CA1 Pyramidal Layer Research Articles

Effects of glucose and glutamine concentration in the formulation of the artificial cerebrospinal fluid (ACSF)

The composition of the ACSF is fundamental in controlling the extracellular environment of the brain slice preparation. ‘Typical’ formulations lack amino acids and contain a higher concentration of glucose (10 mM) than in the cerebrospinal fluid (0.47 – 4.4 mM). We examined the effects of different concentrations of glutamine, the most abundant amino acid in the CSF, and glucose on rat hippocampal slice physiology. Bipolar paired-pulse stimulation was applied to the Schaffer collaterals and population spikes were monitored in the CA1 pyramidal layer for ~1 hour. Addition of glutamine (0.5 mM) to slices superfused with 10 mM of glucose did not enhance population spike amplitude. Higher concentration of glutamine (2-5 mM) resulted in spreading-depression. Decreasing glucose concentration from 10 mM to 5 mM, in the absence of glutamine, attenuated population spikes. Decreasing glucose to 2 mM, in the absence of glutamine, suppressed evoked population spikes. Superfusing brain slices with ACSF containing ‘physiological’ concentrations of both glucose (2 mM) and glutamine (0.5 mM) similarly suppressed population spikes. In separate experiments, during high-K + induced epileptiform activity, glutamine (0.5 mM) did not affect the burst duration, frequency or waveform. These results suggest that the concentration of glucose in ACSF should conservatively be 10 mM in order to maximize paired-pulse population responses while the presence of physiological concentration of glutamine (0.5 mM) has minimal effects on paired-pulse responses and high-K + induced epileptiform activity. These results are discussed in the context of fundamental differences between in vitro brain slice superfusion and in vivo brain perfusion.

Long-term decrease in calbindin-D28K expression in the hippocampus of epileptic rats following pilocarpine-induced status epilepticus

Acquired epilepsy (AE) is characterized by spontaneous recurrent seizures and long-term changes that occur in surviving neurons following an injury such as status epilepticus (SE). Long-lasting alterations in hippocampal Ca(2+) homeostasis have been observed in both in vivo and in vitro models of AE. One major regulator of Ca(2+) homeostasis is the neuronal calcium binding protein, calbindin-D28k that serves to buffer and transport Ca(2+) ions. This study evaluated the expression of hippocampal calbindin levels in the rat pilocarpine model of AE. Calbindin protein expression was reduced over 50% in the hippocampus in epileptic animals. This decrease was observed in the pyramidal layer of CA1, stratum lucidum of CA3, hilus, and stratum granulosum and stratum moleculare of the dentate gyrus when corrected for cell loss. Furthermore, calbindin levels in individual neurons were also significantly reduced. In addition, the expression of calbindin mRNA was decreased in epileptic animals. Time course studies demonstrated that decreased calbindin expression was initially present 1 month following pilocarpine-induced SE and lasted for up to 2 years after the initial episode of SE. The results indicate that calbindin is essentially permanently decreased in the hippocampus in AE. This decrease in hippocampal calbindin may be a major contributing factor underlying some of the plasticity changes that occur in epileptogenesis and contribute to the alterations in Ca(2+) homeostasis associated with AE.

Epileptiform activity in the hippocampus of mice with different predisposition to pinch catalepsy

Experiments on hippocampal slices of CBA mice showed that the probability of spontaneous epileptiform discharges recorded in the field CA1 pyramidal layer is higher in animals predisposed to catalepsy compared to mice with low predisposition to catalepsy. Presumably, some factors determining predisposition to catalepsy modulate synchronization of neuronal activity in the hippocampus; this suggests using hippocampal slices as a model for studies of neurophysiological mechanisms of hereditary predisposition to catalepsy.

Which GABAAReceptor Subunits Are Necessary for Tonic Inhibition in the Hippocampus?

