A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg2+-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg2+-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture.

The complex circuitry of the CA3 region and the abundance of collateral connections has made it difficult to study the mossy fiber pathway in hippocampal slices and therefore to establish the site of expression of long-term potentiation at these synapses. Using a novel cell culture system, we have produced long-term potentiation of the elementary synaptic connections on single CA3 pyramidal neurons following tetanic stimulation of individual dentate gyrus granule cells. As is the case for the hippocampal slice, this potentiation was independent of N-methyl-D-aspartate receptor activation, was simulated by application of forskolin, and its induction did not require any modulatory input. The increase in synaptic strength was accompanied by a reduction in the number of failures of transmission and by an increase in the coefficient of variation of the responses and was prevented by presynaptic injection of an inhibitor of protein kinase A. These findings show that mossy fiber long-term potentiation has a presynaptic locus and that its expression is dependent on protein kinase A.

Distinct short-term plasticity at two excitatory synapses in the hippocampus

A single mossy fiber input contains several release sites and is located on the proximal portion of the apical dendrite of CA3 neurons. It is, therefore, well suited to exert a strong influence on pyramidal cell excitability. Accordingly, the mossy fiber synapse has been referred to as a detonator or teacher synapse in autoassociative network models of the hippocampus. The very low firing rates of granule cells [Jung, M. W. & McNaughton, B. L. (1993) Hippocampus 3, 165–182], which give rise to the mossy fibers, raise the question of how the mossy fiber synapse temporally integrates synaptic activity. We have therefore addressed the frequency dependence of mossy fiber transmission and compared it to associational/commissural synapses in the CA3 region of the hippocampus. Paired pulse facilitation had a similar time course, but was 2-fold greater for mossy fiber synapses. Frequency facilitation, during which repetitive stimulation causes a reversible growth in synaptic transmission, was markedly different at the two synapses. At associational/commissural synapses facilitation occurred only at frequencies greater than once every 10 s and reached a magnitude of about 125% of control. At mossy fiber synapses, facilitation occurred at frequencies as low as once every 40 s and reached a magnitude of 6-fold. Frequency facilitation was dependent on a rise in intraterminal Ca2+ and activation of Ca2+/calmodulin-dependent kinase II, and was greatly reduced at synapses expressing mossy fiber long-term potentiation. These results indicate that the mossy fiber synapse is able to integrate granule cell spiking activity over a broad range of frequencies, and this dynamic range is substantially reduced by long-term potentiation.

Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A)

Synaptic vesicle protein 2 (SV2) is a membrane glycoprotein common to all synaptic and endocrine vesicles. Unlike many proteins involved in synaptic exocytosis, SV2 has no homolog in yeast, indicating that it performs a function unique to secretion in higher eukaryotes. Although the structure and protein interactions of SV2 suggest multiple possible functions, its role in synaptic events remains unknown. To explore the function of SV2 in an in vivo context, we generated mice that do not express the primary SV2 isoform, SV2A, by using targeted gene disruption. Animals homozygous for the SV2A gene disruption appear normal at birth. However, they fail to grow, experience severe seizures, and die within 3 weeks, suggesting multiple neural and endocrine deficits. Electrophysiological studies of spontaneous inhibitory neurotransmission in the CA3 region of the hippocampus revealed that loss of SV2A leads to a reduction in action potential-dependent γ-aminobutyric acid (GABA)ergic neurotransmission. In contrast, action potential-independent neurotransmission was normal. Analyses of synapse ultrastructure suggest that altered neurotransmission is not caused by changes in synapse density or morphology. These findings demonstrate that SV2A is an essential protein and implicate it in the control of exocytosis.

Electrophysiological evidence for a hyperpolarizing, galanin (1-15)-selective receptor on hippocampal CA3 pyramidal neurons

The effects of the 29-amino acid neuropeptide galanin [GAL (1–29)], GAL(1–15), GAL(1–16), and the GAL subtype 2 receptor agonist d-tryptophan2-GAL(1–29) were studied in the dorsal hippocampus in vitro with intracellular recording techniques. GAL(1–15) induced, in the presence of tetrodotoxin, a dose-dependent hyperpolarization in hippocampal CA3 neurons. Most of the GAL(1–15)-sensitive neurons did not respond to GAL(1–29), GAL(1–16), or d-tryptophan2-GAL(1–29). These results indicate the presence of a distinct, yet-to-be cloned GAL(1–15)-selective receptor on CA3 neurons in the dorsal hippocampus.

Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice.

Long-term potentiation (LTP) has been shown to be impaired in mice deficient in the brain-derived neurotrophic factor (BDNF) gene, as well as in a number of other knockout animals. Despite its power the gene-targeting approach is always fraught with the danger of looking at the cumulative direct and indirect effects of the absence of a particular gene rather than its immediate function. The re-expression of a specific gene at a selective time point and at a specific site in gene-defective mutants presents a potent procedure to overcome this limitation and to evaluate the causal relationship between the absence of a particular gene and the impairment of a function in gene-defective animals. Here we demonstrate that the re-expression of the BDNF gene in the CA1 region almost completely restores the severely impaired LTP in hippocampal slices of BDNF-deficient mice. The results therefore provide strong evidence for the direct involvement of BDNF in the process of LTP.

Cooperative interactions in the induction of long-term potentiation and depression of synaptic excitation between hippocampal CA3-CA1 cell pairs in vitro.

The requirement for cooperative interactions between multiple synaptic inputs in the induction of long-term potentiation (LTP) and long-term depression (LTD) has been tested at Schaffer collateral synapses with paired recordings from monosynaptically coupled CA3-CA1 cell pairs in rat hippocampal slice cultures. Tetanization of single presynaptic neurons at 50 Hz (repeated 5-7 times for 300-500 ms each) induced only a transient potentiation (< 3 min) of excitatory postsynaptic potentials (EPSPs). Persistent potentiation (> 15 min) was induced only when single presynaptic action potentials were synchronously paired with directly induced postsynaptic depolarizing pulses (repeated 50-100 times). Tetanus-induced potentiation of extracellularly evoked EPSPs lasting > 4 min could only be obtained if the EPSP was > 4 mV. Because unitary EPSP amplitudes average approximately 1 mV, we conclude that high-frequency discharge must occur synchronously] in 4-5 CA3 cells for LTP to be induced in a common postsynaptic CA1 cell. Asynchronous pairing of presynaptic action potentials with postsynaptic depolarizing current pulses (preceding each EPSP by 800 ms) depressed both naive and previously potentiated unitary EPSPs. Likewise, homosynaptic LTD of unitary EPSPs was induced when the presynaptic cell was tetanized at 3 Hz for 3 min, regardless of their amplitude (0.3-3.2 mV). Homosynaptic LTD of extracellularly evoked Schaffer collateral EPSPs < 4 mV could be induced if no inhibitory postsynaptic potential was apparent, but was prevented by eliciting a large inhibitory postsynaptic potential or by injection of hyperpolarizing current in the postsynaptic cell. We conclude that cooperative interactions among multiple excitatory inputs are not required for induction of homosynaptic LTD of unitary EPSPs.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.

Context-sensitive synaptic plasticity and temporal-to-spatial transformations in hippocampal slices

Hippocampal slices are used to show that, as a temporal input pattern of activity flows through a neuronal layer, a temporal-to-spatial transformation takes place. That is, neurons can respond selectively to the first or second of a pair of input pulses, thus transforming different temporal patterns of activity into the activity of different neurons. This is demonstrated using associative long-term potentiation of polysynaptic CA1 responses as an activity-dependent marker: by depolarizing a postsynaptic CA1 neuron exclusively with the first or second of a pair of pulses from the dentate gyrus, it is possible to “tag” different subpopulations of CA3 neurons. This technique allows sampling of a population of neurons without recording simultaneously from multiple neurons. Furthermore, it reflects a biologically plausible mechanism by which single neurons may develop selective responses to time-varying stimuli and permits the induction of context-sensitive synaptic plasticity. These experimental results support the view that networks of neurons are intrinsically able to process temporal information and that it is not necessary to invoke the existence of internal clocks or delay lines for temporal processing on the time scale of tens to hundreds of milliseconds.

