spontaneous field rhythms were also observed in the hippocampal isolate prepared from young gerbils and rats. Based on these data, we postulate that the spontaneous rhythms represent a fundamental oscillatory state of the hippocampal circuitry isolated from extra-hippocampal inputs.     

Enhancement of the excitatory actions of GABA by barbiturates and benzodiazepines.

Whole cell recordings from hippocampal CA1 pyramidal neurons using electrode chloride concentrations of 12-80 mM demonstrated that the effect of synaptic activation of GABAA receptors was dependent on the transmembrane chloride gradient. When the chloride reversal potential was positive to action potential threshold, GABAA receptor activation was excitatory, and anticonvulsant barbiturates and benzodiazepines enhanced this excitation. Enhancement of GABAergic excitation of interneurons may contribute to the efficacy of these drugs, while enhancement of GABAergic excitation of principal neurons may be an important mechanism of failure, such as occurs in the treatment of neonatal seizures.     

Postnatal development and migration of cholecystokinin-immunoreactive interneurons in rat hippocampus

The development of cholecystokinin-immunoreactive (CCK-IR) interneurons in the rat hippocampus was studied using immunocytochemical methods at the light and electron microscopic levels from early (P0-P8) to later postnatal (P12-P20) periods. The laminar distribution of CCK-IR cell bodies changed considerably during the studied period, which is suggested to be due to migration. CCK-IR cells appear to move from the molecular layer of the dentate gyrus to their final destination at the stratum granulosum/hilus border, and tend to concentrate in the distal third of stratum radiatum in CA1-3. The density of CCK-IR cells is rapidly decreasing during the first 4 postnatal days without any apparent reduction in their total number, therefore it is due to the pronounced growth of hippocampal volume in this period. Axons of CCK-IR interneurons formed symmetrical synapses already at P0, and by far the predominant targets were dendrites of presumed principal cells in all subfields of the hippocampus. These axon arbors began to concentrate around pyramidal cell bodies only at P8, at earlier ages CCK-IR axons crossed stratum pyramidale at right angles, and gave rise to varicose collaterals only outside this layer. The dendrites and somata of CCK-IR cells received synapses already at P0, but those were mostly symmetrical, apart from a few immature asymmetrical synapses. At P4, mature asymmetrical synapses with considerable amounts of synaptic vesicles were already commonly encountered. Thus, the innervation of CCK-IR interneurons apparently develops later than their output synapses, suggesting that they may be able to release transmitter before receiving any considerable excitatory drive. We conclude that CCK-IR cells represent one, if not the major, interneuron type that assists in the maturation of glutamatergic synapses (activation of N-methyl-D-aspartate receptors) via GABAergic depolarization of principal cell dendrites, and may contribute to the generation of giant depolarizing potentials. CCK-IR cells will change their function to perisomatic hyperpolarizing inhibition, as glutamatergic transmission in the network becomes operational.     

Dendritic A-type potassium channel subunit expression in CA1 hippocampal interneurons

Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and potassium channel interacting protein (KChIP) 1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal Ammon's horn (CA1) neurons. Immunohistochemistry was performed on 40 mum rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with a unique dendritic A-type channel complement and ability to control BAPs.