GABA(A) receptors (GABA(A)Rs) assembled of different subunits mediate tonic and phasic inhibition in hippocampal neurons. CA1/CA3 pyramidal cells (PCs) predominantly express alpha5 subunits whereas dentate gyrus granule cells (DGGCs) and molecular layer (ML) interneurons predominantly express delta subunits. Both alpha5- and delta-containing GABA(A)Rs mediate tonic inhibition. We have shown previously that mice lacking alpha5 subunits (Gabra5-/-) have a residual tonic current in CA1/CA3 PCs because of an upregulation of delta subunits, but the basis of the residual tonic current in DGGCs and ML interneurons of mice lacking the delta subunit (Gabrd-/-) is still unknown. We now show that wild-type DGGCs have a small tonic current mediated by alpha5 subunit-containing GABA(A)Rs responsible for approximately 29% of the total tonic current. To better identify the GABA(A)Rs mediating tonic inhibition in hippocampal neurons, we generated mice lacking both alpha5 and delta subunits (Gabra5/Gabrd-/-). Recordings from CA1/CA3 PCs, DGGCs, and ML interneurons in these mice show an absence of tonic currents without compensatory changes in spontaneous IPSCs (sIPSCs), sEPSCs, and membrane resistance. The absence of tonic inhibition results in spontaneous gamma oscillations recordable in vitro in the CA3 pyramidal layer of these mice, which can be mimicked in wild-type mice by blocking alpha5 subunit-containing GABA(A)Rs with 50 nM L-655,708. In conclusion, depending on the cell type, the alpha5 and delta subunits are the principal GABA(A)R subunits responsible for mediating the lion's share of tonic inhibition in hippocampal neurons.

Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5′-Bromo-2′-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ).

Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices

Massive production of free radicals (FR) has been associated with a variety of pathological conditions in the central nervous system (CNS). We have used the FR generating compound hydrogen peroxide (H 2O 2) in organotypic hippocampal slice cultures to model oxidative injury in the brain. Necrotic cell death was monitored for up to 48 h using propidium iodide (PI) and confocal microscopy. A 1 h exposure to H 2O 2 (0.5–2.5 mM) caused a dose-dependent, and region specific cell death in hippocampal slice cultures. Glial cells demonstrated a high degree of vulnerability to H 2O 2. During the initial 3 h post-injury period, regions of the slice where glial cell bodies predominated showed massive cell death. The majority of neurons in the pyramidal layers were spared, though at later time points they appeared damaged as well. Carboxy-dichlorofluorescein imaging revealed a corresponding early increase in ROS generation in glial cells compared to pyramidal neurons. Immunohistochemistry of PI labeled slices identified astrocytes as the cells most sensitive to H 2O 2 toxicity. In dissociated cell cultures of hippocampal astrocytes and neurons, astrocytes also exhibited a significantly higher sensitivity to H 2O 2 than neurons. Hydrogen peroxide-induced cytotoxicity in all regions of the hippocampal slice culture was significantly attenuated by pre-treatment with antioxidants (α-tocopherol and glutathione), and was not prevented by blockade of Ca 2+ influx, or NMDA channel activation. Cyclosporin A, an inhibitor of mitochondrial permeability transition, reduced cytotoxicity in glial areas by more than 50%, while in the CA2–CA3 pyramidal layers a much smaller, but still significant, attenuation of cytotoxicity was observed. Our results suggest that mitochondria are primary targets of H 2O 2 toxicity, particularly in astrocytes.

Abstract 2837: Delayed Calpain Inhibition Is Neuroprotective After Transient Global Brain Ischemia

Introduction/Hypothesis: The delay in neurodegeneration after transient global brain ischemia offers a potentially broad therapeutic window for inhibiting molecular injury mechanisms. One such mechanism, calpain-mediated proteolysis, peaks between 24 and 72 hours after transient forebrain ischemia (TFI) in rats. This study tests the hypothesis that calpain inhibition is neuroprotective when initiated 22 hours after TFI. Methods: Male Long Evans rats (400 – 450 g) were anesthetized (halothane), mechanically ventilated, and instrumented for temperature and hemodynamic monitoring. Each underwent 10 minutes of TFI induced by reversible bilateral carotid occlusion and hypovolemic hypotension (MAP 30 mm Hg). Twenty-two hours after TFI, rats were block randomized (n=6/group) to receive either calpain inhibitor (CEP-3453 (Cephalon), 60 mg/kg IV bolus and 30 mg/kg IV infusion for 50 hours) or vehicle (normal saline IV bolus and infusion for 50 hours). Sham operated rats served as controls. Rats were euthanized 72 hours after injury and brains were processed for immunohistochemistry. Hippocampal CA1 sector calpain activity was analyzed by immunofluorescence using primary antibody that specifically detects calpain-cleaved alpha-spectrin (Ab38). Neurodegeneration was quantified by counting normal appearing Hoechst-stained neuronal nuclei in the hippocampal CA1 pyramidal layer. Ab38 immunofluorescence intensity and normal nuclei counts were compared between vehicle and CEP-3453 treatment groups using a two-tailed Student’s t-test (alpha error 0.05). Results: Baseline and post-injury hemodynamic parameters and temperature were not significantly different between vehicle and CEP-3453 treatment groups. Relative to sham operated controls, mean CA1 sector Ab38 immunofluorescence increased by 263 ± 281% in vehicle treated rats and 68 ± 147% in CEP-3453 treated rats (p=0.17). Normal CA1 pyramidal layer nuclei averaged 10 ± 12% of control in vehicle treated rats and 50 ± 42% of control in CEP-3453 treated rats (p=0.047). Conclusion: These results suggest a causal role for calpains in delayed post-ischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model of transient global brain ischemia.