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13–36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50–65% in the CA3 stratum oriens 110–140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110–120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1–3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.

Hippocampal, but not amygdala, activity at encoding correlates with long-term, free recall of nonemotional information

Participation of two medial temporal lobe structures, the hippocampal region and the amygdala, in long-term declarative memory encoding was examined by using positron emission tomography of regional cerebral glucose. Positron emission tomography scanning was performed in eight healthy subjects listening passively to a repeated sequence of unrelated words. Memory for the words was assessed 24 hr later with an incidental free recall test. The percentage of words freely recalled then was correlated with glucose activity during encoding. The results revealed a striking correlation (r = 0.91, P < 0.001) between activity of the left hippocampal region (centered on the dorsal parahippocampal gyrus) and word recall. No correlation was found between activity of either the left or right amygdala and recall. The findings provide evidence for hippocampal involvement in long-term declarative memory encoding and for the view that the amygdala is not involved with declarative memory formation for nonemotional material.

Experimental diabetes in rats causes hippocampal dendritic and synaptic reorganization and increased glucocorticoid reactivity to stress

We report that 9 d of uncontrolled experimental diabetes induced by streptozotocin (STZ) in rats is an endogenous chronic stressor that produces retraction and simplification of apical dendrites of hippocampal CA3 pyramidal neurons, an effect also observed in nondiabetic rats after 21 d of repeated restraint stress or chronic corticosterone (Cort) treatment. Diabetes also induces morphological changes in the presynaptic mossy fiber terminals (MFT) that form excitatory synaptic contacts with the proximal CA3 apical dendrites. One effect, synaptic vesicle depletion, occurs in diabetes as well as after repeated stress and Cort treatment. However, diabetes produced other MFT structural changes that differ qualitatively and quantitatively from other treatments. Furthermore, whereas 7 d of repeated stress was insufficient to produce dendritic or synaptic remodeling in nondiabetic rats, it potentiated both dendritic atrophy and MFT synaptic vesicle depletion in STZ rats. These changes occurred in concert with adrenal hypertrophy and elevated basal Cort release as well as hypersensitivity and defective shutoff of Cort secretion after stress. Thus, as an endogenous stressor, STZ diabetes not only accelerates the effects of exogenous stress to alter hippocampal morphology; it also produces structural changes that overlap only partially with those produced by stress and Cort in the nondiabetic state.

Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10−14–10−8 M) and des(1–3) IGF-I (10−10–10−8 M) were found to inhibit whereas IGF-II (10−14–10−8 M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.

Ensemble codes involving hippocampal neurons are at risk during delayed performance tests

Multielectrode recording techniques were used to record ensemble activity from 10 to 16 simultaneously active CA1 and CA3 neurons in the rat hippocampus during performance of a spatial delayed-nonmatch-to-sample task. Extracted sources of variance were used to assess the nature of two different types of errors that accounted for 30% of total trials. The two types of errors included ensemble “miscodes” of sample phase information and errors associated with delay-dependent corruption or disappearance of sample information at the time of the nonmatch response. Statistical assessment of trial sequences and associated “strength” of hippocampal ensemble codes revealed that miscoded error trials always followed delay-dependent error trials in which encoding was “weak,” indicating that the two types of errors were “linked.” It was determined that the occurrence of weakly encoded, delay-dependent error trials initiated an ensemble encoding “strategy” that increased the chances of being correct on the next trial and avoided the occurrence of further delay-dependent errors. Unexpectedly, the strategy involved “strongly” encoding response position information from the prior (delay-dependent) error trial and carrying it forward to the sample phase of the next trial. This produced a miscode type error on trials in which the “carried over” information obliterated encoding of the sample phase response on the next trial. Application of this strategy, irrespective of outcome, was sufficient to reorient the animal to the proper between trial sequence of response contingencies (nonmatch-to-sample) and boost performance to 73% correct on subsequent trials. The capacity for ensemble analyses of strength of information encoding combined with statistical assessment of trial sequences therefore provided unique insight into the “dynamic” nature of the role hippocampus plays in delay type memory tasks.