Shunting and hyperpolarizing GABAergic inhibition in the high‐potassium model of ictogenesis in the developing rat hippocampus

Ontogenesis of GABAergic signaling may play an important role in developmental changes in seizure susceptibility in the high-potassium model of ictogenesis in vitro. The age-dependent effects of [K(+)](o) on the reversal potential of the GABA(A)-mediated responses and membrane potential in hippocampal slices in vitro were compared with the effect of GABA(A)-receptors antagonists and GABA(A) modulators on high-potassium induced seizures in the CA3 pyramidal layer of rat hippocampus in vivo. GABA(A) responses were depolarizing at P8-12 and hyperpolarizing at P17-21. In P8-12 rats, GABA(A) responses switch their polarity from depolarizing to hyperpolarizing upon elevation of extracellular potassium. At approximately 10 mM [K(+)](o), activation of GABA(A) receptors produced an isoelectric, purely shunting response characterized by no changes in the membrane potential but an increase in the membrane conductance. In P17-21 rats, the hyperpolarizing GABA(A) driving force progressively increased with elevation of [K(+)](o). In P8-12 rats in vivo, GABA(A)-receptor antagonists did not affect the occurrence of ictal discharges induced by intrahippocampal injection of 10 mM [K(+)](o), but significantly increased seizure duration. Diazepam and isoguvacine completely prevented seizures induced by 10 mM [K(+)](o). In P17-21 rats, GABA(A)-receptor antagonists strongly increased the occurrence of ictal activity induced both by 10 mM [K(+)](o). Taken together, these results suggest that anticonvulsive effects of GABA are because of the combination of shunting and hyperpolarizing actions of GABA. Although shunting GABA is already efficient in the young age group, a developmental increase in the hyperpolarizing GABA(A) driving force likely contributes to the increase in the GABAergic control of seizures upon maturation.

Design-based estimation of neuronal number and individual neuronal volume in the rat hippocampus

Tools recently developed in stereology were employed for unbiased estimation of the neuronal number and volume in three major subdivisions of rat hippocampus (dentate granular, CA1 and CA3 pyramidal layers).The optical fractionator is used extensively in quantitative studies of the hippocampus; however, the classical optical fractionator design may be affected by tissue deformation in the z-axis of the section. In this study, we applied an improved optical fractionator design to estimate total number of neurons on 100μm thick vibratome sections that had been deformed, in the z-dimension, during histological processing.For estimating cell number, it is necessary to randomize only the location of section planes. But, in the local stereological methods, like cell volume estimation, the orientation of sections must also be randomized. We present a detailed application of a method for making vertical sections from the hippocampus. The volume of hippocampal neurons was estimated using the rotator principle on 40μm thick plastic vertical uniform random sections and corrected for tissue shrinkage. Application of the proposed new design should result in more accurate estimates of neuron number and neuronal volumes in tissue sections affected by homogenous non-uniform shrinkage

Ethyl pyruvate attenuates kainic acid‐induced neuronal cell death in the mouse hippocampus

Recent studies have shown that ethyl pyruvate (EP) acts as an anti-inflammatory molecule in several cell lines including RAW264.7 macrophages. However, the potential therapeutic value of EP for the treatment of the pathologic brain has not been investigated fully. In the present study, we examined whether EP has a beneficial effect on KA-induced neuronal cell death. Intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 mug) of KA produced typical neuronal cell death in the CA1 and CA3 pyramidal layers of the hippocampus, and the systemic administration of EP significantly attenuated KA-induced neuronal cell death in these regions. Ethyl pyruvate was found to exert a protective effect when it was injected as late as 12 hr after KA-injection. Moreover, this EP-induced neuroprotection was accompanied by reduced levels of reactive gliosis and COX-2, IL-1beta, and TNF-alpha in the hippocampus. In addition, in passive avoidance tests, KA-induced memory impairment was improved markedly by EP. These results suggest that EP has a therapeutic potential for suppressing KA-induced pathogenesis in the brain.

Cyclothiazide Prolongs Low [Mg2+]–Induced Seizure-Like Events

Here we address the effects of cyclothiazide (CTZ), an allosteric inhibitor of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization, on low [Mg(2+)]-induced seizure-like events (SLEs) recorded from the CA3 pyramidal layer of juvenile rat hippocampal slices. CTZ (100 microM) made the period of tonic-like discharges (161 +/- 18% of control) and the whole SLE (151 +/- 15% of control) longer (in 7 of 9 slices) or induced endless SLE by stabilizing clonic-like bursting (in 2 of 9 slices). CTZ (30 microM) had no significant effects on SLE dynamics (n = 4), whereas 300 microM CTZ induced endless SLEs in four of eight slices. Co-application of CTZ (100 microM) with 100 microM GYKI-52466, the allosteric inhibitor of AMPA receptor function, restrained the effects of CTZ and shortened SLEs and their tonic phases to 37 +/- 4.2 and 47 +/- 4.2% of the control, respectively. Effects of GYKI-52466 and GYKI-52466 with CTZ on SLE dynamics were indistinguishable. 4-aminopyridine (4-AP; 50 microM) alone (n = 5) or in combination with CTZ (n = 6) transformed recurrent SLE pattern into incessant epileptiform activity with patterns distinguishable from those under 100 microM CTZ application. The effect of 4-AP may suggest a role for facilitated presynaptic glutamate release in disrupting recurrent dynamics. In contrast, the self-similar slow-down of low [Mg(2+)]-induced SLE dynamics by CTZ indicate AMPA receptor desensitization as a parameter shaping SLEs.

Alzheimer's Disease-Like Tau Neuropathology Leads to Memory Deficits and Loss of Functional Synapses in a Novel Mutated Tau Transgenic Mouse without Any Motor Deficits

Tau transgenic mice are valuable models to investigate the role of tau protein in Alzheimer's disease and other tauopathies. However, motor dysfunction and dystonic posture interfering with behavioral testing are the most common undesirable effects of tau transgenic mice. Therefore, we have generated a novel mouse model (THY-Tau22) that expresses human 4-repeat tau mutated at sites G272V and P301S under a Thy1.2-promotor, displaying tau pathology in the absence of any motor dysfunction. THY-Tau22 shows hyperphosphorylation of tau on several Alzheimer's disease-relevant tau epitopes (AT8, AT100, AT180, AT270, 12E8, tau-pSer396, and AP422), neurofibrillary tangle-like inclusions (Gallyas and MC1-positive) with rare ghost tangles and PHF-like filaments, as well as mild astrogliosis. These mice also display deficits in hippocampal synaptic transmission and impaired behavior characterized by increased anxiety, delayed learning from 3 months, and reduced spatial memory at 10 months. There are no signs of motor deficits or changes in motor activity at any age investigated. This mouse model therefore displays the main features of tau pathology and several of the pathophysiological disturbances observed during neurofibrillary degeneration. This model will serve as an experimental tool in future studies to investigate mechanisms underlying cognitive deficits during pathogenic tau aggregation.

Temporal Encoding of Place Sequences by Hippocampal Cell Assemblies

Both episodic memory and spatial navigation require temporal encoding of the relationships between events or locations. In a linear maze, ordered spatial distances between sequential locations were represented by the temporal relations of hippocampal place cell pairs within cycles of theta oscillation in a compressed manner. Such correlations could arise due to spike "phase precession" of independent neurons driven by common theta pacemaker or as a result of temporal coordination among specific hippocampal cell assemblies. We found that temporal correlation between place cell pairs was stronger than predicted by a pacemaker drive of independent neurons, indicating a critical role for synaptic interactions and precise timing within and across cell assemblies in place sequence representation. CA1 and CA3 ensembles, identifying spatial locations, were active preferentially on opposite phases of theta cycles. These observations suggest that interleaving CA3 neuronal sequences bind CA1 assemblies representing overlapping past, present, and future locations into single episodes.

Hippocampal Network Hyperactivity After Selective Reduction of Tonic Inhibition in GABAA Receptor α5 Subunit–Deficient Mice

Functionally, gamma-aminobutyric acid receptor (GABAR)-mediated inhibition can be classified as phasic (synaptic) and tonic (extrasynaptic). The GABARs underlying tonic inhibition assemble from subunits different from those responsible for phasic inhibition. We wanted to assess the excitability of hippocampal pyramidal cell (PC) networks following a selective impairment of tonic inhibition. This is difficult to accomplish by pharmacological means. Because the GABAR alpha5 subunits mostly mediate the tonic inhibition in CA1 and CA3 PCs, we quantified changes in tonic inhibition and examined network excitability in slices of adult gabra5-/- mice. In gabra5-/- CA1 and CA3 PCs tonic inhibitory currents were 60 and 53%, respectively, of those recorded in wild type (WT), with no alterations in phasic inhibition. The amount of tonic inhibition recorded in slices was significantly affected by the method of slice storage (interface or submerged chamber). Field recordings in gabra5-/- CA3 pyramidal layer showed an increased network excitability that was decreased by the GABAR agonist muscimol at a concentration that restored the tonic inhibition of gabra5-/- PCs to the WT level without altering phasic inhibition. Through a battery of pharmacological experiments, we have identified delta subunit-containing GABARs as the mediators of the residual tonic inhibition in gabra5-/- PCs. Our study is consistent with an important role of tonic inhibition in the control of hippocampal network excitability and highlights selective enhancers of tonic inhibition as promising therapeutic approaches for diseases involving network hyperexcitability.

Place-Selective Firing of CA1 Pyramidal Cells during Sharp Wave/Ripple Network Patterns in Exploratory Behavior

We observed sharp wave/ripples (SWR) during exploration within brief (<2.4 s) interruptions of or during theta oscillations. CA1 network responses of SWRs occurring during exploration (eSWR) and SWRs detected in waking immobility or sleep were similar. However, neuronal activity during eSWR was location dependent, and eSWR-related firing was stronger inside the place field than outside. The eSPW-related firing increase was stronger than the baseline increase inside compared to outside, suggesting a "supralinear" summation of eSWR and place-selective inputs. Pairs of cells with similar place fields and/or correlated firing during exploration showed stronger coactivation during eSWRs and subsequent sleep-SWRs. Sequential activation of place cells was not required for the reactivation of waking co-firing patterns; cell pairs with symmetrical cross-correlations still showed reactivated waking co-firing patterns during sleep-SWRs. We suggest that place-selective firing during eSWRs facilitates initial associations between cells with similar place fields that enable place-related ensemble patterns to recur during subsequent sleep-SWRs.

Transplantation of cultured astrocytes attenuates degenerative changes in rats with kainic acid-induced brain damage

Viability of astrocyte grafts introduced into CA1 pyramidal layer of the left dorsal hippocampus after injection of kainic acid into this brain region and the effects of these grafts on the hippocampus and amygdala were studied on Wistar rats. In rats with astrocyte grafts the degree of destruction in fields CA1-CA2 of the dorsal and ventral hippocampus, fields CA3-CA4 of the ventral hippocampus, and central and basolateral amygdala was lower compared to animals with kainic acid-induced hippocampal damage and control rats; destructions in the dentate fascia were absent. Our results suggest that astrocyte grafts stimulate neurogenesis in the mature brain of recipient rats with kainic acid-induced brain damage.

Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development

Although organotypic hippocampal slice cultures (OHSCs) are used to study function within the hippocampus, the effect of maintenance in vitro upon protein expression is not fully understood. Therefore, we examined developmental changes in cultures prepared from P8 rats and maintained on porous membranes between medium and atmosphere. Between 7 and 28 days following explantation, altered hippocampal morphology could not be detected despite a significant decrease in both MAP-2c and a mid-range tau isoform by 21 DIV. During the same period, lower GFAP expression was observed, and GFAP labeling suggested a migration of astrocytes to the slice–atmosphere interface. In contrast, levels of the synaptic proteins synaptophysin and PSD-95 were significantly increased, but GAP-43 was not. The preservation of myelinated axons and synapses, along with glial and endothelial cells, was confirmed by ultrastructural analysis. Furthermore, intranuclear inclusion bodies, which are associated with normal aging in vivo, were detected in the CA1 pyramidal layer in cultures older than 14 DIV. When OHSCs were maintained for approximately 3, 4, and 10 weeks, a rise and then fall in the expression of synaptophysin and, especially, PSD-95 were found, and the biphasic trend paralleled by significant changes in Schaffer collateral-evoked excitatory post-synaptic potentials from CA1 neurons. Our data not only describe changes in cytoskeletal, synaptic, and nuclear proteins related to the maintenance of interface OHSCs, but also emphasize the potential of the model for the study of age-related phenomena within the hippocampus.

Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling

Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

Differential expression and regulation of the cAMP‐selective phosphodiesterase type 4A splice variants in rat brain by chronic antidepressant administration

Chronic antidepressant treatment up-regulates the cAMP cascade in limbic brain regions, suggesting that activation of this pathway contributes to the therapeutic efficacy of antidepressants. A role for cAMP signaling is supported by the finding that rolipram, a selective inhibitor of cAMP-specific phosphodiesterases type 4 (PDE4), has antidepressant efficacy in behavioral models of depression and in clinical trials. To elucidate further the role of PDE4 isozymes, we characterized the expression and regulation of PDE4A splice variants (i.e. PDE4A1, PDE4A5, PDE4A8 and PDE4A10) in rat brain by chronic antidepressant treatment. Initial in situ hybridization studies (ISH) revealed high levels of PDE4A1 mRNA in medial septum, diagonal band, olfactory system, hippocampus and cerebellum. PDE4A5 mRNA expression was restricted to the olfactory nuclei, deep cortical layers, dentate and CA1 pyramidal layers. PDE4A10 mRNA was localized in the dentate gyrus and CA1 pyramidal layers. PDE4A8 mRNA was absent in rat brain. We determined the influence of chronic fluoxetine or electroconvulsive seizure (ECS) treatments on PDE4A splice variants expression in various brain regions. ISH analysis indicated that chronic fluoxetine or ECS treatments significantly increased PDE4A1, but not PDE4A5 or PDE4A10, mRNA levels in frontal and parietal cortices. ECS increased PDE4A5 levels in the anterior cingulate and frontoparietal cortices, CA1 and dentate gyrus, whereas chronic fluoxetine or ECS treatment increased PDE4A10 levels in the hippocampus. The differential up-regulation of PDE4A splice variants suggests compensatory region-specific responses to the antidepressant-induced increase in cAMP signaling and suggests that these splice variants may be relevant as targets for antidepressant intervention.

How Good Vibes Turn Bad: Rise of Ictal Events From Oscillatory Network Activity. Focus on “Transient Depression of Excitatory Synapses on Interneurons Contributes to Epileptogenesis During Gamma Oscillations in the Mouse Hippocampal Slice”

small alterations in network properties might destabilize the network. In the first, experimental, part of the study, Traub et al. used high concentrations (10 –100 M) of the metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) to evoke transitions from physiological to pathological oscillatory activity in the CA3 region of the hippocampus. Application of DHPG is known to be epileptogenic in vivo, and, under the in vitro conditions used in this study, DHPG resulted in oscillatory field potentials in the gamma range interspersed with epileptiform burst activity recordable from the CA3 pyramidal layer. In addition, very fast oscillations became apparent immediately before and during the bursts. Thus the DHPG-induced in vitro activity mimicked some of the defining features observed during the onset of ictal events in vivo. Subsequently, intracellular recordings showed that the transitions from gamma oscillations to burst activity were accompanied by an increase in excitatory input and a decrease in GABAergic input to CA3 pyramidal neurons. Conversely, the excitatory synaptic activity onto interneurons of the pyramidal layer sharply declined prior to the occurrence of a population burst, transiently weakening the interneuronal control of the pyramidal cells.